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A New Ciliate Species Tetrahymena farahensis Isolated from the Industrial Wastewater and Its Phylogenetic Relationship with Other Members of the Genus Tetrahymena.

Byline: Muhammad Tariq Zahid Farah Rauf Shakoori Soumble Zulifqar Nusrat Jahan and Abdul Rauf Shakoori

Abstract

Anthropogenic activities are dumping heavy metals into the environment as waste effluents or integral part of some compounds. This has resulted in an increase in the metal concentration beyond the permissible threshold leading to metal toxicity for all forms of life. Metal resistant ciliates remove metal ions from contaminated water mainly by the process of bioaccumulation. This bioaccumulation is due to low molecular weight metal ions chelating proteins known as metallothioneins. In the present study a new species of Tetrahymena (Tetrahymena1.7) is being reported from the local industrial wastewater. Analysis of Tetrahymena 1.7 SS rDNA showed 99% homology to seven different species of the genus Tetrahymena. SS rRNA secondary structure appeared in 40 helices with 18 variations including 17 substitutions and one deletion. All the variations are present in 6 variable lengths namely V2 V3 V4 V7 V8 and V9.

Cytochrome c oxidase subunit 1 (COX1) gene sequence was quite variable with 91% homology to its closest relative T. thermophila. Since this value was higher than the intraspecific variations (=99% homology) Tetrahyemna1.7 has been considered as a new species i.e. Tetrahymena farahensis. Phylogenetic analysis based on both SS rDNA and COX1 using maximum likelihood and neighbor joining methods showed that Tetrahymena farahensis new species was related to T. thermophila and T. malaccensis. Thus it appeared to be a new member of riboset A1 and coxiset A1 on the basis of SS rRNA and COX1 gene respectively.

Key words: Tetrahymena farahensis new species SS rRNA gene COX1 gene cytochrome c oxidase subunit 1 rRNA secondary structure ribotyping.

INTRODUCTION

Among protozoa ciliates are found in almost all types of aquatic habitats including marine freshwater soil and within animal bodies (Small and Gross 1985). Ciliates are thought to have evolved during early Cambrian period well before the evolution of other eukaryotes (Finlay et al. 2000). They are considered as grazers of bacteria and other microbes (Fenchel 1987; Finlay et al. 2000) thus improving the effluents and increasing turnover rate (Nicolau et al. 2001). Ciliates lack cell wall and give a quick response to environmental changes which indicates that ciliates are better pollution detectors compared to bacteria and fungi (Gutierrez et al. 2003).

Tetrahymena is a genus of small ciliates which were previously studied by the names of Leucophyres and Glaucoma (Furgason 1940). Most of the Tetrahymena species are cosmopolitan in distribution. They have eight ciliated membranous structures including four oral one undulating and three adoral membranelles. Macronuclei of all the members of genus Tetrahymena are transcriptionally active (Simon et al. 2008). Tetrahymena species rapidly grow in axenic medium and thus become a model organism for research in physiology and biochemistry (Hill 1972; Elliott 1973). Tetrahymena show closer genetic resemblance to human as compared to yeast model and share a higher degree of functional conservation to human genes (Eisen et al. 2006). This is a valid point to use Tetrahymena instead of other organisms for ecotoxicological studies (Martin-Gonzalez et al. 1999).

Although Tetrahymena is the best studied organism among ciliates yet it is ambiguous to discriminate among species of Tetrahymena on the basis of physical and morphological characteristics (Kher et al. 2011; Gruchy 1955) particularly when grown in different culture media (Corliss 1973; Struder-Kypke et al. 2001). Use of silver staining for morphological identification has been used as primary method for identification (Corliss 1973). Mating technique has also remained an effective tool to identify the cryptic species within genus Tetrahymena as they are reproductively isolated (Doerder et al. 1995; Nanney et al. 1998). This technique however is not only laborious and impractical (Sonneborn 1959) but is also useless for amicronucleate strains of Tetrahymena which do not adopt sexual reproduction (Nanney and McCoy 1976).

Several of molecular biology techniques have also been used to discriminate among Tetrahymena species. Initially these approaches included isozyme mobility and RFLP (Chantangsi et al. 2007). Later it was found that several species of Tetrahymena have identical RFLP pattern (Jerome and Lynn 1996) while in the case of isozyme mobilities similar polymorphism was observed (Nanney et al. 1998). Nucleotide sequences of histones and SSrRNA genes have also been used to discriminate among different species of Tetrahymena but there are several species which have identical SSrRNA genes (Jerome and Lynn 1996; Struder-Kypke et al. 2001). So SSrRNA subunit is considered as too conserved for species identification (Boenigk et al. 2012).

Analysis of cytochrome c oxidase subunit 1 (COX1) technique is an effective approach to identify different species of invertebrates (Folmer et al. 1994). Using 980bp fragment of COX1 as DNA barcode Lynn and Struder-kypke (2006) could successfully identify the reproductively isolated species of Tetrahymena. Intraspecific sequence divergence for T. thermophila was less than 1% while interspecific distance divergence was up to 12%. Similar results from other experiments suggest that sequence divergence of less than 1% might be used to identify different isolates of a species while a divergence higher than this threshold particularly near to 10% can be used to discriminate different species (Chantangsi et al. 2007) and to identify new species.

Phylogenetic relationships among different species have also been established on the basis of DNA-DNA hybridization (Allen and Li 1974) and analysis of SSrRNA gene (Sogin et al. 1986; Jerome and Lynn 1996; Chantangsi and Lynn 2008). There is a need however for a sequence which is variable enough but not up to the extant where homologues are difficult to arrange (Pace et al. 1989). Hebert et al. (2003) have suggested the use of a 650bp long fragment of COX 1 as universal barcode since its variable domains are useful in establishing phylogenetic relationships among Tetrahymena.

In this study we have used the analysis of SS rRNA and COX1 genes for identification of a ciliate protozoan.

MATERIALS AND METHODS

Samples of industrial wastewater collected from Preliminary Tanneries Wastewater Treatment Plant Kasur were brought to the lab for microscopic observation. On the basis of size shape pattern of ciliary lining and pattern of locomotion of the organisms (Edmondson 1966; Curds et al. 1983; APHA 1989) they were identified as Tetrahymena 1.7. Wastewater samples besides ciliates also contained algae fungi and bacteria. Algae were eliminated by keeping the cultures in dark. Fungizone/Amphotericin B (1g/ml) was also added to remove fungal contamination. Ampicillin (100g/ml) kanamycin (50g/ml) and chloramphenicol (20g/ml) were used to inhibit the growth of bacteria when required.

Primarily this culture was maintained in Bold-basal salt medium (Shakoori et al. 2004). Considering that identification of Tetrahymena was not reliable exclusively on the morphological characteristics molecular tools were used to identify and discriminate among different species. SS rRNA and mitochondrial COX 1 genes were used as markers genes for identification up to species level and for further phylogenetic analysis.

DNA barcoding

For this isolated DNA was amplified for SS rDNA and COX1 genes which were afterwards cloned sequenced and analyzed for nucleotide homologies and analyzed for variations.

PCR amplification and nucleotide sequencing of SS rRNA gene

Genomic DNA was isolated from rapidly growing log phase Tetrahymena cells. SS rRNA gene was PCR amplified using the following primers:

EukF (5'-AATATGGTTGATCCTGCCAGT-3')

EukR (5'-TGATCCTTCTGCAGGTTCACCTAC-3').

The reaction mixture (50 l) contained 1X NH4(SO4)2 buffer 2 mM MgCl2 0.2 mM dNTPs (Fermentas #RO181) 0.4 pmol each primer 2.5 U Taq DNA polymerase (Fermentas #EP0402) and 1g genomic DNA. PCR was performed in GeneAmp thermocycler with initial denaturation of 5 min at 94oC followed by 35 cycles each of denaturation at 95oC for 50 sec annealing at 51oC for 45 sec and elongation at 72oC for 50 sec. It was followed by a final elongation step at 72oC for 7 min. PCR product was loaded on 1% agarose gel and electrophoresis was performed at 80V for 45min.

Amplified PCR product of SS rRNA gene was ligated in pTZ57R/T cloning vector using InstaClone PCR cloning kit (#K1214). E.coli DH5a competent cells were transformed with these recombinant plasmid (Sambrook and Russel 2001).

Purified recombinant plasmid (about 300ng) containing SS RNA gene was sent to Macrogen Korea for nucleotide sequencing. Sequence homology was performed by Basic Local Alignment Search Tool (Altschul et al. 1990).

PCR amplification and nucleotide sequencing of COX1 gene A fragment (986nt long) of COX1 gene was PCR amplified using the following primers (Lynn and StrA1/4der-Kypke 2006; Folmer et al. 1994):

CoX-F (5'- TCAGGTGCTGCACTAGC-3')

Cox-R (5'-TAAACTTCAGGGTGACCAAAAAATCA-3')

The composition of the PCR mixture was the same as for SS RNA gene amplification.

PCR was performed in GeneAmp thermocycler with initial denaturation at 94oC for 5min followed by 35 cycles each of denaturation at 94oC for 45sec annealing at 53oC for 35 sec and elongation at 72oC for 40sec; final extension was given for 7 min at 72oC. Amplified PCR product of COX1 gene was ligated in pTZ57R/T cloning vector using InstaClone PCR cloning kit (#K1214). E. coli DH5a competent cells were transformed with recombinant plasmids. This cloned gene was sent to Macrogen Korea for automated sequencing by ABI sequencer.

Phylogenetic analysis

Phylogeny of Tstrahymena 1.7 was studied by drawing phylogenetic tree based on maximum likelihood and neighbour joining methods using SSrRNA gene and COX1 gene sequences. The phylogenetic tree was constructed through Mega 5.2 software (Tamura et al. 2011).

RESULTS AND DISCUSSION

Tetrahymena isolates

Tetrahymena 1.7 was found in two water samples (pH 6.8-7.9 temperature 33-37oC) of Preliminary Tanneries Wastewater Treatment Plant. Ciliates are integral part of land and water ecosystems (Finlay et al. 2000). They have been found in a number of heavy metal polluted wastewater ponds. This shows they have capability to survive in metal stressed environment (Rehman et al. 2006).

Tetrahymena 1.7 cells were recognized by one pointed and the other broader end centrally positioned nucleus ciliated oral groove near the pointed end and the characteristic longitudinal pattern of ciliary linings (Fig. 1). Average size of Tetrahymena varied from 26.5m - 43m in length and 21m35.5m in width. Different stages of asexual reproduction were also clearly visible under the microscope (Fig. 2).

Molecular identification of Tetrahymena 1.7 on the basis of SS DNA analysis

Genomic DNA of Tetrahymena 1.7 was isolated (Fig. 3a) and amplified for small subunit ribosomal RNA gene (see 1.8kb bands in Fig. 3b) and cloned in pTZ57R/T.

Nucleotide sequencing of the gene showed 1753 nucleotides (accession No. HE820726). nBLAST of 1753 nucleotides of SS rRNA gene of Tetrahymena 1.7 showed 99% homology with seven different species of genus Tetrahymena. Comparative analysis of SSrRNA gene sequence with GenBank derived sequences of other Tetrahymena spp. revealed 98.3-99.1% homology of Tetrahymena 1.7 with other members of the genus (Table I). Through multiple alignment of theswe sequences four regions within the SSRNA gene i.e. 268-277 485-488 1329-1343 and 1660-1672 were identified as more variable (Fig. 4). Only one insertion (C) was observed at nucleotide 488. Both transition and transversion mutations were observed however transversions were of relatively higher frequency. Transition TA was observed at 268 277 285 486 and 519 nucleotide positions while transition AT was observed at 487 661 and 1037. There was no CG or GC transition observed in the sequence.

In case of transverions AG mutations were observed at 269 274 672 723 724 1343 1458 1662 1671 and 1674. Similarly CT transversions were observed at 271 276 753 1329 1330 1337 1660 1663 and 1672 positions. While there was only one AC transversion at 513 nucleotide position and two GT transversions at 1373 and 1535 nucleotide positions.

nBLAST results of SS rRNA gene of

Tetrahymena isolate showed 1% divergence with more than seven different species of Tetrahymena including T. malaccensis and T. thermophila. This shows SS rRNA gene is too conserved to resolve the closely related species. The mean sequence divergence of SS rRNA gene for 45 species of Tetrahymena is 1.560.16. Most of the Tetrahymena species showed sequence divergence of 0-2% (Chantangsi et al. 2007). This is insufficient to discriminate among closely related species (Chantangsi and Lynn 2008). The comparison of SS rRNA gene sequence of Tetrahymena 1.7 with other species of genus Tetrahymena showed that the gene was highly conserved with the exception of few intragenic semi-conserved regions which are helpful in establishing the phylogenetic relationships among different members of genus Tetrahymena.

Secondary structure of SS rRNA

The secondary structure models of SS rRNA gene are being used as topographical markers for the analysis of phylogenetic affiliations. These indispensable marker systems have proven to be helpful in exploring potentially useful information among closely and remotely related organisms. Secondary structure models for SS rRNA genes have been documented by various authors (Hirt et al. 1994; Struder-Kypke et al. 2001; Wuyts et al. 2001; Xia et al. 2011; Lee and Gutell 2012). Due to the slowly evolving property of SS rRNA the basic molecular structure of all eukaryotes is conserved throughout evolution except the nine expansion segments (V1-V9). Among these major lengths variable regions V2 V4 and V7 have been reported as fast evolving segments (Hwang et al. 2000; Alkemar and Nygard 2003 2004; Alvares et al. 2004). V6 is thought to be the least variable region in all eukaryotes (Gonzalez and Schmickel 1986).

Table I.- Distance matrix for SS rRNA gene of T. farahensis new species shows its percent identity and divergence with other closely related Tetrahymena species.

###Percent Identity

Divergence###1###2###3###4###5###6###7###8

###1###99.1 98.5 98.5###98.5 98.4###99.1 98.5###1

###2###0.7###99.0 99.0###99.0 98.9###99.8 99.0###2

###3###1.3###1.0###99.5###ggs 99.4###99.0 100.0 3

###4###1.3###1.0###0.5###100.0 99.7 99.0 99.5###4

###5###1.3###1.0###0.5###0.0###99.7###99.0 99.5###5

###6###1.4###1.2###0.6###0.3###0.3###98.9 99.4###6

###7###0.7###0.2###1.0###1.0###1.0###1.0###gg.o###7

###8###1.3###1.0###0.0###0.5###0.5###0.6###1.0###8

###1###2###3###4###5###6###7###8

Secondary structure model of SS rRNAs of Tetraymena 1.7 shown in Figure 5 is folded into 40 helices. Helices numbering system was followed by Nelles et al. (1984). Comparison with other members of genus Tetrahymena showed that all of the variability is contributed by 6 major variable lengths namely V2 V3 V4 V7 V8 and V9 while V1 V5 and V6 regions appear as more conserved with compound single helices. From the conserved arrangement of these helices it can be deduced that all of the mutations are found either in internal or terminal bulges which retained the stable structural configuration in species.

Phylogenetic analysis of Tetraymena 1.7 on the basis of SS rRNA gene

Phylogenetic analysis of Tetraymena 1.7 with other species was performed by drawing phylogenetic tree using neighbour joining method (Fig. 6) on the basis of their SSRNA gene. All different species of Tetrahymena are clustered in different groups termed as ribosets by Nanney et al. (1989). Tetraymena 1.7 appears along with T. malaccensis and T. thermophila which are members of ribose A1. While considering the major grouping T. farahensis appears to be a member of borealis group instead of australis group.

Sogin et al. (1986) using molecular markers worked on the phylogenetic relationships among members of genus Tetrahymena. They are separated into two major groups i.e. australis and borealis (Struder-Kypke et al. 2001). The australis group consists of riboset C which is homogenous while borealis group with riboset A and B is quite heterogeneous (Preparata et al. 1989). Because of very little genetic differences the branches are not strongly supported by high boot strap value (Struder-Kypke et al. 2001). Some species with similar SS rRNA gene can be distinguished on the basis of their morphology (Roque et al. 1970).

Molecular identification of T. farahensis on the basis of COX1 gene

COX1 is more variable and used by the protozologists to discriminate different species of Tetrahymena and other ciliates. A 986 nucleotide fragment of COX1 gene was amplified (Fig. 7) cloned in pTZ57R/T. Cloned fragment of COX1 gene was sequenced from Macrogen Korea using M13 forward and reverse primers. Peaks read through Chromas Lite 2.1 showed a 986 nucleotide fragment of COX1. NCBI nucleotide BLAST showed its 91% homology with T. thermophila with 99% query coverage.

The amplified sequence was submitted to EMBL nucleotide sequence database under accession No. HG710169. Multiple sequence alignment of Tetrahymena 1.7 COX1 with already reported COX1 of different Tetrahymena species showed that the gene is highly variable as compared to SS rRNA (Fig. 8). Homology of COX1 gene of Tetraymena 1.7 with most closely related species of Tetrahymena falls in the range of 89.1-91.1% (Table II).

Table II- Distance matrix for COX1of T. farahensis new species shows its percent identity and divergence with other closely related Tetrahymena species.

Tetrahymena isolates having less than 1% variation in COX1 sequence are considered as same species while sequence divergence between 1 to 12% indicates different species (Lynn and Struder-kypke 2006). Kher et al. (2011) also used COX1 gene as identification marker for Tetrahymena species. However according to the intraspecific divergence in Tetrahymena is likely to increase with spreading geography so it would be safer to say that strains with higher than 5% divergence are separate species. The GC content of the Tetraymena 1.7 COX1 was 27.7% which was slightly higher than T. pigmentosa T. mobilis (25% in both) and T. hyperangularis (26%) (Lynn and Struder-kype 2006). Chantangsi et al. (2007) has also elaborated the role of COX1 in species identification and calculated the value for mean intraspecific divergence for COX1 as 0.95% while interspecific divergence as 10.47%. Thus interspecific divergence is 11 to 58.2 folds higher as compared to intraspecific divergences.

Keeping in view the above parameters Tetrahymena 1.7 can be considered as a new species which is named here as Tetrahymena farahensis after my supervisor Dr. Farah Shakoori. Phylogenetic analysis of T. farahensis on the basis of COX 1 gene sequence COX1 appears to be more variable phylogenetic marker as compared to SS rRNA gene. Figure 7 shows phylogenetic tree inferred on the basis of neighbour joining method using COX1 nucleotide sequences. T. farahensis retains its homology with the same species as in case of SSr RNA gene. The branching pattern is supported by a high boot strap value.

Phylogenetic tree drawn on the basis of T. farahensis COX1 nucleotide sequences clearly shows its close homology with T. malaccensis T. thermophila and T. paravorax.

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Author:Zahid, Muhammad Tariq; Shakoori, Farah Rauf; Zulifqar, Soumble; Jahan, Nusrat; Shakoori, Abdul Rauf
Publication:Pakistan Journal of Zoology
Article Type:Report
Geographic Code:9PAKI
Date:Oct 31, 2014
Words:4716
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