No abstract

No abstract

A B S T R A C TThe European hake Merluccius merluccius is one of the most commercially important and widely distributed fish species, occurring both in European and Mediterranean Sea fisheries. We analyzed the distribution and infection rates of different species of Anisakis in M. merluccius (N = 1130 hakes), by site of infection in the fish host (viscera, dorsal and ventral fillets) from 13 different fishing grounds of the Mediterranean Sea (FAO area 37). The fillets were examined using the UV-Press method. A large number of Anisakis specimens (N = 877) were identified by diagnostic allozymes, sequence analysis of the partial EF1 α-1 region of nDNA and mtDNA cox2 gene. Among these, 813 larvae corresponded to A. pegreffii, 62 to A. physeteris, 1 to A. simplex (s. s.), whereas one resulted as a F1 hybrid between A. pegreffii and A. simplex (s. s.). Remarkably high levels of infection with A. pegreffii were recorded in hakes from the Adriatic/Ionian Sea compared to the fish of similar length obtained from the western Mediterranean fishing grounds. A positive correlation between fish length and abundance of A. pegreffii was observed. Concerning the localization of A. pegreffii larvae in the fish, 28.3% were detected in the liver, 62.9% in the rest of the viscera, 6.6% in the ventral part of the flesh, whereas 2.1% in the dorsal flesh.

Anisakis pegreffii, a recognised etiological agent of human anisakiasis, is a parasite of homeothermic hosts at the adult stage and of ectothermic hosts at the third larval stage. Among distinct factors, temperature appears to be crucial in affecting parasite hatching, moulting and to modulate parasite-host interaction. In the present study, we investigated the gene transcripts of proteins having an antigenic role among excretory secretory products (ESPs) (i.e., a Kunitz-type trypsin inhibitor, A.peg-1; a glycoprotein, A.peg-7; and the myoglobin, A.peg-13) after 24 h, in A. pegreffii larvae maintained in vitro, under controlled temperature conditions. Temperatures were 37 °C and 20 °C, resembling respectively homeothermic and ectothermic hosts conditions, and 7 °C, the cold stress condition post mortem of the fish host. Primers of genes coding for these ESPs to be used in quantitative real-time PCR were newly designed, and qRT-PCR conditions developed. Expression profiles of the genes A.peg-1 and A.peg-13 were significantly up-regulated at 20 °C and 37 °C, with respect to the control (larvae kept at 2 °C for 24 h). Conversely, transcript profiles of A.peg-7 did not significantly change among the chosen temperature conditions. In accordance with the observed transcript profiles, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of the three target ESPs at 37 °C, while only A.peg-13 was observed at 7 °C. The results suggest that temperature conditions do regulate the gene expression profiles of A.peg-1 and A.peg-13 in A. pegreffii larvae. However, regulation of the glycoprotein A.peg-7 is likely to be related to other factors such as the host’s immune response.

A survey on Anisakis simplex (sensu stricto (s.s.)) from blue whiting, Micromesistius poutassou, in the north-eastern Atlantic Ocean revealed the occurrence of high infection levels of third larval stages in visceral organs and flesh. Larvae were genetically identified with a multilocus approach as A. simplex (s.s.). Histochemical, immunohistochemical and ultrastructural observations were conducted on 30 M. poutassou specimens. Gonads, pyloric caeca and flesh harboured encapsulated larvae of A. simplex (s.s.) but no intense host reaction was encountered around the parasite in the above organs. In the liver, the most infected organ, the larvae co-occurred with the coccidian Goussia sp. Within the granuloma around the A. simplex (s.s.) larvae, two concentric layers were recognized, an inner mostly comprising electron-dense epithelioid cells and an outer layer made of less electron-dense epithelioid cells. Macrophages and macrophage aggregates (MAs) were abundant out of the granulomas, scattered in parenchyma, and inside the MAs, the presence of engulfed Goussia sp. was frequent. In liver tissue co-infected with Goussia sp. and A. simplex (s.s.), hepatocytes showed cytoplasmic rarefaction and acute cell swelling. Results suggest that the host-induced encapsulation of A. simplex (s.s.) larvae is a strategic compromise to minimize collateral tissue damage around the larval infection sites, to facilitate the survival of both parasite and host.

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.

No abstract

The third-stage larvae of the parasitic nematode genus Anisakis tend to encapsulate in different tissues including the musculature of fish. Host tissue penetration and degradation involve both mechanic processes and the production of proteins encoded by an array of genes. Investigating larval gene profiles during the fish infection has relevance in understanding biological traits in the parasite’s adaptive ability to cope with the fish hosts’ defense responses. The present study aimed to investigate the gene expression levels of some proteins in L3 of A. simplex (s.s.) infecting different tissues of blue whiting Micromesistius poutassou, a common fish host of the parasite in the NE Atlantic. The following genes encoding for Anisakis spp. proteins were studied: Kunitz-type trypsin inhibitor (TI), hemoglobin (hb), glycoprotein (GP), trehalase (treh), zinc metallopeptidase 13 (nas 13), ubiquitin-protein ligase (hyd) and sideroflexin 2 (sfxn 2). Significant differences in gene transcripts (by quantitative real-time PCR, qPCR) were observed in larvae located in various tissues of the fish host, with respect to the control. ANOVA analysis showed that relative gene expression levels of the seven target genes in the larvae are linked to the infection site in the fish host. Genes encoding some of the target proteins seem to be involved in the host tissue migration and survival of the parasite in the hostile target tissues of the fish host.