DSpace at VNU: Study on Isozymes from Aedes Aegypti Strains of some Residential Areas in Vietnam

V NU JO U R N A L 0 F S C IE N C E . N at . S c i . & Tech . T XIX. N.,4. 2003

STUDY ON ISOZYM ES F R O M A ED ES A E G Y P T I S T R A IN S O F S O M E
R E S ID E N T IA L A R E A S IN V IE T N A M
D o N g o e L ie n , N g u y e n T h i T h u H o a i
N a tio n a l Ư niưersity o f H a n o i
V u Duc H uong
In s titu te o f m a la ria , p a ra s ito lo g y a n d entom ology o f H a n o i
A b stra ct. lsozymes from 4 vvilcỉ- caught collections of Aedes aegypti mơsquito were
compared bv using polyacrvlamide gel electrophoresis. These collections vvere
collected from 4 residential areas of Vietnam (North, South, Centre and Central
highland area). This study revealed that there is an obvious relation between the
insecticide resistance and the expression of isozyme esterase in the mosquito. Some
differences of isozymes between larvae and adults vvere also found.
Key ivords: D erìgue fever, in se cticid e re sista n ce , s tra in , Aedes a e g y p ti%PAG E,
isozyme.
1.

In tr o d u c tio n
Dengue/ Dengue h e m o rrh a g ic fever (D F /D H F ) occurs th ro u g h o u t th e ye a r b u t

p a rtic u la rly in su m m e r tim e (u s u a lly from June to J u ly ). I t e a s ily becomes epidem ic
disease and tends to increase in S outhern region an d C e n tra l h ig h la n d o f V ie tn a m
re ce n tly. One reason fo r th is s itu a tio n is the in se cticid e resistance in dengue v iru s
vectors in w hich Aedes ae gypti is an im p o rta n t one. The m o squito has resisted to
some insecticides such as D D T. hexachloride benzen (o rg a n o c h lo rin e group ) and even
p y re th ro id group w h ic h has been considered as a p o w e rfu l g ro u p is now losing its
effect in some areas o f S o u th e rn region and C e n tra l h ig h la n d o f V ie tn a m [1, 2, 7].
T h e re vvere some dom estic and foreign researches [1 , 2 , 4, 7] s tu d y in g on the
expression o f isozym e com plex in d iffe re n t Aedes a e g y p ti s tra in s in o rd e r to c x p la in
th e re sista n ce a b ility in m o sq u ito . H ow ever, some o p in io n s are c o n tro v e rs ia l.

The ob jective o f th is research is to fin d o u t th e isozym e m a rk e r fo r in se cticid e
resistance by m eans o f s tu d v on th e e le c tro p h o re tic e x p re ssio n o f seve ral isozvmos:
E sterase (EST), O cta n o l dehydrogenase (O D H ), M a la te dehydroge nase (M D H ), A cid
phosphatase (ACP), A lk a lin e phosphatase (A LP ). F rom th is s tu d y , people can have
a m ore a p p ro p ria te m e asurc to choose in se cticid e s fo r each re s id e n tia l area.
T h ere íbre , we have done these w orks:
-

A n a lv z in g th e e le c tro p h o re tic re s u lts on p o ly a c ry la m id e gel o f 5 isozym es

fro m d iffe re n t g e o g ra p h ica l Aedes ae gvpti s tra in s in V ie tn a m .

28


S t u d x on is m y m c s f n n t i (irt/rs a v ỊỊx p ỉi s t r a in s of..

29

C o m p íin n ^ the* r l r r t r o p h n r r tir rx p re s s io n o f r s t r r a s r ỉro m larvíU ' and a d u lts
b\ te e h n iq u e ol |> n lv;irrv|;im i< ir n rl rlo c tro p h o re s is (Ỉ\*\(ÌK ) <)l isozym t\s th e re fo re we
can iin H o u t th e lu*st iso /vim * n u irk c r to in s c c tic id e re sista n co o f m nsquiỉoes.
2.

M a t e r i a ls a n d m o th c id s

B o th la rv .il* íim l íid u lts oĩ ' Aedes ciegypti s tn iin s i\rv cau tfht in these fo llo w in g
(lilT eront rosici(‘nt Ìíìl ỉirons o f V ic tn a m :
• N o rth e rn rc ^ in n : T h in h I j f t v illa g e - T h a n h T r i (lis trie t (T T ). H a n o i; Ly T h a i
To p re c in c t. H oan K ir n i (ỉis tn c t (H K ). H anoi.

- C e n tra l rc iỉio n

Sonịỉ r a u (S O to w n , Phu Yen p ro vin ce ; T u y Hoa to w n (T H ),

Phu Y en: Đ on g Ha tovvn íD H ). Q ua ng T r i p rovince : T rie u Đo v illa g e

• T rie u Phong

d is tr ic t (TP ), Q ua ng T r i p rovince
- S o u th e rn rcg io n : Ben T re to w n (B T ), Ben T re p ro v in c r; D ong X oai to w n (ĐX).
B in h Phuoc p ro v in c v ; B in h ('h a n h v illa g e - Can G io d is t r ir t ( ( ’<’, h H C M c ity ; B in h
T a y p re c in c t - II d ìs tric t. T h u Duc (T D ), H C M .

- Central highland: Thang Loi precinct - Buon Ma Thuot city (HMT), Dac Lac
p ro v in ce ; P lei Can tovvn * Ngoe Hoi ( ỉis tr ic t (N H ), K o n tu m p ro v in c e ; Kon T u m tow n
(K T ), Kon T u n i; Buon T xay tovvn* K ro n g a n a d is tr ic t (K ro ), Dac L a c.pro vince.
The m o sq u ito is isolated by m ethod devéloped by th e In s titu te o f M a la ria ,
P a ra s ito lo g y and K n to m o lo ^v o f H anoi th e n ra ise d fo r new larvat* and a d u lts ( F l)
and fọ r F3 descendents. w h irh th e n can be used in e x p e rim t*n ts . Kive isozymes
chosen

fo r

a n a lv s is

<>n

p o lv a c ry la m id e

gel

are

E stera se

(E S T),

O ctanol

d ch yd ro g cn a se (O I)H ), M a la te dehydrogenase (M D H ), Acicỉ p h osph ata se (AC P),
A lk a lin e phosphatasc' (AI.IM .
M o s q u ito sam ples (íìd u lts o f F l o r F3, la rv a e ) are c riis h r d w ith b u ffe r in ice
in to hom ogenized State. T h e v are th e n c o o lly c e n trifu g e d . T h e s u p e rn a ta n t is
collected fo r u s in g in ele ctro p h o re sis.
M a in c h e m ica l re age nts used in each speciíìc enzym e d e te c tio n are:
• E stera se (K S T): a • n a p h tv l acetate a n d /? - n a p h ty l ace ta te as su b stra te s.
Fíìst R luo KK as ro lo r- producer.
- O cta n o l (loh ydro go nnst1 (O D H ): octanol as s u b s tra tu . c o e n /y m e N A I)*, PMS,
N B T (M T T can bv used in ste a d o f N B T ).
- M a la te dehyđrogenasí* (M D H ): L -m a la te as s u b s tra te , coen/.ym e N A D P * (or
coenzym e N A I)*). I\\1 S and N B T (or M T T ).
• Aeicl phosphatase - A lk a lin e phosph.ìtast* (A LP )
B lue RR.

a - n a p h ty l m onophosphate as s u b s tra te . Fast


I)o Ngoe L ie n , N g u y en T h i T h u H o a i * Vu D u c H u o n g

30

The* p o l y a c r y l a m i d i * ge l is a ( i i s c o n t i n u o u s g e l w h i c h h a v e c o n c c n t r a t i o n oí 4 WI.

a n d 8% or T ‘o a n d 10°ò, size of lOcm X 8cm X 1.5 mm. Tiu* device served for
e le c tro p h o re s is is H oe fer a p p a ra tu s (S an fran cisco. U S A) o r m in iP ro te a n a p p a ra tu s

(HioKad). T he Chemicals W
3. R esu lts and dLscussion
3.1. E le c tr o p h o r e t ic p r o p e r tie s o f e ste ra se
K s tc ra s e ’s e le c tro p h o re tic re s u lt o f m o sq u ito in some a ro a s o f C e n tra l region
and C e n tra l h i^ h la n d is show n in F ig .l.

ỉn this picture. Kro, KT. NH. BMT sampỉes arc Aedes aegypti from Central
h ig h la n c l, T F . Đ U . T H , s c , fro m C e n tra l re gion . A lth o u g h th e same a m o u n ts o f
sam ples w ere loaded in each w e ll o f m ic ro tite r-p la te , we fin d ou t th e esterase
exp re ssio n s th a i

are d iffe re n t.

It

is show n th a t th e a c tiv ity

m o sq u ito in C e n tra l h ig h la n d is s tro n g e r th a n th a t fro m
region.

T h is

re s u lt

co rrcspo nds

to

d ire c t

bioassav

o f estcrast* fro m

m o s q u ito in C e n tra l

re s u lts

of

in s e c ticid e

on

m o squito es w h ic h in d ic a te th a t the a d u lt m o s q u ito in C e n tra l h ig h la n d has much
h ig h e r re s is ta n c e ra titì to p y re th ro id group th a n w h ic h in C e n tra l re g io n (V u Duc
H u o n g e l al. 2003, d a ta not p u b lis h e d ). A d d itin n a lly , th e (a s te s t esterase bands
(KST- 1) in the sc tw o groups are d iffe re n t m a rk e d ly : th e re are a t least 3 bands o f
iso/.ym e este rase KST-1 íro m rtd u lt m o squitoes in C e n tra l h ig h la n d (IìM T , N H , KT)
w h ile th e re a re one o r tw o EST-1 fro m a d u lt m o squito es in C e n tra l re gion .

'

•

Ỉ

S

ể fc a » «
BG

l

i

m

KRo KT NH

í

—

I

S

tề

TP ĐH

Í

S

m

+

TH

sc

i

i ầ

-

' ^

BMT

F ỉg . l: K stera se detc‘cti()iì o f a d u lt m o sq u ito in C e n tra l re g io n and C e n tra l h ig h la n d .
in com parison w ith la rv a (B G ) (on l\ '\ ( l) .
In th is gel, we also a n alyzed a la rv a l sam ple to com pare. A s can be seen from
th e p ic tu re , a ll osterase hands o f la rv a are m uch d a rk e r th a n
m o sq u ito . K iịỉ.2 also m akes i t cle a re r.

those o f a d u lt

.'$1

S tu d y on isozvmes fro rn aedcs (iv ỊỊy p ti s t r a in s o f . "

F tfĩ 2: K sturaso d e te c tio n o f larva in C e n tra l re g io n and

Central h ig h la n d (on PAG ).

T h is p ic tu re is th e esterase com pariso n betw een la rv a e o f some areas in
C e n tra l re g io n and in C e n tra l h ig h la n d .
On th e side o f re g io n a l co m p a riso n . the d iffe re n c e betw een th c la r v a l E S T o f
these tvvo groups is n o t as d e a r as betw een th e a d u lt

EST discussed above.

T h e re ío rc . it can he assum eđ th a t EST e s p e cia lly EST* 1 fro m a d u lt m o s q u ito in
C e n tra l hitfhlanl)i*tw<*cn insíT ticick* re sista n ce and isozyme exp ressio n in la rv a m ay need íu r th e r
research.
O n th e side o f d e v e lo p m e n ta l pe riod c o m p a ris o n , as vve discusseđ above, th e
n u m b e r o f esterase bands and th e ir expression are m uch h ig h e r fro m la rv a th a n
tro in a d u lt m osqư ito. It m ay be due to the fact th a t esterases p la y a m ore im p o rta n t
roli* in p r o t o c t i n g l a r v a in c o m p a r i s o n w i t h a d u l t s b e c a u s e l a r v a e a l w a y s l ĩ v e d in

a q u a tic e n v iro n m e n t vvhere th e re a r r more to x ic s and in s e c tic id e re s id u e th a n in
c o n d itio n vvhere a d u lt m o sq u ito cs live d .

F ig 3: E steraso (le te ctio n of* a d u lt m o squito in N o rth e rn , S o u th e rn , C e n tra l re g io n
and C e n tra l h ig h la n d . in com parison w ith la rv a ( B (j) (on PAG).

Do Ngoe L ie n , N guyên T h i T h u H o a i , Vu D u c H u o n g

32

A fte r a n a ly z in g esterase fro m T h in h L ie t (Tỉ.,* one s tra in o f N o rth e rn re g io n ),
D ong X oai (I)X - S o u th e rn re g io n ) m o sq u ito o f F l and F3 g e n e ra tio n , in c o m p a ris o n
vvith m o s q u ito o f C e n tra l re g io n (SC) and C e n tra l h ig h la n d (B M T ) (F ig .3 ), vve can
ha ve som e fo llo w in g com m en ts:
•

EST-

1 o f C e n tra l h ig h la n d m osquitoes are m ore a c tiv e (esterase bands

e xp re ssio n s are d a rk e r) th a n those o f N o rth e rn and S o u th e rn re g io n sam ples.
E ST-2, 3 o f F3 are a lw a y s m ore a ctive th a n F l (due to b a n d s ’ color).
VVhen c o n tin u o u s ly a n a lv z in g esterase fro m la rv a e in N o rth e rn a n d S o u th e rn
re g io n m o s q u ito (F ig .4 ), we a g a in c o n firm th a t esterase a c tiv ity o f la rv a is m uch
h ig h e r th a n th a t o f a d u lt m o squito . We also see th a t w h ile EST- 4, 2, 1 a c t iv ity fro m
la rv a e is s tro n g e r th a n fro m a d u lts (th e ir bands are h e a v ie r), its EST- 3 a c tiv ity
fro m la rv a e is m a rk e d ly w e a k e r th a n fro m a d u lt m osquitoes.

F ig .4 : K stera se d e te c tio n o f la rv a e m o squito in N o rth e rn , S o u th tT iì re g io n , in
co m p a riso n vvith a d u lts (M ) (on PAG ).
A fte r a ll, it can be th o u g h t th a t isozym e este rase is a good m a rk e r fo r
in s e c tic id e re sista n ce o f th e m o squitoes (e specia lly E S T - 1) (a n d fu rth e rm o re , fo r
research on tw o d e ve lo p m e n ta l p e rio d s o f m osquito, esierase m a rk e r is a ls o use ful
ỉE S T - 3, 4)).

3.2.

E le c tr o p h o r e t ic p r o p c r tie s o f O c ta n o l d e h y d ro g e n a s e a n d

M a la te

d e h y d ro g e n ase
Isozym e octa no l dehydrogenase o f Aedes a e g y p ti s tra in s

in some area o f

N o rth e rn , S o u th e rn , C e n tra l re gion and C e n tra l h ig h la n d has been d e tecte d b u t nơ
s ig n ific a n t d iffe re n c e Vvas found.
Isozym e

m a la te dehvdrogenase o f Aedes a e g yp ti s tra in s

in some area o f

C e n tra l rc ^ io n and C e n tra l h ig h la n d vvas also detected and th (‘ re s u lt o b ta in e d is


S tu íix 0/1 isozymes Ị r o t ì ì (ict/cs n c ịỊx p ỉi s t r a in s of..
s m iil.il tí) \vh ich S ỉiu l s II r t ;il lo u n d in Aedcs íris e ra tu s 2111(1 A r. h c n d iểrs o n ì |H| and
n o ( l i H n v n r e c n n bí* s c c n l ỉ r l v v r e n i v s i s t a n t a n d s e n s i t i v c s t n i i n s .

3.3.

E te v tro p h iằ v c tii'

p r o p e r tie s

of

A c ix ỉ

p h o s p h a ta s e

and

A l k a lin e

p h o s p h (ita s e
No d in V re iu v

in

.1 1ka 11 ni* phosphatase. acid

phosphatast*

betvveen Aedes

oogypti s tn ũ n s oí s o iiìr .irr.i.N 1 1 1 ( V n tr a l region and ( V n t n il h ig h lỉin d w as fou nd .
T h in # th iit can b r n o ti'(l hon* is th a t AC P- 2 is m ore s ta b lc th a n A C P -

1 in

s rp ỉira t mi* gel <>ỉ p l l a r o u m l s.o. OM c o n tr a r y to in s e p a r a t in ^ tf<*l o f p H a r o u n d 9.0.

IU*sitl(*s. no tliH V rrn re in th rs c tw o rn /v m e s is fo u n d betvveen head and th o ra x p a rt
and ab(lonu*n p a rt oỉ t l i r m nsquito.

4. C o n clu sio n
Ba se on t hi* a n a ly s is ()f f> isozym es fro m Aedes a e g yp ti s tra in s in d iffe re n t
rc s iíle n tia l rcg io n s o f V ie tn a m , It IS deduced th a t o n lv este rase (e s p e c ia lly KST- 1)
can 1)1* served as a goođ m a rk e r fo r in s e c tic id e re s is ta n c e « f Aedes a e g y p ti m o sq u ito .
It |> assum able due to the capacity oí’ intensive esterase activity of mosquitoes to
m srctinđc degradation, Iht;ius(» ()f the fact th a t most insecticiỉie has ester linkage
|2. 71. Hovvever, other iso/vm c analysis are also useful in explainin g the insecticide
riỉsistỉince of mosquito.

R eíeren ces
1

Tran Van Tien. Vu Sinh Nam. T rin h Quan Huan, Do Quang Ha. Truông ưyen Ninh.
I)t*n^ue íever. hemorrhagic íever and vector control in Vietnam in recent years.
Prevenũvv Mvdicint’ Journal. Vol. V III (2). 36(1998). pp: 124 (in Vietnamese).
N ntural Sciences and N ational Tochnologv Centre. Plant
Ktìvironm vntnỉ toxicology program V ỈE 197/031, 2001. (in

2

Chem istrv In s titiiti*.
ụroịvcting medicinc,
Vietnnmese).

‘t

T Chareonviriyaphnp, K. Lcrthusnee, Genetic d iffe re n tin tio n of Aedes aegypti

m ainland and Island populations from Southern Thailand, J. American Mosquito
Con tro i Association IS 3(2002). pp: 173 -177.

ỉ

Cì.p. Manchenko. Handbook o f Detection <ỉf Emvmes ttn Electrophoretic Gel, CRC Press,
1994, pp: 40 - 196.

f>

(S

r c Mathews. L E M unsterm ann. Cìenetic D iversity and I)iffe re n tia tio n in N orthern
popuỉation o f the Tree- hok' Mosquito Aedes hendersoni (Diptera: CuỊicidae). Annalis of
fhi' E n to m o lo g ic a l Sí>r/«7v ti/ A m v r i c a . Vol. 76 (3) N o v e m b e r 1983.
l) T Mourva. M .l) (ĩo rh ;iỉr. K Ranerjee, Eíĩect of sublethal dosagưs of insecticide on
C h i k u n g u n y a v i r u s s u s c r p t i h l r a n d r e í r a c t o r y s t r a i n o f A e d e s Qegypti. T he S o u t h e a s t

\sian Journal ()f Troptval medicine and pubỉic Health
S ra m e o .

V25 (3) September 1994*


Do Ngoe L ỉe n , N guyên T h i T h u H o a i , Vu D u c H u o n g
7.

M . M R o d r i g u e z , J.

Bisset,

M.

Rui z, A.

Soca, Cr os s -

r es is t an ce to p y r e t h r o i d

and

o r g a n o p h o s p h a t e insecti ci des. sel ect ion w i t h t e m e p h o s in A e d e s a eg yp ti in Cuba. J.

A m e r ic a n M o s q u ito Control a n d Biology ỉn L a tin A m e r i c a n - a te n th s y m p o s i u m 16 (4):
20 0 0 , pp: 2 9 5 - 3 1 2 .

8.

S.H.Saul, M.fJ.Si nsko, P.R. G rim stad, G.B. Craig, Id e ntifica tion o f sibling species. Aecies
tr is e r ia tu s a n d Ae. hcindersoni bv electrophoresi s. J .M e d .E n t.V o \ 13 (6): 1 977, pp: 7 05*
708.

TAP CHÍ KHO A HOC D H Q G H N . KHTN & CN. T.X1X, N04, 2003_________

N G H IÊ N CỨU IZOZYM CỦA MỘT s ố C H Ủ N G MƯỎI A ED ES AEGYPTI
TẠI MỘT SỐ VÙNG DÂN c ư CỦA VIỆT NAM
ĐỖ N g ọ c L iê n , N g u y ễ n T h ị T h u H o à i
Đ ạ i học Quốc g ia H à N ộ i

Vủ Đửc Hương

Viện sốt rét, K i s in h trũ n g và Côn trù n g T ru n g ương
Sốt D engue/ hay sốt x u ấ t h u y ế t D engue là m ộ t bệnh xẩy ra q u a n h năm ở các
nước n h iệ t đới và á n h iệ t đói nh ư V iệ t N am và v ir u t gây ra bệnh này được tru y ề n
chủ yếu qua m uỗi Aedes ae gypti. Cách tó t n h ấ t đê ph òng bệnh sốt D engue là khống
chế vectơ tru y ề n bệnh này. T u y n h iê n , việc sử d ụ n g các thu ốc d iệ t côn trù n g tro n g
n h iê u năm đă dẫn đến sự k h á n g th u ố c ỏ m uỗi. M ụ c tiê u của ng h iê n cứu này là phân
tích sự tồn tạ i của các izozym liê n quan đến tín h k h á n g th u ố c (i các chủ ng Aedes
ae gypti ỏ m ột sô v ù n g dân cư của V iệ t N am (m iền Bắc, m iền N am , m iền T ru n g và
T â y N guyên).
Các k ế t quả th u được cho th ấ y rà n g các băng esteraza chạy n h a n h n h ấ t (EST*
1) có sự khác b iệ t giữa ch ủ n g n h ạ v thu ốc với c h ủ n g k h á n g thu ốc tro n g k h i các băng
của nhữ ng izozym khá c (octanol dehydrogenaza, m a la t dehydrogenaza, phosphataza
k iể m , phosphataza a x ít) kh ô n g th ấ y có sự khá c b iệ t. Bởi vậy, izozym esteraza có thể
dược coi là m ột c h ỉ th ị rõ rà n g cho tín h kh á n g th u ố c ở m uỗi Aedes ae gypti.