Search

Anisakid nematodes are a globally distributed group of marine mammal parasites. Kogiid whales, including the pygmy sperm whale Kogia breviceps, host an assemblage of specific anisakid species. Currently, three species are known to be specific to kogiid hosts, i.e., Skrjabinisakis paggiae, S. brevispiculata, and the less studied Pseudoterranova ceticola. The aim of this study was to investigate the species diversity of anisakid nematodes sampled from a pygmy sperm whale stranded in 2013 at the edge of its distribution range in the Northeast Atlantic, specifically in the North of Scotland. Nematodes were assigned to genus level based on morphology and identified by sequence analysis of the mtDNA cox2 gene and the rDNA ITS region. The present finding represents the first observation of syntopic occurrence of adult stages of S. brevispiculata, S. paggiae, and P. ceticola in a pygmy sperm whale in the Northeast Atlantic, and represent the northernmost record of these species in this area. Skrjabinisakis brevispiculata was the most abundant species, accounting for 55% of the identified nematodes, predominantly in the adult stage. Anisakis simplex (s.s.) was also abundant, with most specimens in the preadult stage, followed by S. paggiae and P. ceticola. The pygmy sperm whale is rarely documented in Scottish waters, and its occurrence in the area could suggest expansion of its geographic range. The presence of S. brevispiculata, S. paggiae, and P. ceticola in this whale species in this region may indicate a shift in the whole host community involved in the life cycle of these parasites in northern waters. However, it is also plausible that these parasites were acquired while the whale was feeding in more southern regions, before migrating northbound.

Summary: Human dendritic cells (DCs) show remarkable phenotypic changes when matured in the presence of helminth‐derived products. These modifications frequently elicited a polarization towards Th2 cells and regulatory T cells thus contributing to immunological tolerance against these pathogens. In this study, the interaction between DCs and larvae of the zoonotic anisakid nematode Anisakis pegreffii was investigated. A. pegreffii larvae were collected from fish hosts, and monocyte‐derived DCs were cocultured in the presence of the live larvae (L) or its crude extracts (CE). In both experimental conditions, A. pegreffii impacted DC viability, hampered DC maturation by reducing the expression of molecules involved in antigen presentation and migration (ie HLA‐DR, CD86, CD83 and CCR7), increased the phagosomal radical oxygen species (ROS) levels and modulated the phosphorylation of ERK1,2 pathway. These biological changes were accompanied by the impairment of DCs to activate a T‐cell‐mediated IFNγ. Interestingly, live larvae appeared to differently modulate DC secretion of cytokines and chemokines as compared to CE. These results demonstrate, for the first time, the immunomodulatory role of A. pegreffii on DCs biology and functions. In addition, they suggest a dynamic contribution of DCs to the induction and maintenance of the inflammatory response against A. pegreffii. [ABSTRACT FROM AUTHOR]

SUMMARY Ig E sensitization to Anisakis pegreffii in Italian subjects suffering from gastro-allergic anisakiasis ( GAA) (N=5), or showing chronic urticaria ( CU+) after fish consumption (N=100), was investigated. A control group (N=5) was also included. Ig E response was analysed by immunoblotting ( WB) assay, using both excretory/secretory products ( ESPs) and crude extract ( CE) of A. pegreffii larvae. The results were compared with those achieved by the conventional immunological method for Anisakis allergy (ie, immuno CAP). Among the 110 subjects, 28 showed IgE positivity with both WB and iCAP methods; 13 proved IgE reactivity, in WB assay, to ESP antigens of A. pegreffii, here provisionally indicated as Ani s 1 -like, Ani s 7 -like, Ani s 13 -like; only 15 sera have shown IgE- WB reaction to Ani s 7 -like and Ani s 13 -like. iCAP and WB exhibited a high concordance value (κ=1.00) when iCAP value was <0.35 (negative result) and >50.0 (positive result). In the sera samples recorded as positive to Anisakis allergy, Ani s 1 -like was responsible for 46.4% of the sensitivity, while Ani s 7 -like and Ani s 13 -like for 100%. They could be considered as major antigens in the diagnosis of allergic anisakiasis caused by A. pegreffii. [ABSTRACT FROM AUTHOR]

Fish are often speckled with “black spots” caused by metacercarial trematode infection, inducing a host response. Cryptocotyle spp. (Opisthorchiidae) are among the parasites responsible for this phenomenon. So far, the impact on human health is still unknown. In addition, few publications dealing with black spot recovery, identification, distribution and diversity among commercially important fish are available. Moreover, “black spots” have been observed by fishermen on marine fish, revealing an appreciable but unquantified presence in consumed fish. An epidemiological survey of 1,586 fish from seven commercial species (herring, sprat, whiting, pout, dab, flounder, and plaice) was conducted in the Eastern English Channel and the North Sea in January 2019 and 2020. Encysted metacercariae were found in 325 out of 1,586 fish, with a total prevalence of 20.5%. Intensity of infection varied from 1 to 1,104 parasites. The recorded encysted metacercariae were identified either by microscopic examination or with molecular tools. Partial sequences of the mtDNA cox1 gene and of the rDNA ITS region were obtained. Two species of Cryptocotyle, Cryptocotyle lingua (Creplin, 1825) and Cryptocotyle concava (Creplin, 1825) were found. Metacercariae belonging to other trematode families were also identified. Molecular phylogenetic analysis and haplotype network construction were performed to confirm the identification and to study the potential presence of different populations of Cryptocotyle spp. This survey enabled us to describe the distribution of two species of Cryptocotyle in the English Channel and North Sea ecosystems. The observed differences in infestation levels between fish species and geographical areas will contribute to better understanding of the ecology of these parasites.

The genus Anisakis represents one of the most widespread groups of ascaridoid nematodes in the marine ecosystem. Three closely related taxa are recognized in the Anisakis simplex (s. l.) complex: A. pegreffii, A. simplex (s. s.) and A. berlandi. They are widely distributed in populations of their intermediate/paratenic hosts (fish and squids) and definitive hosts (cetaceans). A novel nuclear gene locus, metallopeptidase 10 (nas 10) (451 bp), was sequenced and validated on a total of 219 specimens of the three species of Anisakis, collected in fish and cetacean hosts from allopatric areas included in their ranges of distribution. The specimens of Anisakis were first identified by allozymes and sequence analysis of the mtDNA cox2 and EF1α-1 nDNA. The novel nuclear marker has shown fixed alternative nucleotide positions in the three species, i.e. diagnostic at 100%, permitting the species determination of a large number of specimens analyzed in the present study. In addition, primers to be used for amplification-refractory mutation system (ARMS) PCR of the same gene locus were designed at these nucleotide positions. Thus, direct genotyping determination, by double ARMS, was developed and validated on 219 specimens belonging to the three species. Complete concordance was observed between the tetra-primer ARMS-PCR assays and direct sequencing results obtained for the nas 10 gene locus. The novel nuclear diagnostic marker will be useful in future studies on a multi-locus genotyping approach and also to study possible hybridization and/or introgression events occurring between the three species in sympatric areas.

Eight microsatellite loci, recently developed in the species Anisakis pegreffii, were successfully amplified in Anisakis berlandi, sibling species of the A. simplex (s. l.) complex. They were validated on adult specimens (n = 46) of the parasite species, collected from two individuals of the definitive host, the long-finned pilot whale Globicephala melas from New Zealand waters. Among the eight loci scored, one, Anisl 07132, had null alleles in A. berlandi and was thus excluded from the subsequent genetic analysis. Two loci, Anisl 00314 and Anisl 10535, were monomorphic. In addition, as also previously detected in the other species of the A. simplex (s. l.) complex, the Anisl 7 locus was seen to be sex-linked, showing hemizygosity in male specimens. Differential allele frequency distributions of A. berlandi, with respect to those previously observed in A. pegreffii and A. simplex (s. s.), were found at some microsatellite loci. The Anisl 7 locus provided 100% diagnosis between A. berlandi and A. pegreffii, while others resulted in 99% diagnosis between A. berlandi and the other two species. Simple sequence repeat (SSR) loci also allowed us to estimate the genetic differentiation of A. berlandi from A. pegreffii (F st ≈ 0.45, Dc = 0.82) and A. simplex (s. s.) (F st ≈ 0.57, Dc = 0.73). The results suggest that SSRs provide a set of candidate markers for population genetics analysis of A. berlandi, as well as for the investigation, through a multi-locus genotyping approach, of possible patterns of hybridisation/introgression events between A. berlandi and the other two Anisakis species in sympatric conditions.

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

A retrospective analysis on human anisakiasis in Italy since its first description in 1996 was performed by conducting a literature search. Inclusion criteria based on the presence of a larva and on parasite identification were applied. Epidemiological data and clinical features were analysed. Particular attention was paid to the source of infection. In total, 73 cases were included in the analysis, while 34 were excluded. Cases were reported from eight Italian regions, most frequently Abruzzo, Apulia and Latium. The parasite was detected by endoscopy (51.4%) or laparotomy (48.6%). The site of infection was intestinal (42.5%), gastric (43.8%), oesophageal (1.4%) or ectopic (12.3%). Most of the parasites (71.0%) were identified as Anisakis sp. or A. simplex (s.l.). However, when molecular methods were used (21 cases), A. pegreffii was always identified. In most of the patients (65.7%), the source of infection was raw or undercooked anchovies, followed by “anchovies or sardines” (15.1%), generic “raw seafood” (15.1%), and sardines (1.4%). In only 2 cases (2.7%), the source was not available. This is the first systematic analysis of Italian cases of anisakiasis. The main conclusions derived from the results are: i) attention should be given to the history, in particular when raw marinated anchovies, proven to be the main source of human anisakiasis in Italy, are consumed; ii) in order to assess correct epidemiological data, a confirmed and specific etiological identification should always be sought.

The objective of this opinion was to determine if any wild caught fish species, originating from specific fishing grounds and consumed in the EU/EFTA could be considered free of zoonotic parasites. In this Opinion the term 'fishery products' only refers to fresh finfish. As there are multiple fish species and numerous potential parasites, Anisakis sp. was used as an indicator of zoonotic parasites in marine areas. This parasite species is particularly suited as it is common in marine environments, capable of infecting multiple fish species and is the subject of the majority of published studies. On the rare occasion where Anisakis sp. data were not available, or all tests were negative, other parasites such as Contracaecum osculatum (s.l.) and/or Phocanema spp. were considered. In freshwater systems, all zoonotic parasites were investigated. Consumption, import and landing data were used to determine the most relevant fish species and, where possible, the source fishing areas were identified. The most commonly consumed wild caught fish species in the EU/EFTA include tuna, cod, Alaskan pollock, hake, herring, sardines, mackerel, trout and saithe. Although the majority of these fish are caught in the Atlantic Ocean, the Mediterranean and the Black Sea (37) as well as several areas in the Indian Ocean, imported fish may originate from any global fishing areas, with the exception of Antarctica. Based on the data, at least one zoonotic parasite has been reported in at least one fish species in each of the FAO marine fishing areas. Thus, due to relative low fish host specificity of the zoonotic parasites, the panel concluded that all wild caught fish species may be exposed to and infected with zoonotic parasites. The same applies to freshwater fishing areas, with many areas having multiple studies reporting the presence of zoonotic parasites in the wild caught fish species., (© 2024 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

Dietary changes expose consumers to risks from Anisakis larvae in seafood, leading to parasitic diseases and allergies. Anisakis is recognized by EFSA as a significant hazard, with potential oncogenic implications. Diagnostic advancements, like the Basophil Activation Test (BAT), enhance sensitivity and accuracy in identifying Anisakis sensitization, complementing traditional IgE tests. We conducted a cross-sectional study on patients with allergic symptoms from April 2021 to April 2023 at two outpatient clinics in western Sicily. Our goal was to assess the prevalence of Anisakis -related allergies and to identify risk profiles using specific Anisakis IgE and the BAT, especially in regions with high raw fish consumption. The study evaluated specific Anisakis IgE as a screening tool for Anisakis sensitization, using questionnaires, blood samples, and immuno-allergology analyses. Anisakis -specific IgE values were compared with the BAT results, with statistical analyses including Fisher's exact test and logistic regression. The results showed an 18.5% seroprevalence of Anisakis IgE, while the BAT as a second-level test showed 4.63%, indicating the BAT's superior specificity and accuracy. The study highlighted the importance of the BAT in diagnosing Anisakis sensitization, especially in cases of cross-reactivity with Ascaris and tropomyosin. The findings confirm the BAT's exceptional specificity in identifying Anisakis sensitization and support using Anisakis -specific IgE for population-based risk profiling. The BAT can effectively serve as a confirmatory test.

Maternal parasitemia and placental parasite load were examined in mother-newborn pairs to determine their effect on the congenital transmission of Trypanosoma cruzi . Parasitemia was qualitatively assessed in mothers and newborns by the microhematocrit test; parasite load was determined in the placental tissues of transmitting and non-transmitting mothers by the detection of T. cruzi DNA and by histology. Compared to transmitter mothers, the frequency and prevalence of parasitemia were found to be increased in non-transmitter mothers; however, the frequency and prevalence of parasite load were higher among the transmitter mothers than among their non-transmitter counterparts. Additionally, serum levels of interferon (IFN)-γ were measured by an enzyme-linked immunosorbent assay (ELISA) in peripheral, placental, and cord blood samples. Median values of IFN-γ were significantly increased in the cord blood of uninfected newborns. The median IFN-γ values of transmitter and non-transmitter mothers were not significantly different; however, non-transmitter mothers had the highest total IFN-γ production among the group of mothers. Collectively, the results of this study suggest that the anti- T. cruzi immune response occurring in the placenta and cord is under the influence of the cytokines from the mother's blood and results in the control of parasitemia in uninfected newborns.

Bisphenols and phthalates are wide classes of endocrine disrupting chemicals (EDCs) extensively used as additives in plastic products. In this study, a fast and reliable analytical method based on matrix solid-phase dispersion (MSPD) coupled with LC-MS/MS was developed and optimized for simultaneous determination of 8 bisphenols and 7 phthalates in raw mussel extract. The LC-MS/MS method was tested for linearity (R 2 ), inter- and intra-day repeatability, limit of detection and quantification, both for matrix-free and matrix-matched solutions. The MSPD method was optimized in terms of ratio between sample and sorbent, and the type and quantity of the eluents in order to maximize the recoveries and to minimize matrix effects. The obtained recoveries (values between 75% and 113%), limits of detection (values between 0.048 and 0.36 µg kg -1 ), limits of quantification (values between 0.16 and 1.28 µg kg -1 ), repeatability (RSD% between 1.30% and 8.41%) and linearity (R 2 > 0.998) were satisfactory and suitable for the determination of target micropollutants in food samples. In addition, the low solvent consumption and fast execution make this method ideal for routinely determinations of bisphenols and phthalates in mussels.

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., Competing Interests: If you wish to access the declaration of interests of any expert contributing to an EFSA scientific assessment, please contact interestmanagement@efsa.europa.eu., (© 2024 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

Understanding the genomic underpinnings of thermal adaptation is a hot topic in eco-evolutionary studies of parasites. Marine heteroxenous parasites have complex life cycles encompassing a free-living larval stage, an ectothermic intermediate host and a homeothermic definitive host, thus representing compelling systems for the study of thermal adaptation. The Antarctic anisakid Contracaecum osculatum sp. D is a marine parasite able to survive and thrive both at very cold and warm temperatures within the environment and its hosts. Here, a de novo transcriptome of C. osculatum sp. D was generated for the first time, by performing RNA-Seq experiments on a set of individuals exposed to temperatures experienced by the nematode during its life cycle. The analysis generated 425,954,724 reads, which were assembled and then annotated. The high-quality assembly was validated, achieving over 88% mapping against the transcriptome. The transcriptome of this parasite will represent a valuable genomic resource for future studies aimed at disentangling the genomic architecture of thermal tolerance and metabolic pathways related to temperature stress., (© 2023. Springer Nature Limited.)

The Mediterranean Sea is recognized as a marine biodiversity hotspot. This enclosed basin is facing several anthropogenic-driven threats, such as seawater warming, pollution, overfishing, bycatch, intense maritime transport and invasion by alien species. The present review focuses on the diversity and ecology of specific marine trophically transmitted helminth endoparasites (TTHs) of the Mediterranean ecosystems, aiming to elucidate their potential effectiveness as ‘sentinels’ of anthropogenic disturbances in the marine environment. The chosen TTHs comprise cestodes and nematodes sharing complex life cycles, involving organisms from coastal and marine mid/upper-trophic levels as definitive hosts. Anthropogenic disturbances directly impacting the free-living stages of the parasites and their host population demographies can significantly alter the distribution, infection levels and intraspecific genetic variability of these TTHs. Estimating these parameters in TTHs can provide valuable information to assess the stability of marine trophic food webs. Changes in the distribution of particular TTHs species can also serve as indicators of sea temperature variations in the Mediterranean Sea, as well as the bioaccumulation of pollutants. The contribution of the chosen TTHs to monitor anthropogenic-driven changes in the Mediterranean Sea, using their measurable attributes at both spatial and temporal scales, is proposed.

The rising popularity of undercooked or raw seafood containing larvae of the Anisakis parasite has led to issues of public health concern due to allergic manifestations. We conducted an observational study on the use of an innovative Anisakis allergy diagnostic algorithm in a convenience sample of 53 allergic outpatients recruited in Western Sicily, between April 2021 and March 2022. We included individuals with an anamnesis suggestive of IgE sensitization to Anisakis reporting clinical manifestation in the last month due to allergic reactions after eating fresh fish, or in subjects at high exposure risk with sea products while abstaining from fish ingestion, excluding those with documented fish sensitization. Outpatients were tested via Skin Prick Test, IgE-specific dosage and Basophil Activation Test (BAT). Twenty-six outpatients were diagnosed with Anisakis , while 27 with Chronic Urticaria (CU). We found a seven-fold excess risk for Anisakis (p4) positivity in the Anisakis allergic outpatients, as compared to the CU ones. BAT showed the best diagnostic accuracy (92.45%) and specificity (100%), while specific IgE to Ascaris (p1) documented the best sensitivity (92.31%) but a very low specificity (37.04%). In conclusion, our findings may represent a potentially useful contribution to the future development of updated clinical guidelines.

Introduction: Anisakis pegreffii is a sibling species within the A. simplex (s.l.) complex requiring marine homeothermic (mainly cetaceans) and heterothermic (crustaceans, fish, and cephalopods) organisms to complete its life cycle. It is also a zoonotic species, able to accidentally infect humans (anisakiasis). To investigate the molecular signals involved in this host-parasite interaction and pathogenesis, the proteomic composition of the extracellular vesicles (EVs) released by the third-stage larvae (L3) of A. pegreffii , was characterized., Methods: Genetically identified L3 of A. pegreffii were maintained for 24 h at 37°C and EVs were isolated by serial centrifugation and ultracentrifugation of culture media. Proteomic analysis was performed by Shotgun Analysis., Results and Discussion: EVs showed spherical shaped structure (size 65-295 nm). Proteomic results were blasted against the A. pegreffii specific transcriptomic database, and 153 unique proteins were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis predicted several proteins belonging to distinct metabolic pathways. The similarity search employing selected parasitic nematodes database revealed that proteins associated with A. pegreffii EVs might be involved in parasite survival and adaptation, as well as in pathogenic processes. Further, a possible link between the A. pegreffii EVs proteins versus those of human and cetaceans' hosts, were predicted by using HPIDB database. The results, herein described, expand knowledge concerning the proteins possibly implied in the host-parasite interactions between this parasite and its natural and accidental hosts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Palomba, Rughetti, Mignogna, Castrignanò, Rahimi, Masuelli, Napoletano, Pinna, Giorgi, Santoro, Schininà, Maras and Mattiucci.)

Sibling species of the Contracaecum rudolphii (s.l.) complex are habitual endoparasites of cormorants of the Phalacrocoracidae family, worldwide. In Europe, the two species, C. rudolphii sp. A and C. rudolphii sp. B, have been identified. However, information regarding the occurrence and distribution of these anisakids in cormorants from Spain is scarce. In the present study, 20 specimens of the European Shag, Ph. aristotelis desmarestii , from the western Mediterranean Spanish marine coast were parasitologically analyzed for the presence of nematodes. All hosts were found parasitized with Contracaecum specimens ( n = 1,517). A representative subsample was genetically identified as C. rudolphii sp. A by sequence analysis of the mtDNA cox 2 gene and the ITS1 and ITS2 regions of the rDNA. This represents the first report of C. rudolphii sp. A from the Spanish Mediterranean waters. Population genetic analysis was performed including other C. rudolphii sp. A specimens from the west Sardinian and the Tyrrhenian Sea. At the intraspecific level, a significant genetic differentiation ( Fst ≈ 0.08, p < 0.00001) between the metapopulation from the Spanish Mediterranean coast and that from the Sardinian waters was observed; whereas, no differentiation was found between metapopulations of the parasite from the Spanish and the Tyrrhenian Italian coast. The findings highly support the hypothesis of the adaptation of the life cycle of C. rudolphii sp. A in brackish and marine ecosystems. Furthermore, the results on the population genetics of C. rudolphii sp. A suggest the possible role of the migration routes of wintering populations of cormorants in the Mediterranean Sea in influencing the parasite genetic structure., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Roca-Geronès, Fisa, Montoliu, Casadevall, Tobella, Bas, Palomba and Mattiucci.)

In recent years, a drop in the condition of the European sardine has been observed. Although several causes have been attributed to this issue, as overfishing and climate change, little is known about the link between ascaridoid nematode parasitisation and fish status. In this study, sardines were obtained from four fishing grounds along the Mediterranean (Alboran, Northern Spain, Northern Adriatic, and Aegean), and one location in the Atlantic Ocean (Southern Portugal). After analysing individual fish body condition (by direct tissue fat content measurements and condition indices), and reproductive status (by a detailed gonadal examination) throughout the entire annual cycle, ascaridoids were recognised by combining naked eye and UV-press method along flesh, viscera, and gonads. Afterwards, sequence analysis of the rDNA internal transcribed spacers region (ITS) and the mtDNA cox2 gene were used to identify and characterise the different species of ascaridoids from the fish host in the localities throughout the seasons. The main species found along different areas was Hysterothylacium aduncum , present in the Northern Adriatic (prevalence of 7.6%, mean intensity 1.700), the Atlantic (7.5%, 3.889), and the Northern Spain (3.9%, 1.600). Moreover, few individuals of Anisakis simplex (s.s.) and A. pegreffii were observed in the Atlantic (1.7% and 0.8%, respectively), and the latter species was also found in the Adriatic stock (0.8%). All ascaridoid specimens were found in viscera. Obtained results seem to indicate that in stocks with medium sizes, small variations in length are related to parasite intensity. This study highlights the importance of seasonal parasitological analyses at stock level and, especially, in capital breeders, as relationships between condition and reproduction parameters and parasitism are conditioned by seasonality., Competing Interests: The authors declare no conflict of interest/competing interest., (© 2022 The Authors.)

Anisakiasis is a zoonotic fish-born parasitic disease caused by anisakid nematodes. Paraffin-embedded blocks containing biopsy samples taken from patients suffering gastritis with unknown causes were investigated by real-time PCR, in the Bushehr region, Iran; where human anisakiasis has not been reported, so far. A total of 50 paraffin-embedded blocks were randomly selected from 250 archived blocks of the patients with gastritis. A SYBER green-based real-time PCR targeting the ITS1 region was developed for the identification of Anisakis genus. An 86 bp partial fragment of the Anisakis spp. ITS1 gene was amplified successfully. A total of 3 out of 50 samples (6 %) had positive amplification in the samples and their pathology reports showed a significant finding of moderate chronic gastritis with or without ulcers. In conclusion, the developed qPCR could be used for detecting Anisakis spp. larval DNA in human biopsy blocks. This study showed the hidden human cases of anisakiasis in the Bushehr for the first time., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Nematode parasites of the genus Anisakis (Nematoda, Anisakidae) are considered among the most important biological hazards in seafood products worldwide. In temperate and tropical waters, the most common species appears to be Anisakis typica , generally found around the viscera and sporadically in the flesh of various fish host species. This study investigated the infection sites and genetic diversity of A. typica infecting commercial fishes from the South-West Indian Ocean. Largehead hairtail ( N  = 20) and brushtooth lizardfish ( N  = 72) fished off Tanzania were inspected for anisakid nematodes by UV-press. A subsample of 168 nematodes were identified by sequence analyses of the cox 2 mtDNA gene and ITS region of rDNA. The species A. typica (s.l.) ( N  = 166), Pseudoterranova ceticola (N = 1) and Anisakis paggiae (N = 1) were molecularly identified. Phylogenetic analysis of A. typica (s.l.) sequences based on both genes, indicated the existence of two distinct phylogenetic lineages forming two well-supported clades. The first clade comprised 12 A. typica specimens including individuals from its type locality (central Atlantic Ocean). The second clade comprising 154 specimens, clustered with reference sequences retrieved from GenBank including one apparently undescribed taxon, i.e., Anisakis sp. 1, and A. typica var. indonesiensis . The two reciprocally monophyletic clades are closely related and correspond to two distinct sister species within A. typica (s.l.), presently indicated as A. typica sp. A and A. typica sp. B. Two and four fixed alternative nucleotide substitutions (SNPs), i.e., diagnostic positions, between the two taxa, respectively, were found at the mtDNA cox 2 and the ITS region of rDNA. The genetic data, as well as their occurrence in sympatry, strengthens the hypothesis that the actual specimens represent two distinct gene pools. The occurrence of both A. typica sp. A and A. typica sp. B in the musculature of freshly examined T. lepturus and S. undosquamis, suggests that both species can migrate intra-vitam into the flesh . Although the zoonotic potential of A. typica s.l. is still unclear, the presence of these parasites in the musculature, edible part of the fish, raises health concerns for consumers., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Parasite biodiversity in cetaceans represents a neglected component of the marine ecosystem. This study aimed to investigate the distribution and genetic diversity of anisakid nematodes of the genus Anisakis sampled in cetaceans from the Northeast Atlantic Ocean and the Mediterranean Sea. A total of 478 adults and pre-adults of Anisakis spp. was identified by a multilocus genetic approach (mtDNA cox2, EF1 α - 1 nDNA and nas 10 nDNA gene loci) from 11 cetacean species. A clear pattern of host preference was observed for Anisakis spp. at cetacean family level: A. simplex (s.s.) and A. pegreffii infected mainly delphinids; A. physeteris and A. brevispiculata were present only in physeterids, and A. ziphidarum occurred in ziphiids. The role of cetacean host populations from different waters in shaping the population genetic structure of A. simplex (s.s.), A. pegreffii and A. physeteris was investigated for the first time. Significant genetic sub-structuring was found in A. simplex (s.s.) populations of the Norwegian Sea and the North Sea compared to those of the Iberian Atlantic, as well as in A. pegreffii populations of the Adriatic and the Tyrrhenian Seas compared to those of the Iberian Atlantic waters. Substantial genetic homogeneity was detected in the Mediterranean Sea population of A. physeteris. This study highlights a strong preference by some Anisakis spp. for certain cetacean species or families. Information about anisakid biodiversity in their cetacean definitive hosts, which are apex predators of marine ecosystems, acquires particular importance for conservation measures in the context of global climate change phenomena., (© 2022. The Author(s).)

Objectives: Anisakis pegreffii is a zoonotic parasite requiring marine organisms to complete its life-history. Human infection (anisakiasis) occurs when the third stage larvae (L3) are accidentally ingested with raw or undercooked infected fish or squids. A new de novo transcriptome of A. pegreffii was here generated aiming to provide a robust bulk of data to be used for a comprehensive "ready-to-use" resource for detecting functional studies on genes and gene products of A. pegreffii involved in the molecular mechanisms of parasite-host interaction., Data Description: A RNA-seq library of A. pegreffii L3 was here newly generated by using Illumina TruSeq platform. It was combined with other five RNA-seq datasets previously gathered from L3 of the same species stored in SRA of NCBI. The final dataset was analyzed by launching three assembler programs and two validation tools. The use of a robust pipeline produced a high-confidence protein-coding transcriptome of A. pegreffii. These data represent a more robust and complete transcriptome of this species with respect to the actually existing resources. This is of importance for understanding the involved adaptive and immunomodulatory genes implicated in the "cross talk" between the parasite and its hosts, including the accidental one (humans)., (© 2022. The Author(s).)

The cestode family Gymnorhynchidae Dollfus, 1935 (Trypanorhyncha) comprises three genera and six valid species that are typically intestinal parasites of large pelagic sharks. Members of Gymnorhynchidae show a wide geographic distribution and represent a global sanitary concern because as larvae they infect the edible flesh of several commercially important fishes with some species having allergenic potential. Larval Gymnorhynchidae collected from the muscles of the Atlantic pomfret Brama brama from various localities in the Mediterranean Sea were identified and characterized by combining traditional morphology, scanning electronic microscopy, and molecular analyses using newly generated nuclear 18S and 28S rRNA sequences. Overall, 98 larvae were collected from 20 (100%) Atlantic pomfrets (intensity of infection: 4.9; range: 1-12). High-quality sequences were obtained for 54 larvae. Of these, 11 and 43 larvae were identified as Molicola uncinatus and Gymnorhynchus gigas , respectively. The phylogenetic analysis showed the existence of three main clades within Gymnorhynchidae. The first included species of G. gigas and M. uncinatus from the Mediterranean and Atlantic; the second and third major clades included an unidentified species of Molicola from the Indian Ocean and specimens of Gymnorhynchus isuri from the Mediterranean and Atlantic, respectively. Finally, Chimaerarhynchus rougetae was the basal and most diverging taxon. The phylogenetic analysis suggested that G. gigas is more closely related to the members of Molicola . We demonstrated the coinfection of M. uncinatus and G. gigas from all localities studied and extended the intermediate hosts and geographical range of M. uncinatus by including the Atlantic pomfret and the Tyrrhenian and Ionian Seas, respectively. The present results supported the previous proposal that G. gigas and Molicola species should be included in the same genus. Our study demonstrated the usefulness of the integrative taxonomy for the unequivocal recognition of larval trypanorhynch species, resolving the current difficulties in the taxonomy, and elucidating the poorly known ecological and biological aspects of members of Gymnorhynchidae., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Santoro, Palomba, Aco Alburqueque and Mattiucci.)

Parasitic ascaridoid nematodes occur in a wide range of marine organisms across the globe. Some species of the anisakid family (Ascaridoidea: Anisakidae) can cause gastrointestinal disease in humans (i. e. anisakidosis). Despite their importance as potentially hazardous parasites, the occurrence and infection characteristics of ascaridoids are still poorly known from many host species and geographical areas. This study investigated the diversity and infection levels of ascaridoid parasites in various commercial fish and squid host species off Bangladesh. Fish and squid specimens were visually inspected for nematodes using the UV-press method. Nematodes were assigned to genus level based on morphology and identified by sequence analyses of the entire ITS region and partial 28S rDNA and mtDNA cox2 genes. Third-stage larvae (L3) of Anisakis typica occurred at low prevalence ( P  = 10% and 8%, respectively) in the viscera of Selar crumenophthalmus and Trichiurus lepturus , while Hysterothylacium amoyense occurred in the viscera of Sardinella fimbriata ( P  = 1%) and the viscera and muscle of Harpadon nehereus ( P  = 32%) and T. lepturus ( P  = 76%). Lappetascaris sp. Type A L3 occurred in the mantle of the squid Uroteuthis duvaucelii ( P  = 11%). Anisakis and Lappetascaris species, and H. amoyense were firstly identified in the Bay of Bengal. The potentially zoonotic A. typica was only found in fish viscera. Hysterothylacium amoyense and Lappetascaris sp., both generally regarded as non-zoonotic, occurred at low prevalence in the muscle or mantle of fish or squid, respectively. Since consumption of raw or lightly processed seafood seems to be rare in Bangladesh, the risk of acquiring anisakidosis from consuming fishery products from off Bangladesh appears to be low. Due to its reddish appearance, the visual presence of H. amoyense larvae in fish flesh may represent a food quality issue., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 Published by Elsevier Inc. on behalf of International Association of Food and Waterborne Parasitology.)

Post-mortem examination of a fin whale Balaenoptera physalus stranded in the Mediterranean Sea led to the finding of Bolbosoma balaenae for the first time in this basin. In this work, we describe new structural characteristics of this parasite using light microscopy and scanning electron microscopy approaches. Moreover, the molecular and phylogenetic data as inferred from both ribosomal RNA 18S-28S and the mitochondrial DNA cytochrome oxidase c subunit 1 (cox1) for adult specimens of B. balaenae are also reported for the first time. Details of the surface topography such as proboscis's hooks, trunked trunk spines of the prebulbar foretrunk, ultrastructure of proboscis's hooks and micropores of the tegument are shown. The 18S + 28S rRNA Bayesian tree (BI) as inferred from the phylogenetic analysis showed poorly resolved relationships among the species of Bolbosoma. In contrast, the combined 18S + 28S + mtDNA cox1 BI tree topology showed that the present sequences clustered with the species of Bolbosoma in a well-supported clade. The comparison of cox1 and 18S sequences revealed that the present specimens are conspecific with the cystacanths of B. balaenae previously collected in the euphausiid Nyctiphanes couchii from the North Eastern Atlantic Ocean. This study provided taxonomic, molecular and phylogenetic data that allow for a better characterization of this poor known parasite.

Blastocystis is a common intestinal protist distributed worldwide, infecting humans and a wide range of domestic and wild animals. It exhibits an extensive genetic diversity and, so far, 25 distinct small subunit ribosomal RNA ( SSU rRNA) lineages termed subtypes (STs)) have been characterized; among them, 12 have thus far been reported in humans. The aims of the present study were to detect and genetically characterize Blastocystis sp. in synantropic animals to improve our current knowledge on the distribution and zoonotic transmission of Blastocystis STs in Italy. Samples were collected from N = 193 farmed animals and submitted to DNA extraction and PCR amplification of the SSU rRNA. Blastocystis was detected in 60 samples (31.08%) and successfully subtyped. Phylogenetic analysis evidenced that the isolates from fallow deer, goats, and pigs ( N = 9) clustered within the ST5; those from pheasants ( N = 2) in the ST6; those from chickens ( N = 8) in the ST7; those from sheep ( N = 6) in the ST10; and those from water buffaloes ( N = 9) in the ST14 clade. The comparison between the present isolates from animals and those previously detected in humans in Italy suggested the animal-to-human spillover for ST6 and ST7. The present study represents the widest Blastocystis survey performed thus far in farmed animals in Italy. Further epidemiological studies using molecular approaches are required to determine the occurrence and distribution of Blastocystis STs in other potential animal reservoirs in Italy and to define the pathways of zoonotic transmission.

The southern Pacific Ocean, off the New Zealand coast, has been reported as one sympatric area of the two parasite species Anisakis pegreffii and A. berlandi. Here, a multilocus genotyping approach, based on a panel of eleven DNA microsatellite (SSR) loci plus the sequences analysis of the nuclear nas10 nDNA and the mitochondrial mtDNA cox2 gene loci, was applied to a total of N = 344 adults and larvae of Anisakis spp. from cetacean and fish species, respectively. Out of the newly scored SSR loci, Anisl 15 and Anisl 2 showed fixed alternative alleles between A. pegreffii and A. berlandi resulting as 100% diagnostic loci. Out of SSRs Anisl 00314 and Anisl 7 previously disclosed, two additional loci, i.e., Anisl 4 and Anisl 22, were found to be sex-linked. The Bayesian genotypes clustering approach (STRUCTURE) allowed identification, with a 100% of probability value, N = 208 specimens to the "pure parental" A. pegreffii, N = 133 to the "pure parental" A. berlandi, while one adult and two larval stages showed mixed ancestry between the two groups having, in all cases, a Q-value = 0.50. NEWHYBRIDS analysis assigned (100% of probability) those specimens to their F1 hybrid category. This represents the first evidence of contemporary hybridization between the two parasite species in a sympatric area. The pairwise F ST values estimated at intraspecific and interspecific level, inferred from both SSR loci and mitochondrial mtDNA cox2 sequences, have also demonstrated the existence of two distinct panmictic units in this study area, corresponding respectively to A. pegreffii and A. berlandi. The results obtained support the useful application of a multilocus approach in the identification of sibling species and their hybrid categories in sympatric areas. The possible use of sex-linked SSR loci of the two species of the A. simplex (s. l.), for sex determination of their larval stages, is also suggested., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)