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Description on Tylocephalum salunkhi N. Sp. (Cestoda: Lecanicephalidea) and Study of Conserved Domain across Divergent Phylogenetic Lineages of Class Cestoda

Somnath Waghmare1*, Supugade VB2, Sherkhane AS3, Ramrao Chavan4 and Virendra Gomase5

1Department of Zoology, Nowrosjee Wadia College of Arts and Science, Pune, India

2Department of Zoology, LBS College Satara, MS, India

3The Global Open University, Nagaland, India

4Department of Zoology, Dr. Babasaheb Ambedkar Marathawada University, Aurangabad, India

5Department of CS and IT, Dr Babasaheb Ambedkar Marathwada University, Aurangabad, India

*Corresponding Author:
Somnath Waghmare
Department of Zoology
Nowrosjee Wadia College of Arts and Science, Pune-1, India
Tel: 9881926518
E-mail: [email protected]

Received date: May 19, 2014; Accepted date: October 16, 2014; Published date: October 20, 2014

Citation: Waghmare S, Supugade VB, Sherkhane AS, Chavan R, Gomase V (2014) Description on Tylocephalum salunkhi N. Sp. (Cestoda: Lecanicephalidea) and Study of Conserved Domain across Divergent Phylogenetic Lineages of Class Cestoda. J Bacteriol Parasitol 5:203. doi: 10.4172/2155-9597.1000203

Copyright: © 2014 Waghmare S, etal. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Tylocephalum salunkhi n. sp. cestode parasite of Trygon sephen is described on the basis of type material from Ratnagiri (West Coast of Maharashtra, India). The present worms resemble with Tylocephalum marsupium in having all essential morphological characters. Species having scolex oval, rostellum elongated/rounded, presence of four suckers, mature proglottids are broader than long, testes rounded and excretory canal long tube. But the same differ due to number of testes. Hence, it is new species described.

Keywords

Tylocephalum salunkhi n. sp.; Trygon sephen; MSA; Conserve domain; Phylogenetic analysis

Introduction

Tylocephalum salunkhi n. sp. is an endoparasite, and this tapeworm belonging to the class Cestoda. This is pisian gastrointestinal parasite of family Tetragonocephalidae (Cestoda: Lecanicephalidea), and is the most pathogenic and prevalent species infecting Trygon sephen [1-3].

The genus Tylocephalum was erected by Linton, with its type species T. pingue from Rhinoptera quadriloba at Woods Hole, Shipley et Hornell (1906). Total 19 species of this genus recorded till now. Linton reported T. marsupium from Aetobatis narinari, (Euphrasen, 1790) at Tortugas Chincholikar (1976) added one new species to this genus i.e. T. madhukari from Trygon sp. at Ratnagiri. Jadhav and Shinde described T. singhii from Trygon zugei Muller and Henle, at Bombay, India Wankhede and Jadhav (2003) added new species T. gajanane from Trygon sephen at Bombay (West Coast of India). Later on Pawar et al. added two new species of this genus T. babulae from Trygon zugei and T. shindei from Trygon sephen at Ratnagiri (West Coast of Maharashtra, India).

Phylogenetic analyses have become essential to research on Cestoda class for the evolutionary tree of life. It shows taxonomical classification, identification, and naming of organisms, which is usually richly informed by phylogenetics. Multiple sequence alignments are the resource for the annotation of functional units in proteins called as conserved domain. This Conserved domains can be thought of as distinct functional, structural units of a protein from Cestoda class. In molecular evolution of Cestoda class species such domains may have been utilized as building blocks, and may have been recombined in different arrangements to modulate protein function, which can be determined by sequence and structure analysis [4,5].

Material and Methods

Study of Cestode

Sixty nine cestodes were collected from the intestine of Trygon sephe from Ratnagiri (West Coast of Maharashtra, India), during the period Dec-2006 to Dec-2009. Thirty five cestodes were preserved in hot 4% formalin and specimen were stained with Haris Haematoxyline and Borax carmine stain and passed through various alchoholic grades. Cleared in xylene, mounted in DPX and drawing are made with aid of camera lucida. All measurements are given in the milimeter [6,7].

Sources and sequence information of Cestoda class

We have taken sixty three (63) species of Cestoda class, in which targeted is NADH dehydrogenase subunit 3 protein data were used to observe molecular resemble of related protein by phylogenic analysis [8,9].

Multiple sequence alignment of Cestoda class

Multiple Sequence Alignment (MSA) is conducted by COBALT that aligns protein sequences of similar Cestoda class using a combination of distance matrix and approximate parsimony methods. Numerical setting method is used to study the relative entropy threshold, in bits, that must be met for an alignment column to be displayed in red. A larger number indicates higher degree of conservation. The relative entropy is computed as: Σi fi log2 (fi/pi), where i is residue type, fi is residue frequency observed in the multiple alignment column, and pi is the background residue frequency. Identity setting used for only columns with one residue type will be colored in red [10,11].

Construction of a phylogenetic tree for actin protein

Phylogenetic analyses were performed by Fast minimum evolution algorithm and Neighbor Joining algorithms to allow the reconstruction phylogenetic tree of the molecular evolutionary history of various aligned sequences that are useful to align highly evolved gene families clearing evolutionary relationships such as multiple actin proteins [12,13]. Trees were obtained by the methods fast minimum evolution algorithm and Neighbor Joining algorithms. Evolutionary distance is studied by Grishin (protein) model [14,15] and distance between two sequences modeled as expected fraction of amino acid substitutions per site given the fraction of mismatched amino acids in the aligned region and can be computed for fraction of mismatched amino acids larger than 0.75 [16,17].

Results and Description

Observation of Tylocephalum salunkhi n. sp. (2009)

A new species of the cestode genus Tylocephalum salunkhi n. sp. (2009) obtained from the host Trygon sephen is described. A detailed examination of specimens has allowed us to erect a new species Tylocephalum to accommodate the worm. Microscopic observation shows remarkable differences from other known species of Tylocephalum. The new species is designated as Tylocephalum salunkhi n. sp. (2009) (see keys).

Neck absent 1
Neck present 2
1} Vitellaria granular 3
Vitellaria follicular 4
2} Scolex cushion shaped T. yorkei, Southwell, 1925.
Scolex globular 5
Scolex sub-globular 6
Scolex variable in shape T. dierma, Shipley et Hornell, 1906.
Scolex quadrangular in shape 7
Scolex rounded in shape T. bombayensisJadhav, 1983.
3} Ovary “ H” shaped T. hanumanthraoaeShinde, et.al., 1989.
Ovary compact T. madhukaraeChincholikarand Shinde, 1980
Ovary “U” shaped T. alibagensisBhagwan andMohekar,2003
Ovary lobate T. marsupium Linton, 1916.
Ovary bilobed T. gajananaeWankhedeandJadhav,2003.
4} Scolex circular at the anterior part T. aetiobatidis Shipley et Hornell, 1906.
Scolexglobose T. pingue Linton, 1890.
Anterior region of scolex smaller than posterior region T .minimumSubhapradha, 1955.
Anterior region of scolex larger thanposterior region T. elongatumSubhapradha, 1955.
5} Vagina anterior to cirrus pouch T. mehdiiBhagwanet.al., 2002.
Vagina posterior to cirrus pouch 8
Vagina posterioventral to cirrus pouch T. shindeiPawarandJadhav, 2005.
6} Testes below 20 in number T. salunkhin.sp.
Testes above 20 in number T. squatinaeYamaguti, 1934
7} Vagina dorsolateral to cirrus pouch  T. bonasum Ronald A. Campbell et.al., 1984.
Vagina posterior to cirrus pouch T. aurangabadensisJadhavet.al., 1987.
8} Genital pore lies at marginal T. babulaePawarandJadhav, 2005.
Genital pore Sub-marginal T. shindeiPawarandJadhav 2005.

Tylocephalum salunkhi n.sp. - Comparison keys

The scolex is divided into two regions, anterior and posterior. Presences of four suckers which are oval in shape two are placed towards the anterior side and two are placed towards the posterior side of the scolex. Mature segments are longer than broad. The cirrus pouch is oval, elongated, large, Testes 12-13 in number and pre-ovarian. Ovary bi-lobed, ’V’ shaped. The vitellaria are follicular in shape (Figures 1 and 2).

bacteriology-parasitology-Trygon-sephen

Figure 1: Host Trygon sephen.

bacteriology-parasitology-mature-segment

Figure 2: Scolex, mature segment segments of Tylocephalum salunkhi n. sp. (2009).

Systematic Position

Tylocephalum salunkhi n. sp. (2009)

Class: Eucestoda

Order:Lecanicephalidea

Family:Tetragonocephalidae

Genus:Tylocephalum

Species:Tylocephalum salunkhi n. sp. (2009)

Taxonomy summary

Type species: Tylocephalum salunkhi n. sp. (2009)

Host: Trygon sephen

Habitat: Intestine

Locality: Aurangabad M.S., India

Period of collection: Dec. 2006- Dec.2009

Deposition: Helminthology Research Lab, Dept. Of Zoology, Dr. Babasaheb Ambedkar Marathawada University, Aurangabad.

Evolutionary distance

This study, sixty three NADH dehydrogenase subunit 3 protein from Cestoda class is summarized to study the evolutionary distance. The identification of the origin of NADH dehydrogenase subunit 3 protein, multiple sequences analysis, observing the conserved amino acid residues and reconstruct the phylogenetic tree specify the evolutionary history, relationship of Cestoda with different species (Table 1).

Description Accession No of NADH dehydrogenase subunit 3 protein Identity % E value Total Score
Tylocephalum sp. DTJL-2012 gi|374094534|gb|AEY84602.1| 100 3.00E-71 219
Kotorellapronosoma gi|374094534|gb|AEY84602.1| 74.14 1.00E-47 159
Pachybothriumhutsoni gi|374094534|gb|AEY84602.1| 74.14 1.00E-46 157
Clistobothriummontaukensis gi|374094534|gb|AEY84602.1| 71.55 3.00E-46 156
Diphyllobothriumdendriticum gi|374094534|gb|AEY84602.1| 70.69 2.00E-43 149
Acanthobothrium sp. DTJL-2012 gi|374094534|gb|AEY84602.1| 71.55 5.00E-43 147
Diplogonoporusbalaenopterae gi|374094534|gb|AEY84602.1| 68.97 6.00E-43 147
Diphyllobothriumklebanovskii gi|374094534|gb|AEY84602.1| 68.97 2.00E-42 146
Diphyllobothriumnihonkaiense gi|374094534|gb|AEY84602.1| 68.1 2.00E-41 144
Hymenolepisdiminuta gi|374094534|gb|AEY84602.1| 69.23 3.00E-41 143
Sparganumproliferum gi|374094534|gb|AEY84602.1| 68.97 1.00E-40 141
Anchistrocephalusmicrocephalus gi|374094534|gb|AEY84602.1| 70.69 2.00E-40 141
Haplobothriumglobuliforme gi|374094534|gb|AEY84602.1| 66.38 2.00E-40 141
Proteocephalusmacrocephalus gi|374094534|gb|AEY84602.1| 81.48 2.00E-38 136
Rhodobothrium sp. DTJL-2012 gi|374094534|gb|AEY84602.1| 72.41 2.00E-38 136
Diphyllobothriumstemmacephalum gi|374094534|gb|AEY84602.1| 65.52 3.00E-38 135
Grillotiapristiophori gi|374094534|gb|AEY84602.1| 68.97 5.00E-38 135
Litobothriumnickoli gi|374094534|gb|AEY84602.1| 71.55 2.00E-37 133
Spirometraerinaceieuropaei gi|374094534|gb|AEY84602.1| 68.97 1.00E-36 131
Abothriumgadi gi|374094534|gb|AEY84602.1| 70 2.00E-36 131
Diphyllobothriumklebanovskii gi|374094534|gb|AEY84602.1| 73.68 2.00E-36 130
Dipylidiumcaninum gi|374094534|gb|AEY84602.1| 66.09 5.00E-36 129
Didymobothriumrudolphii gi|374094534|gb|AEY84602.1| 64.6 9.00E-36 129
Tetrabothriuserostris gi|374094534|gb|AEY84602.1| 61.21 1.00E-34 126
Nippotaeniachaenogobii gi|374094534|gb|AEY84602.1| 68.1 2.00E-31 117
Taeniamadoquae gi|374094534|gb|AEY84602.1| 57.98 2.00E-30 115
Taeniaovis gi|374094534|gb|AEY84602.1| 57.76 5.00E-30 114
Versteriamustelae gi|374094534|gb|AEY84602.1| 61.26 7.00E-30 114
Khawiabaltica gi|374094534|gb|AEY84602.1| 65.85 1.00E-29 113
Taeniasolium gi|374094534|gb|AEY84602.1| 56.48 3.00E-29 112
Taeniasaginata gi|374094534|gb|AEY84602.1| 57.14 3.00E-29 112
Caryophyllaeusbrachycollis gi|374094534|gb|AEY84602.1| 65.85 5.00E-29 112
Monobothrioides sp. JB-2012 gi|374094534|gb|AEY84602.1| 63.04 1.00E-28 110
Taeniacrassiceps gi|374094534|gb|AEY84602.1| 63.92 2.00E-28 110
Echinobothriumharfordi gi|374094534|gb|AEY84602.1| 70.53 5.00E-28 109
Khawiaparva gi|374094534|gb|AEY84602.1| 51.67 3.00E-27 107
Taeniataeniaeformis gi|374094534|gb|AEY84602.1| 53.51 3.00E-27 107
Taeniaasiatica gi|374094534|gb|AEY84602.1| 54.31 5.00E-27 106
Taeniapisiformis gi|374094534|gb|AEY84602.1| 58.62 7.00E-27 106
Wenyoniavirilis gi|374094534|gb|AEY84602.1| 46.55 1.00E-26 105
Khawiaarmeniaca gi|374094534|gb|AEY84602.1| 59.04 1.00E-26 105
Breviscolexorientalis gi|374094534|gb|AEY84602.1| 65.85 2.00E-26 105
Taeniatwitchelli gi|374094534|gb|AEY84602.1| 63.79 2.00E-26 105
Echinococcusequinus gi|374094534|gb|AEY84602.1| 65.43 3.00E-25 102
Echinococcusvogeli gi|374094534|gb|AEY84602.1| 64.04 5.00E-25 101
Caryophyllaeidesfennica gi|374094534|gb|AEY84602.1| 61.32 6.00E-25 101
Echinococcuscanadensis gi|374094534|gb|AEY84602.1| 64.04 8.00E-25 101
Khawiasinensis gi|374094534|gb|AEY84602.1| 67.06 8.00E-25 100
Caryophyllaeidesfennica gi|374094534|gb|AEY84602.1| 60.75 9.00E-25 100
Echinococcusortleppi gi|374094534|gb|AEY84602.1| 62.92 2.00E-24 100
Fasciola hepatica gi|374094534|gb|AEY84602.1| 64.63 2.00E-24 100
Fasciolagigantica gi|374094534|gb|AEY84602.1| 65.85 3.00E-24 99.8
Glaridacriscatostomi gi|374094534|gb|AEY84602.1| 69.41 7.00E-24 98.6
Monobothriumhunteri gi|374094534|gb|AEY84602.1| 69.41 9.00E-24 98.2
Khawiasaurogobii gi|374094534|gb|AEY84602.1| 61.54 3.00E-23 97.1
Glaridacriscatostomi gi|374094534|gb|AEY84602.1| 68.24 5.00E-23 96.7
Caryophyllaeuslaticeps gi|374094534|gb|AEY84602.1| 64.13 7.00E-23 96.3
Hydatigeraparva gi|374094534|gb|AEY84602.1| 55.45 7.00E-23 95.9
Taeniamartis gi|374094534|gb|AEY84602.1| 60.34 2.00E-22 94.7
Echinococcusoligarthrus gi|374094534|gb|AEY84602.1| 66.67 1.00E-21 92.8
Opisthorchisviverrini gi|374094534|gb|AEY84602.1| 51.69 1.00E-21 92.8
Taeniamulticeps gi|374094534|gb|AEY84602.1| 62.62 2.00E-21 92.4
Taeniahydatigena gi|374094534|gb|AEY84602.1| 58.62 2.00E-21 92.4

Table 1: Sequences producing significant alignments.

Rectangle tree shows rectangular shaped rooted tree, where root is places in the longest edge. Fast minimum evolution algorithm produce un-rooted tree such as ones shown as radial or force in the tabs below. The rooted trees are created by placing a root in the middle of the longest edge (Figures 3 and 4).

bacteriology-parasitology-evolution-algorithm

Figure 3: Rectangle tree - Fast minimum evolution algorithm model- Phylogenetic study of Raillietina echinobothrida with the help of rendering tree showing the evolutionary difference.

bacteriology-parasitology-rendering-tree

Figure 4: Rectangle tree –Cobalt Model- Phylogenetic study of Raillietina echinobothrida with the help of rendering tree showing the evolutionary difference.

Slanted tree shows similar to rectangle, but with triangular tree shape. Neighbor Joining algorithms produce un-rooted tree such as ones shown as radial or force in the tabs below. The rooted trees are created by placing a root in the middle of the longest edge (Figure 5).

bacteriology-parasitology-model-Phylogenetic

Figure 5: Slanted tree - Grishin (protein) model- Phylogenetic study of Raillietina echinobothrida with the help of rendering tree.

MSA

Multiple sequence alignment analysis shows columns with no gaps are colored in blue or red. The red color indicates highly conserved regions and blue indicates less conserved ones. The Conservation analysis can be used to select a threshold for determining which columns are colored in red (Figure 5). Multiple sequence alignment identify conserved motifs and to predict functional role in the variable sites as well as conserved sites show the sequence divergence profile of these actin proteins, which demonstrate the sequence enrichment strategy of these sequences for adaptation to different physiological systems. Here we observed that from all sequences of actin proteins that Cys, Lys, Asp, (Hydrophilic amino acid) Pro, Gly, (hydrophobic amino acid) which is conserved in all peptides having a common ancestor.

That all of these peptides share eight highly conserved cysteines which were involved in the formation of ß-strands are almost conserved. Cysteine (C) is conserved in all sequences at 8 sites. Multiple sequence alignment by COBALT of various Cestoda class species.

An alignment will display the following symbols denoting the degree of conservation observed in each column. ‘*’ indicate that, the residues in that column are identical in all sequences in the alignment 12 is 12.50 %. ‘:’ indicate that strongly similar, conserved substitutions have been observed, 4 is 4.17 %. ‘.’ indicate that weakly similar, semi-conserved substitutions are observed 1 is 1.04 %.

Conserved domain analysis

Molecular study of Cestoda class species shows one conserved domain is NADH-ubiquinone/plastoquinone oxidoreductase, chain 3 (Figure 6).

bacteriology-parasitology-domain-analysis

Figure 6: Conserved domain analysis shows NADH-ubiquinone/plastoquinone oxidoreductase, chain 3.

Conclusion

The presences of various species of the Cestoda were noted on the basis of actual sighting. Above survey with the primary objective of collecting and identifying the species, Sampling for estimation population of available species and understanding the community structure of Cestodes in different habitat types. Phylogenetic analysis of Cestoda class signifies that NADH-ubiquinone/plastoquinone oxidoreductase, chain 3 protein is important components of Cestoda species, are originated from proteins enriched with different sequence specific substitution strategy for biological needs. Comparative analyses specify that the NADH-ubiquinone/plastoquinone oxidoreductase, chain 3 protein demonstrates how proteins are generated within the nature's testing ground for tailor-made biologic needs. Tracing the natural protein engineering scheme of three NADH dehydrogenase subunit 3 protein enrich our knowledge which in turn helps to molecular phylogeny.

Acknowledgement

Authors are thankful to Department of Zoology, Department of CS and IT, Dr Babasaheb Ambedkar Marathwada University, Aurangabad, India; Principal, Nowrosjee Wadia College of Arts and Science, Pune, India for providing the necessary facilities for research.

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