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.

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.

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 Tolls/Toll like receptors (TLRs), a family of pattern recognition receptors, play important roles in immune responses. Previously, we found that a Toll transcript was increased in a transcriptome library of haematopoietic tissue (Hpt) cells from the red claw crayfish Cherax quadricarinatus post white spot syndrome virus infection. In the present study, a full-length cDNA sequence of Toll receptor (named as CqToll) was identified with 3482 bp which contained an open reading frame of 3021 bp encoding 1006 amino acids. The predicted structure of CqToll protein was composed of three domains, including an extracellular domain of 19 leucine-rich repeats residues, a transmembrane domain and an intracellular domain of 138 amino acids. Tissue distribution analysis revealed that CqToll was expressed widely in various tissues determined from red claw crayfish with highest expression in haemocyte but lowest expression in eyestalk. Importantly, significant lower expression of the anti-lipopolysacchride factor (CqALF), an antiviral antimicrobial peptide (AMP) in crustaceans, but not CqCrustin was observed after gene silencing of CqToll in crayfish Hpt cell cultures, indicating that the CqALF was likely to be positively regulated via Toll pathway in red claw crayfish. Furthermore, the transcription of both an immediate early gene and a late envelope protein gene VP28 of WSSV were clearly enhanced in Hpt cells if silenced with CqToll, suggesting that the increase of WSSV replication was likely to be caused by the lower expression of the CqALF resulted from the loss-of-function of CqToll. Taken together, these data implied that CqToll might play a key role in anti-WSSV response via induction of CqALF in a crustacean C. quadricarinatus.

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.

Gradually increasing atmospheric CO2 partial pressure (pCO2) has caused an imbalance in carbonate chemistry and resulted in decreased seawater pH in marine ecosystems, termed seawater acidification. Anthropogenic seawater acidification is postulated to affect the physiology of many marine calcifying organisms. To understand the possible effects of seawater acidification on the proteomic responses of a marine crustacean brine shrimp (Artemia sinica) three groups of cysts were hatched and further raised in seawater at different pH levels (8.2 as control and 7.8 and 7.6 as acidification stress levels according to the predicted levels at the end of this century and next century, respectively) for 1, 7 and 14 days followed by examination of the protein expression changes via two-dimensional gel electrophoresis. Searches of protein databases revealed that 67 differential protein spots were altered due to lower pH level (7.6 and 7.8) stress in comparison to control groups (pH 8.2) by mass spectrometry. Generally, these differentially expressed proteins included the following: 1) metabolic process-related proteins involved in glycolysis and glucogenesis, nucleotide/amino acid/fatty acid metabolism, protein biosynthesis, DNA replication and apoptosis; 2) stress response-related proteins, such as peroxiredoxin, thioredoxin peroxidase, 70-kDa heat shock protein, Na/K ATPase, and ubiquinol-cytochrome c reductase; 3) immune defence-related proteins, such as prophenoloxidase and ferritin; 4) cytoskeletal-related proteins, such as myosin light chain, TCP1 subunit 2, tropomyosin and tubulin alpha chain; and 5) signal transduction-related proteins, such as phospholipase C-like protein, 14-3-3 zeta, translationally controlled tumour protein and RNA binding motif protein. Taken together, these data support the idea that CO2-driven seawater acidification may affect protein expression in the crustacean A. sinica and possibly also in other species that feed on brine shrimp in the ecosystem, particularly marine food webs.

White spot syndrome virus (WSSV) is a lethal pathogen of shrimp and many other crustaceans, including crayfish. However, the molecular mechanism underlying its cellular entry remains elusive due to the lack of shrimp cell lines for viral propagation. Crayfish hematopoietic tissue (Hpt) cell culture was recently established as a good model for WSSV infection study. Here, we showed that multiple endocytic routes, including clathrin-mediated endocytosis (CME), macropinocytosis and caveolae-mediated endocytosis, were indispensably employed for the viral entry into Hpt cell of the crayfish Cherax quadricarinatus. Intriguingly, cellular autophagic activity was positively correlated with efficient viral entry, in which a key autophagy-related protein, γ-aminobutyric acid receptor-associated protein (Cq-GABARAP), that not only localized but also co-localized with WSSV on the Hpt cell membrane, strongly facilitated WSSV entry by binding to the viral envelope VP28 in a CME-dependent manner that was negatively regulated by Cq-Rac1. Furthermore, cytoskeletal components, including Cq-β-tubulin and Cq-β-actin, bound to both recombinant rCq-GABARAP and WSSV envelope proteins, which likely led to viral entry promotion via cooperation with rCq-GABARAP. Even under conditions that promoted viral entry, rCq-GABARAP significantly reduced viral replication at an early stage of infection, which was probably caused by the formation of WSSV aggregates in the cytoplasm.

White spot syndrome virus (WSSV) is one of the most devastating viral pathogens in both shrimp and crayfish farms, which often causes disease outbreak and leads to massive moralities with significant economic losses of aquaculture. However, limited research has been carried out on the intrinsic mechanisms toward WSSV challenge at the metabolic level. To gain comprehensive insight into metabolic responses induced by WSSV, we applied an NMR approach to investigate metabolic changes of crayfish gill and hepatopancreas infected by WSSV for 1, 6 and 12 h. In gill, an enhanced energy metabolism was observed in WSSV-challenged crayfish samples at 1 h, as marked by increased glucose, alanine, methionine, glutamate and uracil. Afterwards, energy metabolism, lipid metabolism as well as osmoregulation were markedly increased at 6 hpi, as shown by elevated glucose, alanine, methionine, fumarate, tyrosine, tryptophan, histidine, phosphorylcholine, betaine and uracil, whereas no obvious metabolites change was detected at 12 hpi. As for hepatopancreas, disturbed lipid metabolism and induced osmotic regulation was found at 6 hpi based on the metabolic biomarkers such as branched chain amino acids, threonine, alanine, methionine, glutamate, glutamine, tyrosine, phenylalanine, lactate and lipid. However, no obvious metabolic change was shown in hepatopancreas at both 1 hpi and 12 hpi. Taken together, our present results provided essential metabolic information about host-pathogen interactions in crayfish, which shed new light on our understanding of WSSV infection at metabolic level.

To elucidate proteomic changes of Hpt cells from red claw crayfish, Cherax quadricarinatus, we have carried out isobaric tags for relative and absolute quantitation (iTRAQ) of cellular proteins at both early (1 hpi) and late stage (12 hpi) post white spot syndrome virus (WSSV) infection. Protein database search revealed 594 protein hits by Mascot, in which 17 and 30 proteins were present as differentially expressed proteins at early and late viral infection, respectively. Generally, these differentially expressed proteins include: 1) the metabolic process related proteins in glycolysis and glucogenesis, DNA replication, nucleotide/amino acid/fatty acid metabolism and protein biosynthesis; 2) the signal transduction related proteins like small GTPases, G-protein-alpha stimulatory subunit, proteins bearing PDZ- or 14-3-3-domains that help holding together and organize signaling complexes, casein kinase I and proteins of the MAP-kinase signal transduction pathway; 3) the immune defense related proteins such as α-2 macroglobulin, transglutaminase and trans-activation response RNA-binding protein 1. Taken together, these protein information shed new light on the host cellular response against WSSV infection in a crustacean cell culture.