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Hemolin is a distinctive immunoglobulin superfamily member involved in invertebrate immune events. Although it is believed that hemolin regulates hemocyte phagocytosis and microbial agglutination in insects, little is known about its contribution to the humoral immune system. In the present study, we focused on hemolin in Antheraea pernyi ( Ap -hemolin) by studying its pattern recognition property and humoral immune functions. Tissue distribution analysis demonstrated the mRNA level of Ap -hemolin was extremely immune-inducible in different tissues. The results of western blotting and biolayer interferometry showed recombinant Ap -hemolin bound to various microbes and pathogen-associated molecular patterns. In further immune functional studies, it was detected that knockdown of hemolin regulated the expression level of antimicrobial peptide genes and decreased prophenoloxidase activation in the A. pernyi hemolymph stimulated by microbial invaders. Together, these data suggest that hemolin is a multifunctional pattern recognition receptor that plays critical roles in the humoral immune responses of A. pernyi ., Competing Interests: Author YL is employed by Liaoning Applos Biotechnology Co. Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 He, Zhou, Cai, Liu, Zhao, Zhang, Wang and Zhang.)

Hexamerin was originally identified as a storage protein but later confirmed to be involved in many physiological processes. In the present study, we cloned and characterized a novel hexamerin complementary DNA sequence from the Chinese oak silkworm, Antheraea pernyi (Ap-hexamerin), which shows high homology with reported insect methionine-rich hexamerins. The tissue distribution and time course of expression demonstrated that Ap-hexamerin was predominantly synthesized in the fat body and the expression level was significantly increased in response to the microbial challenge, suggesting the relevance of Ap-hexamerin to immune responses. In further immune functional studies, Ap-hexamerin was confirmed to take part in the upregulation of prophenoloxidase (PPO) activation in A. pernyi haemolymph triggered by pathogen-associated molecular patterns (PAMPs). Additional molecular interaction analysis revealed that Ap-hexamerin is capable of binding the PAMPs used in the phenoloxidase assay, suggesting hexamerin in A. pernyi may positively regulate haemolymph PPO activation, acting as a pattern recognition protein., (© 2019 Wiley Periodicals, Inc.)

Pattern recognition receptors (PRRs) are employed in insects to defend against infectious pathogens by triggering various immune responses. Peptidoglycan recognition proteins (PGRPs), a vital family of PRRs, are widely distributed and highly conserved from vertebrates to invertebrates. To date, five PGRP genes have been identified in Antheraea pernyi, but their biochemical roles still remain unknown. In this study, we focused on the immune functions of PGRP-SA in A. pernyi (ApPGRP-SA), which was confirmed to be immune-related according to its significantly up-regulated expression level post microbial injection. In addition, the binding properties of ApPGRP-SA were investigated using a recombinant protein produced in a prokaryotic expression system, revealing that rApPGRP-SA displayed a multi-binding ability to various microbes, including the Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus, Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and fungus Candida albicans, together with their surface pathogen associated molecular patterns (PAMPs). Further studies showed that after recognition, the mixture of rApPGRP-SA/PAMP remarkably stimulated prophenoloxidase (PPO) activation in the hemolymph of A. pernyi in vitro, while the ds-PGRP-SA-treated hemolymph exhibited a lower sensitivity to PAMPs in comparison to the native sample. Moreover, the transcriptional level of the three antimicrobial peptides was also decreased in PGRP-SA knock-down larvae in response to immune-challenge. In summary, we conclude that ApPGRP-SA is a novel identified PGRP in A. pernyi that might act as a broad-spectrum pattern recognition receptor and is involved in the PPO activation system as well as antimicrobial peptide production., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Apolipoproteins are protein components of lipoprotein particles, and are increasingly recognized to be functioning in the innate immune systems of both insects and mammals. Mammalian apolipoprotein B (apoB) is associated with a diverse range of innate immune defenses including suppression of bacterial pathogenesis, virus toxicity neutralization, and inhibition of cytokine releases from immune cells. However, little is known about apoB homologous insect apolipophorin-II/I (apoLp-II/I) in controlling of specific pathogen-host encounters such as microbial infections. In the present study, we describe cDNA cloning and characterization of an apoLp-II/I from Chinese oak silk worm, Antheraea pernyi. The apoLp-II/I cDNA is 10237bp in length, which possesses an open reading frame encoding 3305 amino acids. A consensus cleavage site RGRR presenting from Arg 710 to Arg 713 implies posttranslational cleavage of this protein. Ap-apoLp-II/I shares high sequence identities with apoLp-II/I in lepidoptera and other insects. In addition, considerable similarities also exist between Ap-apoLp-II/I and human apoB, which basically positioned in first 1000 residues of the amino termini. Tissue distribution and time-course expression results demonstrate that Ap-apoLp-II/I transcripts were detected predominantly in the fat body, less in epidermis and rarely in midgut, while the synthetic apoLp-II/I protein was abundant in hemocytes and plasma instead of the fatbody. Expression of Ap-apoLp-II/I was stimulated in response to bacterial challenge. In addition, our preliminary studies established a novel role for Ap-apoLp-II/I in regulating prophenoloxidase activation system. Taken together, apoLp-II/I may play an essential role in innate responses of Antheraea pernyi., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Serpins are a superfamily of proteins engaged in various physiological processes in all kingdoms of life. To date, many striking results have demonstrated serpins are involved in the invertebrate immune system by regulating the proteolytic cascades. However, in most insect species, the immune functions of serpins in response against pathogen invasion remain obscure. In this study, we identified a full-length cDNA sequence of serpin, named serpin-3, from the Chinese oak silkworm Antheraea pernyi. Sequence alignments have indicated that Apserpin-3 might regulate the melanization reaction via inhibiting prophenoloxidases-activating protease(s) in plasma. Furthermore, it was detected to be primarily transcribed within the fat body, epidermis and hemocytes with significant induction following immune-challenge. Further studies have shown that the knockdown of serpin-3 up-regulated the prophenoloxidases cascade stimulated by pathogen in hemolymph, while the addition of recombinant serpin-3 along with the same elicitor led to the suppressed activation of prophenoloxidase. Besides, the injection of dsRNA of serpin-3 caused the elevated expression of antimicrobial peptides. Altogether, we arrived at a conclusion that serpin-3 might act as a negative-regulator in prophenoloxidases activation and inhibit the production of antimicrobial peptides in Antheraea pernyi larvae., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Apolipophorin-III (apoLp-III) is an exchangeable apolipoprotein found in many insect species and functions as a lipid transport vehicle. Recent studies have shown that apoLp-III is a multifunctional molecule involved in not only lipid transportation but also innate immune responses. In the present study, the pattern recognition properties of Antheraea pernyi apoLp-III were investigated. Recombinant Ap-apoLp-III was bound to different species of microbes and further study showed the rAp-apoLp-III is capable of interacting with pathogen associated molecular patterns (PAMPs) on the microbial cell surface. In addition, an Ap-apoLp-III/PAMP mixture stimulated the prophenoloxidase (PPO) activation of A. pernyi hemolymph in vitro, to a greater extent than PAMP alone while Ap-apoLp-III itself failed to activate the PPO system, indicating that Ap-apoLp-III up-regulates PPO activation by combining with PAMP. After pathogen invasion following an injection of Staphylococcus aureus, RNAi-mediated silencing of apoLp-III decreased the transcriptional abundance of three antimicrobial peptide genes. These data suggest that apoLp-III is a versatile pattern recognition receptor and may play important roles in the innate immune responses of Antheraea pernyi., (Copyright © 2016. Published by Elsevier Ltd.)