Critical thermal maxima of early life stages of three tropical fishes: Effects of rearing temperature and experimental heating rate
Introduction
Temperature influences all physiological and cellular processes, governs the pace of metabolic rates in ectothermic organisms, and results in functional constraints at the extremes (Fry, 1971; Pörtner and Farrell, 2008). To compare thermal performance and upper thermal limits for activity between individuals, life stages, populations, or species, different static or dynamic experimental methodologies have been used (Lutterschmidt and Hutchinson, 1997). A popular, dynamic method, referred to as the critical thermal methodology, was developed in the 1940s for reptiles and has since been used with various modifications for other ectothermic vertebrates, invertebrates, and even mammals (Cowles and Bogert, 1944; Hutchinson, 1961; Lutterschmidt and Hutchinson, 1997). In contrast to static methods, where the test subject is exposed to stressful but constant temperatures, the critical thermal methodology includes ramping assays to determine non-lethal endpoints, such as balance disturbances, cessation of locomotory activity, or loss of neuromuscular control (Hoffmann et al., 2013; Lutterschmidt and Hutchinson, 1997). Following Hutchinson's niche concept, these endpoints, where organisms become incapacitated and cannot escape stressful conditions anymore, represent the hard boundaries for a species' survivorship or the so called fundamental niche where a species can exist (Fry, 1971; Gouveia et al., 2014; Hutchinson, 1978).
The potential to draw broader ecological conclusions from critical thermal measurements continues to be attractive to researchers and is, therefore, still commonly used to assess the thermal performance of ectothermic organisms. In recent years, critical thermal maxima (CTmax) have been used, for example, to model the potential for adaptive capacity in ectotherms (Catullo et al., 2015; Geerts et al., 2015; Vinagre et al., 2016), to determine macroecological niche positions (Gouveia et al., 2014), and, in light of anthropogenic climate warming, to estimate global redistributions of ectothermic species across latitudes and construct thermal vulnerability rankings across taxa (Deutsch et al., 2008; Sunday et al., 2012; Vinagre et al., 2019). With climate warming, habitat boundaries are predicted to shift with expected range expansions toward more poleward-oriented areas and range contractions at tropical latitudes (Sunday et al., 2012). Conclusions drawn from these macro-physiological studies are that marine organisms are more sensitive to warming than terrestrial ones (Pinsky et al., 2019), and that, due to limited seasonal variability in temperatures, warming has the most deleterious effects on tropical ectotherms (Huey et al., 2012; Vinagre et al., 2016), including invertebrates (Deutsch et al., 2008; Nguyen et al., 2011), reptiles (Sinervo et al., 2010), amphibians (Duarte et al., 2012), and fishes (Pörtner and Peck, 2010).
The variability in CTmax protocols emerges mainly from using different heating rates, acclimation temperatures and durations, as well as endpoint definitions. In fact, the issue of widely divergent methodologies has already been raised, and the need for standardization has been urged several times throughout recent decades (Chown et al., 2009; Hutchinson, 1961; Lutterschmidt and Hutchinson, 1997). The ramping rates in particular have been in the focus of the debate, as very fast rates can overestimate the maximum thermal tolerance of an organism by creating a lag between the animal's body temperature and that of the environment; whereas, very slow ramping rates may lead to acclimation effects and substantially underestimate thermal tolerance of the organism due to prolonged exposure to thermal stress (Hoffmann et al., 2013; Kingsolver and Umbanhowar, 2018; Terblanche et al., 2011). In fishes, body and water temperatures have been observed to start differing at rates of approximately 18 °C h−1 (Beitinger et al., 2000). However, these rates almost never occur in the natural marine environment, and using lower and more ecologically meaningful rates avoid this issue completely. Surprisingly, previous experimental protocols have sometimes used much higher heating rates (up to 60 °C h−1; literature review of all CTmax studies in sub- and tropical fishes in the supplementary material). Acute thermal stress generally results in increased metabolic demands, and the elevated tissue oxygen demand is met by increases in cardiac performance; hence, the heart is also suspected to be one of the first organs to fail under acute thermal stress (Christen et al., 2018; Ekström et al., 2017). Heart failure and declining cardiac performance at temperatures close to CTmax have been associated with impaired cardiac mitochondrial capacity and respiration efficiency in temperate fishes (Iftikar and Hickey, 2013).
Activity measurements of marker enzymes, such as the aerobic enzyme citrate synthase (CS), and the anaerobic enzyme lactate dehydrogenase (LDH), in cardiac tissue under acute thermal stress have been helpful in providing mechanistic explanations for observed differences in thermal tolerance of fishes (Ekström et al., 2017; O'Brien et al., 2018). More specifically, both enzymes have been used as proxies for aerobic and anaerobic metabolic capacity, respectively (Brijs et al., 2017). CS is a pace-making enzyme in the first step of the citric acid cycle inside the mitochondria where it catalyzes the condensation of acetyl-CoA and oxaloacetate to form citrate. LDH catalyzes the interconversion of pyruvate and lactate concomitantly with the interconversion of NADH and NAD+, which supports the subsequent production of adenosine triphosphate (ATP) in the absence of oxygen. Furthermore, CS is a good marker for mitochondria volume density and can be used to estimate changes in mitochondrial content and help evaluate potential thermal acclimation in larval fishes (Schnurr et al., 2014). Similarly, exposure to warmer temperatures during early development has been previously found to increase LDH activity in spotted wolf fish larvae (Anarhichas minor) and can be an estimate as to the quantitative contribution of anaerobic metabolism to growth and activity (Savoie et al., 2008). In a 2018 study, icefishes that were exposed to acute warming during CTmax trials had higher maximal CS and LDH activities in their cardiac tissue compared to fishes exposed to ambient conditions (O'Brien et al., 2018). According to the authors, this potentially reflects the fishes' ability to increase capacity at the citric acid cycle during exposure to warming and increased usage of lactate as aerobic fuel (O'Brien et al., 2018). Next to metabolic limitations, neuronal failure plays a critical role in reaching CTmax. For example, acute heat stress has been found to affect components of the central nervous system (e.g., the brain) and cause the loss of motor function in juvenile Atlantic cod (Gadus morhua) (Jutfelt et al., 2019).
In fishes, life stage-specific sensitivities to changes in environmental temperatures have been reported for a few – mostly temperate – species (Pörtner et al., 2005; Pörtner and Peck, 2010; Rijnsdorp et al., 2009). Factors contributing to the ontogenetic differences in thermal tolerance of fishes are often attributed to constraints during phases where energy is prioritized for certain tasks, such as fast growth during early ontogeny or during reproduction. Next to allocating most energy to rapid growth, fish larvae are often lacking fully-developed organs and experience changes in body surface area to volume ratios that result in “progressively falling oxygen supply capacity in relation to demand”, which may reduce their upper thermal limits (Pörtner and Peck, 2010). However, acclimation to warmer temperatures can occur during development, for example, via a reduction in mitochondrial density and therefore reduced oxygen demand, which can help an organism to slow down its metabolic rate and ultimately increase its thermal tolerance (Pörtner, 2001). Thermal acclimation strategies have been shown to follow either reversible (e.g., seasonal), developmental (e.g., during early ontogeny), or transgenerational patterns (Angiletta, 2009; Donelson et al., 2017; Munday et al., 2013; Rummer and Munday, 2017). Temperature tolerance in fishes has been observed to be affected by thermal history, with higher temperatures experienced early in development often resulting in increased CTmax (Moyano et al., 2017). This previous research indicates potential life stage-specific sensitivities of fishes to thermal stress, but information on CTmax of fish larvae has so far mostly been compiled for temperate and polar fish species with few information available for early life stages of tropical species (Flynn and Todgham, 2018; Moyano et al., 2017; Spinks et al., 2019). Therefore, more information is required to assess whether developmental acclimation to warmer temperature will elevate CTmax of larval and early juvenile stages of tropical fishes. This knowledge gap and differences in the applied experimental protocols hamper comparative approaches and assessments of potential sensitivity to warming (Rezende et al., 2014).
The effect of different heating rates on CTmax has only rarely been tested in subtropical and tropical fishes (Mora and Maya, 2006). Currently, there exists no standard procedure, particularly regarding the rate of temperature change, for testing CTmax in early life stages of fishes (Moyano et al., 2017). Ideally, heating rates used for evaluating CTmax in tropical species allow comparisons with findings from temperate and polar species and mimic changes in water temperature in the natural habitat. Although not showing much seasonal variation, water temperatures on tropical coral reefs can show considerable diurnal differences in temperature extremes when compared to the surrounding open ocean (1.5–8 °C) (Jimenez et al., 2012; Lowe et al., 2016; McCabe et al., 2010). Extreme diurnal temperature amplitudes of up to 12 °C and maximum temperatures exceeding 36 °C have been reported for reef flats with strong tidal influence, such as Lady Elliot Island (Great Barrier Reef, eastern Australia) (McCabe et al., 2010) and Tallon Island (Kimberly region, northwestern Australia), where the highest amplitudes have been observed over a 10 h ebb tide (Gruber et al., 2017). In tropical coastal waters, daily temperature ranges of up to 6 °C have been measured, with maximum temperatures reaching 35–36 °C (Cape Flattery region, northeastern Australia) (AIMS, 2017; Pusey et al., 2004, 2017). Therefore, tropical coral reef fishes that develop in the open ocean before recruiting to a coral reef (e.g., cinnamon anemonefish, Amphiprion melanopus) may experience less variability in water temperature during early development, compared to coral reef fishes without a pelagic phase (e.g., spiny chromis, Acanthochromis polyacanthus). In riverine habitats even more extreme fluctuations in water temperature have been measured (ranges spanning 10 °C, changing at rates as fast as 2 °C h−1; see references in Norin et al., 2014). Hence, the offspring of catadromic tropical fish species (e.g., barramundi, Lates calcarifer) that develop in the ocean will experience higher variability in water temperature as juveniles and after migrating into rivers.
In this study, we investigated how upper thermal limits of larval and early juvenile stages of three tropical marine fishes, barramundi, cinnamon anemonefish, and spiny chromis, are affected by developmental stage, acclimation to warmer temperatures, and applied experimental heating rates. We hypothesized 1) that exposure to a warmer rearing temperature would result in increased CTmax. We chose CS activity as a proxy for mitochondrial volume densities where potential reductions would indicate developmental acclimation to warming. Therefore, we hypothesized that 2) CS activity would decrease in fishes exposed to a warmer rearing temperature throughout development; whereas, we expected LDH activity, a marker enzyme for anaerobic metabolism, to increase as a result of a quantitative adjustment (higher concentration). Furthermore, we hypothesized that 3) CTmax would increase over early ontogeny; that is, we would observe a positive correlation with body mass. We also aimed to test if 4) faster heating rates would result in higher CTmax. Finally, we hypothesized 5) that fish exposed to acute heat stress would, in contrast to fish maintained at control temperatures, increase the maximum activity of aerobic and anaerobic metabolic marker enzymes. For testing these five hypotheses, we exposed the offspring of three tropical fish species directly after hatch to either current average summer (28.5 °C) or projected mid-century summer temperatures (30.0 °C, +1.5 °C; Hobday and Lough, 2011) and measured CTmax throughout their larval and early juvenile stages. We used three different ecologically-relevant heating rates and tested the activities of CS and LDH throughout ontogeny and after exposure to acute heat stress. We discuss how phenotypic plasticity or species-specific differences in traits related to thermal tolerance can depend on the tested life stage and the chosen heating rate.
Section snippets
Animal husbandry
Three tropical fish species, namely the coral reef-associated spiny chromis damselfish (Acanthochromis polyacanthus, Bleeker, 1855) and cinnamon anemonefish (Amphiprion melanopus, Bleeker, 1852) and the catadromous barramundi (Lates calcarifer, Bloch 1790), were tested for their upper thermal limits throughout their larval and early juvenile phase. All animal husbandry and experimentation took place at the Marine and Aquaculture Research Facilities Unit at James Cook University, Queensland,
CTmax varied among species, over ontogeny, and with different heating rates
CTmax differed significantly between all three tropical fish species (p < 0.001, conditional R2 = 0.82, GLMM; see S2 for detailed model results). After accounting for rearing temperature, body mass, and heating rate, L. calcarifer was able to tolerate the highest temperatures, with a mean CTmax (±s.e.m.) of 40.99 (±0.16)°C. This was 1.87 (±0.43) and 4.04 (±0.17)°C higher than the CTmax of A. melanopus (39.11 ± 0.41 °C) and A. polyacanthus (36.95 ± 0.05 °C), respectively (Fig. 1). The random
Discussion
Our findings suggest that critical thermal maxima (CTmax) of larval and early juvenile stages of the three tropical fishes spiny chromis (A. polyacanthus), cinnamon anemonefish (A. melanopus), and barramundi (L. calcarifer) are i) species- and life stage-specific, ii) not affected by a circa 1.5 °C warmer constant rearing temperature, and iii) significantly elevated by faster heating rates. We did not find warmer rearing temperatures or acute thermal challenges to affect maximum activity of
Conclusions
This study highlights that larval and early juvenile stages of the tropical fishes, spiny chromis (A. polyacanthus), cinnamon anemonefish (A. melanopus), and barramundi (L. calcarifer), differ in their upper thermal tolerance (i.e., CTmax) both between species and between early life stages. CTmax was significantly influenced by body mass and different heating rates but not by warmer rearing temperatures (+1.5 °C). Neither the warmer rearing temperatures nor CTmax tests affected maximum
Funding sources
BI was supported by the Company of Biologists [grant number JEBTF-160307], the German Academic Exchange Service [DAAD, grant number 91585243] and the German Research Foundation [DFG, grant number IL-220/2-1]. JLR was supported by the Australian Research Council [ARC, grant numbers FS110200046, DE150101266] and BI, ATD and JLR received support from the ARC Centre of Excellence for Coral Reef Studies. The funders had no role in study design, data collection and analysis, decision to publish, or
Declaration of interest
The authors declare no competing financial or non-financial interests.
CRediT authorship contribution statement
B. Illing: Conceptualization, Funding acquisition, Methodology, Formal analysis, Investigation, Visualization, Data curation, Writing - original draft. A.T. Downie: Investigation, Writing - review & editing. M. Beghin: Investigation, Writing - review & editing. J.L. Rummer: Supervision, Funding acquisition, Writing - review & editing.
Acknowledgements
The authors would like to thank Jarrod Guppy and Adrien Marc (both Centre for Sustainable Tropical Fisheries & Aquaculture, James Cook University) for providing barramundi larvae through Mainstream Aquaculture Pty., Ltd. Marta Moyano (University of Hamburg) is thanked for inspirational discussions on the CTmax methodology. Similarly, the authors appreciate Cristian Rojas's (CoralCoE, James Cook University) advice on statistical analyses, as well as the excellent and constructive comments from
References (94)
- et al.
Thermal tolerance and thermal sensitivity of heart mitochondria: mitochondrial integrity and ROS production
Free Radic. Biol. Med.
(2018) The effect of environmental factors on the physiology of fish
- et al.
Temperature-dependent physiological and biochemical responses of the marine medaka Oryzias melastigma with consideration of both low and high thermal extremes
J. Therm. Biol.
(2015) - et al.
Thermal stress, thermal safety margins and acclimation capacity in tropical shallow waters - an experimental approach testing multiple end-points in two common fish
Ecol. Indicat.
(2017) - et al.
Ocean warming alters cellular metabolism and induces mortality in fish early life stages: a proteomic approach
Environ. Res.
(2016) - et al.
Effect of the rate of temperature increase of the dynamic method on the heat tolerance of fishes
J. Therm. Biol.
(2006) - et al.
Patterns of catabolic capacities and energy metabolism in developing embryos and yolk-sac larvae of turbot (Scophthalmus maximus L.)
Aquaculture
(2017) - et al.
Does oxygen limit thermal tolerance in arthropods? A critical review of current evidence
Comp. Biochem. Physiol. A. Mol. Integr. Physiol.
(2016) - et al.
Upper thermal limits and warming safety margins of coastal marine species – indicator baseline for future reference
Ecol. Indicat.
(2019) - et al.
Effect of warming rate on the critical thermal maxima of crabs, shrimp and fish
J. Therm. Biol.
(2015)
Vulnerability to climate warming and acclimation capacity of tropical and temperate coastal organisms
Ecol. Indicat.
Australian institute of marine science. Data downloaded 7th November 2017, using temperature loggers from Cape Flattery. Long term monitoring and data Centre
Thermal Adaptation: A Theoretical and Empirical Synthesis
MuMin: Multi-Model Inference. R Package Version 1. 0. 0
Fitting linear mixed-effects models using lme4
J. Stat. Software
Temperature tolerances of North American freshwater fishes exposed to dynamic changes in temperature
Environ. Biol. Fish.
Increased mitochondrial coupling and anaerobic capacity minimizes aerobic costs of trout in the sea
Sci. Rep.
Developmental phenotypic plasticity helps bridge stochastic weather events associated with climate change
J. Exp. Biol.
Response of muscle-based biochemical condition indices to short-term variations in food availability in post-flexion reared sea bass Dicentrarchus labrax (L.) larvae
J. Fish. Biol.
Extending spatial modelling of climate change responses beyond the realized niche: estimating, and accommodating, physiological limits and adaptive evolution: incorporating adaptive capacity into climate change models
Global Ecol. Biogeogr.
Phenotypic variance, plasticity and heritability estimates of critical thermal limits depend on methodological context
Funct. Ecol.
Maximum thermal limits of coral reef damselfishes are size dependent and resilient to near-future ocean acidification
J. Exp. Biol.
Effects of nutrition and temperature on metabolic enzyme activities in larval and juvenile red drum, Sciaenops ocellatus, and lane snapper, Lutjanus synagris
Mar. Biol.
The sea urchin Lytechinus variegatus lives close to the upper thermal limit for early development in a tropical lagoon
Ecol. Evol.
A preliminary study of the thermal requirements of desert reptiles
Bull. Am. Mus. Nat. Hist.
Impacts of climate warming on terrestrial ectotherms across latitude
Proc. Natl. Acad. Sci. Unit. States Am.
Reproductive acclimation to increased water temperature in a tropical reef fish
PloS One
Acclimation to predicted ocean warming through developmental plasticity in a tropical reef fish: thermal acclimation in reef fish
Global Change Biol.
Transgenerational plasticity and climate change experiments: where do we go from here?
Global Change Biol.
Can amphibians take the heat? Vulnerability to climate warming in subtropical and temperate larval amphibian communities
Global Change Biol.
Thermal sensitivity and phenotypic plasticity of cardiac mitochondrial metabolism in European perch, Perca fluviatilis
J. Exp. Biol.
Molar absorption coefficients for the reduced Ellman reagent: reassessmentq
Anal. Biochem.
Lack of coherence in the warming responses of marine crustaceans
Funct. Ecol.
Condition of laboratory-reared and wild-caught larval Atlantic menhaden Brevoortia tyrannus as indicated by metabolic enzyme activities
Mar. Ecol. Prog. Ser.
Thermal windows and metabolic performance curves in a developing Antarctic fish
J. Comp. Physiol. B
An R Companion to Applied Regression
Metabolic recovery in herring larvae following strenuous activity
J. Fish. Biol.
Cube law, condition factor and weight-length relationships: history, meta-analysis and recommendations
J. Appl. Ichthyol.
Rapid evolution of thermal tolerance in the water flea Daphnia
Nat. Clim. Change
Climatic niche at physiological and macroecological scales: the thermal tolerance-geographical range interface and niche dimensionality: physiological and macroecological thermal niches
Global Ecol. Biogeogr.
Metabolism of a tide-dominated reef platform subject to extreme diel temperature and oxygen variations: metabolism of a tide-dominated reef platform
Limnol. Oceanogr.
Projected climate change in Australian marine and freshwater environments
Mar. Freshw. Res.
Upper thermal limits in terrestrial ectotherms: how constrained are they?
Funct. Ecol.
Predicting organismal vulnerability to climate warming: roles of behaviour, physiology and adaptation
Philos. Trans. R. Soc. B Biol. Sci.
An Introduction to Population Biology
Critical thermal maxima in salamanders
Physiol. Zool.
Do mitochondria limit hot fish hearts? Understanding the role of mitochondrial function with heat stress in Notolabrus celidotus
PloS One
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2022, Fish PhysiologyCitation Excerpt :Early life-history traits and development are notoriously susceptible to altered environmental conditions, but in some cases, developmental acclimation can prime later stages for altered environmental conditions. It has been found that the thermal environment of the eggs (Gagliano et al., 2007), embryos, and juvenile early life stages—rearing temperatures—are important (Illing et al., 2020) and will affect the thermal tolerance traits of the adults (Donelson et al., 2011). Increased rearing temperatures in cobia (R. canadum) results in decreased digestion efficiency (Yufera et al., 2019).