SUMMARY

We examine here evaporative water loss, economy and partitioning at ambient temperatures from 14 to 33°C for the monito del monte (Dromiciops gliroides), a microbiotheriid marsupial found only in temperate rainforests of Chile. The monito's standard evaporative water loss (2.58 mg g−1 h−1 at 30°C) was typical for a marsupial of its body mass and phylogenetic position. Evaporative water loss was independent of air temperature below thermoneutrality, but enhanced evaporative water loss and hyperthermia were the primary thermal responses above the thermoneutral zone. Non-invasive partitioning of total evaporative water loss indicated that respiratory loss accounted for 59–77% of the total, with no change in respiratory loss with ambient temperature, but a small change in cutaneous loss below thermoneutrality and an increase in cutaneous loss in and above thermoneutrality. Relative water economy (metabolic water production/evaporative water loss) increased at low ambient temperatures, with a point of relative water economy of 15.4°C. Thermolability had little effect on relative water economy, but conferred substantial energy savings at low ambient temperatures. Torpor reduced total evaporative water loss to as little as 21% of normothermic values, but relative water economy during torpor was poor even at low ambient temperatures because of the relatively greater reduction in metabolic water production than in evaporative water loss. The poor water economy of the monito during torpor suggests that negative water balance may explain why hibernators periodically arouse to normothermia, to obtain water by drinking or via an improved water economy.

INTRODUCTION

Understanding the effects of ambient temperature (Ta) on thermal and metabolic physiology is essential to understanding the thermoregulatory abilities of animals. The effects of Ta on energetics have been well studied for many endothermic mammals and birds, but effects on evaporative water loss are less well documented. For marsupials in particular, there is an extensive data set for body temperature (Tb) and metabolic rate (MR) (e.g. Dawson and Hulbert, 1970; McNab, 1984; McNab, 1986; McNab, 2005; McNab, 2008; Lovegrove, 2000; White and Seymour, 2003; White and Seymour, 2005; Withers et al., 2006) but there are considerably fewer data for total evaporative water loss (EWLT) (e.g. Withers et al., 2006). Consequently, a clear understanding of the factors determining EWLT, including the effects of Ta, is only now being developed for marsupials (e.g. Cooper et al., 2005; Cooper et al., 2009; Withers et al., 2006; Cooper and Cruz Neto, 2009; Cooper et al., 2010; Warnecke et al., 2010; Withers and Cooper, 2009a; Withers and Cooper, 2009b; Withers and Cooper, 2011). Understanding the pattern of change in EWLT with Ta (or, commonly, the lack of change) requires partitioning of EWLT into its respiratory (EWLR) and cutaneous (EWLC) components to understand the effects of Ta on each.

Relative water economy (RWE) is the ratio of metabolic water production (MWP) to EWLT (MacMillen and Hinds, 1983; Hinds and MacMillen, 1986). The effect of Ta on RWE reflects the patterns of Ta on MWP and EWLT (e.g. Bartholomew, 1972; MacMillen and Hinds, 1983; Hinds and MacMillen, 1986; Cooper et al., 2005). For normothermic endotherms, MWP typically increases at low Ta whereas EWLT is relatively constant, hence RWE increases at low Ta. The point of relative water economy (PRWE) is the Ta where MWP=EWLT (i.e. RWE=1), which reflects the relative water balance of a species. For heterothermic endotherms, low Tb during torpor or hibernation markedly decreases RWE (e.g. Cooper et al., 2005; Cooper et al., 2009; Withers and Cooper, 2009a; Withers and Cooper, 2009b; Warnecke et al., 2010). This is potentially important for hibernating species because maintaining water balance is one hypothesis for the necessity of periodic arousal by hibernators (Fisher and Manery, 1967; Speakman and Racey, 1989; Thomas and Cloutier, 1992; Thomas and Geiser, 1997; Humphries et al., 2003).

We examine here the basic physiology of the monito del monte (‘monkey of the mountains’; Dromiciops gliroides Thomas 1894) with particular reference to its water balance and economy. The monito is a small, nocturnal, omnivorous marsupial that is found only in the temperate rainforests of Argentina and Chile (Greer, 1965; Marshall, 1978; Hershkovitz, 1999). Monitos shift diet seasonally, and have a remarkable acclimation of specific enzyme activities for processing fruits and insects (Cortés et al., 2011). They are social, with communal nesting and limited aggressive behaviour (Franco et al., 2011). Their dense, dark fur, reduced ear size, and distinct pouch with only four teats suggest adaptations to a cold and low-productivity environment (Mann, 1955). This enigmatic species, the only living representative of the marsupial order Microbiotheria (Reig, 1955; Mann, 1955; Hershkovitz, 1999), is thought to be more closely related to the Australian marsupials, particularly the Australian pygmy possums, the honey possum and the feathertail glider, than other South American marsupials (Cardillo et al., 2004; Bininda-Emonds et al., 2007; Beck, 2008; Beck et al., 2008; Nilsson et al., 2010).

Early studies indicated that the monito hibernates during winter (Mann, 1955; Greer, 1965; Grant and Temple-Smith, 1987), like pygmy possums and the feathertail glider (Jones and Geiser, 1992; Geiser, 1994; Geiser, 2004). Recent studies have shown that it also uses deep daily torpor, even during summer, as well as multi-day hibernation of up to 6 days duration at Ta of 12.5 and 20°C, with Tb closely approaching Ta (Bozinovic et al., 2004; Nespolo et al., 2010). Bozinovic et al. (Bozinovic et al., 2004) measured MR over a range of Ta and concluded that the monito has a low basal metabolic rate (BMR) and similar-to-expected thermal conductance (C). Cortés et al. (Cortés et al., 2009) described the energetics of the monito at Ta=20°C and Nespolo et al. (Nespolo et al., 2010) further described the bioenergetics of euthermy and daily torpor at Ta=10 and 20°C. Little is known about water balance for the monito. Cortés et al. (Cortés et al., 2009) measured excurrent humidity during respirometric measurement for the monito but did not calculate its EWLT for comparison with other species. We present here the first EWLT data for monitos, the first value for relative water economy of a marsupial hibernator, and non-invasively partition EWLT into cutaneous and respiratory components. We hypothesise that the monito will have a high EWLT and poor RWE, reflecting its mesic habitat and high water content diet, and that deep torpor will reduce both EWLT and RWE so as to compromise water balance and require periodic arousal from hibernation for rehydration.

MATERIALS AND METHODS

Study animals

Eight adult monitos, four male and four female, were captured near Valdivia, Chile (39°48′S, 73°14′W), using wire cage traps modified for placement in trees and shrubs. Traps were baited with banana (see Cortés et al., 2009) and were covered with plastic and lined with Dacron for insulation. Individuals were transported back to the laboratory at Universidad Austral de Chile on the day of capture, and maintained individually in plastic containers (45×30×20 cm) with wood shavings as bedding. They were fed a mixture of baby food (cereal and fruit consommé), honey, fruit and occasionally mealworms. Measurements were made from 30 January to 7 February 2010.

Animal capture, maintenance and experimentation were conducted under permit from Servicio Agrícola y Ganadero, and with approval of the Animal Ethics Committees of Universidad Austral Chile, University of Western Australia and Curtin University.

Respirometry measurements

We measured oxygen consumption (), carbon dioxide production () and EWLT for monitos by flow-through respirometry, at Ta ranging from 14 to 33°C (order of measurements: 30, 20, 14, 33 and 25°C). Seven monitos were measured at Ta=30°C and six were measured at all other Ta. Experiments were conducted for a period of 6–10 h during the monito's rest phase (daytime). Monitos were measured at only one Ta per day, and were allowed a rest period of at least 3 days between successive measurements. To facilitate this rest period, not all individuals were measured at each Ta. Ta was regulated to ±0.1°C using a temperature-controlled cabinet (Pi-Tec Technologies, Santiago, Chile). Body temperature was recorded via the cloaca at the end of experiments, and during torpor bouts (when animals had attained a constant, minimal torpid state), to ±0.1°C using an HH-25TC thermocouple meter (Omega Engineering, Stamford, CT, USA) with a fine plastic-tipped thermocouple wire.

The respirometry system consisted of a mass flow controller (GFC17, Aarlborg, Orangeburg, NY, USA, or CP7509, Cole-Palmer, Vernon Hills, IL, USA) that regulated the inlet flow rate of compressed ambient air (dried with Drierite; W. A. Hammond Drierite Co., Xenia, OH, USA) at approximately 500 ml min−1 at standard temperature and pressure (STPD). Metabolic chambers consisted of 188 cm3 glass tubes with rubber stoppers; inlet and outlet ports were at opposite ends of the chambers, as were the calibration and measurement ports for ventilatory measurements (see below). Excurrent air was passed over a thin-film capacitance relative humidity (RH) probe (MNP45A, Vaisala, Helsinki, Finland) and then dried using Drierite and passed through an oxygen analyser (Foxbox-C, Sable Systems, Las Vegas, NV, USA; or 570A, Servomex, Crowborough, East Sussex, UK) and a carbon dioxide analyser (Foxbox or CA10, Sable Systems). Ambient temperature was monitored during the experiment using the Vaisala probe. Urine and faeces were produced rarely, and were quickly dried by the relatively high air flow rate. Any brief periods of increased RH resulting from urine, faeces or activity could easily be identified on the continuous EWLT trace and were excluded from analyses.

The analog outputs of one Vaisala probe were interfaced to the Foxbox and the O2, CO2, RH and Ta data were logged by a PC via the Foxbox serial output, using a custom-written program (Visual Basic v6, Microsoft, Redmond, WA, USA). The analog outputs of the Servomex, CA-10 and Vaisala probe were interfaced to a PC via an A/D converter (ADC11, Pico Systems, St Neots, Cambs, UK) and logged every 20 s. , and EWLT were calculated for a period of approximately 20 min when physiological variables were constant and minimal (Withers, 2001) using custom-written software (Visual Basic v6). Respiratory exchange ratio (RER) and evaporative quotient (EQ) were calculated as / and EWLT/, respectively. MR was converted to MWP and metabolic heat production (MHP) using the measured RER for that experiment after Withers (Withers, 1992). EWLT was converted to evaporative heat loss (EHL) using 2.4 J mg−1 H2O (McNab, 2002). RWE was calculated as MWP/EWLT, and the point of relative water economy (PRWE) as the Ta at which RWE=1, from the regression of RWE against Ta.

Mass flow meters were calibrated with a Gilian Gillibrator 2 (traceable to a national standard; Clearwater, FL, USA) prior to the commencement of experiments, and this calibration was confirmed during the series of experiments by volumetric replacement of water. The oxygen analysers were two-point calibrated using compressed nitrogen (0% O2) and dry ambient air (20.95% O2). Calibration of the CO2 analysers was achieved using compressed nitrogen (O% CO2) and a certified gas mix (0.011% CO2 in nitrogen; Indura, Santiago, Chile). The calibration of the relative humidity probes was confirmed using 1% RH air (dried with Drierite to approximately 0.005 mg l−1) and 100% RH air (saturated by breathing on the probe).

Ventilatory measurements

Respiratory ventilation was monitored using whole-body plethysmography (Malan, 1973). A pressure transducer (MPX2010, Motorola, Denver, CO, USA) was used to monitor chamber pressure, and its analog output was interfaced to a PC using an ADC11 A/D converter and the values logged using PicoLogger software (Pico Systems). A volume of air (0.05 ml) was repeatedly (approximately five times for each experiment) injected into the system before removing the animal from the chamber at the end of each experiment. The washout characteristics of these calibration pulses were used to convert ventilatory pressure changes to pressures for a virtual closed system, by numerical integration (Szewczak and Powell, 2003). These ‘closed-system’ data were then analysed for respiratory frequency (; min−1) and tidal volume (VT; ml) after Malan (Malan, 1973) using custom-written software (Visual Basic v6). Respiratory minute volume (; ml min−1) was calculated as fR×VT and oxygen extraction efficiency (EO2) was calculated at the time of breathing measurements as EO2=100×(/{[(0.2095+FEO2)/2]×}), where FEO2 is the mean O2 fraction of excurrent air. Ventilatory variables are presented as body temperature and pressure saturated (BTPS), but EO2 was calculated from converted to STPD.

Partitioning of evaporative water loss

We partitioned EWLT using an iterative mathematical model that calculated respiratory and cutaneous evaporative water loss components at each Ta. EWLR (mg g−1 h−1) was calculated from , based on expired humidity (assumed to be 100%) and temperature (Texp; °C), and inspired (chamber) humidity (RHins) and temperature (Ta; °C): EWLR=(60/M)(/1000)[χTexp−(χTaRHins/100)], where M is mass and χ is the water vapour density for inspired air (χTa) and expired air (χTexp) at the appropriate temperature [χ was calculated after Parrish and Putnam (Parrish and Putnam, 1977)]. EWLC (mg g−1 h−1) was calculated from skin temperature (Tskin; °C), cutaneous evaporative resistance (Rskin; s cm−1) and body surface area [As; calculated as 10M0.67 cm2 (Dawson and Hulbert, 1970; Walsberg and King, 1978)]: EWLC=(3.6/R)(As/M)(χTskin−(χTaRHins/100))/Rskin, where χTskin is the water vapour density for air at skin temperature (100% RH) and χTa is the water vapour density for air at ambient temperature/humidity. The values of Tskin, Rskin and Texp that minimised the difference between calculated EWLT (EWLR+EWLC) and measured EWLT were determined by iteratively calculating and summing EWLR and EWLC, for various combinations of Tskin, Rskin and Texp. Texp was varied in steps of 0.01°C from 5°C less than Ta up to Tb (e.g. Jackson and Schmidt-Nielsen, 1964; Schmidt-Nielsen et al., 1970; Collins et al., 1971; Schmid, 1976; Hill, 1978), Tskin in steps of 0.01°C from Ta to Tb (e.g. Bartholomew, 1956; Edwards and Haines, 1978; Webster and Campbell, 1985; Dausmann, 2005) and Rskin in steps of 1 s cm−1 from 100 to 1000 s cm−1 (e.g. Webster and Campbell, 1985; Withers, 1992) (see Discussion). Estimation of EWLC is dependent on the interaction between Tskin and Rskin (i.e. a decrease in Tskin can counterbalance a decrease in Rskin at constant EWLC), so estimation of these parameters is expected to be variable but not to affect the partitioning of EWLT into EWLR and EWLC. The sensitivity of the mathematical partitioning model to potential errors in the iteratively determined Tskin, Texp and Rskin was examined at each Ta by fixing values for two of these variables at the calculated value and determining the effect of varying the third variable on the partitioning calculation.

Statistics

Statistics were calculated in Excel (Microsoft) using v1.8 of statistiXL (www.statistixl.com) and spreadsheet formulae (Zar, 1999). Effects of Ta on physiological variables were investigated for normothermic monitos using least squares regression and one-way ANOVA with a priori contrasts (Cohen, 2008; Withers and Cooper, 2011). Two-way ANOVA was used to compare values for torpid and normothermic monitos. Percentage data were arcsin transformed prior to analysis. All values are presented as means ± s.e.m. Sample size (N) is the number of individuals, and is provided where it differs from that stated previously (e.g. not all individuals entered torpor, so N is less than the stated sample size for some variables); n is the total number of measurements.

RESULTS

Body mass of monitos increased over the study period, resulting in significant differences in mass as successive Ta were measured (F4,26=11.6, P<0.001). Body mass ranged from 24.9±1.00 g at Ta=30°C to 36.4±2.37 g at Ta=20°C.

Monitos remained normothermic at Ta≥30°C, but entered torpor [defined as a of <75% of BMR (Geiser and Baudinette, 1988)] at other Ta (Fig. 1). Body temperature of normothermic monitos was significantly and linearly related to Ta (linear polynomial contrast F1,26=41.9, P<0.001), ranging from 30.1±1.44°C at Ta=14°C to 37.2±0.35°C at Ta=33°C). Thermoneutral Tb (at Ta=30°C; see below) was 34.6±0.34°C. Body temperature of torpid monitos decreased with Ta (F2,24=38.9, P<0.001) and was significantly lower compared with euthermic monitos (F1,24=139, P<0.001) at Ta=14°C (15.2±0.31°C, N=5), 20°C (23.6±0.38°C, N=5) and 25°C (29.0±2.20°C, N=2).

Fig. 1.

Body temperature (Tb), metabolic water production, total evaporative water loss, and wet (Cwet) and dry (Cdry) thermal conductance of monitos. Values are means ± s.e.m. Ta, ambient temperature.

Fig. 1.

Body temperature (Tb), metabolic water production, total evaporative water loss, and wet (Cwet) and dry (Cdry) thermal conductance of monitos. Values are means ± s.e.m. Ta, ambient temperature.

Metabolic water production of normothermic monitos closely mirrored both and , as there was no effect of Ta on RER (F1,26=0.10, P=0.749), which was 0.91±0.014 (N=8, n=31) over all Ta. The lowest MR of 1.42±0.052 ml O2 g−1 h−1 at Ta=30°C, which we consider to be BMR, corresponds to a MWP of 0.89±0.04 mg H2O g−1 h−1. MWP was strongly influenced by Ta (F4,26=61.9, P<0.001), increasing to 2.2±0.12 mg H2O g−1 h−1 at Ta=14°C (Fig. 1). There was a significant linear relationship for MWP at Ta=14 to 30°C (F1,21=8.8, P<0.001), and MWP increased to 1.05±0.06 mg H2O g−1 h−1 at Ta=33°C (significant linear and quadratic contrasts for Ta=14 to 33°C; F1,26≥24.5, P<0.001). MWP of torpid monitos decreased with Ta (F2,18=9.99, P<0.001), and was significantly lower when torpid than normothermic at the same Ta (F1,28=531, P<0.001). There was a significant interaction (F2,28=69.1, P<0.001) between torpor and Ta effects on MWP, with MWP decreasing more below normothermia at lower Ta (Fig. 1).

Standard EWLT (at thermoneutrality, 30°C) was 2.6±0.21 mg H2O g−1 h−1. There was an overall effect of Ta on EWLT (F4,26=10.8, P<0.001; Fig. 1). EWLT did not change between Ta of 14 and 20°C (no significant reverse Helmert, linear or quadratic contrasts; F1,15≤0.871, P≥0.739) but increased at Ta=30°C (F1,26=5.25, P=0.030) and 33°C (F1,26=38.5, P<0.001) and linearly from 25 to 33°C (F1,16=259, P<0.001). EWLT of torpid monitos decreased with Ta (F2,28=4.94, P=0.015) and was significantly lower compared with normothermic monitos at the same Ta (F1,28=85.0, P<0.001). The significant interaction between torpor and Ta effects on EWLT (F2,28=5.72, P=0.008) indicated that EWLT during torpor decreased more below normothermia at lower Ta, with absolute water savings resulting from torpor of 0.68 mg H2O g−1 h−1 at 25°C to 1.70 mg H2O g−1 h−1 at 14°C.

Wet and dry thermal conductances of monitos were influenced by Ta (Cwet, F4,26=15.1, P<0.001; Cdry, F4,26=10.3, P<0.001; Fig. 1). They were constant at Ta from 14 to 25°C (Cwet, F1,15≤0.503, P≥0.489; Cdry, F1,15≤1.98, P≥0.180), but there were significant linear and quadratic components for Ta=14 to 35°C (Cwet, F1,26≥21.2, P<0.001; Cdry, F1,26≥18.1, P<0.001), reflecting the curvilinear increase in Cwet and Cdry at the higher Ta.

In thermoneutrality, was 52.0±6.95 min−1, VT was 0.50±0.088 ml, and χ was 24.8±8.77 ml min−1. There were significant Ta effects for all ventilatory variables (F4,26≥0.367, P≤0.017; Fig. 2). χ decreased markedly with increasing Ta, as demonstrated by a significant linear contrast (F1,26=11.6, P=0.002), with no quadratic effect (P=0.071). VT did not differ between Ta=14 and 25°C, but decreased at 30°C (reverse Helmert contrast, P>0.546 and 0.003, respectively); VT did not differ between Ta=30 and 33°C (Helmert contrast, P=0.741). χ decreased linearly with increasing Ta (F1,26=30.0, P<0.001), with no significant quadratic effect (P=0.497). EO2 increased with Ta (F1,26=7.21, P=0.012), from 12.8% at 14°C to 16.4% at 33°C.

EWLR from the iterative model (Fig. 3) ranged from 1.72±0.31 mg g−1 h−1 at Ta=14°C to 1.63±0.09 mg g−1 h−1 at Ta=30°C and 2.13±0.25 mg g−1 h−1 at Ta=33°C, but there was no significant change between Ta=14 and 33°C (polynomial and reverse Helmert contrasts, F4,26=0.762, P=0.599); the increase in EWTR at Ta=33°C was not significant (P=0.101). EWLC increased slightly but significantly from 0.43±0.01 mg g−1 h−1 at Ta=14°C to 0.47±0.01 mg g−1 h−1 at Ta=20°C (linear contrast, F1,15=6.28, P=0.024), and increased further to 0.95±0.01 mg g−1 h−1 at Ta=30°C (reverse Helmert contrast, F1,26=1288, P<0.001) and 1.41±0.06 mg g−1 h−1 at 33°C (reverse Helmert contrast, F1,26=1288, P<0.001), with linear (F1,26=3179, P<0.001) and quadratic (F1,26=1167, P<0.001) components from Ta=14 to 33°C. EWLR was 77±5% of calculated EWLT at Ta=14°C, and decreased to 63% at Ta=30°C and 59±2% at 33°C, with significant linear (F1,26=30.3, P<0.001) and quadratic (F1,26=9.84, P=0.004) components. Correspondingly, EWLC was approximately 23% of calculated EWLT at Ta=14°C, and increased to 41% at Ta=33°C. Texp increased from 13.3°C at Ta=14°C to 16.4, 24.5, 29.5 and 34.2°C at successively higher Ta values. Tskin increased from 22.8°C at Ta=10°C to 27.6, 30.1, 30.8 and then 36.0°C at successively higher Ta values. Rskin was high at the low Ta values (506, 914 and 659 s cm−1 at Ta=14, 20 and 25°C, respectively), and declined to 381 and 311 s cm−1 at Ta=30 and 33°C, respectively.

Fig. 2.

Respiratory frequency, tidal volume, minute volume and oxygen extraction for normothermic monitos. Values are means ± s.e.m.

Fig. 2.

Respiratory frequency, tidal volume, minute volume and oxygen extraction for normothermic monitos. Values are means ± s.e.m.

The sensitivity analysis of our EWL partitioning model indicated that the % EWLR increased with Texp. Increasing Texp from Ta−5°C to Tb resulted in an EWLR ranging from 53−97% at Ta=14°C to 42−71% at Ta=33°C. The maximum error in our estimated % EWLR resulting from under/overestimation of Texp would range from approximately −24 to +21%. For example, at a Ta of 14°C, a Texp of 9°C rather than the calculated 13.3°C would decrease EWLR from 77 to 53% of EWLT, and a Texp of 30.1°C (Tb) would increase EWLR to 97% of EWLT. At a Ta of 33°C, a Texp of 28°C rather than the calculated 34.2°C would decrease EWLR from 60 to 42% of EWLT, and a Texp of 37.2°C (Tb) would increase EWLR to 71% of EWLT. The % EWLR decreased with higher Tskin. Increasing Tskin from Ta to Tb resulted in a % EWLR ranging from 87–66% at Ta=14°C to 69–55% at 33°C. The maximum error in our estimated % EWLR would range from approximately −12 to +11%. For example, at a Ta of 14°C, a Tskin of 14°C rather than the calculated 22.8°C would increase EWLR to 87% from 77% of EWLT, and a Tskin of 30.1°C (Tb) would decrease EWLR to 66% of EWLT. At a Ta of 33°C, a Tskin=Ta rather than the calculated 36.0°C would increase EWLR to 69% from 60% of EWLT, and a Tskin=37.2°C (Tb) would decrease EWLR to 55% of EWLT. The % EWLR increased with higher Rskin. Increasing Rskin from 100 to 1000 s cm−1 resulted in an EWLR ranging from 48–82% at Ta=14°C to 28–84% at 33°C. The maximum error in our estimated % EWLR would range from approximately −42 to +24%. For example, at a Ta of 14°C, an Rskin of 100 s cm−1 rather than the calculated 506 s cm−1 would decrease EWLR from 77 to 48% of EWLT, and an Rskin of 1000 s cm−1 would increase EWLR to 82% of EWLT. At a Ta of 33°C, an Rskin of 100 s cm−1 rather than the calculated 311 s cm−1 would decrease EWLR from 60% to 28% of EWLT, and an Rskin of 1000 s cm−1 would increase EWLR to 84% of EWLT..

Fig. 3.

Partitioning of total evaporative water loss into respiratory and skin components for normothermic monitos. Values are means + s.e.m.

Fig. 3.

Partitioning of total evaporative water loss into respiratory and skin components for normothermic monitos. Values are means + s.e.m.

There was an effect of Ta on EQ, which increased from 0.61±0.06 at Ta=14°C to 2.14±0.11 at Ta=33°C (Fig. 4). The pattern of Ta response for RWE was described by significant linear (F1,26=142, P<0.001) and quadratic (F1,26=6.57, P=0.0165) contrasts over the Ta range, but from 14 to 30°C was only linear (F1,21=91.2, P<0.001). The PRWE was calculated to be 15.4°C, based on Ta values from 14 to 30°C: RWE=1.70±0.107–0.046±0.005Ta (r2=0.811, F1,23=98.4, P<0.001). RWE of torpid monitos was lower than for normothermic individuals (F2,28=4.67, P=0.018) and was significantly affected by Ta (F1,28=87.7, P<0.001), with a significant interaction (F2,28=18.6, P<0.001) indicating a greater difference between torpid and normothermic RWE at lower Ta. Torpid monitos did not reach a PRWE at low Ta.

DISCUSSION

Thermal, metabolic and hygric responses of monitos to variation in Ta were typical of endotherms in general and other marsupials specifically. The increase in MR with decreasing Ta for thermoregulation directly increased MWP (as RER remained constant). In contrast, EWLT remained constant below thermoneutrality, as has been demonstrated for many other marsupials (e.g. Hinds and MacMillen, 1986; Cooper and Withers, 2002; Larcombe, 2006; Withers and Cooper, 2009a; Withers and Cooper, 2009b). Monitos were thermolabile during normothermia over the 14–33°C Ta range investigated (Fig. 1), with a ΔTbTa of 0.32±0.050. The small standard errors for Tb suggest that this thermolability did not reflect an inability to thermoregulate precisely. Rather, thermolability seems to be a strategy to conserve energy at low Ta. For example, MR would have been approximately 4.51 ml O2 g−1 h−1 at Ta=14°C if Tb had been maintained at thermoneutral Tb=34.6°C (calculated assuming a constant Cwet) rather than the actual 3.53 ml O2 g−1 h−1; this is an energy saving of 22%. Thermolability also reduces both MWP and EWL, and lowers RWE, although the effect is small because the small change in Tb has nearly equivalent effects on MWP and EWLT. For example, the RWE is estimated to be 1.11 at Ta=14°C if Tb was maintained at 34.6°C, whereas RWE is 1.07 at the actual Tb of 30.1°C. The increase in EWLT at Ta within and above the thermoneutral zone suggests that monitos become heat stressed at relatively low Ta, reflecting their cool, mesic habitat and the low fitness advantage of water conservation for this species. EWLT had increased by 22% in thermoneutrality compared with Ta=25°C, and increased further at 33°C by 68%. However, this, even combined with an increased Cdry, was insufficient to prevent a hyperthermia of 3.4°C at Ta=33°C (compared with Tb at thermoneutrality).

Fig. 4.

Evaporative quotient (EWLT/MR) and relative water economy (MWP/EWLT) of normothermic and torpid monitos. Asterisk indicates the point of relative water economy (MWP=EWLT). Values are means ± s.e.m.

Fig. 4.

Evaporative quotient (EWLT/MR) and relative water economy (MWP/EWLT) of normothermic and torpid monitos. Asterisk indicates the point of relative water economy (MWP=EWLT). Values are means ± s.e.m.

Standard EWLT (at 30°C) of monitos conformed to that for other marsupials, being 109% of that predicted for an equivalently sized marsupial [using the minimum variance unbiased estimate (Hayes and Shonkwiler, 2006; Hayes and Shonkwiler, 2007); data set from Warnecke et al. (Warnecke et al., 2010)]. The monitos' EWLT fell well inside the 95% prediction limits for the log-transformed allometric regression for marsupial EWLT, both before and after accounting for phylogenetic history via autoregression (Cheverud and Dow, 1985; Rohlf, 2001) using the mammal ‘supertree’ (Bininda-Emonds et al., 2007). Two other South American marsupials, the gracile mouse opossum [Gracilinanus agilis (Cooper et al., 2009)] and the woolly mouse opossum [Micoureus paraguayanus (Cooper et al., 2010)], also conform closely to the EWLT allometric relationship for Australian marsupials, as do the monito's presumed Australian relatives, the honey possum [Tarsipes rostratus (Cooper and Cruz Neto, 2009)] and the pygmy possum [Cercartetus nanus (Bartholomew and Hudson, 1962)], reflecting the conservative physiology of marsupials.

There was no effect of Ta on EWLT from 14 to 25°C, but EWLT increased at Ta=30 and 33°C. Little is currently known about the relative contribution of EWLR and EWLC components for mammals, particularly below thermoneutrality. Physical partitioning methodologies potentially limit the animal's natural posture and prevent postural changes as a response to varying Ta, they don't completely account for non-respiratory components in estimates of EWLR (e.g. skin or ocular EWL may be included) and they presumably result in some stress to the animal that might modify both MR and EWLT (e.g. Tracy and Walsberg, 2000). Indeed, simply handling an animal to place it in a metabolic chamber has a significant, measurable effect on physiological variables, in particular EWLT, for a considerable period (Hayes et al., 1992; Cooper and Withers, 2009; Page et al., 2011). Invasive partitioning techniques are therefore likely to elevate both EWLR and EWLC, and possibly influence the partitioning of EWLT.

To overcome these issues, we used a non-invasive mathematical modelling approach to partition the EWLT of monitos into EWLR and EWLC, and explain the pattern of EWLT below thermoneutrality. EWLR has previously been calculated for unrestrained animals from respiratory ventilation using measured or assumed expired air temperature (e.g. Chew and White, 1960; Withers et al., 1979; Withers and Williams, 1990; Thomas and Cloutier, 1992; Thomas and Geiser, 1997; Withers and Cooper, 2011); EWLC can then be calculated by difference. The iterative model that we used to solve for the best estimates of EWLR and EWLC, as we did not measure Texp for monitos, found EWLR to be the major component of EWLT at all Ta. Our estimate of 62% EWLR at a thermoneutral Ta=30°C is lower than that reported for deer mice [Peromyscus maniculatus; 88% (Chew, 1955)], similar to that reported for the numbat [Myrmecobius fasciatus; 65% at 25°C (Cooper, 2003)] and house mice and deer mice [Mus musculus and Peromyscus maniculatus; 59–60% (Edwards and Haines, 1978) and 54% (Chew and Dammann, 1961)], and higher than that for Merriam's kangaroo rat [Dipodomys merriami; 38–44% (Tracy and Walsberg, 2000)], the hopping mouse [Notomys alexis; 40% (Withers et al., 1979)] and the pallid bat [Antrozous pallidus; 22% at 26°C (Chew and White, 1960)]. These species differences could reflect differing experimental methodologies (invasive and potentially disturbed/stressed versus non-invasive and basal, and iterative modelling to partition EWLT), or species differences due to particular morphological or environmental selective pressures on EWLR and EWLC, hence the partitioning of EWLT. Both the kangaroo rat and hopping mouse inhabit arid environments, and a low EWLR by enhanced counter-current heat and water exchange might explain their high % EWLC. The large, naked surface area of the wing membranes of bats presumably explains their relatively low EWLR and high EWLC. Further measurements of EWLT partitioning for other species, and a critical evaluation of differing methodologies, seems a fruitful direction for future research.

In the present study, the EWLR of the monito increased slightly (but not significantly; P=0.101) at Ta=33°C, whereas EWLC increased substantially at Ta=30 and 33°C (Fig. 3). Non-respiratory EWL thus appears to be the primary avenue for the monito to enhance thermoregulatory heat loss at high Ta, although it is still only 37–41% of EWLT at Ta=30 and 33°C. EWLT and EWLR are constant at low Ta, with EWLR ranging from 76 to 83% of EWLT. EWLC increases slightly from Ta=14 to 25°C, but is only 17 to 24% of EWTL. Thus, partitioning of EWLT into EWLR and EWLC suggests that the relatively constant EWLT from Ta=14 to 25°C reflects a fairly constant EWLR and EWLC. The constancy of EWLR is despite the increase in at lower Ta (Fig. 2), reflecting the decrease in Texp at lower Ta. The near constancy of EWLC is expected, based on the relatively small change in saturation water vapour pressure between 14 and 25°C.

Our modelling of EWLR and EWLC suggests that Texp was generally similar to Ta, although it was calculated to be 3.6°C less than Ta at 20°C. Texp less than or equal to Ta has been reported for some other small mammals, e.g. kangaroo rats [Dipodomys merriami (Jackson and Schmidt-Nielsen, 1964; Schmidt-Nielsen et al., 1970; Collins et al., 1971)], house and deer mice [Mus musculus, Peromyscus maniculatus (Edwards and Haines, 1978)] and hopping mice [Notomys alexis (Withers et al., 1979)]. A low Texp would reduce the EWLT at low Ta, explaining the relatively constant respiratory component to EWLT (Fig. 3) despite the increase in at low Ta (Fig. 2). Tskin was considerably higher than Ta and lower than Tb at low Ta, and approached Tb at higher Ta, a pattern consistent with measurements for other furred species (e.g. Bartholomew, 1956; Edwards and Haines, 1978). Our calculated values for Rskin ranged from over 500 s cm−1 at Ta<30°C to 300–400 s cm−1 at 30 and 33°C. Estimates of Rskin from other studies at Ta=25–30°C range from approximately 100 to 300 s cm−1 (Chew, 1955; Chew and Dammann, 1961; Edwards and Haines, 1978; Withers et al., 1979; Tracy and Walsberg, 2000). However, simultaneous estimation of Tskin and Rskin in our iterative model is problematic because of their interacting effect on EWLC, so our slightly high estimates of Rskin might reflect an overestimation of Tskin.

The RWE of normothermic monitos increased at lower Ta, reflecting the pattern of increased MWP but constant EWLT. This pattern of RWE increasing at lower Ta is consistent with that observed for other marsupials (e.g. Cooper and Cruz-Neto, 2009; Cooper et al., 2009; Cooper et al., 2010; Warnecke et al., 2010; Withers and Cooper, 2009a; Withers and Cooper, 2009b) and placentals (e.g. MacMillen and Hinds, 1983; Hinds and MacMillen, 1986), resulting in a PRWE where MWP=EWLT. The PRWE of 15.4°C for monitos conforms closely to the PRWE of other marsupials [summary data (Cooper and Withers, 2010), with additional data (Warnecke et al., 2010; Schmidt, 2011)], reflecting its mesic environment and thus little requirement for water conservation during normothermia. The PRWE for the related honey possum [17°C (Cooper and Cruz-Neto, 2009)] was higher than that for the monito, reflecting its smaller (5.4 g) body mass, but the monito had a higher PRWE than the similarly sized (29 g) South American gracile mouse opossum [12°C (Cooper et al., 2009)] and the larger (69 g) woolly mouse opossum [11.3°C (Cooper et al., 2010)] despite its very mesic environment. Further data on the PRWE of South American didelphid marsupials are required to interpret patterns in PRWE, but it does appear that they have less favourable PRWEs than marsupials with an Australian derivation, regardless of current distribution and habitat.

During torpor, EWLT of the monito was reduced to as little as 21% of normothermic EWLT, which is typical of other mammalian hibernators. Torpid gerbils (Gerbillus pusillus) reduce EWLT to 27% of normothermic values (Buffenstein, 1985) and a variety of bat species reduce EWLT during torpor to 14–50% of the normothermic value (Carpenter, 1969; Morris et al., 1994; Hosken and Withers, 1997; Hosken and Withers, 1999). These absolute water savings are generally higher than those measured previously for marsupials that enter torpor daily but do not hibernate [maximum reductions of 32 to 69% of normothermic values (Cooper et al., 2005; Cooper et al., 2009; Withers and Cooper, 2009a; Withers and Cooper, 2009b; Warnecke et al., 2010)], presumably because the lower Tb of hibernators compared with torpidators (e.g. Geiser and Ruf, 1995) increases their absolute hygric and energetic savings.

Torpor has a more profound effect on MWP than EWLT, with the decrease in MR associated with torpor reducing MWP to as little as 6% of normothermic values. The consequence of this greater reduction in MWP than EWLT during torpor is a much less favourable RWE. This pattern for monitos is consistent with other marsupials and also placental mammals (e.g. MacMillen, 1965; Buffenstein, 1985; Cooper et al., 2005; Withers and Cooper, 2009a; Withers and Cooper, 2009b; Warnecke et al., 2010), where torpor provides an absolute water savings but reduces RWE. As MWP and EWLT are the primary avenues of water gain and loss during torpor, the unfavourable RWE during long periods of torpor will presumably result in a long-term negative water balance and could limit torpor duration for hibernators. For example, at Ta=15°C, the RWE of torpid monitos is approximately 0.28, which corresponds to an MWP of 96 mg day−1 (for a 30 g individual with a torpid MR of 0.21 ml O2 g−1 h−1) and an EWLT of approximately 0.444 mg g−1 h−1, or 320 mg day−1. The differential of −224 mg day−1 is equivalent to a 5% mass loss per week, which is not insignificant [torpid Myotis lucifugus lose approximately 4.3% of lean body mass before arousing (Kallen, 1964)]. Torpor bout duration increases for monitos with decreasing Ta to 5 days at Ta=12.5°C in winter (Bozinovic et al., 2004), which is consistent with our prediction of a torpor duration of approximately 1 week based on a critical EWLT of 5% body mass. Thus, our findings for the monito support the idea that the periodic arousals characteristic of hibernators may be at least in part a consequence of water imbalance and the need to drink (Fisher and Manery, 1967; Speakman and Racey, 1989; Thomas and Cloutier, 1992; Thomas and Geiser, 1997).

Acknowledgements

We thank Pablo Cortés and Marcela Franco for their enthusiastic help throughout the study, and the anonymous referees for their useful comments.

FOOTNOTES

FUNDING

This study was funded by an Australian Research Council Discovery grant [DP0665044 to C.E.C. and P.C.W.], and Fondo Nacional de Desarrollo Científico y Technológico grant 3100144 [to R.F.N.].

LIST OF SYMBOLS AND ABBREVIATIONS

     
  • As

    body surface area (cm2)

  •  
  • BMR

    basal metabolic rate (ml O2 g−1 h−1)

  •  
  • BTPS

    body temperature and pressure, saturated

  •  
  • C

    thermal conductance (J g−1 h−1 °C−1)

  •  
  • Cdry

    dry thermal conductance (J g−1 h−1 °C−1)

  •  
  • Cwet

    wet thermal conductance (J g−1 h−1 °C−1)

  •  
  • EO2

    oxygen extraction efficiency (%)

  •  
  • EHL

    evaporative heat loss (J g−1 h−1)

  •  
  • EQ

    evaporative quotient (mg H2O ml−1 O2)

  •  
  • EWLC

    cutaneous evaporative water loss (mg g−1 h−1)

  •  
  • EWLR

    respiratory evaporative water loss (mg g−1 h−1)

  •  
  • EWLT

    total evaporative water loss (mg g−1 h−1)

  •  
  • FEO2

    fractional excurrent oxygen concentration

  •  
  • respiratory rate (breaths min−1)

  •  
  • M

    body mass

  •  
  • MHP

    metabolic heat production (J g−1 h−1)

  •  
  • MR

    metabolic rate (ml O2 g−1 h−1)

  •  
  • MWP

    metabolic water production (mg H2O g−1 h−1)

  •  
  • N

    sample size for individuals

  •  
  • n

    total sample size for measurements

  •  
  • PRWE

    point of relative water economy (°C)

  •  
  • Rskin

    evaporative resistance of the skin (s cm−1)

  •  
  • RER

    respiratory exchange ratio (ml CO2 ml−1 O2)

  •  
  • RH

    relative humidity (%)

  •  
  • RHins

    inspired relative humidity (%)

  •  
  • RWE

    relative water economy (dimensionless)

  •  
  • STPD

    standard temperature and pressure, dry

  •  
  • Ta

    ambient temperature (°C)

  •  
  • Tb

    body temperature (°C)

  •  
  • Texp

    expired air temperature (°C)

  •  
  • Tskin

    skin temperature (°C)

  •  
  • respiratory minute volume (ml min−1)

  •  
  • VT

    tidal volume (ml)

  •  
  • carbon dioxide production rate (ml CO2 g−1 h−1)

  •  
  • oxygen consumption rate (ml O2 g−1 h−1)

  •  
  • χ

    absolute water vapour density (mg cm−3)

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