XXVIII
Congresso Nazionale della Società Italiana di Protistologia ONLUS
GDRE
Meeting Paramecium and its Symbionts, II Holospora Conference
CINAR PATHOBACTER
Kick off meeting
LA LIMONAIA
Pisa (PI), 2 to 8 September 2010
Symposium: Biologically Active Compounds and Protists
New Methodologies in the Investigation of Natural Products. GRAZIANO
GUELLA. Department of Physics and Interdepartmental Centre of Integrative
Biology, University of Trento, I-38050 Povo, (TN), Italy.
The enormous biodiversity developed by Nature during several billion years of
evolution has offered a wealth of chemically diverse compounds (molecular
diversity) that have been selected to modulate biochemical pathways. Although the
challenge of metabolites discovery had been tackled from more than one century, the
modern development of new analytical methodologies allows now the natural
product chemists to cope with the topic from several viewpoints and with better
perspectives than before. Analysis of metabolites has been historically limited to
relatively small numbers of target analytes being, generally, complicated by the
number of analytes, their diversity, and dynamic ranges but today “metabolomic”
approaches to complex sample of natural origin can be carried out through modern
chromatographic methods (mainly HPLC, High Pressure Liquid Chromatography),
spectroscopic techniques (especially Nuclear Magnetic Resonance and Mass
Spectroscopy), synthetic procedures (partial and/or total synthesis of natural
products) and computational calculations (molecular and quantum mechanics). As
metabolites represent the final downstream products of gene transcription, changes in
the metabolome are amplified relative to changes in the transcriptome and the
proteome, and their analysis can provide different but complementary information
about biological function as compared to transcription or expression profiles. The
significant advances that have been made in recent years in the area of technology
development for “metabolomics applications” will be reported together with a
general discussion on the several aspects which currently represent the main
bottlenecks in the field such as our limited ability to identify many of the
components or the lack of known reference compounds.
Structures, Biological Activities and Phylogenetic Significance of Natural Products
from Marine Ciliates of the Genus Euplotes. GRAZIANO DI GIUSEPPE1,
GRAZIANO GUELLA2, FERNANDO DINI1. 1Dipartimento di Biologia; Università
di Pisa, I-56126 Pisa, Italy. 2Dipartimento di Fisica; Università di Trento, I-38050
Povo (TN), Italy.
Cell cultures of the marine ciliates comprising the genus Euplotes have resulted in
the isolation of several classes of terpenoids, including the euplotins, raikovenals,
rarisetenolides, focardins, and vannusals. These terpenoids, whose complex structures
have been elucidated, display activities against other ciliates competing for space and
resources and also possess interesting biological properties such as apoptotic activity.
Generally, the lipophilic nature of these compounds indicates that their principal
targets are likely to be cell membranes, wherein they could play a role in
chemiosmotic control. In order to compare secondary metabolite production with
phylogenetic relationships among Euplotes species, SSUrRNA gene sequences of
representatives belonging to the different marine species were determined and used for
phylogenetic reconstructions. The secondary metabolite profile of ciliates presents an
intraspecific distribution of secondary metabolite combinations which vary among
different population groups. It does not, however, affect the characteristics of a given
morphospecies, thus ensuring the taxonomic value of the families of secondary
metabolites at this level. Moreover, the high number of strains genetically analyzed for
each morphospecies indicates the existence of an internal genetic variability that
undermines the general assumption that morphospecies equate to an evolutionary unit.
Interestingly, these genetic intra-morphospecific groups have their exact counterpart in
the outcome of the chemotaxonomic approach. This analysis shows that different
strains belonging to the same morphospecies but grouped in different genetic clades
are characterized by a different profile of secondary metabolites, both qualitatively and
quantitatively.
Cytotoxic Compounds of the Ciliate Coleps hirtus from Frasassi Caves. F.
BUONANNO1, S. KUMAR2, B. CHANDRAMOHAN2, D. BHARTI2, A. LA
TERZA2, C. ORTENZI1. 1Dipartimento di Scienze dell’educazione e della
formazione, Università di Macerata,I-62100 Macerata, Italy. 2Scuola di Scienze
Ambientali, Università di Camerino, I-62032 Camerino (MC), Italy.
During a survey on the protozoan community present into two main lakes of the
Frasassi caves, “Lago Verde” and “Lago Claudia”, three ciliate species have been
collected and identified by means of both morphological and molecular analyses:
Coleps hirtus, Euplotes aediculatus, and Urocentrum sp.. The first two species were
gradually adapted to growth under the laboratory conditions, however, we were
unable to culture Urocentrum sp. that seem to be strictly adapted to the peculiar
aquatic habitat of the caves characterized by stable temperature, absence of light and
sulfidic (H2S-rich) water. Since a number of ciliates have been shown to be able to
synthesize a large variety of bioactive molecules such as, water soluble cell signaling
proteins (i.e. pheromones) and/or various toxins (secondary metabolites) which are
mainly used for chemical defence against predators, we assayed the capability of the
identified protozoan species to produce (and secrete) such molecules. In particular,
our attention was focused on C. hirtus which was observed to be able to ward off
potential predators by discharging ethanol-soluble compound/s that was/were toxic
for various organisms. In the present study we report some preliminary results
concerning the evaluation of the cytotoxic activity of whole cell ethanolic extract
(EE) obtained from mass cultures of C. hirtus on a panel of seven common species
of free-living freshwater ciliates: Blepharisma japonicum, Climacostomum virens,
Euplotes aediculatus, Oxytricha sp., Paramecium tetraurelia, Spirostomum
ambiguum, Spirostomum teres. Cells were exposed to increasing concentrations of
EE (2-200 μg/ml) at both 1 h and 24 h, and the number of viable cells was
determined by light microscopy. The results indicated that the EE always
significantly affected cell viability, with S. ambiguum, S. teres, P. tetraurelia,
Oxytricha sp., and E. aediculatus showing maximum sensitivity (6.04 µg/ml < LC50
< 33.78 µg/ml), and B. japonicum and C. virens resulting more resistant (87.26
µg/ml < LC50 < 92.25 µg/ml). EE was also fractionated by HPLC on a RP-C18
column where cytotoxic activity resulted associated with compound/s eluted in three
main peaks that will be further characterized by mass spectrometry.
Development of a Laboratory Test for the Identification of Pesticides in Food
Destined for Infants: Assessment of Neurotoxic Effects. A. AMAROLI1, M.G.
ALUIGI2, C. FALUGI2 , M.G. CHESSA1. 1DIPTERIS, Corso Europa 26, 2DIBIO,
Viale Benedetto XV 5, Università degli Studi di Genova, I-16132, Genova, Italy.
One of the most common causes of human illness is pollution, and that associated
with food is one of the most serious. It has been noted for some time, even in
industrialised countries, that food destined for the most vulnerable individuals,
infants and the aged, contains ever increasing quantities of pesticide residues. This
phenomenon is linked to the increased use of these compounds over the last forty
years, up from 0.49 kg per hectare in 1961 to 2 kg in 2004, with the consequence
that these substances are found in the diet of infants, characterised by homogenised
foods, fruit juice, powdered milk, and fresh vegetables (Akland et al., 2000; Berry,
1997; Thomas et al., 1997).The situation seems even more alarming if one refers to
the publications of the World Health Organisation, which document how their
immature organs and detoxification mechanisms make infants particularly sensitive
to exposure to chemical products (Curl et al., 2003).Given these circumstances, we
have developed a laboratory test called PEST Test, based on toxicity biomarkers and
endpoints, to determine the toxic potential of anticholinesterase neurotoxic pesticide
residues in food destined for infants and the relationship between the toxic potential
and possible effects on neurogenesis. In this work the effects of the neurotoxic
organophosphate pesticide chlorpyrifos (CPF) have been studied considering the
literature concentrations on the biological models Dictyostelium discoideum,
Paracentrotus lividus, and Cellule NTera2, as they are compatible with the 3Rs
strategy (Replace, Reduce, Refine of animal experiments) (Russel and Burch,
1959).Our results have revealed that developing organisms are particularly sensitive
to the toxic effects of CPF. In the PEST Test we have developed the measurement of
the cholinesterase inhibition of D. discoideum provides an immediate evaluation of
the presence of pesticides destined for infants and, on the basis of the percentage of
inhibition of its enzymatic activity, the consequence of ingestion on the developing
organism. This research has been funded by the Fondazione CARIGE, 2009-2010
Organic Matter Recycling in a Beach Environment Influenced by Sunscreen
Products and Increased Inorganic Nutrient Supply (Sturla, Ligurian Sea, NW
Mediterranean). FRANCESCA TRIELLI, CRISTINA MISIC, ANABELLA
COVAZZI HARRIAGUE. DIP. TE. RIS., University of Genoa, I-16132 Genoa,
Italy.
The biogeochemical processes on Ligurian beaches are known to closely link the
sediment and the seawater. The intrusion of polluted seawater into the sediment,
together with the input coming from the land, could be a threat to these beach
communities, which are characterised by almost continuous anthropogenic pressure.
Apart from classic chemical pollutants, urban input is known to cause unbalanced
increases in nutrients such as nitrates and phosphates; furthermore, the utilisation of
cosmetic sunscreen products is reaching unexpected levels, thus assuming a
potentially important role in environmental contamination, and very scarce
information has been provided on the effects on the marine environment. Therefore,
the effects of cosmetic sunscreen-products and of increases in inorganic nutrients
(nitrate and phosphate) on organic matter recycling and on the microbial food web
were investigated. A short-term laboratory experiment (17 days duration) was
carried out on microsystems consisting of sediments and seawater collected from the
swash zone of a Ligurian city beach (Sturla). The results revealed that an increase in
micropollutants caused only a transient alteration in the OM recycling processes in
the seawater, while the sedimentary processes followed different pathways in the
different systems, although starting from the same condition. Surprisingly, an
increase in inorganic nutrients did not lead to an increase in the primary biomass or
to significantly higher bacterial abundance, while the sunscreen caused increased
OM recycling, especially devoted to protein and lipid mobilisation, supporting a
growing microbial community. Toxicity tests performed on Euplotes crassus, a
common interstitial protozoan, showed that the protozoa decreased viability
favoured microautotrophic and bacterial increases by reducing the top-down
pressure. The sunscreen, therefore, seemed able to modify the microbial trophic
chain, leading to an unbalanced proliferation of the microbial communities.
Moreover, the reduction in the protozoa abundance could have significant
repercussions on the transfer of energy and material to higher trophic levels.
Analysis of the Performance of an Activated Sludge Process and of the Microbial
Community of its Digestion Tank after Ozone Treatment. LETIZIA MODEO1,
ANNALISA TIEZZI1, CAROLINA CHIELLINI1, CLAUDIA VANNINI1, EMILIO
D’AMATO2, RICCARDO GORI2, CLAUDIO LUBELLO2, GIULIO PETRONI1.
1
Unità di Protistologia-Zoologia, Dipartimento di Biologia, Università di Pisa, I56126, Pisa; 2DICEA, Università di Firenze, I-50139, Firenze, Italy.
Biological treatment with activated sludge (AS) is by far the most used process
for municipal and industrial wastewater. Residue of AS process is termed excess
sludge, whose treatment and disposal is a very important issue both from the
environmental and economic point of view. Sludge ozonation process (SOP) has
been recently applied to reduce excess sludge: ozone has strong cell lytic activity and
can kill the sludge microorganisms and further oxidize the organic substances release
from the cells. SOP acts through a sequence of decomposition processes:
disintegration of suspended solids, solubilization of solids (cells), and mineralization
of organic soluble matter released from microbial cells. A portion of the sludge in
the process reactor is continuously withdrawn, ozonated, and recirculated in the
reactor: a part of the recirculated ozone-treated sludge is oxidized to CO2 while the
rest is turned into new microbial cells; this is known as “lysis-cryptic growth”
process. Sludge matrix includes bacteria, protozoa and metazoans, all of which
contribute to activated sludge function with different growth patterns and rates, and
dependence on environmental conditions. The present study concerns the effects of
ozonation of the aerobic digestion reactor in a WWTP in Sabaudia (LT, Italy). An
analysis of the WWTP performance and of the microbial community associated with
the AS process in the digestion reactor when some sludge is ozonated and
recirculated was carried out by sampling in the digestion tank. As the WWTP
consists of two parallel treatment lines for both wastewater treatment and sludge
treatment, one reactor was used as control (excess sludge was periodically
withdrawn). Concerning ozonated reactor, a volume of sludge equal to that of the
digestion tank was daily withdrawn from the reactor, ozonated, and recirculated in
the reactor at different dosages/periods. Repeatedly collected samples provided
chemical/physical data to estimate excess sludge reduction; bacterial and protozoan
populations were analyzed by means of T-RFLP; microfauna structure was evaluated
through in vivo DIC. Preliminary results of the investigation are presented.
Babesiosis: an Emergent Zoonosis. ALESSANDRA TORINA. Centro Nazionale di
Referenza per Anaplasma, Babesia, Rickettsia e Theilera Istituto Zooprofilattico
Sperimentale della Sicilia, Palermo, Italy.
Babesiosis is caused by intraeritrocytic parasites of genus Babesia and it’s
becoming one of the most interesting emerging zoonosis. Babesia pathogens are
transmitted by tick bite and affect a wide range of animals, causing severe symptoms
related to the massive destruction of red blood cells. The tick genre involved in
Babesia transmission is Ixodes. Occasionally the disease can be transmitted to
humans by the tick bite, by blood transfusion or by trans-placental route. Often the
more severe symptoms were due to a general host immunodeficiency (both natural
and acquired) and the higher number of susceptible hosts is becoming one of the
causes of the disease increasing. The species mainly involved in zoononoses are
Babesia divergens and Babesia microti. Other Babesia species, although less spread,
are cause of human babesioses: WA1 (B. duncani), EU1, MO1 and CA1. Their
classification is not well defined, and maybe they could be strains of the same
species. Interestingly, in Europe the etiological agent responsible of Babesiosis is
mainly B. divergens, while in America is B. microti. This is probably due to the
different diffusion of their specific vectors. In humans babesiosis causes anemia,
fatigue, headache and high fever. The cases ascribed to B. divergens in Europe are
more severe than the ones caused by B. microti in America. In the host immune
response take part both humoral and cellular components, but the g-Interferon
produced by Natural Killer cells seems to have the major role in the parasite growth
control. Unfortunately, human babesiosis is not spread enough to invest money in
the vaccine development, but many results could be obtained by the research on a
vaccine against animal babesiosis. Many studies performed by the National Centre
of Reference to Anaplasma, Babesia, Rickettsia and Theileria, demonstrate that B.
microti was not only detected in Italy, but it is present in non-conventional animals
like cats, that could act as alternative reservoir of the parasite. Furthermore, an
outbreak in cattle caused by B. divergens was detected in Veneto region. It can be
concluded that babesiosis is becoming an emergent zoonosis in Europe, but also in
Italy, and that a good surveillance plan should be carried out to take under control
the parasite and its vector.
Genotyping of Toxoplasma Strains Reveals an Environmental Route to this
Protozoan Infection. M.C. ANGELICI1, C. GIULIANI1, P. DI PINTO2, M.
PUGLIESE1, V. TERIO2, A. DI PINTO2, A. VIMERCATI3, E. MONTEDURO3, G.
M. TANTILLO2. 1 Dipartimento Ambiente e connessa Prevenzione Primaria, Istituto
Superiore di Sanità, Roma, Italy; 2 Dipartimento di Sanità e Benessere degli Animali,
Settore ispezione degli alimenti Facoltà di Medicina Veterinaria, Università di Bari,
Italy; 3 II Clinica Ostetrica del Policlinico, Università di Bari, Italy.
Toxoplasma gondii life-cycle is intricated by the presence of three vital stages
with different biological and evolutionary significance. Oocysts deriving from sexual
stage of parasite spread by cat with feces in environment and corresponding to the
infectious stage of parasite. Human may be infected by ingestion of oocysts
potentially spread in all kinds of environments where being felines and their prey.
Marine and fresh water environments can be contaminated by rivers and waterways
where Toxoplasma cycle is maintained by prey-predator mechanism. We have
demonstrated the presence of Toxoplasma DNA in shellfish hepatopancreas probably
because these animals are able to retain microorganisms by concentration after
filtering a large volume of water. The bivalves were collected in different breeding
areas in the Adriatic shore belonging to the Apulia region in Italy and this is the first
detection of T. gondii DNA in Italian mussels. This result allowed us to carry out a
genotyping of Toxoplasma strains coming from shellfishes, through a PCR-RFLP
analysis, to compare with the genotypes founded during a previous study on
congenital transmission in this Region. The Toxoplasma genotype we founded in the
analyzed amniotic fluids and shellfishes coming from Apulia Region are the same
and belonging only to the type II, the most common agent of congenital
toxoplasmosis. This study suggest that shellfishes could be a risk for toxoplasmosis,
especially during the pregnancy, and that this is further amplified by the eating habits
in South Italy, chiefly in the Apulia Region, where there is large consumption of raw
or undercooked fish, including bivalve molluscs.
Investigation on Susceptibility to Toxoplasma Infection by anHLA-B27 Family
Allele in Italy. ELEONORA SCIMIA, MARIA CRISTINA ANGELICI.
Dipartimento Ambiente e connessa Prevenzione Primaria, Istituto Superiore di
Sanità, Roma, Italy.
The protozoan Toxoplasma gondii is the etiologic agent of the toxoplasmosis.
This infection is very common in Europe where it is present with strains belonging
to different genotypes. The parasite polymorphism shows a different trend from
Nord to South Europe. In Italy, like in Spain and Portugal, it was found a higher
degree of variability then in the rest of Europe. This geographical area corresponds
to the distribution of a very rare allele of the B27 family of the human leukocyte
antigen system (HLAB2702). This study approach is to screen blood samples
coming from pregnant women previously enrolled in a prenatal study for congenital
toxoplasmosis in Italy. These samples derive from two different study groups of
pregnancies: seroconversions without transmission or with proved transmission to
the fetus. Last group provided us with different Toxoplasma genotypes isolated in
Italy. Our research purpose is to investigate on the possible correlation between the
frequencies of this HLA allele and the Toxoplasma infection, targeting a possible
role of the immunity response on the host susceptibility. Furthermore we study the
possible involvement of this allele in the host-parasite relationship in terms of
selection of the Toxoplasma genotype. Analytical method we use is essentially
typing techniques by PCR-RFLP. To identify the sequence of HLA-B2702 we first
carry out amplification of HLA-B27 exon 2 and subsequent nested-PCR with
internal primers. Nevertheless with this approach we could not discriminate between
HLA-B2702 and B2730/B3816 alleles. A further analysis of the amplified exon 2 by
the restriction enzyme SspI and a PCR-RFLP analysis exon 3 by specific primers
and XcmI enzyme allows to identify the HLAB2702. At the moment the results we
obtained are preliminary and do not show a direct correlation between the frequency
of HLA-B2702 and the Toxoplasma infection but a larger number of samples are
under study.
An Overview among Apicomplexa Apical Membrane Antigens-1. ALESSANDRA
TORINA1, ANNALISA AGNONE2, VALERIA BLANDA1, SANTO
CARACAPPA1. 1Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy;
2
Dipartimento di Biopatologia e Biotecnologie Mediche e Forensi, Università di
Palermo, Italy.
The Phylum Apicomplexa contains a number of organisms, some of which
parasitize human or animal species. Phylogenetic studies have demonstrated a
common evolutionary origin, and this aspect reflects in the sharing of key genes for
their surviving. With the aim to add a further step in the vaccine development, some
of them have been extensively studied. Apical Membrane Antigen-1 is a superfamily
of factors found in many protists which act as key factors in the interaction between
the parasite and the host red blood cells (RBCs). They are type-I trans-membran
proteins and they seems very promising as vaccine candidates. Hereby authors
would like to present an overview on AMA-1 and its role in host red blood cells
invasion, focusing on the newly identified AMA-1 of Babesia bigemina (BbigAMA1). AMA-1 from many Babesia parasites (B. bigemina, B. bovis, B. gibsoni and B.
divergens) as well as from Plasmodium (P. falciparum and P. knowlesi), Theileria
(T. annulata and T. parva), and Toxoplasma gondii have been analyzed and
compared. The cladogram obtained by ClustalWanalysis showed a common
phylogenetic origin of the proteins. Interestingly all of them, with the exception of
Theileria species, have a signal peptide. Although the percentage of similarity are
higher among species then among genera, and the predicted tree-dimensional
structures of all the antigens are very similar. Babesia bigemina AMA-1 has been
recently identified. The sharing of specific features as a signal peptide and a transmembrane domain include it in AMA-1 superfamily. In contrast with Plasmodium
falciparum antigen, its sequence resulted to be very conserved among strains, both
geographically near and distant. In other Apicomplexa the characterization of AMA1 has been already performed and its role in host RBCs demonstrated. BbigAMA-1
shares with other Apicomplexa key Cystein residues that were predicted to form
disulfide bonds. The predicted structure homology between BbigAMA-1 and already
identified antigens puts the bases to consider this newly identified protein as a good
babesial vaccine candidate.
Symposium: Biodiversity, Ecology, Morphology and Phylogeny of Protists
Ciliate Systematics: is there a Role for Morphology in the Age of Molecules? ALAN
WARREN. Department of Zoology, Natural History Museum, Cromwell Road,
London, SW7 5BD, UK.
After many decades of relative stability, ciliate systematics has been
revolutionised in the last 30 years. This was initially as a result of ultrastructural
studies, but increasingly from the burgeoning volumes of molecular data. This begs
the question whether morphology still has a significant role in ciliate systematics. In
this talk I will present the case for the continuing importance of morphology using
examples from two recently published studies that investigate respectively the
systematics of: (1) the peritrichs, and; (2) the nassophoreans and phyllopharyngeans.
On the Evolution of Stichotrichia (Ciliophora, Spirotrichea). THIAGO DA SILVA
PAIVA¹, BÁRBARA DO NASCIMENTO BORGES², MARIA LÚCIA HARADA²,
INÁCIO DOMINGOS DA SILVA-NETO¹. ¹Laboratório de Protistologia, Dept. de
Zoologia, Inst. de Biologia, CCS, Universidade Federal do Rio de Janeiro, Brasil;
²Laboratório de Biologia Molecular “Francisco Mauro Salzano”, Universidade
Federal doPará, Brasil.
The systematics of Stichotrichia is generally regarded to be one of the most
confused subjects in ciliate biology, given their complex morphology and the
contradictory phylogenetic hypotheses generated from different data and criteria. We
compare the results of two recent works from our group, which investigate this
subject from two different approaches, focusing on their implications to the
understanding the evolution of the stichotrichs. One, based on phylogenetic analyses
of the 18S-rDNA marker (Paiva et al., 2009 Genet. Mol. Res., 8: 233-246), and the
other on phylogenetic analyses of 135 morphologic characters (Paiva, 2009,
Thesis).The phylogenetic patterns recovered by both kinds of data are consistent in
placing the pseudoamphisiellids and discocephalids as early divergedlineages, but
exhibit mild to large inconsistencies related to the placement of most deep
divergences within the core Stichotrichia. Such deep-diverged lineages usually have
lower statistical support than most shallow nodes, and were sensitive to analytic
criteria and parameter variation (e.g. distance vs. character-based analyses;
Goloboff’s K in implied-weights parsimony). Remarkably, both 18S and
morphological data provided phylogenetic hypotheses with high retention indices,
albeit their data matrices exhibited large amounts of incongruence. Hence, most
analyzed information was to some extent retained as synapomorphies, but mostly
homoplastic due convergences and reversals. This scenario indicates a mosaic
evolution of morphologic features in Stichotrichia. If contextualized with the slowevolving 18S marker (in comparison to the related euplotids), which yields short
branch lengths for most deep divergences within the core Stichotrichia, then it
permits to suppose a rapid radiation of such lineages.
Phylogenetic Study of Urostylid Ciliates (Ciliophora, Urostylida) Based on 18SrDNA Marker. BÁRBARA DO NASCIMENTO BORGES², THIAGO DA SILVA
PAIVA¹, MARIA LÚCIA HARADA², INÁCIO DOMINGOS DA SILVA-NETO¹.
1
Laboratório de Protistologia, Dept. de Zoologia, Inst. de Biologia, CCS,
Universidade Federal do Rio de Janeiro, Brasil; 2Laboratório de Biologia Molecular
“Francisco Mauro Salzano”, Universidade Federal doParà, Brasil.
Urostylid stichotrichs are flexible-body hypotrichous ciliates which present a
mid-ventral complex of paired ventral cirri producing a conspicuous zigzag pattern.
The 18S-rDNA marker was shown to contain enough phylogenetic signal to resolve
the internal relationships of the so called “core urostylids” with relative stability and
high statistical support (Paiva et al., 2009 Genet. Mol. Res., 8: 233-246). Thus, we
here in hypothesize the phylogeny of six urostylid 18S sequences (Caudiholosticha
sylvatica, Hemycicliostyla sphagni, Nothoholosticha sp., Pseudokeronopsis sp.,
Pseudourostyla levis, and a novel [undescribed] urostylid genus) sampled from
Brazilian locations. The DNA was isolated using phenolchloroform method and
the18S gene was isolated via PCR using specific primers. The obtained sequences
were aligned with other from literature using ClustalX implemented in BioEdit
software and the resulted database composed of 113 stichotrich sequences and 891
characters was submitted to phylogenetics analysis using neighbor-joining method
and bootstrap with 1000 replicates as node support using Paup*4b10 software. The
six new sequences group within the core urostylids, which generally exhibited high
support, thus corroborating the literature. C. sylvatica grouped as sister taxon of P.
levis + P. cristata, which were distantly placed from the congener P. franzi. This last
formed a monophylum with H. sphagni, thus suggesting the polyphyly of genus
Pseudourostyla. Notoholosticha spp.formed a consistent monophylum, which was
placed within the cluster of Pseudokeronopsis. As this cluster has strongly (<90%)
supported nodes, it is possible to suppose Notoholosticha is a pseudokeronopsid with
a reduced bicorona. Other urostylid lineages, such as those of uroleptids, Holosticha
and Parabirojimia, departed from other stichotrich nodes, corroborating previous
18S analyses. Interestingly, the branch lengths in the core urostylids are longer than
the observed in most unstable stichotrich nodes groups. This feature is consistent
with the literature, and may suggest a difference in the 18S evolution rate.
Distribution and Abundance of Planktonic Ciliates in Chilka Lake, a Brackish
Water Lagoon on the East Coast of India. C.KALAVATI1, A.V.RAMAN1,
SATEESH NANDURI1, M. RAKHESH1, B.R. SUBRAMANIAN2 1Marine
Biological Laboratory, Department of Zoology, Andhra University,
Visakhapatnam, 2ICMAM Directorate, Ministry of Earth Sciences, NIOT Campus,
Chennai, India.
A one-year study (Jan-Dec 2005) on the planktonic ciliates over a spread of 36
hydrographically differing GPS fixed locations in Chilka Lake, Asia’s largest
brackish water lagoon on the east coast of India, revealed altogether 55 species of
free-living ciliates represented by 7 groups namely, Karyorelicta (2), Spirotrichea
(23), Litostomatea (11), Phyllopharyngea (4), Nassophorea (9), Oligohymenophora
(5) and Colpodea (1). The predominant taxa were Moneuplotes vannus (26.85%),
Prorodon marinus (13.89%) and Halteria sp. (14.10%) constituting up to 50% of the
total ciliate population numerically. In general, the ciliate community consisted of
marine stenohaline components such as Coleps sp., Uronema filificum, Dysteria sp.,
Stentor sp., in the southern part of the Lake (mean salinity 13.9±0.36 PSU); marine
euryhaline species such as Halteria sp., Paramecium sp. Tontonisa sp. and
Holophrya nairi in the central sector (mean salinity 6.45±1.05 PSU); marine
euryhaline species such as Moneuplotes vannus, Halteria sp., and Prorodon marinus
in the north sector (mean salinity 0.8±0.13 PSU) subjected to considerable dilution
through freshwater influx. Trachelocerca phoenicopterus, Prorodon discolor,
Euplotes patella and Coleps sp. were typical of the channel locations (mean salinity
14.85±4.07 PSU). Species mean abundance varied from a minimum of 3 nos.ml-1 to
671 nos.ml-1. There were 14 characterizing species and 28 accidental or rare species
that occurred only sporadically depending on the season and locality. On the basis of
multivariate analysis (hierarchical clustering implemented in PRIMER) using ciliate
abundance data, it was possible to distinguish the 36 locations into 4 clusters/groups.
Group-1 consisted of stations representing the south sector i.e., Rambha Bay (sts. 116); Group-2 the central sector (sts.17-21, 23-25 & 28) and the channel (sts. 34-36)
and Group-3 the northern sector (sts. 22, 26, 27 & 29-33). There was a great measure
of difference in the composition, seasonal succession and numerical abundance of
ciliate populations among the groups/regions examined (ANOSIM, Global R: 0.944
at 0.1%). BioEnv revealed that a combination of salinity, turbidity, pH, total nitrogen
and total phosphorus proved significant (r=0.878) structuring the protozooplankton
abundance.
Symposium: “Nobili - Franceschi” Award
Tubulin Isotypes Contribute to the Diversity of Microtubule Functions in Euplotes
focardii. DANIELA SPARVOLI, PATRIZIA BALLARINI, SANDRA
PUCCIARELLI, SABRINA BARCHETTA, CRISTINA MICELI. Department of
Molecular, Cellular, Animal Biology, University of Camerino, I-62032, Camerino
(MC), Italy.
Ciliated protozoa represent optimal model organisms for studying cell motility
and microtubule-mediated cellular processes, as they are able to assemble 17 diverse
microtubular structures in a single cell. By the genome sequence of ciliates, it
appeared that they possess numerous alpha- and beta-tubulin isotypes that,
differently from the old general view, may have different cell functions. From
several years, we have been involved in the study of tubulins in Euplotes focardii, a
cold adapted ciliate isolated from Antarctica. One, four and two isotypes of alpha-,
beta- and gamma-tubulins were identified, respectively. Among the beta-tubulin
isotypes, the beta-T3 revealed specific characteristics. Beta-T3 antibodies localized
this isotype preferentially at the basis of the cilia and in basal bodies. By iRNA, we
discovered that the silencing of this gene in deciliated cells inhibits cilia reformation,
suggesting that this isotype is necessary for cilia regeneration. Furthermore, the
Euplotes beta-T3 isotype, expressed as fusion protein with GFP in the murine
fibroblast cell line NIH/3T3 localized only at centrosomes, primary cilium, mitotic
spindle and midbody, i.e. microtubule with low turn-over. Since the other E. focardii
isotypes expressed in NIH/3T3 cells fused to GFP do not show the same behaviour,
but co-assemble in cytoplasmic microtubules, we may assume that the beta-T3
behaviour is due to specific characteristics of its sequence and/or conformation that
may confer stability to the microtubular structures including the axoneme.
Transfecting a chimeric construct in NIH/3T3 cells, we also demonstrated that the
amino-terminal domain and not the carboxyl-terminal is responsible of the specific
characteristics of the beta-T3 isotype. By experiments of site-directed mutagenesis,
we showed that the mutation of Ser239 in Cys (present at position 239 in other
isotypes) alters the specific localization properties of the beta-T3 isotype. Further
mutagenesis in our model system are attempted to provide additional insights in the
characterization of the molecular properties responsible for the specific localization
and function of the tubulin isotypes. Moreover, a comparative genomic analysis with
the tubulin isotypes identified in E. crassus, closely related to E. focardii but not
cold adapted, will give us information about the relation between the specific
localization of E. focardii tubulin isotypes and the cold adaptation of this ciliate.
Structure of Euplotes raikovi Pheromone Genes. ANNALISA CANDELORI,
FRANCESCA RICCI, ADRIANA VALLESI. Dipartimento di Scienze Ambientali,
Università di Camerino, I-62032, Camerino (MC), Italy.
In some species of Euplotes, such as E. raikovi, E. nobilii and E. octocarinatus, it
was extensively studied the structure and mechanism of action of the cell typespecific, water-borne signalling proteins (pheromones) that these species evolved to
regulate their switching between the sexual and vegetative stages of the life cycle.
However, little is known about the organization and expression of the genes
encoding these pheromones at level of the transcriptionally active macronuclear
genome. Given this context, we directed our work to clone and characterize the fulllength macronuclear pheromone genes from different mating types of E. raikovi. A
comparative analysis of four complete pheromone-gene sequences specific for
closely homologous pheromones shows that these sequences carry marked variations
only at level of their coding regions. In contrast, the sequences of their 5’ and 3’
non-coding regions appear to be strictly conserved; the 5’ region at an apparently
higher level that the 3’ region. This conservation implies that these regions play a
common, crucial role in the mechanism of expression of the E. raikovi pheromone
genes.
Random Mutagenesis of the Uunicellular Green Alga Chlamydomonas reinhardtii
and Isolation of a gun4 Mutant Impaired in Chlorophyll Biosynthesis and Plastid-tonucleusretrograde Signalling. CINZIA FORMIGHIERI1, MAURO CEOL2,
MANUELA MANTELLI1, JEAN-DAVID ROCHAIX2, ROBERTO BASSI1.
1
Dipartimento di Biotecnologie, University of Verona, Verona, Italy; 2Département
de Biologie Moléculaire, Université de Genève.
Chlamydomonas reinhardtii is widely studied as model organism. All the three
genomes (from nucleus, chloroplast, and mitochondria) are sequenced and
techniques are available for their transformation, thus allowing genetic improvement.
Mutagenesis is a tool to generate strains with improved characteristics with respect
to wt for specific biotechnological application. Moreover it constitutes powerful
approach to understand the physiological role of genes through the analysis of
effects of their deletion on phenotype. Although insertional mutagenesis of the
nuclear genome occurs randomly, it’s the only strategy to obtain stable mutants for
the majority of genes. We generated a nuclear insertion library of C. r. and among
the screened strains we isolated mutants with reduced chlorophyll content per cell.
Characterization of ‘pale green’ helps understanding the regulation of chlorophyll
biosynthesis in algae and to investigate key regulatory factors. Moreover,
chlorophyll biosynthesis in the chloroplast is strictly coordinated with nuclear
expression of chlorophyll-binding proteins and components of the tetrapyrrole
metabolism could be involved in plastid to-nucleus retrograde signaling. An
interesting mutant displays a 50% reduction in chlorophyll with respect to wt upon
knock-out of the gun4 gene.GUN4 is a regulatory subunit of Mg-chelatase, affecting
accumulation of chlorophyll during normal growth conditions. We found that in the
gun4mutant nuclear lhc transcription is no more coordinated with the rate of
chlorophyll synthesis. Nevertheless, accumulation of Lhc proteins is restricted posttranscriptionally due to limited chlorophyll availability, that ultimately has important
effects on the organization of the photosynthetic apparatus. gun4 genes are present in
all oxygenic photosynthetic organisms and the physiological role is likely conserved.
However, we observed differences in C. r. gun4 with respect to Arabidopsis and
Synechocystis gun4 mutants isolated so far, enlightening the evolution of chlorophyll
biosynthesis regulation and retrosignalling.
Molecular Composition and Physiological Effects of R-bodies and Description of
New Strains of Caedibacter. STEFANO GALATI. Unità di Protistologia-Zoologia,
Dipartimento di Biologia, Università di Pisa, I-56126, Pisa, Italy.
The refractile body (R-body) is a proteinaceous ribbon typically coiled within
bacterial cells of the genus Caedibacter, endosymbionts of the ciliate Paramecium.
In Caedibacter, R-bodies are associated with a phenomenon called “killer trait”: it is
the ability of killer paramecia, those harbouring Caedibacter as symbionts, to kill
Caedibacter-free paramecia (sensitives). Released Caedibacter cells carrying an Rbody can be ingested by sensitive paramecia. After ingestion, R-bodies extrude in a
telescopic fashion, disrupt bacterium and food vacuole of sensitive paramecia which
then die. The genetic determinants of R-body synthesis are encoded on
extrachromosomal elements like plasmids or phages. In C. taeniospiralis the
involved genes are rebA, rebB, rebC, and rebD. The aims of this work were: to
describe how the structural R-body components from C. taeniospiralis (RebA,
RebB, RebC) interact to constitute an R-body using SDS-Page and Western Blot; to
test the potential toxicity of recombinant Rbodies on a sensitive strain of
Paramecium through in vivo tests; to molecularly characterize new strains of
Caedibacter species in ciliates applying the full cycle rRNA approach. Biochemical
analyses revealed as major components of recombinant R-bodies RebB and RebA,
while RebC is present only in a minor proportion; probably RebC carries out not
strictly structural functions. The in vivo tests confirmed the absence of toxicity from
the R-body itself and therefore support the hypothesis that it is acting as releasing
vehicle for an unidentified toxin finally responsible for the sensitive Paramecium
death. Although the killer trait is well known since decades and it is supposed to play
an important role in the ecology of certain fresh water habitats, few Caedibacter
have been described so far. As most of these descriptions are from the 80ies,
molecular data of these endosymbionts are scarce. Therefore Paramecium strains
were screened for the presence of Caedibacter and two novel strains of C.
caryophilus were detected, one in the cytoplasm of Paramecium aurelia and the
other in the macronucleus of Spirostomum sp. This work contributes to a better
understanding of the biochemistry of the R-body and its phenotypical effects in the
killer trait and the description of new strains of Caedibacter extend our knowledge
about endosymbiont distribution, occurrence, and host specificity.
Symposium: “Maria Umberta Corrado Delmonte” Award
Benthic Foraminifera as Bioindicators of Pollution: What Can These Protists Tell
Us? A Review of the Italian Experience in the Last Two Decades. FABRIZIO
FRONTALINI, RODOLFO COCCIONI, GIUSEPPE BANCALÀ. Dipartimento di
Scienze della Terra, della Vita e dell'Ambiente, Università di Urbino, I-61029,
Urbino, Italy.
Since the 1950s, numerous studies have demonstrated the value of benthic
foraminifera in detecting ecosystem contamination. The interest in benthic
foraminifera has partly been driven by government policies and programs aimed at
developing suitable, non-invasive bioindicators of marine environmental quality.
This paper accomplishes two things: it reveals that Italian experience has
significantly contributed to the advancement of our understanding of this topic and
summarizes the most important results that have served to greatly improve our
knowledge in this field. Although many issues are still a matter of debate, since it is
difficult to separate natural vs. human-induced pollution and a foraminiferal protocol
has not yet been produced, foraminifera have been proven to be successful
candidates as part of an integrated monitoring program.
Towards an Understanding of the Evolution and Ecology of Host-symbiont
Interactions of Ciliates and their Intracellular Bacteria. MARTINA
SCHRALLHAMMER. Unità di Protistologia-Zoologia; Università di Pisa, I-56126
Pisa, Italy; Abteilung Zoologie, Universität Stuttgart, D-70569 Stuttgart, Germany.
This PhD thesis was accomplished to extend the understanding of the evolution
and ecology of ciliate-bacteria symbioses. Special focus was drawn to ciliates as
natural reservoir of pathogen-related bacteria and on the evolutionary history of
intracellular lifestyles. Evolution of ciliates’ symbionts was studied by molecular
characterization of several intracellular bacteria and their hosts. Analyses of
respective phylogenetic positions allowed conclusions about their potential coevolution with the host. In the computed phylogenetic trees, the characterized
symbionts were placed either basally or in crown position with respect to their
closest relatives, the human pathogens Francisella and Rickettsia. Endosymbionts of
ciliates follow various strategies to ensure their maintenance within host populations.
Ecological and cytological aspects of three very diverse strategies were addressed
using the examples of Polynucleobacter necessarius, Holospora caryophila and
Caedibacter caryophilus. As obligate endosymbiont of different freshwater Euplotes
species, P. necessarius is essential for its ciliate host. Its adaptation to the
intracellular lifestyle was studied by comparison of genome size and reduction with
its closest free-living relative resulting most likely in the discovery of the
evolutionary youngest obligate endosymbiosis described so far. A completely
different strategy to maintain infection of its host population is pursued by H.
caryophila. In vitro infection experiments demonstrated their ability to infect
different host species and to establish rapidly a nuclear infection. C. caryophilus
provides its Paramecium host with a selective advantage by killing uninfected
competitors – a phenomenon known as killer trait. Characterization of several strains
showed that this species is in the progress of radiating into three independent
lineages. The previously supposed narrow host specifity of these bacteria has to be
revised. C. caryophilus is capable of building the R-body, a fascinating structure,
which is of central relevance for the killer trait. An R-body consists of a protein
ribbon which is coiled inside the bacterial cell. In response to a low pH it can unroll
and form a long hollow cylinder. Here, its genetic determinants were characterized
in a second Caedibacter species. Recombinant R-bodies were expressed in
Escherichia coli and tested for functional, pH-dependent unrolling behaviour.
Specific polyclonal antibodies directed against single R-body proteins were
generated and used to analyze the protein composition of recombinant and wild type
R-bodies. Thus, the major proteins involved in the structural composition of the Rbody were identified. The obtained results are first prerequisites to address the
potential of R-bodies for to a future application as nanodevices.
Facultative Associations Between Protists and Rickettsial Symbionts: Morphological
and Molecular Characterization, Functional Implications, and Insights on Emerging
Intracellular Parasites. FILIPPO FERRANTINI. Unità di Protistologia-Zoologia,
Dipartimento di Biologia, Università di Pisa, I-56126, Pisa, Italy.
Rickettsiae and Rickettsia-like organisms (RLOs) are Gram-negative prokaryotes
known as obligate intracellular parasites of a variety of eukaryotic hosts, including
humans. RLOs have been recently reported also in protists, although specific studies
are still lacking. This work was aimed both to investigate the actual diffusion of
RLOs among protists and to get insights into their host range and host shift
capabilities. Protist-borne RLOs were detected by systematic screenings of natural
populations of ciliated protists, and characterized through “full-cycle rRNA
approach” (16S rDNA characterization and use of specifically designed
oligonucleotide probes for in situ detection), electron microscopy and phylogenetic
analysis. Six novel RLOs were found in five different ciliates, namely
Pseudomicrothorax dubius (Nassophorea), Spirostomum minus (Heterotrichea),
Euplotes octocarinatus (Spirotrichea), Paramecium multimicronucleatum
(Oligohymenophorea), and Diophrys oligothrix (Spirotrichea) which harbours two
symbionts. Phylogenetic inferences supported their belonging to the family
Rickettsiaceae as candidate novel genera, except for symbionts of S. minus and P.
multimicronucleatum that branch within the genus Rickettsia. These latter also share
motile flagella, which were never reported among Rickettsiaceae. Symbionts of E.
octocarinatus and one of that of D. oligothrix belong to the same novel bacterial
species, which was further retrieved in three unrelated ciliate hosts, suggesting a
possible host shift through horizontal transfer. Inter- and intraspecific
transmissibility of RLOs was tested using the other symbiont of D. oligotrix as
infector and uninfected conspecific and allospecific (E. harpa) ciliate strains as
hosts: horizontal host shifting has been documented at both levels. Preliminary
additional results indicate that some RLOs can survive in association with labcultured metazoan cell lines. The present work contributed to reveal an unexpected
phylogenetic and morphological diversity among ciliate-borne Rickettsiaceae. The
frequent associations between RLOs and protists, together with the retrieval of the
same bacterial species from different hosts and the reported host shift aptitude,
account for their polyxenic and likely opportunistic nature. Given the
parasitic/pathogenic features of the majority of known Rickettsiaceae, it could be
speculated the possible role of protists as natural reservoir and/or vectors for
potentially hazardous rickettsial pathogens.
Improved Growth in Photobioreactors Using Chlamydomonas reinhardtii Mutants
Selection for Reduced Antenna Size. GIULIA BONENTE1, MANUELA
MANTELLI1, CINZIA FORMIGHIERI1, TOMAS MOROSINOTTO2, ROBERTO
BASSI1. 1Dipartimento di Biotecnologie, Università di Verona, Verona, Italy;
2
Dipartimento di Biologia, Università di Padova, Padova, Italy.
Green algae are promising organisms for biofuels production, due to higher
biomass yield and oil content with respect to plants. However, the exploitation of
mass algal cultures in photobioreactor presents limitations due to adaptation of algae
to low light intensity for optimal growth in low light natural environment consisting
in a large antenna system for photosystems. The high number of chlorophylls per
Reaction Centre produces excess light absorption in surface cell layers, leading to
energy dissipation in heat and suboptimal illumination of deep layers causing energy
consumption by respiration. This reduces the overall productivity. We generated an
insertion mutant library in Chlamydomonas reinhardtii, and screened for clones with
a reduced antenna. Two lines with strongly reduced antenna and low chlorophyll
content were isolated (named az7 and u06). Phenotypical characterization shows that
line u06 differs from line az7 for a higher sensitivity to photo oxidative stress and
displays far lower productivity. Growth tests in multiple overcast layers, simulating
photobioreactors, showed that az7 productivity is strongly improved with respect to
WT in terms of biomass yield. Physiological analysis suggests that the major factor
of increased light use efficiency is a reduction in heat dissipation.
The Effects of Light Irradiation on the Respiration of Living Eukariotyc Cells: Role
of Cytochrome c Oxidase. ALESSANDRO REMEDI. Consiglio Nazionale delle
Ricerche (CNR), Istituto di Biofisica, I-56124, Pisa, Italy.
For a long time some researchers have been investigated the effects of visible-tonear-infrared (Vis-NIR) radiation on eukaryotic cells, reporting a light-induced
increase of cell proliferation parameters (Karu, 1999, 2005; Koutna et al., 2003;
Vladimirov et al., 2004; Wan-Ping Hu et al., 2007, Eells et al. 2003; Whelan et al.,
2003); on the other hand the effect of Visible light can be elicited on isolated
functional mitochondria (Kato et al., 1981;Veshkin, 1991). It has been suggested that
these effects are mediated by the mitochondrial enzyme cytochrome c oxidase,
CcOX. Our goal was to further investigate the effects of Visible irradiation and
elucidate the possible role of CcOX and the in vivo biological mechanism. We
measured O2-consumption rate during light irradiation on Tetrahymena thermophila
cells, which live in light-exposed environments, and aerobically (GLY) and
anaerobically-grown (GLU) Saccharomyces cerevisiae cells. Assay samples,
measured by a thermostatted Clark O2 electrode, were irradiated by a 1-KW Xenon
arc lamp. T. thermophila and aerobically-grown S. cerevisiae respiratory rate
suddenly increases when Visible light is turned on, going back to its previous basal
value when the light is turned off. By scanning the various bands with broadband
filters, blue was most active in eliciting the effect, albeit with different efficiencies
and a smaller but stable effect was measured with orange-red light. Dose-response
curves obtained by irradiating T. thermophila with these two spectral bands, show a
similarity between the light induced respiratory rate increase and the absorption
spectrum of purified CcOX. A lower response to irradiation with visible light was
observed in GLU S. cerevisiae with respect to GLY cells. This result was confirmed
by measures on S. cerevisiae affected by a mutation on CcOX subunity VIb. We also
conducted studies with the CcOX inhibitor NaN3: at the same concentration its
efficacy was lower in irradiated cells with respect to nonirradiated cells. Finally
dose-response curves at different T. Thermophila culture ages indicated that the
basal respiratory rate and the light-induced respiratory rate increase are connected
with the cell physiology. We show herein that cells from widely different taxa
respond to irradiation in a very similar way. These results suggest that the cellular
O2-consumingactivities involved in this phenomenon are ubiquitous and that CcOX
has a role in inducing this effect which is stimulated by light irradiation.
Study of Babesia bigemina Surface Antigens and their Potential Role for Strain
Discrimination. ANNALISA AGNONE. Università degli Studi di Palermo, Palermo,
Italy.
Babesiosis is one of the most common infections of free – living animals
worldwide. Babesia bigemina is an hemoparasite transmitted by the specific tick
vector Rhipicephalus spp. It affects cattle in tropical and subtropical regions causing
severe pathologies. Although the literature concerning bovine babesiosis is rather
wide, thanks to the work carried out by several researchers in the New Continent,
there is no knowledge so far about Italian strains of B. bigemina. The identified or
suspected molecules involved in the erythrocyte invasion are many, however in B.
bigemina very few are characterized. This work concerns some B. bigemina
antigens, that act in different steps of the B. bigemina life cycle. It would be
therefore ideal to include all of them in a subunit vaccine. Thus, in order to add a
further step in developing a vaccine against bovine babesiosis, some Babesia
bigemina surface antigens have been characterized, that can be hypothesized to be
included in a subunit vaccine. Three different surface antigens were sequenced and
analyzed, and their sequences were compared with those of Mexican, Argentinean,
and Australian strains. Gp45 acts in an early stage of host red blood cells. The gene
showed a high degree of polymorphism among Italian and foreign strains, and it has
many features that allow to use it for strain discrimination. AMA-1 is a recently
identified antigen, shared by many Apicomplexa. It conserves a high degree of
sequence homology among strains and it has a potential role in the field of vaccine
development. A peculiar behavior can be ascribed to SBP1, although its
characterization is still in progress.
The Expression Study of the Heat Shock Protein 70 Cytoplasmic Subgroup in
Tetrahymena thermophila. TING YU, SABRINA BARCHETTA, ANTONIETTA
LA TERZA, CRISTINA MICELI. Department of Molecular, Cellular, Animal
Biology, University of Camerino, I-62032, Camerino (MC), Italy.
Heat shock proteins (HSPs) are good candidates for expression regulation and
cell stress response studies due to their fast and high induction ability under various
stress conditions. Although several HSPs genes have been reported as good
phylogenetic markers in ciliates, the expression and functional studies on HSPs were
never carried out comprehensively. A complete survey on Tetrahymena thermophila
macronuclear genome database indicates the presence of total 13 genes under the
HSP70 family. Five genes with high sequence similarity cluster into one subgroup,
and the orthologs of this subgroup from other organisms are generally localized to
cytoplasm and are recognized as the major molecules in cell stress responses against
heat. We performed a series of experiments mainly by real-time PCR to characterize
the transcription profiles of this HSP70 subgroup. Our results indicate that, although
highly similar in their gene sequences, 5 genes showed very different transcription
patterns after the cells being heat-shocked with varied time and temperatures.
Among the 5 genes, HSP70-4, whose transcript was the richest among the five at
normal growth temperature, was not inducible at all heat shock conditions except a
slight (compared to its paralogs) lift at 40°C-a hyperthermic temperature, implying
its unique cellular function compared to the other inducible forms. Different trials at
36, 38, and 40°C showed dramatic increases in the transcript amount for all genes,
indicating that the transcription of this HSP70 subgroup is temperature-dependent.
Time-course analysis carried out at 38°C demonstrated that the accumulation of
transcripts of different paralogs varied through 15min to 90min: transcripts from 2
genes (HSP70-2 & HSP70-3)reached the highest level in only 15 minutes and,
unexpectedly, encountered a rapid turnover afterwards; another 2 genes (HSP70-1 &
HSP70-4) showed small or no change during all sets of exposure time; whereas the
HSP70-5 transcripts remained constant through 15min to 60 min. An inspection of
the un-translated regions of the 5 isoforms proposed a possible mechanism for their
distinct transcription patterns, and a more elaborate experimental analysis by
constructing reporter vector with regulative sequences from gene up- and
downstream regions of the most inducible member, HSP70-2, is ongoing at this
moment. Preliminary results from analysis of the HSP70-2 regulatory sequences
indicate that the 820 nucleotides upstream to HSP70-2 CDS could rapidly induce
both reporter gene transcription and translation to an abundant level, and the
induction can be tightly regulated by a “temperature switch”, suggesting this
promoter a promising applicable tool in protein expression in Tetrahymena. This
study is the first to report the expression pattern of a complete subgroup under the
HSP70 subfamily, and provides many relevant hints for further clarification of the
mechanism of transcription regulation in these eukaryotes.
Symposium: Molecular Biology of Protists
Fundamental Questions and Concepts about Photoreception and the Case of Euglena
gracilis. LAURA BARSANTI, VALTER EVANGELISTA, VINCENZO
PASSARELLI, PRIMO COLTELLI, ANNA MARIA FRASSANITO, PAOLO
GUALTIERI. Istituto di Biofisica, CNR, Via Moruzzi 1, 56124 Pisa, Italy.
The ability to sense light can be considered the most fundamental and presumably
the most ancient property of visual systems. This ability is the basis of phototaxis,
one of the most striking behavioural responses of motile photosynthetic
microorganisms (i.e. microalgae) to light stimuli, which allow them to move toward
or away directional light. In order to fully exploit the information content of light
(intensity, direction, distribution) microorganisms need proper perceiving devices,
termed photoreceptors, which must act as sensor, to perceive wavelength and
direction of light, as transducer, to convert the light signal into chemical and/or
electrical information, but also as amplifier and eventually as transmitters. This
review describes the universal structural, behavioural and physiological features
necessary for the proper functioning of these devices in algae, and how these
features have been investigated by means of different analytical techniques such as
for example microspectroscopy, digital fluorescence microscopy, two photons
FLIM. The insight of the photoreceptive response mechanism is explained using the
unicellular alga Euglena gracilis, in which the different structural, behavioural and
physiological features combine to achieve a concerted, efficient response to light
stimuli.
The DNA Barcode of Ciliated Protists. PAOLA RICCIOLINI, GRAZIANO DI
GIUSEPPE, FERNANDO DINI. Dipartimento di Biologia; Università di Pisa, I56126 Pisa, Italy
With their pervasive distribution and cryptic habits, protists are the least
understood organisms from a biodiversity perspective. Since the types of analysis
that are usually used to identify protists, based primarily on morphological
characters, require expert skills and are particularly difficult, especially due to the
presence of cryptic species, there is a critical need for new diagnostic tools. Recent
investigations have suggested the feasibility of establishing a species identification
system reliant on the analysis of the sequence of the mitochondrial cytochrome c
oxidase subunit 1 gene (cox1), (i.e., DNA barcode). The DNA barcode procedure is
based on the assumption that the sequence divergence of a small DNA fragment
allows species discriminations. We assessed the effectiveness of this procedure in
the most cosmopolitan, ubiquitous, and differentiated group of ciliated protists, the
genus Euplotes. The availability of the largest collection of living strains of Euplotes
currently existing, together with the application of both traditional and innovative
methods to ensure the reliability of the identification of each morphospecies, created
the necessary conditions to allow the evaluation of the DNA barcoding tool in
identify the Euplotes species. Accordingly, 21 strains of the genus Euplotes,
belonging to 8 morphospecies representatives of all habitats, were selected. Novel
PCR primers for Euplotes were developed to amplify approximately 1100-1600 bp
of the cox1 gene and the products obtained were subsequently cloned and sequenced.
All Euplotes strains could be identified to the species level using the cox1 sequence,
with about 50% interspecific sequence divergence. However, strains identified as E.
crassus showed high intraspecific sequence divergence values of 24% and did not
cluster together on a cox1 NJ tree. This may indicate the presence of cryptic species.
Overall, our study demonstrates the feasibility of using the mitochondrial cox1 gene
as a taxonomic marker for ‘barcoding’ and identifying Euplotes species.
Mechanisms Repairing Oxidized Proteins in Euplotes. EUGENIE EMILIE
NGUENG OUMAROU, NICOLETA DOBRI, CLAUDIO ALIMENTI,
PIERANGELO LUPORINI, ADRIANA VALLESI. Dipartimento di Scienze
Ambientali, Università di Camerino, I-62032, Camerino (MC), Italy.
Based on previous observations that aging cultures of E. raikovi and E. nobilii
synthesize increasing concentrations of methionine-oxidized forms of their
signalling pheromones, we focused our attention on the activity of the enzyme
methionine sulfoxide reductase (Msr) that these species, like other aerobic
organisms, utilize to reduce oxidized methionione back to methionines. Both species
were found capable of synthesizing at least two distinct Msr’s of type A for the
reduction of methionines oxidized to the stereoisomeric isoform-S, and one Msr of
type B for the reduction of methionines oxidized to the stereoisomeric isoform-R.
However, the use of anti-Msr antibodies on cell extracts showed that the exposure of
E. raikovi to oxidative stress causes appreciable variations in the cytoplasmic
concentrations of the Msr isoforms, whereas no comparable variation was observed
in similarly stressed E. nobilii cells. We argue that this higher resistance of E. nobilii
to the oxidative stress reflects the ecological adaptation of this Antarctic and Arctic
species to a strongly oxidative environment.
Pheromone-gene Structure and Expression in Two Phylogenetically Early-branching
Euplotes Species, E. raikovi and E. nobilii. FRANCESCA RICCI1, ADRIANA
VALLESI1, CLAUDIO ALIMENTI1, GRAZIANO DI GIUSEPPE2, FERNANDO
DINI2, PIERANGELO LUPORINI1. 1Dipartimento di Scienze Ambientali,
University of Camerino, I-62032, Camerino (MC); 2Dipartimento di Biologia,
University of Pisa, I-56126, Pisa, Italy.
Based on the knowledge of the aminoacid sequences of pheromones purified from
E. raikovi and E. nobilii, we cloned the full-length sequences of species-specific sets
of macronuclear pheromone-genes. It was found that, due to multiple sites of
initiation of transcription and the removal of intron sequences from strictly
conserved regions, each gene synthesizes multiple mRNA’s. Two mRNA’s are
specific for distinct pheromone isoforms and a third species is specific for a new
protein candidate to assist the mechanism of pheromone secretion. It appeared, in
addition, that each cell carries two distinct macronuclear pheromone gene copies
(not a single copy as supposed so far), that differ in their degree of amplification and
in the organization of their intron and regulatory sequences. These new notions
suggest that the two macronuclear pheromone-gene copies derives from the
reorganization of the same micronuclear mat gene locus.
New Insights in the Biology of the Mating-type Systems of the E. crassus-E. minutaE. vannus Species Complex. SERGIO FEDERICI1, ADRIANA VALLESI2,
CLAUDIO ALIMENTI2, GRAZIANO DI GIUSEPPE1, FERNANDO DINI1,
PIERANGELO LUPORINI2. 1Dipartimento di Biologia, University of Pisa, I-56126,
Pisa, Italy; 2Dipartimento di Scienze Ambientali, University of Camerino, I-62032,
Camerino (MC), Italy.
The high-multiple mating type systems of the E. crassus-E. minuta-E. vannus
species complex have traditionally been reported to be genetically determined by
series of alleles at the single locus mat, and these genes would be inherited by a
Mendelian mechanism of hierachical dominance (i. e., mat-1>mat-2>mat-3 and so
forth). Due to the fact that these Euplotes species form mating pairs only after a
period of physical interactions between cells of different mating types, it was in
addition hypothesized that the mating-type factors (pheromones) that are synthesized
by these genes in the transcriptionally active macronucleus are represented by
membrane-bound proteins. Current studies on the structure and mechanism of
expression of these macronuclear pheromone genes in E. crassus contradict this
conventional knowledge. We obtained compelling evidence that, as it occurs in the
phylogenetically more ancient Euplotes species such as E. raikovi and E. nobilii, also
E. crassus synthesizes water-soluble pheromones. In addition, in the case that cells
are heterozyogous at their mat locus, they secrete two structurally distinct
pheromones thus implying that their mat genes are regulated by relationships of codominance and not of hierarchical dominance.
Symposium: Biodiversity, Ecology, Morphology and Phylogeny of Protists
The Mysteries and Challenges of Diatom Life Cycles. MARINA MONTRESOR.
Stazione Zoologica Anton Dohrn, I-80121, Napoli, Italy.
Diatoms are a very diversified group of autotrophic protists responsible for about
25% of the global primary production and play a crucial role in the biogeochemical
cycles of carbon and silica. Diatoms are diploid in their vegetative stage and have a
very peculiar life cycle characterized by the progressive reduction of their cell size as
vegetative division proceeds. This miniaturization process can be arrested by the
onset of the sexual phase within which a larger cell is produced. Moreover, diatoms
include in their life cycle the formation of resting stages. Within this general
framework, there are however considerable differences across marine planktonic
lineages, which reflect adaptation to the planktonic life. Life cycles are the product
of a long evolutionary history and thus represent a key element for our understanding
of species ecology and natural history. A species alternates in its life cycle between
four distinct major phases-Growth, Sexual, Senescence and Quiescence. This implies
that the population of a diatom species will contain cells that undergo different fates,
have differentiation in physiology, and have different functional roles even if they
are genetically identical. Information on life cycle patterns of the different species
and on the factors that regulate transitions amongst distinct life phases are thus
crucial for our understanding of population dynamics in the natural environment. I
will provide an overview on different aspects of ongoing research on diatom life
cycles, outlining their diversity, the implications for ecological and outlining some
major questions and perspectives for future research.
Algal Blooms in Chilka lagoon, East Coast of India. A.V RAMAN1, C.,
KALAVATI1, E. SUNITA BABU1, M. RAKHESH1, B.R. SUBRAMANIAN2.
1
Marine Biological Laboratory, Department of Zoology, Andhra University;
2
Visakhapatnam ICMAM Directorate, NIOT Campus, Chennai, India.
Chilka Lake, Asia’s largest brackish water lagoon and a Ramsar site is under
considerable threat ecologically. Observations (2004-’10) on the phytoplankton
composition and numerical abundance at 36 hydrographically differing locations in
the lagoon revealed dominance of cyanophyceans in particular Microcoleus sp.,
Oscillatoria limnetica and Anabaenopsis sp. Of these, Microcoleus sp. was found in
massive blooms (mean 18,706 nos.ml-1) during February-March 2006 when its
numbers peaked all time high values (4,07,979 nos.ml-1). Hydrographically
conditions in the lagoon were moderate -salinity (mean 10.99 PSU), inorganic
nitrogen (ammonia 3.1, nitrite 0.17, nitrate 0.67 µmol.l-1), DON (24.34 µmol.l-1),
phosphate (0.13 µmol.l-1) and transparency (0.5m). Microcoleus was noticed during
February 2005 also but its intensity was low (mean 2320 nos. ml-1) and the event
somewhat localised. On the basis of multivariate analysis (PRIMER), it was possible
to demonstrate considerable seasonality in phytoplankton abundance attributable to
changes in population structure and prevailing water quality. Essentially, the lagoon
appeared to interchange between periods the cyanophyceans (e.g. Microcoleus sp.)
were in blooms followed by a phase their (cyanophycean) presence was not marked.
A noteworthy feature has been the nearly tenfold decrease in nitrate-N prior to the
onset of blooms both in 2005 and 2006. The period was marked by a rich population
of microphytobenthos (MPB) (chlorophyll 38.3 µg.g-1) which however dwindled
with the onset of cyanophyceans. It would appear from the findings that the MPB
plays a crucial role in the overall nitrogen availability (as N2) to the cyanophycean
outbursts presumably aiding nitrification/denitrification processes associated with
sediment-water fluxes. More recent observations (2009-’10) showed a shift in the
overall phytoplankton composition and the prymnophycean, Phaeocystis sp. is now
the most dominant form. The species which was not found earlier in the lagoon
became somewhat apparent by January, 2009. The massive outburst of Phaeocysits
sp. (mean 274758 nos.ml-1) during January, 2010 appears to suggest the species’
opportunistic nature overpowering Microcoleus sp. and other cyanophyceans noticed
earlier.
A Multidisciplinary Approach to Describe Some Peritrichs (Ciliophora, Peritrichia)
and Mobilids (Ciliophora, Mobilia) from Brazil: a Combined Morphological,
Ecological and Molecular Study. ROBERTO J. P. DIAS1, LAURA R. P. UTZ2,
MARCUS V. X. SENRA3, CARLOS A. G. SOARES3, INÁCIO D. DA SILVANETO1. 1Laboratório de Protistologia, Departamento de Zoologia, Universidade
Federal do Rio de Janeiro, Brazil; 2Faculdade de Biociências, Pontifícia
Universidade Católica do Rio Grande do Sul, Brazil; 3Laboratório de Genética
Molecular de Eucariontes e Simbiontes, Departamento deGenética, Universidade
Federal do Rio de Janeiro, Brazil.
Peritrich ciliates have been traditionally subdivided into two orders, the free
living Sessilida and the parasitic Mobilida. Following the application of molecular
methods, the monophyly of the subclass Peritrichia has recently been questioned.
Peritrichs are one of the most diverse groups of ciliates, comprising over 100 genera
and at least 1000 described species. In this work, we studied the morphology,
ecology, and sequenced the small subunit rRNA of some peritrich ciliates from
marine, freshwater, and terrestrial Brazilian ecosystems. The morphological study of
ten species of peritrich ciliates, eight sessilids, and two mobilids was carried out
using in vivo, protargol-stained and surface topography (scanning electron
microscopy) observations. In the marine habitat (coast of São Paulo State) we found
the sessild species Pseudovorticella sp., Scyphidia sp. as epibiont on polychaetes,
and Vorticella oceanica attached to diatoms. The mobilid genera Urceolaria was
found as epibiont on starfishes. In the freshwater lotic systems (southest Brazil) we
found two species of the sessilid genus Rhabdostyla colonizing oligochaetes and
mosquito larvae, in a polluted stream; the sessilid Epistylis plicatilis from a polluted
stream; one species of Epistylis attached to stonefly nymphs from clean waters; and
the mobilid Trichodina heterodentata infesting tadpoles in an urban stream. In the
terrestrial habitat we found the sessilid ciliate Vorticella gracilis attached to lichens
from the Brazilian Atlantic Forest (southeastern Brazil). We studied the fine
structure of two epibiont species: Vorticella oceanica and Urceolaria sp., and also
investigated the site preference, spatial and temporal occurrence of the epibionts
attached to stonefly nymphs and to oligochaetes, respectively. To shed more light
into the evolutionary relationships within peritrichs, the complete small subunit
rRNA (SSU rRNA) sequences of three sessilids and one mobilid species were used
to construct phylogenetic trees using maximum parsimony, neighbor joining, and
Bayesian analyses. Our results show that the mobilids and sessilids are not sister
groups, supporting that peritrichs do not constitute a monophyletic group.
Morphological, Ultrastructural, and Molecular Analysis of the Ciliate Euplotidium
rosatii n. sp. (Spirotrichea, Hypotrichia) and of its Epixenosomes from Guam Island
(USA). LETIZIA MODEO1, GIULIA LO BARTOLO1, GIULIO PETRONI1,
CHRIS LOBBAN2, FRANCO VERNI1, CLAUDIA VANNINI1. 1Unità di
Protistologia-Zoologia, Dipartimento di Biologia; Università di Pisa, I-56126 Pisa,
Italy; 2Division of Natural Sciences, University of Guam, GU 96923, Mangilao,
USA.
By means of a multidisciplinary approach, the ciliate Euplotidium rosatii n. sp
and its episymbionts, known as epixenosomes (“Verrucomicrobia”) were
characterized from Pacific Ocean. Ciliate morphology was studied through in vivo
observation, Feulgen, and protargol staining. Ultrastructural features of both the
organisms were investigated through scanning (SEM) as well as transmission (TEM)
electron microscopy. Molecular characterization was performed through ribosomal
SSU gene sequencing. Results were then compared with literature data on previously
described species of the Euplotidium genus and their episymbionts; Euplotidium itoi
and Euplotidium arenarium were the only two literature species characterized by
means of a multidisciplinary approach. Although our ciliate presents all of the genus
diagnostic morphological characters, concerning meristic and morphometric data it
does not perfectly fit any of the already described species. SEM analysis supported
protargol staining on the absence of the left marginal cirrus, which occurs in all but
one of the other Euplotidium species. By means of SEM and TEM, epixenosome
superficial morphology and ultrastructure appeared rather reminiscent of the
previously described epixenosomes features. rRNA gene sequences were compared
with on-line NCB database as well as ARB database: ciliate 18S rRNA gene
sequence shows similarity values of 98,66% with E. itoi sequence and 98,54% with
E. arenarium sequence; epixenosomes 16S rRNA gene sequence shows similarities
of 99,32% and 99,17% with the sequences of epixenosomes of E. itoi and of E.
arenarium, respectively. Taken all together, our data propose our ciliate as a new
species of the genus. Results concerning epixenosomes suggest that the same
bacteria species are associated with different species of Euplotidium. Future studies
will clarify whether a coevolution took place between Euplotidium species and their
associated symbionts.
Morphological, Ultrastructural, and Molecular Characterization of Pseudokeronopsis
sp. and Uroleptopsis citrina (Ciliophora, Spirotrichea, Strichotrichia) from Brackish
Water. LETIZIA MODEO, FILIPPO FERRANTINI, VITO BONSIGNORE,
FRANCO VERNI, GIULIO PETRONI. Unità di Protistologia-Zoologia,
Dipartimento di Biologia, Università di Pisa, I-56126, Pisa, Italy.
Two pigmented morphospecies of stichotrichs belonging to the order Urostylida
were discovered in brackish water samples from Sète (Languedoc-Roussillon,
France). Monoclonal cultures of both of them were obtained and a multidisciplinary
study was performed: in vivo observation, protargol staining, scanning (SEM) and
transmission (TEM) electron microscopy were combined with molecular analyses
based on SSU rRNA plus ITS sequences, to achieve species identification and
investigate phylogenetic relationships with related stichotrichs. Results were
compared with literature data on pigmented ciliates of the genera Pseudokeronopsis
and Uroleptopsis. The brick-red morphospecies (strain SFRv1) was compared with
Pseudokeronopsis riccii and Pseudokeronopsis sp. strain Kero, which we previously
studied by means of a multidisciplinary analysis, as well as with Pseudokeronopsis
rubra, Pseudokeronopsis carnea, and Pseudokeronopsis flava, well-known by
morphology and recently characterized from a molecular point of view. Due to its
dark yellow pigmentation, the second morphospecies (strain SFGv1) was compared
with P. flava and with Uroleptopsis citrina. Meristic and morphometric data showed
that SFRv1 does not perfectly coincide with any of the formerly described
Pseudokeronopsis; thus it could be a new species. On the other hand, SFGv1 fits the
description of U. citrina. 18S rRNA and ITS sequences compared with available
sequences on NCBI database supported morphological findings. Phylogenetic trees
based on the cited molecular markers were calculated with ARB program package to
investigate phylogenetic relationships among Urostylida. Molecular analysis pointed
out the partial inadequacy of 18S rRNA gene to fully resolve intra-order
relationships, while the more variable marker ITS allowed the individuation of
species-specific apomorphies which could be used as molecular tools allowing a
quicker identification of these ciliates.
Symposium: Genomic of Protists and of their symbionts
The Genomic Features of Ciliate Endocytobionts. MICHAEL LYNCH. Indiana
University, Bloomington, Indiana USA.
We have been involved in high-throughput genomic sequencing projects
involving three ciliate endocytobionts, all alpha-proteobacters: an unnamed
inhabitant of Ichthyophthirius multifiliis, itself an external parasite of fish (in
collaboration with Robert Coyne); Caedibacter varicaedens, one of the “killer”
bacteria inhabiting Paramecium biaurelia cytoplasm; and Holospora obtusa, a
nuclear inhabitant of Paramecium caudatum (in collaboration with Franz Lang). The
genomes of all three lineages harbor only ~1000 protein-coding genes, and are
substantially lacking in most biosynthetic-pathway genes, including those for aminoacid and nucleotide production. C. varicaedens appears to harbor a very large
number of repetitive elements of unknown function. A broad overview of the
putative comparative biology of these organisms will be given, derived from the
gene content, and in particular with respect to that of other intracellular alpha
proteobacters (including the Rickettsias, Anaplasma, Ehrlichia, and Anaplasma).
A Glimpse into the Population Genetics and Evolution of Noncoding Sequences in
the Paramecium aurelia Species Complex. FRANCESCO CATANIA. Dept. of
Biology, Indiana University, 1001 East Third Street, Bloomington 47405, IN,
USA; Dept. of Animal and Plant sciences, University of Sheffield, Western Bank,
Sheffield S10 2TN, UK.
The remarkable biological features and molecular tools of the ciliate Paramecium
offer an excellent opportunity to study the evolution of genomic architecture, a
product of both neutral and selective processes, and hence, ultimately, of the
population genetic environment. Here, I will first discuss recent findings about the
population genetics of Paramecium, presenting data about the levels of genetic
diversity observed for multiple Paramecium species belonging to a cryptic species
complex, Paramecium aurelia. I will then present the analysis of a 3' UTR
DNAsequence motif that is distinctively conserved in the ribosomal genes of P.
aurelia, and appears to be associated with the evolutionary fate of its host genes.
Finally, I will briefly describe a recently funded project that aims at providing
insights into 1) the rates and mechanisms of origin, evolution, and loss of internal
eliminated sequences (IESs) from protein-coding genes and intergenic regions across
the whole P. aurelia species-complex, and 2) the potential functions of IESs, such as
the capacity to modulate gene expression.
Transcriptomics to Identify New Biomarkers from the Antarctic Ciliate Euplotes
focardii. SANDRA PUCCIARELLI, RAGHUL RAJAN DEVARAJ, GUANG
YANG, SABRINA BARCHETTA, TING YU, PATRIZIA BALLARINI,
CRISTINA MICELI. Department of Molecular, Cellular, Animal Biology,
University of Camerino, I-62032, Camerino (MC), Italy.
The stable cold Antarctic coastal seawaters host a rich variety of eukaryotic
microbes, a large fraction of which is represented by species of ciliates, in particular
Euplotes. Among the Euplotes species isolated from Terra Nova Bay, E. focardii
shows strictly psychrophilic and stenothermal characteristics, including optimal
survival and multiplication rates at 4-5 °C, that denote an ancient colonization of the
Antarctic environment. The successful colonization of the Antarctic habitat by E.
focardii should be related to the evolution of macromolecules able to function at low
temperatures. To understand the molecular basis responsible for cold adaptation, we
started the characterization of the genome and transcriptome from E. focardii. At
present we sequenced ~15Gbases, representing about 7200 genes. To predict the
function of these sequences, we performed a blastx batch operation with Standalone
BLAST. To verify if this stenothermal organism constitutively induces genes
involved in the environmental stress response, we selected the sequences that gave
hits to stress proteins from the transcriptome blastx result file. We found four
different transcripts corresponding to Heat Shock Proteins (HSPs) 70, and two
corresponding to the Heat Shock transcription Factors (HSF). A deeper analysis
revealed the presence of transcripts that shared similarity to metallothioneins, DNA
damage repair factors, proteins induced by oxidative and osmotic stresses, and
proteins responding to centrifugal and shear forces. Moreover, we also found
transcripts that blasted with proteins responding to general stresses. We are currently
investigating the expression variation of these genes determined by environmental
stresses. These preliminary results obtained from the genome and transcriptome of
E. focardii open the way to the characterization of new biomarkers that can be used
for biomionitoring the effects of pollutants and thermal stress on Antarctic
organisms.
Characterization of Lipases from Euplotes focardii with Potential Industrial Value.
GUANG YANG, SANDRA PUCCIARELLI, SABRINA BARCHETTA, TING YU,
CRISTINA MICELI. Department of Molecular, Cellular, Animal Biology,
University of Camerino, I-62032, Camerino (MC), Italy.
Organisms living in cold environments have captured attention for their unique
metabolic properties at low temperature. To identify the molecular mechanisms
responsible for cold-adaptation, we started a genome-scale comparison of protein
sequences from cold-adapted and temperate species of the protozoan genus Euplotes,
including the Antarctic psychrophile E. focardii. These ciliates are characterized by
nuclear dimorphism: 1) the germline micronucleus contains the entire genome as
large chromosomes; and 2) the somatic macronucleus (50 megabases for each
species) contains small linear DNA molecules [nanochromosomes of 1-12 kilobases
(kb)], each of which constitutes a single genetic unit flanked by regulatory regions
and capped by telomeres. We followed two different approaches: i) a pilot
macronuclear nanochromosome library from E. focardii and ii) a transcriptome
analysis by pyrosequencing. To date, we obtained sequences encoding structural
proteins as tubulins, permeases, E1-E2 ATPase, transcription factors, and several
proteins of as yet unknown function. We focused on three members of the lipase
subclass for their important roles in human practices as yogurt and cheese
fermentation, in lipids degradation, and fuel production. The comparison of these
proteins with the orthologous from mesophilic ciliate species revealed the presence
of the canonical amino acid substitutions of psychrophilic protein, i.e. that confer an
increased molecular flexibility of the catalytic site. The increased structural
flexibility may be responsible of the capacity of these proteins to function at low
temperature. We also found an expected increasing of Pro residues in the E. focardii
lipases with respect to those from mesophilic species. By mapping these unique Pro
residues in the predicted 3D-models, we realized that they are responsible for
structural changes of the molecule that enlarge the cavity of the catalytic site, which
may facilitate the access of the substrate. Thus, our comparative genomic approach
appears suitable for “prospecting” for enzymes of industrial value.
Functional Genomics for Tetrahymena beta-tubulins. SABRINA BARCHETTA,
PATRIZIA
BALLARINI,
SANDRA
PUCCIARELLI,
DANIELA
SPARVOLI,CRISTINA MICELI. Department of Molecular, Cellular, Animal
Biology, University of Camerino, I-62032, Camerino (MC), Italy.
Taking the advantage of the availability of the entire genome sequence and
annotation of the ciliate Tetrahymena thermophila, we are studying the cellular
localization and function of new tubulin isotypes. Particular attention is focused on
three beta-tubulin isotypes, the well characterized beta-TT2, and two new divergent
isotypes, named beta-like T1 and T4. By transfecting T. thermophila, we discovered
that only the betaTT2 fused to the GFP is specifically localized in cilia, whereas the
GFPbeta-like T1 and T4 are localized principally at the level of microtubules
involved in nuclear division. These results suggest that ciliary beta-tubulin isotypes
possess intrinsic and different structural properties and/or sequence signals that
specifically deliver them into axonemes and nuclei. We cannot exclude the role of
post-translational modifications in the functional properties of tubulin isotypes, but
the results we have so far suggest relevant roles played by the Cys residues.
Bioinformatic Study on Gene Re-annotation in Plasmodium. RAGHUL RAJAN
DEVARAJ, CRISTINA MICELI. Department of Molecular, Cellular, Animal
Biology, University of Camerino, I-62032, Camerino (MC), Italy.
There is a deluge of whole genome data with 20% to 40% of proteins termed as
hypothetical. These proteins have exciting prospects of being species-specific novel
drug targets. The Plasmodium falciparum genome has more than 55% of the proteins
remaining unannotated, indicating a gap in our understanding of the biology of the
parasite, hence in tackling the major killer disease malaria. In this study, we have reannotated 634 hypothetical proteins based on homology transfer between
P.falciparum, P.vivax and P.knowlesi. All the proteins with more than 50%
similarity were selected which are annotated in one species but not in the other
species or two. Evidence is provided for a handful of proteins using literature data
from publications to support the accuracy of homology based annotation transfer
between closely related species. Since homology based annotation transfer has
limitations, we are now further substantiating the results by (a) COG classification
using COGnitor, (b) assigning functions based on Gene Ontology, and (c) searching
functional domains with pattern databases like SMART and PROSITE. All the
protein sequences are found with high similarity with functional regions or domains
conserved between them. The functional predictions in this study without literature
evidence must be corroborated with results from experimental methods since the
original annotation is insufficient by itself in many of them. Based on these results
we suggest that functions could be assigned to genes in genomes of closely related
species based on homology to a recently annotated genome with better tools and
updated information.
Symposium: Protists and Symbiosis
Cryptic Virulence Effects in a Hidden Host-parasite-competitor System. GISEP
RAUCH1, SASCHA KRENEK2, THOMAS U. BERENDONK2. 1Institute for
Evolution and Biodiversity, University of Münster, Germany; 2Institute for
Hydrobiology, TU Dresden, Germany.
Coevolutionary interactions between hosts and parasites are recognised as main
potential forces influencing the life-history of both antagonists. A key component in
host-parasite interactions is virulence, the host-fitness reduction caused by parasites.
Parasite virulence is influenced by a variety of factors and effects of temperature are
thereby of special interest, as it affects both parasites and hosts and is known to
variably influence virulence on the short-term. We showed experimentally that
higher temperatures clearly increased virulence, using Paramecium tetraurelia
infected with Caedibacter taeniospiralis bacteria. Indeed, the virulence increase was
so strong that although non-infected P. tetraurelia grew better with increasing
temperatures, the fitness of infected hosts decreased with increasing temperatures.
However, a post-experimental survey of our cultures revealed a contamination in
form of Mallomonadaceae sp. flagellates. Removal of the flagellate and reexecution
of the virulence measurement led to markedly changed results. Non-infected P.
tetraurelia populations grew now much slower than infected populations and no
virulence effect was detectable anymore. This may have been due to negative
antibiotic effects used to cure infected P. tetraurelia. In the previous experiment this
antibiotics sensitivity was potentially masked by the presence of flagellates. Thus,
the flagellates had a strong and unexpected impact on the virulence detection,
demonstrating the complexity of presumably simple laboratory systems.
Catching up with Killers: Genomics of Paramecium Killer Endosymbionts. KASIE
T. RAYMANN, THOMAS G. DOAK, MICHAEL LYNCH. Dept. of Biology,
Indiana University, Bloomington IN, USA.
The study of Paramecium endosymbionts began with Sonneborn’s (1938)
observation of killer Paramecium clones that were lethal to sensitive Paramecium
individuals, usually without direct contact. Although Sonneborn didn’t know the
nature of the killing factor, his demonstration of its cytoplasmic transmission quickly
became a influential example of non Mendelian inheritance (Preer 2006).
Sonneborn’s student, John Preer, soon discovered the bacterial nature of such
factors, eventually (with Louise Preer) demonstrating that the bacteria themselves
often harbor elements with phage-like features. Unfortunately, although Paramecium
was an eminent model organism for genetic research only three decades ago, the
surviving academic lineage of Sonneborn and Preer is small, and the system has
languished a bit (Preer1997). However, this work by earlier investigators provides a
solid platform for major breakthroughs in the era of genomics. Killer endosymbionts
of Paramecium are a useful model for studying parasite-host interaction as well as
the mechanisms involved in the killing phenotype. Many strains with the killing
potential produce unique inclusions, called R (refractile) bodies. This R body is a
necessary but insufficient condition for killing in some species of killer
endosymbionts; but there are species of killer endosymbionts that lack the ability to
produce R-bodies and confer the killer phenotype. Also, different killers, kill in
different ways. Currently we have DNA sequence for Caedibacter varicaedens
kappa(stock 7k Schmidit), an R-body containing species, and hope to have a
complete genome assembly within days. The Caedibacter varicaedens project
paralleled the introduction of next-generation sequencing methods, early forms of
which gave very poor assemblies. We are now working to isolate and sequence three
additional species of killer endosymbiont, one species which contains R-bodies (51
kappa) and two that lack R-bodies (299 lambda and 555 mu). Using affinitypurification methods developed by the Preers and others, pure endosymbiont
fractions are isolated, DNA is extracted, and amplified using phi29 strand
displacement amplification. Once we show that these preparationsare not heavily
contaminated, they will be used for 454 complete genome sequencing. We hope that
obtaining the sequenced genomes for each of these endosymbionts (and more) will
be useful, both in characterizing the mechanisms of killing across different
endosymbionts, especially those containing R-bodies, and more generally in
understanding the ways that endosymbionts interact with eukaryotic hosts.
Endosymbiotic Bacteria in Ciliates from Freshwater Habitats: Some "New" and
"Old" Associations. ALESSANDRO RISTORI, GIULIO PETRONI, FRANCO
VERNI, CLAUDIA VANNINI. Dipartimento di Biologia, Università di Pisa, I56126 Pisa, Italy.
Aim of this study is to investigate endosymbiotic associations between bacteria and
freshwater ciliates collected from two different kind of freshwater environments: the
natural sinkhole Pozzo del Merro (Roma, Italy) and the AcquaLatina S.P.A. waste
water treatment plant (Latina, Italy). Isolated ciliates were identified by 18S rRNA
gene sequencing and screened for the presence of bacterial symbionts by FISH with
universal eubacterial probe and probes for major Proteobacteria classes.
Characterization of eventual bacterial symbionts has been undertaken by 16S rRNA
full-cycle approach. Some preliminary results are here presented. Frontonia leucas,
Urostyla grandis, Pattersoniella vitiphila, Prorodon teres, Spirostomum sp.,
Uronema sp., Paramecium tetraurelia, P. nephridiatum, Euplotes eurystomus,
Euplotes sp. were found in the samples, endosymbiotic bacteria being present only in
the latter four. As expected, presence of Caedibacter caryophilus in P. tetraurelia
and of Betaproteobacteria both in E. eurystomus and Euplotes sp. was found.
Preliminary FISH on Euplotes sp. indicated the presence of endosymbiotic bacteria
belonging to the Alphaproteobacteria class, too. BLAST analysis of sequences
retrieved from Euplotes sp. gave in two cases as maximum hits Caedibacter
macronucleorum (98% identity value) and a bacterial endosymbiont of Diophrys sp.
(99%): two endosymbiotic bacteria previously described in different ciliates species.
In a third case no the higher identity value was of 83% with an uncultured
Holosporaceae bacterium (HM138368). As Holosporaceae family comprehends
symbionts of ciliates and amoebae, this last result potentially indicates the discovery
of a new endocitobiotic bacterium. P. nephridiatum was already known to harbor
intracellular bacterial symbionts. FISH performed in the present study confirmed the
presence of symbiotic bacteria, whose 16S rRNA gene characterization is still in
progress.
Molecular and Morphological Characterization of Hosts and Bacterial
Endosymbionts in a Population of Euplotes aediculatus Pierson, 1943 Reveals an
Unexpectedly diverse Community of Cytoplasmic Prokaryotes. VITTORIO
BOSCARO, GIULIO PETRONI, FRANCO VERNI, SERGEI FOKIN, CLAUDIA
VANNINI. Dipartimento di Biologia, Università di Pisa, I-56126 Pisa, Italy.
Aim of this study was the morphological and molecular characterization of
ciliated hosts and bacterial endosymbionts of the population named In. The sample
was taken in a freshwater eutrophic pond near the river Yamuna (Delhi, India) in
February 2002. Morphological analysis of the ciliates has been performed through in
vivo and SEM observations. Morphological features and sequencing of the
SSUrRNA gene of the ciliate identified it as Euplotes aediculatus, a freshwater and
symbiont-containing species of the genus. Preliminary experiments of FISH showed
an unexpected diversity in the community of bacterial endosymbionts inhabiting the
cytoplasm of these ciliate cells. Probes targeting the species Polynucleobacter
necessarius (Betaproteobacteria), the family Rickettsiaceae (Alphaproteobacteria)
and the class Gammaproteobacteria supplied positive results, thus showing the
presence of at least three different bacterial species. Molecular identification of the
symbionts was undertaken by 16S rRNA gene sequence characterization. P.
necessarius is an obligate endosymbiont of E. aediculatus and of other Euplotes
species. In a phylogenetic tree with many previously characterized P. necessarius
sequences, the newly obtained one doesn’t cluster with known E. aediculatus’
symbionts, thus suggesting that P. necessarius was recruited by the host more than
once. Preliminary phylogenetic analysis on a partial sequence of the
Gammaproteobacteria symbionts shows that the sequence of this probably new
genus clusters with the sequences of Caedibacter taeniospiralis, another ciliate
endosymbiont, and of Fangia hongkongensis, forming a sister group clade of the
Francisellaceae family. Rickettsia-like organisms (RLOs) have been just recently
found in ciliates, and only once before in the Euplotes genus; molecular
characterization of this new RLO is currently under investigation. Ultrastructural
observations of some of the bacterial endosymbionts have been performed through
TEM investigations.
GDRE Meeting Paramecium and its symbionts
II Holospora conference
The Holosporas - Challenge and Expectations. HANS-DIETER GÖRTZ. Abteilung
Zoologie, Universität Stuttgart, D-70569 Stuttgart, Germany.
With estimable exactness and accuracy M. W. Hafkine described the biology and
morphology of the genus Holospora and three species, Holospora obtusa, H. elegans
and H. undulata already in 1890 (!). About eigthy years later, D. V. Ossipov and his
students started detailed investigations namely into the ultrastructure and the way of
infection. In the last thirty years the holosporas were phylogenetically localized in
the alphaproteobacteria and cell biological, biochemical and molecular observations
emphasized the uniqueness of these bacteria. On the basis of our present knowledge
and with the tools being available, there is now the chance of studying and
understanding specific biological and molecular features of these obligate
intracellular bacteria. In the talk, some of the most exciting features are addressed
such as the peculiar high-output secretory apparatus: Holospora has the behavior of
depositing proteins in the periplasm before secreting them across the outer
membrane. Further, in the communication of Holospora and Paramecium, the
intracellular transport of bacteria with microtubules as well as actin of the host cell
appear exceptionally efficient and should be investigated. The study and
understanding of many other features, too, such as the mode of crossing the nuclear
envelope and communication of bacteria with host nuclei or the development of
resistance of host populations against Holospora-infections may be of great value.
Genome Evolution of Holospora, Mitochondria and Related alpha-Proteobacteria. B.
FRANZ LANG ROBERT CEDERGREN. Centre for Bioinformatics and Genomics,
Département de biochimie,Université de Montréal, Montréal, Québec, Canada.
A key event in eukaryotic evolution, the symbiotic introduction of mitochondria,
occurred a billion or more years ago. Hence, related phylogenetic inference is
exceedingly difficult, and broad taxon sampling combined with phylogenomic
analyses and the use of adequate phylogenetic models is essential for providing
statistically significant as well as biologically meaningful results. When assuming
only one mitochondrial origin and no or only insignificant lateral transfer of
mitochondrial genes, it is expected that phylogenetic results with data from nuclear
and organelle genomes are similar. In addition, because mitochondria are bacterial
symbionts, the question after the mitochondrial origin has two aspects: (i)where they
first emerged in eukaryotes (either into a non-mitochondriate eukaryotic predecessor,
or as co-founder of the eukaryotic cell itself), and (ii) precisely from which (αproteobacterial) bacterial lineage they derive. To address these questions, we analyze
and compare phylogenies based on nuclear, mitochondrial and bacterial datasets. In
this context, two bacterial endosymbionts of ciliates, Caedibacter and Holospora,
are of particular interest as they appear to branch close to the mitochondrial origin.
The results of our phylogenomic analyses (work in progress, in collaboration with
the M. Lynch group) will be presented. We will further discuss comparative
metabolic and other pathway analyses (using automated prediction tools developed
by the P. Karp group) of Caedibacter, Holospora and related intracellular bacteria
from the Rickettsia/Ehrlichia group.
Secretion of the 63-kDa Periplasmic Protein of the Infectious Holospora obtusa
during Infection and a Possible Function of the Protein. MASAHIRO FUJISHIMA.
Department of Environmental Science and Engineering, Graduate School of Science
and Engineering, Yamaguchi University, Yamaguchi, 753-8512, Japan
Holospora obtusa is a macronucleus-specific endosymbiotic bacterium of the
ciliate Paramecium caudatum. We report the secretion of a 63-kDa periplasmic
protein of an infectious form of the bacterium into the macronucleus of its host.
Indirect immunofluorescence microscopy with monoclonal antibodies against the 63kDa protein demonstrated that, soon after the bacterial invasion into the host
macronucleus, the protein was detected in the infected macronucleus and that levels
of the protein increased dramatically within one day of infection. The use of
inhibitors for host and bacterial protein synthesis illustrated that, in early infection of
H. obtusa, not only the pre-existing but also a newly synthesized 63-kDa protein was
secreted into the host macronucleus. A partial amino acid sequence of the protein
was determined, and a gene encoding the 63-kDa protein was cloned. The deduced
amino acid sequence shows that this protein is a novel protein with two DNAbinding motifs. SDS-DNA PAGE containing calf thymus DNA in the gel and
immunoblotting with a monoclonal antibody specific for the 63-kDa proteins
revealed that the 63kDa proteins can bind DNA. Fractionation of supernatants of the
sonicated infectious forms of this bacterium with a DNA-cellulose column also
showed DNA-binding ability of the 63-kDa protein. These results suggest that the
63-kDa protein functions somehow change the host’s gene expressions by binding to
the host macronuclear DNA.
Host Genotype x Parasite Genotype Interactions in the Paramecium-Holospora
System. ELSA QUILLERY, ALISON B. DUNCAN, SIMON FELLOUS, OLIVER
KALTZ. Institut des Sciences de l'Evolution UMR 5554, Université Montpellier 2,
Montpellier, France.
In host-parasite systems, specialisation of certain parasite genotypes to certain
host genotypes can produce host genotype x parasite genotype (G x G) interactions.
These interactions can contribute to sustained coevolutionary cycles (Red-Queen
dynamics), and, more generally, guarantee the maintenance of genetic variation. We
studied G x G interactions and parasite adaptation to different host genotypes in
experimental populations of the freshwater protozoan Paramecium caudatum and its
bacterial parasite Holospora undulata. (1) A cross-infection experiment revealed
relatively rapid (< 3months) adaptation of the parasite to a given host genotype;
there also was a signature of this adaptation when the parasite was intermittently
cultured on another genotype, indicating the importance of the evolutionary history
for tests of adaptation. (2) In a parasite invasion experiment, we introduced infected
individuals into uninfected populations, and this for different genotypes of the host
recipient population (GRecipient), of the infected carrier individuals (GCarrier) and
the parasite they carried (GParasite). Transmission success (= number of secondary
infections) was significantly influenced by GCarrierx GRecipient and GCarrier x
GParasite interactions. This illustrates thepotentially important role of the carrier
genotype in determining the onset of an epidemic upon arrival in a susceptible
population.
Effect of Temporal and Spatial Environmental Variation on Host and Parasite
Populations. ALISON DUNCAN, ANDY GONZALEZ, OLIVER KALTZ. Institut
des Sciences de l'Evolution (ISEM), Université Montpellier 2, Montpellier, France.
Temporal and spatial environmental variation is predicted to impact population
dynamics and persistence. We subjected microcosm metapopulations of the ciliate
Paramecium caudatum, infected or not with the bacterial parasite Holospora
undulata, to changes in temperature corresponding to permissive (23ºC) or
restrictive (5ºC) conditions. Temporal environmental variation was either random
with no serial correlation in the sequence of changes given, or positively correlated
whereby populations were more likely to experience successive days in a given
environment. Microcosm meta-populations comprised two sub-populations linked by
high or low levels of migration that encountered either spatially heterogeneous
orhomogeneous environmental variation. Populations were said to spatially
homogeneous when sub-populations followed the same sequence of temporal change
or heterogeneous when following different sequences. The parasite was only present
in one of the sub-populations for infected meta-populations on day 1 of the
experiment. We will present results describing how temporal and spatial
environmental variation influences levels of infection and rates of spread to the
uninfected sub-population. We will also describe how these environmental changes
influence both fluctuations and total size for infected and uninfected host
populations.
The Ghost of Parasite Past: Selection against Costly Resistance in Parasite-free
Populations. SIMON FELLOUS, ALISON DUNCAN, OLIVER KALTZ.
Université Montpellier 2 - CNRS, France.
The evolution of resistance against parasites is often accompanied by a reduction
in fitness-relevant life-history traits, such as growth or fecundity. Typically, such
costs of resistance are defined (and measured) as a reduction of fitness in the absence
of the parasite. This leads to the simple prediction that, in populations that become
cleared of the parasite, resistance should decrease and fitness increase, comparable to
levels observed in naive, unexposed populations. We tested this idea in long-term
experimental populations of the ciliate Paramecium caudatum and the parasitic
bacterium Holospora undulata. We isolated uninfected cells from partly infected
populations, from populations that were infected in the past but had lost the parasite
and naïve populations, never exposed to the parasite. We shall present results from
infection tests and growth assays, comparing these three groups.
Holospora and Holospora-like Endosymbiotic Bacteria out of the Paramecium
Genus. SERGEI I. FOKIN. Department of Invertebrate Zoology, St. Petersburg State
University, 199034, St. Petersburg, Russia; Dipartimento di Biologia; Università di
Pisa, I-56122 Pisa, Italy.
Ten Holospora spp. have been found so far in the Paramecum genus, namely:
P. caudatum, P. aurelia, P. bursaria, P. calkinsi, P. nephridiatum and P. putrinum.
Very recently two more examples of bacteria definitely belonging to the genus were
discovered in the Frontonia genus: F. salmastra and F. leucas (Fokin and Görtz,
2009). Although Paramecium and Frontonia are sister genera, we can postulate now
that Holospora are not exclusively endonucleobionts of Paramecium spp. as was
always indicated before (Görtz and Schmidt, 2005). Thus, in the future it can be
expected that some more holosporas in different groups of ciliates will be found.
Indeed, several other bacteria, which have a life cycle similar to Holospora
represented by two morphological forms, have been mentioned from different
ciliates. All these bacteria appear to be infectious, though experimental evidence on
the matter exists only for few of them. There are macronuclear symbionts of
Metopus caudatus, Prorodon teres, Stentor multiformis , S. polymorphus, S. roeselii,
Trithigmostoma cucullulus, Vorticella sp., Spirostomum minus, Trichodina pediculus
and Zoothamnium pelagicum. For the majority of the ciliates Holospora-like bacteria
were just mentioned without serious further investigation or were even shown as
figures without any particular information. According to the morphology of the
infectious form, it is possible that not all of them are holosporas since sometimes the
distribution of the periplasmic part and the tip part morphology deviated quite a bit
from that of “classical” holosporas (Fokin and Görtz, 2009). From another point of
view morphological diversity of the infectious form between different holosporas
also was shown. It is important to stress that some of the mentioned Holospora-like
bacteria were found repeatedly in various locations situated very far from each other.
Reorganization of Macronuclear Architecture in Paramecium caudatum under
Infection with its Endonucleobiont Holospora obtusa. ELENA SABANEYEVA,
KONSTANTIN BENKEN. Department of Cytology and Histology, Faculty of
Biology and Soil Sciences; Saint Petersburg State University, 199034 Saint
Petersburg, Russia.
Holospora obtusa, an alpha-proteobacterium, is an obligatory endobiont of the
ciliate Paramecium caudatum, residing in the host macronucleus. The endonuclear
symbionts affect the nuclear architecture both at the step of their penetration into the
nucleus and in the stably infected cells. At the early steps of the infection the
bacteria cause redistribution of the host chromatin which is easily observed with
phase contrast microscopy. Reproductive, partly differentiated and fully
differentiated infectious forms of H. obtusa were studied using atomic force
microscopy to demonstrate the difference in their affinity to the host chromatin. The
disorganization of the nucleolar apparatus in the stably and heavily infected
macronucleus was demonstrated by means of methyl green – pyronin and AgNOR
staining. Infection with H. obtusa was also shown to change the distribution of
inactivated (phosphorylated) form of cofilin, an actin binding protein. Redistribution
of p-cofilin seems to correlate with nucleolar disorganization. As nucleolus is
believed to be the sequestration centre for temporary inactivated proteins, changes in
p-cofilin distribution might reflect cofilin activation under infection with H. obtusa.
AgNOR staining revealed silver granules predominantly along the bacterial cell wall
strongly suggesting its association with the active ribosomal genes of the host cell.
We believe that endonucleobionts might cause changes in the macronucleus
functioning via their interference with the macronuclear matrix proteins. The most
likely candidate for such interaction may be actin, recently found to be abundant in
the macronucleus. The studies of nuclear actin in endobiont-free and infected
macronuclei are underway.
Host-Isolate Specificity of Infection by Holospora caryophila, Intranuclear
Symbiotic Bacterium of Paramecium. ALEXEY POTEKHIN. Faculty of Biology
and Soil Sciences, St. Petersburg State University, Saint Petersburg, Russia
Holospora are a group of obligate symbiotic bacteria inhabiting specifically
macronucleus or micronucleus of different Paramecium species. Most of Holospora
species are restricted to only one host species with the exception of H. caryophila,
the symbiont of the macronucleus, which was isolated from nature in cells of P.
caudatum and species of the P. aurelia complex (Görtz 2006). Three H. caryophila
isolates (HcD, from P. dodecaurelia strain, HcO, from P. octaurelia strain, HcC,
from P. caudatum strain) were used to infect 44 strains of thirteen Paramecium
species. The observations over the newly infected strains were performed starting in
5-7 days after infection. We considered the symbiotic system formed if the
symbionts passed all stages of their development up to differentiation into normal
infectious form. P. bursaria (5 strains), P. multimicronucleatum (7 strains), P.
woodruffi (1 strain), P. calkinsi (1 strain), P. polycarium (1 strain) were never
infected. Stable combinations between host and symbiont were obtained for strains
of P. octaurelia (HcO, HcD), P. dodecaurelia (HcD), P. novaurelia (HcO, HcD,
HcC), P. quadecaurelia (HcD), P. caudatum (HcC), P. jenningsi (HcO, HcD), and
P. putrinum (HcO, HcD). Thus, the latter two species were recorded as new host
species for H. caryophila. P. caudatum strains were infected heavily only by HcC,
while HcD killed all taken into experiment strains of P. caudatum in a week, and
HcO was never observed in the nuclei of recipient P. caudatum strains and produced
no effects. In some combinations either Holospora tended to get lost from the
infected hosts, or paramecia were too heavily infected and finally were occasionally
killed by the symbionts. Thus, an association, even when formed, can be
deregulated. While strain-isolate specificity of infection could probably be explained
by peculiarities of molecular cross-talk in each combination, differences of
infectivity observed at the host species level may reveal phylogenetic diversity of H.
caryophila isolates. Supported by RFBR 10-04-01192.
Molecular
(re-)
discovery
of
Holospora
caryophila.
MARTINA
SCHRALLHAMMER1,3, ALEXEY POTEKHIN2, GIULIO PETRONI1, HANSDIETER GÖRTZ3. 1Unità di Protistologia-Zoologia, Dipartimento di Biologia,
Università di Pisa, I-56126, Pisa, Italy; 2Faculty of Biology and Soil Sciences, St.
Petersburg State University, Saint Petersburg, Russia; 3Abteilung Zoologie,
Universität Stuttgart, Stuttgart, Germany.
Hafkine described in 1890 highly infectious endosymbionts Holospora, present in
the micro- or macronucleus of certain Paramecium strains. Gromov and Ossipov
(1981) examined organisms identified as two Holospora species, “Holospora
undulata” Hafkine 1890 and “Holospora obtusa” Hafkine 1890 and confirmed the
descriptions of Hafkine and therefore validly described this genus. Cultivation of
bacteria belonging to this genus outside their Paramecium hosts has not yet been
accomplished. Thus, images and descriptions of Holospora species serve as
representatives of their taxonomic types. Phylogenetic relationships among the
different Holospora species are unclear, so far only the 16S rRNA gene sequences of
H. obtusa (1,492 bp) and H. elegans (479 bp) are published in GenBank. Here we
report the isolation of different Paramecium strains naturally infected by Holospora.
Following the traditional taxonomy, these bacteria are identified as Holospora
caryophila. Preliminary analyses reveal that the phylogenetic relationships with the
other molecularly characterized species are quite distant. The molecular
characterization of the type strain of Holospora caryophila (Preer and Preer 1982)
will allow in the future shedding light on the evolutionary relationships among the
newly characterized bacteria and also on the established Holospora species.
POSTER SESSION
Study of the Interactions Between Human Viruses and Protists in Aquatic
Environments. ROBERTA BATTISTINI, MARCO VERANI, GRAZIANO DI
GIUSEPPE, FERNANDO DINI, ANNALAURA CARDUCCI. Department of
Biology, University of Pisa, I-56126, Pisa, Italy.
Some protozoa can be hosts for other organisms such as bacteria, fungi, and
viruses also pathogenic to humans, who have developed resistance mechanisms to
escape phagocytosis and/or digestion by protozoa, in which so they can survive after
being swallowed: thus environmental protozoa can contribute to the spread and
persistence of the microorganisms in water subjected to disinfection treatments. The
interactions of protozoa with some pathogens as Legionella are well known, while
for viruses, some observations suggest a possible role of protozoa as occasional
hosts. In waste water treatment plants (WWTP) this mechanism could be a possible
cause of the virus resistance to disinfection. In order to evidence a possible
interaction between human viruses and protozoa in the environment, a study was
carried out in both experimental and field conditions. A strain of Euplotes
octocarinatus testing negative for adenovirus was chosen for experimental infection
with Adenovirus Type 2 NCPV #00213 and maintained in contact for variable times
(5, 15, 30, 45 min, 1, 3, 6, 24, 48, 72h and 7, 10, 45, 105 days). The samples were
then analyzed by direct immunofluorescence (IF) in 5 replicates. The microscopic
observation showed that viruses enter the protozoa after a contact of 15 minutes and
remained inside the cells until at least 15 days. The fluorescence within cells appears
localize mostly in the cytoplasm below the macronucleus presumably within the
digestive vacuoles, because the cells contain pockets of fluorescence that had the
approximate size, shape and localization of food vacuoles. Subsequently we looking
for the presence of adenovirus in protozoa of a WWTP. Sewages of the plant
oxidation tank were analyzed for the adenovirus presence by real time PCR and all
of them were positive. These preliminary results showed that adenovirus can enter,
probably by phagocytosis, in protozoa and remain inside their cells as evidenced by
IF. The same mechanism seems to occur even in WWTP.
Preliminary Phylogenetic Study of Armophorean (Ciliophora, Armophorea) 18SrDNA and alpha-tubulin Genes. BÁRBARA DO NASCIMENTO BORGES²,
THIAGO DA SILVA PAIVA¹, INÁCIO DOMINGOS DASILVA-NETO¹
1
Laboratório de Protistologia, Dept. de Zoologia, Inst. de Biologia, CCS,
Universidade Federal do Rio de Janeiro, Brasil; 2Laboratório de Biologia Molecular
“Francisco Mauro Salzano”, Universidade Federal doPará, Brasil.
Armophorean are microaerophilic ciliates traditionally classified inclass
Heterotrichea. Molecular phylogenies however, placed them outside the
postciliodesmatophoran heterotrichs, as related either to Spirotrichea or
Litostomatea. The present study aims to construct preliminary phylogenetic
hypotheses on relationships of armophoreans to other major ciliate groups, based on
separate and combined analyses of 18S and alpha-tubulin genes, discussing the
effects of combining both data sets. Sequences of 18S and alpha-tubulin were
equally sampled from the GenBank in order to build data sets encompassing the
ciliate higher taxons. The two sets were aligned independently in the software
ClustalW, and neighbor-joining analyses were conduced in MEGA 4, using 1000
bootstrap replicates as cluster support measure. Rooting was tentatively made at the
Postciliodesmatophora–Intramacronucleata divergence. The 18S analysis resulted in
a tree in which the root placement was possible and corroborated the literature,
considering the reduced taxon sample necessary to keep parity with thealpha-tubulin
data set. Armophoreans were placed as sister taxon of spirotrichs (>80% support),
and diverging after litostomateans. The alphatubulin analysis produced a tree in
which the presumptive root point was impossible, and placed the armophoreans as
sister taxon of Protocruzia, branching off from the phyllopharingeans (<10%
support). Both trees exhibited strong disagreement among higher taxon placement,
thus their consensus had poor resolution. The combined analysis of both data sets
resulted in the placement of armophoreans as sister taxon of the spirotrichs (~40%
support), but diverging after Protocruzia + phyllopharingeans. The average bootstrap
value for the 18S tree was significantly higher than for alpha-tubulin tree (P<0.005;
Mann-Whitney test). Combining both partitions resulted in slight, statistically
insignificant (P=0.1393) decrease of average bootstrap in comparison to 18S and
mild significant increase in comparison to the alpha-tubulin tree (P=0.03). Thus the
results, albeit preliminary, favor the hypothesis of sister relationship among
armophoreans and spirotrichs.
Increased Virulence of Legionella pneumophila versus Acanthamoeba polyphaga
after Chlorine Treatment. A. BUZZIGOLI, B. CASINI, P. VALENTINI, L.
CHIUMIENTO, A. BAGGIANI, F. BRUSCHI, G. PRIVITERA. Dipartimento di
Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia,
Università di Pisa, I-56127, Pisa, Italy
The role of Acanthamoeba spp. is important both in medical and ecological field.
Acanthamoeba spp. are among the most prevalent protozoa found in the
environmental, especially aquatic reservoirs, where this protozoan is a predator of
bacteria and controls bacterial populations, Legionella spp. included. Otherwise this
interaction provides to Legionella an host to parasitize, providing nutrients for
replication and protection against disinfection treatment, especially inside amoebic
cysts. In order to study the effect of chlorine exposure on induction of Legionella
pneumophila virulence genes, we analyzed the expression profile of locus lvh, mip
gene and rtx region, related to amoebal and macrophagic infection, in environmental
strains in vitro exposed to 2.5 ppm of active chlorine. Both strains isolated before
and after the start of a hospital water network hyperchlorination reacted to in vitro
chlorine treatment with an upregulation which appeared more prompt and marked
for post hyperchlorination ones. Some Legionella strains detectable in hospital water
network seemed to be chlorine activated toward a transmissive-phase, leading to the
expression of virulence traits that increased the ability of intracellular infection of
protozoa. All considered Legionella strains were able to infect Acanthamoeba
polyphaga, showing different multiplication capacities within trophozoites.
Evaluation of intracellular growth kinetics in A. polyphaga of different
environmental Legionella strains, either exposed and unexposed to in vitro chlorine
treatment, was assessed by both bacterial count on agar plates and measurement of
the DNA loads by real-time PCR method. Both methods indicated that strains
isolated after water hyperchlorination showed an exponential increase of bacterial
load, reaching a peak of considered values after six days of infection. In contrast,
prehyperchlorination strains displayed low replication ability in Acanthamoeba
polyphaga. Our observations seem to indicate that hyperchlorination enhances the
ability to express more promptly considered virulence genes; this phenomenon may
explain the persistence of Legionella in water network, supporting the critical role of
protozoa in protection of intracellular Legionella from disinfection.
The Peritrich Epibiont Rhabdostyla cf. chironomi Kahl, 1933 (Ciliophora,
Peritrichia) on Chironomid Larvae in the Neotropics: Morphological
Characterization, Site Preference and Spatial Occurrence. ROBERTO J. P. DIAS1,
ADALGISA F. CABRAL2, LAURA R. P. UTZ3, INÁCIO D. DA SILVANETO1,MARTA D´AGOSTO2. 1Laboratório de Protistologia, Departamento de
Zoologia, Universidade Federal do Rio de Janeiro, Brazil; 2Laboratório de
Protozoologia, Departamento de Zoologia, Universidade Federal de Juiz deFora,
Brazil; 3Faculdade de Biociências, Pontifícia Universidade Católica do Rio Grande
do Sul, Brazil.
To demonstrate how environmental factors could influence the spatial distribution
of epibiosis, we investigated the occurrence of the ciliate Rhabdostyla cf. chironomi
(Ciliophora, Peritrichia) attached to Chironomidae larvae in an urban stream in
southeastern Brazil. We identified the ciliates attached to chironomid larvae as
Rhabdostyla chironomi based on the similarity of gross morphological characters
with the species described by Kahl. Since very few characters from live cells were
used by the cited author to describe the species, and one of them (size of the stalk
relative to the body) does not match the character observed for the species collected
here, we suggest that the ciliate epibiont should be referred to as Rhabdostyla cf.
chironomi until a more detailed morphological analyses is carried out. Samples were
collected monthly for 1-year period at five sampling stations along the stream.
Among 24 chironomid genera analyzed, the genus Chironomus was the most
abundant, with 16.84% carrying R. cf. chironomi. Epibiont species were found only
on the ventral tubules of the chironomids. The localization of these ciliates in ventral
tubules may be related to the ventilation behavior shown by chironomids. This
epibiotic relationship was only recorded in sample stations located in a heavily
populated urban area of the stream. Statistical analyses demonstrated significant
correlations between infestation prevalence and environmental parameters such as
rainfall, bacterial density, and host abundance. The occurrence of epibionts on the
larvae was spatially heterogeneous. The results are discussed in terms of possible
factors related to the heterogeneous spatial and temporal occurrence. Better
understanding of the ecological factors responsible for the oscillation of the
epibiont–host relationship is essential to establish future use of this relationship as
water quality indicator in lotic systems. However, the development of techniques to
manage water resources is one of the major challenges faced by economic
development in this century, mainly in tropical areas.
Detection of Toxoplasma gondii DNA in Edible Shellfish. PIETRO DI PINTO¹,
MARIA CRISTINA ANGELICI², VALENTINA TERIO¹, ANGELA DI PINTO¹,
GIUSEPPINA TANTILLO¹. ¹Department of Veterinary Public Health, Faculty of
Veterinary Medicine, Valenzano (Bari), Italy; ²Department of Environment and
Primary Prevention, Istituto Superiore di Sanità, Rome, Italy.
Toxoplasmosis is characterized by the highest human incidence amongst the
parasitic zoonoses as reported in recent Scientific Opinion of the Panel on Biological
Hazards on a request from EFSA on Surveillance and monitoring of Toxoplasma in
humans, foods and animals. Infections by the protozoan parasite Toxoplasma gondii
are widespread in humans and many other species of warm-blooded animals
representing a serious food borne pathogen with a worldwide distribution. Marine
molluscan bivalve shellfish are filter-feeding organisms that represent a potential
vehicle of food borne diseases. Indeed pathogenic microorganisms in the water may
be filtered during feeding, and become concentrated in the digestive glands/tract.
Retail mussels (Mytilus galloprovincialis) sampled between August/2009 and
November/2010 were screened by nested PCR. The DNA of T. gondii was detected
at the first time in five sample of Italian mussel (M. galloprovincialis) produced in
Mar Piccolo (Taranto, Apulia) suggesting the major risk of contamination in
shellfish produced in enclosed basins.
Ciliates Biodiversity from the Frasassi Caves: the Sulfidic Microhabitats of “Pozzo
dei Cristalli”. SANTOSH KUMAR1, BATHRACHALAM CHANDRAMOHAN1,
FEDERICO BUONANNO2, DAIZYBHARTI1, ALFREDO CAMPAGNOLI2,
CLAUDIO ORTENZI2, ANTONIETTA LA TERZA2. 1School of Environmental
Science, University of Camerino, I-62032 Camerino (MC); 2Department of
Educational Sciences and Training, University of Macerata, I-62100 Macerata, Italy.
The sulfide-rich Frasassi cave complex (Genga, AN, Italy) hosts a still largely
uncharacterised microbiota whose study might offers an intriguing view on the
solutions adopted by the different species to survive and interact with each others in
a such harsh environment. Beside the absence of light and low temperatures (12-13
°C), another environmental shaping factors is represented by highly variable
sulphide concentrations (from 0 up to 415 μM H2S). Moreover, up to now, very few
studies attempted to describe ciliate communities from caves as well as their
fluctuation with respect to environmental factors. In this analysis of the ciliate fauna
of Frasassi caves, we focused our attention on a sampling site known as “Pozzo dei
Cristalli” which is highly diversified since it is composed by several microhabitats
represented by small sulfidic (H2S-rich) ponds, streams and spring as well as, deep
and shallow muddy, stagnant lakes. Periodic sampling was realised between
September 2009 and May 2010. Ciliates species were identified by a combinations of
live observations, protargol and silver staining as well as molecular techniques
involving the analysis of 18SrDNA. Our preliminary surveys showed a total of 30
species belonging to three classes, 14 order, and 22 genera. Among them, the most
intriguing species is represented by Urocentrum turbo which is strictly adapted to
high sulphide levels (up to 415 μM), as well as other poorly described species (at
both morphological and molecular level) such as for example Anteholosticha
monilata. Our future goal will be to verify by means of morphological and molecular
techniques, spatio-temporal variations in the ciliate communities along the O2/H2S
gradient in various microhabitats of “Pozzo dei Cristalli”.
Species Criteria in Ciliates: Advantages and Shortcomings of Modern Approaches
Illustrated by Paramecium. IRINA NEKRASOVA, ALEXEY POTEKHIN. Faculty
of Biology and Soil Sciences, St. Petersburg State University, Saint Petersburg,
Russia
In Ciliates the species problem remains a burning issue. A lot of approaches are
currently in use, the most popular among them – mating criterion, RAPD,
sequencing of conservative genes. In Paramecium all these approaches sometimes
bring contradictory results. Multiplication of alleles in three rounds of the whole
genome duplication which took place in evolution of Paramecium and subsequent
species radiation makes interpretation of all existing molecular phylogenies rather
uncertain. Additional criteria are needed to select molecular phylogenies which
would be the most likely to reveal the evolution of Paramecium at different levels.
The only way to find really valid species criteria is to look for “synergetic” effect
given by several approaches applied together. We will present the results of such
complex analysis of about a hundred strains belonging to fifteen P. aurelia sibling
species. We used a new approach to study polymorphism within the P. aurelia
complex – molecular karyotyping of the macronuclear genome by PFGE. Thirteen
types of electrophoretic profiles were observed; isolation of each species or group of
species was accompanied by the appearance of differences in macronuclear genome
molecular organization (Nekrasova et al. 2010). But in three species – P.
dodecaurelia, P. novaurelia, and P. decaurelia – several variants of molecular
karyotypes appeared to be mixed. Results of PFGE analysis support data on
“discordant” strains obtained by Catania et al. (2009): some strains which have been
assigned to a certain species of the P. aurelia complex according to their mating
reactions with the tester strains appeared to belong to other species by molecular
karyotyping as well as by multilocus genetic analysis. Special attention will be given
also to the data obtained by RAPD, to geographic origin of the analyzed strains, to
the classical data on mating abilities, and to the molecular phylogenetic data.
Combination of several approaches may shed more light on divergence and
evolution of Paramecium. Supported by RFBR 10-04-01192.
Recent Contributions to the alpha-taxonomy of Stichotrich Ciliates (Ciliophora,
Stichotrichia) from Brazilian Locations. THIAGO DA SILVA PAIVA, INÁCIO
DOMINGOS DA SILVA-NETO. Laboratório de Protistologia, Dept. de Zoologia,
Inst. de Biologia, CCS, Universidade Federal do Rio de Janeiro, Brasil.
The Stichotrichia are highly diversified hypotrichous ciliates which exhibit a
myriad of divisional morphogenesis patterns, and occur in freshwater, marine, and
soil environments worldwide. They are generally omnivorous and present a body
architecture which may be considered as functionally convergent to that of
microscopic bilateral metazoans. Currently there are circa 600 valid species in the
literature. Among those, some are known as water-quality indicators of the saprobic
system (e.g. Gastrostyla mystacea; Stylonychia pustulata), and some produce
antibiotic substances (e.g. Pseudokeronopsis). It is estimated that > 50% of the
extant species of ciliates are yet unknown to science, and likewise, their roles in
ecosystems are not yet fully understood. Thus, considering the progressive
destruction of natural environments via anthropic activities, and given the lack of
knowledge on extinction rates related to stichotrichs (and to unicellular eukaryonts in
general), efforts on surveying the biodiversity of such organisms in areas where they
are still relatively little known are urgent. Since the year of 2001, we have sampled
from various locations, including the Atlantic and Amazonian forests, Central
plateau, Guanabara Bay, the Jurubatiba Restinga and others. This made possible the
building of a comprehensive and permanently growing collection, containing digital
images, protargol-impregnation slides, and starting recently, molecular data. Several
novel species were identified and some are already published, namely:
Apoamphisiella foissneri, A. jurubatiba, Deviata brasiliensis, Oxytricha marcili,
Parastrongylidium estevesi, Parentocirrus brasiliensis, Pseudokeronopsis
sepetibensis and Pseudourostyla pelotensis. Additionally, new occurrences of rare
and systematically important stichotrich species were also obtained, such as
Hemicycliostyla spp (unpublished) and Strongylidium pseudocrassum. Remarkably,
the rediscovery of this last made possible a thorough systematic reassessment of
genus Strongylidium and related organisms. The augment of alpha taxonomic
information on stichotrichs is expected to improve the knowledge on the natural
history and facilitate ecologic studies on such fascinating organisms.
Paramecium and Stylonychia as Potential Bioindicators of Freshwater Pollution with
Heavy Metals. SVYATOSLAV KOKOTIN, ALEXEY POTEKHIN. Faculty of
Biology and Soil Sciences, St. Petersburg State University, Saint Petersburg, Russia
Heavy metals are among the main and the most dangerous toxicants of rivers,
pools and sewage waters; however, their presence is not always readily detected.
Chemical approaches, though precise, are rather expensive and should be applied
selectively. Thus, a search for bioindicators remains topical. Ciliates being widely
distributed rather large unicellular organisms are still not widely used as such
bioindicators mainly due to the lack of data on their sensitivity even to widespread
toxic substances. We collected more than 200 water samples in more than 80 natural
water reservoirs in Saint Petersburg. Paramecium sp. and Stylonychia sp. (sibling
species S. lemnae and S. mytilus) appeared to be the most common ciliates; they
were met in 28% and 18% of samples respectively. These ciliates can be easily
identified in a water sample even by non-specialists at a low level of magnification.
We obtained the dose-response relationship for lead, copper, cadmium and nickel for
several strains of Stylonychia sp. and P. bursaria. Concentrations of lead, copper,
and cadmium were also measured by inverse voltamperometry in water of 19
reservoirs, where diversity and abundance of ciliates was either maximal or minimal.
Our data showed that concentrations of these metals in water of investigated St
Petersburg reservoirs were approximately 10-100 times lower than concentrations
determined as lethal to ciliates. But when lead, copper and nickel were applied to
ciliates in mixtures, lethal concentrations of each component dropped dramatically
(5-20 times for different metals), getting close to concentrations observed in water
reservoirs. As in nature different pollutants act jointly, even minor concentrations of
some of them, safe in laboratory conditions, become harmful to living organisms.
Thus, we suppose that absence of Paramecium and Stylonychia in a certain aquatic
habitat may indicate that such habitat might be polluted by heavy metals, first of all –
copper and nickel. Supported by the grants of St Petersburg Government to A.P. and
by the International Fogarty Center grant to S.K.
Symbiotic Bacteria Infecting Ciliates in the Branchial Cavity of Shipworms: An
Inter-Kingdom Tripartite Interaction? MARCUS V. X. SENRA 1, ROBERTO J. P.
DIAS 2, INÁCIO D. SILVA-NETO 2, CARLOS A. G. SOARES1. 1Laboratório de
Genética Molecular de Eucariontes e Simbiontes, Departamento de Genética;
2
Laboratório de Protozoologia, Departamento de Zoologia. Inst. de Biologia, CCS,
Universidade Federal do Rio de Janeiro, Brasil.
Teredinidae mollusks have their wood-boring lifestyle supported by the
symbiotic association with the cellulolytic/nitrogen fixing gammaproteobacterium
Teredinibacter turnerae. T. turnerae colonizes bacteriocytes within the invertebrate
gills and besides the nutritional support to the host, this bacterium possibly produces
bioactive compounds in symbiosis. T. turnerae was observed in all Teredinidae
species analyzed worldwide and horizontal transfer of symbionts is speculated.
Although this bacterium can be cultured in vitro, it has never been observed in a free
living form or associated with other substrates in nature. In this work, we have found
three different species of ciliates in association with the mantle cavity of the
mangrove shipworm Neoteredo reynei, including mostly Boveria teredinidi, and few
observations of Metanyctotherus rancureli and Trichodina sp. PCR with specific T.
turnerae primers were performed with bulk DNA of the three ciliates species.
Interestingly the shipworm symbiotic bacterium was detected in B. teredinidi
samples. Freshly collected B. teredinidi were observed under TEM and two different
Gram-negative rod shaped bacterial morphotypes were detected in the ciliate
cytoplasm. It is evident that both morphotypes are well established in the ciliate
cytoplasm and we still do not know if they represent distinct bacterial species or
distinct forms of the same bacterium. It could possibly represent the first detection of
T. turnerae naturally apart from Teredinidae hosts. In vitro phagocytic assays were
performed with B. teredinidi and T. turnerae Gfp+. Intracellular bacteria expressing
Gfp were observed in approximately 70% of the Ciliates after 24h of incubation.
Morphological and molecular characterization of B. teredinidi was also performed
and the 18S rRNA gene sequence was obtained for the first time for the
Thigmotrichida ciliate group. Taken together these data indicate that B. teredini may
play a role in the Teredinidae/T. turnerae system and this is being deeply
investigated.
Fine Structure of Cortex and Endoplasm of the Ciliate Parametopidium
circumlabens (Biggar et Wenrich, 1932) (Cilophora: Armophorea), Endocommensal
of Sea-urchins. INÁCIO D. DA SILVA-NETO. Laboratório de Protistologia,
Departamento de Zoologia, Universidade Federal do Rio de Janeiro, Brazil.
This study concerns Parametopidium circumlabens, found in the digestive tract
of sea-urchins. The somatic infraciliature is formed by dikinetids whose the two
kinetosomes (Ks) are ciliferous; with the anterior Ks are associated a classical ribbon
of transverse microtubules (T1), plus 1 or 2 microtubules which could represent a
second transverse ribbon (T2); the posterior Ks has a ribbon of postciliary
microtubules, bordered by dense material, and a well-striated kinetodesmal fiber.
One or 2 isolated microtubules, located between the two Ks, would be transverse
fibers of the posterior Ks. A dense material, which encircles the basis of Ks, gives
rise to spurs. Longitudinal microtubules run along the left and right sides of the
dikinetids. On the somatic preoral lobe, the dikinetids have two supernumerary
derivates: first, an endoplasmic microfibrillar network with condensation nodes
connects the basis of all the Ks; second, a cylindrical dense axis starts from the left
of the posterior Ks and extends deeply into the endoplasm where it becomes thinner
and thinner, this axis is partially surrounded by a sheet of microtubules on its whole
length. The paroral organelle (diplostichomonad) and the adoral organelles
(paramembranelles) each give rise to a nodded microfibrillar network,
nematodesmata originating from one paroral row surround dorsal naked wall of the
buccal area. P. circumlabens (Armophorea) shares some characters with
Heterotrichida by its somatic dikinetid (except kinetodesmal fiber) and adoral
organelles, and some characters with Clevellandellida by its paroralorganelle, its
kinetodesmal fiber, and microfibrillar network associated to thesomatic dikinetids.
Structure and Ultrastructure of Lichnophora chattoni Villeneuve-Brachon, 1939
(Ciliophora, Spirotrichea), Associated to Zyzzyzus warreni Calder, 1988 (Cnidaria,
Hydrozoa). INÁCIO D. DA SILVA-NETO1,*, CARLOS J. A. CAMPOS2, THIAGO
DA S. PAIVA1, ROBERTO J. P. DIAS1 AND ALVARO E. MIGOTTO3.
1
Laboratório de Protistologia, Departamento de Zoologia, Universidade Federal do
Rio de Janeiro, Brazil; 2Centro de Biologia Marinha/CEBIMar, Universidade de São
Paulo, São Paulo, Brazil; 3Centre for Environment, Fisheries and Aquaculture
Science (Cefas), UK.
A new population of Licnophora chattoni, found in association with a hydroid, is
redescribed using light and electron microscopy. In vivo observations have shown
the presence of numerous bright cortical granules. The flexible and transparent body
ranges in size from 50-65 µm in length and 25-37 μm at its widest part, and has three
distinct regions. The anterior is oval and linked by a neck to the posterior region, the
adhesive disc. On the edge of the ventral region of the body is the adoral zone of
membranelles (~ 81 external and ~ 24 infundibular). The paroral membrane extends
through a groove in the body to the adhesive disc; it is formed by a row of long cilia
arranged in monokinetids. Two dorsal kineties are present, one along the right body
margin and another around the neck. The adhesive disc has a mean diameter of 18
µm, lacks cilia in the area above the velum, which covers a row of dikinetids bearing
long cilia and four dikineties, two or three of which are interrupted on the ventral
surface. 9-12 macronucleus fragments are distributed in the cytoplasm, of which all
but two are connected by conspicuous isthmuses. Two other fragments are located in
the adhesive disc and between those there is an ovate micronucleus. The general
morphology of L. chattoni epibiont on Z. warreni is similar to the original
description, differing in body size and number of fragments of the macronucleus.
The ultrastructural features are very similar to those of L. auerbachii.
Ecto-phosphatase Activity on the Protist Euglena gracilis. THAÍS S. SILVEIRA1,2,
JOSÉ ROBERTO MEYER-FERNANDES2. 1Instituto de Microbiologia Prof. Paulo
de Góes, 2Instituto de Bioquímica Médica; Universidade Federal do Rio de Janeiro,
Brazil
Ecto-enzymes are membrane proteins with active sites facing the external
medium rather than the cytoplasm. They are found both in microorganisms and in
higher eukaryotes. Ecto-phosphatases are a group of ecto-enzymes that generate
inorganic phosphate from phosphorilated substrates and may be involved in cell
nutrition. Euglena gracilis is a model protist for areas as diverse as toxicology
assays, mass production of biotechnological relevant compounds and photo-/
gravitaxis phenomena studies. Here we characterize an ectophosphatase activity
from E. gracilis and investigate this enzyme as a candidate tool for toxicity
bioassays. To evaluate the presence of an ectophosphatase in E. gracilis, this
flagellate was grown in a rich medium, under a 12 h light/12 h dark cycle. Ectophosphatase activity was determined spectrophotometrically measuring the rate of pnitrophenol (p-NP) production from the hydrolysis of the artificial substrate p-nitrophenolphosphate (p-NPP). Intact cells were incubated for 1 h at 25 °C in a
reaction mixture containing 15 mM MES buffer, pH 5.5 and 5 mM p-NPP, as
substrate. E. gracilis show an ecto-phosphatase activity of 20,5 ±2,4 nmol p-NP.(106
cells.h)-1, which is stimulated by copper and inhibited by zinc. Time course of p-NPP
hydrolysis was linear and ecto-phosphatase activity increased linearly with cell
density during the standard 60 min assay. This hydrolysis is preferentially carried in
an acid pH, since activity drops almost 80% in pH 7.0 when compared to pH 4.0.
The supernatant of cells incubated in a reaction mixture without any substrate for 1 h
failed to display a detectable p-NPP hydrolysis when transferred to a complete
reaction mixture, showing that ecto-phosphatases are not released from E. gracilis
surface during the enzymatic assay. The zinc inhibition shown by the ectophosphatase activity described here suggests a possible inhibition by other heavy
metals, one of the most common pollutant tested by E. gracilis biological
parameters.