Ecophysiological biomarkers defining the thermal biology of the Caribbean lobster Panulirus argus

Ecological Indicators 78 (2017) 192–204

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Ecophysiological biomarkers defining the thermal biology of the Caribbean lobster Panulirus argus Gabriela Rodríguez-Fuentes a,c , Margarita Murúa-Castillo d , Fernando Díaz e , Carlos Rosas b,c , Claudia Caamal-Monsreal b,c , Ariadna Sánchez b , Kurt Paschke f , Cristina Pascual b,c,∗ a

Unidad de Química en Sisal, Facultad de Química, Universidad Nacional Autónoma de México, Sisal, Yucatán, Mexico Unidad Multidisciplinaria de Docencia e Investigación, Facultad de Ciencias, Universidad Nacional Autónoma de México, Puerto de Abrigo s/n, Sisal, Yucatán, Mexico c Laboratorio Nacional de Resiliencia Costera (LANRESC), CONACyT, Mexico d Posgrado en Ciencias del Mar y Limnología, Facultad de Química, Sisal, Yucatán, Mexico e Laboratorio de Ecofisiología de Organismos Acuáticos, Departamento de Biotecnología Marina, Centro de Investigación Científica y de Educación Superior de Ensenada, BC, Mexico f Instituto de Acuicultura, Universidad Austral de Chile, Puerto Montt, Chile b

a r t i c l e

i n f o

Article history: Received 18 October 2016 Received in revised form 1 March 2017 Accepted 2 March 2017 Keywords: Thermal biology Panulirus argus Metabolism REDOX system

a b s t r a c t Tropical populations of marine species are predicted to be the most impacted by global warming because they are likely adapted to a narrow range of temperatures in their local environment. In the present study, we investigated the thermal range at which activity metabolic rate (AMR) is maximal, pejus, and critical in the spiny lobster Panulirus argus using different ecophysiological biomarkers. Lobsters were acclimated at constant temperatures (18, 22, 26 and 30 ◦ C; N = 150) at laboratory conditions for 35 days; after that time thermal biology was evaluated. Results obtained demonstrated that, in P. argus the temperature where AMR was maximal does not coincide with temperature that promotes the maximum growth rate (26 ◦ C). In fact, the preferred temperature, determined in a horizontal gradient was around the mean value of 27.5 ◦ C, 5.5 ◦ C above the temperature where animals showed their maximum metabolic scope, growth and thermal preference. Lactate and redox imbalance showed its maximum values in animals acclimated at 30 ◦ C indicating that lobsters where in pejus thermal ranges. Although lobsters are adapted to tolerate temperatures beyond 28 ◦ C, results indicated that temperature and exposure time play important roles in the way organisms respond to thermal challenges. It is likely that the difference in the responses of different ectothermic organisms and the thermal response is linked to the way these effects are measured. These results can help generate predictive models to anticipate changes in the P. argus fishery derived from likely changes in distribution and abundance of this species under warming scenarios of tropical oceans. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction In the last few years there were dozens of papers testing the oxygen- and capacity-limited thermal tolerance (OCLTT) hypothesis (Deutsch et al., 2015; Pörtner, 2010; Pörtner and Knust, 2007) in attempt to validate and/or give new elements to understand how temperature modulates the energy allocation of the aquatic

∗ Corresponding author at: Unidad Multidisciplinaria de Docencia e Investigación, Facultad de Ciencias, Universidad Nacional Autónoma de México, Puerto de Abrigo s/n, Sisal, Yucatán, Mexico. E-mail address: [email protected] (C. Pascual). http://dx.doi.org/10.1016/j.ecolind.2017.03.011 1470-160X/© 2017 Elsevier Ltd. All rights reserved.

organisms. Those studies made on fish (Chen et al., 2015; Gräns et al., 2014; Madeira et al., 2012b; Norin et al., 2014; Rummer et al., 2014), mollusks (Mosrash and Alter, 2015; Oellermann et al., 2012; Sokolova et al., 2011) and crustaceans (Ern et al., 2014, 2015; Lejeusne et al., 2014; Tepolt and Somero, 2014; Vinagre et al., 2014) have reach different conclusions depending in part of the experimental protocols used to acclimate and maintain experimental organisms. Those differences have stimulated intense debates in which acclimation was seriously considered as one of the key element of the experimental protocols that must be addressed in a standardized form before to conclude if OCLTT hypothesis have or not a universal application in all types of aquatic ectotherm organisms (Jutfelt et al., 2014; Pörtner, 2014; Wang and Overgaard, 2007).

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There are studies that, evaluating the OCLTT hypothesis, found that some fish as Fundulus heteroclitus or crustaceans as Macrobrachium rosenbergii can growth as in optimal conditions when exposed to temperatures beyond the temperature range where aerobic scope is maximum (Ern et al., 2014; Healy and Schulte, 2012) or fish as Hippoglosus hippoglosus that have a maximum growth when aerobic scope is lower than observed in higher temperatures (Gräns et al., 2014), failing in predicting that a reduction or increment in aerobic performance is directly related with growth. Aerobic scope can be calculated as the difference between standard (SMR) and maximum metabolic rates (MMR) (Clark et al., 2013). When those metabolic rates are measured in animals acclimated at different temperatures two curves can be obtained (Fry, 1947). The relationship between SMR and temperature is frequently an exponential curve. While the aerobic metabolism and temperature normally shows that, in the extreme temperatures, the difference between SMR and MMR is zero (Ferreira et al., 2014), in optimal temperature ranges such differences are maxima, showing a maximum aerobic scope (AS) (Fry, 1947; Pörtner, 2010; Sokolova et al., 2012). In maximum aerobic scope, lactate levels should be reduced and the antioxidant mechanisms should be in a low level, reflecting low levels of peroxidation (Trübenbach et al., 2013). Further proxies may be useful to estimate aerobic power budget. Given the evidence that there is a strong and predictable relationship between activity and temperature in ectotherms, a certain level of activity may be induced by thermal exposure (Halsey et al., 2015). Halsey et al. (2015) showed in the crab Carcinus maenas that active metabolic rate (AMR) and in consequence, total metabolic rate (MR) were affected by acclimation temperature because there is a direct effect of temperature on the biochemical reactions involved in locomotion. Based on this, we put forward the idea that temperature can be used as a standardized tool to determine activity metabolic rate (AMR) as a proxy of AS, particularly amongst individuals lacking regularly active behavior and considering, from a behavioral perspective, temperature will increase the activity to maximize locomotion in extreme conditions or provoke less activity when temperature provoke a state of aestivation, where the resting metabolic rate (RMR) is forced (Fig. 1). According with the concept that use the critical thermal (CT) maximum (CT-max) or minimum (CT-min) as a tool to evaluate the thermal tolerance of ectotherm species (Hutchison, 1961; Terblanche et al., 2011; Vinagre et al., 2016), and in our experience (Díaz et al., 2006; Noyola et al., 2015; Paschke et al., 2013), thermal tolerance can be evaluated using the dynamic method, where critical thermal maximum (CT-max) and minimum (CT-min) are determined by a gradual increment or reduction of temperature (usually with at a rate close to 1 ◦ C min−1 ) until an end-point is reached. In this method, the rate should be slow enough to permit deep body temperature to equilibrate with water temperature; to follow the test temperature without a significant time lag. According with Hoffmann et al. (2003), slower rates provide sufficient time for hardening a form of phenotypic plasticity that protect cells of subsequent injury, preventing a real evaluation of the acclimation temperature-tolerance relationship. Although the CT limits may occur at different temperatures in different species, there are many reports that has demonstrated that the behavioral response is the same across a diversity of taxa (Salas et al., 2014; Vinagre et al., 2016). For these reasons, CT-max is an excellent index and standard for evaluating the thermal requirements and physiology of aquatic organism living across latitudinal gradients (Cumillaf et al., 2016; Madeira et al., 2012a; Vinagre et al., 2016). Thus, heating rates from about 0.5–1.5 ◦ C min−1 are often used. Measuring CT, the most frequently observed responses when temperature was increased at a rate of 1 ◦ C min−1 were: activity increase in attempt to scape, loss of equilibrium, onset of muscle spasms and after reaching that point, death, few minutes later (Hutchison and

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Fig. 1. Aerobic metabolism of aquatic ectotherms stimulated to be active when animals are placed in temperatures equivalents to 90% CT-max (Red arrow) or stimulated to be low when placed in temperature equivalents to 110% CT-min (blue arrow) (A). This figure was based in the concept, where activity metabolic rate (AMR) = Total Metabolic Rate (MR) or activity is depressed in aestivation temperatures (Halsey et al., 2015). When AMR is calculated (B) a bell shape curve is expected, where the maximum AMR will coincide with highest values of Q10 , where the energy costs are maximum. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Maness, 1979; Lutterschmidt and Hutchison, 1997b). Taking into consideration that total MR = RMR + AMR (Halsey et al., 2015), that under CT evaluations, scape activity can be observed (probably coping the total MR) (Paschke et al., 2013) and that total MR = AMR when AMR is around 90% total MR (Halsey et al., 2015), it is possible to assume that a maximum metabolic rate value can be obtained through weight-specific oxygen consumption when organisms are exposed at temperatures that stimulate scape activities. In this context, AMR = Total MR − RMR can be considered as a proxy of AS, because it reflexes the cost of activity (Fig. 1). So, temperature provoking high (MR) or low (RMR) activities can be also expressed as a Q10 (Halsey et al., 2015) allowing evaluate how temperature is modulating the activity of the metabolic enzymes and the energetic costs of activity induced by temperature (Fig. 1). In this context, higher temperatures increase the proportion of enzymes that have reached their activation energy accelerating the biochemical reactions involved in mitochondrial energy production (including the oxygen consumption), allowing more muscle activity. Consequently, in this study temperature was used with a clearly distinct two-fold aim: as an experimental factor (acclimation period), and as a tool to induce minimum and maximum metabolic activities that resemble the RMR and total MR. In the OCLTT hypothesis, reductions in aerobic scope are suggested to occur when in extreme temperatures, energy demands of the standard metabolism are not satisfy due to incapacity of organisms to supply enough oxygen at cell levels (Pörtner and Farrell, 2008). This hypothesis further states that aerobic scope, growth, activity, maintenance, reproduction and storage are linked due to

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those physiological responses depends of the ATP produced by aerobic metabolism (Sokolova et al., 2012) When temperature is in the optimum range, ATP supply is high enough to cover the maintenance cost and all the physiological demands. Such condition was defined as functional acclimation because ectotherms are in stable state and all their physiological functions are maxima (Pörtner, 2010; Pörtner and Knust, 2007). Beyond the optimum limits, the maintenance cost increases, reducing the ATP available to cover completely those physiological demands. The ranges of temperatures that provoke such conditions are called pejus ranges and reflect the acclimation in protection in which anaerobic metabolism is increased to satisfy the energy demands that the aerobic metabolism cannot support while at the same time the antioxidant defense mechanisms must compensate the excess of ROS produced by the aerobic metabolism (Pörtner, 2010). Beyond the pejus, ectotherms enter into the pessimum range, where all the ATP is theoretically directed to maintenance, activating the repair mechanism (i.e. HSP, HIF1 and HIF2 , etc.) (Pörtner, 2010; Sokolova et al., 2012). Taking into consideration all the biochemical and energetic pathways involved in thermal responses, the evaluation of thermal tolerance of ectothermic organisms should be accomplished, besides the growth rate, with evaluations of total MR, indicators of metabolic pathways used as a source of energy (lactate, glucose, proteins etc.) (Pascual et al., 2003b; Rosas et al., 2004) and some other molecules involved in antioxidant defense mechanism, altogether showing how the ATP is producing optimum interval, how is the antioxidant mechanism in pejus condition and how is the deleterious condition when animals are close to pessimum ranges, and also the form in which they are linked. In thermal studies of aquatic ectotherms thermal preference has been widely used to establish optimal conditions of a given species, because it confirms the hypothesis of co-adaptation, which predicts that preferred temperatures correspond to optimal temperatures, due to the fact that the physiological performance in those conditions is maxima (Angilletta et al., 2002; Norin et al., 2014). Although preferred temperature should be in the same interval of temperatures where total RM is maximum, recent results obtained in barramundi fish (Lates calcarifer) demonstrated that the higher AS was located at 38 ◦ C while preferred temperature of this species was 31–32 ◦ C where growth and locomotion were optimized (Norin et al., 2014). In other study Ern et al. (2014, 2015) have demonstrated that the cardiorespiratory system (e.g., heart and gill ventilation rates) is able to increase oxygen supply proportionally to tissue oxygen demand as temperature increases to critical temperature. Those authors observed that in the freshwater prawn Macrobrachium rosenbergii, the crayfish Astacus astacus and the marine tiger shrimp Penaeus monodon that AS of those species was not significantly reduced in spite of such maximum MR were close to critical temperatures. In this form those species avoid the transition to anaerobic metabolism indicating that factors other than inadequate oxygen delivery must be involved in any loss of ecological performance at high temperature. Although those results could show the thermal plasticity of those crustacean species, results obtained by Ern et al. (2014, 2015) could be limited if is considered that those conclusions were reached in animals acutely exposed at temperature changes. Although the acutely effects of temperature have relevance in studies evaluating the possible consequences of climate change, in the last years few existing studies have examined the chronic effects of exposure to temperature in metabolism of aquatic organisms (Healy and Schulte, 2012). According with Clark et al. (2013) typically in aquatic ectotherms metabolic acclimation occurs within 1–3 weeks, once the physiological mechanisms related with temperature express the new thermal history. In this sense it has been recognized that temperatures to which an animal has previously been exposed can improve its ability to survive hot and

cool and can affect the thermal thresholds at high and low temperatures (Wang and Overgaard, 2007). In this context, conclusions obtained in studies made with acclimated or not acclimated aquatic ectotherms could be strongly different, provoking different perspectives of the consequences of thermal adaptation of organisms from an ecological or evolutive point of view. Thermal limits have received much attention because their investigation provides insights into how climate shapes changes in the distribution, abundance and physiological responses of species (Pörtner, 2001). Furthermore, critical limits are positively related to optimal performance temperatures and are relatively simple to measure (Díaz et al., 2013; Vinagre et al., 2016). Critical thermal limits are considered ecologically relevant because they provide an indication of the activity range for a population under acute exposure conditions and can be used as a potential trigger for cellular activity under extreme thermal conditions (Somero, 2010). This method is supported by the fact that when this is applied, animals are exposed a fast increment of temperature in which they will show their capacity to respond at temperature changes using mechanisms that has acquired during acclimation period. In terms of adaptation, many researchers have observed that thermal window is a useful tool to understand thermal capacities of the species, how different organisms have evolved to colonize determined environments or how will tolerate environmental changes when a climatic perturbation occurs (Cumillaf et al., 2016; Pörtner, 2002, 2010; Pörtner and Farrell, 2008; Pörtner et al., 2005; Vinagre et al., 2016). Spiny lobster (Panulirus argus) inhabits the Caribbean coral reef ecosystem where this species can move through temperatures that oscillate between 23–30 ◦ C moving between the stable environment of coral reef and in the sea grasses where they fed (Rios-Lara et al., 2007). Although some questions remain about the effects of acclimation temperature in lobster, those results suggest that they could be thermally limited in their seasonal migration pattern if there changes in temperature. As colder temperatures than 23 ◦ C are not optimal, there is a potential risk, if the ocean temperatures increase above its tolerance, being trapped between high temperatures in rocky reef ecosystem and low temperatures in deeper zones that are marked by the presence of well-developed thermoclines. In this sense a previous report (Rios-Lara et al., 2007) showed that the P. argus inhabiting Alacranes reef system in the Gulf of Mexico-Caribean Sea zone have a patchy distribution determined by the habitat complexity and where, temperature is one of the environmental cues that determine lobsters distribution. In this context, the present study was designed to define the thermal biology of P. argus including all parts of the curve that can be obtained from the relationship between temperature, RMR and total RM (Fry, 1947; Pörtner, 2010). With this data we defined the optimal zone where AMR was maxima, pejus where a reduction of AMR is observed and the critical threshold responses where CT-max and min were registered. The study was done in acclimated animals considering that during acclimation animals can activate the mechanisms that allow them to show their thermal plasticity. Based on that information it was hypothesized that at temperatures that produce the maximum total MR, lobsters would growth and preferentially select temperatures around this range (Angilletta et al., 2002). Furthermore, we expected that hemolymph proteins, glucose, cholesterol and acylglycerols (AG) follow the same tendency of total RM because those nutrients are used as a source of energy in Crustacea (Rosas et al., 2002). Also, we expect that lactate levels indicate when the total MR is changing from aerobic to anaerobic, both in pejus and pessimus temperature ranges. At the same times, we expect that antioxidant mechanisms, joint with the critical thermal maximum and minimum (CT-max and CT-min), help us to identify the pejus limits giving a complete figure of the spiny lobster thermal biology to provide information in their performance,

G. Rodríguez-Fuentes et al. / Ecological Indicators 78 (2017) 192–204 Table 1 Experimental design and the number of lobsters used to study the effect of temperature on thermal biology of Panulirus argus. Acclimation Temperatures, ◦ C 18 Growth rate Temperature preference and routine hemolymph samples CT-maxa CT-minb RMRc Total MRd Total a b c d

22

26

30

all lobsters all lobsters all lobsters all lobsters 8 8 8 8

5 5 7 7 32

5 5 7 7 32

5 5 7 7 32

5 5 7 7 32

Critical Thermal Maxima. Critical Thermal Minima. Rest metabolic rate, measured in lobster exposed at 110% CT- min temperatures. Total metabolic rate measured in lobsters exposed at 90% CT-max temperatures.

distribution and abundance in the Caribbean Sea, where this species sustains an important fishery.

2. Material and methods 2.1. Animals and experimental conditions Panulirus argus juveniles (N = 140; 47 ± 24 g WW; 3.6 ± 0.7 cm carapace length) were obtained from wild population close to Isla Mujeres (Quintana Roo, Mexico) and transported by road in PVC tubes placed in a 1000 L dark tank with filtered and aerated sea water to the Laboratorio Experimental de Organismos Marinos at Universidad Nacional Autónoma de México (UNAM) located in Sisal, Yucatan, Mexico. Once in laboratory animals were distributed in 600 L tanks with open and aerated seawater flow (25 ◦ C) for a week, before starting the acclimation period. After conditioning period animals were randomly re-distributed in groups of 8 lobsters each in twelve 60 L tanks connected to four re-circulatory UV sterilized sea water systems of 1240 L each that feed three tanks at the same time. Each group of three tanks were maintained a constant temperature of 18, 22, 26 and 30 ◦ C (17.9 ± 0.2 ◦ C; 21.9 ± 0.4 ◦ C; 25.3 ± 0.7 ◦ C; 29.1 ± 0.1 ◦ C), using titanium chillers to temperatures of 18 and 22 ◦ C or titanium heaters for temperatures of 26 and 30 ◦ C. During conditioning and acclimation period (35 d) lobster were feed ad libitum two times a day with a paste elaborated with squid (70%) and crab (30%) meat.

2.2. Experimental design Lobsters were maintained at experimental temperatures by 35 d and studied following the experimental presented in Table 1. After acclimation period all the animals were weighed. After five days 8 lobsters per treatment were used to evaluate temperature preferences. At the end of those evaluations those lobsters were sacrificed and hemolymph and hepatopancreas samples were taken. Data derived of those samples were named as routine condition (RC). After, the rest of lobsters were used to evaluate critical thermal temperatures and to determine resting metabolic rate (RMR) and total MR. After all measurements, sample of hemolymph were obtained to evaluate metabolites (glucose, cholesterol, acyl-glycerides (AG), proteins and lactate), and hepatopancreas was used to assess antioxidant mechanisms SOD = Super oxide-dismutase; GSH = total glutathione; GST = Glutathione-S transferase; LPO = Lipid peroxides and Acetylcholinesterase (AChE) (Table 1).

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2.3. Routine condition (RC) 2.3.1. Growth and total moults Growth rate of lobsters were quantified as changes in cephalothorax length of animals acclimated at experimental temperatures after 35d of experiment. Animals were gently placed in a hand net and carefully measured with a digital Vernier rule. At the same time, every day moults were quantified and lobsters moulted separated of the rest of lobsters when placed in a nest in the same acclimation tank for two days until the exo-skeleton was hard again. During the experiment all lobsters were feed ad libitum. 2.3.2. Temperature preference Thermal preference was determined using the acute method (Reynolds and Casterlin, 1979a,b). A horizontal gradient was constructed from a 400 cm long and 20 cm wide PVC tube which was divided into 20 virtual segments each 20 cm long (Díaz et al., 2006). The water depth was 9 cm and the gradient was formed with a 1000-W heater on one side and a PolyScience IP-35 chiller on the other. The gradient had a 9–35 ◦ C temperature range. An air diffuser hose was placed along the tube to maintain a constant air supply and a constant oxygen concentration of 5–9 mg O2 L−1 and to avoid stratification in the water column. The temperature of each segment was measured using a digital infrared thermometer (Steren HER-425). All organisms that were placed in the gradient were fasted 24 h prior to the experiment to avoid food interference. After 35 d of experimental conditioning, individuals from each acclimation temperature were randomly selected and individually marked with external plastic tags 24 h prior to the experiment. One lobster was introduced into the gradient at the virtual segment corresponding to their acclimation temperature. The location of the organisms and the temperature in each segment were registered every 10 min for a total of 120 min. Eight repetitions were done for acclimation temperature and experimental animals were used only once. The final preferred temperature was graphically determined by the intersection between preferred temperatures and the equality line provided by the acclimation temperature. As a control group, 8 lobsters were placed in the gradient system with the heater and chiller turned off (resulting in a constant temperature of 26 ◦ C along the gradient). This control experiment allowed us to determine that final location was indeed due to temperature selection and not a preference for any particular site inside the tube. 2.3.3. Thermal tolerance CT-max for lobsters were determined using a 40 L aquarium. Temperatures were increased at a rate of 1 ◦ C min−1 (Lutterschmidt and Hutchison, 1997a). A submersible 800W heather equipped with an air stone to avoid temperature stratification in the water column was used to maintain constant increase rates. The criterion to determine the end point of CT-max for lobsters was the loss of righting response (González et al., 2010; Re et al., 2012). The measurements were done for 5 individuals in each treatment. When they reached their end points animals were weighed and sacrificed. Before, samples of hemolymph were taken, followed by samples of hepatopancreas that were used to analyze the antioxidant response. For CT-min the same 40 L aquarium was used. Thermal tolerance to low temperatures in lobsters was determined by introducing a 15 L aquaria filled with sea water in the 40 L aquarium filled with a pre-measured amount of crushed ice that allow a rate of decrease of 1 ◦ C min−1 . In all cases an air stone was used to avoid thermal stratification. Criteria for the determination of the end point of CTmin were the same used for CT-max. When organisms reached this point they were sampled in the same form that in CT-max. The thermal window area of all species was obtained with the CT-max and CT-min data (Brett, 1971) and was expressed in ◦ C2 .

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Table 2 Experimental temperatures used to evaluate the effect of acclimation on RMR and Total MR in P. argus juveniles. Acclimation

◦

Temperatures, ◦ C Thermal limits

Metabolic evaluations

Species

T C

CT-min

CT-max

RMR

Total MR

Panulirus argus

18 22 26 30

11.4 12.8 13.2 14.4

32.9 35.1 36.7 38.7

12.5 14.1 14.5 15.8

29.6 31.6 33.0 34.8

2.4. Hemolymph metabolites, antioxidant mechanisms and AChE

Values of critical thermal maxima and minima (CT-Max; CT-Min), are the mean value of the data reported in the present study. Temperatures used to evaluate RMR and Total MR were calculated from 110% of CT-min and 90% of CT-max, respectively.

The acclimation response ratio (ARR) was calculated as an index to known the magnitude of thermal acclimation of organisms. To do that we used the following equation (Claussen, 1977): ARR =

TCM2 − TCM1 CTMaxorMin = TA2 − TA1 T

(1)

Where CT-max or min2 –CT max or min1 represents the difference between values of critical temperatures (max or min) within the whole acclimation temperature range examined (TA2–TA1). In attempt to estimate how close lobsters live to their upper thermal limits and how close lobsters live to their preferred thermal environment we calculated two indices, both calculated of the difference between CT-max (Vinagre et al., 2016) or preferred temperatures and the maximum habitat temperature. 2.3.4. Total metabolic rate (MR) To obtain MR of lobsters we used temperature to induces respiratory activity in sedentary organisms (Halsey et al., 2015). Respiratory metabolism was measured as oxygen consumption when animals were exposed at 90% of CT-max (Total MR) and 110% of CT-min (RMR), respectively (Table 2). For each trial, acclimated individuals at each experimental temperature were rapidly placed from their acclimation tank in a closed respirometric chamber submerged in a temperature-controlled seawater bath maintained a temperature that induced total MR (90% CT-max) or RMR (110% CT-min) for 5 min. This time was choose considering that after that more exposure time could provoke fatigue and anaerobic metabolism (Norin and Clark, 2016). Initial and final oxygen concentrations were measured with a handheld dissolved oxygen meter (YSI Pro20 Instrument, YSI Incorporated, Yellow Springs, OH, USA) previously calibrated at each experimental temperature. Taking into consideration that this oxygen sensor takes 30–45 min to be stable after a temperature changes, the sensor was calibrated and maintained in the same experimental temperature for 1 h before the oxygen consumption measurements. Records of three chambers filled with water without fish were used as a measured of oxygen consumed by microorganisms in the control chamber. Respiration rate was calculated as shown in equation:



 (V/t)

MO2 = O2(A) − O2(B) ·

M

(2)

where MO2 is respiration rate (mg O2 h−1 g WW−1 ), O2(A) is the initial oxygen concentration in the chamber (mg O2 L−1 ), O2(B) is the final oxygen concentration in the chamber (mg O2 L−1 ), V is the water volume in the chamber minus the volume of water displaced by the animal, t is the time elapsed during measurement (h), and M is body mass of the experimental animal (g WW). Active metabolic rate (AMR) was calculated as: AMR = Total MR − RMR

Values of Q10 were calculated from data of Total Mr and RMR of each acclimation temperature using the equation: Q10 = (Total MR/RMR)(10/T2 − T1) . After oxygen consumption measurements, animals were sampled for metabolites, antioxidant mechanisms and AChE.

(3)

Hemolymph metabolites were measured in groups of 5 lobsters after 35 d of acclimation period (RC), after CT-max and CT-min determinations and after RMR and Total MR measurements. Lobster hemolymph was obtained from the last walking leg sinus with a precooled disposable insulin syringe with a 30G needle. Hemolymph was centrifuged (8000g, 4 ◦ C) for 5 min to obtain plasma. Plasma was placed immediately in liquid nitrogen and stored at −80 ◦ C until analysis. Hemolymph metabolites were analyzed with commercial kits (Lactate with a Trinity Biotech; acylglycerols (AG) with Ellitech TGML5415 kit; cholesterol with Ellitech CHSL5505 kit and glucose with Ellitech GPSL0507 kit) following manufacturer’s instructions. Plasma was later diluted 1:300 for soluble protein determination using a commercial kit (Bio-Rad; Cat. 500-0006) (Bradford, 1976). Determinations were adapted to a microplate using 20 ␮L of plasma and 200 ␮L of enzyme chromogen reagent. Absorbance was recorded using a microplate reader (Benchmark Plus, Bio-Rad) and concentrations were calculated from a standard substrate solution and expressed an mg/ml. Antioxidant defence system and Acetylcholinesterase activity (AChE). Hepatopancreas samples coming from lobster groups were snap-frozen in liquid nitrogen and stored at −80 ◦ C until analysis. Samples were homogenized in cold buffer Tris pH 7.4 at 100 mg tissue/mL using a Potter-Elvehjem homogenizer. Samples were divided in four, each containing enough homogenate to do each test for triplicate. For AChE and Superoxide Dismutase (SOD) activities, samples were centrifuged at 10,000g for 5 min at 4 ◦ C and the supernatant was separated for analysis. Homogenized samples were stored at −80 ◦ C until analysis. Total glutathione (GSH) was measured with a Sigma-Aldrich Glutathione Assay Kit (CS0260). This kit utilizes an enzymatic recycling method with glutathione reductase (Baker et al., 1990). The sulfhydryl group of GSH reacts with Ellman´ıs reagent and produces a yellow coloured compound that is read at 405 nm. Lipid peroxidation (LPO) was evaluated using Peroxi-Detect Kit (PD1, Sigma-Aldrich, USA) following manufacturer’s instructions. The procedure is based on the fact that peroxides oxide iron at acidic pH, Fe3+ ion will form a coloured adduct with xylenol orange that is measured at 560 nm. In order to quantify lipid peroxides only, an extra set of samples were treated with 10 ␮L 10 mM of tryphenolphospine (Banerjee et al., 2003) AChE activity was measured using a modification of the method of (Ellman et al., 1961) adapted to a microplate reader (RodríguezFuentes et al., 2008). Each well contained 10 ␮L of the enzyme supernatant and 180 ␮L of 5, 5 -dithiobis (2 nitrobenzoic acid) (DTNB) 0.5 mM in 0.05 M Tris buffer pH 7.4. The reaction started by adding 10 ␮L of acetylthiocholine iodide (final concentration 1 mM). The rate of change in the absorbance at 405 nm was measured for 120 s. SOD was evaluated using Sigma-Aldrich assay kit (19160) which uses Dojindo’s highly water-soluble tetrazolium salt, WST-1 (2-(4-Iodophenyl)- 3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- 2Htetrazolium,monosodium salt) that produces a water-soluble formazan dye upon reduction with a superoxide anion. The rate of the reduction with O2 are linearly related to the xanthine oxidase (XO) activity, and is inhibited by SOD.Therefore, the IC50 (50% inhibition activity of SOD or SOD-like materials) are determined

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colorimetrically. SOD and AChE activities were reported per mg protein in the sample (Bradford, 1976). 3. Statistical analysis One-way analysis of variance was used to know if acclimation temperature affected the growth rate, CTMin, CTMax, temperature preferred and hemolymph metabolites, antioxidant mechanisms and AChE of lobsters sampled at the end of the acclimation period (Routine condition). Two-way ANOVA were used to know the combined effect of acclimation temperature and temperature exposure of hemolymph metabolites, antioxidant mechanisms and AChE of lobsters after exposure to CTMin, CTMax, RMR and Total RM evaluations. At the end stars-plot were constructed with hemolymph, antioxidant mechanisms and AChE data in attempt to summarize the effects of temperature on physiological conditions of lobsters in routine conditions and measured after metabolism evaluations or after exposure at critical thermal conditions. Results from this section were analysed using the Integrated Biomarker Response (IBR) method (Beliaeff and Burgeot, 2002). In this method the responses are summarized in one general “stress index” and it was applied to assess the effect of temperature in routine, SMR, MMR, CTMax and CTMin conditions. Analysis of data were done following details in (Li et al., 2016): Data were calculated for mean and SD (standard deviation) and were standardized according to the formula Y = (X-m)/S, where Y is the standardized data, X is the data of each biomarker response, m is the mean data of the biomarker, and S is the standard deviation of the biomarker. Values of Z were calculated as Z = Y in the case of activation. The minimum value (Min) was obtained and S was calculated as S = Z + |Min|, where S ≥ 0 and |Min| is the absolute value. Calculation of star plot areas (Ai) were done by the formula Ai = Si /2 sin␤ (Si cos␤ + Si+1 sin␤), where ␤ = Arc tan (Si+1 sin ␣/Si − Si+1 cos ␣), ␣ is 2␲/n radians, and Si is the obtained value of each biomarker. The sum of the area Ai gives the corresponding IBR value IBR = i=1 = Ai, n is the number of the biomarkers. 4. Results 4.1. Routine condition The mean temperature selected in the thermal experiments by lobster acclimated at all temperatures was 27.5 ± 6.6 ◦ C with a range of preferred temperature of spiny lobster of 21–34 ◦ C (Table 3). This value was calculated from 32 lobsters considering that there were no statistical differences between treatments (Table 3) (Fig. 2). CT- min of spiny lobster values were affected by acclimation temperatures with low values in animals acclimated at 18 ◦ C (11.38 ± 0.5 ◦ C) and high in lobsters acclimated at 30 ◦ C (14.4 ± 0.8 ◦ C; p < 0.05). CT- max also changed according with acclimation temperatures with low values in lobster acclimated at 18 ◦ C (32.7 ± 2.5 ◦ C) and high in lobsters acclimated at 30 ◦ C (38.6 ± 1.2 ◦ C) (one way ANOVA repeated measures, P < 0.001 in all cases; Table 3). Preference warming tolerance of the lobsters changed according with acclimation temperature between 18 and 26 ◦ C with a reduction in animals acclimated at 30 ◦ C. So, low values were obtained in lobsters acclimated at 18 ◦ C (0.9 ◦ C) and higher in animals acclimated at 26 ◦ C (2.20 ◦ C). Intermediate values were recorded in lobsters acclimated at 22 ◦ C (1.39 ◦ C) and 30 ◦ C (1.45 ◦ C) (Table 3). In contrast, critical warming tolerance increased according with acclimation temperature with low values in animals acclimated at 18 ◦ C (3.7 ◦ C) and high in lobsters acclimated at 30 ◦ C (9.6 ◦ C) (Table 3). Thermal window of P. argus showed a thermal zone of 276 ◦ C2 where the higher thermal interval was obtained in

Fig. 2. Preferred temperatures, CT- min and CT- max, and thermal window for Panulirus argus acclimated at different temperatures. Isotheoretical temperature indicates when the temperature selected by lobsters and the acclimation temperature are similar. With this line and preferred temperatures, it is possible to calculate final preferendum (arrow: 27.6 ◦ C). Area for maximum and minimum thermal pejus (pejus max and pejus min, respectively) were calculated considering that CT indicates the critical threshold where aerobic scope is close to zero (Sokolova et al., 2012; Cumillaf et al., 2016), and we observed strong changes in antioxidant mechanisms and increments on hemolymph lactate. The optimal zone was identified with the preferred temperatures. Values are mean ± s.d.; n = 32, for lobsters acclimated at 18, 22, 26 and 30 ◦ C; n = 5 for CT- max, n = 5 for CT- min and n = 8 for preferred temperature per each experimental temperature.

pejus min (102.9 ◦ C2 ) than pejus max (36.8 ◦ C2 ). While a reduction on pejus min was observed according with increment of acclimation temperature an increment on pejus max was observed (Fig. 3). ARR of P. argus CT- max varied between 0.37–0.58, with low value when ratio was calculated in the range between 22 and 26 ◦ C and the high in the range between 18 and 22 ◦ C (Fig. 3). Those values were similar to other obtained in tropical crustacean species and higher than observed in temperate crustacean species (Fig. 3). Growth rate of spiny lobster juveniles were affected by temperature (Fig. 3). After 35 d of acclimation the maximum growth rate was observed in animals maintained at 26 ◦ C (0.27 ± 0.05 mm d−1 ) and the minimum in lobsters acclimated at 18 ◦ C (0.14 ± 0.04 mm d−1 ) (P < 0.001; Fig. 4A). Animals maintained at 30 ◦ C showed a growth rate of 0.21 ± 0.07 mm d−1 similar to obtained at 22 ◦ C (P > 0.05; Fig. 4A). Survival was not affected by temperature; 100% survival was recorded in all the treatments. Temperature affected the molting frequency of the lobsters. In the experimental tanks a number of 6, 12, 10 and 17 moults were collected in animals acclimated at 18, 22, 26 and 30 ◦ C, respectively (Fig. 4B). It is interesting to note that the number of moults registered in animals maintained at 18 was 2.8 times lower than the moults registered at 30 ◦ C and the moults registered at 22 were the double of the moults registered at 18 ◦ C (Fig. 4B). When hemolymph metabolites and antioxidant mechanisms were integrated in star plots (Fig. 5A) it was evident that animals acclimated at 18 ◦ C, maintained their hemolymph metabolites low, while at the same time increasing AChE and GST activities and producing high levels of GSH. In contrast, lobsters acclimated at 22 ◦ C showed peaks of glucose with increments of AChE, total GSH and SOD (Fig. 5A). Animals acclimated at 26 ◦ C showed the lower relative values of hemolymph metabolites and antioxidant mechanisms, while in lobsters acclimated at 30 ◦ C peaks of protein, glucose and LPO were observed (Fig. 5A). As a result of the sum of all areas in the star-plot, IBR plot showed that total areas were reduced according to temperature, with the highest values in animal acclimated at 18 ◦ C and the lowest lBR in lobsters acclimated at 26 ◦ C (Fig. 5B). The highest value of IBR observed in animals acclimated at 18 ◦ C was related with the high activity of the antioxidant mechanisms, showed in those organisms (Fig. 5A).

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Table 3 Thermal biology of Panulirus argus including preferred temperature and critical thermal maxima and minima (◦ C) of lobsters acclimated at experimental temperatures during 35 d. Critical warming and preference warming were calculated using 29 ◦ C as a maximum temperature of the habitat (Centro de Ciencias de la Atmósfera, 2014). Values as Mean ± SD. Different letters means statistical differences between treatments. Acclimation Temperature, ◦ C

Preferred n CTMin n CTMax n Maximum Temperature of habitat= Preference Warming Tolerance, ◦ C Critical Warming Tolerance, ◦ C

18

22

26

30

28.10 ± 5.90a 8 11.38 ± 0.49a 5 32.7 ± 2.5a 5 29 ◦ C 0.90 3.7

27.61 ± 9.05a 8 12.75 ± 0.75b 5 35.03 ± 1.02b 5

26.80 ± 5.09a 8 13.2 ± 1.03c 5 36.5 ± 1.2b 5

27.55 ± 6.94a 8 14.4 ± 0.75c 5 38.6 ± 1.18bc 5

1.39 6.03

2.20 7.5

1.45 9.6

Mean ± SD 27.5 ± 6.6 32

Fig. 3. Acclimation response ratio of several temperate and tropical marine crustaceans, including P. argus juveniles (h.1–h.6) obtained in the present study. Temperate species (blue points) classified as: a.1–a.6 (Cuculescu et al., 1998.), b.1–b.5 (Hopkins et al., 2006) and c (McLeese, 1956). Tropical species (red points) classified as: e.1–e.4 (Re et al., 2006), f (González et al., 2010), g.1 and g.2 (Kumlu et al., 2010). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

A

B

Fig. 4. Effect of temperature on growth (A) and total moults registered (B) in Panulirus argus juveniles maintained during 35d in constant conditions. Moult frequency was the result of number of moults registered in each treatment composed by 22 lobsters each. It is interesting to note that many moults does not means higher growth rate. In Crustacea high frequency of moult can indicate also stress. Values of growth rate as mean + SD. Different letters means statistical differences between treatments at P < 0.001 level obtained using one way ANOVA repeated measures.

4.2. Physiological condition at resting metabolic rate (RMR) and total metabolic rate (MR) In the present study, a lower RMR was obtained in lobsters acclimated at 18 and 22 ◦ C than in lobster acclimated at 26 ◦ C and 30 ◦ C (p < 0.05; Fig. 6A). In contrast, a high total MR was registered in animals acclimated at 22 and 26 ◦ C provoking that AMR showed a peak in animals acclimated at 22 ◦ C (P < 0.05; Fig. 6B). Energetic costs associated to activity provoked by temperature (Q10 ) followed the

same tendency, with significant low values in animals acclimated at 18 26 and 30 ◦ C and highin lobsters acclimated at 22 ◦ C (Fig. 6B; P < 0.001). Star-plots showed that lobsters acclimated at 18 ◦ C and after RMR measurements mobilized AG, while activated the AChE. Animals acclimated at 22 ◦ C showed a low mobilization of protein, glucose, AG and cholesterol, and increased AChE, and GST activities. An increment of total GSH was also observed (Fig. 7A). Lobsters acclimated at 26 ◦ C and measured after SMR, mobilized proteins,

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Fig. 5. Biomarker star-plots (A) and the calculated IBR values (B) of metabolites and anti-oxidative mechanisms measured in haemolymph of Panulirus argus after acclimation at different temperatures for 35 d (Routine conditions). SOD = Super oxide-dismutase; GSH = total glutathione; GST = glutathione-S transferase; AChE = Acetylcholinesterase; LPO = Lipid peroxidation.

AG and cholesterol while their antioxidant mechanism were maintained low (Fig. 7A). In contrast, lobsters acclimated at 30 ◦ C and after RMR measurements showed high activity of SOD and high concentration of GSH and LPO joint with an increment of proteins in hemolymph (Fig. 7A). When the Total MR was measured in lob-

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Fig. 6. A) Resting metabolic rate (RMR) and Total Metabolic Rate (MR) of P. argus juveniles acclimated during 35 d at different experimental temperatures. B) Activity metabolic rate calculated as MR − RMR (Halsey et al., 2015), and metabolic costs of activity calculated as Q10 . Values as mean ± SD. Different letters indicate statistical differences between treatments.

sters acclimated at 18 ◦ C and 22 ◦ C, there was an increment in AChE activity (Fig. 7B). In addition, animals acclimated at 22 ◦ C presented high concentration of cholesterol and AG in hemolymph (Fig. 7B). In general, a low activity of the antioxidant mechanisms and low metabolites mobilization was registered in lobsters acclimated at

Fig. 7. Biomarker star-plots (A) and the calculated IBR values (B) of metabolites and anti-oxidative mechanisms measured in haemolymph of Panulirus argus after measurement of RMR and Total MR in lobsters acclimated at different temperatures for 35 d. SOD = Super oxide-dismutase; GSH = reduced glutathione; GST = Glutathione-S transferase; AChE = Acethil-cholinesterase; LPO = Lipo-peroxidation.

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and proteins was observed, while antioxidant mechanisms were maintained without changes (Fig. 8A). In animals acclimated at 26 ◦ C, except glucose, all the metabolites and antioxidant mechanisms were maintained in intermediate concentrations, while in animals acclimated at 30 ◦ C SOD, GSH and GST were activated and haemolymph metabolites were reduced (Fig. 8A). In lobsters exposed at CT-max and acclimated at 18 ◦ C, animals mobilized AG, cholesterol and GST activity while lobsters acclimated at 22 ◦ C presented intermediate levels in the antioxidant biomarkers (Fig. 8B). Lobsters acclimated at 26 ◦ C and exposed at CT-max mobilized cholesterol and showed high levels of LPO while animals exposed at CT-max and acclimated at 30 ◦ C mobilized glucose, lactate and protein with a significant reduction of the anti-oxidant mechanisms (Fig. 8B). After CT-min measurements, maximum values of IBR were obtained in lobsters acclimated at 22 and 26 ◦ C (Fig. 8C) as a reflect of the high concentration of metabolites obtained in lobsters acclimated at 22 ◦ C and high activity of anti-oxidant mechanisms observed in animals acclimated at 26 ◦ C, respectively (Fig. 8B). IBR values obtained after CT-max showed an inverse relationship with IBR from CT-min (Fig. 8C) with lower values obtained in animals exposed at 22 and 26 ◦ C and higher in lobsters acclimated at 18 and 30 ◦ C (Fig. 8B).

5. Discussion

Fig. 8. Biomarker star-plots (A) and the calculated IBR values (B) of metabolites and anti-oxidative mechanisms measured in haemolymph of Panulirus argus after measurement of CT-min and CT-max in lobsters acclimated at different temperatures for 35 d. SOD = Super oxide-dismutase; GSH = reduced glutathione; GST = Glutathione-S transferase; AChE = Acetylcholinesterase; LPO = Lipo-peroxidation.

26 ◦ C (Fig. 7B). In contrast, after Total MR measurements in animals acclimated at 30 ◦ C, a strong mobilization of glucose and lactate was registered in hemolymph, while relatively high concentration of SOD and LPO were registered (Fig. 7B). Peaks of IBR values were obtained in lobsters acclimated at 22 ◦ C for both RMR and Total MR measurements, as a reflect of the high values of activity of antioxidant mechanisms observed in those lobsters (Fig. 7C). Other peak of IBR was obtained in lobsters acclimated at 30 ◦ C after Total MR, reflecting the high concentration of lactate and the activation of antioxidant mechanisms detected in those lobsters (Fig. 7B). 4.3. Physiological condition in critical temperature threshold Star plots showed that animals exposed at CT-min and acclimated at 18 ◦ C mobilized lactate and proteins but maintained their antioxidant defence mechanisms without changes (Fig. 8A). In animals acclimated at 22 ◦ C a high concentration of AG, lactate

This study shows that the maximum AMR temperature (22 ◦ C) of P. argus did not match the temperature (26 ◦ C) where the highest growth rate was observed. Lobsters acclimated between 18 and 30 ◦ C preferred temperatures around 27.5 ◦ C; this is 5.5 ◦ C above the temperature at which the maximum AMR occurred. Lactate is an indicator of anaerobic metabolism activation. Maximum lactate values recorded after maximum activity of animals acclimated at 30 ◦ C suggested that at 30 ◦ C lobsters may be in a sub optimal condition similar to pejus. Therefore, the optimal temperature range for P. argus juveniles may be between 22 ◦ C and 28 ◦ C where maximum AMR was experienced, and temperature selection behaviour and growth were favoured (Fig. 9). The curve describing the relationship between metabolic performance, growth, and temperature thermal preference of P. argus has a bell-shape with an optimal functional plateau between 22 and 28 ◦ C, as observed in other species (Ern et al., 2014). This bell-shape curve was constructed taking into consideration that physiological responses were measured in acute, short and long term exposures and that each type of response showed one specific aspect of the thermal capabilities of the lobsters. So, activity metabolic rate, thermal tolerance and their consequences in physiological conditions were obtained in animals when CTMax, CTMin, RMR and total MR were evaluated in acute term exposures (minutes). Preferred temperatures were evaluated in short term exposures (hours) while growth, moult rate, survival and its consequences in physiological conditions in long term thermal exposures (weeks) (Fig. 8). Therefore, this study suggests that different physiological mechanisms operate in the short, medium and long term, allowing a comprehensive analysis of the type and timing of response related with the thermal tolerance of the lobsters. In routine conditions, lobsters maintained a constant level of blood nutrients at all acclimation temperatures. Lactate levels were also maintained constant, which suggests the existence of mechanisms to maintain homeostasis in a wide range of temperatures (Fig. 8). Although the rate of food intake was not measured in this study, it is likely that lobsters ate more food at high temperatures than at low temperatures. The supply of macronutrients at 30 ◦ C would be expected to correspond to the energy demand of the organism at such temperature, promotes high nutrients levels in hemolymph, following patterns previously observed in penaeid.

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Fig. 9. Aerobic performance and fitness of Panulirus argus acclimated at 18, 22, 26 and 30 ◦ C during 35 d (after Pörtner 2010). Acclimation in functional capacity (AFC) was located between 22 and 28 ◦ C, where lobsters showed maximum activity metabolic rate (AMR) at 22 ◦ C, maximum growth at 26 ◦ C and final temperature preference at 28 ◦ C. In routine conditions, hemolymph metabolites and antioxidant mechanisms where associated with AFC. Acclimation in protection (pejus) were detected between 14–22 ◦ C and between 27 and 30 ◦ C. High levels of hemolymph lactate indicate that the metabolism of lobsters changed from a totally aerobic metabolism to a mix between aerobic and anaerobic metabolism when animals reach 30 ◦ C. This condition marked the limit of pejus and probably the critical threshold temperature (CT). In this context, critical temperatures (CT: Max and Min), could be equivalent to temperature threshold of Pörtner (2010) and Sokolova et al. (2012). SOD: Super-oxide-dismutase; GSH: total glutathione; LPO: Lipid peroxidation; AG: acyl-glycerides; P: protein; Lac: Lactate; Gluco: Glucose; Chol: Cholesterol; AMR: Activity Metabolic Rate; Gr: growth rate.

(Pascual et al., 2004, 2003a, 2012; Rosas et al., 2002). But, how is the relationship between hemolymph metabolites and growth in P. argus lobsters? Within the tolerance range of each species temperature favours growth in crustaceans by shortening the intermolt period, by increasing the size between molts, or both (Burmeister and Saint-Marie, 2010; Hufnagl and Temming, 2011a,b). These responses involve the accumulation of reserves and the production of hormones at different optimal temperatures (Hartnoll, 2001). Therefore, lobsters maintained at 30 ◦ C (showing a high moult rate but lower growth rate than animals maintained at 26 ◦ C) may have experienced a decoupling between the mechanisms associated with moulting and growth. So, at 30 ◦ C a high cholesterol mobilization could be indicating increments on production of moulting hormone, via the synthesis of ␤-estradiol, that had as a consequence a higher moult rate and lower growth (Drach, 1939; Shin and Chin, 1994). As a result, the decrease in time between moults and the inability to store sufficient reserves for 35 d of exposure at 30 ◦ C caused lobsters to grow less than lobsters maintained at 26 ◦ C. When the antioxidant system in lobsters was analysed, it was observed that in the temperatures ranging from 22◦ to 30 ◦ C, LPO levels were not significantly different; the mechanisms involved in maintaining the redox system may thus have controlled ROS levels in this thermal range. In contrast, GSH and GST levels in animals maintained at 18 ◦ C were significantly greater than in other treatments, indicating that the mechanisms associated to the antioxidant system may have been activated to control ROS levels at these temperatures. Accordingly, we can conclude that

Panulirus argus experienced some level of oxidative stress when maintained at 18 ◦ C by 35d; likely a non-favourable temperaturetime condition for these organisms in long term exposures. Scylla serrata individuals acclimated at 28 ◦ C and exposed abruptly to 4 ◦ C for 2 h regulated the antioxidant system, reduced motor activity and increased catalase and glutathione peroxidase (GPX) activity. A reduction in the activity of all enzymes and increased malondialdehyde were subsequently observed. These responses suggest the increase of ROS due to the decreased effectiveness of the antioxidant system (Kong et al., 2007) Acclimation temperature affected AMR in P. argus as was demonstrate in other crustaceans as Carinus maenas, where AMR comprised an important fraction of the total MR (Halsey et al., 2015). Maximum AMR occurred in animals maintained at 22 ◦ C, while intermediate values were recorded at 18 and 26 ◦ C, and low AMR values occurred in animals maintained at 30 ◦ C. Values of Q10 obtained indicates, as predicted (Fig. 1), that higher energetic costs of activity were in animals acclimated at 22 ◦ C, suggesting that, at 22 ◦ C lobsters have their maximum activity performance (Halsey et al., 2015). Although the OCLTT hypothesis (Pörtner, 2010) suggests that AS reflects the aerobic capacity of organisms, and that a maximum AS value should match the maximum values in all physiological functions of ectotherms (i.e. at 22 ◦ C in P. argus), results obtained in the present study using the activity metabolic rate (AMR) as a proxy of AS, indicate that there are other thermal compensatory mechanisms that allow lobsters to growth better during long term exposures at 26 ◦ C. It could be related with different physiolog-

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ical mechanisms involved during short and long term thermal exposures. Results obtained in this study suggests that, depending on the acclimation temperature, joint with muscle activity, other mechanisms as the mobilization of metabolites were used as a compensatory mechanism during forced activity (Fig. 9), suggesting that, in the short-term exposures animals could make different metabolic adjustments to those used during the long-term acclimation. During short-term experiments animals maintained at 26 ◦ C mobilized AG into the tissues while protein and glucose levels remained relatively low. In contrast, animals acclimated at 22 ◦ C maintained low protein, lactate and glucose levels, indicating that these nutrients were not required by the tissues during forced activity. This suggests that at 22 ◦ C, energy production was based on lipids and associated molecules, e.g. cholesterol (Martin-Creuzburg et al., 2012). Lobsters acclimated at 30 ◦ C and measured after total MR showed high lactate production, mobilization of glucose, activation of SOD, and GSH production with subsequent LPO. These responses were also observed in the antioxidant system of lobsters acclimated at 18, 22 and 30 ◦ C, and that were measured after RMR, indicating that the mechanisms operating in pejus min may thus be like those operating in pejus max (Fig. 9), but always during short-term exposures. Although there is no direct evidence linking CT-max and CT-min with the CT proposed by Fry (1947) and used by Pörtner (2010) and Sokolova et al. (2012) to define the OCLTT hypothesis, the measurement of critical limits using methods of temperature increase until the organisms lose their balance is simpler, measurements have little variability and can be used to make comparisons between organisms from different habitats (Cumillaf et al., 2016; Farrell, 2016; Paschke et al., 2013; Vinagre et al., 2016, 2014). As expected for a tropical species such as P. argus, our results suggest that the CT-max interval is greater than the CT-min interval, which are determined by the acclimation temperature (Fig. 9). Depending on the acclimation temperature, P. argus would have a minimum thermal limit between 10.8 ◦ C and 15.2 ◦ C and a maximum between 32.2 and 39.7 ◦ C. Within these temperature ranges, the antioxidant system was insufficient to offset the general physiological condition, and increased blood lactate levels and lipid mobilization were experienced (Fig. 9). Survival in such conditions would be associated with exposure time. It is likely that lactate accumulation and the inability of the redox system to mitigate the effects of temperature cause death in lobsters. Studies in intertidal crustaceans showed that DNA is damaged in extreme conditions, with considerable deleterious consequences for these organisms (Madeira et al., 2016; Vinagre et al., 2016). Thermal studies in marine invertebrates can allow understanding the effect of oceanic warming on these organisms. Therefore, in this study the ARR, the preference, warming tolerance, and the critical warming tolerance were used to examine the ability of lobsters to acclimate to new temperatures. The ARR is an index that defines the rate of change in thermal tolerance (CT-max) after a standardized temperature change (1 ◦ C) (Claussen, 1977). According with the present results, tropical species have generally higher and less variable ARR values than those of temperate species. Therefore, tropical species such as P. argus tend to respond more strongly to changes in temperature but also have less plasticity to withstand such changes (Vinagre et al., 2016). Various tropical species of fish and crustaceans were more vulnerable to warming than temperate species because they are found in temperatures that are more distant to critical thermal limits (Vinagre et al., 2016). Hence, the physiological mechanisms can be activated to adjust to new environmental conditions (González et al., 2010). Because tropical organisms live near their thermal tolerance limits, the physiological mechanisms are less efficient, which has been demonstrated now with juvenile P. argus acclimated at 30 ◦ C.

The preference warming tolerance index (PWTI) is the difference between the preferred temperature for lobsters acclimated at different temperatures and the maximum ambient temperature in the area of distribution of the species (29 ◦ C). Lobsters acclimated at 26 ◦ C had the highest PWTI and therefore appeared to prefer environments with temperatures different from the maximum temperatures that lobsters acclimated at 18, 22 and 30 ◦ C prefer. Acclimation at 26 ◦ C appears to favour growth and behavioural aspects that allow better performance of organisms. An ocean warming scenario that exceeds 26 ◦ C would result in lower growth of lobsters and would favour their preference for higher temperatures, with negative consequences for future populations. In contrast, the critical warming tolerance index (CWTI) suggests that lobsters can tolerate high temperatures even under oceanic warming scenarios. Lobsters acclimated at 30 ◦ C (9.6 ◦ C) had the highest CWTI, suggesting that at 30 ◦ C lobsters do not experience critical thermal conditions even in a warming scenario of +3 ◦ C. However, high temperatures may not favour growth and would cause lobsters to migrate to colder environments. Consequently, P. argus could be trapped between high (>30 ◦ C) and low (18 ◦ C) temperatures that stimulate mechanisms in response to stress. The CWTI indicates that the difference between the CT-max of animals acclimated at 18 ◦ C and the maximum ambient temperature is 3.7 ◦ C. A temperature increase of +3 ◦ C (IPCC, 2014) would negatively affect lobsters found in the limits of their maximum thermal tolerance. Our results indicate that the temperature and exposure time play important roles in the way organisms respond to thermal challenges. It is likely that the difference in the responses of different ectothermic organisms and the thermal response is linked to the way these effects are measured. To get a complete picture of the thermal adaptations, different methods must be used and the analysis of the effects should be performed at different levels of response and exposure times. Using this approach, it was possible to establish a comprehensive scheme of thermal tolerance of the spiny lobster P. argus, species of great commercial importance in the Caribbean Sea. Our results can help generate predictive models to anticipate changes in the P. argus fishery derived from likely changes in distribution and abundance of this species under warming scenarios of tropical oceans (IPCC, 2014). Acknowledgments This study was supported with funding from the Program PAPIIT-UNAM IN223416 awarded to CP and IA200214 awarded to GRF. Master in Science scholarships from the Postgraduate program of Marine Science and Limnology of UNAM, and from CONACYT, were awarded to MM. CONACYT awarded the grant infrastructure I010/186/2014 grant to CR, and the national sabbatical internship scholarship no. 264554 to FD and PASPA-UNAM to CP at ULPGC of Gran Canaria Spain. This study was partially financed by DGCI through the TEMPOXMAR collaboration network. References Angilletta, M.J., Niewiarowski, P.H., Navas, C.A., 2002. The evolution of thermal physiology in ectotherms. J. Therm. Biol. 27, 249–268. Baker, M.A., Cerniglia, G., Zaman, A., 1990. Microtiter Plate Assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples. Anal. Biochem. 190, 360–365. Banerjee, D., Madhusoodanan, U.K., Sharanabasappa, M., Ghosh, S., Jacob, J., 2003. Measurement of plasma hydroperoxide concentration by FOX-1 assay in conjunction with triphenylphosphine. Clin. Chim. Acta 337, 147–152. Beliaeff, B., Burgeot, T., 2002. Integrated biomarker response: a useful tool for ecological risk assessment. Environ. Toxicol. Chem. 21, 316–1322. Bradford, M.M., 1976. A refined and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248.

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