Locomotor Activity and Body Temperature Patterns over a Temperature Gradient in the Highveld Mole-Rat (Cryptomys hottentotus pretoriae)

African mole-rats are strictly subterranean mammals that live in extensive burrow systems. High humidity levels in the burrows prevent mole-rats from thermoregulating using evaporative cooling. However, the relatively stable environment of the burrows promotes moderate temperatures and small daily temperature fluctuations. Mole-rats therefore display a relatively wide range of thermoregulation abilities. Some species cannot maintain their body temperatures at a constant level, whereas others employ behavioural thermoregulation. Here we test the effect of ambient temperature on locomotor activity and body temperature, and the relationship between the two parameters, in the highveld mole-rat. We exposed mole-rats to a 12L:12D and a DD light cycle at ambient temperatures of 30°C, 25°C and 20°C while locomotor activity and body temperature were measured simultaneously. In addition, we investigated the endogenous rhythms of locomotor activity and body temperature at different ambient temperatures. Mole-rats displayed nocturnal activity at all three ambient temperatures and were most active at 20°C, but least active at 30°C. Body temperature was highest at 30°C and lowest at 20°C, and the daily cycle was highly correlated with locomotor activity. We show that the mole-rats have endogenous rhythms for both locomotor activity and body temperature. However, the endogenous body temperature rhythm appears to be less robust compared to the locomotor activity rhythm. Female mole-rats appear to be more sensitive to temperature changes than males, increased heterothermy is evident at lower ambient temperatures, whilst males show smaller variation in their body temperatures with changing ambient temperatures. Mole-rats may rely more heavily on behavioural thermoregulation as it is more energy efficient in an already challenging environment.


Introduction
Daily fluctuations in biological functions is a near universal property of living organisms. In rodents, circadian rhythms such as locomotor activity and body temperatures (T b ), are regulated by internal clocks [1]. The master circadian clock is located in the suprachiasmatic nucleus a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 We investigated this by recording T b and locomotor activity over a range of T a' s and a 12L:12D light cycle. Secondly we examined the endogenous rhythms of both locomotor activity and T b to determine whether the animals exhibit an endogenous rhythm for T b and how it compares with the activity rhythm, and furthermore whether ambient temperature influences the period of these rhythms. We expected to find that T b rhythms would be less robust than that for locomotor activity, but would show similar period lengths than the endogenous rhythms for locomotor activity.

Ethical statement
All experimental procedures were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health [33]. The protocol was approved by the Ethics Committee of the University of Pretoria-clearance number EC073-13. All surgery was performed under Isofluorane by a registered veterinarian, and all efforts were made to minimize suffering.

Capture and housing
Sixteen non-reproductive highveld mole-rats were captured in Centurion, Gauteng (25˚48'14" S; 28˚09'44"E) in South Africa during April/May 2014 using modified Hickman live traps [34] baited with sweet potato. Ten females (mean body mass = 114.50±29.49g) and six males (mean body mass = 132.58±38.72g), originating from 6 colonies, were investigated in this study. One male died (reason unknown) after the first temperature cycle, and was therefore excluded from the analysis.
The animals were transported to the department of Zoology and Entomology, University of Pretoria, Pretoria and housed individually in plastic containers (60x30x30cm) in a light (light phase = 330 lux at floor level (Major tech m940 light meter), dark phase = 0 lux) and temperature controlled room. Containers were lined with wood shavings and each mole-rat was supplied with a nest box and paper tissue for nesting material. The mole-rats were fed an ad libitum diet of sweet potato, which was replaced each day at random times. Bennett and Jarvis (1995) investigated the digestibility and nutritional value of sweet potatoes, and found it to provide animals with adequate nutrition. The well-being of the animals was monitored each day during feeding time. The animals were allowed to acclimatize for a week before commencement of the experimental procedures.

Experimental procedures
Core body temperature was measured with calibrated temperature loggers/iButtons (DS1922L iButtons, MaximIntegrated Products, Dallas, TX, USA) programmed to log a reading hourly with an accuracy of 0.05˚C. The iButtons were covered in surgical wax and were surgically implanted intra-peritonially under isoflourane anaesthesia by a registered veterinarian. The data loggers weigh 3.2g which is below 5% of the body weight of the lightest animal implanted. Animals were pre-medicated with butorphanol for anaesthetic and analgesic purposes at a dose of 2mg/kg given intramuscularly. Anesthesia was induced in animals with 5% Isofluorane using an induction chamber and Isofluorane via a Humphreys ADE circle system machine, and maintained via a face mask on between 2 and 3%. Post-surgery, animals were placed back into their containers and monitored during recovery. All animals were awake and grooming within 15 minutes of being removed from the anesthetic. Animals were monitored closely for 24 hours, where after they were returned to the experimental rooms at the University. The animals resumed normal activity almost immediately, and were allowed to recover from the surgery for two days before commencement of the locomotor activity recordings. Upon completion of the experiment, animals were euthanized with an overdose of halothane, where after the data loggers were extracted and data downloaded.
Locomotor activity was measured using infrared motion detectors (Quest PIR internal passive infrared detector; Elite security products (ESP), Electronic lines, UK) that were fitted onto each plastic container in such a way that the entire floor space of the container was covered. Collective activity measurements were recorded once per minute and captured on a computer situated outside the activity room by the program Vital View (Vital View TM , Minimitter Co., Inc., Sunriver, OR, USA; www.minimitter.com). All animals were subjected consecutively to three different ambient temperatures (30˚C, 25˚C and 20˚C) both on an LD light cycle (12L:12D; light from 07:00-19:00) and under constant darkness cycle (DD). The experimental procedure was as follows: 30˚C LD for 25 days, 30˚C DD for 26 days, 25˚C LD for 25 days, 25˚C DD for 25 days and lastly 20˚C LD for 28 days and 20˚C DD for 29 days. All mole-rats were subjected to all the light and temperature cycles. iButtons were also used to measure soil temperatures on the surface (shade and sun), as well as at 5, 15, 25, 35 and 45cm, the iButtons were placed at a nearby site where they could be left undisturbed for 5 days.

Data analysis
Activity measurements were depicted as double plotted actograms using Actiview Biological Rhythm Analyses 1.2 software (Minimitter Co., Inc., Sunriver, OR, USA; www.minimitter.com), to offer a visual representation of individual activity patterns. As a result of the length of the study, actograms presented in the manuscript are shown separately for each ambient temperature, complete actograms of all animals are presented in the supplementary material (S3 Fig). The data from the first week of each light cycle was excluded from the analysis to exclude potential aftereffects of the previous cycles. Activity counts were summed to hourly values for comparison with the T b readings. For T b , the mean, minimum, maximum and variation was calculated for each 24h period during LD. As a measure of variation, the minimum T b was deducted from the maximum T b . Since data were not normally distributed, non-parametric statistical tests in SPSS 22 (SPSS Inc., Chicago, Illinois) were used for analysis. We used Generalized Linear Mixed Models to evaluate locomotor activity and mean, minimum, maximum and variation in body temperature. Generalised linear model repeated measures were used with gender and the respective ambient temperatures as fixed factors. Body mass was also controlled for. The period lengths for the locomotor activity and body temperature rhythms during constant conditions were determined using the Chi-square periodogram function within the Clocklab software (Clock-Lab™; Actimetrics, Evanston, Illinois, USA). The correlation between the lengths of the endogenous rhythms of locomotor activity and Tb were determined with a non-parametric correlation.

Soil temperatures
Surface and soil temperatures were measured for 5 days during the same time of year that the mole-rats were trapped. The mean ambient temperatures in full sun was comparable to that 5 cm below ground, although the temperature range in full sun was much more extensive. The mean temperatures increased progressively with depth below the ground, while the standard deviation decreased. Minimums, maximums, means and standard deviations of the surface and soil temperatures are listed in Table 1. See supporting information S1 Fig. Soil temperature, for a graphic representation of the temperatures at the different depths over the 5-day period.

Variation in body temperature
Ambient temperature significantly affects the variation between T b max and T b min (F = 49.3, df 1 = 2, df 2 = 1,138, p<0.001). The amount of variation between 20˚C and 25˚C is similar (p = 0.893), but the variation differs significantly between 30˚C and both 20˚C and 25˚C (p<0.001 for both comparisons). Likewise, the minimum T b differed significantly at the different temperatures (F = 134.57, df 1 = 2, df 2 = 1,158, p<0.001), although the minimum T b was comparable at 20˚C and 25˚C (p = 0.123), at 30˚C it was higher than both 20˚C and 25˚C (p<0.001 for both comparisons). Maximum T b also differed significantly at the various ambient temperatures (F = 13.76, df 1 = 2, df 2 = 1,158, p<0.001). Again 20˚C and 25˚C did not differ (p = 0.276), but the maximum T b at 30˚C was significantly higher than at the other two temperatures (both p<0.001, Fig 4).

Circadian cycles in constant conditions
A free-running rhythm for locomotor activity could be calculated for all 15 mole-rats at all three ambient temperatures, albeit rather weak for some. The mean period length (Ƭ) of the locomotor activity rhythms was slightly longer than 24h at all three temperatures (20˚C: 24h14 ± 0h57; 25˚C: 24h09 ± 0h30; 30˚C: 24h20 ± 0h53). Overall, there was no significant difference in the Ƭ of the locomotor activity rhythm at different ambient temperatures (F = 0.87, df 1 = 2, df 2 = 39, p = 0.426) (Figs 2, 3 and 5).
The length of the free-running rhythm of the body temperature could be determined in 12 animals at 20˚C and in 14 animals each at 25˚C and 30˚C. The mean period length of body  Table 2. Mean overall activity counts (±SE) and mean overall body temperatures (C˚) (±SE) for the light and dark phases of the 12L:12D light cycle for highveld mole-rats at the three ambient temperatures tested, as well as separated between genders. Standard deviations are indicated in brackets below. temperature endogenous rhythms was also longer than 24h for all three ambient temperatures (20˚C: 24h10 ± 0h59; 25˚C: 24h23 ± 0h46; 30˚C: 24h10 ± 0h32). The Ƭ of the T b circadian rhythms did not differ at the various ambient temperatures (F = 0.39, df 1 = 2, df 2 = 28, p = 0.68) (Figs 2, 3 and 5). The period lengths of the locomotor activity and Tb rhythms were highly correlated (r = 0.751, p<0.001).

Ambient
The period length of the circadian locomotor activity rhythm was significantly longer in males than in females (F = 10.53, df 1 = 1, df 2 = 33, p = 0.003). This turned out to be as a result of a single male with an exceptionally long circadian period (Fig 5). If this animal is removed from the analysis, there is no difference between males and females (F = 0.63, df 1 = 1, df 2 = 30, p = 0.435). Upon the removal of removal of the animal with the long activity period, the Ƭ of the T b circadian rhythm was similar in males compared to females (F = 0.45, df 1 = 1, df 2 = 26, p = 0.507).

Daily locomotor activity and body temperature
Time of activity is generally assumed to have an adaptive advantage that is species specific [35]. Since mole-rats are subterranean and come above ground very infrequently, light is likely to be of lesser importance in the determination of locomotor activity times and the adaptive significance of robust activity rhythms may be less obvious. However, many mole-rat species investigated do show distinct daily locomotor activity rhythms under specific lighting regimes [15,17,18].
In the current study, all highveld mole-rats displayed more activity during the dark phase of the light cycles and this was true for all ambient temperatures tested. This result is consistent with what has been previously reported for this species [15]. Most other mole-rat species also exhibit nocturnal activity when exposed to a 12L:12D light cycle [15,17,18,36]. A nocturnal activity pattern was observed in both males and females. Although no gender difference was observed in the overall activity counts, the temporal distribution of activity was different for males and females. Activity levels were similar for males and females during the dark phases of all ambient temperatures, but males were more active during the light phases of the higher ambient temperatures, while females were more active during the light phase of the coolest ambient temperature. Sexual dimorphism in locomotor activity is not common in rodents; several other species investigated show no gender differences in locomotor activity patterns, both with and without running wheels [37]. However, some cases of gender differences have been reported, male gerbils show running wheel activity during the day, whereas females do not, but females are overall more active [38], and female Syrian hamsters show higher levels of activity than the males [39].
Our data revealed that locomotor activity and body temperatures (T b ) of the highveld mole-rats are highly correlated, such that the T b rises as activity increases and that both parameters reach a peak at about the same time. Peak values for both activity and body temperature occur at night for nocturnal animals and activity and T b peaks have been reported as being synchronous in rats [1]. Lower body temperatures may occur during inactive periods, for example during sleep [40], but the lowest T b 's for the mole-rats occurred mostly immediately after high levels of activity. Female mole-rats displayed higher T b compared to males at the lower ambient temperatures, while males had higher T b at the highest ambient temperature. As far as we are aware, there are no gender differences in tasks performed in the colony that could account for the difference in T b , nor are there any known physiological differences between males and females. However, no dedicated studies have been performed to look at this phenomenon, and this may be something that should be explored.
As can be seen from our soil temperature results, mole-rats are not exposed to extensive ambient temperature changes in their natural habitat. Hence they may be more sensitive to small temperature changes occurring within the burrow system. Ambient temperature played a significant role in the amount of locomotor activity exhibited by highveld mole-rats. Animals were most active at 20˚C, and became less active as the ambient temperature increased. Another mole-rat species, the Damaraland mole-rat has also been found to display reduced levels of activity at 30˚C when subjected to a similar temperature regime [36]. A reduction in activity levels when the ambient temperature approaches the thermoneutral zone (TNZ) has also been reported for other species such as laboratory mice [41]. In social mole-rat species, the TNZ's typically range between 28-36˚C [6,29,32,42,43]. The TNZ of the highveld mole-rat is around 30-32˚C [44], thus 30˚C falls within this zone.
Within the TNZ of an animal, the basal rate of heat production is equivalent to the rate of heat loss to the environment, therefore no energy is expended to maintain body temperature. In general, animals appear to be comfortable in this zone, and have been shown to prefer ambient temperatures that overlap with this zone for low intensity activities such as resting and sleeping [41]. Small and medium sized mole-rats have been reported to choose ambient temperatures close to 30˚C for resting, while larger mole-rat species preferred slightly cooler ambient temperatures [45]. Reduced activity at higher ambient temperatures may be a mechanism employed to avoid hyperthermia [46] however, temperatures close to the TNZ of animals may simply be energetically less demanding for the animals and high levels of activity are not needed to maintain body temperatures.
At ambient temperatures below the TNZ, animals would need to generate more heat to maintain a constant T b . This can be achieved using behavioural thermoregulation in the form of increased locomotor activity or by huddling to conserve heat [25]. Highveld mole-rats are social animals, and in their natural habitat, they may huddle together in communal nests to conserve body heat. However, in the present study mole-rats were housed individually in order to record individual activity, removing the option of huddling. Hence the increase in locomotor activity at lower T a 's observed here may be induced by the experimental conditions rather than what would occur in nature.

Variation in body temperature
At ambient temperatures overlapping with the TNZ of the animals, the minimum and maximum Tb of all animals were higher than at ambient temperatures below the TNZ. The molerats are likely not exposed to temperatures in their TNZ for extended periods of time in their burrows. Thus at higher ambient temperatures, a theoretical optimal T b may be a relative concept. It may be a trade-off between the energetic costs of reducing Tb by metabolic means or maintaining the Tb at the lowest level behavioural thermoregulation allows for.
Although highveld mole-rats are classified as homeothermic, they still experience temporal variations in body temperature. Our data indicate that highveld mole-rats show increased daily T b variations with decreased ambient temperatures. Although the males showed a difference between the daily maximum and minimum T b , this difference is more pronounced in females, particularly at the lower temperatures. These results may indicate that female highveld mole-rats are more sensitive to changes in ambient temperature than the males. Body temperatures of free-ranging Damaraland mole-rats were also found to vary considerably more during winter compared to summer [6], and the higher variability at cooler ambient temperatures appears to be consistent with the results of the females in our study.
In congruence with the above, female highveld mole-rats showed significantly more variation in their body temperatures compared to the males. Interestingly, there were no difference in the minimum T b 's, but females displayed significantly higher maximum T b 's. Females seem to maintain their T b above a certain minimum, and are able to let their T b vary over a greater range of temperatures than the males. A gender difference was also apparent in Damaraland mole-rats, but there the minimum T b 's differed between the genders, with females showing higher minimum T b 's [6]. Adjustments to thermoregulatory responses appear to be species specific in mole-rats although females seem to be consistently warmer than males.

Circadian rhythms in locomotor activity and body temperature
Highveld mole-rats showed endogenous rhythms of both locomotor activity and body temperature, but the body temperature rhythms were detected in a smaller number of animals. The length of these endogenous rhythms were slightly longer than 24 hours, the majority of the rhythms had lengths close to 24h, with one mole-rat showing very long Ƭ (tau) for its rhythms (almost 28 h). This long Ƭ may potentially be as a result of a genetic mutation, several mutations in other rodent species have been shown to result in either long or short periods for circadian rhythms [47,48]. The length of circadian rhythms may not be critical for the survival of strictly subterranean mammals on a daily basis, however it may affect seasonal behaviours.
Locomotor activity rhythms have previously been described for highveld mole-rats [15], however, this is the first report of body temperature rhythms for this species. Circadian rhythms of body temperature have been observed in naked mole-rats [19] and blind mole-rats [49], but Cape mole-rats showed a lack of an obvious endogenous rhythms for body temperature [20]. Locomotor activity rhythms and T b rhythms of the highveld mole-rats were highly correlated. Since body temperature rises with increased locomotor activity, it may be argued that the body temperature rhythm is merely an effect of the locomotor activity expressed. However, the body temperature rhythm lengths are not exactly equal to those of the locomotor activity rhythms, also not all animals that exhibit discernible locomotor activity rhythms, express body temperature rhythms, suggesting an independent mechanism responsible for the body temperature rhythm. Body temperature rhythms appear to be less stable than the activity rhythms, although both the locomotor activity and body temperature are more arrhythmic in the TNZ.
A large number of other species, both nocturnal and diurnal, terrestrial and subterranean, also exhibit these two rhythms to be closely matched in length [19,[49][50][51]. Neither the tau for locomotor activity nor the T b of highveld mole-rats varied with the different ambient temperatures. This was to be expected since circadian rhythms are temperature compensated [52].

Concluding remarks
Highveld mole-rats could entrain their locomotor activity and body temperature to light cycles, and exhibited circadian rhythms in the absence of external cues. Entrainment and endogenous rhythms of locomotor activity have previously been shown for this species, but this is the first record for body temperature rhythms for highveld mole-rats. The endogenous circadian rhythms for locomotor activity and body temperature were found to be highly correlated. Both the locomotor activity and body temperatures of highveld mole-rats are influenced by ambient temperature, and these two parameters show an inverse relationship. Increased heterothermy is evident at lower ambient temperatures in the females, but not the males. It therefore appears that female highveld mole-rats are more sensitive to changes in ambient temperature than males, but It is not clear why this gender difference in thermoregulation exists. Thermoregulatory abilities of the highveld mole-rats are comparable to what has been reported in other mole-rat species. The relatively stable and moderate environmental conditions in the underground niche may have reduced the selective pressure for physiological thermoregulation in mole-rats. Behavioural thermoregulation may be sufficient and more energy efficient in an already challenging environment.