RESEARCH ARTICLE
Aquatic‐to‐Terrestrial Habitat Shift Reduces Energy Expenditure in Newts PETER KRISTÍN
AND
LUMÍR GVOŽDÍK*
Institute of Vertebrate Biology AS CR, Brno, Czech Republic
ABSTRACT
J. Exp. Zool. 321A:183–188, 2014
Many organisms seasonally modify their standard metabolic rates (SMR). However, the diversity of cues triggering the acclimatization response remains little understood. We examined the influence of experimentally induced aquatic‐to‐terrestrial habitat shift on the thermal sensitivity of SMR in newts. Standard metabolic rates increased with temperature (13–23°C), although consistently lower in terrestrial than aquatic individuals. Motor activity during respirometry trials decreased with temperature at similar rates in both groups. We conclude that in newts, a habitat shift might represent an important modulator of the seasonal acclimatization response in SMR. Lowered SMR suggests the potential to reduce newt maintenance costs and depletion of caloric reserves during the activity‐limited period on land. J. Exp. Zool. 321A:183–188, 2014. © 2013 Wiley Periodicals, Inc. How to cite this article: Kristín P, Gvoždík L. 2014. Aquatic‐to‐terrestrial habitat shift reduces energy expenditure in newts. J. Exp. Zool. 321A:183–188.
Diverse organisms seasonally modify their phenotypes in response to the shift in abiotic conditions. While testing the adaptive significance of acclimatization requires experimentation in the field (Smolinský and Gvoždík, 2013), the joint influence of intercorrelated physical variables prevents understanding of which abiotic factors, or combinations, trigger a particular acclimatization response. This is an important issue because ongoing climate change, besides modifying mean environmental temperatures and precipitation, increases the frequency of weather extremes (Coumou and Rahmstorf, 2012). The resulting unpredictability of seasonal environmental cues complicates the matching of plastic phenotypes with their future optima (Ghalambor et al., 2007). The boom of acclimation studies over the last two decades has substantially improved general knowledge of the reversible plastic responses (reviewed by Whitman, 2009), however it has also caused a publication bias. Some factors, such as ecologically realistic temperature regimes have been consistently studied (Podrabsky and Somero, 2004; Šamajová and Gvoždík, 2010), while other environmental cues remain little understood. Migrating taxa are subjected not only to passive seasonal variations, but also to behaviorally induced shifts in abiotic conditions. A notable example includes some adult amphibians that regularly change between aquatic and terrestrial lifestyles. Given the disparate physical characteristics of air and water (Denny, '93), it is not surprising that this drastic habitat shift induces various morphological, physiological, and behavioral modifications (Walters and Greenwald, '77; Brown et al., '83).
Unfortunately, most studies merely report seasonal changes in phenotypic traits, and thus it remains unclear what primary factors are in fact responsible for the acclimatization response. We examined the plasticity of standard metabolic rates (SMR) in newts. Among temperate amphibians, many species reduce their metabolic rates during summer and fall (Knapp, '74; Harlow, '77). Unfortunately, methodological issues prevent our ability to understand whether the plastic response results from seasonal variations in lifestyle, temperature, reproductive cycle, activity, or a combination thereof (Clarke, '93). The adaptive acclimatization response requires, among others, the presence of predictable environmental cues (Gabriel et al., 2005). The shift between aquatic and terrestrial environments meets the criterion of predictability, and so it can be a suitable candidate cue for activating the seasonal acclimatization of metabolic rates. Accordingly, we hypothesize that the aquatic‐to‐terrestrial habitat
Grant sponsor: Czech Science Foundation; grant number: P506/10/2170. Conflicts of interest: None. Correspondence to: Lumír Gvoždík, Institute of Vertebrate Biology AS CR, Kvetná 8, 603 65 Brno, Czech Republic. E‐mail: [email protected] Received 22 July 2013; Revised 20 November 2013; Accepted 26 November 2013 DOI: 10.1002/jez.1849 Published online 30 December 2013 in Wiley Online Library (wileyonlinelibrary.com).
© 2013 WILEY PERIODICALS, INC.
184
KRISTÍN AND GVOŽDÍK
shift will reduce SMR across a range of ecologically realistic temperatures.
MATERIALS AND METHODS Study Species The alpine newt, Ichthyosaura alpestris (Laurenti, 1768) is an approximately 10‐cm long amphibian distributed from south‐east to north‐west Europe. Usually it has a biphasic lifestyle with an aquatic (April–June) and terrestrial phase, although it sometimes remains in water for the whole year (Fasola and Canova, '92). Aquatic individuals have smooth skin with prominent sexually dichromatic color patterns, whereas the skin of terrestrial newts is uniformly dark with a distinct fine‐granulous texture. Aquatic males possess a low dorsal crest that reduces to an interrupted yellow‐black vertebral stripe during the terrestrial phase. Alpine newts are largely crepuscular and nocturnal animals, although aquatic reproductive individuals are also active during the daytime (Himstedt, '71). Food consists of various invertebrates, mostly oligochaetes and chironomid larvae. Adult newts (Table 1) were captured from a population near Jihlava, Czech Republic, in April 2012. Pairs (male and female) were placed into aquaria (50 30 18 cm high) filled with tap water (18 L) and equipped with water plants (Egeria densa) and a piece of Styrofoam, to allow newts to leave the water. To provide newts with abiotic conditions similar to their natural habitat (Smolinský and Gvoždík, 2013), aquaria were placed outdoors in partially shaded locations until the beginning of July Experimental Setup Ten weeks before the beginning of SMR measurements, aquaria were transferred to the laboratory. Half the aquaria were randomly
Table 1. Newt characteristics in aquatic and terrestrial phases used in metabolic measurements. Phase Traita Snout‐vent length (mm) Body mass (g) Operative temperature (°C)c
Aquatic (n ¼ 16)b
Terrestrial (n ¼ 16)
48.3 1.2 (42–56) 2.79 0.20 (1.75–4.76) 15.3 0.1 (12–21)
47.6 1.2 (42–58) 2.65 0.65 (1.83–4.83) 15.6 0.1 (10–22)
Values are means SE (minimum–maximum). Sex ratio was 1:1 in both groups. c Temperatures were recorded at 30 min intervals in indoor conditions over 10 weeks prior to respirometry trials. a
b
J. Exp. Zool.
selected and the water volume gradually reduced together with a slight elevation (2.5°) of the shorter tank side until 350 mL remained. The land area was covered with filter paper and equipped with a clay pot shelter. Daily checking confirmed that under these conditions, newts became predominantly terrestrial, whereas their counterparts in the remaining aquaria spent most of their time in water. All tanks were placed in a room with a 14:10 hr (light/dark) period and diel thermal regime. Temperature regulation was set to allow for a gradual increase in newt operative temperature (see below) to 22°C during day and a decrease in temperature to 10°C during the night. To assure that both aquatic and terrestrial newts experienced similar thermal conditions (Table 1), four randomly selected tanks were equipped with thermistor probes connected to temperature dataloggers recording at 30 min intervals. Terrestrial operative temperatures were measured inside agar cylinders the same size as the newt body (Navas and Araujo, 2000). Water and paper substrate in terrestrial settings were changed at 3‐day intervals. Water in aquaria was subjected to weekly changes. Except during the measuring period (see below), newts were fed an equal amount of live Tubifex, earthworms, and Chironomus larvae once, or twice a week. All experimental procedures were approved by the Expert Committee for Animal Conservation of the Institute of Vertebrate Biology AS CR (research protocol no. 113/2009). After experiments, all animals were released at the site of their capture. Respirometry Trials In order to measure SMR repeatedly at three temperatures during relatively short time intervals, we applied multi‐channel intermittent flow‐through respirometry (Lighton, 2008). Details of the respirometry system used (Sable Systems, Las Vegas, NV, USA) and its validation are provided elsewhere (Kristín and Gvoždík, 2012). In short, H2O and CO2‐scrubbed (soda lime, silica gel, Drierite, Ascarite, and Drierite) air entered the air pump, pushing it through a Nafion humidifier (ME Series, Perma Pure, Toms River, NJ, USA) to multiplexers (RM‐8 and CBL‐1, Sable Systems) at 120 mL hr1. The multiplexers switched air among eight custom‐made respirometry chambers (60 mL) and baseline. Leaving air flows through a water vapor analyzer (RH‐300, Sable Systems), Nafion dessicator (MD Series, Perma Pure), CO2 analyzer (FoxBox‐C, Sable Systems), CO2 and H2O scrubber (soda lime, silica gel, and Drierite), and O2 analyzer (FoxBox‐C). To maintain stable temperature during trials, respirometry chambers were placed in a temperature‐controlled water bath (accuracy 0.5°C). Newt respiration was measured at 13°C, 18°C, and 23°C—temperatures that newts commonly experience in the field and prefer in laboratory thermal gradients (Šamajová and Gvoždík, 2010; Marek and Gvoždík, 2012). Each individual was measured at each of the three temperatures. To eliminate the influence of a previous temperature on metabolic rates, newts were randomly divided into two groups with a different order of experimental temperatures, that is, 18°C, 13°C, 23°C and 18°C,
METABOLISM OF AQUATIC AND TERRESTRIAL NEWTS 23°C, 13°C. Trials were repeated at weekly intervals. Room temperature was maintained 5°C above the bath temperature to prevent water condensation inside the respirometry system. Bath and room temperatures were recorded at 1 Hz, using calibrated thermistor probes connected to an analog‐to‐digital converter (UI‐2, Sable Systems). For each respirometry trial, we placed eight nonreproductive newts at their postabsorptive state (7 days fasting) individually in respirometry chambers. A respirometry trial lasted 5 hr during a period of daylight (07:00–19:00). Each respirometry chamber was periodically flushed two times per hour (enclosure time ¼ 1,851 sec) and continuously monitored using a webcamera (5 fps; Microsoft LifeCam VX‐2000, Microsoft, Redmond, WA, USA). Motor activity episodes were recorded using a motion video detector (5 sec resolution; iSpy software, available from http:// www.ispyconnect.com). Two activity indices were used for further analyses: immediate activity index, the number of motion events during a measurement interval with the lowest O2 consumption; and the overall activity index, the number of motion events during the whole trial. The body mass of each individual was measured (to 0.01 g) before a trial, using laboratory balances (440‐33N, Kern, Balingen, Germany). To obtain results from intermittent respirometry, minimal oxygen consumption ðV_ O2 Þ and minimal CO2 production ðV_ CO2 Þ values were calculated from peak integrals of sample rates of O2 consumption (MsO2) and CO2 production (MsCO2), divided by chamber enclosure time (Lighton, 2008; Kristín and Gvoždík, 2012) using Expedata software (version 1.3.3, Sable Systems). To calculate sample consumption or production rates, we used the following equation: MsO2 ¼ FR(FiO2–FeO2)/(1–FeO2); MsCO2 ¼ FR (FeCO2–FiCO2), where FR is incurrent flow rate (mL h1), Fe is the fractional concentration of excurrent O2 or CO2, and Fi is a fractional concentration of incurrent O2 or CO2. The lowest V_ O2 and V_ CO2 values of nonmoving individuals (>95% of enclosure time) from each trial were considered estimates of SMR at a given temperature. In 10% of trials, high motor activity violated standard conditions, and thus these data were discarded from further analyses. Statistical Analyses We examined the influence of aquatic and terrestrial phases, and temperature on V_ O2 and V_ CO2 using a general linear mixed model (GLMM). Phase and temperature were set as fixed factors, and individual newt identity as a random grouping factor. To statistically control the effect of body size on V_ O2 and V_ CO2 , we added body mass as a covariate. As we used three experimental temperatures, their linear and quadratic effects were tested using orthogonal polynomial contrasts. The overall activity index was analyzed using the same model, but with count data with Poisson‐ distributed errors. Over‐dispersion was solved by adding an individual observation random effect to the model (Zuur et al., 2012). Analyses were performed in an R programming
185 environment (R Development Core Team, Vienna, Austria) using the “lme4” library.
RESULTS We obtained metabolic data from 32 individuals (Table 1). High motor activity caused a similar number of trials to be discarded from both the aquatic and terrestrial groups (4 and 6 out of 96; Fisher exact test, P ¼ 0.74). Body mass corrected for snout‐vent length was similar in both groups (GLMM, F1,28 ¼ 1.89, P ¼ 0.18). Sex and the interaction between experimental temperature and phase explained little variation in ˙ V O2 (sex: x2 ¼ 0.19, df ¼ 1, P ¼ 0.66; temperature phase: x2 ¼ 3.99, df ¼ 2, P ¼ 0.14), and so were dropped from the minimum adequate model. Oxygen consumption linearly increased with temperature in both phases (t55 ¼ 21.20, P < 0.001; temperature coefficient [Q10] ¼ 2.15 0.10). When body mass was controlled for (F1,28 ¼ 107.25, P < 0.001), terrestrial newts spent less oxygen than aquatic individuals (F1,31 ¼ 6.71, P ¼ 0.015; Fig. 1A). Individual newt identity explained 41% of total variation in V_ O2 (intraclass correlation coefficient), indicating good repeatability of this trait across temperatures. Although V_ CO2 was affected by body mass and temperature (body mass: F1,28 ¼ 68.63, P < 0.001; temperature: F2,55 ¼ 105.62, P < 0.001; Q10 ¼ 2.15 0.14), newt phase explained minor variation in this trait (F1,31 ¼ 0.59, P ¼ 0.45; Fig. 1B). The respiratory exchange ratio V_ CO2 =V_ O2 varied little between both groups (terrestrial phase: 0.93 0.05; aquatic phase: 0.85 0.04; F1,31 ¼1.45, P ¼ 0.24). The overall activity index decreased with rising temperature (x2 ¼ 17.21, df ¼ 2, P < 0.001; Fig. 1C) showing no statistically significant differences between terrestrial and aquatic individuals (x2 ¼ 0.18, df ¼ 1, P < 0.67).
DISCUSSION Although metabolic rates vary seasonally in various taxa, specific factors influencing the plastic response have remained largely unstudied. Our results show that in newts metabolic shift can be affected by their transition from aquatic to terrestrial habitats. This response is consistent across temperatures. As far as we know, this is the first study demonstrating that acclimation of SMR is not only a passive response to changing environmental conditions, but also accompanies an active habitat shift in a seasonally migrating ectotherm. As newts leave water in June, the lower energy demands of terrestrial individuals are consistent with earlier reports of a metabolic decrease during summer in this species (Knapp, '74). The seasonal acclimatization of metabolic rates has been attributed to changes in temperature, reproductive cycle, or activity (see above). Since we controlled all these potentially confounding factors, our study clearly demonstrates that the plastic response is modified by a previously unconsidered influence, the shift from an aquatic to terrestrial habitat. In comparison with published results (Knapp, '74; Harlow, '77), the magnitude of SMR change is J. Exp. Zool.
186
KRISTÍN AND GVOŽDÍK relatively small (ca. 10%) however and it therefore seems likely that the seasonal acclimatization of this trait is affected by habitat shifts in concert with other factors. Among them, temperature is a likely candidate because thermal acclimation of SMR is common in temperate newts and salamanders (Feder, '78; Berner and Puckett, 2010). The interactive influence of a habitat shift with various environmental factors poses an interesting research topic for future studies. As in other acclimation studies (Huey et al., '99; Wilson and Franklin, 2002), the vital issue is the beneficial significance of plasticity in SMR. In amphibians, the major difference between an aquatic and terrestrial lifestyle is the limitation in foraging activity and thus energy gain (Spotila, '72; Feder, '83). From this point of view, the reduction in metabolic rates in the terrestrial phase should be beneficial, reducing maintenance costs across ecologically relevant temperatures, and thus more energy would remain for growth, survival, and reproduction. On the other hand, a high‐ octane life in water reflects newts' high energy demands for reproduction and air‐breathing (Halliday and Sweatman, '76; Šamajová and Gvoždík, 2009). While this hypothetical explanation seems straightforward, it requires empirical verification before conclusive acceptance. Populations of partially migrating newts (Grayson and Wilbur, 2009) provide a suitable model system for fulfilling this task. Contrary to V_ O2 , we found no detectable shift in V_ CO2 . Group means for V_ CO2 followed a similar trend (Figs. 1A, 1B), although their spread was substantially higher than in V_ O2 . Since newts and salamanders have a relatively narrow aerobic scope for activity (Gatten et al., '92), the higher variation likely reflects CO2 release from body fluids during vigorous movements rather than variation in metabolized substrates. It corroborates previous recommendations that the use of V_ CO2 in amphibians provides less reliable data than V_ O2 (Hillman et al., 2009), including the species studied (Kristín and Gvoždík, 2012). Accordingly, V_ CO2 and the respiratory exchange ratio should be interpreted with caution. Motor activity during respirometry trials decreased at higher temperatures. Since motor activity usually accelerates with temperature within a “thermally normal” range in amphibians (Putnam and Bennett, '81), this paradoxical result cannot be explained by the trait's thermal sensitivity. In a terrestrial
3
Figure 1. Continued.
J. Exp. Zool.
Figure 1. The influence of acute temperature on (A) oxygen consumption, (B) CO2 production, and (C) total motor activity during a respirometry trial in aquatic and terrestrial newts. Numbers above bars denote sample sizes. Values are presented as body mass‐adjusted (O2 and CO2) or unadjusted (activity) means SE.
METABOLISM OF AQUATIC AND TERRESTRIAL NEWTS salamander, even relatively mild dehydration reduces foraging behavior (Feder and Londos, '84) and induces postural adjustments to eliminate excessive water loss (Spotila, '72). This suggests that the thermal dependence of motor activity during respirometry trials likely represents newts' innate response for reducing their evaporative water loss at higher temperatures on land. Although acclimation of SMR has been subjected to long‐ term research, our results show the diversity of factors affecting this plastic response is still not fully understood. We demonstrated for the first time that the acclimation of SMR is modified by the transition from an aquatic to terrestrial lifestyle. This cue provides more reliable information about future conditions, and so it seems an even more important elicitor of seasonal shift in SMR than frequently considered factors, such as temperature. Accordingly, the accuracy of this acclimatization response seems less affected by ongoing climate change than others (Chown et al., 2010). The SMR acclimation induced by the habitat shift provides at least two interesting directions for further studies. (i) Given that habitat shifts jointly influence plastic modifications in various morphological, physiological, and behavioral traits (see Introduction), it would be interesting to examine their individual variation, phenotypic integration, and/or trade‐offs between aquatic and terrestrial phases. The focus on the recently highlighted link between SMR and behavior (Careau et al., 2008; Biro and Stamps, 2010) seems especially promising. (ii) Disparate energy costs of living in water and on land provide a physiological perspective on migration‐residency decisions in partially migrating amphibians (Grayson and Wilbur, 2009). Newts are suitable models to examine both research directions.
ACKNOWLEDGMENTS This study was funded by a grant from the Czech Science Foundation (P506/10/2170) and institutional support (RVO: 68081766). This study was performed in accordance with the laws of the countries in which the work was conducted. The Agency for Nature Conservation and Landscape Protection of the Czech Republic issued permission to capture the newts (1154/ZV/2008).
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187 Chown SL, Hoffmann AA, Kristensen TN, et al. 2010. Adapting to climate change: a perspective from evolutionary physiology. Clim Res 43:3–15. Clarke A. 1993. Seasonal acclimatization and latitudinal compensation in metabolism: do they exist? Funct Ecol 7:139–149. Coumou D, Rahmstorf S. 2012. A decade of weather extremes. Nat Clim Chang 2:491–496. Denny MW. 1993. Air and water. The biology and physics of life's media. Princeton: Princeton University Press. Fasola M, Canova L. 1992. Residence in water by the newts Triturus vulgaris, T. cristatus and T. alpestris in a pond in northern Italy. Amphib Reptil 13:227–233. Feder ME. 1978. Environmental variability and thermal acclimation in neotropical and temperate zone salamanders. Physiol Zool 51:7–16. Feder ME. 1983. Integrating the ecology and physiology of plethodontid salamanders. Herpetologica 39:291–310. Feder ME, Londos PL. 1984. Hydric constraints upon foraging in a terrestrial salamander, Desmognathus ochrophaeus (Amphibia, Plethodontidae). Oecologia 64:413–418. Gabriel W, Luttbeg B, Sih A, Tollrian R. 2005. Environmental tolerance, heterogeneity, and the evolution of reversible plastic responses. Am Nat 166:339–353. Gatten RE, Miller K, Full RJ. 1992. Energetics at rest and during locomotion. In: Feder ME, Burggren WW, editors. Environmental physiology of the amphibians. Chicago: Chicago University Press. p 314–377. Ghalambor CK, Mckay JK, Carroll SP, Reznick DN. 2007. Adaptive versus non‐adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments. Funct Ecol 21:394– 407. Grayson KL, Wilbur HM. 2009. Sex‐ and context‐dependent migration in a pond‐breeding amphibian. Ecology 90:306–312. Halliday TR, Sweatman HPA. 1976. To breathe or not to breathe? Newts problem. Anim Behav 24:551–561. Harlow HJ. 1977. Seasonal oxygen metabolism and cutaneous osmoregulation in California newt, Taricha torosa. Physiol Zool 50:231–236. Hillman SS, Withers PC, Drewes RC, Hillyard SD. 2009. Ecological and environmental physiology of amphibians. Oxford: Oxford University Press. Himstedt W. 1971. Diurnal rhythm in salamanders. Oecologia 8:194– 208. Huey RB, Berrigan D, Gilchrist GW, Herron JC. 1999. Testing the adaptive significance of acclimation: a strong inference approach. Am Zool 39:323–336. Knapp W. 1974. Die jahreszeitliche Steuerung der Atmung in Abhangigkeit von Akklimationstemperatur und Experimentaltemperatur bei Triturus alpestris Laur. und Salamandra atra Laur. (Amphibia). Oecologia 15:353–374. Kristín P, Gvoždík L. 2012. Influence of respirometry methods on intraspecific variation in standard metabolic rates in newts. Comp Biochem Physiol A 163:147–151. J. Exp. Zool.
188 Lighton JRB. 2008. Measuring metabolic rates: a manual for scientists. Oxford: Oxford University Press. Marek V, Gvoždík L. 2012. The insensitivity of thermal preferences to various thermal gradient profiles in newts. J Ethol 30:35–41. Navas CA, Araujo C. 2000. The use of agar models to study amphibian thermal ecology. J Herpetol 34:330–334. Podrabsky JE, Somero GN. 2004. Changes in gene expression associated with acclimation to constant temperatures and fluctuating daily temperatures in an annual killifish Austrofundulus limnaeus. J Exp Biol 207:2237–2254. Putnam RW, Bennett AF. 1981. Thermal dependence of behavioral performance of anuran amphibians. Anim Behav 29:502–509. Šamajová P, Gvoždík L. 2009. The influence of temperature on diving behaviourinthealpinenewt,Triturusalpestris.JThermBiol34:401–405. Šamajová P, Gvoždík L. 2010. Inaccurate or disparate temperature cues? Seasonal acclimation of terrestrial and aquatic locomotor capacity in newts. Funct Ecol 24:1023–1030.
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KRISTÍN AND GVOŽDÍK Smolinský R, Gvoždík L. 2013. Does developmental acclimatization reduce the susceptibility to predation in newt larvae? Biol J Linn Soc 108:109–115. Spotila JR. 1972. Role of temperature and water in the ecology of lungless salamanders. Ecol Monogr 42:95–125. Walters PJ, Greenwald L. 1977. Physiological adaptations of aquatic newts (Notophthalmus viridescens) to a terrestrial environment. Physiol Zool 50:88–98. Whitman DW. 2009. Acclimation as adaptive phenotypic plasticity. In: Whitman DW, Ananthakrishnan TN, editors Phenotypic plasticity of insects: mechanisms and consequences. Enfield: Science Publishers. p 675–739. Wilson RS, Franklin CE. 2002. Testing the beneficial acclimation hypothesis. Trends Ecol Evol 17:66–70. Zuur AF, Saveliev AA, Ieno EN. 2012. Zero inflated models and generalized linear mixed models with R. Newburgh: Highland Statistics.
Aquatic‐to‐Terrestrial Habitat Shift Reduces Energy Expenditure in Newts PETER KRISTÍN
AND
LUMÍR GVOŽDÍK*
Institute of Vertebrate Biology AS CR, Brno, Czech Republic
ABSTRACT
J. Exp. Zool. 321A:183–188, 2014
Many organisms seasonally modify their standard metabolic rates (SMR). However, the diversity of cues triggering the acclimatization response remains little understood. We examined the influence of experimentally induced aquatic‐to‐terrestrial habitat shift on the thermal sensitivity of SMR in newts. Standard metabolic rates increased with temperature (13–23°C), although consistently lower in terrestrial than aquatic individuals. Motor activity during respirometry trials decreased with temperature at similar rates in both groups. We conclude that in newts, a habitat shift might represent an important modulator of the seasonal acclimatization response in SMR. Lowered SMR suggests the potential to reduce newt maintenance costs and depletion of caloric reserves during the activity‐limited period on land. J. Exp. Zool. 321A:183–188, 2014. © 2013 Wiley Periodicals, Inc. How to cite this article: Kristín P, Gvoždík L. 2014. Aquatic‐to‐terrestrial habitat shift reduces energy expenditure in newts. J. Exp. Zool. 321A:183–188.
Diverse organisms seasonally modify their phenotypes in response to the shift in abiotic conditions. While testing the adaptive significance of acclimatization requires experimentation in the field (Smolinský and Gvoždík, 2013), the joint influence of intercorrelated physical variables prevents understanding of which abiotic factors, or combinations, trigger a particular acclimatization response. This is an important issue because ongoing climate change, besides modifying mean environmental temperatures and precipitation, increases the frequency of weather extremes (Coumou and Rahmstorf, 2012). The resulting unpredictability of seasonal environmental cues complicates the matching of plastic phenotypes with their future optima (Ghalambor et al., 2007). The boom of acclimation studies over the last two decades has substantially improved general knowledge of the reversible plastic responses (reviewed by Whitman, 2009), however it has also caused a publication bias. Some factors, such as ecologically realistic temperature regimes have been consistently studied (Podrabsky and Somero, 2004; Šamajová and Gvoždík, 2010), while other environmental cues remain little understood. Migrating taxa are subjected not only to passive seasonal variations, but also to behaviorally induced shifts in abiotic conditions. A notable example includes some adult amphibians that regularly change between aquatic and terrestrial lifestyles. Given the disparate physical characteristics of air and water (Denny, '93), it is not surprising that this drastic habitat shift induces various morphological, physiological, and behavioral modifications (Walters and Greenwald, '77; Brown et al., '83).
Unfortunately, most studies merely report seasonal changes in phenotypic traits, and thus it remains unclear what primary factors are in fact responsible for the acclimatization response. We examined the plasticity of standard metabolic rates (SMR) in newts. Among temperate amphibians, many species reduce their metabolic rates during summer and fall (Knapp, '74; Harlow, '77). Unfortunately, methodological issues prevent our ability to understand whether the plastic response results from seasonal variations in lifestyle, temperature, reproductive cycle, activity, or a combination thereof (Clarke, '93). The adaptive acclimatization response requires, among others, the presence of predictable environmental cues (Gabriel et al., 2005). The shift between aquatic and terrestrial environments meets the criterion of predictability, and so it can be a suitable candidate cue for activating the seasonal acclimatization of metabolic rates. Accordingly, we hypothesize that the aquatic‐to‐terrestrial habitat
Grant sponsor: Czech Science Foundation; grant number: P506/10/2170. Conflicts of interest: None. Correspondence to: Lumír Gvoždík, Institute of Vertebrate Biology AS CR, Kvetná 8, 603 65 Brno, Czech Republic. E‐mail: [email protected] Received 22 July 2013; Revised 20 November 2013; Accepted 26 November 2013 DOI: 10.1002/jez.1849 Published online 30 December 2013 in Wiley Online Library (wileyonlinelibrary.com).
© 2013 WILEY PERIODICALS, INC.
184
KRISTÍN AND GVOŽDÍK
shift will reduce SMR across a range of ecologically realistic temperatures.
MATERIALS AND METHODS Study Species The alpine newt, Ichthyosaura alpestris (Laurenti, 1768) is an approximately 10‐cm long amphibian distributed from south‐east to north‐west Europe. Usually it has a biphasic lifestyle with an aquatic (April–June) and terrestrial phase, although it sometimes remains in water for the whole year (Fasola and Canova, '92). Aquatic individuals have smooth skin with prominent sexually dichromatic color patterns, whereas the skin of terrestrial newts is uniformly dark with a distinct fine‐granulous texture. Aquatic males possess a low dorsal crest that reduces to an interrupted yellow‐black vertebral stripe during the terrestrial phase. Alpine newts are largely crepuscular and nocturnal animals, although aquatic reproductive individuals are also active during the daytime (Himstedt, '71). Food consists of various invertebrates, mostly oligochaetes and chironomid larvae. Adult newts (Table 1) were captured from a population near Jihlava, Czech Republic, in April 2012. Pairs (male and female) were placed into aquaria (50 30 18 cm high) filled with tap water (18 L) and equipped with water plants (Egeria densa) and a piece of Styrofoam, to allow newts to leave the water. To provide newts with abiotic conditions similar to their natural habitat (Smolinský and Gvoždík, 2013), aquaria were placed outdoors in partially shaded locations until the beginning of July Experimental Setup Ten weeks before the beginning of SMR measurements, aquaria were transferred to the laboratory. Half the aquaria were randomly
Table 1. Newt characteristics in aquatic and terrestrial phases used in metabolic measurements. Phase Traita Snout‐vent length (mm) Body mass (g) Operative temperature (°C)c
Aquatic (n ¼ 16)b
Terrestrial (n ¼ 16)
48.3 1.2 (42–56) 2.79 0.20 (1.75–4.76) 15.3 0.1 (12–21)
47.6 1.2 (42–58) 2.65 0.65 (1.83–4.83) 15.6 0.1 (10–22)
Values are means SE (minimum–maximum). Sex ratio was 1:1 in both groups. c Temperatures were recorded at 30 min intervals in indoor conditions over 10 weeks prior to respirometry trials. a
b
J. Exp. Zool.
selected and the water volume gradually reduced together with a slight elevation (2.5°) of the shorter tank side until 350 mL remained. The land area was covered with filter paper and equipped with a clay pot shelter. Daily checking confirmed that under these conditions, newts became predominantly terrestrial, whereas their counterparts in the remaining aquaria spent most of their time in water. All tanks were placed in a room with a 14:10 hr (light/dark) period and diel thermal regime. Temperature regulation was set to allow for a gradual increase in newt operative temperature (see below) to 22°C during day and a decrease in temperature to 10°C during the night. To assure that both aquatic and terrestrial newts experienced similar thermal conditions (Table 1), four randomly selected tanks were equipped with thermistor probes connected to temperature dataloggers recording at 30 min intervals. Terrestrial operative temperatures were measured inside agar cylinders the same size as the newt body (Navas and Araujo, 2000). Water and paper substrate in terrestrial settings were changed at 3‐day intervals. Water in aquaria was subjected to weekly changes. Except during the measuring period (see below), newts were fed an equal amount of live Tubifex, earthworms, and Chironomus larvae once, or twice a week. All experimental procedures were approved by the Expert Committee for Animal Conservation of the Institute of Vertebrate Biology AS CR (research protocol no. 113/2009). After experiments, all animals were released at the site of their capture. Respirometry Trials In order to measure SMR repeatedly at three temperatures during relatively short time intervals, we applied multi‐channel intermittent flow‐through respirometry (Lighton, 2008). Details of the respirometry system used (Sable Systems, Las Vegas, NV, USA) and its validation are provided elsewhere (Kristín and Gvoždík, 2012). In short, H2O and CO2‐scrubbed (soda lime, silica gel, Drierite, Ascarite, and Drierite) air entered the air pump, pushing it through a Nafion humidifier (ME Series, Perma Pure, Toms River, NJ, USA) to multiplexers (RM‐8 and CBL‐1, Sable Systems) at 120 mL hr1. The multiplexers switched air among eight custom‐made respirometry chambers (60 mL) and baseline. Leaving air flows through a water vapor analyzer (RH‐300, Sable Systems), Nafion dessicator (MD Series, Perma Pure), CO2 analyzer (FoxBox‐C, Sable Systems), CO2 and H2O scrubber (soda lime, silica gel, and Drierite), and O2 analyzer (FoxBox‐C). To maintain stable temperature during trials, respirometry chambers were placed in a temperature‐controlled water bath (accuracy 0.5°C). Newt respiration was measured at 13°C, 18°C, and 23°C—temperatures that newts commonly experience in the field and prefer in laboratory thermal gradients (Šamajová and Gvoždík, 2010; Marek and Gvoždík, 2012). Each individual was measured at each of the three temperatures. To eliminate the influence of a previous temperature on metabolic rates, newts were randomly divided into two groups with a different order of experimental temperatures, that is, 18°C, 13°C, 23°C and 18°C,
METABOLISM OF AQUATIC AND TERRESTRIAL NEWTS 23°C, 13°C. Trials were repeated at weekly intervals. Room temperature was maintained 5°C above the bath temperature to prevent water condensation inside the respirometry system. Bath and room temperatures were recorded at 1 Hz, using calibrated thermistor probes connected to an analog‐to‐digital converter (UI‐2, Sable Systems). For each respirometry trial, we placed eight nonreproductive newts at their postabsorptive state (7 days fasting) individually in respirometry chambers. A respirometry trial lasted 5 hr during a period of daylight (07:00–19:00). Each respirometry chamber was periodically flushed two times per hour (enclosure time ¼ 1,851 sec) and continuously monitored using a webcamera (5 fps; Microsoft LifeCam VX‐2000, Microsoft, Redmond, WA, USA). Motor activity episodes were recorded using a motion video detector (5 sec resolution; iSpy software, available from http:// www.ispyconnect.com). Two activity indices were used for further analyses: immediate activity index, the number of motion events during a measurement interval with the lowest O2 consumption; and the overall activity index, the number of motion events during the whole trial. The body mass of each individual was measured (to 0.01 g) before a trial, using laboratory balances (440‐33N, Kern, Balingen, Germany). To obtain results from intermittent respirometry, minimal oxygen consumption ðV_ O2 Þ and minimal CO2 production ðV_ CO2 Þ values were calculated from peak integrals of sample rates of O2 consumption (MsO2) and CO2 production (MsCO2), divided by chamber enclosure time (Lighton, 2008; Kristín and Gvoždík, 2012) using Expedata software (version 1.3.3, Sable Systems). To calculate sample consumption or production rates, we used the following equation: MsO2 ¼ FR(FiO2–FeO2)/(1–FeO2); MsCO2 ¼ FR (FeCO2–FiCO2), where FR is incurrent flow rate (mL h1), Fe is the fractional concentration of excurrent O2 or CO2, and Fi is a fractional concentration of incurrent O2 or CO2. The lowest V_ O2 and V_ CO2 values of nonmoving individuals (>95% of enclosure time) from each trial were considered estimates of SMR at a given temperature. In 10% of trials, high motor activity violated standard conditions, and thus these data were discarded from further analyses. Statistical Analyses We examined the influence of aquatic and terrestrial phases, and temperature on V_ O2 and V_ CO2 using a general linear mixed model (GLMM). Phase and temperature were set as fixed factors, and individual newt identity as a random grouping factor. To statistically control the effect of body size on V_ O2 and V_ CO2 , we added body mass as a covariate. As we used three experimental temperatures, their linear and quadratic effects were tested using orthogonal polynomial contrasts. The overall activity index was analyzed using the same model, but with count data with Poisson‐ distributed errors. Over‐dispersion was solved by adding an individual observation random effect to the model (Zuur et al., 2012). Analyses were performed in an R programming
185 environment (R Development Core Team, Vienna, Austria) using the “lme4” library.
RESULTS We obtained metabolic data from 32 individuals (Table 1). High motor activity caused a similar number of trials to be discarded from both the aquatic and terrestrial groups (4 and 6 out of 96; Fisher exact test, P ¼ 0.74). Body mass corrected for snout‐vent length was similar in both groups (GLMM, F1,28 ¼ 1.89, P ¼ 0.18). Sex and the interaction between experimental temperature and phase explained little variation in ˙ V O2 (sex: x2 ¼ 0.19, df ¼ 1, P ¼ 0.66; temperature phase: x2 ¼ 3.99, df ¼ 2, P ¼ 0.14), and so were dropped from the minimum adequate model. Oxygen consumption linearly increased with temperature in both phases (t55 ¼ 21.20, P < 0.001; temperature coefficient [Q10] ¼ 2.15 0.10). When body mass was controlled for (F1,28 ¼ 107.25, P < 0.001), terrestrial newts spent less oxygen than aquatic individuals (F1,31 ¼ 6.71, P ¼ 0.015; Fig. 1A). Individual newt identity explained 41% of total variation in V_ O2 (intraclass correlation coefficient), indicating good repeatability of this trait across temperatures. Although V_ CO2 was affected by body mass and temperature (body mass: F1,28 ¼ 68.63, P < 0.001; temperature: F2,55 ¼ 105.62, P < 0.001; Q10 ¼ 2.15 0.14), newt phase explained minor variation in this trait (F1,31 ¼ 0.59, P ¼ 0.45; Fig. 1B). The respiratory exchange ratio V_ CO2 =V_ O2 varied little between both groups (terrestrial phase: 0.93 0.05; aquatic phase: 0.85 0.04; F1,31 ¼1.45, P ¼ 0.24). The overall activity index decreased with rising temperature (x2 ¼ 17.21, df ¼ 2, P < 0.001; Fig. 1C) showing no statistically significant differences between terrestrial and aquatic individuals (x2 ¼ 0.18, df ¼ 1, P < 0.67).
DISCUSSION Although metabolic rates vary seasonally in various taxa, specific factors influencing the plastic response have remained largely unstudied. Our results show that in newts metabolic shift can be affected by their transition from aquatic to terrestrial habitats. This response is consistent across temperatures. As far as we know, this is the first study demonstrating that acclimation of SMR is not only a passive response to changing environmental conditions, but also accompanies an active habitat shift in a seasonally migrating ectotherm. As newts leave water in June, the lower energy demands of terrestrial individuals are consistent with earlier reports of a metabolic decrease during summer in this species (Knapp, '74). The seasonal acclimatization of metabolic rates has been attributed to changes in temperature, reproductive cycle, or activity (see above). Since we controlled all these potentially confounding factors, our study clearly demonstrates that the plastic response is modified by a previously unconsidered influence, the shift from an aquatic to terrestrial habitat. In comparison with published results (Knapp, '74; Harlow, '77), the magnitude of SMR change is J. Exp. Zool.
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KRISTÍN AND GVOŽDÍK relatively small (ca. 10%) however and it therefore seems likely that the seasonal acclimatization of this trait is affected by habitat shifts in concert with other factors. Among them, temperature is a likely candidate because thermal acclimation of SMR is common in temperate newts and salamanders (Feder, '78; Berner and Puckett, 2010). The interactive influence of a habitat shift with various environmental factors poses an interesting research topic for future studies. As in other acclimation studies (Huey et al., '99; Wilson and Franklin, 2002), the vital issue is the beneficial significance of plasticity in SMR. In amphibians, the major difference between an aquatic and terrestrial lifestyle is the limitation in foraging activity and thus energy gain (Spotila, '72; Feder, '83). From this point of view, the reduction in metabolic rates in the terrestrial phase should be beneficial, reducing maintenance costs across ecologically relevant temperatures, and thus more energy would remain for growth, survival, and reproduction. On the other hand, a high‐ octane life in water reflects newts' high energy demands for reproduction and air‐breathing (Halliday and Sweatman, '76; Šamajová and Gvoždík, 2009). While this hypothetical explanation seems straightforward, it requires empirical verification before conclusive acceptance. Populations of partially migrating newts (Grayson and Wilbur, 2009) provide a suitable model system for fulfilling this task. Contrary to V_ O2 , we found no detectable shift in V_ CO2 . Group means for V_ CO2 followed a similar trend (Figs. 1A, 1B), although their spread was substantially higher than in V_ O2 . Since newts and salamanders have a relatively narrow aerobic scope for activity (Gatten et al., '92), the higher variation likely reflects CO2 release from body fluids during vigorous movements rather than variation in metabolized substrates. It corroborates previous recommendations that the use of V_ CO2 in amphibians provides less reliable data than V_ O2 (Hillman et al., 2009), including the species studied (Kristín and Gvoždík, 2012). Accordingly, V_ CO2 and the respiratory exchange ratio should be interpreted with caution. Motor activity during respirometry trials decreased at higher temperatures. Since motor activity usually accelerates with temperature within a “thermally normal” range in amphibians (Putnam and Bennett, '81), this paradoxical result cannot be explained by the trait's thermal sensitivity. In a terrestrial
3
Figure 1. Continued.
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Figure 1. The influence of acute temperature on (A) oxygen consumption, (B) CO2 production, and (C) total motor activity during a respirometry trial in aquatic and terrestrial newts. Numbers above bars denote sample sizes. Values are presented as body mass‐adjusted (O2 and CO2) or unadjusted (activity) means SE.
METABOLISM OF AQUATIC AND TERRESTRIAL NEWTS salamander, even relatively mild dehydration reduces foraging behavior (Feder and Londos, '84) and induces postural adjustments to eliminate excessive water loss (Spotila, '72). This suggests that the thermal dependence of motor activity during respirometry trials likely represents newts' innate response for reducing their evaporative water loss at higher temperatures on land. Although acclimation of SMR has been subjected to long‐ term research, our results show the diversity of factors affecting this plastic response is still not fully understood. We demonstrated for the first time that the acclimation of SMR is modified by the transition from an aquatic to terrestrial lifestyle. This cue provides more reliable information about future conditions, and so it seems an even more important elicitor of seasonal shift in SMR than frequently considered factors, such as temperature. Accordingly, the accuracy of this acclimatization response seems less affected by ongoing climate change than others (Chown et al., 2010). The SMR acclimation induced by the habitat shift provides at least two interesting directions for further studies. (i) Given that habitat shifts jointly influence plastic modifications in various morphological, physiological, and behavioral traits (see Introduction), it would be interesting to examine their individual variation, phenotypic integration, and/or trade‐offs between aquatic and terrestrial phases. The focus on the recently highlighted link between SMR and behavior (Careau et al., 2008; Biro and Stamps, 2010) seems especially promising. (ii) Disparate energy costs of living in water and on land provide a physiological perspective on migration‐residency decisions in partially migrating amphibians (Grayson and Wilbur, 2009). Newts are suitable models to examine both research directions.
ACKNOWLEDGMENTS This study was funded by a grant from the Czech Science Foundation (P506/10/2170) and institutional support (RVO: 68081766). This study was performed in accordance with the laws of the countries in which the work was conducted. The Agency for Nature Conservation and Landscape Protection of the Czech Republic issued permission to capture the newts (1154/ZV/2008).
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