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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
hj35good@ajou.ac.kr
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 ravitoteja@andc.du.ac.in
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
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gene transcription level. In the present investigation, we studied the acute toxicity of heavy metal -cadmium and the transcriptional changes of hsp70, metallothionein and glutathione peroxidise gene in spirotrich ciliates. The effect of cadmium on the expression of these gene transcripts was investigated by real-time qPCR. The gene expression increased significantly in cadmium treated cells against the control cells indicating that these genes may be involved in cellular defence mechanisms by elevating their gene expression against heavy metal-induced stress.
EVALUATION OF ACUTE TOXICITY OF HEAVY METALS AND THE ACTIVITY OF ANTIOXIDANT ENZYMES IN FRESHWATER CILIATES
Somasundaram S.1, Abraham J.S.1, Toteja R.1, Makhija S.1, Gupta R.2
1 - Acharya Narendra Dev College, University ofDelhi, Delhi, India
2 - Maitreyi College, University ofDelhi, Delhi, India spoorna4@gmail.com; ravitoteja@andc.du.ac.in Heavy metal concentration in the environment is increasing world-wide due to anthropogenic activities and India is no exception to this. Metals are biologically important though increase in the threshold concentration of heavy metal in the environment may interfere with the metabolic activity of organisms. As studies on the cellular level could detect stress reactions earlier in time compared to common physiological methods, and since ciliates offer higher sensitivity towards heavy metals as compared to other microorganisms mainly due to lack of cell wall, ciliates were used as model organism in this study. In the present investigation, the toxicity of heavy metals, viz., Cadmium and Copper was assessed in freshwater ciliates; Tetmemena sp. and Euplotes sp. It was noted that growth rate exhibited inverse relationship with the heavy metal concentration. Mortality rate and various doses of heavy metals (control, LC30, LC50 & LC70) were determined in the ciliates. Generation of reactive oxygen species (ROS) was determined in these ciliates by using specific fluorescent dye in the presence of heavy metals. Increase in ROS production indicated that heavy metal stress induced ROS in living cells. Since it is known that antioxidant enzymes get activated to detoxify ROS, activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were determined in ciliates at various doses of heavy metals. The levels of SOD and GPx were found to be significantly higher in the presence of heavy metals. This study indicates that antioxidant enzymes can be used as an effective
biomarker to evaluate cytotoxic effect of heavy metals.
GENOMIC AND TRANSCRIPTOMIC SURVEY OF OSMOTROPHIC EUGLENID RHAB-DOMONAS COSTATA
Soukal P.1, Hrda S.1, Karnkowska A.1, Strnad H.2, Hroudova M.2, Vlcek C.2, Hampl V.1
1 - Department of Parasitology, Charles University in Prague, Czech Republic
2 - Institute of Molecular Genetics, Academy of Sciences, Czech Republic vlada@natur.cuni.cz.
Euglenids represent a diverse group of protists with diverse modes offeeding including heterotrophy and photosynthesis, e. g. in Euglena gracilis. Until today, there is not available any complete genome sequence of euglenids and this probably represents the biggest gap in the sampling of eukaryotic genomes. We are running genomic and transcriptomic projects on a primary osmotroph Rhabdomonas costata. To avoid bacterial contamination, the DNA sample was prepared from cells picked using laser microdissection and amplified by GenomePlex®. Almost 1GB of reads was generated in four runs on Illumina MiSeq platform. The current assembly is too fragmented to be used for genome annotation, nevertheless the comparison of transcriptomic and genomic data allow us to estimate gene density and the features of introns. Annotation of the mitochondrial backbone metabolism provides the first data on Rhabdomonas mitochondrion.
GENE TRANSFER ACCOMPANYING THE SECONDARY ENDOSYMBIOSIS OF EUGLENID PLASTID
Soukal P.1, Hrda S.1, Karnkowska A.1, Yubuki N.12, Hroudova M.3, Vlcek C.3, Elias M.4, Hampl V.1
1 - Charles University in Prague, Faculty of Science, Department of Parasitology, Prague, Czech Republic
2 - Charles University in Prague, Faculty of Science, Department of Zoology, Prague, Czech Republic
3 - ASCR, v. v. i., Institute of Molecular Genetics, Department ofGenomics andBioinformatics, Prague, Czech Republic
4 - University of Ostrava, Faculty of Science, Life Science Research Centre, Ostrava, Czech Republic vlada@natur.cuni.cz.
Autotrophic euglenids (Euglenophyta) form a monophyletic group with secondary green plastids, which were most probably acquired by their common ancestor. However, the acquisition of the plastid earlier in the evolution of euglenids (plastid-early hypothesis) cannot be ruled out. The process of organelle acquisition is accompanied by
