Accepted 18 December 2001

Transcription

1 The Journl of Experimentl Biology 25, (22) Printed in Gret Britin The Compny of Biologists Limited 22 JEB Accumultion of mmoni in the body nd NH 3 voltiliztion from lkline regions of the body surfce during mmoni loding nd exposure to ir in the wether loch Misgurnus nguillicudtus T. K. N. Tsui 1, D. J. Rndll 2, *, S. F. Chew 3, Y. Jin 1, J. M. Wilson 4 nd Y. K. Ip 1 1 Deprtment of Biologicl Sciences, Ntionl University of Singpore, 1 Kent Ridge Rod, Singpore , Republic of Singpore, 2 Deprtment of Biology nd Chemistry, City University of Hong Kong, Tt Chee Avenue, Kowloon, Hong Kong, Chin, 3 Deprtment of Nturl Sciences, Ntionl Institute of Eduction, Nnyng Technologicl University, 1 Nnyng Wlk, Singpore , Republic of Singpore nd 4 Centro Interdisciplinr de Investigco Mrinh e Ambientl-CIIMAR, Ru do Cmpo Alegre 823, Porto, Portugl *Author for correspondence (e-mil: bhrnd@cityu.edu.hk) Accepted 18 December 21 The wether loch Misgurnus nguillicudtus inhbits rice fields tht experience drought in summer nd mmoni loding during griculturl fertilistion. Exposure of specimens to mmoni led to the ccumultion of mmoni in muscle, liver nd blood. The level of mmoni reched in the plsm ws the highest reported mong fishes. Ammoni ws not detoxified to ure, nd ure excretion rte ws unffected by mmoni exposure. Fish cidified the wter to reduce mmoni loding. Ammoni loding, unlike eril exposure, did not induce glutmine synthesis, nd there ws no ccumultion of glutmine. This is unique observtion different from those reported for other fishes in the literture. An initil switch to prtil mino cid ctbolism led to the ccumultion of lnine nd ws probbly ssocited with decresed rte of mmoni production. Aeril exposure led to decreses in rtes of mmoni nd ure excretion, s well s the ccumultion of tissue mmoni. As the internl mmoni levels incresed, M. nguillicudtus ws ble to excrete some mmoni in the gseous form (NH 3 ). The percentge of Summry mmoni excreted s NH 3 incresed with time of exposure nd with incresing temperture. It ppers tht irbrething through the gut is involved, with the nterior portion of the digestive trct plying centrl role: it becme significntly more lkline in fish exposed to ir or to environmentl mmoni. The skin, which lso becme more lkline during ir exposure, my lso be involved in mmoni voltiliztion in ir-exposed fish. This represents the first report of fish using voltiliztion of NH 3 s prt of defence ginst mmoni toxicity. It cn be concluded tht the min strtegy dopted by M. nguillicudtus confronted with mmoni loding or ir exposure is to tolerte high mmoni levels in the tissues. During periods of elevted tissue mmoni levels, some mmoni is lost by voltiliztion vi ir-brething using the gut. In ddition, some mmoni my be lost cross the skin during ir exposure. Key words: mmoni, mmoni loding, ure, ir exposure, voltiliztion, wether loch, Misgurnus nguillicudtus. Introduction The wether loch Misgurnus nguillicudtus is smll freshwter fish tht lives in rice fields. It is cpble of using its intestine s n dditionl respirtory orgn (McMhon nd Burggren, 1987) to brethe ir. It cn gulp ir, pss it through the digestive trct nd relese it from the vent. This enbles it to live in oxygen-poor wters or to bury itself in mud during long-lsting droughts in the summer. When there is lck of wter, it will bury nd position itself in the soft mud such tht the mouth hs ccess to ir through smll opening, the fish gulping ir occsionlly nd respiring through its intestine. By doing so, this loch cn survive for more thn month until the drought is over. Chew et l. (21) reported tht M. nguillicudtus ws cpble of prtil mino cid ctbolism leding to slight ccumultion of lnine in the first 24 h of eril exposure. Thus, it is possible tht this loch is ble to use certin mino cids s n energy source to sustin musculr ctivities on lnd, s hs been suggested for the mudskipper Periophthlmodon schlosseri (Ip et l., 1993, 21,b). After 24 h of eril exposure, glutmine ccumulted in the muscle nd liver of M. nguillicudtus (Chew et l., 21). Simultneously, mmoni ccumulted to very high levels in ll the tissues exmined. The present study ws undertken to determine whether M. nguillicudtus could excrete NH 3 by

2 652 T. K. N. Tsui nd others voltiliztion during ir exposure. Attempts were mde to evlute the roles of the skin nd the digestive trct in NH 3 voltiliztion. The effects of plcing M. nguillicudtus in concentrted mmoni solutions on mmoni voltiliztion from the gut nd on tissue mmoni levels were lso determined. Mterils nd methods Collection nd mintennce of specimens Mture Misgurnus nguillicudtus (8 15 g) were obtined from the Yuen Long wet-mrket in Hong Kong nd trnsferred to Singpore by ir. They were kept in dechlorinted tp wter t 25 C in the lbortory. No ttempt ws mde to seprte the sexes. Fish were cclimted to lbortory conditions for t lest 48 h before experimenttion. During this period, hlf the wter ws chnged dily nd the fish were fed with commercil fish food. Fish were fsted for 24 h prior to experiments nd were not fed during the experimentl period. Exposure of fish to experimentl conditions Ammoni exposure Fish were exposed to 3 mmol l 1 NH 4 Cl in 2 vols (w/v) of wter, djusted to ph 7.2 using NOH, t 25 C for vrious periods (12, 24 or 48 h). Those collected t the beginning of the experiment nd those exposed to dechlorinted tp wter (ph 7.2) for 48 h served s controls. Aeril exposure Fish exposed to terrestril conditions were plced in individul plstic boxes (2.5 cm 14.5 cm 6. cm) contining thin film of dechlorinted tp wter (5 ml). The boxes were, in turn, plced in fibreglss humidity chmber for the entire period of eril exposure. Determintion of mmoni gs excreted by fish Ammoni exposure Fish were kept individully in conicl flsks contining 1 ml wter or NH 4 Cl (1 mmol l 1 or 2 mmol l 1 ) solution. The flsks were tightly seled such tht they were both irtight nd wtertight, except for the ir inlet nd outlet. Air ws bubbled through the wter in the flsk t constnt rte of 2 l h 1. The flsk ws connected to two test tubes. The first test tube served s wter trp. The second, contining 3 ml of freshly prepred.5 mmol l 1 H 2 SO 4, served to trp ny mmoni gs present in the ir strem, which ws bubbled through the cid. At the end of ech 24 h period, the cid in the trp ws collected nd nlysed for mmoni s described bove. Blnk control experiments were lso run in which nimls were not present in the flsks to ccount for voltiliztion of mmoni from the bthing solutions. In one experiment, fish were plced in smll net cges before being submerged in the flsk to determine whether preventing the fish from gulping ir t the wter surfce would ffect mmoni voltiliztion. Aeril exposure Experiments in which fish were ir-exposed were performed t 2, 25 nd 3 C. Fish were kept initilly s described bove in conicl flsks contining 1 ml of dechlorinted wter. After 24 h, the cid in the trp ws nlysed for mmoni content. The wter in the flsk ws collected for mmoni ssys, nd the flsk ws rinsed. The sme fish ws then immeditely exposed to terrestril conditions for 24 h in the sme pprtus, but with only 1 ml of dechlorinted wter present in the flsk. At the end of the second 24 h period, the cid in the trp ws collected for mmoni ssy nd totlly replenished. The smll volume of wter inside the flsk ws collected, nd the flsk ws rinsed nd refilled with 1 ml of fresh wter. Ammoni production under terrestril conditions ws then monitored for 72 h. To evlute the effects of lrger volume of wter on NH 3 voltiliztion, n experiment ws performed t 25 C with 3 ml, insted of 1 ml, of wter in the flsk. Determintion of blood ph, P CO nd P O Specimens were nesthetised in.2 % 3-minobenzoic cid ethyl ester (MS222; Sigm, USA). The til ws cut, nd rteril blood ws collected in heprinised cpillry tubes. Blood vribles (ph, P CO nd P O ) were determined using n IL 136 blood gs nlyzer. Collection nd preprtion of muscle, liver nd plsm smples To collect muscle nd liver tissue, specimens were killed by blow to the hed. The lterl muscle nd liver were excised nd immeditely freeze-clmped in liquid nitrogen with precooled luminium tongs (Fupel et l., 1972). The entire procedure ws crried out within 1 min. Smples were stored t 8 C until nlysis. The frozen smples were powdered in liquid nitrogen, weighed nd homogenized three times in 5 vols of 6 % trichlorocetic cid (TCA) using n Ik-werk Stufen Ultr- Turrx homogenizer (Jnke & Kunkel, Germny) t mximum speed of 24 revs min 1 for 2 s ech, with 1 s intervls. The homogente ws centrifuged t 1 g t 4 C for 15 min in Beckmn J2-21M/E refrigerted centrifuge (Beckmn Instruments Inc., USA). A second group of specimens ws used for the collection of blood smples. The til of the fish ws cut, nd blood exuding from the severed cudl rtery ws collected in heprinized (sodium heprin) micro-hemtocrit cpillry tubes. The blood smples were centrifuged t 5 g t 4 C for 5 min. The resulting plsm ws collected nd deproteinised immeditely by dding n equl volume of 6 % TCA, followed by centrifugtion t 1 g for 1 min. The superntnt fluid ws kept t 8 C until nlysis. Determintion of mmoni, ure nd free mino cid levels For mmoni nd ure nlyses, the deproteinized smples were neutrlised to ph with 2 mol l 1 KHCO 3. Ammoni content ws determined by the method of Kun nd

3 Ammoni excretion by Misgurnus nguillicudtus 653 Tble 1. Effects of 48 h of mmoni exposure (3 mmol l 1 NH 4 Cl) nd ir exposure on the ph, P O2 nd P CO2 of the blood of Misgurnus nguillicudtus Ammoni- Control exposed Air-exposed ph 7.285±.51 (6) 7.329±.28 (6) 7.528±.17(11)* P O2 (mmhg) 33.5±4.3 (6) 33.5±4.5 (6) 34.4±2. (11) P CO2 (mmhg) 3.1±1.1 (6) 27.4±2.8 (6) 34.5±3.1 (11) 1 mmhg=.133 kp. Vlues re mens ± S.E.M. with number of determintions in prentheses. *Significntly different from the corresponding control, P<.5. Kerney (1974). The chnge in bsorbnce t 34 nm (25 C) ws monitored using Shimdzu UV-16A spectrophotometer. Freshly prepred mmonium chloride (Merck) ws used s stndrd. The ure content in.2 ml of deproteinised smple ws nlysed colorimetriclly ccording to the method of Jow et l. (1999). The difference in bsorbnce of the smple in the presence nd bsence of urese ws used s n estimte of the ure content of the smple. To mesure free ftty cid (FAA) levels, the deproteinised smples were diluted with n equl volume of.2 mol l 1 lithium citrte buffer (Pierce, USA), nd the ph ws djusted to 2.2 with 4 mol l 1 lithium hydroxide. FAA levels were mesured using Shimdzu LC-6A mino cid nlysis system with Shim-pck ISC-7/S154 Li type column. Anlyticl-grde FAA stndrds purchsed from Sigm Chemicl Co. served s references. Results were expressed s µmol g 1 wet mss tissue or µmol ml 1 plsm. Determintion of ure excretion rtes Specimens were exposed to 2 ml of 3 mmol l 1 NH 4 Cl solution (ph 7.2) t 25 C for 48 h. The externl medium ws chnged dily. At 24 h nd 48 h, wter smple ws collected nd nlysed for ure content. Preliminry studies reveled tht ure excretion rte ws liner for t lest 24 h. Ure content ws nlysed colorimetriclly s described bove. The rte of ure excretion ws expressed s µmol dy 1 g 1 fish. Determintion of the ph of wter smples, the skin nd the digestive trct The ph of wter smples nd the surfce of the skin of M. nguillicudtus ws monitored using PHR-146 micro combintion ph electrode (Lzr Reserch Lbortories) nd n Orion 72A ph meter. Preliminry results indicted tht the ph over different regions of the body differed by less thn.1 unit. Subsequently, it ws decided to report the ph obtined from the side of the fish just bove the pectorl fins. The specimen ws then killed, nd the digestive trct ws dissected out. The digestive trct ws divided into three regions: nterior, middle nd posterior. Ech region ws cut open longitudinlly, Ammoni content (µmol g 1 tissue or µmol ml 1 plsm) Ure content (µmol g 1 tissue or µmol ml 1 plsm) B A,b,b nd the ph of the mucosl surfce ws mesured using the micro combintion ph electrode. Sttisticl nlyses Results re presented s mens ± S.E.M. Student s t-test nd one-wy nlysis of vrince (ANOVA) followed by Student Neumn Keul s multiple rnge test were used to compre differences between mens where pplicble. Differences t P<.5 were regrded s sttisticlly significnt. Results Exposure to 3 mmol l 1 NH 4 Cl t ph 7.2 did not ffect blood ph, P O or P CO, but eril exposure resulted in significnt increse in blood ph (Tble 1). Ammoni built up progressively in the muscle nd liver (Fig. 1A) following mmoni exposure.,b,b,b,c b,c,d,d h/c 12 h/n 24 h/n 48 h/n 48 h/c h/c 12 h/n 24 h/n 48 h/n 48 h/c Fig. 1. (A) Effects of vrious periods of mmoni exposure (3 mmol l 1 NH 4Cl) on the concentrtions of mmoni (A) nd ure (B) in the blood plsm (open columns), liver (htched columns) nd muscle (filled columns) of Misgurnus nguillicudtus. Vlues re mens + S.E.M. (N=6). Significntly different from the h/c condition, P<.5; b significntly different from the 12 h/n condition, P<.5; c significntly different from the 24 h/n condition, P<.5; d significntly different from the 48 h/n condition, P<.5. N, 3 mmol l 1 NH 4Cl; C, control.,b

4 654 T. K. N. Tsui nd others Tble 2. Effects of vrious periods of mmoni exposure on the contents of vrious free mino cids (FAAs) nd the totl FAA (TFAAs) in the muscle of Misgurnus nguillicudtus FAA concentrtion (µmol g 1 ) FAA h/c 12 h/n 24 h/n 48 h/n 48 h/c Al 1.3±.3 5.2±.8 3.5±.8 b 2.±.4 b 1.8±.3 b Arg.2±.6.35±.9.2±.1.31±.15.3±.7 Asn.5±.8.65±.19.59±.15.61±.28.59±.1 Asp.1±.3.55±.19.46±.16.14±.3.35±.14 Cys.2±.2.12±.6.5±.3.2±.2 ND Gln 1.5±.2 2.3±.4 3.2±.4 4.1± ±.5 Glu.3±.5 1.3±.6 1.4±.1.91±.26 2.±.3 Gly 4.7± ±.7 2.5±.4 2.4±.5 3.8±.4 His 1.9±.6 2.7±.7 3.±.2 2.3±.3.95±.6 c Ile.28±.1.55±.9.25±.3 b.7±.2 b.46±.8,c,d Leu.51±.4.94±.13.55±.3 b.2±.4,b,c.76±.13,c,d Lys.99± ±.5 1.7±.2.77±.13 b 1.98±.36 Met.83±.2.12±.3.6±.1 b.1±.1,b ND,b,c Orn ND.12±.3.7±.2.2±.2 b ND b Phe.22±.1.51±.9.21±.5 b.23±.2 b.36±.3 b Pro.22±.2.57±.5.5±.4.33±.8 b.47±.2 Ser.56±.7 1.4±.2 1.3±.2 1.1±.2.98±.11 Tu 5.6±.2 3.2±.6 3.2±.3 2.4±.5 3.8±.5 Thr 1.±.2 2.3±.5 2.4±.4 1.1±.3 1.5±.2 Try.1±.1.5±.3.2±.1.3±.3 ND Tyr.13±.1.22±.5.9±.1 b.8±.1 b.15±.2 Vl.41±.3.79±.1.39±.3 b.14±.4,b,c.69±.11,c,d TFAAs 2.3± ± ± ± ±.8 Vlues re mens ± S.E.M. (N=4). N, 3 mmol l 1 NH 4Cl; C, control. ND, not detectble. Significntly different from the h/c vlue, P<.5; b significntly different from the 12 h/n vlue, P<.5; c significntly different from the 24 h/n vlue, P<.5; d significntly different from the 48 h/n vlue, P<.5. Rte of NH 3 voltiliztion (µmol dy 1 ) ph A * B * 1 mmol l 1 NH 4 Cl 2 mmol l 1 NH 4 Cl Ammoni content in the muscle nd liver ws 18.9 nd 17.5 µmol g 1, respectively, fter 48 h exposure. In comprison, the plsm mmoni level rose bruptly in the first 12 h of mmoni exposure nd reched 4.2 µmol ml 1 by 48 h (Fig. 1A). There were no increses in ure level in the muscle, liver or plsm (Fig. 1B). The ure excretion rte of the specimen kept under control condition ws.75±.12 µmol dy 1 g 1 (N=5), which ws unffected by exposure to 3 mmol l 1 NH 4 Cl. The totl free mino cid (TFAA) concentrtions in the muscle (Tble 2), liver (Tble 3) nd plsm (Tble 4) were unffected by mmoni exposure. However, there were minor chnges in the levels of some free mino cids in these tissues. In the muscle, there ws trnsient increse in lnine level from 1.3 to 5.2 µmol g 1 fter 12 h of mmoni exposure, but Fig. 2. Effects of mmoni loding (1 mmol l 1 or 2 mmol l 1 NH 4Cl) on (A) the NH 3 voltiliztion rte (µmol dy 1 ) nd (B) the ph of the surrounding medium of Misgurnus nguillicudtus. Open columns represent the control condition (no fish). Filled columns represent the experimentl condition (with fish). Vlues re mens + S.E.M. (N=4). *Significntly different from the corresponding control condition, P<.5.

5 Ammoni excretion by Misgurnus nguillicudtus 655 Tble 3. Effects of vrious periods of mmoni exposure on the contents of vrious free mino cids (FAAs) nd the totl FAA (TFAAs) in the liver of Misgurnus nguillicudtus FAA concentrtion (µmol g 1 ) FAA h/c 12 h/n 24 h/n 48 h/n 48 h/c Al.59±.4.7±.2.51±.8.69±.1.49±.1 Arg.19±.3.11±.1.7±.2,b.59±.4.1±.5 Asn.48±.15.25±.3.14±.1.15±.1 ND Asp 1.4±.4 1.1±.1.7±.6.65±.7 ND Cys ND.3±.1.4±.1.6±.3 ND Gln 2.3±.7 1.8±.3 2.±.1 3.±.5 1.4±.4 Glu 1.8±.2 3.6±.2 3.2±.2 5.5±.4,b,c 3.3±.4,d Gly.77±.13.78±.15.38±.3.4±.11.75±.6 His.28±.5.34±.3.16±.1,b.16±.1,b.15±.5,b Ile.3±.7.27±.2.18±.2.8±.3.23±.8 Leu.63±.16.45±.3.33±.3.21±.28.4±.14 Lys.83±.1.62±.4.27±.4,b.23±.2,b.33±.9,b Met ND.7±.1.2±.1 b.12±.8 b ND b Orn ND.6±.1.21±.5,b.11±.6 b ND b Phe.26±.5.2±.2.15±.2.8±.1 b.3±.3 c,d Pro.38±.3.29±.4.19±.2.13±.1.48±.9 b,c,d Ser.68±.16.39±.2.2±.2.2±.1.33±.4 Tu 11.8± ±.7 1.5±.7 8.6± ±1.2 Thr 1.2±.3.86±.1.37±.8.28±.3.76±.35 Try ND.35±.2.8±.5 b.11±.4 b.15±.11 b Tyr.11±.1.13±.5.87±.11.89±.29.11±.1 Vl.53±.14.59±.2.35±.5.31±.4.5±.1 TFAA 24.4± ± ±.7 2.9±.8 21.±2.4 Vlues re mens ± S.E.M. (N=4). N, 3 mmol l 1 NH 4Cl; C, control. ND, not detectble. Significntly different from the h/c vlue, P<.5; b significntly different from the 12 h/n vlue, P<.5; c significntly different from the 24 h/n vlue, P<.5; d significntly different from the 48 h/n vlue, P<.5. it decresed therefter. This ws ccompnied by trnsient increses in the levels of some essentil mino cids. Ammoni exposure did not ffect glutmine levels in the muscle, liver or plsm, but led to n increse in the glutmte level in the liver nd decrese in the glutmte level in the plsm. With 2 mmol l 1 NH 4 Cl in the externl medium, the mount of mmoni recovered from the cid trp in the presence of M. nguillicudtus fter 24 h period ws significntly higher thn tht of the blnk without fish (Fig. 2A). However, this difference disppered when the fish ws prevented from ccessing ir (dt not shown). With fish in the medium, the ph recorded in the presence of 2 mmol l 1 NH 4 Cl fter 24 h ws significntly lower thn tht of the blnk without fish (Fig. 2B). For fish exposed to 3 mmol l 1 NH 4 Cl, the mucosl surfce ph of the nterior portion of the digestive trct ws Fig. 3. Effects of eril exposure on the mmoni excretion rte (µmol dy 1 g 1 ) of Misgurnus nguillicudtus. Open columns represent the control condition. Filled columns represent the experimentl condition. S, submerged; T, eril exposure. Vlues re mens + S.E.M. (N=4). *Significntly different from the corresponding control condition, P<.5. Rte of mmoni excretion (µmol dy 1 g 1 ) S S S T S T S T S S Dy 1 Dy 2 Dy 3 Dy 4 Dy 5

6 656 T. K. N. Tsui nd others Tble 4. Effects of vrious periods of mmoni exposure on the contents of vrious free mino cids (FAAs) nd the totl FAA (TFAAs) in the blood plsm of Misgurnus nguillicudtus FAA concentrtion (µmol ml 1 ) FAA h/c 12 h/n 24 h/n 48 h/n 48 h/c Al.29±.4.27±.2.25±.4.28±.2.22±.3 Arg.1±.1.83±.4.7±.1.65±.5.9±.1 d Asn.1±.1.94±.4.9±.1.6±.4,b.13±.1,b,c,d Asp.5±.1.27±.2.22±.2.32±.3.8±.4,b,d Cys.9±.1.13±.1.1±.1.11±.2.9±.1 Gln.23±.2.48±.2.44±.3.38±.3.57±.8,d Glu.16±.2.8±.1.6±.1.9±.1.8±.1 Gly.34±.3.28±.3.24±.2.21±.1.25±.1 His.27±.2.14±.1.11±.1.114±.4.16±.1 Ile.3±.2.29±.4.35±.5.34±.4.29±.3 Leu.49±.3.52±.7.63±.1.58±.6.41±.6 Lys.3±.3.15±.1.16±.1.18±.2.15±.2 Met.5±.1.47±.2.39±.2.3±.2,b.42±.3 Orn.5±.2.23±.4.19±.3.19±.2.15±.2 Phe.1±.1.73±.4.74±.6.66±.5.66±.7 Pro.78±.6.56±.7.62±.3.55±.2.88±.8 b,c,d Ser.2±.4.12±.1.132±.4.93±.5.15±.2 Tu.47±.8.42±.4.27±.3.36±.3.35±.1 Thr.4± ±.75.28±.3.17±.2.43±.4 Try.43±.2.74±.19.52±.4.82±.18.54±.7 Tyr.76±.7.71±.5.68±.8.1±.2.8±.7 Vl.49±.3.5±.6.58±.8.48±.9.48±.4 TFAA 4.6±.4 4.9±.8 4.±.3 3.8±.3 4.1±.2 Vlues re mens ± S.E.M. (N=4). N, 3 mmol l 1 NH 4Cl; C, control. Significntly different from the h/c vlue, P<.5; b significntly different from the 12 h/n vlue, P<.5; c significntly different from the 24 h/n vlue, P<.5; d significntly different from the 48 h/n vlue, P<.5. significntly higher (more lkline) thn tht of the control (Tble 5); however, the ph vlues of the middle nd posterior regions of the intestine were unffected by mmoni exposure (Tble 5). Air exposure cused lrger increse in ph of the nterior intestine thn mmoni exposure in wter. The ph of the skin surfce during eril exposure ws significntly higher thn tht of submerged fish (Tble 5). Aeril exposure led to mrked decrese in the rte of mmoni excretion (Fig. 3). At the sme time, there ws progressive increse in the mount of NH 3 voltilized (Fig. 4). The ph of the film of wter underlying the fish in ir incresed from n initil vlue of 7.2 to between 7.9 nd 8.1. Incresing the environmentl temperture to 3 C incresed the rte of NH 3 voltiliztion by M. nguillicudtus exposed to terrestril conditions (Tble 6). Discussion M. nguillicudtus inhbits muddy swmps nd ponds nd rice fields subject to periodic drying nd hs to solve problems of mmoni toxicity under two situtions. First, to survive drought, M. nguillicudtus burrows into the mud nd brethes ir through hole in the surfce of the mud. In the Rte of mmoni voltiliztion (µmol dy 1 g 1 ) S S S T S T S T S S Dy 1 Dy 2 Dy 3 Dy 4 Dy 5 Fig. 4. Effects of eril exposure on the NH 3 voltiliztion rte (µmol dy 1 g 1 ) of Misgurnus nguillicudtus. Open columns represent the control condition. Filled columns represent the experimentl condition. S, submerged; T, eril exposure. Vlues re mens + S.E.M. (N=4). *Significntly different from the corresponding control condition, P<.5.

7 Ammoni excretion by Misgurnus nguillicudtus 657 Tble 5. Effects of 48 h of mmoni exposure (3 mmol l 1 NH 4 Cl) nd ir exposure on the ph of the skin nd the different prts of the digestive trct of Misgurnus nguillicudtus Control Ammoni-exposed Air-exposed Skin ph 6.65±.9 (5) 6.61±.12 (5) 8.23±.18 (6)* Anterior ph 7.4±.3 (4) 7.93±.8 (5)* 8.23±.1 (6)* Middle ph 7.32±.5 (4) 7.55±.6 (5) 7.68±.1 (6) Posterior ph 7.33±.5 (3) 7.41±.11 (5) 7.7±.13 (6) Vlues re mens ± S.E.M. with number of determintions in prentheses. *Significntly different from the corresponding control, P<.5; significntly different from the nterior digestive trct in the sme condition, P<.5. bsence of wter, mmoni excretion my be impeded, leding to the ccumultion of mmoni in its tissues. Second, mny frmers dd fertilizers to the rice pddies, which my relese mmonium slts into the environment nd expose the loch to high levels of environmentl mmoni. It is well known tht the primry route of mmoni excretion in fish is through the gills (Wood, 1993; Wilkie, 1997). The principl mechnism of mmoni excretion cross the gill epitheli is the non-ionic diffusion of NH 3 down fvourble trnsbrnchil grdient of NH 3 from blood to wter (Rndll nd Wright, 1987). An increse in externl NH 3 concentrtion, if lrge enough, will result in the reversl of the blood-to-wter NH 3 grdient, leding to net NH 3 influx, n inhibition of excretion of endogenous mmoni nd rise in the totl plsm mmoni level in the body (Wood, 1993; Wilkie, 1997). Although the mmoni level in the plsm of M. nguillicudtus in the submerged condition flls within the rnge of those of other fculttive ir-brethers, it ws unexpectedly high in mmoni-exposed (4.2 µmol ml 1 ) nd ir-exposed (5.9 µmol ml 1 ) individuls (Chew et l., 21) fter 48 h of exposure. The mmoni levels in the plsm of Amphipnous euchi, Clris btrchus, Heteropneustes fossilis, Anbs testudineus nd Chnn puncttus rnge from.43 to.89 µmol ml 1 (Sh nd Rth, 1989). The plsm of the tilpi Oreochromis nilotic (in mmoni-loding conditions), the climbing gourmi Anbs scndens nd the snkehed Chnn gchu (in terrestril conditions) contined 1., 1.6 nd 1.6 µmol ml 1 mmoni, respectively (Rndll et l., 1989; Rmswmy nd Reddy, 1983). To our knowledge, no other fish hve been reported to exhibit such high mmoni concentrtion in the plsm under mmoni-loding or terrestril conditions. Tissue mmoni levels incresed mrkedly in M. nguillicudtus exposed to NH 4 Cl in wter or in ir. The levels in the muscle nd liver reched 18.9 µmol g 1 nd 17.5 µmol g 1, respectively, following NH 4 Cl exposure, nd 11.8 nd µmol g 1, respectively, fter 48 h of eril exposure (Chew et l., 21). In the mudskipper Periophthlmodon schlosseri, 3.46 nd 1.64 µmol g 1 mmoni were ccumulted in the liver nd muscle, respectively, fter 24 h of eril exposure (Ip et l., 1993) nd 7.24 µmol g 1 nd 14.8 µmol g 1, respectively, fter 6 dys of exposure to 1 mmol l 1 NH 4 Cl (Rndll et l., 1999). In the portunid crb Necor puber, the muscle mmoni content ws 1.42 µmol g 1 fter 12 h of eril exposure (Durnd nd Regnult, 1998). Clerly, M. nguillicudtus cn tolerte much higher levels of mmoni in its tissues nd orgns thn cn other nimls, nd future reserch on these mmonitolernt processes my increse our understnding of the cellulr mechnisms of mmoni toxicity. For most vertebrtes, such high levels of mmoni would disturb intrcellulr ph nd membrne integrity (Cmpbell, 1991), leding to convulsions nd deth. Working on mmmls, Mrcid et l. (1992) proposed tht mmoni toxicity ws due to the ctivtion of N-methyl-D-sprtte (NMDA)-type glutmte receptors, leding to elevted intrcellulr C 2+ concentrtions nd cell deth. Perhps M. nguillicudtus hs modified NMDA receptors. Levels of free mino cids (FAAs) were vrible in both the mmoni-exposed nd control groups. The fish were not fed during the experiment, nd this my explin the significnt increses in levels of number of FAAs observed fter 48 h of Tble 6. Effects of temperture on the mmoni excretion rte, NH 3 voltiliztion rte, proportion of NH 3 voltilized nd ph of the mbient wter of Misgurnus nguillicudtus on the third dy of ir exposure Rte of excretion of NH + 4 Rte of (NH 3 voltilized 1)/ Temperture Volume of wter into the wter NH 3 voltiliztion (NH + 4 in wter+ ( C) (ml) (µmol dy 1 g 1 ) (µmol dy 1 g 1 ) NH 3 voltilized) ph of wter ±.31 (4).429±.36 (4) 31.8±7.7 (4) 8.5±.24 (4) ±.14 (8).795±.155 (8) 41.±6.3 (8) 8.7±.8 (8) ±.4 (4) 1.67±.43 (4) 56.8±5.6 (4) 8.1±.13 (4) ±.19 (4),b,c.867±.259 (4) 26.2±6.5 (4) c 7.85±.5 (4) Vlues re mens ± S.E.M. with number of determintions in prentheses. Significntly different from vlue t 2 C with 1 ml of wter, P<.5; b significntly different from vlue t 25 C with 1 ml of wter, P<.5; c significntly different from vlue t 3 C with 1 ml of wter, P<.5.

8 658 T. K. N. Tsui nd others fsting (Tble 2). This somewht obscures the FAA response to mmoni exposure. However, it is cler tht there ws trnsient increse in lnine level in the muscle fter 12 h of exposure to 3 mmol l 1 NH 4 Cl. M. nguillicudtus might hve resorted to prtil mino cid ctbolism t the onset of exposure to NH 4 Cl to slow down the rte of internl mmoni production. Ip et l. (21,b) proposed prtil mino cid ctbolism s strtegy for fish to reduce the rte of production of endogenous mmoni during eril exposure. The mino groups of certin mino cids re trnsferred directly or indirectly to pyruvte to form lnine. The resulting crbon chin enters the Krebs cycle nd is prtilly oxidised to mlte, which replenishes pyruvte through the rection ctlysed by mlic enzyme. In this wy, ATP cn be derived from mino cids without producing mmoni. Glutmine hs been found to ply role in mmoni detoxifiction in fish in response to high environmentl mmoni concentrtions (Arillo et l., 1981; Dbrowsk nd Wlsow, 1986; Mommsen nd Wlsh, 1992; Peng et l., 1998). In this regrd, M. nguillicudtus is typicl, for lthough glutmine ccumulted in its muscle fter 48 h of eril exposure, ccumultion of glutmine did not occur in muscle nd liver when specimens were exposed to mmoni. Hence, even if the mechnisms to detoxify endogenous mmoni were present, they were not utilised when the fish were confronted with n influx of exogenous mmoni. The levels of ure in vrious tissues nd the ure excretion rte of M. nguillicudtus exposed to mmoni remined unchnged during mmoni loding. The ctivities of glutmine synthetse nd ornithine ure cycle enzymes were too low to render them effective in detoxifying mmoni (Chew et l., 21). The production of ure s strtegy to detoxify mmoni in response to eril exposure or mmoni loding does not pper to be common phenomenon in teleosts (Ip et l., 21). This is probbly becuse glutmine nd ure production re energy-expensive processes, involving 4 nd 5 moles of ATP (or equivlent), respectively, per mole of product formed (Ip et l., 21). It would pper tht M. nguillicudtus simply tolerted high tissue mmoni levels. In ddition, it lowered the ph of the medium during mmoni exposure. This would reduce the NH 3 concentrtion in the externl medium nd decrese the rte of mmoni influx. Aeril exposure resulted in mrked reduction in the rte of mmoni excretion. Presumbly, mmoni is excreted cross the gills into the wter, nd this is inhibited during ir exposure. A smll mount of mmoni is voltilized into the ir when the niml is submerged (see Fig. 4). The mount excreted by voltiliztion increses when mmoni is dded to the wter nd during eril exposure. NH 3 voltiliztion hs lso been observed in the mngrove killifish Rivulus mrmortus in response to prolonged ir exposure (Frick nd Wright, 22). M. nguillicudtus cn brethe ir using its intestine (McMhon nd Burggren, 1987), gulping ir with its mouth nd blowing gs out of the vent. Our results indicte tht M. nguillicudtus ws cpble of excreting some mmoni in gseous form vi the gut during mmoni-loding. A smll quntity of mmoni ws voltilized from the NH 4 Cl solution in the bsence of fish. This is understndble becuse smll frction (.6 %) of mmoni ws present s NH 3 even t ph 7, nd it would be crried over by the ir current into the cid trp. The frction of NH 3 present in solution decreses with decresing ph. Since there ws significnt reduction in the ph of the externl medium in the presence of fish, the mount of mmoni voltilized should theoreticlly be smller if it ws due only to voltiliztion from the wter. However, tht ws not the cse: voltiliztion incresed in the presence of the fish. Furthermore, M. nguillicudtus stopped voltilizing NH 3 when it ws prevented from ccessing the wter surfce to gulp ir. Hence, it cn be concluded tht the excess NH 3 voltilized represented mmoni excreted from the fish into the ir pssing through its gut nd blown out of the vent. If it hd been excreted cross the skin into the wter, it would hve ppered even when the fish ws forcibly prevented from brething ir. In M. nguillicudtus, the nterior portion of the digestive trct becme more lkline in mmoni-loded nd irexposed fish. Alkliztion would rise the frction of NH 3 in the mmoni excreted into tht region of the digestive trct, nd this would ugment NH 3 voltiliztion. When M. nguillicudtus ws exposed to terrestril conditions, the surfce of the skin lso becme significntly more lkline, nd the ph of the wter underneth the fish ws significntly higher thn tht of the submerged control. Thus, the skin of M. nguillicudtus my lso be site of NH 3 voltiliztion during eril exposure. Similrly, Wieser (1972b) suggested tht, in isopods, mmoni diffuses into the film of wter retined between the pleopods nd is voltilized s result of lkliztion of the solution. Some terrestril crbs hve lso been reported to voltilize NH 3 by lkliztion of urine (De Vries nd Wolcott, 1993; Vrley nd Greenwy, 1994). Although the skin nd digestive trct my both be involved in NH 3 voltiliztion when the fish is moving on lnd, excretion of mmoni through the skin would be ineffective when the fish burrows into the mud. It seems probble tht, when the fish is surrounded by mud, the digestive trct might become the only venue for NH 3 voltiliztion. For certin isopods (Wieser, 1972) nd for M. nguillicudtus exposed to terrestril conditions, the rte of NH 3 voltiliztion increses with temperture. Although the mount of mmoni excreted into the wter remined the sme t different tempertures, the increse in the rte of NH 3 voltiliztion led to increses in totl mmoni excretion t higher tempertures (Tble 6). The observed effects re probbly relted to the direct effect of temperture on the process of voltiliztion. The progressive increse in the mount of NH 3 voltilized by M. nguillicudtus over 3-dy period ws pprently relted to the incresed internl mmoni levels. Besides building up mmoni to very high level in the plsm during eril exposure, the blood ph simultneously becme more lkline. This would led to high level of NH 3 in the blood. The high P NH grdient would fcilitte the efflux of mmoni onto the body surfce. Thus, the increse in mmoni

9 Ammoni excretion by Misgurnus nguillicudtus 659 voltiliztion seen during periods of elevted mmoni level in the wter nd eril exposure re relted to the lkliztion of the gut nd blood nd the increse in totl mmoni concentrtions in the body, ll rising NH 3 levels nd, therefore, mmoni voltiliztion. This is in greement with the observtion of Wright nd O Donnell (1993) on the isopod Porcellio scber tht periodic voltiliztion of NH 3 ws correlted with lrge increse in mmoni levels in the hemolymph nd pleon fluid. The loch Misgurnus nguillicudtus is unusul in tht it tolertes very high mmoni levels in its tissues, but in turn these high levels, together with lkline body surfces, permit significnt mmoni excretion by voltiliztion during eril exposure. References Arillo, A., Mrgiocco, C., Medlodi, F., Mensi, P.nd Schenone, G. (1981). Ammoni toxicity mechnisms in fish: studies on rinbow trout (Slmo girdneri Richrdson). Ecoltoxicol. Environ. Sf. 5, Cmpbell, J. W. (1991). Excretory nitrogen metbolism. In Experimentl nd Metbolic Animl Physiology. Comprtive Animl Physiology, fourth edition (ed. C. L. Prosser), pp New York: Wiley-Interscience. Chew, S. F., Jin, Y. nd Ip, Y. K. (21). The loch Misgurnus nguillicudtus reduces mino cid ctbolism nd ccumultes lnine nd glutmine during eril exposure. Physiol. Biochem. Zool. 74, Dbrowsk, H. nd Wlsow, T. (1986). Sublethl effect of mmoni on certin biochemicl nd hemtologicl indictors in common crp (Cyprinus crpio L.). Comp. Biochem. Physiol. 83C, De Vries, M. C. nd Wolcott, D. L. (1993). Gseous mmoni evolution is coupled to reprocessing of urine t the gills of ghost crbs. J. Exp. Zool. 267, Durnd, F. nd Regnult, M. (1998). Nitrogen metbolism of two portunid crbs, Crcinus mens nd Necor puber, during prolonged ir exposure nd subsequent recovery: comprtive study. J. Exp. Biol. 21, Fupel, R. P., Seitz, H. J., Trnowsk, W., Thiemnn, V. nd Weiss, C. (1972). Problem of tissue smpling from experimentl nimls with respect to freezing techniques, noxi, stress nd nrcosis new method for smpling rte liver tissue nd physiologicl vlues of glycolytic intermedites nd relted compounds. Arch. Biochem. 148B, Frick, N. T. nd Wright, P. T. (22). Nitrogen metbolism nd excretion in the mngrove killifish Rivulus nrmortus. I. The influence of environmentl slinity nd externl mmoni. J. Exp. Biol. 25, Ip, Y. K., Chew, S. F. nd Rndll, D. J. (21). Ammoni toxicity, tolernce nd excretion. In Fish Physiology, vol. 19 (ed. P. A. Wright nd P. M. Anderson), pp New York: Acdemic Press. Ip, Y. K., Lee, C. Y., Chew, S. F., Low, W. P. nd Peng, K. W. (1993). Differences in the responses of two mudskippers to terrestril exposure. Zool. Sci. 1, Ip, Y. K., Lim, C. B., Chew, S. F., Wilson, J. M. nd Rndll, D. J. (21b). Prtil mino cid ctbolism leding to the formtion of lnine in Periophthlmodon schlosseri (mudskipper): strtegy tht fcilittes the use of mino cids s n energy source during locomotory ctivity on lnd. J. Exp. Biol. 24, Jow, L. Y., Chew, S. F., Lim, C. B., Anderson, P. M. nd Ip, Y. K. (1999). The mrble goby Oxyeleotris mrmortus ctivtes heptic glutmine synthetse nd detoxifies mmoni to glutmine during ir exposure. J. Exp. Biol. 22, Kun, E. nd Kerney, E. B. (1974). Ammoni. In Methods of Enzymtic Anlysis, vol. IV (ed. H. U. Bergmeyer nd K. Gwehn), pp New York: Acdemic Press. Mrcid, G., Felipo, V., Hermenegildo, C., Minn, M. D. nd Grisoli, S. (1992). Acute mmoni toxicity is medited by NMDA type of glutmte receptors. FEBS Lett. 296, McMhon, B. R. nd Burggren, W. W. (1987). Respirtory physiology of intestinl ir brething in the teleost fish M. nguillicudtus. J. Exp. Biol. 133, Mommsen, T. P. nd Wlsh, P. J. (1992). Biochemicl nd environmentl perspectives on nitrogen metbolism in fishes. Experienti 48, Peng, K. W., Chew, S. F., Lim, C. B., Kuh, S. S. L., Kok, W. K. nd Ip, Y. K. (1998). The mudskippers Periophthlmodon schlosseri nd Boleophthlmus bodderti cn tolerte environmentl NH 3 concentrtions of 446 nd 36 µm, respectively. Fish Physiol. Biochem. 19, Rmswmy, M. nd Reddy, T. G. (1983). Ammoni nd ure excretion in three species of ir-brething fish subjected to eril exposure. Proc. Ind. Acd. Sci. 92, Rndll, D. J., Wilson, J. M., Peng, K. W., Kok, T. W. K., Kuh, S. S. L., Chew, S. F., Lm, T. J. nd Ip, Y. K. (1999). The mudskipper, Periophthlmodon schlosseri, ctively trnsports NH + 4 ginst concentrtion grdient. Am. J. Physiol. 46, R1562 R1567. Rndll, D. J., Wood, C. M., Perry, S. F., Bergmn, H. L., Mloiy, G. M. O., Mommsen, T. P. nd Wright, P. A. (1989). Ure excretion s strtegy for survivl in fish living in very lkline environment. Nture 337, Rndll, D. J. nd Wright, P. A. (1987). Ammoni distribution nd excretion in fish. Fish Physiol. Biochem. 3, Sh, N. nd Rth, B. K. (1989). Comprtive study of ureogenesis in freshwter ir-brething teleosts. J. Exp. Zool. 252, 1 8. Vrley, D. G. nd Greenwy, P. (1994). Nitrogenous excretion in the terrestril crnivorous crb Geogrpsus gryi: site nd mechnism of excretion. J. Exp. Biol. 19, Wieser, W. (1972). O/N rtios of terrestril isopods t two tempertures. Comp. Biochem. Physiol. 43A, Wieser, W. (1972b). Oxygen consumption nd mmoni excretion in Ligi beudin M.-E. Comp. Biochem. Physiol. 43A, Wilkie, M. P. (1997). Mechnisms of mmoni excretion cross fish gills. Comp. Biochem. Physiol. 118A, Wood, C. M. (1993). Ammoni nd ure metbolism nd excretion. In The Physiology of Fishes (ed. D. H. Evns), pp Boc Rton: Clevelnd Rubber Compny Press. Wright, J. C. nd O Donnell, M. J. (1993). Totl mmoni concentrtion nd ph of hemolymph, pleon fluid nd mxillry urine in Porcellio scber Lttreille (Isopod, Oniscide): reltionships to mbient humidity nd wter vpour uptke. J. Exp. Biol. 176,