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We undertook the first study systematically evaluating the risk of Anisakis-sensitization in Croatian fish-processing workers and potential genetic susceptibility to anisakiasis. Anti-Anisakis IgE seroprevalence and risk factors for 600 employees of Croatian fish processing facilities and 466 blood donor controls, were assessed by indirect ELISA targeted with: recombinant Ani s 1 and Ani s 7 allergens, an Anisakis crude extract, the commercial ImmunoCAP kit, and questionnaires. Genetic susceptibility to anisakiasis was evaluated by genotypisation of human leukocytes alleles (HLA). Anti-Anisakis seropositive and a fraction of negative subjects were also assessed by ELISA and Western Blot (WB) for IgG seroprevalence to Trichinella spp. Overall, the observed anti-Anisakis seroprevalence inferred by indirect ELISA was significantly higher in fish processing workers (1.8%, 95% CI 0.9–3.3%) compared to the controls (0%, 0–0.8%). Seven out of 11 Ani s 1 and Ani s 7-positives and none of selected 65 negative sera, tested positive on whole-Anisakis extract (ImmunoCAP), whereas Anisakis crude extract ELISA detected 3.9% (2.4–6.0%) seropositives in fish processing workers, three (14%) of which showed IgE reactivity to milk proteins. The highest risk associated with Anisakis-sensitization among workers was fishing in the free time, rather than any of attributes related to the occupational exposure. Although no association was observed between anti-Anisakis seropositivity and wearing gloves or protective goggles, the majority of workers (92%) wore protective gloves, minimizing the risk for Anisakis sensitization via skin contact. Six HLA alleles within DRB1 gene were significantly associated with seropositivity under dominant, allelic or recessive models. All sera confirmed negative for anti-Trichinella spp. IgG. The study exhaustively covered almost all marine fish processing workers in Croatia, reflecting real-time Anisakis sensitization status within the industry, already under the influence of wide array of allergens., Author summary Anisakiasis is a human disease caused by ingestion of live Anisakis spp. larvae by raw seafood. Fish processing and aquaculture workers (approximately 60 million people world-wide) are at occupational risk of becoming sensitized to Anisakis. This is the first study systematically evaluating the risk of Anisakis-sensitization in Croatian fish-processing workers and potential genetic susceptibility to anisakiasis in the analysed population. Observed seroprevalence was significantly higher in fish processing workers (1.8%) compared to the controls (0%). Surprisingly, the highest risk associated with Anisakis-sensitization among workers was fishing in their free time, rather than any of attributes related to the occupational exposure. While no association was observed between anti-Anisakis seropositivity and wearing gloves or protective goggles, the majority of workers (92%) wore protective gloves, minimizing the risk for Anisakis sensitization via skin contact. Additionally, seroprevalence to another important food-borne helminth, Trichinella spp. assessed in all Anisakis-positive subjects showed to be negative. Almost all marine fish processing workers in Croatia were part of this research, reflecting real-time Anisakis-sensitization status within the industry. This sets a baseline for the future tracking of sensitization in the sector already under the influence of wide array of allergens.

The blue jack mackerel Trachurus picturatus (Bowdich, 1825) specimens (N = 155) were collected during the MEDITS survey, done along the eastern side, precisely, of the Croatian fishing ground in July 2018. Biometrical analysis of ten morphometric and five meristic characters, as well as genetic analysis proved that the collected specimens were blue jack mackerel. The total length (TL) and weight (W) of all observed specimens ranged from 9.2 to 33.7 cm (12.15 &plusmn, 2.95 cm) and from 5.79 to 384.94 g (17.64 &plusmn, 39.42 g), respectively. All calculated length&ndash, length relationships were linear (r &gt, 0.923). Sex was determined only on two larger specimens (28 cm &lt, TL &lt, 32.8 cm), which were females. In the length&ndash, weight relationship, positive allometry was established (b = 3.1789). Based on 37 partial cytochrome b sequences, the overall haplotype diversity (h) of 0.812 &plusmn, 0.048 and nucleotide diversity (&pi, ) of 0.0064 &plusmn, 0.0007 indicated high levels of haplotype and low nucleotide diversity. The obtained sequences were compared to previously published research within the Northeast Atlantic Ocean and the Mediterranean Sea, confirming the absence of genetic structure among these populations.

The 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.

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.

The Atlantic bluefin tuna (ABFT; Thunnus thynnus) today represents one of the economically most important species for Croatian fisheries industry. Although the most diverse and abundant parasitofauna is usually found in the largest specimens of wild ABFT, the opposite was observed in captivity where parasite populations significantly decline by the end of the farming cycle. Copepod Brachiella thynni, is a skin parasite frequently parasitizing tuna, whose population also decreases in number throughout the rearing process. In order to better understand the immunity mechanisms underlying ABFT reaction to B. thynni infection, we studied expression profiles of immunity related genes; interleukin 1β (il1β), tumour necrosis factors (tnfα1, tnfα2), complement component 4 (c4) and caspase 3 (casp3), in peripheral blood leukocytes (PBLs) during in vitro stimulation by B. thynni protein extracts (i.e. antigens) and in infected tissues at B. thynni parasitation site. Finally, a histopathological analysis of semi-thin and ultra-thin sections of tissues surrounding B. thynii attachment site was performed to evaluate the severity of parasite-induced lesions and identify involved cell lineages. In vitro stimulation of ABFT PBLs with B. thynii antigens caused a dose-depended upregulation of selected genes, among which tnfα1 showed the highest induction by both concentrations of B. thynni protein extract. However, targeted genes were not significantly upregulated in the infected tissue. Also, no significant alterations in ultrastructure of epithelial layers surrounding B. thynii attachment site were noticed, except local tissue erosion, necrosis of squamous epithelium and proliferation of rodlet and goblet cells. Our results suggest that B. thynii has evolved strategies to successfully bypass both innate immune response and the connective-tissue proliferation processes. Therefore, the observed disappearance of this copepod by the end of the rearing process is more likely related to its limited lifespan on the host and its inability to complete the life cycle in the rearing cages, rather than host's reaction.

Background Anisakiasis is an emerging public health problem, caused by Anisakis spp. nematode larvae. Anisakiasis presents as variable and unspecific gastrointestinal and/or allergic clinical symptoms, which accounts for the high rate of misdiagnosed cases. Methodology/Principal findings The aim of this study was to characterize the early cellular (6–72 h p.i.) and molecular (6 h p.i.) immune response and general underlying regulatory mechanism in Anisakis infected rats. Each Sprague-Dawley rat was infected with 10 Anisakis spp. larvae by gastric intubation. Tissues with visible lesions were processed for: i) classic histopathology (HE), immunofluorescence (CD3, iNOS, S100A8/A9), and transmission electron microscopy (TEM); ii) target genes (Il1b, Il6, Il18, Ccl3, Icam1, Mmp9) and microRNA (Rat Immunopathology MIRN-104ZF plate, Quiagen) expression analysis; and iii) global DNA methylation. Histopathology revealed that Anisakis larval migration caused moderate to extensive hemorrhages in submucosal and epimysial/perimysial connective tissue. In stomach and muscle, moderate to abundant mixed inflammatory infiltrate was present, dominated by neutrophils and macrophages, while only mild infiltration was seen in intestine. Lesions were characterized by the presence of CD3+, iNOS+, and S100A8/A9+ cells. The greatest number of iNOS+ and S100A8/A9+ cells was seen in muscle. Il6, Il1b, and Ccl3 showed particularly strong expression in stomach and visceral adipose tissues, but the order of expression differed between tissues. In total, three miRNAs were differentially expressed, two in stomach (miRNA-451 and miRNA-223) and two in intestine (miRNA-451 and miRNA-672). No changes in global DNA methylation were observed in infected tissues relative to controls. Conclusions/Significance Anisakis infection induces strong immune responses in infected rats with marked induction of specific proinflammatory cytokines and miRNA expression. Deciphering the functional role of these cytokines and miRNAs will help in understanding the anisakiasis pathology and controversies surrounding Anisakis infection in humans., Author summary Anisakiasis is a zoonotic disease (infection transmitted between animals and humans) contracted by consumption of raw or undercooked seafood contaminated with Anisakis spp. nematode larvae. Anisakiasis usually presents with variable and unspecific gastrointestinal and/or allergic symptoms, which accounts for the high rate of misdiagnosed cases. Due to changes in dietary habits, such as eating raw or undercooked seafood, anisakiasis is considered an emerging public health problem. Despite the increase in number of reported cases worldwide, mechanisms of immune response to this unspecific human pathogen are poorly known. We have shown that in experimentally infected rats, Anisakis larvae cause severe hemorrhages and necrotic changes of affected tissues in the early phase of infections. Neutrophils and macrophages were abundantly present in tissue lesions, while eosinophils, hallmark of helminth infections, were scarcely present. We have also demonstrated particularly strong expression of several inflammatory genes. Moreover, we give for the first-time insight into putative regulatory mechanism mediated via a distinct class of RNA molecules. Our study may provide new opportunities for better understanding of cellular and molecular response to Anisakis spp., aiming at development of more specific therapeutics and alleviation of pathologies associated with Anisakis spp. infection.

18 pages, 10 figures, 6 tables.-- Arne Levsen ... et al., Harvesting and exploiting limited fisheries resources in a sustainable manner also implies achieving maximum added value from the raw material. However, the presence of parasites in the products may adversely affect consumer perception and/or pose a direct health hazard. As a major stepping-stone of the PARASITE project, an epidemiological survey was carried out to provide the basis for analysis and prediction of consumer exposure risk due to the presence of anisakid nematodes in fish from European wild-catch fisheries. The project consisted of nine separate workpackages (WP) where the exposure risk assessment survey was organized within WP2. The surveillance task also provided the data or samples needed for data management and sample storage (WP3, Biobank), molecular and genetic parasite species identification (WP4), and statistical modelling and inference (WP8). In total 17,760 fish belonging to 16 teleost species were examined for anisakids, with special emphasis on economically and ecologically important species such as Atlantic mackerel, herring, European hake, Atlantic cod and anchovy. The target fish species were sampled at four major European fishing areas including the Barents Sea, North Sea, Baltic Sea, Grand Sole Bank, waters off NW Spain and Portugal, central and western parts of the Mediterranean Sea, and the Adriatic Sea. Thus, the survey represents the largest and most comprehensive epidemiological data compilation of anisakids ever generated in terms of geographical range as well as number of fish host species and sample size. An important requirement of the survey was the use of commonly accepted nematode detection methods, i.e. the UV-press method or artificial digestion, to quantify infection level and spatial distribution of anisakid larvae in the target fish species. The basic layout, set-up and principles of the method, along with a discussion of possible source of errors are described. Additionally, the molecular and genetic markers which were used to identify and characterize different species and populations of anisakids from the targeted fish host species and geographical areas, are reviewed as well. Some basic parasite infection characteristics of each fish host species, and any relationships with the presumably most important infection predictors, i.e. fish host body size and fishing locality, are presented and discussed. Emphasis is put on anisakid occurrence in the flesh of the fish. Based on the findings, a graphical exposure risk profile is introduced, including fish species or products thereof, which due to common processing or preparation practices, are at highest risk to act as source of anisakiasis in Europe, The present work was financially supported by the European Union’s 7th Framework Programme for Research, Technological Development and Demonstration, project Parasite risk assessment with integrated tools in EU fish production value chains (PARASITE), Grant Agreement (GA) no. 312068

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.

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 173.2 mm) ; on average > 2 cm 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.

17 páginas, 7 tablas, 4 figuras.-- This work is licensed under a Creative Commons Attribution 4.0 International License, Anisakiasis is an emerging zoonosis caused by the fish parasitic nematode Anisakis. Spain appears to have the highest reported incidence in Europe and marinated anchovies are recognised as the main food vehicle. Using data on fishery landings, fish infection rates and consumption habits of the Spanish population from questionnaires, we developed a quantitative risk assessment (QRA) model for the anchovy value chain. Spaniards were estimated to consume on average 0.66 Anisakis per untreated (non-frozen) raw or marinated anchovy meal. A dose-response relationship was generated and the probability of anisakiasis was calculated to be 9.56 × 10−5 per meal, and the number of annual anisakiasis cases requiring medical attention was predicted between 7,700 and 8,320. Monte Carlo simulations estimated post-mortem migration of Anisakis from viscera to flesh increases the disease burden by >1000% whilst an education campaign to freeze anchovy before consumption may reduce cases by 80%. However, most of the questionnaire respondents who ate untreated meals knew how to prevent Anisakis infection. The QRA suggests that previously reported figures of 500 anisakiasis per year in Europe is a considerable underestimate. The QRA tool can be used by policy makers and informs industry, health professionals and consumers about this underdiagnosed zoonosis, M. Bao is supported by a PhD grant from the University of Aberdeen and also by financial support of the contract from the EU Project PARASITE (grant number 312068).

00000 ăWOS:000384167300004; International audience; Morphological and molecular tools were combined to resolve the misidentification between Glycymeris glycymeris and Glycymeris pilosa from Atlantic and Mediterranean populations. The ambiguous literature on the taxonomic status of these species requires this confirmation as a baseline to studies on their ecology and sclerochronology. We used classical and landmark-based morphometric approaches and performed bivariate and multivariate analyses to test for shell character interactions at the individual and population level. Both approaches generated complementary information. The former showed the shell width to length ratio and the valve asymmetry to be the main discriminant characters between Atlantic and Mediterranean populations. Additionally, the external microsculpture of additional and finer secondary ribs in G. glycymeris discriminates it from G. pilosa. Likewise, landmark-based geometric morphometrics revealed a stronger opisthogyrate beak and prosodetic ligament in G. pilosa than G. glycymeris. Our Bayesian and maximum likelihood phylogenetic analyses based on COI and ITS2 genes identified that G. glycymeris and G. pilosa form two separate monophyletic clades with mean interspecific divergence of 11% and 0.9% for COI and ITS2, respectively. The congruent patterns of morphometric analysis together with mitochondrial and nuclear phylogenetic reconstructions indicated the separation of the two coexisting species. The intraspecific divergence occurred during the Eocene and accelerated during the late Pliocene and Pleistocene. Glycymeris pilosa showed a high level of genetic diversity, appearing as a more robust species whose tolerance of environmental conditions allowed its expansion throughout the Mediterranean.

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.

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 (PBL). 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.

Economically important sparid fish species, gilthead (Sparus aurata) and white seabream (Diplodus sargus) (Sparidae) are frequently parasitised by Yilmaz Emre Nesrin Emre Ali Aydogdu Ivana Bušelić Lesley R. Smales Ivona Mladineo  diplectanid monogeneans, known to induce severe losses in farming conditions. We have analysed population dynamic of two diplectanid species, Lamellodiscus echeneis and Lamellodiscus ignoratus (Monogenea: Diplectanidae) collected from two bream species in the Beymelek Lagoon (southwest coast of Turkey), comparing it between different host variables (fish size, age and sex) in order to have insight in parasites’ ecology, important for managing parasitosis in the intensive aquaculture system. In seabream (N = 127), L. echeneis prevalence was 46.5 % (exact 95 % confidence limits 38.90- 54.14), mean abundance 5.64 (bootstrap 95 % confidence limits 4.20–7.65) and mean intensity 12.14 (bootstrap 95 % confidence limits 9.49–15.59). In white seabream (N = 102), L. ignoratus prevalence was 24.5 % (exact 95 % confidence limits 16.53– 34.03), mean abundance 1.73 (bootstrap 95 % confidence limits 0.98–3.21) and mean intensity 7.04 (bootstrap 95 % confidence limits 4.60–11.40). Parasites’ parameters differed only between seasons in both hosts and between age categories in gilthead, but not in white seabream.

Usage of bisphenol A (BPA) in production of polycarbonate plastics has resulted in global distribution of BPA in the environment. These high concentrations cause numerous negative effects to the aquatic biota, among which the most known is the induction of endocrine disruption. The focus of this research was to determine the effects of two experimentally determined concentrations of BPA (100 nM and 4 µM) on cellular detoxification mechanisms during the embryonic development (2-cell, pluteus) of the rocky sea urchin (Paracentrotus lividus), primarily the potential involvement of multidrug efflux transport in the BPA intercellular efflux. The results of transport assay, measurements of the intracellular BPA and gene expression surveys, for the first time indicate the importance of P- glycoprotein (P-gp/ABCB1) in defense against BPA. Cytotoxic effects of BPA, validated by the immunohistochemistry (IHC) and the transmission electron microscopy (TEM), induced the aberrant karyokinesis, and consequently, the impairment of embryo development through the first cell division and retardation.

Pro-inflammatory cytokines play an important role in teleost defence against numerous types of pathogens, therefore are often used as biomarkers during various infections. In order to evaluate Atlantic bluefin tuna IL-1β, TNFα1 and TNFα2 induction by PAMPs, we quantified their expression during in vitro stimulation of peripheral blood leukocytes with LPS, Poly I:C. Furthermore, their role in acute and chronic parasitic infection was examined during natural infection of Pseudocycnus appendiculatus (Copepoda) and Didymosulcus katsuwonicola (Digenea), as well as during leukocyte exposure to total protein extracts isolated from two parasite species. Induction of ABT IL- 1β and TNFα2 by PAMPs and protein extracts from D. katsuwonicola and P. appendiculatus, as well as during natural infection with two parasites, suggests these cytokines play an important role in inflammation, being engaged in controlling parasite infections, in contrast to ABT TNFα1. Cellular innate response to the digenean D. katsuwonicola showed rather chronic character, resulting with parasite encapsulation in connective tissue. Mast cells, eosinophils, goblet cells, and occasional rodlet cells found at the site of infection, along with the induction of TNFα2, suggest the presence of a moderate inflammatory reaction that fails to seriously endanger digenean existence. In contrast, copepod P. appendiculatus, attached to the gill epithelium by clamping, caused direct tissue disruption with undergoing necrotic or apoptotic processes, and extensive proliferation of rodlet and goblet cells. Differential expression patterns of target cytokines in tissue surrounding two parasites and in vitro PBL model suggest that quality and quantity of tuna immune response is conditioned by parasite adaptive mechanisms and pathogenicity.

Growth and longevity of Glycymeris nummaria were determined from samples of shells, collected monthly by scuba divers between January 2010 and December 2010 from Mali Ston Bay, eastern Adriatic Sea. Acetate peel replicas of the shell margin (marginal increment analysis) and hinge region of the shell were used to validate an annual periodicity of growth lines and to determine shell age, respectively. Results indicate that G. nummaria forms a narrow growth line after February, when seawater temperatures are at a minimum. Growth lines, visible in the hinge region of all the shells, were used to estimate the age of 76 G. nummaria and provided an estimate of maximum longevity of the species at 20 y. Construction of a von Bertalanffy curve using shell length at age data produced a value of L∞ = 71 mm, a growth parameter (K) of 0.11/y, and time at length 0 was -3.74 y. The growth performance index was 2.74.

Background In countries with elevated prevalence of zoonotic anisakiasis and high awareness of this parasitosis, a considerable number of cases that associate Anisakis sp. (Nematoda, Anisakidae) and different bowel carcinomas have been described. Although neoplasia and embedded larvae were observed sharing the common site affected by chronic inflammation, no association between the nematode and malignancy were directly proved. Similarly, no data are available about the effect of secretory and excretory products of infecting larvae at the host’s cellular level, except in respect to allergenic interaction. Methods To test the mechanisms by which human non-immune cells respond to the larvae, we exposed the fibroblast cell line HS-68 to two Anisakis products (ES, excretory/secretory products; and EC, crude extract) and evaluated molecular markers related to stress response, oxidative stress, inflammation and apoptosis, such as p53, HSP70, TNF-α, c-jun and c-fos, employing cell viability assay, spectrophotometry, immunoblotting and qPCR. Results Both Anisakis products led to increased production of reactive oxygen species (ROS), especially in EC-treated cells. While the ES treatment induces activation of kinases suggesting inflammation and cell proliferation (or inhibition of apoptosis), in EC-treated cells, other signaling pathways indicate the inhibition of apoptosis, marked by strong upregulation of Hsp70. Elevated induction of p53 in fibroblasts treated by both Anisakis products, suggests a significantly negative effect on the host DNA. Conclusions This study shows that in vitro cell response to Anisakis products can result in at least two different scenarios, which in both cases lead to inflammation and DNA damage. Although these preliminary results are far from proving a relationship between the parasite and cancer, they are the first to support the existence of conditions where such changes are feasible. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1895-5) contains supplementary material, which is available to authorized users.