Cloning and sequence analysis of the self-fertilizing fish Rivulus marmoratus immediate early gene c-fos

We have cloned the proto-oncogene c-fos from a self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) after screening of R. marmoratus λGEM-11 genomic DNA library, and sequenced over 12 kb including all exons, introns and the promoter region. The R. marmoratus c-fos gene consisted of one noncoding exon and four exons with high similarity to those of fugu and mammals. We sequenced ≈7 kb of the R. marmoratus c-fos gene promoter region to gain a better understanding of the molecular anatomy of the immediate response of this gene upon cellular damage. In the promoter region, R. marmoratus c-fos gene has seven xenobiotic response elements (XREs) and eight metal response elements (MREs) as well as two estradiol (E2), 4 NFκB, 2 CarG, 2 prolactin (PRL) motifs and one pit1 site, while the 3^′-UTR of this gene contains the estrogen response element (ERE). The seven XRE and eight MRE motifs raise the possibility of its regulation by exposure to environmental pollutants. In this paper, we discuss the gene structure of R. marmoratus c-fos gene and compare its promoter region with those of other organisms' c-fos genes. We propose its potential use in ecotoxicology.     

Genomic cloning and expression of vitellogenin gene from the self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae)

We cloned the vitellogenin gene from the self-fertilizing fish Rivulus marmoratus, and sequenced 12,326 bp. The number of exons of R. marmoratus and rainbow trout vitellogenin genes were different, and also the splicing junctions are different throughout most of the exons and introns but the amino acid similarity of R. marmoratus vitellogenin gene to other species was rather high. In promoter region of R. marmoratus vitellogenin gene, there were several E2 binding sites and the estrogen response element (ERE). We discuss here the gene structure and expression of R. marmoratus vitellogenin gene.     

Cloning of cytochrome P450 1A (CYP1A) genes from the hermaphrodite fish Rivulus marmoratus and the Japanese medaka Oryzias latipes

To use two small fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the Japanese medaka Oryzias latipes (Belloniformes) as testing models in molecular ecotoxicology, we have cloned the cytochrome P450 1A (CYP1A) gene after screening of both genomic DNA libraries, and sequenced 11,863 and 7,243 bp including all the exons and introns with promoter regions, respectively. The Rivulus and the medaka CYP1A gene consisted of seven exons (including non-coding exons) with high homology to mammals. In the promoter region, Rivulus CYP1A gene has seven xenobiotic response elements (XREs) and two metal response elements (MREs), while the Japanese medaka CYP1A gene has six XREs and four MREs. Interestingly, medaka CYP1A gene has a number of MREs at the promoter, which may affect its response on metal exposure. We describe here the gene structure of both fish CYP1A genes.     

Differential distribution of the two cryptic species, Pomatoschistus microps and P. marmoratus, in the lagoons of southern France, with an emphasis on the genetic organisation of P. microps

The presence and the distribution of two cryptic sedentary gobies, Pomatoschistus microps and Pomatoschistus marmoratus, were investigated in several lagoons stretching along the Golfe du Lion, southern France Mediterranean coast, and Corsica. Pomatoschistus microps is the only sedentary Pomatoschistus species in two shallow lagoons with large variations of salinity and temperature. In contrast, P. marmoratus, another sedentary species, can be found preferentially in a deep salty lagoon with small salinity variations. Both species occur sympatrically in the Rhone Delta and hybridise, producing fertile offspring. Using allozymes, competition, linked to fitness, is invoked to explain the distribution pattern of these sibling species.     

基于Ecopath模型的舟山海域褐菖鲉(Sebastiscus marmoratus)生态容量评估

根据2021年渔业资源调查数据构建了含有23个功能组的舟山海域生态系统Ecopath模型,分析了当前舟山海域生态系统总体特征并估算了褐菖鲉在舟山海域的生态容量。结果表明:舟山海域生态系统营养级范围为1.000 (浮游植物和有机碎屑)～4.277 ( 鳐类),石首鱼科、虾类和 鳐类为舟山海域生态系统中的关键种。碎屑食物链和牧食食物链是舟山海域生态系统主要的食物链。碎屑和浮游植物对食物网的贡献率分别为61.32%和38.69%。始于浮游植物和碎屑的营养传递效率分别是9.34%和10.50%,系统总营养传递效率是9.82%。总初级生产量/总呼吸量为2.26,系统连接指数为0.372,系统杂食性指数为0.222。生态系统总体特征反映了舟山海域生态系统的成熟状态较低,生态系统处于不稳定阶段,容易受到外界环境变化的影响。根据模型估算,当褐菖鲉生物量增加至8.6倍时,褐菖鲉达到生态容量0.007 95 t/km²,此时生态系统仍保持平衡,且生态系统总体特征基本稳定。因此,褐菖鲉在舟山海域尚有较大增殖潜力。     

A survey of indicators for reproductive endocrine disruption in Fundulus heteroclitus (killifish) at selected sites in the Chesapeake Bay

Plasma vitellogenin and related parameters in the killifish Fundulus heteroclitus were measured at selected sites in the Chesapeake Bay. In males, vitellogenin was above the detection limit 14% of the time, and detections did not differ between sites or seasons. Few differences in plasma vitellogenin levels were found between sites during fall in either male or female F. heteroclitus, the time of natural gonadal regression for this species. There was some variation in the ratio of male to female F. heteroclitus, but was not consistent at most sites. Significant negative correlations were found between reported sediment polycyclic aromatic hydrocarbons (PAHs) and GSI, and PAHs and plasma vitellogenin in females in both Spring 1999 and Spring 2000. Gonadal anomalies in F. heteroclitus included slight reductions in certain tissue types. Overall, reproductive endocrine disruption in the killifish F. heteroclitus at the sites sampled in the Chesapeake Bay appeared somewhat minimal.     

苯并[a]芘(BaP)对褐菖鲉(Sebasticus marmoratus)肝CYP1A1酶活性、基因表达及蛋白表达的影响

为了了解苯并[a]芘(BaP)对鱼类细胞色素P4501A1(CYP1A1)表达的影响,以褐菖鲉(Sebasticus marmoratus)为实验材料,采用体内实验,研究其在经过不同浓度(0.1、1、10、20、50mg/kg鱼体重量)的BaP诱导后,鱼体肝脏研究CYP1A1基因表达的情况,筛选出后续时间-效应实验中BaP注射的最佳浓度,研究BaP诱导6h、12h、1d、3d、7d后(质量浓度为20mg/kg鱼体重量)鱼体肝脏CYP1A1酶活性、基因表达和蛋白表达的情况。结果表明:剂量-效应实验中,20mg/kg鱼体重量为最佳浓度,此浓度下,基因表达在各组中变化最显著。时间-效应实验中,较空白对照组而言,染毒6h、12h和1d后,EROD酶活性显著增加。3d后开始下降,与对照组相比变化不大,7d后酶活性又发生上调。半定量RT-PCR结果表明,各染毒组与对照组相比,CYP1A1基因表达量都发生了上调,呈现先上升后下降的趋势。其中,6h和12h组相对表达量极显著增加,1d后开始下降且与3d和7d组相比变化不明显。Western blot结果表明,蛋白表达量在染毒12h后表现出显著的诱导效应,随着时间的延长略有回落,但与对照组相比仍有显著性差异。研究表明:BaP对褐菖鲉CYP1A1具有较强的诱导作用。一定质量浓度的BaP注射于褐菖鲉不同的时间后,能诱导褐菖鲉活体EROD酶活性、CYP1A1基因m RNA表达及蛋白表达,并随着时间的延长呈现先诱导后抑制的趋势。这说明BaP作为诱导剂对CYP1A1酶活性和蛋白表达的作用机制可能与调控CYP1A1的转录水平有关。     

Differential gene expression in the brain of Sebastiscus marmoratus in response to exposure to polychlorinated biphenyls (PCBs)

Effects of exposure to polychlorinated biphenyls (PCBs) on Sebastiscus marmoratus were investigated using a suppression subtractive hybridization method. A total of 108 gene sequences were identified as having the potential for being differentially expressed, and 45 could be identified with homologous database sequences. Functions with which they were associated included long-term potentiation and neurotransmitter release, neuroendocrine, mitosis and cell proliferation, energy-related metabolism, general metabolism, signal protein, hemopoiesis system, immune system, and structure. The expression of 17 of these genes was analyzed in the brain using real time fluorescent quantitative PCR. The present study provided a basis for studying the response of fish to PCB exposure and allowed the characterization of new potential neurotoxicol biomarkers of PCB contamination in seawater.     

许氏平鲉(Sebastes schlegelii)EST-SSR标记开发及通用性检测

利用已发表的EST序列设计引物并结合PCR筛选的方法,对许氏平鲉多态EST-SSR引物进行了开发。结果表明:(1)许氏平鲉EST-SSR位点发现率为9.14%,核心序列以二碱基重复出现频率最高,占到33.03%,其中以TG/CA基序最为丰富。(2)共设计引物56对,46对实现有效扩增,其中18对具有多态性。(3)野生群体遗传多样性分析显示,每个位点观测等位基因数(Na)为2—9,平均观测等位基因数为3.89;观测杂合度(Ho)、期望杂合度(He)及平均值分别是0.0333—0.8000、0.0333—0.7927和0.3037、0.3757;每个位点的多态信息含量(PIC)在0.0323—0.7522之间,其中高度多态位点5个,中度多态位点5个;经Bonferroni校正后,3个位点显著偏离Hardy-Weinberg平衡,但两两位点间不存在连锁不平衡现象。(4)许氏平鲉多态EST-SSR引物对朝鲜平鲉、褐菖鲉通用性检测结果表明,引物对二者的通用率和多态率分别为88.89%、100.00%和33.33%、88.89%。研究表明,这些通用引物可用于相关物种的遗传多样性评价、系统进化分析和比较基因组作图等研究。     

缩骨鲫(Carassius auratus sogu var.)MC4R克隆、组织分布以及与生长相关SNPs的筛选

黑色素皮质素受体-4(Melanocortin reporter-4,MC4R)是G蛋白偶联受体超家族成员,在能量调控中发挥重要作用。本实验采用同源克隆以及染色体步移对缩骨鲫(Carassius auratus sogu var.)MC4R基因进行克隆,对其核苷酸序列和推测的氨基酸序列进行生物信息学分析,通过Real-time PCR对其m RNA组织分布进行研究,并对缩骨鲫MC4R编码区潜在的与生长相关的SNPs进行筛选。本实验克隆MC4R基因组基因长度为2854bp,只包含一个外显子,长度为981bp,共编码326个氨基酸,MC4R共有七个跨膜结构,是较为稳定的疏水蛋白。缩骨鲫MC4R与鲫鱼的MC4R氨基酸序列的同源度最高达到99.3%,与MC3R和MC5R同源度相对其他MCRs成员较高。缩骨鲫MC4R m RNA在脑中表达量最为显著,眼中次之,肌肉,卵巢和肾脏中表达较少,在鳃、肠、心脏和肝胰腺中微量表达。在MC4R的CDs区内检测到6个与生长潜在相关的SNPs:两个错义突变(A199G和A881C),4个同义突变(A123G,C231T,A444G和C480T)。本实验可为缩骨鲫生长和发育的分子机制研究以及遗传育种提供基础资料。     

凡纳滨对虾4种血蓝蛋白亚型的特征分析

血蓝蛋白是对虾体内的氧运输蛋白,占血浆中蛋白含量的95%以上.研究表明,它参与了许多生理功能,包括蛋白储运,渗透压调节,蜕皮过程以及骨架形成等等.近年来的研究表明,血蓝蛋白还参与了对虾的先天性免疫反应,具有酚氧化酶,抗菌肽、抗病毒等相关生物活性.本研究从凡纳滨对虾中克隆获得了血蓝蛋白L亚基LvHcL,并对其基因组织结构进行了分析.该基因具有丰富的多态性,含有多个SNP位点.研究中发现该基因亚基至少存在4种亚型,其中一种亚型缺失第二个外显子,仅含有2个外显子.对各个亚型的转录水平分析表明,抗病对虾在病毒感染刺激下,各个亚型的转录水平均会被诱导提高.而普通对虾在病毒感染刺激下,各个亚型的转录水平则被抑制.研究结果表明,血蓝蛋白参与了对虾的抗病毒免疫反应.这些结果不仅有助于深入了解对虾血蓝蛋白的免疫学特点,也有助于促进对无脊椎动物抗病毒免疫机制的认识.     

浒苔光照和盐度胁迫响应基因UpMYB44的克隆与表达分析

为证明R2R3-MYB转录因子在浒苔响应非生物胁迫例如盐度和光照的过程中发挥的重要调控作用,利用实时荧光定量PCR、酵母双杂交系统、亚细胞定位等技术,研究获得了浒苔UpMYB44基因1 437 bp的开放阅读框(ORF)全长序列,编码478个氨基酸,属于典型的R2R3-MYB转录因子并通过烟草叶片转化确定其定位于细胞核,UpMYB44参与浒苔响应光照和盐度的胁迫过程,其中在低光和高盐胁迫下UpMYB44基因的相对表达量升高。筛选出了与UpMYB44互作的UpCPP5蛋白,该蛋白与浒苔的生长和细胞分裂有关,推测UpMYB44可能通过与UpCPP5互作,形成蛋白复合体参与浒苔的增殖过程。研究为日后深入探究MYB类转录因子家族调控藻类生长发育的过程奠定了基础,同时为研究浒苔快速繁殖的机制提供了思路。     

10个菲律宾蛤仔野生群体的遗传多样性研究

对我国沿海地区10个菲律宾蛤仔野生群体线粒体16S r RNA和COI基因部分序列进行测序,分别得到了长度为473bp和632bp的片段。结果表明,16S r RNA 193条序列A+T平均含量为66.6%,共检测到21个变异位点,193个个体具有22种单倍型;COI基因183条序列A+T平均含量为64.8%,共检测到126个变异位点,183个个体具有67种单倍型。基于群体间遗传距离利用Mega5.1软件构建10个群体的NJ树,聚类结果表明,大连群体和荣成群体聚为一支,其余8个群体聚为一支。AMOVA分析表明,大连群体和荣成群体间分化不显著,而荣成、大连群体与其余8个群体间的分化达到极显著水平(P0.01),说明我国沿海的菲律宾蛤仔野生群体存在一定的遗传分化。     

海蜇dmrta2基因的克隆及其在横裂生殖中的表达分析

Dmrt基因家族在后生动物性别决定与分化以及组织和器官的形成等发育过程中发挥着重要作用。为探究dmrt家族基因在海蜇(Rhopilema esculentum)横裂生殖中的功能,本研究以海蜇转录组测序获得的dmrta2基因片段为基础,通过RACE技术获得了海蜇dmrta2基因的cDNA全长,并分析了其在横裂生殖不同时期的表达模式。结果显示海蜇dmrta2基因cDNA全长为1 804 bp,开放阅读框为1 011 bp,所编码蛋白质含336个氨基酸,分子质量为37.25 kDa,理论等电点为9.11。预测海蜇dmrta2编码蛋白为亲水性蛋白质,无跨膜区域,不含信号肽。在33至79位氨基酸之间具有dmrt基因家族保守的DM结构域,与珊瑚、果蝇等物种的同源基因相应区域高度一致。系统进化分析显示,海蜇DMRTA2与鹿角珊瑚DMRTA2和DMRT3以及海葵DMRTA2的亲缘关系最近。原位杂交显示dmrta2基因在海蜇横裂生殖后期主要表达于感觉缘叶原基位置,在初生碟状体中表达于感觉棍位置。研究结果表明,dmrta2基因参与海蜇横裂生殖过程,可能主要与感觉棍神经系统的分化发育相关,研究为进一步解析海蜇等钵水母横裂生殖的分子调控机制提供了基础数据。     

Relationship between heavy metal accumulation and genetic variability decrease in the intertidal crab Pachygrapsus marmoratus (Decapoda; Grapsidae)

The “genetic erosion” hypothesis posits that heavy metal stress is related to a loss of genetic diversity at the population level. The genetic diversity of natural populations can, however, be affected by natural processes as well as by human impact. We studied the relationship between heavy metal bioaccumulation and genetic variability in the intertidal crab Pachygrapsus marmoratus. Tissue samples were collected from 40 individuals inhabiting four polluted and four unpolluted sites along the Tuscan coast (Mediterranean basin), and were examined for four heavy metals (arsenic, As, cadmium, Cd, lead, Pb, and copper, Cu). We also assessed the genetic variability of 235 crabs from the same localities using six microsatellite loci.Our results show that the bioaccumulation levels of these individuals accurately reflect the levels of pollution in their immediate environment, and that heavy metals accumulate more in the hepatopancreas than in the gills. Moreover, populations from polluted sites have significantly less genetic variability, measured as mean standardized d², and a significantly lower percentage of unrelated individuals, than populations from unpolluted sites. This evidence supports the “genetic erosion” hypothesis for metal heavy exposure in natural environments.     

不同内倾角海螺笼对脉红螺的诱捕效果

作者对不同内倾角海螺笼对脉红螺(Rapana venosa)的诱捕效果进行了实验研究。在实验室水槽中,观察了4种不同内倾角(35°、45°、55°和65°)的海螺笼对脉红螺的诱捕效果,同时还研究了光照和温度对海螺笼诱捕效果的影响,为优化海螺笼捕捞技术提供理论依据。实验结果表明:在相同的实验条件下,不同的内倾角对海螺笼诱捕效果影响十分显著(P<0.01),其中内倾角为45°的海螺笼诱捕效果最好,内倾角为35°和55°的海螺笼较好,而内倾角为65°的海螺笼诱捕效果则最差。同时发现,内倾角对脉红螺的个体大小具有选择性,随着内倾角的增大,壳高<7cm的个体占相应笼诱捕总数的百分比越来越小。光照对海螺笼的捕捞效果有十分显著的影响,而温度的影响则不明显。     

Feeding Strategy of Japanese Sand Lance Larvae in Relation to Ciliated Protozoa in the Vicinity of a Thermohaline Front

Larval feeding and survival strategies are described on a Japanese sand lance, Ammodytes personatus Girard, collected in the vicinity of a thermohaline front in the Ise Bay, in comparison with those of other predominant larvae, Hexagrammos spp. and Sebastiscus marmoratus (Cuvier). First-feeding A. personatus larvae (3.1–3.9 mm NL) fed primarily on tintinnid ciliates, subsequently switching to copepod nauplii (4.0–7.9 mm NL larvae) and post-naupliar copepods (8.0–11.3 mm NL larvae). First-feeding Hexagrammos spp. larvae (6.5–6.9 mm NL) fed primarily on post-naupliar copepods, and first-feeding S. marmoratus larvae (<4.0 mm NL), mostly on copepod nauplii. The different food preferences of these species at first-feeding were related to their different mouth widths (0.15–0.19, 0.52–0.56 and 0.32–0.40 mm, respectively) and/or body size (3.1–3.9, 6.5–6.9 and 3.2–3.9 mm NL, respectively). Ciliate-feeding by first-feeding A. personatus larvae was strongly related to the convergence of the larvae and their prey near the thermohaline front, densities of both being greater on the inshore side of the frontal zone. In conclusion, the aggregation of ciliates near the thermohaline front may have improved feeding conditions and survival of first-feeding A. personatus larvae.     

厚壳贻贝(Mytilus coruscus)Hox基因家族的鉴定、进化与表达分析

为探究厚壳贻贝Hox基因家族的定位、进化以及在不同成体组织中的表达模式,基于厚壳贻贝全基因组数据,结合生物信息学方法,鉴定分析厚壳贻贝Hox基因家族成员的理化性质、染色体分布、系统进化关系、基因结构以及表达分析。结果显示,从厚壳贻贝全基因组数据中鉴定出11条Hox基因序列,并分别命名为Hox1-5、Antp、Lox2、Lox4、Lox5、Post1、Post2。对11条Hox序列的基本特征分析发现最长的是编码703个氨基酸的Hox2,最短的是编码190个氨基酸Post2,等电点5.11～10.22,且不均等分布于同一条染色体上。亚细胞定位预测结果显示,除Post1基因在细胞质和细胞核中均有发现,其余10个基因亚细胞均定位在细胞核内。系统进化分析发现,使用厚壳贻贝中鉴定出的101条氨基酸序列进行建树,聚类的结果倾向于四类,而从已鉴定的厚壳贻贝11个基因结合软体动物Hox建树结果分析, Post1和Post2聚为一支, Hox4、Antp、Lox5、Lox2和Lox4聚为一个大类,Hox1、Hox2和Hox3各自聚为一类Hox,此外,Hox5单独聚为一类。厚壳贻贝不同组织的qRT-PCR结...     

Evaluation of food competition and resource partitioning of recruiting fish with permanent residents in a seagrass habitat

ABSTRACT

Recruiting King George whiting Sillaginodes punctatus were studied to assess the potential for food competition with permanent resident fish species in a nursery habitat. Marine migrant post larval S. punctatus (<60?mm TL) consumed primarily harpacticoid copepods and had high (>0.6) diet overlap with permanent resident fish species Favonigobius leteralis and Stigmatopora nigra. Food electivity index indicated that S. punctatus juveniles preferred harpacticoid copepods and amphipods, while juvenile Heteroclinus adelaide and Gymnapistes marmoratus migrating to the nursery habitat targeted larger prey such as amphipods. Preference for larger prey by H. adelaide and G. marmoratus species coupled with differences in prey composition in the stomach was due to mouth size and feeding habits, resulting in different food preferences to S. punctatus. The whiting showed an ontogenetic shift in diet with early settlers (>60?mm TL) consuming less copepods and more amphipods, while previous year recruits (>120?mm TL) consumed polychaete worms. This study indicates that competition for food resources between the new recruiting S. punctatus juveniles and permanent resident juveniles is reduced through differences in temporal and spatial feeding behaviours, mouth morphology, and ontogenetic shift in prey consumption.