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Transcript of Mosquito barcoding new
DNA Barcode Analysis of Mosquito Species from
Pakistan
Muhammad Ashfaq
University of Guelph, Canada
Outline
I. DNA barcoding & its use in species identities
II. DNA barcoding mosquito species from Pakistan
1. Species analysis
2. Distribution analysis
• Why?
AND
• How to identify the biological units?
“In conservation biology it is important to decide on the taxonomic unit deserving Attention”
Biological Species Concept (BSC)
I. DNA barcoding and its use
I. Taxonomic identification Morphological characters Breeding Biology Host plants etc
I. Molecular identification Use of DNA nucleotide data Protein analysis etc
What is molecular identification?
Use of DNA sequence data for individual or species identification
by
• 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 the molecular identification?
The Mitochondrial genome
COI
850 bp658 bp
The candidate gene?
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
A DNA barcode is a short gene sequence taken from standardized portions of the genome, used to identify species
DNA barcoding: towards an inventory of life
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
1. Databasing2. Labeling3. Imaging4. Tissue sampling5. DNA extraction6. PCR7. PCR check8. Sequencing reaction9. Sequencing cleanup10. Sequencing11. Trace editing & submission
Analytical chain
MethodsMethods
BOLD: Barcode of Life Data System
GOAL: Assemble the sequence library – rapidly and inexpensively to identify the organisms
Barcoding: a global initiative
iBOL nodes
Barcoding species from Pakistan
Canadian Centre for DNA Barcoding
NIBGE
Funded by: HEC
:iBOL
Using barcode data for species IDs
• Nucleotide identities/matches
• Distance analysis
• Barcoding gap
• Cluster analysis
MAIMB592-09|HM424125|Isoptera (W)
MATER039-11|Odontotermes obesus (S) MAIMB595-09|HM424126|Isoptera (W)
MAIMB608-09|HM891583|Isoptera (W)
MAIMB586-09|HM424124|Isoptera (W)
MAIMB616-09|HM891589|Isoptera (W)
MAIMB588-09|HM891573|Isoptera (W)
MATER040-11|Odontotermes gurdaspurensis (S) MAIMB648-09|HM424131|Isoptera (W)
MATER050-11|Odontotermes sp. (S) MAIMB598-09|HM891578|Isoptera (W)
MATER019-10|HQ991626|Odontotermes lokanandi (S) MAIMB591-09|HM891575|Isoptera (W)
MAIMB590-09|HM891574|Isoptera (W)
MATER011-10|HQ991622|Coptotermes travian (S) MAIMB638-09|HM891596|Isoptera (W)
MAIMB626-09|HM891591|Isoptera (W)
MAIMB635-09|HM891595|Isoptera (W)
MAIMB629-09|HM891593|Isoptera (W)
MAIMB653-09|HM891600|Isoptera (W)
MAIMB640-09|HM891597|Isoptera (W)
MATER005-10|HQ991620|Microcerotermes sp. (S) MATER014-10|HQ991624|Angulitermes dehraensis (S) MATER032-11|Microtermes obesi (S)
MAIMB645-09|HM891598|Isoptera (W)
MAIMB627-09|HM891592|Isoptera (W)
MATER033-11|Microtermes unicolor (S)75100
9953
100
10072
89
97
57
46
100
100
64
54
100 99
99 68
99
69
0.02
Cluster analysis
Barcoding gap or no gap!
A
B
Identity analysis
NCBI
BOLD
Barcode applications
• Resolving cryptic species complexes
• Estimating species diversity
• Constructing barcode reference libraries
• Resolving commercial disputes
• Detecting invasive species
• Analyzing food chain etc etc
II. Barcoding mosquitoes from Pakistan
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
Progress on mosquito barcoding
Genus: Aedes Genus: Anopheles
Genus: Culex
Known Disease Link
• Aedes – Dengue
• Anopheles – Malaria
• Culex – West Nile Virus, Filariasis, Encephalitis
Dengue transmission cycleDengue transmission cycle
West Nile Virus transmission cycleWest Nile Virus transmission cycle
Current studies
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
Islamabad
Multan
Lahore
Mosquito collection localities
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 Methods
Arial nets Motorized aspirators
Manual catching
Human landing
Pipettes and sieves Traps with CO2 source
Barcode data obtained
Data analysis
• Distance analysis
• Barcode gap analysis
• Cluster analysis
Data analysisSequence alignments
Distance analysis
Histogram of distances Ranked distances
Distance analysis and “barcode gap”
barcode gap
Using ABGD (Automatic Barcode Gap Discovery)
Barcode Gap Analysis (using BOLD)
NN distances
MAMOS171-12| Anopheles culicifacies_A
MAMOS167-12| Anopheles culicifacies
MAMOS173-12| Anopheles annularis
MAMOS147-12| Anopheles splendidus
MAMOS1613-13| Anopheles sp nr dravidicus
MAMOS1227-12| Anopheles stephensi
MAMOS159-12| Anopheles pulcherimus
MAMOS637-12| Anopheles subpictus
MAMOS185-12 | Anopheles peditaeniatus
MADIP347-10| Ochlerotatus pseudotaeniatus
MAMOS086-12| Culicidae
MAMOS1309-12| Aedes albopictus
MAMOS017-12| Aedes aegypti
MAMOS087-12| Aedes sp1pk
MAMOS1230-12| Aedes sp2pk
MADIP300-10| Aedes sp3pk
MAMOS1582-13| Aedes sp4pk
MAMOS1366-12| Mansonia bonneae
MAMOS905-12| Armigeres subalbatus
MAMOS1217-12| Culex tritaeniorhynchus
MAMOS012-12| Culex quinquefasciatus
MADIP334-10| Culex theileri
MAMOS1364-12| Culex perexiguus
MAMOS394-12| Culex fuscocephala
MAMOS207-12| Culex bitaeniorhynchus
MADIP472-11| Culex mimeticus
MAMOS1581-13| Lutzia fuscana
10055
3819
18
49
33
98
50
72
55
50
15
31
23
88
31
30
16
18
15
0.000.010.020.030.040.050.060.07
Anopheles
Aedes
Culex
Cluster analysis of mosquito species
Cluster analysis of Aedes COMPLEX
MADIP347-10| Ochlerotatus pseudotaeniatus
Ochlerotatus pseudotaeniatus (DQ154153)
Aedes iyengari (DQ431717)
MAMOS1309-12| Aedes albopictus
Aedes albopictus (JQ412504)
Aedes caspius (FJ210904)
Aedes vittatus (AY834246)
MAMOS087-12| Aedes sp1pk
MAMOS138-12| Aedes walbus
MAMOS1582-13| Aedes sp4pk
MADIP300-10| Aedes sp3pk
MAMOS1230-12| Aedes sp2pk
Aedes lineatopennis (HQ398909)
MAMOS017-12| Aedes aegypti
Aedes aegypti (HQ688295)
Aedes vexans (AY917213)
MAMOS1366-12| Mansonia bonneae
Aedes fumidus (AY729978)
100
100
100
78
51
14
17
10
1
0.000.020.040.060.08
Aedes aegypti
Aedes albopictus
Aedes sp1pk
Aedes sp2pk
Aedes sp3pk
Aedes sp4pk
Aedes W-albus
Aedes COMPLEX
Ochlerotatus pseudotaeniatus
Species ratio
76%Culex quinquefasciatus
8%
16%
26 species
5%
Genus-wise distribution
Aedes COMPLEX Anopheles COMPLEX
Culex COMPLEX
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