Gene expression in Verson's glands of the fall armyworm suggests their role in molting and immunity.

Verson's glands are segmental pairs of dermal glands attached to the epidermis in lepidopteran larvae. They produce macromolecules during intermolt period and empty them during each molt. Morphological, histochemical, developmental, and protein analysis studies have been conducted to determine the functions of Verson's glands. However, the exact role of Verson's glands remains unclear. In our previous study, a strain of transgenic fall armyworm, Spdoptera frugiperda expressing green fluorescence protein (GFP) and Systemic RNA interference defective protein 1 (SID1) from Caenorhabditis elegans was established to improve RNA interference (RNAi) efficiency. Unexpectedly, we found that GFP fluorescence was significantly brighter in Verson's glands than in other tissues. Also, RNAi efficiency improved more in Verson's glands than in other tissues. We took advantage of improved RNAi efficiency to explore the function of Verson's glands. RNA-seq analysis revealed that genes highly expressed in Verson's glands code for cuticular proteins, molting fluid proteins, hemolymph proteins, and antimicrobial peptides. Injection of dsRNA targeting essential genes, inhibitor of apoptosis (IAP), Actin, and vacuolar-type ATPase (VATPase) interfered with Verson's glands growth. These results revealed that Verson's glands may contribute to hemolymph, cuticle, molting fluid, and immune response during molting. This study also provide useful tools for future research in identifying the physiological role of Verson's glands in lepidopteran insects.

Identification of species-specific juvenile hormone response elements in the fall armyworm, Spodoptera frugiperda.

Juvenile hormones (JH) regulate insect development and reproduction. The JH analogs (JHA) are used as insecticides. However, JHAs are rarely used in managing pests such as the fall armyworm, Spodoptera frugiperda that cause damage during larval stages. The insecticides that antagonize JH action and induce stoppage of feeding and precocious metamorphosis might work better to control these pests. Treating insects with JHA insecticides induces the expression of an early JH response gene, Krüppel homolog 1 (Kr-h1) by working through JH response elements (JHRE) present in its promoter. In this study, we identified JHREs present in the promoter of Kr-h1 gene of a global pest, S. frugiperda, and used them to develop a JHRE-reporter cell platform to screen for JH analogs. JHA, methoprene induced the expression of SfKr-h1 both in vitro and in vivo. JHRE present in the promoters of two SfKr-h1 isoforms, SfKr-h1α and SfKr-h1ß were identified. In Sf9 cells, the knockout of isoform-specific JHRE affected JH response in an isoform-specific manner. We also found that S. frugiperda JHRE (SfJHRE) did not function in the mosquito Aedes aegypti Aag2 cells and Tribolium castaneum TcA cells. Similarly, Ae. aegypti AaJHRE and T. castaneum TcJHRE were only functional in cells derived from these insects. The nucleotide sequence at the 3'end to the conserved core JHRE E-box sequence seems to be responsible for the species specificity observed. Two stable cell lines expressing the luciferase and enhanced green fluorescent protein genes under the control of SfJHRE were established. These cell lines responded well to JHA; these two JHRE-reporter cell lines could be used in screening assays to identify insecticides to manage S. frugiperda and other major pests.

SoxC is Required for Ecdysteroid Induction of Neuropeptide Genes During Insect Eclosion.

In insects, the shedding of the old exoskeleton is accomplished through ecdysis which is typically followed by the expansion and tanning of the new cuticle. Four neuropeptides, eclosion hormone (EH), ecdysis triggering hormone (ETH), crustacean cardioactive peptide (CCAP) and bursicon (Bur) are known to control ecdysis. However, the regulation of these neuropeptide genes is still poorly understood. Here, we report that in the red flour beetle (RFB) Tribolium castaneum and the fall armyworm (FAW) Spodoptera frugiperda, knockdown or knockout of the SoxC gene caused eclosion defects. The expansion and tanning of wings were not complete. In both RFB and FAW, the knockdown or knockout of SoxC resulted in a decrease in the expression of EH gene. Electrophoretic mobility shift assays revealed that the SfSoxC protein directly binds to a motif present in the promoter of SfEH. The luciferase reporter assays in Sf9 cells confirmed these results. These data suggest that transcription factor SoxC plays a key role in ecdysteroid induction of genes coding for neuropeptides such as EH involved in the regulation of insect eclosion.

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda.

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic pest management strategies. The most used transposable element in insect transformation is piggyBac, and piggyBac-based germline transformation has been successfully conducted in model insects. However, it is still challenging to employ this technology in non-model insects that include agricultural pests. This paper reports on germline transformation of a global agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda, using the hyperactive piggyBac transposase (hyPBase). In this work, the hyPBase mRNA was produced and used in place of helper plasmid in embryo microinjections. This change led to the successful generation of transgenic FAW. Furthermore, the methods of screening transgenic animals, PCR-based rapid detection of transgene insertion, and thermal asymmetric interlaced PCR (TAIL-PCR)-based determination of the integration site, are also described. Thus, this paper presents a protocol to produce transgenic FAW, which will facilitate piggyBac-based transgenesis in FAW and other lepidopteran insects.

The genome of the black cutworm Agrotis ipsilon.

The black cutworm (BCW), Agrotis ipsilon, is a worldwide polyphagous and underground pest that causes a high level of economic loss to a wide range of crops through the damage of roots. This species performs non-directed migration throughout East and Southeast Asia seasonally. Lack of a genome information has limited further studies on its unique biology and the development of novel management approaches. In this study, we present a 476 Mb de novo assembly of BCW, along with a consensus gene set of 14,801 protein-coding gene models. Quality controls show that both genome assembly and annotations are high-quality and mostly complete. We focus manual annotation and comparative genomics on gene families that related to the unique attributes of this species, such as nocturnality, long-distance migration, and host adaptation. We find that the BCW genome encodes a similar gene repertoire in various migration-related gene families to the diural migratory butterfly Danaus plexiipus, with additional copies of long wavelength opsin and two eye development-related genes. On the other hand, we find that the genomes of BCW and many other polyphagous lepidopterans encode many more gustatory receptor genes, particularly the lineage-specific expanded bitter receptor genes, than the mono- or oligo-phagous species, suggesting a common role of gustatory receptors (GRs) expansion in host range expansion. The availability of a BCW genome provides valuable resources to study the molecular mechanisms of non-directed migration in lepidopteran pests and to develop novel strategies to control migratory nocturnal pests.

The draft genome of the Asian corn borer yields insights into ecological adaptation of a devastating maize pest.

The Asian corn borer (ACB) is the most devastating pest on maize in the western Pacific region of Asia. Despite broad interests in insecticide resistance, seasonal adaptation, and larval color mimicry regarding the ACB system, lacking of reference genomic information and a powerful gene editing approach have hindered the in-depth studies of these aspects. Here we present a 455.7 Mb draft genome of ACB with 98.4% completeness. Comparative genomics analysis showed an evident expansion in gene families of gustatory receptors (105), which is related to polyphagous characteristics. Based on the comparative transcriptome analysis of resistant and susceptible ACB against Bt Cry1Ab toxin, we identified 26 genes related to Cry1Ab resistance. Additionally, transcriptomics of insects exposed to conditions of low temperature and diapause (LT) vs. room temperature and diapause (RT) provided insights into the genetic mechanisms of cold adaptation. We also successfully developed an efficient CRISPR/Cas9-based genome editing system and applied it to explore the role of color pattern genes in the ecological adaptation of ACB. Taken together, our study provides a fully annotated high-quality reference genome and efficient gene editing system to realize the potential of ACB as a study system to address important biological questions such as insecticide resistance, seasonal adaptation, and coloration. These valuable genomic resources will also benefit the development of novel strategies for maize pest management.

Mutation of P-element somatic inhibitor induces male sterility in the diamondback moth, Plutella xylostella.

BACKGROUND: Genetic manipulation of sex determination pathways in insects provides the basis for a broad range of strategies to benefit agricultural security and human health. The P-element somatic inhibitor (PSI) protein, an exon splicing silencer that promotes male-specific splicing of dsx, plays a critical role in male sexual differentiation and development. The functions of PSI have been characterized in the lepidopteran model species Bombyx mori. However, the molecular mechanism and functions of PSI in Plutella xylostella, a worldwide agricultural pest and taxonomically basal species, are still unknown. RESULTS: Here we identified PxPSI transcripts and analyzed their spatiotemporal expression pattern in P. xylostella. Multiple sequence alignment revealed that PxPSI contains four KH domains and is highly conserved in lepidopterans. We used the CRISPR-Cas9 system to generate mutations of the PxPSI genomic locus. Disruptions of PxPSI caused male-specific defects in internal and external genitals. In addition, we detected female-specific Pxdsx transcripts in PxPSI male mutants. Mutations also caused changes in expression of several sex-biased genes and induced male sterility. CONCLUSION: Our study demonstrates that PxPSI plays a key role in male sex determination in P. xylostella and suggests a potential molecular target for genetic-based pest management in lepidopteran pests. © 2021 Society of Chemical Industry.

Expanding the Toolkit for Genome Editing in a Disease Vector, Aedes aegypti: Transgenic Lines Expressing Cas9 and Single Guide RNA Induce Efficient Mutagenesis.

CRISPR-Cas9 mediated genome editing methods are being used for the analysis of gene function. However, it is hard to identify gene knockout mutants for genes whose knockout does not cause distinct phenotypes. To overcome this issue in the disease vector, Aedes aegypti, a transgenic Cas9/single guide RNA (sgRNA) method, was used to knock out the eye marker gene, kynurenine 3-monooxygenase (kmo), and the juvenile hormone receptor, Methoprene-tolerant (Met). PiggyBac transformation vectors were prepared to express sgRNAs targeting kmo and Met under the control of the U6 promoter. Transgenic Ae. aegypti expressing kmo-sgRNA or Met-sgRNA under the control of the U6 promoter and enhanced green fluorescent protein (eGFP) under the control of the hr5ie1 promoter were produced. The U6-sgRNA adults were mated with AAEL010097-Cas9 adults. The progeny were screened, and the insects expressing eGFP and DsRed were selected and evaluated for mutations in target genes. About 77% and 78% of the progeny that were positive for both eGFP and DsRed in kmo-sgRNA and Met-sgRNA groups, respectively, showed mutations in their target genes.

Caenorhabditis elegans systemic RNA interference defective protein 1 enhances RNAi efficiency in a lepidopteran insect, the fall armyworm, in a tissue-specific manner.

RNA interference (RNAi) is an important tool for gene function studies in insects, especially in non-model insects. This technology is also being developed for pest control. However, variable RNAi efficiency among insects is limiting its use in insects. Systemic RNAi in Caenorhabditis elegans requires systemic RNA interference defective protein 1 (CeSid1). The expression of CeSid1 in insect cell lines was shown to improve RNAi. However, the mechanisms through which this double-stranded RNA (dsRNA) transporter improves RNAi efficiency in insects is not known. We stably expressed CeSid1 in two Spodoptera frugiperda cell lines, Sf9 and Sf17 cells derived from ovary and midgut, respectively. Expression of CeSid1 enhanced RNAi efficiency in ovarian Sf9 cells, but not in midgut Sf17 cells. Reduced accumulation of dsRNA in late endosomes and successful processing dsRNA to siRNA contribute to enhanced RNAi efficiency in Sf9 cells. Transgenic S. frugiperda expressing CeSid1 were produced and tested for RNAi efficiency. RNAi efficiency enhancement due to CeSid1 expression showed tissue specificity. Compared to RNAi efficiency in wild-type S. frugiperda, CeSid1 expressing transgenic S. frugiperda showed a significant improvement of RNAi in tissues such as Verson's glands. In contrast, no improvement in RNAi was observed in tissues such as midgut. The in vitro cell-type specific and in vivo tissue-specific enhancement of RNAi efficiency by CeSid1 in S. frugiperda provides valuable information for improving RNAi in insects such as those belonging to order Lepidoptera where RNAi is variable and inefficient.

Identification and characterization of highly active promoters from the fall armyworm, Spodoptera frugiperda.

The cell lines derived from the fall armyworm (FAW), Spodoptera frugiperda, have been widely used for production of recombinant proteins for applications in both basic research and applications in medicine and agriculture. Promoters from the nucleopolyhedrovirus (NPV) are commonly used in these expression systems. These promoters have some limitations, which may be overcome by using promoters of genes from S. frugiperda. However, information on these promoters is not available. We identified several highly expressed genes from the transcriptomes of S. frugiperda midgut, fat body, epidermis, ovarian cell line (Sf9), and a midgut cell line (Sf17). The activity of potential promoters of 21 highly expressed genes was evaluated in Sf9 and Sf17 cells. Two of these promoters, SfHSC70-P1780 and SfPub-P2009, showed higher activity than commonly used hr5/ie1 (baculovirus enhancer element, hr5 and immediate early gene 1, ie1) promoter. Interestingly, the activity of these two promoters increased after adding hr5 enhancer element. The hr5/SfPub-P2009 promoter performance was evaluated by expressing an exogenous P450 protein in Sf9 cells using a plasmid-based expression system. The activity of this promoter was also evaluated in the FAW by expressing green fluorescence protein using the baculovirus expression system. In both cases, the hr5/SfPub-P2009 promoter performed better than the commonly used hr5/ie1 promoter. These strong endogenous promoters will be useful for studies in S. frugiperda and other lepidopteran insects for multiple applications, including protein expression, genome editing, and transgenic insects.

Identification and functional analysis of promoters of heat-shock genes from the fall armyworm, Spodoptera frugiperda.

The functional information on heat-shock proteins (Hsp) and heat-shock promoters from an important agricultural insect pest, Spodoptera frugiperda, is still lacking. We conducted a genome-wide identification of Hsp genes and identified a total of 21 genes belonging to four major insect Hsp families (small heat-shock proteins, Hsp60, Hsp70, and Hsp90) in S. frugiperda. Expression of most of S. frugiperda (SfHsp) genes could be detected in Sf9 cells, embryos and larval tissues of S. frugiperda. The heat-inducible activity of heat-shock promoters from several SfHsp genes was tested in Sf9 cells and embryos. The promoter of SfHsp70D showed the high constitutive activity in cell line and embryos, while the activity of SfHsp20.15 and SfHsp20.71 promoters was most dramatically induced in Sf9 cells and embryos. In embryos, the heat-induced activity of SfHsp20.71 and SfHsp70D promoters outperformed commercially used ie1 and ie2 promoters. The heat-induced activity of SfHsp70D and SfHsp19.07 promoters were more robust than ie2 promoter in Sf9 cells. These SfHsp promoters with high basal activity or with heat-induced activity from low basal activity, could be used in S. frugiperda or other lepidopteran insects for many applications including transgenesis and genome editing.

Mutation of doublesex in Hyphantria cunea results in sex-specific sterility.

BACKGROUND: The gene doublesex (dsx) plays pivotal roles in sex determination and controls sexually dimorphic development in certain insects. Importantly, it also displays a potential candidate target for pest management due to its sex-specific splicing. Therefore, we used CRISPR/Cas9-mediated gene disruption to investigate the function of dsx in Hyphantria cunea, an invasive forest pest. RESULT: In the present study, we identified the dsx gene from H. cunea which showed a sex-biased expression pattern that was different from other lepidopteran insects. Referring to sex-specific functional analyses in Bombyx mori, we performed a site-specific knockout of the Hcdsx gene by using a CRISPR/Cas9 system, which induced severe abnormalities in external genitalia and some incomplete sex reversal phenotypes, which in turn led to reduced sex-specific fecundity. An alternative splicing pattern of Hcdsx was altered by CRISPR/Cas9-induced mutation, and alterations in splicing affected expression of downstream genes encoding pheromone binding protein 1, vg1 and vg2 (encoding vitellogenin), which contributed to the sex-specific sterility phenotypes in the Hcdsx mutants. CONCLUSION: The Hcdsx gene plays important roles in sexual differentiation in H. cunea. Disruption of Hcdsx induced sex-specific sterility, demonstrating a potential application in control of this pest. © 2019 Society of Chemical Industry.

Mutation of doublesex induces sex-specific sterility of the diamondback moth Plutella xylostella.

DOUBLESEX (DSX): the downstream gene in the insect sex determination pathway, plays a critical role in sexual differentiation and development. The functions of dsx have been characterized in several model insect species. However, the molecular mechanism and functions of sex determination of dsx in Plutella xylostella, an agricultural pest, are still unknown. In present study, we identified a male-specific and three female-specific Pxdsx transcripts in P. xylostella. Phylogenetic analyses and multiple sequence alignment revealed that Pxdsx is highly conserved in lepidopterans. The CRISPR/Cas9 technology was used to induce mutations in the male-specific isoform, the female-specific isoform, and common regions of Pxdsx. Disruptions of Pxdsx sex-specific isoforms caused sex-specific defects in external genitals and partial sexual reversal. In addition, we found that female specific transcripts were detected in PxdsxM male mutants and male-specific transcripts were detected in PxdsxF female mutants. Mutations also caused changes in expression of several sex-biased genes and induced sex-specific sterility. This study demonstrates that Pxdsx plays a key role in sex determination of P. xylostella and suggests novel genetic control approaches for the management of P. xylostella.

The Masc gene product controls masculinization in the black cutworm, Agrotis ipsilon.

Sex determination has been studied in the model lepidopteran species Bombyx mori, but it remains poorly understood in lepidopteran pests. In the present study, we identified and characterized the Masculinizer (Masc) gene in a Noctuidae pest species, Agrotis ipsilon. Sequence analysis revealed that AiMasc encodes a protein of 658 amino acids that has two CCCH-type zinc finger domains and two conserved cysteine residues (Cys-277 and Cys-280). We assessed the masculinizing activity of AiMasc in BmN cells and found that AiMasc induced expression of the male-specific doublesex isoform. Disruption of Masc via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) in A. ipsilon caused abnormalities in abdominal segments and external genitalia, resulting in male-specific sterility. These results suggest that Masc participates in the process of sex determination in A. ipsilon. Successful identification of sex-determination gene in a pest species may enable the development of novel genetic approaches for pest control.

CRISPR/Cas9-based mutation reveals Argonaute 1 is essential for pigmentation in Ostrinia furnacalis.

Ostrinia furnacalis (Lepidoptera: Pyralidae) is one of the most destructive agricultural pests in Asia. Traditional pest-management methods include sex pheromone capture, transgenic crops that produce Bacillus thuringiensis toxin, and pesticides. Although these strategies control pest populations effectively, they also cause negative side effects, including dramatically increased pesticide resistance, severe pollution, and hazards for human health. Recently developed genome editing tools provide new prospects for pest management and have been successfully used in several species. However, few examples have been reported in the agricultural pest O. furnacalis due to a lack in genomic information. In this report, we identified only one transcript of O. furnacalis Argonaute 1 (OfAgo1) gene from the genome and cloned the open reading frame. OfAgo1 presented the maximum expression at the embryo stage or in the fat body during the larval stages. To understand its function, an OfAgo1 mutant was constructed using the Clustered Regularly Interspaced Short Palindromic Repeat/RNA-guided Cas9 nuclease (CRISPR/Cas9). Mutagenesis of OfAgo1 disrupted cuticle pigmentation by down-regulating micro RNAs and pigmentation-related genes. This is the first report for the cloning and functional analysis of OfAgo1, revealing a role of OfAgo1 in cuticle pigmentation. The current report also established a CRISPR/Cas9 system in O. furnacalis, providing a new insight for pest management.

Disruption of sex-specific doublesex exons results in male- and female-specific defects in the black cutworm, Agrotis ipsilon.

BACKGROUND: Doublesex (dsx), the downstream gene in the insect sex-determination pathway, is a key regulator of sexually dimorphic development and behavior across a variety of insects. Manipulating expression of dsx could be useful in the genetic control of insects. However, information on the sex-specific function of dsx in non-model insects is lacking. RESULTS: In this work, we isolated a dsx homolog, which is alternatively spliced into six female-specific and one male-specific isoforms, from an important agricultural pest, the black cutworm, Agrotis ipsilon. Studies on the expression of sex-specific Aidsx mRNA during embryonic development showed that the sixth hour post oviposition is the key stage for sex determination in A. ipsilon. Functional analysis of Aidsx was conducted using a CRISPR/Cas9 system targeting female- and male-specific Aidsx exons. Disruptions of sex-specific Aidsx exons resulted in sex-specific, sexually dimorphic defects in external genitals, gonads and antennae, and expression of sex-specific genes as well as production of offspring in both sexes. CONCLUSION: Our results not only demonstrate that dsx is a key player determining A. ipsilon sexually dimorphic traits, but also provide a potential method for the genetic control of this pest. © 2018 Society of Chemical Industry.

CRISPR/Cas9-mediated Tyrosine hydroxylase knockout resulting in larval lethality in Agrotis ipsilon.

Tyrosine hydroxylase (TH) is involved in insect melanin and the catecholamine biosynthesis pathway. TH as an enzyme catalyzing the conversion of tyrosine to 3,4-dihydroxyphenylalanine is the first step reaction in the pathway. Although TH has been proven to affect the pigmentation of the epidermis and development in many insects, there is no report about physiological function of the TH gene in Agrotis ipsilon. Here we cloned the TH gene from A. ipsilon. Semi-quantitative real-time polymerase chain reaction (PCR) analysis showed that AiTH was expressed at all development stages. Moreover, its high expression levels in the head and epidermis suggest that it is mainly related to pigment deposition and insect development. Then, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system to target the AiTH gene: deletion events were detected at the target sites. Compared with the control group, a few mutants with the phenomenon of narrowing in the egg shell and embryos can develop but cannot hatch; the other hatched embryos were seriously dehydrated after hatching and died within the first day. Quantitative real-time PCR analysis revealed that TH was down-regulated in AiTH mutants. Here, our work demonstrated that AiTH plays an important role in growth and development of newly hatched larvae; meanwhile, it would be a promising target to explore a control strategy for A. ipsilon.

Identification of Key Residues Associated with the Interaction between Plutella xylostella Sigma-Class Glutathione S-Transferase and the Inhibitor S-Hexyl Glutathione.

Glutathione S-transferases (GSTs) are important detoxification enzymes involved in the development of metabolic resistance in Plutella xylostella. Uncovering the interactions between representative PxGSTs and the inhibitor S-hexyl glutathione (GTX), helps in the development of effective PxGST inhibitors for resistance management. As the PxGST most severely inhibited by GTX, PxGSTσ (sigma-class PxGST) adopts the canonical fold of insect GSTs. The formation of the PxGSTσ-GTX complex is mainly driven by H-bond and hydrophobic interactions derived from the side chains of favorable residues. Of the residues composing the active site of PxGSTσ, Lys43 and Arg99 are two hot spots, first reported in the binding of GSH derivatives to GSTs. Such differences indicate the metabolism discrimination of different insect GSTs. Unfavorable interactions between the PxGSTσ active site and GTX are depicted as well. The research guides the discovery and optimization of PxGSTσ inhibitors.

Double-stranded RNA binding protein, Staufen, is required for the initiation of RNAi in coleopteran insects.

RNA interference (RNAi) is being used to develop methods to control pests and disease vectors. RNAi is robust and systemic in coleopteran insects but is quite variable in other insects. The determinants of efficient RNAi in coleopterans, as well as its potential mechanisms of resistance, are not known. RNAi screen identified a double-stranded RNA binding protein (StaufenC) as a major player in RNAi. StaufenC homologs have been identified in only coleopteran insects. Experiments in two coleopteran insects, Leptinotarsa decemlineata and Tribolium castaneum, showed the requirement of StaufenC for RNAi, especially for processing of double-stranded RNA (dsRNA) to small interfering RNA. RNAi-resistant cells were selected by exposing L. decemlineata, Lepd-SL1 cells to the inhibitor of apoptosis 1 dsRNA for multiple generations. The resistant cells showed lower levels of StaufenC expression compared with its expression in susceptible cells. These studies showed that coleopteran-specific StaufenC is required for RNAi and is a potential target for RNAi resistance. The data included in this article will help improve RNAi in noncoleopteran insects and manage RNAi resistance in coleopteran insects.

Identification of yellow gene family in Agrotis ipsilon and functional analysis of Aiyellow-y by CRISPR/Cas9.

The yellow gene family has been identified in several model insects, but yellow genes were poorly identified in non-model insects and the functions of yellow genes are largely unknown. In this study, we identified seven yellow genes in an important agricultural pest Agrotis ipsilon. Each gene encodes a protein containing a major royal jelly domain. Phylogenetic analysis defined these genes as yellow-y, -b, -b2, -c, -d, -e, and -h, respectively. The A. ipsilon yellow genes yellow-b, -b2, and -c were stably expressed in all developmental stages and tissues analyzed, whereas the other four yellow genes had unique expression patterns, suggesting distinct physiological roles of each gene. Using the CRISPR/Cas9 system, we successfully disrupted yellow-y in A. ipsilon and obtained G0 insects with somatic mutations. Unlike the black of wild-type newly hatched larvae and of adults, the mutants were yellow, although in the pupal stage mutant coloration did not differ from wild-type coloration. This phenotype was inherited by G1 offspring. The G0 mutants did not show any growth deficiency compared with control insects; however, a dehydration-like phenotype was observed in newly hatched G1 larvae from sibling crossed mutants. Our results indicate that A. ipsilon yellow-y gene plays a role in body pigmentation and also might function in waterproofing.