Journal of Parasitic Diseases

, Volume 40, Issue 2, pp 330–338 | Cite as

Utilizing ribosomal DNA gene marker regions to characterize the metacercariae (Trematoda: Digenea) parasitizing piscine intermediate hosts in Manipur, Northeast India

  • Voleentina D. Athokpam
  • Donald B. Jyrwa
  • Veena Tandon
Original Article

Abstract

Freshwater fishes in Manipur, Northeast India frequently harbour several types of metacercariae, which based on morphological criteria were identified as Clinostomoides brieni, Euclinostomum heterostomum (Clinostomidae) and Polylekithum sp. (Allocreadiidae). Molecular techniques utilizing PCR amplification of rDNA regions of larger subunit (LSU or 28S), smaller subunit (SSU or 18S) and inter transcribed spacers (ITS1, 2) were used for molecular characterization of these types. Sequences generated from the metacercariae were compared with their related sequences available in public databases; an analysis of the identity matrices and phylogenetic trees constructed was also carried out, which confirmed their identification. Similarly, the sequences generated from Polylekithum sp. were found to be highly similar to the species of the same genus. The rDNA ITS2 secondary structure provided additional confirmation of the robustness of the molecular marker as a tool for taxon-specific characterization.

Keywords

Molecular rDNA ITS2 28S 18S Metacercaria Fish hosts Clinostomidae Allocreadiidae India 

References

  1. Athokpam VD, Tandon V (2013) A survey of metacercarial infections in commonly edible fish and crab hosts prevailing in Manipur, Northeast India. J Parasit Dis. doi:10.1007/s12639-013-0360-z PubMedGoogle Scholar
  2. Bowles J, Blair D, Mcmanus DP (1995) A molecular phylogeny of the human schistosomes. Mol Phylogenet Evol 4:103–109CrossRefPubMedGoogle Scholar
  3. Caetano-Anollés G (2002) Tracing the evolution of RNA structure in ribosomes. Nucleic Acids Res 30:2575–2587CrossRefPubMedPubMedCentralGoogle Scholar
  4. Caffara M, Locke SA, Gustinelli A, Marcogliese DJ, Fioravanti ML (2011) Morphological and molecular differentiation of Clinostomum complanatum and Clinostomum marginatum (Digenea: Clinostomidae) metacercariae and adults. J Parasitol 97(5):884–891CrossRefPubMedGoogle Scholar
  5. Coleman AW (2003) ITS2 is a double-edged tool for eukaryote evolutionary comparisons. Trends Genet 19:370–375CrossRefPubMedGoogle Scholar
  6. Curran SS, Tkach VV, Overstreet RM (2006) A review of Polylekithum Arnold, 1934 and its familial affinities using morphological and molecular data, with description of Polylekithum catahoulensis sp. nov. Acta Parasitolog 51(4):238–248CrossRefGoogle Scholar
  7. Diez B, Pedros-Alio C, Massana R (2001) Study of genetic diversity of eukaryotic picoplankton in different oceanic regions by small-subunit rRNA gene cloning and sequencing. Appl Environ Microbiol 67:2932–2941CrossRefPubMedPubMedCentralGoogle Scholar
  8. Dzikowski R, Levy MG, Poore MF, Flowers JR, Paperna I (2004) Clinostomum complanatum and Clinostomum marginatum (Rudolphi, 1819) (Digenea: Clinostomidae) are separate species based on differences in ribosomal DNA. J Parasitol 90:413–414CrossRefPubMedGoogle Scholar
  9. Ghatani S, Shylla JA, Tandon V, Chatterjee A, Roy B (2012) Molecular characterization of pouched amphistome parasites (Trematoda: Gastrothylacidae) using ribosomal ITS2 sequence and secondary structures. J Helminthol 86:117–124CrossRefPubMedGoogle Scholar
  10. Gibson DI, Jones A, Bray RA (2002) Keys to the Trematoda, vol 1. Natural History Museum, CABIGoogle Scholar
  11. Goswami LM, Prasad PK, Tandon V, Chatterjee A (2009) Molecular characterization of Gastrodiscoides hominis (Platyhelminthes: Trematoda: Digenea) inferred from ITS rDNA sequence analysis. Parasitol Res 104:1354–1358CrossRefGoogle Scholar
  12. Grajales A, Aguilar C, Sánchez JA (2007) Phylogenetic reconstruction using secondary structures of internal transcribed spacer 2 (ITS2, rDNA): finding the molecular and morphological gap in Caribbean gorgonian corals. BMC Evol Biol 7:90CrossRefPubMedPubMedCentralGoogle Scholar
  13. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 4:95–98Google Scholar
  14. Hillis DM, Bull JJ (1993) An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst Biol 42(2):182–192CrossRefGoogle Scholar
  15. Hillis DM, Davis SK (1986) Evolution of ribosomal DNA; fifty million years of recorded history in the frog genus Rana. Evolution 40:1275–1288CrossRefGoogle Scholar
  16. Hillis DM, Dixon MT (1991) Ribosomal DNA: molecular evolution and phylogenetic inference. Q Rev Biol 66:411–453CrossRefPubMedGoogle Scholar
  17. Jaiswal GP (1957) Studies on the trematode parasites of fishes and birds found in Hyderabad State. Zool Jahrb Abt Syst Part I-IV 85(12):1–72Google Scholar
  18. Jhansilakshmibai K, Madhavi R (1997) Euclinostomum heterostomum (Rudolphi, 1809) (Trematoda): life-cycle, growth and development of the metacercaria and adult. Syst Parasitol 38:51–64CrossRefGoogle Scholar
  19. Jones A, Bray RA, Gibson DI (2005) Keys to the Trematoda, vol 2. Natural History Museum, CABIGoogle Scholar
  20. Kanev I, Radev V, Fried B (2002) Family Clinostomidae Lühe, 1901. In: Gibson DI, Jones A, Bray RA (eds) Keys to the Trematoda, vol 1. CABI Publishing and the Natural History Museum, London, pp 113–120Google Scholar
  21. Keller A, Schleicher T, Schultz J, Müller T, Dandekar T, Wolf M (2009) 5.8S–28S rRNA interaction and HMM-based ITS2 annotation. Gene 430:50–57CrossRefPubMedGoogle Scholar
  22. Krüger D, Gargas A (2008) Secondary structure of ITS2 ribosomal RNA provides taxonomic characters for systematic studies—a case in Lycoperdaceae (Basidiomycota). Mycol Res 112:316–330CrossRefPubMedGoogle Scholar
  23. Michot B, Despres L, Bonhomme F, Bachellerie JP (1993) Conserved secondary structures in the ITS2 of trematode pre-rRNA. FEBS Lett 316:247–252CrossRefPubMedGoogle Scholar
  24. Morgan JAT, Blair D (1995) Nuclear rDNA ITS sequence variation in the trematode genus Echinostoma: an aid to establishing relationships within the 37-collar spine group. Parasitology 111:609–615CrossRefPubMedGoogle Scholar
  25. Nguyen TLA, Nguyen TP, Johansen MV, Murrell KD, Phan TV, Dalsgaard A (2009) Prevalence and risks for fish borne zoonotic trematode infections in domestic animals in a highly endemic area of North Vietnam. Acta Trop 112:198–203CrossRefPubMedGoogle Scholar
  26. Petkevičiūtė R, Stunžėnas V, Stanevičiūte G, Sokolov SG (2010) Comparison of the developmental stages of some European allocreadiid trematode species and a clarification of their life-cycles based on ITS2 and 28S sequences. Syst Parasitol 76:169–178CrossRefPubMedGoogle Scholar
  27. Phan VT, Ersboll KA, Nguyen VK, Madsen H, Dalsgaard A (2010) Farm-level risk factors for fishborne zoonotic trematode infection in integrated small-scale fish farms in North Vietnam. PLoS Negl Trop Dis 4:e742CrossRefPubMedPubMedCentralGoogle Scholar
  28. Prasad PK, Tandon V, Biswal DK, Goswami LM, Chatterjee A (2009) Phylogenetic reconstruction using secondary structures and sequence motifs of ITS2 rDNA of Paragonimus westermani (Kerbert, 1878) Braun, 1899 (Digenea: Paragonimidae) and related species. BMC Genom 10(Suppl 3):S25CrossRefGoogle Scholar
  29. Rinaldi L, Perugini AG, Capuano F, Fenizia D, Musella V, Veneziano V, Cringoli G (2005) Characterization of the second internal transcribed spacer of ribosomal DNA of Calicophoron daubneyi from various hosts and locations in southern Italy. Vet Parasitol 131:247–253CrossRefPubMedGoogle Scholar
  30. Schultz J, Maisel S, Gerlach D, Müller T, Wolf M (2005) A common core of secondary structure of the internal transcribed spacer 2 (ITS2) throughout the Eukaryota. RNA 11(4):361–364CrossRefPubMedPubMedCentralGoogle Scholar
  31. Seibel PN, Müller T, Dandekar T, Schultz J, Wolf M (2006) 4SALE—a tool for synchronous RNA sequence and secondary structure alignment and editing. BMC Bioinform 7:498CrossRefGoogle Scholar
  32. Shylla JA, Ghatani S, Chatterjee A, Tandon V (2011) Secondary structure analysis of ITS2 in the rDNA of three Indian paramphistomid species found in local livestock. Parasitol Res 108:1027–1032CrossRefPubMedGoogle Scholar
  33. Subbotin SA, Madani M, Krali E, Sturhan D, Moens M (2005) Molecular diagnostics, taxonomy, and phylogeny of the stem nematode Ditylenchus dipsaci species complex based on the sequences of the internal transcribed spacer rDNA. Phytopathology 95:1308–1315CrossRefPubMedGoogle Scholar
  34. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739CrossRefPubMedPubMedCentralGoogle Scholar
  35. Tandon V, Prasad PK, Chatterjee A, Bhutia PT (2007) Surface fine topography and PCR-based determination of metacercaria of Paragonimus sp. from edible crabs in Arunachal Pradesh, Northeast India. Parasitol Res 102:21–28CrossRefPubMedGoogle Scholar
  36. Tkach VV, Pawlowski J, Sharpilo VP (2000) Molecular and morphological differentiation between species of the Plagiorchis vespertilionis group (Digenea, Plagiorchiidae) occurring in European bats, with a re-description of P. vespertilionis (Müller, 1780). Syst Parasitol 47:9–22CrossRefPubMedGoogle Scholar
  37. Ukoli FMA (1966) On Euclinostomum heterostomum (Rudolphi, 1809). J Helminthol 40:227–234CrossRefPubMedGoogle Scholar
  38. Verma SC (1936) Notes on trematode parasites of Indian birds. Part. I. Allahabad Univ Studies 12:147–188Google Scholar
  39. Vidyarthi RD (1938) New avian trematodes (family: Diplostomidae) from Indian birds. Proc Nat Acad Sci India 8(3):76–84Google Scholar
  40. Wolf M, Achtziger M, Schultz J, Dandekar T, Mueller T (2005) Homology modeling revealed more than 20,000 rRNA internal transcribed spacer 2 (ITS2) secondary structures. RNA 11:1616–1623CrossRefPubMedPubMedCentralGoogle Scholar
  41. Yamaguti S (1971) Synopsis of the digenetic trematodes of vertebrates, vol 1. Keigaku Publishers, Japan, p 1074Google Scholar
  42. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Indian Society for Parasitology 2014

Authors and Affiliations

  • Voleentina D. Athokpam
    • 1
  • Donald B. Jyrwa
    • 1
  • Veena Tandon
    • 1
  1. 1.Department of ZoologyNorth-Eastern Hill UniversityShillongIndia

Personalised recommendations