Genetic differentiation of Artemia franciscana (Kellogg, 1906) in Kenyan coastal saltworks

Abstract

The nature of genetic divergence between the Artemia population native to San Francisco Bay, (SFB) USA and those from the introductions of SFB material in the Kenyan coast (Fundisha and Kensalt) 2 decades ago were investigated using the mitochondrial DNA (mtDNA) and heat shock protein 70 (Hsp70) gene molecular markers. Control samples were obtained from Great Salt Lake (GSL), Utah, USA and Vinh Chau (VC), Vietnam, while Artemia cysts from Tanga, Tanzania were for additional study. The DNA was extracted from 80 single Artemia cysts (10 cysts per sample) using the Chelex protocol for mtDNA analysis while the Promega method was used in the Hsp70 gene analysis. The 1,500 bp fragment of the 12S - 16S region of the mtDNA and a 1,935 bp fragment of the Hsp70 gene were amplified through Polymerase Chain Reaction (PCR) using appropriate primers followed by Restriction Fragment Length Polymorphism (RFLP) digestion based on 6 restriction endonucleases (AluI, HaeIII, HinfI, RsaI, XbaI and HpaII) for mtDNA and 4 restriction endonucleases (AluI, Sau3A, HinfI and RsaI) for the Hsp70 gene. The mtDNA analysis indicated higher haplotype diversity (0.76 ± 0.07) in Artemia samples from Fundisha saltworks while the rest of the samples were monomorphic. The presence of 3 haplotype genotypes in Fundisha (AAAAAA, AAAABA and AAABBA) signified a molecular evidence of a systematic genetic differentiation. However, the haplotype frequencies within Fundisha were statistically insignificant (P > 0.05). The existence of haplotypes AAAAAA and AAAABA in Fundisha, GSL and SFB cyst samples was a molecular evidence of SFB and GSL Artemia strains co-existing in Fundisha saltwork while the Kensalt and Tanga Artemia samples were purely SFB strains. The non-polymorphic DNA fingerprint observed in Kensalt Artemia cysts was probably caused by the non-sequential Artemia culture system, lack of ecological isolation and limited genetic drift. Also, the limited mtDNA fragment analysed might have excluded valuable genetic information from the study. The Hsp70 gene RFLP fingerprint showed lack of unique gene signatures in the Kenyan Artemia samples suggesting that other factors besides Hsp70 could be involved in their superior thermotolerance compared to their SFB ancestors. In conclusion, if indeed there was significant genetic differentiation between the Kenyan Artemia population and their SFB ancestors, then the tools used in the study were not sufficiently adequate to detect this microevolutionary divergence. Further genetical studies based on the larger mtDNA fragment using robust genetic markers are recommended. Also, evolutionary and ecological studies of the heat shock protein family and the stress response are highly encouraged.