Characterization and mechanism of simultaneous degradation of aflatoxin B1 and zearalenone by an edible fungus of Agrocybe cylindracea GC-Ac2.

Contamination with multiple mycotoxins is a major issue for global food safety and trade. This study focused on the degradation of aflatoxin B1 (AFB1) and zearalenone (ZEN) by 8 types of edible fungi belonging to 6 species, inclulding Agaricus bisporus, Agrocybe cylindracea, Cyclocybe cylindracea, Cyclocybe aegerita, Hypsizygus marmoreus and Lentinula edodes. Among these fungi, Agrocybe cylindracea strain GC-Ac2 was shown to be the most efficient in the degradation of AFB1 and ZEN. Under optimal degradation conditions (pH 6.0 and 37.4°C for 37.9 h), the degradation rate of both AFB1 and ZEN reached over 96%. Through the analysis of functional detoxification components, it was found that the removal of AFB1 and ZEN was primarily degraded by the culture supernatant of the fungus. The culture supernatant exhibited a maximum manganese peroxidase (MnP) activity of 2.37 U/mL. Interestingly, Agrocybe cylindracea strain GC-Ac2 also showed the capability to degrade other mycotoxins in laboratory-scale mushroom substrates, including 15A-deoxynivalenol, fumonisin B1, B2, B3, T-2 toxin, ochratoxin A, and sterigmatocystin. The mechanism of degradation of these mycotoxins was speculated to be catalyzed by a complex enzyme system, which include MnP and other ligninolytic enzymes. It is worth noting that Agrocybe cylindracea can degrade multiple mycotoxins and produce MnP, which is a novel and significant discovery. These results suggest that this candidate strain and its enzyme system are expected to become valuable biomaterials for the simultaneous degradation of multiple mycotoxins.

Identification of two galectin-4 proteins (PcGal4-L and PcGal4-L-CRD) and their function in AMP expression in Procambarus clarkii.

Galectins, a family of lectins that bind to ß-galactoside, possess conserved carbohydrate recognition domains (CRDs) and play a crucial role in recognizing and eliminating pathogens in invertebrates. Two galectin-4 genes (PcGal4) isoforms, named PcGal4-L and PcGal4-L-CRD, were cloned from the cDNA library of Procambarus clarkia in our study. PcGal4-L contains an open reading frame (ORF, 1089 bp), which encodes a protein consisting of 362 amino acids including a single CRD and six low complexity regions. The full-length cDNA of PcGal4-L-CRD contains a 483 bp ORF that encodes a protein of 160 amino acids, with a single CRD and a low-complexity region. The difference between the two PcGal4 isoforms is that PcGal4-L has 202 additional amino acids after the CRD compared to the PcGal4-L-CRD. These two isoforms are grouped together with other galectins from crustaceans through phylogenetic analysis. Further study revealed that total PcGal4 (including PcGal4-L and PcGal4-L-CRD) was primarily expressed in the muscle, gills and intestine. The mRNA levels of total PcGal4 in gills and hemocytes were significantly induced after challenge with Aeromonas hydrophila. Both recombinant PcGal4-L and its spliced isoform, PcGal4-L-CRD, could directly bind to lipopolysaccharides, peptidoglycan and five tested microorganisms, inducing a wide spectrum of microbial agglutination. The spliced isoform PcGal4-L-CRD showed a stronger binding ability than PcGal4-L. In addition, when the PcGal4 was knockdown, transcriptions of seven antimicrobial peptides (AMPs) genes (ALF5, ALF6, ALF8, CRU1, CRU2, CRU3 and CRU4) in gills and seven AMPs genes (ALF5, ALF6, ALF8, ALF9, CRU1, CRU3 and CRU4) in hemocytes were significantly decreased. Meanwhile, the survival rate of P. clarkii decreased in the PcGal4-dsRNA group. In summary, these results indicate that PcGal4 can mediate the innate immunity in P. clarkii by bacterial recognition and agglutination, as well as regulating AMP expression, thus recognition and understanding of the functions of galectin in crustaceans in immune resistance.

Functional Analysis of a CTL-X-Type Lectin CTL16 in Development and Innate Immunity of Tribolium castaneum.

C-type lectins (CTLs) are a class of proteins containing carbohydrate recognition domains (CRDs), which are characteristic modules that recognize various glycoconjugates and function primarily in immunity. CTLs have been reported to affect growth and development and positively regulate innate immunity in Tribolium castaneum. However, the regulatory mechanisms of TcCTL16 proteins are still unclear. Here, spatiotemporal analyses displayed that TcCTL16 was highly expressed in late pupae and early adults. TcCTL16 RNA interference in early larvae shortened their body length and narrowed their body width, leading to the death of 98% of the larvae in the pupal stage. Further analysis found that the expression level of muscle-regulation-related genes, including cut, vestigial, erect wing, apterous, and spalt major, and muscle-composition-related genes, including Myosin heavy chain and Myosin light chain, were obviously down-regulated after TcCTL16 silencing in T. castaneum. In addition, the transcription of TcCTL16 was mainly distributed in the hemolymph. TcCTL16 was significantly upregulated after challenges with lipopolysaccharides, peptidoglycans, Escherichia coli, and Staphylococcus aureus. Recombinant CRDs of TcCTL16 bind directly to the tested bacteria (except Bacillus subtilis); they also induce extensive bacterial agglutination in the presence of Ca2+. On the contrary, after TcCTL16 silencing in the late larval stage, T. castaneum were able to develop normally. Moreover, the transcript levels of seven antimicrobial peptide genes (attacin2, defensins1, defensins2, coleoptericin1, coleoptericin2, cecropins2, and cecropins3) and one transcription factor gene (relish) were significantly increased under E. coli challenge and led to an increased survival rate of T. castaneum when infected with S. aureus or E. coli, suggesting that TcCTL16 deficiency could be compensated for by increasing AMP expression via the IMD pathways in T. castaneum. In conclusion, this study found that TcCTL16 could be involved in developmental regulation in early larvae and compensate for the loss of CTL function by regulating the expression of AMPs in late larvae, thus laying a solid foundation for further studies on T. castaneum CTLs.

Full-length transcriptome sequencing and comparative transcriptome analysis of Eriocheir sinensis in response to infection by the microsporidian Hepatospora eriocheir.

As a new generation of high-throughput sequencing technology, PacBio Iso-Seq technology (Iso-Seq) provides a better alternative sequencing method for the acquisition of full-length unigenes. In this study, a total of 22.27 gigabyte (Gb) subread bases and 128,614 non-redundant unigenes (mean length: 2,324 bp) were obtained from six main tissues of Eriocheir sinensis including the heart, nerve, intestine, muscle, gills and hepatopancreas. In addition, 74,732 unigenes were mapped to at least one of the following databases: Non-Redundant Protein Sequence Database (NR), Gene Ontology (GO), Kyoto Encyclopaedia of Genes and Genomes (KEGG), KEGG Orthology (KO) and Protein family (Pfam). In addition, 6696 transcription factors (TFs), 28,458 long non-coding RNAs (lncRNAs) and 94,230 mRNA-miRNA pairs were identified. Hepatospora eriocheir is the primary pathogen of E. sinensis and can cause hepatopancreatic necrosis disease (HPND); the intestine is the main target tissue. Here, we attempted to identify the key genes related to H. eriocheir infection in the intestines of E. sinensis. By combining Iso-Seq and Illumina RNA-seq analysis, we identified a total of 12,708 differentially expressed unigenes (DEUs; 6,696 upregulated and 6,012 downregulated) in the crab intestine following infection with H. eriocheir. Based on the biological analysis of these DEUs, several key processes were identified, including energy metabolism-related pathways, cell apoptosis and innate immune-related pathways. Twelve selected genes from these DEUs were subsequently verified by quantitative real-time PCR (qRT-PCR) analysis. Our findings enhance our understanding of the E. sinensis transcriptome and the specific association between E. sinensis and H. eriocheir infection.

Eriocheir sinensis vesicle-associated membrane protein can enhance host cell phagocytosis to resist Spiroplasma eriocheiris infection.

Vesicle-associated membrane protein (VAMP) belongs to the receptor protein on the membrane of the secretory transport vesicle and involves in host immune function. The intracellular pathogen Spiroplasma eriocheiris could cause Eriocheir sinensis tremor disease. In a previous study, it was found E. sinensis VAMP (EsVAMP) was differently expressed in S. eriocheiris infection by proteomics analysis. This study mainly aims at the function of EsVAMP in the process of the S. eriocheiris infection. The length of EsVAMP gene was 1681 bp, which contained a 395 bp open reading frame, 90 bp 5'-non-coding region (UTR) and 1277 bp 3'-UTR. The results of qPCR showed that EsVAMP was expressed highly in hemocytes and nerves, followed by gills, intestines and hepatopancreas, and lowly expressed in heart and muscles. EsVAMP in hemocytes was up-regulated after S. eriocheiris infection. After EsVAMP over-expression and S. eriocheiris infection, the RAW264.7 cell morphology and cell viability of the experiment group were significantly better than the control group. Meanwhile, the copy number of S. eriocheiris in the experiment group was significantly lower than that in the control group. After EsVAMP and pCMV-Cre-mCherry were ligated and transfected into RAW264.7 cells, it was found that EsVAMP and lysosome co-localized. Meanwhile, the phagocytosed inactivated S. eriocheiris number and phagocytosed efficiency in RAW264.7 cells were increased significantly. The interference experiment was carried out by synthesizing EsVAMP dsRNA to verify that the EsVAMP transcriptions were successfully suppressed. The S. eriocheiris copy number and the mortality of crab increased significantly after EsVAMP RNAi and S. eriocheiris infection. Meanwhile, the phagocytosed inactivated S. eriocheiris number and phagocytosed efficiency in hemocytes decreased significantly after EsVAMP RNAi and S. eriocheiris infection. These results showed that VAMP was involved in the cell phagocytosis to resist pathogen infection.

Toxicity of avermectin to Eriocheir sinensis and the isolation of a avermectin-degrading bacterium, Ochrobactrum sp. AVM-2.

Avermectin is widely used in the prevention and treatment of parasites diseases in aquaculture. However, the residual avermectin has a serious impact on the growth and quality of aquatic animals including Eriocheir sinensis. This study shows that the LC50 of avermectin to E. sinensis for 24, 48, 72 and 96 h was 21.88, 13.40, 9.11 and 7.10 mg/L, respectively. After avermectin stress, the activities of superoxide dismutase (SOD), catalase (CAT) and phenol oxidase (PO) in the hepatopancreas of E. sinensis increased and reached the peak on the 6th day. The content of malondialdehyde (MDA) accumulated with the increase of exposure time and concentration of avermectin. After 15 days of avermectin exposure, hepatopancreas was damaged seriously. These results indicated that avermectin had toxicity to E. sinensis. In order to solve the pollution problem caused by residual avermectin, a degrading bacterium AVM-2 was separated from the sediment of E. sinensis breeding pond. The strain was confirmed to be Ochrobactrum sp by morphology observation, physiological and biochemical identification and 16 S rDNA sequences analysis. When the pH value was 7, the temperature was 30 â„ƒ, the concentration of substrate was low, the quantity of inoculation was high, Ochrobactrum sp. AVM-2 had better degradation effect on avermectin. When the addition of Ochrobactrum sp. AVM-2 was 2.34 × 108 CFU/L, the residual avermectin in muscle and hepatopancreatine significantly decreased, and the degradation rate was about 66%. In summary, Ochrobactrum sp. AVM-2 could be used to solve the residual problem of avermectin and ensure the food safety of E. sinensis.

Risk assessment of multiple-mycotoxin exposure for consumers of chestnuts in Shandong Province markets in China.

In this study, 321 chestnut samples from Shandong Province in China were analysed for the presence of mycotoxins. We screened for 14 mycotoxins including aflatoxins (AFs: AFB1, AFB2, AFG1, AFG2), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), T-2 toxin (T-2), zearalenone (ZEA), ochratoxin A (OTA), fumonisins (FB1, FB2, FB3), and penicillic acid (PeA). Mycotoxins were detected in 56.4% of the samples, and 11 of these mycotoxins were found. Thirty samples from the Shandong Province markets were deemed positive for AFs (9.3%) and had an AFB1 level of >2 µg/kg or a sum of AFB1, AFB2, AFG1, and AFG2 that was >4 µg/kg, which exceed the maximum tolerable level of the European regulations standards (EC/188/2006). The contamination level for total mycotoxins found in chestnuts was in the range of 0.6-2,791.0 µg/kg. The estimated daily intake (EDI) values for each individual mycotoxin and for all of the mycotoxins collectively were calculated by both a deterministic approach and a probabilistic approach. For risk characterisation, dietary exposure to DON, ZEA, FBs, and OTA through consumption of chestnuts, analysed according to both approaches, showed no health risk to Chinese adolescents and adults from exposure to either individual mycotoxins or in combination, but more concern should be paid to the AFs for adolescents and adults at a high consumption level. This is believed to be the first work performing risk assessment of multiple mycotoxins specifically for adolescents, including the recently isolated FBs and PeA, which have recently emerged as mycotoxins of concern, in chestnuts of Shandong Province in China.

A C-type lectin with dual-CRD from Tribolium castaneum is induced in response to bacterial challenge.

BACKGROUND: C-type lectins (CTLs), a group of pattern recognition receptors, are involved in regulating the immune response of insects and could be used as potential targets for pest control. However, information about roles of CTLs in the innate immunity of Tribolium castaneum, a serious, worldwide pest that damages stored grain products, is relatively scarce. RESULTS: Here, a CTL with dual carbohydrate recognition domains (CRDs) containing a highly conserved WHD (Trp53 -His54 -Asp55 ) motif was identified in T. castaneum and named as TcCTL3. Spatiotemporal analysis showed that TcCTL3 was highly expressed in all developmental stages except early eggs, and mainly distributed in central nervous system and hemolymph. The transcript levels of TcCTL3 were significantly increased after lipopolysaccharide (LPS) and peptidoglycan (PGN) stimulation. Recombinant TcCTL3 was able to bind directly to LPS, PGN and all tested bacteria and induce a broad spectrum of microbial agglutination in the presence of Ca2+ . The binding was shown mainly through CRD1 domain of TcCTL3. When TcCTL3 was knocked down by RNA interference, expression of nine antimicrobial peptides (AMPs) (attacin1, attacin2, attacin3, defensins1, defensins2, coleoptericin1, coleoptericin2, cecropins2 and cecropins3) and four transcription factors (TFs) (dif1, dif2, relish and jnk) were significantly decreased under LPS and PGN stimulation, leading to increased mortality of T. castaneum when infected with Gram-positive Staphylococcus aureus or Gram-negative Escherichia coli infection. CONCLUSION: TcCTL3 could mediate the immune response in T. castaneum via the pattern recognition, agglutination and AMP expression. These findings indicate a potential mechanism of TcCTL3 in resisting bacteria and provide an alternative molecular target for pest control. © 2020 Society of Chemical Industry.

Toxicity of deltamethrin to Eriocheir sinensis and the isolation of a deltamethrin-degrading bacterium, Paracoccus sp. P-2.

Deltamethrin is used widely in Eriocheir sinensis aquaculture to remove wild fish and parasites. The residual deltamethrin greatly affects the growth and quality of E. sinensis. In this study, the LC50 of deltamethrin against E. sinensis at 24, 48 and 96 h was determined to be 6.5, 5.0 and 2.8 µg/L, respectively. The enzyme activity and gene transcription of SOD, CAT, and PO in the hepatopancreas of E. sinensis after deltamethrin stimulation showed an increasing tendency, and these enzymes reached their maximum activities at 6-10 d. The MDA content accumulated with increased time of deltamethrin stress. After 15 d of deltamethrin stress, the hepatopancreas of E. sinensis was found to be damaged based on HE staining. These results showed that deltamethrin is highly toxic to E. sinensis. But the half-life of deltamethrin is long and mainly relies on biodegradation. To resolve the pollution of residual deltamethrin, a strain of deltamethrin-degrading bacteria, P-2, was isolated from the sediment of an E. sinensis culture pond. Through morphological observation, physiological and biochemical identification and 16S rDNA sequence analysis, we found that this strain belonged to Paracoccus sp. When the pH was 7, the substrate concentration was low, the inoculation amount was high, and the deltamethrin degradation effect of Paracoccus sp. P-2 was good. The deltamethrin residue in the hepatopancreas and muscle of E. sinensis decreased significantly when Paracoccus sp. P-2 was added at 6.0 × 108 CFU/L. The degradation efficiency of Paracoccus sp. P-2 in the hepatopancreas and muscle was more than 70%. These results showed that Paracoccus sp. P-2, the first deltamethrin-degrading bacterium in aquaculture, could be used to remove residual deltamethrin and improve the food safety of E. sinensis.

A typical C-type lectin, perlucin-like protein, is involved in the innate immune defense of whiteleg shrimp Litopenaeus vannamei.

C-type lectins are a large group of the pattern-recognition proteins, and have been reported to be involved in invertebrate innate immunity, such as cell adhesion, bacterial clearance, phagocytosis, prophenoloxidase activation and encapsulation. Here, a perlucin-like protein (PLP), a typical C-type lectin, was identified from the cDNA library of the shrimp, Litopenaeus vannamei. LvPLP contains a 540 bp open reading frame, encoding a protein of 179 amino acids that includes a single carbohydrate-recognition domain. Phylogenetic analysis showed that LvPLP was clustered into a single group together with other perlucins from molluscs. Quantitative real-time PCR revealed that LvPLP was expressed mainly in the hemocytes, hemolymph, heart and gills. The transcription of LvPLP was significantly induced at 9 h by both Gram- bacteria Vibrio parahaemolyticus and Vibrio anguillarum. Meanwhile, recombinant LvPLP (rLvPLP) bound directly to lipopolysaccharide and peptidoglycan with different affinity. rLvPLP showed a strong ability to bind to Gram+ (Staphylococcus aureus and Bacillus subtilis) and Gram- bacteria (V. parahaemolyticus and V. anguillarum), and could induce agglutination of V. parahaemolyticus and V. anguillarum, but not S. aureus and B. subtilis in the presence Ca2+. Further study showed that when LvPLP was knocked down by RNAi, three phagocytosis-related genes (peroxinectin, mas-like protein and dynamin) and four antimicrobial peptide (AMP) genes (crustin, ALF1, ALF2 and ALF3) were significantly decreased. Altogether, these results demonstrated that LvPLP played a vital role in L. vannamei immune response towards bacterial challenge by binding and agglutinating bacteria and influencing phagocytosis and AMP expression.

Spiroplasma eriocheiris Invasion Into Macrobrachium rosenbergii Hemocytes Is Mediated by Pathogen Enolase and Host Lipopolysaccharide and ß-1, 3-Glucan Binding Protein.

Spiroplasma eriocheiris is a crustacean pathogen, without a cell wall, that causes enormous economic loss. Macrobrachium rosenbergii hemocytes are the major targets during S. eriocheiris infection. As wall-less bacteria, S. eriocheiris, its membrane protein should interact with host membrane protein directly and firstly when invaded in host cell. In this investigation, six potential hemocyte receptor proteins were identified firstly that mediate interaction between S. eriocheiris and M. rosenbergii. Among these proteins, lipopolysaccharide and ß-1, 3-glucan binding protein (MrLGBP) demonstrated to bind to S. eriocheiris using bacterial binding assays and confocal microscopy. Four spiroplasma ligand proteins for MrLGBP were isolated and identified. But, competitive assessment demonstrated that only enolase of S. eriocheiris (SeEnolase) could be a candidate ligand for MrLGBP. Subsequently, the interaction between MrLGBP and SeEnolase was confirmed by co-immunoprecipitation and co-localization in vitro. After the interaction between MrLGBP and SeEnolase was inhibited by antibody neutralization test, the virulence ability of S. eriocheiris was effectively reduced. The quantity of S. eriocheiris decreased in Drosophila S2 cells after overexpression of MrLGBP, compared with the controls. In addition, RNA interference (RNAi) knockdown of MrLGBP made M. rosenbergii more sensitive to S. eriocheiris infection. Further studies found that the immune genes, including MrLGBP and prophenoloxidase (MrproPO), MrRab7A, and Mrintegrin α1 were significantly up-regulated by SeEnolase stimulation. After SeEnolase pre-stimulation, the ability of M. rosenbergii resistance to S. eriocheiris was significantly improved. Collectively, this investigation demonstrated that MrLGBP and pathogen SeEnolase involved in mediating S. eriocheiris invasion into M. rosenbergii hemocytes.

Spiroplasma eriocheiris Enters Drosophila Schneider 2 Cells and Relies on Clathrin-Mediated Endocytosis and Macropinocytosis.

Spiroplasma eriocheiris causes great economic losses in the crustacean aquaculture industry. However, the mechanism of S. eriocheiris infecting host cells has been poorly studied. We established a Spiroplasma-infected Drosophila Schneider 2 (S2) cell model and investigated its pathogenic mechanism. First, S. eriocheiris induced S2 cell apoptosis and necrosis, seriously decreased cell viability, and increased the production of intracellular reactive oxygen species. Further research showed that S. eriocheiris can invade S2 cells, and the number of copies of intracellular spiroplasmas is sharply increased by 12 h postinfection. In addition, S. eriocheiris can cause S2 cells to form typical inclusion bodies and exhibit large vacuoles. Second, S. eriocheiris is internalized into S2 cells and strongly inhibited through blocking clathrin-mediated endocytosis using chlorpromazine and dynasore. Inhibitors of macropinocytosis, protein kinase C and myosin II, cause a significant reduction in S. eriocheiris in S2 cells. In contrast, disruption of cellular cholesterol by methyl-ß-cyclodextrin and nystatin has no effect on S. eriocheiris infection. These results suggest that the entry of S. eriocheiris into S2 cells relies on clathrin-dependent endocytosis and macropinocytosis, but not via the caveola-mediated endocytic pathway. In addition, the intracellular numbers of S. eriocheiris are dramatically reduced after S2 cells are treated with cytoskeleton-depolymerizing agents, including nocodazole and cytochalasin B. Thus, cellular infection by S. eriocheiris is related to microtubules and actin filaments. This research successfully shows for the first time that S. eriocheiris can invade Drosophila S2 cells and provides a process for S. eriocheiris infection.

Proteomic and metabolomic responses in hepatopancreas of whiteleg shrimp Litopenaeus vannamei infected by microsporidian Enterocytozoon hepatopenaei.

Enterocytozoon hepatopenaei (EHP) causes hepatopancreatic microsporidiosis (HPM) in shrimp. HPM is not normally associated with shrimp mortality, but is associated with significant growth retardation. In this study, the responses induced by EHP were investigated in hepatopancreas of shrimp Litopenaeus vannamei using proteomics and metabolomics. Among differential proteins identified, several (e.g., peritrophin-44-like protein, alpha2 macroglobulin isoform 2, prophenoloxidase-activating enzymes, ferritin, Rab11A and cathepsin C) were related to pathogen infection and host immunity. Other proteomic biomarkers (i.e., farnesoic acid o-methyltransferase, juvenile hormone esterase-like carboxylesterase 1 and ecdysteroid-regulated protein) resulted in a growth hormone disorder that prevented the shrimp from molting. Both proteomic KEGG pathway (e.g., "Glycolysis/gluconeogenesis" and "Glyoxylate and dicarboxylate metabolism") and metabolomic KEGG pathway (e.g., "Galactose metabolism" and "Biosynthesis of unsaturated fatty acids") data indicated that energy metabolism pathway was down-regulated in the hepatopancreas when infected by EHP. More importantly, the changes of hormone regulation and energy metabolism could provide much-needed insight into the underlying mechanisms of stunted growth in shrimp after EHP infection. Altogether, this study demonstrated that proteomics and metabolomics could provide an insightful view into the effects of microsporidial infection in the shrimp L. vannamei.

Characterization of cathepsin D from Eriocheir sinensis involved in Spiroplasma eriocheiris infection.

Cathepsin D (catD) belongs to a lysosomal aspartic protease superfamily. The full-length catD cDNA from the Chinese mitten crab Eriocheir sinensis (EscatD) was 2748 bp and contained a 1158-bp ORF encoding a protein of 385 amino acids, including a signal peptide and two N-glycosylation sites. Phylogenetic analysis showed that EscatD was clustered into a single group, together with other catD for crustaceans. Quantitative real-time PCR revealed that EscatD was expressed mainly in the eyes, hemocytes, intestine and nerve and was expressed weakly in heart, muscle and gills. After challenge with Spiroplasma eriocheiris, the expression of EscatD was significantly up-regulated from 1 d to 9 d. The copy number of S. eriocheiris in a silencing EscatD group was significantly higher than those in the control groups during S. eriocheiris infection. Meanwhile, the survival rate of crabs decreased in an EscatD-dsRNA group. We further found that knockdown of EscatD by RNA interference resulted in a downward trend of expression levels of JNK, ERK, relish and p38 during the early stage, as well as a reduction in the expression of five antimicrobial peptides genes, namely, crusrin1, crustin2, ALF1, ALF2 and ALF3. The subcellular localization experiment suggested that recombinant EscatD was mainly located in the cytoplasm. The over-expression in Drosophila S2 cells indicated that EscatD could decrease the copy number of S. eriocheiris and increase cell viability. The above results demonstrated that EscatD plays an important immune role in E. sinensis to S. eriocheiris challenge.

The function of serpin-2 from Eriocheir sinensis in Spiroplasma eriocheiris infection.

Serpin families classified serine protease inhibitors regulate various physiological processes. However, there is not study on the role of serpin in immune responses against Spiroplasma eriocheiris as a novel causative pathogen in the Chinese mitten crab, Eriocheir sinensis. In our study, quantitative real-time PCR (qRT-PCR) revealed that the mRNA transcripts of Esserpin-2 were ubiquitous in every tissue, relative higher expression in hepatopancreas, gill and hemocytes, while the intestine, muscle, heart and nerve showed relative lower expression. Followed by infection with S. eriocheiris, the transcripts of Esserpin-2 were significantly down-regulated from 1 d to 7 d. After double-stranded RNA injection, the transcripts of Esserpin-2 dramatically declined from 48 h to 96 h. The transcripts of proPO were found to be obviously increased after Esserpin-2 silenced, meanwhile, LGBP with no significant difference. The copy number of S. eriocheiris and subsequently the mortality of crabs in a silencing Esserpin-2 group were significantly less than control groups during infection. The subcellular localization experiment suggested that recombinant Esserpin-2 was mainly located in the cytoplasm. Finally, over-expression assay in Drosophila S2 cells indicated that Esserpin-2 could increase copies of S. eriocheiris and result in cell death. These findings demonstrated that Esserpin-2 involved in the innate immune mechanism of E. sinensis in response to S. eriocheiris infection.

Identification and function analysis of ras-related nuclear protein from Macrobrachium rosenbergii involved in Spiroplasma eriocheiris infection.

A ras-related nuclear protein (Ran) protein was obtained from Macrobrachium rosenbergii, named MrRan. Phylogenetic analysis results showed that MrRan was clustered in one group together with other crustaceans. Tissue distribution analysis revealed that MrRan was expressed mainly in gill, intestine and stomach, and expressed weakly in muscle. The MrRan expression levels in gill and hemocyte of prawns were significantly up-regulated after challenged by Spiroplasma eriocheiris. The copy number of S. eriocheiris in MrRan dsRNA injection group was significantly less than control groups during infection. Meanwhile, silencing MrRan obviously increased the survival rate of prawns. The subcellular localization experiment suggested that recombinant MrRan was mainly located in the nucleus, and relatively weak in the cytoplasm. Finally, over-expression in Drosophila S2 cell indicated that MrRan could increase copies of S. eriocheiris and decrease of cell viability. The present study suggested that MrRan participated in regulating the phagocytosis of S. eriocheiris in M. rosenbergii.

Interaction of heat shock protein 60 (HSP60) with microRNA in Chinese mitten crab during Spiroplasma eriocheiris infection.

Heat shock protein 60 from the Chinese mitten crab Eriocheir sinensis (EsHSP60) was previously identified in relation to Spiroplasma eriocheiris infection by isobaric tags for relative and absolute quantitation labelling followed by liquid chromatography-tandem mass spectrometry. In the present study, to validate the immune function of this protein, the cDNA of the EsHSP60 gene was cloned. Various crab tissues were assessed using real-time PCR, which showed that EsHSP60 transcription occurred in all tissues examined. The expression profiles of EsHSP60 in haemolymph at transcription and protein levels when infected with S. eriocheiris were investigated by real-time PCR and Western blot analysis, respectively. A significant increase of EsHSP60 transcription and protein expression appeared post-injection in response to S. eriocheiris infection when compared to the control group. The double-luciferase reporter gene assay showed that the microRNA PC-533-3p interacted with the 3'-untranslated region of EsHSP60 and inhibited the translation of EsHSP60. The expression profiles of PC-533-3p during S. eriocheiris infection were also investigated by real-time PCR. However, the change tendency of PC-533-3p was opposite to that of the EsHSP60 after S. eriocheiris challenge. These data indicate that the EsHSP60 proteins may play an important role in mediating the immune responses of E. sinensis to an S. eriocheiris challenge.

Spiroplasma eriocheiris Adhesin-Like Protein (ALP) Interacts with Epidermal Growth Factor (EGF) Domain Proteins to Facilitate Infection.

Spiroplasma eriocheiris is a novel pathogen found in recent years, causing the tremor disease (TD) of Chinese mitten crab Eriocheir sinensis. Like Spiroplasma mirum, S. eriocheiris infects the newborn mouse (adult mice are not infected) and can cause cataract. Adhesion-related protein is an important protein involved in the interaction between pathogen and host. In this study, the Adhesin-like Protein (ALP) of S. eriocheiris was detected on its outer membrane by using immune electron microscopy, and was found to be involved in the bacterium's infection of mouse embryo fibroblasts (3T6-Swiss albino). Yeast two-hybrid analysis demonstrated that ALP interacts with a diverse group of mouse proteins. The interactions between recombinant partial fibulin7 (FBLN7; including two epidermal growth factor [EGF] domains) and ALP were confirmed by Far-western blotting and colocalization. We synthetized the domains of FBLN7 [EGF domain: amino acids 136-172 and complement control protein (CCP) domain: 81-134 amino acids], and demonstrated that only EGF domain of FBLN7 can interact with ALP. Because the EGF domain has high degree of similarity to EGF, it can activate the downstream EGFR signaling pathway, in key site amino acids. The EGFR pathway in 3T6 cells was restrained after rALP stimulation resulting from competitive binding of ALP to EGF. The unborn mouse, newborn mouse, and the adult mouse with cataract have a small amount of expressed FBLN7; however, none was detected in the brain and very little expression was seen in the eye of normal adult mice. In short, ALP as a S. eriocheiris surface protein, is critical for infection and further supports the role of ALP in S. eriocheiris infection by competitive effection of the EGF/EGFR axis of the target cells.

A novel C-type lectin is involved in the innate immunity of Macrobrachium nipponense.

C-type lectins (CTLs) play important roles in invertebrate innate immunity by recognizing and eliminating pathogens. In the present study, a low-density lipoprotein receptor class A (LDLa) domain-containing CTL was identified from the oriental river prawn Macrobrachium nipponense, designated as MnCTLDcp1. The full-length cDNA of MnCTLDcp1 was composed of 1462 bp, with a 999-bp ORF encoding a 332-aa protein. An LDLa and a single C-type lectin-like domain (CTLD) were found. The mRNA transcripts of MnCTLDcp1 was expressed the highest in heart. After the prawns were challenged by Aeromonas hydrophila and Staphylococcus aureus, the expression level of MnCTLDcp1 in heart and hemocytes were all significantly up-regulated. Sugar binding assay revealed that the MnCTLDcp1 could bind to the glycoconjugates of bacteria surface, such as LPS, PGN and they can compete with bacterial as competitors. The recombinant MnCTLDcp1 agglutinates Gram-positive (S. aureus and Bacillus subtilis) and Gram-negative bacteria (A. hydrophila, Vibrio parahaemolyticus, Escherichia coli and Pseudomonas aeruginosa) in the presence of calcium and also could bind to these bacteria. These results clearly suggested that MnCTLDcp1 functions as a pattern-recognition receptor involved in the innate immunity of M. nipponense.