Jerko Hrabar

Reaction of the mussel Mytilus galloprovincialis (Bivalvia) to Eugymnanthea inquilina (Cnidaria) and Urastoma cyprinae (Turbellaria) concurrent infestation

In total 480 individuals of Mytilus galloprovincialis were sampled monthly from October 2009 to September 2010, at the shellfish farm in the Mali Ston Bay, south Adriatic Sea (Croatia) in order to assess the extent of pathology imposed by two parasites, Eugymnanthea inquilina (Cnidaria) and Urastoma cyprinae (Turbellaria). Although a deteriorating impact on host reproduction or condition index was lacking, we evidenced ultrastructural and functional alteration in host cells at the attachment site. Ultrastructural changes included hemocytic encapsulation of the turbellarian and cell desquamation in medusoid infestation. Caspase positive reaction inferred by immunohistochemistry (IHC) was triggered in cases of turbellarian infestation, in contrast with hydroids, suggesting that the former exhibits more complex host-parasite interaction, reflected in the persistent attempts of the parasite to survive bivalve reaction. We have evidenced that both organisms trigger specific host reaction that although not costly in terms of host reproductive cycle or growth, results in mild tissue destruction and hemocyte activation. A lower degree of tissue reaction was observed in cases of hydroid infestation, compared to turbellarian.

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.

Multixenobiotic Resistance Mechanism in Gills of Reared vs. Wild Mediterranean Mussel Mytilus galloprovincialis (Lamarck, 1819)

Mussel (Mytilus galloprovincialis (Lamarck, 1819)) is directly exposed to sea water contamination that elicits significant physiological and cellular response, although its extent mounted in aquaculture-reared in comparison to wild bivalve populations is scarcely known. Therefore, we have compared contamination biomarkers in mussels from reared (Marina farm) and wild, anthropogenically affected site (Vranjic Bay). While predictably, the levels of metals (Cu, Cd, Pb, Zn, Fe, and Hg) in whole bivalve tissues determined by atomic absorption spectrophotometry resulted in significantly higher concentrations in wild mussels, accompanied by elevated number of apoptotic cells in gills, the activity of multixenobiotic resistance defense mechanism (MXR), measured as the accumulation rate of model substrate rhodamine B (RB) gave contrasting results. The functional RB assay evidenced a lower MXR efflux activity in the gill tissue of wild mussels, indicating two possible scenarios that will need further focus: (1) persisting sea water pollution increased cell damage of bivalve gill cells and consequently led to leakage of the RB into cytoplasm and dysfunctional MXR efflux in wild mussels; or/and (2) a mixture of different toxic compounds present in Vranjic Bay sea water induced oversaturation of MXR efflux, inducing elevated accumulation of the dye. Consequently, it seems that an efficient physiological functioning of MXR in wild mussels is strongly hampered by existence of an unknown quantity of sea water pollutants that may endanger intrinsic organismal defense system and lead toward the enhancement of toxicity.

Geographic and host size variations as indicators of Anisakis pegreffii infection in European pilchard (Sardina pilchardus) from the Mediterranean Sea: Food safety implications

European pilchards are traditionally eaten marinated or salted in the Mediterranean countries often without thermal processing or gutting due to small size. Since ingestion of live third stage Anisakis larvae represents a causing agent in the onset of anisakiasis, the aim of our study was to assess prevalence and intensity of Anisakis infection in European pilchards originating from different Mediterranean regions in a three-year sampling period (2013-2015). A total of 1564 specimens of European pilchard collected from two geographically distinct sampling regions (western Mediterranean and Adriatic Sea) were examined using the UV-Press method, which utilises the fluorescence of frozen anisakids in flattened and subsequently frozen fillets and viscera. A subsample of 67 isolated larvae was identified as A. pegreffii by diagnostic allozyme markers and sequence analyses of the mtDNA cox2 locus. The overall prevalence in pilchards was 12.2% (range 0-44.9% for different sampling points) and mean intensity 1.8. More importantly, we have observed an overall larval prevalence of 1.5% in fillets. The highest prevalence (44.9%) was recorded in pilchards caught in western parts of the Mediterranean. As fish host size was a significant predictor of parasite abundance, it should be highlighted that these pilchards were also the largest (mean total length 173.2mm); on average >2cm larger than the rest of the samples. Other isolated nematode species included Hysterothylacium sp. in viscera, showing almost a double of A. pegreffii prevalence, 20.1%. In summary, our study demonstrates that the presence of A. pegreffii in European pilchards from the Mediterranean Sea is highly influenced by both geographic and host size variation. This implies that, before future risk management measures are developed, these variables should be assessed in order to minimize public health concerns.

Molecular and Cellular Response to Experimental Anisakis pegreffii (Nematoda, Anisakidae) Third-Stage Larval Infection in Rats

Background: Anisakiasis is a zoonotic disease caused by accidental ingestion of live Anisakis spp. third-stage larvae present in raw or undercooked seafood. Symptoms of this emerging infectious disease include mild-to-severe abdominal pain, nausea, and diarrhea. Some patients experience significant allergic reactions. Aims: In order to better understand the onset of anisakiasis, we aimed to: (i) histopathologically describe severe inflammatory/hemorrhagic infection site lesions in Sprague-Dawley rats experimentally infected with Anisakis pegreffii larvae; and (ii) qualitatively and quantitatively characterize the transcriptomes of affected tissues using RNA-Seq. Methodology: The experiment was performed on 35 male rats, sacrificed at 5 time points (6, 10, 18, 24, and 32 h post-infection). Gastric intubation was performed with 10 A. pegreffii larvae (N = 5 infected rats per time point) or 1.5 ml of saline (external control N = 2 rats). 16 pools, seven for muscle tissues and nine for stomach tissues, were created to obtain robust samples for estimation of gene expression changes depicting common signatures of affected versus unaffected tissues. Illumina NextSeq 500 was used for paired-end sequencing, while edgeR was used for count data and differential expression analyses. Results: In total, there were 1372 (855 up and 517 down) differentially expressed (DE) genes in the Anisakis-infected rat stomach tissues, and 1633 (1230 up and 403 down) DE genes in the muscle tissues. Elicited strong local proinflammatory reaction seems to favor the activation of the interleukin 17 signaling pathway and the development of the T helper 17-type response. The number of DE ribosomal genes in the Anisakis-infected stomach tissue suggests that A. pegreffii larvae might induce ribosomal stress in the early infection stage. However, the downstream pathways and post-infection responses require further study. Histopathology revealed severe inflammatory/hemorrhagic lesions caused by Anisakis infection in the rat stomach and muscle tissues in the first 32 h. The lesion sites showed infiltration by polymorphonuclear leukocytes (predominantly neutrophils and occasional eosinophils), and to a lesser extent, macrophages. Conclusion: Understanding the cellular and molecular mechanisms underlying host responses to Anisakis infection is important to elucidate many aspects of the onset of anisakiasis, a disease of growing public health concern.