Download Control of Insect Disease Vectors & Agricultural Pests

Consortium for the Barcode of Life Regional Meeting
Nairobi, Kenya 18-19 October 2006
Control of Disease Vectors
Dr Yvonne-Marie Linton
Natural History Museum, London
Correct species
identification is critical for
effective control of insectborne pathogens and
agricultural pests
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
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
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 morphology
Integrated systematic studies are “the new taxonomy”
98
94
100
95
79
64
67
70
96
75
66
95
87
100
95
95
92
87
An. (C.) christyi
An. (N.) oswaldoi
atropos
sacharovi
persiensis
martinius GB
atroparvus
labranchiae GB
messeae
daciae
maculipennis
melanoon
beklemishevi
quadrimaculatus
maverlius
smaragdinus
inundatus
diluvialis
occidentalis
freeborni
hermsi
earlei
Palaearctic
Nearctic
ITS2 phylogeny Maculipennis Group (745 seqs) (GB GenBank)
New species identified on basis of DNA data and formally
described using integrated description
Annularis Group
1000
600
500
400
300
200
100
nivipes
philippinensis
96
100
ITS2
96
annularis
annularis India*
annularis Viet/Korea/Laos/Camb
annularis Philippines*
philippinensis Vietnam/Laos
nivipes Vietnam/Cambodia/Laos
jamesi Vietnam
99
0.02
maculatus Vietnam
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, USA
May 11-12 2006
• Update meeting of 5 members of the MBI
steering committee to assess success of
pilot study
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
Sigma
COI:
LCO1490F/HCO1490R
Qiagen
500 bp
Archive mosquitoes (QIAmp
micro kit, QIAgen)
1
2
3 4
5
6
7 8 9
10
A
B
A. LCO1490F/HCO2198R
B. LCO1490F/C1J1718MODR
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
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
Optimal DNA extraction from
Museum specimens
100μl GB
2 min
10μl Prot K
Minimum of
12-24hrs in
shaking
incubator @
55oC
QIAgen Blood kit and
magnetic bead DNA
transfer
QIAgen Biosprint, 50μl
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
SI
825
79
NHM
480
69
ICMR
312
UQIC
190
USP
185
RMCA
25
CMU
24
UND
In EtOH
12
50
NHM & SI*
679
Total: 2930
2720
198
12
Zoogeographic Distribution of Culicidae
Genera (approx number of species) per region
14
(200)
14
(190)
23
(880)
24
(820)
18
(560)
20
(520)
MBI
Co-ordinators:
Y. Linton & R. Lane
NHM
SMITHSONIAN
Co-ordinators: R. Harbach (morph)
& Y. Linton (mol)
Co-ordinators: R. Wilkerson
(morph) & D. Foley (mol)
ITM
UND
W. Van Bortel
N. Besansky
India
SE Asia
Africa
Latin America
Australasia
Co-ordinator:
P. Kumar
Co-ordinator:
P. Somboon
Co-ordinator:
M. Coetzee
Co-ordinators:
M. A. Sallum & M. Quinones
Co-ordinator: D. Foley
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!