The crosstalk between Toll and AMPK signaling pathways mediates growth inhibition of Eriocheir sinensis under deltamethrin stress.

Hepatopancreatic necrosis disease (HPND) broke out in 2015 in the Eriocheir sinensis aquaculture region of Xinghua, Jiangsu Province; however, the specific cause of HPND remains unclear. A correlation was found between HPND outbreak and the use of deltamethrin by farmers. In this study, E. sinensis specimens developed the clinical symptoms of HPND after 93 days of deltamethrin stress. The growth of E. sinensis with HPND was inhibited. Adenosine monophosphate-activated protein kinase (AMPK) is a central regulator of energy homeostasis, and its expression was up-regulated in the intestine of E. sinensis with HPND. Growth inhibitory genes (EsCabut, Es4E-BP, and EsCG6770) were also up-regulated in the intestine of E. sinensis with HPND. The expression levels of EsCabut, Es4E-BP, and EsCG6770 decreased after EsAMPK knockdown. Therefore, AMPK mediated the growth inhibition of E. sinensis with HPND. Further analysis indicated the presence of a crosstalk between the Toll and AMPK signaling pathways in E. sinensis with HPND. Multiple genes in the Toll signaling pathway were upregulated in E. sinensis under 93 days of deltamethrin stress. EsAMPK and its regulated growth inhibition genes were down-regulated after the knockdown of genes in the Toll pathway. In summary, the crosstalk between the Toll and AMPK signaling pathways mediates the growth inhibition of E. sinensis under deltamethrin stress.

Expression levels of serine proteases, their homologs, and prophenoloxidase in the Eriocheir sinensis with hepatopancreatic necrosis syndrome (HPNS) and their expression regulation by Runt.

The occurrence of hepatopancreatic necrosis syndrome (HPNS) has seriously affected the sustainable development of Chinese mitten crab (Eriocheir sinensis) farming industry. Limited studies have focused on the immune responses in crabs with HPNS. Serine proteases (SPs) and SP homologs (SPHs) play important roles in the innate immunity of crustaceans. This study investigated the effects of HPNS on the expression levels of genes related to prophenoloxidase (proPO) activation system, and the relationship between Runt transcription factor and the transcriptions of these genes. Eight SPs and five SPHs (SPH1-4, Mas) were identified from E. sinensis. SPs contain a catalytic triad of "HDS", while SPHs lack a catalytic residue. SPs and SPHs all contain a conservative Tryp_SPc domain. Evolutionary analysis showed that EsSPs, EsSPHs, EsPO, and EsRunt were clustered with SPs, SPHs, POs, and Runts of other arthropods, respectively. In crabs with HPNS, the expression levels of six SPs (1, 3, 4, 6, 7, and 8), five SPHs, and PO were significantly upregulated in the hepatopancreas. The knockdown of EsRunt could evidently decrease the expression levels of four SPs (3, 4, 5 and 8), five SPHs (SPH1-4, Mas), and PO. Therefore, the occurrence of HPNS activates the proPO system. Furthermore, the expression levels of partial genes related to proPO system were regulated by Runt. The activation of innate immune system may be a strategy for crabs with HPNS to improve immunity and fight diseases. Our study provides a new understanding of the relationship between HPNS and innate immunity.

A positive feedback loop between two C-type lectins originated from gene duplication and relish promotes the expression of antimicrobial peptides in Procambarus clarkii.

Gene duplication (GD) leads to the expansion of gene families that contributes organisms adapting to stress or environment and dealing with the infection of various pathogens. C-type lectins (CTLs) in crustaceans undergo gene expansion and participate in various immune responses. However, the functions of different CTL produced by GD are not fully characterized. In the present study, two CTL genes (designated as PcLec-EPS and PcLec-QPS, respectively) were identified from Procambarus clarkii. PcLec-EPS and PcLec-QPS originate from GD and the main difference between them is exon 3. PcLec-EPS and PcLec-QPS respectively contains EPS and QPS motif in their carbohydrate recognition domain. The mRNA levels of PcLec-EPS and PcLec-QPS in hemocytes, gills, intestine and lymph underwent time-dependent enhancement after D-Mannose and D-Galactose challenge. Recombinant PcLec-EPS and PcLec-QPS could bind to carbohydrates and microbes, and agglutinate bacteria. The results of experiments on recombinant protein injection and RNA interference indicate that PcLec-EPS and PcLec-QPS can respectively strong recognize and bind D-Mannose and D-Galactose, activate the Relish transcriptional factor, and further upregulate the expression of different antimicrobial peptides (AMPs). In addition, these two CTLs and Relish could positively regulate the expression of each other, suggesting that there is a positive feedback loop between two CTLs and Relish that regulates the expression of AMPs. It may contribute to the expansion of the immune response for host quickly and efficiently eliminating pathogenic microorganisms. This study provides new knowledge for clear understanding the significance and function of different CTL generated by GD in immune defenses in crustacean.

Identification of three novel Spätzle genes in Eriocheir sinensis and their roles during white spot syndrome virus infection.

Proteins of Spätzle family play an essential role in innate immunity in invertebrates by activating the Toll pathway to induce the expression of antimicrobial peptides. However, little is known about the function of Spätzle in in the immune response of the Chinese mitten crab. In the present study, three novel Spätzle genes (named as EsSpz1, EsSpz2, and EsSpz3) were identified from Eriocheir sinensis. The genome structure of EsSpz1 contains two exons and an intron. Three Spätzle proteins all contain a Pfam Spaetzle domain. In the evolution, EsSpz1-3 cluster with other Spätzle proteins from crustaceans. EsSpz1-3 were widely distributed in multiple immune tissues. The expression levels of EsSpz1-3 in the intestine were remarkably upregulated after white spot syndrome virus (WSSV) challenge. The knockdown of EsSpz1-3 remarkably decreased the expressions of crustins and anti-lipopolysaccharide factors during WSSV infection. Moreover, EsSpz1-3 silencing remarkably increased the expression of WSSV envelope protein VP28. These findings suggest that new-found EsSpz1-3 in E. sinensis could promote the synthesis of antimicrobial peptides and inhibit the expression of VP28 during WSSV infection. Our study indicates that EsSpz1-3 in E. sinensis may participate in the innate immune defenses against WSSV by inducing the expression of antimicrobial peptides. This study provides new knowledge for the function of Spätzle in the antiviral immune defense in crustacean.

The forkhead box O transcription factor regulates lipase and anti-microbial peptide expressions to promote lipid catabolism and improve innate immunity in the Eriocheir sinensis with hepatopancreatic necrosis disease.

Eriocheir sinensis is a crustacean with great economic value, but the occurrence of hepatopancreatic necrosis disease (HPND) severely restricts the development of crab aquaculture. Study on the survival mechanism of crabs with HPND is beneficial to provide new strategies for disease prevention and control. The Forkhead box O (FOXO) transcription factor family is involved in various key biological processes of organisms. In this study, a FOXO gene (named as EsFOXO) from E. sinensis was cloned. The full-length cDNA of EsFOXO is 2592 bp containing a 2133 bp open reading frame that encodes 710 amino acids. EsFOXO was widely distributed in multiple immune tissues. Further study found that the expression of EsFOXO in the intestine of crabs with HPND was significantly upregulated compared with that in the normal crabs. However, whether EsFOXO is involved in the immune and metabolic regulation of crabs remains unknown. RNA interference analysis showed that EsFOXO participates in the positive regulation of the expression of two pancreatic lipases, three anti-lipopolysaccharide factors, and three crustins. Results from our research suggest that two strategies are adopted by crabs with HPND for survival under starvation: on the one hand, the synthesis of antimicrobial peptides is increased to improve the innate immunity; on the other hand, the expression of enzymes correlated with lipid catabolism is up-regulated that mobilizes the fat in the crab, going through catabolism. Our study provides more evidence for an in-depth understanding of the survival mechanism of crabs with HPND.