Hormonal Control of Molting in Decapod Crustacea

The involvement of the molting hormone, 20-hydroxyecdysone,in the mediation of molting in decapod crustaceans is brieflyreviewed. Aspects of the secretion and metabolism of its precursor,ecdysone, are discussed. Experiments are described that demonstratethe presence of a molt-inhibiting hormone (MIH) in the sinusglands of juvenile lobsters (Homarus americanus). Assays forMIH include measurement of the molt interval and radioimmunoassayof circulating titers of ecdysteroids in eyestalk-ablated lobsters.This latter assay indicates that sinus gland extracts significantlydecrease the concentration of circulating ecdysteroids 24 hrafter injection. Data are also presented on the circulatingtiters of ecdysteroids during multiple molt cycles of lobstersfollowing eyestalk ablation. These data indicate that theremust be another factor that ultimately regulates the circulatinglevels of the molting hormone.     

Further Studies on Signaling Pathways for Ecdysteroidogenesis in Crustacean Y-Organs

The Y-organs of crustaceans secrete ecdysteroids (molting hormones)and are regulated (negatively) by a neurosecretory peptide,molt-inhibiting hormone (MIH). Signaling path(s) in Y-organswere explored that connect MIH receptors ultimately with suppressionof receptor number for the uptake of cholesterol (ecdysteroidprecursor) and of gene expression of steroidogenic enzymes.Experiments were conducted in vitro with Y-organs of crabs (Cancerantennarius, Menippe mercenaria) and crayfishes (Orconectessp.). It was confirmed in all species that steroidogenesis occursin the absence of external calcium (Ca⁺⁺), but increases toa maximum as Ca⁺⁺ is increased to 1 to 10 mM and is substantiallyinhibited at higher Ca⁺⁺ concentrations. MIH does not requireexternal Ca⁺⁺ for inhibitory action, but inhibition is eliminatedby high Ca⁺⁺concentrations. Several experimental approachesfailed to find evidence of phospholipase C activation, turnoverof inositol triphosphate or diacylglycerol generation connectedwith steroidogenesis. Unbinding or chelation of intracellularCa⁺⁺ with thapsigargin or TMB-8, respectively both caused dose-dependentinhibition of ecdysteroid output. Blockade of Ca⁺⁺ channelswith verapamil, nifedipine or nicardipine also inhibited steroidogenesis;highest doses inhibited profoundly to below Ca⁺⁺-free basallevels. Inhibition also was obtained with all doses of the Ca⁺⁺channel agonist/antagonist (–) BAY K 8644 in crabs, butin crayfishes lower doses were stimulatory. However, if thecrayfish cells were depolarized, allowing greater Ca⁺⁺ influx,the previously stimulatory doses of BAY K 8644 became inhibitory.Y-organ protein kinase C (PKC) is Ca⁺⁺-sensitive. Activationof PKC was uniformly stimulatory in crabs, but inhibitory incrayfishes. Cytochalasin D, which disrupts the actin cytoskeleton,and which causes moderate Ca⁺⁺ influx, stimulated hormone formation.These results are interpreted to indicate a regulatory rolefor Ca⁺⁺ in ecdysteroidogenesis, involving a local, submembranecirculation of Ca⁺⁺ through ion channels and Ca⁺⁺ pumps andinteraction with PKC in phosphorylating key proteins. An optimallocal Ca⁺⁺ environment fostering hormone synthesis is evidentsince too little or too much Ca⁺⁺ is inhibitory. Methyl farnesoate (MF) had no effect on ecdysone productionin crab or crayfish Y-organs in 24-hr incubations with MF at100 pM to 10 µM.     

Hormones in the Lives of Crustaceans: An Overview

We present an overview of the isolation and characterizationof three hormones (or hormone families) important for the growthand development of decapod crustaceans. These hormones includethe ecdysteroids (steroid molting hormones), the hyperglycemichormone neuropeptide family, and the terpenoid methyl farnesoate.Using examples primarily from our laboratory, we describe workon these hormones using various life stages of the lobster (Homarusamericanus) as the principal model.     

Development and utilization of crustacean long-term primary cell cultures: Ecdysteroid effects in vitro

Summary

Long-term maintenance of lobster, Homarus americanus and crayfish, Pacifasticus leniusculus primary cell cultures of testicular and hematopoietic tissues, for 11 and 3 months, respectively, succeeded in a modified Medium 199 supplemented with 10% fetal bovine serum (pH 7.5, 200°C). In addition, NaC1 was used to adjust the lobster culture medium to 1000 mOsm and the crayfish medium to 400 mOsm. Proline concentration was also elevated. Testes were dissociated with 200 U/ml type II collagenase 2–3 days prior to culture.

Lobster hemocytes reacted to 10^?7 M 20-hydroxyecdysone (20-HE) by reducing contact inhibition and increasing invasive behavior one week after hormonal exposure. The presence of 10^?7 M 20-HE caused mesodermal cell death and spermatogonial proliferation in lobster testicular cell cultures within one week. Crayfish testicular mesodermal cells formed vacuoles 5 days after exposure to 10^?8 M 20-HE. These results are discussed in relation to the cellular events that occur in vivo during premolt.     

Hormonal Control of Sexual Differentiation and Reproduction in Crustacea

SYNOPSIS. Sexual differentiation in malacostracan Crustaceais controlled by the androgenic gland hormone (AGH). In males,the primordial androgenic glands (AG) develop and AGH inducesmale morphogenesis. In females, the primordial AG does not developand the ovaries differentiate spontaneously. Implantation ofthe AG into females yields various results, showing that thesensitivity to AGH differs with the species and the receptiveorgans. Purified AGH of the isopod Armadillidium vulgare consistsof at least two molecular forms, which exist as monomeric proteinswith molecular weights of 17,000 ± 800 and 18,300 ±1,000 Da and with isoelectric points of about 4.5 and 4.3, respectively.The antiserum raised against purified AGH makes it possibleto measure AGH activity by immunoassay. Neurohormones control male and female reproduction. In males,they are involved in the maintenance of the male germinativezone and the control of AG activity. In females, the secondaryvitellogenesis is controlled by the vitellogenesis-inhibitinghormone (VIH) and the vitellogenesis- stimulating hormone (VSH).VIH isolated from the lobster Homarus americanus is a peptidewith a molecular weight of 9,135 Da and shows homology to thecrustacean hyperglycemic hormone and moltinhibiting hormone.Involvement of the molting hormone and the juvenile hormone-likecompound in the secondary vitellogenesis have also been suggested.In the amphipod Orchestia gammarella, the vitellogenesis- stimulatingovarian hormone (VSOH) seems to control vitellogenin synthesis     

Characterization and sequence elucidation of a novel peptide with molt-inhibiting activity from the South African spiny lobster, Jasus lalandii

We have isolated a peptide from extracts of sinus glands from a South African spiny lobster species, Jasus lalandii, by high-performance liquid chromatography (HPLC) and identified it as a putative molt-inhibiting hormone (MIH) by (i) an in vitro assay with J. lalandii Y-organs to measure the inhibition of ecdysteroid synthesis and (ii) an immunoassay using antiserum raised against MIH of the edible crab. The MIH of J. lalandii has 74 amino acid residues, a molecular mass of 9006 Da, a free N-terminus and an amidated C-terminus. The full primary sequence has been obtained from sequencing various digest fragments (tryptic, endoproteinase Asp-N, cyanogen bromide) of the unreduced (native) peptide: RFTFDCPGMMGQRYLYEQVEQVCDDCYNLYREEKIAVNCRENCFLNSWFTVCLQATMREHETPRFDIWR SIILKA-NH(2). Structural comparisons with other peptides show that the J. lalandii MIH belongs to the peptide family which includes the crustacean hyperglycemic hormone, molt-inhibiting hormone and vitellogenesis-inhibiting hormone (cHH/MIH/VIH). This novel peptide has 36-43% sequence identity to putative MIHs from other decapod crustaceans and 32-34% identity to the two cHH peptides previously identified in this spiny lobster species. This is the first report of a peptide with MIH activity in the Palinuridae infraorder.     

The crustacean hyperglycemic hormone precursors a and b of the Norway lobster differ in the preprohormone but not in the mature peptide

The neuro-endocrine X-organ sinus-gland complex of crustaceans produces and releases the neuropeptides of the crustacean hyperglycemic hormone (cHH)/molt-inhibiting hormone (MIH)/gonad-inhibiting hormone (GIH) family that regulate important physiological processes, such as growth, reproduction and molting. We cloned two full-length cDNAs encoding the preprocHH-A and preprocHH-B of the Norway lobster Nephrops norvegicus of 132 and 131 amino acid residues. The two cHHs differ in the preprohormone but not in the mature peptide sequence. The mature cHH was expressed in bacteria as GST fusion protein that, in bioassay, shows a hyperglycemic activity similar to that of native cHH present in an eyestalk extract.     

Hemolymph ecdysteroids during the last three molt cycles of the blue crab,Callinectes sapidus: quantitative and qualitative analyses and regulation

The profiles of circulating ecdysteroids during the three molt cycles prior to adulthood were monitored from the juvenile blue crab, Callinectes sapidus. Ecdysteroid patterns are remarkably similar in terms of peak concentrations ranging between 210–330 ng/ml hemolymph. Analysis of hemolymph at late premolt stage revealed six different types of ecdysteroids with ponasterone A (PoA) and 20‐OH ecdysone (20‐OH E) as the major forms. This ecdysteroid profile was consistent in all three molt cycles. Bilateral eyestalk ablation (EA) is a procedure that removes inhibitory neurohormones including crustacean hyperglycemic hormone (CHH) and molt‐inhibiting hormone (MIH) and often results in precocious molting in crustaceans. However, the inhibitory roles of these neuropeptides in vivo have not yet been tested in C. sapidus. We determined the regulatory roles of CHH and MIH in the circulating ecdysteroid from ablated animals through daily injection. A daily administration of purified native CHH and MIH at physiological concentration maintained intermolt levels of ecdysteroids in the EA animals. This suggests that Y organs (YO) require a brief exposure to CHH and MIH in order to maintain the low level of ecdysteroids. Compared to intact animals, the EA crabs did not exhibit the level of peak ecdysteroids, and the major ecdysteroid turned out to be 20‐OH E, not PoA. These results further underscore the important actions of MIH and CHH in ecdysteroidogenesis, as they not only inhibit, but also control the composition of output of the YO activity. © 2009 Wiley Periodicals, Inc.     

Induction and inhibition of an apparent neuronal phenotype in Spodoptera frugiperda insect cells (Sf21) by chemical agents

The goal of this research was to induce neuron-like properties in Sf21 cells, an insect ovarian cell line, which could lead to a new high-throughput insecticide screening method and a way to mass produce insect neuronal material for basic research. This study applied differentiation agents to produce viable neuron-like cells. In the presence of the molting hormone 20-hydroxyecdysone (20-HE), or insulin, in the growth medium, a maximum of ca. 30?% of Sf21 cells expressed an apparent neuronal morphology of unipolar, bipolar, or multipolar axon-like processes within 2?C3?days. Maximal differentiation occurred after 2?days in the presence of 50???M 20-HE or 3?days in 10???M insulin. Both 20-HE and insulin displayed time- and concentration-dependent differentiation with biphasic curves, suggesting that two binding sites or processes were contributing to the observed effects. In addition, combinations of 20-HE and insulin produced apparent synergistic effects on differentiation. Caffeine, a central nervous system stimulant, inhibited induction of elongated processes by 20-HE and/or insulin, with an IC₅₀ of 9 nM for 20-HE, and the inhibition was incomplete, resulting in about one-quarter of the differentiated cells remaining, even at high concentrations (up to 1?mM). The ability to induce a neural phenotype simplifies the studies of insect cells, compared to either the use of primary nervous tissue or genetic engineering techniques. The presence of ion channels or receptors in the differentiated cells remains to be determined.     

三疣梭子蟹蜕皮周期中MIH基因mRNA水平与蜕皮激素浓度变化

甲壳动物的蜕皮过程被认为是由位于眼柄的X器-窦腺复合体(XO-SG)分泌蜕皮抑制激素(MIH)通过调节Y器(YO)合成蜕皮激素而调控的。通过实时荧光定量PCR(qRT-PCR)发现MIH基因在三疣梭子蟹眼柄X器-窦腺复合体中表达最强。采用qRT-PCR分析了MIH基因在三疣梭子蟹蜕皮周期中的表达变化, 结果表明; A期为(0.42±0.08)倍, B期为(1.09±0.09)倍, C期为(1.35±0.16)倍, D₀亚期为(1.00±0.10)倍, D₁亚期(0.78±0.07)倍, D₂亚期为(0.27±0.08)倍, D_3/4亚期为(0.20±0.04)倍。采用高效液相色谱-电喷雾串联质谱(LC-MS/MS)法完成了三疣梭子蟹蜕皮周期中蜕皮激素(20E)浓度变化的测定。A/B期蜕皮激素的浓度较低, 低于仪器检测限0.33 pg, C期为(1.666±0.762) ng/mL, D₀亚期为(4.047±1.5133) ng/mL, D₁亚期为(6.756±4.928) ng/mL, D₂亚期为(8.609±3.827) ng/mL, D₃亚期为(19.534±4.799) ng/mL, D₄亚期为11.616 ng/mL。在三疣梭子蟹蜕皮周期中, MIH基因表达量与血淋巴中蜕皮激素浓度呈现一定拮抗性, 揭示MIH抑制Y器合成蜕皮激素而调控着三疣梭子蟹蜕皮的发生和进行。     

双斑恩蚜小蜂寄生对烟粉虱体内保幼激素和20-羟基蜕皮酮含量的影响

用反相高效液相色谱法（RP-HPLC）测定了烟粉虱Bemisia tabaci体内保幼激素和20-羟基蜕皮酮的滴度。结果显示,被双斑恩蚜小蜂Encarsia bimaculata寄生的烟粉虱高龄若虫体内的保幼激素滴度显著高于（P<0.05）对照即未被寄生的高龄若虫,20-羟基蜕皮酮（20-E）滴度显著低于（P<0.05）对照,与未被寄生的低龄若虫滴度差异不显著,说明双斑恩蚜小蜂寄生后可以将烟粉虱高龄若虫的激素含量保持在低龄若虫的水平,从而调节烟粉虱高龄若虫的生长发育。烟粉虱伪蛹和成虫与对照体内保幼激素含量,烟粉虱伪蛹与对照之间20-E含量差异均不显著（P>0.05）。     

Regulation of the Crustacean Mandibular Organ

The crustacean mandibular organ (MO) produces methyl farnesoate(MF), a juvenile hormone-related compound thought to have rolesin crustacean reproduction and development. Therefore, the controlof MF production by the MO has been of considerable interest.Current evidence indicates that the MO is negatively regulatedby peptides present in the eyestalk (MO inhibiting factor, MO-IH).Several eyestalk neuropeptides have been identified that inhibitMF synthesis by MO incubated in vitro. The amino acid sequencesof these MO-IH peptides are similar to peptides in the crustaceanhyperglycemic hormone (CHH) family of neuropeptides. In addition,there appears to be a compound in the eyestalk that lowers hemolymphlevels of MF in vivo but does not directly affect the MO invitro. The inhibition of MF synthesis by eyestalk peptides involvesthe inhibition of farnesoic acid O-methyl transferase, the lastenzyme in the MF biosynthetic pathway. The activity of thisenzyme is affected by cyclic nucleotides, suggesting that thesecompounds may be involved in the signal transduction pathwaymediating the effects of MO-IH.     

中华绒螯蟹血淋巴20-羟蜕皮酮诱发蜕皮和卵巢发育的作用

通过放射免疫法测定了中华绒螯蟹蜕皮周期血淋巴20-羟蜕皮酮(20-HE)含量的变化。血淋巴20-HE同卵母细胞发育各个阶段有密切的时相关系:在卵母细胞小生长期血淋巴20-HE持续上升,经青春蜕皮进入卵母细胞大生长期后又迅速降低。不能及时青春蜕皮的青春期前雌蟹,稍后仍出现20-HE下降的趋势。对不同实验条件和生理状态下雌蟹的比较表明,20-HE具有诱发蜕皮的特性。卵母细胞早期生长必需高浓度的血淋巴20-HE。外源注射的20-HE有刺激卵巢增重的作用。     

Molecular and biological characterization of molt-inhibiting hormone of Penaeus monodon

The action of molt-inhibiting hormone (MIH) on the inhibition of ecdysone release from the Y-organ of decapod crustacean keeps the animal in the intermolt stage that dominates its molting cycle. MIH is thus one of the major keys in mediating growth and reproduction. This study has isolated cDNA encoding two types of MIH, Pem-MIH1 and Pem-MIH2, from the black tiger shrimp, Penaeus monodon on the basis of sequence homology to MIH from two other shrimp species. The full-length cDNA of Pem-MIH1 was characterized. Pem-MIH1 cDNA harbored 318 bp open reading frame that coded for a translated product containing 28 amino acids of the signal peptide and a putative mature Pem-MIH of 77 amino acids. The recombinant Pem-MIH1 was expressed in Pichia pastoris as a secreted protein. After purification by gel filtration, the purified Pem-MIH1 exhibited the ability to extend molting duration of P. monodon from 11.8 days to 16.3 days suggesting that Pem-MIH1 be responsible for molt-inhibiting function in the shrimp. The attempt to clone Pem-MIH1 and Pem-MIH2 genes was achieved by direct PCR amplification and PCR-based genome walking strategy, respectively. The structure of both Pem-MIH genes, containing three exons interrupted by two introns, was similar to each other and also to that of MIH genes of other crustaceans reported so far. Expression study of Pem-MIH1 and Pem-MIH2 in various tissues of P. monodon revealed the difference in expression patterns. Pem-MIH1 expressed in both the eyestalk and the thoracic ganglia whilst Pem-MIH2 expression was limited to the eyestalk. The expression of MIH in non-eyestalk tissue may suggest additional role of this hormone.     

20-hydroxyecdysone stimulates proliferation of glial cells in the developing brain of the moth Manduca sexta

The steroid hormone 20-hydroxyecdysone (20-HE) controls diverse aspects of neuronal differentiation during metamorphosis in the hawkmoth Manduca sexta. In the present study we have examined the effect of 20-HE on glial cells of the brain during the metamorphic period. The antennal (olfactory) lobe of Manduca provides an ideal system in which to study effects of hormones on glial cells, since three known classes of glial cells participate in its development, and at least one type is critically important for establishment of normal neuronal morphology. These glial cells, associated with the neuropil, form boundaries for developing olfactory glomeruli as a result of proliferation and migration. We determined whether glial cells proliferate in response to 20-HE by injecting a pulse of 20-HE into the hemolymph at different stages of development and monitoring proliferation of all three types of glial cells. Hormone injections at the beginning and end of metamorphic development, when hormone titers are normally low, did not stimulate proliferation of neuropil-associated glial cells. Injections during the period when hormone titers are normally rising produced significant increases in their proliferation. Injections when hormone titers are normally high were ineffective at enhancing their proliferation. One other class of glial cells, the perineurial cells, also proliferate in response to 20-HE. Thus, glial proliferation in the brain is under the control of steroid hormones during metamorphic development. © 1995 John Wiley & Sons, Inc.     

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

In Crustacea, secretion of ecdysteroid 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 mechanism of action of MIH, and (d) responsiveness of Y-organs to MIH. At least 26 MIH/MIH-like sequences have been directly determined by protein sequencing or deduced from cloned cDNA. Recent studies reveal the existence of multiple forms of MIH/MIH-like molecules among penaeids and raise the possibility that molecular polymorphism may exist more generally among MIH (type II) peptides. The hemolymphatic MIH titer has been determined for two species, a crayfish (Procambarus clarkii) and a crab (Carcinus maenas). The data are dissimilar and additional studies are needed. Composite data indicate cellular signaling pathways involving cGMP, cAMP, or both may play a role in MIH-induced suppression of ecdysteroidogenesis. Data from the two species studied in our laboratories (P. clarkii and Callinectes sapidus) strongly favor cGMP as the physiologically relevant second messenger. Ligand-binding studies show an MIH receptor exists in Y-organ plasma membranes, but the MIH receptor has not been isolated or fully characterized for any species. Such studies 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 of Y-organs to MIH. The changes in responsiveness result, at least in part, from changes in glandular phosphodiesterase (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.     

Regulation of dopa decarboxylase gene expression in the larval epidermis of the tobacco hornworm by 20-hydroxyecdysone and juvenile hormone

Dopa decarboxylase (DDC) which converts dopa to dopamine is important for cuticular melanization and sclerotization in insects. An antibody to Drosophila DDC was found to precipitate both DDC activity and a 49-kDa polypeptide synthesized by the epidermis of molting Manduca larvae. Using the Drosophila DDC gene, we isolated the Manduca DDC gene which on hybrid selection produced a 49-kDa translation product precipitable by the Drosophila DDC antibody. The 3.1-kb DDC mRNA appeared 12 hr after head capsule slippage (HCS) and reached maximal levels 7 hr later. Peak expression was twofold higher in melanizing allatectomized larvae and could be depressed to normal levels by application of 0.1 micrograms juvenile hormone I at HCS. Infusion of 1 microgram/hr 20-hydroxyecdysone (20-HE) for 18 hr beginning 2 hr after HCS or addition of 1 microgram/ml 20-HE to the culture medium for 24 hr prevented the normal increase in DDC mRNA. When Day 2 fourth instar epidermis was explanted before the molting ecdysteroid rise and cultured with 1-3 micrograms/ml 20-HE for 17 hr and then for 24 hr in hormone-free medium, DDC expression was three- to fourfold higher than that in epidermis cultured in the absence of hormone. Twelve or more hours of incubation with 20-HE was required for an increase in DDC mRNA, but continuous exposure to 20-HE prevented the increase. In all cultures an initial rapid increase in DDC mRNA was observed which decayed with time in vitro and apparently was associated with the wound response. Thus, ecdysteroid during a larval molt is necessary to program the later expression of DDC, but the subsequent decline of the ecdysteroid is required for this expression to occur.     

Ecdysteroid control of ionic current development in Manduca sexta motoneurons

The steroid hormone 20-hydroxyecdysone (20-HE) regulates several processes during insect metamorphosis. We studied the effects of 20-HE on the development of voltage-sensitive ionic currents of thoracic leg motoneurons of Manduca sexta. The larval leg motoneurons persist throughout metamorphosis but undergo substantial morphological reorganization, which is under the control of 20-HE and accompanied by changes in Ca²⁺ and K⁺ current densities. To determine whether 20-HE controls the changes in Ca²⁺ and K⁺ current levels during postembryonic development, identified thoracic leg motoneurons isolated from late larval and early pupal stages were taken into primary cell culture. Whole-cell Ca²⁺ and K⁺ currents were measured after 1–4 days of steroid hormone incubation. In the presence of 20-HE, peak Ca²⁺ currents of pupal leg motoneurons increased from day 1 to day 4 in vitro. Thus, at culture day 4 the pupal Ca²⁺ current levels were larger in 20-HE–treated than in untreated cells. By contrast, 20-HE did not affect the Ca²⁺ current amplitudes of larval leg motoneurons. Whole-cell K⁺ currents, measured at 4 days in pupal motoneurons, consisted of a fast-activating transient current and a sustained, slowly inactivating current. 20-HE did not affect the amplitude of the transient or sustained currents after 4 days in vitro. Thus, a direct steroid hormone effect may control the proper maturation of voltage-sensitive Ca²⁺ currents in leg motoneurons. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 211–223, 1998     

Expression of crustacean (Callinectes sapidus) molt-inhibiting hormone in insect cells using recombinant baculovirus.

Molt-inhibiting hormone (MIH) negatively regulates the synthesis of ecdysteroid molting hormones by crustacean Y-organs. We report here the expression of blue crab (Callinectes sapidus) MIH in insect cells using recombinant baculovirus. Insect Sf9 cells were transfected with recombinant baculovirus containing a DNA insert encoding the C. sapidus MIH prohormone (signal sequence plus mature hormone). The construct was designed to yield a mature, fully processed recombinant MIH (recMIH). Several baculovirus recombinants showing no contamination with wild-type viral DNA were subsequently analyzed for their ability to direct expression of recMIH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from infected cells revealed time-dependent expression of two proteins of approximately the predicted size for the C. sapidus MIH prohormone and mature hormone. Western blot results (using antiserum against MIH of Carcinus maenas) indicated that the proteins were MIH-immunoreactive. N-Terminal amino acid sequence data and mass spectral analysis indicated the expressed proteins were of the correct sequence and molecular mass. Cell lysates containing the recombinant protein dose-dependently suppressed the synthesis of ecdysteroids by Y-organs in vitro. We anticipate the recombinant peptide will prove useful for studies of the structure and function of MIH.