Introduction
Triploidization is a common technique of chromosome set manipulation that promises benefits associated with reduced fertility or sterility in aquaculture by the incompatibility in homologous chromosome pairing during meiosis I (Lee 2018.). Production of sterile fish finds its use when sexual maturation affects growth and development as in salmon, loach, or catfish production (Janhunen 2016., Park 2006.). Triploidization can reduce aggressive behavior through altered hormonal and metabolic homeostasis, which can also be applied to fishes of the Percidae family (Garner et al. 2008). Also, sterile triploid fish are used as surrogate hosts in primordial germ cell transplantation. The production of pikeperch with diminished fecundity but with acceptable vigor is particularly specific and complicated because pikeperch is considered a highly stress-sensitive fish, and the triploid fish in general appear to be more susceptible to stress (Fraser 2012.). Interspecific hybridization is a commercially used genetic tool in aquaculture to improve disease resistance and environmental tolerance in many species (Jiang 2022.) Nevertheless, thus far the issue of how triploidization would affect the gamete development of originally fertile hybrids was not evaluated.
Material and Methods
Hybrid triploid pikeperch was produced by hybridization of pikeperch ( Sander lucioperca) females with Volga pikeperch males ( Sander volgensis ) and triploidized by inhibiting the first polar body using hydrostatic pressure, 8000 PSI, for 10 min. Larval ploidy level was determined by flow cytometry and chromosome analysis (Káldy 2021). The trial included a hybrid triploid group and three controls, hybrid diploid, pikeperch diploid, and its triploid form. Fish were kept in a recirculation system under natural light at a temperature of 16-22 °C for 330 DPH. Gonads of juvenile fish were fixed in a modified Davidson solution and analyzed using conventional histological techniques, staining with hematoxylin and eosin (Latendresse 2002.).
Results and discussion
Chromosome analysis showed that the triploidization procedure was successful with a 100% rate, in both pressure-treated groups. There were no detected individuals with diploid or mosaic chromosomal structures. In females of both diploid groups, successive development was recorded from the oogonia stage until the cortical alveoli stage, while in males, development from spermatogonia to spermatids was recorded. Among hybrid triploids, there were also two types of gonads. Besides the primordial germ cells - spermatogonia , males showed spermatocytes and spermatids in one of the 5 samples. Hybrid triploids with phenotypically female gonads developed both oocytes (to the same stage as diploids) and spermatids. These fish did not develop normal female gonads but formed an intersex structure. Males, on the other hand, at this stage of development still did not show structural disorders. Triploid pikeperch had uniformed gonads with predominant PPGC and occasional primary oocyte.
From the results of this research, it can be concluded that the gonads of the pikeperch and its hybrid with the Volga pikeperch, in both sexes, develop without a difference, and it seems that they could sexually mature. In the case of triploid pikeperch, a large number of germ cells that did not enter meiosis, with sporadic primary oocytes, and spermatocytes in 100% of the analyzed samples, can justify the conclusion that triploids are infertile due to phenotypically bisexual gonads. While triploid hybrids developed two types of gonads a) pro-male - which did not deviate much morphologically from diploid juvenile testes, neither in shape nor in size, so at this stage of development it cannot be claimed that they are infertile; b) pro-female - due to the large number of spermatocytes and oocytes in the gonad at this stage of development, it can be concluded that they are intersex, and most likely functionally infertile. Further investigation in older age groups of fish is required to confirm these indications.
Acknowledgments
This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant number: 871108 (AQUAEXCEL3.0).
Reference
Lee, H. B., Kim, D. S., Gil, H. W., & Park, I. S. (2018). Physiological responses of diploid and triploid far eastern catfish, Silurus asotus to water temperature stress. Development & Reproduction, 22(2), 165.
Park, I. S., Nam, Y. K., & Kim, D. S. (2006). Growth performance, morphometric traits and gonad development of induced reciprocal diploid and triploid hybrids between the mud loach (Misgurnus mizolepis Günther) and cyprinid loach (Misgurnus anguillicaudatus Cantor). Aquaculture Research, 37(12), 1246-1253.
Latendresse , J. R., Warbrittion , A. R., Jonassen , H., & Creasy, D. M. (2002). Fixation of testes and eyes using a modified Davidson’s fluid: comparison with Bouin’s fluid and conventional Davidson’s fluid. Toxicologic pathology, 30(4), 524-533.
Jiang, G., Li, Q., & Xu, C. (2022). Growth, survival and gonad development of two new types of reciprocal triploid hybrids between Crassostrea gigas and C. angulata. Aquaculture, 559, 738451.
Janhunen , M., Vehviläinen , H., Koskela , J., Forsman , A., & Kankainen , M. (2019). Added value from an added chromosome: Potential of producing large fillet fish from autumn to spring with triploid rainbow trout, Oncorhynchus mykiss. Aquaculture Research, 50(3), 818–825. doi:10.1111/are.13952
Káldy J, Patakiné Várkonyi E, Fazekas GL, Nagy Z,…,& Ljubobratović U. (2021) . Effects of Hydrostatic Pressure Treatment of Newly Fertilized Eggs on the Ploidy Level and Karyotype of Pikeperch Sander lucioperca (Linnaeus, 1758). Life (Basel).;11 (12): 1296. doi: 10.3390/life11121296.
Garner, S. R., Madison, B. N., Bernier, N. J., & Neff, B. D. (2008). Juvenile growth and aggression in diploid and triploid Chinook salmon Oncorhynchus tshawytscha(Walbaum). Journal of Fish Biology, 73(1), 169–185. doi:10.1111/j.1095-8649.2008.01923.x