Ecdysteroid Fluctuations during Embryogenesis in the Giant Freshwater Prawn, Macrobrachium rosenbergii MARCY N. WILDER, TAKUJI OKUMURA, KATSUMI AIDA, AND ISAO HANYU Department
Accepted November 15, 1989 Ecdysteroid levels during the embryogenesis of the giant freshwater prawn, Macrobrarosenbergii, were determined by radioimmunoassay and high-performance liquid chromatography. Ecdysteroids consisting of significant amounts of 20-hydroxyecdysone and high-polarity products (HPP) and lesser amounts of ecdysone and low-polarity products (LPP) were detected in mature ovaries and newly laid eggs. All ecdysteroid groups decreased gradually during the nauplius phase. With the formation of the compound eye and the appearance of the carapace and other body-like structures, marking morphogenesis to the zoeal stage, embryos showed the beginning of a continuous and dramatic increase in ecdysteroid concentrations sustained until larval hatchout. Ecdysteroid levels at hatchout were above 20-fold greater than ecdysteroid levels in newly laid eggs. More specifically, HPP and 20-hydroxyecdysone increased concomitantly, with a decrease in 20hydroxyecdysone only at the end of the embryogenic period, while ecdysone and LPP levels remained low or undetectable. It may be postulated that the presence of ecdysteroids in ovaries and eggs represents a reserve of maternal ecdysteroids which are necessary at the commencement of embryonic development; with the differentiation of embryonic tissue capable of ecdysteroid synthesis, ecdysteroids increase rapidly to play a role in later embryonic development. 8 I!290 Academic Press, Inc. chium
Ecdysteroids play manifold roles in the life cycle of insects. During the larval stages, ecdysteroids produced by the prothoracic glands control molting activity; in reproductive adulthood, ovarian-synthesized ecdysteroids are thought to play a role in the processes of vitellogenesis and maturation. During embryogenesis, reserves of ecdysteroids pending transfer from the follicle cells of the ovaries (Lagueux et al., 1977;Goltzene et al., 1978),and subsequent rises in ecdysteroid titers, possibly due to embryonic prothoracic gland activity (Wentworth and Roberts, 1984; Gande et al., 1979), are apparently involved in the deposition and shedding of the egg cuticle (Bordes-Alleaume and Sami, 1987; Lagueux ef al., 1979; Imboden et al., 1978). In crustaceans, it can be considered that ecdysteroids are present at all stages of development, with a lifetime role in controlling molting processes and possibly repro-
ductive and embryonic events. 20Hydroxyecdysone has been long established as the molting hormone in adult crustacea, first identified in the Australian rock lobster, Jusus lulundei (Hampshire and Horn, 1966). Thereafter, the involvement of ecdysteroids in the larval and juvenile ecdysis of various crustacean species has been reported (Chang and Bruce, 1980, 1981; Spindler and Anger, 1986). It is slightly more recently that research has been undertaken concerning the possible roles of ecdysteroids in oogenesis and embryogenesis. In species such as the shrimp, Puluemon serrutus (Spindler et al., 1987), the shore crab, Curcinus muenus (Lachaise and Hoffman, 1977, 1982), and the spider crab, Acunthonyx lunulutus (Chaix and De Reggi, 1982), fluctuations in ecdysteroid content accompanying embryogenesis have been investigated. As in larval ecdysis, the ecdysteroids principally detected consisted
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of ecdysone, 20-hydroxyecdysone, highpolarity products (HPP) and low-polarity products (LPP), and in some cases, ponasterone A. However, while these species exhibited a similar tendency in that changes in ecdysteroids are correlated with certain embryonic events, patterns of fluctuation are not necessarily in concurrence. In P. serrutus, ecdysteroid levels rise with the completion and formation of the Y-organ midway through embryogenesis, marked by a steadily increasing ratio of 20hydroxyecdysone to ecdysone (Spindler et al., 1987). Hence, it was decided to undertake an investigation on another Palaemonidae species, the Macrobrachium rosenbergii, in order to determine whether a similar trend exists in the embryogenesis of this species. Ecdysteroid determinations were made on embryonic eggs at consecutive intervals throughout the developmental period, as well as on postvitellogenic ovaries, in context of the embryonic stages as delineated by Kwon and Han (1983). MATERIALS
Sampling methods and animals. The giant freshwater prawn, M. rosenbergii, was employed and animals were maintained at 28”. Sampling was conducted at prespawning, on ovaries following the occurrence of the reproductive molt (Day 0), and at postspawning, on eggs attached to the reproductive setae (Days 1,4, 7,10,13,16, and 18). Approximately 300-mg eggs from 6 to 13 individuals at each embryonic stage were taken with pincets, quick-frozen at - 80”, and stored at - 30” until use in ecdysteroid extraction procedures. Embryonic features were observed at each sampling occasion on live eggs by light microscopy. Extraction of e&steroids. An ecdysteroid extraction procedure was developed for experimentation based on the methods of Spindler et al. (1987). Egg mass was determined as frozen wet weight, and 200300 mg per individual were first homogenized in methanol (2.0 ml), followed by centrifugation (lOOOOg, 20 min). The supematant was decanted and saved; following reextraction of the pellet in methanol (1.0 ml), supematants were combined, and methanol was evaporated under reduced pressure. Subsequently, the dried extracts were partitioned twice between chloroform (2.0 ml) and water (1.5 ml). Water layers were combined and the resultant water volume (3.0 ml) was reduced (2.0 ml) and partitioned twice with normal
ET AL. hexane (2.0 ml each). Lastly, water was evaporated to dryness under reduced pressure, and the resultant residue was dissolved in 0.1% gelatin-20 m&f PBS (1.0 ml) for use in radioimmunoassay. Radioimmunoassay (RIA). A double antibody method for ecdysteroid RIA developed by Okumura et al. (1989) was fust validated (see below) for the assay of ovarian and egg extract. Samples (egg or ovary extract material) or ecdysone standards (2-fold serial dilution, 78&lOOO pg/ml) dissolved in gel-PBS were taken in 200~pl aliquots and incubated for 24 hr with 200 ul of first antibody (rabbit anti-20-hydroxyecdysone serum) in a lOOO-fold dilution. One hundred microliters of tritium-labeled ecdysone (approx. 7000 cpm) was then added to each aliquot tube, followed by a second incubation of 24 hr. This was followed by the addition of 200 ~1 second antibody (goat anti-rabbit IgG serum) diluted 60-fold with EDTA-PBS and a third incubation of 24 hr. The fourth day of the assay, aliquots were centrifuged at 3200 rpm, 30 min, 4”. The supematant was discarded via aspiration, and the resulting precipitate was dissolved in 100 ul 0.1 N NaOH. Upon addition of Liquitlour scintillatorf’friton X (2/l) to aliquots, the ecdysteroid concentration as 20-hydroxyecdysone was determined. Assay validation. An ecdysteroid competition curve was determined from egg extract material (267, 134, 66.9,33.4, and 16.7 mg) and parallelism to the standard ecdysone curve (as above) was examined. Subsequently, at binding near 8.5, 30, and 60%, the intraassay coefficients of variation (N = 6) and the interassay coefficients of variation (N = 5) were additionally examined. The rates of recoveries of ecdysone and 20hydroxyecdysone were examined separately by adding known quantities of standards to egg material prior to homogenization. Separation by HPLC. The total ecdysteroid profde corresponding to embryogenesis was obtained according to RIA as above; subsequently, for each embryonic stage and for ovaries, individual extracts in gelPBS were pooled and prepared for separation according to high-performance liquid chromatography (HPLC). Pooled extracts (approx. 5 ml each stage) were extracted with an eightfold volume of methanol. Following centrifugation (3000 rpm, 10 min), the methanol supematant was decanted and saved, and each pellet was reextracted in 0.5 ml methanol. After combining and evaporating the methanol supematants to dryness, the resultant residues for each stage were again amalgamated in 50% HPLC-use high grade methanol. HPLC was performed on a reverse-phase HPLC column (ODS-SOTM, 4.6 X 250 mm, Tosoh Co. Ltd.) employing 50% methanol as the solvent system. The flow rate was set to 0.8 ml/mm, and fractions were collected at one fraction per minute for 50 min. Fractions were dried under reduced pressure, dissolved in gel-PBS, and subjected to RIA analysis.
Statistics. Duncan’s multiple range test was employed in analysis of fluctuations in total ecdysteroid levels as determined according to RIA.
The competition curve for egg extract was parallel to the standard curve as shown in Fig. 1. The intra- and interassay coefficients of variation were respectively 5.8, 5.6, and 7.9%, and 6.6,4.9, and 10.7 at 8.5, 29.5, and 59.5% binding. Recovery rates for ecdysone and 20-hydroxyecdysone were 75.0 and 97.3%, respectively. Embryonic
FIG. 1. Competition curve for egg extract material in serial dilution and standard curve for 20-hydroxyecdysone showing parallelism.
The profile for changes in total ecdysteroid during embryogenesis is shown in Fig. 3. All values, calculated as 20-hydroxyecdysone equivalents, are indicated. Total ecdysteroid in mature ovaries was determined at 26.5 rig/g. From a level of 16.7 rig/g on Day 1, ecdysteroid levels declined gradually (P < 0.01) to 11.3 rig/g until Day 10, which concurred with the appearance of the compound eye. From Day 13, ecdysteroid levels (35.9 rig/g) began to increase dramatically (P C 0.01) until hatchout. On Day 18, concentrations attained levels (483.6 rig/g) more than 20-fold higher than those of freshly spawned eggs. HPLC-RIA
Microscopic observations were carried out for all embryonic stagesinvestigated in this study. Photographic images are shown for Days 1, 10, 13, and 18 in Fig. 2. Day 10 signifies the metamorphosis of embryo from the naupliar stage to the zoeal stage, as the compound eye has appeared. Pulsations of the primitive heart are also apparent. At Day 13, the compound eye has further developed and body-like structures, i.e., carapace, antennae, digestive tract, etc., have taken form. At Day 16, further development of the body has occurred. By Day 18, typically half of the brooded egg mass has hatched to zoeal larvae.
Representative HPLC elution patterns are shown for Day 0 and Day 16 (Fig. 4). The presence of 20-hydroxyecdysone (fractions 11-13) and ecdysone (fractions 17-18) was confirmed by coelution with authentic standards. Other immunoreactive fractions which eluted prior to 20-hydroxyecdysone and subsequent to ecdysone were arbitrarily termed high-polarity products and low-polarity products, respectively. The identities of these fractions are at present under investigation. The overall fluctuations of the four species of detected ecdysteroids are shown in Fig. 5. In ovaries and newly spawned eggs, 20-hydroxyecdysone and high-polarity products were detected at nearly equal levels, approximateIy IO-fold higher than ecdysone. Low-polarity products were not detected to any great extent throughout embryogenesis. As total levels of ecdysteroids decreased gradually from spawning until Day 10, all ecdysteroid groups decreased. HPP remained present in significant amounts, while ecdysone, 20-hydroxyecdysone, and LPP became virtually undetectable. At Day 13, both 20-hydroxyecdysone and HPP began their dramatic increase; however, with further embryonic
FIG. 2. Embryonic stages of M. rosenbergi (a) Day 1; newly laid eggs. (b) Day 10; first appearance of carapace, compound eye. (c) Day 13; further development of body-like appendages, beginning of pulsation of embryonic heart. (d) Day 16; prehatching reduction in yolk.
development, on Day 16, high-polarity products reached levels of 5-fold greater than those of 20-hydroxyecdysone. HPP continued to increase until Day 18, to comprise 99% of total ecdysteroid. Although 20-hydroxyecdysone was detected considerably by Day 16, levels had rapidly declined on Day 18, comprising only 1% of total ecdysteroid. DISCUSSION This investigation has revealed that ecdysteroids are present in mature ovaries and in newly laid eggs, suggestive of a maternal origin. Ecdysteroids were detected as ecdysone, 20-hydroxyecdysone, and high- and low-polarity immunoreactive products; this parallels results for a number of other decapod crustacean species. In P. serrutus, the existence of these four ecdysteroid groups in eggs immediately follow-
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FIG. 3. Total ecdysteroid levels as 20-OHE equivalents (expressed in semilogarismic terms). Ecdysteroid levels in mature ovaries shown as Day 0. Total ecdysteroid levels decrease gradually until Day 10, increasing rapidly around Day 13, culminating in 20-fold higher levels by Day 18 hatchout.
wt) were comparatively of the same order of magnitude. In the spider crab, Acanthonyx iunulatus, 20-hydroxyecdysone and ecdysone were detected in mature ovaries, with the inclusion of HPP and LPP in newly Day 0 spawned eggs (Chaix and De Reggi, 1982). In the shore crab, Carcinus maenus, ecdysone, 20-hydroxyecdysone, and a lowT I-L 10 20 30 'PO polarity ecdysteroid, ponasterone A, were E present in ovaries (Lachaise and Hoffman, LJJ ioo0) HPP 20-OHE E 1977; Lachaise et al., 1981) and in newly LPP g spawned eggs (Lachaise and Hoffman, I E 1982). In the above crab species, total ecjj 75' zi dysteroid levels in ovaries (and in eggs 0. throughout embryogenesis) were on an order of picomoles per milligram wet weight, whereas in M. rosenbergii this was about IO-fold higher in ovaries and newly laid Day 16 eggs (and about IOO-fold higher in the later stages of embryonic development). The IJh -. presence of ecdysteroids in the eggs and 10 20 3b %I ovaries of the Orchestia gammarellus is Retention Time (min) also well known (Blanchet et al., 1979). FIG. 4. Separation of ecdysteroids by reversedM. rosenbergii was in contrast to other phase HPLC followed by RIA. Relative percentages of species regarding its relatively low concenhigh-polarity products (HPP), 20-hydroxyecdysone tration of ovarian ecdysone. Ecdysone was (20-OHE), ecdysone (E), and low-polarity products (LPP) shown for Day 0 (mature ovaries) and Day 16. present, but levels of 20-hydroxyecdysone and HPP were most significant, present at ing spawning has been previously shown lo-fold higher levels. In addition, the pres(Spindler et al., 1987). Total ecdysteroid ence of comparatively high concentrations concentrations in ovaries (26.5 rig/g wet wt) of HPP of strong immunoreactivity in M. and in newly laid eggs (16.9 rig/g wet wt) of rosenbergii differs markedly from the nathe M. rosenbergii and concentrations in the ture of HPP in these other decapod crustaeggs of the P. serratus (approx. 50 rig/g dry ceans. In C. maenus, HPP consisted of 751
FIG. 5. Changes in relative percentages of HPP, ZO-OHE, E, and LPP during embryogeoesis (~a& culated as 20-OHE equivalents).
poorly immunoreactive high-polarity conjugates of ecdysone, 20-hydroxyecdysone, and ponasterone A, and possibly 20,26dihydroxyecdysone. In A. lunulatus, ovarian HPP found concentrated in newly laid eggs was not present to the extent that was ecdysone and 20-hydroxyecdysone. Regarding insecta, the same maternal ecdysteroids classes have been observed. In Locust migratoria, ecdysteroids in newly laid eggs contained in the same concentration as in terminal oocytes immediately prior to ovulation consisted of ecdysone, 20-hydroxyecdysone, and low- and high-polarity products. Low-polarity products were further identified as 2-deoxyecdysone, 2,22-bisdeoxyecdysone, 2,22,25trideoxyecdysone, and 2,14,22,25tetradeoxyecdysone. High-polarity products were determined as conjugates of ecdysone and low-polarity ecdysteroids (Lagueux et al., 1979). A number of reports indicate that maternal ecdysteroids are destined to play a role in embryogenesis, and it is considered here that this is the fate of such ovarian ecdysteroids in M. rosenbergii. The apparent gradual decrease in ecdysteroid observed until Day 10 is possibly due to the utilization of maternal ecdysteroids by the embryo in the metamorphosis to the zoeal stage from the naupliar. Associated with this morphogenesis is the formation of the carapace; ecdysteroids are possibly involved in the secretion of this epicuticular structure. An analogous event in P. serratus is the formation of the first limb buds after a gradual decrease in maternal ecdysteroids (Spindler et al., 1987). It is tempting to draw parallels to the insecta concerning the biosynthetic site of maternal ecdysteroids in M. rosenbergii. In insects, the follicle cells of the ovaries are the determined site of ecdysteroid in reproductive adulthood (Lagueux et al., 1977; Goltzene et al., 1978), as the prothoracic glands have already degenerated (see review, Hagedom, 1983). The spider crab, A.
lunulatus, is very similar in this respect, with the Y-organs degenerating with the pubertal molt (Chaix and De Reggi, 1982). However, in M. rosenbergii, the Y-organ is thought to remain an active synthetic site of ecdysteroids involved in adult molting. While it can be speculated that ecdysteroids intended for embryogenic development are synthesized in the ovaries, ecdysteroids have also been well detected in hemolymph (T. Okumura et al., in preparation); the possibility also exists that ecdysteroid produced by the Y-organ is transported via the hemolymph to be taken in by the ovaries. An investigation on ovarian ecdysteroid levels is in progress. Subsequently, in the 18-day embryonic period of M. rosenbergii, dramatic rises in ecdysteroid levels commenced after Day 10, with further development of the carapace, as well as the appearance of the compound eye, digestive structures, and a beating heart, etc. In P. serratus, ecdysteroid levels increased dramatically with the formation and completion of the Y-organ midway through embryogenesis (Spindler et al., 1987). It seems plausible that a Yorgan-like structure develops around Day 10 in the M. rosenbergii in order to direct further embryonic development. Ecdysteroid profiles of M. rosenbergii appear remarkably similar to those of P. serratus. Ecdysteroid levels are in accordance: In P. serratus, ecdysteroid levels reach approximately 500 and 300 ng ecdysone equivalents per dry weight in an embryonic period of 105 and 35 days, at 19 and 1l”, respectively. Somewhat lower values are obtained if data are corrected according to fresh weight (Spindler et al., 1987), but this does not appear to be a significant deviation from Day 18 ecdysteroid levels in M. rosenbergii of nearly 500 rig/g egg. While LPP and ecdysone fall to low or undetectable concentrations in both prawn species, followed by a subsequent steep increase in 20-hydroxyecdysone and HPP, in the P. serratus, HPP does not exceed levels
of 20-hydroxyecdysone. However, in M. as HPP and 20-hydroxyecdysone increase concomitantly, HPP reaches levels of fivefold greater than those of 20hydroxyecdysone with an observed decrease in 20-hydroxyecdysone. In the crabs, C. maenus (Goudeau and Lachaise, 1983; Lachaise and Hoffman, 1982), A. lunulatus (Chaix and De Reggi, 1982), and in insecta such as the fly, Calliphora erythrocephala (Bordes-Alleaume and Sami, 1987), ovoviviparous cockroach, Nuuphoeta cineru (Imboden et al., 1978), and the common grasshopper, Locust migrutoriu (Sal1 et al., 1983; Lagueux et al., 1979), a different pattern of fluctuations is seen in which four to five peaks of ecdysone are correlated with successive depositions and apolytions of egg cuticles or with embryonic molts. A single ecdysteroid peak is coincident with the formation of the cuticular tracheal system in the fleshfly Sarcophaga bullata (Wentworth and Roberts, 1984). However, all examples suggest a fundamental role in affecting the embryonic epithelium in context of differing developmental strategies. In M. rosenbergii, there does not appear to be evidence that the egg secretes successive cuticles, but we consider that a 20-fold increase in ecdysteroid levels must have a significant physiological role. The steady rise in ecdysteroid concentrations may be in preparation for a dramatic physiological change-perhaps in exuviation at hatching, i.e., a transformation from an egg-encapsulated state, to free-swimming larvae. Again, as differentiation of the Y-organ in crustacea or the prothoracic glands in insectae is thought to belie increases in ecdysteroid titers during embryogenesis, we recognize a necessity in confirming the existence of the putative Y-organ in M. rosenrosenbergii,
However, as 20-hydroxyecdysone is universally considered to be the crustacean hormone with biological activity, the overwhelming presence of HPP, especially dur-
ing the latter stages of development, cannot be disregarded. In M. rosenbergii, it is very possible that HPP is a metabolic by-product of 20-hydroxyecdysone. In C. maenus, peaks in ponasterone A discussed earlier are always followed by subsequent peaks of HPP consisting of conjugated ponasterone A (Lachaise and Hoffman, 1982), suggesting that conjugation is the first degradative step in ecdysteroid metabolism. On the other hand, the rather high concentrations of HPP may indicate that biological activity is not limited to one ecdysteroid, but that according to species and physiological necessity, different ecdysteroids are involved in various processes. In L. migratoria, preceeding deposition of an inner cuticle and a procuticle, ecdysone is hydroxylated to 20hydroxyecdysone and 20,26-dihydroxyecdysone, suggesting that ecdysteroids varying slightly in structure control the secretion of different cuticle types (Lagueux et al., 1979). The identity of HPP in our species is the subject of current investigation-preliminary studies indicate that HPP contains conjugates of ecdysone and 20hydroxyecdysone (M. N. Wilder et al., in preparation). Through this investigation, regarding the roles of ecdysteroids and their economies during embryogenesis, we have developed an awareness that although differences are seen within the crustacea, fundamental similarities exist between our species and other decapod crustacea. Further consideration of the insecta model may prove to be an aid in gaining a more complete understanding of the M. rosenbergii and crustatea in general. ACKNOWLEDGMENTS This study was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.
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