1 Departments of Zoology, 3 Biotechnology & Bioinformatics and 2 Bioinformatics Centre, North-Eastern Hill University, Shillong, 793022 Email: - [email protected].

1Departments of Zoology, 3Biotechnology & Bioinformatics and 2Bioinformatics Centre, North-Eastern Hill University, Shillong, 793022

Email: - [email protected]

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

88thth International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

• 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.

Paragonimus

Egg80-110×48-60µ

Encysted metacercaria ~300- 400µ

Pre-adult Adult: 7.5-12 × 4-6×1-3mm (l: w = 2:1)

• 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.


Distribution of human paragonimiasis

Species of Paragonimus are encountered in Asia, the Americas, and Africa.


Distribution in India


1. P. westermani (Kerbert,1878)- Bengal tiger, Amsterdam Zoo collection from India, Indonesia, China[ Syn. P. edwardsi Gulati, 1926- civet]

2. P. compactus (Cobbold, 1859)- Herpestes edwardsii India (Vevers,1923; Ravikumar et al.,1979); Sri Lanka (Dissanaike and Paramananthan, 1962)

3. P. heterotremus Chen and Hsia, 1964- China, Thailand,

Laos, Vietnam

• Arunachal Pradesh (Narain et al. 2003)

• Manipur (Singh,1996)

4. P. hueitungensis Chung et al.,1975- China

• Manipur (Singh, 2002)

5. P. mungoi, P. pantheri – Orissa (Mishra et al.,1976)-

nomen nudem88thth International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore

Paragonimus species: India

1. Infective stage: metacercaria2. Infective mode: eating raw fresh water

crabs and crayfish with metacercariae3. Infective route: by mouth4. Site of inhabitation: lungs

5. Intermediate hosts: 1st – snail; 2nd

crab, crayfish6. Reservoir hosts: carnivores (tiger, lion,

wolf, fox, dog, leopard, cat etc.)7. Life span: 5-6 years

Life CycleLife Cycle


Pleurocerid snail hosts

Thiarid snail hosts

DiploidTriploid

Paragonimus westermani is the best-known species. Diploid & triploid forms in N.E. Asia: only diploids elsewhere.

??

?

?

?


P. asymmetricusP. bangkokensisP. cheniP. divergensP. fukienensisP. harinasutaiP. heterorchisP. heterotremusP. hokuoensisP. heuitungensisP. iloktsuenensisP. jiangsuensisP. macrorchisP. menglaensisP. microrchisP. minqinensisP. ohiraiP. paishuihoensisP. proliferusP. szechuanensisP. tuanshanensisP. veocularisP. xiangshanensisP. yunnanensis

Euparagonimus cenocopiosus

E. hongzesiensis

Chengdu

Guangzhou

Shanghai

Beijing

Many other species described. Nearly 50 species, mostly in Asia:

over half of these in China alone. This prompted a molecular

taxonomy approach.

(P. westermani not shown here)


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


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.


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.


• 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)


Materials & Methods

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.


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


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.


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.


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.


M 1 2 3 4 5 6

1b

PCR products of Paragonimus metacercaria DNA using primer set 3S - A28 for ITS2

Results

Product size: ~ 500 bp

Final reaction volume = 50μl1.6% agarose gel electrophoresisMarker = Ø x 174 DNA/ HaeIII Digest

PCR amplification and analysis

Amplification conditions:

Initial denaturation at 94ºC =1 minDenaturation at 94ºC = 30 secAnnealing at 55ºC = 38 secExtension at 72ºC = 42 secFinal extension at 72ºC = 10 min

}26 cycles



Sequence motif in PROSITE format (from 5’ to 3’ ends)


Phylogenetic trees of ITS2 sequences of Paragonimid species. (*) = Query sequence

NJ tree

MP tree

BLAST outputs of Paragonimus ITS sequence motifs against NCBI GenBank databaseSpecies motif patterns (5’-3’ ends) Length (bp) No. of best hits Identity

(%) E-value

>Pattern1 G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-G-T-G-G-C-C-A-C-G-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G-C-T-T-A-T-A-A-A-C

50 100 100 8e-19

>Pattern2 C-G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-G-T-G-G-C-C-A-C-G-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G-C-T-T-A-T-A-A-A

50 100 100 8e-19

>Pattern3 G-C-G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-G-T-G-G-C-C-A-C-G-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G-C-T-T-A-T-A-A

50 100 100 8e-19

>Pattern4 T-G-C-G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-G-T-G-G-C-C-A-C-G-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G-C-T-T-A-T-A

50 100 100 8e-19

>Pattern5 T-T-G-C-G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-G-T-G-G-C-C-A-C-G-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G-C-T-T-A-T

50 100 100 8e-19

>Pattern6 A-T-T-G-C-G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-G-T-G-G-C-C-A-C-G-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G-C-T-T-A

50 100 100 8e-19

>Pattern7 T-A-T-T-G-C-G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-G-T-G-G-C-C-A-C-G-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G-C-T-T

50 100 100 8e-19

>Pattern8 A-T-A-T-T-G-C-G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-G-T-G-G-C-C-A-C-G-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G-C-T

50 100 100 8e-19

>Pattern9 C-A-T-A-T-T-G-C-G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-G-T-G-G-C-C-A-C-G-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G-C

50 100 100 8e-19

>Pattern10 G-C-A-T-A-T-T-G-C-G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-G-T-G-G-C-C-A-C-G-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G

50 100 100 8e-19



Predicted ITS2 RNA secondary structures with enthalpies: Indian isolates


Predicted ITS2 RNA secondary structures: Neighbouring countries isolates

Family Paragonimidae: Hypothetical Bayesian analysis phylogeny based upon secondary structure alignment data of ITS2


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.


• ITS sequence motifs

All the sequence motifs were available in all the Paragonimus sequences of different geographical isolates under study.

Validation of motifs showed high percent identity and low E-value scores.


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 of Paragonimus spp need further study with additional molecular markers and barcoding to ascertain intra-specific strain variations, if any.


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.

Acknowledgements


1 Departments of Zoology, 3 Biotechnology & Bioinformatics and 2 Bioinformatics Centre, North-Eastern Hill University, Shillong, 793022 Email: - [email protected].

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