环境DNA分析技术—一种水生生物调查新方法

掌握珍稀濒危和外来入侵物种的分布状况对于物种保护和管理十分重要。环境DNA(Environmental DNA)分析通过收集、分离和分析环境样品中的DNA来检测物种是否存在,是一种低耗、高效、高灵敏度的无损伤性物种监测新技术(e DNA)。本文综述了环境DNA技术的发展、分析方案、优势及存在的问题,主要综述了该方法在外来入侵物种足迹追踪、濒危珍稀水生生物资源调查和物种多样性分析中的研究现状,并对环境DNA分析技术在生物多样性保护研究中的应用前景进行了展望。     

环境DNA在水生生态系统生物量评估中的研究进展

环境DNA (Environment DNA, eDNA)技术因其无创、高效、经济、灵敏等特点在水生生态系统生物评价中愈来愈引起人们的重视。文章围绕eDNA技术的内容、应用现状和面临的挑战3个方面,系统综述了该技术在实验室环境和野外环境水生生态系统生物量评估中的应用进展,探讨其优劣势、生态过程、评估错误等内容,展望了该技术在水生生物量评估领域的应用前景,以期为eDNA技术的相关应用研究提供参考。     

水生食物网研究方法的发展和应用

水生生物在食物网中的相互关系及其变化是生态学研究的重要领域之一,其实质是研究生物之间的捕食与被捕食关系。传统的水生食物网研究方法是基于形态学观察的胃含物分析,具有较大的局限性。随着科学技术的发展,稳定同位素技术、特征脂肪酸组成分析和DNA条形码等分子技术的引入有效的弥补了胃含物分析的不足,并可以更深入的了解水生生物的摄食信息,因此得到了广泛的应用。本文采用文献计量法分析了水生食物网研究方法的发展动态,分别介绍了稳定同位素技术、特征脂肪酸组成分析和DNA条形码技术与胃含物结合应用的研究进展,着重归纳总结了其在水生生物食性研究中的应用现状及发展前景,详细地指出了4种水生食物网研究方法的优势和局限性、适用范畴和对实验样品的需求。胃含物分析作为传统食物网研究方法未来仍是不可缺少的一部分,其向我们传递了最为直接的摄食信息。在此基础上,稳定同位素技术、特征脂肪酸组成分析和DNA条形码等分子技术作为胃含物分析方法的补充手段,将更有利于重建复杂的水生食物网结构,进而为渔业管理提供必要的帮助。     

环境DNA在水域生态中的研究进展

环境DNA(environmental DNA,eDNA)是指生物通过皮肤脱落、唾液、配子、粪便以及分泌物等方式向环境中释放的游离DNA。环境DNA具有敏感性、准确性以及容易操作等诸多优势,更能实时地反映物种多样性以及生物量等,近两三年受到了世界各地学者们的大量关注。水域环境高度复杂,环境DNA在水域生态领域具有重要的应用价值。本文主要从环境DNA在水域生态的应用以及研究方法方面对环境DNA的研究做一小结,同时介绍环境DNA在其他生境的应用,以期为水域生态的研究提供参考。     

水生生物环境丰容技术及其应用研究进展

水生生物作为人类生活的重要组成部分,其在养殖、运输以及展示等过程中福利水平低下现象层出不穷,寻求合适的方法来解决水生生物福利低下的问题,成为保障并提升水生生物福利的一大重要工作。环境丰容作为一项能够通过对圈养动物生存环境进行优化,提升圈养动物福利水平,使得圈养动物获得生理和心理健康,展示其自然行为的技术,成为保障和提升水生生物福利的一个重要手段。目前,与国外相比,国内对水生生物环境丰容技术的研究还处于起步阶段,如何利用环境丰容技术改善水生生物的生存环境,保障并提升水生生物福利水平日益成为研究热点。为此,本文综述了环境丰容的定义、发展历程、环境丰容与动物福利的关系以及在水生生物中的4种主要环境丰容技术。同时,就水生生物环境丰容技术存在的问题进行讨论并提出建议,以期为水生生物环境丰容提供更多方案参考和理论支撑。     

环境DNA技术在水域环境中的应用进展

简述了环境DNA发展进程,介绍了环境DNA应用领域,包括物种及物种多样性研究、生物量评估、珍稀物种和入侵物种监测。归纳了环境DNA研究方法,样品的采集与保存、eDNA捕获、提取、分析。指出了eDNA技术的优势和存在的问题。提出,在今后的环境DNA研究中,一方面要大力发展并推广eDNA技术;另一方面应尽快制定一个统一的eDNA监测方法标准化框架,将eDNA技术和传统调查方法相结合,才能更好地对水生生物进行监测,在水生生态文明建设中发挥更为重要的作用。     

环境DNA技术发展及其在长江流域水生生态学领域的应用研究进展

长江流域鱼类资源丰富、生物多样性高。近年来,受人为干扰、环境变化等因素影响,鱼类资源急剧衰退,全面了解该流域水生生态学信息、进行长江大保护迫在眉睫。随着分子生物学技术的发展,环境DNA技术应运而生,其相比于传统调查方式更加高效、灵敏,应用领域更广;该技术的灵敏性使其非常适合于检测濒危物种、低密度物种入侵、瞬时和隐秘物种的存在,特别是当检测低密度物种的采样工作难以控制时,其敏感性、简便性和降低危害性的优势愈加显现出来。因此,该技术已被广泛应用于食品微生物、生物监测、群落生态学、古环境、保护生物学和生物入侵等领域的研究。介绍了环境DNA定义、发展史、研究方法与优劣势,在此基础上概述了其在长江流域水生生态学领域的应用研究进展,最后展望了环境DNA技术与环境RNA技术相结合的技术革新以及新一代测序手段、大数据及机器智能技术多技术结合助力该领域研究的前景,以期为长江流域持续性生态学监测提供借鉴和参考。     

水生生物近缘种和产地的分子生物学判别

随着分子生物学技术的发展,渔业生物近缘种及产地的判别由形态学深入到了蛋白质和DNA水平。本文综述了目前已经应用于水生生物的相关分子生物学技术(包括色谱、免疫学判别以及蛋白的电泳、DNA的序列分析、PCR-RFLP、RAPD、AFLP、SNP、SSRI、SSR、Real-Time PCRs、pecies-specific primer PCR和PCRlab-on-a-chip等)以及它们各自的优缺点;在把握了这些研究进展的基础上,展望了相关技术的发展趋势。     

多环芳烃对水生动物毒性效应的研究进展

多环芳烃(PAHs)是广泛存在于水体环境中的一类持久性有机污染物,因其具有致癌、致畸和致突变性而备受关注。本文简述了PAHs的定义、分类、来源及其污染现状,并综述了PAHs在水生生物体内蓄积、代谢规律及其对水生生物的毒性效应:在急性毒性层面综述了PAHs对不同水生动物的毒性程度及急性致死效应;在亚急性及慢性毒性层面分别从分子水平、生理生化酶水平、细胞组织水平,综述了:PAHs暴露对水生生物的分子毒性,导致DNA单链断裂、PAHs-DNA加合物的形成;PAHs暴露对水生生物抗氧化酶系统(SOD、CAT、GPx、GSH/GSSG)、外源性有机污染物代谢酶(EROD、GST)活力的影响;PAHs暴露引起的氧化应激、脂质过氧化、组织病理学变化等氧化损伤。本综述目的是便于今后更深入地研究PAHs对水生动物的毒性效应和毒性作用机制,进而更好的控制PAHs污染,保护水生生物和水生生态环境。     

中科院水生所关于水生病毒学研究取得进展

<正>蛙病毒(Ranavirus)是一类能跨种感染全球各地变温动物(包括鱼类、两栖类、爬行类等)的核质大DNA病毒(NCLDVs)成员,但其复制和转录机制在较大程度上仍属未知。近日,中国科学院水生生物研究所水生病毒学学科组基于建立高效蛙病毒重组、基因复制及功能测试系统,以及从鱼类细胞新生DNA中分离特定蛋白、重组病毒亲和层析和纳米荧光素酶Nano Luc互补及质谱分析技术,鉴定并揭示了蛙病毒复制和转录机器的组织架构及相关作用机制。     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.