Based on data from light-loggers on free-ranging urban European blackbirds  we simulated light-at-night exposure in the laboratory and found that it led to reductions in melatonin secretion, which depended on the time of year and time of night (Figure 1). In particular, in winter (Figure 1a) plasma melatonin concentrations were reduced in the beginning and at the end of the night, around the time of evening and morning twilight. In summer, we detected an overall significant main effect of the light-at-night treatment, as melatonin concentrations were lower in the experimental group during the whole night. Overall, our results suggest that light-at-night of very low intensity is able to reduce melatonin release in blackbirds. We speculate that light-induced melatonin suppression may lead to an altered perception of daylength in this species, in particular in winter (late January), when the birds’ reproductive system is reactivated under the influence of increasing daylength . Indeed, in winter the suppression of melatonin in the late night/early morning correlated with the amount of activity just preceding dawn in the birds exposed to light-at-night (Figure 2a): the greater the reduction in melatonin, the greater the amount of morning activity (Figure 3a). The differences in early morning activity could be particularly relevant for the blackbirds’ breeding biology, because at this time the birds’ song activity and mating activity peaks [12, 56].
Suppressed melatonin and increased activity may suggest a physiological mechanism underlying the advanced onset of morning activity and song recorded in several urban bird species in late winter and spring [11, 12, 54, 57]. Based on both, a shorter duration of elevated melatonin and of restfulness at night, blackbirds appeared to have indeed interpreted an urban-like illuminated night as being shorter than a dark night . The findings of an advanced activity onset of birds exposed to light-at-night fit well with considerations from early chronobiology that were based on oscillator theory [58, 59]. In addition to its possible, direct effects on melatonin, nocturnal illumination could also affect the circadian system because it modifies the properties of the diel light signal. The diel light signal is the most powerful synchronizing cue (Zeitgeber) of the circadian system. Light-at-night weakens the strength of the Zeitgeber (given by its amplitude ) because the difference between the darkest and brightest illumination over the course of a day decreases. Using songbirds as study subjects, Aschoff and Wever  have shown that such a decrease in Zeitgeber strength advanced the timing of activity (i.e., the phase angle difference, see also ). Similarly, the differences in melatonin amplitude between summer and winter seasons are also in keeping with early chronobiological observations of changes in the circadian system associated with seasonal change in the Zeitgeber signal [60, 61].
Our findings may have important implications for understanding the control of seasonal processes in urbanized birds, because advanced timing of reproduction has been reported in many species in cities [62, 63]. In particular, European blackbirds from an urban population showed earlier gonadal growth than rural counterparts , and this has been recently linked to the exposure to light-at-night . Could the reduced release of nocturnal melatonin due to light-at-night, as observed in the present study, explain the advanced reproductive physiology of urban birds? Although the direct role of melatonin in photoperiodic time measurement in birds has been long debated [15, 64], new evidence suggests that melatonin can affect some seasonal processes. For example, exogenous melatonin administration can down-regulate the volume of two brain areas involved in seasonal song production, HVC and area X . In addition, melatonin up-regulates GnIH in both the brain  and the gonads  before breeding, thus keeping gonadal sizes and testosterone levels low. We suggest that the decrease in melatonin as a consequence of light-at-night in winter may provide animals with the physiological signal that the night is shorter than the actual photoperiod, and therefore speeds up reproductive activation. We have recently shown that the blackbirds subjected to 0.3 lux of light-at-night developed the reproductive system almost a month earlier than birds kept under dark nights . However, in this former study we found differences in the response of urban and rural blackbirds. Light-at-night had a stronger, accelerating effect on the termination of reproduction in late spring in urban compared to rural birds. In the present study, however, urban and rural birds did not differ in the melatonin response to the night-light stimulation in both seasons. Thus, the melatonin findings reported here can explain overall effects of night-at-light, but do not elucidate differences between urban and rural blackbirds in their gonadal responses to nocturnal illumination. For these differences, it seems that other major factors may be involved. Interestingly, the only interaction between origin and treatment detected in the present study was found for the summer and concerned activity levels. Light at night suppressed activity in urban birds, while it activated rural birds (Additional file 1: Figure S1b).
It is worth to notice that the low nocturnal light levels to which birds in our study were experimentally exposed are similar to those which under natural conditions, occur during full moon. Studies on rodents have shown that a full moon night can impair the properties of the circadian clock and the phase-angle relationship with the external light–dark cycle [41, 66]. However, full moon does not occur as regularly as night-at-light in cities, and it is unlikely that it will affect seasonal rhythms in terrestrial songbirds. Although a single long day is potentially able to alter the response of the avian reproductive axis [67–69], the downstream effects on the time of reproduction (i.e. lay dates) may be limited .
Overall melatonin levels differed seasonally. Both maximum amplitude and average concentration were lower in winter than in summer, irrespective of the light treatment. Seasonal variation in avian melatonin profiles has previously been studied with equivocal results in a number of long-distance migrants and arctic species [35, 70–72]. Data on seasonal change in diel melatonin in non-migratory songbirds, to our knowledge, are limited to the house sparrow (Passer domesticus) . In this species, melatonin amplitude was found to be higher in spring and summer than in winter. Our results, therefore, confirm and expand previous data on seasonal variation in melatonin amplitude in passerine species, but more experimental work is needed to understand the causes and consequences of such variation.
In additional to seasonal differences, individual amplitudes of the diel melatonin secretion tended to be lower in the experimental birds than in the control individuals (Table 1). Perhaps this trend indicates that light-at-night may not only have the capacity to reduce the duration of nocturnal melatonin concentration, but also its diel amplitude. In migratory songbirds, melatonin amplitude has been found to be functionally related to the amount of nocturnal restlessness, or Zugunruhe, such that during times when birds show Zugunruhe at night circulating melatonin concentrations decrease . This decrease in melatonin levels at night has been theorized to reduce the degree of self-sustainment of the circadian clock, allowing for alternative temporal activity patterns such as nocturnal migratory activity . We suggest that light-at-night, via melatonin-suppression, may also reduce self-sustainment of clocks in avian urban-dwellers. Recent data from our study populations partially support this hypothesis . Urban birds seem to have faster but also weaker circadian clocks than rural conspecifics, although it is not clear whether these differences have originated from micro-evolutionary changes or from a phenotypic response to different environmental conditions in the city, such as after-effects of light at night .