Mitochondrial genome of the sweet potato hornworm, Agrius convolvuli (Lepidoptera: Sphingidae), and comparison with other Lepidoptera species.

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.

Characterization and functional study of a Cecropin-like peptide from the Chinese oak silkworm, Antheraea pernyi.

In present study, a Cecropin-like peptide from Antheraea pernyi (ApCec) was cloned and characterized. The full-length ApCec cDNA encoded a protein with 64 amino acids including a putative 22-amino-acid signal peptide, a 4-amino-acid propeptide, and a 38-amino-acid mature peptide. ApCec gene was highly expressed in Malpighian tubules of A. pernyi after induction for 24 h by Escherichia coli in PBS. Pro-ApCec (including propeptide and mature peptide) and M-ApCec (just mature peptide) were synthesized chemically and analyzed by HPLC and mass spectroscopy. The antibacterial activity of M-ApCec is more potent than pro-ApCec against E. coli K12 or B. subtilus in both minimum inhibitory concentration and inhibition zone assays. Hemolytic assay results showed M-ApCec possessed a low cytotoxicity to mammalian cells. The secondary structure of M-ApCec forms α-helical structure, shown by circular dichroism spectroscopy. Transmission electron microscopy analysis suggested that M-ApCec killed bacteria by disrupting bacterial cell membrane integrity. Our results indicate ApCec may play an important role in defending from pathogenic bacteria in A. pernyi, and it may be as a potential candidate for applications in antibacterial drug development and agriculture.

Cathepsin O is involved in the innate immune response and metamorphosis of Antheraea pernyi.

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.

Characterization and function of a cathepsin B in red crayfish (Procambarus clarkii) following lipopolysaccharide challenge.

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.

Gene expression patterns in response to pathogen challenge and interaction with hemolin suggest that the Yippee protein of Antheraea pernyi is involved in the innate immune response.

Yippee was first identified as a protein that physically interacts with the Hemolin protein of Hyalophora cecropia. In this study, we identified a gene with a 366bp open reading frame (ORF) that encodes a 121 amino acid protein containing a conserved Yippee domain. We named this gene Ap-Yippee (Yippee gene from Antheraea pernyi), and investigated the role of the protein in the host immune response. A recombinant Ap-Yippee protein was expressed in Escherichia coli cells, and polyclonal antibodies were produced against the recombinant protein. Real-time PCR and a Western blot analysis revealed that Ap-Yippee is expressed in the hemocytes, Malpighian tubules, midgut, silk gland, epidermis, and fat bodies of A. pernyi, with the highest expression level observed in Malpighian tubules. The fifth instar larvae of A. pernyi were challenged by injecting them with nucleopolyhedrovirus (AP-NPV), the Gram-negative bacterium E. coli, the Gram-positive bacterium Micrococcus luteus, or the entomopathogenic fungus, Beauveria bassiana. These challenges with diverse pathogens resulted in differential expression patterns of the protein. A knockdown of the Ap-Yippee gene by small interfering RNA (siRNA) transfection had a significant influence on the expression of the hemolin in the pupae which was confirmed by qRT-PCR and Western blot. Furthermore, a possible protein-protein interaction between Ap-Yippee and Hemolin was explored by Far-Western blotting. Therefore, our data suggest that the Ap-Yippee protein is involved in a pathway that regulates the immune response of insects.

IDENTIFICATION AND EXPRESSION ANALYSIS OF VITELLOGENIN RECEPTOR FROM THE WILD SILKWORM, Bombyx mandarina.

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.

Molecular characterization of an Apolipophorin-III gene from the Chinese oak silkworm, Antheraea pernyi (Lepidoptera: Saturniidae).

Apolipophorin-III (ApoLp-III) acts in lipid transport, lipoprotein metabolism, and innate immunity in insects. In this study, an ApoLp-III gene of Antheraea pernyi pupae (Ap-ApoLp-III) was isolated and characterized. The full-length cDNA of Ap-ApoLp-III is 687 bp, including a 5'-untranslated region (UTR) of 40 bp, 3'-UTR of 86 bp and an open reading frame of 561 bp encoding a polypeptide of 186 amino acids that contains an Apolipophorin-III precursor domain (PF07464). The deduced Ap-apoLp-III protein sequence has 68, 59, and 23% identity with its orthologs of Manduca sexta, Bombyx mori, and Aedes aegypti, respectively. Phylogenetic analysis showed that the Ap-apoLp-III was close to that of Bombycoidea. qPCR analysis revealed that Ap-ApoLp-III expressed during the four developmental stages and in integument, fat body, and ovaries. After six types of microorganism infections, expression levels of the Ap-ApoLp-III gene were upregulated significantly at different time points compared with control. RNA interference (RNAi) of Ap-ApoLp-III showed that the expression of Ap-ApoLp-III was significantly downregulated using qPCR after injection of E. coli. We infer that the Ap-ApoLp-III gene acts in the innate immunity of A. pernyi.

The complete mitogenome of Bombyx mori strain Dazao (Lepidoptera: Bombycidae) and comparison with other lepidopteran insects.

The complete mitochondrial genome (mitogenome) of Bombyx mori strain Dazao (Lepidoptera: Bombycidae) was determined to be 15,653bp, including 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and a A+T-rich region. It has the typical gene organization and order of mitogenomes from lepidopteran insects. The AT skew of this mitogenome was slightly positive and the nucleotide composition was also biased toward A+T nucleotides (81.31%). All PCGs were initiated by ATN codons, except for cytochrome c oxidase subunit 1 (cox1) gene which was initiated by CGA. The cox1 and cox2 genes had incomplete stop codons consisting of just a T. All the tRNA genes displayed a typical clover-leaf structure of mitochondrial tRNA. The A+T-rich region of the mitogenome was 495bp in length and consisted of several features common to the lepidopteras. Phylogenetic analysis showed that the B. mori Dazao was close to Bombycidae.

Identification of immune response-related genes in the Chinese oak silkworm, Antheraea pernyi by suppression subtractive hybridization.

Insects possess an innate immune system that responds to invading microorganisms. In this study, a subtractive cDNA library was constructed to screen for immune response-related genes in the fat bodies of Antheraea pernyi (Lepidoptera: Saturniidae) pupa challenged with Escherichia coli. Four hundred putative EST clones were identified by suppression subtractive hybridization (SSH), including 50 immune response-related genes, three cytoskeleton genes, eight cell cycle and apoptosis genes, five respiration and energy metabolism genes, five transport genes, 40 metabolism genes, ten stress response genes, four transcription and translation regulation genes and 77 unknown genes. To verify the reliability of the SSH data, the transcription of a set of randomly selected immune response-related genes were confirmed by semi-quantitative reverse transcription-PCR (RT-PCR) and real-time quantitative reverse transcription-PCR (qRT-PCR). These identified immune response-related genes provide insight into understanding the innate immunity in A. pernyi.

Author Correction: Comparative Mitochondrial Genome Analysis of Eligma narcissus and other Lepidopteran Insects Reveals Conserved Mitochondrial Genome Organization and Phylogenetic Relationships.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Characterization of the Complete Mitochondrial Genome of Leucoma salicis (Lepidoptera: Lymantriidae) and Comparison with Other Lepidopteran Insects.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cathepsin L-like protease can regulate the process of metamorphosis and fat body dissociation in Antheraea 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.

Suppressor of cytokine signaling 6 can enhance epidermal growth factor receptor signaling pathway in Bombyx mori (Dazao).

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).

Suppressor of cytokine signaling 2-12 regulates antimicrobial peptides and ecdysteroid signaling pathways in Bombyx mori (Dazao).

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.

Mitochondrial genome of Abraxas suspecta (Lepidoptera: Geometridae) and comparative analysis with other Lepidopterans.

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.

Comparative mitochondrial genome analysis of Daphnis nerii and other lepidopteran insects reveals conserved mitochondrial genome organization and phylogenetic relationships.

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.

A role of tumor susceptibility gene 101 (TSG101) in innate immune response of crayfish Procambarus clarkii.

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.

Characterization and Expression Analysis of Receptor for Activated C Kinase from Silk-producing Insect Antheraea pernyi.

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.

Comparative Mitochondrial Genome Analysis of Eligma narcissus and other Lepidopteran Insects Reveals Conserved Mitochondrial Genome Organization and Phylogenetic Relationships.

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.

Characterization of the Complete Mitochondrial Genome of Leucoma salicis (Lepidoptera: Lymantriidae) and Comparison with Other Lepidopteran Insects.

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.