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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 ana...
I. DNA barcoding and its use• Why?   AND• How to identify the biological units?         Biological Species Concept (BSC)  ...
I.       Taxonomic identification          Morphological characters                Breeding                Biology     ...
What is molecular             identification?Use of DNA sequence data for individual or species  identificationby• Sequenc...
Why molecular identification?• Clear genetic basis• Deformed/broken samples are OK• DNA from fossilized specimens can be u...
Target genes of DNA analysis•   Mitochondrial•   Ribosomal RNA•   Nuclear protein-coding genes•   Satellite DNA/ SSRs•   I...
Problems/ issues•   Different target genes from the same species•   Different regions of the same gene•   No standardized ...
Why not to standardize themolecular identification?
The candidate gene?The Mitochondrial genome                                    COI                           658 bp       ...
Why mtDNA?• Ease of isolation• High copy number• Lack of recombination• Conservation of sequence and structure across  met...
Why COI?•   Relatively well studied at the biochemical level•   Size and structure conserved across all aerobic    organis...
Characteristics of Barcode Regions•   Flanked by conserved regions•   Easy to amplify•   Low intraspecies variability•   D...
DNA barcoding: towards an       inventory of lifeA DNA barcode is a short gene sequencetaken from standardized portions of...
Using DNA Barcodes   Establish reference library of barcodes from    identified voucher specimens   If necessary, revise...
Analytical chain1. Databasing2. Labeling3. Imaging4. Tissue sampling5. DNA extraction6. PCR7. PCR check8. Sequencing react...
Methods
BOLD: Barcode of Life Data System
Barcoding: a global initiative GOAL: Assemble the sequence library – rapidly and inexpensively to identify the organisms
iBOL nodes
Barcoding species from PakistanCanadian Centre for DNA       NIBGEBarcoding                          Funded by: HEC       ...
Using barcode data for species IDs•   Nucleotide identities/matches•   Distance analysis•   Barcoding gap•   Cluster analy...
Cluster analysis                                                     MAIMB592-09|HM424125|Isoptera (W)                    ...
Barcoding gap or no gap!AB
Identity analysisNCBI       BOLD
Barcode applications•   Resolving cryptic species complexes•   Estimating species diversity•   Constructing barcode refere...
II. Barcoding mosquitoes from Pakistan
Mosquito species from the region•   1971: 100 species from West & East Pakistan•   1976-77: 43 species from Lahore area•  ...
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 cycle Dengue transmission cycle                             West Nile Virus transmission cycle        ...
Current studies
Collection Areas• 300 localities• 21 districts of Punjab and few localities from  Peshawar• 2-7 specimens barcoded from ea...
Mosquito collection localities                   Islamabad                           Lahore        Multan
Target Locations•   Urban areas•   Locations in close proximity of    human dwellings, such as:•   Marshes and ponds•   Wa...
Collection MethodsArial nets           Motorized aspirators   Human landingManual catching     Pipettes and sieves     Tra...
Barcode data obtained
Data analysis• Distance analysis• Barcode gap analysis• Cluster analysis
Data analysisSequence alignments
Distance analysis
Distance analysis and “barcode gap”   barcode gap       Histogram of distances       Ranked distancesUsing ABGD (Automatic...
Barcode Gap Analysis (using BOLD)
NN distances
Cluster analysis of mosquito species                                                     100                 MAMOS171-12| ...
Cluster analysis of Aedes COMPLEX                                      100            MADIP347-10| Ochlerotatus pseudotaen...
Aedes COMPLEXAedes aegypti                        Aedes sp1pk                                                  Aedes sp3pk...
Species ratio                                     16%                      8%26 species                           76%     ...
5%
Genus-wise distributionAedes COMPLEX                   Anopheles COMPLEX                Culex COMPLEX
Conclusions• DNA barcodes effectively discriminated the 26  mosquito species• Culex quinquefasciatus was the predominant s...
Acknowledgements Dr. Sohail Hameed, Director NIBGE Dr. Paul Hebert, Director BIO, ON, Canada Dr. Sajjad Mirza, NIBGE S...
Mosquito barcoding new
Mosquito barcoding new
Mosquito barcoding new
Mosquito barcoding new
Mosquito barcoding new
Mosquito barcoding new
Mosquito barcoding new
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  1. 1. DNA Barcode Analysis of Mosquito Species from Pakistan Muhammad Ashfaq University of Guelph, Canada
  2. 2. OutlineI. DNA barcoding & its use in species identitiesII. DNA barcoding mosquito species from Pakistan 1. Species analysis 2. Distribution analysis
  3. 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. 4. I. Taxonomic identification  Morphological characters  Breeding  Biology  Host plants etcI. Molecular identification  Use of DNA nucleotide data  Protein analysis etc
  5. 5. What is molecular identification?Use of DNA sequence data for individual or species identificationby• Sequence comparisons• Constructing phylogenetic trees• Other available tools
  6. 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. 7. Target genes of DNA analysis• Mitochondrial• Ribosomal RNA• Nuclear protein-coding genes• Satellite DNA/ SSRs• Introns• Rare genomic changes
  8. 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. 9. Why not to standardize themolecular identification?
  10. 10. The candidate gene?The Mitochondrial genome COI 658 bp 850 bp
  11. 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. 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. 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. 14. DNA barcoding: towards an inventory of lifeA DNA barcode is a short gene sequencetaken from standardized portions of thegenome, used to identify species
  15. 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
  16. 16. Analytical chain1. Databasing2. Labeling3. Imaging4. Tissue sampling5. DNA extraction6. PCR7. PCR check8. Sequencing reaction9. Sequencing cleanup10. Sequencing11. Trace editing & submission
  17. 17. Methods
  18. 18. BOLD: Barcode of Life Data System
  19. 19. Barcoding: a global initiative GOAL: Assemble the sequence library – rapidly and inexpensively to identify the organisms
  20. 20. iBOL nodes
  21. 21. Barcoding species from PakistanCanadian Centre for DNA NIBGEBarcoding Funded by: HEC :iBOL
  22. 22. Using barcode data for species IDs• Nucleotide identities/matches• Distance analysis• Barcoding gap• Cluster analysis
  23. 23. Cluster analysis MAIMB592-09|HM424125|Isoptera (W) 69 MATER039-11|Odontotermes obesus (S) 99 MAIMB595-09|HM424126|Isoptera (W) MAIMB608-09|HM891583|Isoptera (W) 99 68 MAIMB586-09|HM424124|Isoptera (W) MAIMB616-09|HM891589|Isoptera (W) MAIMB588-09|HM891573|Isoptera (W) 100 99 MATER040-11|Odontotermes gurdaspurensis (S) MAIMB648-09|HM424131|Isoptera (W) 100 MATER050-11|Odontotermes sp. (S) 46 54 MAIMB598-09|HM891578|Isoptera (W) 64 MATER019-10|HQ991626|Odontotermes lokanandi (S) MAIMB591-09|HM891575|Isoptera (W) 100 MAIMB590-09|HM891574|Isoptera (W) MATER011-10|HQ991622|Coptotermes travian (S) MAIMB638-09|HM891596|Isoptera (W) 100 MAIMB626-09|HM891591|Isoptera (W) 53 MAIMB635-09|HM891595|Isoptera (W) 99 MAIMB629-09|HM891593|Isoptera (W) 100 MAIMB653-09|HM891600|Isoptera (W) 72 MAIMB640-09|HM891597|Isoptera (W) 89 MATER005-10|HQ991620|Microcerotermes sp. (S) MATER014-10|HQ991624|Angulitermes57 dehraensis (S) MATER032-11|Microtermes obesi (S) 97 MAIMB645-09|HM891598|Isoptera (W) MAIMB627-09|HM891592|Isoptera (W) 100 75 MATER033-11|Microtermes unicolor (S) 0.02
  24. 24. Barcoding gap or no gap!AB
  25. 25. Identity analysisNCBI BOLD
  26. 26. Barcode applications• Resolving cryptic species complexes• Estimating species diversity• Constructing barcode reference libraries• Resolving commercial disputes• Detecting invasive species• Analyzing food chain etc etc
  27. 27. II. Barcoding mosquitoes from Pakistan
  28. 28. 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
  29. 29. Progress on mosquito barcoding
  30. 30. Genus: Aedes Genus: Anopheles Genus: Culex
  31. 31. Known Disease Link• Aedes – Dengue• Anopheles – Malaria• Culex – West Nile Virus, Filariasis, Encephalitis
  32. 32. Dengue transmission cycle Dengue transmission cycle West Nile Virus transmission cycle West Nile Virus transmission cycle
  33. 33. Current studies
  34. 34. 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
  35. 35. Mosquito collection localities Islamabad Lahore Multan
  36. 36. 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
  37. 37. Collection MethodsArial nets Motorized aspirators Human landingManual catching Pipettes and sieves Traps with CO2 source
  38. 38. Barcode data obtained
  39. 39. Data analysis• Distance analysis• Barcode gap analysis• Cluster analysis
  40. 40. Data analysisSequence alignments
  41. 41. Distance analysis
  42. 42. Distance analysis and “barcode gap” barcode gap Histogram of distances Ranked distancesUsing ABGD (Automatic Barcode Gap Discovery)
  43. 43. Barcode Gap Analysis (using BOLD)
  44. 44. NN distances
  45. 45. Cluster analysis of mosquito species 100 MAMOS171-12| Anopheles culicifacies_A 55 MAMOS167-12| Anopheles culicifacies 18 MAMOS173-12| Anopheles annularis MAMOS147-12| Anopheles splendidus 19 49 38 MAMOS1613-13| Anopheles sp nr dravidicus Anopheles MAMOS1227-12| Anopheles stephensi 98 MAMOS159-12| Anopheles pulcherimus 33 MAMOS637-12| Anopheles subpictus MAMOS185-12 | Anopheles peditaeniatus MADIP347-10| Ochlerotatus pseudotaeniatus MAMOS086-12| Culicidae MAMOS1309-12| Aedes albopictus 15 MAMOS017-12| Aedes aegypti 18 MAMOS087-12| Aedes sp1pk 16 30 MAMOS1230-12| Aedes sp2pk Aedes 50 MADIP300-10| Aedes sp3pk MAMOS1582-13| Aedes sp4pk MAMOS1366-12| Mansonia bonneae MAMOS905-12| Armigeres subalbatus 50 MAMOS1217-12| Culex tritaeniorhynchus 31 31 MAMOS012-12| Culex quinquefasciatus MADIP334-10| Culex theileri 88 72 MAMOS1364-12| Culex perexiguus 23 MAMOS394-12| Culex fuscocephala Culex MAMOS207-12| Culex bitaeniorhynchus 15 55 MADIP472-11| Culex mimeticus MAMOS1581-13| Lutzia fuscana 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00
  46. 46. Cluster analysis of Aedes COMPLEX 100 MADIP347-10| Ochlerotatus pseudotaeniatus Ochlerotatus pseudotaeniatus (DQ154153) Aedes iyengari (DQ431717) 1 100 MAMOS1309-12| Aedes albopictus Aedes albopictus (JQ412504) 10 Aedes caspius (FJ210904) Aedes vittatus (AY834246) 78 MAMOS087-12| Aedes sp1pk MAMOS138-12| Aedes walbus 17 MAMOS1582-13| Aedes sp4pk 51 MADIP300-10| Aedes sp3pk MAMOS1230-12| Aedes sp2pk Aedes lineatopennis (HQ398909) 100 MAMOS017-12| Aedes aegypti Aedes aegypti (HQ688295) 14 Aedes vexans (AY917213) MAMOS1366-12| Mansonia bonneae Aedes fumidus (AY729978)0.08 0.06 0.04 0.02 0.00
  47. 47. Aedes COMPLEXAedes aegypti Aedes sp1pk Aedes sp3pkAedes albopictus Aedes sp2pk Aedes sp4pkAedes W-albus Ochlerotatus pseudotaeniatus
  48. 48. Species ratio 16% 8%26 species 76% Culex quinquefasciatus
  49. 49. 5%
  50. 50. Genus-wise distributionAedes COMPLEX Anopheles COMPLEX Culex COMPLEX
  51. 51. 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
  52. 52. 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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