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Peroxiredoxin (Prx) family members play a critical role in host defense against oxidative stress, and are also involved in immune responses following microbial infection. In the present study, we firstly cloned the cDNA of Peroxiredoxin 5 from Procambarus clarkii (denoted as PcPrx5) and investigated its immune functions towards LPS and Poly I:C exposure. The PcPrx5 cDNA was composed of 564 bp and consisting of 187 amino acid residues which included Prx5-like subfamily domain, AHP1 domain and Redoxin domain. The recombinant protein was expressed in Escherichia coli (Transetta DE3), and anti-Prx5 antibodies were prepared. Tissue specific expression analysis showed that PcPrx5 was ubiquitously expressed in all examined tissues. Further, its mRNA transcript was greatest in hepatopancrease, haemocyte followed by gut and stomach, and was weak in muscle. The LPS and Poly I:C exposure could both significantly up-regulate the transcript level of PcPrx5, however the expression trends were different following LPS and Poly I: C treatments. Further, we investigated the antioxidant role of recombinant PcPrx5 protein in vitro by mixed-function oxidase assay; the results demonstrated a dose-dependent inhibition of DNA damage by PcPrx5. Our results implicate PcPrx5 as an important defense against microbial pathogens and oxidants in P. clarkii., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Cathepsin L is one of the crucial enzyme superfamilies and involved in the immune responses. In the present study, cathepsin L gene from the red crayfish Procambarus clarkii, named PcCTSL, was cloned and characterized. The cDNA fragment of PcCTSL was 1026 bp in length, which encoded a putative protein of 341 amino acid residues with a molecular weight of 37.884 kDa. The theoretical isoelectric point was 5.218. The prepro-cathepsin L was comprised of a typical signal peptide (Met 1 -Ala 18 ), a prodomain proregion peptide (Trp 29 -Phe 89 ) and a mature peptide (Leu 124 -Leu 340 ). Homology analysis indicated that PcCTSL exhibited 53.2%-87.1% identity to other selected species. The recombinant protein of PcCTSL was successfully expressed in Escherichia coli and rabbit anti-PcCTSL polyclonal antibodies were prepared. Real-time quantitative reverse transcription-PCR (qPCR) analysis revealed that the PcCTSL was expressed in all examined tissues, while the greatest mRNA level was observed in hepatopancreas. The expression of PcCTSL mRNA was clearly up regulated in hepatopancreas after challenge by lipopolysaccharide (LPS) and polyriboinosinic polyribocytidylic acid (Poly I:C). RNA interference of PcCTSL affected the gene expression of members of the Toll pathway. Our results suggest that the PcCTSL may play an important role to defend P. clarkii against the pathogens infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The RNA-sequencing followed by de novo assembly generated 61,912 unigene sequences of P. clarkii hepatopancreas. Comparison of gene expression between LPS challenged and PBS control samples revealed 2552 differentially expressed genes (DEGs). Of these sequences, 1162 DEGs were differentially up-regulated and 1360 DEGs differentially down-regulated. The DEGs were then annotated against gene ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Some immune-related pathways such as PPAR signaling pathway, lysosome, Chemical carcinogenesis, Peroxisome were predicted by canonical pathways analysis. The reliability of transcriptome data was validated by quantitative real time polymerase chain reaction (qRT-PCR) for the selected genes. The data presented here shed light into antibacterial immune responses of crayfish. In addition, these results suggest that transcriptomic data provides valuable sequence resource for immune-related gene identification and helps to understand P. clarkii immune functions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Crustaceans express multiple whey acidic protein (WAP) domain containing proteins which are components of host immunity. In the present study, a new double WAP domain containing protein was identified from red swamp crayfish Procambarus clarkii, designated Pc-DWD. The ORF is 387 bp, encoding 128 amino acids consisting of signal peptide of 18 residues, and two tandem WAP domains of 38 and 44 residues. Multiple alignment indicates the presence of conserved motifs in both WAP domains, and phylogenetic analysis shows that Pc-DWD is a new member of the type-IV crustin family. Pc-DWD transcripts were found most abundantly in hemocytes, gills, intestine and heart, and induced by Vibrio anguillarum, Staphylococcus aureus and white spot syndrome virus challenge. RNAi knockdown of Pc-DWD expression led to increased expression of white spot syndrome virus genes and increased crayfish mortality after virus infection. Recombinant Pc-DWD exhibited strong protease inhibitory activity towards commercial subtilicin A and protease K. Pc-DWD inhibited the crude proteases from V. anguillarum and S. aureus cultures and from the crayfish tissue extracts. We infer that Pc-DWD acts in crayfish bacterial and viral immunity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules that play important roles in the innate immunity system of invertebrates, especially in the aspect of antivirus. In the present study, high-throughput small RNA Illumina sequencing systems were used to identify differentially expressed miRNAs (DEMs) from the intestines of Procambarus clarkii that were infected with white spot syndrome virus (WSSV). As a result, 39 known and 12 novel miRNAs were identified in both NG and WG small RNA libraries. Seven DEMs were determined to be involved in the antiviral innate immunity in the intestines of P. clarkii. The results of the target gene predictions of the DEMs showed that the putative target genes of these 7 DEMs are related to tight junctions, vascular smooth muscle contraction regulation of the actin cytoskeleton, focal adhesion, RNA transport, mRNA surveillance, viral carcinogenesis, and Salmonella infection. These results provide theoretical insights for future studies on the antiviral immunity of crustaceans.

Tumor susceptibility gene 101 (TSG101) is a multi-functional gene involved in cell growth and proliferation in vertebrates. However, its role in the innate immune response of crustaceans remains unclear. Here, a TSG101 gene was identified in crayfish Procambarus clarkii with an open reading frame of 1320 bp that encoded a predicted 48.3-kDa protein highly homologous to those in other invertebrates. TSG101 mRNA was highly expressed in stomach and hepatopancreas, and its expression was induced significantly in different tissues (hemocytes, gills and intestine) by lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I: C) with various expression patterns. Recombinant TSG101 protein was expressed in Escherichia coli, and a possible protein-protein interaction between TSG101 and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) was explored by far-western blotting. RNA interference of TSG101 affected the gene expression of members of the Toll pathway. These results suggest that TSG101 is involved in the innate immune responses of P. clarkii., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Antimicrobial peptides (AMPs) are small effectors in host defense by directly targeting microorganisms or by indirectly modulating immune responses. In the present study, two arasin like AMPs, named as Pc-arasin1 and Pc-arasin2, were identified in red swamp crayfish Procambarus clarkii with sequence similarity to the arasins found in Hyas araneus. Both Pc-arasins consisted of signal peptide, N-terminal proline-rich region and C-terminal region containing four conserved cysteine residues. The similarity of two Pc-arasins was 44.44%, and Pc-arasin2 contained several additional residues in the N-terminus. Multiple alignment of arasin family suggested the conservation of the C-terminus and the variation of the N-terminus of Pc-arasins. Both AMPs were found hemocytes-specific, and the expression could be induced the challenge of bacteria, espeacially by the pathogenic bacterium Aeromonas hydrophila. Knockdown of each Pc-arasin expression by double strand RNA would suppress the host immunity against A. hydrophila, and the commercially synthetic Pc-arasins could rescue the knockdown consequence. Both synthetic peptide showed broad antimicrobial activity towards 3 Gram-positive bacterium and 3 Gram-negative bacterium, and the minimal inhibitory concentrations varied from 6.25 μM to 50 μM. These results presented new data about the sequence, expression and function of arasin family, and emphasized the role of this family in host immune response against bacterial pathogens. The characterization of Pc-arasins also provided potential of therapeutic agent development for disease control in aquaculture based on these two newly identified AMPs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNAs) were important post-transcriptional regulators and played vital roles in innate immunity system of invertebrates, especially in the aspect of antivirus. In this study, using high-throughput small RNAs Illumina sequencing system, differentially expressed miRNAs (DEMs) from lymph organs in red swamp crayfish, Procambarus clarkii, infected with white spot syndrome virus, were identified. As a result, 32 known miRNAs and 7 novel miRNAs were identified in crayfish lymph organ small RNAs library of NG and WG. Among them, 7 differentially expressed miRNAs (DEMs) were predicted to be involved in the lymph organ antiviral innate immunity of P. clarkii. Besides, the results showed that putative target genes of these DEMs were related with tight junction, RNA transport, regulation of actin cytoskeleton, focal adhesion, vascular smooth muscle contraction, mRNA surveillance pathway, NOD-like receptor signaling pathway, leukocyte transendothelial migration, and protein processing in endoplasmic reticulum. These results might provide the guiding theoretical foundation for future studies about crustaceans' antiviral innate immunity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017. Published by Elsevier Ltd.)

Serine proteases are involved in many critical physiological processes including virus spread and replication. In the present study, we identified a new clip-domain serine protease (PlcSP) in the crayfish Pacifastacus leniusculus hemocytes, which can interact with the White Spot Syndrome Virus (WSSV) envelope protein VP28. It was characterized by a classic clip domain with six strictly conserved Cys residues, and contained the conserved His-Asp-Ser (H-D-S) motif in the catalytic domain. Furthermore, signal peptide prediction revealed that it has a 16-residue secretion signal peptide. Tissue distribution showed that it was mainly located in P. leniusculus hemocytes, and its expression was increased in hemocytes upon WSSV challenge. In vitro knock down of PlcSP decreased both the expression of VP28 and the WSSV copy number in hematopoietic stem (HPT) cells. Accordingly, these data suggest that the new serine protease may be of importance for WSSV infection into hematopoietic cells., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Calmodulin plays an important role in calcium-dependent signal transduction pathways. In this experiment, a novel calmodulin-like gene (Pc-CaM-L) was identified in the crayfish Procambarus clarkii; it encodes a polypeptide of 145 amino acids. Quantitative real-time PCR analysis revealed that Pc-CaM-L was expressed in all examined tissues, including hepatopancreas, hemocytes, heart, gill, intestine and muscle; the highest Pc-CaM-L expression level was detected in the hepatopancreas. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analysis demonstrated that a recombinant Pc-CaM-L protein was successfully expressed in Escherichia coli. The calcium-binding activity of the purified Pc-CaM-L protein was confirmed by gel mobility shift assay. The expression of Pc-CaM-L was significantly upregulated in gut, gill and hemocytes after lipopolysaccharide or polyinosinic:polycytidylic acid induction. These results suggest that Pc-CaM-L plays a role in the immune response of P. clarkii., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The aim of this study was to evaluate the effects of prebiotics (mannanoligosaccharide and xylooligosaccharide), probiotics (Enterococcus faecalis and Pediococcus acidilactici) and synbiotics for 126 days on the immune responses, hemolymph indices, antioxidant enzymes, and biological responses after a 48-hour Aeromonas hydrophila exposure of sub-adult crayfish (11.45 ± 1.87 g). Most antibacterial activities were observed in the shell mucus of crayfish fed a diet containing xylooligosaccharide + E. faecalis and mannanoligosaccharide + Pediococcus acidilactici against Nocardia brasilience and Vibrio harveyi (p < 0.05). Feeding crayfish a xylooligosaccharide + E. faecalis diet increased protein levels and the activities of alkaline phosphatase and lysozyme in the shell mucus after the feeding trial and 48 h after the A. hydrophila-injection challenge (p < 0.05). The highest ratio of the lactobacillus count to the total viable count was observed in synbiotic diets (p < 0.05). Feeding crayfish a xylooligosaccharide + E. faecalis diet increased the growth rate and the resistance to the A. hydrophila-injection challenge (p < 0.05). These results revealed that feeding crayfish with synbiotic diets was more effective than a single administration with prebiotics and probiotics., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Tolls or Toll-like receptors (TLRs) have an essential role in initiating innate immune responses against pathogens. In this study, a novel Toll gene, PcToll4, was first identified from the intestinal transcriptome of the freshwater crayfish, Procambarus clarkii. The PcToll4 cDNA is 4849bp long with a 3036bp open reading frame that encodes a 1011-amino acid protein. PcToll4 contains a signal peptide, 13 LRR domains, 3 LRR TYP domains, 2 LRR CT domains, an LRR NT domain, a transmembrane region, and a TIR domain. Quantitative RT-PCR analysis revealed that PcToll4 mRNA was detected in all tested tissues, and the expression of PcToll4 in the intestine was significantly upregulated after white spot syndrome virus (WSSV) challenge. Overexpression of PcToll4 in Drosophila Schneider 2 (S2) cells activates the antimicrobial peptides (AMPs) of Drosophila, including metchnikowin, drosomycin, attacin A, and shrimp Penaeidin-4. Results of RNA interference by siRNA also showed that PcToll4 regulates the expressions of 5 anti-lipopolysaccharide factors (ALFs) in the intestine of crayfish. Our findings suggest that PcToll4 is important for the innate immune responses of P. clarkii because this gene regulates the expressions of AMPs against WSSV., (Copyright © 2017. Published by Elsevier B.V.)

Ferritin plays important roles in iron storage, detoxification, and immune response. Here, a ferritin gene (PcFer) was identified in Procambarus clarkii, an economically important freshwater crayfish. Full-length PcFer cDNA was 1022-bp, including a 135-bp 5'-untranslated region (UTR) with a typical iron responsive element, a 374-bp 3'-UTR, and a 513-bp open reading frame encoding a polypeptide of 170 amino acids which contained the Ferritin domain. PcFer has ion binding sites, a ferrihydrite nucleation center, and an iron ion channel. PcFer is phylogenetically closely-related to Pacifastacus leniusculus and Eriocheir sinensis ferritins. Real-time quantitative reverse-transcription PCR analysis showed that PcFer was expressed in all tested P. clarkii tissues, and expressed most in hepatopancreas. After challenge with various heavy metals and lipopolysaccharide, respectively, the hepatopancreatic expression levels of PcFer were markedly upregulated. These results suggest that expression of PcFer might be involved in immune defense and protection of P. clarkii against heavy metal stress., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Triosephosphate isomerase (TIM) is a key enzyme in glycolysis and has been identified as an allergen in saltwater products. In this study, TIM with a molecular mass of 28 kDa was purified from the freshwater crayfish (Procambarus clarkii) muscle. A 90-kDa protein that showed IgG/IgE cross-reactivity with TIM was purified and identified as filamin C (FLN c), which is an actin-binding protein. TIM showed similar thermal and pH stability with better digestion resistance compared with FLN c. The result of the surface plasmon resonance (SPR) experiment demonstrated the infinity of anti-TIM polyclonal antibody (pAb) to both TIM and FLN c. Five linear and 3 conformational epitopes of TIM, as well as 9 linear and 10 conformational epitopes of FLN c, were mapped by phage display. Epitopes of TIM and FLN c demonstrated the sharing of certain residues; the occurrence of common epitopes in the two allergens accounts for their cross-reactivity.

In invertebrates, ficolin-like proteins (FLPs) play important roles in innate immunity against pathogens. Previous studies primarily investigated the functions of FLPs in immune recognition, activation, and regulation. However, limited research has examined the functions of FLPs as immune effectors. In this work, a ficolin-like protein was identified in red swam crayfish (Procambarus clarkii) and designated as PcFLP1. Quantitative RT-PCR and western blot were employed to analyze the distribution and expression profiles of PcFLP1 in the tissues of the crayfish. The results indicated that PcFLP1 was present in all tested tissues, including hemocytes, heart, hepatopancreas, gill, stomach, and mid-intestine. The expression level of PcFLP1 was up-regulated in hemocytes, hepatopancreas and mid-intestines of the crayfish challenged with Vibrio parahaemolyticus. Further study demonstrated that PcFLP1 could protect the hepatopancreatic cells of crayfish from V. parahaemolyticus infection. The recombinant PcFLP1 enhanced bacterial elimination in crayfish, whereas the antibacterial action was inhibited after PcFLP1 was knocked down. Furthermore, PcFLP1 could bound to bacteria and inhibited bacterial replication. These results demonstrated that PcFLP1 plays an important role in the anti-Vibrio immunity of red swamp crayfish., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

White spot syndrome virus (WSSV) is one of the most prevalent and widespread viruses in both shrimp and crayfish aquaculture. MicroRNAs (miRNAs) are crucial post-transcriptional regulators and play critical roles in cell differentiation and proliferation, apoptosis, signal transduction and immunity. In this study, miRNA expression profiles were identified via deep sequencing in red claw crayfish Cherax quadricarinatus haematopoietic tissue (Hpt) cell cultures infected with WSSV at both early (i.e., 1 hpi) and late (i.e., 12 hpi) infection stages. The results showed that 2 known miRNAs, namely, miR-7 and miR-184 play key roles in immunity. Meanwhile, 106 novel miRNA candidates were predicted by software in these combined miRNA transcriptomes. Compared with two control groups, 36 miRNAs showed significantly different expression levels after WSSV challenge. Furthermore, 10 differentially expressed miRNAs in WSSV-exposed Hpt cells were randomly selected for expression analysis by quantitative real-time RT-PCR. Consistent with the expression profiles identified by deep sequencing, RT-PCR showed a significant increase or decrease in miRNA expression in Hpt cells after WSSV infection. Prediction of targets of miRNAs such as miR-7, cqu-miR-52, cqu-miR-126 and cqu-miR-141 revealed that their target genes have diverse biological roles, including not only immunity but also transcriptional regulation, energy metabolism, cell communication, cell differentiation, cell death, autophagy, endocytosis and apoptosis. These results provide insight into the molecular mechanism of WSSV infection and highlight the function of miRNAs in the regulation of the immune response against WSSV infection in crustaceans., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Hemocyte homeostasis-associated-like protein (HHAP) in the freshwater crayfish Pacifastacus leniusculus has a distinct role from that of its homolog PmHHAP in the shrimp Penaeus monodon. Knockdown of PlHHAP in vitro using double-stranded RNA (dsRNA) had no effect on the cell morphology of hematopoietic tissue (HPT) cells. The total hemocyte number and caspase activity were unchanged after PlHHAP knockdown in vivo, in contrast to the results found in shrimp. Moreover, suppression of PlHHAP both in vitro and in vivo did not change the mRNA levels of some genes involved in hematopoiesis and hemocyte homeostasis. Interestingly, bacterial count and scanning electron microscope revealed that depletion of PlHHAP in intestine by RNAi resulted in higher number of bacteria in the crayfish intestine. Together, these results suggest that PlHHAP is not involved in hemocyte homeostasis in the crayfish P. leniusculus but appears to affect the bacterial number in the intestine through an unknown mechanism. Since PlHHAP has different functions from PmHHAP, we therefore named it HHAP-like protein., (Copyright © 2016. Published by Elsevier Ltd.)

Drosophila Toll and mammalian Toll-like receptors (TLRs) are a family of evolutionarily conserved immune receptors that play a crucial role in the first-line defense against intruded pathogens. Activating transcription factor 4 (ATF4), a member of the ATF/CREB transcription factor family, is an important factor that participates in TLR signaling and other physiological processes. However, in crustaceans, whether ATF4 homologs were involved in TLR signaling remains unclear. In the current study, we identified a Toll homolog PcToll2 and a novel ATF4 homolog PcATF4 from Procambarus clarkii, and analyzed the likely regulatory activity of PcATF4 in PcToll2 signaling. The complete cDNA sequence of PcToll2 was 4175 bp long containing an open reading frame of 2820 bp encoding a 939-amino acid protein, and the cDNA sequence of PcATF4 was 2027 bp long with an open reading frame of 1296 bp encoding a 431-amino acid protein. PcToll2 and human TLR4 shared the high identity and they were grouped into a cluster. Furthermore, PcToll2 had a close relationship with other shrimp TLRs that possessed potential antibacterial activity. PcToll2 was highly expressed in the hemocytes, heart and gills, while PcATF4 mainly distributed in gills. Upon challenge with Vibrio parahemolyticus, PcToll2 and PcATF4 together with the antimicrobial peptides of ALF1 and ALF2 were significantly up-regulated in the hemocytes, and the PcATF4 was translocated into the nucleus. After PcToll2 silencing and challenge with Vibrio, the translocation of PcATF4 into the nucleus was inhibited and the expression of ALF1 and ALF2 was reduced, but the expression of PcDorsal and PcSTAT was not affected. Furthermore, after PcATF4 knockdown and challenge with or without Vibrio, the expression of ALF1 and ALF2 was also decreased while the expression of PcToll2 was upregulated. These results suggested that PcToll2 might regulate the expression of ALF1 and ALF2 by promoting the import of PcATF4, instead of the routine transcription factor PcDorsal, into the nucleus participating in the immune defense against Gram-negative bacteria., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Tolls and Toll-like receptors (TLRs) play an important role in host immune defenses by regulating the expression of antimicrobial peptides (AMPs) and cytokines, but the functional differences of crustacean Tolls from Drosophila Tolls or Mammal TLRs are largely unknown. A novel Toll receptor, named PcToll3, was identified from red swamp crayfish, Procambarus clarkii. It was widely expressed in all detected tissues, and its transcript in hemocytes was up-regulated at 12 h after Vibrio parahemolyticus (Vibrio) injection or at 24 h post white spot syndrome virus (WSSV) challenge. After knockdown of PcToll3, the activity of bacterial clearance was inhibited, and the expression levels of AMPs including Crustin1 (Cru1), Anti-lippopolysaccharide factor 1 (ALF1), and Lysozymes1 (Lys1), which could be up-regulated by Vibrio, were all affected. Meanwhile, PcToll3 silencing influenced the expression of myeloid differentiation factor 88 (PcMyd88), tumor necrosis factor-associated factor 6 (PcTRAF6), and PcDorsal, which were the counterparts of Drosophila Toll signaling pathway. Interestingly, PcToll3 silencing inhibited translocation of PcDorsal from cytoplasm to nucleus. Furthermore, the knockdown of PcDorsal also impaired the expression of AMPs after Vibrio challenge. Hence, we concluded that, besides participating in antiviral immunity, PcToll3 might also regulate the expression of Cru1 and Lys1 to participate in anti-Vibrio immune responses by promoting PcDorsal translocation into nucleus., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Fibrinogen-related protein (FREP) family is a large group of proteins containing fibrinogen-like (FBG) domain and plays multiple physiological roles in animals. However, their immune functions in crayfish are not fully explored. In the present study, a novel fibrinogen-like protein (designated as PcFBN1) was identified and characterized from hepatopancreas of red swamp crayfish Procambarus clarkii. The cDNA sequence of PcFBN1 contains an open reading frame (ORF) of 1353 bp encoding a protein of 450 amino acids. Sequence and structural analysis indicated that PcFBN1 contains an FBG domain in C-terminal and a putative signal peptide of 19 amino acids in N-terminal. Semi-quantitative PCR revealed that the main expression of PcFBN1 was observed in hepatopancreas and hemocyte. Temporal expression analysis exhibited that PcFBN1 expression could be significantly induced by heat-killed Aeromonas hydrophila. Tissue distribution and temporal change of PcFBN1 suggested that PcFBN1 may be involved in immune responses of red swamp crayfish. Recombinant PcFBN1 protein binds and agglutinates both gram-negative bacteria Escherichia coli and gram-positive bacteria Micrococcus lysodeikticus. Moreover, binding and agglutination is Ca(2+) dependent. Further analysis indicated that PcFBN1 recognizes some acetyl group-containing substance LPS and PGN. RNAi experiment revealed that PcFBN1 is required for bacterial clearance and survival from A. hydrophila infection. Reduction of PcFBN1 expression significantly decreased the survival and enhanced the number of A. hydrophila in the hemolymph. These results indicated that PcFBN1 plays an important role in the innate immunity of red swamp crayfish as a potential pattern recognition receptor., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cathepsin B is a lysosomal cysteine protease of the papain-like enzyme family with multiple biological functions. In the present study, a cathepsin B gene (named PcCTSB) was cloned and characterized from the red crayfish, Procambarus clarkii. The cDNA fragments of PcCTSB was 990 bp in length. It encoded a putative protein of 329 amino acid residues with predicted molecular weight of 36.4 kDa and isoelectric point of 7.020. Sequence alignment revealed that PcCTSB protein is 53.6%-80.4% identical with those from other 10 species. The predicted tertiary structure of PcCTSB protein was highly similar to that of animals. The results of the phylogenetic analysis indicated that the PcCTSB protein could be clustered with the Eriocheir sinensis cathepsin B protein. The recombinant protein of PcCTSB was expressed successfully in Escherichia coli cells. The mRNA expressions of PcCTSB were detected in all tested tissues, particularly high in the hepatopancreas. After lipopolysaccharide (LPS) challenge, the expression levels of PcCTSB were up-regulated significantly at different time points compared with control. Our results suggested that the PcCTSB might play an important role in defending against the pathogenes infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Apoptosis plays an important role in various biological processes and acts as a host defending mechanism by which infected cells are eliminated to restrict the virus propagation scale. Bax is a crucial pro-apoptotic protein, which mediates the release of cytochrome c from mitochondrion to cytosol in mammalian. However, its role in invertebrate is still obscure. Here, a novel pro-apoptotic protein gene was identified from hemocytes of red swamp crayfish. There was a Bcl-2 domain in the C-terminus of Pc-Bax, which possessed 497 amino acids residues. And an important transmembrane region existed in the C-terminus of Pc-Bax, which implied that Pc-Bax located in mitochondrial membrane. Besides, Pc-Bax was expressed at a relative high level in hemocytes, and a relative low expression levels in hepatopancreas, gills, and intestine. In hemocytes, Pc-Bax transcript was rapidly up-regulated from 12 h to 36 h after WSSV infection. And there was the same trend for Pc-Bax protein expression level in hemocytes after WSSV infection. Results of qRT-PCR testing for VP28 gene showed WSSV replication was obviously enhanced after Pc-Bax knockdown. Meantime, hemocytes apoptosis was suppressed in Pc-Bax knockdown crayfish after WSSV injection, compared with the dsGFP injection group and normal group. Taken together, these results revealed that crayfish hemocytes apoptosis scale was enhanced to suppress WSSV replication by up-regulating Bax protein expression level after WSSV infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

L-type lectins are involved in glycoproteins secretory pathways and are associated with many immune responses. There is growing evidence that L-type lectins are also involved in viral replication. In this study, a novel L-type lectin (named as PcL-lectin) was identified from red swamp crayfish (Procambarus clakii). Gene sequencing and phylogenetic tree analysis results showed that the PcL-lectin was a kind of endoplasmic reticulum Golgi intermediate compartment-53 (ERGIC-53). The expression level of PcL-lectin was significantly down regulated in crayfish after challenged with white spot syndrome virus (WSSV). Recombinant PcL-lectin protein facilitated the replication of WSSV in crayfish. In addition, WSSV replication was decreased when endogenous PcL-lectin was knocked down by RNA interference in crayfish. Furthermore, PcL-lectin may interact with VP24, an envelope protein of WSSV. Our results suggest that PcL-lectin may be required for the multiplication of WSSV, and will pave a new way for the developing of strategies against WSSV infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Crustins are a family of antimicrobial peptides mainly identified in crustaceans and characterized by a whey acidic protein (WAP) domain and an additional glycine-, cysteine-, or proline-rich region. In this study, we identified and characterized PcCru, a new crustin isolated from red swamp crayfish Procambarus clarkii. The open reading frame of PcCru was 333 base pairs long and encoded a 110-residue polypeptide, which contained a signal peptide, a cysteine-rich region, and a WAP domain. The architecture and phylogenetic analysis suggested that PcCru was a new member of the type-I crustin family. PcCru was highly expressed in hemocytes and was significantly induced by viral and bacterial stimulations at both the translational and transcriptional levels. The titer of PcCru in circulating plasma was also increased considerably by bacterial challenge. Recombinant PcCru from both prokaryotic and eukaryotic expression systems were generated, and the proteins exhibited broad-spectrum antimicrobial activity. Furthermore, PcCru protected crayfish from infection by pathogenic bacteria Aeromonas hydrophila in vivo. This study provided new information emphasizing the important role of the crustin family in the crustacean antibacterial immune response., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Chitosan nanoparticles have exhibited potential antibacterial activity or anticancer activity as their unique character. In this study, we investigated the effect of chitosan nanoparticles protect crayfish Procambarus clarkii against WSSV. Chitosan (from crab shell) nanoparticles were prepared by ultrafine milling. The physicochemical properties of the nanoparticles were determined by particle size measure, zeta potential analysis and scanning electron microscope observation. The total hemocyte count (THC), phenoloxidase (PO) and superoxide dismutase (SOD) activity were measured at days 1, 4, 9 and 12, and the survival rate was also recorded after WSSV challenge. The results showed that chitosan nanoparticles could enhance the survival rate of WSSV-challenged crayfish. And crayfish fed diets supplemented with 10 mg/g chitosan nanoparticles (65% mortality) showed a significantly higher survival rate when compared to the control group (100% mortality). The analysis of immunological parameters revealed that 10 mg/g chitosan nanoparticles showed significantly higher level of prophenoloxidase (proPO), superoxide dismutase (SOD) and total hemocyte count (THC) when compared to the control group. It was found that chitosan nanoparticles could inhibit WSSV replication in crayfish. Our results demonstrated that dietary chitosan nanoparticles effectively improve innate immunity and survival of P. clarkii challenged with WSSV., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Crayfish has become one of the most important farmed aquatic species in China due to its excellent disease resistance against bacteria and viruses. However, the antiviral mechanism of crayfish is still not very clear. In the present study, many high-quality sequence reads from crayfish intestine were obtained using Illumina-based transcriptome sequencing. For the normal group (GN), 44,600,142 high-quality clean reads were randomly assembled to produce 125,394 contigs. For the WSSV-challenged group (GW), 47,790,746 high-quality clean reads were randomly assembled to produce 148,983 contigs. After GO annotation, 39,482 unigenes were annotated into three ontologies: biological processes, cellular components, and molecular functions. In addition, 15,959 unigenes were mapped to 25 different COG categories. Moreover, 7,000 DEGs were screened out after a comparative analysis between the GN and GW samples, which were mapped into 250 KEGG pathways. Among these pathways, 36 were obviously changed (P-values < 0.05) and 28 pathways were extremely significantly changed (P-values < 0.01). Finally, five key DEGs involved in the JAK-STAT signaling pathway were chosen for qRT-PCR. The results showed that these five DEGs were obviously up-regulated at 36 h post WSSV infection in crayfish intestine. These results provide new insight into crayfish antiviral immunity mechanisms.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Antimicrobial peptides are important immune effectors involved in mediating innate immune responses against intruding pathogens. Here, we successfully isolated and characterized a novel Type I crustin from the red claw crayfish Cherax quadricarinatus. The full-length cDNA encoded by this gene, designated CqCrs, comprised 608 bp, containing a 5'-untranslated region (UTR) of 55 bp, a 3'-UTR of 229 bp with a poly (A) tail, and an open reading frame (ORF) of 324 bp encoding a polypeptide of 107 amino acids. The deduced amino acid sequence of CqCrs exhibited a configuration typical of other crustacean Type I crustin orthologs, including one signal peptide region at the N-terminus between residues 1 and 16 and a long whey acidic protein (WAP) domain at the C-terminus between residues 60 and 107, along with a WAP-type "four-disulfide core" motif. Phylogenetic analysis showed that CqCrs was clustered first with other crustacean Type I crustins, then with other crustacean Type II crustins, and finally with other crustacean Type III crustins. Transcription of CqCrs was detected in all tissues, especially in immune tissues and was differentially induced in hemocytes post-stimulation with β-1, 3-glucan, lipopolysaccharides (LPS) and peptidoglycans (PG) at selected time-points. To clarify the biological activity of CqCrs, the recombinant CqCrs protein (rCqCrs) was constructed and expressed in Escherichia coli BL21 (DE3). Purified rCqCrs bound to diverse bacteria and inhibited the growth of different microbes to varying degrees. These findings suggest that CqCrs is involved in a specific innate immune recognition and defense mechanisms against bacterial and fungal in C. quadricarinatus., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Down syndrome cell adhesion molecule (Dscam) mediates innate immunity against pathogens in arthropods. Here, a novel Dscam from red claw crayfish Cherax quadricarinatus (CqDscam) was isolated. The CqDscam protein contains one signal peptide, ten immunoglobulin domains, six fibronectin type III domains, one transmembrane domain and cytoplasmic tail. CqDscam phylogenetically clustered with other invertebrate Dscams. Variable regions of CqDscam in N-terminal halves of Ig2 and Ig3 domains, complete Ig7 domain and TM domain can be reshuffled after transcription to produce a deluge of >37,620 potential alternative splice forms. CqDscam was detected in all tissues tested and abundantly expressed in immune system and nerve system. Upon lipopolysaccharides (LPS) and b-1, 3-glucans (Glu) challenged, the expression of CqDscam was up-regulated, while no response in expression occurred after injection with peptidoglycans (PG). Membrane-bound and secreted types of CqDscam were separated on the protein level, and were both extensively induced post LPS challenge. Membrane-bound CqDscam protein was not detected in the serum, but localized to the hemocyte surface by immuno-localization assay. In the antimicrobial assays, the recombinant LPS-induced isoform of CqDscam protein displayed bacterial binding and growth inhibitory activities, especially with Escherichia coli. These results suggested that CqDscam, as one of pattern-recognition receptors (PRRs), involved in innate immune recognition and defense mechanisms in C. quadricarinatus, possibly through alternative splicing., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

European crayfish are sensitive to the crayfish plague pathogen, Aphanomyces astaci, carried by North American crayfish species due to their less effective immune defence mechanisms against this disease. During a controlled infection experiment with a susceptible crayfish species Astacus astacus using three A. astaci strains (representing genotype groups A, B, and E), we investigated variation in their virulence and in crayfish immune defence indicators (haemocyte density, phenoloxidase activity, and production of reactive oxygen species). Experimental crayfish were exposed to two dosages of A. astaci spores (1 and 10 spores mL(-1)). The intensity and timing of the immune response differed between the strains as well as between the spore concentrations. Stronger and faster change in each immune parameter was observed in crayfish infected with two more virulent strains, indicating a relationship between crayfish immune response and A. astaci virulence. Similarly, the immune response was stronger and was observed earlier for the higher spore concentration. For the first time, the virulence of a strain of the genotype group E (isolated from Orconectes limosus) was experimentally tested. Total mortality was reached after 10 days for the two higher spore dosages (10 and 100 spores mL(-1)), and after 16 days for the lowest (1 spore mL(-1)), revealing equally high and rapid mortality as caused by the genotype group B (from Pacifastacus leniusculus). No mortality occurred after infection with genotype group A during 60 days of the experimental trial., (Copyright © 2015 Elsevier Inc. All rights reserved.)

As an ectothermic animal, crayfish immunity and their resistance to pathogen can be significantly affected by environmental factors such as light and temperature. It has been found for a long time that multiple immune parameters of animals and human are circadian-regulated by light-entrained circadian rhythm. Whether temperature also affects the immune rhythm of animals still remains unclear. In the present study, we investigated the effect of temperature cycles on the rhythm of crayfish immunity and their resistance. Survival experiments demonstrated that temperature cycles of 24 °C and 18 °C effectively entrained the circadian rhythm of crayfish resistance to Aeromonas hydrophila in constant dark. After being exposed to temperature cycles, the crayfish injected at different time points exhibited significant difference in resistance to A. hydrophila. Bacterial growth and total hemocyte count (THC) also showed circadian variation in crayfish subjected to temperature cycles, but phenoloxidase (PO) activity didn't show rhythmic change under the same conditions. Quantitative real-time PCR revealed that basal expression of crustin1 and astacidin in crayfish subjected to temperature cycles was circadian-rhythmic, but induced expression by A. hydrophila didn't show the same rhythm. In contrast, crayfish maintained at constant temperature showed completely arrhythmic in bacterial resistance, immune parameters mentioned above and the expression of antimicrobial peptides. The results present here collectively indicated that temperature cycles entrained circadian rhythm of some immune parameters and shaped crayfish resistance to bacteria., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

β-Thymosins participate in numerous biological activities, including cell proliferation and differentiation, wound healing, and anti-inflammatory and antimicrobial activities. Many studies have investigated vertebrate β-thymosins, whereas few reports have focused on invertebrate β-thymosins. In this study, nine isoforms of β-thymosins (PcThy-1 to PcThy-8) were identified from the red swamp crayfish Procambarus clarkii. The isoforms contained different numbers of the thymosin β actin-binding motif. PcThy-1 contained one thymosin β actin-binding motif, whereas PcThy-8 contained eight motifs. Western blot analysis with anti-PcThy-4 antibody showed that three to six isoforms were present in one tissue, and PcThy-4, PcThy-5, PcThy-6, and PcThy-7 were the main isoforms in several tissues. Time course expression analysis of PcThys at the protein level showed that PcThy-4 was upregulated in hemocytes and gills after white spot syndrome virus (WSSV) challenge. PcThy-4, which contained four thymosin β actin-binding motifs, was selected for further research. Tissue distribution analysis by quantitative real-time PCR showed that PcThy-4 was present in tissues of the hemocytes, heart, hepatopancreas, gills, stomach, and intestine at the transcriptional level. Transcriptional expression profiles showed that PcThy-4 was upregulated after WSSV challenge. In vivo RNAi and protein injection assay results showed that PcThy-4 inhibited the replication of WSSV in crayfish and enhanced the survival rate after WSSV infection. Furthermore, PcThy-4 promoted hemocyte phagocytosis of WSSV. Overall, results suggested that PcThys protected crayfish from WSSV infection and played an important role in antiviral immune response., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in shrimp. In this study, MLC with a molecular mass of 18 kDa was purified from crayfish (Procambarus clarkii) muscle. Its physicochemical characterization showed that the purified MLC is a glycoprotein with 4.3% carbohydrate, highly stable to heat, acid-alkali, and digestion, and weakly retains IgE-binding activity when its secondary structure was altered. Serological assays suggested that conformational epitopes predominate over linear epitopes in the purified MLC. Two isoforms of the MLC gene (MLC1 and MLC2) were cloned, and the purified MLC was identified as MLC1. Analysis of the secondary and tertiary structures of the MLCs indicated that MLC1 has four conformational epitopes and three linear epitopes, whereas MLC2 had a major conformational epitope and three linear epitopes. These results are significant for understanding hypersensitization of humans to crayfish.

White spot disease remains a constant threat to aquaculture worldwide. Hyperthermia has been shown to reduce mortality in white spot syndrome virus (WSSV)-infected shrimps, but the mechanism still remains unclear. In this study, we sought to identify host immune factors that contribute to inhibition of WSSV infection during hyperthermia. In WSSV-infected red swamp crayfish Procambarus clarkii (Girard) cultured at 24 ± 1 °C, transcriptional levels of the heat shock protein 70 (Hsp70) gene showed a modest, 2.2-fold increase in haemocytes following 48 h post-infection (hpi). In contrast, in WSSV-infected crayfish cultured at 32 ± 1 °C, Hsp70 gene expression showed a rapid, 19.5-fold induction by 4 hpi. This suggests that Hsp70 plays a positive regulatory role in resistance to WSSV infection during hyperthermia. Furthermore, total haemocyte counts (THC) and phenoloxidase (PO) activity were both significantly increased in WSSV-infected crayfish cultured at 32 ± 1 °C by 48 hpi. Both may be critical for crayfish survival in the late stages of WSSV infection. Collectively, the up-regulation of host protein Hsp70 expression and increase in THC and PO activity suggest that hyperthermia has immunomodulatory effect that enhanced the resistance of P. clarkii to WSSV infection., (© 2014 John Wiley & Sons Ltd.)

Previous studies provide evidences for the possible oxidative damage of toxic environmental pollutants to tissue protein in fish and amphibian, but little information is available about their effects on immunity response in crustacean. In the present study, we evaluated the relationship between oxidative damage and immune response induced by both typical pollutants (viz. copper and beta-cypermethrin), by exposing the freshwater Procambarus clarkii to sub-lethal concentrations (1/40, 1/20, 1/10 and 1/5 of the 96 h LC50) up to 96 h. Five biomarkers of oxidative stress, i.e. reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and protein carbonyl in hepatopancreas, and two immune factors, i.e. phenoloxidase (PO) and hemocyanin in haemolymph were determined. The results indicated that there was a significant increase (P < 0.05) in the contents of ROS, MDA and protein carbonyl accompanied by markedly decreased (P < 0.05) PO and hemocyanin levels in a dose and time dependent manner. The significant and positive correlation (P < 0.01) between protein carbonyls induction and MDA formation was observed in crayfish hepatopancreas at 96 h. The production of these protein carbonyls could significantly depress (P < 0.01) the levels of phenoloxidase and hemocyanin in hemolymph. Higher contents of ROS enhanced the risk of lipid peroxidation, protein carbonylation and immunosuppression of crayfish, and hepatopancreas might play an important role in immune system of crustaceans. Protein oxidation may be one of the main mechanisms for pollution-induced immunotoxicity in P. clarkii., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) play an important role in regulation of anti-microbial peptides (AMPs) expression. A novel vertebrates TLR counterpart named PcToll, was firstly identified from the freshwater crayfish, Procambarus clarkii. Phylogenetic analysis showed that PcToll together with Drosophila melanogaster and Anopheles gambiae Toll9 were clustered with human Tolls. PcToll was mainly expressed in hepatopancreas and gills and it also could be detected in hemocytes, heart, stomach and intestine. PcToll was upregulated in hemocytes and gills post 24 h Vibrio anguillarum challenge. In hepatopancreas and intestine, the highest expression level of PcToll could be observed at 12 h V. anguillarum challenge. In hemocytes, PcToll went up post 24 h Staphylococcus aureus challenge and in gills, the expression level of PcToll showed no obvious change from 2 to 24 h S. aureus challenge. In hepatopancreas post 12 h S. aureus challenge, PcToll was upregulated and it showed obvious upregulation post 12 h S. aureus challenge in intestine. RNAi results showed that PcToll was involved in regulation of crustins (Cru1, Cru2), anti-lipopolysaccharide factor 2 (ALF2) and lysozyme 1 (Lys1) expression. Overexpression of PcToll in Drosophila S2 cells could induce Drosophila Attacin (Atta), Metchnikowin (Mtk), Drosomycin (Drs) and shrimp Penaeidin (PEN4) expression. From the results, it could be speculated that PcToll might play important roles in crayfish innate immune defense., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The hemocytes of the red claw crayfish Cherax quadricarinatus are classified by morphologic observation into the following types: hyalinocytes (H), semi-granulocytes (SG) and granulocytes (G). Density gradient centrifugation with Percoll was developed to separate these three subpopulations of hemocytes. Beads, Escherichia coli, and FITC labeling WSSV were used to investigate the characteristics of granulocytes by using scanning electron microscope, transmission electron microscope, and laser scan confocal microscope. Results showed that granulocytes could phagocytose beads and E. coli by endocytic pathways. WSSV could rely on caveolae-mediated endocytosis to mainly enter into granulocytes. These results could elucidate the mechanism of the innate immunity function of granulocytes, and it also showed the mechanism by which WSSV invaded granulocytes in the red claw crayfish., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

This study aimed to identify differentially expressed genes in Procambarus clarkii crayfish collected from locations of different environmental qualities in the Doñana National Park surrounding areas. The pollution sustained by the crayfish was confirmed by their hepatopancreatic metal concentration. We generated forward and reverse libraries by suppression subtractive hybridization (SSH) to analyze the transcriptional profiles of crayfish from moderately and highly polluted zones in comparison with the control site within the Doñana Biological Reserve. Forty-three differentially expressed genes were detected, and most of them were identified as genes involved in a variety of biological functions, particularly in the innate immune response. To verify the SSH results and assess interindividual variability nine transcripts (ALP, AST, BTF3, CHIT, CTS, ferritin, HC, HC2, and SPINK4) were selected for absolute quantification by real-time qRT-PCR. The qRT-PCR data revealed substantial differences in the absolute amounts of the nine transcripts and confirmed their up- or down-regulation in the polluted sites. Additionally, a positive and significant linear correlation was found between the hepatopancreatic copper concentration and the levels of the transcripts encoding hemocyanins. Finally, the transcriptomic study was complemented with a detailed analysis of SNP profiles of the selected transcripts that revealed point mutations that might underlie adaptive response to environmental stress in P. clarkii. Overall, this work provides novel insights into the molecular pathways that could mediate the response to environmental pollutants in P. clarkii emphasizing the central role of the immune function and thus, should clearly benefit further immunotoxicological research in this organism., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Prebiotics are indigestible fibers that increase beneficial gut commensal bacteria resulting in improvements of the host's health. The beneficial effects of prebiotics are due to the byproducts generated from their fermentation by gut commensal bacteria. In this review, the direct effects of prebiotics on the innate immune system of fish are discussed. Prebiotics, such as fructooligosaccharide, mannanoligosaccharide, inulin, or β-glucan, are called immunosaccharides. They directly enhance innate immune responses including: phagocytic activation, neutrophil activation, activation of the alternative complement system, increased lysozyme activity, and more. Immunosaccharides directly activate the innate immune system by interacting with pattern recognition receptors (PRR) expressed on innate immune cells. They can also associate with microbe associated molecular patterns (MAMPs) to activate innate immune cells. However, the underlying mechanisms involved in innate immune cell activation need to be further explored. Many studies have indicated that immunosaccharides are beneficial to both finfish and shellfish., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Lectins participate in the immune mechanisms of crustaceans. They have been considered as humoral receptors for pathogen-associated molecular patterns; however, some reports suggest that lectins could regulate crustacean cellular functions. In the present study, we purified and characterized a serum lectin (CqL) from the hemolymph of Cherax quadricarinatus by affinity chromatography and determined its participation in the regulation of hemocytes' oxidative burst. CqL is a 290-kDa lectin in native form, constituted by 108, 80, and 29-kDa subunits. It is mainly composed of glycine, alanine, and a minor proportion of methionine and histidine. It showed no carbohydrates in its structure. CqL is composed of several isoforms, as determined by 2D-electrophoresis, and shows no homology with any crustacean protein as determined by Lc/Ms mass spectrometry. CqL agglutinated mainly rat and rabbit erythrocytes and showed a broad specificity for monosaccharides such as galactose, glucose, and sialic acid, as well as for glycoproteins, such as porcine stomach and bovine submaxillary mucin and fetuin. It is a Mn(2+)-dependent lectin. CqL recognized 8% of crayfish granular hemocytes and increased 4.2-fold the production of hemocytes' superoxide anion in vitro assays when compared with non-treated hemocytes. This effect showed the same specificity for carbohydrates as hemagglutination; moreover, superoxide dismutase and diphenyleneiodonium chloride were effective inhibitors of CqL oxidative-activation. The CqL homoreceptor is a 120-kDa glycoprotein identified in the hemocytes lysate. Our results suggest that CqL participates actively in the regulation of the generation of superoxide anions in hemocytes using NADPH-dependent mechanisms., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Using high-throughput Illumina Solexa system, the differential miRNA expressions from hepatopancreas in red swamp crayfish (Procambarus clarkii) fed with diets containing 0 (control) and 75 mg emodin kg(-1) (trial) were identified, respectively. As a result, 13,335,928 raw reads from the control sample and 14,938,951 raw reads from the trial sample were obtained while 13,053,344 (98.77%) and 14,517,522 (98.34%) small RNA were identified, respectively. 106 mature miRNAs (belonging to 68 miRNA gene families) were identified. 35 miRNAs displayed significantly differential expressions between two libraries. Of these, comparing to the control library, 6 miRNAs were significantly up-regulated and 29 miRNAs were significantly down-regulated. Moreover, 5 novel miRNAs (2 from control sample, 3 from trial sample) and target genes were predicted. GO analysis suggested that these miRNAs might be involved in innate immune response, growth, metabolism, cellular process, biological regulation and stimulus response. Our knowledge from this study could contribute to a better understanding of the miRNAs roles in regulating innate immune response and the study of miRNA function in crayfish., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In this study, we identified three white spot syndrome virus (WSSV) strains (WSSV-CN01, WSSV-CN02 and WSSV-CN03) with significant differences in virulence. Among them, WSSV-CN01 caused significant higher and earlier mortality in redclaw crayfish Cherax quadricarinatus, thus was determined as high-virulent, while WSSV-CN02 and WSSV-CN03 were moderate-virulent and low-virulent. By investigating the total number of the circulating haemocytes and the activity of immune relative enzymes, we demonstrated that the different virulent WSSV strains induced distinct immune response in the host. Notably, a dramatic reduction of circulating haemocytes was observed in the crayfish infected with WSSV-CN01 and WSSV-CN02 but not WSSV-CN03. Further analysis revealed that cell death induced by WSSV-CN01 and WSSV-CN02 might be responsible for the decrease of circulating haemocytes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is the activation of prophenoloxidase (proPO) by a proteolytic cascade finalized by the proPO-activating enzyme (ppA), which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly regulated limited proteolysis of downstream processes and are also involved in inflammatory responses to infections for example by activation of interleukin 1ß. Here we show for the first time a link between caspase cleavage of proPO and release of this protein and the biological function of these fragments in response to bacterial infection in crayfish. Different fragments from the cleavage of proPO were studied to determine their roles in bacterial clearance and antimicrobial activity. These fragments include proPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the fragments from the N-terminus after cleavage by caspase-1. The recombinant proteins corresponding to all three of these peptide fragments exhibited bacterial clearance activity in vivo, and proPO-ppA had antimicrobial activity, as evidenced by a drastic decrease in the number of Escherichia coli in vitro. The bacteria incubated with the proPO-ppA fragment were agglutinated and their cell morphology was altered. Our findings show an evolutionary conserved role for caspase cleavage in inflammation, and for the first time show a link between caspase induced inflammation and melanization. Further we give a more detailed understanding of how the proPO system is regulated in time and place and a role for the peptide generated by activation of proPO as well as for the peptides resulting from Caspase 1 proteolysis.

Although a white spot syndrome virus (WSSV) subunit vaccine could significantly enhance the immune response and benefit the shrimp host, its practical application is currently not feasible because of drawbacks in existing expression systems. We generated a transgenic Dunaliella salina (D. salina) strain by introducing the WSSV VP28 gene to produce a novel oral WSSV subunit vaccine. Following transformation of D. salina, VP28 gene expression was assessed by reverse transcription polymerase chain reaction (RT-PCR) assays, enzyme-linked immunosorbent assays (ELISAs), and western blot analysis. The RT-PCR results indicated that the VP28 gene was successfully expressed in D. salina cells. The presence of recombinant VP28 proteins with natural bioactivity was confirmed by western blot analysis and ELISA. Animal vaccination experiments indicated that transgenic D. salina can induce protection against WSSV by oral delivery in crayfish. Our findings indicate that the VP28 gene can be successfully expressed in transgenic D. salina and can be applied as an oral vaccine to protect crayfish against WSSV. We have demonstrated that it is feasible to produce an oral vaccine using D. salina, and thereby provide a new method for controlling other viral diseases in crustaceans.

A new antibacterial peptide called astacidin was characterized from hemocytes of red swamp crayfish Procambarus clarkii, and designated as PcAst. The full-length cDNA of PcAst contained 828 nucleotides with a polyadenylation sequence and a poly-A tail. PcAst encoded a peptide of 43 amino acids, with a signal peptide of 23 amino acids. The mature peptide contained 20 amino acids, among which four were proline/arginine amino acids. Similarity analysis showed that PcAst shared high identity with astacidin 2 from freshwater crayfish Pacifastacus leniusculus. Quantitative real-time PCR analysis showed that PcAst transcripts were mainly distributed in hemocytes and gills. The time-course expression analysis showed that after Vibrio anguillarum and Staphylococcus aureus injection, the transcripts of PcAst were upregulated in the gills. The synthetic small peptide for mature PcAst displayed inhibitory activity against the growth of some Gram-positive and Gram-negative bacteria. This peptide also had a binding ability to bacterial cell wall components, including peptidoglycan, lipopolysaccharide and lipoteichoic acid. PcAst functioned in the bacterial clearance immune reaction after V. anguillarum and S. aureus infection. These results indicate that PcAst has an important function in antibacterial innate immune response in red swamp crayfish P. clarkii., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Arginine kinase (AK) has attracted considerable attention because it has been identified as a shellfish allergen. However, little information is available about AK in crayfish (Procambarus clarkii). In this study, crayfish AK was purified and cloned. Its physicochemical properties, processing stability, and immunological characteristics were analyzed. Crayfish AK was purified by column chromatography, which revealed a single band with molecular mass of 40 kDa; this result was further confirmed by mass spectrometry. The full-length gene sequence of crayfish AK was 1462 bp and encoded a protein of 357 amino acid residues. The results of this study revealed that crayfish AK is a glycoprotein with an isoelectric point of approximately 6.5. Thermal stability assays revealed that crayfish AK easily forms aggregates at temperatures >44°C and was stable at pH 4.0-8.0. SDS-PAGE and dot blotting were used to assess processing stability of purified AK. The results revealed that the IgE-binding activity of crayfish AK is reduced after boiling., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The immune deficiency (IMD) signal pathway mediates immunity against Gram-negative bacteria in Drosophila. Recent studies show that the IMD pathway also involves in antiviral innate immune responses. The functions of the pathway in crustacean immunity are largely unknown. In this paper, two IMDs (FcIMD and PcIMD), one of the key elements of the IMD pathway, were identified from Chinese white shrimp Fenneropenaeus chinensis and red swamp crayfish Procambarus clarkii. Both proteins have a death domain located at the C-terminal. FcIMD was mainly expressed in the gills and stomach and PcIMD was mainly detected in the heart, hepatopancreas, and stomach. FcIMD peaked in hemocytes at 12 h after white spot syndrome virus (WSSV) challenge and it peaked in the gills at 6 h after WSSV challenge, but it was decreased at 2 h and kept the low level to 24 h in hemocytes and no obviously change in gill after Vibrio anguillarum challenge. PcIMD first decreased in hemocytes at 2 h and peaked at 12 h in hemocytes after V. anguillarum challenge. It was also upregulated in gill after bacterial challenge, peaked at 2 h, and decreased at 6 h, and then gradually increased at 12-24 h. PcIMD has no significant change in hemocytes and gill after WSSV challenge. Western blot analysis detected FcIMD protein in all tissues, and immunocytochemical analysis localized FcIMD in the cytoplasm of hemocytes. RNA interference analysis showed that the IMD pathway was involved in regulating the expression of three kinds AMP genes, including crustins, anti-lipopolysaccharide factors and lysozymes, in shrimp and crayfish. They are Cru 1, Cru 2, ALF 1, ALF 2 and Lys 1 in crayfish, and Cru1, Cru 3, ALF 6, ALF 8, and Lys2 in shrimp. These results suggest that although IMD distribution and expression patterns have some differences, the IMD pathway may have conserved function for AMP regulation in shrimp and crayfish immunity against Gram-negative bacteria., (Copyright © 2013 Elsevier Ltd. All rights reserved.)