Biochemical responses and physiological status in the crab Hemigrapsus crenulatus (Crustacea,Varunidae) from high anthropogenically-impacted estuary (Lenga,south-central Chile)

Estuarine environmental assessment by sub-individual responses is important in order to understand contaminant effects and to find suitable estuarine biomonitor species. Our study aimed to analyze oxidative stress responses, including glutathione-S-transferase (GST) activity, total antioxidant capacity (ACAP) and lipid peroxidation levels (TBARS) in estuarine crabs Hemigrapsus crenulatus from a high anthropogenically-impacted estuary (Lenga) compared to low and non-polluted estuaries (Tubul and Raqui), in a seasonal scale (winter–summer), tissue specific (hepatopancreas and gills) and sex related responses. Results showed that hepatopancreas in male crabs better reflected inter-estuary differences. Morpho-condition traits as Cephalothorax hepatopancreas index (CHI) could be used as an indicator of physiological status of estuarine crabs. Discriminant analysis also showed that GST and TBARS levels in summer are more suitable endpoints for establishing differences between polluted and non-polluted sites. These results suggest the importance of seasonality, target tissue, sex and physiological status of brachyuran crabs for estuarine biomonitoring assessment.     

干露胁迫对脊尾白虾(Exopalaemon carinicauda)HSP70和ferritin基因表达的影响

采用常规急性实验方法,研究了不同干露胁迫条件对脊尾白虾(Exopalaemon carinicauda)成活率的影响。结果表明,保持虾体湿润并用冰块降温的P组干露胁迫12h后成活率为75%,显著高于其它各实验组(P<0.05)。采用实时荧光定量PCR方法研究了不同干露胁迫条件对脊尾白虾血细胞和肝胰腺组织中热休克蛋白70(HSP70)和ferritin基因表达的影响。干露胁迫能诱导脊尾白虾血细胞、肝胰腺HSP70基因的表达上调,肝胰腺中的高表达时间相对血细胞中出现的较早;干露胁迫能诱导湿润低温P组脊尾白虾血细胞和肝胰腺ferritin基因的表达上调,并显著高于对照组(P<0.05),而且各组织ferritin基因表达的上调时间具有差异性,血细胞最先上调。其余实验组脊尾白虾各组织ferritin基因表达均下调,并显著低于对照组(P<0.05)。上述结果表明,在脊尾白虾受干露胁迫的耐受范围内,HSP70和ferritin基因发挥抗氧化功能。     

三疣梭子蟹几丁质酶基因(PtCht6)的克隆及其在免疫中的功能分析

几丁质酶(chitinase)在甲壳动物的蜕皮、消化和免疫等很多生物学过程中发挥重要功能,为深入探讨几丁质酶的免疫防御机制,本实验利用RACE技术克隆了三疣梭子蟹(Portunus trituberculatus)PtCht6基因。该基因cDNA全长2736bp,开放阅读框(ORF)2103bp,编码700个氨基酸,具有几丁质酶GH18家族典型特征。利用实时荧光定量技术分析PtCht6在三疣梭子蟹不同组织、不同蜕皮阶段、不同病原感染以及低盐胁迫(11)下的表达特征,结果显示:PtCht6在各组织中均有表达且在肝胰腺中的表达量最高;在不同蜕皮时期的肝胰腺中,其表达量由蜕皮后期(A/B)、蜕皮间期(C)、蜕皮前期(D)依次递减(P0.05);人工感染WSSV和副溶血弧菌后.PtCht6的表达量在肝胰腺中分别于12h、72h达到最大值,在血细胞中均于12h达到最大值,且相较于对照组,整体显著上调表达(P0.05);低盐胁迫能够显著抑制该基因的表达,最高抑制73倍(P0.05)。同时发现,低盐环境下感染WSSV后,该基因达到峰值的时间明显延迟(至少延迟12h.P0.05)。该研究结果预示PtCht6作为免疫因子参与三疣梭子蟹的病原防御,且低盐胁迫在一定程度上抑制了该基因的免疫功能。     

拟穴青蟹(Scylla paramamosain)蛋白二硫键异构酶基因的分子克隆与表达分析

蛋白二硫键异构酶(PDI)是内质网中的关键酶, 参与蛋白合成过程中二硫键的形成、还原和异构。本文首次从拟穴青蟹(Scylla paramamosain)克隆获得了PDI基因cDNA全长, 该序列长度为2015bp, 开放阅读框为1452bp, 编码483个氨基酸。荧光定量PCR检测发现PDI存在于拟穴青蟹的多个组织中; 在拟穴青蟹卵巢发育过程中, PDI基因的表达量在前4个时期逐渐上升, 到了第5期(成熟期)表达量则下降, 表明PDI参与了卵巢发育的蛋白合成过程。免疫组化表明, 拟穴青蟹卵母细胞存在PDI阳性反应, 进一步为该分子参与卵巢发育提供了形态学证据。     

Metallothionein and stress protein levels in shore crabs (Carcinus maenas) along a trace metal gradient in the Fal estuary (UK)

A field study was conducted to assess the potential use of metallothionein (MT) and stress proteins as biomarkers of trace metal exposure. Shore crabs (Carcinus maenas) were collected from five sites: three located in the metal-contaminated Fal estuary (Southwest England) and two control sites located on the south coast of England. Two methods for determining MT levels in crab midgut gland were compared. A direct method in which MT was quantified by spectrophotometric measurement of SH content, and an indirect method in which the metal content (Cu and Zn) of partially purified MT was measured. Stress protein levels (stress-70) were quantified in crab gills using an improved western blotting technique. Elevated Cu and Cd concentrations compared to values in control crabs were found in the midgut glands of crabs from the Fal estuary. No significant variation was seen in Zn concentrations among the five sites. The two techniques used for MT analysis yielded similar results. Metallothionein concentrations were slightly higher in crabs from Restronguet Creek (the most metal-contaminated site) than in crabs from the other four sites, however this difference was not significant (p > 0.05). Stress protein levels did not reflect the metal gradient.     

Biomarkers of biochemical and cellular stress in Carcinus maenas: an in situ field study

Biomarkers are useful diagnostic tools for identifying exposure to physical, chemical, and environmental stresses. In this study a suite of techniques—measurements of metallothionein, lysosomal integrity, and osmoregulatory ability—have been used in an attempt to provide an in situ assessment of environmental quality. Five estuarine sites were studied, two clean sites, two urban sites, and one intermediate. Shore crabs, Carcinus maenas, were collected and haemolymph taken for the lysosomal and osmoregulatory assays. Animals were killed and tissue preserved for metallothionein analysis. Lysosomal integrity was significantly different in crabs from clean sites and the urban sites (n = 40, p < 0.05). The potential value of the biomarker approach is that it could provide data for impact assessment and is applicable in the field.     

斑节对虾GPx3a基因的分子克隆及表达分析

谷胱甘肽过氧化物酶(Glutathione peroxidase,GPx)是生物体内重要的抗氧化酶,能防止过氧化氢对生物体的氧化应激。该研究利用RACE技术获得了斑节对虾(Penaeus monodon)GPx3a(PmGPx3a)的全长c DNA序列,进行了相关生物信息学分析。作者利用荧光定量PCR方法研究了PmGPx3a在斑节对虾不同组织的表达情况。探究了PmGPx3a在不同胁迫条件下(盐度、重金属和细菌)的表达情况。结果表明PmGPx3a c DNA全长1135 bp,其中开放阅读框(ORF)长651 bp,预测编码216个氨基酸。PmGPx3a推导的氨基酸序列与其他动物的GPx3a氨基酸序列具有高度一致性。实时定量PCR结果显示,在高低盐胁迫下,PmGPx3a在肝胰腺中相对表达量都为上升的(p0.05)。在铜、锌、铬胁迫中,鳃中的PmGPx3a的相对表达量总体呈现下降趋势,在肝胰腺中呈现上升趋势。在哈维弧菌(Vibrio harveyi)刺激下,PmGPx3a在血淋巴中的相对表达量总体呈现上升趋势,24 h后表达量最大,显著高于对照组2.2倍(P0.05)。以上研究结果表明,PmGPx3a基因参与了斑节对虾对环境胁迫和氧化应激的适应性反应。     

三疣梭子蟹(Portunus trituberculatus)胞内氯离子通道蛋白基因克隆及其表达分析

本研究克隆了三疣梭子蟹(Portunus trituberculatus)胞内氯离子通道蛋白基因,命名为Pt CLIC。该基因c DNA全长2000bp,5’和3’非编码区(UTR)长分别为76bp和1162bp,开放阅读框长762bp,推测编码253个氨基酸,预测分子量为29.2k Da,理论等电点为5.93。生物信息学分析表明,Pt CLIC基因中未发现跨膜结构域,属于不稳定蛋白;同源性分析表明,Pt CLIC基因编码的氨基酸序列与蚤状溞(Daphnia pulex)CLIC基因的同源性高达83%;系统进化分析表明,三疣梭子蟹与拟穴青蟹首先聚为一支;实时荧光定量RT-PCR分析表明,Pt CLIC基因在选取的所有组织中均有表达,在肝胰腺中的相对表达量最高,且显著高于其它组织(P0.05)。低盐度胁迫显著改变了Pt CLIC基因在三疣梭子蟹鳃和肝胰腺中的表达模式,整体呈先上调后下调的趋势,并发现该基因在低盐耐受和低盐敏感家系中的表达规律差异显著。研究结果表明Pt CLIC基因在三疣梭子蟹渗透压调节中发挥重要作用,可辅助三疣梭子蟹耐低盐品系的选育。     

盐度胁迫对锯缘青蟹血清及组织、器官中PO和SOD活性的影响

采用酶学方法测定了盐度(5、15、25、35)胁迫下锯缘青蟹血清中酚氧化酶(PO)及其肌肉和肝胰腺中超氧化物歧化酶(SOD)活性的变化.结果表明,盐度胁迫显著影响青蟹血清PO活性(p＜0.05).S5、S15低盐度处理组青蟹血清中PO活性显著高于S25、S35高盐度处理组(p＜0.05),同一处理组青蟹血清中PO活性除S15处理组随胁迫时间延长而持续下降外,其余各组呈现出有升有降的变化.各处理组在盐度低于或高于25时,青蟹肌肉中SOD活性升高,且胁迫至72h前,随着胁迫时间延长,SOD活性下降;青蟹肝胰腺中SOD活性随着盐度升高而降低,且同一处理组青蟹肝胰腺中SOD活性则随着胁迫时间的延长而呈现出先降后升的趋势.这表明环境盐度胁迫对青蟹体内SOD活性影响显著,且表现出明显的时间效应性.由此可见,盐度胁迫显著影响青蟹的PO、SOD活性,进而影响青蟹免疫力.     

缢蛏（Sinonovacula constricta）GST和HSP90基因克隆及其在氨氮胁迫下的表达特征分析

克隆获得缢蛏(Sinonovacula constricta)谷胱甘肽S-转移酶(Sc-GSTσ)和热休克蛋白90(Sc-HSP90)基因的cDNA全长,分析了它们的组织表达差异及其在氨氮胁迫下的表达特征。结果表明,Sc-GSTσ的全长cDNA为1 414 bp,含有639 bp的开放阅读框(Open Reading Frame,ORF),编码212个氨基酸,Sc-GSTσ氨基酸序列与其他物种的GST氨基酸序列同源性为31.88%～43.40%;而Sc-HSP90的全长cDNA为2 752 bp,ORF为2 181 bp,编码726个氨基酸,其氨基酸序列与其他物种HSP90的氨基酸序列同源性为76.77%～87.05%。荧光定量PCR分析发现,Sc-GSTσ和Sc-HSP90在缢蛏各组织中均有表达,两者均在肝胰腺中表达量最高。氨氮胁迫后,Sc-GSTσ和Sc-HSP90 mRNA在肝胰腺中表达均显著上调(p<0.05),表明氨氮胁迫引起机体的应激反应,2个基因可能参与机体解毒或防御过程。但胁迫后期表达量下降推测是机体的防御能力有限,不足以完全保护宿主免受应激诱导的细胞损伤。     

缢蛏HAT基因家族的全基因组鉴定及在环境因子、细菌胁迫下的表达分析

组蛋白乙酰化酶(histone acetyltransferases, HATs)在机体发育和环境胁迫响应的调控中发挥着重要作用,但在海洋贝类中研究极少。缢蛏(Sinonovacula constricta)作为典型的滩涂双壳贝类,经常面临多种极端环境和细菌胁迫的挑战,基于全基因组和转录组数据系统分析了缢蛏HAT基因家族(ScHATs)的系统发育关系,以及它们在不同发育时期、不同环境因子和细菌胁迫下的表达模式。结果共鉴定出11个ScHATs基因,根据序列同源性分为GNAT、MYST和p300/CBP三类,每个亚家族中几乎所有的HAT都具有特定的保守结构域和保守基序。基因表达谱和qRT-PCR分析显示,ScHATs在幼虫发育前期表达量普遍高于发育后期;在氨氮胁迫下,ScKAT2B、ScKAT8、ScKAT9-2、Scp300在鳃中的表达量显著变化(P<0.05);在高温胁迫下,ScHATs在鳃和肝胰腺中表现出相似的表达模式,而Scp300在肝胰腺中的表达量显著降低(P<0.05);在副溶血弧菌胁迫下,ScHATs的表达均出现显著上调(P<0.05)。综上结果提示, Sc...     

Effects of molting and environmental factors on trace metal body-burdens and hemocyanin concentrations in the American lobster, Homarus americanus

Hemocyanin concentrations in the hemolymph of marine crustacea are dependent on the molt cycle and on environmental conditions. Studies in our laboratories have found that hemocyanin levels in blue crabs are reduced after ecdysis and under conditions of environmental stress (Engel, Brouwer, & McKenna, 1993. Hemocyanin concentrations in marine crustaceans as a function of environmental conditions. Marine Ecology Progress Series, 93, 233-244). We have extended those studies to include the American lobster, Homarus americanus. Hemolymph and digestive gland tissues from Long Island Sound lobsters were analyzed for hemocyanin, copper, and zinc during different stages of the molt cycle. Hemocyanin, copper and zinc in the hemolymph were highest in premolt stages (D1-D4), and lowest in the postecdysal papershell stages (B1-B2). Concomitantly, copper in digestive glands decreased significantly following ecdysis, but no significant changes in the metals bound to metallothionein (MT) were observed. Copper-MT was the predominant form throughout the molt cycle, presumably because lobsters were obtained from copper-contaminated areas. To examine the effects of environmental factors, intermolt lobsters were collected from locations of different environmental quality along the Atlantic coast, and were analyzed for hemocyanin and trace metals. In general, animals from areas with a history of contamination showed the highest hemocyanin concentrations.     

Heavy metal stress induced hyperglycemia in blue swimmer crab, Portunus pelagicus

The study was to find out the effect of cadmium and mercury on levels of hemolymph glucose, crustacean hyperglycemic hormone (CHH) and hepatopancreas glycogen in the blue swimmer crab Portunus pelagicus. The experiments were performed in both intact and eyestalk ablated crabs. Quantification of CHH was done by the indirect ELISA with the aid of primary anti-Carcinus maenas-CHH antibody. Higher glucose concentration was observed on exposure to 8×10^-6 of cadmium ((825.6±5.42) μg/mL) and 6×10^-6 of mercury ((90.5±6.25) μg/mL) after 48 h and 24 h respectively. Higher level of hemolymph glucose was observed in eyestalk intact crabs on exposure to cadmium and mercury than eyestalk ablated crabs. Decrease in the levels of CHH was observed in both eyestalk intact and ablated crabs on heavy metal exposure. Decline of the hepatopancreas glycogen level was also witnessed with the exposure to heavy metal, which validated its utilization in the production of glucose. Thus this study brings to light, the variations in hemolymph glucose, CHH and hepatopancreas glycogen on heavy metal stress. These carbohydrate metabolites can be used as biomarkers in assessing heavy metal contamination in water bodies.     

脊尾白虾(Exopalaemon carinicauda)ferritin 基因克隆及表达分析

采用RT-PCR及RACE技术获得了总长度为755bp的脊尾白虾ferritin基因cDNA序列。该基因序列5’和3’的非编码区分别为112bp和146bp,开放阅读框497bp,推测编码169个氨基酸,预测蛋白分子量19.1kDa,等电点5.46,该基因命名为Ecfer基因。利用Neighbor-joining法构建系统进化树分析结果表明,Ecfer基因氨基酸序列与无脊椎动物中的甲壳动物聚为一支(同源性为64%—67%)。此外,Ecfer基因氨基酸序列与脊椎动物铁蛋白H链同源性为53%—57%。采用荧光定量RT-PCR研究Ecfer基因在组织中以及经WSSV刺激后血淋巴、肝胰腺中的表达变化情况。研究结果表明,Ecfer在肝胰腺、卵巢、肌肉、鳃和血淋巴中均有分布。注射WSSV后3h和6h,肝胰腺和血淋巴Ecfer基因表达较对照组均显著上调(P<0.05),并具有显著的时间差异性。说明Ecfer基因可能参与了脊尾白虾抵抗WSSV入侵的免疫反应。     

Cadmium-binding proteins in the blue crab, callinectes sapidus: laboratory-field comparison

The blue crab, Callinectes sapidus, is distributed along the east coast of the United States from Cape Cod, Mass., through the Gulf of Mexico, including both relatively unpolluted coastal areas and estuaries contaminated with trace metals. Cadmium is of particular concern because it is concentrated in the digestive glands of blue crabs and can be passed on to consumer organisms. Tissue concentrations and partitioning of trace metals from crabs exposed in the laboratory to 10 ppb dissolved cadmium for 40 days were compared with blue crabs collected from two locations on the Hudson River, NY, Foundry Cove and Haverstraw Bay, Foundry Cove and Haverstraw Bay, both of which have elevated trace metal levels relative to estuarine areas near Beaufort, NC. Crab digestive glands, gills and muscle were removed and analyzed for total cadmium, copper, zinc and nickel concentrations using acid digestion and atomic absorption spectrophotometry, and metal-binding (metallothionein-like) proteins were determined by gel-filtration chromatography. In crabs exposed to cadmium in the laboratory, the cytosolic partitioning was similar to previous investigations at our laboratory where higher levels of cadmium (100 ppb) and shorter exposure times (14 days) were used. The similarity in cadmium partitioning from these two separate experiments indicates dose independence. In crabs from polluted environments the digestive glands contained the highest concentrations of trace metals. Chromatograms of the cytosol from the digestive glands and gills from both field and laboratory exposed crabs showed similar distributions of cadmium, copper and zinc. The gills of both field and laboratory-exposed crabs had metal-binding proteins that contained mostly cadmium, and the digestive glands had metallothionein-like proteins that contained cadmium, copper and zinc. Estimated molecular weights for these proteins were similar to the metallothioneins found in other crustaceans and mammals.     

日本囊对虾Cathepsin L基因的鉴定及其在幼体发育中的表达研究

本研究从日本囊对虾（Marsupenaeus japonica）中获得半胱氨酸蛋白酶家族中的cathepsin L基因,命名为Mj-Cathepsin L。Mj-Cathepsin L基因cDNA序列全长1191bp,其开放阅读框编码的Cathepsin L蛋白前体包含327个氨基酸,由信号肽,蛋白前体和成熟蛋白三部分组成。同源序列比对发现日本囊对虾Cathepsin L蛋白与其他已知物种的cahtepsin L蛋白具有较高的同源性。对Mj-Cathepsin L基因在不同组织以及早期幼体幼体发育阶段的研究发现,Mj-Cathepsin L主要在肝胰腺中大量表达,且在幼体发育不同阶段,其表达水平受发育阶段调控,且Mj-Cathepsin L基因显示出与其五个蜕皮时期相关的表达变化,说明Mj-Cathepsin L有可能在日本囊对虾个体发育过程中扮演不同的重要作用。并通过Western Blot方法在日本囊对虾肝胰腺,胃和肠中检测到Cathepsin L酶原和成熟Cathepsin L两种蛋白存在形式。     

Stress protein expression in Ampelisca abdita (Amphipoda) exposed to sediments from San Francisco Bay

Stress proteins (heat shock proteins, hsps) form part of the cellular protein repair system, and are induced by a wide variety of Stressors. To determine their suitability as tools for assessing sublethal sediment toxicity, we measured levels of members of the stress protein families hsp60 and hsp70 in benthic estuarine amphipods (Ampelisca abdita) exposed to sediments from 23 different sampling sites in San Francisco Bay for 10 d. Concentrations of sediment-associated xenobiotics were determined. Per cent survival was recorded and surviving animals were analysed for stress proteins using western blotting techniques. An inverse correlation (r² = 0.44) was seen between amphipod survival and hsp64 levels, and hsp64 levels were positively correlated with concentrations of total polycyclic aromatic hydrocarbons (PAHs) (r² = 0.5). Principal component analysis revealed that amphipod mortality was linked to a combination of several PAHs (phenanthrene, fluoranthene, pyrene, benzo(a)pyrene) and di-n-butylphthalate at southern San Francisco Bay sites. At northern San Francisco Bay sites, negative correlations were found between hsp64 levels and organotin compounds (MBT, DBT, TBT), and between hsp71 levels and the PAHs, benzo (b,k)fluoranthene and benzo(G,H,I)perylene, suggesting an inhibitory effect of these compounds on stress protein expression.     

缢蛏天然抗性相关巨噬蛋白2(Sc-Nramp2)基因克隆及其在副溶血弧菌刺激下的表达分析

克隆得到缢蛏天然抗性相关巨噬蛋白2（Sc-Nramp2）基因的cDNA全长序列3 681 bp,该基因的开放阅读框有1 776 bp,编码591个氨基酸,预测分子量为65.86 kDa;其结构具有Slc11蛋白家族的典型特征,包括有10个典型的跨膜结构和2个糖基化位点。Sc-Nramp2基因3'-UTR有2个类似于脊椎动物Nramp2中铁反应控制蛋白结合位点;同源性分析表明,Sc-Nramp2和太平洋牡蛎Nramp2-like的同源性最高为71.6%。实时荧光定量PCR结果表明,Sc-Nramp2基因在闭壳肌、外套膜、肝胰腺、斧足、水管和鳃6个组织中均有表达,其中肝胰腺中的表达量最高,其次是鳃,与其他组织均有极显著性差异（P<0.01）;注射副溶血弧菌后,肝胰腺中Sc-Nramp2基因的表达量较对照组显著上调（P<0.01）,且表达量呈现先上升后下降的趋势,在12 h时达到最大表达量,推测Sc-Nramp2基因参与了缢蛏非特异性免疫应答反应。