1 Departments of Zoology, 3 Biotechnology & Bioinformatics and 2 Bioinformatics Centre, North-Eastern Hill Univers

1. Phylogenetic reconstruction using secondary structures and sequence motifs of ITS2 rDNA of Paragonimus westermani (Kerbert, 1878) Braun, 1899 (Digenea: Paragonimidae) and related species P. K. Prasad1, V. Tandon1, D. K. Biswal2, L. M. Goswami1 and A. Chatterjee3 1Departments of Zoology, 3Biotechnology & Bioinformatics and 2Bioinformatics Centre, North-Eastern Hill University, Shillong, 793022 Email: - pramod812@gmail.com 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

2. Paragonimus • Zoonotic lung fluke having diversified effect on its final host. • Over 40 species infect lungs of different mammalian hosts. • ~15 species known to infect humans. Pre-adult Egg 80-110×48-60µ Encysted metacercaria ~300- 400µ Adult: 7.5-12 × 4-6×1-3mm (l: w = 2:1) • P. westermani(Kerbert, 1878) - Best known species, human parasite that can undergo development in >16 different snail species and 50 crustacean species. • Status of prevalence and host range in India- not well documented. 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

3. Distribution of human paragonimiasis Species of Paragonimus are encountered in Asia, the Americas, and Africa. 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

4. Distribution in India 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

5. Paragonimus species: India • P. westermani(Kerbert,1878)- Bengal tiger, Amsterdam Zoo collection from India, Indonesia, China • [Syn. P. edwardsiGulati, 1926- civet] • P. compactus(Cobbold, 1859)- HerpestesedwardsiiIndia (Vevers,1923; Ravikumar et al.,1979); Sri Lanka (Dissanaike and Paramananthan, 1962) • P. heterotremusChen and Hsia, 1964- China, Thailand, Laos, Vietnam • Arunachal Pradesh (Narain et al. 2003) • Manipur (Singh,1996) • P. hueitungensisChung et al.,1975- China • Manipur (Singh, 2002) • P. mungoi, P.pantheri – Orissa (Mishra et al.,1976)- nomennudem 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

6. Life Cycle • Infective stage: metacercaria • Infective mode: eating raw fresh watercrabs andcrayfish with metacercariae • Infective route: by mouth • Site of inhabitation: lungs • Intermediate hosts: 1st – snail; 2ndcrab, crayfish • Reservoir hosts: carnivores (tiger, lion, wolf, fox, dog, leopard, cat etc.) • Life span: 5-6 years 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

7. ? Paragonimus westermani is the best-known species. Diploid & triploid forms in N.E. Asia: only diploids elsewhere. Pleurocerid snail hosts ? ? ? Thiarid snail hosts ? Diploid Triploid 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

8. Many other species described. Nearly 50 species, mostly in Asia: over half of these in China alone. This prompted a molecular taxonomy approach. P. asymmetricus P. bangkokensis P. cheni P. divergens P. fukienensis P. harinasutai P. heterorchis P. heterotremus P. hokuoensis P. heuitungensis P. iloktsuenensis P. jiangsuensis P. macrorchis P. menglaensis P. microrchis P. minqinensis P. ohirai P. paishuihoensis P. proliferus P. szechuanensis P. tuanshanensis P. veocularis P. xiangshanensis P. yunnanensis Beijing Chengdu Shanghai (P. westermani not shown here) Guangzhou Euparagonimus cenocopiosus E. hongzesiensis 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

9. Molecular Taxonomy • Molecular tools- allow quick and accurate identification of genetically distinct but morphologically similar species • Genetic markers in nuclear ribosomal DNA (rDNA) • - to resolve taxonomic issues related to various animal groups • - to find phenotypic variants, geographical isolates • ITS rDNA– widely used region, to explore species boundaries in at least 19 digenean (helminth parasites’) families 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

10. The ribosomal DNA gene cluster • Ribosomal genes and their associated spacers are among the most versatile sequences for phylogenetic analysis. • Useful for diagnostic purposes at the level of species. 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

11. Bioinformatic Tools • Similarity search - BLAST • http://www.ncbi.nlm.nih.gov/blast • Phylogenetic prediction - ClustalW/X • http://www.ebi.ac.uk/clustalw • Phylogenetic trees construction -MEGA (Molecular Evolutionary Genetics Analysis) format, • - Distance methods (Neighbour-Joining, Minimum • Evolution, UPGMA) • - Character- state method (Maximum Parsimony) • Bayesian Analysis - Mrbayes 3.1. 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

12. ITS sequence motifs- useful for development of DNA bar coding; short DNA sequences from a standardized region of genome- diagnostic “biomarker” for species identification • (http://www.barcoding.si.edu/) • Molecular morphometrics- • - traditional morphological and molecular sequence • comparisons by measuring structural parameters. • - Geometrical features, bond energies, base composition etc. • of secondary structure to study phylogenetic relationships of • species • (http://www.bioinfo.rpi.edu/applications/mfold) 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

13. Parasite: • Collected from mountain stream crabs of suspected focal area (Changlang Dist. in Arunachal Pradesh). • Metacercariae isolated from muscles by digestion technique. • DNA isolation: • DNA extracted in FTA card (Whatman’s), punching sample discs • Sample discs- washed with FTA Purification reagent and TE Buffer; used for PCR. Materials & Methods 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

14. DNA amplification and sequencing • ITS2 primers used : • 3S: 5’- GGTACCGGTGGATCACTCGGCTCGTG-3’ (forward) • A28:5’-GGGATCCTGGTTAGTTTCTTTTCCTCCGC-3’ (reverse) • [Designed based on conserved sequences of the 5.8S and 28S genes of Schistosoma spp] • Marker- Phi X 174 DNA/ Hae III Digest in agarose gel • PCR products- purified by Genei Quick PCR Purification Kit; • - sequenced in both directions using • primer set 3S-A28 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

15. Molecular Phylogenetic Analysis • Only unique sequences were used in tree construction. • ITS sequences entered in MEGA for phylogenetic trees construction. • Tree building methods- Maximum Parsimony, Neighbor-Joining, UPGMA, • Minimum Evolution. • Branch support given using 1000 bootstrap replicates in MEGA Bayesian Phylogenetic Analysis • Sequences aligned using Clustal W 2.0.7 and converted to NEXUS file. • Analysis carried out with combined datasets using Mrbayes 3.1. • Cladogram and phylogram with mean branch lengths generated, and read by Tree view V1.6.6. 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

16. Motif identification, testing and validation • Sequences motifs identified from aligned sequences of the data set using PRATT software. • Motifs were expressed using DNA alphabet (A,T,C,G) in PROSITE language. • Validation of motifs were performed for each species using a ‘PATTERN MATCHING’ web application. Evaluation through BLAST analysis • Sequences motifs subjected to BLAST algorithms against nonredundant GenBank database of NCBI (nr). • BLAST outputs analyzed to find perfect pair-wise matches in terms of percent identity and E-values for each species. 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

17. Predicted ITS2 RNA secondary structures and analyses • Secondary structures of ITS2 sequences of various paragonimid species were reconstructed by aligning their sequences using Bioedit (Hall, 1999). • The acquired structures with restrictions and constrains submitted in MFOLD (Zuker, 2003). • RNA structure chosen from different output files with highest negative free energy for various similar structures obtained. 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

18. Results PCR amplification and analysis M 1 2 3 4 5 6 1b PCR products of Paragonimus metacercaria DNA using primer set 3S - A28 for ITS2 Amplification conditions: Initial denaturation at 94ºC =1 min Denaturation at 94ºC = 30 sec Annealing at 55ºC = 38 sec Extension at 72ºC = 42 sec Final extension at 72ºC = 10 min Product size: ~ 500 bp Final reaction volume = 50μl 1.6% agarose gel electrophoresis Marker = Ø x 174 DNA/ HaeIII Digest } 26 cycles 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

19. Sequence motif in PROSITE format (from 5’ to 3’ ends) 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

20. NJ tree MP tree Phylogenetic trees of ITS2 sequences of Paragonimid species. (*) = Query sequence 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

21. BLAST outputs of Paragonimus ITS sequence motifs against NCBI GenBank database 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

22. Predicted ITS2 RNA secondary structures with enthalpies: Indian isolates 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

23. Predicted ITS2 RNA secondary structures: Neighbouring countries isolates 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

24. Family Paragonimidae: Hypothetical Bayesian analysis phylogeny based upon secondary structure alignment data of ITS2 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

25. Discussion • ITS sequences- high species-specific homogeneity. • Primary sequence analysis- • close relationship between query sequence (P. westermani from India) and isolates of related species from neighbouring countries. • Secondary structure analysis- • provided additional information for correct identification of the species. • confirmed the results from primary sequence analysis. 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

26. ITS sequence motifs • All the sequence motifs were available in all the Paragonimussequences of different geographical isolates under study. • Validation of motifs showed high percent identity and low E-value scores. 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

27. Conclusion • The Paragonimus species prevalent in the region is in fact Paragonimus westermani, the most common lung fluke throughout the globe. • ITS2 sequences:- • - reliable tool to identify species and phylogenetic relationships • - potential as species markers. • Different geographical isolates ofParagonimusspp need further study with additional molecular markers and barcoding to ascertain intra-specific strain variations, if any. 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

28. Acknowledgements • DST, DBT, CSIR (GOI)- for Travel Fellowship for InCoB2009. • DIT Project to VT. • AICOPTAX programme (MoE&F, GOI) to VT • DBT Project to VT & AC. • DSA programme (UGC-SAP) in Zoology; • UPE (Biosciences) programme in School of Life Sciences, NEHU, Shillong. • Co-ordinator, Bioinformatics Centre, NEHU. Thank You 8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore