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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
Correct species identification is critical foreffective control of insect-
borne pathogens and agricultural pests
Uses of DNA BarcodesAs 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
New DNA Barcoding Schemes
Mosquitoes (c. 3500 spp) – vectors of malaria, filariasis, dengue, yellow fever, JE, West Nile
virus etcFruit flies (Tephritids) (c. 3000 spp) –
economically devastating pests of fruit crops worldwide
Both have been accepted by CBOL as ‘demonstrator’ projects
MBI inceptive meeting, NHM, London 21-22 Nov 2005
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?
Why barcode mosquitoes?
• Relatively small but diverse group
• Relatively well known
• Actively researched worldwide
• Huge potential impact on parasitic and arboviral disease control
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 morphologyIntegrated systematic studies are “the new taxonomy”
An. (C.) christyiAn. (N.) oswaldoiatropos sacharovi persiensis martinius GBatroparvus labranchiae GB
94
messeae daciae
79
maculipennis melanoon
7067
64
95
98
beklemishevi quadrimaculatusmaverlius smaragdinus inundatus diluvialis 100
8795
9566
75
occidentalis freeborni hermsi earlei 87
92
95
96
100Palaearctic
Nearctic
ITS2 phylogeny Maculipennis Group (745 seqs) (GB GenBank)
New species identified on basis of DNA data and formally described using integrated description
100
200
300400500
1000
600
nivipes philippinensis annularis
Annularis Group
annularis India*
annularis Viet/Korea/Laos/Camb
annularis Philippines*
philippinensis Vietnam/Laosnivipes Vietnam/Cambodia/Laos
jamesi Vietnam
maculatus Vietnam
96100
96
99
0.02
ITS2
Wide geographical sampling reveals new species
Voucher specimens
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
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
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
Smithsonian Institution, USAMay 11-12 2006
• Update meeting of 5 members of the MBI steering committee to assess success of pilot study
Museum specimens from 2000CPV1-1 An. philippinensis
Luksang Kampong, Preah Vihear Province, Cambodia. Human Bait. Linton et al., 2004
GenBank AF546338 mtDNA COI
GenBank AJ674540 nDNA ITS2
Sigma Qiagen
500 bp
COI: LCO1490F/HCO1490R
Archive mosquitoes (QIAmp micro kit, QIAgen)
1 7 8 9 1062 3 4 5A
B
1 7 8 9 1062 3 4 5A
B
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
Optimal DNA extraction from Museum specimens
100μl GB
10μl Prot K
2 min
QIAgen Biosprint, 50μl
Minimum of 12-24hrs in
shaking incubator @
55oC
QIAgen Blood kit and magnetic bead DNA
transfer
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
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
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 12
Zoogeographic Distribution of CulicidaeGenera (approx number of species) per region
14 (190)
24 (820)
14 (200)
18 (560)
20 (520)
23 (880)
NHMCo-ordinators: R. Harbach (morph)
& Y. Linton (mol)
SMITHSONIANCo-ordinators: R. Wilkerson (morph) & D. Foley (mol)
MBICo-ordinators:
Y. Linton & R. Lane
UNDN. Besansky
ITMW. Van Bortel
IndiaCo-ordinator:
P. Kumar
SE AsiaCo-ordinator:P. Somboon
Latin AmericaCo-ordinators:
M. A. Sallum & M. Quinones
AustralasiaCo-ordinator: D. Foley
AfricaCo-ordinator:M. Coetzee
World mosquito workers
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.
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!