A clock for all seasons in the subterranean.

In 1976, Pittendrigh and Daan established a theoretical framework which has coordinated research on circadian clock entrainment and photoperiodism until today. The "wild clocks" approach, which concerns studying wild species in their natural habitats, has served to test their models, add new insights, and open new directions of research. Here, we review an integrated laboratory, field and modeling work conducted with subterranean rodents (Ctenomys sp.) living under an extreme pattern of natural daily light exposure. Tracking animal movement and light exposure with biologgers across seasons and performing laboratory experiments on running-wheel cages, we uncovered the mechanisms of day/night entrainment of the clock and of photoperiodic time measurement in this subterranean organism. We confirmed most of the features of Pittendrigh and Daan's models but highlighted the importance of integrating them with ecophysiological techniques, methodologies, and theories to get a full picture of the clock in the wild. This integration is essential to fully establish the importance of the temporal dimension in ecological studies and tackling relevant questions such as the role of the clock for all seasons in a changing planet.

An increased number of individuals of a potential host facilitates non-photic synchronisation in the haematophagous insect Triatoma infestans.

BACKGROUND: Triatoma infestans (Kissing bug) is the main vector of the parasite causative of Chagas disease in Latin-America. This species shows clear activity rhythms easily synchronised to day-night cycles (photic cycle). The haematophagous nature of these insects lead us to think that they may temporally adapt to the particular activity rhythms of potential hosts (non-photic cycle). Our previous data showed that kissing bugs were weakly affected by the activity-inactivity rhythm of a single host. OBJETIVE: To determine if by increasing the number of individuals of a potential host, T. infestans could increase the likelihood of synchronisation. METHODS: Individual activity rhythms of experimental insects, maintained in constant darkness in light-tight cabinets, localised in a room with 24 rodents, were continuously monitored. Another insect group that served as control was maintained in the same conditions but in a room without rodents. FINDINGS: Most of the experimental insects synchronised, expressing a 24 h period coincident with the activity-inactivity rhythms of the rodents, while the controls free ran with a period significantly longer than 24 h. CONCLUSION: Analogous to what happens with high vs low light intensity in photic synchronisers, a high number of rodents, in contrast to the previous one-rodent experiment, increased the potency of this non-photic zeitgeber.

Temporal Dissociation Between Activity and Body Temperature Rhythms of a Subterranean Rodent (Ctenomys famosus) in Field Enclosures.

Several wild rodents, such as the subterranean tuco-tucos (Ctenomys famosus), switch their time of activity from diurnal to nocturnal when they are transferred from field to the laboratory. Nevertheless, in most studies, different methods to measure activity in each of these conditions were used, which raised the question of whether the detected change in activity timing could be an artifact. Because locomotor activity and body temperature (Tb) rhythms in rodents are tightly synchronized and because abdominal Tb loggers can provide continuous measurements across field and laboratory, we monitored Tb as a proxy of activity in tuco-tucos transferred from a semi-field enclosure to constant lab conditions. In the first stage of this study ("Tb-only group," 2012-2016), we verified high incidence (55%, n = 20) of arrhythmicity, with no consistent diurnal Tb rhythms in tuco-tucos maintained under semi-field conditions. Because these results were discrepant from subsequent findings using miniature accelerometers (portable activity loggers), which showed diurnal activity patterns in natural conditions (n = 10, "Activity-only group," 2016-2017), we also investigated, in the present study, whether the tight association between activity and Tb would be sustained outside the lab. To verify this, we measured activity and Tb simultaneously across laboratory and semi-field deploying both accelerometers and Tb loggers to each animal. These measurements (n = 11, "Tb + activity group," 2019-2022) confirmed diurnality of locomotor activity and revealed an unexpected loosening of the temporal association between Tb and activity rhythms in the field enclosures, which is otherwise robustly tight in the laboratory.

Daylength Shapes Entrainment Patterns to Artificial Photoperiods in a Subterranean Rodent.

Photoperiodism plays an important role in the synchronization of seasonal phenomena in various organisms. In mammals, photoperiod encoding is mediated by differential entrainment of the circadian system. The limits of daily light entrainment and photoperiodic time measurement can be verified in organisms that inhabit extreme photic environments, such as the subterranean. In this experimental study, we evaluated entrainment of circadian wheel-running rhythms in South American subterranean rodents, the Anillaco tuco-tucos (Ctenomys aff. knighti), exposed to different artificial photoperiods, from extremely long to extremely short photophases (LD 21:3, LD 18:6, LD 15:9, LD 9:15, LD 6:18 and LD 3:21). Artificial photoperiods synchronized their activity/rest rhythms and clear differences occurred in (a) phase angles of entrainment relative to the LD cycle and (b) duration of the daily activity phase α. These photoperiod-dependent patterns of entrainment were similar to those reported for epigeous species. Release into constant darkness conditions revealed aftereffects of entrainment to different photoperiods, observed in α but not in the free-running period τ. We also verified if animals coming from summer and winter natural photoperiods entrained equally to the artificial photoperiods by evaluating their phase angle of entrainment, α and τ aftereffects. To this end, experimental animals were divided into "Matching" and "Mismatching" groups, based on whether the experimental photoperiod (short-day [L < 12 h] or long-day [L > 12 h]) matched or not the natural photoperiod to which they had been previously exposed. No significant differences were found in the phase angle of entrainment, α and τ aftereffects in each artificial photoperiod. Our results indicate that the circadian clocks of tuco-tucos are capable of photoperiodic time measurement despite their natural subterranean habits and that the final entrainment patterns achieved by the circadian clock do not depend on the photoperiodic history.

Division of labor in work shifts by leaf-cutting ants.

Foraging rhythms in eusocial insects are determined by the colony´s overall pattern. However, in leaf-cutting ant workers, individual rhythms are not fully synchronized with the colonies' rhythm. The colony as a whole is nocturnal, since most worker activity takes place at night; however some workers forage during the day. Previous studies in individualized ants suggest nocturnal and diurnal workers coexistence. Here observations within the colony, in leaf-cutting ants, showed that workers have differential foraging time preference, which interestingly is associated to body size and differential leaf transportation engagement. Nocturnal ants are smaller and less engaged in leaf transportation whereas diurnal ants are bigger and more engaged in leaf carriage. Mechanisms underlying division of labor in work shifts in ants are still unknown but much can be extrapolated from honeybees; another social system bearing a similar pattern. A collective organization like this favors constant exploitation of food sources while preserving natural individual rhythm patterns, which arise from individual differences, and thermal tolerance, given by the size polymorphism presented by this species.

Telling the Seasons Underground: The Circadian Clock and Ambient Temperature Shape Light Exposure and Photoperiodism in a Subterranean Rodent.

Living organisms anticipate the seasons by tracking the proportion of light and darkness hours within a day-photoperiod. The limits of photoperiod measurement can be investigated in the subterranean rodents tuco-tucos (Ctenomys aff. knighti), which inhabit dark underground tunnels. Their exposure to light is sporadic and, remarkably, results from their own behavior of surface emergence. Thus, we investigated the endogenous and exogenous regulation of this behavior and its consequences to photoperiod measurement. In the field, animals carrying biologgers displayed seasonal patterns of daily surface emergence, exogenously modulated by temperature. In the laboratory, experiments with constant lighting conditions revealed the endogenous regulation of seasonal activity by the circadian clock, which has a multi-oscillatory structure. Finally, mathematical modeling corroborated that tuco-tuco's light exposure across the seasons is sufficient for photoperiod encoding. Together, our results elucidate the interrelationship between the circadian clock and temperature in shaping seasonal light exposure patterns that convey photoperiod information in an extreme photic environment.

Circadian phase and intertrial interval interfere with social recognition memory.

A modified version of the social habituation/dis-habituation paradigm was employed to examine social recognition memory in Wistar rats during two opposing (active and inactive) circadian phases, using different intertrial intervals (30 and 60 min). Wheel-running activity was monitored continuously to identify circadian phase. To avoid possible masking effects of the light-dark cycle, the rats were synchronized to a skeleton photoperiod, which allowed testing during different circadian phases under identical lighting conditions. In each trial, an infantile intruder was introduced into an adult's home-cage for a 5-minute interaction session, and social behaviors were registered. Rats were exposed to 5 trials per day for 4 consecutive days: on days 1 and 2, each resident was exposed to the same intruder; on days 3 and 4, each resident was exposed to a different intruder in each trial. The resident's social investigatory behavior was more intense when different intruders were presented compared to repeated presentation of the same intruder, suggesting social recognition memory. This effect was stronger when the rats were tested during the inactive phase and when the intertrial interval was 60 min. These findings suggest that social recognition memory, as evaluated in this modified habituation/dis-habituation paradigm, is influenced by the circadian rhythm phase during which testing is performed, and by intertrial interval.

Acute effects of light and darkness on the activity and temperature rhythms of a subterranean rodent, the Anillaco tuco-tuco.

Tuco-tucos from Anillaco (Ctenomys aff. knighti), are subterranean rodents that run vigorously on laboratory wheels during the night but are active during the day in semi-natural enclosures, where they surface for foraging and burrow maintenance, under intense sunlight. Several studies have shown that light causes opposite, inhibitory and stimulatory, "masking" effects on the activity levels between nocturnal and diurnal species, respectively. Because of the alternating subterranean/surface activity of tuco-tucos in nature during the day and their ability to shift from diurnal to nocturnal patterns in field-to-lab transitions, we assessed the acute effects of light and darkness on running wheel activity, general activity and body temperature. Adult males and females were kept in a LD 12:12 h regimen and exposed to light and dark pulses to verify masking effects in their rhythms. A first experiment consisted in submitting animals to light pulses of different illuminance during the dark phase. Clear inhibition of wheel-running activity occurred, being the response more pronounced as illuminance of the pulse increased, a response typically seen in nocturnal rodents. A second experiment consisted in submitting animals to light pulses during the dark phase, and later to dark pulses during the light phase. This protocol occurred three times in the conditions: 1) without a wheel, 2) with free access to a wheel, and 3) with a blocked wheel. Wheel running was inhibited and body temperature decreased in most animals during the light pulse, with little to no inhibition on general activity. Dark pulses during the light phase had no effect on wheel-running activity nor on general activity but did affect body temperature. Interestingly, there was a single individual that switched from nocturnal to diurnal when kept without a running wheel, offering an opportunity to test nocturnal and diurnal masking patterns to light in the same individual.

Day and night in the subterranean: measuring daily activity patterns of subterranean rodents (Ctenomys aff. knighti) using bio-logging.

While most studies of the impacts of climate change have investigated shifts in the spatial distribution of organisms, temporal shifts in the time of activity is another important adjustment made by animals in a changing world. Due to the importance of light and temperature cycles in shaping activity patterns, studies of activity patterns of organisms that inhabit extreme environments with respect to the 24-hour cyclicity of Earth have the potential to provide important insights into the interrelationships among abiotic variables, behaviour and physiology. Our previous laboratory studies with Argentinean tuco-tucos from the Monte desert (Ctenomys aff. knighti) show that these subterranean rodents display circadian activity/rest rhythms that can be synchronized by artificial light/dark cycles. Direct observations indicate that tuco-tucos emerge mainly for foraging and for removal of soil from their burrows. Here we used bio-logging devices for individual, long-term recording of daily activity/rest (accelerometry) and time on surface (light-loggers) of six tuco-tucos maintained in outdoor semi-natural enclosures. Environmental variables were measured simultaneously. Activity bouts were detected both during day and night but 77% of the highest values happened during the daytime and 47% of them coincided with time on surface. Statistical analyses indicate time of day and temperature as the main environmental factors modulating time on surface. In this context, the total duration that these subterranean animals spent on surface was high during the winter, averaging 3 h per day and time on surface occurred when underground temperature was lowest. Finally, transport of these animals to the indoor laboratory and subsequent assessment of their activity rhythms under constant darkness revealed a switch in the timing of activity. Plasticity of activity timing is not uncommon among desert rodents and may be adaptive in changing environments, such as the desert where this species lives.

Dissociation of the circadian rhythm of locomotor activity in a 22 h light-dark cycle impairs passive avoidance but not object recognition memory in rats.

We analyzed the effect of dissociation of the circadian rhythm of locomotor activity on the performance in two memory tasks in rats. One group of animals was maintained in a normal 24 h light-dark cycle of 12:12 (T24 group, control). A second group was housed in a 22 h cycle of 11:11 (T22 group, experimental), a condition which is known to produce dissociation of the circadian rhythm of locomotor activity in two components. Both groups were tested on two memory tasks: passive avoidance and object recognition. An additional control group, kept under constant darkness (DD group), was used for a passive avoidance task. Testing occurred 30 min (short-term memory--STM) and 24 h (T24 and DD group) or 22 h (T22 group) (long-term memory--LTM) after training. The T22 group showed impairment on the passive avoidance task (STM and LTM) compared with the T24 and DD groups. On the object recognition task, the T22 and T24 groups performed similarly in all the sessions. In conclusion, circadian rhythm dissociation induced a performance deficit in the passive avoidance task but had no effect on the object recognition task. We suggest that dissociation of the circadian rhythm of locomotor activity may selectively affect some emotional component related to fear and risk evaluation.

An increased number of individuals of a potential host facilitates non-photic synchronisation in the haematophagous insect Triatoma infestans

BACKGROUND Triatoma infestans (Kissing bug) is the main vector of the parasite causative of Chagas disease in Latin-America. This species shows clear activity rhythms easily synchronised to day-night cycles (photic cycle). The haematophagous nature of these insects lead us to think that they may temporally adapt to the particular activity rhythms of potential hosts (non-photic cycle). Our previous data showed that kissing bugs were weakly affected by the activity-inactivity rhythm of a single host. OBJETIVE To determine if by increasing the number of individuals of a potential host, T. infestans could increase the likelihood of synchronisation. METHODS Individual activity rhythms of experimental insects, maintained in constant darkness in light-tight cabinets, localised in a room with 24 rodents, were continuously monitored. Another insect group that served as control was maintained in the same conditions but in a room without rodents. FINDINGS Most of the experimental insects synchronised, expressing a 24 h period coincident with the activity-inactivity rhythms of the rodents, while the controls free ran with a period significantly longer than 24 h. CONCLUSION Analogous to what happens with high vs low light intensity in photic synchronisers, a high number of rodents, in contrast to the previous one-rodent experiment, increased the potency of this non-photic zeitgeber.

Circadian system responses to nocturnal and diurnal hosts in the kissing bug, Triatoma infestans.

Insects express diverse behavioral rhythms synchronized to environmental cycles. While circadian entrainment to light-dark cycles is ubiquitous in living organisms, synchronization to non-photic cycles may be critical for hematophagous bugs that depend on rhythmic hosts. The purpose was to determine whether Triatoma infestans are capable of synchronizing to the circadian rhythms of potential hosts with temporally distinct activity patterns; and, if so, if this synchronization occurs through masking or entrainment. Precise synchronization with the food source may be critical for the insects' survival due to the specific predatory or defensive nature of each host. Kissing bugs were housed in a compartment in constant dark, air-flow-connected to another compartment with a nocturnal or a diurnal host; both hosts were synchronized to a light-dark cycle. The activity rhythms of kissing bugs were modulated by the daily activity rhythms of the vertebrates. Effects were a decrease in the endogenous circadian period, independent of the host being nocturnal or diurnal; in some cases relative coordination occurred and in others synchronization was clearly achieved. Moreover, splitting and bimodality arose, phenomena that were also affected by the host presence. The results indicate that T. infestans were able to detect the non-photic cycle of their potential hosts, an ability that surely facilitates feeding and hinders predation risk. Understanding triatomines behavior is of fundamental importance to the design of population control methods.

Activity levels of female Triatoma infestans change depending on physiological condition.

BACKGROUND: Triatoma infestans (Hemiptera: Reduviidae) is the main vector of Trypanosoma cruzi, the etiological agent of Chagas disease, in South America. Active dispersal of this vector is the principal cause of recolonization of human dwellings previously treated with insecticides. Due to the persistence of vector populations and their movement between habitats, dispersive behavior studies are important for understanding the epidemiology of Chagas disease. The aim of this study was to analyze the relationship among T. infestans females' activity levels according to their physiological conditions. RESULTS: Two groups of insects were used, unfed and fed females. Each was composed of three subgroups in relation to the reproductive state: fifth-stage nymphs, virgin and fertilized females. There was a significant interaction between reproductive and nutritional states among T. infestans female' activity levels. During the experiments, unfed and fed nymphs remained inactive. Virgin females showed a dual behavior in their movement; fasted insects were more active. Fertilized females, both fed and unfed, were always active. CONCLUSION: The reproductive and nutritional conditions of T. infestans females affect their activity levels. When females with different reproductive states remained together, fertilized females showed permanent activity levels, suggesting that this subgroup of females represents the highest epidemiological risk as colonizers of human dwellings.

The Interplay of Energy Balance and Daily Timing of Activity in a Subterranean Rodent: A Laboratory and Field Approach.

The tuco-tuco (Ctenomys aff. knighti) is among the rodent species known to be nocturnal under standard laboratory conditions and diurnal under natural conditions. The circadian thermoenergetics (CTE) hypothesis postulates that switches in activity timing are a response to energetic challenges; daytime activity reduces thermoregulatory costs by consolidating activity to the warmest part of the day. Studying wild animals under both captive and natural conditions can increase understanding of how temporal activity patterns are shaped by the environment and could serve as a test of the CTE hypothesis. We estimated the effects of activity timing on energy expenditure for the tuco-tuco by combining laboratory measurements of metabolic rate with environmental temperature records in both winter and summer. We showed that, in winter, there would be considerable energy savings if activity is allocated at least partially during daylight, lending support to the CTE hypothesis. In summer, the impact of activity timing on energy expenditure is small, suggesting that during this season other factors, such as predation risk, water balance, and social interaction, may have more important roles than energetics in the determination of activity time.

Social modulation of the daily activity rhythm in a solitary subterranean rodent, the tuco-tuco (Ctenomys sp).

South American subterranean rodents are mainly described as solitary and mutual synchronization was never observed among individuals maintained together in laboratory. We report that a single birth event was capable of disrupting the robust nocturnal activity rhythm of singly housed tuco-tucos from north-west Argentina. "Around-the-clock activity" was displayed by 8 out of 13 animals whose cages were closer to the newborn pups. However, experimental exposure to a pup vocalization did not produce a similar effect on the rhythms of adult animals. Our results indicate an effect of social interaction in the expression of biological rhythms even in solitary animals.

Form and function of long-range vocalizations in a Neotropical fossorial rodent: the Anillaco Tuco-Tuco (Ctenomys sp.).

The underground environment poses particular communication challenges for subterranean rodents. Some loud and low-pitched acoustic signals that can travel long distances are appropriate for long-range underground communication and have been suggested to be territorial signals. Long-range vocalizations (LRVs) are important in long-distance communication in Ctenomys tuco-tucos. We characterized the LRV of the Anillaco Tuco-Tuco (Ctenomys sp.) using recordings from free-living individuals and described the behavioral context in which this vocalization was produced during laboratory staged encounters between individuals of both sexes. Long-range calls of Anillaco tuco-tucos are low-frequency, broad-band, loud, and long sounds composed by the repetition of two syllable types: series (formed by notes and soft-notes) and individual notes. All vocalizations were initiated with series, but not all had individual notes. Males were heavier than females and gave significantly lower-pitched vocalizations, but acoustic features were independent of body mass in males. The pronounced variation among individuals in the arrangement and number of syllables and the existence of three types of series (dyads, triads, and tetrads), created a diverse collection of syntactic patterns in vocalizations that would provide the opportunity to encode multiple types of information. The existence of complex syntactic patterns and the description of soft-notes represent new aspects of the vocal communication of Ctenomys. Long-distance vocalizations by Anillaco Tuco-Tucos appear to be territorial signals used mostly in male-male interactions. First, emission of LRVs resulted in de-escalation or space-keeping in male-male and male-female encounters in laboratory experiments. Second, these vocalizations were produced most frequently (in the field and in the lab) by males in our study population. Third, males produced LRVs with greater frequency during male-male encounters compared to male-female encounters. Finally, males appear to have larger home ranges that were more spatially segregated than those of females, suggesting that males may have greater need for long-distance signals that advertise their presence. Due to their apparent rarity, the function and acoustic features of LRV in female tuco-tucos remain inadequately known.

Entrainment of circadian rhythms to irregular light/dark cycles: a subterranean perspective.

Synchronization of biological rhythms to the 24-hour day/night has long been studied with model organisms, under artificial light/dark cycles in the laboratory. The commonly used rectangular light/dark cycles, comprising hours of continuous light and darkness, may not be representative of the natural light exposure for most species, including humans. Subterranean rodents live in dark underground tunnels and offer a unique opportunity to investigate extreme mechanisms of photic entrainment in the wild. Here, we show automated field recordings of the daily light exposure patterns in a South American subterranean rodent, the tuco-tuco (Ctenomys aff. knighti ). In the laboratory, we exposed tuco-tucos to a simplified version of this natural light exposure pattern, to determine the minimum light timing information that is necessary for synchronization. As predicted from our previous studies using mathematical modeling, the activity rhythm of tuco-tucos synchronized to this mostly simplified light/dark regimen consisting of a single light pulse per day, occurring at randomly scattered times within a day length interval. Our integrated semi-natural, lab and computer simulation findings indicate that photic entrainment of circadian oscillators is robust, even in face of artificially reduced exposure and increased phase instability of the synchronizing stimuli.

Effect of circadian phase on performance of rats in the Morris water maze task.

The authors examined spatial working memory in the Morris water maze during the activity and rest periods of Wistar rats. Wheel-running activity was measured continuously as a marker of circadian phase. To minimize possible masking effects on performance, animals were placed in constant dim light the day before testing and tested in similar light conditions. Three experiments were run, each of them using animals varying in their previous experience in the water maze. Half of the animals of each experiment were tested 2 to 3 h after activity onset (active group), and the other half were tested 14 to 15 h after activity onset (inactive group). In the three experiments, a significant phase effect was observed in the animals' performance in the water maze; animals tested in the active phase showed steeper acquisition curves. These phase effects on performance are due to the animals' search pattern and not to a better acquisition and maintenance of spatial information; rats tested in the inactive phase found the platform faster on the first trial of the test, when the information on the location of the platform had not been presented to the animals. This effect vanished as the amount of training in the pool increased. Finally, swimming speed also showed a temporal effect, suggesting the existence of a phase effect for motivation to escape from the water; rats tested during their inactive phase tended to swim faster. All together, the data suggest a modulating effect of the biological clock on performance in the water maze, particularly when the animals are less experienced.

Nocturnal to Diurnal Switches with Spontaneous Suppression of Wheel-Running Behavior in a Subterranean Rodent.

Several rodent species that are diurnal in the field become nocturnal in the lab. It has been suggested that the use of running-wheels in the lab might contribute to this timing switch. This proposition is based on studies that indicate feed-back of vigorous wheel-running on the period and phase of circadian clocks that time daily activity rhythms. Tuco-tucos (Ctenomys aff. knighti) are subterranean rodents that are diurnal in the field but are robustly nocturnal in laboratory, with or without access to running wheels. We assessed their energy metabolism by continuously and simultaneously monitoring rates of oxygen consumption, body temperature, general motor and wheel running activity for several days in the presence and absence of wheels. Surprisingly, some individuals spontaneously suppressed running-wheel activity and switched to diurnality in the respirometry chamber, whereas the remaining animals continued to be nocturnal even after wheel removal. This is the first report of timing switches that occur with spontaneous wheel-running suppression and which are not replicated by removal of the wheel.