A plant-based diet supplemented with Hermetia illucens alone or in combination with poultry by-product meal: one step closer to sustainable aquafeeds for European seabass.

BACKGROUND: Increasing demand for high-value fish species and pressure on forage fish is challenging aquaculture to ensure sustainable growth by replacing protein sources in aquafeeds with plant and terrestrial animal proteins, without compromising the economic value and quality of the final fish product. In the present study, the effects of a plant protein-based diet (CV), two plant-based diets in which graded amounts of plan protein mixtures were replaced with Hermetia illucens meal alone (VH10) or in combination with poultry by-product meal (PBM) (VH10P30), a fishmeal (FM) diet (CF) and an FM diet supplemented with H. illucens (FH10) on growth performance, gut health and homeostasis of farmed subadult European seabass were tested and compared. RESULTS: Fish fed the VH10 and VH10P30 diets showed the highest specific growth rates and lowest feed conversion ratios among the tested groups. Expectedly, the best preservation of PI morphology was observed in fish fed the CF or FH10 diets, while fish fed the CV diet exhibited significant degenerative changes in the proximal and distal intestines. However, PBM supplementation mitigated these effects and significantly improved all gut morphometric parameters in the VH10P30 group. Partial substitution of the plant mixture with insect meal alone or PBM also induced most BBM genes and activated BBM enzymes, suggesting a beneficial effect on intestinal digestive/absorption functions. Regarding intestinal microbiota, fish fed diets containing H. illucens meal (FH10, VH10, VH10P30) had the highest richness of bacterial communities and abundance of beneficial genera such as Lactobacillus and Bacillus. On the other hand, fish fed CV had the highest microbial diversity but lost a significant component of fish intestinal microbiota, the phylum Bacteroidetes. Finally, skin pigmentation most similar to that of farmed or even wild seabass was also observed in the fish groups fed CF, FH10 or VH10P30. CONCLUSION: Plant-based diets supplemented with PBM and H. illucens pupae meal have great potential as alternative diets for European seabass, without affecting growth performance, gut homeostasis, or overall fitness. This also highlights the importance of animal proteins in diets of European seabass, as the addition of a small amount of these alternative animal protein sources significantly improved all measured parameters.

In vivo and in vitro assessment of host-parasite interaction between the parasitic copepod Brachiella thynni (Copepoda; Lernaeopodidae) and the farmed Atlantic bluefin tuna (Thunnus thynnus).

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 1ß (il1ß), 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.

Anisakis Sensitization in the Croatian fish processing workers: Behavioral instead of occupational risk factors?

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.

Interplay between proinflammatory cytokines, miRNA, and tissue lesions in Anisakis-infected Sprague-Dawley rats.

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.

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.

Efficiency of Target Larvicides Is Conditioned by ABC-Mediated Transport in the Zoonotic Nematode Anisakis pegreffii.

Anisakiasis is among the most significant emerging foodborne parasitoses contracted through consumption of thermally unprocessed seafood harboring infective Anisakis species larvae. The efficacy of the currently applied anthelminthic therapy in humans and in model organisms has not proven sufficient, so alternative solutions employing natural compounds combined with chemical inhibitors should be explored. By testing toxicity of the natural monoterpenes nerolidol and farnesol and the conventional anthelminthics abamectin and levamisole in the presence/absence of MK-571 and Valspodar, which inhibit the ABC transporter proteins multidrug resistance protein (MRP-like) and P-glycoprotein (P-gp), we determined the preliminary traits of Anisakis detoxifying mechanisms. We found that Anisakis P-gp and MRP-like transporters have a role in the efflux of the tested compounds, which could be useful in the design of novel anthelminthic strategies. As expected, transporter activation and efflux fluctuated over time; they were synchronously active very early postexposure, whereas the activity of one transporter dominated over the other in a time-dependent manner. MRP-like transporters dominated in the efflux of farnesol, and P-gp dominated in efflux of nerolidol, while both were active in effluxing levamisole. The highest toxicity was exerted by abamectin, a P-gp inhibitor per se, which also elicited the highest oxidative stress in treated Anisakis larvae. We suggest that ß-tubulin, observed for the first time as a core element in Anisakis cuticle, might represent an important target for the tested compounds.

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.

Assessing the risk of an emerging zoonosis of worldwide concern: anisakiasis.

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.

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.

Anisakis pegreffii (Nematoda: Anisakidae) products modulate oxidative stress and apoptosis-related biomarkers in human cell lines.

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.

Combined Use of Morphological and Molecular Tools to Resolve Species Mis-Identifications in the Bivalvia The Case of Glycymeris glycymeris and G. pilosa.

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.

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.

Expression analysis of the Atlantic bluefin tuna (Thunnus thynnus) pro-inflammatory cytokines, IL-1ß, TNFα1 and TNFα2 in response to parasites Pseudocycnus appendiculatus (Copepoda) and Didymosulcus katsuwonicola (Digenea).

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-1ß, TNFα1 and TNFα2 induction by PAMPs, we quantified their expression during in vitro stimulation of peripheral blood leukocytes by LPS and 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-1ß 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.

Population dynamics of two diplectanid species (Monogenea) parasitising sparid hosts (Sparidae).

Economically important sparid fish species, gilthead (Sparus aurata) and white seabream (Diplodus sargus) (Sparidae) are frequently parasitised by 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.

Effect of bisphenol A on P-glycoprotein-mediated efflux and ultrastructure of the sea urchin embryo.

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 (100nM and 4µM) 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.