Journal of Fish Biology (2015) 87, 763–773 doi:10.1111/jfb.12736, available online at wileyonlinelibrary.com
Upper thermal tolerance of wild-type, domesticated and growth hormone-transgenic coho salmon Oncorhynchus kisutch Z. Chen*†, R. H. Devlin‡ and A. P. Farrell*§ *Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, BC, V6T 1Z4, Canada, ‡Center for Aquaculture and Environmental Research, Fisheries and Oceans Canada, 4160 Marine Drive, West Vancouver, BC, V7V 1N6, Canada and §Faculty of Land and Food Systems, University of British Columbia, 2357 Main Mall, Vancouver, BC, V6T 1Z4, Canada (Received 10 October 2014, Accepted 28 May 2015) In coho salmon Oncorhynchus kisutch, no significant differences in critical thermal maximum (c. 26⋅9∘ C, CTmax ) were observed among size-matched wild-type, domesticated, growth hormone (GH)-transgenic fish fed to satiation, and GH-transgenic fish on a ration-restricted diet. Instead, GH-transgenic fish fed to satiation had significantly higher maximum heart rate and Arrhenius breakpoint temperature (mean ± s.e. = 17⋅3 ± 0⋅1∘ C, T AB ). These results provide insight into effects of modified growth rate on temperature tolerance in salmonids, and can be used to assess the potential ecological consequences of GH-transgenic fishes should they enter natural environments with temperatures near their thermal tolerance limits. © 2015 The Fisheries Society of the British Isles
Key words: Arrhenius breakpoint temperature; CTmax ; ECG; heart rate; optimum temperature; restricted ration.
In salmonids, high growth rate has been achieved by domestication or by growth hormone (GH) transgenesis (Du et al., 1992; Devlin et al., 1994, 2001; Fleming et al., 2002; Tymchuk & Devlin, 2005; Tymchuk et al., 2006). In coho salmon Oncorhynchus kisutch (Walbaum 1792), growth-hormone (GH) transgenesis causes many-fold increases in body mass compared with non-transgenic fish of the same age (Devlin et al., 2004). Given the ecological concerns that genetically modified animals may interact with local wild populations by competing for food resources and interbreeding (Tiedje et al., 1989; Devlin et al., 2006; Kapuscinski et al., 2007), empirical data have been collected to study the performance of growth-enhanced animals to help evaluate the potential ecological consequences. These studies include assessments of swimming capacity (Farrell et al., 1997; Lee et al., 2003), anti-predator responses (Sundström †Author to whom correspondence should be addressed. Tel.: +1 778 223 8483; email:
[email protected]
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et al., 2004; Tymchuk et al., 2006), disease resistance (Jhingan et al., 2003) and breeding performance (Bessey et al., 2004; Fitzpatrick et al., 2011; Moreau et al., 2011; Leggatt et al., 2014). Although it is well known that most physiological processes are affected by temperature (Richter & Kolmes, 2005), the thermal performance and limits of animals that have been artificially growth enhanced are still largely unknown. Based on the model of oxygen and capacity-limited thermal tolerance (OCLTT) (Pörtner & Farrell, 2008), a hypothesis is made here that temperature tolerance may be affected in domesticated and GH-transgenic fish. OCLTT is built around thermal effects on maximum capacity of an organism to metabolize aerobically beyond basic maintenance, which is termed aerobic scope (Fry, 1947). The thermal optimum (T opt ) defines the temperature where absolute aerobic scope reaches a peak, while critical temperature (T crit ) defines the thermal extreme where aerobic scope is zero. Because aerobic scope is the difference between routine metabolic rate (RMR) and maximum metabolic rate (MMR), changes in RMR and MMR as a result of growth modification could cause a shift in T opt and T crit . GH-transgenic Atlantic salmon Salmo salar L. 1758 showed a 29% lower aerobic scope than the wild-type control due to the combined effect of elevated RMR and decreased MMR (Deitch et al., 2006). Also, GH-transgenic S. salar and O. kisutch showed elevated metabolic demands during digestion, due to an elevated feed intake, and during exercise (Cook et al., 2000; Lee et al., 2003; Leggatt et al., 2003). Indeed, temperature tolerance has been studied in rainbow trout Oncorhynchus mykiss (Walbaum 1792) showing that the wild-type animals had the ability to tolerant significantly higher temperature than a domesticated strain (Carline & Machung, 2001). Thus, it is expected that the growth-enhanced fish studied here would have an altered thermal performance. To date, there is no existing study of temperature tolerance of GH-transgenic fishes. This study compared the upper thermal performance and tolerance in wild-type, domesticated (selected for fast growth) and GH-transgenic O. kisutch. GH-transgenic fish were divided into two groups. One group was fed to satiation (TF), which had a fast growth rate. The other group was fed with a restricted ration that was equal to the amount consumed by wild-type group (TR), which forced the GH-transgenic fish to have a similar growth rate as the wild-type fish. These four groups of fish were used to compare the effects of strain and ration level, as well as the direct and indirect effects of growth rate. In addition to use the upper critical thermal maximum (CTmax ) as a measure of thermal tolerance, the rate transition temperature for maximum heart rate (f Hmax ) was used to measure upper thermal performance due to the central role of heart in oxygen delivery (Farrell, 2009). Specifically, the Arrhenius breakpoint temperature (T AB ), derived from pharmacologically stimulated heart rate measurements, can approximate T opt for aerobic scope (Casselman et al., 2012; Chen et al., 2013; Ferreira et al., 2014; Muñoz et al., 2014). In addition, the temperature when heart rate stops increasing with temperature and becomes arrhythmic (T ar ) is a good index of cardiac thermal tolerance limits (T crit ) (Ferreira et al., 2014), although it is lower than CTmax (Chen et al., 2013; Muñoz et al., 2014). Thus, the specific objectives were to determine whether (1) CTmax is modified in growth-enhanced fish, especially TF fish and (2) physiological T opt was affected. Strains of O. kisutch were grown at the Fisheries and Oceans Canada, Centre for Aquaculture and Environmental Research (CAER) in West Vancouver, British Columbia (BC), Canada. This bio-contained facility is designed to prevent the escape of fish to the natural environment. Fish were reared under an Animal Use Protocol
© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, 87, 763–773
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(AUP Number: 12-004) approved by Pacific Region Animal Care Committee in accordance with the Canadian Council on Animal Care Guidelines. The wild-type fish were of the Chehalis River stock (from south-western BC) and experimental animals were generated from crosses made in autumn 2011 and subsequently reared in fresh, aerated well water (10⋅0∘ C, range ± 1⋅5∘ C) at CAER. Growth-enhanced domesticated O. kisutch were from a commercial strain that had been selected for enhanced growth for over seven generations and originally derived from the Kitimat River (BC). All fish were fed twice daily. TF, wild-type and domesticated O. kisutch were fed to satiation, while TR O. kisutch were fed the same amount of food as consumed by wild-type fish (to limit their growth rate to that of wild type). In July 2012, when the four groups of fish had a similar body mass, fish were randomly selected from stock tanks for this study. Before each experiment, fish were subjected to a 48 h fasting to ensure a post-prandial state. For CTmax measurements (Fangue et al., 2006), fish were kept in a 40 l container for a 1 h temperature adjustment at 10∘ C. Water temperature was then increased at a rate of 0⋅3∘ C min−1 using water from a heating unit (3016D, Fisher Scientific; www.fishersci.ca) until 22∘ C, and at a rate of 0⋅1∘ C min−1 thereafter. CTmax was recorded as the temperature where fish first could not remain upright for 5 s. To minimize diurnal effects, CTmax was measured in one group daily between 1000 and 1400 hours. Measuring f Hmax in an anaesthetized fish was originally developed by Casselman et al. (2012) as a means to establish a relationship between maximum cardiac function and whole-animal aerobic scope. Following the same protocol, a fish was anaesthetized (85 mg l−1 MS-222 buffered with 170 mg l−1 NaHCO3 ; Sigma-Aldrich; www.sigmaaldrich.com) and placed in the electrocardiogram (ECG) measuring system, where the gills were continuously irrigated with aerated and temperature-controlled fresh water containing a maintenance anaesthetic dose (65 mg l−1 of MS-222 and NaHCO3 ; Sigma-Aldrich). Two silver electrodes were placed on the skin surface (non-penetrating) of the ventral side of the body to capture ECG signal. The electrodes were connected to Animal Bio Amps (AD Instruments Inc.; www.adinstruments.com) that amplified (×1000) and filtered (60 Hz line filter; low-pass: 30–50 Hz; high-pass: 0⋅1–0⋅3 kHz) the ECG signal. The conditioned ECG signal was digitalized using a Powerlab 8/35 data acquisition system and analysed using Labchart 7.0 software (AD Instruments Inc.). Anaesthetized fish were stabilized at the initial temperature (10∘ C) for 1 h prior to intraperitoneal injection of atropine sulphate (1⋅8 mg kg−1 ; Sigma-Aldrich) to block inhibitory vagal tone to the heart and isoproterenol (6 μg kg−1 ; Sigma-Aldrich) to stimulate maximally cardiac 𝛽-adrenoreceptors, which ensured that the heart was beating at a stable f Hmax for that water temperature after a period of 15 min. Incremental heating was started, using 1∘ C increments every 6 min until warming initiated a cardiac arrhythmia. After all experiments, the fish were deeply anaesthetized (100 mg l−1 MS-222 buffered with 200 mg l−1 NaHCO3 ; Sigma-Aldrich) and ventricles were harvested by dissection. A one-way ANOVA with Šídák post hoc analysis was used to analyse differences in condition factor, ventricle mass, CTmax , f Hmax and the rate transition temperatures for f Hmax , (SigmaPlot 11.0, Systat Software; www.sigmaplot.com). In this study, the four groups were size matched at the time of experiment (Table I). Transgenic TF and TR fish had higher conditional factor (K) than the non-transgenic fish (P < 0⋅05). Domesticated fish had the highest mean ± s.e. relative
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Table I. Mean ± s.e. body size and ventricle mass of wild-type, domestic, growth hormone (GH)-transgenic fast growing (TF) and GH-transgenic ration-controlled (TR) Oncorhynchus kisutch. ANOVA with Šídák post hoc analysis showed significant (P < 0⋅05) differences between groups (different lower-case letters)
n Mass (g) LF (cm) K Ventricle (mg) RVM (%)
TF
TR
Wild type
Domesticated
12 36⋅6 ± 3⋅1 13⋅6 ± 0⋅4b 1⋅39 ± 0⋅03a 32⋅4 ± 3⋅0b 0⋅089 ± 0⋅002c
14 36⋅6 ± 2⋅0 14⋅6 ± 0⋅3b 1⋅18 ± 0⋅03b 34⋅8 ± 2⋅5b 0⋅095 ± 0⋅003bc
14 34⋅0 ± 1⋅4 14⋅8 ± 0⋅1b 1⋅05 ± 0⋅02c 33⋅7 ± 1⋅4b 0⋅099 ± 0⋅003b
14 39⋅8 ± 2⋅8 15⋅8 ± 0⋅3a 1⋅00 ± 0⋅04c 44⋅7 ± 3⋅2a 0⋅113 ± 0⋅004a
n, sample size; LF , fork length; K, condition factor; RVM, relative wet ventricle mass to body mass.
ventricle mass (RVM) (0⋅113 ± 0⋅004%, P < 0⋅05), while the TF had the lowest RVM (0⋅089 ± 0⋅002%, P < 0⋅05). CTmax values were centred around 26⋅9∘ C and did not differ significantly among the test groups. Indeed, the numerical spread of CTmax was
