Introduction
The planktivorous filter-feeding Asian carps (bighead carp -Hypophthalmichthys nobilis, silver carp -H. molitrix and their hybrids) had been introduced into more than 30 countries around the world, including Hungary and Lake Balaton. Although the stocking of Hypophthalmichthys spp. to the lake was banned in 1983 by the government, they are still abundant in the lake (despite of that several preceding studies suggested that Hypophthalmichthys spp. are not able to reproduce in this lacustrine environment). The present study is a part of a larger program that analyze the ecological impacts and life history of Hypophthalmichthys spp.in Lake Balaton, including their adverse effects on water quality and native fish populations, the rate of hybridization, growth parameters and feeding habits. The objective of the present analysis was to evaluate the genetic status of filter-feeding Asian carps living in Lake Balaton, by using molecular genetic markers for population genetic analyses and the determination of the proportion of hybrid individuals.
Material and methods
Fin clip samples were collected from 110 Hypophthalmichthys spp individuals captured in Lake Balaton, and from 25 silver carp and 24 bighead carp individuals originating from the live gene bank in Parbatipur, Bangladesh (Mia et al 2005.). DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the protocol of the manufacturer. Ten autosomal microsatellite markers (Hmo01, Hmo02, Hmo03, Hmo13, Hmo33, Hmo34, Hmo36, Hmo37, Hmo39 and Hmo40) were used for the genetic analysis of the individuals as described in Gheyas et al. (2006) with some modifications (primers were fluorescently labeled with FAM, TET and HEX dyes and the length of different microsatellite alleles were assessed by running the multiplexed PCR products against GeneScan™ -500 LIZ® (Applied Biosystems, Foster City, California) size standard on a 3130 Genetic Analyzer. Alleles were visualized using GeneMapper version 4.0 software (Applied Biosystems, Foster City, California). The analysis of mitochondrial cytochrome oxidase I gene was used to identify the maternal linage of individuals. The polymerase chain reactions were carried out as described by Ivanova et al. (2007) with COI-F (5′-TTCTCCACCAACCACAARGAYA
TYGG-3′) and COI-R (5′-CACCTCAGGGTGTCCGAARAAYCARAA-3′) primers. The sequences were determined by BigDye 3.1 sequencing Kit and 3130 Genetic Analyzer (Applied Biosystems, Foster City, California). For data analyses GenAIEx V. 6.5, MEGA7 and DnaSP v5 softwares were used.
Results
The number of microsatellite alleles was found between 4 and 20 with an average of 14 in the Lake Balaton population, 9.3 in silver carp and 9.1 in bighead carp, respectively. Expected heterozygosity values of Lake Balaton stock ranged from 0.59 to 0.91 on different loci within the collected sample group with an average of 0.77, while observed heterozygosity ranged from 0.35 to 0.87 with an average value of 0.68. The expected and observed values differed significantly in case of all markers, indicating that the stock is not in Hardy-Weinberg equilibrium. The stock of Lake Balaton and the bighead carp population showed the signs of light inbreeding. The genetic distances between the populations were between 0.35 and 0.69. The UPGMA dendogram based on Nei's Da distance data showed that the stock of Lake Balaton is between the two gene bank stocks, closer to silver carp. The assignment test (Bayesi method, Rannala and Mountain 1997) and the Structure analysis (Pritchard et al. 2000) revealed that all individuals from Lake Balaton are hybrids with higher proportion of silver carp genome; 86% of the individuals carried silver carp mitochondrial sequences, while only 14% of them had bighead carp maternal line. The numbers of the identified haplotypes were 38 and 8, respectively.
Discussion and conclusion
The results of the microsatellite analyses, complemented with the mitochondrial DNA based maternal line identification clearly showed the hybrid status of the Lake Balaton Hypophthalmichthys spp. stock. Many times there were contradictions between the maternal line typing and the microsatellite based genome distributions, indicating higher level of hybridization within the stock. The microsatellite analyses showed the lack of Hardy-Weinberg equilibrium in the stock suggesting that the individuals are not forming a natural reproductive community. Although the direct stocking of Asian filter feeding carps to the Lake Balaton has been prohibited since 1983, ,the individuals in the stock are typically younger than 33 years old. The recruitment of the stock most probably originates from the artificial hybrid production of the aquaculture companies in the lake's water basin (Boros et al., 2014, Vitál et al. 2015).This hypothesis is confirmed by the microsatellite analyses and the high variance of the mitochondrial haplotypes too. However the occasional natural reproduction of the introduced Asian carps cannot be ruled out..
Acknowledgements
This research was supported by the Hungarian Scientific Research Fund (OTKA) Grant K83893.Special thanks to Dr. David J. Penman for the gene bank samples. Filter feeding Asian carps for this study were provided by the Balaton Fish Management Non-Profit Ltd.
References
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