Aedes aegypti in ho chi minh city viet n

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE 1999 93,581-586 genetic differentiation ‘Institut Pasteur de Ho-Chi-Minh Ville, Laboratoire d’Entomologie Abstract Ae

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1999) 93,581-586

genetic differentiation

‘Institut Pasteur de Ho-Chi-Minh Ville, Laboratoire d’Entomologie

Abstract Aedes aegypti is the principal vector of dengue viruses, responsible for a viral infection that has become a major public health concern in Asia In Viet Nam, dengue haemorrhagic fever was first detected in the 1960s

collected in 1998 in Ho Chi Minh City were subjected to oral infection and isoenzyme polymorphism

belt These results have implications for insecticidal control during dengue outbreaks

Introduction

Dengue epidemics have been reported since 1779

(HIRSCH, 1883) Before the 195Os, epidemics generally

the viruses and their mosquito vectors were displaced

pattern of dengue transmission changed and the disease

particularly those in Asia Increased movement ofhuman

dengue transmission At the same time, the increase in air

traffic further enhanced the spread of dengue viruses

tion of the 4 dengue serotypes), which has resulted in an

increase in dengue incidence, and the emergence of

In South-East Asia, DHF was first described in Manila

disease was first reported in North Viet Nam in 1958,

South Viet Nam in 1960, Singapore in 1960, Penang in

196Os, major dengue epidemics have affected at least 2

million people living around the Red River Delta, with

STEAD et al., 1965; Vo QUI DAI & NGUYEN THI KIM-

THOA, 1967) In this region, the pattern of DHF has

developed since 1975 with a gradual rise in the number of

fatal cases: 119 429 DHF cases were recorded in 1998

versus 19 4 16 in 1975 In 1998, dengue virus was isolated

from 15% of 1467 serum samples from clinical patients,

and in 74% of these cases the virus isolated was denaue-

3 All the DHF cases observed in Ho Chi Minh C& in

1998)

transmission are observed: urban and rural In urban

sporadic cases and local outbreaks are observed There

1915 (STANTON, 1920) In the 192Os, Ae aegyptimade

Address for correspondence: Dr Anna-Bella Failloux, Unite

d’Ecologie des Svstkmes Vectoriels, 25 rue du Dr Roux, 75724

Paris c&Jex 15, -France; phone +j3 1 40613617, fax’+33 1

40613089, e-mail afaillou@pasteur.fr

Ae aegypti invaded Asia during the second half of the 19th Century following the increase in trade and ship- ping The species was first introduced via the seaports and gradually spread inland along rivers and roads Usually, the peak in dengue incidence occurs during the rainy season, coinciding with the highest densities of

Ae aegypti populations In South Viet Nam, dengue cases are observed throughout the year whereas in North Viet Nam, dengue transmission does not occur during the winter In Ho Chi Minh City, DHF cases are mostly reported from June to October A substantial proportion (39.4%) of the mosquitoes collected inside habitations and most of the larvae collected from neridomestic

Institute in Ho Chi Minh City) Only 1% of the collected

of urbanization in providing breeding places suitable for

Ae aegypti

The change in the pattern of dengue transmission in Viet Nam from urban epidemics to endemicity is partly

Urban endemic dengue fever was not known in Asia until the end of the 19th Century although epidemics did

which can survive in urban environments, has led to the

flight range, which restricts its dispersion around breed-

highly urbanized cities therefore tend to differ greatly in

ences may be correlated with traits of medical impor- tance such as competence as a vector for dengue viruses

to dengue 2 virus was analysed to determine the compe-

morphism This indicated the pattern of gene flow and,

Mosquito sawtples (Table 1, Figure) Twenty mosquito samples were collected from Feb- ruary to May 1998 in Ho Chi Minh City and its suburbs:

2 from the 2nd district, 2 from the 7th district, 1 from the 8th district, 5 from Binh Chanh, 2 from Cu Chi, 2 from Hoc Mon, 2 from Nha Be, with the remaining samples from the 6th district, Thu Due, 9th district and Binh Thanh

The samples collected consisted of larvae or pupae which were reared until the imago stage in an insectarium (temperature 25 & l”C, relative humidity 80 f IO%,

582 TRANKHANHTIEN ETAL

1 PHU

2 NHA

3 KIE

4 NBE

5 BIN

6 CHA 7.ABB

8 CUC

9 CHI

10 BCH

11 LOI

12 HA1

13 MON

14 HOC

15 KHA

16 QUA

17 THU

18 BIH

19 THA

20 MYL

05/02198

05102198

07102198

07102198

10102198

1 l/02/98

11102198

19102198

19102198

27102198 05/03/98 05lO3198

20105198

20105198

28105198 19/03/98

25103198

31103198

01104198

28105198

and 12-h light/dark cycle) The resulting FO adults were

allowed to blood feed on mice Batches of eggs were

obtained and hatched and the larvae were reared until the

image stage (Fl) FO adults were stored at -80°C until

analysis forisoenzyme polymorphism and Fl adults were

strain of Ae: aegy@ was collected i; Paea district (Tahiti,

laboratory since 1994 This strain was used as a control

for mosquito oral susceptibility to dengue viruses and as a

mobility control for isoenzyme polymorphism

Experimental infection qf mosquitoes

bne to two infection assays were carried out for each

females were placed in 0.5-litre plastic-screened contain-

ers and starved for 24 h prior to infection They were

allowed to feed for 20 min on an infected artificial meal

maintained at 37°C in glass feeders covered with chicken

skin stretched over thi opening at the base (VAZEILLE-

FALCOZ et aZ., 1999) The infectious meal consisted of 2

parts washed rabbit erythrocytes to 1 part virus suspen-

10”’ MIDso (mosquito infectious dose for 50% for Ae

aegypti individuals) per mL of the dengue type 2 strain

isolated in 1974 from a serum samnle from Bangkok

described elsewhere (VAZEILLE-FALCOZ et al., 1999)

Fully engorged females were incubated at 28°C in small

cardboard containers and were fed on a 10% sugar

solution for 14 days Surviving females were killed and

tested for the presence of dengue virus by indirect

(VAZEILLE-FALCOZ et al., 1999) Prior to the infection of

field-derived populations of Ae aegypti, we determined

the optimal amount of virus necessary to infect an

average of 90% of the Paea strain For such purpose,

serial dilutions of the viral stock were used to infect

mosquitoes The titre of the feeding suspension that gave

90% infected females was estimated and was later used to

infect field populations

Isoenzyvne po&morp&n

Individual mosquitoes were homogenized in 25 L of

(15 000 g, 5 min at +4”C) and half the supernatant

was subjected to starch gel electrophoresis in the Tris-

al., 1988) This made it possible to investigate 10 enzyme svstems oer individual (5 ner eel) We actuallv studied 9 ehzyme systems: esterases (Est, ‘EC 3.1.1.1 )I phospho- glucoisomerase (Pgi, EC 5.3.1.9.), glutamate-oxaloace-

hexokinases (Hk, EC 2.7.1.1.), malate dehydrogenase

(Mpi, EC 5.3.1.8.), malic enzyme (Me, EC 1.1.1.40.)

developed from an isofemale lineage of Ae aegypti ‘Paea’ (Tahiti, French Polynesia) was used as a control Allelic

strain In field populations, alleles were scored in relation

to the most common allele obtained at each locus in the Paea strain

Population diSferentiation

3.1) software (RAYMOND & ROUSSET, 1995) I”, and F,, were estimated as described by WEIR & COCKERHAM (1984) Heterozygote deficits were tested using an exact test procedure (ROUSSET & RAYMOND, 1995) Global

sample Genotypic differentiation was tested by calculat- ing the P value of an F,, estimate The overall significance

of multiple tests was estimated by Fisher’s combined probability test (FISHER, 1970) The number of effective migrants (Nm) was calculated from F,, estimates accord- ing to the relationship: Nm = [( 1 /F,,) - l] /4 (WRIGHT, 1969) Isolation by distance was assessed by determining the significance of the correlation between F,, estimates and geographical distances (SMOUSE et al., 1986; LE- DUC et al., 1992) If necessary, the significance level of each test was adjusted based on the number of tests run,

(HOLM, 1979)

Results Mosquito infection rates For the 15 mosquito samples tested (Table 2), infec- tion rates obtained ranged from 88.8% [QUA (16)] to

sample, if 2 replicates were carried out, the infection rates obtained were compared For PHU (l), NHA (2) and the Paea control, infection rates were homogeneous (P > 0.05) When we compared the rates obtained for the assay with their corresponding controls, only 1 rate

,4EDESAEGYPTI IN HO CHI Mn\rH CITY (WET MM) 583

more details, see Table 1) Scale bar represents 4 km

(17)] In analysis of all the samples, an overall P value

calculated in Fisher’s exact test was more highly signifi-

cant (P = 0.002) than the P value estimated for the

corresponding controls (P = 0,038)

infection rates, 2 groups were set up: one comprised 7

sites[PHU(l),KIE(3),ARR(7),KHA(15),QUA(16),

THU (17) and THA (19)] corresponding to mosquito

populations from tile centre ofHo Chi Mjnh City The

lected in the commuter belt @JHA (2), NBE (4), BIN

rejecred [P = 0,001) for samples from the city centre

whereas the samples collected in the commuter belr were

not differentiated (P = 0.35)

Genetic polymorphism

The 9 enzyme systems studied led to the identification

analysis In the hexokinase system (Hkl, Hk2 and Hk3

loci), only Hk2 was taken into account because Hkl and

and Me were discarded as each had only 1 or 2 alleles

Hk2, Pgi, Mdh and Pgm had allozyme profiles with co-

following genetic analysis is based on these 4 loci

Genotypic association between pairs of loci was ruled

out for aI samples by multiple testing (P > 0.05) The 4

loci analysed were considered to segregate indepen-

dently Analysis of the allele frequencies at these 4 loci

showed significant heterozygote deficit (P < 0.05) in 7 of

80 Bonferroni multjple tests: 5 at the Pgm locus [PHU

the Mdh locus [CHA (6)] and 1 at the Pgi locus [CHI

WI

differentiation of the 20 samDies evaluated bv estimatinn F,, was highly significant (FE = 1-0.099, P 2 10e6) (Tg- ble 4) We investigated the forces generating this pattern

ine to collection site in Ho Chi Minh Citv: samnles from

9 samples collected in the city centre [PHU (I), KIE (3), ARR (7), KHA (15), QUA (16), THU (17), BIH (I@,

differentiated (F,, = +0.07 I, P < 1 Om6) Mosquito sam- ples collected from the commuter b&r fNHA (2), NBE (4), BIN (5), CHA (6), CUC (8), CHI (9), BCH (lo), LOI (1 l), HAI (I2), MON (13) and HOC (14)] were also highly (F, = i-0.125 and significantly differentiated (P < 10v6) Therefore, sites were considered according

to their district Genetic differentiation was high (high F, values) and significant (P < 0.05) for populations col- lected in Cu Chi district (2 sites), Hoc Mon (2) and Binh Chanh (5) whereas in Nha Be district, moquito popula-

Gene flow (Table 4) estimated based on the number of effective migrants, ~Vtn, calculated from F,, estimates, was low for samples from the city centre (N17z = 3.3) and commuter belt (Nm = 1.7) Gene flow was estimated between samples from the same district (i.e., Cu Chi,

genetic exchange was high only for sites from Cu Chi (iVm = 7.31 and sites from Nha Be lNnr = 4-7) We assessed geographical genetic isolation by examining the slope of the regression between log Nm and log distance

TFlAN KHANH TIEN ETAL

% infected females (n) Sample

1 PHU

2 NHA

3 KIE

4 NBE

5 BIN

7 ARR

9 CHI

10 BCH

Replicate

it

i

a

a

a

a

Assay 99.1 (114) 96.5 (145) 96.8 (62) 96.5 (86) 92.2 (179) 93.5 (92)

100 (18) 94.5 (55)

Control

100 (51)

100 (50)

100 (51)

100 (50) 96.1 (52) 94.7 (38) 90.8 (65) 90.8 (65)

P (SD)

1 (0) 0.335 (0.003) 0.503 (0.003) 0.294 (0.003) 0.533 (0~005)

1 (0) 0.332 (0.003) 0.504 (0.004)

11 LO1

12 HA1

15 KHA

16 QUA

17 THU

18 BIH

19 THA All samples

We used a feeding suspension with a final tine of 10” MID,,/mL in all experiments to obtain an average of 90% of infected females in the Paea control strain

P, probability of homogeneity (Fisher exact test); SD, standard deviation

Numbers in bold correspond to significant Pvalues (cO.05)

between F,, estimates and geographical distance The

(b = +O.OOOl) and F,, was nenativelv (b = -0.0004) but

not significantly (P =<-‘0.52) c&related with geographical

distance Thus, the extent of gene flow was not affected

by the geographical distance separating the sites

Discussion

are highly structured in the Ho Chi Minh City area

Populations from the centre of Ho Chi Minh City were

rates

The oral susceptibility of Ae aegypti to dengue 2 virus

considered together Populations from the centre of Ho

whereas those from the commuter belt had homoge-

neous infection rates In the city centre, mosquito surveys

undertaken in 1998 in the Nha Be and 8th districts

showed Ae aegypti and Culex quinquefasciatus to be the

most common species (data provided by the Pasteur

Institute in Ho Chi Minh City) The density of these

mosquitoes rises during the rainy season (June to Sep-

tember) Running water is available in most of the

habitations in the city centre avoiding the need to store

drinking water, but numerous small containers (e.g.,

cans, plastic bottles, tyres) accumulate close to human

habitations Ae aegypti proliferates extensively in such

breeding places This species is considered to be the

principal cause of dengue outbreaks in Ho Chi Minh City

centre because it is present at a higher density than Ae

the commuter belt, rainwater is stored in earthenware

jars, which constitute the main breeding site for both Ae

aeevati and Ae albopictus The strikine differences in

dexgue incidence between the centre (l?- 16%) and the

been shown to be related to the drinking water supply

(MIROVSKY et al., 1965)

DHF mostly occurs ‘in the rainy season when high

densities of Ae aemti are recorded DHF involves an

factors including prior sensitization of the individual’s

immune system to dengue infection Infection with one

serotype but only transient cross-protection against other serotvnes The risk of contractinn DHF is 100 times

primary infections (MONATH, 1994) In Viet Nam, the

breaks involving all 4 serotypes may increase the fre-

gency measure The size of the area treated depends on the number of reported clinical dengue cases If only a few sporadic cases are recorded, spraying is limited to the

‘infected’ houses In densely populated areas, about 10 litres of insecticide are generally required to cover 1 km2 Then heterogeneity of infection rates in the city centre may also be due to differences in insecticidal control Ae aegypti populations are periodically subjected to various degrees of insecticide pressure, generating differentiated populations This idea is supported by the homogeneity

of infection rates obtained for mosquito samples from the commuter belt, which are subjected to fewer insecticidal treatments

mosquito populations from the commuter belt demon- strates that there is little gene flow between mosquito populations As Ae aegyptican disperse over up to 580 m

47 km apart [between CUC (8) and BIH (18)], a few genetic exchanges were recorded These results are consistent with those of C Paupy et al (personal

densely populated agglomerations in Tahiti The proxi- mity of human hosts limits the spread of Ae aegypti

In South Viet Nam, where 80% of the country’s DHF cases were reported in 1998, the most highly populated

million inhabitants, recorded 7211 DHF cases In con- trast, Ben Tre province, with only 1.4 million inhabi- tants, recorded 21087 DHF cases Thus, 1.5% of the

whereas only 0.14% of the inhabitants of Ho Chi Minh

951

586 TRAN KHANH TIEN ET/X

Table 4 Population differentiation of Ae aegypti in Ho Chi Minh City, 1998, as revealed by isoenzyme polymorphism analysis of four enzyme systems

(0.615) (0.001)

(0.367) (0.443)

(0.046) (0.015)

(1.00) (1.00)

“Probability of homogeneity See the text for a description of the analysis

Nm, number of effective migrants; Hk2, hexokinase 2; Mdh, malate dehydrogenase; Pgi, phosphoglucoisomerase; Pgm, phosphoglu- comutase

City were affected by DHF (i.e., one-tenth the propor-

tion in Ben Tre province) These observations demon-

strate the importance of the well-organized mosquito

control in Ho Chi Minh City, which tends to reduce the

size of potentially infected Ae aegyptipopulations there-

by limiting the risk of DHF

Thus, this study shows that in the centre of Ho Chi

Minh City, the extensive genetic differentiation and

heterogeneous infection rates of Ae aegypti populations

are probably due to intensive insecticidal control to

reduce vector density In the commuter belt, Ae aegypti

populations tend to be less genetically differentiated and

to have homogeneous infection rates Further studies on

the pattern of gene flow at various times in the year

(before, during and after the epidemic peak) are re-

quired Similarly, experimental oral infections of Ae

aegypti with other dengue serotypes would be useful as

Viet Nam is an area of dengue hyperendemicity

Acknowledgements

We thank Nadia Monnier for rearing mosquitoes We also

thank Luu Le Loan and Nguyen Huu Cuong, from the Pasteur

Institute in Ho Chi Minh City, for technical assistance We are

indebted to Professor J.-L Durosoir from the Pasteur Institute

in Paris, and to Professor Ha Ba Khiem, Director of the Pasteur

Institute in Ho Chi Minh City, for their support This work

received financial support from the Action Concert&e des Instituts

Pasteur (ACIP no 41175)

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Received 11 June 1999; revised 4 August 1999; accepted for publication 4 August 1999