Anti-lipopolysaccharide factor (ALF) is a small protein with broad-spectrum antimicrobial activity, which has potential application in the disease control. Previously, we isolated seven ALF isoforms from the Chinese shrimp Fenneropenaeus chinensis. In the present study, their distributions in tissues of shrimp were analyzed and the data showed that different isoforms had different expression profiles, which suggested that they might have different functions. Then, the functions of different isoforms were studied by analyzing the antibacterial and antiviral activities of the functional domain of ALFs, the LPS-binding domain (LBD), which were synthesized by chemical methods. Different ALFs showed distinct antibacterial and antiviral activities, which were consistent with their diverse tissue distribution patterns. Sequence analysis on the LBD domain of different isoforms revealed that an identical lysine residue site was specifically conserved in peptides with anti-WSSV activity. In order to confirm whether this lysine residue is critical to the antiviral activity of the peptide, new peptides were synthesized by changing residues at this site. Changing the lysine residue at the specific site to other amino acid residue, the antiviral activity of the peptide apparently decreased. While replacing other residue with a lysine residue at this site in LBD peptide without anti-WSSV activity, the peptide will obtain the antiviral activity to WSSV. These results not only showed us a comprehensive understanding on the function of ALFs from F. chinensis, but also provided clues for the development of ALFs as potential therapeutic drugs to WSSV.

The insulin-like androgenic gland hormone (IAG) is the key regulator in crustacean male sexual differentiation. As a secreted peptide hormone, IAG might perform its biological function through interacting with the membrane receptor. However, the receptor of IAG remains unclear. In the current study, a putative IAG receptor gene (FcIAGR) was identified in Fenneropenaeus chinensis. The deduced amino acid sequence of FcIAGR contained several conserved domains of insulin-like receptor proteins, including two L domains (L1 and L2), a cysteine-rich domain, three fibronectin III domains, a transmembrane domain, and an intracellular tyrosine kinase domain. Tissue distribution and in situ hybridization analysis showed that FcIAGR was predominantly expressed in the androgenic gland and testis in male F. chinensis. Protein colocalization analysis in HEK293 cells showed that FcIAGR could colocalize with both FcIAG1 and FcIAG2, respectively. Yeast two-hybrid assay further confirmed the interactions between FcIAGR and FcIAGs. After a long-term silencing of FcIAGR with double-stranded RNA, most of the germ cells in the testis were arrested at the secondary spermatocytes, whereas those in the control developed into sperm cells. The data indicated that FcIAGR was the receptor of FcIAGs in F. chinensis. The current study provides insight into the mechanism that the insulin-like signaling pathway regulates the male sexual differentiation in Decapoda crustaceans.

Functional communications between nervous, endocrine and immune systems are well established in both vertebrates and invertebrates. Circulating hemocytes act as fundamental players in this crosstalk, whose functions are conserved during the evolution of the main groups of metazoans. However, the roles of the neuroendocrine-immune (NEI) system in shrimp hemocytes during pathogen infection remain largely unknown. In this study, we sequenced six cDNA libraries prepared with hemocytes from Litopenaeus vannamei which were injected by WSSV (white spot syndrome virus) or PBS for 6 h using Illumina Hiseq 4000 platform. As a result, 3444 differentially expressed genes (DEGs), including 3240 up-regulated genes and 204 down-regulated genes, were identified from hemocytes after WSSV infection. Among these genes, 349 DEGs were correlated with innate immunity and categorized into seven groups based on their predictive function. Interestingly, 18 genes encoded putative neuropeptide precursors were induced significantly by WSSV infection. Furthermore, some genes were mapped to several typical processes in the NEI system, including proteolytic processing of prohormones, amino acid neurotransmitter pathways, biogenic amine biosynthesis and acetylcholine signaling pathway. The data suggested that WSSV infection triggers the activation of NEI in shrimp, which throws a light on the pivotal roles of NEI system mediated by hemocytes in shrimp antiviral immunity.

The lipopolysaccharide binding domain (LBD) in anti-lipopolysaccharide factor (ALF) is the main functional element of ALF, which exhibits antimicrobial activities. Our previous studies show that the peptide LBDv, synthesized based on the modified sequence of LBD (named LBD2) from FcALF2, exhibited an apparently enhanced antimicrobial activity. To learn the prospect of LBDv application, the characteristics of LBDv were analyzed in the present study. The LBDv peptide showed higher antimicrobial and bactericidal activities compared with LBD2. These activities of the LBDv peptide were stable after heat treatment. LBDv could also exhibit in vivo antimicrobial activity to Vibrio harveyi. The LBDv peptide was found to bind bacteria, quickly cause bacterial agglutination, and kill bacteria by damaging their membrane integrity. Structure analysis showed that both LBDv and LBD2 held the β-sheet structure, and the positive net charge and amphipathicity characteristic were speculated as two important components for their antimicrobial activity. The cytotoxicity of LBDv was evaluated in cultured Spodoptera frugiperda (Sf9) cells and Cherax quadricarinatus hemocytes. More than 80% cells could survive with the LBDv concentration up to 16 μM. Collectively, these findings highlighted the potential antimicrobial mechanism of LBD peptides, and provided important information for the commercial use of LBDv in the future.