- Email: [email protected]
Effects of warm acclimation on physiology and gonad development in the sea urchin Evechinus chloroticus
Comparative Biochemistry and Physiology, Part A 198 (2016) 33–40
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa
Effects of warm acclimation on physiology and gonad development in the sea urchin Evechinus chloroticus Natalí J. Delorme a,b,⁎, Mary A. Sewell a a b
School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand Institute of Marine Science, Leigh Marine Laboratory, University of Auckland, P.O. Box 349, Warkworth 0941, New Zealand
a r t i c l e
i n f o
Article history: Received 21 November 2015 Received in revised form 17 March 2016 Accepted 23 March 2016 Available online 01 April 2016 Keywords: Climate change Ecophysiology Feeding Growth Reproduction Respiration Scope for growth Sea urchin
a b s t r a c t The physiology of the New Zealand sea urchin Evechinus chloroticus was evaluated through feeding, respiration, growth and gonad growth in adult animals acclimated for 90 days at 18 °C (annual mean temperature) and 24 °C (ambient summer temperature (21 °C) +3 °C). Measured parameters with representative rates of assimilation efficiency were used to calculate scope for growth (SfG) for each treatment. All physiological parameters were negatively affected at 24 °C, showing a decrease in feeding rate which coincided with negative growth and gonad development at the end of the acclimation period, and a decrease in respiration rate suggesting metabolic depression. Histology of gonad samples after the acclimation period also showed no gametic material in animals acclimated at 24 °C. All animals acclimated at 24 °C had negative growth, differing from the calculated SfG which indicated that the animals had sufficient energy for production. The results suggest that calculated SfG in echinoderms should be used together with actual measurements of growth in individuals as, by itself, SfG may underestimate the actual effect of ocean warming when animals are exposed to stressful conditions. Overall, considering the total loss of reproductive output observed in E. chloroticus at higher temperatures, an increase in seawater temperature could dramatically influence the persistence of northern populations of this species, leading to flow-on effects in the subtidal ecosystem. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Environmental conditions play a fundamental role in the normal functioning of animals (Willmer, 1999), with temperature being one of the most important and studied factors. Seawater temperature is the major factor determining optimal functioning in marine invertebrates as they are ectothermic; thus any change in environmental seawater temperature could influence their overall performance (Hochachka and Somero, 2002). An increase in seawater temperature within the optimal temperature range for the animal (i.e., thermal window) will increase animal performance up to a certain point/temperature (i.e., upper pejus temperature), after which performance starts decreasing dramatically (Pörtner et al., 2007; Pörtner and Farrell, 2008). Thermal windows and limits are species-specific, but also depend on the thermal history of the animal (Pörtner, 2002; Somero, 2005, 2010, 2012; Kelly et al., 2012). Generally, increased seawater temperature has been reported to affect feeding, growth, respiration, gonad development and survival in marine invertebrates (Sanford, 2002; Lannig et al., 2006; Pernet et al., 2007; Kemp and Britz, 2008; Gooding et al., 2009; González et al., 2010; McElroy et al., 2012; Zamora and ⁎ Corresponding author at: School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand. E-mail address: [email protected] (N.J. Delorme).
http://dx.doi.org/10.1016/j.cbpa.2016.03.020 1095-6433/© 2016 Elsevier Inc. All rights reserved.
Jeffs, 2012), which may have major consequences for a particular population and marine ecosystem. Overall, increased temperature can affect physiological performance of the animals to the point at which the energy balance is altered and therefore long term survival is compromised. In echinoderms, physiological rates and energy budgets have been measured to determine the condition of the animals under different nutritional and environmental scenarios (e.g., salinity, pH and temperature, Shirley and Stickle, 1982; Hill and Lawrence, 2006; Stumpp et al., 2012; Zamora and Jeffs, 2015; Carey et al., 2016). A common measure of physiological stress/condition in animals is the scope for growth (SfG), which represents the total energy available for production (somatic and gonad growth) considering the total energy input as food and the energy lost through metabolism (Warren and Davis, 1967; Bayne et al., 1979). Values of SfG can be positive, indicating that the animal has enough energy for growth (increasing body size/ weight), zero indicating that the energy input equals the energy expenditure, or negative which indicates that there is not enough energy for growth (decreasing body size/weight) (Naylor et al., 1989; Filgueira et al., 2011). In general, thermal acclimation of echinoderms to temperatures within the species optimum range generally shows positive effects on physiological processes (Siikavuopio et al., 2008, 2012; Gooding et al., 2009; Azad et al., 2011; Watts et al., 2011). However, when animals are exposed to elevated seawater temperatures, processes such as feeding, growth, gonad growth and survival can be negatively
34
N.J. Delorme, M.A. Sewell / Comparative Biochemistry and Physiology, Part A 198 (2016) 33–40
compromised (Siikavuopio et al., 2008; Lawrence et al., 2009; Zamora and Jeffs, 2012; Uthicke et al., 2014), often accompanied by an elevated oxygen consumption (Brockington and Clarke, 2001; Siikavuopio et al., 2008; McElroy et al., 2012; Zamora and Jeffs, 2012; Uthicke et al., 2014; Carey et al., 2016). The New Zealand echinometrid Evechinus chloroticus (Valenciennes 1846) is geographically distributed throughout New Zealand from the Three Kings Island (34°10′S) to the Snares Island (47°60′S) (Fenwick and Horning, 1980; Schiel et al., 1986). Bathymetrically, E. chloroticus is commonly found between 12 and 14 m deep; however, it is also found in low intertidal pools and the shallow subtidal (Dix, 1970a; Barker, 2013). This sea urchin species has an annual breeding cycle, spawning during the austral summer (Nov\\Feb) along its entire distributional range (Dix, 1970b, Barker, 2013,). Due to its broad latitudinal range, E. chloroticus is exposed to a wide range of seawater temperatures, from a minimum of 9.5–10 °C in the South Island during winter, to a maximum of 20–21 °C in the North Island during summer (Garner, 1969). The Intergovernmental Panel on Climate Change (IPCC) has projected an increase in seawater temperature of New Zealand waters of about 0.7–2.3 °C by the year 2090 due to global warming (IPCC, 2014). Previous research has shown that growth of wild E. chloroticus in north-eastern New Zealand varies seasonally, with peak growth in early spring and slowest growth during late spring and early summer when temperatures are higher (Walker, 1981). In the laboratory, thermal acclimation of E. chloroticus (sourced from the South Island of New Zealand) has shown a higher gonad growth as acclimation temperature increased (James and Heath, 2008). However, when acclimated to elevated temperatures (22 and 24 °C), E. chloroticus from both the South Island and the North Island showed a lower rate of increase in gonad growth (James et al., 2009). Nevertheless there is no study to date that has examined the effects of ocean warming on physiological performance and energy balance in E. chloroticus. Here we examined the effect of thermal acclimation at 18 °C (annual mean temperature, and ambient during late spring–early summer) and 24 °C (ambient summer temperature (21 °C) +3 °C) on the physiology (i.e., feeding, respiration, growth and gonad growth) of adult E. chloroticus from a north-eastern population in New Zealand. Further, we used these physiological parameters to calculate SfG in each temperature treatment to determine if SfG is a reliable measure of physiological state and a real representation of the actual production (growth) observed in sea urchins under thermal stress.
seawater temperature was 15.2 ± 0.4 °C (±s.d., n = 336; Leigh Marine Laboratory dataset), while at the time of collection was 15 °C. In the ten days prior to the February collection, average seawater temperature was 19.9 ± 0.3 °C (±s.d., n = 288; Leigh Marine Laboratory dataset), while at the time of collection was 21 °C. At both collection times, the animals were transported about 5 km to the Leigh Marine Laboratory, University of Auckland and held for 3 days in a 280 l tank with ambient flow-through seawater (August: 15 °C; February: 21 °C) to allow the animals to recover from handling stress. Animals collected in August were acclimated for 90 days, whereas animals collected in February were used for experimentation 3 days after collection. The acclimation system consisted of 8 tanks each of 36 l (4 tanks for each acclimation temperature) with flow-through seawater at ambient temperature (15 °C) with aeration provided by an airstone in each tank. The tanks were connected to two heat pumps (Aquahort EH13 12.5 kW, Auckland, New Zealand) which maintained the flowing seawater at the desired temperature. After the recovery period, 12 animals were placed in each tank, resulting in an initial density of 1.7 ± 0.1 and 1.6 ± 0.1 kg m−2 of internal surface area for the 18 and 24 °C treatment respectively (±s.d., n = 4). The animals were maintained in the experimental tanks for 3 days before the seawater temperature of the heating pumps was increased at a rate of 1 °C/day (James and Heath, 2008; James et al., 2009) until the desired acclimation temperatures were reached (18 and 24 °C). These temperatures were maintained in the tanks for 90 days, until November 2013. During the 90-day experimental period the animals were under a photoperiod of 12 h light: 12 h dark and seawater temperature was measured every hour with iBCod 22 l temperature loggers (Thermodata, Brisbane, QLD, Australia). Animals were fed to satiety with Ecklonia radiata and the tanks were cleaned every day. 2.1. Feeding rate Feeding rate of the sea urchins was measured in each tank after an acclimation period of 30 days, and for wild animals during the summer season (ambient temperature of 21 °C). Fronds of the kelp E. radiata were externally dried with paper towels and ~ 100 g (wet weight) of algae was placed into each tank. After 5 days the remaining algae were removed, externally dried and final wet weight determined. To control for natural algal degradation the same procedure was performed in 4 separate tanks without animals with flowing seawater at each temperature maintained by heating pumps. Feeding rate (g kg−1 h−1) was calculated as follows:
2. Material and methods Adult sea urchins were collected from Matheson's Bay, northeastern New Zealand (36°18′17″S; 174°47′51″E) at two separate times (Fig. 1). 1) August 2013: for thermal acclimation of animals at two different temperatures (18 and 24 °C) and to measure physiological performance of these animals (Fig. 1); and 2) February 2014: to determine physiological performance of wild animals during the summer season (Fig. 1). In the two weeks prior to the August collection, average
F¼
½ð F0 Ft Þ ð Fb0 Fbt Þ : WT
ð1Þ
Where F0 is the initial amount of food given, Ft is the amount of food remaining (g), Fb0 and Fbt are the initial and final amount of food in the tanks without animals (g), W is the total weight of the animals in the tank (kg), and T is the duration of the measurement in hours (h).
Fig. 1. Timeline of research relative to the reproductive cycle in Evechinus chloroticus (Walker, 1982). Arrows indicate time of collection of animals (August 2013 for acclimation at 18 and 24 °C, and February 2014 for experimentation on wild animals). Physiological measurements are listed for each collection time. GI0: initial gonad index; GIt: final gonad index.
N.J. Delorme, M.A. Sewell / Comparative Biochemistry and Physiology, Part A 198 (2016) 33–40
separated and their final GI determined (GIt). Gonad index (GI) and the index of increase in GI (Y) were calculated as follow:
2.2. Respiration rate Respiration rates of individual sea urchins were determined after an acclimation period of 30 days (n = 6 urchins per temperature) for urchins that had been starved for 1 week prior to respiration measurements. In addition, respiration rate was determined on wild animals (n = 6) during the summer season (ambient temperature of 21 °C). Respiration rate was determined in individual sea urchins using a double-bottomed plexi-glass respirometry chamber (1.4 l) filled with 1 μm filtered and UV-treated seawater. The sea urchin was placed in the main chamber; a magnetic stir bar with a submersible magnetic stirrer avoided water stratification in the main chamber. Dissolved oxygen concentration (μmol l−1) was recorded every 1 s with a needle type fibre optic oxygen microsensor connected to an oxygen meter MicroxTX3 (PreSens, Regensburg, Germany) and PSt1 (v6.02) software. To keep the temperature constant, the complete system was submersed in a 36 l tank with water at the desired temperature (18 and 24 °C for acclimated animals, and 21 °C for wild animals). Seawater temperature was maintained by a heating unit and a water pump delivering seawater at the desired temperature. The animals were carefully placed in the chamber and oxygen concentration of the water was measured for 2 h (oxygen saturation N 50%). After each respiration measurement, the animal's wet weight, volume and test diameter were measured as described below. In addition, control measurements were performed without animals at each temperature in order to account for background respiration. Respiration rate was calculated as the weight specific oxygen consumption rate (R, mgO 2 kg− 1 h− 1 ) described by Siikavuopio et al. (2008), but with one minor modification to consider background respiration:
R¼
½ðC 0 C t Þ ðC b0 C bt Þ V : WT
35
ð2Þ
Where C0 and Ct are the initial and final oxygen concentrations of the water in the chamber with animals (mgO2 l− 1), Cb0 and Cbt are the initial and final oxygen concentrations of the water in the chamber without animals (mgO2 l− 1), V is the volume of the respirometry chamber minus the volume of the animal (ml), W is the wet weight of the animal (kg), and T is the duration of the measurement (h). 2.3. Growth At the start of the acclimation period, measurements of test diameter (cm) and wet weight (g) of all the animals were recorded in each acclimation tank using callipers and a digital balance (±0.01 g). Growth was determined monthly by measuring test diameter and wet weight of the animals held in each tank, obtaining an average measure per tank at each acclimation temperature. The average measure was used to calculate the percent of change in wet weight and test diameter which were used for the statistical analyses. The animals were starved for 1 week prior to each growth measurement to allow the animals to completely empty their stomachs (i.e., animals fed for 3 weeks before each monthly measurement). 2.4. Gonad growth and histology Gonad growth was measured through the increase in gonad index (Y) which was determined from measurements of initial and final gonad indexes (GIs). After collection of animals and before the temperature experiments were set up, a subsample of 10 animals was used to determine the initial GI (GI0). At the end of the 90 days acclimation period and after the last measurement of growth was performed, a random sample of 10 animals from each acclimation temperature was
GI ¼
100G W
Y ¼ GIt GI 0 :
ð3Þ ð4Þ
Where G is the gonad's wet weight and W is the animal's wet weight. The animals were starved for 1 week prior to GI measurements. The gonads from animals acclimated at 18 and 24 °C were sampled for histology after the 90 days acclimation. In addition, the gonads from wild animals (n = 10) collected during the summer season (February 2014, ambient temperature of 21 °C) were processed for histology. The gonads were fixed in 10% formalin and transferred after 2 days to 70% ethanol for storage. Dehydration, clearing and embedding of the gonads used routine methods for wax histology (Humason, 1979), using haematoxylin and eosin as stains and sectioned at 5–10 μm. Histological sections were observed and photographed using a Leica DMRE compound microscope (Leica Microsystems Inc., Buffalo Grove, IL, USA). 2.5. Scope for growth Scope for growth (SfG) was calculated using the physiological data obtained from feeding and respiration rates measured at day 30 in the different temperature treatments. As the amount of energy lost through faeces and excretion was not measured during the experiment, the approach of Pedersen et al. (2014) was used, based on an assimilation efficiency of 60–80% in laboratory experiments in E. chloroticus (unpublished data). In addition, it has been reported that in echinoderms the loss from excretion is very low (b 1% of total balance) (Hill and Lawrence, 2006; Stumpp et al., 2012; Zamora and Jeffs, 2015). Assimilated food was converted to energy equivalents using the conversion reported by Lamare and Wing (2001) for E. radiata (1 g wet weight = 1.72 kJ). Conversion of respiration measurements to energy equivalents used the oxycaloric coefficient (1 mgO2 = 19.97 J, Winberg, 1960 fide Siikavuopio et al., 2008). Thus, scope for growth (SfG, kJ kg−1 h− 1) was calculated as described by Widdows and Johnson (1988): SfG ¼ A R:
ð5Þ
Where A is the energy gained through the assimilated food (kJ kg− 1 h− 1) and R is the energy lost through respiration (kJ kg−1 h−1). 2.6. Statistical analyses Feeding rate and growth rate (% change in wet weight or test diameter) data were checked for normality and homoscedasticity (Shapiro– Wilk and Levene's tests respectively). Feeding rate was analysed with a one-way ANOVA test using temperature as factor and the feeding rate as the dependent variable. The percent change in wet weight did not meet assumptions of normality; but as it met homoscedasticity assumptions it was analysed using a parametric test (Quinn and Keough, 2002). Both the percent change in wet weight and the percent change in test diameter were analysed with a two-way ANOVA test using temperature and time as factors and the percent change in wet weight and test diameter as the dependent variables. Respiration rate and the increase in gonad index did not meet the assumption of homoscedasticity and therefore were analysed with a Kruskal–Wallis one-way ANOVA on ranks with temperature as factor and respiration rate and increase in gonad as the dependent variables. Significant differences between groups were identified using the a posteriori Tukey test (α = 0.05). All statistical analyses were run using the statistical software included within Sigma Plot 12.5 (SYSTAT Software, Inc., San Jose, CA, USA).
36
N.J. Delorme, M.A. Sewell / Comparative Biochemistry and Physiology, Part A 198 (2016) 33–40
3. Results Seawater temperature in the experimental tanks during the acclimation period was 17.7 ± 0.4 °C (± s.d., n = 2597) and 23.9 ± 0.3 °C (± s.d., n = 2474). Survival in the 18 °C acclimation treatment was 98% as one animal escaped from the tank and fell onto the floor, cracking its test and dying afterwards. In the 24 °C acclimation treatment survival was 96% with two animals which started losing their primary and secondary spines and dying after 2 months of acclimation.
3.1. Feeding rate During the acclimation period, the animals from the 18 °C treatment fed constantly from day 1, whereas the animals from the 24 °C treatment did not feed during the first week of acclimation and then fed on comparatively lower amounts of kelp than the 18 °C treatment. After 30 days of acclimation, the feeding rate was significantly different between treatments (ANOVA: F2,9 = 84.77, P b 0.001). Animals acclimated at 24 °C showed a 73% decrease in feeding rate compared to animals acclimated at 18 °C (P b 0.001, Fig. 2), and a non-significant decrease of 26% compared to control animals at 21 °C (P = 0.327, Fig. 2). Feeding rate from wild animals also showed a decrease in feeding rate of 64% compared to animals acclimated at 18 °C (P b 0.001, Fig. 2).
Fig. 3. Effect of temperature on the respiration rate of Evechinus chloroticus. Animals from 18 and 24 °C were acclimated 30 days to each temperature; whereas Wild-summer (21 °C) correspond to non-acclimated control animals during the summer season (n = 6 for each treatment). All animals were starved for 1 week prior to measurements. Significant differences (P b 0.05) between temperatures are denoted by lower case letters.
3.3. Growth 3.2. Respiration rate Respiration rates of E. chloroticus after 30 days of temperature acclimation at 18 and 24 °C and from wild animals in the summer season (21 °C) were significantly different (Kruskal–Wallis: H = 10.78, d.f. = 2, P = 0.005). Respiration rate increased from 18 to 24 °C; however, this increase was not significant (P N 0.05). The highest respiration rate was observed in non-acclimated animals (21 °C), with a respiration rate of 2.8 mgO2 kg−1 h− 1 higher than animals acclimated at 18 °C (P b 0.05, Fig. 3). No significant differences were observed in the respiration rate between animals acclimated at 18 and 24 °C or between nonacclimated animals (21 °C) and animals acclimated at 24 °C (P N 0.05, Fig. 3).
Initial wet weight and test diameter of the animals in the 8 tanks used for acclimation were not statistically different (Weight: ANOVA: F7,88 = 0.765, P = 0.618; test diameter: ANOVA: F7,88 = 2.096, P = 0.052). During the acclimation period there was a significant increase in wet weight and test diameter at 18 °C compared to animals acclimated at 24 °C (Tables 1 and 2, Fig. 4). At the end of the 90 day acclimation period, animals acclimated at 24 °C decreased their wet weight and their test diameter from the initial condition by 7.2 and 7.8% respectively (Fig. 4). Wet weight and test diameter in these animals were respectively 12.3 and 8.7% lower at the end of the acclimation period than animals acclimated at 18 °C (Fig. 4). No significant differences were found in the percent change in wet weight and the percent change in test diameter with acclimation time and there was no significant interaction between factors (Table 2, Fig. 4).
3.4. Gonad growth and histology
Fig. 2. Effect of temperature on the feeding rate of Evechinus chloroticus. Data represents the mean feeding rate (g kg−1 h−1) ± s.e.m. (n = 4 tanks). Animals at 18 and 24 °C were acclimated for 30 days to each temperature; whereas Wild-summer (21 °C) corresponds to non-acclimated control animals during the summer season. All animals were starved for 1 week prior to measurements. Significant differences (P b 0.05) among temperatures are denoted by lower case letters.
The mean GI of the animals after field collection and prior to thermal acclimation was 2.98 ± 0.40% (±s.e.m., n = 10). After the 90 days of thermal acclimation, the mean final GIs were 4.57 ± 0.37% and 0.50 ± 0.15% for animals acclimated at 18 and 24 °C respectively (±s.e.m., n = 10 for each temperature). The increase in gonad index (Y) was significantly different at the end of the acclimation period in animals acclimated at 18 and 24 °C (Kruskal–Wallis: H = 14.29, d.f. = 1, P b 0.001). Animals acclimated at 24 °C showed a decrease of 2.5% in gonad index from initial conditions (Fig. 5). Gonad growth in these animals was ca. 4% lower than the gonad growth achieved by animals acclimated at 18 °C for 90 days (P b 0.001, Fig. 5). After the 90 day acclimation period, gonads from the animals acclimated at 18 °C were large and yellow-ish in colour; however, the gonads from the animals acclimated at 24 °C were very thin, looked transparent and red/brown-ish in colour. Histology of these gonads showed that at 18 °C and in the non-acclimated animals (21 °C) all animals were mature or partially spawned (Fig. 6a–d). Whereas at 24 °C, gametic material could only be distinguished in 3/10 samples which had degenerating eggs (Fig. 6e); the other 7 sea urchins had only empty gonad tubules with no gametic material in the gonads (Fig. 6f).
N.J. Delorme, M.A. Sewell / Comparative Biochemistry and Physiology, Part A 198 (2016) 33–40
37
Table 1 Mean Wet Weight (WW) and Test Diameter (TD) ± s.e.m. (n = 4 tanks) of Evechinus chloroticus acclimated at different temperatures for 90 days. Time (days)
18 °C
0 30 60 90
24 °C
WW ± s.e.m (g)
TD ± s.e.m. (cm)
WW ± s.e.m. (g)
TD ± s.e.m. (cm)
99.90 ± 3.71 103.22 ± 4.31 104.36 ± 5.18 104.84 ± 4.03
6.01 ± 0.10 5.99 ± 0.10 6.05 ± 0.12 6.06 ± 0.13
98.11 ± 3.49 95.93 ± 3.43 93.52 ± 3.04 91.12 ± 3.59
6.34 ± 0.10 6.05 ± 0.09 5.86 ± 0.14 5.85 ± 0.10
3.5. Scope for growth Scope for growth calculations, assuming an assimilation efficiency of 60–80%, showed that in all treatments the animals have available energy for somatic and gonad production (Table 3); however, the available energy for production is ca. 76% higher in animals acclimated at 18 °C than in animals acclimated at 24 °C (Table 3). On the other hand, animals without previous acclimation taken from the wild during the summer season (February 2014, ambient temperature of 21 °C) showed a scope for growth 27% higher than animals acclimated at 24 °C and 67% lower than animals acclimated at 18 °C (Table 3). The positive values of SfG obtained for animals acclimated at 24 °C do not match the actual values of growth (weight and test diameter) obtained from the experiment, where there was negative growth and no gonad production in 24 °C animals. Null or negative growth (SfG ≤ 0) in E. chloroticus can only be obtained by either decreasing the assimilation efficiency by 9 to 11-fold (≤7.36% at 24 °C; which corresponds to a feeding rate of 0.11 g kg−1 h−1) or increasing the respiration rate 8 to 11-fold higher than that measured.
acclimated at 24 °C showed a lower respiration rate than wild animals during the summer season, it is possible to suggest that animals at 24 °C may be undergoing metabolic depression (Pörtner, 2010, 2012). Therefore, 24 °C could be considered as the temperature of transition from the pejus to the pessimum range in E. chloroticus (Pörtner, 2012; Sokolova et al., 2012; Sokolova, 2013). Animals in the pejus range experience either an increase in maintenance costs to cover demand for stress protection and damage repair, and only storage energy is used to cover these costs; whereas in the pessimum range animals experience a further increase in energy demand for maintenance and an impairment of the aerobic metabolism (Sokolova et al., 2012; Sokolova, 2013). Metabolic depression appears to restore the balance at low turnover rates of energy at the expense of other functions (e.g., reproduction, growth, activity) to ensure short-term survival (Sokolova et al., 2012).
4. Discussion Increasing the seawater temperature to 24 °C, the predicted temperature for northern New Zealand in 2090 (IPCC, 2014), results in decreased feeding rate, negative growth and complete loss of reproductive output in the endemic New Zealand sea urchin E. chloroticus. As respiration rate is maximum at current summer temperatures (21 °C), E. chloroticus in northern New Zealand may already be experiencing temperatures that approach the limits of physiological performance. Respiration rate of wild animals during summer and during the winter season (15 °C, data not shown) was more variable than acclimated animals probably due to the influence of the thermal history of the animals used from the wild. The fact that acclimated animals showed a lower respiration rate than non-acclimated wild animals may be a result of the temperature that wild animals were exposed to prior to the respiration measurements (21 °C). This temperature is in between the temperatures of the two acclimations (18 and 24 °C) and may represent the highest point of respiration of E. chloroticus following a typical performance curve (Pörtner and Farrell, 2008). Considering that animals
Table 2 Summary of statistical analyses performed on the percent of Gained Wet Weight and the percent of Increased Test Diameter from Evechinus chloroticus acclimated at 18 and 24 °C. ANOVA statistic (F), mean square (MS) and p-value (P) are shown for each variable using Temperature and Time as factors. Variable
Factor
df
MS
F
P
% Gained Wet Weight
Temperature (T) Time (t) Txt Residual Temperature (T) Time (t) Txt Residual
1 2 2 18 1 2 2 18
477.365 4.778 23.297 18.570 307.277 2.464 11.428 10.301
25.706 0.257 1.255
b0.001 0.776 0.309
29.830 0.239 1.109
b0.001 0.790 0.351
% Increased Test Diameter
Fig. 4. Effect of temperature on the percent change in wet weight and the percent change in test diameter in Evechinus chloroticus. Data represents the mean change in wet weight and test diameter per tank ± s.e.m. (n = 4 tanks). All animals were starved for 1 week prior to measurements. Significant differences (P b 0.05) between treatments are denoted by lower case letters and detailed in Table 2.
38
N.J. Delorme, M.A. Sewell / Comparative Biochemistry and Physiology, Part A 198 (2016) 33–40 Table 3 Summary of the parameters used for calculations of Scope for Growth (SfG) at the different temperatures (acclimated animals (18 and 24 °C) and wild-summer control animals (21 °C)). Assimilated food (A) was calculated from average feeding rate (FR), using an assimilation efficiency of 60–80% (⁎). Assimilation efficiency (A) and average respiration rate (R) were then converted to energy equivalent (Aenergy and Renergy) which were used for the calculation of the SfG (kJ kg−1 h−1) for each temperature.
FR (g kg−1 h−1) A (g kg−1 h−1) Aenergy (kJ kg−1 h−1) R (mgO2 kg−1 h−1) Renergy (kJ kg−1 h−1) SfG (kJ kg−1 h−1)
18 °C
24 °C
Wild summer-21 °C
5.70 3.42–4.56 5.88–7.84 8.03 0.16 5.72–7.68
1.50 0.90–1.20 1.55–2.06 9.64 0.19 1.36–1.87
2.03 1.22–1.62 2.10–2.79 10.83 0.22 1.88–2.57
⁎ Assimilation efficiency was based on an assimilation efficiency of 60–80% in laboratory experiments in Evechinus chloroticus.
Fig. 5. Effect of temperature acclimation on the gonad index of Evechinus chloroticus. Data represents the mean increase in gonad index (Y) ± s.e.m. (n = 10 animals) at each temperature after 90 days of acclimation. All animals were starved for 1 week prior measurements. Significant differences (P b 0.05) between temperatures are denoted by lower case letters.
Experiments at elevated seawater temperatures in other sea urchins have shown similar patterns of decreased feeding rates and gonad growth (Siikavuopio et al., 2008; Lawrence et al., 2009; Uthicke et al., 2014); the decrease in both growth and gonad development being attributed to the high energy demands of increased metabolism at elevated temperatures (Uthicke et al., 2014). Previous research in E. chloroticus has shown that acclimation for 10 weeks at 18–20 °C is
Fig. 6. Histological sections of gonads of Evechinus chloroticus acclimated at different temperatures. Respectively, (a) and (b): female and male gonads of animals acclimated at 18 °C; (c) and (d): female and male gonads of wild animals (21 °C); (e) and (f): female (with degenerating eggs) and undetermined gonads (empty gonad tubules) of animals acclimated at 24 °C. Labels correspond to ripe ova (O), vitellogenic oocytes (VO), lumen (L) and mature sperm (S). Scale bars = 200 μm.
N.J. Delorme, M.A. Sewell / Comparative Biochemistry and Physiology, Part A 198 (2016) 33–40
optimal for gonad growth, regardless of the study population/location (James et al., 2009). Urchins acclimated at 24 °C had the lowest increase in GI during the spring/early summer when fed an artificial diet (James et al., 2009). In the present study there was a negative effect of acclimation temperature on the percent of change in weight and size; however, there was no effect of time for both acclimation temperatures, which may be a result of the high variability of the data at the different times during acclimation. There was also a negative effect of acclimation temperature on gonad growth in E. chloroticus which decreased by 83% after 90 days of acclimation at 24 °C. Therefore, it is likely that the animals were using the energy available in the gonads to maintain their basic biological functions, since sea urchins allocate a large amount of ingested energy in the gonads (Fernandez and Boudouresque, 2000; Otero-Villanueva et al., 2004). In addition to the decrease in gonad weight reported for E. chloroticus in the present study, and as also previously reported for Echinometra sp. A (acclimated at 31 °C, Uthicke et al., 2014), animals acclimated at 24 °C were not able to produce gametes or generate viable embryos (N. J. Delorme, unpublished). E. chloroticus acclimated at 24 °C is physiologically stressed, resulting in a dramatic loss of wet weight and test size (7 and 8% respectively), and gonad tissue (83%). Negative growth and shrinking have been previously reported for starved E. chloroticus (Dix, 1972) and other sea urchin species (Ebert, 1967; Levitan, 1988; Constable, 1993), which has been related to the reduction in the sutures that hold the plates together rather than calcite resorption (Constable, 1993). The loss of weight and size in an organism is a reflection of a negative energy balance or SfG, with animals having to use their energetic reserves in order to cope with the higher metabolic costs of living under stressful conditions (Filgueira et al., 2011). Previous studies in molluscs have shown that there is a good relationship between actual growth measurements and calculated SfG (e.g., Bayne et al., 1979; Navarro and Winter, 1982; Navarro et al., 2006; Filgueira et al., 2011), and have considered SfG as a reliable measurement of an animal's energetic condition (Navarro et al., 2006). In contrast, in this study SfG at 24 °C indicated sufficient energy for growth after 30 days of acclimation; whereas the sea urchins showed negative growth during the complete duration of the acclimation period. Since the parameters used for calculation of SfG might also vary among individuals, we suggest to complement the use of SfG in echinoderms with actual measurements of growth in individual animals. Although negative SfG has been reported in echinoderms (e.g. Arbacia punculata and Lytechinus variegatus under starvation, Hill and Lawrence, 2006), and matched with negative growth in starfish under salinity stress (Shirley and Stickle, 1982), the SfG calculations here underestimated the real effects of ocean warming on E. chloroticus. In this study, E. chloroticus is estimated to need between 0.16 and 0.22 kJ kg−1 h−1 for metabolic maintenance at temperatures between 21 and 24 °C. We hypothesize that the available energy, shown by a positive SfG, is being reallocated to ensure short-term survival at the expense of growth and reproduction as previously shown for the sea urchin Strongylocentrotus droebachiensis under high pCO2 conditions (Stumpp et al., 2012). Overall performance of E. chloroticus was dramatically decreased when acclimated at 24 °C, which is the maximum temperature that has been recorded in north-eastern New Zealand during summer (February 1974, Leigh Marine Laboratory dataset). In north-eastern New Zealand, seawater temperature has already increased about 0.6 °C in the past 30 years (Schiel, 2013), and a further increase of 0.7–2.3 °C for the year 2090 has been projected (IPCC, 2014). Although animals in the wild are exposed to multiple stressors, temperature is one of the most important factors limiting distribution of species due to its influence on metabolic reactions (Willmer, 1999; Sanford, 2002). The loss of reproductive output at 24 °C in E. chloroticus shown here, combined with the sensitivity of early embryonic stages to environmental stressors (Clark et al., 2009; Delorme and Sewell, 2013, 2014), suggests that there may be changes to the population persistence and
39
distribution of northern populations within the next few decades unless there is acclimatization of E. chloroticus to the new thermal conditions (Bell and Collins, 2008; Somero, 2010), or the limited genetic variation in E. chloroticus (Nagel et al., 2015) is sufficient to facilitate adaptation (Bell and Collins, 2008). Populations of E. chloroticus may become restricted to higher, more southern, latitudes where seawater temperatures are lower during the crucial periods of gonad growth and embryonic and larval development (Delorme and Sewell, 2013, 2014), with the potential loss of an important habitat-determiner (Jones, 1988) from parts of the subtidal ecosystem of north-eastern New Zealand. Acknowledgements Thanks to Errol Murray, Peter Browne, for helping with the experimental set up, to Brady Doak, Richard Taylor for providing diving equipment, to Alwyn Rees and Benn Hanns for providing fresh kelp, to Mark Wilcox for helping with animal collection, to Josefina Peters for helping with gonad histology, and to Leonardo Zamora for helping with animal and data collection. NJD was supported by the Chilean Government (Comisión Nacional de Investigación Científica y Tecnológica, CONICYT). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cbpa.2016.03.020. References Azad, A.K., Pearce, C.M., McKinley, R.S., 2011 Effects of diet and temperature on ingestion, absorption, assimilation, gonad yield, and gonad quality of the purple sea urchin (Strongylocentrotus purpuratus). Aquaculture 317, 187–196. Barker, M., 2013 Evechinus chloroticus. In: John, M.L. (Ed.)Developments in Aquaculture and Fisheries Science vol. 38. Elsevier, pp. 355–368. Bayne, B.L., Moore, M.N., Widdows, J., Livingstone, D.R., Salkeld, P., Crisp, D.J., Morris, R.J., Gray, J.S., Holden, A.V., Newell, R.C., et al., 1979 Measurement of the responses of individuals to environmental stress and pollution: studies with bivalve molluscs. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 286, 563–581. Bell, G., Collins, S., 2008 Adaptation, extinction and global change. Evol. Appl. 1, 3–16. Brockington, S., Clarke, A., 2001 The relative influence of temperature and food on the metabolism of a marine invertebrate. J. Exp. Mar. Biol. Ecol. 258, 87–99. Carey, N., Harianto, J., Byrne, M., 2016 Urchins in a high CO2 world: partitioned effects of body-size, ocean warming and acidification on metabolic rate. J. Exp. Biol. http://dx. doi.org/10.1242/jeb.136101. Clark, D., Lamare, M., Barker, M., 2009 Response of sea urchin pluteus larvae (Echinodermata: Echinoidea) to reduced seawater pH: a comparison among a tropical, temperate, and a polar species. Mar. Biol. 156, 1125–1137. IPCC, 2014. Climate change 2014: impact, adaptation and vulnerability. Working Group II Contribution to the IPCC 5th Assessment Report. Cambridge University Press, Cambridge. Constable, A.J., 1993 The role of sutures in shrinking of the test in Heliocidaris erythrogramma (Echinoidea: Echinometridae). Mar. Biol. 117, 423–430. Delorme, N.J., Sewell, M.A., 2013 Temperature limits to early development of the New Zealand sea urchin Evechinus chloroticus (Valenciennes, 1846). J. Therm. Biol. 38, 218–224. Delorme, N.J., Sewell, M.A., 2014 Temperature and salinity: two climate change stressors affecting early development of the New Zealand sea urchin Evechinus chloroticus. Mar. Biol. 161, 1999–2009. Dix, T., 1970a Biology of Evechinus chloroticus (Echinoidea: Echinometridae) from different localities: 3. Reproduction. N. Z. J. Mar. Freshw. 4, 385–405. Dix, T., 1970b Biology of Evechinus chloroticus (Echinoidea: Echinometridae) from different localities: 1. General. N. Z. J. Mar. Freshw. 4, 91–116. Dix, T., 1972 Biology of Evechinus chloroticus (Echinoidea: Echinometridae) from different localities: 4. Age, growth, and size. N. Z. J. Mar. Freshw. 6, 48–68. Ebert, T.A., 1967 Negative growth and longevity in the purple sea urchin Strongylocentrotus purpuratus (Stimpson). Science 157, 557–558. Fenwick, G., Horning, D., 1980 Echinodermata of the Snares Islands, southern New Zealand. N. Z. J. Mar. Freshw. 4, 437–445. Fernandez, C., Boudouresque, C.-F., 2000 Nutrition of the sea urchin Paracentrotus lividus (Echinodermata: Echinoidea) fed different artificial food. Mar. Ecol. Prog. Ser. 204, 131–141. Filgueira, R., Rosland, R., Grant, J., 2011 A comparison of scope for growth (SFG) and dynamic energy budget (DEB) models applied to the blue mussel (Mytilus edulis). J. Sea Res. 66, 403–410. Garner, D.M., 1969 The seasonal range of sea temperature on the New Zealand shelf. N. Z. J. Mar. Freshw. 3, 201–208.
40
N.J. Delorme, M.A. Sewell / Comparative Biochemistry and Physiology, Part A 198 (2016) 33–40
González, R.A., Díaz, F., Licea, A., Denisse Re, A., Noemí Sánchez, L., García-Esquivel, Z., 2010 Thermal preference, tolerance and oxygen consumption of adult white shrimp Litopenaeus vannamei (Boone) exposed to different acclimation temperatures. J. Therm. Biol. 35, 218–224. Gooding, R.A., Harley, C.D.G., Tang, E., Brown, J.H., 2009 Elevated water temperature and carbon dioxide concentration increase the growth of a keystone echinoderm. Proc. Natl. Acad. Sci. U. S. A. 106, 9316–9321. Hill, S.K., Lawrence, J.M., 2006 Interactive effects of temperature and nutritional condition on the energy budgets of the sea urchins Arbacia punctulata and Lytechinus variegatus (Echinodermata: Echinoidea). J. Mar. Biol. Assoc. UK 86, 783–790. Hochachka, P.W., Somero, G.N., 2002 Biochemical Adaptation: Mechanism and Process in Physiological Evolution. Oxford University Press, New York. Humason, G.L., 1979 Animal Tissue Techniques. W. H. Freeman, San Francisco. James, P.J., Heath, P.L., 2008 The effects of season, temperature and photoperiod on the gonad development of Evechinus chloroticus. Aquaculture 285, 67–77. James, P.J., Heath, P.L., Green, K., Wright, J., 2009 The effect of elevated temperature on roe enhancement of Evechinus chloroticus collected from two latitudes. Aquaculture 288, 273–279. Jones, G.P., 1988 Ecology of rocky reef fish of north-eastern New Zealand: a review. N. Z. J. Mar. Freshw. 22, 445–462. Kelly, M.W., Sanford, E., Grosberg, R.K., 2012 Limited potential for adaptation to climate change in a broadly distributed marine crustacean. Proc. R. Soc. Lond. B Biol. Sci. 279, 349. Kemp, J.O.G., Britz, P.J., 2008 The effect of temperature on the growth, survival and food consumption of the east coast rock lobster Panulirus homarus rubellus. Aquaculture 280, 227–231. Lamare, M.D., Wing, S.R., 2001 Calorific content of New Zealand marine macrophytes. N. Z. J. Mar. Freshw. 35, 335–341. Lannig, G., Flores, J.F., Sokolova, I.M., 2006 Temperature-dependent stress response in oysters, Crassostrea virginica: pollution reduces temperature tolerance in oysters. Aquat. Toxicol. 79, 278–287. Lawrence, J.M., Cao, X., Chang, Y., Wang, P., Yu, Y., Lawrence, A.L., Watts, S.A., 2009 Temperature effect on feed consumption, absorption, and assimilation efficiencies and production of the sea urchin Strongylocentrotus intermedius. J. Shellfish Res. 28, 389–395. Levitan, D., 1988 Density-dependent size regulation and negative growth in the sea urchin Diadema antillarum Philippi. Oecology 76, 627–629. McElroy, D.J., Nguyen, H.D., Byrne, M., 2012 Respiratory response of the intertidal seastar Parvulastra exigua to contemporary and near-future pulses of warming and hypercapnia. J. Exp. Mar. Biol. Ecol. 416–417, 1–7. Nagel, M.M., Sewell, M.A., Lavery, S.D., 2015 Differences in population connectivity of a benthic marine invertebrate Evechinus chloroticus (Echinodermata: Echinoidea) across large and small spatial scales. Conserv. Genet. 16, 965–978. Navarro, J.M., Winter, J.E., 1982 Ingestion rate, assimilation efficiency and energy balance in Mytilus chilensis in relation to body size and different algal concentrations. Mar. Biol. 67, 255–266. Navarro, J.M., Urrutia, G.X., Carrasco, C., 2006 Scope for growth versus actual growth in the juvenile predatory gastropod Chorus giganteus. J. Mar. Biol. Assoc. UK 86, 1423–1428. Naylor, C., Maltby, L., Calow, P., 1989 Scope for growth in Gammarus pulex, a freshwater benthic detritivore. Hydrobiologia 188-189, 517–523. Otero-Villanueva, M.d.M., Kelly, M.S., Burnell, G., 2004 How diet influences energy partitioning in the regular echinoid Psammechinus miliaris; constructing an energy budget. J. Exp. Mar. Biol. Ecol. 304, 159–181. Pedersen, S.A., Hakedal, O.J., Salaberria, I., Tagliati, A., Gustavson, L.M., Jenssen, B.M., Olsen, A.J., Altin, D., 2014 Multigenerational exposure to ocean acidification during food limitation reveals consequences for copepod scope for growth and vital rates. Environ. Sci. Technol. 48, 12275–12284. Pernet, F., Tremblay, R., Comeau, L., Guderley, H., 2007 Temperature adaptation in two bivalve species from different thermal habitats: energetics and remodelling of membrane lipids. J. Exp. Biol. 210, 2999–3014. Pörtner, H.O., 2002 Climate variations and the physiological basis of temperature dependent biogeography: systemic to molecular hierarchy of thermal tolerance in animals. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 132, 739–761. Pörtner, H.O., 2010 Oxygen- and capacity-limitation of thermal tolerance: a matrix for integrating climate-related stressor effects in marine ecosystems. J. Exp. Biol. 213, 881–893.
Pörtner, H.O., 2012 Integrating climate-related stressor effects on marine organisms: unifying principles linking molecule to ecosystem-level changes. Mar. Ecol. Prog. Ser. 470, 273–290. Pörtner, H.O., Farrell, A.P., 2008 Physiology and climate change. Science 322, 690–692. Pörtner, H.O., Peck, L., Somero, G., 2007 Thermal limits and adaptation in marine antarctic ectotherms: an integrative view. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 362, 2233–2258. Quinn, G.P., Keough, M.J., 2002 Experimental Design and Data Analysis for Biologists. Cambridge University Press, Cambridge. Sanford, E., 2002 Water temperature, predation, and the neglected role of physiological rate effects in rocky intertidal communities. Integr. Comp. Biol. 42, 881–891. Schiel, D.R., 2013 The other 93%: trophic cascades, stressors and managing coastlines in non-marine protected areas. N. Z. J. Mar. Freshw. 47, 374–391. Schiel, D.R., Kingsford, M.J., Choat, J.H., 1986 Depth distribution and abundance of benthic organisms and fishes at the subtropical Kermadec Islands. N. Z. J. Mar. Freshw. 20, 521–535. Shirley, T.C., Stickle, W.B., 1982 Responses of Leptasterias hexactis (Echinodermata: Asteroidea) to low salinity. Mar. Biol. 69, 155–163. Siikavuopio, S.I., James, P., Lysne, H., Sæther, B.S., Samuelsen, T.A., Mortensen, A., 2012 Effects of size and temperature on growth and feed conversion of juvenile green sea urchin (Strongylocentrotus droebachiensis). Aquaculture 354–355, 27–30. Siikavuopio, S.I., Mortensen, A., Christiansen, J.S., 2008 Effects of body weight and temperature on feed intake, gonad growth and oxygen consumption in green sea urchin, Strongylocentrotus droebachiensis. Aquaculture 281, 77–82. Sokolova, I.M., 2013 Energy-limited tolerance to stress as a conceptual framework to integrate the effects of multiple stressors. Integr. Comp. Biol. 53, 597–608. Sokolova, I.M., Frederich, M., Bagwe, R., Lannig, G., Sukhotin, A.A., 2012 Energy homeostasis as an integrative tool for assessing limits of environmental stress tolerance in aquatic invertebrates. Mar. Environ. Res. 79, 1–15. Somero, G.N., 2005 Linking biogeography to physiology: evolutionary and acclimatory adjustments of thermal limits. Front. Zool. 2, 1–9. Somero, G.N., 2010 The physiology of climate change: how potentials for acclimatization and genetic adaptation will determine "winners" and "losers". J. Exp. Biol. 213, 912–920. Somero, G.N., 2012 The physiology of global change: linking patterns to mechanisms. Ann. Rev. Mar. Sci. 4, 39–61. Stumpp, M., Trübenbach, K., Brennecke, D., Hu, M.Y., Melzner, F., 2012 Resource allocation and extracellular acid–base status in the sea urchin Strongylocentrotus droebachiensis in response to CO2 induced seawater acidification. Aquat. Toxicol. 110–111, 194–207. Uthicke, S., Liddy, M., Nguyen, H.D., Byrne, M., 2014 Interactive effects of near-future temperature increase and ocean acidification on physiology and gonad development in adult Pacific sea urchin, Echinometra sp. A. Coral Reefs 33, 831–845. Walker, M.M., 1981 Influence of season on growth of the sea urchin Evechinus chloroticus. N. Z. J. Mar. Freshw. 15, 201–205. Walker, M.M., 1982 Reproductive periodicity in Evechinus chloroticus in the Hauraki Gulf. N. Z. J. Mar. Freshw. 16, 19–25. Warren, C.E., Davis, G., 1967 Laboratory studies on the feeding, bioenergetics, and growth of fish. In: Gerking, S.D. (Ed.), The Biological Basis of Freshwater Fish Production. Blackwell Scientific Publications, Oxford, pp. 175–214. Watts, S.A., Hofer, S.C., Desmond, R.A., Lawrence, A.L., Lawrence, J.M., 2011 The effect of temperature on feeding and growth characteristics of the sea urchin Lytechinus variegatus fed a formulated feed. J. Exp. Mar. Biol. Ecol. 397, 188–195. Widdows, J., Johnson, D., 1988 Physiological energetics of Mytilus edulis: scope for growth. Mar. Ecol. Prog. Ser. 46, 113–121. Willmer, P., 1999 Environmental Physiology of Animals. Blackwell Publisher, Massachusetts, USA. Winberg, G.G., 1960 Rate of Metabolism and Food Requirements of Fishes. Fisheries Research Board of Canada, Nanaimo, B.C. Zamora, L.N., Jeffs, A.G., 2012 Feeding, metabolism and growth in response to temperature in juveniles of the Australasian sea cucumber, Australostichopus mollis. Aquaculture 358-359, 92–97. Zamora, L.N., Jeffs, A.G., 2015 Macronutrient selection, absorption and energy budget of juveniles of the Australasian sea cucumber, Australostichopus mollis, feeding on mussel biodeposits at different temperatures. Aquac. Nutr. 21, 162–172.
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa
Effects of warm acclimation on physiology and gonad development in the sea urchin Evechinus chloroticus Natalí J. Delorme a,b,⁎, Mary A. Sewell a a b
School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand Institute of Marine Science, Leigh Marine Laboratory, University of Auckland, P.O. Box 349, Warkworth 0941, New Zealand
a r t i c l e
i n f o
Article history: Received 21 November 2015 Received in revised form 17 March 2016 Accepted 23 March 2016 Available online 01 April 2016 Keywords: Climate change Ecophysiology Feeding Growth Reproduction Respiration Scope for growth Sea urchin
a b s t r a c t The physiology of the New Zealand sea urchin Evechinus chloroticus was evaluated through feeding, respiration, growth and gonad growth in adult animals acclimated for 90 days at 18 °C (annual mean temperature) and 24 °C (ambient summer temperature (21 °C) +3 °C). Measured parameters with representative rates of assimilation efficiency were used to calculate scope for growth (SfG) for each treatment. All physiological parameters were negatively affected at 24 °C, showing a decrease in feeding rate which coincided with negative growth and gonad development at the end of the acclimation period, and a decrease in respiration rate suggesting metabolic depression. Histology of gonad samples after the acclimation period also showed no gametic material in animals acclimated at 24 °C. All animals acclimated at 24 °C had negative growth, differing from the calculated SfG which indicated that the animals had sufficient energy for production. The results suggest that calculated SfG in echinoderms should be used together with actual measurements of growth in individuals as, by itself, SfG may underestimate the actual effect of ocean warming when animals are exposed to stressful conditions. Overall, considering the total loss of reproductive output observed in E. chloroticus at higher temperatures, an increase in seawater temperature could dramatically influence the persistence of northern populations of this species, leading to flow-on effects in the subtidal ecosystem. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Environmental conditions play a fundamental role in the normal functioning of animals (Willmer, 1999), with temperature being one of the most important and studied factors. Seawater temperature is the major factor determining optimal functioning in marine invertebrates as they are ectothermic; thus any change in environmental seawater temperature could influence their overall performance (Hochachka and Somero, 2002). An increase in seawater temperature within the optimal temperature range for the animal (i.e., thermal window) will increase animal performance up to a certain point/temperature (i.e., upper pejus temperature), after which performance starts decreasing dramatically (Pörtner et al., 2007; Pörtner and Farrell, 2008). Thermal windows and limits are species-specific, but also depend on the thermal history of the animal (Pörtner, 2002; Somero, 2005, 2010, 2012; Kelly et al., 2012). Generally, increased seawater temperature has been reported to affect feeding, growth, respiration, gonad development and survival in marine invertebrates (Sanford, 2002; Lannig et al., 2006; Pernet et al., 2007; Kemp and Britz, 2008; Gooding et al., 2009; González et al., 2010; McElroy et al., 2012; Zamora and ⁎ Corresponding author at: School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand. E-mail address: [email protected] (N.J. Delorme).
http://dx.doi.org/10.1016/j.cbpa.2016.03.020 1095-6433/© 2016 Elsevier Inc. All rights reserved.
Jeffs, 2012), which may have major consequences for a particular population and marine ecosystem. Overall, increased temperature can affect physiological performance of the animals to the point at which the energy balance is altered and therefore long term survival is compromised. In echinoderms, physiological rates and energy budgets have been measured to determine the condition of the animals under different nutritional and environmental scenarios (e.g., salinity, pH and temperature, Shirley and Stickle, 1982; Hill and Lawrence, 2006; Stumpp et al., 2012; Zamora and Jeffs, 2015; Carey et al., 2016). A common measure of physiological stress/condition in animals is the scope for growth (SfG), which represents the total energy available for production (somatic and gonad growth) considering the total energy input as food and the energy lost through metabolism (Warren and Davis, 1967; Bayne et al., 1979). Values of SfG can be positive, indicating that the animal has enough energy for growth (increasing body size/ weight), zero indicating that the energy input equals the energy expenditure, or negative which indicates that there is not enough energy for growth (decreasing body size/weight) (Naylor et al., 1989; Filgueira et al., 2011). In general, thermal acclimation of echinoderms to temperatures within the species optimum range generally shows positive effects on physiological processes (Siikavuopio et al., 2008, 2012; Gooding et al., 2009; Azad et al., 2011; Watts et al., 2011). However, when animals are exposed to elevated seawater temperatures, processes such as feeding, growth, gonad growth and survival can be negatively
34
N.J. Delorme, M.A. Sewell / Comparative Biochemistry and Physiology, Part A 198 (2016) 33–40
compromised (Siikavuopio et al., 2008; Lawrence et al., 2009; Zamora and Jeffs, 2012; Uthicke et al., 2014), often accompanied by an elevated oxygen consumption (Brockington and Clarke, 2001; Siikavuopio et al., 2008; McElroy et al., 2012; Zamora and Jeffs, 2012; Uthicke et al., 2014; Carey et al., 2016). The New Zealand echinometrid Evechinus chloroticus (Valenciennes 1846) is geographically distributed throughout New Zealand from the Three Kings Island (34°10′S) to the Snares Island (47°60′S) (Fenwick and Horning, 1980; Schiel et al., 1986). Bathymetrically, E. chloroticus is commonly found between 12 and 14 m deep; however, it is also found in low intertidal pools and the shallow subtidal (Dix, 1970a; Barker, 2013). This sea urchin species has an annual breeding cycle, spawning during the austral summer (Nov\\Feb) along its entire distributional range (Dix, 1970b, Barker, 2013,). Due to its broad latitudinal range, E. chloroticus is exposed to a wide range of seawater temperatures, from a minimum of 9.5–10 °C in the South Island during winter, to a maximum of 20–21 °C in the North Island during summer (Garner, 1969). The Intergovernmental Panel on Climate Change (IPCC) has projected an increase in seawater temperature of New Zealand waters of about 0.7–2.3 °C by the year 2090 due to global warming (IPCC, 2014). Previous research has shown that growth of wild E. chloroticus in north-eastern New Zealand varies seasonally, with peak growth in early spring and slowest growth during late spring and early summer when temperatures are higher (Walker, 1981). In the laboratory, thermal acclimation of E. chloroticus (sourced from the South Island of New Zealand) has shown a higher gonad growth as acclimation temperature increased (James and Heath, 2008). However, when acclimated to elevated temperatures (22 and 24 °C), E. chloroticus from both the South Island and the North Island showed a lower rate of increase in gonad growth (James et al., 2009). Nevertheless there is no study to date that has examined the effects of ocean warming on physiological performance and energy balance in E. chloroticus. Here we examined the effect of thermal acclimation at 18 °C (annual mean temperature, and ambient during late spring–early summer) and 24 °C (ambient summer temperature (21 °C) +3 °C) on the physiology (i.e., feeding, respiration, growth and gonad growth) of adult E. chloroticus from a north-eastern population in New Zealand. Further, we used these physiological parameters to calculate SfG in each temperature treatment to determine if SfG is a reliable measure of physiological state and a real representation of the actual production (growth) observed in sea urchins under thermal stress.
seawater temperature was 15.2 ± 0.4 °C (±s.d., n = 336; Leigh Marine Laboratory dataset), while at the time of collection was 15 °C. In the ten days prior to the February collection, average seawater temperature was 19.9 ± 0.3 °C (±s.d., n = 288; Leigh Marine Laboratory dataset), while at the time of collection was 21 °C. At both collection times, the animals were transported about 5 km to the Leigh Marine Laboratory, University of Auckland and held for 3 days in a 280 l tank with ambient flow-through seawater (August: 15 °C; February: 21 °C) to allow the animals to recover from handling stress. Animals collected in August were acclimated for 90 days, whereas animals collected in February were used for experimentation 3 days after collection. The acclimation system consisted of 8 tanks each of 36 l (4 tanks for each acclimation temperature) with flow-through seawater at ambient temperature (15 °C) with aeration provided by an airstone in each tank. The tanks were connected to two heat pumps (Aquahort EH13 12.5 kW, Auckland, New Zealand) which maintained the flowing seawater at the desired temperature. After the recovery period, 12 animals were placed in each tank, resulting in an initial density of 1.7 ± 0.1 and 1.6 ± 0.1 kg m−2 of internal surface area for the 18 and 24 °C treatment respectively (±s.d., n = 4). The animals were maintained in the experimental tanks for 3 days before the seawater temperature of the heating pumps was increased at a rate of 1 °C/day (James and Heath, 2008; James et al., 2009) until the desired acclimation temperatures were reached (18 and 24 °C). These temperatures were maintained in the tanks for 90 days, until November 2013. During the 90-day experimental period the animals were under a photoperiod of 12 h light: 12 h dark and seawater temperature was measured every hour with iBCod 22 l temperature loggers (Thermodata, Brisbane, QLD, Australia). Animals were fed to satiety with Ecklonia radiata and the tanks were cleaned every day. 2.1. Feeding rate Feeding rate of the sea urchins was measured in each tank after an acclimation period of 30 days, and for wild animals during the summer season (ambient temperature of 21 °C). Fronds of the kelp E. radiata were externally dried with paper towels and ~ 100 g (wet weight) of algae was placed into each tank. After 5 days the remaining algae were removed, externally dried and final wet weight determined. To control for natural algal degradation the same procedure was performed in 4 separate tanks without animals with flowing seawater at each temperature maintained by heating pumps. Feeding rate (g kg−1 h−1) was calculated as follows:
2. Material and methods Adult sea urchins were collected from Matheson's Bay, northeastern New Zealand (36°18′17″S; 174°47′51″E) at two separate times (Fig. 1). 1) August 2013: for thermal acclimation of animals at two different temperatures (18 and 24 °C) and to measure physiological performance of these animals (Fig. 1); and 2) February 2014: to determine physiological performance of wild animals during the summer season (Fig. 1). In the two weeks prior to the August collection, average
F¼
½ð F0 Ft Þ ð Fb0 Fbt Þ : WT
ð1Þ
Where F0 is the initial amount of food given, Ft is the amount of food remaining (g), Fb0 and Fbt are the initial and final amount of food in the tanks without animals (g), W is the total weight of the animals in the tank (kg), and T is the duration of the measurement in hours (h).
Fig. 1. Timeline of research relative to the reproductive cycle in Evechinus chloroticus (Walker, 1982). Arrows indicate time of collection of animals (August 2013 for acclimation at 18 and 24 °C, and February 2014 for experimentation on wild animals). Physiological measurements are listed for each collection time. GI0: initial gonad index; GIt: final gonad index.
N.J. Delorme, M.A. Sewell / Comparative Biochemistry and Physiology, Part A 198 (2016) 33–40
separated and their final GI determined (GIt). Gonad index (GI) and the index of increase in GI (Y) were calculated as follow:
2.2. Respiration rate Respiration rates of individual sea urchins were determined after an acclimation period of 30 days (n = 6 urchins per temperature) for urchins that had been starved for 1 week prior to respiration measurements. In addition, respiration rate was determined on wild animals (n = 6) during the summer season (ambient temperature of 21 °C). Respiration rate was determined in individual sea urchins using a double-bottomed plexi-glass respirometry chamber (1.4 l) filled with 1 μm filtered and UV-treated seawater. The sea urchin was placed in the main chamber; a magnetic stir bar with a submersible magnetic stirrer avoided water stratification in the main chamber. Dissolved oxygen concentration (μmol l−1) was recorded every 1 s with a needle type fibre optic oxygen microsensor connected to an oxygen meter MicroxTX3 (PreSens, Regensburg, Germany) and PSt1 (v6.02) software. To keep the temperature constant, the complete system was submersed in a 36 l tank with water at the desired temperature (18 and 24 °C for acclimated animals, and 21 °C for wild animals). Seawater temperature was maintained by a heating unit and a water pump delivering seawater at the desired temperature. The animals were carefully placed in the chamber and oxygen concentration of the water was measured for 2 h (oxygen saturation N 50%). After each respiration measurement, the animal's wet weight, volume and test diameter were measured as described below. In addition, control measurements were performed without animals at each temperature in order to account for background respiration. Respiration rate was calculated as the weight specific oxygen consumption rate (R, mgO 2 kg− 1 h− 1 ) described by Siikavuopio et al. (2008), but with one minor modification to consider background respiration:
R¼
½ðC 0 C t Þ ðC b0 C bt Þ V : WT
35
ð2Þ
Where C0 and Ct are the initial and final oxygen concentrations of the water in the chamber with animals (mgO2 l− 1), Cb0 and Cbt are the initial and final oxygen concentrations of the water in the chamber without animals (mgO2 l− 1), V is the volume of the respirometry chamber minus the volume of the animal (ml), W is the wet weight of the animal (kg), and T is the duration of the measurement (h). 2.3. Growth At the start of the acclimation period, measurements of test diameter (cm) and wet weight (g) of all the animals were recorded in each acclimation tank using callipers and a digital balance (±0.01 g). Growth was determined monthly by measuring test diameter and wet weight of the animals held in each tank, obtaining an average measure per tank at each acclimation temperature. The average measure was used to calculate the percent of change in wet weight and test diameter which were used for the statistical analyses. The animals were starved for 1 week prior to each growth measurement to allow the animals to completely empty their stomachs (i.e., animals fed for 3 weeks before each monthly measurement). 2.4. Gonad growth and histology Gonad growth was measured through the increase in gonad index (Y) which was determined from measurements of initial and final gonad indexes (GIs). After collection of animals and before the temperature experiments were set up, a subsample of 10 animals was used to determine the initial GI (GI0). At the end of the 90 days acclimation period and after the last measurement of growth was performed, a random sample of 10 animals from each acclimation temperature was
GI ¼
100G W
Y ¼ GIt GI 0 :
ð3Þ ð4Þ
Where G is the gonad's wet weight and W is the animal's wet weight. The animals were starved for 1 week prior to GI measurements. The gonads from animals acclimated at 18 and 24 °C were sampled for histology after the 90 days acclimation. In addition, the gonads from wild animals (n = 10) collected during the summer season (February 2014, ambient temperature of 21 °C) were processed for histology. The gonads were fixed in 10% formalin and transferred after 2 days to 70% ethanol for storage. Dehydration, clearing and embedding of the gonads used routine methods for wax histology (Humason, 1979), using haematoxylin and eosin as stains and sectioned at 5–10 μm. Histological sections were observed and photographed using a Leica DMRE compound microscope (Leica Microsystems Inc., Buffalo Grove, IL, USA). 2.5. Scope for growth Scope for growth (SfG) was calculated using the physiological data obtained from feeding and respiration rates measured at day 30 in the different temperature treatments. As the amount of energy lost through faeces and excretion was not measured during the experiment, the approach of Pedersen et al. (2014) was used, based on an assimilation efficiency of 60–80% in laboratory experiments in E. chloroticus (unpublished data). In addition, it has been reported that in echinoderms the loss from excretion is very low (b 1% of total balance) (Hill and Lawrence, 2006; Stumpp et al., 2012; Zamora and Jeffs, 2015). Assimilated food was converted to energy equivalents using the conversion reported by Lamare and Wing (2001) for E. radiata (1 g wet weight = 1.72 kJ). Conversion of respiration measurements to energy equivalents used the oxycaloric coefficient (1 mgO2 = 19.97 J, Winberg, 1960 fide Siikavuopio et al., 2008). Thus, scope for growth (SfG, kJ kg−1 h− 1) was calculated as described by Widdows and Johnson (1988): SfG ¼ A R:
ð5Þ
Where A is the energy gained through the assimilated food (kJ kg− 1 h− 1) and R is the energy lost through respiration (kJ kg−1 h−1). 2.6. Statistical analyses Feeding rate and growth rate (% change in wet weight or test diameter) data were checked for normality and homoscedasticity (Shapiro– Wilk and Levene's tests respectively). Feeding rate was analysed with a one-way ANOVA test using temperature as factor and the feeding rate as the dependent variable. The percent change in wet weight did not meet assumptions of normality; but as it met homoscedasticity assumptions it was analysed using a parametric test (Quinn and Keough, 2002). Both the percent change in wet weight and the percent change in test diameter were analysed with a two-way ANOVA test using temperature and time as factors and the percent change in wet weight and test diameter as the dependent variables. Respiration rate and the increase in gonad index did not meet the assumption of homoscedasticity and therefore were analysed with a Kruskal–Wallis one-way ANOVA on ranks with temperature as factor and respiration rate and increase in gonad as the dependent variables. Significant differences between groups were identified using the a posteriori Tukey test (α = 0.05). All statistical analyses were run using the statistical software included within Sigma Plot 12.5 (SYSTAT Software, Inc., San Jose, CA, USA).
36
N.J. Delorme, M.A. Sewell / Comparative Biochemistry and Physiology, Part A 198 (2016) 33–40
3. Results Seawater temperature in the experimental tanks during the acclimation period was 17.7 ± 0.4 °C (± s.d., n = 2597) and 23.9 ± 0.3 °C (± s.d., n = 2474). Survival in the 18 °C acclimation treatment was 98% as one animal escaped from the tank and fell onto the floor, cracking its test and dying afterwards. In the 24 °C acclimation treatment survival was 96% with two animals which started losing their primary and secondary spines and dying after 2 months of acclimation.
3.1. Feeding rate During the acclimation period, the animals from the 18 °C treatment fed constantly from day 1, whereas the animals from the 24 °C treatment did not feed during the first week of acclimation and then fed on comparatively lower amounts of kelp than the 18 °C treatment. After 30 days of acclimation, the feeding rate was significantly different between treatments (ANOVA: F2,9 = 84.77, P b 0.001). Animals acclimated at 24 °C showed a 73% decrease in feeding rate compared to animals acclimated at 18 °C (P b 0.001, Fig. 2), and a non-significant decrease of 26% compared to control animals at 21 °C (P = 0.327, Fig. 2). Feeding rate from wild animals also showed a decrease in feeding rate of 64% compared to animals acclimated at 18 °C (P b 0.001, Fig. 2).
Fig. 3. Effect of temperature on the respiration rate of Evechinus chloroticus. Animals from 18 and 24 °C were acclimated 30 days to each temperature; whereas Wild-summer (21 °C) correspond to non-acclimated control animals during the summer season (n = 6 for each treatment). All animals were starved for 1 week prior to measurements. Significant differences (P b 0.05) between temperatures are denoted by lower case letters.
3.3. Growth 3.2. Respiration rate Respiration rates of E. chloroticus after 30 days of temperature acclimation at 18 and 24 °C and from wild animals in the summer season (21 °C) were significantly different (Kruskal–Wallis: H = 10.78, d.f. = 2, P = 0.005). Respiration rate increased from 18 to 24 °C; however, this increase was not significant (P N 0.05). The highest respiration rate was observed in non-acclimated animals (21 °C), with a respiration rate of 2.8 mgO2 kg−1 h− 1 higher than animals acclimated at 18 °C (P b 0.05, Fig. 3). No significant differences were observed in the respiration rate between animals acclimated at 18 and 24 °C or between nonacclimated animals (21 °C) and animals acclimated at 24 °C (P N 0.05, Fig. 3).
Initial wet weight and test diameter of the animals in the 8 tanks used for acclimation were not statistically different (Weight: ANOVA: F7,88 = 0.765, P = 0.618; test diameter: ANOVA: F7,88 = 2.096, P = 0.052). During the acclimation period there was a significant increase in wet weight and test diameter at 18 °C compared to animals acclimated at 24 °C (Tables 1 and 2, Fig. 4). At the end of the 90 day acclimation period, animals acclimated at 24 °C decreased their wet weight and their test diameter from the initial condition by 7.2 and 7.8% respectively (Fig. 4). Wet weight and test diameter in these animals were respectively 12.3 and 8.7% lower at the end of the acclimation period than animals acclimated at 18 °C (Fig. 4). No significant differences were found in the percent change in wet weight and the percent change in test diameter with acclimation time and there was no significant interaction between factors (Table 2, Fig. 4).
3.4. Gonad growth and histology
Fig. 2. Effect of temperature on the feeding rate of Evechinus chloroticus. Data represents the mean feeding rate (g kg−1 h−1) ± s.e.m. (n = 4 tanks). Animals at 18 and 24 °C were acclimated for 30 days to each temperature; whereas Wild-summer (21 °C) corresponds to non-acclimated control animals during the summer season. All animals were starved for 1 week prior to measurements. Significant differences (P b 0.05) among temperatures are denoted by lower case letters.
The mean GI of the animals after field collection and prior to thermal acclimation was 2.98 ± 0.40% (±s.e.m., n = 10). After the 90 days of thermal acclimation, the mean final GIs were 4.57 ± 0.37% and 0.50 ± 0.15% for animals acclimated at 18 and 24 °C respectively (±s.e.m., n = 10 for each temperature). The increase in gonad index (Y) was significantly different at the end of the acclimation period in animals acclimated at 18 and 24 °C (Kruskal–Wallis: H = 14.29, d.f. = 1, P b 0.001). Animals acclimated at 24 °C showed a decrease of 2.5% in gonad index from initial conditions (Fig. 5). Gonad growth in these animals was ca. 4% lower than the gonad growth achieved by animals acclimated at 18 °C for 90 days (P b 0.001, Fig. 5). After the 90 day acclimation period, gonads from the animals acclimated at 18 °C were large and yellow-ish in colour; however, the gonads from the animals acclimated at 24 °C were very thin, looked transparent and red/brown-ish in colour. Histology of these gonads showed that at 18 °C and in the non-acclimated animals (21 °C) all animals were mature or partially spawned (Fig. 6a–d). Whereas at 24 °C, gametic material could only be distinguished in 3/10 samples which had degenerating eggs (Fig. 6e); the other 7 sea urchins had only empty gonad tubules with no gametic material in the gonads (Fig. 6f).
N.J. Delorme, M.A. Sewell / Comparative Biochemistry and Physiology, Part A 198 (2016) 33–40
37
Table 1 Mean Wet Weight (WW) and Test Diameter (TD) ± s.e.m. (n = 4 tanks) of Evechinus chloroticus acclimated at different temperatures for 90 days. Time (days)
18 °C
0 30 60 90
24 °C
WW ± s.e.m (g)
TD ± s.e.m. (cm)
WW ± s.e.m. (g)
TD ± s.e.m. (cm)
99.90 ± 3.71 103.22 ± 4.31 104.36 ± 5.18 104.84 ± 4.03
6.01 ± 0.10 5.99 ± 0.10 6.05 ± 0.12 6.06 ± 0.13
98.11 ± 3.49 95.93 ± 3.43 93.52 ± 3.04 91.12 ± 3.59
6.34 ± 0.10 6.05 ± 0.09 5.86 ± 0.14 5.85 ± 0.10
3.5. Scope for growth Scope for growth calculations, assuming an assimilation efficiency of 60–80%, showed that in all treatments the animals have available energy for somatic and gonad production (Table 3); however, the available energy for production is ca. 76% higher in animals acclimated at 18 °C than in animals acclimated at 24 °C (Table 3). On the other hand, animals without previous acclimation taken from the wild during the summer season (February 2014, ambient temperature of 21 °C) showed a scope for growth 27% higher than animals acclimated at 24 °C and 67% lower than animals acclimated at 18 °C (Table 3). The positive values of SfG obtained for animals acclimated at 24 °C do not match the actual values of growth (weight and test diameter) obtained from the experiment, where there was negative growth and no gonad production in 24 °C animals. Null or negative growth (SfG ≤ 0) in E. chloroticus can only be obtained by either decreasing the assimilation efficiency by 9 to 11-fold (≤7.36% at 24 °C; which corresponds to a feeding rate of 0.11 g kg−1 h−1) or increasing the respiration rate 8 to 11-fold higher than that measured.
acclimated at 24 °C showed a lower respiration rate than wild animals during the summer season, it is possible to suggest that animals at 24 °C may be undergoing metabolic depression (Pörtner, 2010, 2012). Therefore, 24 °C could be considered as the temperature of transition from the pejus to the pessimum range in E. chloroticus (Pörtner, 2012; Sokolova et al., 2012; Sokolova, 2013). Animals in the pejus range experience either an increase in maintenance costs to cover demand for stress protection and damage repair, and only storage energy is used to cover these costs; whereas in the pessimum range animals experience a further increase in energy demand for maintenance and an impairment of the aerobic metabolism (Sokolova et al., 2012; Sokolova, 2013). Metabolic depression appears to restore the balance at low turnover rates of energy at the expense of other functions (e.g., reproduction, growth, activity) to ensure short-term survival (Sokolova et al., 2012).
4. Discussion Increasing the seawater temperature to 24 °C, the predicted temperature for northern New Zealand in 2090 (IPCC, 2014), results in decreased feeding rate, negative growth and complete loss of reproductive output in the endemic New Zealand sea urchin E. chloroticus. As respiration rate is maximum at current summer temperatures (21 °C), E. chloroticus in northern New Zealand may already be experiencing temperatures that approach the limits of physiological performance. Respiration rate of wild animals during summer and during the winter season (15 °C, data not shown) was more variable than acclimated animals probably due to the influence of the thermal history of the animals used from the wild. The fact that acclimated animals showed a lower respiration rate than non-acclimated wild animals may be a result of the temperature that wild animals were exposed to prior to the respiration measurements (21 °C). This temperature is in between the temperatures of the two acclimations (18 and 24 °C) and may represent the highest point of respiration of E. chloroticus following a typical performance curve (Pörtner and Farrell, 2008). Considering that animals
Table 2 Summary of statistical analyses performed on the percent of Gained Wet Weight and the percent of Increased Test Diameter from Evechinus chloroticus acclimated at 18 and 24 °C. ANOVA statistic (F), mean square (MS) and p-value (P) are shown for each variable using Temperature and Time as factors. Variable
Factor
df
MS
F
P
% Gained Wet Weight
Temperature (T) Time (t) Txt Residual Temperature (T) Time (t) Txt Residual
1 2 2 18 1 2 2 18
477.365 4.778 23.297 18.570 307.277 2.464 11.428 10.301
25.706 0.257 1.255
b0.001 0.776 0.309
29.830 0.239 1.109
b0.001 0.790 0.351
% Increased Test Diameter
Fig. 4. Effect of temperature on the percent change in wet weight and the percent change in test diameter in Evechinus chloroticus. Data represents the mean change in wet weight and test diameter per tank ± s.e.m. (n = 4 tanks). All animals were starved for 1 week prior to measurements. Significant differences (P b 0.05) between treatments are denoted by lower case letters and detailed in Table 2.
38
N.J. Delorme, M.A. Sewell / Comparative Biochemistry and Physiology, Part A 198 (2016) 33–40 Table 3 Summary of the parameters used for calculations of Scope for Growth (SfG) at the different temperatures (acclimated animals (18 and 24 °C) and wild-summer control animals (21 °C)). Assimilated food (A) was calculated from average feeding rate (FR), using an assimilation efficiency of 60–80% (⁎). Assimilation efficiency (A) and average respiration rate (R) were then converted to energy equivalent (Aenergy and Renergy) which were used for the calculation of the SfG (kJ kg−1 h−1) for each temperature.
FR (g kg−1 h−1) A (g kg−1 h−1) Aenergy (kJ kg−1 h−1) R (mgO2 kg−1 h−1) Renergy (kJ kg−1 h−1) SfG (kJ kg−1 h−1)
18 °C
24 °C
Wild summer-21 °C
5.70 3.42–4.56 5.88–7.84 8.03 0.16 5.72–7.68
1.50 0.90–1.20 1.55–2.06 9.64 0.19 1.36–1.87
2.03 1.22–1.62 2.10–2.79 10.83 0.22 1.88–2.57
⁎ Assimilation efficiency was based on an assimilation efficiency of 60–80% in laboratory experiments in Evechinus chloroticus.
Fig. 5. Effect of temperature acclimation on the gonad index of Evechinus chloroticus. Data represents the mean increase in gonad index (Y) ± s.e.m. (n = 10 animals) at each temperature after 90 days of acclimation. All animals were starved for 1 week prior measurements. Significant differences (P b 0.05) between temperatures are denoted by lower case letters.
Experiments at elevated seawater temperatures in other sea urchins have shown similar patterns of decreased feeding rates and gonad growth (Siikavuopio et al., 2008; Lawrence et al., 2009; Uthicke et al., 2014); the decrease in both growth and gonad development being attributed to the high energy demands of increased metabolism at elevated temperatures (Uthicke et al., 2014). Previous research in E. chloroticus has shown that acclimation for 10 weeks at 18–20 °C is
Fig. 6. Histological sections of gonads of Evechinus chloroticus acclimated at different temperatures. Respectively, (a) and (b): female and male gonads of animals acclimated at 18 °C; (c) and (d): female and male gonads of wild animals (21 °C); (e) and (f): female (with degenerating eggs) and undetermined gonads (empty gonad tubules) of animals acclimated at 24 °C. Labels correspond to ripe ova (O), vitellogenic oocytes (VO), lumen (L) and mature sperm (S). Scale bars = 200 μm.
N.J. Delorme, M.A. Sewell / Comparative Biochemistry and Physiology, Part A 198 (2016) 33–40
optimal for gonad growth, regardless of the study population/location (James et al., 2009). Urchins acclimated at 24 °C had the lowest increase in GI during the spring/early summer when fed an artificial diet (James et al., 2009). In the present study there was a negative effect of acclimation temperature on the percent of change in weight and size; however, there was no effect of time for both acclimation temperatures, which may be a result of the high variability of the data at the different times during acclimation. There was also a negative effect of acclimation temperature on gonad growth in E. chloroticus which decreased by 83% after 90 days of acclimation at 24 °C. Therefore, it is likely that the animals were using the energy available in the gonads to maintain their basic biological functions, since sea urchins allocate a large amount of ingested energy in the gonads (Fernandez and Boudouresque, 2000; Otero-Villanueva et al., 2004). In addition to the decrease in gonad weight reported for E. chloroticus in the present study, and as also previously reported for Echinometra sp. A (acclimated at 31 °C, Uthicke et al., 2014), animals acclimated at 24 °C were not able to produce gametes or generate viable embryos (N. J. Delorme, unpublished). E. chloroticus acclimated at 24 °C is physiologically stressed, resulting in a dramatic loss of wet weight and test size (7 and 8% respectively), and gonad tissue (83%). Negative growth and shrinking have been previously reported for starved E. chloroticus (Dix, 1972) and other sea urchin species (Ebert, 1967; Levitan, 1988; Constable, 1993), which has been related to the reduction in the sutures that hold the plates together rather than calcite resorption (Constable, 1993). The loss of weight and size in an organism is a reflection of a negative energy balance or SfG, with animals having to use their energetic reserves in order to cope with the higher metabolic costs of living under stressful conditions (Filgueira et al., 2011). Previous studies in molluscs have shown that there is a good relationship between actual growth measurements and calculated SfG (e.g., Bayne et al., 1979; Navarro and Winter, 1982; Navarro et al., 2006; Filgueira et al., 2011), and have considered SfG as a reliable measurement of an animal's energetic condition (Navarro et al., 2006). In contrast, in this study SfG at 24 °C indicated sufficient energy for growth after 30 days of acclimation; whereas the sea urchins showed negative growth during the complete duration of the acclimation period. Since the parameters used for calculation of SfG might also vary among individuals, we suggest to complement the use of SfG in echinoderms with actual measurements of growth in individual animals. Although negative SfG has been reported in echinoderms (e.g. Arbacia punculata and Lytechinus variegatus under starvation, Hill and Lawrence, 2006), and matched with negative growth in starfish under salinity stress (Shirley and Stickle, 1982), the SfG calculations here underestimated the real effects of ocean warming on E. chloroticus. In this study, E. chloroticus is estimated to need between 0.16 and 0.22 kJ kg−1 h−1 for metabolic maintenance at temperatures between 21 and 24 °C. We hypothesize that the available energy, shown by a positive SfG, is being reallocated to ensure short-term survival at the expense of growth and reproduction as previously shown for the sea urchin Strongylocentrotus droebachiensis under high pCO2 conditions (Stumpp et al., 2012). Overall performance of E. chloroticus was dramatically decreased when acclimated at 24 °C, which is the maximum temperature that has been recorded in north-eastern New Zealand during summer (February 1974, Leigh Marine Laboratory dataset). In north-eastern New Zealand, seawater temperature has already increased about 0.6 °C in the past 30 years (Schiel, 2013), and a further increase of 0.7–2.3 °C for the year 2090 has been projected (IPCC, 2014). Although animals in the wild are exposed to multiple stressors, temperature is one of the most important factors limiting distribution of species due to its influence on metabolic reactions (Willmer, 1999; Sanford, 2002). The loss of reproductive output at 24 °C in E. chloroticus shown here, combined with the sensitivity of early embryonic stages to environmental stressors (Clark et al., 2009; Delorme and Sewell, 2013, 2014), suggests that there may be changes to the population persistence and
39
distribution of northern populations within the next few decades unless there is acclimatization of E. chloroticus to the new thermal conditions (Bell and Collins, 2008; Somero, 2010), or the limited genetic variation in E. chloroticus (Nagel et al., 2015) is sufficient to facilitate adaptation (Bell and Collins, 2008). Populations of E. chloroticus may become restricted to higher, more southern, latitudes where seawater temperatures are lower during the crucial periods of gonad growth and embryonic and larval development (Delorme and Sewell, 2013, 2014), with the potential loss of an important habitat-determiner (Jones, 1988) from parts of the subtidal ecosystem of north-eastern New Zealand. Acknowledgements Thanks to Errol Murray, Peter Browne, for helping with the experimental set up, to Brady Doak, Richard Taylor for providing diving equipment, to Alwyn Rees and Benn Hanns for providing fresh kelp, to Mark Wilcox for helping with animal collection, to Josefina Peters for helping with gonad histology, and to Leonardo Zamora for helping with animal and data collection. NJD was supported by the Chilean Government (Comisión Nacional de Investigación Científica y Tecnológica, CONICYT). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cbpa.2016.03.020. References Azad, A.K., Pearce, C.M., McKinley, R.S., 2011 Effects of diet and temperature on ingestion, absorption, assimilation, gonad yield, and gonad quality of the purple sea urchin (Strongylocentrotus purpuratus). Aquaculture 317, 187–196. Barker, M., 2013 Evechinus chloroticus. In: John, M.L. (Ed.)Developments in Aquaculture and Fisheries Science vol. 38. Elsevier, pp. 355–368. Bayne, B.L., Moore, M.N., Widdows, J., Livingstone, D.R., Salkeld, P., Crisp, D.J., Morris, R.J., Gray, J.S., Holden, A.V., Newell, R.C., et al., 1979 Measurement of the responses of individuals to environmental stress and pollution: studies with bivalve molluscs. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 286, 563–581. Bell, G., Collins, S., 2008 Adaptation, extinction and global change. Evol. Appl. 1, 3–16. Brockington, S., Clarke, A., 2001 The relative influence of temperature and food on the metabolism of a marine invertebrate. J. Exp. Mar. Biol. Ecol. 258, 87–99. Carey, N., Harianto, J., Byrne, M., 2016 Urchins in a high CO2 world: partitioned effects of body-size, ocean warming and acidification on metabolic rate. J. Exp. Biol. http://dx. doi.org/10.1242/jeb.136101. Clark, D., Lamare, M., Barker, M., 2009 Response of sea urchin pluteus larvae (Echinodermata: Echinoidea) to reduced seawater pH: a comparison among a tropical, temperate, and a polar species. Mar. Biol. 156, 1125–1137. IPCC, 2014. Climate change 2014: impact, adaptation and vulnerability. Working Group II Contribution to the IPCC 5th Assessment Report. Cambridge University Press, Cambridge. Constable, A.J., 1993 The role of sutures in shrinking of the test in Heliocidaris erythrogramma (Echinoidea: Echinometridae). Mar. Biol. 117, 423–430. Delorme, N.J., Sewell, M.A., 2013 Temperature limits to early development of the New Zealand sea urchin Evechinus chloroticus (Valenciennes, 1846). J. Therm. Biol. 38, 218–224. Delorme, N.J., Sewell, M.A., 2014 Temperature and salinity: two climate change stressors affecting early development of the New Zealand sea urchin Evechinus chloroticus. Mar. Biol. 161, 1999–2009. Dix, T., 1970a Biology of Evechinus chloroticus (Echinoidea: Echinometridae) from different localities: 3. Reproduction. N. Z. J. Mar. Freshw. 4, 385–405. Dix, T., 1970b Biology of Evechinus chloroticus (Echinoidea: Echinometridae) from different localities: 1. General. N. Z. J. Mar. Freshw. 4, 91–116. Dix, T., 1972 Biology of Evechinus chloroticus (Echinoidea: Echinometridae) from different localities: 4. Age, growth, and size. N. Z. J. Mar. Freshw. 6, 48–68. Ebert, T.A., 1967 Negative growth and longevity in the purple sea urchin Strongylocentrotus purpuratus (Stimpson). Science 157, 557–558. Fenwick, G., Horning, D., 1980 Echinodermata of the Snares Islands, southern New Zealand. N. Z. J. Mar. Freshw. 4, 437–445. Fernandez, C., Boudouresque, C.-F., 2000 Nutrition of the sea urchin Paracentrotus lividus (Echinodermata: Echinoidea) fed different artificial food. Mar. Ecol. Prog. Ser. 204, 131–141. Filgueira, R., Rosland, R., Grant, J., 2011 A comparison of scope for growth (SFG) and dynamic energy budget (DEB) models applied to the blue mussel (Mytilus edulis). J. Sea Res. 66, 403–410. Garner, D.M., 1969 The seasonal range of sea temperature on the New Zealand shelf. N. Z. J. Mar. Freshw. 3, 201–208.
40
N.J. Delorme, M.A. Sewell / Comparative Biochemistry and Physiology, Part A 198 (2016) 33–40
González, R.A., Díaz, F., Licea, A., Denisse Re, A., Noemí Sánchez, L., García-Esquivel, Z., 2010 Thermal preference, tolerance and oxygen consumption of adult white shrimp Litopenaeus vannamei (Boone) exposed to different acclimation temperatures. J. Therm. Biol. 35, 218–224. Gooding, R.A., Harley, C.D.G., Tang, E., Brown, J.H., 2009 Elevated water temperature and carbon dioxide concentration increase the growth of a keystone echinoderm. Proc. Natl. Acad. Sci. U. S. A. 106, 9316–9321. Hill, S.K., Lawrence, J.M., 2006 Interactive effects of temperature and nutritional condition on the energy budgets of the sea urchins Arbacia punctulata and Lytechinus variegatus (Echinodermata: Echinoidea). J. Mar. Biol. Assoc. UK 86, 783–790. Hochachka, P.W., Somero, G.N., 2002 Biochemical Adaptation: Mechanism and Process in Physiological Evolution. Oxford University Press, New York. Humason, G.L., 1979 Animal Tissue Techniques. W. H. Freeman, San Francisco. James, P.J., Heath, P.L., 2008 The effects of season, temperature and photoperiod on the gonad development of Evechinus chloroticus. Aquaculture 285, 67–77. James, P.J., Heath, P.L., Green, K., Wright, J., 2009 The effect of elevated temperature on roe enhancement of Evechinus chloroticus collected from two latitudes. Aquaculture 288, 273–279. Jones, G.P., 1988 Ecology of rocky reef fish of north-eastern New Zealand: a review. N. Z. J. Mar. Freshw. 22, 445–462. Kelly, M.W., Sanford, E., Grosberg, R.K., 2012 Limited potential for adaptation to climate change in a broadly distributed marine crustacean. Proc. R. Soc. Lond. B Biol. Sci. 279, 349. Kemp, J.O.G., Britz, P.J., 2008 The effect of temperature on the growth, survival and food consumption of the east coast rock lobster Panulirus homarus rubellus. Aquaculture 280, 227–231. Lamare, M.D., Wing, S.R., 2001 Calorific content of New Zealand marine macrophytes. N. Z. J. Mar. Freshw. 35, 335–341. Lannig, G., Flores, J.F., Sokolova, I.M., 2006 Temperature-dependent stress response in oysters, Crassostrea virginica: pollution reduces temperature tolerance in oysters. Aquat. Toxicol. 79, 278–287. Lawrence, J.M., Cao, X., Chang, Y., Wang, P., Yu, Y., Lawrence, A.L., Watts, S.A., 2009 Temperature effect on feed consumption, absorption, and assimilation efficiencies and production of the sea urchin Strongylocentrotus intermedius. J. Shellfish Res. 28, 389–395. Levitan, D., 1988 Density-dependent size regulation and negative growth in the sea urchin Diadema antillarum Philippi. Oecology 76, 627–629. McElroy, D.J., Nguyen, H.D., Byrne, M., 2012 Respiratory response of the intertidal seastar Parvulastra exigua to contemporary and near-future pulses of warming and hypercapnia. J. Exp. Mar. Biol. Ecol. 416–417, 1–7. Nagel, M.M., Sewell, M.A., Lavery, S.D., 2015 Differences in population connectivity of a benthic marine invertebrate Evechinus chloroticus (Echinodermata: Echinoidea) across large and small spatial scales. Conserv. Genet. 16, 965–978. Navarro, J.M., Winter, J.E., 1982 Ingestion rate, assimilation efficiency and energy balance in Mytilus chilensis in relation to body size and different algal concentrations. Mar. Biol. 67, 255–266. Navarro, J.M., Urrutia, G.X., Carrasco, C., 2006 Scope for growth versus actual growth in the juvenile predatory gastropod Chorus giganteus. J. Mar. Biol. Assoc. UK 86, 1423–1428. Naylor, C., Maltby, L., Calow, P., 1989 Scope for growth in Gammarus pulex, a freshwater benthic detritivore. Hydrobiologia 188-189, 517–523. Otero-Villanueva, M.d.M., Kelly, M.S., Burnell, G., 2004 How diet influences energy partitioning in the regular echinoid Psammechinus miliaris; constructing an energy budget. J. Exp. Mar. Biol. Ecol. 304, 159–181. Pedersen, S.A., Hakedal, O.J., Salaberria, I., Tagliati, A., Gustavson, L.M., Jenssen, B.M., Olsen, A.J., Altin, D., 2014 Multigenerational exposure to ocean acidification during food limitation reveals consequences for copepod scope for growth and vital rates. Environ. Sci. Technol. 48, 12275–12284. Pernet, F., Tremblay, R., Comeau, L., Guderley, H., 2007 Temperature adaptation in two bivalve species from different thermal habitats: energetics and remodelling of membrane lipids. J. Exp. Biol. 210, 2999–3014. Pörtner, H.O., 2002 Climate variations and the physiological basis of temperature dependent biogeography: systemic to molecular hierarchy of thermal tolerance in animals. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 132, 739–761. Pörtner, H.O., 2010 Oxygen- and capacity-limitation of thermal tolerance: a matrix for integrating climate-related stressor effects in marine ecosystems. J. Exp. Biol. 213, 881–893.
Pörtner, H.O., 2012 Integrating climate-related stressor effects on marine organisms: unifying principles linking molecule to ecosystem-level changes. Mar. Ecol. Prog. Ser. 470, 273–290. Pörtner, H.O., Farrell, A.P., 2008 Physiology and climate change. Science 322, 690–692. Pörtner, H.O., Peck, L., Somero, G., 2007 Thermal limits and adaptation in marine antarctic ectotherms: an integrative view. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 362, 2233–2258. Quinn, G.P., Keough, M.J., 2002 Experimental Design and Data Analysis for Biologists. Cambridge University Press, Cambridge. Sanford, E., 2002 Water temperature, predation, and the neglected role of physiological rate effects in rocky intertidal communities. Integr. Comp. Biol. 42, 881–891. Schiel, D.R., 2013 The other 93%: trophic cascades, stressors and managing coastlines in non-marine protected areas. N. Z. J. Mar. Freshw. 47, 374–391. Schiel, D.R., Kingsford, M.J., Choat, J.H., 1986 Depth distribution and abundance of benthic organisms and fishes at the subtropical Kermadec Islands. N. Z. J. Mar. Freshw. 20, 521–535. Shirley, T.C., Stickle, W.B., 1982 Responses of Leptasterias hexactis (Echinodermata: Asteroidea) to low salinity. Mar. Biol. 69, 155–163. Siikavuopio, S.I., James, P., Lysne, H., Sæther, B.S., Samuelsen, T.A., Mortensen, A., 2012 Effects of size and temperature on growth and feed conversion of juvenile green sea urchin (Strongylocentrotus droebachiensis). Aquaculture 354–355, 27–30. Siikavuopio, S.I., Mortensen, A., Christiansen, J.S., 2008 Effects of body weight and temperature on feed intake, gonad growth and oxygen consumption in green sea urchin, Strongylocentrotus droebachiensis. Aquaculture 281, 77–82. Sokolova, I.M., 2013 Energy-limited tolerance to stress as a conceptual framework to integrate the effects of multiple stressors. Integr. Comp. Biol. 53, 597–608. Sokolova, I.M., Frederich, M., Bagwe, R., Lannig, G., Sukhotin, A.A., 2012 Energy homeostasis as an integrative tool for assessing limits of environmental stress tolerance in aquatic invertebrates. Mar. Environ. Res. 79, 1–15. Somero, G.N., 2005 Linking biogeography to physiology: evolutionary and acclimatory adjustments of thermal limits. Front. Zool. 2, 1–9. Somero, G.N., 2010 The physiology of climate change: how potentials for acclimatization and genetic adaptation will determine "winners" and "losers". J. Exp. Biol. 213, 912–920. Somero, G.N., 2012 The physiology of global change: linking patterns to mechanisms. Ann. Rev. Mar. Sci. 4, 39–61. Stumpp, M., Trübenbach, K., Brennecke, D., Hu, M.Y., Melzner, F., 2012 Resource allocation and extracellular acid–base status in the sea urchin Strongylocentrotus droebachiensis in response to CO2 induced seawater acidification. Aquat. Toxicol. 110–111, 194–207. Uthicke, S., Liddy, M., Nguyen, H.D., Byrne, M., 2014 Interactive effects of near-future temperature increase and ocean acidification on physiology and gonad development in adult Pacific sea urchin, Echinometra sp. A. Coral Reefs 33, 831–845. Walker, M.M., 1981 Influence of season on growth of the sea urchin Evechinus chloroticus. N. Z. J. Mar. Freshw. 15, 201–205. Walker, M.M., 1982 Reproductive periodicity in Evechinus chloroticus in the Hauraki Gulf. N. Z. J. Mar. Freshw. 16, 19–25. Warren, C.E., Davis, G., 1967 Laboratory studies on the feeding, bioenergetics, and growth of fish. In: Gerking, S.D. (Ed.), The Biological Basis of Freshwater Fish Production. Blackwell Scientific Publications, Oxford, pp. 175–214. Watts, S.A., Hofer, S.C., Desmond, R.A., Lawrence, A.L., Lawrence, J.M., 2011 The effect of temperature on feeding and growth characteristics of the sea urchin Lytechinus variegatus fed a formulated feed. J. Exp. Mar. Biol. Ecol. 397, 188–195. Widdows, J., Johnson, D., 1988 Physiological energetics of Mytilus edulis: scope for growth. Mar. Ecol. Prog. Ser. 46, 113–121. Willmer, P., 1999 Environmental Physiology of Animals. Blackwell Publisher, Massachusetts, USA. Winberg, G.G., 1960 Rate of Metabolism and Food Requirements of Fishes. Fisheries Research Board of Canada, Nanaimo, B.C. Zamora, L.N., Jeffs, A.G., 2012 Feeding, metabolism and growth in response to temperature in juveniles of the Australasian sea cucumber, Australostichopus mollis. Aquaculture 358-359, 92–97. Zamora, L.N., Jeffs, A.G., 2015 Macronutrient selection, absorption and energy budget of juveniles of the Australasian sea cucumber, Australostichopus mollis, feeding on mussel biodeposits at different temperatures. Aquac. Nutr. 21, 162–172.