Search

The complete mitochondrial genome (mitogenome) of Saturnia japonica (Lepidoptera: Saturniidae) was sequenced and annotated. It is a circular molecule of 15, 376 bp, composed of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNA), and an adenine (A) + thymine (T)-rich region. All protein-coding genes (PCGs) are initiated by the ATN codon except for cytochrome c oxidase subunit 1 (cox1) gene that is seemingly initiated by the CGA codon. Except for cox2 and nad4, which were terminated by incomplete stop codon T or TA, the rest were terminated by canonical stop codon TAA. The A + T-rich region is high conservative, including ‘ATAGA’ motif followed by a 19 bp poly-T stretch, a microsatellite-like element (AT)9 and also a poly-A element, with a total length of 332 bp. The Asn codon was the most frequently used codon, followed by Ile, Leu2, Lys, Met, Phe, and Tyr, while Cys was the least frequently used codon. Phylogenetic relationships analysis based on the 13 PCGs by using maximum likelihood (ML) and neighbor Joining (NJ) revealed that S. japonica belongs to the Saturniidae family. In this study, the annotation and characteristics of the mitogenome of S. japonica were resolved for the first time, which laid a foundation for species classification and the molecular evolution of Lepidoptera: Saturniidae.

The prophenoloxidase (PPO) activation system is an important innate immune defense mechanism in arthropods. Actias selene is a rare and important wild silk insect that can spin high-quality cocoon silk, but, other than its morphology, its molecular mechanism is rarely reported. Here, we report the purification and characterization of a novel

Secretion-associated and ras-related protein 1 (Sar1) is a small GTPase that plays an important role in the transport of protein coated with coat protein complex II vesicles. However, its alternative roles in the biological processes of Procambarus clarkii remain unclear. Here, a sar1 gene (named as Pc-sar1) with an open reading frame of 582 bp from P. clarkii was identified. Pc-sar1 was expressed in all examined tissues with highest expression levels in muscle, which was determined by real-time PCR and western blotting. After the induction of lipopolysaccharide (LPS) and polycytidylic acid (Poly I: C), the transcriptional levels of Pc-sar1 differed in hepatopancreas, gill, muscle and intestine. In contrast, the expression of Pc-sar1 was upregulated by 20-hydroxyecdysone in these four tissues. In addition, the RNA interference of Pc-sar1 significantly affected the expression levels of immune and hormone-related genes. These results indicate that Pc-sar1 is involved in the innate immune response and ecdysteroid signaling pathway.

Background: Antibacterial peptides play important roles in the innate immune system of insects and are divided into four categories according to their structures. Although many antibacterial peptides have been reported in lepidopteran insects, the roles of an attacin-like gene in immune response of Antheraea pernyi remain unclear. Objective: In this study, the cloning and immunological functions of an attacin-like gene from Antheraea pernyi were investigated. Methods: The open reading frame of Ap-attacin-like gene was cloned by PCR using the specific primers and then was ligated to the pET-32a vector to construct the recombinant plasmids Ap-attacin- like-pET-32a. The recombinant Ap-attacin-like protein was expressed in E. coli (BL21 DE3) cells and purified by Ni-NTA affinity chromatography. The expression patterns of Ap-attacin-like in different tissues or under microorganism challenges were investigated by real-time PCR and western blotting. Finally, agar well diffusion assay was performed to determine the antimicrobial activity of the recombinant Ap-attacin-like proteins based on the inhibition rate. Results: The expression level of Ap-attacin-like was highest in the fat body compared with the other examined tissues. The expression of Ap-attacin-like in the fat body was significantly elevated after E. coli, Beauveria bassiana, Micrococcus luteus or Nuclear Polyhedrosis Virus challenges. In addition, the recombinant Ap-attacin-like proteins had obvious antibacterial activity against E. coli. Conclusion: Ap-attacin-like was highly expressed in immune-related tissues and its expression level was significantly induced by different microorganism challenges, suggesting that Ap-attacin-like participated in the innate immunity of A. pernyi.

Ras-related C3 botulinum toxin substrate 1 (Rac1) participates in many biological processes. In this study, a Rac1 gene was identified in the crayfish Procambarus clarkii with an open reading frame of 579 bp that encoded 192 amino acids. This predicted 21.4 kDa protein was highly homologous to those in other invertebrates. Real-time PCR analysis revealed that Pc-Rac1 was expressed in all examined tissues with the highest expression level in hemocytes. The transcriptional expression level of Pc-Rac1 was significantly upregulated in hemocytes and hepatopancreas after lipopolysaccharide (LPS) or polyinosinic: polycytidylic acid (poly I: C) induction. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis suggested that a recombinant Pc-Rac1 protein was successfully expressed in E. coli. Far-western blot analysis demonstrated that Rac1 can interact with the PBD domain of p21-activated kinase 1 (PAK1). RNA interference of Pc-Rac1 affected the mRNA expression levels of immune-related genes lectin, Toll, crustin, TNF, ALF and cactus. These results suggest that Pc-Rac1 is involved in the innate immune responses in P. clarkii.

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.

The complete mitochondrial genome (mitogenome) of Leucoma salicis (Lepidoptera: Lymantriidae) was sequenced and annotated. It is a circular molecule of 15,334 bp, containing the 37 genes usually present in insect mitogenomes. All protein-coding genes (PCGs) are initiated by ATN codons, other than cox1, which is initiated by CGA. Three of the 13 PCGs had an incomplete termination codon, T or TA, while the others terminated with TAA. The relative synonymous codon usage of the 13 protein-coding genes (PCGs) was consistent with those of published lepidopteran sequences. All tRNA genes had typical clover-leaf secondary structures, except for the tRNA

Serine protease inhibitors (Serpins) are a broadly distributed superfamily of proteins with a SERPIN domain and participate in several immune responses. In this study, a serpin-28 gene was identified in B. mori and its role in immune regulation was investigated. This gene has an open reading frame of 1065 bp that encodes a 354-amino acid residue polypeptide containing one SERPIN domain with a predicted molecular weight of 40.3 kDa. Recombinant Bmserpin-28 protein was expressed in Escherichia coli and used to raise rabbit anti-Bmserpin-28 polyclonal antibodies. Quantitative real-time PCR analysis revealed that Bmserpin-28 was expressed in all examined tissues, with maximum expression in the fat body and silk gland. Expression pattern of different developmental stages showed that the highest expression level was in the pupae, while the lowest expression level was recorded at the egg stage. After challenge with four different microorganisms (Escherichia coli, Beauveria bassiana, Micrococcus luteus and B. mori nuclear polyhedrosis virus), the expression pattern of Bmserpin-28 was investigated in fat body and haemocyte samples. A substantial upregulation of Bmserpin-28 expression level was recorded following pathogen challenge in both the tested tissues. Furthermore, RNA interference of Bmserpin-28 resulted in significant upregulation of antimicrobial peptide genes. In summary, our results indicated that Bmserpin-28 may be involved in the innate immunity of B. mori.

The SOCS (Suppressor of cytokine signaling) family members are a potential negative regulator of cytokine signaling pathway and play a key role to maintain immunological functions in animals. SOCS-6 is an important member of the SOCS family, however the functions of this gene have rarely been explored among eukaryotes. Herein, we cloned and expressed SOCS-6 gene from Bombyx mori (Dazao) (BmSOCS-6), and anti-rabbit antibodies were prepared using purified recombinant BmSOCS-6 protein. Under normal physiological conditions, the BmSOCS-6 expression was observed at varied levels in six tissues, with most greatly expressed in fat body and hemocytes. After immune challenge with viral, fungal and bacterial pathogens, the BmSOCS-6 showed distinctly varied expression patterns in tissue, time and microbe dependent manner. By contrast, recombinant BmSOCS-6 protein strongly enhanced the expression of epidermal growth factor receptor (EGFR) pathway related genes, while the depletion of BmSOCS-6 by double stranded RNA suppressed their production. Altogether we concluded that BmSOCS-6 may improve the efficiency of EGFR signaling pathway in B. mori (Dazao).

Cyclic AMP response element binding (CREB) proteins participate in the regulation of many biological processes. However, little is known about their role in immune regulation in the Oak silkworm (Antheraea pernyi). In this study, a CREB gene was identified in A. pernyi and its role in immune regulation was investigated. ApCREB shares conserved signature motifs with other CREB proteins, and includes a typical leucine zipper domain, specific DNA-binding site, nuclear localisation signal (NLS) and cAMP-dependent protein kinase phosphorylation site. Recombinant ApCREB was expressed in Escherichia coli and used to raise rabbit anti-ApCREB polyclonal antibodies. ApCREB mRNA was detected in all examined tissues, with maximum expression in the midgut and integument. Following exposure to four pathogenic microorganisms (Beauveria bassiana, Escherichia coli, Micrococcus luteus, and Antheraea pernyi nuclear polyhedrosis virus), expression of ApCREB was up-regulated by B. bassiana, E. coli and ApNPV, down-regulated by M. luteus. RNA interference of ApCREB affected mRNA expression levels of antimicrobial peptide genes attacin-1, cecropin B, defensin-1, gloverin, and lebocin-2. These findings demonstrate that ApCREB is a CREB homologue that may be involved in innate immunity in A. pernyi.

Cathepsins are a group of protease, located in lysosome and play a vital role in physiological process. Here, we reported cathepsin L-like protease (Ap-cathL), which contained an open reading frame of 1155 bp and encoding 385 amino acid residues protein. The I29 inhibitor domain and peptidase C1A (clan CA of cysteine proteases, papain family C1 subfamily) putative conserved domains were detected in Ap-cathL. Quantitative real-time PCR (qRT-PCR) analysis revealed that Ap-cathL highly expressed in the fat body and midgut. The high expression during the molting stage, pupal stage and following 20E (20-hydroxyecdysone) treatment indicated that it maybe involved in the process of molting and metamorphosis. In addition, depletion of Ap-cathL influenced the expression of apoptosis pathway related genes. The protease inhibitor and RNA interference experiments showed that Ap-cathL was involved in the fat body dissociation of A. pernyi. These results suggest that Ap-cathL may involve in the process of metamorphosis and fat body dissociation of A. pernyi.

Tumor susceptibility gene 101 (TSG101) is a multi-functional gene involved in cell growth and proliferation in vertebrates. However, its role in the innate immune response of crustaceans remains unclear. Here, a TSG101 gene was identified in crayfish Procambarus clarkii with an open reading frame of 1320 bp that encoded a predicted 48.3-kDa protein highly homologous to those in other invertebrates. TSG101 mRNA was highly expressed in stomach and hepatopancreas, and its expression was induced significantly in different tissues (hemocytes, gills and intestine) by lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I: C) with various expression patterns. Recombinant TSG101 protein was expressed in Escherichia coli, and a possible protein-protein interaction between TSG101 and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) was explored by far-western blotting. RNA interference of TSG101 affected the gene expression of members of the Toll pathway. These results suggest that TSG101 is involved in the innate immune responses of P. clarkii.

Suppressors of cytokine signaling (SOCS) are a potent negative regulator of diverse cytokine-related responses to maintain various physiological processes in animals. Here, we obtained the SOCS2-12 gene sequence of Bombyx mori (Dazao) (BmSOCS2-12) from the National Center for Biotechnology Information (NCBI) to study its expression profile in different tissues, as well as in the immune tissues following larval exposure to pathogens. Further, we investigated the role of BmSOCS2-12 in producing antimicrobial peptides (AMPs) and as a regulator of ecdysteroid signaling transduction. The quantitative real-time PCR analysis revealed unequal transcript levels of BmSOCS2-12 in the different tissues, however the gene’s expression was highest in those of fat body and hemocyte. The challenge with pathogens significantly upregulated the transcript level of BmSOCS2-12 in both fat body and hemocyte when compared with the control. By contrast, recombinant BmSOCS2-12 protein injections strongly suppressed the expression of AMPs, while the knockdown of BmSOCS2-12 by double-stranded RNA enhanced their production. Administration of 20-hydroxyecdysone significantly downregulated the BmSOCS2-12 expression in fat body, and the depletion of BmSOCS2-12 enhanced the transcript levels of 20-hydroxyecdysone-responsive genes at 48 h. Altogether, BmSOCS2-12 may have multiple functional roles in the physiology of B. mori (Dazao), since it negatively regulates the expression of AMPs and ecdysteroid signaling transduction.

Calmodulin plays an important role in calcium-dependent signal transduction pathways. In this experiment, a novel calmodulin-like gene (Pc-CaM-L) was identified in the crayfish Procambarus clarkii; it encodes a polypeptide of 145 amino acids. Quantitative real-time PCR analysis revealed that Pc-CaM-L was expressed in all examined tissues, including hepatopancreas, hemocytes, heart, gill, intestine and muscle; the highest Pc-CaM-L expression level was detected in the hepatopancreas. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analysis demonstrated that a recombinant Pc-CaM-L protein was successfully expressed in Escherichia coli. The calcium-binding activity of the purified Pc-CaM-L protein was confirmed by gel mobility shift assay. The expression of Pc-CaM-L was significantly upregulated in gut, gill and hemocytes after lipopolysaccharide or polyinosinic:polycytidylic acid induction. These results suggest that Pc-CaM-L plays a role in the immune response of P. clarkii.

The silkworm, Bombyxmori L., has developed various defense mechanisms that reducethe negative effects of adverse environmental conditions. However, the mechanisms by which many physiological and ecological aspects work, including the functioning of the prophenoloxidase (pro-PO) system in Bombyxmori (BmPPO), are not completely understood. In our previous study, we found that eicosanoids mediated the innate immunity of the Chinese oak silkworm, Antheraeapernyi. To investigate the role of the pro-PO system in the silk-producing insect, Bombyxmori, the transcripts and activity of BmPPO were studied. We found that lipopolysaccharides (LPS) could induce the transcripts of BmPPO1, while eicosanoids could mediatethe induction of BmPPO1 gene expression in hemocytes. Most interestingly, the expression of BmPPO2 had no response to LPS challenge and treatment with eicosanoids. These results support the hypothesis that the B.mori pro-POsystem is associated with the eicosanoids signaling pathway.

Nasonia vitripennis is an important natural enemy of many flies. Pacifastin protease inhibitors (PPIs) play an important role in development and innate immunity of insects. In this study, cDNA sequences of a small pacifastin protease inhibitor in N. vitripennis (NvSPPI) was characterized and its open reading frame (ORF) contains 243bp. Real-time quantitative PCR (RT-qPCR) results revealed that NvSPPI mRNA were detected specifically in the venom apparatus, while they were expressed at low levels in other tissues tested. In the venom apparatus, NvSPPI transcript was highly expressed on the fourth day post eclosion and then declined gradually. The NvSPPI gene was recombinantly expressed utilizing a pGEX-4T-2 vector, and the recombinant products fused with glutathione S-transferase were purified. Inhibition of recombinant GST-NvSPPI to three serine protease inhibitors (trypsin, chymotrypsin, and protease K) were tested and results showed that recombinant NvSPPI could inhibit the activity of trypsin. Meanwhile, we evaluated the influence of the recombinant GST-NvSPPI on the phenoloxidase (PO) activity and prophenoloxidase (PPO) activation of hemolymph from a host pupa, Musca domestica. Results showed PPO activation in host hemolymph was inhibited by recombinant NvSPPI; however, there was no significant inhibition on the PO activity. Our results suggested that NvSPPI could inhibit PPO activation in host hemolymph and trypsin activity in vitro.

C-Type lectins (CTLs) are carbohydrate-recognition proteins and play critical roles in innate immunity. In this study, we characterized a CTL from Antheraea pernyi, named ApCTL, which has two carbohydrate-recognition domains (CRDs). A fragment of 1017 bp cDNA of ApCTL was isolated, which constituted 927 bp open reading frame (ORF), and it encoded putative protein of 308 amino acids. The recombinant protein of ApCTL was expressed in Escherichia coli, and anti-ApCTL antibodies were prepared. ApCTL gene was constitutively expressed in a variety of tissues while it showed a higher expression level in fat body than other tissues. Protein and mRNA levels of ApCTL in fat body and hemolymph were elevated upon nuclear polyhedrosis virus (NPV) and E. coli challenges. Recombinant ApCTL increased the agglutination of E. coli in a Ca2 +-dependent manner and had the bacteriolytic activity on E. coli. Our results suggested that ApCTL may play critical roles in pathogen recognition and could be involved in the innate immunity of A. pernyi.

The hepatopancreas of crustaceans plays an important role in lipid and carbohydrate metabolism, digestion of food, and biogenesis. In this study, the hepatopancreas transcriptome from the red crayfish Procambarus clarkii was characterized for the first time using high-throughput sequencing, producing approximately 41.4 million reads were obtained. After de novo assembly, 57,363 unigenes with an average length of 725bp were identified, Gene Ontology analysis categorized 22,580 as being involved in biological processes, among which metabolic process and cellular process groups were the most highly enriched. A total of 8034 unigenes were assigned to 223 metabolic pathways following mapping against the Kyoto encyclopedia of genes and genomes (KEGG) database. Ecdysteroid receptor (EcR)-mediated signaling pathways were investigated using digital gene expression (DGE) analysis following RNA interference targeting the EcR. A total of 529 differentially expressed genes (DEGs) were identified, including 322 downregulated and 207 upregulated unigenes. Of these, 445 (84.12%) were annotated successfully by alignment with known sequences, many of which were related to catalytic activity and binding functional categories. Using KEGG enrichment analysis, 183 DEGs were clustered into 78 pathways, and six significantly enriched pathways were predicted. The expression patterns of candidate genes identified by real-time PCR were consistent with the DGE results.

Ecdysteroids play an important role in the regulation of molting process in crustaceans. The suppression subtractive hybridization (SSH) technique was employed to search for 20-hydroxyecdysone-responsive genes in the hepatopancreas of the crayfish Procambarus clarkii. Ninety-three putative expressed sequence tags were identified by SSH, including three immune response-related genes, two cell cycle and apoptosis genes, four respiration and energy metabolism genes, four transport-related genes, six metabolism-related genes, two stress response genes, and eight transcription and translation regulation genes. The expression levels of the examined genes were regulated by 20-hydroxyecdysone and varied in the hepatopancreas during the molting stages; data were confirmed by real-time PCR. RNAi of the ecdysteroid receptor had significant effects on the expression levels of 20-hydroxyecdysone-responsive genes. These differentially expressed genes identified by SSH will provide insight into the ecdysteroid signaling pathway in P. clarkii.

Antibacterial peptides play important roles in the innate immune system of insects and are divided into four categories according to their structures. Although many antibacterial peptides have been reported in lepidopteran insects, the roles of an attacin-like gene in immune response of Antheraea pernyi remain unclear.In this study, the cloning and immunological functions of an attacin-like gene from Antheraea pernyi were investigated.The open reading frame of Ap-attacin-like gene was cloned by PCR using the specific primers and then was ligated to the pET-32a vector to construct the recombinant plasmids Ap-attacin- like-pET-32a. The recombinant Ap-attacin-like protein was expressed in E. coli (BL21 DE3) cells and purified by Ni-NTA affinity chromatography. The expression patterns of Ap-attacin-like in different tissues or under microorganism challenges were investigated by real-time PCR and western blotting. Finally, agar well diffusion assay was performed to determine the antimicrobial activity of the recombinant Ap-attacin-like proteins based on the inhibition rate.The expression level of Ap-attacin-like was highest in the fat body compared with the other examined tissues. The expression of Ap-attacin-like in the fat body was significantly elevated after E. coli, Beauveria bassiana, Micrococcus luteus or Nuclear Polyhedrosis Virus challenges. In addition, the recombinant Ap-attacin-like proteins had obvious antibacterial activity against E. coli.Ap-attacin-like was highly expressed in immune-related tissues and its expression level was significantly induced by different microorganism challenges, suggesting that Ap-attacin-like participated in the innate immunity of A. pernyi.

Orthaga olivacea Warre (Lepidoptera: Pyralidae) is an important agricultural pest of camphor trees (Cinnamomum camphora). To further supplement the known genome-level features of related species, the complete mitochondrial genome of Orthaga olivacea is amplified, sequenced, annotated, analyzed, and compared with 58 other species of Lepidopteran. The complete sequence is 15,174 bp, containing 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a putative control region. Base composition is biased toward adenine and thymine (79.02% A+T) and A+T skew are slightly negative. Twelve of the 13 PCGs use typical ATN start codons. The exception is cytochrome oxidase 1 (cox1) that utilizes a CGA initiation codon. Nine PCGs have standard termination codon (TAA); others have incomplete stop codons, a single T or TA nucleotide. All the tRNA genes have the typical clover-leaf secondary structure, except for trnS(AGN), in which dihydrouridine (DHU) arm fails to form a stable stem-loop structure. The A+T-rich region (293 bp) contains a typical Lepidopter motifs 'ATAGA' followed by a 17 bp poly-T stretch, and a microsatellite-like (AT)13 repeat. Codon usage analysis revealed that Asn, Ile, Leu2, Lys, Tyr and Phe were the most frequently used amino acids, while Cys was the least utilized. Phylogenetic analysis suggested that among sequenced lepidopteran mitochondrial genomes, Orthaga olivacea Warre was most closely related to Hypsopygia regina, and confirmed that Orthaga olivacea Warre belongs to the Pyralidae family.

Actias selene (Hübner) is an important silk-spinning moth. Like other moths, it has innate immunity but no acquired immunity. However, there are few studies on immune-related genes of A. selene. Here, differential expression RNAseq experiment was employed to examine the genes related to different metabolic pathways and to explore the immune mechanism of the A. selene post Beauveria bassiana (Bb) and Micrococcus luteus (ML) stimuli. A total of 64,372,921 clean reads were obtained and 39,057 differentially expressed genes (DEGs) were identified. In the Bb vs. PBS group (PBS as the control), 9,092 genes were up-regulated and 4,438 genes were down-regulated; in the ML vs. PBS group, 5,903 genes were up-regulated and 5,175 genes were down-regulated. The KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) analyses of DEGs confirmed that many DEGs were associated with "Metabolism pathway", "cellular process", "cell" and "catalytic activity". Among them, 194 and 149 differentially expressed genes were related to immunity in the Bb vs. PBS group and ML vs. PBS group, respectively. We verified the reliability of the data with reverse transcription quantitative real-time PCR analysis of randomly selected genes. Furthermore, the phylogenetic tree results showed that HSP90, PGRP and MyD88 genes of A. selene were most closely related to Antheraea pernyi (Guérin-Méneville). These results will provide an overview of the molecular mechanism of A. selene resistance to fungal and bacterial infections as well as an evolutionary aspect of these genes. Moreover, the interrelated trophic mechanisms among different groups of organisms are vital to explore, thus this study will lay a foundation for further studies on the innate immune mechanism of saturniid moths, and provide important theoretical basis for studying the relationship between A. selene and other species.

Hemolins play an important role in development and innate immunity in insects. In this study, a hemolin cDNA of 1412bp in Actias selene (As-HEM) was isolated and its open reading frames (ORFs) were 420 amino acid residues. Sequence analysis indicated As-HEM was homologous to those hemolins from other insects species. The recombinant protein of As-HEM was expressed in Escherichia coli, and anti-As-HEM antibodies were prepared. Real-time quantitative PCR (RT-qPCR) and western blot results revealed that mRNA and protein levels of As-HEM were mostly detected in hemocytes and hemolymph. Immune challenge assays showed that both the mRNA and protein levels of As-HEM could be induced significantly post Beauveria bassiana, E. coli, Micrococcus luteus and nuclear polyhedrosis virus challenges. Agglutination assays revealed that recombinant As-HEM could promote the agglutination of E. coli in the presence of calcium. Our results suggested that As-HEM was involved in the innate immunity of A. selene.

The complete mitochondrial genome (mitogenome) of Spilarctia robusta (Lepidoptera: Noctuoidea: Erebidae) was sequenced and analyzed. The circular mitogenome is made up of 15,447 base pairs (bp). It contains a set of 37 genes, with the gene complement and order similar to that of other lepidopterans. The 12 protein coding genes (PCGs) have a typical mitochondrial start codon (ATN codons), whereas cytochrome c oxidase subunit 1 (cox1) gene utilizes unusually the CAG codon as documented for other lepidopteran mitogenomes. Four of the 13 PCGs have incomplete termination codons, the cox1, nad4 and nad6 with a single T, but cox2 has TA. It comprises six major intergenic spacers, with the exception of the A+T-rich region, spanning at least 10 bp in the mitogenome. The nucleotide composition of the genome is greatly A+T biased (81.09%), with a negative AT skewness (-0.007), indicating the presence of fewer As than Ts, similar to other Noctuoidea. The A+T-rich region is 343 bp long, and contains some conserved regions, including an "ATAGA" motif followed by a 19 bp poly-T stretch, a microsatellite-like (AT)9 and a poly-A element, a characteristic shared with other lepidopteran mitogenomes. Phylogenetic analysis, based on 13 PCGs using Maximum likelihood methods revealed that S. robusta belongs to the superfamily Noctuoidea.

Nucleopolyhedroviruses (NPVs) is one group of Baculoviruses. The infection of NPV in silkworm is often lethal. To investigate the effective measures to stop the infection of NPV, we cloned cDNA encoding small heat shock protein 25.4 in Antheraea pernyi (Ap-HSP25.4). The translated amino acid sequence consisted of 223 residues with a calculated molecular mass of 25.4kDa and an isoelectronic point (pI) of 4.93. Quantitative real-time PCR was used to investigate the expression patterns and distribution profiles of Ap-sHSP25.4 before and after challenged with NPV. We found that the inhibitors of eicosanoid synthesis could suppress the transcription of Ap-sHSP25.4 in the fat body in a dose dependent manner. And arachidonic acid induced the expression of Ap-sHSP25.4. Thus, we concluded that sHSPs may be promising candidates to boost insect immunity in practice.

Cathepsin B is a lysosomal cysteine protease of the papain-like enzyme family with multiple biological functions. In the present study, a cathepsin B gene (named PcCTSB ) was cloned and characterized from the red crayfish, Procambarus clarkii . The cDNA fragments of PcCTSB was 990 bp in length. It encoded a putative protein of 329 amino acid residues with predicted molecular weight of 36.4 kDa and isoelectric point of 7.020. Sequence alignment revealed that PcCTSB protein is 53.6%–80.4% identical with those from other 10 species. The predicted tertiary structure of PcCTSB protein was highly similar to that of animals. The results of the phylogenetic analysis indicated that the PcCTSB protein could be clustered with the Eriocheir sinensis cathepsin B protein. The recombinant protein of PcCTSB was expressed successfully in Escherichia coli cells. The mRNA expressions of PcCTSB were detected in all tested tissues, particularly high in the hepatopancreas. After lipopolysaccharide (LPS) challenge, the expression levels of PcCTSB were up-regulated significantly at different time points compared with control. Our results suggested that the PcCTSB might play an important role in defending against the pathogenes infection.

A suppressor of cytokine signaling 2 (SOCS-2) homolog was identified from the red crayfish Procambarus clarkii. Phylogenic analysis showed that P. clarkii SOCS-2 (Pc-SOCS-2) was very similar to that of invertebrates. The sequence contains a central SH2 domain, a conserved SOCS box, and a variable N-terminal domain. Real-time polymerase chain reaction showed that Pc-SOCS-2 mRNA level was higher in muscle and heart than in the other tissues examined. A 40-kDa recombinant protein was successfully expressed and purified from Escherichia coli cells as confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Pc-SOCS-2 was significantly downregulated in the hepatopancreas after 20-hydroxyecdysone injection. Knockdown of Pc-SOCS-2 gene by small interfering RNA transfection in the hepatopancreas increased the expression levels of 20-hydroxyecdysone-responsive genes at 48 hr. Taken together, these results suggest that Pc-SOCS-2 is a negative regulator of ecdysteroid signaling transduction.

Apolipophorin-III (ApoLp-III) is involved in lipid transport and innate immunity in insects. In this study, we isolated ApoLp-III gene (As-ApoLp-III) cDNA from Actias selene Hubner (Lepidoptera: Saturniidae). It contained 660 nucleotides with a putative open reading frame (ORF) of 561 bp encoding 186 amino acid residues. It showed similarities with ApoLp-III proteins from other insect species, especially Antheraea pernyi. The recombinant protein of As-ApoLp-III was expressed in Escherichia coli, and anti-As-ApoLp-III antibodies were prepared. The As-ApoLp-III gene was expressed at high levels in the fat body and integument, and at low levels in the hemolymph, testis, silk gland, malpighian tubule, ovary, and midgut. As-ApoLp-III gene can be upregulated by E. coli, Micrococcus luteus, Beauveria bassiana, and nuclear polyhedrosis virus (NPV) and showed a greater sensitivity for gram-negative bacteria and fungi than for gram-positive bacteria and virus. These data indicated that As-ApoLp-III may play an important role in innate immune responses of Actias selene.

In this study, we sequenced the complete mitochondrial genome of Eligma narcissus and compared it with 18 other lepidopteran species. The mitochondrial genome (mitogenome) was a circular molecule of 15,376 bp containing 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and an adenine (A) + thymine (T) - rich region. The positive AT skew (0.007) indicated the occurrence of more As than Ts. The arrangement of 13 PCGs was similar to that of other sequenced lepidopterans. All PCGs were initiated by ATN codons, except for the cytochrome c oxidase subunit 1 (cox1) gene, which was initiated by the CGA sequence, as observed in other lepidopterans. The results of the codon usage analysis indicated that Asn, Ile, Leu, Tyr and Phe were the five most frequent amino acids. All tRNA genes were shown to be folded into the expected typical cloverleaf structure observed for mitochondrial tRNA genes. Phylogenetic relationships were analyzed based on the nucleotide sequences of 13 PCGs from other insect mitogenomes, which confirmed that E. narcissus is a member of the Noctuidae superfamily.

Colleterial glands (CG) present in the body of adult female of Bombyx mori, which can help adhere eggs on the surface of the host plants. Although this organ has been known for centuries, only morphology and its secretions have been studied. Their gene expression profiles and physiological roles remain largely unknown. Aided by high-throughput next generation sequencing (NGS), we reported the comparative transcriptome analysis of CG isolated from the H9 and the P50 strains of Bombyx mori. A total of 19,896,957 and 20,446,366 clean reads were obtained from CG of H9 and the P50 strains, respectively; then differential expression analysis was performed, and 1,509 differentially expressed genes (DEGs) were identified. Among them, 1,001 genes are up-regulated and 508 genes are down-regulated in P50 individuals compared with H9 individuals. The enrichment of GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) of DEGs confirmed that many DEGs were associated with "Amino acid transport and metabolism", "Nucleotide transport and metabolism", and "Inorganic ion transport and metabolism", 25 of the DEGs related to the "ECM-receptor interaction passway", "sphingolipid metabolism passway", and "amino sugar and nucleotide sugar metabolism passway" were potentially involved in the process of CG development and mucus secretion. According to these data, we hypothesized that CG play an important role in providing favorable physiological environment for the glue secretion formation. In addition, GO enrichment and differential expression analysis of the DEGs in the CG indicate that this gland may be involved in the transporting of small solutes such as sugars, ions, amino acids and nucleotide sugar to the CG. Our findings lay the foundation for further research on CG function.

In this study, we investigated the role of karyopherin alpha 3 in the heat shock response in male silkworm pupae. Karyopherin alpha recognizes the classical nuclear location sequence on proteins and transports them into the nucleus by forming a trimetric complex with karyopherin beta. Three predicted karyopherin alphas (KPNA1, KPNA2 and KPNA3) have been identified from the silkworm Bombyx mori. Pull-down assay result showed that KPNA3 can pull down heat shock transcription factor (HSF) from proteins extracted from tissues using non-denature lysis buffer. After 45 °C heat shock on male B. mori pupae for 30 min, we identified two heat shock protein (HSP) mRNA expression peaks correlating with HSP19.9, HSP20.4 and HSP25.4 at 4 h (peak 1) and 24 h (peak 2). The second peak was eliminated after knockdown of KPNA3. Similar results were obtained following knockdown of HSF, which is the trans-activating factor of heat shock. However, KPNA3 knockdown was not accompanied by the decreased HSF protein levels at 24 h after heat shock which were observed following HSF knockdown. We also expressed recombinant protein GST-KPNA3 and His-HSF in Escherichia coli to perform GST pull-down assay and the result confirmed the interaction between KPNA3 and HSF. We concluded that KPNA3 knockdown eliminates the second heat shock protein peak in the heat shock response of male silkworm pupae by reducing HSF transport into the nucleus.

We isolated a novel defence gene (AsDef) cDNAs from Actias selene (Hubner 1806) (Lepidoptera: Saturniidae). It contained 691 nucleotides with a putative open reading frame of 498 bp encoding 165 amino acid residues with a reeler domain. It showed significant similarities with defence proteins from several other lepidopteran insect species including Samia ricini (Donovan 1798), Antheraea mylitta (Drury 1773), Bombyx mori (Linnaeus 1758), Lonomia oblique (Walker 1855), Hyphantria cunea (Drury 1773) and Manduca sexta (Linnaeus 1763). The recombinant protein of AsDef was expressed successfully in Escherichia coli (Migula 1895) cells, and the anti-AsDef antibody against rabbit was prepared. Results of real-time quantitative Polymerase Chain Reaction (PCR) and western blot showed that AsDef gene was detected at a particularly high levels in fat body, while AsDef protein can only be detected in haemolymph. For immune functions, AsDef gene can be upregulated by E. coli, Micrococcus luteus (Cohn 1872), Bea...

Small heat shock proteins (sHSPs) can regulate protein folding and protect cells from stress. To investigate the role of sHSPs in the silk-producing insect Antheraea pernyi (A. pernyi; Lepidoptera: Saturniidae), cDNA encoding HSP20.8 in A. pernyi, termed Ap-sHSP20.8, was identified as a 564 bp ORF. The translated amino acid sequence encoded 187 residues with a calculated molecular mass of 20.8 kDa and an isoelectronic point (pI) of 5.98; the sequence showed homology to sHSP chaperone proteins from other insects. Ap-sHSP20.8 mRNA transcript expression was abundant in the midgut and fat body and found to be both constitutive and inducible by infectious stimuli. Therefore, Ap-sHSP20.8 may play important roles in A. pernyi immune responses under biotic stress. Furthermore, we found that eicosanoids could mediate the induction of Ap-sHSP20.8 in the fat body and midgut. Our findings show that sHSPs may be promising molecules to target in order to cripple immunity in insect pests.

The vitellogenin receptor (VgR) plays a key role on embryonic development in oviparous animals. Here, we cloned a VgR gene, which was identified from the wild silkworm Bombyx mandarina (BmaVgR) using reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Sequence analysis revealed that BmaVgR is 5,861 bp long with an open reading frame encoded by 1,811 amino acid residues. The predicted amino acid sequence has 99.7 and 98.2% identity with the VgRs of Actias selene and Bombyx mori, respectively. The class B domain sequence of BmaVgR was cloned and expressed in Escherichia coli, and purified by a Ni-NTA column. Polyclonal antibodies were produced against the purified recombinant protein, and titer of the antibody was about 1:12,800 measured by enzyme-linked immunosorbent assay (ELISA). Western blot and RT-qPCR showed that BmaVgR was expressed in the ovary and fat body of female larvae and the ovary of moth, and the expression level was highest at the third day and then declined from third day to seventh in fat body of pupa. After knockdown of the BmaVgR gene through RNA interference (RNAi), other three BmaVgR-related genes (Vg, egg-specific protein, and low molecular weight lipoprotein LP gene) were all downregulated significantly.

In this study, a complete mitochondrial genome (mitogenome) sequence of Abraxas suspecta (Lepidoptera: Geometridae) is isolated and characterized. The complete DNA is 15,547 bp length and contains 2 ribosomal RNA genes, 23 putative transfer RNA (tRNA) genes including an extra tRNAAsn (AUU), 13 protein-coding genes and an adenine (A) + thymine (T)-rich region. The nucleotide composition and gene organization are identical to those of other lepidopteran, except for the presence of an extra copy of trnN (AUU). Of the 38 genes, twenty-five genes (9 PCGs and 16 tRNAs) are encoded by heavy strand (H-strand), while thirteen are encoded by light strand (L-strand). Among the 13 PCGs, 12 PCGs employ ATN as initiation codon, while cytochrome c oxidase subunit 1 (cox1) utilizes CGA as initiation codon. Four of the 13 PCGs have the incomplete termination codon T, while the remainder terminated with the canonical stop codon. All tRNA genes are folded into the typical clover-leaf structure of mitochondrial tRNAs, except for the tRNASer (AGN) gene, in which the DHU arm fails to form a stable stem-loop structure. The A+T-rich region is 532 bp long, and contains some conserved regions, including ‘ATAGA’ motif followed by a 17bp poly-T stretch, a microsatellite-like element (AT)8(AAT)3 and also a poly-A element. A short Phylogenetic analysis based on 13 PCGs using maximum likelihood (ML) and Bayesian inference (BI) revealed that A. suspecta resides in the Geometridae family. We present the method and approach to use moths as model organisms for further genetic and evolutionary biology studies.

Serine protease inhibitors play a key role in the immune system of invertebrates by controlling proteolytic cascades. Besides its importance, the knowledge on immune functions of serpins in most of insects is fragmentary. In the present study, we identified serpin-12 from Antheraea pernyi encoding a predicted 402 amino acid residue protein (Apserpin-12). We expressed the recombinant protein in Escherichia coli and the purified protein was used for the synthesis of rabbit anti-Apserpin-12 polyclonal antibodies and functional studies. Quantitative real-time ploymerase chain reaction (qRT-PCR) analysis revealed that the knock-down of Apserpin-12 enhanced the prophenoloxidase (PPO) cascade stimulated by Micrococcus luteus in hemolymph, whereas addition of recombinant Apserpin-12 protein along with same elicitor led to down-regulate PPO activation. Following different microbial challenge (E. coli, Beauveria bassiana, M. Luteus, and nuclear polyhedrosis virus), the expression of Apserpin-12 mRNA was induced significantly. Furthermore, the Apserpin-12 double-stranded RNA administration elicited the expression of antimicrobial peptides, while the treatment with recombinant protein suppressed their expression. Tissue profile of Apserpin-12 indicated that it is expressed in all examined tissues, that is, hemolymph, malpighian tubules, midgut, silk gland, integument, and fat body with variation in their transcript levels. We concluded that Apserpin-12 may regulate PPO activation and inhibit the production of antimicrobial peptides in A. pernyi, suggesting important role in its immune system.

Antimicrobial peptides (AMPs) play important roles in the insect innate immune response. To investigate the role of a lebocin-like protein in the oak silkworm, Antheraea pernyi, in response to immune challenge, an Ap-lebocin-like gene with an open reading frame of 489 bp was identified. This gene encodes a protein of 162 amino acid residues and belongs to a family of proline-rich antimicrobial peptides. Real-time PCR analysis found that Ap-lebocin-like was expressed in all tested tissues, with the highest expression in the midgut, followed by the epidermis, and the lowest expression in the silk gland. Different transcription patterns of Ap-lebocin-like were observed in the fat body and midgut after injection of Escherichia coli, A. pernyi nucleopolyhedrovirus, Micrococcus luteus, and Beauveria bassiana. An antibacterial activity assay indicated that the Ap-lebocin-like has high antibacterial activity in vitro, with a greater activity toward gram-positive bacteria (Staphylococcus aureus) than toward gram-negative bacteria (E. coli). These results suggested that Ap-lebocin-like participates in the immune response of A. pernyi.

In the present study, we sequenced the complete mitochondrial genome (mitogenome) of Agrius convolvuli (Lepidoptera: Sphingidae) and compared it with previously sequenced mitogenomes of lepidopteran species. The mitogenome was a circular molecule, 15 349 base pairs (bp) long, containing 37 genes. The order and orientation of genes in the A. convolvuli mitogenome were similar to those in sequenced mitogenomes of other lepidopterans. All 13 protein-coding genes (PCGs) were initiated by ATN codons, except for the cytochrome c oxidase subunit 1 (cox1) gene, which seemed to be initiated by the codon CGA, as observed in other lepidopterans. Three of the 13 PCGs had the incomplete termination codon T, while the remainder terminated with TAA. Additionally, the codon distributions of the 13 PCGs revealed that Asn, Ile, Leu2, Lys, Phe, and Tyr were the most frequently used codon families. All transfer RNAs were folded into the expected cloverleaf structure except for tRNASer(AGN), which lacked a stable dihydrouridine arm. The length of the adenine (A) + thymine (T)-rich region was 331 bp. This region included the motif ATAGA followed by a 19-bp poly-T stretch and a microsatellite-like (TA)8 element next to the motif ATTTA. Phylogenetic analyses (maximum likelihood and Bayesian methods) showed that A. convolvuli belongs to the family Sphingidae.

In the present study, the complete sequence of the mitochondrial genome (mitogenome) of Daphnis nerii (Lepidoptera: Sphingidae) is described. The mitogenome (15,247 bp) of D.nerii encodes13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and an adenine (A) + thymine (T)-rich region. Its gene complement and order is similar to that of other sequenced lepidopterans. The 12 PCGs initiated by ATN codons except for cytochrome c oxidase subunit 1 (cox1) gene that is seemingly initiated by the CGA codon as documented in other insect mitogenomes. Four of the 13 PCGs have the incomplete termination codon T, while the remainder terminated with the canonical stop codon. This mitogenome has six major intergenic spacers, with the exception of A+T-rich region, spanning at least 10 bp. The A+T-rich region is 351 bp long, and contains some conserved regions, including 'ATAGA' motif followed by a 17 bp poly-T stretch, a microsatellite-like element (AT)9 and also a poly-A element. Phylogenetic analyses based on 13 PCGs using maximum likelihood (ML) and Bayesian inference (BI) revealed that D. nerii resides in the Sphingidae family.

Cathepsins are key members of mammalian papain-like cysteine proteases that play an important role in the immune response. In this study, a fragment of cDNA encoding cathepsin O proteinase (ApCathepsin O) was cloned from Antheraea pernyi. It contains an open reading frame of 1170bp and encodes a protein with 390 amino acid residues, including a conserved I29 inhibitor domain and a peptidase C1A (clan CA of cysteine proteases, papain family C1 subfamily) domain. Comparison with other previously reported cathepsin O proteins showed identity ranging from 45% to 79%. Quantitative real-time PCR (qRT-PCR) and Western blot analysis revealed that ApCathepsin O was highly expressed in the fat body; furthermore, the high expression during the pupal stage indicated that it might be involved during metamorphosis. After exposure to four different heat-killed pathogens (Escherichia coli, Beauveria bassiana, Micrococcus luteus, and A. pernyi nucleopolyhedrovirus), the expression levels of ApCathepsin O mRNA significantly increased and showed variable expression patterns. This indicates that ApCathepsin O is potentially involved in the innate immune system of A. pernyi. Interestingly, ApCathepsin O expression was upregulated after 20-hydroxyecdysone (20E) injection, which suggested that it might be regulated by 20E. In conclusion, ApCathepsin O is a protease that may play an important role in the innate immune response and metamorphosis of A. pernyi.

The complete mitochondrial genome (mitogenome) of Leucoma salicis (Lepidoptera: Lymantriidae) was sequenced and annotated. It is a circular molecule of 15,334 bp, containing the 37 genes usually present in insect mitogenomes. All protein-coding genes (PCGs) are initiated by ATN codons, other than cox1, which is initiated by CGA. Three of the 13 PCGs had an incomplete termination codon, T or TA, while the others terminated with TAA. The relative synonymous codon usage of the 13 protein-coding genes (PCGs) was consistent with those of published lepidopteran sequences. All tRNA genes had typical clover-leaf secondary structures, except for the tRNASer(AGN), in which the dihydrouridine (DHU) arm could not form a stable stem-loop structure. The A + T-rich region of 325 bp had several distinctive features, including the motif ‘ATAGA’ followed by an 18 bp poly-T stretch, a microsatellite-like (AT)7 element, and an 11-bp poly-A present immediately upstream of tRNAMet. Relationships among 32 insect species were determined using Maximum Likelihood (ML), Neighbor Joining (NJ) and Bayesian Inference (BI) phylogenetic methods. These analyses confirm that L. salicis belongs to the Lymantriidae; and that Lymantriidae is a member of Noctuoidea, and is a sister taxon to Erebidae, Nolidae and Noctuidae, most closely related to Erebidae.

In this study, we sequenced the complete mitochondrial genome of Eligma narcissus and compared it with 18 other lepidopteran species. The mitochondrial genome (mitogenome) was a circular molecule of 15,376 bp containing 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and an adenine (A) + thymine (T) − rich region. The positive AT skew (0.007) indicated the occurrence of more As than Ts. The arrangement of 13 PCGs was similar to that of other sequenced lepidopterans. All PCGs were initiated by ATN codons, except for the cytochrome c oxidase subunit 1 (cox1) gene, which was initiated by the CGA sequence, as observed in other lepidopterans. The results of the codon usage analysis indicated that Asn, Ile, Leu, Tyr and Phe were the five most frequent amino acids. All tRNA genes were shown to be folded into the expected typical cloverleaf structure observed for mitochondrial tRNA genes. Phylogenetic relationships were analyzed based on the nucleotide sequences of 13 PCGs from other insect mitogenomes, which confirmed that E. narcissus is a member of the Noctuidae superfamily.

The receptor for activated C kinase (RACK) is an important scaffold protein with regulatory functions in cells. However, its role in the immune response of Antheraea pernyi to pathogen challenge remains unclear. To investigate the biological functions of RACK in the wild silkworm A. pernyi, cloning was performed and the expression patterns of the RACK gene were analyzed. Sequence analysis revealed that the RACK gene was 1120 bp containing a 960-bp open reading frame. The deduced RACK protein sequence reveals the higher identity with its homologs from other insects. SDS-PAGE and western blot analysis demonstrated successful expression of a 36-kDa recombinant RACK protein in Escherichia coli. The titer of a rabbit-raised antibody against recombinant RACK protein was about 1: 20000, determined by ELISA. Real-time PCR analysis showed that RACK expression was higher in fat bodies than in other examined A. pernyi tissues. The expression of RACK mRNA in fat bodies of fifth larvae of A. pernyi was obviously induced after nucleopolyhedrovirus, E. coli or Beauveria bassiana challenge. However, the expression patterns of RACK were different in response to these pathogens. Our data suggest that RACK may play a role in the innate immune responses of A. pernyi.