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Cloning in sturgeon hybrids (Acipenseridae): experimental reproduction of the first stages of reticular speciation in fish and the method of obtaining all-female offspring

Victor P. Vasil'Ev1, Evgeniy I. Rachek2, Dmitriy Y. Amvrosov2, Anna E. Barmintseva3, Nikolai S. Mugue3 and Ekaterina D. Vasilyeva4*
  • 1 Severtsov Institute of Ecology and Evolution (RAS), Russia
  • 2 Pacific Scientific Research Fisheries Center (TINRO), Russia
  • 3 All-Russian Research Institute Fisheries and Oceanography, Russia
  • 4 Lomonosov Moscow State University, Biological Department, Zoological Museum, Russia

Many sturgeon species have a high ability to form viable and more or less fertile hybrids by artificial crosses (Nikolyukin, 1972). This property is widely used to obtain hybrids with economically valuable traits, of which the most successful are hybrids between the beluga Acipenser huso Linnaeus, 1758 and the sterlet A. ruthenus Linnaeus, 1758 (bester), as well as between the kaluga A. dauricus Georgi, 1775 and the Amur sturgeon A. schrenckii Brandt, 1869. However, the very first karyological studies on this fish revealed that sturgeon species differ in their ploidy levels, and species with the same ploidy often produce normal fertile hybrid progeny capable of successful reproduction, while crossing of sturgeon species with different ploidy levels results in fully sterile or sub-fertile progeny. But it is known that hybrid fish females can be fertile even if they originate from the parent species with significant differences in their karyotypes. All discovered natural clonal species (forms) of vertebrates are of hybrid origin and are represented only by females, which breed using parthenogenesis (reptiles) or gynogenesis (fish, amphibians), in which sperm cells only stimulate the development of oocytes, but true fertilization does not occur. The fecundity of these hybrid females is due to the fact that they produce unreduced oocytes as a result of premeiotic endoreduplication of chromosomes, leading to conjugation of the sister chromosomes (instead of homologous ones) in the first division of meiosis. Thus, the offspring of such females is genetically identical to the mother, i.e. is a clone. Later it was found that the production of unreduced oocytes by artificial hybrids of fish is, although not a frequent phenomenon, but rather ordinary. To date, it is known that artificial interspecific hybrids from different groups of fish (Salmonidae, Cyprinidae, Cyprinodontidae, Orysiatidae, Centrarchidae) can produce unreduced eggs (Vasil’ev and Vasil’eva, 2010), but it was not revealed for sturgeons. However, it should be noted that the analysis of the origin of hexaploidy in sturgeon (370 chromosomes in A. brevirostrum Lesueur, 1818) suggests that hexaploidy resulted from the hybridization of species with different levels of ploidy and the formation of hybrids that produced unreduced eggs (Vasil’ev, 2009). Hybridization, unisexual reproduction, and polyploidy are three related events that underlie the phenomenon of reticular speciation in vertebrates. The complete cycle of reticular speciation involves several stages: 1) interspecific hybridization between diploid bisexual species and the origin of new clonal (gynogenetic in fish) or semiclonal (hybridogenetic) species; 2) back-cross hybridization between clonal or semiclonal species with one of its parents or the third diploid bisexual species resulting in the origin of triploid clonal species; 3) hybridization between clonal triploids and one of parental bisexual species resulting in the origin of tetraploid clonal species or, more important, tetraploid bisexual species by the restoration of bisexuality. Our studies of reticular speciation in Cobitid fishes (Vasil’ev et al., 1989; Vasil’ev and Vasil’eva, 2010), and also the above facts in sturgeons allowed us to set the task of obtaining clonal sturgeon offspring. The positive result of these studies is extremely important in sturgeon aquaculture (to obtain all-female fertile stocks) and in understanding the mechanisms of sturgeon polyploid evolution. Methodology and materials. The experimental crossbreeding to produce clonal offspring in sturgeons was carried out at the Luchegorsk Research Fish-Breeding Station (RFS), Pacific Research Fisheries Center (TINRO-Center). We used eggs from fertile female of the previously obtained hybrid between the sterlet (S) and the kaluga (K) (Rachek and Svirskii, 2010). The development of these hybrid eggs (S x K) was stimulated by using the method of induced gynogenesis: the eggs were inseminated with sperm of the sterlet and the Amur sturgeon, inactivated with ultraviolet radiation. These procedures have been described previously (Badrtdinov et al., 2008, etc.). At the same time, backcrossings between the hybrid female (S x K) and the sterlet were carried out. To confirm the success of sperm inactivation in the experiment (and the absence of male genome in the offspring), microsatellites analysis and karyological studies have been condacted. The head lymphoid organ was used for chromosome slide preparing by previously published karyological method (Vasil′ev and Sokolov, 1980). For karyological analysis we selected metaphase plates in which the chromosome number can be count accurate to 6–10 chromosomes; from one to 23 metaphase plates were analyzed from every studied fish. Five microsatellite loci (AoxD161, Afug41, An20, Afug51, AoxD165) were investigated according to the method described by Barmintseva and Mugue (2013). Results. In backcross hybrids (S x K) x S average chromosome number was about 250. The appearance of progeny with such chromosome number indicates that hybrid female (S × K), used in experimental crosses, produced eggs with chromosome number about 190 (spermatozoa from the sterlet possess about 60 chromosome, a haploid set of 120-chromosomal A. ruthenus). Since the karyotype of A. dauricus includes 250–270 chromosomes (Vasil′ev et al., 2009), this means that hybrid female produces unreduced oocytes (60 + 130 chromosomes), which clearly indicates the possibility of obtaining clonal lines when using inactivated sperm. The insemination of unreduced eggs with UV-inactivated sperm of the sterlet and the Amur sturgeon resulted in a few normal fingerlings without any signs of haploid syndrome. Fourteen of them grew successfully and reached 253-420 mm TL in May 2016. The analysis of five microsatellite loci demonstrated them to be genetically identical to their mother (Fig.), that is, clonal inheritance is observed. Figure. Allelic diversity observed for five microsatellite loci in the hybrid sturgeon female, two males, and progeny obtained by insemination of eggs with inactivated sperm. K – Acipenser dauricus, S – A. ruthenus, AS - A. schrenckii, as – inactivated sperm from A. schrenckii, s – the same from A. ruthenus. Major conclusions. 1. Clonal progeny was firstly obtained in sturgeons. 2. The developed cloning technology is also a method of obtaining all-female offspring, since clones from females must be females. 3. Artificial production of clonal lines from hybrid vertebrates can be considered as experimental reproduction of the first stages of reticular speciation.

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Acknowledgements

Acknowledgements. Scientific investigations of EV are supported by the State Project of ZMMU АААА-А16- 116021660077-3.

References

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Keywords: Clones, Sturgeons, Gynogenesis, Unisexual development, reticular speciation, ploidy

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

Presentation Type: Oral

Topic: GENETICS, GENOMICS AND PHYSIOLOGY

Citation: Vasil'Ev VP, Rachek EI, Amvrosov DY, Barmintseva AE, Mugue NS and Vasilyeva ED (2019). Cloning in sturgeon hybrids (Acipenseridae): experimental reproduction of the first stages of reticular speciation in fish and the method of obtaining all-female offspring. Front. Mar. Sci. Conference Abstract: XVI European Congress of Ichthyology. doi: 10.3389/conf.fmars.2019.07.00053

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Received: 11 May 2019; Published Online: 14 Aug 2019.

* Correspondence: Dr. Ekaterina D Vasilyeva, Lomonosov Moscow State University, Biological Department, Zoological Museum, Moscow, Russia, vas_katerina@mail.ru