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1、2165be seasonal or long-term in nature. Following acute exposure muscle fibres has been found only in a few species of cyprinidThe Journal of Experimental Biology 198, 21652175 (1995)Printed in Great Britain The Company of Biologists Limited 1995thermal acclimation of C-start (escape swimming)in bot

2、h species; however, the magnitude of this responsewas much greater in goldfish (100 %) than in killifish (30 %or less). In goldfish, these changes in the physiologicalto a decline in temperature, muscle power output (see Rome,1990) and consequently locomotor performance (bothsustained and burst-spee

3、d swimming) are severelycompromised (reviewed in Beamish, 1978; Guderley andBlier, 1988; Sidell and Moerland, 1989; Bennett, 1990;Johnston et al. 1991).Long-term exposure to cold temperature inducesacclimatory changes in muscle contractile properties andenzyme activities, and these have been interpr

4、eted asenhancing locomotor performance at low temperatures(Loughna et al. 1983). Long-term acclimatory responses in redfish (Johnston et al. 1975; Heap et al. 1985, 1986; Crockfordand Johnston, 1990; Fleming et al. 1990; Gerlach et al. 1990;Hwang et al. 1990; Goldspink et al. 1992) and has beendemon

5、strated to be absent in others (Penney and Goldspink,1981; Sidell et al. 1983; Heap et al. 1985; Sidell and Johnston,1985; Johnston and Wokoma, 1986; Moerland and Sidell,1986). Since the contractile properties of fast twitch muscle arethought to limit maximum locomotor speed at the organismallevel (

6、Wardle, 1975; Marsh and Bennett, 1985, 1986; Marsh,1990; Swoap et al. 1993), the absence of acclimation at themuscle level suggests that organismal escape performance mayMany teleost fish species are often exposed to changes intemperature. These changes may be relatively acute, forexample during mig

7、ration, flooding, tidal changes, etc, or mayduring cold acclimation are well known and documented inseveral species of fish (see Johnston and Dunn, 1987).However, temperature acclimation of white (fast twitch)performance and the physiological properties of fast twitchmuscle fibres that underlie it w

8、ere investigated in thesespecies at the molecular (myosin isoform expression),biochemical (myofibrillar ATPase activity), cellular(contractile kinetics) and organismal levels of organisation.Peptide maps were obtained for fast muscle myosin heavychains, isolated from 10 C- and 35 C-acclimated fish.D

9、ifferent myosin heavy chain isoforms were expressed inresponse to a change in acclimation temperature ingoldfish, but myosin heavy chain isoform expression wasunaffected by acclimation temperature in killifish.Compared with fish acclimated to 35 C, acclimation to10 C increased the activity of fast m

10、uscle myofibrillarATPase assayed at 10 C fivefold in goldfish and only 50 %in killifish. Muscle twitch contraction time at 10 Cproperties of fast twitch fibres during 10 C acclimationresulted in a six- to eightfold increase in the speed andturning velocity of fish performing C-starts at 10 C. Bycomp

11、arison, the somewhat smaller acclimatory response ofkillifish fast muscle properties was accompanied by only aminor (50 % or less) adjustment in locomotor performance.Thermal acclimatory responses of fast muscle at themolecular, biochemical and cellular levels of organisationare clearly reflected in

12、 alterations in organismal escapeperformance.Key words: thermal acclimation, C-start, fish, goldfish, Carassiusauratus, killifish, Fundulus heteroclitus, muscle physiology,kinematics, polyploidy.IntroductionGoldfish (Family Cyprinidae, Carassius auratus) andkillifish (Family Cyprinodontidae, Fundulu

13、s heteroclitus)were acclimated to 10, 20 and 35 C for 4 weeks. TheSummaryTHE THERMAL ACCLIMATION OF BURST ESCAPE PERFORMANCE IN FISH: AN INTEGRATED STUDY OF MOLECULAR AND CELLULAR PHYSIOLOGY ANDORGANISMAL PERFORMANCETIMOTHY P. JOHNSON* ANDDepartment of Ecology and Evolutionary Biology, The UniverAcc

14、epted 2 June 1995(slow twitch) muscle and sustained swimming behaviour*Present address: School of Applied Sciences, University of Glamorgan, Pontypridd, Mid-Glamorgan CF37 1DL, UKdecreased significantly in response to acclimation to 10 CALBERT F. BENNETTsity of California, Irvine, CA 92717, USAnot a

15、cclimate at cold temperatures. In any event, the2166acclimation of burst escape speed in fish has not beeninvestigated. It was therefore the aim of this investigation todetermine to what degree thermal acclimatory responses ofmuscle at the molecular and cellular levels are capable ofameliorating the

16、 detrimental effects of exposure to coldtemperature on burst escape locomotor performance.In this paper, we examine the effects of acute temperatureexposure and thermal acclimation on the C-start, an escaperesponse in teleost fish initiated by the Mauthner cell (Zottoli,1977). This locomotor behavio

17、ur is ideal for this investigationas it is highly stereotyped and involves the simultaneousrecruitment of all motor units, including all the fast twitchfibres. The C-start consists of two stages (Eaton et al. 1977).In stage 1, the fish bends into a C as a result of thesimultaneous contraction of all

18、 muscle fibres along one side ofthe body; stage 2 consists of an S-shaped propulsive stroke(see Fig. 1A; Eaton et al. 1977; Nissanov and Eaton, 1989). Inthis study, the thermal dependence of C-start performance andthe underlying physiology of fast muscle function (myosinisoform expression, myofibril

19、lar ATPase activity, twitchcontraction kinetics) were investigated in one species knownto demonstrate significant thermal acclimatory ability in thesephysiological parameters (goldfish) and compared with anotherspecies (killifish), previously shown to lack similar responses.Materials and methodsFish

20、Goldfish (Family Cyprinidae, Carassius auratus) wereobtained from a local fish farm (Bayou Aquatics, Ontario, CA,USA) and killifish (Family Cyprinodontidae, Fundulusheteroclitus) from Woods Hole Marine Biological Laboratory(MA, USA). All animals were of similar size, with a mean totallength of 6.40.

21、8 cm (S.D., N=75) for goldfish and 6.80.8 cm(N=64) for killifish. Goldfish were maintained in fresh waterand killifish in 100 % sea water under ambient light conditions(16 h:8 h light:dark). Fish were divided into three groups andkept at 10, 20 and 35 C (0.5 C) for at least 4 weeks prior toexperimen

22、tation. All animals were fed ad libitum with fishflake and frozen bloodworms. For experimental procedures,fish were rapidly killed by a blow to the head followed byimmediate decapitation.Polyacrylamide gel electrophoresisMuscle samples from 10 C- and 35 C-acclimated fish wereanalysed using electroph

23、oretic techniques.SDSpolyacrylamide gel electrophoresis (SDSPAGE) wasperformed using small vertical slab gels(80 mm373 mm30.5 mm; Bio-Rad Mini-PROTEAN II).SDSPAGE gels were run using separating gels containing arange of acrylamide concentrations of 10 % to 20 %, with astacking gel of 4 %. Alkali ure

24、a gels were run to characterisethe myosin light chains (MLCs) of actomyosin prepared frommyofibril samples. MLCs were separated on gels containingT. P. JOHNSON AND A. F. BENNETT8 mol l21urea, 10 % acrylamide at pH 8.6 (Perrie and Perry,1970). Actomyosin was prepared from myofibril samplesaccording t

25、o Huriaux et al. (1990). For SDSPAGE and alkaliurea gels, protein samples were prepared for electrophoresis byboiling for 3 min in a solution containing 60 mmol l21TrisHCl, pH 6.76, 2 % SDS, 10 % glycerol, 1 %mercaptoethanol and 0.002 % Bromophenol Blue (Crockfordand Johnston, 1990).Myofibril sample

26、s were also characterised using isoelectricfocusing on 125 mm365 mm30.4 mm gels (Bio-Rad model111 Mini IEF Cell), containing Bio-lyte carrier ampholytes(pH range 3.55.2, 2.25 %, pH range 3.59.5, 0.75 %) (Bio-Rad), 2 % Nonidet P-40, 1 % mercaptoethanol, 4.85 %acrylamide and 0.15 % N,N -methylene-bis-

27、acrylamide(Crockford and Johnston, 1990). Samples were prepared bydissolving myofibrils in 8 mol l21urea, 2 % Nonidet P-40 and1 % mercaptoethanol.Peptide mapping was used to characterise myosin heavychains (MHCs). SDSPAGE gels (7.5 %) of myofibril samples(approximately 5 mg ml21) were run according

28、to themethodology described above, stained briefly in CoomassieBlue (30 s) and washed with distilled water. MHC bands werecut from the gel, chopped finely and homogenised with a glasspestle, in 400 ml of a medium containing 50 mmol l21TrisHCl(pH 7.4) and 0.5 % SDS, and left to stand for 60 min (Croc

29、kfordet al. 1991). The samples were spun in an Eppendorf bench-top centrifuge for 15 min and the top 100200 ml of buffer wascarefully removed, avoiding contamination with gel fragments.MHCs purified in this way ran as single bands on re-electrophoresis. Samples of MHCs were digested with Type1-S chy

30、motrypsin (from bovine pancreas, Sigma; 200 ng ofenzyme per 50 ml sample) at 20 C for 30 min. Digestion wasstopped by the addition of 2-mercaptoethanol (2 %) and SDS(2 %) and heating to 100 C for 3 min (Crockford et al. 1991).The resulting peptides were resolved using 18 % to 22 %SDSPAGE gels and st

31、ained using a Bio-Rad silver stain pluskit.Myofibrillar ATPase activityAfter the fish had been killed, the fast (white) epaxial musclewas rapidly removed from the entire length of the fish, takingcare to avoid slow twitch muscle fibres along the lateral line.Myofibrils were prepared according to Joh

32、nston et al. (1975)in a solution containing 1 % (w/v) Triton X-100, 100 mmol l21NaCl, 5 mmol l21EDTA, 10 mmol l21TrisHCl, pH 7.2 at0 C. The protein concentration of the final sample wasdetermined using a Bio-Rad detergent-compatible proteinassay kit. A sample of myofibrils was suspended in glycerola

33、nd stored at 280 C for electrophoretic analysis (describedabove). Myofibrils (approximately 0.2 mg ml21) were pre-incubated at 10, 20 and 35 C for 2 min in a solution containing30 mmol l21TrisHCl, 1.17 mmol l21EGTA, 1.17 mmol l21MgSO4, 2.8 mmol l21CaCl2, pH 7.4 at assay temperature.Following the add

34、ition of 6 mmol l21ATP, the activity ofmyofibrillar ATPase was assayed by measuring the amount ofphosphate produced in 2 min. The reaction was terminated bythe addition of 33 % (v/v) of 10 % trichloroacetic acid at 0 C.The final concentration of inorganic phosphate was determinedby the method of Fis

35、ke and Subbarrow (1925). To determinethe thermal stability of the myofibrillar ATPase, enzymeactivities were monitored following pre-incubation at hightemperature (40 C) for between 5 min and 1 h. ATPase activitywas then determined at 40 C according to the methodsdescribed above.Twitch contraction k

36、ineticsFast muscle twitch contraction kinetics were measured insitu in freshly killed fish using a modification of the methoddescribed by Archer et al. (1990). The experiments wereperformed in a Perspex temperature-controlled (0.1 C)chamber with a base of Sylgard elastomer on which to securethe spec

37、imen with dissection pins. Experimental preparationsof goldfish were continually immersed in Ringers solutioncontaining 119 mmol l21NaCl, 2.7 mmol l21KCl, 1 mmol l21MgCl2, 1.8 mmol l21CaCl2, 20 mmol l21NaHCO3and10 mmol l21sodium pyruvate (Johnson et al. 1994).Preparations of killifish were maintaine

38、d in Ringers solutiondesigned for marine teleosts (Altringham and Johnston, 1988)and containing 132.5 mmol l21NaCl, 2.6 mmol l21KCl,1 mmol l21MgCl2, 2.7 mmol l21CaCl2, 18.5 mmol l21NaHCO3, 3.2 mmol l21NaH2PO4and 10 mmol l21sodiumpyruvate. The pH of the Ringers solution was maintained at7.4 by bubbli

39、ng with 95 % O2:5 % CO2. The abdominal cavitywas exposed with a lateral incision, the viscera were carefullyremoved and the abdominal myotomes pinned back.Abdominal membranes were removed to expose the fastmuscle myotomes and a suction electrode was placed on thefourth or fifth spinal nerve close to

40、 its origin from the spinalcolumn. During the contraction of the appropriate myotome,force was monitored using a force transducer made from twoEntran ESU-060-1000 strain gauges bonded to a spring steelbar. A pin, bonded to the surface of the transducer, was insertedinto the adjacent myotome next to

41、the posterior myoseptum.Output signals from the transducer were modified using acustom-designed bridge amplifier and displayed and storedusing a Metrabyte A/D converter interfaced to an IBM-PC.Single, supramaximal stimuli (typically 0.10.5 ms duration,2030 V) were administered via the suction electr

42、ode using aGrass S48 stimulator connected to a current amplifier. Thepreparation was allowed to recover for at least 10 min betweenstimuli. Twitch contraction time (Tc, the time from the onsetof force production to 50 % relaxation) was measured inpreparations from all acclimation groups and both spe

43、cies attemperatures encompassing the acclimation regimes.High-speed video analysisFish were videotaped performing C-starts (escaperesponses) in an area 25 cm320 cm315 cm using a NACHSV-400 high-speed video system at 400 frames s21.Experimental trials were performed in an insulated glassaquarium mark

44、ed on the base with a 2 cm32cm32 cm grid.Fish were filmed ventrally by placing a front-surface mirror at45 to the horizontal below the tank. Illumination was providedby a combination of 500 W photofloods (Smith Victor) and2167Acclimation of burst escape performancesynchronized strobes (NAC). Tempera

45、ture was controlled(0.5 C) using submersible aquarium heaters and Neslabcryocool immersion coolers. C-starts were elicited either bystriking the side of the tank or by dropping a small weight intothe tank above the fish. Only C-starts performed from astationary position were recorded and analyzed. T

46、rials wereperformed over a broad range of temperatures within thecritical thermal limits for the different acclimation groups andspecies. Critical thermal maximum was defined as thetemperature at which fish lost equilibrium and critical thermalminimum as the temperature at which fish failed to respo

47、nd totactile stimulation. Fish recovered completely when returnedto more moderate temperatures. Water temperature waschanged at a rate not exceeding 10 C h21. Animals wereplaced in the experimental arena at the acclimation temperatureand allowed to adjust to their new surroundings for no less than1

48、h. After recording C-starts at this temperature, the water waseither cooled or heated to the next trial temperature. Withinacclimation groups, the order in which trials were performed(i.e. above or below acclimation temperature first) wasrandomized from fish to fish. C-starts at acclimationtemperatu

49、re were recorded again at the end of the experimentto determine repeatability. Up to three C-starts were recordedper fish at each temperature.C-start sequences were downloaded from the video and storedas digital images on an IBM-PC installed with a PC-VISIONplusframegrabber card (Imaging Technology Inc., MA, USA). Videoframes were downloaded from the first detectable signs ofmovement by the fish following the stimulus, until the animalmoved beyond the field of view or slowed significantly; onlysequences in which both stage 1 and stage 2 o

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