Analyses of mitochondrial amino acid sequence datasets support the proposal that specimens of Hypodontus macropi from three species of macropodid hosts represent distinct species.

BACKGROUND: Hypodontus macropi is a common intestinal nematode of a range of kangaroos and wallabies (macropodid marsupials). Based on previous multilocus enzyme electrophoresis (MEE) and nuclear ribosomal DNA sequence data sets, H. macropi has been proposed to be complex of species. To test this proposal using independent molecular data, we sequenced the whole mitochondrial (mt) genomes of individuals of H. macropi from three different species of hosts (Macropus robustus robustus, Thylogale billardierii and Macropus [Wallabia] bicolor) as well as that of Macropicola ocydromi (a related nematode), and undertook a comparative analysis of the amino acid sequence datasets derived from these genomes. RESULTS: The mt genomes sequenced by next-generation (454) technology from H. macropi from the three host species varied from 13,634 bp to 13,699 bp in size. Pairwise comparisons of the amino acid sequences predicted from these three mt genomes revealed differences of 5.8% to 18%. Phylogenetic analysis of the amino acid sequence data sets using Bayesian Inference (BI) showed that H. macropi from the three different host species formed distinct, well-supported clades. In addition, sliding window analysis of the mt genomes defined variable regions for future population genetic studies of H. macropi in different macropodid hosts and geographical regions around Australia. CONCLUSIONS: The present analyses of inferred mt protein sequence datasets clearly supported the hypothesis that H. macropi from M. robustus robustus, M. bicolor and T. billardierii represent distinct species.

An aspartyl protease inhibitor orthologue expressed by Parelaphostrongylus tenuis is immunogenic in an atypical host.

Parelaphostrongylus tenuis is a neurotropic nematode common in white-tailed deer (Odocoileus virginianus) of eastern North America. This parasite is the causative agent of a debilitating neurologic disease in atypical hosts, including domestic livestock. In order to identify proteins of potential significance in the host-parasite relationship, a cDNA library was produced from adult P. tenuis mRNA. Screening the library with antisera from infected red deer (Cervus elaphus elaphus) and immunized AO strain rats, we identified clones with sequence similarities to aspartyl protease inhibitors from several parasitic nematodes. Antibody that was generated against this recombinant protein of P. tenuis (Pt-API-1) detected the native protein in E/S products, in muscle and gonad, and on the surface of the cuticle of adult male and female P. tenuis. The native protein was detected in internal structures of first-stage (L1) and third-stage (L3) larvae. Reverse transcription-PCR confirmed expression of Pt-api-1 in L1, L3, and adult male and female worms. Expression of Pt-API-1 throughout the life cycle of P. tenuis suggests an essential function. Antibodies specific for recombinant Pt-API-1 were detected by enzyme-linked immunosorbent assay in sera from 12 red deer experimentally infected with P. tenuis. Antibodies were detected within 28 to 56 days postinfection. Responses were sustained or biphasic in animals with patent infections, consistent with expression of Pt-API-1 by L1. Our results are compatible with findings in other parasitic nematodes showing that aspartyl protease inhibitors are highly immunogenic.

A preliminary analysis of proteolytic activity of excretory-secretory products from Cyathostominea.

The excretory-secretory product (ESP) derived from Cyathostominea in vitro was assessed, in terms of subunit composition, and proteolytic activity using as substrates azocasein and two synthetic fluorogenic peptides. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved 13 subunits, and the presence of the protein cysteine proteinase activator dithiothreitol (DTT) revealed 21 subunits. DTT also enhanced azocaseinolysis, and hydrolysis of carbobenzoxy-phenylalanyl-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-NHMec) and carbobenzoxy-arginyl-arginine-7-amido-4-methylcoumarin (Z-Arg-Arg-NHMec). At the optimum pH of 5.5, hydrolysis of Z-Phe-Arg-NHMec was three-fold greater than that of Z-Arg-Arg-NHMec suggesting that the proteolytic specificities of the ESP are more like those of papain or cathepsin L, rather than cathepsin B. In SDS-PAGE gelatin gels, DTT was a requirement for proteolysis by the ESP. Optimum resolution was at pH=5.5, resolving six bands ranging from 114-20kDa. Cysteine proteinase inhibitors abolished all gelatinolytic activity at the pH values tested. Such data indicate the presence of cysteine-class proteinases in the ESP of Cyathostominea.

Characterization of Chabertia ovina by isoenzyme gel electrophoresis: comparative study with Oesophagostomum venulosum.

The glucose-6-phosphate dehydrogenase (G6PD, EC.1.1.1.49), glucose phosphate isomerase (GPI, EC.5.3.1.9), and malate dehydrogenase (MDH, EC.1.1.1.37) isoenzymatic patterns of Chabertia ovina were determined by starch-gel electrophoresis. The G6PD and GPI isoenzymatic patterns were characterized by the existence of three phenotypes: (1) a single and slow anodic band, (2) a single and fast anodic band, and (3) a large spot matching its migration with bands 1 and 2. These three phenotypes may be explained as the existence of only one gene locus for the G6PD and GPI in C. ovina. Allelic frequencies and the Hardy-Weinberg test were determined. This test indicated that the population was not in Hardy-Weinberg equilibrium. The MDH isoenzymatic pattern of C. ovina was characterized by the presence of two bands with anodic and cathodic migration. Furthermore, comparative isoenzyme studies were carried out between Oesophagostomum venulosum and C. ovina. The different G6PD, GPI, and MDH isoenzymatic patterns observed for the two species allowed us to distinguish them and, therefore, to use isoenzymatic patterns as a diagnostic tool to discriminate these species.

Evidence for hybridisation between Paramacropostrongylus iugalis and P. typicus (Nematoda:Strongyloidea) in grey kangaroos, Macropus fuliginosus and M. giganteus, in a zone of sympatry in eastern Australia.

Specimens of Paramacropostrongylus iugalis and P. typicus, collected from eastern (Macropus giganteus) and western (M. fuliginosus) grey kangaroos in New South Wales and Queensland, were examined morphologically and electrophoretically at 4 enzyme loci previously demonstrated to be diagnostic between the 2 species. Collections of P. iugalis from M. giganteus from outside the zone of sympatry of the 2 kangaroo species conformed electrophoretically and morphologically with previous studies. Within the zone of sympatry, the 2 nematode species were distinguishable electrophoretically, with most P. iugalis occurring in M. giganteus and all P. typicus occurring in M. fuliginosus. Some specimens of P. iugalis were identified in M. fuliginosus and, in both host species, nematodes were encountered with electrophoretic profiles intermediate between P. iugalis and P. typicus. The frequent occurrence in these specimens of heterozygotes suggested that the genetic barriers between the 2 nematode species were not complete and that genetic interchange (i.e. hybridisation) was occurring.

An electrophoretic analysis of patterns of speciation in Cloacina clarkae, C. communis, C. petrogale and C. similis (Nematoda:Strongyloidea) from macropodid marsupials.

An electrophoretic study was conducted on Cloacina clarkae, C. communis, C. petrogale and C. similis based on 19 enzyme loci. C. communis was widely distributed in Macropus robustus, showing some genetic variation among populations but occasionally switching to other macropodid hosts (M. agilis, M. antilopinus). C. similis occurred in members of the Petrogale penicillata complex, Macropus dorsalis and Thylogale billardierii, but showed no evidence of genetic differentiation in spite of its occurrence in different host species and in geographically distinct regions of Australia. C. clarkae from Macropus eugenii was genetically indistinguishable from C. similis and was considered synonymous with it. C. petrogale occurred in a similarly diverse range of hosts and geographical regions to C. similis, but was represented electrophoretically as 4 distinct genetic species, 1 in Petrogale assimilis, a second in P. lateralis purpureicollis, a third in Macropus parryi in Queensland and a fourth in M. eugenii in South Australia. Although the host and geographical ranges of C. similis and C. petrogale are analogous, the genetic uniformity of the former and diversity of the latter illustrate the incomplete understanding we have of the immediate causes of speciation in nematodes.

Studies on the effect of fenbendazole and mebendazole on some enzymes of swine kidney worm Stephanurus dentatus.

The effect of fenbendazole and mebendazole on the activity of some enzymes of the homogenates of swine kidney worm Stephanurus dentatus was investigated. Fenbendazole at 10(-5) M inhibited malate oxidation by 49% and 51% and oxaloacetate reduction by 33% and 40% whereas, mebendazole at 10(-5) M diminished malate oxidation by 25% and 35% and oxaloacetate reduction by 12% and 14% in male and female S. dentatus, respectively. Lactate dehydrogenase activity was inhibited by 45% and 50% in male and female worm respectively by 10(-5) M fenbendazole. Aldolase activity in both male and female S. dentatus was inhibited by 10(-5) M fenbendazole and mebendazole. Fenbendazole at 10(-5) M caused moderate inhibition of acid and alkaline phosphomonoesterases but mebendazole did not show a significant effect on these enzymes. Cholinesterase activity was not affected significantly with either compound. The possible mode of action of the two compounds is compared.

Sibling species within Macropostrongyloides baylisi (Nematoda: Strongyloidea) from macropodid marsupials.

Macropostrongyloides baylisi from four different species or subspecies of host were analysed electrophoretically at 27 enzyme loci. The results revealed the existence of two species, one in Macropus giganteus and the other in M. robustus robustus, M.r. erubescens and M.r. parryi, that had fixed genetic differences at 33% of loci. Populations of nematodes from two subspecies of M. robustus, M.r. robustus from Queensland and M.r. erubescens from South Australia, had fixed genetic differences at two (7.4%) of 27 loci and were considered to belong to the same species. No fixed genetic differences were detected between nematodes from M. parryi and M.r. robustus. A discriminant function analysis of morphological data assigned 96% of specimens to groups defined on the basis of the host species or subspecies from which they were obtained. This separation of Ma. baylisi into host-specific groups did not, however, totally correlate with the electrophoretic data. The species of M. baylisi in M. giganteus was genetically more distinct from the sibling species in M. robustus/M. parryi than to a related but morphologically dissimilar nematode, Ma. yamagutii from M. fuliginosus. This suggests an evolutionary parallel between host and parasite at the genetic level which is not reflected by morphological differences.

Detection by allozyme electrophoresis of cryptic species of Hypodontus macropi (Nematoda: Strongyloidea) from macropodid marsupials.

Allozyme electrophoresis of 98 Hypodontus macropi from eight different species of hosts using 24 enzymes revealed a complex of at least six sibling species, with 15-50% fixed genetic differences between taxa. Except for the taxon parasitizing Macropus rufus/M. robustus, pairs of parasite taxa were, in each case, sympatric at each locality examined, thus supporting the conclusion that they represent valid species. The existence of a series of host-specific nematode taxa explains many of the inconsistencies noted previously in the host distribution of H. macropi. Comparison of parasite allozyme phenograms with host phylogeny suggests that four of the speciation events could be attributable to cospeciation and two to host switching. A clear case of host switching between M. rufus/M. robustus and M. fuliginosus was found.

Demonstration of collagenase activity in adult Strongyloides ratti (Nematoda:Strongyloididae) and its absence in the infective larvae.

Larvae and adults of Strongyloides ratti were examined for collagenolytic activity on 14C proline-labelled, native, guinea-pig skin collagen substrate. The activity was measured by determining either the amount of hydroxyproline released or the amount of radioactivity in the solubilized fraction of the collagen substrate. Bacterial collagenase was used for enzyme control and trypsin served as substrate control. No collagenolytic activity was found in living larvae, their extracts or metabolites. The collagenolytic activity of the metabolites of adult worms appeared weak, whereas that of the extracts of the adults was pronounced. It is suggested that collagenase is active in the adult females at the time of migration in the intestinal mucosa during oviposition.