中国科学院昆明动物研究所机构知识库

鱇（鱼良）白鱼（Anabarilius grahami）和滇池金线鲃（Sinocyclocheilus grahami）属于云南喀斯特地貌特有土著鱼类，分别是抚仙湖和滇池流域的特有种，人类活动导致抚仙湖湖体鱇（鱼良）白鱼种群急剧衰退、滇池湖体滇池金线鲃种群消失。为拯救两种珍稀鱼类，人工增殖放流相继开展，至今，两个物种野外种群数量均有一定程度恢复，但放流种群遗传结构、遗传多样性等还鲜有研究，同时，放流的科学性、合理性未进行过系统评估。本研究基于线粒体细胞色素b（CYTb）基因和微卫星数据，对鱇（鱼良）白鱼和滇池金线鲃塘养种群和回捕种群遗传结构、遗传多样性进行分析，评价增殖放流效果，并在此基础上，设计一系列试验探讨提高滇池金线鲃放流效果的方法，为科学、合理地开展增殖放流提供指导意见。鱇（鱼良）白鱼14个种群CYTb基因和微卫星结果显示：鱇（鱼良）白鱼现今湖体种群遗传多样性介于最古老的种群与人工繁殖最差种群之间。中国科学院昆明动物研究所珍稀鱼类保育研究基地有效保存了两个遗传多样性最高的种群，其次为九溪和火焰山养殖场种群，最差的种群来自于禄充、海口以及从海口引种到保育基地繁殖的F1代种群。放流前遗传多样性评估的缺失，使遗传多样性较低的群体进入湖体，对湖体原有野生种群造成遗传稀释和遗传渐渗，最终致使湖体种群遗传多样性下降。哈迪-温伯格平衡（Hardy-Weinberg equilibrium）检验表明，除保育研究基地2个种群（EFCC004、EFCC005）以及湖体部分采样点种群外，多数种群7个微卫星位点有3-4个位点显著偏离哈迪-温伯格平衡，因各种群未出现明显遗传分化，可采用种群内杂交恢复部分位点的平衡。通过对滇池金线鲃19个种群CYTb基因遗传分析研究发现，滇池金线鲃具有较低的线粒体基因多样性和较高的核基因多样性。中国科学院昆明动物研究所珍稀鱼类保育研究基地繁殖滇池金线鲃子代CYTb基因核苷酸多态性高于亲本。微卫星分析结果显示：人工繁殖技术的改进，可使滇池金线鲃微卫星遗传多样性高于野生群体，早期繁殖F1代种群遗传多样性低于技术成熟后繁殖种群，相应地，早期放流滇池湖体种群遗传多样性低于后期放流盘龙江种群。此外，回捕种群遗传多样性较放流种群略低，提示放流种群部分个体因受选择压力遭到淘汰。哈迪-温伯格平衡检验显示，放流和回捕种群绝大多数位点均不偏离哈迪-温伯格平衡，说明种群遗传结构良好，合理的增殖放流有利于维持和恢复各位点哈迪-温伯格平衡。遗传分化结果显示，承龙水苑种群与其他滇池金线鲃种群分化较高，不宜向滇池湖体投放该群体。光周期对滇池金线鲃仔稚鱼生长试验结果表明：光照时间长短对滇池金线鲃仔鱼生长影响不显著，但对滇池金线鲃稚鱼生长影响明显。越冬前，持续光照有利于滇池金线鲃稚鱼生长；越冬期间，持续光照对滇池金线鲃稚鱼造成光毒害，致使生长缓慢而不及自然光照和持续黑暗试验组且出现较大比例畸形。此外，光周期影响稚鱼神经丘和味蕾的数量，持续黑暗有利于积累更多神经丘和味蕾从而易于食物获取。因此，人工养殖条件下应模拟野生环境，为仔稚鱼提供遮蔽物，同时，放流地点应选择环境丰富度高且具备躲避物的水域，提高放流苗种存活率。滇池金线鲃种内杂交结果显示：杂交优势体现于河流型与龙潭型杂交组合间，旧寨龙潭和牧羊河种群之间的杂交可获得最大生长速度。生长速度受生长相关基因控制：Ghrelin和IGF-1基因的表达对滇池金线鲃生长起促进作用；MSTN和SST1基因的表达则起抑制作用。牧羊河、三百亩水库和旧寨龙潭种群遗传分化较小，且种群内部结构复杂，塘养条件下部分个体生长快速，可能由种群内部杂交产生。虽然牧羊河和承龙水苑种群遗传分化较高，但杂交优势并不明显，提示两者杂交可能出现部分杂交不亲和性，为避免种群间杂交造成遗传衰退，应禁止向牛栏江投放牧羊河种群。为提高滇池金线鲃雌性比例，用以保护数量稀少的种群，分别通过紫外线灭活滇池金线鲃精子和软鳍新光唇鱼（Neolissochilus benasi）精子诱导滇池金线鲃雌核发育。灭活滇池金线鲃精子诱导雌核发育结果显示：灭活5分钟，单倍体开始出现（11.8%），且随着时间延长，单倍体比例随之升高，40分钟达到52.8%；除40分钟照射组外，其他各组均有一定比例的三倍体出现，所占比例在7%至21%之间。灭活软鳍新光唇鱼精子诱导雌核发育结果显示：灭活10分钟，不经过冷休克即可获得90%以上雌核发育二倍体，而冷休克诱导二倍体率却略有降低；灭活时间达到20分钟，雌核发育二倍体比例降低至30%左右，单倍体率大幅增加。无论灭活时间长短，受精率（10%左右）和出膜率（30%左右）均很低。 紫外线灭活滇池金线鲃精子实验揭示，滇池金线鲃种群内可能存在三倍体，于是对4个种群的800个个体进行检测，均发现一定比例不育个体，且不育个体性腺不发育，而以脂肪组织代替。其中，牧羊河亲本种群及其繁殖子二代不育个体占2%左右，2008年和2010年繁殖种群不育个体占比较高，分别占9.0%和11.5%。对2008年繁殖不育种群进行倍性检测，不育种群中既有二倍体，又有三倍体，比率约为4:1，不育个体体型与正常雌性差异较小，但显著大于正常雄性。亲缘关系分析表明，不育个体来源广泛，种群存在四倍体几率较低。增殖放流中，不育个体可能造成放流群体食物资源、空间生态位竞争，但可考虑繁殖部分不育个体用于增殖放流种群标记。

Anabarilius grahami and Sinocyclocheilus grahami, are endemic to the karst area in Yunnan province of China and distribute in Lake Fuxian and Dianchi basin respectively. The population of A. grahami had dramatic decrease in Lake Fuxian, and S. grahami had disappeared in Lake Dianchi by human activities. In order to save these two creatures, enhancement was carried out after successful artificial breeding. Up to now, the quantities of these two fish in the wild have partly recovered. However, few studies focus on genetic structure and diversity of releasing populations. At the same time, the releasing scientificalness and validity had never evaluated systematically. In this study, we analysed the genetic structure and diversity of population under artificial rearing and recaptured based on mitochondrial CYTb gene and microsatellite data, and evaluated the effects of enhancement. Furthermore, a series of tests had conducted to provide scientific and rational measures in artificial enhancement and releasing of S. grahami. Based on the data of CYTb and SSR of 14 populations, the genetic diversity levels of A. grahami in Lake Fuxian are between the oldest population and the worst artificial population at present. The best two genetic diversity populations are protected in the endangered fish conservation center (EFCC), Kunming Institute of Zoology, Chinese Academy of Sciences, then the fishery of Jiuxi and Huoyanshan, the lowest diversity are found in fishery of Luchong, Haikou and first generation population in the endangered fish conservation center which introduced from Haikou. Due to deficiency of genetic test before releasing, lower genetic diversity population had release to Lake Fuxian, genetic dilution and introgression had affected original wild population and lead to the decline of genetic diversity. The results of Hardy-Weinberg balance test reflected that 3 to 4 loci deviated in total of 7 loci at most populations except EFCC004, EFCC005 and part of samples recapture from the Lake. For the lower genetic differentiation, crossbreeding could be taken to recovery balance of part locus. Genetic analysis based on 19 populations reflected that S. grahami have relatively lower diversity in mitochondrial gene and higher diversity in nuclear gene (SSR data). Nucleotide polymorphism of CYTb gene found in first generation was higher than parental strains in the endangered fish conservation center, Kunming Institute of Zoology, Chinese Academy of Sciences. Improvement of artificial breeding technology made a contribution to microsatellite diversity. First generation in early stage had a relatively lower diversity compare to technique was well developed and corresponding to the recapture sample from Lake Dianchi and Panlong river. Besides, the recapture populations were not as good as than in releasing populations in genetic diversity which pointed out that part of individuals suffered natural and artificial selection and end up with elimination. Hardy-Weinberg test reflected that the genetic structure were reasonable because most of the loci had no deviation, appropriate enhancement and releasing could maintain and recovered the balance of loci. Higher genetic differentiation was found between Chenglong vauclusian spring and other populations, thus, this population should not release to Lake Dianchi.According to data collected from photoperiod trial, photoperiod had no significant effects on larvae but dramatically influenced on juvenile of S. grahami. Before winter, continuous light promoted the growth of fries. However, phototoxicity caused a lower growth rate in 24Light:0Dark group compare to natural photoperiod and continuous dark during the period of winter, and led to a higher ration of deformity. Moreover, continuous dark was beneficial to the formation of neuromasts and taste buds for finding food easier. As a troglophile species, simulation field environment should be considered to provide suitable shelter for larvae and juvenile under artificial culture and chose high environmental richness sites for releasing. Crossbreeding results reflected that heterosis were found between rheophilic and limnophilic, using parental strains of Muyang river as female and Jiuzhai vauclusian spring as male could achieve a greatest growth rate. Growth performance was controlled by growth-related genes: expression of Ghrelin and IGF-1 could promote growth, but on the contrary, expression of MSTN and SST1 could inhibit growth. For the complicated genetic structure, crossbreeding might occur in intra-population of Muyang river, Sanbaimu reservoir and Jiuzhai vauclusian spring which made part of individual grow faster in artificial rearing. Although the distance between Muyang river and Chenglong vauclusian spring was the largest, heterosis was not distinct. Cross-incompatibility might be responsible for the growth of hybrids between these two populations. Thus, individuals produced by Muyang river should be prohibited to release in Niulan river to avoid hybrid deperession. In order to rescue nearly extinct populations, we need to enhance the ratio of female individuals in production. We obtained gynogenesis diploid in S. grahami using ultraviolet (UV)-irradiated S. grahami and Neolissochilus benasi sperm. Results based on ultraviolet (UV)-irradiation S. grahami sperm reflected that haploid occurred when irradiation last 5 minutes (11.8%) and as the time expand, the ratio of haploid rising and the biggest ratio was 52.8% at 40 minutes. Besides treatment of 40 minutes, a ratio range from 7% to 21% of triploid had found. Results based on ultraviolet (UV)-irradiation N. benasi sperm reflected that 10 minutes of UV irradiation could obtain more than 90 percent of gynogenesis diploid by spontaneous generation and cold shock could decline this ratio slightly. When the irradiation time reach at 20 minutes, the ratio of gynogenesis diploid drop to 30% and haploid ratio had a sharp rise. No matter how long the irradiation last, both of the fertilization rate (about 10%) and hatching rate (about 30%) were very low. Based on the data obtained from offspring after UV irradiation on S. grahami sperm, we found there are triploid individuals in artificial populations. After four populations (800 individuals) checking, sterile individuals were found with gonadal dysgenesis and replaced by adipose tissue. In these four Muyang river populations, parents introduced from wild and its F2 generation had about 2% sterile individual ratio and the ratio was higher in F1 generations of breeding in 2008 and 2010 with 9.0% and 11.5% respectively. The ploidy test in 2008 breeding population reflected that both of diploid and triploid were exist (the ratio of diploid: triploid was about 4:1). The body size measured by standard length and body weight reflected that sterile individuals were as bigger as normal female and significant bigger than male. Genetic relationship reflected that sterile individuals came from different parents, and there was little probability that tetroploid existed in natural populations. On one hand, sterile individuals might be compete food and ecological niche with their fertile companions. On the other hand, we could consider using sterile individuals as a method of tag-released.