Mladineo Ivona, Trumbić Željka, Radonić Ivana, Vrbatović Anamarija, Hrabar Jerko, Bušelić Ivana
Laboratory of Aquaculture, Institute of Oceanography & Fisheries, Šetalište Ivana Meštrovića 63, 21000 Split, Croatia.
University Department of Marine Studies, University of Split, Ruđera Boškovića 33, 21000 Split, Croatia.
Int J Parasitol. 2017 Mar;47(4):215-223. doi: 10.1016/j.ijpara.2016.11.003. Epub 2017 Jan 3.
The genus Anisakis includes nine species which, due to close morphological resemblance even in the adult stage, have previously caused many issues in their correct identification. Recently observed interspecific hybridisation in sympatric areas of two closely related species, Anisakis simplex sensu stricto (s.s.) and Anisakis pegreffii, has raised concerns whether a F1 hybrid generation is capable of overriding the breeding barrier, potentially giving rise to more resistant/pathogenic strains infecting humans. To assess the ecological significance of anisakid genotypes in the Adriatic Sea, an allopatric area for the two above-mentioned species, we analysed data from PCR-RFLP genotyping of the ITS region and the sequence of the cytochrome oxidase 2 (cox2) mtDNA locus to discern the parental genotype and maternal haplotype of the individuals. Furthermore, using in silico genome-wide screening of the A. simplex database for polymorphic simple sequence repeats or microsatellites in non-coding regions, we randomly selected potentially informative loci that were tested and optimised for multiplex PCR. The first panel of microsatellites developed for Anisakis was shown to be highly polymorphic, sensitive and amplified in both A. simplex s.s. and A. pegreffii. It was used to inspect genetic differentiation of individuals showing mito-nuclear mosaicism which is characteristic for both species. The observed low level of intergroup heterozygosity suggests that existing mosaicism is likely a retention of an ancestral polymorphism rather than a recent recombination event. This is also supported by allopatry of pure A. simplex s.s. and A. pegreffii in the geographical area under study.
异尖线虫属包括九个物种，由于即使在成虫阶段形态也极为相似，此前在其正确鉴定方面引发了诸多问题。最近在两个密切相关的物种——狭义异尖线虫（Anisakis simplex sensu stricto，s.s.）和佩氏异尖线虫（Anisakis pegreffii）的同域分布区域观察到种间杂交现象，这引发了人们对F1杂交一代是否能够突破繁殖障碍的担忧，因为这可能会产生更具抗性/致病性的毒株感染人类。为了评估在亚得里亚海（上述两个物种的异域分布区域）异尖线虫基因型的生态意义，我们分析了来自ITS区域的PCR-RFLP基因分型数据以及细胞色素氧化酶2（cox2）线粒体DNA位点的序列，以辨别个体的亲本基因型和母本单倍型。此外，通过对狭义异尖线虫数据库进行全基因组范围内的非编码区多态性简单序列重复或微卫星的计算机筛选，我们随机选择了潜在的信息位点，并对其进行多重PCR测试和优化。为异尖线虫开发的第一组微卫星显示出高度多态性、敏感性，并且在狭义异尖线虫和佩氏异尖线虫中均能扩增。它被用于检测表现出线粒体-核镶嵌现象的个体的遗传分化，这是两个物种的共同特征。观察到的群体间低水平杂合性表明，现有的镶嵌现象可能是祖先多态性的保留，而非近期的重组事件。在本研究的地理区域中狭义异尖线虫和佩氏异尖线虫的异域分布也支持了这一点。
