JP7515909B2 - Method for diagnosing biological rhythms in fish - Google Patents

Description

The present invention relates to a method for non-invasively diagnosing the circadian rhythms of fish by analyzing the mRNA of fish contained in environmental RNA in the water in which the fish are kept.

A method for determining the circadian rhythms of fish is known to involve quantifying mRNA of clock genes in tissues.
Many internal rhythms are generated by fluctuations in clock genes with an approximately 24-hour cycle, and these internal rhythms can be inferred by quantifying mRNA.
However, the most common method for diagnosing the circadian rhythms of fish involves anesthetizing the fish to be diagnosed, euthanizing them, and then measuring the expression levels of genes in the fish's bodies. This method places a heavy burden on the experimenter and is in keeping with animal ethics, so improvements were needed.

Therefore, the present inventors came up with the idea of a method for non-invasively determining the circadian rhythms of fish by measuring the mRNA of the clock gene per2 contained in the breeding water in which the fish are kept by quantitative PCR.
We then measured the amount of mRNA for the clock gene per2 in the breeding water in which the yellowfin grouper was kept every six hours. The mRNA amount in the yellowfin grouper showed expression fluctuations, with more during the day and less at night (Figure 1).
This result is consistent with the expression fluctuations of the per2 gene in each peripheral tissue of the yellowfin grouper, supporting the idea that the environmental RNA in the breeding water in which the yellowfin grouper is kept reflects the yellowfin grouper's internal rhythm.
Therefore, the method for quantifying fish mRNA contained in environmental RNA from the breeding water in which fish are kept can be performed with just the simple procedure of collecting the breeding water in which the fish are kept, and is a non-invasive diagnostic method that does not require contact with the living fish. Compared to conventional methods, this method places less of a burden on the experimenter and is an excellent method that respects animal welfare.

As a method for quantitatively analyzing such environmental RNA, Patent Document 1 discloses an ecosystem survey method for quantitatively evaluating the number of individuals and biomass of species living in an aquatic environment from the environmental RNA contained in the aquatic environment.
Environmental RNA is expected to be transported shorter distances than environmental DNA, making it more likely to accurately detect only those species actually present near the sampling site, making it suitable for ecosystem survey methods.
Furthermore, the invention described in this document also makes it possible to investigate biological species living in aquatic environments without directly capturing the species, enabling non-invasive detection.
The document also discloses that environmental RNA is extracted by obtaining aquatic environment samples by collecting surface or deep water from the sea, rivers, lakes, ponds, swamps, fish farms, aquariums, etc., or by collecting mud from the bottom of these samples, and further discloses a method in which the aquatic environment sample is filtered and then the RNA or DNA contained in the filtrate is extracted using the phenol/chloroform method, the AGPC (acid guanidinium thiocyanate-phenol-chloroform extraction) method, or a commercially available RNA or DNA extraction reagent.

However, the method described in the document, which involves obtaining an aqueous environment sample, filtering the aqueous environment sample, and extracting RNA or DNA contained in the filtrate, is difficult to implement in environments with high rearing densities, such as aquaculture tanks.
In other words, aquaculture tanks generally tend to have high stocking densities in order to prioritize profit efficiency, but as a result, the water contains a lot of impurities such as excrement and leftover food.
When such aquaculture water is used as a sample, the filtration filter easily becomes clogged, making it difficult to extract RNA or DNA contained in the filtrate.

Patent Publication No. 2021-108593

In order to solve the above problems, the present invention aims to provide a method for non-invasively diagnosing the biological rhythms of fish by obtaining a sample (breeding water) that contains as little impurities as possible, such as excrement and residual food, even in environments with high breeding densities, such as aquaculture tanks, and analyzing the mRNA of fish contained in environmental RNA from the breeding water in which fish are raised.

The method for diagnosing biological rhythms in fish according to the present invention comprises the steps of:
The floating mucus of fishes floating in the breeding water,
Collected using polyester felt,
Polyester felt containing floating mucus,
The extraction reagent is stored at the bottom of the sampling tube, which is fitted with a check valve, and the reagent is passed through the check valve.
Remove the breeding water from the polyester felt containing the floating mucus.
This method is characterized by quantitatively analyzing the mRNA contained in the collected floating mucus.

Compared to conventional methods, this method involves simple operations and makes it possible to diagnose the biological rhythms of fish without directly touching the fish.
In addition, the method can be implemented at a lower cost than filtration-based methods, and in particular, when RNA is collected using polyester felt, the RNA yield is greater and the success rate of analysis is higher than when using reins or cotton swabs.

A graph showing the daily expression fluctuation of the per2 gene mRNA of the grouper in the breeding water. A photo of the mucus floating in the breeding water. A photo of polyester felt used as a substitute for a drawing A graph comparing the amount of RNA that can be collected in one trial using the filtration method and the non-filtration method (by filtration material). Photograph of gene expression analysis using RT-PCR of RNA extracted from floating mucus. A photo of the sequence analysis of DNA amplified by RT-PCR. FIG. 1 shows an embodiment of a container for biological rhythm diagnosis with an integrated extraction reagent and an example of its use.

In the method for diagnosing biological rhythms in fish according to the present embodiment, floating mucus (FIG. 2) floating on the water surface in a culture tank in which fish are cultured was collected as an object to be analyzed.
In the present invention, floating mucus is used as a term meaning "mucus that is released from the surface of a fish's body and floats on the water surface."
In addition, in this embodiment, the floating mucus was collected from the water in an aquaculture tank where fish are cultivated, but similarly good results can be obtained if the floating mucus is collected from the water in a breeding tank where fish are simply raised rather than cultivated. Therefore, in this invention, the term "aquaculture tank" is used to include the "breeding tank", and "breeding water" is used to mean the water stored in both tanks.

In this embodiment, polyester felt 1 was used as the felt-like fibrous body for collecting suspended mucus (FIG. 3).
In the present invention, the term "felt-like fibrous body" is used to mean "a mass of entangled fibers."
The felt-like fibrous body is made up of intertwined fibers forming a mass of fibers, which enables the body to reliably trap only the floating mucus from the breeding water.

Any type of fiber can be used as the felt-like fiber as long as it does not dissolve in the extraction reagent. For example, petroleum-based fibers can be suitably used.
In particular, polyester-based resin fibers whose main component is polyester (e.g., polyethylene terephthalate) or polypropylene-based resin fibers whose main component is polypropylene are lightweight and easy to handle, and are suitable for entangling only the floating mucus from the breeding water.
Therefore, in this embodiment, polyester felt, which is a mass of entangled polyester resin fibers, was used as the felt-like fibrous body.

After removing the rearing water from the polyester felt, the polyester felt was placed in a 5 ml tube containing RLT buffer (350 μl, QIAGEN, Netherlands).
The 5 ml tube was mixed by inversion (room temperature, 20 minutes), and then centrifuged (25°C, 8,000 g, 1 minute) to collect the polyester felt and supernatant at the bottom of the container.
Only the supernatant was transferred to a 1.5 ml microtube, and 70% ethanol (400 μl) was added and thoroughly mixed by pipetting.
The obtained sample was transferred to RNeasy Mini Spin Columns (QIAGEN), and environmental RNA was purified according to the manufacturer's protocol and eluted with nuclease-free water (30 μl).

Generally, when using water from aquaculture tanks that contains a lot of excrement and leftover food, the filtration time is long, and it takes a long time to extract mRNA.
However, in this embodiment, by using polyester felt to collect only the floating mucus from the breeding water in the aquaculture tank, there is no need to filter the breeding water, and the sampling time, which previously took approximately 60 minutes including filtration time, can be significantly reduced to approximately 5 minutes.
Another advantage is that it can be carried out at a lower cost than filtration methods.
Furthermore, the method of collecting RNA using polyester felt was found to result in a higher RNA yield (Figure 4) and a higher success rate of analysis than the method using reins and cotton swabs.

Figure 5 is a photograph showing gene expression analysis of RNA extracted from floating mucus by RT-PCR. In the Mucus (floating mucus) sample at the top of the figure, clear expression of the per2 gene (mgPer2) of the yellow spotted grouper was observed.
FIG. 6 is a photograph showing the sequence analysis of the gene sequence of DNA amplified by RT-PCR.
The DNA fragment obtained in this example showed high homology with the genes of the grouper, which belongs to the same genus as the spotted grouper.

In addition, when diagnosing the biological rhythms of fish, a container with an integrated extraction reagent can be used to remove the breeding water from the polyester felt.
In this embodiment, a container for biological rhythm diagnosis was used, which was configured by disposing a check valve at the middle stage of a sampling tube with an extraction reagent stored at the bottom (FIG. 7).
After the felt-like fibrous body is placed in the container, the rearing water contained in the felt-like fibrous body is removed by passing it through a check valve.
Furthermore, the check valve allows only the felt-like fibrous bodies with suspended mucus attached to drop into the extraction reagent stored at the bottom of the container (Figure 7).
If the sampling tube used as the container for biological rhythm diagnosis has a sealable structure, it is possible to prevent contamination with foreign matter and the handling procedure can be very simple.

1 Polyester felt

Claims (3)

The floating mucus of fishes floating in the breeding water,
The fibers are collected by entangling them in a felt-like fibrous mass, which is a mass of entangled fibers.
A method for diagnosing the biological rhythms of fish, characterized by quantitatively analyzing mRNA contained in collected floating mucus. The floating mucus of fishes floating in the breeding water,
The fibers are collected by entangling them in polyester felt, which is a mass of intertwined fibers.
A method for diagnosing the biological rhythms of fish, characterized by quantitatively analyzing mRNA contained in collected floating mucus. The floating mucus of fishes floating in the breeding water,
The fibers are collected by entangling them in polyester felt, which is a mass of intertwined fibers.
The polyester felt with the floating mucus entangled therein is
The extraction reagent is stored at the bottom of the sampling tube, which is fitted with a check valve, and the reagent is passed through the check valve.
The breeding water is removed from the polyester felt in which the floating mucus has been entangled,
A method for diagnosing the biological rhythms of fish, characterized by quantitatively analyzing mRNA contained in collected floating mucus.