The document discusses the impact of anthropogenic climatic and physiographic changes on ecosystems and biodiversity, with a focus on lepidopterans (moths and butterflies) as key indicators for bioassessment. It highlights the methodology of DNA barcoding using mitochondrial genes for rapid identification and assessment of genetic divergence in lepidopteran species, emphasizing the urgent need for a comprehensive DNA barcode library. The document also acknowledges contributions from various individuals and institutions supporting the research efforts in this field.

1. Organized by
Department of Zoology and Environmental Sciences, Punjabi University, Patiala (Pb.) – 147
002, India

2. Dr A P Singh
Associate Professor in Zoology (Entomology),
SGGS College (affiliated to Panjab University), Chandigarh (U.T.) - 160 019,
India
E-mail: apsingh_60@yahoo.co.in

3.  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

4.  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.

5.  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.

7.  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.

8.  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.

9. 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.

10. 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

11. Figures DNA Barcoding
DNA Barcode
Chromatorogram
Chromatogram (Magnified)

12. Moths and Butterflies