Population parameters and mito-nuclear mosaicism of Anisakis spp. in the Adriatic Sea.

Recombinant genotypes of A. simplex sensu stricto (s.s.) and A. pegreffii, two species of Anisakis simplex complex found in sympatric waters of the Mediterranean Sea, are believed to be a product of interspecific hybridisation and/or DNA introgression. In contrast, such events within an allopatric area as the Adriatic Sea are unlikely to occur and therefore observed recombination should be assessed more closely. We have genotyped 525 anisakids collected from migratory and non-migratory fish of the southern part of the Adriatic Sea, inferring its omniparentage at nuclear (ITS locus) and matrilineage at mitochondrial locus (cox2). The aim was to address the presence and cause of the recombination within the population and to test its genetic structure under admixture theory. Population parameters, i.e. prevalence, and mean abundance and intensity of anisakids were also evaluated to contribute for future epidemiological risk assessments. As a result, we have inferred the presence of A. pegreffii, A. typica and A. ziphidarum in the Adriatic, lacking type species A. simplex s.s. at both nuclear and mitochondrial locus. A. pegreffii population shows a high level of admixture and heterogeneity and a recent demographic expansion from a small population size. We argue that the observed recombinant genotypes in the Adriatic are a product of ancestral polymorphism and consequent remote genetic introgression.

Anisakis simplex complex: ecological significance of recombinant genotypes in an allopatric area of the Adriatic Sea inferred by genome-derived simple sequence repeats.

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.

Autochthonous Bacterial Isolates Successfully Stimulate In vitro Peripheral Blood Leukocytes of the European Sea Bass (Dicentrarchus labrax).

Commercially available probiotics are routinely administered as feed supplements in aquaculture important species. Among them, the European sea bass (Dicentrarchus labrax) is the most widely reared fish in the Mediterranean, whose rearing systems are highly variable between countries, affecting at some level the sustainability of production. After random isolation of autochthonous gut bacteria of the sea bass, their identification and pathogenicity testing, we have selected three potentially probiotic isolates; Pseudoalteromonas sp., Alteromonas sp., and Enterovibrio coralii. Selected isolates were tested and their immunostimulative efficiency was compared with a commercially available Lactobacillus casei isolate, inferring inflammatory, apoptotic and anti-pathogen response of sea bass' peripheral blood leukocytes. Phagocytic activity, respiratory burst, and expression of lysozyme, Mx protein, caspase 3, TNF-α, IL-10 genes was measured 1, 3, 5, and 12 h post-stimulation by four bacterial isolates to evaluate early kinetics of the responses. Best immunostimulative properties were observed in Pseudoalteromonas-stimulated leukocytes, followed by Alteromonas sp. and L. casei, while Enterovibrio coralii failed to induce significant stimulation. Based on such in vitro assay intestinal autochthonous bacterial isolates showed to have better immunostimulative effect in sea bass compared to aquaculture-widely used L. casei, and further steps need to engage tank and field feeding trials to evaluate long-term prophylactic suitability of the chosen isolates. A panel of biomarkers that represent pro-/anti-inflammatory, pro-/anti-apoptotic, and anti-bacteria/viral responses of the fish should be taken into consideration when evaluating the usefulness of the potential probiotic in aquaculture.