Effect of temperature on energetic balance and fatty acid composition of early juveniles of Octopus maya

Journal of Experimental Marine Biology and Ecology 445 (2013) 156–165

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Effect of temperature on energetic balance and fatty acid composition of early juveniles of Octopus maya Javier Noyola a, Maite Mascaró b, Claudia Caamal-Monsreal b, Elsa Noreña-Barroso c, Fernando Díaz d, Denise Re d, Adolfo Sánchez b, Carlos Rosas b,⁎ a

Departamento de Biología, Facultad de Ciencias, Universidad Nacional Autónoma de México, Circuito exterior Cd. Universitaria, México, 04510, D.F., Mexico Unidad Multidisciplinaria de Docencia e Investigación, Facultad de Ciencias, Universidad Nacional Autónoma de México, Puerto de Abrigo s/n, Sisal, Yucatán, Mexico Facultad de Química, Universidad Nacional Autónoma de México, Unidad de Química-Sisal, Puerto de Abrigo s/n, Sisal, Yucatán, Mexico d Departamento de Bioecnologia Marina, Centro de Investigacion Cientifica y de Educacion Superior de Ensenada. Carretera Ensenada-Tijuana # 3918. Col Pedregal Playitas. CP 22860. Ensenada B.C., Mexico b c

a r t i c l e

i n f o

Article history: Received 20 July 2012 Received in revised form 16 April 2013 Accepted 17 April 2013 Available online 22 May 2013 Keywords: Energetic balance Fatty acids Octopus maya Temperature adaptation

a b s t r a c t Octopus maya is an endemic species located on the continental shelf of the Yucatán peninsula. This area is located between the Caribbean Sea and the Gulf of Mexico, two ecosystems linked by the Yucatán Channel, in which the temperature in the benthos community is regulated by summer upwelling. Despite being part of a tropical ecosystem, O. maya's niche on the Yucatán peninsula is relatively cold, favoring both growth and reproduction of this octopus species. The present study was designed to test the effects of temperature on the energy balance of early juveniles of O. maya in an attempt to determine the physiological mechanisms related to adaptation at relatively low temperatures that are common in the Yucatán peninsula and favor growth of this tropical octopus species. Fatty acid profiles were also analyzed in attempt to understand how experimental temperatures modulate the lipid metabolism of O. maya, and to explore if fatty acid profiles can be used as a tool to explain the physiological mechanisms that support growth of this cephalopod species in the Yucatán peninsula environment. Early juveniles (age: 20 days) of O. maya were individually acclimated at 18, 22, 26 and 30 °C during 20 days. Growth rate, survival, ingestion rate, and fasting and feeding oxygen consumption were measured at the end of the experiment. Growth rate was affected by temperature, with high growth rates for animals maintained between 18 and 26 °C, and low rates for octopuses maintained at 30 °C. Routine and total metabolism increased exponentially with temperature, while apparent heat increment (RAHI) was higher for octopuses maintained at 18 °C than in the rest of the treatments. The energy challenge into biomass production was higher for octopuses maintained at 18 to 22 °C than 26 °C or 30 °C, demonstrating that low temperatures favor growth, probably due to several factors acting simultaneously: 1) a reduction of basal metabolism and consequent reduction in overall activity; 2) more time available for digestion and thus a better utilization of nutrients from food (fatty acids, among others); and 3) an increased time period in which to eat a greater quantity of food. Our study provided evidence that adaptive mechanisms such as a high ingestion rate and efficient use of fatty acids, among others, operate at low temperatures, beyond the mechanistic effect that temperature has on the enzymatic kinetics of O. maya. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Due to their inherently high energy efficiencies, cephalopods can present high growth rates because of their ability to sustain continuous hyperplastic and hypertrophic muscle growth (Semmens et al., 2011). Cephalopods, being ectothermal animals, are strongly influenced by temperature because it plays an important role in the regulation of embryo development, growth patterns and reproduction (Aguado-Giménez and García-García, 2003; André et al., 2008, 2009; Caveriviére et al., 1999; Katsanevakis et al., 2005a, 2005b; Leporati et al., 2007; Miliou et al., ⁎ Corresponding author. Tel.: +52 988 9131009. E-mail address: [email protected] (C. Rosas). 0022-0981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jembe.2013.04.008

2005). According to (Pörtner et al., 2005), natural selection should in general favor those individuals which are energy efficient, grow fast and reproduce successfully: this is the case for cephalopods (Arkhipkin, 1992; Mangold, 1983; Wells and Clark, 1996). The levels at which these processes operate are interdependent as they all take a share of the organism's energy budget. Hence, the fitness of an organism (i.e. growth and reproductive success) depends on energy turnover and is largely influenced by temperature and its variability (Pörtner and Farrell, 2008). Farias et al. (2009) based on Pörtner et al. (2005) propose three forms of adaptation to explain why a sub-polar octopus species (E. megalocyathus at 12 °C) had a higher growth efficiency than a tropical octopus species (O. maya at 28 °C): (1) a reduction of basal metabolism allowing for increased energy allocated to growth at lower

J. Noyola et al. / Journal of Experimental Marine Biology and Ecology 445 (2013) 156–165

temperatures (i.e. temperature adaptation);(2) the maximum growth rate may evolve inversely with the length of a period of high temperatures (i.e., counter-gradient variation) (Schmidt-Nielsen, 1990); and 3) a mixed strategy involving both of the above. We hypothesized that cephalopod adaptations may be affected by constraints that vary among species. Results observed for E. megalocyathus and those previously reported for O. vulgaris and O. pallidus (Katsanevakis et al., 2005a, 2005b; Leporati et al., 2007; Miliou et al., 2005) revealed that this octopus species could have a counter-gradient adaptation. The fact that a higher scope for growth was observed in sub-polar octopus species when compared to tropical species suggested that cephalopods have physiological adaptations that allow them to exploit fully environmental conditions in a wide temperatures interval. These adaptations imply that cephalopod growth is maximized in low temperatures by a highenergy-turnover: at low temperature, owing to a reduction in basal metabolism, there is an increment on physiologically useful energy, which enhances food conversion, and finally favors an excess of energy to be used for growth. In this study these concepts are acknowledged as Pörtner's hypothesis (Pörtner and Farrell, 2008; Pörtner et al., 2005). Polyunsaturated fatty acids (PUFA), consisting of 20 carbon atoms, are known as precursors of eicasonoids, which have a role of assisting in blood clotting, immune responses, inflammatory responses, cardiovascular tone, renal function, neural function and reproduction of cephalopods (Miliou et al., 2006). Arachidonic acid (ARA; 20:4n−6) is the primary precursor of eicosanoids in mammals and fish, although Miliou et al. (2006) note that ARA is not an essential fatty acid for O. vulgaris. In the same study, the authors also determined the existence of a relationship between temperature, body weight and fatty acids, based on their finding that at low temperatures O. vulgaris has sufficient n−3 HUFA levels for large individuals, and at warm temperatures for small individuals. The essentiality of n−3 highly unsaturated fatty acids (HUFA) in fish diets is directly related to their role as components of bio-membrane phospholipids, a role which is well illustrated in homeoviscous regulation processes whereby fish restructure their bio-membranes in response to changes in the environmental temperature (Hazel, 1984). Although cold acclimation resulted in reduced proportions of saturated fatty acids and increased proportions of polyunsaturated ones in fish (Tocher, 1995), in cephalopods a reduction in PUFA was related to their use as a source of energy for membrane synthesis, even at low temperatures (Miliou et al., 2006). Octopus maya, a benthic octopus with holo-benthic hatchlings, is endemic to the Yucatan Peninsula (Solis-Ramirez, 1997). After eclosion, hatchlings pass through transitional stages during the first 15 days, which are characterized by yolk internal absorption, changes in digestive gland and a holo-benthic behavior. After this period, the digestive gland is completely functional, and juveniles are completely developed (Moguel et al., 2010). The Yucatán peninsula in Mexico is a transition site between the Caribbean Ocean and the Gulf of Mexico, characterized by a summer upwelling that reduces the temperatures in summer, maintaining a relatively low temperature in the distribution zone of this endemic octopus species (Zavala-Hidalgo et al., 2003, 2006). On the eastern Yucatán shelves, persistent upwelling is observed due to favorable winds throughout the year with a cold sea surface temperature (5 and 30 m depth) and a large chlorophyll-a content along the inner shelf of the Yucatán peninsula from May to September (Enriquez et al., 2010). Although the Yucatán peninsula is located in a tropical zone, the niche occupied by O. maya is characterized by temperatures that fluctuate between 21 and 26 °C (Zavala-Hidalgo et al., 2006), due to the upwelling phenomena. In a previous study in which the effect of temperature on thermal preference and thermal window of O. maya juveniles was studied (Noyola et al., 2013), a final thermal preferendum of approximately 23.4 °C was observed. In the same study, thermal windows (obtained as the difference between maximum and minimum critical thermal values) were observed to be within the range of 20 to 21 °C in animals maintained between 18 and 26 °C, but reduced to 17.5 °C when octopuses were acclimated at 30 °C. This demonstrates that temperatures higher than 26 °C reduce the thermal

157

tolerance of this species. In fact, we have been incubating O. maya embryos between 24 and 26 °C in the five years of hatchling production in our laboratory because at higher temperatures, the metabolism of embryos is accelerated, and yolk is consumed before the embryo reaches the age and size suitable for hatching (Moguel et al., 2010). Other studies also demonstrate that wild O. maya populations, when influenced by upwelling cold streams, are larger and spawn all year; this is in contrast to octopus populations that live in other zones of the Yucatán peninsula where upwelling effects are not so marked, suggesting that differences in temperature might not only be modulating embryo development (as we observed in laboratory conditions; Uriarte et al., 2012), but also the growth rate and reproduction of octopuses. These observations suggest that, dependent on the zone of upwelling influence, O. maya in the Yucatán peninsula could use the energetic shift (temperature adaptation; Pörtner et al., 2005) provoked by low temperatures for growth and reproduction. An understanding of this adaptation could be helpful in formulating different climatic scenario models that allow prediction of changes in wild populations and the consequences for octopus fisheries and/or aquaculture (Higgins et al., 2012). In this context, the present study was designed to test the hypothesis of Pörtner et al. (2005), exploring the effects of temperature on the energy balance of early juveniles of O. maya in an attempt to understand which physiological mechanisms relate to adaptation at the relatively low temperatures that are common in the Yucatán peninsula and which favor the growth of this tropical octopus species. Fatty acids profiles were also analyzed in an attempt to determine how experimental temperatures modulate the lipid metabolism of O. maya and to explore if fatty acid profiles can be used as a tool to explain the physiological mechanisms that support growth of this cephalopod species in the environmental conditions of the Yucatán peninsula. 2. Material and methods 2.1. Organisms Two hundred early octopus juveniles (O. maya) were obtained from hatchlings spawned under controlled conditions from wild females acclimated in the maturation area of the National University of Mexico (UNAM), located in Sisal, Yucatán, Mexico. 2.2. Animals and rearing conditions O. maya with one-day post-hatchings (n = 200; 0.102 ± 0.001 g) were placed in 30 l plastic tanks connected to re-circulatory seawater system, at a density of 50 octopus/tank. Clean Melongena corona bispinosa (Mollusca; Gastropoda) shells were provided as refuges. Animals were fed twice per day with a shrimp-squid paste (30–70%) bound by gelatine at a ratio of 30% of body weight (Quintana et al., 2011; Rosas et al., 2008). After the conditioning period (20 days), octopuses (n = 200, 0.5 ± 0.15 g) were weighed and individually placed in 500 ml containers. Each hatchling was blotted dry with a paper towel to avoid data overestimation due to water content, and was individually placed in a plastic box and weighed in an electro balance (±0.01 g). Containers were provided with two windows covered with plastic mesh (5 mm) on each side and placed into a re-circulatory seawater system (salinity of 32; O2 higher than 5 mg/L; pH > 8, and temperature between 24 and 26 °C). A clean M. corona bispinosa shell was placed in each container as a refuge. Four groups, each with 50 octopuses, were randomly assigned to experimental temperatures: 18, 22, 26 and 30 °C. Temperature in the re-circulatory seawater systems was controlled using chillers and submersible titanium heaters, all connected to digital controls (±0.3 °C) with a thermocouple sensor. Water quality was maintained through the use of mechanical filtration (5 μm) and UV light prior to entering the culture system, and water flow was maintained at 20 L h − 1. Low light intensity (30 lx cm − 2) was provided by two 30 W fluorescent bulbs covered with a red filter in order to moderate stress levels (Koueta and Boucaud-Camou, 2003).

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2.3. Growth, feed intake and absorption efficiency Each octopus was individually weighed at days 1 and 20 of the experiment, and the data used to calculate individual growth (g) and daily growth coefficient: −1

DGC; % d

¼ ½ðLnW2–LnW1Þ=t100

where W2 and W1 are the final and initial wet weights of the octopuses, Ln the natural logarithm, and t the number of experimental days. Mortality was also noted for all the temperature treatments. Feed intake and oxygen consumption experiments were carried out simultaneously using the respirometer system. Four 4 h after placing the food in each container, uneaten food was removed, dried and weighed to determine intake and feed conversion. The ingestion rate (I) was calculated as the difference between the delivered feed and that which still remained 4 h after being offered to the animals, and corrected by the percentage of leached nutrients. The leaching percentage of the pastes was measured using the shaking method (Obaldo et al., 2002). For this, 2 g of diet were placed in 250-mL flasks placed in a horizontal shaker at each experimental temperature for2 h. After that time, all the water was passed through a paper filter to separate the remaining paste from the leached water. The leached and original feed samples were dried in a convection oven at 60 °C for 48 h, and then cooled in a desiccator. Dried feed samples were weighed and analyzed for dry matter retention. 2.4. Oxygen consumption and nitrogen excretion The effect of temperature on oxygen consumption was measured in 7 individuals from each experimental temperature. Oxygen consumption (VO2) was measured using a continuous flow respirometer, comprising respirometric chambers connected to a well aerated re-circulating system (Rosas et al., 2008). Juveniles were placed in 90 ml chambers with an approximate flow rate of 0.1 l min−1. All animals were allowed to acclimatize to the chambers for 1–1.5 h before measurements were made. They were offered a M. corona bispinosa shell as shelter. A chamber without an octopus (with a shelter) was used as a control both during routine metabolism and feeding metabolism measurements. To do that, control chamber was feed with a similar ration used to feed octopus. Measurements of dissolved oxygen (DO) were recorded for each chamber (entrance and exit) every minute using oxygen sensors attached to flow-cells that were connected by optical fibre to an Oxy 10 miniamplifier (PreSens©, Germany). The sensors were calibrated for each experimental temperature using saturated seawater (100% DO) and a 5% sodium sulphate solution (0% DO). Fasting metabolism was obtained from measurements taken every minute for 60 min after the conditioning period. Afterwards, octopuses were fed, taking into consideration that half of the ration (15% of wet weight (ww) day−1) was given two times a day to complete a total ration of 30% of wet weight (ww) day −1. Oxygen consumption measurements during the feeding phase were taken every minute until the oxygen consumption returned to pre-feeding values. At the end of the experiment octopuses were weighed. Oxygen consumption was calculated as the difference in DO concentrations between the input and output of each chamber, with the water flow being timed. Routine metabolism (Rrout) was estimated from the VO2 (mg g−1 h−1) of fasting octopuses. The apparent heat increase (RAHI; J g−1 h−1) was estimated from the difference between the VO2 of fasting octopuses and the maximum value attained after feeding, taking into account the time needed to reach the oxygen consumption peak after feeding. The effects of temperature on the oxygen consumption rate (Rtot) of O. maya were investigated using Q10 values estimated by the Van't Hoff equation: 10=ðt2–t1Þ

Q 10 ¼ ðK2=K1Þ

;

where K1 and K2 are the respiration rates at the temperature t1 and t2. The conversion factor of an oxygen consumption of 13.6 J mg−1 was used to transform fasting and postprandial VO2 to J g−1 wet weight (ww) (Lucas, 1993). The RAHI value was calculated taking into consideration that octopuses were fed twice a day during the extent of the experiment. In consequence absolute RAHI was calculated as: RAHI ¼ ½ðVO2 Maf−VO2 bf Þ2TRP where RAHI is the metabolic rate (joules g dw−1 day−1), obtained from the difference between the oxygen consumption maximum (Maf) after and before feeding (bf) (mg O2 h−1 g−1 dw), and 2TRP is the time taken to reach the peak (h) obtained from octopuses fed twice per day. 2.5. Activation energy An Arrhenius plot (natural logarithm of the metabolic rate [Rtot] as a function of inverse temperature) (Clarke and Fraser, 2004) was used to determine if the metabolism of O. maya has a mechanistic behavior, and whether it is possible to calculate the activation energy using the Boltzman factor as eEi/KT (Gillooly et al., 2001), where Ei is the activation energy, K is the Boltzman constant (8.6618 × 10 −5 eV) and T is the slope of the Arrenhius plot. 2.6. Energy balance Energy budget was estimated using the following equations (Lucas, 1993): As ¼ R þ Pg where As is assimilated energy, Rtot indicates respiration (Rtot = Rrout + RAHI), and Pg is the energy invested in growth, all of them expressed as kJ g−1 day−1. Energy produced (Pg) was obtained using the actual growth rate of all octopuses obtained during the experimental time (20 days). The value of 10.1 kJ g−1 dw was used to transform the growth data into production units (Pg; J g−1 day−1 live weight; (Rosas et al., 2007, 2008). Assimilated, respiratory and production gross efficiencies were calculated as As/I × 100, R/I × 100 and Pg/I × 100, respectively. Respiratory (R) and production net efficiencies (Pg E) were calculated as R/As × 100 and Pg/As × 100, respectively. 2.7. Fatty acids Octopus samples from each experimental temperature (N = 5for each) were frozen (− 80 °C) for fatty acid determination and then freeze-dried prior to analysis and preservation. Lipids were extracted with chloroform: methanol (2:1, v/v), according to the Folch extraction procedure (Folch et al., 1957). Lipid extracts were saponified with 20% KOH:Methanol (w:v) and free fatty acids were recovered in hexane from the acidified saponifiable fraction (pH = 1–2). Fatty acid methyl esters. (FAMEs) were obtained by esterification with 10%BF3 in methanol (Fluka, 15716) for 60 min at 80 °C. FAMEs were analyzed by capillary gas chromatography in a Perkin Elmer Clarus 500 GC equipped with a Perkin Elmer Elite-WAX(30 m 9 0.25 mm 9 0.25 lm film thickness, crossbond–PEG) capillary column and a flame ionization detector. Hydrogen was used as carrier gas with a flow rate of 40 mL min −1. Injector and detector temperatures were programmed to 280and 250 °C, respectively. Column temperature was programmed from 40 to 200 °C at 20 °C min −1and from 200 to 250 °C at 2.5 °C min −1. Individual FAMEs were identified by comparing retention times with reference standards (Supelco 37 Comp. FAME Mix, 47885-U). Results were reported as area percentages.

J. Noyola et al. / Journal of Experimental Marine Biology and Ecology 445 (2013) 156–165

2.8. Statistical analysis Because food intake, metabolism and biomass production summarize the energy balance equation, we analyzed differences in ingestion rate (I, joules g dw − 1 day − 1), basal respiratory metabolism (Rrout, joules g dw − 1 day − 1), apparent heat increase (RAHI; joules g dw−1 day−1) and production rate (Pg, joules g dw−1 day−1) amongst individuals maintained at different temperatures to test the influence of acclimation temperature on energy balance in O. maya juveniles. We tested the simultaneous response of the 4 variables to acclimation temperature (fixed factor with 4 levels) on the basis of a triangular matrix produced by Euclidean distances amongst samples (n = 28; 7 individuals × 4 temperature levels), using permutation methods (Anderson et al., 2008). Prior to the analysis, a square root transformation of the 4 variables was performed in order to bring together extreme values. Because all variables were measured in the same units, values were neither centred nor standardized in any other way. Unrestricted permutation of raw data (999 permutations) was used to generate the empirical distribution of the pseudo-F statistic under the null hypothesis and calculate p values associated with the observed value of the test statistic (Anderson et al., 2008). After testing for the main term in the model, pairwise comparisons between the 4 groups were performed using a direct multivariate analogue of the univariate t statistic (pseudo-t statistic; Anderson et al., 2008). The significance of the observed pseudo-t statistic was assessed using unrestricted permutations. A Principle Component Analysis (PCA) was used to visualize relationships among the samples in the multivariate space, and identify the variables with the highest loadings in the first principle component. A second PCA was performed to explore differences in fatty acids profiles from animals maintained at different temperatures. The score plots obtained after the generation of the two first principal components were used to determine groups based on experimental temperatures. The analysis was conducted using the fatty acids matrix. PCA was run iteratively, selecting variables based on analysis of the anti-image matrix and higher (>0.4) communalities. Univariate ANOVAs followed by Tukey´s pair-wise comparisons were performed on fatty acid data in order to establish differences between temperature treatments (Zar, 1999). 3. Results 3.1. Ingestion rate Ingestion rate varied with acclimation temperature, showing low values amongst octopuses maintained at 18 to 26 °C and high values amongst those maintained at 30 °C (Table 1). Individuals maintained at 30 °C showed an ingestion rate around 50% of dry weight, which was 190% higher than that registered amongst individuals maintained at lower temperatures (Table 1). 3.2. Growth and survival Growth varied markedly with temperature. Octopuses maintained at 18 °C showed the highest DGC values, followed by octopus maintained at 22 and 26 °C, and finally by those maintained at 30 °C (Table 2). Values of DGC of animals maintained at 18 °C were 128% higher than those observed in animals maintained at 30 °C (Table 2). Survival was also affected by temperature, with higher values for individuals maintained between 22 and 30 °C than those maintained at 18 °C (Table 2). 3.3. Oxygen consumption Oxygen consumed by octopuses was strongly affected by temperature. Routine oxygen consumption registered at 30 °C was the highest, with values 3.26 times higher than that registered at 18 and 22 °C

159

Table 1 Ingestion rates expressed as % wet (ww) and dry body weight (dw, g) of early O. maya juveniles maintained at 4 acclimation temperatures (°C). Both wet (ww) and dry weight (dw, g) of octopuses in each case are also given. Values as mean ± SD; n = 7. Juvenile O. maya T

ww

18 22 26 30

1.72 1.80 1.59 0.61

Ingestion rate dw

± ± ± ±

0.29 0.58 0.23 0.20

0.34 0.36 0.32 0.12

% ww ± ± ± ±

0.06 0.12 0.05 0.04

3.76 3.27 3.27 10.12

% dw ± ± ± ±

1.00 1.84 0.41 2.46

18.80 16.35 16.35 50.59

± ± ± ±

4.99 9.19 2.03 12.29

(Table 3). An intermediate value of Rrout was registered at 26 °C (Table 3). After the fasting period, octopuses acclimated at four temperatures were fed, and an increment in oxygen consumption was registered (Fig. 1). After feeding, both the maximum oxygen consumption (Table 3) and the time taken to reach this value were temperaturedependant. It took 97, 22, 23 and 15 min for octopuses to reach that maximum at 18, 22, 26 and 30 °C, respectively (Fig. 1). The maximum oxygen consumption registered after octopuses maintained at 30 °C had finished feeding was 3.6 times higher than that observed in those maintained at 22 °C (Fig. 2A). Apparent heat increment (RAHI) was also affected by temperature (Fig. 2A). Values registered in animals maintained at 30 °C showed an RAHI 200% higher than that obtained in those maintained at 26 °C (Table 3). Total oxygen consumption (Rtot = Rrout + RAHI) was affected by temperature, with results indicating that temperature modulates the respiratory metabolism in an exponential form (Fig. 2b). The relationship between Rtot and temperature was described by the Arrhenius plot (Fig. 3). Oxygen consumed by O. maya maintained at 18 and 22 °C did not differ significantly. As a result, the relationship between oxygen consumption and 1/KT was not linear (Fig. 3). The Q10 values were approximately 1 for increments between 18 and 22 °C, and 18 and 26 °C. For increments between 18 and 30 °C and 22 and 26 °C, Q10 values were 2.4 and 2.8, respectively. Values higher than 2 were registered for increments between 22 and 30 °C (Q10 = 4.3), and 26 and 30 °C (Q10 = 6.6) (Table 4). 3.4. Energy balance Temperature significantly affected the partial energy balance of O. maya juveniles. The PCA performed on the variables that summarize energy balance showed that the first principle component explained 85% of total data variation (λ = 328), whereas the second explained only 10% (λ = 38.9). The variables with the highest loadings for the first principle component were production (0.816) and routine metabolism (−0.576), indicating that this axis represents a production of energy to growth gradient. Samples were clearly ordered along the first principle component, and the order was consistent with temperature acclimation (Fig. 4): octopuses maintained at 18 and 22 °C shared positions amongst high production values and low routine metabolism values; those maintained at 26 °C were located at Table 2 Initial and final weight (g), growth rate (DGC % day−1) and survival (%) of early O. maya juveniles maintained at 4 acclimation temperatures (°C) during 20 days. Values are mean + SD; the number of individuals (n) is indicated in each case. Temperature

Initial weight n Final weight n DGC n Survival

18

22

26

30

0.49 ± 0.08 53 1.11 ± 0.24 31 4.1 ± 1.1 31 58

0.58 ± 0.21 50 1.12 ± 0.40 42 3.4 ± 1.6 42 84

0.53 ± 0.15 52 1.03 ± 0.26 52 3.4 ± 1.2 52 100

0.41 ± 0.13 49 0.58 ± 0.19 48 1.8 ± 1.5 48 98

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Table 3 Oxygen consumption of early O. maya uveniles maintained at 4 acclimation temperatures (°C). Respiration was measured as mg O2 h−1 g of wet weight of individuals at fasting (Rroutine), maximum after feeding (Maximum), apparent heat increment (RAHI) and total (Rtotal). Values are mean + SD; n = 7. Temperature 18 Rroutine Maximum RAHI Rtotal

0.36 1.28 0.25 0.61

22 ± ± ± ±

0.08 0.51 0.02 0.05

26

0.31 0.46 0.24 0.55

± ± ± ±

0.04 0.05 0.02 0.03

0.66 0.84 0.17 0.84

30 ± ± ± ±

0.1 0.11 0.02 0.06

1.45 1.75 0.33 1.78

± ± ± ±

0.11 0.09 0.02 0.07

intermediate production and routine metabolism values; and those kept at 30 °C were situated at the extreme left of the axis, where production of energy to growth had the lowest values. Results of the statistical analysis showed the simultaneous response of variables summarizing energy balance in O. maya juveniles was significantly affected by acclimation temperature (pseudo-F = 21.4; p = 0.001; 999 unique permutations). Subsequent pair-wise comparisons showed that octopuses maintained at 18 and 22 °C had a statistically similar response, but were both significantly different from those kept at 26 and 30 °C (Table 5). Differences in energy balance between octopuses maintained at 26 and 30 °C were also significant (Table 5). Production of energy to growth (Pg) was affected by temperature, with low values in octopuses maintained at 30 °C. A mean value of 1465 ± 356 joules day−1 was calculated from the Pg values obtained in individuals maintained at 18 to 22 °C (Table 6). Of the total ingested energy, octopuses maintained between 18 and 22 °C channelled 22 to 24% of their ingested energy (I) to Rtot. In contrast, those maintained at 26 and 30 °C channelled 49 and 96% of I to Rtot, respectively (Table 6). Consequently, of the total ingested energy, that channelled to biomass production was higher in animals maintained between 18 and 22 °C (59 and 66%) than was observed in animals maintained at 30 °C (18%). The same trend was observed when Rtot and Pg were calculated as a function of assimilated energy (Table 6).

3.5. Fatty acids The predominant fatty acid in octopuses maintained at experimental temperatures was palmitic acid (C16:0) with values of 40, 46, 40 and 33% for animals maintained at 18, 22, 26 and 30 °C, respectively. Of the total fatty acids of O. maya, 73 to 77% were unsaturated fatty acids (UFA) with maximum values registered in those animals maintained at 22 °C (Table 7). Monounsaturated fatty acids (MUFA) had values of between 17 and 23%, with high values for octopuses maintained at 18 °C (23%), while polyunsaturated fatty acids (PUFA) had values of between 6 and 15%, with maximum values for octopuses maintained at 22 °C (Table 7). The factor 1 axis of the PCA conducted using all fatty acid data absorbed 46% of the total variance, correlating negatively (p b 0.01) with C14:0, C18:2n6, C22:0 and C22:6n3, separating animals maintained at 18 and 30 from those maintained at 22 to 26 °C (Fig. 5).The factor 2 axis absorbed 27% of the variance and correlated positively (p b 0.05) with C14:1, C15:1 and C18:3n6, separating animals maintained at 18 from those maintained at 30 °C (Fig. 5). Temperature affected PUFA levels in O. maya juveniles, with high values for animals maintained at 22 and 26 °C (Fig. 6). Of the PUFAs, DHA and arachidonic acid followed the same tendency: high values for animals maintained at 22 and 26 °C and low for the remaining treatments (p b 0.05). Temperature did not affected UFA, MUFA or EPA levels (p > 0.05; Fig. 6). Temperature did affect the relationship between n-6 PUFA and the energy channelled to production (P) (Fig. 7). Animals with higher values of P and maintained at between 18 and 22 °C showed values of n − 6 PUFA higher than 1.13%, while animals maintained at 30 °C showed both low values of P and low values of n− 6 PUFA (0.63%) (p b 0.05; Fig. 7). 4. Discussion The temperature range of our experiments covered the same temperature range within which the species is mostly encountered in the Yucatán peninsula. Thus, we considered that energy balance calculated

3

18°C

2.5

Oxygen consumption (mg O2 h-1 g ww)

Oxygen consumption (mg O2 h-1 g ww)

3

max

2

F

1.5 1 0.5

22°C

2.5 2

max F

1.5 1 0.5 0

0 0

50

100

150

200

250

0

300

50

100

3

26°C

Oxygen consumption (mg O2 h-1 g-1 ww)

Oxygen consumption (mg O2 h-1 g-1 ww)

3

max

2.5 F

2

150

200

250

200

250

300

Time (min)

Time (min)

1.5 1 0.5

30°C F

2.5

max

2 1.5 1 0.5 0

0 0

50

100

150

Time (min)

200

250

300

0

50

100

150

300

Time (min)

Fig. 1. Oxygen consumption by early O. maya juveniles maintained at 4 acclimation temperatures. Values as mean ± SD of measurements taken every minute; n = 7 at each temperature level. F = feed; max = maximum oxygen consumption registered after feed.

Oxygen consumption (mg O2 h-1 g-1 ww)

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2.5

161

A b´ b

2

a´ 1.5

Rrut

a´

Rmax

a

1

a´

a

RAHI

a

0.5

0 18

22

26

30

Temperature (°C) 2.00

Total Oxygen consumption (mg O2 h-1 g-1 ww )

1.80

B

1.60 1.40 1.20 1.00 0.80 0.60

VO2 = 0.05e 0.11T R² = 0.76

0.40 0.20 0.00 15

17

19

21

23

25

27

29

31

Temperature (°C) Fig. 2. Oxygen consumption by early O. maya juveniles maintained at 4 acclimation temperatures. A: routine metabolism (Rrout) before feeding, maximum after feeding (Rmax) and the difference (RAHI = Rmax − Rrout). Values are mean ± SD. B: Total oxygen consumption (Rtot = Rrout + RAHI).

for this study reflects in a realistic form the use and destination of the ingested energy of O. maya individuals exposed to their natural temperature ranges. The results obtained in our study demonstrated that, as expected, O. maya juveniles are well adapted to use the energetic shift proposed by Pörtner et al. (2005), for which relatively low temperatures enhance the growth as a consequence of improving the mechanisms involved in utilization of the ingested energy. O.maya, as for most of the cephalopods, has high growth rates because of its high

Ln Total oxygen consumption (mg O2 h-1 g-1 ww)

1.00

0.50

0.00 3.250

3.300

3.350

3.400

3.450

-0.50

-1.00

levels of energy turnover (Wells and Clark, 1996); thesis supported by a high protein turnover derived from a high ingestion rate (Farias et al., 2009; Miliou et al., 2005; Onthank and Cowles, 2011; Rosas et al., 2011). In such circumstances, and without food limitations, both wild and laboratory cephalopods can produce high biomass in a relatively short time (Baeza-Rojano et al., 2010; Estefanell et al., 2011). In the Yucatán peninsula, O. maya apparently has no food limitations because of the benefits for the ecosystem of the seasonal upwelling (Enriquez et al., 2010), which saves energy, at the same time, via a reduction in maintenance metabolism due to a temperature reduction provoked by cold water entrance during the summer upwelling. These results help to extend the hypothesis of Pörtner et al. (2005) to this particular tropical environment where upwelling appears as a temperature modulator factor. Taking into account that as consequences of climatic changes, upwelling can be altered due to amplitude or frequency of meteorological oscillations (storms, hurricanes, etc.) that can affect the temperature where O. maya lives, this species appears as a candidate

Table 4 Q10 values calculated for early O. maya juveniles maintained at 4 acclimation temperatures.

-1.50

-2.00

Temperature (1/KT x 103)

Fig. 3. Total oxygen consumption by early O. maya juveniles maintained at 4 acclimation temperatures, and expressed as an Arrhenius plot, with a fitted least-squares regression.

Temperature, C

18

22

26

30

18 22 26 30

– – – –

0.8 – – –

1.5 2.8 – –

2.4 4.3 6.6 –

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J. Noyola et al. / Journal of Experimental Marine Biology and Ecology 445 (2013) 156–165

40

Table 6 Energy balance of O. maya juveniles acclimated to 4 temperatures. Values are expresses as mean ± SD (joules g dw−1 day−1); n = 7, or otherwise indicated.

Temp 18 22 26 30

Temperature

20

PC2

18

0

-20 -60

-40

-20

0

20

40

PC1 Fig. 4. Results for the Principal Components Analysis on ingestion rate (I), basal respiratory metabolism (Rrout), apparent heat increase (RAHI) and production rate (Pg, all in joules g dw − 1 day− 1) amongst early O. maya juveniles maintained at 4 acclimation temperatures.

for monitoring if such environmental changes could affect Yucatán peninsula ecosystem. Although a maximum growth rate was obtained in animals maintained between 18 and 22 °C, maximum survival rates were recorded in octopuses maintained between 22 and 26 °C, suggesting that the optimal range of temperature encompasses both temperature ranges. Taking into consideration that previous results showed a final preferendum of 23.4 °C for O. maya (Noyola et al., 2013), we propose that around these temperatures a maximum physiological performance can be observed when energetic balance is measured. According to Onthank and Cowles (2011), assimilation efficiency (AS/I, %), the portion of energy ingested that is invested into the respiratory metabolism and biomass production (R + Pg), is useful metrics in determining the physiological performance of cephalopods. As other cephalopods, O. maya showed high values of AS/I % demonstrating that, independently of temperature, the mechanisms to transform energy from food into biomass and metabolic energy is highly efficient in this species. Other high values of AS/I % were showed for O. maya. Van Heukelem (1976) showed values of As/I % between 64 and 69% for juveniles while Rosas et al. (2007) found values round 90% in animals fed crab. In other octopus species high As /I % values were also obtained. In polar octopus Pareledone charcoti Daly and Peck (2000) showed values between 95 and 97%, similar to those showed by Enteroctopus megalocyathus pre adults (71%; Pérez et al., 2006). In O. vulgaris As/I % high values were reported by Petza et al. (2006) with values that oscillate between 83 and 90.7% depending of the characteristics of individuals studied. It is interesting to note that in the present study we registered AS/I% values higher than 100% in animals maintained at 26 and 30 °C. Although we have no explication about that results it is possible that ingestion rate measurements could be underestimated in animals maintained at high temperatures

Table 5 Results of pair-wise comparisons amongst O. maya juveniles maintained at 4 acclimation temperatures (18, 22, 26 and 30 °C). Values of the pseudo-t statistic are given together with the probability of obtaining values alike or lower under the null hypothesis of no differences. The number of unique permutations used to produce the empirical distribution for each test is also given. Comparison

pseudo-t

P

# permutations

18 18 18 22 22 26

1.04 2.49 7.05 1.72 5.63 5.81

0.324 0.002 0.001 0.043 0.002 0.001

758 757 755 781 776 777

vs vs vs vs vs vs

22 26 30 26 30 30

Ingestion (I) 2661 ± 624 Respiratory metabolism (R) 553 ± 120 Rroutine 27 ± 3 RAHI Rtotal 580 ± 126 Production (Pg) 1567 ± 381 AS = R + Pg 2147 AS/I, % 81 Rtot/I, % 22 Pg/I, % 59 Rtot/AS, % 27 Pg/AS, % 73

22

26

30

2076 ± 317

2165 ± 322

2407 ± 160

493 ± 57 6±1 499 ± 57

1054 ± 163 4±1 1058 ± 164

2316 ± 178 6±1 2322 ± 178

1364 ± 475 1863 90 24 66 27 73

1272 ± 265 2331 108 49 59 45 55

432 ± 140 2753 114 96 18 84 16

producing in consequence high value of AS/I%. Although the ingestion rate measurements were done carefully results obtained in the present study suggest that other more precise methods should be used to estimate the ingestion rate in animals maintained at high temperatures, because in such condition temperature can affect the lixiviation process, that with method used now was not quantified. The results obtained in our study showed low values of Q10 obtained for animals maintained at between 18 and 22 °C, and 18 to 26 °C, suggesting that compensatory mechanisms are operating, presumably to reduce basal metabolism at low temperatures (Pörtner and Farrell, 2008). In fact, we observed that animals maintained at

Table 7 Total lipid fatty acid composition (% of total fatty acids) of early O. maya juveniles maintained at 4different acclimation temperatures. Values are mean ± SD. Fatty acids

18 °C

22 °C

26 °C

30 °C

C14:0 C14:1 C15:0 C15:1 C16:0 C16:1 C17:0 C17:1 C18:0 C18:1n9 C18:2n6 C18:3n6 C18:3n3 C20:0 C20:1n9 C20:2 C20:3n3 C20:3n6 C20:4n6 C20:5n3 C22:0 C22:1n9 C22:2 C22:6n3 UFA MUFA PUFA ARA EPA + DHA/PUFA n−3 n−6 n−3/n−6 EPA DHA DHA/EPA

2.08 + 0.43 0.37 + 0.12 0.64 + 0.15 0.05 + 0.03 39.75 + 2.63 1.09 + 0.25 3.09 + 0.05 3.37 + 0.82 27.88 + 0.81 6.06 + 2.78 0.66 + 0.16 0.37 + 0.00 0.36 + 0.02 0.50 + 0.13 9.96 + 1.71 0.68 + 0.24 0.34 + 0.04 0.08 + 0.02 0.11 + 0.04 0.35 + 0.08 0.30 + 0.15 2.14 + 0.78 3.47 + 1.08 0.84 + 0.54 74.24 + 14 23.03 + 7.23 7.26 + 1.81 0.11 + 0.04 0.16 2.09 + 0.12 1.14 + 0.06 1.84 0.35 + 0.08 0.84 + 0.54 2.42

3.49 + 0.11 0.03 + 0.01 0.91 + 0.21 0.00 + 0.00 46.18 + 14.85 0.90 + 0.66 2.70 + 0.00 5.12 + 1.42 23.01 + 8.91 4.83 + 1.72 1.54 + 0.35 0.00 + 0.00 0.36 + 0.15 0.43 + 0.00 9.99 + 0.00 0.82 + 0.56 0.36 + 0.00 0.07 + 0.06 0.32 + 0.21 0.28 + 0.10 0.57 + 0.09 0.41 + 0.11 9.70 + 1.88 1.43 + 0.53 77.30 + 11 21.29 + 5.72 14.87 + 6.95 0.32 + 0.21 0.12 2.81 + 0.17 1.85 + 0.19 1.52 0.28 + 0.10 1.43 + 0.53 5.18

2.63 + 1.25 0.00 + 0.00 0.66 + 0.21 0.00 + 0.00 40.96 + 16.56 0.45 + 0.17 3.14 + 0.32 3.65 + 1.56 25.21 + 7.72 4.81 + 1.65 1.44 + 0.62 0.00 + 0.00 1.23 + 0.00 0.48 + 0.12 7.08 + 1.40 1.23 + 0.65 0.36 + 0.22 0.09 + 0.02 0.37 + 0.18 0.39 + 0.15 0.62 + 0.23 1.82 + 0.21 5.92 + 2.95 1.47 + 0.35 73.70 + 23.4 17.81 + 4.38 12.48 + 4.8 0.37 + 0.18 0.15 3.90 + 0.15 1.81 + 0.27 2.16 0.39 + 0.15 1.47 + 0.35 3.77

1.54 + 0.14 0.01 + 0.01 0.49 + 0.37 0.00 + 0.00 33.41 + 8.47 0.70 + 0.24 4.23 + 0.60 4.47 + 0.72 34.75 + 8.91 4.35 + 0.79 0.48 + 0.08 0.00 + 0.00 0.26 + 0.09 0.01 + 0.01 7.72 + 2.13 0.53 + 0.30 0.12 + 0.07 0.06 + 0.04 0.15 + 0.03 0.30 + 0.04 0.05 + 0.03 1.55 + 0.28 3.56 + 1.59 0.84 + 0.06 74.48 + 31.8 18.80 + 8.43 6.30 + 2.17 0.15 + 0.03 0.18 1.72 + 0.05 0.63 + 0.04 2.71 0.30 + 0.04 0.84 + 0.06 2.82

UFA = Unsaturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = Polyunsaturated fatty acids; ARA (arachidonic acid) = C20:4n6; EPA = C20:4n6; DHA = C22:6n3.

Production (Pg) (J g dw-1 day-1)

J. Noyola et al. / Journal of Experimental Marine Biology and Ecology 445 (2013) 156–165

6 5 4

18°C

3

Factor 2

2 1

22 - 26°C

0 -1 -2

30°C

2500

2000

18°C 22°C

1500

26°C

1000

30°C

500

0

-3

0

*

0.5

1

1.5

2

2.5

n-6 PUFA (%)

-4 -5 -8

163

-6

-4

-2

0

2

4

6

Fig. 7. Relationship between production energy (j g dw−1 day−1) and n−6 PUFA (%) of early O. maya juveniles maintained at 4 acclimation temperatures. Vertical bars indicate ± SD of production mean values; horizontal bars indicate ± SD of n−6 PUFA mean values; asterisks indicate statistical differences at p b 0.05.

8

Factor 1 Fig. 5. Results for the Principal Components Analysis for composition of total fatty acids of the early O. maya juveniles maintained at 4 acclimation temperatures.

120

35

100

30

80

25

MUFA (%)

UFA (%)

18 and 22 °C showed high ingested energy values and a low metabolism, suggesting that, at those temperatures, mechanisms to save energy are operating. Although we do not currently have sufficient information concerning these mechanisms, there is evidence which indicates that cold adaptation is related to the form in which RNA transcription is edited, creating functional diversity in the octopus at the level of the nervous system (Garret and Rosenthal, 2012). In coleoid cephalopods, A-to-I RNA editing adds a layer of complexity to the proteome. A clear advantage of this strategy is that it allows for more options: different isoforms can be expressed in response to different conditions. Exactly how organisms exercise these options

is largely unknown (Garret and Rosenthal, 2012) but results obtained in the present study suggest that this type of mechanism could also be operating at a respiratory pathway level in O. maya. In the present study, temperatures of 22 to 30 °C did not provoke significant differences in the absolute AHI of animals, showing that in this range of temperatures the energetic costs of the feeding process were not affected. In contrast, animals maintained at 18 °C had high AHI values, suggesting that at low temperatures the energy requirement of all the behavioral, physiological and biochemical processes involved in feeding were strongly affected, because a more extended period of time was invested in reaching the peak at that temperature than was observed for the other treatments. Although these results may be considered negative for the octopus energetics, it was also

60 40 20

20 15 10 5

0 15

20

25

30

0 15

35

20

25

0.6

20

0.5

b

15 10

b a

a

5 20

25

30

30

35

0.3 0.2

0 15

35

20

2.5

0.6

2

0.5

b

b

1.5

25

Temperature (°C)

ARA (%)

DHA (%)

35

0.4

Temperature (°C)

a

a

0.5 0 15

30

0.1

0 15

1

25

Temperature (°C)

EPA (%)

PUFA (%)

Temperature (°C)

0.4

b b

0.3 0.2

a

a

0.1 20

25

Temperature (°C)

30

35

0 15

20

25

30

35

Temperature (°C)

Fig. 6. Fatty acids profile of early O. maya juveniles maintained at 4 acclimation temperatures. Values are mean ± SD. Different letters indicate statistical differences at p b 0.05.

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determined that, at 18 °C, O. maya ingested more energy and had a higher biomass production than observed in the other treatments, probably due to several factors acting simultaneously:1) a reduction of basal metabolism and consequent reduction in overall activity; 2) a longer time available for digestion and therefore a better utilization of nutrients from food; and 3) an increased time period to eat a greater quantity of food. Measuring the effects of temperature on the specific dynamic action, or SDA (called here AHI), of O. vulgaris, Katsanevakis et al. (2005b) found that SDA magnitude was not affected by temperature for animals maintained at 20 and 28 °C, suggesting that the energetic cost associated with the mechanical and biochemical transformations of ingested food was the same at both temperatures. The authors also concluded that a higher ingestion rate ought to be observed in animals maintained at 20 °C, in order to explain why a temperature of 20 °C favors a similar growth rate to that observed at 28 °C (Katsanevakis et al., 2005a). O. maya is well adapted to the relatively low temperatures found in the Yucatán peninsula. In addition to the energy adaptations revealed by the present study, there are other mechanisms possibly involved in the form in which temperature modulates the growth of O. maya. The results we obtained showed that the fatty acids profile changed according to experimental temperatures, suggesting that processes other than feeding or respiratory mechanisms could be operating to promote the growth rate of animals at temperatures between 22 and lower than 26 °C. These results reinforce the hypothesis that around those temperatures (Noyola et al., 2013) O. maya displays its maximum physiological performance. As shown by the PCA, three groups were formed: animals maintained at 18 °C, animals maintained at 22 and 26 °C, and animals maintained at 30 °C.These groups were separated mainly by differences in PUFA levels. Maximum levels of PUFA, and particularly of DHA and arachidonic acid, were registered in animals maintained at 22 and 26 °C, suggesting that at these temperatures the fatty acids demand was lower than observed at 18 or 30 °C. A similar relationship between temperature and PUFA levels was observed in O. vulgaris juveniles, for which animals maintained at low (15 °C) and high (25 °C) temperatures showed lower PUFA levels than observed in animals maintained at 20 °C (Miliou et al., 2006). Taking into account growth rate results obtained in previous studies (Miliou et al., 2005) those authors concluded that the decreases in EPA and DHA observed at the low temperature reflect a higher demand for EPA and DHA to support membrane synthesis of faster growing large octopuses, rather than a response enabling the maintenance of membrane permeability and plasticity. We observed a high growth rate and energy production for animals maintained at 18 °C, reinforcing the hypothesis that suggests that the effects of temperature on growth rate appear to play an important role in the fatty acid composition of octopuses (Farías et al., 2011; Miliou et al., 2006; Navarro and Villanueva, 2000, 2003). This hypothesis was newly confirmed by the construction of a relationship between energy channelled to production and the n−6 PUFA series, suggesting that animals maintained at between 18 and 26 °C have a lipid metabolism that permits octopuses to maintain levels of n−6 PUFA higher than 0.6%, a level that appears as a limit for growth of O. maya. The Arrhenius plot obtained in the present study showed that the metabolism of O. maya changes with temperature but not in a mechanistic form, as was predicted by (Gillooly et al., 2001). Adaptive mechanisms such as high ingestion rate, efficient use of fatty acids, among others, are operating beyond a mechanistic effect of temperature on enzymatic kinetics. In light of the results obtained in the present study, we can conclude that O. maya energetics is favored by low temperatures due to the mechanisms that have evolved in the Yucatán peninsula ecosystem, in which temperature is regulated by oceanographic phenomena. The challenge will be, on the one hand, to establish the physiological and biochemical mechanisms that have allowed the octopuses to adapt to a relatively cold environment and, on the other hand, to determine if it is possible to use O. maya as a species to monitor climatic change.

Acknowledgments The present paper was financed by the PAPIIT program (IN 212012) at the National University of Mexico. Financial support was also provided by the CONACYT program (CB2010-01, project No. 150810). [SS]

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