Keith Vickerman

Biology of African trypanosomes in the tsetse fly

African trypanosomes present several features of interest to cell biologists. These include: a repressible single mitochondrion with a large mass of mitochondrial DNA, the kinetoplast; a special organelle, the glycosome, which houses the enzymes of the glycolytic chain; a surface coat of variable glycoprotein which enables the parasite to evade the mammalian hosts immune response; and a unique flagellum‐to‐host attachment mechanism associated with novel cytoskeletal elements. Trypanosome development during the life cycle involves cyclical activation and repression of genes controlling these activities. Understanding the complexity of parasite development in the tsetse fly vector is especially challenging but may help to suggest new methods for the control of trypanosomiasis.

The evolutionary expansion of the trypanosomatid flagellates.

The trypanosomatids combine a relatively uniform morphology with ability to parasitise a very diverse range of hosts including animals, plants and other protists. Along with their sister family, the biflagellate bodonids, they are set apart from other eukaryotes by distinctive organisational features, such as the kinetoplast-mitochondrion and RNA editing, isolation of glycolysis enzymes in the glycosome, use of the flagellar pocket for molecular traffic into and out of the cell, a unique method of generating cortical microtubules, and bizarre nuclear organisation. These features testify to the antiquity and isolation of the kinetoplast-bearing flagellates (Kinetoplastida). Molecular sequencing techniques (especially small subunit ribosomal RNA gene sequencing) are now radically reshaping previous ideas on the phylogeny of these organisms. The idea that the monogenetic (MG) trypanosomatids gave rise to the digenetic (DG) genera is losing ground to a view that, after the bodonids, the African trypanosomes (DG) represent the most ancient lineage, followed by Trypanosoma cruzi (DG), then Blastocrithidia (MG), Herpetomonas (MG) and Phytomonas (DG), with Leptomonas (MG), Crithidia (MG), Leishmania (DG) and Endotrypanum (DG) forming the crown of the evolutionary tree. Vast genetic distances (12% divergence) separate T. brucei and T. cruzi, while the Leishmania species are separated by very short distances (less than 1% divergence). These phylogenetic conclusions are supported by studies on RNA editing and on the nature of the parasite surface. The trypanosomatids seem to be able to adapt with ease their energy metabolism to the availability of substrates and oxygen, and this may give them the ability to institute new life cycles if host behaviour patterns allow. Sexual processes, though present in at least some trypanosomatids, may have played only a minor part in generating diversity during trypanosomatid evolution. On the other hand, the development of altruistic behaviour on the part of some life cycle stages may be a hitherto unconsidered way of maximising fitness in this group. It is concluded that, owing to organisational constraints, the trypanosomatids can undergo substantial molecular variation while registering very little in the way of morphological change.

A quick, simple method for purifying Leishmania mexicana amastigotes in large numbers

A rapid method for the bulk isolation of purified Leishmania mexicana mexicana amastigotes from parasite-induced lesions in experimentally infected mice is described. The procedure includes purification steps based on differences in net cell charge, lysis susceptibility and buoyant density between parasite and host cells. Yields of up to 2 x 10(10) untransformed amastigotes with minimal contamination with host cells and cell debris can be obtained. At least 90% of the purified amastigotes are viable as judged by light and electron microscopy, the staining of their lysosomes with acridine orange, their ability to transform to promastigotes and their infectivity to macrophages in vivo and in vitro.

Ribosomal RNA Phylogeny of Bodonid and Diplonemid Flagellates and the Evolution of Euglenozoa

Abstract Euglenozoa is a major phylum of excavate protozoa (comprising euglenoids, kinetoplastids, and diplonemids) with highly unusual nuclear, mitochondrial, and chloroplast genomes. To improve understanding of euglenozoan evolution, we sequenced nuclear small-subunit rRNA genes from 34 bodonids (Bodo, Neobodo, Parabodo, Dimastigella-like, Rhynchobodo, Rhynchomonas, and unidentified strains), nine diplonemids (Diplonema, Rhynchopus), and a euglenoid (Entosiphon). Phylogenetic analysis reveals that diplonemids and bodonids are more diverse than previously recognised, but does not clearly establish the branching order of kinetoplastids, euglenoids, and diplonemids. Rhynchopus is holophyletic; parasitic species arose from within free-living species. Kinetoplastea (bodonids and trypanosomatids) are robustly holophyletic and comprise a major clade including all trypanosomatids and most bodonids (‘core bodonids’) and a very divergent minor one including Ichthyobodo. The root of the major kinetoplastid clade is probably between trypanosomatids and core bodonids. Core bodonids have three distinct subclades. Clade 1 has two distinct Rhynchobodo-like lineages; a lineage comprising Dimastigella and Rhynchomonas; and another including Cruzella and Neobodo. Clade 2 comprises Cryptobia/ Trypanoplasma, Procryptobia, and Parabodo. Clade 3 is an extensive Bodo saltans species complex. Neobodo designis is a vast genetically divergent species complex with mutually exclusive marine and freshwater subclades. Our analysis supports three phagotrophic euglenoid orders: Petalomonadida (holophyletic), Ploeotiida (probably holophyletic), Peranemida (paraphyletic).

Changes in oxidative metabolism and ultrastructure accompanying differentiation of the mitochondrion in Trypanosoma brucei

Abstract A method is described for obtaining optimal growth and morphological transformation of Trypanosoma brucei 792G in a monophasic blood lysate medium starting from populations of bloodstream trypanosomes containing over 90 per cent intermediate and stumpy forms. Transformation was accompanied by: (1) an increase in length of the trypanosomes from 13·8 (± 3·0) μm to 23·0 (± 2·5) μm; (2) an increase in the kinetoplastic index from 1·0 to greater than 2·0; (3) development of the mitochondrion from a single abflagellar canal to a network of subpellicular canals, with outgrowth of the post-kineto-plastic region of the mitochondrion; (4) replacement of some of the tubular mitochondrial cristae by plate-like cristae; (5) the acquisition of succinoxidase, succinate-cytochrome c reductase, and glycerophosphate-cytochrome c reductase activities; (6) a marked increase in proline oxidase and NADH-cytochrome c reductase activities; (7) loss of the surface coat of the flagellate and concomitant reduction in the smooth-membrane systems lying between the nucleus and flagellar pocket; (8) reduction in infectivity of the trypanosomes to the mammalian host. Although growth in primary culture continued up to 100 h, transformation was completed in 48 h. All the respiratory enzyme activities tested were insensitive to cyanide throughout transformation. Division of trypanosomes appeared to be taking place throughout the transformation process. Cyanide sensitivity developed only after subculture of the trypanosomes into a biphasic medium.

Phylogeny and Classification of Cercomonadida (Protozoa, Cercozoa): Cercomonas, Eocercomonas, Paracercomonas, and Cavernomonas gen. nov.

Cercomonads (=Cercomonadida) are biflagellate gliding bacterivorous protozoa, abundant and diverse in soil and freshwater. We establish 56 new species based on 165 cultures, differential interference contrast microscopy, and 18S and ITS2 rDNA sequencing, and a new genus Cavernomonas studied by scanning electron microscopy. We fundamentally revise the phylogeny and classification of cercomonad Cercozoa. We describe 40 Cercomonas species (35 novel), six Eocercomonas (five novel), two Cavernomonas, and 18 Paracercomonas species (14 novel). We obtained additional cercomonad clade A (Cercomonas, Eocercomonas, Cavernomonas) sequences from multiple environmental DNA libraries. The most commonly cultivated genotypes are not the commonest in environmental DNA, suggesting that cercomonad ecology is far more complex than implied by laboratory cultures. Cercomonads have never been isolated from saline environments, although some species can grow in semi-saline media in the laboratory, and environmental DNA libraries regularly detect them in coastal marine sediments. The first ultrastructural study of an anaerobic cercozoan, Paracercomonas anaerobica sp. nov., a highly divergent cercomonad, shows much simpler ciliary roots than in clade A cercomonads, a ciliary hub-lattice and axosome, and mitochondria with tubular cristae, consistent with it being only facultatively anaerobic. We also describe Agitata tremulans gen. et sp. nov., previously misidentified as Cercobodo (=Dimastigamoeba) agilis Moroff.

Respiration of Leishmania mexicana amastigotes and promastigotes

Promastigotes of Leishmania mexicana mexicana recently derived from amastigotes by transformation in vitro respired at a rate (17 nmol O2/min per 10(8) parasites) 4-5 times higher than that of amastigotes, but when the difference in cell protein content between the two preparations was taken into account the rates were not significantly different (32 nmol O2/min per mg protein). The respiration of both amastigotes and promastigotes was sensitive to cyanide, azide, antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide and high concentrations of amytal, but insensitive to rotenone and salicyl-hydroxamic acid, indicating that the two developmental forms possess a similar cytochrome-containing respiratory chain. D-Glucose and non-esterified fatty acids stimulated promastigote respiration and amastigote transformation to promastigotes in vitro; possibly these substances are important exogenous energy substrates for both forms of the parasites. Amino acids (incuding L-proline) and proteins did not appear to be used as energy substrates. The respiration rate of promastigotes was found to rise significantly upon continued sub-culture in vitro; at the same time cell size and protein content increased.

An estimate of the size of the metacyclic variable antigen repertoire of Trypanosoma brucei rhodesiense

A group of 27 variable antigen type (VAT)-specific monoclonal antibodies (McAbs) have been made against metacyclic forms of a cloned stock of Trypanosoma brucei rhodesiense. In combination, these labelled in immunofluorescence 99.3% of trypanosomes in salivary probes from tsetse flies. The 0.7% of unlabelled trypanosomes were believed to be uncoated forms. The ability of a mixture of antibodies to kill metacyclics in vitro by complement-mediated lysis, thus neutralizing their infectivity for mice, was tested. The antibody mixture consisted of 24 McAbs plus 3 VAT-specific rabbit antisera. In 12 replicate experiments this mixture of antibodies prevented infection of mice. Parallel controls showed that neutralization was probably antibody-mediated and VAT specific. However, we have not been able to repeat these results on a long-term basis; this may be due to a loss of neutralizing activity by one of the McAbs. The successful neutralization experiments indicate that the number of VATs in the metacyclic repertoire of one stock of T. b. rhodesiense is limited to at most 27.

Loss of variable antigen during transformation of Trypanosoma brucei rhodesiense from bloodstream to procyclic forms in the tsetse fly

A pleomorphic line of Trypanosoma brucei rhodesiense expressing a single variable antigen was used to quantify the rate of loss of the surface coat from bloodstream forms transforming to procyclics in the tsetse fly, Glossina morsitans, and in in vitro culture. Loss of variable antigen occurred at similar rates in the crop and anterior portion of the midgut of tsetse flies and in in vitro culture, but in the posterior portion of the fly midgut it occurred 2–3 times faster. The posterior portion of the midgut is the most important site for transformation of bloodstream-form trypanosomes to procyclics, and the dynamics of at least one component of this process are therefore not accurately paralleled in vitro.

The diversity and ecological significance of Protozoa

The unicellular eukaryotes are currently grouped in the kingdom Protista, together with their multicellular relatives. The inclusion of protozoa, algae and water moulds in a single taxon has resulted in nomenclatural problems, academic homelessness, and a reduction in their teaching. There are around 40 000 described protozoan protist species. Protozoa are principally grazers of bacteria, increasing mineralization and making nutrients more available to other organisms; most are aquatic, but they are also widespread animal parasites and symbionts. Their biomass, role in food chains, roles as mutualists and pathogens, and value as biomonitors are reviewed. To assess the role of protozoa in ecosystems more accurately, the current poor taxonomic standards in ecological work on protozoa must be improved. The manpower to respond to existing and new challenges in the field is declining as protozoology disappears from university courses and this problem needs to be addressed.