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

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

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

9. Life in the field
Life in the field

10. 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

11. SI 825 79 NHM 480 69 ICMR 312 UQIC 190 USP 185 12 RMCA 25 CMU 24 UND
Specimen source
Archive specimens
Extracted DNA
In EtOH SI
825 79
NHM
480 69
ICMR
312
UQIC
190
USP
185 12
RMCA 25
CMU 24
UND 50
NHM & SI*
679
Total: 2930
2720
198

13. Museum collections are critical to MBI success

14. NHM SMITHSONIAN MBI UND ITM India SE Asia Latin America Australasia
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
P. Somboon
Latin America
M. A. Sallum & M. Quinones
Australasia
Co-ordinator: D. Foley
Africa
M. Coetzee
World mosquito workers

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

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

17. Archive mosquitoes 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

18. Non-destructive sampling of rare specimens

19. 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

20. 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

21. 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!

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

23. 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

24. 654 records generated so far
Pie chart showing genus composition of 654 Culicidae barcode records (ICMR + NHM)

25. Genus
No. of species
(154)
No. of records
(654)
No. of haplotypes
(384)
Pi (mean nucleotide diversity / site)
Aedeomyia 1
Aedes 12 40 20
Anopheles 98
529
314
Armigeres 2
Culex 22 39 27
Culiseta 4
Ficalbia
Heizmannia
Malaya 6
Mansonia
Mimomyia
Orthopodomyia
Tripteroides
Uranotaenia
Verrallina 3

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

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

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

29. 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

30. Voucher specimens

31. Processing museum specimens
Pinned specimens left in humidifier overnight.
MALE
FEMALE
Abdomen removed
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
Specimens taken from type locality or as near to as possible

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

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

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

35. Species complexes

36. Limits of barcodes in Anopheles
bwambae
arabiensis / gambiae

37. 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!!!!

38. Working together for a brighter future