BI OLOGY OF AMAZONI AN MOSQUI TOES. I I I . ESTERASE I SOZYMES I N ANOPHELES DARLI NGI .
Joselita M.M. dos Santos ( * ) Eucleia P.B. Contel ( ** ) Warwick E. Kerr ( *** )
SUMMARY
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(*) Instituto Nacional de Pesquisas da Amazônia, Manaus-AM.
(**) Faculdade de Medicina de Ribeirão Preto, Departamento de Genética. (***) Universidade Federal do Maranhão, Departamento de Biologia.
1NTR0DÜCTION
Ar nophe l e s da r l i ng i i s a mo s q u i t o s p e c i e s c l o s e l y a s s o c i a t e d wi t h ma n i n t he Amazon r e g i o n . Th u s i t i s i mpor t a nt t o o b t a i n d a t a o n i t s b i o l o g y , e c o l o g y , e t h o l o g y a n d
enet i cs . I n p r e v i o u s p a p e r s we h a v e r e p o r t e d d a t a o n t he b i o l o g y a n d e t h o l o g y of t hi s mos qui t o ( Sa nt os e t al . , 1981a ; 1981 b ) . I n t he p r e s e n t p a p e r we s t udy s i x e s t e r a s e
s ozymes , p a r t i c u l a r l y t he g e n e t i c a s p e c t s of Es t e r a s e 2.
Wh i t t ,
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19 76 ) a n dzyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
c ul i c i f a c i e s ( Duba s h e t a l . , 19 8 2 ) , a mo n g o t h e r s . Th e o b j e c t i v e o f t he p r e s e n t s t udy wa s t o a n a l y s e e s t e r a s e i s o z y me s i n A. darl i ngi d u r i n g d i f f e r e n t d e v e l o p me n t a l s t a g e s , a n d t o e s t i ma t e t he g e n e f r e q u e n c i e s i nzyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 3 s a mp l e c o l l e c t e d i n t he Ama z o n r e g i o n . An i n t e r e s t i n g r e s ul t t h a t wa s a l r e a d y publ i s
h-e d i s t hh-e h-e s t i ma t h-e of t hh-e n u mb h-e r o f ma l h-e s t ha t ma t h-e wi t h a d h-e t h-e r mi n h-e d f h-e ma l h-e by me a n s o f t he a l l e l e f r e q u e n c i e s .
MATERI AL AND METHODS
Th e e s p e c i e s us e d i n t hi s s t u d y , Ano phe l e s da r l i ng ! , wa s c o l l e c t e d a t t wo
d i f f e r e n t l oc a l i t i e s ( Km 137 a n d Km 182) a l o n g t he Ma n a u s - Bo a Vi s t a Hi g h wa y ( BR,
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)7h), Al l d e v e l o p me n t a l s t a ge s we r e us e d f or t he o n t o g e n e t i c s t u d y . La r v a e we r e r e a r e d byt he me t h o d s o f Fr e i r e & Fa r i a ( 1 9^ 7) a n d Ra bba ni e t al . ( 1 9 7 6 ) , s l i g h t l y modi f i e d. I ndi vi dua l s we r e s t u d i e d by e l e c t r o p h o r e s i s a t a l l d e v e l o p me n t a l s t a g e s . 1st , 2n d a n d 3r d i n s t a r l a r va e we r e r e s p e c t i v e l y , p o o l e d t o g e t h e r , wh e r e a s ^ t h i n s t a r l ar v a e , p u p a e a n d a d u l t s we r e h o mo g e n i z e d i n d i v i d u a l l y . La r v a e a n d p u p a e we r e homogeni -z e d i n 15 ul d i s t i l l e d wa t e r , a n d a d u l t s i n 20 ul d i s t i l l e d wa t e r . Th e homoge na t e was c e n t r i f u g e d a t 3, 5 0 0 r pm f or 5 mi n u t e s a t r o o m t e mp e r a t u r e . Se ve r a l b u f f e r s ys t e ms wer e t e s t e d a n d Tr i s - HCl , pH 8. 6 wa s s e l e c t e d ( Smi t h e t a l . , 19 7 1, s l i g h t l y mo d i f i e d ) .
El e c t r o p h o r e s i s wa s c a r r i e d o u t by t h e t e c h n i q u e o f St a i n e r £ J o h n s o n ( 1 9 7 3 ) , wi t h s ome mo d i f i c a t i o n s , o n p o l y a c r y l a mi d e g e l . 7 g Cy a n o g u m- ^ 1 we r e d i s s o l v e d i n 100 ml 0. 02 M Tr i s - HCl b u f f e r , pH 8 . 6 , a n d t he r e a c t i on wa s c a t a l y z e d by t he s t e p wi s e ad-d i t i o n o f O. Tr ol TMED a nad-d 0. ^ 3 ml 10¾ a mmo n i u m p e r s u l f a t e . El e c t r o p h o r e s i s wa s car r i ead-d o u t a t
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h9 f or a p e r i o d of a p p r o x i ma t e l y 3 h o u r s wi t h a d i f f e r e n c e of 170 V be t we e n t he e n d s of t he g e l , unt i l t he d y e us e d a s a mi g r a t i o n c ont r ol ( Br omophe nol Bl u e ) had mi g r a t e d a p p r o x i ma t e l y 10 c m f r o m t he o r i g i n .Es t e r a s e a c t i v i t y wa s c h a r a c t e r i z e d us i ng na pht hol e s t e r s by t he me t h o d s of St a i n e r & J o h n s o n ( 19 7 3 ) : 20 mg Fa s t Bl u e RRa n d 2 ml o f s u b s t r a t e s o l u t i o n ( l g est er d i s s o l v e d i n 100 ml 1: 1 wa t e r : a c e t o n e , v / v ) . Th e f o l l o wi n g s u b s t r a t e s we r e t e s t e d : a - na pht hyl a c e t a t e , p r o p i o n a t e , s t e a r a t e a n d l a u r a t e ; ( 3- napht hyl a c e t a t e , pr opi
ona-t e a n d s ona-t e a r a ona-t e .
Onl y ^ t h - i n s t a r l a r va e we r e u s e d t o d e t e r mi n e p o s s i b l e g e n e t i c v a r i a b i l i t y and, c o n s e q u e n t l y , ge ne f r e q u e n c y , s i n c e t he i r i s o z y me s s h o we d t he be s t r e s o l u t i o n . We a n a l y s e d 1^3 l a r va e f r o m 1 5o v i p o s i t i o n s s e l e c t e d a t r a n d o m.
RESULT I
Si x r e g i o n s o f e s t e r a s e a c t i v i t y , a l l o f t he m e l e c t r o n e g a t i v e , we r e det ect ed us i ng d i f f e r e n t na pht hol e s t e r s u b s t r a t e s . Th e y we r e d e n o mi n a t e d e s t e r a s e - 1 t hr ough 6 a c c o r d i n g t o e l e c t r o p h o r e t i c mo b i l i t y , t he l owe r n u mb e r s b e i n g g i v e n t o t he es t er as es
of hi ghe r mo b i l i t y ( Fi gur e l ) .
ESTERASE I SOZYMES OF ANOPHELES DARLI NGI DURI NG ONTOGENETI C DEVELOPMENT
Th e r e s u l t s o f t he e l e c t r o p h o r e t i c a n a l y s i s o f e n z y me s h a v i n g e s t e r a s e a c t i v i t y i n
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Ano phe l e s da r l i ng i d u r i n g t he d i f f e r e n t d e v e l o p me n t a l p h a s e s , d e t e c t e d wi t h a - a n d B-napht yl a c e t a t e a n d a - a n dzyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
8 - na pht yl p r o p i o n a t e , a r e d e s c r i b e d b e l l o w. Th e i s oz yme pr of i l es o b t a i n e d wi t h t he f our s u b s t r a t e s we r e q u i t e s i mi l a r , a n d s i mi l a r i t y wa s hi gher wh e n c t - na pht hyl a c e t a t e a n d p r o p r i o n a t e we r e u s e d .Es t e r a s e s 1 a n d 2 s h o we d t he h i g h e s t s t a i n i n g i n t e n s i t y i n t he l a r va l s t a g e s , b u t r educed s t a i n i n g i n p u p a e a n d a d u l t s wi t h a l l f our s u b s t r a t e s . No e s t e r a s e-2 a c t i v i t y was d e t e c t e d i n p u p a e wh e n S- na pht hyl p r o p i o n a t e wa s u s e d . Th e o p p o s i t e o c c u r r e d wi t h e s t e r a s e s 3 a n d
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h, wh i c h we r e mo s t d e e p l y s t a i n e d i n p u p a e a n d a d u l t s . No e s t e r a s e - 3 or e s t e r a s e-4 a c t i v i t y wa s d e t e c t e d i n 1s t i ns t a r l a r v a e wh e n a - n a p h t h y l a c e t a t e wa s us ed, or i n a n y l a r va l s t a g e wi t h 8- n a p h t h y l a c e t a t e a n d p r o p i o n a t e .Es t e r a s e s 5 a n d 6 we r e d e t e c t e d d u r i n g a l l d e v e l o p me n t a l s t a g e s wh e n a - na pht hyl acet at e a n d p r o p i o n a t e we r e u s e d , but o n l y i n s ome s t a g e s wi t h t he o t h e r t wo s ubs t r a -t es .
Es t e r a s e - 1 s h o we d g r o wi n g s t a i n i n g i nt e ns i t y i n t he l a r va l s t a g e s , wi t h h i g h i nt ens i t y i n p u p a e a n d r e d u c e d ( ma ny t i me s a b s e n t ) i n t e n s i t y i n a d u l t s . Es t e r a s e - 2
had e x c e l e n t s t a i n i n g i nt e ns i t y i n t he l a r va l s t a g e s , a n d e s t e r a s e s 3 a n d
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k i n ge ne r a l had gr owi ng s t a i n i n g i nt e ns i t y f r o m t he l a r va l s t a g e s t o t he a d u l t p h a s e r e ga r dl e s s oft he s ubs t r a t e u s e d . Es t e r a s e 5 a nd 6 we r e t he we a k l y s t a i n e d wi t h a n y o f t he s ubs t r a -t es , a n d i n ma n y a n a l y s e s we r e n o -t v i s u a l i z e d a -t a l l i n -t he ge l wh e n 8- na pht hyl a c e t a t e a nd p r o p i o n a t e we r e u s e d .
Si g n i f i c a n t c h a n g e s i n s t a i n i n g i nt e ns i t y o c c u r r e d d u r i n g t he pupa l s t a ge f or s o me of t he i s o z y me s . Es t e r a s e 3 a n d A s h o we d i n c r e a s e d s t a i n i n g i n t e n s i t y a t pupa t i on and r e ma i ne d we l l v i s i b l e t h r o u g h o u t t he pupa l s t a g e ( Fi gur e 2 ) .
Es t e r a s e s 1 a n d 2 ( t he mo s t i nt e ns e l y s t a i n e d i s o z y me s i n t h e l a r va l a na l ys e s ) c ont i nue d t o s h o w c o n s i d e r a b l e s t a i n i n g d u r i n g p u p a t i o n . Ho we v e r , t h e i nt e ns i t y d e c r e as ed a f t e r a b o u t 10 h o u r s , a n d n e i t h e r e s t e r a s e wa s d e t e c t e d a p p r o x i ma t e l y 20 hour s af t er p u p a t i o n . On t h e o t h e r h a n d , a mo r e d e t a i l e d a n a l y s i s o f t h e s e d a t a s e e ms t o s how t ha t e s t e r a s e - 1 a n d e s t e r a s e-2 a p p e a r wi t h h i g h e r i n t e n s i t y d u r i n g t he 4 t h l a r va l s t age, d i s a p p e a r i n o l d e r p u p a e ( da r k pupa e ) a n d r e a p p e a r wi t h l ow i nt e ns i t y i n a d u l t s . Thi s p h e n o me n o n n e e d s f u r t h e r d e t a i l e d i n v e s t i g a t i o n .
Pupa l a n a l y s i s s h o we d t h a t , a s p u p a e g r o w o l d e r , t he e l e c t r o p h o r e t i c pr of i l e of e s t e r a s e i s oz yme s i s ve r y s i mi l a r t o t ha t d e t e c t e d i n a d u l t s . No d i f f e r e n c e s we r e de t e c t e d b e t we e n a d u l t ma l e s a n d f e ma l e s .
ELECTROPHORETI C VARI ANTS OF ANOPHELES DARLI NGI
Th e i s o e s t e r a s e p r o f i l e o f Ano phe l e s da r l i ng i i s c o mp l e x , wi t h s i x mo l e c u l a r
f or ms wh o s e s t a i n i n g i nt e ns i t y v a r y a c c o r d i n g t o d e v e l o p me n t . Va r i a t i o n i n e l e c t r op r e t l c mo b i l i t y wa s o b s e r v e d i n f our o f t h e s e s i x e s t e r a s e s .
Es t e r a s e
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-1: mo s t l a r va l a n d pupa l a n a l y s e s s h o we d o n e or t wo b a n d s f or t hfs e n z y me . I n s ome i n d i v i d u a l s , t he e l e c t r o p h o r e t i c p r o f i l e s we r e c h a r a c t e r i z e d by the a p p e a r a n c e of t hr e e b a n d s . Th i s i s t ypi c a l of h e t e r o z y g o u s f or a d i me r e n z y me , whose s y n t h e s i s d e p e n d s o n t wo c o d o mi n a t a l l e l e s . Ho we v e r , o u r o b s e r v a t i o n s wi t h r espect t o t hi s v a r i a b i l i t y c a n n o t be c o n s i d e r e d d e f i n i t i v e , b o t h b e c a u s e o f t he s ma l l number o f a n a l y s e s a n d b e c a u s e o f t he e x i s t a n c e of a n o v e r l a p b e t we e n t he s h o we s t ba nd of t hi st ypi c a l h e t e r o z y g o t e p r o f i l e a n d t he f a s t e s t b a n d o f t he f o r m d e n o mi n a t e d e s t e r a s e - 2. Es t e r a s e - 2 : o f a l l i s oz yme s d e t e c t e d a t t he d i f f e r e n t d e v e l o p me n t a l s t a ge s , es t e r a s e - 2 s howe d t he s h a r p e s t v a r i a t i o n , wi t h t h r e e d i f f e r e n t p h e n o t y p e s pe r f e c t l y di s-t i n g u i s h a b l e o n s-t he q e l . Th e f i r s s-t , r e p r e s e n s-t e d by a s i n g l e b a n d of f a s s-t mobi l i s-t y, was d e s i g n a t e d e s t e r a s e - 2 F; t he s e c o n d , a l s o r e p r e s e n t e d by a s i n g l e b a n d , but of sl ow mo b i l i t y , wa s d e n o mi n a t e d e s t e r a s e - 2 S, a n d t he t h i r d , wi t h t wo b a n d s r unni ng si mul t a ne ous l y a n d s h o wi n g t he s a me mo b i l i t y a s t he b a n d s t ha t o c c u r r e d i n d i v i d u a l l y i n t p h e n o t y p e s d e s c r i b e d e a r l i e r wa s d e n o mi n a t e d e s t e r a s e - 2 FS( Fi g u r e 3 ) • Th i s i s oz yme f or n wa s a l s o s t u d i e d i n s ome o f i t s g e n e t i c a s p e c t s . Pr e l i mi n a r y a n a l y s e s we r e a l s o done us i ng a f l u o r o g e n i c s u b s t r a t e ( ^- me t hyl umbe l 1i f e r yI p r o p i o n a t e ) , wh i c h a l s o r eveal s p o l y mo r p h i c v a r i a t i o n i n e s t e r a s e - 2 . Th i s t ype of a n a l y s i s wi t h a f l u o r o g e n i c s ubs t r
a-t e wa s d o n e mo r e i n d e p a-t h i n a-t he s a-t u d y o f a n
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A, d a r li ng i s a mp l e c o l l e c t e d f r om ot her t e s i n t he Ama z o n r e gi on ( Cont e l e t a l . , 19 8 * 0 .Es t e r a s e s 3 a n d s t a i n i n g i n t e n s i t y wa s ve r y l ow i n l a r v a e , a n d t hi s pr even-t e d a mo r e p r e c i s e a n a l y s i s o f even-t he p o s s i b l e v a r i a n even-t s o f even-t h e s e even-t wo i s o z y me s . I n pupae a n d a d u l t s , e s t e r a s e - 3 s h o ws o n e t o t wo b a n d s wi t h d i f f e r e n t e l e c t r o p h o r e t i c mobi l i t y1
( s l ow a n d f a s t ) wh i c h a r e l o c a t e d v e r y c l o s e t o t he e s t e r a s e - ** r e g i o n . Th i s ( es t er as e**) i s f r e q u e n t l y r e p r e s e n t e d by a s i n g l e b a n d wi t h v a r i a t i o n s i n e l e c t r o p h o r e t i c mobi -l i t y. An o t h e r b a n d -l e s s t hi c k t ha n t he c o mmo n o n e wa s o c c a s i o n a -l -l y d e t e c t e d i n t h« e s t e r a s e - ** r e g i o n , s u g g e s t i n g t ha t i t i s p r o b a b l y a s e c o n d a r y i s o z y me . An a l y s i s of t h e s e t wo r e g i o n s wa s a l s o h a mp e r e d by o c c a s i o n a l o v e r l a p p i n g .
Es t e r a s e s 5 a n d 6 : a l t h o u g h t he r e wa s e v i d e n c e of e l e c t r o p h o r e t i c va r i a nt s of t he s e t wo e n z y me s i n At h - i n s t a r l a r va e i t wa s n o t p o s s i b l e t o e s t a b l i s h wh e t h e r t he t ype of c ont r ol h a d g e n e t i c b a s i s b e c a u s e t he t wo i s oz yme s we r e we a k l y s t a i n e d o n t he g e l s .
GENETI C DATA AND ESTI MATES OF THE GENE FREQUENCI ES OF THE POLYMORPHI C ESTERASE- 2 SYSTEM
The r e s ul t s o b t a i n e d ( t hr e e d i s t i n c t e l e c t r o p h o r e t i c p h e n o t y p e s ) s u g g e s t t hat e s t e r a s e - 2 i s c o n t r o l l e d by t wo a l l e l e s a t t he s a me l o c u s , Es t 2* S a n d Es t 2 * F. The Es t 2 * F a l l e l e d e t e r mi n e s t he e s t e r a s e wi t h f a s t mo b i l i t y a n d Es t 2~S t he e s t e r a s e wi t h
wer e d e n o t e d
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Es t * 2 S/ Es t * 2 S, Es t 2 * F/ Es t 2 * S a nd Es t 2 * F/ Es t * 2 F.Our r e s ul t s i ndi c a t e t ha t t he f r e q u e n c y o f t he Es t 2 * S a l l e l e i s
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0 . 5 2 1. We a l s o obs er ved t ha t p h e n o t y p e d i s t r i b u t i o n a g r e e s wi t h t he d i s t r i b u t i o n p r e d i c t e d by r a n d o mmat i ng, a s s h o wn i n Ta b l e 1.
DISCUSSION AND CONCLUSIONS
Th e a n a l y s i s o f s e ve r a l i s oz yme s y s t e ms i n An o p h e l e s ha s i ndi c a t e d t ha t t he s e mos qui t oe s a r e ve r y
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
us e f ul f or t he s t u d y o f b i o c h e mi c a l g e n e t i c s a n d d e v e l o p me n t ( Ve d -Br at £ Wh i t t , 19 7 6 ) . A r e l a t i v e l y l a r ge n u mb e r o f e s t e r a s e s h a s b e e n v i s u a l i z e d i n mos qui t oe s b y gel e l e c t r o p h o r e s i s . Fr e y v o g e l e t a l . (19^ 8) ha ve s h o wn 19 e s t e r a s e bands i n Ano phe l e s s t e phe ns i a n dzyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
2k i n Cul e x t a r s a l i s . Ni n e e s t e r a s e f o r ms ha ve be e n r e c ogni z e d i n Ano phe l e s a l bi ma nus ( Ve dBr a t & Wh i t t , 19 7 5 ) . Br i e ge l & Fr e yvoge l ( 1 9 7 0 have d e t e c t e d 12 e s t e r a s e f or ms i n Cu l e x p i p i e n s f a t f g a ns , a n d Na r a n g e t a l . ( 19 79 b ) det ect ed 7 e s t e r a s e f or ms i n A. n u n e z t o v a r i .I n t he p r e s e n t s t u d y , 6 e s t e r a s e f or ms we r e v i s u a l i z e d d u r i n g t h e d e v e l o p me n t o f A. da r l i ng i , s o me o f wh i c h i n d i c a t i n g a l l e l i c v a r i a t i o n a n d c h a n g e s i n g e n e e x p r e s s i o n .
Es t er as es 1 a nd 2 , t h a t s t a i n e d mo r e d e e p l y i n l a r v a e , r e v e a l e d c y c l i c e x p r e s s i o n ; e s t er as es 3 a n d k, t h a t s t a i n e d mo r e d e e p l y i n p u p a e a n d a d u l t s , we r e r a r e l y pr e s e nt i n l ar vae, a n d e s t e r a s e s 5 a n d 6 p r e s e n t a t a l l d e v e l o p me n t a l s t a g e s , we a k l y s t a i n e d i n pupae a n d a dul t s .
Si mi l a r r e s u l t s ha ve b e e n r e p o r t e d f or e n z y me s o f Dr o s o phi l a a nd o t h e r i n s e c t s , s uch a s i s oc i t r a t e d e h y d r o g e n a s e , h e x o k i n a s e , d e s o x y r i b o n u c l e a s e , a l k a l i n e p h o s p h a t a s e, e s t e r a s e , a my l a s e , g lu c o s e - 6 - p h o s p h a t e de hy dr oge nas e , and ac on' i t as e ( Wa gne r £ Se l a nde r , 1971
») .
As f a r a s t he c y c l i c e x p r e s s i o n o f t he g e n e c o n t r o l l i n g e s t e r a s e 1 s y n t h e s i s , s i -mi l ar r e s ul t s we r e o b t a i n e d by Ve d Br a t £ Wh i t t ( 1975) i n Ano phe l e s a l b i ma n u s : t he enzymes s h o we d a v e r y we a k b a n d b e f o r e p u p a t i o n , wa s n o t d e t e c t e d o n t he ge l d u r i n g t he f i r s t 2 t o 3 h o u r s o f t he pupa l s t a g e a n d wa s v i s u a l i z e d up t o 18 h o u r s a f t e r p u p a t i o n . The a u t h o r s e x p l a i n e d t hi s f a c t i n t e r ms o f t he r e g u l a t i o n o f e c d y s o n e c o n c e n t r a t i o n , pos t ul a t i ng t h a t t hi s i s oz yme f o r m p a r t i c i p a t e s i n e c d y s o me me t a b o l i s m. I t i s p o s s i b l e t hat e s t e r a s e s 1 a n d 2 i n t he pupa l s t a g e o f A. da r l i ng i ha ve s o me r e g u l a t o r y f unc t i on on e c dys one l e v e l s . Th e a p p e a r a n c e o f t h i s i s oz yme d u r i n g t he a d u l t p h a s e ma y be i nt er ^ pr et ed t o b e d u e t o i t s p a r t i c i p a t i o n i n l i pi d d e g r a d a t i o n . I n c o n t r a s t t o t he b e h a v i o r
of e s t e r a s e s 1 a n d 2 , e s t e r a s e s 3 a n d
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
h a r e mo r e d e e p l y s t a i n e d i n p u p a e a n d a d u l t , t hus s u g g e s t i n g t he y ma y p l a y a r ol e i n t he ma i n t e n a n c e o f l ow l e ve l s of j u v e n i l e hor moi ns e c t s c a n a s s u r e nor ma l me t a mo r p h o s i s . Pa n t e l o u r i s a n d Do wn e r (
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
19 6 9 ) a l s o det ect ed c a r b o x y l e s t e r a s e f or ms i nzyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Dr o s o phi l a I mmi g r a nt s t ha t we r e mo r e d e e p l y s t a i n e d i n pupa e anda d u l t s . Th e s e d a t a ma y ha ve i mp l i c a t i o n s f or t h o s e i n t e r e s t e d i n t he us e o f j uveni l e h o r mo n e a n d i t s a n a l o g s a s i n s e c t - c o n t r o l l i n g a g e n t s . Wh i t mo r e e t a l. ( 1972) suggest t ha t i ns e c t s ha ve a b i o c h e mi c a l r e p e r t o r y t ha t not o n l y p e r mi t s t he d e g r a d a t i o n of f o r e i g n mo l e c u l e s ( s uc h a s DDT, f or e x a mp l e ) b u t t ur ns t h e m r e s i s t a n t by me a n s of t hei r o wn h o r mo n e s wh e n n e c e s s a r y . Gi l mo u r ( 1 9 6 5 , q u o t e d by c h a mb e r s , 19 73) advanced t he h y p o t h e s i s t h a t s ome i ns e c t c a r b o x y l e s t e r a s e s r e p r e s e n t a n i mpor t a nt def ens e a g a i n s t o r g a n o p h o s p h a t e s a n d p o s s i b l e o t h e r i n s e c t i c i d e s . Pa s t e u r £ Si n e g r e ( 1975) show-e d a n i nc r show-e a s show-e i n t hshow-e f r show-e q u show-e n c y o f t hshow-e Es t- 2 ^ ' a l l e l e i n p o p u l a t i o n s of Cul ex pi pi e ns t ha t we r e l e s s s e n s i t i v e t o a n o r g a n a p h o s p h a t e i n s e c t i c e d e .
Es t e r a s e s 5 a n d 6 , wh i c h a r e p r e s e n t i n a l l d e v e l o p me n t a l s t a g e s , ma y ha ve a mo r e g e n e r a l i z e d f u n c t i o n t ha n t he e s t e r a s e s t h a t a r e c h a r a c t e r i s t i c s o f a par t i cul ar d e v e l o p me n t a l s t a g e . Es t e r a s e s t ha t a r e p r e s e n t t h r o u g h o u t d e v e l o p me n t ha ve r epor t ed i n An o p h e l e s a l bi ma nus ( Ve dBr a t £ Wh i t t , 1 9 7 5 ) , Dr o s o phi l a ( Pa n t e l o u r i s e t a l. , 1969) Be r g e r £ Ca n t e r , 1 9 7 3 ) , a n d Pl o di a i nt e r punc t e l l a ( Pa t a r y a s e t al . , 19 7 0 ) - Our d a t a d i d not d e mo n s t r a t e t he e x i s t e n c e o f i s oz yme f or ms t ha t a p p e a r i n a s i n g l e phase o f d e v e l o p me n t o n l y , i n c o n t r a s t t o t he d a t a r e p o r t e d by Ve d Br a t £ Wh i t t ( 1976) in Ano phe l e s a l bi ma nus , wh e r e s ome i s o z y me s a p p e a r o n l y i n l a r va e a n d o t h e r s i n pupa e . Qu a l i t a t i v e d i f f e r e n c e s i n t he e l e c t r o p h o r e t i c p r o f i l e o f e s t e r a s e s d u r i n g t he var i ous d e v e l o p me n t a l p h a s e s ha ve b e e n a l s o o b s e r v e d i n o t h e r i n s e c t s , s uc h a s b e e s , for e x a mp l e ( Cont e l £ Me s t r i n e r , 1 9 7 5 ) . i n t he p r e s e n t s t u d y , n o q u a l i t a t i v e di f f er ences we r e d e t e c t e d i n t he i s oz yme f or ms o f ma l e s a n d f e ma l e s , i n a g r e e me n t wi t h obs e r va t i ons by Br i e qe l £ Fr e yvoge l ( 1971) i n Ae de s a e g y p t i .
Ho s t of t he s i x e s t e r a s e i s o z y me s o f A. da r l i ng i s h o we d e l e c t r o p h o r e t i c var i ant Si , The v a r i a t i o n o b s e r v e d f or t hi s e n z y me g r o u p i s c o n s i s t e n t i n a wi d e v a r i e t y o f or gan-i s ms : Api s me l l i f e r a ( Me s t r i n e r £ Co n t e l , 19 7 2 ) , Fundul us he t e r o c l i t us ( Hol me s £ Wh i t t , 1 9 7 0 ) , Dr o s o phi l a wi l l i s t o ni ( Aya l a e t a l . , 1972) Pe r a my s c us l e uc opus( Wi l -mo t £ Un d e r h i l l , 19 7 3 ) . Dr o s o phi l a ps e udo o bs c ur a ( Le wont i n £ Hu d d y , 19 6 6 ) , Col i as e u r y t h e me ( Bur ns £ J o h n s o n , 19 6 7 ) , Ano phe l e s a l bi ma nus ( Ve dBr a t £ Wh i t t , 1975) , Ae-de s a e g y pt i ( Sa ul e t a l . , 19 7 6 ) , a n d Ano phe l e s punc t i pe nni s ( Na r a ng & Ki t z mi l l e r ,
19 71a a n d 19 7 1b ) .
Es t e r a s e-1 s h o we d e l e c t r o p h o r e t i c v a r i a n t s i n mo s t o f t he l a r va e s t u d i e d . One or t wo r e g i o n s , a n d o c c a s i o n a l l y t h r e e , i ndi c a t i ng a c t i v i t i e s , we r e f r e q u e n t l y vi s ual
-i z e d . Th e a p p e a r a n c e o f t h r e e b a n d s -i n t he e s t e r a s e-1 f o r m ma y be i n t e r p r e t e d t o be t he h e t e r o z y g o u s f or p h e n o t y p e a p r o t e i n wi t h d i me r i c s t r u c t u r e .
Es t e r a s e - 2 v a r i a n t s c o n s i s t i n g of t hr e e d i s t i n c t p h e n o t y p e s we r e a l s o obs er ved a t d i f f e r e n t d e v e l o p me n t a l s t a g e s : a p h e n o t y p e wi t h a s i n g l e , f a s t b a n d ; a s e c ond one wi t h a s i n g l e , s l o w b a n d , a n d a t hi r d o n e wi t h t wo s i mu l t a n e o u s b a n d s , o n e o f whi ch wa s f a s t , a n d t he o t h e r s l o w. Th e s e d a t a s u g g e s t t h a t t he g e n e t i c c ont r ol o f t hese e n z y me o c c u r s t h r o u g h t wo c o d o mi n a n t a l l e l e s a n d t ha t t he e n z y me ha s a mo n o me r i c s t r
I n mo s t o f t he pupa l a n d a d u l t a n a l y s e s , e s t e r a s e
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
-3 s howe d t wo v a r i a n t s , a f a s t oneand a s l ow o n e - Ho we v e r} a s pr e vi o u s l y c o mme n t e d , t he i n t e r p r e t a t i o n o f t hi s v a r i a b i l i-ty i s d i f f i c u l t , ma i n l y b e c a u s e o f t he o v e r l a p p i n g o f e s t e r a s e-3 a nd e s t e r a s e -
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
hbands, a p h e n o me n o n a l r e a d y o b s e r v e d i n
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
A. nune z t o v a ri b y Na r a n g e t a l . ( 1 9 7 9 a ) . Mor e concl us i ve d a t a ma y be o b t a i n e d by us i ng d i f f e r e n t t ype s o f s u b s t r a t e s a n d i n h i b i t o r s .The us e o f t he s e s u b s t a n c e s a n d o f o t h e r k i n d s o f b u f f e r s ma y p r o v i d e d a t a f or be t t e r car act er i z a t i o n of t h e d t f f e r e n t i s o z y me s . As a l r e a d y i n d i c a t e d , we a r e now u s i n g a l a r ge r number of s u b s t r a t e s .
The v a r i a t i o n o b s e r v e d wi t h r e s pe c t t o e s t e r a s e s 5 a n d 6 s u g g e s t s g e n e t i c c o n -t rol r a -t he r -t ha n s e c o n d a r y c h a n g e s , i . e . , p o s -t - -t r a n s l a -t i o n a l c h a n g e s . Ho we v e r , -t he anal ys i s o f t hi s v a r i a t i o n wa s d i f f i c u l t b e c a u s e t he s e i s oz yme s s t a i n e d v e r y we e k l y i n t he gel . Th i s i s a q u e s t i o n we i nt e nd t o a p p r o a c h i n l a t e r s t u d i e s .
The a n a l y s e s o f At h - i n s t a r l a r va e wi t h r e s pe c t t o t he Es t 2 l oc us s ugge s t t h a t t he t hr e e e l e c t r ophor e t i c v a r i a n t s a r e p h e n o t y p i c e x p r e s s i o n s o f t he a c t i vi t i e s o f t wo a l l e l e s , Est 2*S a nd Es t 2 * F t ha t a r e l o c a t e d o n a n a u t o s o me a n d a r e e x p r e s s e d c o d o mt n a n t l y i n
he t e r oz ygot e s . Th e f r e q u e n c i e s o f t h e s e a l l e l e s we r e p r a c t i c a l l y i de nt i c a l ( Es t 2*S =
0.521)
a nd t he o b s e r v e d p h e n o t y p e d i s t r i b u t i o n i n t he p o p u l a t i o n a g r e e d wi t h t he expect ed t h e o r e t i c a l d i s t r i b u t i o n . Th e i s oz yme p r o f i l e of t he h e t e r o z y g o t e , t wo equal l y s t a i ne d b a n d s , s ugge s t t ha t e s t e r a s e-2 ha s a mo n o me r i c s t r u c t u r e .The r e s u l t s r e p o r t e d i n t he p r e s e n t p a p e r o n t he b a s i s of t h e ge ne f r e q u e n c i e s obs er ved f or t he Es t 2 ~ F a n d Es t 2* S a l l e l e s a n d o f t he p o s s i b l e t ype s o f ma t i n g a l s o demons t r at ed t ha t Ano phe l e s da r l i ng i f e ma l e s a r e mo n o g a mo u s , o r , e v e n i f a f e ma l e ma -t es wi -t h -t wo ma l e s s he wi l l be f e c u n d a -t e d by -t he s p e r ma -t o z o a o f o n l y o n e . ( Sa n-t os e t al . , 1981a ) . I n t ha t o c c a s i o n , t he x2
v a l u e o b t a i n e d by t he a u t h o r s , wh e n t he h y p o -t hesi s of a f e ma l e b e i n g f e r -t i 1 i z e d by mo r e -t ha n o n e ma l e wa s c o n s i d e r e d , wa s h i g h l y s i gni f i cant ( P < 0. 00? ) . Wh e n t he h y p o t h e s i s of a s i n g l e ma l e f e r t i l i z i n g a f e ma l e was c o n s i d e r e d , t he x2
v a l u e s s h o we d t h a t t h e d i f f e r e n c e s o b t a i n e d we r e n o t s t a t i s t i -cal l y d i f f e r e n t ( 0 . 3 0 < P < 0 . 50 ) f or m t hos e e x p e c t e d . Th e r e f o r e , t he y s u g g e s t e d t hat e a c h f e ma l e i s f e r t i l i z e d b y o n e ma l e o n l y .
ACKNOWLEDGEMENT S
RESUMO
Seis isoenzimas de esterase foram estudadas durante o desenvolvimento de Anopheles darlingi usando eletroforese em gel de poliacrilamida e dois substratos diferentes,
a-naftilacetato e a-naftliproprlonato. Esterases
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
5 e 6 foram detectados em todos ose st ági o s do desenvolvimento, esterases 1 e 2 foram coradas com maior intensidade em larva, enquanto esterases 3 e 4 foram melhor visualizadas em pupa e adulto. Foram observadas grandes diferenças na intensidade de algumas isoenzimas durante o estágio de pupa,
Quatro das se is isoenzimas revelaram variações na mobilidade eletroforética. A
esterase 1 foi a escolhida para estudos genéticos, em virtude de ter sido a isoenz ima
o i i •
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
FIG. 1
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
- An o p h e l e s d a r l i n g i e s t e r a s e i s o z y me s v i s u a l i z e d o n g e l s t a i n e d a t pH 8. 6 u s i n g a - n a p h t h y l a c e t a t e a s s u b s t r a t e . Sl o t s 1 t o 13 we r e f i l l e d wi t h 4 t h i n s t a r l a r v a e e x t r a c t s , a n d t he o t h e r s we r e f i l l e d wi t h p u p a e e x t r a c t s .1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
FIG. 2 - Es t e r a s e i s o z y me s f r o m p u p a e wi t h d i f f e r e n t a g e s : 3 h r s ( s a mp l e 1 ) , 5 h r s ( s a mp l e s 2 a n d 3 ) , 8 h r s ( 4 - 6 ) , 12 h r s ( 7 - 9 ) , 15 h r s ( 1 0 - 1 3 ) , 20 h r s ( 14 a n d 1 5 ) , a n d 24 h o u r s ( s a mp l e 16 a n d 1 7 ) .
# m
Est 2* S Est 2* FO
1 2 3 4 5 6
References
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