Peritrophin was first isolated from insect peritrophic membrane (PM) and was thought to protect insects from invasion of microorganisms and to stimulate digestion of food. In this study, a peritrophin-like gene (EsPT) was obtained from Eriocheir sinensis. The full length cDNA of EsPT was 1232 bp, which contained 1005 bp ORF encoding a protein of 334 amino acids, including a 22 amino acid signal peptide, and 3 conserved chitin binding type 2 domains (ChtBD2) characterized by having a 6-cysteine motif. Phylogenetic analysis showed that EsPT was clustered together with 2 insect peritrophin-44-like proteins (MdP44L from Musca domestica and CcP44L from Ceratitis capitata), an insect chitin binding peritrophin-A domain containing protein (CfPT from Coptotermes formosanus) and a crustacean peritrophin (MnPT from Macrobrachium nipponense). Tissue distribution analysis revealed that EsPT was mainly expressed in hepatopancreas, intestine and hemocytes. The expression of EsPT is regulated by lipopolysaccharide, peptidoglycan, Staphylococcus aureus, Vibrio parahaemolyticus and Aeromonas hydrophila challenge. The recombinant EsPT could bind to different microbes, and enhanced the clearance of V. parahaemolyticus in vivo. In crabs, silencing of EsPT by siRNA suppressed the elimination of V. parahaemolyticus and increasing number of bacteria, finally upregulated the expression of anti-lipopolysaccharide factor (ALF) and clip domain serine proteases (cSP). The results might indicate that EsPT was involved in the anti-bacterial innate immunity of crabs.

C-type lectins (CTLs) have crucial functions in recognizing and eliminating pathogens in innate immunity. This study identified a novel low-density lipoprotein receptor class A (LDLa) domain-containing CTL, designated as EsCTLDcp, from the Chinese mitten crab Eriocheir sinensis. The EsCTLDcp cDNA is 1258 bp long, with a 975 bp open reading frame that encodes a 324-amino acid protein. EsCTLDcp contains a signal peptide, an LDLa, and a single C-type lectin-like domain. EsCTLDcp was only expressed in the hepatopancreas of normal crabs, and its expression was regulated following crab challenge with pathogen-associated molecular patterns and with bacteria. The recombinant EsCTLDcp agglutinates Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Vibrio parahaemolyticus and Aeromonas hydrophila) in the presence of calcium. rEsCTLDcp also binds to various bacteria including S. aureus, Bacillus thuringiensis, Bacillus subtilis, Escherichia coli, Vibrio natriegens, V. parahaemolyticus, and A. hydrophila. The rEsCTLDcp protein helped the crabs clear the virulent Gram-negative bacterium V. parahaemolyticus in vivo, as well as interacted with VP24, an envelope protein of white spot syndrome virus (WSSV). These data suggest that EsCTLDcp functions as a pattern-recognition receptor involved in the innate immunity of E. sinensis.

Myeloid differentiation factor 88 (MyD88) is a universal and essential adapter protein that participates in the activation of the Toll-like receptor (TLR)/interleukin-1 receptor-mediated signaling pathway. In this study, two MyD88 genes (HcMyD88-1 and HcMyD88-2) were identified from triangle-shell pearl mussel (Hyriopsis cumingii). Both HcMyD88-1 and HcMyD88-2 proteins were determined to have a death domain at the N-terminal and a TIR domain at the C-terminal. Both HcMyD88-1 and HcMyD88-2 genes were mainly expressed in the hepatopancreas of healthy mussels. HcMyD88-1 and HcMyD88-2 slightly responded to Gram-negative bacterial challenge. Upon bacterial challenge with Gram-positive Staphyloccocus aureus, HcMyD88-1 and HcMyD88-2 transcription levels remarkably increased at 2 and 6h, respectively. Overexpression of HcMyD88-1 and HcMyD88-2 proteins in Drosophila Schneider 2 cells led to the activation of antimicrobial peptide genes. These results indicated that HcMyD88-2 had higher activity than HcMyD88-1 during the activation of attacin A, drosomycin, and metchnikowin genes, suggesting that HcMyD88 genes may play a role in antibacterial innate immune defense.

As pattern recognition receptors (PRRs), C-type lectins (CTLs) play crucial roles in recognizing and eliminating pathogens in innate immunity. In this study, a novel CTL (HcCUB-Lec) was identified from the triangle sail mussel Hyriopsis cumingii. The full-length of HcCUB-Lec cDNA was 1558 bp with an open reading frame of 1281 bp that encodes a putative protein of 426 amino acid residues, including an N-terminal signal peptide, a complement Uegf Bmp1 (CUB) domain, a single carbohydrate recognition domain (CRD), and a transmembrane domain. Quantitative real-time PCR analysis revealed that HcCUB-Lec transcript was distributed in all examined tissues with the highest levels in hepatopancreas and was significantly upregulated in gills and hepatopancreas after immune challenge with Staphyloccocus aureus and Vibrio parahaemolyticus. When HcCUB-Lec was silenced by RNAi, the expression levels of three antimicrobial peptides, including whey acidic protein (HcWAP), defensin (HcDef), and lysozyme (HcLyso), were dramatically decreased in gills. The recombinant HcCUB-Lec and its individual CUB and CRD domains can bind with Gram-positive bacteria (S. aureus and Bacillus subtilis), Gram-negative bacteria (V. parahaemolyticus and Aeromonas hydrophila), and polysaccharides (lipopolysaccharide and peptidoglycan). Moreover, rHcCUB-Lec and its domains could also agglutinate S. aureus and V. parahaemolyticus in the presence of Ca2+ and can clear V. parahaemolyticus in H. cumingii. Results of this study suggest that HcCUB-Lec acts as an antimicrobial PRR that participates in the innate immune responses of H. cumingii.

The Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway is associated with the innate immune system and plays crucial roles in the mediation of immune response to viral infections. In this study, three STAT isoform cDNAs were cloned from the red swamp crayfish Procambarus clarkii, and they were designated as PcSTATa, PcSTATb, and PcSTATc. PcSTATa and PcSTATb were generated through the alternative splicing of the last exon, and PcSTATc was produced by intron retention. PcSTATa, PcSTATb, and PcSTATc contained 2382, 2337, and 2274 bp open reading frames encoding proteins with 793, 778, and 757 amino acid residues, respectively. Domain prediction analysis revealed that three isoforms of PcSTATs contain a STAT interaction domain, a STAT all-alpha domain, a STAT DNA binding domain, and a Src-homology 2 domain. The mRNA transcripts of three PcSTAT isoforms were detected in all examined tissues of male and female crayfish. The expression levels of the three PcSTAT isoforms in the hemocytes, gills, and intestines significantly changed after the white spot syndrome virus (WSSV) challenge. PcSTAT silencing by dsRNA interference could positively regulate the expression levels of three anti-lipopolysaccharide factors (PcALF1, PcALF2, and PcALF6) and two crustins (PcCrus1 and PcCrus2) and negatively regulate the expression levels of three ALFs (PcALF3, PcALF4, and PcALF5) and two crustins (PcCrus3 and PcCrus4). These results suggest that all three PcSTAT isoforms are involved in the host defense against WSSV infection.

Spätzle, an extracellular ligand of the Toll receptor, is involved in the innate immunity of crustaceans. In this study, four Spätzle genes were cloned from Macrobrachium nipponense and designed as MnSpz1, MnSpz2, MnSpz2-isoform, and MnSpz3. The coding region of the four Spätzle genes all contained one intron and two exons, and they were predicted to be produced by gene duplication based on sequence similarities and phylogenetic tree. The predicted MnSpz1, MnSpz2, and MnSpz3 proteins all contained a signal peptide and a Spätzle domain. No signal peptide but a Spätzle domain existed in MnSpz2-isoform because of frameshift mutation caused by 50 bp nucleotide deletion compared with MnSpz2. Quantitative real-time polymerase chain reaction (RT-qPCR) analysis showed that MnSpz1, MnSpz2, and MnSpz3 were expressed in all the detected tissues of M. nipponense, and MnSpz2 was found to be the major isoform in the heart, gills, stomach, and intestine. After stimulation by Vibrio parahaemolyticus, Staphylococcus aureus, or White spot syndrome virus (WSSV), the expression levels of MnSpz1, MnSpz2, and MnSpz3 changed. Given the high similarities among MnSpz1-3, RNA interference (RNAi) using dsRNA of MnSpz1 inhibited the expression of the three Spätzle genes (MnSpz1, MnSpz2 and MnSpz3). Silencing of MnSpz1-3 down-regulated the expression levels of nine antimicrobial peptide (AMP) genes in M. nipponense. After Knockdown of MnSpzs, the number of V. parahaemolyticus, S. aureus and WSSV copies in M. nipponense increased significantly in vivo. Our results suggest that Spätzles are involved in the innate immunity of M. nipponense. The expansion of MnSpz genes through gene duplication is beneficial to enhance the innate immune defense ability of M. nipponense.

Dorsal is a Rel/NF-κB transcription factor, which forms a key part of the Toll pathway. Lysozyme is a ubiquitous enzyme that degrades bacterial cell walls. In this study, a Dorsal homolog was cloned and characterized from triangle sail mussel Hyriopsis cumingii, namely, HcDorsal. Dorsal consisted of 3041 bp, including a 1938 bp open reading frame encoding a 645 amino acid protein. The deduced HcDorsal protein contained a Rel homology domain and an Ig-like, plexin, transcription factor domain. Analysis of expression patterns showed that HcDorsal was highly expressed in the hepatopancreas of H. cumingii. The expression level of HcDorsal continuously increased after Vibrio parahaemolyticus stimulation. When HcDorsal was knocked down by siRNA interference, two phage lysozyme genes (HcLyso1 and HcLyso2) obtained by horizontal gene transfer were significantly downregulated in hemocytes of mussels. Furthermore, knockdown of HcLyso1 and HcLyso2 could weaken V. parahaemolyticus clearance ability. Recombinant HcLyso1 and HcLyso2 proteins accelerated the bacterial clearance in vivo in mussels and evidently inhibited the growth of V. parahaemolyticus. These results suggested that HcDorsal could be activated after V. parahaemolyticus stimulation and then modulate the immune response through the transcriptional regulation of HcLyso1 and HcLyso2, thereby playing a protective role in mussels.

Lysozyme (Lyz) is an alkaline enzyme that hydrolyzes mucopolysaccharides in bacteria and is highly conserved vertebrates and invertebrates. In this study, a c-type lysozyme gene (named ToLyzC) from the obscure puffer Takifugu obscurus was cloned and characterized. The full-length cDNA of ToLyzC was 432 bp, encoding 143 amino acids, with a predicted molecular mass of 16.2 kDa and a theoretical pI of 8.86. The depicted protein sequence contained a LYZ1 domain from 16 to 142 amino acids, seven conserved cysteine residues. Phylogenetic analysis indicated that ToLyzC clustered with Lyzs from other teleost fishes. Quantitative real-time PCR analysis revealed that ToLyzC mRNA was mainly expressed in the liver. The transcript level of ToLyzC gene was significantly upregulated after Staphylococcus aureus and Vibrio harveyi challenge. The optimal pH and temperature of recombinant ToLyzC protein (rToLyzC) lytic activity was detected to be 7.5 and 35 °C, respectively. rToLyzC exhibited significant antibacterial and bacterial binding activities against S. aureus, Aeromonas hydrophila, V. harveyi, and Edwardsiella tarda at different time points. In addition, the morphological changes of V. harveyi cells treated with rToLyzC were observed under scanning electron microscope, which further confirmed the antibacterial and bacteriolytic activity of rToLyzC. Taken together, our current study indicated that ToLyzC is involved in the immune response to bacterial infection in obscure puffers.

Tumor necrosis factor (TNF) is one of the most important cytokines involved in various biological processes in vertebrates and invertebrates. In the present study, a new member of the TNF superfamily (named EsTNFSF) was identified from the Chinese mitten crab (Eriocheir sinensis). The full-length cDNA of EsTNFSF is 2462 bp and encodes a polypeptide with 499 amino acids. The deduced EsTNFSF protein contained a transmembrane region and a conserved extracellular C-terminal TNF domain. Phylogenetic analysis indicated that EsTNFSF was closely related to other TNFSFs from crustaceans. Quantitative real-time PCR analysis showed that EsTNFSF was expressed in all the tissues examined, and the highest expression was found in the hepatopancreas. The mRNA levels of EsTNFSF in hemocytes underwent a time-dependent and variable degree of enhancement after stimulation with lipopolysaccharide, peptidoglycan, Staphylococcus aureus, and Vibrio parahaemolyticus. Functionally, EsTNFSF knockdown by siRNA suppressed the transcriptional levels of c-Jun N-terminal kinase and two antimicrobial peptides, anti-lipopolysaccharide factor and crustin. Furthermore, purified recombinant EsTNFSF protein accelerated the bacterial clearance in vivo and inhibited the growth of V. parahaemolyticus and S. aureus in vitro. The results revealed that EsTNFSF, as an inducible immune response gene, plays a crucial role in the antibacterial immune defense of E. sinensis.

C-type lectins play crucial roles in innate immunity. In the present study, a novel C-type lectin gene, designated as MrCTL, was identified from Macrobrachium rosenbergii. MrCTL contains 2 carbohydrate-recognition domains (CRDs), namely MrCRD1 and MrCRD2. The MrCRD1 contains a QEP motif and MrCRD2 contains a motif of EPD. MrCTL was mainly expressed in the hepatopancreas. The expression level of MrCTL in hepatopancreas was significantly upregulated after a challenge with Vibrio parahaemolyticus or White spot syndrome virus (WSSV). The recombinant MrCTL, MrCRD1 and MrCRD2 have an ability to agglutinate both Gram-negative (V. parahaemolyticus) and Gram-positive bacteria (Staphylococcus aureus) in a calcium dependent manner. The recombinant MrCTL, MrCRD1 and MrCRD2 bind directly to all tested microorganisms. All these results suggested that MrCTL may have important roles in immune defense against invading pathogens in prawns.

Calnexin (Cnx) is a membrane-bound lectin chaperone of the endoplasmic reticulum. In this study, a novel Cnx homologue from the obscure puffer Takifugu obscurus was characterized, tentatively named ToCnx. The cDNA of ToCnx was 1803 bp, and it contained an open reading frame encoding a polypeptide of 600 amino acid residues with a calculated molecular weight of 67.5 kDa. Multiple alignment of the deduced amino acid sequences of ToCnx and other related fish Cnxs revealed that ToCnx had typical characteristics of fish Cnxs. Sequence comparison and phylogenetic tree analysis showed that ToCnx had the closest relationship with Cnxs from Takifugu flavidus and Takifugu rubripes. ToCnx transcripts were detected in all the tissues examined, and they were mainly expressed in the liver, kidney, and intestine. Upon Vibrio harveyi, Edwardsiella tarda, and Aeromonas hydrophila infection, ToCnx transcripts were all significantly upregulated in the kidneys. The recombinant calreticulin domain of ToCnx (rToCnx) was prepared by prokaryotic expression. In the absence of calcium, rToCnx was able to bind three Gram-negative bacteria (V. harveyi, E. tarda, and A. hydrophila) and two bacterial saccharides, such as lipopolysaccharide and peptidoglycan. In the presence of calcium, rToCnx could agglutinate all the detected microorganisms. In addition, rToCnx possessed the effect of inhibiting the growth of three microbe strains. These observations suggested that ToCnx is an important participant in host immune defense against bacteria.

ADP-ribosylation factors (Arfs) are small GTP-binding proteins that have an essential function in intracellular trafficking and organelle structure. To date, little information is available on the Arfs in the economically important giant freshwater prawn Macrobrachium rosenbergii and their relationship to viral infection. Here we identified two Arf genes from M. rosenbergii (MrArf1 and MrArf2) for the first time. Phylogenetic analysis showed that MrArf1, together with MjArf1 from shrimp Marsupenaeus japonicus belonged to Class I Arfs. By contrast, MrArf2 didn't not match any of the Arfs classes of I/II/III, although it could be clustered with an Arf protein from M. japonicas called MjArfn, which may represent an analog of the Arf. MrArf1 was ubiquitously expressed in all the examined tissues, with the highest transcription level in the hepatopancreas, whereas MrArf2 was only highly expressed in the hepatopancreas and exhibited very low levels in the heart, stomach, gills and intestine. The expression level of MrArf1 in the gills was down-regulated post 24 h WSSV challenge, and reached the maximal level at 48 h. MrArf1 in the hepatopancreas went up from 24 to 48 h WSSV challenge. MrArf2 transcript in the gill also went down at 24 h and then was upregulated at 48 h WSSV challenge. MrArf2 increased significantly in the hepatopancreas 24 h after infection and then went down at 48 h WSSV challenge. RNAi results showed that knockdown of MrArf1 or MrArf2 could inhibit the expression of the envelope protein gene vp28 of the WSSV. So, it could be speculated that MrArf1 and MrArf2 might play important roles in the innate immune system against WSSV infection.

L-type lectins contain a leguminous lectin domain and bind to high-mannose type oligosaccharides. In the secretory pathway, L-type lectins play crucial functions in the trafficking, sorting, and targeting of maturing glycoproteins. This study identified two novel L-type lectins, designated as EsERGIC-53 and EsVIP36, from the Chinese mitten crab Eriocheir sinensis. The complete nucleotide sequence of ERGIC-53 cDNA was 1955 bp, containing a 1506 bp open reading frame (ORF) encoding a putative protein of 501 deduced amino acids. The full-length cDNA of VIP36 was 3474 bp with a 984 bp ORF encoding a 327-amino acid peptide. The deduced ERGIC-53 and VIP36 proteins contained a putative signal peptide and an L-type lectin-like domain. Phylogenetic analysis showed that ERGIC-53 and VIP36 belonged to different clades of L-type lectin family. Reverse transcription PCR showed that ERGIC-53 and VIP36 were expressed in all tested tissues. Quantitative real-time RT-PCR analysis revealed that ERGIC-53 and VIP36 transcripts in hepatopancreas were significantly induced at various time points after infection with lipopolysaccharide (LPS), peptidoglycan (PGN), Staphylococcus aureus, Vibrio parahaemolyticus, and Aeromonas hydrophila. A bacterium-binding experiment showed that both ERGIC-53 and VIP36 could bind to different microbes. Sugar binding assay revealed that these lectins could also bind to the glycoconjugates of bacteria surface, such as LPS, PGN, d-Mannose, and N-Acetyl-d-mannosamine. Moreover, these two L-type lectins agglutinated bacteria in a calcium-dependent manner, and both exerted the ability of facilitating the clearance of injected bacteria V. parahaemolyticus in the crab. Our results suggested that ERGIC-53 and VIP36 functioned as pattern recognition receptors in the immune system of E. sinensis.

Integrins belong to a superfamily of conserved α β heterodimeric cell surface receptors that have critical function in cell migration, differentiation, and survival. In this study, an integrin called EsIntegrin was identified from Chinese mitten crab Eriocheir sinensis. EsIntegrin cDNA is 4415 bp long with a 2457 bp open reading frame that encodes an 818 amino acid protein. EsIntegrin contains a signal peptide, an integrin beta subunit (N-terminal portion of extracellular region) INB domain, an epidermal growth factor (hEGF) domain, an integrin B tail domain, a transmembrane region, and an integrin b cyt domain. EsIntegrin was mainly expressed in hemocytes and the heart, with a relatively lower expression level in gills, nerves, intestine, hepatopancreas, muscles, and eyestalk. When healthy crabs were challenged with LPS, PGN, Staphyloccocus aureus, or Vibrio parahaemolyticus, EsIntegrin expression level was upregulated significantly. Recombinant EsIntegrin has agglutination activity to Gram-positive (e.g., S. aureus and Bacillus subtilis) and Gram-negative bacteria (e.g., V. parahaemolyticus and Aeromonas hydrophila) in the presence of calcium. Furthermore, rEsIntegrin could not only bind to various bacteria such as S. aureus, Micrococcus luteus, B. subtilis, Bacillus megaterium, Bacillus thuringiensis, V. parahaemolyticus, Vibrio anguillarum, A. hydrophila, Vibrio natriegens, and Escherichia coli, but this compound also helped crabs in clearing virulent Gram-negative bacterium, V. parahaemolyticus, in vivo. These data suggested that EsIntegrin might function as cellular receptor that is involved in anti-bacterial immunity from E. sinensis.

Myeloid differentiation factor 88 (MyD88) is a universal and essential adapter protein that participates in the activation of the Toll-like receptor/interleukin-1 receptor-mediated signaling pathway. In the present study, a new MyD88 gene (named EsMyD88) was identified in the Chinese mitten crab Eriocheir sinensis. The cDNA of EsMyD88 was 2210 bp long with a 1416 bp open reading frame that encoded a protein with 472 amino acids. Predicted EsMyD88 protein had a death domain at the N-terminal and a TIR domain at the C-terminal. BLASTP and phylogenetic analysis results showed that EsMyD88 was clustered in one group together with other crustaceans MyD88 (SpMyD88, FcMyD88, LvMyD88, and LvMyD88-1). EsMyD88 was detected in all the examined tissues of healthy crabs, and was mainly expressed in the hemocytes and nerves. When normal crabs were challenged with lipopolysaccharide, peptidoglycan, Staphylococcus aureus, Vibrio parahaemolyticus, or Aeromonas hydrophila, the expression levels of EsMyD88 significantly increased either in the hepatopancreas or hemocytes. Results of the pull-down assay showed that EsMyD88 could bind to downstream cytosolic adaptor EsTube. Overexpression of EsMyD88 protein in Drosophila Schneider 2 cells led to the activation of antimicrobial peptide genes. RNA interference assay showed that EsMyD88 is involved in regulating the transcription of ALF1 and ALF2, Cru1 and Cru2, and Lys in crab challenged with V. parahaemolyticus. All the results mentioned earlier indicated that EsMyD88 gene has a key function in antibacterial innate immune defense.