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.

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.

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.

饲料蛋白对翘嘴红鲌蛋白质周转代谢的影响

选择健康的翘嘴红(体重12.84±0.60g)为试验鱼,以红鱼粉为蛋白源,配制5个蛋白水平(31.04%、35.51%、40.89%4、6.62%、50.33%)的等能、等必需氨基酸(EAA)平衡关联度的半精制饲料;又以豆粕替代鱼粉,大豆蛋白分别替代0、13.5%、27%、40.5%、54%的鱼粉蛋白,配制5个EAA关联度的等蛋白(40%)、等能(20MJ.kg-1)的半精制饲料,探讨饲料蛋白水平和大豆蛋白对鱼粉蛋白的替代对翘嘴红肌肉和肝胰脏蛋白质周转代谢的影响。8周饲养结果表明,饲料蛋白水平对翘嘴红的特定增重率(SGR)具有显著性影响(P<0.05),40.89%饲料蛋白组的SGR显著高于31.04%、35.51%饲料蛋白组(P<0.05),但与46.62%和50.33%饲料蛋白组没有显著性差异(P>0.05);饲料蛋白水平对白肌、肝胰脏蛋白质生长率具有相似的影响,40.89%饲料蛋白组的白肌、肝胰脏蛋白质生长率分别达到2.13%.d-1,显著高于31.04%和35.51%两组(P<0.05),但与46.62%和50.33%饲料蛋白组无显著性差异(P>0.05)。饲料蛋白水平的增加促进了翘嘴红的生长和蛋白质的合成。肌肉蛋白质合成率(Ks)、蛋白质降解率(Kd)随饲料蛋白水平的增加而增加(P<0.05),肌肉蛋白质的增加归因于蛋白质合成的增加较降解的增加更占优势,蛋白质的沉积率在适宜蛋白水平时最高。当大豆蛋白分别替代13.5%2、7.0%4、0.5%的鱼粉蛋白时,翘嘴红的SGR与对照组差异不显著(P>0.05),而且都显著高于54.0%替代组(P<0.05)。白肌蛋白质生长率(Kg)也显著受到大豆蛋白替代的影响(P<0.01),当饲料中大豆蛋白对鱼粉蛋白替代量为40.5%时,与对照组相比,Kg极显著下降(P<0.01)。随着饲料中大豆蛋白对鱼粉蛋白替代量的增加,白肌Ks随之降低,当饲料中大豆蛋白对鱼粉蛋白替代量达54%时,与对照组相比,肌肉Ks显著下降(P<0.05)。白肌Kd与白肌Ks的变化趋势相似(P<0.1),而PDE没有受到饲料中大豆蛋白对鱼粉蛋白替代的影响(P>0.05)。可见随着大豆蛋白对鱼粉蛋白替代量的提高,氨基酸平衡关联度逐渐降低,蛋白质合成逐渐降低(P<0.05),生长下降,饲料必须氨基酸平衡程度的变化没有改变蛋白质的沉积效率。

相同EAA模式下不同日粮蛋白水平对草鱼肌肉、肝胰脏蛋白周转代谢的影响

在日粮必需氨基酸模式相同条件下 ,研究不同日粮蛋白水平对草鱼肌肉、肝胰脏蛋白质周转代谢的影响。其中周转代谢参数包括 :蛋白质的合成速率 (FSR)、降解速率 (FDR)、生长速率 (FGR)、合成能力 (CS)、合成的翻译效率 (KRNA)和蛋白质沉积效率 (PRE)。试验结果表明 :1.日粮蛋白水平的增加促进了草鱼的生长 ,促进了肌肉、肝胰脏蛋白质的增长。饲料转化效率和肌肉蛋白质沉积效率在适宜蛋白水平时最高。2.肌肉FSR、FDR与日粮蛋白水平呈正相关关系 ;肝胰脏蛋白FSR不受日粮蛋白水平的影响 ,蛋白质FDR与日粮蛋白水平呈负相关关系。3.肌肉蛋白FGR的增加归因于蛋白质合成的增长较降解的增长更占优势 ,以及蛋白质合成的KRNA 的提高 ;肝胰脏蛋白FGR的增加归因于蛋白质降解的减少。4.肌肉、肝胰脏蛋白CS不受日粮蛋白水平的影响。5.肌肉PRE(生长占合成的比例 )在适宜蛋白质水平 (30 % )时最高 ,而且在该水平下饲料系数最低 。

香鱼肝胰腺cDNA文库的构建和鉴定

以香鱼正常肝胰腺为材料,构建cDNA文库。初始文库库容大于1.3×107cfu/ml,扩增文库的滴度大于1.1×106cfu/ml,平均插入片段约为1.0 kb。从文库中随机挑选297个cDNA克隆测序,得到232个功能已知EST序列,功能包括代谢、蛋白质合成、细胞与机体防御、信号转导以及细胞结构与运动等5个方面。随机测序获得香鱼弹性蛋白酶（Elastase,ELA）编码序列,cDNA序列全长957个核苷酸,3’-末端具有polyA尾,编码一个由268个氨基酸组成28.7 ku大小蛋白。序列分析表明,香鱼ELA的N端15个氨基酸为信号肽,成熟肽具有丝氨酸蛋白酶家族的典型结构特征。香鱼ELA与斜带石斑鱼、大西洋鳕和金头鲷的ELA氨基酸序列同源性最高,达76%~77%。系统进化树分析表明,香鱼ELA与其他鱼类ELA紧密成簇。RT-PCR检测显示,香鱼ELA基因mRNA在香鱼肝胰腺、脾、肠组织中均大量表达。

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.

酵母水解物对青鱼幼鱼生长性能、肌肉品质及肝胰脏抗氧化指标和组织形态的影响

本试验旨在研究饲料中添加不同水平酵母水解物(YHY)对青鱼(Mylopharyngodon pi ceus)幼鱼生长性能、全鱼及肌肉氨基酸组成以及肝胰脏抗氧化指标和组织形态的影响。在基础饲料中分别添加0、0.5%、1.0%、1.5%、2.0%和2.5%的YHY,配制成6种等氮等能的试验饲料[分别命名为YHY0(对照)、YHY0.5、YHY1.0、YHY1.5、YHY2.0和YHY2.5]。选择初始体重为(5.53±0.14)g的青鱼幼鱼720尾,随机分为6组,每组4个重复,每个重复30尾鱼,在室内循环水系统中进行为期8周的养殖试验。结果表明:1)青鱼幼鱼的特定生长率(SGR)呈先上升后下降的趋势,蛋白质效率(PER)上升后逐渐平缓。YHY1.5和YHY2.0组SGR显著高于对照组(P<0.05),且与YHY2.5组相比无显著差异(P>0.05);YHY1.5、YHY2.0和YHY2.5组PER显著高于对照组(P<0.05)。2)饲料中添加不同水平YHY对青鱼幼鱼全鱼的干物质、粗蛋白质和粗灰分含量无显著影响(P>0.05)。全鱼粗脂肪含量呈上升趋势,在YHY2.5组达到最大,显著高于对照组(P<0.05)。YHY添加水平对青鱼幼鱼肌肉的干物质、粗蛋白质和粗脂肪含量无显著影响(P>0.05)。YHY2.0和YHY2.5组肌肉粗灰分含量显著高于对照组(P<0.05)。3)与对照组相比,饲料中添加YHY显著增加了青鱼幼鱼全鱼以及肌肉中的必需氨基酸以及呈味氨基酸含量(P<0.05),同时,显著提高了肌肉中各种氨基酸的氨基酸价(AAS)、化学价(CS)以及必需氨基酸指数(EAAI)(P<0.05)。4)青鱼幼鱼肝胰脏总抗氧化能力(T AOC)及总超氧化物歧化酶(T SOD)活性均呈先升高后降低的变化趋势。在YHY添加水平为1.5%时,青鱼幼鱼肝胰脏的T AOC及T SOD活性均达到最高,显著高于对照组(P<0.05)。YHY对青鱼幼鱼肝胰脏过氧化氢酶(CAT)活性无显著影响(P>0.05),有利于改善肝胰脏细胞空泡化现象。综上所述,基于SGR和PER的数据分析,在饲料中添加1.57%~2.02%的YHY能提高青鱼幼鱼的生长性能,同时,YHY能提高青鱼幼鱼全鱼及肌肉的氨基酸含量、肝胰脏抗氧化指标,改善肌肉品质,并且不会对青鱼幼鱼的肝胰脏组织形态产生不良影响。

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.