A novel nuclear marker and development of an ARMS-PCR assay targeting the metallopeptidase 10 (nas 10) locus to identify the species of theAnisakis simplex(s. l.) complex (Nematoda, Anisakidae)

Abstract

The genus Anisakis represents one of the most widespread groups of ascaridoid nematodes in the marine ecosystem. Three closely related taxa are recognized in the Anisakis simplex (s. l.) complex: A. pegreffii, A. simplex (s. s.) and A. berlandi. They are widely distributed in populations of their intermediate/paratenic hosts (fish and squids) and definitive hosts (cetaceans). A novel nuclear gene locus, metallopeptidase 10 (nas 10) (451 bp), was sequenced and validated on a total of 219 specimens of the three species of Anisakis, collected in fish and cetacean hosts from allopatric areas included in their ranges of distribution. The specimens of Anisakis were first identified by allozymes and sequence analysis of the mtDNA cox2 and EF1α-1 nDNA. The novel nuclear marker has shown fixed alternative nucleotide positions in the three species, i.e. diagnostic at 100%, permitting the species determination of a large number of specimens analyzed in the present study. In addition, primers to be used for amplification-refractory mutation system (ARMS) PCR of the same gene locus were designed at these nucleotide positions. Thus, direct genotyping determination, by double ARMS, was developed and validated on 219 specimens belonging to the three species. Complete concordance was observed between the tetra-primer ARMS-PCR assays and direct sequencing results obtained for the nas 10 gene locus. The novel nuclear diagnostic marker will be useful in future studies on a multi-locus genotyping approach and also to study possible hybridization and/or introgression events occurring between the three species in sympatric areas.

Molecular Identification of Zoonotic Parasites of the Genus Anisakis (Nematoda: Anisakidae) from Fish of the Southeastern Pacific Ocean (Off Peru Coast)

“…In addition, all the specimens identified as A. pegreffii (N = 288) by mtDNA cox2 sequences analysis, showed a genotype pattern belonging to the A. pegreffii species at the tetra-primer ARMS-PCR method, having the diagnostic nucleotide positions at 373 and 117 bp as those given by Palomba et al [31] for the species A. pegreffii. Further, no heterozygote genotypes at those positions were found in the samples of Anisakis spp.…”

First report on molecular identification of Anisakis simplex in Oncorhynchus nerka from the fish market, with taxonomical issues within Anisakidae

“…The detection of A. simplex helminths in minced fish increases the probability of infection of the human population, so there should be tight control over the distribution of such products in the food markets (Bao et al, 2017). Dead A. simplex larvae can save allergenic properties even after prolonged storage in frozen form (−20 ± 2°C for 11 months) (Rodríguez-Mahillo et al, 2010). WHO recognized A. simplex as the parasite with the largest number of known allergens 14 'Ani s' proteins, demonstrated strong cross-reactivity (especially Ani s 2 (paramyosin) and Ani s 3 (tropomyosin)) to homologous proteins of other nematodes and invertebrates and resistant to pepsin (Aibinu et al, 2019;Baird et al, 2014).…”

“…Three sibling species were distinctly separated from each other and this was also strongly supported by bootstrap values ( Yamada et al, 2017 ). Third, even gene loci coding metallopeptidase enables detection of fixed nucleotide positions (SNP) demonstration that A. pegreffii , A. simplex (s. s.), and A. berlandi are independent ( Palomba et al, 2020 ). Fourth, biology of helminths cannot be the reason for determine A. simplex and A. pegreffii into one species complex.…”

“…The whole anisakids and decomposed worms in thermally processed food can provoke severe allergic reactions range from hives and angioneurotic edema to anaphylactic shock (Audicana et al, 2002;Hoshino and Narita, 2011). Anisakidosis is a common disease in Holland, Germany, France, Italy, Spain, Greece, USA, countries of the Pacific coast of Latin America, China, Japan, Thailand, in the Russian Far East; Japan has the highest incidence (up to 1,000 cases per year) (Baird et al, 2014;Nieuwenhuizen and Lopata, 2014;Pravettoni et al, 2012;Yera et al, 2018). In Russia, the problem of anisakidosis remains insufficiently studied.…”

“…In current taxonomy there is a complex of species A. simplex sensu lato (Mattiucci et al, 1997;Nascetti et al, 1983Nascetti et al, , 1986 which is made up of the three sibling species: A. simplex sensu stricto, A. pegreffii, and A. berlandi (Mattiucci et al, 2014). But, according to the available literature data a clear division of species (A. simplex, A. pegreffii, and A. berlandi) has been repeatedly demonstrated on phylogenetic trees reconstructed using nuclear ITS rDNA (Pekmezci et al, 2014;Tunya et al, 2020) and mitochondrial cox2 gene (Anshary et al, 2014;Barcala et al, 2018;Mattiucci et al, 2009;Setyobudi et al, 2011;Valentini et al, 2006) DNA markers, and finally complete mitochondrial genomes (Yamada et al, 2017). Also A. typica (Diesing, 1860) is suspicious with its uncertain position on different phylogenetic reconstructions (Iñiguez et al, 2009;Sardella and Luque, 2016).…”