The Role of Chitooligosaccharidolytic β-N-Acetylglucosamindase in the Molting and Wing Development of the Silkworm Bombyx mori

The insect glycoside hydrolase family 20 β-N-acetylhexosaminidases (HEXs) are key enzymes involved in chitin degradation. In this study, nine HEX genes in Bombyx mori were identified by genome-wide analysis. Bioinformatic analysis based on the transcriptome database indicated that each gene had a distinct expression pattern. qRT-PCR was performed to detect the expression pattern of the chitooligosaccharidolytic β-N-acetylglucosaminidase (BmChiNAG). BmChiNAG was highly expressed in chitin-rich tissues, such as the epidermis. In the wing disc and epidermis, BmChiNAG has the highest expression level during the wandering stage. CRISPR/Cas9-mediated BmChiNAG deletion was used to study the function. In the BmChiNAG-knockout line, 39.2% of female heterozygotes had small and curly wings. The ultrastructure of a cross-section showed that the lack of BmChiNAG affected the stratification of the wing membrane and the formation of the correct wing vein structure. The molting process of the homozygotes was severely hindered during the larva to pupa transition. Epidermal sections showed that the endocuticle of the pupa was not degraded in the mutant. These results indicate that BmChiNAG is involved in chitin catabolism and plays an important role in the molting and wing development of the silkworm, which highlights the potential of BmChiNAG as a pest control target.


Introduction
Crop pests pose a serious challenge to agricultural production and food security [1,2]. As such, target-based insecticides have high specificity and safety to non-target organisms and the environment; thus, they have attracted considerable attention [3][4][5][6][7][8]. Chitin is a long-chain polymer of N-acetylglucosamine that is present in many insect tissues including the epidermis, integument, trachea, salivary gland, and intestinal peritrophic membrane. Chitin serves as a protective and supporting polysaccharide in insect exoskeletons and the peritrophic membrane [9,10]. Abnormal chitin synthesis and degradation impairs insect growth and development, and can cause mortality [11]. Chitin metabolism-related enzymes could be promising targets for insect pest control [12].

Genome-Wide Identification and Expression Profiles of the HEX Genes in B. mori
To identify HEX genes in B. mori, the amino acid sequences from previously characterized HEXs from three species (N. lugens, O. furnacalis, and T. castaneum) were used to search the B. mori genome and transcriptome databases. A total of nine genes encoding proteins that contain the GH20 domain were identified in B. mori (Table 1). Different HEX genes contained different exon numbers ranging from one to 15 ( Figure 1A). Domain analysis showed that all putative HEXs contain GH20b and GH20 domains except for BmHexD-like, which only had the GH20 domain. Only BmHex-C, BmGlcNase1, and BmChiNAG have a putative signal peptide, and BmFDL-B has a transmembrane helix at the N-terminus ( Figure 1B). The GH20 HEX enzymes have two minimal model architectures: model A containing at least a non-catalytic GH20b domain and the catalytic one (GH20), and model B with only the catalytic GH20 domain. In model A, the non-catalytic domain GH20b is required for expression and to stabilize GH20 enzymes [15]. Therefore, most HEX genes in B. mori belong to model A and only BmHexD-like belongs to model B. To decipher the sequence conservation, a multiple sequence alignment of these HEXs was done and the GenBank accession numbers used in the multiple alignments are listed in Table S1. The results showed that the conserved catalytically active sites were located in the GH20 domain regions and the mean similarity between these HEX genes was 29.02% ( Figure S1). To categorize the evolutionary relationship of putative HEXs in silkworms with their homologous protein from other species, we compiled all 53 HEXs by a BLAST search and analyzed their phylogeny. Table S2 shows the GenBank accession numbers and the protein sequences used in the phylogenetic tree. Evolutionary analysis showed that these HEXs could be classified into five major groups, namely NAGI group, NAGII group, a fused lobes (FDL) group, a Hex group, and a HexD-like group ( Figure 1C). We analyzed the temporal expression profiles and tissue distribution of these HEX genes in silkworms based on a transcriptome database, namely SilkDB. The results showed that they are mainly expressed in the epidermis, head, silk gland, reproductive organs, and trachea ( Figure 1D). Specifically, BmFDL-B, BmHex-B, and BmHex-C were highly expressed in the anterior silk gland or middle silk gland. BmFDL-A, BmFDL-C, BmHex-B, BmHex-C, and BmHexD-like were highly expressed in reproductive organs. BmFDL-A and BmFDL-C were specifically expressed in the testis during all testing periods. BmHex-A was highly expressed in the epidermis during the period of the rapid growth of the silkworm. BmChiNAG was highly expressed during the larval wandering stage and mainly expressed in chitin-rich tissues such as the epidermis, head, and anterior silk gland. Compared to the other seven genes, BmChiNAG had the highest expression level in the epidermis at the larval wandering stage and showed a periodic trend that included high transcript levels peaking before each molt ( Figure 1E). Coincidentally, BmChiNAG was grouped with the enzymatically characterized NAGI group and previous HEXs studies on Nilaparvata lugens and Tribolium castaneum also showed that the NAGI group's genes are essential for the insects to successfully molt [16,23]. Therefore, to explore a potential target for insect pest control, BmChiNAG was selected for further study.
domain GH20b is required for expression and to stabilize GH20 enzymes [15]. Therefore, most HEX genes in B. mori belong to model A and only BmHexD-like belongs to model B. To decipher the sequence conservation, a multiple sequence alignment of these HEXs was done and the GenBank accession numbers used in the multiple alignments are listed in Table S1. The results showed that the conserved catalytically active sites were located in the GH20 domain regions and the mean similarity between these HEX genes was 29.02% ( Figure S1).

Sequence Analysis and Expression Patterns of BmChiNAG
To determine the sequence information of BmChiNAG, we cloned its open reading frame (ORF) from the silkworm epidermis and analyzed its sequence in detail. BmChi-NAG possesses an ORF of 1791 bp ( Figure S2), coding a BmChiNAG protein consisting of 596 amino acids with an estimated molecular mass (MM) and isoelectric point (pI) of 68.33 kDa and 5.26, respectively. Domain analysis revealed that the BmChiNAG protein contained a conserved GH20 catalytic domain (residues 211-554), an additional GH20b domain (residues 67-187), and a conserved catalytic motif (HMGGDEV×××CW). Based on the structure of O. furnacalis, Hex1 comparison domain models of BmChiNAG were constructed using SWISS-MODEL ( Figure 2A). Conserved active site residues were also found in the BmChiNAG protein sequence ( Figure 2B). Multiple sequence alignment illustrated that ChiNAGs from B. mori and other insects are highly conserved especially at the GH20 catalytic region ( Figure S3). For example, the deduced protein sequence of BmChiNAG shared a high similarity (74.62 and 72.77%) with T. ni and O. furnacalis compared with A. aegypti (36.11%). However, at the GH20 catalytic region, the BmChiNAG shared a higher similarity (82.14, 81.19 and 42.73%) with T. ni, O. furnacalis, and A. aegypti. Table S3 shows the species and GenBank accession numbers used in the multiple alignments. mis at the larval wandering stage and showed a periodic trend that included high transcript levels peaking before each molt ( Figure 1E). Coincidentally, BmChiNAG was grouped with the enzymatically characterized NAGI group and previous HEXs studies on Nilaparvata lugens and Tribolium castaneum also showed that the NAGI group's genes are essential for the insects to successfully molt [16,23]. Therefore, to explore a potential target for insect pest control, BmChiNAG was selected for further study.

Sequence Analysis and Expression Patterns of BmChiNAG
To determine the sequence information of BmChiNAG, we cloned its open reading frame (ORF) from the silkworm epidermis and analyzed its sequence in detail. BmChiNAG possesses an ORF of 1791 bp ( Figure S2), coding a BmChiNAG protein consisting of 596 amino acids with an estimated molecular mass (MM) and isoelectric point (pI) of 68.33 kDa and 5.26, respectively. Domain analysis revealed that the BmChiNAG protein contained a conserved GH20 catalytic domain (residues 211-554), an additional GH20b domain (residues 67-187), and a conserved catalytic motif (HMGGDEV×××CW). Based on the structure of O. furnacalis, Hex1 comparison domain models of BmChiNAG were constructed using SWISS-MODEL ( Figure 2A). Conserved active site residues were also found in the BmChiNAG protein sequence ( Figure 2B). Multiple sequence alignment illustrated that ChiNAGs from B. mori and other insects are highly conserved especially at the GH20 catalytic region ( Figure S3). For example, the deduced protein sequence of BmChiNAG shared a high similarity (74.62 and 72.77%) with T. ni and O. furnacalis compared with A. aegypti (36.11%). However, at the GH20 catalytic region, the BmChiNAG shared a higher similarity (82.14, 81.19 and 42.73%) with T. ni, O. furnacalis, and A. aegypti. Table S3 shows the species and GenBank accession numbers used in the multiple alignments.  qRT-PCR was performed to determine the tissue and temporal expression patterns of BmChiNA. A total of 12 tissues from silkworm on day 3 of the fifth instar were analyzed to determine the tissue-specific expression profiles. The mRNA of BmChiNAG was transcribed in all of the tested tissues, with the highest mRNA expression levels in the wing disc, followed by the hemocyte, epidermis, and head ( Figure 2C). We analyzed the temporal expression patterns in the wing disc and epidermis from day 1 of the fourth instar larva to day 1 of the pupa. The data showed that BmChiNAG expression was highest during the larval wandering stage both in the wing disc and the epidermis. (Figure 2D,E).

Effect of Functional Defects of BmChiNAG on the Wing Development
We previously constructed a BmChiNAG-knockout line in which the BmChiNAG gene was knocked out by CRISPR/cas9 [42]. A 38-base deletion on the third exon of the BmChiNAG gene resulted in premature termination of translation ( Figure S4). This caused the loss of the additional GH20b catalytic domain and the GH20 catalytic domain, and led to the failure of protein function. Accordingly, we used the BmChiNAG-knockout line to study the functional defects of BmChiNAG.
The lack of BmChiNAG severely impaired wing development in 39.2% of females with the BmChiNAG/+ heterozygote genotype ( Table 2). Mutant pupae exhibited tiny wings that did not cover the meta-thorax and their weights were significantly lower than those of normal-winged individuals ( Figure 3A,E). The area of mutanted wing was 77.34% smaller than normal ( Figure 3B,F). The wing membrane was wrinkled and the wing veins were both shorter and thicker than the control ( Figure 3C,G). To fully understand the precise roles of BmChiNAG in wing formation, the ultrastructure of a cross-section of the forewing was observed by scanning electron microscopy (SEM). In the normal wing structure, the upper wing membrane protrudes outward to form smooth, hollow, tube-like wing veins. Compared with the normal wing structure, the upper and lower wing membrane of the BmChiNAG/+ heterozygous were separated and wrinkled, and the wing veins were uneven in thickness and larger in diameter ( Figure 3D). Taken together, the BmChiNAG is required for normal wing membrane stratification and wing vein structure.

Effects of Functional Defects of BmChiNAG on the Morphology of Larvae and the Molting Process
The BmChiNAG mutant homozygotes exhibited multiple morphological abnormalities compared to the wild-type. First, the lack of BmChiNAG resulted in blackening of the head of the fifth instar larva and a smaller larval body size ( Figure 4A,D). Second, all of the BmChiNAG homozygous mutant larvae could not properly molt to the pupa. Their head capsule could not split and eventually they died at about 10 days into the pupal stage ( Figure 4B). Additionally, the weight of the mutant pupae was significantly lower than that of the wild-type pupae ( Figure 4E). Third, the pupa skin sections showed that the epidermal cells and endocuticle of the pupal skin were not degraded in the mutant ( Figure 4C). The skin of the homozygous pupae was significantly thicker than that of the wild-type ( Figure 4F).

Effects of Functional Defects of BmChiNAG on the Morphology of Larvae and the Molting Process
The BmChiNAG mutant homozygotes exhibited multiple morphological abnormalities compared to the wild-type. First, the lack of BmChiNAG resulted in blackening of the head of the fifth instar larva and a smaller larval body size ( Figure 4A,D). Second, all of the BmChiNAG homozygous mutant larvae could not properly molt to the pupa. Their head capsule could not split and eventually they died at about 10 days into the pupal stage ( Figure 4B). Additionally, the weight of the mutant pupae was significantly lower than that of the wild-type pupae ( Figure 4E). Third, the pupa skin sections showed that the epidermal cells and endocuticle of the pupal skin were not degraded in the mutant ( Figure  4C). The skin of the homozygous pupae was significantly thicker than that of the wildtype ( Figure 4F). Insects undergo periodic molting during their lifetime in order to ensure normal growth and development. Due to the knockout of BmChiNAG, the fifth instar larvae failed to molt successfully. To determine if BmChiNAG expression levels were affected by ecdysone given that ecdysone is closely related to insect molting and metamorphosis development, the fifth instar larvae were injected with 20E (20-hydroxyecdysone) and changes of the mRNA levels of BmChiNAG at different post-injection time points were observed. The expression levels of BmChiNAG were significantly increased after treatment with 20E ( Figure 4G). This indicated that BmChiNAG expression is regulated by 20E at the larval stage. Due to molting, including a series of continuous processes such as the secretion of molting fluid, the formation of a new epidermis and the shedding of the old epidermis occur, in which a variety of enzymes are involved. During molting processes, chitinases (Cht) and β-N-acetyl-hexosaminidases (HEX) are responsible for the hydrolysis of chitin to chitosan oligosaccharides and monosaccharides, respectively; phenol oxidase (PPO1) and tyrosine hydroxylase (Th) play a key role in the hardening of insect exoskeletons, while dopa decarboxylase (Ddc) and acetyltransferase (At) are involved in the tanning of newly formed cuticles [43]. To further study the effects of functional defects of BmChiNAG on the molting process, we explored the expression changes of these five molting-related genes in the BmChiNAG mutant homozygous epidermis. The qPCR results showed that compared to the control group, the expression levels of these molting-related genes were all significantly decreased in the mutant group ( Figure 4H). These results clearly indicate that BmChiNAG affects the expression of molting-related genes. Insects undergo periodic molting during their lifetime in order to ensure normal growth and development. Due to the knockout of BmChiNAG, the fifth instar larvae failed to molt successfully. To determine if BmChiNAG expression levels were affected by ecdysone given that ecdysone is closely related to insect molting and metamorphosis development, the fifth instar larvae were injected with 20E (20-hydroxyecdysone) and changes of the mRNA levels of BmChiNAG at different post-injection time points were observed.

Discussion
Chitin metabolism is an important process of successful insect molting and metamorphosis [11,44,45]. If chitin digestion and degradation in the old cuticle are hindered, insect growth and development will be blocked [10]. The key genes that affect insect molting are potential insect control targets [6,7,44]. HEXs are enzymes involved in chitin catabolism during insect growth [30]. Multiple HEX genes as a gene family are present in insects. Their roles have been studied in numerous insect species but a comprehensive and systematic study of all HEX genes only in three insect species has been conducted. There are four HEX genes in T. castaneum (Coleoptera) [16], five in D. melanogaster (Diptera) [22], and 11 in N. lugens (Homoptera) [23]. We identified nine HEX genes in B. mori and phylogenetic analysis grouped these HEX proteins into five major groups (Figure 1). The numbers of HEX genes differ among insect species, implying that the HEX family has undergone frequent gene loss and duplication [16]. Furthermore, the mean similarity between the HEX genes of B. mori was low (29.02%). These findings suggest that functional divergence of insect HEXs genes occurred as a result of species differentiation as they evolved in various habitats with different requirements. This can also be seen from spatial and temporal expression patterns. HEX family genes of some species have varied spatiotemporal expression patterns, with high abundance in specific tissues or development stages [16,23,43]. In B. mori, BmFDL-B, BmHex-B, and BmHex-C are highly expressed in the silk gland and this indicates that they may be involved in the formation of cocoon silk [41]. BmFDL-A, BmFDL-C, BmHex-B, BmHex-C, and BmHexD-like were highly expressed in reproductive organs, indicating that these genes may be involved in gametogenesis and fertilization [21,22]. BmChiNAG had high transcript levels in chitin-rich tissues and showed a cyclic trend with high transcript levels peaking before each molt. This suggests that BmChiNAG may be important in chitin catabolism and molting. Previous study showed that only the insects injected with dsRNA targeting the NAGI group's genes exhibited lethal phenotypes in N. lugens and T. castaneum [16,23]. Coincidentally, phylogenetic analysis in this study showed that BmChiNAG was grouped with the enzymatically characterized NAGI group. These results indicated that the genes of the NAGI group are essential for the insects to successfully molt and may be preferred targets for novel pesticides.
In 1927, the BmChiNAG protein was first purified from B. mori integument tissue and this enzyme was shown to react with chitooligosaccharides to produce GlcNAc [46]. In the present study, BmChiNAG seemed to be involved in chitin catabolism and played a crucial role in B. mori metamorphosis. First, BmChiNAG was highly expressed in chitin-rich tissues, such as the wing disc, epidermis, and head ( Figure 2). Specific high expression levels of BmChiNAG were also observed during the metamorphosis period in the wing disc and critical period of molting in the epidermis [47]. Second, BmChiNAG knockout impaired wing development in 39.2% of female moths with the BmChiNAG/+ heterozygote genotype (Figure 3). Wing-mutated individuals had small and curly wings during pupal and adult stages compared with wild-type B. mori. Studies in O. furnacalis, T. castaneum, and D. melanogaster also demonstrated that NAGs were important for the normal formation of adult wings. Moth wings are mainly made up of protein and chitin, which have a sandwichlike structure involving one protein layer sandwiched between two chitin layers [48]. The chitin fibers contributed to the mechanical strength of the lightweight and rigid wing [49]. In this study, the ultrastructure of a cross-section of the forewing showed that the lack of BmChiNAG affected the stratification of the wing membrane and the formation of the normal wing vein structure. This suggested that BmChiNAG may affect wing development by affecting chitin metabolism. Third, the BmChiNAG homozygous mutants had molting defects and eventually died. Relatively low expression of molting-related genes was caused by the lack of BmChiNAG (Figure 4). Molting is an essential insect developmental process in which a variety of enzymes are involved. Chitinases and HEXs are responsible for the hydrolysis of chitin to chitosan oligosaccharides and monosaccharides, respectively [9]. The lack of BmChiNAG may affect the complete hydrolysis of chitosan oligosaccharides into monosaccharides, which may inhibit the expression of BmCht and thus disrupt chitin degradation. Previous studies have shown that when the chitin metabolism-related NAG was suppressed, new chitin biosynthesis was also inhibited [28]. We speculate that this indirectly affected the expression of several genes related to cuticle tanning and hardening. The insect cuticle is composed of the envelope, the epicuticle (outer), the exocuticle (medial), and the endocuticle (inner). Among these, the exocuticle and endocuticle are composed of chitin and protein complexes [50]. The pupa skin sections showed that the endocuticle of pupa skin in the BmChiNAG homozygous mutants was not degraded, indicating that the BmChiNAG gene appears to be involved in chitin catabolism and plays a vital role in the molting process. Therefore, BmChiNAG may be a valuable insecticidal target because normal periodic molting and flight ability are key factors underlying insect growth, movement, and reproduction [51].
Some HEXs inhibitors have been uncovered via structure-based virtual screening [7,38,[52][53][54] but they are all based on the crystal structure information of OfHex1. This is because the OfHex1 of O. furnacalis is the only insect-derived HEX with crystal structure information. Thus, no HEXs inhibitors have been developed for the control of agricultural pests [30]. A major issue involving the application of HEXs inhibitors as pesticides is selectivity. Insecticides should consider the toxicity risks to non-target organisms such as humans and some economic insects [11]. B. mori is a lepidopteran model insect in various life science research due to its many basic physiological processes conserved among insects [42,55,56]. Thus, identifying the crystal structure information and regulatory mechanisms of HEXs in B. mori can provide clues to target-based insecticide design.
In summary, this study characterized nine HEX genes in B. mori using genome databases and focused on one of them, namely BmChiNAG. BmChiNAG is involved in insect chitin catabolism and plays an important role in successful molting and wing development. Future studies are needed to clarify the crystal structure information of BmChiNAG and to consider the crystal structures of targetable HEXs.

Experimental Animals
The control strain Dazao was obtained from the State Key Laboratory of Silkworm Genome Biology (Southwest University, Chongqing, China). The BmChiNAG-knockout line, in which the BmChiNAG gene was knockout by CRISPR/cas9, was obtained from a previous study [42]. Specifically, the single-guide RNA (sgRNA) target was predited by the online tool (http://crispr.dbcls.jp/, accessed on 15 July 2021) to obtain the target sites. Subsequently, the GeneArt™ Precision gRNA Synthesis Kit (Thermo Fisher Scientific, Shanghai, China) was used to synthesize sgRNA according to the manufacturer's manual. The eggs from Bombyx mori within 2 h of oviposition were collected and fixed on a microscope slide (Promega, USA). Then, the eggs were microinjected under a stereomicroscope (SZX-ILLK200, Olympus) with an individual injection volume of 10 nL mixture of sgRNA (500 ng/µL) and Cas9 protein (500 ng/µL, Thermo Fisher Scientific, Shanghai, China). Among the injected embryos (n = 240), 52.1% hatched and all individuals survived to the adult stage. The genomic DNA was extracted from the adult's wings and then PCR-amplified as well as sequenced to screen out the chimeric individuals of the BmChiNAG gene. The chimeric individuals were crossed with wild-type individuals to produce the progeny and then screen out the heterozygous mutant individuals with a loss of function of the BmChiNAG gene, and the heterozygous mutants were self-crossed to obtain homozygous mutant individuals.
These B. mori strains were reared on fresh mulberry leaves at 25 • C under a 12:12 h (L:D) photoperiod. Adults mated and laid eggs at room temperature.

Multiple Sequence Alignments, Phylogenetic Analysis, and 3D Structure Prediction
The multiple sequence alignment of HEXs and homologous proteins downloaded from NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 23 June 2021) were created by Clustal X and Jalview software, and a phylogenetic tree was constructed by MEGA 7 software with the Neighbor-joining method with 1000 bootstrap values to confirm the reliability of the branching. The 3D structure of BmChiNAG was predicted by SWISS-MODEL online (http://swissmodel.expasy.org/interactive, accessed on 23 June 2021). PyMOL bioinformatics software was used to highlight the conserved domains and motifs of the 3D structures.

RNA Isolation and cDNA Synthesis
To analyze tissue-specific expression, 12 tissues (head, midgut, fat body, Malpighian tubule, silk gland, wing disc, hemocyte, epidermis, nerve, muscle, and male and female reproductive organs) were dissected from silkworms on day 3 of the 5th instar. The wing disc and epidermis of B. mori from day 1 of the 4th instar larva to day 1 of the pupae samples were prepared to detect the temporal expression patterns. RNA was extracted and purified by an SV Total RNA Isolation System Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. After integrity confirmation by agarose gel electrophoresis and concentration determination by NanoDrop TM 2000 (Thermo Fisher, Wilmington, DE, USA), 2 µg RNA was used to synthesize the first-strand cDNA using the SuperScript TM III Reverse Transcriptase System (Invitrogen, Carlsbad, CA, USA).

Molecular Cloning of BmChiNAG
The mRNA full-length sequence of BmChiNAG was obtained from B. mori full-length transcripts [60]. The open reading frame (ORF) of BmChiNAG was predicted using the ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html, accessed on 27 June 2021). Polymerase chain reaction (PCR) was employed to clone the ORF of BmChiNAG with gene-specific primers (Table S4). cDNA synthesized from total RNA isolated from the epidermis at the larval wandering stage was used as the template. PCR was performed by initially denaturing the cDNA template for 2 min at 98 • C followed by 35 cycles, each consisting of 10 s at 98 • C, 15 s at 60 • C, 110 s at 68 • C, and a final extension step of 10 min at 70 • C. The PCR products were purified using an E.Z.N.A. TM Gel Extraction Kit (OMEGA, Norcorss, GA, USA) and the purified product was subcloned into a pEASY ® -Blunt Zero Cloning Kit (TransGen Biotech, Beijing, China) for sequencing from both directions.

Quantitative Real-Time PCR (qRT-PCR)
qPCR was performed to analyze the relative mRNA expression levels of selected genes. A Bio-rad CFX96 sequence detection system with an iTaqSYBRGreen (Bio-rad, Hercules, CA, USA) was used to assess the transcript level of the gene of interest. The relative expression level of each gene was calculated by the 2 −∆∆Ct method and normalized to the abundance of the eif4A gene. Comparisons of target gene expressions were performed using Student's t-test. The primers used for qRT-PCR are listed in Table S5.

20-Hydroxyecdysone (20E) Induction
Silkworm larvae at day 2 of the 5th instar were injected with 20E (Sigma Aldrich, St. Louis, MO, USA) to investigate its effect on the expression of BmChiNAG. Larvae were divided into two groups and each group contained >36 larvae. The 20E was diluted with 75% ethanol at a concentration of 10 µg/µL. Ten uL 20E (10 µg/µL) was injected with a glass capillary and 10 uL 75% ethanol was used as a control. One hour after injection, the silkworm was allowed to feed on mulberry leaves as normal. Epidermis samples were collected at 6 h, 12 h, 24 h, and 48 h post-injection.

Morphology Observation of Wings and Pupa Skins
The wing morphology of B. mori was observed using an encoded stereo microscope (M205A, Leica) and then Image-J was used to calculate the surface area of the wings. The wing and pupal skin were sectioned using a freezing microtome (HM525 NX, Thermo) to obtain serial sections of 3 µm thickness and then their ultrastructure was both observed and photographed by a fluorescence microscope (DP80, Olympus) as well as scanning electron microscopy (SU3500, HITACHI).

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.