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. 2012 Oct 7;38(1):22–26. doi: 10.1007/s12639-012-0184-2

Molecular characterization of the Indian poultry nodular tapeworm, Raillietina echinobothrida (Cestoda: Cyclophyllidea: Davaineidae) based on rDNA internal transcribed spacer 2 region

Ramnath 1, D B Jyrwa 1, A K Dutta 1, B Das 1, V Tandon 1,✉
PMCID: PMC3909599  PMID: 24505172

Abstract

The nodular tapeworm, Raillietina echinobothrida is a well studied avian gastrointestinal parasite of family Davaineidae (Cestoda: Cyclophyllidea). It is reported to be the largest in size and second most prevalent species infecting chicken in north-east India. In the present study, morphometrical methods coupled with the molecular analysis of the second internal transcribed spacer (ITS2) region of ribosomal DNA were employed for precise identification of the parasite. The annotated ITS2 region was found to be 446 bp long and further utilized to elucidate the phylogenetic relationships and its species-interrelationships at the molecular level. In phylogenetic analysis similar topology was observed among the trees obtained by distance-based neighbor-joining as well as character-based maximum parsimony tree building methods. The query sequence R. echinobothrida is well aligned and placed within the Davaineidae group, with all Raillietina species well separated from the other cyclophyllidean (taeniid and hymenolepid) cestodes, while Diphyllobothrium latum (Pseudophyllidea: Diphyllobothriidae) was rooted as an out-group. Sequence similarities indeed confirmed our hypothesis that Raillietina spp. are neighboring the position with other studied species of order Cyclophyllidea against the out-group order Pseudophyllidea. The present study strengthens the potential of ITS2 as a reliable marker for phylogenetic reconstructions.

Keywords: Cestode, ITS2, Phylogeny, Raillietina echinobothrida, rDNA

Introduction

The Raillietina group of the cyclophyllidean eucestodes conventionally treated as one genus viz. Raillietina Fuhrmann, 1920 (Davaineidae), comprises a large assemblage of over 200 known species and is considered a monophyletic group. Members of the genus Raillietina Megnin, 1881 are obligate, widely-dispersed cestode parasites of avian and mammalian hosts and display a wide range of body forms, life histories, and host associations. Of these, R. echinobothrida is amongst the most prevalent pathogenic cestode parasites of birds throughout the world (Schmidt 1986; Khalil et al. 1994; Mariaux 1996, 1998; Hoberg et al. 1997, 1999) and earns the distinction of being the second most prevalent helminth recorded from the domestic fowl in north-east India (Yadav and Tandon 1991). The adult tapeworm is found in the intestinal lumen of its definitive host, Gallus domesticus and causes characteristic hyperplastic enteritis associated with the formation of granuloma on the intestinal wall of the infected birds (Soulsby 1982), whereas the larval stage resides in various tissues of the insect intermediate host, Tetramorium spp. (Jones and Horsfall 1935; Horsfall 1938; Gogoi and Hazarika 1975). The taxonomy of Cyclophyllidea traditionally depends upon the accurate descriptions of small differences in the size and shape of the scolex, rostellum (unarmed or armed with rows of hooks), suckers and gravid proglottids (Sawada 1965). However, with a rich resource of bourgeoning data the molecular approach is proving to be more rapid, sensible and reliable than that based on morphological data alone for phylogenetic analysis (Brooks and Hoberg 2001).

The rDNA internal transcribed spacer (ITS) sequence data have revolutionized the phylogenetic analysis as a powerful tool in resolving remarkable taxonomic issues and discriminating genera and species across a large variety of organisms, because these regions ITS spacers are more variable and more informative than other parts of the rDNA locus (Hershkovitz and Lewis 1996; Coleman 2003). Furthermore, the ITS2 markers have also been proposed for use in species barcoding and DNA microarrays, thus increasing the detection power of closely related species (Park et al. 2007; Engelmann et al. 2009). In recent years, several molecular tools including sequence data related to conserved domains have unveiled their utility in molecular identification, taxonomy, systematics and phylogeny of floral, faunal and microbial species, too (Huang et al. 2006; Prasad et al. 2009a, b; Wiemers et al. 2009; Yao et al. 2010; Sharma et al. 2012). Since Woese and Fox (1977), the nuclear ribosomal repeat cistrons have also been widely implied for parasites (Littlewood and Olson 2001; Waeschenbach et al. 2007; Jyrwa et al. 2009; Goswami et al. 2009; Tandon et al. 2010; Prasad et al. 2011; Ghatani et al. 2012). Molecular phylogeny has advanced our understanding of comparative studies in basic and applied biology and more generally of their position within the phylum Platyhelminthes (Brooks and Hoberg 2001; Tandon et al. 2010).

The objective of the present study was to broaden the molecular and genetic information on the invasive tapeworm, R. echinobothrida, supplementing the morphological observation. In this study, we assess the validity of the selected ITS2 fragment of R. echinobothrida in discriminating closely related cestode species. Further validation needs to be well established, so as to exploit the ITS2 as well as other markers for robust molecular characterization of this avian parasite.

Materials and methods

The parasite

Live tapeworms (R. echinobothrida) were collected from the small intestine of naturally infected and freshly sacrificed domestic chicken in Shillong and its suburbs, Meghalaya. This species is characterized by the presence of a heavily armed rostellum with two rows of hooklets and four circular suckers on the scolex (Soulsby 1982).

Isolation of genomic DNA and amplification of ITS2 region

Genomic DNA (gDNA) from a single adult worm was isolated using standard phenol/chloroform/iso-amyl alcohol method (Sambrook and Russel 2001). DNA integrity was analyzed on 0.8 % (w/v) agarose gel containing 0.05 % ethidium bromide. ITS2 sequences were obtained by PCR amplification of gDNA using universal flatworm (Schistosoma) primers (3S: 5′-GGTACCGGTGGATCACTCGGCTCGTG-3′ as the forward primer and A28: 5′-GGGATCCTGGTTAGTTTCTTTTCCTCCGC-3′ as the reverse primer) following Bowles et al. (1995). In brief, PCR amplification was performed in a 25 μl reaction mixture containing 1× PCR buffer, 1.5 mM MgCl2, 200 μM dNTPs, 10 pmol of each primer, 1 U of Taq DNA polymerase and 30–50 ng of gDNA using an automated thermal cycler (AB Veriti™-4375786). The cycling conditions were as follows: initial denaturation at 95 °C for 5 min followed by 35 cycles of 95 °C for 30 s, 55 °C for 45 s, 72 °C for 60 s and final extension at 72 °C for 10 min. The amplicons were resolved on 1.5 % (w/v) agarose gel containing 0.05 % ethidium bromide. PCR products were gel eluted and purified using Sigma gel elution kit (GenElute™-NA1111). The amplicons were resuspended in nuclease-free water and sequenced on an automated sequencer at The Centre for Genomic Applications (TCGA), New Delhi.

Sequence analysis and construction of phylogenetic trees

Nucleotide sequences were aligned using ClustalW and BioEdit softwares (Thompson et al. 1994). After alignment, ambiguous regions (i.e., those containing gaps) were removed. Analysis was carried out on all substitutions. Sequences were deposited in GenBank and their accession numbers obtained.

Phylogenetic trees were constructed by comparing the ITS2 sequences of R. ehinobothrida with ITS2 sequences of other cyclophyllidean cestodes available in GenBank using BLAST (Basic Local Alignment Search Tool; http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences were entered, edited, and aligned using ClustalX, in MEGA 5.05 (Tamura et al. 2011). For tree construction distance-based neighbor-joining (NJ) as well as the character-based maximum parsimony (MP) methods were used with branch support was given using 1000 bootstrap replicates.

The evolutionary history was inferred using the NJ method (Saitou and Nei 1987) and a consensus tree inferred from 100 bootstrap replicates taken to represent the evolutionary history of taxa analyzed. The evolutionary distances were computed using the maximum composite likelihood (MCL) method and are in the units of the number of base substitutions per site. The MP tree was obtained using the Close-Neighbor-Interchange algorithm (Nei and Kumar 2000) with search level 3 in which the initial trees were obtained with the random addition of sequences (100 replicates). All positions containing gaps and missing data were eliminated.

Results and discussion

The PCR-amplified product was successfully obtained using the trematode universal primers and is shown in Fig. 1. Sequence analysis of the ITS2 PCR product revealed the size of 600 bp with flanking regions of 5.8S and 28S region; the annotated product ITS2 region was found to be 446 bp in length spanning the ITS2 region. The query sequence was compared with ITS2 sequences of other cyclophyllidean cestodes available in public domain. The ITS2 region of the latter revealed a wide range in size, i.e., 367–938 bp in length. All these species used in the study along with their GenBank accession numbers are listed in Table 1.

Fig. 1.

Fig. 1

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PCR-amplified product of ITS2-gel picture (Lane 1 DNA Ladder (100 bp), Lane 2 purified product)

Table 1.

Cestode species used in this study and GenBank accession numbers for their corresponding ITS2 sequences

Species Country GenBank accession no. Order: family Annotated ITS2 region bp
Raillietina echinobothrida a India JN797628 Cyclophyllidea: Davaineidae 446
Raillietina dromaius Australia AY382320.1 Cyclophyllidea: Davaineidae 830
Raillietina chiltoni Australia AY382319.1 Cyclophyllidea: Davaineidae 781
Raillietina beveridgei Australia AY382318.1 Cyclophyllidea: Davaineidae 938
Raillietina australis Australia AY382317.1 Cyclophyllidea: Davaineidae 841
Hymenolepis diminuta China JF766714.1 Cyclophyllidea: Hymenolepididae 791
Hymenolepis nana Mexico HM536187.1 Cyclophyllidea: Hymenolepididae 531
Hymenolepis microstoma Japan AB494478.1 Cyclophyllidea: Hymenolepididae 501
Taenia hydatigena China FJ886761.1 Cyclophyllidea: Taeniidae 367
Taenia saginata China AY825542.1 Cyclophyllidea: Taeniidae 733
Taenia multiceps China FJ886762.1 Cyclophyllidea: Taeniidae 507
Diphyllobothrium latum b France DQ768176.1 Pseudophyllidea: Diphyllobothriidae Incomplete
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aQuery sequence generated as per this study

bUsed as out-group

Phylogenetic trees were obtained by comparing the ITS2 query sequences of R. echinobothrida with those of other cestode species available in GenBank; Diphyllobothrium latum (a representative of the order Pseudophyllidea and a parasite of piscivorous mammals) was taken as the out-group. Similar topology was observed among the trees obtained by distance-based (NJ) as well as character-based (MP) tree building methods in phylogenetic analysis (Fig. 2). The species included in the analysis mainly clustered into three major clades: the Davaineidae, Hymenolepididae, Taeniidae, with D. latum (Pseudophyllidea: Diphyllobothriidae) at the base of the branch rooted as an out-group, which may indicate its early evolutionary history. The query sequence R. echinobothrida is well aligned and placed within the Davaineidae group, with all species of Raillietina well separated from the taeniid and hymenolepid cestodes (Fig. 2).

Fig. 2.

Fig. 2

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NJ and MP phylogenetic trees of Raillietina spp. and other cyclophyllidean cestode spp. based on ITS sequences (* Query sequence generated as per this study; ** Used as out-group)

It is now widely accepted that rDNA ITS2 region is a useful tool for differentiation of taxa at the level of phylogeny due to the fact that ITS sequences are variable (Wolf et al. 2005). Because of its fast rate of evolution the ITS2 region continues to be applied for phylogenetic reconstructions at the species and genus levels (Alvarez and Wendel 2003; Wolf et al. 2005; Ashokan and Pillai 2008). In context of the cestode group of parasites the orders Cyclophyllidea, Pseudophyllidea, Caryophyllidea and Trypanorhyncha have been assessed on the basis of morphological, ultrastructural and ontogenetic characters (Hoberg et al. 1997, 2001; Beveridge et al. 1999; Bray et al. 1999) and several controversial phylogenetic disputes of cestode parasites have been published (Haukisalmi et al. 2001; Literak et al. 2006). Since in contrast to the bordering regions of ribosomal subunits; sequence is not conserved evolutionarily, thus genetic differentiation is detectable even in closely related groups of organisms. Sequence similarities indeed confirmed our hypothesis that Raillietina spp. are neighboring the position with other studied species of Order Cyclophyllidea against the out-group Order Pseudophyllidea. Morphological variation is either limited or the homology of morphological features remains unclear in tapeworms of the genus Raillietina. Hence, molecular characterization supplements and validates unambiguous identification at species level.

Acknowledgments

Authors thank the Department of Biotechnology (Government of India), New Delhi, for their financial support for the study. Thanks are also due to the Head, Department of Zoology, NEHU, for providing infrastructural facilities.

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