Heat dissipation in subterranean rodents: the role of body region and social organisation

Tested animals

Altogether 73 individuals from seven species of subterranean rodents differing in body mass, phylogenetic relatedness, and sociality were studied (Table 1). All animals were adult non-breeders, or their breeding history was unknown in solitary species, but none of them showed signs of recent breeding, which may theoretically influence measured parameters. For the purpose of this study, we used the following taxa. African mole-rats (Bathyergidae): the social Ansell’s mole-rat Fukomys anselli (Burda, Zima, Scharff, Macholán & Kawalika 1999) occupies the miombo in a small area near Zambia’s capital Lusaka; another social species of the genus Fukomys is named here as Fukomys “Nsanje” because founders of the breeding colony were captured near town Nsanje in south Malawi. Although we used name Fukomys darlingi (Thomas 1895) for mole-rats from this population in previous studies (e.g.^38,49), its taxonomic status is still not resolved; the social common mole-rat Cryptomys hottentotus hottentotus (Lesson, 1826) occurs in mesic and semi-arid regions of southern Africa; the solitary Cape dune mole-rat Bathyergus suillus (Schreber, 1782) inhabits sandy soils along the south-western coast of South Africa; and the solitary Cape mole-rat Georychus capensis (Pallas, 1778) occupies mesic areas of the South Africa⁵⁰. In addition, we studied the social coruro Spalacopus cyanus (Molina, 1782) (Octodontidae) occupying various habitats in Chile⁵¹; and the solitary Upper Galilee Mountains blind mole rat Nannospalax galili (Nevo, Ivanitskaya & Beiles 2001) (Spalacidae) from Israel⁵². Further information about the species including number of individuals used in the study, their physiology and ecology is shown in Table 1.

All experiments were done on captive animals. Georychus capensis, C. hottentotus, and B. suillus, were captured about four months before the experiment, and kept in the animal facility at the University of Pretoria, South Africa (temperature: 23 °C; humidity: 40–60%, photoperiod: 12L:12D). The animals were housed in plastic boxes with wood shavings used as a bedding. Cryptomys hottentotus and G. capensis were fed with sweet potatoes; B. suillus with sweet potatoes, carrots, and fresh grass. Fukomys anselli, F. “Nsanje”, N. galili, and S. cyanus were kept for at least three years in captivity (or born in captivity) before the experiment in the animal facility at the University of South Bohemia in České Budějovice, Czech Republic (temperature: African mole-rats 25 °C, N. galili and S. cyanus 23 °C; humidity: 40–50%, photoperiod: 12L:12D). The animals were kept in terraria with peat as a substrate and fed with carrots, potatoes, sweet potatoes, beetroot, apple, and rodent dry food mix ad libitum.

Experimental design

We measured T_b and T_s in all species at six T_as (10, 15, 20, 25, 30 and 35 °C). Each individual of all species was measured only once in each T_a. Measurements were conducted in temperature controlled experimental rooms in České Budějovice and Pretoria. Each animal was tested on two experimental days.

The animals were placed in the experimental room individually in plastic buckets with wood shavings as bedding. On the first day, the experimental procedure started at T_a 25 °C. They spent 60 min of initial habituation in the first T_a after which T_b and T_s were measured as described in the following paragraphs. The T_a was then increased to 30 °C and 35 °C, respectively. After the experimental room reached the focal T_a, the animals were left minimally 30 min in each T_a to acclimate, and the measurements were repeated. Considering their relatively small body size, tested animals were very likely in thermal equilibrium after this period because mammals of a comparable body mass are thermally equilibrated after similar period of acclimation^53,54,55,56. On the second day, the procedure was repeated with the initial T_a 20 °C and decreasing to 15 °C and 10 °C, respectively. The time span between the measurements of the same individual in different T_a was at least 150 min. Between experimental days, the animals were kept at 25 °C in the experimental room (individuals of social species were housed together with their family members).

Body temperature measurements

We used two sets of equipment to measure animal T_b and T_s. In B. suillus, G. capensis, and C. hottentotus, T_b was measured by intraperitoneally injected PIT tags (< 1 g, LifeChip with Bio-Thermo Technology; Destron Fearing Corp., Dallas, Texas, USA, accuracy 0.5 °C, resolution 0.1 °C). A vet injected the tags under anaesthesia (Isoflurane) three months before the experiment. The tags were calibrated by the manufacturer and were read using a Global Pocker Reader EX (Destron Fearing Corp., Dallas, Texas, USA). T_s was measured by FLIR SC325 thermal camera (FLIR Systems, Inc., Wilsonwille, Oregon, USA; sensitivity < 50 mK, accuracy ± 2%, frame rate 31 fps, calibrated by the manufacturer).

Core body temperatures of F. “Nsanje”, F. anselli, N. galili, and S. cyanus were measured using a RET-2 temperature probe (Physitemp Instruments LLC., Clifton, New Jersey, USA; tip diameter 3.2 mm, inserted at least 2 cm in the rectum, accuracy 0.1 °C) connected to Thermalert TH-8 (Physitemp Instruments LLC., Clifton, New Jersey, USA; resolution 0.1 °C). This procedure took less than 30 s. The apparatus was verified against a thermometer (EL-USB-2-LCD+; Lascar Electronics Ltd., Salisbury, UK; overall accuracy 0.45 °C) calibrated by an accredited laboratory. Both means of measuring T_b have been shown to provide almost identical results in small mammals including one species belonging to African mole-rats^57,58,59. Surface temperature was measured using a Workswell WIRIS thermal imaging system (ver. 1.5.0, Workswell s.r.o., Praha, Czech Republic, sensitivity 30 mK, accuracy ± 2%, frame rate 9 fps, calibrated by the manufacturer). Both thermal cameras were calibrated prior to measurement of each animal, and we used the same software to process the raw thermograms (see below).

To measure T_s, a focused radiometric video of the animal was taken with its different body parts exposed perpendicularly to the camera. More specifically, the animals were held hanging by the loose skin around the tail, and their dorsal and ventral body regions were sequentially exposed to the camera. This procedure took less than 30 s. To ensure unbiased T_s measurements, all fans in the experimental room were switched off during measurements, and a non-uniformity correction (type of calibration of the camera) was performed just before measuring each animal. Since the seven tested species were of different sizes, and the lenses of the two thermal cameras had different focal lengths, and thus field of views, the animals were filmed at different distances from the camera to fully utilise the resolution of the two cameras. Distances of animals to the camera lens were 38–42 cm for N. galili, F. anselli, F. “Nsanje”, and S. cyanus, 53–57 cm for C. hottentotus, 64–68 cm for G. capensis, and 86–92 cm for B. suillus. Room air temperature and humidity were monitored throughout the trials (EL-USB-2-LCD+; Lascar Electronics Ltd., Salisbury, UK; calibrated by a certified laboratory). Humidity in both labs ranged from 47 to 72% during all experiments.

To assess the heat dissipation (which is related mainly to insulative properties of the integument) of each species regardless of their actual value of T_b, we introduced T_diff parameter defined as the difference between T_b and T_s of a particular body region of each individual (the dorsum, the venter, and the feet) at each T_a.

Handling stress may influence T_b and may also evoke vasoconstriction on the periphery and thus potentially affect T_s⁵⁸. To test the potential influence of handling procedures employed in our study on T_s and T_b, we carried out several simple experiments. Firstly, we measured T_b of three individuals of F. mechowii (species not included in the present study, but in³¹) by intraperitoneal probes (G2-HR E-Mitter, Starr Life Sciences Corp., Oakmont, Pennsylvania, USA; the only species with these probes in our breeds), and found no significant differences in T_b prior to or after the 30 s handling period (T_b was 34.0 ± 0.6 °C and 34.1 ± 0.7 °C before and after handling, respectively; paired t-test: t = −0.42, df = 3, p = 0.70). Secondly, to exclude the possibility of a long-term effect of repeated handling on T_b, we simulated the measurement procedure with another three individuals of the same species with intraperitoneally injected PIT tags obtaining T_bs without direct contact. We placed mole-rats singly in a bucket and after 150 min we measured their T_b. Subsequently, we lifted them for a period of one min and returned them into the bucket. After 150 min, we again measured their T_bs. Lifting of the mole-rats and temperature measurement was repeated once again (Note that 150 min is the minimal period between two manipulations of each individual in our experiment). The T_bs did not change during these three subsequent measurements (Generalized Least Squares model [GLS]: F = 0.7, p = 0.544; T_b values were 33.14 ± 0.6, 33.13 ± 0.77 and 33.23 ± 0.92 °C). Thirdly, to rule out a possible effect of handling on T_s, we measured dorsal T_s in ten individuals of F. “Nsanje” (species included in the present study) before and after the handling period which took usually less than 30 s. Similarly, we did not find significant differences (T_s was 30.5 ± 1.1 °C and 30.3 ± 1.1 °C before and after the 30 s handling, respectively; paired t-test: t = 1.78, df = 9, p = 0.11). It should be noted that all animals are accustomed to this handling, as they undertake it on a weekly basis during routine activities, such as weighing, cleaning of terraria, and miscellaneous behavioural and physiological experiments. We therefore suggest that the different approaches in obtaining T_b, and different time in captivity for experimental animals did not affect our results substantially.

Thermogram processing and analysis

The thermographic camera produces (sequences of) images in the infrared range, so-called thermograms. Sharp and high contrast radiometric thermograms of the dorsum, the venter and one hind foot as a representative of the feet, were captured from the raw radiometric video in the software CorePlayer (ver. 1.7.70.320; Workswell s.r.o., Praha, Czech Republic; https://workswell-thermal-camera.com/workswellcoreplayer), and processed into non-radiometric thermograms by specifying temperature calculation parameters in the software ThermoFormat (Workswell s.r.o., Praha, Czech Republic; https://workswell-thermal-camera.com/workswell-thermoformat). These parameters were entered as follows: fur emittance was set to 0.97 (e.g.⁶⁰); air humidity, air temperature, and reflected temperature were entered as means of experimental room humidity and air temperature during the course of T_s measurement of each species at given T_a; distance was set as the distance of camera lens from the animal. Although Šumbera et al.³¹ identified a few other body surface areas through which body heat is mainly passively dissipated in mole-rats (peripalpebral, nose, and ear area), we did not include them in our study due to their relatively small area, and thus very low contribution to overall heat dissipation.

Processed thermograms were used to infer mean T_s of the whole ventral and dorsal body region, and the feet necessary for the calculation of T_diff. The analysed body regions were manually marked out with a polygonal region of interest in the CorePlayer (see Fig. 1 as an example for polygon of the whole venter). The region of interest was adjusted to fit onto the part of the body region of the particular individual perpendicularly oriented to the camera lens. Additionally, the venter was divided in the anterior–posterior axis into five areas (Fig. 1: 1—between the front legs, 2—to the widest part of the chest, 3—to the end of the rib cage, 4—to the hips, and 5—between hind legs without the anogenital area).

Data analyses

In all analyses performed in this study, T_diff was used as a dependent variable assuming Gaussian error distribution. In all models described below, body region (the dorsum, the venter, and feet), social organisation (social and solitary), and ventral area (1–5) were treated as explanatory categorical variables, whereas T_a was treated as a covariate. Means ± SD are given throughout the text. The sex of the tested animals was not included as an explanatory variable, mainly because of a relatively low number of males and females tested. Nevertheless, we did not expect any differences because there are no remarkable sex differences in thermal biology of subterranean rodents apart from body mass if females are not pregnant or lactating pups²⁵. To test our hypotheses, we fitted the models on subsets of data divided according to body region and selected T_a values. All analyses were carried out in R⁶¹ and all figures were edited using the program Inkscape 0.92 (https://inkscape.org/).

T_b, T_s, and T_diff over the T_a gradient

Prior to testing of our hypotheses, we performed the following basic statistical exploratory analyses of T_b and T_s. For each species, we firstly calculated a piecewise linear regression implemented in the R package Segmented⁶² to assess whether there is a breakpoint T_a, at which the regression curve characterising the change in T_b (expressed as mean values for each T_a for each species) for a given species changes its slope. Secondly, we calculated a linear regression to characterise a change in T_s (expressed as mean values for each T_a for each species) in response to increasing T_a. We used Bonferroni procedure to correct the α of the tests since we calculated one piecewise linear regression for T_b for each of the seven species (adjusted α = 0.0071) and 21 linear regressions for T_s (adjusted α = 0.0024).

For each species separately, we tested whether T_diff differs amongst the three body regions, i.e. the dorsum, the venter, and the feet, using GLS marginal models implemented in the nlme package⁶³. One GLS model was calculated for each of three T_as − 10 and 35 °C as extremes in order to obtain information about T_diff in cold and hot conditions, respectively, and 30 °C which represents TNZ of all the studied species (Table 1). In all GLS models, the body region was the explanatory variable, and the identity of tested individuals was included to avoid pseudo-replication. We used the Bonferroni procedure to correct the significance level of the tests because we tested each T_a separately for seven species and three T_as (adjusted α = 0.0024), and then performed a similar post-hoc comparison 13-times (adjusted α = 0.0038).

For each species separately, we first calculated three linear regressions to test whether the slope characterising the change of T_diff in response to increasing T_a for each of the three body regions is different from zero. For each body region, T_diff value for a given T_a was calculated as the mean obtained from all individuals within a given species, for which we obtained a complete set of T_s values from all three body regions at all six T_as. Afterwards, a homogeneity-of-slopes model was calculated to test whether these three regression lines are parallel to each other, i.e. there is no interaction between body region and T_a. The function “emtrends” implemented in the R package emmeans⁶⁴ was used to calculate post-hoc comparisons to find differences between the slopes for the dorsum, the venter, and the feet in these models. We used the Bonferroni procedure to correct the significance level of the tests we performed for each of the seven species: (a) three similar regression models (adjusted α = 0.0024) and (b) altogether seven homogeneity-of-slope models with a post-hoc test (adjusted α = 0.0071).

T_diff of the three body regions in solitary and social species

To assess the effect of the social organization on T_diff (expressed as a mean value for each T_a for each species) in each of the three body regions, we fitted Generalized Linear Mixed Models in a Bayesian framework using Markov Chain Monte Carlo sampling algorithm (MCMC) implemented in the R package MCMCglmm⁶⁵. This approach was used to account for non-independence in T_diff measurements arising due to shared phylogenetic ancestry, and therefore a random effect of phylogeny was included in all models. We controlled for phylogenetic relatedness using the subset tree of the mammal phylogeny by Upham et al.⁶⁶. The set of subtrees was retrieved via an online tool: vertlife.org/phylosubsets/. The maximum credibility tree was then inferred using the R package phangorn⁶⁷, and taxa labels were changed using the R package phytools⁶⁸ in order to merge the taxa with the comparative dataset.

Parameter estimates for fixed and random effects of each model were obtained from sampling posterior distributions by running 2,500,000 MCMC iterations with a burn-in period of 20% and a thinning interval sampling each 1000th iteration. Estimates of each parameter are thus based on 2000 samples from a posterior distribution. In all models, we used weakly informative priors for variance components (V = 1, ν = 0.02), and a default prior specification for fixed effects, i.e. mean centred on zero with a very large variance (µ = 0, V = 10⁸), to let the posterior be determined mostly by the information in the data. Mixing and convergence of MCMC chains were assessed by visual inspection of both time-series and density plots, as well as by calculating autocorrelation among successive MCMC samples. There was no apparent trend among MCMC samples in time-series plots, and autocorrelation was low in each fitted model. Effective sample size was ~ 2000 for all estimated parameters in all models, suggesting generally good mixing and convergence properties of MCMC chains.

For each of the three body regions separately, we fitted a model of the formula: T_diff ~ T_a + social organisation + T_a: social organisation (the same T_diff datasets as for linear regressions were used for these models). Beside the random effect of the phylogeny accounting for evolutionary history of the studied taxa, we included also a random effect of species to account for species-specific effects on the variability in T_diff in response to T_a. This model allowed us to test whether the slopes for the change in T_diff as a response to increasing T_a differ between solitary and social species. Interaction term was considered only when its effect in a model was significant (see highest posterior density (HPD) intervals and/or pMCMC in Table 3), and the deviance information criterion (DIC) indicated a better fit (lower DIC) of the model with than without interaction. If the effect of interaction was not significant, we ran the model without the interaction term, and interpreted only the effects of T_a and social organization, respectively.

T_diff along the venter in solitary and social species

For each species individually, we tested whether T_diff differs among the five ventral areas using GLS models. One GLS model was calculated for each T_a of 10, 30 and 35 °C. In all GLS models, the ventral area was the explanatory variable (a factor with five levels: ventral area 1—ventral area 5, see Fig. 1). We used the Bonferroni procedure to correct the significance level of the tests since we tested each of three T_as separately for seven species (adjusted α = 0.0024). In all GLS models, an identity of tested individuals was included to avoid pseudo-replication.

In addition, for each of the three T_a, we fitted one MCMCglmm of the form: T_diff ~ ventral area + social organisation + ventral area: social organisation, with random effects of the phylogenetic relatedness and species. This model allowed us to test whether the pattern of mean change in T_diff (calculated as the mean from all individuals of each species entering GLS analysis mentioned above) across five ventral areas differed between solitary and social species. The effect of interaction term was considered significant if the HPD intervals did not include zero (see HPD intervals and/or pMCMC in Table 4), and DIC of the model with interaction was lower than that of the model without it.

Ethical approval

The experimental procedures in the Czech Republic were approved by the University of South Bohemia Animal Welfare committee and Ministry of Education, Youth and Sports of the Czech Republic (Permission no. MSMT-26065/2014-12). The experimental procedures in South Africa were approved by University of Pretoria Ethics Committee (Permission no. EC069-16). All procedures were performed in accordance with relevant guidelines and regulations.

Source: Ecology - nature.com