Agarose Hydrogel-Boosted One-Tube RPA-CRISPR/Cas12a Assay for Robust Point-of-Care Detection of Zoonotic Nematode Anisakis.

Rapid and accurate detection of the zoonotic nematode Anisakis is poised to control its epidemic. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-associated assay shows great potential in the detection of pathogenic microorganisms. The one-tube method integrated the CRISPR system with the recombinase polymerase amplification (RPA) system to avoid the risk of aerosol pollution; however, it suffers from low sensitivity due to the incompatibility of the two systems and additional manual operations. Therefore, in the present study, the agarose hydrogel boosted one-tube RPA-CRISPR/Cas12a assay was constructed by adding the CRISPR system to the agarose hydrogel, which avoided the initially low amplification efficiency of RPA caused by the cleavage of Cas12a and achieved reaction continuity. The sensitivity was 10-fold higher than that of the one-tube RPA-CRISPR/Cas12a system. This method was used for Anisakis detection within 80 min from the sample to result, achieving point-of-care testing (POCT) through a smartphone and a portable device. This study provided a novel toolbox for POCT with significant application value in preventing Anisakis infection.

A novel rhabdovirus detected in Anisakis larvae distributed in the coastal areas of Japan: Viral genome analysis and possible coevolutionary relationship between virus and host nematodes.

In the last decade, it has become evident that various RNA viruses infect helminths including Order Ascaridida. However, there is still no information available for viruses infecting Anisakis. We herewith demonstrate the presence of a novel rhabdovirus from Anisakis larvae detected by next-generation sequencing analysis and following RT-PCR. We determined the nearly all nucleotide sequence (12,376 nucleotides) of the viral genome composed of seven open reading frames, and we designated the virus as Suzukana rhabdo-like virus (SkRV). BLASTx search indicated that SkRV is a novel virus belonging to the subfamily Betanemrhavirus, rhabdovirus infecting parasitic nematodes of the Order Ascaridida. SkRV sequence was detectable only in the total RNA but not in the genomic DNA of Anisakis, ruling out the possibility of SkRV being an endogenous viral element incorporated into the host genomic DNA. When we individually tested Anisakis larvae obtained from Scomber japonicus migrating in the coastal waters of Japan, not all but around 40% were SkRV-positive. In the phylogenetic trees of Betanemrhavirus and of the host Ascaridida nematodes, we observed that evolutional distances of viruses were, to some extent, parallel with that of host nematodes, suggesting that viral evolution could have been correlated with evolution of the host. Although biological significance of SkRV on Anisakis larvae is still remained unknown, it is interesting if SkRV were somehow related to the pathogenesis of anisakiasis, because it is important matter of public health in Japan and European countries consuming raw marine fishes.

Prevalence of Anisakis larvae in cultured mackerel Scomber japonicas in Japan and the relationship between the intensity of Anisakis infection in cultured mackerel and fish fatness.

Incidences of food poisoning caused by Anisakis have increased in Japan, and a significant number of anisakiasis cases in Tokyo attributed to the consumption of mackerel (Scomber japonicus) have been reported. There are two types of cultured mackerel in Japan: those cultured fully from controlled parent fish eggs and those cultured from wild juveniles collected from the sea. In this study, we aimed to investigate the prevalence of Anisakis larvae in cultured mackerel (184 fish) in 15 products and identified the species using molecular analysis to evaluate the risk of food poisoning. In total, 1567 Anisakis larvae were detected in 70 of 130 mackerel in 10 products; however, Anisakis larvae were not detected in 54 mackerel using artificially reared juveniles in 5 products. Moreover, 277 larvae were detected in fish muscle, and 98.6 % (273/277 larvae) were molecularly identified as Anisakis simplex sensu stricto (A. simplex). Conversely, 1043 Anisakis pegreffii larvae were identified genetically and/or morphologically but only 2 larvae were identified in the muscle. There was no significant relationship between the host coefficient of fatness and the infection intensity of Anisakis larvae in individual fish (Spearman's rank correlation coefficient test, P > 0.05). Based on the results of the analysis of the cytochrome c oxidase subunit2 (cox2) gene of A. simplex and A. pegreffii detected in this study, we attempted to estimate the catch area of the juveniles (Pacific stock and Tsushima Warm Current stock). The clusters on the phylogenetic tree of the cox2 gene of A. pegreffii from the mackerel presumed to be the two above mentioned geographic distributions were not separated and these geographic origins could not be estimated. This study revealed that mackerel cultured using wild juveniles are likely to be contaminated with Anisakis larvae, which can be detected not only in the visceral organs, but also in the muscle. Anisakis infection in cultured mackerel did not influence fish growth and evaluating the intensity of Anisakis based on the fatness level of the mackerel was complicated. To prevent anisakiasis caused by the consumption of mackerel cultured using wild juveniles, it is important to steadily control Anisakis through heating and freezing.

Phylogenetic and phylogeographic analyses of Anisakis simplex sensu stricto (Nematoda: Anisakidae) from the common minke whale in Korean waters.

The genus Anisakis is among the most significant parasites to public health, as it causes anisakiasis, a parasitic infection in humans resulting from consuming raw or undercooked seafood. Although the infection status of i>Anisakis in second intermediate hosts, such as marine fishes and cephalopods, and humans have been severally reported in Korea, no information about the definitive host in Korean waters is available. In 2014, 2 adult gastric nematodes were collected from a common minke whale (Balaenoptera acutorostrata) found in the East Sea, Korea. These worms were identified as A. simplex sensu stricto (s.s.) by comparing the mitochondrial COX2 marker with previously deposited sequences. Phylogenetic and phylogeographic analyses of A. simplex (s.s.) worldwide revealed 2 distinct populations: the Pacific population and the European waters population. This is the first report on adult i>Anisakis and its definitive host species in Korea. Further studies on Anisakis infection in other cetacean species and marine mammals in Korean seas are warrantedi>Anisakis.

A case of hepatic anisakidosis caused by Anisakis pegreffii mimicking liver cancer.

Extra-gastrointestinal anisakidosis is rare. We herein report an Anisakis pegreffii infection in a patient with hepatic anisakidosis diagnosed based on its molecular identification. A 71-year-old male patient had a hepatic tumor presenting as a low-density area of 20 mm in diameter in segment 6 of the liver on abdominal ultrasonography, computed tomography, and magnetic resonance imaging. The surgically resected pathological specimen revealed a necrotizing eosinophilic granuloma containing nematode larvae, possibly an Anisakis larva. Molecular and phylogenetic analysis demonstrated Anisakis larvae belonging to A. pegreffii. The present results will help identify and characterize unknown Anisakis species in histological sections.

Proteomic characterization of extracellular vesicles released by third stage larvae of the zoonotic parasite Anisakis pegreffii (Nematoda: Anisakidae).

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.

Prevalence and abundance of Anisakis larvae in ready-to-eat mackerel products in Japan.

The risk of contracting anisakiasis from consuming ready-to-eat (RTE) mackerel products in Japan was investigated by examining the prevalence and abundance of Anisakis simplex and its sibling species in these products. From 2019 to 2021, a total of 448 RTE mackerel products were purchased in Japan. Anisakis larvae were isolated from 244 of the 448 samples (54 %), and live larvae were isolated from 161 of the 448 samples (36 %). In total, 3170 Anisakis larvae, which included 919 live larvae, were isolated. The isolated Anisakis larvae consisted of 3118 A. simplex (s. s.), 27 A. pegreffii, and 25 hybrid genotype (A. simplex [s. s.] × A. pegreffii) larvae. No A. berlandi larvae were isolated. The prevalence of larvae in samples of mackerel caught in the Southern Japan region and Sea of Japan was much lower than that in mackerel caught in other areas. Both the prevalence of Anisakis larvae in all samples and their abundance in larvae-positive samples exhibited specific seasonal variations, being high in spring.

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.

Molecular identification and infection levels of Anisakis species (Nematoda: Anisakidae) in the red scorpionfish Scorpaena scrofa (Scorpaenidae) from the Aegean Sea.

The red scorpionfish Scorpaena scrofa (Scorpaenidae) is a high commercial value marine fish species along the Mediterranean coasts. Anisakiasis is a fish-borne parasitic zoonoses caused by Anisakis larvae in consumers. To date, there are only a few epidemiological studies on the presence and molecular identification of Anisakis larvae infecting S. scrofa. A total of 272 S. scrofa captured from the Gulf of Izmir in the Turkish Aegean coasts (FAO 37.3.1) were examined for Anisakis larvae between March 2019 and March 2020. The prevalence, mean intensity and mean abundance of Anisakis larvae were 9.6% (95% CI 6.5-13.7%), 2.8 (95% CI 1.88-5.19), and 0.27 (95% CI 0.15-0.56), respectively. All Anisakis larvae were collected from the viscera and body cavity of S. scrofa. Anisakis pegreffii, A. typica, and A. ziphidarum were genetically identified by RFLP analysis of the ITS region. These species were also confirmed by cox2 sequence analysis. A weak positive and statistically significant correlation between the total length (ρS 0.204; p = 0.001) and total weight (ρS 0.200; p = 0.001) of S. scrofa and the number of Anisakis larvae was observed. This survey presents the first molecular detection of A. typica and A. ziphidarum in S. scrofa. Thus, this fish species is a new host for A. typica and A. ziphidarum. This is also the first report of the presence of A. ziphidarum in the Aegean Sea.

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%.

Anisakiasis Annual Incidence and Causative Species, Japan, 2018-2019.

Using data from 2018-2019 health insurance claims, we estimated the average annual incidence of anisakiasis in Japan to be 19,737 cases. Molecular identification of larvae revealed that most (88.4%) patients were infected with the species Anisakis simplex sensu stricto. Further insights into the pathogenesis of various anisakiasis forms are needed.

Distribution and genetic diversity of Anisakis spp. in cetaceans from the Northeast Atlantic Ocean and the Mediterranean Sea.

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.

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.

Intraspecific Genetic Variation of Anisakis typica in Indian Mackerel Caught from the Gulf of Thailand, Samut Songkhram Province.

Anisakis nematodes infecting Indian mackerel (Rastrelliger kanagurta) were initially discovered in Thailand in our preliminary investigation. Nevertheless, the species of Anisakis collected has not been determined nor has its genetic variation been researched. Thus, this study aimed to molecularly identify the species of Anisakis specimens using the internal transcribed spacer (ITS) region of ribosomal DNA sequences. In addition, the intraspecific genetic variation was also determined using mitochondrial cytochrome oxidase subunit II (COII) gene sequences. The phylogenetic relationships of the ITS region classified all samples into Anisakis typica; however, the genetic variation between them could not be distinguished. By contrast, the phylogenetic tree analysis of the COII region identified all samples as A. typica, with 17 different haplotypes by 66 polymorphic sites and five of the substitutions resulted in amino acid change. Additionally, the distribution pattern of the COII region can be separated into two groups between South America and Asian countries. All our haplotypes belong to Asian countries. Compared with the two genetic markers used in this investigation, COII appears to be a better candidate for studying genetic variation sensitive to environmental changes and intermediate or definitive host behavioral changes.

Anisakis spp, DNA detection in paraffin-embedded tissue biopsies recovered from patients with gastritis using real-time PCR in Bushehr, Persian Gulf, Iran.

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.

Multigene phylogenetic analysis reveals non-monophyly of Anisakis s.l. and Pseudoterranova (Nematoda: Anisakidae).

The nematode genera Anisakis s.l. and Pseudoterranova (Anisakidae) include causative agents of anisakiasis and pseudoterranovosis, parasitic diseases resulting from eating undercooked or raw fish or squid. Species in both genera have thus attracted considerable attention especially in public health and taxonomic studies. The phylogenetic relationships of these genera within the subfamily Anisakinae, however, remain to be investigated with dense taxonomic sampling. In this study, we collected an anisakid third-stage larva, and identified it morphologically and molecularly as Pseudoterranova ceticola. Phylogeny of 15 anisakine species, including the newly collected specimen of Ps. ceticola, was reconstructed based on sequences of three mitochondrial (cox1, cox2, and 12S rRNA) and two nuclear (ITS and 28S rRNA) regions. The obtained tree suggested the non-monophyly of Anisakis s.l. and Pseudoterranova. Anisakis s.l. was divided into two groups, which are distinguished from each other by the shape of the ventriculus. Based on phylogenetic relationships and morphology, three species with a shorter ventriculus ("A." brevispiculata, "A." paggiae, and "A." physeteris) were assigned to the genus Skrjabinisakis, as recently proposed. Pseudoterranova ceticola was distantly related to the monophyletic Ps. decipiens species complex. Although the phylogenetic position of the type species Ps. kogiae has not been investigated due to a lack of sequence data, this species may morphologically and ecologically resemble Ps. ceticola, inferring a close kinship between the two species.

A miRNAs catalogue from third-stage larvae and extracellular vesicles of Anisakis pegreffii provides new clues for host-parasite interplay.

Anisakids are widespread marine parasites of medical, veterinary and economic relevance. They infect marine natural hosts but humans can accidentally acquire the fish-borne zoonosis anisakiasis by ingesting infected raw fishes or mollusks. Among the several species described, Anisakis pegreffii is one of the main etiological agent of the disease, in particular in the Mediterranean area. Despite the growing evidence of miRNAs involvement in host-parasite interplay, and the emerging role of exosomal microvesicles in shuttling them between different cell types (and sometime across species), no information on miRNAs from any Anisakis species is presently available. In this study we isolated extracellular vesicles (EVs) released by Anisakis pegreffii infective third-stage larvae (L3) and analyzed by RNA-seq small RNAs from both L3 and EVs. We showed by nanoparticle tracking analysis that L3 release in culture medium particles of size compatible with the one of extracellular vesicles. A catalogue of 156 miRNAs from A. pegreffii was compiled by sequence comparison to evolutionary close species and miRNA prediction software. Using differential expression analysis, we identified a small number of highly abundant miRNAs in larvae and extracellular vesicles fractions whose potential biological relevance may deserve future investigation. Finally, A. pegreffii miRNAs were compared to those described in other parasitic helminths and predicted targets among human genes were searched, suggesting their potential involvement during infection.

De novo transcriptome assembly and annotation of the third stage larvae of the zoonotic parasite Anisakis pegreffii.

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).

Molecular Diagnosis of Pseudoterranova decipiens Sensu Stricto Infections, South Korea, 2002‒2020.

Human Pseudoterranova decipiens larval infections were diagnosed by molecular analysis of mitochondrial cox1 and nd1 genes in 12 health check-up patients in South Korea during 2002-2020. Based on high genetic identity (99.3%-100% for cox1 and 96.7%-98.0% for nd1), we identified all 12 larvae as P. decipiens sensu stricto.

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.