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

lsozymes from 4 vvilcỉ- caught collections of Aedes aegypti mơsquito were compared bv using polyacrvlamide gel electrophoresis.. These collections vvere collected from 4 residential area

V N U 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 IS O Z Y M E S F R O M A E D E S A E G Y P T I S T R A I N S O F S O M E

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 niư ersity o f H a n o i

V u D u c H u o n g

In s titu te o f m a la ria , p a ra s ito lo g y a n d e n tom o logy o f H a n o i

A b s tra c t 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 ivo rd s: D erìgue feve r, in s e c tic id e re s is ta n c e , s tr a in , Aedes a e g y p ti% PAG E,

isozym e

1 I n t r o d u c t i o n

Dengue/ Dengue h e m o rrh a g ic feve r (D F /D H F ) occurs th ro u g h o u t th e ye a r b u t

p a r tic u la r ly in s u m m e r tim e (u s u a lly from Ju n e to J u ly ) I t e a s ily becomes e p id e m ic disease and tends to increase in S o u th e rn re gion a n d C e n tra l h ig h la n d o f V ie tn a m

re c e n tly One reason fo r th is s itu a tio n is th e in s e c tic id e re sista n ce in dengue v iru s

vectors in w hich Aedes a e g yp ti is an im p o rta n t one The m o sq u ito has re siste d to

some in se cticid e s such as D D T he xa ch lo rid e benzen (o rg a n o c h lo rin e g ro u p ) 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 lo sin g 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 som e d o m e stic an d fo re ig n researches [ 1 , 2 , 4, 7] s tu d y in g on th e

exp ressio n o f isozym e c o m p le x in d iffe re n t Aedes a e g y p ti s tr a in s in o rd e r to c x p la in

th e re s is ta n c e a b ilit y in m o s q u ito H ow ever, some o p in io n s a re c o n tro v e rs ia l

The o b je ctive o f th is re se a rch is to fin d o u t th e isozym e m a rk e r fo r in s e c tic id e

re sista n ce b y m eans o f s tu d v on th e e le c tro p h o re tic e x p re s s io n o f se ve ra l isozvm os:

E sterase (EST), O c ta n o l d e h ydroge nase (O D H ), M a la te d e h yd ro g e n a se (M D H ), A cid

phosph ata se (AC P), A lk a lin e phosph ata se (A L P ) F ro m th is s tu d y , pe ople can have

a m ore a p p ro p ria te m e a su rc to choose in se cticid e s fo r each re s id e n tia l area

T h e re íb re , 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 r e n t g e o g ra p h ic a l Aedes a e g vp ti 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 (ir t /r s a v Ị Ị x p ỉi s t r a i n s of 2 9

C o m p íin n ^ the* r l r r t r o p h n r r t ir rx p re s s io n o f r s t r r a s r ỉro m la rvíU ' and a d u lts b\ te e h n iq u e ol |> n lv ;irrv |;im i< ir n r l 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 is o /v im * n u ir k c r to in s c c tic id e re sis ta n c o o f m n sq u iỉo e s

2 M a t e r i a l s 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 ca u tfh t in these fo llo w in g (lilT e ro n t rosici(‘nt Ìíìl ỉiron s o f V ic tn a m :

• N o rth e r n rc ^ in n : T h in h I j f t v illa g e - T h a n h T r i (lis tr ie t (T T ) H a n o i; L y T h a i

To p re c in c t H oan K ir n i ( ỉis tn c t (H K ) H a n o i

- C e n tra l rc iỉio n Sonịỉ r a u ( S O to w n , Phu Yen p ro v in c e ; T u y Hoa to w n (T H ), Phu Y en: Đ o n g Ha tovvn íD H ) Q u a n g T r i p ro vin ce : T r ie u Đo v illa g e • T r ie u Phong

d is t r ic t (T P ), Q u a n g T r i p ro vin ce

- S o u th e rn rc g 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 (Đ X)

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 tr ic 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 c e ; P le i C an tovvn * Ngoe Hoi ( ỉis tr ic t (N H ), K o n tu m p ro v in c e ; Kon T u m to w n (K T ), Kon T u n i; Buon T xa y tovvn* K ro n g a n a d is tr ic t (K ro ), Dac L a c p ro vin ce

T h e m o s q u ito is is o la te d by m e thod devéloped by th e In s t it u te o f M a la ria ,

P a ra s ito lo g y an d K n to m o lo ^ v o f H a n o i th e n ra is e d fo r new la rva t* and a d u lts ( F l) and fọ r F3 descendents w h ir h th e n can be used in e x p e rim t*n ts Kive isozym es chosen fo r a n a lv s is <>n p o lv a c ry la m id e gel are E ste ra se (E S T ), O cta n o l

d c h y d ro g c n a s e (O I)H ), M a la te de hydrogenase (M D H ), Acicỉ p h o sp h a ta se (A C P ),

A lk a lin e phosphatasc' (AI.IM

M o s q u ito sa m p le s (íìd u lts o f F l o r F3, la rv a e ) are c r iis h r d w ith b u ffe r in ice

in to ho m og enize d 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

co lle cte d fo r u s in g in e le c tro p h o re s is

M a in c h e m ic a l re a g e n ts used in each speciíìc en zym e d e te c tio n are:

• E ste ra se (K S T ): a • n a p h tv l a ce tate a n d /? - n a p h ty l a c e ta te as su b s tra te s

Fíìst R luo KK as ro lo r- produ cer

- O c ta n o l (lo h yd ro g o n n st1 (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 (T ) a • n a p h ty l m onophosphate as su b stra te , Kast Blue RR.

- A lk a lin e phosph.ìtast* (A L P ) a - n a p h ty l m o no p h o sp h a te as s u b s tra te Fast

B lue RR

30 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

The* p o l y a c r y l a m i d i * g e l i s 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 o e fe r a p p a ra tu s (S a n fra n cisco U S A ) o r m in iP ro te a n a p p a ra tu s (HioKad) T h e Chemicals W<T(* bought from SIGMA C o m p a n y

3 R e s u lt s and dLscussion

3.1 E le c t r o p h o r e t ic p r o p e r t ie 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 s q u ito in som e 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 sho w 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 g io n A lth o u g h th e sam e a m o u n ts o f sam ples w ere loaded in each w e ll o f m ic r o tite r - p la te , we fin d o u t th e esterase

e xp re s s io n s th a i a re d iffe re n t It is show n th a t th e a c tiv ity o f estcrast* fro m

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

re g io n T h is re s u lt c o rrcsp o n d s to d ire c t b io a ssa v re s u lts o f in s e c tic id e on

m o sq u ito e s w h ic h in d ic a te th a t th e a d u lt m o s q u ito in C e n tra l h ig h la n d has m uch

h ig h e r re s is ta n c e r a titì to p y r e th r o id gro u p 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 (K S T- 1) in th e sc tw o group s are d iffe re n t m a rk e d ly : th e re a re a t least 3 bands o f iso/.ym e e ste ra se KST-1 íro m rtd u lt m o squito es 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 sq u ito e s in C e n tr a l re g io n

F ỉg l: K ste ra se detc‘cti()iì o f a d u lt m o s q u ito in C e n tra l re g io n an d C e n tra l h ig h la n d

in co m p a riso n w ith la rv a (B G ) (on l \ '\ ( l)

In th is gel, we also a n a lyze d a la r v a l sa m p le to co m p a re A s can be seen from

th e p ic tu re , a ll oste rase hands o f la rv a are m uch d a r k e r th a n those o f a d u lt

m o s q u ito K iịỉ.2 also m a kes i t c le a re r

S tu d y on isozvmes fro rn aedcs ( iv ỊỊy p ti s t r a i n s o f " .'$1

F tfĩ 2 : K stu ra so 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 P AG )

T h is p ic tu re is th e este ra se co m p a riso n betw een la rv a e o f som e a re a s 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 c o m p a ris o n the d iffe re n c e b e tw e e n th c la r v a l E S T o f these tvvo group s is n o t as d e a r as betw een th e a d u lt EST discu ssed above

T h e re ío rc it can he assum eđ th a t EST e s p e c ia lly EST* 1 fro m a d u lt m o s q u ito in

C e n tra l hitfh lan< l has a c lo st1 re la tio n w ith th e p y r e th r o id re s is ta n c e T h e r e la tio n l)i*tw<*cn in síT ticick* re s is ta n c e and isozym e e xp re ssio n in la rv a m ay need í u r t h e r research

O n th e side o f d e v e lo p m e n ta l p e rio d c o m p a ris o n , as vve discusseđ above, th e

n u m b e r o f esterase b a n d s an d t h e ir exp ressio n are m u ch h ig h e r fro m la r v a th a n tro in a d u lt m osqư ito It m a y be du e to th e fact th a t e ste rase s p la y a m ore im p o r ta n t

roli* i n p r o t o c t i n g l a r v a i n 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 i n

a q u a tic e n v iro n m e n t vvhere th e re a r r m ore to x ic s an d 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 s q u ito c s liv e d

F ig 3: E ste ra so (le te c tio n of* a d u lt m o sq u ito in N o rth e rn , S o u th e rn , C e n tra l re g io n

an d C e n tra l h ig h la n d in co m p a riso n w ith la rv a ( B ( j) (on PAG )

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

A f t e r a n a ly z in g este ra se fro m T h in h L ie t (Tỉ.,* one s tr a 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 s q 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 co m m e n ts:

• EST- 1 o f C e n tra l h ig h la n d m osquitoes are m o re a c tiv e (e stera se bands

e x p re s s io n s a re 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 S T -2, 3 o f F3 are a lw a y s m ore a c tiv e 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 este ra se 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 sq u ito We also see th a t w h ile EST- 4, 2, 1 a c t iv it y 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 o squito es

F ig 4 : K ste ra se d e te c tio n o f la rv a e m o sq u ito in N o rth e r n , S o u th tT iì re g io n , in

c o m p a ris o 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 e ste ra se is a good m a rk e r fo r

in s e c tic id e re s is ta n c e o f th e m o squito es (e sp e cia lly E S T - 1) (a n d fu r th e rm o r e , fo r

research on tw o d e v e 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 u se fu l

ỉE S T - 3, 4)).

3.2 E le c t r o p h o r e t ic p r o p c r t ie 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 r o g e n ase

Iso zym e o cta n o l d e h ydroge nase o f Aedes a e g y p ti s tra in s in some area o f

N o r th e r n , 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 has been d e te cte d b u t nơ

s ig n ific a n t d iffe re n c e Vvas fou nd

Iso zym e m a la te de hvdroge nase o f Aedes a e g y p ti s tr a 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 d e te cte d an d 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 i n s of

s m iil.il tí) \v h ic h 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| an d

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 tr o p h iằ v c tii' p r o p e r tie s o f A c ix ỉ p h o s p h a ta s e a n d A l k a l i n e

p h o s p h (ita s e

No d in V r e 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 # t h iit can b r n o ti'(l hon* is th a t A C P- 2 is m ore s ta b lc th a n A C P - 1 in

s r p 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 r n /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 c lu sio n

Ba se on t hi* a n a ly s is ()f f> isozym es fro m Aedes a e g y p ti s tra in s in d iffe r e n t

rc s iíle n tia l rc g io n s o f V ie tn a m , It IS deduced th a t o n lv e ste ra se (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 s q u ito

It |> assum able due to the capacity oí’ intensive esterase a c tiv ity of mosquitoes to

m srctin đ c degradation, Iht;ius(» ()f the fact th a t most insecticiỉie has ester lin kag e

|2 71 Hovvever, other iso/vm c analysis are also useful in e x p la in in g the insecticide

riỉsistỉince of mosquito.

R e íe r e n c e s

I)t*n^ue íever hem orrhagic íever and vector control in V ietnam in recent years

Prevenũvv Mvdicint’ Journal. Vol V III (2) 36(1998) pp: 124 (in Vietnamese)

ụroịvcting medicinc, Ktìvironm vntnỉ toxicology program V ỈE 197/031, 2001 (in

Vietnnmese)

m ainland and Island populations from Southern T h ailand, J Am erican M osquito

Con tro i Association IS 3(2002) pp: 173 -177.

1994, pp: 40 - 196.

f> r c M athews L E M unsterm ann Cìenetic D ive rsity and I)iffe re n tia tio n in N orthern

fhi' E n t o m o l o 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.

(S l) T M ourva 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 h e S o u t h e a s t

S r a m e o

D o Ngoe L ỉe n, N gu yê n 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 Bi sset , M Rui z, A Soca, C r o s s - r e s i s t a n c e to p y r e t h r o i d a n d

o r g a n o p h o s p h a t e i nsect i ci des s el ect i on w i t h t e m e p h o s in A e d e s a e g y p ti in Cuba J

A m e r ic a n M o s q u ito Control a n d B io lo g y 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):

2 0 0 0 , pp: 2 9 5 - 3 1 2

8 S.H.Saul, M f J Si nsko, P.R G rim stad, G.B Craig, Id e n tifica tio n o f sibling species Aecies

tr is e r ia t u s a n d Ae h cindersoni bv e l ect r ophor esi s J M e d E n t V o \ 13 (6): 1 9 77 , pp: 7 0 5 * 708.

TAP CHÍ KHO A H OC D H Q G H N K H TN & CN T.X1X, N04, 2003 _

N G H IÊ N CỨU IZOZYM CỦA MỘT s ố C H Ủ N G MƯỎI A E D E S AEGYPTI

TẠI MỘT SỐ VÙNG DÂN c ư CỦA V IỆT 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 tr ù n g T ru n g ương

Sốt D engue/ h a y sốt x u ấ t h u y ế t D engue là m ộ t bệnh xẩ y ra q u a n h năm ở các nước n h iệ t đới và á n h iệ t đói n h ư V iệ t N am và v i r u t gây ra bệnh n à y được tru y ề n

ch ủ yếu qua m u ỗi Aedes a e g yp ti Cách tó t n h ấ t đê p h ò n g bệnh sốt D engue là khố ng

chế vectơ tru y ề n bệnh này T u y n h iê n , việc sử d ụ n g các th u ố c d iệ t côn tr ù 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 n g h iê n cứu này là phân

tíc h sự tồn tạ i của các izo zym liê n quan đến tín h k h á n g th u ố c (i các c h ủ n g Aedes

a e g yp ti ỏ m ột sô v ù n g dân cư của V iệ t N am (m iề n Bắc, m iền N am , m iề n 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 e ste ra za chạy n h a n h n h ấ t (EST* 1) có sự khác b iệ t giữa c h ủ n g n h ạ v th u ố c với c h ủ n g k h á n g th u ố c tro n g k h i các băng của nh ữ ng izozym kh á c (o cta n o l dehydroge naza , m a la t d e h ydroge naza , phosphataza

k iể m , ph o sp h a ta za a x ít) k h ô n g th ấ y có sự kh á c b iệ t Bởi vậ y, izozym este raza có thể

dược coi là m ộ t c h ỉ t h ị rõ rà n g cho tín h k h á n g th u ố c ở m u ỗ i Aedes a e g yp ti.