Isolation and the pathogenicity characterization of Decapod iridescent virus 1 (DIV1) from diseased Macrobrachium nipponense and its activation on host immune response.

The high morbidity and mortality of Macrobrachium nipponense occurred in several farms in China, with cardinal symptoms of slow swimming, loss of appetite, empty of intestine, reddening of the hepatopancreas and gills. The pathogen has been confirmed as Decapod Iridescent Virus 1 (DIV1), namely DIV1-mn, by molecular epidemiology, histopathological examination, TEM observation, challenge experiment, and viral load detection. Histopathological analysis showed severe damage in hepatopancreas and gills of diseased prawns, exhibited few eosinophilic inclusions and pyknosis, and TEM of diseased prawns revealed that icosahedral virus particles existed in hepatopancreas and gill, which confirmed the disease of the farmed prawns caused by the DIV1 infection. Besides, challenge tests showed LD50 of DIV1 to M. nipponense was determined to be 2.14 × 104 copies/mL, and real-time PCR revealed that M. nipponense had a very high DIV1 load in the hemocytes, gills and hepatopancreas after infection. Furthermore, qRT-PCR was undertaken to investigated the expression of six immune-related genes in DIV1-infected M. nipponense after different time points, and the results revealed UCHL3, Relish, Gly-Cru2, CTL, MyD88 and Hemocyanin were significantly up-regulated in hemocytes, gills and hepatopancreas, which revealed various expression patterns in response to DIV1 infection. This study revealed that DIV1 infection is responsible for the mass mortality of M. nipponense, one of the important crustacean species, indicating its high susceptibility to DIV1. Moreover, this study will contribute to exploring the interaction between the host and DIV1 infection, specifically in terms of understanding how M. nipponense recognizes and eliminates the invading of DIV1.

rpoS involved in immune response of Macrobrachium nipponens to Vibrio mimicus infection.

Vibrio mimicus is a pathogenic bacterium that cause red body disease in Macrobrachium nipponense, leading to high mortality and financial loss. Based on previous studies, rpoS gene contribute to bacterial pathogenicity during infection, but the role of RpoS involved in the immune response of M. nipponense under V. mimicus infection remains unclear. In this study, the pathogen load and the RNA-seq of M. nipponense under wild-type and ΔrpoS strain V. mimicus infection were investigated. Over the entire infection period, the ΔrpoS strain pathogen load was always lower than that of the wild-type strain in the M. nipponense hemolymph, hepatopancreas, gill and muscle. Furthermore, the expression level of rpoS gene in the hepatopancreas was the highest at 24 hours post infection (hpi), then the samples of hepatopancreas tissue infected with the wild type and ΔrpoS strain at 24 hpi were selected for RNA-seq sequencing. The results revealed a significant change in the transcriptomes of the hepatopancreases infected with ΔrpoS strain. In contrast to the wild-type infected group, the ΔrpoS strain infected group exhibited differentially expressed genes (DEGs) enriched in 181 KEGG pathways at 24 hpi. Among these pathways, 8 immune system-related pathways were enriched, including ECM-receptor interaction, PI3K-Akt signaling pathway, Rap1 signaling pathway, Gap junction, and Focal adhesion, etc. Among these pathways, up-regulated genes related to Kazal-type serine protease inhibitors, S-antigen protein, copper zinc superoxide dismutase, tight junction protein, etc. were enriched. This study elucidates that rpoS can affect tissue bacterial load and immune-related pathways, thereby impacting the survival rate of M. nipponense under V. mimicus infection. These findings validate the potential of rpoS as a promising target for the development of a live attenuated vaccine against V. mimicus.

Effects of temperature and CO2 concentration on the early stage nucleation of calcium carbonate by reactive molecular dynamics simulations.

It is significant to investigate the calcium carbonate (CaCO3) precipitation mechanism during the carbon capture process; nevertheless, CaCO3 precipitation is not clearly understood yet. Understanding the carbonation mechanism at the atomic level can contribute to the mineralization capture and utilization of carbon dioxide, as well as the development of new cementitious materials with high-performance. There are many factors, such as temperature and CO2 concentration, that can influence the carbonation reaction. In order to achieve better carbonation efficiency, the reaction conditions of carbonation should be fully verified. Therefore, based on molecular dynamics simulations, this paper investigates the atomic-scale mechanism of carbonation. We investigate the effect of carbonation factors, including temperature and concentration, on the kinetics of carbonation (polymerization rate and activation energy), the early nucleation of calcium carbonate, etc. Then, we analyze the local stresses of atoms to reveal the driving force of early stage carbonate nucleation and the reasons for the evolution of polymerization rate and activation energy. Results show that the higher the calcium concentration or temperature, the higher the polymerization rate of calcium carbonate. In addition, the activation energies of the carbonation reaction increase with the decrease in calcium concentrations.

Pathogenicity of Plesiomonas shigelloides causing mass mortalities of largemouth bass (Micropterus salmoides) and its induced host immune response.

The outbreak of mass mortality of M. salmoides occurred in an aquaculture farm in Jiangsu province of China, showing signs of skin ulceration and haemorrhages. The bacteria were isolated from diseased largemouth bass, and identified as Plesiomonas shigelloides based on morphological, physiological and biochemical features, as well as 16S rRNA gene sequence analysis. The pathogenicity of P. shigelloides was determined by challenge experiments, and the median lethal dosage (LD50) of the isolate NJS1 for M. salmoides was calculated as 1.6 × 105 CFU/mL at 7 d post-infection. Histopathological analysis revealed that extensive necrosis, vacuolization and inflammation were presented in the kidney, liver and gill of the diseased fish. Detection of virulence-related genes showed that P. shigelloides NJS1 was positive for astA, astB, astD, astE, actP and 6 ahpA. Additionally, the host defensive response of M. salmoides infected by P. shigelloides was analyzed by quantitive real-time PCR (qRT-PCR), and the results showed that the expression levels of Cas3, Hep1, HIF, IgM, IL15 and TGF were significantly up-regulated in head kidney, liver and spleen in different hours post-infection, which revealed varying expression profiles and clear transcriptional activation of immune related genes. The results suggested that P. shigelloides was an etiological element in the mass mortalities of M. salmoides and this study provided deeper insights for the pathogenesis and host defensive system in P. shigelloides invasion.

The pathogenic characterization of Citrobacter freundii and its activation on immune related genes in Macrobrachium nipponense.

Out breaks of mass mortalities occurred in Macrobrachium nipponense farms in Jintan county, Jiangsu Province. The bacterial isolates from M. nipponense exhibited the same phenotypic traits and biochemical characteristics, and were identified as Citrobacter freundii according to biochemical characteristics and molecular identification. The infection test revealed that the strain YG2 was pathogenic to M. nipponense, and the half lethal dose (LD50) was 3.35 × 105 CFU/mL at 7 d post-infection. Detection of virulence genes indicated that YG2 was positive for cfa, ureG, ureF, ureE, ureD, viaB, ompX, and LDH. Furthermore, the results of extracellular enzyme analysis revealed that the strain can produce protease, amylase, lecithin, urease, and hemolysin. Antibiotic resistance results showed that the isolate was resistant to ampicillin, cefazolin, cephalothin, cefoxitin, aboren, doxycycline, neomycin, penicillin, erythromycin, and vancomycin. The expression level of MyD88, α2M, CDSP, and Relish were detected in hepatopancreas, hemolymph, gills and intestine tissues by quantitive real-time PCR (qRT-PCR), and clear transcriptional activation of these genes were observed in M. nipponense after C. freundii infection. These results revealed pathogenicity of C. freundii and its activation of host immune response, which will provide a scientific reference for the breeding and disease prevention in M. nipponense culture.

Pathogenicity of Aeromonas veronii causing mass mortalities of Odontobutis potamophila and its induced host immune response.

Aeromonas veronii is a freshwater bacterium associated with many diseases in aquatic animals. However, few cases of A. veronii infection were reported in Odontobutis potamophila, which has been becoming a promising fish species in China in recent years. In this study, the dominant bacteria were isolated from diseased O. potamophila showing signs of hemorrhage on fins, ulceration on the dorsal and abdomen. The representative isolate Stl3-1was identified as A. veronii based on analysis of its morphological, physiological, and biochemical features, as well as 16S rRNA and gyrB gene sequences. The median lethal dosage (LD50) of the isolate Stl3-1 for O. potamophila was determined as 4.5 × 105 CFU/mL. Histopathological analysis revealed that the isolate Stl3-1caused considerable histological lesions in the fish, including tissue cell degeneration, necrosis, and inflammatory cell infiltrating. Detection of virulence-related genes showed that A. veronii Stl3-1 was positive for exu, ompA, lip, flaH, hlyA, aer, flgM, tapA, act, flgA, gcaT and flgN. Additionally, quantitive real-time PCR (qRT-PCR) was also undertaken to analyses the host defensive response in O. potamophila infected by A. veronii. The immune-related gene expressions in O. potamophila during experimental infection were monitored at different point of time, and the results showed that the expression levels of MHC II, Myd88, TLR, and SOD were significantly up-regulated in liver, gill, spleen, and head kidney. The results revealed that A. veronii was a pathogen causing mass mortalities of O. potamophila and will contribute to better understanding the host defensive response against A. veronii infection.

New insights into the early stage nucleation of calcium carbonate gels by reactive molecular dynamics simulations.

The precipitation of calcium carbonate (CaCO3) is a key mechanism in carbon capture applications relying on mineralization. In that regard, Ca-rich cementitious binders offer a unique opportunity to act as a large-scale carbon sink by immobilizing CO2 as calcium carbonate by mineralization. However, the atomistic mechanism of calcium carbonate formation is still not fully understood. Here, we study the atomic scale nucleation mechanism of an early stage amorphous CaCO3 gel based on reactive molecular dynamics (MD) simulations. We observe that reactive MD offers a notably improved description of this reaction as compared to classical MD, which allows us to reveal new insights into the structure of amorphous calcium carbonate gels and formation kinetics thereof.

Aeromonas hydrophila associated with red spot disease in Macrobrachium nipponense and host immune-related gene expression profiles.

In September 2018, a serious disease causing high mortality with red spot syndrome occurred in a Macrobrachium nipponense aquaculture farm in Jintan County, Jiangsu Province, China. In this study, a pathogenic isolate 5-S3 was isolated from diseased M. nipponense and was identified as Aeromonas hydrophila by phenotypically and molecularly. The pathogenicity of the isolate 5-S3 to M. nipponense was determined by challenge experiments. Results of artificial challenge showed A. hydrophila was pathogenic to M. nipponense, the LD50 was 9.58 × 104 CFU/mL, and histopathological analysis revealed that the hepatopancreas of infected M. nipponense exhibited obvious inflammatory responses to A. hydrophila infection. The isolate showed significant phenotypical activities such as the lecithinase, esterase, caseinase and hemolysin which are indicative of their virulence potential. Besides, virulence genes such as aerA, act, fla, ahpß, alt, lip, eprCAI, hlyA, acg and gcaT were detected in the isolate 5-S3. Subsequently, the immune-related genes expression in M. nipponense were evaluated by quantitative real-time PCR (qRT-PCR), and the results showed that the expression levels of dorsal, relish, crustin1, crustin2, anti-lipopolysaccharide factors 1 (ALF1), anti-lipopolysaccharide factors 2 (ALF2), hemocyanin, i-lysozyme and prophenoloxidase were significantly up-regulated in hepatopancreas of M. nipponense after A. hydrophila infection, the stat, p38, crustin3, anti-lipopolysaccharide factors 3 (ALF3) genes had no significant change during the infection. The present results reveal that A. hydrophila was an etiological agent causing red spot syndrome and mass mortality of M. nipponense and the influence of A. hydrophila infection on host immune genes.

Multi-biomarker assessment in the giant freshwater prawn Macrobrachium rosenbergii after deltamethrin exposure.

Deltamethrin (DM) is a synthetic pyrethroid used for agricultural purposes to control insects. However, its extensive use contaminates the aquatic environment and results in serious health problems in aquatic organisms. Knowledge about the toxic effect of DM in freshwater prawns is limited; therefore, this study aims to assess the toxicity of DM in Macrobrachium rosenbergii based on multiple biomarkers. Four-day acute toxicity tests showed that DM was highly toxic to M. rosenbergii with the 24 h, 48 h, 72 h and 96 h LC50 values to be 1.919, 0.603, 0.539, and 0.449 µg/L, respectively. According to 96 h LC50, prawns were exposed to DM at three concentrations (0.02, 0.08, and 0.32 µg/L) for 4 days, and then moved into fresh water for decontamination to investigate the toxic effect of DM in M. rosenbergii. At low concentration (0.02 µg/L and 0.08 µg/L), DM did not cause obvious histopathological damage to hepatopancreas and gill tissue, while at high concentration (0.32 µg/L), the histopathological harm was serious and the damage did not recover to the initial level after 7-day decontamination. 0.02 µg/L DM exposure did not induce significant changes in most of the biomarkers except the increased lactate dehydrogenase (LDH) activity, lactic acid (LD) level, and the first increased then decreased mRNA expression of immune-related genes, indicating the stimulation of DM on energy production and immunity. 0.08 µg/L and 0.32 µg/L DM exposure resulted in varying degrees of damage on prawns, but overall, their toxic effects showed similar trends based on the biomarkers. Increase in malonaldehyde (MDA) and hydrogen peroxide (H2O2) content and decrease in superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity after DM exposure demonstrated the oxidative stress caused by DM. The significantly increased acid phosphatase (ACP), alkaline phosphatase (AKP), LDH activity and LD level indicated hepatopancreatic dysfunction and respiration disruption. The first increased and then decreased expression pattern of immune-related genes indicated the immunosuppression caused by DM. After 7-day decontamination in freshwater, the activity/level of the biomarkers partly recovered. This study revealed the severe toxic effect of DM on Macrobrachium rosenbergii based on multiple biomarkers, providing fundamental knowledge for the establishment of DM toxicity assessment system with proper parameters in freshwater crustaceans.

Macitentan Attenuates Chronic Mountain Sickness in Rats by Regulating Arginine and Purine Metabolism.

Chronic mountain sickness (CMS) is a high altitude complication with high rates of morbidity and mortality. CMS is characterized by high-altitude polycythemia (HAPC) and high-altitude pulmonary hypertension (HAPH). In this study, macitentan, a dual endothelin receptor antagonist, was used to treat CMS, and the induced metabolomics changes were studied. CMS was induced in rats in a hypobaric hypoxia chamber (simulating a 5500 m plateau) for 4 weeks. Macitentan was administered in the third and fourth weeks (30 mg·kg-1·day-1). At the end of the follow-up period, we performed echocardiography, measured hemodynamic parameters and hematocrit, and performed histological staining. Furthermore, ultraperformance liquid chromatography-mass spectrometry (UPLC-MS)-based metabolic analysis was applied to explore metabolic changes associated with hypobaric hypoxia, with or without macitentan. qRT-PCR and kits for the determination of xanthine oxidase (XO) activity were used for validation experiments. Macitentan was effective in attenuating CMS, including CMS-induced right ventricle hypertrophy, HAPC, and HAPH. The levels of 48 metabolites were significantly changed in the CMS model group compared to the control group. Of these changes, 21 were reversed by treatment with macitentan. Enrichment analysis revealed that the purine metabolism pathway, as well as the arginine/proline metabolism pathway, might be the key pathways adjusted by macitentan. Furthermore, we verified macitentan played a beneficial role by directly regulating the expression of arginine1 and arginine2 in the arginine/proline metabolic pathway, and the activity of xanthine oxidase in the purine metabolic pathway. In conclusion, this study demonstrated that macitentan significantly ameliorated CMS in rats, and the mechanism was attributed to the reversion of the disorder in purine and arginine/proline metabolism, via direct regulation of XO activity and arginine1/2 expression. These findings are expected to provide new insights into the therapeutics and mechanism of macitentan in CMS.

Pathogenicity of Aeromonas hydrophila causing mass mortalities of Procambarus clarkia and its induced host immune response.

Outbreaks of mass mortalities among cultured Procambarus clarkia occurred in a commercial hatchery during the spring of 2019 in Jiangsu province of China. Here, we exploit the pathogenicity and immune response of Aeromonas hydrophila (GPC1-2), which was isolated from diseased P. clarkia. Crayfish challenged showed similar pathological signs to the naturally diseased P. clarkia, lethal dose 50% (LD50) of the strain GPC1-2 to P. clarkia was 3.8 × 106 CFU/mL. Detection of virulence-associated genes by PCR indicated that the strain GPC1-2 carried hlyA, aerA, alt, ast, act, aha, ahp, ahpA, and ahpB. Histopathological analysis of hepatopancreas revealed that the hepatic tubule lumen and the gap between the hepatic tubules became larger, and the brush border disappeared in the P. clarkia infected by GPC1-2. Quantitive real-time PCR (qRT-PCR) was undertaken to measure mRNA expression levels for six immune-related genes in P. clarkia after A. hydrophila infection. The expression level of proPO, NOS, ALF1, TLR2, PX, and AST were detected in hemolymph, hepatopancreas, gill and intestine tissues, and clear transcriptional activation of these genes were observed in the infected individuals. These results revealed pathogenicity of A. hydrophila and its activation of host immune response, which will provide a scientific reference for the breeding and disease prevention in P. clarkia culture.

Ursolic Acid Improves Monocrotaline-Induced Right Ventricular Remodeling by Regulating Metabolism.

Pulmonary arterial hypertension (PAH) is a progressive and malignant disease characterized by pulmonary small arteries and right ventricle (RV) remodeling that can lead to severe RV dysfunction and death. The current therapeutic targets for RV dysfunction, which is strongly linked to mortality, are far from adequate. Therefore, we investigated the effect of ursolic acid (UA), a pentacyclic triterpenoid carboxylic acid, on PAH-induced RV remodeling and its underlying mechanism. We established a PAH model by injecting Sprague Dawley rats with monocrotaline (MCT, 60 mg/kg, ip), as verified by echocardiography and hemodynamic examination. Proteomic analysis was performed on RV samples using a Q Exactive high-field mass spectrometer, followed by KEGG enrichment analysis. The effect of 4 weeks of UA (50 mg/kg) treatment on RV remodeling was explored based on ultrasound, hemodynamic parameters, and histological changes, with the mechanism verified in vivo and in vitro by qRT-PCR and western blotting. RV hypertrophy, fibrosis, increased apoptosis, and abnormal metabolism were induced by MCT and suppressed by UA via a mechanism that changed the expression of key markers. UA also attenuated the Phenylephrine-induced hypertrophy of neonatal rat ventricular myocytes and upregulated peroxisome proliferator-activated receptor-alpha (PPARα), a key fatty acid metabolism regulator, and its downstream factor carnitine palmitoyl transferase 1b. In conclusion, UA exerts beneficial effects on PAH-induced RV dysfunction and remodeling by regulating PPARα-dependent fatty acid metabolism.

Impact of Aeromonas hydrophila and infectious spleen and kidney necrosis virus infections on susceptibility and host immune response in Chinese perch (Siniperca chuatsi).

Co-infections with pathogenic microorganisms are common in aquaculture, resulting in more serious economic losses than single-pathogen infection. Infection of Aeromonas hydrophila (A. hydrophila) often occurs together with infectious spleen and kidney necrosis virus (ISKNV) in Chinese perch (Siniperca chuatsi) culture ponds. In this study, A. hydrophila and ISKNV were inoculated into Chinese perch to mimic individual infection, secondary infection, and mixed infection. The effects of concurrent infections on the susceptibility and the immune response of the host and changes in bacterial and viral load were studied. The results showed relatively complex interaction between ISKNV and A. hydrophila for different infection modes, acting in an antagonistic or synergistic manner. The experimental groups infected with a mixture of ISKNV and A. hydrophila showed higher mortality rate than groups infected with single-pathogen or secondary infection groups, suggesting a synergistic lethal effect of A. hydrophila and ISKNV co-infection. Serious clinical symptoms and obvious histopathological changes were observed in moribund fish under the mixed-infection condition. In addition, obviously higher mortalities were caused by secondary bacterial infections than the number caused by secondary viral infections. ISKNV-primary infection increased the mortality caused by secondary bacterial infections, but A. hydrophila-primary infection did not significantly increase the mortality caused by secondary viral infections. Co-infected fish showed high expression levels of IRF1, Mx, Viperin, Hepcidin, TNFα, and IL-1ß mRNAs relative to the levels in healthy fish, which suggested that the co-infection of these two pathogens activated the host immune system and caused host inflammation. These results of infection with A. hydrophila and ISKNV provided the theoretical basis to analyze the pathogenic effects and interaction between pathogens, and could facilitate design of strategies for clinical prevention and control measures of outbreak of fulminant hemorrhagic disease and bacterial sepsis in Chinese perch.

Transcriptome analysis and immune-related genes expression reveals the immune responses of Macrobrachium rosenbergii infected by Enterobacter cloacae.

Macrobrachium rosenbergii is an important cultural species in China and other Southeast Asian countries. However, Enterobacter cloacae infection has caused a great economic loss in M. rosenbergii culture industry. The immune responses of M. rosenbergii to the E. cloacae infection is not fully characterized. To investigate the immune response of M. rosenbergii against E. cloacae, we performed transcriptome analysis of the M. rosenbergii hepatopancreas with and without E. cloacae infection using RNA-seq. After assembly and annotation, 29,731 high quality unigenes were obtained from RNA-seq data. Differential expression analysis revealed the existence of 2498 significantly differently expressed genes (DEGs) at 12 h post infection, with 1365 up-regulated and 1133 down-regulated genes. Among these DEGs, some well-known immune-related genes were up-regulated significantly, including C-type lectin 1, lectin 3, anti-lipopolysaccharide factor 2, Cu/Zn superoxide dismutase and heat shock protein 70. GO analysis demonstrated 24 biological process subcategories, 14 cellular component subcategories, and 12 molecular function subcategories that were enriched among these DEGs, and some DEGs were clustered into immune related subcategories such as immune system process, response to stimulus, biological adhesion, and antioxidant activity. These DEGs were enriched into 216 KEGG pathways including a core set of immune correlated pathways notably in phagosome and lysosome. In addition, 5 up-regulated and 5 down-regulated immune-related DEGs were selected for further validation by quantitative real-time PCR and the results showed consistence with the RNA-seq data. Additionally, the expression level of six selected immune-related genes (ALF2, CLEC1, LEC3, hemocyanin1, HSP70 and SOD) based on the transcriptomic data were monitored at different point of time in hepatopancreas, gill, hemolymph and intestine. Results revealed these immune-related genes were significantly up-regulated in different tissues from 6 to 24 h after E. cloacae infection. Overall, these results provided valuable information for further studying the immune response of M. rosenbergii against E. cloacae infection.

Insights into carbohydrate metabolism from an insulin-like peptide in Macrobrachium rosenbergii.

This study identified an insulin-like peptide (ILP) in Macrobrachium rosenbergii termed Mr-ILP and further investigated its function through glucose injection and RNAi. With the analysis of five other glucose metabolism related genes, this study shed light on the molecular mechanism of carbohydrate metabolism in crustaceans. Mr-ILP shared the typical skeleton with six conserved cysteine and mainly expressed in neuroendocrine system. In M. rosenbergii, the elevated hemolymph glucose concentration after glucose injection returned to basal levels in short time, implying an efficient regulatory system in carbohydrate metabolism. Hyperglycemic related genes answered the elevated hemolymph glucose concentration quickly with significant decreased expression level, while Mr-ILP showed delayed response. Instead, glycolysis increased after glucose injection, which indicated glycolysis might play an important role in lowering the abnormally high glucose level. In vivo silencing of Mr-ILP, by injecting the prawns with double-stranded RNA (dsRNA) for 21 days reduced its expression by approximately 75%. Accordingly, glycogen synthase decreased and the trehalose and glycogen level in the hepatopancreas were significantly reduced, indicating the function of Mr-ILP in oligosaccharide and polysaccharide accumulation. When Mr-ILP was silenced, the expression of hyperglycemic related genes were enhanced, but the hemolymph glucose level was not elevated significantly, which might attribute to the increased glycolysis to keep a balanced glucose level in hemolymph.

Virulence properties of Vibrio vulnificus isolated from diseased zoea of freshness shrimp Macrobrachium rosenbergii.

Macrobrachium rosenbergii is one of the most economically important freshwater shimp, with fast growth and high nutrient content in the agricultural development of China. However, it had been suffering diseases infection, causing mass death and great economic losses. In the present study, a bacteria strain was isolated from the diseased zoea of M. rosenbergii and was identified as Vibrio vulnificus by biochemical characteristics and 16S rRNA homologous analysis. The infection test showed that the strain GXFL1-3 was pathogenic to zoea and postlarva of M. rosenbergii, and the half lethal dose (LD50) were 1.16 × 106 CFU/mL and 1.45 × 106 CFU/mL, respectively. Detection of virulence-associated genes by PCR indicated that GXFL1-3 was positive for fur, OmpU, acfA, flaA, vvhA, vvp and tcp, the detection of extracellular enzymes and hemolysin showed that GXFL1-3 was positive for protease, amylase, lecithin, urease and hemolysin activity, further supporting its pathogenicity. A duplex PCR for rapid detection of V. vulnificus was established. Only V. vulnificus could amplify two specific bands of flaA and fur, while the other six strains of Vibrio were negative. The minimum detectable amount of template was 2.4 × 103 CFU/mL through sensitivity test.

Changes of microRNAs expression profiles from red swamp crayfish (Procambarus clarkia) hemolymph exosomes in response to WSSV infection.

MicroRNAs (miRNAs) as short noncoding RNAs play important regulatory roles in diverse biological processes by degrading the target mRNAs, and could be delivered by exosomes. WSSV is a highly pathogenic and prevalent virus, and has brought high mortality of P. clarkia. Till present, no studies focus on the miRNAs changes in exosomes during WSSV infection. To understand the different virulence of WSSV on miRNAs expression in P. clarkia hemolymph exosome, the deep sequencing was performed to compare the small RNA libraries from the hemolymph exosome of P. clarkia individuals with or without WSSV infections. From the TEM observations, NTA and Western Blot analysis, the extracted exosomes were well identified with classic characteristics. The 209 conserved miRNAs and 250 novel miRNAs were identified from the small RNA libraries. In response to WSSV infection, there were about 98 miRNAs significantly up-regulated and 59 miRNAs significantly down-regulated. The target genes prediction, GO and KEGG enrichment analysis revealed that some target genes of P. clarkia miRNAs were grouped mainly into the categories of biological regulation, immune system process, signal pathway and other more functions. This is the first report of comprehensive identification of P. clarkia hemolymph exosome miRNAs being differentially regulated in response to WSSV infection. These results will help to understand the hemolymph exosome miRNAs response to different virulence WSSV infection.

The pathogenicity characterization of non-O1 Vibrio cholerae and its activation on immune system in freshwater shrimp Macrobrachium nipponense.

Outbreaks of mass mortalities among cultured Macrobrachium nipponense occurred in a commercial hatchery during the autumn of 2017 in Jiangsu province, P. R. China, and non-O1 Vibrio cholerae was isolated and identified as causal agents of M. nipponense, with a LD50 value 4.09 × 104 CFU/mL. Detection of virulence-associated genes by PCR indicated that XL1 was positive for Mp, HlyA, RtxA, OmpU, Ace, Zot and T6SS. Furthermore, the results of extracellular enzyme analysis revealed that the strain can produce lecithinase, amylase, gelatinase and hemolysin. Histopathological analysis revealed that the hepatic tubule lumen and the gap between the hepatic tubules became larger, and the brush border disappeared in the hepatopancreas. Quantitive real-time PCR (qRT-PCR) was undertaken to measure mRNA expression levels for thirteen immune related genes in M. nipponense after non-O1 V. cholerae infection. The transcriptional analysis of these immune related genes demonstrated that the expression levels of dorsal, relish, p38, crustin1, crustin2, crustin3, hemocyanin, i-lysozyme, anti-lipopolysaccharide factors 1, anti-lipopolysaccharide factors 2, prophenoloxidase were significantly up-regulated in hemolymph of M. nipponense post-infection. These results revealed varying expression profiles and clear transcriptional activation of these immune related genes in hemolymph, which will contribute to better understand the pathogenesis and host defensive system in non-O1 V. cholerae invasion.

Pathogenicity of non-O1/ O139 Vibrio cholerae and its induced immune response in Macrobrachium rosenbergii.

Outbreaks of mass mortalities occurred in Macrobrachium rosenbergii farms in Gaoyou county, Jiangsu Province of China. The bacterial isolates from M. rosenbergii exhibited the same phenotypic traits and biochemical characteristics, and were identified as non-O1/O139 Vibrio cholerae according to biochemical characteristics and molecular identification. In challenge test, M. rosenbergii infected with non-O1/O139 V. cholerae GXFL1-4 developed similar pathological signs to the naturally diseased prawns, and LD50 of the strain to M. rosenbergii was 4.5 × 106 CFU/mL at 96 h post-infection. Histopathological analysis revealed that hepatopancreas and intestines of diseased M. rosenbergii exhibited obvious inflammatory responses to non-O1/O139 V. cholerae infection. Detection virulence factors of the strain GXFL1-4 showed that the bacteria produced caseinase, lipase, amylase, lecithinase and hemolysin, and carried toxR, hlyA, ompW, ompU, hap, rtxA and rtxC virulence related genes, supporting the strong virulence to M. rosenbergii. Additionally, the immune related gene expression in M. rosenbergii evaluated by qRT-PCR analysis showed that HSP70, Crustin, Lysozyme, TRL1, ALF1, Lectin, Peroxinectin, proPO and SOD immune related genes were significantly up-regulated at 6 and 12 h after infection with GXFL1-4. The results of our study suggested that non-O1/O139 V. cholerae was an etiological element in the mass mortalities of M. rosenbergii and this study provided preliminary insights into the diversity in the immune response of M. rosenbergii to the bacterial invasion.

Excessive use of enrofloxacin leads to growth inhibition of juvenile giant freshwater prawn Macrobrachium rosenbergii.

Giant freshwater prawn Macrobrachium rosenbergii is an economically important species. However, its growth retardant have brought serious economic losses in recent years. Antibiotics abuse is suggested as a reason for M. rosenbergii's growth retardant, while few studies focused on the toxic effect of antibiotics on M. rosenbergii. To investigate the effect of enrofloxacin, a widely used antibiotic, on juvenile M. rosenbergii, a 14 days exposure study was carried out within 0.2, 1 and 5 mg/L enrofloxacin and followed by 7 days decontamination. Results showed that during the test period, enrofloxacin had the largest accumulation in juvenile shrimp at day 3, and gradually decreased at day 7 and 14, and almost all the drugs are cleared after 3 days decontamination. Short-term exposure to low dose enrofloxacin can promote the growth of juveniles. High dose enrofloxacin inhibited the growth of juvenile shrimp, to gill and liver damage, and induced apoptosis of the hepatopancreatic cells. These adverse effects was possibly caused by enrofloxacin-induced oxidative stress. Moreover, we also found the damage caused by high concentrations of enrofloxacin was irreversible in the short term. Collectively, these data indicated that enrofloxacin did affect the juvenile shrimp growth and development, and high level enrofloxacin abuse may contributed to M. rosenbergii's growth retardant.