The phylogenetic position of Udonella (Platyhelminthes).

Phylogenetic analysis of molecular data from complete 18S rRNA and partial 28S rRNA genes, of a variety of platyhelminths, places the enigmatic Udonella caligorum firmly as a monopisthocotylean monogenean. Both maximum parsimony and a modified distance measure, operating under a maximum likelihood model, gave identical solutions for each data set. These data further support morphological evidence from ultrastructural studies indicating the neodermatan affinities of Udonella, namely shared features in sensory receptors, surface tegument, sperm structure and spermiogenesis. The molecular data reject the class Udonellidea and the placement of udonellids as sister-group to the Neodermata. As shown previously with molecular data, the monogeneans appear as a paraphyletic assemblage comprising strongly monophyletic Monopisthocotylea and Polyopisthocotylea. Their relationships with the trematodes and cestodes are not resolved with 28S rDNA or 18S rDNA alone.

Comparison of the large subunit ribosomal DNA of Neospora and toxoplasma and development of a new genetic marker for their differentiation based on the D2 domain.

The latest release of the large subunit ribosomal database contains 429 sequences, yet only 10 (six nuclear and four mitochondrial) are derived from parasites of the phylum Apicomplexa. Three of these (all Toxoplasma gondii) were previously contained in the 1994 release of the database. As an initiative towards an understanding of ribosomal gene organization in the Apicomplexa, the primary sequence of the large subunit (LSU) rDNA of Neospora caninum is presented, and compared with a consensus sequence derived for the LSU rDNA of T. gondii. Nucleotide differences observed between these two taxa in the D2 expansion segment (or domain) (also called the C1/C1' region) of the LSU rDNA were incorporated into a primer that forms the basis of a species-specific polymerase chain reaction (PCR) for N. caninum. The D2 domain of the LSU rDNA, therefore, represents a new genetic marker that can be used for the differentiation and identification of Neospora from other cyst-forming coccidia.

Gnathostomulida--an enigmatic metazoan phylum from both morphological and molecular perspectives.

On the basis of few and contentious morphological characters Gnathostomulids have been thought to be the sister-group of either the Platyhelminthes or the Syndermata (Rotifera + Acanthocephala). We provide a full 18S rDNA sequence for a species of Gnathostomula and attempt to resolve its position among the Metazoa, on the basis of molecular evidence. Sixty sequences, representing 30 nominal phyla and including new entoproct and gastrotrich sequences, were used to reconstruct phylogenies using maximum-parsimony, neighbor-joining, and minimum evolution models. We were unable to support either of the morphological hypotheses outright and, moreover, our data supported more strongly a third possible relationship with the gnathostomulids as a member of the Nematoda + Chaetognatha clade. Superficially, as active benthic, vermiform creatures with sclerotized cuticular jaws, they fit a predicted ancestral form of the Nematoda + Chaetognatha clade and, as such, would arguably be members of the Ecdysozoa. The molecular data at least call for a reevaluation of the morphological data and a denser sampling of the lesser phyla. Data from morphology and molecules act synergistically in estimating phylogeny; morphology alone provided limited phylogenetic signal and alternative phylogenetic hypotheses, whereas the molecular solution suggested an alternative topology which, when interpreted in the light of comparative anatomy, may suggest previously unconsidered possibilities.

A robust molecular phylogeny of the Tricladida (Platyhelminthes: Seriata) with a discussion on morphological synapomorphies.

The suborder Tricladida (Platyhelminthes: Turbellaria, Seriata) comprises most well-known species of free-living flatworms. Four infraorders are recognized: (i) the Maricola (marine planarians); (ii) the Cavernicola (a group of primarily cavernicolan planarians); (iii) the Paludicola (freshwater planarians); and (iv) the Terricola (land planarians). The phylogenetic relationships among these infraorders have been analysed using morphological characters, but they remain uncertain. Here we analyse the phylogeny and classification of the Tricladida, with additional, independent, molecular data from complete sequences of 18S rDNA and 18S rRNA. We use maximum parsimony and neighbour-joining methods and the characterization of a unique gene duplication event involving the Terricola and the dugesiids to reconstruct the phylogeny. The results show that the Maricola is monophyletic and is the primitive sister group to the rest of the Tricladida (the Paludicola plus the Terricola). The Paludicola are paraphyletic since the Terricola and one paludicolan family, the Dugesiidae, share a more recent common ancestor than the dugesiids with other paludicolans (dendrocoelids and planariids). A reassessment of morphological evidence may confirm the apparent redundancy of the existing infraorders Paludicola and Terricola. In the meantime, we suggest replacing the Paludicola and Terricola with a new clade, the Continenticola, which comprises the families Dugesiidae, Planariidae, Dendrocoelidae and the Terricola.

The cysteine proteinases of Schistosoma mansoni cercariae.

Based on substrate preferences, cercariae of Schistosoma mansoni were seen to express both cathepsin L and cathepsin B cysteine proteinases, although the former activity was many-fold greater. Two cathepsin L activities identified in cercarial extracts by zymography co-migrated with activities in extracts of 3 h and 24 h schistosomula and in extracts of adult worms. Since these enzymes have been implicated in haemoglobin digestion by adult worms, they may perform a similar function in schistosomula. Immunolocalization using scanning electron micrographs showed that cathepsin L and cathepsin B proteinases were present in the cercarial post-acetabular glands. In addition, cercarial serine proteinase activities considered to facilitate skin penetration efficiently cleaved the substrates Z-Gly-Pro-Arg-NHMec and Z-Gly-Pro-Lys-NHMec. Cercariae release most of this serine proteinase activity when induced to secrete the contents of their acetabular glands. In contrast, newly transformed 3 h and 24 h schistosomula did not express this activity.

Parasite speciation within or between host species?--phylogenetic evidence from site-specific polystome monogeneans.

Partial nuclear 28S ribosomal RNA and mitochondrial cytochrome c oxidase subunit I (COI) gene sequences (953 and 385 nucleotides, respectively) of one fish monogenean (outgroup) and six polystome monogeneans (four Polystomoides spp. from the oral cavities and urinary bladders of freshwater turtles in Australia and Malaya, two Neopolystoma spp. from the urinary bladder and conjunctival sac of a freshwater turtle in Australia) were used to examine the question of whether congeneric species infecting different sites in the same host species have speciated in that host by adapting to different sites, or whether species infecting a particular site in one host have given rise to species infecting the same site in different hosts. Results show unequivocally that congeneric species infecting the same site, even of host species belonging to different suborders and occurring on different continents, are more closely related than congeneric species infecting different sites of the same host species. This is interpreted as meaning that speciation has not occurred in one host. Morphological evolution of polystomes has been very slow: few differences between species and even genera have evolved over a period of at least 150 Myr, and this is matched by low substitution rates of nucleotides, and the ambiguous position of species of different genera, depending on whether COI or 28S rDNA sequences are used.

Host-like sequences in the schistosome genome.

A series of recent papers has indicated that widespread genomic rearrangements take place in the genome of schistosomes during the life cycle of the parasite. These results have been controversial since genomic rearrangements are not common in eukaryotes, probably because excessive genome plasticity would carry a heavy evolutionary price. Here, Karen Clough, Alec Drew and Paul Brindley present data that ostensibly support the concept of widespread genomic rearrangements, but for which they suggest a different interpretation. They conclude that artefactual contamination of schistosome genome preparations with host DNA can probably explain the Southern hybridization results which led to the original hypothesis of developmental, genomic rearrangements.

Characterization of the cathepsin-like cysteine proteinases of Schistosoma mansoni.

Adult Schistosoma mansoni parasites synthesize and secrete both cathepsin L and cathepsin B cysteine proteinases. These cysteine proteinase activities, believed to be involved in hemoglobin digestion by adult schistosomes, were characterized by using specific fluorogenic peptide substrates and zymography. Both cathepsin L- and B-like activities with pH optima of 5.2 and 6.2, respectively, predominated in soluble extracts of worms, and both these activities were secreted by adult worms into the culture medium. The specific activity of cathepsin L was about double that of cathepsin B when each was assayed at its pH optimum, and moreover, the specific activities of cathepsins L and B in extracts of female schistosomes were 50 to 100% higher than in extracts of male schistosomes. Analysis of the primary structure of two cloned S. mansoni cathepsins L, here termed cathepsin L1 and cathepsin L2, revealed that they are only 44% similar and that cathepsin L2 showed more identity (52%) with human cathepsin L than with schistosome cathepsin L1. Moreover, differences in their active site, propeptide region, and potential for glycosylation suggest separate functions for schistosome cathepsin L1 and cathepsin L2.

Characterization and cloning of the cathepsin L proteinases of Schistosoma japonicum.

Adult Schistosoma japonicum parasites synthesize and secrete both cathepsin L and cathepsin B cysteine proteinases. The specific activities of cathepsin L were many-fold higher than that of cathepsin B. The cDNAs encoding two distinct cathepsin L proteinases, here termed cathepsin L1 and L2, were isolated. The deduced amino acid sequences of the mature cathepsin L1 and L2 were approximately 41% identical, and moreover, S. japonicum cathepsin L2 showed more similarity with human cathepsin L than with schistosome cathepsin L1. Schistosome cathepsin L proteinases may be involved in the digestion of hemoglobin obtained from host erythrocytes. However, since we detected their presence in schistosome eggs, the release of these enzymes by eggs trapped in the liver and other organs may be associated with the granulomatous responses which characterize the pathology of human schistosomiasis.

Digestion of haemoglobin by schistosomes: 35 years on.

Adult schistosomes obtain nutrients by digesting haemoglobin, which they obtain from ingested host red blood cells. Here John Dalton, Angela Smith, Karen Clough and Paul Brindley argue that a cathepsin L proteinase recently identified in their laboratory as the predominant cysteine proteinase activity of Schistosoma mansoni may play the leading role in haemoglobin digestion. They discuss the importance of elucidating the roles of both cathepsin B and cathepsin L in the digestion of haemoglobin, since both should be considered important targets to which novel schistosomicidal agents could be directed.

Adult Schistosoma mansoni express cathepsin L proteinase activity.

This report presents the deduced amino acid sequence of a novel cathepsin L proteinase from Schistosoma mansoni, and describes cathepsin L-like activity in extracts of adult schistosomes. Using consensus primers specific for cysteine proteinases, gene fragments were amplified from adult S. mansoni cDNA by PCR and cloned. One of these fragments showed marked identity to Sm31, the cathepsin B cysteine proteinase of adult S. mansoni, whereas another differed from Sm31 and was employed as a probe to isolate two cDNAs from an adult S. mansoni gene library. Together these cDNAs encoded a novel preprocathepsin L of 319 amino acids; this zymogen is predicted to be processed in vivo into a mature, active cathepsin L proteinase of 215 amino acids. Closest homologies were with cathepsins L from rat, mouse, and chicken (46-47% identity). Southern hybridization analysis suggested that only one or a few copies of the gene was present per genome, demonstrated that its locus was distinct from that of Sm31, and that a homologous sequence was present in Schistosoma japonicum. Because these results indicated that schistosomes expressed a cathepsin L proteinase, extracts of adult S. mansoni were examined for acidic, cysteine proteinase activity. Based on rates of cleavage of peptidyl substrates employed to discriminate between classes of cysteine proteinases, namely cathepsin L (Z-phe-arg-AMC), cathepsin B (Z-arg-arg-AMC) and cathepsin H (Bz-arg-AMC), the extracts were found to contain vigorous cathepsin L-like activity.(ABSTRACT TRUNCATED AT 250 WORDS)