Molecular mechanisms regulating molting in a crustacean Halagowder Devaraj and Ayithan Natarajan Unit of Biochemistry, Department of Zoology, University of Madras, Tamil Nadu, India Periodic shedding of exoskeleton is associated with growth in crustaceans. The mechanisms that control this phenomenon may also control the growth and dif- ferentiation processes. Eyestalk ablation has been tra- ditionally carried out to shorten the duration of the molt cycle and to influence the growth, reproduction and other metabolic activities of crustaceans [1]. Lack of adequate knowledge on the molecular physiology of eyestalk ablation, excessive loss of hemolymph, labor intensiveness, increased incidence of shrimp mor- tality and reduction in life span restrict the applicabil- ity of the eyestalk ablation technique to modern aquaculture practices. Efforts therefore have been made to understand the molecular physiology of mol- ting and its ramifications in aquaculture practices. As the growth in crustaceans is not continuous because of the rigid exoskeleton, it is often shed to allow periodic growth [2]. Molting is controlled by a complex interplay of hormones, in particular, the negative regulation of molt-inhibiting hormone (MIH) from X-organ sinus gland (XO-SG) complex which suppresses the synthesis or secretion of molting hor- mones (ecdysteroids) from the Y-organ [3,4]. As crustacean growth consists of tandem prolifera- tive and growth arrest phases, we investigated the expression of the growth arrest-specific protein (Gas7) in the molting process of crustaceans. Gas7 was pri- marily characterized in NIH 3T3 cultured fibroblast cells which enter a quiescent state following serum deprivation [5]. It promotes G 0 arrest and is preferen- tially expressed in differentiated neuronal cells and peripheral nerves in mouse and other animals [6] Individual Gas7 genes have been implicated in a vari- ety of biological functions, including the control of Keywords crustaceans; ecdysteroid; growth arrest- specific protein (Gas7); molt-inhibiting hormone; X-organ sinus gland complex Correspondence H. Devaraj, Unit of Biochemistry, Department of Zoology, University of Madras, Life Sciences Building, Guindy Campus, Chennai 600 025, Tamil Nadu, India Fax: +91 44 22301003 Tel: +91 44 22200574 E-mail: hdrajum@yahoo.com (Received 20 September 2005, revised 15 December 2005, accepted 22 December 2005) doi:10.1111/j.1742-4658.2006.05117.x Crustacean growth and development is characterized by periodic shedding (ecdysis) and replacement of the rigid exoskeleton. Secretions of the X-organ sinus gland complex control the cellular events that lead to growth and molting. Western blot and ELISA results showed a progressive increase in growth arrest-specific protein (Gas7) from early postmolt stage to a maximum at late postmolt stage. Phosphorylation of ERK2, a down- stream signaling protein, was also identified in the subsequent stages. ERK2 phosphorylation resulted in the expression of molt-inhibiting hor- mone (MIH). Specific ERK inhibitors (PD98059 and UO126) exhibited the ability to reduce the molting duration of Fenneropenaeus indicus from 12–14 days to 7–8 days, suggesting that the ERK1 ⁄ 2 signaling pathway is responsible for the expression of MIH, which controls the molt cycle. We have identified the stage-specific expression of Gas7 ( 48 kDa) in the X-organ sinus gland complex of eyestalk which is involved in the down- stream signaling of the ERK1 ⁄ 2 pathway regulating the expression of MIH during the molt cycle of the white shrimp, F. indicus. These are the first data showing an association between the Gas7 signal-transduction process and regulation of the molt cycle and provides an alternative molecular intervention mechanism to the traditional eyestalk ablation in crustaceans. Abbreviations ERK, extracellular signal-regulated kinase; Gas7, growth arrest-specific protein 7; MIH, molt-inhibiting hormone; XO-SG complex, X-organ sinus gland complex. FEBS Journal 273 (2006) 839–846 ª 2006 The Authors Journal compilation ª 2006 FEBS 839 microfilament organization [6], nerve cell growth or differentiation [7], tyrosine kinase receptor activity [7], and the negative [8] and positive [7] control of cell cycling in human Schwann cells. Receptor tyrosine kinases play a central role in transducing the external signals across cell membranes into the intracellular signaling systems, which in turn lead to cell proliferation, differentiation, and other responses in human Schwann cells [7]. Receptor tyro- sine kinases transduce the signals via extracellular signal-regulated kinases (ERKs), the serine ⁄ threonine protein kinases belonging to the family of mitogen- activated protein kinases in cardiac myocytes [9]. ERKs play an important role in the downstream regu- lation of several cellular processes, such as prolifer- ation and differentiation, and directly modulate cellular functions that influence gene transcription and translation in cancer cells [10]. The role of Gas7 has not been studied so far in crus- taceans. As the XO-SG complex is a structural and functional homologue of vertebrate hypothalamus, which is involved in the secretion of neurohormones that control growth and differentiation processes such as molting [11], we focused on the expression of Gas7 in the XO-SG complex and its role in the regulation of MIH expression. Therefore this study aimed to illustrate the signal- transduction pathway in the regulation of the molt cycle in Fenneropenaeus indicus and to devise a mole- cular intervention technology as an alternative to the traditional eyestalk ablation process. Results Western blot analysis Western blot analysis of extracts prepared from the eyestalk of F. indicus during different molting stages showed a single dominant band of Gas7 only in the early postmolt (A) and late postmolt (B) stages (Fig. 1A). The immunoreactive band of Gas7 had a molecular mass of  48 kDa. It was prominently detec- ted in the XO-SG complex using rabbit polyclonal anti-Gas7 serum, but was not observed in other stages of molting (Fig. 1A). Immunohistochemical localization Immunohistochemical analysis of eyestalk neural gan- glia sections showed a highly positive immunoreactive expression of Gas7 in the XO-SG complex of the eye- stalk of F. indicus only in the early postmolt (A) and late postmolt (B) stages but not in the intermolt (C), early premolt (D 0 and D 1 ) and late premolt (D 2-3 ) sta- ges using antibody to Gas7 (Fig. 2). The immunoreac- tivity of Gas7 was observed prominently as a cluster of neurosecretory cell bodies or group of cell mass exclusively over the neurosecretory centers of the XO-SG complex. Most of the Gas7-expressing neuro- secretory cell bodies were found in the peripheral regions and the neuropile regions of eyestalk neural ganglia and more adjacent to the medulla terminalis X-organ and sinus gland. A B Fig. 1. Western blot analysis of proteins from the eyestalk of F. indicus. (A) The supernatant from the XO-SG complex of the eyestalk of F. indicus was subjected to SDS ⁄ PAGE (10% gel) and electroblotted on to nitrocellulose membrane. The  48-kDa Gas7 was detected only in the early postmolt and late postmolt stages by western blot analysis using rabbit Gas7 polyclonal antibody. (B) Protein samples were extracted from the eyestalk of different molting stages of F. indicus and subjected to SDS ⁄ PAGE (10% gel); proteins were transferred on to nitrocellulose membrane. Phosphorylation of ERK2 protein was found in the intermolt and early premolt stages by a shift in electrophoretic mobility on west- ern blot analysis using rabbit ERK1 ⁄ 2 antibody, goat anti-rabbit IgG–horseradish peroxidase conjugate and developed with 3,3¢-di- aminobenzidine tetrahydrochloride substrate. KD, kDa; M, broad range of standard protein markers run on SDS ⁄ 10% polyacryl- amide gel, electroblotted on to nitrocellulose membrane, and stained with Ponceau S red; A, early postmolt; B, late postmolt; C, intermolt; D 0 , early premolt; D 1 , early premolt; D 2-3 , late premolt. Gas7 and the ERK1 ⁄ 2 signaling pathway in MIH expression H. Devaraj and A. Natarajan 840 FEBS Journal 273 (2006) 839–846 ª 2006 The Authors Journal compilation ª 2006 FEBS ELISA Gas7 concentrations were determined by direct ELISA using antibody to Gas7. A progressive increase from the early postmolt stage to a maximum at the late postmolt stage followed by a sudden fall in the sub- sequent stages (Fig. 3) was observed. The results are consistent with the results of western blot and immu- nohistochemical analysis. Concentrations of the immunoreactively expressed MIH protein were also quantified by direct ELISA from the extracts prepared from the XO-SG complex of the eyestalk from control groups as well as groups treated with specific inhibitors (PD98059 and UO126). In control stages, MIH concentrations were increased significantly from early postmolt to a maximum at intermolt, but were dramatically reduced to a mini- mum at early premolt and late premolt stages. In the case of the PD98059-treated and UO126-treated stages (intermolt and early premolt), MIH concentrations were reduced to a basal level (Fig. 4A,B). Phosphorylation of ERK2 Phosphorylation or activation of ERK2 protein was found only in intermolt and early premolt by a shift in mobility on western blot analysis (Fig. 1B). No shift in electrophoretic mobility was observed in early postmolt, late postmolt, early premolt and late premolt stages. Fig. 2. Immunohistochemical localization of Gas7 expression was observed only in (A) early postmolt and (B) late postmolt stages in the eyestalk tissue of F. indicus using rabbit polyclonal antibody to Gas7. (N), Negative control treated with normal goat serum not showing expression of Gas7; ME, medulla externa; MI, medulla interna; MT, medulla terminalis; SG, sinus gland; XO, X-organ; NSC, neurosecretory cell bodies. Scale bar represents 100 lm. Fig. 3. Quantitative determination of Gas7 concentrations in extracts prepared from the XO-SG complex of F. indicus by direct ELISA using rabbit polyclonal antibody to Gas7. The line diagram represents high concentrations of Gas7 immunoreactivity only in early postmolt, maximum at late postmolt, and basal level in subse- quent stages. Values are expressed as mean ± SD (n ¼ 5). H. Devaraj and A. Natarajan Gas7 and the ERK1 ⁄ 2 signaling pathway in MIH expression FEBS Journal 273 (2006) 839–846 ª 2006 The Authors Journal compilation ª 2006 FEBS 841 Treatment with inhibitors and blocking of ERK2 activation Phosphorylation of ERK2 and expression of MIH were studied in both control and inhibitor-treated (PD98059 and UO126) stages. Phosphorylation of ERK2 was detected as a shift in mobility during the intermolt and early premolt stages of control shrimps, whereas PD98059-treated stages (intermolt and early premolt) showed no shift in ERK2 protein mobility using rabbit anti-ERK1 ⁄ 2 IgG (Fig. 5A). Mouse anti-phospho- ERK1 ⁄ 2 IgG specifically detected only phosphorylated ERK2 protein in control groups of intermolt and early premolt stages, but the antibody could not detect the ERK2 protein in UO126-treated stages (Fig. 5B). Like- wise, the immunoreactive band of MIH was also detec- ted only in the control stages, i.e. early postmolt, late postmolt, intermolt and early premolt, but not in the PD98059-treated and UO126-treated stages (intermolt and early premolt) (Fig. 6A,B). The results are consis- tent with the results of direct ELISA. Fig. 4. Effect of (A) PD98059 and (B) UO126 on MIH concentra- tions of F. indicus. MIH concentrations were determined by direct ELISA in extracts prepared from control (Untreated) and PD98059 or UO126 treated stages of F. indicus using anti-r-Pej-MIH IgG. The bar diagram shows the highly significant concentrations (P<0.0001; analysis of variance) of MIH in the intermolt stage of the control samples, but PD98059 and UO126 treated stages (inter- molt and early premolt) show very low concentrations of MIH. Values are expressed as mean ± SD (n ¼ 5). A B Fig. 5. Effect of (A) PD98059 and (B) UO126 on the phosphoryla- tion of the ERK2 protein from the XO-SG complex. (A) Protein sam- ples from the XO-SG complex of different molting stages were run on SDS ⁄ 10% polyacrylamide gel and electroblotted on to nitrocellu- lose membrane. Phosphorylation of the ERK2 protein was detected by a shift in electrophoretic mobility on western blot analysis only in control stages (intermolt and early premolt), but PD98059-treated stages did not show ERK2 phosphorylation using rabbit antibody to ERK1 ⁄ 2. (B) Mouse anti-phosphoERK1 ⁄ 2 IgG specifically detected only phosphorylated ERK2 protein in control groups of intermolt and early premolt stages, but the antibody could not detect the ERK2 protein in UO126-treated stages. KD, kDa; M, broad range of standard protein markers run on SDS ⁄ 10% polyacrylamide gel, electroblotted on to nitrocellulose membrane, and stained with Ponceau S red; A, early postmolt; B, late postmolt; C, intermolt; D 0 , early premolt; D 1 , early premolt; D 2-3 , late premolt. Gas7 and the ERK1 ⁄ 2 signaling pathway in MIH expression H. Devaraj and A. Natarajan 842 FEBS Journal 273 (2006) 839–846 ª 2006 The Authors Journal compilation ª 2006 FEBS Molting duration of F. indicus The inhibitor-treated (PD98059 and UO126) stages showed an absence of MIH, which reduces the molting duration of F. indicus. The shrimps in the control groups required  12–14 days to complete the molt cycle, whereas in those injected with either PD98059 or UO126 the duration of the molt cycle was reduced to  7–8 days (Fig. 7). The available data also show a correlative and significant change in the shortening of the molt cycle which leads to frequent molting and growth of shrimps. Discussion Gas7 is a multifunctional protein involved in the mat- uration of neurons and release of neurotransmitters [5]. The stage-specific expression of the Gas7 gene using RT-PCR of Gas7 mRNA and coimmunoprecipi- tation of Sky receptor has been identified in the early and late postmolt stages of Penaeus monodon, suggest- ing that Gas7 may act as a ligand for tyrosine kinase Sky receptor (unpublished work). ERK2 is part of the Ras ⁄ Raf ⁄ MEK downstream signaling pathway for many growth factors that act by binding to tyrosine kinase receptors in cardiac myocytes [19]. The mito- gen-activated protein kinase pathway is involved in a number of cellular changes inducing growth and differ- entiation in human Schwann cells [7]. In cardiac myo- cytes, protein kinase C generates a positive feedback loop to ERK2 phosphorylation [19], and this effect has also been demonstrated in P. monodon (unpub- lished work). In decapod crustaceans, the growth and differenti- ation processes are controlled by a complex interplay of neuropeptides that are stage specific and inducible [3,4]. To elucidate the functional necessity of Gas7 in MIH expression, highly selective ERK inhibitors (PD98059 and UO126) were used, which completely inhibited the Gas7-dependent downstream signaling during ERK2 phosphorylation. ERK2 is active only when it is phosphorylated [7]. Inhibition of ERK2 with ERK ⁄ MEK inhibitors (PD98059 and UO126) appears A B Fig. 6. Effect of (A) PD98059 and (B) UO126 on  7–8-kDa MIH from the XO-SG complex. Protein samples prepared from the XO-SG complex of the eyestalk of F. indicus were run on SDS ⁄ 15% polyacrylamide gel. Immunoblotting was performed using anti-r-Pej-MIH IgG in different molting stages. The  7–8-kDa MIH protein was detected only in the control stages (early post- molt, late postmolt, intermolt and early premolt), whereas shrimps treated with PD98059 and UO126 (intermolt and early premolt sta- ges) show an absence of MIH immunoreactivity. KD, kDa; M, broad range of standard protein markers run on SDS ⁄ 15% polyacrylamide gel, electroblotted on to nitrocellulose membrane, and stained with Ponceau S red; A, early postmolt; B, late postmolt; C, intermolt; D 0 , early premolt; D 1 , early premolt; D 2-3 , late premolt. Fig. 7. Molt cycle duration of F. indicus in controls and shrimps injected with specific inhibitors (PD98059 and UO126) on day 3 after the first molt was recorded as hours between the first molt and the second molt. Values are mean ± SD (n ¼ 5). The highly significant (P < 0.0001, analysis of variance) reduction in molt cycle duration was observed only in the D 1 stage. H. Devaraj and A. Natarajan Gas7 and the ERK1 ⁄ 2 signaling pathway in MIH expression FEBS Journal 273 (2006) 839–846 ª 2006 The Authors Journal compilation ª 2006 FEBS 843 to inhibit ERK phosphorylation, which can be seen as shift in the mobility of ERK2 protein during inter- molt and early premolt stages, resulting in the down- regulation of MIH expression. The data show that phosphorylation of ERK2 results in activation of tran- scription factors inside the nucleus that modulate MIH gene transcription and translation. The shrimps treated with ERK inhibitors showed a shortening of the intermolt and early premolt periods, which suggests changes in the periodicity of the mol- ting process. In addition, the molt cycle duration of F. indicus was reduced dramatically from 12 ± 2 days to 7 ± 1 days when the shrimps were injected with ERK inhibitors. Thus, application of these inhibitors significantly reduced the molt cycle duration, as they inhibit MIH by interfering with downstream signals from Gas7. Hence, these data clearly indicate that transcription of Gas7 occurs through co-ordinated events involving activation of the ERK signal (Fig. 8). These are the first data to show an association between the Gas7 gene and molting processes in crus- taceans mediated by the expression of MIH through the ERK1 ⁄ 2 signaling pathway. Reduction of the molt cycle by ERK inhibitors has an application potential in the aquaculture industry. Experimental procedures Experimental animals The Indian white shrimp F. indicus was selected for this study because of its nonseasonal availability and easy main- tenance. They were obtained from the coastal region of Kovalam, Chennai, India. They were maintained in the laboratory at a temperature of 28 ± 2 °C under natural photoperiod (12 h light ⁄ 12 h darkness) in plastic tanks con- taining filtered, continuously aerated seawater (26–28% salinity). The stocking density was 10 shrimps per tank in accordance with the water quality. The filtered seawater was changed everyday, and the animals were fed every day ad libitum with commercial feed pellets. Identification of molting stages The different molting stages of F. indicus were determined from morphological changes in the setae of the uropod and pleopod, carapace and exoskeleton, on the basis of criteria established by Vijayan et al. [12]. The pleopod and uropod of the same animal were removed and placed on a clean micro slide; the slide was then covered with a rectangular coverslip. The slide was kept under a light microscope and the various molting stages such as early postmolt (A), late Gas7 Gas7 binding to Sky receptor (Stage A and B) PD98059 UO126 Inhibition of ERK2 Phosphorylation (Stage C and D 0 ) Inhibition of MIH expression (Stage C and D 0 ) Phosphorylation of ERK2 (Stage C and D 0 ) P P Ras Raf MEK1 ERK1/2 MIH MIHGene MIHGene Expression of MIH (Stage C and D 0 ) Sky receptor phosphorylation (Stage B and C) Fig. 8. Role of Gas7 in the molt cycle of F. indicus. Sky receptor dimerization and autophosphorylation is mediated by Gas7 during early postmolt (A) and late postmolt (B) stages. Activation of the Ras ⁄ Raf ⁄ MEK pathway subsequent to Sky receptor activa- tion results in ERK2 phosphorylation and expression of MIH during intermolt (C) and early premolt (D 0 ) which suppresses the ecdysteroid concentrations. Inhibition of ERK2 phosphorylation by PD98059 and UO126 suppresses MIH expression and increases ecdysteroid concentrations, which results in shortening of the molt cycle. Gas7 and the ERK1 ⁄ 2 signaling pathway in MIH expression H. Devaraj and A. Natarajan 844 FEBS Journal 273 (2006) 839–846 ª 2006 The Authors Journal compilation ª 2006 FEBS postmolt (B), intermolt (C), early premolt (D o and D 1 ) and late premolt (D 2-3 ) were identified. Extraction of eyestalk neural ganglia The eyestalks were clipped from different molting stages of live shrimps. The eyestalks containing whole peduncular neural ganglia (XO-SG complex) were dissected from the surrounding exoskeleton of the eyestalk under a Carl Zeiss (Go ¨ ttingen, Germany) Stereo Zoom dissection microscope. They were homogenized in lysis buffer containing 135 mm NaCl, 20 mm Tris ⁄ HCl, 2 mm EDTA and 1 mm phenyl- methanesulfonyl fluoride, pH 7.4, as described by Watson et al. [13], with slight modifications. The total homogenate was microfuged at 8000 g,4°C for 10 min, and the super- natant was recovered in different eppendorf tubes. They were stored at )20 °C until subsequent analysis. Western blot analysis The proteins were separated by SDS ⁄ PAGE (10% gel) for the detection of Gas7 [14] and electroblotted [15] on a nitrocellulose membrane at 25 V ⁄ 130mA for 4 h at 4 °C. The membrane was incubated overnight at 4 °C with rabbit polyclonal antibody to Gas7 (1 : 1000 dilution; gift from S. Lin-Chao, Institute of Molecular Biology, Academia Sinica, Taiwan) followed by incubation for 2 h at room tempera- ture with goat anti-rabbit IgG (1 : 2000 dilution) conju- gated with horseradish peroxidase. Finally, Gas7 was detected by incubation with 3,3¢-diaminobenzidine tetra- hydrochloride as chromogenic substrate by the method of Ju et al. [5]. Immunohistochemistry Paraffin sections (15 lm) were prepared by conventional methods [16]. The tissue sections were dewaxed in xylene and rehydrated in descending alcohol series and washed in NaCl ⁄ P i (pH 7.4). After being blocked with 3% BSA ⁄ Tris ⁄ NaCl ⁄ Tween, the sections were incubated separately with Gas7 antibody (1 : 1000 dilution) for 16–18 h at 4 °C. After being washed, goat anti-rabbit IgG–horseradish conjugate (1 : 2000 dilutions) was applied to the sections for 2 h at room temperature. Subsequently, Tris ⁄ NaCl (pH 7.6) con- taining 0.05% 3,3¢-diaminobenzidine tetrahydrochloride and 0.01% H 2 O 2 were added as substrate for color development. The reaction was stopped with Tris ⁄ NaCl, dehydrated in an ascending alcohol series, cleared in xylene, and mounted with DPX permount for observation [17]. ELISA ELISA was performed to analyze the concentrations of Gas7 present in the eyestalk as well as to study the concentrations of MIH in the presence of inhibitors (PD98059 and UO126) in the different stages of F. indicus as described by Shih et al. [17]. In this assay, the specific pro- tein samples were diluted with coating buffer and coated on to 96-well microtiter plates. The primary antiserum used was anti-Gas7 (1 : 1000 dilution). Goat anti-rabbit IgG–horse- radish peroxidase conjugate (1 : 2000 dilution) was used as secondary antibody. Thereafter, 3,3¢,5,5¢-tetramethylbenzi- dine was used for color development. The reaction was stopped with 1 m H 2 SO 4 , and the absorbance of the protein was measured spectrophotometrically at 450 nm. Phosphorylation of ERK2 Phosphorylation of ERK2 was detected by a shift in elec- trophoretic mobility as described by Li et al. [7]. The protein samples of different molting stages of F. indicus were run on SDS ⁄ 10% polyacrylamide gels and transferred to nitrocellu- lose membrane (25 V, 130mA for 4 h). The membrane was treated with antibodies raised in rabbit against ERK1 ⁄ 2 pro- tein (primary antibody, 1 : 1000 dilution; Chemicon Inter- national, Temecula, CA, USA) followed by incubation with goat anti-rabbit IgG (1 : 2000 dilution) coupled with horse- radish peroxidase. The membrane was later incubated with 3,3¢-diaminobenzidine tetrahydrochloride substrate. Treatment with inhibitors (PD98059 and UO126) and blocking of ERK2 activation The cultivable white shrimps (F. indicus) were purchased, and two groups of animals at different molting stages were maintained in the laboratory (salinity 26–28%, pH 8.1, and temperature 28 ± 2 °C). Each group contained 10–15 shrimps, of which, one group from the intermolt and early premolt stages were injected with specific inhibitors, and the other served as control. PD98059 and UO126 (Chem- icon International) are highly selective in vivo inhibitors of the ERK kinase cascade. A 50 lm solution was diluted in dimethyl sulfoxide and injected into test shrimps via the arthrodial joints. The control groups were injected with dimethyl sulfoxide only [18]. The injected shrimps were maintained until they reached the second molt of the same stage. Then, the neural ganglia were dissected and homogenized in lysis buffer on ice. The supernatant was recovered after centrifugation at 10 000 r.p.m. for 10 min. The extracts prepared from the XO-SG complex of inhibitor-injected and control shrimps were run on SDS ⁄ 10% polyacrylamide gel and electroblot- ted on to nitrocellulose membrane. Then, the membranes were probed separately with rabbit anti-ERK1 ⁄ 2, mouse anti-phosphoERK1 ⁄ 2 and anti-r-Pej-MIH IgG. In addition, the molting behaviors and molting duration were observed in the control and inhibitor-treated shrimps, and the data were recorded. H. Devaraj and A. Natarajan Gas7 and the ERK1 ⁄ 2 signaling pathway in MIH expression FEBS Journal 273 (2006) 839–846 ª 2006 The Authors Journal compilation ª 2006 FEBS 845 Statistical analysis Mean and standard deviation were calculated. Differences between molting stages were analyzed using one-way analy- sis of variance on spss version 10.0 software (SPSS Inc., Chicago, IL, USA). Acknowledgements This work was supported by grants from the Depart- ment of Science and Technology (DST), UGC-DRS program, COSIST program and Department of Bio- technology (DBT) (BT PR3992 ⁄ AA2 ⁄ 03 ⁄ 202 ⁄ 2003), New Delhi. We gratefully acknowledge Dr Sue Lin- Chao, Institute of Molecular Biology, Academia Sinica, Taiwan, for the gift of Gas7 antibody and Dr Tsuyoshi Ohira, Department of Applied Biological Chemistry, University of Tokyo, Japan, for the gift of MIH anti- body. We also acknowledge Mr A. Anand Kumar for his help in the preparation of the manuscript. References 1 Chang ES (1995) Physiological and biochemical changes during the molt cycle in decapod crustaceans: an over- view. J Exp Mar Biol Ecol 193, 1–14. 2 Keller R (1992) Crustacean neuropeptides: structures, function and comparative aspects. Experientia 48, 439– 447. 3 Soumoff C & O’Connor JD (1982) Repression of Y-organ secretory activity by molt-inhibiting hormone in the crab, Pachygrapsus crassipes. Gen Comp Endocri- nol 48, 432–439. 4 Mattson MP & Spaziani E (1985) Characterization of molt-inhibiting hormone (MIH) action on crustacean Y-organ segments and dispersed cells in culture and a bioassay for MIH activity. J Exp Zool 236, 93–101. 5 Ju YT, Chang ACY, She BR, Tsaur ML, Hwang HM, Chao CCK, Cohen SN & Lin-Chao S (1998) gas7 :a gene expressed preferentially in growth-arrested fibro- blasts and terminally differentiated Purkinje neurons affects neurite formation. Proc Natl Acad Sci USA 95, 11423–11428. 6 Brancolini C, Bottega S & Schneider C (1992) Gas 2, a growth arrest-specific protein, is a component of the microfilament network system. J Cell Biol 117, 1251– 1261. 7 Li R, Chen J, Hammonds G, Phillips H, Armanini M, Wood P, Bunge R, Godowski PJ, Sliwkowski MX & Mather JP (1996) Identification of gas6 as a growth factor for human Schwann cells. J Neuro Sci 16, 2012–2019. 8 Del Sal G, Collavin L, Ruaro ME, Saccone S, Della Valle G & Schneider C (1994) Structure, function, and chromosome mapping of the growth suppressing human homologue of the murine gas1 genes. Proc Natl Acad Sci USA 91, 1848–1852. 9 Bogoyevitch MA, Glennon PE, Andersson MB, Clerk A, Lazou A, Marshall CJ, Parker P & Sugden PH (1994) Endothelin-1 and fibroblast growth factors sti- mulate the mitogen-activated protein kinase signaling cascade in cardiac myocytes. J Biol Chem 269, 1110– 1119. 10 Osborn MT & Chambers TC (1996) Role of stress-acti- vated ⁄ c-Jun NH 2 -terminal protein kinase pathway in the cellular response to adriamycin and other chemo- therapeutic drugs. J Biol Chem 271, 30950–30955. 11 Cooke IM & Sullivan RE (1982) Hormones and neuro- secretion. In The Biology of Crustacea: Structure and Function (Atwood HL & Sandeman DC, eds), vol 3, pp 205–290. Academic Press, New York. 12 Vijayan KK, Sunil Kumar MK & Diwan AD (1997) Studies on molt staging, molting duration and molting behavior in Indian white shrimp, Penaeus indicus Milne Edwards (Decapoda: Penaeidae). J Aqua Trop 12, 53–64. 13 Watson RD, Lee KJ, Borders KJ, Dircksen H & Lilly KY (2001) Molt-inhibiting hormone immunoreactive neurons in the eyestalk neuroendocrine system of the blue crab, Callinectes sapidus. Arthropd Struct Dev 30, 69–76. 14 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4 . Nature 227, 680–686. 15 Towbin H, Staehelin T & Gordon J (1979) Electro- phoretic transfer of proteins from polyacrylamide gel to nitrocellulose sheets. Procedure and some applications. Proc Natl Acad Sci USA 76, 4350–4354. 16 Presnell JK & Screibman MP (1997) Humason’s Animal Tissue Techniques. Johns Hopkins University Press, Baltimore. 17 Shih TW, Suzuki Y, Nagasawa H & Aida K (1998) Immunohistochemical identification of hyperglycemic hormone and molt-inhibiting hormone-producing cells in the eyestalk of the kuruma prawn, Penaeus japonicus. Zool Sci 15, 389–397. 18 Strohm C, Barancik T, Bruhl ML, Kilian SA & Schaper W (2000) Inhibition of the ER Kinase cascade by PD98059 and UO126 counteracts ischemic precondition- ing in pig myocardium. J Cardiovasc Pharmacol 36, 218–229. 19 Kometiani P, Li J, Gnudi L, Kahn BB, Askari A & Xie Z (1998) Multiple signal transduction pathways link Na + ⁄ K + -ATPase to growth-related genes in cardiac myocytes. The role of Ras and mitogen-activated pro- tein kinases. J Biol Chem 273, 15249–15256. Gas7 and the ERK1 ⁄ 2 signaling pathway in MIH expression H. Devaraj and A. Natarajan 846 FEBS Journal 273 (2006) 839–846 ª 2006 The Authors Journal compilation ª 2006 FEBS . Molecular mechanisms regulating molting in a crustacean Halagowder Devaraj and Ayithan Natarajan Unit of Biochemistry, Department of Zoology,. postmolt, maximum at late postmolt, and basal level in subse- quent stages. Values are expressed as mean ± SD (n ¼ 5). H. Devaraj and A. Natarajan Gas7 and the