Morphological and molecular characterization of Anisakis larvae (Nematoda: Anisakidae) in Beryx splendens from Japanese waters

The third-stage (L3) larvae of Anisakis, which are the etiological agents of human anisakiasis, have been categorized morphologically into Anisakis Type I larvae and Anisakis Type II larvae. Genetic analysis has allowed easy identification of these larvae: Anisakis Type I larvae include the species Anisakis simplex sensu stricto, Anisakis pegreffii, Anisakis simplex C, Anisakis typica, Anisakis ziphidarum, and Anisakis nascettii, whereas Anisakis Type II larvae include the species Anisakis physeteris, Anisakis brevispiculata, and Anisakis paggiae. Since human consumption of raw fish and squid is common in Japan, we investigated Anisakis L3 larvae in 44 specimens of Beryx splendens from Japanese waters. A total of 730 Anisakis L3 larvae collected from B. splendens were divided morphologically into 4 types: Type I, Type II, and 2 other types that were similar to Anisakis Type III and Type IV described by Shiraki (1974). Anisakis Type II, Type III, and Type IV larvae all had a short ventriculus, but their tails were morphologically different. In addition, data from genetic analysis indicated that Anisakis Type II, Type III, and Type IV larvae could be identified as A. physeteris, A. brevispiculata, and A. paggiae, respectively. Therefore, A. physeteris, A. brevispiculata, and A. paggiae can be readily differentiated not only by genetic analysis but also by morphological characteristics of L3 larvae.     

Molecular characterization of Anisakis larvae from fish caught off Sardinia

Anisakis spp. larvae are parasitic, and potentially zoonotic, nematodes transmitted by marine fish and cephalopods, which are the main intermediate hosts of the third larval stage. The accidental consumption of infected raw or poorly cooked fish may cause gastroenteric diseases and allergies in humans. The aim of the present study was to use polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to define the occurrence, species variability, and host preferences of Anisakis spp. larvae in fish caught off the coast of Sardinia. Necropsy was used on 285 samples; 552 Anisakis spp. L3 larvae were isolated from 87 fish that tested positive for this nematode. Anisakis pegreffii was most frequently encountered (90.6%), with a primary preference for Scomber scombrus, Zeus faber, and Trachurus mediterraneus. In contrast, the prevalence of Anisakis physeteris was only 1.3%. A hybrid genotype of Anisakis simplex sensu stricto and Anisakis pegreffii was also observed, which confirms the results of previous studies carried out in the western Mediterranean. Interestingly, no Anisakis simplex s.s. larvae were recovered. These results indicate that the diversity of Anisakis species is low in Sardinia waters, probably because of its geographic position.     

Genetic markers in ribosomal DNA for the identification of members of the genus Anisakis (Nematoda: ascaridoidea) defined by polymerase-chain-reaction-based restriction fragment length polymorphism

Polymerase-chain-reaction-based restriction fragment length polymorphism analysis was performed to establish genetic markers in rDNA, for the identification of the three sibling species of the Anisakis simplex complex and morphologically differentiated Anisakis species, i.e. Anisakis physeteris, Anisakis schupakovi, Anisakis typica and Anisakis ziphidarum. Different restriction patterns were found between A. simplex sensu stricto and Anisakis pegreffii with two of the restriction endonucleases used (HinfI and TaqI), between A. simplex sensu stricto and A. simplex C with one endonuclease (HhaI), and between A. simplex C and Aniskis pegreffii with three endonucleases (HhaI, HinfI and TaqI), while no variation in patterns was detected among individuals within each species. The species A. physeteris, A. schupakovi, A. typica and A. ziphidarum were found to be different from each other and different from the three sibling species of the A. simplex complex by distinct fragments using 10-12 of the endonucleases tested. The polymorphisms obtained by restriction fragment length polymorphisms have provided a new set of genetic markers for the accurate identification of sibling species and morphospecies.     

Genetic identification of Anisakis larvae in European hake from Atlantic and Mediterranean waters for stock recognition

The occurrence of seven species of the larval parasitic nematode Anisakis , which can be used as a biological tag for hake Merluccius merluccius stocks throughout their geographical range, is reported. Hake were collected from 14 localities in the Mediterranean Sea and the Atlantic Ocean. Anisakis larvae ( n  = 1950), which were recovered, were identified to species by means of genetic markers (allozymes). Within Anisakis type I, the larvae of A. pegreffii , A. simplex s.s ., A. typica and A. ziphidarum were detected, while within Anisakis type II, A. physeteris , A. brevispiculata and Anisakis sp. were identified. There were significant differences in the relative proportions of the various Anisakis species identified in hake samples from the Mediterranean Sea and Atlantic Ocean, suggesting the existence of different stocks of M. merluccius in European waters.     

Restriction fragment length polymorphisms of Anisakinae larvae

The analysis of restriction fragment length polymorphisms (RFLPs) was applied to distinguish several kinds of Anisakinae larvae, Anisakis larvae (type I) collected from two different paratenic hosts, Anisakis larvae (type II) and Contracaecum larvae. The patterns of the two different paratenic host-derived DNA of Anisakis larva (I) were exactly the same in hybridized fragments generated by six endonucleases. The quite different patterns in RFLPs of genomic DNA were observed among the Anisakis larva (I), Anisakis larva (II) and Contracaecum larvae. The results suggest that the RFLPs analysis may be useful for distinguishing Anisakinae larvae and clarifying the relationships between Anisakis larvae and their adult worms.     

Molecular characterization of larval anisakid nematodes from marine fishes of Madeira by a PCR-based approach, with evidence for a new species

One-hundred and fifteen anisakid larvae from 3 different fish hosts, Aphanopus carbo, Scomber japonicus, and Trachurus picturatus, caught in Madeiran waters, were identified by PCR-RFLP. Three distinct species were identified in A. carbo, namely Anisakis simplex sensu srricto, Anisakis pegreffii, and Anisakis ziphidarum; 5 in S. japonicus, i.e., A. simplex s.s., A. pegreffii, Anisakis physeteris, Anisakis typica, and A. ziphidarum; and 3 in T. picturatus, i.e., A. simplex s.s., A. pegreffii, and A. typica. Anisakis simplex s.s. was the most frequent species in both A. carbo and S. japonicus (54% and 23.5%, respectively). Anisakis pegreffii and A. physeteris occurred with a frequency of 20.6% in S. japonicus, whereas in T. picturatus the most frequent species was A. typica (41.9%), followed by A. simplex s.s. (32.3%). Furthermore, A. carbo and S. japonicus were infected by an apparently undescribed taxon, provisionally named Anisakis sp. A. Based on estimations of the genetic distance, this new taxon seems to be more similar to A. ziphidarum (0.0335) than to other species of the genus.     

Distribution of Anisakis species larvae from fishes of the Japanese waters

Human anisakiasis is caused by the consumption of raw, marinated or undercooked fish and squid infected with nematodes of the genus Anisakis Dujardin, 1845. In view of food safety, this study was carried out to examine the distribution of Anisakis species in marine fishes within Japanese waters. Seven fish species from six localities were collected and examined for Anisakis infection. Morphological and molecular (ITS region and mtDNA cox2 gene) characterization revealed the presence of two, among the three sibling species of Anisakis simplex, viz. A. simplex sensu stricto (s.s.) and A. pegreffii. Distribution data were collated with the results from the previous researches to better understand Anisakis distribution in Japanese waters. Distributions of Anisakis species were found to be locality-specific rather than host-specific, particularly between the two major species, A. simplex s.s. and A. pegreffii. Anisakis simplex s.s. is mainly found in fishes from northern Japan to Pacific sides, whereas A. pegreffii is in fishes from the Sea of Japan to East China Sea sides.     

Molecular identification of Anisakis species from Pleuronectiformes off the Portuguese coast

Anisakid nematodes belonging to the Anisakis simplex complex are highly prevalent in several fish species off the coast of Portugal and are an important zoonotic problem in the Iberian Peninsula. Two reproductively isolated sibling species of the Anisakis simplex complex were identified from Pleuronectiformes inhabiting the Portuguese coast using restriction fragment length polymorphism (RFLP). Recombinant genotypes corresponding to presumptive Anisakis simplex sensu stricto and Anisakis pegreffii hybrids were also detected by this technique, as well as the species Anisakis typica. Although 25 species of Pleuronectiformes were investigated, Anisakis spp. larvae were only found in seven: Arnoglossus imperialis, Arnoglossus laterna, Lepidorhombus boscii, Citharus linguatula, Platichthys flesus, Dicologlossa cuneata and Solea senegalensis. The occurrence of hybrids in relatively sedentary fishes such as the Pleuronectiformes suggests that the Portuguese coast may constitute an area of hybridization and, therefore, is of particular interest for the study of the process of hybridization and speciation for these anisakids.     

A new species of Anisakis Dujardin, 1845 (Nematoda, Anisakidae) from beaked whales (Ziphiidae): allozyme and morphological evidence

Larvae and adults of Anisakis, recovered from the beaked whales Mesoplodon layardii and Ziphius cavirostris from the Mediterranean Sea and South African waters, were analysed morphologically and by molecular markers (allozymes). A new Anisakis species was identified, showing fixed allele differences at a number of loci from the other Anisakis spp. tested (A. simplex complex, A. physeteris). The lack of hybrid or recombinant genotypes in mixed infections with A. pegreffii, A. simplex C and A. physeteris, as well as the high values of genetic distance (average D_Nei = 1.65 from the members of the A. simplex complex, and D_Nei = 3.09 from A. physeteris) showed that the new species is reproductively isolated. This new Anisakis species is morphologically different from the other Anisakis retained by Davey (1971) as either good species or species inquirendae. The name Anisakis ziphidarum n. sp. is proposed for the new species.     

Molecular phylogenetics and diagnosis of Anisakis, Pseudoterranova, and Contracaecum from northern Pacific marine mammals

Individual specimens of Anisakis, Pseudoterranova, and Contracaecum collected from marine mammals inhabiting northern Pacific waters were used for comparative diagnostic and molecular phylogenetic analyses. Forty-eight new sequences were obtained for this study of 14 Anisakis taxa, 8 Pseudoterranova taxa, 4 Contracaecum taxa, and 4 outgroup species. Partial 28S (LSU) and complete internal transcribed spacer (ITS-1, 5.8S, ITS-2) ribosomal DNA was amplified by the polymerase chain reaction and sequenced. Sequences of ITS indicated that Pseudoterranova specimens from Zalophus californianus (California sea lion), Mirounga angustirostris (northern elephant seal), Phoca vitulina (harbor seal), Enhydra lutris (sea otter), and Eumetopias jubatus (Steller's sea lion) exactly matched P. decipiens s. str., extending the host and geographic range of this species. Anisakis from northern Pacific marine mammals were most closely related to members of the A. simplex species complex. Comparison of Anisakis ITS sequences diagnosed the presence of A. simplex C in 2 M. angustirostris hosts, which is a new host record. Anisakis specimens from Phocoena phocoena (harbor porpoise), Lissodelphis borealis (Pacific rightwhale porpoise), and E. jubatus included 3 ITS sequences that did not match any known species. Contracaecum adults obtained from Z. californianus were most closely related to C. ogmorhini s.l. and C. rudolphii, but ITS sequences of these Contracaecum specimens did not match C. ogmorhini s. str. or C. margolisi. These novel Anisakis and Contracaecum ITS sequences may represent previously uncharacterized species. Phylogenetic analysis of LSU sequences revealed strong support for the monophyly of Anisakinae, Contracaecum plus Phocascaris, Pseudoterranova, and Anisakis. Phylogenetic trees inferred from ITS sequences yielded robustly supported relationships for Pseudoterranova and Anisakis species that are primarily consistent with previously published phenograms based on multilocus electrophoretic data.     

The value of external morphology in the identification of larval anisakid nematodes: a scanning electron microscope study

We studied larval nematodes of four genera of the Anisakidae using a scanning electron microscope (SEM). The anterior and posterior extremities and cuticular structures of the 3rd-stage larvae (L3) of Anisakis type I, Pseudoterranova decipiens, Contracaecum type B and Hysterothylacium were examined. The 4th-stage larvae (L4) of Anisakis type I, P. decipiens, recovered after infection into laboratory rats, and the L3 and L4 of Anisakis type I larvae from human were also examined in the same way. There were generic differences in the shape and size of the lip bulges, external papillary structures, the appearance of the boring tooth, the width and depth of the grooves and ridges of the cuticle and the caudal structures of the L3. In Anisakis type I and P. decipiens L3, changes were seen in the anterior extremity, cuticle and posterior extremity after molting to the L4. Similar changes can be expected in larvae infecting man. The L4 of Anisakis type I from rat and man were similar, while the L4 of Anisakis type I and P. decipiens showed differences. These ultrastructural differences might be of value in the identification of fragments recovered during endoscopy in man.     

Electrophoretic identification of Anisakis sp. larvae (Ascaridida: Anisakidae) from Clupea harengus L. in Baltic Sea

On the basis of electrophoretic studies carried out on 15 gene-enzyme systems, 80 Anisakis sp. larvae from the herring Clupea harengus, fished in South Baltic Sea and Gdanisk Bay, were identified as Anisakis simplex B. This is the first record of Anisakis simplex B in the Baltic Sea. The spawning migration of the herrings from the North Sea to the Baltic Sea and the distribution of A. simplex B are briefly discussed.     

Population structure of three species of Anisakis nematodes recovered from Pacific sardines (Sardinops sagax) distributed throughout the California Current system

Members of the Anisakidae are known to infect over 200 pelagic fish species and have been frequently used as biological tags to identify fish populations. Despite information on the global distribution of Anisakis species, there is little information on the genetic diversity and population structure of this genus, which could be useful in assessing the stock structure of their fish hosts. From 2005 through 2008, 148 larval anisakids were recovered from Pacific sardine (Sardinops sagax) in the California Current upwelling zone and were genetically sequenced. Sardines were captured off Vancouver Island, British Columbia in the north to San Diego, California in the south. Three species, Anisakis pegreffii, Anisakis simplex 'C', and Anisakis simplex s.s., were identified with the use of sequences from the internal transcribed spacers (ITS1 and ITS2) and the 5.8s subunit of the nuclear ribosomal DNA. The degree of nematode population structure was assessed with the use of the cytochrome c oxidase 2 (cox2) mitochondrial DNA gene. All 3 Anisakis species were distributed throughout the study region from 32°N to 50°N latitude. There was no association between sardine length and either nematode infection intensity or Anisakis species recovered. Larval Anisakis species and mitochondrial haplotype distributions from both parsimony networks and analyses of molecular variance revealed a panmictic distribution of these parasites, which infect sardines throughout the California Current ecosystem. Panmictic distribution of the larval Anisakis spp. populations may be a result of the presumed migratory pathways of the intermediate host (the Pacific sardine), moving into the northern portion of the California Current in summer and returning to the southern portion to overwinter and spawn in spring. However, the wider geographic range of paratenic (large piscine predators), and final hosts (cetaceans) can also explain the observed distribution pattern. As a result, the recovery of 3 Anisakis species and a panmictic distribution of their haplotypes could not be used to confirm or deny the presence of population subdivision of Pacific sardines in the California Current system.     

Ani s 10, a new Anisakis simplex allergen: cloning and heterologous expression

Anisakiasis is a human disease caused by accidental ingestion of larval nematodes belonging to the Anisakidae family. Anisakiasis is often associated with a strong allergic response. Diagnosis of A. simplex allergy is currently carried out by test based on the IgE reactivity to a complete extract of L3 Anisakis larvae although the specificity of these diagnostic tests is poor. Improving the specificity of the diagnostic test is possible using purified recombinant allergens. A new Anisakis allergen, named Ani s 10, was detected by immunoscreening an expression cDNA library constructed from L3 Anisakis simplex larvae. The new allergen was overproduced in Escherichia coli; it is a protein of 212 amino acids and it was localized as a 22 kDa protein band in an ethanol fractionated extract from the parasite. Ani s 10 has no homology with any other described protein, and its sequence is composed by seven almost identical repetitions of 29 amino acids each. A total of 30 of 77 Anisakis allergic patients (39%) were positive both to rAni s 10 and natural Ani s 10 by immunoblotting. The new allergen could be useful in a component-resolved diagnosis system for Anisakis allergy.     

Anisakid parasites of the pouting (Trisopterus luscus) from the Cantabrian Sea coast, Bay of Biscay, Spain

An epidemiological survey was undertaken of anisakids in 139 specimens (length: 13.2-24.5 cm) of pouting or bib (Trisopterus luscus) captured off the coast of northern Spain in the Cantabrian Sea. Third-stage larvae of two species of nematodes, Anisakis larvae type I and Hysterothylacium aduncum, were isolated. One adult female H. aduncum was also detected in the intestine of one pouting. Total prevalence of anisakids was 88.5%. Hysterothylacium aduncum and Anisakis showed, respectively, prevalence of 87.8% and 22.3%, mean intensity of 19.7 and 3.5, and mean abundance of 17.3 and 0.8. Analysis of infection parameters as a function of host length revealed a much higher prevalence in pouting specimens with length < 20 cm (94.4% for H. aduncum; 28.0% for Anisakis) than in those with length > or = 20 cm (65.6% for H. aduncum; 3.1% for Anisakis). The high mean intensity of Anisakis in muscle of parasitized pouting (5.9) may pose human health risks, although these are minimized by eating only thoroughly cooked pouting, as is the custom in Spain.     

Infestation by larvae of Anisakis simplex A and Anisakis physeteris in fish species of the Italian seas

Data on the occurrence of larvae of Anisakis simplex A and Anisakis physeteris in marine fishes from Italian waters are reported. The larvae have been identified by multilocus electrophoresis using biochemical keys. Considerations on the life-history pattern of these species in the Mediterranean Sea are advanced.     

Diagnosis of a case of gastric anisakidosis by PCR-based restriction fragment length polymorphism analysis

A set of genetic markers, based on PCR-RFLPs of three diagnostic restriction enzymes (Hhal, Hinfl and Taql), which proved to be suitable for the identification of the species of the genus Anisakis, was used for the first molecular identification of a larva obtained by endoscopy in a case of gastric anisakidosis, in a 51 year old woman from Southern Italy. The analysis of the restriction profiles obtained allowed the larva to be identified as Anisakis pegreffii, one of the three sibling species of the A. simplex complex. PCR-RFLP proved to be a cost-effective and reliable tool for the exact identification of Anisakis larvae recovered from infected humans.     

Cross-reactivity between antigens of Anisakis simplex s.l. and other ascarid nematodes

A study of the cross-reactivity among somatic and excretory-secretory antigens of the third stage larvae of Anisakis simplex s.l. and somatic antigens of other ascarid nematodes (Ascaris lumbricoides, A. suum, Toxocara canis, Anisakis physeteris, Hysterothylacium aduncum and H. fabri) was carried out by immunoblotting. It was revealed a high degree of cross-reactivity among ascarids in the 30 and > 212 kDa range by using sera against somatic and excretory-secretory antigens of A. simplex s.l. It has been revealed also specific components of the Anisakis genus (< 7.2, 9, 19 and 25 kDa) that will be interesting in diagnosis.     

Genetic relationships among Anisakis species (Nematoda: Anisakidae) inferred from mitochondrial cox2 sequences, and comparison with allozyme data

The genetic relationships among 9 taxa of Anisakis Dujardin, 1845 (A. simplex (sensu stricto), A. pegreffii, A. simplex C., A. typica, A. ziphidarum, A. physeteris, A. brevispiculata, A. paggiae, and Anisakis sp.) were inferred from sequence analysis (629 bp) of the mitochondrial cox2 gene. Genetic divergence among the considered taxa, estimated by p-distance, ranged from p = 0.055, between sibling species of the A. simplex complex, to p = 0.12, between morphologically differentiated species, i.e., A. ziphidarum and A. typica. The highest level was detected when comparing A. physeteris, A. brevispiculata, and A. paggiae versus A. simplex complex (on average p = 0.13) or versus A. typica (on average p = 0.14). Sequence data from the newly identified Anisakis sp. poorly aligned with other Anisakis species but was most similar to A. ziphidarum (p = 0.08). Phylogenetic analyses based upon Parsimony and Bayesian Inference, as well as phenetic analysis based upon Neighbor-Joining p-distance values, generated similar tree topologies, each well supported at major nodes. All analyses delineated two main claides, the first encompassing A. physeteris, A. brevispiculata, and A. paggiae as a sister group to all the remaining species, and the second comprising the species of the A. simplex complex (A. simplex (s.s.), A. pegreffii and A. simplex C), A. typica, A. ziphidarum, and Anisakis sp. In general, mtDNA-based tree topologies showed high congruence with those generated from nuclear data sets (19 enzyme-loci) and with morphological data delineating adult and larval stages of the Anisakis spp.; however, precise positioning of A. typica and A. ziphidarum remain poorly resolved, though they consistently clustered in the same clade as Anisakis sp. and the A. simplex complex. Comparison of anisakid data with those currently available for their cetacean-definitive hosts suggests parallelism between host and parasite phylogenetic tree topologies.