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 R E S ID E N T IA L A R E A S IN V IE T N A M

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 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 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

,

*

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

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 .

' • Ỉ S l i í I S Í S i -

ể f c a » « m — t ề m + i ầ ' ^ BG KRo KT NH TP ĐH TH s c BMT

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

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

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,

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

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 .11ka 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 t r a l re g io n and ( V n t n il h ig h lỉin d w as fo u n d . 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

1 Tran Van Tien. Vu Sinh Nam. T rin h Quan Huan, Do Quang Ha. Truông ưyen N inh.

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).

2 Chem istrv In s titiiti*. N ntural Sciences and N ational Tochnologv Centre. Plant ụroịvcting medicinc, Ktìvironm vntnỉ toxicology program V ỈE 197/031, 2001. (in Vietnnmese).

‘t T C hareonviriyaphnp, K. Lcrthusnee, Genetic d iffe re n tin tio n o f Aedes aegypti 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.

ỉ Cì.p. Manchenko. Handbook o f Detection <ỉf Emvmes ttn Electrophoretic Gel, CRC Press, 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 popuỉation o f the Tree- hok' Mosquito Aedes hendersoni (Diptera: CuỊicidae). Annalis of

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

\sian Journal ()f Troptval medicine and pubỉic Health V25 (3) September 1994*

S r a m e o .

D o Ngoe L ỉe n

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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.