Pathology of the liver, kidney and gonad of flounder (Platichthys flesus) from a UK estuary impacted by endocrine disrupting chemicals.

The Mersey estuary is highly contaminated with xenobiotics compared to the nearby Dee estuary. Male flounder, a migratory flatfish caught in the Mersey frequently contains high blood concentrations of the oestrogen controlled, female protein vitellogenin, suggesting that Mersey flounder have been exposed to endocrine disrupting contaminants. Males caught from the Dee contain lower blood vitellogenin levels. Preliminary histopathological examination of 410 flounder from both estuaries, focuses on the liver, kidney and gonads. Hepatic tubular vacuolation, foci of cellular alteration, and hepatocellular tumours were seen. Renal and gonadal pathology included the presence of two Mersey fish with enlarged and abnormal glomeruli, three phenotypic male Mersey flounder with unilateral intersex gonads and one male with bilateral, abnormal ovarian follicular components in the testis. These initial results provide pathological evidence of xenobiotic exposure in flounder sampled from both estuaries. Preliminary findings from flounder sampled from Millport, an offshore site in Southwest Scotland, showed no histopathological evidence of xenobiotic exposure.     

Do endocrine disrupting chemicals threaten Mediterranean swordfish? Preliminary results of vitellogenin and Zona radiata proteins in Xiphias gladius.

Endocrine Disrupting Chemicals (EDCs) have the potential to alter hormone pathways that regulate reproductive processes in wildlife and fishes. In this research the hypothesis that Mediterranean top predator species (such as large pelagic fish) are potentially at risk due to EDCs is investigated. These marine organisms tend to accumulate high concentrations of EDCs such as polyhalogenated aromatic hydrocarbons (PHAHs). The potential effects of EDCs on a fish species of commercial interest, the top predator Xiphias gladius (swordfish), were investigated using vitellogenin (Vtg) and Zona radiata proteins (Zrp) as diagnostic and prognostic biomarkers. Dramatic induction of typically female proteins (Vtg and Zrp) was detected by ELISA and Western Blot in adult males of the species. These results are the first warning of the potential risk for reproductive function of Mediterranean top predators, and suggest the need for continuous monitoring of this fragile marine environment.     

日本蟳繁殖生物学的初步研Ⅱ.雄性繁殖习性及胚胎发育

根据精巢形态、切片组织学观察及扫描电镜观察 ,可将日本虫寻的精巢发育划分为三期 :未发育期、发育期与成熟期。通过扫描电镜观察可知 ,它的精子为无鞭毛精子 ,呈圆球形 ,大小为 30 μm。并运用实地调查、解剖、分析样本及光镜观察等手段 ,对其性腺形状、交配行为及胚胎发育等作了初步的描述。     

Do endocrine disrupting chemicals threaten Mediterranean swordfish? Preliminary results of vitellogenin and Zona radiata proteins in Xiphias gladius

Endocrine Disrupting Chemicals (EDCs) have the potential to alter hormone pathways that regulate reproductive processes in wildlife and fishes. In this research the hypothesis that Mediterranean top predator species (such as large pelagic fish) are potentially at risk due to EDCs is investigated. These marine organisms tend to accumulate high concentrations of EDCs such as polyhalogenated aromatic hydrocarbons (PHAHs). The potential effects of EDCs on a fish species of commercial interest, the top predator Xiphias gladius (swordfish), were investigated using vitellogenin (Vtg) and Zona radiata proteins (Zrp) as diagnostic and prognostic biomarkers. Dramatic induction of typically female proteins (Vtg and Zrp) was detected by ELISA and Western Blot in adult males of the species. These results are the first warning of the potential risk for reproductive function of Mediterranean top predators, and suggest the need for continuous monitoring of this fragile marine environment.     

半滑舌鳎(Cynoglossus semilaevis)Patched 1基因的克隆和表达分析

Hedgehog(Hh)信号通路在脊椎动物和非脊椎动物发育过程中细胞信号传导方面发挥重要的作用。Patched 1 (Ptc1)基因是Hh的受体,通过和Hh配体结合调节Hh信号通路。本研究克隆和鉴定了半滑舌鳎的Ptc1基因(csptc1),该基因cDNA全长为5212 nt,编码蛋白包括1543个氨基酸残基,序列比对和进化分析显示该蛋白在进化过程中较为保守。荧光定量显示该基因在脑,肝脏,心脏,鳃,肠,脾脏、肾脏和性腺组织中普遍表达,在精巢中的表达水平显著高于卵巢。精巢中的原位杂交信号主要位于该基因主要在初级精母细胞,次级精母细胞和支持细胞中,而在卵母细胞中的信号较弱。Csptc1基因在卵巢中的甲基化水平高于在雄鱼和伪雄鱼精巢中。这一研究结果可能说明csptc1基因参与了 Desert Hedgehog (DHH)基因维持雄鱼和伪雄鱼的生殖细胞系以及精子发生等生物学过程。     

Isolation and characterization of a SEPALLATA-like gene,ZjMADS1, from marine angiosperm Zostera japonica

In flowering plants, floral homeotic MADS-box genes, which constitute a large multigene family, play important roles in the specification of floral organs as defined by the ABCDE model. In this study, a MADS-box gene, ZjMADS1, was isolated and characterized from the marine angiosperm Zostera japonica. The predicted length of the ZjMADS1 protein was 246 amino acids (AA), and the AA sequence was most similar to those of the SEPALLATA (SEP) subfamily, corresponding to E-function genes. Southern blot analysis suggested the presence of two SEP3-like genes in the Z. japonica genome. ZjMADS1 mRNA levels were extremely high in the spadices, regardless of the developmental stage, compared to other organs from the reproductive and vegetative shoots. These results suggest that the ZjMADS1 gene may be involved in spadix development in Z. japonica and act as an E-function gene in floral organ development in marine angiosperms.     

Cloning, expression and characterization of serine palmitoyltransferase (SPT)-like gene subunit (LCB2) from marine Emiliania huxleyi virus (Coccolithovirus)

The authors have isolated and characterized a novel serine palmitoyltransferase(SPT)-like gene in marine Emiliania huxleyi virus(EhV-99B1).The open-reading frame(ORF) of EhV99B1-SPT encoded a protein of 496 amino acids with a calculated molecular mass of 96 kDa and Ip 6.01.The results of sequence analysis showed that there was about 31%-45% identity in amino acid sequence with other organisms.The maximum likelihood phylogenetic tree suggested that the EhV99B1-SPT gene possibly horizontally transferred from the eukaryote.Hydrophobic profiles of deduced amino acid sequences suggested a hydrophobic,globular and membrane-associated protein with five transmembrane domains(TMDs) motifs.Several potential N-linked glycosylation sites were presented in SPT.These results suggested that EhV99B1-SPT was an integral endoplasmic reticulum membrane protein.Despite lower sequence identity,the secondary and three-dimensional structures predicted showed that the "pocket" structure element composed of 2α-helices and 4βsheets was the catalytic center of this enzyme,with a typical conserved "TFTKSFG" active site in the N-terminal region and was very close to those of prokaryotic organisms.However,the N-terminal domain of EhV99B1-SPT most closely resembled the LCB2 catalysis subunit and the C-terminal domain most closely resembled the LCB1 regulatory subunit of other organisms which together formed a spherical molecule.This "chimera" was highly similar to the prokaryotic homologous SPT.For a functional identification,the EhV99B1-LCB2 subunit gene was expressed in Escherichia coli,which resulted in significant accumulation of new sphingolipid in E.coli cells.     

Differential gene expression in mummichogs (Fundulus heteroclitus) following treatment with pyrene: comparison to a creosote contaminated site

Mummichogs, Fundulus heteroclitus, an estuarine fish with a relatively small home range found along the eastern coast of the United States are well-suited to monitoring contaminant effects, including those of polycyclic aromatic hydrocarbons (PAHs). One of the common PAHs in estuaries is pyrene. We report here on efforts to develop multiple biomarkers of pyrene exposure in this species. Adult male mummichogs were exposed in the laboratory to the weak aryl hydrocarbon receptor (AhR) agonist pyrene at 0, 30, or 50 microg/L in 7-day static renewal exposures. The RNA was extracted from livers and alterations in mRNA expression were assessed by subtractive hybridization and differential display in order to produce multiple biomarkers of pyrene exposure. Genes demonstrating differential expression were confirmed by quantitative-PCR (Q-PCR) and include cytochrome P-450 1A (CYP1A), a putative hepatocyte growth factor activator, a X-ray inducible retrotransposon, and several expressed sequenced tags (ESTs). Some of these genes represent new biomarkers of pyrene exposure and potential biomarkers of PAH exposure. Therefore, similar changes were investigated at a Superfund site in Charleston, SC. Mummichogs from a creosote contaminated site and from a reference site (North Inlet National Estuarine Research Reserve near Georgetown, SC) were trapped, RNA extracted from the livers, and Q-PCR performed. Many of the genes differentially expressed following pyrene exposure were not altered at the creosote contaminated site in comparison to the reference site. However, CYP1A and an EST were induced. CYP1A induction at Diesel Creek indicates that this population of fish does not demonstrate refractory CYP1A phenotypes observed at several sites with high levels of AhR agonists. Ultimately, we anticipate that the use of multiple biomarkers of PAH exposure will provide useful information on the potential effects of toxicants.     

Comparison of the ectosymbionts and parasites of an introduced crab,Charybdis japonica,with sympatric and allopatric populations of a native New Zealand crab,Ovalipes catharus (Brachyura: Portunidae)

The success of biological invaders is often attributed to escape from specialist enemies in their natural range, such as predators and parasites. For enemy escape to have direct consequences in competitive interactions, invaders need to be less vulnerable to enemies than native competitors in the region they invade, but first the presence of these enemies must be established. We investigated the macroparasite and ectosymbiont fauna of the recently introduced portunid crab Charybdis japonica, and compared it with sympatric and allopatric populations of the native New Zealand portunid, Ovalipes catharus. A total of 468 crabs (350 O. catharus and 118 C. japonica) were collected from six harbours throughout New Zealand (Whangarei, Waitemata, Nelson, Lyttelton, Dunedin, and Bluff) and the identity, incidence and prevalence of ectosymbionts and parasites were compared among the different populations. Charybdis japonica and O. catharus harboured different ectosymbionts. Serpulid polychaete tubes occurred on the exoskeleton of 85.4% of C. japonica examined, but were absent from O. catharus. The bryozoan, Triticella capsularis occurred on 97.4% of O. catharus but was not found on C. japonica. Few endoparasites were present in either species. An unidentified juvenile ascaridoid nematode occurred in the hindgut of 5.9% of C. japonica, but was not found in sympatric populations of O. catharus. A second, unidentified species of ascaridoid nematode occurred in 7.1% of O. catharus from Nelson, but was not present in specimens from the five other harbours sampled. Melanised lesions were observed in the muscle tissue of almost half (46.6%) of the C. japonica examined. Histological examination showed these to be of two types: (1) spherical bodies resembling melanised trematode metacercariae; and (2) lesions consistent with wound repair. Lesions were not observed in O. catharus. Although the identity of parasites and epibionts carried by each species differed, both C. japonica and O. catharus had relatively low parasite species richness. We could not test whether the introduced portunid, C. japonica, is any less vulnerable to parasite enemies than the New Zealand portunid, O. catharus.     

Trophic accumulation and depuration of mercury by blue crabs (Callinectes sapidus) and pink shrimp (Penaeus duorarum)

Mercury concentrations in blue crabs (Callinectes sapidus) collected from an area of mercury-contaminated sediments in Lavaca Bay, TX, USA, are more than an order of magnitude greater than concentrations in penaeid shrimp from the same area. Laboratory feeding experiments using mercury-contaminated fish as food showed that both blue crabs and pink shrimp (Penaeus duorarum) could accumulate mercury concentrations comparable to those in their food in 28 days. Calculated mercury assimilation efficiencies averaged 76% for blue crabs and 72% for pink shrimp. Significant depuration of mercury by blue crabs was not observed during a subsequent 28-day period, but pink shrimp lost mercury at a rate of about 0.012 day-1. Model calculations predict biomagnification factors of mercury of about two to three at steady state for both species. The large difference in observed concentrations of mercury in field-collected blue crabs and penaeid shrimp does not result from differences in efficiency of mercury assimilation from their food or from differences in excretion rates. It is more likely the result of differences in residence times in the contaminated area and of differences in feeding habits.     

中国沿海10 种方蟹16S rRNA 基因序列分析及系统发育研究

中国沿海10种方蟹线粒体16S rRNA基因部分片段的序列长度为517 bp～533 bp。它们的核苷酸序列A、T、G、C的含量相似,A+T的含量(69.8%～76.0%)明显高于G+C的含量;10种方蟹的16S rRNA基因序列比对获得541 bp的同源序列(含插入/缺失位点),除插入/缺失位点外共检测到146个变异位点,其中81个为简约信息位点。4种厚蟹与2种近方蟹的遗传距离(0.054～0.085)都显著小于与其他方蟹之间的遗传距离,甚至明显小于与4种厚蟹原本属于同一相手蟹科的2种相手蟹之间的遗传距离(0.105～0.155);而基于16S rRNA基因片段序列采用NJ法构建的系统进化树的拓扑结构也显示,原本属于相手蟹科的侧足厚蟹、天津厚蟹、日本仿厚蟹和伍氏仿厚蟹没有与2种相手蟹聚为一支,而是最终与属于弓蟹科的2种近方蟹聚为一大支,且有高达99%的支持率。结果表明,4种厚蟹与2种近方蟹的亲缘关系相对较近,而与2种相手蟹等其他方蟹的亲缘关系则相对较远。因此,研究结果支持将4种厚蟹从相手蟹科移到弓蟹科。此外,属于相手蟹科的2种相手蟹聚为一支,属于方蟹科的白纹方蟹和属于斜纹蟹科的瘤突斜纹蟹又各自成为一支;表明16S rRNA基因的分子数据支持其形态学分类结果的正确性,提示上述4科蟹类可能分别为单系。     

雨生红球藻八氢番茄红素脱氢酶pds基因上游序列的分离和分析

在某些蓝藻、绿藻和高等植物中,八氢番茄红素脱氢酶是β-胡萝卜素合成过程中的一个关键酶之一,应用巢式PCR方法从雨生红球藻中克隆了八氢番茄红素脱氢酶基因约1kb的5’上游序列。通过生物信息学方法进行序列分析,发现pds基因上游序列中包含了一些可能的顺式元件,如ABRE调控元件、C-repeat/DRE调控元件等。同时,构建了由pds-启动子控制报告基因的重组载体,并转化到雨生红球藻细胞中,进行了报告基因瞬间表达的检测。结果表明,克隆的pds启动子区域具有启动子活性,可以驱使报告基因进行瞬间表达。     

大珠母贝外套膜基因PMMG1的克隆、表达及其特征分析

EF-hand结构域在真核生物细胞内钙离子吸收和运输中发挥重要作用,在贝类中可能参与贝壳和珍珠质的形成。根据已报道的贝类中具有EF-hand结构域的碱基序列设计简并引物,从大珠母贝外套膜cDNA文库中筛选得到PMMG1基因(大珠母贝外套膜基因1)。PMMG1基因全长618 bp,开放读码框编码140个氨基酸,N-端的22个氨基酸肽段为信号肽。PMMG1氨基酸序列与合浦珠母贝PFMG1的一致性为56%,预测有两个EF-hand结构域。选取编码PMMG1成熟蛋白的cDNA序列插入pET-32a质粒构建表达载体,通过IPTG诱导,Ni2+-NTA亲和层析柱纯化,成功获得预期大小的融合蛋白。凝胶电泳迁移率的变化证明PMMG1蛋白具有结合Ca2+/Mg2+的活性,组织特异性表达表明PMMG1基因在外套膜的表达量远高于其他组织。大珠母贝外套膜基因PMMG1的克隆与表达研究为进一步研究该蛋白在珍珠质矿化中的作用、探讨珍珠质形成的分子生物学机制奠定了一定基础。     

6-磷酸山梨醇脱氢酶(gutD)基因克隆、测序和表达

本研究根据6-磷酸山梨醇脱氢酶基因(gutD)两端序列设计引物，以假单胞杆菌(Pseudonmonas XM)的总DNA为模板通过PCR的方法克隆gutD基因并进行序列分析，将克隆得到的gutD基因与原核表达载体pBV220连接并转化大肠杆菌(Escherichia coli)JM101后检测其蛋白表达及菌株生长耐盐性。     

Food partitioning of leaf-eating mangrove crabs (Sesarminae): Experimental and stable isotope (C and N) evidence

The feasibility of mangrove leaves as a full diet for sesarmid crabs has been questioned for decades. Since these leaves are nitrogen-poor, sesarmids probably obtain nitrogen from other sources to sustain growth. The aim of this study was to assess the food partitioning of the sesarmid species Neoepisesarma versicolor with emphasis on nitrogen allocation. The preference for animal tissue when crabs were pre-fed diets of different nitrogen content was determined in the laboratory. Furthermore, the possible in situ diet composition of N. versicolor was established from carbon and nitrogen stable isotope signature (δ¹³C and δ¹⁵N) of freshly caught individuals and their potential food sources, using a concentration-dependent mixing model. N. versicolor showed significantly higher feeding preferences for fish meat when pre-fed leaf material without than with access to meat, indicating that this crab species can meet its nitrogen demand by ingesting animal tissue. The stable isotope mixing model based on in situ materials suggests that the diet of N. versicolor consists of ∼60% leaves in terms of biomass, leaving ∼40% for other sources such as animal tissue and benthic microorganisms. The biomass contribution from animal tissues, in form of e.g. other crustaceans and fish carcasses, was found to account for ∼15%. Despite the relative low biomass fraction, animal food sources may contribute with up to half of the nitrogen in the diet of N. versicolor. The quantity of ingested sediment most likely exceeds that of animal tissues. However, due to the low concentration of assimilable microalgae and other microorganism, we propose that sediment associated sources are less important as a nitrogen source for N. versicolor than hitherto presumed.     

长牡蛎nacrein 基因的克隆、结构及进化分析

首次在长牡蛎(Crassostrea gigas)中克隆得到一种新的贝壳基质蛋白nacrein-like protein F3的全长c DNA序列。nacrein-like protein F3基因c DNA全长1499bp,其中编码区长度为1242bp,编码一条含413个氨基酸残基的多肽链。氨基酸序列比对和结构域分析均表明其为合浦珠母贝(Pinctada fucata)nacrein的同源蛋白,含有1个保守的α-碳酸酐酶结构域,但由于重复结构域的插入,α-碳酸酐酶结构域被间隔成2个亚结构域。系统进化分析显示nacrein-like protein F3与贻贝(Mytilus californianus)nacrein-like protein进化关系最近。此外,在软体动物中,双壳纲nacrein-like proteins进化速度相对较快,推测与寒武纪时期剧烈的环境变化有关,如影响贝壳形成的海水化学变化。     

Molecular characterization and expression of the SiUCP2 gene in sea urchin Strongylocentrotus intermedius

Uncoupling protein 2 (UCP2) is a proton transporter located in the inner mitochondrial membrane, and inhibits the formation of adenosine triphosphate and reacti...