L. Paggi

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.

Electrophoretic studies on the Anisakis simplex complex (Ascaridida:Anisakidae) from the Mediterranean and North-East Atlantic.

Abstract Nascetti G., Paggi L., Orecchia P., Smith J.W., Mattiucci S. and Bullini L. 1986. Electrophoretic studies on the Anisakis simplex complex (Ascaridida: Anisakidae) from the Mediterranean and North-East Atlantic. International Journal for Parasitology 16: 633–640. The genetic variation of the sibling species Anisakis simplex A and A. simplex B was investigated by electrophoretic analysis of 22 gene-enzyme systems. The two species are reproductively isolated and no gene flow takes place between them. Three loci, Sod, Adk-2 and Lap-1, show distinct alleles in A. simplex A and A. simplex B, allowing their reliable identification both at the larval and adult stages. A fourth locus, Got, appears to be diagnostic at the 95% level. The value of Neis genetic distance found between A. simplex A and A. simplex B is 0.28. Parameters of genetic variability (He, P, A) are given for both species. The geographic distribution of A. simplex A and A. simplex B appears to be mainly Mediterranean for the former, and mainly North Atlantic for the latter. Several paratenic hosts (fish and squid) and one cetacean definitive host are identified for each of the two species. The names A. pegreffii and A. simplex are tentatively proposed for A. simplex A and A. simplex B respectively.

Genetic evidence for three species within Pseudoterranova decipiens (nematoda, ascaridida, ascaridoidea) in the north atlantic and norwegian and barents seas

Genetic variation of 1017 specimens of codworm, Pseudoterranova decipiens, collected from fish and seals at 23 sampling locations in the North Atlantic and Norwegian and Barents Seas, was analysed on the basis of 16 enzyme loci. Three reproductively isolated species, provisionally designated P. decipiens A, B and C, were detected, showing distinct alleles at the following loci: Mdh-1, 6Pgdh, Np, Pgm, Est-2 (between species A and B); Mdh-3, 6Pgdh, Np, Sod-1, Adk, Pgm, Est-2, Mpi (between A and C); Mdh-1, Mdh-3, Sod-1, Adk, Pgm, Est-2, Mpi (between B and C). One F1 hybrid was observed between P. decipiens A and B, but this apparently does not lead to any gene exchange between the two species, which do not show any evidence of introgression. No hybrids or introgressed individuals were observed between P. decipiens C and either A or B. Genetic distances among conspecific populations were low (average Neis D 0.001-0.005), even though they were collected thousands of kilometres apart, indicating high levels of gene flow within each of the three species. The values of Neis index D were 0.44 between P. decipiens A and B, 0.57 between B and C, and 0.79 between A and C. Estimated evolutionary divergence times, using Neis formula, range from 2 to 4 million years. Differences between P. decipiens A, B and C were also found with respect to genetic variability, morphology, geographical distribution and hosts. Mean heterozygosity values of 0.08, 0.05 and 0.02 were obtained for P. decipiens A, B and C, respectively. Preliminary morphological examination of adult males, previously identified by multilocus electrophoresis, revealed differences in the relative size and pattern of caudal papillae. P. decipiens B is widespread in the study area, whereas P. decipiens A was found only in the North-East Atlantic and Norwegian Sea. In this area P. decipiens A is most common in the grey seal, Halichoerus grypus, while the common seal, Phoca vitulina, is the main host for P. decipiens B. In Canadian Atlantic waters, where P. decipiens A is apparently absent, P. decipiens B infects both grey and common seals; a few specimens were also found in the hooded seal, Cystophora cristata. The only definitive host so far identified for P. decipiens C is the bearded seal, Erignathus barbatus; P. decipiens C appears to be widespread, occurring in both the North-West Atlantic and Barents Sea.

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 (Stellers 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.

Three sibling species within Contracaecum osculatum (nematoda, ascaridida, ascaridoidea) from the atlantic arctic-boreal region: Reproductive isolation and host preferences

Genetic variation within and between population samples from 22 locations of the Atlantic Arctic-Boreal region, including 1657 specimens morphologically assigned to Contracaecum osculatum, was electrophoretically analysed at 17 loci. Highly significant deviations from the Hardy-Weinberg equilibrium were found at various loci in several samples, owing to the existence of three distinct gene pools within C. osculatum (sensu lato) from the study area. These gene pools correspond to three biological species (provisionally designated A, B and C), characterized by distinct genotypes at several diagnostic loci. Reproductive isolation between C.osculatum A, B and C is confirmed by the lack of F1, recombinant, or backcross genotypes in sympatric areas, despite the occurrence of multiple infections. Mean heterozygosity per locus is on average 0.11 in species A, 0.10 in B and 0.07 in C. High levels of gene flow were found within each of the three species, the values of Nm (number of migrant individuals) ranging from 3.41 (C. osculatum C) to 5.77 (C. osculatum A). Average Neis genetic distance is 0.46 between A and B, 0.50 between A and C and 0.77 between B and C. From these values, times of evolutionary divergence from 2 to 4 million years can be estimated. Genetic relationships among populations and species of the C. osculatum complex are illustrated by principal component analysis. The role of both geographical isolation and host preferences in the speciation of C. osculatum (sensu lato) is discussed. A morphological distinction of the three species has not yet been possible (sibling species). However, there is evidence that the name C. osculatum (sensu stricto) should be used for species C, which shows a geographical distribution and definitive host corresponding to the neotype of C. osculatum (sensu stricto). Finally, a comparison is made between the members of the C. osculatum complex from the Atlantic Arctic-Boreal region and those of the Pseudoterranova decipiens complex from the same area, as to: (i) times of evolutionary divergence, (ii) geographical distribution, and (iii) host preferences.

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

Abstract 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. osculatum 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.

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 stricto, 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.

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.

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.

Occurrence and molecular identification of Anisakis spp. from the North African coasts of Mediterranean Sea

Larval forms of the genus Anisakis were reported infecting several fish species from the North African coasts of central Mediterranean Sea. Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis was used to investigate the occurrence of larval forms of different Anisakis species in teleost fishes and squid from North African coasts of the Mediterranean Sea and to establish the geographical and host range of these parasites in this area. A total of 282 Anisakis larvae were identified by PCR-RFLP from 13 teleost fish species and one cephalopod species captured at different sites off the Algerian, Tunisian and Libyan coasts. The type I larvae were found with a frequency of 93.62% and were identified as belonging to the following species: Anisakis simplex s.str., Anisakis pegreffii, A. simplex s.str/A. pegreffii hybrids and Anisakis typica. The type II larvae were found to belong to Anisakis physeteris, with the frequency of 6.38%. The record of A. simplex s.str/A. pegreffii hybrids, previously recorded from the Spanish and Portuguese Atlantic coasts and the Alboran Sea, extends their geographic distribution to the Tunisian coasts. The occurrence of A. simplex s.str. and hybrids away from their known area of distribution may predict the successful use of Anisakis larvae for tagging Scomber scombrus fish stocks for fisheries management purposes. Moreover, the results reported provide valuable information regarding the diversity of Anisakis species in the study area, indicating that several Anisakis sibling and morphospecies coexist in the North African coasts of the Mediterranean Sea.