pheromone in the green shore crab carcinus maenas L. - CiteSeerX

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Indices biochimiques et milieux marins. Journées du GABIM, Brest, 18-20 Nov. 1981 Publi. CNEXO (Actes Colloq.) n. 14, 1982, p. 35 à 46

THE METABOLISM OF ECDYSONE AND ITS PUTATIVE ROLE AS THE FEMALE SEX PHEROMONE IN THE GREEN SHORE CRAB CARCINUS MAENAS L.

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by F. Buchholz Institut für Meereskunde an der Universitat Kiel, D2300 Kiel, Germany (FRG)

A B S T R A C T -Both male and female crabs excrete large amounts of ecdysteroids and related metabolites during al1 major stages of the molting cycle.

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A sex specific pattern in the metabolic pathways could not be established.

These findings contradict the hypothesis that ecdysone functions as the female sex pheromone.-

R E S U M E -Les Crabes des deux sexes excrètent de grandes quantités d1ecdyst&roides et de leursmétabolites au cours des principales phases de la mue. Les voies du métabolisme ne présentent aucune différence liée au sexe. Ces résultats sont en désaccord avec l'hypothèse selon laquelle l'ecdysone jouerait le rôle de phéromone sexuelle femelle.-

K E Y

W O R D S : Carcinus maenas, ecdysteroids, metabolism, pheromone sex

M O T S

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-C

L E S : Carcinus maenas, ecdysteroides,métabolisme, phéromone sexuelle

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INTRODUCTION

Chemical communication p l a y s an important r o l e i n marine communities, however, only a few compounds showing e i t h e r a t t r a c t i v e o r r e p e l l e n t a c t i v i t y are chemically c h a r a c t e r i z e d s o f a r . The s t e r o i d ecdysone, t h e common molting hormone of t h e c r u s t a c e a n s , seems t o be one of t h e s e exceptions. KITTREDGE e t a l . (1971) concluded £rom t h e i r b e h a v i o r a l experiments t h a t ecdysone a d d i t i o n a l l y a c t s a s t h e s e x u a l pheromone i n brachyuran c r a b s . The hormone i s r e l e a s e d by females t o a t t r a c t t h e males ( d i s c u s s e d i n DUNHAM, 1978). Ecdysone and i t s r e l a t e d compounds are t h e o n l y s t e r o i d s i d e n t i f i e d s o f a r i n t h e phylum of t h e i n v e r t e b r a t e s . Ecdysone w a s f i r s t found i n i n s e c t s and c h a r a c t e r i z e d by BUTENANDT and KARLSON (1954). C u r r e n t l y it i s b e l i e v e d t o be t h e p r e c u r s o r of t h e a c t i v e molting hormone, which b e a r s an a d d i t i o n a l hydroxyl group, s i t u a t e d on t h e s i d e c h a i n : 20-OH-ecdysone. A number of s t u d i e s on t h e b i o s y n t h e s i s , metabolism and mode of a c t i o n of t h e e c d y s t e r o i d s are a l r e a d y published ( f o r r e f s e e HOFFMANN 1981 )

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KITTREDGE e t a l . (1971) were a b l e t o induce mating behavior i n male c r a b s of d i f f e r e n t s p e c i e s by adding ecdysone t o t h e medium. Only ready-to-molt females a r e a t t r a c t i v e and c o p u l a t i o n can o n l y t a k e p l a c e i f t h e females are f r e s h l y molted (RYAN, 1966). Accordingly, two f u n c t i o n s of t h e s t e r o i d seem obvious : it i s a c t i v e i n t e r n a l l y , e l i c i t i n g molting, and a t t h e same time s e r v e s as an e x t e r n a l a t t r a c t a n t s i g n a l l i n g t h e preparedness t o molt and t h u s t o c o p u l a t e . But t h i s assumption i m p l i e s one p r e r e q u i s i t e . To avoid i n t r a s p e c i f i c confusion o n l y ready-to-molt, a d u l t females should r e l e a s e ecdysone. Consequently a marked d i f f e r e n c e i n t h e mode of t h e metabolism and t h e e x c r e t i o n of t h e s t e r o i d must e x i s t between t h e two sexes. We can t r y t h i s arqument w i t h t h e h e l p of biochemical methods such a s Thin-Layer-Chromatography ( T L C ) , Radioimmunoassay ( R I A ) and r a d i o t r a c e r experiments. The e x c r e t i o n of t h e f r e e hormone w a s mainly s t u d i e d by SEIFERT (1982) of Our working group, whereas i t s metabolism i s t h e s u b j e c t of t h e p r e s e n t publication.

MATERIAL AND METHODS

Mature C. maenas of both sexes of a narrow s i z e range, 27 f 3 mm, were used. They were c o l l e c t e d on t h e i s l a n d of Nordstrand. I n t h e experiment d e p i c t e d i n Fig. 2 t h e s i z e c l a s s was 22 2 1 mm. A l 1 animals were k e p t under c o n t r o l l e d aquarium c o n d i t i o n s (ADELUNG & PONAT, 1980) and underwent one molt b e f o r e being taken i n t o t h e experiment. Molt s t a g i n g was done according t o ADELUNG (1971). The r e s u l t i n g s t a g e s can a l s o be expressed i n terms of t h e c l a s s i c a l Drach-scheme, see SPINDLER, e t a l . ( 197 4 )

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I n j e c t i o n s were performed by i n s e r t i n g t h e needle of a 10/ u l Hamilton Syringe (701) through t h e membrane s e p a r a t i n g Coxa and Basiischium of a walking l e g . Ecdysone was purchased from Simes,

Milano. The g e n e r a l l y l a b e l l e d 3~-ecdysone, s p e c i f i c a c t i v i t y 63 C i / m M o l , was produced by t h e Zoecon Corporation, USA. I t was p u r i f i & from a u t o l y s i s products by TLC before use. Generally 1 ecdysone was i n j e c t e d . I n t h e case of t h e tracer experiments/ug this amount contained between 78 and 84 l a b e l . Animals were e x t r a c t e d by a n-butanol/water p a r t t i o n Has described by ADELUNG (1971). E x t r a c t s were s t r e a k e d i n narrow bands ont0 Silicagel-TLC p l a t e s (Merck 11798, 0.25 mm, K i e s e l g e l 6 0 F 254) and w e r e developed i n an e q u i l i b r a t e d chamber i n chloroform/methanol, 4 : 1. The r e s o l u t i o n w a s g r e a t l y enhanced by using precoated high performance p l a t e s with a "concentration zone". A l 1 of t h e described m e t a b o l i t e s appeared w e l l s e p a r a t e d and highly r e ~ r o d u c i b l ea s could be v e r i f i e d by s t a t i s t i c a l treatment (BUCHHOLZ, 1980). I n t h e f i r s t experiment (sec Fig. 1 ) 22 f r a c t i o n s were scraped o f f t h e p l a t e and e l u t e d t h r e e t i m e s w i t h t h e s o l v e n t , which then w a s evaporated and t h e r e s i d u e subjected t o R&ioimmunoassay using t h e antibody ~ # 3( s p e c i f i c a t i o n and technique see GOODWIN, 1977 and BUCHHOLZ, 1980).

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I n t h e c a s e of t h e r a d i o t r a c e r experiments 100 ug of ecdysone and 20-OH-ecdysone were added t o t h e homogenate / a s c a r r i e r and TLC-Standard. The developed p l a t e s were read with a Berthold r a d i o t h i n l a y e r scanner II. I n a second r u n t h e r a d i o a c t i v i t y could be q u a n t i f i e d by t h e b u i l t i n i n t e g r a t i o n system. Ecdysone and 20-OH-ecdysone were i d e n t i f i e d on t h e p l a t e s by W-scanning a t 254 m, and a d d i t i o n a l l y by a d e r i v a t i s a t i o n procedure : t h e r e s p e c t i v e bands were e l u t e d and a c e t y l a t e d i n a c e t i c anhydride/ p y r i d i n e , 1 : 1 , f o r 75 min a t 2 2 O ~ . The products were separated by TLC i n chloroform/ethanol 4 : 1 , and t h e bands i d e n t i f i e d by radioscanner and W - l i g h t a t 254 nm. The bands w e r e compared w i t h t h o s e of correspondingly d e r i v a t i z e d , r a d i o l a b e l l e d standard hormones. 3 ~ - 2 0 - 0 ~ - e c d ~ s o n es,p e c i f i c a c t i v i t y 3.8 Ci/mE401, was purchased from NEM f o r t h i s purpose. A l 1 o t h e r m e t a b o l i t e s were i d e n t i f i e d by t h e i r Rf-values. The seawater i n which t h e c r a b s were kept w a s e x t r a c t e d t h r e e t i m e s with water s a t u r a t e d n-butanol which was washed twice w i t h butanol s a t u r a t e d water. The y i e l d of t h e procedure was 75 8 % , determined i n t r i p l i c a t e . The e x t r a c t s were subjected t o TLC and evaluated a s described. RESULTS AND DISCUSSION I n o r d e r t o t e s t whether u n a l t e r e d ecdysone was excreted d i r e c t l y , f o u r male c r a b s were kept i n a c o n t a i n e r with seawater f o r 36 hours. The water was e x t r a c t e d and t h e condensed e x t r a c t s subjected t o TLC. The 22 f r a c t i o n s were t e s t e d f o r ecdysone a c t i v i t y by RIA. The chromatographie s e p a r a t i o n s t e p was introduced t o exclude m i s i n t e r p r e t a t i o n of t h e r e s u l t s caused by u n s p e c i f i c c r o s a r e a c t i o n of t h e antiserum with unknown substances. The main R I A a c t i v i t y found, corresponded e x a c t l y t o t h e cochromatographing 20-OH-ecdysone standard. The t i t e r w a s s o unexpectedly high t h a t t h e upper l i m i t of d e t e c t i o n was surpassed by f a r . Therefore t h e a b s o l u t e v a l u e s could n o t be determined with s u f f i c i e n t p r e c i s i o n (Fig. 1 ) . Calculated from d a t a of SEIFERT (1982) animals of t h e same molting s t a g e (D2/D3) e x c r e t e about 80 ng d u r i n g t h e 36 hour period. No important c r o s s r e a c t i o n was found besides some minor peaks i n t h e v i c i n i t y of t h e main peak i n d i c a t i n g t h e presence of c l o s e l y r e l a t e d degradation products of ecdysone.

5

4 41°

E-OH E

15

2O

FRACTION NUMBER

Fig.1 TLC-separation of an extract of aquarium-water in which 4 male crabs in late molting stages, D /D , were kept for 36h. Determination by ~idi&mmunoassa~.

h AFTER INJECTION Fig.2 The relative hormone-contents (value after 15 min 100%) in the hemolymph, and excreted hormone-activity in the aquarium water after injection of 1 ug ecdysone. Determination by RIA and ~ritium-labdlled hormone.

I n a f u r t h e r set of experiments t h e r e t e n t i o n timeof ecdysone i n t h e organism was determined by i n j e c t i o n of 1 ug/animal. I n Fig. 2 t h e upper curve shows t h e diminishing v a l u e s of / Tritium a c t i v i t y i n hemolymph samples taken from 12 i n j e c t e d c r a b s of both sexes. The excreted Tritium a c t i v i t y , found i n t h e aquarium water t h e animals were kept i n , i s represented by t h e curve which r u n s mirrori n v e r t e d t o t h e l a t t e r . The r a t e of e l i m i n a t i o n i s r a t h e r slow, a s 50% of t h e a c t i v i t y i s s t i l l p r e s e n t a f t e r 16h. The s o l i d curve shows t h e rate of e l i m i n a t i o n t e s t e d by i n j e c t i o n of u n l a b e l l e d ecdysone determined by RIA. A s t e e p drop i s evident. This can be explained by t h e fact t h a t t h e i n j e c t e d ecdysone i s converted r a p i d l y t o 20-OH-Ecdysone (see below). The used antibody i s only h a l f a s s e n s i t i v e t o 20-OH-ecdysone a s t o ecdysone. Therefore t h e conversion adds t o t h e e l i m i n a t i o n and t h i s causes t h e s t e e p e r s l o p o f t h e graph. The l a t t e r test w a s necessary, because it w a s assumed e a r l i e r (ADELUNG, 1967) t h a t t h e i n j e c t i o n of exogenous ecdysone s t i m u l a t e s t h e n a t u r a l s y n t h e s i z i n g s t r u c t u r e s t o produce endogenous hormone. A s t h e curve does n o t rise again a f t e r t h e i n i t i a l drop, such a p o s i t i v e feedback mechanism can now be excluded. I n t h e following t h e r e s u l t s of t h e main s e t of experiments a r e described. The i n a c t i v a t i o n and e x c r e t i o n of t h e molting hormone was i n v e s t i g a t e d i n d e t a i l . To t h i s end ecdysone, t h e metabolic precursor of 20-OH-ecdysone, was i n j e c t e d and by employing r a d i o a c t i v e label t h e m e t a b o l i s a t i o n of t h e i n j e c t e d hormone was examined. According t o t h e slow e l i m i n a t i o n r a t e t h e c r a b s i n t h i s experiment were maintained f o r 36 h a f t e r i n j e c t i o n and then e x t r a c t e d . The water t h e animals were kept i n w a s a l s o examined. I n Fig. 3 t h e m e t a b o l i t e s demonstrated on a radiochromatogram are depicted. I n both diagrams shown, a s u b s t a n t i a l peak corresponding t o 20-OH-ecdysone i s seen b e s i d e s unchanged ecdysone. I n a d d i t i o n , t h r e e minor peaks P -Pg are apparent i n both e x t r a c t s . I n t h e water e x t r a c t s an a d i t i o n a l unpolar peak P3.1 i s evident. Apart from t h e s e o t h e r minor m e t a b o l i t e s appear, b u t only t h e described ones a r e chosen f o r f u r t h e r study a s t h e s e are encountered i n s u b s t a n t i a l amounts i n a l 1 extracts.

d

Fig.3.l.

Whole-animal axtract

Fig.3.2.

Extract of aquarium water

Rradio-TLC ch-tograms of metabolites in 3~-ecdysone injected mail8 ancl their tquarium water.

If the rate of metabolisation is observed with increasing time of incubation (see Fig. 4 ) , the activity bound to 20-OH-ecdysone after six hours amounts to 50%. The other metabolites show little activity. After 36 h the animals contain only 6% unmetabolized ecdysone. The overall percentage of the minor derivatives increases to 18% of the 3H-activity. If the composition of these metabolites other than ecdysone are observed in more detail, their relative proportion appears to be changing with the duration of the incubation (Fig. 5). At first the unpolar derivatives emerge, but after 36 h the proportion is shifted towards the polar and for this reason slow moving substances.

overall derivati sation

minor metaboli tes

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h INCUBATED

Fig.4 Percentage of metabolized 3~-ecdysone in wholeanimal extracta with increaaing incubation-times. n = 3, mean and standard deviation indicated.

Proportion of metabolites other than ;hS-OH-ecdysone with increasigg incubationtimee

In the course of Our studies of the molt physiology in C. maenas the changing titer of the ecdysones was determined in relation to the molting cycle (ADELUNG, 1971). By RIA 1 found a steady increase of the hormone concentration in the hemolymph towards a sharp rise just prior to molting (BUCHHOLZ, 1980; see also ANDRIEUX, 1976). This peak was followed by a steep drop, occuring about one day before the molt. The question was then, whether the rate of conversion and excretion as well as the

relative proportion of the metabolites would also change during the molting cycle. In respect to elucidate the function of the pheromone it is of particular interest, whether a sex-specific difference in the metabolism related to the cycle can be demonstrated. Accordingly, in the following injection experiments three groups of crabs of different molting phases were used. The groups consisted of three male and three female crabs showing the stages of intermolt, early, and late premolt respectively. IV considering . the results, the extractsof whole animals contain 76 ,6% 20-OH-ecdysone and 16 f 4% of the other metabolites. No significant sex-specific as well as stage dependent differences appear between the investigated groups. The crabs of each group were kept together for the standard period of 36 h in seawater, which was extracted and analysed as described. The results are shown in Fig. 6. The white bars represent the portion of 20-OH-ecdysone and the black areas the overall percentage of the other described metabolites. The difference of the bars from 100% accounts for unmetabolized excreted ecdysone.

9

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0 '

100%

100% l

1

I

1 I I

1 1

3

X

D3

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VI11

D,

g

IV

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Fig. 6 Percentage of metabolized 3~-ecdysonein extracts of aquarium-water. Comparison of the two sexes of crabs in postmolt, early and late premolt stages. White bars: 20-OH-ecdysone; black bars: minor metabolites.

It can be noted that the metabolites other than 20-OH-ecdysone constituate a substantial part of the excreted hormone derivatives, In the earlier stages more than half of the derivatised ecdysone falls to the share of the unknown metabolites. Their relative amount does not Vary significantly throughout the molting cycle. If 20-OH-ecdysone is considered, a stage dependent difference is obvious. Shortly before molting at least double as much 20-OH-ecdysone is generated than in the earlier stages. The high interna1 hormone titer at this stage is a sign for a high demand for 20-OH-ecdysone. Therefore the conversion rate increases, and consequently more metabolised ecdysone is found in the excreta. This derivatisation pattern is common to both sexes. An increase in the conversion rate was also noted by LA CHAISE et al. (1976) in C. maenas and McCARTHY and SKINNER (1 977) in Gecarcinus lateralis.

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The last diagram, Fig. 7, represents data from the same experiment. Here the percentages of the minor metabolites are considered, whereas 20-OH-ecdysone is omitted. The derivatives found

EXTRACTS

50%

Fig.7 Proportion of metabolites other thaq 20-OH-ecdysone in whole-animal extracts a p extracts of the aquarium water, 36h after injection of H-ecdysone. Comparison of the two sexes of crabs in postmolt, early and late prmolt stages.

i n animals and t h e i r e x c r e t a are a g a i n compared. No major d i f f e r e n c e s between t h e female and t h e male groups a r e apparent i n t h e p a t t e r n s of m e t a b o l i t e s . Besides t h i s r e s u l t some i n t e r e s t i n g d e t a i l s can t o be pointed o u t : I n t h e water e x t r a c t t h e unpolar d e r i v a t i v e Pj dominates, accounting f o r up t o two t h i r d s of t h e o v e r a l l a c t i v i t y . I t i s t h e one m e t a b o l i t e which d i s a p p e a r s q u i c k l y from t h e animal e x t r a c t s w i t h i n c r e a s i n g i n c u b a t i o n time a s a l r e a d y shown i n diagram 5. I n t h e male animals w i t h t h e h i g h e s t molting s t a g e X, t h e p r o p o r t i o n i s s h i f t e d towards d e r i v a t i v e P The same tendency appears on t h e s i d e of t h e female c r a b s , y e t male animals a r e probably s l i g h t l y more advanced i n t h e i r molting s t a g e .

th

S t e r o i d s l i k e t h e e c d y s t e r o i d s can be e x t r a c t e d s e l e c t i v e l y with n-butanol. P o l a r s u b s t a n c e s which show a higher s o l u b i l i t y i n water remain a t l e a s t p a r t l y i n t h e e x t r a c t e d water phases. For t h i s reason samples w e r e taken from t h e e x t r a c t e d water phases and t h e i r r a d i o a c t i v i t y determined. Apparently (see Tab. 1 ) t h e remaining a c t i v i t y i n t h e animal e x t r a c t s does n o t change s i g n i f i c a n t l y with 8% s t a y s i n t h e e x t r a c t s . The p i c t u r e t h e molting stage.About 28 i s d i f f e r e n t concerning t h e remaining a c t i v i t y encountered i n t h e e x t r a c t s of t h e aquarium w a t e r , where a s i g n i f i c a n t i n c r e a s e w i t h t h e p r o g r e s s i n g molting s t a g e can be noted. I t was n o t p o s s i b l e t o f u r t h e r a n a l y s e t h e n a t u r e of t h e s e e x c r e t e d substances which c a r r y up t o h a l f of t h e i n j e c t e d a c t i v i t y . Therefore t h e r e s u l t s of t h i s p a r t of t h e d e r i v a t i s a t i o n experiments have t o be considered preliminary

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