WO2014020129A2

WO2014020129A2 - Allatostatin-b peptides for inducing the settlement of lophotrochozoan marine larvae

Info

Publication number: WO2014020129A2
Application number: PCT/EP2013/066233
Authority: WO
Inventors: Gáspár JÉKELY; Elizabeth Williams; Markus CONZELMANN
Original assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Current assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Priority date: 2012-08-01
Filing date: 2013-08-01
Publication date: 2014-02-06
Anticipated expiration: 2015-02-01
Also published as: WO2014020129A3

Abstract

The present invention relates to a method of inducing or enhancing the settlement and/or growth of one or more larva of Lophotrochozoan marine invertebrates, such as molluscs, in aquaculture. The method comprises culturing the one or more larva in a composition, whereby the composition comprises or consists essentially of one or more peptides derived from an allatostatin-b polypeptide. Further, peptides derived from an allatostatin-b polypeptide and compositions comprising same are provided.

Description

Allatostatin-B peptides for inducing the settlement of Lophotrochozoan marine larvae

The present invention relates to a method of inducing or enhancing the settlement and/or growth of one or more larva of Lophotrochozoan marine invertebrates, such as molluscs, in aquaculture. The method comprises culturmg the one or more larva in a composition, whereby the composition comprises or consists essentially of one or more peptides derived from an allatostatin-b polypeptide. Further, peptides derived from an allatostatin-b polypeptide and compositions comprising same are provided.

Metazoan life cycles show a great diversity in larval, juvenile and adult forms and in the timing and ecological context of the transitions between these forms. Neuroendocrine signals involving hormones and neuropeptides generally underlie these transitions in all investigated animal species, but no neuropeptide has been found to regulate these transitions across different phyla (1, 2). Given these limitations, the origins of animal life cycle transitions are unclear.

In Lophotrochozoan marine invertebrates, the typical life cycle consists of a planktonic larva that settles to the ocean floor and metamorphoses into a benthic juvenile. This transition is often triggered by chemical cues from the environment (10). The apical organ, an anterior cluster of larval sensory neurons (11), has been implicated in the larval to juvenile transition in Lophotrochozoan marine invertebrates (12, 13). For example, the ciliated larvae of the annelid genus Platynereis, like the larval stages of other marine invertebrates, are planktonic and spend several days in the open water. Subsequently, the larvae settle at suitable settlement sites, and grow into benthic juvenile worms (metamorphosis). The early planktonic swimming phase and the process of settlement are regulated by a very simple nervous system in these larvae (see Jekely (2008) Nature 456, 395-399). However, the neural mechanisms underlying the transformation of environmental cues into habitat and morphological change during settlement and metamorphosis are poorly understood. Since most animal phyla are marine, understanding these mechanisms in marine organisms is needed for a more comprehensive view of metazoan life cycle evolution.

With the depletion of natural stocks in the world's oceans, there is a growing interest in Lophotrochozoan marine invertebrate aquaculture. Several species, including various scallops, oysters and abalones, are being cultured for commercial purposes. Lophotrochozoan marine aquaculture represents a large and fast growing industry. The production of oysters and miscellaneous marine molluscs amounted to 5.6 million tonnes worldwide in 2004 with an annual rate of growth of 5.3 %. In Europe, mollusc aquaculture production was 658,000 tonnes in 2008, contributing to 26 % of the volume of the total European aquaculture production (FAO (2010) Fisheries and Aquaculture Circular No. 1061/1). The limiting factor in mollusc aquaculture is the generation of spat that settle on the culture substrate. Most of mollusc aquaculture is based on natural spat collection that is highly vulnerable to algal blooms, and sensitive to climatic conditions or pollutions. For this reason, inland hatcheries are increasingly used for the major commercial species (scallops, mussels, oysters). Such commercially operating hatcheries therefore represent a growing market worldwide and hatchery-produced spat are expected to supply an ever-increasing portion of the global aquaculture industry (FAO (2010) Fisheries and aquaculture technical paper 500/1).

A bottleneck in the aquaculture production of these Lophotrochozoan marine invertebrates, like molluscs, is the induction of the settlement and growth of the planktonic larval stages into settled juveniles ("spat"). Andersen (2011), Can. J. Zool. 89 (579-598) suggests optimization of the culture environment (flow rates, light conditions, temperature, feed concentration) to improve settlement. Magnesen (2007) reports on a land-based raceway nursery to increase yield and spat production (see Magnesen (2007), Aquacultural Engineering 36, 149-158.

An improved efficiency of spat production and a faster larval growth in commercial hatcheries could have a significant impact on the aquaculture industry. Such an improved efficiency would potentially also allow the hatchery production of spat for species that traditionally rely on natural spat collection in the sea (e.g. oyster). An expanded sector of sustainable aquaculture could relieve pressure on natural fisheries. This could have beneficial environmental impacts, since many natural populations of molluscs are currently overfished and endangered (e.g. abalone). The aquaculture industry can also benefit local economies. Moreover, hatchery-produced spat is sometimes used to restock depleted mollusc populations. Such a use could help the recovery of local fisheries (FAO (2010) Fisheries and Aquaculture Circular No. 1061/1; FAO (2010) Fisheries and aquaculture technical paper 500/1. Enhancing the settlement and growth of Lophotrochozoan marine invertebrate larvae would therefore greatly facilitate and advance aquaculture.

Thus, the technical problem underlying the present invention is the provision of means and methods to facilitate and advance commercial aquaculture of marine invertebrates, in particular of molluscs and shellfish for nourishment and/or food consumption.

The technical problem is solved by provision of the embodiments characterized in the claims. Accordingly, the present invention relates to a method of inducing or enhancing the settlement and/or growth of one or more larva of Lophotrochozoan marine invertebrates comprising culturing said one or more larva in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide.

It was found that the herein provided peptides derived from an allatostatin-b polypeptide can be advantageously used in the aquaculture of marine invertebrates. Therefore, the induction or enhancement of the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates may relate to the induction or enhancement of the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates in aquaculture, comprising culturing said one or more larva in aquaculture in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide.

Accordingly, the present invention relates to a method of inducing or enhancing the settlement and/or growth of one or more larva of Lophotrochozoan marine invertebrates, wherein the induction or enhancement of the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates relates to the induction or enhancement of the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates in aquaculture, comprising culturing said one or more larva in aquaculture in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide.

In other words, the present invention relates to a method of inducing or enhancing the settlement and/or growth of one or more larva of Lophotrochozoan marine invertebrates in aquaculture, said method comprising culturing said one or more larva in aquaculture in a composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide.

As shown herein in Example 4 and Figure 30, the prior art FLamide peptide induced sinking of swimming marine invertebrate larvae, however, the prior art peptides do not induce or enhance settlement of the larvae. It is important that the term "Settlement" as used herein is different to "sinking of swimming", because "settlement" involves attachment of the larvae to a surface, like a substrate or sediment or sculture vessel surface.

In an alternative aspect, the present invention relates to a method of inducing or enhancing the settlement and/or growth of one or more larva of marine invertebrates in aquaculture, comprising culturing said one or more larva in aquaculture in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide. All explanations and definitions given herein in relation to the "method of inducing or enhancing the settlement and/or growth of one or more larva of Lophotrochozoan marine invertebrates comprising culturing said one or more larva in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide" apply, mutatis mutandis, in this context.

The term "aquaculture" as used herein is well known in the art. The term relates, for example, to the farming of aquatic organisms, such as the marine invertebrates as defined herein (e.g. Lophotrochozoan marine invertebrates like trochozoans (such as mollusks and annelids)). Therefore, "aquaculture" is also referred to as "aquafarming" and both terms can be used interchangeably herein. Aquaculture as used herein may involve cultivating saltwater populations of marine invertebrates under controlled conditions (in contrast to commercial fishing). Envisaged herein is a specific type of aquaculture, termed "mari culture" which refers particularly to the cultivation of marine invertebrates, like the marine Lophotrochozoan invertebrates as defined herein. Mariculture may involve said cultivation in the open ocean, an enclosed section of the ocean (i.e. in a semi-natural environment), or in tanks, ponds or raceways which are filled with saline water, like brackish water or seawater. Often bivalves are cultivated in brackish water, while mollusks are often cultivated in seawater.

In relation to the culturing of edible molluscs as defined herein, like shellfish (e.g. bivalves/mussels), the term„aquaculture" refers to„aquaculture" for industrial purpose or commercial purpose, like for food production or for food industry. Therefore,„aquaculture" refers to culturing mollusc larva as a source of food (like food for humans). In other words, „aquaculture" refers to the culturing of mollusc larva for consumption (like for human consumption).„Aquaculture" for conservational environmental purpose (like aquaculture as part of an environmental program, such as restoration of coral reefs, or like an aid in conserving endangered species is not contemplated herein. It is to be understood that the term „shellfish" refers to edible„shellfish".

In relation to the culturing of annelids as defined herein, the term„aquaculture" refers to „aquaculture" for industrial purpose or commercial purpose, like for bait production or for bait industry. Therefore,„aquaculture" refers to culturing annelid larva as a source of bait (like fish bait). In other words,„aquaculture" refers to the culturing of annelid larva for use as bait (like fish bait). Again,„aquaculture" for conservational environmental purpose (like aquaculture as part of an environmental program, such as restoration of coral reefs, or like an aid in conserving endangered species is not contemplated herein. „Aquaculture" as used herein may involve the culturing of the marine invertebrate larvae in hatcheries. A hatchery is usually a facility where eggs are hatched under artificial conditions. As used herein the term "hatchery" refers especially to a facility where marine invertebrate larvae are cultured from the "egg stage" to the larval stage or even to the "spat" stage (sometimes even to the mature (adult) stage). Hatcheries can be used to cultivate molluscs to sell for food eliminating the need to find the molluscs in the wild. They may provide some species outside of their natural season. In such a case, they may raise the mollusc until the molluscs are ready to be eaten. The term "saline water" as used herein refers to water that contains a significant concentration of dissolved salts (particularly NaCl), like brackish water or seawater. The concentration of the dissolved salts is often expressed in parts per million (ppm) of salt. For example, if water has a concentration of 10,000 ppm of dissolved salts, then one percent (10,000 divided by 1,000,000) of the weight of the water comes from dissolved salts. Based on the salinity concentration level saline water may be classified in three categories. Slightly saline water contains around 1,000 to 3,000 ppm. Moderately saline water contains roughly 3,000 to 10,000 ppm. Highly saline water has around 10,000 to 35,000 ppm of salt. Seawater usually has a salinity of roughly 35,000 ppm. Thus, saline water to be used herein may contain a concentration of dissolved salts of from about 1,000 to 50,000 ppm, such as about 10,000 to about 35,000 ppm. If seawater is to be used, the concentration of dissolved salts is of from 34,000 to 36,000 ppm, like about 35,000 ppm.

Also brackish water may be used in aquaculture herein. Brackish water is water that has more salinity than fresh water, but usually not as much as seawater. Brackish water may cover a range of salinity regimes. It is characteristic of many brackish surface waters that their salinity can vary considerably over space and/or time. If brackish water is to be used, the concentration of dissolved salts may therefore range from about 500 to about 35,000 ppm.

Surprisingly, it has been found herein for the first time that peptides can induce settlement and growth of Lophotrochozoan marine invertebrate larvae. As shown in Example 1 and Figure 11, peptides derived from an allatostatin-b precursor peptide induce ciliar closure of the invertebrate larvae, which, in turn results in sinking of the larvae. Unexpectedly, the allatostatin-b peptides did not only affect swimming behaviour of the larvae, as it has been observed with other unrelated peptides (see Conzelmann (2011) PNAS 108, E1174-E1183) the allatostatin-b peptides induced settlement of the larvae, i.e. the cilia-driven attachment of the apical side to the bottom of the culture vessel; see Figure 21. In contrast, the peptides disclosed in Conzelmann (2011; loc. cit.) do not induce settlement; see Example 4 and Figure 30. Cnidarian peptides unrelated to allatostatin-b are known to play a role in the metamorphosis of Cnidarian larvae (see Takahashi (2011) J Amino Acids Article ID 424501), Gajewski (1996) Roux's Arch Dev Biol 205, 232-242; Iwao (2002) Coral Reefs 21, 127-129; Plickert (2003) Int J Dev Biol 47, 439-450; Erwin (2010) Coral Reefs 29, 929-939). The PhD thesis of Eisuke Hayakawa (2006) describes conserved peptides in Hydra. However, it was not suggested that these Cnidarian peptides might be useful to induce settlement of Lophotrochozoa.

As shown in Example 6 and Fig. 33, cnidarian GLWamides, and diverse protostome Wamides (GWamides, MIPs (allatostatin-b peptides), form a cluster of homologous peptides. These peptides share an amidated Trp residue which is preceded by a small aliphatic residue. Yet, protostome MIPs have another conserved Trp (W-Xg-g-Wamide motif) that is lacking from LWamides and GWamides (Fig. 40). Although GWamides and MIPs (allatostatin-b peptides) cluster together, only MIPs (the herein provided peptides derived from an allatostatin-b polypeptide as defined and explained herein) are capable of inducing settlement as defined herein, i.e. only MPs (peptides derived from an allatostatin-b polypeptide) induce attachment peptides and GWamide peptides do not induce settlement, i.e. the larvae do not attach to a surface.

The settlement of the herein provided peptides derived from an allatostatin-b polypeptide was followed by enhanced growth and feeding of the larvae. Thus, these results provide evidence that allatostatin-b derived peptides can be used to trigger and accelerate the development of Lophotrochozoan marine invertebrate larvae to settled juveniles (spat) and mature animals, facilitating and enhancing aquaculture of Lophotrochozoan marine invertebrates such as molluscs and cephalopods.

The term "settlement" as used herein, refers to the transition from a planktonic form into a benthic form during the life cycle of a Lophotrochozoan marine invertebrate. "Settlement" comprises two features, namely, as a first feature, the reduction in the activity of cilia of the Lophotrochozoan marine invertebrates, in particular the more frequent and longer closures of all cilia in the ciliary bands, and, as a second feature, attachment to a surface of the Lophotrochozoan marine invertebrates, of the Lophotrochozoan marine invertebrates characterized by the cilia-driven attachment of the lateral or apical side of the Lophotrochozoan marine larva(e) to the culture vessel or sediment or substrate, like to the surface (e.g. bottom or wall) of the culture vessel or sediment surface or substrate surface. The term "ciliar closure" as used herein refers to the the fact that ciliary become less active, because the cilia close more frequently for longer periods. The reduced activity of cilia triggers sinking of the larvae. The attachment is characterized by the cilia-driven attachment of the apical side of the Lophotrochozoan marine larva(e) to a surface, the bottom of the culture vessel or sediment surface or substrate, i.e. the larvae are no longer "swimming" but are "sessile". "Sessile" can mean in this context that settled larvae no longer swim. For example, the settled larvae no longer move in a three-dimensional, but move in or along a two-dimensional direction, e.g. they crawl on the surface as defined above. „Crawling" may mean that the larvae crawl on the surface using their cilia. "Sessile" can mean that the settled larvae do not move or move only very little from their place of settlement. The settled larvae canfirmly attach to the surface as defined above (e.g. wall or bottom of the culture vessel or sediment surface or substrate surface. "Firm attachment" can involve irreversible attachment. For example, some species secrete a gluey material to promote attachment. This can be seen as an example of irreversible attachment.

"Settled larvae" can show a substrate exploratory behaviour, for example, they may move in small jumps. The substrate exploratory behaviour may include frequent contact with the substrate/substrate surface.

As mentioned above, "Settled larvae" can show a "sessile" behaviour (i.e. can be sessile), like crawling on the surface (e.g. substrate surface and the like)/ substrate etc.

Both features, i.e. the reduction of cilia activity and the attachment of the Lophotrochozoan marine larva(e) to the surface (e.g. bottom or wall of the culture vessel or sediment surface or substrate surface), need to be observed in order to qualify the behavior of larvae as settlement. If marine invertebrate larvae "settle", they attach to the bottom or wall (or generally surface) of the culture vessel or sediment surface or substrate surface as describe above.

If the culturing of the marine invertebrate larvae is in a "non-natural environment", the culturing of the marine invertebrates in a composition may involve culturing in a culture vessel (e.g. tanks, ponds or raceways which may be filled with saline water, like brackish water or seawater.). Accordingly, the marine invertebrates may„settle", i.e. attach to the bottom or wall (or generally surface) of said culture vessel(s).

If the culturing of the marine invertebrate larvae is in a "natural environment" (like in the open ocean) or in a„semi-natural environment" (like an enclosed section of the ocean), the marine invertebrates may„settle", i.e. attach to the sediment surface or substrate. Examples of such a sediment surface or substrate are sand bottoms, rocky outcrops, coral, bay mud, culch and the like. The term "growth" as used herein, refers to the increase in size of an animal and a progression through consecutive developmental stages (e.g. the addition of new segments). Growth can be determined by measuring the length of an animal, or by scoring morphological progression through development.

The induction of the settlement and growth of the planktonic larval stages results in the metamorphosis of the planktonic larvae into settled juveniles ("spat"). An assay for determining whether (a) Lophotrochozoan marine invertebrate Iarva(e) (or a population of Lophotrochozoan marine invertebrate larva(e) settles is provided in the appended example and may be performed as follows: the swimming activity of the larvae can be assayed using video microscopy and tracking the larvae, either in vertical or horizontal chambers. A significant reduction in average swimming speed or the percent of time that larvae swim on average can be regarded to satisfy condition 1) of settlement. The behaviour of the larvae can be further monitored and their contact with the substrate can be scored. A significant increase in the number of larvae that show contact with the substrate can be considered to satisfy condition 2) of settlement.

The benthic zone is the ecological region at the lowest level of a body of water such as an ocean or a lake, including the sediment surface and some sub-surface layers. Organisms living in this zone are called benthos. They generally live in close relationship with the substrate bottom; many such organisms are permanently attached to the bottom. The superficial layer of the soil lining the given body of water, the benthic boundary layer, is an integral part of the benthic zone, as it greatly influences the biological activity which takes place there. Examples of contact soil layers (which are exemplary sediment surface or substrates mentioned above) include sand bottoms, rocky outcrops, coral, and bay mud.

In context of the terms "inducing settlement of one or more larva of Lophotrochozoan marine invertebrates" or "inducing growth of one or more larva of Lophotrochozoan marine invertebrates" it is envisaged that (an) individual larva(e) of a given population of larvae of Lophotrochozoan marine invertebrates start settling and/or growing upon culturing in a composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide. In accordance with the above, it is envisaged that a (preferably statistically significantly) higher portion/percentage of larvae of a given population of larvae of Lophotrochozoan marine invertebrates start settling and or growing upon culturing in said composition compared with a control population of Lophotrochozoan marine invertebrate larvae (control larvae being cultured in a composition in the absence of the peptide(s) derived from an allatostatin-b polypeptide). For example, one or more larvae of a given population cultured in said composition may start settling and/or growing, whereas none of the larvae of a given control population starts settling and/or growing.

In context of the terms "enhancing settlement of one or more larva of Lophotrochozoan marine invertebrates" or "enhancing growth of one or more larva of Lophotrochozoan marine invertebrates" it is envisaged that a (preferably statistically significantly) higher portion/percentage of larvae of a given population of larvae of Lophotrochozoan marine invertebrates settles and/or grows upon cultaring in said composition compared with a control population of Lophotrochozoan marine invertebrate larvae (control larvae being cultured in a composition in the absence of the peptide(s) derived from an allatostatin-b polypeptide).

In the prior art a mean metamorphosis ratio of 20 % was described (see Andersen (2011), loc. cit.). It is envisaged herein that at least 20 % 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % and up to 100 % of a given population of Lophotrochozoan marine invertebrate larvae settle and/or grow upon culturing in a composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide. Thus, the mean settlement/metamorphosis ratio in a given population of Lophotrochozoan marine invertebrate larvae is increased by culturing in said composition compared with of a control population of Lophotrochozoan marine invertebrate larvae (control larvae being cultured in a composition in the absence of the peptide(s) derived from an allatostatin-b polypeptide).

Further, it is envisaged that the time until the above indicated percentage of larvae settle and/or grow is (preferably statistically significantly) reduced by culturing in said composition compared with of a control population of Lophotrochozoan marine invertebrate larvae (control larvae being cultured in a composition in the absence of the peptide(s) derived from an allatostatin-b polypeptide). For example, the larvae cultured in said composition may settle and/or grow within 1 days whereas larvae of a control population may settle and/or grow within 5 days.

The herein found capacity of allatostatin-b peptides to induce settlement and growth of Lophotrochozoan marine invertebrate larvae was unexpected, because the function of said peptide in Lophotrochozoan marine invertebrates was, up to now, not known. Unrelated peptides were shown to act as neuropeptides and to influence swimming behaviour (see Conzelmann, loc. cit.). Yet, allatostatin-b peptides were not identified or proposed as potential neuropeptides therein. Thus, a potential role of allatostatin-b peptides in neuronal signalling in Lophotrochozoan marine invertebrates was not even proposed in the art. As shown herein in Example 1 and Fig. 12, allatostatin-b peptides do, in contrast to prior art peptides as disclosed in Conzelmann (loc. cit.) not influence ciliar beat frequency (control ciliary beats per second; 15.47 s.e.m. 0.87 n=l l, with 20 μΜ allatostatin-b peptide: 15.8 s.e.m. 0.48 n=15).Further, they exert their activity via a specific allatostatin-b receptor; see Example 1 and Fig. 3. Unrelated peptides do not bind to said receptor indicating that such unrelated peptides do not induce settlement.

In insects, life cycle transitions are regulated by juvenile hormones and ecdysones and link various larval or juvenile stages to a reproductive adult via successive molts (3). The levels of these hormones are regulated by allatostatin and neuropeptides secreted from specialized neurosecretory cells {4-6). Similar peptides have also been identified in marine mollusks and annelids, but their functions are, as mentioned, not known (7-9). Given the distant relationship of marine invertebrates and insects and the completely different life-cycle in the marine environment, the prior art provided no hint that allatostatin-b derived peptides could induce settlement or growth of marine Lophotrochozoan invertebrate larvae.

As shown in Figure 3D and 3E the herein provided allatostatin-b peptides are surprisingly capable of activating allatostatin-b receptors in a cross-species specific manner in distantly related Lophotrochozoan marine invertebrates like Platynereis and Capitella. This supports that the herein provided allatostatin-b peptides can be used to induce settlement and/or growth of larvae of diverse Lophotrochozoan marine invertebrates, like annelids, and molluscs.

Herein, a family of Lophotrochozoan marine invertebrate small neuropeptides was identified and characterised, which trigger the settlement and metamorphosis of marine annelid larvae. It was found that these peptides are conserved in other Lophotrochozoan marine invertebrates, like molluscs, including commercially relevant species that are produced in large quantities in aquaculture facilities in Europe and worldwide. These peptides can be used to speed up and make larval settlement and growth more efficient in commercially relevant molluscs where spat is produced in inland hatcheries. The effects of the mollusc peptides on larval settlement and growth can, based on the teaching of the present invention, be easily applied to molluscs, for example using two species, the great scallop Pecten maximus with commercial relevance, and the sea hare Aplysia c lifornica, an established laboratory model. Using settlement inducing peptides could help to overcome a major bottleneck in Lophotrochozoan marine invertebrate aquaculture, make the industry more productive, and reduce the reliance on natural spat collection, thereby potentially also contributing to the recovery of natural stocks.

Herein, the G-protein coupled receptor for allatostatin-b derived peptides was identified. The receptor was activated by the peptide in the nanomolar concentration range in cell culture assays. Using electron microscopy the neurosecretory neurons that produce and release allatostatin-b derived peptides were characterised. It was found that these neurons are both neurosecretory and chemosensory. Without being bound by theory, this indicates that allatostatin-b derived peptides have a role in translating environmental chemical signals into settlement behaviour. As note above, it was found that allatostatin-b derived peptides and their receptors are widely distributed among other Lophotrochozoan marine invertebrates, including commercially relevant molluscs, and thus represent an ancient peptide family with a conserved role in the regulation of the planktonic to benthic transition during the life cycle of Lophotrochozoan marine invertebrates. Since annelid and mollusc larvae are similar to each other both ecologically and evolutionarily, the herein provided findings provide means to induce and enhance the settlement and growth of commercially relevant Lophotrochozoan marine invertebrates, like molluscs produced in inland hatcheries.

It is one advantage of the herein provided method that settlement and growth of Lophotrochozoan marine invertebrate larvae can be induced or enhanced without the need for genetic modification (which is a concern for many seafood consumers), simply through culturing the larvae in a composition comprising or consisting essentially of one or more peptides derived from allatostatin-b polypeptide (e.g. through the one-time exposure to the herein described peptides derived from allatostatin-b polypeptide.

Further, it was shown herein that the conserved tryptophane (W) amino acid residue at or near the C-terminus of the allatostatin-b peptides is important for the capacity of the peptides to induce settlement and growth of the Lophotrochozoan marine invertebrate larvae. Mutated peptides having the conserved W amino acid residue at/near the C-terminus replaced with an alanine residue lost the capacity to activate the allatostatin-b receptor (see Example 1 and Fig. 3D, E). These mutants also lost the capacity to induce settlement and/or growth of the larvae (see Example 1, Fig. 13 E, F and Fig. 16 E, F).

While it was found herein that the W amino acid residue at near the C-terminus of the allatostatin-b peptides is important for the capacity of the peptides to induce or enhance settlement/growth of Lophotrochozoan marine invertebrate larvae, the peptides to be used herein maintain said capacity even if the W amino acid residue is not the last residue at the C- terminus of the peptides; see Example 2 and Fig. 20. This is surprising because allatostatin-b peptides produced by enzymatic cleavage of the allatostatin-b precursor in the larvae usually have a C-terminal W amino acid residue. Such naturally occurring peptides derived from precursor proteins by enzymatic cleavage usually also have an amidated C-terminal W amino acid residue. Herein it was unexpectedly found that the amidation of the C-terminal W amino acid residue is dispensible for the capacity to induce or enhance settlement growth of the larvae; see Example 2 and Fig. 20. The peptide(s) derived from allatostatin-b to be used in accordance with the present invention may be a fragment of an allatostatin-b polypeptide. Exemplary allatostatin-b polypeptides are described herein below. The fragment may have any size. Envisaged herein are, for example, fragments having a length of from 2 to 48 amino acids. Further, the use of fragments is envisaged that have or consist of the consensus motif from N to C terminus (X)_nW_a(X)_nZWb(X)n, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_a may be absent. Z may also be L or M. The potential length of the fragments/peptides to be used herein and the consensus motif are described below in more detail.

As mentioned, the methods of the present invention comprise culturing one or more larva of Lophotrochozoan marine invertebrates in a composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide. The term "culturing" as used herein encompasses any method known in the art for culturing cultivating Lophotrochozoan marine invertebrate larvae, provided that the culturing nvolves the use of one or more peptides derived from an allatostatin-b polypeptide. The term "culturing" implies that the larvae are contacted with the one or more peptides. The term "contacted" is used in a wide meaning and may comprise any culturing in the presence of the one or more peptides. The term "culturmg" does however not necessarily imply that the marine Lophotrochozoan invertebrate larvae are cultured during their entire development into juvemle(s)/spat(s) in the presence of the peptide(s) derived from an allatostatin-b polypeptide. For the purpose of the present invention, it may suffice that the larvae are cultured in the presence of an amount of the peptide(s) sufficient to induce or enhance settlement and/or growth (i.e. to induce development into a juvenile) for a relatively short period of time, and that the larvae are, upon induction or enhancement of settlement/growth not cultured in the presence of the peptide or not contacted with the peptide.

In context of "culturing" the larva(e) in the herein described composition, including "contacting" the larvae with the peptide(s), it is to be understood that the culturing/contacting may be simply made by growing the larva(e) in a composition comprising or consisting essentially of the one or more peptides derived from an allatostatin-b polypeptide. For example, the composition may comprise water (like saline water, e.g. brackish water or (natural) seawater) and the larvae may be cultured or grown in said water. The water may comprise or consist essentially of the one or more peptides derived from an allatostatin-b polypeptide before the larva(e) are added to the water. Vice versa, the larvae may be first added to the water and the peptide(s) derived from an allatostatin-b polypeptide may be added subsequently. It is also envisaged that the larvae are cultured in water that comprised or consisted essentially of the one or more peptides derived from an allatostatin-b polypeptide before the larva(e) were added to the water and that the peptides derived from an allatostatin-b polypeptide are again added after the larva(e) were added. For example, it may be that the peptides derived from an allatostatin-b polypeptide are consumed during culturing (e.g. by uptake through the larvae or other organisms, degradation and the like), and that the peptides are (again) added to maintain the desired concentration which is sufficient to induce or enhance settlement and/or growth of the larvae.

The peptides derived from an allatostatin-b polypeptide may, for example, be added to the water in form of a forage, comprising nutrients like phytoplankton and/or zooplankton. It is also envisaged that the peptides derived from an allatostatin-b polypeptide may be added to the water as such, e.g. in form of powder consisting or consisting essentially of said peptides.

It is to be understood that the larvae are cultured under suitable conditions taking advantage of the prior art knowledge on the cultivation of Lophotrochozoan marine invertebrate larvae. Exemplary conditions for culturing Lophotrochozoan marine invertebrate larvae are, for example, described in Andersen (2011), loc. cit. and Torkildsen (2004) Aquaculture International 12, 489-507, which are incorporated herein by reference. Such culturing conditions include, for example, culturing as untreated batch cultures, culturing as chloramphenicol-treated batch cultures, culturing as flow-through cultures with filtered water and culturing as flow-through cultures with water from a biofilter.

It is envisaged herein that the larvae may be cultured in a composition comprising or consisting essentially of structurally identical peptides derived from allatostatin polypeptide. For example, the composition may comprise only one of the following non-limiting exemplary peptides AWMKNNIAW (SEQ ID NO: 38), AWGDNNMRVW (SEQ ID NO: 39), AWNKNSMRVW (SEQ ID NO: 40), AWKGQSARVW (SEQ ID NO: 41), GWNGNSMRVW (SEQ ID NO: 42), KWGSNSMRVW (SEQ ID NO: 43), GWADNNMRVW (SEQ ID NO: 44), RKWSKFSSW (SEQ ID NO: 45), WKNMAVW (SEQ ID NO: 46), WKQMASW (SEQ ID NO: 47), WKQMSVW (SEQ ID NO: 48), WKEMSVW (SEQ ID NO: 49), WKQMAVW (SEQ ID NO: 50), WKQMATW (SEQ ID NO: 51), AWNKNSMRVWP (SEQ ID NO: 52), AWNKNSMRVW A (SEQ ID NO: 53), AW, VW, TW, SW, or NW. The composition may comprise exemplary peptides MW, LW or QW.

It is also envisaged that the larvae may be cultured in a composition which comprises or consists essentially of a mixture (i.e. one or more) structurally non-identical peptides. Such structurally non-identical peptides may have a certain amino acid sequence in common (i.e. an identical "core" sequence) and may only be modified at the N-terminus and/or the C-terminus adjacent in that further amino acid residues are added to the "core" sequence. Such a "core" sequence may be any of the peptide sequences described further below, like the non-limiting exemplary peptides AWMK NIAW (SEQ ID NO: 38), AWGDNNMRVW (SEQ ID NO:39), AWNKNSMRVW (SEQ ID NO: 40), AWKGQSARVW (SEQ ID NO: 41), GWNGNSMRVW (SEQ ID NO: 42), KWGSNSMRVW (SEQ ID NO: 43), GWADNNMRVW (SEQ ID NO: 44), RKWSKFSSW (SEQ ID NO: 45), WKNMAVW (SEQ ID NO: 46), WKQMASW (SEQ ID NO: 47), WKQMSVW (SEQ ID NO: 48), WKEMSVW (SEQ ID NO: 49), W QMAVW (SEQ ID NO: 50), WKQMATW (SEQ ID NO: 51), AWNKNSMRVWP (SEQ ID NO:52), or AWNKNSMRVWA (SEQ ID NO: 53). The "core" sequence may even be shorter and comprise or consist of only two amino acids, like AW, VW, TW, SW, or NW. The core sequences may comprise or consist of MW, LW or QW. Structurally non-identical peptides may (in addition or in the alternative to adding amino acids at the C-terminus and/or the N-terminus as outlined above) also have an identical amino acid sequence with the exception that one or more amino acids are deleted/inserted/replaced as further explained below. Structurally non-identical peptides may, for example, merely have a consenus motif in common, whereby the consensus motif from N to C terminus is (XJ_nWapQ_nZW_biX)!_!, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_a may be absent. Z may be L, M or Q. Thus, the use of a mixture of any of the herein provided structurally non-identical peptides is envisaged in context of the present invention.

The allatostatins (AST) are known as a family of insect neuropeptides of at least three peptide groups: A-type allatostatins, which have the common C-terminal sequence Y/FXFGLamide; the B-type (cricket-type) allatostatins which have the common C-terminal sequence, W(X(6))W amide, and C-type allatostatins which have a non-amidated C terminus, and a structure unrelated to other allatostatins. "Allatostatins" are also known under the synonyms '^yoinhibiting peptide (MIP)" or "prothoracicostatic peptide (PTSP"); these terms can be used interchangeably herein. The A-type allatostatins are inhibitory insect neuropeptides discovered in cockroaches through their capacity to inhibit juvenile hormone biosynthesis. B- type allatostatins inhibit the biosynthesis of juvenile hormones in crickets. C-type allatostatin was first discovered in the moth, Manduca sexta and decreases the frequency of spontaneous crop muscle contractions. Accordingly, insect neuropeptide allatostatins primarily inhibit juvenile hormone biosynthesis.

Orthologous peptides are known in various taxa; however, their function is largely not known in the art.

The term "allatostatin-b" as used herein refers primarily to (a) polypeptide(s) of Lophotrochozoan marine invertebrates (i.e. the allatostatin-b polypeptides are of marine Lophotrochozoan invertebrate origin). The "allatostatin-b" polypeptides belong to the "B-type allatostatins" of Lophotrochozoan marine invertebrates having a consensus motif from N to C terminus (X)_nW_a(X)_nZWb(X)n, whereby X is any amino acid residue, n is an integer of from 0 to 10 and Z is A, V, T, S or N, and whereby W_a may be absent. Z may be L, M or Q.

Exemplary "allatostatin-b" polypeptides are shown in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 4 (allatostatin-b polypeptide of Capitella teleta), SEQ ID NO:6 (allatostatin-b polypeptide of Aplysia californica), SEQ ID NO: 8 (allatostatin-b polypeptide of Crassostrea gigas), SEQ ID NO: 10 (allatostatin-b polypeptide of Lottia gigantea), SEQ ID NO: 12 (allatostatin-b polypeptide of Lymnaea stagnalis), SEQ ID NO: 14 (allatostatin-b polypeptide of Biomphalaria glabrata), SEQ ID NO: 16 (allatostatin-b polypeptide of Tritonia diomedea), SEQ ID NO: 18 (allatostatin-b polypeptide of Mytilus californianus), SEQ ID NO:20 (allatostatin-b polypeptide of Euprymna scolopes), SEQ ID NO:22 (allatostatin-b polypeptide of Idiosepius paradoxus), or SEQ ID NO:24 (allatostatin-b polypeptide of Doryteuthis pealeii). A further exemplary "allatostatin-b" polypeptide is shown in SEQ ID NO: 37 (allatostatin-b polypeptide of Hirudo medicinalis).

The above "allatostatin-b" polypeptides of marine Lophotrochozoan invertebrates are known or expected to be cleaved into fragments in their natural environment (i.e. in the organism they originate from); see Figure 2. The "allatostatin-b" polypeptides are cleaved by prohormone convertases into fragments which may be used in accordance with the present invention; see Chun (1994) Neuron, Volume 12, Issue 4, 831-844.

Thus, the above "allatostatin-b" polypeptides of marine Lophotrochozoan invertebrates are "allatostatin-b" precursor polypeptides. Peptides derived therefrom (such as fragments of allatostatin-b polypeptides) exert the biological activity of e.g. inducing or enhancing the settlement and/or growth of larvae of marine Lophotrochozoan invertebrates or specifically binding to the allatostatin-b receptor.

As illustrated in Figure 3, "allatostatin-b" polypeptides of marine Lophotrochozoan invertebrates (i.e. the "allatostatin-b" precursor polypeptides) have a common cleavage site which is designated by two basic amino acids amino acids "K " or "RK" or "KK" or "RR". Fragments of the "allatostatin-b" polypeptides of marine Lophotrochozoan invertebrates to be used in accordance with the present invention, may, thus, be fragments of "allatostatin-b" polypeptides, whereby these fragments are generated by cleavage of the "allatostatin-b" polypeptides at the common cleavage site (designated by characteristic dibasic cleavage site such as "KR" or "RK" or "KK" or "RR"). The terms "allatostatin-b polypeptide" and "allatostatin-B precursor polypeptide" can be used interchangeably herein. Such peptide fragments are structurally similar or even identical to fragments of the "allatostatin-b" polypeptides of marine Lophotrochozoan invertebrates occurring in nature. It can easily be deduced from the herein provided "allatostatin-b" precursor polypeptides and fragments thereof, that the first N-terminal amino acid of exemplary fragments to be used in accordance with the present invention is the amino acid following a cleavage site (like "KR" or "RK" or "KK" or "RR") or a signal peptide cleavage site. The last C-terminal amino acid of exemplary fragments is the amino acid just before a cleavage site (like "KR" or "RK" or "KK" or "RR"). Thus, the fragments to be used herein comprise or consist of the amino acid sequence lying within cleavage sites like "KR" or between a signal peptide and a dibasic cleavage site.

Allatostatin-b peptides which may, for example, be obtained by cleavage of allatostatin polypeptides by prohormone convertases usually have a C-terminal glycine residue. Such peptides derived from an allatostatin-b polypeptide by e.g. prohormone convertases may be used in accordance with the present invention. In a natural environment, the C-terminal glycine residue is often converted by amidation into the characteristic amidation signature. Therefore, peptides having an amidated C-terminal amino acid are envisaged herein.

The following exemplary fragments may be used in accordance with the present invention (cf. Fig. 13 and 16):

AWMKNNIAW (SEQ ID NO: 38), AWGDNNMRVW (SEQ ID NO: 39), AWNKNSMRVW (SEQ ID NO: 40), AWKGQSARVW (SEQ ID NO: 41), GWNGNSMRVW (SEQ ID NO: 42), KWGSNSMRVW (SEQ ID NO: 43), GWADNNMRVW (SEQ ID NO: 44), RKWSKFSSW (SEQ ID NO: 45), WKNMAVW (SEQ ID NO: 46), WKQMASW (SEQ ID NO: 47), WKQMSVW (SEQ ID NO: 48), WKEMSVW (SEQ ID NO: 49), WKQMAVW (SEQ ID NO: 50), WKQMATW (SEQ ID NO: 51), AWNKNSMRVWP (SEQ ID NO: 52), or AWNKNSMRVW A (SEQ ID NO: 53).

Further exemplary fragments (or peptides comprising or consisting of these peptides) to be used in accordance with the present invention are as follows:

VNNWNQFPAW (SEQ ID NO: 54), RWSSLGTW (SEQ ID NO: 55), SWLDRLISANNNW (SEQ ID NO: 56), WKSMSNSW (SEQ ID NO: 57), RWSSLSAW (SEQ ID NO: 58), KWNQVGVW (SEQ ID NO: 59), RWSSVSAW (SEQ ID NO: 60), GWNNLQSW (SEQ ID NO: 61), PWSSFKSW (SEQ ID NO: 62), RNPWHSLSTW (SEQ ID NO: 63), AWKSSYLNTW (SEQ ID NO: 64), WTNSGLITW (SEQ ID NO: 65), KWNQFITW (SEQ ID NO: 66), ASDKGWNGFTTW (SEQ ID NO: 67), NKDWSSLSTW (SEQ ID NO: 68), GQNKDWSSLTTW (SEQ ID NO: 69), GHDRDWNS LTTW (SEQ ID NO: 70), ANKDWS SLSTW (SEQ ID NO: 71), NDWSALSTW (SEQ ID NO: 72), A N DWASLTTW (SEQ ID NO: 73), ANDRDWNSLTTW (SEQ ID NO: 74), AKGNNWSGLTTW (SEQ ID NO: 75), DWNSLTTW (SEQ ID NO: 76), ANKDWSGLTTW (SEQ ID NO: 77), GNKDWSGLTTW (SEQ ID NO: 78), GKNDWSGLTTW (SEQ ID NO: 79), GNNDWSGLTTW (SEQ ID NO: 80), DIKGWNGLTTW (SEQ ID NO: 81), TTW, WAQLSTW (SEQ ID NO: 82), QWNAFSSW (SEQ ID NO: 83), WKQMAVW (SEQ ID NO: 84), WKDMPVW (SEQ ID NO: 85), WSDMGVW (SEQ ID NO: 86), WKDMGVW (SEQ ID NO:87), W EMSVW (SEQ ID NO: 88), , EMSVW (SEQ ID NO: 89), WKQMSVW (SEQ ID NO: 90), PSWSSTGFSSW (SEQ ID NO: 91), WNSK AVW (SEQ ID NO: 92), DADTWDSMAAW (SEQ ID NO: 93), SPDTXDSMAAW (SEQ ID NO: 94), SPDTWDSMAAW (SEQ ID NO: 95), DWNSLQAW (SEQ ID NO: 96), DWDSLAAW (SEQ ID NO: 97), DRLDTWNSMNTW (SEQ ID NO: 98), SPNTWDSMAAW (SEQ ID NO: 99), NGDTWD SMS AW (SEQ ID NO: 100), DWDSLQAW (SEQ ID NO: 101), DADTWDSMSAW (SEQ ID NO: 102), RAGDTWDSMSAW (SEQ ID NO: 103), DWDSLQAW (SEQ ID NO: 104), or DWDSLAAW (SEQ ID NO: 105).

Further exemplary peptides to be used herein are:

GWKQGASYSW (SEQ ID NO: 106), AWNKNNMRVW (SEQ ID NO: 107),

GWKDSSMRVW (SEQ ID NO: 108), WGKNNLRVW (SEQ ID NO: 109),

GWHGNGVRQW (SEQ ID NO: 110), AWAKNNMRVW (SEQ ID NO: 111),

KWGGNNNMRVW (SEQ ID NO: 112), KWGANSMRVW (SEQ ID NO: 113),

KWGGSNTMRTW (SEQ ID NO: 114), GWKNNNMRVW (SEQ ID NO:l 15),

RWGGNDMRVW (SEQ ID NO: 116), SWKTNVMRVW (SEQ ID NO: 117),

AWVGDKSLSW (SEQ ID NO: 118).

Further exemplary peptides to be used herein are:

KWGSDRMGLW (SEQ ID NO: 119), KWTSGKMGMW (SEQ ID NO: 120), KWD SRNMGMW (SEQ ID NO: 121), RWGPEGMW (SEQ ID NO: 122), KWASGSMGMW (SEQ ID NO: 123).

The peptides to be used herein, like the above described exemplary fragments derived from allatostatin-b polypeptide(s), often comprise a C-terminal tryptophane (W) amino acid residue. The C-terminal tryptophane (W) amino acid residue may be the last C-terminal amino acid residue of the peptide.

Naturally occurring peptide fragments of allatostatin-b polypeptides often have a C-terminal glycine residue which is preceded by a tryptophane (W) amino acid residue. Said C-terminal glycine residue often gives rise to amidation of the tryptophane (W) amino acid residue, resulting in a peptide fragment with an amidated tryptophane (W) amino acid residue. Thus, the herein provided peptides comprising a C-terminal tryptophane (W) amino acid residue may have an amidated C-terminal tryptophane residue.

As mentioned, it was found that an tryptophone (W) amino acid residue near or at the C- terminus is important for the capacitiy of the peptides to induce settlement and or growth of the larvae of marine Lophotrochozoan invertebrates. Further, said tryptophone (W) amino acid residue may be preceded by an aliphatic amino acid residue, such as alanine, valine, leucine, or isoleucine. Thus, the peptide to be used herein may comprise or consist of one of the following exemplary peptides: AW or VW.

Yet, said tryptophone (W) amino acid residue may also be preceded by other amino acids. The peptide to be used herein may, therefore, comprise or consist of one of the following exemplary peptides: TW, SW, or NW. The peptide may also comprise or consist of the peptide LW, MW or QW.

Though the use of fragments of the "allatostatin-b" polypeptides of Lophotrochozoan marine Lophotrochozoan invertebrates, for example those having a C-terminal tryptophane amino acid residue (which is optionally amidated), is envisaged herein, the peptides to be used herein are not limited to such fragments. As shown herein, also peptides which are, for example, modified in that additional amino acids are added to the C-terminus or to the N- terminus of the fragments are capable of inducing settlement and/or growth. It is also shown herein that peptides can induce settlement and/or growth of larvae of marine Lophotrochozoan invertebrates independent of the fact whether the C-terminal amino acid is amidated. Therefore, the use of parts/portions of the above provided fragments or of further modified peptides is contemplated herein and within the scope of the present invention, wherein the peptides may have an amidated C-terminal amino acid residue.

In accordance with the above, (a) peptide(s) to be used herein has or consists of the consensus motif from N to C terminus (X)_nW_a(X)_nZW_b(X)n, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_a may be absent. (A) peptide(s) to be used herein preferably has or consists of the consensus motif from N to C terminus (X)_aW_a(X)nZWb(X)n, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N. Z may, in one alternative, be L, M or Q. n may be any natural number, for example, of from 0 to 15 amino acids for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 For example, the central amino acid residue X may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues. Preferably, the central amino acid X is of from 2 to 13, more preferably of from 4 to 11 amino acids. Most preferably, the central amino acid is of from 6, 7 or 8 amino acids. A corresponding peptide to be used herein may, accordingly, have or may consist of the consensus motif from N to C terminus (X)_nW_a(X)oZWb(X)_n, whereby Z is A, V, T, S or N, and whereby W_amay be absent. Z may, in one alternative, be L, M or Q.

Further exemplary peptide(s) to be used herein may have or may consist of the consensus motif from N to C terminus

(X)„W_a(X)₂ZW (X)_n, (X)_nW_a(X)₃ZW_b(X)_n, (X)_nW_a(X)₄ZW_b(X)_I1, POnWatXJsZWbfX)., (X)„W_a(X)₆ZW_b(X)_n, (X)_nW_a(X)₇ZW^, (X)nWa(X)_gZW_b(X)_n, (X)_nW_a(X)₉ZW_b(X)_n, or (X)_nW_a(X)₁₀ZW_b(X)„, (X)_nW_a(X)_nZW_b(X)_n, (X)_nWa(X)₁₂ZW_b(X)_n, (X)_nW_a(X)₁₃ZW_b(X)_n, (X)_nW_a(X)i4ZW_b(X)„, (X)_nW_a(X)i₅ZW_b(X)_m whereby Z is A, V, T, S or N. Z may, in one alternative, be L, M or Q. As indicated above, W_a may be absent. Applying the above teaching, further exemplary peptide(s) to be used herein having or consisting of the consensus motif from N to C terminus as defined herein can easily be obtained and are within the scope of the present invention.

The C-terminal amino acid residues X may, for example, be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino residues. If, for example, the C-terminal amino acid residue X is 0, the tryptophane amino acid residue is the last amino acid residue at the C-terminus, i.e. the tryptophane amino acid residue is the C-terminal amino acid of the peptide. If, for example, the C-terminal amino acid residue X is 1, the tryptophane amino acid residue is the penultimate amino acid residue at the C-terminus of the peptide, i.e. the tryptophane amino acid residue is at or near the C-terminus of the peptide. The same explanation/definition applies mutatis mutandis to other values of the C-terminal amino acid X (e.g. if the C- terminal amino acid residues X are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues).

The N-terminal amino acid residue X may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues.

For example, the C-terminal amino acid residue(s) X may be proline and/or alanine. If, for example, the C-terminal amino acid residue X is 1, X may be proline or alanine. A corresponding peptide to be used herein may, accordingly, have or may consist of the consensus motif from N to C terminus (X)_nW_a(X)_nZW_b(Ala)₁, whereby Z is A, V, T, S or N, and whereby W_a may be absent. Z may, in one alternative, be L, M or Q. A further corresponding peptide to be used herein may have or may consist of the consensus motif from N to C terminus (X)_nW_api)_nZW_b(?T0) whereby Z is A, V, T, S or N, and whereby W_a may be absent. Z may, in one alternative, be L, M or Q. Exemplary peptides provided in the appended example which induce settlement and/or growth of larvae of marine Lophotrochozoan invertebrates are AWNK SMRVWP-amide (SEQ ID NO. 189) and AWNKNSMRVWA-amide (SEQ ID NO: 190). These peptide(s) or likewise the non- amidated peptides (i.e. AWNKNSMRVWP (SEQ ID NO: 52) or AWNKNSMRVWA (SEQ ID NO: 53)) may be used herein.

Generally, the amino acid X according to the above formula/consensus motif may be any amino acid, like any amino acid residue occurring at a corresponding position in a natural allatostatin-b polypeptide (i.e. an allatostatin-b precursor polypeptide as exemplarily described herein).

In accordance with the above, the peptides derived from allatostatin-b polypeptides (like fragments of allatostatin-b polypeptides) may have a C-terminal tryptophane amino acid residue, i.e. the tryptophane amino acid residue is the last amino acid residue at the C- terminus of the peptide. The peptides derived from allatostatin-b polypeptides may also have a tryptophane amino acid residue near the C-terminus of the peptide. For example, from N- terminus to C-termiuns, the tryptophane amino acid residue may be the penultimate amino acid of the peptide, the tryptophane amino acid residue may be the third-to-last amino acid of the peptide, the tryptophane amino acid residue may be the fourth-to-last amino acid of the peptide, the tryptophane amino acid residue may be the fifth-to-last amino acid of the peptide, the tryptophane amino acid residue may be the sixth-to-last amino acid of the peptide, the tryptophane amino acid residue may be the seventh-to-last amino acid of the peptide, the tryptophane amino acid residue may be the eighth-to-last amino acid of the peptide, the tryptophane amino acid residue may be the ninth-to-last amino acid of the peptide, the tryptophane amino acid residue may be the tenth-to-last amino acid of the peptide, the tryptophane amino acid residue may be the eleventh-to-last amino acid of the peptide, the tryptophane amino acid residue may be the twelvth-to-last amino acid of the peptide, the tryptophane amino acid residue may be the thirteenth-to-last amino acid of the peptide, or the tryptophane amino acid residue may be the fourteenth-to-last amino acid of the peptide, or the tryptophane amino acid residue may be the fifteenth-to-last amino acid of the peptide, or the tryptophane amino acid residue may be the sixteenth-to-last amino acid of the peptide, and the like.

The peptide provided or to be used herein may have a length of from 2 to about 50 (like 48) amino acids. For example, the peptide may consist of from 2 to 48 amino acids, like of from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids. Preferably, the peptide provided or to be used herein consists of from 4 to 16, or 5 to 15 amino acids. More preferably, the peptide provided or to be used herein consists of from 6 to 14, 7 to 13 or 7 to 12 amino acids. Most preferably, the peptide provided and/or to be used herein consists of 8, 9, 10 or 11 amino acids. Shorter peptides are preferred, because they may more easily be synthesized by chemical synthetic methods. The term "having a length of from" as used herein can interchangeably used with the term "consisting of from".

The term "allatostatin-b polypeptide" may refer to an allatostatin-b polypeptide of (a) marine Lophotrochozoan invertebrate(s), for example, allatostatin-b polypeptides having a sequence as deposited in corresponding public databases (e.g NCBI or EMBL). Exemplary "allatostatin-b" polypeptides to be used herein (e.g. from which peptides to be used in methods for inducing or enhancing the settlement and/or growth of marine Lophotrochozoan invertebrate larvae can be derived, such as fragments thereof and/or peptides having or consisting of the consensus motif (X)„W_a(X)_nZWb(X)_n as defined above) have been described above and are shown in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 4 (allatostatin-b polypeptide of Capitella teleta), SEQ ID NO:6 (allatostatin-b polypeptide of Aplysia califomica), SEQ ID NO: 8 (allatostatin-b polypeptide of Crassostrea gigas)SEQ ID NO: 10 (allatostatin-b polypeptide of Lottia gigantea), SEQ ID NO: 12 (allatostatin-b polypeptide of Lymnaea stagnalis), SEQ ID NO: 14 (allatostatin-b polypeptide of Biomphalaria glabrata), SEQ ID NO: 16 (allatostatin-b polypeptide of Tritonia diomedea), SEQ ID NO: 18 (allatostatin-b polypeptide of Mytilus californianus), SEQ ID NO:20 (allatostatin-b polypeptide of Euprymna scolopes), SEQ ID NO:22 (allatostatin-b polypeptide of Idiosepius paradoxus), or SEQ ID NO:24 (allatostatin-b polypeptide of Doryteuthis pealeii). A further exemplary "allatostatin-b" polypeptide is shown in SEQ ID NO: 37 (allatostatin-b polypeptide of Hirudo medicinalis).

Thus, the term "allatostatin-b polypeptide" may refer to one of the above exemplary polypeptides or to an orthologous polypeptide of any of the above exemplary allatostain-b polypeptide. Means and methods for deterniining/obtaining isolating such orthologous allatostatin-b polypeptide(s) from marine Lophotrochozoan invertebrates, like annelids or molluscs (e.g. Pecten maximus), are described further below.

These allatostatin-b poly peptides may be encoded by a nucleic acid sequence shown in SEQ ID NO:l (nucleic acid encoding allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 3 (nucleic acid encoding allatostatin-b polypeptide of Capitella teleta), SEQ ID NO: 5 (nucleic acid encoding allatostatin-b polypeptide of Aplysia califomica), SEQ ID NO: 7 (nucleic acid encoding allatostatin-b polypeptide of Crassostrea gigas), SEQ ID NO:9 (nucleic acid encoding allatostatin-b polypeptide of Lottia gigantea), SEQ ID NO: 11 (nucleic acid encoding allatostatin-b polypeptide of Lymnaea stagnalis), SEQ ID NO: 13 (nucleic acid encoding allatostatin-b polypeptide of Biomphalaria glabrata), SEQ ID NO: 15 (nucleic acid encoding allatostatin-b polypeptide of Tritonia diomedea), SEQ ID NO: 17 (nucleic acid encoding allatostatin-b polypeptide of Mytilus californianus), SEQ ID NO: 19 (nucleic acid encoding allatostatin-b polypeptide of Euprymna scolopes), SEQ ID NO:21 (nucleic acid encoding allatostatin-b polypeptide of Idiosepius paradoxus), or SEQ ID NO:23 (nucleic acid encoding allatostatin-b polypeptide of Doryteuthis pealeii). A further exemplary nucleic acid encoding an "allatostatin-b" polypeptide is shown in SEQ ID NO: 36 (nucleic acid encoding allatostatin-b polypeptide of Hirudo medicinalis).

The present invention is not limited to peptides derived from the above exemplary allatostatin-b polypeptide(s). Peptides derived from further, potentially yet to be identified allatostatin-b polypeptide(s) may be used herein without deferring from the gist of the present invention. Such allatostatin-b polypeptide(s) may be orthologous allatostatin-b polypeptide(s), i.e. orthologues of the herein above provided and described exemplary "allatostatin-b" polypeptides. Thus, the term "allatostatin-b polypeptide" may also refer to such (an) orthologous allatostatin-b polypeptide^), like allatostatin-b polypeptides from Pecten maximus or allatostatin-b polypeptides from annelid species (like annelids used as baits, in particular annelid baits which are commercially grown in aquaculture. Such annelids are described in more detail further below and comprise annelids belonging to the family Nereidae or belonging to the family Arenicolidae. For example, the annelid may belong to the genus Nereis, such as Nereis virens, or the annelid may belong to the genus Arenicola, such as Arenicola defodiens or Arenicola marina. For example, the annelid may be Nereis diversicolor.

The term "allatostatin-b polypeptide" may also refer to (an) orthologous allatostatin-b polypeptide(s) from further annelid species as described below, like annelids belonging to the genus Perinereis, like Perinereis cultrifera, Perinereis nuntia or Perinereis brevicirrus. The annelid may belong to the family Glyceridae, and may, for example, belong to the genus Glycera (like Glycera dibranchiate). The annelid may belong to the family Nephtyidae, and may, for example, belong to the genus Nephtys, like Nephtys hombergi. The annelid may belong to the family Eunicidae, and may, for example, belong to the genus Marphysa, like Marphysa sanguinea or Marphysa leidyi. The annelid may belong to the family Onuphyidae, and may, for example, belong to the genus Onuphis, like Onuphis teres. The annelid may belong to the family Eunicidae Lumbrinereidae, and may, for example, belong to the genus Lumbrinereis, like Lumbrinereis impatiens.

Again, the use of peptides derived from orthologous allatostatin-b polypeptides of the above annelids in the methods of the present invention is contemplated herein. All definitions and explanations given herein in relation to "allatostatin-b polypeptides" , "peptides derived from allatostatin-b polypeptides" and the like in context of the herein provided methods apply, mutatis mutandis, to the use of such orthologous allatostatin-b polypeptides in accordance with the present invention.

Peptides to be used in methods for inducing or enhancing the settlement and/or growth of marine Lophotrochozoan invertebrate larvae (such as fragments thereof and/or peptides having or consisting of the consensus motif (X)_nW_a(X)_nZWt,(X)_n as defined above) can readily be derived from such orthologous allatostatin-b polypeptide(s). The identification of orthologous polypeptides and corresponding nucleic acids is routine in the art, as further described below. Orthologous polypeptides may be found and identified e.g. in marine Lophotrochozoan invertebrates, for example, animals belonging to the trochozoa. The animals may, for example, belong to the annelid phylum, or to the mollusc phylum. The animal may be an annelid (e.g. belonging to the genus Platynereis or belonging to the genus Capitella, such as Platynereis dumerilii or Capitella teleta as exemplified herein) or it may be a mollusc (e.g. belonging to the genus Aplysia, such as Aplysia californica, or belonging to the genus Crassostrea, such as Crassostrea gigas, or belonging to the genus Pecten, like Pecten maximus).

The orthologous polypeptides may particularly be found and identified in marine Lophotrochozoan invertebrates of commercial value, like molluscs belonging to the class bivalvia or molluscs belonging to the class cephalopoda. Exemplary bivalve(s) from which orthologous polypeptides may be obtained/identified belong to the Pteriomorphia subgroup or may belong to the genus Pecten, Crassostrea Ruditapes, Anadara, Perna, Patinopecten, Mytilis or Mercenaria, such as Pecten maximus, Ruditapes philippinarum, Crassostrea gigas, Anadara granosa, Perna viridis, Patinopecten yessoensis, Mytilis edulis, Mytilis galloprovincialis, Perna canaliculus and Mercenaria mercenaria. Exemplary cephalopod(s) from which orthologous polypeptides may be obtained/identified belong to the genus Sepia or Octopus, like Sepia officinalis or Octopus vulgaris.

As mentioned, the term "allatostatin-b polypeptide" may refer to (an) allatostatin-b polypeptide of (a) marine Lophotrochozoan invertebrate(s) and may refer to polypeptides which are orthologous or similar to the exemplary polypeptides disclosed herien above. The terms "orthologous"/"similar" are described herein below.

The allatostatin-b polypeptide (from which peptide fragments for use in accordance with the present invention may be derived) may be an allatostatin-b polypeptide selected from the group consisting of (a) a polypeptide comprising or consisting of an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence identified or obtained from an marine Lophotrochozoan invertebrate as defined above;

(b) a polypeptide having or consisting of an amino acid sequence identified or obtained from an marine Lophotrochozoan invertebrate as defined above;

(c) a polypeptide as defined in (a) or (b) wherein one or more amino acids are deleted, inserted, added or substituted, and whereby said polypeptide has allatostatin-b biological activity;

(d) a polypeptide encoded by a nucleic acid molecule encoding a peptide having or consisting of an amino acid sequence identified or obtained from an marine Lophotrochozoan invertebrate as defined above;

(e) a polypeptide comprising or consisting of an amino acid sequence encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (a) or (d) and whereby said polypeptide has allatostatin-b biological acitvity;

(f) a polypeptide having at least 40 % similarity to the polypeptide of any one of (a) to (e), and whereby said polypeptide has allatostatin-b biological acitvity; and

(g) a polypeptide comprising or consisting of an amino acid sequence encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (d) and (e).

In accordance with the above, the allatostatin-b polypeptide (from which peptide fragments for use in accordance with the present invention may be derived) may be an allatostatin-b polypeptide selected from the group consisting of

(a) a polypeptide comprising or consisting of an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO:l (nucleic acid encoding allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 3 (nucleic acid encoding allatostatin-b polypeptide of Capitella teleta), SEQ ID NO: 5 (nucleic acid encoding allatostatin-b polypeptide of Aplysia californica), SEQ ID NO: 7 (nucleic acid encoding allatostatin-b polypeptide of Crassostrea gigas), SEQ ID NO:9 (nucleic acid encoding allatostatin-b polypeptide of Lottia gigantea), SEQ ID NO: 11 (nucleic acid encoding allatostatin-b polypeptide of Lymnaea stagnalis), SEQ ID NO: 13 (nucleic acid encoding allatostatin-b polypeptide of Biomphalaria glabrata), SEQ ID NO: 15 (nucleic acid encoding allatostatin-b polypeptide of Tritonia diomedea), SEQ ID NO: 17 (nucleic acid encoding allatostatin-b polypeptide of Mytilus californianus), SEQ ID NO: 19 (nucleic acid encoding allatostatin-b polypeptide of Euprymna scolopes), SEQ ID NO:21 (nucleic acid encoding allatostatin-b polypeptide of Idiosepius paradoxus), and SEQ ID NO:23 (nucleic acid encoding allatostatin-b polypeptide of Doryteuthis pealeii);

(b) a polypeptide having or consisting of an amino acid sequence as depicted in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 4 (allatostatin- b polypeptide of Capitella teleta), SEQ ID NO:6 (allatostatin-b polypeptide of Aplysia californica), SEQ ID NO: 8 (allatostatin-b polypeptide of Crassostrea gigas), SEQ ID NO: 10 (allatostatin-b polypeptide of Lottia gigantea), SEQ ID NO: 12 (allatostatin-b polypeptide of Lymnaea stagnalis), SEQ ID NO: 14 (allatostatin-b polypeptide of Biomphalaria glabrata), SEQ ID NO: 16 (allatostatin-b polypeptide of Tritonia diomedea), SEQ ID NO: 18 (allatostatin-b polypeptide of Mytilus californianus), SEQ ID NO:20 (allatostatin-b polypeptide of Euprymna scolopes), SEQ ID NO:22 (allatostatin-b polypeptide of Idiosepius paradoxus), and SEQ ID NO:24 (allatostatin- b polypeptide of Doryteuthis pealeii);

(d) a polypeptide encoded by a nucleic acid molecule encoding a peptide having or consisting of an amino acid sequence as depicted SEQ ID NO: 2 (allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 4 (allatostatin-b polypeptide of Capitella teleta), SEQ ID NO:6 (allatostatin-b polypeptide of Aplysia californica), SEQ ID NO:8 (allatostatin-b polypeptide of Crassostrea gigas)SEQ ID NO: 10 (allatostatin-b polypeptide of Lottia gigantea), SEQ ID NO: 12 (allatostatin-b polypeptide of Lymnaea stagnalis), SEQ ID NO: 14 (allatostatin-b polypeptide of Biomphalaria glabrata), SEQ ID NO: 16 (allatostatin-b polypeptide of Tritonia diomedea), SEQ ID NO: 18 (allatostatin-b polypeptide of Mytilus californianus), SEQ ID NO:20 (allatostatin-b polypeptide of Euprymna scolopes), SEQ ID NO:22 (allatostatin-b polypeptide of Idiosepius paradoxus), and SEQ ID NO:24 (allatostatin- b polypeptide of Doryteuthis pealeii);

As mentioned, the allatostatin-b polypeptide may be one of the following exemplary polypeptides shown in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii); SEQ ID NO: 4 (allatostatin-b polypeptide of Capitella teleta); SEQ ID NO:6 (allatostatin-b polypeptide of Aplysia californica),or SEQ ID NO:8 (allatostatin-b polypeptide of Crassostrea gigas). The allatostatin-b polypeptide (from which peptide fragments for use in accordance with the present invention may be derived) may, accordingly, be an allatostatin-b polypeptide selected from the group consisting of

(a) a polypeptide comprising or consisting of an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO:l (nucleic acid encoding allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 3 (nucleic acid encoding allatostatin-b polypeptide of Capitella teleta), SEQ ID NO: 5 (nucleic acid encoding allatostatin-b polypeptide of Aplysia californica), and SEQ ID NO: 7 (nucleic acid encoding allatostatin-b polypeptide of Crassostrea gigas));

(b) a polypeptide having or consisting of an amino acid sequence as depicted in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 4 (allatostatin- b polypeptide of Capitella teleta), SEQ ID NO: 6 (allatostatin-b polypeptide of Aplysia californica), and SEQ ID NO:8 (allatostatin-b polypeptide of Crassostrea gigas);

(d) a polypeptide encoded by a nucleic acid molecule encoding a peptide having or consisting of an amino acid sequence as depicted in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 4 (allatostatin-b polypeptide of Capitella teleta), SEQ ID NO: 6 (allatostatin-b polypeptide of Aplysia californica), and SEQ ID NO: 8 (allatostatin-b polypeptide of Crassostrea gigas));

In accordance with the above, the allatostatin-b polypeptide may be a polypeptide shown in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii). Thus, the allatostatin-b polypeptide (from which peptide fragments for use in accordance with the present invention may be derived) may, accordingly, be an allatostatin-b polypeptide selected from the group consisting of

(a) a polypeptide comprising or consisting of an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO:l (nucleic acid encoding allatostatin-b polypeptide of Platynereis dumerilii);

(b) a polypeptide having or consisting of an amino acid sequence as depicted in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii);

(d) a polypeptide encoded by a nucleic acid molecule encoding a peptide having or consisting of an amino acid sequence as depicted in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii);

The allatostatin-b polypeptide may be a polypeptide shown in SEQ ID NO: 4 (allatostatin-b polypeptide of Capitella teleta). The allatostatin-b polypeptide (from which peptide fragments for use in accordance with the present invention may be derived) may, accordingly, be an allatostatin-b polypeptide selected from the group consisting of

(a) a polypeptide comprising or consisting of an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO: 3 (nucleic acid encoding allatostatin-b polypeptide of Capitella teleta);

(b) a polypeptide having or consisting of an amino acid sequence as depicted in SEQ ID NO: 4 (allatostatin-b polypeptide of Capitella teleta);

(d) a polypeptide encoded by a nucleic acid molecule encoding a peptide having or consisting of an amino acid sequence as depicted in SEQ ID NO: 4 (allatostatin-b polypeptide of Capitella teleta);

The allatostatin-b polypeptide may be a polypeptide shown in SEQ ID NO:6 (allatostatin-b polypeptide of Aplysia californica). The allatostatin-b polypeptide (from which peptide fragments for use in accordance with the present invention may be derived) may, accordingly, be an allatostatin-b polypeptide selected from the group consisting of

(a) a polypeptide comprising or consisting of an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO: 5 (nucleic acid encoding allatostatin-b polypeptide of Aplysia californica);

(b) a polypeptide having or consisting of an amino acid sequence as depicted in SEQ ID NO:6 (allatostatin-b polypeptide of Aplysia californica);

(d) a polypeptide encoded by a nucleic acid molecule encoding a peptide having or consisting of an amino acid sequence as depicted in SEQ ID NO:6 (allatostatin-b polypeptide of Aplysia californica);

The allatostatin-b polypeptide may be a polypeptide shown in SEQ ID NO: 8 (allatostatin-b polypeptide of Crassostrea gigas). The allatostatin-b polypeptide (from which peptide fragments for use in accordance with the present invention may be derived) may, accordingly, be an allatostatin-b polypeptide selected from the group consisting of (a) a polypeptide comprising or consisting of an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO:7 (nucleic acid encoding allatostatin-b polypeptide of Crassostrea gigas);

(b) a polypeptide having or consisting of an amino acid sequence as depicted in SEQ ID NO: 8 (allatostatin-b polypeptide of Crassostrea gigas);

(d) a polypeptide encoded by a nucleic acid molecule encoding a peptide having or consisting of an amino acid sequence as depicted in SEQ ID NO: 8 (allatostatin-b polypeptide of Crassostrea gigas);

The term "allatostatin-b polypeptide" from which a peptide for use in the present invention may be derived, may refer to a polypeptide having at least 40 % similarity to a polypeptide as defined in section (a) to (e) of the above-described specific aspect of the present invention. The "allatostatin-polypeptide" has preferably essentially the same biological activity as a polypeptide having 100 % similarity to a polypeptide as indicated in section (a), (b) or (d), i.e. a polypeptide being essentially identical to a polypeptide having an amino acid sequence as depicted in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 4 (allatostatin-b polypeptide of Capitella teleta), SEQ ID NO:6 (allatostatin-b polypeptide of Aplysia californica), SEQ ID NO: 8 (allatostatin-b polypeptide of Crassostrea gigas). Methods for determining the biological activity of (an) allatostatin-b polypeptide(s) are described herein.

The peptides to be used in accordance with the present invention may be obtained by (enzymatic) cleavage of the herein provided allatostatin-b polypeptide(s), by chemical or biotechnological synthesis (like recombinant production).

Thus, the "allatostatin-b polypeptide(s)" from which a peptide for use in the present invention may be derived, or the peptides derived from the allatostatin-b polypeptide(s), may further comprise a heterologous polypeptide, for example, (an) amino acid sequence(s) for identification and/or purification of the recombinant protein (e.g. amino acid sequence from C-MYC, GST protein, FLAG peptide, HIS peptide and the like), an amino acid sequence used as reporter (e.g. green fluorescent protein, yellow fluorescent protein, red fluorescent protein, luciferase, and the like), or antibodies/antibody fragments (like scFv). A person skilled in the art knows that for determination of similarity as described herein only a part of the allatostatin-b polypeptide polypeptides is to be used, whereby said part is allatostatin-b polypeptide.

It is also envisaged herein that an "allatostatin-b polypeptide" as defined herein or a peptide derived from an "allatostatin-b polypeptide" as defined herein, though being of, for example, marine Lophotrochozoan invertebrate origin as described above, may be modified in order to change certain properties of the polypeptide. For example, such a modified "allatostatin-b polypeptide" (or a modified peptide derived from an allatostatin-b polypeptide) may exhibit increased biological activity as defined herein or increased stability when compared to the "original" allatostatin-b polypeptide (i.e. an allatostatin-b polypeptide as produced in a healthy, non-transgenic organism, e.g. the exemplary allatostatin-b polypeptide of marine Lophotrochozoan invertebrates as defined and described above).

For example, the polypeptide having the amino acid sequence as shown in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 4 (allatostatin-b polypeptide of Capitella teleta), SEQ ID NO:6 (allatostatin-b polypeptide of Aplysia californica), SEQ ID NO: 8 (allatostatin-b polypeptide of Crassostrea gigas), can be considered as an "original" allatostatin-b polypeptide. A polypeptide having the amino acid sequence as shown in SEQ ID NO: 10 (allatostatin-b polypeptide of Lottia gigantea), SEQ ID NO: 12 (allatostatin-b polypeptide of Lymnaea stagnalis), SEQ ID NO: 14 (allatostatin-b polypeptide of Biomphalaria glabrata), SEQ ID NO: 16 (allatostatin-b polypeptide of Tritonia diomedea), SEQ ID NO: 18 (allatostatin-b polypeptide of Mytilus californianus), SEQ ID NO:20 (allatostatin-b polypeptide of Euprymna scolopes), SEQ ID NO:22 (allatostatin-b polypeptide of Idiosepius paradoxus), SEQ ID NO:24 (allatostatin-b polypeptide of Doryteuthis pealeii), or SEQ ID NO: 37 (allatostatin-b polypeptide of Hirudo medicinalis) can be considered as an "original" allatostatin-b polypeptide. A "modified" allatostatin-b polypeptide may have (an) insertion(s), (a) deletion(s), additions(s) and/or (an) substitution(s) of one or more amino acids as described below in more detail.

Likewise, for example, a modified peptide derived from an allatostatin-b polypeptide may exhibit increased biological activity as defined herein or increased stability when compared to the "original" peptide derived from an allatostatin-b polypeptide (i.e. a peptide derived from an allatostatin-b polypeptide as produced in a healthy, non-transgenic organism, e.g. the peptides derived from the exemplary allatostatin-b polypeptide of marine Lophotrochozoan invertebrates as defined and described above).

For example, the following peptides can be considered as "original" peptides derived from (an) allatostatin-b polypeptide: AWMKNNIAW (SEQ ID NO: 38), AWGDNNMRVW (SEQ ID NO: 39), AWNKNSMRVW (SEQ ID NO: 40), AWKGQSARVW (SEQ ID NO: 41), GWNGNSMRVW (SEQ ID NO: 42), KWGSNSMRVW (SEQ ID NO: 43), GWADNNMRVW (SEQ ID NO: 44), RKWS FSSW (SEQ ID NO: 45), WKNMAVW (SEQ ID NO: 46), WKQMASW (SEQ ID NO: 47), WKQMSVW (SEQ ID NO: 48), WKEMSVW (SEQ ID NO: 49), WKQMAVW (SEQ ID NO: 50), WKQMATW (SEQ ID NO: 51).

Further peptides peptides which can be considered as "original" peptides derived from (an) allatostatin-b polypeptide are:

VNNWNQFPAW (SEQ ID NO: 54), RWSSLGTW (SEQ ID NO: 55), SWLDRLISANNNW (SEQ ID NO: 56), WKSMSNSW (SEQ ID NO: 57), RWSSLSAW (SEQ ID NO: 58), KWNQVGVW (SEQ ID NO: 59), RWSSVSAW (SEQ ID NO: 60), GWNNLQSW (SEQ ID NO: 61), PWSSFKSW (SEQ ID NO: 62), RNPWHSLSTW (SEQ ID NO: 63), AWKSSYLNTW (SEQ ID NO: 64), WTNSGLITW (SEQ ID NO: 65), KWNQFITW (SEQ ID NO: 66), ASDKGWNGFTTW (SEQ ID NO: 67), NKDWSSLSTW (SEQ ID NO: 68), GQNKDWS SLTTW (SEQ ID NO: 69), GHDRDWNSLTTW (SEQ ID NO: 70), ANKDWSSLSTW (71), NDWSALSTW ((SEQ ID NO: 72), ANNKDWASLTTW (SEQ ID NO: 73), ANDRDWNSLTTW (SEQ ID NO: 74), AKGNNWSGLTTW (SEQ ID NO:75), DWNSLTTW (SEQ ID NO: 76), ANKDWSGLTTW (SEQ ID NO:77), GNKDWSGLTTW (SEQ ID NO: 78), GKNDWSGLTTW (SEQ ID NO: 79), GNNDWSGLTTW (SEQ ID NO: 80), DIKGWNGLTTW (SEQ ID NO: 81), TTW, WAQLSTW (SEQ ID NO: 82), QWNAFSSW (SEQ ID NO: 83), WKQMAVW (SEQ ID NO: 84), WKDMPVW (SEQ ID NO: 85), WSDMGVW (SEQ ID NO: 86), WKDMGVW (SEQ ID NO: 87), WKEMSVW (SEQ ID NO: 88), KEMSVW (SEQ ID NO: 89), WKQMSVW (SEQ ID NO: 90), PSWSSTGFSSW (SEQ ID NO: 91), WNSKMAVW (SEQ ID NO: 92), DADTWDSMAAW (SEQ ID NO: 93), SPDTWDSMAAW (SEQ ID NO: 95), DWNSLQAW (SEQ ID NO: 96), DWDSLAAW (SEQ ID NO: 97), DRLDTWNSMNTW (SEQ ID NO: 98), SPNTWD SMAAW (SEQ ID NO: 99), NGDTWDSMSAW (SEQ ID NO: 100), DWDSLQAW (SEQ ID NO: 101), DADTWDSMSAW (SEQ ID NO: 102), RAGDTWDSMSAW (SEQ ID NO: 103), DWDSLQAW (SEQ ID NO: 104), or DWDSLAAW (SEQ ID NO: 105). Also the following peptides peptides can be considered as "original" peptides derived from (an) allatostatin-b polypeptide: KWGSDRMGLW (SEQ ID NO: 119), KWTSGKMGMW (SEQ ID NO: 120), KWD SRNMGMW (SEQ ID NO: 121), RWGPEGMW (SEQ ID NO: 122), KWAS GSMGMW (SEQ ID NO: 123).

It has been found in the present invention that the capacity of the peptides to induce settlement and/or growth of marine Lophotrochozoan invertebrate larvae is not species- specific, i.e. the herein provided peptides can induce settlement and/or growth of marine Lophotrochozoan invertebrate larvae in a cross-species-specific manner; see Example 1 and Fig. 3. Therefore, the peptides to be used herein may be used for inducing settlement and/or growth of a wide range of closely to distantly related marine Lophotrochozoan invertebrate larvae of e.g. the Lophotrochozoan syperphylum, such as annelids and molluscs.

Thus, it is envisaged that the peptide derived form an "allatostatin-b polypeptide" isolated/obtained from a specific marine Lophotrochozoan invertebrate organism may also be used for the induction or enhancement of settlement and/or growth of distantly related organisms; for example, peptides derived from an allatostatin-b polypeptide, which is isolated/obtained from Platynereis dumerilii (or likewise from Capitella teleta), may be used for inducing or enhancing settlement and/or growth of one or more larva of a mollusc species, like the herein described commercially relevant mollusc species.

Closely related organisms may, in particular, be organisms which form a subgroup of a species, e.g. different races of a species. Also organisms which belong to a different species but can be subgrouped under a common genus can be considered as closely related. Less closely related organisms belong to different genera subgrouped under one family. Distantly related organisms belong to different families. The taxonomic terms "race", "species", "genus", "family" and the like are well known in the art and can easily be derived from standard textbooks. Based on the teaching provided in the present invention a skilled person is therefore easily in the position to identify "closely related" or "distantly related" organisms.

It is also envisaged that peptides derived from an allatostatin-b polypeptide, which is isolated/obtained from a specific marine Lophotrochozoan invertebrate species is used for inducing or enhancing settlement and/or growth of one or more larva of said specific marine Lophotrochozoan invertebrate species. For example, (a) peptide(s) derived from an polypeptide allatostatin-b polypeptide having the amino acid sequence as shown in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii) may be used for inducing or enhancing settlement and/or growth of one or more larva of Platynereis dumerilii. The same explanation/definition applies, mutatis mutandis, to peptide(s) derived from an polypeptide allatostatin-b polypeptide of (or obtained/isoloated from) other marine Lophotrochozoan invertebrate species, such as peptides derived from the allatostatin-b polypeptide having the amino acid sequence as shown in SEQ ID NO: 4 (allatostatin-b polypeptide of Capitella teleta), SEQ ID NO:6 (allatostatin-b polypeptide of Aplysia californica), SEQ ID NO:8 (allatostatin-b polypeptide of Crassostrea gigas), SEQ ID NO: 10 (allatostatin-b polypeptide of Lottia gjgantea), SEQ ID NO: 12 (allatostatin-b polypeptide of Lymnaea stagnalis), SEQ ID NO: 14 (allatostatin-b polypeptide of Biomphalaria glabrata), SEQ ID NO:16 (allatostatin-b polypeptide of Tritonia diomedea), SEQ ID NO: 18 (allatostatin-b polypeptide of Mytilus californianus), SEQ ID NO:20 (allatostatin-b polypeptide of Euprymna scolopes), SEQ ID NO:22 (allatostatin-b polypeptide of Idiosepius paradoxus), or SEQ ID NO:24 (allatostatin-b polypeptide of Doryteuthis pealeii) or SEQ ID NO: 37 (allatostatin-b polypeptide of Hirudo medicinalis) or peptides derived from orthologous allatostatin-b polypeptides as described and defined herein, such as the orthologous Pecten maximus allatostatin-b polypeptide.

Exemplary peptides derived from orthologous allatostatin-b polypeptides may be derived from orthologous allatostatin-b polypeptides of marine Lophotrochozoan invertebrates of commercial value (e.g. molluscs belonging to the class bivalvia or molluscs belonging to the class cephalopoda). Exemplary bivalve(s) from which orthologous polypeptides may be obtained/identified belong to the Pteriomorphia subgroup or belong to the genus Pecten, Crassostrea, Ruditapes, Anadara, Perna, Patinopecten, Mytilis or Mercenaria, such as Pecten maximus, Ruditapes philippinarum, Crassostrea gigas, Anadara granosa, Perna viridis, Patinopecten yessoensis, Mytilis edulis, Mytilis galloprovincialis, Perna canaliculus and Mercenaria mercenaria. Exemplary cephalopod(s) from which orthologous polypeptides may be obtained/identified belong to the genus Sepia or Octopus, like Sepia officinalis or Octopus vulgaris.

The nucleic acid molecule encoding the allatostatin-b polypeptide (or peptide derived therefrom) may be comprised in a recombinant vector in which said nucleic acid molecule is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells. Expression of said nucleic acid molecule comprises transcription of the nucleic acid molecule into a translatable mRNA. Regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the lambda PL, lac, trp, tac, ara, phoA, tet or T7 promoters in E. coli. Possible regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells or yeast, are well known in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals effecting termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally associated or heterologous promoter regions. Examples for regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoters in yeast or the CMV_? SV40, RSV (Rous sarcoma virus) promoters, CMV enhancer, SV40 enhancer or a globin intron in mammalian and other animal cells. Apart from elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the coding region.

Methods which are well known to those skilled in the art can be used to construct recombinant vectors (see, for example, the techniques described in Sambrook (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory NY and Ausubel (1989), Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY). In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDVl (Pharmacia), pCDM8, pRc/CMV, pcDNAl, pcDNA3, pPICZalpha A (Invitrogen), or pSPORTl (GffiCO BRL). Furthermore, depending on the expression system that is used, leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the culture medium may be added to the coding sequence of the nucleic acid molecule.

The present invention also relates to vectors, particularly plasmids, cosmids, viruses, and bacteriophages that are conventionally employed in genetic engineering, comprising a nucleic acid molecule encoding the the allatostatin-b polypeptide (or peptide derived therefrom). Preferably, said vector is an expression vector and/or a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses or bovine papilloma virus may be used for delivery of the polynucleotides or vector of the invention into targeted cell populations.

The vectors containing the nucleic acid molecules encoding the allatostatin-b polypeptide (or peptide derived therefrom) can be transfected into the host cell by well known methods, which vary depending on the type of cell. Accordingly, the invention further relates to a cell comprising said nucleic acid molecule or said vector. Such methods, for example, include the techniques described in Sambrook (1989), loc. cit. and Ausubel (1 89), loc. cit. Accordingly, calcium chloride transfection or electroporation is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts (Sambrook (1989), loc. cit.). As a further alternative, the nucleic acid molecules and vectors of the invention can be reconstituted into liposomes for delivery to target cells. The nucleic acid molecule or vector of the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extra-chromosomally. Accordingly, the present invention also relates to a host cell comprising the nucleic acid molecule and/or the vector of this invention. Host cells for the expression of polypeptides are well known in the art and comprise prokaryotic cells as well as eukaryotic cells, e.g. E. coli cells, yeast cells, invertebrate cells, CHO cells, CHO-K1 cells, HEK 293 cells, Hela cells, COS-1 monkey cells, melanoma cells such as Bowes cells, mouse L-929 cells, 3T3 cell lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines and the like.

In a further aspect, the present invention comprises methods for the preparation of the allatostatin-b polypeptide (or peptide derived therefrom), comprising culturing the (host) cell of this invention and isolating said the allatostatin-b polypeptide (or peptide derived therefrom) from the culture as described herein. In general, the allatostatin-b polypeptide (or peptide derived therefrom) may be produced by recombinant DNA technology, e.g. by cultivating a cell comprising the described nucleic acid molecule or vectors which encode the the allatostatin-b polypeptide (or peptide derived therefrom) and isolating the allatostatin-b polypeptide (or peptide derived therefrom) from the culture. The allatostatin-b polypeptide (or peptide derived therefrom) may be produced in any suitable cell culture system including prokaryotic cells, e.g. E. coli BL21, KS272 or JM83, or eukaryotic cells, e.g. Pichia pastoris, yeast strain X-33 or CHO cells. Further suitable cell lines known in the art are obtainable from cell line depositories like the American Type Culture Collection (ATCC).

The term "prokaryotic" is meant to include bacterial cells while the term "eukaryotic" is meant to include yeast, higher plant, insect and mammalian cells. The transformed hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth.

Herein provided is a process for the preparation of a the allatostatin-b polypeptide (or peptide derived therefrom) described above comprising cultivating a cell under conditions suitable for expression of the the allatostatin-b polypeptide (or peptide derived therefrom) and isolating said protein/polypeptide from the cell or the culture medium.

The allatostatin-b polypeptide (or peptide derived therefrom) as provided herein may comprise a chemically reactive group, for example when said the allatostatin-b polypeptide (or peptide derived therefrom) is part of a "fusion protein"/"fusion construct". As also described above, the the allatostatin-b polypeptide (or peptide derived therefrom) can be prepared by recombinant expression in a transformed cell in several ways according to methods well known to the person skilled in the art, for example: (i) direct expression in the cytoplasm with the help of an N-terminal Met residue / start codon (ii) secretion via an N- terminal signal peptide, for example OmpA, PhoA (Monteilhet (1993) Gene. 1993 125:223- 228), mellitin (Tessier (1991) Gene 98: 177-183), interleukin 2 (Zhang (2005) J Gene Med 7: 354-365), hGH (Pecceu (1991) Gene 97(2):253-258) and the like, followed by intracellular cleavage resulting in the mature N-terminus, such as Ala or Pro; (iii) expression as a fusion protein with another soluble protein, e.g., maltose-binding protein at the N-terminus and with a protease cleavage site interspersed (Kapust and Waugh (2000) Protein Expr. Purif. 19:312- 318), followed by specific protease cleavage in vitro or in vivo, thus releasing the allatostatin- b polypeptide (or peptide derived therefrom) with its mature N-terminus. Another suitable fusion partner is the SUMO protein, which can be cleaved by SUMO protease. Further fusion partners include, without limitation, glutathion-S-transferase, thioredoxin, a cellulose-binding domain, an albumin-binding domain, a fluorescent protein (such as GFP), protein A, protein G, an intein and the like (Malhotra (2009) Methods Enzymol. 463:239-258).

With the means and methods provided herein it is possible to manufacture and provide for the herein disclosed (i) allatostatin-b polypeptide (or peptide derived therefrom) and (ii) peptides/proteins comprising said allatostatin-b polypeptide (or peptide derived therefrom) which comprise (a) further molecule(s) of interest, like a useful protein, a protein segment or a small molecule. Accordingly, the present invention also provides for a method for the preparation and/or manufacture of a the allatostatin-b polypeptide (or peptide derived therefrom) as comprised in conjugates.

These methods comprise (as one step) the cultivation of the (host) cell as provided herein above and (as a further step) the isolation of the allatostatin-b polypeptide (or peptide derived therefrom) and/or polypeptide conjugate from the culture or from said cell. This isolated the allatostatin-b polypeptide (or peptide derived therefrom) as well as the isolated conjugate may than be further processed. For example, the allatostatin-b polypeptide (or peptide derived therefrom) or conjugate may be chemically linked or coupled to a molecule of interest. Furthermore and as an alternative, the molecule of interest may be enzymatically conjugated e.g. via transglutaminase (Besheer (2009) J Pharm Sci. 98:4420-8) or other enzymes (Subul (2009) Org. Biomol. Chem. 7:3361-3371) to said the allatostatin-b polypeptide (or peptide derived therefrom) or conjugate.

The allatostatin-b polypeptide (or peptide derived therefrom) or conjugate comprising same can be isolated (inter alia) from the growth medium, cellular lysates, periplasm or cellular membrane fractions. The isolation and purification of the expressed polypeptides of the invention may be performed by any conventional means (Scopes (1982) "Protein Purification", Springer, New York, NY), including ammonium sulphate precipitation, affinity purification, column chromatography, gel electrophoresis and the like and may involve the use of monoclonal or polyclonal antibodies directed, e.g., against a tag fused with the the allatostatin-b polypeptide (or peptide derived therefrom) of the invention. For example, the protein can be purified via the Strep-tag II using streptavidin affinity chromatography. Substantially pure polypeptides of at least about 90 to 95 % homogeneity (on the protein level) are preferred, and 98 to 99 % or more homogeneity are most preferred. Depending upon the host cell / organism employed in the production procedure, the allatostatin-b polypeptide (or peptide derived therefrom) of the present invention may be glycosylated or may be non- glycosylated.

The peptide derived from allatostatin-b polypeptide may comprise additional amino acid stretches which may, as such, not contribute to the biological activity of the herein provided peptide. The further amino acid sequences/amino acid residues may, for example, be useful as linkers. Such peptide linkers may be composed of flexible residues like glycine or serine. Such peptide linkers could have a different length of from, for example, 5 to 15 amino acids. A peptide with the amino acid sequence GGGSGGGSGGGS (SEQ ID NO: 124) would be an example for a flexible linker that could be used.

The peptide derived from allatostatin-b polypeptide can also be prepared via chemical peptide synthesis techniques including both solution methods and solid phase methods.

Solid phase synthesis in which the C-terminal amino acid of the polypeptide sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence can be used as an exemplary synthetic method for preparing the peptides. Techniques for solid phase synthesis are described by Merrifield, et al, 1963 J Am Chem Soc 85:2149-2154. Automated systems for performing solid phase peptide synthesis are commercially available.

Solid phase synthesis is usually started from the carboxy-terminal end (i.e., the C-terminus) of the polypeptide by coupling a protected amino acid via its carboxyl group to a suitable solid support. The solid support used is not a critical feature provided that it is capable of binding to the carboxyl group while remaining substantially inert to the reagents utilized in the peptide synthesis procedure. For example, a starting material can be prepared by attaching an amino- protected amino acid via a benzyl ester linkage to a chloromethylated resin or a hydroxymethyl resin or via an amide bond to a benzhydrylamine (BHA) resin or p- methylbenzhydrylamine (MBHA) resin. Materials suitable for use as solid supports are well known to those of skill in the art and include, but are not limited to, the following: halomethyl resins, such as chloromethyl resin or bromomethyl resin; hydroxymethyl resins; phenol resins, such as 4-(a-[2,4-dimethoxyphenyl]-Fmoc-aminomethyl)phenoxy resin; tert-alkyloxycarbonyl-hydrazidated resins; and the like. Such resins are commercially available and their methods of preparation are known to those of ordinary skill in the art. The acid form of the peptides may be prepared by the solid phase peptide synthesis procedure using a benzyl ester resin as a solid support. The corresponding amides may be produced by using benzhydrylamine or methylbenzhydrylamine resin as the solid support. A perso skilled in the art will recognize that when the BHA or MBHA resin is used treatment with anhydrous hydrofluoric acid to cleave the peptide from the solid support produces a peptide having a terminal amide group.

The a-amino group of each amino acid used in the synthesis should be protected during the coupling reaction to prevent side reactions involving the reactive a-amino function. Certain amino acids also contain reactive side-chain functional groups (e.g. sulfhydryl, amino, carboxyl, hydroxyl, etc.) which must also be protected with appropriate protecting groups to prevent chemical reactions from occurring at those sites during the peptide synthesis. Protecting groups are well known; see, for example, The Peptides: Analysis, Synthesis, Biology, Vol. 3: Protection of Functional Groups in Peptide Synthesis (Gross and Meienhofer (eds.), Academic Press, N.Y. (1981)).

A properly selected a-amino protecting group will render the a-amino function inert during the coupling reaction, will be readily removable after coupling under conditions that will not remove side chain protecting groups, will not alter the structure of the peptide fragment, and will prevent racemization upon activation immediately prior to coupling. Similarly, side chain protecting groups must be chosen to render the side chain functional group inert during the synthesis, must be stable under the conditions used to remove the a-amino protecting group, and must be removable after completion of the peptide synthesis under conditions that will not alter the structure of the peptide.

Coupling of the amino acids may be accomplished by a variety of techniques known to those of skill in the art. Typical approaches involve either the conversion of the amino acid to a derivative that will render the carboxyl group more susceptible to reaction with the free N- terminal amino group of the peptide fragment, or use of a suitable coupling agent such as, for example, Ν,Ν'-dicyclohexylcarbodimide (DCC) or Ν,Ν'- diisopropylcarbodiimide (DIPCDI). Frequently, hydroxybenzotriazole (HOBt) is employed as a catalyst in these coupling reactions.

Generally, synthesis of the peptide is commenced by first coupling the C-terminal amino acid, which is protected at the N-amino position by a protecting group such as fluorenylmethyloxycarbonyl (Fmoc), to a solid support. Prior to coupling of an Fmoc-amino acid, the Fmoc residue has to be removed from the polymer. Fmoc-amino acid can, for example, be coupled to the 4-(a-[2,4-dimethoxyphenyl]-Fmoc-amino-methyl)phenoxy resin using Ν,Ν'-dicyclohexylcarbodimide (DCC) and hydroxybenzotriazole (HOBt) at about 25°C for about two hours with stirring. Following the coupling of the Fmoc protected amino acid to the resin support, the α-amino protecting group is removed using 20% piperidine in DMF at room temperature.

After removal of the ct-amino protecting group, the remaining Fmoc-protected amino acids are coupled stepwise in the desired order. Appropriately protected amino acids are commercially available from a number of suppliers (e.g., Novartis (Switzerland) or Bachem (California)). As an alternative to the stepwise addition of individual amino acids, appropriately protected peptide fragments consisting of more than one amino acid may also be coupled to the "growing" peptide. Selection of an appropriate coupling reagent, as explained above, is well known to a person skilled in the art.

Each protected amino acid or amino acid sequence is introduced into the solid phase reactor in excess and the coupling is carried out in a medium of dimethylformamide (DMF), methylene chloride (CH2CI2), or mixtures thereof. If coupling is incomplete, the coupling reaction may be repeated before deprotection of the N-amino group and addition of the next amino acid. Coupling efficiency may be monitored by a number of means well known to those of skill in the art. A preferred method of monitoring coupling efficiency is by the ninhydrin reaction. Peptide synthesis reactions may be performed automatically using a number of commercially available peptide synthesizers such as the Applied Biosystems ABI 433A peptide synthesizer (Foster City, CA).

The peptide can be cleaved and the protecting groups removed by stirring the insoluble carrier or solid support in anhydrous, liquid hydrogen fluoride (HF) in the presence of anisole and dimethylsulfide at about 0°C for about 20 to 90 minutes, preferably 60 minutes; by bubbling hydrogen bromide (HBr) continuously through a 1 mg 10 ml suspension of the resin in trifluoroacetic acid (TFA) for 60 to 360 minutes at about room temperature, depending on the protecting groups selected; or by incubating the solid support inside the reaction column used for the solid phase synthesis with 90% trifluoroacetic acid, 5% water and 5% triethylsilane for about 30 to 60 minutes. Other deprotection methods well known to those of skill in the art may also be used.

The peptides can be isolated and purified from the reaction mixture by means of peptide purification well known to a person skilled in the art. For example, the peptides may be purified using known chromatographic procedures such as reverse phase HPLC, gel permeation, ion exchange, size exclusion, affinity, partition, or countercurrent distribution. The following relates to allatostatin-b polypeptide(s) to be used in accordance with the present invention. The term "allatostatin-b" and "allatostatin-b polypeptide" has been described herein above in detail. The meaning of the term "polypeptide" and "nucleic acid sequence(s)/molecule(s)" are well known in the art and are used accordingly in context of the present invention. For example, "nucleic acid sequence(s)/molecule(s)" as used herein refer(s) to all forms of naturally occurring or recombinantly generated types of nucleic acids and/or nucleic acid sequences/molecules as well as to chemically synthesized nucleic acid sequences/molecules. This term also encompasses nucleic acid analogs and nucleic acid derivatives such as e.g. locked DNA, PNA, oligonucleotide thiophosphates and substituted ribo-oligonucleotides. Furthermore, the term "nucleic acid sequence(s)/molecules(s)" also refers to any molecule that comprises nucleotides or nucleotide analogs.

Preferably, the term "nucleic acid sequence(s)/molecule(s)" refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The "nucleic acid sequence(s)/molecule(s)" may be made by synthetic chemical methodology known to one of ordinary skill in the art, or by the use of recombinant technology, or may be isolated from natural sources, or by a combination thereof. The DNA and RNA may optionally comprise unnatural nucleotides and may be single or double stranded. "Nucleic acid sequence(s)/molecule(s)" also refers to sense and anti-sense DNA and RNA, that is, a nucleotide sequence which is complementary to a specific sequence of nucleotides in DNA and/or RNA.

Furthermore, the term "nucleic acid sequence(s)/molecule(s)" may refer to DNA or RNA or hybrids thereof or any modification thereof that is known in the state of the art (see, e.g., US 5525711, US 4711955, US 5792608 or EP 302175 for examples of modifications). The nucleic acid molecule(s) may be single- or double-stranded, linear or circular, natural or synthetic, and without any size limitation. For instance, the nucleic acid molecule(s) may be genomic DNA, cDNA, mRNA, antisense RNA, ribozymal or a DNA encoding such RNAs or chimeroplasts (Colestrauss, Science (1996), 1386-1389). Said nucleic acid molecule(s) may be in the form of a plasmid or of viral DNA or RNA. "Nucleic acid sequence(s)/molecule(s)" may also refer to (an) oligonucleotide(s). wherein any of the state of the art modifications such as phosphothioates or peptide nucleic acids (PNA) are included.

The nucleic acid sequence encoding allatostatin-b polypeptide of other species than the herein provided nucleic acid sequences encoding allatostatin-b polypeptides can be identified by the skilled person using methods known in the art, e.g. by using hybridization assays or by using alignments, either manually or by using computer programs such as those mentioned herein below in connection with the definition of the term "hybridization" and degrees of similarity. The nucleic acid sequence encoding for orthologues of an allatostatin-b polypeptide may be at least 40% similar to the nucleic acid sequence as shown in SEQ ID NO:l (nucleic acid encoding allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 3 (nucleic acid encoding allatostatin-b polypeptide of Capitella teleta), SEQ ID NO: 5 (nucleic acid encoding allatostatin-b polypeptide of Aplysia californica), SEQ ID NO:7 (nucleic acid encoding allatostatin-b polypeptide of Crassostrea gigas), SEQ ID NO:9 (nucleic acid encoding allatostatin-b polypeptide of Lottia gigantea), SEQ ID NO: 11 (nucleic acid encoding allatostatin-b polypeptide of Lymnaea stagnalis), SEQ ID NO: 13 (nucleic acid encoding allatostatin-b polypeptide of Biomphalaria glabrata), SEQ ID NO: 15 (nucleic acid encoding allatostatin-b polypeptide of Tritonia diomedea), SEQ ID NO: 17 (nucleic acid encoding allatostatin-b polypeptide of Mytilus californianus), SEQ ID NO: 19 (nucleic acid encoding allatostatin-b polypeptide of Euprymna scolopes), SEQ ID NO:21 (nucleic acid encoding allatostatin-b polypeptide of Idiosepius paradoxus), SEQ ID NO:23 (nucleic acid encoding allatostatin-b polypeptide of Doryteuthis pealeii) or SEQ ID NO: 36 (nucleic acid encoding allatostatin-b polypeptide of Hirudo medicinalis)..

More preferably, the nucleic acid sequence encoding for orthologues of the above described allatostatin-b polypeptide(s) is at least 45 %, 50 %, 55 %, 60 %, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, even more preferably at least 95%, 96%, 97% or 98% similar to the nucleic acid sequence as shown in SEQ ID NO:l (nucleic acid encoding allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 3 (nucleic acid encoding allatostatin-b polypeptide of Capitella teleta), SEQ ID NO: 5 (nucleic acid encoding allatostatin-b polypeptide of Aplysia californica), SEQ ID NO: 7 (nucleic acid encoding allatostatin-b polypeptide of Crassostrea gigas), SEQ ID NO:9 (nucleic acid encoding allatostatin-b polypeptide of Lottia gigantea), SEQ ID NO: 11 (nucleic acid encoding allatostatin-b polypeptide of Lymnaea stagnalis), SEQ ID NO: 13 (nucleic acid encoding allatostatin-b polypeptide of Biomphalaria glabrata), SEQ ID NO: 15 (nucleic acid encoding allatostatin-b polypeptide of Tritonia diomedea), SEQ ID NO: 17 (nucleic acid encoding allatostatin-b polypeptide of Mytilus californianus), SEQ ID NO: 19 (nucleic acid encoding allatostatin-b polypeptide of Euprymna scolopes), SEQ ID NO:21 (nucleic acid encoding allatostatin-b polypeptide of Idiosepius paradoxus), SEQ ID NO:23 (nucleic acid encoding allatostatin-b polypeptide of Doryteuthis pealeii) or SEQ ID NO: 36 (nucleic acid encoding allatostatin-b polypeptide of Hirudo medicinalis), wherein the higher values are preferred.

Most preferably, the nucleic acid sequence encoding for orthologs of the above described allatostatin-b polypeptide(s) is at least 99% similar to the nucleic acid sequence as shown in SEQ ID NO:l (nucleic acid encoding allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 3 (nucleic acid encoding allatostatin-b polypeptide of Capitella teleta), SEQ ID NO:5 (nucleic acid encoding allatostatin-b polypeptide of Aplysia californica), SEQ ID NO:7 (nucleic acid encoding allatostatin-b polypeptide of Crassostrea gigas), SEQ ID NO: 9 (nucleic acid encoding allatostatin-b polypeptide of Lottia gigantea), SEQ ID NO: 11 (nucleic acid encoding allatostatin-b polypeptide of Lymnaea stagnalis), SEQ ID NO: 13 (nucleic acid encoding allatostatin-b polypeptide of Biomphalaria glabrata), SEQ ID NO: 15 (nucleic acid encoding allatostatin-b polypeptide of Tritonia diomedea), SEQ ID NO: 17 (nucleic acid encoding allatostatin-b polypeptide of Mytilus californianus), SEQ ID NO: 19 (nucleic acid encoding allatostatin-b polypeptide of Euprymna scolopes), SEQ ID NO:21 (nucleic acid encoding allatostatin-b polypeptide of Idiosepius paradoxus), or SEQ ID NO:23 (nucleic acid encoding allatostatin-b polypeptide of Doryteuthis pealeii) or SEQ ID NO: 36 (nucleic acid encoding allatostatin-b polypeptide of Hirudo medicinalis).

The term "orthologous protein" "orthologous polypeptide" or "orthologous gene'Vorthologous nucleic acid molecule" as used herein refers to proteins/polypeptides and genes/nucleic acid molecuels, respectively, in different species that are similar to each other because they originated from a common ancestor. As mentioned above, the use of such an "orthologous protein' '/"orthologous polypeptide" or "orthologous gene'Vorthologous nucleic acid molecule" is envisaged in context of the present invention.

Hybridization assays for the characterization of orthologues of known nucleic acid sequences are well known in the art; see e.g. Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989).

The term "hybridization" or "hybridizes" as used herein may relate to hybridizations under stringent or non-stringent conditions. If not further specified, the conditions are preferably non-stringent. Said hybridization conditions may be established according to conventional protocols described, e.g., in Sambrook (2001) loc. cit; Ausubel (1989) loc. cit., or Higgins and Hames (Eds.) "Nucleic acid hybridization, a practical approach" IRL Press Oxford, Washington DC, (1985). The setting of conditions is well within the skill of the artisan and can be determined according to protocols described in the art. Thus, the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as, for example, the highly stringent hybridization conditions of 0.1 x SSC, 0.1% SDS at 65°C or 2 x SSC, 60°C, 0.1 % SDS. Low stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may, for example, be set at 6 x SSC, 1% SDS at 65°C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. In accordance with the present invention, the terms "similarity" or "percent similarity" or "identical" or "percent identity" or "percentage identity" or "sequence identity" ("similarT'similarity" and "identical/"identity" can be used interchangeably herein) in the context of two or more nucleic acid sequences refers to two or more sequences or subsequences that are the same, or that have a specified percentage of nucleotides that are the same (preferably at least 40 % identity, more preferably at least 45 %, 50 %, 55 %, 60 %, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identity, most preferably at least 99% identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 75% to 90% or greater sequence identity may be considered to be substantially identical. Such a definition also applies to the complement of a test sequence. Preferably the described identity exists over a region that is at least about 15 to 25 nucleotides in length, more preferably, over a region that is at least about 50 to 100 nucleotides in length and most preferably, over a region that is at least about 800 to 1400 nucleotides in length, or the full length of a sequence as shown in SEQ ID NO:l (nucleic acid encoding allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 3 (nucleic acid encoding allatostatin-b polypeptide of Capitella teleta), SEQ ID NO: 5 (nucleic acid encoding allatostatin-b polypeptide of Aplysia californica), SEQ ID NO:7 (nucleic acid encoding allatostatin-b polypeptide of Crassostrea gigas), SEQ ID NO:9 (nucleic acid encoding allatostatin-b polypeptide of Lottia gigantea), SEQ ID NO: 11 (nucleic acid encoding allatostatin-b polypeptide of Lymnaea stagnalis), SEQ ID NO: 13 (nucleic acid encoding allatostatin-b polypeptide of Biomphalaria glabrata), SEQ ID NO: 15 (nucleic acid encoding allatostatin-b polypeptide of Tritonia diomedea), SEQ ID NO: 17 (nucleic acid encoding allatostatin-b polypeptide of ytilus californianus), SEQ ID NO: 19 (nucleic acid encoding allatostatin-b polypeptide of Euprymna scolopes), SEQ ID NO:21 (nucleic acid encoding allatostatin-b polypeptide of Idiosepius paradoxus), SEQ ID NO;23 (nucleic acid encoding allatostatin-b polypeptide of Doryteuthis pealeii) or SEQ ID NO: 36 (nucleic acid encoding allatostatin-b polypeptide of Hirudo medicinalis).

Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as known in the art.

Although the FASTDB algorithm typically does not consider internal non-matching deletions or additions in sequences, i.e., gaps, in its calculation, this can be corrected manually to avoid an overestimation of the % identity. CLUSTALW, however, does take sequence gaps into account in its identity calculations. Also available to those having skill in this art are the BLAST and BLAST 2.0 algorithms (Altschul, (1997) Nucl. Acids Res. 25:3389-3402; Altschul (1993) J. Mol. Evol. 36:290-300; Altschul (1990) J. Mol. Biol. 215:403-410). The BLASTN program for nucleic acid sequences uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLOSUM62 scoring matrix (Henikoff (1989) PNAS 89:10915) uses alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

In order to determine whether a nucleotide residue in a nucleic acid sequence corresponds to a certain position in the nucleotide sequence of e.g. SEQ ID NO:l (nucleic acid encoding allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 3 (nucleic acid encoding allatostatin-b polypeptide of Capitella teleta), SEQ ID NO: 5 (nucleic acid encoding allatostatin-b polypeptide of Aplysia californica), SEQ ID NO:7 (nucleic acid encoding allatostatin-b polypeptide of Crassostrea gigas), SEQ ID NO:9 (nucleic acid encoding allatostatin-b polypeptide of Lottia gigantea), SEQ ID NO: 11 (nucleic acid encoding allatostatin-b polypeptide of Lymnaea stagnalis), SEQ ID NO: 13 (nucleic acid encoding allatostatin-b polypeptide of Biomphalaria glabrata), SEQ ID NO: 15 (nucleic acid encoding allatostatin-b polypeptide of Tritonia diomedea), SEQ ID NO: 17 (nucleic acid encoding allatostatin-b polypeptide of Mytilus califomianus), SEQ ID NO: 19 (nucleic acid encoding allatostatin-b polypeptide of Euprymna scolopes), SEQ ID NO:21 (nucleic acid encoding allatostatin-b polypeptide of Idiosepius paradoxus), SEQ ID NO:23 (nucleic acid encoding allatostatin-b polypeptide of Doryteuthis pealeii) or SEQ ID NO: 36 (nucleic acid encoding allatostatin-b polypeptide of Hirudo medicinalis), the skilled person can use means and methods well-known in the art, e.g., alignments, either manually or by using computer programs such as those mentioned herein. For example, BLAST 2.0, which stands for Basic Local Alignment Search Tool BLAST (Altschul (1997), loc. cit.; Altschul (1993), loc. cit.; Altschul (1990), loc. cit.), can be used to search for local sequence alignments. BLAST, as discussed above, produces alignments of nucleotide sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying similar sequences. The fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP). An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cut-off score set by the user. The BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance. The parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.

Analogous computer techniques using BLAST (Altschul (1997), loc. cit.; Altschul (1993), loc. cit.; Altschul (1990), loc. cit.) are used to search for identical or related molecules in nucleotide databases such as GenBank or EMBL. This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score which is defined as:

% sequence identity x % maximum BLAST score

100 and it takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1-2% error; and at 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules. Another example for a program capable of generating sequence alignments is the CLUSTALW computer program (Thompson (1994) Nucl. Acids Res. 2:4673-4680) or FASTDB (Brutlag (1990) Comp. App. Biosci. 6:237-245), as known in the art.

The explanations and definitions given herein above in respect of "similarity of nucleic acid sequences" apply, mutatis mutandis, to "amino acid sequences", in particular an amino acid sequence as depicted in in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 4 (allatostatin-b polypeptide of Capitella teleta), SEQ ID NO:6 (allatostatin-b polypeptide of Aplysia califomica), SEQ ID NO: 8 (allatostatin-b polypeptide of Crassostrea gigas), SEQ ID NO: 10 (allatostatin-b polypeptide of Lottia gigantea), SEQ ID NO: 12 (allatostatin-b polypeptide of Lymnaea stagnalis). SEQ ID NO: 14 (allatostatin-b polypeptide of Biomphalaria glabrata), SEQ ID NO: 16 (allatostatin-b polypeptide of Tritonia diomedea), SEQ ID NO: 18 (allatostatin-b polypeptide of Mytilus californianus), SEQ ID NO:20 (allatostatin-b polypeptide of Euprymna scolopes), SEQ ID NO:22 (allatostatin-b polypeptide of Idiosepius paradoxus), SEQ ID NO:24 (allatostatin-b polypeptide of Doryteuthis pealeii) or SEQ ID NO: 37 (allatostatin-b polypeptide of Hirudo medicinalis).

The polypeptide to be used in accordance with the present invention may have at least 40 % homology/similarity/identity to the polypeptide having the amino acid sequence as depicted in in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 4 (allatostatin-b polypeptide of Capitella teleta), SEQ ID NO:6 (allatostatin-b polypeptide of Aplysia californica), SEQ ID NO: 8 (allatostatin-b polypeptide of Crassostrea gigas), SEQ ID NO:10 (allatostatin-b polypeptide of Lottia gigantea), SEQ ID NO:12 (allatostatin-b polypeptide of Lymnaea stagnalis), SEQ ID NO: 14 (allatostatin-b polypeptide of Biomphalaria glabrata), SEQ ID NO: 16 (allatostatin-b polypeptide of Tritonia diomedea), SEQ ID NO:18 (allatostatin-b polypeptide of ytilus californianus), SEQ ID NO:20 (allatostatin-b polypeptide of Euprymna scolopes), SEQ ID NO: 22 (allatostatin-b polypeptide of Idiosepius paradoxus), SEQ ID NO:24 (allatostatin-b polypeptide of Doryteuthis pealeii) or SEQ ID NO: 37 (allatostatin-b polypeptide of Hirudo medicinalis).

More preferably, the polypeptide has at least 45 %, 50 %, 55 %, 60 %, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% similarity/identity to the polypeptide having the amino acid sequence as depicted in in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 4 (allatostatin-b polypeptide of Capitella teleta), SEQ ID NO:6 (allatostatin-b polypeptide of Aplysia californica), SEQ ID NO:8 (allatostatin-b polypeptide of Crassostrea gigas), SEQ ID NO: 10 (allatostatin-b polypeptide of Lottia gigantea), SEQ ID NO: 12 (allatostatin-b polypeptide of Lymnaea stagnalis), SEQ ID NO: 14 (allatostatin-b polypeptide of Biomphalaria glabrata), SEQ ID NO: 16 (allatostatin-b polypeptide of Tritonia diomedea), SEQ ID NO: 18 (allatostatin-b polypeptide of Mytilus californianus), SEQ ID NO:20 (allatostatin-b polypeptide of Euprymna scolopes), SEQ ID NO:22 (allatostatin-b polypeptide of Idiosepius paradoxus), SEQ ID NO:24 (allatostatin-b polypeptide of Doryteuthis pealeii) or SEQ ID NO: 37 (allatostatin-b polypeptide of Hirudo medicinalis), wherein the higher values are preferred.

Most preferably, the polypeptide has at least 99% similarity/identity to the polypeptide having the amino acid sequence as depicted in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii), SEQ ID NO: 4 (allatostatin-b polypeptide of Capitella teleta), SEQ ID NO:6 (allatostatin-b polypeptide of Aplysia californica), SEQ ID NO:8 (allatostatin-b polypeptide of Crassostrea gigas), SEQ ID NO: 10 (allatostatin-b polypeptide of Lottia gigantea), SEQ ID NO: 12 (allatostatin-b polypeptide of Lymnaea stagnalis), SEQ ID NO: 14 (allatostatin-b polypeptide of Biomphalaria glabrata), SEQ ID NO: 16 (allatostatin-b polypeptide of Tritonia diomedea), SEQ ID NO: 18 (allatostatin-b polypeptide of Mytilus californianus), SEQ ID NO:20 (allatostatin-b polypeptide of Euprymna scolopes), SEQ ID NO:22 (allatostatin-b polypeptide of Idiosepius paradoxus), SEQ ID NO:24 (allatostatin-b polypeptide of Doryteuthis pealeii) or SEQ ID NO: 37 (allatostatin-b polypeptide of Hirudo medicinalis). The allatostatin-b polypeptide (or peptides derived therefrom) may have one or more amino acids deleted, inserted, added and/or substituted provided that allatostatin-b polypeptide maintains essentially the biological activity which is characteristic of the herein above described allatostatin-b polypeptides. Preferably, any such deletions, insertions, additions and/or substitutions (in this context particularly substitutions) are conservative, i.e. amino acids are substituted by amino acids having the same or similar characteristics. For example, a hydrophobic amino acid will preferably be substituted by another hydrophobic amino acid and so on.

The above allatostatin-b peptides (or peptides derived therefrom) may optionally be altered so as to form non-peptide analogs, including but not limited to replacing one or more bonds with less labile bonds, cyclization and the like. Such a modification may be advantageous, because it may help removing the peptides from the water/aquaculture after the settlement and/or growth was induced or enhanced. It may either allow recycling of the peptides, or it may prevent release of the peptides to the environment. An N-terminal biotinylation via a Cys residue may be advantageous to isolate/recover the peptides from the water/aquaculture via streptavidin beads after the settlement and/or growth was induced or enhanced.

A "peptidomimetic organic moiety" can optionally be substituted for (an) amino acid residue(s) in an allatostatin-b polypeptide (or peptides derived therefrom) both as conservative and as non-conservative substitutions. These moieties are also termed "non- natural amino acids" and may optionally replace (an) amino acid residue(s), (an) amino acid(s) or act as (a) spacer group(s) within the peptides in lieu of the substituted/replaced amino acids.

The peptidomimetic organic moieties may have steric, electronic or configurational properties similar to the substituted/replaced amino acid(s). Such peptidomitnetics may be used to replace amino acids in the essential positions, and are considered conservative substitutions. Such similar properties are not necessarily required. The only restriction on the use of peptidomimetic organic moiety is that the an allatostatin-b polypeptide (or peptides derived therefrom) at least substantially retains its biological activity as compared to the native/original allatostatin-b polypeptide (or peptides derived therefrom) as provided and described herein.

Peptidomimetic organic moieties may be used to inhibit degradation of the allatostatin-b polypeptide (or peptides derived therefrom) by enzymatic or other degradative processes. The peptidomimetics may be produced by organic synthetic techniques. Non-limiting examples of suitable peptidomimetics include D amino acids of the corresponding L amino acids, tetrazol (Zabrocki et al., J. Am. Chem. Soc. 110:5875 5880 (1988)); isosteres of amide bonds (Jones et al., Tetrahedron Lett. 29: 3853 3856 (1988)); LL 3 amino 2 propenidone 6 carboxylic acid (LL Acp) (Kemp et al., J. Org. Chem. 50:5834 5838 (1985)). Similar analogs are shown in Kemp et al., Tetrahedron Lett. 29:5081 5082 (1988) as well as Kemp et al., Tetrahedron Lett. 29:5057 5060 (1988), Kemp et al., Tetrahedron Lett. 29:4935 4938 (1988) and Kemp et al., J. Org. Chem. 54: 109 115 (1987). Further peptidomimetics are described in Nagai and Sato, Tetrahedron Lett. 26:647650 (1985); Di Maio et al., J. Chem. Soc. Perkin Trans., 1687 (1985); Kahn et al., Tetrahedron Lett. 30:2317 (1989); Olson et al., J. Am. Chem. Soc. 112:323 333 (1990); Garvey et al., J. Org. Chem. 56:436 (1990). Other exemplary peptidomimetics are hydroxy 1,2,3,4 tetrahydroisoquinoline 3 carboxylate (Miyake et al., J. Takeda Res. Labs 43 :53 76 (1989)); 1,2,3,4 tetrahydro- isoquinoline 3 carboxylate (Kazmierski et al., J. Am. Chem. Soc. 133 :2275 2283 (1991)); histidine isoquinolone carboxylic acid (HIC) (Zechel et al., Int. J. Pep. Protein Res. 43 (1991)); (2S, 3S) methyl phenylalanine, (2S, 3R) methyl phenylalanine, (2R, 3S) methyl phenylalanine and (2R, 3R) methyl phenylalanine (Kazmierski and Hruby, Tetrahedron Lett. (1991)).

Exemplary, non-natural amino acids include commercially available beta-amino acids (beta3 and beta2), homo-amino acids, cyclic amino acids, aromatic amino acids, Pro and Pyr derivatives, 3 -substituted Alanine derivatives, Glycine derivatives, ring-substituted Phe and Tyr Derivatives, linear core amino acids or diamino acids.

Any part of the allatostatin-b polypeptide (or peptides derived therefrom) may be chemically modified, i.e. changed by addition of functional groups. The modification may be performed during synthesis of the molecule if a chemical synthetic process is followed, for example by adding a chemically modified amino acid. However, chemical modification of an amino acid when it is already present in the molecule ("in situ" modification) is also envisaged.

The amino acid(s) of any of the allatostatin-b polypeptide (or peptides derived therefrom) can be modified according to any of the following exemplary types of modification (in the peptide conceptually viewed as "chemically modified"). Non-limiting exemplary types of modification include carboxymethylation, acylation, phosphorylation, glycosylation or fatty acylation. Ether bonds can optionally be used to join the serine or threonine hydroxyl to the hydroxyl of a sugar. Amide bonds can optionally be used to join the gmtamate or aspartate carboxyl groups to an amino group on a sugar (Garg and Jeanloz, Advances in Carbohydrate Chemistry and Biochemistry, Vol. 43, Academic Press (1985); Kunz, Ang. Chem. Int. Ed. English 26:294-308 (1987)). Acetal and ketal bonds can also optionally be formed between amino acids and carbohydrates. Fatty acid acyl derivatives can optionally be made, for example, by acylation of a free amino group (e.g., lysine) (Toth et al., Peptides: Chemistry, Structure and Biology, Rivier and Marshal, eds., ESCOM Publ., Leiden, 1078-1079 (1990)).

As used herein the term "chemical modification" when referring to an allatostatin-b polypeptide (or peptide derived therefrom), refers to an allatostatin-b polypeptide (or peptide derived therefrom) wherein at least one of its amino acid residues is modified either by natural processes, such as processing or other post-translational modifications, or by chemical modification techniques which are well known in the art. Examples of the numerous known modifications typically include, but are not limited to: acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristylation, pegylation, prenylation, phosphorylation, ubiquitination, or any similar process.

Further, amino acids may be added to the herein above described allatostatin-b polypeptide(s) (or peptides derived therefrom). For example, at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 and up to 300 amino acids (or even more amino acids) may be added to the N-terminus of the allatostatin-b polypeptides (or peptides derived therefrom). In addition, or in the alternative, a at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 and up to 300 amino acids (or even more amino acids) may be added to the C-terminus of the allatostatin-b polypeptides (or peptides derived therefrom) without deferring from the gist of the present invention.

The biological activity of the peptides to be used herein (i.e. the peptides derived from (an) allatostatin polypeptide(s)) can be assayed in tissue culture experiments where the allatostatin-b G-protein coupled receptor is expressed in cell culture (e.g. CHO-K1 cells) also expressing a calcium-sensitive bioluminescent fusion protein. Receptor activation can then be measured using standard GPCR-activation assays using a fluorescent plate reader. Alternatively, the activity of the peptides can be determined in biological assays where larvae are incubated in the presence of the peptide and their swimming and settlement behavior (including surface contacts) is quantified.

Moreover, the biological activity of the allatostatin polypeptide precursor (from which the peptides to be used herein are derived) can be determined by its processing as substrate the signal ecognition particle for signal-peptide containing preproteins, prohormone convertases or alpha-amydating enzymes. As mentioned above, the present invention relates to a method of inducing or enhancing the settlement and/or growth of one or more larva of marine Lophotrochozoan invertebrates comprising culturing said one or more larva in a composition, said composition comprising or consisting essentially of one or more peptides derived from allatostatin-b.

Thus, the present invention relates to a method of inducing the settlement of one or more larva of marine Lophotrochozoan invertebrates comprising culturing said one or more larva in a composition, said composition comprising or consisting essentially of one or more peptides derived from allatostatin-b. In an alternative, the present invention relates to a method of inducing the growth of one or more larva of marine Lophotrochozoan invertebrates comprising culturing said one or more larva in a composition, said composition comprising or consisting essentially of one or more peptides derived from allatostatin-b. In an alternative, the present invention relates to a method of enhancing the settlement of one or more larva of marine Lophotrochozoan invertebrates comprising culturing said one or more larva in a composition, said composition comprising or consisting essentially of one or more peptides derived from allatostatin-b. In an alternative, the present invention relates to a method of enhancing the settlement and/or growth of one or more larva of marine Lophotrochozoan invertebrates comprising culturing said one or more larva in a composition, said composition comprising or consisting essentially of one or more peptides derived from allatostatin-b. In an alternative, the present invention relates to a method of inducing the settlement and growth of one or more larva of marine Lophotrochozoan invertebrates comprising culturing said one or more larva in a composition, said composition comprising or consisting essentially of one or more peptides derived from allatostatin-b. In an alternative, the present invention relates to a method of enhancing the settlement and growth of one or more larva of marine Lophotrochozoan invertebrates comprising culturing said one or more larva in a composition, said composition comprising or consisting essentially of one or more peptides derived from allatostatin-b.

The explanations and definitions given herein in relation to "inducing or enhancing the settlement and/or growth", "marine Lophotrochozoan invertebrates", "culturing in a composition", "peptides derived from allatotatin-b" and the like apply, mutatis mutandis, to the above described aspects of the herein provided method of the invention.

Because marine larvae of cephalopods (the paralarvae) do not settle, the aspects of the present invention relating to the induction or enhancement of the growth of one or more larva of marine invertebrates is particularly relevant for cephalopod larvae, i.e. wherein the marine invertebrate larva(e) or animal(s) stemming therefrom belong(s) to the class cephalopoda. The composition provided and to be used herein (e.g. for culturing the one or more larva of marine Lophotrochozoan invertebrates) may further comprise water. Because the marine Lophotrochozoan larvae grow under natural conditions in saline water, the water may be saline water, such as brackish water or (natural) seawater.

The term "saline water" as used herein refers to water that contains a significant concentration of dissolved salts (particularly NaCl), like brackish water or seawater. The concentration of the dissolved salts is often expressed in parts per million (ppm) of salt. For example, if water has a concentration of 10,000 ppm of dissolved salts, then one percent (10,000 divided by 1,000,000) of the weight of the water comes from dissolved salts. Based on the salinity concentration level saline water may be classified in three categories. Slightly saline water contains around 1,000 to 3,000 ppm. Moderately saline water contains roughly 3,000 to 10,000 ppm. Highly saline water has around 10,000 to 35,000 ppm of salt. Seawater usually has a salinity of roughly 35,000 ppm. Thus, saline water to be used herein may contain a concentration of dissolved salts of from about 1,000 to 50,000 ppm, such as about 10,000 to about 35,000 ppm. If seawater is to be used, the concentration of dissolved salts is of from 34,000 to 36,000 ppm, like about 35,000 ppm.

The peptide derived from an allatostatin-b polypeptide is present in the composition in a concentration sufficient to induce or enhance the settlement and/or growth of the one or more larva. A concentration sufficient to induce or enhance the settlement and/or growth of the one or more larva is, for example a concentration of from 5 nM to 100 μΜ, of from 10 nM to 100 μΜ, preferably of from 100 nM to 100 μΜ. For example, the peptide may be present in the composition in a concentration of from 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μΜ, 2 μΜ, 3 μΜ, 4 μΜ, preferably of from 5 μΜ, 6 μΜ, 7 μΜ, 8 μΜ, 9 μΜ, 10 μΜ, 15 μΜ, 20 μΜ, 25 μΜ, 30 μΜ, 35 μΜ, 40 μΜ, 45 μΜ,50 μΜ, 55 μΜ, 60μΜ, 65 μΜ, 70 μΜ, 75 μΜ, 80 μΜ, 85 μΜ, 90 μΜ, 95 μΜ, to 100 μΜ.

Based on the teaching of the present application, a person skilled in the art is easily in the position to determine the concentration of peptide(s) derived from an allatostatin-b polypeptide that is sufficient to induce or enhance the settlement and/or growth of the one or more larva. Assays for determining the settlement and or growth of the larvae are described above and provided in the appended example. A person skilled in the art is readily in the position to employ such exemplary assays to determine the minimum, optimal and maximum concentration that will induce or enhance settlement and or growth of the larvae. Such a minimum/ optimal/maximum concentration can be considered as a sufficient concentration of peptide(s) derived from an allatostatin-b polypeptide to induce or enhance the settlement and/or growth of the one or more larva.

The herein provided method may further comprise growing mature one or more animal stemming from the one or more larva of marine Lophotrochozoan invertebrates and harvesting the mature one or more animal. The term "marine Lophotrochozoan invertebrates" as used herein refers primarily to invertebrate animals which live in the sea (saline water, such as sea- water as defined above). The one or more larva or one or more animal stemming therefrom may belong to the Lophotrochozoan superphylum. The Lophotrochozoan superphylum was established based on molecular data and includes the group of trochozoans and of the lophophorata

The Lophotrochozoa comprise two groups, the trochozoans and the lophophorata. The "trochozoans" are a unified taxonomic group in that they produce a "trochophore" larva (or "trochophore-like larva") as explained further below. The Trochozoa include the Mollusca (molluscs), Annelida (annelids), Nemertea, and Sipuncula. Previously, also arthropods were considered to be trochozoans, even though the arthropods do not produce trochophore larvae, because both annelids and arthropods are segmented. However, recent research showed important differences between trochozoans and arthropods. Consequently, arthropods are now placed among the Ecdysozoa.

The term "larva of marine Lophotrochozoan invertebrates" as used herein refers, in accordance with the above, to a (developing) larva of invertebrate animals which live in the sea, like trochozoans (such as molluscs and annelids). Thus, "larva" is the invertebrate animal in a larval stage. In accordance with the above, the larva(e) may be (a) "planktonic larva(e)". Upon growth and metamorphosis, the larva develops first into a "juvenile" ("spat") and then into a mature animal (i.e. the juvenile animal or mature animal "stems from" the larva). The terms "juvenile" and "spat" are used herein interchangeably. The term "mature animal" as used herein refers to a mature marine Lophotrochozoan invertebrate animals that has developed from the larva and is therefore no longer in the larval stage. The term "mature animal" as used herein may, for example, refer to sexually mature/pubescent animals. The (developing) larvae may be defined as„(developing) larvae that are capable of passing/pass through the stage of a trochophore larva or trochophore-like larva".

The larva of marine Lophotrochozoan invertebrates as defined herein may be a healthy larva, i.e. in no need of a medical (prophylactic) treatment.

The term "larva" as used herein refers to various larval stages of the marine Lophotrochozoan invertebrates. Depending on the marine Lophotrochozoan invertebrate to be used herein, different terms of the "larva" may be used, as it is commonly known in the art. For example, if the larva belongs to the annelid phylum, the larva may be a trochophore larva, a metatrochophore larva or a nectochate larva. If the larva belongs to the class bivalvia, the larva may be a trochophore larva or a veliger larva. If the larva belongs to the class cephalopoda, the larva may be a paralarva.

In accordance with the above, the one or more larva or one or more mature animal stemming therefrom may belong to the annelid phylum, or the mollusc phylum. The annelid may belong to the genus Platynereis or the genus Capitella, such as Platynereis dumerilii or Capitella teleta. The mollusc may belong to the genus Aplysia, such as Aplysia californica.

The molluscs may belong to the class bivalvia or the molluscs may belong to the class cephalopoda. The bivalve or mollusc may be commercially relevant. Exemplary bivalves to be used herein may belong to the Pteriomorphia subclass or to the Heterodonta subclass.

Pteriomorphia is a subclass of marine bivalve molluscs containing several major families, like the Arcoida, Ostreoida, Pectinoida, Limoida, Mytiloida, and Pterioida. This group includes mussels, scallops, pen shells, and oysters. Most commercially relevant bivalves may be found in the family Arcoidea (like the genus Anadara), Ostreoidea (like the genera Pecten, Crassostrea, Patinopecten), or Mytiloida (like the genera Perna or Mytilis). Accordingly, exemplary bivalves to be used herein may belong to any of Arcoida, Ostreoida, Pectinoida, Limoida, Mytiloida, and Pterioida, such as Arcoidea, Ostreoidea, or Mytiloida.

The Heterodonta subclass includes mostly saltwater bivalve molluscs, like clams and cockles. Commercially relevant bivalves are primarily found in the order Veneroida (like the genera Ruditapes or Mercenaria). Accordingly, exemplary bivalves to be used herein may belong to the Veneroida.

Exemplary bivalves to be used herein may belong to the genus Pecten, Ruditapes, Crassostrea, Anadara, Perna, Patinopecten, Mytilis or Mercenaria. Non-limiting examples of bivalves are Pecten maximus, Ruditapes philippinarum, Crassostrea gigas, Anadara granosa, Pema viridis, Patinopecten yessoensis, Mytilis edulis, Mytilis galloprovincialis, Pema canaliculus or Mercenaria mercenaria.

Herein peptides are provided which are or are derived from an allatostatin-b polypeptide. Exemplary peptides have been described above in context of methods of inducing or enhancing the settlement and/or growth of one or more larva of marine Lophotrochozoan invertebrates. The explanations and definitions given herein above in said context apply, mutatis mutandis, to the following text passages describing peptides which are or are derived from an allatostatin-b polypeptide.

Accordingly, the peptide provided herein may have or consist of the consensus motif from N to C terminus (X)_nW_a(X)_nZWb(X)n, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_a may be absent. The peptide may, for example, have a length of from 2 to 48 amino acids. For example, the peptide may consist of from 2 to 33 amino acids, like of from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 46, 47, or 48 amino acids.

The peptide may consist of a fragment of an allatostatin-b polypeptide. Exemplary allatostatin-b polypeptide are described herein. For example, the peptide may comprise or consist of a peptide selected from the group consisting of AW, VW, TW, SW, and NW.. In the alternative, the the peptide may comprise or consist of the peptide MW, LW or QW. The peptide may comprise or may consist of a peptide like AWMKNN1AW (SEQ ID NO: 38), AWGDNNMRVW (SEQ ID NO: 39), AWNKNSMRVW (SEQ ID NO: 40), AWKGQSARVW (SEQ ID NO: 41), GWNGNSMRVW (SEQ ID NO: 42), RKWSKFSSW (SEQ ID NO: 45), WKNMAVW (SEQ ID NO: 46), WKQMASW (SEQ ID NO: 47), WKQMSVW (SEQ ID NO: 48), WKEMSVW (SEQ ID NO: 49), WKQMAVW (SEQ ID NO: 50), WKQMATW (SEQ ID NO: 51), AWNKNSMRVWP (SEQ ID NO: 52), or AWNKNSMRVW A (SEQ ID NO: 53).

Further, the peptide may comprise a C-terminal tryptophane (W) amino acid residue. The C- terminal residue of the peptide (like the C-terminal tryptophane (W) amino acid residue) may be amidated.

Moreover, herein provided is an antibody specifically binding to/specifically recognizing the herein above provided and defined peptide(s). As one advantageous property the herein provided antibodies are cross-species specific, i.e. they can bind to (poly) peptides of various species. It has been found herein that antibodies specifically binding to the above peptides may be generated using small (i.e. the peptides consist of 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, preferably 5 or less amino acids, like 4, 3, or 2 amino acids) amidated peptides as immunogens; see Example 3. Said immunogenic peptides comprise or consist of a sequence from N-terminus to C-terminus RY, GW, FV, FL and DL. Also the peptides derived from an allatostatin-b polypeptide as defined herein may be used or considered as "immunogenic peptides" without deferring from the gist of the present invention. The respective definitions and explanations given herein above in relation to "peptides derived from an allatostatin-b polypeptide" apply, mutatis mutandis, in this context. The immunogenic peptide may have one or more additional amino acids added at the N-terminus and the C-terminus. Accordingly, the present invention provides immunogenic peptides having or consisting of the consensus motif from N-terminus to C-terminus (X)_mRY(X)_n, (X)_mGW(X)_n, (X)_mFV(X)_n, (X)_mFL(X)„, (X)_mDL(X)]j, whereby X may be any amino acid (such as a cysteine amino acid residue), and whereby m is an integer of from 0 to 8 and whereby n is an integer of from 0 to 8. Preferably, n is zero. The immunogenic peptides may consist of 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, preferably 5 or less amino acids, like 4, 3, or 2 amino acids). The C-terminal amino acid residue of said immunogenic peptides may be amidated. Exemplary immunogenic peptides are CRYamide, CGWamide, CFVamide, CFLamide or CDLamide. The antibodies produced by using said immunogenic peptides specifically recognize the epitope RY, GW, FV, FL and DL, respectively.

As mentioned, it was found herein that two-amino-acid amidated peptides can be used to generate specific antibodies. This finding challenges previous notions about the immunogenic potential of peptide sequences. Usually, it is considered in the art that up to 5 amino acid peptides (i.e. peptides consisting of 5 or less amino acids) are not immunigenic at all. It is believed that the strong immunogenicity of the above provided and defined peptides may be due to their C-terminal amidation (see, for example, the exemplary immunogenic peptides CRYamide, CGWamide, CFVamide, CFLamide or CDLamidein the context of two amino acid residues with an additional cysteine residue at the N-terminus). We believe that it is not the amide group that is recognized alone, since all peptides have it. Still, no cross-reactivity was observed. The importance of amidation, and not the two residues alone, is supported by the observation that such two-amino-acid motifs can be found in thousands of other proteins (61,717 DL, 32,582 FV, 9,459 GW and 18,792 RY in the Capitella predicted proteome); yet, no strong background staining was observed.

Overall, the data provided herein indicate that the antibodies strongly and specifically bind to the amidated peptides that were used for immunization. First, the stringent affinity purification protocol that was employed together with the peptide-blocking experiments showed that the antibodies strongly bind to the short amidated peptides. Second, the specific neuronal stainings in tissues corresponding to the expression patterns of the precursor genes in Platynereis showed that the antibodies specifically bind to the respective peptides.

The strategy employed herein to generate highly reactive, specific cross-species antibodies can be applied to other conserved neuropeptides present in various taxonomic breadth as well. With the increasing sampling of metazoan genomes and transcriptomes, and the accumulation of data from understudied groups (e.g. hemichordates, platyhelminths, priapulids), further conserved peptide motifs may be identified. Further sampling will also allow to identify other groups where the antibodies described here could be used as neuronal markers. Given the brevity of the sequences, cross reactivity to more neuropeptide types cannot be excluded. It is therefore important to scrutinize the available transcriptome, genome, and mRNA expression, in order to properly evaluate the immunoreactivity.

Finally, C-terminal amidation is commonly used in the art for immunization for peptides that derive from an internal part of the protein, to keep the peptide closer to its natural state. Our results caution that such an unnatural terminal amide in internal peptide sequences may trigger an undesired immune response, and potentially cause cross-reactivity to naturally occurring amidated peptides.

With the DLamide, FVamide and RYamide antibodies projections to larval ciliary bands were observed. It was recently shown that the neurons expressing these neuropeptides also innervate the ciliary band in Platynereis larvae, and the neuropeptides regulate the activity of cilia. All three neuropeptides increased the beating frequency of cilia and inhibited ciliary arrests, and thereby influenced the swimming depth of the planktonic larvae. In Capitella all three neuropeptides were detected in the ciliary band nerve. In Pecten and Phestilla larvae FVamide and RYamide immunoreactivity was found in a nerve running along the ciliated velum. RYamide neurons also seems to innervate locomotor cilia in bryozoan and cnidarian larvae.

In sum, the data herein show that specific cross-species reactive antibodies recognize the conserved neuropepeptide motifs DLamide, FVamide, FLamide, GWamide and RYamide. These antibodies can be used in a wide range of marine invertebrates, including annelids, moUusks, and bryozoans. Further genomic and transcriptomic sampling could identify further animal groups where these peptide motifs are conserved and where the herein provided antibodies could be used. The data also highlights the good antigenic potential of very short amidated peptide motifs. The ongoing sampling of neuropeptide diversity will allow the development of other similar antibodies to further enriching this important toolbox of comparative neurobiology. The sampling across diverse marine Lophotrochozoan larvae demonstrates the broad utility of these antibodies..

In accordance with the above, immunogenic peptides can be used to produce antibodies that specifically bind to epitopes of the herein above provided and defined allatostatin-b polypeptide(s) and/or peptides derived from an allatostatin-b polypeptide. Exemplary epitopes that can be recognized by the antibodies are AW, VW, TW, SW, or NW, Further epitopes may be MW, LW or QW. Accordingly, the present invention provides immunogenic peptides having or consisting of the consensus motiv from N-terminus to C-terminus (X)_mAW(X)_n, (X)_mVW(X)„, (X)_mTW(X)_n, (X)_mSW(X)_n, (X)_m W(X)„, (X)_mQW(X)_n, whereby X may be any amino acid (such as a cysteine amino acid residue), and whereby m is an integer of from 0 to 8 and whereby n is an integer of from 0 to 8. The immunogenic peptides may consist of 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, preferably 5 or less amino acids, like 4, 3, or 2 amino acids). The C-terminal amino acid residue of said irnmunogenic peptides may be amidated. Examplary immunogenic peptides are CAWamide, CVWamide, CTWamide, CSWamide, CNWamide. Further exemplary immunogenic peptides are CMW amide, CLW amide or CQWamide. The antibodies generated by using such immunogens can be used to detect the expression level of allatostatin-b derived peptides (e.g. endogenous peptides). An exemplary antibody specifically binding to the VW epitope of allatostatin-b-derived peptides (or allatostatin-b polypeptide) has been used in Example 1. Such antibodies are specifically useful as research tools or for purification of the herein provided peptides (like peptides derived from an allatostatin-b polypeptide).

The herein provided antibodies may also be comprised in a composition, such as a diagnostic composition. Also a kit comprising such a composition, such as a diagnostic composition, and corresponding uses of the kit are envisaged in context of the present invention.

The present invention also relates to an antibody/antibodies as defined above or the above composition comprising said antibody/antibodies for the preparation of a diagnostic kit for use in the methods of the present invention.

The antibody may be a polyclonal antibody, a monoclonal antibody, a full antibody (immunoglobulin), a F(ab)-fragment, a F(ab)2-fragment, a single-chain antibody, a chimeric antibody, a CDR-grafted antibody, a bivalent antibody-construct, a bispecific single chain antibody, a synthetic antibody or a cross-cloned antibody and the like. Based on the teaching of the present invention, polyclonal or monoclonal antibodies or other antibodies (derived therefrom) can be prepared using, inter alia, standard immunization protocols; see Ed Harlow, David Lane, (December 1988), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; or Ed Harlow, David Lane, (December 1998), Portable Protocols (Using Antibodies): A Laboratory Manual 2^nd edition, Cold Spring Harbor Laboratory.

For example, immunization may involve the intraperitoneal or subcutaneous administration of the immunogenic peptide as defined above (and/or fragments, isoforms, homologues and so on) as defined herein to a mammal (e.g. rodents such as mice, rats, hamsters and the like). Also fragments of the immunogenic peptide as defined above may be used, wherein the fragment preferably contains the amino acid sequence RY, GW, FV, FL, DL, AW, VW, TW, SW, or NW. The fragment may also contain the amino acids LW, MW, or QW. Zero or at least one and up to eigth amino acid may be adjacent to one or both side(s) of the above sequences. Corresponding fragments to be used as immunogenic peptides may be derived from the herein described and defined allatostatin-b polypeptides. The immunogenic peptides may be prepared by enzymatic digestion e.g. of allatostatin-b polypeptides or by chemical synthesis.

Methods known in the art can be used for the preparation and screening of antibodies that specifically bind to or specifically recognize the herein above provided and described allatostatin-b polypeptides (or peptides derived therefrom). For example, antibodies specifically recognizing binding to the allatostatin-b polypeptides (or peptides derived therefrom) may be affinity purified. ELISA is commonly used for screening sera and/or assaying affinity column fractions. Western Blots can be used to demonstrate that the antibody can detect the actual protein of interest and to evaluate whether the antibody only recognizes the protein of interest, or if it cross-reacts with other proteins.

A person skilled in the art is in the position to apply and to adapt the teaching of the above documents for the generation and validation of antibodies specifically binding to or specifically recognizing the herein provided peptides like allatostatin-b polypeptides (or peptides derived therefrom) as defined herein in context of the present invention.

Further, the present invention relates to a composition comprising or consisting essentially of one or more of the herein above described and defined peptides. The composition may, for example, be used in aquaculture of larvae of marine Lophotrochozoan invertebrates, whereby said one or more peptides comprised in the composition is capable of inducing or enhancing the settlement of one or more larva of marine Lophotrochozoan invertebrates.

The composition may optionally further comprise nutrients, such as phytoplankton or zooplankton, for feeding one or more larva of marine Lophotrochozoan invertebrates. The composition may optionally comprise phytoplankton and zooplankton. The composition may be a forage or may comprise a forage.

The composition may optionally further comprise one or more biocides (e.g. antifouling agents), disinfectants, pesticides, ingredients for the treatment of soil and/or water (e.g. ingredients for adjusting the pH value), anorganic fertilizers, organic fertilizers, hormones, microbial products, vitamins and/or enzymes. The pesticide may be a herbicide, a fungicide or insecticide or piscizide.

In case the larva(e) of marine Lophotrochozoan invertebrates is(are) in need of a medical (prophylactic) treatment, the composition may, in addition to the peptide defined and provided above (like the peptide derived from allatostatin-b), comprise one or more medicaments for treating a disease of the one or more larva of marine Lophotrochozoan invertebrates. Non-limiting examples of the one or more medicaments to be used are an antibiotic, a vaccine, a parasitizide, an anaesthetic or an immune stimulant.

Further, the present invention provides water as used in aquaculture of one or more larva of marine Lophotrochozoan invertebrates

(i) comprising one or more peptide as defined herein, wherein the peptide is preferably present in the water in a concentration sufficient to induce or enhance the settlement of one ore more larva of marine Lophotrochozoan invertebrates.; or

(ii) comprising the composition as defined herein, wherein the peptide(s) as comprised in the composition are preferably present in the water in a concentration sufficient to induce or enhance the settlement of Lophotrochozoan marine larvae.

The "concentration sufficient to induce or enhance the settlement of one ore more larva of marine Lophotrochozoan invertebrates" has been defined and explained above. Thus, the method may, for example,

(i) comprise one or more peptide as defined herein, wherein the peptide is present in the water in a concentration of from 5 nM to 100 μΜ; or

(ii) comprise the composition as defined herein, wherein the peptide(s) as comprised in the composition are present in the water in a concentration of from 5 nM to 100 μΜ.

All explanations and definitions given herein above in relation to, for example, the peptide(s), the composition, the larva of marine Lophotrochozoan invertebrates, the concentration sufficient to induce or enhance the settlement and/or growth of the Iarva(e) and exemplary concentrations of the peptide, apply, mutatis mutandis, in the context of the water to be used in aquaculture of one or more larva of marine Lophotrochozoan invertebrates. The term "aquaculture" as used herein is well known in the art. The term relates, for example, to the farming of aquatic organisms, such as the marine Lophotrochozoan invertebrates as defined herein (e.g trochozoans like mollusks and annelids). Therefore, "aquaculture" is also referred to as "aquafarming" and both terms can be used interchangeably herein. Aquaculture as used herein involves cultivating saltwater populations under controlled conditions (in contrast to commercial fishing). Envisaged herein is a specific type of aquaculture, termed "mariculture" which refers particularly to the cultivation of marine organisms, like the marine Lophotrochozoan invertebrates as defined herein. Mariculture may involve said cultivation in the open ocean, an enclosed section of the ocean, or in tanks, ponds or raceways which are filled with saline water, like seawater.

As mentioned, „aquaculture" may involve the use of hatcheries. For example, bivales (mussels), like oysters and the like may be cultivated as follows in hatcheries:

Bivalves naturally often grow in estuarine bodies of brackish water. In aquaculture, the temperature and salinity of the water are controlled (or at least monitored), so as to induce spawning and fertilization, as well as to speed the rate of maturation.

Three methods of cultivation are commonly used. In each case oysters are cultivated to the size of "spat," the point at which they attach themselves to a substrate. The substrate is known as a "culch" or "cultch". The loose spat may be allowed to mature further to form "seed" oysters with small shells. In either case (spat or seed stage), they are then set out to mature. The maturation technique is where the cultivation method choice is made.

In one method the spat or seed oysters are distributed over existing oyster beds and left to mature naturally. Such oysters will then be collected using the methods for fishing wild oysters, such as dredging.

In the second method the spat or seed may be put in racks, bags, or cages(or they may be glued in threes to vertical ropes) which are held above the bottom. Oysters cultivated in this manner may be harvested by lifting the bags or racks to the surface and removing mature oysters, or simply retrieving the larger oysters when the enclosure is exposed at low tide. The latter method may avoid losses to some predators, but is more expensive

In the third method the spat or seed are placed in a culch within an artificial maturation tank. The maturation tank may be fed with water that has been especially prepared for the purpose of accelerating the growth rate of the oysters. In particular the temperature and salinity of the water may be altered somewhat from nearby ocean water. The carbonate minerals calcite and aragonite in the water may help oysters develop their shells faster and may also be included in the water processing prior to introduction to the tanks. This latter cultivation technique may be the least susceptible to predators and poaching, but is the most expensive to build and to operate. The Pacific oyster C. gigas is the species most commonly used with this type of farming.

As mentioned above, the herein provided peptides derived from an allatostatin-b polypeptide are useful in food production, like production of molluscs (especially edible molluscs, like (edible) seafish) or useful in bait production, like annelid production for use as bait.

Accordingly, the present invention relates to the following aspects:

A method for obtaining one or more molluscs for nourishment, comprising the steps

(a) culturing one or more mollusc larva in a composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide as defined herein;

(b) inducing or enhancing settlement of the one or more larva;

(c) developing mature molluscs;

(d) harvesting mature molluscs.

The step (c) of developing mature molluscs may involve allowing the larva of step (a) or (b) to mature. In other words the step (c) of developing mature molluscs may involve allowing said larva to develop into a mature mollusc.

A method for obtaining one or more shellfish for nourishment, comprising the steps

(a) culturing one or more shellfish larva in a composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide as defined in herein;

(b) inducing or enhancing settlement of the one or more larva;

(c) developing mature shellfish;

(d) harvesting mature shellfish.

The step (c) of developing mature shellfish may involve allowing the larva of step (a) or (b) to mature. In other words the step (c) of developing mature shellfish may involve allowing said larva to develop into a mature shellfish.

(b) inducing or enhancing growth of the one or more larva;

(c) developing mature molluscs;

(d) harvesting mature molluscs. The step (c) of developing mature molluscs may involve allowing the larva of step (a) or (b) to mature. In other words the step (c) of developing mature molluscs may involve allowing said larva to develop into a mature mollusc.

(a) culturing one or more shellfish larva in a composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide as defined herein;

(b) inducing or enhancing growth of the one or more larva;

(c) developing mature shellfish;

(d) harvesting mature shellfish.

The term "nourishment" as used herein refers to "consumption", in particular "human consumption". In other words, the mollusc or shellfish obtained in accordance with the above aspects of the present invention is intended to serve as food. The food is to be consumed, in particular by (a) human(s).

The one or more shellfish may belong to the class bivalvia as herein.

The one or more mollusc may belong to the class bivalvia as herein.

The one or more mollusc may belong to the class cephalopoda as defined herien.

A method for obtaining one or more annelids for use as bait, comprising the steps

(a) culturing one or more annelid larva in a composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide as defined herein;

(b) inducing or enhancing settlement of the one or more larva;

(c) developing mature annelids;

(d) harvesting mature annelids;

(e) preparing harvested mature annelids for use as a bait.

The step (c) of developing mature annelid may involve allowing the larva of step (a) or (b) to mature. In other words the step (c) of developing mature annelids may involve allowing said larva to develop into a mature annelids.

A method for obtaining one or more annelids for use as bait, comprising the steps (a) culturing one or more annelid larva in a composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide as defined in herien;

(b) inducing or enhancing growth of the one or more larva;

(c) developing mature annelids;

(d) harvesting mature annelids;

(e) preparing harvested mature annelids for use as a bait.

The method may be for obtaining one or more annelids for use as fish bait.

The annelid to be used/obtained in accordance with the present invention may be a commercially relevant annelid, such as annelids used as baits, particularly annelids grown in aquaculture for bait production. The main annelids which is commercially grown as bait in the UK is Nereis virens. Also widely used as bait worm and cultivated are Arenicola defodiens or Arenicola marina. Accordingly, the annelid may belong to the family Nereidae or may belong to the family Arenicolidae. For example, the annelid may belong to the genus Nereis, such as Nereis virens, or the annelid may belong to the genus Arenicola, such as Arenicola defodiens or Arenicola marina. For example, the annelid may be Nereis diversicolor.

Further annelids contemplated herein are which are commonly used as baits may belong to the genus Perinereis, like Perinereis cultrifera, Perinereis nuntia or Perinereis brevicirrus. The annelid may belong to the family Glyceridae, and may, for example, belong to the genus Glycera (like Glycera dibranchiate). The annelid may belong to the family Nephtyidae, and may, for example, belong to the genus Nephtys, like Nephtys hombergi. The annelid may belong to the family Eunicidae, and may, for example, belong to the genus Marphysa, like Marphysa sanguinea or Marphysa leidyi. The annelid may belong to the family Onuphyidae, and may, for example, belong to the genus Onuphis, like Onuphis teres. The annelid may belong to the family Eunicidae Lumbrinereidae, and may, for example, belong to the genus Lumbrinereis, like Lumbrinereis impatiens.

The use of peptides derived from orthologous allatostatin-b polypeptides of the above annelids in the methods of the present invention is contemplated herein.

The annelid may belong to the genus Platynereis or the genus Capitella, such as Platynereis dumerilii or Capitella teleta;

Preferably, the mollusc (like shellfish) as defined herein is an edible mollusc (edible shellfish).

As used herein, the term "shellfish" is intended to have its standard meaning as understood in the art, namely referencing exoskeleton-bearing aquatic invertebrates that are used as food and/or are intended for (human) consumption. Thus, the term shellfish expressly references only edible species of molluscs. For avoidance of any doubt, the term shellfish as used herein does not encompass any non-edible mollusc, or any mollusc, not intended for (human) consumption. Similarly, references to the general terms mullosc, and analogous terms as used in connection with the invention are understood to refer exclusively to edible species of mulloscs. Accordingly, when used in connection with the invention, the terms mollusc, may be used interchangeably with the terms "edible mollusc."

As explained above, it has been found herein that the peptides derived from the allatostatin-b polypeptide interact with a specific receptor. Accordingly, the corresponding allatostatin-b- peptide/ allatostatin-b-peptide-receptor complex is provided. Therefore, the present invention provides a protein complex comprising a first protein interacting with a second protein, wherein

(i) the first protein is a peptide derived from an allatostatin-b polypeptide as defined herein; and

(ii) the second protein is allatostatin-b-peptide-receptor, wherein said allatostatin-b- peptide-receptor is

a) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO: 34;

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO:35;

(c) a polypeptide as defined in (a) or (b) wherein one or more amino acids are deleted, inserted, added or substituted, wherein said polypeptide is capable of interacting with peptide derived from an allatostatin-b polypeptide;

(d) a polypeptide encoded by a nucleic acid molecule encoding a peptide having an amino acid sequence as depicted in SEQ ID NO:35;

(e) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (a) or (d) and encoding allatostatin-b-peptide-receptor or a functional fragment or functional derivative thereof, wherein said polypeptide is capable of interacting with a peptide derived from an allatostatin-b polypeptide;

(f) a polypeptide having at least 60 % homology/identity to the polypeptide of any one of (a) to (e), whereby said polypeptide is allatostatin-b-peptide-receptor or a functional fragment or functional derivative thereof, wherein said polypeptide is capable of interacting with a peptide derived from an allatostatin-b polypeptide;

(g) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (d) and (e); or

(h) a fusion protein containing any of (a) to (g).

The present invention also provides a method for making the above protein complex, comprising the steps of:

(a) providing said first protein and said second protein; and

(b) contacting said first protein with said second protein.

Further, the present invention provides a method for selecting modulators of the above protein complex, said method comprising:

(a) contacting said first protein with said second protein in the presence of one or more test compounds; and

(b) detecting an interaction between said first protein and said second protein.

The contacting step may be conducted in vitro or may be conducted within a host cell.

The above protein complex may also be used to screen and identify peptides derived from an allatostatin-b polypeptide to be used herein. For example, if a protein/peptide forms a complex with the allatostatin-b-peptide-receptor (e.g. shown in SEQ ID NO. 34), the peptide is likely derived from an allatostatin-b polypeptide and, therefore, capable of inducing or enhancing settlement/growth of marine Lophotrochozoan larvae.

Accordingly, the present invention provides a method for screening test peptides, comprising the steps of

(a) contacting the test peptide with the allatostatin-b-peptide-receptor as defined above (e.g. the receptor shown in SEQ ID NO. 34);

(b) evaluating the presence of an interaction between said test peptide and the allatostatin-b- peptide-receptor; and

(c) selecting the test peptide that interacts with the allatostatin-b-peptide-receptor.

The selected peptides may be further validated for their capacity to induce/ or enhance settlement/growth of marine Lophotrochozoan larvae, e.g. via the herein above described assays to determine the respective biological activity. In accordance with the above, the present invention relates to the following aspects:

A method of inducing or enhancing the settlement and or growth of one or more Lophotrochozoan larva of marine invertebrates comprising culturing said one or more larva in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide is a fragment of an allatostatin-b polypeptide.

A method of inducing or enhancing the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates in aquaculture, comprising culturing said one or more larva in aquaculture in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide is a fragment of an allatostatin-b polypeptide.

A method of inducing or enhancing the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates comprising culturing said one or more larva in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide has a length of from 2 to 48 amino acids.

A method of inducing or enhancing the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates in aquaculture, comprising culturing said one or more larva in aquaculture in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide has a length of from 2 to 48 amino acids.

A method of inducing or enhancing the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates comprising cu turing said one or more larva in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide is a fragment of an allatostatin-b polypeptide, and wherein said peptide derived from an allatostatin-b polypeptide has a length of from 2 to 48 amino acids.

A method of inducing or enhancing the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates in aquaculture, comprising culturing said one or more larva in aquaculture in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide is a fragment of an allatostatin-b polypeptide, and wherein said peptide derived from an allatostatin-b polypeptide has a length of from 2 to 48 amino acids.

A method of inducing or enhancing the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates comprising culturing said one or more larva in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide has or consists of the consensus motif from N to C terminus (X)_nW_a(X)nZWb(X)_n, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_a may be absent.

A method of inducing or enhancing the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates in aquaculture, comprising culturing said one or more larva in aquaculture in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide has or consists of the consensus motif from N to C terminus (X)_nW_a(X)_nZWb(X)n, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_a may be absent.

A method of inducing or enhancing the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates comprising culturing said one or more larva in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide is a fragment of an allatostatin-b polypeptide, and wherein said peptide derived from an allatostatin-b polypeptide has or consists of the consensus motif from N to C terminus (X)_nW_a(X)nZW_b(X)n, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_a may be absent.

A method of inducing or enhancing the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates in aquaculture, comprising culturing said one or more larva in aquaculture in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide is a fragment of an allatostatin-b polypeptide and wherein said peptide derived from an allatostatin-b polypeptide has or consists of the consensus motif from N to C terminus (X)_nW_a(X)_nZWb(X)_n, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_a may be absent.

A method of inducing or enhancing the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates comprising culturing said one or more larva in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide has a length of from 2 to 48 amino acids and wherein said peptide derived from an allatostatin-b polypeptide has or consists of the consensus motif from N to C terminus (X)nWa(X)_nZWb(X)n, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_a may be absent.

A method of inducing or enhancing the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates in aquaculture, comprising culturing said one or more larva in aquaculture in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide has a length of from 2 to 48 amino acids and wherein said peptide derived from an allatostatin-b polypeptide has or consists of the consensus motif from N to C terminus (X)_nW_a(X)nZWb(X)n, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_a may be absent.

A method of inducing or enhancing the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates comprising culturing said one or more larva in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide is a fragment of an allatostatin-b polypeptide and wherein said peptide derived from an allatostatin-b polypeptide has a length of from 2 to 48 amino acids and wherein said peptide derived from an allatostatin-b polypeptide has or consists of the consensus motif from N to C terminus (X)nW_a(X)nZWb(X),i, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_amay be absent.

A method of inducing or enhancing the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates in aquaculture, comprising culturing said one or more larva in aquaculture in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide is a fragment of an allatostatin-b polypeptide and wherein said peptide derived from an allatostatin-b polypeptide has a length of from 2 to 48 amino acids and wherein said peptide derived from an allatostatin-b polypeptide has or consists of the consensus motif from N to C terminus (X)_nW_a(X)_nZW_b(X)n, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_a may be absent.

The present invention also relates to the following aspects:

A method of inducing or enhancing the settlement and/or growth of one or more larva of marine invertebrates in aquaculture, comprising culturing said one or more larva in aquaculture in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide is a fragment of an allatostatin-b polypeptide.

A method of inducing or enhancing the settlement and/or growth of one or more larva of marine invertebrates in aquaculture, comprising culturing said one or more larva in aquaculture in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide has a length of from 2 to 48 amino acids.

A method of inducing or enhancing the settlement and/or growth of one or more larva of marine invertebrates in aquaculture, comprising culturing said one or more larva in aquaculture in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide is a fragment of an allatostatin-b polypeptide, and wherein said peptide derived from an allatostatin-b polypeptide has a length of from 2 to 48 amino acids.

A method of inducing or enhancing the settlement and/or growth of one or more larva of marine invertebrates in aquaculture, comprising culturing said one or more larva in aquaculture in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide has or consists of the consensus motif from N to C terminus (X)_nWa(X)_nZWb(X)n, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_a may be absent.

A method of inducing or enhancing the settlement and/or growth of one or more larva of marine invertebrates in aquaculture, comprising culturing said one or more larva in aquaculture in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide is a fragment of an allatostatin-b polypeptide and wherein said peptide derived from an allatostatin-b polypeptide has or consists of the consensus motif from N to C terminus (X)_nW_a(X)nZWb(X)n, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_a may be absent.

A method of inducing or enhancing the settlement and/or growth of one or more larva of marine invertebrates in aquaculture, comprising culturing said one or more larva in aquaculture in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide has a length of from 2 to 48 amino acids and wherein said peptide derived from an allatostatin-b polypeptide has or consists of the consensus motif from N to C terminus (X)_nW_a(X)_nZWb(X)_n, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_a may be absent.

A method of inducing or enhancing the settlement and/or growth of one or more larva of marine invertebrates in aquaculture, comprising culturing said one or more larva in aquaculture in a composition, said composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide, wherein said peptide derived from an allatostatin-b polypeptide is a fragment of an allatostatin-b polypeptide, and wherein said peptide derived from an allatostatin-b polypeptide has a length of from 2 to 48 amino acids, and wherein said peptide derived from an allatostatin-b polypeptide has or consists of the consensus motif from N to C terminus (X)_nW_a(X)nZWb(X)n, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_a may be absent.

It is to be understood that in the above disclosed aspects of the present invention, allatostain-b polypeptide is that shown in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii).

In the alternative, the allatostain-b polypeptide is that shown in SEQ ID NO: 4 (allatostatin-b polypeptide of Capitella teleta).

In the alternative, the allatostain-b polypeptide is that shown in SEQ ID NO:6 (allatostatin-b polypeptide of Aplysia californica).

In the alternative, the allatostain-b polypeptide is that shown in SEQ ID NO:8 (allatostatin-b polypeptide of Crassostrea gigas).

It is to be understood that in the above disclosed aspects of the present invention, the peptide derived from allatostain-b polypeptide comprises or consists of AW.

In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of VW.

In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of TW.

In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of SW.

In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of NW.

It is to be understood that in the above disclosed aspects of the present invention, the peptide derived from allatostain-b polypeptide comprises or consists of AWMKNNIAW (SEQ ID NO: 38).

In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of AWGDNNMRVW (SEQ ID NO: 39)

In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of AWNKNSMRVW (SEQ ID NO: 40).

In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of AWKGQSARVW (SEQ ID NO: 41).

In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of GWNGNSMRVW (SEQ ID NO: 42).

In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of KWGSNSMRVW (SEQ ID NO: 43).

In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of GWADNNMRVW (SEQ ID NO: 44).

In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of RKWSKFSSW (SEQ ID NO: 45). In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of WK MAVW (SEQ ID NO: 46).

In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of WKQMASW (SEQ ID NO: 47).

In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of WKQMSVW (SEQ ID NO: 48).

In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of WKEMSVW (SEQ ID NO: 49).

In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of WKQMAVW (SEQ ID NO: 50).

In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of WKQMATW (SEQ ID NO: 51).

In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of AWNKNSMRVWP (SEQ ID NO: 52).

In the alternative, the peptide derived from allatostain-b polypeptide comprises or consists of AWN NSMRVWA (SEQ ID NO: 53).

In accordance with the above, the present invention relates to the following items:

1. Method of inducing or enhancing the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates said method comprising culturing said one or more larva in a composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide.

2. The method of item 1, wherein the induction or enhancement of the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates relates to the induction or enhancement of the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates in aquaculture, said method comprising culturing said one or more larva in aquaculture in a composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide. The method of item 1 or 2, wherein said peptide derived from an allatostatin-b polypeptide is a fragment of an allatostatin-b polypeptide. The method of any one of items 1 to 3, wherein said peptide derived from an allatostatin-b polypeptide has a length of from 2 to 48 amino acids. The method of any one of items 1 to 4, wherein said peptide derived from an allatostatin-b polypeptide has or consists of the consensus motif from N to C terminus (X)_nW_a(X)nZW_b(X)_n, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_amay be absent. The method of any one of items 1 to 5, wherein said peptide derived from an allatostatin-b polypeptide comprises or consists of a peptide selected from the group consisting of AW, VW, TW, SW, and NW. The method of any one of items 1 to 6, wherein said peptide derived from an allatostatin-b polypeptide comprises or consists of a peptide selected from the group consisting of

AWMKNNIAW (SEQ ID NO. 38), AWGDNNMRVW (SEQ ID NO: 39), AWNKNSMRVW (SEQ ID NO: 40), AWKGQSARVW (SEQ ID NO. 41), GWNGNSMRVW (SEQ ID NO: 42), KWGSNSMRVW (SEQ ID NO: 43), GWADNNMRVW (SEQ ID NO: 44), RKWSKFSSW (SEQ ID NO: 45), WKNMAVW (SEQ ID NO: 46), WKQMASW (SEQ ID NO. 47), WKQMSVW (SEQ ID NO: 48), WKEMSVW (SEQ ID NO. 49), WKQMAVW (SEQ ID NO: 50), WKQMATW (SEQ ID NO. 51), AWNKNSMRVWP (SEQ ID NO: 52), and AWNKNSMRVW A (SEQ ID NO. 53). The method of any one of items 1 to 7, wherein said peptide derived from an allatostatin-b polypeptide comprises a C-terminal tryptophane (W) amino acid residue. The method of any one of items 1 to 8, wherein the C-terminal amino acid residue of said peptide derived from an allatostatin-b polypeptide is amidated. The method of any one of items 1 to 9, wherein said allatostain-b polypeptide is shown in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii); or SEQ ID NO: 4 (allatostatin-b polypeptide of Capitella teleta); or SEQ ID NO:6 (allatostatin-b polypeptide of Aplysia californica); or SEQ ID NO:8 (allatostatin-b polypeptide of Crassostrea gigas); or SEQ ID NO: 10 (allatostatin-b polypeptide of Lottia gigantea); or SEQ ID NO: 12 (allatostatin-b polypeptide of Lymnaea stagnalis); or SEQ ID NO: 14 (allatostatin-b polypeptide of Biomphalaria glabrata); or SEQ ID NO: 16 (allatostatin-b polypeptide of Tritonia diomedea); or SEQ ID NO: 18 (allatostatin-b polypeptide of Mytilus californianus); or SEQ ID NO:20 (allatostatin-b polypeptide of Euprymna scolopes); or SEQ ID NO:22 (allatostatin-b polypeptide of Idiosepius paradoxus); or SEQ ID NO:24 (allatostatin-b polypeptide of Doryteuthis pealeii); or an orthologous polypeptide of any of the above allatostain-b polypeptide. The method of any one of items 1 to 10, wherein said one or more larva or one or more animal belong to the annelid phylum or belong to the mollusc phylum. A method for obtaining one or more molluscs for nourishment, comprising the steps

(a) culturing one or more mollusc larva in a composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide as defined in items 3 to 10;

(b) inducing or enhancing settlement and/or growth of the one or more larva;

(c) developing mature molluscs;

(d) harvesting mature molluscs. A method for obtaining one or more shellfish for nourishment, comprising the steps

(a) culturing one or more shellfish larva in a composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide as defined in items 3 to 10;

(b) inducing or enhancing settlement and/or growth of the one or more larva;

(c) developing mature shellfish;

(d) harvesting mature shellfish. A method for obtaining one or more annelids for use as bait, comprising the steps

(a) culturing one or more annelid larva in a composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide as defined in items 3 to 10;

(b) inducing or enhancing settlement and/or growth of the one or more larva;

(c) developing mature annelids;

(d) harvesting mature annelids;

(e) preparing the harvested mature annelids for use as bait. 15. The method of any one of items 11 to 14, wherein said one or more mollusc or one or more shellfish belongs to the class bivalvia, for example, to the genus Pecten, Crassostrea, Ruditapes, Anadara, Perna, Patinopecten, Mytilis or Mercenaria, like Pecten maxirnus, Ruditapes philippinarum, Crassostrea gigas, Anadara granosa, Perna viridis, Patinopecten yessoensis, Mytilis edulis, Mytilis galloprovincialis, Perna canaliculus and Mercenaria mercenaria; or

wherein said mollusc belongs to the genus Aplysia, such as Aplysia californica; or wherein the one or more mollusc belongs to the class cephalopoda, for example, to the genus Sepia or Octopus, like Sepia officinalis or Octopus vulgaris; or

wherein the annelid belongs to the family Nereidae, for example to the genus Nereis, such as Nereis virens, or wherein the annelid belongs to the family Arenicolidae, for example, to the genus Arenicola, such as Arenicola defodiens or Arenicola marina; or wherein the annelid belongs to the genus Platynereis, such as Platynereis dumerilii, or to the genus Capitella, or Capitella teleta.

The present invention is further illustrated by reference to the following non-limiting figures and examples. Unless otherwise indicated, established methods of recombinant gene technology were used as described, for example, in Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001) ) which is incorporated herein by reference in its entirety.

The Figures show:

Figure 1. Segment addition and cephalic metamorphosis in Platynereis dumerilu after settlement of the larvae.

Outline (left) and SEM (right) images of (A) 3 -segment, (B) 4-segment (C) 5-segment and (D) 7-segment larvae showing the morphological changes occurring during growth and cephalic metamorphosis. During cephalic metamorphosis the first pair of parapodia are transformed into the posterior pair of tentacular cirri. Chaetae are lost from the first chaetigerous segment, which is then incorporated into the head. At completion of cephalic metamorphosis, all tentacular cirri point in an anterior-lateral direction, pgz - posterior growth zone. Scale bars: 100 um.

Figure 2. AST-B neuropeptides in Platynereis and Capitella.

(A) Schematic drawing of the respective AST-B precursor proteins. The N-terminal signal peptide and the peptides (grey) flanked by basic cleavage sites are shown. (B) AST-B peptides of Platynereis. (C) AST-B peptides of Capitella. (B-C) Peptides that were used in pharmacological assays are highlighted in bold. Asterisks in (B-C) indicate sequences that were used for control peptides where tryptophans were substituted for alanin residues.

Figure 3. AST-B/Sex peptide receptor signaling Is conserved in annelids.

(A) Multiple alignment of AST-B peptide sequences with the basic cleavage sites (KR and/or R), the amidation signature Gly, the conserved tryptophan residues and aliphatic residues. (B) Dose-response curve of the Pdu-AST-B receptor to Pdu-AST-B7. (C) Activation of the Pdu- AST-B receptor by Pdu-AST-B7 exclusive of other Platynereis neuropeptides. (D) Activation of the Pdu-AST-B receptor by Ala-substituted Pdu-AST-B7 and Cte-AST-Bl peptides. (E) Activation of the Cte-AST-B receptor by Ala-substituted Cte-AST-Bl and Pdu-AST-B7 peptides. The positive control in (C-D) represents activation of GPR109a with 100 μΜ nicotinic acid. Data are represented as mean ± s.e.m.

Figure 4. Neighbor-joining phylogenetic tree of AST-B/Sex peptide receptors.

The receptors that have been deorphaned in (6, 16) are underlined. The bootstrap values at selected nodes are indicated.

Figure 5. AST-B is expressed in sensory-neurosecretory cells in Platynereis and Capitella.

(A) SEM image of a 48 hpf Platynereis larva. (B) Whole-mount in situ hybridization of Pdu- AST-B in 48 hpf larva, counterstained for acTubulin. (C) Immunostaining for AST-B, counterstained for acTubulin. (D) Close-up view of mitotracker-filled AST-B chemosensory neurons in the apical organ. (E) TEM reconstruction of the ventral pair of AST-B-expressing neurons in a 72 hpf Platynereis larva. (F) Immunostaining of a Capitella larva with AST-B antibody, counterstained for acTubulin. (G) Whole-mount in situ hybridization for Pdu-AST- B-receptor in 48 hpf larva, counterstained for acTubulin. (H) Average expression patterns of Pdu-AST-B and Pdu-AST-B receptor in 48 hpf larvae projected onto a common reference by image registration. (A) is a ventral view, (B-D, F-H) are anterior views, (E) is a dorsal view. Arrowheads in (B-D) point at the same median AST-B expressing cells. Arrows in (B, C) point at the cells that have been reconstructed by TEM and are shown in (E). The scale bar in (A-C, F-G) is 50 urn, in (D, H) is 20 urn in (E) is 10 urn.

Figure 6. Antibody specificity test of the AST-B antibody and the cross-species reactive VW-amide antibody.

(A) AST-B immunostaining. (B) AST-B immunostaining following preincubation of the antibody with 5 mM Pdu-AST-B 7 peptide for 2h. (C) Irmnunostaining with a cross-species reactive VW-amide antibody. (D) Immunostaining with a cross-species reactive VW-amide antibody following preincubation with 5 mM VW-amide peptide for 2h. All images are anterior views of 48 hpf Platynereis larvae, counterstained for acTubulin. The same confocal microscopy and image processing parameters were applied for all images. Scale bar: 50 μιη.

Figure 7. Spatial expression of Platynereis AST-B throughout development.

(A, C-G) Whole-mount in situ hybridization for the Pdu-AST-B precursor counterstained for acTubulin (B-D, F-I) Immunostaining with an AST-B antibody, counterstained for acTubulin (grey) (left panels). Developmental stages are indicated. (A, B, D, F) are anterior views, (C, E, G-I) are ventral views. Scale bar in (A-H) 50 um, in (I) 200 urn.

Figure 8. Correlation of light and electron microscopy identifies the two ventral AST-B expressing sensory-neurosecretory neurons.

(A) Close-up view of the two ventral AST-B neurons labeled by AST-B immunostaining on a 72 hpf Platynereis larva. Arrows point at the long dendrites that project apically between the gland cells. (B) Neurosecretory projections of the two ventral AST-B neurons with dense- cored vesicles. (C) Reconstruction of the two ventral AST-B neurons in relation to the three large secretory gland cells and the cuticle border. The apical microvilli are shown in yellow and red. (D) Apical sensory ending of a ventral AST-B neuron. Arrow points at the basal body of the sensory cilium, arrowheads indicate apical microvilli. The dashed line indicates the border of the cuticle. (E) Dendrite cross-section of the two ventral AST-B neurons at the level of the secretory gland cells. Arrows point at the dendrites. GC: gland cell. Scale bar in (B, D) is 1 um, in (E) 5 um.

Figure 9. Identification of the dye-filling neurons as AST-B expressing neurons.

(A) Mitotracker dye-filling of apical organ chemosensory neurons with long microvilli in a 37 hpf larva, DIC image (left) mitotracker (middle), merged image (right), (B) AST-B immunostaining in a 48 hpf non-ablated larva (C) AST-B immunostaining in a 48 hpf larva where the right mitotracker-filled neuron (c.f. Fig. 2D) was ablated (C) AST-B immunostaining in a 48 hpf larva where both mitotracker-filled neuron (c.f. Fig. 2D) were ablated. Scale bar 25 um.

Figure 10. Gene expression profiling by image registration of AST-B and AST-B- receptor expressing neurons.

Image registration of average expression patterns of (A) Pdu-AST-B precursor (B) Pdu-AST- B-receptor (C) Pdu-phc2, (D) Pdu-dimmed and (E) Pdu-otp, projected on the axonal scaffold visualized by acTubulin staining (white). (F-O) Pairwise coexpression of the indicated genes. The overlap in the average gene expression 3D image stacks registered to the same reference template is shown. All images are of 48 hpf Platynereis larvae. Scale bar: 50 um. Figure 11. AST-B triggers larval settlement in Platynereis via the AST-B receptor.

(A) Percentage of time where cilia are closed in uninjected larvae, and in AST-B-rec mismatch (MM), AST-B-rec-startl and AST-B-rec-start2 morpholino-injected larvae, exposed to 5 μΜ Pdu-AST-B7 peptide (unless otherwise indicated). (B) Representative kymographs of ciliary beating and ciliary closures in control and morpholino injected Platynereis larvae exposed to 5 μΜ Pdu-AST-B7. Kymographs were generated from line selections perpendicular to the beating cilia, as described (19, 20). (C) Swimming trajectories of control larvae (left column) and larvae in the presence of 5 μΜ Pdu-AST-B7 (right column). Red dots indicate the end of tracks. Tracks were generated from a 4 sec video at 30 frames per second. (D) Angular histograms of the displacement vectors of swimming tracks for control larvae, larvae in the presence of 5 μΜ PDu-AST-B7, larvae after washout of peptide and larvae in the presence of 20 uM Pdu-AST-B7 (W9A) peptide. In (D) the P values of a x2-test comparing the number of upward and downward swimming larvae are indicated: ***p < 0.001. n>100 larvae (55-60 hpf). In (A), data are shown as mean + s.e.m. P values of an unpaired / test are indicated: *P < 0.05, **P < 0.01, and ***P < 0.001, n>10 larvae (48 hpf).

Figure 12. Pdu- AST-B has no effect on ciliary beat frequency (CBF).

Ciliary beat frequency in uninjected control larvae in the presence of different concentrations of Pdu-AST-B7 peptide, and in AST-B-Rec mismatch morpholino and AST-B-Rec-startl morpholino injected larvae in the presence of 5 μΜ Pdu-AST-B7 peptide. Data are shown as mean + s.e.m. n > 10 48hpf larvae.

Figure 13 Angular histograms of the displacement vectors of swimming tracks for control larvae and larvae in the presence of the indicated peptide.

P values of a x2-test comparing the number of upward and downward swimming larvae are indicated: *P < 0.05, ***P < 0.001. n>100 Platynereis larvae (55-60hpf).

Figure 14. AST-B morpholinos efficiently knock down AST-B expression in Platynereis. (A) Larva injected with AST-B mismatch- 1 morpholino, immunostained with AST-B antibody (B) Larva injected with AST-B startl morpholino, immunostained with AST-B antibody (C) Larva injected with AST-B mismatch2 morpholino, immunostained with AST-B antibody (D). Larva injected with AST-B start2 morpholino, immunostained with AST-B antibody All images are anterior views of 6 dpf larvae, counterstained for acTubulin. Identical confocal microscopy and image processing parameters were applied for all images. (A-D) are anterior views, (E-F) are ventral views. Scale bar: 100 μχη. Figure 15. Characterization of Platynereis larval feeding.

Differential interference contrast (top) and AF488 fluorescent filter (bottom) light micrographs of example 7 and 10 day old Platynereis control larvae (A, B) and Pdu- AST-B morpholino knockdown larvae (C, D), Feeding larvae have autofluorescent Tetraselmis sp. algae in the gut. Scale bar: 100 um.

Figure 16. AST-B induces metamorphosis in annelids.

(A) SEM images of the larval (left) and juvenile (right) stages of Platynereis and Capitella.

(B) % of larvae feeding at 7-9 dpf following injection of mismatch and Pdu-AST-B start morpholinos (C) % of larvae feeding at 10-12 dpf following injection of mismatch and Pdu- AST-B start morpholinos (D) % of 25 dpf larvae with 3, 4, 5 segments, or complete cephalic metamorphosis following exposure to 5 uM Pdu-AST-B peptides, from 4 dpf on. (E) Fraction of Capitella larvae metamorphosed over time in response to different Cte-AST-B and Ala- substituted peptides. Data are shown as mean mean ± s.e.m. Significance of t-test (B,C) or 2- way ANOVA (E,F) is shown as * if p<0.05; *** if p<0.001. hpi: hours post induction. Scale bars in (A) 100 urn.

Figure 17. Body length of AST-B morpholino injected larvae.

Mean body length of mismatch morpholino and AST-B morpholino injected larvae at 7-9 and 10-12 dpf. Data are shown as mean +/- s.e.m.

Figure 18. Altered gut coloration in larvae exposed to AST-B.

Example larvae from experiments described in Figure 4D. (A) Control DMSO treated 8 dpf larva. (B) 8 dpf larva treated with 5 um AST-B. (C) Control DMSO treated 1 month old juvenile (D) 1 month old juvenile treated with 5 μηι AST-B. Note darker coloration of the gut. At 1 month AST-B treated juvenile is bigger and has developed more segments. All treated larvae expressed this gut phenotype to different extents. Scale bar: 100 um.

Figure 1 . Segmentation and cephalic metamorphosis in Platynereis.

% of 25 dpf larvae with 3, 4, 5 segments, or complete cephalic metamorphosis. Larvae were reared in natural seawater at a density of 3 larva ml and fed with Tetraselmis sp. from 6 days onwards.

Figure 20, Modified AST-B peptides trigger settlement

% of Platynereis larvae crawling in the presence of different modified AST-B peptides (20 uM). All peptides are modified versions of AST-B peptide 7.

N>72 larvae for each condition Figure 21.AST-B treatment triggers crawling of the larvae in the culture dish. Example frame of a video showing larvae crawling on the bottom of a plastic petri dish. Control larvae swim and would be out of focus and therefore not visible in this assay.

Percent of 2.5 dpf larvae touching the bottom of a plastic petri dish in the presence of 5 μΜ AST-B-7 peptide.

Figure 22. Two amino-acid amidated motifs are conserved in neuropeptides across phyla.

Multiple sequence alignment of neuropeptides with a conserved C-terminus found in various eumeatzoan species for DLamides (A), FVamides (B), FLamide (D) GWamides (D) and RYamides (E) including annelids (Platynereis, Capitella, Helobdella), mollusks (Lottia), platyhelminthes (Schmidtea, Macrostonum), nematodes (Caenorhabditis), arthropods (Cancer, Drosophila, Apis) and cnidarians (Podocoryne, Hydractinia). The conserved C- termini that were used for antibody production are highlighted in black. Sequence information obtained from [19-26]. RPCH: red pigment concentrating hormone; AKH: adipokinetic hormone.

Figure 23. Correspondence of antibody signals with the respective precursor mR A expression in Platynereis.

(A-E) Immunostaining with the DLamide (A), FVamide (B), FLamide (C), GWamide (D) and RYamide (E) antibodies counterstained for acetylated tubulin. (Α'-Ε') mRNA in situ hybridization counterstained for acetylated tubulin for DLamide (A), FVamide (B), FLamide (C), GWamide (D) and RYamide (E) neuropeptide precursors. All images are anterior views of 48 hpf Platynereis larvae. Asterisks indicate cells that show a spatial correspondence with the mRNA in situ hybridization signals in A'-E\ Scale bars: 50 um.

Figure 24. Blocking of immunostaining signals with peptide pre-incubation.

(A-E) Regular immunostaining (upper panel), and stainings with an antibody that was pre- incubated with the corresponding synthetic Platynereis full length neuropeptide (bottom panel) for DLamide (A), FVamide (B), FLamide (C), GWamide (D) and RYamide (E). Samples were counterstained for acetylated tubulin (cyan). All images are anterior views of 72 hpf Platynereis larvae. Scale bar: 50 um.

Figure 25. DLamide immunoreactivity in Capitella larvae.

(A) Anterior view of an early Capitella larva and (B) ventral view of a late Capitella larva stained with the DLamide antibody counterstained for acetylated tubulin. Scale bars: 50 um. Figure 26. FVamide immunoreactivity in annelid and mollusk larvae.

Irnmunostainings with the FLamide antibody counterstained for acetylated tubulin in (A) an early Capitella larva, anterior view, (B) a late Capitella larva, ventral view, (C) a Pecten veliger larva, lateral view, and (D, E) a Phestilla larva, dorsal (D) and ventral (E) views. Scale bars: 50 um.

Figure 27. FLamide immunoreactivity in annelid and mollusk larvae.

Irnmunostainings with the FLamide antibody counterstained for acetylated tubulin in (A) a late Capitella larva, ventral view, and (B) a Phestilla larva. Scale bars: 50 um.

Figure 28. GWamide immunoreactivity in annelid, mollusk and crustacean larvae.

Immunostainings with the GWamide antibody counterstained for acetylated tubulin in (A) an early Capitella larva, anterior view (B) a late Capitel larva, ventral view, (C) a Pecten veliger larva, lateral view, (D) a Phestilla larva, dorsal view, (E) and a crustacean larva, ventral view. Scale bars: 50 um.

Figure 29. RYamide immunoreactivity in annelid, bryozoan, mollusk. crustacean and cnidarian larvae.

Irnmunostainings with the RYamide antibody counterstained for acetylated tubulin in (A) an early Capitella larva, anterior view, (B) a late Capitella larva, ventral view, (C) a Cryptosula larva, anterior view, (D) a Pecten veliger larva, anterior view, (E) a Pecten veliger larva counterstained with the anti-FVamide antibody, anterior view, (F) a Phestilla larva, dorsal and (G) ventral view, (H) a crustacean larva, ventral view, (I) a Clava planula larva, lateral view, and (I) an Aurelia planula larva, lateral view, and (K) close-up of a sensory neuron. Scale bars: 50 um, unless otherwise indicated.

Figure 30. Prior art FLamide peptide, which is not a peptide derived from an allatostatin-h polypeptide, does not induce settlement

Figure 31. RGW amide triggers downward vertical migration but not settlement.

(A) Vertical distribution of 2 day old Platynereis larvae treated with 5μΜ RGWamide for 10 min (right) compared to control larvae (teft), showing that RGWa induces downward vertical migration of larvae. Assays were performed in a 25cm high vertical migration setup.

(B) Ciliary beat frequency of immobilized 48 hpf Platynereis larvae treated with increasing concentrations of VWa (Ast-B 7) which does not increase ciliary beat frequency. RGWa treatment on 38 hpf larvae significantly increases ciliary beat frequency, suggesting that downwards vertical migration observed in (A) is a result of active downward swimming and not caused by a cessation of cilia, hpf = hours post fertilization. (C) Larval settlement assay of 2 day old larvae performed in a 24-well plate. AST-B-7 treatment led to a sustained surface contact whereas RGWa did not, indicating that RGWa does not directly trigger settlement, dpf = days post fertilization.

(D) Horizontal swimming assay performed in a 24-well plate shows that RGWa does not reduce swimming speed. This indicates that RGWa does not directly trigger settlement.

p- values of an un-paired t-test are indicated: *** P < 0.001

Fig. 32. BLOSUM62 cluster map of metazoan pNP families.

Nodes correspond to pNPs and are colored based on taxonomy. Edges correspond to BLAST connections of p-value>le-5.

Kg. 33. PAM30 map of the Central Cluster (CC).

The largest cluster in the PAM30 map was defined using linkage clustering and optimized separately. Nodes correspond to pNPs and are colored based on taxonomy. Edges represent BLAST connections of p-value>le-5. Color-code as in Fig. 1.

Fig. 34. Phyletic distribution of metazoan pNP families.

The families that are part of the CC are shown in red. Ancestral bilaterian (*), protostome (+), deuterostome (o) and chordate (-) families are indicated. Pdf, leucokinin, TRH, and PTH may be ancestral bilaterian, motilin, MCH, endothelin ancestral chordate, based on GPCR distribution.

Fig. 35. BLOSUM62 cluster map of class A neuropeptide GPCRs.

Nodes correspond to class A GPCRs and are colored based on taxonomy. Edges represent

BLAST connections of p-value>le-30. Color-code same as in Fig. 1.

Fig. 36. Large cohesive sequence clusters, repeat length and distribution of R I FA I amides and Wamides in pNP CLANS maps.

(A) Individual clusters in the BLOSUM62 map were determined by linkage clustering (minimum 3 linkages) and are shown in different colors. Only clusters with more than 30 sequences are shown. The Central Cluster is shown in red. (B) The largest cluster in the BLOSUM62 map was defined by linkage clustering (minimum 3 linkages). This subset of sequences was further optimized and color-coded for taxonomy. (C) Different repeat lengths of pNPs are indicated in different colors on the PAM30 CLANS cluster map. Only those pNPs were colored that had at least two amidated peptides of the same length flanked by dibasic cleavage sites. The color code for the different repetitive peptide lengths is shown. (D) PAM30 clustering showing the Central Cluster with the mapping of RFamide, RYamide and Wamide terminal motifs. Fig. 37. Cluster analysis of pNPs and class B neuropeptide GPCRs.

(A) A BLOSUM62 cluster map of pNPs was colored to highlight the indicated amidated termini in the mature neuropeptides. The individual amidated termini and the family they belong to are listed in the table. (B) BLOSUM62 cluster map of class B neuropeptide GPCRs. Nodes correspond to class B GPCR sequences and are colored based on taxonomy. Edges represent BLAST connections of p-value>le-50. (C) BLOSUM62 cluster map of prokineticin/astakine/colipase, (D) ΡΊ Η/trunk/noggin and (E) neuroparsin/IGFBP domains. Representatives of the indicated families were clustered and colored based on taxonomy. Edges represent BLAST connections of p-value>le-5.

Fig. 38. Phyletic distribution of metazoan neuropeptide GPCR families.

Phyletic distribution of metazoan class A and class B neuropeptide GPCR families. Class B GPCRs are indicated as (B). Ancestral bilaterian (*), protostome (+), deuterostome (o) and chordate (-) families are indicated.

Fig. 39. Structure of placozoan and lophotrochozoan opioid pNPs.

(A) Schematic structure of pNPs from the placozoan Trichoplax adhaerens. (B) Schematic structure of the Platynereis dumerilii (annelid), Lottia gigantea and Haliotis asinina (mollusks) opioid pNPs. SPs are shown in blue, peptides with a C-teminal Gly in green, dibasic cleavage sites in red, Cys residues in yellow. The sequence logos show the conservation of residues in the predicted mature peptides.

Fig. 40. Multiple alignments of pNP families.gl05

The multiple alignments for the selected pNP families were generated either with Muscle or Cobalt. GenBank/SwissProt or JGI identifiers and full species names are shown. The multiple alignments were visualized with Jalview. The sequences are colored according to the Clustalx color scheme, using varying conservation cutoff. Short motifs were identified using MEME. The sequences shown in Fig. 40 relate to SEQ ID NOs: 196 to 426. The Examples illustrate the invention.

Example 1: AIlatostatin-B peptides induce the settlement of Lophotrochozoan marine larvae.

Materials and Methods

Gene identification

Platynereis genes were identified from EST sequences generated from a full-length normalized cDNA library from mixed larval stages. Capitella genes were identified at the JGI Genome Portal (nttp://genome.jgi.doe.gov) (34).

Receptor deorphanization

Platynereis and Capitella sex peptide receptor orthologs were cloned into a pcDNA3.1+ vector (Invitrogen) with Hindlll and Not! The Platynereis receptor was PCR amplified from larval cDNA using the primers

ACAATAAAGCTTGCCACCATGATGGAAGTAAGCTATTCAAATGGAAATG (SEQ ID NO: 32) (including Hindlll site and Kozak consensus) and ACAATAGCGGCCGCTTAAATATTTGTAGTTTTAGTCGTGTGATCG (SEQ ID NO: 33) (including Notl site), the Capitella receptor clone used was a synthetic construct (GenScript). CHO-K1 cells stably expressing a calcium- sensitive bioluminescent fusion protein (17) were transfected and receptor activation was measured as previously described (18). As positive control, we used the GPR109a receptor stimulated with 100 μΜ nicotinic acid. Measurements were performed using a fluorescent plate reader. The area under each calcium transient (measured for one minute) was calculated using Ascent software (Thermo Electron Corporation) and expressed as integrated luminescence units (relative units).

Animal culture

Larvae were obtained from established breeding cultures for Platynereis (55), Capitella (36). Antibodies and tissue staining

Rabbit antisera against Platynereis AST-B and against the conserved C-terminal VW-amide motif were affinity purified using the respective peptides coupled to a SulfoLink resin (Thermo Scientific, Rockford, USA) via an N-terminal Cys (CAWNKNSMR VW-amide (SEQ ID NO: 427) and CVW-amide). A detailed protocol for preparing antibodies specifically recognizing the conserved DL-amide motif, FV-amide motif, FL-amide motif, GW-amide motif or RY-amide motif is provided in Example 3. This protocol was used for the preparation of the antibodies specifically recognizing the VW-amide motif as used in Example 1. In situ hybridization was performed as previously described (38). Dye filling and laser ablation

50 μg mitotracker red FM (Invitrogen, special packaging) was freshly dissolved in 100 μΐ DMSO. The solution was added to 30 hpf larvae at 1 :500 dilution and incubated for 1 h for optimal dye filling. Single larvae were mounted on a glass slide with 2 layers of adhesive tape on both sides in 20 ul natural seawater and covered with a coverslip to immobilize them. Dye- filled neurons were ablated on an Olympus F VI 000 confocal microscope equipped with a 355 nm pulsed laser (teem photonics) coupled via air and controlled by the SIM Scanner. The ablated larvae were recovered and fixed at 48 or 52 hpf and processed for AST-B immunostaining.

Light microscopy and image registration

Confocal imaging and image registration were performed as previously described (20). A reference generated based on DAPI staining of nuclei was used for registration. For coexpression analysis the image stacks of the average registered gene expression patterns were compared. Coexpression was determined based on overlap in the image stacks following thresholding of the individual average gene expression signals.

JEM

72 hpf Platynereis larvae were frozen using a high-pressure freezer (BAL-TEC HPM 010, Balzers, Liechtenstein) and quickly transferred to liquid nitrogen. Frozen samples were treated with a substitution medium containing 2% osmium tetroxide in acetone and 0.5% uranyl acetate in a cryosubstitution unit (Leica EM AFS-2). The samples were cryosubstited over a regime of gradually rising temperatures and embedded in Epon. 0 nm serial sections were cut on a Reichert Jung Ultracut E microtome. The sections were collected on single- slotted copper grids (NOTSCH-NUM 2x1 mm, Science Service) with Formvar support film, contrasted with uranyl acetate and Reynold's lead citrate, and carbon coated to stabilized the film. Image acquisition of serial sections was performed at a pixel size of 3.87 nm on a FEI TECNAI Spirit transmission electron microscope equipped with an UltraScan 4000 4X4k digital camera using the image acquisition software Digital Micrograph (Gatan Software Team Inc.) Stitching and alignment were done using the TrakEM2 software. All structures were segmented manually as area-lists, which were exported into 3Dviewer and Imaris.

SEM

Samples were fixed with 3% glutaraldehyde in 0.1 M phosphate buffer pH 7.2, rinsed in phosphate buffer, further fixed with 1% osmium tetroxide in water and dehydrated in an ascending ethanol series over several days. Critical point drying with carbon dioxide was performed in a Polaron E 3000. The samples were coated with gold-palladium in a Balzers MED 010. Images were taken on a Hitachi S-800 Scanning electron microscope.

Behavior

Vertical larval swimming and ciliary beating assays were performed and analyzed as previously described (20).

Peptide treatment

Peptide treatment experiments were carried out in Nunclon 6-well tissue culture dishes, with 10 ml sterile filtered seawater (FSW) per well. Each control and peptide treatment was replicated across three wells, with 30 larvae per well.

For Platynereis, 5 uM synthetic peptides were added at 4 days after fertilization. Larvae were also fed at 4 days with 5 μΐ Tetraselmis sp. algal culture. At 25 days after fertilization (21 days after peptide addition), larvae were scored for number of segments and cephalic metamorphosis (as described in Fig. 1.).

Capitel larvae were collected upon emergence from the brooding tube and kept for 3 days in Nunclon 6-well tissue culture dishes, as described above. 10 μΜ synthetic peptides were added at 3 days after emergence from the tubes. Wells were scored for metamorphosis (defined as loss of cilia and swimming, emergence of hook-shaped chaetae, crawling and tube secretion) at 12, 24, 36, 48, 72 and 96 h after peptide addition.

Wells were scored for metamorphosis (defined as loss of apical tuft, cessation of swimming, and emergence of feeding tentacles) at 12, 24 and 48 h after peptide addition.

Morpholino injections

For microinjections, fertilized Platynereis eggs developing at 16°C were rinsed -1 h after fertilization with sterile filtered seawater (FSW) in a 100 um sieve to remove the egg jelly, followed by treatment with 70 μg/ml proteinase K for 1 min to soften the vitellin envelope. Injections were carried out using Eppendorf Femtotip II needles with a Femtojet microinjector (Eppendorf, Germany) on a Zeiss Axiovert 40 CL inverted microscope equipped with a Luigs & Neumann micromanipulator. The temperature of the developing zygotes was maintained at 16°C throughout injection using a Luigs & Neumann Badcontroller V cooling system and a Roth Cyclo 2 water pump.

Two translation-blocking morpholinos (MO's) (with two corresponding 5 bp mismatch control MO's) were designed to target the Pdu-AST-B gene and two translation-blocking MO's (with one corresponding 5 bp mismatch control MO) were designed to target the Pdu- AST-B receptor gene. Morpholinos with the following sequences were purchased from Gene Tools, USA: Pdu-ASTB-start MOl TGATAGTGACGCGATCCATTGGACT (SEQ ID NO: 25)

Pdu-ASTBR-start MOl TCCATCATTTTGAATGTTGAATGCA (SEQ ID NO:29) Pdu-ASTBR-mism MOl

(SEQ ID NO: 31) Pdu-ASTBR-start M02 GTCAATGAGGTCACAAACATCCAAC (SEQ ID NO: 30)

Nucleotides complementary to the start codon (ATG) are underlined; nucleotides altered in mismatch control morpholinos are highlighted in bold MOs were diluted in water with 12 μg/μl fluorescein dextran (M_r 10,000, Invitrogen) as a fluorescent tracer. 0.6 mM MOs were injected with an injection pressure of 600 hP a for 0.1 s and a compensation pressure of 35 hPa. Injected zygotes were kept in Nunclon 6- well plates in 10 ml FSW and their development was monitored daily. 48 hpf injected larvae were used for ciliary resting measurements in the presence of 5 μΜ synthetic Pdu-AST-B peptide or DMSO as control. Larvae were fed 5 μΐ Tetraselmis sp. algal culture at 6 dpf. Feeding in 7-14 dpf injected larvae was assessed by checking for the presence of fluorescent Tetraselmis spp. algae in the gut using a Zeiss Axioimager Zl microscope with a AF488 fluorescent filter and a 20X objective. 48 hpf and 6 dpf larvae injected with Pdu-AST-B start and mismatch morpholinos were fixed in 4% paraformaldehyde in IX PBS with 0.1% Tween-20 for immunostaining with the AST- B antibody in order to assess morpholino specificity and effectiveness.

Results

Herein the function of AST-B neuropeptides in the Lophotrochozoan marine annelid models Platynereis dumerilii and Capitella teleta (Fig. 2) was identified. Platynereis belongs to the errant annelids (errantia), and Capitella to the sedentary annelids (Sedentaria), representing a very deep evolutionary divergence, encompassing most annelid diversity (14). BLAST searches with annelid AST-B precursors retrieved mollusk AST-B precursors (e- value 6e-15; also called myoinhibiting peptide, MIP or prothoracicostatic peptide, PTSP). A multiple alignment of AST-B-amide peptides shows the conservation of the last Trp residue preceding the amidation signature Gly, as well as a small, often aliphatic residue before that Trp moiety (Fig. 3A).

The first Trp (position 1 or 2) is present in all protostome peptides (Fig. 3A, Fig. 2, (IS)). Overall, these data argue for the homology of the annelid and mollusc precursors, revealing an ancient AST-B peptide family that traces back to the last common ancestor of eumetazoans. In insects, AST-B peptides signal via a G-protein coupled receptor, the sex peptide receptor (SPR) (6, 16). We identified the orthologs of insect SPRs in both Platynereis and Capitell (Fig. 4) and tested whether these receptors are, as in insects, activated by AST-B peptides. Ligand stimulation in CHO cells co-expressing the receptor, a bioluminescent Ca²⁺ reporter, and a promiscuous G-protein (17, 18) showed that annelid AST-B peptides are specific and potent agonists for these receptors in the nanomolar range (EC5o=10.15 nM) (Fig. 3B-E). From 13 different Platynereis neuropeptides (19, 20) tested with the Platynereis receptor, we detected calcium signals only in the presence of Pdu- AST-B (Fig. 3C). Consistently, Capitella Cte- AST-B activated the Capitella sex peptide receptor ortholog (Fig. 3E).

The AST-B peptides from the two species cross-activated the receptor from the other species (Fig. 3D, E). Substitution of the last, but not the first Trp residue with Ala in both the Platynereis and the Capitella synthetic peptides resulted in a loss of activity (Fig. 3D, E). This shows that the most conserved C-terminal Trp residue is indispensable for receptor activation.

Whole-mount in situ hybridization in Platynereis larvae (Fig. 5A) revealed Pdu-AST-B precursor expression in sensory cells of the apical organ (Fig. 5B). Immunostaining with a specific AST-B antibody (Fig. 6 and Supplementary material) showed that the projections of these cells terminate in the apical nerve plexus (Fig. 5C), a region of strong neurosecretory activity (20, 21). Pdu-AST-B was expressed in the median brain and paired cells in the trunk throughout larval development (Fig. 7). To characterize the morphology of AST-B expressing neurons in Platynereis at an ultrastructural level, we traced the entire volume of two ventral AST-B cells (Fig 5 B,C arrows) using serial sectioning transmission electron microscopy (TEM). In a dataset of 664 sections (50 nm) we identified the two ventral AST-B neurons based on the position of their cell bodies relative to large adjacent secretory gland cells, and the characteristic shape and position of their dendrites and axons (Fig. 5E, fig. 8). Both neurons have a sensory dendrite with a cilium and long apical microvillar extensions surrounding the central cilium (fig. 8). These extensions run at the basal side of the cuticle in a subcuticular space, allowing direct sensory contact with the seawater. The axons project to the dense apical nerve plexus of the larva, where they branch extensively. The branched axons are full of dense-cored vesicles, indicating that the AST-B sensory neurons are neurosecretory (fig. 8). To investigate the sensory modality of the AST-B neurons we performed dye-filling experiments on live larvae. In nematodes, dye-filling is the exclusive property of chemosensory neurons (22). Upon incubation of the larvae with the fluorescent dye mitotracker, we observed labeling in several neurons in the larval episphere with long apical microvilli, including two prominently labeled apical organ cells (Fig. 5D and fig. 9). Laser ablation of these two cells and subsequent AST-B immunostaining identified them as the two median AST-B expressing neurons (arrowheads in Fig. 5B-D and fig. 8). These results indicate that the apical organ AST-B expressing cells are indeed in direct contact with the seawater and are likely to be chemo sensory. In Capitella larvae, a similar immunolabeling of AST-B expressing cells in the apical organ and nerve plexus was observed (Fig. 5F). A specific and cross-reactive antibody against the short peptide VW-amide was developed (fig. 6) to label AST-B expressing neurons. The expression of the Pdu-AST-B-receptor was characterized. It was found to be expressed in the apical organ of the Platynereis larva (Fig. 5G).

An expression profiling on the Platynereis AST-B and AST-B-receptor cells was performed using in situ hybridization and image registration (20, 23). The AST-B-receptor expressing cells were found to be positioned interspersed between the AST-B neurons, with little overlap (Fig. 5H). These cells may thus respond to AST-B neuropeptides released at the neurosecretory plexus. The neurosecretory cell marker prohormone convertase phc2 (21) and the transcription factors dimmed (dimm) and orthopedia (otp) (21) (fig. 10) were analyzed. In Drosophila, dimm directs the differentiation of neuroendocrine neurons and is co-expressed with AST-B in the median brain (24, 25). In vertebrates, Otp is required for the terminal differentiation of hypothalamic neuropeptidergic neurons (26). Image registration revealed that the Pdu-AST-B and Pdu-AST-B-receptor expressing neurons also express phc2, and partially overlap with otp and dimm expression (fig. 10). This molecular fingerprint confirms the neurosecretory character of the Pdu-AST-B expressing neurons, and indicates that the Pdu- AST-B-receptor expressing cells also have neurosecretory output, otp and dimm was found to be co-expressed broadly in the Platynereis apical organ, strengthening the evolutionary link between neurosecretory centres across bilateria (21). The molecular similarities between the AST-B neurons of both Platynereis and Drosophila indicate that these neurons represent a conserved neurosecretory cell type in the median brain of protostomes.

The chemosensory-neurosecretory AST-B neurons in Platynereis may play a role in the detection and transduction of environmental cues during larval settlement and metamorphosis. Therefore it was investigated whether AST-B functions as a regulator of larval settlement and metamorphosis in Lophotrochozoan marine invertebrates like annelids. First, 2-day-old Platynereis larvae were incubated in synthetic AST-B peptides and larval swimming behavior was tested. At this stage, larvae have a solely pelagic lifestyle and swim with their ciliary bands (27). When cilia beat, larvae swim upwards. At regular intervals the entire ciliary band arrests, allowing the larvae to sink. The alternation of sinking and upward swimming keeps the larvae in the water column (20). AST-B peptide treatment strongly inhibited ciliary activity, triggering longer and more frequent ciliary arrests (Fig. 11 A and B). The In contrast to other peptides (20), the AST-B peptide did not alter ciliary beat frequency (fig. 12), This strong ciliary inhibition led to larval sinking in vertical columns, shortly after addition of the peptide (Fig. 11C, D), an effect that could be reversed by washout (Fig. 11D). Other Platynereis AST-B peptides also triggered larval sinking (fig. 13, cf. fig. 2). In accordance with the receptor activation results (Fig. 3D), substitution of the last (W9A), but not the first (W2A), Trp residue to Ala rendered the peptide inactive in the vertical assay (Fig. 1 ID and fig. 13).

Larvae treated with AST-B in a petri dish showed settlement behavior, characterized by the active, cilia-driven attachment of the apical side to the bottom of the dish (Figure 21) Untreated control larvae were freely swimming in the water, and did not show surface attachment behavior (Figure 21). Similar sinking and substrate attachment behaviors can be observed when annelid larvae encounter natural settlement cues (28, 29).

To test whether the observed effects of synthetic AST-B application on cilia were due to signaling via the AST-B-receptor, the receptor was knocked down by microinjecting two different translation-blocking morpholinos and a control mismatch morpholino into fertilized eggs. Following injections with the start morpholinos, an effect of AST-B on ciliary closures was no longer observed in 48 hpf larvae, indicating that AST-B triggers settlement behavior signaling via the AST-B-receptor (Fig. 11 A, B).

Following settlement, Platynereis larvae undergo metamorphosis, characterized by a series of physiological, behavioral and morphological changes spanning several weeks (27). Larvae start feeding at approximately 6 days and subsequently develop additional body segments at the posterior growth zone. After the development of the

segment, larvae undergo cephalic metamorphosis, during which the first chaetigerous segment loses its chaetae, develops a posterior pair of tentacular cirri and is added to the head (fig. 15). The role of Pdu-AST-B in the regulation of each of these steps was investigated. First, start-site blocking morpholinos against the Pdu-AST-B precursor were microinjected and the larval feeding at 7-12 days post fertilization (dpi) was scored. The morpholinos efficiently knocked down Pdu-AST-B expression, as shown by immunostainings with the AST-B antibody on 6 dpf larvae (fig. 14). Most larvae injected with a control morpholino started feeding on the green alga Tetraselmis sp. detectable by chlorophyll fluorescence in the gut at 7 days (fig. 15). Significantly fewer larvae injected with the AST-B morpholinos were feeding by 7-9 dpf (Fig. 16B).

A developmental delay as a reason for these effects on feeding was excluded, since 6 dpf larvae in both groups had the same size (fig. 17) and morphology and no detectable differences in the nervous system based on acetylated tubulin stainings (fig. 14). Larvae treated with AST-B peptide showed altered gut coloration, indicating that similar to insects, AST-B may play a role in the regulation of Platynereis gut physiology (fig. 18) (30). These findings show that Pdu-AST-B plays a role in the post-settlement initiation of feeding, but is not a regulator of early larval growth.

Following settlement and the initiation of feeding, larval and juvenile development is plastic, and is determined by culture density and food availability. Juvenile worms reared at a density of 3 larvae/ml and fed regularly with algae develop the 5^th trunk segment between 16-18 dpf, and the 6^th segment between 26-28 dpf (fig. 19). Unfed larvae never develop the 4th segment, dying after 1 month (n=90). We investigated whether Pdu-AST-B also plays a role in the regulation of juvenile growth and cephalic metamorphosis. Since morpholinos could not be used in these late stages, juveniles were soaked instead in AST-B peptides to score the effect on the addition of new segments and cephalic metamorphosis. Both segmentation and cephalic metamorphosis were accelerated by sustained exposure to AST-B peptides (Fig. 16D). However, these effects on juvenile growth and metamorphosis may be indirect since Pdu-AST-B regulates feeding.

To test if AST-B peptides can also directly induce annelid metamorphosis, independent of effects on feeding, Capitella larvae were exposed to Capitella AST-B peptides. Contrary to Platynereis, Capitella larvae undergo rapid settlement and metamorphosis, characterized by the loss of cilia, and the development of a worm-like body (31) (Fig. 16A). Incubation of stage 9 Capitella larvae (3 days after emerging from the brooding tubes) in AST-B peptide efficiently triggered settlement and metamorphosis within one day (Fig. 16E). Since the larvae were not fed during these experiments, indirect effects via feeding can be excluded. These results indicate that AST-B is a general regulator of settlement and metamorphosis in Lophotrochozoan marine invertebrate larvae such as annelids. Unrelated Cnidarian GLW- amides have been shown to trigger metamorphosis in coral larvae and some hydrozoans (32, 33).

These results indicate that the apical organ of annelids is an ancient neuroendocrine gland that both senses the external marine environment and regulates internal neuroendocrine signals. Herein AST-B and its receptor were identified as key components of the neural signaling regulating settlement and metamorphosis of Lophotrochozoan marine invertebrate larvae. Example 2: Settlement experiments with Platynereis AST-B7 peptide with different modifications at the C-terminus

A further set of experiments with mod fied peptides was performed. The peptides tested are: AWNKNSMRVW-carboxy (SEQ ID NO: 40), AWNKNSMRVWP-arnide (SEQ ID NO: 189), and AWN NSMRVWA-amide (SEQ ID NO: 190).

Platynereis larvae were obtained from a breeding culture. Freely swimming 2 day old larvae were placed in vertical perplex tubes in natural seawater. Larval swimming was recorded with a DMK 21BF03 camera (The Imaging Source) at 30 frames/sec. The tubes were illuminated laterally with red light-emitting diode (LED) lights that larvae are unable to detect at the stages studied. Larval swimming videos were analyzed using custom ImageJ macros and Perl scripts. We analyzed the directionality of larval swimming tracks and plotted the behavior of a group of larvae on circular histograms.

All of these peptides were effective in a settlement assay on Platynereis larvae at 20 μΜ concentration (Fig. 22).

The carboxy and C-terminally extended peptides also induced settlement which indicates that a C-terminal W amino acid residue is not absolutely required. The results suggest that the peptides bind the receptor not by fitting an amidated W into a binding pocket, but probably more in a 'sideways' orientation.

Example 3: Antibodies against two-amino-acid conserved invertebrate neuropeptide epitopes

The most extensively used antibodies recognize the neuropeptide RFamide and the monoamine transmitter serotonin. These antibodies label respective neuron-populations and their axons and dendrites in a large number of species across various animal phyla.

Antibodies that show specific immunoreactivity across a broad range of species are valuable tools for comparative neuroanatomy in non-model organisms. Nonetheless, the current antibody repertoire for non-model invertebrates is limited. For example, antibodies against serotonin commonly label cell bodies and their projections, allowing comparative studies of neurodevelopment and neuroanatomy across diverse species and phyla (e.g. A Hay-Schmidt, Proc Biol Sci 2000). The most extensively used antibodies recognize the neuropeptide RFamide and the monoamine transmitter serotonin. These antibodies label respective neuron- populations and their axons and dendrites in a large number of species across various animal phyla. The commonly used antibody is against FMRFamide, a neuropeptide first discovered in mollusks [1, 2]. Similar RFamide neuropeptides were later found to be widespread among eumetazoans [3-5]. The FMRFamide antibody has been extensively used in invertebrate neuroanatomy due to the broad distribution of RFamide-like peptides and the cross-reactivity of the antibody with various RFamides [6]. The FMRFamide antibody labels distinct neuronal subsets and their projections, and can be applied as a standard histology reagent to increase morphological resolution [11], as a marker to clarify phylogenetic relationships within phyla [7, 8, 9], or to study the evolution of nervous system architecture between related groups [10].

Neuropeptides are signaling molecules that are translated as precursor molecules, typically consisting of an N-terminal signal peptide and multiple copies of similar peptide-motifs, flanked by dibasic cleavage sites (Lys and Arg residues). The precursor is cleaved and often further modified to yield shorter active neuropeptides [12, 13]. Alpha-amidation is the most common post-translational modification, where a C-terminal glycine is enzymatically converted into an amide group. This modification protects the small peptides from degradation and is critical for receptor binding (e.g. Han et al 2002 PNAS) [14, 15]. Amidation is also thought to confer high immunogenic potential to short neuropeptides [16- 18] and antibodies raised against amidated peptides are highly specific for the amidated peptide moiety [18]. The C-terminal residues in amidated neuropeptides are often highly conserved across different species and even phyla [19](refs). However, the short peptide motifs were considered non-immunogenic. Surprisingly, it was found herein that such short and conserved amidated peptide motifs show sufficient immunogenic potential.The resulting antibodies provided herein can advantageously be used as neuronal markers across a wide range species, similarly to the FMRFamide antibody.

Herein specific neuronal antibodies against the two-amino-acid amidated peptide motifs in five conserved neuropeptides, DLamide, FVamide, FLamide, GWamide and RYamide are provided. These antibodies recognize specific subsets of neurons and their projections in cnidarian, annelid, mollusk, bryozoan and crustacean larvae. The antibody stainings revealed that the neuropeptidergic innervation of locomotor cilia is a general feature of ciliated larvae.

Methods

Animal husbandry

Capitella teleta was kept as a breeding culture at 18°C in mud [35]. Larvae were dissected from their brood tubes and raised to the stage needed. Platynereis dumerilii was kept as a breeding culture at 18°C as previously described [36]. Generation of polyclonal neuropeptide antibodies

The amidated peptides, coupled to an adjuvant (lipoadjuvant Pam3) via an N-terminal additional Cysteine (i.e. CRYamide, CGWamide, CFVamide, CFLamide CDLamide), were used to immunize rabbits. Sera were affinity-purified on the respective peptide epitopes using a SulfoLink resin (Thermo Scientific, Rockford, USA) that allows the coupling of Cysteine containing peptides via a disulfide bond. After coupling of 1 mg peptide epitope to 2 ml resin in Coupling Buffer (CB; 50 mM Tris pH 8.5, 5 mM EDTA), the resin was washed 3 times with 10 ml CB. Excess reactive sites were blocked by incubating the resin in 2 ml 50 mM Cysteine for 45 min, followed by 3 washes with 1M NaCl and 3 washes with 25 ml phosphate buffered saline (PBS). 25 ml serum was applied to the resin and incubated overnight to facilitate antibody binding. After the serum was allowed to flow through, the resin was washed 5 times with 25 ml PBS followed by a wash with 15 ml 0.5 M NaCl / PBS and again 2 times 10 ml PBS. The antibodies were eluted and fractionated with 8 times 1 ml of 100 mM glycine pH 2.7, 8 times 1 ml of 100 mM glycine pH 2.3 and 8 times 1 ml of 100 mM glycine pH 2.0. The fractions were neutralized by directly collecting them in an adequate volume (about 40, 75 and 95 μΐ for the different pH solutions) 1M Tris-HCl pH 9.5. The protein concentration of each fraction was determined, and the first two fractions of the pH 2.7 peak (usually fractions 2 and 3) were discarded, since these contain the lowest affinity antibodies. The peak fractions and the end-of-peak fractions were pooled, and concentrated, if necessary, using Vivaspin centrifugation tubes with a molecular weight cut-off of lOkDa (Sartorius, Gottingen, Germany). Antibodies were stored in 50% glycerol at -20 DC for mid-term (up to 1 year), and -80°C for long-term storage.

Immunohistochemistry

For immunostainings. larvae were fixed in 4% formaldehyde in PTW (PBS + 0.1% Tween- 20) for 2 h and stored in 100% Methanol at -20 °C until use. After stepwise re-hydration to PTW, samples were permeabilized with proteinase-K treatment (100 μg ml in PTW, 1-3 min). To stop proteinase-K activity, larvae were rinsed with Glycine buffer (5 μg/ml in PTW) and post-fixed in 4% formaldehyde in PTW for 20 min followed by 2 times 5 minutes washes in PTW and 2 times 5 minutes washes in THT (0.1 M Tris-HCl pH 8.5 + 0.1% Tween-20). Larvae and antibodies were blocked in 5% sheep serum in THT for 1 h. Primary antibodies were used at a final concentration of 1 μ§/ηι1 for rabbit neuropeptide antibodies and at 0.5 g/ml for mouse anti-acetylated tubulin antibody (Sigma, Saint Louis, USA) and incubated overnight at 6 °C. Weakly bound primary antibodies were removed by two 10 min washes in 1 M NaCl in THT, followed by 5 times 30 min washes in THT. Larvae were incubated overnight at 6 °C in the dark in 1 μg ml anti-rabbit Alexa Fluor® 647 antibody (Invitrogen, Carlsbad, CA, USA) and in 0,5 μ§/ΐηΙ anti-mouse FITC antibody (Jackson Immuno Research, West Grove, PA, USA) and then washed 6 times 30 min with THT-buffer, and mounted in 87% glycerol including 2.5 mg/ml of the anti-photobleaching reagent 1,4- diazabicyclo[2.2.2]octane (Sigma, St. Louis, MO, USA). Pecten larvae were additionally treated with 4% Paraformaldehyde in PBS with 50 uM EDTA pH 8,0 for 1 h to decalcify their shells before the immunostaining procedure (performed as described above). For cnidarian larvae we used a mouse anti-tyrosylated tubulin antibody (Sigma, Saint Louis, USA) at 1 μg/πll. For immunostaining with multiple rabbit primary antibodies in the same sample, antibodies were directly labeled with a fluorophore using the Zenon® Tricolour Rabbit IgG Labeling Kit (Invitrogen, Carlsbad, CA, USA) and used in combination with mouse anti- acetylated tubulin antibody.

For blocking experiments we preincubated the antibodies in 5 mM of the respective full length Platynereis peptides (YYGFNNDLamide, AHRFVamide, VFRYamide, RGWamide) for 2h before immunostainings.

Whole mount RNA in situ hybridization

The RNA in situ hybridizations were carried out as previously described [37]. Microscopy and Image Processing.

Images were taken on an Olympus Fluoview-1000 confocal microscope using a 60x water- immersion objective and the appropriate laser lines to capture fluorescent signals. Signals from RNA in situ hybridizations (nitro blue tetrazolium chloride/5-Bromo-4-cloro-3-indolyl phosphate precipitate) were imaged with reflection confocal microscopy as described [37]. Images were processed with Imaris 6.4 (BitPlane Inc, Saint Paul, USA) and nageJ 1.45 [38] software.

Abbreviations

hpf: hours post fertilization; RPCH: red pigment concentrating hormone; AKH: adipokinetic hormone; NPF: short neuropeptide F; NPY: short neuropeptide; CB: coupling buffer; PBS: phosphate buffered saline; PTW: phosphate buffered saline; THT: 0.1 M Tris-HCl pH 8.5 + 0.1% Tween-20.

Results

Neuropeptides show a broad phyletic distribution in invertebrates, including DLamide, FVamide, FLamide, GWamide and RYamide. These neuropeptides show strong conservation of the two carboxy terminal amino acids and are alpha-amidated at their C-termini. Specific, affinity purified polyclonal antibodies were develped against each of these rwo-amino-acids amidated motifs. Antibody reactivity and specificity was tested both by peptide preincubation experiments and by showing a close correlation between the immunostaining signals and mRNA expression patterns of the respective precursors in the annelid model Platynereis, Also the usefulness of these antibodies was demonstrated by performing irnmunostainings in a broad range of species, including cnidarians, annelids, mollusks, a bryozoan, and a crustacean. In all cases tested, the antibodies labeled distinct neuronal populations and their axonal projections. In cnidarian, annelid, mollusks and bryozoan ciliated larvae some of the antibodies revealed peptidergic innervation of locomotor cilia.

Five specific, cross-species-reactive antibodies were developed recognizing conserved two- amino-acid amidated neuropeptide epitopes. These antibodies allow specific labeling of peptidergic neurons and their projections in a broad range of invertebrates. The comparative survey across several marine phyla revealed the broad occurrence of peptidergic innervation of larval ciliary bands, hinting at a general role of these neuropeptides in the regulation of ciliary swimming.

Generation of specific antibodies against two-amino-acid amidated neuropeptide epitopes We set out to develop antibodies against the conserved two-amino acid amidated C-termini of DLamide, FVamide, FLamide, GWamide and RYamide neuropeptides (Figure 22). Rabbits were immunized with the short amidated peptides extended with an N-terminal Cys to allow coupling to a carrier during the immunizations. We also used the Cys residue to couple the peptides to a resin and affinity purify the antibodies from the respective sera. We employed a high stringency affinity purification protocol including high salt washes and low pH elution to obtain high-affinity antibody fractions.

Next we tested the reactivity of the affinity purified neuropeptide antibodies in whole mount irnmunostainings on larvae of the marine annelid model Platynereis dumerilii. We found labeling for all antibodies in a subset of neurons and their axons in the larval episphere (Figure 23 A-D). To test the specificity of our antibodies, we pre-incubated them in the synthetic amidated full length Platynereis peptides. This treatment led to a complete block of the signal for the anti-DLamide, anti-FVamide and anti-RYamide antibodies (Figure 24 A,C,E) and a strong reduction in signal intensity for the anti-FLamide and anti-GWamide antibodies (Figure 24 B, D). These results show that the peptides bind to the respective antibodies and prevent further binding to epitopes in the tissue.

The specificity of the antibodies is further supported by the close correlation between the cell body positions revealed by immunostairiing and the expression patterns of the respective precursor mRNAs (Figure 23, bottom panels, asterisks). A recently described set of antibodies raised against full length Platynereis DLamide, FVamide, FLamide and RYamide also show very similar neuronal signals [19]. Overall, our specificity tests in Platynereis demonstrate that the antibodies raised against the two-amino-acid amidated motifs are remarkably specific and can be used to obtain high- quality tissue stainings. To test the utility of our antibody collection as cross-species-reactive neuronal markers, next we performed immunostainings on a variety of marine larvae.

DLamide immunoreactivity in annelids

DLamide neuropeptides have been described from the errant annelid (Errantia) Platynereis and the sedentary annelids (Sedentaria) Capitella and Helobdella (Ref, Figure 22A). Since errant and sedentary annelids encompass most of annelid diversity [27], DLamide is potentially widely distributed in the phylum. To test if our DLamide antibody can be used as a pan-annelid marker, we also tested its reactivity in Capitella. In Capitella larvae we found staining in neurons of the apical organ. These neurons show a flask-shaped morphology typical of sensory cells and project to the larval ciliary band (Figure 25, arrow) in a similar fashion observed for Platynereis larvae (cf. Figure 23 A). We also found strong staining in the ventral nerve cord in older Capitella larvae (Figure 25). The specific reactivity of the DLamide antibody in both Errantia and Sedentaria reveals its usefulness as a pan-annelid neuroanal marker.

FVamide and FLamide immunoreactivity in annelids and mollusks

FVamides and FLamides have been described in annelids, mollusks and platyhelminths. In the annelids Platynereis and Capitella there is one FVamide neuropeptide precursor, whereas there are three different precursors in the mollusk Lottia gigantea (Figure 22B, C) [24], FLamides either are encoded by a distinct precursor and are expressed in distinct cell, as in annelids, or co-occur on the same precursor with FVamides, as in mollusks. Regardless of the number of precursor genes, the conserved FVamide and FLamide epitopes could allow the labeling of all FVamide and FLamide expressing neurons in annelids and mollusks. We tested the reactivity of both antibodies on Capitella larvae and on larvae of the bivalve mollusk Pecten maximus and the nudibranch mollusk Phestilla sibogae. In Capitella, we found FVamide immunoreactivity in neurons in apical organ that projected to the ciliary band (Figure 26A, cf. Figure 23B), and also in the ventral nerve cord (Figure 26B). The FLamide antibody also labeled the ventral nerve cord in Capitella (Figure 27 A). In Pecten veliger larvae, the FVamide antibody labeled a small number of neurons some of which projected to the ciliated velum (Figure 26C). In Phestilla both antibodies showed strong staining in the cerebropleural ganglion between the eyes (Figure 26D, 27B). FVamide also labeled two nerves running along the ciliated velum (Figure 26E). GW mide immunoreactivity in annelids, mollusks and crustaceans

GWamides are present in annelids, mollusks (APGWamides), platyhelminthes, crustaceans (as red pigment concentrating hormone, RPCH) and insects (as adipokinetic hormone, AKH, Figure 22D). Even though the sequence similarity is limited, the annelid and mollusk GWamide precursors are the likely lophotrochozoan orthologs of arthropod RPCH and AKH neuropeptide precursors. The orthology is also supported by sequence similarity outside the repetitive GWamide motifs. The precursor C-termini in both the arthropod and lophotrocozoan sequences contain an additional predicted peptide that likely forms a disulfide bridge.

We tested our GWamide antibody in the annelid Capitella, in the mollusks Pecten and Phestilla and in an unidentified crustacean larva collected from a plankton sample (Figure 28). In Capitella we found staining similar to Platynereis, in a small number of neurons in the apical organ (Figure 28 A, cf Figure 23D) and staining in the ventral nerve cord of older larvae (Figure 28B), but no ciliary innervation. In the mollusk larvae the GWamide antibody also labels a small number of neurons and their projections (Figure 28C, D). In the crustacean larva the antibody labels a pair of neurons on two sides of the labrum (Figure 28E).

RYamide immunoreactivity in cnidarian, annelid, bryozoan, mollusk and crustacean larvae RYamide neuropeptides have been described in various marine phyla including cnidarians, annelids, molluscs, platyhelmithes and crustaceans. RYamides are also present in terrestrial invertebrates such as nematodes and insects (Figure 22E). In cnidarians, platyhelminthes and nematodes, RYamide neuropeptides co-occur with RFamides in the same precursor [22, 23, 28], whereas in most other phyla they originate from a distinct precursor. Given the broad phyletic distribution of RYamides and the observation that it often derives from a distinct precursors expressed in different cells than RFamide (Conzelmann et al), the RYamide antibody could have a great value for comparative neuroanatomical studies. To explore the potential of the RYamide antibody, we tested its reactivity in cnidarians, annelids, mollusks and bryozoans.

In Capitella larvae we found RYamide staining in individual sensory neurons in the apical organ. As for DLamide and FVamide. these neurons projected to the ciliary band nerve (Figure 29 A, cf. Figure 23E), suggesting a role for RYamides in regulating ciliary activity in Capitella. We also found a strong staining in the ventral nerve cord in older Capitella larvae (Figure 29B). In larvae of the bryozoan Cryptosula sp. we detected strong RYamide immunoreactivity in the nerve nodule and in nerves projecting to the ciliary band (Figure 29C). In Pecten larvae (Figure 29D) we detected two pairs of neurons and their projections along the ciliated velum. We also co-stained Pecten larvae for RYamide and FVamide, using pre-incubation of the primary antibodies with differently labeled fluorescent secondary antibodies. This demonstrates that the antibodies can also be used in combination. In Phestilla larvae we detected strong RYamide signal in the cerebropleural ganglion between the eyes and the pedal ganglion, as well as the nerves connecting these ganglia, and also in nerves running to the ciliated velum (Figure 29F, G). In the cnidarians Aurelia and Clava we found staining of sensory neurons in the ciliated planula larvae (Figure 29 I-K). These neurons had sensory morphology with sensory dendrites projecting to the surface of the ciliated neuroectoderm (Figure 29K), and basal projections running along the basal side of the ciliated neuroectoderm. These results show that the RYamide antibody is a widely applicable neuronal marker across several invertebrate phyla. It is to be noted that the RYamide antibody may cross react with invertebrate neuropeptides belonging to the NPF/NPY (short neuropeptide F, NPF; short neuropeptide Y, NPY) family, that sometimes have a C-tcrminal RYamide, such as in Apis melifera and Bombyx mori NPFs [31].

Example 4: Prior art FLamidc peptide, which is not a peptide derived from an allatostatin-b polypeptide, does not induce settlement

52 hpf Platynereis larvae were exposed to FLamide and allatostatin-B peptides in a petri dish with natural seawater. The % of larvae that showed extended contact with the bottom surface of the dish was counted.

In contrast to the control and FL-amide treated larvae that show little surface contact, 75% of allatostatin-b treated larvae showed sustained surface contact; see Figure 30.

Example 5: RGWamide triggers downward vertical migration but not settlement Materials and Methods

Peptide treatment

Peptide treatment experiments for sustained surface contact were carried out in Nunclon 6- well tissue culture dishes, with 10 ml sterile filtered seawater (FSW) per well. The 3-amino- acid chemically synthetised, terminally amidated RGW-amide peptide was used in a final concentration of 5 JJM.

Peptide treatment experiments for scoring ciliary beating were carried out in FSW, with the larvae placed in a chamber formed between a microscope slide and a coverslip separated by 3 layers of adhesive tape. Recordings of ciliary beating and arrest were performed with a Zeiss Axioimager microscope and a DMK 21BF04 camera (The Imaging Source) at 60 frames/sec. To assay vertical swimming and sinking freely swimming larvae in 25 cm-high vertical tubes were recorded with a DMK 21BF03 camera (The Imaging Source) at 30 frames/sec. The tubes were illuminated laterally with red light-emitting diode (LED) lights that larvae are unable to detect at the stages studied.

Results

RGWamide treatment does not trigger annelid larval settlement

Since RGWamide also belong to the Wamide family of peptides, we wanted to see if this peptide is able to trigger annelid larval settlement, similarly to allatostatin-B. In vertical swimming experiments we observed that RGWamide treatment triggered rapid downward movement of larvae (Fig. 31 A). However, we did not observe an increased freqeunce of sustained surface contact (Fig. 32). RGWamide treatment also significantly increased the beating frequence of cilia, in contrast to allatostatin-B, (Fig. 3 IB) suggesting that the strong downward movement in the vertical column is due to an upregulation of ciliary activity together with downward orientation. In conclusion, even though RGWamide is a member of the Wamide family, it is not able to trigger the two hallmarks of larval settlement, the inhibition of cilia and sustained surface contact, it is therefore not a settlement inducing peptide. As shown in Figure 31, prior art RGWamide triggered downward vertical migration but not settlement.

Example 6: A global view of the evolution and diversity of metazoan neuropeptide signaling

Methods

pNPs were retrieved using a combination of strategies. UniProt sequences annotated with the GO term GO:0007218 (neuropeptide signaling pathway) were collected. Transmembrane proteins and proteins lacking a SP were removed. NCBI sequences were retrieved with the query 'neuropeptide NOT receptor'. Non-neuropeptide sequences (e.g. neuropeptide processing enzymes) were removed. UniProt was also searched for proteins with a SP using SignalP4 and containing the motif

[ |R][K|R]\w{3}G[K|R][K|R]\w{3}G[K|R][K|R]\w{3}G[K|R][K|R] to retrieve repetitive pNPs of amidated neuropeptides. Manually curated lists of pNPs from B. floridae, S. kowalevskii, T, adhaerens, Capitella teleta, Helobdella robusta, and L. gigantea were also created, either by species-specific searches or based on the literature. pNP lists from mass spectrometry studies were also added. Sequences were clustered using CLANS2 (32) to identify all major pNP families. Members from each cluster were used as queries in PSI- BLAST searches in the protein NR database at the NCBI using varying e- value cutoff (1 to le-5) and either the BLOSUM62 or the PAM30 matrix. Newly detected sequences were examined and false positive matches were removed. The NCBI EST collection est.other (excluding human and mouse) was also searched, ESTs were translated using ESTScan and screened for the presence of a SP. Sequences without a SP, spurious matches, toxins and antimicrobial peptides were removed. Adiponectins were also removed because the collagen domain showed many spurious matches to repetitive peptides. Redundancy was reduced to 95% identity using CD-HIT.

Class A GPCRs with the Interpro domains IPRO 19427 or IPR000276 or IPR017452 were downloaded from Uniprot and used to search the C. teleta, H. robusta, and L. gigantea predicted proteins (e-value le-20). The combined set was reduced to 75% redundancy. The resulting 16,123 GPCRs were clustered with Clans. Linkage clustering (minimum 10 links at e-value le-20; minimum 10 sequences) was performed to identify coherent clusters of which neuropeptide receptor clusters were manually selected. These sequences were filtered with HMMTOP and only sequences with 7 transmembrane domains and an extracellular N- terminus were retained. Class B sequences with the domains IPRO 17981 or IPR001879 or IPR000832 or IPR017983 from Uniprot were enriched with C. teleta, H. robusta, and L. gigantea sequences, filtered with HMMTOP, and clustered with Clans to select neuropeptide receptors. For the final clustering 1465 Class A and 547 Class B receptors were used. All sequences were annotated with the full classification, retrieved based on the NCBI Taxonomy identifier (taxid), using a bio-perl script. A custom perl script was used to annotate pNPs with the three last amino acids of the amidated peptides preceding a G[K|R][K|R] motif. The length of the predicted amidated peptides flanked by dibasic cleavage sites was also included in the description for repetitive pNPs. At least two peptides flanked by dibasic cleavage sites had to have the same length. Sequences were clustered with CLANS2. CLANS performs all- against-all BLAST and represents sequences by nodes in a graph, placed randomly in a three dimensional space. Clustering is performed using attractive forces proportional to the negative logarithm of the BLAST p- values, and a uniform repulsive force. pNPs and GPCRs were clustered with a p- value cutoff of le-5 and le-40, respectively. Clustering was first performed in 3D and then the maps were collapsed to 2D for easier representation. Taxonomy, amidated motifs, and the length of the neuropeptide repeats were mapped on the cluster maps using the 'Sequence groups' tool. To read the Clans files (Datasets 1-3) install Clans (32) and run the command line command: java -Xmx4000m -jar /your install directory/CLANS.jar -load Clans_jGle. Multiple alignments were generated by ClustalW, Muscle or Cobalt. Motifs were identified with MEME.

Dataset SI. Clans file of the 6225 pNPs analyzed.

The file contains all sequences (between the lines <seq> and </seq>), annotations, and the BLAST p-value matrix (between the lines <hsp> and </hsp>). The cluster map can be visualized with Clans at http://134.34.129.6/programs/clans/index.php using the command: java -Xmx2000m -jar ./CLANS.jar -load clans infile.

Dataset S2, Clans file of the 1465 class A neuropeptide GPCRs analyzed.

The file contains all sequences (between the lines <seq> and < seq>), annotations, and the

BLAST p-value matrix (between the lines <hsp> and < hsp>). The cluster map can be visualized with Clans using the command: java -Xmx2000m -jar ./CLANS.jar -load clans_infile.

Dataset S3. Clans file of the 547 class B neuropeptide GPCRs analyzed.

The file contains all sequences (between the lines <seq> and </seq>), annotations, and the

BLAST p-value matrix (between the lines <hsp> and </hsp>). The cluster map can be visualized with Clans using the command: java -Xmx2000m -jar ./CLANS.jar -load clans infile.

Dataset S5. pNPs identified from Trichopl x adhaerens, Branchiostoma floridae, Saccoglossus kowalevskii and Petromyzon marinus.

The used sequences of the NPs (Dataset SI), class A neuropeptide GPCRs (Dataset S2), class B neuropeptide GPCRs (Dataset S3) and pNPs (Dataset S5) were retrieved from the UniProt and GenBank public databases.

The term "pNP" is an abbreviation of the term "proneuropeptide". The term "GPCR" is an abbreviation of the term "G-protein coupled receptor". The terms "pNP" and "proneuropeptide" can be used interchangeably herein. The terms "GPCR" and "G-protein coupled receptor" can be used interchangeably herein. These terms are known in the art and, inter alia, descried in the following references:

Liu F, Baggerman G, Schools L, Wets G: The construction of a bioactive peptide database in Metazoa. J Proteome Res 2008, 7:4119-4131.

Hewes RS, Taghert PH: Neuropeptides and neuropeptide receptors in the Drosophila melanogaster genome. Genome Res 2001 , 11:1126-1142.

Results

Neuropeptides are diverse neuron-secreted peptides with neuromodulatory, neurotransmitter or hormonal functions. Most neuropeptides signal via GPCRs (1), with a few exceptions (2- 8). As modulators of neuronal activity, neuropeptides contribute to the generation of different outputs from the same neuronal circuit in a context-dependent manner (9), or orchestrate complex motor programs (10). Many neuropeptides act as hormones, and are released into the haemolymph by neurohemal organs, such as the vertebrate pituitary gland, or the insect corpora cardiaca (11). These peptide hormones regulate various aspects of physiology, including growth, metabolism and reproduction. Active neuropeptides are generated from an inactive pNP, which contains a single or multiple copies of active peptides. Active peptides are commonly short, with only a few families adopting well-defined, but unrelated folds (e.g. prolactin, glycoprotein hormones). pNPs have a signal peptide (SP) and enter the secretory apparatus where dedicated proteases cleave them at mono- or dibasic cleavage sites (12), and where the maturing peptides are often further modified (13). pNPs are ubiquitous in eumetazoans, and genomics and mass spectrometry revealed the full neuropeptide repertoire of several species (14-20). Given their wide occurrence in metazoans, and importance in neuronal regulation, a global pNP phylogeny would further our knowledge of nervous system evolution. pNP phylogenetics is challenging as pNP evolution has patterns and constraints different from the evolution of folded proteins (21). pNPs are often repetitive, with the number and length of repeats changing during evolution, or the sequences diverging into distinct peptides within a precursor (21-23). Conserved sequence stretches in pNPs often constitute only a few residues corresponding to biologically active short peptides (24-27). Neuropeptides showing such limited conservation were nevertheless shown to be ligands of orthologous GPCRs in different phyla, confirming that the pNPs are orthologous (28-30). The high diversity and repetitive sequence of pNPs hampers multiple alignment-base molecular phylogeny analyses. Although a pNP catalogue exists (31), it is not clear how the diverse families are related. Furthermore, the paucity of pNP information from placozoans, ambulacrarians, and cephalochordates, obscures the evolutionary origins of distinct pNP families. We have performed a comprehensive analysis of pNP and neuropeptide GPCR evolution using sequence- similarity based clustering. Clustering and mining of poorly studied genomes clarified the interrelatedness and origin of pNP families. GPCR clustering revealed several bilaterian orthologous groups containing receptors for orthologous peptides. These results provide a global view on the evolution of metazoan neuropeptide signaling and uncover the stable evolutionary association of GPCR-ligand pairs.

Cluster-representation ofpNP and GPCR diversity

A non-redundant dataset of 6225 pNPs (Methods and Dataset SI) from 10 animal phyla belonging to approximately 80 families were clustered based on all-against-all sequence similarity (BLAST p-values) (32) using either the BLOSUM62 or the PAM30 matrix. Clustering recovered all known families (Fig. 32), several of them unique with no connections to other families (e.g. prolactin, galanin, CART), or only few spurious hits to unrelated clusters (e.g. CHH to relaxin). However, 22 of 80 families were strongly connected to form one large Central Cluster (CC; Figs. 32, 33 and 36). In the CC some sequences were only indirectly connected via a network of transitive BLAST connections. The core of the CC contained repetitive pNPs that give rise to short, amidated neuropeptides (e.g. FMRFamides, MIP, LWamide). The term "MIP" as used herein refers to peptides derived from "allatostatin- b polypeptide". Several peripheral groups (e.g. NPFF, GnlH, TRH) were connected to the core, but not to other derived families, representing independent divergences from the more ancestral sequences of the core (Figs. 32, 33 and 36).

The clustering of repetitive pNPs may not reflect evolutionary relatedness but spurious BLAST matches due to identical repeat length and reoccurring dibasic cleavage sites. To exclude this possibility I mapped the length of the neuropeptide repeats of a subset of pNPs to the cluster map. Several pNPs with the same repeat length were far from each other in the map, indicating that repeat-length alone does not explain the observed clustering (Fig. 36C). To test if clustering correlates with short terminal amidated motifs I mapped the occurrence of such motifs. I identified 32 3-amino-acid amidated motifs specific to a certain cluster (Figs. 36A 37A).

To analyze the phyletic distribution of pNP families, I projected taxonomic information onto the cluster map, distinguishing 11 metazoan clades (Figs. 32, 33). I also performed sensitive similarity searches to detect distant homologs, allowing the reconstruction of pNP repertoire at key nodes of the metazoan tree (Fig. 34). I also clustered class A (rhodopsin) and class B (secretin) neuropeptide GPCRs, revealing several orthologous clusters (p-value < le-50, Figs. 35, 37.fi, and 38), and the phyletic breadth and time of origin of different families (Fig. 38). The combined analyses of pNP and GPCR distribution allowed a detailed reconstruction of the evolution of neuropeptide signaling in metazoa.

Placozoan sequences reveal the deep origin of CC pNPs

Database searches identified three pNPs in the neuron-less placozoan, T. adhaerens (Fig. 9 A and Dataset S5). These pNPs have a SP, and repetitive short sequences flanked by dibasic cleavage sites, preceded by the amidation signature glycine. They showed BLAST hits to repetitive pNPs from various bilaterians, including Famides (XP_002117813), mollusk PRQFVamide (XP_002116174.1), or S. kowalevsUi Samide (XP_002112824.1), and mapped to the CC (Fig. 32). The T. adhaerens genome contains enzymes for pNP processing and several GPCRs. Orthologs of the relaxin and glycoprotein hormone GPCRs could be identified (Fig. 35), but no relaxin- or glycoprotein-hormone-like pNP was found. No pNP sequence or neuropeptide GPCR could be identified in the sponge Amphimedon queenslandica despite the fact that sponges contain pNP processing enzymes.

Ancient eumetazoan pNP families

The last common ancestor of eumetazoans had an extended pNP repertoire with short amidated peptides (Wamide, R[FY]amide), insulin-related peptide, prokineticin (33, 34), and glycoprotein hormone. FMRFamide-like peptides (FLP) form a heterologous group of F/Yamides with many families of unclear relatedness (35, 36). The pNP map clarifies the relationships of FLPs (Figs. 32, 33, 36S, and 36D). Cnidarian RFamides map near the repetitive bilaterian R[FY] amides in the CC, revealing an ancestral eumetazoan R[FY] amide orthology group (Figs. 33, 36D). FLPs with several repeats could also be identified in the cephalochordate, B. floridae, and the hemichordate S. L·walevskii, but not in vertebrates, indicating that vertebrates have a structurally more derived pNP complement. [LV]Wamides constitute another eumetazoan orthology group within the CC (Figs. 32, 33 and 36£>). This group contains cnidarian GLWamides, and diverse protostome Wamides (GWamides, MPs), which cluster together and share an amidated Trp residue preceded by a small aliphatic residue. Protostome MIPs have another conserved Trp (W-X6-₈-Wamide motif) that is lacking from LWamides and GWamides (Fig. 40). Wamides at the core of the CC connect to peripheral families. GWamides connect to AKH and RPCH (Fig. 33). These three families are present in arthropods, mollusks and annelids and share a C-terminal segment with a disulphide bond (37). These pNPs in turn connect to the ancestral bilaterian GnHR/corazonin family, suggesting a complex scenario of duplication and gene loss for the origin of these families from repetitive Wamides (38). A relationship between GnHR and AKH has been proposed based on shared sequence features (38) and the relatedness of the receptors (39, 40). The clustering of GnRH/AKH receptors in the GPCR map confirms this (Fig. 35).

Glycoprotein hormones and prokineticins also trace back to the stem eumetazoan. Glycoprotein hormones have two related subunits belonging to the Cys-knot superfamily that also includes TGF-β, NGF, PDGF, and the BMP antagonists gremlin/DAN (41)(pfam:PF00007). Cys-knot domains are also present in several multi-domain proteins (e.g. mucins). PSI-BLAST searches identified a glycoprotein hormone in the sea anemone Nematostella vectensis, showing highest similarity to arthropod bursicons (Fig. 32 and Fig. 40). This is consistent with the presence of glycoprotein-hormone receptor-like sequences in cmdarians (42)(Fig. 35). A bursicon-like sequence is also present in the sea urchin Strongylocentrotus purpuratus, but not in other deuterostomes. Prokineticins/astakines consist of a Cys-rich domain that is also found in colipases (34) and as a C-terminal domain in the Cys-rich Wnt antagonists, the dickkopf-related proteins. Dickkops have an additional N- terminal domain. The prokmeticin/colipase domain is also present in Hydra magnipapillata (e.g. XPJ)02160463.2) and N. vectensis (XP_001641384.1) independently of the dickkopf N- terminal domain and is potentially a precursor of the cognate domain in prokineticins (Fig. 31C).

The presence of two insulin-related peptides in N. vectensis indicates that this family is also ancestral to eumetazoans. A group of cnidarian GPCRs was also identified that clustered with receptors for the chordate insulin-like peptide, relaxin (Fig. 35), suggesting that relaxin receptors may be ancestrally involved in insulin-related peptide signaling. A greatly extended pNP repertoire in urbilateria

Several pNP families have previously been shown to be ancestral to bilaterians including the tachykinins (43), corticotropin-releasing factors (CRF) (44), calcitonin (45), neuromedin- U/pyrokinin (46), allatostatin-C/somatostatin (47), cholecystokinin/sulfakinin (48), pedal peptide/orcokinin (49), vasopressin/oxytocin (50), GnRH/corazonin/AKH (40, 51), 7B2 (52), and PY NPF (53). Many of these families form well-connected clusters in the pNP map with both protostome and deuterostomes sequences (Fig. 32), with some exceptions, where sequence conservation is limited (neuromedin-U/pyrokinin, calcitonin/DH31, cholecystokinin/sulfakinin, allatostatin-C/somatostatin; Fig. 40).

The GPCR map revealed several orthologous clusters of protostome and deuterostome sequences with orthologous neuropeptide ligands, confirming the above relationships. These included the class A receptors for tachykinin, neuromedin-U/pyrokinin, vasopressin/oxytocin, GnRH/corazonin AKH, allatostatin-C/somatostatin, and cholecystokinin/sulfakinin (Fig. 35). The class B GPCRs for calcitonin DH31 and CRF/DH44 also formed bilaterian-wide clusters (Fig. 375). Members of several of these families and their putative receptors could also be identified in S. kowalevskii andB.floridae (Figs. 32, 35 31B and Datasets S2, S3, S5).

The urbilaterian origin of four further pNP families was revealed by sequence searches, and was partly supported by GPCR clustering. The evidence for opioid-like peptides in protostomes has been controversial (54), and no pNP has yet been described in any invertebrate. Database searches identified proenkephalin-like pNPs in annelid and mollusk ESTs. HHpred searches with the Lottia gigantea and Platynereis dumerilii pNPs identified the nociceptin/proenkephalin family as homologs (probability 94.6%, p=6.5E-07 and 81%, p=1.8E-05, respectively). The opioid peptides produced from these pNPs are longer than their vertebrate counterparts, are often amidated, and share the N-terminal motif YGx[FL]+ (+ is a hydrophobic residue; Fig. 395, Fig. 40). Vertebrate opioid pNPs have a segment after the SP with six Cys (22). 6 to 8 Cys following the SP are also present in the annelid and mollusk opioid pNPs, although not in conserved positions. These similarities establish the homology of the lophotrochozoan pNPs with the vertebrate opioid family. No protostome opioid receptor could be identified.

Sensitive similarity searches also identified deuterostome and ecdysozoan orthologs of lophotrochozoan luqins (55). Luqins retrieved a S. purpuratus sequence that identified a S. kowalevskii pNP. Both ambulacrarian sequences have an RWamide motif and a proline-rich C-terminal peptide with two conserved Cys residues (Fig. 40). PSI-BLAST searches also revealed the homology of luqins and insect RYamide pNPs. Luqin and RYamide pNPs have two R[YF] amide peptides directly following the SP and also share the C-terminal peptide with the two conserved Cys residues (56). In the GPCR map mollusk luqin and insect RYamide receptors cluster together with two GPCRs from S. purpuratus that likely represent deuterostome luqin receptor orthologs, confirming the ancestral presence of luqin signaling in bilaterians. Another ancestral bilaterians family, first described in lophotrochozoans, is achatin (57). Homologs of mollusk achatin could be identified in annelids, in S. kowalevskii, and B. floridae (Dataset S5). Achatins share the GF[GAF][DNG] motif (Fig. 40). No achatin receptor has yet been described.

Database searches and the GPCR map also revealed the orthology of allatotropin and orexin pNPs (Fig. 35). A S. kowalevskii sequence (Dataset S5), identified by PSI-BLAST using arthropod allatotropin queries, shares a conserved C-terminal domain with protostome allatotropins. This sequence also contains an orexin peptide directly after the SP (Fig. 40) (58). The allatotropin and orexin receptors cluster together, and this cluster also contains a S. kowalevskii receptor. These results establish allatotrop i/orexin and their receptors as orthologs representing an ancient bilaterian family.

Several other orthologous GPCR pairs for seemingly unrelated peptide families were also recovered, suggesting that the corresponding pNPs also represent orthologous, ancestral bilaterian families. These include the CCHamide/neuromedin-B, neuropeptide-S/CCAP, and allatostatin-A galanin receptors. Of these families the orthology of CCAP/NPS is supported by a shared N-terminal K-R-x-F-x-N motif (Fig. 40). CCHamides (59, 60) are related to annelid and mollusk excitatory peptide pNPs (61). This family is also related the LI 1 pNPs of annelids, mollusks, nematodes (62) and crustaceans, as shown by PSI-BLAST. Ll l/CCHamide/EP peptides share the signature sequence Cys-X6,s-Cys-X-Gly-X2,3 with an internal disulphide bond (Fig. 40) and represent two paralogous families, ancestrally present in protostomes. The orthology of LI 1/CCHamide/EP and neuromedin-B is not recognizable at the pNP level, but are listed as orthologs based on the receptor evidence. Likewise, an orthologous relationship between allatostatin-A and galanin is not evident at the pNP level, but is supported by the orthology of their receptors and a similar role in the regulation of lipid storage (63), among other functions. Neuromedin-B and galanin receptors are present in B. floridae, but no pNP orthologs were found. Identifying the ligands for these receptors may reveal intermediate sequences that could reinforce these relationships.

Other GPCRs also formed bilaterian orthologous clusters, even though their known peptide ligands have a more limited distribution. Leucokinins were first described in arthropods, and sequence searches identified homologs in nematodes, mollusks and annelids. These peptides share the FxxW[GA]-NH₂ motif (Fig. 40) and connect to the CC (Fig. 33). The GPCR map revealed the presence of an ambulacrarian leucokinin receptor, indicating that leucokinin-like peptides may also be present in ambulacraria. GPCR clustering revealed another bilaterian GPCR family, the orphan GPR83 receptors. These may be receptors for an as yet unidentified ancient bilaterian peptide. TRP receptor orthologs are also present in protostomes (Figs. 35, 37B), The ancestry of TRP has been traced to the stem deuterostome (49), but may be urbilaterian. Among the class B GPCRs, the vertebrate PTH receptor has orthologs in invertebrates. PTH could only be identified in vertebrates, but the broader distribution of the receptors indicates that this family also has urbilaterian origin. Likewise, a S, purpuratus ortholog of protostome Pdf receptors suggests a deeper origin for this family.

GPCRs for most F Yamide peptide families cluster together, including sulfakinin/CCK, QRFP, NPFF/GnlH, SIFa, NPY, NPF, sNPF, PrRP, RYamide, luqin, kisspeptin, and allatotropin receptors (Fig. 35). These GPCRs likely represent stem bilaterian duplicates. Within this large GPCR cluster, bilaterian orthologous groups can be recognized for sulfakinin/CCK, orexin/allatotropi n , and RYa/luqin pNP pairs, in agreement with pNP classification. GPCR orthologjes further suggest that NPFF/GnlH/SIFamide and PrRP/sNPF pNPs likewise represent ancestral bilaterian families, and that vertebrate kisspeptins may have invertebrate orthologs. Vertebrate NPFF and GnlH pNPs are paralogs as indicated by sensitive similarity searches, an intermediate sequence from the cyclostome Paramyxine atami, and a shared PQRFamide motif. A B. floridae NPFF/GnlH could also be identified. NPFF and GnlH receptors cluster with several B. floridae GPCRs and are connected to protostome SIFamide receptors, indicating that these Famide families are likely orthologs. The receptors for PrRP and sNPF pNPs also cluster together, in the vicinity of NPY/NPF receptors (Fig. 35), in agreement with a distant relationship between the sNPF, NPF, and NPY pNP families (64), Protostome- and deuterostome-specific pNPs

Some pNPs are restricted to protostomes, either tracing back to the protostome stem or present in one or two phyla only (Fig. 34). Ancient protostome pNPs include proctolin (65), prohormone-2, and -4, and myomodulin/myosuppressin. The protostome origin of proctolin and myomodulin/myosuppressin is also supported by the GPCR map (prohormone-2, and -4 receptors are not known). Arthropod myosuppressins are muscle inhibitory peptides (66). Their receptors cluster with mollusk and annelid receptors indicating that orthologous peptides are present in lophotrochozoans. The likely orthologs are the myomodulins (67), myoactive peptides found in mollusks, annelids and nematodes (68) that share an LR[MLF]- NH₂ motif with myosuppressins (Fig. 40). The biochemical characterization of these lophotrochozoan receptors could confirm this connection. Some pNPs are more restricted phyletically, including arthropod ADF (15), neuroparsin, and PTTH, mollusk R3-14, ecdysozoan CHH/ion transport peptide, and lophotrochozoan fulicin and pleurin. Cnidarians, platyhelminthes, echinoderms and nematodes also have several pNPs with no similarity to sequences from other phyla (not all listed in Fig. 34).

The arthropod PTTHs are related to the extracellular signaling molecule trunk (69) that is a member of an ancient bilaterian family, since trunk orthologs could be identified in annelids, mollusks, and in B. floridae. Trunk is distantly related to the TGF-beta inhibitors, noggins, which are also present in placozoans and sponges (Fig. 37Z>). PTTH represents an arthropod- specific paralog of trunk, and is listed as an arthropod-specific pNP (Fig. 34). The Cys-rich neuroparsins show similarity to the insulin-like growth factor binding protein (pfam:PF00219; HHpred probability 99.5% and ρ=1Ε-18), a domain found in various multidomain proteins (e.g. HTRA serine proteases), and represent an arthropod-specific duplication and stand-alone version of this widespread domain (Fig. 37E).

Several pNP families are only found in the deuterostomes. Some originated either in the chordate or the deuterostome stem lineage (Fig. 34). Ambulacrarian orthologs reveal the stem deuterostome origin of some pNPs. Secretogranin-3 could be identified in S. walevskii, S. purpuratus, and B. floridae. Several members of the FAM55/neurexophilin family, first described in vertebrates (70), could also be identified in S. purpuratus, S. kowalevskii, and B. floridae (Fig. 32, 34). B. floridae orthologs revealed the stem chordate origin of orexigenic neuropeptide QRFP-like/26RFa and CART (CART; Fig. 40, Datasets S5). GPCR clustering suggests a stem-chordate origin of further families, where the pNPs are only known in vertebrates. These include motilin/ghrelin, MCH/Mgrp, and endothelin. The presence of glucagon and gastric inhibitory peptide receptor orthologs in C. intestinalis suggests that these families originated before the vertebrate-urochordate divergence. Other vertebrate-specific pNPs show no resemblance to any family, and represent likely vertebrate innovations (Fig. 32, 34). The history of the GPCRs supports this for urotensin, adrenomedullin, PACAP/VIP, neuropeptide-B/-W, and neurotensin. Growth hormone, VIP, glucagon, and adrenomedullin could also be identified in the lamprey Petromyzon marinus indicating that these originated along the vertebrate stem.

A striking pattern in the evolution of pNPs is the interrelatedness of several pNPs in the CC and the independent derivation of several families from the CC. This indicates that a large fraction of metazoan pNP families are deep paralogs. Importantly, only a clustering approach could reveal this pattern, since many of the derived families are not similar to each other, and the homologies are only revealed by indirect links in a network of BLAST interactions. The broad network of interactions in the highly diverse CC demonstrates the unique pattern of sequence evolution of pNPs. pNPs may evolve more freely in sequence space than globular proteins, where key conserved residues can be identified across very distant homologs. pNPs at two sides of the CC may not show any sequence similarity, except for the SP and cleavage sites, yet be related transitively, via a network of strong sequence similarity. The paralogous nature of several Y/Famide pNPs in the CC is further supported by the close relationship of their receptors.

The finding of repetitive pNPs belonging to the CC in the placozoan T. adhaerens indicates that this class of pNPs was present in the common ancestor of placozoans and eumetazoans and a large diversity of pNPs evolved from such ancestral sequences via successive phases of clade-specific derivations. The presence of pNPs in placozoans indicates that neuropeptide signaling may predate the origin of nervous systems. Although the phylogenetic position of placozoans is not fully resolved, they may be the sister group to the eumetazoans, potentially representing a primitive neuron-less organism (71). If this is the case, then the study of placozoan neuropeptidergic cells may give unique insights into the evolutionary origin of neuronal signaling. The role of neuropeptide signaling in T. adhaerens is unclear, but may involve paracrine communication between sensory cells and effector cells to regulate ciliary crawling, or digestive enzyme secretion. The last common ancestor of eumetazoans had various small amidated peptides (RFamide, RYamide, Wamide), a glycoprotein hormone, prokineticin, and insulin-related peptide. In vertebrates, the glycoprotein hormones of the pituitary are under the control of short, amidated peptides (TRH, GnRH) and regulate sexual development, reproduction, growth and metabolism. Insulin-related peptides are conserved regulators of growth and metabolism. In cnidarians, external stimuli are directly translated into neuroendocrine signals by chemosensory-neurosecretory cells releasing small amidated peptides to regulate growth and metamorphosis (72). Wamides may have been ancestrally involved in mediating life cycle transitions triggered by chemosensory cues (73). RFamides may have ancient roles in muscle control (74), ciliary locomotion (27, 62), and food intake (75). The role of glycoprotein hormones and insulin-like peptides in cnidarians is unclear, but they may be part of a small- peptide/glycoprotein/insulin module in the regulation of growth, metabolism or sexual maturation.

The combined analysis of pNPs and neuropeptide GPCRs identified 27 ancestral urbilaterian pNP-receptor families pointing at a hitherto unknown sophistication of neuropeptidergic systems in the urbilaterian. These pNPs regulate several aspects of physiology, including sexual behavior and reproduction (GnRH, achatin, oxytocin, GnlH/SIFamide), diuresis (CRF/diuretic hormone, calcitonin, vasopressin), gut and heart activity (achatin, luqin, orcokinin), pain perception (opioid), and food intake (NPY, kinins, neuromedin-U, galanin/allatostatin-A, orexin/allatotropin). These pNPs may have originated concomitantly with the origin of a complex bilaterian body plan having a through gut with novel controls for food intake and digestion, excretory and circulatory systems, light-controlled reproduction (50), a centralized nervous system (76), and complex reproductive behavior (77).

In protostomes and deuterostomes further pNPs regulating for example gut motility and feeding (myomodulin, CART, 26RFa, MCH, motilin/ghrelin) evolved independently. Vertebrates invented more than 20 families, including PACAP/VIP/GHRG, neurotensin and leptin. A full analysis of the timing of these vertebrate innovations will require a better lamprey genome assembly. The stable association of receptor-ligand families across bilateria revealed the long-term coevolution of receptor-ligand pairs. References referred to in Example 3

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The present invention refers to the following nucleotide and amino acid sequences:

The sequences provided herein are available in the NCBI database and can be retrieved from www at ncbi.nlm.nih,gov/sites/entrez?db=gene; Theses sequences also relate to annotated and modified sequences. The present invention also provides techniques and methods wherein homologous sequences, and variants of the concise sequences provided herein are used. Preferably, such "variants" are genetic variants.

Gene sequences

The following sequences are either synthetic or derive from short read assemblies, and were therefore not submitted to a sequence database.

SEQ ID o. 1 :

Nucleotide sequence encoding Platynereis dumerilii allatostatin-b polypeptide. The coding region ranges from nucleotide 215 to nucleotide 1075.

>Pdu-VWamide neuropeptide precursor B63-J08_VWamide_full_sequence [Platynereis dumerilii].

CTGCAGCGCCCACTGCATAAGTGCGTGGGACAGTATTTCAACAGACAGCCACTAT

AAATATATCGAAGAAAGTTTAATCCTGTTAGTTGGAGATAAGAGGGAGAGAAGG

AACTAGTGTTTGGAACAACTCTTTATAGGAAACAAGGAACACACAAAGAAGCAA

CAAGGAAGAAAAGGAAAGAATATTAATACAACCAAGGGCCCTAGCAGTCCAATG

GATCGCGTCACTATCACCTGCTTCTCCCTCTGTCTGGCGTCAGTCCTCATCCCCCT

GGTTCACAGCGAAGAAAATGTCGATCTGGACGAAGACAAAAGGGCGTGGATGAA

GAACAATATCGCGTGGGGTAAAAGAGGGTGGAAGCAGGGCGCCTCCTACTCTTG

GGGCAAACGAGACTCAGAGGGTGACGGCTTGATGAGCGATGAAGAGAAAAGGG

CGTGGAACAAGAACAACATGAGAGTGTGGGGCAAACGCAGCGATGAAGACGAC

AAAAGAGGCTGGAAAGATAGCTCGATGAGAGTGTGGGGAAAACGAGCTGGCGA GGACGACAATAACAAACGCTGGGGTAAGAACAACCTCAGAGTCTGGGGTAAACG

AGCTGATGACTTGGAAGTCCTGGAAGACAAGAGAGCTTGGGGCGATAACAACAT

GAGAGTGTGGGGCAAACGAAGCGATTTGGAAGATGACAAAAGGGCGTGGAATA

AGAACTCGATGAGAGTGTGGGGGAAGAGAGACATGGAAGAGGATGAAGACAAC

AAGAGGGCGTGGAAAGGTCAAAGTGCACGAGTATGGGGTAAGAGAGCGGACGA

GGACGACAAACGAGGATGGAACGGTAACTCAATGAGGGTGTGGGGTAAACGTGG

TTGGCACGGCAACGGAGTCCGACAATGGGGAAAACGCCTCCACCTGGATGATGT

GGAACCTATCCTAGATGATGAAGAAAGCAAAAGAGCCTGGGCTAAAAATAACAT

GAGGGTTTGGGGAAAGAGATCGACAGACAATGTAAGGAACATGAAAGCGGTCGT

AGCCGAACCCGCTGAAGTTGCGGCTGATGCAGAGTCTGCAAAAAGCAGTTAACA

ACTGAATGAACATCTAGCAAAATCCCTCTCCTACTTTCTCTTCCTTACATCTCCTC

TCCAGTCTCCAAGGCTCTCTCTTCCTTGATGATTTAGACTCTGATGCAGCTCCGGG

TCAACGACATACCATCTAACTCTGATGACCTTTCCTTTCGATTCGATGCAATACGG

TTGAATACAACGTGTGAAAAAACTTGATACGACTTTCATATATGAATTGACTTTG

AATTGAGGTGATGATTTTAATTGAAATGAATTTTGAAATGATTATGTGATGTGAC

ATAGACCAACTAAGAGCATGCACCTGTATTCCATGTGTAAGACCTTGACAACAAG

CATTGAAATGCTGCAATCAATGATGACATTTAAGTTGAAGACAATTTTGAGTTGC

AGTAGAATACGATACCAGTCCATTGTTATTTTGACCAATGTATCTCCTTATGATAA

TACCAGTGGGAAGCCATAAAACTAAAAAAACTTGTCAATAATAGATGGCTGACTT

TAATGACACTTATTAATGATTGTGTTTTATACACTAAAGTAACGATTTTGTACAAA

GGAAAAAAAAAAGAACTGCAAAATATACATGTTrCCTCTTTGAGTAACCAATGTT

GAGTTACCAAGTGGTGCCTCAAGGAACCAAATAAAATACTGCAAATATATTTGAC

ATGGACTTGCAGTTGTCAGTTAAAAGTGTTTATCCATGTGTTACATCTACACGTTA

CATTTTCGCAATTAAAATTTTGCTTGTTT SEQ ID No. 2:

Amino acid sequence of Platynereis dumerilii allatostatin-b polypeptide.

MDRVTITCFSLCLASVLIPLVHSEENVDLDEDK AWMKJSiNIAWGKRGWKQGASYS

WGKRDSEGDGLMSDEEKRAWNKNNMRWGKRSDEDDKRGWKDSSMRVWGKRA

GEDDNNKRWGKNNLRVWGKRADDLEVLEDKRAWGDNNMRVWG RSDLEDDKR

AWNKNSMRVWGKliU⁾MEEDEDNKRAWKGQSARVWGKRADEDDKRGWNGNSMR

VWGKRGWHGNGVRQWGKRLHLDDVEPILDDEESKRAWA NNMRVWGB RSTDNV

RNMKA WAEP AEVA AD AES AKS S

SEQ ID No. 3:

Nucleotide sequence encoding Capitella teleta allatostatin-b polypeptide. The coding region ranges from nucleotide 52 to nucleotide 672.

>gi| 161315101 |gb|EY648053.1 |EY648053 CAWW9228.fwd CAWW Capitella sp. I ECS- 2004 Subtracted Late Library, Embryos >48hr after egg deposition Capitella teleta cDNA clone CAWW9228 5', mR A sequence

CGGAATTCCCGGGATGACGAATTGGCGAAGCGCAAGTGGGGTTCCAACAGCATG AGAGTATGGGGCAAAC

GCGCTGACGACAACAAACGCAAATGGGGCTCAAACTCTATGCGTGTGTGGGGCA AAAGAGCCGATGATGA

CATGGACGAGAGCAAGAGAGGATGGAAGAACAATAACATGAGAGTGTGGGGAA AGAGAGCCGATGACGAG

ATTGACGAAGACAAGAGAAAGTGGGGATCGAACAGCATGAGAGTCTGGGGCAA GAGATCGGCCGATGATG

ACGCCGAACTGGCAGCTGCCGTACCTCACGCTATCGTCAAGAGAAGTTTGGACAG CGAGGAGTTTACTGA

TGATATGGAAAAACGACGATGGGGAGGCAACGACATGCGAGTTTGGGGCAAACG GCGCTCGCGGGCTGAT

GGTCCAAAAAGAAGCTGGAAAACTAATGTCATGCGCGTCTGGGGTAAGCGCGGA TGGGCGGACAACAACA

TGCGAGTCTGGGGAAAGAGGGCTGACGAGGGAGCCGAGAAGAGGGCGTGGGTC GGTGACAAGTCGCTTTC

ATGGGGCAAGAGATCCGACAACGAAGTCATCAGGAATTTATTAGCAGAACAGGA GAAAGAAAACGAGCTG

AGAGAATACCTTGGCAGTCGGTTATACGAAGATGAAGTTTAATCGTCCCACTACC CACGTGACCACCCGT

GTGAAGTGTGACGTCATAGCCATCAATGAGGTCATCGCGCATGCGCAGCANAAC CGGAAGTTACTCCACA

TACCATGGAGACAGCGATCTCAATATCACTTTTTCTATTTCTCTCTAAAATTTTTTC

GCGTTCTGCGCTT

GTGAAACGGACTCCTTTTGTTC

SEQ ID No. 4:

Amino acid sequence of Capitella teleta allatostatin-b polypeptide.

MRVWGKRADDNKRKWGSNSMRVWGKRADDDMDESKJ GW NNM

DEIDEDKRKWGSNSMRVWG

KRSADDDAELAAAWHAIVKRSLDSEEFTDDMEKRRWGGNDMRVWGKRRSRADGP KRSWKTNVMRVWGB R

GWADNNMRWGKJIADEGAEKRAWGDKSLSWGKRSDNEVIRNLLAEQEK^NELR EYLGSRLYEDEV*

SEQ ID No. 5:

Nucleotide sequence encoding Aplysia californica allatostatin-b polypeptide. The coding region ranges from nucleotide 1 to nucleotide 720.

>gi|121379702|gb|EB256780.1 |EB256780 CNSNOl-C-002639-501 Normalized CNS library (juvenile 1) Aplysia californica cDNA clone CNSNOl-C-002639 5', mRNA sequence CTGTCGGACTCTCCGGCCAGCGGGGCTGACGTGCCCCCTCTCCCCTCATCTGCAG CGACGAACGCCGCCG

TGGACAAAGAATGGCTGCGTCAGAAGCTGGAAGAAGGACAATTTCTACCACAAC AGGACAAACGATGGGG

AGGTATCAATTCTTGGATGACGCACCGTCTCGGCGGACCCTCCGAGAGGGATTCG AGCCAGGACTCTCTG GATAAACAACTGCTCGTGAACAACGTCCAGAACTACGATGACTCGAGTAAGAGG AAGTGGTCGAAATTCT

CGTCTTGGGGGAAGCGCGACGCTTCGGAGGAAACGCCTGAAGGTGGTGAGGGTG AAGACGGACTCGGCGC

TGTAAAGAAGTGGAAGAATATGGCGGTGTGGGGGAAACGGGCAGAAGATGGTTT AGGCAAGAGATGTAAG

CAGATGGCCACTTGGGGCAAGAGAGAAGACGGTGATGTTTTAGGGTTGGGGACG GACAAGAGATGGAAAC

AAATGGCCTCGTGGGGTAAACGATTAGATGATTCTGACCGAGATAAGAAATGGA AACAAATGTCTGTTTG

GGGAAAGAGGGAGGATAACGGGGAACCACTGGACAAAAAGTGGAAACAAATGA GTGTGTGGGGGAAGAGA

GATACTTTAGATGACCCGGAGAAAAGATGGAAGCAGATGGCAGTCTGGGGGCAA AGGCAAGGTTTAGATG

ACAGAAACGATAAAAAAAAAAA SEQ ID No. 6:

Amino acid sequence of Aplysia californica allatostatin-b polypeptide.

MSDSPASGADVPPLPSSAATNAAVDKEWLRQKLEEGQFLPQQDKRWGGINSWMTH

RLGGPSERDSSQDSL

D QLLVNNVQNYDDSS -RKWSKFSSWG RDASEETPEC^EGEDGLGAVKKW NM AVWGKRAEDGLGKRCK

QMATWGKREDGDVLGLGTDKRWKQMASWG RLDDSDRDKKWKQMSVWGKRED

N GEPLDK WKQMS VWGKR

DTLDDPEKRWKQMAVWGQRQGLDDRNDKKK

SEQ ID No. 7:

Nucleotide sequence encoding Crassostrea gigas allatostatin-b polypeptide. The coding region ranges from nucleotide 33 to nucleotide 677.

>gi|313359302|gb|HS211341.1 |HS211341 CCTS10477. CCTS Crassostrea gigas mixed adult tissues library 4 normalized Crassostrea gigas cDNA clone CCTS 10477 5', mRNA sequence

GT CATGGGTCAGACTGGCTTGTAAATTATAGTTGAGATTTTTTTCGGTTACTTTT CAATATTCCCTCAC

ACAAGAACCTGGAATAATGCTTTGCCATCTAGAGCAGCTGCTTCTCTCGTGTATA GTGCTTTGCCTCGTT

AAAGTTTGTGCTAGCCTAAAGGCACAAGATGAAGCCTCTGTCAATGACCACGACA TTGTAAGGAGACAAG

CAATGGGTCATAGTTTCGGTGATGAACTCGAAGGGTTTGATCTGGATCCGAAGAG AGTCAATAATTGGAA

TCAATTCCCGGCCTGGGGAAAAAGATTATCTAAACGCAGATGGTCTTCTCTGGGG ACCTGGGGAAAAAGG AGTTGGCTGGATCGACTTATCAGTGCAAACAATAACTGGGGTAAGCGATGGAAG TCCATGTCCAACTCAT

GGGGGAAACGCCAAGCCCCTTCGGAATTCGACGGTTTAAGCGACGATTATATTAA C ATTA A GAAG AGATC

TGTCGACTCTAAATCCAGCCACAATTCTAGGAACAAGCGATCGATACCTACCGAG CTGTCCCCCGAGCAA

AATGAAGAAAAGAGAAGATGGTCGTCATTGTCGGCGTGGGGAAAAAGAAGCGAC GATGACGAGAAGCGAA

GATGGTCCTCTCTTTCAGCTTGGGGTAAAAGGAGTAACCCAGAGCATA

SEQ ID No. 8:

Amino acid sequence of Crassostrea gigas allatostatin-b polypeptide.

MRPFSVTFQYSLTQEPGIMLCHLEQLLLSCIVLCLVKVCASLKAQDEASVNDHDIVRR QAMGHSFGDELE

GFDLDPKRVNNWNQFPAWGK LSKRRWSSLGTWGKRSWLDRLISANNNWGKRWK SMSNS WGKRQ AP S EFD

GLSDDYINIK RSVDS SSHNSRNKRSffTELSPEQNEEKRRWSSLSAWGKRSDDDEK

RRWSSLSAWGKR

SNPEH

SEQ ID No. 9:

Nucleotide sequence encoding Lottia gigantea allatostatin-b polypeptide. The coding region ranges from nucleotide 166 to nucleotide 783.

>gi|163518426|gb|FC762217.1 |FC762217 CBBN13169.fwd CBBN Lottia gigantea 3_}4,5,6.5d Larvae (M) Lottia gigantea cDNA clone CBBN13169 5', mRNA sequence

ACAAGTGTTATACTATGACAAGAACTACCATATAAAGACAATATCTATCGTGGAT CGATTGAAAATAATT

TTTAATTCAAGCTAAGGCAACGCTTCTACCAAGTTAATTTTGGTGATCAAATTTGT GACGGGACCCTGCA

TATATCTGCTTGTGAATTTTGATTCCTGAAAATAAACAGAACTGGAAGTCTGGAA AGAAGTTATCACCAT

CCAATGGATTTAAAGACTATACTCTGCCTTATTATATATTCCTTATTATTACAAAT ATCACACGCAGAGG

AACAACTCGCCAACGACATAGAACTTTCAAATTCTCTGAACCCTGTGGACAAACG

TTATTTGAACACGTGGGGTAAACGATGGAACCCACGATACAATTTAAGAGGGTAT CAGAGAATGCCAATT TGGGCAAAGAGATGGACGAATTCGGGGTTAATTACGTGGGGTAAGCGAAGTGCT GATACTGAAATTCCCA

TTCATAAACGAAAATGGAATCAGTTTATCACGTGGGGTAAACGTAGTGGAGTGCC ATCTATTGTTAAACG

TAGTGTAGGTGATGAGCTTGTACCGTGGGGTAAAAACAAAGACACTCTACCGGA ACTCAATACATCTAGT

GATAATTTAGACAATAAACTAATAGATCTTGAAACTACACCTTCTACAGATAAAC TTTCTTTAGATGAGA

AAAGAGCCAGTGATAAAGGATGGAATGGATTTACAACGTGGGGAAAACGTGCTA

ATAAAGACTGGTCGTC

GTTGTCTACCTGGGG

SEQ ID No. 10:

Amino acid sequence of Lottia gigantea allatostatin-b polypeptide.

MKINRTGSLERSYHHPMDLKTILCLIIYSLLLQISHAEEQLANDIELSNSLNPVDKJ AW KSSYLNTWGKR

WNPRYNLRGYQRMPIWAKRWTOSGLITWGKRSADTEIPIHKRKWNQFITWGKRSGV PSrVKRSVGDELVP

WGKJ^KDTLPELNTSSDNLDNKLIDLETTPSTDKLSLDEKRASDKGWNGFTTWGKRA NKDWSSLSTW

SEQ ID No. 11:

Nucleotide sequence encoding Lymnaea stagnalis allatostatin-b polypeptide. The coding region ranges from nucleotide 22 to nucleotide 858.

>gi| 14832045 l |gb|ES580344.1|ES580344 FPS019.CR_G09 LSGlO-01 Lymnaea stagnalis cDNA clone FPS019 G09 5', mRNA sequence

TAGTTACGGGGGAAGTCAAGTTTGGTAGTCAGGTTTTTTTTTTCTCCCTCCGTCTC CATTGCAAGTCGGC

ATAGTCGTGTGCCGTTGATCGCGAGTTACAGCCCCTTTGGAATAGTGAAACTATT AAACAGAACCAAAAT

GTATTTATCAGGGAGAATGCCCAGGGATTCAAGTCAGTTCAACGCACTTAGGTTA

ACATTACTTCAAATA

TTTCTCTTATCCCTAACCCAAGACACGTCGTCGGCAGCAAGGACGTTGCACCTAC CCAGTGTCATCAACG

AACTGCCGTCCAAGGCTATCACCGACTCGACAGAACTTTCCCAACAGAACACTTT CGGCAACGTTCATTC AGATTTGGCGAAAGAGCCAGGCGCGTACATTCATACATTTAGGGACTTCGATGAC GCAACTCAAGAGACA

GACACCAGCTTAAGAGGCACCCAAAATTGGTCGGAGCTGTCGAAGTGGGTCAAA GGGGATAGCTATCCAG

AATCGGATAACCAGTCGTCGGAGAACAACTCCCAGAACAAGCGCCAGTGGAACG CGTTCTCTTCATGGGG

CAAGAGGTCGGCCGAGGATGAGAATAGCAGTGGACTGGATGAGCAGAAACGTTG GAAGCAGATGGCCGTT

TGGGGAAAGAGATACGCTGATCCAGATCATGAAAAGAAGTGGAAGGACATGCCT GTATGGGGAAAACGAG

ACATGGATACTGATATGGATAAAAGATGGAGTGACATGGGTGTCTGGGGCAAAC GAGGCATGAATACTGA

TATGGATAAAAGATGGAAAGACATGGGTGTCTGGGGGAAAAGAGACATGGATAC

TGATATGGATAAAAGA

TGGAGTGACATGGGTGTCT

SEQ ID No. 12:

Amino acid sequence of Lymnaea stagnalis allatostatin-b polypeptide. >lcl| Sequence 1 ORF:22..858 Frame +1 Lymnaea stagnalis mollusk Euthyneura

MVWFFFSPSVSLA.SRHS VPLIASYSPFGTV LLNRTKMYLSGRMPRDSSQFNALRLT LLQIFLLSLTQ

DTSSAARTLHLPSVINELPSKAITDSTELSQQNTFGNVHSDLAKEPGAYIHTFRDFDDA TQETDTSLRGT

QN WSELSKWVKGDS YPESDNQS S ENN S QNKRQWNAF S S WGKRS AEDENS SGLDEQ KRWKQMAVWGKRYAD

PDHEKKWKDMPWGKRDMDTDMDKRWSDMGWGKRGMNTDMDKRWKDMGV WGKRDMDTDMDKRWSDMGV

SEQ ID No. 13:

Nucleotide sequence encoding Biomphalaria glabrata allatostatin-b polypeptide. The coding region ranges from nucleotide 322 to nucleotide 489.

>gi|54424269|gb|CV548090.1|CV548090 1742BUN46F10 BgORESTES uninfected NHM 1742 Brain Biomphalaria glabrata cDNA clone ZBA4873 similar to prothoracicostatic peptide precursor, mRNA sequence

GGGCAAAAGAGACTTTGATCCTGAATTGGAGAAGAAATGGAAAGAGATGGCTGT

TTGGGGCAAAAGAGAT

TTCGATCCTGAATTAGAAAAAAAATGGAAAGAAATGTCAGTATGGGGCAAAAGA GACTTTAATCCTGAAT

TAGAGAAGAAATGGAAAGAAATGTCAGTATGGGGCAAAAGAGACTTTAACCCTG AATTAGAGAAGAAACG GAAAGAAATGTCTGTTTGGGGCAAAAGAGACTTTAATCCTGAATTAGAGAAAAA ATGGAAAGAAATGTCA

GTATGGGGCAAAGGAGACTTTGATCCTGAACTAGAGAAGAAATGGAAGCAGATG TCCGTGTGGGGCAAAC

GAGGTGTGGACAACAACGGAAAACAGACCATACGACACACACGAAAATGGAGG TGGGCAAATCTAGGCGC

GAAGCGACCCAGTTGGAGCAGTACAGGTTTCTCCAGCTGGGGGAAAAGATCAGA

AGACCTTGACCTTCAC

GATGTCAAGGAATACCTTCAGAAAACATTACCTGTCTCAGAGCTAGAAGAGACA CCACAAGAGGCTGGTG

AGTTGAAATCACAAGATCAAGGGACGCCTCTGTCCAAGACGTAATGTGCCACAA AAGTCACAAGGTGTGG

GGGGGCTTCAGGAAGTGTCAGTTTCACCCACGTCACGTGATACAATGTCCAGCCA

GCAGACTGTACTATT

TGTTTACACTTGTACA

SEQ ID No. 14:

Amino acid sequence of Biomphalaria glabrata allatostatin-b polypeptide. >lcl| Sequence 1 ORF:47..604 Frame +2 Biomphalaria glabrata mollusk Euthyneura

MAWGK DFDPELEKKWKEMSWGKRDFNPELEKKWKEMSVWGKRDFNPELEK KPvKEMSVWGKRDFNPEL

EKKWKEMSWGKGDFDPELEKKWKQMSVWGK GVDNNGKQTIRHT WRWANL GAKRPSWSSTGFSSWGK

RSEDLDLHDVKEYLQKTLPVSELEETPQEAGELKSQDQGTPLSKT* SEQ ID No. 15:

Nucleotide sequence encoding Tritonia diomedea allatostatin-b polypeptide. The coding region ranges from nucleotide 70 to nucleotide 528.

>gi|308074691 |gb|EV285739.1|EV285739 Tdi-CNS_18_M12.g Central Nervous System (CNS) from one juvenile Tritonia diomedea Tritonia diomedea cDNA, mRNA sequence GGATNNNNNNNNNNNNNNNNNNNNN^

NAAAGTGGAACAGTAAAA

TGGCGGTCTGGGGCAAAAGAGATAGTCCGGTGGACTTAGAGAAAAAGTGGAACA GTAAAATGGCGGTCTG

GGGCAAAAGAGATAGTCCGATGGACTTAGAGAAAAAGTGGAACAGTAAAATGGC GGTCTGGGGCAAAAGA

GATAGTCCGGTGGATTTAGAGAAAAAGTGGAACAGTAAAATGGCGGTGTGGGGC AAAAGAGATAGTCCGG

TGGACTTAGAGAAAAAATGGAATGATAAAATGGCGGTGTGGGGCACAAGAGGTC TTTCGGATTTAGAAAA CAAGTGGATGATTGTGCAACCATTGGGGAAGGAATTAGATGCAGTGAGCAGAGG ACTTTATTCATCTAGT

GACAAACCATCGAGCACAGGTGATATGTTGGTGCCGGAGACAGCACTAGAACAG TTACAGCTGCAGGACA

GTGCCGGTCTCCTGCAGGACAACAAAGAAATTCCCTTGG SEQ ID No. 16:

Amino acid sequence of Tritonia diomedea allatostatin-b polypeptide. >lcl|Sequence 1 ORF:70..528 Frame +1 Tritonia diomedea mollusk Euthyneura

AWGKJ DSPVDLEKKWNSKMAWGKRDSPMDLEKKWNSKMAVWGKRDSPVD LEKKWNSKMAVWGKRDSP

VDLEKKWNDKMAVWGTRGLSDLENKWMIVQPLGKELDAVSRGLYSSSDKPSSTGD

MLVPETALEQLQLQD

SAGLLQDNKEIPL

SEQ ID No. 17:

Nucleotide sequence encoding Mytilus californianus allatostatin-b polypeptide. The coding region ranges from nucleotide 24 to nucleotide 275.

>gi|145885551|gb|ES391133.1 [ES391133 MUS13-F16.xld-t SHGC-MUS Mytilus californianus cDNA 5', mRNA sequence

GGGATTCTGAATTAGACAATGACATGGATAAACGTAAATGGAATCAAGTCGGAG TCTGGGGAAAAAGGAG

TGAACCTGACAAGAGAAGATGGTCTTCTGTATCAGCATGGGGAAAACGAGGATG GAACAACCTCCAGTCA

TGGGGAAAACGCCCATGGTCATCATTCAAATCATGGGGAAAACGTAATCCATGG CATTCATTGTCAACAT

GGGGCAAACGATCTGTGCCAGTTGAGAGTGACCAAGATGCCACCTACACAGCCC AAGCTGTTTGAAAGGG

GAAATAACTCGACATGTACAGATTCCATTATACCAATTAGCAATAAACAAATTTC GTGACAAAGTATATA

CTTCGCTATATTGTAATTTGTCTTAAAGAATCTTTT ATGTATTTTAGTGAAATGTA ATTTGATTTTCGA

GCCTGCTGACATTTAATTGAAAGGATGCTAAACTATCACCTCAGTCCCGGCAATC TAATTAAATGTTTGT

TAATATGTTGTGTAGGACTAAGTGCGTCCGGTAATGGCGGACATAATAAGTTGTT TAAAAGCTGCTTCAT

TTCAGGAATAT AAGTCACGAAACAT TAGAATGACAGAAGAATGATTTTTGTTC TAAAACTGTTGATGT

ATTGAGTATGCACACGAGGAATGTAATTATTATT AAGGAGAAAGTTGTTTTTCT CTTTTTTTCTATTTT AATCATTTGTTTTTTGTAACCTTTAGTTCAATTTATTTCGAATTCTAAGAAGGTTTT TCTGTCGGTATAC

CGAATGATAGAGATGGCAAGTACTTGTATGACAATTCATAGACTTATACATACGA ACTTAACTTTTTGAT

TTTTTAAAGTCTATGGTTGATAGAAATTACNATAT

SEQ ID No. 18:

Amino acid sequence of Mytilus calitbrnianus allatostatin-b polypeptide.

MDKRKWNQVGWGKRSEPDKRRWSSVSAWGKilGWNNLQSWGiCRPWSSFKSWG

KR PWHSLSTWG RSVPV

ESDQDATYTAQAV*

SEQ ID No. 19:

Nucleotide sequence encoding Euprymna scolopes allatostatui-b polypeptide. The coding region ranges from nucleotide 205 to nucleotide 687. Frame -3

>gi[84432649|gb|DW267246.1 (DW267246 UI-S-GN1 -abx-e-12-0-UI.sl UI-S-GN1 Euprymna scolopes cDNA clone Ul-S-GN 1 -abx-e- 12-0-UI 3', mRNA sequence

TTTTTTTTTTTTTTTTTTAC ATATTAATAATAA

GTATGTTTTAAGACAGCTT CTCTCTAAATACATTTTGGACATGTCCTTCGTGATG TAATTCTTCAAACC

TCGGGGCATATCCATTTTCAACGACAATCTCTCCAAACAGTCAAATGTATATATT GTTTTCGGCTCATCG

CCCTGACTTAGAGTTCTTTCCACTGCTTCTTTTCATCATATTTTTTACTTCTGAGCC TAGCTTTTCGTCT

TCATCACTAGCAGCAATGTCCCTCTTGCCCCAAGCGGCCAGTGAATCCCAATCCT TCTTGTCTTTTGCGT

T CCGGTTCGTTTACCCCAAGCTTGAAGAGAGTTCCAGTCACGTTTATCTTCAGAA TCAGCGTTTCGTTT

ACCCCAGGCGGCCATCGAGTCCCAGGTGTCTGGGCTTCTTTTACCCCAGGCGGCC ATCGAGTCCCAGGTG

TCTGGGCTTCT TTACCCCAGGCGGCCATCGAGTCCCAGGTGTCAGGGCTTCTTTT ACCCCAGGCGGCCA

TCGAGTCCCAGGTGTCTGGGCT CTTTTACCCCAGGCGGCCATCGAGTCNCAGGT

ACCCCAGGCCGCCATAGAATCCCAAGTGTCCGCATCACGTTTACCCCAAGTTCCC ATGGAATCNCACGTG TCGGGATTTCGTTTACCCAGGCATGCATGGAACCCACGTGTCAGGGTTTCGTTTAC CC

SEQ ID No. 20:

Amino acid sequence of Euprymna scolopes allatostatin-b polypeptide.

MGTWGKRDADTWDSMAAWGKRSPDTXDSMAAWGKRSPDTWDSMAAWGK SPD

TWDSMAAWGKRSPDTWDS

MAAWGK SPDTWDSMAAWGKIWADSEDKRDWNSLQAWGKRTGNAKDKKDWDS

LAAWGKRDIAASDEDEKL

GSEVKNMMKRSSGKNSKSGR*

SEQ ID No. 21 :

Nucleotide sequence encoding Idiosepius paradoxus allatostatin-b polypeptide. The coding region ranges from nucleotide 93 to nucleotide 723.

>gi|117940898|gb|DB915757.1 |DB915757 DB915757 Northern pygmy squid cDNA library Idiosepius paradoxus cDNA clone Ip_aB_020_G04 5', mRNA sequence

AAAGTGAAAGCAAGAGCTTAAAGCCACTTTCTGTTATAAAGGCAGAAAACTTAA AATCGTGGGCCAAACG

TAGCACAACCAATGGCAAGGTCCTGAATGAAATACGCCAGGCTGTGGCTAGAGG CGTGTTTCCCGCAGCT

GTGGGCCCCATAGACAAATCCGGTGACAAATATATGAGCTTCCTTCAGCATTGGT TAAAAAATAACGGCT

ACCCAGCCACAATGGCAAACTTGTGGGCCAGAAATACCGGCTTTAAATCCCGCGT AACTCGAGAGATTAA

TAATAACGACGACGACCTACCTGACAAAAAGGATAGACTTGATACATGGAATTCT ATGAATACTTGGGGT

AAACGAAGCCCGAATACTTGGGACTCTATGGCCGCTTGGGGTAAGCGTAACGGT GATACCTGGGACTCGA

TGTCCGCTTGGGGTAAACGAAATGGTGATACCTGGGATTCGATGTCCGCCTGGGG TAAGCGAAATGGAGA

TACCTGGGACTCGATGTCTGCCTGGGGTAAGCGAAACGGCGACACTTGGGATTCG ATGTCCGCCTGGGGT

AAGCGAAACGGCGACACTTGGGACTCGATGTCCGCCTGGGGTAAGCGAAATGGT GATACCTGGGACTCTA

TGTCCGCCTGGGGAAAGAGGAATGCTGACAACGCCGGAGACAAACGCGACTGGG

ACTCTCTACAGGCCTG

GGGGAAACGTAACATTGCTCCAAA

SEQ ID No. 22: Amino acid sequence of Idiosepius paradoxus allatostatin-b polypeptide.

MNEIRQAVA GVFPAAVGPIDKSGDKYMSFLQHWLKN GYPATMANLWARNTGFK

SRVTREINNNDDDLP

DKKDRLDT SMN G]a SPNTm)SMAAWGKRNGDTWDSMSAWGKl^^TGDTTO SMSAWGKRNGDTWDSMSA

WGKJ GDTWDSMSAWGKRNGDTWDSMSAWGKRNGDTWDSMSAWGKRNADNA GDKRDWDSLQAWG RNIAP

SEQ ID No. 23:

Nucleotide sequence encoding Doryteuthis pealeii allatostatin-b polypeptide. The coding region ranges from nucleotide 75 to nucleotide 422.

>gi|342665597|gb|JK333766.1 |JK333766 oyl93b05.yl Woods Hole Squid Stellate Ganglia cDNA Library Doryteuthis pealeii cDNA, mRNA sequence

CGTCCGAGAGAAACGGTGACACTTGGGACTCCTTGTCGGCCTGGGGTAAGCGAA GCCCCAACACCTGGGA

TTCTATGTCAGCCTGGGGCAAGAGAAACGGTGACACCTGGGATTCTATGTCAGCC TGGGGTAAACGAGAT

GCAGATACCTGGGATTCTATGTCGGCCTGGGGTAAGAAGAGAGCGGGTGATACTT GGGATTCGATGTCCG

CTTGGGGTAAGAGAAATGCTGACTCTGGAAACAAACGTGACTGGGACTCTCTTCA GGCTTGGGGAAAACG

CGGTAAGGACAAGAAAGATTGGGATTCTCTTGCTGCCTGGGGTAAGAGGAACAT TGCTGGAAATGGAAAT

GATGAGATAGGCTCACAAGTTCAAAGTTTGATGAAAAGAAGCAGCGGAAAAAAT GCCAAGTCAAGACGAT

GAGCCGATATAAAATGGATCAATATGTATAAACTTGGCTGTTTGGAGAGACTCTC GTTGGAAATGGTCAT

GTCAAGGTTATTGGCCAATAGCAACAACAACACCAGAGGCATGTCCAGACTGAA CTTAGAGAGAAAGCGC

TCTTATTATATTGTATTATTAT AATATTATTATTATTAT GATTT ATTATTATTAT TATCATTATTAT

TATTCAATAAAAACTGCAGTCGAACTTGTTAAAAAAAAAAAAAAAAAAAG SEQ ID No. 24:

Amino acid sequence of Doryteuthis pealeii allatostatin-b polypeptide.

MSAWGKRNGDTWDSMSAWGK.RDADTWDSMSAWGKKRAGDTWDSMSAWGKRN

ADSGNKRDWDSLQAWGKRG

KDKKDWDSLAAWGKRNIAGNGNDEIGSQVQSLMKRSSG NAKSRR*

The following are amino acid sequences of peptides to be used herein which are derived from Aplysia californica allatostatin-b polypeptide:

RKWSKFSSW-amide (SEQ ID NO: 125)

W MAVW-amide (SEQ ID NO: 126)

WKQMATW-amide (SEQ ID NO: 127)

WKQMASW-amide (SEQ ID NO: 128)

WKQMSVW-amide(SEQ ID NO: 129)

WKEMSVW-amide (SEQ ID NO: 130)

WKQMAVW-amide (SEQ ID NO: 131)

WKQMATW-amide (SEQ ID NO: 132)

WKQMSVW-amide (SEQ ID NO: 133)

The following are amino acid sequences of peptides to be used herein which are derived from

Lottia gigantea allatostatin-b polypeptide:

AWKSSYLNTW-amide (SEQ ID NO: 134)

WTNSGLITW-amide (SEQ ID NO: 135)

KWNQFITW-amide (SEQ ID NO: 136)

ASDKGWNGFTTW-amide (SEQ ID NO: 137)

NKDWSSLSTW-amide (SEQ ID NO: 138)

GQNKDWSSLTTW-amide (SEQ ID NO: 139)

GHDRDWNSLTTW-amide (SEQ ID NO: 140)

ANKDWSSLSTW-amide (SEQ ID NO: 141)

NDWSALSTW-amide (SEQ ID NO: 142)

ANNKDWASLTTW-amide (SEQ ID NO: 143)

ANDRDWNSLTTW-amide (SEQ ID NO: 144)

AKGNNWS GLTTW-amide (SEQ ID NO: 145)

DWNSLTTW-amide (SEQ ID NO: 146)

ANKDWSGLTTW-amide (SEQ ID NO: 147)

GNKDWSGLTTW-amide (SEQ ID NO: 148)

GKNDWSGLTTW-amide (SEQ ID NO: 149)

GNKDWSGLTTW-amide (SEQ ID NO: 150)

GNNDWSGLTTW-amide (SEQ ID NO: 151)

DIKGWNGLTTW-amide (SEQ ID NO: 152)

TTW-amide

WAQLSTW-amide (SEQ ID NO: 153)

The following are amino acid sequences of peptides to be used herein which are derived from Lymnaea stagnalis allatostatin-b polypeptide:

QWNAFSSW-amide (SEQ ID NO: 154) WKQMAVW-amide (SEQ ID NO: 155)

W DMPVW-amide (SEQ ID NO: 156)

WSDMGVW-amide (SEQ ID NO: 157)

W DMGVW-amide (SEQ ID NO: 158)

Biomphalaria glabrata allatostatin-b polypeptide:

WKEMSVW-amide (SEQ ID NO: 159)

KEMSVW-amide (SEQ ID NO: 160)

WKQMSVW-amide (SEQ ID NO: 161)

PSWSSTGFSSW-amide (SEQ ID NO: 162)

The following are amino acid sequences of peptides to be used herein which are derived from Tritonia diomedea allatostatin-b polypeptide:

WNSKMAVW-amide (SEQ ID NO: 163)

Crassostrea gigas allatostatin-b polypeptide:

VNNWNQFPAW-amide (SEQ ID NO: 164)

RWSSLGTW-amide (SEQ ID NO: 165)

SWLDRLISANNNW-amide (SEQ ID NO: 166)

WKSMSNSW-amide (SEQ ID NO: 167)

RWSSLSAW-amide (SEQ ID NO: 168)

Mytilus californianus allatostatin-b polypeptide:

KWNQVGVW-amide (SEQ ID NO: 169)

RWSSVSAW-amide (SEQ ID NO: 170)

GWNNLQSW-amide (SEQ ID NO: 171)

PWSSF SW-amide (SEQ ID NO: 172)

RNPWHSLSTW-amide (SEQ ID NO: 173)

Eupryrnna scolopes allatostatin-b polypeptide:

DADTWDSMAAW-amide (SEQ ID NO: 174)

SPDTXDSMAAW-amide (SEQ ID NO: 175)

SPDTWDSMAAW-amide (SEQ ID NO: 176)

DWNSLQAW-amide (SEQ ID NO: 177) DWDSLAAW-amide (SEQ ID NO: 178)

Idiosepius paradoxus allatostatin-b polypeptide:

DRLDTWNSMNTW-amide (SEQ ID NO: 179)

SPNTWDS AAW-amide (SEQ ID NO: 180)

NGDTWDS SAW-amide (SEQ ID NO: 181)

DWDSLQAW-amide (SEQ ID NO: 182)

Doryteuthis pealeii allatostatin-b polypeptide:

NGDTWDSMSAW-amide (SEQ ID NO: 183)

DADTWDSMSAW-amide (SEQ ID NO: 184)

RAGDTWDSMSAW-amide (SEQ ID NO: 185)

DWDSLQAW-amide (SEQ ID NO: 186)

DWDSLAAW-amide (SEQ ID NO: 187)

The following are modified amino acid sequences of peptides to be used herein which are derived from an allatostatin-b polypeptide:

AWNKNSMRVW-carboxy (SEQ ID NO: 188)

AWNKNSMRVWP-amide (SEQ ID NO: 189)

AWNKNSMRVWA-amide (SEQ ID NO: 190)

SEQ ID No. 25:

Nucleotide sequence of translation-blocking morpholino 1 (Pdu-ASTB-start MOl) to target the Pdu-AST-B gene (the Pdu-AST-B is shown in SEQ ID NO: 1). Nucleotides complementary to the start codon (ATG) are underlined.

TGATAGTGACGCGATCCATTGGACT

SEQ ID No. 26:

Nucleotide sequence of 5 bp mismatch control MO 1 to target the Pdu-AST-B gene (Pdu- ASTB-mism MOl). Nucleotides altered in mismatch control morpholinos are highlighted in bold

TG TAGTGAC^CG|rCgA¾TGGACT

SEQ ID No. 27: Nucleotide sequence of translation-blocking morpholino 2 (Pdu-ASTB-start M02) to target the Pdu-AST-B gene (the Pdu-AST-B gene is shown in SEQ ID NO: 1). Nucleotides complementary to the start codon (ATG) are underlined.

CTAGTTCCTTCTCTCCCTCTTATCT

SEQ ID No. 28:

Nucleotide sequence of 5 bp mismatch control M02 to target the Pdu-AST-B gene (Pdu- ASTB-mism M02). Nucleotides altered in mismatch control morpholinos are highlighted in bold

SEQ ID No. 29:

Nucleotide sequence of translation-blocking morpholino 1 to target the Pdu-AST-B receptor gene (Pdu-ASTBR-start MOl). The Pdu-AST-B receptor gene is shown in SEQ ID NO: 34. Nucleotides complementary to the start codon (ATG) are underlined.

TCCATCATTTTGAATGTTGAATGCA

SEQ ID No. 30:

Nucleotide sequence of translation-blocking morpholino 2 to target the Pdu-AST-B receptor gene (Pdu-ASTBR-start M02). The Pdu-AST-B receptor gene is shown in SEQ ID NO: 34. Nucleotides complementary to the start codon (ATG) are underlined.

GTCAATGAGGTCACAAACATCCAAC

SEQ ID No. 31:

Nucleotide sequence of 5 bp mismatch control MO 1 to target the Pdu-AST-B receptor gene Pdu-ASTBR-mism MOl. The Pdu-AST-B receptor gene is shown in SEQ ID NO: 34. Nucleotides altered in mismatch control morpholinos are highlighted in bold

SEQ ID No. 32:

Nucleotide sequence of primer for PCT amplification of the Platynereis receptor from larval cDNA including Hindlll site and Kozak consensus

ACAATAAAGCTTGCCACCATGATGGAAGTAAGCTATTCAAATGGAAATG SEQ ID No. 33:

Nucleotide sequence of primer for PCT amplification of the Platynereis receptor from larval cDNA

ACAATAGCGGCCGCTTAAATATTTGTAGTTTTAGTCGTGTGATCG SEQ ID No. 34:

Nucleotide sequence encoding Capitella teleta AST-B receptor

AAGCTTGCCACCATGACCACCACTGCTGCCGCTGCTGTCGCCAAAATGAACGACT

CGGTAGCGTTCACTCCTTCTCACTCACCATTAGCCTCAACGGACGAGGCCTACAG

GAACCAATCGTTGTACCCTGACTACGGATTTCACGGATGCGTGAATGTATCGCAA

AATATATCGTGTACTCCCTTTCATTTGGAGACTGACAGAGCTTTCACTCGATACTC

AGTGGTAGTGTATGGTTTCGTCTCGCCAGCACTGGTCATCTTCACGCTAATGACCA

ACAGTCTGGTGTGTCTTGTACTGATCAAGAAACATATGCGCACCCCCACTAATGT

CATGCTGCTTGCCATGGCTGTCTCGGACATGCTGACCGGGGTCTGGTCGGTGCCC

TGCTTCATCTATTTTTACACCCTTGGCCACTACCATGATTGGGTACCGTATGATTG

GTGCATGCCGTATTTGGTACTTTACGAGTATATGCCGACCGTTTTCCACACAGCCT

CTATCTGGTTGACTGTCGCGCTAGCAGTTCAGCGCTATATCTATGTCTGCCACTCG

ATGAAGGCAAAGCGGTGGTGTACCATCCCGAACGCCATCAAAGGTGTGCTCCTCA

TCTACGCTTTGGCCTTCGCTTCACAGATCAACCGCTTCGTTGAGAATGATTTCCTC

CCCATCAGCGTCTACTCAACCGTCAATTCGAGCAAACTCGTGCAAGCATGCCAGA

TCGAATTAATCGGCTTTGTACAACAGCATGACGTTGTGTATTTTTCTATCTACTGG

TGGTCTAGGGTCATCTTCATCCACTTAATTCCCTGCACGTCTCTGGTGATACTGAA

CTCATTACTGAT TGCACCATGAAGAAAGCACAACGCCGTCGCGACCAACTTCTC

AAGCAGAACCGCAAATCCGAGTCTCGTCGTCTCCAGGAAAGCAACTGCACCACC

GCAATGCTGGTTACGGTGGTCGGAGTCTTCCTCTTGGTGGAGTTTCCCTTGGCTGT

CTTCTTCATCCTGATGATCTTCGAGAACACCTTCAACGTAGTACTGATCAATGACA

ACATTAGGCATGAGGCGTCGCTCTTTATTAATCTGGCGATTTTGCTTAGCTACCCG

ATTAATTTTTTCATT ATTGTGGTATGTCTCGCCAGTTCCGTGAAACCTTTATGCG

GCTCTTCAAACCCGGGACCCCCGCTTTGGACCGTGAGCACTCGCAGTACATCTCC

CTCGCGACTGAGAACGGAGCCAAGTCTTATGAAACAAAAGACACTGCGATATGA

GCGGCCGCTCTCCCTATAGTGAGTCGTATTA

SEQ ID No. 35:

Amino acid sequence of Capitella teleta AST-B receptor

MTTTAAAAVAKMNDSVAFTPSHSPLASTDEAYRNQSLYPDYGFHGCVNVSQ NISCTPFHLETDRAFTRYS

VVWGFVSPALWTLMT SLVCLVLIKKHMRTPTNVMLLAMAVSDMLTGV WSVPCFIYFYTLGHYHDWV

PYDWCMPYLVLYEYMPTVFHTASIWLTVALAVQRYIYVCHSMKAKRWCTIP NAIKGVLLIYALAFASQIN

RFVENDFLPISVYSTVNSSKLVQACQIELIGFVQQHDWYFSIYWWSRVIF1HLI PCTSLVILNSLLICT MK.KAQRRRDQLLKQNRKSESRRLQESNCTTAMLVTVVGVFLLVEFPLAVFFI LMIFENTFNWLINDNLR

HEASLFINLAILLSYPINFFIYCGMSRQFRETFMRLFKPGTPALDREHSQYISLAT

ENGAKSYETKDTAI

*

SEQ ID No. 36:

Amino acid sequence of Hirudo medicinalis allatostatin-b polypeptide.

>gi |312549773 jgb |FP618166.1 |FP618166 FP618166 Hirudo medicinalis normalized library

Hirudo medicinalis cDNA, mRNA sequence ORF:150..740 Frame +3

CAATAATTGTCAATCATCAATGACTGTCTCCCTAATGATGATAATATAAAAATAA ATTTAGCAAAACCTT

AAAAAAGTCTCTACTGGTATTTGCT TTGCGTCTCTCGATATTTTCCTCCAAAGAT TTCAGTGCAAGGGG

ATCTATCTTATGGCTTCATACATAAACCCGGTGGCCGTATTCTTGTTGCTGAGTTC TTCAGTCCTGTTGG

TCTCCCATGGTGTCTCGGCCAAGGACGTCGAATATGCAGAATCTGGCATCTATGA

TGAATCTCATCCCAA

AGCAGAAAACACGGAATCTCATCAGAATAAACGAAAGTGGGGCTCTGACAGGAT GGGACTCTGGGGAAAA

CGATTCGAAATGAACGACGACGACTCTGCCACTACGGAAGTGGAGAAAAAGAAG TGGACTTCCGGGAAAA

TGGGCATGTGGGGTAAGAGATCACTCGCTGACTGGCAAAAAAGAAAGTGGGACT CCAGGAACATGGGCAT

GTGGGGCAGAAAAAAATCGCTACCGGTAGCGACGGCGACAGTTAGAGACATCGA CTATTACGGCGATTAC

TACGACGGAAATGACATGATTTACACTGAGAAAAGAAGATGGGGTCCCGAGGGC ATGTGGGGTAAGAGGT

TGGATAGTGCCATTAATCAGATAAGATCTCAAAAGAGCAAGTGGGCGAGTGGAA GCATGGGCATGTGGGG

AAAGAGATCGTACGTTGGCCGGGACAATGCGACTGAATAGGAAAAAAGACCCTA

AACGACTCTGATTTTG

AATTGTTTATTTCGAGTCTGGGGCGAATA

SEQ ID No. 37:

Amino acid sequence of Hirudo medicinalis allatostatin-b polypeptide.

>lcl|Sequence 1 ORF:150..740 Frame +3 MASYINPVAVFLLLSSSVLLVSHGVSAKDVEYAESGIYDESHPKAENTESHQNKRKW GSDRMGLWGKRFE

MNDDDSATTEVEKKKWTSGKMGAWGKRSLADWQKRKWDSRNMGMWGRKKSLP VATATVRDIDYYGDYYDG

NDMIYTEK.RRWGPEGMWGKRLDSAINQIRSQKSKWASGSMGMWGKRSYVGRDNA

TE*

The following are amino acid sequences of peptides to be used herein which are derived from Hirudo medicinalis allatostatin-b polypeptide:

KWGSDRMGLW-amide (SEQ ID NO: 191)

KWTSGKMGMW-amide (SEQ ID NO: 192)

KWD SRNMGMW-amide (SEQ ID NO: 193)

RWGPEGMW-amide (SEQ ID NO: 194)

WASGSMGMW-amide (SEQ ID NO: 195)

As used herein, the terms "comprising'V'including" or grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. Thus, the terms "comprising"/"including"/'¾aving" mean that any further component (or likewise features, integers, steps and the like) can be present.

The term "consisting of means that no further component (or likewise features, integers, steps and the like) can be present.

The term "consisting essentially of or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic characteristics of the composition to be used in accordance with the present invention. Thus, the term "consisting essentially of means that specific further components (or likewise features, integers, steps and the like) can be present, namely those not materially affecting the essential characteristics of the composition. In other words, the term "consisting essentially of (which can be interchangeably used herein with the term "comprising substantially"), allows the presence of other components in the composition to be used herein in addition to the mandatory components (the one or more peptides derived from an allatostatin-b polypeptide), provided that the essential characteristics of the composition are not materially affected by the presence of other components. All references cited herein are fully incorporated by reference. Having now fully described the invention, it will be understood by a person skilled in the art that the invention may be practiced within a wide and equivalent range of conditions, parameters and the liek, without affecting the spirit or scope of the invention or any embodiment thereof.

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Claims

2. The method of claim 1, wherein the induction or enhancement of the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates relates to the induction or enhancement of the settlement and/or growth of one or more Lophotrochozoan larva of marine invertebrates in aquaculture, said method comprising culturing said one or more larva in aquaculture in a composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide.

3. The method of claim 1 or 2, wherein said peptide derived from an allatostatin-b polypeptide is a fragment of an allatostatin-b polypeptide.

4. The method of any one of claims 1 to 3, wherein said peptide derived from an allatostatin-b polypeptide has a length of from 2 to 48 amino acids.

5. The method of any one of claims 1 to 4, wherein said peptide derived from an allatostatin-b polypeptide has or consists of the consensus motif from N to C terminus (X)_nW_a(X)_nZWb(X)n, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_a may be absent.

6. The method of any one of claims 1 to 5, wherein said peptide derived from an allatostatin-b polypeptide comprises or consists of a peptide selected from the group consisting of AW, VW, TW, SW, and NW.

7. The method of any one of claims 1 to 6, wherein said peptide derived from an allatostatin-b polypeptide comprises or consists of a peptide selected from the group consisting of

AWMKNNIAW (SEQ ID NO: 38), AWGDNNMRVW (SEQ ID NO: 39), AWNKNSMRVW (SEQ ID NO: 40), AWKGQSARVW (SEQ ID NO: 41), GWNGNSMRVW (SEQ ID NO. 42), KWGSNSMRVW (SEQ ID NO: 43) GWADNNMRVW (SEQ ID NO: 44), RKWSKFSSW (SEQ ID NO: 45), WKNMAVW (SEQ ID NO: 46), WKQMASW (SEQ ID NO: 47), W QMSVW (SEQ ID NO: 48), W EMSVW (SEQ ID NO: 49), WKQMAVW (SEQ ID NO: 50), WKQMATW (SEQ ID NO: 51), AWNKNSMRVWP (SEQ ID NO: 52), and AWNKNSMRVWA (SEQ ID NO. 53).

8. The method of any one of claims 1 to 7, wherein said peptide derived from an allatostatin-b polypeptide comprises a C-terminal tryptophane (W) amino acid residue.

9. The method of any one of claims 1 to 8, wherein the C-terminal amino acid residue of said peptide derived from an allatostatin-b polypeptide is amidated.

10. The method of any one of claims 1 to 9, wherein said allatostain-b polypeptide is shown in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii); or SEQ ID NO: 4 (allatostatin-b polypeptide of Capitella teleta); or SEQ ID NO:6 (allatostatin-b polypeptide of Aplysia californica); or SEQ ID NO:8 (allatostatin-b polypeptide of Crassostrea gigas); or SEQ ID NO: 10 (allatostatin-b polypeptide of Lottia gigantea); or SEQ ID NO: 12 (allatostatin-b polypeptide of Lymnaea stagnalis); or SEQ ID NO: 14 (allatostatin-b polypeptide of Biomphalaria glabrata); or SEQ ID NO: 16 (allatostatin-b polypeptide of Tritonia diomedea); or SEQ ID NO: 18 (allatostatin-b polypeptide of Mytilus californianus); or SEQ ID NO:20 (allatostatin-b polypeptide of Euprymna scolopes); or SEQ ID NO:22 (allatostatin-b polypeptide of Idiosepius paradoxus); or SEQ ID NO:24 (allatostatin-b polypeptide of Doryteuthis pealeii); or an orthologous polypeptide of any of the above allatostain-b polypeptide.

1 1. The method of any one of claims 1 to 10, wherein said composition further comprises water, such as saline water like brack water or seawater.

12. The method of any one of claims 1 to 1 1, wherein said peptide derived from an allatostatin-b polypeptide is present in the composition in a concentration sufficient to induce or enhance the settlement and/or growth of the one or more larva.

13. The method of claim 12, wherein said peptide derived from an allatostatin-b polypeptide is present in the composition in in a concentration of from 5 nM to 100 μΜ.

The method of any one of claims 1 to 13, further comprising growing mature one or more animal stemming from the one or more larva and harvesting the mature one or more animal.

15. The method of any one of claims 1 to 14, wherein said one or more larva or one or more animal stemming therefrom belong to the trochozoa.

16. The method of any one of claims 1 to 15, wherein said one or more larva or one or more animal belong to the annelid phylum, or to the mollusc phylum.

17. The method of claim 16, wherein said annelid belongs to the genus Platynereis or the genus Capitella, such as Platynereis dumerilii or Capitella teleta; or wherein said mollusc belongs to the genus Aplysia, such as Aplysia californica.

18. The method of claim 16, wherein said mollusc belongs to the class bivalvia or the class cephalopoda.

19. The method of claim 18, wherein said bivalve belongs to the genus Pecten, Crassostrea, Ruditapes, Anadara, Pema, Patinopecten, Mytilis or Mercenaria.

20. The method of claim 19, wherein said bivalve is selected from the group consisting of Pecten maximus, Ruditapes philippinarum, Crassostrea gigas, Anadara granosa, Perna viridis, Patinopecten yessoensis, Mytilis edulis, Mytilis galloprovincialis, Perna canaliculus and Mercenaria mercenaria

21. The method of claim 18, wherein said cephalopod belongs to the genus Sepia or Octopus.

22. The method of claim 21, wherein said cephalopod is Sepia officinalis or Octopus vulgaris.

23. A peptide having or consisting of the consensus motif from N to C terminus (X)_nWa(X)_nZW_b(X)_n, whereby X is any amino acid residue, n is an integer of from 0 to 15 and Z is A, V, T, S or N, and whereby W_a may be absent.

24. The peptide of claim 23, wherein said peptide has a length of from 2 to 48 amino acids.

25. The peptide of claim 23 or 24 consisting of a fragment of allatostatin-b.

26. The peptide of any one of claims 23 to 25, wherein said peptide comprises or consists of a peptide selected from the group consisting of AW, VW, TW, SW, and NW.

27. The peptide of any one of claims 23 to 26, wherein said peptide comprises or consists of a peptide selected from the group consisting of

AW K IAW (SEQ ID NO: 38), AWGDNNMRVW (SEQ ID NO: 39), AWNKNSMRVW (SEQ ID NO: 40), AW GQSARVW (SEQ ID NO: 41), GWNGNSMRVW (SEQ ID NO: 42), KWGSNSMRVW (SEQ ID NO: 43), GWADNNMRVW (SEQ ID NO: 44), R WSKFSSW (SEQ ID NO: 45), WKNMAVW (SEQ ID NO: 46), WKQMASW (SEQ ID NO: 47), WKQMSVW (SEQ ID NO: 48), WKEMSVW (SEQ ID NO: 49), W QMAVW (SEQ ID NO: 50), WKQMATW (SEQ ID NO: 51), AWNKN SMRVWP (SEQ ID NO: 52), and AWNKNSMRVW A (SEQ ID NO. 53).

28. The peptide of any one of claims 23 to 27, wherein said peptide comprises a C-terminal tryptophane (W) amino acid residue.

29. The peptide of any one of claims 23 to 28, wherein the C-terminal amino acid residue is amidated.

30. Antibody specifically binding to the peptide of any one of claims 23 to 29.

31. Composition comprising or consisting essentially of one or more peptides according to claims 23 to 29.

32. The composition of claim 31 for use in aquaculture of larvae of Lophotrochozoan marine invertebrates, whereby said one or more peptides comprised in the composition is capable of inducing or enhancing the settlement and/or growth of one or more larva of Lophotrochozoan marine invertebrates.

33. The method of any one of claims 1 to 22, or the composition of claim 31 or 32, wherein said composition optionally further comprises nutrients, such as phytoplankton and/or zooplankton, for feeding one or more larva of Lophotrochozoan marine invertebrates.

34. The method of any one of claims 1 to 22 and 33, or the composition of any one of claims 31 to 33, wherein said composition optionally further comprises one or more biocides (e.g. antifouling agents), disinfectants, pesticides, ingredients for the treatment of soil and/or water (e.g. ingredients for adjusting the pH value), anorganic fertilizers, organic fertilizers, hormones, microbial products, vitamins and/or enzymes.

35. The method of claim 34, or the composition of claim 34, wherein said pesticide is a herbicide, a fungicide or insecticide or piscizide.

36. The method of any one of claims 1 to 22, and 31 to 35, or the composition of any one of claims 31 to 35, wherein said composition comprises, in addition to the peptide, one or more medicaments for treating a disease of the one or more larva of Lophotrochozoan marine invertebrates.

37. The method of claim 36, or the composition of claim 36, wherein said medicament is an antibiotic, a vaccine, a parasitizide, an anaesthetic and/or an immune stimulant.

38. Water as used in aquaculture of one or more larva of Lophotrochozoan marine invertebrates

(i) comprising one or more peptide as defined in any one of claims 23 to 29, wherein the peptide is preferably present in the water in a concentration sufficient to induce or enhance the settlement and/or growth of one ore more larva of Lophotrochozoan marine invertebrates; or

(ii) comprising the composition as defined in any one of claims 31 to 37, wherein the peptide(s) as comprised in the composition are preferably present in the water in a concentration sufficient to induce or enhance the settlement and/or growth of one ore more larva of Lophotrochozoan marine invertebrates, for example in a concentration from 5 nM to 100 μΜ.

39. The water of claim 38,

(i) comprising one or more peptide as defined in any one of claims 23 to 29, wherein the peptide is present in the water in a concentration of from 5 nM to 100 μΜ; or

(ii) comprising the composition as defined in any one of claims 31 to 37, wherein the peptide(s) as comprised in the composition are present in the water in a concentration offrom 5 nM to 100 μΜ.

40. An allatostatin-b polypeptide selected from the group consisting of

(b) a polypeptide having or consisting of an amino acid sequence as depicted in SEQ ID NO:2 (allatostatin-b polypeptide of Platynereis dumerilii); (c) a polypeptide as defined in (a) or (b) wherein one or more amino acids are deleted, inserted, added or substituted, and whereby said polypeptide has allatostatin-b biological activity;

(f) a polypeptide having at least 50 % similarity to the polypeptide of any one of (a) to (e), and whereby said polypeptide has allatostatin-b biological acitvity; and

41. A method for obtaining one or more molluscs for nourishment, comprising the steps

(a) culturing one or more mollusc larva in a composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide as defined in claims 3 to 10;

(b) inducing or enhancing settlement of the one or more larva;

(c) developing mature molluscs;

(d) harvesting mature molluscs.

42. A method for obtaining one or more shellfish for nourishment, comprising the steps

(a) culturing one or more shellfish larva in a composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide as defined in claims 3 to 10;

(b) inducing or enhancing settlement of the one or more larva;

(c) developing mature shellfish;

(d) harvesting mature shellfish.

43. A method for obtaining one or more molluscs for nourishment, comprising the steps

(b) inducing or enhancing growth of the one or more larva;

(c) developing mature molluscs; (d) harvesting mature molluscs.

44. A method for obtaining one or more shellfish for nourishment, comprising the steps

(b) inducing or enhancing growth of the one or more larva;

(c) developing mature shellfish;

(d) harvesting mature shellfish.

45. The method of any one of claims 41 to 44, wherein the one or more mollusc or one or more shellfish belongs to the class bivalvia as defined in any one of claims 18 to 20.

46. The method of claim 43, wherein the one or more mollusc belongs to the class cephalopoda as defined in any one of claims 18, 21 and 22.

47. A method for obtaining one or more annelids for use as bait, comprising the steps

(a) culturing one or more annelid larva in a composition comprising or consisting essentially of one or more peptides derived from an allatostatin-b polypeptide as defined in claims 3 to 10;

(b) inducing or enhancing settlement of the one or more larva;

(c) developing mature annelids;

(d) harvesting mature annelids;

(e) preparing the harvested mature annelids for use as bait.

48. A method for obtaining one or more annelids for use as bait, comprising the steps

(b) inducing or enhancing growth of the one or more larva;

(c) developing mature annelids;

(d) harvesting mature annelids;

(e) preparing the harvested mature annelids for use as bait.

49. The method of claim 47 or 48 for obtaining one or more annelids for use as fish bait.

50. The method of any one of claims 47 to 49, wherein the annelid belongs to the family Nereidae or belongs to the family Arenicolidae.

5 . The method of any any one of claims 47 to 50, wherein the annelid belongs to the genus Nereis, such as Nereis virens, or wherein the annelid belongs to the genus Arenicola, such as Arenicola defodiens or Arenicola marina.

52. The method of any one of claims 47 to 51, wherein the annelid belongs to the genus Platynereis or the genus Capitella, such as Platynereis dumerilii or Capitella teleta;