AU599569B2 - Methods for blocking blowfly oviposition - Google Patents

Description

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LCt I It I C C C C I I CI Name of Applicants Address of Applican 14 PtrLc-r 'S$r The Patentc Act 1952 r1AP-T, 1 0AMJP*L R(ce F-Q-UE-E-NS--A-N'D-a1ydts 8-t--L-u-c--arQue e-ns-l-and-, -4O0674usra-1-a t tI Actual Inventors JS~ MARTIN J. RICE, STEPHEN SEXTON A.M. ESMAIL Address for Service :-GR-A*N-T-AAMS-&-C)M

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COMPLETE SPECIFICATION FOR THE INVENTION ENTITLED: 4 "METHODS FOR BLOCKING BLOWFLY OVIPOSITION"1 The following statement is a full description of the invention including the best method of~ performing it known to us: -1- 7; IADDRESS FOR SERVICE SC. Adams ALTERED TO: The Commissioner of Patents, Commonwealth of Australia.

THIS INVENTION relates to methods for blocking blowfly oviposition (and blowfly maggot development) and preparation therefor.

Infestation of living animals with fly maggots i.e.

myiasis is the cause of considerable, world wide stock loss and human suffering. Lucilia cuprina (Wied.), the primary Australian blowfly, causes losses of over $110 million annually. At present, the main protective treatments against blowflies are "Diazanon" and "Vetrazin" both are fairly toxic and dangerous to handle and are expensive, and insect resistance to "Diazanon" is being shown in some areas.

The blowfly is attracted to 3heep by bacterial odours from wet dermal lesions, it feeds on serous exudates and then probes the wool with its ovipositor. Gustatory receptors on the tarsi, mouth-parts and the ovipositor monitor the site for oviposition. Such involvement of gustatory receptors suggests that antifeedant chemicals might influence oviposition.

Extracts of neem, Azadirachta indica A. Juss, are antifeedants for many phytophagous insects, insecticidal and growth retarding effects have also been noted. The most active agent is Azadirachtin, a tetranortriterpenoid, concentrated in the leaves and kernels of neem.

Azadirachtin inhibits feeding in insect species from 7 orders and causes developmental abnormalities in 4 orders.

It reduces egg deposition by dried fruit beetles and leaffolder moths, and damages oocytes, reducing egg production A and hatching. Azadirachtin has proven a useful natural pesticide for phytophagous pests.

It is an object of the present invention to provide dose-dependant, blocking effects on sheep blowfly oviposition.

It is a preferred object to provide blocking effects to blowfly maggot development.

2 i i 1 It is a further preferred object to provide such effects using naturally occurring chemical extracts which have a low toxicity to humans and to the animals being treated.

Other preferred objects will become apparent from the following description.

Broadly, the invention is concerned with a method of blocking blowfly oviposition using ne rally occurring chemical extracts from the seeds or kernels of meliaceous plants such as neem, rosewood, white cedar or red cedar, said extracts being crudely expressed or solvent separated.

Such extracts are referred to in the following description and claims as "extracts as hereinbefore described".

Solvent separation may involve extraction in alcohol (e.g.

methanol and/or ethanol) or extraction in other suitable inert carriers.

In one aspect the invention resides in a method of blocking blowfly oviposition including the steps of mixing the extracts as hereinbefore described from the seeds or kernels of meliaceous plants with an inert carrier, and applying the mixture to the fleece, skin or coat of an animal to be treated.

Preferably the invention, in a second aspect, resides in a method of blocking blowfly oviposition including the steps of mixing solvent-separated extracts from the seeds or kernels of neem plants with an inert carrier, and applying the mixture to the fleece, skin or coat of an animal to be treated.

In a third aspect, the present invention further resides in a method for blocking blowfly maggot development using the extracts and steps as aforementioned. As will be understood, the said blocking of development results from one or more of repellent effects, feeding reduction and growth and moulting disruption.

In a further aspect, the invention resides in a 3 -I i i i imethod of protecting animals against cattle ticks, including the steps of mixing the extracts as hereinbefore described from the seeds or kernels of meliaceous plants with an inert carrier, and applying the mixture to the fleece, skin or coat of an animal to be treated.

To enable the invention to be fully understood, a number of preferred embodiments will now be described.

A. Effects of Meliaceous Extracts on Blocking Blowfly Oviposition Two different extracts of neem kernels were tested: crude expressed oil (CN) and a 30% methanolic extract (AZT). L. cuprina from a sheep strike at Julia Creek, Queensland, were reared at 28 2CC for about ten generations. Females from generations 4-10 were mated and made gravid by protein feeding. Groups of 10 or 20 were placed in gauze-covered cages of about 1 x 10-2 m 3 with water and sucrose supplied. Each oviposition test had two semicircular pads of 2 mm Wettex(R), with 10 evenlyspaced 2 mm holes, fitted in the base of an 85mm plastic petri dish. Treatment pads received 2mL of AZT dilutions, the alcohol 3a r I was evaporated, and 10 mL of sheep's plasma added (pH adjusted to 9.5 with KOH). Control pads received no AZT. Other test pads received 0.5 mL of CN dilutions in vegetable oil, or pure CN; control pads received mL vegetable oil. The oil was evenly spread over the surface of pads soaked in sheep's plasma. After exposure to the flies for 24 or 38 h, egg batches on the pads were counted; full batches (50-300 eggs) scored 1, small batches (10-50) scored 0.2, batches less than 10 did not score.

The first experiment compared different doses of AZT in a no choice situation. Four groups of flies were tested: control (no AZT); 20,100 and 600 ppm AZT.

In each case both oviposition pads had the same treatment.

Ao$ The pads were removed after about 24 h and egg batches counted. All four groups were then retested with AZT-free pads, and egg batches counted after about 24 h. In Table fit 1, row A gives the batches laid when the flies were tested t 'first with AZT doses, row B gives the batches laid after- 'f wards when the flies were retested with pads having no AZT.

Control pads received 64% of their batches on day 1, 36% on day 2. In contrast the tr6ated pads showed an AZT-doserelated decrease in batches on day 1, and an increase on day 2. With 600 ppm AZT, only 6% of batches were -p on I S day 1, 94% on day 2. It is considered these results repres ent a dose-related inhibition of oviposition by AZT. That Sis, egg batches are retained by the flies with increasing frequency as they are tested by increasing AZT concentrations. On subsequent exposure to AZT-free pads retained batches are laid, enabling inhibition to be quantif;ed 99 1 30 (row C in Table Inhibition of oviposition is a behavr °i ioural expression of a dose-related change in sensory input causes by the action of AZT on specific sensory receptors (as hereinbefore described).

The first experiment reveals another effect of increasing AZT concentration: a reduction in total batches -4- 'r laid (rows D and E in Table In contrast to inhibition, reduction is high at 20 ppm AZT, rising to only 55% at 600 ppm. It is likely that reduction is based on a different mechanism to inhibition, as discussed below.

In a second experiment a series of choices were offered between a pad treated with an AZT concentration and an AZT-free pad. The blowflies showed preferences when selecting an oviposition site, these were then expressed as the deterrence value of each AZT concentration (column 4 in Table At 200 ppm AZT demonstrated 100% deterrence, all batches were laid on the AZT-free pads.

With decreasing concentration, deterrence decreased, 84% at 100 ppm, 42% at 50 ppm, 0% at 20 ppm. The slight preference for 20 ppm is an artefact of the relatively c low fly numbers. In summary, blowflies show substantial t c oviposition deterrence to 50 ppm AZT, rising to total c- deterrence at 200 ppm. This relationship is similar to that found for inhibition but differs from that for t Ct reduction.

The third experiment was similar to the second except that the choices offered were between two pads ti t having different AZT concentrations. Oviposition was I largely on the lower of the following: 200 verses 100; 100 versus 20; and 50 versus 20 ppm AZT (Table The edges of the oviposition pads were within 3 mm of each otier, the results thus illustrated the blowflies' fine gustatory discrimination between adjacent AZT concentrations. That deterrence was higher between 50 and 20 ppm 30 than between 50 and 0 ppm may be an artefact of low numbers.

A fourth experiment involved testing CN dilutions in inert vegetable oil against controls treated with the oil alone. At 1% (1 x 10 4ppm) CN 21% deterrence was recorded (n 130), at 10% (1 x 105ppm) CN 77% deterrence (n 120), and at 100% CN 94% deterrence (n 170). These

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results suggest that CN is approximately 1 x 10 3 times as effective as AZT.

This series of experiments has shown the anti-ovipositant action of neem extracts on L. cuprina.

At least three effects are involved: reduction, inhibition and deterrence.

It was found that oviposition reduction has the lowest threshold, 20 ppm AZT, substantially reducing total egg batches. This concentration does not substantially evoke inhibition or deterrence, suggesting that reduction is an internal effect, caused by ingested AZT.

It could be an endocrine effect as topically applied and ingested azadirachtin disrupts insect growth by altering hormone balance. That reduction reaches only 55% at 600 ppm AZT is not suprising, as at such high concentrations the antifeedant considerably decreases ingestion.

Oviposition inhibition is an effect due to retention of eggs that would otherwise have been laid, until sensory input confirms that a suitable substrate is available. Inhibition can be considered as temporal choice making. In contrast, oviposition deterrence is spatial choice making, the flies comparing adjacent sites for suitability. Both inhibition and deterrence have similar AZT-doze-responses, probably because they depend upon the same sensory information. Both became significant between 20 and 100 ppm, are high at 100 ppm, and are total or almost so by 200-600 ppm AZT. It appears likely that inhibition and deterrence depends upon the action S, of azadirachtin on the gustatory sensilla of the blowfly.

30 In a locust, acadirachtin is known to stimulate certain deterrent receptor cells of the mouthparts and palps. In larval Lepidoptera, 0.1 M (56 X 10 3 ppm) azadirachtin causes high frequency discharges in deterrent-sensitive neurones of the medial sensillum styloconicum of the maxillary palps.

Preliminary results from the application of neem -6- I- lg1 extract to sheep have shown that it is well retained by the fleece, even under very wet field conditions.

The extracts used were not pure azadirachtin but the results suggest that a properly formulated preparation of pure azadirachtin would also offer a means of protecting sheep from L. cuprina.

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s4 ts t I t -7- .now S C TABLE 1 INHIBITION AND REDUCTION OF BLOWFLY OVIPOSITION BY AZADIRACHITA EXTRACT

CONTROL

0 EXTRACT TREATMENT (ppm) A. EGG BATCHES ON TREATED PADS 59.6 (64%) 37.4 (59%) 25.6 (41%) 13.4 2.6 37.6 39.2(94%) B. SUBSEQUENT BATCHES 34.2 (36%) ON CLEAN PADS C. INHIBITION D. TOTAL A B E. REDUCTION 0 93.8 0 8 63.0 33 59 51.0 46 91 41.8 A. Egg batches from 120 gravid Lucilia cuprina after 24 h with treated pads; 50-300 eggs 1 batch; 10-50 eggs 0.2 batch.

B. Egg batches from same flies re-tested over the following 24 h with untreated pads.

Control A% Treatment A% S. x 100 Control A-- Control D Treatment D Control D -r

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TABLE 2 DETERRENCE OF BLOWFLY OVIPOSITION BY AZADIRACHTA EXTRACT NUMBER TREATMENT EGG BATCHES DETERRENCE OF FLIES CHOICE OFFERED 1 LAID %2.

ppm AZT 9.2 versus CONTROL 8.4 ppm AZT 2.2 versus 42 CONTROL 5.4 ppm AZT 1.2 versus ppm AZT 8.4 100 ppm AZT 1.4 versus 84 CONTROL 16.0 100 ppm AZT 0.4 versus 78 ppm AZT 3.4 200 ppm AZT 0.0 versus +100 CONTROL 12.8 200 ppm AZT 1.4 versus 66 100 ppm AZT Two treated pads in same Choice 2 Choice 1 Choice 1 Choice 2 dish 100 offered for 48 h.

B. Effects of Mleee.eoaesExtracts on the Feeding Behaviour and Development of Larvae of Lucilia cuprina 2 mL of known dilutions of AZT, neem oil, rosewood leaf extract, red and white cedar leaf extracts etc. were applied to Wettex(

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pads (as previously described) and the solvent evaporated. Control pads were prepared by treatment with 2 mL of the solvent alone. Pads were then placed in the bases of a divided plastic petri dish, where 10 mL of fresh, defibrinated sheep's plasma was added to each of the two pads. In "choice" experiments one pad had the meliaceous extract the other solvent-only; the control dishes having two solvent-only pads. In "non-choice" experiments both pads had the same extract treatment, St controls again having two solvent-only pads.

Fifty first instar larvae of Lucilia cuprina (Wied.) were added to the centre of each dish and allowed to disperse over the pads, in a uniformly low illuminance t apparatus. Their distribution over the two pads, was then recorded several times per day, over the next 2-3 days. The dishes were then placed on top of sawdust, the maggots crawling from the pads into the sawdust to pupate. The numbers of pupae and numbers of adult blowflies ecloded were also recorded. In some experiments the weight of the pupae and the sex of the flies were recorded.

This method enables both the effects of meliaceous extracts on larval feeding choice and on larval/pupal development to be quantified. Both are important criteria from the applied, sheep-blowfly control viewpoint: deterrence of larval feeding protects sheep against maggot damage S 30 and blocking of development reduces blowfly numbers and potential for damaging sheep.

SI. CHOICE EXPERIMENT A AZT Cone. on AZT Pupae Ave Wt Blowflies ~D '0 r 000\ \CT-;cA i Cd 7t O control 47 28mg 39 17 22 10 ppm 0 35 29 13 3 ppm 2 38 28 19 8 11 ppm 0 33 27 18 4 14 100 ppm 0 18 26 0 200 ppm 0 10 24 0 600 ppm 0 3 22 0 maggots distributed on pads 45% v AZT dilutions in sheep's plasma proved deterrent to maggot feeding down to 10 ppm.

Even though maggots were given a choice of substrate and largely confined themselves to untreated pads, their wanderings took them across the AZT treated pads.

This was clearly sufficient for them to collect a deletrious dose of AZT, even 10 ppm considerably reducing the number of maggots developing to adult blowflies. Even the slight exposure to 100 ppm AZT was sufficient to totally block development to adulthood. Although the tests show a skew in sex ratio this was not confirmed in a subsequent experiment.

II. CHOICE EXPERIMENT B This was a similar experiment to that described above (Choice Experiment However, it involved replicates of each test, involving 2250 maggots (the previous experiment tested 350 maggots).

-11- ~ccl- i 1L-.rru~~-rw-ur~ -racl--UP-- I rrm~- AZT Conc. on AZT Pupating Ecloding ppm (Adults) 0 control 82 1 48 74 33 92 13 38 22 8 22 2 5 8 0 100 0 66 200 1 34 0 600 0 26 1 *maggots distributed on pads 51% v 49% r 15 The results confirm those found in the previous experiment and extend them to show that even 1 ppm AZT in sheep's plasma, though not deterrent of feeding, causes a 29% reduction in the number of adults ecloded. This is a similar effect to one noted previously in oviposition experiments, the metabolic action of AZT continues to a lower threshold than the behavioural action of the chemicals.

t .At 5 ppm significant behavioural deterrence is shown, with insignificant numbers of maggots on the AZT "t treated pad at 10 ppm and higher. At 20 ppm and higher 25 insignificant numbers of larvae are able to develop through to adulthood.

III, CHOICE EXPERIMENT C This was similar to the two previous experiments (Choice Experiments A and B) and involved 3 replicates each having 33 maggots. A 10% suspension of homogenised calf's liver was mixed into the sheep's plasma, to increase the survival of maggots.

-12- 'X r AZT Cone. on AZT Exloding ppm (Adults) 0 (control) 98 1 49 68 33 73 38 39 21 57 11 2 100 2 11 200 1 13 600 4 1 48% v 52% on the two pads.

In the presence of liver extract the AZT is slightly less effective. Threshold effects for deterrence (1 ppm) and inhibition of development 1 ppm) being the same, however really high deterrance is only found at 20 ppm (rather than 10 ppm) and virtually complete inhibition of development is at 50 ppm rather than 20 ppm (as was found in Choice Experiment It is apparent that improving the nutritional qualities of the maggot diet is able in some way to slightly offset the effectivness of AZT.

IV. NON-CHOICE EXPERIMENT D This was similar to the three previous experiments, B and C) except that both pads in the base of the plastic petri dish received the same extract treatment. The maggots were forced to feed on the concentration of AZT in sheep's plasma offered. 3 replicates using 33 maggots each were used.

AZT Cone. ppm Pupae Flies O (control) 51 38 31 18 9 3 0 0 6% DYsoxylon Fraseranum leaves 6 2 an ethanolic extract of rosewood.

PhLI-3 -13- When maggots are forced (by the non-choice situation) to feed on AZT containing sheep's plasma their development to pupae and to adults is inhibited by lower concentrations than in the choice situations, as would be expected. Even 0.5 ppm AZT more than halves the number of maggots reaching adulthood. 5 ppm AZT is sufficient to prevent any maggots reaching adulthood.

9ks extract of a related meliaceous tree (rosewood Dysoxylon fraseranum) was also shown to be very effective in the non-choice situation in preventing maggots developing to pupae and adult flies. The Dysoxylon extract was very economical to prepare and it may be that this and other meliaceous extracts will prove the materials of choice against sheep blowflies and other insect pests, in some situations.

V. FIELD EXPERIMENT E A methanolic mixture of purified neem oil, plus a spreading agent was applied at the rate of about 1 L per sheep, to 6 numbered, merino ewes with fleece at about half length. Another, similar set of ewes were treated with the same material without neem oil. The 12 sheep were allowed to graze freely in a large grassy paddock at Yeerongpilly D.P.I. Farm, Brisbane, over the period December 12, 1984 to January 16, 1985. During this time they were subject to typically hot, wet windy weather.

Fleece snips (approximately 20 g each) were taken from all 12 sheep and used in non-choice maggot experiments.

The fleece samples (numbered as from their parent sheep) were soaked in sheep's plasma and each had 40 first instar Lucilia cuprina maggots applied.

On the control wool snips there was an average of 53% larval mortality and 5% pupal mortality. Approximately 50% of the larvae reached adulthood. On the treated weathered wool snips there was 94% larval mortality and 100% pupal mortality, with no maggots reaching adulthood.

Neem oil in the formulation used is able to retain its anti-developmental potency against Lucilia cuprina larvae and pupae under rigorous field conditions.

VI. NON-CHOICE EXPERIMENT o A powdered, dewaxed alcoholic extract of neem seeds was tested against first instar larvae (technique as in Experiment D).

Marked inhibition of larval development was shown by 250 ppm and above. Whilst this was not as potent as AZT it is a very economical material to prepare.

VII. SUMMARY AZT and other meliaceous extracts (including rosewood) have powerful inhibitory effects on the feeding behaviour of maggots down to about 1-5 ppm. They have marked effects on the development of larvae to pupae and adults at concentrations of 1 ppm and lower. One formulation of such an extract has been shown to retain its anti-maggot activity under field conditions on sheep fleece.

The extracts from the meliaceous plants provide a two-pronged attack against blowflies. They block oviposition in the fleece and are potent against the immature blowflies. In addition, the extracts are nontoxic to humans or the sheep and do not suffer the handling problems of the chemicals presently in use.

C. Deterrence of Ambush Behaviour of Larvae of the Cattle Tick (Boophilus microplus) by Extracts of Meliaceous Plants The effectiveness of the extracts of the meliaceous plants against blowfly oviposition stimulated the suggestion that the extracts may also be effective against other stock pests and, in particular, the cattle tick Boophilus microplus.

A 30% methanolic, dewaxed, solvent extract of neem seeds (Azadirachta indica A. Juss.), and 30% ethanolic ALn 15 -o extract of rosewood (Dysoxylon fraseranum) and white cedar (Melia azedaracht) were applied to the terminal 10 mm of mm bamboo toothpicks. The toothpicks were pressed into a basal stand of modelling clay, with their treated tips uppermost. Control picks received pure solvent dips and were placed alternately with the extract-treated picks.

The base of the apparatus was surrounded with deionised water containing 0.1% detergent (to prevent the larvae dispersing). 200-300 10 day old B. microplus larvae (obtained from C.S.I.R.O. laboratory, Long Pocket, Brisbane Yeerongpilly strain) were placed on the base. As they could not disperse because of the water moat, they were forced to either climb the toothpicks or remain on the base. Most of the ticks were very active, moving at a r-te of 2-50 mm per minute, exploring the base and the toothpicks. After an hour they began to slow down and to settle at the tops of the toothpicks in typical ambusher fashion. The numbers of larvae resting on the tips of the toothpicks were counted 15-50 times at irregular intervals to 2 hours) during daytime for the following 7 days.

Numbers exceeding 50 were counted as Control X±S.E. 28.1 5.2 n A.Z.T. 30 X+S.E. 3.7 2.3 n Melia 30 X+S.E. 20.5 4.1 n Dysox 30 X±S.E. 4.6 3.8 n (Note: Where denotes "Mean", denotes "Standard Error") Extracts of neem and rosewood show promise as deterrents of the larvae of cattle ticks microplus) whilst white cedar extracts were not so effective. The result demonstrates a use of meliaceous extracts as protectants of stock against B. microplus.

It is believed that the extracts also act on the gustatory receptors of the cattle ticks (as has been shown above on the blowflies) to act as deterrents for tick 16 strike on cattle.

As the extracts can be easily mixed in a suitable inert carrier, the extracts can be applied to the sheep fleece or to the cattle skin by either spraying or pouring the mixture onto the animals to be treated.

The various examples described have been given by way of illustrative examples only and various changes and modifications may be made thereto without departing from the scope of the present invention defined in the appended claims.

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Claims (5)

1. 2. A method of blocking blowfly oviposition including the steps of mixing solvent-separated extracts from the seeds or kernels of neem plants with an inert carrier, and applying the mixture to the fleece, skin or coat of an animal to be treated.
2. 3. A method as claimed in Claim 1 or Claim 2 wherein: the extracts are extracted in a solvent comprising an ancohol carrier.
3. 4. A method as claimed in Claim 3 wherein: the alcohol is methanol and/or ethanol. A method as claimed in any one of Claims 1 to 4 wherein: the mixture blocks blowfly maggot development on the treated animal.
4. 6. A method as claimed in any one of Claims 1 to 4 wherein: the mixture acts to protect the treated animals against cattle ticks and the like stock pests.
5. 7. A method of protecting animals against cattle ticks, including the steps of mixing the extracts as hereinbefore described from the seeds or kernels of meliaceous plants with an inert carrier, and applying the mixture to the fleece, skin or coat of an animal to be treated, DATED this twenty-sixth day of February, 1990. MARTIN JAMES RICE By his Patent Attorneys PETER MAXWELL ASSOCIATES ci 0 18