Cloning and expression of alpha class glutathione S-transferase gene from the small hermaphroditic fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae)

In order to assess its potential as a biomarker of aquatic pollution, an alpha class glutathione S-transferase gene (GSTα gene) was cloned from the small hermaphroditic fish Rivulus marmoratus. The R. marmoratus GSTα gene spanned 1.3 kb, consisting of 6 exons encoding 221 amino acid residues. It showed high similarity to zebrafish GST. We named this R. marmoratus GSTα gene as rm-GSTα. The cDNA of the rm-GSTα gene was also investigated for its phylogeny, tissue-specific and chemical-induced expression. Rm-GSTα was subcloned into a 6 × His-tagged pCR^®T7 TOPO TA expression vector to produce the recombinant 6 × His-tagged rm-GST protein. This will be used in future to raise an rm-GSTα antibody for use in the study of phase II metabolism involved in detoxification. We also exposed R. marmoratus to 300 μg/l of 4-nonylphenol in water, and found approximately 4-fold induction of R. marmoratus GSTα mRNA in the treated animals. In this paper, we discuss the characteristics of the R. marmoratus GSTα gene as well as its potential use in relation to environmental pollution.     

大型溞(Daphnia magna)线粒体基因组的测定与序列分析

利用Long-PCR及普通PCR技术,结合引物步移法测序,经拼接、组装后获得中国淮河大型溞(Daphnia magna)线粒体基因组全序列,并对其基因组成、结构特点等进行初步分析.大型溞线粒体基因组全长为14948 bp,A、T、G、C各碱基含量分别为32.37%、34.73%、15.58%、17.31%,表现出明显的AT偏倚.13个蛋白质编码基因、22个t RNA基因、2个rRNA基因和1个D-loop区(控制区)的排列方式与典型的节肢动物线粒体基因组的排列方式略有不同.除COI基因以CTG、ATP8基因以GTG、ND3基因以ATC以及ND6基因以ATT为起始密码子以外,其余9个蛋白质编码基因均以ATG作为起始密码子;9个蛋白质编码基因具有典型的完全终止子TAG或TAA,COI、COII、ND4和ND5等基因采用不完全终止密码子T.基因组包含9个基因间隔区,共81 bp,长度1~62 bp;13个基因重叠区,共77 bp,重叠碱基数在1~27 bp之间,最大重叠在16 S rRNA和tRNA~(Val)基因之间.在预测的22个tRNA基因的二级结构中,发现只有tRNA~(Ser1)基因未能形成完整的二级结构,其他21个基因均可形成典型的三叶草结构.16 S rRNA和12 S rRNA基因长度分别为1373 bp和752 bp,D-loop区全长289 bp.本研究结果为探讨大型溞在溞属中的系统学地位及其与其他种间的系统发生关系等问题提供了数据资源.     

Copper toxicity in the marine copepod Tigropus japonicus: Low variability and high reproducibility of repeated acute and life-cycle tests

The intertidal copeopod Tigriopus japonicus, which is abundant and widely distributed along the coasts of Western Pacific, has been suggested to be a good marine ecotoxicity testing organism. In this study, a series of experiments were conducted to investigate the reproducibility and variability of copper (Cu) sensitivity of T. japonicus so as to evaluate its potential to serve as an appropriate test species. To understand the seasonal variation of Cu sensitivity, individuals of T. japonicus were collected from the field in summer and winter, and subjected to standard 96 h acute (static renewal) toxicity tests. 96 h-LC50 values of T. japonicus collected from the two seasons were marginally different (p = 0.05), with an overall coefficient of variation (CV) of 33%. Most importantly, our results indicated that chronic Cu sensitivity of T. japonicus was highly reproducible. The CVs of intrinsic rates of increase in the population of the control and Cu treatment (10 μg Cu l⁻¹) groups were only 10–11% between 10 runs of a standardised complete life-cycle test. Moreover, different Cu(II) salts generally resulted in a similar 96 h-LC50 value while Cu(I) chloride was consistently slightly less toxic than Cu(II) salts. Given such a high reproducibility of toxic responses, it is advocated to use T. japonicus as a routine testing organism.     

Insights into the diversity,distribution and phylogeny of three ergasilid copepods (Hexanauplia: Ergasilidae) in lentic water bodies of the Morava river basin,Czech Republic

Ergasilidae are a globally distributed group of copepods parasitising fish. Despite their common occurrence in European freshwaters, there is relatively little molecular data available. In this study, we recorded three ergasilid copepod species parasitising six fish species (families Cyprinidae, Percidae, Centrarchidae) in lentic water bodies along the River Morava in the Czech Republic: Ergasilus sieboldi infecting fish gills, Paraergasilus longidigitus infecting nasal cavities and Neoergasilus japonicus infecting fins. This represents a further occurrence of N. japonicus as a non-native European species, and the first report of P. longidigitusis from Central Europe waters. While strong niche selection potentially offers a free niche for each of these ergasilids in all hosts, parasite co-infections were relatively rare, with species distribution among hosts reflecting host preference. This study provides the first molecular data for E. sieboldi and P. longidigitus as native European ergasilid copepods. Phylogenetic analysis based on combined 18S and 28S rDNA showed a close relationship between P. longidigitus with other Paraergasilus species, supporting a monophyletic origin of this genus. The phylogenetic position of E. sieboldi was not clearly resolved, highlighting the need for more data and further intensive genetic investigations of other ergasilid species.     

A comparative study of toxicity identification using Daphnia magna and Tigriopus japonicus: Implications of establishing effluent discharge limits in Korea

In Korea, the new permission criteria for industrial effluents based on Daphnia magna acute toxicity tests will be gradually implemented starting from 2011. Thus, in this study, toxicity assessment and identification using a marine species (Tigriopus japonicus) and the freshwater species (D. magna) was comparatively investigated. Effluent from an acid mine drainage treatment plant showed acute toxicity toward both organisms due to low pH, which was removed by neutralization of the effluent. Additionally, evaluation of the effluent of an electronics company revealed that Cu was attributable to the observed toxicity, and the effluent was more toxic toward T. japonicus than D. magna. Moreover, effluents from a metal plating factory were acutely toxic toward D. magna (6.50 TU), while they were not toxic against T. japonicus. Toxicity identification revealed that the high level of Cl⁻ (12,841 mg L⁻¹) was the cause of toxicity. Thus, the effluents had no effect on the marine species, T. japonicus. These findings suggest that a marine species rather than a freshwater species is more desirable for toxicity assessment of industrial effluent discharged into the saltwater, and thus should be considered in the legislation of toxicity-based discharge limits in Korea.     

Molecular cloning and β-naphthoflavone-induced expression of a cytochrome P450 1A (CYP1A) gene from an anadromous river pufferfish, Takifugu obscurus

In recent years, there has been a decline in the wild populations of river pufferfish, Takifugu obscurus. Besides overexploitation for commercial purposes, environmental pollution is believed to have contributed to its decline. However, almost no information exists about genes involved in metabolism of xenobiotics by this species. Nevertheless, there is interest in fugu fishes, since they possess the smallest genome among vertebrates. We cloned and characterized the full-length cDNA sequence of a cytochrome P450 1A (CYP1A) gene from T. obscurus. Phylogenic relationship of T. obscurus CYP1A was also compared to other fish species. The tissue distribution and time-dependant induction of CYP1A mRNA were studied by real-time PCR in T. obscurus exposed to an aryl hydrocarbon receptor (Ahr) agonist, β-naphthoflavone (BNF). The greatest basal expression in livers of control as well as BNF-treated individuals. However, brain, gill, gonad, intestine, and kidney also expressed CYP1A. Muscles expressed the least CYP1A. The results of the time-course study revealed induction in brain and gills after 6 h and at 12 h in most tissues. Except for gills, all other organs retained induced expression of CYP1A mRNA up to 96 h.     

Cloning and developmental expression of kinesin superfamily7 (kif7) in the brackish medaka (Oryzias melastigma), a close relative of the Japanese medaka (Oryzias latipes)

kif7 is a member of the kinesin superfamily members which are molecular motor proteins that move along microtubules in a highly regulated manner through ATP hydrolysis. In this paper, we report on the cloning of the Oryzias melastigma kif7 (omkif7) using primers designed according to the Japanese medaka (Oryzias latipes) database. The cloned omkif7 has an open reading frame of 3762bp and is deduced to encode a polypeptide of 1254 amino acids that possesses the putative ATP-binding and microtubule-binding motifs in its motor domain at the N-terminal region. We characterized the cloned omkif7 by comparison with the zebrafish kif7. Both omkif7 and zebrafish kif7 are shown to be expressed in all embryonic stages and adult tissues examined with higher expression level in the testis and ovary. Whole-mount in situ hybridization revealed that the expression of omkif7 is ubiquitous during the early stages of embryonic development, but became more restrictive and localized to the brain, fin bud and eye at later development. This study suggested that the brackish O.melastigma can serve as a good seawater model organism for developmental studies by utilizing the resources developed from its close relative of the Japanese medaka.     

三种裸腹溞的16S rDNA序列分析与系统分类探讨

为了检验已记录的3种裸腹溞(发头裸腹溞(Moina irrasa)、短型裸腹溞(Moina brachiata)、微型裸腹溞(Moina micrura))的系统分类,用试剂盒法分别提取3种裸腹溞的基因组DNA.利用特异性引物,通过PCR扩增了3种裸腹溞的16S r DNA部分序列,并与来自Gen Bank中每个种类相似度较高的裸腹溞属序列进行分析.结果表明,3种裸腹溞的平均种间相似度为88.7%,碱基中A+T含量均明显高于G+C含量.本研究的发头裸腹溞的16S r DNA序列与Gen Bank所下载的多刺裸腹溞(Moina macrocopa)的16S r DNA序列相似度为99%,遗传距离(K2P双参考模型)为0.5%,属种内范围;两个地区的短型裸腹溞测得的16S r DNA序列与Gen Bank下载的欧洲短型裸腹溞的16S r DNA序列序列相似度相对较低(88%~90%),遗传距离较大(13.2%~13.5%左右),已达到属内种间分化水平.基于16S r DNA构建的NJ树和贝叶斯树也支持以上结论.结果表明,本研究的发头裸腹溞可能为多刺裸腹溞,本研究用的短型裸腹溞与Gen Bank下载的欧洲短型裸腹溞已经达到种间分化的标准.由于缺乏物种形态资料和其他分子标记的对比,3种裸腹溞的分类地位还需进行更深入的探讨.     

Copper toxicity in the marine copepod Tigropus japonicus: Low variability and high reproducibility of repeated acute and life-cycle tests

The intertidal copeopod Tigriopus japonicus, which is abundant and widely distributed along the coasts of Western Pacific, has been suggested to be a good marine ecotoxicity testing organism. In this study, a series of experiments were conducted to investigate the reproducibility and variability of copper (Cu) sensitivity of T. japonicus so as to evaluate its potential to serve as an appropriate test species. To understand the seasonal variation of Cu sensitivity, individuals of T. japonicus were collected from the field in summer and winter, and subjected to standard 96 h acute (static renewal) toxicity tests. 96 h-LC50 values of T. japonicus collected from the two seasons were marginally different (p = 0.05), with an overall coefficient of variation (CV) of 33%. Most importantly, our results indicated that chronic Cu sensitivity of T. japonicus was highly reproducible. The CVs of intrinsic rates of increase in the population of the control and Cu treatment (10 μg Cu l⁻¹) groups were only 10–11% between 10 runs of a standardised complete life-cycle test. Moreover, different Cu(II) salts generally resulted in a similar 96 h-LC50 value while Cu(I) chloride was consistently slightly less toxic than Cu(II) salts. Given such a high reproducibility of toxic responses, it is advocated to use T. japonicus as a routine testing organism.     

Comparative petroleum hydrocarbons levels and biochemical responses in mussels from hydrothermal vents (Bathymodiolus azoricus) and coastal environments (Mytilus galloprovincialis)

Aliphatic hydrocarbons and PAHs in the whole soft tissues of Bathymodiolus azoricus from three Mid-Atlantic Ridge hydrothermal vents (Menez-Gwen, Lucky Strike and Rainbow), and Mytilus galloprovincialis from three contaminated coastal sites in South Portugal were analysed, and its effects on the digestive gland microsomes mixed-function oxygenase system (MFO) were assessed.Aliphatic hydrocarbons levels were present in the same magnitude in both coastal and hydrothermal environments, while the UCM (unresolved complex mixture) for coastal mussels were higher than in vent mussels. In general, significantly higher PAHs concentrations were found in coastal mussels, compared to B. azoricus where low molecular weight PAHs (2–3 rings) represented the majority of PAHs contrarily to what was observed in M. galloprovincialis. The MFO components were present in both mussel species, and were detected in vent mussels for the first time. However this system seems to have different roles in species from these contrasting environments. In coastal mussels MFO responded to hydrocarbon contamination while response in hydrothermal organisms appeared to be related mainly to endogenous factors.     

广东流溪河水库异尾轮属(Trichocerca)的分布模式及其影响因素

异尾轮虫是热带亚热带地区轮虫的重要优势类群.为了解其在水库中的分布模式和潜在影响因素,于2013-2014年对广东流溪河水库河流区、过渡区及湖泊区的异尾轮虫群落及环境因子进行逐月调查.2年共采集到异尾轮虫11种,其中对棘异尾轮虫(Trichocerca similis)、圆筒异尾轮虫(T.cylindrica)、暗小异尾轮虫(T.pusilla)和刺盖异尾轮虫(T.capucina)为优势种,主要出现在丰水期,具有明显的季节分布模式.2013-2014年河流区、过渡区和湖泊区异尾轮虫丰度平均值分别为142.9、102.5和53.6 ind./L,3个水域的异尾轮虫总丰度在夏季(5-9月)较高.从水库上游到下游,异尾轮属的物种丰富度和Simpson多样性指数呈现递减趋势,体现了群落对环境的分区和稳定性变化的响应.群落丰度组成的主成分分析表明,主要优势种的丰度全年分布差异较大,而多数种类全年分布差异小.冗余分析表明,叶绿素a浓度、晶囊轮虫丰度、水温+磷酸盐浓度分别为影响河流区、过渡区和湖泊区异尾轮虫群落结构的关键变量,而降雨则是影响整个库区异尾轮虫群落结构的潜在变量.     

一株淡水红藻的形态及分子系统发育研究

以采自山西晋祠泉的一株淡水红藻JC1712001为实验材料,对其进行分子系统发育分析和形态特征鉴定,同时利用分子数据对其进行生物地理学研究.结果显示,该株淡水红藻JC1712001与美国、西班牙地区报道的马赫拉熊野藻（Kumanoa mahlacensis Kumano）遗传距离最近,基于rbcL、COI和UPA序列的遗传距离均为0.基于3个序列,利用贝叶斯法、最大似然法和邻接法3种方法构建的系统发育树具有高度一致的系统发育关系,JC1712001均与西班牙及美国地区已报道的马赫拉熊野藻聚为一支,支持率都在97%以上,支持其为马赫拉熊野藻,同时也表明3个基因的高度保守性.形态特征的观察也支持这个结果.马赫拉熊野藻在欧洲、亚洲、北美洲均有分布,RASP软件构建的地理起源图谱显示,欧洲类群地理起源更为古老.马赫拉熊野藻为稀有种,本文的研究结果丰富了我国淡水红藻的分布,为其系统发育和地理起源研究提供了依据.     

中国鱼腥藻属的两个新记录种

迄今为止,全球范围共报道出100多种鱼腥藻,中国记录报道的仅有50余种,但有些种类的描述仍较模糊.最近在福建、浙江等地进行野外调查时,发现了多种丝状蓝藻.其中就有2种鱼腥藻在中国尚未报道:威格鱼腥藻(Anabaenaviguieri Denis et Frémy 1923)和史密斯鱼腥藻(Anabaena smithii(Komárek)Watanabe 1992).     

汾河太原河段水华藻类分类及分子系统研究

2012-2016年,每年的春、夏、秋季对汾河太原河段进行浮游植物样品采集.通过对样品的形态观察和描述,共鉴定出5种水华优势种,5月发生的裸藻水华优势种为裸藻属的膝曲裸藻(Euglena geniculata)和血红裸藻(E.sanguinea).而7-9月发生的微囊藻水华优势种为微囊藻属的铜绿微囊藻(Microcystis aeruginosa)、挪氏微囊藻(M.novacekii)和惠氏微囊藻(M.wesenbergii).分离纯化共得到11株单克隆水华藻,其中铜绿微囊藻8株,挪氏微囊藻2株,血红裸藻1株.运用cpcBA-IGS、gyrB和cpSSU基因序列构建分子系统发育树,进一步确定水华藻的系统分类地位,结果表明cpcBA-IGS是研究汾河太原河段铜绿微囊藻分类很好的分子标记,而cpSSU基因可很好地区分血红裸藻和其他裸藻种.     

Identification of genes expressed in juvenile Haliotis rufescens in response to different copper concentrations in the north of Chile under controlled conditions

This study reports molecular markers potentially associated with resistance or sensitivity to the impact of copper in juvenile red abalone, Haliotis rufescens, in the north of Chile under experimental conditions. Genomic analysis was made applying subtractive hybridization libraries (SSH) to identify genes up-and down regulated during cooper exposure in abalone over periods of 12 and 168 h exposed to 2.5 and 10 μg/L of Cu⁺². Results obtained from the SSH library revealed 368 different sequences regulated by copper, that correspond to eight major physiological functions. The validation of these sequences obtained by SSH as well as their expression kinetics were made by PCR in real time on 14 potential genes regulated by metal stress. This study provides information for the characterization of potential genomic markers that may be used in future environmental monitoring and to investigate new mechanisms of stress to copper in this commercially important marine species.     

A primitive odobenine walrus of Early Pliocene age from Japan

Abstract The well preserved cranium of Protodobenus japonicus, a new genus and species of odobenine walrus, is from the lower part of the Tamugigawa Formation at Ooshima-mura, Higashi Kubiki-gun, Niigata Prefecture, central Japan. The lower part of the formation that yielded P. japonicus is Early Pliocene, dating approximately from 5.0 to 4.9 Ma. The skull of P. japonicus is generally shaped like that of the modern walrus, Odobenus rosmarus (Linnaeus, 1758), but is much more primitive, especially in the dentition. Protodobenus japonicus is derived from the Imagotariinae. It is similar to such primitive fossil walruses as Prorosmarus alleni from the Western Atlantic and Aivukus cedrosensis from Baja California, and has some similarities as well as significant differences from them. Protodobenus japonicus could have evolved directly into Odobenus in the North Pacific Ocean, and could have dispersed directly to the Arctic Ocean. This is contrary to the scenario proposed by Repenning and Tedford, in which primitive odobenids, like Aivukus cedrosensis, migrated to the Atlantic Ocean through the Central American Seaway and returned to the North Pacific Ocean as O rosmarus via the Arctic about 0.6 Ma. Regarding the first lower premolar of the living walrus, Fay concluded after studying fetal O. rosmarus that the first premolar (P₁) of the mandible is absent, and that the lower tooth row consists of: C₁ P₂, P₃, P₄. Protodobenus japonicus has in its mandible I _1-2 C₁ P₂, P₃, P₄, and this is the primitive pinniped formula. Pinnipeds typically lose the molars, not the anterior premolars, and the present author believes that the correct tooth formula for O rosmarus is C₁ P₁, P₂, P₃. Its skull, palate, and mandible suggest that P. japonicus had morphology pre-adapted to an early stage of benthic suction feeding, but it probably was unable to rely on that method of feeding. The discovery of P. japonicus demonstrates that the odobenine lineage (true walruses) goes back in time at least 5 Ma. Other more aberrant odobenine walruses, such as Aivukus and Prorosmarus, and pseudo-walruses of the subfamily Dusignathinae, are more distantly removed from the lineage leading to living walruses than has previously been suspected.     

Modelling Potential Field Sources in the Gelibolu Peninsula (Western Turkey) Using a Markov Random Field Approach

In this study, a Markov Random Field (MRF) approach is used to locate source boundary positions which are difficult to identify from Bouguer gravity and magnetic maps. As a generalized form of Markov Chains, the MRF approach is an unsupervised statistical model based algorithm and is applied to the analysis of images, particularly in the detection of visual patterns or textures. Here, we present a dynamic programming based on the MRF approach for boundary detection of noisy and super-positioned potential anomalies, which are produced by various geological structures. In the MRF method, gravity and magnetic maps are considered as two-dimensional (2-D) images with a matrix composed of N₁ × N₂ pixels. Each pixel value of the matrix is optimized in real time with no a priori processing by using two parameter sets; average steering vector (θ) and quantization level (M). They carry information about the correlation of neighboring pixels and the locality of their connections. We have chosen MRF as a processing approach for geophysical data since it is an unsupervised, efficient model for image enhancement, border detection and separation of 2-D potential anomalies. The main benefit of MRF is that an average steering vector and a quantization level are enough in evaluation of the potential anomaly maps. We have compared the MRF method to noise implemented synthetic potential field anomalies. After satisfactory results were found, the method has been applied to gravity and magnetic anomaly maps of Gelibolu Peninsula in Western Turkey. Here, we have observed Anafartalar thrust fault and another parallel fault northwest of Anafartalar thrust fault. We have modeled a geological structure including a lateral fault, which results in a higher susceptibility and anomaly amplitude increment. We have shown that the MRF method is effective to detect the broad-scale geological structures in the Gelibolu Peninsula, and thus to delineate the complex tectonic structure of Gelibolu Peninsula.