© 2001 by The Society for Integrative and Comparative Biology
Molecular Cloning, Expression, and Tissue Distribution of Crustacean Molt-Inhibiting Hormone1
1 Department of Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294
2 Department of Microbiology and Center for Macromolecular Crystallography, University of Alabama at Birmingham, Birmingham, Alabama 35294
3 Department of Biological Sciences, University of North Carolina at Wilmington, Wilmington, North Carolina 28403
4 Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242
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In crustaceans, secretion of ecdysteroid molting hormones by Y-organs is regulated by molt-inhibiting hormone (MIH), a neuropeptide produced by the X-organ/sinus gland complex of the eyestalks. The current review considers recent research on MIH, with a primary focus on MIH of brachyurans (crabs). New data on the production of recombinant MIH (rMIH) are also included. Available data indicate the MIH gene of brachyurans encodes a 113 amino acid prohormone composed of a 35 residue signal peptide and a 78 residue mature MIH. The primary structure of MIH is highly conserved among brachyurans. The MIH transcript is detectable in eyestalk neural ganglia throughout the molt cycle of the blue crab, Callinectes sapidus. Stage-specific changes in the abundance of MIH mRNA in C. sapidus eyestalks are generally consistent with the hypothesis that MIH negatively regulates ecdysteroid production during the molt cycle. MIH transcripts have also been detected in the brain of two species. Recombinant MIH was produced using prokaryotic (pET vector/Escherichia coli) and eukaryotic (baculovirus/insect cells) expression systems. Recombinant MIH produced in E. coli was of the predicted size and was MIH immunoreactive; it did not have MIH bioactivity. Polyclonal antisera raised against the prokaryotically expressed rMIH bound specifically to neurosecretory cells in the X-organ, their associated axons, and axon terminals in the sinus gland. Recombinant MIH expressed using the baculovirus system was of the predicted size, was MIH immunoreactive, and inhibited ecdysteroid production by Y-organs in vitro.
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INTRODUCTION |
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Crustacean postembryonic development, including cycles of molting and regeneration, is controlled by C-27 steroid hormones termed ecdysteroids. Ecdysteroids are synthesized and secreted by paired Y-organs, ectodermally-derived endocrine glands located in the anterior cephalothorax. The ecdysteroidogenic activity of Y-organs is negatively regulated (inhibited) by molt-inhibiting hormone (MIH), a neuropeptide produced in the crustacean eyestalk (reviews: Skinner, 1985
; Fingerman, 1987; Watson et al., 1989; Chang, 1993; Lachaise et al., 1993).
In decapod crustaceans, the protocerebrum extends into the eyestalks as a series of four neural ganglia. The most proximal ganglion, the medulla terminalis, contains a cluster of neurosecretory cells collectively termed the X-organ. Axons extend distally from X-organ somata and end in the sinus gland, a collection of bulbous axon terminals from which MIH is thought to be released (Bliss and Welsh, 1952; Passano, 1953; review: Skinner, 1985). Thus, ablation of the eyestalks leads to an increase in the hemolymph ecdysteroid titer and precocious molting (Chang and Bruce, 1980; Hopkins, 1982), while injection of eyestalk extract into eyestalk-ablated animals lowers the ecdysteroid titer and delays molting (Bruce and Chang, 1984; Chang et al., 1987). Further, eyestalk or sinus gland extracts, or purified MIH, suppress ecdysteroid production by Y-organs in vitro (Soumoff and O'Connor, 1982; Watson and Spaziani, 1985; Mattson and Spaziani, 1985; Webster, 1993). These and related observations have led to a working model for the endocrine regulation of crustacean molting: It is hypothesized that during much of the molting cycle, MIH from the X-organ/sinus gland complex inhibits ecdysteroid synthesis by Y-organs and thus suppresses molting; a molting sequence is thought to be initiated when MIH secretion diminishes or stops and ecdysteroid production by Y-organs increases.
The neuroendocrine regulation of crustacean molting is under active investigation for a variety of reasons. From the standpoint of comparative physiology, the crustacean system is proving to be a useful model for study of the regulation of steroidogenesis and the structure and function of neurosecretory systems. Like vertebrate steroidogenic glands, the Y-organs utilize cholesterol as a precursor for steroidogenesis (Watson and Spaziani, 1985) and are controlled by a regulatory peptide. Y-Organs offer the advantage over vertebrate gonads and adrenal cortices of being structurally simple and histologically homogeneous; MIH provides the potentially insightful contrast of inhibiting steroidogenesis—the principal regulatory peptides of vertebrate glands stimulate steroidogenesis. In addition, the ready accessiblity and experimental tractability of the X-organ/sinus gland complex have made it an important model for study of the structure and function of neurosecretory cells (Durand, 1956; Andrews et al., 1971; Cooke et al., 1977; Keller et al., 1994). Indeed, study of invertebrate systems, including the X-organ/sinus gland complex, provided insights crucial to the understanding of neuroendocrine systems in general (see Scharrer and Scharrer, 1954). From the standpoint of applied science, manipulation of the crustacean neuroendocrine system holds the prospect of providing a method for controlling the growth of wild and cultured species, an attractive possibility for fisheries managers and the aquaculture industry.
The current article discusses recent research on crustacean MIH, with a primary focus on MIH of brachyurans. It includes a review of recent findings from our laboratories on the cloning of cDNAs encoding MIH, and the tissue sites and developmental patterns of MIH gene expression. Also included are new data on the expression of recombinant MIH in Escherichia coli.
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MATERIALS AND METHODS |
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Animals
Callinectes sapidus were purchased from local fishermen in Weeks Bay, AL or Wilmington, NC, or from Gulf Specimen Marine Laboratories, Inc. (Panacea, FL). They were maintained in recirculating seawater tanks as previously described (Lee and Watson, 1995
), and staged according to established criteria (Drach and Tchernigovtzeff, 1967; Stevenson, 1972). Cancer magister were collected from Yaquina Bay, Newport, OR, shipped by air to the University of Alabama at Birmingham, and used immediately upon arrival. For RNA extraction or immunocytochemistry, eyestalks were clipped from crabs and transferred to Pantin's saline (Pantin, 1934); soft tissues were removed and non-neural tissues trimmed away. For some experiments, samples of gill, claw muscle, hepatopancreas, gonad, and thoracic ganglion were also removed to serve as controls. All tissues were quick frozen in liquid nitrogen and stored at –80°C until analysis. For one experiment, hemolymph was drawn by syringe from the bases of walking legs, added to two volumes of methanol, and stored at –20°C until analysis.
Molecular cloning and sequence analysis
Complementary DNAs encoding MIH of C. sapidus and C. magister were cloned as previously described (Lee et al., 1995; Umphrey et al., 1998). MIH amino acid sequence data were compared (clustal alignment, PAM250 distance tables) using MegAlign software from DNA STAR.
Determination of sites of MIH mRNA expression
Sites of MIH mRNA expression in C. sapidus were determined by Northern blot as described by Lee et al. (1995). Total RNA was extracted using TRIzol Reagent (GIBCO-BRL), then separated electrophoretically on a 1.2% agarose/0.66 M formaldehyde gel, transferred to a Nytran membrane (Schleicher and Schuell), and crosslinked by uv radiation (254 nm, 125 mJ total energy). A 253 base-pair probe was generated by PCR amplification of a portion of the cloned C. sapidus MIH cDNA, then labeled by random priming (Prime-a-Gene Labeling Kit, Promega). Prehybridization, hybridization, and wash conditions were as described (Lee et al., 1995). Blots were exposed to Kodak X-OMAT film at –80°C.
Determination of MIH mRNA levels during a molt cycle
Steady state MIH mRNA levels were determined during a molt cycle as described by Lee et al. (1998). Eyestalk neural ganglia were pooled by stage (4–8 eyestalks per sample) for the following stages of the molt cycle: intermolt (C4), early premolt (D1), mid-premolt (D2), late premolt (D3), very late premolt (D3/D4) and postmolt (A/B). Total RNA was extracted and MIH mRNA detected by Northern blot as described above. After exposure to the MIH probe, the membrane was washed in boiling 0.1x SSC/0.1% SDS, then reexposed to film to ensure that the MIH cDNA probe had been stripped. To control for unequal loading and transfer of RNA, the stripped membrane was reprobed as described above using a radiolabeled fragment of an actin cDNA from the lobster, Homarus gammarus (El Haj et al., 1997). The hybridization signal density on experimental and control autoradiograms was determined densitometrically using a Personal Densitometer and ImageQuant software, both from Molecular Dynamics. To determine the normalized steady state level (abundance) of MIH mRNA, the absorbance obtained using the MIH cDNA probe was divided by that obtained using the actin probe. Data were expressed relative to the MIH/actin ratio in postmolt (stage A/B) neural ganglia.
Determination of the hemolymph ecdysteroid titer
Hemolymph samples (diluted 1:3 with methanol) were centrifuged (15 min, 1,500 g, 4°C) and the supernatant saved. Pellets were extracted two additional times with 2 ml methanol as described above, and the supernatants combined with the first. The combined supernatants were dried under a gentle stream of nitrogen, and the dried residue dissolved in 1 ml methanol. Aliquots were removed from samples, dried under vacuum, and their ecdysteroid content determined by radioimmunoassay as previously described (Lee et al., 1998).
Expression of recombinant molt-inhibiting hormone (rMIH) in Escherichia coli
For expression of rMIH, a PCR fragment encoding the mature MIH peptide was amplified from a previously cloned C. sapidus MIH cDNA (Lee et al., 1995). The 5' primer was designed to create a NdeI restriction site containing an initiation codon. The 3' primer introduced a stop codon and BamHI restriction site. The PCR fragment was digested with NdeI and BamHI, and cloned into the appropriate sites of the pET15b vector (Novagen). The construct was designed to yield an expressed fusion protein composed of rMIH with a polyHis tag at the N-terminus. E. coli (strain BL21(DE3)) were transformed with MIH/pET15b, and the sequence and reading frame of the insert were confirmed by DNA sequencing using the dideoxy chain termination method (Sequenase DNA Sequencing Kit, United States Biochemical Corporation). Protein expression was induced by adding 0.5 mM IPTG, and the cells harvested after 4 hr. The cells were disrupted by sonication, samples separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and gels stained using Coomassie Blue R-250 (Hames, 1986). Subsequent fractionation of extracts by centrifugation revealed the expressed protein was present in insoluble inclusion bodies. The inclusion bodies were denatured with 6 M guanidine-HCl and purified on a nickel affinity column using a protocol provided by the supplier (Novagen). The protein was refolded on the column by gradually reducing the concentration of guanidine-HCl. The refolded MIH fusion protein was dialyzed to 20 mM HEPES, 150 mM NaCl, 5 mM ß-mercaptoethanol, 10% glycerol, and its purity determined by SDS-PAGE. The MIH immunoreactivity of the expressed protein was assessed by Western blot (Harlow and Lane, 1988), using as primary antibody polyclonal antiserum raised against MIH of Carcinus maenas (Dircksen et al., 1988).
Production of anti-rMIH antiserum
Anti-rMIH immune sera were produced in rabbits according to standard protocols (Harlow and Lane, 1988). One week before immunization, blood was withdrawn from an ear vein and preimmune serum prepared. Rabbits were immunized subcutaneously with 50 µg rMIH in buffer:Freund's complete adjuvant (1:1) (Pierce). Six and twelve weeks later, animals received a boost of 25 µg rMIH in buffer:Freund's incomplete adjuvant (1:1) (Pierce). The binding properties of anti-rMIH immune sera were characterized by Western blot (Harlow and Lane, 1988) and immunocytochemistry (see below).
Immunocytochemistry
Methods for whole mount immunocytochemistry were adapted from those described by Beltz and Kravitz (1983). Dissected eyestalk neural ganglia were fixed for 14 to 18 hr in 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). After fixation, ganglia were rinsed 6 times over 6 hr in 0.1 M sodium phosphate buffer (pH 7.4), 0.3% Triton X-100, 0.1% sodium azide (PTA), and the neurilemma teased away. Ganglia were then incubated for 22 hr at 4°C with anti-rMIH (diluted 1:100 in PTA). Following primary antibody treatment, ganglia were rinsed 6 times over 6 hr in PTA, then incubated for 13–16 hr at 4°C with secondary antibody (goat anti-rabbit IgG FITC conjugate (Sigma) diluted 1:25 in PTA). Subsequently, ganglia were rinsed 6 times over 6 hr in 0.1 M sodium phosphate buffer (pH 7.4), then briefly in 0.004 M sodium carbonate buffer (pH 9.5). For conventional immunofluorescence microscopy, ganglia were mounted in glass depression slides in glycerol:0.020 M sodium carbonate buffer, pH 9.5 (4:1); coverslips were elevated with modeling clay. For confocal immunofluorescence microscopy, ganglia were mounted as above except the mounting medium contained the antioxidant n-propyl gallate (8%). Controls included omitting the primary antibody, using control rabbit serum as primary antibody, and omitting the secondary antibody. Slides were viewed using a Leitz fluorescence microscope, a Bio-Rad MRC 1000 confocal microscope, or an Olympus Fluoview confocal microscope.
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RESULTS AND DISCUSSION |
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MIH gene and peptide
Recent progress has been made in understanding the structure of the MIH gene and peptide. Klein et al. (1993)
cloned a cDNA encoding MIH of the shore crab, Carcinus maenas. Our laboratory cloned cDNAs encoding MIH of the blue crab, Callinectes sapidus (Lee et al., 1995) and the Dungeness crab, Cancer magister (Umphrey et al., 1998). Chan et al. (1998) used PCR-based genomic DNA walking to clone an MIH gene from the crab, Charybdis feriatus. More recently, Lu et al. (2000) reported the Cancer pagurus genome contains at least two copies of the MIH gene. For each crab studied, the MIH gene encodes a 113 amino acid prohormone (proMIH) composed of a 35 residue signal peptide and a 78 amino acid mature MIH. The studies by Chan et al. (1998) and Lu et al. (2000) further revealed that the MIH genes of C. feriatus and C. pagurus consist of three exons and two introns, and span approximately 4.3 kb. Sequence analysis of the 5' flanking regions showed several potential binding sites for known transcription factors (Chan et al., 1998; Lu et al., 2000).
Amino acid sequences of MIH peptides from brachyurans are shown in Figure 1. For purpose of comparison, residues identical to those of C. sapidus MIH are shaded. C. sapidus MIH shows high sequence identity (79–85%) with MIH from other brachyurans. Each peptide contains the six cysteine residues characteristic of the crustacean hyperglycemic hormone (CHH) peptide family (Keller, 1992); 54 of the 78 amino acids are common to all. The amino acid sequence of the proMIH signal peptide is also highly conserved among brachyurans (Umphrey et al., 1998; Chan et al., 1998). C. sapidus MIH shows lower sequence identity to MIH from astacurans (lobsters and crayfish) and shrimp (see Umphrey et al., 1998; Chan et al., 1998). The latter observation is consistent with the hypothesis of greater evolutionary divergence between brachyurans and astacurans than within brachyurans (Schram, 1982).
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The observed conservation of MIH primary structure among crabs is consistent with previous findings that MIH from one crab species is capable of inhibiting ecdysteroid production by Y-organs from another (Webster, 1986, 1993
). Although the MIH receptor has not been isolated or biochemically characterized for any crustacean species, this interspecific bioactivity strongly suggests that the hormone-binding domain of the MIH receptor is likely to be highly conserved among brachyurans (see Webster, 1993).
Tissue sites of MIH gene expression
Tissue sites of MIH gene expression in C. sapidus were identified by Northern blot. Using a fragment of the cloned MIH cDNA as probe, Northern blot analysis revealed specific hybridization to a single band of RNA in eyestalk but not muscle, gill, thoracic ganglion, or hepatopancreas (Fig. 2). A similar pattern of tissue-specific expression of MIH mRNA was observed for C. magister: RT-PCR with MIH-specific primers yielded a PCR product of the predicted size from eyestalk but not hepatopancreas, gonad, gill or muscle RNA (Umphrey et al., 1998). Using RT-PCR, Chan et al. (1998) detected expression of the MIH gene in eyestalks and brain of C. feriatus. Transcripts were not detected in the ovary, hepatopancreas, muscle or epidermis. The presence of MIH transcripts in the brain of C. feriatus is consistent with a previous report that mRNA encoding an MIH-like peptide is present in the brain of white shrimp, Penaeus vannamei (Sun, 1995). The finding of nontraditional sites of MIH mRNA expression raises the intriguing possibility that MIH may have alternate modes of action (e.g., as a neuromodulator or neurotransmitter), influence physiological process other than molting, or both. The occurrence of multiple modes of action and disparate physiological influences is common among vertebrate neuropeptides (see Hökfelt, 1991). The recent report by Chung et al. (1999) suggests it may also be true for CHH-family neuropeptides.
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30 µg/lane) from eyestalk neural ganglia (lane 1), muscle (lane 2), gill (lane 3), thoracic ganglia (lane 4), and hepatopancreas (lane 5) was separated on a denaturing gel (1.2% agarose/0.66 M formaldehyde). After blotting and uv-crosslinking, the membrane was probed with a [32P]dCTP-labeled fragment of a cloned C. sapidus MIH cDNA. Positions of the RNA size markers, as determined by ethidium bromide staining, are shown at left (from Lee et al., 1995In a study of MIH gene expression during development, MIH transcripts were detected (by RT-PCR) in fertilized eggs, pre-hatched embryos, and 1-day-old, 2-day-old, and 3-day-old larvae of C. feriatus (Chan et al., 1998
). Whole mount in situ hybridization revealed the MIH mRNA was found in larval brain, but not in larval eyestalks. By contrast, Webster and Dircksen (1991) observed MIH immunoreactivity in the X-organ/sinus gland complex of prezoeal and zoeal stage C. maenas larvae.
MIH mRNA levels during the molt cycle
We have recently investigated MIH gene expression during a molt cycle of C. sapidus (Lee et al., 1998). MIH mRNA was detected in eyestalk neural ganglia by Northern blot. The MIH transcript was detectable in eyestalks from all stages tested. However, densitometric analysis of autoradiograms revealed stage-specific changes in the level of MIH mRNA during the molt cycle (Fig. 3A). The level of MIH mRNA dropped steadily during premolt (D1–D4), reaching a minimum in D3/D4, then increased ten-fold in postmolt (A/B), and remained elevated during intermolt (C4). Stage-specific changes in the level of MIH mRNA were accompanied by significant fluctuations in the hemolymph ecdysteroid titer (Fig. 3B). The ecdysteroid titer increased steadily to a peak of 377.0 ng/ml in D3 of premolt, then dropped to 120.0 ng/ml in D4 (just prior to molting), and was low during postmolt (A/B, 4.4 ng/ml) and intermolt (C4, 3.3 ng/ml).
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The above pattern of changes in MIH mRNA levels is generally consistent with the model that MIH negatively regulates ecdysteroid synthesis during the molt cycle. However, data from two stages did not appear to fit the model. First, the ecdysteroid titer had begun to rise by D1 of premolt, yet MIH mRNA levels were elevated during D1. Second, the drop in the ecdysteroid titer between D3 and D4 occurred when the level of MIH mRNA appeared to be decreasing. One possible explanation is that the level of MIH mRNA may not precisely track the titer of the MIH peptide in hemolymph (i.e., that there is a time lag between changes in peptide secretion and changes in gene expression). Another is that MIH is not the sole regulator of ecdysteroid synthesis (see Skinner, 1985
; Webster and Keller, 1989; Hopkins, 1992).
Comparative data on MIH gene expression during the molt cycle are scant. MIH mRNA was detected in eyestalk neural ganglia of C. feriatus throughout the molt cycle, but the analysis was qualitative not quantitative (Chan et al., 1998). Another study concluded that the level of MIH mRNA in eyestalks of the prawn Penaeus japonicus did not change significantly during the molt cycle, and suggested that regulation of MIH was post-transcriptional (Ohira et al., 1997). Our data do not exclude the possibility of post-transcriptional regulation of MIH in C. sapidus, but the results strongly suggest that changes in MIH mRNA levels play a critical role in the regulation of molting. It is not clear whether the apparent difference between C. sapidus and P. japonicus is a result of methodological or species differences.
Expression of recombinant MIH (rMIH)
Although much has been learned about the structure and function of MIH, progress has been impeded by the lack of sufficient quantities of purified neuropeptide. Thus, production of recombinant hormone in high yield would be desirable. We have used eukaryotic (baculovirus/insect cells) and prokaryotic (pET vector/E. coli) expression systems to produce rMIH. For expression of rMIH in E. coli, a PCR fragment encoding MIH of C. sapidus was subcloned into the pET 15b vector (Novagen). The construct was designed to yield rMIH with a polyHis tag at the N-terminus. E. coli were transformed with MIH/pET15b and expression induced with IPTG. Electrophoretic analysis of cell lysates showed that induced cells, but not uninduced cells, contained a prominent protein of the predicted size (10.4 kD, Fig. 4). Fractionation studies revealed the expressed protein was present in insoluble inclusion bodies (data not shown). The inclusion bodies were denatured with 6 M guanidine-HCl in binding buffer and purified by affinity chromatography (Fig. 4). Western blot analysis, using as probe antiserum raised against MIH of C. maenas, showed the expressed protein was MIH immunoreactive (data not shown). The protein did not, however, possess MIH bioactivity, i.e., it did not suppress in vitro ecdysteroid production by Y-organs of C. sapidus or Menippe mercenaria (data not shown). The results suggest that the prokaryotically expressed rMIH is not properly folded or lacks postranslational modifications required for biological activity.
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Polyclonal antisera raised against prokaryotically expressed rMIH (anti-rMIH) bound specifically to neurosecretory cell bodies in the blue crab X-organ, their associated axons, and terminals in the sinus gland (Fig. 5). While we have not done extensive cell counts, our initial impression is that anti-rMIH labels more cell bodies in the blue crab X-organ than does antiserum raised against MIH of C. maenas. Confirmation and final interpretation of these results will require additional studies of the binding properties of anti-rMIH.
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Preliminary results of baculovirus expression of rMIH have been reported (Lee and Watson, 1998
). Briefly, a baculovirus expression system was used to generate rMIH in insect Sf9 cells: the recombinant peptide was of the predicted size, was MIH-immunoreactive, and suppressed dose-dependently ecdysteroid production by Y-organs in vitro. The results will be reported in full elsewhere (Lee and Watson, in preparation). We anticipate that eukaryotically expressed rMIH and antiserum raised against both recombinant peptides will be useful experimental tools for enhancing understanding of the structure and function of crustacean MIH.
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ACKNOWLEDGMENTS |
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We thank Shawn Williams (University of Alabama at Birmingham) and Dr. Stephen Kempf (Auburn University) for their assistance with confocal microscopy, Michael Vickery (University of Alabama at Birmingham) for his assistance with MIH sequence analysis, Dr. Heinrich Dircksen (University of Bonn) for providing antiserum raised against MIH of Carcinus maenas, and Dr. E. S. Chang (University of California) for providing the actin probe. The research was supported by NSF grants IBN-9419916 (RDW) and IBN-9603547 (ES), NOAA/MS-AL Sea Grant NA56RG0129 (RDW), a Student Research Grant from the Alabama Academy of Science (KJL), and a Sigma Xi Grant-in-Aid of Research (KJL).
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FOOTNOTES |
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1 From the Symposium on Recent Progress in Crustacean Endocrinology: A Symposium in Honor of Milton Fingerman presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 4–8 January 2000, at Atlanta, Georgia.
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