Identification and Functional Analysis of the Pre-piRNA 3' Trimmer in Silkworms.

PIWI-interacting RNAs (piRNAs) play a crucial role in transposon silencing in animal germ cells. In piRNA biogenesis, single-stranded piRNA intermediates are loaded into PIWI-clade proteins and cleaved by Zucchini/MitoPLD, yielding precursor piRNAs (pre-piRNAs). Pre-piRNAs that are longer than the mature piRNA length are then trimmed at their 3' ends. Although recent studies implicated the Tudor domain protein Papi/Tdrkh in pre-piRNA trimming, the identity of Trimmer and its relationship with Papi/Tdrkh remain unknown. Here, we identified PNLDC1, an uncharacterized 3'-5' exonuclease, as Trimmer in silkworms. Trimmer is enriched in the mitochondrial fraction and binds to Papi/Tdrkh. Depletion of Trimmer and Papi/Tdrkh additively inhibits trimming, causing accumulation of ∼35-40-nt pre-piRNAs that are impaired for target cleavage and prone to degradation. Our results highlight the cooperative action of Trimmer and Papi/Tdrkh in piRNA maturation.

Studying the role of Bombyx mori molybdenum cofactor sulfurase in Bombyx mori nucleopolyhedrovirus infection.

Molybdenum cofactor sulfurase (MoCoS) is a key gene involved in the uric acid metabolic pathway that activates xanthine dehydrogenase to synthesise uric acid. Uric acid is harmful to mammals but plays crucial roles in insects, one of which is the immune responses. However, the function of Bombyx mori MoCoS in response to BmNPV remains unclear. In this study, BmMoCoS was found to be relatively highly expressed in embryonic development, gonads and the Malpighian tubules. In addition, the expression levels of BmMoCoS were significantly upregulated in three silkworm strains with different levels of resistance after virus infection, suggesting a close link between them. Furthermore, RNAi and overexpression studies showed that BmMoCoS was involved in resistance to BmNPV infection, and its antivirus effects were found to be related to the regulation of uric acid metabolism, which was uncovered by inosine- and febuxostat-coupled RNAi and overexpression. Finally, the BmMoCoS-mediated uric acid pathway was preliminarily confirmed to be a potential target to protect silkworms from BmNPV infection. Overall, this study provides new evidence for elucidating the molecular mechanism of silkworms in response to BmNPV infection and new strategies for the prevention of viral infections in sericulture.

Cocoonase is indispensable for Lepidoptera insects breaking the sealed cocoon.

Many insects spin cocoons to protect the pupae from unfavorable environments and predators. After emerging from the pupa, the moths must escape from the sealed cocoons. Previous works identified cocoonase as the active enzyme loosening the cocoon to form an escape-hatch. Here, using bioinformatics tools, we show that cocoonase is specific to Lepidoptera and that it probably existed before the occurrence of lepidopteran insects spinning cocoons. Despite differences in cocooning behavior, we further show that cocoonase evolved by purification selection in Lepidoptera and that the selection is more intense in lepidopteran insects spinning sealed cocoons. Experimentally, we applied gene editing techniques to the silkworm Bombyx mori, which spins a dense and sealed cocoon, as a model of lepidopteran insects spinning sealed cocoons. We knocked out cocoonase using the CRISPR/Cas9 system. The adults of homozygous knock-out mutants were completely formed and viable but stayed trapped and died naturally in the cocoon. This is the first experimental and phenotypic evidence that cocoonase is the determining factor for breaking the cocoon. This work led to a novel silkworm strain yielding permanently intact cocoons and provides a new strategy for controlling the pests that form cocoons.

Structural basis for juvenile hormone biosynthesis by the juvenile hormone acid methyltransferase.

Juvenile hormone (JH) acid methyltransferase (JHAMT) is a rate-limiting enzyme that converts JH acids or inactive precursors of JHs to active JHs at the final step of JH biosynthesis in insects and thus presents an excellent target for the development of insect growth regulators or insecticides. However, the three-dimensional properties and catalytic mechanism of this enzyme are not known. Herein, we report the crystal structure of the JHAMT apoenzyme, the three-dimensional holoprotein in binary complex with its cofactor S-adenosyl-l-homocysteine, and the ternary complex with S-adenosyl-l-homocysteine and its substrate methyl farnesoate. These structures reveal the ultrafine definition of the binding patterns for JHAMT with its substrate/cofactor. Comparative structural analyses led to novel findings concerning the structural specificity of the progressive conformational changes required for binding interactions that are induced in the presence of cofactor and substrate. Importantly, structural and biochemical analyses enabled identification of one strictly conserved catalytic Gln/His pair within JHAMTs required for catalysis and further provide a molecular basis for substrate recognition and the catalytic mechanism of JHAMTs. These findings lay the foundation for the mechanistic understanding of JH biosynthesis by JHAMTs and provide a rational framework for the discovery and development of specific JHAMT inhibitors as insect growth regulators or insecticides.

Cloning, expression, and characteristic analysis of the novel ß-galactosidase from silkworm, Bombyx mori.

ß-Galactosidase is a critical exoglycosidase involved in the hydrolysis of lactose, the modification and degradation of glycoprotein in vivo. In this study, the ß-galactosidase gene of silkworm (BmGal), whose cDNA comprises 11 exons and contains an intact ORF of 1,821 bp, was cloned. The protein sequence of BmGal showed high similarity with other known insect ß-galactosidases. No activity of the BmGal expressed in Escherichia coli or Pichia pastoris was detected while it was successfully expressed with high enzyme activity in baculovirus expression system in silkworm, and the electrophoresis result revealed that the BmGal showed activity in oligomer mode. Enzyme activity assay showed that its optimum pH was 8.4 and its optimum temperature was 40 °C. What is more, we found that iron ions can stimulate the activity of the enzyme while cobalt, nickel, or lead ions can inhibit its activity significantly. Besides, the temporal-spatial transcription pattern of the BmGal mRNA level was analyzed, which showed that BmGal was transcribed at the highest level in the fifth larval instar but relatively low level in the pupal and adult stage, and the highest transcriptional level of BmGal was found in testis among all the tissues concerned.

Structure-function analysis of silkworm sucrose hydrolase uncovers the mechanism of substrate specificity in GH13 subfamily 17 exo-α-glucosidases.

The domestic silkworm Bombyx mori expresses two sucrose-hydrolyzing enzymes, BmSUH and BmSUC1, belonging to glycoside hydrolase family 13 subfamily 17 (GH13_17) and GH32, respectively. BmSUH has little activity on maltooligosaccharides, whereas other insect GH13_17 α-glucosidases are active on sucrose and maltooligosaccharides. Little is currently known about the structural mechanisms and substrate specificity of GH13_17 enzymes. In this study, we examined the crystal structures of BmSUH without ligands; in complexes with substrates, products, and inhibitors; and complexed with its covalent intermediate at 1.60-1.85 Å resolutions. These structures revealed that the conformations of amino acid residues around subsite -1 are notably different at each step of the hydrolytic reaction. Such changes have not been previously reported among GH13 enzymes, including exo- and endo-acting hydrolases, such as α-glucosidases and α-amylases. Amino acid residues at subsite +1 are not conserved in BmSUH and other GH13_17 α-glucosidases, but subsite -1 residues are absolutely conserved. Substitutions in three subsite +1 residues, Gln191, Tyr251, and Glu440, decreased sucrose hydrolysis and increased maltase activity of BmSUH, indicating that these residues are key for determining its substrate specificity. These results provide detailed insights into structure-function relationships in GH13 enzymes and into the molecular evolution of insect GH13_17 α-glucosidases.

Heat shock protein 70, glutamate dehydrogenase, and angiotensin-converting enzyme of Bombyx mori mediate the cell attachment of Cypovirus 1.

Dendrolimus punctatus causes great damage to pine forests worldwide. Dendrolimus punctatus cypovirus 1 (DpCPV-1) is an important pathogen of D. punctatus. However, the mechanism of DpCPV-1 cell entry has not been elucidated. In this study, we revealed that both GTase and MTase domains of VP3 (B-spike) and VP4 (A-spike) of DpCPV-1 interacted with the midgut proteins of Bombyx mori. Binding and competition assays revealed that GTase, MTase and VP4 played roles as viral attachment proteins. Far-Western blotting and LC-MS/MS analyses identified that heat shock protein 70 (BmHSP70), glutamate dehydrogenase (BmGDH), and angiotensin-converting enzyme (BmACE) in the midgut proteins as ligand candidates of the viral attachment proteins, and this was further verified by co-immunoprecipitation and fluorescence co-localization assays. Viral binding to the host midgut in vitro was inhibited by pre-treating B. mori midgut proteins with anti-BmHSP70, anti-BmGDH, anti-BmACE antibodies singly and in combination. Incubating DpCPV-1 virions with prokaryotically expressed BmHSP70, BmGDH, and BmACE also decreased viral attachment to the host midgut. In vivo bioassays revealed that viral infection in Helicoverpa armigera was partially neutralized by BmHSP70, BmGDH, and BmACE. Taking together, we concluded that HSP70, GDH, and ACE mediate DpCPV attachment and entry via binding to the viral attachment proteins, VP3 and VP4. The findings provide foundation for further understanding the entry mechanisms of cypoviruses.

Milligram scale expression, refolding, and purification of Bombyx mori cocoonase using a recombinant E. coli system.

Silk is one of the most versatile biomaterials with signature properties of outstanding mechanical strength and flexibility. A potential avenue for developing more environmentally friendly silk production is to make use of the silk moth (Bombyx mori) cocoonase, this will at the same time increase the possibility for using the byproduct, sericin, as a raw material for other applications. Cocoonase is a serine protease utilized by the silk moth to soften the cocoon to enable its escape after completed metamorphosis. Cocoonase selectively degrades the glue protein of the cocoon, sericin, without affecting the silk-fiber made of the protein fibroin. Cocoonase can be recombinantly produced in E. coli, however, it is exclusively found as insoluble inclusion bodies. To solve this problem and to be able to utilize the benefits associated with an E. coli based expression system, we have developed a protocol that enables the production of soluble and functional protease in the milligram/liter scale. The core of the protocol is refolding of the protein in a buffer with a redox potential that is optimized for formation of native and intramolecular di-sulfide bridges. The redox potential was balanced with defined concentrations of reduced and oxidized glutathione. This E.coli based production protocol will, in addition to structure determination, also enable modification of cocoonase both in terms of catalytic function and stability. These factors will be valuable components in the development of alternate silk production methodology.

Bmcas-1 plays an important role in response against BmNPV infection in vitro.

Apoptosis, as one kind of innate immune system, is involved in host response against pathogens innovation. Caspases play a vital role in the execution stage of host cell apoptosis. It has been reported that Bmcaspase-1 (Bmcas-1) has a close relationship with Bombyx mori nucleopolyhedrovirus (BmNPV) infection for its differentially expressed patterns after viral infection. However, its underlying response mechanism is still unclear. The significant differential expression of Bmcas-1 in different tissues of differentially resistant strains revealed its vital role in BmNPV infection. To further validate its role in BmNPV infection, budded virus (BV)-eGFP was analyzed after knockdown and overexpression of Bmcas-1 by small interfering RNA and the pIZT-mCherry vector, respectively. The reproduction of BV-eGFP obviously increased at 72 h after knockdown of Bmcas-1, and decreased after overexpression in BmN cells. Moreover, the conserved functional domain of Cas-1 among different species and the closed evolutionary relationship of Cas-1 in Lepidoptera hinted that Bmcas-1 might be associated with apoptosis, and this was also validated by the apoptosis inducer, Silvestrol, and the inhibitor, Z-DEVD-FMK. Therefore, Bmcas-1 plays an essential antiviral role by activating apoptosis, and this result lays a fundament for clarifying the molecular mechanism of silkworm in response against BmNPV infection and breeding of resistant strains.

Gene mapping reveals the association between tyrosine protein kinase Abl1 and the silk yield of Bombyx mori.

The Z chromosome of the silkworm contains a major gene that influences silk yield. This major locus on chromosome Z accounts for 35.10% of the phenotypic variance. The location and identification of the gene have been a focus of silkworm genetics research. Unfortunately, identification of this gene has been difficult. We used extreme phenotype subpopulations and selected from a backcross population, BC1 M, which was obtained using the high-yield strain 872B and the low-yield strain IS-Dazao as parents, for mapping the gene on the chromosome Z. The candidate region was narrowed down to 134 kb at the tip of the chromosome. BmAbl1 in this region correlated with silk gland development by spatiotemporal expression analysis. This gene was differentially expressed in the posterior silk glands of the high- and low-yield strains. In BmAbl1, an insertion-deletion (indel) within the 10th exonic region and an SNP within the 6th intronic region were detected and shown to be associated with cocoon shell weight in 84 Bombyx mori strains with different yields. Nucleotide diversity analysis of BmAbl1 and its 50 kb flanking regions indicated that BmAbl1 has experienced strong artificial selection during silkworm domestication. This study is the first to identify the genes controlling silk yield in the major QTL of the Z chromosome using forward genetics.

Identification of the in vitro antiviral effect of BmNedd2-like caspase in response to Bombyx mori nucleopolyhedrovirus infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most serious pathogens in sericulture, and the underlying antiviral mechanism in silkworm is still unclear. Bombyx mori Nedd2-like caspase (BmNc) has been identified as a candidate antiviral gene from previous transcriptome data, since it is differentially expressed in the midgut of differentially resistant silkworm strains following BmNPV infection. However, the molecular mechanism by which BmNc responds to BmNPV is unknown. In this study, the relationship between BmNc and BmNPV was confirmed by its significantly different expression in different tissues of differentially resistant strains after BmNPV infection. Moreover, the antiviral role of BmNc was confirmed by the significantly higher fluorescence signals of BV-eGFP after knockdown of BmNc in BmN cells, and a reduced signal after overexpression. This was further verified by the capsid gene vp39 expression, DNA copy number, and GP64 protein level in the RNAi and overexpression groups. Furthermore, the antiviral phenomenon of BmNc was found to be associated with apoptosis. In brief, BmNc showed a relatively high expression level in the metamorphosis stages, and the effect of BmNc on BmNPV infection following RNAi and overexpression was eliminated after treatment with the inducer, Silvestrol, and the inhibitor, Z-DEVD-FMK, respectively. Therefore, it is reasonable to conclude that BmNc is involved in anti-BmNPV infection via the mitochondrial apoptosis pathway. The results provide valuable information for elucidating the molecular mechanism of silkworm resistance to BmNPV infection.

Bombyx mori histone methyltransferase BmAsh2 is essential for silkworm piRNA-mediated sex determination.

Sex determination is a hierarchically-regulated process with high diversity in different organisms including insects. The W chromosome-derived Fem piRNA has been identified as the primary sex determination factor in the lepidopteran insect, Bombyx mori, revealing a distinctive piRNA-mediated sex determination pathway. However, the comprehensive mechanism of silkworm sex determination is still poorly understood. We show here that the silkworm PIWI protein BmSiwi, but not BmAgo3, is essential for silkworm sex determination. CRISPR/Cas9-mediated depletion of BmSiwi results in developmental arrest in oogenesis and partial female sexual reversal, while BmAgo3 depletion only affects oogenesis. We identify three histone methyltransferases (HMTs) that are significantly down-regulated in BmSiwi mutant moths. Disruption one of these, BmAsh2, causes dysregulation of piRNAs and transposable elements (TEs), supporting a role for it in the piRNA signaling pathway. More importantly, we find that BmAsh2 mutagenesis results in oogenesis arrest and partial female-to-male sexual reversal as well as dysregulation of the sex determination genes, Bmdsx and BmMasc. Mutagenesis of other two HMTs, BmSETD2 and BmEggless, does not affect piRNA-mediated sex determination. Histological analysis and immunoprecipitation results support a functional interaction between the BmAsh2 and BmSiwi proteins. Our data provide the first evidence that the HMT, BmAsh2, plays key roles in silkworm piRNA-mediated sex determination.

Krüppel homolog 1 represses insect ecdysone biosynthesis by directly inhibiting the transcription of steroidogenic enzymes.

In insects, juvenile hormone (JH) and the steroid hormone ecdysone have opposing effects on regulation of the larval-pupal transition. Although increasing evidence suggests that JH represses ecdysone biosynthesis during larval development, the mechanism underlying this repression is not well understood. Here, we demonstrate that the expression of the Krüppel homolog 1 (Kr-h1), a gene encoding a transcription factor that mediates JH signaling, in ecdysone-producing organ prothoracic gland (PG) represses ecdysone biosynthesis by directly inhibiting the transcription of steroidogenic enzymes in both Drosophila and Bombyx Application of a JH mimic on ex vivo cultured PGs from Drosophila and Bombyx larvae induces Kr-h1 expression and inhibits the transcription of steroidogenic enzymes. In addition, PG-specific knockdown of Drosophila Kr-h1 promotes-while overexpression hampers-ecdysone production and pupariation. We further find that Kr-h1 inhibits the transcription of steroidogenic enzymes by directly binding to their promoters to induce promoter DNA methylation. Finally, we show that Kr-h1 does not affect DNA replication in Drosophila PG cells and that the reduction of PG size mediated by Kr-h1 overexpression can be rescued by feeding ecdysone. Taken together, our data indicate direct and conserved Kr-h1 repression of insect ecdysone biosynthesis in response to JH stimulation, providing insights into mechanisms underlying the antagonistic roles of JH and ecdysone.

A Matrix Metalloproteinase Mediates Tracheal Development in Bombyx mori.

The trachea of insects is a tubular epithelia tissue that transports oxygen and other gases. It serves as a useful model for the studying of the cellular and molecular events involved in epithelial tube formation. Almost all of the extracellular matrix can be degraded by Matrix metalloproteinases (MMPs), which is closely related to the processes of development and regeneration. The regulation of trachea by MMPs is roughly known in previous studies, but the detailed regulation mechanism and involved gene function are not fully explored. In this article, we found MMP1 expressed highly during tracheal remodeling, and knocked out it makes the tracheal branch number reduced in Bombyx mori. In trachea of transgenic BmMMP1-KO silkworm, the space expanding of taenidium and epidermal cells and the structure of apical membrane were abnormal. To explore the underlying mechanism, we detected that DE-cadherin and Integrin ß1 were accumulated in trachea of transgenic BmMMP1-KO silkworm by immunohistochemistry. Moreover, 5-Bromo-2'-Deoxyuridine (BrdU) labeling showed that knockout of BmMMP1 in silkworm inhibited tracheal cell proliferation, and BmMMP1 also regulated the proliferation and migration of BmNS cells. All of the results demonstrated that BmMMP1 regulates the development of the tracheal tissue by expanding the space of tracheal cuticles and increases the number of tracheal branches by degrading DE-cadherin and Integrin ß1.

Structural and biochemical insights into the catalytic mechanisms of two insect chitin deacetylases of the carbohydrate esterase 4 family.

Insect chitin deacetylases (CDAs) catalyze the removal of acetyl groups from chitin and modify this polymer during its synthesis and reorganization. CDAs are essential for insect survival and therefore represent promising targets for insecticide development. However, the structural and biochemical characteristics of insect CDAs have remained elusive. Here, we report the crystal structures of two insect CDAs from the silk moth Bombyx mori: BmCDA1, which may function in cuticle modification, and BmCDA8, which may act in modifying peritrophic membranes in the midgut. Both enzymes belong to the carbohydrate esterase 4 (CE4) family. Comparing their overall structures at 1.98-2.4 Å resolution with those from well-studied microbial CDAs, we found that two unique loop regions in BmCDA1 and BmCDA8 contribute to the distinct architecture of their substrate-binding clefts. These comparisons revealed that both BmCDA1 and BmCDA8 possess a much longer and wider substrate-binding cleft with a very open active site in the center than the microbial CDAs, including VcCDA from Vibrio cholerae and ArCE4A from Arthrobacter species AW19M34-1. Biochemical analyses indicated that BmCDA8 is an active enzyme that requires its substrates to occupy subsites 0, +1, and +2 for catalysis. In contrast, BmCDA1 also required accessory proteins for catalysis. To the best of our knowledge, our work is the first to unveil the structural and biochemical features of insect proteins belonging to the CE4 family.

Efficient production of recombinant SARS-CoV-2 spike protein using the baculovirus-silkworm system.

In the case of a new viral disease outbreak, an immediate development of virus detection kits and vaccines is required. For COVID-19, we established a rapid production procedure for SARS-CoV-2 spike protein (S protein) by using the baculovirus-silkworm expression system. The baculovirus vector-derived S proteins were successfully secreted to silkworm serum, whereas those formed insoluble structure in the larval fat body and the pupal cells. The ectodomain of S protein with the native sequence was cleaved by the host furin-protease, resulting in less recombinant protein production. The S protein modified in furin protease-target site was efficiently secreted to silkworm serum and was purified as oligomers, which showed immunoreactivity for anti-SARS-CoV-2 S2 antibody. By using the direct transfection of recombinant bacmid to silkworms, we achieved the efficient production of SARS-CoV-2 S protein as fetal bovine serum (FBS)-free system. The resultant purified S protein would be useful tools for the development of immunodetection kits, antigen for immunization for immunoglobulin production, and vaccines.

Molecular survey of the phosphoserine phosphatase involved in L-serine synthesis by silkworms (Bombyx mori).

Phosphoserine phosphatase (PSP) catalyses the synthesis of l-serine via the phosphorylated pathway by facilitating the dephosphorylation of phosphoserine. A cDNA encoding PSP from the silkworm Bombyx mori (bmPSP) was isolated using reverse transcription-PCR and then sequenced. The resulting clone encoded 236 amino acids with a molecular weight of 26 150, exhibiting 14-60% sequence identity with other PSPs. The recombinant PSP was overexpressed in Escherichia coli and purified. Kinetic studies showed that bmPSP possessed activity toward l-phosphoserine, and Asp20, Asp22 and Asp204 in bmPSP were found to be critical for modulating bmPSP activity. Real-time PCR analysis provided evidence that the amount of bmpsp transcript was reduced in middle silk glands of a sericin-deficient silkworm strain. These findings revealed that bmPSP may play important roles in synthesizing one-carbon donors of l-serine, which is abundant in silk, as well as other cell metabolites in B. mori.

Regulation of genes for ubiquitination and SUMO-specific protease involved in larval development of the silkworm, Bombyx mori.

Protein modifications with highly conserved small proteins, such as ubiquitin (Ub) and small ubiquitin-like modifier (SUMO), regulate various cellular processes; however, the contribution of these protein modifications to larval development in insects has not yet been elucidated. We investigated the regulation of genes for these protein modifications in the posterior silk gland (PSG) during larval development of the silkworm Bombyx mori. We found that several genes encoding enzymes (E1, E2, and E3) for ubiquitination and SUMO-specific protease were upregulated by 20-hydroxyecdysone (20E), and, consistently, increases in ubiquitinated proteins were observed during the fourth molting stage. An injection of 20E into larvae at the fourth feeding stage induced higher expression levels of these E1, E2, and E3 genes and ecdysis approximately one day earlier than in mock-treated larvae. The expression of the fibroin heavy-chain gene (fibH) was simultaneously suppressed approximately one day earlier in 20E-injected larvae. The treatment of cultured PSG with 20E also induced these genes, which could be categorized into at least two types: those induced by a high dose of 20E, or by a pulse of 20E. In contrast to the 20E treatment, the administration of PR-619, an inhibitor of Ub- and SUMO-specific proteases in larvae, delayed ecdysis and prolonged the expression of fibH. These results suggest that the regulation of genes for ubiquitination and SUMO-specific protease is involved in the larval development of B. mori.

Functional analysis of novel sulfotransferases in the silkworm Bombyx mori.

Sulfoconjugation plays a vital role in the detoxification of xenobiotics and in the metabolism of endogenous compounds. In this study, we aimed to identify new members of the sulfotransferase (SULT) superfamily in the silkworm Bombyx mori. Based on amino acid sequence and phylogenetic analyses, two new enzymes, swSULT ST1 and swSULT ST2, were identified that appear to belong to a distinct group of SULTs including several other insect SULTs. We expressed, purified, and characterized recombinant SULTs. While swSULT ST1 sulfated xanthurenic acid and pentachlorophenol, swSULT ST2 exclusively utilized xanthurenic acid as a substrate. Based on these results, and those concerning the tissue distribution and substrate specificity toward pentachlorophenol analyses, we hypothesize that swSULT ST1 plays a role in the detoxification of xenobiotics, including insecticides, in the silkworm midgut and in the induction of gametogenesis in silkworm ovary and testis. Collectively, the data obtained herein contribute to a better understanding of SULT enzymatic functions in insects.

Specific distribution of expression and enzymatic activity of cholesterol biosynthetic enzyme DHCR24 orthologs in the phytophagous insect.

Insects must intake sterol compounds because of their inability to synthesize cholesterol de novo. In phytophagous insects, enzymatic conversion of phytosterols to cholesterol involving 24-dehydrocholesterol reductase (DHCR24) exerts to acquire cholesterol. Here, we reported the presence of two DHCR24 homologs in the silkworm Bombyx mori, BmDHCR24-1 and -2, with several transcript variants. Consistent with the data of spatial expression analyses by RT-PCR, predominant enzymatic activity of DHCR24 was observed in B. mori larval midgut whereas weak activity was observed in the other tissues examined. In addition, BmDHCR24-1 expression in HEK293 cells showed an enzymatic activity, but BmDHCR24-2 did not, although both BmDHCR24s were localized in the endoplasmic reticulum, where the mammalian DHCR24s are located to exert their enzymatic activities. The present data indicated that BmDHCR24-1 but not BmDHCR24-2 contributes to conversion of phytosterols to cholesterol mainly in the midgut of the phytophagous lepidopteran larvae.