The detection of Anisakis simplex s.s./A. pegreffii putative hybrids has been a controversial issue in spite of the fact that natural hybridization is an extended process across free living and parasitic organisms. Differential traits of biomedical and ecological importance, such as invasive and allergenic potential have been demonstrated in both cryptic species. Therefore, in this work, we discuss about the potential for hybridization between these anisakid species in sympatric zones, implementing a multi-marker Restriction fragment length polymorphism (RFLP) genotyping approach based on the ribosomal DNA internal transcribed spacer 1 (ITS1), the mitochondrial cytochrome C oxidase 2 (Cox-2) and a new nuclear marker, the highly conserved β-tubulin gene (β-TUB). The two cryptic species differed at least in 7 bp in the β-TUB gene and some larvae with heterozygous genotypes at the 7 diagnostic nucleotide positions were found. Taxonomic, population and genealogical analyses served to support the occurrence of hybridization between both species. Predicted restriction endonucleases enzymes were assayed for Cox-2 and β-TUB markers. The implemented multi-marker PCR-RFLP allowed us to detect the two pure parental species, F1 hybrids, hybrid backcrossed progeny and individuals with nuclear-mitochondrial discordance, being a useful, simple and reproducible procedure in any laboratory for epidemiological studies.

Anisakis simplex ss / A. pegreffii的检测尽管自然杂交是跨越自由生活和寄生生物的扩展过程，但假定的杂交仍是一个有争议的问题。在这两个隐性物种中均已证明了具有生物医学和生态重要性的差异性状，例如侵入性和致敏性。因此，在这项工作中，我们讨论了同胞带区域中这些anisakid物种之间杂交的潜力，基于核糖体DNA内部转录间隔子1（ITS1），线粒体实现了多标记限制性片段长度多态性（RFLP）基因分型方法细胞色素C氧化酶2（Cox-2）和新的核标记物，高度保守的β-微管蛋白基因（β-TUB）。β-TUB基因中的两个隐性物种至少相差7 bp，并且在7个诊断核苷酸位置发现了一些具有杂合基因型的幼虫。分类学，种群和家谱分析有助于支持两种物种之间杂交的发生。分析了预测的限制性核酸内切酶的Cox-2和β-TUB标记。已实施的多标记PCR-RFLP技术使我们能够检测到两个纯亲本物种，F1杂种，杂种回交后代以及核线粒体不一致的个体，这在任何流行病学研究实验室中都是有用，简单且可重复的过程。分析了预测的限制性核酸内切酶的Cox-2和β-TUB标记。已实施的多标记PCR-RFLP技术使我们能够检测到两个纯亲本物种，F1杂种，杂种回交后代以及核线粒体不一致的个体，这在任何流行病学研究实验室中都是有用，简单且可重复的过程。分析了预测的限制性核酸内切酶的Cox-2和β-TUB标记。已实施的多标记PCR-RFLP技术使我们能够检测到两个纯亲本物种，F1杂种，杂种回交后代以及核线粒体不一致的个体，这在任何流行病学研究实验室中都是有用，简单且可重复的过程。