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).

De novo transcriptome assembly and analysis of differential gene expression following peptidoglycan (PGN) challenge in Antheraea pernyi.

Antheraea pernyi is not only an important economic insect, it is increasingly employed as a model organism due to a variety of advantages, including ease of rearing and experimental manipulation compared with other Lepidoptera. Peptidoglycan (PGN) is a major component of the bacterial cell wall, and interactions between PGN and A. pernyi cause a series of physiological changes in the insect. In the present study, we constructed cDNA libraries from a A. pernyi PGN-infected group and a control group stimulated with phosphate-buffered saline (PBS). The transcriptome was de novo assembled using the Trinity platform, and 1698 differentially expressed genes (DEGs) were identified, comprising 894 up-regulated and 804 down-regulated genes. To further investigate immune-related DEGs, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were performed. GO analysis identified major immune-related GO terms and KEGG enrichment indicated gene responses to three pathways related to the insect immune system. Several homologous genes related to the immune response of the A. pernyi fat body post-PGN infection were identified and categorised. Taken together, the results provide insight into the complex molecular mechanisms of the responses to bacterial infection at the transcriptional level.

A small heat shock protein 21 (sHSP21) mediates immune responses in Chinese oak silkworm Antheraea pernyi.

Small heat shock proteins (sHSPs) are conserved among insects and play an important role in the regulation of many biological processes, including temperature stress, abiotic stress, immune responses, metamorphosis, and embryo development. Antheraea pernyi is an economically valuable silk-producing moth and source of insect food containing high-quality protein. The aim of this study was to quantify expression of the ApsHSP21 gene in response to pathogen-associated molecular patterns (PAMPs) and nucleopolyhedrovirus (NPV) challenge. The deduced ApsHSP21 protein sequence consists of 186 residues with a calculated molecular mass of 21.0 kDa and an isoelectronic point (pI) of 6.63. The protein contains a conserved α-crystallin domain (ACD), and includes two casein kinase II phosphorylation sites, a protein kinase C phosphorylation site, two tyrosine kinase phosphorylation sites, and various polypeptide binding sites. Phylogenetic analysis revealed that ApsHSP21 is closely related to homologs from other insects. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis revealed that expression of ApsHSP21 was significantly up-regulated at different timepoints following simulated pathogen challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), glucan, and NPV. The results suggest sHSP21 is involved in innate immune responses 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 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.