Introduction

Internal Transcribed spacers are widely used to resolve the phylogenetic relationship for closely related taxa due to their relatively rapid evolution rate (Baldwin, 1992; Schlotterer et al, 1994; Mai and Coleman, 1997; Weekers et al, 2001; Oliver et al, 2002, Ashokan and Pillai, 2008). Internal transcribed spacer refers to a piece of non-functional RNA situated between structural ribosomal RNA on a common precursor transcript. Many parasitologists used internal transcribed spacers and ribosomal DNA as tool to resolve the controversial phylogenetic dispute of cestod parasites (Skerikova et al, 2007; Luo et al, 2003; Literak et al, 2006; Kralova et al, 2001;Magnish et al, 2002; Wickstron et al, 2005; Gao et al, 2007; Logan et al, 2004; Haukisalmi et al, 2008 ). Skerikova et al, 2007 used 18SrDNA to investigate phylogeny of European species of tapeworm genus Proteocephalus. The study of Luao et al (2003) in the genus Digramma and Ligula (Pseudophyllidea) using the entire internal transcribed spacer of the ribosomal DNA showed low level of nucleotide variation between the two genera may imply that cestodes in the genus Digramma is synonym of Ligula. The phylogenetic study of 20 samples of pseudophyllidea cestodes of family Diphyllobothridea from different hosts and geographical region using ITS2 rRNA shows controversy in the previous phylogeny of these groups (Logan et al, 2004). The phylogenetic study of Paranoplocephala alternata and P.artica using ITS1 shows they are sister taxa and the latter species probably diverged from P.alternata in eastern Beringia (Haukisalmi et al, 2008).

The transcripts folding structure of the ITS2 provide some signals that guide the ribosomal coding region when they are processed into small, 5.8S and large ribosomal RNA (Van der Sande et al, 1992; Van Nues et al, 1995). The potential to predict the folding structure has enhanced the role of ITS in phylogenetic studies, since it is important to guide reliable sequence (Michot et al, 1999). The secondary structure can be predicted by many methods like electron microscopy (Gonzales et al, 1990) chemical and structure probing (Yeh and Lee,1992; Van Nues et al, 1995 ) and computer software program (e.g. mfold and sfold) which utilize minimum free energy values (Zuker and Steigler, 1981). By using mfold software a secondary structure for the ITS2 with 4 domains (I~IV) has been proposed for green algae, flowering plants, fruit flies, parasitic flat worms, gastropods and mouse (Schlottere et al, 1994; Mai and Coleman, 1997; Michot et al, 1999, Coleman and Vacquiere, 2002; Gottschling and Plotner, 2004). A highly conserved sequence is situated around a central loop and at the apex of a long stem in the 3´- half (Joseph et al, 1999). Due to high rate of sequence variation of transcribed spacers this may exhibit dramatic size variation and extensive sequence divergence even among moderately distant species (Michot et al, 1983; Furlong and Made, 1983). Nevertheless, the presence of phylogenetically conserved secondary structure elements in the 5´ externally transcribed spacer was recently revealed by the comparative analysis of a limited set of vertebrate sequence (Michot and Bachellerie, 1991).The coding region which has been most widely used in mites is the ITS2. Molecular phylogenetic study in mite Tetranchus (Navajas et al, 1992) agreed closely with morphology but sequence two sympatric species of Eotetrnchus from different hosts show substantial genetic divergence.

The over all literature cited shows that ITS2 are widely used to resolve the phylogeny of cestodes and many other invertebrates. Utility of ITS2 in phylogenetic study is limited with regards to particular species of cestodes belongs to limited families. Thus the present investigation was concentrated to reveal the phylogeny of cestodes from different families using ITS2 and 5.8S rDNA genes.

Materials and Methods

ITS2 and 5.8S rDNA sequences of 12 cestodes from eucestoda belongs to different orders (Pseudocephalidea, Cyclocephalidea, Proteocephalidea, Anaplocephalidea and Cyclocephalidea) that are deposited in GenBank were investigated. The accession numbers of ITS2 and 5.8S gene are:-

Figure 1

Sequence alignment

Motif finding

Secondary structure prediction

RNA fold

Phylogenetic analysis

Results

Sequence analysis of ITS2 and 5.8S regions

The length of ITS2 of 12 selected cestodes ranged in size 444bp and 677bp. Twelve dispersed but unambiguously conserved sequence segments encompassing about a third of the ITS2 length have been identified (Fig1). They were interspersed with variable regions and gaps where size variations accumulate. The characteristics of sequences for each isolates are shown in the table 1. The length variations were observed with maximum length being 677bp in Silurotaenia siluri and minimum 444bp in Chrepdobothrium erasi. The ITS2 sequence of ligula and Digramma shows similarity in length, bases energy level and number of stems. The G+C content for the 2 regions of rDNA (5.8S and ITS2) of all the isolates ranged from 13.75% to 16.18%. For ITS2 regions the sequence identities ranges, with maximum 91.2% similarity between Digramma interrupta and minimum similarity between Bothricephalus acheilognatha and Stylesia globipunctata (Table3). ITS2 region shows simple tandem repeats were present at various locations along the ITS2. The sequence similarity is more towards the 5´ end and with dispersed conserverdness in the middle than towards the 3´ end.

Figure 2

Table1. Ribosomal DNA ITS2 and 5.8S ribosomal DNA gene sequence data in 12 cestodes species

Figure 3

Fig2: Phylogenetictree of cesstodes species ITS2 sequence

Figure 4

Secondary structure in ITS2 and 5.8S regions

Secondary structures of ITS2 and 5.8S regions were given in Table 2. The secondary structure of the cestodes from different hosts belonging to different order was classified into four groups based on the analysis of conserved stem and loops (Fig 2). Class I includes Septilifer maringaensis (Stem >15), class II includes Ligula sps,

Digramma interrupta, Stylesia gllobipunctata, Proteocephalus thymalis, Taenia saginata, Bothricephalus acheilognatha, Zygobothrium megacephalum (Stem Between 18 to 20), class III includes Thomassioscolex didelphidis, Chrepidobothrium erasi and Mesocestodes litteratus (Stem between 15 to 17), class IV includes Silurotaenia siluri (Stem >20).The common motifs identified in ITS2 shows repetition of ATGT many times (Table 4). Apart from the common conserved motifs shared among the species that are categorized into different classes, variable regions also do exists. The observed similarities at the secondary structural level (Fig3) are further reflected at energy level and of ITS2 and 5.8S region of various selected species of cestodes. The secondary structure features of various species were classified into four groups (Fig 2). Class I includes (Mesocestodes litteratus and Septilifer maringaensis), Class II includes (Stylesia gllobipunctata and Taenia saginata), Class III includes (Bothricephalus acheilognatha, Chrepidobothrium erasi, Thomassioscolex didelphidis, Zygobothrium megacephalum and Silurotaenia siluri), Class IV includes (Ligula sp and Digramma interrupta

Figure 5

Table2: Secondary Structural Feature of rDNA ITS2 and 5.8S rDNA gene in 12 Cestod species.

Figure 6

Table 3: Minimum and maximum pair wise genetic distance (in %) Within and among analyzed Cestodes species

Figure 7

Figure 10

Fig 4: Two−dimensional histogram of base pairs in sequence of Ligula spp and

Figure 9

Table 4: Sequence Repeats found in the ITS2 sequence

Discussion

Tapeworms are a group of parasitic worms that live in the intestinal tracts of some animals including man. Several different species of tapeworms can infect human. Tapeworm infection pose serious public problem in many less developed countries due to poor sanitation conditions (Garcia et al, 2003). The tapeworms are biologically known as

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cestodes. The phylogeny of cestodes is one of the most controversial one in front of systematists. Monophyly of euestoda was firmly corroborated by the study of Hoberg et al (1997). The molecular study reveals that the morphological charcter based classifications of the cestodes are controversial (Lao et al, 2003). The low level of nucleotide relation between the two genera may imply that cestodes in the genus Digramma are paraphyletic to the Ligula genus, and Digramma is synonym of ligula (Lao et al, 2003). In our study it is observed that ligula and Digramma shows high similarity in nucleotide sequence, negative free energy, number of stems and the 2DHistogram (Fig.4). It indicates that Ligula and Digramma phylogeny to be realigned.

In the present investigation, the ITS2 and 5.8S rDNA sequence reflected the trend observed in phylogeny. The more distantly related the less was the convergence at the ITS2 level (Figure-1 and 2). However accumulated substitutions in ITS2 leading to length variation also had a profound effect on the conserved ness among the structure. The length of ITS2 is more or less similar in all the isolates except Stylesia gllobipunctata, Mesocestodes litteratus, Taenia saginata, Chrepidobothrium erasi and Septilifer maringaensis, where it is smaller than others. This may be due to insertion affected by many factors including genetic drifts, rate of unequal cross over, relative number of and size of repeats, gene conversion, immigration and the number of loci. (Levinson and Gutman., 1987). The length variation in Taenai saginata may be due to deletion, less tandem repeats and genetic drifts. In all other isolates there is high level of sequence conservation even in the tandem repeats. The conservation is more predominant in the case of ITS2 than 5.8S sequence of all isolates selected. This conservation was further reflected at the secondary structure and energy level. The predicted features of ITS2 using RNADRAW are given in the table 1. ITS2 RNA structure from Bothricephalus acheilognatha have the highest negative free energy (-62.9 K.cal ), followed by Proteocephalus thymalis Zygobothrium megacephalum (-50.16 K.cal.) and remain selected species shows free energy -47± 0.45. Visual comparison shows that this is related to the trend in the cladogram given in the figure 1 and2. This convergence at secondary structural level among a few species from different hosts may be due to the evolutionary pressure on ITS2 to maintain the RNA secondary structure involved in post -transcriptional processing of rRNA (Shinohara et al, 1999). Secondary structure prediction for ITS2 regions shows that these domains base pair to be form a core region central to several stem features implying that conserved ness is more important for the proper rRNA folding pattern (Wesson et al, 1992). The conserved ness is more obvious in the case ITS2 indicating ITS2 provide a better understanding the deep phylogeny of the lower taxa (Navajas and Fenton, 2000). Studies in Tetranchus urticae revealed very low level of variation at the ITS2 locus in this species. In Casava green mite (Mononychellus progresivus) Navajas et al. (1994) found a similar pattern of variation with intraspecific diversity being lows for ITS2. In the present investigation all the isolates studied shows sequence variation in ITS2 regions. The sequence variation is more in ITS2 than 5.8S rDNA. The result suggests that the difference and conserved ness observed between ITS2 and 5.8S of different isolates are not 'neutral' and are not simple accumulated random nucleotide changes, but bare a significant functional trend. The study of Wesson et al. (1992) in mosquito genera found that intra spacer variable regions appear to co-evolve and that ITS2 variation is constrained to some extent by its secondary structure. Study by Van der Sande et al. (1992) in yeast have demonstrated that ITS2 is essential for the correct and efficient processing and maturation of certain ribosomal unit and finally for the efficient functionary of the rDNA cluster.

Conclusion

The selected 12 cestodes species from different hosts present world wide and they cause diseases of various extensions in their hosts. This study reveals although sequence variation was found in ITS2 sequence this did not correlate well with morphology and host suggesting that all the sequences studied belong with a single polymorphic species. All the selected species shows sequence alignment considerably and reflected the similarity in energy level and secondary structure. While the difference between the species is to rank them in different orders is substantiated through motif structure and different loops in the secondary structure of both ITS2 and 5.8S rRNA