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Molecular identification of Pseudodiscus collinsi from wild Indian elephant (Elephas maximus indicus) based on ITS-2 Cover

Molecular identification of Pseudodiscus collinsi from wild Indian elephant (Elephas maximus indicus) based on ITS-2

Helminthologia
Volume 62 (2025): Issue 3 (September 2025)
By: A. Anaswara,  S. Arun,  R. Geethu,  N. C. Sreenidhi,  S. Suriya,  S. Harshit,  P. K. Binoy,  V. Anju,  C. K. Deepa,  K. G. Ajith Kumar and  R. Ravindran  
Open Access
|Nov 2025

Figures & Tables

Fig. 1.

Pseudodiscus collinsi: A. Acetyl alum carmine stained adult intestinal fluke (o.s.: Oral sucker; o.p.: Oral pouch; oes.: Oesophagus; i.c.: Intestinal caeca; gp.: Genital pore; vt.g.: Vitelline glands; ut.: Uterus; tes.: Testes; o.: Ovary; sh.gl.: Shell gland; v.s.: Ventral sucker) B. Line diagram of adult Pseudodiscus collinsi C. Oral pouches of adult Pseudodiscus collinsi.
Pseudodiscus collinsi: A. Acetyl alum carmine stained adult intestinal fluke (o.s.: Oral sucker; o.p.: Oral pouch; oes.: Oesophagus; i.c.: Intestinal caeca; gp.: Genital pore; vt.g.: Vitelline glands; ut.: Uterus; tes.: Testes; o.: Ovary; sh.gl.: Shell gland; v.s.: Ventral sucker) B. Line diagram of adult Pseudodiscus collinsi C. Oral pouches of adult Pseudodiscus collinsi.

Fig. 2.

PCR amplification ITS-2 (~400 bp) of P. collinsi (Lane M: 100 bp plus ladder, Lane 1,2: P. collinsi samples, Lane N: Negative control).
PCR amplification ITS-2 (~400 bp) of P. collinsi (Lane M: 100 bp plus ladder, Lane 1,2: P. collinsi samples, Lane N: Negative control).

Fig. 3.

Phylogenetic analysis based on ITS-2 of P. collinsi. The evolutionary history was inferred using the Maximum Likelihood (ML) method. The optimal tree is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the Kimura 2-parameter method and are in the units of the number of base substitutions per site. The rate variation among sites was modelled with a gamma distribution (shape parameter = 1). This analysis involved 25 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There was a total of 335 positions in the final dataset. Evolutionary analyses were conducted in MEGA 11 (Tamura et al., 2021).
Phylogenetic analysis based on ITS-2 of P. collinsi. The evolutionary history was inferred using the Maximum Likelihood (ML) method. The optimal tree is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the Kimura 2-parameter method and are in the units of the number of base substitutions per site. The rate variation among sites was modelled with a gamma distribution (shape parameter = 1). This analysis involved 25 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There was a total of 335 positions in the final dataset. Evolutionary analyses were conducted in MEGA 11 (Tamura et al., 2021).

Primer details and cycling conditions_

GenePrimer nameSequenceNo of base pairs (bp)Product size (bp)Cycling conditionsReferences
ITS2Forward primer GA15′-AGA ACA TCG ACA TCT TGA AC-3′20~400 bp Denaturation: 95°C - 1 min Annealing: 55°C - 2 min Extension: 74°C - 1 min30sec } 1 cycle \left. {\matrix{ {{\rm{ Denaturation: }}{{95}^^\circ }{\rm{C}}\;{\rm{ - }}\;1{\rm{min}}} \hfill \cr {{\rm{ Annealing: }}{{55}^^\circ }{\rm{C}}\;{\rm{ - }}\;2{\rm{min}}} \hfill \cr {{\rm{ Extension: }}{{74}^^\circ }{\rm{C}}\;{\rm{ - }}\;1{\rm{min}}30{\rm{sec}}} \hfill \cr } } \right\}\quad 1{\rm{ cycle }}Anderson and Barker, 1998; Luton et al., 1992
Reverse primer BD25′-TAT GCT TAA ATT CAG CGG GT-3′20 Denaturation: 95°C - 30sec Annealing: 55°C - 30sec Extension: 74°C - 1 min30sec Final extension: 74°7  - min }30 cycles \left. {\matrix{ {{\rm{ Denaturation: }}{{95}^^\circ }{\rm{C}}\;{\rm{ - }}\;30{\rm{sec}}} \hfill \cr {{\rm{ Annealing: }}{{55}^^\circ }{\rm{C}}\;{\rm{ - }}\;30{\rm{sec}}} \hfill \cr {{\rm{ Extension: }}{{74}^^\circ }{\rm{C}}\;{\rm{ - }}\;1{\rm{min}}30{\rm{sec}}} \hfill \cr {{\rm{ Final extension: }}{{74}^^\circ }7\;{\rm{ - }}\;{\rm{min}}} \hfill \cr } } \right\}30{\rm{ cycles }}
DOI: https://doi.org/10.2478/helm-2025-0029 | Journal eISSN: 1336-9083 | Journal ISSN: 0440-6605
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Language: English
Page range: 254 - 258
Submitted on: Apr 9, 2025
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Accepted on: Oct 17, 2025
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Published on: Nov 26, 2025
Published by: Slovak Academy of Sciences, Institute of Parasitology
In partnership with: Paradigm Publishing Services
Publication frequency: Volume open
Keywords:
Amphistomes,
Digenetic trematode,
Pseudodiscus collinsi,
Indian elephant,
ITS-2
Related subjects:
Life sciences,
Zoology,
Ecology,
Life sciences, other,
Medicine,
Clinical medicine,
Microbiology, virology and infection epidemiology

© 2025 A. Anaswara, S. Arun, R. Geethu, N. C. Sreenidhi, S. Suriya, S. Harshit, P. K. Binoy, V. Anju, C. K. Deepa, K. G. Ajith Kumar, R. Ravindran, published by Slovak Academy of Sciences, Institute of Parasitology
This work is licensed under the Creative Commons Attribution 4.0 License.

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