Norepinephrine induces transient modulation of the physiological responses of whiteleg shrimp, Litopenaeus vannamei

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1 Aquulture 254 (26) Norepinephrine indues trnsient modultion of the physiologil responses of whiteleg shrimp, Litopeneus vnnmei Shinn-Pyng Yeh, Hung-Tien Chiu, Winton Cheng Deprtment of Aquulture, Ntionl Pingtung University of Siene nd Tehnology, Pingtung 9121, Tiwn Reeived 1 August 25; reeived in revised form 2 Novemer 25; epted 2 Novemer 25 Astrt Levels of gluose, ltte, N +,K +,Cl, protein, nd oxyhemoynin in the hemolymph nd its osmollity were mesured when the whiteleg shrimp, Litopeneus vnnmei ( g), were individully injeted with sline or norepinephrine t 1 8,1 7, or 1 6 mol shrimp 1. Results showed tht elevtions of hemolymph gluose nd ltte ourred t etween 2 nd 4 h, inreses in hemolymph osmollity, N +, nd totl protein ourred t 2 h, nd redution in hemolymph Cl nd K + ourred t 2 h fter in shrimp whih hd reeived NE t N1 8, N1 8, nd 1 6 mol shrimp 1, respetively. All physiologil prmeters hd returned to the ontrol vlues 2 4 h fter reeiving the norepinephrine. The injetion of norepinephrine t N1 8 mol shrimp 1 lso signifintly deresed the oxyhemoynin/ protein rtio of L. vnnmei t 2 h s result of elevtion of hemolymph protein. These results suggest tht stress-induing norepinephrine uses trnsient period of modultion of energy metolism nd osmoregultion, nd respirtory response in L. vnnmei s it dpts to n environmentl stress. 25 Elsevier B.V. All rights reserved. Keywords: Litopeneus vnnmei; Norepinephrine; Gluose; Ltte; Osmollity; Ion; Protein; Oxyhemoynin 1. Introdution Whiteleg shrimp (Litopeneus vnnmei), whih is nturlly distriuted long Pifi osts of Centrl nd South Ameri, ws introdued to Asi in 1985, nd hs eome primry speie urrently eing ultured in Thilnd, Tiwn, nd Chin (Lin et l., 199). For more thn 1 yers, ommeril shrimp frming, minly sed on lol speies suh s the tiger shrimp, Peneus monodon nd kurum shrimp, Mrsupeneus jponius, hs deresed due to disese outreks ssoited with teri nd viruses. Therefore, L. vnnmei hs reeived muh ttention, nd y 23, out 3369 h of Corresponding uthor. Tel.: x6224; fx: E-mil ddress: winton@mil.npust.edu.tw (W. Cheng). ponds hd een onverted to the ulture of this speie in Tiwn. Severl iogeni mines whih funtion minly s neuroregultors (i.e., neurotrnsmitters nd neuromodultors), inluding serotonin, dopmine (DA), otopmine, histmine, nordrenline (norepinephrine, NE), drenline (epinephrine), tryptmine, nd tyrmine hve een identified nd quntittively mesured in the ryfish, Pifstus leniusulus, nd other depod rustens (Eloffson et l., 1982; Fingermn nd Nghushnm, 1992; Kuo et l., 1995). They re distriuted in the rusten nervous system nd hve diverse rry of physiologil effets (Kuo nd Yng, 1999; Tierney et l., 23; Chiu et l., in press). NE hs een reported to stimulte the hyperglyemi effets in M. rosenergii, nd to t on the trget tissue nd not through the rusten hyperglyemi hormone (CHH) /$ - see front mtter 25 Elsevier B.V. All rights reserved. doi:1.116/j.quulture

2 694 S.-P. Yeh et l. / Aquulture 254 (26) (Kuo nd Yng, 1999). Studies in mrine, freshwter, nd terrestril rustens hve indited tht iogeni mines re involved in ioni nd osmoti regultion, nd suh studies were reviewed y Morris (21). Using HPLC with n eletrohemil detetor, NE ws deteted in the eyestlk, thori gngli, nd rin of P. monodon t onentrtions of 26.62±4.93 pmol eyestlk 1, 1.54±.15 μmol (g protein) 1 nd 6.71±1.98 μmol (g protein) 1, respetively (Kuo et l., 1995). Hemoynin, respirtory pigment, represents 8 95% of the totl protein onentrtion in the hemolymph of depod rustens (Jeuniux, 1971). Hemoynin hs een reported to inrese under hypoxi in the lue r, Cllinetes spidus (DeFur et l., 199), nd in the shrimp, Crngon rngon (Hgermn, 1986), nd to inrese under hypo-osmoti stress in the shore r, Crinus mens (Boone nd Shoffeniels, 1979), nd in C. spidus (Mson et l., 1983). Hemoynin nd hemolymph protein re ffeted y slinity in the fleshy prwn, Peneus hinensis (Chen et l., 1993). Environmentl ftors suh s temperture, ph, oxygen, nd ron dioxide re known to influene the oxygen ffinity of hemoynin in the lue r, C. spidus, whih ffets oxygen trnsport (Burnett, 1992). Severl physiologil nd iohemil responses in rustens hve een reported to our under stressful onditions. The relese of iogeni mines is elieved to e proto-stress response. In the present pper, we ttempted to exmine severl physiologil prmeters inluding oxyhemoynin, protein, gluose, ltte, Cl, N +, nd K + onentrtions in the hemolymph nd its osmollity of L. vnnmei following n injetion of NE. 2. Mterils nd methods 2.1. Litopeneus vnnmei L. vnnmei juveniles ( g) were otined from ommeril frm in Pingtung, southern Tiwn, nd limted in the lortory for 2 weeks efore experimenttion. During the limtion period, shrimp were fed twie dily with formulted shrimp diet (Shint Feed Compny, Pingtung, Tiwn). For 1 dy prior to the experiment, the shrimp were not fed. For the experiment of physiologil prmeter ssys, tests were rried out in 1 replite test groups onsisting of 1 shrimp eh in 2-l PVC tnks ontining 1 l of n erted test solution. No signifint differenes in weight were oserved mong the tretment groups. During the experiments, the wter temperture ws mintined t 27±1 C nd ph , while slinity ws mintined t 2. Among the A, B, C, D /D 1 nd D 2 /D 3 stges of L. vnnmei, hemolymph osmollity, nd Cl,N +,K +, protein, nd oxyhemoynin levels were the highest t stge D /D 1, nd lowest t stge A, respetively. The ft tht the hemolymph osmollity, nd protein, Cl, N +, nd K + levels were lower during the postmolt, nd were higher during intermolt nd erly premolt stges ws onsidered to e ssoited with wter uptke t the time of molting (Cheng et l., 22). All shrimp used in this study were individully exmined to determine molt stges (Roertson et l., 1987), nd only those t stge C (intermolt) were used in order to minimize intrinsi vritions Test solutions Norepinephrine hydrohloride (A7256, Sigm Chemil, St. Louis, MO, USA), neurotrnsmitter, ws used in the study. Norepinephrine hydrohloride ws dissolved in sterile sline (.85% NCl) to onentrtions of 5 1 4,5 1 3,or5 1 2 mol l 1, respetively, efore injetion (Kuo et l., 1995) Effet of norepinephrine on the physiologil prmeters of L. vnnmei L. vnnmei ws individully injeted in the ventrl sinus of the ephlothorx with either 5 1 4, 5 1 3,or5 1 2 mol l 1 of n NE solution (2 μl) to hieve doses of 1 8, 1 7, nd 1 6 mol shrimp 1, respetively. The shrimp tht reeived sline (2 μl) served s the ontrol group. There were 4 tretments (sline, 1 8,1 7, nd 1 6 mol NE shrimp 1 ) with 5 smpling times (2, 4, 8, 16, nd 24 h). Ten shrimp for eh tretment nd time were used for the studies. In ddition, 1 shrimp with no tretment were used s the ontrol group. At the eginning nd 2, 4, 8, 16, nd 24 h fter the injetion, hemolymph (1 μl) ws withdrwn from the ventrl sinus of eh shrimp into 1-ml sterile syringe (25 guge) ontining.9 ml of n ntiogulnt solution (3 mm trisodium itrte,.34 M sodium hloride, 1 mm EDTA; ph 7.55, with the osmollity djusted with NCl to 78 mosm kg 1 ). The diluted hemolymph ws entrifuged t 664 g t 4 C for 15 min, the superntnt fluid ws olleted s the stok solution, nd gluose nd ltte were immeditely nlyzed. Gluose onentrtions were mesured with oupled gluose oxidse nd peroxidse retion using Sigm dignostis gluose kit (t. no ). The optil density t 55 nm ws mesured using n ELISA plte reder, nd gluose onentrtions were

3 S.-P. Yeh et l. / Aquulture 254 (26) lulted from stndrd urve of known gluose onentrtions. Ltte onentrtions were mesured using the olorimetri ltte oxidse nd peroxidse method with Sigm dignostis ltte kit (t. no ). The optil density t 54 nm ws mesured using n ELISA plte reder, nd ltte onentrtions were lulted from stndrd urve of known ltte onentrtions. Hemolymph osmollity nd medium osmollity were mesured y injeting 2-μl smple into miro-osmometer (Model 33; Advne Instruments, Norwood, MA, USA). To determine N +,K +, nd Cl levels, 1 μl of hemolymph smple ws immeditely injeted into n Ion-Seletive Eletrode Anlyzer (Medi EsyLyte PLUS, Bedford, MA, USA). Hemolymph protein ws determined with the Bio- Rd Protein Assy Kit no. 5-6 (Bio-Rd Lortories, Rihmond, CA, USA) using ovine lumin (with moleulr weight of 66,) s the stndrd (Brdford, 1976). To mesure oxyhemoynin, 1 μl of hemolymph ws immeditely diluted with 9 μl of distilled wter, nd the sorne ws mesured t 335 nm (hrteristi of oxyhemoynin) using Hithi (Tokyo, Jpn) U-21 spetrophotometer. The onentrtion of oxyhemoynin ws lulted sed on the methods of Nikerson nd Vn Holde (1971) nd Hgermn (1983). The rtio of oxyhemoynin to protein ws lulted y dividing the onentrtion of oxyhemoynin (mmol l 1 ) y tht of protein (mmol l 1 ), whih ws onverted from mg ml 1 to mmol l 1 y dividing y 66 (Chen nd Cheng, 1993) Sttistil nlysis A multiple-omprison (Tukey's) test ws onduted to ompre the signifint differenes mong tretments using the SAS omputer softwre (SAS, 21). The perentge dt of oxyhemoynin to protein were normlized using rsine trnsformtion efore nlysis. For sttistilly signifint differenes, it ws required tht p Results At time h, the hemolymph gluose level ws.85 ±.4 mmol l 1. After 2 h, gluose levels hd signifintly inresed to 155.4%, 194.2%, nd 238.8% for the shrimp tht hd reeived NE t 1 8,1 7, nd 1 6 mol shrimp 1, respetively, s ompred to the level of shrimp tht hd reeived sline. The inrese in the gluose level of shrimp following NE tretment ppered to hve een dose dependent. After 4 h, the gluose level hd further signifintly inresed to 128.5%, 133.9%, nd 198.8% for the shrimp tht hd reeived NE t 1 8,1 7, nd 1 6 mol shrimp 1, respetively, s ompred to the level in shrimp tht hd reeived sline. However, no signifint differene in 3. A Sline 1-8 mole shrimp mole shrimp mole shrimp B Sline 1-8 mole shrimp mole shrimp mole shrimp -1 Hemolymph gluose (µmole ml -1 ) d Hemolymph ltte (µmole mi -1 ) d d Fig. 1. Hemolymph gluose (μmol ml 1 ) (A) nd ltte (μmol ml 1 ) (B) of Litopeneus vnnmei whih reeived sline or norepinephrine t 1 8, 1 7,or1 6 mol shrimp 1. Eh r represents the men vlue from 1 smples with the stndrd error. Brs with different letters signifintly differ (p.5).

4 696 S.-P. Yeh et l. / Aquulture 254 (26) hemolymph gluose ws oserved mong the 4 tretments from 8 to 24 h (Fig. 1A). Hemolymph ltte ontents of shrimp whih hd reeived NE t 1 8,1 7, nd 1 6 mol shrimp 1 were signifintly higher thn those of prwns whih hd reeived the sline ontrol from 2 to 4 h. The inrese in ltte ontents of shrimp following NE tretment t 2 nd 4 h ppered to hve een dose dependent. Hemolymph ltte ontents fter 2 h inresed y 16.3%, 217.6% nd 269.%, nd fter 4 h inresed y 178.5%, 231.6% nd 265.1% in shrimp tht hd reeived NE t 1 8, 1 7, nd 1 6 mol shrimp 1, respetively, s ompred to sline ontrol shrimp. However, no signifint differene in ltte ontents ws oserved mong the 4 tretments from 8to24h(Fig. 1B). Hemolymph osmollity nd sodium levels of shrimp whih hd reeived NE t 1 7, nd 1 6 mol shrimp 1 were signifintly higher thn those of the sline ontrol shrimp fter 2 h. Hemolymph osmollity hd inresed y 13.5%, 14.8%, nd 15.2%, nd hemolymph sodium hd inresed y 12.2%, 13.9%, nd 14.1% in Fig. 2. Hemolymph osmollity (mosm kg 1 ) (A), Cl (μmol ml 1 ) (B), N + (μmol ml 1 ) (C), nd K + (μmol ml 1 ) (D) of Litopeneus vnnmei whih reeived sline or norepinephrine t 1 8, 1 7, or 1 6 mol shrimp 1. Sttistil desriptions re the sme s those in Fig. 1.

5 S.-P. Yeh et l. / Aquulture 254 (26) Oxyhemoynin (µmole ml -1 ) A Sline 1-8 mole shrimp mole shrimp mole shrimp B Sline 1-7 mole shrimp mole shrimp mole shrimp -1 Totl protein (mg ml -1 ) Rtes of oxyhemoynin to protein (%) C Sline 1-8 mole shrimp mole shrimp mole shrimp Fig. 3. Hemolymph protein levels (mg ml 1 ) (A), oxyhemoynin (μmol ml 1 ) (B), nd the rtio of oxyhemoynin to protein (%) (C) of Litopeneus vnnmei whih reeived sline or norepinephrine t 1 8,1 7,or1 6 mol shrimp 1. Sttistil desriptions re the sme s those in Fig. 1.

6 698 S.-P. Yeh et l. / Aquulture 254 (26) shrimp tht hd een dministrted NE t 1 8,1 7, nd 1 6 mol shrimp 1, respetively, ompred to the sline ontrol shrimp. However, no signifint differenes in hemolymph osmollity or sodium were oserved mong the 4 tretments from 4 to 24 h (Fig. 2A, C). Hemolymph hloride levels were signifintly lower in shrimp whih hd reeived NE t 1 6 mol shrimp 1 thn in shrimp whih hd reeived sline nd NE t 1 8 nd 1 7 mol shrimp 1, nd hd deresed to 95.5% ompred to the level of shrimp tht hd reeived sline fter 2 h. However, no signifint differenes in hemolymph hloride were oserved etween the eginning nd 2 h fter in shrimp whih hd reeived NE t 1 6 mol shrimp 1, nd mong the 4 tretments from 4 to 24 h (Fig. 2B). Hemolymph potssium of shrimp 2 h fter hving reeived NE t 1 6 mol shrimp 1 hd signifintly deresed ompred to shrimp whih hd reeived sline nd NE t 1 8 mol shrimp 1. Hemolymph potssium hd deresed to 82.6% ompred to the sline ontrol shrimp. However, hemolymph potssium levels did not signifintly differ mong the 4 tretments from 4to24h(Fig. 2D). Although, no signifint differene in hemolymph oxyhemoynin levels ws noted in the shrimp 2 h fter hving reeived NE t 1 6 mol shrimp 1, the level ws slightly higher thn those tht hd reeived sline nd NE t 1 8,1 7 mol shrimp 1. However, no signifint differenes in hemolymph oxyhemoynin levels were oserved mong the 4 tretments from 4 to 24 h (Fig. 3A). Hemolymph protein ws signifintly higher in shrimp tht hd reeived NE t 1 6 mol shrimp 1 thn in shrimp whih hd reeived NE t 1 8,1 7 mol shrimp 1 ; its level fter 2 h ws higher thn tht of shrimp whih hd reeived the sline ontrol. Hemolymph protein hd inresed y 136.6%, 136.8%, nd 156.1% in shrimp tht hd een dministrted NE t 1 8,1 7,nd1 6 mol shrimp 1, respetively, ompred to the sline ontrol shrimp. However, no signifint differenes in hemolymph protein were oserved mong the 4 tretments from 4 to 24 h (Fig. 3B). The oxyhemoynin/ protein rtio ws signifintly lower in shrimp tht hd reeived NE t 1 8,1 7, nd 1 6 mol shrimp 1 thn in shrimp whih hd reeived sline, nd hd signifintly deresed to 72.6%, 73.5%, nd 74.4%, respetively, ompred to tht of shrimp whih hd reeived sline fter 2 h. However, no signifint differenes in the hemolymph oxyhemoynin/protein rtio were oserved mong the 4 tretments from 4 to 24 h (Fig. 3C). 4. Disussions Stress-indued neuroendorine hnges re thought to divert n orgnism's energy resoures wy from physiologil funtions tht do not immeditely help the niml overome thret nd survive to those tht do (Mule nd Vnderkooi, 1999). To meet the heightened energy demnds of stressed nimls, glyogen, due to its esy vilility for energy prodution, my e rpidly tolized resulting in losses of those reserves in tissues; onsequently resulting in signifint elevtion in lood gluose levels (Ferrndo nd Andreu, 1991). Hyperglyemi s seondry stress response hs een doumented in mny speies of rusten in response to wide rnge of stresses (Reddy et l., 1996; Kuo nd Yng, 1999). It is known tht CHH, synthesized nd relesed from the X-orgn sinus glnd omplex in rustens, is n importnt neurohormone involved in gluose metolism. For hyperglyemi responses to old shok in the freshwter gint prwn, M. rosenergii, mong the iogeni mines dministered, Kuo nd Yng (1999) indited tht the hyperglyemi responses indued y injetion of NE (norepinephrine) nd OA (otopmine) were nerly identil etween the intt nd eyestlk-lted prwns. They, therefore, suggested tht hyperglyemi effets of NE nd OA diretly ffet the trget tissues nd re not medited through the CHH. Hemolymph gluose levels signifintly inresed in shrimp L. vnnmei, tht hd reeived dopmine, nd the effets ppered to hve een dose dependent (Chiu et l., in press). A similr trend of hemolymph gluose inrese ws lso oserved in shrimp whih hd reeived NE in the present study. These fts led us to hypothesize tht stresses indue the relese of NE in the hemolymph nd ffet the trget tissue in L. vnnmei, resulting in hyperglyemi response. However, the mode of tion is unknown nd ould e hieved in numer of wys yet to e determined. Inresed energeti requirements during stressful onentrtions neessitte n inrese in ATP through the opertion of the retion sequene normlly leding to the formtion of exess pyruvte. Under hypoxi onditions, the impired oxidtion of pyruvte in the mitohondri leds to lti id formtion. A lrge proportion of lti id n diffuse into the lood, thus inresing the onentrtion of lti id in the lood (Krlsson nd Jos, 1982). In rustens, homeostsis of hemolymph gluose is mintined y n intertion etween CHH relese nd rohydrte metolites in the hemolymph. During inreses in glyolyti flux, ltte my use the relese of CHH

7 S.-P. Yeh et l. / Aquulture 254 (26) y positive feedk mehnism. The hormone then stimultes glyogenolysis, thus inresing gluose vilility. The exess gluose relesed then suppresses CHH relese from the X-orgn sinus glnd omplex y negtive feedk (Sntos nd Keller, 1993). For L. vnnmei whih reeived DA, oth the hemolymph gluose nd hemolymph ltte levels signifintly inresed from 2 to 4 h, nd then ll hd returned to the initil level fter 8 h (Chiu et l., in press). Similr results were lso oserved in shrimp whih reeived NE in the present study. The fts suggest tht hemolymph gluose nd ltte re involved in the regultion of CHH relese in L. vnnmei resulting from n NE relese effet on the trget tissue under stress. Further reserh is needed to exmine the reltionship etween CHH nd NE vrition in the hyperglyemi response of shrimp. Neuroregultor involvement in osmoregultory proesses in rustens ws reviewed y Morris (21) nd Tierney et l. (23). The perirdil orgns nd, more reently, the sinus glnd hve oth een shown to relese osmoregultory ompounds. In C. spidus, Kmemoto nd Oym (1985) demonstrted tht extrts from the perirdil orgns stimulte N + uptke y gill tissue. Also, DA nd OA elevte AMP levels, nd pplitions of duitytyl AMP elevte N + uptke in gill tissue, suggesting tht the mines t through this seond messenger pthwy to inrese N + uptke (Lohrmnn nd Kmemoto, 1987). DA nd AMP ting s first nd seond messengers, respetively, to inrese N + uptke were supported y susequent reserh using gill tissue from C. mens (Sommer nd Mntel, 1988), the purple shore r, Leptogrpsus vriegtes (Morris nd Edwrds, 1995), nd the Chinese mitten r, Eriohier sinensis (Mo et l., 1998). Findings of the ove studies suggest tht AMP indues N + flux y stimulting N + /K + - ATPse tivity in gill memrnes; however, AMP my lso indue N + flux vi V-ATPse (Riestenptt et l., 1995). In rustens, gills re importnt orgns for respirtion s well s for osmoregultion (Mntel nd Frmer, 1983; Pequeux, 1995). Morris (21) indited tht euryhline mrine rs utilize pil memrne rnhil N + /H + nd Cl /HCO 3 exhnges powered y sl memrne N + /K + -ATPse, ut in freshwter rustens, n pil V-ATPse provides for the eletrogeni uptke of Cl in exhnge for HCO 3. The HCO 3 is provided y roni nhydrse whih filittes CO 2 exretion, while NH + 4 sustitutes for K + in sl ATPse nd for H + in the pil exhnge. For C. mens trnsferred to diluted sewter, there ws rpid inrese in DA in the hemolymph (Ztt, 1987), nd inreses in N + /K + -ATPse tivity nd energy metolism (Sommer nd Mntel, 1988). L. vnnmei reeiving 1 6 mol shrimp 1 DA showed trnsient elevtions in osmollity, nd N + nd Cl levels (Chiu et l., in press). In the present study, however, shrimp reeiving 1 6 mol shrimp 1 NE showed trnsient elevtions in osmollity nd N +, nd dereses in K + nd Cl levels suggesting tht ute modifition of osmollity nd ions my involve neuroendorine ontrol in L. vnnmei under environmentl stresses. The present study indites tht fter 2 h, NE dministered to L. vnnmei indued notle protein elevtion, oxyhemoynin/ protein rtio redution, nd n invrile oxyhemoynin onentrtion in the hemolymph. However, no signifint differenes in hemolymph protein, oxyhemoynin, or the oxyhemolymph/ protein rtio were oserved mong the 4 tretments fter 4 h. Chiu et l. (in press) indited tht fter 2 h, DA dministered to L. vnnmei indued notle protein elevtion, oxyhemoynin/ protein rtio redution, nd n invrile oxyhemoynin onentrtion in the hemolymph. Those fts suggest tht DA nd NE promote trnsient moiliztion of protein whih diffuses into the hemolymph of shrimp, resulting in redution in the oxyhemoynin/ protein rtio. A rhythmiity of physiologil proesses under the influenes of yli hnges in the photoperiod regime hs often een reported. In the present study, hemolymph ltte onentrtions in L. vnnmei whih hd reeived sline were lower fter 4 h thn those of shrimp t other smpling times. Two hours fter n NE injetion t 1 6 mol shrimp 1, L. vnnmei hemolymph Cl levels were lower thn those of shrimp reeiving the sline ontrol nd NE t 1 7 nd 1 8 mol shrimp 1, nd did not differ signifintly from the ontrol t h. The fts suggest tht hnges in the rhythmiity of physiologil proesses do our, nd tht NE my inhiit the rhythmi hnge in Cl regultion. In onlusion, the present study doumented tht L. vnnmei reeiving NE t 1 6 mol shrimp 1 experiened trnsient elevtions in osmollity, nd gluose, ltte, N +, nd protein levels, s well s redutions in Cl,K +, nd the oxyhemoynin/protein rtio levels in the hemolymph. Norepinephrine plys n importnt role in neurotrnsmission nd uses energy metolism, osmoregultion, nd respirtory response modultion. Further work on NE nd other hormones will provide etter understnding of the response of shrimp to stressors in their environment, nd in prodution systems.

8 7 S.-P. Yeh et l. / Aquulture 254 (26) Aknowledgements The pper ws supported y grnt (NSC B-2-3) from the Ntionl Siene Counil, Repuli of Chin. Referenes Boone, W.R., Shoffeniels, E., Hemoynin synthesis during hypo-osmoti stress in the shore r Crinus mens (L.). Comp. Biohem. Physiol. 63B, Brdford, M.M., A rpid nd sensitive method for the quntittion of mirogrm quntities of protein using the priniple of protein dye inding. Anl. Biohem. 72, Burnett, L.E., Integrted funtion of the respirtory pigment hemoynin in rs. Am. Zool. 32, Chen, J.C., Cheng, S.Y., Studies on hemoynin nd hemolymph protein levels of Peneus jponius sed on sex, size nd moulting yle. Comp. Biohem. Physiol. 16B, Chen, J.C., Nn, F.H., Cheng, S.Y., Sheen, S.S., Effets of mient mmoni on mmoni-n nd protein onentrtions in hemolymph nd mmoni-n exretion of Peneus hinensis. Mr. Eol., Prog. Ser. 98, Cheng, W., Liu, C.H., Yn, D.F., Chen, J.C., 22. 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