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.     

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.     

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 Nei's 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 Nei's 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 Nei's 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.     

Nematode infestation of fillets from Atlantic cod, Gadus morhua, off eastern Canada

The prevalence and intensity of larval nematodes in fillets of Atlantic cod, Gadus morhua, were examined and compared with similar data from a previous survey conducted about 30 yr ago. Anisakis simplex occurred more often in the nape of the fillet, whereas Pseudoterranova decipiens was the predominant species in napeless fillets. The results suggest an increase in both the prevalence and intensity of P. decipiens in fillets of cod, especially those originating from the Gulf of St. Lawrence and coastal Nova Scotia.     

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.     

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.     

On the ecology and distribution of Pseudoterranova decipiens C (Nematoda: Anisakidae) in an intermediate host, Hippoglossoides platessoides, in northern Norwegian waters.

The distribution of Pseudoterranova decipiens C helps demonstrate the ecological basis of this genetically defined sibling species. In northern Norwegian waters the major fish intermediate host is Hippoglossoides platessoides. Overall prevalence, mean intensity and intensity range in H. platessoides were 15%, 16.5 and 1-165, respectively. Outside the range of its only known definitive host, the seal Erignathus barbatus, the parasite was not found in the same intermediate host.     

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.     

Polychaeta collected by U. D. Gaikwad at Ratnagiri, south of Bombay

A collection of polychaete worms, obtained by Mr U. D. Gaikwad from sandy and rocky shores near the Ratnagiri Marine Biological Station which is some 240 km south of Bombay, included 50 species. Among them were seven new species namely: Intoshella indica, Hololepidella maculata, Lepidasthenia tubicola. Nereis (Nereis) gaikwadi, Polydora gaikwadi, Cirratulus indicus, and Sobellaria simplex The remaining species belong to the Indo-west Pacific fauna but several have not previously been recorded from India itself. Glycera decipiens Marenzeller, Dorvillea matsushimaensis (Okuda & Yamada) and Mesochaetopterus japonicus Fujuwara, known only from Japan, have been re-described. Nicolea gracilibranchis Grube is shown to be a synonym of the widely distributed N. venustula (Montagu) and Terebella yappensis Okuda, originally described as a variety of T. ehrenbergi, is raised to species rank.     

Gastrointestinal helminths of the cetaceans Phocoena spinipinnis (Burmeister, 1865) and Cephalorhynchus eutropia (Gray, 1846) from the southern coast of Chile.

Gastrointestinal helminths of the cetaceans Cephalorhynchus eutropia and Phocoena spinipinnis accidentally entangled in gillnets off the coast of Queule, Chile, were identified from 1989 to 1990. Pseudoterranova sp., Polymorphus (Polymorphus) cetaceum and Synthesium tursionis occurred in both cetaceans. Additionally, Anisakis sp. and Braunina cordiformis were found in C. eutropia, and Anisakis simplex was identified from P. spinipinnis. The species with the highest prevalence and mean intensity of infection in P. spinipinnis and C. eutropia were P. (P.) cetaceum and B. cordiformis, respectively. The diet of both cetaceans consists mainly of fishes.     

Integrative taxonomy of Leptonetela spiders (Araneae,Leptonetidae), with descriptions of 46 new species

Extreme environments,such as subterranean habitats,are suspected to be responsible for morphologically inseparable cryptic or sibling species and can bias biodiversity assessment.A DNA barcode is a short,standardized DNA sequence used for taxonomic purposes and has the potential to lessen the challenges presented by a biotic inventory.Here,we investigate the diversity of the genus Leptonetela Kratochvíl,1978 that is endemic to karst systems in Eurasia using DNA barcoding.We analyzed 624 specimens using one mitochondrial gene fragment (COI).The results show that DNA barcoding is an efficient and rapid species identification method in this genus.DNA barcoding gap and automatic barcode gap discovery (ABGD) analyses indicated the existence of 90 species,a result consistent with previous taxonomic hypotheses,and supported the existence of extreme male pedipalpal tibial spine and median apophysis polymorphism in Leptonetela species,with direct implications for the taxonomy of the group and its diversity.Based on the molecular and morphological evidence,we delimit and diagnose 90 Leptonetela species,including the type species Leptonetela kanellisi (Deeleman-Reinhold,1971).Forty-six of them are previously undescribed.The female of Leptonetela zhai Wang & Li,2011 is reported for the first time.Leptonetela tianxinensis (Tong & Li,2008) comb.nov.is transferred from the genus Leptoneta Simon,1872;the genus Guineta Lin & Li,2010 syn.nov.is a junior synonym of Leptonetela;Leptonetela gigachela (Lin & Li,2010)comb.nov.is transferred from Guineta.The genus Sinoneta Lin & Li,2010 syn.nov.is a junior synonym of Leptonetela;Leptonetela notabilis (Lin & Li,2010) comb.nov.and Leptonetela sexdigiti (Lin & Li,2010) comb.nov.are transferred from Sinoneta;Leptonetela sanchahe Wang & Li nom.nov.is proposed as a replacement name for Sinoneta palmata (Chen et al.,2010) because Leptonetela palmata is preoccupied.     

Sporothrix brunneoviolacea and Sporothrix dimorphospora, two new members of the Ophiostoma stenoceras-Sporothrix schenckii complex

Sporothrix inflata is a saprobic member of the Ophiostoma stenoceras-Sporothrix schenckii species complex, reported mainly from soil. Ophiostoma bragantinum, an ascomycete described from Brazil, has been proposed as its possible teleomorph. Previous studies revealed that Sporothrix inflata is phenotypically and genetically variable, suggesting the existence of cryptic species. During a continued survey on the biodiversity of microfungi from different countries, seven isolates morphologically similar to S. inflata were obtained from soil samples collected in Spain and USA. In this study their phenotypic features and phylogenetic relationships were assessed. DNA sequence data of two nuclear loci revealed that these isolates correspond to two unnamed clades in S. inflata s.l., one of which also included the type strain of Humicola dimorphospora, a species that traditionally has been considered a synonym of S. inflata. These two groups are proposed herein as Sporothrix brunneoviolacea sp. nov. and Sporothrix dimorphospora comb. nov. S. brunneoviolacea is characterized phenotypically by the production of a diffusible violet-brown pigment in culture and mostly globose, pigmented, lateral blastoconidia. On the other hand S. dimorphospora lacks diffusible pigments and shows mostly subglobose to obovoid pigmented lateral blastoconidia. In contrast to the type strain of S. inflata S. brunneoviolacea and S. dimorphospora assimilate raffinose. The phylogenetic analysis suggested that the proposed anamorph-teleomorph connection between S. inflata and O. bragantinum might not be correct.     

The importance of sculpin (Myoxocephalus scorpius) as intermediate host and transmitter of the sealworm Pseudoterranova decipiens.

A total of 186 sculpins (Myoxocephalus scorpius) were caught at Vega, Nordland, Norway, and examined for sealworm larvae (Pseudoterranova decipiens). Over 80% of the fish were infected. Very high infections were found with a maximum of nearly 300 larvae in one sculpin. Infections of this magnitude have so far not been reported from sculpins or any other fish species of this size. The importance of the sculpin in the life cycle and in the transmission of P. decipiens to seals or other fish species is briefly discussed.     

Larval anisakids parasitizing the blue whiting, Micromesistius poutassou, from Motril Bay in the Mediterranean region of southern Spain

A total of 301 blue whiting, Micromesistius poutassou Risso, 1826, ranging in length from 17 to 28 cm, from Motril Bay (Mediterranean coast, south Spain) were examined for anisakid nematodes, as these fish are common items in the Spanish Mediterranean diet. Three anisakid species were morphologically identified with a total prevalence of 10.63%. Anisakis simplex s.l. Rudolphi, 1809 had a prevalence value of 6.65%, compared with 2.66% for A. physeteris Baylis, 1923 and 2.33% for Hysterothylacium aduncum Rudolphi, 1802. Variations in prevalence values with season and host size are discussed. Allozyme markers (leucine aminopeptidase-1) were used to identify anisakid nematodes assigned to the A. simplex complex and all examined larvae were found to correspond genetically to A. pegreffii Nascetti et al., 1986.     

我国大陆细蜉属Caenis 修订及一新种描述(蜉蝣目:细蜉科)

我国大陆细蜉属Caenis共知5种,其中短铗细蜉Caenis parviforcipis(Zhou et al.)n．comb．由短尾蜉属Brachycercus转移而来;中华细蜉Caenis sinensis Gui et al．稚虫形态在本文中首次描述;另外从标本来看,已知的黑铗细蜉Caenis nigroforceps Zhou et al．实际为近岸细蜉Caenis rivulorum Eaton的新异名;本文描述1新种：花斑细蜉Caenis melanoleuca,sp．nov．;第5种黑点细蜉Caenis nigropunctata Klapalek也根据标本得到了确认。文中还给出了以上5种成虫和4种稚虫的检索表。所有标本都保存在南京师范大学生命科学学院。     

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.     

中国豆科和樟科几种植物学名的订正

在编写《Flora of China》过程中,根据标本和文献的查阅,对国产豆科和樟科几种植物学名进行修订。本文记述5个新组合、4个新异名和一个恢复的种名。5个新组合是:垂果山蚂蝗Desmodium strigilosum var.pendenticarpum(C.Z.Gao Q.R.Lai) P.H.Huang,心叶山蚂蝗Desmodium flexuosum var.cordifoliolatum(P.C.Li) P.H.Huang,密毛长柄山蚂蝗Podocarpium densum(C.Chen et X.J.Cui)P.H.Huang,东北长柄山蚂蝗Podocarpium podocarpum var.mandshuricum(Maxim.) P.H.Huang和尖叶长柄山蚂蝗Podocarpium podocarpum var.japon-icum(Matsum.)P.H.Huang.4个新异名如下:李氏木姜子Litsea lii C.E.Chang及其变种能汉木姜子var.nunkao-tahangensis(Liao) Liao作为竹叶木姜子L.pseudoelongata Liou Ho的新异名,竹头角木姜子L.akosensis var.chitouchiaoensis Liao作为台湾木姜子L.hayatae Kanehira的新异名和密毛山蚂蝗Desmodium densum (C.Chen et X.J.Cui)H.Ohashi作为密毛长柄山蚂蝗Podocarpium densum (C.Chen et X.J.Cui)P.H.Huang的新异名。由于Litsea pedicellata一名被占用,恢复Litsea taiwaniana Kamikoti作为黄肉树合法的种名。     

Nematodes of elasmobranch fishes from the southern coast of Brazil

New records for nematode species recovered from elasmobranch fishes in Brazil are established and new systematical arrangements proposed. Parascarophis sphyrnae Campana-Rouget, 1955 from the spiral valve of Sphyrna zygaena is referred for the first time in South America as a new host record. Procamallanus (S.) pereirai Annereaux, 1946, from the spiral valve of Raja castelnaui is reported parasitizing an elasmobranch host. Nematode larvae of the genera Anisakis, Contracaecum, Pseudoterranova and Raphidascaris are listed from the stomach and spiral valves of several hosts. Anisakidae larvae previously referred in Brazil in the genus Phocanema should be reallocated in Pseudoterranova. Nematodes of the genera Anisakis, Contracaecum, Pseudoterranova and Raphidascaris are reported for the first time parasitizing elasmobranchs in Brazil.