Genetic Diversity And Physiology Of The Cavefish (Triplophysa Xiangxiensis)

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.