POSTER PRESENTATION

‘NATIONAL CONFERENCE ON MAN AND ENVIRONMENT’

OCTOBER 15 – 16, 2012

Organized by Department of Zoology and Environmental Sciences, Punjabi University,

Patiala (Pb.) – 147 002, India

TOPICRapid Impact Assessment of Climatic and Physiographic Changes

on Flagship Group Lepidoptera by DNA Barcoding

Dr A P SinghAssociate Professor in Zoology (Entomology),

SGGS College (affiliated to Panjab University), Chandigarh (U.T.) - 160 019, India

E-mail: apsingh_60@yahoo.co.in

INTRODUCTION

The global/regional climatic changes and local physiographic changes are anthropogenic

These changes have immense impacts on health of various ecosystems and the biodiversity, including insects, supported by them.

The Impact Assessment, is difficult, costly, time consuming and may involve several methods. One of these methods is called, bioassessment

Major focus of Bioassessment studies are specific mega-diverse biological groups, the insects being one of them.

Among insects, the Lepidoptera is a known flagship group for bioassessment, as shows good species diversity (with over 1,60,000 described species ;Kristensen, et.al.,2007), intraspecific genetic divergence and wide geographical distribution.

Lepidopterans are recognized as moths, butterflies and skippers. Many of these are described as biodiversity indicators, ecological indicators, and substantial number of species are pests of agricultural crops, horticultural trees and forest plants. These features further enhance their bioassessment potentials.

Bioassessment involves sampling of Lepidopterans for making observations such as species extinction, changed distributional limits and population sizes, and genetic divergence within species. For this rapid identifications of collected samples is required.

Rapid identifications and assessment of genetic divergence within species involve the mitochondrial CO1 gene based DNA barcoding (Hajibabaei, et. al., 2004, 2006, Desalle, 2006). Additional mitochondrial genes such as nad1 ,nad4 ,nad5 ,nad6 ,COII, and 16s rRNA are employed for assessing intraspecific variations.

Global barcode database of the order Lepidoptera is available on BOLDSystems (http://www.boldsystems.org). It is regularly updated by submissions from all parts of the world. At the moment, BOLDSystems has 74,947 species with barcodes and 6, 70,267 specimens with barcodes for the Lepidoptera.

AIMS

To expose zoologists and Environmentalists to methodology of bioassessment of impacts of climatic and physiographic changes.

To highlight Lepidoptera as one of the flagship group for such impact studies.

To throw light on DNA Barcoding as a chief tool to be adopted for rapid bioassessment studies.

To highlight mitochondrial genes – CO1, COII, nad1, nad4, nad5, nad6 and 16s rRNA, as the chief tools for diagnosis and delimitation of species, and to assess genetic divergence within species.

MATERIAL AND METHODS

Order Lepidoptera of Class Insecta comprise the material for the present studies. Butterflies and moths will be collected by following their collection protocols; and two

right legs will be excised from each sample and preserved in ethyl alcohol, labeled and stored in refrigerator for DNA Barcoding.

DNA Barcoding is based on Mid region of the mitochondrial COI gene which is amplified through PCR by employing:

- Universal Insect Specific Primers (Hebert, et. al., 2003) LCO : 5’-GGTCAACAAATCATAAAGATATTGG-3’, and HCO : 5’-TAAACTTCAGGGTGACCAAAAAATCA-3’

- Lepidoptera Specific Primers (Hajibabaei, et. al., 2006) LepF : 5-ATTCAACCAATCATAAAGATATTGG-3, and LepR: (5-TAAACTTCTGGATGTCCAAAAAATCA-3, or Enh_Lep R1: 5-CTCCWCCAGCAGGATCAAAA-3’

Intraspecific variations are studied by additional mitochondrial genes – nad1 (Miller, et.al., 2009), nad4 (Gomez, et.al.,2009, Liu, et. al., 2010 ), nad5 (Meraner, et.al., 2008), nad6 (Silva-Brandao, et. al., 2011), COII (Liu, et. al., 1992), and 16s rRNA (Raffiudin, et. al., 2011); and for their amplification, specific primers are used according to respective protocols.

The sequences for the identified and described species are submitted along with collateral information including photograph to web-repositories (www.BOLDSystems.org;

www.ncbi.nih.gov).These add into already existing database. Later, submission of sequences of unknown specimens helps in immediate arrival at the

conclusion whether species is already known or novel to science and how much genetic closeness it has with different species.

% divergence or genetic distances are estimated through multiple sequence alignment in MEGA version 5 software to delimit species, and to assess population variations within species.

Flow Chart of DNA Barcoding

CONCLUSIONS

Till majority Lepidopteran species are barcoded and submitted on global database, rapid identification of Lepidopterans vis-à-vis prompt bioassessment of impacts of climatic changes and physiographic changes is not possible.

There is an urgent need for developing a comprehensive DNA barcode library for the Lepidopterans.

As the rate of submissions is picking up pace and good amount of funds are being diverted by states to DNA Barcoding, rapid bioassessment of impacts on Lepidoptera will soon become reality. This will help in its effective conservation.

ACKNOWLEDGEMENTS

Thanks are due to Principal SGGS College, Chandigarh for allowing the author to attend and present this poster in the present conference.

Sincere thanks are also due to Dr Virash Gupta (Professor in Microbiology; PAU, Ludhiana; for introduction to RAPD and Cloning) and Dr Sanil George (Scientist, Rajiv Gandhi Centre of Biotechnology, Trivandrum; for exposure to technique of Bar-coding) for respective reasons.

Dr Jagbir Singh Kirti (Professor in Entomology, Punjabi University, Patiala), needs special thanks, as has been my real mentor during my research for doctorate degree.

REFERENCES

1. Desalle, R.(2006).Species Discovery versus Species Identification in DNA Barcoding Efforts: Response to Rubinoff. Conservation Biology. 20 (4): 1545–1547

2. Gomez P.L.M., Giraldo, C., Lopez, A. and Uribe, S. (2009). Molecular and morphological differentiation of Oleria makrena(Hewitson) and Oleria fumata (Haensch) (Lepidoptera, Ithomiinae). Neotrop Entomol. 38: 616-623.

3. Hajibabaei, M., Janzen, D.H., Burns, J.M., Hallwach, W. and Hebert, P.D.N. (2006). DNA barcodes distinguish species of tropical Lepidoptera. Proc. Natl. Acad. Sci. USA. 103: 968-971.

4. Hebert PDN, Penton EH, Burns JM, Janzen DH and Hallwachs W. (2004). Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc. Natl. Acad .Sci. USA. 101:14812-14817.

5. Hebert, P. D. N., Ratnasingham, S. and DeWaard, J. R. (2003) 'Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species', Proceedings of the Royal Society B: Biological Sciences 270(0): 596-99.

6. Kristensen, N.P., Scoble, M.J. and Karsholt, O. (2007). Lepidoptera phylogeny and systematics: the state of inventorying moth and butterfly diversity. Zootaxa. 1688: 699-747.

7. Liu, H. & Beckenbacch, A.T. (1992). Evolution of the mitochondrial cytochrome oxidase II gene among 10 orders of insects. Molecular Phylogenetic Evol. 1(1):41-52.

8. Liu, Y., Hou, M. and Wu, K. (2010). Gentic variation of mitochondrial DNA in Chinese populations of Pectinophora gossypiella (Lepidoptera: Gelechiidae). Environmental Entomology. 39 (4): 1344-1351.

9. Meraner A, Brandstätter A, Thaler R, Aray B, Unterlechner M, Niederstätter H, Parson W, Zelger R, Dalla Via J and Dallinger R. (2008). Molecular phylogeny and population structure of the codling moth (Cydia pomonella) in Central Europe: I. Ancient clade splitting revealed by mitochondrial haplotype markers. Mol Phylogenet Evol 4: 825-837. 

10. Miller NJ, Dorhout DL, Rice ME and Sappington TW (2009) Mitochondrial DNA variation and range expansion in Western bean cutworm (Lepidoptera, Noctuidae): No evidence for a recent population bottleneck. Environ Entomol 38:274-280.

11. Raffiudin, R., Winnie, R.M and Samudra, M. (2010). Intraspecific variations of 16s mitochondrial gene sequence of yellow rice stem borer, Scirpophaga incertulus (Lepidoptera: Crambidae) from west Java. Biotropia. 18(1): 24-30.

12. Silva-Brandao, K.l., Lyra, M.L., et.al. (2011). Exploitation of mitochondrial nad6 as a complimentary marker for studying population variability in Lepidoptera. Genetics and Molecular Biology. 34 (4): 719-725.

IMPORTANTS WEB LINKS

www.nhm.ac.uk Portal of natural history museum

www.tolweb.org/tree Database of phylogenies; tree of life project

www.sp2000 Cataloguing of world’s biota

www.itis.usda.gov Integrated taxonomic identification system

www.gbif.org Global biodiversity information facility

www.all-species.org All species foundation aimed at making inventory of species on earth in next 25 years

www.leptree.net Genomic inspired international community collaboration dedicated to lepidopteran phylogeny

www.biosis.org Excellent taxonomy & nomenclature resource

www.discoverlife.org Free online tool for identification

www.identifylife.org Collaborative project providing ways to identify organisms

www.systematicbiology.org For taxonomy of all groups

www.lepbarcoding.org Tree of life project

www.barcodeoflife.org CBOL; Consortium of barcode of life

www.BOLDSystem.org Barcode of life data system

www.eol.org Encyclopedia of life

www.ncbi.nih.gov/Genbank National center for biotechnology information (NCBI), USA

www.bolnet.ca Canadian barcode of life network

Figures DNA Barcoding

DNA Barcode

Chromatorogram

Chromatogram (Magnified)

Moths and Butterflies