Comparative Biochemistry and Physiology, Part A 152 (2009) 139–148

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Comparative Biochemistry and Physiology, Part A

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Review

Crustacean molt-inhibiting hormone: Structure, function, and cellular mode of action

Teruaki Nakatsuji a, Chi-Ying Lee b, R. Douglas Watson a,⁎a Department of Biology, University of Alabama at Birmingham, AL 35294, USAb Department of Biology, National Changhua University of Education, Changhua, 50058, Taiwan, ROC

⁎ Corresponding author. Department of Biology, UniverBirmingham, AL 35244, USA. Tel.: +1 205 934 2031; fax: +

E-mail address: rdwatson@uab.edu (R.D. Watson).

1095-6433/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.cbpa.2008.10.012

a b s t r a c t

a r t i c l e i n f o

Article history:

In Crustacea, secretion of e Received 21 August 2008Received in revised form 15 October 2008Accepted 15 October 2008Available online 30 October 2008

Keywords:Molt-inhibiting hormoneMIHEcdysteroidY-organMoltingCrustacean hyperglycemic hormone familyPhosphodiesterase

cdysteroid molting hormones by Y-organs is regulated, at least in part, by molt-inhibiting hormone (MIH), a polypeptide neurohormone produced by neurosecretory cells of the eyestalks.This article reviews current knowledge of MIH, with particular emphasis on recent findings regarding the (a)structure of the MIH peptide and gene, (b) levels of MIH in eyestalks and hemolymph, (c) cellular mechanismof action of MIH, and (d) responsiveness of Y-organs to MIH. At least 26 MIH/MIH-like sequences have beendirectly determined by protein sequencing or deduced from cloned cDNA. Recent studies reveal the existenceof multiple forms of MIH/MIH-like molecules among penaeids and raise the possibility that molecularpolymorphism may exist more generally among MIH (type II) peptides. The hemolymphatic MIH titer hasbeen determined for two species, a crayfish (Procambarus clarkii) and a crab (Carcinus maenas). The data aredissimilar and additional studies are needed. Composite data indicate cellular signaling pathways involvingcGMP, cAMP, or both may play a role in MIH-induced suppression of ecdysteroidogenesis. Data from the twospecies studied in our laboratories (P. clarkii and Callinectes sapidus) strongly favor cGMP as thephysiologically relevant second messenger. Ligand-binding studies show an MIH receptor exists in Y-organplasma membranes, but the MIH receptor has not been isolated or fully characterized for any species. Suchstudies are critical to understanding the cellular mechanism by which MIH regulates ecdysteroidogenesis.Rates of ecdysteroid synthesis appear also to be influenced by stage-specific changes in the responsiveness ofY-organs to MIH. The changes in responsiveness result, at least in part, from changes in glandularphosphodiesterase (PDE) activity. The PDE isotype (PDE1) present in Y-organs of C. sapidus is calcium/calmodulin dependent. Thus, calcium may regulate ecdysteroidogenesis through activation of glandular PDE.

© 2008 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1402. Structure of the MIH peptide and gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

2.1. MIH peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1402.2. MIH gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

3. MIH transcript and peptide levels during the molting cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1423.1. MIH transcript levels in eyestalk tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1423.2. MIH Peptide Levels in Sinus Glands and Hemolymph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

4. Cellular mechanism of action of MIH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1434.1. Cellular signaling pathways in Y-organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1434.2. MIH receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

5. Responsiveness of Y-organs to MIH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1456. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

sity of Alabama at Birmingham,1 205 975 6097.

l rights reserved.

140 T. Nakatsuji et al. / Comparative Biochemistry and Physiology, Part A 152 (2009) 139–148

1. Introduction

Aspects of crustacean growth and postembryonic development,including molting and regeneration, are controlled by C-27 steroidhormones termed ecdysteroids (Skinner, 1985; Lachaise et al., 1993).Ecdysteroids are produced by paired Y-organs, ectodermally-derivedendocrine glands located in the anterior cephalothorax (Skinner,1985;Spaziani, 1990). In general, the level of ecdysteroids in hemolymph islow throughout intermolt (stages C1–4), rises during premolt (stagesD0–4), typically peaking in D2–3, then falls prior to molting, resulting ina low level of ecdysteroids during ecdysis (stage E) and postmolt(stages A–B) (see Skinner, 1985). The synthesis of ecdysteroids by Y-organs is negatively regulated (inhibited) by a peptide neurohormone,molt-inhibiting hormone (MIH). MIH is produced in a cluster ofeyestalk neurosecretory cell soma (the X-organ) and released fromtheir associated axon terminals in the neurohemal sinus gland(Skinner, 1985; Lachaise et al., 1993). Thus, ablation of the eyestalksleads to enhanced ecdysteroid secretion by Y-organs, an increase inthe ecdysteroid titer, and precocious molting (Keller and Schmid,1979; Chang and Bruce, 1980; Hopkins, 1983), while injection ofeyestalk extract or synthetic MIH into eyestalk-ablated animals lowersthe ecdysteroid titer and delays molting (Bruce and Chang, 1984;Chang et al., 1987; Nakatsuji and Sonobe, 2004). In vitro studiesindicate MIH acts directly on Y-organs to suppress synthesis ofecdysteroids (Soumoff and O'Connor, 1982; Watson and Spaziani,1985a; Mattson and Spaziani, 1985a; Webster 1986; Schoettker andGist, 1990) and uptake of lipoprotein-bound cholesterol, the biosyn-thetic precursor of ecdysteroids (Watson and Spaziani, 1985a,b; Kangand Spaziani, 1995a,b). Based on these and related findings, alongstanding model for molt control in crustaceans suggests thatMIH from the X-organ/sinus gland complex inhibits Y-organs duringmuch of the molting cycle (principally intermolt), and that a moltingsequence is initiated when MIH secretion diminishes (Skinner, 1985).While the model remains valuable as a base from which testablehypotheses can be formed, several lines of evidence suggest it isincomplete. As examples, recently published data indicate thehemolymphatic MIH titer does not strictly conform to predictions ofthe model (Nakatsuji and Sonobe, 2004; Chung and Webster, 2005),and compounds other than MIH have been implicated, either directlyor indirectly, in the regulation of Y-organs (Webster and Keller, 1986;Webster, 1993; Dell et al., 1999; Yu et al., 2002).

Fig. 1. Multiple sequence alignment of representative MIH/MIH-like peptide precursors. Sianalyses of Chf-MIH, Mee-MIHA, Mee-MIHB, Pem-MIH1, Pem-MIH2, Pem-SGP-C1, Pem-SGP-C2Katayama et al. (2003) to be functionally critical for MIH activity are shown below the sequeMIHB, Pej-MIHC, Lis-MIH, Trc-MIH) at positions 48, 56, and 61. Amino acid residues are numfrom the following sources: Cam—Carcinus maenas (Klein et al., 1993); Cas—Callinectes sapidet al., 1998); Orl—Orconectes limosus (Bulau et al., 2005); Prc—Procambarus clarkii (Nagasawa eMee—Metapenaeus ensis (Gu and Chan, 1998; Gu et al., 2002); Liv—Litopenaeus vannamei (Ccurvirostris (accession number: AF312978); Lis—Litopenaeus stylirostris (accession number: AFN-terminal ends of mature peptides of Trc-MIH and Lis-MIH are lacking.

Amino acid sequence data place MIH in the crustacean hypergly-cemic hormone (CHH) family of peptides. The family includes (inaddition to CHH and MIH), vitellogenesis-inhibiting hormone (VIH)and mandibular organ-inhibiting hormone (MOIH) (Keller, 1992;Wainwright et al., 1996; Van Herp, 1998). Based on preprohormonestructure, the CHH family of peptides can be divided into two groups,one group containing CHH, and the other containing MIH, VIH, andMOIH. Preprohormones of the former group (type I peptides) containa CHH precursor related peptide (CPRP) that is cleaved out duringprocessing; preprohormones of the latter group (type II peptides) lackthe CPRP (Lacombe et al., 1999).

The cellular mechanism of action of MIH remains an area of activeresearch. Radioreceptor binding assays, using [125I]MIH as ligand,indicate MIH receptors are present in Y-organ membrane preparations(Webster,1993; ChungandWebster, 2003), but theMIH receptorhas notbeen isolated or thoroughly characterized for any crustacean species.Available data indicateMIH-receptor occupancy is linked to activation ofone ormore cyclic nucleotide cell signalingpathways (cAMP or cGMP, orboth) (Mattson and Spaziani, 1986; Sedlmeier and Fenrich, 1993; Saïdiet al.,1994;Nakatsuji et al., 2006a,b). Additional cell signaling pathways,including calcium signaling, have been linked directly or indirectly toMIH action (see Spaziani et al.,1999, 2001; Imayavaramban et al., 2007).

Several studies suggest that ecdysteroid production may beregulated not only by the level of circulating MIH, but also by theresponsiveness of Y-organs to MIH (Sefiani et al., 1996; Chung andWebster, 2003; Nakatsuji and Sonobe, 2004). Recent results indicatethat stage-specific changes in responsiveness of Y-organs to MIHresult, at least in part, from stage-specific changes in cyclic nucleotidephosphodiesterase (PDE) activity (Nakatsuji et al., 2006a).

The present article reviews current knowledge of MIH, withparticular emphasis on recent findings regarding the (a) structure oftheMIHpeptideand gene, (b) levels ofMIH ineyestalks andhemolymph,(c) cellular mechanism of action of MIH, and (d) responsiveness of Y-organs to MIH.

2. Structure of the MIH peptide and gene

2.1. MIH peptide

At least 26 MIH/MIH-like sequences have been identified fromdecapod crustaceans of various taxa, with primary structure ranging

x cysteine residues are indicated by asterisks (⁎). Sites of intron insertion, revealed by, Live-MIH1, and Liv-MIH2, are marked by arrows (↓). Conserved residues proposed bynces. Note the substitutions often occurring in the peptides of group B (Mee-MIHB, Pej-bered relative to the first residue (+1) of the mature peptides. Sequences analyzed areus (Lee et al., 1995); Cap—Cancer pagurus (Lu et al., 2001); Chf—Charybdis feriatus (Chant al., 1996); Pej—Penaeus japonicus (Ohira et al., 1997; 2005; Yamano and Unuma, 2006);hen et al., 2007); Pem—Penaeus monodon (Yodmuang et al., 2004); Trc—Trachypenaeus312976). Note that data regarding the signal peptide of Orl-MIH, and signal peptides and

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from 74 to 79 amino acids. Distinguishing primary sequence char-acteristics include the presence of 6 cysteine residues at strictlyconserved positions (Fig. 1). Formation of 3 disulfide bonds by thesecysteine residues (Cys7–Cys44, Cys24–Cys40, and Cys27–Cys53) is criticalfor stabilizingmolecular structure (Katayama et al., 2001, 2003). Otherconserved residues present in the majority of MIH/MIH-likesequences are those at positions 12, 13, 19, 20, 48, 56, 59, 61, 62, 69,72, 73, and 76 (see Fig.1; Katayama et al., 2003; and discussion below).

The C-terminal end of MIH appears more variable than the N-terminal end. Where relevant data exist, the C-terminus is either free(Cam-MIH, Webster, 1991; Cap-MIH, Chung et al., 1996; Pej-MIHA,Yang et al., 1996) or amidated (Prc-MIH, Nagasawa et al., 1996; Orl-MIH, Bulau et al., 2005; Jal-MIH, Marco et al., 2000), and the N-terminus is always unblocked. In many cases, the primary structure ofMIH/MIH-like molecules was deduced from cloned cDNA sequencesand possible terminal modifications are thus unknown. The C-terminus of Orl-MIH, as directly determined by peptide sequencing,is amidated (Ala75–NH2) instead of the Ala75–Gly76–Arg77 predicted bythe cDNA sequence (Bulau et al., 2005). On the other hand, the C-termini of Cam-MIH, Cap-MIH, and Pej-MIHA are free (Webster, 1991;Chung et al., 1996; Yang et al., 1996) and the same as predicted by thecDNA sequences (Klein et al., 1993; Ohira et al., 1997; Lu et al., 2001).The functional significance of an amidated C-terminus in MIH has notbeen determined. For CHH, the C-teminal amide affects secondarystructure and is involved in conferring hyperglycemic activity(Katayama et al., 2002; Mosco et al., 2008).

The existence of multiple molecular forms of CHH family peptidesis well-documented, in particular for the type I CHH peptides (Soyez,

Fig. 2. Phylogenic analysis of MIH/MIH-like sequences. The phylogenic tree was generatedbootstrap replicates. Sequences analyzed are from the following sources: Jal—Jasus lalandii (Mmagister (Umphrey et al., 1998); Scs—Scylla serrata (accession number: AY083797); Fec—Fenet al., 2002); the remainder of the sequences are from sources indicated in the legend of Fi

1997; 2003). Recent studies in penaeids have revealed that thephenomenon of molecular polymorphism is also present in type IIpeptides. Thus, there are 4MIH/MIH-likemolecules identified so far inPenaeus monodon (Krungkasem et al., 2002; Yodmuang et al., 2004), 3for Penaeus japonicus and Fenneropenaeus chinensis (Yang et al., 1996;Ohira et al., 1997; Wang and Xiang, 2003; Ohira et al., 2005; Yamanoand Unuma, 2006), and 2 for Litopenaeus vannamei and Metapenaeusensis (Gu and Chan, 1998; Gu et al., 2002; Chen et al., 2007).

Some of the con-specific peptides are highly similar in primarysequence (e.g., Pem-SGP-C1 and Pem-SGP-C2 are 97% identical; Fec-NP1 is 95% and 93% identical to Fec-NP2 and Fec-NP3, respectively),while others differ considerably (e.g., Pej-MIHA is 73% identical to bothPej-MIHB and Pej-MIHC;Mee-MIHA andMee-MIHB are 70% identical;Liv-MIH1 and Liv-MIH2 are 71% identical; Pem-MIH1 and Pem-MIH 2are 84% identical). A phylogenic tree for MIH/MIH-like sequences isshown in Fig. 2. Penaeid sequences are separated from the groupconsisting of brachyuran MIH, the group consisting of astacoideanMIH, and Jal-MIH. Penaeid peptides can be further separated into 2subgroups, A and B (Fig. 2).

Where relevant data are available, the molt-inhibiting activity ofpeptides in subgroup A is more potent than that of con-specificpeptides in subgroup B (i.e., Mee-MIHA vs. Mee-MIHB, Gu et al., 2002;Pej-MIHA vs. Pej-MIHB, Ohira et al., 2005). Thus, it has been proposedthat peptides of different subgroups might diverge functionally, withpeptides of subgroup A acting physiologically as MIH, and those ofsubgroup B playing regulatory roles other than or in addition toinhibition of molting (Gu et al., 2002; Ohira et al., 2005). Possible rolesfor the peptides of subgroup B have also been proposed. For example,

using MEGA3. The confidence values of the branching pattern were tested on 1000arco et al., 2000); Gel—Gecarcinus lateralis (accession number DQ473354); Crm—Cancerneropenaeus chinensis (Wang and Xiang, 2003); Pem—Penaeus monodon (Krungkasemg. 1.

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based on their sequence similarity to vitellogenesis-inhibitinghormone (VIH) and the pattern of variation of transcript levels infemale animals at various stages of ovarian development, it has beensuggested that subgroup B peptides may be involved in regulation ofgonad maturation (Gu et al., 2002; Ohira et al., 2005). A recent reportsuggestedMee-MIHB acts as a gonad-stimulating hormone inM. ensis.Thus, recombinant Mee-MIHB increased vitellogenin gene expressionboth in vitro and in vitro, while RNA interference (using Mee-MIHBdouble-stranded RNA) reduced vitellogenin gene expression (Tiu andChan, 2007). By contrast, in previous experiments, neither Pej-MIHBnor Pej-MIHA had a significant effect on vitellogenin gene expressionin an in vitro ovarian assay (Tsutsui et al., 2005).

In a structural study of Pej-MIHA, 13 surface-located residues wereproposed to be functionally critical for Pej-MIHA activity (Katayamaet al., 2003). These residues arewell-conserved in the penaeid peptidesof subgroup A (as well as brachyuran and astacoidean peptides); in thepeptides of subgroup B, 3 of the residues (Glu48, Ala56, andGlu61/Asp61)are often replaced by residues having significantly different properties(Gln48/Phe48, Ser56, and Ala61) (Fig.1). The replacementsmight explainthe between-group difference in molt-inhibiting potency.

Data from the above cited studies show multiple MIH/MIH-likemolecules in penaeid species, but additional studies are needed toconclusively determine whether there is functional divergence amongthe peptides. Recent studies in the white shrimp L. vannamei revealedthat gene expression of Liv-MIH1 and Liv-MIH2 varied during the moltcycle in a manner suggesting both peptides act physiologically as MIH(Chen et al., 2007; see Section 3.1, below). These data are consistentwiththe proposition that peptides of subgroup A, towhich Liv-MIH1 and Liv-MIH2 belong, function as MIHs, and supports the emerging consensusthat multiple MIHs exist in a given species, as exemplified by thepresence of multiple subgroup A peptides in each of the 3 species, L.vannamei, P. monodon, and F. chinensis (Krungkasem et al., 2002; Wangand Xiang, 2003; Yodmuang et al., 2004; Chen et al., 2007).

2.2. MIH gene

MIH/MIH-like peptide precursors have been determined mainlythrough conceptual translation of their cDNA sequences. Theprecursor sequences contain a signal peptide of various lengths (22–35 residues) and a mature MIH/MIH like peptide. The length of thesignal peptide of brachyuran precursors is typically longer than thoseof astacoidean or penaeid precursors (35 vs. 22–29 residues) (Fig. 1).

The structure of several genes encoding MIH/MIH-like peptide hasbeen elucidated, including Chf-MIH, Mee-MIHA, Mee-MIHB, Pem-SGP-C1, Pem-SGP-C2, Pem-MIH1, Pem-MIH2, Liv-MIH1, and Liv-MIH2; the 3exon/2 intron organization of these genes appears to be highlyconserved (Chan et al., 1998; Gu and Chan, 1998; Gu et al., 2002;Krungkasem et al., 2002; Yodmuang et al., 2004; Chen et al., 2007).Intron I is inserted between the codons for the 6th and 7th amino acidresidues of the signal peptide for penaeid genes (an exception is Mee-MIHB, where, because of a relatively shorter signal peptide for penaeidgroup B precursors, the insertion occurs between those for the 1st and2nd residues of the signal peptide), and is between the codons for the12th and 13th residues of the signal peptide for brachyuran genes.Intron II is invariably inserted after the second base of the codon forthe 41st residue of the mature peptide for all genes (Fig. 1). Thus, ExonI contains the sequence for the 5′-untranslated region (UTR) and a partof the signal peptide, Exon II encodes the remaining part of the signalpeptide and the N-terminal half (up to the 40th residue) of the maturepeptide, and Exon III contains the sequences for the remaining C-terminal half of the mature peptide and the 3′-UTR.

The 5′ regulatory region of the MIH gene has been characterized inonly two species, the crabs Charybdis feriatus and Cancer pagurus. ForChf-MIH, several potential transcription binding elements (SP1, Pit-1,CREB, and TATA) were identified in a putative promoter region, andthe functionality of the promoter was demonstrated by successfully

driving the expression of reporter proteins (Chan et al., 1998). CREB,TATA, and an ecdysone-responsive element CF1/USP were located in aputative promoter region of Cap-MIH, (Lu et al., 2000).

The spatial patterns of expression of MIH/MIH-like peptide genesvary considerably. When analyzed by Northern blot analysis, Cas-MIHand Cap-MIH transcripts were detected only in the eyestalk ganglia(Lee et al., 1995; Lu et al., 2001), but Cap-MIH transcripts were alsodetected in several extra-eyestalk nerve tissues when analyzed bynested PCR assay (Lu et al., 2001). Data reported for penaeid genes aresimilarly variable but correspond well with the results of phylogenicanalyses. Thus, transcripts of subgroup A peptides (Pej-MIHA, Mee-MIHA, Pem-MIH1, Pem-MIH2, Pem-SGP-C1, Pem-SGP-C2, Liv-MIH1,and Liv-MIH2) are mainly or exclusively expressed in the eyestalkganglia, whereas those of subgroup B peptides (Mee-MIHB, Pej-MIHB)predominantly in extra-eyestalk tissues (e.g., the thoracic ganglia,abdominal ganglia, brain). The pattern of differential tissue expressionis consistent with the hypothesis that peptides of different groupsmight play different physiological roles.

3. MIH transcript and peptide levels during the molting cycle

3.1. MIH transcript levels in eyestalk tissue

Several studies have assessedMIH transcript abundance in eyestalkganglia, or MIH peptide levels in sinus glands or hemolymph. Lee et al.(1998) used densitometric scans of Northern blots to determine MIHmRNA abundance in eyestalk tissue of the crab, Callinectes sapidus;hemolymphatic ecdysteroid titers were determined by radioimmu-noassay for the same animals. The level of MIH mRNA was elevatedduring postmolt (A/B) and intermolt (C4), stages when the ecdysteroidtiter was low, then decreased during premolt stages D1–D3, a timewhen the ecdysteroid titer increased (Fig. 3). Chen et al. (2007) usedreal-time PCR to determine the steady-state levels of two putativeMIHtranscripts (Liv-MIH1 and Liv-MIH2) in eyestalk ganglia of the Pacificwhite shrimp (L. vannamei). The levels of Liv-MIH1 and Liv-MIH2 wereelevated during postmolt (A and B), intermolt (C) and early premolt(D1'), then progressively declined, reaching a low during premolt stageD2D3, a time when the hemolymphatic ecdysteroid titer wassignificantly elevated. The results from the two studies cited aboveare generally consistent with the hypothesis that MIH negativelyregulates ecdysteroid synthesis during a molt cycle. By contrast, Ohiraet al. (1997) useddensitometric scans ofNorthern blots tomonitorMIHtranscript (Pej-SGP-IV, also termed Pej-MIHA) levels in eyestalk tissueof the kuruma prawn (P. japonicus), and concluded that transcriptabundance did not change significantly during a molt cycle. Likewise,Chung and Webster (2003) observed no difference in MIH transcriptlevels (as determined by real-time RT–PCR) between X-organs ofintermolt (C4) and premolt (D2) green crabs (Carcinus maenas).

3.2. MIH Peptide Levels in Sinus Glands and Hemolymph

Using a sandwich enzyme-linked immunoassay, Nakatsuji et al.(2000) determined that the MIH content of crayfish (Procambarusclarkii) sinus glands increased by approximately 2-fold betweenintermolt and early premolt, remained elevated through middlepremolt, and then dropped again during late premolt. It washypothesized that MIH accumulates in P. clarkii sinus glands duringearly andmiddle premolt as a result of a decrease in secretion, and thatthe drop in stored MIH observed during late premolt was a result ofincreased secretion of the neurohormone. By contrast, Chung andWebster (2003) observed no change in theMIH content of sinus glandsduring a molt cycle of C. maenas.

Determination of the hemolymphatic MIH titer has been a per-sistent challenge in the field of crustacean endocrinology. To date, MIHlevels have been determined during a molt cycle for only two species:the crayfish P. clarkii and the crab C. maenas. MIH was quantified in

Fig. 4. Changes in the hemolymphatic MIH and ecdysteroid titers during the molt cycleof P. clarkii. Hemolymph was collected from 13–34 animals at each stage during themolt cycle. (A) The MIH titer was determined by time-resolved fluoroimmunoassay. (B)The ecdysteroid titer was determined by radioimmunoassay and expressed in ecdysoneequivalents. Results were expressed as mean±SE and analyzed by t-test. Valuessignificantly different from the intermolt value are as indicated (Pb0.05 (⁎) andPb0.001 (⁎⁎⁎)). From Nakatsuji and Sonobe, 2004; used by permission of the publisher.

Fig. 3. MIH mRNA levels and ecdysteroid titer during a molt cycle of C. sapidus. (A) MIHmRNA was detected by Northern blot using an MIH cDNA probe, and hybridizationsignal intensity determined densitometrically. Data were normalized to account forunequal RNA loading using a lobster actin probe, and expressed relative to the MIH/actin ratio in postmolt (A/B). Eyestalk neural ganglia were pooled by stage (4–8eyestalks per sample); samples were analyzed individually. For stages represented bymultiple samples (n=2–3), mean+SE were determined. Data were analyzed by ANOVA(p=0.03). (B) Ecdysteroids in the hemolymph of individual crabs were quantified byradioimmunoassay. Bars represent mean+SE (n=2–9; n=1 for D4). Data were analyzedby ANOVA. From Lee et al., 1998; used by permission of the publisher.

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hemolymph of P. clarkii by time-resolved fluoroimmunoassay (usingaffinity purified antibodies raised against synthetic fragments ofMIH);hemolymphatic ecdysteroids were quantified in the same animals byradioimmunoassay (Nakatsuji and Sonobe, 2003; 2004). The level ofMIH in hemolymph was high during intermolt, dropped significantlyduring early premolt, then rose and remained elevated through theremainder of the molting cycle (Fig. 4A). The drop in hemolymphaticMIH seen during early premolt coincidedwith a statistically significantincrease in the level of hemolymphatic ecdysteroids; thereafter, theecdysteroid titer continued to rise, reaching a peak during middlepremolt, then dropped in late premolt (prior to ecdysis) and was lowduring postmolt (Fig. 4B). The results are consistent with the hypothe-sis that MIH suppresses ecdysteroid production during intermolt andpostmolt, and that a drop in the level of MIH in hemolymph duringearly premolt permits the increase in ecdysteroid production seenduring that same stage. However, the observation that hemolymphaticMIH is elevated during middle and late premolt (when ecdysteroidlevels are also elevated) was not predicted by the longstanding modelof MIH action. This seeming contradiction may be accounted by theobservation that Y-organs of P. clarkii are poorly responsive to MIHduring middle and late premolt (Nakatsuji and Sonobe, 2004; see alsoSection 5, below). Subsequent to the above report, MIHwas quantifiedin hemolymph of C. maenas by radioimmunoassay (using polyclonalantisera raised against purified MIH) (Chung and Webster, 2005). Nosignificant changes in the level of MIH in hemolymph were observed

during the molting cycle, excepting a significant surge during latepremolt, just prior to molting. We cannot explain the discrepanciesbetween the above studies. Interpretation of the results is complicatedby differences between MIH assay methods and by the fact that dataare available from only two species.

4. Cellular mechanism of action of MIH

4.1. Cellular signaling pathways in Y-organs

Existing data indicate that cellular signaling pathways involvingcAMP, cGMP, or both play a role in MIH-mediated regulation ofecdysteroidogenesis. Mattson and Spaziani (1985b) reported thatadding eyestalk extract (containing MIH activity) to incubations ofcrab (Cancer antennarius) Y-organs resulted in a 4-fold increase inintracellular cAMP (detectable 1 h after treatment and sustainedthrough 6 h of incubation). Mattson and Spaziani (1985b) furtherobserved that a cAMP analog, or agents that increased intracellularcAMP, each mimicked the inhibitory action of MIH, while a cGMPanalog did not (Mattson and Spaziani, 1985b). They concluded thatcAMP mediates MIH-induced suppression of ecdysteroid production.Intracellular cGMP levels were not measured.

By contrast, Sedlmeier and Fenrich (1993) reported that incubationof crayfish (Orconectes limosus) Y-organs in eyestalk-conditioned

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medium resulted in an increase in intracellular cGMP content within2 h, but had no effect on cAMP content, and that a cGMP analog dose-dependently suppressed ecdysteroid production. They concluded thatMIH-induced suppression of ecdysteroid production is mediated bycGMP, not by cAMP. Our laboratory has studied the link of cyclicnucleotide cell signaling to MIH action in Y-organs of two species: C.sapidus (Nakatsuji et al., 2006b) and P. clarkii (Nakatsuji et al., 2006a).We found that addition of recombinant MIH to incubation mediumsignificantly enhanced intracellular cGMP accumulation in Y-organs ofC. sapidus, but had no effect on intracellular cAMP (Fig. 5) (Nakatsujiet al., 2006b). In companion experiments, a cGMP analog (8-Br-cGMP)significantly suppressed ecdysteroid production by Y-organs of C.sapidus, but neither cAMP analogs (db-cAMP or 8-Br-cAMP) nor anactivator of adenylyl cyclase (forskolin) had a detectable effect onecdysteroidogenesis (Nakatsuji et al., 2006b). Likewise, addition ofsyntheticMIH to incubationmedium, increased cGMP content, but notcAMP content, in Y-organs of crayfish, P. clarkii (Nakatsuji et al.,2006a). Thus, results from our laboratory also indicateMIH suppressesecdysteroidogenesis via a cGMP second messenger.

Consistent with this conclusion, Saïdi et al. (1994) reported thatadding purified MIH to incubations of Y-organs from intermolt greencrabs (C. maenas) produced a large (20-fold) and sustained (≥60 min)increase in intracellular cGMP, but had no effect on cAMP. However, thissame group also observed that adding purifiedMIH to incubations of Y-organs from premolt C. maenas produced not only a large (60-fold) andsustained (≥60min) increase in intracellular cGMP, but also a smaller (2-fold) and more transient (1–4 min) increase in cAMP. They suggestedthat cAMP might act cooperatively with cGMP in premolt Y-organs.Interpretation of the combined results from all sources is hindered bythe fact that the matter has been studied in only a few species.

Fig. 5. Effect of recombinant MIH on intracellular cyclic nucleotide levels in Y-organs ofC. sapidus. Two days after eyestalk ablation, paired Y-organs were removed from crabsand incubated (25 °C) in 0.3 ml culture medium, the experimental gland in mediumcontaining recombinant MIH (10 nM)+IBMX (1mM) (●), and the contralateral (control)gland in medium containing vehicle+ IBMX (1 mM) (○). At the times indicated, thelevels of glandular cAMP (A) and cGMP (B) were determined by an enzymeimmunoassay. Results are expressed as mean±SE of three individual assays. FromNakatsuji et al., 2006b; used by permission of the publisher.

Cell signaling molecules other than cyclic nucleotides have alsobeen implicated, directly or indirectly, in MIH action. Among these,calcium appears to play a critical role. Mattson and Spaziani (1986)preloaded dispersed Y-organ cells with 45Ca+, and found that additionof eyestalk extract (containing MIH activity) elicited a statisticallysignificant efflux of radiolabeled calcium into incubationmedium. Theauthors hypothesized that a decrease in intracellular calciummight beinvolved in MIH-induced suppression of ecdysteroidogenesis. Con-sistent with that hypothesis are the observations that calciumantagonists (lanthanum or ruthenium red), an intracellular calciumchelator (TMB-8), a calmodulin inhibitor (trifluorperazine), or calciumchannel blockers (verapamil, nifedipine, or nicardipine) individuallysuppressed basal ecdysteroid production by Y-organs in vitro, orenhanced the suppressive effect of MIH, or both (Mattson andSpaziani, 1986; reviews: Spaziani et al., 1999, 2001). Needed areexperiments designed to measure the intracellular calcium concen-tration in Y-organ cells in the presence and absence of MIH.

In related experiments, treatment of crab (C. antennarius) Y-organswith the calcium ionophore A23187 (which increases intracellular Ca+)stimulated ecdysteroid production in a dose-dependent manner(Mattson and Spaziani, 1986). This and other findings led to the hy-pothesis that an increase in intracellular calcium may stimulatethe surge in ecdysteroidogenesis that results in the premolt peak inthe hemolyphatic ecdysteroid titer (Mattson and Spaziani, 1986). Thecause of the hypothesized increase in intracellular calcium is unknown.One possibility is that it is elicited bya specific ligand; the existence of apositive regulator of ecdysteroidogenesis in crustaceans has been longhypothesized (e.g., see Skinner, 1985). Another possibility is that thehypothesized increase in intracellular calcium is a consequence of thegeneral increase in hemolymphatic Ca+ that occurs during premolt asCa+ is mobilized from the old exoskeleton (see Greenaway, 1985).Existing data suggest elevated intracellular calcium may be linked toenhanced ecdysteroidogenesis through activation of at least twointracellular enzymes, protein kinase C (PKC) and cyclic nucleotidephosphodiesterase (PDE). Regarding PKC, the results are mixed,depending on species. Adding an activator of PKC (phorbol 12-myristate 13-acetate) to incubations of crab (C. antennarius) Y-organsdose-dependently stimulated ecdysteroid production, and the enzymewas Ca+-sensitive (Mattson and Spaziani, 1987). By contrast, addingphorbol 12-myristate 13-acetate to incubations of crayfish (Orconectesimmunis) Y-organs suppressed ecdysteroid production (Spaziani et al.,2001). The reason for the difference between species is unknown.Regarding PDE, the results consistently show that calciumactivates theenzyme (an effect that lowers intracellular cyclic nucleotide levels andenhances ecdysteroidogenesis). Thus, adding a PDE inhibitor (3-isobutyl-1-methyl-xanthine, IBMX) to incubations of Y-organs sup-pressed ecdysteroid secretion (Mattson and Spaziani 1985b, 1986),while adding a calcium ionophore (A23187) stimulated ecdysteroidsecretion, an effect that was blocked by inhibiting PDE with IBMX(Mattson and Spaziani, 1986). Moreover, in cell-free Y-organ prepara-tions, PDE activity was dose-dependently increased by calcium (10−7–10−4 M) and suppressed by a calmodulin inhibitor (Mattson andSpaziani, 1986). To summarize, available data suggest MIH may sup-press ecdysteroidogenesis, in part, by promoting calciumefflux fromY-organs, and that an increase in intracellular calcium leads to enhancedecdysteroidogenesis through activation of PKC, or PDE, or both.Although the hypothesized increase in intracellular calcium does notappear to be directly linked to MIH receptor occupancy, the abovefindings are included here because recent data indicate changes in PDEactivity may underlie stage-dependent changes in the responsivenessof Y-organs to MIH (Nakatsuji et al., 2006a, see Section 5., below).

4.2. MIH receptor

Data on the MIH receptor are scant. Receptor-binding assays (using[125I]MIH as ligand) revealed the existence of high-affinity, specific,

Fig. 6. Effect of a phosphodiesterase inhibitor (IBMX) on the responsiveness of Y-organsto MIH during a molt cycle. Y-Organs were removed from crayfish in various stages ofthe molt cycle and incubated in vitro (6 h, 25 °C). One gland of a pair was incubated inmedium containing MIH (4 nM, open bars), IBMX (1 mM, striped bars), or both (darkbars), the contralateral gland in medium containing vehicle. At the end of incubation,the ecdysteroid content of the mediumwas determined by RIA. Results are expressed aspercent inhibition relative to vehicle controls. Bars represent mean±SE (n=3–11). Datawere analyzed by ANOVA and Tukey's multiple comparisons. For treatment with MIH,among the multiple comparisons, the following stages were significantly different fromintermolt: early premolt (Pb0.01⁎), middle premolt (Pb0.0001⁎⁎⁎), and late premolt(Pb0.001⁎⁎). For treatment with IBMX alone or MIH+IBMX, no comparison wassignificantly different from intermolt (PN0.05). From Nakatsuji et al., 2006a; used bypermission of the publisher.

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and saturable MIH binding sites (indicative of MIH receptors) onplasma membranes of C. maenas Y-organs (Webster, 1993). Asazumaet al. (2005) subsequently reported the chemical cross-linking of 125I-labeled recombinantMIH to a 70 kDa protein in Y-organs of the prawn,Marsupenaeus japonicas, but activation of the protein by MIH has notbeen demonstrated. Thus, while progress has been made, the MIHreceptor has not been fully characterized for any crustacean species.

Studies of cellular signaling in Y-organs can provide insight into thenature of the MIH receptor. As noted above, available data suggestMIH-induced suppression of ecdysteroidogenesis is mediated bycellular signaling pathways involving cAMP, cGMP, or both. For thetwo species studied in our lab (C. sapidus and P. clarkii), the datastrongly favor cGMP as the physiologically relevant second messengerof MIH. The enzymes that regulate synthesis of cGMP, guanylylcyclases, fall into two major classes: the cytoplasmically localized,soluble guanylyl cyclases (sGC) and the membrane associated,receptor guanylyl cyclases (rGC) (Wedel and Garbers, 2001). Conven-tional sGC are heterodimers that can be activated by nitric oxide(Wedel and Garbers, 2001). Atypical sGC, partially sensitive orinsensitive to nitric oxide, have also been described (Morton, 2004).Receptor guanylyl cyclases, which exist as homodimers, are integralmembrane proteins with five signature domains: extracellular,transmembrane, kinase-like, dimerization, and cyclase catalytic(Wedel and Garbers, 2001). The binding of a specific ligand to theextracellular domain of the rGC activates the intracellular cyclasedomain. In our hands, a NO donor (sodium nitroprusside) did notsuppress ecdysteroid production by blue crab Y-organs in vitro (Hanand Watson, unpublished). In addition, persuasive data indicate CHHenhances cGMP levels in crustacean muscle by activating an rGC, andhas no effect on sGC activity (Goy,1990). Thus, our working hypothesisis that the MIH receptor is an rGC.

As part of studies to test this hypothesis, we have cloned from C.sapidus Y-organs a cDNA (CsGC-YO1) encoding an rGC (Zheng et al.,2006). Immunocytochemical studies, using as primary antibodyantipeptide antibodies raised against the extracellular domain ofCsGC-YO1, confirm that CsGC-YO1 is a membrane-associated protein(Zheng et al., 2008). Pretreating Y-organs with the antipeptideantibodies significantly blunts the suppressive effect of MIH onecdysteroidogenesis (Zheng et al., 2008). Quantitative RT–PCRrevealed that expression of the CsGC-YO1 transcript in Y-organs iselevated during intermolt (a stage when MIH is thought to activelysuppress ecdysteroidogenesis) (Zheng et al., 2008). The combinedresults are consistent with the hypothesis that CsGC-YO1 is an MIHreceptor. However, an analysis of the tissue distribution of the CsGC-YO1 transcript (using end point RT–PCR) revealed the transcript wasexpressed not only in Y-organs, but also in several of the other tissuesexamined, including ventral nerve cord, thoracic ganglion, and brain(Zheng et al., 2006). This pattern of expression was not predicted bycurrent understanding ofMIH action (i.e., Y-organs are the only knowntarget tissues for MIH). One explanation is that MIH is not theactivating ligand for CsGC-YO1. It is possible, for example, that CSGC-YO1 is a receptor for crustacean hyperglycemic hormone (CHH).Previous studies indicate that Y-organs possess distinct CHH receptors,and addition of CHH (in high dose) to Y-organ incubations suppressesecdysteroid production (Webster, 1993). Liu et al. (2004) have recentlycloned a cDNA encoding a putative CHH receptor (PcGC-M2) frommuscle of the crayfish, P. clarkii. Like the CsGC-YO1 transcript, thePcGC-M2 transcript shows wide tissue distribution. However, thetissue distribution pattern of CsGC-YO1 does not precisely track thetissue distribution pattern of PcGC-M2. Another interpretation ofthe observed tissue distribution pattern of the CsGC-YO1 transcript isthat MIH affects target tissues other than Y-organs. Pleiotropic effectsof polypeptide hormones arewidely observed in both invertebrate andvertebrate systems. Conclusive identification of the activating ligandfor CsGC-YO1 will require studies designed to test the ability of MIH(and other candidate ligands) to bind and activate the receptor.

Alternative hypotheses exist regarding the nature of the MIHreceptor. Kim et al. (2004) hypothesize that the MIH receptor is a Gprotein-coupled receptor. They propose a model whereby the bindingof MIH to its receptor leads to G-protein-mediated activation ofadenylyl cyclase. A resulting increase in intracellular cAMP, in turn, issuggested to promote Ca+/calmodulin-mediated activation of nitricoxide synthase (NOS) and release of nitric oxide (NO). NO-mediatedactivation of an sGC is suggested to produce an increase in intracellularcGMP that leads to suppression of ecdysteroidogenesis. Consistentwith predictions of the model, NOS (transcript and protein) and sGC(transcript) are present is Y-organs of Gecarcinus lateralis (Kim et al.,2004; Lee et al., 2007). However, while the roles of Ca+ and cyclicnucleotide signaling in regulation of ecdysteroidogenesis are notcompletely settled, much of the available data (see Section 4.1, above)do not conform to predictions of the model. Experiments designed todetermine whether MIH stimulates a sequential increase in intracel-lular cAMP and cGMP and activates NOS and sGC in Y-organs, andwhether NO suppresses ecdysteroidogenesiswill be critical tests of thehypothesis.

In summary, lack of information on the structure and function ofthe MIH receptor remains a major gap in our understanding of theregulation of crustacean molting. The MIH receptor has not beenisolated and fully characterized for any crustacean species. Relevantdata on cell signaling pathways linked to ecdysteroidogenesis aredisparate and do not provide conclusive evidence regarding the natureof the receptor.

5. Responsiveness of Y-organs to MIH

It has become increasingly clear that rates of ecdysteroid secretionare influenced not only by MIH, but also by stage-specific changes inthe responsiveness of the Y-organs to MIH. Sefiani et al. (1996)observed a marked decline in the sensitivity of shrimp (Penaeusvannamei) Y-organs to sinus gland extract during premolt stages D2

and D3. A decline in the responsiveness of Y-organs to MIH duringmiddle and late premolt was subsequently reported for crabs (Chung

Fig. 7. Changes inphosphodiesterase activity in Y-organs during amolt cycle of P. clarkii. Y-Organs were removed from crayfish in various stages of the molt cycle and glandular PDEactivity determined using [3H]cGMP as substrate. Results are expressed as mean±SE(n=4–8). Data were analyzed by ANOVA and Tukey's multiple comparisons. Among themultiple comparisons, middle premolt was significantly different from intermolt andpostmolt (Pb0.001). From Nakatsuji et al., 2006a; used by permission of the publisher.

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and Webster, 2003) and crayfish (Nakatsuji and Sonobe, 2004). Thecellular mechanism(s) underlying the changes in responsiveness of Y-organs to MIH are not well understood. Radioligand binding assaysrevealed no change in the number of MIH receptors in Y-organs duringa molt cycle in C. maenas (Chung and Webster, 2003), indicating thatchanges in sensitivity to MIH are likely due to events downstream ofreceptor binding and may involve changes in intracellular signalingpathways. The observation that 3-isobutyl-1-methylxanthine (IBMX),a nonselective inhibitor of cyclic nucleotide phosphodiesterase (PDE),suppressed ecdysteroid secretion by Y-organs (Mattson and Spaziani,1985b) suggests that intracellular PDE activity may be involved inregulation of Y-organ responsiveness.

To assess the possible involvement of PDE in determining the re-sponsiveness of Y-organs to MIH, Y-organs were removed from crayfish(P. clarkii) at various stages of the molt cycle and incubated in vitro withMIH, IBMX, or both (Nakatsuji et al., 2006a). The responsiveness of Y-organs to MIH alone was high during the intermolt stage, declined inearly premolt, was low during middle and late premolt, and thenincreased during postmolt (Fig. 6), confirming previous reports of stage-specific changes in responsiveness. When IBMX was included in theincubation medium along with MIH, the responsiveness of premolt Y-organs toMIHwas restored to a level similar to that observed in intermoltY-organs (Fig. 6). The results suggest that stage-specific changes in theresponsivenessofY-organs toMIHmaybea resultof changes inglandularPDE activity. Assays of PDE activity in P. clarkii Y-organs revealed enzymeactivity was low during intermolt stage, rose to a peak during middlepremolt, and then declined again by postmolt (Fig. 7). The combinedresults are consistentwith the hypothesis that PDE activity plays a centralrole in determining the responsiveness of Y-organs to MIH during themolt cycle. PDE activity in Y-organs of P. clarkiiwas strongly inhibited by8-methylmethoxy-IBMX (a selective inhibitor of PDE1) (Nakatsuji et al.,2006a). Our tentative conclusion is that the PDE isotype present in Y-organs of P. clarkii is PDE1. PDE1 enzymes are calcium/calmodulin-dependent, and catalyze the hydrolysis of cAMP and cGMP; they arerecognized as keyenzymes involved in the complex interactions betweenthe cyclic nucleotide and calcium second messenger systems (review:Kakkar et al.,1999). The conclusion that Y-organs ofP. clarkii contain PDE1is consistent with the observations of Mattson and Spaziani (1986) thatecdysteroid production by C. antennarius Y-organs in vitrowas enhancedby a calcium ionophore (A23187), and that a calmodulin inhibitor(trifluoperazine) lessened the inhibitory effect of sinus gland extract on

ecdysteroidogenesis. Mattson and Spaziani (1986) hypothesized thatcalcium/calmodulin increases PDE activity, thereby antagonizing theinhibitory effect ofMIH on ecdysteroid production. Thus, calciummay beinvolved in the regulation of responsiveness of Y-organs to MIH throughactivation of glandular PDE.

6. Summary

Since the observation by Zeleny (1905) that eyestalk ablationaccelerates molting, and the subsequent findings by Brown andCunningham (1939) that eyestalk-mediated control of molting ishormonal, much has been learned of the structure and function of thecrustacean molt-inhibiting hormone (MIH). This review is focused oncurrent knowledge of the (a) structure of the MIH peptide and gene,(b) levels of MIH in eyestalks and hemolymph, (c) cellular mechanismof action of MIH, and (d) responsiveness of Y-organs to MIH.

Application of the methods of molecular biology and proteinbiochemistry has yielded considerable information on the structure oftheMIHpolypeptide and gene. Amino acid sequence data placeMIH in thecrustacean hyperglycemic hormone (CHH) family of peptides. Theexistence of multiple molecular forms of type I CHH family peptides iswell established. Recent reports indicatemultiplemolecular forms ofMIH/MIH-like peptides (type II peptides) are present in penaeids. The possibilitythat multiple forms of MIH exist in Brachyura and Astacidea should becarefully considered. Also needed are additional experiments designed totest the hypothesis that there is functional divergence among themultiplestructural variants.

Hemolymphatic MIH titers have been reported for only two species(the crayfish P. clarkii and the crab C. maenas). The data are dissimilar.For P. clarkii, the data suggest that a drop in hemolymphatic MIH duringearly premolt permits the increase in ecdysteroid production that drivesmolting (Nakatsuji and Sonobe, 2003; 2004). By contrast, for C. maenas,the results show no change in the level of MIH during premolt (Chungand Webster, 2005). Additional data from other species are needed.Given the sequence similarity between MIH and other CHH familypeptides, and because the level of CHH in hemolymph is significantlyhigher than the level of MIH in hemolymph, the specificity of the assaysused to quantify MIH will be of critical importance.

Regarding the cellularmodeof action ofMIH,we think the compositedata strongly favor the hypothesis that MIH-induced suppression ofecdysteroidogenesis is mediated by a cGMP second messenger. How-ever, alternative hypotheses exist, and a role for cAMP cannot be ruledout. Conclusive identification and thorough characterization of theMIHreceptor will be critical steps in resolving these issues.

Stage-specific changes in the responsiveness of Y-organs to MIHappear to play an underappreciated role in the regulation of ecdysteroidproduction. Recent data indicate that changes in glandular responsive-ness toMIH are due, at least in part, to changes in glandular PDE activity.Because glandular PDE activity is Ca+/calmodulin dependent, it seemsclear that an understanding of the mechanisms that underlie theregulation of free calcium levels in Y-organs is critical to a comprehen-sive understanding of the regulation of ecdysteroidogenesis.

Acknowledgments

Research from our laboratories was supported by the NationalScience Foundation (IBN-0213047), the U.S. Department of Commerce/National Oceanic and Atmospheric Administration through TheUniversity of Southern Mississippi (NA06OAR4170078), and theNational Science Council (93-2311-B-018-001; 94-2311-B-018-001).

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