Hepatopancreatic Necrosis Disease Research Articles

The crosstalk between Toll and AMPK signaling pathways mediates growth inhibition of Eriocheir sinensis under deltamethrin stress

Hepatopancreatic necrosis disease (HPND) broke out in 2015 in the Eriocheir sinensis aquaculture region of Xinghua, Jiangsu Province; however, the specific cause of HPND remains unclear. A correlation was found between HPND outbreak and the use of deltamethrin by farmers. In this study, E. sinensis specimens developed the clinical symptoms of HPND after 93 days of deltamethrin stress. The growth of E. sinensis with HPND was inhibited. Adenosine monophosphate-activated protein kinase (AMPK) is a central regulator of energy homeostasis, and its expression was up-regulated in the intestine of E. sinensis with HPND. Growth inhibitory genes (EsCabut, Es4E-BP, and EsCG6770) were also up-regulated in the intestine of E. sinensis with HPND. The expression levels of EsCabut, Es4E-BP, and EsCG6770 decreased after EsAMPK knockdown. Therefore, AMPK mediated the growth inhibition of E. sinensis with HPND. Further analysis indicated the presence of a crosstalk between the Toll and AMPK signaling pathways in E. sinensis with HPND. Multiple genes in the Toll signaling pathway were upregulated in E. sinensis under 93 days of deltamethrin stress. EsAMPK and its regulated growth inhibition genes were down-regulated after the knockdown of genes in the Toll pathway. In summary, the crosstalk between the Toll and AMPK signaling pathways mediates the growth inhibition of E. sinensis under deltamethrin stress.

Integrated pathological, proteomic and metabolomic analyses reveal significant changes of Eriocheir sinensis hepatopancreatic in response to the microsporidian Hepatospora eriocheir infection

Infection by Hepatospora eriocheir would cause serious disease, hepatopancreatic necrosis disease (HPND), and lead to significant economic losses in Eriocheir sinensis. In this study, the response of E. sinensis during H. eriocheir infection was explored by PCR detection, physiological determination (Masson staining and Calcofluor White staining), electron microscopy, and integrated proteome and metabolome analyses. Pathological and electron microscopy analysis demonstrated that H. eriocheir infection could severely damage the E. sinensis hepatopancreas tissue collagen fibers, muscle fibers, and the cells ultrastructure. Base on the PCR detection and Calcofluor White staining results selected the H. eriocheir positive or negative E. sinensis hepatopancreas used to proteome and metabolome analyses. Using a 2-fold change as a physiologically significant benchmark, total of 370 significantly changed proteins (225 up-regulated proteins and 145 down-regulated proteins) and 386 significantly different metabolites (217 metabolites up-regulated and 169 metabolites down-regulated) in crab hepatopancreas during H. eriocheir infection were identified by proteomic and metabolomic analysis, respectively. Biological analysis of these significantly changed proteins and metabolites showed that the lipid metabolism (for example, glycerophospholipid metabolism, carnitine metabolism, lysolecithin metabolism, biosynthesis of unsaturated fatty acids, sphingolipid metabolism, and linoleic acid metabolism) and energy metabolism (such as tricarboxylic acid cycle (TCA cycle), oxidative phosphorylation, and ATP-binding cassette transporter (ABC transporters)) of E. sinensis hepatopancreas were disproportionally affected by H. eriocheir infection. Meanwhile, several key immune-related processes, proteins or metabolites were activated to against the H. eriocheir infection, for example apoptosis, phagocytosis, oxidative stress, C-type lection, β-1, 3-glucan binding protein, vitellogenin receptor, arginine, leucine, aspartic acid, lysine. This study could provide an insightful view into the response mechanism in E. sinensis hepatopancreas during H. eriocheir infection.

Inhibited expression of amylases and cellulases in crabs with hepatopancreatic necrosis disease and their regulation by EcR and HR38

Hepatopancreatic necrosis disease (HPND) is a serious threat to the stable and sustainable development of the Chinese mitten crab farming industry. Studies have found that the hepatopancreas is the main lesion organ in HPND crabs, capable of causing metabolic abnormalities and non-feeding (starvation state). Here, four amylases (named as EsAmy1-4) and four cellulases (endo-beta-1, 4-glucanase, named as EsGlu1-4) that relate to carbohydrate metabolism were cloned from Eriocheir sinensis. Our studies found that the expressions of EsAmy1-4 and EsGlu1-4 were significantly decreased in the hepatopancreas of crabs with HPND. Moreover, the expressions of two important regulatory factors of carbohydrate metabolism, hormone receptor 38 (EsHR38) and ecdysone receptor (EsEcR), were remarkably decreased in the hepatopancreas of crabs with HPND. Further studies found that knockdown of EsHR38 and EsEcR could down-regulated the expressions of EsAmy1-4 and EsGlu1-4. In addition, downregulation of EsEcR could decrease the expression of EsHR38. Hence, HPND causes down-regulation of EsEcR expression, inhibits the transcription of EsHR38, further inhibits the expression of EsAmy1-4 and EsGlu1-4, and then leads to the abnormal carbohydrate metabolism. The abnormalities of carbohydrate metabolism may result in the abnormal food intake in crabs. This study provides new evidence for further understanding the mechanism of HPND.

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.

Rapid detection of Penaeus vannamei diseases via an improved LeNet

Shrimp disease is a greatly important factor in the culture of Penaeus vannamei, the shrimp species with the highest yield in the world aquaculture industry. Hepatopancreatic necrosis disease (HPND, 37 %), red body disease (RBD, 26 %), and whitish muscle disease (WMD, 18 %) were the most common Penaeus vannamei diseases, all of which are usually classified and identified by two kinds of detection (manual detection and germs purifying method). Most of these detections suffer from the class low accuracy, too complex, or too costly. In this study, we tackle this situation with an improved LeNet, which includes modifying model parameters and computational methods. More particularly, this study proposes a convolutional neural networks (CNN) model that is based on LeNet network framework and can reduce parameters and accelerate calculation. To offer improvements in classification and identification, we increase the number of feature maps. Meanwhile, to firstly take denoise and then strengthen characteristic in pretreatment, HSV color space conversion and Gaussian noise with a level of 20 are led into. We conclude that the model generates the precision at about 96.1 % when the weight parameter learning rate is 0.002 and the number of iterations is 120 after being trained and validated. The study has made tremendous progress in the rapid detection of Penaeus vannamei diseases by providing an effective technological path and suggesting the possibility of realizing early disease warnings in future works.

Integrative Analysis of Transcriptome and Metabolome Reveals Molecular Responses in Eriocheir sinensis with Hepatopancreatic Necrosis Disease.

Simple SummaryThere are still many doubts about the etiology and pathogenic mechanisms of Chinese mitten crab hepatopancreatic necrosis disease (HPND). In the present study, new insights into the molecular responses of diseased crabs were found by integrating transcriptomics and metabolomics. The results show that the immune system, metabolic capacity, antioxidant system, detoxification capacity, and nervous system of HPND-affected crabs were impacted, and these changes may be related to the pathological mechanism of HPND. Furthermore, we suspect that functional hypoxia may be involved in the progression of HPND, which provides new insights into the pathogenic mechanism of HPND. In conclusion, based on our findings, we further confirmed that HPND could be mainly caused by abiotic factors.Hepatopancreatic necrosis disease (HPND) is a highly lethal disease that first emerged in 2015 in Jiangsu Province, China. So far, most researchers believe that this disease is caused by abiotic factors. However, its true pathogenic mechanism remains unknown. In this study, the effects of HPND on the metabolism and other biological indicators of the Chinese mitten crab (Eriocheir sinensis) were evaluated by integrating transcriptomics and metabolomics. Our findings demonstrate that the innate immunity, antioxidant activity, detoxification ability, and nervous system of the diseased crabs were affected. Additionally, metabolic pathways such as lipid metabolism, nucleotide metabolism, and protein metabolism were dysregulated, and energy production was slightly increased. Moreover, the IL-17 signaling pathway was activated and high levels of autophagy and apoptosis occurred in diseased crabs, which may be related to hepatopancreas damage. The abnormal mitochondrial function and possible anaerobic metabolism observed in our study suggested that functional hypoxia may be involved in HPND progression. Furthermore, the activities of carboxylesterase and acetylcholinesterase were significantly inhibited, indicating that the diseased crabs were likely stressed by pesticides such as pyrethroids. Collectively, our findings provide new insights into the molecular mechanisms altered in diseased crabs, as well as the etiology and pathogenic mechanisms of HPND.

16S rRNA gene sequencing analysis reveals an imbalance in the intestinal flora of Eriocheir sinensis with hepatopancreatic necrosis disease

Hepatopancreas necrosis disease (HPND) is a highly fatal disease that first appeared in Jiangsu Province, China, in 2015, and later spread to many other provinces, which had a severe impact on the culture of Chinese mitten crab (Eriocheir sinensis). Here, changes in the intestinal flora of healthy and HPND-affected Chinese mitten crabs were compared via 16S rRNA sequencing. Our findings indicated that Firmicutes, Bacteroidota, and Proteobacteria were the three dominant phyla in both healthy and HPND-affected crabs and exhibited no significant differences in α-diversity (richness p = 0.0892; evenness and diversity p = 0.0630). Furthermore, there were no significant changes in the abundance of Proteobacteria between the experimental groups. However, the abundance of Bacteroidota in the HPND group was significantly higher than that of the control group (HPND: 30.12%, Control: 16.60%), whereas the abundance of Firmicutes was significantly lower (HPND: 29.90%, Control: 50.55%). At the genus level, the abundance of Candidatus Bacilloplasma, Desulfovibrio, Bacteroides, and Aeromonas also differed significantly between groups (P < 0.05). Collectively, our study confirms an imbalance in the gut microbiota of Chinese mitten crabs with HPND and we speculate that this alteration may affect the metabolism and immune function of these organisms. Furthermore, we suspect that the structural changes in the intestinal flora of sick crabs observed in our study may be related to HPND.

The forkhead box O transcription factor regulates lipase and anti-microbial peptide expressions to promote lipid catabolism and improve innate immunity in the Eriocheir sinensis with hepatopancreatic necrosis disease

Eriocheir sinensis is a crustacean with great economic value, but the occurrence of hepatopancreatic necrosis disease (HPND) severely restricts the development of crab aquaculture. Study on the survival mechanism of crabs with HPND is beneficial to provide new strategies for disease prevention and control. The Forkhead box O (FOXO) transcription factor family is involved in various key biological processes of organisms. In this study, a FOXO gene (named as EsFOXO) from E. sinensis was cloned. The full-length cDNA of EsFOXO is 2592 bp containing a 2133 bp open reading frame that encodes 710 amino acids. EsFOXO was widely distributed in multiple immune tissues. Further study found that the expression of EsFOXO in the intestine of crabs with HPND was significantly upregulated compared with that in the normal crabs. However, whether EsFOXO is involved in the immune and metabolic regulation of crabs remains unknown. RNA interference analysis showed that EsFOXO participates in the positive regulation of the expression of two pancreatic lipases, three anti-lipopolysaccharide factors, and three crustins. Results from our research suggest that two strategies are adopted by crabs with HPND for survival under starvation: on the one hand, the synthesis of antimicrobial peptides is increased to improve the innate immunity; on the other hand, the expression of enzymes correlated with lipid catabolism is up-regulated that mobilizes the fat in the crab, going through catabolism. Our study provides more evidence for an in-depth understanding of the survival mechanism of crabs with HPND.

Toll-crustin signaling pathway is activated in the Eriocheir sinensis with hepatopancreatic necrosis disease (HPND)

The occurrence of hepatopancreatic necrosis disease (HPND) puts pressure on the healthy aquaculture and development of Chinese mitten crab, Eriocheir sinensis. Research on the mechanism of innate immunity regulation in crabs with HPND is limited. We studied the effects of HPND on the expression of Toll receptor and crustin gene, as well as the relationship between Toll and the synthesis of crustin in E. sinensis. The full-length cDNA and genome structure of six Tolls (EsToll1–6) and five crustins (EsCru1–5) were acquired by gene cloning. The genome of EsToll1–6 can be divided into three types, namely, typical, intronless, and vertebrate-like Tolls. Predicted protein domains showed that all the Tolls and crustins contained the conserved Toll-interleukin1-resistance (TIR) and whey acidic protein (WAP) domain, respectively. Evolutionary analysis results displayed that EsToll1–6 and EsCru1–5 clustered with other Toll receptors and crustin proteins from crustaceans, respectively. EsToll3–6 and EsCru3–5 were widely distributed in multiple immune tissues. EsToll1–5 and EsCru1,2,4,5 expressions in the intestine of crabs with HPND significantly increased. The knockdown of EsToll1–6 could evidently decrease the expressions of different crustins that were positively regulated by HPND. The Toll-crustin signaling pathway was activated in diseased crabs, which may be related to the starvation caused by HPND. Crabs in a state of starvation due to HPND activate their innate immune system. This may be a strategy for the body to protect itself against microbial infection. Our study provides new insights for HPND research.

Characterization of vB_VpaP_MGD2, a newly isolated bacteriophage with biocontrol potential against multidrug-resistant Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a major foodborne pathogen and is also pathogenic to shrimp. Due to the emergence of multidrug-resistant V. parahaemolyticus strains, bacteriophages have shown promise as antimicrobial agents that could be used for controlling antibiotic-resistant strains. Here, a V. parahaemolyticus phage, vB_VpaP_MGD2, was isolated from a clam (Meretrix meretrix) and further characterized to evaluate its potential capability for biocontrol. Podophage vB_VpaP_MGD2 had a wide host range and was able to lyse 27 antibiotic-resistant V. parahaemolyticus strains. A one-step growth curve showed that vB_VpaP_MGD2 has a short latent period of 10 min and a large burst size of 244 phages per cell. Phage vB_VpaP_MGD2 was able to tolerate a wide range of temperature (30°C-50°C) and pH (pH 3-pH 10). Two multidrug-resistant strains (SH06 and SA411) were suppressed by treatment with phagevB_VpaP_MGD2 at a multiplicity of infection of 100 for 24 h without apparent regrowth of bacterial populations. The frequency of mutations causing bacteriophage resistance was relatively low (3.1 × 10-6). Phage vB_VpaP_MGD2 has a double-stranded DNA with a genome size of 45,105 bp. Among the 48 open reading frames annotated in the genome, no lysogenic genes or virulence genes were detected. Sequence comparisons suggested that vB_VpaP_MGD2 is a member of a new species in the genus Zindervirus within the subfamily Autographivirinae. This is the first report of a member of the genus Zindervirus that can infect V. parahaemolyticus. These findings suggest that vB_VpaP_MGD2 may be a candidate biocontrol agent against early mortality syndrome/acute hepatopancreatic necrosis disease (EMS/AHPND) caused by multidrug-resistant V. parahaemolyticus in shrimp production.

Hepatopancreatic necrosis disease (HPND) in Chinese mitten crab Eriocheir sinensis tightly linked to low concentration of two insecticides

Hepatopancreatic necrosis disease (HPND) in Chinese mitten crab <i>Eriocheir sinensis</i> tightly linked to low concentration of two insecticides

Transcriptome analysis of hepatopancreas of Eriocheir sinensis with hepatopancreatic necrosis disease (HPND).

Hepatopancreatic necrosis disease (HPND) is a newly emerging disease in the Chinese mitten crab, Eriocheir sinensis, which has resulted in large economic losses. However, the underlying cause of this disease remains unclear. To better understand the pathogenesis and pathogenic mechanism of HPND, we compared the transcriptome differences of the hepatopancreas of E. sinensis with and without HPND. The analysis yielded > 30 million reads for each sample of three test (with HPND) and three control groups (without HPND). We observed 978 downregulated genes and 644 upregulated genes. Among the gene ontology categories “biological process,” “cellular component,” and “molecular function”, the subcategories cellular process, single-organism process, biological regulation, metabolic process, cell part, organelle, organelle part, binding, and catalytic were enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that “metabolism of xenobiotics by cytochrome P450,” “drug metabolism—cytochrome P450,” “chemical carcinogenesis,” and “material metabolism” were the “five” most significantly enriched pathways in the hepatopancreas of E. sinensis with HPND. The results revealed that material metabolic abnormalities and drug effects from the external environment might be associated with HPND in the Chinese mitten crab. Considering the wide use of pyrethroids for pond cleaning in Xinghua city, we speculated that pyrethroids might cause HPND in the Chinese mitten crab. Our study provided useful information about the cause and pathogenetic mechanisms of HPND and could help to prevent this disease in production practice.

Transcriptomic analysis reveals that hepatopancreatic necrosis disease in Eriocheir sinensis (Chinese mitten crabs) may be the result of autophagy and apoptosis

Massive economic losses have been caused by Hepatopancreas necrosis disease (HPND) to the Chinese mitten crab (Eriocheir sinensis) culture industry; however, the actual cause and pathological mechanisms is unknown. In this study, hepatopancreatic and intestinal cells of the diseased crabs showed distinctive pathological changes which were characteristic of autophagy and apoptosis, but no pathogens were observed. To understand the molecular pathogenesis, a transcriptomic analysis was conducted; approximately 21,523 (26,224) unigenes had COG (KEGG) annotations. The analysis of differentially expressed genes (DEGs) with fold changes >2 and P < 0.05 between diseased crabs with mild symptoms (XHAF-1) and diseased crabs with severe symptoms (XHAF-2), and crabs without visible symptoms (XHAF-CK) in hepatopancreas showed that DEGs increased with the degree of hepatopancreatic necrosis. The DEGs of XHAF-1 vs XHAF-CK (XHAF-2 vs XHAF-CK) were enriched into 1191 (1366) gene ontology (GO) terms, including the positive regulation of phagocytosis, autophagic vacuole assembly and negative regulation of glial cell apoptotic processes. The DEGs of XHAF-1 vs XHAF-CK (XHAF-2 vs XHAF-CK) were enriched into 32 (45) KEGG pathways including autophagy and the p53 signaling pathway. Furthermore, the expression levels of genes related to autophagy and apoptosis were altered in diseased crabs, suggesting that HPND may be the result of autophagy and apoptosis. Moreover, expression of genes related to retinol metabolism disregulated, implying that the hepatopancreas of crabs with HPND turn from golden yellow/light yellow to almost white were associated with retinol metabolism dysregulation. These findings provide new clues in understanding of the etiology and pathogenesis of HPND.

Comparative LC-MS based non-targeted metabolite profiling of the Chinese mitten crab Eriocheir sinensis suffering from hepatopancreatic necrosis disease (HPND)

Hepatopancreatic necrosis disease (HPND) is a newly emerged disease in the Chinese mitten crab, and has caused significant economic damage to crab farmers. However, the exact pathogenesis of crab HPND has not been elucidated. To understand the metabolic shifts in the pathogenesis of the disease, we used LC-MS for metabolite profiling of the hepatopancreas of crabs with HPND. Diseased (DC) and healthy crabs (HC) from the ponds with disease occurrence, and healthy crabs (HHC) from adjacent ponds free from HPND, were analyzed. Histopathologic characteristics and potential pathogens in the hepatopancreas of the healthy and diseased crabs were preliminary investigated. Cellular damage or necrosis, including cell swelling, rupturing, vacuole formation and nuclear fragmentation were observed in diseased crabs. Pathogen screening revealed that, Hepatospora eriocheir, a parasite which was ever deemed to be the agent of the disease before, was unlikely the cause of HPND. Partial least squares discriminant analysis (PLS-DA) score plots revealed a significant metabolic difference between DC and HC in positive ionization modes (R2 = 0.96807, Q2 = 0.74558), while HC and HHC were grouped closely together. Fourty-five differential metabolites were identified and used for further functional pathway analyses. Two potential pathogenic factors, including fatty acid metabolic abnormalities and high concentrations of propamocarb (a widely used pesticide in vegetables), were found to be likely associated with HPND in the Chinese mitten crab. The identified metabolites and regulation pathways, and screening of potential pathogens in diseased crabs, should provide useful information for the prevention of the disease in the future.

Screening of pathogens from a biosecured pacific white shrimp (Penaeus vannamei) farm in Kattur, Tamil Nadu

Screening for presence of various pathogens were carried out in one of the biosecurity maintained shrimp farms in Kattur Village of Tamil Nadu. Sampled shrimps exhibited pinkish discolouration of the body, pale hepatopancreas and antennal cut indicating bacterial infection. Similarly, there was also large scale size variation and reduced growth compared to the days of culture. Samples were screened for bacterial and four (including three OIE-listed) viral pathogens. The samples were also subjected to histopathological investigations. Based on the biochemical tests, the isolated bacteria was identified as Vibrio parahaemolyticus. Metalloprotease gene-specific PCR further confirmed the isolates to be V. parahaemolyticus. PCR was carried out to further investigate the early mortality syndrome/acute hepatopancreatic necrosis disease (EMS/AHPND) strain status of these isolates and were found to be negative. The samples were found to be positive for white spot syndrome virus (WSSV) by second step PCR and infectious hypodermal and haematopoietic necrosis virus (IHHNV) by direct PCR. All these were negative for monodon baculovirus, yellow head virus and gill associated virus. Infection of samples by WSSV and IHHNV were further confirmed by histopathology. The finding of the present study indicated the reduction in growth and size variation due to bacterial infection by V. parahaemolyticus and viral infection by IHHNV. Though bisosecurity was maintained in the farm, the pathogens are suspected to be transmitted through the seed or improper pond preparation.

Pond sludge and increased pH cause early mortality syndrome/acute hepatopancreatic necrosis disease (EMS/AHPND) in cultured white shrimp

Microcosm experiments simulating the occurrence of early mortality syndrome/acute hepatopancreatic necrosis disease (EMS/AHPND) in white shrimp production ponds were performed in 30-L aquariums. Healthy white shrimp, Litopenaeus vannamei, were reared in aquariums containing EMS/AHPND-free hatchery or pond water. Raw pond sludge, collected from shrimp ponds where EMS/AHPND had occurred, was added to some test aquariums, while others were treated with sterilized pond sludge. In some aquariums, water pH was increased from 7.5 to 8.8. Microcosms with stable pH (around 7.5) and/or autoclaved sludge served as controls. The combination of raw sludge and increased pH induced EMS/AHPND and killed white shrimp, whereas raw sludge/stable pH and autoclaved sludge/increased pH combinations did not affect healthy shrimp. Thus, EMS/AHPND outbreaks are due not only to the causative agent but also to environmental stresses such as pH fluctuation. These findings contribute to improved management in shrimp production farms.

A Meta-Transcriptomics Survey Reveals Changes in the Microbiota of the Chinese Mitten Crab Eriocheir sinensis Infected with Hepatopancreatic Necrosis Disease.

Infection of the freshwater Chinese mitten crab Eriocheir sinensis with hepatopancreatic necrosis disease (HPND) has been a major problem in the crab-cultivated Chinese Province of Jiangsu since 2015. To explore the etiology of HPND, meta-transcriptomic libraries of the hepatopancreata from crabs with and without HPND were constructed. Comparison analyses showed that there were no statistically significant differences in viral and microsporidial communities in the hepatopancreata of diseased and healthy crabs. Bacteroidetes, Proteobacteria, and Firmicutes were the most dominant bacterial phyla in the hepatopancreata of healthy crabs, with a combined prevalence of 93%. However, a decrease in bacterial diversity and a striking shift in the microbial composition were found in the hepatopancreata of crabs infected with HPND. Tenericutes was the most prevalent bacterial phylum in diseased crabs (31.82%), whereas its prevalence was low in healthy crabs (0.02%). By contrast, the prevalence of Bacteroidetes was significantly lower in crabs with HPND (3.49%) than in crabs without HPND (41.04%). We also found that the prevalence of Actinobacteria was higher in crabs with HPND (16.70%) than in crabs without the disease (4.03%). The major bacterial family within the Tenericutes phylum in crabs with HPND was detected by polymerase chain reaction and determined to be Mycoplasmataceae. In conclusion, there were striking changes in the microbiota of diseased and healthy crabs. Specifically, the prevalence of bacteria belonging to Tenericutes and Actinobacteria phyla increased, whereas the prevalence of bacteria belonging to the Bacteroidetes phylum decreased in crabs with HPND, clearly pointing to an association with HPND.

Impact of farm management on expression of early mortality syndrome/acute hepatopancreatic necrosis disease (EMS/AHPND) on penaeid shrimp farms in Thailand

Asian shrimp farming industry has experienced massive production losses due to a disease caused by toxins of Vibrio bacteria, known as early mortality syndrome/acute hepatopancreatic necrosis disease (EMS/AHPND) for the last 5years. The disease can cause up to 100% cumulative pond mortality within a week. The objective of this study was to identify factors associated with AHPND occurrence on shrimp farms. A case-control study was carried out on shrimp farms in four provinces of Thailand. Factors related to farm characteristics, farm management, pond and water preparation, feed management, post-larvae (PL) shrimp and stock management were evaluated. Multivariable logistic regression analysis identified factors affecting AHPND occurrence at the pond level. Chlorine treatment, reservoir availability, use of predator fish in the water preparation, culture of multiple shrimp species in one farm and increased PL stocking density contributed to an increased risk of AHPND infection, while delayed first day of feeding, polyculture and water ageing were likely to promote outbreak protection. Additionally, the source of PL was found to be associated with AHPND occurrence in shrimp ponds, which requires further study at the hatchery level. Identification of these factors will facilitate the development of effective control strategies for AHPND on shrimp farms.

A novel method of real-time reverse-transcription loop-mediated isothermal amplification developed for rapid and quantitative detection of a new genotype (YHV-8) of yellow head virus

A new genotype of yellow head virus (YHV), designated as YHV-8, was found in farmed shrimp Fenneropenaeus chinensis suffering suspectedly from EMS/AHPNS (early mortality disease/acute hepatopancreatic necrosis disease) in China in 2012. In this study, a one-step, real-time reverse-transcription loop-mediated isothermal amplification (rRT-LAMP) assay was developed for better detection of both genotypes of YHV-1 and YHV-8. A set of six specific primers was successfully designed targeting a conserved region of the YHV genome. The LAMP reaction was optimized to contain 8mmoll(-1) Mg(2+) and 1·4mmoll(-1) dNTPs, and to be performed at 58°C for 60min. The detection sensitivity of the rRT-LAMP method was as low as 7×10(0) copies per reaction. The specificity of the method was validated by the absence of any cross-reaction with the RNA samples extracted from other shrimp viruses (Taura syndrome virus, white spot syndrome virus, infectious hypodermal and haematopoietic necrosis virus, hepatopancreatic parvovirus) and specific pathogen-free (SPF) shrimp. The resulting standard curves showed high correlation coefficient values. Furthermore, the test of farm samples showed that YHV was detected in three of 111 Litopenaeus vannamei, six of eight Fenneropenaeus chinensis, five of 19 Macrobrachium rosenbergii and none of the nine Marsupenaeus japonicus. These results suggest that this assay is applicable widely as a new rapid and sensitive detection method in the research of YHV. In this study, we designate a new genotype of yellow head virus (YHV) as YHV genotype 8 (YHV-8) which was detected in diseased shrimp in China. A rapid, sensitive and specific rRT-LAMP detecting method for both YHV-8 and YHV-1 has been established. It is anticipated that this novel assay will be instrumental for diagnosis and surveillance of the virulent genotypes of YHV.

First case of hepatopancreatic necrosis disease in pond‐reared Chinese mitten crab, Eriocheir sinensis, associated with microsporidian

An epidemic of hepatopancreatic necrosis disease (HPND) with a high mortality rate (40%-50%) recently occurred in the cultured Chinese mitten crab, Eriocheir sinensis, which is a very important economic crustacean species in China. Histology revealed infection by a microsporidian parasite within the cytoplasm of the epithelial cells of the hepatopancreas. Numerous discrete inclusions in the infected cells and presumably free parasite spores were also observed. By negative staining using electron microscopy, a typical morphology of spores was observed with a protuberant front of the anchoring disc. Infection was confined to the epithelial cells of the hepatopancreas, with no other organ implicated. By sequencing the PCR products using specific primers based on conserved regions of microsporidian small subunit (18S) ribosomal DNA, it was revealed that the parasite from HPND ponds had 99% sequence identity to that of Hepatospora eriocheir. Phylogentic analysis also placed the microsporidian in the same lineage as H.eriocheir. This study reported the first case of widespread infections of H.eriocheir associated with HPND found in the pond-reared Chinese mitten crab, E.sinensis. The description of microsporidian in this important commercial host is fundamental for future consideration of factors affecting stock health and sustainability.