A multigene approach to study ecological and evolutionary aspects of two parasites species Anisakis simplex (s. s.) and Anisakis pegreffii
Author(s)
Acerra, Virginia
Date Issued
June 17, 2016
Type
Doctoral Thesis
Abstract
The genus Anisakis includes species of nematodes, endoparasitic, characterized by a complex
biological cycle. The nematode eggs are released into the sea in the faeces by marine mammals;
shellfish (Euphasiacea, Mysidacea, etc.) are the first intermediate host; various species of fish and
cephalopod molluscs are intermediate/paratenic hosts; finally, the cetaceans, are the definitive
ones. Some Anisakis species are zoonotic, and therefore could be dangerous for the humans, who,
eating raw or undercooked fish, can become an accidental host, acquiring a zoonosis, the
anisakiasis.
The taxonomy of helminth endoparasites group has been revolutionized by molecular genetic
studies, identifying the existence of different sibling species within morphospecies. Among the
"cryptic species", A. pegreffii and A. simplex (s. s.) are the species considered in this research
study. They show different distribution ranges that overlap along the Atlantic coasts of Spain and
Portugal, in the Alboran Sea and in NW Pacific Ocean (China Sea and Japan Sea). In some areas
of sympatry and in numerous other areas of allopatry, recent studies have highlighted the presence
of phenomena of hybridization between the two taxa.
The aims of the current study were: 1) obtaining data regarding the prevalence and the
distribution of Anisakis species in the flesh of European hake caught in the Mediterranean and NE
Atlantic waters and providing epidemiological data for a risk assessment related to this zoonotic
parasite in sea food (“Genetic identification and distribution of the larval parasites Anisakis pegreffii
and A. simplex (s. s.) in fish tissues of Merluccius merluccius from Tyrrhenian Sea and Spanish
Atlantic coast: implications for food safety”. Cipriani et al. 2015);
2) verifying that the distribution of Anisakis spp. larvae in the viscera and musculature of
European anchovy, in order to: i) quantify the intra vitam migration and document the post-mortem
migration of the zoonotic parasite, A. pegreffii in fish stored at different temperature conditions and
at various time intervals; ii) provide epidemiological data both in viscera and musculature of the
anchovy to assess the human infection risk posed by A. pegreffii (“Larval migration of the zoonotic
parasite Anisakis pegreffii (Nematoda: Anisakidae) in European anchovy, Engraulis encrasicolus:
Implications to seafood safety.” Cipriani et al. 2016);
3) a multi marker nuclear loci analysis of several nematodes of Anisakis spp. collected from
allopatric and sympatric areas of the two species (A. pegreffii and A. simplex s. s.) was carried out in
order to: i) correctly identify specimens belonging to the parental taxa (A. pegreffii and A. simplex s.
s.); ii) compare the assignment of each Anisakis specimen obtained from biparentally inherited
codominant nuclear markers (allozyme loci) and the sequence analysis of a nuclear DNA locus (i.e.
partial sequence of the elongation factor 1 alpha1 gene) with respect to the assignment obtained, on
the same specimens, by means of the single molecular marker extensively used to distinguish the
two species (i.e. the analysis of ITS region of rDNA); iii) recognize hybrid categories between two
taxa; and iiii) distinguish introgression or current hybridization phenomena between two species
(“No more time to stay ‘single’: a multi-marker nuclear genotyping approach for the identification
of Anisakis pegreffii, A. simplex (s. s.), and their hybrids (Namatoda: Anisakidae). Mattiucci et al.
2016).
According to this approach: to detect significant differences in the density values of the species
A. pegreffii and A. simplex s. s. Both Anisakis species occurred in the viscera and flesh of the fish.
However, significant differences were found in the infection levels in terms of both the
geographical origin of the fish and the infection site of the two Anisakis species. For instance, the
overall parasite burden of A. pegreffii reported in Tyrrhenian Sea was significantly lower than
Atlantic area. A. simplex s. s. infection exhibited significantly higher values than A. pegreffii in both
the viscera and the flesh of hake;
Experiments carried out to evaluate the potential migration intra-vitam e post-mortem in E.
encrasicolus associated with temperature and timing of fish’ conservation simulating different
conditions of fish’ storage before consumption. These series of experiments showed that the
migration post mortem of larvae of A. pegreffii is statistically correlated to the temperature and
timing of fish’conservation. Three different categories of markers are used: allozyme, EF 1 alpha1
nDNA e ITS rDNA. Analyses of results using the three indicators reported above have shown: only
few subjects reported a hybrid genotype F1, sampled in sympatry areas. At the contrary, results
based on the only ITS classified more subjects as hybrids. Therefore our results suggested some
issues regarding the use of a single indicator to identify the types of A. pegreffii e A. simplex s. s.
and their hybrids.
Additional information
Dottorato di ricerca in Ecologia e gestione delle risorse biologiche