【作者】 姚雁鸿；

【导师】 余来宁；

【作者基本信息】 华中农业大学 ， 水产养殖， 2012， 博士

【摘要】 湘西盲高原鳅(Triplophysa xiangxiensis)属鲤形目(Cypriniformes)、鳅科(Cobitidae)、高原鳅属(Triplophysa)鱼类,分布于湖南龙山县火岩乡多个溶洞的地下河中,属当地特有洞穴鱼类,1986年湘西盲高原鳅首次被杨干荣等描述并被定名为湘西盲条鳅(Noemacheilinae xiangxiensis sp.nov.);1992年中国科学院昆明动物研究所的陈银瑞等对模式标本分析后,根据新的分类系统重新定名为湘西盲高原鳅(Triptophysa xiangxiensis Yang et al.)。湘西盲高原鳅是典型的洞穴鱼类,具有典型洞穴鱼类的一般特征：全身裸露无鳞,身体呈半透明状,从外表可见内脏器官和皮下血管,视觉器官完全退化。洞穴鱼类在进化生物学、比较生理学研究上有巨大的价值,然而普遍处于濒危之中。目前对湘西盲高原鳅研究较少,本文对湘西盲高原鳅微卫星、线粒体DNA中D-LOOP基因和Cyt-b基因进行了分析,查明其种群遗传多样性；同时对其生境水环境进行了调查并分析了其在饥饿、低溶氧条件下的生理代谢,为对该洞穴鱼的保护提供了依据,具体结果及内容如下：1.湘西盲高原鳅遗传多样性的微卫星分析利用筛选的16对微卫星标记对来自于湖南湘西龙山县乌龙山3个不同的洞穴的盲高原鳅群体进行遗传多样性及遗传分化分析。通过计算多态信息含量、平均杂合度、等位基因数、遗传距离、基因流、F-统计量等参数,评估各盲高原鳅群体遗传多样性和各群体间遗传分化。16个微卫星标记在3个群体中共检测出83个等位基因。每个座位检测到3～8个等位基因不等。3个群体各个多态位点的平均观测杂合度分别为0.3625～0.9465,平均期望杂合度为0.5386～0.9065。3个群体多态微卫星位点的PIC平均为0.2632、0.2313、0.3035。3个群体的遗传多样性均较低,分子变异方差分析(AMOVA)结果表明,遗传变异大部分(92.84%)来自群体内,仅有7.16%的变异来自于群体间,数据表明3个群体处于未分化状态,遗传一致性较大。2.基于线粒体Cytb基因和D-loop序列变异的湘西盲高原鳅遗传多样性分析运用线粒体Cyt b基因序列和d-loop基因序列对来自湖南湘西龙山县乌龙山3个不同的洞穴的群体的82尾湘西盲高原鳅遗传多样性及遗传分化进行了分析。湘西盲高原鳅线粒体Cyt b基因序列片段长1140bp,线粒体控制区序列片段长934bp,在控制区中发现6个多态位点且全部为单一变异位点,序列中检测到7个单倍体,单倍型多样性指数介于0.230到0.282,核苷酸多样性指数介于0.00009到0.00032。3个群体的遗传多样性均较低,所有样本的Cyt b序列完全一致,没有发现变异,基于d-loop的分子变异方差分析(AMOVA)结果表明,湘西盲高原鳅遗传变异系数FST=-0.0089,为负值,总遗传变异中,年度种群间变异占-0.89%,各年度种群内变异占101.57%,变异主要来自种群内部。3.湘西盲高原鳅栖息地水质季节变化研究本研究对湘西盲高原鳅的生境进行了调查,分析了湘西盲高原鳅所在洞穴暗河的主要环境因子年度变化。发现该暗河水质年度变化较小,水体主要离子年度波动平缓,水温基本稳定,在12--16℃波动,水体偏碱性,硬度较高,DO稳定在6mg/L左右,水体一直很清澈。4.饥饿和再投喂对湘西盲高原鳅体组成及肠道消化酶活性的影响研究了湘西盲高原鳅经不同饥饿时间处理,再恢复喂食后的生长情况。结果表明：5个月饥饿期导致鱼体重损失约23%,恢复投喂后1个月产生了部分补偿生长效应；饥饿30d对鱼体蛋白质、灰分基本没影响；而脂肪含量显著下降(p<0.05),水分含量轻微上升；恢复投喂后,各饥饿处理组体组分基本在5天内恢复到对照组水平。饥饿期间肠道脂肪酶、蛋白酶、淀粉酶均表现出明显下降,饥饿前10天下降最快,以后逐渐趋于平稳,恢复投喂后基本在5天内恢复到正常水平,只有30天饥饿组脂肪酶在10天内恢复到正常水平。5.湘西盲高原鳅耗氧率及低氧条件下呼吸代谢研究本实验用流水装置研究了湘西盲高原鳅的耗氧率与体重、水温的关系及昼夜变化,同时研究了在溶氧逐渐降低过程中湘西盲高原鳅呼吸反应。结果表明：1.湘西盲高原鳅的耗氧率随体重增加而下降,随水温增加而升高,耗氧率没有昼夜变化；2.在4.89-7.68mg/L的正常溶解氧(DO)范围内,其呼吸频率(fR)、吸水量(VSR)、鳃通量(Vc)以及氧利用率(Eo2)变化差异不显著(P>0.05)；当DO水平降低至2.58mg/L后,湘西盲高原鳅的fR、US.R及VG显著增加,而E02显著下降(P<0.05)：当DO水平下降至2.58mg/L时,湘西盲高原鳅的耗氧率(Vo2)达到最高值。更多还原

【Abstract】 The cavefish Triptophysa xiangxiensis, a species of ray-finned fish in the genus Triplophysa, occurs in feihu cave of western Hunan Province of china. it was first described in1987by yang et al.. The fish shares many characters with other cave-dwelling Triplophysa species, such as the reduction of eyes, scaleless or with rudimentary scales and colorless or with shallow dark bars and blotches body, developed barbells. Cave-dwelling fish has great value on the comparative physiology and regressive evolutionary studying. Most cave environments must be considered to be under threat. Cave-dwelling fish also must be considered to be under threat. The limited knowledge of Triptophysa xiangxiensis would be unfavorable to its protection. In this study, first we used mitochondrial DNA (mtDNA) D-loop sequences, Cytochrome b(cytb)gene sequences and microsatellite to examine the genetic structure of the Triplophysa xiangxiensis populations in feihu cave of western Hunan Province of china; second, the habitat of T. xiangxiensis were investigated;then we examined the influence of reduced oxygen concentrations on the respiratory metabolism and some of the physiological response to starvation and subsequent re-feeding. The information generated in this study will contribute to the knowledge of this endangered Triplophysa xiangxiensis species and would be favorable to its protection. The contents are as follows:1. Microsatellite analysis of population genetic diversity in Triplophysa xiangxiensisIn this study, the genetic diversity and genetic differentiation of a total of82individuals from three T. xiangxiensis populations which obtained from three caves in wulongshan mountain were evaluated using mitochondrial DNA (mtDNA) D-loop sequences and Cytochrome b(cytb) gene.1140bp of the mitochondrial cytochrome b and934bp of control region were sequenced for82individuals of T.xiangxiensi. No genetic variation were found in cytochrome b, and all sequences were identical. In the control region,6polymorphic sites were found from all samples, and all singleton variable Seven haplotypes were detected among the sequences, with one of them (haplotype1) being popular and represented by73individuals. The remaining haplotypes were represented by one to three individuals. As for annual samples, sequences of all individuals from year2008were completely identical except one. Four and five haplotypes were determined from specimens of year2009and2010, respectively. The haplotype and nucleotide diversisty ranged from0.230to0.282and from0.00009to0.00032, respectively. All of which revealed that the genetic diversity in these three T. xiangxiensis populations were low. The AMOVA analysis indicates that100.89%of the genetic variability occurred within annual temporary samples, and negative value of0.89%was contributed from among temporary samples. The network constructed by sequences of control region displayed a star-like topology, with the haplotype1in center and the other haplotypes connecting with it by one step of mutation reveals.2. Low variation in mitochondrial DNA of the cavefish, Triplophysa xiangxiensisIn this study, A total of103individuals from three T. xiangxiensis populations which obtained from three caves in wulongshan mountain were studied using16pairs of microsatellite markers. Using polymorphism information content (PIC), mean heterozygosity (H), number of effective alleles and F-statistics, the genetic diversity and genetic differentiation were evaluated. A total of83different alleles were detected in all examined loci. The number of alleles ranged from3to8, with an average number of about5per locus. The observed (HO) and expected heterozygosity(HE) ranged from0.3625to0.9465and from0.5386to0.9065, respectively. The polymorphism information content for these three populations were0.2632、0.2313、0.3035. All of which revealed that the genetic diversity in these three T. xiangxiensis populations were low. The analysis of molecular variance(AMOVA) indicated that almost majority of the variance in the T. xiangxiensis was within stocks(92.84%),and7.16%was among stocks. The result of AMOVA、F-statistics, Nei’s genetic distance and genetic identity indicated that genetic difference was relatively small and genetic differentiation was low, with high genetic identity between all two populations. The information generated in this study will contribute to the conservation of this endangered T. xiangxiensis species.3. Study the habitat of T. xiangxiensis within one yearIn this study, the habitat of T. xiangxiensis were investigated. The main conclusions have been summarized as follows:(1).The main environmental factors of the sub river changes very little within the year.(2) The water quality is in Grade I except total hardness and Fe according to groundwater quality standard. The water is very clear within the year.(3) The water temperature is mainly steady at12-16℃within the year.(4) The water is slightly alkaline and DO is about6mg/L4. Effects of starvation and subsequent re-feeding on biochemical composition and digest enzymes of T. xiangxiensisBecause of high variation of food availability in cave aquatic systems, cave-dwelling fish may experience serve starvation during their growth, which may lead to variation in body weight, main biochemical composition of carcass and digestive enzyme activities in fish. The present experiment was designed to study the effects of starvation and subsequent re-feeding on body weight, main biochemical composition of muscle, intestine digestive enzyme activity in T. xiangxiensis cultured in basement aquarium at water temperature16-18℃.The fish were divided into three groups, of which three groups were prepared for sampling during a different periods of starvation (0d,10d,20d and30d), and the others were prepared for sampling during a different period of re-feeding (5d,10d,15d). Each treatment was assigned to duplicate aquariums. The results were as follows:body weight decreased to about23%after5-month-starvation; T. xiangxiensis exhibited partial compensatory growth in one month after re-feeding; There was no difference in protein and ash content during the30-day-starvation (P>0.05). The lipid content of muscle decreased significantly in10-day-starvation (P<0.05) and got back to normal level after5-day-re-feeding.(P>0.05); The water content of muscle increased slightly in10-day-starvation (P<0.05).The activities of digest enzyme (protease、amylase、lipase) in the intestine all decreased significantly in first5-day-starvation and then decreased gradually. The activities of digest enzyme got back to normal level after5-day-re-feeding except lipase of s30. The activities of lipase of s30got back to normal level after10-day-re-feeding.5. Effects of Hypoxia on the Rate of Oxygen Consumption of Respiratory Metabolism of T. xiangxiensisIn this study, the diurnal rhythms of oxygen consumption and the oxygen consumption rate in relation to body weight、water temperature in T. xiangxiensis were examined. We also examined the influence of reduced oxygen concentrations on the respiratory metabolism. The results showed that (1) The oxygen consumption rate(VO2)of the T. xiangxiensis decreases with the body weight inereasing;(2) The water temperature affects of oxygen consumption rate in T. xiangxiensis,it increases gradually with water rising at6-23℃;(3) Diurnal rhythms of the oxygen consumption rate were not found;(4) Normal DO levels (4.89-7.68mg/L) did not affect significantly the respiration of T. xiangxiensis in terms of respiratory frequency (fR), respiratory stroke volume (VS.R), gill ventilation (Vg) and oxygen extraction efficiency (EO2)(P>0.05); while DO level declined to2.58mg/L fR, VS.R and VG increased significantly and EO2decrease significantly (P<0.05); The oxygen consumption rate (VO2) of T. xiangxiensis reached the peak when DO levels declined to2.58mg/L.更多还原