Black soldier fly and method for producing black soldier fly

Abstract

An aspect of the present disclosure is to provide a black soldier fly having improved digestibility. Another object of another aspect of the present invention is to provide a method for producing a black soldier fly having improved digestibility, and a method for improving digestibility of a black soldier fly. Yet another aspect of the present invention is to provide a black soldier fly that is easy to collect and has excellent digestibility. In addition, another aspect of the present invention is to provide a method for producing a black soldier fly which is easily collected and has excellent digestibility.SOLUTION: A black soldier fly, wherein an expression level of at least one laccase family gene selected from the group consisting of a laccase 2 gene and a laccase 1 gene is reduced as compared with a wild type, and / or a function of an expression product of the laccase family gene is reduced as compared with a wild type.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an American soldier fly and a method for producing an American soldier fly. [Background technology]

[0002] Because black soldier flies have a wide range of feeding habits and thrive even when fed discarded food waste, they are attracting attention as a new protein resource. Black soldier flies are produced in factories using various food and agricultural waste as feed, and their use is also progressing, for example, as a new aquaculture feed ingredient to replace fishmeal. The final stage larvae of black soldier flies are the heaviest of all stages and have a soft outer skin, making them suitable as a feed ingredient for processing when using black soldier flies as feed for livestock and aquaculture. For this reason, final stage larvae are currently collected in factories that produce black soldier flies for feed. On the other hand, the outer skin of black soldier flies in the pre-pupa and later stages is hard and less digestible, resulting in lower protein utilization efficiency in livestock and aquaculture. For this reason, black soldier flies in the pre-pupa and later stages are not actively used.

[0003] Furthermore, a gene called laccase2, which is a diphenol oxidase, is widely conserved in insects. The laccase2 gene plays an important role in hardening and coloring the cuticle. For example, Non-Patent Document 1 describes that reducing the expression level of the laccase2 gene in the red flour beetle by RNAi caused the insect's body to soften and subsequently die. Furthermore, Non-Patent Documents 2 to 7 describe that reducing the expression level of the laccase2 gene by RNAi in the bean bug, N. plebeius, P. platypus, Anopheles sinensis, cotton boll weevil, German-winged green bug, sweet potato weevil, or Aedes albopictus mosquito suppresses cuticle formation or cuticle pigmentation.

[0004] Furthermore, a gene encoding a diphenol oxidase called Laccase1 is also widely conserved in insects. Regarding the Laccase1 gene, for example, Non-Patent Document 8 reports that knocking down the Laccase1 gene in Drosophila melanogaster extends its lifespan under conditions of high iron concentrations. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] ArakaneY, et al., “Laccase 2 is the phenoloxidase generequired for beetle cuticle tanning.” Proc Natl Acad SciU S A. 2005 Aug 9;102(32):11337-42. [Non-patent document 2] FutahashiR, et al., “Laccase2 is required for cuticular pigmentationin stinkbugs.” Insect Biochem Mol Biol. 2011Mar;41(3):191-6. [Non-patent document 3] Du, MH, et al., “Suppression of Laccase 2 severely impairscuticle tanning and pathogen resistance during the pupal metamorphosis ofAnopheles sinensis (Diptera: Culicidae).” ParasitesVectors 10, 171 (2017). [Non-patent document 4] FirminoAAP, et al., “RNAi-Mediated Suppression of Laccase2Impairs Cuticle Tanning and Molting in the Cotton Boll Weevil (Anthonomus grandis).”Front Physiol. 2020 Nov 16;11:591569. [Non-patent document 5] Nishide, Y, et al., “Diversity and function of multicopperoxidase genes in the stinkbug Plautia stali.” Sci Rep10, 3464 (2020). [Non-patent document 6] OlivierChristiaens, et al., “RNA interference: a promising biopesticide strategy against the African Sweet potato Weevil Cylas brunneus.”Scientific Reports December 2016 6(1):1-11 [Non-Patent Document 7] Wu X, et al. “Laccase2 is required for sclerotization and pigmentation of Aedes albopictus eggshell.” Parasitol Res.2013May;112(5):1929-34. [Non-patent document 8] LangM, et al., “Multicopper oxidase-1 is a ferroxidase essential for ironhomeostasis in Drosophila melanogaster.”, Proc Natl Acad Sci US A. 2012 Aug14;109(33):13337-42. Summary of the Invention [Problem to be solved by the invention]

[0006] As mentioned above, black soldier fly larvae are actively used as feed for black soldier fly aquaculture. However, because black soldier fly larvae have the tendency to burrow into food, a process of physically separating the larvae from the food is required when recovering the larvae from the rearing tank. Furthermore, when food waste is used as black soldier fly feed, even if the larvae and food are separated by sieving or other methods, small pieces of plastic of the same size and specific gravity as the larvae may be mixed in, making precise separation difficult. Furthermore, the timing at which black soldier fly larvae reach prepupation varies among individuals, so by the optimal time for recovery when the majority of larvae are at their maximum size, some individuals will have reached prepupation, which can also be a problem when recovering larvae.

[0007] On the other hand, the prepupae of the American soldier fly tend to actively separate from their food, simplifying the process of separating them from the food. However, the prepupae of the American soldier fly have a hard outer shell, and are poorly digestible when ingested by farmed organisms such as fish, reducing their value as a feed ingredient. If the digestibility of the American soldier fly prepupae could be improved, the prepupae, which are inexpensive to collect, could be used as a feed ingredient, potentially improving productivity.

[0008] Therefore, one aspect of the present invention aims to provide a black soldier fly with improved digestibility. Another aspect of the present invention aims to provide a method for producing a black soldier fly with improved digestibility, and a method for improving the digestibility of a black soldier fly. Yet another aspect of the present invention aims to provide a black soldier fly that is easy to recover and has excellent digestibility. In addition, another aspect of the present invention aims to provide a method for producing a black soldier fly that is easy to recover and has excellent digestibility. [Means for solving the problem]

[0009] The inventors discovered that when the expression level of the laccase 2 gene was reduced by RNAi in the prepupae of the American soldier fly, digestibility by the digestive enzymes of red sea bream was improved. They also discovered that when the expression level of the laccase 1 gene was reduced by RNAi in the American soldier fly, the larval period was extended and digestibility by the digestive enzymes of red sea bream was improved compared to individuals that had prepupated at the same time, leading to the completion of the present invention.

[0010] That is, the present invention relates to, for example, the following inventions. [1] The expression level of at least one laccase family gene selected from the group consisting of the laccase 2 gene and the laccase 1 gene is reduced compared to the wild type, and / or the function of the expression product of the laccase family gene is reduced compared to the wild type. [2] the laccase 2 gene is a gene that has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 10 and encodes a protein having diphenol oxidase activity; The American soldier fly described in [1], wherein the laccase 1 gene is a gene that has a sequence identity of 80% or more with the amino acid sequence shown in SEQ ID NO: 3 and encodes a protein having diphenol oxidase activity. [3] The American soldier fly according to [1] or [2], wherein the expression level of the laccase family gene is reduced compared to the wild type. [4] An American soldier fly, in which the expression level of a laccase 2 gene is reduced compared to the wild type, and / or the function of the expression product of the gene is reduced compared to the wild type. [5] The American soldier fly described in [4], in which the expression level of the laccase 2 gene is reduced compared to the wild type. [6] An American soldier fly, in which the expression level of a laccase 1 gene is reduced compared to the wild type, and / or the function of the expression product of the gene is reduced compared to the wild type. [7] The American soldier fly described in [6], in which the expression level of the laccase 1 gene is reduced compared to the wild type. [8] A feed containing the American soldier fly according to any one of [1] to [7]. [9] A method for producing American soldier fly, comprising a step of reducing the expression level of at least one laccase family gene selected from the group consisting of laccase 2 gene and laccase 1 gene compared to the wild type, and / or reducing the function of the expression product of the laccase family gene compared to the wild type.

[10] the laccase 2 gene is a gene that has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 10 and encodes a protein having diphenol oxidase activity; The method for producing the laccase 1 gene according to [9], wherein the laccase 1 gene is a gene that has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 and encodes a protein having diphenol oxidase activity.

[11] The method according to [9] or

[10] , wherein the expression level of the laccase family gene is reduced compared to the wild type.

[12] A method for producing American soldier fly, comprising a step of reducing the expression level of laccase 2 gene compared to the wild type and / or reducing the function of the expression product of the gene compared to the wild type.

[13] A method for producing the American soldier fly described in

[12] , which includes a step of reducing the expression level of the laccase 2 gene compared to the wild type.

[14] A method for producing American soldier fly, comprising a step of reducing the expression level of laccase 1 gene compared to the wild type and / or reducing the function of the expression product of the gene compared to the wild type.

[15] A method for producing the American soldier fly described in

[14] , which includes a step of reducing the expression level of the laccase 1 gene compared to the wild type.

[16] A method for improving the digestibility of American soldier fly, comprising a step of reducing the expression level of at least one laccase family gene selected from the group consisting of laccase 2 gene and laccase 1 gene compared to the wild type, and / or reducing the function of the expression product of the laccase family gene compared to the wild type.

[17] the laccase 2 gene is a gene that has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 10 and encodes a protein having diphenol oxidase activity; The method according to

[16] , wherein the laccase 1 gene is a gene encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 and having diphenol oxidase activity.

[18] The method according to

[16] or

[17] , wherein the expression level of the laccase family gene is reduced compared to that of the wild type.

[19] A method for improving the digestibility of American soldier fly, comprising a step of reducing the expression level of laccase 2 gene compared to the wild type and / or reducing the function of the expression product of the gene compared to the wild type.

[20] The method described in

[19] , which comprises a step of reducing the expression level of the laccase 2 gene compared to the wild type. [twenty one] A method for improving the digestibility of American soldier fly, comprising the step of reducing the expression level of the laccase 1 gene compared to the wild type and / or reducing the function of the expression product of the gene compared to the wild type. [twenty two] The method described in

[21] , which comprises a step of reducing the expression level of the laccase 1 gene compared to the wild type. [twenty three] The method according to any one of

[16] to

[22] , wherein the digestibility includes protein digestibility. [twenty four] The method according to any one of

[16] to

[23] , wherein the digestibility is that in fish. [twenty five] The method according to

[24] , wherein the fish is red sea bream. [Effects of the Invention]

[0011] According to one aspect of the present invention, it is possible to provide an American solider fly with improved digestibility. According to another aspect of the present invention, it is possible to provide a method for producing an American solider fly with improved digestibility, and a method for improving the digestibility of an American solider fly. According to yet another aspect of the present invention, it is possible to provide an American solider fly that is easy to collect and has excellent digestibility. In addition, according to another aspect of the present invention, it is possible to provide a method for producing an American solider fly that is easy to collect and has excellent digestibility. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a graph showing the results of analyzing the expression level of laccase 1 in black soldier fly larvae that were not injected with dsRNA (control) and black soldier fly larvae that were injected with dsRNA of the laccase 1 gene (RNAi treatment). [Figure 2] 1 shows representative photographs of a prepupa of an American soldier fly that was not injected with dsRNA (a normal prepupa), and a larva of an American soldier fly that was injected with dsRNA of the laccase 1 gene (a larva with delayed molting). [Figure 3] 1 is a graph showing the results of analyzing the total amino acid content in prepupae of American solider fly that were not injected with dsRNA (control) and in larvae of American solider fly that were injected with dsRNA of the laccase 1 gene (RNAi treatment). [Figure 4] 10 is a graph showing the results of analyzing the amounts of essential amino acids (A) and non-essential amino acids (B) in black soldier fly prepupae (control) that were not injected with dsRNA, and black soldier fly larvae that were injected with dsRNA of the laccase 1 gene (RNAi treatment). [Figure 5]1 is a graph showing the results of analyzing the expression level of the laccase 2 gene in black soldier fly larvae that were not injected with dsRNA (control) and black soldier fly larvae that were injected with dsRNA of the laccase 2 gene (RNAi treatment). [Figure 6] 1 shows representative photographs of black soldier fly prepupae that were not injected with dsRNA (normal prepupae), and black soldier fly prepupae that were injected with dsRNA of the laccase 2 gene (prepupae with softened cuticle). [Figure 7] 1 is a graph showing the results of analyzing the total amino acid amount in prepupae of American solider fly that were not injected with dsRNA (control) and prepupae of American solider fly that were injected with dsRNA of the laccase 2 gene (RNAi treatment). [Figure 8] 10 is a graph showing the results of analyzing the amount of essential amino acids in prepupae of American solider fly (control) that were not injected with dsRNA (A), and in prepupae of American solider fly (RNAi-treated, softened) that were injected with dsRNA of the laccase 2 gene (B). [Figure 9] 10 is a graph showing the results of analyzing the amount of each non-essential amino acid in prepupae of American solider fly (control) that were not injected with dsRNA (A), and in prepupae of American solider fly (RNAi-treated, softened) that were injected with dsRNA of the laccase 2 gene (B). DETAILED DESCRIPTION OF THE INVENTION

[0013] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0014] [American soldier fly] In the solider soldier fly of this embodiment, the expression level of at least one laccase family gene selected from the group consisting of laccase 1 gene and laccase 2 gene is reduced compared to the wild-type, and / or the function of the expression product of the laccase family gene is reduced compared to the wild-type. In the solider soldier fly of this embodiment, the expression level of the laccase 1 gene may be reduced compared to the wild-type, and / or the function of the expression product of the gene may be reduced compared to the wild-type. Hereinafter, in this specification, "laccase family gene" means at least one selected from the group consisting of laccase 1 gene and laccase 2 gene. In the solider soldier fly of this embodiment, the expression level of the laccase 1 gene may be reduced compared to the wild-type, and / or the function of the expression product of the gene may be reduced compared to the wild-type. Therefore, the solider soldier fly of this embodiment has improved digestibility compared to the wild-type solider soldier fly. As used herein, "digestibility" refers to the ease of digestion (decomposition) by digestive enzymes possessed by organisms other than the American solider fly, and the digestion may be the digestion (decomposition) of proteins, carbohydrates, fats, nucleic acids, etc. into peptides, amino acids, oligosaccharides, monosaccharides, fatty acids, nucleotides, or the digestion (decomposition) of proteins. The American solider fly according to this embodiment has particularly improved protein digestibility compared to wild-type American solider flies, as shown in the examples described below.

[0015] Furthermore, the black soldier fly according to the present embodiment is easy to collect and has superior digestibility compared to untreated wild-type black soldier fly prepupae. For example, in the black soldier fly according to the present embodiment, when the laccase family gene is the laccase 2 gene, as described above, the process of separating the black soldier fly prepupae from food is simple, and the black soldier fly has superior digestibility compared to untreated wild-type black soldier flies. Therefore, the black soldier fly according to the present embodiment is easy to collect and has superior digestibility. Furthermore, in the black soldier fly according to the present embodiment, when the laccase family gene is the laccase 1 gene, the black soldier fly undergoes delayed molting, extending the larval period. This makes it difficult to confuse the black soldier fly larvae according to the present embodiment with prepupae when collecting them. Furthermore, as described above, the larvae have superior digestibility compared to prepupae. Therefore, even in this case, the black soldier fly according to the present embodiment is easy to collect and has superior digestibility.

[0016] The digestibility may be, for example, digestibility in any organism, digestibility in aquaculture organisms, digestibility in organisms other than aquaculture organisms, or digestibility in fish, amphibians, reptiles, birds, mammals, etc. Examples of fish include red sea bream, yellowtail, yellowtail, tuna, horse mackerel, pufferfish, and shrimp. Examples of amphibians include frogs and salamanders. Examples of reptiles include turtles, crocodiles, snakes, and lizards. Examples of birds include chickens, ducks, geese, turkeys, quails, pheasants, parakeets, and canaries. Examples of mammals include cows, pigs, sheep, goats, horses, rabbits, dogs, cats, hamsters, mice, rats, monkeys, and humans. The digestibility is preferably digestibility in aquaculture organisms, more preferably fish, and even more preferably red sea bream.

[0017] The American black soldier fly (scientific name: Hermetia illucens) is an insect of the family Syrphidae in the order Diptera. The form of the American black soldier fly according to this embodiment may be a larva, pre-pupa, pupa, or adult, and is preferably a pre-pupa from the viewpoint of more significantly achieving the effects of the present invention. Furthermore, when the form of the American black soldier fly according to this embodiment is a pre-pupa, the process of physically separating the pre-pupa of the American black soldier fly from the rearing tank becomes easier. Furthermore, from the viewpoint of superior digestibility compared to other forms, the American black soldier fly according to this embodiment may be a larva.

[0018] As used herein, the term "gene" refers to a DNA region that includes a region (transcribed region) that is transcribed into an RNA molecule (e.g., mRNA) in a cell. In other words, the laccase 2 gene may include not only the transcribed region but also regulatory regions located upstream or downstream of the transcribed region and untranslated regions (5'UTR, 3'UTR), or may consist of only the transcribed region. The transcribed region may include not only exons but also introns, and may be what is known as cDNA.

[0019] The laccase1 gene is a diphenoloxidase widely conserved among insects and plays an important role in iron metabolism and immune responses. Specifically, laccase1 is present on the basement membrane side of the digestive organs and Malpighian tubules. By binding iron, it is thought to be involved in iron metabolism as a ferroxidase, regulating iron accumulation and oxidative stress. Furthermore, in the American soldier fly, reducing the expression level of the laccase1 gene and decreasing the function of its expression product cause delayed molting. The laccase2 gene is a diphenoloxidase widely conserved among insects and plays an important role in hardening and pigmenting the cuticle. Insects such as the American soldier fly have a tough membrane covering their cuticle, which protects against contamination from the outside world and maintains the insect's morphology. The cuticle's main components are chitin and protein, and the cuticle of insects such as the American soldier fly is characterized by its hardness and brown to black color. Specifically, laccase 2 is thought to play a role in hardening the cuticle by being involved in the cross-linking of chitin, the main component of the cuticle, with proteins, and between proteins, and to play a role in coloring the cuticle by being involved in melanin synthesis, etc. Wild-type black soldier fly larvae are soft and white, but when they reach the prepupae stage, their cuticle becomes hard and black.

[0020] It is known that reducing the expression level of the laccase 1 gene in Drosophila compared to the wild type extends lifespan under certain conditions (conditions with a high iron concentration) (although no such reports have been published for the American solider fly). However, extending lifespan and the larval period due to delayed molting are not necessarily associated with improved digestibility, making the above-mentioned effect of the present invention unexpected. It is also known that reducing the expression level of the laccase 2 gene in the American solider fly compared to the wild type prevents hardening and discoloration of the cuticle. However, the absence of hardening of the cuticle (softening of the cuticle) is not necessarily associated with improved digestibility, making the above-mentioned effect of the present invention unexpected. The American solider fly of this embodiment may have a reduced expression level of a laccase family gene compared to the wild type. Furthermore, the American solider fly of this embodiment may have a reduced expression level of the laccase 1 gene compared to the wild type. The American solider fly of this embodiment may have a reduced expression level of the laccase 2 gene compared to the wild type.

[0021] The laccase 1 gene includes, for example, a nucleotide sequence that transcribes an mRNA consisting of the nucleotide sequence shown in SEQ ID NO: 1, a nucleotide sequence that transcribes an mRNA consisting of the nucleotide sequence shown in SEQ ID NO: 1, a nucleotide sequence that transcribes an mRNA consisting of the nucleotide sequence shown in SEQ ID NO: 2, a nucleotide sequence that expresses a protein consisting of the amino acid sequence shown in SEQ ID NO: 3, etc. The nucleotide sequence shown in SEQ ID NO: 1 is the nucleotide sequence of the mRNA of the laccase 1 gene of the American solider fly (GenBank accession number: XM_038054340.1). The nucleotide sequence shown in SEQ ID NO: 2 is the nucleotide sequence of the CDS of the laccase 1 gene of the American solider fly. The amino acid sequence shown in SEQ ID NO: 3 is the amino acid sequence of the laccase 1 protein of the American solider fly (GenBank accession number: XP_037910268.1).

[0022] The laccase 1 gene may be, for example, a gene containing a nucleotide sequence that transcribes mRNA consisting of a nucleotide sequence having 80% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1. Alternatively, the laccase 1 gene may be, for example, a gene that has 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 3 and encodes a protein having diphenol oxidase activity. The sequence identity may be, for example, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or even 100%. Sequence identity will be described later. Furthermore, since reducing the expression level of the laccase 1 gene, for example, by RNAi or the like, caused delayed molting, it is believed to be a protein required for normal molting in American soldier fly larvae. A protein required for normal molting in American soldier fly larvae means a protein whose expression level and / or function, when reduced compared to the wild type, either extends the time until molting (causing delayed molting) or prevents molting from occurring.

[0023] The regulatory region may be, for example, a transcription regulatory region or a translation regulatory region. Examples of the transcription regulatory region include a promoter, an enhancer, and a silencer. Examples of the translation regulatory region include a ribosome binding region.

[0024] The black soldier fly of this embodiment may or may not have a mutation that reduces the expression level of the laccase 1 gene compared to the wild type and / or reduces the function of the expression product of the gene compared to the wild type (hereinafter also referred to as a "laccase 1 gene mutation"). In other words, the black soldier fly of this embodiment may have a wild-type genome or a mutant genome (mutant black soldier fly).

[0025] Reducing the expression level of the laccase 1 gene compared to the wild type may mean reducing the amount of mRNA of the laccase 1 gene compared to the wild type, or reducing the amount of the expression product (protein) encoded by the laccase 1 gene. In other words, reducing the expression level of the laccase 1 gene compared to the wild type means reducing the expression level of the protein encoded by the laccase 1 gene.

[0026] If the insect of this embodiment does not have a laccase 1 gene mutation, the expression level of the laccase 1 gene can be reduced by, but is not limited to, a method of introducing antisense RNA into the insect or a method using RNA interference (RNAi).

[0027] The method using RNAi can be carried out, for example, by introducing an RNAi molecule against the laccase 1 gene into an American solider fly. Examples of the RNAi molecule against the laccase 1 gene include nucleic acids such as double-stranded (ds) RNA, siRNA, shRNA, and microRNA (miRNA) that have a base sequence corresponding to the laccase 1 gene or a part thereof. The RNAi molecule may be the above-mentioned nucleic acid itself, or a vector that expresses the above-mentioned nucleic acid. Therefore, the American solider fly according to this embodiment may have an RNAi molecule against the laccase 1 gene introduced therein. The length of the RNAi molecule may be, for example, 20 bases or more, 30 bases or more, 50 bases or more, 70 bases or more, 100 bases or more, 150 bases or more, 180 bases or more, 200 bases or more, 1000 bases or less, 900 bases or less, 800 bases or less, 700 bases or less, or 650 bases or less, or may be 20 to 2000 bases, 30 to 2800 bases, 50 to 1500 bases, 100 to 1200 bases, 150 to 1000 bases, 200 to 1000 bases, 300 to 800 bases, 300 to 700 bases, 200 to 650 bases, or 300 to 650 bases. Specific examples of RNAi molecules against the laccase 1 gene include, but are not limited to, dsRNA containing a portion of the base sequence shown in SEQ ID NO: 2, dsRNA containing the base sequence shown in SEQ ID NO: 9, or dsRNA consisting of the base sequence shown in SEQ ID NO: 9.

[0028] The above-mentioned antisense RNA or RNAi molecule can be obtained by a method using known chemical synthesis methods, a method using in vitro enzyme transcription, or the like.

[0029] When the insect according to this embodiment has a laccase 1 gene mutation, the method for introducing the mutation is not particularly limited, and for example, known gene editing techniques can be used. Examples of gene editing techniques include, but are not limited to, methods using zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR / Cas9.

[0030] The laccase1 gene mutation may be introduced into the laccase1 gene, and may be introduced into the transcription region, the regulatory region, the non-coding region, or the like of the laccase1 gene. The laccase1 gene mutation may be a substitution, deletion, insertion, and / or addition of 1 or more, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, or 30 or more bases in the laccase1 gene base sequence. The laccase1 gene mutation may be a substitution, deletion, insertion, and / or addition of 100 or fewer, 80 or fewer, 60 or fewer, 50 or fewer, or 40 or fewer bases in the laccase1 gene base sequence. The laccase1 gene mutation may be a deletion of the entire laccase1 gene.

[0031] The laccase 1 gene mutation may be introduced, for example, in the region corresponding to the 1st to 3177th bases in the base sequence shown in SEQ ID NO: 1, or in the region corresponding to the 1st to 2580th bases in the base sequence shown in SEQ ID NO: 2.

[0032] The nucleotide sequence of the laccase 1 gene having a laccase 1 gene mutation may include, for example, a nucleotide sequence that transcribes mRNA consisting of a nucleotide sequence having 99% or less sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, a nucleotide sequence that transcribes mRNA consisting of a nucleotide sequence having 99% or less sequence identity to the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence that expresses a protein consisting of an amino acid sequence having 99% or less sequence identity to the amino acid sequence shown in SEQ ID NO: 3. The 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.

[0033] As used herein, sequence identity refers to the percentage (%) of matching bases or residues when the base sequences or amino acid sequences being compared are aligned (multiple alignment). Multiple alignment refers to an alignment of base sequences with appropriate gaps inserted so that corresponding base or amino acid sequence portions are aligned, making the base sequences or amino acid sequences comparable to each other. For multiple alignment, known multiple alignment creation programs can be used. For example, Clustal W, Clustal X, BLAST, etc., can be suitably used.

[0034] The expression level of the laccase1 gene in the black soldier fly according to this embodiment can be analyzed by measuring the amount of RNA (e.g., mRNA) expressed from the laccase1 gene and the amount of protein produced from the RNA. The expression level of the laccase1 gene in the black soldier fly according to this embodiment can be analyzed by, for example, RT-PCR, RNA-seq, Northern blot, etc. when measuring the amount of RNA, and by, for example, Western blot, etc. when measuring the amount of protein. The expression level of the laccase1 gene in the black soldier fly according to this embodiment may be reduced compared to the wild-type, and may be reduced by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%.

[0035] The function of the expression product of the laccase 1 gene in the black soldier fly according to the present embodiment can be analyzed by, for example, comparing the time required for the black soldier fly according to the present embodiment to that of a wild-type black soldier fly when the black soldier fly is in the pre-pupa, pupa, or adult stage. The time required for the black soldier fly according to the present embodiment to molt is longer than that of a wild-type black soldier fly. Furthermore, analysis can be performed by, for example, preparing samples containing laccase 1 from the black soldier fly according to the present embodiment and a wild-type black soldier fly, reacting the samples with a substrate for measuring diphenol oxidase activity, and comparing the color change and absorbance change, etc.

[0036] The function of the expression product of the laccase 1 gene in the American soldier fly in this embodiment may 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 may be reduced by less than 100%.

[0037] Examples of the laccase 2 gene include a nucleotide sequence that transcribes an mRNA consisting of the nucleotide sequence shown in SEQ ID NO: 12, a nucleotide sequence that transcribes an mRNA containing the nucleotide sequence shown in SEQ ID NO: 12, the nucleotide sequence shown in SEQ ID NO: 11, a nucleotide sequence that expresses a protein containing the amino acid sequence shown in SEQ ID NO: 10, and the like. The nucleotide sequence shown in SEQ ID NO: 12 is the nucleotide sequence of the mRNA of the laccase 2 gene of American solider fly (GenBank accession number: XM_038066390.1). The nucleotide sequence shown in SEQ ID NO: 11 is a portion of the nucleotide sequence of the CDS of the laccase 2 gene of American solider fly. The amino acid sequence shown in SEQ ID NO: 10 is a portion of the amino acid sequence of the laccase 2 protein of American solider fly (GenBank accession number: XP_037922318.1).

[0038] The laccase 2 gene may be, for example, a gene containing a nucleotide sequence that transcribes mRNA consisting of a nucleotide sequence having 80% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 12. Alternatively, the laccase 2 gene may be, for example, a gene that has 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 10 and encodes a protein having diphenol oxidase activity. The sequence identity may be, for example, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or may be 100%.

[0039] The RNAi molecule against the laccase 2 gene may be one in which the "laccase 1 gene" in the above description is replaced with the "laccase 2 gene." Specific examples of the RNAi molecule against the laccase 2 gene are not particularly limited, but may be, for example, a dsRNA containing a part of the base sequence shown in SEQ ID NO: 12, a dsRNA containing the base sequence shown in SEQ ID NO: 18, or a dsRNA consisting of the base sequence shown in SEQ ID NO: 18.

[0040] The laccase 2 gene mutation may be introduced, for example, in the region corresponding to the 1st to 4926th bases in the base sequence shown in SEQ ID NO: 12, or in the region corresponding to the 1st to 1551st bases in the base sequence shown in SEQ ID NO: 11.

[0041] The function of the expression product of the laccase 2 gene in the black soldier fly according to the present embodiment can be analyzed by, for example, using image analysis software to compare the body color of the black soldier fly according to the present embodiment with that of a wild-type black soldier fly when the black soldier fly is in a pre-pupa, pupa, or adult state. Furthermore, analysis can be performed by, for example, preparing samples containing laccase 2 from the black soldier fly according to the present embodiment and the wild-type black soldier fly, reacting the samples with a substrate for measuring diphenol oxidase activity, and comparing the resulting color change and absorbance change.

[0042] The other laccase 2 gene may be similar in configuration to the laccase 1 gene. That is, the other laccase 2 gene may be configured in the above description by replacing "laccase 1 gene" with "laccase 2 gene." In this specification, a "laccase family gene" refers to at least one gene selected from the group consisting of the laccase 1 gene and the laccase 2 gene, and an embodiment in which the "laccase 1 gene" is replaced with "laccase family gene" can be applied.

[0043] The digestibility of the black soldier fly according to this embodiment may be improved compared to that of wild-type black soldier flies. For example, protein digestibility can be analyzed using a method similar to that described in <1-6. In vitro digestion with red sea bream digestive enzymes> in the Examples below. More specifically, the method is as follows: black soldier flies are freeze-dried, excised, and incubated with a solution containing a digestive enzyme from a given organism. The sample is then deproteinized and subjected to an amino acid analyzer for amino acid analysis under high-resolution analytical conditions. Similarly, the digestibility of carbohydrates, fats, and nucleic acids can be analyzed by freeze-drying black soldier flies, excising them, and incubating them with a solution containing a digestive enzyme from a given organism, and analyzing the decomposition of the corresponding substances using known methods.

[0044] The digestibility of the American soldier fly of this embodiment may be improved compared to the wild type, and may be improved by, for example, 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more.

[0045] The black soldier fly according to this embodiment has improved digestibility and can be used as feed for any living organism. A feed according to one 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 used as is, or may be dried and powdered, for example.

[0046] The content of American soldier fly in the feed according to this embodiment may be set appropriately depending on the organism to which the feed is given, but may be, for example, 0.1% by mass or more, 1% by mass or more, 10% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, on a dry weight basis, relative to the total mass of the feed, or may be 100% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less.

[0047] The feed according to the present embodiment may contain ingredients suitable for organisms other than the black soldier fly according to the present embodiment, such as lipids, proteins, carbohydrates, vitamins, and minerals, and more specifically, may contain soybeans, wheat flour, corn, wheat germ, seaweed powder, concentrated alfalfa, fish meal, fish oil, spirulina, chitosan, kale, seaweed, 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).

[0048] [Method for producing American soldier flies] The method for producing a black soldier fly according to this embodiment includes a step of reducing the expression level of at least one laccase family gene selected from the group consisting of laccase 1 gene and laccase 2 gene compared to the wild type and / or reducing the function of an expression product of the laccase family gene compared to the wild type (a step of reducing the expression of the laccase family gene). The method for producing a black soldier fly according to this embodiment may include a step of reducing the expression level of the laccase 1 gene compared to the wild type and / or reducing the function of an expression product of the gene compared to the wild type (a step of reducing the expression of the laccase 1 gene). The method for producing a black soldier fly according to this embodiment may include a step of reducing the expression level of the laccase 2 gene compared to the wild type and / or reducing the function of an expression product of the gene compared to the wild type (a step of reducing the expression of the laccase 2 gene). The method for producing a black soldier fly according to this embodiment preferably includes a step of reducing the expression level of a laccase family gene compared to the wild type. More preferably, the method for producing a black soldier fly according to this embodiment includes a step of reducing the expression level of the laccase 1 gene compared to the wild type. The method for producing an American soldier fly according to this embodiment more preferably includes a step of reducing the expression level of the laccase 2 gene compared to that of the wild type.

[0049] The production method according to this embodiment includes the steps described above, and thus can produce black soldier flies with improved digestibility compared to the wild-type. Furthermore, the production method according to this embodiment can produce black soldier flies that are easy to collect and have superior digestibility compared to untreated wild-type black soldier flies prepupae.

[0050] The above step in the production method according to this embodiment (the step of reducing the laccase 2 gene) may be performed without or with the introduction of a mutation into the laccase 2 gene. The above step in the production method according to this embodiment (the step of reducing a laccase family gene or the step of reducing the laccase 1 gene) is the same as that for the laccase 2 gene whether the gene is a laccase family gene or a laccase 1 gene, and an embodiment in which "laccase 2 gene" is read as "laccase family gene" or "laccase 1 gene" can be applied.

[0051] When the above step is not performed by introducing a mutation into the laccase 2 gene, the step of reducing the expression level of the laccase 2 gene compared to the wild type may be performed, for example, by introducing antisense RNA into the American solider fly, or by RNA interference (RNAi). Preferably, RNA interference (RNAi) is used. RNAi can be performed by introducing an RNAi molecule against the laccase 2 gene as described above into the American solider fly. Whether the gene targeted in the above step is a laccase family gene or a laccase 1 gene, it is the same as the laccase 2 gene, and the production method according to this embodiment can be applied in an embodiment in which "laccase 2 gene" is read as "laccase family gene" or "laccase 1 gene."

[0052] The form of the American soldier fly into which the antisense RNA or RNAi molecule is introduced may be an adult, pupa, pre-pupa, larva, egg, or embryo, and it is preferable to introduce it into a larva.

[0053] When the above step is carried out by introducing a mutation into the laccase 2 gene, it may be carried out, for example, by a known gene editing technique. Gene editing techniques are not particularly limited, but examples include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR / Cas9, with CRISPR / Cas9 being preferred. The mutations introduced into the laccase 2 gene are as described above. Whether the gene targeted in the above step is a laccase family gene or a laccase 1 gene, the process is the same as the laccase 2 gene, and the production method according to this embodiment can be applied in an embodiment in which "laccase 2 gene" is read as "laccase family gene" or "laccase 1 gene."

[0054] It is known that insects other than the American solider fly (e.g., red flour beetle) can die depending on the degree of reduction in the expression level of the laccase 2 gene by RNAi. Therefore, for the American solider fly, by appropriately adjusting the amount of reduction in the expression level of the laccase 2 gene and the amount of reduction in the function of the expression product of the gene in the reduction step to a level that does not cause the American solider fly to die, it is possible to create a strain of American solider fly with improved digestibility. The same applies to laccase family genes or laccase 1 genes as to laccase 2 genes.

[0055] The production method according to this embodiment may further include a step of rearing American solider fly in which the expression level of the laccase 2 gene has been reduced compared to the wild type and / or the function of the expression product has been reduced compared to the wild type (hereinafter also referred to as the "rearing step"). In the rearing step, whether the gene is a laccase family gene or a laccase 1 gene is the same as the laccase 2 gene, and the production method according to this embodiment can also be applied in an embodiment in which "laccase 2 gene" is read as "laccase family gene" or "laccase 1 gene".

[0056] By including a rearing step in the production method according to this embodiment, the expression level of the laccase 2 gene can be more sufficiently reduced and / or the function of the expression product of the gene can be reduced. The same applies to the laccase 2 gene whether the gene is a laccase family gene or a laccase 1 gene. Furthermore, the rearing step can produce black soldier fly larvae or prepupae with improved digestibility, and in the case of prepupae in particular, recovery is easy, allowing for reduced production costs. Black soldier fly larvae or prepupae with improved digestibility can be used as feed for rearing. Accordingly, one embodiment also provides a method for producing feed containing black soldier flies.

[0057] The conditions for the rearing step may be set appropriately depending on the type or morphology of the American 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 may be, for example, a photoperiod of 16 hours of light and 8 hours of darkness.

[0058] The production method according to this embodiment may further include a step of mating American solider flies in which the expression level of the laccase 2 gene has been reduced compared to the wild type and / or the function of the expression product of the gene has been reduced compared to the wild type (hereinafter also referred to as the "mating step"). In the mating step, whether the gene is a laccase family gene or a laccase 1 gene is the same as the laccase 2 gene, and the production method according to this embodiment can be applied in an embodiment in which "laccase 2 gene" is read as "laccase family gene" or "laccase 1 gene".

[0059] The method for producing the laccase 2 gene according to this embodiment includes a crossbreeding step, which can reduce the expression level of the laccase 2 gene and / or change the degree of functional impairment of the expression product of the laccase 2 gene compared to the wild type. The same applies to the laccase 2 gene whether the gene is a laccase family gene or a laccase 1 gene.

[0060] The mating step may involve mating two black soldier flies in which the expression level of the laccase 2 gene has been reduced compared to the wild type and / or the function of the expression product of the gene has been reduced compared to the wild type, or it may involve mating a black soldier fly in which the expression level of the laccase 2 gene has been reduced compared to the wild type and / or the function of the expression product of the gene has been reduced compared to the wild type with another black soldier fly in which the expression level of the laccase 2 gene has been reduced compared to the wild type and / or the function of the expression product of the gene has been reduced compared to the wild type. In the mating step, whether the gene is a laccase family gene or a laccase 1 gene is the same as the laccase 2 gene, and the production method according to this embodiment can be applied in an embodiment in which "laccase 2 gene" is read as "laccase family gene" or "laccase 1 gene".

[0061] In the production method according to this embodiment, when a mutation is introduced into the laccase 2 gene, the mating step is not particularly limited, and may involve mating the black soldier flies so that they become homozygous or heterozygous for the mutation in the laccase 2 gene. In the mating step, whether the gene is a laccase family gene or a laccase 1 gene is the same as the laccase 2 gene, and the production method according to this embodiment can be applied in an embodiment in which "laccase 2 gene" is read as "laccase family gene" or "laccase 1 gene."

[0062] Mating to produce a heterozygote may be performed by mating with an individual that does not have a mutation in the laccase 2 gene. Whether the gene is a laccase family gene or a laccase 1 gene, it is the same as the laccase 2 gene, and the production method according to this embodiment can be applied in an embodiment in which "laccase 2 gene" is read as "laccase family gene" or "laccase 1 gene."

[0063] [Method for improving the digestibility of American soldier flies] The method for improving the digestibility of black soldier flies according to this embodiment includes a step of reducing the expression level of a laccase family gene compared to the wild type and / or reducing the function of the expression product of the gene compared to the wild type (reducing step). The method for improving the digestibility of black soldier flies according to this embodiment includes a step of reducing the expression level of a laccase 1 gene compared to the wild type and / or reducing the function of the expression product of the gene compared to the wild type (reducing step). The method for improving the digestibility of black soldier flies according to this embodiment includes a step of reducing the expression level of a laccase 2 gene compared to the wild type and / or reducing the function of the expression product of the gene compared to the wild type (reducing step).

[0064] Specific aspects of the method for improving the digestibility of black soldier flies according to this embodiment are the same as those of the method for producing black soldier flies according to this embodiment.

[0065] As described above, digestion may be the digestion (decomposition) of proteins, carbohydrates, fats, nucleic acids, and the like into peptides, amino acids, oligosaccharides, monosaccharides, fatty acids, and nucleotides, or may be protein digestion (decomposition). As shown in the examples below, the American soldier fly according to this embodiment has particularly improved protein digestibility and overall improved digestibility compared to wild-type American soldier flies. Therefore, the digestibility in the method for improving the digestibility of American soldier flies according to this embodiment may also include protein digestibility. Protein digestibility can be evaluated using digestive enzymes. The digestive enzymes are not particularly limited as long as they are capable of decomposing proteins into peptides or amino acids, and examples include trypsin, chymotrypsin, elastase, aminopeptidase, and carboxypeptidase. Furthermore, the origin of these enzymes is not particularly limited and may be derived from fish, amphibians, reptiles, birds, mammals, or the like, with fish-derived enzymes being particularly preferred.

[0066] In the method for improving the digestibility of black soldier flies according to this embodiment, the digestibility may be that of any organism, may be that of a farmed organism, may be that of an organism other than a farmed organism, or may be that of fish, amphibians, reptiles, birds, mammals, etc. Specific species of organisms are as described above, and the digestibility is preferably that of a farmed organism, more preferably fish, and even more preferably red sea bream. [Example]

[0067] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0068] [Test Example 1: Test on laccase 1 gene] (Evaluation of the digestibility of prepupae of American soldier fly) <1-1. Rearing American soldier flies> In 2013, American soldier fly larvae were collected in Tsukuba, Ibaraki Prefecture, and colonies were reared at the National Agriculture and Food Research Organization (NARO) according to the following procedure. Immediately after hatching, larvae were fed artificial diet and reared in plastic containers until they reached the prepupal stage, at which point their body color turned black. The prepupa were then removed from the plastic container and placed in another plastic container with coffee grounds for pupation. Newly emerged adults were transferred to mesh cages for mating and oviposition. Rearing was carried out under conditions of 27°C, 60% RH, and a 16-hour light / 8-hour dark photoperiod.

[0069] <1-2. Synthesis of double-stranded RNA for RNA interference> Double-stranded RNA (dsRNA) of the laccase 1 gene of American solider fly was prepared as follows. Primers Hi-Laccase1-F (SEQ ID NO: 4) and Hi-Laccase1-R (SEQ ID NO: 5) shown in Table 1 were designed based on the nucleotide sequence (SEQ ID NO: 1) of the mRNA "XM_038054340.1" of the laccase 1 gene of American solider fly registered in the NCBI protein database. Total RNA was extracted from American solider fly larvae using RNeasy® reagent (Qiagen). Next, template cDNA was synthesized using the total RNA, SuperScript III® reagent (Invitrogen), and an oligo-dT primer. Using the synthesized cDNA as a template, DNA consisting of the nucleotide sequence shown in SEQ ID NO: 8 was amplified by PCR using TaKaRa Ex Taq Hot Start Version (Takara Bio Inc.) and primers T7-Hi-Laccase1-F (SEQ ID NO: 6) and T7-Hi-Laccase1-R (SEQ ID NO: 7) shown in Table 1. The amplified DNA was confirmed, and dsRNA containing the nucleotide sequence corresponding to SEQ ID NO: 9 was synthesized using this DNA and the T7 RiboMAX™ Express RNAi system (Promega). The synthesized dsRNA was stored at -80°C.

[0070] [Table 1]

[0071] <1-3. Evaluation of the decrease in laccase 1 expression level by dsRNA injection> Seven-day-old American soldier fly larvae were rinsed with distilled water and their body surfaces gently wiped to remove water. The dsRNA solution of the laccase 1 gene obtained in 1-2 above was adjusted to a concentration of 3 μg / μl, and 5 μl was injected using a syringe (needle volume: 10 μl, needle length: 50 mm). The same volume of PBS solution was injected as a control.

[0072] The expression level of the laccase 1 gene was evaluated by quantitative real-time PCR using total RNA extracted from larvae 7 days after injection and primers Hi-Laccase1-F and Hi-Laccase1-R. Total RNA was extracted from larvae 7 days after injection using RNeasy® reagent (Qiagen). As a control, the expression level of the laccase 1 gene was evaluated in the same manner in larvae not injected with dsRNA. The expression level is a relative expression level normalized by the actin expression level. The results are shown in Figure 1. In Figure 1, "****" indicates statistical significance at p<0.0001.

[0073] As shown in Figure 1, it was confirmed that the expression level of the laccase 1 gene was significantly reduced in the black soldier fly larvae injected with dsRNA of the laccase 1 gene compared to the black soldier fly larvae that were not injected with dsRNA.

[0074] <1-4. Creation of softened prepupae and delayed molting larvae> Fourteen-day-old American soldier fly larvae were rinsed with distilled water and their body surfaces gently wiped to remove water. Five microliters of the laccase 1 gene dsRNA solution obtained in 1-2 above was injected using a syringe (needle volume: 10 μl, needle length: 50 mm). The American soldier fly larvae were then reared on an artificial diet under the same conditions as in 1-1. Five days later, the body color and weight of these larvae were evaluated. As a control, prepupae without dsRNA injection were also evaluated for body color and weight in the same manner. Evaluations were conducted on 22 individuals of each type. Photographs of representative individuals are shown in Figure 2. In Figure 2, the "delayed molting larva" refers to a larva injected with the laccase 1 gene dsRNA solution, and the "normal prepupae" refers to a prepupae without dsRNA injection.

[0075] As shown in Figure 2, all 22 prepupae of black soldier fly that were not injected with dsRNA became black, but 32% (7 out of 22) of the larvae of black soldier fly that were injected with dsRNA for the laccase 1 gene remained white larvae due to delayed molting. Six black prepupae that were not injected with dsRNA and five white larvae that were injected with dsRNA were sampled and subjected to the following digestibility evaluation test.

[0076] <1-5. Preparation of red sea bream digestive enzyme mixture> Red sea bream (300-400 g) fed a compound feed (Nisshin Marubeni Corporation) were abdominally opened, and the intestinal contents were squeezed out and centrifuged (20,000 × g, 5 min, 0°C) to obtain the supernatant. This supernatant was then dialyzed against H3BO3-KCl-NaOH buffer (pH 8.2; Bower and Bates, 1955) at 20°C to hydrolyze proteins and remove the resulting amino acids. The indigestible material was then removed again by centrifugation, and the resulting supernatant was frozen in liquid nitrogen and stored at -80°C until use. Just before use, the mixture was thawed and centrifuged (20,000 × g, 5 min, 0°C) to obtain the supernatant, which was used as the red sea bream digestive enzyme mixture.

[0077] <1-6. In vitro digestion using red sea bream digestive enzymes> The prepupae or larvae of black soldier fly collected in 1-4 were freeze-dried, and the seventh and eighth somites were excised and placed in a tube (2.0 mL Safe-Lock, Eppendorf AG) with 570 μL of potassium chloride-sodium hydroxide buffer (pH 7.75), 30 μL of red sea bream digestive enzyme mixture, and 10 μL of 0.6% sodium azide solution. The tube was incubated at 25°C for 14 hours in an aluminum block shaking incubator (WSC-2630, ATTO) with shaking at 1300 rpm. After incubation, the tube was centrifuged (20,000 × g, 5 minutes, 0°C). 190 μL of the supernatant was mixed with 10 μL of 100% trichloroacetic acid, and the precipitate was removed by centrifugation to deproteinize the supernatant. The resulting supernatant was subjected to amino acid analysis using a JLC-500 / V amino acid analyzer (JEOL Ltd.) under high-resolution analytical conditions. The results are shown in Figures 3 and 4. In Figures 3 and 4, "RNAi treatment" refers to larvae injected with a dsRNA solution of the laccase 1 gene, and "control" refers to prepupae that were not injected with dsRNA. In Figure 3, "**" indicates statistical significance at p<0.01.

[0078] As shown in Table 2, there was no significant difference in dry weight between the cut samples.

[0079] [Table 2]

[0080] As shown in Figure 3, the total amino acid content of larvae injected with the dsRNA solution of the laccase 1 gene was significantly increased compared to untreated prepupae, despite no difference in dry weight. Furthermore, as shown in Figure 4, the total amino acid content of larvae injected with the dsRNA solution of the laccase 1 gene was significantly increased compared to untreated prepupae, despite no difference in dry weight. The significant difference in amino acid content between the two groups was likely due to delayed molting in the black soldier fly injected with the dsRNA solution of the laccase 1 gene, preventing the cuticle from hardening, while the untreated black soldier fly molted and became a prepupae with a hard cuticle. Based on the above, the suppression of laccase 1 gene expression in black soldier flies improves their digestibility, increases the total amino acid content without disrupting the balance of the amino acid content, and therefore the black soldier flies of the present invention are considered to be of high quality as feed ingredients. Furthermore, suppression of laccase 1 gene expression delays molting in black soldier flies, extending the larval period, making it difficult to confuse black soldier fly larvae with prepupae during collection. As a result, the black soldier flies are easy to collect and have superior digestibility compared to untreated wild-type black soldier fly prepupae.

[0081] [Test Example 2: Test on laccase 2 gene] <2-1. Rearing American soldier flies> American soldier flies were reared in the same manner as in 1-1.

[0082] <2-2. Synthesis of double-stranded RNA for RNA interference> Primers tLac2_ex2_8F (SEQ ID NO: 13) and tLac2_ex2_428R (SEQ ID NO: 14) shown in Table 3 were designed based on the nucleotide sequence (SEQ ID NO: 12) of the mRNA "XM_038066390.1" of the laccase 2 gene of American solider fly, registered in the NCBI protein database. Furthermore, using cDNA synthesized as in 1-2 as a template, DNA consisting of the nucleotide sequence shown in SEQ ID NO: 17 was amplified by PCR using TaKaRa Ex Taq Hot Start Version (Takara Bio Inc.) and primers T7-tLac2_ex2_26F (SEQ ID NO: 15) and T7-tLac2_ex2_409R (SEQ ID NO: 16) shown in Table 3. Subsequently, dsRNA containing the nucleotide sequence corresponding to SEQ ID NO: 18 was synthesized as in 1-2. The synthesized dsRNA was stored at -80°C.

[0083] [Table 3]

[0084] <2-3. Evaluation of the decrease in laccase 2 expression level by dsRNA injection> The dsRNA solution of the laccase 2 gene was injected into American solider fly larvae in the same manner as in 1-3, except that the dsRNA solution of the laccase 2 gene obtained in 2-2 was used instead of the dsRNA solution of the laccase 1 gene.

[0085] The expression level of the laccase 2 gene was evaluated in the same manner as in 1-3, except that primers tLac2_ex2_8F and tLac2_ex2_428R were used instead of primers Hi-Laccase1-F and Hi-Laccase1-R. The expression levels are relative expression levels normalized by actin expression levels. The results are shown in Figure 5. When primers tLac2_ex2_8F and tLac2_ex2_428R were used in quantitative real-time PCR, DNA consisting of the base sequence shown in SEQ ID NO: 19 was amplified. In Figure 5, "**" indicates statistical significance at p<0.01.

[0086] As shown in Figure 5, it was confirmed that the expression level of the laccase 2 gene was significantly reduced in the black soldier fly larvae injected with dsRNA of the laccase 2 gene compared to the black soldier fly larvae that were not injected with dsRNA.

[0087] <2-4. Preparation of prepupae with softened epidermis> Final-instar larvae of American soldier fly were rinsed with distilled water, and the body surface was gently wiped to remove water. 5 μl of the laccase 2 gene dsRNA solution (3000 ng / mL) obtained in 2-2 was injected into the body cavity of 24 final-instar larvae of American soldier fly using a syringe (needle volume: 10 μl, needle length: 50 mm). These final-instar larvae of American soldier fly were then reared on an artificial diet under the same conditions as in 2-1, and their phenotypes were observed after prepupal molting. Photographs of representative individuals are shown in Figure 6. In Figure 6, "prepupa with softened cuticle" refers to a prepupa injected with the laccase 2 gene dsRNA solution, and "normal prepupa" refers to a prepupa not injected with dsRNA.

[0088] As shown in Figure 6, the prepupae of black soldier flies that were not injected with dsRNA turned black, but 8 out of 24 prepupae of black soldier flies that were injected with dsRNA of the laccase 2 gene were observed to have turned white. Three prepupae that were not injected with dsRNA and three white prepupae that were injected with dsRNA were sampled and subjected to the following digestibility evaluation test.

[0089] <2-5. Preparation of red sea bream digestive enzyme mixture> A red sea bream digestive enzyme mixture was prepared in the same manner as in 1-5.

[0090] <2-6. In vitro digestion using red sea bream digestive enzymes> Amino acid analysis was performed on the black soldier fly prepupae sampled in 2-4 in the same manner as in 1-6. The results are shown in Figures 7 to 9. In Figure 7, "RNAi treatment" refers to prepupae injected with a dsRNA solution of the laccase 2 gene, and "control" refers to prepupae not injected with dsRNA. Figure 8 is a graph showing the results of analyzing the amounts of essential amino acids in black soldier fly prepupae not injected with dsRNA (control) (A) and black soldier fly prepupae injected with dsRNA of the laccase 2 gene (RNAi treatment, softened) (B). Figure 9 is a graph showing the results of analyzing the amounts of non-essential amino acids in black soldier fly prepupae not injected with dsRNA (control) (A) and black soldier fly prepupae injected with dsRNA of the laccase 2 gene (RNAi treatment, softened) (B).

[0091] As shown in Table 4, there was no significant difference in dry weight between the cut samples.

[0092] [Table 4]

[0093] As shown in Figure 7, the total amino acid content of prepupae injected with the laccase 2 gene dsRNA solution was significantly increased compared to untreated prepupae, despite the same dry weight. Furthermore, as shown in Figures 8 and 9, the total amino acid content of prepupae injected with the laccase 2 gene dsRNA solution was significantly increased compared to untreated prepupae, despite the same dry weight. No amino acids were significantly decreased. Because the hardened cuticular membrane of the American soldier fly accounts for less than 1% of its body weight, the significant difference in amino acid content between the two prepupae is not likely due solely to the effect on the cuticle caused by the reduced expression of the laccase 2 gene.

Claims

1. The American soldier fly has a reduced expression level of at least one laccase family gene selected from the group consisting of the laccase 2 gene and the laccase 1 gene compared to the wild type, and / or the function of the expression product of the laccase family gene is reduced compared to the wild type.

2. the laccase 2 gene is a gene that has a sequence identity of 80% or more with the amino acid sequence shown in SEQ ID NO: 10 and encodes a protein having diphenol oxidase activity; The American soldier fly according to claim 1, wherein the laccase 1 gene is a gene that has a sequence identity of 80% or more with the amino acid sequence shown in SEQ ID NO: 3 and encodes a protein having diphenol oxidase activity.

3. The American soldier fly according to claim 1 or 2, wherein the expression level of the laccase family gene is reduced compared to that of a wild type.

4. An American soldier fly, in which the expression level of a laccase 2 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.

5. The American soldier fly according to claim 4, wherein the expression level of the laccase 2 gene is reduced compared to that of the wild type.

6. An American soldier fly, in which the expression level of a laccase 1 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.

7. The American soldier fly described in claim 6, in which the expression level of the laccase 1 gene is reduced compared to that of the wild type.

8. A feed comprising the black soldier fly of claim 1 or 2.

9. A method for producing American soldier flies, comprising a step of reducing the expression level of at least one laccase family gene selected from the group consisting of the laccase 2 gene and the laccase 1 gene compared to the wild type, and / or reducing the function of the expression product of the laccase family gene compared to the wild type.

10. the laccase 2 gene is a gene that has a sequence identity of 80% or more with the amino acid sequence shown in SEQ ID NO: 10 and encodes a protein having diphenol oxidase activity; The method for producing the laccase 1 gene according to claim 9, wherein the laccase 1 gene is a gene encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 and having diphenol oxidase activity.

11. The method according to claim 9 or 10, wherein the expression level of the laccase family gene is reduced compared to that of a wild type.

12. A method for producing American soldier fly, comprising a step of reducing the expression level of laccase 2 gene compared to the wild type and / or reducing the function of the expression product of said gene compared to the wild type.

13. A method for producing the American soldier fly described in claim 12, comprising a step of reducing the expression level of the laccase 2 gene compared to that of the wild type.

14. A method for producing American soldier fly, comprising a step of reducing the expression level of laccase 1 gene compared to the wild type and / or reducing the function of the expression product of said gene compared to the wild type.

15. A method for producing the American soldier fly described in claim 14, comprising a step of reducing the expression level of the laccase 1 gene compared to the wild type.

16. A method for improving the digestibility of American soldier fly, comprising a step of reducing the expression level of at least one laccase family gene selected from the group consisting of laccase 2 gene and laccase 1 gene compared to the wild type, and / or reducing the function of the expression product of the laccase family gene compared to the wild type.

17. the laccase 2 gene is a gene that has a sequence identity of 80% or more with the amino acid sequence shown in SEQ ID NO: 10 and encodes a protein having diphenol oxidase activity; The method according to claim 12, wherein the laccase 1 gene is a gene encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 and having diphenol oxidase activity.

18. The method according to claim 16 or 17, wherein the expression level of the laccase family gene is reduced compared to that of the wild type.

19. A method for improving the digestibility of American soldier fly, comprising a step of reducing the expression level of laccase 2 gene compared to the wild type and / or reducing the function of the expression product of said gene compared to the wild type.

20. The method according to claim 19, comprising a step of reducing the expression level of the laccase 2 gene compared to the wild type.

21. A method for improving the digestibility of American soldier fly, comprising a step of reducing the expression level of laccase 1 gene compared to the wild type and / or reducing the function of the expression product of said gene compared to the wild type.

22. The method of claim 21, comprising a step of reducing the expression level of the laccase 1 gene compared to the wild type.

23. 18. The method of claim 16 or 17, wherein the digestibility comprises protein digestibility.

24. 18. The method of claim 16 or 17, wherein the digestibility is the digestibility in fish.

25. 25. The method of claim 24, wherein the fish is red sea bream.

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