Ingestion of fishing gear and Anisakis sp. infection in a beached Indo-Pacific finless porpoise (Neophocaena phocaenoides) in the Jeju Island, Republic of Korea: findings from post-mortem computed tomography and necropsy.

BACKGROUND: Human fishing activities have significantly affect environmental concern for marine ecosystems, conservation of marine mammals, and human health. Coastal cetaceans are highly vulnerable to ingestion of fishing gear, bycatching, or entanglement, all of which can be fatal for these animals. In particular, certain coastal dolphins and porpoises are heavily impacted by fishing gear such as angling gear or stownet, as their food often overlap with the target fish species of human fisheries. CASE PRESENTATION: This study presents a case of an Indo-Pacific finless porpoise (Neophocaena phocaenoides) beached on the coast of Jeju Island, Republic of Korea, with ingestion of fishing gear and severe Anisakis infection. Although this species inhabits waters ranging from the Persian Gulf to Taiwan, several stranded carcasses have been reported on Jeju Island in recent years. Post-mortem computed tomography revealed a bundle of four fishing hooks in the forestomach, along with nylon lines and steel lines with connectors, which were assumed to be angling gear for Jeju hairtail (Trichiurus lepturus). Further necroscopic investigation revealed that the forestomach contained a large number of Anisakis spp. (Nematoda: Anisakidae). Histological examination revealed a thickened forestomach wall with pinpoint and volcanic ulcerations, a thickened layer of stratified squamous epithelium, and infiltrated stroma in the squamous epithelium. CONCLUSIONS: This study emphasizes the urgent need to address the impact of fishing activities on marine mammals, marine litter pollution, and the bycatch problem in Korean seawater. In addition, the occurrence of N. phocaenoides in seawater around Jeju Island should be raised in future geographical ecology or veterinary pathology studies and when its distribution is updated.

The response of Anisakis simplex (s. s.) to anthelmintics - Specific changes in xenobiotic metabolic processes.

Anisakiasis is a parasitic disease transmitted through the consumption of raw or undercooked fish and cephalopods that are infected with larvae of Anisakis simplex (sensu stricto) or Anisakis pegreffii. The purpose of this study was to investigate how A. simplex (s. s.) responds to the influence of anthelmintics such as ivermectin (IVM) and pyrantel (PYR). In vitro experiments were conducted using larvae at two developmental stages of A. simplex (s. s.) (L3 and L4) obtained from Baltic herring (Clupea harengus membras). Larvae were cultured with different concentrations of IVM or PYR (1.56, 3.125, and 6.25 µg/mL) for various durations (3, 6, 9, and 12 h) under anaerobic conditions (37 °C, 5% CO2). The gene expression of actin, ABC transporter, antioxidant enzymes, γ-aminobutyric acid receptors, and nicotinic acetylcholine receptors, as well as the oxidative status were analyzed. The results showed that A. simplex (s. s.) L3 stage had lower mobility when cultured with PYR compared to IVM. The analysis of relative gene expression revealed significant differences in the mRNA level of ABC transporters after treatment with IVM and PYR, compared to the control group. Similar patterns were observed in the gene expression of antioxidant enzymes in response to both drugs. Furthermore, the total antioxidant capacity (TAC) and glutathione S-transferase (GST) activity were higher in the treatment groups than in the control group. These findings suggest a relationship between the expression of the studied genes, including those related to oxidative metabolism, and the effectiveness of the tested drugs.

Variation in the levels of anisakid infection in the European anchovy Engraulis encrasicolus (Linnaeus) from the Bay of Biscay during the period 2000-2023 (ICES Subarea 8).

The European anchovy Engraulis encrasicolus is one of the most important commercial species in the Bay of Biscay (ICES Subarea 8), and our analysis focused on the analysis of the temporal mean abundance, prevalence, and intensity of Anisakis spp. larvae species in anchovies from ICES Subarea 8 in the years 2000, 2001, 2014-2016, and 2019-2023. Prevalence in adult individuals of anchovy was only 1% in 2000 but increased to 90% in 2014. Since 2015, the prevalence has decreased, and the number of individuals affected in 2023 accounted for 17.6%. The mean abundance showed a similar trend, with a peak of 3.79 nematodes/anchovy in 2014, falling to 0.21 in 2023. The species A. simplex sensu stricto and A. pegreffii were identified by PCR/SANGER sequencing and PCR/RLFP techniques in 2019 and 2020. Anisakis simplex (s.s.) was the most abundant species and, according to the results returned by these two techniques, it accounted for an average of 62.4% and 52.1% of total nematodes in 2019 and 2020, respectively. The results of studies monitoring infection levels in anchovies showed that the mean abundance and prevalence changed over the course of the study period and that the proportion of different species of Anisakis is also subject to variation from year to year.

Zoonotic Marine Nematode Infection of Fish Products in Landlocked Country, Slovakia.

Fish products in Slovakia have been heavily infected with Anisakis spp. larvae, which causes human anisakiasis. We found larvae in all tested samples of frozen Atlantic herring. Anisakid allergen t-Ani s7 testing revealed 2 positive cases in humans, signaling need for health authorities to closely monitor zoonotic marine parasites, even in inland areas.

Morphological and molecular identification of third-stage larvae of Anisakis typica (Nematoda: Anisakidae) from Red Sea coral trout, Plectropomus areolatus.

Anisakidosis is a foodborne zoonotic infection induced by members of the family Anisakidae via the consumption of raw or undercooked fish such as sushi and sashimi. Identifying anisakid larval species is critical for the epidemiology and diagnosis of diseases caused by them. This study aimed at identifying Anisakis larvae collected from marine fish in Egyptian waters based on morphological characteristics and molecular analysis. Thirty marine fish coral trout, Plectropomus areolatus, were collected from Hurghada, Red Sea, Egypt, to investigate larval nematodes of the genus Anisakis. The larvae were detected encapsulated in the peritoneal cavity and muscle of the fish host. This examination revealed that anisakid larvae naturally infected 19 fish specimens with a prevalence of 63.33% and a mean intensity of 4.1 ± 0.40. Most of them (68 larvae: 71.57%) were found in the musculature. Morphological and morphometric analyses using light and scanning electron microscopy revealed a head region with a prominent boring tooth, inconspicuous lips, and a characteristic protruded cylindrical mucron. All larvae in this study possessed the same morphology as Anisakis Larval type I. Molecular analysis based on ITS region using maximum likelihood and Bayesian phylogenetic methods confirmed them as Anisakis typica. This is the first study to identify A. typica larvae from the commercial fish coral trout P. areolatus in Egyptian waters using morphological and molecular methods.

Anisakid parasites (Nematoda: Anisakidae) in 3 commercially important gadid fish species from the southern Barents Sea, with emphasis on key infection drivers and spatial distribution within the hosts.

Northeast Arctic cod, saithe and haddock are among the most important fisheries resources in Europe, largely shipped to various continental markets. The present study aimed to map the presence and distribution of larvae of parasitic nematodes in the Anisakidae family which are of socioeconomic and public health concern. Fishes were sourced from commercial catches during winter or spring in the southern Barents Sea. Samples of fish were inspected for nematodes using the UV-press method while anisakid species identification relied on sequencing of the mtDNA cox2 gene. Anisakis simplex (s.s.) was the most prevalent and abundant anisakid recorded, occurring at high infection levels in the viscera and flesh of cod and saithe, while being less abundant in haddock. Contracaecum osculatum (s.l.) larvae, not found in the fish flesh, showed moderate-to-high prevalence in saithe, haddock and cod, respectively. Most Pseudoterranova spp. larvae occurred at low-to-moderate prevalence, and low abundance, in the viscera (Pseudoterranova bulbosa) and flesh (Pseudoterranova decipiens (s.s.) and Pseudoterranova krabbei) of cod, only 2 P. decipiens (s.s.) appeared in the flesh of saithe. Body length was the single most important host-related factor to predict overall abundance of anisakid larvae in the fish species. The spatial distribution of Anisakis larvae in the fish flesh showed much higher abundances in the belly flaps than in the dorsal fillet parts. Trimming of the flesh by removing the belly flaps would reduce larval presence in the fillets of these gadid fish species by 86­91%.

Analysis of potential drivers of spatial and temporal changes in anisakid larvae infection levels in European hake, Merluccius merluccius (L.), from the North-East Atlantic fishing grounds.

We analysed the spatial and temporal variability of Anisakis larvae infection in hake (Merluccius merluccius) from the North-East Atlantic from 1998 to 2020 and the potential drivers (i.e., environmental and host abundance) of such variation. The results showed that hake from separate sea areas in the North Atlantic have marked differences in temporal abundance levels. Hake larger than 60 cm were all parasitized in all ICES (International Council for the Exploration of the Sea) subareas 6, 7, and 8. The belly flaps were the most parasitized parts of the flesh, accounting for 92% of the total. Individuals of Anisakis simplex, Anisakis pegreffii, Anisakis spp. and a hybrid of Anisakis simplex × pegreffii were genetically identified, and Anisakis simplex as the most abundant (88-100%). An ecological niche model of Anisakis occurrence in fishes in the NE Atlantic was built to define the thermal optimum and environmental ranges for salinity, depth, chlorophyll concentration, and diffuse attenuation. The temporal variability of anisakid infection in fishes in the last two decades indicated an increase in the NE Atlantic at an annual rate of 31.7 nematodes per total number of specimens examined per year. This rise in infection levels could be triggered by the increase in intermediate host fish stocks, especially hake in the area.

Prevalence and molecular identification of zoonotic Anisakis and Pseudoterranova species in fish destined to human consumption in Chile.

Zoonotic larvae of the family Anisakidae found in several fish species represent a serious risk in public health since they may cause food-borne anisakidosis in humans. Chile has culinary preferences including eating raw fish in many traditional preparations. In the present study, a total of 180 fish specimens representing three different fish species, i.e., Chilean hake (Merluccius gayi), snoek (Thyrsites atun), and sea bream (Brama australis), were caught at central coast of Chile. Parasitological examination was performed on musculature and abdominal cavity for subsequent extraction and quantification of anisakid larvae. Estimation of infection parameters, such as prevalence, was performed indicating 100% (CI: 0.94-1.0) prevalence of anisakid L3 in Chilean hakes and snoeks. Moreover, sea breams reached a prevalence of 35% (CI: 0.23-0.48). Prevalence of anisakid larvae in muscle was also analyzed showing values of 18.6% (CI: 0.097-0.309) in Chilean hakes, 15% (CI: 0.07-0.26) in snoeks, and 1.7% (CI: 0-0.089) in sea breams. Meanwhile, prevalence of anisakid larvae in internal organs showed highest values for peritoneum (100% and 83.3%) for snoeks and Chilean hakes, respectively, for liver (96.7%) and gonads (86.6%) in Chilean hakes, and for intestine (98.3%) in snoeks. Molecular analysis of collected anisakid L3 unveiled presence of two potentially zoonotic nematode species, i.e., Pseudoterranova cattani and Anisakis pegreffii. P. cattani was found in Chilean hakes and snoeks being the first molecular host species report for Chilean snoeks. Besides, A. pegreffii was also identified in these species being the first molecular report on this regard. These findings are relevant for better understanding of epidemiology of anisakiasis in Chilean coasts and for public health issues considering potential risk of human population due to its culinary preferences in eating raw fish.

Demographic history and population genetic structure of Anisakis pegreffii in the cutlassfish Trichiurus japonicus along the coast of mainland China and Taiwan.

Studying the genetic diversity of nematode parasite populations is crucial to gaining insight into parasite infection dynamics and informing parasite phylogeography. Anisakiasis is a zoonotic disease caused by the consumption of infectious third-stage larvae (L3) of Anisakis spp. carried by marine fish. In the present study, a total of 206 mitochondrial DNA sequences (cytochrome c oxidase 2, cox2) were used to study the genetic diversity, genetic structure, and historical demography of twelve A. pegreffii populations from Trichiurus japonicas along the coast of mainland China and Taiwan. Two distinct evolutionary lineages of A. pegreffii and no significant genealogical structures corresponding to sampling localities suggested that isolation in the marginal seas shaped their patterns of phylogeographic distribution along the coast of mainland China and Taiwan during glaciation with lower sea levels. Furthermore, pairwise FST values and AMOVA did not indicate any significant genetic differentiation among groups with no relation to the geographic area, which might be attributed to fewer barriers to gene flow as well as large population sizes. The results of the neutrality test, mismatch distribution, and Bayesian skyline plot analyses showed that entire population underwent population expansion during the late Pleistocene. Analysis of the demographic history revealed that A. pegreffii underwent historical lineage diversification and admixture due to secondary contact based on ABC analysis. The present research represents the first definitive population structure and demographic history across sampling locations of A. pegreffii along the coast of mainland China and Taiwan.

Increasing intensities of Anisakis simplex (Rudolphi, 1809 det. Krabbe, 1878) larvae with weight and sea age in returning adult Atlantic salmon, Salmo salar L., of coastal waters of Norway.

Ninety wild Atlantic salmon, Salmo salar L., (1.5-10.3 kg) were caught in the Namsen Fjord near the mouth of River Namsen, mid-Norway, and examined for the presence and distribution of Anisakis simplex (Rudolphi, 1809 det. Krabbe, 1878) larvae by digestion of the viscera and muscles in a pepsin/HCl solution. All salmon were migrating spawners after 1-4 years of feeding in the Atlantic Ocean. All 90 Atlantic salmon had A. simplex larvae in the viscera, and all, except two, had A. simplex larvae in the musculature. The number of A. simplex larvae in each fish varied between 3 and 181, and the total mean number of nematode larvae was 44.5. The intensity of A. simplex larvae was positively correlated with increasing weight and sea age of the host. However, the proportion of larvae in the muscle fillets decreased with increasing host weight and sea age. Atlantic salmon females had more A. simplex larvae than males. In all the fish examined, 70.2% of the A. simplex larvae were found in the viscera and 29.8% in the musculature. The majority (93%) of the larvae in the musculature occurred in the hypaxial sections anterior to the anus. As A. simplex larvae commonly occur in the musculature of wild Atlantic salmon, consumption of unfrozen, raw or semi-raw musculature represents a risk for humans developing anisakiasis.

Anisakid and Raphidascaridid parasites in Trachurus trachurus: infection drivers and possible effects on the host's condition.

This study investigated the distribution of nematode larvae of Anisakidae and Raphidascarididae (genera Anisakis and Hysterothylacium) in Trachurus trachurus (Linnaeus, 1758) in the Ligurian and central-northern Tyrrhenian Seas. The relationship between the number of parasites and the length and weight parameters of the fish was assessed, and the possible effect of the parasites on the condition factor was evaluated. A total of 190 T. trachurus specimens were collected in July 2019. Parasites were found in 70 individuals. A total of 161 visible larvae were collected in the viscera. Morphological analysis revealed the presence of Anisakis spp. in 55 fish and Hysterothylacium spp. in 15 fish, while 5 fish showed coinfection with both genera. The specimens subjected to PCR (n = 67) showed that 85% of the Anisakis larvae analyzed belonged to the species A. pegreffii, while the remaining 15% belonged to hybrids of A. pegreffii-A. simplex (s.s.). A total of 58% (n = 7) of the Hysterothylacium larvae analyzed belonged to the species H. fabri, while 42% belonged to the species H. aduncum. Our results support the hypothesis that infection with these parasites does not affect the condition of the fish host analyzed, and that body size and depth are major drivers in determining infection levels with Anisakid and Raphidascaridid nematodes.

The occurrence of Anisakis spp. in Australian waters: past, present, and future trends.

As one of the world's megadiverse countries, Australian biodiversity is vital for global biodiversity. Nematodes belonging to the genus Anisakis (family Anisakidae) are an important part of this biodiversity due to their ability to be repeatedly transmitted among their intermediate hosts before reaching the top of the food pyramid. Therefore, they have a significant impact on the community structures of various ecosystems. In addition, globally, they are known to be of medical and veterinary significance. The aim of this article is to provide an update on the current knowledge about these important parasites in Australia. Since 1916, a total of 234 records of Anisakis spp. from various hosts and localities have been found in Australia. It is estimated that the occurrence of Anisakis spp. and their health impacts in at least 84, 98.5, and 95% of Australian marine mammals, fish, and water birds, respectively, have not been documented yet. The results of this study suggest Australia is perhaps home to the most diverse Anisakis fauna. Available information is dominated by reports of these parasites in fish hosts, many of them among edible fish. Given the popularity of seafood in Australia and the occurrence of infectious stages of Anisakis spp. in edible fish, all stakeholders should be made aware of the occurrence, prevalence, and survival of Anisakis spp. in seafood. Also, as more pet owners feed their pets with a variety of fish and seafood products, it is important for veterinarians to be aware of seafood transmitted Anisakis spp. in pet animals. This study also highlights several important knowledge gaps: (i) The detailed life cycle of Anisakis spp. in Australia is not known. Detecting their first intermediate hosts is important for better management of crustacean zooplankton populations in our waters. (ii) Research on Anisakis spp. in Australia has been restricted to limited taxonomical studies and should extend to other aspects of these important parasites. (iii) The capacity to identify parasite taxa to species is especially important for resolving biological diversity around Australia; however, opportunities to formally train in parasite taxonomy are rare and diminishing. There is a need to train researchers with taxonomy skills. (iv) Given the vast range of biodiversity in Australia and the broad host-specificity of Anisakis spp., particularly in the larval stages, the full range of their intermediate hosts remains unknown. (v) The health impacts of the infection of the intermediate/definitive hosts with Anisakis spp. are not fully understood. Thus, one of the important areas for future studies is investigating the pathogenicity of Anisakis spp. in affected animals. This is a crucial yet unknown factor for the conservation of some endangered species in Australia.

Genetic identification of Anisakis spp. (Nematoda: Anisakidae) from cetaceans of the Southwestern Atlantic Ocean: ecological and zoogeographical implications.

Adult Anisakis Dujardin, 1845 were found in two specimens of killer whale Orcinus orca and one specimen of franciscana Pontoporia blainvillei stranded from off the coast of Buenos Aires Province, Argentina. Genetic identification of the nematodes (N = 144) was performed by sequence analysis of the mitochondrial (mtDNA cox2) and the nuclear (nas 10 nDNA) gene loci. Anisakis pegreffii and Anisakis berlandi were detected in the two individuals of O. orca, while Anisakis typica and A. pegreffii were identified in P. blainvillei. Morphological and morphometric analysis also carried out on adult specimens of A. pegreffii and A. berlandi has allowed to underlining the usefulness of genetic/molecular markers in their recognition. This represents the first record of A. pegreffii in O. orca and P. blainvillei and of A. berlandi in O. orca. This is also the first sympatric and syntopic occurrence, as adults, of A. pegreffii and A. berlandi from the Austral Region of the Atlantic Ocean waters. These results provide insights into the knowledge of the host ranges and geographical distribution of these parasites in the basin waters of the region. Pontoporia blainvillei showed low abundance values of infection with Anisakis spp., which is the general pattern for coastal dolphins in the area, whereas O. orca harboured higher abundance of Anisakis spp. than those previously recorded among cetacean species in the Argentine Sea. Differences in the Anisakis spp. distribution and their parasitic loads, observed among the three host specimens, are discussed in relation to the oceanographic parameters, as well as to the host ecology. The usefulness of genetic/molecular markers in the recognition of adults of the sibling species A. pegreffii and A. berlandi with considerable overlapping in morphometric and morphological characters was underlined. The distribution of Anisakis species from Southwestern Atlantic waters is discussed in relation to their value as indicators for studies on the zoogeography of their hosts at a regional-scale level.

Estimation of the number of Anisakis larvae in commercial fish using a descriptive model based on real-time PCR.

BACKGROUND: Seafood parasitation by Anisakis (Anisakidae) larvae has been reported in most of the oceans and seas worldwide. The presence of these nematodes in commonly consumed fish represents a potential hazard for consumers as they can provoke gastrointestinal symptoms and allergic reactions. In the present work, the capacity of a SYBR Green qPCR protocol to quantify Anisakis larvae in commercial fish was evaluated using experimentally spiked samples with different numbers (0-50) of A. simplex third-stage larvae (L3). To verify the agreement of the obtained results, 25 naturally infected fish specimens of Atlantic blue whiting underwent a parallel visual inspection. RESULTS: The logarithmic behavior of the Cq data obtained from the experimentally spiked samples allowed the development of a descriptive mathematical model that correlates the Cq value with the number of Anisakis larvae (R2 = 0.9908, CV = 2.37%). In the commercial blue whiting specimens there was a high correlation between the results of the molecular technique and the visual inspection (R2 = 0.9912); the Bland-Altman analysis showed that 94% of the differences were within the limits of agreement (-4.98 and 6.68), indicating the reliability of the descriptive mathematical model based on the SYBR Green qPCR technique. CONCLUSION: The descriptive function presented based on the SYBR Green qPCR assay is promising as a sensitive and accurate tool for measuring the Anisakis larval load in commercial fish, with a potential application not only in the food industry but also in prevention programs for public health. © 2020 Society of Chemical Industry.

Immunohistopathological response against anisakid nematode larvae and a coccidian in Micromesistius poutassou from NE Atlantic waters.

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

World-wide prevalence of Anisakis larvae in fish and its relationship to human allergic anisakiasis: a systematic review.

The infective stage of Anisakidae nematodes responsible for allergic reactions in humans is found in a variety of edible fish and cephalopods. The identification of geographical regions that are high risk for infected seafood may help prevent allergic reactions in humans. Despite an abundance of published literature which has identified anisakid larvae in an array of edible seafood as well as scattered reports of human allergic anisakiasis, the relationship between the two has not been fully explored. Therefore, a systematic spatio-temporal study was conducted to determine the prevalence of Anisakis spp. in fish from January 2000 to August 2020 firstly to explore the relationship between fish infection and cases of allergic anisakiasis and secondly to use fish infection data to map potential allergic anisakiasis 'hot spots'. A systematic literature search for original English text articles was conducted through search engines, Web of Science, Scopus, PubMed, Science Direct and Google Scholar. Out of 3228 articles which describe anisakid infection in fish, 264 were used for data extraction. Of 904 articles describing allergic anisakiasis, 37 were used for data extraction. A qualitative summary of the extracted data was performed using equal interval method (ArcMap software) in order to compare the global distribution of Anisakis-infected fish. Of the 152-identified fish hosts, five families were most commonly infected with Anisakis spp. These included Lophiidae (86.9%), Trichiuridae (77.05%), Zeidae (70.9%), Merlucciidae (67.8%) and Gadidae (56.8%). The hot spot areas for allergic anisakiasis were North and northeast of Atlantic Ocean, southwest of USA, west of Mexico, south of Chile, east of Argentina, Norway, UK and west of Iceland (confidence 99%). The highest rate of allergic anisakiasis was in Portugal and Norway with the prevalence rate of 18.45-22.50%. Allergologists should consider allergic anisakiasis as a public health issue particularly in high-risk countries where high prevalences in fish have been demonstrated.

Occurrence of larval anisakids in horse mackerel (Trachurus trachurus) caught in Portuguese waters.

The third-stage larvae (L3) of the Anisakidae family are parasitic nematodes with zoonotic impact and are frequently encountered in the organs and musculature of various fish intended for human consumption. Since Anisakis simplex (s.s.) and A. pegreffii are the major aetiological agents of human disease, this study aims to combine the morphological and molecular data on the recovered anisakid larvae to contribute to a simplified morphological distinction of those species and conducted a survey of anisakid larvae infection in horse mackerel (Trachurus trachurus). Here, 116 horse mackerel caught in Portuguese waters were analysed for the presence of L3 of anisakids, and 3148 larvae were collected, of which only 30% were retrieved during visual inspection. As such, visual inspection does not appear to be very effective in anisakid detection. A prevalence of 84.5% of infected fish was found, and the mean intensity and mean abundance were 32.1 and 27.1 parasites per fish, respectively. The morphological and molecular analyses of 196 L3 randomly chosen from the total sample of parasites demonstrated the presence of L3 of mostly Anisakis spp., with only one L3 of Hysterothylacium aduncum. Relative frequencies of 62.9% for A. pegreffii and 37.1% for A. simplex (s.s.) were obtained. The morphometry differences between these two sibling species were evaluated, and the results demonstrated significant differences between the length of the ventriculus and the length of the oesophagus. Precisely, A. simplex (s.s.) has a longer oesophagus and ventriculus than A. pegreffii. As such, these differences may be used to distinguish the two species through morphological analysis.

Recent increase of ulcerative lesions caused by Anisakis spp. in cetaceans from the north-east Atlantic.

Species of Anisakis typically infect the stomach of cetaceans worldwide, often causing ulcerative lesions that may compromise the host's health. These nematodes also cause anisakiasis or allergic reactions in humans. To assess the risks of this emerging zoonosis, data on long-term changes in Anisakis infections in cetaceans are necessary. Here, we compare the prevalence and severity of ulcerative lesions caused by Anisakis spp. in five cetacean species stranded along the north-west Spanish coast in 2017-2018 with published data from 1991-1996. Open ulcers were found in 32/43 short-beaked common dolphins, Delphinus delphis; 3/5 striped dolphins, Stenella coeruleoalba; 1/7 bottlenose dolphins, Tursiops truncatus; and 1/3 harbour porpoises, Phocoena phocoena meridionalis; a single individual of long-finned pilot whale, Globicephala melas, was found uninfected. In common dolphins, the mean abundance of open ulcers per host was 1.1 (95% confidence interval: 0.8-1.3), with a maximum diameter (mean ± standard deviation) of 25.4 ± 16.9 mm. Stomachs with scars or extensive fibrosis putatively associated with Anisakis were detected in 14 and five animals, respectively. A molecular analysis based on the mitochondrial cytochrome c oxidase II gene using 18 worms from three cetacean species revealed single or mixed infections of Anisakis simplex sensu stricto and Anisakis pegreffii. Compared with the period 1991-1996, we found a strong increase of prevalence, abundance and extension of ulcerative lesions in most cetacean species. Anisakis populations could have increased in the study area over the last decades, although we cannot rule out that a higher environmental stress has also boosted the pathological effects of these parasites.

A new species of Contracaecum (Nematoda: Anisakidae) found parasitizing Nannopterum brasilianus (Suliformes: Phalacrocoracidae) and Hoplias argentinensis (Characiformes: Erythrinidae) in South America: morphological and molecular characterization of larval and adult stages.

Nematode species of the genus Contracaecum Railliet & Henry, 1912 have been reported around the world in many species of fish-eating birds and seals. Here, Contracaecum jorgei n. sp. is morphologically described using light and scanning electron microscopy for adults and fourth-stage larvae (L4) found in the bird Nannopterum brasilianus and third-stage larvae (L3) found in the freshwater fish Hoplias argentinensis, both from the province of Córdoba, Argentina. Additionally, sequences of cytochrome c oxidase subunit II were obtained from these specimens and molecular phylogenetic analysis was used to determine its relationships within the genus. The present species is distinguished from other species by the number and disposition of cephalic papillae; shape and size of the interlabia; length of the spicules; and number and arrangement of papillae in the posterior end of the male. Furthermore, in the molecular analyses, sequences obtained from adult L4 and L3 specimens of C. jorgei n. sp. were similar and grouped, forming an independent lineage, thus confirming it as a distinct species. Thus, morphological characteristics associated with molecular data support the proposal of a new species.

Novel polymorphic microsatellite loci in Anisakis pegreffii and A. simplex (s. s.) (Nematoda: Anisakidae): implications for species recognition and population genetic analysis.

The species of Anisakis constitute one of the most widespread groups of ascaridoid nematodes in the marine ecosystem. Three closely related taxa are recognised in the A. simplex (s. l.) complex, i.e. A. pegreffii, A. simplex (s. s.) and A. berlandi. They are distributed in populations of their intermediate/paratenic (fish and squids) and definitive (cetaceans) hosts. A panel of seven microsatellite loci (Anisl 05784, Anisl 08059, Anisl 00875, Anisl 07132, Anisl 00314, Anisl 10535 and Anisl 00185), were developed and validated on a total of N = 943 specimens of A. pegreffii and A. simplex (s. s.), collected in fish and cetacean hosts from allopatric areas within the range of distribution of these parasite species. In addition, the locus Anisl 7, previously detected in those Anisakis spp., was investigated. The parasites were first identified by sequence analysis of the EF1 α-1 nDNA. The panel of the microsatellites loci here developed have allowed to: (i) detect diagnostic microsatellite loci between the two species; (ii) identify specimens of the two species A. pegreffii, A. simplex (s. s.) in a multi-marker nuclear genotyping approach; (iii) discover two sex-linked loci in both Anisakis species and (iv) estimate levels of genetic differentiation at both the inter- and intra-specific level.