Molecular and evolutionary basis for survival, its failure, and virulence factors of the zoonotic nematode Anisakis pegreffii.

Parasitism is a highly successful life strategy and a driving force in genetic diversity that has evolved many times over. Accidental infections of non-targeted hosts represent an opportunity for lateral host switches and parasite niche expansion. However, if directed toward organisms that are phylogenetically distant from parasite's natural host, such as humans, it may present a dead-end environment where the parasite fails to mature or is even killed by host immunity. One example are nematodes of Anisakidae family, genus Anisakis, that through evolution have lost the ability to propagate in terrestrial hosts, but can survive for a limited time in humans causing anisakiasis. To scrutinize versatility of Anisakis to infect an evolutionary-distant host, we performed transcriptomic profiling of larvae successfully migrating through the rat, a representative model of accidental human infection and compared it to that of larvae infecting an evolutionary-familiar, paratenic host (fish). In a homeothermic accidental host Anisakis upregulated ribosome-related genes, cell division, cuticle constituents, oxidative phosphorylation, in an unsuccessful attempt to molt to the next stage. In contrast, in the paratenic poikilothermic host where metabolic pathways were moderately upregulated or silenced, larvae prepared for dormancy by triggering autophagy and longevity pathways. Identified differences and the modelling of handful of shared transcripts, provide the first insights into evolution of larval nematode virulence, warranting their further investigation as potential drug therapy targets.

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.

Acting locally - affecting globally: RNA sequencing of gilthead sea bream with a mild Sparicotyle chrysophrii infection reveals effects on apoptosis, immune and hypoxia related genes.

BACKGROUND: Monogenean flatworms are the main fish ectoparasites inflicting serious economic losses in aquaculture. The polyopisthocotylean Sparicotyle chrysophrii parasitizes the gills of gilthead sea bream (GSB, Sparus aurata) causing anaemia, lamellae fusion and sloughing of epithelial cells, with the consequent hypoxia, emaciation, lethargy and mortality. Currently no preventive or curative measures against this disease exist and therefore information on the host-parasite interaction is crucial to find mitigation solutions for sparicotylosis. The knowledge about gene regulation in monogenean-host models mostly comes from freshwater monopysthocotyleans and almost nothing is known about polyopisthocotyleans. The current study aims to decipher the host response at local (gills) and systemic (spleen, liver) levels in farmed GSB with a mild natural S. chrysophrii infection by transcriptomic analysis. RESULTS: Using Illumina RNA sequencing and transcriptomic analysis, a total of 2581 differentially expressed transcripts were identified in infected fish when compared to uninfected controls. Gill tissues in contact with the parasite (P gills) displayed regulation of fewer genes (700) than gill portions not in contact with the parasite (NP gills) (1235), most likely due to a local silencing effect of the parasite. The systemic reaction in the spleen was much higher than that at the parasite attachment site (local) (1240), and higher than in liver (334). NP gills displayed a strong enrichment of genes mainly related to immune response and apoptosis. Processes such as apoptosis, inflammation and cell proliferation dominated gills, whereas inhibition of apoptosis, autophagy, platelet activation, signalling and aggregation, and inflammasome were observed in spleen. Proteasome markers were increased in all tissues, whereas hypoxia-related genes were down-regulated in gills and spleen. CONCLUSIONS: Contrasting forces seem to be acting at local and systemic levels. The splenic down-regulation could be part of a hypometabolic response, to counteract the hypoxia induced by the parasite damage to the gills and to concentrate the energy on defence and repair responses. Alternatively, it can be also interpreted as the often observed action of helminths to modify host immunity in its own interest. These results provide the first toolkit for future studies towards understanding and management of this parasitosis.

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.

Gastric lesions in dolphins stranded along the Eastern Adriatic coast.

Stranded cetaceans are often found with gastric lesions associated with the presence of parasites; most frequently, nematodes of the genus Anisakis and the heterophyd digenean trematode Pholeter gastrophilus. In this study, we present histopathology mainly (but not exclusively) related to these 2 parasite species. Macroscopically, lesions associated with the presence of Anisakis spp. were characterised by the presence of ulcers within the gastric mucosa, while the digenean P. gastrophilus was found within large submucosal fibrotic nodules in the gastric wall. Anisakis-induced alterations included severe ulcerative gastritis with mixed inflammatory infiltrate often associated with colonies of bacteria, and mild to moderate granulomatous gastritis with eosinophilic infiltrate. P. gastrophilus-associated lesions were characterised by fibrogranulomatous gastritis with mixed inflammatory infiltrate. Additionally, immunohistochemical (IHC) analysis of P. gastrophilus lesions was consistent with the histopathologic findings, revealing inflammation-mediated stimulation. IHC-positive localisation of CD3+, iNOS+ and caspase-3+ cells suggests intensive accumulation of cytotoxic T-cells, proinflammatory cytokines and execution-phase of cell apoptosis at the parasitized area. In contrast, mechanical damage, rather than visible inflammatory response could be observed at the site of attachment of Braunina cordiformis recorded in 4 animals. Lesions not associated with the presence of parasites were mostly characterised by focal loss of superficial epithelial cells and accumulation of brown hemosiderin-like pigment or fibrous gastritis with lymphoplasmacytic infiltrate. In light of these results, we argue that observed 'tolerant' host-parasite interactions that led toward gastric lesions do not represent the cause of death and stranding of cetaceans included in this study.

A case report of Anisakis pegreffii (Nematoda, Anisakidae) identified from archival paraffin sections of a Croatian patient.

BACKGROUND: This is the first report of anisakiasis in a Croatian patient, evidenced from an archival paraffin-embedded and hematoxylin-eosin stained tissue section. Anisakiasis has been only suspected in the country based on previously detected anti-Anisakis IgE seroprevalence in the healthy coastal population, as well as an acute case where pathohistological and serological findings suggested the diseases, but the migrating larva has not been retrieved. CASE PRESENTATION: Seventy years-old female, operated in 1998 for pulmonary carcinoma, was admitted to the General hospital Sibenik, Croatia in 2003, because of gastric pain and nausea that lasted for couple of days. She was showing good general condition, full mobility and lucidity, subfebrile status. Abdominal palpation inferred acute pain in paraumbilical and ileocecal region. Exploratory right pararectal laparotomy revealed a hardened, 5 cm-long structure, located intraluminally in the sigmoid colon, not perforating colon serosa. The process has been dissected and sent for patohistological diagnosis. Results showed a 2 mm-long whitish nematode spiralised in muscular layer of colon mucosa surrounded by granulomatous inflammation. CONCLUSION: After genomic DNA isolation of the nematode from the histological section, and amplification at the mitochondrial cytochrome oxidase 2 locus, etiological agent has been identified as Anisakis pegreffii. Used methodology suggests that screening of archival suspicious sections is feasible in order to study epidemiology of this zoonotic disease poorly recognised in Croatia.

Application of compound mixture of caprylic acid, iron and mannan oligosaccharide against Sparicotyle chrysophrii (Monogenea: Polyopisthocotylea) in gilthead sea bream, Sparus aurata.

We have evaluated the therapeutic effect of a compound mixture of caprylic acid (200 mg/kg fish), organic iron (0.2% of diet) and mannan oligosaccharide (0.4% of diet) in gilthead sea bream, Sparus aurata Linnaeus, infected with Sparicotyle chrysophrii Beneden et Hesse, 1863 in controlled conditions. One hundred and ten reared and S. chrysophrii-free fish (197 g) located in a cement tank were infected by the parasite two weeks following the addition of 150 S. chrysophrii-infected fish (70 g). Growth parameters and gill parasitic load were measured in treated against control fish after a ten-week-period. Differences in final weight, feed conversion ratio, specific growth rate and feed efficiency were not statistically significant between the experimental groups, suggesting no evident effect with respect to fish growth during the study period. Although the prevalence of S. chrysophrii was not affected by the mixture at the end of the experiment, the number of adults and larvae was significantly lower. The mean intensity encompassing the number of adults and larvae was 8.1 in treated vs 17.7 in control fish. Individual comparisons of gill arches showed that the preferred parasitism site for S. chrysophrii it the outermost or fourth gill arch, consistently apparent in fish fed the modified diet and in control fish. In conclusion, the combined application of caprylic acid, organic iron and mannan oligosaccharide can significantly affect the evolution of infection with S. chrysophrii in gilthead sea bream, being capable of reducing adult and larval stages of the monogenean. However, no difference in growth improvement was observed after the trial period, potentially leaving space for further optimisation of the added dietary compounds.

Development and validation of a mixed-tissue oligonucleotide DNA microarray for Atlantic bluefin tuna, Thunnus thynnus (Linnaeus, 1758).

BACKGROUND: The largest of the tuna species, Atlantic bluefin tuna (Thunnus thynnus), inhabits the North Atlantic Ocean and the Mediterranean Sea and is considered to be an endangered species, largely a consequence of overfishing. T. thynnus aquaculture, referred to as fattening or farming, is a capture based activity dependent on yearly renewal from the wild. Thus, the development of aquaculture practices independent of wild resources can provide an important contribution towards ensuring security and sustainability of this species in the longer-term. The development of such practices is today greatly assisted by large scale transcriptomic studies. RESULTS: We have used pyrosequencing technology to sequence a mixed-tissue normalised cDNA library, derived from adult T. thynnus. A total of 976,904 raw sequence reads were assembled into 33,105 unique transcripts having a mean length of 893 bases and an N50 of 870. Of these, 33.4% showed similarity to known proteins or gene transcripts and 86.6% of them were matched to the congeneric Pacific bluefin tuna (Thunnus orientalis) genome, compared to 70.3% for the more distantly related Nile tilapia (Oreochromis niloticus) genome. Transcript sequences were used to develop a novel 15 K Agilent oligonucleotide DNA microarray for T. thynnus and comparative tissue gene expression profiles were inferred for gill, heart, liver, ovaries and testes. Functional contrasts were strongest between gills and ovaries. Gills were particularly associated with immune system, signal transduction and cell communication, while ovaries displayed signatures of glycan biosynthesis, nucleotide metabolism, transcription, translation, replication and repair. CONCLUSIONS: Sequence data generated from a novel mixed-tissue T. thynnus cDNA library provide an important transcriptomic resource that can be further employed for study of various aspects of T. thynnus ecology and genomics, with strong applications in aquaculture. Tissue-specific gene expression profiles inferred through the use of novel oligo-microarray can serve in the design of new and more focused transcriptomic studies for future research of tuna physiology and assessment of the welfare in a production environment.

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.

Cross-fertilization as a reproductive strategy in a tissue flukes Didymosulcus katsuwonicola (Platyhelmintes: Didymozoidae) inferred by genetic analysis.

Mitochondrial DNA locus cytochrome oxidase I was used to asses intraspecific genetic diversity of a didymozoid species Didymosulcus katsuwonicola. Adult forms of this species live encapsulated in pairs in the gills of the reared Atlantic bluefin tuna (Thunnus thynnus). The life cycle of this food-borne parasites and its migration in the host tissues after releasing from the digestive tract to the definitive site in the gills are unknown. Our goal was to assess whether two encysted didymozoids share the same haplotype, indicative of a common maternal origin, as well as the extent of cross- in respect to self-fertilization strategy. Intraspecific comparison showed high haplotype diversity, while the presence of two matching haplotypes within a single cyst encompassed only 17% of sampled individuals. This infers that cross-fertilization between paired individuals within the cyst is more common mechanism than self-fertilization. Such hermaphroditic parasite's trait suggests the existence of intricate infection and reproduction mechanisms, presumably as an adaptation for successful fulfillment of their indirect life cycle through dissemination of genetically more diverse and consequently more fit offspring.

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.

Ecology and genetic structure of zoonotic Anisakis spp. from adriatic commercial fish species.

Consumption of raw or thermally inadequately treated fishery products represents a public health risk, with the possibility of propagation of live Anisakis larvae, the causative agent of the zoonotic disease anisakidosis, or anisakiasis. We investigated the population dynamics of Anisakis spp. in commercially important fish-anchovies (Anisakis), sardines (Sardina pilchardus), European hake (Merluccius merluccius), whiting (Merlangius merlangus), chub mackerel (Scomber japonicus), and Atlantic bluefin tuna (Thunnus thynnus)-captured in the main Adriatic Sea fishing ground. We observed a significant difference in the numbers of parasite larvae (1 to 32) in individual hosts and between species, with most fish showing high or very high Anisakis population indices. Phylogenetic analysis confirmed that commercial fish in the Adriatic Sea are parasitized by Anisakis pegreffii (95.95%) and Anisakis simplex sensu stricto (4.05%). The genetic structure of A. pegreffii in demersal, pelagic, and top predator hosts was unstructured, and the highest frequency of haplotype sharing (n = 10) was between demersal and pelagic fish.

Characterization of three pro-inflammatory cytokines, TNFα1, TNFα2 and IL-1ß, in cage-reared Atlantic bluefin tuna Thunnus thynnus.

Atlantic bluefin tuna (BFT) (Thunnus thynnus) is of great economic significance for world aquaculture and therefore it is necessary to ensure optimal and sustainable conditions for the farming of this species. Intensive culture of fish may be limited by infectious diseases that can impact on growth performance and cause heavy losses. However, to date there are no reports of cloning and expression analysis of any major immune genes of Atlantic BFT although some immune genes are known in other BFT species. Therefore the aim of this study was to characterize the first cytokine molecules in Atlantic BFT, through: 1) Isolation of full-length cDNA and gene sequences of TNFα1, TNFα2 and IL-1ß, 2) comparison of these molecules to known sequences in other vertebrates, especially teleost fish, by multiple sequence alignment, phylogenetic tree analysis and homology modeling; 3) Quantification of in vivo expression of these cytokines in selected tissues in reared BFT over the duration of the farming process. The results indicated that these three cytokines could have value for monitoring Atlantic BFT health status. Curiously, the liver seemed to be an important site of cytokine production during poor health conditions in this species, perhaps reflecting its role as an important organ involved in fish defenses.

An inside out journey: biogenesis, ultrastructure and proteomic characterisation of the ectoparasitic flatworm Sparicotyle chrysophrii extracellular vesicles.

BACKGROUND: Helminth extracellular vesicles (EVs) are known to have a three-way communication function among parasitic helminths, their host and the host-associated microbiota. They are considered biological containers that may carry virulence factors, being therefore appealing as therapeutic and prophylactic target candidates. This study aims to describe and characterise EVs secreted by Sparicotyle chrysophrii (Polyopisthocotyla: Microcotylidae), a blood-feeding gill parasite of gilthead seabream (Sparus aurata), causing significant economic losses in Mediterranean aquaculture. METHODS: To identify proteins involved in extracellular vesicle biogenesis, genomic datasets from S. chrysophrii were mined in silico using known protein sequences from Clonorchis spp., Echinococcus spp., Fasciola spp., Fasciolopsis spp., Opisthorchis spp., Paragonimus spp. and Schistosoma spp. The location and ultrastructure of EVs were visualised by transmission electron microscopy after fixing adult S. chrysophrii specimens by high-pressure freezing and freeze substitution. EVs were isolated and purified from adult S. chrysophrii (n = 200) using a newly developed ultracentrifugation-size-exclusion chromatography protocol for Polyopisthocotyla, and EVs were characterised via nanoparticle tracking analysis and tandem mass spectrometry. RESULTS: Fifty-nine proteins involved in EV biogenesis were identified in S. chrysophrii, and EVs compatible with ectosomes were observed in the syncytial layer of the haptoral region lining the clamps. The isolated and purified nanoparticles had a mean size of 251.8 nm and yielded 1.71 × 108 particles · mL-1. The protein composition analysis identified proteins related to peptide hydrolases, GTPases, EF-hand domain proteins, aerobic energy metabolism, anticoagulant/lipid-binding, haem detoxification, iron transport, EV biogenesis-related, vesicle-trafficking and other cytoskeletal-related proteins. Several identified proteins, such as leucyl and alanyl aminopeptidases, calpain, ferritin, dynein light chain, 14-3-3, heat shock protein 70, annexin, tubulin, glutathione S-transferase, superoxide dismutase, enolase and fructose-bisphosphate aldolase, have already been proposed as target candidates for therapeutic or prophylactic purposes. CONCLUSIONS: We have unambiguously demonstrated for the first time to our knowledge the secretion of EVs by an ectoparasitic flatworm, inferring their biogenesis machinery at a genomic and transcriptomic level, and by identifying their location and protein composition. The identification of multiple therapeutic targets among EVs' protein repertoire provides opportunities for target-based drug discovery and vaccine development for the first time in Polyopisthocotyla (sensu Monogenea), and in a fish-ectoparasite model.

Re-evaluation of certain aspects of the EFSA Scientific Opinion of April 2010 on risk assessment of parasites in fishery products, based on new scientific data. Part 1: ToRs1-3.

Surveillance data published since 2010, although limited, showed that there is no evidence of zoonotic parasite infection in market quality Atlantic salmon, marine rainbow trout, gilthead seabream, turbot, meagre, Atlantic halibut, common carp and European catfish. No studies were found for greater amberjack, brown trout, African catfish, European eel and pikeperch. Anisakis pegreffii, A. simplex (s. s.) and Cryptocotyle lingua were found in European seabass, Atlantic bluefin tuna and/or cod, and Pseudamphistomum truncatum and Paracoenogonimus ovatus in tench, produced in open offshore cages or flow-through ponds or tanks. It is almost certain that fish produced in closed recirculating aquaculture systems (RAS) or flow-through facilities with filtered water intake and exclusively fed heat-treated feed are free of zoonotic parasites. Since the last EFSA opinion, the UV-press and artificial digestion methods have been developed into ISO standards to detect parasites in fish, while new UV-scanning, optical, molecular and OMICs technologies and methodologies have been developed for the detection, visualisation, isolation and/or identification of zoonotic parasites in fish. Freezing and heating continue to be the most efficient methods to kill parasites in fishery products. High-pressure processing may be suitable for some specific products. Pulsed electric field is a promising technology although further development is needed. Ultrasound treatments were not effective. Traditional dry salting of anchovies successfully inactivated Anisakis. Studies on other traditional processes - air-drying and double salting (brine salting plus dry salting) - suggest that anisakids are successfully inactivated, but more data covering these and other parasites in more fish species and products is required to determine if these processes are always effective. Marinade combinations with anchovies have not effectively inactivated anisakids. Natural products, essential oils and plant extracts, may kill parasites but safety and organoleptic data are lacking. Advanced processing techniques for intelligent gutting and trimming are being developed to remove parasites from fish.

Helminthic host defense peptides: using the parasite to defend the host.

Parasitic helminths are destined to share niches with a variety of microbiota that inevitably influence their interaction with the host. To modulate the microbiome for their benefit and defend against pathogenic isolates, helminths have developed host defense peptides (HDPs) and proteins as integral elements of their immunity. These often exert a relatively nonspecific membranolytic activity toward bacteria, sometimes with limited or no toxicity toward host cells. With a few exceptions, such as nematode cecropin-like peptides and antibacterial factors (ABFs), helminthic HDPs are largely underexplored. This review scrutinizes current knowledge on the repertoire of such peptides in helminths and promotes their research as potential leads for an anti-infective solution to the burgeoning problem of antibiotic resistance.

Assessing Portuguese health risks: Anisakiks parasite in Atlantic chub mackerel (Scomber colias) sold in Portuguese markets.

Anisakiosis is a significant zoonotic disease caused by parasitic nematodes of the Anisakis genus. It can be contracted by humans through the consumption of raw or undercooked fish contaminated with the parasite, leading to gastrointestinal and allergic symptoms. While anisakiosis is not frequently documented in Portugal, the presence of allergic reactions to Anisakis in Spain suggests ongoing exposure in the Iberian Peninsula. To address this concern, the Interdisciplinary Centre of Marine and Environmental Research in Porto, Portugal, in collaboration with the Biology Centre of Czech Academy of Sciences in Ceske Budejovice, Czech Republic, has proposed a project entitled 'Assessing Portuguese Health Risks: Anisakis Parasite in Atlantic Chub Mackerel (Scomber colias) Sold in Portuguese Markets' under the European Food Risk Assessment Fellowship Programme. The primary objective of the project is to gather valuable epidemiological data on the host, Atlantic chub mackerel (S. colias) and the parasitic nematode (Anisakis spp.) with the focus on assessing contamination levels and evaluating potential health risks associated with anisakiosis in the Portuguese population. By conducting this research, the project aims to contribute to the understanding of anisakiosis and its impact on public health in Portugal. Investigation of the presence of the Anisakis parasite in Atlantic chub mackerel sold in Portuguese markets will provide crucial insights into the risks associated with consuming raw or undercooked fish. Ultimately, our findings will aid in the development of preventive measures and guidelines to ensure the well-being of the Portuguese population.

In vitro culture of the zoonotic nematode Anisakis pegreffii (Nematoda, Anisakidae).

BACKGROUND: Anisakiasis is a foodborne disease caused by the third-stage larvae (L3) of two species belonging to the genus Anisakis: Anisakis pegreffii and Anisakis simplex sensu stricto. Both species have been the subject of different -omics studies undertaken in the past decade, but a reliable in vitro culture protocol that would enable a more versatile approach to functional studies has never been devised. In nature, A. pegreffii shows a polyxenous life-cycle. It reproduces in toothed whales (final host) and disseminates embryonated eggs via cetacean faeces in the water column. In the environment, a first- (L1) and second-stage larva (L2) develops inside the egg, and subsequently hatched L2 is ingested by a planktonic crustacean or small fish (intermediate host). In the crustacean pseudocoelom, the larva moults to the third stage (L3) and grows until the host is eaten by a fish or cephalopod (paratenic host). Infective L3 migrates into the visceral cavity of its paratenic host and remains in the state of paratenesis until a final host preys on the former. Once in the final host's gastric chambers, L3 attaches to mucosa, moults in the fourth stage (L4) and closes its life-cycle by becoming reproductively mature. METHODS: Testing two commercially available media (RPMI 1640, Schneider's Drosophila) in combination with each of the six different heat-inactivated sera, namely foetal bovine, rabbit, chicken, donkey, porcine and human serum, we have obtained the first reliable, fast and simple in vitro cultivation protocol for A. pegreffii. RESULTS: Schneider's Drosophila insect media supplemented with 10% chicken serum allowed high reproducibility and survival of adult A. pegreffii. The maturity was reached already at the beginning of the third week in culture. From collected eggs, hatched L2 were maintained in culture for 2 weeks. The protocol also enabled the description of undocumented morphological and ultrastructural features of the parasite developmental stages. CONCLUSIONS: Closing of the A. pegreffii life-cycle from L3 to reproducing adults is an important step from many research perspectives (e.g., vaccine and drug-target research, transgenesis, pathogenesis), but further effort is necessary to optimise the efficient moulting of L2 to infective L3.

Fecundity, in vitro early larval development and karyotype of the zoonotic nematode Anisakis pegreffii.

The in vitro life cycle of zoonotic helminths is an essential tool for -omic translational studies focused on disease control and treatment. Anisakiosis is an emerging zoonosis contracted by the ingestion of raw or undercooked fish infected with the third stage larvae (L3) of two sibling species Anisakis simplex sensu stricto (s.s.) and Anisakis pegreffii, the latter being the predominant species in the Mediterranean basin. Recently, in vitro culture of A. pegreffii has been developed to enable fast and large-scale production of fertile adults. However, the conditions for larval development from hatching to infective L3 were not fulfilled to complete the cycle. Herein, we used a Drosophila medium supplemented with chicken serum and adjusted different osmolarities to maintain the culture of L3 hatched from eggs for up to 17 weeks. The highest survival rate was observed in the medium with the highest osmolarities, which also allowed the highest larval exsheathment rate. Key morphological features of embryogenesis and postembryogenesis studied by transmission electron microscopy revealed that the excretory gland cell is differentiated already up to 48 h post-hatching. Extracellular vesicles and cell-free mitochondria are discharged between the two cuticle sheets of the second stage larvae (L2). Contemporarly cultivated, two populations of adult A. simplex s.s. and A. pegreffii reached an average production of 29,914.05 (± 27,629.36) and 24,370.96 (± 12,564.86) eggs/day/female, respectively. The chromosome spreads of A. pegreffii obtained from mature gonads suggests a diploid karyotype formula of 2n = 18. The development of a reliable protocol for the in vitro culture of a polyxenous nematode such as Anisakis spp. will serve to screen for much needed novel drug targets, but also to study the intricated and unknown ecological and physiological traits of these trophically transmitted marine nematodes.

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.