Mosquito Oostatic Hormone - ACS Symposium Series (ACS

Chapter 13 Mosquito Oostatic Hormone A Trypsin-Modulating Oostatic Factor 1

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Dov Borovsky , David A. Carlson , and Donald F. Hunt 1

Florida Medical Entomology Laboratory, University of Florida-IFAS, Vero Beach, FL 32962 Insects Affecting Man and Animal Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Gainesville, FL 32604 Department of Chemistry, University of Virginia, Charlottesville, VA 22901

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Mosquito oostatic hormone, a trypsin modulating oostatic factor (TMOF) has been purified from the ovaries of female Aedes aegypti. The hormone is a decapeptide of molecular weight 1047.6. The primary sequence has been determined using mass spectra recorded on a quadrupole Fourier transform instrument. Injection of synthetic analogs into mosquitoes, biting midges, flies and fleas inhibited proteolytic enzyme biosynthesis in the midgut. The target tissue of the hormone is midgut epithelial cells, and binding of [H]TMOF to midgut receptor was demonstrated in vivo. TMOF exhibits homology to mammalian, plant and several viral proteins. This is the first insect peptide hormone to share homology simultaneously with viral, plant and mammalian proteins. These results indicate that trypsin synthesis in mosquitoes and other insects may be regulated by sequence-related TMOFs that bind to midgut specific receptors. 3

Knowledge and u n d e r s t a n d i n g o f how hormones r e g u l a t e c e l l u l a r p r o c e s s e s i n mosquitoes may be o f g r e a t importance f o r c o n t r o l l i n g t h e i r r e p r o d u c t i o n . C o n c e i v a b l y , new t y p e s o f i n s e c t i c i d e s may be d e v e l o p e d t h a t i n t e r f e r e w i t h hormonal r e g u l a t i o n , as f o r example, does t h e j u v e n i l e hormone a n a l o g (methoprene). A l t h o u g h c o n s i d e r a b l e r e s e a r c h has been d e v o t e d i n e l u c i d a t i n g how b i o s y n t h e s i s o f midgut p r o t e a s e s i s i n i t i a t e d i n mosquitoes, v e r y l i t t l e h a s been done t o determine how t h i s process i s terminated. Thus, i t i s i m p o r t a n t t o i n v e s t i g a t e t h e r o l e o f hormones i n t h e c o n t r o l o f d i g e s t i o n . A n t i g o n a d o t r o p i n s o r f a c t o r s t h a t i n h i b i t egg development, such as o o s t a t i c hormones and t r y p s i n m o d u l a t i n g o o s t a t i c f a c t o r (TM0F), have been demonstrated i n t h e c o c k r o a c h e s , B l a t t e l a germanica and B l a t t a o r i e n t a l i s , (1) t h e eye gnat, Hippelates c o l l u s o r (2.), decapod c r u s t a c e a n s (3.) , and t h e house f l y , Musca domestica, (4.5). I n mosquitoes, Meola and L e a (6) and E l s e and

0097-6156/91A)453-0133$06.00/0 © 1991 American Chemical Society Menn et al.; Insect Neuropeptides ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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J u d s o n (7) s i m i l a r l y demonstrated an o v a r y - p r o d u c e d humoral factor secreted during vitellogenesis that i n h i b i t e d yolk d e p o s i t i o n i n l e s s developed f o l l i c l e s . However, t h e work o f M e o l a and L e a (6.) and E l s e and J u d s o n (7) was done b e f o r e a d u l t p r e v i t e l l o g e n i c f o l l i c u l a r growth was r e c o g n i z e d as a s e p a r a t e , hormonally-regulated process preceding v i t e l l o g e n e s i s (8.9). T h e r e f o r e , we r e p e a t e d some o f t h e i r work by i m p l a n t i n g o v a r i e s from b l o o d - f e d donors i n t o females t h a t had r e a c h e d the r e s t i n g s t a g e ( i . e . , completed p r e v i t e l l o g e n i c growth 3-5 days a f t e r emergence) and t h e n f e d them b l o o d . C o n v e r s e l y , we i m p l a n t e d o v a r i e s t h a t had r e a c h e d the p r e v i t e l l o g e n i c r e s t i n g s t a g e i n t o r e c i p i e n t s t h a t were b l o o d f e d 24 h e a r l i e r . I n b o t h c a s e s , the l a r g e r o v a r i e s i n h i b i t e d the growth o f the l e s s mature o v a r i e s ( T a b l e I ) . These r e s u l t s c o n f i r m e a r l i e r o b s e r v a t i o n s , i n d i c a t i n g t h a t the o v a r y s e c r e t e s a humoral o o s t a t i c f a c t o r t h a t i n h i b i t s egg development i n l e s s mature o v a r i e s . T a b l e I . I n h i b i t i o n o f Egg Development i n P r e v i t e l l o g e n i c by V i t e l l o g e n i c O v a r i e s Female Ae. aegypti f e d sugar f o r 3 days

Number o f females (n)

a. B l o o d f e d 24 h and i m p l a n t e d w i t h an o v a r y (50 um) removed from s u g a r - f e d donor 3-5 days o l d and a n a l y z e d 48 h l a t e r

25

b. Implanted with ovaries (150 um) removed from donors f e d on b l o o d f o r 24 h. Females were f e d b l o o d and a n a l y z e d 48 h l a t e r

30

Donor o v a r y yolk size (um ± SEM)

50 ±

400

10

+50

Ovaries

Recipient yolk size (um ± SEM)

380 ±

80

50 +

20

O v a r i e s from s u g a r - f e d females (3 days o l d ) had r e a c h e d the p r e v i t e l l o g e n i c stage. O v a r i e s w i t h y o l k s i z e < 70 um d i d n o t move p a s t the p r e v i t e l l o g e n i c s t a g e . Mechanisms o f

Inhibition

General. S e v e r a l mechanisms o f i n h i b i t i o n have been s u g g e s t e d . I n Rhodnius p r o l i x u s , o o s t a t i c hormone p r o d u c e d by the abdominal n e u r o s e c r e t o r y organs i n h i b i t s the a c t i o n o f JH on v i t e l l o g e n i c f o l l i c l e c e l l s , p r e v e n t i n g the o v a r y from a c c u m u l a t i n g v i t e l l o g e n i n from the hemolymph (10.11). I n the house f l y

Menn et al.; Insect Neuropeptides ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Mosquito Oostatic Hormone


M. domestica o o s t a t i c hormone seems t o i n h i b i t t h e r e l e a s e o r s y n t h e s i s o f egg development n e u r o s e c r e t o r y hormone (EDNH) ( 1 2 ) , b u t i n m o s q u i t o e s t h e hormone seems t o a c t d i r e c t l y on t h e o v a r y (6). R. prolixus o o s t a t i c hormone, w h i c h has been p a r t i a l l y p u r i f i e d ( 1 3 ) , i s a s m a l l p e p t i d e o f M, 1,411 as d e t e r m i n e d on Sephadex G-50. K e l l y e t a l . (5) p r e p a r e d a crude e x t r a c t o f o o s t a t i c hormone from M. domestica, i n j e c t e d i t i n t o the autogenous mosquito Aedes atropalpus, and d e m o n s t r a t e d i n h i b i t i o n o f b o t h egg development and e c d y s t e r o i d b i o s y n t h e s i s . They s u g g e s t e d t h a t o o s t a t i c hormone f u n c t i o n s a t a p o i n t subsequent t o r e l e a s e o f EDNH. They were u n a b l e , however, t o e x c l u d e t h e p o s s i b i l i t y t h a t r e l e a s e o f EDNH, i t s e l f , was i n h i b i t e d . Mosquito s p e c i f i c . S i n c e 1985 my l a b o r a t o r y has b e e n i n v o l v e d i n the p u r i f i c a t i o n and s e q u e n c i n g o f Aedes aegypti "oostatic hormone". The e a r l i e s t r e s u l t s from my l a b o r a t o r y (14) i n d i c a t e d t h a t t h e mosquito o v a r y was a r i c h s o u r c e f o r " o o s t a t i c hormone". S t a r t i n g w i t h 4,800 p a i r s o f o v a r i e s I have p u r i f i e d t h e hormone 7 , 0 0 0 - f o l d u s i n g low p r e s s u r e chromatography. The hormone was f o u n d t o be a s m a l l p e p t i d e - l i k e m o l e c u l e o f M, 2,200 a t pH 4.5. I n j e c t i o n o f t h e p u r i f i e d p e p t i d e i n t o female m o s q u i t o e s i n h i b i t e d y o l k d e p o s i t i o n and v i t e l l o g e n i n s y n t h e s i s (14; T a b l e II). Table

II.

I n h i b i t i o n o f Egg Development w i t h Hormone

O o s t a t i c hormone (nmol)

2.86 1.45 0.24 Control

Number o f females

17 18 17 10

Purified

Oostatic

Yolk length (um ± SE)

53 82 101 186

± ± ± ±

7 8 5 4

Female A. aegypti were f e d b l o o d on a c h i c k e n , i m m e d i a t e l y i n j e c t e d w i t h o o s t a t i c hormone, and 24 h l a t e r a n a l y z e d f o r egg development. M, o f o o s t a t i c hormone was u s e d as 1047.6 b a s e d on mass s p e c t r a a n a l y s i s o f t h e p u r i f i e d hormone f o r nmol calculations. A c t i v i t y o f t h e hormone i n t h e o v a r y i n c r e a s e d r a p i d l y f o l l o w i n g b l o o d f e e d i n g and r e a c h e d a maximum a t 48 h ( 1 4 ) . The hormone d i d n o t b l o c k t h e r e l e a s e o f EDNH from t h e mosquito b r a i n and thus, we assumed t h a t the hormone a c t s d i r e c t l y on t h e o v a r y e i t h e r by p r e v e n t i n g p i n o c y t o s i s o r by i n h i b i t i n g e c d y s t e r o i d b i o s y n t h e s i s (14). I t took s e v e r a l years o f experimentation to f i n d o u t t h a t t h e p r i m a r y t a r g e t o f the f a c t o r t h a t we c a l l e d " o o s t a t i c hormone" was n o t t h e o v a r y . Injection of partially p u r i f i e d " o o s t a t i c hormone" i n t o female Ae. aegypti i n h i b i t e d egg development, p r o t e o l y t i c enzyme a c t i v i t y ( t r y p s i n and c h y m o t r y p s i n - l i k e enzymes) and b l o o d d i g e s t i o n i n t h e midgut (15). Controls i n j e c t e d with i n s u l i n chain A d i d not a f f e c t these processes. A s i n g l e i n j e c t i o n o f " o o s t a t i c hormone"


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p r e p a r a t i o n c a u s e d 1.7-1.5 f o l d r e d u c t i o n i n t r y p s i n - l i k e enzymes a c t i v i t y as measured w i t h a - N - b e n z o y l - D L - a r g i n i n e - p - n i t r o a n i l i d e (BApNA), w i t h a 10 h d e l a y i n peak a c t i v i t y ( 1 5 ) . I n c o l l a b o r a t i o n w i t h Dr. Y. S c h l e i n o f t h e Hebrew U n i v e r s i t y o f J e r u s a l e m , Department o f P a r a s i t o l o g y , a q u a n t i t a t i v e and s e n s i t i v e method t o d e t e r m i n e c o n c e n t r a t i o n s o f t r y p s i n and c h y m o t r y p s i n - l i k e enzymes i n i n s e c t s was d e v e l o p e d (16) . The a s s a y i s b a s e d on t h e s p e c i f i c b i n d i n g o f [ 1 , 3 - H ] d i i s o p r o p y l f l u o r o p h o s p h a t e (DFP) t o t r y p s i n and c h y m o t r y p s i n - l i k e enzymes. T r y p s i n - l i k e enzymes c a n be d e t e r m i n e d s p e c i f i c a l l y i n t h e p r e s e n c e o f 10 mM TPCK ( t o s y l a m i d e - 2 - p h e n y l e t h y l c h l o r o m e t h y l ketone; chymotrypsin i n h i b i t o r ) and c h y m o t r y p s i n - l i k e enzymes c a n be d e t e r m i n e d i n t h e p r e s e n c e o f 10 mM TLCK ( t o s y l - L - l y s i n e c h l o r o m e t h y l k e t o n e HC1; trypsin inhibitor). The a s s a y s e n s i t i v i t y i s l i m i t e d o n l y b y t h e s p e c i f i c a c t i v i t y o f DFP: a v a i l a b l e commercial p r e p a r a t i o n s have sp. a c t . o f 35 Ci/mmol and thus the lower l i m i t o f d e t e c t i o n i s 65 ng o f e i t h e r t r y p s i n o r c h y m o t r y p s i n . U s i n g t h i s method I f o l l o w e d t h e b i o s y n t h e s i s o f t r y p s i n - l i k e enzymes ( c h y m o t r y p s i n l i k e enzymes a r e r e s p o n s i b l e f o r o n l y 7% o f the p r o t e o l y t i c a c t i v i t y ) i n t h e midgut o f female Ae. aegypti. A 4-fold r e d u c t i o n i n [1, 3- H] d i i s o p r o p y l p h o s p h o r y l - t r y p s i n - l i k e d e r i v a t i v e s was n o t e d a f t e r " o o s t a t i c hormone" t r e a t m e n t ( 1 5 ) . I n j e c t i o n o f " o o s t a t i c hormone" i n t o d e c a p i t a t e d and o v a r i e c t o m i z e d females, a n i m a l s t h a t do n o t s y n t h e s i z e e c d y s t e r o i d s b u t s y n t h e s i z e p r o t e a s e i n t h e i r midgut, i n h i b i t e d t r y p s i n - l i k e enzyme b i o s y n t h e s i s and b l o o d d i g e s t i o n i n t h e i r midgut, i n d i c a t i n g t h a t " o o s t a t i c hormone" p r o b a b l y i n h i b i t s midgut c e l l s t h a t s y n t h e s i z e t r y p s i n , and n o t t h e o v a r y o r t h e b r a i n ' s e n d o c r i n e system T a b l e I I I ( 1 5 ) . Ae. aegypti "oostatic hormone" i s n o t s p e c i e s s p e c i f i c and i n j e c t i o n s o f t h e hormone i n t o Culex quinquefasciatus, Culex nigripalpus and Anopheles albimanus cause i n h i b i t i o n o f egg development, b l o o d d i g e s t i o n and s y n t h e s i s o f t r y p s i n - l i k e enzymes ( 1 5 ) . S i n c e t h r e e o f t h e s e mosquito s p e c i e s a r e i m p o r t a n t v e c t o r c a r r i e r s o f d i s e a s e ( y e l l o w f e v e r , m a l a r i a and e n c e p h a l i t i s ) , i t i s i m p o r t a n t t o f i n d o u t t h e e x a c t mechanism t h a t t e r m i n a t e s v i t e l l o g e n e s i s and b l o o d digestion.


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P u r i f i c a t i o n and Sequencing To answer t h e q u e s t i o n whether o u r hormone a c t s on t h e o o c y t e s and i s a t r u e " o o s t a t i c hormone", o r whether i t s f u n c t i o n i s t o modulate midgut s e r i n e p r o t e a s e s and, thus s h o u l d be c a l l e d T r y p s i n M o d u l a t i n g O o s t a t i c F a c t o r (TMOF), we p u r i f i e d t h e "hormone" t o a p p a r e n t homogeneity, sequenced i t and s y n t h e s i z e d s y n t h e t i c analogs. The "hormone" was i s o l a t e d from 30,000 p a i r s o f o v a r i e s , 4 days a f t e r female Ae. aegypti were f e d a b l o o d meal. O v a r i e s were e x t r a c t e d i n d i s t i l l e d water c o n t a i n i n g 1 mM p h e n y l m e t h y l s u l f o n y l f l u o r i d e (PMSF), a c i d i f i e d t o pH 4.5 and h e a t t r e a t e d . The e x t r a c t was c e n t r i f u g e d and t h e s u p e r n a t a n t p u r i f i e d u s i n g DEAE, C r e v e r s e d phase h i g h p r e s s u r e l i q u i d chromatography (HPLC) and g e l f i l t r a t i o n on TSK G2000 SW column. About 549 nmol o f p u r i f i e d "hormone" were o b t a i n e d from 30,000 p a i r s o f Ae. aegypti ovaries (i.e., 6 g of soluble protein) after l8


13.

BOROVSKY E T AL.


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T a b l e I I I . E f f e c t o f O o s t a t i c Hormone on T r y p s i n S y n t h e s i s i n D e c a p i t a t e d and O v a r i e c t o m i z e d Female Ae. aegypti


Female Ae. aegypti were f e d b l o o d on a c h i c k e n and then

No. of females

8

Trypsinlike a c t i v i t y (BApNA) (nmol/min/midgut ± SE)

a.

Immediately decapitated

25

3.74 ± 0.2

b.

Immediately d e c a p i t a t e d and injected with o o s t a t i c hormone (1.45 nmol)

25

1.63 ± 0.1

c.

Immediately d e c a p i t a t e d and injected with i n s u l i n chain A (1.45 nmol)

25

3.94 ± 0.3

25

5.96 ± 0.4

Immediately ovariectomized and i n j e c t e d w i t h o o s t a t i c hormone (1.45 nmol)

25

0.22 ± 0.1

f . Immediately ovariectomized and i n j e c t e d w i t h i n s u l i n chain A (1.45 nmol)

25

5.23 ± 0.2

d. A s s a y e d later e.

a

24 h

T e n female Ae. aegypti midguts were removed and a n a l y z e d f o r t r y p s i n a c t i v i t y 24 h l a t e r . R e s u l t s t a k e n from ( 1 5 ) . the f i v e - s t e p p u r i f i c a t i o n p r o c e d u r e . F o r sequence a n a l y s i s " o o s t a t i c hormone" was rechromatographed on r e v e r s e d phase HPLC and a pure p e p t i d e (2.6 nmol) was c o l l e c t e d a t 18.8 min and s e n t t o Dr. Donald F. Hunt, Department o f C h e m i s t r y , U n i v e r s i t y o f V i r g i n i a , C h a r l o t t e s v i l l e , VA who r a n t h e sample u s i n g mass s p e c t r o m e t r y w i t h quadrupole F o u r i e r t r a n s f o r m ( 1 7 ) . Bombardment o f t h e sample (20-50 pmol) i n t h i o g l y c e r o l w i t h 6 kV cesium i o n s d e t e c t e d an abundant (M + H ) i o n a t m/z 1047.6. D a t a o b t a i n e d from c o l l i s i o n a c t i v a t e d d i s s o c i a t i o n s p e c t r a r e c o r d e d on a t r i p l e quadrupole mass s p e c t r o m e t e r s u p p o r t e d t h e s t r u c t u r e o f " o o s t a t i c hormone" as NH -Tyr-Asp- Pro-Ala-Pro-Pro-Pro-Pro-Pro-Pro-COOH. The e a r l i e r d a t a o f M, 2000 was b a s e d on g e l f i l t r a t i o n , and was +

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INSECT NEUROPEPTIDES: CHEMISTRY, BIOLOGY, AND ACTION

n o t a c c u r a t e due t o the p o l y p r o l i n e t h a t i n t e r a c t e d w i t h the column m a t r i x and gave a l a r g e a p p a r e n t M, (14) . A p e p t i d e h a v i n g the i d e n t i c a l sequence was s y n t h e s i z e d u s i n g s t a n d a r d automated s o l i d phase p e p t i d e t e c h n i q u e s ( 1 8 ) . The s y n t h e t i c p e p t i d e was t h e n chromatographed u s i n g C reversed phase HPLC. N a t i v e " o o s t a t i c f a c t o r " c o e l u t e d w i t h the s y n t h e t i c p e p t i d e i n d i c a t i n g t h a t " o o s t a t i c hormone" s t r u c t u r e i s NH -TyrAsp-Pro-Ala-(Pro) -COOH. A C o r e y - P a u l i n g - K o l t u n (CPK) atomic model o f the f a c t o r i n d i c a t e s t h a t the c a r b o x y l end o f the molecule probably e x h i b i t s a left-handed h e l i c a l conformation i n s o l u t i o n due t o the s i x p r o l i n e r e s i d u e s ( 2 3 ) . 18

2

6


P r o p e r t i e s o f S y n t h e t i c Hormone Mode o f a c t i o n . The p o s s i b i l i t y t h a t mosquito o o s t a t i c f a c t o r a c t s as an i n h i b i t o r o f midgut t r y p s i n and c h y m o t r y p s i n enzymes u s i n g the same mechanisms as o t h e r i n h i b i t o r s (TLCK, TPCK, soybean t r y p s i n i n h i b i t o r ) (16) was a l s o i n v e s t i g a t e d . These i n h i b i t o r s b i n d t o the a c t i v e s i t e and p r e v e n t h y d r o l y s i s o f substrate. Female Ae. aegypti were f e d b l o o d on a c h i c k e n and 24 h l a t e r , when t r y p s i n and c h y m o t r y p s i n a c t i v i t i e s a r e a t a maximum, midguts (5 p e r group) were removed, homogenized i n 50 mM T r i s - H C l b u f f e r , pH 7.9, c e n t r i f u g e d a t 12,000 g f o r 20 min a t 4°C, and the s u p e r n a t a n t s c o l l e c t e d . One group was i n c u b a t e d w i t h 20 ug (19.5 nmol) o f s y n t h e t i c o o s t a t i c f a c t o r i n 100 ul i n c u b a t i o n m i x t u r e (a 7 - f o l d h i g h e r q u a n t i t y t h a n i s u s e d in vivo) f o r 30 min and then [ H]DFP added, whereas c o n t r o l s were i n c u b a t e d o n l y w i t h [ H]DFP. A t the end o f i n c u b a t i o n b o t h groups were a n a l y z e d f o r [ H ] D I P - t r y p s i n and c h y m o t r y p s i n - l i k e d e r i v a t i v e s ( 1 6 ) . No d i f f e r e n c e was found between the two groups ( T a b l e IV) w h i c h i n d i c a t e s t h a t o o s t a t i c f a c t o r does n o t b i n d t o the a c t i v e s i t e and does n o t i n h i b i t enzyme a c t i v i t y , b u t p r o b a b l y the b i o s y n t h e s i s o f t r y p s i n and c h y m o t r y p s i n . 3

3

3

T a b l e IV.

Does S y n t h e t i c O o s t a t i c F a c t o r I n h i b i t T r y p s i n l i k e Chymotrypsinlike A c t i v i t i e s ?

Female Ae. aegypti midguts were a n a l y z e d f o r serine protease a c t i v i t y 24 h a f t e r a b l o o d meal w i t h a. O o s t a t i c f a c t o r (20 ug) b. W i t h o u t o o s t a t i c f a c t o r

and

3

No. o f Groups

[ H ] D I P - t r y p s i n and chymotrypsin (cpm/midgut ± SEM)

3 3

47,330 ± 2,959 49,891 ± 3,341

S i x groups o f female Ae. aegypti (5 p e r group) were f e d b l o o d , the midguts were d i s s e c t e d out, and a n a l y z e d 24 h l a t e r i n the p r e s e n c e and absence o f s y n t h e t i c o o s t a t i c f a c t o r f o r [ H]DIPt r y p s i n and c h y m o t r y p s i n - l i k e enzymes SOURCE: Reprinted from ref. 16. Copyright 1988 Wiley-Liss 3


13.

BOROVSKY ET AL.


139

Species S p e c i f i c i t y . O o s t a t i c hormone i s n o t s p e c i e s s p e c i f i c ( 1 4 ) , when Ae. aegypti s y n t h e t i c o o s t a t i c hormone was i n j e c t e d i n t o C. v a r i i p e n n i s , Ae. aegypti, An. quadrimaculatus, Stomoxys a l c i t r a n s , Ctenocephalides f e l i s (Bouche) and Lutzomyia anthophora ( A d d i s ) s y n t h e s i s o f [ 1 , 3 - H ] D I P - t r y p s i n - l i k e isozymes was r e d u c e d ( T a b l e V ) . 3

Target Tissue. To s t u d y whether midgut c e l l s have " o o s t a t i c hormone" s p e c i f i c r e c e p t o r s the hormone was l a b e l e d w i t h [ H ] a c e t i c a n h y d r i d e to a s p e c i f i c a c t i v i t y o f 0.53 Ci/mmol, and a l i q u o t s o f 780 pmol i n 0.5 ul were i n j e c t e d i n t o s u g a r - f e d female Ae. aegypti. A t i n t e r v a l s f o l l o w i n g the i n j e c t i o n , midguts were removed, washed 3 times i n t i s s u e c u l t u r e medium TC 199 (GIBCO L a b o r a t o r i e s ) , homogenized i n 1 N NaOH (100 u l ) , c e n t r i f u g e d a t 12,000g and the s u p e r n a t a n t a n a l y z e d i n a l i q u i d s c i n t i l l a t i o n counter. Fifteen-minutes a f t e r i n j e c t i n g " o o s t a t i c hormone" the r a t e o f b i n d i n g t o gut r e c e p t o r was 8.7 pmol/h/gut, the b i n d i n g r a t e i n c r e a s e d to 13 pmol/h/gut a t 1 h and d e c l i n e d t h e r e a f t e r t o 4 pmol/h/gut between 2-4 h, t o 0.9 pmol/h/gut a t 8 h, 0.13 pmol/h/gut a t 30 h and 0.0089 pmol/h/gut a t 96 h. At peak b i n d i n g 1.7%-2.1% o f i n j e c t e d " o o s t a t i c hormone" bound t o gut r e c e p t o r . These r e s u l t s i n d i c a t e t h a t the gut has hormone s p e c i f i c r e c e p t o r and t h a t the b i n d i n g i s a dynamic, r e c e p t o r s p e c i f i c process. No b i n d i n g t o o t h e r t i s s u e s ( b r a i n , t h o r a x , f a t body, o v a r y and M a l p i g h i a n t u b u l e s ) was d e t e c t e d 24 h a f t e r i n j e c t i n g the "hormone". Thus, a more a p p r o p r i a t e name f o r " o o s t a t i c hormone" i s T r y p s i n M o d u l a t i n g O o s t a t i c F a c t o r (TMOF). The r a p i d d e c l i n e i n b i n d i n g 1 h a f t e r i n j e c t i n g TMOF i n d i c a t e s t h a t the f a c t o r i s r a p i d l y removed from the hemolymph o r m e t a b o l i z e d i n t o an i n a c t i v e m o l e c u l e . Thus, a b i n d i n g a s s a y o f TMOF to midgut r e c e p t o r i s a b e t t e r way t o e x p r e s s i t s a c t i v i t y .


3

Homology. An o n - l i n e computer s e a r c h (MATCH and PIR a c c e s s e s the N a t i o n a l Biomedical, N a t i o n a l Bioresearch, Medical Research F o u n d a t i o n ) f o u n d t h a t TMOF has s i g n i f i c a n t homology t o mammalian, p l a n t and s e v e r a l v i r a l p r o t e i n s t h a t a r e e i t h e r s y n t h e s i z e d by double s t r a n d e d DNA v i r u s e s ( E p s t e i n B a r r v i r u s and Herpes s i m p l e x v i r u s ) o r s i n g l e s t r a n d e d RNA v i r u s e s . ( A b e l s o n murine l e u k e m i a v i r u s , a v i a n s p l e e n n e c r o s i s v i r u s , r u b e l l a v i r u s , s i m i a n T - l y m p h o t r o p i c v i r u s and HIV-2). This close homology (7 t o 8 amino a c i d s out o f 10) may i n d i c a t e t h a t TMOF d i f f e r s from o t h e r i n s e c t hormones t h a t e x h i b i t homology o n l y t o v e r t e b r a t e p r o t e i n s . P r o t h o r a c i c o t r o p i c hormone (PTTH), and l e u c o s u l f a k i n i n e x h i b i t homology w i t h human i n s u l i n , g a s t r i n and c h o l e c y s t o k i n i n , r e s p e c t i v e l y (19.20). Model. B l o o d d i g e s t i o n p l a y s a key r o l e i n the s u r v i v a l o f v e c t o r a r t h r o p o d s and i n the t r a n s m i s s i o n o f many i m p o r t a n t d i s e a s e s such as m a l a r i a , dengue and Lyme d i s e a s e (21.22). Collating t h e s e o b s e r v a t i o n s , a model f o r the c o n t r o l o f t r y p s i n b i o s y n t h e s i s i n the mosquito i s p r o p o s e d . A f t e r the b l o o d meal the midgut e p i t h e l i a l c e l l s s t a r t t o s y n t h e s i z e t r y p s i n i n c o n c e r t w i t h the n e u r o e n d o c r i n e system t h a t s y n t h e s i z e s o o s t a t i c hormone, as was p r o p o s e d f o r Rhodnius by Davey and K u n s t e r ( 1 1 ) , w h i c h accumulates i n the d e v e l o p i n g o v a r y as a prohormone. The


140


T a b l e V. E f f e c t o f S y n t h e t i c O o s t a t i c Hormone on T r y p s i n B i o s y n t h e s i s i n M o s q u i t o e s , F l e a s , F l i e s and S a n d f l i e s

Females were f e d b l o o d and t h e n

C. f e l i s (Bouche) a. Immediately i n j e c t e d w i t h o o s t a t i c hormone (477 pmol) b. C o n t r o l

(not injected)

3

No. o f females

[1,3- H]DIP t r y p s i n p e r midgut (cpm) a

11

1,876

10

3,192

14

5,644

9

10,385

4

1,802

8

2,492

16

41,042

10

60,868

17

13,625

7

99,167

6

10,186

10

25,999


S. c a l c i t r a n s c.

Immediately i n j e c t e d w i t h o o s t a t i c hormone (4.77 nmol) d. Immediately i n j e c t e d with saline L. anthophora e. Immediately i n j e c t e d w i t h o o s t a t i c hormone (477 pmol) f. A.

Control

(not injected)

quadrimaculatus

g. Immediately i n j e c t e d w i t h o o s t a t i c hormone (10 nmol) h. Immediately i n j e c t e d with saline A. aegypti i . Immediately i n j e c t e d w i t h o o s t a t i c hormone (2.87 nmol) j . Immediately i n j e c t e d with saline C. v a r i i p e n n i s k. Immediately i n j e c t e d w i t h o o s t a t i c hormone (286 pmol) 1. Immediately i n j e c t e d with saline

a

Females were a n a l y z e d 24 h a f t e r the b l o o d meal f o r [1,3-3H]DIPtrypsinlike derivatives. O o s t a t i c hormone c o n c e n t r a t i o n s a r e b a s e d on M, 1047.6 d e r i v e d from mass s p e c t r a a n a l y s i s . R e s u l t s a r e average o f 2 d e t e r m i n a t i o n s .


13. BOROVSKY ET AL.


141

active hormone is released into the hemolymph 24 h later, and signals the midgut epithelial cells to stop the synthesis of trypsin-like enzymes which ceases 55 h later. Acknowledgment. This work was partially supported by The Lady Davis Foundation visiting Professorship award to D.B., USDA research grant CRCR 1-2394 to D.B., and by the National Institutes of Health Grant, GM 37537, to D.F.H., Instrument development funds (to D.F.H.) from the Monsanto Co., Center for Innovative Technology (BIO-87006), and the National Science Foundation (CHE8618780) are also gratefully acknowledged.


Literature Cited 1. Iwanov, P. P.; Mescherskaya, K. A. Zool. Jb (Physiol.) 1935, 55, 281-348. 2. Adams, T. S.; Mulla, M. S. Ann. Entomol. Soc. Am. 1967, 60. 1117-1182. 3. Carlisle, D. B.; Knowles, F. Cambridge Monogr. Exp. Biol. 1959, 10, 1-120. 4. Adams, T. S.; Hintz, A. M.; Pomonis, J.G. J. Insect Physiol. 1968, 14, 983-993. 5. Kelly. T. J.; Birnbaum, M. J.; Woods, C. W.; Borkovec, A. B. J. exp. Zool. 1984, 229, 491-496. 6. Meola, R; Lea, A. O. J. Med. Entomol. 1972, 9, 99-103. 7. Else, J. G.; Judson, C. L. J. Med. Entomol. 1972, 9 527530. 8. Gwadz, R.; Spielman, A. J. Insect Physiol. 1973, 19, 14411448. 9. Hagedorn, H. H.; Turner, S.; Hagedorn, E. A.; Pontecorvo, D,; Greenbaum, P.; Pfeifer, D.; Wheelock, G.; Flanagan, T. R. J. Insect Physiol. 1977, 23, 203-206. 10. Davey, K. G. In Comparative Endocrinology: Gailland, P. J.; Boer, H. H., Eds.; Elsevier: Amsterdam, 1978; pp. 13-16. 11. Davey, K. G.; Kunster, J. E. Can. J. Zool. 1981, 59, 761764. 12. Adams, T. S. In Advances in Invertebrate Reproduction; Clark, W. H., Jr; Adams, T. S., Eds.; Elsevier: Amsterdam, 1981; pp. 109-125. 13. Liu, T. P.; Davey, K. G. Gen. Comp. Endocrinol. 1974, 24, 405-408. 14. Borovsky, D. Arch. Insect Biochem. Physiol. 1985, 2, 333349. 15. Borovsky, D. Arch. Insect Biochem. Physiol. 1988, 7, 187210. 16. Borovsky, D.; Schlein, Y. Arch. Insect Biochem. Physiol. 1988, 8, 249-260. 17. Hunt, D. F.; Yates, J. R., III; Shabanowitz, J.; Winston, S.; Hauer, C. R. Proc. Natl. Acad. Sci., USA 1986, 83, 6233-6237. 18. Barany, G.,; Merrifield, R. B. In The Peptides: Gross, E.; Meienhofer, J., Eds.; Academic: New York, 1979, Vol. 2, pp. 1-284.



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19. Nagasawa, H.; Kataoka, H.; Isogai, A.; Tamura, S.; Suzuki, A.; Ishizaki, H.; Mizoguchi, A.; Fujiwara, Y.; Suzuki, A. Science 1984, 296, 1344-1345. 20. Nachman, R. J.; Holman, G. M.; Haddon, W. F.; Ling, N. Science 1986, 234, 71-73. 21. Borovsky, D.; Spielman, A. In Host Regulated Developmental Mechanisms in Vector Arthropods, Proceedings Vero Beach Symposium; Borovsky, D., Spielman, A., Eds.; University of Florida - IFAS, Florida Medical Entomology Laboratory, 1986, pp. 1-217. 22. Borovsky, D.; Spielman, A. In Host Regulated Developmental Mechanisms in Vector Arthropods, Proceedings Vero Beach Symposium; Borovsky. D.; Spielman, A., Eds.; University of Florida - IFAS, Florida Medical Entomology Laboratory, 1989, pp. 1-324. 23. Dickerson, R. E.; Geis, I. The structure and action of proteins; Harper and Row, New York, 1969; pp. 1-119. RECEIVED September 21, 1990


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