initial presentation of the congress

Initial Presentation of the Congress

Dear Protistologist,

On behalf of “Grupo Especializado de Protistología” and “Groupment de

Protistologues de Langue Française”, we have the pleasure to announce you

the conjoint celebration in 2008 of their respective meetings. The gathering,

called First Spanish-French Congress on Protistology, will be held in Seville,

from 4th to 6th June. The aim of this meeting is to promote Protistology in

Europe through scientific exchange. Official language is English. We shall

make up an attractive scientific program and encourage the active

participation of young researchers (pre-, post-docs); the city will provide

participants with nice spring weather, history, and charm.

The scientific program will be divided in three main axes, Evolution of

protists, Biology of free-living protists, and Biology of parasitic protists, with

oral presentations and posters. Topics should be set up according to received

abstracts. If financial support allows it, two granted talks, opening and closing

the meeting, each given by an outstanding protistologue during 60 minutes,

are foreseen. Oral communications will be held in morning and afternoon

sessions. Every session will include one talk, given by a senior researcher

during 45 minutes, five secondary talks, given by young researchers during 20

minutes. All submitted abstracts should be considered as potential for oral

presentations; selection will be carried out by the scientific committee of the

meeting. Non-selected abstracts should be presented as posters. A session for

poster discussion will be included also.

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Organizing Committee

Grupo Especializado de Protistología (GEP), Sociedad Española de Microbiología (SEM). Groupement des Protistologues de Langue Française (GPLF). The Federation of European Protistological Societies (FEPS), a non-profit association of national and cross-national European Societies of Protistology and less formal national groups of Protistology of diverse European countries, supports the meeting President: Eduardo Villalobo. Departamento de Microbiología, Universidad de Sevilla. SPAIN. GEP-SEM. Vice-Presidents: Aurelio Serrano. Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC, Sevilla. SPAIN. GEP-SEM. Isabelle Desportes Museum National d'Histoire Naturelle, Paris. FRANCE. GPLF.

Scientific Committee

-Dr. Antonio Torres, Universidad de Sevilla, Sevilla. SPAIN. -Dr Gabriel Gutiérrez, Universidad de Sevilla, Sevilla. SPAIN. -Dr. Federico Valverde, Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC, Sevilla. SPAIN. -Dr. Susana Serrano, Universidad Complutense de Madrid, Madrid. SPAIN. -Dr. Juan Carlos Gutiérrez, Universidad Complutense de Madrid, Madrid. SPAIN. -Dr. Luis Miguel Ruiz Pérez, Instituto de Parasitología y Biomedicina López-Neyra, CSIC, Granada. SPAIN. -Dr. Loïc Morin, Université de Paris Sud, Orsay. FRANCE. -Dr. Anne Baroin-Tourancheau, Université de Paris Sud, CNRS, Orsay. FRANCE. -Dr. Purificación López García, Université de Paris Sud, CNRS, Orsay. FRANCE. -Dr. Nancy Guillén, Institut Pasteur, CNRS, Paris. FRANCE.

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Abbreviated Scientific Program Wednesday, 4th June -12:00-14:00 h. Registration. -15:00-15:15 h. Welcome talk by Dr. Eduardo Villalobo (President of the Congress), Dr. Loïc Morin (President of GPLF), and Dr. Aurelio Serrano (President of GEP-SEM). -15:15-16:15 h. Opening lecture by Dr. Andrés Aguilera. AFTERNOON, SESSION I. MOLECULAR/CELLULAR BIOLOGY, AND PHYSIOLOGY OF PARASITIC PROTISTS. Chairperson: Dr. Luis Miguel Ruiz Pérez -16:30-18:00 h. Oral presentations and discussions. -18:00-18:30 h. Poster pasting. -20:00-22:00 h. Welcome cocktail at Patio del Rectorado, Universidad de Sevilla. Thursday, 5th June. MORNING, SESSION II. MOLECULAR/CELLULAR BIOLOGY, AND PHYSIOLOGY OF FREE-LIVING PROTISTS. Chairperson: Dr. Geneviéve Bricheux -09:00-10:00 h. Main talk by Dr Philippe Bouchard. -10:00-10:30 h. Coffee break. -10:30-12:30 h. Oral presentations and discussions. -12:30-13:30 h. Posters discussion session. I Jornadas Internacionales de Tratamiento y Reutilización de Aguas Residuales: El Papel de los Protistas (only in spanish). Dr. Susana Serrano and Dr. Blanca Pérez Uz. Moderadora: Pilar Calvo -13:30-15:00 h. Lunch and coffee. AFTERNOON, SESSION III. ECOLOGY AND BIOTECHNOLOGY OF FREE-LIVING PROTISTS. Chairperson: Dr. Juan Carlos Gutiérrez -15:00-17:30 h. Conferences on the Role of Protists on Wastewater Treatment and Reuse. Dr. Purificación López García, Dr. António Luís Pereira do Amaral and Dr. Lucía Arregui. -17:30-19:00 h. Oral presentations and discussions. -20:30 h. “Tapas tour” in the city centre. Friday, 6th June MORNING, SESSION IV. BIOMEDICINE AND BIOTECHNOLOGY OF PARASITIC PROTISTS. Chairperson: Dr. Federico Valverde -09:00-10:00 h. Main talk by Dr. Naveed Ahmed Khan. -10:00-10:30 h. Coffee break. -10:30-12:00 h. Oral presentations and discussions. -12:00-13:30 h. Posters discussion session. -13:30-15:00 h. Lunch and coffee. AFTERNOON, SESSION V. PROTIST EVOLUTION. Chairperson: Dr. Loïc Morin -15:00-15:15. Closing talk by Dr. Ernesto García López (Vice-president Sociedad Española de Microbiología) -15:15-16:00 h. Main talk by Béatrice Lecroq. -16:00-18:00 h. Oral presentations and discussions. -18:00-19:00 h. Closing lecture by Dr. David Moreira. -20:15-21:15 h Visit to Reales Alcázares.

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Congress Venue

The Congress will be held in Seville (SW, Spain) at

"ESCUELA TÉCNICA SUPERIOR DE INGENIERÍA

INFORMÁTICA (ETSII), UNIVERSIDAD DE SEVILLA",

situated in "Campus Reina Mercedes" (Avenida Reina

Mercedes, 41012 Sevilla). We will meet in the "Salón

de Actos" of the "Escuela", with capacity for more

than 300 people, provided with audiovisual media,

and located in the first floor of the building.

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INDEX OF ABSTRACTS BY AUTHORS

OPENING LECTURE

Aguilera, Andrés 9

CLOSING LECTURE

Moreira, David 11

SESSION I. MOLECULAR/CELLULAR BIOLOGY, AND PHYSIOLOGY OF PARASITIC PROTISTS. Chairperson: Luis Miguel Ruiz Pérez

Kohl, Linda 13

López Farfán, Diana 14

Aranguren, Raquel 15

Arroyo, Francisco T. 16

Marques, Adam 17

Marques, Adam 18

Weber, Christian 19

SESSION II. MOLECULAR/CELLULAR BIOLOGY, AND PHYSIOLOGY OF FREE-LIVING PROTISTS. Chairperson: Geneviève Bricheux

Bouchard, Philippe 21

Baroin-Tourancheau, Anne 22

Ruger Herreros, Carmen 23

Damaj, Raghida 24

Serrano, Aurelio 25-26

Siddiqui, Ruqaiyyah 27

Aubusson-Fleury, Anne 28

Bricheux, Geneviève 29

Calvo, Purificación 30

Gallego, Andrea 31

Palomino Gutiérrez, Carmen 32

SESSION III. ECOLOGY AND BIOTECHNOLOGY OF FREE-LIVING PROTISTS Conferences on the Role of Protists on Wastewater Treatment and Reuse. Chairperson: Juan Carlos Gutiérrez

López García, Purificación 34

Pereira do Amaral, António Luís 35

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INDEX OF ABSTRACTS BY AUTHORS

Arregui, Lucía 36

Amaro, Francisco 37

Fajon, Céline 38

Aguilar, María 39

Canals, Oriol 40

Chatzinotas, Antonis 41

Conty, Ana 42

Serrano, Susana 43

SESSION IV. BIOMEDICINE AND BIOTECHNOLOGY OF PARASITIC PROTISTS. Chairperson: Federico Valverde

Khan, Naveed Ahmed 45-46

Guillen, Nancy 47

Siddiqui, Ruqaiyyah 48

Moktar, Norhayati 49

Gestal, Camino 50

Maíllo, Pedro Andrés 51-52

Umar, Nor Aini 53

SESSION V. PROTIST EVOLUTION. Chairperson: Loïc Morin

Lecroq, Béatrice 55

Albi Rodríguez, Tomás 56

Lara, Enrique 57

Valverde, Federico 58

Amar, Laurance 59

Hernández-Piñero, Sara 60

Lecroq, Béatrice 61

Marande, William 62

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Opening Lecture

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Understanding transcription-associated genome instability in Saccharomyces cerevisiae Andrés Aguilera. CABIMER-Universidad de Sevilla, Av. Americo Vespucio s/n, 41092 SEVILLA, Spain. [email protected] Transcription takes place on the same substrate as replication, repair and recombination and, not surprisingly, there is a functional and physical interconnection between these processes. A number of conserved factors with a role in mRNP biogenesis, transcription and RNA export show a strong effect on different types of genetic instability, and recent experimental evidence suggests that transcription-mediated hyper-recombination can be mediated by replication-fork impairment. A comparative molecular and genetic analysis of replication fork-progression impairment as a source of hyper-recombination, as well as data on new factors governing repair and hyper-mutation in association with transcription will be presented. Specific emphasis will be made on the relevance of the formation of R-loops as a source of transcription-associated genomic instability.

Opening Lecture FSFCP, Sevilla '08

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Closing Lecture

FSFCP, Sevilla '08

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An overview of the new phylogeny of eukaryotes David Moreira. Ecologie, Systématique et Evolution. CNRS Université Paris-Sud. [email protected] The use of molecular information, in particular 18S rRNA sequences, in the late 80s initiated a revolution in the study of the evolutionary relationships among eukaryotes. The first molecular phylogenies showed a stepwise emergence of the different eukaryotic phyla, which supported the idea of a progressive evolution of eukaryotes from very simple forms (the amitochondriate Archezoa) to the complex multicellular groups (animals, plants and fungi). Nevertheless, this paradigm has been challenged in the last decade thanks to the use of alternative molecular markers, especially conserved protein sequences. All known eukaryotes appear to derive from mitochondriate ancestors, which places the origin of mitochondria very early in eukaryotic evolution. Moreover, the topology of the first 18S rRNA-based trees has been demonstrated to be strongly affected by tree reconstruction artefacts, in particular the long branch attraction, responsible of the basal emergence of several fast evolving groups. Instead of a stepwise branching pattern, all the eukaryotic phyla appear to have originated in a short period of time, namely, in a massive radiation (the 'Big Bang hypothesis'). This makes the inference of the relative order of emergence of these phyla a very difficult task since the phylogenetic signal for the internal nodes is very limited. The use of improved methods and massive sequence data sets, and the search of rare shared characters are being used to address this problem. A current widespread view of the eukaryotic tree divides the eukaryotic diversity in a few super-groups: Opisthokonta, Amoebozoa, Plantae, Chromalveolata, Excavata and Rhizaria. Some of them are well supported but others are still hypothetical. I will discuss the evidence available for each one of them, with special attention to the data coming from our own research.

Closing Lecture FSFCP, Sevilla '08

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Session I

Molecular/Cellular Biology and Physiology of Parasitic Protists

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Trypanosome serine-threonine phosphatase: a regulator of nuclear positioning? Gallet C, Demonchy R, Mohamed M, Bastin P, Grellier P and Linda Kohl. Biologie Fonctionnelle des Protozoaires, Museum National d'Histoire Naturelle, Paris. [email protected] Phosphorylation and dephosphorylation of cellular proteins are important steps in the control of major biological events in many eukaryotes. It was shown previously that treatment of Trypanosoma cruzi with CalyculinA, an inhibitor of PP1 and PP2a, caused a change in morphology from trypomastigotes to amastigotes, accompanied by the nucleus and the kinetoplast coming closer together. At the onset of this project only one PP1 gene was known. In view of the difficulties to genetically manipulate T.cruzi, we decided to investigate the protein and its roles in the related organism T. brucei. Using a GFP fusion, we determined that TbPP1 is localised in the cytoplasm. Silencing of the expression of this gene by RNA interference showed that the protein is not essential. Indeed, the knockdown cells grew at a normal rate, but they displayed a clear phenotype with the nucleus and the kinetoplast in unusual close proximity. Moreover the endocytic compartment, normally situated between nucleus and kinetoplast, was also mislocalised. We determined that the nuclear position was faulty: it was found too posterior when compared to wild type (or control 29-13) cells. PP1 could therefore be an important regulator of nuclear positioning in trypanosomes.

Session I, Oral FSFCP, Sevilla '08

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An RNA polymerase II specific subunit is necessary for the RNA polymerase I transcription in Trypanosoma brucei Diana López-Farfán, Peñate X, Landeira D, Wentland A, Vidal I and Navarro M. . Instituto de Parasitología y Biomedicina López-Neyra, CSIC. [email protected] Trypanosoma brucei is the etiologic agent of human African trypanosomiasis also known as African sleeping sickness. Changes in the Variant Surface Glycoprotein (VSG) type on the surface allow the parasite to elude the host immune antibody response, ensuring a persistent infection. The VSG transcription demonstrates monoallelic expression, with ability to switch transcription between telomeric VSG expression sites. In the insect mid-gut stage of the parasite, the procyclin family of glycoproteins covers the surface. VSG and procyclin transcription is mediated by RNA polymerase I (pol I) instead of pol II, the polymerase committed to mRNA production in eukaryotes, such an unusual feature may be accomplished by the recruitment of specific subunits or cofactors that permit pol I to transcribe protein coding RNAs. Towards our goal of identification of RNA pol I subunits, we developed an in vivo functional approach to measure transcriptional activity in a dual reporter cell line, employing inducible RNA interference for the knockdown of different polymerases subunits. This system allows to distinguish between pol I and pol II transcription in vivo. We find that transcription mediated by pol I requires a dissociable subunit of the pol II complex. This subunit was found to interact with two pol I-specific subunits, TbRPA1 and TbRPB6z in co-immunoprecipitation experiments. Pol I-specific transcription was affected upon RNAi depletion in run-on assays using permeabilized cells. In addition, in-vitro transcription assays suggest that VSG promoter activity is enhanced after addition of the recombinant pol II subunit. Together, our results represent the first in vivo example of a functional RNA polymerase chimera consisting of a core pol I complex that recruits a specific pol II subunit.


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Protozoan parasites in bivalve molluscs: aims and profits of a national reference laboratory for molluscs diseases Raquel Aranguren, Gestal C, Novoa B and Figueras A. Instituto de Investigaciones Marinas,CSIC. LNR Moluscos Bivalvos. [email protected] Bivalve molluscs aquaculture is an increasing activity in several European countries. Culture conditions of the Bivalve molluscs (high stock densities and operating procedures) increases an stress situation which leads to high economical looses due to the appearance of different diseases. The European Union has published a council Directive (2006/88/CE) in which it is established a normative on animal health conditions for placing on the market, imports and transits of aquaculture animals and their products and prevention and control of certain diseases in aquatic animals. This Directive establishes a list with three protozoan parasites infecting bivalve molluscs causing important economic looses already known to be presented in certain zones of the UE that must be notified to the competent authorities in case of suspicion or detection within the UE. These three protozoan parasites are Marteilia refringens; a parasite infecting flat oysters, (O. edulis) and mussels (Mytilus sp.), Bonamia ostreae; a parasite infecting flat oysters (O. edulis) and Perkinsus olseni; infecting different clam species. Nowadays there are no effective tools to fight against molluscs diseases. However, one of the aims of a National Reference Laboratory for Molluscs diseases designated by UE (Directive 95/70/CE) is to developed rapid diagnostic techniques (PCR) for the detection of these parasites to control the infection and put the stock on the market before high mortalities could be registered. All Member States have to designate its competent authority and a National Reference Laboratory for the purposes of the Directive and notify the Commission thereof.


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Histological study of the infection process of Colletotrichum acutatum on strawberry plants Francisco T. Arroyo1, Moreno J2, Daza P2, Torreblanca J2 and Romero F1. 1IFAPA Centro Las Torres-Tomejil and 2Departamento Biología Celular, Universidad de Sevilla. [email protected] Anthracnose caused by Colletotrichum acutatum Simmonds, is a major disease of the cultivated strawberry (Fragaria xananassa Duch.) in Europe. Lesions can occur all over the plant but anthracnose crown rot is specially severe leading to wilt and death. Based on differences of susceptibility of strawberry tissues to this fungus, a study of the infection process on crowns, leaves and petioles was carried out. The infection process by Colletotrichum acutatum, in the different strawberry structures previously cited, was examined via light microscopy and scanning and transmission electron microscopy. Crowns, petioles, adaxial and abaxial surfaces of strawberry leaves were inoculated with a conidial suspension of the fungus. The initial stages of infection, from spore adhesion to germination on plant surface, were similar in the tissues studied. However, strategy of colonization and invasion of the fungus in crowns, petioles and leaves was different. In this way, after penetration of plant cuticle we could observe that: 1) the pathogen invaded the leaves occupying the intramural spaces in most of the cases, and some epidermal cells; 2) the colonization of the crown tissues by the fungus was characterized by intracellular and intercellular growth in the cortex, medulla and vascular system resulting in cell colapse and necrosis; 3) on petioles, the fungus developed intercellular and intracellular growth in the cortex and the epidermal layer, but not in the vascular tissues. Moreover, this strategy was maintained through 30 days after inoculation; the behaviour of the pathogen was very similar, and we could only find the fungus in the same tissues previously cited on crowns, petioles and leaves.

Session I, Poster FSFCP, Sevilla '08

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The dance of Enteromyxum leei (Diamant, Lom and Dykova, 1994), Mixozoa Le Breton A, Krasteva D and Adam Marques. Laboratoire Ecosystèmes Lagunair, Université Montpellier II. [email protected] Perkinsosis is a notifiable disease affecting several species of clam in Galicia (NW Spain) caused by the protozoan parasite Perkinsus olseni. The two classical diagnostic methods recommended by the Office International des Epizooties (OIE) in the Manual of Diagnostic Tests of Aquatic Animals, histology and incubation on Ray's fluid thioglicollate medium (RFTM) are tedious and need highly qualified personnel. In order to develop faster and cheaper diagnosis techniques, classical methods are compared with a nested PCR assay. Five different clam species of trade interest cultured in Galicia were employed for the detection of P. olseni. Each clam was simultaneously processed for histopathology and PCR. In addition, 150 clams were subjected to RFTM incubation. A Nested PCR on Internal Transcribed Spacer (ITS) region was carried out and product identity was corroborated by in situ hybridization and sequencing. Results from the different techniques were compared for diagnostic sensitivity, specificity and number of false positives compared with a gold standard consistent in the other techniques under comparison. Positive Predictive Value (PPV) and Negative Predictive Value (NPV) were also calculated and κ- value statistic was used to measure the level of agreement of the PCR assay and the combination of the classical diagnostic methods. Nested PCR was the most sensitive method considering all the samples. PCR is the diagnostic method that gives the lowest number of false negative results. PPV and NPV results indicate than PCR is suitable for screening of low-parasitized populations of clams as experiments of eradication of P. olseni. PCR method is cheaper and faster than histopathology and incubation on RFTM. Histology possesses a higher PPV, suggesting that this technique is strongly recommended for confirmatory diagnosis of P. olseni. In addition, histology can give a general vision of the health status of the animal. Results from incubation on RFTM suggest that this method has low specificity and reveals great number of false positives.


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Gregarines/Myriapods Chilopoda, an example of host parasitizes co-evolution Krasteva D and Adam Marques. Laboratoire Ecosystèmes Lagunair, Université Montpellier II. [email protected] Myriapods are among oldest terrestrial invertebrates on the planet. There are two principal evolutionary paths: Diplopoda and Chilopoda. The Diplopoda have two pair of legs per segment and they are herbivores and detritivors. The Chilopoda have only one pair of legs per segment and they are predators.Among the parasites which they lodge, the gregarines are abundant. They are clustered in Dactilophoridae family and show a close specificity with the various genus of Chilopoda. Dactilophoridae are characterised by multiple rhizoid prolongations, which penetrate inside the intestinal host cells. They constitute a differentiation of epimerite. It's allows the fixation of the gregarines on multiple intestinal cells and some times this structure can be seen directly. The size, the shape and the number of these rhizoids allow to distinguish the species and to group them in genus. There is a remarkable parallelism between Chilopoda family and genus of gregarines. Dactilophoridae parasitize 3 families of Chilopods in particular. Each gregarine genus is specific to a genus of Chilopod: Grebnickiella parasitizes exclusively Scolopendra, Dactilophorus-Cryptops and Echinomera-Lithobius. These observations permit to isolate Trichorhynchidae from Dactilophoridae and confirm the early evolutive divergence between Scutigeromorpha and Scolopendromorpha.


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The asparagine-rich antigen (ariel) encoding genes are up regulated in pathogenic Entamoeba histolytica infecting liver Christian Weber and Guillén N. Unité Biologie Cellulaire du Parasitisme, Institut Pasteur-INSERM. [email protected] Amoebiasis is a parasitic infection of the human intestine and liver that causes 50 million clinical cases per year leading to 100,000 deaths. The causative agent of amoebiasis is Entamoeba histolytica, a protozoan belonging to the most ancient eukaryote organisms. A genome project concerning E. histolytica and its non-pathogenic relative Entamoeba dispar is actually completed. The life cycle of E. histolytica is made up of two stages, the cyst that is the contaminant form and the vegetative trophozoite. Among the infected individuals only 10% experience amoebiasis. Hepatic liver abscess formation is the most common complication of amoebiasis. The first question that we have addressed was to see whether there are differences in gene expression between E. histolytica strain HM-1:IMSS isolated from liver and long term axenic cultivation that has lost virulence. Hybridization on microarrays and bioinformatic analysis demonstrated that 37 specific genes are overexpressed in parasites recently isolated from liver infection at very high rates (2 to 6 fold changes). Among them, the asparagine-rich E. histolytica antigen (ARIEL-1), which is absent in the non-pathogenic related species Entamoeba dispar, stand out. Ariel proteins are homologue to an amoebic membrane protein called SREHP (serine rich) that is important for the attachment of the amoeba to the host cells. From bioinformatics analysis ariel genes are presented in eight copies at the genome level but precedent data appeared contradictory in regard to different versions of the genome annotation. As for the number of different ariel encoding genes, the first study proved eight distinct proteins with 2 to 16 octapeptide repeats and with corresponding molecular masses between 11 and 23 kDa.For all of them, the central part contains a variable number of octapeptide repeats but the Carboxyl and Amino termini are conserved. We determined the number of distinct ariel genes expressed in E. histolytica (RACE and PCR) and clone different versions into a suitable vector to express them in bacteria. Further molecular characterization, by RNA interference and anti-sense technologies, is underway. It will allow the production of parasite strains blocked in these different factors and for the analysis of liver abscesses formation.


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Session II

Molecular/Cellular Biology and Physiology of Free-Living Protists

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Membrane skeleton in ciliates at the post-genomic level: the multigenic family of epiplasmins Philippe Bouchard. Laboratoire Microorganismes: Génome et Environnement, Université Blaise Pascal. [email protected]. Cytoskeleton is largely considered as the cytoplasm manager, defining cell shape and cell motility. Its association with membranes is essential. A well documented example implies the actin cytoskeleton and lessons can be drawn from the “amoeboid displacement”. Tubulin made cytoskeleton is also in close interaction with membranes to produce forces that help cells to move. In Ciliates, actin is underrepresented as a main contributor to cell shape and movement while tubulin is largely represented in cilia and sub-membranous systems. Epiplasm has been extensively studied as an original sub-membranous cytoskeleton system in Ciliates. Such a strategy to sustain membranes is also evidenced in the euglenoid flagellate. To date, protistologists success in partial molecular characterisations of epiplasmic components. But links to their origins, biochemical, structural characteristics or behaviour and specific functions are very elusive. Specific ciliates genome programs are now in progress and extensive dataset are available. In this talk, we investigate the question of epiplasm in the two ciliates Paramecium and Tetrahymena. It appears that one of the most efficient tool used in the epiplasm field is a methodology that can compare protein at the structural level. This method, known as “hydrophobic clusters analysis” (HCA), helps us to describe the multigenic family of epiplamins in Paramecium. We show that this multigenic family is interspecific since epiplasmins can be indentified in the genome of Tetrahymena. Structural links can now be evidenced with EpC, one of the main components of the epiplasm in Tetrahymena. In this way, the question of a parenthood of epiplasmins and intermediate filaments proteins is asked.

Session II, Oral FSFCP, Sevilla '08

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Ciliates utilize the lys63-linked polyubiquitynation reaction Grisvard J, Aubusson-Fleury A, Lemullois M and Anne Baroin-Tourancheau. Université Paris-Sud, UMR CNRS 8080. [email protected] Post-translational modifications of proteins by ubiquitin are implicated in diverse biological processes. Conventional polyubiquitination through Lys48 targets the proteins to 26S proteasomes for degradation. Noncanonical polyubiquitination through Lys63 on the other hand serves as a signalling mechanism for a number of regulatory functions. Lys63 polyubiquitin chains have been implicated in several non-proteolytic cellular processes such as plasma membrane protein endocytosis (Galan, J. M. and Haguenauer-Tsapis, R. 1997 EMBO J., 16: 5847-5854), ribosome function (Spence et al., 2000, Cell, 102: 67-76), DNA postreplication repair (Broomfield et al., 1998, PNAS, 95: 5678-5683 ) and IB kinase activation (Deng et al., 2000, Cell, 103: 3351-361). So far, only one ubiquitin-conjugating enzyme (Ubc), Ubc13, is known to mediate the assembly of these chains and it requires an Ubc inactive variant (UEV) as cofactor. The identification of a structural and functional homolog of Uev family proteins (ShUev) in Sterkiella histriomuscorum supports an ancient origin of this complex (Villalobo et al. 2002, Mol. Biol. Evol. 19: 39-48). To address this point in Sterkiella, we searched for the Ubc13 partner of ShUev. Sterkiella contains a single Ubc13 gene. We showed that ShUbc13 interacts with ShUev in a yeast two-hybrid analysis. In addition, homology modelling based on the human or yeast Ubc13/Uev crystallized heterodimer reveals that the putative Sh(Ubc13/Uev) complex depicts a high 3D structural conservation. Expression analyses at the mRNA and protein level were performed in vegetative, starved encysted and excysting cells. Both genes showed a similar pattern of expression in agreement with their co-functioning. But whereas mRNA levels were shown to vary according to the E-E stage, no change in Ubc13 overall protein level was observed. Ubc13 could be detected in cytoplasm and with a dotted pattern in several other subcellular localizations, which included the plasma membrane, the organelles membranes, the cil tip and the nucleus. Our data confirm that the Ubc13/Uev complex is highly conserved in the eukaryotic kingdom and support an ancient and ubiquitous signalling role in the cell. Putative pathways will be discussed.


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The RCO1/RCM1 complex regulates transcriptional adaptation to light in the fungus Neurospora crassa Carmen Ruger-Herreros, Olmedo M, Corrochano LM. Departamento de Genética. Universidad de Sevilla. [email protected] The gene con-10 of the ascomycete fungus Neurospora crassa is expressed during conidiation or after illumination of vegetative mycelia. Photoactivation of con-10 is transient and disappears after two hours of light due to photoadaptation. The rco mutants (regulation of conidiation) were isolated by the abundant expression of con-10 in vegetative mycelia. The genes rco-1, encoding a putative gene repressor, and the gene rco-3, encoding a putative glucose sensor are required for the repression of con-10 in vegetative mycelia. The predicted RCO1 polypeptide is a homolog of Tup1, a protein that mediates transcriptional repression together with the protein Ssn6 in Saccharomyces cerevisiae. The Neurospora homolog of ssn6 is rcm-1 (regulation of conidiation and morphology). The genes rco-1 and rcm-1 are not regulated by light. We have observed that a deletion of rco-1 or the inactivation of rcm-1 by RIP result in an enhanced and sustained gene photoactivation, suggesting that both proteins are part of a repressor complex that regulates transcriptional adaptation to light. We are currently attempting to determine the subcelular localization of RCO1 using two different strategies. First, detection of RCO1 by immunoblotting in citoplasmic and nuclear protein extracts obtained by cell fractionation. Second, by the introduction in the Neurospora genome of a translational fusion of rco-1 to GFP.


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Differential localisation of epiplasmins in the cortex of Paramecium Raghida Damaj, Aubusson-Fleury A1, Coffe G, Bricheux G and Bouchard P. Laboratoire Microorganismes: Génome et Environnement, Université Blaise Pascal and 1Université de Paris-Sud. [email protected] Paramecium has many specialised cortical territories which define a global body plan. Epiplasmins are original proteins constitutive of the under-membrane skeleton known as the epiplasm. They belong to a large multigenic family of 51 members and we show that the alteration of epiplasm by RNAi induces abortion of cell division. Multivariate in silico analysis of epiplasmins has revealed that these proteins can be sub-grouped. Here we report the question of a functional repartition of epiplasmins in the cortical unit of Paramecium. By expression of GFP tagged protein, we show that some epiplasmins define a pericinetosomal territory while others define a core epiplasm. Finally it appears that another group of epiplasmins is peripheric, delimiting each cortical unit. We can also highlight, by electron microscopy, a partition of the epiplasm into two layers, one rather cytoplasmic, the other rather membranous. We thus propose that there are specific functional localisations of different epiplasmins ensuring the organisation of cortical units and the success of general cell division.


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Transcriptional regulation of Chlamydomonas reinhardtii proton translocating pyrophosphatase by environmental conditions Herrera-Palau R, Pérez-Castiñeira JR, Valverde F and Aurelio Serrano. Instituto de Bioquímica Vegetal y Fotosíntesis. CSIC-Universidad de Sevilla. [email protected] Proton-pumping pyrophosphatases (H+-PPase, EC 3.6.1.1) are integral membrane proteins that use the chemical bond energy of inorganic pyrophosphate (PPi) to generate a transmembrane electrochemical potential. Located in endomembranes, they are structurally simpler than F-, P- and V-ATPases, and -as was recently demonstrated- exhibit a broad distribution among bacteria, archaea, protists and plants. Two biochemically different classes of H+-PPases differing in K+ sensitivity, namely stimulated (type I) and insensitive (type II) to this cation, were identified (1,2). Higher levels of this protein were observed under energetic stress in the photosynthetic bacterium Rhodospirillum rubrum and the plant Arabidopsis thaliana. Moreover, rice seeds show both transcript induction and increased level of the H+-PPase protein under anoxia and thermal chock. Therefore, a role for H+-PPase under energetic stress conditions allowing ATP saving was proposed (2). The photosynthetic protist Chlamydomonas reinhardtii (Chlorophyceae) possess a single vacuolar type I H+-PPase and two non-homologous soluble PPases (sPPases) located in energetic organelles, chloroplasts and mitochondria (3). This microalga is, therefore, a relatively simple appropriate system to analyze the responses of the two main PPases classes to diverse environmental conditions (nutrient availability, stress, etc). Semiquantitative RT-PCR and Western blots were used respectively to analyze transcript and protein PPase levels under different trophic regimes (phototrophy, mixotrophy and heterotrophy) and abiotic stresses, as well as along circadian cycle experiments. H+-PPase clearly shows a light-dependent transcriptional induction and higher activity levels in phototrophic cells, even though no changes of protein levels were produced by the trophic regimes. Differences in the apparent Mm of the immunodetected protein bands under heterotrophic conditions suggest possible post-translational modifications. Moreover, RT-PCR experiments show H+-PPase gene undergoes a circadian cycle response that was not observed for sPPase and V-ATPase genes. On the other hand, ionic stress causes a clear induction of the H+-PPase transcript more prominent under acute salt stress. All these results strongly suggest a functional substitution of V-ATPase by H+-PPase in response to severe energetic stress, under which the vacuolar acidification should be largely maintained at the expense of PPi generated in cell anabolism thus allowing a substantial energy (and ATP) saving. Supported by BFU2007-61887 (MCI) and PAIDI group BIO-261 (JA) grants.


25

1. Drozdowicz YM, Rea PA. (2001) Trends Plant Sci. 6:206-211. 2. Serrano A, Perez-Castiñeira JR, Baltscheffsky M, Baltscheffsky H (2007) IUBMB Life 59:76-83. 3. Gómez-Garcia MR, Losada M, Serrano A (2006) Biochem. J. 395:211-221.


26

Galactose-binding protein (GBP)-mediated encystation in Balamuthia mandrillaris Ruqaiyyah Siddiqui, Pavlopoulo E and Khan NA. School of Biological and Chemical Sciences, Birbeck College, University of London. [email protected] Balamuthia mandrillaris is an emerging protozoan pathogen that can produce fatal granulomatous encephalitis. A difficulty in treating this infection arises due to the ability of B. mandrillaris to undergo a phenotypic switch from the infective trophozoite stage to the dormant encysted stage, which is resistant to the recommended levels of antimicrobial agents. In addition to possible drug resistance, cysts may also reactivate following antimicrobial chemotherapy leading to recurrence of infection. Progress toward more effective therapies is hampered by our poor knowledge of the mechanisms involved with B. mandrillaris encystment. Our studies have shown that a galactose-binding protein (GBP), expressed on the surface membranes of B. mandrillaris is required for parasite binding to and cytotoxicity of human brain microvascular endothelial cells. Here, we tested the involvement of GBP in B. mandrillaris encystation. Using axenic cultures, an in vitro encystment assay was performed by incubating B. mandrillaris trophozoites in RPMI-1640 for 48h at 37ºC, in the presence of exogenous sugars. The addition of galactose resulted in high levels of B. mandrillaris encystation, compared with the control (75% versus 40% respectively), while fucose, glucose, xylose and mannose had no effect. Galactose increased B. mandrillaris encystation in a concentration-dependent manner. Encystation was blocked using cytochalasin D indicating the role of actin polymerisation. The rho kinase inhibitor, Y27632 only partially inhibited encystation suggesting the involvement of other Rho GTPases such as Cdc42 and Rac1 pathways. Sodium orthovanadate inhibited, while Genistein promoted encystation suggesting that protein tyrosine kinases play a role in B. mandrillaris differentiation. LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), which are signaling molecules responsible for numerous cellular responses, also inhibited encystation of B. mandrillaris in a concentration-dependent manner, compared with the controls. For all inhibitors, there was no effect on the number and/or viability of trophozoites after 2 days of culture in the encystation medium containing drugs, suggesting that the inhibitory effect of the drugs on encystation was not due to their toxic effect on trophozoites. Overall, these results suggest that B. mandrillaris encystation require the activities of protein tyrosine kinases, Rho GTPases and PI3K. A complete understanding of encystation in B. mandrillaris may help identify targets for therapeutic interventions.


27

Spatio-temporal pattern of basal body duplication during division in Paramecium aurelia: new facts and hypotheses Ruiz* F, Koll* F andAnne Aubusson-Fleury. Biologie Cellulaire 4, Université Paris-Sud and *Centre Genetique Moleculaire, CNRS-Gif sur Yvette. In Paramecium tetraurelia, the pattern of basal body duplication during division is known to occur through 2 successive waves of basal body (BB) and cortical unit (CU) proliferation running from the cell equator towards the poles allowing to define invariant, duplicated and hyperduplicated cell territories (Iftode et al, 1989, Development, 105: 191). In order to unambiguously distinguish parental from neo-formed basal bodies, we used cells expressing a centrin-GFP transgene, which display a specific labelling of basal bodies (Ruiz et al, 2005, Cur Biol, 15:2097). Under controlled culture conditions, autogamy yielded the loss of the transgene in such a way that by the first two post-autogamous divisions, all parental BBs still retained the GFP-labelling while all new BBs were unlabelled. Immunocytochemical observation of such dividing cells demonstrated the following points. 1) During a first wave which generates the set of new CUs, new BBs always appear anteriorly, whether in two BBs or in single BB unit, but never between two paired BBs as previously suggested. Then epiplasmic scales form around each BB. 2) In the invariant anterior field, CUs with paired BBs do not duplicate but a wave of disassembly and reassembly of the anterior BB of each pair progresses from the anterior suture towards the rear of the kineties. Thus all CUs will successively pass through a stage with only one BB during division. 3) For particular CUs, a second wave of BB duplication restores the paired BBs pattern by addition of a new BB anterior to the existing one. These observations provide a measure of the stability of basal bodies through at least two cell generations and the results show that: 1) all BBs do not duplicate during the first proliferation wave even in hyperduplicated zones. This observation suggests that local constraints act on BB assembly. 2)The cell pattern emerges from a combination of two kinds of processes , a proliferative wave which yields the new set of singled BB units and a reshaping one which restores the final pattern. The fact that the reshaping process may occur in the absence of proliferative one (Romero and Torres, Development, 1993, 117: 1099) demonstrate that these two processes, which both include BB proliferation, are driven under different regulatory controls.

Session II, Poster FSFCP, Sevilla '08

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Multifunctional Pcars and the membrane skeleton Geneviève Bricheux, Aubusson-Fleury* A, Coffe G, Donnadieu F, Damaj R, Lemullois M*, Vellet A and Bouchard P. Laboratoire Microorganismes: Génome et Environnement, Université Blaise Pascal and *Université Paris-Sud. [email protected] Two kinds of epiplasm have been mainly reported. One described in the ciliate genera Paramecium and Tetrahymena (epiplasmin) and the other one described in Euglenoïd flagellates and some ciliates (P. dubius), made of proteins known as articulins. Using cross reacting antibodies, it has been postulated that articulins also exist in P. tetraurelia. This has been investigated using the numerous available genomic dataset. HCA analyses help us to design articulin's modules that have been used for BLAST analysis. We were able to characterize two types of proteins we called Pcar-large and Pcar-small (Protein containing articulin repeats). Pcar-large has been localized in the cortex of P. tetraurelia, to the base of cilia, at the level of the transition basal body/axoneme. This material localizes around the terminal plate in connection with the epiplasmic layer. This protein harbor two structural characteristics: one module with articulin repeats and another one with an accumulation of alkaline residues. Such an accumulation of alkaline residues has been demonstrated in MAPs. Combined with the localisation results, these data indicate that this protein may represent linkers between microtubules and epiplasm. This type of protein is also found in Tetrahymena. Searching for new members of Pcar, we have investigated similarities within other genomes. Interestingly, it appears that these proteins have a wide representation across different phyla.


29

Ultrastructure and sugar present in the cyst wall of of Vorticella microstoma resting cyst Purificación Calvo1, Lara C2 and Sanchez-Aguayo I3 1Departamento de Microbiología, 2Departamento de Bioquímica and 3Departamento de Biología Celular, Universidad de Sevilla. [email protected] The light and electron microscopy study of Vorticella microstoma resting cyst shows that the cyst wall consists of four morphologically distinct layers (ectocyst, mesocyst, endocyst and granular layer) like oxitrich ciliates and the peritrich ciliate Opisthonecta henneguyi. The ectocyst is a thin layer, not smooth, with a big protuberance being plug-like. Te mesocyst is the thickest layer and is made of a compact material. The endocyst, like the ectocyst, is a thin layer, although less dense. The granular layer varies in thickness and connects the endocyst with the cytoplasm. Kinetosomes are maintained in the mature resting cyst, whereas cilia are resorbed. We have analysed the carbohydrate residues present in the cyst wall of the peritrich ciliate Vorticellla microstoma by light inmunocytochemistry, using six different lectins. The endocyst has a great variety of saccharide residues since it binds to WGA, PNA, DBA, MAA and UEA lectins. The carbohydrate residues present in this layer might function as receptors during excystment process. The mesocyst presents a strong staining with the WGA lectin showing the presence of (N-acetylglucosamine)n residues, probably chitin. The ectocyst binds to Con A, suggesting that it contains alfa-D-glucosyl and/or alfa-D-mannosyl residues.The granular layer did not stain with any of thelectins tested.


30

Apoptosis in Tetrahymena hermophila induced by different environmental stressors Andrea Gallego, Martín-González A, de Lucas MP and Gutiérrez JC. Departamento. Microbiología-III, Universidad Complutense de Madrid. [email protected] Unlike in mammals, microbial apoptosis is a quite rare process with unknown biological significance. The exposure of Tetrahymena thermophila to several pollutants or stress conditions; heavy metals (Cd, Cu or Zn, 24h), oxidants (menadione 2h or paraquat 24h) or nutritional stress (starvation, 48h or 4 days) produces, in certain proportion of cell population, several morphological, physiological and molecular changes, which represent apoptotic hallmarks. Treatments with campthotecin and doxorubicin, a well-known inducers of apoptosis, also cause similar cell modifications. Cu or Zn increasing concentrations originates a decreasing in cell size but an increasing cell complexity, indicating a cellular blebbing. On the contrary, Cd exposures do not induce these changes. Using annexin-V conjugated with FITC and quantification by flow cytometry, we have detected that metal treatments produce a phosphatidylserine membrane translocation in some cells. The proportion of these cells increases under high concentrations and prolonged (24h) stress treatments. Mitochondria are crucial targets in both pathways of programmed cell death type I. By confocal microscopy and the specific fluorophore JC-1, we have observed a progressive decrease in mitochondrial membrane potential, proportional to the increasing of toxic agent concentration. Metallic treatments also generate abundant reactive oxygen species (ROS), detected using flow cytometry and the fluorophore dihyhidrorodamine. ROS production was higher, at a Cu or Zn toxic concentrations (LC50). However, similar Cd treatments cause a drastic increasing of cell necrosis. Nuclear changes are another important apoptotic targets. By DAPI fluorescence microscopy, we have detected that diverse metal treatments produce the breakdown of macronuclear membrane and the formation of 1-3 apoptotic macronuclear bodies (AMB). The cellular percentage showing AMB is correlated with the metal concentration. Apoptosis is also characterized by DNA ladder formation. Single metal treatments generate two types of DNA fragmentation patterns; short treatments (8h) originate DNA smears with a size range of 1230 bp to lower than 154 bp, while a longer treatments (24h)originate two groups of DNA fragments (1033-517 and 220 to <154 bp). In binary metal exposures, the generation of DNA ladders decrease drastically. Finally, we have also analysed the expression, under above mentioned stressors, of some apoptotic key genes; met-8 encoding a metacaspase, cas-1 and cas-2 encoding two caspase-like proteins and endo G encoding an endonuclease, involved in the typical apoptotic DNA fragmentation. Supported by the project CGL2005-00548 (MEC).


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Preliminary identification of nonsense-mediated decay genes in ciliates Carmen Palomino-Gutiérrez, Alcázar-Limón F, Hernández-Piñero S, Molero R and Villalobo E. Departamento de Microbiología, Universidad de Sevilla. [email protected] In all the eukaryotes so far studied, a quality control system, called nonsense-mediated decay (NMD), detects and degrades mRNAs with premature stop codons (PTC). This system should avoid the translation of truncated proteins, which sometimes are deleterious for cells. Many proteins are required for NMD, some of which are common to all living organisms, while other are group-specific. Essentially, the the NMD core is composed of seven proteins, product of genes smg1-7 (supresor with morphogenetic effect on genitalia) Paramecium tetraurelia and Tetrahymena thermophila, two ciliated protists, possess deviant genetic codes in which canonical TAA y TAG stop codons codify glutamine and TGA is the actual stop codon. How NMD operate in ciliates is unknown. To shed some light on NMD in ciliates, we have undertaken the search for genes homologous to NMD genes of organisms with canonical genetic codes. Particularly, we have searched for smg1-7 genes in P tetraurelia and T thermophila. In these ciliates, we have found gene sequences with similarity to smg2-4 of baker’s yeast. We have also found gene sequences with similarity to genes encoding Y14 and MAGOH. These proteins are part of the exon-junction complex, which also play an important role in NMD. By means of RT-PCR, we have determined that genes homologous to SMG2-4, Y14, and MAGOH are expressed in P. tetraurelia and/or T. thermophila.


32

Session III

Ecology and Biotechnology of Free-Living Protists

Conferences on the Role of Protists

on Wastewater Treatment and Reuse

FSFCP, Sevilla '08

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Metagenomics, a powerful approach to the study of microbial communities Purificación López García. Unité d’Ecologie, Systématique et Evolution, Université Paris-Sud. [email protected] Environmental microbiology has experienced a revolution in the past two decades with the development and massive application of molecular methods based on the amplification, cloning and sequencing of conserved phylogenetic markers, notably small subunit rRNA (SSU RNA) genes, to characterize the microbial diversity present in natural samples. This approach has led to the detection of an unforeseen diversity of prokaryotic and, more recently, eukaryotic microbes in all possible kinds of biotopes. In the case of bacteria and archaea, a large amount of the phylotypes identified are very divergent from known cultivated members, forming clades that, in agreement with their phylogenetic depth, likely correspond to new phyla or Candidate Divisions. The extent of this undescribed diversity is far from trivial, and many of these as yet uncultivated groups are abundant in many ecosystems and may play key ecological roles. One of the challenges ahead is to link particular environmental lineages to their functional roles. Metagenomics (environmental genomics or community genomics) is a promising approach to provide additional information about the gene content and, in some cases, genome structure of uncultivated communities. In only few years, the metagenomic study of a variety of ecosystems, from soils and oceans to wastewater treatment has revealed its potential to highlight the importance of certain metabolisms in the environment, sometimes leading to the revision of key biogeochemical cycles, including the N and C cycles. Some metagenomic studies point out to a remarkable role of aerobic ammonium oxidation members by Group I crenarchaeota in soils and oceans, that of carbon monoxide oxidation in oceans or that of anaerobic ammonium oxidation by members of the Planctomycetales in sludge treatment plants. With the spreading use of pyrosequencing, complementing more classic strategies based on shotgun sequencing and large-insert genomic library analyses, metagenomics is becoming an essential source of information about microbial ecology and evolution in natural ecosystems.

Session III, Oral FSFCP, Sevilla '08

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Wastewater protozoa survey by image processing and analysis methodologies António Luís Pereira do Amaral. Departamento de Engenharia Química e Biológica, Instituto Superior de Engenharia de Coimbra. [email protected] Protozoa and metazoan are considered good indicators of the treatment quality in activated sludge systems being sensitive to physical, chemical and operational processes. Therefore, it is possible to correlate the predominance of certain species or groups and operational parameters of the plant. This work presents a semi-automatic image analysis procedure for the recognition of the metazoan and protozoa species most frequently found in WWTP by determining morphological data and multivariable analysis with discriminant analysis and neural network techniques. The non-stalked organisms' identification obtained individual recognition percentages above 80% with insignificant misclassification errors (usually bellow 1%) therefore yielding good overall recognition performances (above 80%). Furthermore, a closer look revealed a slight misclassification problem between the Monogononta and Digononta assessment. Although the overall results were not satisfactory for the stalked organisms, the identification ability of the biological indicators Opercularia and V. microstoma has attained some degree of confidence in accurately establishing their presence on WWTP. Furthermore, good identification percentages were obtained for the most significant Carnivorous and large stalk Colonial whilst the less significant Vorticella and small stalk Colonial only attained reasonable identification percentages. The main protozoa and metazoa groups (flagellates, ciliates, sarcodines, and metazoa) and protozoan ciliates (carnivorous, crawling, swimming, sessile and non-ciliated) recognition attained good overall recognition performances (above 95%, except for crawling ciliates), with global protozoa and metazoa groups assessment around 95%. The overall recognition performance for the plant operating conditions assessment were quite fair for the effluent quality, aeration and nitrification evaluation (above 80%) and good on the sludge age determination (91.7%). Close related to the overall results, the assessment of critical conditions such as low effluent quality, low aeration, and fresh sludge attained also good overall recognition performance levels around 90%. From these results it may be inferred that the current methodology can be used to predict at some extent, critical WWTP conditions in a feasible time period (within few hours) and without the need of specialized personnel in protozoology. The acquisition of the protozoa and metazoa images can be performed in a one to two hours period for a total of one to two hundred images, whilst the images treatment, data determination and analysis in two to three hours.


35

Microscopic characterization of activated sludge flocs from full-scale plants with advanced biological nitrogen removal systems Lucía Arregui, Pérez-Uz B, Calvo P, Salvadó H, Fernández N, Rodriguez E, Zornoza A and Serrano S. Departamento de Microbiología III, Universidad Complutense de Madrid. [email protected] Protozoa and metazoan are considered good indicators of the treatment quality in activated sludge systems being sensitive to physical, chemical and operational processes. Therefore, it is possible to correlate the predominance of certain species or groups and operational parameters of the plant. This work presents a semi-automatic image analysis procedure for the recognition of the metazoan and protozoa species most frequently found in WWTP by determining morphological data and multivariable analysis with discriminant analysis and neural network techniques. The non-stalked organisms' identification obtained individual recognition percentages above 80% with insignificant misclassification errors (usually bellow 1%) therefore yielding good overall recognition performances (above 80%). Furthermore, a closer look revealed a slight misclassification problem between the Monogononta and Digononta assessment. Although the overall results were not satisfactory for the stalked organisms, the identification ability of the biological indicators Opercularia and V. microstoma has attained some degree of confidence in accurately establishing their presence on WWTP. Furthermore, good identification percentages were obtained for the most significant Carnivorous and large stalk Colonial whilst the less significant Vorticella and small stalk Colonial only attained reasonable identification percentages. The main protozoa and metazoa groups (flagellates, ciliates, sarcodines, and metazoa) and protozoan ciliates (carnivorous, crawling, swimming, sessile and non-ciliated) recognition attained good overall recognition performances (above 95%, except for crawling ciliates), with global protozoa and metazoa groups assessment around 95%. The overall recognition performance for the plant operating conditions assessment were quite fair for the effluent quality, aeration and nitrification evaluation (above 80%) and good on the sludge age determination (91.7%). Close related to the overall results, the assessment of critical conditions such as low effluent quality, low aeration, and fresh sludge attained also good overall recognition performance levels around 90%. From these results it may be inferred that the current methodology can be used to predict at some extent, critical WWTP conditions in a feasible time period (within few hours) and without the need of specialized personnel in protozoology. The acquisition of the protozoa and metazoa images can be performed in a one to two hours period for a total of one to two hundred images, whilst the images treatment, data determination and analysis in two to three hours.


36

Metallothionein promoters from the ciliate Tetrahymena thermophila: tools to design heavy metal whole cell biosensors Francisco Amaro, Turkewitz A*, Martín-González A and Gutiérrez JC. Departamento de Microbiología III, Universidad Complutense de Madrid and *Departmentof Molecular Genetics & Cell Biology. University Chicago [email protected] In environmental biomonitoring, global parameters such as bioavailability, toxicity and genotoxicity cannot be tested using molecular recognition or chemical analysis, but can only be assayed using whole cells. Obviously, in these bioassays the question to be resolved is not "What toxicants does the sample contain?, but rather "How toxic is the sample?. A whole-cell biosensors (WCB) uses the whole prokaryotic or eukaryotic cell as a single reporter incorporating both bioreceptor and transducer elements. It typically combines a promoter-operator, which acts as the sensing element, with a reporter gene coding for an easily detectable protein. Ciliates, as eukaryotic microbes without cell wall, might offer a higher sensitivity to environmental pollutants, and therefore, a faster cellular response (Gutiérrez et al., 2003). The main heavy metal resistance mechanism in Tetrahymena thermophila involves sequestration by proteins from the metallothionein (MT) family (Díaz et al., 2007). In T. thermophila and related species, MT gene transcription is dramatically induced when cells are exposed to heavy metals. By using two different vigorous MT promoters, from MTT1 and MTT5 genes, we have constructed two different promoter-reporter gene fusions, to develop two different WCB based on recombinant cell lines of T. thermophila. Cells of this ciliate were transformed (by electroporation or biolistically) to express chimerical promoter-reporter gene fusions consisting of; (a)- the firefly luciferase gene under the control of either MTT1 or MTT5 promoter, it was constructed using the system pBTU1/BTU2 designed to replace the BTU1 locus by homologous recombination (these constructs are denominated as TtPMTT1::Luc or TtPMTT5::Luc); or (b)- consisting of the Green Fluorescence Protein (GFP) (as reporter gene) linked to either the ORFs of MTT1 or MTT5 genes under the control of the MTT1 promoter, it was constructed by using the vector pVGF.1 (pTtMTT1::MTT1::GFP or pTtMTT1::MTT5::GFP). At present, we are carrying out the evaluation and validation of these WCBs under different heavy metals and other stress conditions. Both fluorescent and bioluminescent WCBs produce a measurable signal even at low metal concentration. Bioluminiscent WCBs were more sensitive to CdCl2 than fluorescent WCBs, indicating a higher sensitivity as reporter gene. WCBs carrying the fusion protein MTT5::GFP showed higher levels of cadmium tolerance, indicating that the MT linked to GFP is still able to bind metal ions. Supported by the project CGL2005-00548 (MEC).


37

Effects of diuron and glyphosate on periphytic microbial communities and Enterobacteria: experimental approaches Céline Fajon, Forestier C*, Vermerie M* and Bohatier J. Laboratoire de Microbiologie: Génome et Environnement, Université Blaise Pascal and *Université d’Auvergne. [email protected] Natural ecosystems are widely influenced by human activities, especially agricultural practices that include the extensive uses of xenobiotics such as herbicides. The phenylurea herbicide diuron and the glyphosate are frequently detected in European streams and rivers and their concentrations range from 0.05 to 20.3 µg/l (IFEN, 2004). Bye soil leaching and overland flow, most of these organic molecules leads to surface water contamination. Their toxic properties mean that these contamination pose significant toxicological risk to resident aquatic organisms. Because of their physiological characteristics, microalgae represent potential primary targets for herbicide in lotic ecosystems. The released of organic compounds from the altered algal communities can indirectly disturb the heterotrophic bacterial community that play a key role in aquatic environments, and consecutively all the living microorganisms involved in the trophic chain. Therefore the modifications induced by the presence of organic compounds could provide selective pressure that promote the emergence of water-borne pathogens, either microorganisms naturally able to growth in water or coming from faecal sources. Most of the bacteria in lotic ecosystems are associated with biofilms on liquid - solid surfaces. They represent environments with special features that render them most exposed to xenobiotics. We will propose to determine the influence of diuron and glyphosate on the development of periphytic microbial communities within biofilms by experimental approaches. We focused on physiological and taxonomic modifications related directly and/or indirectly to herbicide contamination Natural biofilms were grown on glass substratum and incubated into a French river during two weeks and microbial periphytic communities will be exposed to herbicide contamination in laboratory under experimental conditions. The effect of herbicide on microalgal, bacterial and Enterobacteria communities will be appreciated by measurements of metabolic activities and analysis of diversity by molecular approaches (TTGE, FISH). The periphytic microbial communities collected on glass substratum have been first characterized before their herbicide exposition. The bacterial density (1.7- 3.5 cell/cm²) is comparable to those reported in literature. The analysis of the bacterial community structure by FISH reveals the dominance of bacteria related to CFB group. The presence of specific prokaryotic pathogens, i.e. Enterobacteria, was detected by specific PCR using the genomic DNA from the total biomass as template.


38

Protostelids (Amoebozoa) from Mediterranean environments María Aguilar and Lado C. Departamento de Micología, Real Jardin Botánico (CSIC), Madrid. [email protected] Protostelids are Amoebozoans that produce simple fruiting bodies, known as sporocarps, that are comprised of a single acellular stalk and one to a few spores. Their trophic stage varies from uninucleate amoeboid or amoeboflagellate cells to multinucleate reticulate plasmodia. Most protostelids appear to be members of the taxon Mycetozoa that also includes the myxomycetes and the dictyostelid cellular slime molds (Spiegel et al 1995, Baldauf & Doolittle, 1997). A few other species of protosteloid amoebae are found in other taxa of Amoebozoa. It is remarkable that Europe, one of the most studied territories of the world in terms of biodiversity, has hardly been studied for this group (Aguilar et al 2007). No extensive works have taken place in the Mediterranean Basin, one of the world biodiversity hotspots. The objective of this work is to provide a survey of the protostelid species present in this area and a first approximation to their ecology in Mediterranean climate. The sampling took place in 13 localities, in the provinces of Madrid, Cuenca, Guadalajara, Toledo y Ávila (Spain). A total of 100 samples from three different microhabitats were collected and cultured: ground litter (layer of plant debris extending over the soil surface), aerial litter (assemblage of dead but still attached parts of standing plants) and bark. Accumulation curves were used to estimate the maximum number of species to be expected (Schnittler 2001). This estimator was compared to the number of species recovered from our samples to calculate the extent to which the survey was exhaustive. A canonical correspondence analysis (CCA) was performed using the species as dependent variables and environmental factors as independent variables. To test the null model of the independence between the species data matrix and the environmental data matrix a Monte Carlo test of 1000 permutations has been carried out. This work has been supported by the Research Project CGL2005-00320/BOS of the Ministry of Education and Science of Spain. Aguilar M, Lado C & Spiegel FW (2007). Mycological Research 111 : 863-872. Baldauf SL & Doolittle WF (1997). Proc. Natl. Acad. Sci. USA 94: 12007-12012. Schnittler M (2001). Mycologia 93: 653-669. Spiegel FW, Lee SB & Rusk SA (1995). Can. J. Bot. 73: 738-746.


39

Detection and diversity of free-living protozoa in waters from cooling towers and hot water systems Oriol Canals, Araujo RM* and Salvadó H. Departamento de Biología Animal and *Departamento de Microbiología, Universitat de Barcelona. [email protected] To study the prevalence and diversity of free-living protozoa, potential hosts of bacteria such as Legionella spp., in urban water systems, we analysed 175 samples from public buildings through direct observation and culture. Of these, 143 samples were collected from sanitary-hot water systems, and 32 from cooling towers. Free-living ciliates, flagellates and amoeba were observed in 39 of the 143 samples (27.7%) from sanitary hot water systems, and in 7 of the 32 (21.9%) samples from cooling towers. Amoebae showed the highest prevalence, being detected in 42 samples from a total of 175 (24%). In the samples that presented protozoa, amoebae were detected in 42 from a total of 46 cases (91.3%). Among others, we identified amoebae from the genus Acanthamoeba and members of the families Valhkamphidae and Hartmannellidae. Flagellates were observed in 24 of the 46 samples (52.1%) that presented protozoa, and we also identified, among others, individuals from the family Bodonidae. Finally, ciliates were detected in 3 samples from a total of 46 (6.5%), Colpoda steinii being the only species detected. On results on the prevalence of free-living amoeba in urban waters (24%) are similar to those published by Shoff et al (J. of Water and Health, 6, 99-104, 2008) in the USA, who reported a prevalence of 19.4 %, although these findings differ substantially from the 89% prevalence described in a study of tap water in the UK by Kilvington et al (Invest. Ophthalmol. Vis. Sci. 45, 165-169, 2004). This work was supported by MEC GCL2005-01465

Session III, Poster FSFCP, Sevilla '08

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Protistan grazing stimulates bacterial benzene degradation under nitrogen limitation Antonis Chatzinotas, Goedden O and Harms H. Department of Environmental Microbiology, UFZ-Helmholtz Centre for Environmental Research, Leipzig. [email protected] The possibility to stimulate bacterial contaminant degradation via the recycling of limiting inorganic nutrients by protistan grazing was tested in controlled laboratory experiments. To this end, nitrogen-limited microcosms containing benzene-degrading bacteria, a bicosoecid flagellate and benzene were used. During the grazing experiment, bacteria consumed approximately three times more benzene than in a control containing cycloheximide inactivated protists. The specific bacterial benzene degradation rate was significantly increased as long as high numbers of active protists were present. While benzene degradation in the control stopped after 284 hours at a high residual benzene concentration, it continued to completion after 795 hours in the grazing experiment. The stimulation was a consequence of nitrogen remineralization by grazing protists as could be demonstrated in a separate nitrogen release experiment. Nutrient recycling by bacterivorous protists may thus stimulate the growth-coupled contaminant degradation by heterotrophic bacteria in nutrient-limited systems such as contaminated groundwater. Future research, especially with a focus on natural attenuation processes, should regard bacterial contaminant degradation much more under an ecological perspective that also accounts for the fate of the biodegradation-derived biomass and its function as a reservoir of elements essential for ongoing biodegradation.


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Ciliates in Mediterranean shallow lakes: the influence of macrophytes and eutrophication Ana Conty and Bécares E. Departamento de Ecología, Universidad de León. [email protected] Nineteen lakes were sampled during the summers of 2003 and 2004 in the community of Castilla y León (Northwest Spain), to study the influence of macrophytes and eutrophication in the communities of ciliates in shallow Mediterranean lakes. Chlorophyll a concentration (Chla) was higher correlated to ciliate density than total phosphorous. The slope of the regression between the abundance of ciliates and Chla showed greater abundance of ciliates than those lakes of higher latitudes, having similar values to those found in subtropical zones. Some values of ciliate densities were between the highest ever found in shallow lakes, with values higher than 625 cells mL-1. Ciliate carbon biomass constituted an average of 39% of total zooplankton biomass, reaching 71% in hypereutrophic lakes. Prostomatids and oligotrichs dominated in lakes with low Chla, whereas scuticociliates and small oligotrichs dominated in hypereutrophic ones. Macrophytes influenced ciliate community structure, dominated by oligotrichs, prostomatids and scuticociliates in lakes with few macrophytes and by peritrichs, hypotrichs, pleurostomatids and gymnostomatids in lakes with abundant vegetation. One third of the ciliate taxa were not euplanktonic, demonstrating the importance of benthos and periphyton in these lakes. It was also stated the influence of eutrophication in the size-structure of the ciliates. Medium size ciliates (20-40 micrometers) were dominant in lakes with low Chla, whereas the smallest (<20 micrometers) and the biggest ranges (>100 micrometers) dominated in lakes with higher Chla. Evidences support the hypothesis that ciliates exerted a strong predation pressure on bacteria, since a positive relation between ciliates and “bacterial filaments” was found. The structure of the ciliate community seemed to be mainly controlled by bottom-up factors (resources), although size-structure was also significantly influenced by top-down variables (predation), especially to the abundance of big cladocera.


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Guidelines for identification of ciliates in wastewater treatment plants (IWA Publishing, 2008) Susana Serrano, Arregui L, Pérez-Uz B, Calvo P and Guinea A. Departamento de Microbiología III, Universidad Complutense de Madrid. [email protected] Ciliates are useful tools for the control of the wastewater biological reactors. These microorganisms can be used to check the state of the biological community and as bioindicators of operational plant parameters, however, to be successful on the target to identify these microorganisms certain knowledge and experience are necessary. A book is presented here with the main purpose to provide a feasible mean to identify ciliates in wastewater treatment plants. A simple and easy-to-use key is proposed and brought up to date with the latest taxonomic modifications published in this group. Brief morphological descriptions of every species with detailed drawings and pictures are included as well as information on environmental conditions associated to the presence/absence of different species or communities in these controlled environments. Description of the most common methods for sampling, counting and staining ciliates and a short glossary with terms frequently used about ciliate cellular structures is also provided. This book would be useful for undergraduate and graduate students as well as professionals and researchers interested on management and lab control of wastewater treatment plants or polluted environments.


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Session IV

Biomedicine and Biotechnology of Parasitic Protists.

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Mechanisms associated with traversal of the blood-brain barrier by Acanthamoeba Naveed Ahmed Khan, Martazavi P, Kim KS*, Stins M* and Goldsworthy G. School of Biological and Chemical Sciences, Birkbeck College, University of London and *Johns Hopkins University School of Medicine, Baltimore. [email protected] Granulomatous amoebic encephalitis is a fatal human infection. However, its pathogenesis and pathophysiology remains unclear. Here, it is shown that the African migratory locust can be used as a model to study Acanthamoeba pathogenesis. Up to 100% of mature adult locusts, injected intra-abdominally with a suspension of 106 parasites were killed within 14 days. It was determined that the parasites disseminate via haematogenous spread, as demonstrated by the recovery of amoebae in the locust haemolymph, muscles, fat bodies, but not faeces. To determine association of amoebae with the central nervous system, locust brains were dissected out, and incubated with chlorhexidine (100 µM; final concentration) at 37oC for 60 min to kill extracellular amoebae. The brains were washed a further three times, homogenised and cultured for the presence of amoebae. Brain lysates from amoebae-infected locusts were positive for A. castellanii, whereas brain lysates from PYG-injected (control) locusts showed no organisms. Our findings support the notion that haematogenous spread is a pre-requisite for parasite invasion of the central nervous system, but it is not clear how circulating amoebae cross the blood-brain barrier. To this end, we have developed an in vitro model of the blood-brain barrier by isolating human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We used primary HBMEC to study interactions with pathogenic Acanthamoeba, and identified several determinants, both from the host side and the parasite side, which are responsible for increased blood-brain barrier damage/permeability. Using adhesion assays, it was observed that parasite adhesion to HBMEC is mediated by a mannose-binding protein that is expressed on the surface of Acanthamoeba. This initial binding of Acanthamoeba results in HBMEC cell cycle arrest as observed by inhibition of expression of genes including cyclins F, G1 and cyclin dependent kinase 6, GADD45A and p130 Rb that encode proteins important for cell cycle progression. Western blotting assays confirmed further that Acanthamoeba abolished pRb phosphorylations leading to cell cycle arrest at G1-to-S transition. Longer incubations result in Acanthamoeba-mediated host cell death as determined by cytotoxicity assays. Next, we identified phosphatidylinositol 3-kinase (PI3K) as a major HBMEC signalling molecule activated in response to Acanthamoeba. In HBMEC, Acanthamoeba increased Akt phosphorylations, a downstream effector of PI3K. Acanthamoeba-mediated HBMEC cell death was inhibited in a concentration dependent manner using LY294002 i.e., a PI3K inhibitor. HBMEC transfected with dominant-negative PI3K (∆p110K) were significantly less susceptible to Acanthamoeba-mediated

Session IV, Oral FSFCP, Sevilla '08

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HBMEC cell death. In an attempt to identify contact-independent mechanisms associated with Acanthamoeba pathogenesis, it was shown that Acanthamoeba serine proteases increase HBMEC permeability, which may lead to perturbations of the blood-brain barrier. Future studies will continue to identify both host and parasite factors involved in the pathogenesis and pathophysiology of Acanthamoeba encephalitis. Knowledge of such factors is crucial for the rational development of therapeutic interventions.


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Human tumour necrosis factor modulates gene expression of genes encoding ribosomal proteins and endocytosis markers in Entamoeba histolytica Blazquez S, Labruyère E, Weber C and Nancy Guillén. Unité Biologie Cellulaire du Parasitisme, Institut Pasteur. [email protected] Tumour necrosis factor (TNF) is a pleiotropic pro-inflammatory cytokine. In mammalian cells, after binding of the ligand to its receptor, it is internalised by clathrin-mediated endocytosis, the endosomes fuse with the lysosome to degrade the cytokine and allow receptor recycling. The signalling pathways induced, lead to cell death by initiation of the caspase cascade and activation of transcription factors modulating gene expression (cell survival or enhancement of the inflammatory response). Entamoeba histolytica is the etiological agent responsible for human amoebiasis. TNF, is a key element modulating the inflammatory response during amoebiasis. TNF is not cytotoxic for E. histolytica, rather it has been shown to have a chemoattractant and chemokinetic effect on E. histolytica trophozoïtes. In this work, we investigated the cellular and molecular effects of TNF on E. histolytica by cellular methods and on microarrays. We first determined that TNF bound to the trophozoites and that this binding was specific. The uptake of TNF occurred in vesicles in a potentially active manner, suggesting that E. histolytica uptake of TNF could be by receptor-dependent endocytosis. We then compared RNA extracted from trophozoites in presence of TNF with RNA extracted from parasites that had not been in contact with TNF. We showed that TNF could modulate gene expression of E. histolytica. Genes encoding proteins involved in intracellular trafficking and signal transduction were down-regulated. Interestingly, genes encoding ribosomal proteins were up-regulated and correlated with an stress-like response.


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Effect of free versus liposomal-complexed pentamidine isethionate on biological charactersitics of Acanthamoeba castellanii Ruqaiyyah Siddiqui, Syed A, Tomas S, Prieto J* and Khan NA. School of Biological and Chemical Sciences and *School of Pharmacy, Birkbeck College, University of London. [email protected] Acanthamoeba keratitis is a painful infection with blinding consequences. Current treatment against Acanthamoeba keratitis requires early diagnosis followed by topical application of drugs. Treatment can last up to a year, and even then, infection can recur. The overall aim of the present study was to determine whether liposomes enhance therapeutic value of anti-amoebic drugs. The liposomes consisted of l-α-phosphatidylcholine, and cholesterol or ergosterol in a molar ratio of 1:5. An anti-amoebic drug, pentamidine isethionate was incorporated into phospholipid vesicles using the dehydration-rehydration procedure so that the final drug to lipid ratio was 1:5. The effect of drug alone (P), liposomal cholesterol-P (Pc ) or liposomal ergosterol-P (Pe) and controls were determined on parasite binding to human cells, encystation, and Acanthamoeba-mediated host cell death. At 10 µg/ml, liposomal drug was 12 times more effective than free drug at preventing Acanthamoeba binding to human cells. In addition, liposomal drug was 1.5 times more effective in reducing Acanthamoeba-mediated human cell cytotoxicity, compared with the drug alone. Notably, free or liposomal drug had no effect on the encystation of Acanthamoeba. These studies suggest that liposomal-complexed anti-amoebic drugs may have promise in the treatment of Acanthamoeba keratitis.


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Evaluation of gram-chromotrope Kinyoun staining technique in detecting Microsporidia spores in faecal specimen Norhayati Moktar, Al-Mekhlafi A, Fatmah MS, Noor Aini U, Anisah N. Department of Parasitology, Universiti Kebangsaan Malaysia. [email protected] Microsporidiosis is a new emerging opportunistic infection and has been widely recognized as an important in human especially in immunocompromised individuals. This study aimed to evaluate the modification of the usual Gram-chromotrope stain technique developed in-house known as Gram-chromotrope Kinyoun in comparison with Weber Modified Trichrome staining technique which is considered as the reference technique. Two hundred and ninety (290) faecal specimens received by Microbiology Diagnostic Laboratory of Hospital Universiti Kebangsaan Malaysia were examined for the presence of microsporidial spores using both techniques. Using the reference staining technique microsporidial spores were detected in 17.2% of the faecal specimens. The sensitivity and specificity of Gram-chromotrope Kinyoun compared to the reference technique were 98% and 98.3% respectively. The positive predictive value was 92.5% and the negative predictive value was 99.6%. The aggrement between the reference technique and Gram-chromotrope Kinyoun staining technique was statistically significant by Kappa statistics (K=0.941, P<0.001). In conclusion Gram-chromotrope Kinyoun staining technique has high sensitivity and specificity in the detection of microsporidial spores in faecal specimens. Therefore it is recommended to be used in the diagnosis of intestinal microsporidiosis.


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Detection of Perkinsus olseni (Lester & Davis, 1981) in clams employing a nested PCR assay Balseiro P, Aranguren R, Camino Gestal, Novoa B and Figueras A. Departamento de Patología de Organismos Marinos, Instituto de Investigaciones Marinas (CSIC), LNR Moluscos Bivalvos, Vigo. [email protected] Perkinsosis is a notifiable disease affecting several species of clam in Galicia (NW Spain) caused by the protozoan parasite Perkinsus olseni. The two classical diagnostic methods recommended by the Office International des Epizooties (OIE) in the Manual of Diagnostic Tests of Aquatic Animals, histology and incubation on Ray's fluid thioglicollate medium (RFTM) are tedious and need highly qualified personnel. In order to develop faster and cheaper diagnosis techniques, classical methods are compared with a nested PCR assay. Five different clam species of trade interest cultured in Galicia were employed for the detection of P. olseni. Each clam was simultaneously processed for histopathology and PCR. In addition, 150 clams were subjected to RFTM incubation. A Nested PCR on Internal Transcribed Spacer (ITS) region was carried out and product identity was corroborated by in situ hybridization and sequencing. Results from the different techniques were compared for diagnostic sensitivity, specificity and number of false positives compared with a gold standard consistent in the other techniques under comparison. Positive Predictive Value (PPV) and Negative Predictive Value (NPV) were also calculated and κ-value statistic was used to measure the level of agreement of the PCR assay and the combination of the classical diagnostic methods. Nested PCR was the most sensitive method considering all the samples. PCR is the diagnostic method that gives the lowest number of false negative results. PPV and NPV results indicate than PCR is suitable for screening of low-parasitized populations of clams as experiments of eradication of P. olseni. PCR method is cheaper and faster than histopathology and incubation on RFTM. Histology possesses a higher PPV, suggesting that this technique is strongly recommended for confirmatory diagnosis of P. olseni. In addition, histology can give a general vision of the health status of the animal. Results from incubation on RFTM suggest that this method has low specificity and reveals great number of false positives.

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Study of the parasites from teleosts fishes of aquaculture interest in Catalonia (NE Spain) Pedro Andrés Maíllo1,2, Salvadó H1,2, Marques A3 and Gracia MP1,2. 1Departamento de Biología Animal, Universitat de Barcelona, 2 Xarxa de Referència de Recerca i Desenvolupament, Aqüicultura-Generalitat de Catalunya and 3Université Montpellier II. [email protected] The present study was undertaken to fill a gap of information about parasites from teleost fishes in Catalonia (NE Spain) and forms part of a ongoing investigation on the parasites from fishes of aquaculture interest. A total of 216 eels (Anguilla anguilla L., 1758; mean length: 34.15±0.37 cm; mean weight 75.48±2.45 g), 192 grey mullets (Mugil cephalus L., 1758; mean length: 17.4±1.3 cm, mean weight 50.6±11.0 g) and 90 golden grey mullet (Liza aurata (Risso, 1810); mean length: 33.5±2.9 cm; mean weight 324.8±72.8 g) were examinated in search of parasites.Eels were captured from three brackish coastal lagoons within the Ebre Delta (Western Mediterranean): l'Encanyissada, la Tancadaand el Canal Vell. All three lagoons are connected to Mediterranean sea by channels and they have some freshwater inputs whose volume may be relevant. Grey mullets were captured in l'Encanyissada lagoon. Golden grey mullet specimens were captured in the Mediterranean Sea, off the coasts of Barcelona (FAO-37-1 zone).The parasites showed several locations on the host: scales, gill arches, gill lamellae, messenteria vessels, kidneys and gallbladder. Different species were observed by light and electron microscopy (TEM and SEM).In eels were determined: a ciliata, Trichodina pediculatus (gills, prevalence: 0.02%); a coccidian, Eimeria anguillae (intestine, prev: 7.69-9.06%); two myxozoan: Myxidium giardi (gills, prev: 15.4-15.86% - kidney, prev: 20.5-44,38%),and Myxobolus sp (kidney, prev: 7.69-14.72%). M. giardi occurred in all the analyzed samples.In grey mullets were determined: a ciliata, Trichodina sp. (gills, prev: 2.20%), four Myxozoan, Myxobolus ichkeulensis (gill arches, prev: 52.75%), Myxobolus bizerti (gill lamellae, prev: 5.27%), Myxobolus sp1. (fins, prev: 39.01%), and Myxobolus sp2 (gallbladder, prev:14.29%).In golden grey mullet were determined Myxobolus spinacurvatura (messenteria vessels, prev: 42.86%).Myxobolus genus is the main histozoic myxozoa affecting gills. They can cause serious pathological effects on tissues. The most common species were M. ichkeulensis, whose cysts, elongate (4mm lenght) and irregular contour, can cause gill damage. Parasite found in this study may represent a potential risk in fish farms. Both Trichodina sp. and Myxidium giardi or Myxobolus can cause lesions to gills that serve as a gateway to viral and bacterial secondary infections. This work was supported by Xarxa de Referència de Recerca i Desenvolupament


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Ultrastructural characteristics of the genus Myxobolus BÜTSCHLI, 1882 (Myxozoa) infecting the gills of Mugil cephalus Pedro Andrés Maíllo1,2, Marques A3 and Gracia MP1,2. 1Departamento de Biología Animal, Universitat de Barcelona, 2 Xarxa de Referència de Recerca i Desenvolupament, Aqüicultura-Generalitat de Catalunya and 3Université Montpellier II. [email protected] This study presents the ultrastructure and development stages of genus Myxobolus, that infects the gills of grey mullet (Mugil cephalus). The parasite was observed in 52.75% of the examinated fishes. Specimens were captured in a brackish coastal lagoon (l’Encanyissada) within the Ebre delta (Western Mediterranean). The parasite forms elongate cysts masses distributed at the base of arches gills. Number of cysts were variable but, generally, we found from three to eleven. Length of cysts was 1 to 4.0 mm. Spores of this histozoic Myxozoa presents two peripheral valvogenic cells, a pair of capsulogenic cells and a single binucleate sporoplasm, characteristic of Myxobolus genus. Transmision electron microscopy (TEM) revealed that polysporous trophozoite presents an external layer of connective tissue (host tissue) in contact with a single trophozoite membrane. Trophozoite endoplasm contains different developing stages: germinative cells, immature and mature spores. Spores are quite spherical (lenght 10.5 - 11.3 µm; width 9.0 - 11.0 µm), and they have from nine to eleven sutural marks along the sutural edge. Polar capsules were oval and did not exceed the mid-length of the spore (length 3.28 µm, width: 2.09 µm). They showed four to five filaments turns. Myxobolus species are characterized by its relativelly strict site specificity. Based on the ultrastructural morphology and specificity to the host, we conclude that this species is Myxobolus ichkeulensis (Bahri & Marques, 1996). This work was supported by Xarxa de Referència de Recerca i Desenvolupament en Aqüicultura de la Generalitat de Catalunya (Autonomic Government).


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Species Identification of Intestinal Microsporidia Using IFA-MAbs Nor Aini Umar, Al-Meklafi A, Fatmah MS, Anisah N, Norhayati M. Department of Pathology, Universiti Kebangsaan Malaysia. [email protected] The aetiologic agents for microsporidiosis are eukaryotic obligate, intracellular spore-forming protozoan parasites. Enterocytozoon bineusi and E. intestinalis are the primary microsporidium species associated with humans. The species identification of these organisms is only possible using Transmission Electron Microscopy (TEM), molecular technique and immunofluorescent antibody assays (IFA) using specific monoclonal and polyclonal antibodies. In this study 50 faecal specimens which were found positive and another 50 negative for microsporidial spores using Weber Modified Trichrome staining technique were then tested using IFA-MAbs. Out of these 100 faecal specimens examined, 56% were detected positive for intestinal microsporidiosis. The majority of this microsporidia spores identified by IFA-MAbs were E. bineusi 42(75%) followed by E. intestinalis 7 (12.5%). Mixed infection of both species is found in 7 (12.5%) of the faecal specimens. Using Weber Modified Trichrome staining technique as a reference test, the sensitivity and specificity of IFA-MAbs in detecting microsporidial spores were 98% and 86% respectively. The positive predictive value and the negative predictive value were 87.5% and 97.7% respectively. The agreement between Weber Modified Trichrome staining technique and IFA-MAbs was statistically significant by Kappa statistics (K=0.840; P<0.001).


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Session V

Protist Evolution

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A new genus of Xenophyophores (Foraminifera) from Japan Trench: morphological description, molecular phylogeny, and elemental analysis Béatrice Lecroq1, Goodday A2, Tsuchiya M3, and Pawlowski J1. 1Department of Zoology and Animal Biology, University of Geneva, 2National Oceanographic Center, UK and 3Japan Agency Marine-Earth Science Technology. [email protected] The deep-sea floor is inhabited by a number of unusual and enigmatic taxa unknown in shallow waters. These include the xenophyophores, a group of giant protists that construct fragile agglutinated tests. Here we describe Shinkaiya lindsayi, a new genus and species of xenophyophore collected by the submersible Shinkai 6500 at 5435 m water depth near the Japan Trench. The phylogenetic analysis performed with its complete SSU rDNA sequence confirms that S. lindsayi is a foraminiferan, closely related to another xenophyophore Syringammina corbicula Richardson, 2001 and to a monothalamous foraminiferan Rhizammina algaeformis Brady, 1879. In terms of morphology, the new genus resembles Syringammina but its test wall is thicker, softer and more weakly cemented. Moreover, the SSU rDNA sequences of the two genera are highly divergent. Mass spectra analyses reveal unusually high concentrations of some elements. The granellare system (the cytoplasm and the organic sheath that encloses it) is apparently devoid of barite crystals, which are usually abundant as intracellular inclusions in xenophyophores, but is rich in mercury. Fecal pellets retained in the stercomare system concentrate heavy metals including uranium. Based on a comparison of the compositions of the agglutinated test wall, the granellare, the stercomare and the surrounding sediment, we discuss the impact of xenophyophores on their habitat.

Session V, Oral FSFCP, Sevilla '08

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Non-homologous proteins of diverse phylogeny are involved in polyphosphate metabolism of photosynthetic protists Tomás Albi-Rodríguez and Serrano Aurelio. Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla. [email protected] Inorganic polyphosphates (polyP) are linear polymers of ortophosphate (Pi) residues linked by high energy, phosphoanhydride bonds. The ubiquitous occurence of polyPs suggests that the may have important metabolic, regulatory and stress-tolerance roles. We have performed the first systematic survey of genes involved in polyP metabolism with photosynthetic microorganisms, both prokaryotes (anoxygenic photobacteria, cyanobacteria) and eukaryotes (microalgae, tallophytic algae, mosses). All main photobacterial groups exhibit genes encoding polyP-kinases (PPK, EC 2.7.4.1) and exopolyphosphatases of the Ppx-GppA family (PPX, EC 3.6.1.11). Cyanobacterial heterocystous strains possess in addition polyP-glucokinase (PPGK, EC 2.7.1.63) encoding genes. These genes have been validated by heterologous expression and protein characterization. Interestingly, orthologs of cyanobacterial PPK and PPX genes were identified and found to be expressed in diverse photosynthetic protists (green and red microalgae, like Ostreococcus tauri, Galdieria sulphuraria and Cyanidioschyzon merolae, among others), tallophytic red algae (Porphyra yezoensis), mosses (Physcomitrella patens) and higher plants, thus indicating that they have been functionally preserved during the evolution of the photosynthetic lineage. In some species paralog genes were found. Many of these eukaryotic genes, in contrast to their prokaryotic counterparts, encode precursor proteins with N-terminal signal/transit peptides and nuclear localization signals, suggesting its localization in specific subcellular compartments. It is interesting to note at this respect that some of them appear to be fusion proteins with domains involved in nucleic acid biochemistry (e.g., NUDIX, in the Cy. merolae PPK; citidine-deaminase, in a P. patens PPX paralog). On the other hand, genes for non-homologous PPX proteins of the DHH-DHHA2 phosphoesterase family and a closely related family II sPPase -not previously reported in phototrophs and eukaryotes, respectively- were also identified in representative species of diverse microalgal groups (Prasinophyceae, Dinophyceae, Bacillariophyceae, Haptophyceae) and some of them were functionally characterized. The molecular phylogeny of these proteins and their relationships with functional orthologs of non-photosynthetic organisms is discussed. Supported by BFU2007-61887 (MCI) and PAIDI group BIO-261 (JA) grants.


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Pan-oceanic distribution of new highly diverse clades of deep-sea diplonemids Enrique Lara, Moreira D, Vereshchaka A and López-García P. Unité d'Ecologie, Systématique et Evolution, Université Paris-Sud. [email protected] Molecular rRNA gene surveys reveal a considerable diversity of microbial eukaryotes in different environments. Even within a single clade, the number of distinct phylotypes retrieved often goes beyond previous expectations. Here, we have used specific 18S rRNA PCR primers to investigate the diversity of diplonemids, a poorly known group of flagellates with only a few described species. We analysed surface and deep-sea plankton samples from different oceanic regions, including the water-column in the Marmara Sea. We retrieved a large diversity of diplonemid phylotypes, most of which formed two novel distinct clades without cultured representatives. Although most diplonemid phylotypes appeared to be ubiquitous in oceans, they showed a marked stratified distribution through the water column, being very scarce or absent in surface waters. The small and specific diplonemid diversity found in surface samples and the fact that most sequences of uncultured diplonemids found in other studies came from deep-sea environments, suggest that the two major uncultured diplonemid clades group species preferentially inhabit the deep ocean.


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An evolutionary conserved photoperiod signal in algae and plants Serrano G, Herrera-Palau R, Serrano A and Federico Valverde. Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla. [email protected] Chlamydomonas reinhardtii is a chlorophyte alga with a robust circadian clock that controls several crucial developmental processes (1). In higher plants one of the major events regulated by the circadian clock is the floral transition, which determines the onset of the reproductive programme that produces flowers. Because flowering is essential for the reproductive success of the plant, it is regulated by a complex system that integrates multiple external and internal inputs (2). In the model plant species Arabidopsis thaliana, the product of the gene CONSTANS (CO) regulates photoperiodic flowering, which is one of the most important and widely distributed traits to optimize flowering time among distantly related plants (3). A functional genomic study has shown that Chlamydomonas presents a single copy of a gene homologous to CO (CrCO) in its recently sequenced genome (4). Expression of this gene in plant vectors under different promoters complements the late flowering phenotype of co- mutants and causes early flowering in diverse genetic backgrounds. This functional complementation hints to the evolutionary conservation of the light signals that promote this phase transition. On the other hand, the CrCO gene in Chlamydomonas seems to be involved in an output pathway regulated by the circadian clock and the photoperiod, controlling several crucial responses such as reproduction, germination or starch accumulation. Employing a fusion of an inducible promoter (the ammonia-repressed nitrate reductase promoter) to CrCO coding sequence, we have studied its influence on development. On the other hand, antisense CrCO mutants have a strong growth arrest phenotype that may cause the termination of the algal culture. In the light of these results we have studied the effect of photoperiod on algal development and situated CrCO as a key modulator of light-dependent developmental processes.References.1. Mittag, M., Kiaulehn and Johnson, H. (2005) The Circadian Clock in Chlamydomonas reinhardtii. What Is It For? What Is It Similar To? Plant Physiol. 137, 399-409.2. Bäurle, I. and Dean, C. (2006) The Timing of Developmental Transitions in Plants. Cell 125, 655-664.3. Kobayashi, Y. and Weigel, D. (2007) Move on up, it's time for change-mobile signals controlling photoperiod-dependent flowering. Genes Dev. 21, 2371-2384.4. Merchant, S.S. et al. (2007) The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions. Science 318, 245-251.


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Content and evolution of a non-coding genomic fraction in a context of whole-genome duplication: the Paramecium tetraurelia case Chen C-L1,2, Zhou H2, Liao J-Y2, Qu L-H2 and Laurence Amar1. 1CNRS-UMR 8080-Université Paris Sud and 2Key Laboratory of Gene Engineering of the Ministry of Education, China. [email protected] Despite the fact that non-coding DNA (ncDNA) serves essential functions and that whole-genome duplications (WGD) are now known as widespread evolutionary events, ncDNA content and evolution in a WGD context is not well documented yet. Here we specifically analyze the ncDNA of the genome of the unicellular eukaryote Paramecium tetraurelia to the formation of which three WGDs contributed. We first used motif-based programs and experimental screens to characterize >400 non-coding RNA (ncRNA) genes that group into 176 sets on the basis of a common WGD origin. All genes but two paralogs map within the intergenic DNA. No set but two harbor genes evolved from the two older duplicates while the loss of duplicates arisen from the two following WGDs resulted in shrunk sets of 1-2 ncRNA genes in most cases. We then used a comparative genomic approach to characterize introns and sequences adjacent to protein-coding paralogs arisen from the last WGD. The 50-bp and 75-bp sequences that flank respectively the gene stop and start codons display nucleotide substitutions at levels lower than in further separated regions, with markedly low levels for the sequences flanking the highly transcribed genes. Introns known to be 25 bases on average display a sprawling distribution of sequence identity from 40% to 100%. Highly conserved introns appeared as landmarks of evolutionary constrained protein domains. Finally we used structure-based programs to characterize 188 intergenic sequences that may encode a conserved RNA stable secondary structure. One half of these sequences should identify untranslated regions (UTRs) sequences since they lay adjacent, or close, to protein-coding genes, and the other, putative ncRNA genes to be tested. Combined with previously published protein-coding gene data, our data provide first integrative overview of the interplaying coding and non-coding fractions of the P. tetraurelia genome.


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Characterization and phylogeny of folate/biopterin genes in ciliates Sara Hernández-Piñero, Palomino-Gutiérrez C, El Mounadi K, Morin L*, Baroin-Tourancheau A*, Torres A and Villalobo E. Departamento de Microbiología, Universidad de Sevilla and *Laboratoire de Biologie Cellulaire 4, Université Paris-Sud. [email protected] Folate is an important cofactor for all living organisms, since it participates in one-carbon transfer reactions. Only bacteria, though not all, fungi and plants have the ability to synthesize folate. Despite its importance in cellular metabolism, animals are unable to synthesize folate, necessitating of exogenous uptake, which is usually mediated by the reduced folate carrier (RFC), though other transporters have been characterized. Some eukaryotic parasites, such as kinetoplastids and apicomplexans, salvage folate from medium, though the latter organisms also synthesize folate de novo. In these parasites, salvage is mediated by folate/biopterin transporters (FBT), which has also been identified in plants and cyanobacteria. We took the advantage of the availability of genomic resources in several ciliates to search for FBT homologues. In this poster, we show the identification and partial characterization of putative FBT-coding genes in Paramecium tetraurelia and Tetrahymena thermophila. We also report the identification of FBT in non-cyanobacterial bacteria, and in both plastid-bearing and plastid-lacking protists. Interestingly, FBT homologues have not been identified in metazoans and fungi A phylogenetic tree built with all obtained FBTs confirm ciliate sequences belong to FBT family. The tree pattern is incongruent with established eukaryotic phylogenies, therefore we raise and discuss two evolutionary scenarios explaining FBT phylogeny; the first assumes gene paralogy while the second one assumes gene pseudoparalogy.

Session V, Poster FSFCP, Sevilla '08

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Hidden eukaryotic diversity in enigmatic deep sea protists Béatrice Lecroq1, Goodday A2, Cedhagen T3 and Pawlowski J1. 1Department of Zoology and Animal Biology, University of Geneva, Switzerland, 2National Oceanographic Center, UK and 3Department of Marine Ecology, University of Aarhus, Denmark. [email protected] Komokiacea are enigmatic protists extremely diverse and abundant in the deep-sea. They consist in a complex system of fine branching tubules sometimes covered by sediment creating a mudball structure. Because of their featureless external appearance they were frequently ignored or overlooked. Komokiacea are currently classified within Foraminifera but their taxonomic position still remains unresolved at a molecular level. Here we report the SSU rDNA sequences obtained from two komokiacean species: Normanina conferta and Septuma ocotillo from Southern Ocean. Although our study failed to univocally determine the origin of komokiaceans, it reveals an unexpectedly huge eukaryotic diversity associated with these organisms. This diversity seems much higher that in sediment samples collected in the vicinity of examined specimens. The komokiacean skeletons possibly serve as a habitat for a large variety of protists, creating hot-spots of euakaryotic diversity at the deep-sea bottom.


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Protist molecular diversity and phylogeny using small-and large-subunit ribosomal RNA genes amplified from environmental samples William Marande, López-García P and Moreira D. Unité d'Ecologie, Systématique et Evolution, Université de Paris-Sud. [email protected] The resolution of the phylogenetic relationships between the major eukaryotic groups is still a challenging question for evolutionary biologists. The small subunit (SSU) ribosomal DNA (rDNA) was the first marker used to address this duty. However, in recent years, several studies demonstrated that the SSU rDNA does not provide sufficient information to reconstruct, with confident statistical support, the global phylogeny of eukaryotes. Today, the analysis of EST libraries and the complete genome sequencing efforts allow retrieving very large data sets for phylogenomic analyses, generally based on long concatenations of numerous conserved protein sequences. However, this approach is severely limited by the poor number of taxa represented with such a large data principally due to the difficulty to obtain cultures suitable for cDNA or genome library construction. We investigated an alternative approach that consist of increasing the SSU rDNA phylogenetic signal with the large subunit rDNA and having a large taxon sampling by the use of complete ribosomal operon sequences amplified from environmental DNA and cultured species. For the first time, we show that complete eukaryotic ribosomal clusters can be amplified from environmental samples with high efficiency. We have constructed SSU rDNA libraries in parallel with ribosomal cluster libraries from 4 different environmental DNA samples. The statistical comparison of the two types of libraries reveals that they are not significantly different. This result indicates that our environmental analyses made by PCR are not systematically biased by the primer pairs used nor by the size of the PCR product, but it reflects the DNA content of the samples, at least for the dominant species. In addition, we have sequenced more than 60 complete SSU-LSU clusters in order to increase the sampling for almost all the eukaryotic supergroups. Maximum likelihood phylogenetic analyses for 150 eukaryotic SSU-LSU combined sequences yielded globally strong statistical support and recover the monophyly of the accepted supergroups as the Opisthokonts. Our results illustrate that combined SSU-LSU rDNA analyses can be easily applied to environmental diversity surveys, increasing the probability of a correct taxonomic affiliation even for very divergent sequences. Phylogenetic analysis based on these two markers can help to resolve several ambiguous regions of the eukaryotic tree and identify important taxa for subsequent multi-gene analysis.


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LIST OF PARTICIPANTS IN THE FSFCP FAMILY NAME FIRST NAME INSTITUTION EMAIL

AGUILERA ANDRÉS UNIVERSIDAD DE SEVILLA-CABIMER (SPAIN) [email protected]

SERRANO SUSANA UNIVERSIDAD COMPLUTENSE DE MADRID (SPAIN) [email protected]

LÓPEZ FARFÁN DIANA CAROLINA INSTITUTO DE PARASITOLOGÍA Y BIOMEDICINA LÓPEZ-NEYRA (SPAIN) [email protected]

VALVERDE ALBACETE FEDERICO INSTITUTO DE BIOQUÍMICA VEGETAL Y FOTOSÍNTESIS (SPAIN) [email protected]

ARRÉGUI GARCÍA-ROVES LUCIA UNIVERSIDAD COMPLUTENSE DE MADRID (SPAIN) [email protected]

CANALS ORIOL UNIVERSITAT DE BARCELONA (SPAIN) [email protected]

DAZA NAVARRO PAULA UNIVERSIDAD DE SEVILLA [email protected]

ARROYO CORDERO FRANCISCO IFAPA CENTRO LAS TORRES-TOMEJIL (SPAIN) [email protected]

SERRANO AURELIO INSTITUTO DE BIOQUÍMICA VEGETAL Y FOTOSÍNTESIS (SPAIN) [email protected]

CALVO PILAR UNIVERSIDAD COMPLUTENSE DE MADRID (SPAIN) [email protected]

MARQUES ADAM UNIVERSITÉ MONTPELLIER II (FRANCE) [email protected]

GUILLEN-AGHION NANCY INSTITUT PASTEUR (FRANCE) [email protected]

WEBER CHRISTIAN INSTITUT PASTEUR (FRANCE) [email protected]

AGUILAR GONZÁLEZ MARÍA REAL JARDÍN BOTÁNICO (SPAIN) [email protected]

PÉREZ UZ BLANCA UNIVERSIDAD COMPLUTENSE DE MADRID (SPAIN) [email protected]

KHAN NAVEED AHMED UNIVERSITY OF LONDON (UNITED KINGDOM) [email protected]

RUGAIYYAH SIDDIQUI UNIVERSITY OF LONDON (UNITED KINGDOM) [email protected]

LÓPEZ GARCÍA PURIFICACIÓN UNIVERSITÉ PARIS-SUD (FRANCE) [email protected]

MOREIRA FERNÁNDEZ DAVID UNIVERSITÉ PARIS-SUD (FRANCE) [email protected]

MARANDE WILLIAM UNIVERSITÉ PARIS-SUD (FRANCE) [email protected]

LARA ENRIQUE UNIVERSITÉ PARIS-SUD (FRANCE) [email protected]

LADO CARLOS REAL JARDÍN BOTÁNICO (SPAIN) [email protected]

GALLEGO ANDREA UNIVERSIDAD COMPLUTENSE DE MADRID (SPAIN) [email protected]

AMARO FRANCISCO UNIVERSIDAD COMPLUTENSE DE MADRID (SPAIN) [email protected]

MORIN LOÏC UNIVERSITÉ PARIS-SUD (FRANCE) [email protected]

AMAR LAURENCE UNIVERSITÉ PARIS-SUD (FRANCE) [email protected]

BAROIN TOURANCHEAU ANNE UNIVERSITÉ PARIS-SUD (FRANCE) [email protected]

AUBUSSON-FLEURY ANNE UNIVERSITÉ PARIS-SUD (FRANCE) [email protected]

KOHL LINDA MUSEUM NATIONAL D'HISTOIRE NATURELLE (FRANCE) [email protected]

FSFCP, Sevilla '08

63

FAMILY NAME FIRST NAME INSTITUTION EMAIL

GUTIÉRREZ JUAN CARLOS UNIVERSIDAD COMPLUTENSE DE MADRID (SPAIN) [email protected]

MOKTAR NORHAYATI UNIVERSITI KEBANGSAAN MALAYSIA (MALAYSIA) [email protected]

UMAR NOR AINI UNIVERSITI KEBANGSAAN MALAYSIA (MALAYSIA) [email protected]

BOUCHARD PHILIPPE UNIVERSITÉ BLAISE PASCAL (FRANCE) [email protected]

DAMAJ RAGHIDA UNIVERSITÉ BLAISE PASCAL (FRANCE) [email protected]

FAJON CÉLINE UNIVERSITÉ BLAISE PASCAL (FRANCE) [email protected]

BRICHEUX GENEVIÈVE UNIVERSITÉ BLAISE PASCAL (FRANCE) [email protected]

CONTY ANA UNIVERSIDAD DE LEÓN (SPAIN) [email protected]

PEREIRA DO AMARAL ANTONIO LUIS INSTITUTO SUPERIOR DE ENGENHARIA DE COIMBRA (PORTUGAL) [email protected]

LÓPEZ-ARCHILLA ANA ISABEL UNIVERSIDAD AUTÓNOMA DE MADRID (SPAIN) [email protected]

LECROQ BEATRICE UNIVERSITY OF GENEVA (SWITZERLAND) [email protected]

MAÍLLO BELLÓN PEDRO ANDRES UNIVERSITAT DE BARCELONA (SPAIN) [email protected]

MOLINA PEREZ SILVIA AGUAS DEL HUESNA, SL (SPAIN) [email protected]

MORALES RODRIGUEZ MIRIAM UNIVERSIDAD DE LA LAGUNA (SPAIN) [email protected]

GESTAL MATEO CAMINO INSTITUTO DE INVESTIGACIONES MARINAS (SPAIN) [email protected]

ARANGUREN RUIZ RAQUEL INSTITUTO DE INVESTIGACIONES MARINAS (SPAIN) [email protected]

CHATZINOTAS ANTONIS UFZ-HELMHOLTZ CENTRE FOR ENVIRONMENTAL RESEARCH (GERMANY) [email protected]

VILLALOBO EDUARDO UNIVERSIDAD DE SEVILLA (SPAIN) [email protected]

TORRES RUEDA ANTONIO UNIVERSIDAD DE SEVILLA (SPAIN) [email protected]

CALVO RUIZ PURIFICACIÓN UNIVERSIDAD DE SEVILLA (SPAIN) [email protected]

PALOMINO GUTIÉRREZ CARMEN UNIVERSIDAD DE SEVILLA (SPAIN) [email protected]

GARCÍA LÓPEZ ERNESTO CENTRO DE INVESTIGACIONES BIOLÓGICAS (SPAIN) [email protected]

RUGER HERREROS CARMEN UNIVERSIDAD DE SEVILLA (SPAIN) [email protected]

ALCÁZAR LIMONES FERMÍN UNIVERSIDAD DE SEVILLA (SPAIN) [email protected]

HERNÁNDEZ PIÑERO SARA UNIVERSIDAD DE SEVILLA (SPAIN) [email protected]

BAENA SAAVEDRA ANTONIO UNIVERSIDAD DE SEVILLA (SPAIN) [email protected]

LÓPEZ GARCÍA ANTONIO A. UNIVERSIDAD DE SEVILLA (SPAIN) [email protected]

MOLERO PAYÁN RAFAEL UNIVERSIDAD DE SEVILLA (SPAIN) [email protected]

SERRANO BUENO GLORIA INSTITUTO DE BIOQUÍMICA VEGETAL Y FOTOSÍNTESIS (SPAIN) [email protected]

RUIZ PÉREZ LUIS MIGUEL INSTITUTO DE PARASITOLOGÍA Y BIOMEDICINA LÓPEZ-NEYRA (SPAIN) [email protected]

FSFCP, Sevilla '08

64

OUR GRATITUDE TO ALL THAT HAVE SUPPORTED THE CONGRESS

OUR GRATITUDE ALSO TO OUR SOCITIES

initial presentation of the congress

Documents