Diversity of MrTolls and their regulation of antimicrobial peptides expression during Enterobacter cloacae infection in Macrobrachium rosenbergii.

Toll/Toll-like receptor (TLR) is an important pattern recognition receptor that plays an important role in the immunity of animals. Six Toll genes were identified in Macrobrachium rosenbergii, namely, MrToll, MrToll1, MrToll2, MrToll3, MrToll4, and MrToll5. SMART analysis showed that all six Tolls have a transmembrane domain, a TIR domain, and different number of LRR domains. The phylogenetic tree showed that six Tolls were located in six different branches. Among these six Tolls, only MrToll4 contains the QHR motif, which is similar to insect Toll9. MrToll4 belongs to V-type/scc Toll with only one LRRCT domain. MrToll1 and MrToll5 are classical P-type/mcc Toll with two LRRCT domains and an LRRNT. MrTolls were distributed in the hemocytes, heart, hepatopancreas, gills, stomach, and intestine. During the infection of Enterobacter cloacae, the expression level of MrToll and MrToll1-4 was upregulated in the intestine of M. rosenbergii. RNA interference experiments showed that the expression of most antimicrobial peptide (AMP) genes was negatively regulated by MrTolls during E. cloacae infection. On the contrary, crustin (Cru) 3 and Cru4 were inhibited after the knockdown of MrToll, and Cru1 and Cru4 were significantly downregulated with the knockdown of MrToll4 during E. cloacae challenge. These results suggest that MrTolls may be involved in the regulation of AMP expression in the intestine during E. cloacae infection.

A novel C-type lectin (SpccCTL) suppresses MCRV replication by binding viral protein and regulating antiviral peptides in Scylla paramamosain.

Pattern recognition receptors (PRRs) play a crucial role in the recognition and activation of innate immune responses against invading microorganisms. This study characterizes a novel C-type lectin (CTL), SpccCTL. The cDNA sequence of SpccCTL has a full length of 1744 bp encoding a 338-amino acid protein. The predicted protein contains a signal peptide, a coiled-coil (CC) domain, and a CLECT domain. It shares more than 50 % similarity with a few CTLs with a CC domain in crustaceans. SpccCTL is highly expressed in gills and hemocytes and upregulated after MCRV challenge, suggesting that it may be involved in antiviral immunity. Recombinant SpccCTL (rSpccCTL) as well as two capsid proteins of MCRV (VP11 and VP12) were prepared. Pre-incubating MCRV virions with rSpccCTL significantly suppresses the proliferation of MCRV in mud crabs, compared with the control (treatment with GST protein), and the survival rate of mud crabs is also significantly decreased. Knockdown of SpccCTL significantly facilitates the proliferation of MCRV in mud crabs. These results reveal that SpccCTL plays an important role in antiviral immune response. GST pull-down assay result shows that rSpccCTL interacts specifically with VP11, but not to VP12. This result is further confirmed by a Co-IP assay. In addition, we found that silencing SpccCTL significantly inhibits the expression of four antimicrobial peptides (AMPs). Considering that these AMPs are members of anti-lipopolysaccharide factor family with potential antiviral activity, they are likely involved in immune defense against MCRV. Taken together, these findings clearly demonstrate that SpccCTL can recognize MCRV by binding viral capsid protein VP11 and regulate the expression of certain AMPs, suggesting that SpccCTL may function as a potential PRR playing an essential role in anti-MCRV immunity of mud crab. This study provides new insights into the antiviral immunity of crustaceans and the multifunctional characteristics of CTLs.

Duox mediated ROS production inhibited WSSV replication in Eriocheir sinensis under short-term nitrite stress.

Nitrite stress and white spot syndrome virus (WSSV) infection are major problems threatening the sustainable and healthy development of Eriocheir sinensis. Some studies have found that nitrite stress can lead to the production of reactive oxygen species (ROS), whereas synthetic ROS plays a vital role in the signaling pathway. However, whether nitrite stress influences the infection of crabs by WSSV remains unclear. NADPH oxidases, including NOX1-5 and Duox1-2, are important for ROS production. In the present study, a novel Duox gene (designated as EsDuox) was identified from E. sinensis. The studies found that nitrite stress could increase the expression of EsDuox during WSSV infection and decrease the transcription of the WSSV envelope protein VP28. Moreover, nitrite stress could increase the production of ROS, and the synthesis of ROS relied on EsDuox. These results indicated a potential "nitrite stress-Duox activation-ROS production" pathway that plays a negative role in WSSV infection in E. sinensis. Further studies found that nitrite stress and EsDuox could promote the expression of EsDorsal transcriptional factor and antimicrobial peptides (AMPs) during WSSV infection. Moreover, the synthesis of AMPs was positively regulated by EsDorsal in the process of WSSV infection under nitrite stress. Furthermore, EsDorsal played an inhibitory role in the replication of WSSV under nitrite stress. Our study reveals a new pathway for "nitrite stress-Duox activation-ROS production-Dorsal activation-AMP synthesis" that is involved in the defense against WSSV infection in E. sinensis during short-term nitrite stress.

Glutathione S-transferase gene diversity and their regulation by Nrf2 in Chinese mitten crab (Eriocheir sinensis) during nitrite stress.

Eriocheir sinensis is one of the most important economic aquatic products in China. However, nitrite pollution has become a serious threat to the healthy culture of E. sinensis. Glutathione S-transferase (GST) is an important phase II detoxification enzyme, which plays a leading role in the cellular detoxification of exogenous substances. In this study, we obtained 15 GST genes (designated as EsGST1-15) from E. sinensis, and their expression and regulation in E. sinensis under nitrite stress were studied. EsGST1-15 belonged to different GST subclasses. EsGST1, EsGST2, EsGST3, EsGST4, and EsGST5 belonged to Delta-class GSTs; EsGST6 and EsGST7 are Theta-class GSTs; EsGST8 is a mGST-3-class GST; EsGST9 belonged to mGST-1-class GSTs; EsGST10 and EsGST11 belonged to Sigma-class GSTs; EsGST12, EsGST13, and EsGST14 are Mu-class GSTs; EsGST15 is a Kappa-class GST. Tissue distribution experiments showed that EsGSTs were widely distributed in all detected tissues. The expression level of EsGST1-15 was significantly increased in the hepatopancreas under nitrite stress, indicating that EsGSTs were involved in the detoxification of E. sinensis under nitrite stress. Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a transcription factor that can activate the expression of detoxification enzyme. We detected the expression of EsGST1-15 after interfering with EsNrf2 in the hepatopancreas of E. sinensis with or without nitrite stress. Results showed that EsGST1-15 were all regulated by EsNrf2 with or without nitrite stress. Our study provides new information about the diversity, expression, and regulation of GSTs in E. sinensis under nitrite stress.