A morphological and molecular study of Pseudocorynosoma Aznar, Pérez Ponce de León and Raga 2006 (Acanthocephala: Polymorphidae) from Mexico with the description of a new species and the presence of cox 1 pseudogenes.

Pseudocorynosoma tepehuanesi n. sp., is described from the intestine of the ruddy duck Oxyura jamaicensis Gmelin, 1789 from single locality from northern Mexico. The new species is mainly distinguished morphologically from the other five described species of Pseudocorynosoma from the Americas (P. constrictum, type species, P. peposacae, P. anatarium, P. enrietti and P. iheringi) associated with waterfowl species by possessing a proboscis with 15 longitudinal rows with 7-8 hooks each, a trunk expanded anteriorly and by having smaller lemniscus. Partial sequences of the mitochondrial gene cytochrome c oxidase subunit I (cox 1) and the large subunit (LSU) of ribosomal DNA including the domains D2+D3 were used independently to corroborate the morphological distinction between the new species and other two congeneric species (P. constrictum and P. anatarium) from North America. The genetic divergence estimated among the new species and the other two species ranged from 15 to 18% for cox 1 and from 3.2 to 4% for LSU. The cox 1 alignment shows 24 sequences from P. anatarium with abnormalities, which were defined as pseudogenes due the presence of insertions, deletions and premature stop codons. Maximum likelihood and Bayesian inference analyses with each data set showed that the acanthocephalans from ruddy duck represent an independent clade with strong bootstrap support and posterior probabilities. The phylogenetic tree inferred with cox 1 gene placed all the pseudogenes from P. anatarium in single clade suggesting that those genes arose after speciation process within genus Pseudocorynosoma. The morphological evidence, plus the monophyly in both phylogenetic analyses indicate that the acanthocephalans collected from intestine of the ruddy duck from northern Mexico represent a new species.

Validating the systematic position of Profilicollis Meyer, 1931 and Hexaglandula Petrochenko, 1950 (Acanthocephala: Polymorphidae) using cytochrome c oxidase (Cox 1).

Members of the Polymorphidae (Acanthocephala) are distributed worldwide as endoparasites of marine mammals, fish-eating birds, and waterfowl. The family contains 10 genera, with approximately 127 species. Polymorphids are characterized by having a spinose trunk with a bulbous proboscis, double-walled proboscis receptacle, long lemnisci, and 4 tubular cement glands. The taxonomic position of several genera within Polymorphidae has been controversial when considering morphological and ecological characters. The mitochondrial coding gene cytochrome-c oxidase representing species of 5 genera of polymorphids (Corynosoma, Lühe, 1904, Hexaglandula Petrochenko, 1950, Southwellina Witenberg 1932, Polymorphus Luhë, 1911, and Profilicollis Meyer, 1931) were sequenced to determine the sister-group relationships among 2 particular genera, i.e., Hexaglandula, and Profilicollis. Maximum likelihood and Bayesian analyses showed that Polymorphidae is a monophyletic assemblage, and that 3 major clades are present. Our results provide support for the idea that Hexaglandula represents an independent lineage, whereas, in the case of Profilicollis, there is no conclusive evidence that they are not members of Polymorphus. The analyses also confirm that Polymorphus is paraphyletic, suggesting that the genus represents a complex of species that should be reexamined and reclassified using morphological, ecological, and molecular data. Our observations suggest that decapods (intermediate hosts for the 2 genera under study) were independently colonized at least twice during the evolutionary history of the group.

An acanthocephalan parasite increases the salinity tolerance of the freshwater amphipod Gammarus roeseli (Crustacea: Gammaridae).

Studies of the influence of parasites on host fitness generally conclude that parasites have a strong negative effect on their hosts. In this study, we have investigated experimentally the role of Polymorphus minutus, an acanthocephalan parasite, on the salinity tolerance of the freshwater amphipod Gammarus roeseli, one of its intermediate hosts. Unexpectedly, P. minutus-infected gammarids were more tolerant to salinity stress than uninfected ones. The mean lethal salt concentrations for 50% mortality of hosts tested were 17.3 (infected) and 9.7 g/L (uninfected). The parasitic load (one or two parasites per host) did not affect the result. The size of hosts had no significant influence on the salinity tolerance of either infected or uninfected gammarids. The mobility of all types of gammarid decreased when the salinity exceeded 9.0 g/L, but there was no significant difference between infected and uninfected gammarids. We discuss the higher salinity tolerance of infected amphipods in relation to O(2) consumption and osmoregulation. Finally, we demonstrate that the salinity tolerance is enhanced in the parasitized amphipod but without a significant change in behavior or an osmoregulatory adjustment.

Evidence for the hypothesis of strain formation in Pomphorhynchus laevis (Acanthocephala): an investigation using mitochondrial DNA sequences.

A hypothesis has been erected stating that in the British Isles the acanthocephalan, Pomphorhynchus laevis can be separated into 3 strains, an English, Irish and marine strain. Ecological and morphological evidence exists in support of this hypothesis. An investigation at the molecular level was conducted in order to test the validity of the existing evidence. A mitochondrial gene, subunit I of cytochrome c oxidase was partially sequenced from 3 Irish populations of P. laevis, 1 Scottish population and 3 English populations. P. laevis sequences from brown trout from Ireland, England and Scotland were very similar, showing a mean sequence divergence of 0.7%. Sequences from two populations of P. laevis from English chub and bullhead were also similar to each other (0.35% divergence). These two groups of sequences, the brown trout group and the chub/bullhead group were 2.2% different. These data confirm the existence of at least 2 strains in Ireland and Britain, although there is evidence to suggest that these strains are defined by their host species rather than by their geographical distributions.

Proteolytic enzymes of Pomphorhynchus laevis and in three other acanthocephalan species.

Cystacanths and adults of the acanthocephalan Pomphorhynchus laevis were found to release proteolytic enzymes during in vitro culture. Culture media in which cystacanths had been kept contained a trypsinlike collagenolytic proteinase exhibiting a molecular mass of 29 kDa on gelatin substrate gel electrophoresis and a pH optimum at pH 9.0. Samples of adults possessed a trypsinlike collagenolytic proteinase with a molecular mass of 26 kDa and showed a pH optimum at 9.0. Leucine aminopeptidase activity was also present in culture media of adults. An apparent molecular mass of 92 kDa and a pH optimum at pH 8.5 were determined for this proteolytic enzyme. It was concluded that the trypsinlike proteinases of both stages were necessary for the complete and quick perforation of the fishes' intestinal wall, whereas the leucine aminopeptidase seemed to have a nutritional function in the terminal stages of protein hydrolysis at the surface of the worm's body wall. Cystacanths of 3 other species of fish-parasitizing Acanthocephala (Acanthocephalus anguillae, Acanthocephalus lucii, Paratenuisentis ambiguus) did not show any histolytic activity.

Leucine aminopeptidase activity in adults of Pomphorhynchus laevis (Acanthocephala): a histochemical study.

In frozen sections of the acanthocephalan Pomphorhynchus laevis, which is a frequent intestinal parasite of cyprinid and salmonid fishes, leucine aminopeptidase (APase) was localized histochemically in outer parts of the presomal bulbus as well as in all layers and most nuclei of the metasomal body wall. Enzyme activity visualized at pH 6.5 using L-leucyl-4-methoxy-2-naphtylamide as the substrate was also associated with ovarian balls, immature larvae, and the testes. The results are discussed with respect to the possible function of APases and the proposed sites of amino acid uptake in tissues of P. laevis.

Molecular systematics in the acanthocephalan genus Echinorhynchus (sensu lato) in northern Europe.

New biological species and high levels of inter- and intraspecific genetic divergence were discovered in an allozyme study of some North European members of the acanthocephalan genus Echinorhynchus (sensu lato), parasites of fish and malacostracan crustaceans. (i) A strong differentiation between the marine E. gadi and the fresh- and brackish-water E. salmonis (genetic identity I congruent to 0) supports a generic distinction between these taxa; however, the subdivision would not entirely concur with the concepts of Echinorhynchus (sensu stricto) and Metechinorhynchus suggested earlier. (ii) Samples of E. gadi from the Baltic, Norwegian and North Seas included three distinct, partially sympatric biological species (spp. I-III; I congruent to 0.5). (iii) E. bothniensis, previously only known from the northern Baltic Sea, represents a complex of freshwater taxa with an intermediate host relationship to the 'glacial relict' Mysis spp. and with a distributional and host analogy to the North American E. leidyi. A population in a northern lake in the Barents Sea basin is closely related to E. bothniensis of the Baltic area, but is probably specifically distinct; the divergence between these populations (I congruent to 0.6) is similar to that between their Mysis host species. (iv) Considerable intraspecific differentiation (FST = 0.25), probably reflecting post-glacial population bottlenecks, was found between Baltic and nearby lacustrine E. bothniensis, and between Atlantic and Baltic E. gadi sp. I.

Cystathionine beta-synthase and gamma-cystathionase in helminths.

The activities of gamma-cystathionase and cystathionine beta-synthase were investigated in a range of gastrointestinal, free-living and entomophagous nematodes. Although nematode gamma-cystathionase used the same range of substrates as the mammalian hepatic enzyme, its activity was extremely low and there were significant interspecies variations with respect to the relative order of active substrates. Like the mammalian liver enzyme, nematode cystathionine beta-synthase showed activity in the directions of both cystathionine synthesis and the forward and reverse "L-serine sulphhydrase" reactions. However, the most important feature of the survey was the widespread ability of nematode cystathionine beta-synthase to catalyse the non-mammalian "activated L-serine sulphhydrase" reaction (L-cysteine + R-SH----cysteine thioether + H2S). Additional survey work revealed that the ability to catalyse the activated L-serine sulphhydrase reaction was almost universal amongst nematodes. Activated L-serine sulphhydrase activity was also demonstrated in the acanthocephalan Pomphorhynchus laevis but was absent from cestodes and digeneans.

Specificity of the surface aminopeptidase in Moniliformis moniliformis (Acanthocephala).

The hydrolysis of various oligopeptides in solution by intact Moniliformis moniliformis was examined using paper chromatographic analysis of the incubation medium. In the presence of transport inhibitors, the respective peptide sub-units and/or amino acid residues accumulated in the bathing medium. Only peptides with serine, methionine, leucine or alanine at the NH2-terminal end of the peptide were hydrolysed. There was no hydrolysis when these amino acids were located internally or at the COOH-terminus indicating genuine aminopeptidase activity of the class, alpha-aminoacylpeptide hydrolase. Hydrolysis was negligible when the NH2-terminus was arginine, aspartic acid, glutamic acid, glycine, histidine, lysine, phenylalanine, proline, tryptophan, tyrosine, or valine. In separate experiments, mediated uptake of 0.1 mM 3H-leucine by the worms in 2 min was inhibited 100% by 5 mM unlabelled leucine or tri-serine, but only partially inhibited by 5 mM Ser-Gly (66%), 10 mM Ser-Gly (74%), 5 mM Leu-Leu (69%), 10 mM Leu-Leu (70%), 5 mM Leu-Gly (58%) or 5 mM Met-Met (69%). Because the inhibitions produced by 5 mM Leu-Leu plus 5 mM Met-Met (79%) or 5 mM Leu-Leu plus 5 mM Ser-Gly (76%) were not additive, a single enzyme is indicated. The name serine aminopeptidase is proposed because of its preference for serine.

A study of phosphoenolpyruvate carboxykinase from Moniliformis dubius (Acanthocephala).

Procedures have been developed for the extraction and subsequent purification of the enzyme phosphoenolpyruvate carboxykinase (GTP) (PEPCK) from Moniliformis dubius (Acanthocephala), a parasite of the rat small intestine. This is believed to be the first purification of PEPCK from an invertebrate animal. The enzyme, when purified to homogeneity as indicated by electrophoretic criteria, has a molecular weight of 73 700. Kinetic studies indicated that the enzyme had a pH optimum of 5.5 and required Mn2+ as the divalent cation. The apparent Km values determined for the substrates of the carboxylation reaction were low compared with the published values for purified PEPCK from vertebrate sources. Several competitive inhibitors were found and their Ki values determined. The possible regulation of PEPCK activity in M. dubius is discussed with reference to the observed kinetic parameters.