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time, mostly due to: (i) its peculiar habitat, (ii) the presence of cytoplasmic bacterial endosymbionts, (iii) a complex life-cycle with unusual morphologies, and (iv) a presumably mutualistic association with its animal host. Moreover, several lines of evidence suggest that the renal sac of an individual tunicate host harbors a complex community of diverse Nephromyces lineages: the multiple-infection hypothesis. We decided to further investigate this hypothesis and elucidate the phylogenetic position of Nephromyces within Apicomplexa by using deep sequencing of renal sacs in order to better understand the developmental dynamics of this symbiosis. Our initial analyses revealed the presence of eleven apicoplast genomes in one single renal sac. Surprisingly, there was considerable sequence divergence among the apicoplast genomes, although their gene content and order was highly conserved. We then used the phylogenetic information contained within 27 apicoplast proteins to infer Nephromyces' phylogenetic placement within Apicomplexa. These preliminary results support the idea that molgulid renal sacs are complex ecosystems inhabited by a diverse community of different Nephromyces lineages. In addition, we clarified the phylogenetic position of Nephromyces as sister to 'core' apicomplexans (Hematozoa+Coccidia), and highlighted the significant intra-clade divergence among these apicoplasts. Future efforts will focus on elucidating the metabolic contribution of each partner to the dynamics of this complex symbiotic system.
PHYLOGENY, DIVERSITY AND EVOLUTION OF AMOEBOZOA: NEW FINDINGS AND NEW PROBLEMS Smirnov Alexey
DepartmentofInvertebrate Zoology, Faculty ofBiology, St. Petersburg State University, St. Petersburg, Russia [email protected]
Amoebozoa is a major group of protists that unifies naked and testate lobose amoebae, varioseans, slime molds and pelobionts. The diversity and systematics of some groups, like slime molds and testate amoebae, possessing stable morphological characters - fruiting bodies or tests - was relatively well studied, also some evolutionary traits derived from morphology were further disproved by molecular phylogeny. In contrast, naked amoeboid protists with no constant shape remain relatively poorly known, molecular phylogeny nearly entirely rearranged earlier views on their evolutionary relationships. SSU-based phylogeny shows that naked amoebae belong to three different amoebozoan lineages and the most of organisms formerly known
as protostelids are dispersed among different groups. The basic finding was the split of naked amoebae in two lineages, named Tubulinea and Discosea and the fact that testate lobose amoebae form just a single branch within Tubulinea. The basal branching of Amoebozoa could not be properly resolved with SSU trees. Multigene data help to clarify many essential questions, e.g. they show that Discosea does not appear to be a monophyletic group. However, with the increment of taxon sampling multigene trees seem to experience the same problems and artifacts as SSU trees did. Recent studies of extreme and exotic habitats revealed a number of new species, especially among the smallest amoebozoans, some were found to be key taxa for rooting Amoebozoan tree. Genbank contains numerous unnamed sequences belonging to new amoebozoan lineages. This indicates that environmental diversity of Amoebozoa is still in much unexplored. Supported with RSF grant 14-14-00474.
TRANSCRIPTOME ANALYSIS OF NAEGLE-RIA FOWLERICYSTS AND TROPHOZOTIES BY NEXT-GENERATION SEQUECING Sohn Hae-Jin1, Seong Gi-Sang1, Kang Heekyoung1, Jung Suk-Yul2, Shin Ho-Joon1, Kim Jong-Hyun3
1 - DepartmentofMicrobiology, Ajou University School of Medicine, Republic of Korea
2 - Department of Biomedical Laboratory Science, Molecular Diagnostics Research Institute, Namseoul University, Republic of Korea
3 - Institute ofAnimalMedicine, College of Veterinary Medicine, GyeongsangNational University, Republic of Korea
Naegleria fowleri, ubiquitous pathogenic amoeba causing the fatal primary amoebic meningo-encephalitis (PAM) in experimental animal and humans, is predominantly living in the ponds, lakes, rivers and swimming pools. N. fowelri trophozoites are encysted under unfavorable conditions such as cold temperature, starvation and desiccation. However, the information in differential expression genes between cysts and trophozoites of N. fowelri is very limited. In this study, RNA-sequencing libraries from N. fowleri cysts and trophozoites were investigated by Next-Generation Sequencing (NGS) analysis. In the NGS database, the assembly procedure resulted in mean full length of 11, 254 nucleotides in total 42,220 transcript contigs and 37.21 % of C+G contents. RNA sequencing indicated that upregulated 143 genes in cysts showed 2 folds expression in comparison with trophozoites and 163 genes were downregulated. These genes were found to participate in Kyoto Encyclopedia
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of Genes and Genomes (KEGG) pathway. KEGG pathway included metabolisms(131), cellular processes(43), environmental Information processing^), genetic information processing(66) and organismal systems(20). On the other hands, by analysis of 10,713 sequences via the gene ontology database, their annotations included biological processes(1,069) which were cellular process(228), metabolic process(214) and single organisms process(193), molecular functions(415) containing catalytic activity(195) and binding(186) and cellular components (923) possessing cells(240) and cell parts(225). Increased differential expression trans-criptome levels in N. fowleri cysts compared to trophozoites were mainly categorized as serine/ threonine protease, kinase, and lipid metabolisms related protein. Finally, this study may provide new insights into the environmental resistant genes or pathogenic related genes in N. fowleri survival and infectivity. Kewords: Naegleria fowleri, cyst, trophozoite, Next-Generation Sequencing, transcriptome.
AMOEBICIDAL ACTIVITY OF CHLORINE DIOXIDE AGAINST PATHOGENIC ACAN-THAMOEBA CASTELLANII, A. POLYPHGA AND NAGELERIA FOWLERI Sohn Hae-Jin1, Song Kyoung-Ju2, Seong Gi-Sang1, Kang Heekyoung1, Kim Jong-Hyun3, Kim Jong-Rak2, Shin Ho-Joon1
1 - Department ofMicrobiology, Ajou University School of Medicine, Republic of Korea
2 - PurgoFarm Co. Ltd, Republic of Korea
3 - Institute ofAnimal Medicine, College of Veterinary Medicine, GyeongsangNational University, Republic of Korea
Pathogenic free-living amoebae, Acanthamoeba spp. and Naegleria fowleri, are widely distributed in soil and water. Several species of Acanthamoeba and N. fowleri can cause serious human infections such as granulomatous amoebic encephalitis (GAE) or primary amoebic meningoencephalitis (PAM) which almost results in death. Recently, there has been an increased incidence of Acanthamoeba keratitis (AK), particularly in contact lens wearers. Chlorine dioxide (ClO2), yellow-green gas is a powerful disinfectant which is 2.5 and 500,000 times more effective than chlorine-based disinfectants and alcohol, respectively. In this study, we examined the amoebicidal effect of ClO2 gas against A. castellanii, A. polyphagia and N. fowleri trophozoites or cysts. ClO2 gas was induced from "Puristic" that it was a ready-to-use product using a tubing stick without necessitating the ClO2 gas generation on site.
Amoebic trophozoites or cysts were exposed to ClO2 gas (0.064ppmv/min) for 12- 48 hr. Amoebae maintained for 12 - 48 hr without exposure to ClO2 gas were used for the control groups. By microscopic examination, amoebic viability was assessed, and then growth rates were estimated. The results showed that the viability of amoebae was effectively inhibited by treatment with ClO2 gas, as which their viability were assessed by re-cultivation with each flesh medium. The growth rates of A. castellanii and A. polyphaga trophpzoites which were exposed to ClO2 gas for 24 hr were decreased by 50 and 60 % respectively. Whereas, the growth rate of N. fowleri exposed with ClO2 gas for 24 hr was completely inhibited. Furthermore, the actin mRNA levels of amoebae checked by RT-PCR were significantly reduced by treatment of CO2 gas. Similarly, the growth rate of A. castellanii and A. polyphaga cysts exposed with ClO2 gas for 12 hr was significantly inhibited. Taken together, it is suggested that ClO2 gas has amoebicidal effect on A. castellanii, A. polyphaga and N. fowleri. Therefore, the chlorine dioxide is proposed as an effective agent for the prevention and control of Acanthamoeba and N. fowleri infections. Keywords: Acanthamoeba castellanii, A. polyphaga, Naegleri fowleri, chlorine dioxide, amoebicidal effect.
MOLECULAR DEFENCE MECHANISMS IN SPIROTRICH CILIATES TO PREVENT HEAVY METAL TOXICITY
Somasundaram S.1, Abraham J.S.1, Toteja R.1, Gupta R.2, Makhija S.1
1 - Acharya Narendra Dev College, University ofDelhi, Delhi, India
2 - Maitreyi College, University ofDelhi, Delhi, India [email protected]
Heavy metal pollution of water is of major concern these days especially in developing countries like India. Anthropogenic activity elevates the concentration of such elements, mainly in aquatic systems. About 85 percent of the water pollution is caused by domestic and industrial sources. A wide range of contaminants are continuously introduced into the water and among these contaminants, heavy metals due to their toxicity, accumulation and non-degradable nature, constitute one of the most dangerous groups. Prolonged exposure to heavy metals such as cadmium, copper, lead and zinc disturb normal biological function and evoke cellular stress response. Due to stress condition, apart from the release of antioxidant enzymes, other proteins like Heat Shock Proteins (HSPs) and Metallothioneins (Mts) are the major ones that get induced and these proteins trigger changes at the
