High‐throughput SNP‐genotyping analysis of the relationships among Ponto‐Caspian sturgeon species

Legally certified sturgeon fisheries require population protection and conservation methods, including DNA tests to identify the source of valuable sturgeon roe. However, the available genetic data are insufficient to distinguish between different sturgeon populations, and are even unable to distinguish between some species. We performed high‐throughput single‐nucleotide polymorphism (SNP)‐genotyping analysis on different populations of Russian (Acipenser gueldenstaedtii), Persian (A. persicus), and Siberian (A. baerii) sturgeon species from the Caspian Sea region (Volga and Ural Rivers), the Azov Sea, and two Siberian rivers. We found that Russian sturgeons from the Volga and Ural Rivers were essentially indistinguishable, but they differed from Russian sturgeons in the Azov Sea, and from Persian and Siberian sturgeons. We identified eight SNPs that were sufficient to distinguish these sturgeon populations with 80% confidence, and allowed the development of markers to distinguish sturgeon species. Finally, on the basis of our SNP data, we propose that the A. baerii‐like mitochondrial DNA found in some Russian sturgeons from the Caspian Sea arose via an introgression event during the Pleistocene glaciation.     

鲟分枝杆菌病及其病原研究

20092010年间,我国人工养殖的中华鲟（Acipenser sinensis）、史氏鲟（Acipenser schrencki）和杂交鲟（hybrid sturgeon:A. baeri-A. gueldenstaedtii）暴发了细菌性疾病。患病鲟通过组织切片观察,病原菌的分离、鉴定以及组织样品中病原菌的检测,结果显示从19条患病鲟中分离到49株分枝杆菌。病原菌经过多个保守基因的测序分析和部分生理生化特征的鉴定,共发现有7种分枝杆菌,分别为龟分枝杆菌（Mycobacterium chelonae）、海分枝杆菌（Mycobacterium marinum）、戈登氏分枝杆菌（Mycobacterium gordonae）、偶发分枝杆菌（Mycobacterium fortuitum）、苏尔加分枝杆菌（Mycobacterium szulgai）、猪分枝杆菌 （Mycobacterium porcinum）和Mycobacterium arpuense。在诊断过程中发现两种或三种分枝杆菌同时存在于同一样品中,分子生物学的诊断结果表明分枝杆菌复合感染十分常见,而海分枝杆菌是分枝杆菌复合感染中最为常见的分枝杆菌。分离的病原菌对斑马鱼的攻毒试验结果表明在以上7种分枝杆菌中海分枝杆菌的毒力最强。以上结果表明海分枝杆菌是鲟分枝杆菌病的主要致病菌,分枝杆菌复合感染是鲟分枝杆菌病的主要感染形式。研究中史氏鲟和中华鲟的分枝杆菌病,以及在病鱼体内分离的猪分枝杆菌和M. arupense在国内外均尚未见报道。       

Meiotic gynogenesis in the stellate,Russian sturgeon and sterlet

Diploid gynogenetic progenies were obtained in the stellate sturgeon Acipenser stellatus, Russian sturgeon A. gueldenstaedtii, and sterlet A. ruthenus by means of insemination of the eggs with UV-irradiated spermatozoa and suppression of the second meiotic division by heat shock. The gynogenetic nature of experimental fish was confirmed by RAPD-PCR analysis of DNA. Effective photoreactivation of UV-induced lesions of spermatozoa was shown in the case of illumination of the fertilized eggs with visible light. This phenomenon should be taken into account when determining the doses of irradiation that allow inactivation of the male chromosomes and incubating gynogenetic embryos. Gynogenetic stellate and Russian sturgeons are viable and can be reared in order to study the mechanism of sex determination in sturgeons.     

Effects of Temperature on the Early Development, Growth, and Survival of Shortnose Sturgeon, Acipenser brevirostrum, and Atlantic Sturgeon, Acipenser oxyrhynchus, Yolk-Sac Larvae

We reared shortnose and Atlantic sturgeons at different temperatures after hatch and measured yolk utilization rate and efficiency (YUE), maximum standard length, survival and development of escape response. Newly hatched Atlantic sturgeon, were smaller in size, more efficient at utilizing yolk (incorporating yolk to body tissue) and reached developmental stages sooner than shortnose sturgeon reared at the same temperatures (13–15°C). Within each species, decreasing temperature delayed yolk absorption, escape initiation, time to reach maximum size, and time to 100% mortality. However, YUEs and the size of the larvae at these 'stages' were independent of rearing temperature for both species. These results suggest that even as temperature drives metabolic processes to speed up development, these two species are still extremely efficient at transferring yolk energy to body tissues. The lower efficiencies experienced by larval shortnose may reflect difference in yolk quality between the two species and/or the Atlantic sturgeon's higher conversion efficiency. The ability of these two sturgeon species to develop successfully and efficiently under a wide range in temperatures may provide a competitive advantage over more stenothermic species and explain their persistence through evolutionary time.     

Meiotic Gynogenesis in the Stellate and Russian Sturgeons and Sterlet

Diploid gynogenetic progenies were obtained in the stellate sturgeon Acipenser stellatus, Russian sturgeon, A. gueldenstaedtii, and sterlet A. ruthenus by means of insemination of the eggs with UV-irradiated spermatozoa and suppression of the second meiotic division by heat shock. The gynogenetic nature of experimental fish was confirmed by RAPD-PCR analysis of DNA. Effective photoreactivation of UV-induced lesions of spermatozoa was shown in the case of illumination of the fertilized eggs with visible light. This phenomenon should be taken into account when determining the doses of irradiation that allow inactivation of the male chromosomes and incubating gynogenetic embryos. Gynogenetic stellate and Russian sturgeons are viable and can be reared in order to study the mechanism of sex determination in sturgeons.     

鲟鱼分子鉴定方法的整合应用

鲟形目物种是国家重点保护水生野生动物和CITES附录物种。其人工养殖种群数量众多, 种类丰富, 产品贸易量大, 但种类鉴定困难。本文在厘清当前鲟鱼商业类群的基础上, 通过分析现有种类鉴定方法, 整合了线粒体DNA遗传分析、SNP分析和微卫星DNA分析的鉴定方法, 探讨其鉴定国际贸易所涉鲟鱼的可行性。结果表明: 上述3种方法整合应用可在11种纯种鲟鱼及其正反杂交产生的杂交鲟范围内进行盲检。当前共有贸易鲟鱼36种, 其中杂交鲟14种, 杂交鲟的亲本共涉及9种鲟鱼。整合方法可准确鉴定小体鲟(Acipenser ruthenus)、达氏鳇(Huso dauricus)、施氏鲟(Acipenser schrenckii)、欧洲鳇(Huso huso)、闪光鲟(Acipenser stellatus)、高首鲟(A. transmontanus)两两杂交所产生的杂交鲟, 小体鲟为母本与纳氏鲟(Acipenser naccarii)或富氏鲟(A. fulvescens)或中华鲟(A. sinensis)产生的杂交鲟, 纯种的达氏鳇、高首鲟、富氏鲟和中华鲟, 但无法准确鉴定纯种的西伯利亚鲟(Acipenser baerii)和俄罗斯鲟(A. gueldenstaedti)以及父母本涉及此两种鲟鱼的杂交鲟。由于已开发的分子标记仍有限, 上述结果是对当前CITES贸易所涉鲟鱼鉴定的最大范围, 可以满足一些鲟鱼野生种群保护、贸易产品检测、种质资源管理等情景下的鉴定需求。     

Outmigration pathways of stocked juvenile European sturgeon (Acipenser sturio L., 1758) in the Lower Rhine River,as revealed by telemetry

Working towards a future Rhine Sturgeon Action Plan the outmigration pathways of stocked juvenile European sturgeon (Acipenser sturio L., 1758) were studied in the River Rhine in 2012 and 2015 using the NEDAP Trail system. A total of 87 sturgeon of 3 to 5 years old (n = 43 in 2012, n = 44 in 2015) were implanted with transponders and released in May and June in the river Rhine at the Dutch‐German border, approximately 160 km from the sea. In total three sturgeons (3%) were found dead on river banks within seven days after the release. Based upon their wounds these sturgeons were likely hit by ship‐propellers. Tracking results were obtained from 57 (66%) of the sturgeons, of which 39 (45%) indicated movement into the Port of Rotterdam. Here the sturgeons remained for an average of two weeks, which suggests they spent time to acclimatize to higher salinities before entering the North Sea. Of the 45 (52%) sturgeons that were confirmed to have entered the North Sea, ten (22%) were recaptured (mainly by shrimpers and gill‐nets) close to the Dutch coastline; nine were alive and were released. From the results we obtained the preferred outmigration pathways, movement speeds and an indication of impacting factors (i.e. ship propellers and bycatch). Bycatches provided also localisations information in the coastal area. A next step to complete this work would be to assess habitat selection in freshwater and downstream migration of young of the year (YOY sturgeons) in the Lower Rhine.     

Genetic variation and relationships of seven sturgeon species and ten interspecific hybrids

Background

Sturgeon cultivation is important for both industry and aquaculture in China. To date, more than 17 species or strains have been farmed for fillets and caviar production. Crossbreeding among different sturgeon species is frequent and the F2 hybrids are fertile. However, large-scale farming can have negative impacts on wild populations i.e. escape of exotic sturgeons and must be taken into consideration. Escape of exotic sturgeons can cause severe ecological problems, including threatening native sturgeon species once the exotic varieties become established or hybridize with native individuals. However, little is known about their genetic resources and variation.

Methods

Genetic diversity and introgression of seven sturgeon species were analyzed using mitochondrial DNA cytochrome oxidase subunit I (COI) and nine microsatellite markers. This study included 189 individuals from seven sturgeon species and 277 individuals from ten lineages of F2 hybrid strains.

Results

MtDNA COI sequences (632 bp long) were generated from 91 individuals across the 17 sturgeon strains and produced 23 different haplotypes. Haplotype diversity was high (h = 0.915 ± 0.015) and nucleotide diversity was low (π = 0.03680 ± 0.00153) in the seven sturgeon species and ten interspecific hybrids. Phylogenetic analyses resulted in almost identical tree topologies, and different haplotype structures were mainly related with sturgeons of different female parents. Analysis of molecular variance revealed that 81.73% of the genetic variance was due to matrilineal differences, while 9.40% resulted from strain variation. Pairwise Fst values obtained with POLYSAT software, were high among strains and ranged from 0.031 to 0.164. Admixture analysis assigned seven distinct groups and ten genotypes of admixed clusters composed of hybrid strains using STRUCTURE when assuming K = 7.

Conclusions

The interspecific mtDNA gene tree corresponded to the expected taxonomic divisions. These relationships were also supported by the results from the microsatellite analysis and contributed to unambiguously identify seven sturgeon species and ten F2 hybrid strains from sturgeon farms in China. Moreover, we found that introgressive hybridization is pervasive, exists in both purebred and hybrid sturgeons, and may reflect widespread mismanagement in sturgeon breeding in China.     

Status of sturgeon aquaculture and sturgeon trade in China: a review based on two recent nationwide surveys

The authors reviewed the aquacultural history of Acipenseriformes in China, related the legal status and examined the current status of the cultured species or hybrids, origins of seedlings, quantities of production, geographic distribution in farming, and the sustainability for both restocking programmes and human consumption. The census shows that since 2000, the production of cultured sturgeons in China appears to have become the largest in the world. As of 2000, the rapid growth of sturgeon farming in China mainly for commercial purposes has shifted harvests in the Amur River from caviar production to the artificial culture of sturgeon seedlings. This dramatic development has also caused a series of extant and potential problems, including insufficient market availability and the impact of exotic sturgeons on indigenous sturgeon species. Annual preservation of sufficient higher‐age sturgeons should be a national priority in order to establish a sustainable sturgeon‐culture industry and to preserve a gene pool of critically endangered sturgeon species to prevent their extinction.     

Screening and identification of female-specific DNA sequences in octaploid sturgeon using comparative genomics with high-throughput sequencing

In this study, six candidate female-specific DNA sequences of octaploid Amur sturgeon (Acipenser schrenckii) were identified using comparative genomic approaches with high-throughput sequencing data. Their specificity was confirmed by traditional PCR. Two of these sex-specific sequences were also validated as female-specific in other eight sturgeon species and two hybrid sturgeons. The identified female-specific DNA fragments suggest that the family Acipenseridae has a ZZ/ZW sex-determining system. However, one of the two DNA sequences has been deleted in some sturgeons such as Sterlet sturgeon (Acipenser ruthenus), Beluga (Huso huso) and Kaluga (H. dauricus). The difference of sex-specific sequences among sturgeons indicates that there are different sex-specific regions among species of sturgeon. This study not only provided the sex-specific DNA sequences for management, conservation and studies of sex-determination mechanisms in sturgeons, but also confirmed the capability of the workflow to identify sex-specific DNA sequences in the polyploid species with complex genomes.     

Organization of the diencephalon ventrothalamic area in the sturgeons

Cytoarchitectonics of ventrothalamic area of the diencephalon of the sturgeons was studied in four species of the sturgeons, the great sturgeon Huso huso L., the Kura sturgeon Acipenser guelden-staedtii persicus n. Kurensis Belyaeff, the Caspian sturgeon, Ac. stellatus Pall., and the barbel sturgeon Ac. nudioventris Lov. using Nissl staining and Bilschowski impregnation techniques in Viktorov’s modification. The ventral thalamus of the sturgeons was found to include seven nuclei: ventromedial, ventrolateral, intermediate and ventral thalamic nuclei, thalamic eminence, and nuclei of ventral and medial optic tracts. The main attention was paid to comparative analysis of this area in the sturgeons and other actinopterygian fish. The structures common to all ray-finned fish or only to the lower teleosts and ganoids as well as characterizing only the sturgeons.     

Sensitivity of North American sturgeons and paddlefish to fishing mortality

Sturgeons and paddlefish exhibit unusual combinations of morphology, habits, and life history characteristics, which make them highly vulnerable to impacts from human activities, particularly fisheries. Five North American sturgeons (shortnose, Gulf, pallid, Alabama, and green sturgeon) are listed as endangered or threatened by management authorities. Managers have instituted fishery closures for the three other species of North American sturgeons (Atlantic, white, and shovelnose) and paddlefish because of low stock abundance at some point in this century. Reproductive potential in four species I examined (Atlantic, white, and shortnose sturgeon, and paddlefish) is more sensitive to fishing mortality than it is for three other intensively-fished coastal species in North America: striped bass, winter flounder, and bluefish. The sturgeons and paddlefish are generally longer-lived than the three other coastal species, and also have an older age at full maturity, lower maximum fecundity values, and older ages at which 50% of the lifetime egg production is realized with no fishing mortality.     

Past and current status of sturgeons in the upper and middle Danube River

Of the six species of sturgeons native to the Danube basin, five occurred in the upper and middle Danube. Among anadromous sturgeons were the large winter races of beluga, Huso huso, Russian sturgeon, Acipenser gueldenstaedtii, and stellate sturgeon, A. stellatus, which ascended the middle, and sometimes also the upper Danube, to spawn. Due to overfishing, followed by severe habitat alteration including damming and pollution, these anadromous sturgeons are critically endangered or extirpated from the upper and middle Danube. Acipenser gueldenstaedtii and A. nudiventris are represented only as resident non-migratory races with very small populations. The most abundant and widely distributed species is the sterlet, A. ruthenus, although it is presently limited to the middle Danube. Its population increased in some sections of the middle Danube during the past 15 years, presumably because of improving water quality, but this species remains at risk because of continuing habitat degradation.     

New strategies for brood stock management of sturgeon in the Sea of Azov basin in response to changes in patterns of spawning migration

The sturgeon stock in the Sea of Azov is presently maintained by artificial reproduction allowing for the harvest of sturgeons in this basin. Shortened spawning migrations in regulated rivers and long-term artificial selection of the ripest fish at the hatcheries have resulted in high functional maturity of sturgeon spawners in the sea near the river mouth. The traditional technology of artificial sturgeon reproduction is non-effective under modern ecological conditions. The use of ecological and hormonal methods to control seasonal propagation of stellate sturgeon, A.stellatus , Russian sturgeon, A.gueldenstaedti , beluga, Huso huso , and sterlet, A.ruthenus , make it possible to stagger the sexual cycle of breeders from the various biological groups for early (5 months) or later (6 months) spawning. Use of these methods preserves the natural heterogeneity of sturgeon populations maintained through stocking, and also allows for a considerable increase in the quantity of fry reared at the same production base.
The use of the synthetic analogue of mammalian gonadotropin-releasing hormone (Gn-RHa) is better for inducing maturity of the ripest brood fishes, especially for stellate sturgeon, in comparison with the traditional pituitary injections. The schedule of injections was determined for the various species and seasons of spawning.
Short-term rearing of larvae in ponds (no more than 15 days) and subsequent adaptation to sea water in brackish lagoons (1–5 %o) allows for repeated use of nursery ponds in sturgeon hatcheries and a considerable increase in the quantity and survival of sturgeon juveniles in the sea.
The restoration of the sturgeon population structure in the Sea of Azov is not possible without developing a genetic collection of the fish under a controlled culture regime.     

Genetic monitoring of progeny derived from natural spawning of Acipenser ruthenus Linnaeus, 1758 and Acipenser stellatus Pallas, 1771 as well as their hybrids in the Lower Volga river during the period 2017 to 2019

The state of natural spawning of sturgeons in the Lower Volga River was investigated through genetic monitoring of juveniles (larvae, fingerlings, subadults) captured at seven traditionally used monitoring locations. Sampling was performed during the period 2017–2019. In total, 460 individuals were caught and genotyped using a set of six microsatellite loci (Afug41, Afug51, Afug135, An20, AoxD161, AoxD165) and mtDNA control region sequencing. The observed species-specific microsatellite alleles revealed the presence of juvenile sturgeons originating from natural spawning also included some hybrids between Acipenser stellatus and A. ruthenus. Thus, the majority of sterlet breeding takes place in natural spawning grounds but also sterlet hybridizes with a small number of stellate sturgeons. Juveniles of other sturgeon species were not identified, and it is concluded that natural spawning activities of Russian sturgeon, beluga and ship sturgeon were insignificant or even absent in the Lower Volga during the observation period.     

Embryonic development of adriatic sturgeon,Acipenser naccarii (Bonaparte 1836), in farming conditions: a guide

The sequence and timing of the embryonic development of Adriatic sturgeon (Acipenser naccarii) are described at a constant temperature (17 ± 1 °C), from fertilisation to hatch in a hatchery. Fertilised eggs, obtained from one female and two males, were held in a flow‐through system. On the first day, embryos were sampled every 20 min during the first 12 h, and every 40 min in the following 12 h. Embryos were sampled hourly on the second day. On the third and fourth days, embryos were sampled every 2 h, and every 3 h on the fifth and sixth days. The first cleavage furrow appeared at 1 h 40 min postfertilisation. Blastulation concluded at 12 HPF (hours postfertilisation), when the dorsal blastopore lip formed. The completion of gastrulation was at 18 HPF, closing of the neural tube at 23 HPF and formation of the s‐shape heart at 31 HPF. The mass hatch occurred at 120 HPF. A. naccarii is a relatively common sturgeon species in aquaculture and possesses similar developmental patterns to those of other sturgeons; hence, we can use this species as a model for further studies about sturgeon species which are more difficult to breed artificially. Information about the embryonic development of the Adriatic sturgeon could assist restocking programmes.     

五种鲟鱼线粒体控制区异质性和系统发育分析

利用保守引物得到五种鲟鱼的线粒体DNA（mtDNA）控制区（D-loop）全长,长度在795~813 bp。序列中包括了CBS（conserved sequence block）和TAS（termination-associated sequence）区域。利用最大似然法、最大简约法和贝叶斯法构建了系统发育树,发育树分成两枝,呈现明显的生物地理分布。分析表明,现有的鳇属鱼类不是单系群起源。五种鲟鱼D-loop序列都存在长度和数目不等串联重复序列,长度在78~82 bp之间,重复序列拷贝数在4~6次不等,因此造成了mtDNA广泛的异质性现象。不同种类的重复序列单元十分相似,达氏鳇和史氏鲟重复序列单元相似度为82.93%,西伯利亚鲟和俄罗斯鲟重复序列单元相似度为90.59%。在串联重复序列后是一段不完全重复序列。通过与已有同种的重复序列比对发现不同鲟鱼重复序列相同,不同地理区域相同物种的重复序列可能发生过分子内重组。这些表明重复序列在鲟鱼进化上具有相关意义,推测重复序列可能产生在种分化前,重组发生在种分化后。     

Sex steroids in the serum of Caspian sturgeons and their specific cytosol binding in brain and gonads during the migratory cycle

Testosterone (T) and estradiol 17β (E2) concentrations were measured in the serum of Russian, stellate and beluga sturgeons. Hormone concentrations were comparatively low during the feeding period at sea and did not differ between seasons or species. At the beginning of the spring spawning migration into freshwater, both of the spring and winter races of Russian sturgeon ( Acipenser gueldenstaedtii ) and beluga sturgeon ( Huso huso ), as well as the spring-summer race of stellate sturgeon ( Acipenser stellatus ), exhibited a marked elevation of serum T, despite differences in their degrees of gonadal development. There were no differences in specific cytosol binding of androgens and estrogens in the forebrain of either migratory form of Russian sturgeon, while the specific binding of sex steroids was higher in the hypothalamus of the spring form. The data indicate that T and its receptors may control aspects of anadromous migration in sturgeons.     

Triple hybrids obtained by artificial hybridization of the Russian sturgeon Acipenser gueldenstaedtii with the hybrid of the starred sturgeon A. stellatus and the great sturgeon A. huso (Acipenseridae): The kind of inheritance of some morphological characters and fertility of the parental hybrid form

Results are described of experimental crossing for obtaining of triple hybrids of the Russian sturgeon Acipenser gueldenstaedtii, the great sturgeon A. huso, and the starred sturgeon A. stellatus and of comparative analysis of some quantitative morphological characters in these hybrids, in the progeny of parental species and of reciprocal hybrids of starred sturgeon and great sturgeon. By most investigated characters, in triple hybrids, the states of the paternal species (Russian sturgeon) are inherited, by some other characters-of great sturgeon-the state of starred sturgeon is not inherited. Therefore, on the basis of external appearance, they may be identified as the hybrid of Russian sturgeon with great sturgeon. The observed segregation by the form of gill rakers into paternal, maternal, or intermediate variants leads to a conclusion of nonmonogenous inheritance of a wide form and a conical form of gill rakers in sturgeons. On the whole, the obtained results clearly demonstrate how, due to effects of matrocliny and partocliny, and different combination of dominant and recessive alleles of the genes determining the diagnostic characters, the hybrid individuals may acquire a different extent of similarity and differences from the parental species. The investigated triple hybrids are an interesting example of mutual leveling of divergent traits in the course of combination of different genomes within limits of one closely related Atlantic group of sturgeons. As a result, a certain averaged habitus is formed which might take place also in case of formation of polyploidy species in the process of evolution.     

Population viability analysis of the Danube sturgeon populations in a Vortex simulation model

Populations of six sturgeon species in the Danube River (beluga, Russian sturgeon, stellate sturgeon, sterlet, ship sturgeon and Atlantic sturgeon) have experienced severe decline during the last several decades, mostly due to the unsustainable fishery, river fragmentation and water pollution. Present lack of knowledge on basic sturgeon demography, life history and relative effects of different negative factors is further hindering implementation of efficient policy and management measures. In the present study, population viability analysis in a Vortex simulation model has been conducted in order to assess the state of the six Danube sturgeon species, their future risk of extinction and to determine the most suitable conservation and management measures. Population viability analysis has revealed a large sensitivity of the Danube sturgeon populations to changes in the natural mortality, fecundity, age at maturity and spawning frequency. It was also confirmed that the sturgeons are highly susceptible to even moderate levels of commercial fishery, and that their recovery is a multi-decadal affair. Stocking with adult individuals was shown to produce considerably greater effect on population persistence than stocking with juveniles, but the latter approach is probably still preferable since it avoids many inherent problems of aquaculture cultivation. This study represents the first population viability analysis of the Danube sturgeons.