Yu-Xuan Sun

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.

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

Abstract 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. HighlightsFull‐length cDNA of Serine Protease Inhibitor14 was identified in Antheraea pernyi.Apserpin‐14 expressed ubiquitously in all tested tissues and developmental stages.Apserpinn‐14 mRNA in fat body and hemocyte up regulate after microbial challenge.Apserpin‐14 regulates prophenol oxidase activation.Apserpin‐14 was found to regulate antimicrobial peptides.

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

ABSTRACT 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. HighlightsA cathepsin L‐like protease (Ap‐cathL) was first characterized from A. pernyi.The spatial and temporal expression profiles of Ap‐cathL were investigated.20E (molting hormone) induces Ap‐cathL expression in the fat body.Ap‐cathL RNAi influenced the expression of apoptosis‐related genes.Ap‐cathL functions in the fat body dissociation via inhibitor and RNAi experiments.

Characterization and functional analysis of serpin-1 like gene from oak silkworm Antheraea pernyi

Serpins are a broadly distributed family of proteases found in various organisms that play an important role in regulating the immune response. Here, we identified a serpin-1 gene from Antheraea pernyi that encodes a 279 amino acid protein with a molecular weight of 30.8 kDa. We expressed the recombinant Ap-serpin-1 protein in Escherichia coli and used the purified protein to prepare rabbit anti-Ap-serpin-1 polyclonal antibodies. We calculated the enzyme-linked immunosorbent assay titer of the antibody as 1:128000. Quantitative real-time polymerase chain reaction analysis revealed that Ap-serpin-1 was expressed in all examined tissues, including hemolymph, malpighian tubules, midgut, silk gland, integument and the fat body; the highest Ap-serpin-1 expression levels was detected in the fat body. We next investigated the expression patterns of Ap-serpin-1 in both fat body and hemolymph samples, following treatment with E. coli, Beauveria bassiana, Micrococcus luteus and nuclear polyhedrosis virus (NPV). We reported that NPV and M. luteus significantly enhanced Ap-serpin-1 expression in the fat body. While, in the hemolymph samples, treatment with B. bassiana and M. luteus was shown to upregulate Ap-serpin-1 expression at 24 h induction. Altogether, our results suggest that Ap-serpin-1 is involved in the innate immunity of A. pernyi.

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.

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.

Identification and function of cAMP response element binding protein in Oak silkworm Antheraea pernyi

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.

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 genes 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.

Molecular cloning, expression, and characterization of E2F transcription factor 4 from Antheraea pernyi

The E2F transcription factor family is distributed widely in eukaryotes and has been well studied among mammals. In the present study, the E2F transcription factor 4 (E2F4) gene was isolated from fat bodies of Antheraea pernyi and sequenced. E2F4 comprised a 795 bp open reading frame encoding a deduced amino acid sequence of 264 amino acid residues. The recombinant protein was expressed in Escherichia coli (Transetta DE3), and anti-E2F4 antibodies were prepared. The deduced amino acid sequence displayed significant homology to an E2F4-like protein from Bombyx mori L. Quantitative real-time polymerase chain reaction analysis revealed that E2F4 expression was highest in the integument, followed by the fat body, silk glands, and haemocytes. The expression of E2F4 was upregulated in larvae challenged by bacterial (Escherichia coli, Micrococcus luteus), viral (nuclear polyhedrosis virus), and fungal (Beauveria bassiana) pathogens. These observations indicated that E2F4 is an inducible protein in the immune response of A. pernyi and probably in other insects.

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

&NA; 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). HighlightsA BmSOCS‐6 gene was firstly identified from B. mori (Dazao).BmSOCS‐6 ubiquitously expressed in different tissue as well as in developmental stages.BmSOCS‐6 transcript in fat body and hemocytes was up‐regulated following pathogen challenge.BmSOCS‐6 was involved in the regulation of epidermal growth factor receptor pathway.