1. Control of Disease Vectors Dr Yvonne-Marie Linton Natural History Museum, London Consortium for the Barcode of Life Regional Meeting Nairobi, Kenya 18-19 October 2006

2. Correct species identification is critical for effective control of insect- borne pathogens and agricultural pests

3. Uses of DNA Barcodes As a diagnostic tool: for identifying regulated species: –disease vectors, agricultural pests, invasive species –protected species, CITES listed, trade-sensitive for general scientific research –ecological studies, inventories As a “Triage” tool for flagging potential new species (undescribed and cryptic) To assist in taxonomic research

4. New DNA Barcoding Schemes Mosquitoes (c. 3500 spp) – vectors of malaria, filariasis, dengue, yellow fever, JE, West Nile virus etc Fruit flies (Tephritids) (c. 3000 spp) – economically devastating pests of fruit crops worldwide Both have been accepted by CBOL as ‘demonstrator’ projects

6. MBI inceptive meeting, NHM, London 21-22 Nov 2005

7. Can DNA barcoding work in mosquitoes? Can a short region of DNA (720bp of COI) really enable us to identify all known species? Can it help identify unknown species?

8. Why barcode mosquitoes? Relatively small but diverse group Relatively well known Actively researched worldwide Huge potential impact on parasitic and arboviral disease control

9. Overcoming the global taxonomic impediment DNA characters are easier to obtain and compare, making the discovery of new species more rapid BUT sequence data is effectively useless unless meshed with a strong taxonomic framework based on morphology Integrated systematic studies are “the new taxonomy”

10. An. (C.) christyi An. (N.) oswaldoi atropos sacharovi persiensis martinius GB atroparvus labranchiae GB 94 messeae daciae 79 maculipennis melanoon 70 67 64 95 98 beklemishevi quadrimaculatus maverlius smaragdinus inundatus diluvialis 100 87 95 66 75 occidentalis freeborni hermsi earlei 87 92 95 96 100 Palaearctic Nearctic ITS2 phylogeny Maculipennis Group (745 seqs) (GB GenBank) New species identified on basis of DNA data and formally described using integrated description

11. 100 200 300 400 500 1000 600 nivipesphilippinensisannularis Annularis Group annularis India* annularis Viet/Korea/Laos/Camb annularis Philippines* philippinensis Vietnam/Laos nivipes Vietnam/Cambodia/Laos jamesi Vietnam maculatus Vietnam 96 100 96 99 0.02 ITS2 Wide geographical sampling reveals new species

12. Voucher specimens

13. Outcome of the MBI inceptive meeting MBI objectives: To generate DNA barcodes for at least 5 specimens of each species of 80% of the World Culicidae within two years

14. Major obstacles to objectives How many Culicid species are there? Where will we find them? Where would the specimens come from? –Frozen DNA collections? –Field collected samples? –Museum specimens? We realised that for the project to be a success in such a short time frame, museum specimens would have to play a major role

15. To meet our objectives A pilot study to assess the feasibility of getting usable DNA from Museum specimens must be undertaken - If success rate was higher than 50% we could go ahead with the project! An up-to-date taxon list was needed! Species distributions needed to be established

16. Smithsonian Institution, USA May 11-12 2006 Update meeting of 5 members of the MBI steering committee to assess success of pilot study

17. Museum specimens from 2000 CPV1-1 An. philippinensis Luksang Kampong, Preah Vihear Province, Cambodia. Human Bait. Linton et al., 2004 GenBank AF546338 mtDNA COI GenBank AJ674540 nDNA ITS2 SigmaQiagen 500 bp COI: LCO1490F/HCO1490R

18. Archive mosquitoes (QIAmp micro kit, QIAgen) A. LCO1490F/HCO2198R B. LCO1490F/C1J1718MODR 1.An. gambiae – 1938 2.An. minimus – 1998 3.An. gambiae – 1936 4.St. aegypti – 1973 5.St. aegypti – 1954 6.St. aegypti – 1916 7.C. quinquefasciatus -1969 8.Neg. extraction 9.An. gambiae – 2001 10.Neg. PCR

19. Optimal DNA extraction from Museum specimens 100μl GB 10μl Prot K 2 min QIAgen Biosprint, 50μl Minimum of 12-24hrs in shaking incubator @ 55 o C QIAgen Blood kit and magnetic bead DNA transfer

20. Priority order of sequencing? 1.Field collected samples less than 10 years old (silica gel or pinned) 2.Mosquitoes stored individually in >80% ETOH and less than 10 years old 3.Mosquito specimens from pinned collections >10 years old 4.Slide mounted larvae/pupae Specimens from as wide a geographical range as possible will be used

21. Culicidae species list 3,449 formally recognised species as at July 1 2006 Quantitative counts of museum holdings 2930/3449 in 9 collections 85% of all currently known taxa are available to MBI

22. Specimen sourceArchive specimensExtracted DNAIn EtOH SI82579 NHM48069 ICMR312 UQIC190 USP18512 RMCA25 CMU24 UND50 NHM & SI*679 Total: 2930272019812

23. Zoogeographic Distribution of Culicidae Genera (approx number of species) per region 14 (190) 24 (820) 14 (200) 18 (560) 20 (520) 23 (880)

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

25. MBI strategy summary Primarily reliant on museum specimens but fresh is better! To actively include global members of the mosquito community as collaborators Donations of specimens will be acknowledged in the BOLD database All specimens will be identified and voucher specimens stored where possible Access to the data will be immediate and free.

26. Current status of MBI –We have a updated species list –We have knowledge of species distribution –We can get good quality sequenceable DNA from museum specimens –We have tested the utility of the barcoding primers across many Culicidae genera –We have access to 85% of all the known taxa –We need your help! BUT WE ARE READY TO GO!