Toll-interacting protein participates in immunity and development of the lepidopteran insect Antheraea pernyi.

Toll-interacting protein (Tollip) participates in multiple biological processes. However, the biological functions of Tollip proteins in insects remain to be further explored. Here, the genomic sequence of tollip gene from Antheraea pernyi (named Ap-Tollip) was identified with a length of 15,060 bp, including eight exons and seven introns. The predicted Ap-Tollip protein contained conserved C2 and CUE domains and was highly homologous to those tollips from invertebrates. Ap-Tollip was highly expressed in fat body compared with other determined tissues. As far as the developmental stages were concerned, the highest expression level was found at the 14th day in eggs or the 3rd day of the 1st instar. Ap-Tollip was also obviously regulated by lipopolysaccharide, polycytidylic acid or 20E in different tissues. In addition, the interaction between Ap-Tollip and ubiquitin was confirmed by western blotting and pull-down assay. RNAi of Ap-Tollip significantly affected the expression levels of apoptosis and autophagy-related genes. These results indicated that Ap-Tollip was involved in immunity and development of A. pernyi.

A Protein Asteroid with PIN Domain in Silkworm Bombyx mori Is Involved in Anti-BmNPV Infection.

Nuclease is a type of protein that degrades nucleic acids, which plays an important role in biological processes, including RNA interference efficiency and antiviral immunity. However, no evidence of a link between nuclease and Bombyx mori nucleopolyhedrovirus (BmNPV) infection in silkworm B. mori has been found. In this study, a protein asteroid (BmAst) containing the PIN domain and XPG domain was identified in silkworm B. mori. BmAst gene was highest expressed in hemocytes and fat body of the 5th instar larvae, and high expression in the pupa stage. The transcriptional levels of the BmAst gene in 5th instar larvae were significantly induced by BmNPV or dsRNA. After knocking down BmAst gene expression by specific dsRNA, the proliferation of BmNPV in B. mori was increased significantly, whereas the survival rate of larvae was significantly lower when compared with the control. Our findings indicate that BmAst is involved in silkworm resistance to BmNPV infection.

Complete biodegradation of the oldest organic herbicide 2,4-Dichlorophenoxyacetic acid by engineering Escherichia coli.

After nearly 80 years of extensive application, the oldest organic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) has caused many problems of environmental pollution and ecological deterioration. Bioremediation is an ideal method for pollutant treatment. However, difficult screening and preparation of efficient degradation bacteria have largely hindered its application in 2,4-D remediation. We have created a novel engineering Escherichia coli with a reconstructed complete degradation pathway of 2,4-D to solve the problem of screening highly efficient degradation bacteria in this study. The results of fluorescence quantitative PCR demonstrated that all nine genes in the degradation pathway were successfully expressed in the engineered strain. The engineered strains can quickly and completely degrade 0.5 mM 2, 4-D within 6 h. Inspiring, the engineered strains grew with 2,4-D as the sole carbon source. By using the isotope tracing method, the metabolites of 2,4-D were found incorporated into the tricarboxylic acid cycle in the engineering strain. Scanning electron microscopy showed that 2,4-D had less damage on the engineered bacteria than the wild-type strain. Engineered strain can also rapidly and completely remedy 2,4-D pollution in natural water and soil. Assembling the metabolic pathways of pollutants through synthetic biology was an effective method to create pollutant-degrading bacteria for bioremediation.

A Kazal-type serine protease inhibitor mediates innate immunity in wild silkworm Actias selene Hübner.

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 KSPI gene from A. selene (AsKSPI, which can negatively regulate PPO activation. Its open reading frame (ORF) was 291 bp, encoding 96 amino acids. Real-time quantitative PCR (RT-qPCR) showed that AsKSPI mRNA was significantly expressed in the fat body. Immunostimulatory tests showed that the mRNA levels of AsKSPI in the fat body were up-regulated following injection of Micrococcus luteus, Escherichia coli, Beauveria bassiana, and nuclear polyhedrosis virus (NPV). Enzyme activity experiments showed that the purified recombinant AsKSPI could inhibit the activation of PPO in hemolymph of A. selene, but did not affect phenoloxidase (PO) activity after PPO had been activated. So, AsKSPI could regulate the innate immunity of A. selene through the PPO cascade. These findings will contribute to the understanding of the immune mechanism of wild silkworm and provide a basis for better protection and utilization of special economic insect resources.

Characterization and Phylogenetic Analysis of the Complete Mitochondrial Genome of Saturnia japonica.

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

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

Immunological Function of the Antibacterial Peptide Attacin-Like in the Chinese Oak Silkworm, Antheraea pernyi.

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.

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.

Characterization of the complete mitochondrial genome of Orthaga olivacea Warre (Lepidoptera Pyralidae) and comparison with other Lepidopteran insects.

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.

The RNA-seq approach to discriminate gene expression profiles in response to Beauveria bassiana and Micrococcus luteus microbial pathogens on Actias selene (Lepidoptera: Saturniidae).

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.

Identification and function of a lebocin-like gene from the Chinese oak silkworm, Antheraea pernyi.

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.

How Preferred Brands Relate to the Self: The Effect of Brand Preference, Product Involvement, and Information Valence on Brand-Related Memory.

This study adopted the paradigm of the self-reference effect to explore how brand preference, product involvement, and information valence affects brand-related memory by three experiments. Experiment 1 examined memory differences between positive/negative information of self-/other-preferred brands. Results showed increased memory of positive words (i.e., the effect of information valence) in the self-preferred brand group, yet memory of self-preferred brands was poorer than that of other-preferred brands. Experiment 2 examined effects of degree of brand preference and information valence, and revealed a positive association between degree of preference and memory of brand-related positive words. Experiment 3 explored the effects of brand preference and product involvement. Results showed that the memory of high-preference brands was stronger in the high-involvement group. Additionally, product involvement demonstrated a significant positive correlation with memory. The observed effects of information valence, especially in self-preference (Experiment 1) and high-preference (Experiment 2) conditions, can be explained by self-schema and mnemic neglect theories. The increased memory of highly preferred brands in a high-involvement condition can be explained by intimacy and self-expansion models (Experiment 3).

Characterization of small GTPase Rac1 and its interaction with PAK1 in crayfish Procambarus clarkii.

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.

Characterization and functional analysis of serpin-28 gene from silkworm, Bombyx mori.

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.

Comparative transcriptome analysis of silkworm, Bombyx mori colleterial gland suggests their functional role in mucous secretion.

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.

Role of Antheraea pernyi serpin 12 in prophenoloxidase activation and immune responses.

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.

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.

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.

Serpin-14 negatively regulates prophenoloxidase activation and expression of antimicrobial peptides in Chinese oak silkworm Antheraea pernyi.

Genes encoding proteins of serpins superfamily are widely distributed in invertebrates. In insects, serpins play important roles in regulating immune responses and other physiological processes. Here, we report the cloning and characterization of cDNA of Apserpin-14 from Chinese oak silkworm (Antheraea pernyi). The Apserpin-14 gene contains 1206 bp open reading frame, encoding a predicted 401 amino acid residue protein. We expressed the recombinant Apserpin-14 protein in Escherichia coli and then purified protein was used to prepare rabbit anti-Apserpin-14 polyclonal antibodies. Quantitative real-time PCR analysis revealed that mRNA level of Apserpin-14 was highest in the fat body, whereas, among developmental stages the 5th instar and pupal stage showed greatest expression. Furthermore, Escherichia coli, Beauveria bassiana, Micrococcus luteus and nuclear polyhedrosis virus challenge enhanced Apserpin-14 transcript in both the fat body and hemocyte. Recombinant Apserpin-14 added to hemolymph inhibited spontaneous melanization and suppressed prophenoloxidase activation stimulated by M. luteus, but did not affect phenoloxidase (PO) activity. Injection of recombinant Apserpin-14 protein into A. pernyi larvae significantly reduced the transcript levels of antimicrobial peptides in the fat body, while its depletion by double stranded RNA enhanced their expression. We concluded that Apserpin-14 likely involved in regulation of proPO activation and production of antimicrobial peptides, implying its important role in the innate immune system of A. pernyi.