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!