Corrigendum. The correct sentence should read: to the reversed seasonal temperatures (15 C in winter, 5 C in summer).

Transcription

1 Corrigendum Dy, N. nd Butler, P. J. (25). The effets of limtion to reversed sesonl tempertures on the swimming performne of dult rown trout Slmo trutt. J. Exp. Biol. 28, On pge 2686 n error ppered in the seond sentene of the seond prgrph of the Results setion in oth the on-line nd print versions of this pper, whih reds: However in oth winter nd summer, the Urits of these groups were signifintly higher thn those for the groups limted to the reversed sesonl tempertures (5 C in winter, 15 C in summer). The orret sentene should red: However in oth winter nd summer, the Urits of these groups were signifintly higher thn those for the groups limted to the reversed sesonl tempertures (15 C in winter, 5 C in summer). The uthors pologise for ny inonveniene used.

2 The Journl of Experimentl Biology 28, Pulished y The Compny of Biologists 25 doi:1.1242/je The effets of limtion to reversed sesonl tempertures on the swimming performne of dult rown trout Slmo trutt N. Dy* nd P. J. Butler Shool of Biosienes, The University of Birminghm, Edgston, Birminghm B15 2TT, UK *Author for orrespondene (e-mil: n.dy@hm..uk) Aepted 4 My 25 Adult rown trout (Slmo trutt) were limtised to nd mintined t sesonl tempertures (5 C in winter; 15 C in summer) nd limted to reversed sesonl tempertures (15 C in winter; 5 C in summer) while exposed to the nturl (i.e. sesonlly vrile) photoperiod. The men ritil swimming speeds (U rit ) of nimls limtised to the sesonl tempertures were similr, ut more thn 3% greter thn those for fish limted to the reversed sesonl tempertures. The lower vlues of U rit tht ompnied limtion to reversed sesonl tempertures ppered lrgely to result from the inility of white musle to funtion mximlly, sine the onentrtions of ltte nd mmoni in white musle of fish swum to U rit t reversed sesonl Summry tempertures were signifintly lower thn those in fish swum t sesonl tempertures. These oservtions, together with iohemil nd morphometri ttriutes of musle tissue, suggest tht swimming ility is influened, t lest in prt, y sesonl ftors other thn temperture. These dt hve importnt implitions for the design of experiments using fish tht experiene preditle, usully sesonl, hnges in their nturl environment (temperture, dissolved oxygen, hnges in wter levels, et.). Key words: rown trout, Slmo trutt, swimming, morphometry, temperture. Introdution Critil swimming speed (U rit ; Brett, 1964) is n inditor of swimming pity nd n therefore reflet the physiologil sttus of tive fish (see Plut, 21). In some speies, U rit is redued when fish re suddenly exposed to tempertures tht re signifintly higher or lower thn their limtion temperture (Rndll nd Bruner, 1991), while other speies re more tolernt (Jones nd Sidell, 1982). However, with full limtion, swimming performne n e improved t lower tempertures, s is the se for the goldfish, Crssius urtus (Rome et l., 1985), ut often remins lower thn tht oserved in individuls of the sme speies tht re limted to higher temperture. At lower tempertures, the metoli rte is redued nd this my redue loomotory pity, due to deresed ontrtile rtes in oth the red nd white swimming musles nd the hert (Vornnen, 1994). As ompenstory response to exposure to low environmentl temperture, iohemil nd morphologil hnges our in the slow oxidtive (red) musle fires in vriety of speies inluding slmonids. These hnges inlude inreses in the reltive proportion nd in the pillry density of these fires, inreses in oth mitohondril densities nd mitohondril riste surfe densities (nd hene eroi enzyme tivity), dereses in the length of the diffusion pth etween the sroplsmi nd mitohondril omprtments, nd hnges in the proportions of musle enzymti nd myosin hevy hin isoforms (see Johnston, 1982; Jones nd Sidell, 1982; Blier nd Guderley, 1988; Egginton nd Sidell, 1989; Londrville nd Sidell, 1996; Cordiner nd Egginton, 1997; Egginton nd Cordiner, 1997; Guderley nd St-Pierre, 22; Wte, 22). In ddition, limtion to lower tempertures is often ompnied y n inrese in the lipid ontent of eroi musle, whih my enhne oxygen trnsport s well s ting s n intrellulr oxygen store (Hoofd nd Egginton, 1997). In most studies where swimming performne hs een investigted, the wter tempertures nd photoperiods under whih experimentl nimls hve een mintined hve een quoted. However, few hve mentioned t wht time of the yer suh experiments hve een performed or whether wter qulity (ph, dissolved ions, et.) ws mintined onstnt throughout the experimentl period. Also, it is rrely indited whether the experiments were rried out t sesonlly pproprite tempertures nd photoperiods. Previous studies (Butler et l., 1992; Beumont et l., 1995; Dy nd Butler, 1996) hve reveled tht dult rown trout, Slmo trutt, n mintin their swimming performne (s determined y U rit ) independently of the sesonl temperture to whih they were limtised (5 C in winter; 15 C in summer). In these experiments, no ttempt ws mde to ontrol

3 2684 N. Dy nd P. J. Butler photoperiod, so tht ll nimls were lso exposed to the nturl (i.e. sesonl) light/drk yle. Similr results were otined for white rppie (Pomonis nnulris) limted to three different tempertures nd exposed to five different photoperiods (Smiley nd Prsons, 1997). To dte, this ltter study, together with tht rried out y Kolok (1991) on lrgemouth ss, Miropterus slmoides, pper to e the only ones where the determintion of U rit hs een performed under onditions where oth photoperiod nd environmentl temperture hve een mnipulted. However, there hve een other studies on the effets of seson nd therml limtion on the loomotory pprtus. For exmple, Guderley et l. (21) demonstrted the effets of these ftors on the speed of loomotion of the three-spine stiklek (Gsterosteus uletus) during experimentlly eliited strtle responses, nd Kilrski et l. (1996) demonstrted the effets of seson on short-term therml limtion nd on hnges in the inner mitohondril memrnes of oxidtive ( red ) skeletl musle of ruin rp (Crssius rssius). The primry purpose of the present study ws to see whether the ility of rown trout to mintin swimming performne t 5 C or 15 C ws independent of seson while other environmentl vriles (wter qulity, stoking density nd the vilility of food) were mintined onstnt nd the nimls exposed to the nturl (i.e. sesonlly hnging) photoperiod (Butler et l., 1992; Butler nd Dy, 1993; Dy nd Butler, 1996). Briefly, fish were limtised to oth sesonl tempertures (5 C in winter, 15 C in summer) or limted to reversed sesonl tempertures (15 C in winter, 5 C in summer) in moving wter nd then swum in vrile-speed wter hnnel up to their U rit. Tissue smples were susequently tken for iohemil nd morphometri nlysis to determine if ny oservle differenes in dt otined from these nlyses ould e relted to ny differenes in U rit. A preliminry report of prt of this study ws given in Dy nd Butler (1999). Mterils nd methods Animl husndry Adult (non-reeding) rown trout Slmo trutt L. (mss g; Tle 1) were otined from the Ledmill Trout Frm, Hthersge, Deryshire, UK. As in our previous study (Dy nd Butler, 1996), the fish were initilly pled in lrge (2 m dimeter) irulr glss fire trining tnk (for detils, see Butler et l., 1992) tht ws ontinully supplied (12 l h 1 ) with dehlorinted Birminghm tp wter ([C 2+ ]=13 2 µmol l 1 ). In the middle of this tnk ws smller tnk (dimeter, 1 m; volume 4 l). Together, the two formed.5 m wide.6 m deep hnnel through whih erted wter flowed t pproximtely.25 m s 1. Severl openended plsti tues (internl dimeter, 1 m; length, 55 m) were suspended mid-wter in the hnnel to provide shelter for the fish. They invrily preferred to hide in these ut, to do so, they hd to swim ontinuously ginst the diretion of flow in order to mintin their sttion. The presene of oth tues nd flowing wter ensured tht individuls were suffiiently trined for the susequent swimming experiments. The nimls were left for minimum of two weeks in this tnk. After this initil period, the fish were trnsferred to similr tnk supplied with ontinuous (3 l min 1 ) supply of rtifiil lkewter ([C 2+ ] 25 µmol l 1 ;), whih ws identil to tht used in our previous studies (Butler et l., 1992; Dy nd Butler, 1996). The fish were left in this wter for minimum of further 8 weeks. For nimls tht were mintined t reversed sesonl tempertures, wter tempertures were initilly either inresed or deresed y pproximtely 1. C per dy until the required limtion temperture hd een otined. All fish were exposed to the nturl (i.e. sesonlly vrile) photoperiod, within lortory with lrge windows, nd fed twie dily to stiety on floting food pellets [Minstrem trout diet, B.P. Nutrition (UK) Ltd]. Experimentl design Dt were otined from four groups of 12 resting fish nd four omprle groups of fish tht were swum up to their U rit. Eh group ws limtised to one of the sesonl tempertures (5 C in winter, 15 C in summer) or limted to one of the reversed sesonl tempertures (15 C in winter nd 5 C in summer) (Tle 1). During the winter, ll experiments were performed etween mid-novemer nd Jnury, while during the summer, experiments were performed from mid-june to August. These periods orrespond pproximtely to minimum nd mximum dylengths of the plerti sesonl photoperiod nd the minimum nd mximum tempertures experiened y stokfish t the fish frm (Fig. 1). Tle 1. Tretments nd physil hrteristis of rown trout used in the present study Alimtion Totl Mss Gondosomti Condition N Seson temperture ( C) length (mm) (g) index (% ody mss) ftor 12 Winter ± ± ±.9* 1.7± ± ± ± ±.7 12 Summer ± ± ±.8 1.6± ± ±9.6.63±.5* 1.55±.6 Gondosomti index=1 [totl gond mss (g)]/[ody mss (g)]%. Condition ftor=1 [ody mss (g)/[fork length (mm)] 3. *Signifint differene etween groups mintined t sesonl tempertures (P<.5; N=12).

4 Sesonlity nd swimming performne in trout 2685 Dy length (h)/wter temperture ( C) Dy length Men temperture of wter t trout frm Experimentl periods N D J F M A M J J A S O Month Fig. 1. The nnul vrition in dy length nd wter temperture t Ledmill Trout Frm, from where stoks of dult rown trout were otined, nd the periods when ll experiments were performed. Arrows indite strt of the limtion periods, width of olumns indite experimentl periods nd height of olumns indite sesonl tempertures. Experimentl protool nd removl of tissue smples for iohemil nd morphometri studies After limtistion or limtion, nimls tht were to e smpled were pled in vrile-speed, Blzk-type wter hnnel for 6 dys (Butler et l., 1992). Photoperiod nd temperture, qulity nd rte of supply of the wter were identil to those experiened y the nimls during the limtion period. After this experimentl period, eh fish ws either smpled t rest or fter eing swum to its U rit. The ltter ws determined y swimming for 15 min t.2 m s 1 nd then for the sme time t inresing.1 m s 1 inrements until the niml esed to swim (Butler et l., 1992). The fish ws then immeditely overed with sheet of wet fom (whih prevented it from struggling) efore eing removed from the wter hnnel nd killed (y low to the hed followed y destrution of the rin). The fish ws then quikly weighed nd the hert rpidly exised. Red musle, white musle nd liver were immeditely removed, weighed nd freeze-lmped with luminium tongs tht hd een pre-ooled in liquid N 2. Red musle ws removed s thin 5 7 m strip posteriorly from the left flnk nd finishing 3 m from the se of the til fin. White musle ws tken s longitudinl lok of tissue, pproximtely 6 m in length nd 1 m in dimeter, from deep epxil (dorso-lterl) musle in the sme region. All tissue smples tht were to e used for susequent iohemil nlyses were removed, proessed nd stored in liquid N 2 within pproximtely 9 s from the time of deth of the niml. These were lter ground to fine powder under liquid N 2, prior to susequent iohemil nlysis. Determintion of gondosomti indies nd ondition ftors The ondition ftor for eh fish ws determined nd the gonds removed nd weighed for the determintion of the gondosomti index (Anderson nd Gutreuter, 1983) (see Tle 1). Musle morphometry The reminder of the red nd white musles from the left flnk of eh niml ws disseted out seprtely nd weighed. The umulted musle msses for oth red musle nd white musle were lulted nd then douled. Preliminry investigtions hd shown tht ontrlterl differenes etween totl msses for oth red nd white musles did not vry y more thn ±3.5%. For detiled morphometri studies, whole loks of tissue ontining oth red musle nd white musle were removed from the right flnk of the niml (within 5 min fter deth) y diretly utting deep through the skin in the region of the lterl line. These were then quikly oted in Tissue-Tek mountnt medium (Gurr) nd frozen in smll (5 ml) plsti eker ontining isopentne tht hd een previously ooled in liquid N 2. Setions of musle (thikness, 1 µm) were ut t 2 C in ryostt (Bright Instruments, UK) nd stined for lkline phosphtse tivity t room temperture y the method of Zid et l. (1984) so tht lood pillries eme visile. These were then exmined under mirosope fitted with mer luid tthment (Crl Zeiss) whih projeted imges onto digitising tlet (GTCO Corportion, Rokville, USA). All imges were proessed using Sigm Sn PC digitising softwre (Jndel Sientifi Cliforni, USA). Men musle fire ross-setionl res, tissue pillry densities, nd the men numer of pillries per musle fire were determined with the id of rndomly pled unised smpling ounting frme (Egginton, 199). Musle iohemistry Phosphofrutokinse (PFK) nd itrte synthse (CS) tivities were ssyed t 15 C in musle smples tken from resting nimls. PFK ws ssyed ording to the method of Su nd Storey (1994) t 34 nm using Shimdzu UV-16A spetrophotometer fitted with CPS24A temperture ontroller (Shimdzu Corp., Jpn). CS tivity ws determined in the sme smples t 412 nm y the method of Srere et l. (1963), s modified y Hnsen nd Sidell (1983). PFK ws extrted y homogenising musle smples on ie (3 15 s ursts t 2 5 r.p.m. with Ultr-Turrx T25 homogeniser) in n extrtion uffer ontining 75 mmol l 1 Tris, 1 mmol l 1 EDTA, 2 mmol l 1 MgCl 2 nd 2 mmol l 1 DTT (ph 7.4). This medium (minus the DTT) ws lso used to extrt CS. Prior to nlysis, ll smples were lrified y entrifugtion t 3 g for 5 min t 15 C. It should e noted tht preliminry ssys for oth of these enzymes were rried out t 5 C ut, for some reson, the dt otined were highly vrile etween liquots of tissue homogente. There ws no sign of ondenstion t 5 C nd the vriility ws eliminted when liquots of homogenised smples were nlysed t 15 C. All vlues of enzyme tivities re given per unit wet mss. Musle glyogen nd free gluose levels were determined

5 2686 N. Dy nd P. J. Butler y the method of Keppler nd Deker (1974). For determintion of ltte onentrtions, 1 12 mg smple of frozen, powdered tissue ws homogenised with ie-old 1 mol l 1 perhlori id (dilution ftor 1:5, mss:volume). After entrifugtion t 8 g for 1 min, the superntnt ws neutrlised with 2 mol l 1 KOH nd then ssyed t 34 nm nd 25 C y the method of Gutmn nd Whlefeld (1974). Totl musle lipid ontent ws determined y the method of Bligh nd Dyer (1959). For totl mmoni onentrtion [Tmm], weighed (pproximtely 1 mg) portions of frozen, powdered red musle nd white musle were homogenised nd deproteinised in ie-old 1 mol l 1 perhlori id (dilution ftor 1:5 mss:volume) nd then entrifuged t 1 g for 2 min to remove preipitted proteins. The superntnt ws neutrlised with 2 mol l 1 KHCO 3 (Kun nd Kerney, 1984) nd then nlysed for totl mmoni ontent ([NH 3 ]+[NH 4+ ]) using the Sigm 171-A dignosti kit (Dy nd Butler, 1996). Sttistil nlyses All dt were nlysed y nlysis of vrine (ANOVA). Between-tretment omprisons were mde using the post-ho Tukey multi-omprison test (Zr, 1984), nd signifine ws tken to e when P<.5. When ny vrile is quoted s eing different from nother, this mens tht the differene is sttistilly signifint. All mens re plotted with their stndrd errors (S.E.M.). Results The nimls The gondosomti indies (GS) for three of the four groups of fish were similr; the GS for fish mintined t 15 C in summer ws greter thn those for fish mintined t 5 C in summer, 5 C in winter nd 15 C in winter. Condition ftor did not differ etween the four groups of nimls (Tle 1). Swimming performne There ws no differene etween the U rit s of the two groups of fish swum t sesonl tempertures (5 C in winter, 15 C in summer; Tle 2). However, in oth winter nd summer, the U rit s of these groups were signifintly higher thn those for the groups limted to the reversed sesonl tempertures (5 C in winter, 15 C in summer). In winter, fish limted to 15 C exhiited 32% lower U rit ompred with those limtised to 5 C, while in summer, fish limted to 5 C showed 3% lower U rit thn those fish mintined t 15 C. At reversed sesonl tempertures, the men U rit of fish swum t 15 C in winter ws 11% lower thn tht for fish swum t 5 C in summer (Tle 2). Morphometry Msses of hert, red musle nd white musles While there ws little effet of seson or temperture on the mss of the white musle, fish limted to 5 C hd greter mounts of oth red nd hert musles thn those limted to 15 C, nd the differene ws greter in winter (Tle 2). Tle 2. Men vlues (± S.E.M.) for U rit, nd morphometry nd enzyme tivity of red nd white musles in dult rown trout Winter Winter Summer Summer U rit (ody lengths s 1 ) 1.95± ± ±.6 2.1±.12 Musle mss (g) Red musle 11.8± ± ± ±.48 White musle ± ± ± ±19.45 Hert.63±.2.47±.3.55±.1.39±.1 Men fire ross-setionl re (µm 2 ) Red musle 1414±78 14± ±69 156±41 White musle 4442±275, 4463± ± ±76 Cpillry density (pillries mm 2 ) Red musle 1512± ±6 272± ±3 White musle 23±8 28±1 222±17, 231±19 Cpillry-to-fire rtio Red musle 2.36±.6 2.9± ± ±.4 White musle 2.5± ± ± ±.12 CS tivity (µmol min 1 g 1 wet mss) Red musle 22.35± ± ± ±.68 White musle 4.12± ±.32, 3.2± ±.15 PFK tivity (µmol min 1 g 1 wet mss) Red musle 4.1± ± ± ±.61 White musle 21.85± ± ± ±3.45 Vlues with sme letters re not signifintly different from one nother (P<.5; N=12).

6 Sesonlity nd swimming performne in trout 2687 Thus, the mounts of red nd hert musles in fish limtised to 5 C in winter were 42% nd 61% greter, respetively, thn those in fish limtised to 15 C in summer, ut in fish limted to 5 C in summer, the mounts of red nd hert musles were only 2% nd 41% greter, respetively, thn those in fish limtised to 15 C in summer. Morphometry of red nd white musles Agin, there ws little systemti differene in ny of the mesured prmeters in white musle etween the different groups (Tle 2), wheres men fire ross-setionl re of the red musle showed similr pttern to the mount of red musle (see ove). In fish limtised to 5 C in winter, men fire ross-setionl re of the red musle ws 34% greter thn in fish limtised to 15 C in summer, ut in fish limted to 5 C in summer, men fire ross-setionl re of the red musle ws only 13% greter thn tht in fish limtised to 15 C in summer (Tle 2). Although pillry density ws gretest in red musle of fish limted to 5 C in summer nd lowest in fish limtised to 5 C in winter, the men numer of pillries per musle fire ws pproximtely 15% greter in red musle of fish limted to 5 C thn in those limted to 15 C, irrespetive of seson (Tle 2). Enzyme tivities The tivities of CS were pproximtely 5.5 times greter in red musle thn in white musle (Tle 2), nd in oth musles showed similr ptterns mong the groups of fish to those seen with the mount of red musle nd men fire ross-setionl re for red musle (see ove). In fish limtised to 5 C in winter, CS tivity in red nd white musles ws 65% nd 82%, respetively, higher thn in those of fish limtised to 15 C in summer, ut in fish limted to 5 C in summer, CS tivity in red nd white musles ws only 28% nd 34% higher thn in those of fish limtised to 15 C in summer (Tle 2). The tivities of PFK were etween 5 nd 5.5 times greter in white musle thn in red musle in the vrious groups of fish (Tle 2), ut within eh musle, the tivities were similr in ll groups of fish, exept in those limted to 15 C in summer. In this group, tivity of PFK ws pproximtely 8% higher thn tht in the other groups (Tle 2). Metoli sustrtes nd metolites In resting fish, the onentrtions of glyogen in the liver were 7 1 times greter thn those in white musle nd 8 18 times greter thn those in red musle (Fig. 2). In resting fish, there ws no differene in the onentrtions of glyogen in the red musles etween ny of the groups of fish, wheres in white musle nd liver, the onentrtions were gretest in fish limtised to 5 C in winter nd lowest in fish limtised to 15 C in summer. In ll tissues, the onentrtions of glyogen were lower in fish tht hd swum to U rit thn in those t rest, ut, fter swimming to U rit, the onentrtions were not s low in fish limted to reversed sesonl tempertures s in those limtised to sesonl tempertures (Fig. 2). Similrly, in resting fish, the onentrtion of free gluose Glyogen (µmol g 1 wet mss) A B C Sesonl tempertures Reversed sesonl tempertures Red musle Winter White musle Liver ws gretest in liver nd lowest in red musle (Fig. 3), ut, in this se, the onentrtions in eh tissue were similr in resting fish nd were higher in fish swum to U rit. However, the onentrtions fter swimming to U rit were not s gret in fish limted to reversed sesonl tempertures s in those limtised to sesonl tempertures (Fig. 3). The onentrtions of lipid in red musle of resting fish were etween 6 nd 13 times higher thn those in white musle (Fig. 4). In white musle of resting fish, lipid onentrtions differed little etween groups of fish, exept for those limted to 5 C in summer, in whih it ws pproximtely 2% lower thn in the other groups. However, in red musle of resting fish, those limted to 5 C hd greter onentrtions of lipid thn those limted to 15 C, nd the differene ws greter in summer. Lipid onentrtion in the red musle of resting fish limtised to 5 C in winter ws 11% greter thn tht in fish limtised to 15 C in summer, ut in resting fish limted to 5 C in summer, lipid Summer Fig. 2. Men vlues (± S.E.M.) of glyogen onentrtions in red musle, white musle nd liver of dult rown trout smpled t rest or fter swimming up to their ritil swimming speed (U rit ). For the pproprite omprisons, vlues with the sme letters re not signifintly different from one nother (P<.5; N=12).

7 2688 N. Dy nd P. J. Butler Gluose (µmol g 1 wet mss) A B C Sesonl tempertures Reversed-sesonl tempertures Red musle e d d e Winter White musle Liver onentrtion ws only 83% greter thn tht in fish limtised to 15 C in summer. The onentrtions of lipid were lower in oth red nd white musles in ll groups of fish swum to U rit ompred to those in the groups of resting fish, exept in the white musle of those limted to 5 C in summer, nd the proportionl redution ws similr in eh se (Fig. 4). In resting fish, the onentrtions of ltte were etween 15 nd 2% greter in white musle thn in red musle nd, within eh musle, the onentrtions were similr in ll groups of fish (Fig. 5A,B). The onentrtions of ltte in oth musles of fish tht hd swum to U rit were higher thn in those t rest nd, in red musle, the onentrtions were similr in ll groups. However, in white musle, the onentrtions of Summer Fig. 3. Men vlues (± S.E.M.) of free gluose onentrtions in red musle, white musle nd liver of dult rown trout smpled t rest or fter swimming to ritil swimming speed (U rit ). For the pproprite omprisons, vlues with the sme letters re not signifintly different from one nother (P<.5; N=12). Totl tissue lipid (% wet mss) A B Sesonl tempertures Reversed-sesonl tempertures Winter Red musle White musle ltte in the fish swum to U rit were 45 55% lower in those limted to reversed sesonl tempertures ompred with those in fish limtised to sesonl tempertures (Fig. 5). A similr pttern to tht seen with ltte onentrtions ourred with totl mmoni [Tmm] in the musles, with greter [Tmm] present in oth musles in fish swum to U rit ompred with those in resting fish (Fig. 5C,D). However, in white musle, [Tmm] of fish swum to U rit were lower in those limted to reversed sesonl tempertures thn in those limtised to sesonl tempertures. Disussion In the present study, where oth morphometri nd swimming performne dt were otined, it is importnt tht the experimentl nimls used were ll of similr size. It my lso hve een importnt tht they were ll in similr stte of sexul mturity, lthough pulished dt orrelting hnges in sttes of sexul mturity to hnges in morphometri vriles re sprse. The ondition ftor for eh of the four groups of fish tht were used in this study did not differ. In ddition, the GSI of three of these groups ws similr, the exeption eing the GSI of the group mintined t 15 C in summer, whih ws 2 3% higher thn the GSIs for the other groups. The hnge in GSI is smll ompred with those in Summer Fig. 4. Men vlues (± S.E.M.) of totl lipid levels in red musle nd white musle of dult rown trout smpled t rest or fter swimming to ritil swimming speed (U rit ). For the pproprite omprisons, vlues with the sme letters re not signifintly different from one nother (P<.5; N=12).

8 Sesonlity nd swimming performne in trout 2689 Sesonl tempertures Ltte (µmol g 1 wet mss) A B Reversed-sesonl tempertures Red musle White musle Winter Summer Ammoni (µmol g 1 wet mss) C D Winter Red musle White musle Summer Fig. 5. Men vlues (± S.E.M.) of ltte nd mmoni onentrtions in red musle nd white musle of dult rown trout smpled t rest or fter swimming to ritil swimming speed (U rit ). For the pproprite omprisons, vlues with sme letters re not signifintly different from one nother (P<.5; N=12). pulished dt. For exmple, Tylor et l. (1996) reported tht the GSI for the dult rinow trout Onorhynhus mykiss used in their experiments differed y more thn 6-fold, from.48 for fish mintined t 4 C to 3.6 for fish limted to 18 C. Swimming performne The present study lerly demonstrtes tht limtion to reversed sesonl tempertures lowers swimming performne (U rit ) in dult rown trout. This ppers to e due, t lest in prt, to redution in the use of white musle, s the inreses in ltte nd mmoni in white musle in the fish swimming to U rit t reversed sesonl tempertures were not s gret s those oserved in fish swum t sesonl tempertures. Thus, this redution in swimming performne with limtion to reversed sesonl tempertures nnot e explined y unusully high levels of plsm nd musle mmoni, s ours in fish exposed to low ph (Beumont et l., 2). In ddition, the nimls held t reversed sesonl tempertures were smpled fter hving limted to these onditions over n extended period. Thus, other, omprtively long-term, hnges must hve een responsile for the differenes in swimming performne etween the groups of fish limtised to sesonl nd limted to reversed sesonl tempertures. Effets of limtistion to sesonl tempertures on musle morphometry nd iohemistry There is inresing evidene tht limtion to low temperture uses mny fish to undergo ntomil nd iohemil hnges to their loomotory pprtus. In the urrent study, the pillry-to-fire rtio, men ross-setionl fire re nd mss of red musle were signifintly greter in fish limtised to 5 C in winter thn in those limtised to 15 C in summer. The greter mss of red musle in fish limtised to 5 C in winter thn in those limted to 15 C in summer ws the result of n inrese in men fire ross-setionl re of this tissue. This phenomenon of ulking-up of eroi musle t low temperture hs een demonstrted in numer of speies inluding Crssius urtus (Johnston nd Luking, 1978) nd Morone sxtilis (Jones nd Sidell, 1982). As oserved in other speies, inluding rinow trout (Egginton nd Cordiner, 1997), the pillry to red musle fire rtio inresed with limtion to the lower temperture in the present study, lthough the pillry density per unit re deresed due to the inrese in fire dimeter. Suh hnges, together with the rdi hypertrophy, ould potentilly inrese lood flow to eroi musle, whih would enhne eroi metolism of this tissue during winter. White musle hnged little with limtistion to the lower winter temperture or to 5 C in summer. This is somewht surprising, sine it suggests tht white musle hs low plstiity in response to hnges in ioti onditions. Undoutedly, white musle must dpt to environmentl temperture in some wy, sine there is no differene in U rit etween nimls mintined t the two sesonl tempertures, despite lk of hnge in the speed t whih white musle is reruited (pproximtely 1 ody length s 1 ; Dy nd Butler, 1996).

9 269 N. Dy nd P. J. Butler The phenomenon of inresed eroi enzyme tivity t lower tempertures is well known nd hs een demonstrted in numer of speies (see Introdution), where it ppers to e relted to n enhnement in the ility of the fish to utilise lipid s n energy soure t low tempertures (Hzel nd Prosser, 1974). The urrent study demonstrtes tht, when ssyed t the sme temperture (15 C), CS tivities in oth red nd white musles were greter in winter fish t 5 C thn in summer fish t 15 C (see lso Bttersy nd Moyes, 1998), nd the reverse ws true for PFK. Unfortuntely, it did not provide n indition of in situ (physiologil) tivities of CS nd PFK in the winter fish. For this, tivities of oth of these enzymes should hve een determined t 5 C for the winter fish ut, s we hve previously indited (see Results), we were unle to otin onsistent dt t this temperture. Therefore, the dt only demonstrte hnges in solute pity nd do not provide evidene for true therml ompenstion. Nthnilides (1996) used the term EQ 1 to desrie the extent to whih the tivity of n enzyme inreses with every 1 C derese in limtion temperture nd ompred this with the Q 1 of the enzyme. If EQ 1 equls Q 1, there is perfet ompenstion. Assuming Q 1 of 1.46 for CS (Nthnilides, 1996), the rtio of EQ 1 /Q 1 is 1.13 for sesonlly limted rown trout nd.88 for those t 5 nd 15 C in summer. Thus, this nlysis lerly demonstrtes tht ompenstion of CS tivity is greter t low sesonl temperture thn t low non-sesonl temperture. Effets of limtion to reversed sesonl tempertures on musle morphometry nd iohemistry The differenes etween the vriles mesured in fish t sesonl nd reversed tempertures indite tht environmentl temperture is not the only ftor responsile for these oservtions. Sesonl effets lso seem to e t work. For exmple, PFK levels in nimls t 15 C were onsiderly lower in winter thn in summer nd indeed were no different from those oserved in nimls limted to 5 C in winter. In ddition, t 5 C, the pillry density of red musle ws higher in summer ompred with in winter, proly due to the oserved derese in fire size. The tivities of CS in red nd white musle, the msses of red nd hert musle nd men red musle fire ross-setionl re were ll lower in fish limted to 5 C in summer thn in those limtised to the sme temperture in winter. This suggests tht, t the lower temperture, full therml ompenstion is only possile during winter. It would pper, therefore, tht the hnges in musle morphometry nd iohemistry tht ompny limtion to reversed sesonl tempertures re inomplete, when ompred with hnges oserved in fish limted to sesonl tempertures, or, t the very lest, tke longer thn the durtion of limtion period used in the present study. Further evidene for this omes from study y Kilrski et l. (1996) on the mitohondril morphometris of oxidtive musle of ruin rp tht were limted for 6 weeks to 5 C nd 25 C in oth winter nd summer. In this study, it ws demonstrted tht t 5 C the surfe density of outer mitohondril memrne per musle ws higher in summer thn in winter. At 25 C the surfe density of inner mitohondril memrne per fire nd the surfe density of the inner mitohondril memrne were higher in summer thn in winter. In ddition, Bouhrd nd Guderley (23) studied the time ourse of limtion, s determined y red musle mitohondril enzyme tivity nd respirometry, of groups of rinow trout tht were either wrm limted (wter temperture rised from 5 C to 15 C during winter) or old limted (wter temperture lowered from 15 C to 5 C during summer). They found tht wrm limtion ppered to e ompleted within 8 weeks wheres old limtion ppered to e inomplete fter 1 weeks. The only other omprle iohemil dt for the effets of reversed sesonl tempertures on enzyme tivity onern the three-spine stiklek, Gsterosteus uletus (Guderley et l., 21). In this study, fish were limted to 8 C nd 23 C in oth spring nd utumn, nd enzyme tivities were determined (t 1 C nd 2 C) for petorl nd xil musle. It ws oserved tht, in spring, the highest level of PFK tivity ws oserved in xil musle from fish limted to the lower temperture in spring nd ssyed t 2 C (there ws no differene t 1 C nd no dt were presented for the utumn experiment). This is the opposite of tht oserved in the urrent study for rown trout limted to 15 C in winter. Guderley et l. (21) lso demonstrted tht for petorl (i.e. eroi) musle (in ontrst to wht ws oserved in xil musle) there ws no signifint differene etween the levels of CS tivity etween the two groups of fish tht were limted to 8 C nd 23 C in the spring. This gin ws the reverse of wht we oserved in the rown trout, where the highest levels in red musle ourred t 5 C. In spite of these ontrditory dt (whih my e relted to differenes in life yle or txonomy), oth of these studies do suggest tht sesonlly hnging environmentl temperture is not the only ftor tht influenes hnges in musle morphology nd physiology. Wht is the primry influene on swimming performne? The iohemil nd morphologil dt in the present study provide evidene tht full therml ompenstion does not our in the fish tht re limted to reversed sesonl tempertures. Thus, there must hve een ftor or ftors in ddition to temperture tht were responsile for the differenes in U rit etween the groups of fish limtised to sesonl nd limted to reversed sesonl tempertures. In ft, with few possile exeptions (e.g. Sturnes et l., 1994), there ppers to e little experimentl evidene to support the ide tht temperture lone n t s zeitgeer in fish, lthough it n, in onjuntion with photoperiod, influene physiologil hnges (MCormik et l., 22). Seddon nd Prosser (1997) onluded tht limtion to environmentl temperture ws not n ll or none phenomenon nd my depend on vriety of sesonl ftors, inluding time of olletion, nutritionl nd reprodutive stte, nd irnnul yles.

10 Sesonlity nd swimming performne in trout 2691 Photoperiod is proly the most importnt environmentl ue influening hnges in the loomotory pprtus nd U rit of rown trout, sine it hs previously een demonstrted to e the dominnt zeitgeer for severl endogenous rhythms nd physiologil hnges in other fish speies inluding irdin rhythmiity in hert rte (Penne nd Le Brs, 1988) nd the timing of smolting in ndromous slmonids (Hor, 1988; Duston nd Sunders, 199). In ddition, studies on the effets of light exposure on sexul mturtion of the stiklek (G. uletus) hve reveled endogenous dily nd nnul rhythms of hnging photoretivity (Bggermn, 1985). In this speies, the onset of sexul mturtion n e experimentlly triggered y short (2 h) exposure to light during the sotophse in winter. Beuse of its preditle nnul vrition, photoperiod undoutedly funtions s synhroniser of suh rhythms in the nturl yle. Assuming tht suh rhythms exist in rown trout, it would e intriguing to perform swimming experiments, suh s those desried in the present study, on fish tht were sujeted to oth reversed sesonl tempertures nd reversed sesonl photoperiods. Wht other ftors my influene swimming performne? In ddition to photoperiod, there re numer of nturlly ourring, sesonlly hnging environmentl ftors tht my hve influened dpttion of the loomotory pprtus of rown trout to unsesonl tempertures. One exmple is the nnul nd semi-nnul vrition in the erth s geomgneti field (see Mlin et l., 1999) to whih nimls, inluding slmonids, re known to e sensitive (see e.g. Yokoi et l., 23; Dieel et l., 2). So, it is oneivle tht hnges in geomgnetism ould e n exogenous ue tht, proly in omintion with others, ffets hnges in the loomotory pprtus of rown trout. Indeed, there is inresing evidene tht in migrtory nimls, inluding slmonids, photoreeption nd geomgneti detetion re inextrily linked (Deutshlnder et l., 1999). Endogenous iologil loks my lso ply n importnt role, sine their powerful influene on orgnisms is well known. For exmple, Sether et l. (1996) demonstrted tht rti hr (Slvelinus lpinus) showed sesonl hnges in food onsumption nd growth tht were endogenously driven, eing unffeted y experimentl mnipultion of photoperiod. Perhps it is the regulrity of one or more of these internl loks tht prevents the loomotory pprtus of rown trout from eoming fully dpted to unsesonl environmentl tempertures. The present study is the first to investigte the effets of limtion to reversed sesonl tempertures on U rit nd the morphometris nd iohemistry of the loomotory pprtus of slmonid fish. It illustrtes the importne of ensuring tht experiments performed on fish suh s rown trout should e performed under onditions of temperture nd photoperiod tht pproximte those ourring in the nturl environment t the time of the yer when they re performed. It is only under these onditions tht the dt produed from suh studies will e of relevne to nimls in their nturl environment. This work ws funded y n NERC grnt wrded to P.J.B. Referenes Anderson, R. O. nd Gutreuter, S. J. (1983). Length, weight nd ssoited struturl indies. In Fisheries Tehniques (ed. L. A. Nielson nd D. L. Johnson), pp Bethesd, Mrylnd: Amerin Fisheries Soiety. Bggermn, B. (1985). The roles of dily nd nnul iologil rhythms in the photoperiodi regultion of the reeding seson in the Stiklek Gsterosteus uletus L. Behviour 93, 1-7. Bttersy, B. J. nd Moyes, C. D. (1998). Influene of limtion temperture on mitohondril DNA, RNA, nd enzymes in skeletl musle. Am. J. Physiol. 275, R95-R912. Beumont, M. W., Butler, P. J. nd Tylor, E. W. (1995). Plsm mmoni in rown trout (Slmo trutt) exposed to su-lethl ph opper in soft idi wter nd its reltionship to deresed swimming performne. J. Exp. Biol. 198, Beumont, M. W., Tylor, E. W. nd Butler, P. J. (2). 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