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Dr Yvonne-Marie Linton
Natural History Museum, London
Malaria kills a child every 30 seconds in Africa
Dengue MalariaEncephalitis
Yellow fever
WN virus
Filariasis
The changing face of mosquito-borne disease
Man
Parasite / Virus
Parasite / Virus
Mosquito
To generate DNA barcodes for 80% of the World Culicidae within two years
No less than 5 individuals per species
MBI objectives
Zoogeographic Distribution of CulicidaeGenera (approx number of species) per
region
14 (190)
24 (820)
14 (200)
18 (560)
20 (520)
23 (880)
3457 species in 43 genera
Life in the field
Life in the field
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
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
0 100 200 300 400 500 600 700 800 900 1000
ANOPHELES
AEDEOMYIA
AYURAKITIA
CULISETA
FICALBIA
HEIZMANNIA
JOHNBELKINIA
MALAYA
MIMOMYIA
ORTHOPODOMYIA
SABETHES
TOPOMYIA
TRIPTEROIDES
VERRALLINA
Number of species per genera
Taxa in Hand Missing taxa
Museum collections are critical to MBI success
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
But…..can you get DNA from old specimens?
Mosquitoes 1998-2001
Sigma Qiagen
500 bp
ITS2: ITS2A/ITS2B
Sigma Qiagen
500 bp
COI: LCO1490F/HCO1490R
Archive mosquitoes
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
Non-destructive sampling of rare specimens
MBI sequencing strategy
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
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
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!
$£$£ MBI Sponsors $£$£
• CBOL
• Colciencias, Colombia
• Christ Church Canterbury
• Great British public!!
…so six weeks ago we started……
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
Aedeomyia
Aedes
Anopheles
Armigeres
Culex
Culiseta
Ficalbia
Heizmannia
Malaya
Mansonia
Mimomyia
Orthopodomyia
Tripteroides
Uranotaenia
Verrallina
Pie chart showing genus composition of 654 Culicidae barcode records (ICMR + NHM)
654 records generated so far
Genus No. of species (154)
No. of records(654)
No. of haplotypes(384)
Pi (mean nucleotide diversity / site)
Aedeomyia 1 1 1
Aedes 12 40 20 0.10470
Anopheles 98 529 314 0.11287
Armigeres 1 2 2 0.00236
Culex 22 39 27 0.09216
Culiseta 4 4 4 0.12734
Ficalbia 1 1 1
Heizmannia 1 1 1
Malaya 1 6 4 0.01746
Mansonia 2 2 2 0.12944
Mimomyia 1 1 1
Orthopodomyia 1 1 1
Tripteroides 1 1 1
Uranotaenia 2 2 2 0.13570
Verrallina 2 3 3 0.07853
0
5
10
15
20
25
Aedeo
myia
(6)
Aedes
(913
)
Anoph
eles(
460)
Armige
res(
58)
Culex(7
65)
Culiset
a(38)
Ficalbia(
8)
Heizman
nia(40)
Mala
ya(1
2)
Man
sonia
(23)
Mim
omyia
(44)
Orthopo
domyia
(38)
Tripte
roide
s(12
2)
Urano
taenia
(220
)
Verra
llina(
95)
Graph showing % species barcoded so far in 15 of 43 Culicid genera
Graph showing mean numbers of records per species by genus
0
1
2
3
4
5
6
7
Anopheles barcode record quality (529 records; ICMR – 20, NHM – 509; 154 species)
Sequence Length: 98.1% over 500bp; 58.4% 658-700bp
Sequence Quality:• 100% are sequenced in both directions• All bases have been manually verified• Min. 85% overlap• No records with missing/ambiguous bases
included
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
Voucher specimens
Processing museum specimensPinned 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 BOLDSpecimens taken from type
locality or as near to as possible
Tree of Culicidae
4020
ANCLAVIG
ER
4024ANCLAVIGER
4099CLAVIGER
4096CLAVIGER
4021ANCLAVIGER
4022ANCLAVIGER
3987ANCLAVIGER4023ANCLAVIGER4097CLAVIGER4098CLAVIGER3978ANCLAVIGER
5001ANSPLENDIDUS
5002ANSPLENDIDUS
Anophelessplendidus
4904ANBARBIROSTRIS
4907ANBARBIROSTRIS6201nigerrimus
6160707bp6197nigerrimus
3226ANFARAUTI-6
3227ANFARAUTI-63225ANFARAUTI-63228ANFARAUTI-66193.6194.
1300ANDIRUS1301ANDIRUS
1996ANTESSELLATUS1998ANTESSELLATUS2000ANTESSELLATUS1997ANTESSELLATUS1355ANKOCHI
2008ANKOCHI2006ANKOCHI2007ANKOCHI
5427.5442.5434.5413.
1034ANNIMPE1035ANNIMPE1037ANNIMPE1038ANNIMPE1033ANNIMPE
1036ANNIMPE1039ANNIMPE
AnophelesjeyporiensisvoucherVCRCMMA12506AnophelesjeyporiensisvoucherVCRCMMA12507
AnophelesjeyporiensisvoucherVCRCMMA12502
AnophelesjeyporiensisvoucherA12479VCRCMM
AnophelesjeyporiensisvoucherA12500VCRCMM4942ANJEYPORIENSIS
2193ANJEYPORIENSIS4937ANJEYPORIENSIS
4938ANJEYPORIENSIS
4940ANJEYPORIENSIS
4939ANJEYPORIENSIS
4941ANJEYPORIENSIS
2192ANJEYPORIENSIS
3981ANMARTERI3985ANMARTERI
3984ANMARTERI
3983ANMARTERISOGDIANUS
3982ANMARTERI
3980ANMARTERI
3979ANMARTERI
1094ANPSEUDOBARBIROSTRIS
1096ANPSEUDOBARBIROSTRIS
1962ANSAMARENSIS
1960ANSAMARENSIS
1964ANSAMARENSIS
Anophelesaitkenii1963ANFRANCISCOI
2037ANFRANCISCOI
2038ANFRANCISCOI
1985ANFRANCISCOI
2036ANFRANCISCOI
2039ANFRANCISCOI
4906ANBARBIROSTRIS2
4911ANBARBISROSTRIS2
4912ANBARBIROSTRIS2
1057ANBARBIROSTRIS
4930ANCAMPESTRIS
Anophelesbarbirostris6155(IN114-3)
6158(IN123-3) 1097ANPEDITAENIATUS
2041ANPEDITAENIATUS
2042ANPEDITAENIATUS
1098ANPEDITAENIATUS
1099ANPEDITAENIATUS
1965ANPEDITAENIATUS
1971ANPEDITAENIATUS
1972ANPEDITAENITUS
4910ANPEDITAENIATUS
4909ANPEDITAENIATUS
Anophelespeditaeniatus
KS2-6(6)2707bpSPNOV
KS2-6-6-3707bpSPNOV
JP1-3ANHYRSPNOV1
JP1-4ANSPNRSINENSIS
JP27-1ANHYRCSPNOV1
KS51-100ANHYRCSPNOV1
KS2-7(1)1ANSPNRSINENSIS
JP7-3ANSPNRSINENSIS
KS2-6(3)sinensis710bp
1014ANPARALIAE
1019ANPARALIAE
5193ANPARALIAE
5192ANPARALIAE
5188ANPARALIAE
5190ANPARALIAE
1010ANSINENSIS
1012ANSINENSIS
1011ANSINEN
SIS
1013ANSIN
ENSIS
1022ANCR
AWFO
RDII
5715ANLESTERISP1
1021ANLESTERI1
KS2-3(6)ANYATSUSHIROENSIS
KS7(6)4ANYATSUSHIRO
ENSIS
KS8(16)ANPU
LLUS
KS8(10)AN
PULLU
S
4077ANPSEUD
OPICTUS
3677hyrcanusCOI
3554hyrcanusCOI
3326ANH
YR
CAN
US
3324ANH
YR
CAN
US
3552hyrcanusCO
I
3559hyrcanusCO
I
3557hyrcanusCO
I
3684hyrcanusCO
I
3325AN
HY
RC
AN
US
3551hyrcanusCO
I
3555hyrcanusCO
I
3556hyrcanusCO
I
4864ANSM
ARAG
DIN
US
4865ANSM
ARAG
DIN
US
4867ANSM
ARAG
DIN
US
4868AN
SM
AR
AG
DIN
US
4859AN
SM
AR
AG
DIN
US 4847A
NM
AV
ER
LIUS Anophelesvaruna
5115ANVAR
UN
A
21AN
VA
RU
NA
5114AN
VA
RU
NA
220gambiae
214gambiae
131ANSTEPHENSI
135ANSTE
PHE
NSI
146ANSTE
PHE
NSI
148AN
STE
PH
EN
SI
137AN
STE
PH
EN
SI
136AN
ST
EP
HE
NS
I
Anophelesstephensi
147AN
ST
EP
HE
NS
I145 A
NS
TE
PH
EN
SI
AnophelesstephensivoucherA
13574VC
RC
MM
AnophelesstephensivoucherV
CR
CM
MA
13668c
AnophelesstephensivoucherV
CR
CM
MA
13667
AnophelesstephensivoucherV
CR
CM
MA
11467
2720AN
PE
RY
AS
SU
I
2721AN
PE
RY
AS
SU
I
2717AN
PE
RY
AS
SU
I
2718AN
PE
RY
AS
SU
I
2719AN
PE
RY
AS
SU
I
4735ANHERM
SI
4767ANH
ER
MSI
4766ANH
ER
MS
I
4734AN
HE
RM
SI
4736AN
HE
RM
SI
4765AN
HE
RM
SI
4732AN
HE
RM
SI
4733AN
HE
RM
SI
4706AN
FRE
EB
OR
NI
4705AN
FRE
EB
OR
NI
4703AN
FR
EE
BO
RN
I
4701AN
FR
EE
BO
RN
I
4702AN
FR
EE
BO
RN
I
3927ANBEKLEM
ISHEVI
3951ANBEKLEM
ISHEVI
3937ANBEK
LEMISH
EVI
3941ANBEK
LEMISH
EVI
3921AN
BE
KLE
MIS
HE
VI
3922AN
BE
KLE
MIS
HE
VI
3942AN
BE
KLE
MIS
HE
VI
3929AN
BE
KLE
MIS
HE
VI
3956AN
BE
KLE
MIS
HE
VI
3923AN
BE
KLE
MIS
HE
VI
3924AN
BE
KLE
MIS
HE
VI
3919AN
BE
KLE
MIS
HE
VI
3925AN
BE
KLE
MIS
HE
VI
3825AN
BE
KLE
MIS
HE
VI
3957AN
BE
KLE
MIS
HE
VI
3940AN
BE
KLE
MIS
HE
VI
3930AN
BE
KLE
MIS
HE
VI
3953AN
BE
KLE
MIS
HE
VI
3938AN
BE
KLE
MIS
HE
VI
3935AN
BE
KLE
MIS
HE
VI
4871AN
EA
RLE
I4873A
NE
AR
LEI
4869AN
EA
RLE
I3576A
NM
ES
SE
AE
3577AN
ME
SS
EA
E3574A
NM
ES
SE
AE
3575AN
ME
SS
EA
E3573A
NM
ES
SE
AE
B2503D
AC
IAE
3671DA
CIA
E3453AN
PERSIEN
SIS
3459AN
PE
RS
IEN
SIS
3309AN
PE
RS
IEN
SIS
3280AN
PE
RS
IEN
SIS
3277AN
PE
RS
IEN
SIS
3448AN
PE
RS
IEN
SIS
3281AN
PE
RS
IEN
SIS
3456AN
PE
RS
IEN
SIS
3 28 2AN
PE
RS
IEN
SIS
3319AN
PE
RS
IEN
SIS
3315AN
PE
RS
IEN
SIS
3303AN
PE
RS
IEN
SIS
3560.
3314AN
PE
RS
IEN
SIS
3316AN
PE
RS
IEN
IS
3317AN
PE
RS
IEN
SIS
3279AN
PE
RS
IEN
SIS
3441AP
ER
SIE
NS
IS
3457AN
PE
RS
IEN
SIS
3458AN
PE
RS
IEN
SIS
3455AN
PE
RS
IEN
SIS
3442AN
PE
RS
IEN
SIS
3318ANPER
SIENS
IS
3302ANPER
SIENS
IS
3304PERSIEN
SIS
3443ANPER
SIENSIS
3306PERSIENSIS
3308ANPERSIENSIS
2742AN
SA
CH
AR
OV
I
2739AN
SA
CH
AR
OV
I
2740AN
SA
CH
AR
OV
I
2741ANSAC
HA
RO
VI3579AN
AT
RO
PA
RV
US
3662AN
AT
RO
PA
RV
US
5983AN
AT
RO
PA
RV
US
3582AN
ATR
OP
AR
VU
S
3587AN
ATR
OP
AR
VU
S
5982AN
ATR
OP
AR
VU
S
3599ATR
OP
ARVU
S
3770ANATR
OP
ARV
US
3658ANATR
OP
ARV
US
3767ANATR
OPARVUS
3588ANATR
OPARVUS
3595ANATR
OPARVUS
3586ANATROPARVUS
2773ANM
ELANO
ON
2871715bpmelanoon(G
R-)
2770715bpmelanoon(G
R-)
2881715bpmelanoon(G
R-)
2772ANMELANOON
2776ANMELANOON
2774ANMELANOON
2775ANMELANOON
2777ANMELANOON
3564Anmaculipennisss
3287ANM
ACULIPENN
IS
2931ANMACULIPENNIS
3566Anmaculipennsss
3288ANMACULIPENNIS
3307ANMACULIPENNIS
3445ANMACULIPENNIS
3562Anmaculipennisss
3563Anmaculipennis
3508ANMACULIPENNIS
3123ANMACULIPENNIS
3322ANMACULIPENNIS
3039ANMACULIPENNIS
3661ANMACULIPENNIS
3752ANMACULIPENNIS
3050ANMACULIPENNIS
3286ANMACULIPENNIS
3451ANMACULIPENNIS3140ANMACULIPENNIS
3449ANMACULIPENNIS 3463ANMACULIPENNIS 3285ANMACULIPENNIS3450ANMACULIPENNIS3323ANMACULIPENNIS
3455ANMACULIPENNIS
3124ANMACULIPENNIS
3509ANMACULIPENNIS
4853ANATROPOS
117ANCHRYSTII
118ANCHRYSTII
5053
ANPAMPANAI
5055ANPAMPANAI
5054ANPAMPANAI 4914
ANSAWADW
ONPORNI
4915
ANSAWADW
ONGPORNI
4917ANSAW
ADWONGPORNI
2206MACCOMPLEX
5043ANMACULATUS-K
4919ANMACULATUS-K
5020ANMACULATUS-K
5021ANMACULATUS-K
1896ANSALBAII
5370ANWILLMORI
5371ANWILLMORI
5658ANDRAVIDICUS
2207MACCOMPLEX
5206ANMACULATUS
5236ANMACULATUS
5234ANMACULATUS
5254ANMACULATUS5253ANPSEUDOWILLMORI
5659ANPSEUDOWILLMORI
Anophelesjamesii
5076ANJAMESII
5077ANJAMESII
5710ANJAMESII
5079ANJAMESII
5078ANJAMESII
5080ANJAMESII5081ANJAMESII
4896ANNIVIPES
5045ANNIVIPES
5033ANNIVIPES
4950ANNIVIPES
4951ANNIVIPES
4952ANNIVIPES
1899ANPHILIPPINENSIS
4106ANSUPERPICTUS
4107ANSUPERPICTUS
4104ANSUPERPICTUS
4105ANSUPERPICTUS
5044ANKARWARI
5046ANKARWARI
5047ANKARWARI
5048ANKARWARI
2208ANACONITUS
2349ANACONITUS
2347ANACONITUS
2345ANACONITUS2210ANACONITUS2209ANACONITUS2211ANACONITUS 3604ANLOUNIBOSI
2343ANCUCPHONGENSIS2340ANCUCPHONGENSIS
2344ANCUCPHONGENSIS
1059ANANNULARIS1060ANANNULARIS1061ANANNULARIS1062ANANNULARIS
Anophelesannularis
1067ANFLAVIROSTRIS1068ANFLAVIROSTRIS1066ANFLAVIROSTRIS1072ANFLAVIROSTRIS1069ANFLAVIROSTRIS
AnophelesculicifaciesAnophelesculicifaciesisolateRameswaram
2202ANMINIMUSC2346ANMINIMUSC2204ANMINIMUSC
2205ANMINIMUSC
AnophelesminimusAnophelesfluviatilis
AnophelesfluviatilisvoucherA12467VCRCMM
AnophelesfluviatilisvoucherVCRCMMA12471
AnophelesfluviatilisvoucherA12482VCRCMM
AnophelesfluviatilisvoucherA12478VCRCMM
AnophelesfluviatilisvoucherVCRCMMA12501c
4085ANALGERIENSIS
24ANPHAROENSIS27ANPHAROENSIS
28ANPHAROENSIS
23ANPHAROENSIS
25ANPHAROENSIS
26ANPHAROENSIS1207ANVAGUS1217ANVAGUS
1208ANVAGUS1212ANVAGUS
1209ANVAGUS
1213ANVAGUS
1221ANVAGUS
AnophelesvagusisolatePondicherry
2699ANBRAZILIENSIS2696ANBRAZILIENSIS
2702ANBRAZILIENSIS
2700ANBRAZILIENSIS
2697ANBRAZILIENSIS
2698ANBRAZILIENSIS
2701ANBRAZILIENSIS
9014ANMARAJOARA2444ANNUNEZTOVARI
245ANRANGELI
259ANRANGELI
256ANRANGELI
264ANRANGELI
258ANTRIANNULATUS
257ANTRIANNULATUS
267ANTRIANNULATUS335ANBENARROCHIB
374ANBENARROCHIB
380ANBENARROCHIB
563ANBENARROCHIB
562ANBENARROCHIB
393ANBENARROCHIB
9001ANOSWALDOID
9013ANOSWALDOID
9002ANOSWALDOID
9012ANOSWALDOID
8997ANOSWALDOID
9003ANOSWALDOID
9011ANOSWALDOID
8999ANOSWALDO
ID
9000ANO
SWALDO
ID
9007ANO
SWALDO
ID
9005ANO
SW
ALD
OID
9010AN
OS
WA
LDO
ID
Anophelessubpictus
AnophelessubpictusvoucherVCRCMMA115372422ANSPNRSUBPICTUS
2439ANSPNRSUBPICTUS
2437ANSPNRSUBPICTUS
2438ANSPNRSUBPICTUS
1917ANNRSUBPICTUS
2412ANSPNRSUBPICTUS
2415ANSPNRSUBPICTUS
2421ANSPNRSUBPICTUS
2418ANSPNRSUBPICTUS
2419ANSPNRSUBPICTUS
5067ANSPNRSUBPICTUS
1918ANSPNRSUBPICTUS
1919ANSPNRSUBPICTUS
2413ANSPNRSUBPICTUS
2431ANSPNRSUBPICTUS
2416ANSPN
RSUBPICTUS
2417ANSPN
RSUBPICTUS
2434ANSPN
RSUBPICTUS
2432ANSPN
RS
UBP
ICTU
S
2436ANSPN
RS
UBP
ICTU
S
2420AN
SP
NR
SU
BP
ICTU
S
2433AN
SP
NR
SU
BP
ICTU
S
2435AN
SP
NR
SU
BP
ICTU
S
AnophelessubpictusvoucherVCRCM
MA11536
AnophelessubpictusvoucherVCRCM
MA11535
1100ANINDEFINITUS
1177ANLITORALIS
1111ANINDEFINITUS
1178ANLITORALIS
1176ANLITO
RALIS
1103ANIN
DE
FINITU
S
1179ANLITO
RALIS
1180AN
LITO
RA
LIS
1175AN
LITO
RA
LIS
1149AN
IND
EFIN
ITU
S
2674ANSUNDAICUS-4
2683ANSUNDAICUS-4
982ANSUNDAICUS
984SUND
AICUS
983ANSU
ND
AICU
S
1056ANSU
ND
AICU
S
1055ANSU
ND
AICU
S
11ANEPIRO
TICUS
7ANEPIR
OTIC
US
6AN
EP
IRO
TICU
S
12AN
EP
IRO
TICU
S
9AN
EP
IRO
TIC
US
3subpictuscoi
1911AN
SU
BP
ICTU
S
1912AN
SU
BP
ICT
US
8AN
SU
BP
ICT
US
47HC
MV
IET
NA
M
1subpictuscoi2
su
bp
ictu
sc
oi
1914AN
SU
BP
ICT
US
1915AN
SU
BP
ICT
US
1913AN
SU
BP
ICT
US
4AN
SU
BP
ICT
US
5subpictuscoi
6154AN
BA
RB
IRO
STR
ISG
P
1058AN
BA
RB
IRO
ST
RIS
2310AN
BA
RB
IRO
ST
RIS
GP
2336AN
BA
RB
IRO
ST
RIS
GP
54ANBWAMBAE
57ANBWAMBAE
57bwambae
54bw
amba
e
59bw
amba
e
60bw
amba
e
60bw
amba
ecoi
99gam
biaeco
i
102ANGAMBIAE
223gambiae
2548arabiensis
216g
ambia
e
181a
rabie
nsis
172A
rabi
ensis
2537
gam
biae
210A
rabi
ensi
s
79Ar
abie
nsis
101A
NG
AM
BIA
E
81A
NA
RA
BIE
NS
IS20
4gam
biae
174a
rabi
ensi
s
193a
rabi
ensi
s
189a
rabi
ensi
s22
7gam
biae
168a
rabi
ensi
s10
3AN
GA
MB
IAE
170a
rabi
ensi
s22
6gam
biae
179a
rabi
ensi
s16
2AN
AR
AB
IEN
SIS
175a
rabi
ensi
s12
6AN
GA
MB
IAE
184a
rabi
ensi
s
98ga
mbi
aeco
i
178g
ambi
ae
169a
rabi
ensi
s
2534
gam
biae
166a
rabi
ensi
s
100A
NG
AM
BIA
E
55bw
amba
e
183a
rabie
nsis
187A
rabie
nsis
83ANARABIE
NSIS
80arabiensis
203arabiensis
180arabiensis
53bwambae
185Arabiensis
163arabiensis
113gambiaecoi176arabiensis
104ANGAMBIAE173Arabiensis55ANBWAMBAE6410ANARABIENSIS
6412ANARABIENSIS84ANARABIENSIS
85ANARABIENSIS
Cul
exbr
evip
alpi
siso
late
Mam
alap
usi
Cul
exbr
evip
alpi
siso
late
Pond
iche
rry
Cul
exbr
evip
alpi
siso
late
Lond
amun
dasi
Och
lero
tatu
spor
tono
voen
sisi
sola
teM
uthu
pe
Och
lero
tatu
spor
tono
voen
sisi
sola
teM
arav
ak
Och
lero
tatu
swar
divo
uche
rA12
563V
CR
CM
M
Och
lero
tatu
swar
diis
olat
eCor
inga
Och
lero
tatu
swar
divo
uche
rA12
564V
CR
CM
M
Och
lero
tatu
swar
diiso
late
Mut
hupe
t
Aede
omyi
acat
astic
ta
2574
AEZA
MM
ITTI
II
2575
AEZA
MM
ITTI
I
Aede
svitt
atus
isol
ateP
ondi
cher
ry
2983
CUL
ISET
ASPP
3952
CUL
ISET
ASPP
3856
MAL
GEN
UR
OST
RIS
3858
MAL
GEN
UR
OST
RIS
3857
MAL
GEN
URO
STR
IS
3860
MALG
ENUROSTR
IS
3859
MAL
GEN
URO
STR
IS
Mala
yage
nuro
stris
Fica
lbiam
inim
a
Orth
opod
omyia
anop
helo
ides
Trip
tero
idesa
rano
ides
Urano
taen
iaatra
Urano
taen
iabico
lor
Man
sonia
annu
lifera
Man
sonia
unifo
rmis
Urano
taen
iarec
ondi
ta
1959
AEPAICIL
IUS
Och
lerot
atus
pseu
dota
enia
tusv
ouch
erA12
344
Aedes
iyeng
ari
Mim
omyia
cham
berla
ini
Heizm
annia
discr
epan
sisola
teKar
nata
ka
Heizm
annia
chan
di
4322
AEAE
GYP
TI
4324
AEAE
GYP
TI
4323
AEAEGYPTI
Aedesa
egyp
ti2246AEAEGYPTI
2243AEAEGYPTI
2258
MANSONIASP
Aedesvexa
nsAedesfu
midus
Ochlero
tatuscogil
liisolateKarn
ataka
Armige
ress
ubalb
atusis
olate
Karna
taka
Armige
ress
ubalb
atusis
olateC
horao
Armigeress
ubalbatus
Verrallin
aindicaiso
latePondicherry
Verrallin
alugubrisiso
lateChoraoVerrallin
alugubrisiso
lateMuthupet
3164
AECRETINUS
3166
AECRETINUS
3165AECRETINUS
3167AECRETINUS
3168AECRETINUS
3169AECRETINUS
2229AESIMPSONI
2230AESIMPSONI
2095AEAFRICANUS
2093AEAFRICANUS
2094AEAFRICANUS
Aede
salb
opict
us
4317
AEAL
BOPI
CTU
S
4319
AEAL
BOPI
CTU
S
4321
AEALBO
PICTU
S
3158
AEALBOPIC
TUS
4320AEALBOPICTUS
4318AEALBOPICTUS
4302AEALBOPICTUS
4815AEALBOPICTUS
4300AEALBOPICTUS
4816AEALBOPICTUS4817AEALBOPICTUS4821AEALBOPICTUS4303AEALBOPICTUS
4301AEALBOPICTUS
4822AEALBOPICTUS
4823AEALBOPICTUS
Culexmalayi
CulexpluvialisvoucherVCRCMMA09239c
Culexminor
2985CULISETASPPCulexrubithoracis
Culexnigropunctatus
CulexpallidothoraxvoucherA12348VCRCMMCulexinfantulus
Culexfuscanus
CulextritaeniorhynchusisolatePondicherry
Culextritaeniorhynchusisolation-sourcebr
Culextritaeniorhynchus
CulextritaeniorhynchusisolateMarvakkadu6032CULEXSPCulexvishnui
Culexvishnuiisolation-sourceirrigationca
CulexpseudovishnuiisolatePondicherry
Culextritaeniorhynchusisolation-sourceirCulexwhitmoreivoucherA12334VCRCMM
CulexbitaeniorhynchusvoucherA12558VCRCMM
CulexbitaeniorhynchusisolatePondicherryCulexinfulaCulexmurrelli
Culexfuscocephala
Culexhutchinsoni
Culexpipiensquinquefasciatus
2212CXQUINQUEFASCIATUS
2084CXPIPIENSMOLESTUS2086CXPIPIENSMOLESTUS
2214CXQUINQUEFASCIATUS2213CXQUINQUEFASCIATUS2096CXQUINQUEFASCIATUS2088CXPIPIENSMOLESTUS5435707bp
6088CULISETASPCulexgelidusCulexsitiensvoucherA12560VCRCMMCulexsitiensvoucherVCRCMMA12561CulexsitiensvoucherA13575VCRCMMCulexsitiensvoucherA13577VCRCMM
0.01
gambiaeAedes albopictus
Culex and Aedes
Anopheles
Good illustration of how divergence within species is much lower than between species.
CulexbrevipalpisisolatePondicherry CulexbrevipalpisisolateLondamundasi
CulexbrevipalpisisolateMamalapusi 2088CXPIPIENSMOLESTUS
5435707bp 2096CXQUINQUEFASCIATUS 2213CXQUINQUEFASCIATUS 2214CXQUINQUEFASCIATUS 2086CXPIPIENSMOLESTUS 2084CXPIPIENSMOLESTUS 2212CXQUINQUEFASCIATUS Culexpipiensquinquefasciatus
Culexhutchinsoni Culexfuscocephala
Culexmurrelli Culexinfula
CulexbitaeniorhynchusvoucherA12558VCRCMM CulexbitaeniorhynchusisolatePondicherry
CulexwhitmoreivoucherA12334VCRCMM CulexpseudovishnuiisolatePondicherry
Culextritaeniorhynchusisolation-sourceir Culexvishnui Culexvishnuiisolation-sourceirrigationca
6032CULEXSP CulextritaeniorhynchusisolateMarvakkadu
Culextritaeniorhynchus CulextritaeniorhynchusisolatePondicherry
Culextritaeniorhynchusisolation-sourcebr 6088CULISETASP Culexgelidus
CulexsitiensvoucherA12560VCRCMM CulexsitiensvoucherVCRCMMA12561
CulexsitiensvoucherA13575VCRCMM CulexsitiensvoucherA13577VCRCMM
Culexfuscanus Culexinfantulus
Culexnigropunctatus CulexpallidothoraxvoucherA12348VCRCMM
Culexrubithoracis 2985CULISETASPP
Culexminor Culexmalayi
CulexpluvialisvoucherVCRCMMA09239c OchlerotatusportonovoensisisolateMuthupe
OchlerotatusportonovoensisisolateMaravak OchlerotatuswardivoucherA12563VCRCMM OchlerotatuswardiisolateCoringa
OchlerotatuswardivoucherA12564VCRCMM OchlerotatuswardiisolateMuthupet Aedeomyiacatasticta
2574AEZAMMITTIII 2575AEZAMMITTII
AedesvittatusisolatePondicherry 2983CULISETASPP
3952CULISETASPP 3856MALGENUROSTRIS 3858MALGENUROSTRIS 3857MALGENUROSTRIS
3860MALGENUROSTRIS 3859MALGENUROSTRIS Malayagenurostris
Ficalbiaminima Orthopodomyiaanopheloides
Tripteroidesaranoides Uranotaeniaatra
Uranotaeniabicolor Mansoniaannulifera
Mansoniauniformis Uranotaeniarecondita
1959AEPAICILIUS OchlerotatuspseudotaeniatusvoucherA12344
Aedesiyengari Mimomyiachamberlaini
HeizmanniadiscrepansisolateKarnataka Heizmanniachandi 4322AEAEGYPTI 4324AEAEGYPTI 4323AEAEGYPTI
Aedesaegypti 2246AEAEGYPTI
2243AEAEGYPTI 2258MANSONIASP
Aedesvexans Aedesfumidus
OchlerotatuscogilliisolateKarnataka ArmigeressubalbatusisolateKarnataka
ArmigeressubalbatusisolateChorao Armigeressubalbatus
VerrallinaindicaisolatePondicherry VerrallinalugubrisisolateChorao
VerrallinalugubrisisolateMuthupet 3164AECRETINUS 3166AECRETINUS 3165AECRETINUS 3167AECRETINUS 3168AECRETINUS 3169AECRETINUS
2229AESIMPSONI 2230AESIMPSONI
2095AEAFRICANUS 2093AEAFRICANUS 2094AEAFRICANUS
Aedesalbopictus 4317AEALBOPICTUS
4319AEALBOPICTUS 4321AEALBOPICTUS
3158AEALBOPICTUS 4320AEALBOPICTUS 4318AEALBOPICTUS 4302AEALBOPICTUS 4815AEALBOPICTUS 4300AEALBOPICTUS 4816AEALBOPICTUS 4817AEALBOPICTUS 4821AEALBOPICTUS 4303AEALBOPICTUS 4301AEALBOPICTUS 4822AEALBOPICTUS 4823AEALBOPICTUS
54100
7310
54
100
69
87100
2616
7
100
100
5577
56100
43
62
47
3
100
6161
100
35
5
10
3
92
6
4
3
2
65
2
8538
4344
10042
100
5
308
0
65100
97
100
293
1
0
7421
161
5326
0
4060
57100
100
38
20
39100
10089
52
100
100
68100
77
5
3068
491950
35
83
100
3
1
2
5
0
0
0
0
0
8
0.02
Aedes albopictus
Aedes cretinus
Aedes aegypti
Culex pipiens s.l.
Ochlerotatus
Culicine mosquitoes
Aedine mosquitoes
Utility of barcodes in Anopheles 2208ANACONITUS
2349ANACONITUS
2347ANACONITUS
2345ANACONITUS
2210ANACONITUS
2209ANACONITUS
2211ANACONITUS
3604ANLOUNIBOSI
2343ANCUCPHONGENSIS
2340ANCUCPHONGENSIS
2344ANCUCPHONGENSIS
1059ANANNULARIS
1060ANANNULARIS
1061ANANNULARIS
1062ANANNULARIS
Anophelesannularis
1067ANFLAVIROSTRIS
1068ANFLAVIROSTRIS
1066ANFLAVIROSTRIS
1072ANFLAVIROSTRIS
1069ANFLAVIROSTRIS
Anophelesculicifacies
AnophelesculicifaciesisolateRameswaram
2202ANMINIMUSC
2346ANMINIMUSC
2204ANMINIMUSC
2205ANMINIMUSC
Anophelesminimus
Anophelesfluviatilis
AnophelesfluviatilisvoucherA12467VCRCMM
AnophelesfluviatilisvoucherVCRCMMA12471
AnophelesfluviatilisvoucherA12482VCRCMM
AnophelesfluviatilisvoucherA12478VCRCMM
AnophelesfluviatilisvoucherVCRCMMA12501c
100
89100
9
1
100
97
100
99
30
4490
99
99
9830
31
63
99
89
18
5
0.01
Mis-ID (aconitus)
Sp. nov
Unknown larvae
Species complexes
Limits of barcodes in Anopheles 54ANBWAMBAE 57ANBWAMBAE
57bwambae 54bwambae 59bwambae
60bwambae 60bwambaecoi
99gambiaecoi 102ANGAMBIAE
223gambiae 2548arabiensis
216gambiae 181arabiensis 172Arabiensis 2537gambiae 210Arabiensis 79Arabiensis
101ANGAMBIAE 81ANARABIENSIS
204gambiae 174arabiensis 193arabiensis 189arabiensis 227gambiae 168arabiensis 103ANGAMBIAE
170arabiensis 226gambiae 179arabiensis 162ANARABIENSIS 175arabiensis 126ANGAMBIAE 184arabiensis 98gambiaecoi
178gambiae 169arabiensis 2534gambiae 166arabiensis
100ANGAMBIAE 55bwambae 183arabiensis 187Arabiensis
83ANARABIENSIS 80arabiensis
203arabiensis 180arabiensis 53bwambae 185Arabiensis 163arabiensis 113gambiaecoi 176arabiensis
104ANGAMBIAE 173Arabiensis 55ANBWAMBAE
6410ANARABIENSIS 6412ANARABIENSIS
84ANARABIENSIS 85ANARABIENSIS
88252265
1878
30
34
46
12
64
66
4051
220
2
7
18
37
59
0.001
bwambae
arabiensis / gambiae
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!!!!
Working together for a brighter future