Caspase, an aspartate specific proteinase mediating apoptosis, plays a key role in immune response. In our previous study, the expression of a caspase gene was up-regulated in a transcriptome library from the haematopoietic tissue (Hpt) cells of red claw crayfish Cherax quadricarinatus post white spot syndrome virus (WSSV) infection. To further reveal the effect of caspase on WSSV infection, we cloned this caspase gene (denominated as CqCaspase) with an open reading frame of 1062 bp, which encoded 353 amino acids with a caspase domain (CASc) containing a p20 subunit and a p10 subunit. Tissue distribution analysis indicated that the mRNA transcript of CqCaspase was widely expressed in all tested tissues with the highest expression in Hpt, while the lowest expression in muscle. To further explore the effect of CqCaspase on WSSV replication, recombinant protein of CqCaspase (rCqCaspase) was delivered into Hpt cells followed by WSSV infection, which resulted in a significantly decreased expression of both an immediate early gene IE1 and a late envelope protein gene VP28 of WSSV, suggesting that CqCaspase, possibly by the enhanced apoptotic activity, had a strong negative effect on the WSSV replication. These data together indicated that CqCaspase was likely to play a vital role in immune defense against WSSV infection in a crustacean C. quadricarinatus, which shed a new light on the mechanism study of WSSV infection in crustaceans.

Influenza A virus non-structural-1A binding protein (named as Ns1abp) was originally identified as a host protein from human that bound to the viral NS-1 protein. In our previous study, the expression of an Ns1abp-like gene (denoted as CqNs1abp-like gene) was found to be up-regulated in a transcriptome library from the haematopoietic tissue (Hpt) cells of red claw crayfish Cherax quadricarinatus post white spot syndrome virus (WSSV) infection. To elucidate the role of CqNs1abp-like gene involved in WSSV infection, we cloned the CqNs1abp-like gene in which the open reading frame was 2232 bp, encoding 743 amino acids with two typical domains of one BTB (Broad-Complex, Tramtrack and Bric a brac) domain at N-terminal and six Kelch domains at C-terminal. The gene expression profile showed that the mRNA transcript of CqNs1abp-like gene was widely expressed in all the tested tissues with highest expression in nerve, relatively high expression in Hpt and lowest expression in eyestalk. Importantly, both the WSSV entry and the viral replication were significantly reduced in Hpt cells after gene silencing of CqNs1abp-like gene. By using protein pull-down assay, we found that the recombinant BTB domain, six Kelch domains and CqNs1abp-like intact protein were all bound to the WSSV envelope protein VP28, respectively, in which the BTB domain showed slightly less binding affinity than that of the six Kelch domains or the recombinant intact protein. Besides, the WSSV entry into Hpt cells was clearly decreased when the virus was pre-incubated with the recombinant BTB domain, six Kelch domains, or the recombinant CqNs1abp-like intact protein, respectively, suggesting that the CqNs1abp-like gene was likely to function as a putative recognition molecular towards WSSV infection in a crustacean C. quadricarinatus. Taken together, these data shed new light on the mechanism of WSSV infection and a putatively novel target on anti-WSSV infection in crustacean farming.

It is well known that iron is an essential element for all living organism. The intracellular iron availability is also important for the host's innate immune response to various pathogens, in which the iron homeostasis can be regulated by ferritin due to its iron storage property. In this study, a full-length cDNA sequence of ferritin (named as CqFerritin) was identified with 1410 bp from red claw crayfish Cherax quadricarinatus, which contained an open reading frame of 513 bp, encoding 170 amino acids with a conserved ferritin domain. Tissue distribution analysis demonstrated that CqFerritin was widely expressed in various tissues with high presence in haemocyte, haematopoietic tissue (Hpt) and heart, while lowest expression in hepatopancreas. In addition, loss-of-function of CqFerritin by gene silencing resulted in significantly higher expression of an envelope protein VP28 of white spot syndrome virus (WSSV) in red claw crayfish Hpt cell cultures, indicating the potential antiviral response of CqFerritin. To further explore the effect on WSSV replication by CqFerritin, recombinant CqFerritin protein (rCqFerritin) was transfected into Hpt cells followed by WSSV infection. Importantly, the replication of WSSV was obviously decreased in Hpt cells if transfected with rCqFerritin protein, suggesting that CqFerritin had clearly negative effect on WSSV infection. Furthermore, intracellular accumulation of iron ions was found to promote the WSSV replication in a dose-dependent manner, illustrating that the iron level regulated by CqFerritin was likely to be vital for WSSV infection in red claw crayfish. Taken together, these data suggest that CqFerritin plays an important role in immune defense against WSSV infection in a crustacean C. quadricarinatus.

The gradual increase of CO2 concentration in the atmosphere, absorbed by the ocean surface water through air to sea equilibration termed ocean acidification (OA), leads to the decline of pH in seawater. It is not clear so far how the composition of fatty acids, particular the immune-related, in marine crustacean and the subsequent energy supply in marine ecosystem are affected by OA. The brine shrimp Artemia sinica is an open and common feed that provide essential fatty acids for mariculture. In this study, the fatty acids profiles of brine shrimp cultured under different lower pH levels of CO2 driven seawater were investigated. The results showed a significant reduction of the proportion of total saturated fatty acids under the pH7.6 within one week. Meanwhile, the percentage of total monounsaturated fatty acids was significantly decreased at day 14 under pH7.8, and this percentage gave a significant increase of proportion within one week under pH7.6. Furthermore, the relative content of total polyunsaturated fatty acids (PUFAs) was found to be clearly increased with exposure to different seawater acidification at day 1, suggesting that the brine shrimp immune response was likely to be affected by acidified seawater as the PUFAs have been well known to be involved in immunomodulatory effects through alterations on cell membrane fluidity/lipid mediators and gene expression of cell signaling pathways. Notably, eicosapentaenoic acid and docosahexaenoic acid, which have essential effect on various physiological processes such as inflammatory cytokines production and cell structural stability, were strongly increased under two lower pH treatments within one week and with the significant increase at day 1 under pH7.6. These data clearly supported the hypothesis that OA might affect fatty acids composition, likely also the innate immunity, in crustacean and the subsequent energy transfer by food-chain system in the marine ecosystem.

White spot syndrome virus (WSSV) is a lethal pathogen of shrimp and many other crustaceans, which has been causing huge economic losses in global aquaculture. Laminin receptor (LR) is a cell surface receptor which participates in the interactions between cells as well as cells and extracellular matrix. Previously, we found that a CqLR-like gene was responsive to WSSV infection in the hematopoietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus. To further reveal the role of CqLR-like gene involved in WSSV infection, the full-length cDNA of CqLR-like gene was cloned with 1000 bp, and the open reading frame encoded 308 amino acids with a conserved laminin-binding domain. Importantly, both the WSSV entry and viral replication were strongly reduced in Hpt cells after loss-of-function of CqLR-like gene by gene silencing. Protein interaction assay demonstrated that the recombinant CqLR-like protein could bind to WSSV virion in vitro by enzyme-linked immunosorbent assay and the binding affinity was in a dose-dependent manner. Furthermore, recombinant CqLR-like protein was found to bind to WSSV envelop protein VP28, but not other envelop proteins tested including VP19, VP24, and VP26, by pull down assay in HEK293T cells. In regarding to that LR is mainly localized on many types of cells’ membrane, these data together suggested that CqLR-like protein was likely to function as a putative recognition molecule towards WSSV and act in the viral entry into a crustacean host cell, which may benefit the elucidation of the WSSV pathogenesis and further the pharmaceutical target for the possibly effective control of WSSV disease.