BSSP Spring Meeting 1999 ~ Contributed papers

Predatory behaviour of the heterotrophic microflagellate Paraphysomonas vestita

Keith Davidson. Dunstaffnage Marine Laboratory, University of the Highlands and Islands.

The predatory behaviour of a phagotrophic microflagellate, Paraphysomonas vestita, when presented with different phytoplankton prey species was investigated in laboratory culture. Paraphysomonas ingested Isochrysis galbana more rapidly than Pavlova lutheri although both species are prymnesiophytes of similar size and were available at similar carbon:nitrogen ratio. Maximum rate of ingestion of prey and maximum rate of microflagellate growth (in terms of cell number or volume increase) were related to the inoculum prey concentration in a hyperbolic manner. However, in transient conditions these functions did not continue to hold, and ingestion of prey by Paraphysomonas was found to be better represented by a hyperbolic function of the predator/prey ratio. In transient conditions, the threshold prey concentration varied between prey species and for a single species when the prey inoculum concentration changed. However, a single threshold prey/predator ratio was observed for both prey species studied. Such relationships have consequences for our understanding of the predatory behaviour of microflagellates and for the formulation of mathematical models to predict the dynamics of microbial food webs, as the dynamics of ratio dependent systems differ from those based on simple prey concentrations. A temporal decoupling existed between ingestion of prey and subsequent microflagellate cell division. Division was therefore not a simple function of prey density. It was possible to account for this processing time by relating division to the prey/predator ratio using a function incorporating a time lag, potentially negating the need to simulate intracellular processes to predict division rate.

Seasonal dynamics of the protozooplankton in a maritime Antarctic lake undergoing eutrophication

Helen G. Butler. British Antarctic Survey.

Signy Island in the maritime Antarctic has 16 freshwater lakes with a range of trophic status. Fur seals were virtually absent from the island in the 1960s but now visit in numbers exceeding 15,000 per year and occupy several lake catchments. Lakes such as Heywood Lake are enriched by seal excrement and contain high concentrations of nutrients and chlorophyll a. The aims of this project were to carry out the first year-round study of protozooplankton dynamics in Heywood Lake and to assess the impact of seal-induced nutrient enrichment by comparison with previous data for the lake. Results show marked seasonal variations in abundance and diversity of protozooplankton, peaking during the austral summer. In winter the entire water column of the lake became anoxic and the plankton was dominated by a small number of taxa. Heterotrophic nanoflagellates (HNF) dominated protozoan biomass for most of the year, with abundances varying between 1.8 x 105 and 2.5 x 107 l-1. Ciliates reached a maximum density of 4.7 x 104 l-1 in December 1995, the only time that their biomass exceeded that of HNF. Numbers of naked amoebae ranged from undetectable to 4.8 x 104 l-1, but they made only a small contribution to total biomass. An estimated 89 species of protozoa were observed over the study period, most of which appear to be cosmopolitan. Comparison of the current status of the lake with three decades of data collected on Signy Island reveals changes in seasonal patterns and planktonic community structure and an increase in microbial abundance.

Carbon dynamics of a mixotrophic flagellate

Harriet Jones. Centre for Population Biology, Imperial College.

Mixotrophic flagellates have many options available with regard to resource acquisition and allocation. The mixotrophic Chrysophyte Ochromonas sp. can obtain carbon via photosynthesis, by eating prey, or by taking up dissolved organic carbon. This carbon is used for cell maintenance, chlorophyll production, ingestion and assimilation of prey, cell division, cell biovolume, or it can be released as dissolved organic carbon. Data will be shown to demonstrate the dynamics of carbon acquisition and allocation, how it varies with their environment and also within a 24 h cycle. Experimental data will be compared with theoretical models to demonstrate two different types of mixotroph behaviour using Ochromonas sp. and the mixotrophic haptophyte Chrysochromulina brevifilum.

Biodiversity databases - why are they relevant for protistologists?

Sue Brandt. Centre for Plant Diversity and Systematics, School of Plant Sciences, The University of Reading.

The U.N. Convention on Biological Diversity, and the increasing use of the World Wide Web as a virtual library has led to several initiatives attempting to organise species-based information on the Internet. These include the Species 2000 project and various regional initiatives such as the European Register of Marine Species (ERMS) and the North American Integrated Taxonomic Information System (ITIS). These projects draw on the databases produced by international collaborations of taxonomists, who work together to provide a "responsible" consensus view of the taxonomy within their group. Examples of such databases include the Smithsonian/Wilson World List of Mammals, FishBase and the ILDIS World Database of Legumes. These all contain a global list of the species within their taxon, together with synonymic indexing.

With the adoption of Species 2000 at the heart of the OECD MegaScience Forum's Global Biodiversity Information Facility (GBIF), there is now a great need for protistologists to join the taxonomic database community to prevent the emergence of "an Index of All Known Species (Excluding Protists)".

Freshwater choanoflagellate cysts

Barry S.C. Leadbeater* and Serguei A. Karpov**. *School of Biological Sciences, University of Birmingham; **Biological Institute, Saint Petersburg State University, Russia.

There are a number of reports of cyst formation in freshwater choanoflagellates but most are uncertain in detail and there is no complete study that satisfies all the criteria needed to confirm cyst formation. Recently, an isolate of Desmarella has routinely produced cysts in culture. This has permitted an experimental study of encystment and excystment and a thorough investigation of the ultrastructure of cyst formation. Batch cultures of Desmarella inoculated with motile vegetative cells start to produce cysts within 3 days during the exponential phase of growth. Cyst production proceeds until late stationary phase when there is a preponderance of cysts. Transfer of cysts to fresh medium results in limited excystment. Encystment involves the production of electron-dense fibrillar wall material, firstly around the neck of the cell and then around the posterior end. As the wall material is deposited the neck of the cell elongates and the dicyosome rotates from the horizontal to vertical plane. The number of mitochondrial profiles seen in individual sections of cells increases. Finally the neck of the cell is retracted, the flagellum and collar tentacles are withdrawn and the bottom of the neck of the cyst wall is sealed with a diaphragm of wall material. Excystment, which has not be observed directly, appears to involve the disruption of the wall at the base of the neck, the remainder of the wall remains intact.

The potential use of GFP-expressing bacterial cells for measuring protozoan grazing rates

Jackie Parry and Karen Heaton. Division of Biological Sciences, I.E.N.S., Lancaster University.

Two Escherichia coli strains (DH5a and HB101) have been transformed, using plasmid pUV-BAD, to express green fluorescent protein (GFP). This protein originates from the bioluminescent jelly fish Aequorea victoria and emits green light when excited by blue light. The use of these live fluorescing bacterial cells has been evaluated as a potential marker for the determination of protozoan grazing rates. A range of protozoan species, both marine and freshwater, have been employed in this study together with three analytical methods.

The first method is the most commonly used direct method i.e. to count the number of fluorescent prey cells within a protozoan cell, using epifluorescence microscopy. These experiments have to be very short, as when the pH reduces to 2-3 (e.g. inside a food vacuole) the fluorescence disappears. The use of these cells will be compared the use of DTAF-stained cells and fluorescent microspheres.

The second and third methods determine grazing rate indirectly, by monitoring the decrease in the fluorescence of the prey population (due to pH inactivation - digestion) in the presence of a protozoan, over time. The use of a fluorescent spectrophotometer has been evaluated for this purpose, but the method has been found to be complicated, time consuming, requires a large sample volume and is extremely tedious. Experiments carried out in multiwell plates and automatically analysed by a multilable, multitask, plate counter are currently being evaluated.

Myosin and motility in the apicomplexan protozoa

Conrad King, David Bruce and *John Sleep. Biology Department, University College London and *MRC Muscle and Cell Motility Group, King's College London.

Previous studies have implicated a role for actin/myosin in powering cell gliding ,surface bead translocation and target cell invasion in this important group of parasites. Using Eimeria sporozoites myosin could be detected in cell extracts (heavy chain about 93kD) and in association with the pellicle. Genomic DNA from E.tenella was probed for myosin genes using primers for the ATP binding site in PCR studies and a single positive clone was subsequently obtained (closest similarity to a myosin II gene from Dictyostelium).The trophozoites of Gregarina provide an ideal model system to elucidate the molecular dynamics of bead translocation. A variety of sizes can be used (86µm ->2 µm diameter). In the latter case optical tweezers were used to restrain bead movement . A model was developed involving the 'non-processive' action of the putative myosin molecules and might account for the variety of motile manifestations found in these protozoa.

<

Actin-based motility in the net slime mould Labyrinthula: search for the motor(s)

Terry Preston and Conrad King. Biology Department, University College London.

Labyrinthula displays 2 types of microtubule-independent motility - filopodial based growth at the periphery of the colony & gliding of spindle cells within the extensive actin-based cytoplasmic network. We are particularly interested in looking for the myosin(s) involved in these 2 motile phenomena. Inhibitors of actin dynamics (cytochalasin D) and myosin function (BDM) affected filopodia and gliding respectively. Western blotting on cell extracts probed with anti-peptide antibodies against myosin heavy chain sequences revealed only a single polypeptide of approx 100kDa.

We isolated total genomic DNA from Labyrinthula and used PCR to search for the myosin repertoire of this organism. Degenerate primers to the highly conserved ATP-binding site of myosin were used. Four different positive clones were obtained and their sequences compared with the myosin data base. As we have isolated 4 different gene sequences so far this is evidence for at least 4 distinct myosins. This is probably indicative of the variety of myosin-based functions carried out in this organism.

Myxidium sp. from lesser weever fish Trachinus vipers: cell-within- cell organization and sporogony

E.U. Canning, A. Curry, C.L. Anderson and B. Okamura. Imperial College, London.

A species of Myxidium commonly infects the gall bladder of lesser weever fish Trachinus vipers. The small plasmodia attach themselves to the gall bladder wall by inserting fine pseudopodia between cells, stopping short at the tight junction between epithelial cells. Nuclear division in each plasmodium is followed by endogenous formation of two secondary cells enveloped by endoplasmic reticulum. Secondary cells can form tertiary cells. Proliferation of plasmodia is effected by release of secondary cells, which then become primary plasmodia, to repeat the process. Nutrition of plasmodia is by uptake of membranes and fluid from the bile into large food vacuoles. These are present in primary, secondary and tertiary cells. Sporogony is disporous, each plasmodium producing two sets of sporogonic cells by endogeny (2 valvogenic, 2 capsulogenic and a binuclate sporoplasm per set). Only the primary plasmodial cell has food vacuoles. Mature spores are typical of Myxidium with a flask-shaped polar capsule at each end of an elongate spore and a central sporoplasm. The spore valves are covered with close-packed rows of very fine villus-like processes and the valves overlap at the central suture.

Development and survival of Leishmania mexicana in the sandfly Lutzomyia longipalpis

Matthew E Rogers, Michael L Chance and Paul A Bates. Division of Molecular Biology and Immunology, Liverpool School of Tropical Medicine.

The parasitic protozoon Leishmania mexicana occupies two niches in its life cycle: the phagolysosomal system of mammalian macrophages and the alimentary tract of the sandfly vector. Within the vector the parasite undergoes a series of morphological transformations, multiplies and migrates to the anterior midgut. This culminates in the differentiation of the mammal-infective metacyclic promastigote form. In order to understand how Leishmania responds to this environment, we have defined several parasite stages in the L. mexicana/Lu. longipalpis experimental system using precise morphological criteria. Infections were generated by feeding sandflies on a parasite/blood mixture. The main categories observed were: amastigotes, procyclics, nectomonads, short nectomonads, haptomonads, premetacyclics, metacyclics and paramastigotes. The relative prevalence and location of each type varied through the course of the in vivo infection in a well defined series of peaks, suggesting precursor-product relationships. Application of these categories to cultures demonstrated that it is also possible to model the same progression in vitro. The validity of the in vivo model was tested by feeding sandflies on a parasite/plasma mixture i.e. lacking the erythrocyte component. This led to alterations in both the numbers and developmental sequence observed. Expression of parasite chitinase was investigated in vitro, and was detected in RNA isolated by Northern blot and RT-PCR.

A species of microsporidium with characters of Nosema and Vaiyimorpha which is highly pathogenic to Diamondback moth, Plutelia xylostelia

E.U. Canning, A. Curry, S. Cheney, N.J. Lafranchi-Tristem and Md. A. Haque. Imperial College, London.

Plutella xylostella is probably the most serious pest of cruciferous plants in the tropics. A microsporidium introduced into a laboratory colony via eggs of P. xylostella received from Malaysia caused 100% mortality at all doses investigated (1.5 x 106 - 1.5 x 103 spores/larva) and reduced food consumption by 95% at the highest dose (Haque et al, 1999). All stages of merogony are diplokaryotic by repeated budding and binary fission. The predominant sporogonic phase is disporoblastic, producing two diplokaryotic spores. These stages are characteristic of the genus Nosema. In addition, the parasite frequently initiates an octosporoblastic sequence within a sporophorous vesicle. This is a character of the genus Vairimorpha. The initial stages are normal with separation of the nuclei and formation of secretions in the episporontal space. Thereafter the development is chaotic and no spores are formed. Phylogenetic analysis of this species in comparison with a range of Nosema spp. and Vairimorpha spp., using sequences of the 16 rDNA, placed the new microsporidium clearly within a clade of Nosema spp. from Lepidoptera. It is suggested that the dimorphic life cycle is ancestral, that the octosporoblastic sequence is retained by true Vairimorpha spp., lost completely by true Nosema spp. and partially lost by species such as the one in P. xylostella.

Haque, Md. A. et al. (1999). Entomopathogenicity of Vairimorpha sp. (Microsporidia) in the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae). Bull.Ent.Res. (in press)