Abstracts and Proceedings

Spring Conference 2006:
Incorporating A Special Symposium on Molecular Ecology and Protozoa

This meeting took place at Coventry University, on March 30th-April 1st 2006. Abstracts and proceedings from this meeeting are given below.

Opening the black box of marine heterotrophic flagellates

Ramon Massana

Institut de Cičncies del Mar (CSIC), Passeig Marítim de la Barceloneta 37-49, 08003 Barcelona, Catalonia, Spain. ramonm@icm.csic.es

Heterotrophic flagellates are ubiquitous protists that play key roles in planktonic marine food webs. They are the main consumers of prokaryotes and participate directly in nutrient remineralization. Both direct measurements and size-fractionation grazing experiments have revealed that these assemblages are numerically dominated by very small cells of 2-3 µm in diameter, which mostly remain unidentified. Recent molecular surveys of marine picoeukaryotes have revealed a huge diversity and the presence of novel lineages. Here I will show that some of these novel lineages, in particular some organisms forming novel clades within the stramenopile radiation (MAST, Marine Stramenopiles), account for a significant part of heterotrophic flagellates assemblages. Phylogenetic probes against different MAST groups have been designed and optimized for FISH to follow the abundance and distribution in the environment of the corresponding organisms, and together with different experimental approaches to allow to infer their ecological role. Tools to open the black box of marine heterotrophic flagellates, which are composed of diverse groups with diverse functions, start to be available.

Heterotrophic flagellates within marine picoplankton: predation and parasitism

Guillou Laure, Chambouvet Aurélie. Station Biologique de Roscoff, Place Georges Teissier, BP74, 29682 Roscoff- France

Novel and unsuspected diversity was recently discovered within extremely small marine eukaryotes by culture-independent surveys (ciPCR). These techniques mostly consist on the direct amplification of genes (usually the SSU rDNA) from pooled environmental genomic DNA without the need to identify and isolate responsible organisms. Within them, the novel alveolates group I and group II, were systematically reported from a wide range of marine ecosystems, i.e. from the sea surface to deep sediments, getting through deep anoxic permanent waters or hydrothermal sediments. Based upon sequence comparison, the whole group II was suspected to be synonymous to Syndiniales, an order exclusively composed of marine parasites. Are extremely small marine eukaryotes composed of marine parasites? Based upon direct observations (Fluorescent in situ hybridizations), we demonstrated that a large number of novel alveolate Group II are parasitoids in marine coastal waters. Live cycle, host specificity, and ecological impact of such parasitoids in marine waters will be then discussed.

Flow cytometric enumeration of DNA-stained planktonic protists in contrasting regions of the Atlantic Ocean

Mikhail V. Zubkov

Background: Protists are the key primary producers and consumers of biomass in the world ocean. While flow cytometric enumeration of phototrophic protists (PP) is possible due to autofluorescence of their photosynthetic pigments, heterotrophic protists (HP) are usually counted fixed, stained under microscope. The aim of the present study was to test the practicality of enumerating fixed, DNA-stained HP in contrasting regions of the Atlantic Ocean.

Methods: Paraformaldehyde-fixed, SYBR Green I DNA-stained oceanic protists were enumerated using a standard flow cytometer (FACSort, BD) at an enhanced flow rates to provide adequate numbers of counted cells. HP and PP were discriminated using the extra red chlorophyll-derived fluorescence of the latter. For comparing with stained PP counts, the unstained fixed and unfixed picoplanktonic PP were enumerated using picophytoplankton and nanophytoplankton protocols.

Results: The relationship between counts of oceanic stained and unstained fixed and unfixed picoplanktonic PP was statistically close to 1:1, confirming the accuracy of stained protist counting and good discrimination of PP from HP cells. The vertical distribution of HP in the surface mixed layer was similar in different regions of the ocean with maximum abundances of 500-1100 cells ml-1 that coinciding with maxima of PP and/or cyanobacterial abundances. In agreement with earlier studies in more productive aquatic environments a significant correlation (correlation coefficient 0.84, P<0.0001), was found between the HP and total prokaryote numbers, with an average ratio ~1300 prokaryotes to 1 HP cell.

Conclusion: The present study showed that it is practically possible to enumerate stained protists in oceanic waters, including oligotrophic gyres, by flow cytometry.

Marrying function and genes in protists, an interdisciplinary approach.

C. D. Lowe, D. J. S. Montagnes, S. J. Kemp, School of Biological Sciences, University of Liverpool, UK

We used an interdisciplinary approach to assess phylogenetic and functional variation in two free-living microeukaryotes. We assessed phylogenetic and functional diversity among 11 strains of the model flagellate Oxyrrhis marina using 5.8S ITS rDNA and growth responses to salinity; strains formed four phylogentic clades and displayed two growth responses to salinity: one group achieved highest growth rates at high salinities, and the other grew best at low salinities. No clear correlation between molecular, ecophysiological, or geographical differences occurred. However, salinity tolerance was associated with habitat: intertidal strains grew best at high salinities; open-water strains grew best at low salinities. Extending this approach we examined salinity tolerance in the Brachionus plicatilis (Rotifera) species complex (admittedly, not protists but useful models, nonetheless). Ecophysiological and biochemical responses revealed an ion-regulatory mechanism, sensitive to salinity, which differed between sibling species. Molecular techniques indicated variation in, and changes in expression of, genes associated with this mechanism, again indicating sibling species differences. Such genetic variation might account for ecologically relevant differences in salinity tolerance between sibling species. We suggest that interdisciplinary studies like these offer a means to assess relationships between phylogenetic and functional variation and characterise the mechanisms underpinning important ecological traits.

Molecular Phylogeny and Morphological Evolution in Social Amoebas

Prof. Sandie Baldauf

The social amoebas (Dictyostelia) are an ancient group that display a unique form of true multicellularity in a wide range of structural forms. While the genome sequence of Dictyostelium discoideum is now complete, there is little molecular data from the rest of the group. We have constructed the first molecular phylogeny of Dictyostelia using two independent molecular markers and including nearly all known taxa. We find that dictyostelid taxonomy requires major revision at the deepest levels. A mapping of all well-documented morphological characters onto the phylogeny identifies novel evolutionary patterns in cell-cell signalling and development including independent evolution of complex morphologies.

The use of fast-evolving genetic markers to map global distribution of Protozoa

David Bass

Some ecological and evolutionary processes appear to be fundamentally different between microbes and larger organisms. A key example is the Ubiquitous Dispersal Hypothesis (UDH), which states that microbes are globally distributed, and will grow wherever on the planet conditions suit them. The UDH has been upheld for protists by most morphological and some molecular analyses, but most evolutionary markers used to investigate microbial biogeography evolve too slowly to test this hypothesis adequately. Here we use two fast evolving markers to map and compare the global diversity of four protist groups by screening multiple environmental gene libraries of the rapidly-evolving ITS rDNA of several cercomonad strains (Cercozoa) and a unique fast-evolving 18S rDNA region of cyclotrichids (Alveolata). Even at this high level of resolution many identical sequences were retrieved from widely separated global sampling sites implying rapid global dispersal. Many cercomonads show high levels of physiological adaptability, with identical ITS sequences recovered from both marine and non-marine environments, whereas cyclotrichid distributions appear to be more dependent on ecological conditions. Our results support both elements of the UDH for many lineages - global dispersal and environmental selection. We anticipate that protozoan communities generally comprise a large diversity of coexisting but genetically distinct strains and are rapidly renewable and open to invasion on a global scale.

Evidence for cosmopolitan ecotypes within the ciliate Cyclidium glaucoma

Susan Browna , Bland J. Finlaya1, Genoveva F. Estebana, Tom Fenchelb and Kerstin Hoef-Emdenc

  1. Centre for Ecology and Hydrology, Winfrith Technology Centre, Winfrith Newburgh, Dorchester, Dorset DT2 8ZD, UK
  2. Marine Biological Laboratory, University of Copenhagen, Strandpromenaden 5, DK-3000, Helsingřr, Denmark
  3. Botanisches Institut, Universität zu Köln, Gyrhofstr. 15, 50931 Köln, Germany

Many protistan morphospecies have been demonstrated to harbour a high degree of genetic diversity. Whilst the morphotype itself is assumed to have been conserved as an adaptive peak, the ecological significance of the cryptic genetic diversity within a morphospecies is poorly understood.

In this study, ribosomal DNA (rDNA) sequence variation within the common and ubiquitous ciliate morphospecies Cyclidium glaucoma has been investigated. A total of 54 sequences from both clonal cultures and environmental samples (using a specific primer) have been obtained from 47 locations over seven continents. Sequence comparisons reveal three lineages which correlate with the salinities of the habitats from which the sequences were obtained. Ribotypes isolated from hyperhaline habitats formed a discrete sub-cluster. But there is no detectable geographic pattern in the distribution of C. glaucoma ribotypes, for example, sequences originating from Argentina, Peru, Japan, Morocco, Russia and Ukraine were identical, as were those from Denmark and Australia. The significance of these results will be discussed; do the rDNA sequence clusters represent cosmopolitan ecotypes?

Survival strategies of malaria parasites in the mammalian host - recent work on dendritic cell function in infected mice

Stephen Phillips, Caterina Di Lorenzo, Owain Millington, Cathy Rush, Angela Grierson, Jim Brewer, Paul Garside

Division of Infection and Immunity, Glasgow Biomedical Research Centre, University of Glasgow and Centre for Biophotonics, Strathclyde Institute for Biomedical Sciences, University of Strathclyde

Acute and to a lesser degree chronic asexual blood stage malaria infections are associated with a reduced response to heterologous antigens such as vaccines and also to the parasite itself. Dendritic cells as antigen presenting cells play a central role in the induction of a primary immune response to an antigen. We have been investigating the interaction between asexual blood stage malaria parasites and murine dendritic cells (DCs) to see if the parasites modulate DC function. Most studies have been with the rodent malaria Plasmodium chabaudi and have investigated the DCs exposed in vitro and in vivo to the parasite. Experiments will be described which demonstrate both in vitro and in vivo that the parasite reduces DC activation but nonetheless these cells can activate T cells to some extent. In spite of T cell activation in vivo T cell expansion is impaired and their migration into follicles to provide B cell help is reduced. Malarial pigment (haemozoin) is one factor which contributes to modulation of DCs.

Do temperature-food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina

Susan A Kimmance1*, David Atkinson2, David J S Montagnes2

  1. Present address, Plymouth Marine Laboratory, Prospect Place, Plymouth PL1 3DH, United Kingdom,
  2. School of Biological Sciences, University of Liverpool, Biosciences Building, Crown Street, Liverpool, L69 7ZB, United Kingdom

The consequence of interactions between temperature and food concentration for protistan population dynamics and estimates of aquatic productivity are relatively unknown, primarily because we lack adequate parameters for models. Using the heterotrophic flagellate Oxyrrhis marina, we demonstrate the importance of considering temperature and food concentration, in combination, to determine the responses of grazing rate, specific growth rate, cell volume, specific production, and yield. By applying iterative curve-fitting to data obtained from multiple temperature-food concentration combinations, we produced phenomenological models of grazing rate, specific growth rate, and cell volume. We then compared predictions from a simple predator-prey simulation model that applied either our derived equations or a single exponential (Q10) relationship to the specific growth and ingestion responses at 20şC. Our results demonstrate the complex effects of food and temperature in combination on production parameters, and we argue that these should be considered in aquatic ecosystem simulation models.

The Molecular Phylogeny of Microsporidia: Clades Based on Habitat and Host.

H.Elizabeth McClymont*, A.M.Dunn*, R.S.Terry*, D.Rollinson#, D.T.J.Littlewood# and J.E.Smith* (*School of Biology, University of Leeds and #the Natural History Museum, London).

Novel molecular information on Microsporidia in land and freshwater Gastropods reveals that they fall in two distinct areas of the phylogenetic tree of Microsporidia. One group falls close to a genus of mammalian-infective microsporidia and the other close to genera that have been described mostly from insect hosts. However, how much do we know about the Phylum Microspora in terms of the phylogenetic relationships and the ecological groups? How many new species are there which are yet to be discovered? Can we rely on a phylogeny based on one gene - the ribosomal DNA? Is it premature to draw broad evolutionary inferences from existing phylogenies and host associations? We will present a new phylogenetic analysis including the new microsporidia identified in African and British freshwater snails and common British Slugs and review research being carried out into the systematics of the Microsporidia.

What's it all about? The Choanoflagellate Lorica

Barry Leadbeater School of Biosciences, University of Birmingham

Conventionally the choanoflagellate lorica has been seen as a basket made up of siliceous costae with an appearance and properties more suited to a taxonomist than for its important role in the marine environment. In fact the siliceous basket is a framework for the deployment of a very thin organic investment which enables the cell to concentrate a current of water on to its collar of tentacles thereby enhancing its feeding potential. In many species, increase in the efficiency of water propulsion is achieved in much the same way as a chimney creates a draught of air above a fire or oven. Variations in the size, shape and degree of silicification of the lorica, together with the positioning of the organic investment, have allowed choanoflagellates to diversify on a global scale and colonise all manner of marine environments.

Cells in baskets, cells that can count Cells that do somersault, cells that bounce The finer points of choanoflagellate lorica structure and assembly

Rebecca Atkinson, Rong Cheng and Barry Leadbeater School of Biosciences, University of Birmingham

The choanoflagellate lorica is a framework of costae (longitudinal, transverse and spiral) containing rod-shaped units called costal strips made of silica. Costal strips are produced individually within the parent cell and are transferred to the top of the collar where they are stored until a complete set is produced. Cell division then takes place and one daughter, the juvenile, is pushed backwards out of the lorica taking with it the accumulated strips. Within 1-2 minutes a new lorica is assembled. The order of costal strip production, the logistics of accumulation and the well-defined movements of the collar tentacles determine precisely the substructure of the mature lorica. A combination of light and electron microscopy and growth experiments involving silica depletion have revealed the intricacies of the underlying logistics and mechanism of lorica assembly.

Apicomplexan Cell Surface Motility

Conrad King Biology Department, University College London, WC1E 6BT

Involvement of actin and myosin at the pellicle is responsible for target cell invasion,overt gliding and bead translocation. Models based on Plasmodiumwould locate myosin to the more internal inner membrane complex whilst the actin is more closely associated with the plasmalemma. Due to low Reynold's Numbers inertial forces are negligible and motility will only be observed at that instance when force generation between cell and substratum/bead occurs. In studies on 2 µm bead translocation (0->10µm s-1) in Gregarina very erratic bead movement occured with long periods of zero movement. Also in laser-trap experiments only occasionally could the bead escape from the force. These results point to the erratic generation of force at the cell surface with respect to intensity and time. The best hypothesis to explain this would be polymerisation of actin leading to an F-Actin template. Ignorance of the in-vitro details of actin polymerisation raises the question of whether these rates could match the in-vivo motility rates seen in some genera (up to 60µm s-1 in Porospora).

Genetic analysis of trypanosomes

Wendy Gibson, Lori Peacock, Vanessa Ferris, Katherine Williams and Mick Bailey Schools of Biological Sciences and Clinical Veterinary Science, University of Bristol, Bristol BS8 1UG, UK

Trypanosoma brucei undergoes genetic exchange in its insect vector the tsetse fly by an unknown mechanism. To visualize the production of hybrids in the fly, one parental trypanosome clone was transfected with a gene encoding Green Fluorescent Protein (GFP) and the other with a gene encoding Red Fluorescent Protein (RFP). Genetic crosses of red and green parental trypanosomes produced progeny with red, green, yellow or no fluorescence, consistent with Mendelian segregation and reassortment of the reporter genes. Hybrid genotypes of progeny clones were confirmed by microsatellite and molecular karyotype analysis. Yellow hybrids were easily visualized in the fly and were observed only in flies with mixed red and green salivary gland infections. Although most infected flies had a co-infection of red and green trypanosomes in the midgut, yellow trypanosomes were not observed in the midgut or proventriculus. We conclude that genetic exchange occurs among trypanosomes in the salivary glands and that the only prerequisite for genetic exchange is co-infection of a salivary gland with both parental trypanosomes. The high frequency of hybrid production observed in this cross, together with the easy visualization of hybrids, means we now have an optimum system for further exploration of genetic exchange in trypanosomes.

Glyphosate toxicity to protozoa and metazoa

Humphrey G Smith and SJ Coupe, The James Starley Building, Coventry University, CV1 5FB.

Permeable paving systems (PPS) reduce flooding and pollution in urban water. Biodegradation processes involving the mixed microbial community in PPS are critical to water quality improvements. If herbicides are sprayed onto permeable pavements during routine landscaping, the extent of attenuation of these compounds by PPS, and also their discharge into receiving soils and waters is currently unknown. It is also unknown whether commonly used glyphosate containing herbicides disrupt the oil biodegradation processes and inhibit biofilm development. The effect of glyphosate on common PPS protozoa and metazoa was analysed in a series of in-vitro tests. The ciliate Colpoda cucullus and a single species of nematode were isolated from pavement effluent and grown using an oil degrading gram-negative bacterium as food. This bacterium was initially isolated from an oil degrading permeable pavement, and was subsequently grown in bulk culture on flooded agar plates comprising mineral nutrients (Bushnell-Haas) with oil as the sole carbon and energy source. The sensitivity of these organisms to a range of glyphosate concentrations was assessed by direct counts of the number of live individuals remaining in a set volume of culture after exposure to the contaminant. Colpoda cucullus was shown to be very sensitive to glyphosate with an LD50 of around 50 mg/l. The lowest applied concentration of 24 mg/l was also shown to significantly cause mortality. The resistance of nematodes was much greater however with an LD50 of around 1000 mg/l. Some live nematodes were observed with glyphosate concentrations up to 24000 mg/l, but a significant level of mortality compared with non-glyphosate controls was observed with only 240 mg/l. The concentration of glyphosate recommended for domestic use is 2400 mg/l, indicating that the effect of this compound on different members of the PPS microbial community and PPS water treatment, is likely to be highly variable.

The effect of glyphosate containing herbicides on protozoan community structure

Stephen J Coupe and HG Smith The James Starley Building, Coventry University, CV1 5FB.

Research into the effect of herbicides on the microbial communities growing in permeable pavement systems (PPS) has demonstrated that glyphosate containing compounds can significantly increase protozoan mortality. The effect of glyphosate on mixed microbial communities from oil degrading PPS was analysed in-vitro. The ability of PPS decomposer organisms to degrade glyphosate was measured using mineral oil alone, with glyphosate alone and in other treatments using a mixture of these compounds. The measurement of biodegradation was by CO2 evolution. Glyphosate was added at a concentration of 2400 mg/l, which is the dose recommended by the manufacturer. The effect on protozoa was measured by both quantitative and qualitative analyses of the community structure over a 75 day test period. Results showed that glyphosate was rapidly degraded by PPS organisms as CO2 levels were significantly higher than cultures where no oil or glyphosate was added. The highest levels of CO2 however, were observed where oil and glyphosate were added together, showing that PPS microbes can degrade a range of different organic contaminants even when they are added simultaneously. Despite the stimulation of decomposer organisms by glyphosate, the effect on protozoa was pronounced, and reduced the total number of protozoa compared to oil-only cultures throughout the test period. Glyphosate reduced the total protozoan count by at least one order of magnitude during the first three weeks of the experiment. Common ciliates such as Colpoda cucullus were absent from glyphosate-dosed cultures but were abundant when oil was added alone. In total, 13 species of testate amoeba were recorded from glyphosate treated cultures and testates were the only organisms identified microscopically except for nematodes. Without glyphosate, four testate species were recorded in addition to ciliates and microflagellates (implying that the other nine testate species were present but at concentrations below the levels of detection). It is not known at present whether the critical factor for testate dominance in glyphosate cultures is due to protection from the herbicide by the test or because of reduced competition. The effect of glyphosate on PPS protozoa was to increase the number of larger and more slow-growing species at the expense of more common organisms that have been routinely recorded in previous PPS effluent and culture experiments. The biodegradation of glyphosate containing herbicides in culture indicates that if this compound is physically immobilised, it will not persist within PPS in the long-term. The effects of herbicides on protozoan community structure may not become clear immediately, but must be monitored, to ensure that the vital role protozoa play in biodegradation is not disrupted by these changes.

The stimulation of oil degrading microbes by targeted nutrient addition: Protozoan colonisation studies.

Gillian E Spicer, Stephen J Coupe, Humphrey G Smith The James Starley Building, Coventry University, CV1 5FB.

The development of a self-fertilising geotextile mat designed to provide a sustained slow-release of inorganic nutrients for the growth of oil degrading micro-organisms in permeable pavement systems (PPS) is reported. The mat is produced from a geotextile spun from polymer fibres containing spherical phosphated polymer beads, this releases phosphate upon contact with water at a desirable level for microbial growth. The nutrient containing geotextile system works very effectively as increased microbial activity has been observed in experiments conducted in model PPS. An experiment was performed to monitor the colonisation of this nutrient dosed geotextile by protozoa over the initial eight weeks of a 24 week experiment. Following the addition of oil and simulated rainfall, protozoa both in PPS effluent and on harvested samples of geotextile were enumerated using direct counts and identified where possible. The screening of geotextile samples by SEM was also performed to provide information of the quantity of biofilm accumulating on the geotextile over the time course. Results showed that protozoa were found in much higher numbers in PPS effluent with 1.5 x 104 per ml than on the geotextile with 1.0 x 102 per ml despite the presence of nutrients on the phosphated fibres. The species richness of protozoa was also higher in effluent than on harvested geotextiles. Eight species of protozoa were recorded from effluent and three from geotextiles. SEM results showed a clear increase in the amount of biomass accumulated on the geotextiles changing from small clusters of bacteria at the beginning of the experiment to considerable bacterial and fungal aggregations in the latest samples. Previous work has demonstrated that PPS geotextiles do become covered with microbial growth after a period of six months attaining a density of 8.0 x 103 and with the presence of 13 species. The results from the current experiment indicate that insufficient time had elapsed for sufficient biofilm accumulation to support a number of protozoa similar to that observed previously. These confirm previous results demonstrating that protozoa are originally brought along on pavement building materials, in particular granite, and gradually colonise other PPS surfaces such as geotextiles. As sampling continues it is hoped that increases in protozoan cell densities, and species richness on the phosphate rich geotextiles, will be monitored over the remaining 16 weeks of the experiment.

Haemoglobin degradation possibly is the first target of a medicinal plant template, inhibiting growth of plasmodium falciparum in infected erythrocytes

Feiz Haddad M.H.1, Wright C.W.2, Dodson H.I3

  1. School of Medicine, Department of Mycoparasitology
  2. Gendishapour Medical Science Ahvaz University, Ahvaz, Iran
  3. School of Pharmacy, Department of Biomedical Sciences, the University of Bradford, Bradford, UK

The Plasmodium sp. requires amino acids for the synthesis of their proteins and for energy metabolism. Haemoglobin serves as the major source of amino acids for the parasite. Haemoglobin degradation releases heme and globin that generates amino acids. The heme moiety does not appear to be metabolised or recycled but is toxic to the parasite, which is converted to an insoluble inert polymer known as the malaria pigment, hemozoin. This conversation can be inhibited in the presence of malaria standard drugs such as chloroquine.

Here we have shown, using Sodium Dodecyl Sulphide Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Transmission Electron Microscopy (TEM) the accumulation of hemoglobin and heme in the cytoplasm of parasite cells when the cells are treated with both malaria standard drugs and the medicinal plant template. The SAS-PAGE gels showed that in the malaria parasites treated with quinine, chloroquine, amadiaquine and plant template, most of the FPIX is associated with a protein that has a molecular mass of 16 kDa, i.e. Identified to that of haemoglobin. As the TEM studies showed morphological changes with food vacuole (FV) enlargement, disintegration of malaria pigment and erosion properties on the FV cell membrane.

These results suggest that possibly haemoglobin degradation in the FV is a primary target for the plant template and standard drugs as a part of their parasite killing mechanism.

Posters

Effects of a plant template and its analogues agaainst Giardia intestinalis in vitro

Feiz Haddad M.H.1, Wright C.W.2, Dodson H.I3

  1. School of Medicine, Department of Mycoparasitology,
  2. Gendishapour Medical Science Ahvaz University, Ahvaz, Iran
  3. School of Pharmacy, Department of Biomedical Sciences the University of Bradford, Bradford, UK

New drugs are very urgently needed for the treatment of number protozoal diseases such as giardiasis. The potential value of some synthetics from a plant template were evaluated against G. intestinalis and compared with standard drugs. Compounds that inhibit parasite attachment and/or growth are considered potentially active. Antigiardial activity of the plant template and its seven derivatives were tested. Results showed that two had MIC less than 20 µM and the remainder between 40 and 177 µM. The MIC of metronidazole, emetine hydrochloride and berberine chloride as standard drugs were 1.85, 4.51, and 5.86 µM respectively. The 2-Methoxy and 7-nitro analogues had MICs of similar magnitude to the plant template, however, 2-Fluoro analogue was more than twice as potent. But 11-one and 3-n-acetyl analogues were less than half as active. Apparently, the substitution of fluorine at position 2 increased antigiardial activity to more than two fold while a methoxy group in the same position does not change the activity. The n-acetyl group at position 3 and ketone (-C=O) at position 11 reduced antigiardial activity. While the 9-nitro and 7,9-dinitro with 9.95 and 17.36 µM were the most potent analogues with 10 and 5 fold more activity than the parent compound respectively. Placing the nitro group at position 9 resulted in a marked increase in activity while at position 7 decreased dramatically to 79.6 µM. Therefore, it is obvious that the type and status of the substitution groups on the structure of the plant template is fundamental to antigiardial activity.

Fluorescently-labelled heterotrophic flagellates and their applicability to grazing experiments

Martin-Cereceda1 M, Smith1 VH, Guinea2 A and Novarino3 G

  1. Department of Ecology and Evolutionary Biology, Haworth Hall, 1200 Sunnyside Avenue University of Kansas Lawrence, KS 66045 USA cereceda@ku.edu
  2. Departamento de Microbiologia III, F. Biologia, UCM, Madrid, Spain
  3. Department of Zoology, Natural History Museum, Cromwell Road, London SW7 5BD, UK

Fluorescent live flagellates were designed to investigate flagellate grazing by other protists. Probes of this kind often involve undesirable effects such as killing of the prey, which may affect palatability by the predator. Our initial premises for the design of the fluorescent flagellates were that the fluorescent molecule should: i) allow viability of the flagellate; ii) not fade, at least for a period long enough to carry out grazing assays; iii) label the flagellate without masking the cytoplasm. Several fluorochromes were tested, and a 5µg/l solution of Hoechst 33342 (a DNA-binding fluorescent dye) gave the best results. This made it possible to monitor live grazing of flagellates for at least 48 hours and carry out error-free and precise enumerations of flagellate cells inside the predator cell. Several grazing experiments were performed with Hoechst 33342-stained fluorescent flagellates and the most significant results were obtained using Goniomonas amphinema as the fluorescent flagellate prey and Oxyrrhis marina as the predator. Results showed that after 2 hours of contact between the fluorescent prey and the predator 30% of the O. marina population had preyed upon G. amphinema. Our results suggest that this is a rapid and inexpensive method for the direct observation of ingestion of heterotrophic flagellates by other protists. The fact that the flagellates remain alive while the predation occurs could make this method very well suited to studying trophic interactions within the microbial food web.

Characterization & Purification of Planktonic Protozoan Feeding Receptor

Emma C. Wootton & Emily C. Roberts Department of Biological Sciences, University of Wales Swansea, UK

Through selective feeding, protists play a fundamental role in structuring bacterial and phytoplankton communities within the marine environment. Although recent evidence indicates that protozooplankton can select food based on cell surface properties of their prey, the underlying mechanisms are poorly understood.

Previously, using haemagglutination experiments, we identified a Ca2+-dependent, mannose-binding lectin (MBL) on the marine dinoflagellate Oxyrrhis marina. Feeding experiments, involving live and bead prey, demonstrated the employment of this lectin as a feeding receptor, used for recognizing and selecting prey. Here, we present initial characterization of O. marina MBL, which will aid purification of this protein.