Spring Meeting - University of East Anglia, April 2001

BSSP 2001 Spring Meeting - Invited Papers

  1. PLENARY LECTURE
  2. OCEANOGRAPHIC PROTOZOA
  3. NEW TECHNOLOGIES AND ADVANCES IN THE STUDIES OF PARASITIC PROTOZOA.
  4. BSSP 2001 Spring Meeting - Contributed Papers
  5. BSSP 2001 Spring Meeting - Posters

ABSTRACTS

PLENARY LECTURE

THE LARGE SCALE PHYLOGENY AND CLASSIFICATION OF PROTOZOA AND OTHER PROTISTS.

T. Cavalier-Smith, Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS.

The past few years have seen a revolution in our understanding of protist evolution, overturning earlier ideas on the position of the root of the eukaryotic tree. I shall explain this and attempt a new synthesis, integrating recent molecular and morphological evidence on the large-scale structure of the eukaryote tree. There is now compelling evidence that the infrakingdom Alveolata (Ciliophora, Sporozoa, dinoflagellates and protalveolates) had a photosynthetic common ancestor and that they are sisters of the kingdom Chromista. Alveolates and chromists form a clade (chromalveolates) that arose by the symbiogenetic uptake of a red alga. I shall discuss the evolutionary implications of our complete sequencing of the genome of the enslaved relict red algal nucleus that is now the nucleomorph of cryptomonad chromists. I shall present new rRNA trees bearing on the phylogeny of alveolates and on the basal branches of the Protozoa. I shall outline reasons for grouping Metamonada, Parabasalia, Euglenozoa, Percolozoa and Loukozoa together as the Excavata and argue that excavates, chromalveolates and plants form a major branch of the eukaryotes. I shall present extensive new data on molecular phylogeny of Cercozoa and suggest that they are related to Retaria (Radiolaria and Foraminifera) and that this composite group is related to plants/chromalveolates/excavates. I shall give new molecular evidence on the diversity of Choanozoa and their closer relationship to animals than to fungi; all three groups comprise a robust opisthokont clade to which Apusozoa are sisters. The position of Amoebozoa is the least clear, since they branch quite close to the likely position of the root of the eukaryote tree, which I shall discuss. Amoebozoa comprise Mycetozoa, Archamoebae and Lobosa and are probably either sisters of the opisthokont/apusozoan clade or of all other eukaryotes combined.

OCEANOGRAPHIC PROTOZOA

GRAZING OF AUTOTROPHIC AND HETEROTROPHIC PROTISTS BY ZOOPLANKTON

Peter Verity, Skidaway Institute of Oceanography.

The conceptual basis for our understanding of pelagic food webs during the first 75 years of the twentieth century was comparatively simple and, in retrospect, impossibly naive. The cornerstone of the euphotic zone was thought to be large phytoplankton, typically equated with the algal class called diatoms, which were eaten by large zooplankton such as copepods and krill, which in turn fed fish. The incomplete nature of this dogma was illuminated by Pomeroy (1974), and a quarter century of study since has supported the current paradigm that in fact small protists are generally the workhorses of the euphotic zone.

This talk will consider some of the methods and technology which permitted this research; review grazing rates by microzooplankton on auto- and heterotrophic protists from polar to tropical regions; relate this grazing to abiotic and biotic (e.g. prey availability) factors; present evidence that prey choice is not random; and discuss linkages between plankton community structure and biogeochemical cycling in the oceans.

MICROBIAL DYNAMICS DURING MONSOONAL UPWELLING.

Peter Burkill and Elaine Fileman Plymouth Marine Laboratory, Prospect place, West Hoe, Plymouth PL1 3DH, UK,

Textbooks state that upwelling biology is rather simple. Typically, upwelling fertilises surface waters; diatoms flourish and are grazed by copepods that are then eaten by fish. Such a simple foodchain is ecologically efficient and is one of the reasons why ca ~50% of man's fisheries are associated with upwelling. We were able to study upwelling under monsoonal Beaufort 9-11 conditions in the Arabian Sea. What we found was starkly different. There were some diatoms but they were few and rather emaciated. Instead, there were impressive concentrations of smaller algal taxa. Flagellates and other protozoa were present but did not increase in numbers during our 10 day study. What was happening? The talk will present our views.

CHARACTERISATION OF PROTOZOAN PREY BY FLOW CYTOMETRY AND ITS APPLICATION TO THE QUANTIFICATION OF PROTOZOAN HERBIVORY IN THE NE ATLANTIC OCEAN.

Glen Tarran, Claire Stelfox-Widdicombe and Elaine Fileman Plymouth Marine Laboratory, Prospect Place, West Hoe, Plymouth PL1 3DH.

Microzooplankton are an important component of oceanic food webs and are capable of grazing all of the daily primary production. The processes involved with grazing are complex but we are generally limited to quantifying grazing on the total phytoplankton community.

Flow cytometry is a generic technology, which is well suited to the rapid analysis and quantification of phytoplankton. It also enables characterisation of components of the phytoplankton community based on their light scattering and fluorescence characteristics, such that different size classes and groups such as coccolithophores and cryptophytes can be discriminated.

Using a dilution approach to study grazing and flow cytometry we have quantified the abundance and predation upon sub-populations within the phytoplankton community to study the contribution of different phytoplankton groups and size classes to protozoan diet during a cruise in the Northeast Atlantic Ocean. The way in which flow cytometry is used in theses studies along with results from the cruise will be presented with a look forward to ways in which techniques can be refined and applied to additional aspects of grazing mediated processes.

THE EFFECT OF SILICON AND NITROGEN LIMITATION ON COASTAL MICROBIAL PLANKTON.

Emily Roberts, Dunstaffnage Marine Laboratory, Keith Davidson, Scottish Association for Marine Science, Dunstaffnage Marine Laboratory and Linda Gilpin, Napier University.

Increased anthrpogenic N loading is characteristic of many coastal areas, whereas Si loading has remained constant or decreased. These changes in nutrient input dynamics may result in a shift in inorganic N:Si ratios, favouring the development of Si limitation in certain coastal areas. In June 2000 the mesocosm facilities at Trondheim Marine Biological Station were used to investigate the influence of N:Si ratio on microbial plankton dynamics. Natural seawater was enriched to produce N:Si ratios of 4:1 and 1:1 in order to obtain Si limited and (balanced) "Redfield" nutrient conditions, respectively. Changes in cell densities, nutrient concentrations, and particulate carbon and nitrogen were assessed by sampling every two days over a 16 day period. Biomass of all autotrophic planktonic groups (diatoms, phototrophic nanoflagellates and mixo- autotrophic dinoflagellates) was found to be greater under Si limitation. Although there were fewer diatom cells under Si limitation, biomass was higher compared to Redfield conditions due to an increased content per cell. Protozooplankton (heterotrophic nanoflagellate, heterotrophic dinoflagellate and ciliate) abundance and biomass was greater under Si limitation. Of all the heterotrophic groups present, heterotrophic nanoflagellates had the greatest predicted grazing impact potentially causing the observed oscillations in bacterial abundance. The effect of N:Si ratio on plankton species composition will be discussed. A shift in nutrient limitation from N to Si appears to alter the marine microbial food web resulting in increased planktonic food available to higher trophic levels and a more abundant microbial loop community.

A MICRO-HABITAT OF THE OPEN OCEAN WHICH SINKS FAST AND CHANGES RAPIDLY.

Richard Lampitt, SOC, Southampton, UK Marine Snow.

Marine snow is a class of large inanimate particle found throughout the world's oceans. Fragile, and occurring at low abundances, these particles have proved to be difficult to record and collect but are now thought to be the principal vehicles by which material is transported from the site of primary production in the upper sunlit zone of the water column to the deep sea. Due to their rapid sinking rate, these large aggregated particles link the top and bottom of the ocean in a much closer temporal sense than had been thought the case until very recently providing an explanation for seasonal changes in the appearance of the deep-sea floor and for the seasonal reproduction which is carried out by some species of deep sea benthic animals. This seasonal change in the marine snow pool is primarily due to variations in the larger particles (6.3 - 9.9mm), the smaller ones only demonstrating slight seasonality.

The use of microelectrode techniques has demonstrated that there can be strong chemical gradients within marine snow particles creating microhabitats for the microbiota which can not be found anywhere else in the water column. Severe oxygen depletion has been measured in some snow particles and it is very likely that the free methane which is found in the upper water column has been created in anoxic regions of snow aggregates. Such chemical gradients will thus have a very major affect on interactions between the different components of the microbial community.

It now seems that the rates of production and destruction of marine snow are high so as to facilitate diurnal changes in the particle pool well below the upper mixed layer of the ocean. If these rapid changes over time periods of a few hours are a common feature, the pressures to take advantage of any microhabitats will be very great and the methods to examine them should reflect this.

PROTOZOAN ASSIMILATION OF BACTERIAL PROTEINS AND DNA: COMPARISON OF MIXOTROPHIC AND HETEROTROPHIC FLAGELLATES.

Mikhail Zubkov, Plymouth Marine Laboratory, Prospect place, West Hoe, Plymouth PL1 3DH, UK, Eckart Zöllner and Klaus Jürgens, Max Planck Institute of Limnology, PO Box 165, D-24302 Plön, Germany.

Assimilation of bacterial biomass by three species of phagotrophic flagellates was studied using radioactive tracer techniques and short-term feeding experiments. Macromolecules of two different bacterial strains and natural limnic bacterioplankton were pulse-chase-labelled with one of the following precursors 3H-thymidine, 35S- / 14C-methionine or 14C-leucine, before bacteria were fed to flagellates and radioactive labels were traced into flagellate macromolecules. The concentrations of prey and predators were monitored by flow cytometry. The aim of the work was to compare assimilation efficiencies of different bacterial macromolecules by mixotrophic (Ochromonas) and heterotrophic (Spumella and Bodo) flagellates. We observed that flagellate assimilation efficiency of bacterial DNA (mean 15-30%, depending on flagellate species) was lower than of bacterial proteins (mean 26-68%). Protozoan assimilation efficiencies did not differ significantly whether the bacterial prey were growing or long-term starvation cultures. Heterotrophic flagellate species had similar assimilation efficiencies of bacterial proteins, when either leucine (26-42%) or methionine (31-41%) was used as a tracer. In contrast the mixotrophic flagellate assimilated significantly more residues of labelled methionine (68%) than of labelled leucine (54%). Methionine was apparently incorporated as an intact molecule and most likely the mixotrophic flagellate preferentially assimilated methionine as a source of reduced sulphur, being able to synthesise other amino acids. Our results suggest that labelled proteins are more appropriate than labelled DNA for studying transfer of bacterial biomass within food webs.

HETEROTROPHIC PROTOZOA AND THE TRANSFORMATION OF DMSP TO DMS IN THE OCEANS

Stephen Archer, Claire Widdicombe, Peter Burkill, Plymouth Marine Laboratory, Prospect Place, Plymouth, UK, Gill Malin, Michael Steinke and Peter Liss. School of Environmental Sciences, University of East Anglia Norwich UK.

Laboratory experiments have demonstrated that grazing may be a key process by which the phytoplankton osmolyte dimethylsulphoniopropionate (DMSP) is transformed to dissolved dimethyl sulphide (DMS). Heterotrophic protozoa are often the major grazers of phytoplankton in the oceans, yet there is limited information on the role they play in DMS production in natural waters. During a series of research cruises we have used a modification of the dilution approach to determine the production rates of DMSP, the grazing pressure exerted by heterotrophic protozoa on DMSP-containing phytoplankton and the extent of grazing-mediated production of dissolved DMSP and DMS. Our results indicate that heterotrophic protozoa play an important role in determining the fate of DMSP production and transform sufficient DMSP to the dissolved phase to support a daily turnover of the dissolved DMSP+DMS standing stock. How relevant this transformation pathway is to the concentration of DMS in surface waters and therefore the potential flux of DMS to the atmosphere will be discussed in light of the results of a recent multidisciplinary research cruise on DMS biogeochemistry.

HERBIVOROUS PROTISTS AND CHEMICAL DEFENCE IN PHYTOPLANKTON - DO DIMETHYL SULPHIDE (DMS) AND RELATED COMPOUNDS PLAY A ROLE IN TROPHIC INTERACTIONS?

Michael Steinke, Gill Malin and Peter Liss, School of Environmental Sciences, University of East Anglia, Norwich, UK, Stephen Archer and Peter Burkill, Plymouth Marine Laboratory, Prospect Place, Plymouth, UK.

Marine waters are an important source of biogenic trace gases including dimethyl sulphide (DMS) which is of considerable importance in the global sulphur cycle, affects atmospheric acidity and is thought to play a significant role in climate regulation. The precursor of DMS is dimethylsulphoniopropionate (DMSP), an osmolyte in various phytoplankton taxa, such as the haptophytes and dinophytes. Grazing by microzooplankton can greatly stimulate DMS production but this process is poorly understood. However, the enzyme DMSP lyase is believed to play a key role in the cleavage of phytoplankton DMSP. This enzyme has been found in heterotrophic bacteria and marine phytoplankton, where DMSP lyase activities are species- and strain-specific. Selective grazing of microzooplankton can result in various DMS production rates, hence, it influences the ultimate flux of DMS to the atmosphere. Acrylate, the by-product of DMSP cleavage, has been suggested as a chemical deterrent of microzooplankton grazing, because grazing of the heterotrophic dinoflagellate Oxyrrhis marina on high-lyase strains of the coccolithophorid Emiliania huxleyi was reduced compared to grazing on strains with low DMSP lyase activity. Biogenic trace gases may also be important as infochemicals in marine tritrophic interactions similar to the production of volatiles by higher plants which attract mutualistic carnivores as part of an inducible defence. Such indirect defences have not been investigated for marine plankton. Regulatory mechanisms for grazing on phytoplankton are likely to be important for the success of certain species in dominating phytoplankton assemblages. Furthermore, this might represent a so far unrecognised regulation mechanism within the marine foodweb that results in the production of climate-relevant gases.

NEW TECHNOLOGIES AND ADVANCES IN THE STUDIES OF PARASITIC PROTOZOA.

USING TOXOPLASMA GONDII AND MAMMALIAN MICROARRYS TO EXPLORE THE HOST-INTRACELLULAR PATHOGEN INTERFACE

Jennifer M. Fitzpatrick and James W. Ajioka. Dept. of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK.

Interactions between host and intracellular pathogen are a complex series of physiological changes in both the host and pathogen. Studies of the cellular and molecular interplay between host and intracellular pathogen, even in the most defined in vitro system, is technically challenging because of problems associated with differentiating host and pathogen derived macromolecules. Current evidence suggests that host cell functions are altered after invasion and subsequent growth of T. gondii. DNA microarrays afford the opportunity to investigate large-scale patterns of transcriptional gene expression associated with intracellular pathogenicity. The combination of microarray technology with the exploitation of phylogenetic distance between host and intracellular pathogen allows the simultaneous monitoring of transcriptional events in the host-intracellular pathogen interaction. Data from micorarrays of T. gondii and mammalian cDNAs hybridised with RNA prepared from T. gondii tachyzoite infected human foreskin fibroblasts demonstrate the feasibility of developing a model system for investigating host-intracellular pathogen relationships.

VACCINATION WITH A NOVEL STAGE-REGULATED SURFACE PROTEIN FROM LEISHMANIA DONOVANI CONFERS PROTECTION AGAINST VISCERAL LEISHMANIASIS IN BALB/C MICE

Stager S. 1, Alexander J. 3, Brombacher F.4, Smith D.F.2 and Kaye P.M. 1 1 London School of Hygiene and Tropical Medicine, Dept. of Infectious Diseases, London, 2 Imperial College of Science, Technology and Medicine, Dept. of Biochemistry, London, 3 Department of Immunology, University of Strathclyde, Glasgow, 4 Department of Immunology, Medical School, University of Cape Town, Cape Town.

Leishmania. donovani-derived Hydrophilic Acylated Surface Protein B1 (HASPB1; formerly known as GBP in L. major) is one of the few defined antigens described for L.donovani, the causative agent of visceral leishmaniasis. Immunisation of BALB/c mice with rHASPB1 with or without IL-12 results in a significant reduction in parasite burden in both liver and spleen, and is effective against challenge for at least 3 months. Vaccination with rHASPB1 in the absence of IL-12 is more effective in promoting protection in the spleen. Preliminary studies of the mechanisms underlying the immune response to HASPB1 show that HASPB1 vaccination induces IL-12 production by dendritic cells (DC). Vaccine-induced protection also correlates with an antigen-specific IgG1 response and IFNg production by HASPB1-specific CD8+ T cells. These data suggest that DC priming of CD8+ T-cells underlies the efficacy of rHASPB1 vaccination. Surprisingly, vaccination with rHASPB1 fails to confer protection in IL-4 and IL-4Ra deficient mice, suggesting further complexity in the regulation of vaccine-induced immunity.

IDENTIFICATION OF ANTIMICROBIAL AGENT TARGETS IN APICOMPLEXANS

Craig Roberts Department of Immunology, Strathclyde Institute of Biomedical Sciences, University of Strathclyde, Glasgow, G4 ONR

.

Current antimicrobial agents for the treatment of diseases caused by Apicomplexan parasites are inadequate due to toxicity, intolerance, allergy, non-specificity and the development of parasite drug resistance. In addition, certain lifecycle stages of Apicomplexan parasites are unaffected by current treatments which therefore allow a reservoir of disease to persist within the infected host. A number of recently described plant like biochemical pathways in these organism may provide potential targets for new, better drugs. Two pathways that are promising in this respect are the shikimate pathway and type II fatty acid biosynthesis. We have recently demonstrated that inhibition of these pathways by glyphosate or triclosan, respectively inhibits the growth of these parasites. The use of EST and genome projects have greatly facilitated the quest for the enzymes of these pathways in Apicomplexans.

MOLECULAR EPIDEMIOLOGY OF PARASITIC DISEASE: THE TRANSMISSION AND PATHOGENESIS OF TOXOPLASMA GONDII.

Judith.E.Smith School of Biology, Leeds University, Leeds, LS2 9JT, UK and Geoff Hide Division of Biological Sciences, University of Salford, Salford, M5 4WT, UK.

Pathogenesis of parasitic disease ultimately depends on the interaction between host susceptibility/resistance genes and parasite virulence genes. Our interests are in determining the basis of parasite virulence through cell and population level studies as exemplified by our work on Toxoplasma gondii. Toxoplasma is a zoonotic parasite which affects all mammals and birds and may be transmitted through a sexual cycle in the cat, via carnivory or via congenital infection. Toxoplasma isolates are designated as virulent or avirulent on the basis of in vivo and in vitro growth characteristics. Early genetic analysis divided reference isolates into either two or three clades, with all virulent isolates belonging to a single lineage (type 1). However, specific epidemiological surveys suggest that type 1 isolates are rare and the majority of disease is due to type 2 isolates. In order to analyse transmission and virulence at the population level we therefore require polymorphic markers that can be used to type strains directly from field samples. We have developed a novel PCR based approach to amplify mobile genetic elements (MGEs)to assay positional variation of MGEs in different isolates. The technique enables us to differentiate between type 2 reference isolates and has sufficient sensitivity and specificity to enable amplification directly from infected tissue samples. We have initially applied MGE-PCR to analysis of congenital infection and abortion in sheep. Our results suggest a common source of toxoplasma infection within the population studied and demonstrate a high rate of congenital transmission in this species. The MGE technique is a generic method. Markers have also been developed for the African trypanosome T.brucei, and are able to differentiate between human and animal isolates derived from a single focus of disease.

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