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Dr Yvonne-Marie Linton Natural History Museum, London

Dr Yvonne-Marie Linton Natural History Museum, London. Malaria kills a child every 30 seconds in Africa. WN virus. Dengue. Malaria. Encephalitis. Filariasis. Yellow fever. The changing face of mosquito-borne disease. Mosquito. Parasite / Virus. Parasite / Virus. Man. MBI objectives.

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Dr Yvonne-Marie Linton Natural History Museum, London

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  1. Dr Yvonne-Marie Linton Natural History Museum, London

  2. Malaria kills a child every 30 seconds in Africa

  3. WN virus Dengue Malaria Encephalitis Filariasis Yellow fever

  4. The changing face of mosquito-borne disease

  5. Mosquito Parasite / Virus Parasite / Virus Man

  6. MBI objectives To generate DNA barcodes for 80% of the World Culicidae within two years No less than 5 individuals per species

  7. Zoogeographic Distribution of CulicidaeGenera (approx number of species) per region 14 (200) 14 (190) 23 (880) 18 (560) 24 (820) 20 (520) 3457 species in 43 genera

  8. Life in the field Life in the field

  9. Combined collections 2765 Culicid taxa located = 85% • NHM = Natural History Museum, UK • SI = Smithsonian Institution, USA • ICMR = Indian Centre for Medical Research, India • UQIC = University of Queensland Insect Collection • USP = University of Sao Paolo, Brazil • RMCA = Royal Museum for Central Africa, Belgium • CMU = University of Chiang Mai, Thailand • UND = University of Notre Dame, USA

  10. Museum collections are critical to MBI success

  11. MBI Co-ordinators: Y. Linton & R. Lane NHM Co-ordinators: R. Harbach (morph) & Y. Linton (mol) SMITHSONIAN Co-ordinators: R. Wilkerson (morph) & D. Foley (mol) ITM W. Van Bortel UND N. Besansky India Co-ordinator: P. Kumar SE Asia Co-ordinator: P. Somboon Africa Co-ordinator: M. Coetzee Latin America Co-ordinators: M. A. Sallum & M. Quinones Australasia Co-ordinator: D. Foley World mosquito workers

  12. But…..can you get DNA from old specimens?

  13. Sigma Qiagen Sigma Qiagen ITS2: ITS2A/ITS2B COI: LCO1490F/HCO1490R 500 bp 500 bp Mosquitoes 1998-2001

  14. A. LCO1490F/HCO2198R B. LCO1490F/C1J1718MODR An. gambiae – 1938 An. minimus – 1998 An. gambiae – 1936 St. aegypti – 1973 St. aegypti – 1954 St. aegypti – 1916 C. quinquefasciatus -1969 Neg. extraction An. gambiae – 2001 Neg. PCR Archive mosquitoes

  15. Non-destructive sampling of rare specimens

  16. MBI sequencing strategy • Field collected samples less than 10 years old (silica gel or pinned) • Mosquitoes stored individually in >80% ETOH and less than 10 years old • Mosquito specimens from pinned collections >10 years old • Slide mounted larvae/pupae

  17. Wide utility of barcoding primers Successfully amplified whole barcode region from 17 of 43 genera (Aedes problematic) 10 internal primer sets developed for concantenation of degraded DNA

  18. So…..what next? • We had the technical ability (DNA/primers) • We had the specimens • We had CBoL’s support • We had the enthusiasm ……….but we needed the money!

  19. $£$£ MBI Sponsors $£$£ • CBOL • Colciencias, Colombia • Christ Church Canterbury • Great British public!! …so six weeks ago we started……

  20. First strategy • Target DNA collections in the NHM & SI 12,200 Culicidae DNA extractions curated & databased (9,700 NHM; 2,500 SI) - Frozen collections modernised to barcode tubes - All data linked to voucher specimens & collection records where available

  21. 654 records generated so far Pie chart showing genus composition of 654 Culicidae barcode records (ICMR + NHM)

  22. Graph showing % species barcoded so far in 15 of 43 Culicid genera

  23. Graph showing mean numbers of records per species by genus

  24. Anopheles barcode record quality (529 records; ICMR – 20, NHM – 509; 154 species) Sequence Length: 98.1% over 500bp; 58.4% 658-700bp Sequence Quality: • 100% are sequenced in both directions • All bases have been manually verified • Min. 85% overlap • No records with missing/ambiguous bases included

  25. Anopheles barcode record quality (529 records; ICMR – 20, NHM – 509; 154 species) Vouchers: • 79.4% of all records have associated voucher material (immature skins, isofamily, remaining pinned specimen) • 100% have frozen DNA vouchers • 89 specimens (16.8%) are from type series

  26. Voucher specimens

  27. Pinned specimens left in humidifier overnight. Abdomen removed MALE FEMALE Terminalia clipped & stored in tube of glycerol attached to pin. Remainder of abdomen used for DNA extraction Abdomen used for DNA extraction Barcode label with unique DNA barcode tube number added to specimen All data from pinned specimen labels entered into database for BOLD Processing museum specimens Specimens taken from type locality or as near to as possible

  28. Tree of Culicidae Anopheles Aedes albopictus gambiae Good illustration of how divergence within species is much lower than between species. Culex and Aedes

  29. Culex pipiens s.l. Culicine mosquitoes Ochlerotatus Aedine mosquitoes Aedes aegypti Aedes cretinus Aedes albopictus

  30. Utility of barcodes in Anopheles Unknown larvae Sp. nov Mis-ID (aconitus)

  31. Species complexes

  32. Limits of barcodes in Anopheles bwambae arabiensis / gambiae

  33. So after six weeks…….. • 654 records processed for 154 species - Over 20% of Anopheles species completed - Representatives of 15 Culicid genera - 5.5% of job done! • 2,400 specimens have been sequenced, and await processing …………..so, lots still to do!!!!

  34. Working together for a brighter future

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