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Genetic monitoring of natural sturgeon spawning in the lower Volga for the period 2017-2018

Alexey A. Sergeev1*, Anna E. Barmintseva1, Natalia I. Chavychalova2, Maria E. Tolochkova1 and Nikolai S. Mugue1
  • 1 All-Russian Research Institute Fisheries and Oceanography, Russia
  • 2 Federal State Budgetary Scientific Institution Caspian Fisheries Research Institute, Russia

Sturgeons of the Caspian Sea were a valuable commercial fishery object, but at present time its commercial fishing is prohibited because of the threat of extinction. Natural sturgeon populations are partially supported by restocking of juveniles, produced by state-operated sturgeon hatcheries. The dam of Volgograd hydroelectric station has blocked spawning migration routes and cut the majority of historical sturgeon spawning grounds in the Volga River, however, natural sturgeon spawning still exists below the dam. Previously this area was important breeding grounds for all Caspian sturgeon species: the beluga (Huso huso Linnaeus, 1758), the Russian sturgeon (Асipenser gueldenstaedtii Brand, 1833), the stellate sturgeon (Асipenser stellatus Pallas, 1771), the sterlet (Асipenser ruthenus Linnaeus, 1758), and almost extinct the ship sturgeon (Асipenser nudiventris Lovetsky,1828) (Vlasenko, Veschev,2008). The study of the natural sturgeon spawning effectiveness is important for the development of conservation measures. The aim of this work was to investigate the state of natural sturgeon spawning in the lower Volga. For this purpose, genetic monitoring techniques were used - analysis of mitochondrial and microsatellite markers of sturgeon fry and juveniles caught in the natural environment. This report presents the results of observations for 2017-2018. In total, we examined 376 individuals from six locations. The collection of sturgeon fry and juveniles was carried out during the entire spring-summer period. In 2017, the sturgeon fry collecting was held near the village Enotayevka (Fig. 1) - 94 sturgeon larvae were examined. In the spring-summer period 2018, the sturgeon fry and juveniles were caught in the Zamyany, Staritsa and Boundin locations (259 individuals in total), and in the river expeditions in locations 1 and 2 of lower Volga (24 individuals). In September 2018 during the sea expedition in the northern part of the Caspian Sea, four juvenile sturgeon individuals of the stellate sturgeon were caught. DNA was isolated and purified using the silica-based method (Ivanova et al., 2006). The mtDNA control region (D-loop) was amplified with primers DL651 (АTCTTААCАTCTTCАGTG) and M13АHR3 (TCАCАCАGGАААCАG-CTАTGАCАTАCCАTААTGTTTCАTCTАCC) (Mugue et al., 2008). Amplification was carried out in a total volume of 15 μL (30 mM Tris-HCl (pH 8.6), 16.6 mM (NH4)2SO4, 2.5 mM MgCl2, 0.6 mM dNTP, 3 pmol of each primer, about 100 ng DNA, and 0.5 units of Taq polymerase, Eurogen) under the following reaction conditions: initial DNA denaturation at 95°C for 2 min; 42 cycles of denaturation at 94°C for 20 s, primer annealing at 52°C for 45 s, and DNA extension at 72°C for 30 s; and final extension at 72°C for 10 min, using the PCR-primers. Sequencing was performed on an ABI 3500 automated sequencer using the BigDye v.3.1 sequencing kit from same primers. Processing of chromatograms and multiple sequence alignment was carried out in the Geneious 6.0.5. (Kearse et al., 2012). Sturgeon juveniles were genotyped using a set of five informative microsatellite loci (Afug41, Afug51, An20, AoxD161, AoxD165) described previously for other sturgeon species (Zane et al., 2002; Welsh, Blumberg, and May, 2003; Henderson-Arzapalo and King, 2002). Amplifications were performed in a total volume of 15 µL [70 mM Tris–HCl (pH 8.6); 16.6 mM (NH4)2SO4; 1.8 mM MgCl2; 200 µL of each deoxyribonucleoside triphosphate; 1 pmol primer, labeled at 5' end with either FAM, HEX, or TAMRA fluorescent dye; 4 pmol reverse (unlabeled) primer; 50–100 ng DNA template; and 0.8 units Taq polymerase (Sileks)]. The reaction conditions included initial DNA denaturation for 1 min at 94°C, followed by eight cycles of denaturation at 95°С for 20 s, and primer annealing at 58°С in the first cycle for 25 s; in each subsequent cycle, the annealing temperature was reduced by 0.5°С and primer extension at 65°С for 40 s, followed by 25 cycles of denaturation at 95°С for 20 s, primer annealing at 54°С, primer extension at 65°С for 40 s, and final extension at 65°С for 10 min. The electrophoretic separation of amplification products was performed using the ABI3100/3130 The allele sizes were determined using the GeneMarker software (Softgenetics, USA). The list of species-specific alleles was obtained from previous studies (Barmintseva, Mugue, 2013). We performed species identification of sturgeon fry and juveniles by DNA sequencing of the control region and define their mitochondrial haplotype, which could reveal an approximate number of females participated in breeding. The use of microsatellite analysis should have shown allelic polymorphism as a parameter for estimation the gene pool diversity of sturgeon species spawning in the Volga. Unexpectedly, allelic analyses revealed the presence of juvenile sturgeons from natural spawning were of hybrid origin (Table 1). They were hybrids of stellate sturgeon and sterlet. In this study we have not screened juveniles possessing A. ruthenus mtDNA with microsatellite markers, therefore we can not confirm than reciprocal hybrids (A. ruthenus female x A. stellatus male) does not occur on the natural spawning grounds According to the analysis of mitochondrial haplotypes, at least 16 females of stellate sturgeon came to spawn in the Volga in 2017-2018, where they produced about the same quantity of hybrid offspring with sterlet as pure stellate sturgeons. According to the two-year genetic monitoring, therefore, the mass in the Volga sterlet reproduces in natural spawning grounds and hybridizes with a small number of coming stellate sturgeons. The spawning volume of other sturgeon species (Russian sturgeon, beluga, and ship sturgeon) is either insignificant or was absent during these periods of observation. The process of natural hybridization revealed the catastrophically depressed state of sturgeon spawning in the Volga River when sturgeons that came to spawn do not find a partner of their own species and are forced to hybridize with the abundant sterlet individuals. Our finding indicates that mature stellate sturgeons can occasionally reach natural spawning grounds, but its hybridization with A. ruthenus will further deteriorate the already depressed population of A. stellatus in the Caspian Sea. * Figure 1. The map of sample collection locations. Green marks – sturgeon fry collecting locations, blue marks 1 and 2 – locations of river expeditions for collecting sturgeon fingerlings. * Authors Alexey A. Sergeev and Anna E. Barmintseva contributed equally.

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Acknowledgements

We would like to acknowledge all researchers of the Laboratory of fish reproduction (FSBSI «CaspNIRKh», Astrakhan, Russia) especially Mr. Stanislav A. Vlasenko, Mr. Sergey S. Fomin and teams of research vessels «Dafnia» and «Ecologist» for assistance in collecting samples.

References

Barmintseva A.E., Mugue N.S. (2013) The use of microsatellite loci for identification of sturgeon species (Acipenseridae) and hybrid forms. Russian Journal of Genetics, 49 (9), 950-961. doi: 10.1134/S1022795413090032 Ivanova, N.V., deWaard, J., and Hebert, P.D.N. (2006) An inexpensive, automation-friendly protocol for recovering high-quality DNA. Molecular Ecology Notes, 6, 998—1002. doi: 10.1111/j.1471-8286.2006.01428.x Kearse M., Moir R., Wilson A., Stones-Havas S., Cheung M., Sturrock S., Buxton S., Cooper A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Meintjes, P., and Drummond, A. (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data, Bioinformatics, 28 (12), 1647—1649. doi: 10.1093/bioinformatics/bts199 Mugue N.S., Barmintseva A.E., Rastorguev S.M., Mugue V.N., and V. A. Barmintsev (2008) Polymorphism of the Mitochondrial DNA Control Region in Eight Sturgeon Species and Development of a System for DNA-Based Species Identification. Russian Journal of Genetics, 44 (7), 913-920. doi:10.1134/S1022795408070065 Vlasenko A.D., Veschev P.V. (2008) Scales of natural reproduction of sturgeons in the lower reaches of the Volga river in present environmental conditions. Fisheries Issues, V. 9, №4 (36), 912-925 (in Russian). Zane, L., Patarnello T., Ludwig, A., Fontana F. and Congiu L. (2002) Isolation and characterization of microsatellites in the Adriatic sturgeon (Acipenser naccarii), Mol. Ecol. Notes, 2, 586–588. doi: 10.1046/j.1471-8278 .2002.00328.x Welsh, A.B., Blumberg, M., and May, B. (2003) Identification of microsatellite loci in lake sturgeon, Acipenser fulvescens, and their variability in green sturgeon, A. medirostris, Mol. Ecol. Notes, 3, 47–55. doi: 10.1046/j.1471-8286 .2003.00346.x Henderson-Arzapalo A. and King, T.L. (2002) Novel microsatellite markers for Atlantic sturgeon (Acipenser oxyrinchus) population delineation and broodstock management, Mol. Ecol. Notes, 2002, 2, 437–439. doi: 10.1046/j.1471-8278 .2002.00262.x

Keywords: Genetic monitoring, Wild spawning control, Sturgeons, Volga (river), molecular genetics, sterlet (Acipenser rhutenus L.), Stellate sturgeon, mtDNA, Microsatellite (or short tandem repeat [STR])

Conference: XVI European Congress of Ichthyology, Lausanne, Switzerland, 2 Sep - 6 Sep, 2019.

Presentation Type: Oral

Topic: THREATS AND CONSERVATION

Citation: Sergeev AA, Barmintseva AE, Chavychalova NI, Tolochkova ME and Mugue NS (2019). Genetic monitoring of natural sturgeon spawning in the lower Volga for the period 2017-2018. Front. Mar. Sci. Conference Abstract: XVI European Congress of Ichthyology. doi: 10.3389/conf.fmars.2019.07.00135

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Received: 17 Jun 2019; Published Online: 14 Aug 2019.

* Correspondence: Mr. Alexey A Sergeev, All-Russian Research Institute Fisheries and Oceanography, Moscow, Russia, aleks.sergeyev@gmail.com