Ecdysteroid titers during the molt cycle of Orchestia cavimana (Crustacea, Amphipoda)

GENERAL

AND

COMPARATIVE

Ecdysteroid

ENDOCRINOLOGY

65, 23-33 (1987)

Titers during the Molt Cycle of Orchestia (Crustacea, Amphipoda)

cavimana

F. GRAF AND J. I? DELBECQUE “Cytologie

et physiologie

des Arthropodes,” Facultk des Sciences, CNRS No. 674, Boulevard Gabriel, F-21100

Universitk de Dijon, Dijon, France

Unit@ associke

au

Accepted July 10. 1986 Ecdysteroids

were analyzed during a molt cycle in adult males of the crustacean Orlevels were determined in the hemolymph and in whole bodies, using radioimmunoassay. Results show a single and sharp peak at the end of Dl stage, reaching 810 pg eq/pl in the hemolymph (a 230-fold increase compared to the middle of intermolt). From B stage to the beginning of Dl , levels are very low but increase regularly and significantly. The amplitude and the temporal position of the peak are discussed in detail, in relation to the precision of the staging (17 different stages can be easily made in Orchestia) and to the cuticle cycle (the hormonal peak occurs ca. 10 hr before the beginning of cuticle synthesis at D2). Preliminary experiments, using monoclonal antibodies during the period of low ecdysteroid titers or high-performance liquid chromatography followed by polyclonal RIA during the peak period, suggest that the immunoreactive hormone in 0. cavimana behaves like 20-hydroxyecdysone. However, other minor compounds have been detected (some unknown, others migrating like ecdysone and ponasterone A in HPLC). o 1987 chestia

cavimana;

Academic

Press. Inc.

In Crustaceans which have an indeterposterior caeca of Orchestia cavimana minate growth, most of the physiological (Graf, 1969; Graf and Michaut, 1980; Graf processes (and not only those related to “and Meyran, 1983, 1985), several questions growth) are linked to the periodic shedding arose about the possible role of ecdysteof the exoskeleton, i.e., to the molting roids. Though MH variations have been decycle. Among the six different factors pro- scribed in a very close species, Orchestia posed for molt control (review in Skinner, gamarellus (Blanchet et al., 1976, 1979), 1985), only ecdysteroids (i.e., molting hor- we thought it was necessary to reinvestimones or MH) have been chemically char- gate these fluctuations in our model 0. caacterized and can be precisely measured. vimana, in order to make more precise inThe knowledge of ecdysteroid titers ap- terpretations of its physiology. Moreover, pears now to be a prerequisite to every this species is exemplary through the possiphysiological investigation in such animals, bility of a precise determination of a great particularly when growth is concerned. number of stages, which can be exactly However, variations in molting hormone correlated with the cuticular deposition levels have been investigated in less than (Graf, 1969, 1986) in a relatively short molt 20 crustacean species, and, in spite of sev- cycle (17 stages in a 46-day mean cycle). eral common characteristics, the results MATERIALS AND METHODS show some inconsistencies; particularly, Animals. The talitridean amphipod 0. cavimana the exact position of the MH peak(s) in comparison with the stages of cuticular de- Heller is the only European terrestrial amphipod living in moist biotopes along streams. This may be position is not clear (see Discussion). called a “hygrophile aerial life” in order to denote that In our investigations on calcium metaboit is not dependent on aquatic medium. Although this lism and on epithelial differentiation of the species is rare in France (Graf and Sellem, 1977). it 23

0016-6480/87 $1.50 Copyright 0 1987 by Academic Press. Inc. All rights of reproduction m any form reserved.

24

GRAF AND DELBECQUE

has been easily raised in the laboratory for 20 years in The hormonal concentrations were determined with a large subaquatic moss-grown terrarium, with a daily the radioimmunoassay (RIA) of De Reggi et al. (1975). cycle of 12 hr electric light (Graf, 1969). by competition with an iodinated radioactive analog. In order to avoid possible hormonal differences be- , generally against polyclonal antibodies showing a 2.2tween various age classes (Stevenson et al., 1979) or fold better sensitivity for ecdysone than for 20-hydroxyecdysone (Hirn and Delaage, 1980). Some verifibetween sexes (Hoarau and Hirn, 1978; Baldaia er al., 1984). experiments have been performed on 18- to cations in the hemolymph series, particularly during 20-mm male adults only. the period of low ecdysteroid titers, were made using The determination method of periods, phases, a monoclonal antibody, highly specific for 20-hydroxstages, and substages is that of Graf (1969, 1986). ac- yecdysone (EC-19 clone, from immunotech, Marcording to the principles of Drach (1939) and Drach seille-Luminy, France). From a methodological viewand Tchemigovtzeff (1969), based upon the careful point, comparison of the results obtained with antiobservation of the morphogenesis of the dactylopodite bodies having a wide specificity and those having a high specificity for 20-hydroxyecdysone could estiand the propodite of the third pereiopod (reference appendage). Because the claw formation in the dactymate the proportion of this hormone among the other ecdysteroids. However, the results obtained in our lopodite is very precocious (beginning with the dactylian apolysis at C2), the events of the early morphoexperiments with the two antibodies were perfectly genesis of the claw divide the intermolt period C into compatible (suggesting that 20-hydroxyecdysone is four main stages. The formation of spines and the se- the main circulating hormone) and they have generally cretion of the epicuticle and then the procuticle at the been regrouped in the same means ? standard errors. level of the propodite divide the premolt period D (beIn both cases, dialysis was used to separate free and bound radioactivity; then radioactivity measurements ginning with the generalized apolysis) into seven main stages. However, since cuticulogenesis is not synchrowere compared to standard curves obtained with calibrated solutions of pure 20-hydroxyecdysone and thus nous between the reference appendage and the whole body, it must be emphasized that the beginning of the the results have been expressed in picogram equivalents of 20-hydroxyecdysone per microliter hemoD2 stage is characterized by the initiation of epicutilymph or per milligram fresh weight. cule deposition in the whole body, whereas it is correlated with the initiation of procuticule secretion in the Each sample was analyzed at least in duplicate and often more, until appropriate dilutions and reproducpropodite of the reference appendage. For the whole molt cycle, 17 stages have been con- ible results were found. Seven to ten different samples sidered: the Dl I” stage has been divided into two, re- were analyzed for each stage in the hemolymph series, spectively, eD1”’ and 1Dl”’ (early and late); the D2 three to eight samples in the case of whole animals. Eventually, means were compared with Student’s t stage into three, respectively, e, m, 1D2 (early, middle, and late). Animals immediately after ecdysis test. HPLC. Analysis of the predominant immunoreachave been considered to belong to the A stage (early postmolt) and those 24 to 36 hr after ecdysis to the B tive ecdysteroids was done on two hemolymph pools stage (late postmolt). The mean duration of the from the peak period, using reverse-phase high-performance liquid chromatography (HPLC) followed by molting cycle in our experimental conditions was RIA (with polyclonal antibodies) on l-ml collected about 46 days. fractions. HPLC conditions were the following: Hormonal measurements. Hemolymph was colMerck RP 18 Lichro-Cart column (7 urn; 25 cm X lected from the pericardial cavity, dorsally between 4mm); two Waters M-6000 pumps with an M-740 conthe seventh and the eighth tergites, with graduated, troller and a WISP injector; the solvent was methanol microcapillary glass tubes. Individual samples, 45% in water at 1 mlimin during 12 min and then stepranging from 4 to 18 ul (mean: 8 ul), were eventually gradient to methanol 60% during 13 min, and lastly pooled to about 50 (~1, particularly for stages showing methanol 100%. Between calibration and ecdysteroid low ecdysteroid titers. The samples were then diluted in 500 (~1methanol and the ecdysteroids extracted by analysis in biological samples, five blank injections sonication and centrifugation (10,OOOg; 5 min); the and runs were made in order to avoid any contamination, the last control run being collected and assayed MH-containing supernatants were then separated with RIA. from the pellets. Whole animals (average fresh weight: 150 mg) were RESULTS always analyzed individually, after three successive sonications and centrifugations in methanol (600 ~1 at Immunoreactivity of the hemolymph. every step). Then, the supematants, for whole animals The variations in ecdysteroid titers in 0. as well as for hemolymph, were evaporated under nicavimana (Fig. 1) first present a period of trogen and the extracts suitably diluted in 0.1 M citrate buffer, pH 6.2. basal titers from A to Cl stages: the con-

ECDYSTEROIDS

IN Orchestiu

25

hemolymph

FIG. 1. Ecdysteroid titers in the hemolymph of Orchestia cavimana, expressed as pg eq 20-hydroxyecdysoneikl. Abscissa represents the interval between two successive ecdyses (E) and shows the different stages from A to D3 (substages e for early, m for middle, and 1 for late) related to a mean time scale in days. Each point represents the average of 7 to 10 samples and vertical bars indicate SEM (when absent, they are inferior to the size of the point).

centration during the A stage (2.1 2 0.2 pg 20-hydroxyecdysone equiv./pl hemolymph; SEM) does not statistically differ (P = 0.4) with B and Cl stages (e.g., 2.3 -+ 0.4 pg eq/kl at Cl). The results were at the limit of sensitivity when using the classical polyclonal RIA (taking account of our dilution conditions). However, the values were confirmed using the monoclonal antibodies and were significantly different from the background (about 0.2 ? 0.1 pg eq/pl). During a second period, from C2 to Dl’b, titers remain very low but increase progressively: the concentration at C2 (3.5 rt 0.5 pg eq/kl) is significantly different

from A-B-Cl values (P < 0.05) and the concentrations at C3 (5.1 t 0.4), C4 (7.7 * 0.6), Dl’a (13.0 -+ 2.2), and Dl’b (25.4 t 4.1) are significantly different between themselves at P < 0.05. Finally, after Dl’b, there is a very sharp peak with a strong increase from Dl” to the end of Dl”’ (810 & 45 pg eq/kl, the highest individual specimen being measured at 1040 pg eq/$) followed by a strong decrease down to the end of the molt cycle (27 pg eq/kl at D3, i.e., less than 10 hr before ecdysis). The precise staging method in 0. cavimana enables us to assume that the MH peak occurs exactly at the end of the Dl”’

26

GRAF

AND

stage (LDI”‘): at the beginning of Dl”’ (eDl”‘), i.e., approximately 24 hr earlier, the titer is significantly lower (320 t40 pg eq/Fl, P < 0.05) and the same is true at the next step (eD2), about 24 hr later, where the levels are approximately half reduced (436 & 45 pg eq/pl, significantly different at P < 0.05). More generally, all the successive values measured from D 1 ‘b to LD2 are significantly different at P < 0.05. Thus the phenomenon appears to be very quick. Moreover, the amplitude of the variation is remarkable: the mean value at the peak is 230-fold higher than the intermolt titer at C2 and 385-fold higher than the mean basal titer. Note also that the strong decrease before ecdysis cannot be attributed to water absorption in this terrestrial animal. It evidences an ecdysteroid half-life of several hours only. Immunoreactivity of whole animals. The variations in ecdysteroid concentrations in whole animals (Fig. 2) appear similar to those of hemolymph, except just after ecdysis; the lowest concentrations indeed have been found not during the postmolt but at the beginning of the intermold period: 6.5 ? 1.8 pg eq/mg at Cl vs 13.8 + 1.4 pg eq/mg at the A stage (these values are significantly different between themselves at P < 0.05 and also significantly different from the background of the polyclonal RIA). From C2, however, as in the hemolymph, a weak but progressive increase is observed until Dl’a, and then the concentrations strongly increase to reach a maximum also in late Dl ’ “, at 295 2 25 pg eq/mg. After the peak, the MH decrease in whole animals is at least as fast as in the hemolymph until D2 (67 t 10 pg eq/mg), but weaker near the ecdysis (14 ? 3 pg eq/mg at D3). A comparison of the ecdysteroid concentrations near ecdysis suggests two remarks. First, the MH concentrations related to the fresh weight do not statistically differ from D3 to A stages; unlike other crustaceans, this animal indeed does not swallow water

DELBECQUE

to shed its cuticle, so it does not gain but loses weight. However, the old cuticle, which is strongly reabsorbed in 0. cavimana, has a weak ecdysteroid content, about three-fold less than the remaining whole body after similar extraction; so, by considering the sum of the newly ecdysed animal plus the old cuticle, the total ecdysteroid concentrations are slightly reduced at a calculated value of about 12 pg eq/mg (Fig. 2, asterisk). The other remark concerns the hormonal decrease between A and Cl stages; the weak gain of fresh weight by the animals during this period cannot be responsible for the ecdysteroid fall. Identification of ecdysteroids. As a preliminary attempt to identify the immunoreactive ecdysteroids, several observations were done. First, using the polyclonal RIA at the period of high ecdysteroid titers, the results obtained with the same extracts analyzed at various dilutions were compatible when related to reference curves of standard 20-hydroxyecdysone and not with those of ecdysone. A complementary observation was made at the period of low ecdysteroid titers by comparing the data obtained using polyclonal and monoclonal antibodies; though the respective means were sometimes significantly different from a statistical viewpoint, they differed by less than 1.5 pg eq/pl during the whole period from A to C3 stages. Taking into account the very different specificities and sensibilities of the two antibodies, these data suggest that the main immunoreactive hormone is 20-hydroxyecdysone or a very close product, during the period of low ecdysteroid titers. A similar conclusion was obtained from another kind of experiment, involving HPLC followed by RIA of hemolymph pools during the peak; as shown in Fig. 3, the greatest part of the immunological response migrated like standard 20-hydroxyecdysone. However, other compounds were evidenced, giving a weaker but signif-

ECDYSTEROIDS

IN Orchestia

27

250

200

150

100

1 ..... r ..........

51

t

,(........ v”’ .............. (......... (........................... ...........

1, i*.*

FIG. 2. Ecdysteroid titers in the whole animals, expressed as pg eq 20-hydroxyecdysoneimg fresh wt. Each point represents the average of 3 to 8 samples. The last point (asterisk) represent the calculated concentration of whole bodies just after ecdysis plus the old cuticles. See Fig. I for other details.

icant response; some were unknown, others were found to be eluted at the retention times of ecdysone and of ponasterone A, respectively. DISCUSSION

The present study describes the molting hormone titers in adult males of the crustacean 0. cavimana. Though from a global viewpoint, our results are in agreement with comparable observations on other crustaceans and insects, several differences exist, principally the amplitude of hormonal changes and the position of the peak, in methodological and physiological respects. It is first important to stress that this

study presents a great number of hormonal measurements. 0. cavimana indeed is a species enabling a very precise seriation of the different stages (up to 17 in this study), based upon morphological observations. Moreover, this species can be easily raised under standard conditions, thus allowing more numerous and more reproducible samplings: in particular, it has been possible to make hormonal measurements both on hemolymph and on whole animals (Fig. 4). The precision of hormonal measurements due to species convenience is also strengthened by the RIA method itself. The crucial step in the RIA procedure is the separation of free and bound radioactivity

28

GRAF AND DELBECQUE

tb

i0

min

FIG. 3. Analysis of an hemolymph extract near the hormonal peak (eD2) by HPLC followed by RIA on I-min collected fractions. The left ordinate represents the immunoreactivity expressed as a percentage of the response measured on an aliquot before the separation, the right one its correspondance in pg eq/kl. The abscissa represents the time in min. Arrows indicate the retention times of reference 20-hydroxyecdysone (Z&Y), makisterone A (M), ecdysone (E), and ponasterone A (P), respectively.

(e.g., Walgraeve et al., 1986 in Artemia). In this study, this separation was performed by dialysis, which is known to give the most reproducible and sensitive results (Hirn and Delaage, 1980). Moreover, the lowest ecdysteroid concentrations have been verified using two different antibodies, including a very specific and sensitive monoclonal one. The observed amplitude of ecdysteroid variations in 0. cavimana, which reaches a ratio of 230 from C2 to the peak, is a remarkable fact, evidenced by a precise staging and the accuracy of RIAs. The mean amplitude observed in the hemolymph of other crustaceans is about a sixfold increase (review in Spindler et al., 1980) and the highest values described until now are about a 125 (Andrieux et al., 1976, in Carcinus) or 150-fold increase (Jegla et al., 1983, in Orconectes). In insects, which generally show greater ecdysteroid variations than crustaceans, such an amplitude is more frequent (review in Smith, 1985); however, though ecdysteroid peaks in insects are often higher than in Orchestia, basal titers are also more elevated. Undoubtedly, the difference observed in crustaceans is due in part to specific peculiarities, but probably also to the various

methods used, and in particular, to an incomplete seriation, pooling several substages, leading to peaks having a truncated shape. Similarly, the exact position of the ecdysteroid peak, which may vary according to species, strongly depends also upon the precision of staging. In 0. cavimana, the MH peak has been situated precisely at the end of Dl”‘, which is an original result since most of previous studies observed the peak at the middle of D2 (e.g., Willig and Keller, 1973, in Orconectes; Blanchet et al., 1976, 1979, in Orchestia gamarellus; Chang and O’Connor, 1978, in Pachygrapsus; Hoarau and Hirn, 1978, in Helleria; Baldaia et al., 1984, in Palaemon) or in D2-D3 or D3 (Adelung, 1969; Spindler et al., 1974; Andrieux et al., 1976 and Lachaise et al., 1976, in Carcinus; McCarthy and Skinner, 1977, and McCarthy, 1982, in Gecarcinus; Charmantier-Daures and De Reggi, 1980, in Pachygrapsus; Chaix et al., 1981, in Acanthonyx; Soumoff and Skinner, 1983, in Callinectes). Few studies localized the peak earlier, i.e., at the middle of Dl in Spheroma (Charmantier et al., 1976) or at Dl” in Ligia (Girard and Maissiat, 1983). The observations which best resemble ours concern Orconectes sanborni (Stevenson et al., 1979), in which the peak is situated in Dl”’ -D2 (but in fact, these stages were pooled) and Orconectes limosus (Jegla et al., 1983), in which the peak is situated at the beginning of D2 (but in this case, the whole Dl period was pooled). Our observations in 0. cavimana also show that the peak is relatively sharp, since less than 24 hr after the maximal titers, concentrations are already half reduced. Of course, here again, a precise staging method evidences a phenomenon impossible to observe when stages spread over several days or weeks. A last remark concerning the staging method is related to the physiological problem of cuticulogenesis. In most studies on crustaceans, stage determination is made according to the principles of Drach

ECDYSTEROIDS

IN

29

Orchestia

100.. 1 .z ? : T

-

so-

T a

20~

AB

CI

cz

c3

co

Do Dt’a

Dib

DJ’

y;“eD;

,Ds

~,;,,,,.,....,....,,...~..~...~...~...~.~...~~days 5

:...

“‘......,

F intermolt

ipOStmO/t~

\hz;;:iy

iw;:iz.d

premo’t

FIG. 4. Comparison of RIA results obtained from hemolymph and rithmic scale on the ordinate axis, which evidences the regular increase intermolt period and the change of concentration predominance at the abscissa as Fig. 1, on which the two successive apolyses are indicated.

(1939, 1944), on a reference appendage. However, cuticulogenesis in this appendage is not synchronous with that of the whole body. For example, Demeusy (1979) has shown that the epipodite of the third maxillipede (reference appendage generally used in Decapoda) appears to be in a Dl’ stage when the rest of the body is only at C4. This observation can explain why most of results in Decapoda localize the hormonal peak relatively later than in 0. cavimana. One can stress that the studies situating the hormonal peak close to that of Orchestia used a staging method grounded upon cuticle formation on the whole body. In Orchestiu also, there is heterochrony between the reference appendage and the rest of the body (Graf, 1969, 1986) which has

whole bodies, using a logaof concentrations during the end of Dl’ and at D3. Same See Fig. 2 for other details.

been taken into account (see Materials and Methods). In our opinion, such a remark is true not only for crustaceans, but also for other arthropods, particularly for insects, often staged by morphological criteria. It is generally admitted that there is a relationship between the increase of ecdysteroid concentrations and the synthesis of the new cuticle in crustaceans (Willig and Keller, 1973) as in other arthropods. However, the significance of such a relation is sometimes indefinite. Our results, demonstrating that the MH peak in 0. cavimana occurs about 10 hr before the beginning of D2 (which corresponds to the beginning of epicuticle deposition), leads to the conclusion that this peak really triggers the primary steps of cuticulogenesis, i.e., the epi-

30

GRAF

AND

cuticle deposition. This interpretation fits with the experiments of ecdysteroid injection or of in vitro cultures showing that the epidermal response to ecdysteroids is generally clearly visible at the cuticular level in about 24 hr (see the review of Spinder et al., 1980, for crustaceans). It is also consistent with several observations in insects. In Tenebrio, for example, the ecdysteroid peak has been timely correlated with epicuticle deposition in the abdominal integument (Delbecque et a/.. 1978). However, in insects as in crustaceans, the MH peak sometimes appears to occur earlier or later (see the review of Riddiford, 198.5), depending partly on the interspecific differences, but probably also on staging difficulties due to the asynchrony of the development between the whole body and a particular reference organ. Later steps of cuticulogenesis, on the contrary, though representing the greater part of cuticle synthesis, occur during the titer decrease. After hormonal stimulation, high levels are no longer necessary for cuticle synthesis: however. it is noticeable that even a few hours before ecdysis (D3), the hemolymph levels remain as high as those in Dl’b. It appears obvious in 0. cavimana that dactylian as well as generalized apolyses occur at relatively low MH levels, several days before the peak. Such an observation, which is frequent among crustaceans, appears unusual among insects; Tenebvio, an insect which shows a similar precocious apolysis in its last larval instar (Delbecque et al., 1978) is generally considered to be an exception (see the review of Riddiford, 1985). At least temporally, the determination of such apolyses is different from that of cuticle secretion, which does not exclude the possibility of induction by lowlevel fluctuations of ecdysteroids. In 0. cavimuna, a low but regular increase of hormonal titers has been evidenced during the intermolt period (see Figs. 1 and 2); this regular increase appears even more spaced with a semilog scale

DELBECQUE

(Fig. 4) allowing the superimposition of both types of measures. Apart from the great peak in Dl”‘, no other small peak was detected during the dactylian apolysis or the generalized one. These results are in agreement with most previous studies on crustaceans, except for Ligia (Girard and Maissiat, 1983), showing a significant peak at the dactylian apolysis, and for Uca (Hopkins, 1983), presenting a transitory peak at the generalized apolysis. Of course, the possibility of missing a very transient peak always exists, even in Orchestiu, since staging was more spaced during the intermolt and individual samples were pooled. However, in insects, small ecdysteroid peaks generally occur during the last larval instar and are clearly correlated with the commitment to metamorphosis (review in Riddiford, 1985). a function which does not exist in crustaceans that are closer to ametabolic insects, like Thrrmobia (Rojo de la Paz et al., 1983). Another interesting observation can be made about the low ecdysteroid levels, particularly well evidenced with the semilog superimposition of the results (Fig. 4). During the greater part of the molt cycle, ecdysteroid concentrations appear slightly higher in the whole body than in the hemolymph: the situation suddenly reverses between Dl’b and Dl” which corresponds to the beginning of the MH peak and undoubtedly to an activation of the Y organs. At this period, secretion of ecdysteroids into the hemolymph exceeds storage in the tissues. Before Dl” as well as after D3, it is the contrary in that ecdysteroids appear to be cleared more rapidly from hemolymph than from other tissues. It is noteworthy that during the whole premolt period the hemolymph ecdysteroids remain at a relatively higher level than other tissues. Obviously, the postmolt diminution in 0. cavimuna continues that of the premolt. This seems particularly evident in whole animals: Concentrations are at the lowest in the hemolymph at A and B stages, while they continue to decrease in other tissues.

ECDYSTEROIDS

It is important to remind here that Orchestin is a terrestrial animal. The ecdysteroid decrease is not due to a massive absorption of water, as often occurs in crustaceans. Moreover, the cuticle of this animal is strongly reabsorbed, and the values of MH concentrations after ecdysis. related to the fresh weights with or without the old cuticle, are very similar. In our opinion, these favorable conditions allow a reliable comparison of the results. This is generally not the case in other crustaceans. for which the loss of fresh weight due to the casting of the old cuticle, often weakly reabsorbed. and the gain of hemolymph volume, due to a massive absorption of water at ecdysis, completely disturb the concentrations. The last point to be discussed refers to the nature of the immunoreactive material detected by RIA. The use of various antibodies which differ in their specificity and/ or various dilutions with a given antibody gives a better idea of the major hormone detected (see O’Connor, 1985). Our results with this approach, even at the lowest levels by using a polyclonal and a monoclonal antibody, indicate that the immunoreactive response behaves essentially like 20-hydroxyecdysone. The same conclusion has been obtained during the peak by HPLC-RIA, showing that the major immunoreactive compound present in the hemolymph migrates like 20-hydroxyecdysone, two other less abundant products migrating like ecdysone and ponasterone A, and some other products being unidentified. Though such a pattern is shared by several other crustaceans (review in Spindler et al., 1980; 1984) our results do not exclude that isomeric compounds of 20-hydroxyecdysone, having similar chromatographic or immunoreactive properties (e.g., inokosterone) may also be present. Finally, the question remains open why 0. ccwitmtuz does not transform injected radioactive ecdysone into 20-hydroxyecdysone (a problem under investigation, mentioned in Connat and Diehl, 1986), in spite

31

IN Orclzesticr

of the probable occurence of the two hormones in the animal. ACKNOWLEDGMENTS We are indebted to Dr. M. De Reggi supply of RIA antigens and antibodies Mathelin for technical assistance.

for the kind and to Mrs.

REFERENCES Adelung. D. (1969). Die Ausschuttung und funktion von Hautungshormon wahrend eines Zwischenhautungs-lntervalls bei der Strandkrabbe Ctrrc~irlrrs ,t~t,e,~c,., L. Z. Ntrtrwfitd~. B 24, l447- 1455. Andrieux. N.. Porcheron. P.. and Berreur-Bonnenfant. J. t 1981). Etudes quantitative et qualitative des ecdysteroides presents in vivo chez le crabe Cd~i,rrr.\ I)IU~,~OJ sain ou parasite par Sacculina carcini. AK/I. Zoo/. Evp. Geii. 122, YY- 108. Andrieux. N.. Porcheron. P.. Berreur-Bonnenfant. J.. and Dray, F. t 1976). Determination du taux d’ecdysone au tours du cycle d’intermue chez le crabe Ctr~i)r~!.\ )~~e/r~s: Comparaison entre individus sains et parasites par Srrc~crr/i))o c,tr~i~i. C.R. Ac,trd. Sci. Ptrris 283. l42Y- 1432. Baldaia. L.. Porcheron. P.. Coimbra. J.. and Cassier. P. t 1984). Ecdysteroids in the shrimp Prr/tre)))o)r .sc’~~/rr.\: Relations with molt cycle. Ge/r. Co/rip. E/ldoc~rirlo/. 55, 437-443. Blanchet. M.-F., Porcheron. P.. and Dray. F. (1976). Etude des variations du taux des ecdysones au tours du cycle d’intermue chez le male d’0rclzestitr~tr)~)urr~r//~r Pallas (Crustace Amphipode) par dosage radioimmunologique. C.R. Actrd. Sc,i. Pmi~ D 283. 65 I-654. Blanchet. M. E. Porcheron. P.. and Dray. F. (1979). Variations du taux des ecdysteroides au tours des cycles de mue et de vitellogenese chez le Crustace Amphipode. Orc.lrr.\tirr~tr)>)trrc,//r/.r fnt. J. f/~wrt. Rqnwd. 1, l33- 139. Chaix. J. C.. Marvaldi. J.. and Secchi. J. t 1981). Variations of ecdysone titer and hemolymph major proteins during the molt cycle of the spider crab Actrtttltortys lirtttrltrtrrs. Camp. Biocltcn~. Ph~.sio/. (B) 69, 709-714. Chang. E. S.. and O’Connor. J. D. t 1978). ftr hv secretion and hydroxylation of a-ecdysone as a function of the crustacean molt cycle. Gets. Cotttp.Gtdocrind. 36. IS I - 160. Charmantier. G.. Olle. M.. and Trilles. J.-P. t 1976). Aspects du dosage de I’ecdysterone chez Spherotntr ~erratuttl(Crustacea. Isopoda, Flabellifera) et premiers resultats. C.R. Actrd. Sci. Pa/?.s 283, 1329-1331. Charmantier-Daures, M.. and De Reggi. M. (1980). Aspects preliminaires des variations hemolymphatiques du taux d’ecdysteroides chez Ptr-

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