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Bombyx mori development: the context for its endocrine system
BIOCHIMIE, 1~79, 61, 147-162.
Bombyx mori development : the context for its endocrine system. Madeleine COULON.
Laboratoire de Biomdtrie. Universitd Claude Bernard Lyon I, 43, Boulevard du 11 Novembre 1918, 69621 Villeurbanne, France.
R~sum~.
Summary.
Chez les i n s e c t e s trois t y p e s principaux d'horm o n e s sont 6tudi6s : n e u r o h o r m o n e s (brain-hormone), h o r m o n e s st6roides, h o r m o n e s juv6niles. Un bilan quantitatif pour c h a c u n de c e s q r o u p e s d ' h o r m o n e s est dress6 c h e z Bombyx mori, tout a u l o n g du d 6 v e l o p p e m e n t : o v o c y t e , ceuf, larve, chrysalide.
Quantitative data on n e u r o h o r m o n e s (brainh o r m o n e s ) steroid h o r m o n e s , juvenile h o r m o n e s , in a n insect : Bombyx mot/ are outllined. The variations of h o r m o n a l contents throuqhout larv a l a n d p u p a l d e v e l o p m e n t , in eqqs a n d ovoc y t e s are a n a l y z e d . Key words: Brain-hormones, Eedysteroids, Juvenile hormones, Egg, Larvae, Pupae, Bombyx mort.
En~docrine r e g u l a t i o n of the i n v e r t e b r a t e s develo p m e n t has been . p r i m a r i l y d e m o n s l r a t e d in insects. H o w e v e r p r o g r e s s in t h i s field have y i e l d to a compl.ex s c h e m a of regula¢ion. SeweraJ h o r mones o c c u r d u r i n g larval, p u p a l , i m a g i n a l develo p m e n t and d u r i n g embryogenesis. T h r e e t y p e s of h o r m o n e s are des.cribed and s t u d i e d generally. Tlle n e u r o h o r m o n e s s y n t h e s i zed in different cells of the nervous system w h i c h t r a n s i t in n e u r o h e m a l organ ( c o r p o r a c a r d i a c a for i:nstance). The s t e r o i d h o r m o n e s : ecdysteroi,ds p r o d u c e d in different tissues ( p r o t h o r a c i c gland, f o l l i c u l a r cells for instance). A n d l h e juvenile h o r mones s y n t h e s i z e d b y l h e corpora-all'ata. The target tissues can be defined an'd these h o r m o n e s regulate m a n y p a r t i c u l a r ph.ases of d e v e l o p m e n t . E ~ d o c r i n e c o n t r o l of molt a n d m e t a m o r p h o s i s have been d e m o n s t r a t e d in Bombyx mort by ligations a n d s u r g i c a l a b l a t i o n s [1, 2] as w e l l as en'doc r i n e r e g u l a t i o n of .determinism of alia,pause [3]. The a i m of this p a p e r is to b r i n g t o g e t h e r re.cent data on B o m b y x mort h o r m o n a l situation p a r t i c u l a r l y in focusing on q u a n t i t a t i v e d e t e r m i n,ation of the d i f f e r e n t k n o w n h o r m o n e s . Most of the d a l a c o n c e r n l a r v a l a n d p u p a l stages a n d also eggs development, the i n t e r v e n t i o n of h o r m o n e s a f t e r e m e r g e n c e is still a m a t t e r of question in B o m b y x mort. 1. THE NEUROHORMONES.
In insects different types of n e u r o s e c r e t o r y cells have been d e s c r i b e d in b r a i n and nervous
ganglions. In Bombyx n e u r o s e c r e t o r y cells of the p a r s - i n t e r c e r e b r a l i s and n e u r o s e c r e t o r y cells of suboesophagea)l ga,nglion are the most w e l l k n o w n . A n e u r o h o r m o n e s y n t h e s i z e d by n e u r o s e e r e t o r y cells of the suboesophageal ganglion is i m p l i c a t e d in t h e d e t e r m i n a t i o n of e m b r y o n a r y d i a p a u s e (in the female). N e u r o s e c r e t o r y cells of the p a r s intercerebralis synthesized brainhormones (B.H.). F r o m Bombyx b r a i n s extracts, three active fractions of high molecu, lar w e i g h t can be s e p a r a l e d [4]. P u r i f i c a t i o n of p o s s i b l e different c o m p o n e n t s w i t h c h a r a c t e r i s t i c s of g l y c o p r o t e i n has been rep o r t [5]. Starting w i t h b r a i n of Anthereae pernyi an active f r a c t i o n was o b t a i n e d t h a t m a y be mucop o l y s a c c h m ' i d e [6]. It a p p e a r s that p r o t h o r a c o t r o p i c h o r m o n e (PTTH, n e u r o h o r m o n e s t i m u l a t i n g the p r o t h o r a c i c gland) is p r o t e i n a c e o u s but that at Jeasl two p o l y p e p t i d e s m a y have s i m i l a r activity. Bioassay for b r M n - h o r m o n e s necessitates a large n u m b e r of w h o l e b r a i n s [7]. The p r e s e n c e of o t h e r b r a i n - h o r m o n e s as w e l l as the difficulty to obtain a great a m o u n t of s y n c h r o n i z e d b r a i n mat e r i a l m a k e s the vMidity of the b i o a s s a y for P T T H questionable. F u r t h e r m o r e this b i o a s s a y m e a s u r e s the a m o u n t of h o r m o n e s p r e s e n t in the b r a i n , w h i c h does not p r e s u m e of its c o n t e n l ,at the site of action. 1.1. Larval development. As m a n y insects several types of n e u r o s e c r e t o r y cells are d e s c r i b e d in B o m b y x mort p a r s - i n t e r e e -
148
M. Coulon.
r e b r a l i s . A cells [8] or A' cells [9] can be c o m p a r e d to A cells of Locusta [10] p r o d u c i n g p r o t h o r a c o t r o p i c h o r m o n e . The c h a n g e s of n e u r o h o r m o n e content in these cells d u r i n g l a r v a l d e v e l o p m e n t have b e e n e s t i m a t e d b y n e u r o s e c r e t o r y cell activity ; because of the s i m i l a r i t y of Bombyx and Locusta b r a i n cells ,it is s u p p o s e d that the most of the o b s e r v e d v a r i a t i o n s .are d u e to fluctuations of P T T H s y n t h e s i s a n d or secretion. V a r i a t i o n s of n u m b e r of n e u r o s e c r e t o r y granules c o n t a i n e d in A' ceils are d e s c r i b e d (electron m i c r o s c o p i c cytob i o m e t r y ) . A c t i v i t y c y c l e of these ceils d u r i n g w h o l e larxral d e v e l o p m e n t is d e s c r i b e d (CoubleFoussal, Coulon in p r e p a r a t i o n ) . Briefly : the numb e r of n e u r o s e c r e t o r y granules p e r surface unit is m a x i m u m at each l a r v a l e c d y s i s w h e r e after it decreases : a s u d d e n release of n e u r o s e c r e t o r y produets is o b s e r v e d in c o i n c i d e n c e w i t h the first food intake. S y n t h e s i s a,ctivity of the cells is i n d i cated b y an i n c r e a s e of granules n u m b e r ; ,later this n u m b e r is stable d a r i n g m i d d l e stage suggesting a b a l a n c e b e t w e e n s y n t h e s i s a c t i v i t y and release of p r o d u c t s . Before ,larval e c d y s i s neurosec r e t o r y granules are seen a c c u m u l a t e d in the cells. D u r i n g l a r v a l d e v e l o p m e n t this A' cells neuros e c r e t o r y a c t i v i t y i n Bombyx b r a i n can be d i s t u r b e d b y agents s u c h as fasting [9] and X r a y s [Coul~le-Foussal, Coulon in p r e p r a t i o n ] . In this exper i m e n t a l situation a r e t e n t i o n of n e u r o s e c r e t o r y p r o d u c t s is o b s e r v e d in the cells. In Bombyx l a r v a e two n e u r o s e c r e t o r y cells have been o b s e r v e d in the frontal ganglion [11] ; h o w e ver n o t h i n g is k n o w n about ,developmental of t h e i r activity. I n .an other L e p i d o p t e r a Manduca sexta, n e u r o s e c r e t i o n is a c c u m u l a t e d in these two cells dur,ing fasting, the r e l e a s e d at t i m e of food i n t a k e [12]. 1.2. Pupal developmenl. In L e p i d o p t e r a w i t h p u p a l d i a p a u s e (Hyalopthora, Philosamia, Anthereae for instance) the b r a i n is r e q u i r e d for f u r t h e r development. Brainless p u p a e of Bombgx b e c o m e ¢ d a u e r ~ p u p a e ( p e r m a n e n t p u p a e ) [13] the d e v e l o p m e n t of these p u p a e r e s u m e after i m p l a n t a t i o n of brain. N e u r o h o r m o n e s c o n t e n t in h e a d a n d t h o r a x a b d o m e n of Bombgx pupae ( b i o a s s a y : S a m i a test) is a l w a y s greater in female t h a n in male [7]. The b r a i n n e u r o s e c r e t o r y cells ( e x c e p t suhoesophageal ganglion) release t h e i r s e c r e t i o n s as soon as the l a r v a l - p u p a l moult occurs. D u r i n g p u p a l d e v e l o p m e n t b r a i n h o r m o n e s c o n t e n t alveays h i g h e r in e a r l y p u p a e . In female o v a r i e c t o m i z e d d u r i n g the 4th i n s t a r t h e BH level in p u p a e is g r e a t e r t h a n in n o r m a l female (male c a s t r a t i o n has no effect) ;
BIOCHIMIE, 1979, 61, n" 2.
this result w o u l d i n d i c a t e that a p a r t of BH c o u l d be used b y o v a r i e s for m a t u r a t i o n a n d / o r vitellogenesis in oocytes [7]. 2.
THE
ECDYSTEROIDS
: STEROIDS
HORMONES.
The first i n s e c t h o r m o n e w a s iso'lated a n d crystallized from Bombyx mort p u p a e a n d c a l l e d eed y s o n e [14]. A s e c o n d bi(~logical active s u b s t a n c e was s e p a r e d and o b t a i n e d p u r e in still s m a l l e r amount f r o m Bombyx p u p a e : e c d y s t e r o n e [15]. T h r o u g h extensive c h e m i c a l studies f o l l o w e d b y X r a y c r y s t a l l o g r a p h i c analysis, the s t r u c t u r e w a s e s t a b l i s h e d [16], a n d c o n f i r m e d b y synthesis. Eed y s o n e is a C~2T sterol of e x c e p t i o n a l l y p o l a r char a c t e r . E c d y s t e r o n e is r e c o g n i z e d as 2 0 - h y d r o x y ecdysone. It is a d m i t t e d that p r o t h o r a c i c g l a n d s y n t h e s,izes e e d y s o n e in Bombyx mori [17] :and Mandaca [18]. In in vitro culture of Bombyx p r o t h o r a c i c gland, the m a x i m u m release a c t i v i t y takes p l a c e at the end of the 5 th i n s t a r [19]. In p r o t h o r a c i c gland of this insect a p o s s i b l e i n t e r m e d i a t e between c h o l e s t e r o l and e c d y s o n e is d e t e c t e d (3~h y d r o x y 5a cholestan-6-one) [20]. Efficient conversion of e c d y s o n e to e c d y s t e r o n e has o b t a i n e d in ligated a b d o m e n of Bombyx [213. It has been t e n t a t i v e l y suggested that oenocytes m a y be the site of e c d y s o n e to e c d y s t e r o n e c o n v e r s i o n [191. F i r s t e c d y s o n e assay on Bombyx l a r v a e has been p e r f o r m e d w i t h the b i o a s s a y (Ca l l i p h o r a test) [221. The v a r i a t i o n of the s t e r o i d h o r m o n e content has been m e a s u r e d b y r a d i o i m m u n o a s s a y (RIA) t h r o u g h o u t Bombyx mori d e v e l o p m e n t from h a t c h i n g to e m e r g e n c e [23, 24, 25, 261. 2.1. Larval development. I m p o r t a n t changes of h a e m o l y m p h e c d y s o n e t i t e r are o b s e r v e d along the l a r v a l d e v e l o p m e n t . D u r i n g each l a r v a l i n s t a r feeding p e r i o d a low level of e c d y s o n e is o b s e r v e d ; a b r i e f p e a k o c c u r s a s h o r t time before the b e g i n n i n g of m o u l t i n g w h e n the l a r v a e stops f e e d i n g and then e e d y s o n e falls before ecdysis. The e c d y s o n e level i n c r e a s e s again soon after the s i l k w o r m start s p i n n i g t h e r e cocoon at the end of the 5 th instar. Then it r e a c h a p e a k s t r e c h i n g on about 2-3 days. I n other Lepid o p t e r a as Philosamia cynthia [253 a n d Manduca sexta [27] h o r m o n a l .peak is o b s e r v e d at t h e same t i m e of the 5 th instar. In Bombyx mort the s t e r o i d s content v a r i a t i o n has been p a r t i c u l a r l y s t u d i e d at h a t c h i n g and first food intake. D u r i n g last d e v e l o p m e n t , e m b r y o eats yolk and serosa (which contained ecdysteroids). E c d y s t e r o i d s level in l a r v a e is high at h a t c h i n g
B o m b y x endocrinology. [23] ; d u r i n g the first h o u r s of feeding, this level falls ( e x t r a c t i o n on l a r v a e in toto, the RIA is effected after thin l a y e r c h r o m a t o g r a p h y : TLC). This d a t a can be r e l a t e d w i t h e l i m i n a t i o n of egg ecdystero'ids (essentialy of ~ma¢ernal origin) a n d w i t h the outset of the p r o g r a m of l a r v a l d e v e l o p m e n t [28, and Coulon in p r e p a r a t i o n ] . By RIA, e c d y s o n e a n d e c d y s t e r o n e are t i t r a t e d in Bombyx h a e m o l y m p h . It is v e r y i m p o r t a n t to k n o w the c o n v e r s i o n r h y t h m of e c d y s o n e to ecdysterone an.d t h e i r m e t a b o l i s m . After i n j e c t i o n of [3H] e c d y s o n e to Bombyx 5 th i n s t a r larvae, the most e c d y s o n e p a r t (50 p e r cent) is c o n v e r t e d into e c d y s t e r o n e in 15 m n [21] (TLC a n d l i q u i d c h r o m a t o g r a p h y ) this c o n v e r s i o n is m a x i m u m at spinning at the same time the c a t a b o l i s m of these two h o r m o n e s decreases simul¢aneous to the endogenous h o r m o n e s p e a k [25]. Different c o m p o n e n t s are p r e s e n t in w e a k quantity, p a r t i c u ' l i a r l y conjugates and glycosides w h i c h ave e l i m i n a t e d into gut. D u r i n g 2rd a n d 3 ~a i n s t a r s r a d i o c h r o m m a t o g r a p h i c m e t h o d after i n j e c t i o n of aH e c d y s o n e p e r m i t s the o b s e r v a t i o n of m a x i m u m c o n v e r s i o n of e c d y s o n e to ecdys¢erone d u r i n g t h e p e a k of endogenous h o r m o n e s for each i n s t a r ; the p e r c e n tage of p o l a r c o m p o n e n t s is m i n i m u m a~ the same time [24]. In larvae, X r a y s can d i s t u b e d the synthesis of s t e r o i d s h o r m o n e s a n d the .conversion of e c d y s o n e to e c d y s t e r o n e [23, 24]. 2.2. Pupal development. After
radioimmunoassay
on
haemolymph
of
Bombyx mori p u p a e a large p e a k of e c d y s o n e and e c d y s t e r o n e is d e t e c t e d [25] d u r i n g the first nine days of this stage ; the content is the s~me in m a l e a n d female. A c o m b i n a t i o n of TLC a n d RIA a n a l y s i s of the h a e m o l y m p h samples "was p e r f o r m e d on Bombyx pupae. It a p p e a r s t h a t no or v e r y little BIA activity p u p a e is f o u n d in the e c d y s t e r o n e f r a c l i o n [251. In Anthereae p u p a e the c o n v e r s i o n of e c d y sone to e c d y s t e r o n c is o b s e r v e d (after i n j e c t i o n ~of [3HI ecdysone). The m e t a b o l i s m of these h o r m 0 nes is the same as in l a r v a e [20]. After bioass,ay ( C a l l i p h o r a test) on in toto g r o u n d p u p a e , one p e a k is d e t e c t e d in male p u p a e w h e r e a s t w o p e a k s are o b s e r v e d in female p u p a e [30]. The s e c o n d p e a k is r e l a t e d to o v a r y a c t i v i t y since il d i s a p p e a r e d in ovariec,tomiz~d female. Mor e o v e r in ovaries imp'lanted in a male pupae, mature o v o c y l e s c o n t a i n the same q u a n t i t y of ecdys t e r o i d s t h a n in ovocytes d e v e l o p e d in female pup a e although t h e r e is no e e d y s o n e ,in h a e m o l y m p h at l h e same t i m e (Lamy, Coulon in p r e p a r a t i o n ) . BIOCHIMIE, 1979, 61, n ° 2.
149
2.3. The ecdystero'ids conten.t of {he ovocyte. In eggs a n d ovocytes RIA a n d b i o s s a y m e a s u r e ment of e c d y s o n e c o n t e n t i n c l u d e s p r e c u r s o r s a n d d e g r a d a t i o n p r o d u c t s . T h e r e f o r e w e refere as ecd y s t e r o i d s to designate .all RIA active m a t e r i a l [31, 3 2 ] . I n Locusta [31] Lagueux d e m o n s t r a t e d that foll i c u l a r .cells s y n t h e s i z e ecdystero'/ds. In Bombyx mort although these cells have an i m p o r t a n t met a b o l i s m activity, t h e r e is no evidence for them s y n t h e s i z i n g ecdystero~ds. E c d y s t e r o ' i d s s y n t h e s i zed in o v a r y are a c c u m u l a t e d in o v o c y t e s [33]. Total ecdystero'/ds level changes w i t h ovocy{e devel o p m e n t a n d it passes b y a m a x i m u m . 2.4. The ecdystero'/ds content of the egg and e m b r y o . The egg, closed system, is f o r m e d of two p a r t s : y o l k and e m b r y o . E m b r y o d e v e l o p m e n t occurs in the m i d d l e y o l k ; u n f e r t i l i z e d egg y o l k c o n t a i n s e c d y s t e r o i d s . E m b r y o g e n e s i s begins in the p r e s e n c e of e c d y s t e r o i d s . E n d o c r i n e organs differentiate but w e d o not k n o w at w i c h stage they are functional. F i r s t b i o a s s a y ( C a l l i p h o r a test) show the p r e sence of e c d y s t e r o i d s in Bombyx mort eggs [34], a p a r t of w h i c h is e c d y s o n e conjugates as it has been d e m o n s t r a t e d b y e n z y m a t i c t r a i t m e n t . These p r e v i o u s results h a v e been c o n f i r m e d b y c h r o m a t o g r a p h i c s t u d y (high p r e s s u r l i q u i d c h r o m a t o g r a p h y : HPLC) c o u p l e d w i t h b i o a s s a y s h o w i n g no e c d y s t e r o n e but e c d y s o n e a n d conjugates, [35]. Bo2ubyx eggs w o u l d c o n t a i n e d also 2-deoxy a ecd y s o n e [36]. D u r i n g the w h o l e d e v e l o p m e n t of Bombyx m o n o v o l t i n eggs, ecdystero~d level has been m e a s u r e d from l a y i n g to h a t c h i n g [37t (by r a d i o i m m u n o a s s a y ) . Very early at the developm e n t of these eggs e o d y s t e r o i d conten,t is h i g h ; durin,g the d i a p a u s e this level d e c r e a s e s w h e r e a s v a r i a t i o n s of few a m p l i t u d e are o b s e r v e d d u r i n g p o s t - d i a p a u s e . W e can say that the most p a r t of e e d y s t e r o i d s p r e s e n t .are l o c a l i z e d in the y o l k a n d serosa a n d r e m a i n i n g p a r t takes p l a c e in e m b r y o at leaM for late stages. In m o n o v o l t i n e m b r y o , increase of h o r m o n a l content is o b s e r v e d before emb r y o n a r y molt. A f t e r w a r d s y o l k and serosa are eat a n d w e observe a great i n c r e a s e of e m b r y o e c d y s t e r o i d s con.tent (Coulon). 3. T H E JUVENILE HORMONES ( J H ) .
J'uvenile h o r m o n e h a s been isolated from male imago a b d o m e n of s i l k m o t h Hyalophora cecropia [38, 39, 40]. T h r e e JtI w e r e purified, the s t r u c t u r e a n d stereo c h e m i s t r y of the first p u r e JH isolated is JH I [41, 42] : .methyl 12, 14-dihomojuvenate. The o t h e r c o m p o n e n t in ¢ c e c r o p i a oil >> is JH II 11
M. Coulon.
150
[43, 44] : methyl 12- homojuvenate, JH III is methyl juvenate : methyl 10, l l - e p o x y f a r n a s o a t e . They have .a t e r p e n o i d stru.cture. The c o n v e r s i o n from one type of JH to a n o t h e r c a n n o t occur. The c o r p o r a allata synthesize JH molecules. I n Bombyx mort c o r p o r a allata are two glands ~ i t h lympho'/d structure [45] w h i c h have a secretory cycle. JH molecules w e r e recovered from m e d i u m in w h i c h corpora allata from newly emerged male eeeropia had been cultivated ; but these molecules were n o l p r e s e n t in med,ium w i t h freshly excized corpo.ra allala. 3.1. The larval development. Very little i n f o r m a t i o n is available on JH meas u r m e n t because ,there w e r e no efficient quantitative t e c h n i q u e u n t i l these last years. I n Bombyx mort Morohoshi studies the f u n c t i o n s of b r a i n c o r p o r a allata system (by ablation a n d i m p l a n t techniques) but this author give rarely n u m b e r e d data therefore it is very ,difficult to take out conclusions. In the s i l k w o r m Hyalophora ceeropia two m e t h o d s [47, 48] agree i n s h o w i n g j u v e n i l e h o r m o n e s to be high early i n larval i n s t a r a n d fall off as the n ex¢ moR a p p r o a c h e d . I n Bombyx no a n y data on JH c o n t e n t v a r i a t i o n s are avalaihle. I n an other Lepidoptera Manduca sexta [49] three JH are extracted a n d assayed (.by chromatograp h i q u e t e c h n i q u e s : TLC, ,HP~C, gas l i q u i d chrom a t o g r a p h y : GLC) ; in early 4 th instar three hormones are present, J'H III in less quantity. At beg i n n i n g of 5 th i n s t a r the JH I is more i m p o r t a n t than JH II and JH III but t h e i r levels decrease quickly. These authors .point out that in s i l k w o r m Hyalophora in earlier i n s t a r JH I is the only type revealed. 3.2. The pupal development. In Bombyx mort no any q u a n t i t a t i v e ,data on JH content are avai'lable d u r i n g p u p a l development, I n Galleria pupae (bioassay : Galleria test) no juvenile h o r m o n e was .detected d u r i n g the greater part of the p u p a l stage but an increase of the JH level is observed two days before emergence [50] (in this species ovogenesis takes place i n imago). In m a n y insects ovocyfes development is controlled by j u v e n i l e h o r m o n e a n d b r a i n h o r m o n e specially in species in w h i c h ovogenesis takes place i n the imago. I n Bomb!Ix .and other Lepidoptera in w k i c h ovocyte d e v e l o p m e n t takes place i n pupae, JH appears to have no effect on synthesis of vitellogenin by fat-body. After removal o f c o r p o r a allata at larvo-pupal ecdysis, the ovocytes
BIOCHIMIE,
1979, 61, n ° 2.
n u m b e r is r e d u c e d but vitellogenesis is not suppressed [51]. 3.3. The juvenile hormone content in ovocyte and egg. Just before l a y i n g bioassay has detected juvenile h o r m o n e i n Hyalophora cecropia eggs [48] this JH is o r i g i n a l i n g from female silkmoth corpora allata. An increase of JH level i n eggs, by early i n j e c t i o n or topical a p p l i c a t i o n leads to abn o r m a l embryogenesis [52]. I n Bombyx egg no data are available on JH level.
Conclusion. Diagrams of two e n d o c r i n e regulation in insects are ,drawn figure 1.
A
diuretic h.... £
[ V
Target tissues
.........
i
(-Ve ,:._v
ovary
FIG. 1. - - I)iagrams of two endocrine regulations in insects. A : G e n e r a l e n d o c r i n e c o n t r o l . B : e n d o c r i n e c o n t r o l of ovoeyte development. [] Site of synthesis of an hormone. -+ hormone. -+ other substance. PTTH pTocothoraeotropic hormone. BH brain h o r m o n e . E ecdysone. 20-E ecdysterone : 20-hydroxyecdysone. JH juvenile hormone. NSC-PIC neuroseeretory cell's of the pars-intercerebralis. Proth.gl. prothoraeie gland. CA corpora a ll,ata. CC corpora cardiaca. FC Follicular ceils. FB Fat body.
Fat body is a target tissue for h o r m o n e s just like the e p i d e r m a l cells or i m a g i n a l disks [53] (fig. 1A). In Bombyx mori fat body is the site of i n t e n s e
151
Bombyx endocrinology. proteosynthesis: It is of the most interest to k n o w the r e l a t i o n s h i p b e t w e e n the v a r i a t i o n s of h o r m o nal level (ecdysone a n d YH) and the v a r i a t i o n s of p r o t e o s y n t h e s i s in fat body. The d e v e l o p m e n t of B o m b y x mort is discontinuous, d u r i n g l a r v a l stage each m o l t constitutes a p h y s i o l o g i c a l starvation. The c o m p a r i s o n in fat b o d y of p r o t e o s y n t h e s i s rec o v e r i e s after molt and after an e x p e r i m e n t a l starvation p e r i o d and t h e i r relation to e n d o c r i n e modifications in both systems could b r i n g to progress k n o w l e d g e in some problems. In B o m b y x d a u e r p u p a e (brainless p u p a e a r r e s t t h e i r d e v e l o p m e n t and ,present c o n s e q u e n t l y a p u p a l diapause) proteosynthesis in ovaries s p e c i a l l y and e n d o c r i n e context are m o d i f i e d [54] (fig. 1B). D u r i n g diapanse the m e t a b o l i s m is modified in a closed m e d i u m : the egg. V a r i a t i o n s of the enz y m a t i c p o o l are s p e c i a l l y detected, and w e h a v e o b s e r v e d v a r i a t i o n s of the steroids c o n t e n t in the egg d u r i n g this diapause. The silk gland synthesizes two p a r t i c u l a r proteins, it is possible that this gland w o u l d be a target tissue for h o r m o n e s a n d that a part of its metabolism w o u l d be c o n t r o l l e d by e n d o c r i n e system. We have t h e r e f o r e the p o s s i b i l i t y to study relationship b e t w e e n m a n y different p r o t e o s y n t h e s i s and e n d o c r i n e regulation in m a n y systems of B o m b y x mori. It ap,pears that B o m b y x mori can be a good syst e m to u n d e r s t a n d en,docrine regulation of the o v a r y a c t i v i t y (fig. 1B) because it is d e v e l o p e d in pupae w h i c h has no n u t r i t o n a l relation w i t h the e n v i r o n m e n t . T h e synthesis are effected f r o m the p r e c u r s o r s a c c u m u l a t e d d u r i n g l a r v a l life w h i c h can be k n o w n . The synthesis of v i t e l l o g e n i n in fat body appears i n d e p e n d a n t of J H in B o m b y x , but it s h o u l d be a d v a n t a g e o u s to u n d e r s t a n d the mec h a n i s m of t h e r e g u l a t i o n of vitel]ogenin up-take by ovary. We h a v e seen that p r o t e o s y n t h e s i s in o v a r y is not i n d e p e n d a n t of BH ; it is possible to modifie the pool of p r e c u r s o r s (if the larval devel o p m e n t is d i s t u r b e d ) and to study the regulation of ovocyte d i f f e r e n t i a t i o n in relation w i t h endoc r i n e system in this e x p e r i m e n t a l situation. In B o m b y x mori w e h a v e m a n y q u a n t i t a t i v e data on h o r m o n a l levels t h r o u g h o u t the different stages of its d e v e l o p m e n t . At b r i e f interval, it w i l l be possible to d e t e r m i n e by n e w assay (RIA) the v a r i a t i o n s of t h e J H c o n t e n t in h a e n m l y m p b , t h r o u g h o u t l a r v a l and pupal d e v e l o p m e n t . T h e r e fore the r e l a t i o n b e t w e e n e c d y s o n e s and j u v e n i l e h o r m o n e s w i l l be studied. B o m b y x mori appears
BIOCHIMIE, 1979, 61, n ° 2.
to be a good m o d e l to m a k e progress in k n o w ledge of e n d o c r i n e regulation of whcrle developm e n t of the insects. REFER'ENCY. 1. F u k u d a , S. (1944.) J. of the Fac. of Sc. Imp.. Univ., 6, 47'8-532.
2. Bounhiol, J. J. (1958) Th6se, Paris. 3. Fukuda, S. (1963) Bull. Sot. Zool., 2, 15.1-179. 4. Is.hizaki, H. ~ Ichik,awa, M. (19.67) Biol. Bull. Mar. Biol. Lab., Woods I~oIe, 133~ 355-368. 5. Yamazaki, M. ~ Kobayashi, K. (1969) J. Insect. Physiol., 15, 1981'-199~. 6. Williams, C. M. (10~7)In ¢ Insects and Physiology ~, Oliver and Boyd, E ~ n b u r g and London, 133-139.
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15. Karlson, P. (1956) Ann. 8el. Zool. Biol. Anita., 18, 125-137. 16. Huter, RI. a ttoppe, W. (1965) Chem. Bet., 98, 2403~424. 17. Chino, H. a Sakurai, S. (19'74) Science, 183, 529-530. 18. King, D. S., Bollenba,ch'er, W. E., Borst, D. W., Vedeckis, W. V., O Connor, J. D., I~tycheriach, P. I. a Gilbert, b. I. (1974) Proe. Nat. Acad. Set. USA, 71, 7~3-79~. 19. Ruhtand, U. & l~omer, F. (1977) Wilhelm Roux's Archives, 181, 123-134. 20. S.akural, S., I k~k,awa, N., Oh.taki, T., • Chino, H. (I977) Science, 198, 627~-629. 21. M,oriyama, H., N,akan, ishi, K., King, D. S., Okauchi, T., S'iddall, J. B. ,~ Kafferl, W. (1'970) Gem Comp. Endocrinol., 15, 80-87. 92. Shaaya, E. • Karlson, P. (1066) Develop. Biol., 11, 424-452. 23. Coulon, M. (1977) Dev. Growth and Differ, 19, 181185. 24. Coulon, M. • Feyereisen, R. (1977) C. R. Acad. Sci. Paris, 284, 1689-169~. 25. Calvez, B., Hirn, M~. ~ De P~ggi, (19 ) Febs Letters, 71, 57-61. 26. Coulon, M., Calvez, B., De Reggi, M., Legay. J. M. Hirn, M. (1978) Experientia, in press. 27. Bollenbaeher, W. E., V~deekis, W. V. • C~lbert, L. I. (1975) Develop. Biol., ~4, 4~6-53. 28. Coulon, M. (1977) Ann. Zool. Ecol. Anita., 9, 649-663. 29. Cherbas, L. ~ Cherbas, P. (19q0) Biol. Bull., 138, 115-1'28.
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Bombyx mori development : the context for its endocrine system. Madeleine COULON.
Laboratoire de Biomdtrie. Universitd Claude Bernard Lyon I, 43, Boulevard du 11 Novembre 1918, 69621 Villeurbanne, France.
R~sum~.
Summary.
Chez les i n s e c t e s trois t y p e s principaux d'horm o n e s sont 6tudi6s : n e u r o h o r m o n e s (brain-hormone), h o r m o n e s st6roides, h o r m o n e s juv6niles. Un bilan quantitatif pour c h a c u n de c e s q r o u p e s d ' h o r m o n e s est dress6 c h e z Bombyx mori, tout a u l o n g du d 6 v e l o p p e m e n t : o v o c y t e , ceuf, larve, chrysalide.
Quantitative data on n e u r o h o r m o n e s (brainh o r m o n e s ) steroid h o r m o n e s , juvenile h o r m o n e s , in a n insect : Bombyx mot/ are outllined. The variations of h o r m o n a l contents throuqhout larv a l a n d p u p a l d e v e l o p m e n t , in eqqs a n d ovoc y t e s are a n a l y z e d . Key words: Brain-hormones, Eedysteroids, Juvenile hormones, Egg, Larvae, Pupae, Bombyx mort.
En~docrine r e g u l a t i o n of the i n v e r t e b r a t e s develo p m e n t has been . p r i m a r i l y d e m o n s l r a t e d in insects. H o w e v e r p r o g r e s s in t h i s field have y i e l d to a compl.ex s c h e m a of regula¢ion. SeweraJ h o r mones o c c u r d u r i n g larval, p u p a l , i m a g i n a l develo p m e n t and d u r i n g embryogenesis. T h r e e t y p e s of h o r m o n e s are des.cribed and s t u d i e d generally. Tlle n e u r o h o r m o n e s s y n t h e s i zed in different cells of the nervous system w h i c h t r a n s i t in n e u r o h e m a l organ ( c o r p o r a c a r d i a c a for i:nstance). The s t e r o i d h o r m o n e s : ecdysteroi,ds p r o d u c e d in different tissues ( p r o t h o r a c i c gland, f o l l i c u l a r cells for instance). A n d l h e juvenile h o r mones s y n t h e s i z e d b y l h e corpora-all'ata. The target tissues can be defined an'd these h o r m o n e s regulate m a n y p a r t i c u l a r ph.ases of d e v e l o p m e n t . E ~ d o c r i n e c o n t r o l of molt a n d m e t a m o r p h o s i s have been d e m o n s t r a t e d in Bombyx mort by ligations a n d s u r g i c a l a b l a t i o n s [1, 2] as w e l l as en'doc r i n e r e g u l a t i o n of .determinism of alia,pause [3]. The a i m of this p a p e r is to b r i n g t o g e t h e r re.cent data on B o m b y x mort h o r m o n a l situation p a r t i c u l a r l y in focusing on q u a n t i t a t i v e d e t e r m i n,ation of the d i f f e r e n t k n o w n h o r m o n e s . Most of the d a l a c o n c e r n l a r v a l a n d p u p a l stages a n d also eggs development, the i n t e r v e n t i o n of h o r m o n e s a f t e r e m e r g e n c e is still a m a t t e r of question in B o m b y x mort. 1. THE NEUROHORMONES.
In insects different types of n e u r o s e c r e t o r y cells have been d e s c r i b e d in b r a i n and nervous
ganglions. In Bombyx n e u r o s e c r e t o r y cells of the p a r s - i n t e r c e r e b r a l i s and n e u r o s e c r e t o r y cells of suboesophagea)l ga,nglion are the most w e l l k n o w n . A n e u r o h o r m o n e s y n t h e s i z e d by n e u r o s e e r e t o r y cells of the suboesophageal ganglion is i m p l i c a t e d in t h e d e t e r m i n a t i o n of e m b r y o n a r y d i a p a u s e (in the female). N e u r o s e c r e t o r y cells of the p a r s intercerebralis synthesized brainhormones (B.H.). F r o m Bombyx b r a i n s extracts, three active fractions of high molecu, lar w e i g h t can be s e p a r a l e d [4]. P u r i f i c a t i o n of p o s s i b l e different c o m p o n e n t s w i t h c h a r a c t e r i s t i c s of g l y c o p r o t e i n has been rep o r t [5]. Starting w i t h b r a i n of Anthereae pernyi an active f r a c t i o n was o b t a i n e d t h a t m a y be mucop o l y s a c c h m ' i d e [6]. It a p p e a r s that p r o t h o r a c o t r o p i c h o r m o n e (PTTH, n e u r o h o r m o n e s t i m u l a t i n g the p r o t h o r a c i c gland) is p r o t e i n a c e o u s but that at Jeasl two p o l y p e p t i d e s m a y have s i m i l a r activity. Bioassay for b r M n - h o r m o n e s necessitates a large n u m b e r of w h o l e b r a i n s [7]. The p r e s e n c e of o t h e r b r a i n - h o r m o n e s as w e l l as the difficulty to obtain a great a m o u n t of s y n c h r o n i z e d b r a i n mat e r i a l m a k e s the vMidity of the b i o a s s a y for P T T H questionable. F u r t h e r m o r e this b i o a s s a y m e a s u r e s the a m o u n t of h o r m o n e s p r e s e n t in the b r a i n , w h i c h does not p r e s u m e of its c o n t e n l ,at the site of action. 1.1. Larval development. As m a n y insects several types of n e u r o s e c r e t o r y cells are d e s c r i b e d in B o m b y x mort p a r s - i n t e r e e -
148
M. Coulon.
r e b r a l i s . A cells [8] or A' cells [9] can be c o m p a r e d to A cells of Locusta [10] p r o d u c i n g p r o t h o r a c o t r o p i c h o r m o n e . The c h a n g e s of n e u r o h o r m o n e content in these cells d u r i n g l a r v a l d e v e l o p m e n t have b e e n e s t i m a t e d b y n e u r o s e c r e t o r y cell activity ; because of the s i m i l a r i t y of Bombyx and Locusta b r a i n cells ,it is s u p p o s e d that the most of the o b s e r v e d v a r i a t i o n s .are d u e to fluctuations of P T T H s y n t h e s i s a n d or secretion. V a r i a t i o n s of n u m b e r of n e u r o s e c r e t o r y granules c o n t a i n e d in A' ceils are d e s c r i b e d (electron m i c r o s c o p i c cytob i o m e t r y ) . A c t i v i t y c y c l e of these ceils d u r i n g w h o l e larxral d e v e l o p m e n t is d e s c r i b e d (CoubleFoussal, Coulon in p r e p a r a t i o n ) . Briefly : the numb e r of n e u r o s e c r e t o r y granules p e r surface unit is m a x i m u m at each l a r v a l e c d y s i s w h e r e after it decreases : a s u d d e n release of n e u r o s e c r e t o r y produets is o b s e r v e d in c o i n c i d e n c e w i t h the first food intake. S y n t h e s i s a,ctivity of the cells is i n d i cated b y an i n c r e a s e of granules n u m b e r ; ,later this n u m b e r is stable d a r i n g m i d d l e stage suggesting a b a l a n c e b e t w e e n s y n t h e s i s a c t i v i t y and release of p r o d u c t s . Before ,larval e c d y s i s neurosec r e t o r y granules are seen a c c u m u l a t e d in the cells. D u r i n g l a r v a l d e v e l o p m e n t this A' cells neuros e c r e t o r y a c t i v i t y i n Bombyx b r a i n can be d i s t u r b e d b y agents s u c h as fasting [9] and X r a y s [Coul~le-Foussal, Coulon in p r e p r a t i o n ] . In this exper i m e n t a l situation a r e t e n t i o n of n e u r o s e c r e t o r y p r o d u c t s is o b s e r v e d in the cells. In Bombyx l a r v a e two n e u r o s e c r e t o r y cells have been o b s e r v e d in the frontal ganglion [11] ; h o w e ver n o t h i n g is k n o w n about ,developmental of t h e i r activity. I n .an other L e p i d o p t e r a Manduca sexta, n e u r o s e c r e t i o n is a c c u m u l a t e d in these two cells dur,ing fasting, the r e l e a s e d at t i m e of food i n t a k e [12]. 1.2. Pupal developmenl. In L e p i d o p t e r a w i t h p u p a l d i a p a u s e (Hyalopthora, Philosamia, Anthereae for instance) the b r a i n is r e q u i r e d for f u r t h e r development. Brainless p u p a e of Bombgx b e c o m e ¢ d a u e r ~ p u p a e ( p e r m a n e n t p u p a e ) [13] the d e v e l o p m e n t of these p u p a e r e s u m e after i m p l a n t a t i o n of brain. N e u r o h o r m o n e s c o n t e n t in h e a d a n d t h o r a x a b d o m e n of Bombgx pupae ( b i o a s s a y : S a m i a test) is a l w a y s greater in female t h a n in male [7]. The b r a i n n e u r o s e c r e t o r y cells ( e x c e p t suhoesophageal ganglion) release t h e i r s e c r e t i o n s as soon as the l a r v a l - p u p a l moult occurs. D u r i n g p u p a l d e v e l o p m e n t b r a i n h o r m o n e s c o n t e n t alveays h i g h e r in e a r l y p u p a e . In female o v a r i e c t o m i z e d d u r i n g the 4th i n s t a r t h e BH level in p u p a e is g r e a t e r t h a n in n o r m a l female (male c a s t r a t i o n has no effect) ;
BIOCHIMIE, 1979, 61, n" 2.
this result w o u l d i n d i c a t e that a p a r t of BH c o u l d be used b y o v a r i e s for m a t u r a t i o n a n d / o r vitellogenesis in oocytes [7]. 2.
THE
ECDYSTEROIDS
: STEROIDS
HORMONES.
The first i n s e c t h o r m o n e w a s iso'lated a n d crystallized from Bombyx mort p u p a e a n d c a l l e d eed y s o n e [14]. A s e c o n d bi(~logical active s u b s t a n c e was s e p a r e d and o b t a i n e d p u r e in still s m a l l e r amount f r o m Bombyx p u p a e : e c d y s t e r o n e [15]. T h r o u g h extensive c h e m i c a l studies f o l l o w e d b y X r a y c r y s t a l l o g r a p h i c analysis, the s t r u c t u r e w a s e s t a b l i s h e d [16], a n d c o n f i r m e d b y synthesis. Eed y s o n e is a C~2T sterol of e x c e p t i o n a l l y p o l a r char a c t e r . E c d y s t e r o n e is r e c o g n i z e d as 2 0 - h y d r o x y ecdysone. It is a d m i t t e d that p r o t h o r a c i c g l a n d s y n t h e s,izes e e d y s o n e in Bombyx mori [17] :and Mandaca [18]. In in vitro culture of Bombyx p r o t h o r a c i c gland, the m a x i m u m release a c t i v i t y takes p l a c e at the end of the 5 th i n s t a r [19]. In p r o t h o r a c i c gland of this insect a p o s s i b l e i n t e r m e d i a t e between c h o l e s t e r o l and e c d y s o n e is d e t e c t e d (3~h y d r o x y 5a cholestan-6-one) [20]. Efficient conversion of e c d y s o n e to e c d y s t e r o n e has o b t a i n e d in ligated a b d o m e n of Bombyx [213. It has been t e n t a t i v e l y suggested that oenocytes m a y be the site of e c d y s o n e to e c d y s t e r o n e c o n v e r s i o n [191. F i r s t e c d y s o n e assay on Bombyx l a r v a e has been p e r f o r m e d w i t h the b i o a s s a y (Ca l l i p h o r a test) [221. The v a r i a t i o n of the s t e r o i d h o r m o n e content has been m e a s u r e d b y r a d i o i m m u n o a s s a y (RIA) t h r o u g h o u t Bombyx mori d e v e l o p m e n t from h a t c h i n g to e m e r g e n c e [23, 24, 25, 261. 2.1. Larval development. I m p o r t a n t changes of h a e m o l y m p h e c d y s o n e t i t e r are o b s e r v e d along the l a r v a l d e v e l o p m e n t . D u r i n g each l a r v a l i n s t a r feeding p e r i o d a low level of e c d y s o n e is o b s e r v e d ; a b r i e f p e a k o c c u r s a s h o r t time before the b e g i n n i n g of m o u l t i n g w h e n the l a r v a e stops f e e d i n g and then e e d y s o n e falls before ecdysis. The e c d y s o n e level i n c r e a s e s again soon after the s i l k w o r m start s p i n n i g t h e r e cocoon at the end of the 5 th instar. Then it r e a c h a p e a k s t r e c h i n g on about 2-3 days. I n other Lepid o p t e r a as Philosamia cynthia [253 a n d Manduca sexta [27] h o r m o n a l .peak is o b s e r v e d at t h e same t i m e of the 5 th instar. In Bombyx mort the s t e r o i d s content v a r i a t i o n has been p a r t i c u l a r l y s t u d i e d at h a t c h i n g and first food intake. D u r i n g last d e v e l o p m e n t , e m b r y o eats yolk and serosa (which contained ecdysteroids). E c d y s t e r o i d s level in l a r v a e is high at h a t c h i n g
B o m b y x endocrinology. [23] ; d u r i n g the first h o u r s of feeding, this level falls ( e x t r a c t i o n on l a r v a e in toto, the RIA is effected after thin l a y e r c h r o m a t o g r a p h y : TLC). This d a t a can be r e l a t e d w i t h e l i m i n a t i o n of egg ecdystero'ids (essentialy of ~ma¢ernal origin) a n d w i t h the outset of the p r o g r a m of l a r v a l d e v e l o p m e n t [28, and Coulon in p r e p a r a t i o n ] . By RIA, e c d y s o n e a n d e c d y s t e r o n e are t i t r a t e d in Bombyx h a e m o l y m p h . It is v e r y i m p o r t a n t to k n o w the c o n v e r s i o n r h y t h m of e c d y s o n e to ecdysterone an.d t h e i r m e t a b o l i s m . After i n j e c t i o n of [3H] e c d y s o n e to Bombyx 5 th i n s t a r larvae, the most e c d y s o n e p a r t (50 p e r cent) is c o n v e r t e d into e c d y s t e r o n e in 15 m n [21] (TLC a n d l i q u i d c h r o m a t o g r a p h y ) this c o n v e r s i o n is m a x i m u m at spinning at the same time the c a t a b o l i s m of these two h o r m o n e s decreases simul¢aneous to the endogenous h o r m o n e s p e a k [25]. Different c o m p o n e n t s are p r e s e n t in w e a k quantity, p a r t i c u ' l i a r l y conjugates and glycosides w h i c h ave e l i m i n a t e d into gut. D u r i n g 2rd a n d 3 ~a i n s t a r s r a d i o c h r o m m a t o g r a p h i c m e t h o d after i n j e c t i o n of aH e c d y s o n e p e r m i t s the o b s e r v a t i o n of m a x i m u m c o n v e r s i o n of e c d y s o n e to ecdys¢erone d u r i n g t h e p e a k of endogenous h o r m o n e s for each i n s t a r ; the p e r c e n tage of p o l a r c o m p o n e n t s is m i n i m u m a~ the same time [24]. In larvae, X r a y s can d i s t u b e d the synthesis of s t e r o i d s h o r m o n e s a n d the .conversion of e c d y s o n e to e c d y s t e r o n e [23, 24]. 2.2. Pupal development. After
radioimmunoassay
on
haemolymph
of
Bombyx mori p u p a e a large p e a k of e c d y s o n e and e c d y s t e r o n e is d e t e c t e d [25] d u r i n g the first nine days of this stage ; the content is the s~me in m a l e a n d female. A c o m b i n a t i o n of TLC a n d RIA a n a l y s i s of the h a e m o l y m p h samples "was p e r f o r m e d on Bombyx pupae. It a p p e a r s t h a t no or v e r y little BIA activity p u p a e is f o u n d in the e c d y s t e r o n e f r a c l i o n [251. In Anthereae p u p a e the c o n v e r s i o n of e c d y sone to e c d y s t e r o n c is o b s e r v e d (after i n j e c t i o n ~of [3HI ecdysone). The m e t a b o l i s m of these h o r m 0 nes is the same as in l a r v a e [20]. After bioass,ay ( C a l l i p h o r a test) on in toto g r o u n d p u p a e , one p e a k is d e t e c t e d in male p u p a e w h e r e a s t w o p e a k s are o b s e r v e d in female p u p a e [30]. The s e c o n d p e a k is r e l a t e d to o v a r y a c t i v i t y since il d i s a p p e a r e d in ovariec,tomiz~d female. Mor e o v e r in ovaries imp'lanted in a male pupae, mature o v o c y l e s c o n t a i n the same q u a n t i t y of ecdys t e r o i d s t h a n in ovocytes d e v e l o p e d in female pup a e although t h e r e is no e e d y s o n e ,in h a e m o l y m p h at l h e same t i m e (Lamy, Coulon in p r e p a r a t i o n ) . BIOCHIMIE, 1979, 61, n ° 2.
149
2.3. The ecdystero'ids conten.t of {he ovocyte. In eggs a n d ovocytes RIA a n d b i o s s a y m e a s u r e ment of e c d y s o n e c o n t e n t i n c l u d e s p r e c u r s o r s a n d d e g r a d a t i o n p r o d u c t s . T h e r e f o r e w e refere as ecd y s t e r o i d s to designate .all RIA active m a t e r i a l [31, 3 2 ] . I n Locusta [31] Lagueux d e m o n s t r a t e d that foll i c u l a r .cells s y n t h e s i z e ecdystero'/ds. In Bombyx mort although these cells have an i m p o r t a n t met a b o l i s m activity, t h e r e is no evidence for them s y n t h e s i z i n g ecdystero~ds. E c d y s t e r o ' i d s s y n t h e s i zed in o v a r y are a c c u m u l a t e d in o v o c y t e s [33]. Total ecdystero'/ds level changes w i t h ovocy{e devel o p m e n t a n d it passes b y a m a x i m u m . 2.4. The ecdystero'/ds content of the egg and e m b r y o . The egg, closed system, is f o r m e d of two p a r t s : y o l k and e m b r y o . E m b r y o d e v e l o p m e n t occurs in the m i d d l e y o l k ; u n f e r t i l i z e d egg y o l k c o n t a i n s e c d y s t e r o i d s . E m b r y o g e n e s i s begins in the p r e s e n c e of e c d y s t e r o i d s . E n d o c r i n e organs differentiate but w e d o not k n o w at w i c h stage they are functional. F i r s t b i o a s s a y ( C a l l i p h o r a test) show the p r e sence of e c d y s t e r o i d s in Bombyx mort eggs [34], a p a r t of w h i c h is e c d y s o n e conjugates as it has been d e m o n s t r a t e d b y e n z y m a t i c t r a i t m e n t . These p r e v i o u s results h a v e been c o n f i r m e d b y c h r o m a t o g r a p h i c s t u d y (high p r e s s u r l i q u i d c h r o m a t o g r a p h y : HPLC) c o u p l e d w i t h b i o a s s a y s h o w i n g no e c d y s t e r o n e but e c d y s o n e a n d conjugates, [35]. Bo2ubyx eggs w o u l d c o n t a i n e d also 2-deoxy a ecd y s o n e [36]. D u r i n g the w h o l e d e v e l o p m e n t of Bombyx m o n o v o l t i n eggs, ecdystero~d level has been m e a s u r e d from l a y i n g to h a t c h i n g [37t (by r a d i o i m m u n o a s s a y ) . Very early at the developm e n t of these eggs e o d y s t e r o i d conten,t is h i g h ; durin,g the d i a p a u s e this level d e c r e a s e s w h e r e a s v a r i a t i o n s of few a m p l i t u d e are o b s e r v e d d u r i n g p o s t - d i a p a u s e . W e can say that the most p a r t of e e d y s t e r o i d s p r e s e n t .are l o c a l i z e d in the y o l k a n d serosa a n d r e m a i n i n g p a r t takes p l a c e in e m b r y o at leaM for late stages. In m o n o v o l t i n e m b r y o , increase of h o r m o n a l content is o b s e r v e d before emb r y o n a r y molt. A f t e r w a r d s y o l k and serosa are eat a n d w e observe a great i n c r e a s e of e m b r y o e c d y s t e r o i d s con.tent (Coulon). 3. T H E JUVENILE HORMONES ( J H ) .
J'uvenile h o r m o n e h a s been isolated from male imago a b d o m e n of s i l k m o t h Hyalophora cecropia [38, 39, 40]. T h r e e JtI w e r e purified, the s t r u c t u r e a n d stereo c h e m i s t r y of the first p u r e JH isolated is JH I [41, 42] : .methyl 12, 14-dihomojuvenate. The o t h e r c o m p o n e n t in ¢ c e c r o p i a oil >> is JH II 11
M. Coulon.
150
[43, 44] : methyl 12- homojuvenate, JH III is methyl juvenate : methyl 10, l l - e p o x y f a r n a s o a t e . They have .a t e r p e n o i d stru.cture. The c o n v e r s i o n from one type of JH to a n o t h e r c a n n o t occur. The c o r p o r a allata synthesize JH molecules. I n Bombyx mort c o r p o r a allata are two glands ~ i t h lympho'/d structure [45] w h i c h have a secretory cycle. JH molecules w e r e recovered from m e d i u m in w h i c h corpora allata from newly emerged male eeeropia had been cultivated ; but these molecules were n o l p r e s e n t in med,ium w i t h freshly excized corpo.ra allala. 3.1. The larval development. Very little i n f o r m a t i o n is available on JH meas u r m e n t because ,there w e r e no efficient quantitative t e c h n i q u e u n t i l these last years. I n Bombyx mort Morohoshi studies the f u n c t i o n s of b r a i n c o r p o r a allata system (by ablation a n d i m p l a n t techniques) but this author give rarely n u m b e r e d data therefore it is very ,difficult to take out conclusions. In the s i l k w o r m Hyalophora ceeropia two m e t h o d s [47, 48] agree i n s h o w i n g j u v e n i l e h o r m o n e s to be high early i n larval i n s t a r a n d fall off as the n ex¢ moR a p p r o a c h e d . I n Bombyx no a n y data on JH c o n t e n t v a r i a t i o n s are avalaihle. I n an other Lepidoptera Manduca sexta [49] three JH are extracted a n d assayed (.by chromatograp h i q u e t e c h n i q u e s : TLC, ,HP~C, gas l i q u i d chrom a t o g r a p h y : GLC) ; in early 4 th instar three hormones are present, J'H III in less quantity. At beg i n n i n g of 5 th i n s t a r the JH I is more i m p o r t a n t than JH II and JH III but t h e i r levels decrease quickly. These authors .point out that in s i l k w o r m Hyalophora in earlier i n s t a r JH I is the only type revealed. 3.2. The pupal development. In Bombyx mort no any q u a n t i t a t i v e ,data on JH content are avai'lable d u r i n g p u p a l development, I n Galleria pupae (bioassay : Galleria test) no juvenile h o r m o n e was .detected d u r i n g the greater part of the p u p a l stage but an increase of the JH level is observed two days before emergence [50] (in this species ovogenesis takes place i n imago). In m a n y insects ovocyfes development is controlled by j u v e n i l e h o r m o n e a n d b r a i n h o r m o n e specially in species in w h i c h ovogenesis takes place i n the imago. I n Bomb!Ix .and other Lepidoptera in w k i c h ovocyte d e v e l o p m e n t takes place i n pupae, JH appears to have no effect on synthesis of vitellogenin by fat-body. After removal o f c o r p o r a allata at larvo-pupal ecdysis, the ovocytes
BIOCHIMIE,
1979, 61, n ° 2.
n u m b e r is r e d u c e d but vitellogenesis is not suppressed [51]. 3.3. The juvenile hormone content in ovocyte and egg. Just before l a y i n g bioassay has detected juvenile h o r m o n e i n Hyalophora cecropia eggs [48] this JH is o r i g i n a l i n g from female silkmoth corpora allata. An increase of JH level i n eggs, by early i n j e c t i o n or topical a p p l i c a t i o n leads to abn o r m a l embryogenesis [52]. I n Bombyx egg no data are available on JH level.
Conclusion. Diagrams of two e n d o c r i n e regulation in insects are ,drawn figure 1.
A
diuretic h.... £
[ V
Target tissues
.........
i
(-Ve ,:._v
ovary
FIG. 1. - - I)iagrams of two endocrine regulations in insects. A : G e n e r a l e n d o c r i n e c o n t r o l . B : e n d o c r i n e c o n t r o l of ovoeyte development. [] Site of synthesis of an hormone. -+ hormone. -+ other substance. PTTH pTocothoraeotropic hormone. BH brain h o r m o n e . E ecdysone. 20-E ecdysterone : 20-hydroxyecdysone. JH juvenile hormone. NSC-PIC neuroseeretory cell's of the pars-intercerebralis. Proth.gl. prothoraeie gland. CA corpora a ll,ata. CC corpora cardiaca. FC Follicular ceils. FB Fat body.
Fat body is a target tissue for h o r m o n e s just like the e p i d e r m a l cells or i m a g i n a l disks [53] (fig. 1A). In Bombyx mori fat body is the site of i n t e n s e
151
Bombyx endocrinology. proteosynthesis: It is of the most interest to k n o w the r e l a t i o n s h i p b e t w e e n the v a r i a t i o n s of h o r m o nal level (ecdysone a n d YH) and the v a r i a t i o n s of p r o t e o s y n t h e s i s in fat body. The d e v e l o p m e n t of B o m b y x mort is discontinuous, d u r i n g l a r v a l stage each m o l t constitutes a p h y s i o l o g i c a l starvation. The c o m p a r i s o n in fat b o d y of p r o t e o s y n t h e s i s rec o v e r i e s after molt and after an e x p e r i m e n t a l starvation p e r i o d and t h e i r relation to e n d o c r i n e modifications in both systems could b r i n g to progress k n o w l e d g e in some problems. In B o m b y x d a u e r p u p a e (brainless p u p a e a r r e s t t h e i r d e v e l o p m e n t and ,present c o n s e q u e n t l y a p u p a l diapause) proteosynthesis in ovaries s p e c i a l l y and e n d o c r i n e context are m o d i f i e d [54] (fig. 1B). D u r i n g diapanse the m e t a b o l i s m is modified in a closed m e d i u m : the egg. V a r i a t i o n s of the enz y m a t i c p o o l are s p e c i a l l y detected, and w e h a v e o b s e r v e d v a r i a t i o n s of the steroids c o n t e n t in the egg d u r i n g this diapause. The silk gland synthesizes two p a r t i c u l a r proteins, it is possible that this gland w o u l d be a target tissue for h o r m o n e s a n d that a part of its metabolism w o u l d be c o n t r o l l e d by e n d o c r i n e system. We have t h e r e f o r e the p o s s i b i l i t y to study relationship b e t w e e n m a n y different p r o t e o s y n t h e s i s and e n d o c r i n e regulation in m a n y systems of B o m b y x mori. It ap,pears that B o m b y x mori can be a good syst e m to u n d e r s t a n d en,docrine regulation of the o v a r y a c t i v i t y (fig. 1B) because it is d e v e l o p e d in pupae w h i c h has no n u t r i t o n a l relation w i t h the e n v i r o n m e n t . T h e synthesis are effected f r o m the p r e c u r s o r s a c c u m u l a t e d d u r i n g l a r v a l life w h i c h can be k n o w n . The synthesis of v i t e l l o g e n i n in fat body appears i n d e p e n d a n t of J H in B o m b y x , but it s h o u l d be a d v a n t a g e o u s to u n d e r s t a n d the mec h a n i s m of t h e r e g u l a t i o n of vitel]ogenin up-take by ovary. We h a v e seen that p r o t e o s y n t h e s i s in o v a r y is not i n d e p e n d a n t of BH ; it is possible to modifie the pool of p r e c u r s o r s (if the larval devel o p m e n t is d i s t u r b e d ) and to study the regulation of ovocyte d i f f e r e n t i a t i o n in relation w i t h endoc r i n e system in this e x p e r i m e n t a l situation. In B o m b y x mori w e h a v e m a n y q u a n t i t a t i v e data on h o r m o n a l levels t h r o u g h o u t the different stages of its d e v e l o p m e n t . At b r i e f interval, it w i l l be possible to d e t e r m i n e by n e w assay (RIA) the v a r i a t i o n s of t h e J H c o n t e n t in h a e n m l y m p b , t h r o u g h o u t l a r v a l and pupal d e v e l o p m e n t . T h e r e fore the r e l a t i o n b e t w e e n e c d y s o n e s and j u v e n i l e h o r m o n e s w i l l be studied. B o m b y x mori appears
BIOCHIMIE, 1979, 61, n ° 2.
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