Rhythm-Specific Mutations in Drosophila rajasekari Altered Adult Locomotor Activity Rhythm but Not Eclosion Rhythm

The early and late strains for phase angle difference (Φ) of adult locomotor activity in Drosophila rajasekari were developed by artificial selection; these strains differed in Φ, activity pattern, activity level, free-running period (τ) in constant darkness (DD) and light induced phase shifts from those of the wild type (Joshi, 1998). The present studies were designed to determine whether or not the psi-mutations for adult locomotor activity rhythm had also altered the fundamental properties of the eclosion rhythms in these strains. The circadian rhythms of eclosion have been studied in the wild type, the early and late strains. In contrast to the effects on the locomotor activity rhythms in the early and late strains, the psi-mutations have no apparent effect on the eclosion median in light-dark cycles of 12 : 12 h, on τ in DD, light induced phase shifts or subjective light sensitivity in these strains. Thus the psi-mutations for the adult locomotor activity rhythms in D. rajasekari appear to be rhythm-specific mutations altering the locomotor rhythms but not the eclosion rhythms.     

Selection for Phase Angle Difference of the Adult Locomotor Activity in Drosophila rajasekari Affects the Activity Pattern,Free-running Period,Phase Shifts and Sensitivity to Light

Laboratory selection for the phase angle difference (Ψ, the time from lights-off in a 24 h light–dark cycle to an activity onset) of the adult locomotor activity in Drosophila rajasekari reared in LD (light:dark cycles) of 12:12 h for 59 generations resulted in the early and late strains which differed in Ψ value by about 8 h. The selection affected the activity pattern in LD 12:12; in contrast to the wild-type, which had a broad plateau of activity pattern, the early strain exhibited a biphasic activity pattern with morning and evening peaks, whereas the late strain had a single evening peak which extended for 6 h in the dark. The selection significantly decreased and increased the activity level per cycle in LD 12:12, continuous darkness (DD) and continuous light (LL) in the early and late strains respectiv ely when compared to that of the wild-type (P < 0.01). The free running period (τ) in DD was shortened in the early strain and lengthened in the late strain by the shortening and lengthening of the activity phases respectively, the rest phases remained unchanged in these strains from that of the wild-type. Phase response curves (PRCs) were measured for light pulses in all strains, the PRC for the early strain was characterized by larger phase shifts when compared to the PRC for the late or for the wild type flies. The ability of τ to be progressively lengthened by increasing intensity of LL was increased and decreased in the early and late strains respectively. Moreover, the threshold light intensity of LL to generate arrhythmicity was apparently decreased in the early strain and increased in the late strain, suggesting that the selection for Ψ had differently affected the subjective light sensitivity in these strains.     

Multi-Oscillatory Control of Eclosion and Oviposition Rhythms in Drosophila melanogaster: Evidence from Limits of Entrainment Studies

The eclosion and oviposition rhythms of flies from a population of Drosophila melanogaster maintained under constant conditions of the laboratory were assayed under constant light (LL), constant darkness (DD), and light/dark (LD) cycles of 10:10 h (T20), 12:12 h (T24), and 14:14 h (T28). The mean (±95% confidence interval; CI) free-running period (τ) of the oviposition rhythm was 26.34 ± 1.04 h and 24.50 ± 1.77 h in DD and LL, respectively. The eclosion rhythm showed a τ of 23.33 ± 0.63 h (mean ± 95% CI) in DD, and eclosion was not rhythmic in LL. The τ of the oviposition rhythm in DD was significantly greater than that of the eclosion rhythm. The eclosion rhythm of all 10 replicate vials entrained to the three periodic light regimes, T20, T24, and T28, whereas the oviposition rhythm of only about 24 and 41% of the individuals entrained to T20 and T24 regimes, respectively, while about 74% of the individuals assayed in T28 regimes showed entrainment. Our results thus clearly indicate that the τ and the limits of entrainment of eclosion rhythm are different from those of the oviposition rhythm, and hence this reinforces the view that separate oscillators may regulate these two rhythms in D. melanogaster.     

Effects of psi-mutations on the Oviposition Rhythm of Drosophila rajasekari

The psi -mutations affected the circadian rhythm of locomotor activity in the early and late strains of Drosophila rajasekari (Joshi, 1999a). The present study was designed to determine the effects of psi -mutations on the oviposition rhythm of the early and late strains. Oviposition rhythms were studied in light-dark cycles of 12 :12 h in which the light intensity of photophase was 0.001, 0.01, 0.1, 1, 10, 100 or 1000 lux. The oviposition rhythm of wild type was unimodal at or above 10 lux with a peak before lights-off, while it was bimodal at lower light intensities. The early strain was unimodal at all light intensities with a peak after lights-on at or above 10 lux, and around the mid-day at or below 1lux. The late strain was rhythmic at 100 and 1000 lux with a peak after the lights-off, weakly rhythmic at 10 lux and arrhythmic at or below 1 lux. Free running period in constant darkness was shortest in the early and longest in the late strain. Threshold light intensity of constant light to generate arrhythmicity was lowest in the early and highest in the late strain, apparently the photic sensitivity of the clock photoreceptors was differentially altered by these mutations. Thus the psi -mutations for locomotor rhythmicity affected the oviposition rhythm too, suggesting that the same circadian oscillator might be controlling these both rhythms.     

Effects of psi-mutations on the Oviposition Rhythm of Drosophila rajasekari

The psi -mutations affected the circadian rhythm of locomotor activity in the early and late strains of Drosophila rajasekari (Joshi, 1999a). The present study was designed to determine the effects of psi -mutations on the oviposition rhythm of the early and late strains. Oviposition rhythms were studied in light-dark cycles of 12 :12 h in which the light intensity of photophase was 0.001, 0.01, 0.1, 1, 10, 100 or 1000 lux. The oviposition rhythm of wild type was unimodal at or above 10 lux with a peak before lights-off, while it was bimodal at lower light intensities. The early strain was unimodal at all light intensities with a peak after lights-on at or above 10 lux, and around the mid-day at or below 1lux. The late strain was rhythmic at 100 and 1000 lux with a peak after the lights-off, weakly rhythmic at 10 lux and arrhythmic at or below 1 lux. Free running period in constant darkness was shortest in the early and longest in the late strain. Threshold light intensity of constant light to generate arrhythmicity was lowest in the early and highest in the late strain, apparently the photic sensitivity of the clock photoreceptors was differentially altered by these mutations. Thus the psi -mutations for locomotor rhythmicity affected the oviposition rhythm too, suggesting that the same circadian oscillator might be controlling these both rhythms.     

Entrainment of eclosion rhythm in Drosophila melanogaster populations reared for more than 700 generations in constant light environment

In this paper, we report the results of our extensive study on eclosion rhythm of four independent populations of Drosophila melanogaster that were reared in constant light (LL) environment of the laboratory for more than 700 generations. The eclosion rhythm of these flies was assayed under LL, constant darkness (DD) and three periodic light-dark (LD) cycles (T20, T24, and T28). The percentage of vials from each population that exhibited circadian rhythm of eclosion in DD and in LL (intensity of approximately 100 lux) was about 90% and 18%, respectively. The mean free-running period (τ) of eclosion rhythm in DD was 22.85 ± 0.87 h (mean ± SD). Eclosion rhythm of these flies entrained to all the three periodic LD cycles, and the phase relationship (ψ) of the peak of eclosion with respect to “lights-on” of the LD cycle was significantly different in the three periodic light regimes (T20, T24, and T28). The results thus clearly demonstrate that these flies have preserved the ability to exhibit circadian rhythm of eclosion and the ability to entrain to a wide range of periodic LD cycles even after being in an aperiodic environment for several hundred generations. This suggests that circadian clocks may have intrinsic adaptive value accrued perhaps from coordinating internal metabolic cycles in constant conditions, and that the entrainment mechanisms of circadian clocks are possibly an integral part of the clockwork.     

Entrainment Properties of the Locomotor Activity Rhythm of Drosophila melanogaster Under Different Photoperiodic Regimens

In this paper we report the results of an experiment to assess how closely repeated brief light pulses (LPs) mimic the effects of 12:12 h light/dark (LD) cycles (PPc). The locomotor activity rhythm of individual fruit flies from a laboratory population of Drosophila melanogaster was monitored under four different photoperiodic regimens, created using 12 h of light and 12 h of darkness or brief light pulses (LPs). The phase relationship (Ψ) and the stability (precision) of the locomotor activity rhythm during entrainment were estimated in order to compare the state of the circadian clocks under the four different photoperiodic regimens. The flies (n = 72) were subjected to four different LD cycles: (i) 12 h of light and 12 h of darkness (complete photoperiod, PPc); (ii) a single brief LP of 15 min duration presented close to the onset of activity (SLP-1); (iii) a single brief LP of 15 min duration administered close to the offset of activity (SLP-2); and (iv) two brief LPs administered 12 h apart (skeleton photoperiod, PPs). The locomotor activity rhythm of the flies was first monitored under constant darkness (DD) for about 10 days and then under the four different photoperiodic regimens for about 10 days, and finally in DD for the remainder of the experiment. The Ψ of the locomotor activity rhythm and its precision under PPc and PPs did not differ significantly, but they were significantly different from the SLP-1 and SLP-2 conditions. The results provide interesting insights into photoentrainment mechanisms of circadian clocks in D. melanogaster, and suggest that skeleton photoperiods, but not single brief LPs, mimic the actions of complete photoperiods.     

Internal Desynchronisation in Blow Fly (Calliphora vicina) Locomotor Activity Rhythms: Evidence for a Complex Circadian Pacemaker

The majority of blow flies (Calliphora vicina) display circadian locomotor activity rhythms that free-run with an unchanging period (τ) in darkness (DD), or entrain to a light-dark cycle (LD 1:23). However, a minority produce more complex patterns (spontaneous changes in τ, arrhythmicity, or 'split' rhythms) in DD, or undergo rhythm dissociation ('internal desynchronisation') when the light pulses of LD 1:23 initially illuminate the subjective night. These patterns are interpreted as evidence for a complex, multioscillatory and multicellular, structure of the insects' circadian system, and this complexity is discussed in terms of the neuronal architecture of the fly's brain.     

Effect of light intensity on the phase and period response curves in the nocturnal field mouse Mus booduga

The effect of light intensity on the phase response curve (PRC) and the period response curve (τRC) of the nocturnal field mouse Mus booduga was studied. PRCs and τRCs were constructed by exposing animals free-running in constant darkness (DD), to fluorescent light pulses (LPs) of 100 lux and 1000 lux intensities for 15min duration. The waveform of the PRCs and τRCs evoked by high light intensity (1000 lux) stimuli was significantly different compared to those constructed using low light intensity (100 lux). Moreover, a weak but significant correlation was observed between phase shifts and period changes when light stimuli of 1000 lux intensity were used; however, the phase shifts and period changes in the 100 lux PRC and τRC were not correlated. This suggests that the intensity of light stimuli affects both phase and period responses in the locomotor activity rhythm of the nocturnal field mouse M. booduga. These results indicate that complex mechanisms are involved in entrainment of circadian clocks, even in nocturnal rodents, in which PRC, τRC, and dose responses play a significant role.     

Does the difference in the timing of eclosion of the fruit fly Drosophila melanogaster reflect differences in the circadian organization?

The eclosion rhythm of a laboratory population of Drosophila melanogaster was studied under 12h light, 12h dark (LD 12:12) cycles. Although most of the flies were found to eclose just after “lights on” in LD 12:12, termed within gate (WG) flies, a few flies were found to eclose nearly 10h after peak eclosion, termed outside gate (OG) flies. The circadian parameters of the clocks controlling oviposition rhythms in the WG and the OG flies were estimated to understand the cause of such differences in the timing of eclosion. The distribution of the fraction of individual flies exhibiting single, multiple, and no significant period in the WG flies was significantly different from distribution in the OG flies. Compared to the WG flies, more OG flies were found to exhibit oviposition rhythm with multiple periodicity, whereas more WG flies exhibited an oviposition rhythm with a single significant period. The fraction of flies with arrhythmic oviposition was similar in both the WG and the OG flies. Free-running period τ in constant darkness (DD) and the phase angle difference ψ in LD 12:12 for the oviposition rhythm of WG and OG flies were significantly different. These results suggest that the differences in the time of eclosion between the flies eclosing within the gate and outside the gate of eclosion are probably due to differences in the circadian system controlling eclosion, which is reflected by the differences in their oviposition rhythm. (Chronobiology International, 18(4), 601-612, 2001)     

The circadian system of Trypanosoma cruzi-infected mice

The effects of Chagas disease on the mammalian circadian system were studied in Trypanosoma cruzi-infected C57-Bl6J mice. Animals were inoculated with CAI or RA strains of T. cruzi or vehicle, parasitism confirmed by blood specimen visualization and locomotor activity rhythms analyzed by wheel-running recording. RA-strain infected mice exhibited significantly decreased amplitude of circadian rhythms, both under light-dark and constant dark conditions, probably due to motor deficiencies. CAI-treated animals showed normal locomotor activity rhythms. However, in these mice, reentrainment to a 6h phase shift of the LD cycle took significantly longer than controls, and application of 15min light pulses in DD produced smaller phase delays of the rhythms. All groups exhibited light-induced Fos expression in the suprachiasmatic nuclei. We conclude that the main effect of T. cruzi infection on the circadian system is an impairment of the motor output from the clock toward controlled rhythms, together with an effect on circadian visual sensitivity.     

Many Circadian Oscillators Regulate Developmental and Behavioural Events in the Flesh-Fly, Sarcophaga Argyrostoma

In the flesh-fly, Sarcophaga argyrostoma, the initiation of larval wandering, pupal eclosion, and the induction of pupal diapause by seasonal changes in night length, are all regulated by circadian oscillators. They differ, however, in several respects. The rhythm of larval wandering shows a free-running period (S) of about 20 hr and a steady-state phase-relationship to the light cycle (±) in which maximum activity occurs at dusk or in the night; that for pupal eclosion shows ± close to 24 hr and ± close to dawn; and that for diapause induction ± longer than 24 hr and a photoinducible phase (φ_i) late in the subjective night. The three oscillators are, therefore, considered to be functionally separate. In addition, adult locomotor activity, the deposition of cuticular growth layers on thoracic apodemes, and the duration of larval wandering, are possibly regulated by further, distinct, oscillators. The circadian system in S. argyrostoma, therefore, contains at least three, and probably as many as six, known circadian pacemakers.     

Locomotor activity rhythms in cave fishes from Chapada Diamantina, northeastern Brazil (Teleostei: Siluriformes)

Previous studies on the locomotor activity of troglobitic (exclusively subterranean) species have shown that circadian rhythmicity may be reduced in populations evolving in the absence of zeitgebers such as daily cycles of light and temperature; therefore, circadian activity rhythms, although not infradian nor ultradian rhythms, seem to have been selected by external, ecological factors. We studied the locomotor activity of a highly specialized Heptapteridae catfish (undescribed genus and species) from Chapada Diamantina, NE Brazil, compared to another specialized Brazilian troglobitic heptapterid, Taunayia sp. Locomotor activity was continuously measured in the laboratory with an infra-red photocell system. Seven specimens of the new genus were tested, each one during 14 consecutive days according to the following schedule: three days in DD → seven days in LD (12:12 h) → four days in DD. Data were submitted both to fast Fourier transform periodogram followed by Siegel's test of significance and Lombs - Scargle periodogram techniques in order to identify spectral composition of the time series. In general, results were similar to those obtained for Taunayia sp.: (a) for most specimens, absence of significant circadian components in locomotor activity under DD; (b) for all specimens, significant circadian components under LD, with higher levels of activity during the dark phase, as expected for species belonging to nocturnal epigean taxa; (c) for most specimens, no residual oscillations recorded when free-running conditions were reinstalled. Circadian locomotor activity detected under LD may thus be interpreted as a direct, masking effect of the LD cycle. This suggests a pattern for highly specialized troglobitic species, isolated for a long time in the subterranean habitat, with a progressive reduction of circadian time-keeping mechanisms. Our studies also demonstrate the potential of subterranean organisms for investigation of the origin, evolution, functioning and genetics of circadian rhthmicity.     

A case for multiple oscillators controlling different circadian rhythms in Drosophila melanogaster

A population of the fruit fly Drosophila melanogaster was raised in periodic light/dark (LD) cycles of 12:12 h for about 35 generations. Eclosion, locomotor activity, and oviposition were found to be rhythmic in these flies, when assayed in constant laboratory conditions where the light intensity, temperature, humidity and other factors which could possibly act as time cue for these flies, were kept constant. These rhythms also entrained to a LD cycle of 12:12 h in the laboratory with each of them adopting a different temporal niche. The free-running periods (tau) of the eclosion, locomotor activity and oviposition rhythms were significantly different from each other. The peak of eclosion and the onset of locomotor activity occurred during the light phase of the LD cycle, whereas the peak of oviposition was found to occur during the dark phase of the LD cycle. Based on these results, we conclude that different circadian oscillators control the eclosion, locomotor activity and oviposition rhythms in the fruit fly D. melanogaster.     

Locomotor Rhythms in the Fiddler Crab, Uca subcylindrica

The locomotor activities of individual specimens of Uca subcylindrica (Stimpson) collected from semi-arid, supratidal habitats in south Texas and northeastern Mexico were studied in the laboratory using periodogram analysis. When crabs were placed under constant darkness (DD) or constant illumination (LL), free-running circadian rhythms were observed in the activity recordings. The locomotor activity of strongly rhythmic crabs in LL has an average period length of 24.4 h. Crabs held in DD express motor rhythms with periods of approximately 24.0 h. In LL the most common wave form for activity is unimodal, while under DD it is bimodal. Recordings under natural illumination (NL) revealed that both period length and the time of maximum activity (phasing) varied through the year. During winter months, the crabs are primarily diurnal with peaks in activity occurring between 0900 and 2100 h and possess a circadian rhythm with a 23.9 h period. During summer, crabs were nocturnal with maximal activity between 1300 and 0600 and a circadian period closer to 24.0 h. In these experiments, the rhythmic locomotor activities of U. subcylindrica are best described as “circadian”. This is unusual for a genus known for its expression of circatidal and circalunidian rhythms.     

Circadian Rhythms of Locomotor Activity in Lycosa tarentula (Araneae, Lycosidae) and the Pathways of Ocular Entrainment

Lycosa tarentula is a ground-living spider that inhabits a burrow where it awaits the appearance of prey or conspecifics. In this study, circadian rhythms of locomotor activity were examined as well as the ocular pathway of entrainment. Thirty-three adult virgin females were examined under constant darkness (DD); all of them exhibited robust circadian rhythms of locomotor activity with a period averaging 24.1h. Fourteen of these spiders were studied afterwards under an LD 12:12 cycle; they usually entrained to in the first or second day, even when the light intensity was as low as 1 lx. During the LD cycle, locomotor activity was generally restrained to the darkness phase, although several animals showed a small amount of diurnal activity. Ten males were also examined under LD; they were also nocturnal, but were much more active than the females. Seven females were examined under constant light (LL); under this they became arrhythmic. Except for the anterior median eyes (OMAs), all the eyes were capable of entraining the locomotor activity to an LD cycle. These results demonstrate that under laboratory conditions and low light intensities locomotor activity of Lycosa tarentula is circadian and in accordance with Aschoff's 'rule'. Only OMAs are unable to entrain the rhythm; the possible localization of circadian clock is therefore discussed.     

Role of Proinflammatory Cytokines on Lipopolysaccharide-Induced Phase Shifts in Locomotor Activity Circadian Rhythm

We previously reported that early night peripheral bacterial lipopolysaccharide (LPS) injection produces phase delays in the circadian rhythm of locomotor activity in mice. We now assess the effects of proinflammatory cytokines on circadian physiology, including their role in LPS-induced phase shifts. First, we investigated whether differential systemic induction of classic proinflammatory cytokines could explain the time-specific behavioral effects of peripheral LPS. Induction levels for plasma interleukin (IL)-1α, IL-1β, IL-6, or tumor necrosis factor (TNF)-α did not differ between animals receiving a LPS challenge in the early day or early night. We next tested the in vivo effects of central proinflammatory cytokines on circadian physiology. We found that intracerebroventricular (i.c.v.) delivery of TNF-α or interleukin IL-1β induced phase delays on wheel-running activity rhythms. Furthermore, we analyzed if these cytokines mediate the LPS-induced phase shifts and found that i.c.v. administration of soluble TNF-α receptor (but not an IL-1β antagonistic) prior to LPS stimulation inhibited the phase delays. Our work suggests that the suprachiasmatic nucleus (SCN) responds to central proinflammatory cytokines in vivo, producing phase shifts in locomotor activity rhythms. Moreover, we show that the LPS-induced phase delays are mediated through the action of TNF-α at the central level, and that systemic induction of proinflammatory cytokines might be necessary, but not sufficient, for this behavioral outcome. (Author correspondence: dgolombek@unq.edu.ar)     

Circadian Rhythm in the Locomotor Activity of a Surface-Dwelling Millipede Syngalobolus sp.

Locomotor activity of the surface-dwelling millipede Syngalobolus sp. was recorded under laboratory conditions. Infra-red diodes were used to detect the locomotor activity in an oval shaped chamber, which was connected with an event recorder. The results of 11 individuals showed that the millipedes entrained to light/dark (LD12:12 h) conditions with negative phase angle difference (-83.2 ± 24.72 min). The millipedes showed a clear-cut free-running rhythm with a period (t) of 23.8 ± 1.0 h (n = 9) in constant darkness (DD). The period in continuous light (LL) was relatively greater (25.2 ± 0.1 h; n = 3) than that in DD.     

Possible evidence for shift work schedules in the media workers of the ant species Camponotus compressus

The locomotor activity rhythm of the media workers of the ant species Camponotus compressus was monitored under constant conditions of the laboratory to understand the role of circadian clocks in social organization. The locomotor activity rhythm of most ants entrained to a 24 h light/dark (12:12 h; LD) cycle and free-ran under constant darkness (DD) with circadian periodicities. Under entrained conditions about 75% of media workers displayed nocturnal activity patterns, and the rest showed diurnal activity patterns. In free-running conditions these ants displayed three types of activity patterns (turn-around). The free-running period (τ) of the locomotor activity rhythm of some ants (10 out of 21) showed period lengthening, and those of a few (6 out of 21) showed period shortening, whereas the locomotor activity rhythm of the rest of the ants (5 out of 21) underwent large phase shifts. Interestingly, the pre-turn-around τ of those ants that showed nocturnal activity patterns during earlier LD entrainment was shorter than 24 h, which became greater than 24 h after 6-9 days of free-run in DD. On the other hand, the pre-turn-around τ of those ants, which exhibited diurnal patterns during earlier LD entrainment, was greater than 24 h, which became shorter than 24 h after 6-9 days of free-run in DD. The patterns of activity under LD cycles and the turn-around of activity patterns in DD regime suggest that these ants are shift workers in their respective colonies, and they probably use their circadian clocks for this purpose. Circadian plasticity thus appears to be a general strategy of the media workers of the ant species C. compressus to cope with the challenges arising due to their roles in the colony constantly exposed to a fluctuating environment.     

Presence of circadian rhythms in the locomotor activity of a cave-dwelling millipede Glyphiulus cavernicolus sulu (Cambalidae, Spirostreptida)

The locomotor activity of the millipede Glyphiulus cavernicolus (Spirostreptida), which occupies the deeper recesses of a cave, was monitored in light-dark (LD) cycles (12h light and 12h darkness), constant darkness (DD), and constant light (LL) conditions. These millipedes live inside the cave and are apparently never exposed to any periodic factors of the environment such as light-dark, temperature, and humidity cycles. The activity of a considerable fraction of these millipedes was found to show circadian rhythm, which entrained to a 12:12 LD cycle with maximum activity during the dark phase of the LD cycle. Under constant darkness (DD), 56.5% of the millipedes (n = 23) showed circadian rhythms, with average free-running period of 25.7h ± 3.3h (mean ± SD, range 22.3h to 35.0h). The remaining 43.5% of the millipedes, however, did not show any clear-cut rhythm. Under DD conditions following an exposure to LD cycles, 66.7% (n = 9) showed faint circadian rhythm, with average free-running period of 24.0h ± 0.8h (mean ± SD, range 22.9h to 25.2h). Under constant light (LL) conditions, only 2 millipedes of 11 showed free-running rhythms, with average period length of 33.3h ± 1.3h. The results suggest that these cave-dwelling millipedes still possess the capacity to measure time and respond to light and dark situations. (Chronobiology International, 17(6), 757-765, 2000)