Two new species of Contracaecum Railliet & Henry, 1912 (Nematoda: Anisakidae), C. fagerholmi n. sp. and C. rudolphii F from the brown pelican Pelecanus occidentalis in the northern Gulf of Mexico.

DNA sequencing of the nuclear ribosomal DNA internal transcribed spacers (ITS) and mitochondrial rrnS and cox2 genes, and analysis of polymorphisms in restriction profiles in the ITS and rrnS, were used to characterise anisakid nematodes belonging to Contracaecum Railliet & Henry, 1912 infecting the brown pelican Pelecanus occidentalis (L.) in Galveston Bay, Texas and Sarasota Bay, Florida. Molecular data led to the detection of two new species: Contracaecum fagerholmi n. sp., which was also supported by clear morphological evidence, and Contracaecum rudolphii F, a new cryptic species within the Contracaecum rudolphii Hartwich, 1964 complex. Bayesian phylogenetic analysis demonstrated that C. fagerholmi and C. rudolphii F form two well-separated clusters, with C. fagerholmi being closely related to Contracaecum bioccai Mattiucci et al., 2008 and C. rudolphii F being included in the C. rudolphii complex. C. fagerholmi can be readily differentiated morphologically from all of its congeners, other than C. microcephalum (Rudolphii 1809) and the five currently recognised members of the C. rudolphii complex (C. rudolphii A, B, C, D and E). C. fagerholmi differs from C. microcephalum in the length of the spicules and the shape of the distal tip of the spicules, and from C. rudolphii (sensu lato) in the shape and size of the ventro-lateral and dorsal lips and by having interlabia which are not distally bifurcate. Further studies are needed to determine which morphological characteristics can be used to distinguish the cryptic species of the C. rudolphii complex in order to assign them with formal names. The recovery of a third species, C. bioccai, from the brown pelican confirms its occurrence in this host and extends its known geographical distribution.

Genetic characterization of members of the genus Contracaecum (Nematoda: Anisakidae) from fish-eating birds from west-central Florida, USA, with evidence of new species.

Specimens of Contracaecum spp. from Phalacrocorax auritus and Pelecanus occidentalis from Florida were characterized by sequencing of the small subunit of the mitochondrial ribosomal RNA gene (rrnS) and by PCR-based RFLP analysis of the same gene and of the internal transcribed spacers (ITS) of nuclear ribosomal DNA. Analyses of the rrnS sequence data using the MP and UPGMA approaches yielded trees with similar topologies, delineating 3 main clusters. Specimens from Ph. auritus, morphologically assigned to C. rudolphii (s.l.), were part of the cluster comprising also the other 2 species of the C. rudolphii complex (A and B), but representing a genetically distinct group, potentially corresponding to a distinct lineage within the complex, provisionally named as C. rudolphii C. The second cluster comprised 5 individuals from P. occidentalis, which formed a genetically relatively homogeneous group. The rrnS data indicate that these specimens (indicated as Contracaecum sp. 1) are clearly genetically different from the morphologically most closely related species, i.e. C. rudolphii (s.l.) and C. microcephalum, and could represent a new species. The third cluster comprised 7 specimens from P. occidentalis morphologically assigned to C. multipapillatum (s.l.). These were shown to be genetically homogeneous and related to but quite distinct from C. multipapillatum from Greece, although additional studies are needed to assess their status. PCR-RFLP based markers for the quick identification of these taxa are provided.

Practical PCR tools for the delineation of Contracaecum rudolphii A and Contracaecum rudolphii B (Ascaridoidea: Anisakidae) using genetic markers in nuclear ribosomal DNA.

Using genetic markers defined previously in the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA), PCR-coupled restriction fragment length polymorphism (PCR-RFLP) and specific PCR assays were established for the specific detection of each of two morphologically indistinguishable operational taxonomic units (Contracaecum rudolphii A and Contracaecum rudolphii B) within Contracaecum rudolphii (s.l.) and their differentiation from Contracaecum septentrionale, a closely related congener. Application of these tools to C. rudolphii (s.l.) adults from Phalacrocorax carbo sinensis (the Eurasian subspecies of the great cormorant) from Qinghai Lake in China, revealed C. rudolphii B to infect this host. This is the first report of C. rudolphii B in P. carbo sinensis outside of Europe (where it was originally detected), supporting the proposal that this species has a broad geographical distribution. Together with other methods, each of these molecular tools will be useful for investigating the ecology of C. rudolphii A and C. rudolphii B as well as C. septentrionale.

Intestinal anisakiasis in Italy: case report.

A case of intestinal anisakiasis caused by Anisakis sp. larva type I in a woman from Italy who consumed raw marinated anchovies, is reported. The diagnosis was based on the morphological features characteristic of anisakid larval stages, which were readily recognized in a large granuloma removed after emergency surgical treatment.

Occurrence of recombinant genotypes of Anisakis simplex s.s. and Anisakis pegreffii (Nematoda: Anisakidae) in an area of sympatry.

The anisakid nematode populations collected from fish and stranded cetaceans along from Iberian Peninsula waters were morphologically identified as corresponding to the Anisakis simplex complex. In order to realise their molecular identification and to analyse the extent of genetic variation, the entire ITS (ITS1, 5.8S rDNA gene and ITS2) and the mitochondrial small subunit of rRNA were pcr-amplified and sequenced. Digestions of the amplified its region with HinfI and HhaI allowed the identification of three different genotypes, belonging to A. simplex s.s., A. pegreffii and a yet not described recombinant genotype. The ITS sequences of the recombinant genotypes showed the presence of heterozygotes C/T at position 240 and 256 of the aligned sequence. Otherwise, the analysis of mtDNA sequences showed the existence of a different parental origin for recombinant genotypes. In order to check if they can be the products of a polymorphism normally occurring both in A. pegreffii and in A. simplex s.s., and/or the existence of an incomplete concerted evolution, three samples were also collected as controls in isolated geographic areas, where sympatric coexistence between A. simplex s.s. and A. pegreffii does not occur. The results supports the hypothesis that the recombinant individuals may be a product of interspecific hybridisation, and describe the Iberian Peninsula waters as a hybrid zone for the two sibling species.

Genetic evidence for two sibling species within Contracaecum ogmorhini Johnston & Mawson, 1941 (Nematoda: Anisakidae) from otariid seals of boreal and austral regions.

Genetic variation of Contracaecum ogmorhini (sensu lato) populations from different otariid seals of the northern and southern hemisphere was studied on the basis of 18 enzyme loci as well as preliminary sequence analysis of the mitochondrial cyt b gene (260 bp). Samples were collected from Zalophus californianus in the boreal region and from Arctocephalus pusillus pusillus, A. pusillus doriferus and A. australis from the austral region. Marked genetic heterogeneity was found between C. ogmorhini (sensu lato) samples from the boreal and austral region, respectively. Two loci (Mdh-2 and NADHdh) showed fixed differences and a further three loci (Iddh, Mdh-1 and 6Pgdh) were highly differentiated between boreal and austral samples. Their average genetic distance was D(Nei) = 0.36 at isozyme level. At mitochondrial DNA level, an average proportion of nucleotide substitution of 3.7% was observed. These findings support the existence of two distinct sibling species, for which the names C. ogmorhini (sensu stricto) and C. margolisi n. sp., respectively, for the austral and boreal taxon, are proposed. A description for C. margolisi n. sp. is provided. No diagnostic morphological characters have so far been detected; on the other hand, two enzyme loci, Mdh-2 and NADHdh, fully diagnostic between the two species, can be used for the routine identification of males, females and larval stages. Mirounga leonina was found to host C. ogmorhini (s.s.) in mixed infections with C. osculatum (s.l.) (of which C. ogmorhini (s.l.) was in the past considered to be a synonym) and C. miroungae; no hybrid genotypes were found, confirming the reproductive isolation of these three anisakid species. The hosts and geographical range so far recorded for C. margolisi n. sp. and C. ogmorhini (s.s.) are given.

SSCP-based identification of members within the Pseudoterranova decipiens complex (Nematoda: Ascaridoidea: Anisakidae) using genetic markers in the internal transcribed spacers of ribosomal DNA.

The anisakid nematodes morphologically corresponding with Pseudoterranova decipiens sensu lato (s.l.) (Krabbe, 1878) from different seal or sea lion hosts and geographical origins, previously identified as Pseudoterranova krabbei, P. decipiens (s.s.), P. bulbosa, P. azarasi and P. cattani by multilocus enzyme electrophoresis, were characterized using a DNA approach. Also a population of P. decipiens (s.l.) from Chaenocephalus aceratus, the blackfin icefish, from Antarctica and another from Osmerus eperlanus, the European smelt, from Germany were included in the study. The first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of ribosomal DNA (rDNA) were amplified by PCR from individual nematodes and analysed by single-strand conformation polymorphism (SSCP), followed by selective sequencing. While no variation in single-stranded ITS-1 and ITS-2 profiles was detected among samples representing each of the species or populations (with the exception of slight microheterogeneity), SSCP analysis of the ITS-2 amplicons allowed the unequivocal differentiation of all of the 5 sibling species of P. decipiens (s.l.) examined, which was supported by sequence differences in ITS rDNA. Samples representing the P. decipiens (s.l.) population from O. eperlanus had the same SSCP profile as those of P. decipiens (s.s.), which was supported by a lack of nucleotide difference in the ITS between them, suggesting that the former represented P. decipiens (s.s.). Based on SSCP results and ITS sequence data, P. decipiens (s.l.) from C. aceratus was genetically most distinct with respect to all other members of Pseudoterranova examined, which indicated that it may represent P. decipiens E (based on geographical origin) or a distinct species. These findings and the molecular approach taken should have important implications for studying the life-cycles, transmission patterns, epidemiology and population genetics of these anisakid nematodes, and the diagnosis of their infections.

Genetic markers in the study of Anisakis typica (Diesing, 1860): larval identification and genetic relationships with other species of Anisakis Dujardin, 1845 (Nematoda: Anisakidae).

Genetic variation at 21 gene-enzyme systems was studied in a sample of an adult population of Anisakis typica (Diesing, 1860) recovered in the dolphin Sotalia fluviatilis from the Atlantic coast of Brazil. The characteristic alleles, detected in this population, made it possible to identify as A. typica, Anisakis larvae with a Type I morphology (sensu Berland, 1961) from various fishes: Thunnus thynnus and Auxis thazard from Brazil waters, Trachurus picturatus and Scomber japonicus from Madeiran waters, Scomberomorus commerson, Euthynnus affinis, Sarda orientalis and Coryphaena hippurus from the Somali coast of the Indian Ocean, and Merluccius merluccius from the Eastern Mediterranean. Characteristic allozymes are given for the identification, at any life-stage and in both sexes, of A. typica and the other Anisakis species so far studied genetically. The distribution of A. typica in warmer temperate and tropical waters is confirmed; the definitive hosts so far identified for this species belong to delphinids, phocoenids and pontoporids. The present findings represent the first established records of intermediate/paratenic hosts of A. typica and extend its range to Somali waters of the Indian Ocean and to the Eastern Mediterranean Sea. A remarkable genetic homogeneity was observed in larval and adult samples of A. typica despite their different geographical origin; interpopulation genetic distances were low, ranging from D(Nei)=0.004 (Eastern Mediterranean versus Somali) to D(Nei)=0.010 (Brazilian versus Somali). Accordingly, indirect estimates of gene flow gave a rather high average value of Nm = 6.00. Genetic divergence of A. typica was, on average, D(Nei)=1.12 from the members of the A. simplex complex (A. simplex s.s, A. pegreffii, A. simplex C) and D(Nei)=1.41 from A. ziphidarum, which all share Type I larvae; higher values were found from both A. physeteris (D(Nei)=2.77)

Electrophoretic detection of population variation within Contracaecum ogmorhini (Nematoda: Ascaridoidea: Anisakidae).

This study examined genetic variation among specimens of Contracaecum ogmorhini from different otariid hosts and geographical origins using a polymerase chain reaction (PCR)-based mutation detection approach. The first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of ribosomal DNA (rDNA) were amplified individually by PCR, scanned for sequence variation by single-strand conformation polymorphism (SSCP), and samples displaying variable SSCP profiles were subjected to cycle sequencing. While C. ogmorhini individuals from Arctocephalus pusillus pusillus (CoAPP) from South Africa and those from Arctocephalus pusillus doriferus (CoAPD) from Australia had very similar SSCP profiles for both ITS-1 and ITS-2, individuals of C. ogmorhini from Zalophus californianus (CoZC) from Pacific Canada could be unequivocally distinguished based on their profiles. In accordance with SSCP results, both CoAPP and CoAPD had identical ITS consensus sequences, whereas CoZC differed in sequence from both CoAPP and CoAPD populations by 0.2% (one base in the ITS-1) and 0.7% (two bases in the ITS-2). Based on the nucleotide difference in the ITS-2 sequence, a PCR-linked restriction fragment length polymorphism (RFLP) could be employed to distinguish individuals representing CoZC from those of both CoAPP and CoAPD. The findings suggest that C. ogmorhini may represent a complex of at least two species.

Mutation scanning for sequence variation in three mitochondrial DNA regions for members of the Contracaecum osculatum (Nematoda: Ascaridoidea) complex.

Anisakid nematodes of seals from different geographical origins, previously identified by multilocus enzyme electrophoresis as Contracaecum osculatum A (CoA), C. osculatum B (CoB), C. osculatum C (CoC), C. osculatum D (CoD), C. osculatum E (CoE) and C. osculatum baicalensis (Cob), were characterised genetically using a mutation scanning approach, in order to define genetic markers for their specific identification and differentiation. Three mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit I (COI), and the small and large subunits of rRNA (ssrRNA and IsrRNA, respectively) were amplified separately from individual nematodes by polymerase chain reaction (PCR), analysed by single-strand conformation polymorphism (SSCP), and samples displaying sequence variability were subjected to sequencing. Forty-six haplotypes were defined for 62-66 individuals (representing the six members of C. osculatum). All taxa except CoD and CoE could be identified, or delineated from one another, by nucleotide differences in the COI, ssrRNA and/or IsrRNA sequences. For all three mtDNA regions, 4 (10.5%), 7 (18.4%), 15 (39.5%) and 11 (28.9%) of 38 nucleotide positions were considered diagnostic (fixed) and could thus unequivocally delineate CoA, CoB, CoC and Cob. The lack of an unequivocal nucleotide difference in any of the three mtDNA sequences between CoD and CoE was in accordance with previous ribosomal DNA sequence data but inconsistent with multilocus enzyme electrophoretic data. Using all fixed nucleotide positions, CoA, CoD/E and CoB were genetically more similar to Cob than each was to CoC, similar to previous findings. In spite of not being able to distinguish among all six taxa of C. osculatum, the present study demonstrated clearly the usefulness and attributes of the mutation scanning approach for investigating population genetic structures of species of parasitic nematodes.

Genetic divergence and reproductive isolation between Anisakis brevispiculata and Anisakis physeteris (Nematoda: Anisakidae)s.

In order to assess the taxonomic status of Anisakis brevispiculata Dollfus, 1966 population samples of this taxon from central and south-eastern Atlantic ocean were compared at 22 enzymatic loci with samples belonging to Anisakis physeteris Baylis, 1923 from the Mediterranean sea and central-eastern Atlantic ocean. Very low interpopulational genetic divergence was observed both within A. brevispiculata (average D(Nei) = 0.008) and within A. physeteris (D(Nei) = 0.009) despite the geographic distance among the samples, indicating high levels of gene flow in both taxa. On the other hand, the average genetic distance between A. brevispiculata and A. physeteris was found to be D(Nei) = 0.80, a value generally observed between well differentiated congeneric species. The reproductive isolation between A. brevispiculata and A. physeteris is indicated by the following observations: (1) no F(1) hybrids or recombinant genotypes were until now observed; and (2) the two Anisakis species do not seem to share their definitive hosts. The main definitive host of A. brevispiculata is the pygmy sperm whale (Kogia breviceps), while for A. physeteris it is the sperm whale (Physeter catodon). Only adult males differ slightly in spicule length, while females and larval stages are not differentiated morphologically. Both A. brevispiculata and A.physeteris show a type II larva. The correct recognition of A. brevispiculata from A. physeteris and from other Anisakis species studied, in either sexes and at any life stage, is made easy by allozyme markers (e.g. Icdh, Gapdh, Sod-1, Np, Aat-2, Adk-2, fEst-2, PepB, PepC-2, Mpi). Diagnostic keys, which can be used for routine identification in the field of these Anisakis worms, based on genetic markers, are given.

Allozyme and PCR-RFLP markers in anisakid nematodes, aethiological agents of human anisakidosis.

In the framework of the researches granted by MURST COFIN97, studies on anisakid nematodes, aethiological agents of human anisakidosis, were carried out. The project was aimed to implement the knowledge on the systematics, genetics, ecology and epizootiology of species of the genera Anisakis and Pseudoterranova by applying genetic markers obtained from multilocus allozyme electrophoresis and from PCR-based techniques. The results obtained by allozyme studies allowed to extend the geographic distribution and to detect new definitive and intermediate/paratenic hosts of two sibling species of the A. simplex complex, i.e. A. simplex s.s. and A. pegreffii and to characterise the species A. simplex C, a new sibling species within the A. simplex complex as well as a new species belonging to the genus Anisakis, A. ziphidarum. Combined allozyme and morphological analyses provided markers for the identification of the four sibling species of the Pseudoterranova decipiens complex and their nomenclatural designations. New markers based on PCR-RFLP analysis were used for the identification of sibling species of the Anisakis simplex complex and of another four species of the genus. Genetic markers based on three diagnostic restriction enzymes allowed the identification as A. pegreffii of a larva obtained by endoscopy in a case of human anisakidosis in Southern Italy.

Phylogenetic relationships among species of contracaecum Railliet & Henry, 1912 and Phocascaris Høst, 1932 (Nematoda:Ascaridoidea) based on nuclear rDNA sequence data.

Nuclear-encoded large-subunit ribosomal DNA sequences were used to infer a phylogenetic hypothesis for 17 taxa (16 nominal species) of the genera Contracaecum and Phocascaris. Phylogenetic trees based on these data have been used to assess the validity of the taxonomic distinction between these genera, which was based on the presence or absence of certain structural features, rather than on explicit hypotheses of evolutionary history. Phylogenetic hypotheses based on parsimony, likelihood, and neighbor-joining analyses of these sequence data strongly support the hypothesis that species of Phocascaris are nested within the clade of Contracaecum species hosted by phocid seals, and are more closely related to species of the Contracaecum osculatum complex than to other Contracaecum species. Alternative tree topologies representing Phocascaris as not nested within the C. osculatum complex were significantly worse interpretations of these sequence data. Phylogenetic analysis also provides strong support for the monophyly of all taxa (Contracaecum and Phocascaris) from phocid seals, which is consistent with Berland's (1964) proposal that such species form a natural group; however, his proposal to recognize all species in phocid seals as Phocascaris, with all species from birds as Contracaecum would result in a paraphyletic Contracaecum, according to the molecular phylogenetic hypothesis.

Assessing sequence variation in the internal transcribed spacers of ribosomal DNA within and among members of the Contracaecum osculatum complex (Nematoda: Ascaridoidea: Anisakidae).

The anisakid nematodes of seals from different geographical origins, previously identified as Contracaecum osculatum A, C. osculatum B, C. osculatum C, C. osculatum D, C. osculatulm E and C. osculatum baicalensis by multilocus enzyme electrophoresis, were characterised using a DNA approach. The first and second internal transcribed spacers (ITS-1, ITS-2) of ribosomal DNA (rDNA) were individually amplified by polymerase chain reaction (PCR) and analysed by single-strand conformation polymorphism (SSCP) and sequencing. SSCP analyses allowed the unequivocal differentiation of all taxa except C. osculatum D from C. osculatum E. While C. osculatum D and C. osculatum E had identical ITS sequences, each of the other four taxa had distinct sequences, with interspecific differences ranging from 0.3% to 2.3%. C. osculatum C was genetically the most distinct taxon with respect to all other members of the species complex.

Pseudoterranova decipiens species A and B (Nematoda, Ascaridoidea): nomenclatural designation, morphological diagnostic characters and genetic markers.

Five genetically distinct and reproductively isolated species have been detected previously within the morphospecies Pseudoterranova decipiens from the Arctic-Boreal, Boreal and Antarctic. Morphological analysis was carried out on male specimens identified by genetic (allozyme) markers, allowing the detection of significant differences at a number of characters between two members of the P. decipiens complex, namely P. decipiens A and B. On the basis of such differences, the nomenclatural designation for the two species is discussed. The names Pseudoterranova krabbei n. sp. and P. decipiens (sensu stricto) are proposed for species A and B, respectively. Morphological and genetic differentiation between the two species is shown using multivariate analysis. Allozyme diagnostic keys for routine identification of the four members of the P. decipiens complex, namely P. decipiens (s.s.), P. krabbei, P. bulbosa and P. azarasi, irrespective of sex and life-history stage, are provided.

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.

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.

Genetic and ecological data on the Anisakis simplex complex, with evidence for a new species (Nematoda, Ascaridoidea, Anisakidae).

Isozyme analysis at 24 loci was carried out on anisakid nematodes of the Anisakis simplex complex, recovered from various intermediate/paratenic (squid, fish) and definitive (marine mammals) hosts from various parts of the world. A number of samples were found to belong to A. simplex sensu stricto and Anisakis pegreffii, widely extending the geographic ranges and the number of hosts of these 2 species. In addition, a new distinct gene pool was detected, showing different alleles with respect to A. simplex s. str and A. pegreffii at 5 diagnostic loci (99% level). Samples with this gene pool were assigned to a new species, provisionally labeled A. simplex C. Reproductive isolation between A. simplex C and the other 2 Anisakis species was directly assessed by the lack of hybrid and recombinant genotypes in mixed samples from sympatric areas, i.e., Pacific Canada for A. simplex C+A. simplex s. str., South Africa and New Zealand for A. simplex C+A. pegreffii, even when such samples were recovered from the same individual host. Similar levels of genetic divergence were observed among the three species (DNei from 0.36 to 0.45). At the intraspecific level, Canadian Pacific and Austral populations of A. simplex C were found to be genetically rather differentiated from one another (average DNei = 0.08), contrasting with the remarkable genetic homogeneity detected within both A. simplex s. str. and A. pegreffii (average DNei about 0.01). Accordingly, a lower amount of gene flow was estimated within A. simplex C (Nm = 1.6) than within the other 2 species (Nm = 5.4 and 17.7, respectively). Anisakis simplex C showed the highest average values of genetic variability with respect to both A. simplex s. str. and A. pegreffii, e.g., expected mean heterozygosity. Hr = 0.23, 0.16, and 0.11, respectively, in the 3 species. Data on geographic distribution and hosts of the 3 members so far detected in the A. simplex complex are given. Their ecological niche is markedly differentiated, with a low proportion of hosts shared. Intermediate and definitive hosts of A. simplex s. str. and A. pegreffii appear to belong to distinct food webs, benthodemersal, and pelagic, respectively; this would lead to different transmission pathways for the parasites.

Two new members in the Contracaecum osculatum complex (Nematoda, Ascaridoidea) from the Antarctic.

The genetic structure of adults and larvae of Contracaecum osculatum (sensu lato) from the Antarctic is analyzed on the basis of 24 enzyme loci. Significant deviations of genotype frequencies from the Hardy-Weinberg equilibrium were found, even in samples recovered from the same host. These data indicate that two distinct, reproductively isolated species coexist in C. osculatum (sensu lato) samples from the Antarctic. They were provisionally designated C. osculatum D and E, as they do not correspond to any of the three species previously detected in this complex from the Atlantic Arctic Boreal region (C. osculatum A, B and C). An allozyme diagnostic key for the identification of the five members of the C. osculatum complex, at the larval and adult stage and in both sexes, is given. Species D and E were found to be genetically quite variable: average P99 = 84.3, A = 3.3 and He = 0.23. Both showed high values of intraspecific gene flow: Nm = 4.6 and 6.1 respectively; similar values were found for the Arctic-Boreal C. osculatum A, B and C. The most related members of the complex are the Antarctic species E and the Arctic-Boreal species A (DNei = 0.21), while the most differentiated ones are the Arctic-Boreal species B and C (DNei = 0.76). The evolutionary divergence of C. osculatum C started more than 3 million years ago, in a Pliocene refugium (Baltic Sea). As to the other C. osculatum species, their evolutionary divergence took place during Pleistocene, when this complex achieved a bipolar distribution. This process involved two distinct colonizations of the marine Antarctic region by ancestors of the northern hemisphere, about 1.5 and 1 million years ago, giving origin to C. osculatum D and E respectively.

Present occurrence of Anguillicola novaezelandiae (Nematoda: Dracunculoidea) in Europe and its development in the intermediate host.

An examination of a sample of European eels, Anguilla anguilla (L.), collected from Lake Bracciano near Rome in 1993, the only known European locality with the occurrence of the introduced swimbladder nematode Anguillicola novaezelandiae Moravec et Taraschewski, 1988, revealed for the first time the presence of two Anguillicola species, A. novaezelandiae and A. crassus. In view of the investigations carried out by current authors in Bracciano Lake in the years 1982-1992, it is apparent that the latter species has been introduced into the lake quite recently, where it quickly became a dominant species. The development of A. novaezelandiae was experimentally studied in the copepod intermediate host, Cyclops strenuus, for the first time. The copepods were infected with nematode second-stage larvae at 21-22 degrees C; fully developed infective third-stage larvae were obtained 13 days p.i. The general morphology of individual larval stages of A. novaezelandiae was similar to that of larvae of the related species A. crassus.