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