DNA Barcode Analysis of Mosquito Species from Pakistan Muhammad Ashfaq University of Guelph, Canada
OutlineI. DNA barcoding & its use in species identitiesII. DNA barcoding mosquito species from Pakistan 1. Species analysis 2. Distribution analysis
I. DNA barcoding and its use• Why? AND• How to identify the biological units? Biological Species Concept (BSC) “In conservation biology it is important to decide on the taxonomic unit deserving Attention”
I. Taxonomic identification Morphological characters Breeding Biology Host plants etcI. Molecular identification Use of DNA nucleotide data Protein analysis etc
What is molecular identification?Use of DNA sequence data for individual or species identificationby• Sequence comparisons• Constructing phylogenetic trees• Other available tools
Why molecular identification?• Clear genetic basis• Deformed/broken samples are OK• DNA from fossilized specimens can be used• Identification of immature stages, or concealed stages possible• Quick and unbiased• Reliable
Target genes of DNA analysis• Mitochondrial• Ribosomal RNA• Nuclear protein-coding genes• Satellite DNA/ SSRs• Introns• Rare genomic changes
Problems/ issues• Different target genes from the same species• Different regions of the same gene• No standardized comparisons• No single database to perform sequence comparisons• Species consensus/ conclusions – difficult
Why not to standardize themolecular identification?
The candidate gene?The Mitochondrial genome COI 658 bp 850 bp
Why mtDNA?• Ease of isolation• High copy number• Lack of recombination• Conservation of sequence and structure across metazoa• Range of mutational rates in different regions of the molecule
Why COI?• Relatively well studied at the biochemical level• Size and structure conserved across all aerobic organisms• Mix of variable and conserved regions• Largest of the three CO subunits• Broad spectrum of substitutional rates
Characteristics of Barcode Regions• Flanked by conserved regions• Easy to amplify• Low intraspecies variability• Discontinuous variation between species• Long enough to work in all groups• Short enough for single reads
DNA barcoding: towards an inventory of lifeA DNA barcode is a short gene sequencetaken from standardized portions of thegenome, used to identify species
Using DNA Barcodes Establish reference library of barcodes from identified voucher specimens If necessary, revise species limits Then: Identify unknowns by searching against reference sequences Look for matches (mismatches) against ‘library on a chip’ Before long: Analyze relative abundance in multi- species samples
Mosquito species from the region• 1971: 100 species from West & East Pakistan• 1976-77: 43 species from Lahore area• 1978-79: 30 species from Central Punjab• 1997: 30 species from Indian Punjab• 2007: 63 species from India – Culex tritaeniorhynchus was reported as the predominent species in Punjab, Pakistan No definite number of mosquito species from Pakistan available
Target Locations• Urban areas• Locations in close proximity of human dwellings, such as:• Marshes and ponds• Water drums• Abandoned locations• Underconstruction buildings• Junkyards• Sub-ground water catchment areas• Gutters• Waste bins near hospitals and petrol-pumps
Collection MethodsArial nets Motorized aspirators Human landingManual catching Pipettes and sieves Traps with CO2 source
Conclusions• DNA barcodes effectively discriminated the 26 mosquito species• Culex quinquefasciatus was the predominant species in our collections comprising 76% of the total• There were seven species in Aedes complex which made only a fraction of the collected specimens• The distribution patterns did not reveal localization of a species in one specific area• Similarity of members of Aedes complex warrants use of molecular methods for vector population forecasts
Acknowledgements Dr. Sohail Hameed, Director NIBGE Dr. Paul Hebert, Director BIO, ON, Canada Dr. Sajjad Mirza, NIBGE Several faculty members around Pakistani universities for providing specimens and help in identifications My lab staff and students Higher Education Commission for funding
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