Mitochondrial DNA variation, effective female population size and population history of the endangered Chinese sturgeon, Acipenser sinensis

The anadromous Chinese sturgeon (Acipenser sinensis), mainly endemic to the Yangtze River in China, is an endangered fish species. The natural population has declined since the Gezhouba Dam blocked its migratory route to the spawning grounds in 1981. In the near future, the completion of the Three Gorges Dam, the world's largest hydroelectric project, may further impact this species by altering the water flow of the Yangtze River. Little is currently known about the population genetic structure of the Chinese sturgeon. In this study, DNA sequence data were determined from the control region (D-loop) of the mitochondrial genome of adult sturgeons (n = 106) that were collected between 1995-2000. The molecular data were used to investigate genetic variation, effective female population size and population history of the Chinese sturgeon in the Yangtze River. Our results indicate that the reduction in abundance did not change genetic variation of the Chinese sturgeon, and that the population underwent an expansion in the past. AMOVA analysis indicated that 98.7% of the genetic variability occurred within each year's spawning populations, the year of collection had little influence on the diversity of annual temporary samples. The relative large effective female population size (N-ef) indicates that good potential exists for the recovery of this species in the future. Strikingly, the ratio of N-ef to the census female population size (N-f) is unusually high (0.77-0.93). This may be the result of a current bottleneck in the population of the Chinese sturgeon that is likely caused by human intervention.

Species identification of Chinese sturgeon using acoustic descriptors and ascertaining their spatial distribution in the spawning ground of Gezhouba Dam

This study has developed an improved subjective approach of classification in conjunction with Step wise DFA analysis to discriminate Chinese sturgeon signals from other targets. The results showed that all together 25 Chinese sturgeon echo-signals were detected in the spawning ground of Gezhouba Dam during the last 3 years, and the identification accuracy reached 90.9%. In Stepwise DFA, 24 out of 67 variables were applied in discrimination and identification. PCA combined with DFA was then used to ensure the significance of the 24 variables and detailed the identification pattern. The results indicated that we can discriminate Chinese sturgeon from other fish species and noise using certain descriptors such as the behaviour variables, echo characteristics and acoustic cross-section characteristics. However, identification of Chinese sturgeon from sediments is more difficult and needs a total of 24 variables. This is due to the limited knowledge about the acoustic-scattering properties of the substrate regions. Based on identified Chinese sturgeon individuals, 18 individuals were distributed in the region between the site of Gezhouba Dam and Miaozui reach, with a surface area of about 3.4 km(2). Seven individuals were distributed in the region between Miaozui and Yanshouba reach, with a surface area of about 13 km(2).

Effects of damming on population sustainability of Chinese sturgeon, Acipenser sinensis: evaluation of optimal conservation measures

The numbers of spawning sites for Chinese sturgeon have been drastically reduced since the construction of the Gezhouba Dam across the Yangtze River. This dam has blocked migration of Chinese sturgeon to their historic spawning ground causing a significant decline of the Chinese sturgeon population. We conducted a VORTEX population viability analysis to estimate the sustainability of the population and to quantify the efficiency of current and alternative conservation procedures. The model predicted the observed decline of Chinese sturgeon, resulting from the effect of the Gezhouba Dam. These simulations demonstrated the potential interest of two conservation measures: increasing spawning area and reducing predation on sturgeon eggs. The simulations also demonstrated that the actual restocking program is not sufficient to sustain sturgeon population as the artificial reproduction program induce the loss of more wild mature adults that the recruitment expected by the artificial reproduction.

Using drift nets to capture early life stages and monitor spawning of the Yangtze River Chinese sturgeon (Acipenser sinensis)

P>A sampling system for capturing sturgeon eggs using a D-shaped bottom anchored drift net was used to capture early life stages (ELS) of Chinese sturgeon, Acipenser sinensis, and monitor annual spawning success at Yichang on the Yangtze River, 1996-2004, before and just after the Three Gorges Dam began operation. Captured were 96 875 ELS (early life stages: eggs, yolk-sac larvae = eleuthero embryos, and larvae); most were eggs and only 2477 were yolk-sac larvae. Most ELS were captured in the main river channel and inside the bend at the Yichang spawning reach. Yolk-sac larvae were captured for a maximum of 3 days after hatching began, indicating quick dispersal downstream. The back-calculated day of egg fertilization over the eight years indicated a maximum spawning window of 23 days (20 October-10 November). Spawning in all years was restricted temporally, occurred mostly at night and during one or two spawning periods, each lasting several days. The brief temporal spawning window may reduce egg predation by opportunistic predators by flooding the river bottom with millions of eggs. During 1996-2002, the percentage of fertilized eggs in an annual 20-egg sample was between 63.5 to 94.1%; however, in 2003 the percentage fertilized was only 23.8%. This sudden decline may be related to the altered environmental conditions at Yichang caused by operation of the Three Gorges Dam. Further studies are needed to monitor spawning and changes in egg fertilization in this threatened population.

Molecular and expression characterization of two somatostatin genes in the Chinese sturgeon, Acipenser sinensis

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species and also an important resource for the sturgeon aquaculture industry. SMART cDNA was synthesized from the hypothalamus of Chinese sturgeon, and the full-length cDNAs of two somatostatin (SS) genes were cloned and sequenced. The first cDNA (AsSS1) encodes a 116-amino acid protein that contains the SS14 sequence at its C-terminal extremity. AsSS1 shows high identity to that of human and other vertebrates. The second cDNA (AsSS2) encodes a 111-amino acid protein that contains the somatostatin variant [Pro(2)]-SS14 at its C-terminal extremity. Both the two SS mRNAs were expressed in brain and pituitary with different mRNA levels. But in peripheral tissues, AsSS2 was more widely distributed than AsSS1. High mRNA levels of AsSS2 were found in liver, kidney and heart, while low mRNA levels of AsSS2 were also detected in ovary. Throughout embryogenesis and early larval development only AsSS2 mRNAs were detected. Furthermore, in the hypothalamus of one to five year-old Chinese sturgeon, AsSS2 but not AsSS1 maintained stable expression. The mRNA distribution suggests that the Chinese sturgeon AsSS2 products play important physiological functions in adult fish as well as in cell growth and organ differentiation in embryo and larva development. (C) 2009 Elsevier Inc. All rights reserved.

Molecular characterization of Chinese sturgeon gonadotropins and cellular distribution in pituitaries of mature and immature individuals

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species, and also an important resource for the sturgeon aquaculture industry. To understand molecular characterization of Chinese sturgeon gonadotropins (GTHs), we cloned the full-length cDNAs of gonadotropin subunits common alpha (GTH-alpha), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from a pituitary cDNA library of mature female. Two subtypes of GTH-alpha were identified. The nucleotide sequences of A. sinensis common alpha I (AsGTH-alpha I), common alpha II (AsGTH-alpha II), FSH beta (AsFSH beta) and LH beta (AsLH beta) subunit cDNAs are 345, 363, 387 and 414 bp in length, and encode mature peptides of 115, 121, 129 and 138 aa, respectively. Then, three polyclonal antibodies were prepared from the in vitro expressed AsGTH-alpha I, AsFSH beta and AsLH beta mature proteins, respectively. Significant expression differences were revealed between immature and mature sturgeon pituitaries. Western blot detection and immunofluoresence localization revealed the existence of three-gonadotropin subunits (AsGTH-alpha, AsFSH beta and AsLH beta) in mature sturgeon pituitaries, but only AsFSH beta was detected in immature individual pituitaries during early stages in the sturgeon life, and obvious difference was observed between males and females. In males, AsFSH beta was expressed in 4-year-old individuals, whereas in females, AsFSH beta was just expressed in 5-year-old individuals. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Establishment of a Chinese sturgeon Acipenser sinensis tail-fin cell line and its susceptibility to frog iridovirus

Chinese sturgeon Acipenser sinensis, a cartilaginous ganoid, is a 'living fossil' on a deeply isolated evolutionary branch. A cell line was established from Chinese sturgeon tail-fin tissue (CSTF) . These epithelial CSTF cells grew well in Dulbecco's modified Eagle's medium at 25 degrees C. Karyotypic analysis revealed a normal diploid karyotype with 2n = 264 and large numbers of punctate chromosomes. A strain of frog iridoviruses [Rana grylio virus (RGV)] was used to test the susceptibility of this cell line to infection. Infection was confirmed by cytopathic effect, immunofluorescence and electron-microscope observations, which detected the viral antigens or particles in the cytoplasm of RGV-infected cells. Molecular analysis further suggested that c. 550 bp DNA fragment could be cloned from the RGV-infected CSTF cells' DNA with major capsid protein gene polymerase chain reaction primers. Furthermore, after transfection with pEGFP vector DNA, the CSTF cell line produced significant fluorescent signals indicating its utility in exogenous studies.

Isolation and characterization of microsatellites in Chinese sturgeon, Acipenser sinensis

From (GATA)(n) and (AAAG)(n) enriched genomic libraries for the Chinese sturgeon (Acipenser sinensis), 50 primer pairs were developed using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Forty-six primer pairs exhibited highly polymorphic with two to 11 alleles per locus, while the rest four displayed monomorphic. These markers yielded 246 alleles in a survey of eight specimens of wild A. sinensis. Average observed heterozygosity ranged from 0.13 to 1.00. These loci should provide sufficient levels of genetic diversity to allow parentage analysis for artificial stocking management and delineation of fine-scale population structure.

Microsatellites assessment of Chinese sturgeon (Acipenser sinensis Gray) genetic variability

Four microsatellites were used to examine the genetic variability of the spawning stocks of Chinese sturgeon, Acipenser sinensis, from the Yangtze River sampled over a 3-year period (1999-2001). Within 60 individuals, a total of 28 alleles were detected over four polymorphic microsatellite loci. The number of alleles per locus ranged from 4 to 15, with an average allele number of 7. The number of genotypes per locus ranged from 6 to 41. The genetic diversity of four microsatellite loci varied from 0.34 to 0.67, with an average value of 0.54. For the four microsatellite loci, the deviation from the Hardy-Weinberg equilibrium was mainly due to null alleles. The mean number of alleles per locus and the mean heterozygosity were lower than the average values known for anadromous fishes. Fish were clustered according to their microsatellite characteristics using an unsupervised 'Artificial Neural Networks' method entitled 'Self-organizing Map'. The results revealed no significant genetic differentiation considering genetic distance among samples collected during different years. Lack of heterogeneity among different annual groups of spawning stocks was explained by the complex age structure (from 8 to 27 years for males and 12 to 35 years for females) of Chinese sturgeon, leading to formulate an hypothesis about the maintenance of genetic diversity and stability in long-lived animals.

Ontogenetic behavior and migration of Chinese sturgeon, Acipenser sinensis

The Chinese sturgeon, Acipenser sinensis, is an anadromous protected species that presently only spawns in the Yangtze River. Using laboratory experiments, we examined the behavioral preference of young Chinese sturgeon to physical habitat (water depth, illumination intensity, substrate color, and cover) and monitored their downstream migration. Hatchling free embryos were photopositive, preferred open habitat, and immediately upon hatching, swam far above the bottom using swim-up and drift. Downstream migration peaked on days 0-1, decreased about 50% or more during days 2-7, and ceased by day 8. Days 0-1 migrants were active both day and night, but days 2-7 migrants were most active during the day. After ceasing migration, days 8-11 embryos were photonegative, preferred dark substrate and sought cover. Free embryos developed into larvae and began feeding on day 12, when another shift in behavior occurred-larvae returned to photopositive behavior and preferred white substrate. The selective factor favoring migration of free embryos upon hatching and swimming far above the bottom may be avoidance of benthic predatory fishes. Free embryos, which must rely on yolk energy for activity and growth, only used 19 cumulative temperature degree-days for peak migration compared to 234 degree-days for growth to first feeding larvae, a 1 : 12 ratio of cumulative temperature units. This ratio suggests that sturgeon species with large migratory embryos, like Chinese sturgeon, which require a high level of energy to swim during migration, may migrate only a short time to conserve most yolk energy for growth.

Growth of juvenile Chinese sturgeon Acipenser sinensis fed live and formulated diets

Triplicate groups of 3.8-g juvenile Chinese sturgeon Acipenser sinensis were reared for 8 weeks in indoor flow-through systems on one of four diets: a natural diet consisting solely of live tubificid worms, a semimoist practical diet, a dry practical diet, and a purified diet. The formulated diets were prepared in the laboratory and had protein contents of 47-50%. Except for the group fed the purified diet, fish showed high survival (94-96%) and growth (final weight, 41-45 g). Survival and specific growth rate did not differ significantly between groups fed the natural, semimoist, and dry practical diets, but were significantly (P < 0.05) lower in fish fed the purified diet. Proximate analysis showed that fish fed purified diet had lower protein and lipid levels but a higher moisture content than fish fed other diets. Our results demonstrated that growth and survival of cultured juvenile Chinese sturgeon fed practical diets were comparable with those fed live tubificid worms. However, Chinese sturgeon fed a purified diet showed inferior growth and survival.

Liver proteome profiling of juvenile Chinese sturgeon (Acipenser sinensis) using GeLC-MS/MS approach

Chinese sturgeon (Acipenser sinensis), mainly distributed in the Yangtze River, has been listed as a grade I protected animal in China because of a dramatic decline in population owing to loss of natural habitat for reproduction and interference by human activities. Understanding the proteome profile of Chinese sturgeon liver would provide an invaluable resource for protecting and increasing the stocks of this species. In this study, we have analyzed proteome profiles of juvenile Chinese sturgeon liver using a one-dimensional gel electrophoresis coupled to LC-MS/MS approach. A total of 1059 proteins and 2084 peptides were identified. The liver proteome was found to be associated with diverse biological processes, cellular components and molecular functions. The proteome profile identified a variety of significant pathways including carbohydrate metabolism, fatty acid metabolism and amino acid metabolism pathways. It also established a network for protein biosynthesis, folding and catabolic processes. The proteome profile established in this study can be used for understanding the development of Chinese sturgeon and studying the molecular mechanisms of action under environmental or chemical stress, providing very useful omics information that can be applied to preserve this species.