Genome mapping in aquatic animals: Progress and future perspectives

The genome of aquatic animals is poorly understood and information from different taxonomic groups is sketchy. While there have been intensive genomic studies on some fish models, investigations on other fishes and invertebrates have been scarce. Yet there are recently some coordinated studies on genome mapping in a number of aquaculture animals of economic importance. This review summarizes information available on genome mapping of the important fish models and aquaculture animals. The future perspectives of this field of studies are discussed.

Determination of microcystin-LR and its metabolites in snail (Bellamya aeruginosa), shrimp (Macrobrachium nipponensis) and silver carp (Hypophthalmichthys molitrix) from Lake Taihu, China

This paper describes seasonal changes of microcystin-LR (MC-LR) and its glutathione (MC-LR-GSH) and cysteine conjugates (MC-LR-Cys) in three aquatic animals - snail (Bellamya aeruginosa), shrimp (fMacrobrachium nipponensis) and silver carp (Hypophthalmichthys molitrix) collected from Lake Taihu, China. MC-LR, MC-LR-GSH, and MC-LR-Cys were determined by liquid chromatography electrospray ionization mass spectrum (LC-ESI-MS). The mean MC-LR concentrations in the hepatopancreas of snail and shrimp and liver of silver carp were 6.61, 0.24, and 0.027 mu g g(-1) dry weight (DW), respectively: while the average MC-LR-Cys concentrations were 0.50, 0.97, and 5.72 mu g g(-1) DW, respectively. MC-LR-GSH was usually not detectable in these samples. The above results suggest that: (1) in aquatic animals, especially fish, the main excretion form of MC-LR could be MC-LR-Cys, but not MC-LR-GSH, whereas MC-LR-Cys might play an important role in detoxication of MC-LR and (2) that efficiency of MC-LR-Cys formation differs among species. The main detoxication pathway of MC-LR in aquatic animals is suggested as follows: when MC-LR enters into liver/hepatopancreas, it firstly conjugates with polypeptide or protein (including GSH, PP-1 and 2A) containing Cys residues, perhaps also some free cysteine; subsequently, MC-LR-Cys is degraded from these polypeptide or protein; and finally is excreted from animals by the compound of MC-LR-Cys. (C) 2009 Elsevier Ltd. All rights reserved.

Metallothionein-2 gene from the mandarin fish Siniperca chuatsi: cDNA cloning, tissue expression, and immunohistochemical localization

The metallothionein-2 (MT-2) gene was isolated from the mandarin fish, one of the most important industrial aquatic animals in China, by using rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of MT-2 comprised 60 amino acids and showed approximately 62.3% identity to human metallothionein. Its promoter region was amplified by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The MT-2 gene consists of 3 exons and 2 introns, extending approximately 900 bp of genomic sequence. Phylogenetic analysis clearly demonstrated that MT-2 formed a clade with fish metallothionein. The promoter region contained 5 putative metal-regulatory elements (MREs) and 1 TATA box. Real-time quantitative RT-PCR analysis revealed that MT-2 transcripts were significantly increased in the brain and gills and were stable in the muscles, liver, and trunk kidney in Cd2+-stimulated fish. Western blotting analysis demonstrated that the protein of the MT-2 gene was expressed mainly in the gills, liver, heart, trunk kidney, muscle, and intestine; it was weakly detected in the brain and head kidney. Moreover, the MT-2 protein was immunohistochemically detected in the cytoplasm in the liver and trunk kidney. All the above results revealed that the mandarin fish MT-2 would be a useful biomarker for metal pollution. (C) 2008 Published by Elsevier Inc.

Environmental signals: Synthetic humic substances act as xeno-estrogen and affect the thyroid system of Xenopus laevis

According to outdated paradigms humic substances (HS) are considered to be refractory or inert that do not directly interact with aquatic organisms. However, they are taken up and induce biotransformation activities and may act as hormone-like substances. In the present study, we tested whether HS can interfere with endocrine regulation in the amphibian Xenopus laevis. In order to exclude contamination with phyto-hormones, which may occur in environmental isolates, the artificial HS 1500 was applied. The in vivo results showed that HS 1500 causes significant estrogenic effects on X. laevis during its larval development and results of semi-quantitative RT-PCR revealed a marked increase of the estrogenic biomarker estrogen receptor mRNA (ER-mRNA). Furthermore, preliminary RT-PCR results showed that the thyroid-stimulating hormone (TSH beta-mRNA) is enhanced after exposure to HS1500, indicating a weak adverse effect on T3/T4 availability. Hence, HS may have estrogenic and anti-thyroidal effects on aquatic animals, and therefore may influence the structure of aquatic communities and they may be considered environmental signaling chemicals. (c) 2005 Elsevier Ltd. All rights reserved.

Physiological and biochemical microcystin-RR toxicity to the Synechococcus elongatus

Freshwater Microcystis may form dense blooms in eutrophic lakes. It is known to produce a family of related cyclic hepatopeptides (microcystins, MC) that constitute a threat to aquatic ecosystems. Most toxicological studies of microcystins have focused on aquatic animals and plants, with few examining the possible effects of microcystins on phytoplankton. In this study we chose the unicellular Synechococcus elongatus (one of the most studied and geographically most widely distributed cyanobacteria in the picoplankton) as the test material and investigated the biological parameters: growth, pigment (chlorophyll-a, phycocyanin), photosynthetic activity, nitrate reductase activity, and protein and carbohydrate content. The results revealed that microcystin-RR concentrations above 100 mug (.) L-1 significantly inhibited the growth of Synechococcus elongatus. In addition, a change in color of the toxin-treated algae (chlorosis) was observed in the experiments. Furthermore, MC-RR markedly inhibited the synthesis of the pigments chlorophyll-a and phycocyanin. A drastic reduction in photochemical efficiency of PSII (F-v/F-m) was found after a 96-h incubation. Changes in protein and carbohydrate concentrations and in nitrate reductase activity also were observed during the exposure period. This study aimed to evaluate the mechanisms of microcystin toxicity on a cyanobacterium, according to the physiological and biochemical responses of Synechococcus elongatus to different doses of microcystin-RR. The ecological role of microcystins as an allelopathic substance also is discussed in the article. (C) 2004 Wiley Periodicals, Inc.

A review of advances in research on marine molluscan antimicrobial peptides and their potential application in aquaculture

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. They are usually characterized by their small-size, heat-stability and broad range of antimicrobial activity. This review covers research advances on marine mollusc AMPs, specifically those isolated from mussels, scallops, oysters, venerid clams and abalone, which mainly include MGD, mytilin, myticin, mytimycin, big defensin, and RPD-1. Their structural characteristics, antibacterial activity, and expression pattern as well as peptide distribution and their release following microbial challenge are also discussed. In addition, the prospect of the application of AMPs as food additives or their use in immunostimulation to prevent diseases of aquatic animals, as well as their potential hazards, are also discussed.

Aquatic resource conservation - The first Yangtze finless porpoise successfully born in captivity

Background. As the sole freshwater subspecies of finless porpoise (Neophocaena phocaenoides), the Yangtze finless porpoise (N. p. asiaeorientalis) lives only in the middle and lower reaches of the Yangtze River and its appended Poyang and Dongting Lakes. As a result of human activity on the river, including over and illegal fishing, pollution, transportation and dam construction, the population of Yangtze finless porpoises has been steadily and rapidly decreasing during the past several decades, which leads the animal to be endangered. Methods. For saving this unique animal from extinction, three corresponding measures, in situ conservation, ex situ conservation, and intensifying breeding research in captivity, were proposed and have been implemented since the 1980s. Results. After successfully rearing the animals in captivity for almost nine years, the first Yangtze finless porpoise was successfully born in captivity on July 5, 2005. The calf is male, with a body length of 69 cm. This is the first freshwater cetacean ever born in captivity. Conclusion. The successful birth of this calf confirms that it is possible to breed the Yangtze finless porpoise in captivity. Furthermore, this will greatly benefit the conservation efforts, and also greatly bolster our on-going efforts to study the reproductive biology of these animals. Recommendation. More studies and efforts are expected to establish a sustainable, captive colony of the Yangtze finless porpoise, which will not only greatly expand our knowledge about the reproduction biology of this animal, but also help to redeem the wild population through a careful yearly 'soft releasing' process.

三峡大坝的修建对库区动物的影响

世界上最大的大坝—三峡大坝―于2003年6月正式建成并蓄水，三峡水库的水位由蓄水前的84m上升至蓄水后的139m。三峡大坝的修建与蓄水，已经显著地改变了三峡库区陆地生态系统和水域生态系统的生态环境。三峡大坝的修建将如何影响生活在这一区域（包括陆地和水域）的野生动植物资源，是个亟待解决的问题。 对于陆地生态系统，三峡大坝的修建，使三峡库区的生态环境进一步破碎化（Fragmentation）和岛屿化（Island）。为了了解三峡大坝的修建对三峡库区野生动物所产生的影响，我们选择适应性强且对环境变化敏感的啮齿动物为研究对象，在三峡库区选取两个岛屿（洛碛岛和皇华岛）及其对岸为研究点，在野外调查了啮齿动物的群落组成、种群密度与分布，并用胃容物分析和稳定性同位素分析方法研究了两个研究点优势种啮齿动物的食物来源与组成。结果表明，两个研究点间啮齿动物群落组成有很大差异，这主要是由于啮齿动物分布的地域性差异引起的。在同一个研究点，岛屿上的啮齿动物群落组成与对岸的没有差异，但在岛屿上的啮齿动物种群密度却明显高于其对岸的，这说明随着库区水位的大幅度提升，岛屿的面积急剧减小，使原本栖息在水边的啮齿动物不得不向岛屿的中上部迁移，致使岛屿上的啮齿动物的种群密度迅速增加。啮齿动物的食物来源有四种：C3非豆科植物，C3豆科植物，C4植物及水生生物。不同种啮齿动物的食物种类组成也不同，四川短尾鼩（Anourosorex squamipes）的食物中有水生生物的组成成分，但其它啮齿动物食物中不含水生生物。不同食物来源在啮齿动物的整体食物中所占比例也不同，并呈明显的季节性变化，这种变化与田间的农作物种类密切相关。动物组织的稳定性同位素组成不仅可以示踪动物的食物来源，也反映了动物的生存状况。我们的结果显示：对岸啮齿动物的稳定性同位素组成相对集中，并且不同种啮齿动物间没有重叠。这一方面说明对岸啮齿动物的食物来源相对丰富，啮齿动物可以选择各自喜食食物，在此环境下的啮齿动物种间竞争相对较弱，至少是种间食物竞争较弱。岛屿上啮齿动物的稳定性同位素值分布范围远大于在对岸的，并且种间稳定性同位素组成有明显的重叠部分。稳定性同位素组成的重叠意味着有相似的食物来源，也即栖息生境相似。这说明岛上的啮齿动物种间存在较剧烈的食物竞争关系。在资源限制情况下，多种啮齿动物不得不利用共同的食物来源。换句话说，岛屿化过程将加剧岛屿上啮齿动物的种间竞争。 洛碛岛上的四川短尾鼩因为可以利用水生食物来源，这使得它在岛屿化过程中处于有利地位。随着岛屿化进程的加剧，四川短尾鼩在岛屿上的优势将更加明显。而皇华岛屿上的褐家鼠的食物来源单一，会因岛屿面积的进一步减小，食物来源更加缺乏，它们将不得不改其食物组成或面临消失。而白腹巨鼠的栖息环境靠近江边，将因水位的上升进一步上移，它们的栖息环境与普通田鼠的发生重叠，使得白腹巨鼠与普通田鼠发生栖息地的竞争与食物竞争，并面临更多的人为因素干扰。 对于水域生态系统，长江是我国最大的河流，也是我国淡水鱼类最丰富的区域。三峡大坝的修建已经显著改变了三峡库区的水文特征。为了了解三峡大坝的修建是否会改变三峡库区的有机物组成，并进而影响到三峡库区水生生物的食物来源和组成，我们选择了三个水文特征不同的研究点（洛碛江段、皇华城江段和茅坪江段）调查了三峡库区的常见鱼种类组成，并用稳定性同位素方法研究了三峡库区洪水前后的有机物组成变化与鱼类的食物网模型，用稳定性同位素划分了鱼类食物网结构及鱼类体长与其肌肉δ15N值间的关系。结果表明，三峡大坝的修建，已经显著改变了皇华城和茅坪江段的水文特征，同时也“干扰”了生活在这一区域的鱼类。适应于流水环境的鱼类在库区回水处和大坝附近几乎消失，而喜欢静态环境的其它鱼类却得到极大的发展，例如鲢鱼（H. molitrix）和草鱼（ C. idellus）。 洪水前后，三峡库区的有机物组成成分有明显改变。洪水前，水中有机物主要以河流自身生产力产生的有机物为主（浮游植物、藻类等）;洪水后，水中有机物主要以外来有机物为主（陆地植物、土壤有机物和从上游带来的有机物）。对于三个食物网模型：河流连续体模型（RCC）、脉冲模型（FPC）和河流生产力模型（RPM），河流生产力模型能更好的解释三峡库区的水生生物的食物来源，即三峡库区的水生生物的食物主要来源于河流自身生产力产生的有机物。但外来有机物作为水生生物的一种辅助食物来源，在洪水期间起到不可忽视的作用。 在本研究中，鱼类体长与其δ15N值间的关系与选取鱼的种类有关，比如南方大口鲇（Silurus asotus）的体长与其肌肉的δ15N值呈明显负相关关系（R2≈0.5），而鱖鱼（Siniperca）、铜鱼（Coreius guichenoti）和草鱼（Ctenopharyngodon idellus）的体长与其肌肉的δ15N值却呈正相关关系。 三峡库区常见鱼类主要以杂食性和广普性鱼类为主，食物网结构十分复杂。通过胃容物分析和稳定性同位素分析，三峡库区水生生物营养级间的判别值约为3.1‰。依据鱼类肌肉的δ15N值，三峡库区的常见鱼类可以划分为四个营养级：草食性鱼类（herbivorous fish）、初级杂食性鱼类（1ºomnivorous fish）、次级杂食性鱼类（2ºomnivorous fish）和食鱼性鱼类（piscivorous fish）。营养级间没有明确的分界限，鱼类的营养级从2到4.8，是连续分布的营养级结构，从而更真实的反映了自然界中动物的捕食关系和在食物网中的位置。 自然丰度变化的稳定性同位素(Stable isotope)作为一种天然的示踪物，在动物生态学上已经得到广泛应用。动物的稳定性同位素可以清楚的示踪动物的食物来源、食物组成、栖息地情况和生存状况等多种信息，结合传统的胃容物分析，或其它的粪便分析、储藏物分析等，稳定性同位素技术在动物生态学研究方面必将得到更广泛的应用。

Characterization of Sarcocystis species in domestic animals using a PCR-RFLP analysis of variation in the 18S rRNA gene: a cost-effective and simple technique for routine species identification

Thirteen restriction endonucleases were used to investigate nucleotide sequence variation in the 18S rRNA DNA of 88 individuals from ten Sarcocystis taxa collected as cysts from their intermediate hosts, swine, cattle and water buffalo. A DNA sequence of

Comparative responses in rare minnow exposed to 17 beta-estradiol during different life stages

Present in the excrement of humans and animals, 17 beta-estradiol (E-2) has been detected in the aquatic environment in a range from several nanograms to several hundred nanograms per liter. In this study, the sensitivities of rare minnows during different life stages to E-2 at environmentally relevant (5, 25, and 100 ng l(-1)) and high (1000 ng l(-1)) concentrations were compared using vitellogenin (VTG) and gonad development as biomarkers under semistatic conditions. After 21 days of exposure, VTG concentrations in whole-body homogenates were analyzed; the results indicated that the lowest observed effective concentration for VTG induction was 25 ng l(-1) E-2 in the adult stage, but 100 ng l(-1) E-2 in the larval and juvenile stages. After exposure in the early life stage, the larval and juvenile fish were transferred to clean water until gonad maturation. No significant difference in VTG induction was found between the exposure and control groups in the adults. However, a markedly increased proportion of females and appearance of hermaphrodism were observed in the juvenile-stage group exposed to 25 ng l(-1) E-2. These results showed that VTG induction in the adult stage is more sensitive than in larval and juvenile stages following exposure to E-2. The juvenile stage may be the critical period of gonad development. Sex ratio could be a sensitive biomarker indicating exposure to xenoestrogens in early-life-stage subchronic exposure tests. The results of this study provide useful information for selecting sensitive biomarkers properly in aquatic toxicology testing.

Hexabromocyclododecane-induced developmental toxicity and apoptosis in zebrafish embryos

Hexabromocyclododecane (HBCD) is widely used as a brominated flame retardant, and has been detected in the aquatic environment, wild animals, and humans. However, details of the environmental health risk of HBCD are not well known. In this study, zebrafish embryos were used to assess the developmental toxicity of the chemical. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to various concentrations of HBCD (0, 0.05, 0.1, 0.5, and 1.0 mg L-1) until 96 h. Exposure to 0.1, 0.5, and 1.0 mg L-1 HBCD significantly increased the malformation rate and reduced survival in the 0.5 and 1.0 mg L-1 HBCD exposure groups. Acridine orange (AO) staining showed that HBCD exposure resulted in cell apoptosis. Reactive oxygen species (ROS) was significantly induced at exposures of 0.1, 0.5, and 1.0 mg L-1 HBCD. To test the apoptotic pathway, several genes related to cell apoptosis, such as p53, Puma, Apaf-1, caspase-9, and caspase-3, were examined using real-time PCR. The expression patterns of these genes were up-regulated to some extent. Two anti-apoptotic genes, Mdm2 (antagonist of p53) and Bcl-2 (inhibitor of Bax), were down-regulated, and the activity of capspase-9 and caspase-3 was significantly increased. The overall results demonstrate that waterborne HBCD is able to produce oxidative stress and induce apoptosis through the involvement of caspases in zebrafish embryos. The results also indicate that zebrafish embryos can serve as a reliable model for the developmental toxicity of HBCD. (C) 2009 Elsevier B.V. All rights reserved.