California-Davis, Bodega Bay, CA USA. *Author for correspondence Accepted 14 September 2009

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1 7 The Journl of Experimentl Biology, 7-8 Pulished y The Compny of Biologists doi:./je.89 Molt cycle regultion of protein synthesis in skeletl muscle of the lckck lnd cr, Gecrcinus lterlis, nd the differentil expression of myosttin-like fctor during trophy induced y molting or unweighting J. A. Covi, B. D. Bder, E. S. Chng nd D. L. Mykles, Deprtment of Biology, Colordo Stte University, Fort Collins, CO 8 USA nd Bodeg Mrine Lortory, University of Cliforni-Dvis, Bodeg By, CA 99 USA Author for correspondence (don@lmr.colostte.edu) Accepted Septemer 9 SUMMARY In decpod crustcens, clw muscle undergoes trophy in response to elevted ecdysteroids while thorcic muscle undergoes trophy in response to unweighting. The signling pthwys tht regulte muscle trophy in crustcens re lrgely unknown. Myosttin is negtive regultor of muscle growth in mmmls, nd myosttin-like cdna is preferentilly expressed in muscle of the lnd cr, Gecrcinus lterlis (Gl-Mstn). Contrry to prediction, levels of Gl-Mstn mrna decresed drmticlly in oth the clw closer nd weighted thorcic muscles when molting ws induced y either eyestlk ltion (ESA) or multiple lim utotomy (MLA). However, the effect of molt induction ws greter in the clw muscle. By lte premolt, Gl-Mstn mrna in the clw muscle decresed 8% nd 9% in ESA nd MLA nimls, respectively, nd ws negtively correlted with ecdysteroids. Gl-Mstn mrna in thorcic muscle decresed 8% nd 8% in ESA nd MLA nimls, respectively, ut ws only wekly correlted with ecdysteroid. Clw nd thorcic muscles lso differed to vrying extents in the expression of ecdysteroid receptor (Gl-EcR nd Gl- RXR), elongtion fctor- (Gl-EF-), nd clpin T (Gl-ClpT) in response to molt induction, ut levels of the four trnscripts were not correlted with ecdysteroid. The downregultion of Gl-Mstn expression in premolt clw muscle coincided with - nd -fold increses in protein synthesis in the myofirillr nd solule protein frctions, respectively. Furthermore, the rte of the increse in the synthesis of solule proteins ws greter thn tht of myofirillr proteins during erly premolt (.:, solule:myofirillr), ut the two were equivlent during lte premolt. By contrst, Gl-Mstn mrna incresed -fold nd Gl-ClpT mrna decresed % in unweighted thorcic muscle; there ws little or no effect on Gl-EF-, Gl-EcR, nd Gl-RXR mrna levels. These dt indicte tht Gl-Mstn expression is negtively regulted y oth ecdysteroids nd lod-ering contrctile ctivity. The downregultion of Gl- Mstn in clw muscle my induce the elevted protein turnover ssocited with remodeling of the contrctile pprtus during molt-induced trophy. The upregultion of Gl-Mstn in unweighted thorcic muscle suggests tht this fctor is lso involved in disuse trophy when hemolymph ecdysteroid levels re low. Key words: myosttin, muscle trophy, protein synthesis, trnsltion, myofirillr protein, solule protein, clpin, ecdysone receptor, retinoid X receptor, elongtion fctor, molting, unweighting, utotomy, eyestlk ltion, lim regenertion. INTRODUCTION Skeletl muscle is highly dptle tissue. In mmmls, muscle mss is regulted y n rry of physiologicl nd pthologicl conditions, such s nervous ctivity, hormones, pssive stretching, disuse nd disese (reviewed y Fvier et l., 8; Tisdle, 9). In decpod crustcens, skeletl muscle retins gret del of plsticity in the dult (reviewed y Mykles, 997), which is exemplified in reversile trophy tht occurs under two disprte conditions. One of these is the trophy of clw muscle s prt of developmentl progrm leding up to ecdysis, or molting (reviewed y Mykles nd Skinner, 98). A reduction in mss fcilittes withdrwl of the lrge closer muscle in the propodus through the nrrow si-ischil segment t ecdysis (reviewed y Mykles, 999). Both C + -dependent nd uiquitin-dependent proteolytic systems re elevted in trophic clw muscle (reviewed y Mykles, 999). Clpins (Clp) degrde myofirillr proteins nd cdnas encoding three G. lterlis clpins (Gl-ClpB, -M nd -T) hve een chrcterized; eyestlk ltion cuses trnsient increse in Gl- ClpT nd ecdysone receptor (Gl-EcR) expression (Kim et l., ). An extensive remodeling of the contrctile pprtus is coincident with clw muscle trophy, ut no chnge in fier phenotype occurs (Ismil nd Mykles, 99; Mykles nd Skinner, 98; Mykles nd Skinner, 98). Protein synthesis is elevted during premolt (Skinner, 9). It is hypothesized tht the resulting increse in protein turnover fcilittes rerrngement of the myofilment lttice in trophic muscle (Mykles, 997). The fully reversile trophy of the clw muscle during premolt is induced y elevted ecdysteroid titers in the hemolymph nd does not occur in leg or thorcic muscles (reviewed y Mykles nd Skinner, 98; Mykles, 999). Clw nd thorcic muscles express different ssemlges of Gl-RXR (retinoid X receptor) isoforms, suggesting tht the two muscles differ in response to ecdysteroids (Kim et l., ). Reversile trophy of thorcic muscles occurs during intermolt in response to unweighting, which is cused y utotomy, or voluntry loss, of the ssocited lim in three decpod species (Crcinus mens Linneus 78, Uc pugiltor Bosc 8 nd Procmrus spp.) (Moffett, 987; Schmiege et l., 99). Continued trophy despite coordinted movement of the regenerting lim ud indictes tht mss of thorcic muscles is not strictly regulted y ctivity. Furthermore, trophy of thorcic muscles in response to lim utotomy does not pper to involve

2 Regultion of crustcen muscle trophy 7 ecdysteroid-dependent signling, s precocious molting is not induced if less thn five lims re utotomized in G. lterlis (Skinner nd Grhm, 97). Ultrstructurl exmintion of trophic thorcic muscle demonstrtes tht the reduction in fier re is ssocited with disruption of srcomere orgniztion, loss of orgnelles, nd incresed erosion of myofilments (Schmiege et l., 99). This stnds in contrst to the highly ordered restructuring of muscle fiers in the premolt clw (Ismil nd Mykles, 99; Mykles nd Skinner, 98), nd suggests tht proteolytic regultion in trophic thorcic muscle differs from tht of the clw muscle. A myosttin-like protein is expressed in G. lterlis (Gl-Mstn), nd my e involved in regulting muscle mss in crustcens. Myosttin, memer of the trnsforming growth fctor- (TGF- ) superfmily of cytokines, is negtive regultor of muscle mss in mmmls (reviewed y Lee, ; Rodgers nd Grikipti, 8), the function of which is conserved in t lest one species of lower verterte (Medeiros et l., 9). Expression in non-muscle tissues does, however, suggest roder regultory role for myosttin, especilly in lower vertertes (reviewed y Rodgers nd Grikipti, 8). Like other TGF- fmily memers, myosttin is secreted s ltent utocrine/prcrine fctor, nd is ctivted y proteolytic clevge of the propeptide; the mture peptide inds to ctivin receptors in the plsm memrne s homodimer (reviewed y Herpin et l., ; Lee, ). Activtion of these serine/threonine kinse receptors initites signling through Smd trnscription fctors (reviewed y Liu, ; Xu, ) tht initite shift in the lnce etween nolic nd ctolic pthwys in skeletl muscle, therey cusing net loss of muscle protein. In mmmls, myosttin stimultes uiquitin-dependent protein degrdtion nd inhiits trget of rpmycin (TOR)-dependent protein synthesis (reviewed y Mtsks nd Ptel, 9; Tisdle, 9). Fier trophy is induced y elevted expression of myosttin (Amirouche et l., 9; Durieux et l., 7; Reisz-Porszsz et l., ; Zimmers et l., ). Conversely, fier hypertrophy is induced y reduced myosttin signling (Mgee et l., ; Medeiros et l., 9; Nktni et l., 8; Qio et l., 8). The Gl-Mstn cdna encodes 97-minocid () polypeptide tht includes - mture peptide domin which hs % identity nd 9% similrity to humn myosttin (Covi et l., 8). The function of this puttive crustcen myosttin is unknown, ut its predominnt expression in skeletl muscle of the lnd cr suggests role in regulting muscle mss. The regultory systems responsile for inducing nd reversing the trophy of skeletl muscle in crustcens re lrgely unknown. Atrophy of clw muscle during premolt is triggered y ecdysteroids, wheres trophy of thorcic muscle during intermolt is ecdysteroidindependent nd my e triggered y reduced lod. The purpose of this study ws to exmine the role of Gl-Mstn in the trophy of clw nd thorcic muscle. We hypothesized tht Gl-Mstn expression would increse in trophic skeletl muscle, nd tht n Mstn/Smd signling pthwy would upregulte the expression of ecdysteroid receptor nd Gl-ClpT. Expression of elongtion fctor (Gl-EF- ), housekeeping gene, would not e ffected. Molting ws induced y eyestlk ltion (ESA) or multiple lim utotomy (MLA) (reviewed y Mykles, ; Skinner, 98). Gene expression ws ssessed in clw closer, weighted thorcic nd unweighted thorcic muscles over the molting cycle. Trnscripts for Gl-Mstn (Covi et l., 8), ecdysteroid receptor (Gl-EcR nd Gl-RXR) (Kim et l., ; Kim et l., ), Gl-EF- (Kim et l., ) nd Gl-ClpT (Kim et l., ) were quntified y reltime reverse trnscription-polymerse chin rection (qrt-pcr). Protein synthesis ws mesured y incorportion of [ S]methionine in clw closer muscle cultured in vitro. Protein synthesis nd Gl- Mstn mrna were inversely correlted with hemolymph ecdysteroid levels. By contrst, unweighting incresed Gl-Mstn expression in thorcic muscle from intermolt nimls. Regultion of muscle trophy clerly differs etween the two muscles. However, nlysis of the dt suggests tht Gl-Mstn regultes protein turnover in moltinduced nd disuse muscle trophies. MATERIALS AND METHODS Animls nd molt induction Adult mle Gecrcinus lterlis (Fréminville 8) used in [ S]methionine incorportion experiments were otined from Bermud nd mintined s descried previously (Skinner, 9); molting ws induced y MLA. Adult nimls used in qrt-pcr experiments were otined from the Dominicn Repulic nd mintined t C in 7 9% reltive humidity. It is relevnt to note tht G. lterlis from Bermud, Puerto Rico nd the Dominicn Repulic ll experience similr trophy of clw musculture during premolt. Thus, differences etween Bermud nd Dominicn Repulic popultions re ssumed to e insignificnt. Intermolt nimls were kept in communl (7 nimls per cge) plstic cges lined with spen edding wetted with p.p.t. Instnt Ocen (Aqurium Systems, Mentor, OH, USA). Lim-utotomized nimls were kept in individul qurt size (~ l) plstic cges lined with snd wetted with p.p.t. Instnt Ocen. Cges for lim-utotomized nimls were shielded from room lighting with cloth, s constnt drkness shortens the premolt period (Bliss nd Boyer, 9). Animls were mintined under h: h light:drk cycle nd fed iceerg lettuce, crrots nd risins twice weekly. Animls used in qrt-pcr experiments were kept in cptivity for less thn months prior to hrvesting of tissues. Precocious molting is induced y ESA nd utotomy of t lest five wlking legs (reviewed y Mykles, ; Skinner, 98). Regenertion of utotomized lims occurs in two phses: sl growth, which occurs during intermolt, nd proecdysil growth (Hopkins, 99). Progression through premolt in eyestlk-lted nd multiple lim-utotomized crs ws monitored y mesuring the length of the lim regenerte t the position of the third wlking leg nd clculting the regenertion index [R-index; length of regenerte /crpce width (Bliss, 9)]. In G. lterlis, the R-index rnges etween nd (Hollnd nd Skinner, 97; Yu et l., ). Eyestlk-lted nimls missing more thn three lims y the time of dissection were not used. All eight wlking lims were utotomized for the MLA experiments, nd the weighted thorcic muscle hrvested in this experiment ws ssocited with the two intct chelipeds. ESA ws done s descried previously (Covi et l., 8). Orgn culture Penicillin nd streptomycin were otined from Sigm (St Louis, MO, USA). All other regents used in [ S]methionine incorportion experiments were the sme s descried previously (Mykles, 99). [ S]methionine (specific ctivity ~.7 Bq mmol ) ws purchsed from ICN Biochemicls (Cost Mes, CA, USA). Autotomized clws were pcked in crushed ice for h to induce polysis (O Brien et l., 98). Clw closer muscle ws cultured s descried previously (Mykles, 99). Briefly, tissue ws equilirted for min in filter-sterilized supplemented culture medium contining mmol l Hepes NOH (ph 7.), mmol l NCl,. mmol l KCl, 8.8 mmol l CCl,.8 mmol l MgSO,. mmol l sodium phosphte,. mmol l D-glucose,. mmol l sodium citrte,. mmol l pyruvic cid, mmol l glutmine,. mmol l L-lnine,. mmol l L-rginine,. mmol l L- sprgine,. mmol l sprtic cid,. mmol l L-cystine,

3 7 J. A. Covi nd others. mmol l L-glutmic cid,. mmol l glycine,. mmol l L- histidine,. mmol l L-isoleucine,. mmol l L-leucine,. mmol l L-lysine,. mmol l L-phenyllnine,. mmol l L- proline,. mmol l L-serine,. mmol l L-threonine,. mmol l L-tryptophn,. mmol l L-vline, penicillin ( i.u. ml ), nd streptomycin ( g ml ). After equilirtion, the medium ws replced with fresh medium contining [ S]methionine (.7 Bq ml ) nd the culture ws incuted on n oritl shker for h. Equilirtion of rdioctively-leled mino cid occurs within h (Skinner, 9) (M. Hire nd D.L.M., dt not shown). After incution, tissues were frozen in liquid N nd stored t 8 C. Muscles were homogenized in volumes of uffer A ( mmol l Tris HCl, ph 7.; mmol l KCl; mmol l EDTA; mmol l dithiothreitol) with Polytron homogenizer for s t high speed. The homogentes were centrifuged t, g for min t C. The solule protein in the superntnt frction ws dilyzed ginst two -l chnges of mmol l mmonium cette, frozen, lyophilized, nd dissolved in ml SDS smple uffer (Medler et l., 7) t C for min. The myofirillr protein in the pellet otined from centrifugtion ws wshed three times with ml uffer A nd extrcted with five volumes. mol l NCl nd mmol l sodium phosphte (ph 7.) t C for min. The solule nd myofirillr protein solutions were clrified y centrifugtion t, g for min t C. The filter pper disk ssy (Bollum, 98) ws used to quntify the incorportion of rdioctivity into protein. Protein concentrtion ws determined y fluorescence emission spectroscopy (Avruch nd Wllch, 97). Smples ( g) of solule nd myofirillr proteins were pplied to. cm squres of MM pper nd ir dried. Filters were wshed riefly in % trichlorocetic cid (TCA) t room temperture, wshed in % TCA t 9 C for min, rinsed in 9% ethnol t room temperture, nd ir dried. Filters were plced in vils contining Ecoscint O (Ntionl Dignostics, Atlnt, GA, USA) nd the rdioctivity ws mesured with Beckmn LS 7 liquid scintilltion counter. Incorportion ws expressed s nmol [ S]Met mg protein h. For utordiogrphy, proteins were seprted using % SDS-PAGE (Medler et l., 7) nd exposed to X-ry film t 8 C. RNA isoltion nd quntittive rel-time RT-PCR All regents were of moleculr iology grde. Animls were nesthetized y immersion in crushed ice for min prior to dissection. Clw closer nd thorcic muscles were hrvested within min, frozen in liquid N nd stored t 8 C. Weighted nd unweighted thorcic muscle ws determined y the presence or sence, respectively, of the ssocited pereiopod. Sufficient time hd elpsed fter utotomy for sl lim regenerte to hve formed. Totl RNA ws isolted using TRIzol regent (Invitrogen, Crlsd, CA, USA) ccording to the mnufcturer s protocol. Briefly, totl RNA ws treted with DNse I (Invitrogen) to remove genomic DNA. After DNse tretment, totl RNA ws sujected to phenol:chloroform:isomyl lcohol extrction (::), followed y precipittion of RNA to remove DNse, slts nd digested DNA. Totl RNA ws precipitted with volume of isopropnol nd resuspended in l of RNA Storge Solution (Amion, Austin, TX, USA). Nucleotide concentrtion ws determined y sornce t nm fter : dilution with Tris-EDTA (TE) uffer (Integrted DNA Technology), nd.7 g ws used for l cdna synthesis rection using SuperScript III reverse trnscriptse (Invitrogen) nd oligo-dt()vn primer ( mol l ). Resulting cdna ws treted with RNse H (New Englnd Biols, Ipswich, MA, USA) to remove complementry RNA. Quntittive nlysis of Gl-Mstn [GenBnk EU8 (Covi et l., 8)], Gl-EF- [GenBnk AY (Kim et l., )], Gl- EcR [GenBnk AY97 (Kim et l., )], Gl-RXR [GenBnk DQ78 (Kim et l., )] nd Gl-ClpT [GenBnk AY9 (Kim et l., )] ws conducted using LightCycler FstStrt DNA Mster Plus SYBR Green I rection mix nd LightCycler 8 therml cycler (Roche Applied Science, Indinpolis, IN, USA). Rections consisted of l first strnd cdna, l SYBR Green Mster Mix,. l ech of mmol l forwrd primer nd mmol l reverse primer, nd l PCR-grde wter. PCR conditions included n initil denturtion t 9 C for min, followed y cycles of denturtion t 9 C for s, nneling t gene-specific temperture (Tle ) for s, nd extension t 7 C for s. Gl-Mstn primers were designed using the end of the mture peptide domin (Covi et l., 8), nd, thus, mplified ll splice vrints tht encoded full-length mture peptide. Primers for RXR were designed using sequence common to ll known splice vrints (Kim et l., ). At the end of ech run, n nlysis of the PCR product melting temperture ws conducted. Trnscript concentrtions were clculted with LightCycler 8 softwre (Roche; version.) using stndrds curves produced y seril dilution of purified PCR product ( g l to ng l ). The clirtor templte used to verify consistency etween runs contined cdna pooled from clw muscle, thorcic muscle, hert, heptopncres, eyestlk gngli, thorcic gngli, testis nd ovry. Ecdysteroid levels in the hemolymph were quntified y rdioimmunossy s descried previously (Medler et l., ). Sttisticl nlyses Sttisticl nlysis ws performed using JMP.. softwre (SAS Institute, Inc., Cry, NC, USA). qrt-pcr dt were log trnsformed prior to nlysis to reduce differences in vrince from the men. T m, melting temperture. Tle. Primers used for qrt-pcr Nme Sequence T m ( C) Product size cef F -TTCTATGCCTTTGGCCGTGTCTTCTC- 9 p cef R -TGATGGTGCCCGTCTTAACCAGATAC- RcEcR F -CACGAAGAATGCCGTGTACCAGTGTA- 7 p RcEcR R -CATCTGCTTCAGTTGGCTGCTCAAAC- MstnIMP F -GCTGTCGCCGATGAAGATGT- 8 p MstnIMP R -GTGTGGCTAGAATGAGAGATGTG- qgl-rxr F -CTCAGGCAAGCACTATGGCGT- p qgl-rxr R -TCAAGCACTTCTGGTAGCGGCAG- RcClpT F -TCTCTGATGTGCTGTGCCATAACTCC- 7 p RcClpT R -TGATGCAGAGACCTGTGACCATTCTG-

4 Regultion of crustcen muscle trophy 7 Vrinces mong log-trnsformed dt were determined to e equivlent using Brown Forsythe tests (P<.). Mens for trnscript undnce were compred mong developmentl stges for individul muscle types using nlysis of vrince (ANOVA). Post-hoc multiple comprisons were mde using Tukey Krmer HSD tests. A pired t-test ws used to compre mens for trnscript undnce etween clw nd thorcic muscles, or weighted nd unweighted thorcic muscles, t ech developmentl stge. All dt not plotted s individul points re presented s men ± s.e.m. nd the level of significnce ws set t. for ll sttisticl nlyses. RESULTS Scles for presenttion of dt Dt re presented s function of three vriles: time, R-index nd molt stge. Time ws used to descrie events following ESA nd ecdysis, ut did not llow comprisons with premolt nimls tht were induced to molt y MLA. R-index ws used to compre the effects of molt induction y ESA nd MLA during premolt. The molt stge clssifictions developed y Drch (Drch, 99), nd lter modified y Skinner on G. lterlis (Skinner, 9), were used to integrte nlyses sed on time nd R-index. Effects of molt induction y MLA on clw muscle protein synthesis For necdysil, or intermolt (stge C ) nimls, [ S]methionine incorportion into solule protein ws.7-fold greter thn incorportion into myofirillr proteins (.7 vs. nmol [ S]Met mg protein h ; Fig. A; P<.). During erly premolt (R-index 8 to ), incorportion egn to increse, reching synthetic rtes for myofirillr nd solule proteins tht were nd times greter (P<.), respectively, y mid premolt thn those in clw muscles from intermolt nimls (Fig. A). The stoichiometry etween the synthesis of solule nd myofirillr proteins chnged during the lte D stge of premolt; the synthesis of solule protein incresed t greter rte thn myofirillr protein (. to ) during the first hlf of stge D, ut the reltive increse in synthetic rtes for the two protein pools were equivlent therefter (Fig. B). Slopes for liner regressions of myofirillr nd solule synthetic rtes t R< nd R> re significntly different (P<.), nd the synthesis of solule nd myofirillr proteins within the sme muscle ws highly correlted (Fig. B). Overll, the synthesis of solule protein during premolt ws out.-fold greter thn tht of myofirillr protein (Fig. A). SDS-polycrylmide gel electrophoresis showed no quntittive or qulittive chnges in the protein composition of solule nd myofirillr preprtions during premolt (Fig. A,B). Autordiogrphs showed incresed glol synthesis of solule nd myofirillr proteins (Fig. C,D). There were no qulittive differences in the proteins synthesized t ny molt stge. Furthermore, the reltive synthesis of ech of the proteins in the solule nd myofirillr frctions did not chnge during the premolt period (Fig. C,D). Effects of ESA nd MLA on hemolymph ecdysteroids levels Titers of hemolymph ecdysteroids incresed in response to oth ESA nd MLA, ut were higher in ESA nimls t ll R-index vlues smpled (compre Fig. A nd Fig. A). ESA induced five-fold increse in ecdysteroids over the first h tht ws followed y much slower nd prolonged increse; y weeks post-esa, the hemolymph ecdysteroid level ws -fold higher thn tht oserved in intct nimls (Fig. A; R.). During the third week following ESA, ecdysteroids incresed rpidly gin, nd were -fold greter thn levels in intct nimls y dy (Fig. A; R 9.9). The initil increse in hemolymph ecdysteroids oserved in response to MLA ws more grdul thn tht oserved for ESA. Men ecdysteroid levels were only 7-fold greter thn in intct nimls ± dys fter MLA (Fig. A; R.). Hemolymph ecdysteroids incresed rpidly in lte premolt (R.), reching concentrtion tht ws 8-fold greter on verge thn tht in intermolt nimls. Ecdysteroid levels decresed to <7 ng ml in postmolt nimls nd remined low for t lest dys following ecdysis (Fig. A). Effects of ESA on gene expression in clw nd weighted thorcic muscles ESA hd smll or only trnsient effects on Gl-EF- expression during the first two weeks post-esa. The copy numer of mrna trnscripts for Gl-EF- did not chnge significntly in clw muscle during the first dys following ESA (Fig. C; R 7). However, nmol [ S]Met mg protein h A Solule protein Myofirillr protein C D D D - Myofirillr (log nmol [ S]Met mg protein h )... B R > R > y=.9x. y=.79x. r =.7 r =.899 Intct Lim ud growth (R-index)... Solule (log nmol [ S]Met mg protein h ) Fig.. Effects of molting on synthesis of solule nd myofirillr proteins in clw muscles in vitro. Clw muscles from intermolt (R-index 7) nd premolt (Rindex 7 ) were incuted for h in medium contining [ S]methionine; incorportion of rdioctivity ws quntified y the filter pper disk ssy nd expressed s nmol [ S]Met mg protein h. (A) Protein synthesis s function of R-index in nimls stimulted to molt y multiple leg utotomy. Best fit lines using exponentil functions re shown (solule, r.; myofirillr, r.8). (B) Synthesis of myofirillr proteins s function of the synthesis of solule proteins. Corresponding molt stges sed on epiderml chnges (Drch, 99) re given ove the sciss; correltion etween Drch stges nd lim regenertion is sed on Hollnd nd Skinner (Hollnd nd Skinner, 97) nd Skinner (Skinner, 98).

5 7 J. A. Covi nd others Fig.. Autordiogrphs showing synthesis of solule nd myofirillr proteins in clw muscles from intermolt nd premolt lnd crs. Muscles were incuted nd processed s descried in the legend to Fig.. Smples of solule ( g; A,C) nd myofirillr proteins ( g; B,D) were seprted on % SDS-polycrylmide gels. One pir of gels (A,B) ws stined with Coomssie Blue (CB); second pir ws dried nd exposed to X-ry film (film) for 8 weeks (C) or weeks (D). Lne, smples from necdysil (An), or intermolt, muscle; lnes f, smples from proecdysil, or premolt, muscles (Rindex.,., 8. nd.). A, ctin; MHC, myosin hevy chin; P, prmyosin; Tm, tropomyosin; TnI, troponin-i; nd TnT, troponin-t. Positions of moleculr mss stndrds, in kd, re indicted on the left. y dys post-esa (R ), Gl-EF- levels hd incresed.-fold reltive to tht in intct nimls (Fig. C). In thorcic muscle, Gl- EF- copy numer decresed y % during the first h following ESA nd returned to intct levels within weeks (Fig. C). These levels re similr to previously pulished vlues (Kim et l., ). Trnscript copy numer for Gl-EF- ws consistently lower in thorcic muscle thn in clw muscle t ll molt stges (Fig. C). ESA cused lrge reduction in Gl-Mstn expression, ut the timing nd extent of this reduction differed etween clw nd thorcic muscles. No significnt chnge in the numer of copies of Gl-Mstn mrna occurred in clw muscle during the first dys following ESA (Fig. D; R 8). However, the eginning of downwrd trend in Gl-Mstn trnscript undnce in clw muscle ws pprent t dy 7 post-esa (R ). By dy (R 9.9), Gl- Mstn trnscript level hd decresed y 8% (Fig. D). No significnt chnge in Gl-Mstn copy numer ws pprent until dy post- ESA in thorcic muscle (R 9.9), nd the mgnitude of the decrese in copy numer (8%) ws smller thn tht oserved for clw muscle (Fig. D). This difference in the response to ESA is supported y sttisticl comprison of trnscript undnce in the two muscle tissues t ech molt stge; trnscript copy numer for Gl-Mstn ws significntly lower in thorcic muscle in intct nd erly D premolt nimls (R 7.), ut ws significntly higher in thorcic muscle t R (Fig. D). Effects of ESA on the expression of the suunits of the ecdysteroid receptor (Gl-EcR nd Gl-RXR) were dependent on tissue nd suunit type. ESA hd no significnt effect on trnscript undnce of Gl- EcR in clw muscle, ut induced stedy nd significnt increse in thorcic muscle (Fig. B). By dy (R 9.9), Gl-EcR trnscript levels hd incresed y.-fold reltive to the level in intct nimls (Fig. B). It is relevnt to note tht vrinces round the men for dys nd following ESA were higher reltive to those t lter time points nd for other gene trnscripts (Fig. ). Consequently, for Gl-EcR trends spnning these erlier time points could e oscured y the high degree of vrition mong individuls during the immedite response to ESA. Copy numer for Gl-EcR in thorcic muscle ws significntly lower thn tht of clw muscle t low nd high R-index vlues. Trnscript copy numer for Gl-RXR did not chnge significntly in clw muscle during the first dys following ESA (Fig. E; R 8). Gl-RXR trnscript levels dropped % etween dy nd 7 post-esa (R 7. nd, respectively), nd slowly returned to norml levels therefter (Fig. E). A similr trend ws pprent in thorcic muscle, lthough the drop etween dy nd 7 post-esa ws slightly lrger (%) nd the susequent return to norml levels ws not oserved (Fig. E). It is importnt to note tht the qrt-pcr primers mplified region conserved mong ll known isoforms. Therefore, the dt re the sum of ll Gl-RXR vrints, which differ in expression etween clw nd thorcic muscles (Kim et l., ). After trnsient decrese, ESA cused modest increse in Gl- ClpT expression in clw nd thorcic muscles; this ws followed y decrese t lter premolt stges. Trnscript undnce for Gl-

6 Regultion of crustcen muscle trophy 77 Ecdysteroids (ng ml ) EF- (log copies µg totl RNA ) RXR (log copies µg totl RNA ) A ESA C, E,,,,,, Intct Men Post- R-vlue ESA (d) ,,, C D D D Lim ud growth (R-index) EcR (log copies µg totl RNA ) Mstn (log copies µg totl RNA ) Clp T (log copies µg totl RNA ) B ESA, D F,, Intct,,,,,,,,,,,,, c C D D D Lim ud growth (R-index) Fig.. Effects of eyestlk ltion on hemolymph ecdysteroids (A) nd gene expression (B F) in clw nd weighted thorcic muscles. (A) Hemolymph ecdysteroid titers were quntified y rdioimmunossy. Dt re expressed s function of R-index; inset delinetes the reltionship etween the R-index nd time following ESA. Aundnce of mrna trnscripts, quntified y qrt- PCR, re plotted s function of R-index for ecdysone receptor (Gl-EcR; B), elongtion fctor (Gl-EF-; C), myosttin (Gl-Mstn; D), retinoid X receptor (Gl-RXR; E), nd clpin T (Gl-ClpT; F). Intermolt nimls re plotted s seprte dt points to the left of the R-index scle (Intct). Open circles nd dshed line, thorcic muscle; closed dimonds with solid line, clw muscle. Stndrd error mesurements for R-index mens re not displyed, ut re less thn or equl to.. Shred letters indicte no significnt difference for multiple comprisons mong clw muscle dt. Shred numers indicte no significnt difference for multiple comprisons mong thorcic muscle dt. Asterisks indicte significnt difference etween clw nd thorcic muscle mens t the sme molt stge. Scles for ordintes re identicl, ut the rnges re shifted to ccommodte vried levels of expression of different genes. Corresponding molt stges re given ove the sciss s in Fig.. Dt re presented s men ± s.e.m. (N ). ClpT decresed y % during the first h following ESA in oth muscles, ut this decrese ws only sttisticlly significnt in thorcic muscle (Fig. F). Copy numer for Gl-ClpT returned to the levels in intct nimls in oth tissues y dy post-esa (R 7.). Trnscript levels in clw muscle incresed y.7-fold etween dys nd post-esa (R. nd., respectively) wheres levels in thorcic muscle incresed y.7-fold (Fig. F). Trnscript undnce decresed etween dy nd dy (R. nd 9.9, respectively) post-esa in oth muscles, ut this drop ws only sttisticlly significnt in thorcic muscle (Fig. F). Effects of MLA on gene expression in clw nd thorcic muscles The expression of Gl-EcR nd Gl-RXR ws exmined following MLA. Levels of mrna were similr etween clw nd thorcic muscles t ll molt stges except dys postmolt for oth Gl-EcR nd Gl-RXR (Fig. B,E). Gl-EcR trnscript levels in oth muscles incresed y two- to threefold during premolt, decresed y 9% etween lte premolt nd dys postmolt, nd remined low for t lest dys fter ecdysis (Fig. B). No significnt chnge in Gl- RXR mrna ws oserved in either muscle over molting cycle (Fig. E). However, levels of oth Gl-EcR nd Gl-RXR trnscripts were significntly higher in thorcic muscle from -dy postmolt nimls (Fig. B,E). In response to MLA, the undnce of Gl-EF- trnscripts decresed y % in clw muscle nd % in thorcic muscle during the erly D stge of premolt, ut this chnge ws only sttisticlly significnt for the thorcic muscle (Fig. C; R ). Gl-EF- trnscript undnce in oth muscles returned to intct levels y the lte D stge of premolt (Fig. C; R ). Gl-EF- levels were generlly lower in clw muscle thn thorcic muscle (Fig. C). Trnscript undnce for Gl-Mstn decresed in oth clw nd thorcic muscle following MLA, ut this decrese ws more pronounced in clw muscle. This is illustrted y direct comprison etween clw nd thorcic muscles t specific molt stges; Gl-Mstn mrna levels for clw muscle were significntly greter thn tht of thorcic muscle during the erly D stge of premolt (R ), ut significntly less thn thorcic muscle during the lte D stge of premolt (R.) nd dys postmolt (Fig. D). Gl-Mstn copy numer in the high R-index group ws 9% lower thn tht of intct nimls for clw muscle nd 8% lower for thorcic muscle (Fig. D; intct vs R.). Gl-Mstn trnscript levels incresed over tenfold in clw nd fivefold in thorcic muscle etween lte premolt (R.) nd dys postmolt groups (Fig. D). A lrge decrese in

7 78 J. A. Covi nd others Ecdysteroids (ng ml ) EF- (log copies µg totl RNA ) RXR (log copies µg totl RNA ) A MLA Ecdysis MLA Ecdysis B C,,, E A I C D D D - B C Intct, Lim ud growth (R-index) Postmolt (dys) D, Clp T (log copies µg totl RNA ) F,,,,,,, c c,,, A I C D D B C Intct D - c c,,, Lim ud growth (R-index) Postmolt (dys) Fig.. Effects of multiple lim utotomy on hemolymph ecdysteroids (A) nd gene expression (B F) in clw nd weighted thorcic muscles. (A) Hemolymph ecdysteroid titers were quntified y rdioimmunossy. Dt re expressed s function of R-index. Postmolt mesurements of hemolymph ecdysteroids were < ng ml ; error rs for postmolt re excluded, ecuse mesurements were often elow the detection limit of ng ml. Aundnce of mrna trnscripts, quntified y qrt-pcr, for the Gl-EcR (B), Gl-EF- (C), Gl-Mstn (D), Gl-RXR (E) nd Gl-ClpT (F) re plotted s function of R-index for premolt nimls nd dys following ecdysis for postmolt nimls. Dt for intermolt nimls re plotted seprtely to the left of the R-index scle (Intct). Open circles nd dshed line, thorcic muscle; closed dimonds with solid line, clw muscle. Stndrd error mesurements for R-index mens re not displyed, ut re less thn or equl to.. Shred letters indicte no significnt difference for multiple comprisons mong clw muscle dt. Shred numers indicte no significnt difference for multiple comprisons mong thorcic muscle dt. Asterisks indicte significnt difference etween clw nd thorcic muscle mens t the sme molt stge. Scles for ordintes re identicl, ut the rnges were shifted to ccommodte vried levels of expression mong genes. Corresponding molt stges re given ove the sciss s in Fig.. Dt re presented s men ± s.e.m. Smple sizes vried with molt stge (intct, N 8; R. nd., N ; R., N 9; dys nd dys postmolt, N ). EcR (log copies µg totl RNA ) Mstn (log copies µg totl RNA ) Gl-Mstn trnscript undnce occurred etween nd dys postmolt for oth muscles; drop of 8% occurred in clw nd drop of 7% occurred in thorcic muscle (Fig. D). Chnges in the undnce of Gl-ClpT trnscripts differed etween clw nd thorcic muscles in response to MLA. The trnscript copy numer for Gl-ClpT trnscript did not chnge significntly in thorcic muscle during premolt (Fig. F). In clw muscle, Gl-ClpT mrna incresed significntly during premolt; copy numer t lte premolt (R.) ws.-fold greter thn tht t erly premolt (R ; Fig. F). This is supported y comprison of clw nd thorcic muscle t specific R-index vlues; trnscript levels for clw muscle were significntly less thn tht of thorcic muscle during erly premolt nd significntly greter during lte premolt (Fig. F; R vs R.). However, ecuse trnscript undnce decresed y % in clw muscle during erly premolt, the lte premolt vlue ws not significntly higher thn tht of the intct nimls. Reltionship etween Gl-Mstn mrna nd hemolymph ecdysteroid levels Aundnce of Gl-Mstn mrna ws generlly correlted with circulting ecdysteroids, ut this reltionship ws much stronger in MLA nimls thn in ESA nimls. Gl-Mstn trnscript undnce ws high t low ecdysteroid levels, nd decresed significntly s ecdysteroid titers incresed during premolt (Fig. ). Gl-Mstn mrna in clw muscle ws negtively correlted with

8 Regultion of crustcen muscle trophy 79 Mstn (copies µg totl RNA ).e+.e+.e+.e+.e+.e+.e+ A B Mstn (log copies µg totl RNA ) Mstn (log copies µg totl RNA ) Ecdysteroids (log pg µl ) Ecdysteroids (log pg µl ) log copies µg totl RNA EF. Mstn NSD EcR NSD RXR Unweighted Weighted <. ClpT Fig.. Effects of unweighting on gene expression in thorcic muscle of intermolt lnd crs. Comprison of Gl-EF-, Gl-Mstn, Gl-EcR, Gl-RXR nd Gl-ClpT trnscript undnce etween unweighted (lck) nd weighted (gry) thorcic muscles from the sme nimls. Dt presented s men ± s.e.m.; N. P-vlues for significnt differences re given ove ech pirwise comprison; NSD, no significnt difference t...e+ mrna undnce in weighted thorcic muscle during premolt nd postmolt (Fig. 7). 8 Ecdysteroids (pg µl ) Fig.. Reltionship etween Gl-Mstn mrna nd hemolymph ecdysteroid levels. Regressions of Gl-Mstn trnscript undnce vs ecdysteroid titers include ll intermolt nd premolt dt used in Fig. D nd Fig. D. Animls were stimulted to molt y MLA (A) or ESA (B). Log trnsformed dt re plotted in the insets. Open circles nd dshed line, thorcic muscle; solid circles nd solid line, clw muscle. Correltion coefficients for liner regressions in insets re s follows: (A) clw muscle, r.; thorcic muscle, r.7; (B) Clw muscle, r.9; thorcic muscle, r.8. hemolymph ecdysteroids in nimls induced to molt y MLA (Fig. A inset). A similr reltionship ws evident in ESA nimls, ut vriility ws much higher t ecdysteroid levels etween nd pg l (Fig. B) nd levels of Gl-Mstn mrna were wekly correlted with hemolymph ecdysteroids (Fig. B inset). If intct nimls were excluded from the nlysis, the correltion coefficient for liner fit to the log vs log dt for ESA clw muscle incresed to.7, wheres the fit for thorcic muscle did not chnge pprecily (dt not shown). The negtive correltion etween hemolymph ecdysteroids nd Gl-Mstn mrna ws weker for thorcic muscle thn clw muscle in oth MLA nd ESA experiments (Fig. A,B insets). Expression of Gl-EcR, Gl-RXR. Gl-ClpT, nd Gl-EF- ws not correlted with ecdysteroid concentrtion (dt not shown). Effects of unweighting on gene expression in thorcic muscle Lim utotomy induced specific chnges in gene expression in the corresponding thorcic muscle of intermolt nimls. Unweighting hd little or no effect on expression of Gl-EF-, Gl-EcR nd Gl- RXR in thorcic muscle (Fig. ). However, trnscript levels of oth Gl-Mstn nd Gl-ClpT were significntly ltered in response to unweighting; Gl-Mstn mrna incresed threefold, wheres Gl- ClpT mrna decresed y more thn % (Fig. ). Aundnce of Gl-Mstn mrna in unweighted thorcic muscle prlleled Gl-Mstn DISCUSSION The primry gol of the present study ws to ssess the role of myosttin-like protein, Gl-Mstn, in regulting the trophy of clw closer muscle during premolt nd thorcic muscle fter lim utotomy. The dt show tht () protein synthesis in clw muscle increses in two distinct stges during premolt; () trnscript level for Gl-Mstn decreses drmticlly in skeletl muscles during oth premolt nd postmolt; () trnscript level for Gl-Mstn in trophic clw muscle is negtively correlted with hemolymph ecdysteroids during premolt; () trnscript level for Gl-Mstn in intermolt nimls is significntly elevted in unweighted thorcic muscles; nd () trnscript levels for Gl-ClpT, Gl-EF-, Gl-EcR, nd Gl-RXR in skeletl muscle re not correlted with Gl-Mstn trnscript undnce or hemolymph ecdysteroids. Together these dt provide evidence supporting the hypothesis tht Gl-Mstn medites reversile trophy of skeletl muscle in response to elevted ecdysteroids nd unweighting, ut through two distinct mechnisms. Skeletl muscle mss is determined y the lnce etween protein synthetic nd degrdtion rtes. In generl, muscle trophy results from the net loss of protein cused y incresed protein degrdtion, decresed protein synthesis, or comintion of the two (reviewed y Sndri, 8; Tisdle, 9). The lrge increse in protein synthesis tht occurs in the clw during molt-induced trophy is n exception to this rule, s protein degrdtion must increse to n even greter extent to effect the reduction in mss tht occurs during the premolt period. This seems counterproductive, especilly in considertion of the incresed energetic costs ssocited with upregulted nolic processes. In mmmls, incresed protein turnover occurs during remodeling of skeletl muscle undergoing fier trnsformtion nd crdic muscle undergoing rosiglitzone-induced hypertrophy (Festucci et l., 9; Termin nd Pette, 99). We suggest tht incresed protein turnover is necessry for the extensive remodeling of the srcomere tht is oserved s myofirils re reduced in size (Mykles, 997). In the isomorphic clws of lnd cr, G. lterlis, nd the mjor clw of fiddler cr, Uc pugnx, there is preferentil loss of out thin myofilments for every thick myofilment, which results in

9 8 J. A. Covi nd others Mstn (log copies µg totl RNA )... MLA,, Ecdysis,, A I C D D D - B C Intermolt Lim ud growth (R-index) Postmolt (dys) Fig. 7. Effects of molting on Gl-Mstn expression in weighted nd unweighted thorcic muscles. Aundnce of Gl-Mstn mrna trnscripts is plotted s function of R-index for premolt nimls nd dys following ecdysis for postmolt nimls. Molting ws induced y MLA.Dt from intermolt nimls re plotted s seprte points to the left of the R-index scle (Intermolt). This is the sme intermolt dt s in Fig.. Open circles nd dshed line, weighted thorcic muscle; this is the sme dt s in Fig. D. Closed circles nd solid line, unweighted thorcic muscle. Stndrd error mesurements for R-index mens re not displyed, ut re less thn or equl to.. Shred numers indicte no significnt difference for multiple comprisons mong weighted muscle dt. For unweighted muscle dt different letters indicte significnt difference for multiple comprisons. Asterisks indicte significnt difference etween unweighted nd weighted thorcic muscle mens t the sme molt stge. Corresponding molt stges re given ove the sciss s in Fig.. Dt re presented s men ± s.e.m. Smple sizes vried with molt stge (intct, N 8; R. nd., N ; R., N 9; dys nd dys postmolt, N ) Only one mesurement ws mde for the unweighted group during lte premolt. decrese in the thin:thick myofilment rtio from ~9: to ~:; this corresponds to % to % decrese in the rtio of ctin to myosin hevy chin (Ismil nd Mykles, 99; Mykles nd Skinner, 98; Mykles nd Skinner, 98). The loss of thin myofilments increses the thick myofilment pcking density etween % nd 7% (Ismil nd Mykles, 99; Mykles nd Skinner, 98). It is generlly thought tht exchnge of myofirillr proteins etween srcomeric nd cytoplsmic pools occurs t the periphery of the srcomere (Belcstro et l., 99; Russell et l., 99) nd is the rte-limiting step in the incorportion of newly-synthesized protein into the contrctile pprtus (Feng et l., 9). Thus, high protein turnover rte would fcilitte rerrngement of the myofilments within the myofiril. Synthesis of solule nd myofirillr proteins incresed during proecdysis (premolt), confirming results from previous study on G. lterlis y Skinner (Skinner, 9). Skinner oserved.- fold increse in the incorportion of [- C]leucine into totl protein from skeletl muscle during erly to mid premolt (pooled smples from stges D to D ) tht ws reversed in lte premolt or erly postmolt. The present study expnds on this work y quntifying protein synthesis in the solule nd myofirillr protein frctions, nd provides more detiled exmintion of protein synthesis during stges D to D. The increse in protein synthesis tht occurs in trophic clw muscle peks during mid to lte premolt. During premolt, de novo synthesis of myofirillr nd solule protein in the clw increses pproximtely - nd -fold, respectively (Fig. A; rnging from R to ). Pek vlues indicte tht the rte of protein synthesis my increse s much s -fold in oth protein pools y lte D (Fig. A). Dt presented in Fig. nd y Skinner (Skinner, 9) indicte tht protein synthesis reches mximum in clw muscle etween the lte D nd D stges of premolt, nd egins to decrese prior to ecdysis. This coincides with pek in hemolymph ecdysteroids t stge D (Skinner, 98). The decrese in protein synthesis during lte premolt my e regulted y the lrge drop in circulting ecdysteroids t stge D (Skinner, 98). Muscle trophy, nd ssocited remodeling of the contrctile pprtus, is completed y this stge (Skinner, 98). The increse in protein synthesis tht occurs in trophic clw muscle ppers to involve two distinct phses of regultion. The reltionship etween protein synthesis in solule nd myofirillr protein pools chnges significntly in lte D of premolt (Fig. B). The reltive increse in protein synthetic rte for the two protein pools is nerly equivlent in nimls with n R-index of more thn, ut preferentil incorportion of [ S]methionine into solule protein occurs in nimls with n R-index less thn (.:, solule:myofirillr rtio; Fig. B). Although there is gret del of evidence indicting tht proteolytic systems re upregulted in trophic clw muscle (reviewed y Goll et l., ; Mykles, 998) (see lso Krmerov et l., ; Murphy et l., 7), it is s yet uncler wht proteins re preferentilly synthesized during erly premolt. Whtever the cse, it is cler tht the preferentil incorportion into solule protein erly in premolt must involve mny proteins, s there re no ovious chnges in the pool of rdioctively-leled proteins during premolt (Fig. C). Incresed cpcity for protein synthesis during premolt involves n increse in riosoml RNA content. Levels of rrna increse y % in epidermis, digestive glnd nd skeletl muscle in G. lterlis during premolt (Skinner, 98). Since copy numer ws expressed s function of totl RNA, seemingly stedy-stte levels of n mrna my ctully reflect nerly % increse in undnce during premolt. Given tht only one out of five of the trnscripts exmined decresed during the period when rrna content in muscle ws incresing, it is resonle to suggest tht either glol increse in trnscription or decrese in RNA degrdtion occurs in skeletl muscle during premolt. Decresed incorportion of [ H]uridine into rrna in epithelil tissue during premolt suggests tht stility of riosomes increses in this tissue during premolt (Skinner, 9). Ecdysteroids my stimulte protein degrdtion y ctivting clpins tht degrde myofirillr proteins (for reviews, see Mykles, 998; Mykles, 999), ut this ctivtion does not pper to involve trnscriptionl regultion. Levels of Gl-ClpM nd Gl-ClpB mrna do not chnge fter ESA (Kim et l., ), nd, lthough Gl-ClpT mrna levels increse threefold in trophic clw muscle during erly premolt, trnscript levels initilly decrese y % to % fter molt induction (Fig. F, Fig. F). Thus, there is no significnt difference etween intermolt nd premolt (lte D to D ) levels of Gl-ClpT mrna. These dt differ somewht from tht of Kim et l. (Kim et l., ), who reported trnsient increse in Gl-ClpT mrna in clw dy fter ESA. The reson for this discrepncy is not cler. However, given the modest chnges in Gl- ClpT mrna levels oserved during molt-induced trophy of the clw (Fig. F, Fig. F), nd the significnt decrese in Gl-ClpT mrna oserved during trophy of thorcic muscle induced y lim utotomy (Fig. ), we conclude tht Gl-ClpT ctivity is not regulted t the trnscriptionl level in trophic crustcen muscle. Ecdysteroids pper to control Gl-Mstn expression in skeletl muscle over much of the molting cycle. Gl-Mstn mrna levels decrese y t lest 8% in thorcic muscle nd 8% in clw muscle

10 Regultion of crustcen muscle trophy 8 during premolt (Fig. D, Fig. D). Gl-Mstn trnscript undnce in clw muscle is lso negtively correlted with ecdysteroid levels over the sme period (Fig. ). The reltively low correltion etween hemolymph ecdysteroids nd Gl-Mstn expression in thorcic muscle my result, in prt, from greter suppression t low ecdysteroid levels indictive of intermolt nimls; Gl-Mstn expression in intermolt nimls is % greter in clw muscle thn in thorcic muscle. At dys postmolt, when ecdysteroid titers re low, Gl-Mstn mrna levels increse in thorcic nd clw muscle y - nd - fold, respectively (Fig. D). In view of these dt, it seems resonle to suggest tht Gl-Mstn is negtively regulted y ecdysteroid during premolt nd erly postmolt. However, the regultion of Gl-Mstn expression lso ppers to involve ecdysteroid-independent signls. Despite the presence of unchnging nd extremely low ecdysteroid levels following ecdysis, Gl-Mstn mrna in thorcic nd clw muscles decreses y 7% nd 8%, respectively, etween dys nd dys postmolt (Fig. D). A direct link etween ecdysteroid signling nd expression of Gl-Mstn remins to e estlished. Autotomy-induced muscle trophy differs from molt-induced trophy oth in its regultory origin nd trnscriptionl profile. In response to the loss of lim, the thorcic muscle trophies rpidly nd more thn hlf of the mss is lost; it remins in n trophied stte until new lim is regenerted nd ecomes fully functionl fter molting (Moffett, 987). However, unlike trophy of the clw during premolt, trophy of thorcic muscle in response to lim utotomy does not pper to e regulted y ecdysteroids, s trophy occurs during intermolt when ecdysteroid levels re low (Fig. A, Fig. A). The intct innervtion nd muscle structure (Moffett, 987) suggests tht the mechnism my e response to physicl unweighting. Wht is cler is tht the regultion of Gl-Mstn expression differs etween molt-induced trophy nd utotomyinduced trophy of the thorcic muscle. Although Gl-Mstn mrna decreses s much s 8% in weighted thorcic muscle nd 9% in clw muscle during premolt, levels increse y % in unweighted thorcic muscles (Fig. ). Atrophy in unweighted thorcic muscle my e very similr to some forms of disuse trophy oserved in mmmls (reviewed y Fvier et l., 8). An interesting question is whether elevted levels of Gl-Mstn trnscript in unweighted thorcic muscle re correlted with decrese in glol protein synthesis. The regultion of Mstn expression in crustcen muscles induced y ESA nd MLA differs from tht in mmmlin muscles induced to trophy y vrious tretments or conditions. In mmmls, Mstn mrna levels re elevted in muscle trophies induced y diseses (Crneiro et l., 8; Costelli et l., 8), denervtion (Liu et l., 7; Sho et l., 7), unloding (Allen et l., 9; Rerdon et l., ; Wehling et l., ), glucocorticoid (M et l., ) nd ging (Leger et l., 8). Glucocorticoids stimulte Mstn trnscription vi response elements in the promoter region (M et l., ) (for reviews, see Schkmn et l., 8; Tisdle, 9). Glucocorticoids lso stimulte protein degrdtion nd inhiit protein synthesis in mmmlin skeletl muscles vi Mstn signling (for reviews, see Schkmn et l., 8; Tisdle, 9). Moreover, trophy induced y dexmethsone is prevented y deletion of the Mstn gene in knockout mice (Gilson et l., 7). By contrst, Mstn expression in crustcen muscles is inversely correlted with ecdysteroid levels (Fig. ), nd Gl-Mstn mrna is reduced during molt-induced trophy (Fig. D, Fig. D). Although Mstn expression in mmmlin nd crustcen muscles responds differently to steroid hormones, the dt presented here support the hypothesis tht Mstn inhiits protein synthesis in oth groups of orgnisms through the trget of rpmycin complex (TORC). TORC, component of the insulin/insulin-like growth fctor pthwy controlling growth in eukryotic cells nd is serine/threonine protein kinse; it regultes protein synthesis t the trnsltionl level through the phosphoryltion of S kinse nd EF-inding protein- (for reviews, see Proud, 7; Wullschleger et l., ). This sme pthwy medites nutrient-dependent growth in insects (for review, see Mirth nd Riddiford, 7). There is growing recognition tht TORC is centrl to the effects of growth fctors, nutrients, contrction nd ging on mmmlin muscle mss (reviewed y Drummond et l., 9; Miyzki nd Esser, 9; Sndri, 8). Recent studies indicte tht Mstn is negtive regultor of this pthwy in mmmlin muscle. Mstn inhiits signling vi TORC in rt skeletl muscle (Amirouche et l., 9) nd protein synthesis in C C muscle cells (Tylor et l., ). Furthermore, Mstn reduces phosphoryltion of Akt (McFrlne et l., ), which could led to inctivtion of TORC (Proud, 7). Conversely, Mstn inhiition stimultes protein synthesis vi the TORC pthwy in mouse skeletl muscle (Welle et l., 9). In decpod crustcens, elevted ecdysteroids hve only smll effect on gene expression of myofirillr protein, suggesting tht trnscriptionl regultion hs reltively minor contriution to chnges in protein levels occurring over the molting cycle (El Hj, 999; Medler et l., ; Whiteley nd El Hj, 997; Whiteley et l., 99). However, incresed protein synthesis is correlted with incresed riosoml RNA nd ctivity in muscles from premolt nimls (El Hj et l., 99; Skinner, 98). The downregultion of Gl-Mstn is ssocited with n increse in glol synthesis of oth solule nd myofirillr proteins (Figs, ). Tken together, these dt provide evidence to suggest tht Gl-Mstn, either directly or indirectly, suppresses protein synthesis. Further studies re needed to estlish whether Gl-Mstn inhiits trnsltion vi TORC. For future studies using molt induction, it is relevnt to note tht n exmintion of ecdysteroidogenesis nd gene expression in skeletl muscle demonstrtes tht ESA nd MLA stimulte similr, ut not identicl, responses. Both ESA nd MLA cuse precocious molting, ut the two differ in the timing of hormonl cues nd premolt processes. ESA triggers n cute response y the Y-orgn, resulting in elevted hemolymph ecdysteroid levels within dy of ESA nd shortened premolt period (Skinner nd Grhm, 97). By contrst, intermolt nimls induced y MLA enter premolt weeks fter the procedure (Hollnd nd Skinner, 97), nd ecdysteroids increse more grdully. Expression of Gl-EcR follows similr pttern to tht reported y Kim et l. (Kim et l., ) during the first dys fter ESA, wherein trnsient increse is oserved in clw muscle nd trnsient decrese is oserved in thorcic muscle. Expression of Gl-EcR converges somewht etween these tissues over period of weeks. By contrst, consistent increse in Gl-EcR mrna is oserved in oth clw nd thorcic muscle during premolt induced y MLA. In ddition, levels of Gl-RXR mrna remin unchnged throughout the molt cycle induced y MLA, ut decrese trnsiently during stge D in ESA nimls. These dt confirm tht skeletl muscle is responsive to chnges in ecdysteroids over molting cycle, nd tht the rte of chnge in ecdysteroid titers ffects the expression of responsive genes, including those for ecdysteroid receptor suunits. In summry, Gl-Mstn is differentilly regulted in two distinct trophic tissues: premolt clw muscle nd unweighted thorcic muscle. The reduction in Gl-Mstn trnscript in premolt clw nd thorcic muscle is correlted with incresing ecdysteroid levels. These dt provide support for the hypothesis tht ecdysteroids repress Gl-Mstn expression during premolt when ound to the