Genetic relationships among species of Contracaecum Railliet & Henry, 1912 and Phocascaris Höst, 1932 (Nematoda: Anisakidae) from pinnipeds inferred from mitochondrial cox2 sequences, and congruence with allozyme data

The genetic relationships among 11 taxa, belonging to the genus Contracaecum (C. osculatum A, C. osculatum B, C. osculatum (s.s.), C. osculatum D, C. osculatum E, C. osculatum baicalensis, C. mirounga, C. radiatum, C. ogmorhini (s.s.), C. margolisi) and Phocascoris (Phocoscris cystophorae), parasites as adults of seals, were inferred from sequence analysis 1519 bp) of the mitochondrial cytochrome c oxidase subunit II (mtDNA cox2) gene. Phylogenetic analyses obtained from Parsimony (MP) and Neighbour-Joining (NJ) K2P distance values generated similar topologies, each well supported at major nodes. All analyses delineated two main clades: the first encompassing the parasites of the phocid seals, i.e. the C. osculatum species complex, C. osculatum boicolensis, C. mirounga and C. radiatum, with the latter two species forming a separate subclade; the second including the parasites of otarids, i.e. C. ogmorhini (s.s.) and C. margolisi. An overall high congruence between mtDNA inferred tree topologies and those produced from nuclear data sets (20 allozyme loci) was observed. Comparison of the phylogenetic hypothesis here produced for Controcaecum spp. plus Phocascaris with those currently available for their definitive hosts (pinnipeds) suggests parallelism between hosts and parasite phylogenetic tree topologies.     

A new anisakid species parasitizing the imperial cormorant Phalacrocorax atriceps from the North Patagonian Coast, Argentina

The anisakid species Contracaecum chubutensis n. sp. was found parasitizing the imperial cormorant Phalacrocorax atriceps at 2 different localities, Bahía Bustamante (45 degrees 11'S, 66 degrees 30'W) and Puerto Madryn (42 degrees 47'S, 65 degrees 02'W) on the Patagonian coast. Morphometrical analysis and further studies of adult specimens of C. chubutensis n. sp. and fourth-stage larvae of Contracaecum sp. were done using light and scanning electron microscopy. The possession and disposition of 7 pairs of tail papillae differentiate this new species from C. osculatum, C. radiatum, C. plagiaticium, and C. mirounga. Double-sized spicules separate it from C. variegatum and C. pelagicum. Moreover, the paracloacal papillae disposition and tail shape differentiate this new species from C. travassosi. The presence of bifurcated interlabia separate it from C. ogmorhini and C. margolisi. Three well-marked lip notches, a sharp tail shape, and the of the transversal paracloacal papillae disposition distinguishes C. chubutensis n. sp. from C. magnipapillatum and C. septentrionale. The species here described can be differentiated from C. caballeroi by its bifurcated interlabia, longer spicules, and for possessing more precloacal papillae pairs. It can be separated from C. rudolphii by a thinner interlabia at the base, deeper lip notches, a sharper tail, the disposition of transversal paracloacal papillae, and greater distance between both papillae of the first subventral distal papillae pair. In addition, fourth-stage larvae recovered from the same host and localities were identified and described as Contracaecum sp. Prevalence of adult parasites was 66.7 %, mean intensity was 4.0, and mean abundance was 7.1.     

Selected mitochondrial genes as species markers of the Arctic Contracaecum osculatum complex

This study, aimed at testing the hypothesis that some mitochondrial genes can serve as species-specific markers, involved a comparison of the sequence variance of selected mitochondrial DNA genes of the Arctic Contracaecum osculatum species (C. osculatum A, C. osculatum B and C. osculatum C). We compared differences between five complete (ND2, CYTB, ND3, ND4L and ND6) and three partial (CO1, CO3 and ND5) protein-coding genes. The total length of the sequence of each of the 13 specimens was 4830?bp. The sample consisted of C. osculatum L3 larvae collected from Reinhardtius hippoglossoides and Gadus ogac from the Barents Sea and Davis Strait. The K2P distance values between the species ranged within 0.06-0.12, the intraspecific variability (0.01-0.03) proving 3-6 times lower. The lowest interspecific divergence was observed between C. osculatum A and C. osculatum B, whereas the highest intraspecific diversity was typical of C. osculatum C. Among the C. osculatum species studied, the highest nucleotide diversity was recorded in the CYTB, CO3 and ND5 genes. These genes may be useful in species identification of the very closely related Contracaecum sibling 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.     

Allozyme and morphological identification of shape Anisakis,Contracaecum and Pseudoterranova from Japanese waters (Nematoda,Ascaridoidea)

Allozyme markers were used to identify anisakid nematodes from marine Japanese waters, morphologically assigned to three species complexes: Anisakis simplex (s. l.), Contracaecum osculatum (s. l.) and Pseudoterranova decipiens (s. l.). Samples assigned to A. simplex (s. l.) were found to correspond genetically to A. simplex sensu stricto, those of C. osculatum (s. l.) to C. osculatum A. No morphological characters are yet available to distinguish sibling species of these two complexes. As to the P. decipiens complex, two distinct species were detected: the first corresponded to P. decipiens C, previously recovered in the northern Atlantic, the second to P. decipiens D from Japan. The two species are genetically well differentiated, with five of the 19 loci tested showing distinct fixed alleles. Their reproductive isolation was proved by the lack of hybrids or recombinants in sympatric samples recovered from the same definitive host, Erignathus barbatus. P. decipiens D was found to correspond morphologically to Porrocaecum azarasi, previously considered a synonym of P. decipiens. Accordingly, the name Pseudoterranova azarasi (Yamaguti & Arima, 1942) n. comb. is proposed for P. decipiens D. Similarly, P. decipiens C fits in general morphology, type-locality and host with Ascaris bulbosa, also previously considered a synonym of P. decipiens. The name Pseudoterranova bulbosa (Cobb, 1888) n. comb. is proposed for P. decipiens C.     

Ascaridoidea: a simple DNA assay for identification of 11 species infecting marine and freshwater fish,mammals, and fish-eating birds

Eleven species belonging to superfamily Ascaridoidea, which infect marine and freshwater fish, mammals, and fish-eating birds, were analyzed using a PCR-RFLP method. The following species were investigated: Anisakis pegreffi, A. physeteris, and A. simplex (parasites of fish and mammals), Contracaecum osculatum, C. radiatum, and C. rudolphi (parasites of mammals and fish-eating birds), Hysterothylacium aduncum (a parasite of fish), Porrocaecum angusticolle, P. crassum, P. depressum, and P. ensicaudatum (parasites of fish-eating birds). PCR-amplified rDNA regions encompassing ITS1, 5.8S rDNA, and ITS2 produced on templates of genomic DNA isolated from all investigated species were digested with TaqI, AluI, BsuRI, and RsaI endonucleases. Restriction patterns showed that endonuclease TaqI is the most useful enzyme for identification of all investigated species. No variations in restriction patterns within each species were detected. Therefore, we propose that the PCR-RFLP assay described in this report may be used for identification of marine and freshwater parasites from superfamily Ascaridoidea.     

Comparison of a manual and an automated method to estimate the number of uterine eggs in anisakid nematodes: to Coulter or not to Coulter. Is that the question?

Studies reporting numbers of eggs in vagina and utero in nematodes often give little information of the technique used for the estimations. This situation hampers comparison among studies, because, so far, differences in estimations provided by different techniques have not been assessed. This note examines whether a manual method based on visual counts in aliquots and an automated method using a Coulter counter yield equivalent estimations of egg numbers in vagina and utero of 3 anisakid nematode species (Anisakis simplex, Pseudoterranova decipiens, and Contracaecum osculatum). The number of eggs from 50 females per nematode species was estimated using both techniques. The automated and manual methods yielded similar egg counts (correlation coefficients >0.9 in the 3 species), but the methods were not always statistically equivalent. The automated method was more precise and seemed less dependent on egg density, whereas the manual method was less time-consuming (contrary to previous perceptions) and less expensive. Despite the higher precision of automated counts, the manual technique seemed to produce similar estimates; thus, it may be particularly useful in developing countries where nematode parasitism is prevalent in humans and domestic animals, but scientific resources are limited.     

Contracaecum ogmorhini复合种线粒体rrnL基因的多态性

本项目应用聚合酶链式反应单链构象多态性(PCR SSCP) 及DNA序列分析的方法, 研究了南半球Contracaecum ogmorhini两个不同种群和北半球C. margolisi一个种群在线粒体核糖体大亚基( rrnL) 基因部分序列的差异及种群遗传关系。结果显示: 南半球C. ogmorhini 两个种群在种群间及种群内的rrnL 序列相似性很高; 而北半球种群与南半球种群的rrnL序列相似性则相对较低, 它们之间rrnL 序列种间遗传差异大于种内遗传变异, 而且存在着可区分两个姊妹种的2个碱基差异。rrnL 序列分析结果支持北半球C. margolisi 相对于南半球C. ogmorhini而言, 的确是一个不同的物种。     

Multiplex PCR for the identification of Anisakis simplex sensu stricto, Anisakis pegreffii and the other anisakid nematodes

A multiplex PCR method was established for the rapid identification of Anisakis simplex sensu stricto, A. pegreffii, A. physeteris, Pseudoterranova decipiens, Contracaecum osculatum and Hysterothylacium aduncum. The sequence alignment of the internal transcribed spacer 1 region (ITS-1) between A. simplex s. str. and A. pegreffii showed a high degree of similarity, but only two C-T transitions were observed. To differentiate A. simplex s. str. from A. pegreffii, an intentional mismatch primer with an artificial mismatched base at the second base from the primer 3' end was constructed. This intentional mismatch primer, which produced a PCR band only from A. pegreffii DNA, was able to differentiate the two morphologically indistinguishable sibling species of A. simplex. Specific forward primers for other anisakid species were also designed based on the nucleotide sequences of the ITS region. The multiplex PCR using these primers yielded distinct PCR products for each of the anisakid nematodes. The multiplex PCR established in this study would be a useful tool for identifying anisakid nematodes rapidly and accurately.     

Parasite-related lesions in a bearded seal, Erignathus barbatus.

A free-ranging adult male bearded seal (Erignathus barbatus) killed by polar bears was salvaged and examined at necropsy. Significant findings included diffuse intrahepatic bile duct fibrosis and chronic cholangitis; multiple nodules of chronic fibrosing pancreatitis; and gastric ulcers. Trematode eggs (Family Campulidae) were found in the pancreatic nodules. These eggs and the trematodes that produced them probably caused the pancreatic and liver lesions. Phocanema decipiens and Contracaecum osculatum were found in the stomach lumen; several P. decipiens were attached to gastric ulcers. Sarcocystis sp. is reported for the first time in a bearded seal.     

Molecular identification of two strains of third-stage larvae of Contracaecum rudolphii sensu lato (Nematoda: Anisakidae) from fish in Poland

Contracaecum sp. larvae (L3) from fish were identified using nucleotide sequences of the internal transcribed spacers ITS-1 and ITS-2 of the ribosomal DNA. The nematode larvae originated from fish in a freshwater situation (crucian carp Carassius carassius, from Selment Wielki Lake in Mazury, northeastern Poland) and a brackish-water region (Caspian round goby Neogobius melanostomus from the Baltic Sea, Gdafisk Bay at the Polish coast). Two strains (Contracaecum rudolphii A and B) of Contracaecum rudolphii senso lato, a parasite common at the adult stage in fish-eating birds, were identified. In fish from the freshwater site, only the strain temporarily designated C. rudolphii B was identified; in the brackish-water region, both strains were found, suggesting that fish serve as paratenic host for both genotypes. Contracaecum rudolphii sensu lato has been recorded in several species of fish-eating birds in Poland, particularly in the great cormorant, Phalacrocorax carbo, in which the abundance is highest. The results, although based on a restricted number of larvae, suggest that the life cycles of both genotypes can be completed in the Polish region and that at least one of them, C. rudolphii B, can develop both in fresh and brackish water.     

Coexistence and genetic variability of Contracaecum rudolphii A and Contracaecum rudolphii B (Nematoda: Anisakidae) in cormorants, Phalacrocorax carbo sinensis, in the Baltic region

Although numerous genetically isolated entities within Contracaecum rudolphii sensu lato are presently defined, information on the distribution and ecology of these groups is nonetheless in demand. In the present study, information based upon DNA sequence data (restriction analysis and sequencing of rDNA) on the distribution of the species C. rudolphii A and C. rudolphii B in the cormorant Phalacrocorax carbo sinensis from the Baltic region (Poland and Finland) is provided. These data corroborate previous work that identified C. rudolphii A in brackish water regions and C. rudolphii B in fresh water sites, although mixed infections also occur. The 2 species may inhabit the same host specimen. One rare heterozygote of the species was recorded.     

Levels of infection of gastric nematodes in a flock of great cormorants (Phalacrocorax carbo) from Lake Biwa, Japan

A high prevalence (86.7%) of various species of nematodes was observed in the stomach of great cormorants living in Lake Biwa, Japan. There were varying numbers of adults belonging to two common genera, Eustrongylides Jagerskiold 1909 (Nematoda: Dioctophymatidae) and Contracaecum Railliet & Henry 1912 (Nematoda: Anisakidae). The first included common adenophorean nematodes comprising a single species, Eustrongylides tubifex and the second comprised ascaroid nematodes that contained four named species: Contracaecum rudolphii Hartwich, 1964, Contracaecum microcephalum Yamaguti, 1961, Contracaecum multipapillatum Drasche, 1882 and Contracaecum chubutensis Garbin, 2008. After the prevalence and intensity of the infection had been noted, both types of nematodes were frequently observed to penetrate the mucosa and intrude into the wall of the glandular stomach, where they caused gross haemorrhage and ulceration. The Eustrongylides sp. was predominantly found in a nodular lesion of the proventricular wall, while Contracaecum spp. were observed either free in the lumen of the proventriculus or, on occasion, deeply penetrating its wall. Of the Contracaecum spp., C. rudolphii was the most prevalent. Grossly, large numbers of nematodes were present in infected stomachs (for C. rudolphii intensity was 1-34 and 3-57 nematodes in male birds and 1-21 and 1-32 in females; for C. microcephalum 1-2 and 1 in male birds and 1-2 in females; for C. multipapillatum 2 in male cormorants and no infection in females; for C. chubutensis 1-2 and 1 in male birds and 1-5 and 1 in females and for E. tubifex 1-5 nematodes in male birds and 2-8 in females). Ulcerative inflammation and hyperaemia were the most common pathological presentations, especially in areas that had been invaded by parasites. Microscopically, varying degrees of granulomatous inflammatory reactions were seen, in addition to degenerated nematodes which appeared to have deeply penetrated mucosal surfaces and were surrounded by fibrous connective tissues.