Florence Huby-Chilton

First complete large subunit ribosomal RNA sequence and secondary structure for a parasitic nematode: phylogenetic and diagnostic implications

The complete sequence and secondary structure of the large subunit of nuclear ribosomal RNA(LSUrRNA) were determined for the parasitic nematode Labiostrongylus bipapillosus (order Strongylida). Its LSU rRNA sequence was shorter (by 18 bp) than that of the free-living nematode, Caenorhabditis elegans (order Rhabditida), the only other species within the Nematoda for which a complete LSU rRNA sequence has been determined. Interspecific differences in sequence were greater in the 12 D domains compared with the core segments, with the secondary structure being maintained by partial or complete compensatory base pair changes. The magnitude of interspecific sequence difference in each D domain (except for D6 and D12) was similar, suggesting that several domains contain informative genetic markers for phylogenetic studies of the phylum Nematoda at different taxonomic levels. The LSU rRNA may also provide species-specific markers for the identification of some bursate nematodes of veterinary and medical importance.

Genetic variation within species of the nematode genus Cloacina (Strongyloidea : Cloacininae) parasitic in the stomachs of rock wallabies, Petrogale spp. (Marsupialia : Macropodidae) in Queensland

Four morphospecies of Cloacina, parasitic nematodes in the stomachs of rock wallabies (Petrogale spp.) from Queensland, were compared genetically using sequence data of the two internal transcribed spacers of nuclear ribosomal DNA (rDNA). The results suggest that two geographically isolated populations of C. ernabella from P. purpureicollis were genetically distinct. Based on the autapomorphic species concept, these two C. ernabella populations represented different species. For the three other nematode morphospecies, there were genetic differences among individuals of a morphospecies present in different species of host. The results suggest that each may represent a complex of sibling species, with a different species present in each species of rock wallaby examined for that morphospecies. In the C. caenis and C. pearsoni complexes, the lineage present in P. purpureicollis from western Queensland represents a sister taxon to those in the P. pencillata complex from the east coast. In the C. robertsi complex, the taxon parasitic in P. persephone represents the sister taxon to those in the P. pencillata complex and in P. purpureicollis. C. robertsi was found for the first time in P. purpureicollis from Winton in central Queensland, suggesting contact in the recent past between populations of P. purpureicollis and a member of the P. penicillata complex.

Single-strand conformation polymorphism analysis of genetic variation in Labiostrongylus longispicularis from kangaroos

Single‐strand conformation polymorphism (SSCP) analysis was employed to screen for sequence heterogeneity in the second internal transcribed spacer (ITS‐2) of ribosomal (r) DNA of Labiostrongylus longispicularis,a parasitic strongylid nematode occuring in some species of kangaroo in different geographical regions of Australia. The results showed that most of the nematodes screened had different SSCP profiles, which were subsequently shown to correspond to polymorphisms and/or an indel in the ITS‐2 sequence. These variable sites related mainly to unpaired regions of the predicted secondary structure of the precursor rRNA molecule. SSCP profiles could be used to distinguish L. longispicularisin Macropus robustus robustus(New South Wales) from L. longispicularisin Macropus robustus erubescensand Macropus rufus(South Australia). This difference corresponded to a transversional change in the ITS‐2 sequence at alignment position 82. The study demonstrated clearly the effectiveness of SSCP analysis for future large‐scale population genetic studies of L. longispicularisin order to test the hypothesis that L. longispicularisfrom different geographical regions represents multiple sibling species.*

Genetic variation within the Hypodontus macropi (Nematoda: Strongyloidea) complex from macropodid marsupial hosts in Australia

Genetic variation was investigated in the strongylid nematode Hypodontus macropi from macropodid marsupials using the second internal transcribed spacer of ribosomal DNA. A total of 547 specimens from ten species of hosts, representing all of the known hosts of the parasite, from across the Australian continent was examined. Phylogenetic analyses revealed distinct genetic clades in each of Macropus agilis, M. dorsalis, M. rufogriseus, M. bicolor, Petrogale persephone, Thylogale billardierii and T. stigmatica. A further clade contained all specimens from M. robustus and M. rufus, together with two examples of host switching by nematodes into M. fuliginosus. The latter clade was subdivided into three subclades, one comprising specimens occurring in M. robustus erubescens, M. rufus and M. fuliginosus, the second in M. r. woodwardi and the third in M. r. robustus suggesting a relationship between the subclades and the subspecies of M. robustus. The extent of the genetic differences and the fact that several of them occur in broad sympatry suggests that H. macropi as currently defined morphologically may represent as many as ten cryptic species. Limited evidence was found for co‐speciation between hosts and parasites; rather most relationships were better explained by host switching.

Detection and identification of Tetratrichomonas in a preputial wash from a bull by PCR and SSCP.

Motile Tritrichomonas foetus-like trichomonads were found during microscopic examination of a wet mount sample of a preputial wash collected from a bull. Staining of the organisms with a modified Wright-Giemsa stain revealed that several had four anterior flagella of unequal length instead of the three anterior flagella of equal length characteristic of T. foetus. Limited propagation of these organisms was achieved in InPouch medium but no growth occurred in modified Diamonds medium. The 5.8S rRNA gene and the flanking internal transcribed spacers were amplified from the trichomonad gDNA of two preputial wash samples and a fecal sample taken from the affected bull. Amplicons were subjected to single-strand conformation polymorphism (SSCP) analyses. The SSCP banding patterns of the amplicons from gDNA of the preputial wash samples were different from those of a T. foetus control sample. These unknown trichomonads were not detected in the fecal sample. The gDNA extracted from preputial washes was also subjected to PCR using primers developed to amplify the 16S rDNA of the non-T. foetus trichomonads, Tetratrichomonas and Pentatrichomonas spp. Amplicons were produced from the gDNA of the two preputial washes but not from the T. foetus gDNA control sample. The 16S rDNA sequences obtained from the trichomonads in the two preputial washes samples were 100% similar to that of a Tetratrichomonas species previously isolated from an Angus bull from the United States.

Molecular evidence for a cryptic species within the parasitic nematode Macroponema comani (Strongyloidea: Cloacininae)☆

Nematodes resembling Macroponema comani, a common parasite of eastern grey kangaroos, Macropus giganteus, in eastern Australia were collected from an unexpected host species, the northern wallaroo, Macropus robustus woodwardi, in the Northern Territory, representing a highly disjunct occurrence. Although these specimens showed no morphological differences when compared with Ma. comani from M. giganteus, sequencing of the first and second internal transcribed spacers ITS-1 and ITS-2 of the nuclear ribosomal DNA revealed seven base pair differences in each spacer region between the two taxa. These differences included a number of autapomorphies. Sequences from both taxa differed significantly from those of Ma. beveridgei, a common parasite of the common wallaroo, Macropus robustus robustus and the euro, M. robustus erubescens. Based on these findings, the specimens in M. r. woodwardi are considered to represent a crypic species.

Phylogenetic relationships of species within the tribe Labiostrongylinea (Nematoda: Cloacinidae) from Australian marsupials based on ribosomal DNA spacer sequence data.

Parasitic nematodes of the tribe Labiostrongylinea (Family Cloacinidae) occur in the stomachs of a wide variety of potoroid and macropodid marsupials in Australia, Papua Indonesia and Papua New Guinea. The aim of the present study was to infer the evolutionary relationships of the five genera of labiostrongyline nematodes that occur in Australian potoroids and macropodids using sequence data of the nuclear first and second internal transcribed spacers of ribosomal DNA. The phylogenetic analyses resulted in the separation of the Labiostrongylinea into two major groups reflecting coevolution between hosts and parasites. Two nematode species belonging to the genus Potorostrongylus formed a sister group to the remaining species of the Labiostrongylinea. This genus occurs exclusively in potoroid marsupials, which are considered to be basal to the macropodid marsupials. The second major group included species of Labiostrongylus, Labiosimplex, Labiomultiplex and Parazoniolaimus, all of which occur in macropodids. These species formed two distinct clades, one predominating in the host genera Thylogale and Onychogalea, and the second in the genus Macropus, which includes the more recent macropodids. However, there is also evidence of colonisation by both nematode clades of relatively unrelated hosts. In addition, genetic differences among individuals of Lm. eugenii from geographically isolated populations of M. eugenii, and among Ls. longispicularis from different subspecies of M. robustus suggest the existence of sibling species that may have arisen by allopatric speciation. The broad coevolutionary relationship between the labiostrongyline nematodes and their marsupial hosts therefore represents a mixture of potential cospeciation and colonisation events.

Analysis of genetic variation in Globocephaloides populations from macropodid marsupials using a mutation scanning-based approach

Three species of Globocephaloides, parasitic nematodes occurring in macropodid marsupials in different areas of Australia, were characterized by the sequences of the first and second internal transcribed spacers (ITS‐1 and ITS‐2) of nuclear ribosomal DNA. Samples were subjected to PCR‐coupled SSCP analysis and targeted sequencing, in order to assess genetic variation within and among individuals from different host species. Both SSCP and sequence data supported the current classification of morphospecies. Contrary to a previous hypothesis that cryptic species exist within the Globocephaloides trifidospicularis “complex”, no or minor (0.2%) variation was detected among individuals from different hosts or geographical origins. Within G. macropodis populations, there was a consistent difference in both the ITS‐1 (5.2%) and ITS‐2 (7.1%) sequences between individuals derived from Macropus agilis and those from Macropus dorsalis. Although the results suggested that G. macropodis from each host species represented sibling species, future morphological study of individuals representing each G. macropodis genotype is warranted to provide further support for this hypothesis. (Nucleotide sequence data have been deposited in the GenBank database under accession nos. GQ131400‐GQ131409.)

Bighorn Sheep, a New Host Record for Parelaphostrongylus odocoilei (Nematoda: Protostrongylidae)

Larval nematodes with a dorsal spine on the tail were recovered from fecal samples of California bighorn sheep (Ovis canadensis californiana) in northeastern Washington State, USA. The identity of these dorsal-spined larvae (DSL) was established by single-strand conformation polymorphism (SSCP) analyses of a partial fragment of the first internal transcribed spacer of the ribosomal DNA. The SSCP profiles of individual DSL from bighorn sheep were compared with those of DSL of five protostrongylid species (Parelaphostrongylus andersoni, P odocoilei, P. tenuis, Elaphostrongylus rangiferi, and Muellerius capillaris) but were identical to only those of P. odocoilei. This study represents the first confirmed identification of P. odocoilei in bighorn sheep.

A Method for Extracting Genomic DNA from Individual Elaphostrongyline (Nematoda: Protostrongylidae) Larvae and Differentiation of Elaphostrongylus Spp. from Parelaphostrongylus Spp. by PCR Assay

This article reports a rapid and effective method for the extraction and purification of genomic DNA (gDNA) from individual first-stage larvae (L1) of elaphostrongyline nematodes that had been stored frozen or fixed in 95% ethanol for 1 to 5 years. The method was highly effective for L1s of all 6 species of elaphostrongylines, based on polymerase chain reaction (PCR) amplification of a partial fragment of the first internal transcribed spacer (ITS-1) of the ribosomal DNA. Differences were detected in the sizes of partial ITS-1 amplicons between the 2 elaphostrongyline genera, Elaphostrongylus and Parelaphostrongylus. The reliability of the ITS-1 PCR assay was tested by using L1s of unknown identity from Newfoundland and Labrador, Canada. The ability to consistently isolate gDNA from individual L1s, together with a simple PCR-based method to distinguish between Parelaphostrongylus and Elaphostrongylus, have important implications for diagnostic testing and for conducting epizootiological studies on these parasites of veterinary importance.