A multi-restriction fragment length polymorphism genotyping approach including the beta-tubulin gene as a new differential nuclear marker for the recognition of the cryptic species Anisakis simplex s.s. and Anisakis pegreffii and their hybridization events

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Highlights

  • Hybrids showed heterozygous genotypes at the 7 β-TB diagnostic positions.

  • More hybrids were detected with the ITS1 marker than with the β-TB marker.

  • Multi-marker approach identified F1 hybrids, hybrid backcrossing and introgression.

  • All F1 hybrids were encountered in the sympatric areas.

  • Network showed three distinct but closely related subpopulations.

Abstract

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.

Introduction

Hybridization is recognized as a significant process in the evolution of free-living organisms, but the role and extent that this process can play in the evolution of parasites remains obscure (Arnold, 2004; Criscione et al., 2007; King et al., 2015). Natural hybridization has been recorded across parasitic organisms such as the species pairs Schistosoma mansoni Sambon, 1907, and S. rodhaini Brumpt, 1931, Ascaris lumbricoides Linnaeus, 1758, and A. suum Goeze, 1782, or Anopheles gambiae s.s. Giles, 1902, and A. coluzzii Coetzee and Wilkerson, sp. n., among others (Detwiler and Criscione, 2010; King et al., 2015; Steinauer et al., 2010). Knowledge of such hybridization events is critical from an epidemiological perspective and such events can lead to changes in key pathogenic and transmission traits. In that sense, extensive introgression between Anopheles species has been shown, suggesting enhanced vectorial capacity and adaptation to humans as primary hosts resulted from interspecific genetic exchange. Recent studies have demonstrated rapid adaptive introgression of the insecticide resistance mutation Vgsc-L10114 F from A. gambiae to A. coluzzii, and the potential of closely related interspecific hybrid crosses to enhance the fitness of malaria transmission in comparison with parental species (King et al., 2015; Norris et al., 2015). Evidence of hybridization between animal parasitic Haemonchus nematodes resulted in interspecies introgression of anthelmintic resistance alleles from H. contortus into the H. placei genetic background (Chaudhry et al., 2015).

The genetic population structure of a species is revealed by the distribution of the genetic variation across specimens over different spatial scales and it is highly influenced by the study methodology, i.e. isoenzymes versus DNA and single versus multiple markers, which can be nuclear or mitochondrial. The markers used should be able to accurately determine whether the shared polymorphisms detected are due to processes of incomplete lineage sorting, historical introgression or contemporary hybridization.

Amongst the parasites responsible for foodborne diseases, nematodes within the genus Anisakis Dujardin, 1845, mainly A. simplex s.s. Nascetti et al., 1986, and A. pegreffii Campana-Rouget and Biocca, 1995 (Nematoda, Anisakidae), are the causative agents of: i) ulcerative, inflammatory and / or hemorrhagic lesions of the stomach in some teleost fish and marine mammals, which can sometimes lead to death (Buchmann and Mehrdana, 2016; Hrabar et al., 2017); ii) the fish-borne illness in humans known as anisakiosis, which ranges from self-limiting gastrointestinal forms to severe systemic allergic reactions; since the 1960s, a growing number of cases have been reported worldwide in spite of misdiagnosis and under-reporting (Audicana and Kennedy, 2008). High infection prevalence by the third larval stage of anisakid nematodes has been found in epidemiological surveys in fish, the source of human infection; these figures vary depending on the fish species and size, capture area, capture season, etc. (Abattouy et al., 2014; Mladineo et al., 2017a, 2017b; Molina-Fernández et al., 2015).

Differentiation between the cryptic species A. simplex s.s. and A. pegreffii in sympatric areas via molecular markers has given rise to specimens with combined genetic patterns between these two species. Since its first detection in 2003 (Abollo et al., 2003), many authors have found these recombinant genotypes in areas of sympatry at variable percentages (Abattouy et al., 2016; Cipriani et al., 2015; Hermida et al., 2012; Martín-Sánchez et al., 2005; Umehara et al., 2006). Nevertheless, the detection of these putative hybrids has been the cause of some controversy in terms of its interpretation due to: a. the fact that adult hybrids are rarely found, b. the recovery of recombinant larval forms within allopatric areas (Cavallero et al., 2014, 2012; Chaligiannis et al., 2012; Mladineo et al., 2017a, 2017b; Pekmezci, 2014), c. the use of a single marker for molecular ecology studies (Anderson, 2001; Chaligiannis et al., 2012; Lee et al., 2009). However, adult hybrid individuals have been recovered from cetaceans (Cavallero et al., 2014; Umehara et al., 2006), and migratory aquatic mammalians and fish migration could justify the finding of recombinant larvae outside sympatric areas (Abattouy et al., 2016; Mladineo et al., 2017a, 2017b). In addition, the use of markers with the power to elucidate the causes of shared polymorphisms has helped to study similar situations in other parasitic species (Criscione et al., 2007; Steinauer et al., 2008).

Differential traits of biomedical importance, such as invasive and allergenic potential have been demonstrated in both cryptic species (Arcos et al., 2014; Romero et al., 2013), highlighting the importance of their taxonomic differentiation. Hybridization has ecological and evolutionary consequences due to the introgression of novel genes, what could result in the evolution of more pathogenic genotypes or genotypes with reduced host specificity (King et al., 2015; Seehausen, 2004).

This fact highlights the need to focus on apparent hybridization phenomena in A. simplex complex.

Beta-tubulin (β-TUB) is a nuclear gene that encodes for one of the two essential subunits of the microtubules present in eukaryotic cells, which constitute one of the main components of the cytoskeleton of these cells (Weisenberg, 1972). Although this protein has a very ancient origin, its sequence is highly conserved in metazoans, including nematodes. The differences are mostly pronounced in the carboxy-terminal parts of the protein, but the N-terminal fragment of the sequence has minimal variation (Kwa et al., 1994). This high degree of conservation would be imposed by the structural limitations of the assembly and disassembly of microtubules (Burns, 1991), and could be used for species identification (Hansen et al., 2013; Tydén et al., 2013).

Our objectives were to: (1) describe the genetic variability in the β-TUB gene between A. simplex s.s. and A. pegreffii and within these species, (2) examine the usefulness of this gene, alone and in combination with the ribosomal DNA (rDNA) internal transcribed spacer (ITS) 1-5,8S-ITS2 (rDNA ITS) and the mitochondrial DNA cytochrome C oxidase 2 (mtDNA Cox-2) for the differentiation of both species (3) develop a multi-marker RFLP genotyping approach that simplifies its use and (4) discuss the hybridization potential between both species in sympatric zones.

Section snippets

Parasites, fish and geographical areas

Anisakis larvae were collected from blue whiting (Micromesistius poutassou Risso, 1827), European hake (Merluccius merluccius Linnaeus, 1758) and anchovy (Engraulis encrasicolus Linnaeus, 1758) caught off the coasts of the Iberian Peninsula (FAO 27.VIIIc, 27.IXa, and 37.1.1), Italy and Croatia (FAO 37.2.1), and in the Northeast Atlantic Ocean (Little Sole Bank fishing ground, FAO 27.VIIh), provided by a trusted dealer on the morning of the experimental infection and delivered on ice. We

Anisakis simplex s.l. collected and identified by PCR-RFLP of ITS region of rDNA

A total of 596 larvae collected from fish caught in Iberian waters, and in the North Atlantic ocean and the Adriatic sea, were identified by PCR-RFLP as A. simplex s.s. (292, 49.0 %), A. pegreffii (238, 39.9 %) or A. simplex s.s./A. pegreffii (As/Ap) mixed genotype (66, 11.1 %). The proportions of these genotypes in each geographical area are shown in Fig. 1. Thirty-two percent (21/66) of the putative hybrids only displayed the sum of the RFLP band patterns of both Anisakis species with one

Discussion

Here we describe the first molecular analysis of A. simplex s.l. from different fishes and locations across Mediterranean and Atlantic waters, using β-tubulin sequences as a nuclear marker. A. simplex s.s. and A. pegreffii differed at least at 7 bp in the β-tubulin gene: positions 33, 75, 86, 269, 297, 394 and 496 of the studied region. Position 451 was not consistent throughout all specimens examined making the interpretation of this nucleotide ambiguous, while it may be of interest in the

Funding

The study was funded by the Junta de Andalucía (research group BIO-176), Spain.

CRediT authorship contribution statement

Magdalena Gómez-Mateos: Investigation, Formal analysis, Visualization. Gema Merino-Espinosa: Validation, Supervision. Victoriano Corpas-López: Writing - review & editing. Adela Valero-López: Supervision, Funding acquisition. Joaquina Martín-Sánchez: Conceptualization, Methodology, Writing - original draft.

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

Acknowledgements

We want to thank Dolores Molina Fernández her expertise in graphics edition. The results of this paper are part of the doctoral thesis of Magdalena Gómez-Mateos Pérez.

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