Electrophoretic comparison of Rugopharynx longibursaris Kung and R. omega Beveridge (Nematoda: Strongyloidea), with the description of R. sigma n. sp. from pademelons,Thylogale spp. (Marsupialia: Macropodidae)

An electrophoretic comparison of the nematodes Rugopharynx longibursaris and R. omega, both from Macropus rufogriseus in south-eastern Australia, revealed fixed genetic differences at 4.5% of the 23 enzyme loci examined. The electrophoretic data do not therefore reject the null hypothesis that the two taxa are conspecific. R. longibursaris was found in Tasmania and in the western mainland population of M. rufogriseus, while R. omega occurred only in the eastern mainland population. Implications for the taxonomic status of the western host population are considered. Specimens formerly assigned to R. omega, from Thylogale stigmatica from Queensland, were found to differ at 45.0% of enzyme loci from specimens from M. rufogriseus. Morphological examination revealed differences in the shape of the buccal capsule, the position of the deirid, the morphology of the spicule tip and the presence of a gubernaculum. A new species, R. sigma, is erected for specimens from T. stigmatica, T. thetis and T. calabyi.     

Restriction endonuclease mapping of a plasmid that confers oncogenicity upon Agrobacterium tumefaciens strain B6-806

The 26 SmaI digest fragments of pTi-B6-806 plasmid have a total molecular weight (121 × 10⁶) which accounts for the size of the plasmid as determined by contour length measurements. We have determined the physical arrangement of all SmaI digest fragments with reference to HpaI digest fragments. Hybridization of individual labeled SmaI digest fragments to HpaI digest fragments (cellulose nitrate transfers) allowed the latter to be ordered and located the SmaI boundary fragments. Recleavage of isolated HpaI fragments with SmaI revealed the SmaI fragments located within each HpaI fragment. The order of these internal SmaI fragments within a given HpaI fragment was determined by partial digestion of the latter with SmaI and hybridization of the resulting fragments with SmaI boundary fragments. From the sizes of partial digest fragments containing each boundary, the order of occurrence of SmaI fragments from each end was deduced. The complete map of the SmaI digest fragments is presented. The map of the HpaI digest fragments is presented with the following ambiguity: The order of fragments 12, 15, and 16, which map within SmaI fragment 1, was not determined. The SmaI digest fragments that contain DNA sequences transferred to plant cells during tumor induction, fragments 3b and 10c, were found to be contiguous on the physical map.     

Tumor-inducing (Ti) plasmids of Agrobacterium share extensive regions of DNA homology.

Labeled Ti plasmid DNAs from diverse Agrobacterium strains were hybridized to Southern blots of pTi-B6-806 plasmid DNA digest fragments of known map order. The map location of DNA sequences common to all Ti plasmids was found to be extensive, consistent with the view that Ti plasmids have evolved from a common ancestral plasmid.     

Lack of evidence for co-speciation in a parasitic nematode of grey kangaroos

Multilocus enzyme electrophoresis was used to compare specimens of the parasitic nematode Cloacina obtusa from the stomach of the eastern grey kangaroo, Macropus giganteus and the western grey kangaroo, M. fuliginosus. Allelic variation among nematodes was detected at 17 (85%) of 20 loci, but there was only a single fixed genetic difference (at the locus for isocitrate dehydrogenase, IDH) between C. obtusa from M. fuliginosus and those from M. giganteus in areas where each host occurred in allopatry. However, this fixed difference was not apparent within the zone of host sympatry. Although electrophoretic data indicate genetic divergence among allopatric populations of C. obtusa in the two host species, the magnitude of the electrophoretic difference (5%) between these populations does not refute the hypothesis that C. obtusa represents a single species. The 'usual' situation for parasitic helminths of grey kangaroos is that pairs of parasite species occur in the two host species. This situation differs for C. obtusa, where there has been a lack of speciation following a speciation event in its macropodid marsupial hosts. This finding suggests that a speciation event in the host does not necessarily lead to a speciation event for all its parasites and further highlights our lack of understanding of which processes drive speciation in parasites.     

Secretory phospholipase A2 receptor-mediated activation of cytosolic phospholipase A2 in murine bone marrow-derived mast cells

The current study examined the signal transduction steps involved in the selective release of arachidonic acid (AA) induced by the addition of secretory phospholipase A2 (sPLA2) isotypes to bone marrow-derived mast cells (BMMC). Overexpression of sPLA2 receptors caused a marked increase in AA and PGD2 release after stimulation of BMMC, implicating sPLA2 receptors in this process. The hypothesis that the release of AA by sPLA2 involved activation of cytosolic PLA2 (cPLA2) was next tested. Addition of group IB PLA2 to BMMC caused a transient increase in cPLA2 activity and translocation of this activity to membrane fractions. Western analyses revealed that these changes in cPLA2 were accompanied by a time-dependent gel shift of cPLA2 induced by phosphorylation of cPLA2 at various sites. A noncatalytic ligand of the sPLA2 receptor, p-amino-phenyl-alpha-D-mannopyranoside BSA, also induced an increase in cPLA2 activity in BMMC. sPLA2 receptor ligands induced the phosphorylation of p44/p42 mitogen-activated protein kinase. Additionally, an inhibitor of p44/p42 mitogen-activated protein kinase (PD98059) significantly inhibited sPLA2-induced cPLA2 activation and AA release. sPLA2 receptor ligands also increased Ras activation while an inhibitor of tyrosine phosphorylation (herbimycin) inhibited the increase in cPLA2 activation and AA release. Addition of partially purified sPLA2 from BMMC enhanced cPLA2 activity and AA release. Similarly, overexpression of mouse groups IIA or V PLA2 in BMMC induced an increase in AA release. These data suggest that sPLA2 mediate the selective release of AA by binding to cell surface receptors and then inducing signal transduction events that lead to cPLA2 activation.     

Molecular approaches for studying ascaridoid nematodes with zoonotic potential, with an emphasis on Toxocara species

Species-specific identification of ascaridoid nematodes at any developmental stage is a prerequisite for detailed investigation of the life cycles, systematics and epidemiology of this important group, and is also crucial for the diagnosis of associated infections. The morphological identification of some species and/or their larval stages can, however, present considerable difficulty. Recently, PCR-based methods, using genetic markers in the internal transcribed spacers (ITS) of ribosomal DNA, have been shown to provide reliable alternatives to more traditional methods for the specific identification of nematodes. This article provides an account of recent research on the development of PCR-based methods (utilizing ITS sequences) for the specific identification of ascaridoid nematodes of zoonotic potential, for the diagnosis of infections, and for the analysis of genetic variation within and among individual nematodes and their populations. Prospects for using these diagnostic and analytical tools to investigate epidemiological and population genetic questions relating to ascaridoid parasites are also discussed.     

Evolutionary relationships of trichostrongyloid nematodes (Strongylida) inferred from ribosomal DNA sequence data

The evolutionary relationships of 21 species of trichostrongyloid nematodes were determined by use of sequence data of the second internal transcribed spacer of the ribosomal DNA aligned according to secondary structure information. Irrespective of the method of analysis used, the topologies of the phylogenetic trees derived from the molecular data differed with respect to all four hypotheses proposed previously for the evolutionary relationships of the different subfamilies within the Trichostrongylidae based on morphological data. Thus, the molecular data set did not resolve the conflict between the four previous proposals for the subfamilial relationships. Nonetheless, all trees derived from the molecular data showed strong support for the exclusion of the genera Filarinema and Amidostomum from the clade containing the species within the family Trichostrongylidae. This represents a major difference from the most recent proposal of the systematics of the Trichostrongyloidea in which these two genera were included within the Trichostrongylidae. Therefore, the molecular data support an earlier systematic framework in which Filarinema and Amidostomum were considered to be sister groups of the Trichostrongyloidea.     

Co-evolutionary relationships between the nematode subfamily Cloacininae and its macropodid marsupial hosts

Morphologically based phylogenies of the cloacinine genera Cyclostrongylus, Macropostrongylus, Pharyngostrongylus, Popovastrongylus, Rugopharynx, Thallostonema, Wallabinema, and Zoniolaimus were constructed and compared with the phylogeny of their respective macropodid hosts. These comparisons show some evidence of co-speciation. However, there was little consistency among trees of different nematode genera, parasite species were scattered amongst hosts and basal parasite taxa were, in some instances, parasitic in hosts belonging to derived clades. A cladistic analysis, using as characters 208 cloacinine nematode species found in 23 species of host, produced a tree largely resembling that of the host tree but with significant differences explainable by host switching among macropodids occupying similar habitat. Nematodes were moderately host-specific, but some species occurred in three or more distantly related host species indicating a degree of host switching. The results are more consistent with the hypothesis of a colonisation of macropodid hosts by cloacinine nematodes rather than a prolonged period of co-speciation although alternative interpretations of the data are also considered.