Peroxiredoxin-2 gene in Antheraea pernyi modulates immune functions and protect DNA damage.

Peroxiredoxins have been shown to protect insects from oxidative damage and to play a role in the immune system. In the present study, we cloned and characterized the Antheraea pernyi peroxiredoxin 2 (ApPrx-2) gene, then assessed its functional roles. The ApPrx-2 gene has a 687 bp open reading frame that encodes a protein with 288 amino acid residues. Quantitative real-time PCR analysis revealed that the mRNA levels of ApPrx-2 were highest in the hemocytes. Immune challenge assay revealed that ApPrx-2 transcription could be induced after microbial challenge. A DNA cleavage assay employing recombinant ApPrx-2 protein and a metal-catalyzed oxidation system showed that rApPrx-2 protein could protect supercoiled DNA against oxidative stress. The protein antioxidant activity of rApPrx-2 was examined, and it was found that rApPrx-2 exhibited a high level of antioxidant activity by removing H2O2. In addition, ApPrx-2 knockdown larvae had higher H2O2 levels and a lower survival rate when compared to controls. Interestingly, the antibacterial activity was significantly higher in ApPrx-2 depleted larvae compared with control. Overall, our findings indicate that ApPrx-2 may be involved in a range of physiological functions of A. pernyi, as it protects supercoiled DNA from oxidative stress and regulates antibacterial activity.

Insight into crustacean cathepsins: Structure-evolutionary relationships and functional roles in physiological processes.

Cathepsins belong to a group of proteins that are present in both prokaryotic and eukaryotic organisms and have an extremely high degree of evolutionary conservation. These proteins are functionally active in extracellular environments as soluble enzymatic proteins or attached to plasma membrane receptors. In addition, they occur in cellular secretory vesicles, mitochondria, the cytosol, and within the nuclei of eukaryotic cells. Cathepsins are classified into various groups based on their sequence variations, leading to their structural and functional diversification. The molecular understanding of the physiology of crustaceans has shown that proteases, including cathepsins, are expressed ubiquitously. They also contain one of the central regulatory systems for crustacean reproduction, growth, and immune responses. This review focuses on various aspects of the crustaceans cathepsins and emphasizes their biological roles in different physiological processes such as reproduction, growth, development, and immune responses. We also describe the bioactivity of crustaceans cathepsins. Because of the vital biological roles that cathepsins play as cellular proteases in physiological processes, they have been proposed as potential novel targets for the development of management strategies for the aquaculture industries.

MicroRNAs reshape the immunity of insects in response to bacterial infection.

The interaction between bacteria and insects can significantly impact a wide range of different areas because bacteria and insects are widely distributed around the globe. The bacterial-insect interactions have the potential to directly affect human health since insects are vectors for disease transmission, and their interactions can also have economic consequences. In addition, they have been linked to high mortality rates in economically important insects, resulting in substantial economic losses. MicroRNAs (miRNAs) are types of non-coding RNAs involved in regulating gene expression post-transcriptionally. The length of miRNAs ranges from 19 to 22 nucleotides. MiRNAs, in addition to their ability to exhibit dynamic expression patterns, have a diverse range of targets. This enables them to govern various physiological activities in insects, like innate immune responses. Increasing evidence suggests that miRNAs have a crucial biological role in bacterial infection by influencing immune responses and other mechanisms for resistance. This review focuses on some of the most recent and exciting discoveries made in recent years, including the correlation between the dysregulation of miRNA expression in the context of bacterial infection and the progression of the infection. Furthermore, it describes how they profoundly impact the immune responses of the host by targeting the Toll, IMD, and JNK signaling pathways. It also emphasizes the biological function of miRNAs in regulating immune responses in insects. Finally, it also discusses current knowledge gaps about the function of miRNAs in insect immunity, in addition to areas that require more research in the future.

The Potential Biological Roles of Circular RNAs in the Immune Systems of Insects to Pathogen Invasion.

Circular RNAs (circRNAs) are a newly discovered class of endogenously expressed non-coding RNAs (ncRNAs). They are highly stable, covalently closed molecules that frequently exhibit tissue-specific expression in eukaryotes. A small number of circRNAs are abundant and have been remarkably conserved throughout evolution. Numerous circRNAs are known to play important biological roles by acting as microRNAs (miRNAs) or protein inhibitors ('sponges'), by regulating the function of proteins, or by being translated themselves. CircRNAs have distinct cellular functions due to structural and production differences from mRNAs. Recent advances highlight the importance of characterizing circRNAs and their targets in a variety of insect species in order to fully understand how they contribute to the immune responses of these insects. Here, we focus on the recent advances in our understanding of the biogenesis of circRNAs, regulation of their abundance, and biological roles, such as serving as templates for translation and in the regulation of signaling pathways. We also discuss the emerging roles of circRNAs in regulating immune responses to various microbial pathogens. Furthermore, we describe the functions of circRNAs encoded by microbial pathogens that play in their hosts.

Antheraea pernyi Suppressor of Cytokine Signaling 2 Negatively Modulates the JAK/STAT Pathway to Attenuate Microbial Infection.

The Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway has been shown to govern various physiological processes, including immune responses, hematopoiesis, cell growth, and differentiation. Recent studies show that suppressors of cytokine signaling (SOCS) proteins attenuate JAK-STAT signaling in mammals; however, their functions are less clear in lepidopteran insects. Here, we report a full-length sequence of SOCS-2 from the Chinese oak silkworm Antheraea pernyi (designated as ApSOCS-2) and study its biological role in immune responses via the JAK-STAT pathway. ApSOCS-2 expression was high in the fat bodies and hemocytes of A. pernyi fifth instar larvae. After pathogen infection with nucleopolyhedrovirus, Beauveria bassiana, Escherichia coli, and Microccus luteus, ApSOCS-2 mRNA was strongly increased compared to the control group. To elucidate the possible involvement in innate immunity, we measured antimicrobial peptide genes expression profiles in the fat body of A. pernyi. In contrast, recombinant ApSOCS-2 protein administration significantly reduced the AMPs transcription, while the depletion of ApSOCS-2 by RNAi increased their expression. Furthermore, we observed higher antibacterial activity and lower bacterial replication in dsApSOCS-2-treated larvae. The ApSOCS-2 transcription level was reduced in STAT depleted A. pernyi larvae challenged by M. luteus. The ApSOCS-2 RNAi data sets were also subjected to transcriptomic analysis, which suggests that ApSOCS-2 is a key regulator of immune function. Taken together, our data suggest that ApSOCS-2 is required for the negative regulation of AMPs transcripts via the JAK-STAT pathway in the insect.

Molecular Identification of Two DNA Methyltransferase Genes and Their Functional Characterization in the Anti-Bacterial Immunity of Antheraea pernyi.

Under different physiological conditions, such as microbial infection, epigenetic mechanisms regulate genes at the transcription level in living organisms. DNA methylation is a type of epigenetic mechanism in which DNA methyltransferases modify the expression of target genes. Here, we identified a full-length sequence of DNMT-1 and DNMT-2 from the Chinese oak silkworm, A. pernyi, which was highly similar to the homologous sequences of Bombyx mori. ApDNMT-1 and ApDNMT-2 have unique domain architectures of insect DNMTs, highlighting their conserved functions in A. pernyi. ApDNMT-1 and ApDNMT-2 were found to be widely expressed in various tissues, with the highest levels of expression in hemocytes, the ovary, testis, and fat bodies. To understand the biological role of these genes in microbial resistance, we challenged the fifth instar larvae of A. pernyi by administrating Gram-positive and Gram-negative bacteria and fungi. The results revealed that transcript levels of ApDNMT-1 and ApDNMT-2 were increased compared to the control group. The inhibition of these genes by a DNMTs inhibitor [5-azacytidine (5-AZA)] significantly reduced bacterial replication and larvae mortality. In addition, 5-AZA treatment modified the expression patterns of antimicrobial peptides (AMPs) in the A. pernyi larvae. Our results suggest that ApDNMT-1 and ApDNMT-2 seem to have a crucial role in innate immunity, mediating antimicrobial peptide responses against bacterial infection in A. pernyi.

Integrins in the Immunity of Insects: A Review.

Integrins are a large group of cell-surface proteins that are classified as transmembrane proteins. Integrins are classified into different types based on sequence variations, leading to structural and functional diversity. They are broadly distributed in animals and have a wide range of biological functions such as cell-to-cell communication, intracellular cytoskeleton organization, cellular signaling, immune responses, etc. Integrins are among the most abundant cell surface proteins in insects, exhibiting their indispensability in insect physiology. Because of their critical biological involvement in physiological processes, they appear to be a novel target for designing effective pest control strategies. In the current literature review, we first discuss the discovery and expression responses of integrins against various types of pathogens. Secondly, we examine the specific biological roles of integrins in controlling microbial pathogens, such as phagocytosis, encapsulation, nodulation, immune signaling, and so on. Finally, we describe the possible uses of integrins to control agricultural insect pests.

The Emerging Role of STING in Insect Innate Immune Responses and Pathogen Evasion Strategies.

Emerging evidence reveals that the stimulator of the interferon genes (STING) signaling pathway in insects and other animal cells helps them to sense and effectively respond to infection caused by numerous types of microbial pathogens. Recent studies have shown that genomic material from microbial pathogens induces the STING signaling pathway for the production of immune factors to attenuate infection. In contrast, microbial pathogens are equipped with various factors that assist them in evading the STING signaling cascade. Here we discuss the STING signaling pathway different animal groups compared to human and then focus on its crucial biological roles and application in the microbial infection of insects. In addition, we examine the negative and positive modulators of the STING signaling cascade. Finally, we describe the microbial pathogen strategies to evade this signaling cascade for successful invasion.

Transcriptome Sequencing Highlights the Regulatory Role of DNA Methylation in Immune-Related Genes' Expression of Chinese Oak Silkworm, Antheraea pernyi.

Antheraea pernyi is an important lepidopteran used as a model insect species to investigate immune responses, development, and metabolism modulation. DNA methylation has recently been found to control various physiological processes throughout the life of animals; however, DNA methylation and its effect on the physiology of insects have been poorly investigated so far. In the present study, to better understand DNA methylation and its biological role in the immune system, we analyzed transcriptome profiles of A. pernyi pupae following DNA methylation inhibitor injection and Gram-positive bacteria stimulation. We then compared the profiles with a control group. We identified a total of 55,131 unigenes from the RNA sequence data. A comparison of unigene expression profiles showed that a total of 680 were up-regulated and 631 unigenes were down-regulated in the DNA-methylation-inhibition-bacteria-infected group compared to the control group (only bacteria-injected pupae), respectively. Here, we focused on the immune-related differentially expressed genes (DEGs) and screened 10 genes that contribute to immune responses with an up-regulation trend, suggesting that microbial pathogens evade host immunity by increasing DNA methylation of the host genome. Furthermore, several other unigenes related to other pathways were also changed, as shown in the KEGG analysis. Taken together, our data revealed that DNA methylation seems to play a crucial biological role in the regulation of gene expression in insects, and that infection may enhance the host genome DNA methylation by a yet-unknown mechanism.

Identification and the immunological role of two Nimrod family genes in the silkworm, Bombyx mori.

In animals, immune signaling pathways and effector molecules participate in attenuating microbial infection. Recent work has shown that the Nimrod family proteins can directly bind to bacteria, and this binding leads to bacterial phagocytosis. Although the Nimrod gene family has been reported in many non-drosophilids, their functions remain unexplored in most insect species. Here, we report two members (Nimrod-B and Draper) of the Nimrod gene family from Bombyx mori and analyzed their role in immunity. The two genes were ubiquitously expressed in the tested tissues; but, they transcribed preferentially in immune tissues. The developmental profiles showed that BmNimrod-B and BmDraper transcription levels were highest in the pupal stages. Challenge with microbial pathogens induced the transcription levels of all two genes at different time points. Knockdown of BmDraper decreased the bacterial clearance and increased their replication relative to the control group, whereas, BmNimrod-B suppression had a non-significant effect on them. Furthermore, the mortality rate was increased after BmDraper silencing. The knockdown of these genes did not significantly affect the production of antimicrobial peptides following E. coli infection. Taken together, the Nimrod family genes play a crucial role in host defense by positively regulating the antibacterial immune response in silkworm B. mori.

Suppressor of cytokine signalling 6 is a potential regulator of antimicrobial peptides in the Chinese oak silkworm, Antheraea pernyi.

The SOCS/CIS is a family of intracellular proteins distributed widely among living organisms. The members of this family have extensively been studied in mammals and have been shown to regulate various physiological processes. In contrast, the functional roles of SOCS/CIS family proteins are unknown in most invertebrates, including insects. Here, we retrieved a full-length open reading frame (ORF) of SOCS-6 from Chines oak silkworm, Antheraea pernyi (Designated as ApSOCS-6), using the RNA-seq database. The predicted ApSOCS-6 amino acid sequence comprised an N-terminal SH2 domain and a C-terminal SOCS-box domain. It shared the highly conserved structures of the SOCS proteins with other lepidopteran species. ApSOCS-6 mRNA transcript was detected in all the tested tissues of the A. pernyi larvae; however, the highest mRNA levels were found in the larval hemocytes, fat bodies, and integuments. The mRNA transcript levels of ApSOCS-6 were increased in the A. pernyi larval hemocytes and fat bodies after a challenge by the Gram-positive bacteria, M. luteus, Gram-negative bacteria, Escherichia coli, Virus, ApNPV, and Fungus, B. bassiana. After the knockdown of ApSOCS-6, we found a significant increase in bacterial clearance and a decrease in the relative replication of bacteria. To evaluate the possible cause of enhanced antibacterial activity, we measured antimicrobial peptides expression in the fat body of A. pernyi larvae. The production of AMPs was strongly increased in the B. cereus infected larval fat bodies following silencing of ApSOCS-6. Our data indicate that ApSOCS-6 negatively regulates the expression of AMPs in immune tissues in response to the B. cereus challenge.

The identification of nuclear factor Akirin with immune defense role in silkworm, Bombyx mori.

Akirins, highly conserved nuclear factors, regulate diverse physiological processes such as innate immunity. The biological functions of Akirins have extensively been studied in vertebrates and many invertebrates; however, there is no report so far on lepidopteran insects. In the present study, we identified and characterized a novel Akirin from the silkworm, Bombyx mori (designated as BmAkirin), and explored its potential roles in innate immunity. The expression analysis revealed the unequal mRNA levels of BmAkirin in all the tested tissues; however, the gene's transcription level was highest in testis, followed by ovaries and hemocytes. It also had significant expression levels at the early stages of embryonic development. Expression of BmAkirin in fat bodies and hemocytes exhibited an increase in various degrees when challenged with virus, fungus, Gram-negative bacteria, and Gram-positive bacteria. Immunofluorescence analysis showed BmAkirin protein was prominently localized in the nucleus. Knockdown of BmAkirin strongly reduced the expression of AMPs and decreased the survival ability of larva upon immune stimulation. Moreover, the bacterial clearance ability of larvae was also decreased following the depletion of BmAkirin. Collectively, our results demonstrate that BmAkirin plays an indispensable role in the innate immunity of Bombyx mori (B. mori) by positively modulating AMPs expression in vivo.

A review on the DNA methyltransferase family of insects: Aspect and prospects.

The DNA methyltransferase family contains a conserved set of DNA-modifying enzymatic proteins. They are responsible for epigenetic gene modulation, such as transcriptional silencing, transcription activation, and post-transcriptional modulation. Recent research has revealed that the canonical DNA methyltransferases (DNMTs) biological roles go beyond their traditional functions of establishing and maintaining DNA methylation patterns. Although a complete DNA methylation toolkit is absent in most insect orders, recent evidence indicates the de novo DNA methylation and maintenance function remain conserved. Studies using various molecular approaches provided evidence that DNMTs are multi-functional proteins. However, still in-depth studies on their biological role lack due to the least studied area in insects. Here, we review the DNA methylation toolkit of insects, focusing on recent research on various insect orders, which exhibit DNA methylation at different levels, and for which DNMTs functional studies have become available in recent years. We survey research on the potential roles of DNMTs in the regulation of gene transcription in insect species. DNMTs participate in different physiological processes by interacting with other epigenetic factors. Future studies on insect's DNMTs will benefit to understand developmental processes, responses to various stimuli, and adaptability of insects to different environmental conditions.

A hemocyte-specific cathepsin L-like cysteine protease is involved in response to 20-hydroxyecdysone and microbial pathogens stimulation in silkworm, Bombyx mori.

Cathepsin L protease belongs to the papain-like cysteine proteases family, plays indispensable roles in animals' pathological and physiological processes. However, little is known about Cathepsin L in silkworm, Bombyx mori. Herein, a novel Cathepsin L-like (Cat L-like) was cloned and identified from silkworm by the rapid amplification of cDNA ends (RACE). Cat L-like contains an intact open reading frame (ORF) of 1 668 bp and encodes 556 amino acid residues, consisting of a signal peptide, typical cathepsins' inhibitor_I29, and pept_C1 domain. Cat L-like is specifically and highly expressed in hemocytes. The cathepsin (including Cathepsin L, B, and H) crude extract from hemocytes had typical substrate specific catalytic activities and were sensitive to pH and temperature. Cat L-like up-regulated considerably after 20-hydroxyecdysone (20-E) administration, indicating that Cat L-like may be regulated by insect hormone. The responses of Cat L-like against bacterial infection suggest it may play essential roles in silkworm immunity. Overall, our studies provide a theoretical basis and insights to further investigate the functions of Cat L-like and in insects' innate immunity mechanisms.

Bmintegrin ß1: A broadly expressed molecule modulates the innate immune response of Bombyx mori.

Integrins are transmembrane glycoproteins that are broadly distributed in living organisms. As a heterodimer, they contain an α and a ß subunit, which are reported to be associated with various physiological and pathological processes. In the present study, a 2502 bp full-length cDNA sequence of Bmintegrin ß1 was obtained from the silkworm, Bombyx mori. Bmintegrin ß1 belongs to the ß subunit of the integrin family and contains several typical structures of integrins. Gene expression profile analysis demonstrated that Bmintegrin ß1 was ubiquitously expressed in all tested tissues and organs, with the maximum expression levels in fat body and hemocytes. The immunofluorescence results showed that Bmintegrin ß1 was located in the cell membrane and widely distributed in fat bodies and different types of hemocytes. Bmintegrin ß1 expression was remarkably increased after challenging with different kinds of bacteria and pathogen-associated molecular patterns (PAMPs). Further investigation revealed that Bmintegrin ß1 could participate in the agglutination of pathogenic bacteria possibly through direct binding with the relative bacteria and PAMPs. Altogether, this study provides a novel insight into the immune functional features of Bmintegrin ß1.

The mitochondrial genome of Smerinthus planus (Lepidoptera: Sphingidae) and its comparative analysis with other Lepidoptera species.

In the present study, the complete mitochondrial genome of Smerinthus planus Walker (Lepidoptera: Sphingidae) was sequenced and analyzed to add additional traits for expanding our knowledge on systematics and phylogenetics of world-wide studied Sphingidae moths. The mitochondrial genome is a circular double-stranded DNA molecule, 15368 bp in size. It includes 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, twenty-two transfer RNA (tRNA) genes, and an adenine (A) + thymine (T) rich region. All the PCGs start with the typical ATN start codons, except for the nad5 gene, which initiates with TTA. The codon usage analysis revealed that Phe, Ile, Lys, Leu, Asn, and Tys were the most common amino acids, while Cys and Trp were least common. Among the 13 PCGs, nine genes harbor the complete termination codon TAA, whereas the remaining four genes (nad1, cob, nad4, and nad3) terminate with TAG. The A+T rich region of S. planus is 318 bp. This region displays the highest A+T rich content, accounting for 91.50%, with both AT skew (-0.09) and GC skew (-0.26) are negative. Like other Lepidopterans, the A+T-rich region of the S. planus also contains some conserved regions, including the motif 'ATAGA' followed by an 18 bp poly-T stretch, a microsatellite-like (AT)8 and a poly-A element. Phylogenetic relationships, based on nucleotide sequences from the genomes of 31 species, confirmed that S. planus belong to the Sphingidae family. This study is aimed to improve the mitochondrial genome database of moths and provide valuable information for studying the genetic evolution and phylogeny of Lepidopteran species.

Molecular characterization of a heat shock protein 21 (Hsp21) from red swamp crayfish, Procambarus clarkii in response to immune stimulation.

Small heat shock proteins are a molecular chaperone and implicated in various physiological and stress processes in animals. However, the immunological functions of Hsp genes remain to elucidate in the crustaceans, particularly in red swamp crayfish, Procambarus clarkii. Here we report the cloning of heat shock protein 21 from the P. clarkii (hereafter Pc-Hsp21). The open reading frame of Pc-Hsp21 was 555 base pairs, encoding a protein of 184 amino acid residues with an alpha-crystallin family domain. Quantitative real-time PCR (qRT-PCR) analysis revealed a constitutive transcript expression of Pc-Hsp21 in the tested tissue, with the highest in hepatopancreas. The transcript abundance for this gene enhanced in hepatopancreas following immune challenge with the lipopolysaccharide, peptidoglycan, and poly I:C compared to the control group. The depletion of Pc-Hsp21 by double-stranded RNA altered transcript expression profiles of several genes in hepatopancreas, genes involved in the crucial immunological pathways of P. clarkii. These results suggest that Pc-Hsp21 plays an essential biological role in the microbial stress response by modulating the expression of immune-related genes in P. clarkii.

Characterization of the cathepsin D in Procambarus clarkii and its biological role in innate immune responses.

Cathepsin D belongs to aspartic protease family, produced in the rough endoplasmic reticulum, and then transported to lysosomes, where it participates in various physiological processes. Despite its importance, only a few reports available on the functional role of cathepsin D in crustaceans. Herein, we cloned a cDNA fragment of cathepsin D from the hepatopancreas of the red swamp crayfish, Procambarus clarkii (Pc-cathepsin D) for the first time. It included 1158 base pairs open reading frame, encoding a protein of 385 amino acids. Multiple alignment analysis confirmed the presence of aspartic proteinase active sites and N glycosylation sites. Pc-cathepsin D mRNA expression was high in the gills followed by gut, heart, hepatopancreas of P. clarkii. At different time points post-infection with lipopolysaccharides, peptidoglycan, or polyinosinic polycytidylic acid, Pc-cathepsin D mRNA expression significantly enhanced compared with the control group. Knockdown of the Pc-cathepsin D by double-stranded RNA, strikingly, changed the expression of all the tested P. clarkii immune-associated genes, including Pc-Toll, Pc-lectin, Pc-cactus, Pc-anti-lipopolysaccharide factor, Pc-phospholipase, and Pc-sptzale. Altogether, these results suggest that Pc-cathepsin D is needed to confer innate immunity against microbial pathogens by modulating the expression of crucial transcripts that encode immune-associated genes.

The red swamp crayfish, Procambarus clarkii cathepsin C, participates in the innate immune response to the viral and bacterial pathogens.

The cathepsin C, a lysosomal cysteine protease, involves the modulation of immune and inflammatory responses in living organisms. However, the knowledge on cathepsin C in red swamp crayfish (Procambarus clarkii), a freshwater crustacean with economic values, remained unclear. In the present study, we provide identification and molecular characterization of cathepsin C from P. clarkii. (Hereafter Pc-cathepsin C). The Pc-cathepsin C cDNA contained a 1356 bp open reading frame that encoded a protein of 451 amino acid residues. The deduced amino acid sequence comprised of cathepsin C exclusion domain and pept_C1 domain, and also catalytic residues (Cys248, His395 and Asn417). Analysis of the transcriptional patterns of the Pc-cathepsin C gene revealed that it was broadly distributed in various tissues of P. clarkii, and it was more abundant in the hepatopancreas and gut. Following a challenge with viral and bacterial pathogen-associated molecular patterns, the expression of Pc-cathepsin C was strongly enhanced at different time points. The knockdown of Pc-cathepsin C, altered the expression of immune-responsive genes, suggesting its immunoregulatory role in P. clarkii. This study has identified and provided the immunoregulatory function of Pc-cathepsin C, which will contribute to further investigation of the molecular mechanism of cathepsin C in crustaceans.

Niemann-Pick type C1 regulates cholesterol transport and metamorphosis in silkworm, Bombyx mori (Dazao).

NPC1 is a large glycoprotein with 13 transmembrane-spanning domains, which plays a crucial biological role in cholesterol transport and metamorphosis of animals. However, the physiological functions of this gene have rarely been elucidated in insects. Here, we isolated the NPC1 gene from Bombyx mori (BmNPC1), sequenced and evaluated its physiological functions. BmNPC1 comprised of 3702 bp open reading frame, encoding a protein of 1233 amino acid residues. The recombinant protein was expressed, and anti-BmNPC1 antibodies were synthesized. Immunofluorescence assay revealed that BmNPC1 protein localized in the cytoplasm of the cells. The qRT-PCR analysis showed that BmNPC1 expression was most significant in the testis, followed by the malpighian tubules, hemocytes, and ovary. The knockdown of BmNPC1 by double-stranded RNA caused the accumulation of cholesterol in the cells. Furthermore, suppression of this gene influenced the expression of ecdysone-responsive genes and also prevented the molting in B. mori (Dazao) larvae. Overall, BmNPC1 may have different biological roles in the physiology of silkworm, B. mori (Dazao), since it regulates the cholesterol transport and molting process.