Search

Methanotrophs in marine sediments and overlying water attenuate the emissions of methane into the atmosphere and thus play an important role for the global cycle of this greenhouse gas. However, gas released from natural hydrocarbon seeps are not pure methane but commonly mixed hydrocarbons. Currently, how methanotrophic bacteria behave in the co-presence of methane and heavier hydrocarbons remains unknown. In this paper, the bacteria were cultured aerobically in fresh sediment samples (collected from Bohai Bay in eastern China) at 28°C under the atmospheres of pure methane and methane+ethane+propane mixed gas, respectively. The prevailing terrigenous n-alkanes and fatty acids in the original sediment samples varied consistently after incubations, confirming the proceeding of aerobic bacterial activities. The real-time quantitative PCR assay and sequencing of the 16S rRNA and particulate methane monooxygenase (pmoA) genes revealed the changes of microbe communities to a methanotroph-dominating structure after incubations. Particularly, after incubations the family Methylococcaceae (type I methanotrophs) became dominant with proportions higher than 40%, whereas Methylocystaceae (type II methanotrophs) nearly disappeared in all incubated samples. Moreover, the species of methanotrophs from the samples treated with pure methane were dominated by Methylobacter luteus, whereas Methylobacter whittenburyi took the predominant proportion in the samples treated with mixed gas. The phenomenon suggests that some methanotrophs may also utilize ethane and propane. Collectively, this study may help to gain a better understanding of the effects and contributions of microbial activities in marine hydrocarbon seep ecosystems.

Spiroplasma eriocheiris (S. eriocheiris) infection causes a significant economic loss in Penaeus vannamei (P. vannamei) culture industry. However, the response of P. vannamei hemocytes to S. eriocheiris infection has not been extensively studied. In this study, we conducted full-length transcriptome and long non-coding RNA (lncRNA) analyses of P. vannamei hemocytes by a challenge test with S. eriocheiris. Following assembly and annotation, there were 8077 high-quality unigenes. A total of 1168 differentially expressed genes (DEGs) were obtained, including 792 up-regulated and 376 down-regulated genes by differential expression analysis. Gene ontology (GO) enrichment analysis showed that the up-regulated DEGs were mainly clustered into immune system process, defense response, cell cycle and organelle organization. On the other hand, the down-regulated DEGs included that genes that were mainly clustered into metabolic processes related to organic compounds, metabolic process and cellular metabolic process. Protein-protein interaction (PPI) network analysis of DEGs indicated that the pivotal gene interactions were connected to stress response, immune system process and cell cycle. The lncRNA analysis identified multiple lncRNAs, which were highly co-expressed with the immune-related genes, such as lncRNA transcript-12631 and transcript-12631, suggesting that lncRNAs may be involved in the regulation of immune defense in shrimp hemocytes. Additionally, 20 hub unigenes and putative lncRNAs related to immune system were validated by quantitative real-time PCR (qRT-PCR), validating the reliability of RNA-Seq. This study revealed a close connection between the immune and metabolic systems of S. eriocheiris infected P. vannamei.

In recent years, Cyprinid herpesvirus 2 (CyHV-2) has severely affected the culture of crucian carp Carassius auratus gibelio, causing massive economic losses. However, the responses of crucian carp during infection with CyHV-2 have not been explored. In this study, physiological and biochemical indicators and enzyme activities of crucian carp before and after infection with CyHV-2 were analyzed. The number of white blood cells in the diseased fish was significantly increased. Compared to control group, red blood cell content, hemoglobin content, mean corpuscular volume, mean corpuscular hemoglobin content and mean corpuscular hemoglobin concentration in the experimental group were significantly decreased. In the diseased fish group, the levels of the glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, alkaline phosphatase, lactate dehydrogenase and urea nitrogen in the blood were also significantly increased compared to the control group. In addition, the activity of serum total antioxidant capacity, acid phosphatase, glutathione-S transferase and catalase in diseased fish were significantly higher than in the control group. LC-Q/TOF-MS analysis platform was used in this study for the first time to detect changes in the serum metabolome of crucian carp infected with CyHV-2. A total of 79 differential metabolites have been identified. Significant increases in concentrations were found for several carbohydrates (glucose, mannose, ribose, etc.), fatty acids (pentadecanoic acid, arachidonic acid, palmitic acid, eicosapentaenoic acid, hydroxylic tetracarbonate alkenoic acid, linoleic acid and its derivatives) and most of the amino acids and their derivatives. However, guanine, cytosine, uridine and VB2 were significantly decreased. KEGG pathways enrichment showed that amino acid metabolism and lipid metabolism were severely influenced. The above results provide a theoretical basis for the infection mechanism of CyHV-2

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.

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.

Transferrin, a member of the iron binding superfamily protein, plays an extremely important role in the transport of iron in the biological process of cells. The result of preliminary proteomic study on E. sinensis hemocytes infected Spiroplasma eriocheiris showed the expression of transferrin (EsTF) and ferrin (EsFe) significantly changed. In addition, other reports have confirmed that transferrin, ferritin and iron are involved in the immune response of hosts. In order to validate the immune function of EsTF, the whole length of EsTF was successfully amplified by the gene cloning and RACE technique. The results showed that the full-length cDNA of the EsTF gene was 2748 bp, including a 2193 bp open reading frame which encodes 730 amino acids. The result of bioinformatics analysis showed EsTF contains two highly conserved TR_FER domains. Evolutionary analysis showed that EsTF has a close genetic relationship with other TFs of invertebrates. In addition, EsTF mRNA was highly transcripted in nerve and intestine tissues, followed by hemocytes. The expression of EsTF, EsFe1 and EsFe2 increased after exogenous supplemental of iron under the concentration of 100 nmol/L in water. After exogenous supplement of iron and injection with S. eriocheiris, these three gene transcription of mRNA levels were higher than that of PBS group, while lower than the S. eriocheiris group and the iron group. Besides, the copy number of S. eriocheiris in the experimental group was significantly reduced, and the death rate decreased. As can be seen, iron made transferrin and ferritin return to normal levels during the infection of S. eriocheiris and help the host maintain normal immunity levels to resist S. eriocheiris. These results further demonstrated that EsTF, EsFe1, EsFe2 and iron play a role in the immune defense mechanism of the crabs to resist S. eriocheiris infection.

As a new-found aquaculture pathogen, Spiroplasma eriocheiris, has resulted in inconceivable economic losses in aquaculture. In the infection of S. eriocheiris, the Procambarus clakii hemocytes have indicated to be major target cells. What was designed to examine in our study is the hemocytes' immune response at the protein levels. Before the pathogen was injected and after 192 h of post-injection, the differential proteomes of the crayfish hemocytes were analyzed immediately by isobaric tags for relative and absolute quantization (iTRAQ) labeling, followed by liquid chromatogramphytandem mass spectrometry (LC-MS/MS). This research had identified a total of 285 differentially expressed proteins. Eighty-three and 202 proteins were up-regulated and down-regulated, respectively, caused by the S. eriocheiris infection. Up-regulated proteins included alpha-2-macroglobulin (α2M), vitellogenin, ferritin, etc. Down-regulated proteins, involved with serine protease, peroxiredoxin 6, 14-3-3-like protein, C-type lectin, cdc42 homolog precursor, etc. The prophenoloxidase-activating system, antimicrobial action involved in the immune responses of P. clarkii is considered to be damaged due to S. eriocheiris infection. The present work could lay the foundation for future research on the proteins related to the susceptibility/resistance of P. clarkii to S. eriocheiris. In addition, it is helpful for our understanding molecular mechanism of disease processes in crayfishes.

The Chinese mitten crab, Eriocheir sinensis, is an important economic aquatic product in China. Oxygen is essential for the crab, hypoxia is a natural phenomenon common in the process of aquaculture and will bring a harmful impact on the growth, development and disease outbreak of crab. To understand the effect of hypoxia on the crab, the proteome changes of E. sinensis hemocytes in the hypoxia (1.5 ± 0.5 mg/L of DO) and the normoxia groups (6.0 ± 0.5 mg/L of DO) were obtained using iTraq labeling and affinity enrichment followed by high-resolution LC-MS/MS analysis. In total, 664 differentially expressed proteins were reliably quantified (567 up-regulated and 97 down-regulated) using 1.2-fold change in expression as a physiologically significant benchmark. GO annotation, KEGG annotation, etc. were used to analysis these significantly different expression proteins. Many pathways or biological process were identified in the process of E. sinensis hemocytes response hypoxia, such as tricarboxylic acid (TCA) cycle, cell apoptosis, VEGFR/MAPK pathway, prophenoloxidase system (proPO system), phagocytosis, and so on. Ten selected genes from these different expression proteins were verified by qRT-PCR analysis. Finally, the activity of several major antioxidant enzymes (phenoloxidase (PO), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA)) were analysis, the results showed that hypoxia stress significantly increased these enzymes activity. These study could serve as a basis to understand the relationship between E. sinensis hemocytes and hypoxia stress, and also provide reference to study hypoxia stress in other crustaceans.

Spiroplasma eriocheiris is a novel pathogen similar to the Spiroplasma mirum and also had an ability to infect the newborn mice and caused cataract. Our study was designed to study how S. eriocheiris infects mouse 3T6-Swiss albino cells and to elucidate the cellular molecular pathogenesis of Spiroplasma. FCM analysis and MTT analysis clearly shown that S. eriocheiris could induce 3T6 cell apoptosis and cause cell viability decreased seriously. Immunofluorescence experiments and TEM analysis shown that S. eriocheiris can invade 3T6 cells and form typical inclusion bodies and exhibit vacuolization in vitro. S. eriocheiris-oxytetracycline protection assay show that the infective bacteria already were detected at 1h post infection, and sharply increased at 12h after the bacteria infection. To further study the infection mechanism of S. eriocheiris, global mRNA and microRNA (miRNA) expression profiling were analyzed after the cells infected with the bacteria. A total of 619 non-redundant annotated transcripts (183 up-regulated and 436 down-regulated) and 22 miRNAs (8 up-regulated and 14 down-regulated) were differential expression after 6h S. eriocheiris infection compared to control group. Integrated analysis shown that homologous genes from differential expression miRNA targets and the differential expression genes of the mRNA microarray were major focused on two important pathways focal adhesion and MAPK signaling pathway. To validate the results of microarray, eight focal adhesion (β-Catenin, Parvin, Grb2 and ERK) and MAPK signaling pathway (FGFR, Grb2, ERK, MKK3, p38 and JNK) genes and the housekeeping gene GAPDH were assayed by qPCR and Western blot to confirm the results. Eight miRNAs (miR-143-3p, miR-214-5p, miR-322-3p, miR-328-5p, miR-351-5p, miR-466h-5p, miR-503-5p and miR-30c-1-3p) and the housekeeping gene U6 miRNA were assayed by qPCR to confirm the results of microarray. All the results help us better understand the infection mechanism of S. eriocheiris.

Spiroplasma eriocheiris, which causes tremor disease in Chinese mitten crab Eriocheir sinensis, has led to huge economic losses in aquaculture. Immunoproteomics, a new scientific technique combining proteomics and immunological analytical methods, provided the direction of our research on S. eriocheiris. The aim of our study was to identify the proteome, antigen proteins and antigen membrane proteins of S. eriocheiris. A total of 780 S. eriocheiris proteins were identified by the LC-MS/MS technique. Based on immunoproteomics, 51 proteins and 7 proteins in S. eriocheiris were identified by anti-S. eriocheiris serum and negative serum respectively (six proteins in common). Thus, 45 antigenic proteins in S. eriocheiris were identified; among them, molecular chaperone DnaK, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ATP synthase subunit beta and enolase can be considered as immunogenic proteins. Similarly, 32 membrane proteins and 6 membrane proteins were identified by anti-S. eriocheiris serum and negative serum respectively (two proteins in common). Thus, 30 antigenic membrane proteins in S. eriocheiris were identified; three of them have been reported as surface proteins including pyruvate kinase, enolase and GAPDH. All of these proteins may play key roles in the pathogeny and can be used in the future for diagnoses and prevention. Significance and Impact of the Study Spiroplasma eriocheiris is a novel pathogen causing the tremor disease in Chinese mitten crab Eriocheir sinensis. This is the first time LC-MS/MS was used to identify the proteome, antigen protein and antigen membrane protein of S. eriocheiris. The results can certainly provide valuable information towards the identification of virulent proteins or diagnosis of pathogenic mechanisms.

The Chinese mitten crab Eriocheir sinensis is an important economic crustacean that has been exposed to various diseases. Spiroplasma eriocheiris , isolated from tremor-diseased E. sinensis , was first identified as a lethal pathogen of freshwater crustaceans. To understand the pathogenesis of S. eriocheiris to E. sinensis , the transcriptomic profiles of haemocytes in the experimental and control groups at 1 d and 7 d post-injection were obtained using Illumina HiSeq 2500. These results showed that 40,358,724, 44,462,112, 45,516,576 and 37,713,728 paired-end clean reads were obtained from the cDNA libraries of DZ1 (the control group at 1 d), DZ7 (the control group at 7 d), SY1 (the experimental group at 1 d) and SY7 (the experimental group at 7 d), respectively. In total, 106,641 unique transcript fragments (unigenes) were assembled, with an average length of 710 bp. On the first day of stimulation, 33,084 up-regulated transcripts and 19,208 down-regulated transcripts were found in the experimental group compared with those in the control group. On the seventh day of stimulation, 40,198 up-regulated transcripts and 12,032 down-regulated transcripts were found in the experimental group compared with those in the control group. Some canonical immune-related pathways were identified via KEGG pathway analysis, including complement and coagulation cascades, the VEGF signalling pathway, the Wnt signalling pathway, natural killer cell-mediated cytotoxicity, the MAPK signalling pathway, neuroactive ligand-receptor interactions, and the Lysosome pathway. We found important immune-related genes (GNPTAB, MASP2, F7, F5, NFATC, TRAF6, MAP3K5, and TRa) in the KEGG pathway, and those genes were confirmed by qRT-PCR analysis. In addition, the significantly enriched neuroactive ligand-receptor interaction pathway was associated with intense paroxysmal tremors of infected crabs. Our results provide valuable information for the further analysis of the mechanisms of E. sinensis defence against S. eriocheiris invasion.

The characteristics of the hydrate-bearing layer are important parameters for gas hydrate exploration and exploitation, which can be provided by laboratory analysis. We have carried out a systematic analysis of modern instruments, including the laser grain size analyzer, X-ray computed tomography, scanning electron microscopy, X-ray diffraction (XRD), Raman spectroscopy, gas chromatography, and isotope mass spectrometry, on the hydrate-bearing sediment samples recovered from the Shenhu area in the South China Sea in 2016. We focused on the structure, hydration number, and gas composition of the gas hydrate samples. We also evaluated the effects of sediments on the occurrence and distribution of gas hydrate. Our results showed that the sediments are much finer than those recovered from the Shenhu area in 2007. Some samples are found to contain calcareous microfossils and foraminifera, which are beneficial for dispersed gas hydrate formation. Whereas the only visible hydrate sample (FC4) has not found any bioclast in the sediments. Based on the Raman data and the XRD analysis, the gas hydrates demonstrate typical structure I (sI) hydrates, with cage occupancies of methane molecules higher than 99.3% in large cages and 81.5%–91.4% in small cages, respectively. The crystal lattice parameter is 11.89 Å with the hydration number being approximately 6.0. Methane is obviously the dominant component, ranging from 97.6% to 99.95% in these samples. Comprehensive study of carbon and hydrogen isotopes suggests that the hydrate-bound gases are from biogenic and thermogenic gases in the study area.

Spiroplasma eriocheiris, a novel pathogen of Chinese mitten crab Eriocheir sinensis tremor disease, has led into catastrophic economic losses in aquaculture. S. eriocheiris invaded the hemocytes in the early stage, then invaded nerve tissue and caused typically paroxysmal tremors of pereiopod in the late stage of infection. The purpose of this study was to detect the infection mechanism of hemocytes in the early stage and thoracic ganglion in the late stage of S. eriocheiris infection at the protein level. Hemocytes and thoracic ganglion were collected at 24 h and 10 d after injection (the crabs with typical paroxysmal tremors of the pereiopod), respectively. TMT was performed with isobaric markers, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). In hemocytes, 127 proteins were up-regulated and 85 proteins were down-regulated in 2747 quantified proteins. Many proteins and process including proPO system proteins, hemolymph coagulation system proteins and lectins were differently expressed in hemocytes and involved in the early immune process of E. sinensis against S. eriocheiris infection. Meanwhile, 545 significantly different expression proteins (292 down-regulated and 253 up-regulated protein including a number of immune-associated, nervous system development and signal transmission related proteins) were identified in thoracic ganglion in the late stage of S. eriocheiris infection. The qRT-PCR analysis results shown that the selected significantly changed proteins in hemocytes and thoracic ganglion were consistent with the TMT proteomics. This paper reported for the first time to study the responses of crab hemocyte and thoracic ganglion against the S. eriocheiris infection at different stages. These findings help us understand the infection mechanism of S. eriocheiris at different stage with the different tissue.

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 β-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.

As one of the most devastating diseases in Eriocheir sinensis, tremor disease (TD) is caused by Spiroplasma eriocheiris and dramatically increases the economic losses of crab aquaculture. With the development of biotechnology, metabolomics plays an important role in the screening of metabolites related to physiological and pathological changes. In this study, the different metabolites induced by S. eriocheiris were investigated in hemocytes of crab E. sinensis using metabolomics based on GC/MS among diverse phenotypes groups (tremor group, no-symptom group, and control group). Compared with the tremor group, 42 differential metabolites (26 up-regulated and 16 down-regulated) were significantly different in the no-symptom group. The KEGG pathway enrichment analysis showed that the differential metabolites were enriched in “ABC transporters”, “Vitamin B6 metabolism”, “Ascorbate and aldarate metabolism” and “Starch and sucrose metabolism”. The cellobiose and α-ketoglutarate (AKG) were chosen to verify the function of these two metabolites. The crabs were fed with dietary cellobiose (1, 5, and 10 mg/g diet weight) or AKG (1, 5, and 10 mg/g diet weight) before S. eriocheiris injection. The results show that all three concentrations of cellobiose and AKG could reduce the mortality of crabs and decrease the copies of S. eriocheiris in the hemocytes. The histopathological sections of the hindgut of E. sinensis show that cellobiose and AKG affect the connective tissue of the outer layer of muscle. Overall, these results provide insight into TD control and the sustainable development of aquaculture.

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.

Spiroplasma eriocheiris, as a novel aquaculture pathogen, has led into catastrophic economic losses in aquaculture. The Macrobrachium rosenbergii hemocytes were major target cells in S. eriocheiris infection. Our study was designed to examine the hemocytes' immune response at the protein levels. The differential proteomes of the prawn hemocytes were analyzed immediately prior to injection with the pathogen, and at 192 h post-injection by isobaric tags for relative and absolute quantization (iTRAQ) labeling, followed by liquid chromatogramphytandem mass spectrometry (LC–MS/MS). A total of 69 differentially expressed proteins were identified. Forty-nine proteins were up-regulated and 20 proteins were down-regulated resulting from a S. eriocheiris infection. Up-regulated proteins included vertebrate gliacolin-like protein, vitellogenin, Gram-negative binding protein 1, alpha2 macroglobulin isoform 2 (a2M), etc. Down-regulated proteins, involved with beta-1,3-glucan-binding protein (BGBP), immunoglobulin like, Rab7, lipopolysaccharide and β-1,3-glucan (LGBP), actin-related protein, etc. Selected bioactive factors (tachylectin, α2M and vitellogenin, BGBP, C-type lectin, LGBP and Rab7) were verified by their immune roles in the S. eriocheiris infection using real-time PCR. The present work could serve as a basis for future studies on the proteins implicated in the susceptibility/resistance of M. rosenbergii to S. eriocheiris, as well as contribute to our understanding of disease processes in prawns. Biological significance This is the first time using an iTRAQ approach to analyze proteomes of M. rosenbergii mobilized against S. eriocheiris infection and substantiated the hemocytes' proteomic changes in M. rosenbergii using an infection model. The results reported here can provide a significant step forward toward a more complete elucidation of the immune relationship between M. rosenbergii and the pathogen S. eriocheiris.

To investigate use of posterior groove of palatovaginal (PV) canal as an anatomic landmark in determining the location of PV canal in axial computed tomography (CT) images of pterygopalatine fossa (PPF). A total of 20 skull specimens were examined in this analysis. Each skull was scanned by CT with and without a probe inserted through PV canal to measure the anatomic structures. CT images of 70 patients were used for comparing the rate of correct location of PV canal between the conventional method (using the vidian canal as a landmark) and the method of using the posterior groove as a landmark. Two skulls were dissected using endoscopy to further reveal the advantage of the posterior groove as a landmark. In all 20 skull specimens, the groove showed the morphology of a narrow groove and elliptical fossa in 24 and 16 sides, respectively. In CT images, the angle from PV canal and the posterior groove to the hard palate was 53.14° ± 5.48° and 20.93° ± 6.28°, respectively, which was significantly different (P ≤ 0.05). The rate of correct location of PV canal was statistically significantly higher with the method of posterior groove as a landmark than the conventional method (70.7 vs 49.3 %, P

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.

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.

A pattern recognition protein (PRP), lipopolysaccharide and β-1,3-glucan binding protein (LGBP), cDNA was cloned from the hemocytes of red claw crayfish, Cherax quadricarinatus by the techniques of homology cloning and RACE. Analysis of nucleotide sequence revealed that the full-length cDNA of 1456 bp has an open reading frame of 1095 bp encoding a protein of 364 amino acids including a 15 amino acid signal peptide. The calculated molecular mass of the mature protein (349 aa) was 39.92 kDa with an estimated pI of 4.50. Sequence comparison of the deduced amino acid sequence of C. quadricarinatus LGBP (named as CqLGBP) showed a high identity of 82%, 77%, 76% and 74% with Procambarus clarkii LGBP, Homarus gammanus BGBP, Penaeus monodon BGBP and Litopenaeus stylirostlis LGBP, respectively. The CqLGBP sequence contains: (1) two putative integrin-binding motifs, (2) a glucanase motif, (3) two putative N-glycosylation sites, (4) one protein kinase C phosphorylation site, and (5) a putative recognition motif for β-1,3-linkage of polysaccharides. The recombinant CqLGBP were expressed in Escherichia coli BL21 with pGEX-6P-2 expression vector. The titer of rabbit anti-CqLGBP serum was above 1:12,800. Microorganism binding assay showed CqLGBP binds two kinds of crustacean pathogens Spiroplasma eriocheiris and Aeromonas hydrophila. Quantitative RT-PCR method was used to quantify the variation of mRNA transcription level during artificial infection with S. eriocheiris and A. hydrophila. A significant enhancement of CqLGBP transcriptions was apparent post-injection in response to bacterial infection compared to the control group. These data should be helpful to better understand the function of CqLGBP in the crayfish immune system.

Spiroplasmas are small, wall-less, helical, and motile bacteria classified within the class Mollicutes. Spiroplasma has been identified as a lethal pathogen of four freshwater crustaceans in previous studies. Here, the fifth crustacean host of spiroplasma was found. A novel disease of oriental river prawn Macrobrachium nipponense appeared in the summer of 2012 in Jiangsu province of China. Morphological observation, molecular biological methods and infection experiments identified the pathogen as a spiroplasma. The agent isolated from diseased prawns was able to pass through membrane filters with pores 220 nm in diameter and cultivated by R2 medium. A 16S rRNA complete sequence was cloned from the isolation and alignment results revealed that the spiroplasmas from freshwater crustaceans were highly related. Phylogenetic analysis of these sequences showed that the five freshwater crustacean spiroplasma strains had a close relationship with Spiroplasma mirum . The pathogenicity of the agent, evaluated by the mortalities, was determined to be 65% through a challenge experiment. The above results indicated that M. nipponense is another new spiroplasma host in aquatic crustaceans, thus further indicating that more attention should be paid to this pathogen.

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.

Spiroplasma eriocheiris, a pathogen that causes mass mortality of Chinese mitten crab Eriocheir sinensis, is a wall less bacteria and belongs to the Mollicutes. This study was designed to investigate the effects of colchicine on S. eriocheiris growth, cell morphology, and proteins expression. We found that in the presence of colchicine, the spiroplasma cells lost their helicity, and the length of the cells in the experimental group was longer than that of the control. With varying concentrations of the colchicine treatment, the total time to achieve a stationary phase of the spiroplasma was increased, and the cell population was decreased. The virulence ability of S. eriocheiris to E. sinensis was effectively reduced in the presence of colchicine. To expound the toxical mechanism of colchicine on S. eriocheiris, 208 differentially expressed proteins of S. eriocheiris were reliably quantified by iTRAQ analysis, including 77 up-regulated proteins and 131 down-regulated proteins. Especially, FtsY, putative Spiralin, and NADH oxidase were down-regulated. F0F1 ATP synthase subunit delta, ParB, DNABs, and NAD(FAD)-dependent dehydrogenase were up-regulated. A qRT-PCR was conducted to detect 7 expressed genes from the iTRAQ results during the incubation. The qRT-PCR results were consistent with the iTRAQ results. All of our results indicate that colchicine have a strong impact on the cell morphology and cellular metabolism of S. eriocheiris.

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.

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.

Spiroplasma eriocheiris is a pathogen that causes mass mortality in Chinese mitten crab, Eriocheir sinensis. S. eriocheiris causes tremor disease and infects almost all of the artificial breeding crustaceans, resulting in disastrous effects on the aquaculture economy in China. S. eriocheiris is a wall-less helical bacterium, measuring 2.0 to 10.0 μm long, and can swim up to 5 μm per second in a viscous medium without flagella by switching the cell helicity at a kink traveling from the front to the tail. In this study, we showed that S. eriocheiris performs chemotaxis without the conventional two-component system, a system commonly found in bacterial chemotaxis. The chemotaxis of S. eriocheiris was observed more clearly when the cells were cultivated under anaerobic conditions. The cells were polarized as evidenced by a tip structure, swimming in the direction of the tip, and were shown to reverse their swimming direction in response to attractants. Triton X-100 treatment revealed the internal structure, a dumbbell-shaped core in the tip that is connected by a flat ribbon, which traces the shortest line in the helical cell shape from the tip to the other pole. Sixteen proteins were identified as the components of the internal structure by mass spectrometry, including Fibril protein and four types of MreB proteins.

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.

Spiroplasma eriocheiris is an emerging pathogen in freshwater crustaceans. In recent years, Eriocheir sinensis, Procambarus clarkii, Litopenaeus vannamei, Macrobrachium rosenbergii and Macrobrachium nipponense had been infected by this pathogen in China. An immunochromatographic strip test using gold nanoparticles was developed for rapidly detecting this pathogen. The strip test based on the principle of sandwich immunoassay by the specific combination between the pathogen and polyclonal antibody on a nitrocellulose membrane. Positive samples were displayed as red lines at the test and control zones of the nitrocellulose membrane, while negative samples resulted in a red line only at the control zone. The limit of detection was proved to be 106 Color Change Unit/ml. The test strip could be visually detected within 15 min and do not have cross-reaction with other aquatic bacteria. This test strip allows on-site rapid detection of S. eriocheiris in crustacean without the requirement of specialized equipment and professional personnel. The one-step test strips developed in our study had high sensitivity, specificity, reproducibility and stability. In conclusion, this method was proved to be convenient, feasible, rapid and effective for detecting S. eriocheiris.

Spiroplasma eriocheiris is as a novel pathogen of Chinese mitten crab Eriocheir sinensis tremor disease. The hemocytes have been shown to be major target cells in S. eriocheiris infection. The aim of this study was to examine the hemocytes' immune response at the protein levels. The differential proteomes of the crab hemocytes were analyzed immediately prior to injection with the pathogen, and at 10 d post-injection by isobaric tags for relative and absolute quantization (iTRAQ) labeling, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1075 proteins were identified by LC-MS/MS and de novo sequencing data. Using a 1.2-fold change in expression as a physiologically significant benchmark, 76 differentially expressed proteins (7.07%) were reliably quantified by iTRAQ analysis. Thirty-five (3.26%) proteins were up-regulated and 41 (3.81%) proteins were down-regulated resulting from a S. eriocheiris infection. Approximately 20 differential proteins in hemocytes were involved in the stress and immune responses. Up-regulated proteins included alpha-2-macroglobulin (α2M), prostaglandin D synthase (GST), ferritin, and heat shock protein 60. Down-regulated proteins included two lectins (mannose-binding protein and hemocytin), three kinds of serine proteinase inhibitors (two serpins and pacifastin), three different kinds of serine proteases, mitogen-activated protein kinase kinase (MAPKK), and two thioredoxins (Trx), crustin, etc. Selected bioactive factors (α2M, GST, ferritin, tubulin, crustin, thioredoxin, clip domain serine protease and serpin) are verified by their immune roles in the S. eriocheiris infection using Real-time PCR. The variation trend of immune gene's mRNA expression is similar with the result of iTRAQ, except the tubulin. The prophenoloxidase-activating system, antimicrobial action and antioxidant system involved in the immune responses of E. sinensis is believed to be a resistance to S. eriocheiris infection. This is the first report of the proteome response of crab hemocytes against S. eriocheiris infection. These findings contribute to our understanding of tremor disease processes in crabs, and provide the first evidence to promote a search for potential biomarkers of the disease.

Spiroplasma is a novel pathogen of commercially exploited crustaceans. To more full clarify its pathogenic mechanism at the molecular level in vitro , a spiroplasma-infected hemocytes model was developed as described here, using a modified method of hemocytes culture from the Chinese mitten crab Eriocheir sinensis . It has been standardized by employing L-15 growth medium supplemented with 15% fetal bovine serum along with 0.15% glucose, 0.75% NaCl, antibiotics (100 U ml −1 penicillin, 100 U ml −1 streptomycin) and a suitable pH of 7.20–7.40, incubated at 28 °C without the requirement for 5% carbon dioxide. Cytopathic effects of Spiroplasma eriocheiris in the cultures, including cell debris and cellular exudates, were observed as early as 36 h post-inoculation. The green fluorescent dye Alex-488 was used as an immunoflourescence marker of S. eriocheiris to study its adhesion to, and infection of, the host cell. At about 24-h post-inoculation, a large number of spiroplasmas were observed infecting the host hemocytes cells, which became slug-like and widely distributed in the culture. Intracellular inclusion bodies containing spiroplasmas, and a large number of free spiroplasmas released from the ruptured cells, were observed using transmission electron microscopy. All the results show that S. eriocheiris can invade E. sinensis hemocytes in vitro , and this can provide a significant step forward toward further study of the relationship between the novel pathogen spiroplasma and its hosts in laboratory experimental studies.

The lipopolysaccharide and β-1,3-glucan binding protein (LGBP), one of the pattern recognition proteins, plays an important role in the innate immune response of invertebrates. A 1,506 bp full-length cDNA of a LGBP gene was cloned and characterized from the oriental river prawn Macrobrachium nipponense (named as MnLGBP). Analysis of the nucleotide sequence revealed that the cDNA clone has an open reading frame of 1,119 bp, encoding a protein of 372 amino acids including a 21-aa signal peptide. The calculated molecular mass of the mature protein (351 aa) was 39.9 kDa with an estimated pI of 4.63. The MnLGBP sequence contains: (1) two putative integrin-binding motifs, (2) a glucanase motif, (3) two putative N-glycosylation sites, (4) one protein kinase C phosphorylation site, and (5) a putative recognition motif for β-1,3-linkage of polysaccharides. Sequence comparison based on the deduced amino acid sequence of MnLGBP showed varied identity of 89, 76 and 74% with those of Macrobrachium rosenbergii LGBP, Marsupenaeus japonicus β-1,3-glucan binding proteins, and Fenneropenaeus chinensis LGBP, respectively. Quantitative RT-PCR results showed that MnLGBP was expressed in nerve, intestine, muscle, gill, heart, haemocytes and at the highest level in hepatopancreas. After challenge with the pathogen, Aeromonas hydrophila and Vibrio parahaemolyticus, the expression of MnLGBP mRNA was significantly upregulated in the hepatopancreas compared to the control group. At the same time, the mRNA level of MnproPO increased dramatically at 48 h after injection of bacteria. These data should be helpful to better understand the function of MnLGBP in the prawn immune system.

Instead of the usual host, the goldfish, cyprinid herpesvirus 2 (CyHV-2), has been detected in the Prussian carp (Carassius gibelio) from Hungary, the Czech Republic and mainland China in recent years, which has caused the massive mortality of C. gibelio and appears to be spreading worldwide. From May to July of 2011 and 2012, the massive mortality of C. gibelio was observed in many aquatic breeding ponds in northern Jiangsu province, eastern China. Moribund fish showed petechial and eccymotic hemorrhages in gills and around the base of the fins and white color of fins. Histological examination revealed that tissues from gill, kidney and spleen presented symptoms of infiltration of hemocytes and the lesions. Transmission electron microscopy of these cells demonstrated naked herpes-like virus nucleocapsids (95–110 nm) in their nuclei, while the cytoplasm contained multiple aggregates of enveloped viral particles (170–200 nm). CyHV-2 infected gill, kidney and spleen and multiplied or enveloped in both nucleus and cytoplasm of leukocytes or hematopoietic cells. Maturation of CyHV-2 occurred in the cytoplasm, particularly associated with Golgi apparatus, indicating that the virus from C. gibelio probably contains viral glycoproteins. PCR method was used for detecting the virus by using CyHV-2 primers and produced positive results in the tissues of gill, kidney and spleen but negative results in the fins, liver and intestine, which coincided with histological and morphological results. The present work presents the ultrastructural pathology of this serious disease in C. gibelio caused by CyHV-2. It will be helpful to further understand the mechanism of CyHV-2 infection of this new fish host.

The superoxide dismutases (SODs) are important antioxidant enzymes to remove excess biologically reactive oxygen intermediates, and thus avoid adverse conditions. In this study, an extracellular copper–zinc superoxide dismutase (ecCuZnSOD) gene and a mitochondrial manganese superoxide dismutase (mtMnSOD) gene were cloned from hemocytes of the freshwater crayfish, Procambarus clarkii . The open reading frame (ORF) of ecCuZnSOD is 612 bp encoding a 204 amino acid (aa) protein with a 24 aa signal peptide, whereas the ORF of mtMnSOD is 654 bp and encodes a 218 aa protein with a 20 aa mitochondrial-targeting sequence in the N-terminus. P. clarkii ecCuZnSOD and mtMnSOD proteins showed high similarity with ecCuZnSODs and mtMnSODs from other crustaceans, respectively. Both ecCuZnSOD and mtMnSOD of P. clarkii were highly expressed in hepatopancreas, hemocytes and gill; lower expression levels were seen in intestine, stomach, nerve, heart and muscle. The mRNA expressions of three SODs of P. clarkii (ecCuZnSOD, mtMnSOD and cytosolic MnSOD (cytMnSOD)) were studied after Spiroplasma eriocheiris and Aeromonas hydrophila stimulations. After S. eriocheiris challenge, the mRNAs of three SODs in hemocytes and gill were up-regulated, and ecCuZnSOD also increased in the hepatopancreas. However, the mtMnSOD and cytosolic MnSOD in the hepatopancreas were down-regulated. After A. hydrophila challenge, the mRNA expressions of three SODs in hepatopancreas and hemocytes were up-regulated and down-regulated in gill. The total SOD activity and CuZnSOD activity were increased compared to the control group with both S. eriocheiris and A. hydrophila challenges. The results may indicate that the SODs of P. clarkii have important roles in innate immune responses against S. eriocheiris and A. hydrophila .

Superoxide dismutase (SOD, EC 1.15.1.1) is an enzyme catalyzing the dismutation of the superoxide anion into molecular oxygen and hydrogen peroxide. In the present study, the full length cDNA of extracellular copper–zinc superoxide dismutase (MnECSOD) from Macrobrachium nipponense was cloned (GenBank accession no. JX045662). Analysis of the nucleotide sequence revealed that the MnECSOD cDNA consisted of 817 bp with an open reading frame (ORF) of 597 bp encoding a polypeptide of 198 amino acids with a putative signal peptide of 19 amino acids. The mature protein had a predicted molecular weight of 18.71 kDa with an estimated p I of 4.87. Four copper binding sites, 4 zinc binding sites and 2 cysteines involved in the formation of the disulfide bridge were conserved in the protein. Sequence comparison showed that the MnECSOD protein had 86% and 55% similarity to that of freshwater prawn Macrobrachium rosenbergii and white shrimp Litopenaeus vannamei , respectively. After challenge with Aeromonas hydrophila , the expression of MnECSOD mRNA in the hepatopancreas was increased responsively. After a drastic decrease at 12 h, the expression level increased again by 6.75-fold compared to that in the control group at 48-h post-infection. These results may indicate that MnECSOD was involved in the innate immune responses of M. nipponense .

Spiroplasma is an emerging pathogen in the commercially exploited freshwater crayfish, Procambarus clarkii . In this article, spiroplasma infection of crayfish was confirmed for the first time by polymerase chain reaction (PCR), which gave positive results from all the tissue samples dissected from moribund P. clarkii including gill, hepatopancreas, cardiac muscle and intestinal tract. Histological examination showed systemic inflammatory reactions in the above tissues, with hypertrophied nuclei and phagocytic cell formation, hemocyte aggregation, epithelial cell shedding, vacuolar degeneration and focal necrosis. Tissue alterations were evaluated semi-quantitatively by the degree of tissue change (DTC). DTC values determined for infected tissues were significantly greater than the respective controls, and cardiac muscle was the most severely damaged organ, with highest DTC value. In addition, an in situ hybridization (ISH) based on oligonucleotide probes to detect the pathogen directly in the tissue, allowing pathogen–lesion correlation, was established. With the advantages of better tissue penetration, potentially more specific and stable, two oligonucleotide probes were designed. Positive reactions to the probes were observed within tissues where PCR and histopathology analysis had previously suggested the presence of the causative agent within these lesions. ISH revealed that hemocytes and connective tissues were the two main targets of spiroplasma in P. clarkii . Results in this article could facilitate the study of spiroplasma infection mechanism in crayfish, and enhance the early diagnosis of the novel pathogen.

Tremor disease (TD) has been found to be a lethal epidemic in Chinese mitten crab during recent years. A new species of spiroplasma, Spiroplasma eriocheiris (S. eriocheiris), was identified as the pathogen causing TD. In order to acquire appropriate biological tools for characterizing this newly discovered pathogen, we developed a new S. eriocheiris specific monoclonal antibody (mAb) 6H7. The antibody showed high binding affinity to S. eriocheiris (K(a)=6.25×10(8) M(-1)) and it bound to the adhesin-like protein (ALP41) of S. eriocheiris in Western blot analysis. Screening of a commercially available 12-mer linear peptide library by using 6H7 as bait led to isolation of a consensus sequence (FQGINHYNQMER). The clone displaying this sequence exhibited a significant, dose-dependent binding to 6H7 and inhibited the binding of S. eriocheiris to 6H7, suggesting a similar epitope between the sequence and S. eriocheiris. Homology searches and multiple sequence alignments indicated moderate homology between the consensus peptides and the ALP of S. eriocheiris. Thus, an internal image of 6H7 binding epitope might be buried in ALP41 of S. eriocheiris. In conclusion, 6H7 is a promising mAb to identify S. eriocheiris and the consensus sequence can be used in future research on the characterization of S. eriocheiris and its pathogenesis.

Spiroplasma eriocheiris is known to cause tremor disease in the Chinese mitten crab Eriocheir sinensis; however, the molecular characterization of this pathogen is still unclear. S. eriocheiris has the ability to invade and survive within mouse 3T6 cells. The invasion process may require causing damage to the host cell membrane by chemical, physical or enzymatic means. In this study, we systematically characterized a novel lysophospholipase (lysoPL) of S. eriocheiris TDA-040725-5T. The gene that encodes lysoPL in S. eriocheiris (SE-LysoPL) was cloned, sequenced and expressed in Escherichia coli BL21 (DE3). Enzymatic assays revealed that the purified recombinant SE-LysoPL hydrolysed long-chain acyl esterases at pH 7 and 30 °C. SE-LysoPL was detected in the membrane and cytoplasmic protein fractions using the SE-LysoPL antibody in Western blot. The virulence ability of S. eriocheiris was effectively reduced at the early stage of infection (m.o.i.=100) by the SE-LysoPL antibody neutralization test. To the best of our knowledge, this is the first study to identify and characterize a gene from S. eriocheiris encoding a protein exhibiting lysoPL and esterase activities. Our findings indicate that SE-LysoPL plays important roles in the pathogenicity of S. eriocheiris.

A microsporidian parasite, Hepatospora eriocheir, is an emerging pathogen for the Chinese mitten crab Eriocheir sinensis. Currently, there is scant information about the way it transmits infection in the crustacean of commercial importance, including its pathogenesis, propagation and infection route in vivo. In this study, chromogenic in situ hybridization (ISH) and quantitative real-time PCR (qPCR) assays were developed to address this pressing need, and we provided an advance in the detection methods available. Pathogens can be seen in situ with associated lesions using ISH. Positive hybridization signals were noted inside the epithelial cells of the hepatopancreas, and putative free parasite spores were observed within the tubule lumen, which were associated with lesions detected by electron microscopy and haematoxylin and eosin (H&E) analysis. qPCR allows the determination of parasite loads in infected tissues, which is important for understanding disease progression and transmission. The hepatopancreas displayed the biggest statistical copy numbers among different tissues of infected crabs, confirming a tissue-specific pathogen infection characteristic. The qPCR assay also proved to be suitable for the diagnosis of asymptomatic carrier crabs. Combination of the two methods could facilitate the study of H. eriocheir infection mechanism in E. sinensis, enhance the early diagnosis of the pathogen and improve the management of microsporidian diseases in commercial crustaceans.

Post-translational modifications such as acetylation are an essential regulatory mechanism of protein function. Spiroplasma eriocheiris, with no cell wall and a helical structure, is a novel pathogen of freshwater crustacean. There is no other evidence of acylation (such as succinylation and propionylation) except acetylation genes in S. eriocheiris concise genome. So the acetylation may play an important role in S. eriocheiris. Here, we conducted the first lysine acetylome in S. eriocheiris. We identified 2567 lysine acetylation sites in 555 proteins, which account for 44.69% of the total proteins in this bacterium. To date, this is the highest ratio of acetylated proteins that have been identified in bacteria. Fifteen types of acetylated peptide sequence motifs were revealed from the acetylome. Forty-five lysine-acetylated proteins showed homology with acetylated proteins previously identified from Escherichia coli, Vibrio parahemolyticus and Mycobacterium tuberculosis. Notably, most proteins in glycolysis and all proteins in the arginine deiminase system were acetylated. Meanwhile, the cell skeleton proteins (Fibril and Mrebs) were all acetylated the observed acetylation also played an important role in cell skeleton formation. The results imply previously unreported hidden layers of post-translational regulation in lysine acetylation that define the functional state of Spiroplasma. Biological significance This is the first time to analyze PTM of Spiroplasma. This is the highest ratio of acetylated proteins that have been identified in bacteria. S. eriocheiris lysine acetylome reveals acetylated proteins related to metabolism and helical structure. These data provide an important resource to elucidate the role of acetylation in Spiroplasma cellular physiology.

Global warming and increased nutrient fluxes cause cyanobacterial blooms in freshwater ecosystems. These phenomena have increased the concern for human health and ecosystem services. The mass occurrences of toxic cyanobacteria strongly affect freshwater zooplankton communities, especially the unselective filter feeder Daphnia. However, the molecular mechanisms of cyanobacterial toxicity remain poorly understood. This study is the first to combine the established body growth rate (BGR), which is an indicator of life-history fitness, with differential peptide labeling (iTRAQ)-based proteomics in Daphnia magna influenced by microcystin-producing (MP) and microcystin-free (MF) Microcystis aeruginosa. A significant decrease in BGR was detected when D. magna was exposed to MP or MF M. aeruginosa. Conducting iTRAQ proteomic analyses, we successfully identified and quantified 211 proteins with significant changes in expression. A cluster of orthologous groups revealed that M. aeruginosa-affected differential proteins were strongly associated with lipid, carbohydrate, amino acid, and energy metabolism. These parameters could potentially explain the reduced fitness based on the cost of the substance metabolism.

Flow cytometry provides rapid and reproducible methods for analyzing crustacean cellular immune responses to pathogens. We used this method to investigate the hemocytes sub-populations of freshwater prawn Macrobrachium rosenbergii and their immune responses to a novel pathogen spiroplasma MR-1008. M. rosenbergii inoculated with 100 μl spiroplasma strain MR-1008 in logarithmic phase (10(8) spiroplasmas ml(-1)) were examined for total hemocytes count (THC) and changes in differential involvement of hemocytes sub-populations during 1-28 d after inoculation. The results showed that THC was dramatically lowered 1 d after inoculation, and it obviously increased at the 5 d after inoculation; thereafter, a high level of THC was maintained to 15 d. Three morphologically distinct hemocytes sub-populations including granular cells (GC), semigranular cells (SGC) and hyaline cells (HC) could be identified by flow cytometry, and the proportions of the 3 kinds of cell categories varied obviously during the infection of spiroplasma suggesting differential involvement according to the pathogen. The flow cytometry used in this study confirmed that the semigranular cells were the main hemocytes involved in the cellular defense against spiroplasma in the M. rosenbergii.

Hemocytes from the red swap crayfish Procambarus clarkii were classified into three subpopulations: hyalinocytes (H), semigranulocytes (SG) and granulocytes (G) by morphologic observation and flow cytometry (FCM). The primary hemocyte culture was then established for studies on the in vitro propagation of Spiroplasma eriocheiris isolated from naturally infected P. clarkii . Grace's insect medium supplemented with 15% fetal bovine serum (FBS), along with 100 U ml − 1 penicillin, 100 μg ml − 1 streptomycin, with a final pH of 7.2–7.4, incubated at 28 °C, supported the best survival in primary cultures. However, the granulocytes dehisced rapidly in culture medium, potentially impacting the survival of the other cell types. A two-step density gradient centrifugation with Percoll was developed to separate the hemocytes. When cultured separately, hyalinocytes and semigranulocytes maintained higher viability (> 75%) after 16 days of incubation compared with granulocytes, which degraded over 2–6 days. After a challenge with S. eriocheiris , cytopathic effects (CPE) of the cultured hemocytes were observed as early as 48 h post-inoculation, and as the infection progressed, CPE became more apparent, with cell debris and cellular exudates in inoculated cultures. Cell lysis was noticeable within 60 h after challenging. A quantitative real-time RT-PCR was conducted to detect the immune responses during the challenging process at 2 h, 4 h, 8 h, 10 h, 24 h, 48 h and 60 h, respectively. Clear time-dependent expression patterns of the peroxinectin gene (referred as Pcpxin ), recently isolated from the crayfish hemocytes, and heat shock protein 70 (HSP70) gene were observed after S. eriocheiris challenge. The results above should be helpful in promoting research with S. eriocheiris , including elucidation of pathogenesis, host pathogen interaction and the defense mechanisms, and ultimately lead to prevention of this crustacean disease.

The Chinese mitten crab Eriocheir sinensis is one of the most important freshwater aquaculture crustacean species in China. MicroRNAs (miRNAs) are small non-coding RNAs that are important effectors in the intricate host-pathogen interaction network. To increase the repertoire of miRNAs characterized in crustaceans and to examine the relationship between host miRNA expression and pathogen infection, we used the Illumina/Solexa deep sequencing technology to sequence two small RNA libraries prepared from haemocytes of E. sinensis under normal conditions and during infection with Spiroplasma eriocheiris. The high-throughput sequencing resulted in approximately 30,975,151 and 30,826,277 raw reads corresponding to 12,077,088 and 16,271,545 high-quality mappable reads for the normal and infected haemocyte samples, respectively. Bioinformatic analyses identified 735 unique miRNAs, including 36 that are conserved in crustaceans, 134 that are novel to crabs but are present in other arthropods (PN-type), and 565 that are completely new (PC-type). Two hundred twenty-eight unique miRNAs displayed significant differential expression between the normal and infected haemocyte samples (p 0.0001). Of these, 133 (58%) were significantly up-regulated and 95 (42%) were significantly down-regulated upon challenge with S. eriocheiris. Real-time quantitative PCR (RT-qPCR) experiments were preformed for 10 miRNAs of the two samples, and agreement was found between the sequencing and RT-qPCR data. To our knowledge, this is the first report of comprehensive identification of E. sinensis miRNAs and of expression analysis of E. sinensis miRNAs after exposure to S. eriocheiris. Many miRNAs were differentially regulated when exposed to the pathogen, and these findings support the hypothesis that certain miRNAs might be essential in host-pathogen interactions. Our results suggest that elucidation of the molecular mechanisms responsible for miRNA regulation of the host's innate immune system should help with the development of new control strategies to prevent or treat S. eriocheiris infections in crustaceans.

Primary culture of hemocyte from the giant freshwater prawn, Macrobrachium rosenbergii , was established for studies on the in vitro propagation of the novel nature pathogen spiroplasma MR-1008 . In this context, a modified method of hemocyte culture from this crustacean has been standardized by employing Leibovitz-15(L-15) growth medium supplemented with 15% Fetal Bovine Serum (FBS) along with 0.1% glucose, 0.5% NaCl and antibiotics (100 U ml − 1 penicillin, 100 U ml − 1 streptomycin,1 μg ml − 1 amphotericin b) and a suitable pH of 7.20–7.40. In this medium, the hemocytes can be subcultured twice, including significant cell proliferation and viability of up to 12 days. Susceptibility of the hemocytes was investigated by challenging with spiroplasma MR-1008. Cytopathic effects (CPE) were observed as early as 48 h post-inoculation; and as the infection progressed, CPE became more apparent, with cell debris and cellular exudates present in inoculated cultures. Cell lysis was noticeable within 60 h after infection. A quantitative real-time RT-PCR was conducted to detect the immune related gene expression during the infection process at 2, 4, 12, 24, 48, and 60 h, respectively. The expression levels of lipopolysaccharide and β-1,3-glucan-binding protein (LGBP), prophenoloxidase (proPO), α-2-macroglobulin (α2M) and copper/zinc SOD (Cu/Zn-SOD) genes in hemocytes of M. rosenbergii demonstrated that these immune genes were induced by challenge with spiroplasma MR-1008. The primary hemocyte culture can be used to investigate the interaction between host and pathogen in vitro, promote enhanced understanding of spiroplasma MR-1008, and ultimately lead to prevention this crustacean disease.

A series of mass mortalities of the cultured blood clam, Tegillarca granosa, occurred in the Yueqing Bay of China from 2005 to 2009. An obligate intracellular prokaryote, designated as rickettsia-like organism (RLO), was frequently found in the moribund or dead blood clam sample during ultrastructural examination. These organisms were usually round, ellipsoid or occasionally dumbbell-shaped, ranged from approximately 0.28 to 0.71 µm in size and had a trilaminar cell wall. Two reproductive modes of organisms, transverse binary fission and budding, were observed. The organisms were able to form eosinophilic inclusions. Most inclusions were found within epithelial and connective tissues of the mantle, gills and digestive tube. The biological and morphological characteristics indicate that these organisms may belong to the family Rickettsiaceae. RLOs exhibited significant pathogenicity. Cytopathological examinations revealed extensive necrosis and destruction in the infected cell. The degree of tissue destruction was positively related to the number of RLO inclusions in the tissues, and the cytopathological effects were positively related to the number of intracellular RLO. RLOs and their inclusions were discovered throughout different disease areas and in different time periods. The infection intensity of the RLOs was positively correlated with the mortality rate of clams. Therefore, RLO infection might be associated with mass mortalities of cultured blood clams in the Yueqing Bay.

Disease epizootics in freshwater cultured crustaceans, including freshwater prawns (Macrobrachium rosenbergii), gained high attention recently in Jiangsu, China due to intensive development of freshwater aquaculture and their susceptibility to massive mortalities. Morphological observation indicated that the pathogen in diseased M. rosenbergii had a helical morphology and lacked a cell wall. The agent could infect hemocytes and all the connective tissues of the organs such as hepatopancreas, digestive tract, and cardiac muscle. The agent was able to pass through membrane filters with pores 220 nm in diameter and could be cultivated by R2 medium. The pathogenicity of the agent was evaluated by Koch's postulate and the mortalities produced by the pathogen were determined to be 80% through experimental infection. The phylogenetic position was investigated by analysis of the 16S rRNA complete sequence. The result revealed that the pathogen had a close relationship with other freshwater spiroplasmas and was situated in the Mirum clade together with Spiroplasma mirum in a phylogenetic tree. Taken in total, the findings demonstrated that the pathogen isolated from diseased M. rosenbergii was a spiroplasma microorganism. This is the fourth report of a spiroplasma pathogen in commercially exploited crustaceans and indicates that spiroplasma pathogens have spread rapidly in aquatic crustaceans; thus, requiring more substantial research attention.

A motile bacterium, designated strain TDA-040725-5T, was isolated from the haemolymph of a Chinese mitten crab, Eriocheir sinensis, with tremor disease. Based on 16S rRNA gene sequence analysis, the strain was phylogenetically distinct from other spiroplasmas but was closely related to Spiroplasma mirum ATCC 29335T. Cells of strain TDA-040725-5T were variable in length and shape, helical and motile, as determined by phase-contrast light microscopy. Examination by electron microscopy revealed wall-less cells delimited by a single membrane. The strain grew in M1D or R-2 liquid media at 20–40 °C, with optimum growth at 30 °C. Doubling time at the optimal temperature was 24 h. The strain catabolized glucose and hydrolysed arginine but did not hydrolyse urea. The DNA G+C content was 29.7±1 mol%. The genome size was ~1.4–1.6 Mbp. Serological analysis, performed using the deformation test, did not reveal any reciprocal titres ≥320, indicating that strain TDA-040725-5T had minimal cross-reactivity to strains of recognized species of the genus Spiroplasma. Based on this evidence, strain TDA-040725-5T ( = CCTCC M 207170T = DSM 21848T) represents a novel species of the genus Spiroplasma, for which the name Spiroplasma eriocheiris sp. nov. is proposed, belonging to the novel Spiroplasma serological group XLIII.