Threatened chronotopes: can chronobiology help endangered species?

Pittendrigh and Daan's 1976 article "Pacemaker structure: A clock for all seasons" marks the foundation of modern seasonal chronobiology. It proposed the internal coincidence model comprised of a Morning (M) and Evening (E) oscillator, which are coupled but synchronized separately by dawn and dusk. It has become an attractive model to explain the seasonal adaptation of circadian rhythms. Using the example of the European hamster, this article connects the classical entrainment concept to species decline and, ultimately, conservation concepts. Seasonality of this species is well studied and circannual rhythms have been described in at least 32 parameters. The European hamster is listed as critically endangered on the International Union for Conservation of Nature (IUCN) red list. Changes in the temporal structure of the environment (the chronotope) caused by climate change and light pollution might be responsible for the global decline. The article shows that classical chronobiological concepts such as the internal coincidence model (Pittendrigh and Daan Pittendrigh and Daan, J Comp Physiol [a] 106:333-355, 1976) are helpful to understand the (chronobiological) causes of the decline and can potentially support species conservation. Knowing the species' physiological limitations as well as its adaptation capacities can potentially prevent its extinction at a time when classical conservation concepts have reached their limits.

Morphometric parameters predict body fat proportions in common hamsters.

Common hamsters (Cricetus cricetus) are hibernators that rely both on body fat reserves and food stores for the winter period. They face an ongoing population decline in most parts of their distribution and recently were classified as critically endangered. Knowledge on individual body fat proportions in this species is of particular interest for conservation, because it could contribute to better understand the high plasticity in overwintering strategies, overwinter mortality rates, individual variations in reproductive output, and give information on the animals' health state. To calculate body fat proportions, we validated a method that can be applied in the field without the use of anesthesia. To develop this method, we first analyzed the body fat in carcasses of common hamsters using Soxhlet extractions and measured four morphometric parameters (body mass, head length, tibia length, foot length). The morphometric measurements were then integrated in a linear regression model to predict body fat proportions based on the measured values. The morphometric variables yielded an explained variance (adjusted R 2) of 96.42% and body fat proportions were predicted with a mean absolute error of 1.27 ± 0.11% from measured values. We applied the model to predict body fat for available field data, which consistently produced reliable values. By measuring the four morphometric parameters and following the provided instructions, body fat proportions can be reliably and noninvasively estimated in captive or free-ranging common hamsters. Furthermore, the method could be applicable to other rodents after species-specific validation.

Tardiness Increases in Winter: Evidence for Annual Rhythms in Humans.

Annual rhythms in humans have been described for a limited number of behavioral and physiological parameters. The aim of this study was to investigate time-of-year variations in late arrivals, sick leaves, dismissals from class (attendance), and grades (performance). Data were collected in Dutch high school students across 4 academic years (indicators of attendance in about 1700 students; grades in about 200 students). Absenteeism showed a seasonal variation, with a peak in winter, which was more strongly associated with photoperiod (number of hours of daylight) compared with other factors assessed (e.g., weather conditions). Grades also varied with time of year, albeit differently across the 4 years. The observed time-of-year variation in the number of sick leaves was in accordance with the literature on the seasonality of infectious diseases (e.g., influenza usually breaks out in winter). The winter peak in late arrivals was unexpected and requires more research. Our findings could be relevant for a seasonal adaptation of school schedules and working environments (e.g., later school and work hours in winter, especially at higher latitudes where seasonal differences in photoperiod are more pronounced).

Melatonin-independent Photoperiodic Entrainment of the Circannual TSH Rhythm in the Pars Tuberalis of the European Hamster.

Adaptation of biological rhythms to a seasonal environment in circannual mammals is achieved via the synchronization of intrinsic circannual rhythms to the external year by photoperiod. In mammals, the photoperiodic information is integrated to seasonal physiology via the pineal hormone melatonin regulation of pars tuberalis (PT) TSHß expression and its downstream control of hypothalamic dio2 gene expression. In the circannual European hamster, however, photoperiodic entrainment of the circannual clock is possible in pinealectomized animals. The present study explores whether the TSHß expression in the PT and the downstream hypothalamic pathways are regulated by photoperiod in European hamsters in the absence of melatonin. All animals were kept on an accelerated photoperiodic regime, which compressed the natural year to a 6-month cycle. Sham-operated European hamsters and half of the pinealectomized European hamsters entrained their annual cycle in reproduction, body weight, and activity pattern to this cycle, whereas the other half of the pinealectomized animals followed only each second cycle. In all animals, PT TSHß and hypothalamic dio2 expressions were higher in hamsters displaying a summer physiological state than in those in winter state. Moreover, in agreement with their seasonal state, reproductive animals (summer state) showed higher expression of rfrp and lower expression of kiss1-genes encoding central regulators of the reproductive axis-than those animals in reproductive quiescence (winter state), indicating the hypothalamic integration of the photoperiodic signal even in pinealectomized animals. The appropriate occurrence of a well-characterized activity pattern indicative of a so-called sensitive phase to short photoperiod suggested that the SCN constructs the melatonin-independent photoperiodic message. This message is sufficient to entrain the circannual rhythm in TSHß expression in the PT and the downstream hypothalamic neuroendocrine pathway through a yet unknown pathway. These results reinforce the hypothesis that the PT is the site for the integration of circannual and photoperiodic information.

A circannual clock drives expression of genes central for seasonal reproduction.

Animals living in temperate zones anticipate seasonal environmental changes to adapt their biological functions, especially reproduction and metabolism. Two main physiological mechanisms have evolved for this adaptation: intrinsic long-term timing mechanisms with an oscillating period of approximately 1 year, driven by a circannual clock [1], and synchronization of biological rhythms to the sidereal year using day length (photoperiod) [2]. In mammals, the pineal hormone melatonin relays photoperiodic information to the hypothalamus to control seasonal physiology through well-defined mechanisms [3-6]. In contrast, little is known about how the circannual clock drives endogenous changes in seasonal functions. The aim of this study was to determine whether genes involved in photoperiodic time measurement (TSHß and Dio2) and central control of reproduction (Rfrp and Kiss1) display circannual rhythms in expression under constant conditions. Male European hamsters, deprived of seasonal time cues by pinealectomy and maintenance in constant photoperiod, were selected when expressing a subjective summer or subjective winter state in their circannual cycle of body weight, temperature, and testicular size. TSHß expression in the pars tuberalis (PT) displayed a robust circannual variation with highest level in the subjective summer state, which was positively correlated with hypothalamic Dio2 and Rfrp expression. The negative sex steroid feedback was found to act specifically on arcuate Kiss1 expression. Our findings reveal TSH as a circannual output of the PT, which in turn regulates hypothalamic neurons controlling reproductive activity. Therefore, both the circannual and the melatonin signals converge on PT TSHß expression to synchronize seasonal biological activity.

Dual control of seasonal time keeping in male and female juvenile European hamsters.

In contrast to photoperiodic rodent species, adult circannual European hamsters (Cricetus cricetus) do not rely on melatonin as transducer of the photoperiodic message. Instead, seasonal entrainment involves a special circadian organisation which characterizes a photoperiod-sensitive phase. When days shorten a precise activity pattern ("summer pattern") switches to a weak or arrhythmic "winter pattern". At the very same day gonadal regression is initiated and the circannual clock is reset. In contrast to this difference in photoperiodic time measurement, the broad time span in which offspring are born and the birth-season dependent timing of puberty is similar to photoperiodic rodents. We investigated how juvenile European hamsters measure photoperiod to situate themselves at the proper position in the annual cycle. Activity and 6-sulphatoxymelatonin (aMT6s) excretion were recorded in pups of five litters born at different seasons. Pups of all litters showed an activity pattern identical with the adults' summer pattern until postnatal day 78, suggesting that the pathway known to reset the circannual clock in adults is functional. The synchronous start of reproduction in yearlings supports this. However, since puberty and gonadal regression occurred before the switch in the activity pattern, the timing of reproduction in the birth year must be controlled by other means. As in photoperiodic species melatonin might be involved, since the aMT6s excretion showed daily and seasonal rhythms from early life on.

Photoperiod can entrain circannual rhythms in pinealectomized European hamsters.

In mammals, the pineal hormone melatonin is thought to be essential to process environmental photoperiodic information. In this study, we demonstrate in a circannual species, the European hamster Cricetus cricetus, the existence of a melatonin-independent second pathway. In 4 physiological parameters (reproduction, body weight, activity pattern, body temperature), a large majority of pinealectomized European hamsters were entrained to an accelerated photoperiodic regime. It compressed the natural variations in the photoperiod to a 6-month cycle, which allowed us to record up to 6 complete physiological cycles during the life span of the individuals. We show further that whether a pinealectomized animal is able to entrain to changes in the photoperiod is influenced by the season of pinealectomy. The results do not disprove that melatonin is capable of entraining a circannual rhythm, but they show clearly that melatonin is not necessary, demonstrating another melatonin-independent pathway for circannual entrainment by the photoperiod. In view of these new insights, a revision of the original literature revealed that probably the melatonin-independent pathway plays an important role in most circannual mammals but only a minor role in photoperiodic species. Thus, the present work provides also the first evidence for different synchronization mechanisms in photoperiodic and circannual species.

Duper: a mutation that shortens hamster circadian period.

Three animals born to homozygous tau mutant (τ(ss), "super short") Syrian hamsters showed extremely short free-running periods of locomotor activity (τ(DD) of approximately 17.8 hours). Inbreeding produced 33 such "super duper" animals, which had a τ(DD) of 18.09 ± 0.05 hours, which was shorter than that of τ(ss) hamsters (20.66 ± 0.07 hours, p < 0.001). To test the hypothesis that a gene (Duper) is responsible for a 2-hour shortening of τ(DD), we backcrossed super duper hamsters to unrelated τ(ss) animals. The F(1) pups uniformly had a τ(DD) similar to that of τ(ss) hamsters (19.89 ± 0.15 hours), but F(2) animals showed a 1:1 ratio of the 18- to 20-hour phenotypes. In contrast, the F(1) of a cross between super duper hamsters and τ(ss) animals presumed heterozygous for duper showed a 1:1 ratio of 18- to 20-hour phenotypes, and inbreeding of the super duper F(1) offspring uniformly produced F(2) pups with extremely short τ(DD) (17.86 ± 0.5 hours). We isolated the duper mutation on a wild-type background through crossing of super duper with wild-type animals. Restriction digests identified short-period F(2) pups that lack the mutant CK1ε allele, and these animals had a mean τ(DD) of 23.11 ± 0.04 hours. τ(DD) of duper hamsters born and raised in DD was significantly shorter than in hamsters raised in 14L:10D (21.92 ± 0.12 hours, p < 0.0001). τ(DD) shortened twice as much in τ(s) and τ(ss) hamsters than in wild-type animals that were homozygous for duper, indicating the presence of epistatic interactions. Assortment of phenotypes in the F(2) generation fit the expected distribution for expression of duper as recessive (χ(2) = 6.41, p > 0.1). Neither CK1ε nor CK1δ coding region base sequences differed between super duper and τ(ss) hamsters. The growth rate of super duper mutants is similar to that of τ(ss) animals but slightly but significantly reduced at particular postweaning time points. We conclude that duper represents a new mutation that substantially reduces τ(DD) and has significant effects on physiology and metabolism.

Phase shift of the circannual reproductive rhythm in European hamsters by 2 days of long photoperiod.

OBJECTIVES AND DESIGN: In European hamsters a circannual clock drives the seasonal changes in the reproductive state. Its resetting by photoperiod is clearly phase dependent. In mid subjective winter a 1-month pulse of long photoperiod (LP) advances the onset of the reproductive phase of animals maintained in constant short photoperiod (SP) by up to 1.5 months. The present study investigated whether shorter pulses, i.e. 8, 4 or 2 days LP-pulses are still effective to phase shift the circannual rhythm. MAIN FINDINGS: All pulses induced gonadal development after a similar time relative to the offset of the pulse and earlier than in the control group. Thus, they all shared a similar effectiveness. CONCLUSIONS: In European hamsters a very brief LP-pulse can phase shift the reproductive rhythm but its strength is not determined by its duration at least not in the tested range.

Circannual phase response curves to short and long photoperiod in the European hamster.

This study investigated in male European hamsters (Cricetus cricetus ) whether entrainment of circannual rhythms follows the principles of the nonparametric entrainment model. In 2 experiments the times of the year when long (LP) or short photoperiod (SP) are able to synchronize the reproductive cycle were determined, by recording phase response curves (PRCs). A total of 28 groups of 10 hamsters were synchronized by SP, before being subjected to 2 converse experiments: a) 14 groups were transferred to constant LP, only interrupted by SP for 1 month (SP-pulse), the pulse being increasingly delayed between groups by 2 weeks or 1 month steps; and b) the remaining 14 groups stayed in constant SP interrupted by LP for 1 month (LP-pulse) at different phases of the cycle. In a 3rd experiment 5 groups of 10 European hamsters were subjected to constant LP interrupted by 1-month SP-pulses in regular non-365-day zeitgeber intervals (circannual T-cycles) differing between groups (c). The reproductive state was checked every 2 or 4 weeks. The PRCs revealed that an SP-pulse had a very strong phase-resetting capability of -180 degrees to at least +81 degrees in subjective summer (a). During subjective winter when the animals hibernate, a SP-pulse had only weak effectiveness (a) whereas an LP-pulse advanced the circannual clock by up to +41 degrees (b). In the latter conditions a further advance of up to +156 degrees was achieved by the decrease in photoperiod at the return to SP conditions, which terminated the reproductive phase already after 4 to 5 weeks. In different circannual T-cycles the animals entrained for at least 2 cycles (c). In conclusion, 1) the circannual rhythm of European hamsters can be entrained by one photoperiodic signal per cycle, 2) the decrease in photoperiod is most important for its synchronization, and 3) as in circadian clocks the resetting of circannual clocks follows the principles of the nonparametric entrainment model.

Asymmetric control of short day response in European hamsters.

The European hamster (Cricetus cricetus) is a circannual species in which the synchronization of the circannual cycle to the natural year occurs during 2 annual phases of sensitivity. Around the summer solstice, the animals are sensitive to a shortening of photoperiod. During this sensitive phase, pronounced changes in circadian output parameters are observed, indicating a different functional state of the circadian system. This special state is assumed to be necessary to develop the extreme sensitivity to short day length in European hamsters during this phase. In natural conditions, the animals are able to recognize the shortening of photoperiod already in mid-July, when the photoperiod is reduced only by 30 min. To investigate the short-day response in sensitive European hamsters on the basis of the 2-coupled oscillator model of Pittendrigh and Daan (1976), daily activity and the reproductive state of European hamsters were recorded after an asymmetrical reduction of photoperiod from long (LD 16:08) to short (LD 08:16) photoperiods. The activity pattern of the animals showed an immediate response to the short photoperiod at the day of transfer when the night was extended only into the evening, but there was a significant delay in the response time when the night was extended into the morning. Thus, the evening oscillator E is more important in inducing the photoperiodic response than the morning oscillator M. Moreover, the broad intragroup variation in the latter conditions strongly suggests that the changes in the activity pattern were endogenously induced and that the animals were not able to recognize a lengthening of the night into the morning. Gonadal regression started in both groups 3 weeks after the change in the activity pattern, indicating that this process is initiated when the circadian system has received the short-day signal either through changes in photoperiod or through the circannual clock.

Seasonal variations in circadian rhythms coincide with a phase of sensitivity to short photoperiods in the European hamster.

European hamsters (Cricetus cricetus) show pronounced seasonal changes in their physiology and behavior. The present study provides a detailed analysis of the temporal relationship between seasonal cycles of reproduction and body mass and seasonal changes of two circadian parameters, i.e., locomotor activity and 6-sulphatoxymelatonin (aMT6s) excretion, in individual animals kept under natural light conditions. Our results demonstrate a characteristic pattern of locomotor activity and aMT6s excretion observed around the summer solstice, i.e., from mid-May to mid-July. During this time, locomotor activity was characterized by a high level of activity and an early activity onset, while the nightly elevation of melatonin was reduced to baseline levels. These seasonal changes in aMT6s excretion and locomotor activity were only loosely related to changes in the reproductive status of the animals, but correlated well with a period of the annual cycle during which the animals were sensitive to short days. They may therefore reflect a specific state of the circadian pacemaker system within the SCN and can thus be a valuable tool to further characterize molecular and physiological mechanisms of photoperiodic time measurements in European hamsters.

European hamsters (Cricetus cricetus) show a transient phase of insensitivity to long photoperiods after gonadal regression.

Annual rhythms of body weight and reproduction in the European hamster (Cricetus cricetus) are the result of an interaction between seasonal changes in day length (photoperiod) and seasonal changes in the responsiveness of animals to these photoperiods. The present study demonstrates that under natural conditions European hamsters are not able to perceive long photoperiods (i.e., a 16L:8D cycle) before mid-November. This is an important difference to other hamster species, in which regrowth of the gonads can be stimulated by exposure to long photoperiods at any stage of gonadal regression. The experiments also demonstrate the existence of an annual phase of sensitivity to long photoperiods that starts around mid-November and extends until March/April. During this phase of sensitivity, exposure to a long photoperiod (16L:8D) induced gonadal regrowth within 3 wk. Additional experiments with an accelerated photoperiodic lighting regimen indicated that a photoperiod of approximately 13 h is necessary to stimulate gonadal regrowth. Under natural light conditions in Stuttgart (48.46 degrees N), a photoperiod of 13 h is reached by the beginning of April, which fits well with the finding that the majority of animals kept under a natural light:dark cycle had well-developed gonads by the end of April. Nevertheless, these animals showed a rather variable timing of gonadal regrowth, ranging from early January to late April. This is most likely the result of two processes: first, an endogenous mechanism (photorefractoriness) that induces gonadal recrudescence without any photoperiodic information while the animals are still in their hibernation burrows, and second, a direct stimulatory effect of long photoperiods.