BSSP 2001 Spring Meeting - Posters
INFLUENCE OF MICRO-ZOOPLANKTON IN MARINE PRODUCTIVITY
Aditee Mitra, SAMS, Dunstaffnage Marine Laboratory, Keith Davidson, SAMS, Dunstaffnage Marine Laboratory,
and Kevin Flynn, University of Wales, Swansea.
Micro-zooplankton play an important role in the dynamics of marine ecosystem as they provide a link for nutrients to pass up the food chain through their ingestion of phytoplankton and bacterial prey and are preyed upon by meso-zooplankton and metazoan grazers. These species are, therefore, potentially a very important component of food web models. However, many planktonic ecosystem models fail to include a representation of grazing micro-zooplankton as a separate entity, either including them in the same compartment as the phytoplankton or separately in an extremely simplified form. Based on current understanding of micro zooplankton physiology, mathematical models of varying degrees of complexity with unlimited prey have been developed using the graphical modelling package (Powersim Constructor). Where possible parameters have been obtained from literature data, others have been estimated on physiological grounds. A detailed steady state sensitivity analysis was conducted to determine the validity of model structure, parameterisation and the robustness of model predictions to changes in parameter values. The output of the different models has been tuned to each other to optimise parameter values using the software package Powersim Solver, which uses numerical Monte Carlo methods. The dynamics of the different micro-zooplankton models are now being studied in transient rather than steady state conditions. The model behaviour in both steady state and transient conditions will be presented and discussed.
GROWTH DYNAMICS AND THE POLYMORPHIC ADAPTATIONS OF A BACTERIVOROUS MARINE SCUTICOCILIATE
Blanca Pérez-Uz, Mercedes Martín-Cereceda and Almudena Guinea. Dept. Microbiología III. Fac. Biología. Universidad Complutense de Madrid, Madrid, Spain.
The life cycle of a marine scuticociliate Urocryptum tortum has been studied. This ciliate, recently redescribed, has a polymorphic life cycle with three different stages previously unknown. The succession of forms is an adaptation to the bacterial prey presence. The growth dynamics leading these morphological changes has been monitored under experimental conditions in monoxenic culture. The trophont is the actively feeding form appearing at the exponential growth phase; it requires a minimum bacterial concentration of 108 bacteria ml-1 to keep growth. Under this bacterial concentration, conjugation processes take place. At the end of the exponential growth phase or beginning of the stationary growth phase a non-divisional morphogenesis process in the exconjugants leads to a non-feeding migratory form, the tomite (or theront). This tomite can be reversed to the trophont stage if the bacterial concentration increases over 108 bacteria ml-1; if no bacterial patch is found between 20-60 hours this stage will give place to cyst. Cysts appear associated into flocs and excystment resumes only when bacterial concentration is over 108 bacteria ml-1.
ARE PROTOZOA RESPONSIBLE FOR CONTROLLING PICOCYANOBACTERIAL ABUNDANCE IN EUTROPHIC WATERS?
Katie Harper, Jackie Parry, John Smith, Lancaster University, Lancaster, LA1 4YQ, and John Day, CCAP, Windermere Laboratory, Far Sawrey, Ambleside, Cumbria, LA22 OLP.
Picocyanobacteria (<3 mm) are known to be limited by nutrients in oligotrophic waters but the controlling factors in eutrophic waters are far less clear. The community structure and abundance of picocyanobacteria in Esthwaite Water, a eutrophic lake, were analysed during the summer of 2001 using HIP1 PCR and epifluorescence microscopy, respectively. Results from this study, and whole water enrichment experiments, showed no significant correlation between the abundance of picocyanobacteria and either heterotrophic flagellates or ciliates. It was the dinoflagellates which increased in abundance when whole water was enriched with various picocyanobacterial 'HIP 1 types'. The interaction between these picocyanobacteria and representative dinoflagellate isolates (from the CCAP) has thus been examined under controlled laboratory conditions.
THE POTENTIAL HARBOURING OF COLIFORM BACTERIA BY PROTOZOA IN BIOFILMS
Joanna English, Jackie Parry, Lancaster University, Lancaster, LA1 4YQ and Roger Pickup, CEH Windermere, Ferry House, Far Sawrey, Ambleside, LA22 0LP.
Protozoa have been nicknamed the"Trogan Horses of the Microbial World" since certain pathogenic bacteria e.g. Legionella spp. and Vibrio cholera, can survive and replicate within their cells. Other bacteria can survive a passage through a protozoan cell, without replication e.g. coliform bacteria, but the impact of this process on both participants has not been fully evaluated. Abundances of both participants can be high within natural biofilms and this poster discusses the distribution of various faecal coliform species and protozoan species within young epilithic biofilms over time, and the effect these bacterial strains have on the functioning of the protozoan cells.
A ROLE FOR MICROFAUNA IN THE ECOLOGY OF NATURAL MICROBIAL BIOFILM COMMUNITIES
S. A. Symmons1, P. Gilbert1, R. Ledder1, D. G. Allison1, D. Charbonneau2 and A. J. McBain1 1University of Manchester, Manchester, UK; 2Procter and Gamble, Cincinnati, Ohio, USA.
Biofilm, removed from the horizontal pipe section of a domestic kitchen sink contained a dense and diverse microflora composed of aerobic (i.e. Pseudomonadaceae and micrococci), facultative and strictly anaerobic bacteria (SRB). Microscopy revealed substantial populations of protozoa (Colpoda maupasi), bdelloid rotifers (Habrotrocha) and rhabditid nematodes. Microcosm communities were established by repeatedly inoculating drain biofilm into constant depth film fermenters (CDFFs, pan depth 5 mm) with anaerobic gassing for the first 48 h. A feast / famine regime, using artificial dishwater and tap water respectively, enabled quasi-steady-states to be achieved after c. 14 d. CDFF pan samples were reproducible and closely reflected the species composition of the original samples, including the micro-fauna. Sectioned biofilm revealed stratification with rotifers and protozoa distributed throughout the depths of biofilm and nematodes confined to upper areas. Pure culture studies with different bacterial drain isolates, together with the selected microfauna demonstrated not only prey preferences on the part of the rotifers and nematodes, but also extreme difficulties in separating C. maupasi from particular bacterial types. Many studies now recognise an intracellular association between some bacteria and predatory protozoa such as acanthamoeba. Accordingly we attempted to 'cure' populations of ciliates from their associated bacteria. Various treatments involving combinations of amikacin (100µg/ml), polymyxin B (150u/ml), lysozyme and Tris-EDTA sufficient to cure acanthamoeba of extracellular bacteria failed in this instance to eliminate Klebsiella pneumoniae from cultures of C. maupasi. Growth (24h) on killed Escherichia coli in the presence of seven different antibiotics gave aseptic protozoal cell lines, but these could only be re-cultured in the presence of viable prey. Strong prey preferences, together with intimate commensalism between bacteria and fauna suggest that the latter play a key role in biofilm community architecture.
HOW DOES OPISTHONECTA MATIENSIS CONJUGATE ?
Mercedes Martín-Cereceda, Blanca Pérez-Uz and Almudena Guinea. Departamento de Microbiología III, F. Biología, Universidad Complutense de Madrid, Madrid, Spain.
The nuclear events occurring during the conjugation process of the new free-swimming peritrich Opisthonecta matiensis are studied. The process firstly involves an asymmetric division resulting in two size-different cells. The macroconjugant is similar to a vegetative cell (45-72 µm length) whilst the microconjugant is much smaller (15-25 µm length) and has conspicuous aboral cilia. New data about the infraciliature, myonem system and binary fission of microconjugants are reported. Microconjugants contact the ventral area of macroconjugants by the aboral pole, and become enclosed completely. Occasionally, more than one microconjugant can enter the macroconjugant, and all seem to be viable. Once inside, the microconjugant micronucleus undergoes a mitotic division while the macroconjugant micronucleus remains undivided. At this stage macronuclei of both conjugants are still unaltered. All the micronuclei undergo two posterior meiotic divisions thus forming four haploid nuclei in the macroconjugant and eight haploid nucleic in the microconjugant. Then, the microconjugant plasmatic membrane dissapears. Micronuclei meiotic divisions are all synchronised and linked to the beginning of degeneration in both conjugant macronuclei. Only one of the haploid nuclei of each conjugant remains viable (pronuclei) fussing together to produce the synkarion. We have observed three postzygotic divisions of the synkarion yielding eight diploid nuclei. One of these becomes the micronucleus of the postconjugant cell, and the other seven are supposed to be macronuclear primordia (anlagen) segregated to daughter cells by binary fission. This aspect is currently being investigated since our observations on this species indicate that only three anlagen are formed. Results are compared with those of a stalked peritrich (Opercularia sp.) and of a species of the same genus (Opisthonecta henneguyi); differences observed are discussed.
THE MICROBIAL ECOLOGY OF A SANDY, TEMPERATE SHORE.
Phillip Cowie, University Marine Biological Station, Millport, Isle of Cumbrae.
The sheltered beach of Kames Bay, Isle of Cumbrae, Scotland has been studied since the 1800s. Initial research focused on the study of the macrofaunal and meiofaunal species and communities within the beach. One area of research which has lagged behind the others is the study of the microbial communities occupying the porous system of the sand body (the interstitial habitat). This study is examining the relationship between components of the microbial food-web (bacteria, heterotrophic protozoa - especially gymnamoebae), abiotic factors (temperature, pH, salinity, grain size, particulate organic carbon) and meiofauna (nematodes & copepods). Samples are being collected on a bi-monthly basis from the upper intertidal, lower intertidal and subtidal oxic sediments of Kames Bay.
A NOVEL AUTOMATED METHOD FOR DETERMINING PROTOZOAN GRAZING RATES
Heaton, K. and Jackie Parry, Department of Biological Sciences, Lancaster University, Lancaster, UK.A novel automated method for the determination of protozoan grazing rates has been devised employing a GFP-expressing bacterium as the tracer prey. After ingestion by the protozoan, acidification in the food vacuole leads to loss of the fluorescence within the prey cells and hence, a decrease in the overall fluorescence of the culture occurs over time. This decrease is recorded by an automated multi-task plate reader (Victor 1420) and an Excel macro transforms the results, reducing the data generation time for grazing experiments from several weeks to 5 minutes. Examples of the use of this method are displayed.
GENETIC EXCHANGE IN TRYPANOSOMA BRUCEI
Wendy Gibson School of Biological Sciences, University of Bristol, Bristol BS8 1UG.
In the order Kinetoplastida, sexual reproduction has been conclusively demonstrated in only one trypanosome species, Trypanosoma brucei. The process has never been observed directly and so many details of the mechanism are obscure. The process takes place in the tsetse fly vector, probably within the salivary glands, but it is not an obligatory part of the lifecycle of the trypanosome. Analysis of several different experimental crosses shows that genetic markers are inherited in a Mendelian fashion, but no haploid intermediate stage has been identified and it is not known whether T. brucei can undergo meiosis. The kinetoplast DNA is inherited uniparentally with respect to the maxicircles (equivalent to the mitochondrial genome of other eukaryotes) and biparentally with respect to the minicircles. Together these results suggest that both meiosis and fusion are involved in genetic exchange, but not necessarily in that order. To get any further towards unraveling what happens during genetic exchange, we need to see the process within the fly. This means pinpointing the developmental stage and the region of the fly where genetic exchange takes place. To do this, we need to be able to visualize hybrids in situ and distinguish them from parental trypanosomes. In recent experiments we have exploited the fluorescent reporter gene, GFP, under control of the bacterial Tet repressor. One parental clone has been transformed with both GFP and repressor constructs, so that it does not express GFP. Following mating with a wild-type parent and assuming that meiosis occurs, segregation of the reporter and repressor genes should give rise to a proportion of hybrid progeny which have only the GFP gene and are fluorescent. In such an experimental cross, fluorescent trypanosomes with hybrid genotypes have been observed in trypanosome-infected salivary glands.
STUDIES ON A NOVEL TOXOPLASMA GONDII DENSE GRANULE PROTEIN
Fiona L. Henriquez, Russell E. Lyons and Craig W Roberts University of Strahclyde, Department of Immunology, Glasgow G4 ONR
At present ten dense granule proteins have been identified in Toxoplasma gondii, including 2 NTPases and 8 proteins of unknown function designated GRA1-8. They are released after parasitic invasion of the host cell after which they are targeted to a specific site, or in some cases specific sites, within or on the parasitophorous vacuole. GRA3 apparently targets two sites, the intravacuolar network (IVN) of the PV, and the parasitophorous vacuole membrane (PVM). We have recently identified an EST (Expression Sequence Tag) that putatively encodes a protein that shares 100% identity with the C-terminal of GRA3. It is hypothesised that this protein, named GRAHOM, encodes a novel dense granule. As GRAHOM would also be recognised by the antibodies previously used to immunolocalise GRA3, this may explain the apparent dual localisation of GRA3. It appears likely that GRAHOM may be targeted to one of these sites and GRA3 to the other according to their N-terminal sequences. To determine this, we are currently working to express and purify, as recombinant fusion proteins, the common C-terminal of GRA3 and GRAHOM and the unique N-terminals of each. Production of antibodies to each of these proteins will facilitate their immunolocalisation. Expression has been optimised for all three proteins using a variety of expression vectors. To achieve this, for two of these proteins (5' unique potion of GRA3 and the common portion), co-transformation with RIG plasmid, which contains tRNAs for rare codons has been necessary. The 5' unique portion of GRAHOM has been successfully expressed in large quantities as a his-tagged fusion protein, isolated using nickel agarose affinity purification and used to generate polyclonal sera. This sera recognises a protein of 64kDa in T. gondii.
THE EVOLUTIONARY RELATIONSHIPS OF AUSTRALIAN TRYPANOSOMES
Patrick Hamilton 1, Jamie Stevens2, Wendy Gibson1 1 School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK 2 School of Biological Sciences, University of Exeter, UK EX4 4PS.
Trypanosomes form an early branch of the eukaryote tree and are now ubiquitous parasites of vertebrates, transmitted by a variety of ectoparasite vectors. Molecular phylogenies for the genus Trypanosoma, based on the small subunit ribosomal RNA gene (ssu rRNA), indicate an ancient divergence between the human infective trypanosomes T. cruzi and T. brucei. In these phylogenies, trypanosomes from Australian vertebrates are only represented by three trypanosomes; one from a kangaroo Macropus giganteus giganteus, one from a common wombat Vombatus ursinus ursinus and T. binneyi from the platypus. The phylogenetic position of these trypanosomes has been influential in the construction of theories on trypanosome evolution.
We have undertaken a survey of trypanosomes from Australian vertebrates. This has resulted in ten new trypanosome isolates: five from platypus, two from the common wombat, one from a wallaby, one from a European Rabbit and one from a Currawong (bird). The trypanosomes from the three marsupials fell in different clades, and were more closely related to trypanosomes from non-Australian mammals than to each other, suggesting that Australian marsupial trypanosomes have divergent evolutionary origins. The Currawong trypanosome clustered with T. bennetti, an avian trypanosome from North America.