Effect of Chromium on Oxidative Damage and Antioxidant Capacity of Ctenopharyngodon idellus

[Objective] This study aimed to investigate the effect of chromium on ox- idative damage and antioxidant capacity of Ctenopharyngodon idellus （grass carp）. [Method] The grass carps were treated with hexavalent chromium （Cr^6＋） solution at concentrations of 0, 7.23, 14.47, 28.94 mg/L, and then the content of malondialde- hyde （MDA）, the level of total antioxidative capacity （T-AOC） and the activity of gtu- tathione-S-transferase （GST） in the hepatopancreas of grass carp were determined after 96 hours in different treatment groups. [Result] The content of MDA presented increasing trend with the increase of exposure Cr^6＋ concentrations. The activity of T-AOC increased firstly, then decreased with the increasing Cr^6＋ exposure concentra- tions, showing that the level of T-AOC was induced in tow and medium concentrat ions （7.23 and 14.47 mg/L）, but inhibited in high concentrations （28.94 mg/L）. Among the exposure groups, the level of T-AOC in medium concentration group （14.47 mg/L） was significantly higher than the control （P〈0.05）. Except the low concentration groups （7.23 mg/L） of which the GST activity was slightly induced, the GST activities of the other groups all showed downward trend with increasing Cr^6＋ levels, and the activity of GST in 28.94 mg/L group was significantly lower than the control group （P〈0.05）. [Conclusion] Cr^6＋ could cause large oxidative damage in the hepatopancreas of grass carp, thus poisoning it, and Cr^6＋ may further damage the organizational structure and physiological function of grass carp.

Starvation on changes in growth and fatty acid composition of juvenile red swamp crawfish, Procambarus clarkii

The effect of starvation on physiological and biochemical indicators of body, muscle and hepatopancreas were investigated for juvenile red swamp crawfish （Procambarus clarkii）, and the results showed the effect was obvious. In a 70-day starvation experiment, mean body weight, exuvial rate, abdominal muscle ratio and hepatosomatic index decreased significantly. The percentage of protein, fat, ash and moisture were also significantly affected. The protein content decreased sharply at Day 30 onward and the percentage of fat decreased rapidly from Day 50 and on. The percentages of ash and moisture started to increase significantly at Day 40 and 30 respectively. After the starvation experiment, the hepatopancreatic total and neutral lipid content decreased slightly from 23.90% to 20.39% and 73.75% to 70.35%, while the hepatopancreatic polar lipid content increased from 23.81% to 27.32%. On the other hand, all the muscular total, neutral and polar lipid contents, and the muscular fatty acid compositions did not change significantly except for the hepatopancreatic fatty acid composition. The relative percentages of 14：0, 16：0, 16：1n-7 and 18：1n-9 were decreased by 55.17%, 11.11%, 8.5% and 6.70% from the initial values, and the relative percentages of 18：2n-6, 18：3n-3, 20：4n-6, 20：5n-3 and 22：6n-3 increased obviously from 5.2%, 3.9%,4, 2.4%, 10.6%, and 13.7% to 7.1%, 5.0%, 2.8%, 11.5%, and 14.4% respectively. Preference of utilization of fatty acids from hepatopancreas during starvation was as follows： 14：0〉16：0〉16：1n-7〉18：1n-9, and the conservation of highly unsaturated fatty acids was in the order of： 18：2n-6 〉18：3n-3〉20：4n-6〉20：5n-3〉22：6n-3.

Mercury (Hg^(2+)) effect on enzyme activities and hepatopancreas histostructures of juvenile Chinese mitten crab Eriocheir sinensis

We studied the effects of mercury (Hg2+) on antioxidant and digestive enzyme activities in terms of LC50 value and on hepatopancreas histostructures of juvenile Chinese mitten crabs Eriocheir sinensis in 40-day exposure to various concentrations of Hg2+ (0, 0.01, 0.05, 0.10, 0.20, and 0.30 mg/L). The results show that the activities of superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) significantly increased in the concentrations of 0.01 and 0.05 mg/L, while that of enzyme decreased in 0.10, 0.20 and 0.30 mg/L treatments. Meanwhile, Hg2+ disrupted the histostructures of the hepatopancreas, causing decreases in activities of pepsin, tryptase, amylase, and cellulose, which are synthesized in the hepatopancreas. Moreover, as the Hg2+ concentration increased, the survival rate of the crabs decreased, worst at 56.57% in 0.30 mg/L. Therefore, although crabs are able to tolerate low levels of mercury pollution, high levels lead to cellular injury and tissue damage in hepatopancreas, which then loses some of its vital physiological functions such as absorption, storage, and secretion.

Impact of ocean acidification on physiology and microbiota in hepatopancreas of Pacific oyster Crassostrea gigas

The hepatopancreas is an important tissue involved in various biological metabolism for mollusks,but its responses to ocean acidifi cation(OA)have not been well evaluated.In this study,the oysters were cultured in simulated conditions by continuously bubbling with ambient air(pH=8.10)or air-CO_(2)(pH=7.50)for up to two months,and the variations on the antioxidant capacity,digestive ability,and microbiota composition in hepatopancreas of Crassostrea gigas were analyzed.The results show that although superoxide dismutase and glutathione responded quickly to OA stress,the antioxidant capacity of the hepatopancreas was inhibited,as revealed by the decrease of the total antioxidant capacity,which led to an upward trend of the malondialdehyde,demonstrating that the oxidative damages were accumulated under the OA process.The determination of the digestive ability manifested as the decrease of pepsin activity and the recovery of lipase and amylase activity after long-term acidifi cation,which may be helpful to improve the adaptability of oysters.In addition,analysis on 16S rDNA amplicon revealed that the total species abundance and diversity of the hepatopancreas microbiota experienced a dynamic change,but fi nally it decreased greatly after long-term acidifi cation.The structure of the hepatopancreas microbiota was changed drastically with the change of the dominant species from aerobic to the anaerobic and facultative anaerobic bacteria,and the abnormal proliferation of some species,such as genus of Mycoplasma and order Clostridiales,which may aggravate the adverse eff ects of OA on the physiological functions of the hepatopancreas.As a result,our fi ndings enrich our understanding of the accumulated oxidative damage and adaptive digestive ability in oyster hepatopancreas caused by OA.For the fi rst time,the changes of the hepatopancreas microbiota under long-term acidifi cation conditions are described,proving a good reference for the study of the response and adaptation mechanisms of bivalve mollusks in a wide range of oceans OA.

Transcriptome Analysis Provides New Insights into Host Response to Hepatopancreatic Necrosis Disease in the Black Tiger Shrimp Penaeus monodon

The hepatopancreatic necrosis disease(HPND)is a common shrimp disease.Aquaculture of the black tiger shrimp Penaeus monodon has suffered with the frequent outbreak of HPND.Understanding the molecular mechanisms of host response to HPND is of pivotal importance for improving its farming performance.In this study,the RNA-Seq platform was utilized to investigate transcriptomic changes of the hepatopancreas in pond-cultured P.monodon with HPND symptoms.A total of 62071 genes with an average length of 953 bp were obtained,and a lot of simple sequence repeat(SSR)loci related to these genes were identified.Totally,5204 differentially expressed genes(DEGs)were detected between healthy shrimp and HPND shrimp,with 3399 genes upregulated and 1,805 genes down-regulated.These genes had a wide range of biological functions,and several well-known immunerelated genes including integrin alpha 5,integrin beta 1,C type lectin and catalase were among the DEGs.Cell signaling pathways including the extracellular matrix(ECM)receptor interaction,phagosome and lysosome pathways were significantly upregulated in HPND-infected shrimps,indicating their involvement in the immune responses of shrimp against HPND.The data obtained in this study offers new insights into the molecular mechanisms of shrimp host response to HPND disease,and provides a resource for molecular marker development and genetic breeding studies of P.monodon.

Sublethal Antimony (III) Exposure of Freshwater Swamp Shrimp (Macrobrachium Nipponense): Effects on Oxygen Consumption and Hepatopancreatic Histology

This study was an attempt to realize the effects of antimony on freshwater swamp shrimp (Macrobrachium nipponense). An experiment of this nature, which have not previously been carried out in this species. First, median lethal concentrations were determined in acute toxicity tests. The 96-h LC50 value was 6.748 (5.728-7.950) mg Sb/l for adult M. nipponense and 1.635 (1.271-2.103) mg Sb/l for juvenile M. nipponense. Juvenile M. nipponense were exposed to 4 different sublethal levels of antimony (0.1, 0.4, 0.8, and 1.2 mg Sb/l) over a 7-d test period and a 7-d recovery period. After 30 min (acute), there was an increase in the amount of oxygen consumed in all exposed groups. On days 3, 7, and 14, decreases in oxygen consumption were significant (p < 0.05) for the higher-exposure level groups (0.8 and 1.2 mg/l). Light microscopy investigations showed histopathological alterations in the hepatopancreas which correlated with exposure concentrations. The alterations included degenerative changes in the lumen, a reduction in the lumen volume, and injury to epithelial cells in the histoarchitecture of hepatopancreas.

Environmental stress upon hepatopancreatic cells of freshwater prawns (Decapoda: Caridea) from the floodplain of ParanáRiver

In order to evaluate the influence of stressed environments on hepatopancreatic cells of fre- shwater prawns, Macrobachium borellii Nobili, 1896 and Palaemonetes argentinus Nobili 1901, (Crustacea, Decapoda, Palaemonidae) were collected at three different aquatic environments with different relationship to urban development in Argentina. Furthermore the effects of several cypermethryn concentration on hepa- topancreatic cell of M. borellii and P. argentinus were evaluated in a laboratory assays. The “N&#176;1” lake (Santa Fe Argentina) which is more affected by the anthropogenic pressure and “Don Felipe” lake which is still not strongly by human activities were the studied sites from the floodplain of Paran&#225;river. While Alejandra lake was the intermedia effects sites. Different damaged ultra-structures were found in F- and R-cells of prawns in the stressed lake. The predominant features were: disrupted the microvillous border, swelled mitochondria, reduction of endoplasmic reticulum, dyctiosomes, glycoproteins, desna-turalization of vacuole membrane and premature autolysis. Moreover the F-cell number was higher in the environment near to city than in the others sites. Similar effects were observed in the cypermethryn assays. The observations clearly indicate that the ultrastructure of midgut gland in the both palaemonids varies depending on the site from which animals are collected and the biocid presence. So, in this case it can be stated that the hepatopancreas histology of fre- shwater prawns is a good tool to monitor the impact of a stressed environment upon freshwater prawns.

Analysis of DNA Methylation Level of Portunus trituberculatus Subjected to Low Salinity with Methylation-Sensitive Amplification Polymorphism

In this study,the methylation-sensitive amplification polymorphism(MSAP)was used to compare the genomic DNA methylation level of muscle,gill and hepatopancreas of Portunus trituberculatus subjected to salinity 12 for 30 days to illustrate the epigenetic mechanism of osmoregulation.Thirty primers were used to analyze the difference of methylation level of different tissues.The results showed that the baseline methylation level of muscle,hepatopancreas and gill was 47.31%,22.94%and 17.69%,respectively.After exposed to low salinity stress,the methylation epiloci changed in the three tissues.Both demethylation and methylation processes occurred under low salinity stress.The methylation ratio decreased in muscle and gill but increased in hepatopancreas.These results indicated that DNA methylation is tissue-specific when P.trituberculatus responds to low salinity.

Transcriptome of Eriocheir sinensis under air exposure

Chinese mitten crab,Eriocheir sinensis,is an economically important crab in China.Air exposure is a common stress for E.sinensis,especially during the harvest and transportation.Several studies have investigated the effects of air exposure stress on E.sinensis at physiological and molecular levels.However,the common and distinct mechanisms adopted by male and female crabs to cope with air exposure stress remain unclear.In this study,we performed a comparative transcriptome analysis from hepatopancreatic tissue of female and male Chinese mitten crabs in response to air exposure stress.In total,428 and 1322 diff erentially expressed genes(DEGs)were identifi ed in female and male crabs under air exposure,respectively.Our results showed that the transcriptional levels of several glycolysis related genes and anti-apoptotic proteins were up-regulated in both female and male crabs in response to air exposure.Moreover,our findings indicated that female E.sinensis might preferentially increase the expression of heat shock proteins(HSPs)to deal with air exposure stress,while male E.sinensis tend to resist air exposure stress via increasing antioxidant enzyme expression.Overall,this study provides novel insights into the molecular mechanisms underlying the air exposure stress response of E.sinensis.

Construction of full length cDNA expression library of hepatopancreas of Penaeus monodon

mRNA was isolated from the hepatopancrease of shrimp Penaeus monodon with a PolyAT-tract System 1000 Kit. By using mRNA as template, double - strand cDNA with EcoR I/Xho I ends was synthesized by using a ZAP Express cDNA Synthesis Kit. The cDNA was inserted into the lambda ZAP Express vector predigested with EcoR l/Xho Ⅰ, and the recombinant DNA was in vitro packaged into lambda phage with GigapackⅢ Gold packaging extracts. These recombinant phages were then used to transfect E. coli XL1 - Blue MRF', and finally a cDNA expression library was constructed. The library is 7.2 × 10~5pfu in capacity and its recombination ratio is higher than 99 % . The size of the inserted cDNAs was determined by EcoR l/Xho I digestion of 9 phagemids prepared by in vivo excision of plaques selected randomly from amplified cDNA library . The longest inserted cDNA is about 1.6 kb in length. The complete sequence (about 1.2 kb) of actin cDNA was amplified from the library by PCR reveals that this library contains full-length cDNAs of Penaeus monodon hepatopancreas and is available for screening and expression of shrimp genes.

Acute toxicity of organochlorine insecticide endosulfan to the giant freshwater prawn Macrobrochium rosenbergii

Endosulfan, an organochlorine pesticide, is highly toxic and effective at controlling pests in agriculture, horticulture, and public health programs. In this study, static bioassays were used to evaluate the toxicity of endosulfan to freshwater prawns( Macrobrachium rosenbergii) of various lengths(1.5±0.03,4±0.08, and 7±0.06 cm). Additionally, the activities of peroxidase(POD), acid phosphatase(ACP),alkaline phosphatase, acetylcholinesterase(AChE), and Na + /K +-ATPase were analyzed to refl ect the effects of endosulfan exposure. The 96 h LC 50 of endosulfan for prawns 1.5, 4, and 7 cm long were 1.86, 4.53,and 6.09μg/L, respectively, improved tolerance to endosulfan with growth. The POD activities of test organisms exposed to low concentrations of endosulfan were inhibited, indicating the presence of oxygen damaged tissue. Moreover, a notable decrease in AChE activity was observed due to overstimulation of neurotransmission, which might result in abnormal behavior. The effect caused by endosulfan on phosphatase production in the hepatopancreas of prawns 1.5, 4, and 7 cm long was different because the ability of nonspecifi c immune regulation increased with growth. The 96 h LC 50 values obtained in this study could be used in the formulation of water-quality criteria in China. Moreover, the changes in enzymes activities of M. rosenbergii under stress of endosulfan could be applied in the establishment of early warning indicators for bio-safety.

Effects of transport stress on immune response, physiological state, and WSSV concentration in the red swamp crayfish Procambarus clarkii

Transport is an essential part of the aquaculture and research of the main freshwater aquaculture crayfish Procambarus clarkii in China.However,transport is often accompanied by a low survival rate.Assessing the physiological state of P.clarkii before and after transport may discover the cause of this high mortality rate.In this study,ice-cold and exposed-to-air transport methods were compared using an array of parameters,including relative expression level of heat shock protein 70(HSP70),content of serum glucose and cortisol,immune parameters(enzyme and immune-related genes),and white spot syndrome virus(WSSV)concentration were investigated to understand the physiological state of P.clarkii before and after transport,as well as the cause of dying crayfish on days 5 and 7 after transport stress.Histological sections of hepatopancreas,gills,and intestines reflected pathological changes.The survival rate of crayfish with ice-cold transport was significantly higher than that with exposed-to-air transport,and mortality peaked at 3-9 days after transport stress.A prolonged response to oxidative stress and short-term immunosuppression was present after transport,and the trend of the WSSV concentration in the hepatopancreas was similar to the mortality rate of P.clarkii.The contents of serum glucose and cortisol,antioxidant enzymes and immune-related indexes,and the concentration of WSSV in hepatopancreas of dying crayfish were significantly higher than those of vibrant crayfish on the 5th and 7th days after transport.The hepatopancreas,intestines,and gills of dying crayfish had varying degrees of damage,and the hepatopancreas and intestines were severely damaged.The results suggested that the death of P.clarkii after transport stress is caused by oxidative stress,the imbalance of reactive oxygen species regulation,and decreased WSSV resistance,which eventually led to irreversible tissue damage.The increase of WSSV in the body of crayfish might be the direct cause of crayfish death.

Effects of water-borne copper on the gills and hepatopancreas of Macrobrachium rosenbergii

We focused on elucidating the toxic effects of water-borne copper on the giant freshwater prawn Macrobrachium rosenbergii.After seven days of exposure to copper(Cu2+)at concentrations ranging from 0.01 mg L–1 to 0.5 mg L–1,three isozymes,malate dehydrogenase,alkaline phosphatase and esterase,were analyzed and compared using polyacrylamide electrophoresis(PAGE)and biochemical staining.The results indicated that the electrophoretic patterns of the isozymes showed a copper-concentration-related difference.Low doses of copper stimulated strong expression of the three isozymes.Electrophoretic patterns of malate dehydrogenase and alkaline phosphatase gradually became weaker or even lost as the level of copper increased.In contrast,esterase patterns exhibited an increased molecular heterogeneity at higher copper concentrations.A transmission electron microscope was used to study ultrastructure differences in the gills and hepatopancreas of M.rosenbergii,and the results showed significant structural damage at increased levels of copper compared with the control group.The basement mem-branes and mitochondira in the gills were seriously damaged,the cuticle electron density distribution was not homogeneous,and an infolded basement membrane,circularized nucleus,disintegrated nuclear membrane,and decreased mitochondria number and size were observed in the gills.Similarly,flowing out of karyoplasms,partly falling microvilli,decreased mitochondrion,partly disappeared mitochondrial cristae,and a thinned matrix were observed in the hepatopancreas.These findings indicate that exposure to elevated copper levels might damage the ultrastructure of the gills and hepatopancreas of M.rosenbergii and might further weaken their normal physical activities.Isozymes were quite sensitive to environmental stress and changes in isozyme elctrophoretic patterns might be effective biomarkers of environmental contamination.

Transcriptomic response to ammonia-N stress in the hepatopancreas of swimming crab Portunus trituberculatus

The swimming crab Portunus trituberculatus is an important farmed species in China.Ammonia-N represents a major physiological challenge for crab culture and the hepatopancreas plays a major role in physiological adaptation processes.To better understand the molecular mechanism of the crab in response to ammonia-N stress,we performed a transcriptome analysis in the hepatopancreas of P.trituberculatus challenged with ammonia-N stress(5 mg/L),using the high-throughput sequencing technology.In total,52,280 unigenes were obtained from the hepatopancreas of P.trituberculatus,with an average length of 678 bp.Functional categorization and pathways showed some diiferentially expressed genes were involved in amino acid and nucleobase metabolism,energy metabolism,antioxidation,immune response,reproduction,moulting and material transport.The differential expression patterns of eight randomly selected annotated genes were validated by quantitative real-time PCR(qPCR).Results revealed a substantial number of genes modified by ammonia-N stress and a few significant ammonia acclimation pathways,which will serve as an invaluable resource for revealing the molecular basis of physiological adaptation mechanism in P.trituberculatus.