upper thermal limits of growth in brook trout and their ... · for brook trout growth (baldwin,...

RESEARCH ARTICLE

Upper thermal limits of growth in brook trout and their relationshipto stress physiologyJoseph G Chadwick1 and Stephen D McCormick12

ABSTRACTDespite the threat of climate change the physiological mechanismsresponsible for reduced performance at high temperatures remainunclear for most species Elevated but sublethal temperatures mayact via endocrine and cellular stress responses to limit performancein important life-history traits such as growth Here brook trout(Salvelinus fontinalis) subjected to chronically elevated or dailyoscillating temperatures were monitored for growth and physiologicalstress responses Growth rate decreased at temperatures above 16degCand was negative at 24degC with an estimated upper limit for positivegrowth of 234degC Plasma cortisol increased with temperature and was12- and 18-fold higher at 22 and 24degC respectively than at 16degCwhereas plasma glucose was unaffected by temperature Abundanceof heat shock protein 70 (HSP70) in the gill increased with temperatureand was 11- and 56-fold higher at 22degC and 24degC respectively than at16degC Therewas no relationship between temperature and plasmaClminusbut there was a 53 and 80 decrease in gill Na+K+-ATPase activityand abundance at 24degC in comparison with 16degC Daily temperatureoscillations of 4degC or 8degC (19ndash23degC or 17ndash25degC) were compared with21degC controls Growth rate decreased with temperature and was 43and 35 lower by length and mass respectively in the 8degC dailyoscillation treatment than in the controls There was no effect oftemperature oscillation on plasma cortisol or glucose levels Incontrast gill HSP70 abundance increased with increasing dailyoscillation and was 40- and 700-fold greater at 4degC and 8degC dailyoscillation respectively than in the constant temperature controls Inindividuals exposed to 17ndash25degC diel oscillations for 4 days and thenallowed to recover at 21degC gill HSP70 abundance was still elevatedafter 4 days recovery but not after 10 days Our results demonstratethat elevated temperatures induce cellular and endocrine stressresponses and provide a possible mechanism by which growth islimited at elevated temperatures Temperature limitations on growthmay play a role in driving brook trout distributions in the wild

KEY WORDS Climate change Temperature toleranceOsmoregulation Cortisol Glucose Heat shock protein

INTRODUCTIONClimate change is one of the largest ecological challenges thatconservationists face today and will provide many obstacles tothose attempting to manage natural resources In spite of the

increasing attention to the ecological impacts of climate change it isstill unclear what physiological mechanisms are important to theresponse of individual species to climate change (Wikelski andCooke 2006 Portner and Farrell 2008 Portner et al 2009)Several integrative approaches indicate a decrease in aerobic scopegrowth and swimming performance at elevated temperatures that areassociated with cellular and physiological stress responses (Portnerand Farrell 2008 Sokolova 2013) Although somatic growth is akey aspect of population persistence our understanding of themeans by which temperature impacts growth is limited Sublethaltemperatures may act via the stress response to inhibit growth inindividuals thus constraining populations

The eastern brook trout Salvelinus fontinalis (Mitchill 1814) isan iconic cold-water species of North America and for many streamsystems the most abundant vertebrate Brook trout may beparticularly sensitive to increased water temperatures in responseto climate change as local populations are spatially constrained tostream networks Previous brook trout habitat models suggest globalwarming will lead to a significant loss of habitat throughout thespecies range with increasing impacts felt by southern populations(Meisner 1990 Flebbe et al 2006) These models currently rely onuntested assumptions of the upper temperature limit of persistenceand would be improved with information on the upper thermallimits for growth in this species There are well-defined effects oftemperature on growth rates for most freshwater salmonids (Brett1979) and a number of studies have found 13ndash16degC to be optimalfor brook trout growth (Baldwin 1956 McCormick et al 1972Hokanson et al 1973 Dwyer et al 1983 McMahon et al 2007)but the upper limits for growth in brook trout have yet to bedetermined Recent field studies indicate that brook trout are notpresent in waters above a 24-daymeanmaximum temperature of 22degC(Wehrly et al 2007) Additional work in a lentic system suggeststhat populations are limited by temperatures above 20degC (Robinsonet al 2010) Laboratory and field-based studies suggest the upperincipient lethal temperature for brook trout is 253degC (Fry et al1946 Fry 1951 Wehrly et al 2007) The disparity between thelethal temperature and the temperature limits seen in nature suggeststhat sublethal temperature effects play a crucial role in limiting fishpopulations perhaps through their impact on food consumptionand growth

The majority of studies designed to investigate the impacts ofelevated temperature on physiology and whole-animal performanceuse acute (hours) or chronic (days or weeks) elevated fixedtemperatures While important these constant exposureexperiments are inconsistent with what most species experience inthe wild Daily variations in temperature are a normal part of life formost habitats and as the climate warms many species willexperience daily temperature oscillations that feature stressfullyelevated temperatures Daily temperature oscillations have beenshown to affect heat shock proteins (HSPs) and metabolicadjustments in several salmonids (Callaghan et al 2016) ThereReceived 14 April 2017 Accepted 25 August 2017

1Graduate Program in Organismic amp Evolutionary Biology University ofMassachusetts Amherst Amherst MA 01003 USA 2US Geological SurveyLeetown Science Center Conte Anadromous Fish Research Laboratory OneMigratory Way Turners Falls MA 01376 USA

Author for correspondence (smccormickusgsgov)

SDM 0000-0003-0621-6200

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is conflicting evidence on the impact of oscillating temperatures onthe growth of fishes (Hokanson et al 1977 Thomas et al 1986Meeuwig et al 2004 Shrimpton et al 2007) which may relate tothe highest temperature fish experience and the magnitude ofthermal variationThere is increasing interest in utilizing mediators of the stress

response such as increased plasma cortisol glucose lactate andHSP expression as indicators of both individual- and population-level responses to environmental stressors including elevatedtemperature (Wendelaar Bonga 1997 Iwama et al 1999 2004Lund et al 2002 Wikelski and Cooke 2006 Wendelaar Bonga1997) Experimental warming of juvenile Chinook salmon andrainbow trout resulted in elevated circulating cortisol and glucoselevels (Meka and McCormick 2005 Quigley and Hinch 2006)Similarly adult sockeye salmon exhibited elevated plasma cortisoland lactate levels in response to increased temperature and exercise(Steinhausen et al 2008) A variety of studies have demonstratedelevated HSPs in response to elevated temperature in a variety ofsalmonid species in the laboratory (DuBeau et al 1998 Smithet al 1999 Mesa et al 2002 Lund et al 2003 Rendell et al2006 Stitt et al 2014) and in the wild (Werner et al 2005) Recentlaboratory and field work from our group indicate that acutetemperature thresholds for increased gill HSP70 (207degC) andplasma glucose (212degC) in brook trout are similar to their proposedthermal ecological limit of 210degC (Chadwick et al 2015)Sublethal temperatures may act via the stress response to inhibitgrowth in individuals (Iwama et al 1999) potentially limitingpopulations (Portner 2010)Based on the studies cited above we predicted that temperatures

above the optimum would result in a relatively steep decline ingrowth rate and that oscillating temperatures would result indecreased growth compared with constant temperatures with thesame mean temperature We also predicted that observed decreasesin growth due to temperature treatment would be accompanied byincreased endocrine (plasma cortisol) andor cellular (HSP70)stress responses In order to test these predictions we exposed brooktrout to constant elevated temperatures (target temperatures of 1618 20 22 or 24degC) or to daily temperature oscillations of 0 4 or 8degC(constant 21degC 19ndash23degC or 17ndash25degC) for 24 days In addition tomeasuring growth we collected blood and gill tissue to assessbiomarkers for stress including gill HSP70 plasma cortisol andglucose We also examined gill Na+K+-ATPase (NKA) activity andabundance and plasma Clminus to explore the relationship betweentemperature and osmoregulation A third experiment was conductedto examine acute responses and recovery from daily temperaturevariations Brook trout were exposed to the same daily temperaturevariations as above for 1 day or 4 days and then sampled 1 h 2 4and 10 days later to determine the recovery period following dailytemperature oscillations

MATERIALS AND METHODSFish stockJuvenile (0+) brook trout were obtained from the Sandwich StateHatchery (Sandwich MA USA) and brought to the ConteAnadromous Fish Research Center (Turners Falls MA USA) inJuly 2009 for the chronic elevated temperature study and in July2010 and 2011 for the oscillating temperature studies Fish werehoused in 17 m diameter tanks (150ndash200 fish per tank) suppliedwith 4 l minminus1 of chilled Connecticut River water (16plusmn2degC) andgiven supplemental aeration Fish were fed to satiation (FinfishStarter Zeigler Bros Gardners PA USA) twice daily withautomatic feeders and maintained under natural photoperiod

Rearing and experiments were carried out in accordance withUSGS Animal Care Guidelines under IACUC 9070 Fish were usedin the same year they were obtained so that 0+ fish of similar sizewere used in all studies During all experiments fish were fed acommercial trout pellet (42 protein 16 fat Finfish Gold ZieglerBros)

Experiment 1 chronic temperature growth studyIn August 2009 one week before the start of the experiment 80 fish[124ndash158 cm fork length (FL)] were removed from their rearingtank and lightly anesthetized with buffered and neutralized tricainemethanesulfonate (50 mgMS-222 lminus1 pH 70) While anesthetizedthe fish were measured for length (nearest 01 cm) and mass (nearest01 g) and implanted with a passive integrated transponder (PIT) taginserted into the abdominal cavity via a small (sim5 mm) incision inthe ventral surface just rostral to the pelvic fins After recoveringfrom the anesthetic the fish were divided randomly into five 09 mdiameter experimental tanks (N=16 per tank) The tanks weresupplied with Turners Falls city water at a rate of 09 l minminus1 and thetemperature was maintained at 16degC with 800 W titanium bayonetheaters Each tank was provided with supplemental aeration Fishwere fed to satiation twice daily throughout the experiment Thetanks were isolated from the rest of the laboratory so that the dailyfeeding was the only disturbance that the fish experienced At eachfeeding fish were carefully observed so that food was offered onlyuntil the point where it was no longer being consumed (when one ortwo pellets would drop to the bottom of the tank) The amount offeed consumed at each feeding was measured to the nearest 01 g

Feed was withheld from the fish for 24 h prior to the start of theexperiment and at any other time that length and mass weremeasured Seven days after PIT tags were implanted fish were againmeasured for length and mass and returned to their appropriate tankWater temperatures were then elevated at a rate of 2degC hminus1 until thetarget temperatures of 16 18 20 22 or 24degC were reached Eachtank was maintained at its specific target temperature for theremainder of the study The water temperature of each tank wasmeasured and recorded every 15 min using Hobo temperatureloggers (Onset Computer Corporation Bourne MA USA) Waterflow rate and feeding regime were as described with a 75 waterchange every 4 days Dissolved oxygen levels were measured dailyand were always above 90 saturation

The fish were measured for length and mass every 8 days for24 days On the eighth day half of the fish (N=8 per tank) were killedusing a lethal dose of anesthetic (100 mg MS-222 lminus1 pH 70) sothat blood and tissue samples could be taken The remainder of thefish (N=8 per tank) were sampled after 24 days Blood was collectedfrom the caudal vessels using 1 ml ammonium heparanized syringeswithin 5 min of tank disturbance The blood was spun at 3200 g for5 min at 4degC and the plasma was aliquoted and stored at minus80degC Abiopsy of four to six gill filaments was taken from the first arch andimmersed in 100 microl of ice-cold SEI buffer (150 mmol lminus1 sucrose10 mmol lminus1 EDTA 50 mmol lminus1 imidazole pH 73) and stored atminus80degC The liver was removed weighed (nearest 00001 g) andstored at minus80degC After sampling the gills and blood the remainingcarcass was reweighed and then dried to a constant mass (48 h) at60degC to obtain dry mass for the calculation of conversion efficiency

Experiment 2 oscillating temperature growth studyIn October 2010 90 fish (120ndash169 cm FL) were moved from theirrearing tank to one of nine 06 m diameter experimental tanks(N=10 per tank) and allowed to acclimate for one week prior to thestart of the experiment The fish were fed to satiation once daily and

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the tanks were supplied with 16degC Turners Falls city water at a rateof 08 l minminus1 The water temperature was increased from 16degC to21degC over 48 h and then maintained at 21degC for 5 days Each tankreceived additional heated (34degC) city water as needed to achievethe desired temperature The heated water flowed through solenoidvalves (Granzow Inc Charlotte NC USA) that were controlled byOmega cn7500 controllers (Omega Engineering Inc Stamford CTUSA) with resistance thermometer input installed on each tank Thecontrollers were optimized to the testing conditions andprogrammed to pulse the solenoid valves open and shut atvarying frequency to either maintain a set point or to achieve anew set point within a predetermined time frame Each tank wasprovided with supplemental aerationThe temperature regimes chosen for this study were based on

temperature records from known brook trout streams in westernMassachusetts (Chadwick et al 2015) Feed was withheld from thefish for 24 h prior to the start of the experiment On the first day ofthe experiment all of the fish were lightly anesthetized (50 mg MS-222 lminus1 pH 70) so that length and mass could be recordedAdditionally each fish received a unique paint mark on either theanal or caudal fin for individual identification purposes Fish wereallowed to recover before being returned to their experimental tanksThe three temperature treatments (three tanks per treatment) wereinitiated that evening Treatment 1 was a control group and wasmaintained at 21degC throughout the study Treatment 2 consisted of adaily 4degC oscillation that fluctuated between 19degC and 23degCTreatment 3 consisted of a daily 8degC oscillation that fluctuatedbetween 17degC and 25degC Treatments 2 and 3 were designed suchthat the daily low temperature occurred at 0600 h and the daily highat 1800 h so that the daily mean was 21degC These temperaturetreatments were repeated daily for 24 days Water temperatures weremeasured and recorded every 15 min using Hobo pendenttemperature loggers (Onset Computer Corporation) Fish were fedto satiation once daily and the amount of feed offered was measuredby weight (nearest 01 g) Feeding occurred between 1100 h and1200 h when all treatments were at 21degC In this and all otherexperiments dissolved oxygen levels were measured daily and werealways above 90 saturation On day 6 a valve remained open inone of our three control tanks and the subsequent temperature spikekilled all of the fish in the tankEvery 8 days the fish were measured for length and mass as

described On day 8 four fish per tank were killed using a lethaldose of anesthetic (100 mg MS-222 lminus1 pH 70) so that bloodplasma and gill tissues could be sampled FL (nearest 01 cm) andmass (nearest 01 g) were recorded for each fish Blood plasma gilltissue and liver were collected as described above The remainingfish (N=6 per tank) were sampled after 24 days All samplingoccurred between 0900 h and 1100 h

Experiment 3 oscillating temperature acute exposure andrecoveryIn September 2011 160 fish (114ndash160 cm FL) were moved fromtheir rearing tank to one of 10 06 m diameter experimental tanks(N=16 per tank) and allowed to acclimate for 11 days prior to the startof the experiment The fish were fed to satiation once daily and thetanks were supplied with 18degC Turners Falls city water at a rate of08 l minminus1 The water temperature was increased from 18degC to 21degCover 4 days and then maintained at 21degC until the start of theexperiment The water temperature in each tank was regulated asdescribed above Each tankwas provided with supplemental aerationFeed was withheld from the fish for 24 h prior to the start of the

experiment There were five temperature treatments (two tanks per

treatment) used in this study Treatment 1 was a control group andwas maintained at 21degC throughout the study Treatment 2 consistedof a daily 4degC oscillation that fluctuated between 19degC and 23degCTreatment 3 consisted of a daily 8degC oscillation that fluctuatedbetween 17degC and 25degC Treatments 2 and 3 were repeated daily for4 days Treatments 4 and 5 featured the same temperatureoscillations as Treatments 2 and 3 but these fish only experiencedthis fluctuation on 1 day and were otherwise kept at 21degC On thelast day of exposure each treatment reached its peak temperature andwas allowed to return to 21degC where they were held for theremainder of the study in order to investigate recovery time fromelevated temperatures Temperature treatments were initiated in themorning so that peak temperature was reached in the afternoon aswould occur in nature

Four fish per tank (N=8 per treatment) were sampled 1 h afterreaching peak temperature on the fourth day of treatment Fish werekilled using a lethal dose of anesthetic (100 mgMS-222 lminus1 pH 70)so that tissues could be sampled as described Four fish per tankwere sampled 2 4 and 10 days after initial sampling as describedAll fish were fed to satiation on the morning after initial sampling(1 day) and at 3 5 7 and 9 days and the amount of feed offered wasmeasured by weight (nearest 01 g)

Physiological parametersBlood for hematocrit measurement was collected in heparanizedmicro-hematocrit capillary tubes and centrifuged at 13500 g for5 min in a micro-hematocrit centrifuge and read on a micro-capillary reader (DamonIEC Division Needham MA USA)

Plasma Clminus was measured by silver titration using a digitalchloridometer (Labconco Kansas City MO USA) Plasma glucosewas measured by enzymatic coupling with hexokinase and glucose6-phosphate dehydrogenase Plasma cortisol levels were measuredby a competitive enzyme immunoassay validated for use insalmonids (Carey and McCormick 1998) Sensitivity as definedby the dosendashresponse curve was 1ndash400 ng mlminus1 The lowerdetection limit was 03 ng mlminus1 Using a pooled plasma samplethe mean intra-assay variation was 72 (N=6) and the mean inter-assay variation was 118 (N=6)

NKA activity in gill homogenates was determined using atemperature-regulated microplate method (McCormick 1993) Gillbiopsies were homogenized in 150 microl of SEID (SEI buffer and 01deoxycholic acid) Ouabain-sensitive ATPase activity wasmeasured by coupling the production of ADP to NADH usinglactic dehydrogenase and pyruvate kinase in the presence andabsence of 05 mmol lminus1 ouabain Samples (10 microl) were run induplicate in 96-well microplates at 25degC and read at a wavelength of340 nm for 10 min on a THERMOmax microplate reader usingSOFTmax software (Molecular Devices Menlo Park CA USA)Protein concentration of the homogenate was determined using aBCA protein assay

Gill HSP70 and NKA protein abundance were measured aspreviously described (Pelis and McCormick 2001 Chadwick et al2015) The remaining homogenate from the gill NKA activity assaywas diluted with an equal volume of 2times Laemmli buffer heated for15 min at 60degC and stored at minus80degC Thawed samples were run on a75 SDS-PAGE gel at 25 microg per lane with 5 microg Precision Plusprotein standards in a reference lane (Bio-Rad LaboratoriesHercules CA USA) For each gel two additional referencesamples were run one for HSP70 and one for NKA analysisFollowing electrophoresis proteins were transferred to ImmobilonPVDF transfer membranes (Millipore Bedford MA USA) at 30 Vovernight in 25 mmol lminus1 Tris and 192 mmol lminus1 glycine buffer at

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pH 83 PVDF membranes were blocked in phosphate-bufferedsaline with 005 Triton X-100 (PBST) and 5 non-fat dried milkfor 1 h at room temperature rinsed in PBST and probed with anHSP70 antibody (AS05061 Agrisera Vaumlnnaumls Sweden) diluted120000 in PBST and 5 non-fat dried milk for 1 h at roomtemperature This antibody is specific to the inducible isoform ofsalmonid HSP70 and does not recognize the constitutive isoform(Rendell et al 2006) After rinsing in PBST blots were exposed togoat anti-rabbit IgG conjugated to horseradish peroxidase diluted110000 in PBST and 5 non-fat dried milk for 1 h at roomtemperature rinsed again in PBST exposed to chemiluminescentsolutions and then exposed to X-ray film (RPI Mount Prospect ILUSA) After imaging blots were rinsed in stripping solution(625 mmol lminus1 Tris 2 SDS 100 mmol lminus1 β-mercaptoethanolpH 67) for 30 min at 50degC to remove antigen Blots were reblockedand reprobed using an NKA α-subunit antibody (α5 IowaHybridoma Bank Iowa City IA USA) diluted 110000 followedby goat anti-mouse IgG conjugated to horseradish peroxidasediluted 110000 following the same protocol This antibody hasbeen widely used in studies on teleost fish and has been previouslyvalidated for use in salmonids (McCormick et al 2009) Digitalphotographs were taken of individual gels and band-stainingintensity was measured using ImageJ (NIH Bethesda MD USA)protein abundance is expressed as a cumulative 8-bit grayscalevalue The HSP70 and NKA reference lanes on each gel were usedto correct for inter-blot differences

StatisticsDaily growth rate in mass was calculated as 100[(natural log of endmass)ndash(natural log of start mass)]number of days Daily growthrate in length was calculated as [(end fork length)ndash(start forklength)]number of days Hepatosomatic index was calculated as100(liver massbody mass) All data are presented as meansplusmnsemIndividually marked fish were used in the study and individualswere treated as an independent replicate in all ANOVA analysesGrowth rate as a function of experimental treatment was similar inthe first 8-day and last 16-day intervals so only growth for the entire24 day experiment is presented For this reason one-way ANOVAwas used to examine the impact of temperature treatment on growthat 24 days for Experiments 1 and 2 Two-way ANOVAwas used toexamine the impact of temperature treatment and duration onphysiological parameters for all three experiments Several of thephysiological parameters (plasma cortisol gill HSP70 and NKAabundance) did not meet the assumption of homogeneity ofvariance so the data for these parameters were ranked prior toANOVA In Experiments 2 and 3 we tested for tank effects using anested ANOVA and found no significant tank effects (Pgt03) withthe exception of plasma Clndash in Experiment 2 (tank effect P=0006)As there was minor variation in this parameter (range 128ndash141 mmol lminus1) and no significant treatment effect we judged thistank effect to be biologically unimportant For all analyses theprobability of establishing statistical significance was Ple005 Priorresearch has established non-linear responses to temperature forsome of the physiological parameters in this study (Chadwick et al2015) Therefore when significant effects were detected byANOVA we subsequently conducted both linear and non-linear(first- and second-order) regression analyses using individual data(not means) and presented the result with the highest R2 valueWhere appropriate Tukeyrsquos HSD post hoc test was used todetermine differences among individual groups All statisticalanalyses were performed using Statistica 60 (Statsoft Inc TulsaOK USA)

RESULTSExperiment 1 chronic temperature growth studyMean water temperatures varied slightly from the targettemperatures and were 155 177 200 224 and 244degC duringthe 24 days (Fig 1A) Therewas one mortality in the 20degC treatmenton day 16 and one in the 24degC treatment observed on day 23Throughout 24 days daily growth rate in length was highest at 16degC(0104 mm dayminus1) and decreased significantly with temperature to alow at 24degC (minus0017 mm dayminus1) (Fig 1B) Similarly daily growthrate in mass was highest at 16degC (32 mass dayminus1) and decreasedsignificantly with temperature to a low at 24degC (minus09 mass dayminus1) (Fig 1C) Our regression model indicates that the

Temperature (degC)

16 18 20 22 24

Gro

wth

rate

(mm

day

ndash1)

ndash002

0

002

004

006

008

010

012 B

235degC

Day

Tem

pera

ture

(degC

)

14

16

18

20

22

24

26

0 4 8 12 16 20 24

A

16 18 20 22 24

Spe

cific

gro

wth

rate

( m

ass

dayndash

1 )

ndash1

0

1

2

3

4

234degC

C

Fig 1 Influence of temperature on growth rate of brook trout Watertemperature recorded every 20 min in each of the five temperature treatments(A) and their impact on linear (B) and specific growth rate in mass (C) in brooktrout Values are meansplusmnsem of 7ndash8 fish per treatment Regression lines forB and C were R2=082 and R2=081 respectively (Plt000001)

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upper limit for positive growth by length (00 mm dayminus1) and mass(00 mass dayminus1) for juvenile brook trout is 235degC and 234degCrespectively (Fig 1BC) As temperature increased the amount offeed consumed decreased (861 774 570 458 and 250 g dryfeed tankndash1) as did conversion efficiency (0432 0401 03470347 0213 and ndash0931 g dry fish g dry feedndash1) Hepatosomaticindex decreased with temperature and was 55 lower at 24degC thanat 16degC (Table 1) There was no effect of treatment length onhepatosomatic index (R2=050 temperature Plt001 treatmentlength P=021)Plasma cortisol levels were lowest at 16degC (13 ng mlminus1) and

increased with temperature to a peak of 234 ng mlminus1 at 24degC

(Fig 2A) There was a significant effect of temperature (Plt00001)no effect of treatment duration (P=054) but a significant interactionon plasma cortisol (Plt0001) Although there was a significanteffect of temperature on plasma glucose (P=0001) there was nodistinct pattern and no effect of treatment duration (P=060interaction P=0011 Fig 2B) Abundance of the inducibleisoform of HSP70 in gill tissue increased with temperature andwas 107- and 560-fold higher after 24 days at 22degC and 24degCrespectively than at 16degC (Fig 2C) There was no effect oftreatment length on gill HSP70 abundance (temperature Plt00001treatment duration P=087 interaction P=035)

Gill NKA activity decreased with temperature and after 24 dayswas 53 lower at 24degC than at 16degC (Fig 3A) Gill NKA activity

Table 1 Hematocrit plasma [Clminus] and hepatosomatic index (HSI) inbrook trout after 8 and 24 days of temperature treatment

Temperature(oC)

Hematocrit ()Plasma [Clminus](mmol lndash1)

HSI ()day 24Day 8 Day 24 Day 8 Day 24

155 32plusmn08 32plusmn08 124plusmn17 130plusmn08 27plusmn011177 34plusmn09 34plusmn11 126plusmn19 128plusmn08 25plusmn008200 35plusmn08 31plusmn07 129plusmn21 131plusmn16 29plusmn013224 32plusmn11 30plusmn07 126plusmn26 131plusmn17 18plusmn021244 34plusmn09 18plusmn12 124plusmn25 126plusmn32 12plusmn008

Temperature is the mean water temperature over the 24 days (Experiment 1)Values are meansplusmnsem of 7ndash8 fish per treatment The asterisks indicate asignificant difference from the 155degC control group

16 18 20 22 24

Pla

sma

gluc

ose

(mm

ol lndash

1 )

0

1

2

3

4

5

6 B16 18 20 22 24

Pla

sma

corti

sol (

ng m

lndash1)

0

10

20

30

A

16 18 20 22 24

Gill

HS

P70

abu

ndan

ce(8

-bit

cum

ulat

ive

gray

scal

e

103 )

0

5

10

15

20

25

Temperature (degC)

C

8 days

24 days

Fig 2 Influence of temperature on physiological stress responses inbrook troutPlasma cortisol (A) glucose (B) and gill HSP70 (C) levels in brooktrout subjected to constant temperature treatments for 8 and 24 days Valuesare meansplusmnsem of 7ndash8 fish per treatment Only significant regression linesare shown and had R2=018 P=00075 (plasma cortisol day 8) R2=050Plt000001 (plasma cortisol day 8) and R2=057 Plt000001 (gill HSP70)

16 18 20 22 24

Gill

Na+

K+ -

ATP

ase

activ

ity(micro

mol

AD

P m

gndash1

prot

ein

hndash1 )

0

1

2

3

4 A

Temperature (degC)16 18 20 22 24

Gill

Na+

K+ -

ATP

ase

abun

danc

e(8

-bit

cum

ulat

ive

gray

scal

e

103 )

0

5

10

15

20

25

30

35 B

8 days 24 days

Fig 3 Influence of temperature on gill Na+K+-ATPase (NKA) in brooktrout Gill Na+K+-ATPase (NKA) activity (A) and abundance (B) in brook troutsubjected to constant temperature treatments for 8 and 24 days Values aremeansplusmnsem of 7ndash8 fish per treatment Only significant regression lines areshown and had R2=037 Plt000001 (gill NKA activity) R2=021 P=00030(gill NKA abundance day 8) and R2=058 Plt000001 (gill HSP70)

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also decreased with treatment length (temperature Plt0001treatment duration Plt0001 interaction P=052) Similarly gillNKA abundance decreased with temperature and was 80 lower at24degC than at 16degC after 24 days (Fig 3B) There was no relationshipbetween treatment length and gill NKA abundance (temperaturePlt0001 treatment duration P=016 interaction P=0034)Hematocrit levels were between 30 and 35 in all treatmentsexcept after 24 days at 24degC where they decreased to a low of 18(Table 1 temperature Plt0001 treatment duration Plt0001interaction Plt0001) There was no relationship betweentemperature and plasma Clndash levels which were similar among alltreatments (Table 1 temperature P=026 treatment durationP=002 interaction P=073)

Experiment 2 oscillating temperature growth studyOn the first day of the growth study our heating systemmalfunctioned and peak temperatures were not achievedhowever this was the only time when there was a significantdeviation from our target temperatures (Fig 4AB) There were twomortalities during the experiment one occurred on day two in oneof our control tanks and the other on day eight in one of the 8degCoscillation tanks

In individuals sampled at 24 days growth rate (mm dayminus1)declined with increased temperature oscillation (Fig 5A P=003)and was 23 and 43 lower at 4degC and 8degC oscillationrespectively than in the 21degC control Specific growth ratewas 10 and 35 lower in the 4degC (176 mass dayminus1) and8degC (128 mass dayminus1) oscillations than in the 21degC(195 mass dayminus1) control statistical significance was notdetected by ANOVA (P=007) but was detected by regressionanalysis (Fig 5B P=0026) Feed consumed decreased withincreasing temperature oscillation (1043plusmn105 994plusmn35 and 814plusmn11 g dry feed tankndash1 P=0041) Conversion efficiency was similarat 0degC and 4degC (0259plusmn0001 and 0278plusmn0009 g dry fish gndash1 dryfeed respectively) but was lowest at 8degC oscillation (0218plusmn0033 g dry fish gndash1 dry feed) 16 lower than in the 21degC control(P=022) Hepatosomatic index increased over the duration of thestudy but there were no differences between any of the treatmentgroups (Table 1 temperature P=034 treatment duration Plt001)

Time of day (h)00

0002

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)

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25A

Day

17

19

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25

0 4 8 12 16 20 24

B

Day

17

19

21

23

25

0 2 4 6 8 10 12 14

C

Fig 4 Oscillating temperature treatments used in experiments 2 and 3Water temperature recorded every 10 min in each representative tank of eachof the three temperature treatments (AB) Experiment 2 (C) Experiment 3

0 2 4 6 8

Gro

wth

rate

(mm

day

ndash1)

0

002

004

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008

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a

ab

b

A

Daily temperature oscillation (degC)

0 2 4 6 8

Spe

cific

gro

wth

rate

( m

ass

dayndash

1 )

10

12

14

16

18

20

22 B

Fig 5 Influence of oscillating temperatures on growth rate of brook troutEffect of oscillating temperature treatments for 24 days on linear (A) andspecific growth rate in mass (B) in brook trout Values are meansplusmnsem of 14ndash15 fish per treatment (4ndash5 fish per tank) Means with the sameletters were not significantly different from one another (Pgt005 Tukeyrsquos HSDpost hoc test) Regression lines for A and B were R2=016 (P=00066) andR2=011 (P=0026) respectively

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There was no effect of temperature treatment or duration onplasma cortisol levels (Fig 6A temperature P=085 durationP=0087 interaction P=055) There was no effect of treatmenttemperature or duration on plasma glucose (Fig 6B temperatureP=036 treatment duration P=006 interaction P=027) GillHSP70 increased with magnitude of temperature oscillation(Fig 6C temperature Plt0001 treatment duration P=0005interaction P=0072) and was 40- and 700-fold greater at 4degC and8degC oscillation respectively than in the 21degC control There wasno significant effect of oscillating temperature treatment on gillNKA activity plasma chloride or hematocrit (Pgt005 data notshown)

Experiment 3 oscillating temperature acute exposure andrecoveryWe were close to achieving our target temperatures and there wasminimal deviation from the planned daily temperature fluctuations(Fig 4C) There was no effect of temperature treatment on plasmacortisol (Fig 7A temperature P=025 treatment duration P=009)or plasma glucose (Fig 7B temperature P=025) Plasma glucoselevels did increase over the course of the study (Fig 7B treatmentduration Plt001) but this occurred in all temperature treatmentsThere was a significant effect of temperature treatment and durationon gill HSP70 abundance (Fig 7C temperature Plt0001 treatmentduration Plt0001 interaction Plt0001) At 1 h gill HSP70


0 2 4 6 8

Pla

sma

corti

sol (

ng m

lndash1)

0

5

10

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25Day 8

Day 24

A

0 2 4 6 8

Pla

sma

gluc

ose

(mm

ol lndash

1 )

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8B

0 2 4 6 8

Gill

HS

P70

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ndan

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-bit

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ive

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0

10

20

30

40

a b

c

C

Fig 6 Influence of oscillating temperatures on physiological stressresponses of brook trout Plasma cortisol (A) glucose (B) and gill HSP70 (C)levels in brook trout subjected to oscillating temperature treatments for 8 and24 days Values are meansplusmnsem of 7ndash8 fish per treatment Means with thesame letters were not significantly different from one another (Pgt005 TukeyrsquosHSD post hoc test) Regression lines for HSP70 at day 8 and 24 were R2=080(Plt00001) and R2=077 (Plt00001) respectively

1 48 96 240P

lasm

a co

rtiso

l (ng

mlndash1

)0

5

10

15

20

2521degC23degC 1 day23degC 4 days25degC 1 day25degC 4 days

A

1 48 96 240

Pla

sma

gluc

ose

(mm

ol lndash

1 )

0

2

4

6

8 B

Hours after peak temperature1 48 96 240

Gill

HS

P70

abu

ndan

ce(8

-bit

cum

ulat

ive

gray

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e

103 )

0

10

20

30

40

d

d

cba

dd

ca

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aa

C

cd

Fig 7 Recovery of physiological stress responses from oscillatingtemperatures Plasma cortisol (A) glucose (B) and gill HSP70 (C) levels inbrook trout subjected to oscillating temperature treatments 1 and 4 days andthen sampled 1 48 96 and 240 h after treatment Values are meansplusmnsem of7ndash8 fish per treatment Means with the same letters were not significantlydifferent from one another (Pgt005 Tukeyrsquos HSD post hoc test) All drawnregression lines were statistically significant (glucose Plt00001 HSP70Plt0018)

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abundance corresponded with magnitude and duration of dailytemperature oscillation (control lt 4degC oscillation 1 day lt 4degCoscillation 4 days lt 8degC oscillation 1 day lt 8degC oscillation4 days) After fish were returned to control conditions gill HSP70abundance decreased over time to those of the control treatedindividuals At 4 days HSP70 abundance in individuals exposed to4degC oscillation had recovered to control levels whereas thoseexposed to 8degC oscillation remained elevated At 10 days therewere no significant differences among the treatment groups Therewas no effect of oscillating temperature on hematocrit (temperatureP=016 data not shown)

DISCUSSIONOur results indicate a decline in growth rate as temperature increasesabove 16degC in brook trout and that the upper limit for their positivegrowth is 235degC These results are in line with an extensiveliterature suggesting optimal growth of brook trout between 13degCand 16degC (Baldwin 1956 Hokanson et al 1973 Dwyer et al1983) None of these studies incorporated enough treatments abovethe optimal temperature to adequately describe brook trout growth atelevated temperatures Nor did they test temperatures high enoughto determine the upper limit for growth in brook trout Wehrly et al(2007) reported that brook trout are not found in waters above a24 day mean maximum temperature of 22degC despite the fact that thelethal temperature in this species is 253degC (Fry et al 1946 Fry1951 Wehrly et al 2007) The fact that the ecological limit is moreclosely associated with temperature limitations on growth than it iswith the lethal temperature suggests that temperature limitations ongrowth may play a key role in determining brook trout distributionsIn the current study individually marked fish and near continuous

monitoring of temperature were used to provide a clear indication ofthe relationship between temperature and growth It should be notedthat lsquotank effectsrsquo (uncontrolled variables that affect an entire tank)can have impacts on growth rate in long-term experiments (Speareet al 1995) Because even small differences in temperatureaccumulated over several weeks can impact growth we chose touse a broad range of temperatures and a regression approach ratherthan replicate a small number of temperatures This allowed us tomore accurately determine the shape of the growth curve and relateit to physiological parameters Using the same tank system as inExperiment 1 we found no evidence of tank effects in Experiment 2at either constant or fluctuating temperatures The accuracy of thisapproach is further supported by the strong correspondence betweenthe specific growth rate of 195 dayndash1 found for nominaltemperature 21degC in Experiment 2 and 175 dayndash1 that waspredicted for this temperature by the growth curve from Experiment1 (Fig 2C)It should also be noted that behavior of fish within tanks may have

a role to play in growth and physiological responses Individualhierarchies can develop within tanks (Johnsson et al 1996) andtemperature itself may affect these hierarchies This may explain thegreater variation in growth at intermediate temperatures used in thepresent study (22degC Fig 1C) than at the optimal (16degC) or highesttemperatures (24degC) However we found no fin damage in fish athigher temperatures that would indicate a high level of aggressionwas occurring under these conditionsIn spite of a well-developed literature in fish demonstrating

decreased growth at elevated temperatures the mechanism fortemperature control of growth remains unclear Induction of theendocrine stress response is likely to be involved in limiting growthat high temperature In the current study we observed an increase inplasma cortisol levels as temperature increased above 16degC with

highest plasma cortisol levels at 22degC and 24degC This effect oftemperature on plasma cortisol was greater at 24 days than it was at8 days Increased cortisol in response to elevated temperatures(Mesa et al 2002 Quigley and Hinch 2006) elevated temperatureand angling (Meka and McCormick 2005) elevated temperatureand salinity (Gonccedilalves et al 2006) and elevated temperature andexercise (Steinhausen et al 2008) has been reported in a number ofsalmonids and together with our findings implicate temperature asan endocrine stressor In the current study plasma cortisol levelswere elevated at temperatures where growth was decreased Cortisolis an important regulator of metabolism and elevated levels mayimpact growth through several pathways Elevated plasma cortisol isknown to increase metabolism as it heightens gluconeogenesis inthe liver and raises rates of catabolic pathways such as glycolysisand proteolysis (Vanderboon et al 1991 Mommsen et al 1999)This increased metabolic rate is important during times of stress as itprovides the necessary energy needed by vital organs to maintainhomeostasis but it also diverts resources away from anabolicpathways necessary for growth In the laboratory cortisoladministration has been shown to increase aerobic and anaerobicmetabolism in cutthroat trout and rainbow trout (Morgan andIwama 1996 De Boeck et al 2001) More recently cortisolinjection resulted in decreased growth in wild largemouth bass andincreased standard metabolic rate in laboratory-reared largemouthbass (OrsquoConnor et al 2011) Furthermore exposure to a dailystressor resulted in increased metabolic rate and decreased aerobicscope in green sturgeon (Lankford et al 2005)

In the current study we observed decreased feeding attemperatures that were high enough to induce a cortisol responseThis finding is in agreement with a growing literature thatdemonstrates a negative relationship between cortisol and appetiteand feeding rates in fish In rainbow trout and channel catfishexogenous cortisol administration resulted in decreased feeding andgrowth rates (Gregory and Wood 1999 Peterson and Small 2005Madison et al 2015) We have found that 30 day exogenouscortisol treatment of 8 microg gminus1 resulted in a 75 reduction in growthrate of juvenile brook trout (L Vargas-Chacoff and SDMunpublished results) The estimated plasma cortisol at the midpointof this treatment was 26 ng mlminus1 similar to the levels seen with thehighest temperature treatment in the present study (22 ng mlminus1)indicating that the observed levels of plasma cortisol in the currentstudy have the potential to affect growth in brook trout Chronicstress and dietary cortisol reduced feed intake and conversionefficiency in sea bass (Leal et al 2011) Atlantic salmon smolts andrainbow trout exhibited suppressed feeding following an acuteconfinement stressor (Pankhurst et al 2008ab) The exactmechanism for the suppression of feeding by cortisol is still underinvestigation and while there is evidence that cortisol reducesplasma ghrelin levels (Pankhurst et al 2008ab) it is likely thatother neuroendocrine factors controlling appetite are also affectedIn addition to the direct effects of cortisol it is possible thatstimulation of corticotropin-releasing factor (CRF) andadrenocorticotropic hormone (ACTH) in response to thermalstress may also affect appetite and growth in fish (Bernier andPeter 2001)

Cortisol elevated in response to high temperature may alsoinfluence growth through the growth hormoneinsulin-like growthfactor I (GHIGF-I) axis Exogenous cortisol has been shown toreduce IGF-I mRNA and plasma levels in tilapia (Kajimura et al2003) In channel catfish cortisol administration resulted in reducedgrowth and plasma IGF-I and increased plasma GH (Peterson andSmall 2005) In contrast Madison et al (2015) found that cortisol

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treatment reduced growth in rainbow trout while decreasing plasmaGH and with no change in plasma IGF-I These findings add to anextensive literature on the effects of starvation in salmonids (Deaneand Woo 2009) Nutritional restriction often leads to liver GHresistance a condition in which the downregulation of GH receptorresults in decreased plasma IGF-I despite elevated plasma GH (Grayet al 1992 Perez-Sanchez et al 1994 Pierce et al 2005) Indeedit is possible that the observed effects of cortisol on the GHIGF-Iaxis may be a secondary response to reduced food intakeCortisol modulates aspects of metabolism such as increasing

plasma glucose levels via gluconeogenesis in the liver (Vanderboonet al 1991 Wendelaar Bonga 1997 Mommsen et al 1999) In thecurrent study we found that long-term exposure to high temperaturecaused increased plasma cortisol without accompanying increasesin plasma glucose However recent work from our laboratorydemonstrated increased plasma glucose levels above 212degC inbrook trout exposed to acute temperature increases (Chadwick et al2015) Vanlandeghem et al (2010) observed an increase in plasmaglucose in largemouth bass 1 h after heat shock but not after 6 h Itis possible that glucose responds to acute but not chronictemperature elevations in brook trout and that we missed thesignal due to the timing of our sampling Increases in cortisol may infact induce increased glucose production which is matched byincreased glucose utilization resulting in no net increase in plasmaglucose It is also possible that fish at elevated temperatures simplydepleted their hepatic glycogen stores as a result of this chronicthermal stressor Indeed we observed a 55 decrease inhepatosomatic index after 24 days at 24degC compared with 16degCcontrolsIn the current study elevated temperature also induced a cellular

stress response Gill HSP70 levels increased with temperature andwere 11- and 56-fold higher at 22degC and 24degC respectively thanthey were at 16degC There was relatively little gill HSP70 at 20degC butat 22degC expression had been induced suggesting a threshold forinduction of the HSP70 response of between 20degC and 22degC Acute(hours) temperature exposures indicate a threshold for HSP70induction of 207degC in brook trout (Chadwick et al 2015)Interestingly the threshold for the heat shock response is similar toboth the upper limits for growth as well as to their upper ecologicallimit (Wehrly et al 2007) Acute exposure to elevated temperaturehas been shown to increase red blood cell HSP70 abundance abovecontrol levels in brook trout at 25degC but not at lower temperatures(Lund et al 2003) however they found increased HSP70mRNA at22degC in a variety of tissues It is likely that the assay used in theirstudy was not as sensitive as ours due to the use of an antibody thatrecognized the constitutive and inducible isoforms of HSP70 butthere are also likely to be tissue-specific differences in HSPexpression patterns In Atlantic salmon a close relative with greaterthermal tolerance than brook trout the threshold for induction ofHSP70 and HSP30 was found to be between 22degC and 25degC (Lundet al 2002) Acute exposure to 25degC or 26degC resulted in increasedhepatic HSP70 levels in rainbow trout and Chinook salmon butthese studies were not designed to determine temperature thresholdsfor induction (Mesa et al 2002 Rendell et al 2006) In addition tothese laboratory studies field-based studies have observed apositive relationship between water temperature and HSP levels inbrook trout rainbow trout and Atlantic salmon (Lund et al 2002Werner et al 2005 Feldhaus et al 2010 Chadwick et al 2015)Here we showed decreased growth at temperatures high enough

to induce gill HSP70 The HSP response is not without energeticcosts and may therefore impact growth It has been suggested thatthe synthesis of HSPs consumes an inordinate amount of cellular or

organismal nutrient stores and could occupy enough of thetranscriptional and translational machinery within the cell tohinder other essential biochemical pathways including thoseassociated with growth (Feder and Hofmann 1999) If this is truethen one might expect to see an attenuated HSP response inindividuals subjected to nutritional restriction In a number ofspecies starved fish exhibit increased HSP levels under non-stressful temperature conditions (Cara et al 2005 Yengkokpamet al 2008 Piccinetti et al 2012) but when given a temperatureincrease the HSP response in starved fish is less than that of fed fish(Deng et al 2009 Piccinetti et al 2012 Han et al 2012) Viantet al (2003) reported a correlation between HSP induction andreduced metabolic condition in juvenile steelhead trout Currentlythe metabolic cost of mounting an HSP response represents a gap inour understanding of the cellular stress response

Our findings of decreased growth with increased temperatureoscillation are similar to those reported in Lahontan cutthroat troutwhere growth declined with increasing magnitude of dailyoscillation around a mean of 18degC (Meeuwig et al 2004) Intheir study mass growth ratewas 24 and 52 lower in the 6degC and12degC daily oscillation treatments respectively than in 18degCcontrols A study in rainbow trout used several constanttemperature treatments each with a corresponding 4degC dailyoscillation treatment around the same mean temperature(Hokanson et al 1977) and found that specific growth rate wassimilar across most of the constant and oscillating treatments but atthe highest mean temperature (22degC) specific growth rate in theoscillating treatment (212 g dayminus1) was substantially lower than inthe constant control (394 g dayminus1) (Hokanson et al 1977) This isin agreement with work in coho and Atlantic salmon that utilizedrelatively low peak temperatures of 17degC and 20degC respectively(Shrimpton et al 2007 Thomas et al 1986) and found nodifference in growth between oscillating and constant temperaturetreatments We propose that oscillating temperatures that involvedaily exposure to stressful temperatures (eg those that induce anendocrine andor cellular stress response) will have negative effectson growth rate whereas those that are below the threshold forinducing stress will have little or no effect on growth In streamscontaining brook trout in northeastern USA daily variations intemperature can be as high as 6degC (JGC and SDM unpublishedobservations) Therefore the temperature variations in theselaboratory studies including the current study feature temperatureoscillations that are ecologically relevant The consistency of thesefindings give validity to the idea that daily temperature variationsthat are elevated beyond the thermal optimum result in reducedgrowth It seems likely that these effects of daily temperature ongrowth will also occur in fish in the wild Thus predictive models ofthe impact of climate change on fish (and other ectotherms) will beimproved by the inclusion of daily variations in temperature andtheir impact on growth and metabolism

We were surprised that we did not observe an endocrine stressresponse in our oscillating temperature experiment especially inlight of the clear HSP70 response Elevated plasma cortisol wasclearly shown in individuals exposed to the chronically elevatedtemperatures of Experiment 1 and we have observed elevatedplasma cortisol and glucose levels in wild brook trout sampled atsites with elevated temperatures (Chadwick et al 2015) This is inaddition to a literature that has shown elevations in plasma cortisoland glucose in response to elevated temperatures in other salmonids(Quigley and Hinch 2006 Steinhausen et al 2008) In a separateexperiment from their growth study Thomas et al (1986) observedelevated plasma cortisol in individuals exposed to a daily oscillation

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of 65ndash20degC when compared with more moderate daily oscillationsand constant temperature controls However redband troutsubjected to 8degC oscillations of 8ndash16degC or 18ndash26degC did notexhibit elevated plasma cortisol levels (Cassinelli and Moffitt2010) It is also possible that there was an endocrine stress responseinduced during our growth study and that we missed the signal dueto the timing of our sampling In Atlantic salmon plasma cortisoland glucose have been shown to return to baseline levels withinhours following an acute crowding stressor (Carey and McCormick1998) In our growth study we sampled fish in the morning whenwater temperatures had been below 21degC for at least 9 h potentiallygiving them time to recover from any thermal stress experiencedduring the previous day However in our acute exposures wesampled after 1 h at peak temperatures and still did not observeelevated plasma cortisol or glucose suggesting that these oscillatingtemperature treatments were not severe or long enough to induce anendocrine stress response It is possible that plasma cortisol willonly be elevated (or detectably different) when temperatures arehigh enough to induce severe and long-term reductions in foodconsumption and growth It should also be noted that there arediurnal (circadian) rhythms in plasma cortisol in fish that areindependent of temperature (Audet and Claireaux 1992) that mayhave affected our ability to detect temperature impacts on cortisolIn addition to thermal impacts on brook trout growth and stress

physiology we also explored the influence of elevated temperatureon osmoregulation Gill NKA activity (50) and abundance (80)decreased with temperature and were lowest at 24degC The greaterimpact on abundance than activity suggests that the activity per unitmolecule NKA is greater at elevated temperatures Despite thisthere were no differences in plasma Clndash levels in our temperaturetreatments and no effect of oscillating temperature on gill NKAactivity Elevated temperature has been shown to reduce the lengthof the smolt window as characterized by decreased gill NKAactivity and seawater tolerance in anadromous salmonids(McCormick et al 1996 1999) although this may be morerelated to temperature impacts on development than it is totemperature effects on osmoregulation per se Similarly elevatedtemperature also reduces survival and gill NKA activity in sockeyesalmon during their spawning migration (Crossin et al 2008) Aninverse relationship between temperature and gill NKA activity hasbeen observed in cod (Staurnes et al 1994) halibut (Jonassen et al1999) and pupfish (Stuenkel and Hillyard 1980) but not in turbot(Burel et al 1996) It is plausible that changes in enzyme kinetics oralterations in behavior at elevated temperatures lessen the demandfor both gill NKA activity and abundance although moreexploration in these areas is clearly neededUnderstanding the impact of temperature on animal performance

has been a goal of environmental physiology for decades (Fry1971) The threat of climate change and introduction of the oxygen-and capacity-limited thermal tolerance (OCLTT Portner 2010)have focused renewed interest on the relationship between animalphysiology and their distributions in nature In spite of this interestthere has been relatively little work in fish that characterizes therelationship between elevated temperature and decreasing foodconsumption and growth Our results indicate that the decrease ingrowth with elevated temperature is relatively gradual and not asharp decrease that characterizes many hypothetical performancecurves This distinction is important as it shapes our understandingof the response to temperature and the mechanisms behind it Such agradual decrease may not be consistent with the catastrophic loss ofprotein function which has been suggested to be involved withdeclining performance at high temperature (Portner 2010) One

possible explanation for this more gradual decrease in foodconsumption and growth rate is that animals are protectingthemselves from a dramatic decrease in aerobic scope that wouldoccur after a large meal The metabolic costs of digesting a meal canbe as high as basal metabolic rate and even higher at elevatedtemperature (Brett 1979) and thus could limit aerobic scopeMaintaining a safety margin of aerobic scope may be crucial forpredator avoidance and thus important to survival This hypothesisis consistent with the lsquovoluntaryrsquo nature of lower food consumptionat higher temperature Digestive inefficiencies and greater costs ofdigestion may play a synergistic role in this scenario

Here we demonstrated reduced growth in juvenile brook troutheld at constantly elevated temperatures These treatments alsoinduced the cellular and endocrine stress responses at thresholdssimilar to those for significant decreases in growth Growth is animportant aspect of life history that affects the reproductive capacityof an individual In salmonids a clear relationship between bodysize and fecundity has been demonstrated (Thorpe et al 1984) andan inverse relationship between temperature and reproduction hasbeen described in wild brook trout (Robinson et al 2010)Furthermore reduced body size may increase an individualrsquosvulnerability to predation and decrease its ability to establishterritory and exploit food resources Taken together the impact ofelevated temperature on growth in brook trout individuals mayprovide a mechanism by which populations are limited by elevatedtemperature We fully acknowledge that the response of any speciesto a changing environment is dynamic and that changes in growthrate represent only one of many potential responses Elevatedtemperatures may impact behavior feeding and predator avoidance(along with a host of other aspects of physiology) as well asaffecting most other species with which brook trout interactNonetheless our results present evidence of a sublethal pathwaythrough which elevated temperatures will affect animal distribution

AcknowledgementsWe thank Ken Simmons Craig Lodowsky and the staff of the Sandwich StateHatchery for providing juvenile brook trout We thank Michael OrsquoDea for help inrearing the fish and in running the gill Na+K+-ATPase activity assay and AmyRegishfor her help in running the cortisol assayWe thank Andy Danylchuk and Paul Sievertfor their comments on an early draft of this manuscript and two anonymousreviewers for their comments which helped improve the final version Certainportions of this manuscript were taken directly from the Masterrsquos thesis of JGC Jrwho owns the copyright

Competing interestsThe authors declare no competing or financial interests

Author contributionsConceptualization JGC SDM Methodology JGC SDM Validation JGCSDM Formal analysis JGC Resources SDM Data curation JGC Writing -original draft JGC Writing - review amp editing JGC SDM Supervision SDMProject administration SDM Funding acquisition JGC SDM

FundingThis research was supported by the National Science Foundation through aGraduate Research Fellowship to JGC Additional funding for this work came fromthe Organismic amp Evolutionary Biology Program at the University of MassachusettsAmherst in the form of a student research grant

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various blood parameters in brook trout (Salvelinus fontinalis) Can J FishAquat Sci 49 870-877

Baldwin N S (1956) Food consumption and growth of brook trout at differenttemperatures Trans Am Fish Soc 86 323-328

Bernier N J and Peter R E (2001) The hypothalamicndashpituitaryndashinterrenal axisand the control of food intake in teleost fish Comp Biochem Physiol B BiochemMol Biol 129 639-644

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Brett J R (1979) Bioenergetics and growth Fish Physiol 8 599-675Burel C Person-Le Ruyet J Gaumet F Le Roux A Severe A and BoeufG (1996) Effects of temperature on growth and metabolism in juvenile turbotJ Fish Biol 49 678-692

Callaghan N I Tunnah L Currie S and MacCormack T J (2016) Metabolicadjustments to short-term diurnal temperature fluctuation in the rainbow trout(Oncorhynchus mykiss) Physiol Biochem Zool 89 498-510

Cara J B Aluru N Moyano F J and Vijayan M M (2005) Food-deprivationinduces HSP70 and HSP90 protein expression in larval gilthead sea bream andrainbow trout Comp Biochem Physiol B Biochem Mol Biol 142 426-431

Carey J B and McCormick S D (1998) Atlantic salmon smolts are moreresponsive to an acute handling and confinement stress than parr Aquaculture168 237-253

Cassinelli J D and Moffitt C M (2010) Comparison of growth and stress inresident redband trout held in laboratory simulations of montane and desertsummer temperature cycles Trans Am Fish Soc 139 339-352

Chadwick J G Nislow K H and McCormick S D (2015) Thermal onset ofcellular and endocrine stress responses correspond to ecological limits in brooktrout an iconic cold water fish Conserv Physiol 3 cov017

Crossin G T Hinch S G Cooke S J Welch D W Patterson D A JonesS R M Lotto A G Leggatt R A Mathes M T Shrimpton J M et al(2008) Exposure to high temperature influences the behaviour physiology andsurvival of sockeye salmon during spawning migration Can J Zool 86 127-140

Deane E E Woo N Y S (2009) Modulation of fish growth hormone levels bysalinity temperature pollutants and aquaculture related stress a review RevFish Biol Fish 19 97-120

De Boeck G Alsop D and Wood C (2001) Cortisol effects on aerobic andanaerobic metabolism nitrogen excretion and whole-body composition injuvenile rainbow trout Physiol Biochem Zool 74 858-868

Deng D-F Wang C F Lee S Bai S and Hung S S O (2009) Feeding ratesaffect heat shock protein levels in liver of larval white sturgeon (Acipensertransmontanus) Aquaculture 287 223-226

DuBeau S F Pan F Tremblay G C and Bradley T M (1998) Thermal shockof salmon in vivo induces the heat shock protein HSP 70 and confers protectionagainst osmotic shock Aquaculture 168 311-323

Dwyer W P Piper R G and Smith C E (1983) Brook trout growth efficiency asaffected by temperature Prog Fish-Cult 45 161-163

Feder M E and Hofmann G E (1999) Heat-shock proteins molecularchaperones and the stress response Evolutionary and ecological physiologyAnnu Rev Physiol 61 243-282

Feldhaus J W Heppell S A Li H and Mesa M G (2010) A physiologicalapproach to quantifying thermal habitat quality for Redband Rainbow Trout(Oncorhynchus mykiss gairdneri) in the south Fork John Day River OregonEnviron Biol Fishes 87 277-290

Flebbe P A Roghair L D and Bruggink J L (2006) Spatial Modeling toproject southern Appalachian trout distribution in a warmer climate Trans AmFish Soc 135 1371-1382

Fry F E J (1951) Some environmental relations of the speckled trout (Salvelinusfontinalis) Proc NEAtlantic Fish Conf 1 1-29

Fry F E J (1971) The effect of environmental factors on the physiology of fish InFish Physiology Volume VI Environmental Relations and Behavior (ed W SHoar and D J Randall) pp 1-98 New York Academic Press

Fry F E J Hart S A and Walker K F (1946) Lethal temperature relations for asample of young speckled trout Salvelinus fontinalis Univ Toronto Stud BiolSer 54 9-35

Gonccedilalves J Carraccedila S Damasceno-Oliveira A Fernandez-Duran B DiazJ Wilson J and Coimbra J (2006) Effect of reduction in water salinity onosmoregulation and survival of large Atlantic salmon held at high watertemperature N Am J Aquac 68 324-329

Gray E S Kelley K M Law S Tsai R Young G and Bern H A (1992)Regulation of hepatic growth-hormone receptors in coho salmon (Oncorhynchus-Kisutch) Gen Comp Endocrinol 88 243-252

Gregory T R and Wood C M (1999) The effects of chronic plasma cortisolelevation on the feeding behaviour growth competitive ability and swimmingperformance of juvenile rainbow trout Physiol Biochem Zool 72 286-295

Han D Huang S S Y Wang W-F Deng D-F and Hung S S O (2012)Starvation reduces the heat shock protein responses in white sturgeon larvaeEnviron Biol Fishes 93 333-342

Hokanson K E F McCormick J H Jones B R and Tucker J H (1973)Thermal requirements for maturation spawning and embryo survival of the brooktrout Salvelinus fontinalis J Fish Res Board Can 30 975-984

Hokanson K E F Kleiner C F and Thorslund T W (1977) Effects of constanttemperatures and diel temperature fluctuations on specific growth and mortalityrates and yield of juvenile rainbow trout Salmo gairdneri J Fish Res Board Can34 639-648

Iwama G K Vijayan M M Forsyth R B and Ackerman P A (1999) Heatshock proteins and physiological stress in fish Am Zool 39 901-909

Iwama G K Afonso L O B Todgham A Ackerman P and Nakano K(2004) Are HSPs suitable for indicating stressed states in fish J Exp Biol 20715-19

Johnsson J I Jonsson E and Bjornsson B T (1996) Dominance nutritionalstate and growth hormone levels in rainbow trout (Oncorhynchus mykiss) HormBehav 30 13-21

Jonassen T M Imsland A K and Stefansson S O (1999) The interaction oftemperature and fish size on growth of juvenile halibut J Fish Biol 54 556-572

Kajimura S Hirano T Visitacion N Moriyama S Aida K and Grau E G(2003) Dual mode of cortisol action on GHIGF-IIGF binding proteins in thetilapia Oreochromis mossambicus J Endocrinol 178 91-99

Lankford S E Adams T E Miller R A and Cech J JJr (2005) The cost ofchronic stress Impacts of a nonhabituating stress response on metabolicvariables and swimming performance in sturgeon Physiol Biochem Zool 78599-609

Leal E Fernandez-Duran B Guillot R Rıos D and Cerda-Reverter J M(2011) Stress-induced effects on feeding behavior and growth performance of thesea bass (Dicentrarchus labrax) a self-feeding approach J Comp Physiol BBiochem Syst Environ Physiol 181 1035-1044

Lund S G Caissie D Cunjak R A Vijayan M M and Tufts B L (2002) Theeffects of environmental heat stress on heat-shock mRNA and protein expressionin Miramichi Atlantic salmon (Salmo salar) parr Can J Fish Aquat Sci 591553-1562

Lund S G Lund M E A and Tufts B L (2003) Red blood cell HSP 70 mRNAand protein as bioindicators of temperature stress in the brook trout (Salvelinusfontinalis) Can J Fish Aquat Sci 60 460-470

Madison B N Tavakoli S Kramer S andBernier N J (2015) Chronic cortisoland the regulation of food intake and the endocrine growth axis in rainbow troutJ Endocrinol 226 103-119

McCormick S D (1993) Methods for nonlethal gill biopsy andmeasurement of Na+K+ -ATPase activity Can J Fish Aquat Sci 50 656-658

McCormick J H Jones B R and Hokanson K E F (1972) Effects oftemperature on growth and survival of young brook trout Salvelinus fontinalisJ Fish Res Board Can 29 1107-1112

McCormick S D Shrimpton J M and Zydlewski J D (1996) Temperatureeffects on osmoregulatory physiology of juvenile anadromous fish In GlobalWarming Implications for Freshwater and Marine Fish (ed C M Wood and D GMcDonald) pp 279-301 Cambridge Cambridge University Press

McCormick S D Cunjak R A Dempson B OrsquoDea M F and Carey J B(1999) Temperature-related loss of smolt characteristics in Atlantic salmon(Salmo salar) in the wild Can J Fish Aquat Sci 56 1649-1667

McCormick S D Regish A M and Christensen A K (2009) Distinctfreshwater and seawater isoforms of Na+K+-ATPase in gill chloride cells ofAtlantic salmon J Exp Biol 212 3994-4001

McMahon T E Zale A V Barrows F T Selong J H and Danehy R J(2007) Temperature and competition between bull trout and brook trout A test ofthe elevation refuge hypothesis Trans Am Fish Soc 136 1313-1326

Meeuwig M H Dunham J B Hayes J P and Vinyard G L (2004) Effects ofconstant and cyclical thermal regimes on growth and feeding of juvenile cutthroattrout of variable sizes Ecol Freshw Fish 13 208-216

Meisner J D (1990) Potential loss of thermal habitat for brook trout due to climaticwarming in 2 southern ontario streams Trans Am Fish Soc 119 282-291

Meka J M and McCormick S D (2005) Physiological response of wild rainbowtrout to angling impact of angling duration fish size body condition andtemperature Fish Res 72 311-322

Mesa M G Weiland L K andWagner P (2002) Effects of acute thermal stresson the survival predator avoidance and physiology of juvenile fall chinooksalmon Northwest Sci 76 118-128

Mommsen T P Vijayan M M and Moon T W (1999) Cortisol in teleostsdynamics mechanisms of action and metabolic regulation Rev Fish Biol Fish9 211-268

Morgan J D and Iwama G K (1996) Cortisol-induced changes in oxygenconsumption and ionic regulation in coastal cutthroat trout (Oncorhynchus clarkiclarki) parr Fish Physiol Biochem 15 385-394

OrsquoConnor C M Gilmour K M Arlinghaus R Matsumura S Suski C DPhilipp D P and Cooke S J (2011) The consequences of short-term cortisolelevation on individual physiology and growth rate in wild largemouth bass(Micropterus salmoides) Can J Fish Aquat Sci 68 693-705

Pankhurst N W King H R and Ludke S L (2008a) Relationship betweenstress feeding and plasma ghrelin levels in rainbow trout Oncorhynchus mykissMar Freshwater Behav Physiol 41 53-64

Pankhurst N W Ludke S L King H R and Peter R E (2008b) Therelationship between acute stress food intake endocrine status and life historystage in juvenile farmed Atlantic salmon Salmo salar Aquaculture 275 311-318

Pelis R M and McCormick S D (2001) Effects of growth hormone and cortisolon Na+-K+-2Cl(minus) cotransporter localization and abundance in the gills of Atlanticsalmon Gen Comp Endocrinol 124 134-143

Perez-Sanchez J Marti-Palanca H and Le Bail P-Y (1994) Homologousgrowth-hormone (Gh) binding in gilthead sea bream (Sparus-Aurata) - effect offasting and refeeding on hepatic Gh-binding and plasma somatomedin-likeimmunoreactivity J Fish Biol 44 287-301

Peterson B C and Small B C (2005) Effects of exogenous cortisol on the GHIGF-IIGFBP network in channel catfish Domest Anim Endocrinol 28 391-404

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Piccinetti C C Ricci L A Tokle N Radaelli G Pascoli F Cossignani LPalermo F Mosconi G Nozzi V Raccanello F et al (2012) Malnutritionmay affect common sole (Solea solea L) growth pigmentation and stressresponse molecular biochemical and histological implications Comp BiochemPhysiol A Mol Integr Physiol 161 361-371

Pierce A L Shimizu M Beckman B R Baker D M and Dickhoff W W(2005) Time course of theGHIGFaxis response to fasting and increased ration inchinook salmon (Oncorhynchus tshawytscha) Gen Comp Endocrinol 140192-202

Portner H-O (2010) Oxygen- and capacity-limitation of thermal tolerance amatrixfor integrating climate-related stressor effects in marine ecosystems J Exp Biol213 881-893

Portner H O and Farrell A P (2008) Ecology physiology and climate changeScience 322 690-692

Portner H O Farrell A P Knust R Lannig G Mark F C and Storch D(2009) Adapting to climate change response Science 323 876-877

Quigley J T and Hinch S G (2006) Effects of rapid experimental temperatureincreases on acute physiological stress and behaviour of stream dwelling juvenilechinook salmon J Therm Biol 31 429-441

Rendell J L Fowler S Cockshutt A and Currie S (2006) Development-dependent differences in intracellular localization of stress proteins (HSPs) inrainbow trout Oncorhynchus mykiss following heat shock Comp BiochemPhysiol D Genomics Proteomics 1 238-252

Robinson J M Josephson D C Weidel B C and Kraft C E (2010)Influence of variable interannual summer water temperatures on brook troutgrowth consumption reproduction and mortality in an unstratified adirondacklake Trans Am Fish Soc 139 685-699

Shrimpton J M Zydlewski J D and Heath J W (2007) Effect of dailyoscillation in temperature and increased suspended sediment on growth andsmolting in juvenile chinook salmon Oncorhynchus tshawytscha Aquaculture273 269-276

Smith T R Tremblay G C and Bradley T M (1999) Characterization of theheat shock protein response of Atlantic salmon (Salmo salar) Fish PhysiolBiochem 20 279-292

Sokolova I M (2013) Energy-limited tolerance to stress as a conceptualframework to integrate the effects of multiple stressors Integr Comp Biol 53597-608

Speare D J Macnair N and Hammell K L (1995) Demonstration of tank effecton growth of juvenile rainbow trout (Oncorhynchus mykiss) during an ad libitumfeeding trial Am J Vet Res 56 1372-1379

Staurnes M Rainuzzo J R Sigholt T and Joslashrgensen L (1994) Acclimationof Atlantic cod (Gadus-Morhua) to cold-water - stress-response osmoregulation

gill lipid-composition and gill Na-K-Atpase activity Comp Biochem Physiol APhysiol 109 413-421

Steinhausen M F Sandblom E Eliason E J Verhille C and Farrell A P(2008) The effect of acute temperature increases on the cardiorespiratoryperformance of resting and swimming sockeye salmon (Oncorhynchus nerka)J Exp Biol 211 3915-3926

Stitt B C Burness G Burgomaster K A Currie S McDermid J L andWilson C C (2014) Intraspecific variation in thermal tolerance and acclimationcapacity in brook trout (Salvelinus fontinalis) physiological implications for climatechange Physiol Biochem Zool 87 15-29

Stuenkel E L and Hillyard S D (1980) Effects of temperature and salinity on GillNa+-K+ ATPase activity in the pupfish Cyprinodon salinus Comp BiochemPhysiol A Physiol 67 179-182

Thomas R E Gharrett J A Carls M G Rice S D Moles A and Korn S(1986) Effects of fluctuating temperature onmortality stress and energy reservesof juvenile coho salmon Trans Am Fish Soc 115 52-59

Thorpe J E Miles M S and Keay D S (1984) Developmental rate fecundityand egg size in Atlantic salmon Salmo salar L Aquaculture 43 289-305

Vanderboon J Vandenthillart G E E J and Addink A D F (1991) Theeffects of cortisol administration on intermediary metabolism in teleost fishCompBiochem Physiol A Physiol 100 47-53

Vanlandeghem M M Wahl D H and Suski C D (2010) Physiologicalresponses of largemouth bass to acute temperature and oxygen stressors FishManag Ecol 17 414-425

Viant M R Werner I Rosenblum E S Gantner A S Tjeerdema R S andJohnson M L (2003) Correlation between heat-shock protein induction andreduced metabolic condition in juvenile steelhead trout (Oncorhynchus mykiss)chronically exposed to elevated temperature Fish Physiol Biochem 29 159-171

Wehrly K E Wang L Z and Mitro M (2007) Field-based estimates of thermaltolerance limits for trout Incorporating exposure time and temperature fluctuationTrans Am Fish Soc 136 365-374

Wendelaar Bonga S E (1997) The stress response in fish Physiol Rev 77591-625

Werner I Smith T B Feliciano J and Johnson M L (2005) Heat shockproteins in juvenile steelhead reflect thermal conditions in the Navarro Riverwatershed California Trans Am Fish Soc 134 399-410

Wikelski M and Cooke S J (2006) Conservation physiology Trends Ecol Evol21 38-46

Yengkokpam S Pal A K Sahu N P Jain K K Dalvi R Misra S andDebnath D (2008) Metabolic modulation in Labeo rohita fingerlings duringstarvation HSP70 expression and oxygen consumption Aquaculture 285234-237

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is conflicting evidence on the impact of oscillating temperatures onthe growth of fishes (Hokanson et al 1977 Thomas et al 1986Meeuwig et al 2004 Shrimpton et al 2007) which may relate tothe highest temperature fish experience and the magnitude ofthermal variationThere is increasing interest in utilizing mediators of the stress

response such as increased plasma cortisol glucose lactate andHSP expression as indicators of both individual- and population-level responses to environmental stressors including elevatedtemperature (Wendelaar Bonga 1997 Iwama et al 1999 2004Lund et al 2002 Wikelski and Cooke 2006 Wendelaar Bonga1997) Experimental warming of juvenile Chinook salmon andrainbow trout resulted in elevated circulating cortisol and glucoselevels (Meka and McCormick 2005 Quigley and Hinch 2006)Similarly adult sockeye salmon exhibited elevated plasma cortisoland lactate levels in response to increased temperature and exercise(Steinhausen et al 2008) A variety of studies have demonstratedelevated HSPs in response to elevated temperature in a variety ofsalmonid species in the laboratory (DuBeau et al 1998 Smithet al 1999 Mesa et al 2002 Lund et al 2003 Rendell et al2006 Stitt et al 2014) and in the wild (Werner et al 2005) Recentlaboratory and field work from our group indicate that acutetemperature thresholds for increased gill HSP70 (207degC) andplasma glucose (212degC) in brook trout are similar to their proposedthermal ecological limit of 210degC (Chadwick et al 2015)Sublethal temperatures may act via the stress response to inhibitgrowth in individuals (Iwama et al 1999) potentially limitingpopulations (Portner 2010)Based on the studies cited above we predicted that temperatures

above the optimum would result in a relatively steep decline ingrowth rate and that oscillating temperatures would result indecreased growth compared with constant temperatures with thesame mean temperature We also predicted that observed decreasesin growth due to temperature treatment would be accompanied byincreased endocrine (plasma cortisol) andor cellular (HSP70)stress responses In order to test these predictions we exposed brooktrout to constant elevated temperatures (target temperatures of 1618 20 22 or 24degC) or to daily temperature oscillations of 0 4 or 8degC(constant 21degC 19ndash23degC or 17ndash25degC) for 24 days In addition tomeasuring growth we collected blood and gill tissue to assessbiomarkers for stress including gill HSP70 plasma cortisol andglucose We also examined gill Na+K+-ATPase (NKA) activity andabundance and plasma Clminus to explore the relationship betweentemperature and osmoregulation A third experiment was conductedto examine acute responses and recovery from daily temperaturevariations Brook trout were exposed to the same daily temperaturevariations as above for 1 day or 4 days and then sampled 1 h 2 4and 10 days later to determine the recovery period following dailytemperature oscillations

MATERIALS AND METHODSFish stockJuvenile (0+) brook trout were obtained from the Sandwich StateHatchery (Sandwich MA USA) and brought to the ConteAnadromous Fish Research Center (Turners Falls MA USA) inJuly 2009 for the chronic elevated temperature study and in July2010 and 2011 for the oscillating temperature studies Fish werehoused in 17 m diameter tanks (150ndash200 fish per tank) suppliedwith 4 l minminus1 of chilled Connecticut River water (16plusmn2degC) andgiven supplemental aeration Fish were fed to satiation (FinfishStarter Zeigler Bros Gardners PA USA) twice daily withautomatic feeders and maintained under natural photoperiod

Rearing and experiments were carried out in accordance withUSGS Animal Care Guidelines under IACUC 9070 Fish were usedin the same year they were obtained so that 0+ fish of similar sizewere used in all studies During all experiments fish were fed acommercial trout pellet (42 protein 16 fat Finfish Gold ZieglerBros)

Experiment 1 chronic temperature growth studyIn August 2009 one week before the start of the experiment 80 fish[124ndash158 cm fork length (FL)] were removed from their rearingtank and lightly anesthetized with buffered and neutralized tricainemethanesulfonate (50 mgMS-222 lminus1 pH 70) While anesthetizedthe fish were measured for length (nearest 01 cm) and mass (nearest01 g) and implanted with a passive integrated transponder (PIT) taginserted into the abdominal cavity via a small (sim5 mm) incision inthe ventral surface just rostral to the pelvic fins After recoveringfrom the anesthetic the fish were divided randomly into five 09 mdiameter experimental tanks (N=16 per tank) The tanks weresupplied with Turners Falls city water at a rate of 09 l minminus1 and thetemperature was maintained at 16degC with 800 W titanium bayonetheaters Each tank was provided with supplemental aeration Fishwere fed to satiation twice daily throughout the experiment Thetanks were isolated from the rest of the laboratory so that the dailyfeeding was the only disturbance that the fish experienced At eachfeeding fish were carefully observed so that food was offered onlyuntil the point where it was no longer being consumed (when one ortwo pellets would drop to the bottom of the tank) The amount offeed consumed at each feeding was measured to the nearest 01 g

Feed was withheld from the fish for 24 h prior to the start of theexperiment and at any other time that length and mass weremeasured Seven days after PIT tags were implanted fish were againmeasured for length and mass and returned to their appropriate tankWater temperatures were then elevated at a rate of 2degC hminus1 until thetarget temperatures of 16 18 20 22 or 24degC were reached Eachtank was maintained at its specific target temperature for theremainder of the study The water temperature of each tank wasmeasured and recorded every 15 min using Hobo temperatureloggers (Onset Computer Corporation Bourne MA USA) Waterflow rate and feeding regime were as described with a 75 waterchange every 4 days Dissolved oxygen levels were measured dailyand were always above 90 saturation

The fish were measured for length and mass every 8 days for24 days On the eighth day half of the fish (N=8 per tank) were killedusing a lethal dose of anesthetic (100 mg MS-222 lminus1 pH 70) sothat blood and tissue samples could be taken The remainder of thefish (N=8 per tank) were sampled after 24 days Blood was collectedfrom the caudal vessels using 1 ml ammonium heparanized syringeswithin 5 min of tank disturbance The blood was spun at 3200 g for5 min at 4degC and the plasma was aliquoted and stored at minus80degC Abiopsy of four to six gill filaments was taken from the first arch andimmersed in 100 microl of ice-cold SEI buffer (150 mmol lminus1 sucrose10 mmol lminus1 EDTA 50 mmol lminus1 imidazole pH 73) and stored atminus80degC The liver was removed weighed (nearest 00001 g) andstored at minus80degC After sampling the gills and blood the remainingcarcass was reweighed and then dried to a constant mass (48 h) at60degC to obtain dry mass for the calculation of conversion efficiency

Experiment 2 oscillating temperature growth studyIn October 2010 90 fish (120ndash169 cm FL) were moved from theirrearing tank to one of nine 06 m diameter experimental tanks(N=10 per tank) and allowed to acclimate for one week prior to thestart of the experiment The fish were fed to satiation once daily and

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gray

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103 )

0

5

10

15

20

25

30

35 B

8 days 24 days


3980


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Time of day (h)00

0002

0004

0006

0008

0010

0012

0014

0016

0018

0020

0022

0000

00

Tem

pera

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(degC

)

17

19

21

23

25A

Day

17

19

21

23

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0 4 8 12 16 20 24

B

Day

17

19

21

23

25

0 2 4 6 8 10 12 14

C


0 2 4 6 8

Gro

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rate

(mm

day

ndash1)

0

002

004

006

008

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a

ab

b

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Spe

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gro

wth

rate

( m

ass

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1 )

10

12

14

16

18

20

22 B


3981


Journal

ofEx

perim

entalB

iology




0 2 4 6 8

Pla

sma

corti

sol (

ng m

lndash1)

0

5

10

15

20

25Day 8

Day 24

A

0 2 4 6 8

Pla

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ose

(mm

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1 )

0

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0

10

20

30

40

a b

c

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0

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20

30

40

d

d

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ca

cd

aa

C

cd


3982


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ofEx

perim

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iology









3983


Journal

ofEx

perim

entalB

iology








3984


Journal

ofEx

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entalB

iology














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Journal

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entalB

iology





















































3986


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ofEx

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iology




























3987


Journal

ofEx

perim

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iology




Time of day (h)00

0002

0004

0006

0008

0010

0012

0014

0016

0018

0020

0022

0000

00

Tem

pera

ture

(degC

)

17

19

21

23

25A

Day

17

19

21

23

25

0 4 8 12 16 20 24

B

Day

17

19

21

23

25

0 2 4 6 8 10 12 14

C


0 2 4 6 8

Gro

wth

rate

(mm

day

ndash1)

0

002

004

006

008

010

a

ab

b

A


0 2 4 6 8

Spe

cific

gro

wth

rate

( m

ass

dayndash

1 )

10

12

14

16

18

20

22 B


3981


Journal

ofEx

perim

entalB

iology




0 2 4 6 8

Pla

sma

corti

sol (

ng m

lndash1)

0

5

10

15

20

25Day 8

Day 24

A

0 2 4 6 8

Pla

sma

gluc

ose

(mm

ol lndash

1 )

0

2

4

6

8B

0 2 4 6 8

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ce(8

-bit

cum

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gray

scal

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103 )

0

10

20

30

40

a b

c

C


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a co

rtiso

l (ng

mlndash1

)0

5

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A

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(mm

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1 )

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gray

scal

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0

10

20

30

40

d

d

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dd

ca

cd

aa

C

cd


3982


Journal

ofEx

perim

entalB

iology









3983


Journal

ofEx

perim

entalB

iology








3984


Journal

ofEx

perim

entalB

iology














3985


Journal

ofEx

perim

entalB

iology





















































3986


Journal

ofEx

perim

entalB

iology




























3987


Journal

ofEx

perim

entalB

iology




0 2 4 6 8

Pla

sma

corti

sol (

ng m

lndash1)

0

5

10

15

20

25Day 8

Day 24

A

0 2 4 6 8

Pla

sma

gluc

ose

(mm

ol lndash

1 )

0

2

4

6

8B

0 2 4 6 8

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-bit

cum

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gray

scal

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103 )

0

10

20

30

40

a b

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C


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(mm

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3982


Journal

ofEx

perim

entalB

iology









3983


Journal

ofEx

perim

entalB

iology








3984


Journal

ofEx

perim

entalB

iology














3985


Journal

ofEx

perim

entalB

iology





















































3986


Journal

ofEx

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entalB

iology




























3987


Journal

ofEx

perim

entalB

iology









3983


Journal

ofEx

perim

entalB

iology








3984


Journal

ofEx

perim

entalB

iology














3985


Journal

ofEx

perim

entalB

iology





















































3986


Journal

ofEx

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entalB

iology




























3987


Journal

ofEx

perim

entalB

iology








3984


Journal

ofEx

perim

entalB

iology














3985


Journal

ofEx

perim

entalB

iology





















































3986


Journal

ofEx

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entalB

iology




























3987


Journal

ofEx

perim

entalB

iology














3985


Journal

ofEx

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entalB

iology





















































3986


Journal

ofEx

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entalB

iology




























3987


Journal

ofEx

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entalB

iology





















































3986


Journal

ofEx

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entalB

iology




























3987


Journal

ofEx

perim

entalB

iology




























3987


Journal

ofEx

perim

entalB

iology

upper thermal limits of growth in brook trout and their ... · for brook trout growth (baldwin,...

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