Enrique M. Rodríguez,* Laura S. López Greco,* Daniel A. Medesani,* Hans Laufer, and Milton Fingerman

Transcription

1 General and Comparative Endocrinology 125, (2002) doi: /gcen , available online at on Effect of Methyl Farnesoate, Alone and in Combination with Other Hormones, on Ovarian Growth of the Red Swamp Crayfish, Procambarus clarkii, during Vitellogenesis Enrique M. Rodríguez,* Laura S. López Greco,* Daniel A. Medesani,* Hans Laufer, and Milton Fingerman *Animal Physiology Laboratory, FCEyN, University of Buenos Aires, Ciudad Universitaria, C1428EHA Buenos Aires, Argentina; Department of Molecular and Cell Biology, The University of Connecticut, Storrs, Connecticut 06268; and Department of Ecology and Evolutionary Biology, Tulane University, New Orleans, Louisiana Accepted August 20, 2001 The effect of methyl farnesoate (MF) on the ovaries of female red swamp crayfish, Procambarus clarkii, undergoing vitellogenesis was determined both in vivo and in vitro. The in vivo assay showed a positive effect of MF on oocyte growth when injected alone and in combination with 17 -estradiol, but not in combination with JHIII or 17 -hydroxyprogesterone. A higher level of incorporation of labeled leucine was induced by MF on isolated pieces of ovary. The same effect was seen when ovary and mandibular organ (MO) were coincubated. These results suggest that MF stimulated the synthesis of vitellin in the ovary of crayfish. In vitro, 17 -hydroxyprogesterone completely suppressed the stimulatory action of the MO on the ovary, suggesting a competitive inhibition between 17 -hydroxyprogesterone and MF on the ovary and/or a negative feedback by that steroid on the MO. Key Words: methyl farnesoate; hormones; crayfish; vitellogenesis. INTRODUCTION Methyl farnesoate (MF), a sesquiterpenoid compound secreted by the mandibular organs (MO) has been identified by Laufer et al. (1987) as the juvenile hormone of crustaceans. Structurally, it is the unepoxidated form of the juvenile hormone III (JHIII) of insects and has been shown to have roles in both growth and reproductive processes. High hemolymphatic levels of endogenous MF were found in both vitellogenic females and reproductively active males (see review by Laufer et al., 1993). Stimulatory in vivo effects of MF on ovarian maturation of the red swamp crayfish, Procambarus clarkii, throughout all the vitellogenic ovarian stages has been reported (Laufer et al., 1998). MF was also able to produce growth of penaeid oocytes in vitro (Tsukimura and Kamemoto, 1991). Also, some stimulatory effects of MF on molting have been seen in crustaceans, both in vitro (Tamone and Chang, 1993) and in vivo (summarized by Homola and Chang, 1997). A mandibular organ inhibiting hormone (MOIH), secreted by the sinus gland in the eyestalks, has been identified and sequenced in some crustaceans species (Liu et al., 1997; Webster, 1998). However, practically no attempts have been made to assay the effect of MF in combination with other hormones shown to have reproductive functions. The current knowledge about the neurohormonal regulation of reproduction and growth has been summarized (Charmantier et al., 1997; Fingerman, 1997, /02 $35.00

2 Effect of Methyl Farnesoate on Crayfish 35 among others), but several pieces of this puzzle are still waiting to be identified and functionally characterized, especially those involving the interactions between the components of the multiple hormonal system involved. Among the neurotransmitters having a demonstrated role in reproduction, the stimulatory effect of serotonin has been reported in several studies, by stimulation of the secretion of a gonadal stimulating hormone (GSH) from brain and thoracic ganglia (reviewed by Fingerman, 1997). Some other hormones possibly play a role in crustacean reproduction. Among them, some vertebratelike steroids such as progesterone and estradiol have been detected in the hemolymph of several crustacean species, their levels changing in correlation with the oocyte maturation cycle (Fingerman et al., 1995). A close correlation was also noted between a rise in hemolymphatic steroids and vitellogenin (Quinitio et al., 1994; Shih, 1997). Stimulatory effects on growth of shrimp oocytes were seen in vitro with 17 -hydroxyprogesterone, MF, and JHIII (Tsukimura and Kamemoto, 1991). The objective of this study was to assay in the crayfish, P. clarkii, the effect of MF alone and in combination with other reproductive factors. In vivo assays involved injection of selected combinations of various compounds into females that had already entered active vitellogenesis. In addition, some in vitro assays were carried out to test for possible interactions among the assayed compounds. METHODS In Vivo Experiments Adult female P. clarkii, averaging mm in carapace length (corresponding to a weight of g), were obtained from a local seafood dealer. Ten females were sacrificed at the beginning of the experiment (June 5, 2000) to evaluate the degree of oocyte growth (initial control), according to Kulkarni et al. (1991). During the assay, the animals were kept in recirculating freshwater aquaria, equipped with biological filtration. Six hours before being injected, crayfish were fed ad libitum on commercial, hormone-free pellets for shrimp (Burris Mill & Feed, Inc., Franklinton, LA). The photoperiod (12L:12D h) and temperature (24 1 ) were held constant throughout each experiment to evaluate only the effect of the injected chemicals on reproduction and growth. Ten females were randomly assigned to each of the following experimental groups, each group being in a separate aquarium: (A) control (crayfish saline ethanol 5% v/v), (B) MF, (C) MF JH III, (D) MF 17 -hydroxyprogesterone, and (E) MF 17 -estradiol. The biological isomer all-trans MF was extracted and purified by Dr. Hans Laufer, according to the procedure described previously (Laufer et al., 1987). All remaining drugs were purchased from Sigma Chemical Co., St. Louis, Missouri. All chemicals were dissolved in crayfish saline solution (van Harreveld, 1936). Methyl farnesoate, JHIII, 17 -hydroxyprogesterone, and 17 -estradiol were first dissolved in ethanol. The control group received crayfish saline solution plus the amount of ethanol used as carrier (5% v/v). MF was injected, both alone and in combination with the other chemicals, at a dose of 10 8 mol (near g)/crayfish, in accord with the levels previously used in the same species (Laufer et al., 1998). The doses used for the other chemicals were similar to those employed in previous studies with several crustacean species: 10 7 mol/crayfish for JHIII, 17 -estradiol, and 17 -hydroxyprogesterone (Nagabhushanam et al., 1980, 1983). The injection volume was 100 l in all cases. All chemical solutions were prepared weekly and were injected in the abdominal muscle by means of 1-ml syringes (27G needle). Since the hormones used are relatively quickly degraded by crustaceans (Chang, 1987; Young et al., 1992), injections were given twice a week, for a period of 3 weeks. The crayfish were inspected daily and those that molted were isolated individually, to avoid cannibalism in the aquaria. The water in the pans that contained the isolated crayfish was the same as that used in the recirculating system and was replaced every day. At the end of the assay, all the animals were weighed, their gonads were then quickly dissected out and finally weighed. The gonadosomatic index (GSI) was calculated as fresh weight of ovary/whole crayfish 100. In addition, the same weighed gonads from both the initial and the concurrent controls were fixed

3 36 Rodríguez et al. overnight in Bouin s solution, at 5, to estimate the mean oocyte diameter (MOD) to check the vitellogenic stage according to the scale described by Kulkarni et al. (1991). Further histological processing was the same as that used in that study. MOD were then measured by means of a 8 Zeiss micrometric ocular lens, calibrated against a Leitz Wetzlar plate with 10- m spacing, on a representative section of each ovary. Every oocyte whose nucleus was visible in the section was measured. The proportion of molted animals was also calculated for each experimental group. In Vitro Experiments The effect of some of the assayed chemicals on oocyte growth was evaluated in vitro with regard to the incorporation of leucine into acid-precipitable proteins, by isolated pieces of ovary. In all cases, 10-ml sterile vials were used, containing 2 ml of Medium 199 (as powdered medium with l-glutamine and Earle s salts; Sigma Chemical Co.) dissolved in Van Harreveld s (1936) crustacean saline, modified to compensate for the salts already present in the culture medium. In addition, as in previous studies (Sarojini et al., 1995c; Rodríguez et al., 2000), 6 mg of penicillin G per 100 ml of medium was added to prevent bacterial growth; and the ph was adjusted to 7.4 with 0.5 N NaOH. All vials were incubated for 24 h in an orbital shaker (50 60 rpm), at a temperature of 25 1 in constant darkness. Ten vials were used for all of the experimental groups, each vial receiving a piece of ovary (approximately cm) from a different female. An aliquot of 30 l from the tritiated leucine stock solution (1:10 dilution from 1 mci/ml; Sigma Chemical Co.) was added to each vial, so that the total activity in each vial was 3 Ci, the same as that used by Eastman-Reks and Fingerman (1985). The ovaries also were processed according to the method used by these authors. Briefly, the ovaries were homogenized in 2 ml cold 10% trichloroacetic acid (TCA) and centrifuged at 5000g for 10 min at 4 ; pellets were washed twice with cold TCA, resuspended, and decanted onto a Millipore suction filtration funnel. The filter paper discs (nitrocellulose; 0.22 m) were then air-dried and submerged in the fluor for radioactive determination. The protein concentration in the ovaries was measured in an aliquot of the ovarian extracts by means of a Lowry micro-method for determination of total proteins (Sigma Chemical Co.). Uptake of labeled leucine by an ovary was expressed as CPM/ g protein. First experiment. Three concentrations of methyl farnesoate were assayed on isolated pieces of ovary. Final concentrations in the experimental vials were 1.5, 5.0, and 15.0 M (volume added 20 l), the lowest concentration being close to that expected in hemolymph immediately after injection in the in vivo assay. A control series having only the concentration of ethanol presented as solvent in the highest concentration of methyl farnesoate (9.4 l/ml) was also run. Second experiment. To evaluate both the effect on oocyte growth of a mandibular organ in the culture medium and any effect produced by some of the substances assayed in vivo, the following experimental groups were run: (A) ovary muscle, ethanol added; (B) ovary muscle, JHIII added; (C) ovary muscle, 17 -hydroxyprogesterone added; (D) ovary muscle, 17 -estradiol added; (E) ovary mandibular organ, ethanol added; (F) ovary mandibular organ, JHIII added; (G) ovary mandibular organ, 17 -hydroxyprogesterone added; and (H) ovary mandibular organ, 17 -estradiol added. The mandibular organs were identified by the descriptions in several reports, especially that of Yudin et al. (1980). The organs were carefully dissected out, together with the small anterior portion of the chitinous tendons where they were closely attached, and added to the same vial as that having the ovary of the same female. In the control series, a piece of muscle taken from the chelipeds, similar in size to a mandibular organ, was added. Each vial received an aliquot of 15 l from the stock solution of each hormone assayed. The concentration of each of the hormones added to the vials was 15 M, similar to that expected in the in vivo assays immediately after injection. Statistical Analyses A one-way ANOVA, followed by planned comparisons of each treatment against controls (Sokal and Rohlf, 1981), was applied to analyze MOD and the results from the first in vitro experiment, and a twoway ANOVA was used to analyze the results from the second in vitro experiment, with kinds of tissue and hormones as factors. The Fisher exact test (Sokal and

4 Effect of Methyl Farnesoate on Crayfish 37 Rohlf, 1981) was used to compare the proportions of molted crayfish. RESULTS Mortality throughout the in vivo assay averaged 26%. Mean MOD of initial control indicated that crayfish were starting into the midvitellogenic stage at the beginning of the assay, whereas the mean value for saline, concurrent control, revealed that the animals were well advanced in the midvitellogenic stage at the end of the experiment, according to the staging system of Kulkarni et al. (1991). Few animals molted during the assay; no significant differences (P 0.05) in the proportions of molting were found with respect to the saline control group. Final number of crayfish are shown in Fig. 1, together with the GSI values obtained at the end of the assay. All the experimental groups showed a GSI value significantly higher (P 0.05) than that of the initial control (Fig. 1). However, only the groups injected with MF alone and MF plus 17 - estradiol showed a GSI significantly higher (P 0.05) than that of the saline concurrent control. Figure 2 shows the results obtained from the first in vitro experiment. All tested concentrations of MF showed a value higher than that of the control, but only the highest caused a significantly higher (P 0.05) growth of the ovary, with respect to the control group. The results of the second in vitro experiment are shown in Fig. 3. No significant differences (P 0.05) were found in the series having only ovary, that is, between each hormone tested and the saline control. The same comparison for the other series (ovary plus mandibular organ) yielded significantly lower values than the saline plus mandiular organ group, for both 17 -hydroxyprogesterone and 17 -estradiol. Although the values for even the mandibular organ JHIII group were lower than those for the saline plus mandibular organ group, the addition of the mandibular organ to the culture medium produced per se (in FIG. 1. Gonadosomatic indexes ( standard error), at the end of the in vivo assay. Small asterisks indicate significant differences (P 0.05) with respect to the initial control; large asterisks indicate the same with respect to the saline, concurrent control. MF, methyl farnesoate. The numbers of crayfish alive at the end of the assay are indicated between parentheses. The doses used were 10 8 mol/ crayfish for MF and 10 7 for the remaining hormones. FIG. 2. Uptake of labeled leucine by oocytes at the end of the first in vitro assay. Mean values ( standard errors) are indicated. An asterisk indicates a significant difference (P 0.05) with respect to the control. saline control) a significantly higher (P 0.05) uptake of leucine, in comparison to the group that had only ovary. This significant difference (P 0.05) was maintained when JHIII and 17 -estradiol were added, but not in the group receiving progesterone, where the positive effect produced by the mandibular organ in the other groups was abolished.

5 38 Rodríguez et al. FIG. 3. Uptake of labeled leucine by oocytes at the end of the second in vitro assay. Mean values ( standard errors) are indicated. Asterisks indicate significant differences (P 0.05) with respect to the control of the same series; signs indicate significant differences (P 0.05) between series, for a given treatment. The concentration of each hormone was 15 M. DISCUSSION The injection of MF during the 3-week in vivo assay (Fig. 1) showed a clear induction of ovarian growth, similar in magnitude to that reported by Laufer et al. (1998) in the same species assayed in June (as in this study) by feeding daily on pellets enriched with MF. The dose of MF employed in both cases was similar (about 2 g/animal), although in the current study we administrated only two doses each week, since neither digestive nor absorptive processes were mediating the intake of the hormone. We saw a stimulatory effect of MF in vitro (first in vitro experiment, Fig. 2), with regard to higher leucine incorporation into oocyte proteins. The incorporation of labeled leucine into vitellin subunits has been demonstrated in crab ovaries, by means of the same protocol used in this study (Eastman-Reks and Fingerman, 1985). The results of coincubation of mandibular organs and ovary, with no hormone added (second in vitro experiment; Fig. 3), were consistent with the data in Figs. 1 and 2, when the fact that MF is the main hormone secreted by MO is taken into account (Laufer et al., 1987). However, the possibility of cosecretion of other hormone(s) by MO to some extent should be taken into account, i.e., farnesoic acid (Chang, 1993) and some vertebrate-like steroids, as suggested for Homarus americanus (Couch et al., 1987; Waddy and Aiken, 2000). In this sense, however, no direct effect of the assayed steroids on the ovary were seen, as discussed below. A previous study reporting the in vitro effect of MF, JHIII, and several steroids on the oocytes of the shrimp Penaeus vannamei was done by Tsukimura and Kamemoto (1991). These authors found a higher oocyte diameter in pieces of ovary incubated with MF, JHIII, or 17 -hydroxyprogesterone at concentrations ranging from 10 pm to 100 nm. The evidence currently available indicates that in penaeids endogenous and exogenous vitellogenesis both take place in the ovary. In addition to the vitellin synthesized by oocytes, the ovarian follicular cells produce its precursor, vitellogenin, which is taken up to a great extent by the oocytes during secondary vitellogenesis (reviewed in Yano, 2000). A similar pattern was reported for the crayfish P. clarkii with a quantitative enzyme-linked immunosorbent assay (Tuberty and Fingerman, 1998). Although the pattern for the synthesis of yolk proteins in the ovary seems to be similar in P. vannamei and P. clarkii, in the later species only MF exerted a stimulatory effect in vitro, among all compounds tested, albeit at a concentration of 15 M, this concentration being 10-fold higher than that injected in the in vivo assay. MF could theoretically have caused the effect observed in vitro (Fig. 2) by stimulating the secretion of other hormone(s) by the incubated ovary, mainly the secretion of steroids by follicular cells, as suggested by other authors (Quackenbush, 1994; Yano, 2000). However, from the second in vitro experiment (Fig. 3), it is clear that no direct stimulatory effect on the ovary by 17 -hydroxyprogesterone or 17 -estradiol (administered with no mandibular organ added) took place during the assay. Nevertheless, we cannot discard the possibility that any other gonadotropic hormone which the same gonads eventually could produce (such as ecdysteroids, which were not assayed in this study) is being secreted in response to MF. In any case, the hypothesis that MF/MO products directly stimulate the synthesis of vitellin in the ovary of P. clarkii (i.e., vitellin synthesis in oocytes and/or vitellogenin synthesis in follicular cells) seems plausible and should be tested.

6 Effect of Methyl Farnesoate on Crayfish 39 Nevertheless, 17 -progesterone was able to produce, when injected alone, significant oocyte growth in vivo, during early vitellogenesis (Rodríguez et al., 2001) and in the present study, involving mid-vitellogenic oocytes; 17 -estradiol plus MF also produced significant ovarian growth in vivo, even more than that produced by MF alone. Vertebrate-like steroids have been shown to induce significant oocyte growth in several crustaceans (Nagabhushanam et al., 1980, 1983; Yano, 1985, 1987). In any case, though, the dynamics of steroid secretion should be considered also. A high positive correlation between steroid and vitellogenin hemolymphatic levels has been reported for shrimp and crabs (Quinitio et al., 1994; Shih, 1997), but correlation between increased levels of progesterone and vitellogenesis is not necessarily the rule, as reviewed by Fingerman et al. (1993). Moreover, a total inhibition was caused by 17 hydroxyprogesterone on the inductive effect of MO on oocyte growth, as shown by the results from the second in vitro experiment (Fig. 3). These results clearly show that in vitro suppression of oocyte growth induced by MO products was exerted by progesterone (Fig. 3). To a lesser extent, an inhibitory effect of 17 -estradiol was also seen in vitro, partially reducing the effect of MO added to the ovary. Two main possibilities arise from these results. The first is the possibility of the steroids antagonizing the MO products at the target (ovarian) level. Since 17 hydroxyprogesterone was able to antagonize the in vivo effect of exogenous MF, this possibility seems likely, at least for that steroid. The second possibility has to do with the steroids exerting a negative feedback on mandibular organ (for MF secretion) and/or eyestalks (for MOIH secretion). In this sense, feedback mechanisms, especially those produced by the hormones secreted in the last step, are essential for the fine tuning of the secretion rates of all the hormones involved in neuroendocrine reflexes. However, although feedback systems have been studied extensively in vertebrates, very few have been documented in invertebrates. An alternative explanation for the lack of stimulation observed in vivo (Fig. 1) in almost all the combinations of MF with other compounds could be the induction of esterases and other P450-dependent enzymes by lypophilic hormones, but the exception of MF plus 17 -estradiol does not support this possibility. The complexity of the relationships between the several hormones acting on reproduction is another critical point to be considered, especially since the functional relationships between the secretion of GSH and/or inhibition of GIH with the secretion and actions of MF are still very far from being elucidated. ACKNOWLEDGMENTS We thank Carina López (CONICET, Argentina) for helping with the histological preparations and Dr. David Hurley (Tulane University, USA) for technical support. This work was partially supported by a grant from ANPCYT, Argentina (PICT ). REFERENCES Chang, E. S. (1987). Chemistry of crustacean hormones that regulate growth and reproduction. In Recent Advances in Marine Biotechnology (M. Fingerman, R. Nagabhushanam, and M.-F. Thompson, Eds.), Vol. I, pp Oxford & IBH Publishing Co., New Delhi. Chang, E. S. (1993). Comparative endocrinology of molting and reproduction: Insects and crustaceans. Annu. Rev. Entomol. 38, Charmantier, G., Charmantier-Daures, M., and Van Herp, F. (1997). Hormonal regulation of growth and reproduction in crustaceans. In Recent Advances in Marine Biotechnology, (M. Fingerman, R. Nagabhushanam, and M.-F. Thompson, Eds.), Vol. I, pp Oxford & IBH Publishing Co., New Delhi. Couch, E. F., Hagino, N., and Lee J. W. (1987). Changes in estradiol and progesterone immunoactivities in tissues of the lobster, Homarus americanus, with developing and immature ovaries. Comp. Biochem. Physiol. A 85, Eastman-Reks, S. B., and Fingerman, M. (1985). In vitro synthesis of vitellin by the ovary of the fiddler crab Uca pugilator. J. Exp. Zool. 233, Fingerman, M. (1995). Endocrine mechanisms in crayfish, with emphasis on reproduction and neurotransmitter regulation of hormone release. Am. Zool. 35, Fingerman, M. (1997). Roles of neurotransmitters in regulating reproductive hormone release and gonadal maturation in decapod crustaceans. Invertebr. Reprod. Dev. 31, Fingerman, M., Nagabhushanam, R., and Sarojini, R. (1993). Vertebrate-type hormones in crustaceans: Localization, identification and functional significance. Zool. Sci. 10, Homola, E., and Chang, E. S. (1997). Methyl farnesoate: Crustacean juvenile hormone in search of function. Comp. Biochem. Physiol. B 117,

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