Allatal Stimulation and Inhibition in Locusts - ACS Symposium Series

Chapter 15 Allatal Stimulation and Inhibition in Locusts Downloaded by CALIFORNIA INST OF TECHNOLOGY on January 20, 2018 | http://pubs.acs.org Publication Date: January 25, 1991 | doi: 10.1021/bk-1991-0453.ch015

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Shalom W. Applebaum , Michal Gadot , Jacquelyn Hirsch , and Fathi Abd El-Hadi l

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Department of Entomology, Faculty of Agriculture, The Hebrew University, Rehovot, Israel Department of Zoology, Institute of life Sciences, The Hebrew University, Jerusalem, Israel

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The ability of the adult female locust to synthesize juvenile hormone (JH) is dependent on allatal maturation after adult emergence and by long-term (slow) and short-term (rapid) modulation of the competent corpora allata (CA). Allatal maturation is described in terms of increasing levels of terminal epoxidase and methyl transferase and to an increasing capacity of the CA to respond to allatotropin. Mature CA respond equally to farnesoic acid, the penultimate precursor of JH. Evidence exists that lateral neurosecretory cells of the brain are involved in long-term modulation of the mature CA. Short-term modulation is attributed to cerebral allatotropin and to ovarian allatostatin, which overrides allatotropic stimulation at the end of vitellogenesis. The rate-limiting step in JH biosynthesis, under short-term control, is concerned with mevalonate synthesis. This is concluded from in vitro factorial experiments with two 3-hydroxy-3-methyl-glutaryl CoA reductase inhibitors: hydroxymethylglutarate and mevinolin, with and without allatotropin and mevalonate. The s y n t h e s i s o f j u v e n i l e hormone (JH) i n t h e i n s e c t c o r p o r a a l l a t a (CA) i s c o n t r o l l e d by an a r r a y o f i n h i b i t o r y and s t i m u l a t o r y factors o r i g i n a t i n g i n d i f f e r e n t p a r t s o f t h e r e t r o c e r e b r a l complex and i n the o v a r y . The n a t u r e o f t h e f a c t o r s i n v o l v e d and t h e i r i n t e r a c t i o n s seem d i s s i m i l a r i n d i f f e r e n t i n s e c t groups, and i t i s premature, b a s e d on what i s known t o date ( 1 . 2 ) , t o f o r m u l a t e a u n i f i e d model o f a l l a t a l c o n t r o l f o r a l l i n s e c t s . D i f f e r e n c e s may t r u l y e x i s t , however t h e y may be a consequence o f i n f o r m a t i o n i n a d e q u a t e t o g e n e r a l i z e from. R a t h e r , we p r e s e n t h e r e i n a model o f a l l a t a l c o n t r o l i n t h e a d u l t female l o c u s t d u r i n g r e p r o d u c t i v e m a t u r a t i o n , w h i c h may s e r v e

0097-6156/91/0453-0152$06.00/0 © 1991 American Chemical Society Menn et al.; Insect Neuropeptides ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Downloaded by CALIFORNIA INST OF TECHNOLOGY on January 20, 2018 | http://pubs.acs.org Publication Date: January 25, 1991 | doi: 10.1021/bk-1991-0453.ch015

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as a r e f e r e n c e p o i n t f o r e v a l u a t i n g o t h e r i n s e c t g r o u p s . Most o f the work c i t e d from our l a b o r a t o r y has been p e r f o r m e d w i t h the A f r i c a n migratory l o c u s t (Locusta m i g r a t o r i a m i g r a t o r i o i d e s ) . The b a s i c m e t h o d o l o g i e s o f m e a s u r i n g JH t i t e r and rates of biosynthesis will be mentioned h e r e o n l y b r i e f l y . JH titer is d e t e r m i n e d by e i t h e r radioimmunoassay (3) o r p r e f e r a b l y by gas chromatography c o u p l e d w i t h m a s s - s p e c t r o m e t r y ( 4 - 6 ) . A l t e r n a t i v e l y , JH b i o s y n t h e s i s can be d e t e r m i n e d w i t h e x c i s e d CA i n v i t r o by the radiochemical (RCA) method ( 7 ) . JH values obtained by titering r e f l e c t the e q u i l i b r i u m o f r a t e o f JH s y n t h e s i s , i t s d e g r a d a t i o n and c l e a r a n c e from the hemolymph, i t s t i s s u e uptake and e x c r e t i o n . The r a t e o f de novo s y n t h e s i s , as measured by the RCA method, i s a r g u e d t o c o r r e l a t e w e l l w i t h JH hemolymph t i t e r (8.9) . As we s h a l l l a t e r show, the c o r r e l a t i o n o f i n v i t r o s y n t h e s i s and i n v i v o t i t e r s i s not always maintained in L. migratoria under all experimental situations. Three-Component Model o f C o n t r o l o f JH

Biosynthesis

D e v e l o p m e n t a l M a t u r a t i o n i s a p r o g r e s s i v e a c q u i s i t i o n o f competence o f the CA t o s y n t h e s i z e JH, which n o r m a l l y p r o c e e d s i n c o n j u n c t i o n w i t h d e v e l o p m e n t a l changes a s s o c i a t e d w i t h i n c r e a s e i n g l a n d volume (10.11). B i o s y n t h e t i c c a p a c i t y i n c r e a s e s markedly d u r i n g the f i r s t 2-3 days a f t e r a d u l t emergence (12.13). T o t a l c a p a c i t y o f the CA i s e x p r e s s e d and can be q u a n t i f i e d when a l l r a t e - l i m i t i n g s t e p s i n the pathway o f JH s y n t h e s i s are e i t h e r f u l l y a c t i v a t e d o r b y p a s s e d , and no i n h i b i t o r s p r e s e n t . I n p r a c t i c e t h i s i s done by i n c u b a t i n g CA i n vitro i n the presence of farnesoic acid (FA) , the penultimate p r e c u r s o r o f JH. Exogenous FA supplements the endogenous p o o l w i t h i n the CA and e l i c i t s a s i g n i f i c a n t i n c r e a s e i n s y n t h e s i s , c o n s i s t e n t with the suggestion t h a t the l a s t two steps i n JH biosynthesis ( e p o x i d a t i o n and m e t h y l a t i o n ) are s u b s t r a t e limited, rather than b e i n g r a t e - l i m i t e d by d e f i c i e n c y o f the enzymes i n v o l v e d ( 8 ) . Long-Term M o d u l a t i o n o f JH s y n t h e s i s i s a s c e r t a i n e d by m e a s u r i n g the b a s a l r a t e s i n v i t r o . B a s a l r a t e s o f e x c i s e d CA a r e l i n e a r f o r a t l e a s t 8 h r ( 1 4 ) . B a s a l r a t e s i n c r e a s e g r a d u a l l y d u r i n g the first gonadotrophic c y c l e as v i t e l l o g e n e s i s i s a t t a i n e d and progresses. They continue to increase even after the CA have matured d e v e l o p m e n t a l l y and a t t a i n e d maximal competence. A d e c r e a s e i n b a s a l r a t e s i s o b s e r v e d a f t e r d e p o s i t i o n o f the f i r s t b a t c h o f eggs and i n o l d n o n - v i t e l l o g e n i c f e m a l e s , w h i l e the l e v e l o f a l l a t a l competence s t i l l remains h i g h (13.14). Short-Term Modulation allatotropic and allatostatic is superimposed on the b a s a l a c t i v i t i e s o f d e v e l o p m e n t a l l y mature CA. Locust c e r e b r a l a l l a t o t r o p i n was first demonstrated i n v i t r o in m e t h a n o l i c b r a i n and corpora cardiaca (CC) extracts (14.15) and a l l a t o t r o p i n I was l a t e r p a r t i a l l y p u r i f i e d and c h a r a c t e r i z e d ( 1 6 ) . A l l a t o t r o p i n I i s found i n h i g h c o n c e n t r a t i o n s i n the b r a i n , where i t i s presumably s y n t h e s i z e d , and i n c o r p o r a c a r d i a c a (CC) t o where i t is presumably transported and released as required. The exact l o c a t i o n o f a l l a t o t r o p i n s y n t h e s i s i n the b r a i n i s s t i l l unknown. Ovarian a l l a t o s t a t i n r a p i d l y d e p r e s s e s a l l a t o t r o p i n - s t i m u l a t e d JH synthesis ( 1 7 ) . T h i s a l l a t o s t a t i c f a c t o r i s methanol s o l u b l e and

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

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INSECT NEUROPEPTIDES: CHEMISTRY, BIOLOGY, AND ACTION

resistant to b o i l i n g decay r a p i d l y i n v i t r o


S i t e s o f Synthesis

(16). A l l a t o t r o p i c / a l l a t o s t a t i c i n t h e absence o f t h e e f f e c t o r .

modulations

and R e l e a s e

The o r i g i n and t r a n s p o r t o f t h e " m a t u r a t i o n f a c t o r s " i n t h e a d u l t l o c u s t has been examined by e v a l u a t i n g t h e i n v i v o and i n v i t r o e f f e c t s o f s u r g i c a l interventions (Figure 1). Electrocoagulation o f the median n e u r o s e c r e t o r y c e l l s (MNC) o f t h e p a r s i n t e r c e r e b r a l i s o f 2-day o l d a d u l t female L. m i g r a t o r i a p r e v e n t e d t h e normal i n c r e a s e i n b o t h CA volume and e p o x i d a s e a c t i v i t y a s s o c i a t e d w i t h JH s y n t h e s i s ( 1 8 ) . E l e c t r o s t i m u l a t i o n o f t h e MNC o f 1-day o l d f e m a l e s enhanced JH biosynthesis i n v i t r o w i t h i n 2-3 days o f t h e s t i m u l a t i o n , w h i l s t cautery o f the c e r e b r a l axonal t r a c t s o f these c e l l s (the i n t e r n a l cardiaca tract TCC-I), prevented both normal development and enhancement by e l e c t r o s t i m u l a t i o n ( 1 9 ) . E l e c t r o c o a g u l a t i o n o f t h e TCC-I i n 8-day o l d f e m a l e s , i n which t h e CA a r e f u n c t i o n a l l y mature, d i d n o t a f f e c t t h e i r i n v i t r o a c t i v i t y ( 2 0 ) . These f i n d i n g s s u g g e s t t h a t t h e MNC r e g u l a t e t h e development o f t h e CA d u r i n g t h e f i r s t days a f t e r a d u l t emergence, as would be e x p e c t e d f o r t h e h y p o t h e t i c a l maturation f a c t o r s . Their p o s s i b l e routes o f transmission are not o b v i o u s . No d i r e c t nervous c o n n e c t i o n has been d e s c r i b e d between t h e MNC and t h e CA i n L. m i g r a t o r i a (21.22). F u r t h e r m o r e , t r a n s e c t i o n o f the NCA-I i n newly-emerged females i m p a i r e d t h e i r b a s a l a c t i v i t y , b u t did n o t e f f e c t subsequent JH s y n t h e s i s i n vivo (23.24). These o b s e r v a t i o n s a r e c o n s i s t e n t w i t h the p o s s i b i l i t y t h a t the m a t u r a t i o n f a c t o r s are humorally transmitted to the disconnected CA. On t h e o t h e r hand, some e v i d e n c e i n d i c a t e s t h a t a x o n a l t r a n s m i s s i o n does have an e f f e c t on CA m a t u r a t i o n . Thus, t h e a s s y m e t r y i n CA volume and a c t i v i t y between t h e p a i r e d CA (11.25) i s more c o n s i s t e n t w i t h n e u r a l c o n t r o l o f CA development. The f a c t o r s o f l o n g - t e r m m o d u l a t i o n most p r o b a b l y o r i g i n a t e i n the l a t e r a l n e u r o s e c r e t o r y c e l l s (LNC) o f t h e p r o t o c e r e b r u m and a r e t r a n s p o r t e d v i a t h e NCC-II t o t h e CC and from t h e r e v i a t h e NCA-I t o the CA. JH s y n t h e s i s i s f i r s t r e d u c e d as an immediate r e s p o n s e t o e l e c t r o c o g u l a t i o n o f t h e LNC i n v i t e l l o g e n i c L. m i g r a t o r i a f e m a l e s , but thereafter recovers. O n l y a f t e r 30 h r does a f i n a l decline commence. NCA-I t r a n s e c t i o n i s a more m i l d t r e a t m e n t , and t h e i n i t i a l transient reduction i s n o t evoked. Here t o o t h e i n v i t r o basal a c t i v i t y o f t h e CA d e c l i n e s a f t e r 30 h r o f n e r v e t r a n s e c t i o n (20) suggesting that a stimulatory c o n t r o l f a c t o r i s n e u r a l l y transmitted. I n S c h i s t o c e r c a g r e g a r i a . t h e d e c r e a s e i n CA a c t i v i t y a f t e r n e r v e t r a n s e c t i o n t a k e s about 6 h r (26), which i s s t i l l w i t h i n t h e range o f l o n g - t e r m m o d u l a t i o n . D e s t r u c t i o n o f t h e NCC-I has no e f f e c t on t h e b a s a l i n v i t r o a c t i v i t y o f t h e CA i n v i t e l l o g e n i c f e m a l e s , i n d i c a t i n g that t h e MNC do n o t p a r t i c i p a t e i n l o n g - t e r m modulation. The denervation o f mature CA s p e c i f i c a l l y reduce t h e i r b a s a l l e v e l o f a c t i v i t y , b u t n o t t h e i r competence, as t h e s e g l a n d s c a n be s t i m u l a t e d by FA t o t h e same degree as c o n t r o l i n t a c t CA ( 9 ) , i n d i c a t i n g t h e independence o f l o n g - t e r m m o d u l a t i o n and t h e m a t u r a t i o n p r o c e s s . The b a s a l a c t i v i t y o f CA from d i a p a u s i n g f e m a l e s o f t h e S a v i o s t r a i n o f L. m i g r a t o r i a . d e n e r v a t e d i n v i v o (NCA-I t r a n s e c t i o n ) , d e c r e a s e d o v e r a f o u r days p e r i o d , a f t e r w h i c h t h e y were f o u n d t o be inactive when a s s a y e d i n v i t r o (27). This i n d i c a t e s an a x o n a l a c t i v a t i n g f a c t o r whose e f f e c t on b a s a l a c t i v i t y decays o v e r s e v e r a l days a f t e r n e r v e t r a n s e c t i o n .


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LNC

F i g u r e 1. Schematic r e p r e s e n t a t i o n o f t h e main f e a t u r e s o f t h e locust brain and r e t r o c e r e b r a l complex. MNC = median neurosecretory cells; LNC = l a t e r a l neurosecretory c e l l s ; TCC-I a x o n a l i n t e r c e r e b r a l t r a c t s from t h e MNC l e a d i n g t o t h e NCC-I, a v e n t r a l p a i r o f n e r v e s which i n n e r v a t e t h e CC. TCC-II = t h e a x o n a l i n t e r c e r e b r a l t r a c t s from t h e LNC l e a d i n g t o t h e NCC-II, a d o r s a l p a i r o f n e r v e s which i n n e r v a t e t h e CC. G-CC - t h e g l a n d u l a r l o b e o f t h e CC; S-CC = the s e c r e t o r y neurohemal l o b e o f t h e CC. NCA-I a r e a p a i r o f n e r v e s which l e a d t o t h e CA. The NCA-II l e a d t o t h e subesophageal ganglion.



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S h o r t - t e r m m o d u l a t i o n o f l o c u s t CA a c t i v i t y was based on i n d i r e c t e v i d e n c e u n t i l the r e c e n t i s o l a t i o n and p a r t i a l p u r i f i c a t i o n of a l l a t o t r o p i n I (16) . T h i s i s most p r o b a b l y the same f a c t o r d e s c r i b e d from the b r a i n and CC o f a d u l t L. m i g r a t o r i a f e m a l e s ( 1 5 ) . Allatotropin causes a transient stimulation of the rate of JH synthesis i n the mature CA in vitro. In c o n t r a s t to long-term a c t i v a t i n g f a c t o r s , the e f f e c t o f a l l a t o t r o p i n I decays r a p i d l y , within an hour of i t s absence (14) . H i g h levels of in vivo a l l a t o t r o p i n - s t i m u l a t e d JH s y n t h e s i s would t h e r e f o r e n o t be d e t e c t e d by the i n v i t r o system. A l l a t o t r o p i c s t i m u l a t i o n might a l s o e x p l a i n the s u s t a i n e d h i g h l e v e l s o f a c t i v i t y o c c a s i o n a l l y o b t a i n e d with transplanted CA, where no nervous connections remain (28) . A t r a n s i e n t i n v i v o s t i m u l a t i o n o f JH s y n t h e s i s t h a t i s n o t r e t a i n e d during the i n v i t r o a s s a y may e x p l a i n the i n c o n s i s t e n c i e s f o u n d between i n v i v o and in vitro JH synthesis i n s o l i t a r ious and g r e g a r i o u s L. m i g r a t o r i a ( 6 ) . T i t e r d e t e r m i n a t i o n s o f a l l a t o t r o p i n I are n o t y e t p o s s i b l e , but semi- q u a n t i t a t i v e d e t e r m i n a t i o n s , b a s e d on dose-response curves, suggest that high levels of cerebral a l l a t o t r o p i n I are found a t a l l s t a g e s o f mature l o c u s t s (14), though l e s s i s found i n the b r a i n s o f newly f l e d g e d l o c u s t s (Abd E l - H a d i , F.; Applebaum, S.W., unpublished data). I n d i a p a u s i n g females o f the S a v i o s t r a i n o f L. m i g r a t o r i a . the b a s a l a c t i v i t y o f the CA i s t o t a l l y i n h i b i t e d i n v i v o . as e s t a b l i s h e d by the a r r e s t o f o o c y t e development and absence o f JH in the hemolymph (27.). However, t h e s e same g l a n d s a r e i m m e d i a t e l y a c t i v a t e d when t h e y are i n c u b a t e d i n v i t r o ( i . e . , d e n e r v a t e d and removed from t h e i r n a t u r a l m i l i e u ) , i n d i c a t i n g t h a t t h e i r m a t u r a t i o n has n o t been r e t a r d e d . In v i v o d e n e r v a t i o n o f the g l a n d s (NCA-I t r a n s e c t i o n ) i n diapausing females e l i c i t s e l e v a t e d JH titer and induces oocyte development, i n d i c a t i n g t h a t t h i s s h o r t - t e r m i n h i b i t i o n i s a x o n a l l y imposed. Mode o f

Action

The development and d e p o s i t i o n o f eggs i s c y c l i c i n l o c u s t s , w i t h the f i r s t c y c l e accompanied by p a r a l l e l m a t u r a t i o n o f the CA (11.14). The l a t t e r has u s u a l l y been e v a l u a t e d by d e t e r m i n i n g JH p r o d u c t i o n i n vitro i n the p r e s e n c e o f FA (8.13.29), b u t a l l a t a l r e s p o n s e to a l l a t o t r o p i n would seem t o be more a p p r o p r i a t e f o r p o r t r a y i n g the p h y s i o l o g i c a l s t a t e o f CA m a t u r a t i o n . The r a t e s o f JH synthesis e l i c i t e d by FA and a l l a t o t r o p i n are e q u i v a l e n t o n l y i n mature CA. Immature CA o f newly emerged a d u l t f e m a l e s e x h i b i t b o t h low b a s a l a c t i v i t y and low competence. Some s m a l l s t i m u l a t i o n o f JH s y n t h e s i s is obtained i n response to exogenous FA, i n d i c a t i n g that the endogenous FA s u p p l y i s l i m i t e d r e l a t i v e t o the c a p a c i t y o f the terminal enzymes. However, immature CA are no stimulated by a l l a t o t r o p i n , p o s s i b l y because they are d e f i c i e n t i n r a t e - l i m i t i n g enzymes, or alternatively, that they have not yet developed responsiveness t o the hormone ( F i g u r e 2 ) . R e s p o n s i v e n e s s i s u s u a l l y meant t o d e s c r i b e the abundance o f hormone r e c e p t o r s , b u t no s t u d i e s on a l l a t o t r o p i n r e c e p t o r s have been c o n d u c t e d i n i n s e c t CA. Considerable differences are found in the rates of JH s y n t h e s i z e d by mature CA o f d i f f e r e n t l o c u s t females and between the



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F i g u r e 2. C u m u l a t i v e JH s y n t h e s i s i n v i t r o b y CA o f immature (2-3 days) a d u l t female l o c u s t s . The f i r s t 5 h r demonstrate basal s y n t h e s i s . I = a d d i t i o n o f e i t h e r 5 0 u l f a r n e s o i c a c i d (FA) ( • ) o r a l l a t o t r o p i n (0) f o r an a d d i t i o n a l 4 h r i n c u b a t i o n . I I = t r a n s f e r of the a l l a t o t r o p i n - t r e a t e d CA t o FA-supplemented media, and F A - t r e a t e d CA t o a l l a t o t r o p i n - s u p p l e m e n t e d media. V a l u e s a r e t h e mean ± SE o f 11 r e p l i c a t e s . The e r r o r b a r s o f t h e 1 h r i n c u b a t i o n have been p l a c e d above and b e s i d e t h e v a l u e s i n o r d e r n o t t o o b s c u r e them.



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two g l a n d s o f each l o c u s t (11.14.18) . About the same degree o f v a r i a t i o n a l s o e x i s t s i n the competence o f t h e s e g l a n d s and a good c o r r e l a t i o n was found between the b a s a l and F A - i n d u c e d r a t e s of s y n t h e s i s ( 1 3 ) . Such a c o r r e l a t i o n was however n o t found i n s i m i l a r e x p e r i m e n t s w i t h 8-day o l d females ( 9 ) . The b i o s y n t h e t i c m a t u r a t i o n o f the CA i n v o l v e s the e l a b o r a t i o n o f key enzymes a t d i f f e r e n t l e v e l s o f the b i o s y n t h e t i c pathway o f JH. The first of these are r a t e - l i m i t i n g enzymes, early in the b i o s y n t h e t i c pathway, amenable t o a c t i v a t i o n or i n h i b i t i o n . The second o f these, terminal e p o x i d a s e and methyl-transferase, are u s u a l l y r e g a r d e d as n o n - r a t e - l i m i t i n g . T h e i r i n c r e a s e i n a c t i v i t y is d i r e c t l y c o r r e l a t e d t o the i n c r e a s e i n CA volume (18.30). When the amount o f t h e s e enzymes i s m a r g i n a l , as presumably o c c u r s i n immature CA, t h i s a l s o r e s t r i c t s the t o t a l c a p a c i t y o f the CA to s y n t h e s i z e JH. When mature CA are a c t i v a t e d , the l e v e l s o f r a t e - l i m i t i n g enzymes s u p p l y s u f f i c i e n t p r e c u r s o r FA f o r maximal a c t i v i t y o f the t e r m i n a l e p o x i d a s e and m e t h y l - t r a n s f e r a s e . T h i s can be d e m o n s t r a t e d by the r a p i d i n v i t r o e f f e c t o f a l l a t o t r o p i n on de novo s y n t h e s i s o f JH from acetate (31). Reserpine treatment retards the development of allatal competence o f l o c u s t females f o r s e v e r a l weeks, b u t b a s a l rates appear to be normal (32). T h i s i n d i c a t e s t h a t development o f the two key enzyme systems a r e not dependent one on the o t h e r . R e s e r p i n e a c t s i n v e r t e b r a t e systems by d e p l e t i n g monoamine t r a n s m i t t e r s u b s t a n c e s i n the c e n t r a l nervous system (.33) and has been shown to a c t i v a t e peptidergic neurosecretory pathways in insects (34.35). The d i f f e r e n t i a l r e s p o n s e to r e s e r p i n e i n the l o c u s t CA s u g g e s t s t h a t p a r t o f the p r o c e s s o f d e v e l o p m e n t a l m a t u r a t i o n may be dependent on neurosecretion. O t h e r t h a n the d e s c r i p t i o n o f n e u r a l and humoral c o n n e c t i o n s and i n t e r a c t i o n s , which has been d e t a i l e d above, l i t t l e i s known o f the mode o f a c t i o n o f l o n g - t e r m m o d u l a t i n g f a c t o r s on b a s a l a c t i v i t y o f the CA. B a s a l a c t i v i t y i s m a r g i n a l i n mature n o n v i t e l l o g e n i c o l d e r females and no c o r r e l a t i o n can be found i n such c a s e s between b a s a l and F A - i n d u c e d a c t i v i t y . The l a t t e r i s o f t e n h i g h , i n d i c a t i n g t h a t competence i s r e t a i n e d ( i . e . , t h a t the n o n - r a t e l i m i t i n g terminal enzymes a r e abundant and a c t i v e ) . We i n t e r p r e t such l o n g - t e r m c o n t r o l o f the b a s a l a c t i v i t y o f the g l a n d s i n terms o f an equilibrium between a c t i v e and i n a c t i v e forms o f the r a t e - l i m i t i n g enzyme (or enzymes). We r e g a r d t h i s e q u i l i b r i u m as s u b j e c t to g r a d u a l change d u r i n g the c y c l e s o f p r o d u c t i v i t y . Short-term modulation is superimposed by allatotropic and a l l a t o s t a t i c f a c t o r s on the b a s a l , l o n g - t e r m modulated JH s y n t h e s i s . The basal activity of l o c u s t CA i s u s u a l l y below the maximal potential for JH synthesis and is amenable to allatotropic s t i m u l a t i o n . The s t r u c t u r e o f l o c u s t a l l a t o t r o p i n has not y e t been determined, but i t i s known t o be a p e p t i d e , presumably o f low molecular weight. Various observations indirectly support the p r o b a b i l i t y t h a t i t i s h u m o r a l l y t r a n s m i t t e d . CA e x c i s e d from mature v i r g i n female l o c u s t s and from s i b l i n g mated females ( T a b l e 1) do not d i f f e r i n t h e i r b a s a l a c t i v i t y and r e s p o n s i v e n e s s t o a l l a t o t r o p i n , but o o c y t e development i s r e t a r d e d i n the v i r g i n females (.36) . T h i s i m p l i e s t h a t a l t h o u g h m a t u r a t i o n and l o n g - t e r m a c t i v a t i o n o f the CA


15.

APPLEBAUM ET AL.

159



a r e u n a f f e c t e d by mating, a l l a t o t r o p i n s t i m u l a t e s JH s y n t h e s i s by the CA i n vivo, hastening the maturation of oocytes. Allatotropic s t i m u l a t i o n has been shown t o decay r a p i d l y , so t h a t when the CA a r e e x c i s e d and i n c u b a t e d i n i t s absence, they r e v e r t t o b a s a l r a t e s o f JH p r o d u c t i o n , w h i c h do n o t a c c u r a t e l y r e p r e s e n t the i n v i v o s t a t e o f a c t i v i t y . The v a r i a t i o n i n b a s a l a c t i v i t y o f i n d i v i d u a l g l a n d s has been a t t r i b u t e d to random changes i n the p h y s i o l o g i c a l l e v e l of a c t i v a t i o n o f the g l a n d s , due to a p u l s a t i l e o n / o f f mechanism (18), b u t h e r e too i t i s more p r o b a b l e t h a t t h e s e d i f f e r e n c e s r e f l e c t the i n h e r e n t v a r i a b i l i t y i n the m a t u r i t y o f the g l a n d s .

Table

1.

E f f e c t o f M a t i n g on Oocyte S i z e and JH

Treatment

A B C

Oocyte S i z e (mm)

5.8±0.4 1.7±1.2 1.1±0.7

Synthesis

In V i t r o

B a s a l JH S y n t h e s i s Allatotropin-stimulated (pmole/CA.hr) JH s y n t h e s i s (pmole/CA.hr)

29.8±10.8 20.0± 8.1 40.1±16.8

75.6±24.0 65.8±20.6 81.6132.5

V a l u e s a r e the mean o f 5 r e p l i c a t e s 1 SE. Females and males caged t o g e t h e r ; 20 days a f t e r a d u l t emergence. Females and males s e p a r a t e d by s c r e e n mesh; 20 days a f t e r a d u l t emergence. C= Females i s o l a t e d from males i n s e p a r a t e c u l t u r e room; 20 days a f t e r a d u l t emergence.

A= B=

C e r e b r a l l e v e l s of a l l a t o t r o p i n are h i g h a f t e r maturation and hemolymph l e v e l s a r e presumably s u b j e c t t o c o n t r o l by a r e l e a s i n g f a c t o r . N e u r o p a r s i n s A and B are c e r e b r a l p e p t i d e s p r o d u c e d i n the MNC (37) which a r e t r a n s p o r t e d to the CC, from where t h e y have been isolated (38) and t h e i r sequences d e t e r m i n e d ( 3 9 ) . Semi-isolated n e u r o p a r s i n A r e t a r d e d o o c y t e growth when i n j e c t e d and a n t i s e r u m to neroparsin A induced green pigmentation and precocious sexual m a t u r a t i o n , b u t n e i t h e r had any e f f e c t on i n v i t r o (40) o r i n v i v o JH levels ( G i r a r d i e , unpublished data, c i t e d i n 40). I t i s t h e r e f o r e u n l i k e l y t h a t n e u r o p a r s i n A r e d u c e s hemolymph l e v e l s o f JH. I t may perhaps be i n v o l v e d i n i n h i b i t i n g the r e l e a s e o f a l l a t o t r o p i n i n a p p r o p r i a t e p h y s i o l o g i c a l s i t u a t i o n s . A l l the d a t a a t hand s u g g e s t t h a t the mature CA i n f a c t s y n t h e s i z e JH a t a b a s a l r a t e w h i c h i s o n l y a p a r t o f t h e i r p o t e n t i a l , and t h a t t h e y a r e t h e n a c t i v a t e d by allatotropin selectively. The d i r e c t e f f e c t o f an a l l a t o s t a t i c o v a r y - d e r i v e d f a c t o r was f i r s t r e p o r t e d i n a survey of v a r i o u s t i s s u e e x t r a c t s o f v i t e l l o g e n i c l o c u s t females on JH s y n t h e s i s i n v i t r o ( 1 6 ) . We have r e c e n t l y found



160


(17, H i r s c h , J . ; Applebaum, S.W., u n p u b l i s h e d ) t h a t t h i s a l l a t o s t a t i c factor i s degraded by t r y p s i n and p r o t e i n a s e K, b u t n o t by chymotrypsin. Ovarian a l l a t o s t a t i n i s absent i n the p r e v i t e l l o g e n i c ovary. I t s content i s h i g h e s t i n t h e o v a r y o f a female b e a r i n g o o c y t e s o f 3 mm l e n g t h and l a t e r d e c r e a s e s . Thereafter, ovarian a l l a t o s t a t i n accumulates i n t h e hemolymph. I n v i t r o . i t o v e r r i d e s a l l a t o t r o p i c s t i m u l a t i o n , and we s u g g e s t t h a t t h i s may a l s o be t h e s i t u a t i o n i n vivo with ovarian-derived a l l a t o s t a t i n p a r t i c i p a t i n g i n the n e g a t i v e c o n t r o l o f CA a c t i v i t y a t t h e end o f t h e g o n a d o t r o p h i c cycle (Figure 3). As was p r e v i o u s l y s t a t e d , t h e amount o f JH p r o d u c e d by mature CA i s dependent on t h e a c t i v a t i o n s t a t e o f t h e p r e s u m p t i v e r a t e - l i m i t i n g enzymes, b u t n o t h i n g i s known o f how a l l a t o t r o p i n a c t i v a t e s t h e s e enzymes o r how a l l a t o s t a t i n i n a c t i v a t e s them. E x p e r i m e n t s u t i l i z i n g p r e c u r s o r s were d e s i g n e d i n o r d e r t o i d e n t i f y t h e r e g u l a t o r y enzymes r e s p o n s i b l e f o r r a t e - l i m i t a t i o n o f JH s y n t h e s i s . FA, t h e p e n u l t i m a t e p r e c u r s o r o f JH, e n t e r s a f t e r t h e r a t e - l i m i t i n g s t a g e and a c t i v a t e s competent CA o f v i t e l l o g e n i c f e m a l e s . We have r e c e n t l y shown t h a t mevalonate (17), one o f t h e f i r s t p r o d u c t s w i t h i n t h e b i o s y n t h e t i c pathway, a l s o a c t i v a t e s mature CA and by d e f i n i t i o n i s t h e r e f o r e s i t u a t e d a f t e r the r a t e - l i m i t i n g stage. Comparative d o s e - r e s p o n s e c u r v e s w i t h t h e s e two p r e c u r s o r s show t h a t t h e more removed t h e exogenous precursor i s from JH, t h e h i g h e r the concentrations r e q u i r e d f o r s t i m u l a t i o n : FA i s a c t i v e i n t h e m i c r o m o l a r range, w h i l s t mevalonate a c t s i n t h e m i l l i m o l a r range. M e v a l o n a t e i s p r o d u c e d by t h e a c t i o n o f 3-hydroxy-3-methylglutaryl (HMG) coenzyme A r e d u c t a s e . Two s p e c i f i c inhibitors of HMG-CoA r e d u c t a s e - mevinolin and h y d r o x y m e t h y l g l u t a r a t e - were therefore included i n factorial i n vitro e x p e r i m e n t s , w i t h and w i t h o u t mevalonate and/or a l l a t o t r o p i n . H y d r o y x m e t h y l g l u t a r a t e i s a s t r u c t u r a l a n a l o g o f HMG-CoA r e d u c t a s e , as i s t h e f u n g a l m e t a b o l i t e m e v i n o l i n . Both a r e c o m p e t i t i v e i n h i b i t o r s o f t h e v e r t e b r a t e enzyme, and reduce cholesterol biosynthesis ( 4 1 ) • We found that c o n c e n t r a t i o n s o f 10 M m e v i n o l i n and lOOmM HMG d e p r e s s e d t h e b a s a l l e v e l o f JH s y n t h e s i s s i g n i f i c a n t l y , and a l l a t o t r o p i n was u n a b l e i n b o t h c a s e s t o c i r c u m v e n t t h i s i n h i b i t i o n . I n c o n t r a s t , mevalonate i n c r e a s e d t h e JH s y n t h e s i s t o l e v e l s s i g n i f i c a n t l y h i g h e r t h a n t h e basal levels, approaching those elicited by a l l a t o t r o p i n (Abd E l - H a d i , F. ; Applebaum, S.W., u n p u b l i s h e d d a t a ) ( F i g u r e 4 ) . These r e s u l t s s t r o n g l y s u g g e s t t h a t mevalonate s y n t h e s i s i s r a t e - l i m i t i n g JH s y n t h e s i s . We a r e s t i l l l e f t with the n e c e s s i t y o f d i r e c t l y identifying the r e g u l a t o r y enzymes and how t h e i r activity i s c o n t r o l l e d a t t h e m o l e c u l a r l e v e l . HMG-CoA r e d u c t a s e i s an o b v i o u s c a n d i d a t e f o r t h i s r o l e . I n v e r t e b r a t e s , i t ' s a c t i v i t y i s modulated by r e v e r s i b l e p h o s p h o r y l a t i o n ( 4 2 ) . However, i n e x p e r i m e n t s c a r r i e d out i n v i t r o with CA o f t h e c o c k r o a c h D i p l o p t e r a p u n c t a t a , no c o r r e l a t i o n was e v i d e n t between a c t i v i t y o f t h e enzyme and JH s y n t h e s i s (43). The l o c u s t CA seems t o be a p r e f e r r e d system f o r e x a m i n i n g a l l a t o t r o p i c r e g u l a t i o n o f JH s y n t h e s i s , b u t pure s y n t h e t i c allatotropin i s essential for definitively e s t a b l i s h i n g the presumptive r o l e o f reductase i n the r e g u l a t i o n o f a l l a t a l a c t i v i t y . E f f o r t s t o p u r i f y and sequence a l l a t o t r o p i n a r e i n p r o g r e s s .



15.

APPLEBAUM ETAL.


161

oocyte length ( m m ) F i g u r e 3. I n h i b i t i o n o f JH s y n t h e s i s i n v i t r o by a l l a t o t r o p i n s t i m u l a t e d CA o f v i t e l l o g e n i c l o c u s t s . § • = ovarian extract; 0 — 0 = hemolymph e x t r a c t . V a l u e s a r e t h e mean o f 7-12 r e p l i c a t e s .

80

60

40 -C

< I

20

1

-20

6

8

F i g u r e 4. D i f f e r e n t i a l JH s y n t h e s i s i n v i t r o (stimulated/inhibited minus b a s a l rates) i n the presence o f m e v i n o l i n o r HMG ± allatotropin o r mevalonate. 1 = control (no a d d i t i o n s ) ; 2 = a l l a t o t r o p i n ; 3 = mevanolate; 4 = m e v i n o l i n ; 5 = HMG; 6 = HMG + a l l a t o t r o p i n ; 7 - m e v i n o l i n + a l l a t o t r o p i n ; 8 = HMG + mevalonate; 9 - m e v i n o l i n + mevalonate. V a l u e s a r e t h e mean ± SE o f 10-14 replicates.


162


Acknowledgments


The work cited from the authors laboratory was supported in part by the S.A. Schonbrunn Research Endowment Fund and by grant no. 87-5 from the German-Israeli Agricultural Research Agreement. J. Hirsch is the recipient of a Mauerberger Foundation Fund Fellowship. Literature Cited 1. Tobe, S.S.; Stay, B. Adv. Insect Physiol. 1985, 18, 305-432. 2. Feyereisen, R. In Comprehensive Insect Physiology, Biochemistry and Pharmaco1ogy; Kerkut, G.A.; Gilbert, L.I., Eds.; Pergamon: Oxford, 1985; Vol. 7, pp. 391-429. 3. Granger, N.A.; Goodman, W.G. Insect Biochem. 1983, 13, 333-340. 4. Mauchamp, B.; Couillaud, F.; Malosse, C. Anal. Biochem. 1985, 145, 251-256. 5. Rembold, H.; Lackner, B. J. Chromatog. 1985, 323, 355-361. 6. Dale, J.F.; Tobe, S.S. J. Insect Physiol. 1986, 32, 763-769. 7. Tobe, S.S.; Pratt, G.E. Biochem. J. 1974, 144, 107-113. 8. Tobe, S.S.; Pratt, G.E. In The Juvenile Hormones; Gilbert, L.I., Ed.; Plenum: New York, 1976; pp 147-163. 9. Couillaud, F.; Mauchamp, B.; Girardie, A.; De Kort, S. Arch. Insect Biochem. Physiol. 1988, 7, 133-143. 10. Johnson, R.A.; Hill, L. J. Insect Physiol. 1975, 21, 1517-1519. 11. Ferenz, H.-J.; Kaufner, I. In Juvenile Hormone Biochemistry; Pratt, G.E.; Brooke, G.T., Eds. Elsevier/North Holland: Amsterdam. 1981; pp 135-145. 12. Tobe, S.S.; Stay, B. Gen. Comp. Endocrinol. 1977, 31, 138-147. 13. Gadot, M.; Applebaum, S.W. Mol. Cell. Endocrinol. 1986, 48, 69-76. 14. Gadot, M.; Applebaum, S.W. Arch. Insect Biochem. Physiol. 1985, 2, 117-129. 15. Ferenz, H.-J.; Diehl, I. Z. Naturforsch. 1983, 38c, 856-858. 16. Gadot, M.; Rafaeli, A.; Applebaum, S.W. Arch. Insect Biochem. Physiol. 1987c 4, 213-223. 17. Applebaum, S.W.; Hirsch, J . ; Abd El-Hadi, F.; Moshitzky, P. In: Progress in Comparative Endocrinology; Epple, A; Scanes, C.G.; Stetson, M.H. Eds. Wiley-Liss: New York. 1990; pp 186-192. 18. Pratt, G.E.; Pener, M.P. J. Insect Physiol. 1983, 29, 33-39. 19. Tobe, S.S.; Girardie, J . ; Girardie, A. J. Insect Physiol. 1982, 28, 867-872. 20. Couillaud, F.; Girardie, A. Int. J. Invertebr. Reprod. Develop. 1985, 8, 303-315. 21. Rademakers, L.H.P.M. Cell Tissue Res. 1977, 184, 381-395. 22. Poras, M. C. R. Acad. Sc. Paris 1983, 297, 483-488. 23. Couillaud, F.; Mauchamp, B.; Girardie, A. Experientia 1985, 41, 1165-1167. 24. Couillaud, F.; Mauchamp, B.; Girardie, A. J. Insect Physiol. 1987, 33, 223-228. 25. Tobe, S.S. Can. J. Zool. 1977, 55, 1509-1514. 26. Tobe, S.S.; Chapman, C.S.; Pratt, G.E. Nature (London) 1977, 286, 728-730. 27. Baehr, J.C.; Caruelle, J.P.; Poras, M. Int. J. Invertebr. Reprod. Develop. 1986, 10, 143-150. 28. Pener, P.M. Entomol. Exp. Appl. 1968, 11, 94-100.



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29. Applebaum, S.W.; Gadot, M. In Insect Neurochemistry and Neurophysiology; Borkovec, A.B.; Gelman D.B., Eds.; Humana: Clifton, 1986; pp 281-284. 30. Tobe, S.S.; Pratt, G.E. Life Sciences 1975, 17, 417-422. 31. Gadot, M.; Goldman, A.; Cojocaru, M.; Applebaum, S.W. Mol. Cell. Endocrinol. 1987b, 49, 99-107. 32. Gadot, M.; Factor, O.; Applebaum, S.W. Arch. Insect Biochem. Physiol. 1987a, 4, 17-28. 33. Stitzel, R.E. Pharmacol Rev. 1977, 28, 179-205. 34. Evans, P.D. Adv. Insect Physiol. 1980, 15, 317-473. 35. Orchard, I. In Insect Neurochemistry and Neurophysiology: Borkovec, A.B.; Kelly, T.J., Eds.; Plenum: New York, 1984; pp 115-134. 36. Engelmann, F. The Physiology of Insect Reproduction; Pergamon: Oxford, 1970. 37. Girardie, J . ; Boureme, D.; Girardie, A. Insect Biochem. 1987a, 17, 29-36. 38. Girardie, J . ; Faddoul, A.; Girardie, A. Insect Biochem. 1985, 15, 85-92. 39. Girardie, J . ; Girardie, A.; Huet, J.-C.; Pernollet, J.-C. FEBS Letters 1989, 245, 4-8. 40. Girardie, J . ; Boureme, D.; Couillaud F.; Tamarelle, M.; Girardie, A. Insect Biochem. 1987b, 17, 977-983. 41. Fears, R. In 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase; Sabine, J.R., Ed.; CRC: Boca Raton, 1983; pp 189-208. 42. Kennelly, P.J.; Rodwell, V.W. J. Lipid Res. 1985, 26, 903- 914. 43. Feyereisen, R.; Farnsworth, D.E. Mol. Cell. Endocrinol. 1987 53, 227-238. RECEIVED

October 10, 1990


Allatal Stimulation and Inhibition in Locusts - ACS Symposium Series

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