Black soldier fly, method for breeding black soldier flies, and method for rearing black soldier flies.

Abstract

The present invention aims to provide a breedable black soldier fly that is low in fat and has an increased ratio of defatted meal weight to the total weight of fat and defatted meal (protein, etc.). The present invention also aims to provide a method for producing a breedable black soldier fly that is low in fat and has an increased ratio of defatted meal weight to the total weight of fat and defatted meal (protein, etc.), and a method for breeding said black soldier fly. [Solution] A black soldier fly in which the expression level of the acetyl-CoA carboxylase gene is reduced compared to the wild type, and / or the function of the expression product of the above gene is reduced compared to the wild type.

Description

Technical Field

[0001] The present invention relates to American Mizua, a method for producing American Mizua, and a method for raising American Mizua.

Background Art

[0002] American Mizua is expected as a next-generation protein resource in the aquaculture industry, and its development and utilization are underway. On the other hand, since fish oil necessary for the growth of fish needs to be added to fish feed, when American Mizua is mixed into fish feed, the fat stored in American Mizua becomes redundant. Therefore, when American Mizua is mixed into fish feed, a processing operation for separating the protein and fat of American Mizua is required.

[0003] The acetyl-CoA carboxylase (ACC) gene is a gene that functions in the fatty acid synthesis pathway. For example, Non-Patent Document 1 describes that when spirotetramat, a function inhibitor of ACC, is administered to Drosophila suzukii (a species closely related to Drosophila) adults, the eggs laid by females are likely to dry out and have a lethal effect on larvae. Non-Patent Document 2 describes that cyclic ketoenol-based insecticides inhibit the function of ACC derived from insects. Furthermore, Non-Patent Document 3 describes that in Drosophila melanogaster, the homozygous mutant of ACC is recessive lethal and the heterozygous mutant of ACC is viable.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

[0005] As mentioned above, when using black soldier flies as feed for fish farming, processing is required to separate the defatted meal (containing crude protein, crude fiber, and ash, etc.) from the fat (crude fat), which is time-consuming. Therefore, there is a need to create black soldier flies that are low in fat and easy to process, but currently no useful strains of black soldier flies with such properties exist.

[0006] Therefore, the present invention aims to provide a breedable black soldier fly that is low in fat and has an increased ratio of defatted meal weight (protein, etc.) to the total of fat weight and defatted meal weight. The present invention also aims to provide a method for producing a breedable black soldier fly that is low in fat and has an increased ratio of defatted meal weight (protein, etc.) to the total of fat weight and defatted meal weight, and a method for breeding said black soldier fly. [Means for solving the problem]

[0007] The inventors of the present invention have found that by reducing the expression level of the ACC gene or the function of the ACC protein in the larvae of the black soldier fly (USA) through RNAi or by feeding them spirotetramat, an inhibitor of ACC function, the larvae become low in fat, the ratio of the weight of defatted meal (protein, etc.) to the total weight of fat and defatted meal increases, and they can be reared, thus completing the present invention.

[0008] In other words, the present invention relates to, for example, the following inventions. [1] Black soldier fly (Erythrina argiolus) in which the expression level of the acetyl-CoA carboxylase gene is reduced compared to the wild type, and / or the function of the expression product of the above gene is reduced compared to the wild type. [2] Feed containing the black soldier fly described in [1]. [3] A method for producing black soldier flies, comprising the steps of reducing the expression level of the acetyl-CoA carboxylase gene compared to the wild type, and / or reducing the function of the expression product of the above gene compared to the wild type. [4] The method according to [3], comprising the step of reducing the expression level of the acetyl-CoA carboxylase gene compared to the wild type, wherein the step is performed by administering an RNAi molecule to the gene. [5] The method according to [3] or [4], comprising the step of reducing the function of the expression product of the acetyl-CoA carboxylase gene compared to the wild type, wherein the step is performed by administering a functional inhibitor of the expression product of the gene. [6] The method according to [5], wherein the functional inhibitor is spirotetramat. [7] [1] The method for rearing black soldier flies described above, A method comprising feeding a diet containing fat recovered from black soldier flies. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a breedable black soldier fly that is low in fat and has an increased ratio of defatted meal weight (protein, etc.) to the total of fat weight and defatted meal weight. According to the present invention, it is also possible to provide a method for producing a breedable black soldier fly that is low in fat and has an increased ratio of defatted meal weight (protein, etc.) to the total of fat weight and defatted meal weight, and a method for breeding said black soldier fly. [Brief explanation of the drawing]

[0010] [Figure 1] This figure shows the results of evaluating the expression level of the acetyl-CoA carboxylase (ACC) gene in black soldier fly larvae that were not injected with dsRNA targeting the ACC gene (control group) and black soldier fly larvae that were injected with dsRNA targeting the ACC gene to induce ACC RNAi (ACC RNAi). [Figure 2] These are photographs of black soldier fly larvae that were not injected with dsRNA for the ACC gene (control group), and black soldier fly larvae that were injected with dsRNA for the ACC gene to induce ACC RNAi (ACC RNAi). [Figure 3] This figure shows the results of evaluating the body weight of black soldier fly larvae that were not injected with dsRNA for the ACC gene (control group) and black soldier fly larvae that were injected with dsRNA for the ACC gene to induce ACC RNAi (ACC RNAi). [Figure 4] This figure shows the results of evaluating the fat mass of black soldier fly larvae that were not injected with dsRNA for the ACC gene (control group) and black soldier fly larvae that were injected with dsRNA for the ACC gene to induce ACC RNAi (ACC RNAi). [Figure 5]This is a figure showing the results of evaluating the weight of defatted meal (protein, etc.) in larvae of the American whitefly that did not receive injection of dsRNA against the ACC gene (control group), and in larvae of the American whitefly in which dsRNA against the ACC gene was injected to induce ACC RNAi (ACC RNAi). [Figure 6] This is a figure showing the results of evaluating the ratio of each weight to the total weight of fat weight and defatted meal (protein, etc.) weight in larvae of the American whitefly that did not receive injection of dsRNA against the ACC gene (control group), and in larvae of the American whitefly in which dsRNA against the ACC gene was injected to induce ACC RNAi (ACC RNAi). [Figure 7] This is a photograph of larvae of the American whitefly that did not ingest spirotetramat (control group), and larvae of the American whitefly that ingested spirotetramat (SPT). [Figure 8] This is a figure showing the results of evaluating the body weights of larvae of the American whitefly that did not ingest spirotetramat (control group), and larvae of the American whitefly that ingested spirotetramat (SPT). [Figure 9] This is a figure showing the results of evaluating the fat weights in larvae of the American whitefly that did not ingest spirotetramat (control group), and larvae of the American whitefly that ingested spirotetramat (SPT). [Figure 10] This is a figure showing the results of evaluating the weights of defatted meal (protein, etc.) in larvae of the American whitefly that did not ingest spirotetramat (control group), and larvae of the American whitefly that ingested spirotetramat (SPT). [Figure 11] This is a figure showing the results of evaluating the ratio of each weight to the total weight of fat weight and defatted meal (protein, etc.) weight in larvae of the American whitefly that did not ingest spirotetramat (control group), and larvae of the American whitefly that ingested spirotetramat (SPT). [Figure 12]This is a graph showing the results of evaluating the average weight of larvae of the American midge (SPT) that fed on artificial feed 1 mixed only with spirotetramat, and larvae of the American midge (SPT + fat) that fed on artificial feed 2 mixed with spirotetramat and fat recovered by squeezing wild-type American midges. [Figure 13] This is a graph showing the results of predicting the domain structure of the ACC protein of the American midge by comparing the amino acid sequences of the ACC proteins of Drosophila melanogaster and Trichoplusia ni with the amino acid sequence of the ACC protein of the American midge.

Mode for Carrying Out the Invention

[0011] Hereinafter, the mode for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[0012] 〔American midge〕 The American midge according to this embodiment has a reduced expression level of the acetyl-CoA carboxylase (ACC) gene compared to the wild type, and / or the function of the expression product of the above gene is reduced compared to the wild type. Therefore, the American midge according to this embodiment has a lower fat content compared to the wild-type American midge, and the ratio of the weight of defatted meal (protein, etc.) to the total weight of fat weight and defatted meal weight has increased, and it can also be reared.

[0013] Here, in this specification, the "wild-type" American midge means an American midge that has a wild-type ACC gene and has not been exposed to substances that can affect the expression level of the gene and the function of the expression product of the gene.

[0014] The black soldier fly (scientific name: Hermetia illucens) is an insect belonging to the family Fabidae in the order Diptera. The morphology of the black soldier fly in this embodiment may be larva, prepupa, pupa, or adult, but from the viewpoint of more significantly exhibiting the effects of the present invention, the larval stage is preferred.

[0015] In this specification, "gene" means a DNA region that includes a region (transcription region) that is transcribed into an RNA molecule (e.g., mRNA) in a cell. That is, an ACC gene may include not only the transcription region but also regulatory regions located upstream or downstream of the transcription region, as well as untranslated regions (5'UTR, 3'UTR), or it may consist only of the transcription region. The transcription region may include not only exons but also introns, and may be so-called cDNA.

[0016] The acetyl-CoA carboxylase (ACC) gene is a biotin-dependent enzyme that catalyzes the carboxylation of acetyl-CoA to produce malonyl-CoA.

[0017] As mentioned above, it is known that inhibiting the function of the ACC protein or homozygous loss-of-function mutants of the ACC gene are lethal in insects. Furthermore, while heterozygous mutants of the ACC gene are known to be rearable in insects, it is not known that these insects are actually low in fat and that the ratio of defatted meal (protein, etc.) to the total of fat weight and defatted meal weight increases. Therefore, the unexpected result is that reducing the expression level of the ACC gene in the black soldier fly compared to the wild type, or reducing the function of the ACC protein compared to the wild type, results in insects that are not lethal, are rearable (survivable), and are actually low in fat with an increased ratio of defatted meal (protein, etc.) to the total of fat weight and defatted meal weight.

[0018] The ACC gene includes, for example, a nucleotide sequence containing the nucleotide sequence shown in SEQ ID NO: 2, a nucleotide sequence that transcribes mRNA consisting of the nucleotide sequence shown in SEQ ID NO: 34, a nucleotide sequence that expresses a protein consisting of the nucleotide sequence shown in SEQ ID NO: 4, or an amino acid sequence shown in SEQ ID NO: 5. The nucleotide sequence shown in SEQ ID NO: 2 is the nucleotide sequence on the genome of the ACC gene of the black soldier fly; the nucleotide sequence shown in SEQ ID NO: 3 is the nucleotide sequence of the mRNA of the ACC gene of the black soldier fly; the nucleotide sequence shown in SEQ ID NO: 4 is the nucleotide sequence of the CDS of the ACC gene of the black soldier fly; and the amino acid sequence shown in SEQ ID NO: 5 is the amino acid sequence of the ACC protein of the black soldier fly.

[0019] The regulatory region may be, for example, a transcriptional regulatory region or a translational regulatory region. Examples of transcriptional regulatory regions include promoters, enhancers, and silencers. Examples of translational regulatory regions include ribosome-binding regions.

[0020] The black soldier fly according to this embodiment does not have a mutation that reduces the expression level of the ACC gene compared to the wild type, and / or reduces the function of the expression product of the above gene compared to the wild type (hereinafter also referred to as "ACC gene mutation"), but it may have such a mutation.

[0021] Reducing the expression level of the ACC gene compared to the wild type may mean reducing the amount of ACC gene mRNA compared to the wild type, or it may mean reducing the amount of the expression product (protein) encoded by the ACC gene. In other words, reducing the expression level of the ACC gene compared to the wild type means reducing the expression level of the protein encoded by the ACC gene.

[0022] If the black soldier fly according to this embodiment does not have an ACC gene mutation, the expression level of the ACC gene can be reduced by, for example, introducing antisense RNA into the insect or using RNA interference (RNAi), although this is not particularly limited.

[0023] The RNAi method can be carried out, for example, by introducing an RNAi molecule targeting the ACC gene into a black soldier fly. Examples of RNAi molecules targeting the ACC gene include nucleic acids such as double-stranded (ds)RNA, siRNA, shRNA, and microRNA (miRNA) that correspond to or are part of the nucleotide sequence of the ACC gene. The RNAi molecule may be the nucleic acid itself or a vector that expresses the nucleic acid. Therefore, the black soldier fly according to this embodiment may have an RNAi molecule targeting the ACC gene introduced into it. Specific examples of RNAi molecules targeting the ACC gene are not particularly limited, but may include, for example, a dsRNA containing the nucleotide sequence shown in SEQ ID NO: 9, or a dsRNA consisting of the nucleotide sequence shown in SEQ ID NO: 9.

[0024] Antisense RNA or RNAi molecules as described above can be obtained by known chemical synthesis methods, in vitro enzyme transcription, or other methods.

[0025] If the black soldier fly according to this embodiment does not have an ACC gene mutation, the function of the ACC gene expression product can be reduced by, for example, a method using a functional inhibitor of the ACC gene expression product. More specifically, this can be done by feeding the black soldier fly with the functional inhibitor included in its feed.

[0026] Examples of inhibitors of ACC gene expression products include cyclic ketoenol drugs such as spirotetramat, spirodiclofen, and spiromesifen.

[0027] If the black soldier fly according to this embodiment has an ACC gene mutation, the method for introducing the mutation is not particularly limited, but for example, known gene editing techniques can be used. The gene editing techniques are not particularly limited, but examples include methods using zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR / Cas9.

[0028] An ACC gene mutation only needs to be introduced into the ACC gene, and may be introduced into the transcription region, regulatory region, non-coding region, etc. of the ACC gene. Furthermore, an ACC gene mutation may be a substitution, deletion, insertion, and / or addition of 1 or more bases, 2 or more bases, 3 or more bases, 5 or more bases, 10 or more bases, 20 or more bases, 30 or more bases, 40 or more bases, 50 or more bases, 100 or more bases, 200 or more bases, 400 or more bases, 600 or more bases, 800 or more bases, 1000 or more bases, 2000 or more bases, 3000 or more bases, 4000 or more bases, 5000 or more bases, 6000 or more bases, 7000 or more bases, or 8000 or more bases in the base sequence of the ACC gene. ACC gene mutations may be substitutions, deletions, insertions, and / or additions of 8200 bases or less, 8000 bases or less, 7000 bases or less, 6000 bases or less, 5000 bases or less, 4000 bases or less, 3000 bases or less, 2000 bases or less, 1000 bases or less, 800 bases or less, 600 bases or less, 400 bases or less, 200 bases or less, 100 bases or less, 80 bases or less, 60 bases or less, 50 bases or less, or 40 bases or less in the nucleic acid sequence of the ACC gene. ACC gene mutations may also be deletions of the entire length of the ACC gene.

[0029] Since the domain structures of the ACC protein in Drosophila melanogaster and Trichoplusia ni have been elucidated, the inventors compared their amino acid sequences with those of the ACC protein in the black soldier fly to predict the domain structure of the ACC protein in the black soldier fly. The results are shown in Figure 13.

[0030] The BT domain of the ACC protein is a crucial region for the interaction between the biotin carboxylase domain (BC domain) and the carboxytransferase domain (CT domain) of the ACC protein. The BC domain uses ATP to convert carbonate ions (HCO3) into carbonate ions. - The BC domain catalyzes the activation of the BC domain and its binding to biotin. This reaction leads to the formation of carboxybiotin. The CT domain also catalyzes the transfer of a carboxyl group from carboxybiotin to acetyl-CoA, producing malonyl-CoA. The interaction between the BC domain and the CT domain is crucial for the ACC protein to function properly.

[0031] ACC gene mutations may be introduced into the BC domain region of the ACC protein, the CT domain region of the ACC protein, and / or the BT domain region of the ACC protein.

[0032] As shown in Figure 13, the BC domain region of the ACC protein corresponds to amino acid residues 149 to 654 of the amino acid sequence shown in SEQ ID NO: 5. As shown in Figure 13, the CT domain region of the ACC protein corresponds to amino acid residues 1683 to 2227 of the amino acid sequence shown in SEQ ID NO: 5. As shown in Figure 13, the BT domain region of the ACC protein can be predicted to correspond to amino acid residues 654 to 781 of the amino acid sequence shown in SEQ ID NO: 5.

[0033] The ACC gene sequence having an ACC gene mutation may include, for example, a nucleotide sequence containing a nucleotide sequence having 99% or less sequence identity with the nucleotide sequence shown in SEQ ID NO: 2, a nucleotide sequence that transcribes mRNA consisting of a nucleotide sequence having 99% or less sequence identity with the nucleotide sequence shown in SEQ ID NO: 3, a nucleotide sequence that transcribes mRNA consisting of a nucleotide sequence having 99% or less sequence identity with the nucleotide sequence shown in SEQ ID NO: 4, a nucleotide sequence having 99% or less sequence identity with the nucleotide sequence shown in SEQ ID NO: 4, or a nucleotide sequence that expresses a protein consisting of an amino acid sequence having 99% or less sequence identity with the amino acid sequence shown in SEQ ID NO: 5. The above sequence identity may be, for example, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less. Alternatively, the above sequence identity may be, for example, 1% or more, 5% or more, or 10% or more.

[0034] In this specification, sequence identity refers to the percentage (%) of matching bases or residues when the base sequences or amino acid sequences being compared are multiple-aligned. Multiple alignment refers to aligning base sequences or amino acid sequences by inserting appropriate gaps so that corresponding base sequences or amino acid sequence portions are aligned, in order to make them comparable to each other. For multiple alignment, known multiple alignment programs can be used. For example, Clustal W, Clustal X, and the BLAST program can be suitably used.

[0035] The expression level of the ACC gene in the black soldier fly according to this embodiment can be analyzed by, for example, RT-PCR, RNA-seq, or Western blotting. The expression level of the ACC gene in the black soldier fly according to this embodiment only needs to be reduced compared to the wild type, but it may be a reduction of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, and may also be a reduction of less than 100%, 98% or less, 95% or less, 90% or less, 80% or less, 70% or less, or 60% or less.

[0036] The function of the ACC gene expression product in the black soldier fly according to this embodiment can be analyzed, for example, by preparing a sample containing ACC protein from the black soldier fly according to this embodiment and the wild-type black soldier fly, using acetyl-CoA as a substrate with the sample, and comparing the amount of malonyl-CoA produced.

[0037] More specifically, samples containing ACC protein, acetyl-CoA, and 14 Mix with 1C-labeled sodium bicarbonate (NaHCO3) and react them together. 14 The function of the expression product of the ACC gene can be analyzed by measuring the amount of 13C-labeled malonyl-CoA produced. For example, this can be carried out by referring to Japanese Patent Publication No. 2011-521940.

[0038] The function of the ACC gene expression product in the black soldier fly according to this embodiment only needs to be reduced compared to the wild type, but may be reduced by, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, or it may be reduced by less than 100%, 98% or less, 95% or less, 90% or less, 80% or less, 70% or less, or 60% or less.

[0039] The fat weight of the black soldier fly according to this embodiment only needs to be reduced compared to the wild type. For example, it is sufficient if the reduction is more than 0% per unit of dry body weight, and it may be a reduction of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more, or a reduction of 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, or 50% or less.

[0040] The fat weight of the black soldier fly according to this embodiment may be, for example, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 18% or less, 15% or less, 13% or less, or 10% or less per dry body weight.

[0041] The weight of defatted meal (protein, etc.) of the black soldier fly according to this embodiment should be increased compared to the wild type, but for example, an increase of more than 0% per unit of dry body weight is sufficient, and the increase may be 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more, and may also be 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, or 50% or less.

[0042] The weight of the defatted meal (protein, etc.) of the black soldier fly according to this embodiment may be, for example, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 82% or more, 85% or more, 87% or more, or 90% or more per dry body weight.

[0043] The fat weight (percentage of fat weight) relative to the total of the fat weight and defatted meal (protein, etc.) weight of the black soldier fly according to this embodiment may be, for example, 20% or less, 15% or less, 10% or less, or 5% or less, or it may be 1% or more, 2% or more, or 3% or more.

[0044] In this embodiment, the weight of defatted meal (the ratio of the weight of defatted meal) to the total weight of fat and defatted meal (protein, etc.) of the black soldier fly may be, for example, 80% or more, 85% or more, 90% or more, or 95% or more, or 99% or less, 98% or less, or 97% or less.

[0045] The fat weight and defatted meal (protein, etc.) weight per black soldier fly according to this embodiment can be analyzed, for example, by the same method as described in the examples below. More specifically, they can be analyzed by the following method.

[0046] Black soldier flies are frozen at -20°C, then dried in a freeze-dryer for 24 hours. Each dried black soldier fly is then pulverized to obtain a powder (meal). The black soldier fly meal is defatted using hexane at 25°C in a ratio of 100 mg:1 mL. The weight of the recovered fat and defatted meal is measured, and the fat weight (mg / fly) and defatted meal weight (mg / fly) per black soldier fly are determined. The main components of dried black soldier flies are lipids, proteins, crude fiber, and ash. Defatted meal is obtained by defatting and pulverizing dried larvae, and contains crude protein, crude fiber, and ash. In defatted meal, the mass of fat relative to the total mass of defatted meal may be 25% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, or 5% by mass or less, or 1% by mass or more.

[0047] The black soldier fly according to this embodiment is low in fat, and the ratio of the weight of defatted meal (protein, etc.) to the total weight of fat and defatted meal is increased. The feed according to this embodiment contains the black soldier fly according to this embodiment. The form of the black soldier fly in the feed according to this embodiment is not particularly limited and may be as is, or dried and powdered, etc.

[0048] The content of black soldier flies in the feed according to this embodiment may be set appropriately depending on the organism being fed. For example, it may be 0.1% or more, 1% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, or 60% or more on a dry weight basis relative to the total mass of the feed, or it may be 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, or 50% or less.

[0049] The feed according to this embodiment may contain components suitable for organisms to be fed, in addition to the black soldier fly according to this embodiment. Such components may include, for example, lipids, proteins, carbohydrates, vitamins and minerals, and more specifically, soybeans, wheat flour, corn, wheat germ, seaweed powder, concentrated alfalfa, fish meal, fish oil, spirulina, chitosan, kale, nori, carotenoids, garlic, vitamins (choline chloride, vitamin E, vitamin C, inositol, vitamin B5, vitamin B2, vitamin A, vitamin B1, vitamin B6, vitamin B3, folic acid, vitamin D3, biotin), and minerals (Ca, Fe, Mg, Zn, Mn, Cu, I).

[0050] [Method for breeding the black soldier fly] The method for producing black soldier flies according to this embodiment includes a step of reducing the expression level of the ACC gene compared to the wild type, and / or reducing the function of the gene expression product compared to the wild type (reduction step). The method for producing black soldier flies according to this embodiment preferably includes a step of reducing the expression level of the ACC gene compared to the wild type.

[0051] The production method according to this embodiment, by including the above steps, makes it possible to produce black soldier flies that are lower in fat and have a higher ratio of defatted meal (protein, etc.) weight to the total weight of fat and defatted meal compared to wild-type black soldier flies, and that can also be reared.

[0052] In the production method according to this embodiment, the reduction step may be performed by introducing a mutation into the ACC gene, or it may not be performed by introducing a mutation into the ACC gene.

[0053] If the reduction step described above is not performed by introducing a mutation into the ACC gene, the step of reducing the expression level of the ACC gene compared to the wild type may be performed, for example, by administering antisense RNA to black soldier flies, or by RNA interference (RNAi). Preferably, it is performed by RNA interference (RNAi). The reduction step described above may be performed by administering an RNAi molecule targeting the ACC gene to black soldier flies as described above.

[0054] The form of the black soldier fly into which the above-mentioned antisense RNA or RNAi molecule is introduced may be an adult, pupa, prepupa, larva, egg, or embryo, but it is preferable to introduce it into the larva.

[0055] If the reduction process described above is not performed by introducing a mutation into the ACC gene, the process of reducing the function of the ACC gene expression product compared to the wild type may be performed, for example, by administering an inhibitor of the function of the ACC gene expression product as described above.

[0056] When the above process is carried out by introducing a mutation into the ACC gene, it may be carried out by, for example, a known gene editing technology. The gene editing technology is not particularly limited, but examples include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR / Cas9, with CRISPR / Cas9 being preferred. The mutation to be introduced into the ACC gene is as described above.

[0057] The method for producing the product according to this embodiment may further include a step of rearing black soldier flies in which the expression level of the ACC gene is reduced compared to the wild type, and / or the function of the expression product is reduced compared to the wild type (hereinafter also referred to as the "rearing step").

[0058] The production method according to this embodiment, by including a rearing step, can more effectively reduce the expression level of the ACC gene and / or the function of the gene expression product. Furthermore, the rearing step allows for the production of low-fat black soldier flies, and since processing work to separate fat from defatted meal (protein, etc.) is unnecessary, manufacturing costs can also be reduced. The black soldier flies according to this embodiment can be used as feed. Therefore, in one embodiment, a method for producing feed containing black soldier flies is also provided.

[0059] The conditions in the rearing process can be set appropriately depending on the species or morphology of the black soldier fly, but the temperature may be, for example, 25°C to 30°C. The relative humidity (RH) may be, for example, 60% to 80%. The photoperiod is not particularly limited, but for example, it may be a photoperiod of 16 hours of light and 8 hours of dark.

[0060] The method for producing the product according to this embodiment may further include a step of mating black soldier flies in which the expression level of the ACC gene is reduced compared to the wild type, and / or the function of the expression product of the above gene is reduced compared to the wild type (hereinafter also referred to as the "mating step").

[0061] By including a crossbreeding step in the production method according to this embodiment, it is also possible to change the degree of reduction in the expression level of the ACC gene and / or the degree of reduction in the function of the gene expression product compared to the wild type.

[0062] The mating process may involve mating black soldier flies in which the expression level of the ACC gene has been reduced compared to the wild type, and / or the function of the expression product of the above gene has been reduced compared to the wild type, or mating black soldier flies in which the expression level of the ACC gene has been reduced compared to the wild type, and / or the function of the expression product of the above gene has been reduced compared to the wild type, with black soldier flies that do not have these characteristics.

[0063] The mating process is not particularly limited when a mutation is introduced into the ACC gene in the production method according to this embodiment. It may involve mating the black soldier flies to become homozygous for the mutation in the ACC gene, or to become heterozygous.

[0064] To produce heterozygotes, crosses may be made with individuals that do not have mutations in the ACC gene.

[0065] [How to raise black soldier flies] The method for rearing black soldier flies according to this embodiment includes feeding them a feed containing fat recovered from black soldier flies.

[0066] The fat recovered from the black soldier fly refers to the fat extracted from the body of the black soldier fly, and can also be described as black soldier fly fat extract.

[0067] Methods for recovering fat from black soldier flies include, for example, physical and chemical methods. Physical methods include, for example, pressing and centrifugation. Chemical methods include, for example, extraction using organic solvents. Examples of organic solvents include hexane, acetone, methanol, and chloroform.

[0068] A preferred method for recovering fat from black soldier flies used as feed is a physical method, and more preferably a pressing method.

[0069] The amount of fat recovered from black soldier flies in a feed containing fat recovered from black soldier flies may be, for example, 1% or more by mass, 2% or more by mass, 3% or more by mass, 4% or more by mass, 5% or more by mass, 6% or more by mass, 7% or more by mass, 8% or more by mass, 9% or more by mass, or 10% or more by mass relative to the total mass of the feed, or it may be 50% or less by mass, 40% or less by mass, 30% or less by mass, or 20% or less by mass.

[0070] Other rearing conditions for the black soldier fly according to this embodiment may be as described in the rearing process above. [Examples]

[0071] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0072] [Example 1: RNAi experiment on the ACC gene in black soldier fly] <1. Rearing of the Black Soldier Fly> In 2013, black soldier fly larvae were collected in Tsukuba City, Ibaraki Prefecture, and colonies were reared at the National Agriculture and Food Research Organization (NARO) according to the following procedure. Immediately after hatching, the larvae were fed artificial feed (containing glucose, molasses, yeast, and cornmeal) (prepared by NARO based on Chia-Ming Liu et al., Journal of Insects as Food and Feed, 2024) and reared in plastic containers until they reached the prepupal stage when their body color turned black. Next, the prepupa were removed from the plastic containers and kept in another plastic container along with coffee grounds for pupation. Newly emerged adults were moved to a mesh cage for mating and egg-laying. The above rearing was carried out under conditions of 27°C, 60% RH, and a photoperiod of 16 hours of light and 8 hours of dark.

[0073] <2. Synthesis of double-stranded RNA for RNA interference> (Searching for ACC in the black soldier fly) The double-stranded RNA (dsRNA) of the ACC gene of the American midge was prepared as follows. The ACC (DmACC) gene of Drosophila melanogaster registered in Flybase (https: / / flybase.org / ) was searched, and the amino acid sequence of DmACC (FlyBase: CG11198, SEQ ID NO: 1) was obtained. In NCBI BLAST, based on the amino acid sequence of DmACC, the ACC (HiACC) of the American midge was searched against the genomic sequence of the American midge, iHerIll2.2.curated.20191125. As a result, the nucleotide sequence (SEQ ID NO: 2), mRNA nucleotide sequence (SEQ ID NO: 3), and cDNA nucleotide sequence (SEQ ID NO: 4) of the HiACC gene were obtained.

[0074] <Synthesis of double-stranded RNA for RNA interference> The double-stranded RNA (dsRNA) of the ACC gene of the American midge was prepared as follows. Based on the nucleotide sequence of the mRNA of the ACC gene of the American midge (SEQ ID NO: 3), the primers Fw_T7_HiACC (SEQ ID NO: 6) and Rv_T7_HiACC (SEQ ID NO: 7) shown in Table 1 were designed. Also, total RNA was extracted from the larvae of the American midge using the RNeasy (registered trademark) reagent (manufactured by Qiagen). Next, using the total RNA, PrimeScript (registered trademark) II 1st strand cDNA Synthesis Kit reagent (manufactured by Takara Bio) and oligo dT primers, cDNA serving as a template was synthesized. Using the synthesized cDNA as a template, TaKaRa Ex Taq Hot Start Version (manufactured by Takara Bio), and the primers Fw_T7_HiACC (SEQ ID NO: 6) and Rv_T7_HiACC (SEQ ID NO: 7) shown in Table 1, DNA consisting of the nucleotide sequence shown in SEQ ID NO: 8 was amplified by PCR. The amplified DNA was confirmed, and using the DNA and the T7 RiboMAX (trademark) Express RNAi system (manufactured by Promega), dsRNA containing the nucleotide sequence corresponding to SEQ ID NO: 9 was synthesized to obtain a dsRNA solution of the ACC gene. The dsRNA solution was stored at -20°C.

[0075]

Table 1

[0076] <Introduction of double-stranded RNA for RNA interference into larvae> The 7-day-old larvae of the American burying beetle were rinsed with distilled water, and the body surface was gently wiped to remove water. The dsRNA solution of the ACC gene obtained in 2. above was adjusted to a concentration of 1 μg / μl with a PBS solution, and 1 μl was injected with a syringe (needle volume: 10 μl, needle length: 50 mm). The control group was injected with the same amount of PBS solution.

[0077] <3. Evaluation of reduction in ACC expression level> The evaluation of the expression level of the ACC gene was performed by quantitative real-time PCR using total RNA extracted from one larva (N = 6) 14 days after injection and primers of Fw_HiACC (SEQ ID NO: 10) and Rv_HiACC (SEQ ID NO: 11). Also, the expression level of the ACC gene was normalized by the expression level of the actin gene. Total RNA was extracted from the larvae 14 days after injection using the RNeasy (registered trademark) reagent (manufactured by Qiagen). As a control, the expression level of the ACC gene was evaluated in the same manner for each larva (N = 8) injected with PBS instead of dsRNA. The results are shown in Figure 1. Furthermore, photographs of the larvae of the American burying beetle injected with dsRNA against the ACC gene (ACC RNAi) 14 days after injection and the larvae of the American burying beetle injected with PBS instead of dsRNA against the ACC gene (control group) 14 days after injection are shown in Figure 2.

[0078] As shown in Figure 1, compared to black soldier fly larvae that were not injected with dsRNA for the ACC gene (control group in Figure 1), black soldier fly larvae injected with dsRNA for the ACC gene (ACC RNAi in Figure 1) showed a significantly reduced expression level of the ACC gene. Furthermore, as shown in Figure 2, black soldier fly larvae injected with dsRNA for the ACC gene (hereinafter also referred to as "ACC RNAi larvae") were smaller in body size compared to black soldier fly larvae injected with PBS instead of dsRNA for the ACC gene (hereinafter also referred to as "control group larvae").

[0079] <4. Evaluation of body weight, fat weight, defatted meal (protein, etc.) weight, and the ratio of fat weight to defatted meal (protein, etc.) weight> Black soldier fly larvae were frozen at -20°C, then dried in a freeze-dryer for 24 hours. The dried larvae (control group: 11 individuals, ACC RNAi: 8 individuals) were individually ground into a powder (meal). The weight of the meal was measured, and the average dry body weight of the larvae (mg / larva) was calculated and shown in Figure 3.

[0080] Fat was extracted from each dried larva using hexane at 25°C with a ratio of meal:hexane = 100 mg:1 mL. The weight of the extracted fat and the remaining meal (defatted meal) was measured. In the defatted meal, the mass of fat relative to the total mass of the defatted meal was 5% by mass or less. The average fat weight of the larva (mg / larva) was calculated and is shown in Figure 4. The average weight of the defatted meal (mg / larva) was calculated and is shown in Figure 5. Furthermore, the ratio of the weight of each to the total weight of fat and defatted meal (protein, etc.) was calculated and is shown in Figure 6.

[0081] As shown in Figures 2 and 3, the ACC RNAi larvae had a lower body weight compared to the control group larvae. Also, as shown in Figure 4, the ACC RNAi larvae had a lower fat weight compared to the control group larvae. Furthermore, as shown in Figure 5, the ACC RNAi larvae had a lower defatted meal (protein, etc.) weight compared to the control group larvae. On the other hand, as shown in Figure 6, the ACC RNAi larvae had a lower ratio of fat weight to the total of fat weight and defatted meal (protein, etc.) weight, and a higher ratio of defatted meal (protein, etc.) weight compared to the control group larvae.

[0082] These findings suggest that reducing the expression level of the ACC gene through RNAi or other means can yield farmable black soldier flies that are low in fat and have an increased ratio of defatted meal weight to the total weight of fat and defatted meal (protein, etc.).

[0083] [Example 2: Feeding experiment of ACC functional inhibitors in black soldier fly] <1. Rearing of the Black Soldier Fly> After fertilization, the larvae of the black soldier fly were reared under the same conditions as in Example 1, 1. above, by feeding them a standard artificial feed (containing glucose, molasses, yeast, and cornmeal) (prepared by the National Agriculture and Food Research Organization, based on Chia-Ming Liu et al., Journal of Insects as Food and Feed, 2024) until the 6th day (6 days old). Subsequently, from the 7th day of age, the above artificial feed was replaced with a feed containing spirotetramat (SPT, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), an inhibitor of ACC function, and the larvae were reared for at least one more week. As a control, black soldier fly larvae were reared without replacing the feed with one containing spirotetramat. Figure 7 shows photographs of black soldier fly larvae reared using feed containing spirotetramat (hereinafter also referred to as "larvae that ingested spirotetramat") and black soldier fly larvae reared without switching to feed containing spirotetramat (hereinafter also referred to as "larvae in the control group").

[0084] The feed containing spirotetramat was prepared by dissolving spirotetramat (CAS RN: 203313-25-1) in ethanol and mixing it at a ratio of 1:1000 to the total mass of the feed. In addition, 10% of the total mass of the feed was added to the spirotetramat-containing feed, which was obtained by pressing and recovering fat from wild-type black soldier flies.

[0085] <2. Evaluation of body weight, fat weight, defatted meal (protein, etc.) weight, and the ratio of fat weight to defatted meal (protein, etc.) weight> Black soldier flies (control group: 4 individuals, spirotetramato-fed group: 5 individuals) reared for more than one week under the conditions described in Example 2, Section 1 were evaluated for body weight, fat weight, defatted meal (protein, etc.) weight, and the ratio of each weight to the total of fat weight and defatted meal (protein, etc.) weight. Each indicator was evaluated in the same manner as in Example 1. In the defatted meal, the mass of fat relative to the total mass of the defatted meal was 5% by mass or less. The results are shown in Figures 8 to 11.

[0086] As shown in Figures 7 and 8, no significant difference in body weight was observed between larvae that ingested spirotetramat (SPT in Figures 7 and 8) and larvae in the control group (control group in Figures 7 and 8). Furthermore, as shown in Figure 9, larvae that ingested spirotetramat (SPT in Figure 9) had lower fat weight compared to larvae in the control group (control group in Figure 9). In addition, as shown in Figure 10, larvae that ingested spirotetramat (SPT in Figure 10) had higher defatted meal (protein, etc.) weight compared to larvae in the control group (control group in Figure 10). Finally, as shown in Figure 11, larvae that ingested spirotetramat (SPT in Figure 11) had a lower ratio of fat weight to the total of fat weight and defatted meal (protein, etc.) weight, and a higher ratio of defatted meal (protein, etc.) weight compared to larvae in the control group (control group in Figure 11).

[0087] The results from Example 2 suggest that reducing the function of ACC protein with an ACC inhibitor (such as spirotetramato) can yield farmable black soldier flies that are low in fat and have an increased ratio of defatted meal weight to the total weight of fat and defatted meal (protein, etc.).

[0088] [Example 3: Investigation of the effects of fat recovered from wild-type black soldier fly] Artificial feed 1, which was mixed with spirotetramatode alone, and artificial feed 2, which was mixed with spirotetramatode and fat extracted from wild-type black soldier flies, were prepared.

[0089] After collecting the fertilized eggs, the larvae of the black soldier fly were fed a standard artificial feed (prepared by the National Agriculture and Food Research Organization, based on Chia-Ming Liu et al., Journal of Insects as Food and Feed, 2024) until day 6 (6 days old) under the same conditions as in Example 2, section 1 above. From day 7 onwards, they were fed artificial feeds 1 and 2 and reared for another two weeks. After that, the body weight of the black soldier flies reared on each artificial feed was measured. The results are shown in Figure 12.

[0090] As shown in Figure 12, when comparing the body weight of black soldier fly larvae raised on artificial feed 1 (SPT in Figure 12) and black soldier fly larvae raised on artificial feed 2 (SPT + fat in Figure 12), the black soldier fly larvae raised on artificial feed 2 had a higher body weight.

[0091] These results suggest that lipids recovered from black soldier flies can rescue growth inhibition in black soldier flies where ACC gene expression levels are reduced compared to the wild type, and / or ACC protein function is reduced compared to the wild type.

Claims

1. A black soldier fly in which the expression level of the acetyl-CoA carboxylase gene is reduced compared to the wild type, and / or the function of the expression product of said gene is reduced compared to the wild type.

2. A feed comprising the black soldier fly described in claim 1.

3. A method for producing black soldier flies, comprising the steps of reducing the expression level of the acetyl-CoA carboxylase gene compared to the wild type, and / or reducing the function of the gene expression product compared to the wild type.

4. The method according to claim 3, comprising the step of reducing the expression level of the acetyl-CoA carboxylase gene compared to the wild type, wherein the step is performed by administering an RNAi molecule to the gene.

5. The method according to claim 3, comprising the step of reducing the function of the expression product of the acetyl-CoA carboxylase gene compared to the wild type, wherein the step is carried out by administering a functional inhibitor of the expression product of the gene.

6. The method according to claim 5, wherein the functional inhibitor is spirotetramat.

7. A method for rearing black soldier flies according to claim 1, A method comprising feeding a diet containing fat recovered from black soldier flies.

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