Evaluation of the physiological status of the Pacificsardine, Sardinops sagax caeruleus, acclimated to differentthermal regimes based on selected blood parameters

Marcel Martínez-Porchas &

Mónica Hernández-Rodríguez &

Sharon Z. Herzka & Luis E. Gutierrez-Millán

Received: 23 March 2010 /Accepted: 23 November 2010 /Published online: 8 December 2010# Springer Science+Business Media B.V. 2010

Abstract Despite the widespread recognition of theimportance of temperature in the population dynamicsand distribution of Pacific sardine (Sardinops sagaxcaeruleus), few studies have examined the species’physiological response to temperature under con-trolled conditions. The effect of fluctuating thermalregimes on blood plasma cortisol, glucose, aspartateaminotransferase (ALT), alanine aminotransferase

(AST) and red blood cell (RBC) counts of Pacificsardine was evaluated to monitor physiologicalresponse. Sardines from the southern subpopulationthat inhabits waters off Baja California, Mexico, werecollected during fall 2008. Acclimation to fourfluctuating temperature regimes (two symmetric andtwo asymmetric) and a constant, optimal temperatureoccurred over 20 d. Symmetric and asymmetricregimes were designed to simulate regional summer(18–23°C) and winter (13–18°C) temperature rangeswithin 24 h. In the optimal regime (OR), sardineswere acclimated to 18°C. In symmetric regimes,sardines were exposed for 7 h to the high and lowtemperatures, while in asymmetric regimes exposureto the high temperature was shorter (4 vs.10 h). Bloodsampling took place during the low and hightemperature exposure, and we tested for differencesin blood parameters compared to OR. Plasma cortisoland glucose concentration of sardines from summerregimes were only significantly higher at 23°C. ASTactivities were significantly higher than OR during thehigh and low temperatures of both summer treat-ments. No differences were found in individualsexposed to the winter regimes. ALT activities weresignificantly higher than OR only during the high andlow temperature of the symmetric summer regime.RBC were significantly higher than OR for bothsummer regimes. Sardines from southern subpopula-tion exhibit a negative physiological response to hightemperatures, yet they can acclimate to the lower(≤18°C) temperatures typical of the California Current

Environ Biol Fish (2011) 91:39–49DOI 10.1007/s10641-010-9757-z

M. Martínez-PorchasCentro de Investigación en Alimentacióny Desarrollo (CIAD),Km 0.6 Carretera a La Victoria,83304 Hermosillo, Sonora, Mexico

M. Hernández-Rodríguez (*)Departamento de Acuicultura (CICESE) México,Carretera Ensenada-Tijuana No. 3918,22860 Ensenada, Baja California, Mexicoe-mail: mhernand@cicese.mx

S. Z. HerzkaDepartamento de OceanografíaBiológica (CICESE) México,Carretera Ensenada-Tijuana No. 3918,22860 Ensenada, Baja California, Mexico

L. E. Gutierrez-MillánDepartamento de Investigaciones Científicas yTecnológicas de la Universidad de Sonora,83000 Hermosillo, Sonora, Mexico

Present Address:M. Hernández-RodríguezDepartment of Aquaculture,P.O. Box: 434844, San Diego, CA 92143-4844, USA

System. Temperatures ≥23°C may be considered adetrimental sublethal temperature, particularly forlong exposure periods.

Keywords Sardinops sagax caeruleus .

Subpopulation . Sardine physiology . Stress indicators .

Blood parameters . Temperature

Introduction

Forage species are a key trophic component of theCalifornia Current System, an upwelling ecosystem ofthe western margin of North America (Emmett et al.2005; Horn and Ferry-Graham 2006). The currentrange of the Pacific sardine (Sardinops sagax caer-uleus) extends from British Columbia, Canada, to thesouthern Pacific coast of the Baja California peninsulaand the Gulf of California (McFarlane and Beamish2001; Nevárez-Martínez et al. 2001). Pacific sardinescurrently support important fisheries off the westerncoast of North America and within the Gulf ofCalifornia (Emmett et al. 2005; Sweetnam 2006). Inaddition to serving as forage for many species of fish,marine mammals and birds, sardines are also animportant food source for human consumption andlivestock and aquaculture feeds (Frimodt 1995;Schweigert 2002).

Pacific sardine populations exhibit a high level oftemporal and spatial variation in abundance (seereview in Checkley et al. 2009). For almost a century,researchers have used a variety of approaches toevaluate the relationship between environmental con-ditions and their distribution, population dynamicsand stock structure (Radovich 1982; Marr 1960;Cisneros-Mata et al. 1995; García-Fontes et al.1996; McFarlane and Beamish 2001; Smith 2005).Geographic shifts in its distribution and abundancehave been related to oceanographic regime shifts, ElNiño Southern Oscillation events and overfishing,among others (Radovich 1982; Francis et al. 1998;Lluch-Belda et al. 1991; Chavez et al. 2003).

Studies indicate temperature plays an important rolein the species’ abundance, distribution and populationdynamics, either directly or indirectly (e.g., Marr 1960;Lluch-Belda et al. 1991; McFarlane and Beamish2001; Félix-Uraga et al. 2004). For example, thecollapse of the northern sardine population thatinhabited Canadian, US and Mexican waters during

the late 1940s has been linked to decadal-scaletemperature shifts in the pelagic environment in theregion, although overfishing may have contributed tothe collapse (McFarlane et al. 2005). Key criteriacurrently used to distinguish among subpopulations inthe California Current System are spawning tempera-ture and egg abundance, as well as region-specificlanding data and sea surface temperature (SST; seereview in Smith 2005). Seasonal migration patternshave also been closely linked with temperature (Félix-Uraga et al. 2004), and satellite-derived SSTs havebeen used to characterize the temporal and spatialdistribution of spawning habitat (Reiss et al. 2008).

Climate change can impact populations of smallpelagic fish through a variety of direct or indirectmechanisms, including variations at the base of thefood web due to changes in productivity, modifica-tion of spatial and temporal abundance patterns, andalterations of recruitment levels due to oceano-graphic shifts (Freón et al. 2009). Specifically,temperature variations in the pelagic environmentdue to climate change could influence the distribu-tion and abundance of Pacific sardine. In particular,Pacific sardine from Ensenada may undergo dailythermal fluctuations from 18 to 23°C in summer, butit has been also documented that they can thrive inlower thermal fluctuations such as 13 to 18°C, whichare common in northern waters during summer or inEnsenada during winter (Martínez-Porchas et al.2009a). However, periods of exposure to higher orlower temperatures could not be always the same,because they can move vertically in the watercolumn, influenced by a variety of environmentalfactors (Emmett et al. 2005). Evaluation of thephysiological response of sardines to temperaturevariations and exposure periods could thus contrib-ute to the understanding of the species’ distributionat a variety of scales, and is also necessary forpredicting the potential response of the species todifferent climate change scenarios.

Despite the widespread recognition of the impor-tance of temperature in the population dynamics anddistribution of Pacific sardines, very few studies haveexamined the species’ physiological response totemperature under controlled laboratory conditions(see Lasker and Theilacker 1962; Lasker 1962, 1964for studies on larvae; Martínez-Porchas et al. 2009a).Hence, there is very limited information on the effectof temperature on the physiological status of S. sagax.

40 Environ Biol Fish (2011) 91:39–49

Recently, Hernández-Lopez (2006) found that theexposure of sardines to lethal and critical temper-atures (>24°C) caused dysfunction of the liver andkidneys; in addition Martínez-Porchas et al. (2009a)also found that the same specie´s avoided to introducein temperatures higher than 23°C in a thermalgradient and preferred to undergo lower temperatures(until 13°C). It is well recognized that fish areparticularly susceptible to temperature due to its effecton rates of feeding, metabolism, growth and excre-tion, as well as other physiological responses (Brett1979; Linton et al. 1997).

The measurement of blood parameters has been auseful tool for evaluating stress levels and physiolog-ical status of fishes (Hattingh 1977; Wells et al. 1984;Morales et al. 2004; Öner et al. 2008). However, thereare a limited number of studies assessing the responseof blood parameters of marine pelagic and schoolingspecies to stressful conditions. For instance, Marçaloet al. (2006, 2008) found that the European sardine(Sardina pilchardus) exhibited increasing cortisol andglucose levels after being exposed to purse seinefishing and captivity.

The aim of this research was to evaluate theeffect of temperature on specific blood parametersof sardine acclimated to different thermal regimesin order to study their physiological and stressresponse. Cortisol and glucose play a major role inthe bioenergetics of organisms, and are recognizedas indicative of a stress response such as exposureto a thermal challenge. Cortisol stimulates theformation and secretion of glucose in differenttissues (Iwama et al. 1999), and both parametersincrease in response to the greater energy demandcaused by stress (King et al. 2006; Lima and Ribeiro2006). Aminotransferase enzymes (aspartate amino-transferase and alanine aminotransferase) are foundin the liver, kidney, gills, intestine, brain, and otherorgans, and play a key role in fish protein andcarbohydrate metabolism. These enzymes leak intoblood plasma as a consequence of tissue damage ordysfunction (Ciereszko et al. 1998; Oluah 1999).They had been extensively used to monitor thephysiological status of fishes exposed to stressfulconditions (Wells et al. 1986; Poledník et al. 2008).Another response that can help evaluate the effect oftemperature is red blood cells (RBC) count. Eryth-rocytes play a major role in oxygen transport andCO2 removal. RBC increase in response to a higher

oxygen demand due to an increase in temperature orexposure to stressful conditions.

Materials and methods

Sardines 19.0±1.0 cm standard length (SL; mean ±standard deviation [SD]) and 68.5±3.5 g (mean ± SD)were captured by the live bait fishery that operates inTodos Santos Bay in Baja California, Mexico, duringSeptember 2008. Sardines were caught at 19–20°C.Based on Félix-Uraga et al.’s (2004) study relatingfishery catches to sea surface temperature (SST) alongthe species’ distribution, and what is currently knownwith regard to its population structure and verticalpatterns (Smith 2005) the sardines we used in ourexperiments belong to the southern subpopulation, orthe so-called “temperate group” (caught at SST 17–22°C). Sardines from the “cold group” are caught offnorthern Baja California, California and the PacificNorthwest at SSTs of 13 to 17°C, and those from the“warm group” are caught off the southern BajaCalifornia Peninsula (>22°C) (Félix-Uraga et al.2004).

Sardines are extremely delicate, and massivemortality can occur due to handling. We developeda protocol that substantially reduced the mortality ofsardines during transport to the laboratory. Shortlyfollowing capture, sardines were transported in 25 Lplastic bags (25 sardines/bag) to the Marine FishCulture Laboratory at the Centro de InvestigaciónCientífica y de Educación Superior de Ensenada(CICESE). The water within the bags was maintainedat 20°C and dissolved oxygen (DO) was >7 mg L−1.Upon arrival to the laboratory, sardines were intro-duced into 1500 L tanks (21°C, DO 5.90 mg L−1, pH7.8, total ammonia nitrogen [TAN] 0.12 mg L−1). Atetracycline treatment was administered (50 mg L−1)for 3 h during each of two d to eliminate parasites andpathogens. On the third day, a herbal antibacterial(Melafix®) treatment was administered for 5 h(0.05 mL L−1) to further eliminate pathogens andheal injuries caused during capture and transport. Inorder to minimize stress and allow for a recoveryperiod, sardines were maintained in the laboratory for1 month under a 300% daily water exchange rate anda 12:12 photoperiod. They were fed ad libitum with atrout diet (42% protein; 1 mm pellet size, Silvercup®).Survival was monitored daily and dead fish were

Environ Biol Fish (2011) 91:39–49 41

immediately removed from the tanks to avoid anegative effect on water quality.

Thermal acclimation

Sardines were introduced into 300 L circular tanks (4tanks/treatment; 40 individuals·tank−1) and acclimatedto five thermal regimes. Two of the regimes weresymmetrical (the same amount of time was spent atthe high and low temperature during each 24 hperiod), two were asymmetrical (longer exposure tothe high daily temperature, see below) and a fifth wasa constant optimal temperature regime. The tempera-ture ranges used for symmetric and asymmetricregimes were selected based on the seasonal temper-ature fluctuations typical of Todos Santos Bay, thetemperature range at which sardines from differentsubpopulations are captured along the Pacific (Félix-Uraga et al. 2004) and considering that sardinesexhibit vertical migration over a daily cycle (Emmettet al. 2005).

In symmetric and asymmetric regimes, sardineswere exposed to fluctuating temperatures during thecourse of each 24 h period, while in the fifth regimesardines were maintained at a constant 18°C (optimalregime, OR). The OR temperature was establishedbased on prior thermoregulation experiments, whichindicated 18°C was the temperature preference ofsardines independent of previous acclimation temper-ature (Hernández, unpubl. data).

In the symmetrical winter regime (SWR), temper-ature fluctuated from 13 to 18°C. Water temperaturewas maintained at a constant 13°C during 7 h,gradually increased to 18°C over the course of 5 h,remained constant at 18°C for 7 h and decreased to13°C over 5 h (7-5–7-5) (Fig. 1). The symmetricalsummer regime (SSR) was similar, but the range oftemperatures was 18–23°C. During each 24 h period,water temperature was increased or kept constantusing titanium heaters (1000 Watt). A decrease intemperature was achieved by injecting cool water(4°C, 3 L·min−1) with a submersible pump from anexternal 800 L reservoir. The pump and heaters wereconnected to an automatic controller (Chrontrol XT,ChronTrol Corp.) programmed to turn them on andoff according to the time of the day. The temperaturein the experimental tanks was monitored every 15 minwith automatic temperature loggers (StowAway®TidbiT™, Onset).

Sardines exposed to the asymmetric thermalregimes also simulated winter and summer tempera-ture conditions (AWR and ASR, respectively). Thesame temperature ranges used for symmetricalregimes were used (13–18°C and 18–23°C). Howev-er, in the asymmetrical thermal regimes sardines wereexposed to the low and high temperatures for 4 and10 h, respectively (4-5–10-5) (Fig. 1).

Sardines were acclimated to each thermal regime for20 d prior to evaluation of blood parameters. Theacclimation period was established as a chronic expo-sure so that sardines could be acclimated and avoid a“general alarm reaction” (Martinez-Porchas et al.2009b) based also on previous data regarding theacclimation period required for this particular species(Hernández unpubl. data). The high and low temper-atures of the symmetrical thermal regimes wereprogrammed to occur at 12:00 and 00:00, respectively.In the asymmetric thermal regimes the high and lowtemperature exposure began at 12:00 and 21:00,respectively. The light was turned on from 7:00 to19:00 for all treatments. The transition from light todark and vice versa was carried out gradually over a30 min period to minimize stress.

Physicochemical conditions in the thermal regimetanks were similar to those maintained during theacclimation period (DO 6.03 mg L−1, pH 7.8, TAN0.12 mg L−1); DO and pH were monitored by anoxygen meter (YSI 55; YSI Incorporated®) and a pHmeter (Hanna instruments®) whereas TAN wasmeasured by a colorimetric method (HACH DR/4000; Hach, Co.). Sardines were fed ad libitum threetimes a day with the same trout food. Excess food,faeces and dead animals were removed from the tanksevery day in order to maintain optimal water qualityconditions. Given that the inside of the tanks used forthermal regime experiments was white, only whitecolored nets were used to extract experimentalorganisms prior to blood sampling to minimize thestress due to capture.

Blood sampling

After 20 d of acclimation, all fish were sacrificed andblood samples were collected from sardines at themidpoints of the highest and lowest temperature ofeach thermal regime. Sardines were anesthetized with0.3 ml L−1 of 2-phenoxyethanol for 5 min prior toextracting blood using the heart puncture technique

42 Environ Biol Fish (2011) 91:39–49

(Schreck and Moyle 1990). Blood samples wereplaced in heparinized microtainer tubes and centri-fuged at 10 000 rpm during 10 min to obtain plasma.Glucose, cortisol, alanine and aspartate aminotrans-ferase (ALT and AST, respectively) in blood plasmawere measured from samples obtained at the high andlow temperature, while red blood cells (RBC) countswere performed only on blood sampled at the hightemperatures.

Cortisol was measured with an ELISA kit(NEOGEN ®, #402710). The cortisol sample wasextracted from plasma with ethyl ether, preparedwith enzyme conjugate (Cortisol Enzyme Conjugate[HRP]) and the substrate (K-Blue Substrate [TMB])was added to the sample. Absorbance at 650 nmwas read with a microplate spectrophotometer(Beckman AD200) after a 30 min incubation periodat 25°C. The concentration of cortisol (ng mL−1)was determined using a standard curve derivedfollowing the kit’s instructions.

Glucose concentration (mg dL−1) was analyzedusing the glucose-oxidase method and a prefabricatedkit (Randox ® Gluc-Pap, GL 1021). A subsample of10 μL of plasma were mixed with 1 ml of glucosereagent from the kit, incubated during 25 min and readat 500 nm. Results were compared with a blank and astandard of known concentration (100 mg dL−1).

Different concentrations of the original standard wereprepared by dilution (75, 50 and 25 mg dL−1). Theabsorbance at each concentration was read at 500 nmand a standard curve was derived relating glucoseconcentration to absorbance (r2=0.977). The concen-tration of glucose in plasma was obtained using thefollowing equation:

mg=dL ¼ Abs� sample

Abs� standard

� �Standard concentration

AST (EC 2.6.1.1) concentrations were determinedusing the GOT/ASAT UV method described by Sizerand Jenkins (1962) for the measurement of amino-transferase enzymes. A subsample of 0.1 mL ofplasma was mixed with 2.0 mL of α-oxoglutarate +L-aspartate and the change in absorbance (ΔAbs) wasregistered every minute at 340 nm for 5 min. Themean ΔAbs·min−1 was multiplied by 1746 to obtainunits of enzyme per liter of plasma (U L−1). Toanalyze ALT (ALT EC 2.6.1.2, AL 1200), 0.1 mL ofplasma subsample were added to 2.0 mL of α-oxoglutarate + L-alanine. The mean change inabsorbance (ΔAbs) at 340 nm per minute wasmultiplied by 1746 (U L−1).

To estimate the number of red blood cells(cells·mL−1), a whole blood sample was placed in a

12162024

Optimal Regime

12162024

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12162024

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Tem

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(°C

)

Time (Days)

12162024

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12162024

1 2 3 64 5 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Asymmetric Summer Regime

Fig. 1 Thermal regimes towhich sardines (S. sagax)were acclimated. Tempera-tures in the acclimationtanks were measured withthermographs. The arrowsindicate sampling points

Environ Biol Fish (2011) 91:39–49 43

Thoma pipette (Propper), diluted 1:200 withHendrick’s solution (Hendrick 1952) and bloodcells were counted in a hematocytometer (HausserScientific 3100).

Statistical analyses

To evaluate for differences in the values of bloodparameters between treatments and sampling times(the high or low temperature period of the fourtreatments), we used a two-way ANOVA with anested design. We nested for sampling temperaturewithin treatments and used a post-hoc test to test fordifferences between treatments. To test for differencesin blood parameters of sardines collected during thehigh or low temperature periods of each cyclingtreatment vs. the optimal temperature treatment, weused two separate one-way ANOVAs. ALT data weresquare root transformed prior to analysis so as tocomply with the assumptions of normality (testedwith the Shapiro-Wilk’s test) and homogeneity ofvariance (Levene test). Post-hoc multiple comparisonswere carried using a Dunnett’s test (2-sided). Pairwisecomparisons were used to evaluate whether a givenparameter differed significantly from the OR treat-ment. A chi-square test was used to compare thesurvival response of animals exposed to differentthermal acclimations.

Results

Sardine survival during the 20 d thermal acclimationperiod was 96, 84, 94, 68 and 80% for OR, SWR,AWR, SSR and ASR, respectively; however, nosignificant differences were observed among treat-ments (p=0.10 chi-square). Survival was lowest in thethermal acclimation treatments that included thehighest temperatures (SSR and ASR). Between thesetwo treatments, survival was highest in the asymmet-ric regime, in which the amount of time sardines spentat the highest temperature was shorter. There were nosignificant differences in total ammonia nitrogen,nitrite, pH and salinity levels among thermal regimes.Dissolved oxygen levels (5.95–6.04 mg L−1)remained within an optimal range in all cases(Table 1).

The two-way nested analysis of variance indicatedsampling temperature (F=13.5, p=0.00), but not the

temperature regime (F=1.0, p=0.47) had a significanteffect on cortisol levels. The cortisol levels weresignificantly higher when measured at the highesttemperature of each treatment compared to thoseobserved at the lower temperatures (18 and 13°C).When compared to the cortisol levels from sardinesacclimated to the constant optimal temperature (OR),no statistical differences were relative to 18°Cindependent of acclimation regime (≈18 ng·mL−1;p≥0.99). However, cortisol levels were significantlylower at 13°C of SWR and AWR (p=0.01 and p=0.04, respectively). In sardines sampled during thehighest temperature (23°C), cortisol levels were 58%(p=0.00) and 38% (p=0.02) higher for the ASR andSSR, respectively, than those sampled in the OR(Fig. 2).

Glucose levels were also affected by the samplingtemperature (F=15.9, p=0.00) but not by acclimationregime (F=0.7, p=0.58). Glucose levels were higherin sardines sampled at the highest temperature of eachtreatment compared to those sampled at the lowertemperature. Compared to the OR, glucose concen-trations were significantly higher (p≤0.03) at 23°C inboth summer regimes; specifically, concentrationswere 58% and 25% higher during the high tempera-ture period of SSR and ASR, respectively, comparedto OR (Fig. 2). No significant differences (p>0.40)were observed among glucose levels of OR sardinesand sardines acclimated to both winter regimes (SWRand AWR) and sampled at 13 or 18°C.

In contrast, two-way ANOVA indicated AST levelswere affected by acclimation regime (F=78.2, p=0.00),but not by sampling temperature (F=0.4, p=0.78). Theenzyme levels were higher in sardines acclimated atboth summer regimes (SSR and ASR) compared tothose acclimated to any of the winter regimes (SWR andAWR). Compared to OR (100 UL−1), sardines fromboth summer regimes had significantly greater (p=0.00)AST values (139–170 UL−1 and 150–180 UL−1 forASR and SSR, respectively), independent of samplingtemperature (18 or 23°C). Similar values of enzymeactivity were observed for optimal and winter regimes(≈100 UL−1, p=0.98).

ALT levels were highly variable (CV=0.6). Nev-ertheless, results were similar to those for AST;acclimation regime affected the enzyme levels (F=25.5, p=0.01), but sampling temperature did not havea significant effect on ALT (F=0.3, p=0.84). How-ever, this effect was only observed for sadines

44 Environ Biol Fish (2011) 91:39–49

0

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**

** ** ****

** **

** ** ****

SWR AWR SSR ASR SWR AWR SSR ASR

SWR AWR SSR ASR SWR AWR SSR ASR

Fig. 2 Mean ± SD of plasma cortisol, glucose, aspartate andalanine aminotransferases (AST & ALT) in sardines acclimatedto fluctuating thermal regimes. White bars represent the optimaltemperature acclimation regime (18°C). Gray bars indicatesymmetric and asymmetric winter regimes (SWR & AWR), andthe black bars represent the values of the symmetric and

asymmetric summer regimes (SSR & ASR). Blood sampleswere taken during the high and low temperatures of the winter(13 and 18°C) and summer (18 and 23°C) regimes. Asterisksindicate statistical differences (**) (P<0.05) compared with theoptimal regime

Table 1 Mean ± standard deviation of water quality parametersmeasured in fluctuating thermal acclimation tanks in whichPacific sardines were maintained for 20 d. Measurements weretaken daily. Temperatures in parentheses indicate the constant

temperature to which sardines were exposed in the optimalthermal regime or the low and high temperatures used insymmetrical and asymmetrical thermal regimes. DO Dissolvedoxygen, TAN Total ammonia nitrogen

Optimalregime(18°C)

Symmetrical winterregime (13–18°C)

Asymmetrical winterregime (13–18°C)

Symmetrical summerregime (18–23°C)

Asymmetrical summerregime (18–23°C)

DO (mg L−1) 6.02±0.11 6.04±0.06 6.01±0.04 5.92±0.11 5.95±0.05

TAN (mg L−1) 0.12±0.10 0.12±0.10 0.09±0.06 0.16±0.12 0.14±0.03

NO2 (mg L−1) 0.08±0.05 0.11±0.10 0.10±0.08 0.10±0.08 0.11±0.02

pH 7.82±0.05 7.77±0.05 7.80±0.04 7.80±0.08 7.82±0.05

Salinity (‰) 33.5±1.0 33.6±0.5 33.1±1.6 33.6±0.6 33.6±0.5

Total time at thehighest temperature(h)

– 140 140 80 80

Environ Biol Fish (2011) 91:39–49 45

aclimated to the SSR. One-way ANOVA’s indicatedno significant differences in ALT levels betweentreatments (10 UL−1, F=1.89, p=0.71) and OR,except for the SSR regime,which showed significantgreater values (p=0.03) during the high and lowtemperatures of each treatment (18 ad 23°C) and OR(Fig. 2).

Red blood cell (RBC) concentrations were lower insardines exposed to both winter regimes (SWR andAWR) compared with OR; however significant differ-ences were only observed among the OR and theSWR. The highest RBC were found during bothsummer regimes, but there were no significant differ-ences (F=18.43, p=0.11) when compared with OR(Fig. 3).

Discussion

The effect of thermal regimes on the stressful andphysiological status of sardines was observed in theblood parameters. The levels measurements of glu-cose and cortisol were useful to evaluate the stressfulstatus of fishes, whereas the AST, ALT and RBCrevealed their physiological status. Increases inglucose and cortisol were oserved in stressed sardines,while increases in AST and ALT revealed a deter-iotated physiological status. Cortisol and glucosewere affected by sampling temperature, while amino-transferase enzymes and erithrocytes were influencedby the chronic exposure to the different thermalregimes.

In our experiments, mean values of cortisol,glucose and ALT activity were not significantlydifferent when sampled at 18°C compared with OR,independent of acclimation regime. This is consistentwith results obtained in previous studies, in whichPacific sardine chose temperatures of 18°C in athermal gradient independently of the previous accli-mation temperature (Martínez-Porchas et al. 2009a).In contrast, we found that the high temperature usedduring the summer acclimation regime (23°C) didhave a significant effect on the physiologicalperformance of S. sagax caeruleus. The levels ofcortisol, glucose were significantly higher whenmeasured at 23°C compared to 18°C; AST and ALTwere higher at low and high temperatures of bothsummer regimes and RBC counts had higher values.In addition, sardines from the summer symmetric andasymmetric regimes showed lower survival than inthe optimal regime and both winter regimes (AWRand SWR).

Increases in the levels of these blood parametershave been observed in response to adverse environ-mental conditions, such as stressful temperatures,thermal shock, handling, air exposure and others(Thomas et al. 1986; King et al. 2006; Choi et al.2007). Adverse conditions can cause a stress responsein fish, leading to an increased energy demand toallow for organismal and cellular level responses to astressor challenge (Iwama et al. 1999). The increasedenergy demand caused by the high temperatureexposure was reflected in the higher concentrationsof cortisol and glucose exhibited by sardines exposedto the symmetric and asymmetric summer regimes.Likewise, several studies have reported increases inboth parameters in teleost fishes when exposed totemperatures outside the preferred range or otherstressors (Thomas et al. 1986; Le-Morvan et al. 1995;King et al. 2006; Lima and Ribeiro 2006).

The negative effect of high temperature on sardinephysiology was also reflected in ASTand ALTactivities,which were higher in sardine from both summer regimes(SSTand ASR) than in the optimal regime. High stressfultemperaturesmay cause injuries to teleost tissues, causingthe release of AST and ALT to the bloodstream inresponse to organ dysfunction and cellular lysis (Oluah1999; Choi et al. 2007). Hernández-Lopez (2006)demonstrated that high stressful temperatures can affectthe tissue integrity of the Pacific sardine when exposedto 25, 26 and 27°C; histological analysis indicated high

Fig. 3 Mean ± SD of red blood cell concentration ofsardines subjected to an optimal temperature regime (whitebar), symmetric and asymmetric fluctuating winter regimes(SWR & AWR, gray bars) and the symmetric and asymmetricsummer regimes (SSR & ASR, black bars). Asterisks indicatestatistical differences in fluctuating temperature regimes (**)(P<0.05) compared with the optimal regime

46 Environ Biol Fish (2011) 91:39–49

temperatures caused degeneration of hepatic cells. Inaddition, the increased activity of AST and ALT at 23°Cmay be at least partly related to the increase in cortisolconcentrations, because high cortisol levels can causetissue degeneration in teleosts (Dickhoff 1989).

Although AST and ALT activities were high whenmeasured at 18 or 23°C, the intensity of the responsewas higher in the symmetric regime. Sardines wereexposed to 23°C for a total of 140 h in SSR compared to80 h during the ASR. These results are consistent withthe observation that Pacific sardine from the southernsubpopulation actively avoid temperatures >22.5°Cwhen introduced into a horizontal thermal gradient(Martínez-Porchas et al. 2009a). The responses of ASTand ALT were not transient with the temperature ofthermal regimes, as was the case for cortisol andglucose, because release of cortisol and glucosedepend upon endocrine activity and aminotransferaselevels are mainly related with injuries and organmalfunctions.

The highest concentration of red blood cells was alsoobserved in sardines exposed to the summer regimes.High temperatures cause an increase in metabolic rate,which is supported through biochemical oxidation path-ways using the oxygen molecules transported byerythrocytes (Wittenberg andWittenberg 1989). Lecklinet al. (1995) acclimated rainbow trout (Oncorhynchusmykiss) to different temperatures (4, 11 and 18°C) andalso observed that the number of erythrocytes increasedas a function of acclimation temperature (0.71, 0.77 and1.07×109cells·mL−1 respectively).

Taken together, the response of blood parameterswe measured at 23°C indicate that higher temper-atures are likely to have a negative effect on thephysiological performance of Pacific sardines. This isconsistent with the results of Martínez-Porchas et al.(2009a), who found that Pacific sardines from thesouthern subpopulation have upper incipient lethaltemperature (UILT) near 24°C. Thus, physiologicalperformance was negatively affected at 23°C becauseit was close to their lethal temperature. In addition,van der Lingen (1994) found that respiration rates ofSardinops sagax from the Benguela Current Systemincreased exponentially with temperature between 10and 22°C. Higher temperatures thus require a higherenergy demand, which in turn can lead to the stressresponse we observed.

In contrast, exposure to a low temperature (13°C)did not have a negative physiological response, at

least as indicated by the blood parameters wemeasured. The significant decrease in cortisol con-centration in sardines exposed to 13°C compared to18°C does not suggest a stress response, but rather anacclimation capacity to a lower energy demand. Alower energy demand requires a lower amount ofglucose, which in turn requires a lower cortisol levelto stimulate the release of glucose to the bloodstream(Begg and Pankhurst 2004). If 13°C had beenstressful for sardines, the expected response wouldhave been an increase in cortisol and glucose levels,as has been observed in other experiments withteleosts (Le-Morvan et al. 1995).

In the Mexican Pacific, sardines are captured atSSTs higher than 23°C. For example, in the Gulf ofCalifornia sardine are captured in waters with a SSThigher than 28°C (Lluch-Belda et al. 1991; Félix-Uraga et al. 2004). However, sardines in the Gulf ofCalifornia are known to exhibit large (up to 300 m)vertical migrations in response to warm surfacewaters (M. Nevárez, pers. comm.) and that theyactively seek regions of the gulf in which there ispersistent upwelling (Hammann et al. 1988; Nevárez-Martínez et al. 2001).

In contrast, sardines showed an optimal perfor-mance at lower temperatures (13 and 18°C). Sardinesare typically captured at 13–18°C (Félix-Uraga et al.2004), which is consistent with the absence of a stressresponse and an agreement with our observations.Based on the blood parameters we measured and thethermal behavior of sardines from the same locality(in a previous year) and acclimated to similar thermalregimes (Martínez-Porchas et al. 2009a), it can beassumed that sardines inhabiting Ensenada (duringfall) are able to acclimate to the lower temperatures(13–18°C) that are characteristic of the CaliforniaCurrent System and which includes the thermalhabitat of the cold subpopulation (13–17°C) (Félix-Uraga et al. 2004). Considering only temperature,Pacific sardines from Ensenada can withstand temper-atures as low as 13°C without exhibiting a stressresponse. The results contribute to the findings thatrevealed that phenotypic and slight genetic differ-ences among the different subpopulations are higherto the extreme limits of the distribution and almostimperceptible in among the subpopulations of thecenter (Hedgecock et al. 1989; Lacomte et al. 2004;De la Cruz-Agüero and García-Rodríguez 2005) suchas the temperate and cold groups.

Environ Biol Fish (2011) 91:39–49 47

The sardines studied in this experiment showed tobe susceptible to high temperatures (≥23°C) whereasthey exhibited a great physiological performance atlower temperatures; that could be a particular charac-teristic from sardines of the temperate subpopulations.Also, the measured blood parameters showed to beconfident indicators of the physiological status of thespecies. Nonetheless, it is important to continuestudying the effect of temperature and other factorson the physiological performance of the differentsubpopulations to continue differentiating among thediverse groups and contribute to the knowledge of thespecies’ dynamics and distribution.

Acknowledgments This work was supported by the FederalGovernment of Mexico, through regular funding of the ConsejoNacional de Ciencia y Tecnología (CoNaCyT) grant P 46060-Z.Lydiah Ladah (Department of Biological Oceanography,CICESE) provided temperature data collected with thermo-graphs at 10 m during July-August 2005 and December-January 2005-2006 in Todos Santos Bay. We also extent ourgratefulness to Francisco Valenzuela, Luis Murillo, NorbertoFlores, José Carballo, Adrian Celaya and José Espinoza fortheir technical support.

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