Identification of acid phosphatase (ShACP) from the freshwater crab Sinopotamon henanense and its expression pattern changes in response to cadmium.

Acid phosphatase(ACP) is an important immune enzyme in crustacean humoral immunity. At present, the research on ACP mainly focuses on the biochemical properties of the enzyme, while few studies on gene expression. In this study, ShACP was cloned and the effect of cadmium stress on the expression and function of ShACP in the freshwater crab Sinopotamon henanense was studied. Analysis of the ShACP sequence and tissue distribution results showed that the cDNA sequence of ShACP was 1629 bp, including 48 bp 5' untranslated region, 1209 bp open reading frame region, and 372 bp 3' untranslated region, encoding 402 amino acids. ShACP contained multiple phosphorylation sites and mainly played a role in the hemolymph. Under low-concentration cadmium stress, the body improved immunity by enhancing the expression of ShACP, while high-concentration cadmium stress inhibited the expression of ShACP. ShACP can promote the phagocytosis of hemocytes, while cadmium stress reduced the phagocytosis of hemocytes. This study provides a theoretical basis for further research on the immune system of crabs and is of great significance for the study of crustacean immune responses under heavy metal stress.

Cloning, identification, and functional characterization of novel prophenoloxidases (ShproPO) from the freshwater crab Sinopotamon henanense in response to cadmium exposure and Aeromonas hydrophila infection.

Prophenoloxidase (proPO) is essential in the prophenoloxidase-activating system (proPO-AS) which is important for defense against foreign infection in crustaceans. However, most studies have focused on expression in the presence of a single pathogenic bacterium, and very few have addressed the presence of environmental contaminants simultaneously, such as cadmium (Cd) and Aeromonas hydrophila. Our study aimed to investigate the function of proPO in the freshwater crab Sinopotamon henanense and the changes in its expression by Cd and infection of A. hydrophila. A novel proPO from the hemocytes of S. henanense (ShproPO) was found in this research, the full-length cDNA of ShproPO was 2620 bp of encoding a protein of 678 amino acids containing three typical hemocyanin domains. The ShproPO protein could be found in both the granular (GHc) and the semi-granular hemocytes (SGHc). The ShproPO mRNA was found to be abundantly expressed in hemocytes and could be influenced by A. hydrophila infection. These results indicate that ShproPO could be involved in the antibacterial process. Further research found that low concentrations of Cd could promote its expression after infection with A. hydrophila. Therefore, it was hypothesized that Cd disrupted the response of crabs to A. hydrophila infection. Subsequently, PO enzyme activity was found to be significantly reduced through in vivo RNA interference with ShproPO, and the results suggested that ShproPO is likely to be a key enzyme in the melanization response. Finally, ShproPO was found to significantly enhance the phagocytosis of A. hydrophila-infected hemocytes by in vitro recombination, confirming that ShproPO is involved in hemocyte-mediated melanization and phagocytosis. Our findings reveal completely new insight into the immunotoxicity of Cd and the immune function of ShproPO in S. henanense.

A novel type II crustin in the innate immune response of the freshwater crab (Sinopotamon henanense) against infection and its expression changes by cadmium.

Antibacterial peptide (AMP), an effector of the innate immune system, is an essential component of invertebrate innate immunity. Crustin is a family of antimicrobial peptides that are widely studied in crustaceans. Here we report a novel crustin (designated Shcrustin) from the freshwater crab Sinopotamon henanense. The results revealed that the full-length cDNA of Shcrustin was 691 bp with an open reading frame (ORF) of 510 bp. Phylogenetic analysis of the Shcrustin sequence showed that it clustered with type II crustin. Shcrustin exists in different tissues, among which the highest expression level is found in the gills. After the bacterial challenge, the expression of Shcrustin increased in hemocytes or gills. However, crustin expression was suppressed in the presence of cadmium (Cd). To elucidate the biological activity of Shcrustin, we constructed a recombinant Shcrustin protein. Purified rShcrustin could bind to a variety of bacteria and inhibit the growth of different bacteria indicating that Shcrustin has inhibitory activity against gram-positive and gram-negative bacteria. In addition, the phagocytic rate of hemocytes toward bacteria decreased after the interference of Shcrustin expression by RNA interference, suggesting that Shcrustin may be involved in such a process. Therefore, we conclude that Shcrustin may be involved in the innate immunity of S. henanense by binding to bacteria and promoting hemolymph phagocytosis to clear invading pathogens. It is an important immune effector against pathogen infection. In the presence of Cd, it may alter the expression of Shcrustin and suppress its immune function.

Clone, identification and functional characterization of a novel toll (Shtoll1) from the freshwater crab Sinopotamon henanense in response to cadmium exposure and Aeromonas hydrophila infection.

Toll is essential in innate immune system which is important for defense against bacterial, fungal and viral infections in invertebrates. Our previous study showed that cadmium (Cd) could change the expression pattern of ShToll3 in the epithelium (gills and midgut from the freshwater crab Sinopotamon henanense) infected by Aeromonas hydrophila. To investigate the diverse innate immune roles of crustacean homolog Tolls, in this study, we cloned Shtoll1 from S. henanense. The full-length cDNA of Shtoll1 was 4746 bp, with an ORF of 3033 bp encoding a putative protein of 111 amino acids, a 5'-untranslated region of 255 bp and a 3'-untranslated region of 1713 bp. Phylogenetic analysis showed that ShToll1 was clustered into the group of DmToll1, DmToll 4 and DmToll 5. In addition, the tissue distribution results showed that Shtoll1 was expressed widely in different tissues, with the highest expression in heamocytes. Besides, Shtoll1 expressions were upregulated in heamocytes and hepatopancreas after A. hydrophila infection. At the same time, the increase of Shtoll1 expressions were examined in heamocytes in response to Cd exposure and A. hydrophila infection in combination. Through western blotting and immunohistochemical analysis, the ShToll1 expressions in heamocytes were increased in response to A. hydrophila and Cd independently as well as in combination. Moreover, the mRNA level of three antimicrobial peptides (AMPs) alf5, alf6, and c-lys, which possibly responded to Cd and A. hydrophila stimulation through Shtoll1, were analyzed. Thus, we conclude that Cd expand the susceptibility of ShToll1 to A. hydrophila infection in heamocytes. This suggest that ShToll1 may contribute to the innate immune defense of S. henanense against A. hydrophila and Cd in heamocytes.