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. 2025 May 14;33(4):1345–1356. doi: 10.1111/1744-7917.70070

SV2B is a crucial factor for early larval development in the silkworm, Bombyx mori

Yuting Zhang 1,2, Zhiping Xing 1,2, Hui Dong 1,2, Tao Lu 1,2, Yuping Deng 1,2, Zhipeng Li 1,2, Bo Hu 1,2,✉, Anjiang Tan 1,2,✉
PMCID: PMC13440980  PMID: 40369800

Abstract

Synaptic vesicle glycoprotein 2B (SV2B) gene plays a crucial role in neuromodulation and neurotransmission and is a key regulator of synaptotagmin trafficking. However, physiological functions of this gene in insects remain poorly understood. In this study, we investigated the function of the BmSV2B gene in growth and development of silkworms. Tissue expression profiling revealed that BmSV2B is highly expressed in head and midgut. A phylogenetic tree and sequence alignment demonstrated that this gene is highly conserved among lepidopteran insects. Knockout of BmSV2B using the clustered regularly interspaced small palindromic repeats (CRISPR) / CRISPR‐associated nuclease 9 (Cas9) system resulted in smaller body size compared to the wild type (WT) strain. In the BmSV2B mutants, the levels of triacylglycerol were dramatically lower than that in WT. Furthermore, we found that deletion of BmSV2B extended the developmental time of larvae and led to early larval death. High‐throughput RNA sequencing and quantitative real‐time polymerase chain reaction analysis showed that the expression levels of juvenile hormone‐degrading genes, digestive genes, 20‐hydroxyecdysone ‐response genes and forkhead box O (FOXO) were significantly affected by the absence of BmSV2B. Taken together, BmSV2B is essential for early larval development in silkworms and could serve as a potential target for insecticides, offering a more effective approach to pest control management.

Keywords: Bombyx mori, body size, CRISPR/Cas9, insecticide target, synaptic vesicle glycoprotein 2B

Using the CRISPR/Cas9 system, we successfully knocked out BmSV2B and found that the resulting mutants displayed a significantly smaller body size compared to wild type silkworms. Furthermore, the deletion of BmSV2B not only extended the duration of larval development but also resulted in early larval mortality.

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Introduction

Synaptic vesicle glycoprotein 2 (SV2) proteins are members of the major facilitator superfamily, a large family of membrane transporters widely expressed in almost all living organisms (Stout et al., 2019). The SV2 family comprises 3 members, SV2A, SV2B, and SV2C, which share 61%−64% sequence homology and 80% structural homology in mammals (Bajjalieh et al., 1994). Like other proteins in the major facilitator superfamily, SV2s possess 12 transmembrane domains, with cytosolic N‐ and C‐termini (Stout et al., 2019). These isoforms are expressed in different patterns across various regions of the brain and other tissues, suggesting specialized roles in different neural circuits (Bajjalieh et al., 1994). SV2 proteins play a critical role in neuromodulation and neurotransmission and are well‐known as crucial regulators of synaptotagmin trafficking (Liu et al., 2024b). In mice, the absence of SV2A results in death approximately 3 weeks after birth due to severe seizures (Crowder et al., 1999), while the depletion of SV2B has no effect on growth, and the knockout of SV2C exhibits lower dopamine release and develops a Parkinson's‐like phenotype (Janz et al., 1999; Dunn et al., 2017). The SV2B protein plays an essential role in the formation and maintenance of the glomerular capillary wall (Fukusumi et al., 2015). Additionally, SV2B knockout (KO) mice show protection against oxidative stress induced by Aβ25−35 and against a reduction in choline acetyltransferase (ChAT) activity within the hippocampus (Detrait et al., 2014). The absence or dysfunction of SV2B can lead to impaired neurotransmission and has been associated with various neurological disorders. For example, studies in animal models have indicated that the loss of SV2B can lead to motor deficits and cognitive impairments (Olson et al., 2015). Additionally, SV2B has been identified as a potential target for therapeutic interventions in epilepsy, as certain antiepileptic drugs interact with SV2B to enhance its function (Löscher et al., 2016). SV2B is thought to interact with other synaptic proteins to facilitate the fusion of synaptic vesicles with the presynaptic membrane, thereby promoting the release of neurotransmitters into the synaptic cleft (Yao et al., 2011). In a Drosophila model of Parkinson's disease (PD), SV2C plays a crucial role in mediating the inverse association between smoking and PD (Hill‐Burns et al., 2013; Olsen et al., 2023). However, the physiological functions and underlying mechanisms of SV2 proteins in other insects remain largely unknown.

Body size is a key trait for all organisms, controlling various aspects of survival and reproduction in individuals’ adaptation to their living environment (Koyama & Mirth, 2018; Texada et al., 2020; Yuan et al., 2020). Genes associated with the regulation of body size have been identified in multiple mammals and insects (Kemper et al., 2012; Nijhout & Callier, 2015). Several pathways have been examined in Drosophila melanogaster, including the insulin/insulin‐like growth factor and target of rapamycin signaling pathways, which contribute to the control of body size in response to nutrition (Hyun, 2013; Texada et al., 2020). For example, overexpression of DILP2 increases organismal size by increasing the cell size and cell number of individual organs (Brogiolo et al., 2001), and mutations in dTOR inhibit larval growth (Zhang et al., 2000). Forkhead box O (FOXO), a downstream transcription factor of the insulin‐signaling pathway, functions as a regulator of organ and body growth in insects. For instance, upregulation of dFOXO during early larval stages suppresses larval growth, and overexpression of dFOXO during the 3rd larval instar results in the development of adults with reduced body size in D. melanogaster (Kramer et al., 2003). In Tribolium castaneum, knockdown of FOXO led to a small body size phenotype, resulting from a reduction in growth rate (Lin et al., 2018). Similarly, BmHippo mutation induces smaller body size (Xu et al., 2018), and transgenic overexpression of Mlx and Mondo increases the larval body size in Bombyx mori (Qin et al., 2024). However, the role of SV2 proteins in body size has not been studied in insects.

In the present study, we investigated the functional role of the homolog of the SV2B gene in the lepidopteran model silkworm, B. mori, an economically important insect. Here, BmSV2B was identified by cloning and sequencing, and bioinformatic analysis and the expression profile in tissues were also analyzed. We knocked out the BmSV2B gene using a transgenic clustered regularly interspaced small palindromic repeats (CRISPR) / CRISPR‐associated nuclease 9 (Cas9) system. The silkworm larvae lacking BmSV2B exhibited a smaller body size, delayed development, and larval lethality. High‐throughput RNA sequencing (RNA‐seq) and quantitative real‐time polymerase chain reaction (qRT‐PCR) analysis revealed significant alterations in the transcript levels of digestive enzymes, 20‐hydroxyecdysone (20E)‐responsive genes, juvenile hormone (JH)‐degrading genes, and FOXO in the mutants. Our data demonstrate that BmSV2B is essential for regulation of insect growth and development, and can serve as a target gene for controlling lepidopteran pests.

Materials and methods

Silkworm strains

Nistari, a nondiapausing silkworm strain, was used for all experiments. Larvae were reared on mulberry leaves with a standard environment of 75% relative humidity (RH), 25 °C, and 12 h light : 12 h dark cycle (Xing et al., 2025).

Phylogenetic analysis

The SV2B sequences of B. mori and other species were obtained from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/). DNAMAN (https://www.dnaman.com/) was utilized to align the amino acid sequences (Larkin et al., 2007). The neighbor‐joining method, along with 1000 bootstrap replications, was used to construct the phylogenetic tree. Phylogenetic analyses were conducted using MEGA X (https://www.megasoftware.net/) (Tamura et al., 2011).

RNA isolation and complementary DNA (cDNA) synthesis

TRIzol Reagent (Invitrogen, USA) was utilized to extract the total RNAs. A NanoDrop 1000 Spectrophotometer (Thermo, USA) was used to quantify the RNA. Subsequently, the first‐strand cDNAs were synthesized using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Japan) following the manufacturer's instructions.

Quantitative real‐time PCR (qRT‐PCR) analysis

The expression levels of the BmSV2B gene were analyzed by qRT‐PCR according to our previous methods (Yang et al., 2021). Gene‐specific primers were designed using the Primer3web version 4.1.0 (https://bioinfo.ut.ee/primer3/). B. mori ribosomal protein 49 (Bmrp49) was used as the internal reference gene for analyzing BmSV2B expression. The reaction conditions were carried out following the manufacturer's instructions. The 2−ΔΔCT method was used to calculate the relative messenger RNA (mRNA) levels of BmSV2B. All assays were performed with 3 independent biological replicates. All methods and data were identified according to the guidelines for the minimum information for the publication of qRT‐PCR experiments (Bustin et al., 2009). All primers are listed in Table S1.

Plasmid construction and genetic transformation

A binary transgenic CRISPR/Cas9 system was employed to construct mutants, following our previous methods (Xing et al., 2025). The plasmids pBac [HR5IE1‐EGFP‐nos‐Cas9], pBac [HR5IE1‐EGFP‐3×P3‐Cas9] and pBac [IE1‐DsRed‐U6‐sgRNA] (U6‐sgRNA) were constructed according to previous methods (Li et al., 2023; Xing et al., 2025). Single guide RNAs (sgRNAs) were selected using CRISPR direct (http://crispr.dbcls.jp/). The Plasmid Midi Kit (Qiagen, Frankfurt, Germany) and phenol‐chloroform were used to extract and purify the plasmids. Microinjection was carried out using an Eppendorf microinjector. A mixture of transfection plasmids and piggyBac helper plasmids was co‐injected into fertile eggs laid within 8 h by microinjection. G1 positive larvae were screened using fluorescence microscopy (Nikon AZ100, Tokyo, Japan) based on the fluorescent markers enhanced green fluorescent protein (EGFP) and DsRed. Heterozygous F1 progeny from the cross of nos‐Cas9 or 3×P3‐Cas9 lines with the U6‐sgRNAs line were used in subsequent experiments.

Mutagenesis analysis

TransDirect® Animal Tissue PCR Kit (TransGenBiotech, Haidian, Beijing, China) was used to extract genomic DNA of F1 individuals. The genomic PCR conditions were as follows: 94 °C for 3 min, 35 cycles of 98 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, followed by a final extension period at 72 °C for 10 min. The PCR products were ligated into the PJET 1.2 vector and sequenced. All primers are listed in Table S1.

Total triacylglycerol (TAG) level measurement

Third‐instar silkworm larvae, with mulberry leaves removed, were dissected in phosphate‐buffered saline (PBS). Dissections were performed with 3 larvae per replicate, for a total of 3 replicates. The samples were then frozen in liquid nitrogen and stored at −80 °C. The frozen samples were homogenized in PBS on ice. The protein concentration of the samples was measured using the BCA Protein Assay Kit (Sangon Biotech, Shanghai, China), and the TAG level was detected using the TG Content Assay Kit (Sangon Biotech, Shanghai, China).

Glucose and trehalose contents measurements

Hemolymph (10−50 µL) of both wild type (WT) and BmSV2B mutant silkworms were collected into tubes. The glucose content was determined using the Glucose Content Assay Kit (Sangon Biotech, Shanghai, China) according to the instructions. Similarly, the trehalose content was determined using the Trehalose Content Assay Kit (Sangon Biotech, Shanghai, China) according to the instructions.

Body weight and length measurements

To measure the body mass, silkworm larvae were provided with enough fresh mulberry leaves. Starting from 0 h after hatching, the larvae were weighed once every 24 h (with 30 1st instar larvae in each group) for 2 consecutive days. From the 2nd molting (designated as L3D0), the larvae were weighed individually once every 24 h for 3 consecutive days using an electronic analytical balance (Sartorius, Göttingen, Germany) with a precision of 0.001 g. For the body length measurements, starting from 0 h after hatching, the body length of each larva was measured individually once every 24 h for 3 consecutive days. From the 2nd molting (designated as L3D0), the body length of each larva was also measured individually once every 24 h for another 3 consecutive days, with the reading accuracy reaching 0.1 cm.

High‐throughput RNA‐seq

The midgut of silkworm larvae in L3D2 were dissected. The NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) was used generate to RNA‐seq libraries according to the instructions. After RNA‐seq, the reads were quality‐checked, filtered, and mapped to the reference silkworm genome database (http://www.silkdb.org/silkdb/) using bowtie 2. The abundance of gene transcripts was measured by fragment per kilobase of exon per million fragments mapped (FPKM). The differentially expressed genes (DEGs) were identified by NOIseq method. Gene Ontology (GO) annotation of DEGs was analyzed by the Blast2GO software. Pathway assignments were determined following the Kyoto Encyclopedia of Genes and Genomes (KEGG) database using tBLASTn.

Statistical analysis

The results of multiple experiments are presented in the form of mean ± standard deviation, and the differences were evaluated using a two‐tailed Student's t‐test with GraphPad Prism 8 software (GraphPad, San Diego, CA, USA). The following values were used: ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant.

Results

Characterization and expression profiles of BmSV2B

The full length of BmSV2B sequence consists of 1 611 nucleotides, encoding 536 amino acids. Protein structure analysis showed that BmSV2B is a 12‐transmembrane protein (Fig. S1A). A phylogenetic tree, constructed from SV2B amino acid sequences of B. mori and 11 other species, showed that insect SV2B is widely divergent from human SV2B (Fig. S1B). Sequence alignment also demonstrated that the protein sequences are highly conserved among lepidopteran insects (Fig. S2). These results can serve as a foundation for transferring the transgenic system from the silkworm to other lepidopteran insects, thereby enhancing the efficacy of biological control strategies. The relative mRNA expression levels of BmSV2B were analyzed using qRT‐PCR. These results revealed that the BmSV2B gene is highly expressed in the head and midgut tissues (Fig. 1A). The expression levels of BmSV2B increase during larval development, peaking in the head at the 3rd d of the 5th instar stage (L5D3) and in the midgut at L5D1 (Figs. 1B and C). Additionally, we measured the expression of BmSV2B during starvation to explore its role in the silkworm's response to nutritional stress. Our results revealed that the expression of BmSV2B significantly decreased at 6, 12, 24, and 48 h following starvation treatment of the larvae (Fig. 1D), suggesting its potential involvement in regulating energy balance and nutrient metabolism.

Fig. 1.

Fig. 1

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Tissue expression profile and relative expression levels of BmSV2B after starvation. (A) Tissue expression profile of BmSV2B from the wild type (WT) at 5th instar d 3 (L5D3) were determined by qRT‐PCR. (B, C) The expression level of BmSV2B in head and midgut were analyzed in silkworm larvae from L2D2 to L5D4 in 3 biological replicates. (D) Relative expression levels of BmSV2B in the midgut of silkworm larvae during the starvation treatment from 6 to 48 h were determined in 3 biological replicates (each replicate containing 3 silkworms). The results are presented as the mean ± standard deviation, and ***P < 0.001. qRT‐PCR, quantitative real‐time polymerase chain reaction.

Targeted BmSV2B mutagenesis using the CRISPR/Cas9 system

The BmSV2B gene, located on the 9th chromosome, contains 11 exons and 10 introns. We have identified 2 sgRNA target sequences within the open reading frame. A binary transgenic CRISPR/Cas9 system was operated. In this system, U6 promoter was used to drive the expression of sgRNA targets, and nos or 3×P3 promoter was utilized to control the expression of the Cas9 protein (Figs. 2A and 3A). Two 20‐base pair (bp) sgRNA targets, designated as TS1 and TS2, are located in exon 4 and exon 7, respectively (Figs. 2B and 3B). A total of 320 fertile eggs were injected, of which 237 (74.1%) hatched and 198 larvae (62.0%) survived to adulthood. These adult insects were sib‐mated or crossed with WT, yielding a total of 2 broods (G1) (Table S2). The expression levels of BmSV2B between the WT and the mutant strains were measured by qRT‐PCR (Figs. 2C and 3C). The results showed that the transcript levels of BmSV2B were significantly decreased in the mutants. Furthermore, genomic mutagenesis was analyzed by PCR using the genomic DNA from the mutants, and different numbers of bases were deleted at the 2 target sites (Figs. 2D and 3D).

Fig. 2.

Fig. 2

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Clustered regularly interspaced small palindromic repeats (CRISPR) / CRISPR‐associated nuclease 9 (Cas9)‐mediated knockout and mutations detection. (A) Construction of piggyBac‐based transgenic plasmids. Expression of the 2 BmSV2B single guide RNAs (sgRNAs) is driven by 2 U6 promoters with DsRed as a fluorescent marker. Expression of the Cas9 under the control of a nos promoter is indicated by enhanced green fluorescent protein (EGFP). (B) Schematic depiction of the BmSV2B locus and sgRNA targeting sites. Exons are shown in black boxes and introns are marked with polyline. The sgRNA targeting sites, TS1 and TS2, are located on the sense strand of exon‐4 and exon‐7, respectively. (C) The relative messenger RNA (mRNA) expression levels of BmSV2B were analyzed in the midguts of the wild type (WT) and ΔSV2B at L1D2 and in newly hatched WT and ΔSV2B, respectively (ΔSV2B was referred to as the BmSV2B mutant). Three biological replicates were carried out. Bars represents mean ± standard deviation and statistical comparisons between 2 groups were performed using two‐tailed Student's t‐test (***P < 0.001). (D) Mutations detection of BmSV2B in offspring after crossing nos‐Cas9 and U6‐sgRNAs transgenic silkworm lines. The sgRNA targeting sequences are in black, and the adjacent protospacer motif (PAM) sequences are in red. Lowercase letters represent the bases inserted during the repair process, and the symbol “‐” represents the bases that are deleted.

Fig. 3.

Fig. 3

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Mutagenesis induced by the transgenic clustered regularly interspaced small palindromic repeats (CRISPR) / CRISPR‐associated nuclease 9 (Cas9) system. (A) Schematics of the plasmids pBac [IE1‐DsRed2‐U6‐BmSV2B sgRNAs] and pBac [IE1‐EGFP‐3×P3‐Cas9]. (B) Genomic structure of BmSV2B. The single guide RNA (sgRNA) target sequence is in green and the protospacer adjacent motif (PAM) sequences are in red. (C) The relative messenger RNA (mRNA) expression levels of BmSV2B in wild type (WT) and ΔSV2B insects. Rp49 was used as an internal reference gene for the analyses of BmSV2B expression. The error bars represent the mean ± standard deviation values. Significant difference is denoted using asterisks (Student's t‐test, *P < 0.05, ***P < 0.001). (D) Mutagenesis analysis by polymerase chain reaction (PCR). Using PCR‐based amplification and sequencing, we targeted regions in ΔSV2B and WT silkworms defined by sgRNAs TS1 and TS2. A range of large fragment deletions were identified in the ΔSV2B silkworms.

Fig. 4.

Fig. 4

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BmSV2B mutation reduced the body size of silkworm larvae. (A) BmSV2B mutations showed smaller body size at the 1st instar larval stage (L1D2). (B) Larval body weight changes during development. (C) Larval body length changes during development. The results are presented as the mean ± standard deviation. Asterisks indicate significant differences between WT and ΔSV2B (Student's t‐test, ***P < 0.001, ns, non‐significant). WT, wild type.

BmSV2B mutation caused developmental defects

Under the same feeding conditions, the BmSV2B mutant larvae, which are the offspring from the cross‐breeding of the nos‐Cas9 and U6‐sgRNAs transgenic silkworms, exhibited significantly reduced body sizes compared to the WT individuals on L1D2 (Fig. 4A). Similarly, the body sizes of the BmSV2B mutant offspring after crossing 3×P3‐Cas9 and U6‐sgRNAs transgenic silkworm lines were significantly smaller than WT individuals on L3D1 and L3D3 (Fig. 5A). Furthermore, we measured larval body weight and length, and found that the weights and lengths of the BmSV2B mutant larvae were dramatically reduced during the same developmental period (Figs. 4B, 4Cand 5B, 5C). We further determined the contents of TAG of WT and BmSV2B mutants. The result showed that the total TAG levels in BmSV2B mutants were lower than those in WT on L3D3 (Fig. 6A). The contents of glucose and trehalose did not significantly change (Figs. 6B and C). When WT larvae had entered the 3rd d of the 4th instar (L4D3), the development of the BmSV2B mutant larvae was delayed and mutants arrested at the 3rd instar period (Fig. 6D). All BmSV2B mutant offspring from the cross‐breeding of the nos‐Cas9 and U6‐sgRNAs transgenic silkworm lines died before the 2nd instar, and the offspring of 3×P3‐Cas9 and U6‐sgRNAs transgenic silkworms died before the 4th instar (Fig. 6E).

Fig. 5.

Fig. 5

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Effects of clustered regularly interspaced small palindromic repeats (CRISPR) / CRISPR‐associated nuclease 9 (Cas9) knock out BmSV2B in the silkworm. (A) Photographs of ΔSV2B and WT silkworms in L3D1 and L3D3 showed smaller size of mutants. (B, C) BmSV2B mutation reduced the body size. Larval body weight and length changes during development. The results are presented as the mean ± standard deviation. ***P < 0.001. WT, wild type.

Fig. 6.

Fig. 6

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The mutation of BmSV2B led to developmental defects. (A) Relative triacylglycerol (TAG) level in the silkworms from WT and ΔSV2B (L3D3) were determined in 3 biological replicates (each replicate contains tissue from 3 animals). (B, C) The contents of glucose and trehalose in WT and ΔSV2B. (D) Growth cycle. The stages of larval development in WT and ΔSV2B (L1, 1st instar; L2, 2nd instar; L3, 3rd instar; L4, 4th instar; L5, 5th instar; P, pupae). L1‐P stages are marked in red, blue, orange, green, purple, and gray sequentially through the developmental timeline. The dashed lines at time points 72, 144, 216, 288, and 408 h indicate the transition checkpoint of WT. The curve between different stages shows the molting ratio. (E) Survival statistics of larvae in WT and ΔSV2B. The results are presented as the mean ± standard deviation. **P < 0.01; ns, non‐significant. WT, wild type.

RNA‐seq and qRT‐PCR analysis in BmSV2B mutant silkworms

To elucidate the molecular mechanisms of BmSV2B in regulating growth and development, RNA‐seq analysis of the midgut of silkworm larvae was conducted. The expression levels of 3 033 genes were significantly affected in the midgut of BmSV2B mutant strains. Among these genes, 2 483 genes were upregulated and 550 genes were downregulated (Fig. 7A). GO enrichment analysis showed that metabolic processes, binding and catalytic activity changed significantly in the midgut of BmSV2B mutant strains and KEGG pathways enrichment analysis revealed that Sphingolipid signaling pathway, Neurotrophin signaling pathway, ErbB signaling pathway were remarkably affected (Figs. 7B and C). We analyzed the expression levels of the digestive enzymes (Alpha‐amylase, Beta‐fructofuranosidase, Trypsin 1, Trypsin 2, Trypsin 3, and Lipase 1), 20E‐responsive genes (ecdysone receptor [EcR], Ultraspiracle [USP], broad‐complex core protein [Br‐C], ecdysone‐induced protein 74A [E74A], beta‐Fushi tarazu factor 1 [βFTZ‐F1], ecdysone‐induced protein 75A [E75A], ecdysone‐induced protein 75B [E75B], hormone receptor 3 [HR3], hormone receptor 4 [HR4]), juvenile hormone degradation genes (juvenile hormone esterase [JHE], juvenile hormone epoxide hydrolase [JHEH], juvenile hormone diol kinase [JHDK]) and FOXO using qRT‐PCR. These results showed that the relative mRNA levels of the 6 digestive enzymes were significantly decreased in the mutants (Fig. 8A). Similarly, the transcript levels of E75A, E75B, HR3, EcR, JHE, and JHDK were also dramatically reduced in the mutants (Figs. 8B and C). Conversely, the expression of FOXO was significantly upregulated in the mutants (Fig. 8D). These results indicate that BmSV2B controls growth and development by regulating the expression of the digestive enzymes, 20E‐responsive genes, JH‐degrading genes, and FOXO.

Fig. 7.

Fig. 7

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RNA sequencing (RNA‐seq) analysis of the midgut in the BmSV2B mutants relative to the WT. (A) Statistical chart of significantly differentially expressed genes (DEGs) in WT and ΔSV2B silkworms. (B) GO enrichment analysis revealed significant differences in genes within the midgut of ΔSV2B compared to WT silkworms at L3D2. (C) The top 10 enriched KEGG pathways, which exhibited significant changes in the midgut of ΔSV2B compared to WT silkworms at L3D2. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; WT, wild type.

Fig. 8.

Fig. 8

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The relative messenger RNA (mRNA) expression of digestive genes (A), juvenile hormone‐degrading genes (B), 20‐hydroxyecdysone (20E)‐response genes (C), and forkhead box O (FOXO) (D) was analyzed in WT and ΔSV2B insects. The 2−ΔΔCT method was used for quantification of the gene expression level. The cycle threshold (Ct) values for the genes were normalized to the Ct values of Rp49. The results are presented as the mean ± standard deviation, and asterisks represent *P < 0.05 and ***P < 0.001; ns, non‐significant. WT, wild type.

Discussion

In mammals, SV2B is implicated in Alzheimer's disease pathogenesis, and SV2B knockout mice exhibit protection against toxicity and cognitive deficits induced by injected Aβ oligomers (Heese et al., 2001). In this study, we found that SV2B is highly conserved in lepidopteran insects and is particularly highly expressed in the midgut. Deletion of this gene resulted in reduced body size, impaired development, and larval death. RNA‐seq and qRT‐PCR analyses revealed that the mutation in BmSV2B affected the expression of digestive enzymes, 20E‐responsive genes, JH degradation genes, and FOXO. These results suggest that BmSV2B plays a crucial role in growth and development in silkworms.

In vertebrates, the synaptic vesicle glycoprotein 2 (SV2) family, which includes the 3 paralogs SV2A, SV2B, and SV2C, consists of a 12‐transmembrane protein that is ubiquitously present on every secretory vesicle, including synaptic vesicles, and play a pivotal role in neurotransmission (Stout et al., 2019). In this study, BmSV2B was identified as a 12‐transmembrane protein, and a phylogenetic tree revealed that SV2B is evolutionarily conserved, suggesting that its functional role may be highly conserved from mammals to insects. In mammals, SV2B is most strongly expressed in the trigeminal and motor nuclei, with very little expression in the globus pallidus, dentate gyrus of the hippocampus, cerebellum, and substantia nigra pars reticulata (Bajjalieh et al., 1994). Surprisingly, BmSV2B was found to be highly expressed in the midgut, indicating that this gene may play a significant role in growth and development in the midgut of silkworms.

Complex mechanisms regulate body size in insects (Texada et al., 2020). Inhibition of digestive enzymes can block development and reduce body size in insects (Ashouri & Farshbaf Pourabad, 2021). For instance, inactivation of β‐glucosidase by the inhibitor conduritol B epoxide decreased glucose concentration in the midgut and resulted in approximately a 25% loss of body weight in Coptotermes formosanus (Zhang et al., 2012). In Spodoptera frugiperda, treatment with trypsin inhibitors or knockdown of these genes by double‐stranded RNA (dsRNA) led to a significant reduction in body weight (Hafeez et al., 2021). Similarly, the mRNA expression levels of Alpha‐amylase, Beta‐fructofuranosidase, Trypsin 1, Trypsin 2, Trypsin 3, and Lipase 1 were significantly decreased in the BmSV2B‐deficient silkworms compared to the control. FOXO, a downstream transcription factor of the insulin‐signaling pathway, plays a crucial role in body growth. Overexpression of FOXO in silkworms resulted in a smaller body size (Lu et al., 2019). Here, the transcriptional level of FOXO was markedly increased in the BmSV2B mutants. Taken together, these results suggest that BmSV2B controls body size by positively regulating the expression of digestive enzyme genes and negatively mediating the expression of FOXO.

Two major hormones, JH and 20E, play crucial roles in regulating insect growth and development through their precisely coordinated levels, which are regulated by biosynthesis and degradation genes (Dubrovsky, 2005; Tan et al., 2005; Daimon et al., 2012; Li et al., 2019). In this study, we observed that the expression levels of JH degradation genes, JHE and JHDK, were significantly reduced. Our previous studies have shown that overexpression of JHE promotes larval development, while depletion of JHE results in the extension of larval stages (Zhang et al., 2017). Similarly, suppression of JHDK increased the JH titer and extended larval growth in Heortia vitessoides Moore (Lyu et al., 2019). Meanwhile, we also found that the mRNA levels of 20E‐response genes, E75A, E75B, HR3, and EcR, were dramatically decreased. In D. melanogaster, loss of the ecdysteroid‐inducible E75A orphan nuclear receptor reduced the ecdysteroid titer and led to developmental delay and arrest (Bialecki et al., 2002). Knockdown of HR3 and HR4 genes resulted in severe developmental delay in Sogatella furcifera (Long et al., 2023). These findings suggest that SV2B controls larval growth and development by regulating the expression of genes of JH and 20E pathways.

Chemical pesticides were the major methods of controlling agricultural pests. However, insect pests have developed serious resistance to multiple insecticides according to the Arthropod Pesticide Resistance Database (APRD 2016). Consequently, new insecticide targets are needed to control agricultural pests. B. mori is not only an economically important insect, but also a model lepidopteran insect (Zhang et al., 2024). Numerous studies have reported that silkworms have currently been used to search insecticide targets for pest control (Tan et al., 2022; Shen et al., 2024; Liu et al., 2024a). In this study, the absence of BmSV2B in silkworms resulted in larval death and the protein sequences were highly conserved in lepidopteran insects. These findings suggest that SV2B could serve as an eco‐friendly, novel insecticide target for the management of lepidopteran pests.

In conclusion, this study marks the first use of transgenic CRISPR/Cas9 technology in insects to elucidate the function of the SV2B gene. The results confirm the importance of SV2B for growth and development in B. mori. Depletion of BmSV2B not only inhibited normal growth and development but also led to larval death. This suggests that small molecule inhibitors or nucleic acid‐based pesticides targeting SV2B could be utilized in future control strategies for lepidopteran pests.

Disclosure

The authors have no conflict of interest.

Supporting information

Fig. S1 Schematic depiction and phylogenetic analysis of the BmSV2B.

Fig. S2 Amino acid sequence alignment of BmSV2B between insects and other lepidopteran species.

Table S1 The primers used in this paper.

Table S2 Efficiency statistics of embryo microinjection.

INS-33-1345-s001.docx (2.1MB, docx)

Acknowledgments

This work was supported by the National Science Foundation of China (31925007 and 32000333) and the Natural Science Foundation of Jiangsu Province (BK20221292).

Contributor Information

Bo Hu, Email: hubo2022@just.edu.cn.

Anjiang Tan, Email: atan@just.edu.cn.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1 Schematic depiction and phylogenetic analysis of the BmSV2B.

Fig. S2 Amino acid sequence alignment of BmSV2B between insects and other lepidopteran species.

Table S1 The primers used in this paper.

Table S2 Efficiency statistics of embryo microinjection.

INS-33-1345-s001.docx (2.1MB, docx)

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