Methyl-CpG binding domain (MBD)2/3 specifically recognizes and binds to the genomic mCpG site with a ß-sheet in the MBD to affect embryonic development in Bombyx mori.

Methyl-CpG (mCpG) binding domain (MBD) proteins especially bind with methylated DNA, and are involved in many important biological processes; however, the binding mechanism between insect MBD2/3 and mCpG remains unclear. In this study, we identified 2 isoforms of the MBD2/3 gene in Bombyx mori, MBD2/3-S and MBD2/3-L. Binding analysis of MBD2/3-L, MBD2/3-S, and 7 mutant MBD2/3-L proteins deficient in ß1-ß6 or α1 in the MBD showed that ß2-ß3-turns in the ß-sheet of the MBD are necessary for the formation of the MBD2/3-mCpG complex; furthermore, other secondary structures, namely, ß4-ß6 and an α-helix, play a role in stabilizing the ß-sheet structure to ensure that the MBD is able to bind mCpG. In addition, sequence alignment and binding analyses of different insect MBD2/3s indicated that insect MBD2/3s have an intact and conserved MBD that binds to the mCpG of target genes. Furthermore, MBD2/3 RNA interference results showed that MBD2/3-L plays a role in regulating B. mori embryonic development, similar to that of DNA methylation; however, MBD2/3-S without ß4-ß6 and α-helix does not alter embryonic development. These results suggest that MBD2/3-L recognizes and binds to mCpG through the intact ß-sheet structure in its MBD, thus ensuring silkworm embryonic development.

Homeobox protein A1-like and DNA methylation regulate embryo-specific Zinc finger protein 615 gene expression and embryonic development in the silkworm Bombyx mori.

DNA methylation and transcription factors play roles in gene expression and animal development. In insects, DNA methylation modifies gene bodies, but how DNA methylation and transcription factors regulate gene expression is unclear. In this study, we investigated the mechanism that regulates the expression of Bombyx mori Zinc finger protein 615 (ZnF 615), which is a downstream gene of DNA methyltransferase 1 (Dnmt1), and its effects on the regulation of embryonic development. By progressively truncating the ZnF 615 promoter, it was found that the -223 and -190 nt region, which contains homeobox (Hox) protein cis-regulatory elements (CREs), had the greatest impact on the transcription of ZnF 615. RNA interference (RNAi)-mediated knockdown and overexpression of Hox family genes showed that Hox A1-like can enhance the messenger RNA level of ZnF 615. Further studies showed that Hox A1-like regulates ZnF 615 expression by directly binding to the -223 and -190 nt region of its promoter. Simultaneous RNAi-mediated knockdown or overexpression of Hox A1-like and Dnmt1 significantly inhibited or enhanced the regulatory effect of either gene alone on ZnF 615 expression, suggesting that both DNA methylation of gene bodies and binding of transcription factors to promoters are essential for gene expression. RNAi-mediated knockdown of Hox A1-like and Dnmt1 showed that the embryonic development was retarded and the hatching rate was decreased. Taken together, these data suggest that Hox A1-like and DNA methylation enhance the expression of ZnF 615, thereby affecting the development of B. mori embryos.

DNA methylation-mediated expression of zinc finger protein 615 affects embryonic development in Bombyx mori.

Cell division and differentiation after egg fertilization are critical steps in the development of embryos from single cells to multicellular individuals and are regulated by DNA methylation via its effects on gene expression. However, the mechanisms by which DNA methylation regulates these processes in insects remain unclear. Here, we studied the impacts of DNA methylation on early embryonic development in Bombyx mori. Genome methylation and transcriptome analysis of early embryos showed that DNA methylation events mainly occurred in the 5' region of protein metabolism-related genes. The transcription factor gene zinc finger protein 615 ( ZnF615) was methylated by DNA methyltransferase 1 (Dnmt1) to be up-regulated and bind to protein metabolism-related genes. Dnmt1 RNA interference (RNAi) revealed that DNA methylation mainly regulated the expression of nonmethylated nutrient metabolism-related genes through ZnF615. The same sites in the ZnF615 gene were methylated in ovaries and embryos. Knockout of ZnF615 using CRISPR/Cas9 gene editing decreased the hatching rate and egg number to levels similar to that of Dnmt1 knockout. Analysis of the ZnF615 methylation rate revealed that the DNA methylation pattern in the parent ovary was maintained and doubled in the offspring embryo. Thus, Dnmt1-mediated intragenic DNA methylation of the transcription factor ZnF615 enhances its expression to ensure ovarian and embryonic development.

Dynamic transcriptome analysis of Bombyx mori embryonic development.

Bombyx mori has been extensively studied but the gene expression control of its embryonic development is unclear. In this study, we performed transcriptome profiling of six stages of B. mori embryonic development using RNA sequencing (RNA-seq). A total of 12 894 transcripts were obtained from the embryos. Of these, 12 456 transcripts were shared among the six stages, namely, fertilized egg, blastoderm, germ-band, organogenesis, reversal period, and youth period stages. There were 111, 48, 41, 54, 77, and 107 transcripts specifically expressed during the six stages, respectively. By analyzing weighted gene correlation networks and differently expressed genes, we found that during embryonic development, many genes related to DNA replication, transcription, protein synthesis, and epigenetic modifications were upregulated in the early embryos. Genes of cuticle proteins, chitin synthesis-related proteins, and neuropeptides were more abundant in the late embryos. Although pathways of juvenile hormone and the ecdysteroid 20-hydroxyecdysone, and transcription factors were expressed throughout the embryonic development stages, more regulatory pathways were highly expressed around the organogenesis stage, suggesting more gene expression for organogenesis. The results of RNA-seq were confirmed by quantitative real-time polymerase chain reaction of 16 genes of different pathways. Nucleic acid methylation and seven sites in histone H3 modifications were confirmed by dot blot and western blot. This study increases the understanding of the molecular mechanisms of the embryonic developmental process and information on the regulation of B. mori development.

Transcriptomic analysis reveals the regulation network of BmKrüppel homolog 1 in the oocyte development of Bombyx mori.

Krüppel homolog 1 (Kr-h1), a zinc finger transcription factor, is involved in the metamorphosis and adult reproduction of insects. However, the role of Kr-h1 in reproduction of holometabolic insects remains to be elucidated. The regulation network of Kr-h1-associated genes in the reproduction in Bombyx mori was investigated in this study. The higher expression level of BmKr-h1 in the ovaries was detected during the late pupal stage and adults. RNA interference (RNAi)-mediated depletion of BmKr-h1 in the female at day 6 of pupae resulted in abnormal oocytes at 48 h post-double-stranded RNA treatment, which showed less yolk protein deposition and partially transparent chorion. RNA-seq and subsequent differentially expressed transcripts analysis showed that knockdown of BmKr-h1 caused a decrease in the expression of 2882 genes and an increase in the expression of 2565 genes in the oocytes at day 8 of pupae. Totally, 27 genes coding for transcription factors were down-regulated, while six genes coding for other transcription factors were up-regulated. BmKr-h1 bound to the Kr-h1 binding site of the transcription factors AP-1 (activating protein-1) and FOXG1 to increase their messenger RNA transcripts in the BmN cells, respectively. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses of that positively co-expressed with AP-1 and FOXG1 transcripts showed mainly enrichment in the metabolic-related pathways, the nutrient absorption and the yolk protein absorption processes. These data suggested that BmKr-h1 might directly regulate the metabolic-related pathways, the nutrient absorption and the yolk protein absorption processes or probably through AP-1 and /or FOXG1 to regulate oocyte development.

Analyses of microRNAs and transcriptomes in the midgut of Spodoptera litura feeding on Brassica juncea.

Spodoptera litura is a destructive agricultural pest in tropical and subtropical areas. Understanding the molecular mechanisms of S. litura adaptation to its preferred host plants may help identify target genes useful for pest control. We used high-throughput sequencing to characterize the expression patterns of messenger RNAs (mRNAs) and microRNAs (miRNAs) in the midgut of S. litura fed on Brassica juncea for 6 h and 48 h. A total of 108 known and 134 novel miRNAs were identified, 29 miRNAs and 237 mRNAs were differentially expressed at 6 h of B. juncea feeding, 26 miRNAs and 433 mRNAs were differentially expressed at 48 h. For the mRNAs, the up-regulated genes were mostly enriched in detoxification enzymes (cytochrome P450, esterase, glutathione S-transferase, uridine diphosphate-glucuronosyl transferase), while the down-regulated genes were mostly enriched in proteinases and immune-related genes. Furthermore, most detoxification enzymes begin to up-regulate at 6 h, while most digestion and immune-related genes begin to up- or down-regulate at 48 h. Eighteen and 37 differently expressed transcription factors were identified at 6 h and 48 h, which may regulate the functional genes. We acquired 136 and 41 miRNA versus mRNA pairs at 6 h and 48 h, respectively. Some down-regulated and up-regulated miRNAs were predicted to target detoxification enzymes and proteinases, respectively. Real-time quantitative polymerase chain reaction of nine randomly selected miRNAs and 28 genes confirmed the results of RNA-seq. This analyses of miRNA and mRNA transcriptomes provides useful information about the molecular mechanisms of S. litura response to B. juncea.

Genomic divergences between the two polyphagous Spodoptera relatives provide cues for successful invasion of the fall armyworm.

The fall armyworm Spodoptera frugiperda recently invaded China, ravaging crops in many provinces. Deciphering the possible genetic basics for its successful invasion is critical for innovative and specific control for this gluttonous pest. Here we generated comparative genomic analyses between S. frugiperda and its native relative, S. litura, which differs in host preference, locomotivity and production behavior. We demonstrated that S. frugiperda genes are enriched in taste sensory perception and nervous system, obviously different from those of S. litura. Potential host adaptation genes showed generally an elevated ratio of non-synonymous substitution rate to synonymous substitution rate, suggesting a faster evolution during the divergence of the two species. Focusing on these sets of genes, we identified 23 genes being under positive selection in S. frugiperda. Among them are two notable genes involved in sensory perception, gustatory receptor (GR) and an acetaldehyde oxidase, which are important for host detection in invasion and expansion processes. Another two genes are mitochondrial adenosine triphosphate synthase ß subunit and ferritin heavy chain, which may be associated with the enhanced locomotivity and resistance, which fascinated long-distance migration needed for invasion and rapid expansion. Another interesting gene is chorion protein, in which positive selection sites in S. frugiperda were found and a replacement in one site is predicted to affect the protein function, which might be associated with competent reproductivity in S. frugiperda to ensure genetic resources for expansion.

Signal transducer and activator of transcription is involved in the expression regulation of ecdysteroid-induced insulin-like growth factor-like peptide in the pupal wing disc of silkworm, Bombyx mori.

In insects, 20-hydroxyecdysone (20E) and insulin-like growth factor-like peptides (IGFLPs) regulate the development of imaginal discs. However, how IGFLPs are up-regulated to impact the development of the pupal wing disc is still unclear. In this study, we investigated the expression regulation of IGFLP in the pupal wing disc of silkworm, Bombyx mori. We confirmed that B. mori IGFLP (BmIGFLP) was mainly expressed in the pupal wing disc and the expression of BmIGFLP could be significantly induced by 20E. Bioinformatics analysis of BmIGFLP promoter sequence revealed three cis-regulation elements (CREs) of signal transducer and activator of transcription (STAT), which is a key component in the Janus-activated kinase / STAT pathway. Luciferase activity assays showed that two CREs enhanced the transcriptional activity of BmIGFLP. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that BmSTAT proteins in the nuclear extracts of B. mori pupal wing discs and BmN cells could only bind to the STAT CRE3, indicating that STAT CRE3 activated by BmSTAT enhances BmIGFLP expression at pupal stages. Although 20E could not enhance the expression of BmSTAT, 20E enhanced the nucleus translocation of BmSTAT to bind with the STAT CRE3 in the BmIGFLP promoter. The increase of transcriptional activity of the STAT CRE3 by overexpression of BmSTAT and addition of 20E in BmN cells confirmed this result. Taken together, all data indicate that BmSTAT is one of the transcription factors activating 20E-induced BmIGFLP expression in the pupal wing disc.

Identification and functional characterization of doublesex gene in the testis of Spodoptera litura.

Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific SldsxM and female-specific SldsxF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/µL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at G0 generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.

Expression profiles of glutathione S-transferase superfamily in Spodoptera litura tolerated to sublethal doses of chlorpyrifos.

Chlorpyrifos (CPF) is a broad-spectrum organophosphate insecticide. Glutathione S-transferases (GSTs) in insects are a family of detoxification enzymes and they play critical roles in CPF detoxification. Spodoptera litura is one of the most destructive agricultural pests in tropical and subtropical areas in the world. In this study, 37 Slgsts from 46 unique transcripts of gsts in S. litura transcriptome data, including eight previously reported GSTs, were identified and their expression patterns in susceptible and 12-generation-CPF-treated strains were analyzed to understand the roles of these Slgsts in sublethal doses of CPF tolerance. The results indicate that the members of the S. litura GST superfamily could be distinguished into three major groups: one group, including six cytosolic Slgsts (SlGSTe1, SlGSTe3, SlGSTe10, SlGSTe15, SlGSTo2 and SlGSTs5) and two microsomal Slgsts (SlMGST1-2 and SlMGST1-3), was directly responsible for CPF induction in both 12-generation-treated and susceptible strains; the second group, including three cytosolic Slgsts (SlGSTe13, SlGSTt1 and SlGSTz1) and one microsomal Slgst (SlMGST1-1), was induced only in the 12-generation-treated strain; the third group, including eight cytosolic Slgsts (two epsilon, three delta, one omega, one zeta and one unclassified Slgst), was expressed 1.52-5.15-fold higher in the 12-generation-treated strain than in the susceptible strain.

A novel cytochrome P450 CYP6AB14 gene in Spodoptera litura (Lepidoptera: Noctuidae) and its potential role in plant allelochemical detoxification.

Cytochrome P450 monooxygenases (P450s) play a prominent role in the adaptation of insects to host plant chemical defenses. To investigate the potential role of P450s in adaptation of the lepidopteran pest Spodoptera litura to host plant allelochemicals, an expressed sequence data set derived from 6th instar midgut tissues was first mined. One sequence identified from the S. litura 6th instar midgut EST database was determined by phylogenetic analysis to belong to the CYP6AB P450 subfamily, and named CYP6AB14. Dietary supplementation of S. litura larvae with either xanthotoxin (XAN), coumarin (COU) and flavone (FLA) led to elevated CYP6AB14 transcript levels in both midgut and fat body tissues. Injection of CYP6AB14-derived double-stranded RNA (dsRNA) into S. litura individuals significantly reduced CYP6AB14 transcript levels, and resulted in increased developmental abnormalities and higher mortality rates among XAN, COU and FLA-fed larvae. Our results strongly suggest a key role for CYP6AB14 in plant allelochemical detoxification in S. litura.

Detoxification of insecticides, allechemicals and heavy metals by glutathione S-transferase SlGSTE1 in the gut of Spodoptera litura.

Insect glutathione S-transferases (GSTs) play important roles in detoxifying toxic compounds and eliminating oxidative stress caused by these compounds. In this study, detoxification activity of the epsilon GST SlGSTE1 in Spodoptera litura was analyzed for several insecticides and heavy metals. SlGSTE1 was significantly up-regulated by chlorpyrifos and xanthotoxin in the midgut of S. litura. The recombinant SlGSTE1 had Vmax (reaction rate of the enzyme saturated with the substrate) and Km (michaelis constant and equals to the substrate concentration at half of the maximum reaction rate of the enzyme) values of 27.95 ± 0.88 µmol/min/mg and 0.87 ± 0.028 mmol/L for glutathione, respectively, and Vmax and Km values of 22.96 ± 0.78 µmol/min/mg and 0.83 ± 0.106 mmol/L for 1-chloro-2,4-dinitrobenzene, respectively. In vitro enzyme indirect activity assay showed that the recombinant SlGSTE1 possessed high binding activities to the insecticides chlorpyrifos, deltamethrin, malathion, phoxim and dichloro-diphenyl-trichloroethane (DDT). SlGSTE1 showed higher binding activity to toxic heavy metals cadmium, chromium and lead than copper and zinc that are required for insect normal growth. Western blot analysis showed that SlGSTE1 was induced in the gut of larvae fed with chlorpyrifos or cadmium. SlGSTE1 also showed high peroxidase activity. All the results together indicate that SlGSTE1 may play an important role in the gut of S. litura to protect the insect from the toxic effects of these compounds and heavy metals.

Transcriptomic analysis of developmental features of Bombyx mori wing disc during metamorphosis.

BACKGROUND: Wing discs of B. mori are transformed to pupal wings during the larva-to-pupa metamorphosis with dramatic morphological and structural changes. To understand these changes at a transcriptional level, RNA-seq of the wing discs from 6-day-old fifth instar larvae (L5D6), prepupae (PP) and pupae (P0) was performed. RESULTS: In total, 12,254 transcripts were obtained from the wing disc, out of which 5,287 were identified to be differentially expressed from L5D6 to PP and from PP to P0. The results of comprehensive analysis of RNA-seq data showed that during larvae-to-pupae metamorphosis, many genes of 20E signaling pathway were up-regulated and those of JH signaling pathway were down-regulated. Seventeen transcription factors were significantly up-regulated. Cuticle protein genes (especially wing cuticle protein genes), were most abundant and significantly up-regulated at P0 stage. Genes responsible for the degradation and de novo synthesis of chitin were significantly up-regulated. There were A and B two types of chitin synthases in B. mori, whereas only chitin synthase A was up-regulated. Both trehalose and D-fructose, which are precursors of chitin synthesis, were detected in the hemolymph of L5D6, PP and P0, suggesting de novo synthesis of chitin. However, most of the genes that are related to early wing disc differentiation were down-regulated. CONCLUSIONS: Extensive transcriptome and DGE profiling data of wing disc during metamorphosis of silkworm have been generated, which provided comprehensive gene expression information at the transcriptional level. These results implied that during the larva-to-pupa metamorphosis, pupal wing development and transition might be mainly controlled by 20E signaling in B. mori. The 17 up-regulated transcription factors might be involved in wing development. Chitin required for pupal wing development might be generated from both degradation of componential chitin and de novo synthesis. Chitin synthase A might be responsible for the chitin synthesis in the pupal wing, while both trehalose and D-fructose might contribute to the de novo synthesis of chitin during the formation of pupal wing.

De novo characterization of transcriptome and gene expression dynamics in epidermis during the larval-pupal metamorphosis of common cutworm.

Larval cuticle is degraded and replaced by the pupal counterpart during larval-pupal metamorphosis in the holometabolous insects. In addition to the extrinsic transformation, the epidermis goes through significant changes at molecular levels. To elucidate the intrinsic mechanism of epidermal metamorphosis, the dynamics of chitin content in the cuticle was examined in an important agricultural lepidopteran, the common cutworm, and the transcriptome was analyzed using Illumina sequencing technology. Gene expression profiles during the metamorphosis were further studied by both the digital gene expression (DGE) system and real-time quantitative PCR. The results showed that the chitin content decreased in prepupae and then increased in pupae. A total of 58 million sequencing reads were obtained and assembled into 70,346 unigenes. Over 9000 unigenes were identified to express differentially during the transformation process. As compared with the 6th instar feeding larvae, the most significant changes took place in the proteasome and metabolic pathways in prepupae and pupae, respectively. The cytochrome P450s, VHDLs, chitinase, serine protease and genes involved in sex pheromone biosynthesis changed their mRNA levels remarkably. Three chitinolytic enzymes (chitinase, ß-N-acetylglucosaminidase and chitin deacetylase) showed distinct mRNA expression patterns, the former two enzymes revealed the highest expression in prepupae, however the latter one showed its climax mRNA level in pupae. The gene expression patterns suggest that chitinase and ß-N-acetylglucosaminidase may be responsible for the degradation of larval cuticles, whereas chitin deacetylase may help to degrade the pupal counterparts. Gene expression dynamics also implied that the chitin of pupal cuticle might be formed by recycling of the degraded chitin of larval cuticle rather than through de novo synthesis. The 20E-induced nuclear receptors seem to be important factors regulating chitin metabolic enzymes during the cuticle remodeling. Our data provide a comprehensive resource for exploring the molecular mechanism of epidermal metamorphosis in insects.

Analysis of expressed sequence tags and characterization of a novel gene, Slmg7, in the midgut of the common cutworm, Spodoptera litura.

BACKGROUND: Out of total 3,081 assembled expressed sequence tags (ESTs) sequences representing 6,815 high-quality ESTs identified in three cDNA libraries constructed with RNA isolated from the midgut of Spodoptera litura, 1,039 ESTs showed significant hits and 1,107 ESTs did not show significant hits in BLAST searches. It is of interest to clarify whether or not these ESTs that did not show hits function in S. Litura. RESULTS: Twenty "no-hit" ESTs containing at least one putative open reading frame were selected for further expression analysis. The results from northern blot analysis showed that six of the selected ESTs are expressed in the larval midgut of this insect at different levels, suggesting that these ESTs represent true mRNA products, whereas the other 14 ESTs could not be detected. Homologues of the four larval midgut-predominant genes (Slmg2, Slmg7, Slmg9 and Slmg17) were detected in the genomes of other lepidopteran insects but not in Drosophila melanogaster. A novel gene, Slmg7, is expressed at a high level specifically in the midgut during each of the larval stages. Slmg7 is a single copy gene and encodes a 143-amino acids protein. The SLMG7 protein was localized to the cytoplasm of Spli-221 cells. CONCLUSIONS: Six ESTs from the no hit list are transcribed into mRNA and are mainly expressed in the midgut of S. litura. Slmg7 is a novel gene that is localized to the cytoplasm.

Cloning and expression analysis of six small heat shock protein genes in the common cutworm, Spodoptera litura.

Small heat shock proteins (sHsps) are probably the most diverse in structure and function among the various superfamilies of stress proteins. To explore the diverse functions of insect sHsps, six sHsp cDNAs were cloned from the midgut cDNA library of Spodoptera litura, and a phylogenetic tree was constructed based on the conserved α-crystalline domains. The expression patterns in different developmental stages and tissues, as well as in response to both thermal and 20-hydroxyecdysone (20E) induction, were studied by real-time quantitative PCR. Based on sequence characteristics and phylogenetic relationships, the six SlHsps were classified into three independent groups: BmHsp20.4 like proteins (SlHsp19.7, 20.4, 20.7, 20.8), BmHsp26.6 like protein (SlHsp20), and BmHsp21.4 like protein (SlHsp21.4). All the SlHsps showed highest expression in the Malpighian tubules. The four BmHsp20.4 like protein genes were up-regulated by thermal stress and showed expression variation with development. SlHsp20 exhibited lower expression levels in both egg and larval stages than in pupal and adult stages. SlHsp21.4 retained a constant expression level during all life stages. The expression of both SlHsp20.4 and SlHsp20.8 was significantly up-regulated by 20E. These results indicate that sHsps play diverse functions in S. litura: the BmHsp20.4 like proteins are involved in both thermal adaptation and development; SlHsp20 does not respond to temperature stress but possibly plays a role in metamorphosis; SlHsp21.4 may have no direct relationship with either thermal response or development.

The sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCPx) is involved in cholesterol uptake in the midgut of Spodoptera litura: gene cloning, expression, localization and functional analyses.

BACKGROUND: Sterol carrier protein-2/3-oxoacyl-CoA thiolase (SCPx) gene has been suggested to be involved in absorption and transport of cholesterol. Cholesterol is a membrane component and is a precursor of ecdysteroids, but cannot be synthesized de novo in insects. However, a direct association between SCPx gene expression, cholesterol absorption and development in lepidopteran insects remains to be experimentally demonstrated. RESULTS: An SCPx cDNA (SlSCPx) cloned from the common cutworm, Spodoptera litura, was characterized. The SlSCPx cDNA encoded a 535-amino acid protein consisting of a 3-oxoacyl-CoA thiolase (SCPx-t) domain and a SCP-2 (SCPx-2) domain. SlSCPx mRNA was expressed predominately in the midgut, while SlSCPx-2 mRNA was detected in the midgut, fat body and epidermis and no SlSCPx-t mRNA was detected. A 58-kDa full-length SCPx protein and a 44-kDa SCPx-t protein were detected in the midgut of sixth instar larvae when the anti-SlSCPx-t antibody was used in western blotting analysis; a 16-kDa SCP-2 protein was detected when anti-SlSCPx-2 antibody was used. SlSCPx protein was post-translationally cleaved into two smaller proteins, SCPx-t and SCPx-2. The gene appeared to be expressed into two forms of mRNA transcripts, which were translated into the two proteins, respectively. SlSCPx-t and SlSCPx-2 proteins have distinct and different locations in the midgut of sixth instar larvae. SlSCPx and SlSCPx-t proteins were detected predominately in the cytoplasm, whereas SlSCPx-2 protein was detected in the cytoplasm and nuclei in the Spli-221 cells. Over-expression of SlSCPx and SlSCPx-2 proteins enhanced cholesterol uptake into the Spli-221 cells. Knocking-down SlSCPx transcripts by dsRNA interference resulted in a decrease in cholesterol level in the hemolymph and delayed the larval to pupal transition. CONCLUSION: Spatial and temporal expression pattern of this SlSCPx gene during the larval developmental stages of S. litura showed its specific association with the midgut at the feeding stage. Over-expression of this gene increased cholesterol uptake and interference of its transcript decreased cholesterol uptake and delayed the larval to pupal metamorphosis. All of these results taken together suggest that this midgut-specific SlSCPx gene is important for cholesterol uptake and normal development in S. litura.