Postprandial increases in nitrogenous excretion and urea synthesis in the giant mudskipper Periophthalmodon schlosseri

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1 The Journl of Experimentl Biology 27, 15-2 Published by The Compny of Biologists 24 doi:1.1242/jeb Postprndil increses in nitrogenous excretion nd ure synthesis in the gint mudskipper Periophthlmodon schlosseri Yuen K. Ip 1, *, Chit K. Lim 1, Serene L. M. Lee 1, Wi P. Wong 1 nd Shit F. Chew 2 1 Deprtment of Biologicl Science, Ntionl University of Singpore, Kent Ridge, Singpore 11754, Republic of Singpore nd 2 Nturl Sciences, Ntionl Institute of Eduction, Nnyng Technologicl University, 1 Nnyng Wlk, Singpore 71, Republic of Singpore *Author for correspondence (e-mil: dbsipyk@nus.edu.sg) Accepted 1 June 24 The objective of this study ws to determine the effects of feeding on the excretory nitrogen (N) metbolism of the gint mudskipper, Periophthlmodon schlosseri, with specil emphsis on the role of ure synthesis in mmoni detoxifiction. The mmoni nd ure excretion rtes of P. schlosseri incresed 1.7- nd 1.92-fold, respectively, within the first h fter feeding on guppies. Simultneously, there were significnt decreses in mmoni levels in the plsm nd the brin, nd in ure contents in the muscle nd liver, of P. schlosseri t h post-feeding. Thus, it cn be concluded tht P. schlosseri ws cpble of unloding mmoni originlly present in some of its tissues in nticiption of mmoni relesed from the ctbolism of excess mino cids fter feeding. Subsequently, there were significnt increses in ure content in the muscle, liver nd plsm (1.9-, nd 1.2-fold, respectively) t h post-feeding, nd the rte of ure synthesis pprently incresed 5.8-fold between h nd h. Incresed ure synthesis might hve occurred in the liver of P. schlosseri becuse the gretest increse in ure content ws observed therein. The excess ure ccumulted in the body t h ws completely excreted between nd 12 h, nd the percentge of wste- Summry N excreted s ure-n incresed significntly to 2% during this period, but never exceeded 5%, the criterion for ureotely, mening tht P. schlosseri remined mmonotelic fter feeding. By 24 h, 2.7% of the N ingested by P. schlosseri ws excreted, out of which 22.% ws excreted s ure-n. This is the first report on the involvement of incresed ure synthesis nd excretion in defense ginst mmoni toxicity in the gint mudskipper, nd our results suggest tht n mple supply of energy resources, e.g. fter feeding, is prerequisite for the induction of ure synthesis. Together, increses in nitrogenous excretion nd ure synthesis fter feeding effectively prevented postprndil surge of mmoni in the plsm of P. schlosseri s reported previously for other fish species. Consequently, contrry to previous reports, there were significnt decreses in the mmoni content of the brin of P. schlosseri throughout the 24 h period postfeeding, ccompnied by significnt decrese in brin glutmine content between 12 h nd 24 h. Key words: mino cids, mmoni, mmoni excretion, feeding, glutmine, mudskipper, nitrogen metbolism, ornithine ure cycle, Periophthlmodon schlosseri, ure. Introduction When confronted with eril exposure, tropicl ir-brething fishes would be expected to hve difficulties excreting mmoni, resulting in n ccumultion of toxic mmoni in the body. In the mid-19s, there ws concerted effort to extend the known principles of mmoni detoxifiction by ure synthesis in Africn lungfishes nd mphibins to these irbrething fishes (Grhm, 1997). Mlcolm Gordon nd coworkers (Gordon et l., 198, 199; Gordon, 197) were the first to investigte if induction of ure synthesis occurred in mudskippers during eril exposure. Mudskippers (Periophthlmus spp., Boleophthlmus spp., Scrtelos spp. nd Periopthlmodon spp.) re euryhline nd mphibious gobioid teleosts (Order: Perciformes, nd Fmily Gobiide) usully found in mngrove swmps nd esturies. They re highly dptble to different environmentl conditions (Clyton, 199; Chew et l., 24). The mud deposited by the river t the estury forms suitble hbitt for these mudskippers to thrive nd build their burrows. During the breeding seson, the femle lys eggs inside the burrow, nd the mle stys therein to tke cre of the developing embryos. The gint mudskipper Periophthlmodon schlosseri cn be found on muddy shores in esturies nd in the tidl zone of rivers in Singpore (Ip et l., 199), Indonesi, New Guine, Indi, Peninsulr Mlysi, Srwk nd Thilnd (Murdy, 1989). It is crnivorous nd cn grow up to 27 cm in length. Periophthlmodon schlosseri is the only species of mudskipper tht hs not been found outside the tropics. It cn survive eril

2 1 Y. K. Ip nd others exposure much better thn other mudskippers (Ip et l., 199; Kok et l., 1998), in prt due to its specilized gill morphology nd morphometry (Low et l., 1988, 199; Wilson et l., 1999, 2). While other mudskipper species build burrow on soft mud nd dispper into the burrow twice dy during high tides, P. schlosseri builds its burrow on high ground nd usully swims long the wter s edge when the tide is high. Gordon et l. (199, 1978) reported tht when the mudskipper Periophthlmus sorbinus ws exposed to terrestril conditions for 12 h, ure production incresed more thn threefold. However, Gregory (1977) could not detect ctivity of some ornithine ure cycle (OUC) enzymes, including crbmoyl phosphte synthetse (CPS), rgininosuccinte synthetse nd rgininosuccinte lyse from the liver of Periophthlmus expeditionium, Periophthlmus grcilis, nd Scrtelos histophorous. It ws therefore concluded tht ure ws produced in livers of these mudskippers through uricolysis, involving urte oxidse, llntoinse nd llntoicse. The ctivities of rginse nd urte oxidse in their livers re high enough to ccount for the rte of ure excretion (Gregory, 1977). Working on the mudskippers Periophthlmus modestus (s P. cntonensis) nd Boleophthlmus pectinitrostris, Morii (1979) nd Morii et l. (1978, 1979) reported tht mmoni ws not detoxified to ure in these mudskippers during eril exposure. Iwt et l. (1981) nd Iwt (1988) lso reported tht ure production remined unchnged in P. modestus exposed to environmentl mmoni or terrestril conditions. When P. modestus ws exposed to 15 N-lbelled mmoni, ure-n ws only slightly lbelled (Iwt nd Deguichi, 1995). Recently, Lim et l. (21) confirmed tht no N-cetylglutmte ctivted CPS ctivity could be detected (detection limit=.1 µmol min 1 g 1 ) from the liver mitochondri of Boleophthlmus bodderti. Tking ll these results together, it cn be concluded tht ure synthesis de novo my not occur in Periophthlmus spp., Scrtelos spp. or Boleophthlmus spp. To dte, the only mudskipper tht possesses full complement of heptic OUC enzymes, in spite of uncertinty on the type of mitochondril CPS present, is the gint mudskipper P. schlosseri (Lim et l., 21). However, similr to other mudskipper species, detoxifiction of mmoni to ure does not occur in P. schlosseri confronted with dverse environmentl conditions such s eril exposure (Ip et l., 199; Lim et l., 21), lkline environmentl ph (Chew et l., 2) nd environmentl mmoni (Peng et l., 1998; Rndll et l., 1999). Insted, P. schlosseri dopts other strtegies to defend ginst mmoni toxicity (Ip et l., 21, in press; Rndll et l., 24; Chew et l., 24). It is cpble of ctively excreting NH + 4 ginst n mmoni concentrtion (Rndll et l., 1999; Ip et l., 24) or in medium with lkline ph (Chew et l., 2), mnipulting the ph of the externl environment (Chew et l., 2; Ip et l., 24), nd ltering the phospholipid composition of its skin to reduce the influx of NH during environmentl mmoni exposure (Ip et l., in press). In ddition, it cn detoxify mmoni to glutmine when exposed to high concentrtions of environmentl mmoni (Peng et l., 1998), nd reduce mmoni production nd undergo prtil mino cid ctbolism during eril exposure (Ip et l., 21b, Lim et l., 21). With the development of ll these mechnisms, it remins n enigm s to why there is still the need to express the OUC in the liver of dult P. schlosseri. Although the expression of OUC is known to occur in fish embryos (Depeche et l., 1979; Wright, 1995; Chdwick nd Wright, 1999; Terjesen et l., 2), the presence of functionl OUC in the liver of dult teleosts is rre, except for the Lke Mgdi tilpi Alcolpi grhmi (Rndll et l., 1989), the gulf todfish Opsnus bet (Mommsen nd Wlsh, 1989; Anderson nd Wlsh, 1995) nd certin ctfishes (Heteropneustes fossilis nd Clris btrchus) from Indi (Sh nd Rth, 1994; Sh et l., 1997, 1999; for contrry view on C. btrchus, see Ip et l., 24b; Chew et l., 24). Being the only mudskipper tht is crnivorous (other species re either herbivorous or omnivorous), we suspected tht the presence of the OUC in P. schlosseri could be relted to its high protein diet (mngrove crbs nd smll fishes), nd is involved in the fish s defence ginst postprndil mmoni toxicity. A postprndil surge in plsm mmoni level is known to occur in severl fish species (Kushik nd Teles, 1985; Wicks nd Rndll, 22). Therefore, this study ws undertken to determine the effects in P. schlosseri of feeding on nitrogen (N) excretion nd metbolism, with specil emphsis on the role of ure synthesis in mmoni detoxifiction. The hypothesis tested ws tht feeding would induce incresed ure synthesis nd ure excretion in this mudskipper. Mterils nd methods Animls Periophthlmodon schlosseri Plls ( 11 g body mss) were cptured t Pontin, Mlysi, nd trnsferred to Singpore. They were mintined in plstic quri in 5% (15 slinity) sewter t 25 C in the lbortory, nd the sewter ws chnged dily. No ttempt ws mde to seprte the sexes. The fish were cclimted to lbortory conditions for 1 week. During the dpttion period, P. schlosseri were fed smll guppies (Poecili reticult). Food ws withdrwn 9 h prior to experiments, which gve sufficient time for the gut to be emptied nd high probbility tht feeding would then occur. All experiments were performed under 12 h:12 h drk:light regime. Feed nlysis The wet mss of guppies ws obtined to the nerest mg. Smples of guppies were then freeze-dried nd the dry mss recorded. Subsequently, they were nlyzed for nitrogen (N) nd crbon (C) using Eurovector EA11 Elementl Anlyzer (Miln, Itly) equipped with the Cllidus softwre. BBOT (C 2 H 2 N 2 O 2 S) stndrd obtined from Eurovector ws used s stndrd for comprison. In ddition some smples were extrcted in 7% ethnol for 24 h to remove non-protein

3 Feeding nd nitrogen metbolism in P. schlosseri 17 N-compounds, before freeze-drying for nitrogen nd crbon nlyses. The difference in vlues between smples with nd without ethnol extrction reveled the combined contribution of mmoni, ure, free mino cids (FAAs), purines nd pyrimidines to the N nd C contents of the guppy. To determine the content of mmoni, ure, FAAs nd protein-bound mino cids (PAAs) in guppies, smples were weighed, ground to powder in liquid nitrogen, nd homogenized using n Ultr-Turrx homogenizer (Jnke nd Kundel, Stufeni, Stufen, Germny) in 5 volumes (w/v) of % trichlorocetic cid (TCA) t 24 r.p.m. three times for 2 s ech, with 1 s intervl between ech homogeniztion. The homogente ws centrifuged t 1 g t 4 C for 15 min to obtin the superntnt nd precipitted proteins. The ph of the superntnt ws djusted to 5.5. with 2 mol l 1 KHCO. Ammoni ws ssyed using the method of Bergmeyer nd Beutler (1985). Ure ws determined colorimetriclly by the method of Jow et l. (1999). For FAA nlysis, the superntnt obtined ws djusted to ph 2.2 with 4 mol l 1 lithium hydroxide nd diluted ppropritely with.2 mol l 1 lithium citrte buffer (ph 2.2). FAAs were nlyzed using Shimdzu LC-A mino cid nlysis system (Kyoto, Jpn) with Shim-pck ISC-7/S154 Li-type column (Kyoto, Jpn). The precipitted proteins were hydrolyzed with 4 mol l 1 methnesulfonic cid contining.2% -(2-minoethyl)indole (Pierce, Rockford, IL, USA) under vcuum in Pierce hydrolysis tubes t 115 C for 22 h by the method of Simpson et l. (197). The hydrolyste ws centrifuged, djusted to ph 2.2 with 4 mol l 1 lithium hydroxide nd diluted ppropritely with.2 mol l 1 lithium citrte buffer (ph 2.2) for nlysis by the Shimdzu LC-A mino cid nlysis system. Despite performing complete FAA nd PAA nlyses on the guppy smples, only the contents (µmol g 1 wet mss) of free nd protein-bound rginine, totl FAA nd totl PAA re presented here. Feeding the nimls Specimens were divided into two groups. The first group of control fish ws not fed while the second group of experimentl fish ws llowed to feed on guppies to 1.5% of their body mss d libitum. The experiment ws considered to commence (time = h) when the fish stopped feeding upon stition. The fed fish nd control fish were gently trnsferred to individul tnks contining 8 ml of 5% sewter. The ctul mss of feed consumed by the fish ws then clculted by subtrcting the mss of ny leftover food from the initil mss of food given to the fish. Collection of wter, tissue smples nd feed for nlyses Wter smples ( ml) were collected t h intervls during the subsequent 24 h period post-feeding, cidified with 7 µl of 1 mol l 1 HCl, nd kept t 4 C until nlysis. At,,, 12 nd 24 h, fish were killed by strong blow to the hed, nd the lterl muscle, liver, gut nd brin quickly excised. The gut ws removed, flushed well with wter, nd divided into two hlves longitudinlly. The excised tissues nd orgns (<1 g) were immeditely freeze-clmped in liquid nitrogen using precooled tongs (Fupel et l., 1972). Frozen smples were kept t 8 C until nlysis. Blood smples were collected from the severed cudl peduncle into heprinized cpillry tubes, nd centrifuged t 5 g nd 4 C for 5 min to obtin the plsm. The plsm ws deproteinized by the ddition of n equl volume (v/v) of ice-cold % TCA nd centrifuged t 1 g nd 4 C for 15 min. The resulting superntnt ws kept t 25 C until nlysis. The frozen smples were weighed, ground to powder in liquid nitrogen, nd homogenized using the Ultr-Turrx homogenizer in 5 volumes (w/v) of ice-cold % perchloric cid t 24 r.p.m. three times, 2 s ech, 1 s intervl between ech homogeniztion. The homogente ws centrifuged t 1 g t 4 o C for min, nd the superntnt obtined ws kept t 25 C until nlysis. Determintion of mmoni nd ure concentrtions in wter smples Ammoni in wter smples ws determined by the method of Anderson nd Little (198), nd ure content ws nlyzed s described by Jow et l. (1999). The rtes of mmoni or ure excreted were expressed s µmol N h 1 g 1 wet mss of the fish. Determintion of mmoni, ure nd glutmine in tissues smples The deproteinized tissue smples were djusted to ph..5 with 2 mol l 1 KHCO. Ammoni nd ure contents were determined using the method of Bergmeyer nd Beutler (1985) nd Jow et l. (1999), respectively. Ure content in the brin ws not determined becuse of the smll size of the brin smple. Glutmine ws determined by the method of Mecke (1985). Results were expressed s µmol g 1 wet mss tissue or µmol ml 1 plsm. Sttisticl nlyses Results re presented s mens ± stndrd errors of the men (S.E.M.). Two-til Student s t-test nd one-wy nlysis of vrince followed by Duncn s multiple-rnge test were used to evlute differences between mens, where pplicble. Arcsine trnsformtion ws pplied to percentge dt before sttisticl nlysis. Differences with P<.5 were regrded s sttisticlly significnt. Results For every 1 g wet mss of guppy (N=), there ws.25 g of freeze-dried mteril, of which 8.82±.17% nd 42.±1.2% of the dry mss were N nd C, respectively. After ethnol extrction, the percentges of dry mss represented by N nd C were 8.48±.25 nd 4.9±2.1, respectively, indicting tht proteins were the mjor contributor of N. The mmoni nd ure contents (µmol g 1 wet mss) of guppies were 2.±.1

4 18 Y. K. Ip nd others Ammoni excreted (µmol h 1 g 1 wet mss) * * 9,b Fig. 1. The mount of mmoni excreted (µmol N h 1 g 1 wet mss fish) by unfed (control; white squres) nd fed (grey squres) Periophthlmodon schlosseri t h intervls during the 24 h period post-feeding. Vlues re mens + S.E.M. (N=4). *Significntly different from the corresponding control vlue, P<.5. Significntly different from h, P<.5; b significntly different from h, P<.5. Ure-N excreted (µmol h 1 g 1 wet mss) * * *, 9 *, 9 12 d b, *, Fig. 2. The mount of ure excreted (µmol N h 1 g 1 wet mss fish) by unfed (control; white squres) nd fed (grey squres) Periophthlmodon schlosseri t h intervls during the 24 h period post-feeding. Vlues re mens + S.E.M. (N= 4). *Significntly different from the corresponding control vlue, P<.5. Significntly different from h, P<.5; b significntly different from h, P<.5; c significntly different from 9 h, P<.5; d significntly different from 9 12 h, P<.5. nd 1.1±.1, respectively. The free nd proteinbound rginine contents were.5±.19 nd 7±2, respectively. The totl PAA content (149±58 µmol g 1 wet mss) ws 4-fold greter thn the totl FAA content (44± µmol g 1 wet mss). The contribution of rginine to the totl FAA nd totl PAA contents were 1.2% nd 4.9%, respectively. The men body mss of P. schlosseri (N=17) nd the wet mss of the guppies ingested were 95.8±.9 g nd.7±. g, respectively. After feeding, significnt increse in the mmoni excretion rte occurred immeditely between nd h. The mmoni excretion rte of the experimentl nimls ws gretest between nd h (.18 µmol N h 1 g 1 wet mss fish), nd ws pproximtely 1.7-fold greter thn the corresponding control vlue (Fig. 1). Overll, the mount of mmoni excreted within the 24 h period for fed fish (19.5 µmol N 24 h 1 g 1 wet mss) ws significntly greter thn the unfed control (1.8 µmol N 24 h 1 g 1 wet mss). The rte of ure excretion lso incresed immeditely fter feeding, nd the increse lsted for 12 h (Fig. 2). The gretest ure excretion rte ws observed t 12 h, reching.714 µmol N h 1 g 1 wet mss, which ws 2.14-fold greter thn the corresponding control vlue (Fig. 2). The ure excretion rtes between 12 nd 21 h were comprble to the corresponding controls, but incresed significntly gin t h postfeeding (Fig. 2). The mount of ure excreted within the 24 h period for fed specimens (.74 µmol N 24 h 1 g 1 wet mss) ws significntly greter thn in the unfed control (2.8 µmol N 24 h 1 g 1 wet mss). Specificlly between nd 12 h, the mount of totl-n excreted s ure-n incresed to 2%, which ws pproximtely 1.-fold greter thn the corresponding control vlue (Fig. ). Ammoni content of the muscle remined reltively unchnged during the 24 h period postfeeding (Fig. 4). In contrst, there ws 2.2-fold increse in mmoni content in the liver t h (Fig. 4), nd slight but significnt increse in mmoni content in the gut t h nd h (Fig. 4). In the brin, there were significnt decreses in the mmoni content throughout the 24 h period postfeeding (Fig. 4). The mmoni concentrtion in the plsm decresed significntly t h nd returned to the norml level therefter (Fig. 4). The ure contents of the muscle nd liver of P. schlosseri t h post-feeding were significntly lower thn the corresponding h control vlue, but by h hd incresed significntly (Fig. 5). There ws lso significnt increse in plsm ure

5 Feeding nd nitrogen metbolism in P. schlosseri 19 *,,b *,,b Nitrogen excreted s ure-n (%) Fig.. The percentge of totl-n excreted s ure-n by unfed (control; white squres) nd fed (grey squres) Periophthlmodon schlosseri t h intervls during the 24 h period post-feeding. Vlues re mens + S.E.M. (N=4). *Significntly different from the corresponding control vlue, P<.5. Significntly different from h, P<.5; b significntly different from h, P<.5; c significntly different from 9 h, P<.5; d significntly different from 9 12 h, P< ,b (control) b b,c b,c b 12 b,c b,c b 24 Fig. 4. Ammoni content (µmol g 1 wet mss tissue or µmol ml 1 plsm) in muscle (white brs), liver (grey brs), gut (blck brs), brin (htched brs) nd plsm (crosshtched brs) of Periophthlmodon schlosseri during the 24 h period post-feeding. Vlues re mens + S.E.M. (N= 4), except for brin nd plsm (N=). Significntly different from h control, P<.5; b significntly different from h vlue, P<.5; c significntly different from h vlue, P< ,b ,b 1..5 (control),b b b,c c 12 c c 24 Fig. 5. Ure content (µmol g 1 wet mss tissue or µmol ml 1 plsm) in muscle (white brs), liver (grey brs), gut (blck brs) nd plsm (htched brs) of Periophthlmodon schlosseri during the 24 h period post-feeding. Vlues re mens + S.E.M. (N= ), except for gut (N=4). Significntly different from h control, P<.5; b significntly different from h vlue, P<.5; c significntly different from h vlue, P<.5.

6 2 Y. K. Ip nd others concentrtion in fed fish t h (Fig. 5). By contrst, the ure content of the gut remined reltively constnt throughout the 24 h period post-feeding (Fig. 5). Feeding hd no significnt effect on the glutmine content in muscle of P. schlosseri, but the glutmine content in the liver significntly decresed t h nd h. In ddition, brin glutmine content significntly decresed in the fed fish t 12 h nd 24 h post-feeding (Fig. ). By contrst, there ws significnt increse in the glutmine content in the gut t h post-feeding (Fig. ). Discussion Feeding induced instntneous increses in mmoni nd ure excretion in P. schlosseri After consumption of protein-contining mel, FAAs produced by the ctions of proteses in the limentry trct nd peptidses in the intestinl mucosl cells (Mthews, 1975) enter the circultion. The mjority of these mino cids, in excess of wht is required for protein synthesis, re ctbolized in the liver (Cmpbell, 1991), relesing mmoni nd resulting in momentrily increse in plsm mmoni level in fish (Kushik nd Teles, 1985; Wicks nd Rndll, 22), ssocited with chnges in mmoni excretion in fish during the postprndil period; the rte of mmoni excretion usully increses between 2 h nd 11 h post-feeding (vn Weerd et l., 1995; Dosdt et l., 199; Gelineu et l., 1998). In rinbow trout Onchorhynchus mykiss, the plsm mmoni concentrtion increses to 7 µg ml 1 or 2.7 µmol ml 1 8 h fter feeding (Wicks nd Rndll, 22). By contrst, the rtes of mmoni nd ure excretion in the gint mudskipper incresed by 1.7- nd 1.92-fold, respectively, within the first h post-feeding. In fct, the gretest rte of mmoni excretion ws observed t h nd returned bck to norml t 9 h. Since there were significnt decreses in mmoni levels in plsm nd brin of P. schlosseri t h post-feeding, these results indicte tht P. schlosseri hd unloded the mmoni originlly present in some of its tissues immeditely fter feeding (probbly initited by the feeding ction involved), in nticiption of mmoni being relesed by ctbolism of excess mino cids. In ddition, there ws 1.4-fold increse in mmoni excretion over the 24 h period, with the mjority excreted between 12 h nd 21 h. This unique pttern of mmoni excretion is prt of novel phenomenon: unlike in other fish species (Kushik nd Teles, 1985; Wicks nd Rndll, 22), there ws no postprndil surge in mmoni concentrtion in the plsm of P. schlosseri during the 24 h post-feeding period. Other fctors contributing to this novel phenomenon were incresed synthesis nd excretion of ure (see below) in P. schlosseri fter feeding (control) b b A significnt increse in the rte of ure excretion in P. schlosseri lso occurred immeditely fter feeding, nd lsted for 12 h. At h post-feeding, the ure contents in the muscle nd liver decresed by.% nd 74.7%, respectively. Tken together, these results indicte tht feeding induced n instntneous increse in the rte of nitrogenous excretion (mmoni + ure) in P. schlosseri. A hypotheticl P. schlosseri weighing 7 g would hve consumed.5 g of guppies or.84 mmol of N. By 24 h postfeeding, totl of.52 mmol (clculted from Figs 1 nd 2) or 2.7% of the ingested N would hve been excreted by this 7 g fish. Out of this.52 mmol N excreted, 22.% (.11 mmol) would be ure-n, wheres in control (unfed) fish only 1.1% of the wste-n would be excreted s ure- N. The percentge of ure-n ctully excreted incresed to 2% between nd 12 h in the fed fish, nd since there were significnt increses in ure content in the muscle nd liver t h fter the initil unloding of ure t h, it cn be deduced tht n increse in ure production hd occurred in P. schlosseri between h nd h postfeeding. Incresed ure synthesis in P. schlosseri fter feeding The ure excretion rte incresed pproximtely twofold in P. schlosseri t 12 h post-feeding. In ddition, the ure contents of muscle, liver nd plsm incresed 1.9-, nd 1.2- fold, respectively, t h, before returning bck to control vlues t 12 h. These results suggest tht ure production incresed in P. schlosseri fter feeding. Furthermore, it is pprent tht the incresed rte of ure 12 b,b,c 24 b,b,c Fig.. Glutmine contents (µmol g 1 wet mss tissue) in the muscle (white brs), liver (grey brs), gut (blck brs) nd brin (htched brs) of the gint mudskipper, Periophthlmodon schlosseri during the subsequent 24 h period post-feeding. Vlues re mens + S.E.M. (N=4), except for the brin (N=). Significntly different from h control, P<.5; b significntly different from h vlue, P<.5; c significntly different from h vlue, P<.5.

7 Feeding nd nitrogen metbolism in P. schlosseri 21 production must hve been greter thn tht of ure excretion, in order for ure to be ccumulted in the tissues of the fed fish. To mintin the concentrtion of ure in the body of the control niml t stedy stte, the rte of ure excretion must blnce the rte of ure production. This implies tht the hourly rte of ure production in P. schlosseri t h ws. µmol h 1 g 1 [.21 ure-n/( 2 N); from Fig. 2], or 2.52 µmol h 1 for hypotheticl 7 g fish. Upon feeding, 7 g fish would hve excreted 2.1 µmol ure between h nd h (.57 ure-n g 1 7 g/2 N; from Fig. 2). Bsed on vlues of 42 g muscle, 2 g liver nd 2 ml plsm in 7 g fish (Lim et l., 21), the excess mount of ure ccumulted between h nd h cn be clculted s (.47 µmol g 1 42 g) + (1.18 µmol g 1 2 g) + (.9 µmol ml 1 2 ml) or 2.9 µmol 7 g 1 fish (from Fig. 5). Thus, the mount of ure produced by 7 g fish during this h period post-feeding is equl to the sum of the mount excreted nd the mount ccumulted in the body, which is or 44 µmol. The hourly rte of ure production in 7 g specimen between h nd h postfeeding is therefore 44 µmol h 1 or 14.7 µmol h 1, which mens tht the ure production rte incresed 5.8-fold (=14.7 µmol h 1 vs µmol h 1 ), with the production of.5 µmol ure in excess, within this h period. Ure cn be produced vi uricolysis, rgininolysis or the OUC (Cmpbell, 197), but only production vi the OUC cn be regrded s synthetic process. Uric cid cn be produced vi purine ctbolism; however, purine, together with other ethnol-extrctble nitrogenous compounds, only hd minor contribution to the totl-n in guppies (.2% of dry mss, or.2 µmol N per.5 g guppies, of which t lest 2. µmol N ws contributed by FAA). Therefore, degrdtion of purine from ingested guppies pprently could not ccount for the mount of 58.1 µmol ure produced during the 24 h period post-feeding (see bove). The mounts of rginine present s FAA nd PAA in.5 g of guppies consumed by 7 g P. schlosseri were.28 nd 8.7 µmol, respectively. Since one mole of rginine gives rise to one mole of ure, t most 9 µmol of ure could be produced through rgininolysis, bsed on the highly unlikely ssumption tht both free nd protein-bound rginine were selectively nd completely ctbolized in preference to other mino cids. Even then, this (9 µmol) could ccount for only 7% of the 58.1 µmol ure produced during the 24 h period. Therefore, it cn be concluded tht mjor portion of the ure produced by P. schlosseri fter feeding ws ctully synthesized de novo vi the OUC. Ure synthesis is likely to hve occurred in the liver becuse the gretest increse in ure content ws observed therein. Since there is 2 g of liver in 7 g fish, the rte of ure synthesis in the liver between h nd h post-feeding is equl to 14.7 µmol h 1 /( min 2 g) or.12 µmol min 1 g 1, which is close to the highest CPS ctivities reported for the liver of P. schlosseri (.117 µmol min 1 g 1 ; Lim et l., 21). Attempts hd been mde previously to elucidte the mechnisms dopted by P. schlosseri to meliorte mmoni toxicity during exposure to terrestril conditions (Ip et l., 199, 21b; Lim et l., 21), lkline environmentl ph (Chew et l., 2) or environmentl mmoni (Peng et l., 1998; Rndll et l., 1999; Ip et l., 24). Despite possessing ll the enzymes for ure synthesis de novo in its liver, P. schlosseri is pprently incpble of detoxifying mmoni to ure when exposed to these experimentl conditions. Thus, our results represent the first report on the involvement of incresed ure synthesis nd excretion in the defense ginst postprndil mmoni toxicity in the gint mudskipper. Since ll previous studies (Peng et l., 1998; Rndll et l., 1999; Chew et l., 2; Ip et l., 199, 21b; Lim et l., 21) on P. schlosseri were performed using fsted specimens, our results suggest tht, perhps, n mple supply of energy resources, e.g. fter feeding, is prerequisite for the induction of ure synthesis. P. schlosseri cn survive eril exposure much better thn other species of mudskippers (Ip et l., 199; Kok et l., 1998) prtly becuse of its specilized gill morphology nd morphometry (Low et l., 1988, 199; Wilson et l., 1999, 2); but fusions of secondry lmelle would impose inefficiency in the brnchil excretion of mmoni in wter. Therefore, in ddition to n increse in the rte of mmoni excretion fter feeding, incresed ure synthesis is essentil in preventing postprndil surge of mmoni. However, P. schlosseri remined mmonotelic throughout the 24 h period post-feeding. With the excess ure ccumulted in the body t h being completely excreted between nd 12 h, the percentge of wste-n excreted s ure-n incresed significntly to 2%, but never exceeded 5%, the criterion for ureotely. In this respect, P. schlosseri is different from the mmonotelic, but ureogenic, slender Africn lungfish Protopterus dolloi, which becomes ureotelic fter feeding (Lim et l., in press). Ammoni nd glutmine contents in brin of P. schlosseri decresed significntly fter feeding The mechnisms involved in defense ginst postprndil mmoni toxicity in P. schlosseri were so effective tht the mmoni level in the brin decresed throughout the 24 h period. Consequently, P. schlosseri exhibited phenomenon different from other fishes with respect to the response of brin glutmine content to feeding. It is well known tht fish brins re protected from mmoni toxicity by glutmine synthetse (Mommsen nd Wlsh, 1991), nd tht mmoni levels in the fish brin increses significntly fter feeding, leding to incresed glutmine synthesis nd its ccumultion therein (Wicks nd Rndll, 22; Lim et l., in press). By contrst, feeding led to significnt decrese in the brin glutmine level of P. schlosseri between 12 h nd 24 h. This observtion is consistent with the fct tht there ws n bsence of ny postprndil mmoni surge in the plsm throughout the 24 h period post-feeding.

8 22 Y. K. Ip nd others Conclusion The rtes of mmoni nd ure excretion incresed significntly in P. schlosseri fter feeding. It would pper tht P. schlosseri ws cpble of unloding mmoni nd ure from its body (over h period post-feeding) in nticiption of n increse in mmoni production from ctbolism of excess mino cid. In ddition, the rte of ure synthesis incresed 5.8-fold between h nd h. These dpttions effectively prevented postprndil surge of mmoni in P. schlosseri, s hs been reported previously for other fish species. Consequently, there were significnt decreses in the mmoni content in the brin of P. schlosseri throughout the 24 h period post-feeding. In ddition, unlike in other fish species, the brin glutmine content decresed significntly fter feeding (12 24 h). Similr to P. schlosseri, certin dult teleosts such s the lrgemouth bss Micropterus slmoides nd the plinfin midshipmn Porichthys nottus re known to be ureogenic (for review, see Anderson, 21), lthough it hs been suggested tht the OUC nd CPS in these mmonotelic fishes my not hve significnt physiologiclly function (Anderson, 21). Perhps future work should im to elucidte if ure synthesis de novo plys n importnt role in defense ginst postprndil mmoni toxicity in these ureogenic fishes s reported herein for the gint mudskipper P. schlosseri. References Anderson, P. M. (21). Ure nd glutmine synthesis: Environmentl influences on nitrogen excretion. In Fish Physiology vol. 2, Nitrogen Excretion (ed. P. A. Wright nd P. M. Anderson), pp New York: Acdemic Press. Anderson, P. M. nd Little, R. M. (198). Kinetic properties of cynse. Biochemistry 25, Anderson, P. M. nd Wlsh, P. J. (1995). 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