1. DNA Barcode Analysis of
Mosquito Species from
Pakistan
Muhammad Ashfaq
University of Guelph, Canada
2. Outline
I. DNA barcoding & its use in species identities
II. DNA barcoding mosquito species from
Pakistan
1. Species analysis
2. Distribution analysis
3. 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”
4. I. Taxonomic identification
Morphological characters
Breeding
Biology
Host plants etc
I. Molecular identification
Use of DNA nucleotide data
Protein analysis etc
5. What is molecular
identification?
Use of DNA sequence data for individual or species
identification
by
• Sequence comparisons
• Constructing phylogenetic trees
• Other available tools
6. 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
7. Target genes of DNA analysis
• Mitochondrial
• Ribosomal RNA
• Nuclear protein-coding genes
• Satellite DNA/ SSRs
• Introns
• Rare genomic changes
8. 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
9. Why not to standardize the
molecular identification?
11. 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
12. 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
13. 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
14. DNA barcoding: towards an
inventory of life
A DNA barcode is a short gene sequence
taken from standardized portions of the
genome, used to identify species
15. 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
32. 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
38. Collection Areas
• 300 localities
• 21 districts of Punjab and few localities from
Peshawar
• 2-7 specimens barcoded from each locality
• A total of 1425 specimens
• 190 larvae
• 1235 adults
41. 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
42. Collection Methods
Arial nets Motorized aspirators Human landing
Manual catching Pipettes and sieves Traps with CO2 source
58. 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
59. 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
