Psi-Mutation Affects Phase Angle Difference, Free-Running Period and Phase Shifts in Aedes krombeini (Stegomyia)

A new mutation, designated as psi-mutant, affecting the timing of the circadian oviposition rhythm was discovered the in natural population of Aedes krombeini . This mutation advanced the phase of the oviposition median in an entraining light-dark cycle of 12:12 h by ca. 7.0 h and shortened the free running period t in constant darkness (DD) by ca. 4.0 h. Early oviposition in psi-mutants was also observed when while free-running in DD they were subjected to 24-h temperature cycles (29°C for 12 h and l8°C for l2 h). When the phase response curves (PRCs) for light pulses against DD as background were measured, the PRC for the psi-mutant had large delaying phase shifts, whereas, that of the wild strain had small delaying phase shifts.     

Psi-Mutation Affects Phase Angle Difference,Free-Running Period and Phase Shifts in Aedes krombeini (Stegomyia)

A new mutation, designated as psi-mutant, affecting the timing of the circadian oviposition rhythm was discovered the in natural population of Aedes krombeini. This mutation advanced the phase of the oviposition median in an entraining light-dark cycle of 12:12 h by ca. 7.0 h and shortened the free running period t in constant darkness (DD) by ca. 4.0 h. Early oviposition in psi-mutants was also observed when while free-running in DD they were subjected to 24-h temperature cycles (29°C for 12 h and l8°C for l2 h). When the phase response curves (PRCs) for light pulses against DD as background were measured, the PRC for the psi-mutant had large delaying phase shifts, whereas, that of the wild strain had small delaying phase shifts.     

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.     

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.     

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)     

A Mutation in Drosophila simulans that Lengthens the Circadian Period of Locomotor Activity

The length of the Thr-Gly repeat within the period gene of Drosophilids, coevolves with its immediate flanking region to maintain the temperature compensation of the fly circadian clock. In Drosophila simulans, balancing selection appears to maintain a polymorphism in this region, with three repeat lengths carrying 23, 24 or 25 Thr-Gly pairs, each in complete linkage disequilibrium with a distinctive flanking region amino acid moiety. We wondered whether separating a specific length repeat from its associated flanking haplotype might have functional implications for the circadian clock. We fortuitously discovered a population of flies collected in Kenya, in which a chimeric Thr-Gly haplotype was segregating that carried the (Thr-Gly)24 repeat, but the flanking region of a (Thr-Gly)23 allele. One of the five isofemale lines that carried this 'mutant' Thr-Gly sequence showed a dramatically long and temperature-sensitive free-running circadian period. This phenotype was mapped to the X chromosome, close to the D. simulans per gene, but there was also a significant effect of a modifying autosomal locus or loci. It seems remarkable that such a mutant phenotype should be discovered in a screen of chimeric Thr-Gly regions.     

Zebrafish Temperature Selection and Synchronization of Locomotor Activity Circadian Rhythm to Ahemeral Cycles of Light and Temperature

In addition to light cycles, temperature cycles are among the most important synchronizers in nature. Indeed, both clock gene expression and circadian activity rhythms entrain to thermocycles. This study aimed to extend our knowledge of the relative strength of light and temperature as zeitgebers for zebrafish locomotor activity rhythms. When the capacity of a 24∶20°C (thermophase∶cryophase, referred to as TC) thermocycle to synchronize activity rhythms under LL was evaluated, it was found that most groups (78%) synchronized to these conditions. Under LD, when zebrafish were allowed to select the water temperature (24°C vs. 20°C), most fish selected the higher temperature and showed diurnal activity, while a small (25%) percentage of fish that preferred the lower temperature displayed nocturnal activity. Under conflicting LD and TC cycles, fish showed diurnal activity when the zeitgebers were in phase or in antiphase, with a high percentage of activity displayed around dawn and dusk (22% and 34% of the total activity for LD/TC and LD/CT, respectively). Finally, to test the relative strength of each zeitgeber, fish were subjected to ahemeral cycles of light (T=25 h) and temperature (T=23 h). Zebrafish synchronized mostly to the light cycle, although they displayed relative coordination, as their locomotor activity increased when light and thermophase coincided. These findings show that although light is a stronger synchronizer than temperature, TC cycles alone can entrain circadian rhythms and interfere in their light synchronization, suggesting the existence of both light‐ and temperature‐entrainable oscillators that are weakly coupled.     

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.     

Responses of the Circadian Locomotor Activity Rhythm of Mus Booduga to Shifts in LD Schedules

The responses of the field mouse Mus booduga to shifts in schedules of LD cycles were monitored and the results were interpreted with the help of a PRC constructed for the same species. The results reveal that, M. booduga reentrained faster with a lesser number of transients after delay shifts than advance shifts, thus exhibiting “asymmetry effect.” A positive correlation was observed between the number of transients and the number of hours of shift. In most of the shifts, the sign of the transients (negative for delaying transients and positive for advancing transients) coincided with the direction of the shift. Interestingly, 11 and 12 h of advance shifting resulted in delaying transients. An 11-h advance shift can also be interpreted as a 13-h delay. Reentrainment through delaying transients is faster as compared to reentrainment through advancing transients. Thus, this animal might have taken a “shorter route,” as proved by the fact that an 11-h advance shift has evoked delaying transients. But a 13-h advance shift evoked only advancing transients. This prompts us to speculate that there may be a “phase jump” in M. booduga. Further, irrespective of whether L or D has been doubled in a 12-h shift, both evoked only delaying transients.     

Varying the Length of Dim Nocturnal Illumination Differentially Affects the Pacemaker Controlling the Locomotor Activity Rhythm of Drosophila Jambulina

Photic entrainment of animals in the field is basically attributed to their exposure to the dimly lit nights flanked by the dawn and dusk twilight transitions. This implicates the functional significance of the dimly lit nights as that of the twilight transitions. Recently, the authors have demonstrated that the dimly lit night at 0.0006 lux altered the attributes of the circadian rhythm of locomotor activity of Drosophila jambulina. The present study examined whether the durations of such dimly lit nights affect the entrainment and free-running rhythmicity of D. jambulina. Flies were subjected for 10 days to two types of 24-h lighting regimes in which the photophase (L) was at 10 lux for all flies but the scotophase, which varied in duration from 9 to 15?h, was either at 0 lux (D phase) for control flies or 0.0006 lux (the artificial starlight or S phase) for experimental flies. Thereafter, they were transferred to constant darkness (DD) to compare the after-effects of the dimly lit nights on the period (τ) of free-running rhythm in DD with that of the completely dark nights. Control flies were entrained by all LD cycles, but the experimental flies were entrained only by five LS cycles in which the duration of the S phases ranged from 10 to 14?h. The two LS cycles with very short (9?h) and long (15?h) S phases rendered the flies completely arrhythmic. Control flies started activity shortly before lights-on and continued well after lights-off. The experimental flies, however, commenced activity several hours prior to lights-on but ended activity abruptly at lights-off as the result of a negative masking effect of nocturnal illumination. Length of the midday rest was considerably shorter in the control than in the experimental flies in each lighting regime. The active phase in the control flies was predictably shortened; nonetheless, it was invariable in the experimental flies as the nights lengthened. Transfer from lighting regimes to DD initiated robust free-running rhythmicity in all flies including the arrhythmic ones subjected to LS cycles with 9 and 15?h of scotophases. The τ was profoundly affected by the nocturnal irradiance of the prior entraining lighting regime, as it was always shorter in the experimental than in the control flies. Thus, these results indisputably demonstrate the changes in fundamental properties of the circadian pacemaker of D. jambulina were solely attributed to the extremely dim nocturnal irradiance. This strain of D. jambulina is entrained essentially by the dimly lit natural nights, since it is never exposed to the prevailing photic cues such as the twilight transitions or bright photoperiod, owing to the dense vegetation of its habitat. (Author correspondence: drdsjoshi08@gmail.com)     

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (≡24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal''s endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.     

Stocking Density Affects Circadian Rhythms of Locomotor Activity in African Catfish,Clarias gariepinus

The effect of stocking density on the locomotor activity of African catfish C. gariepinus under different light regimes was investigated. C. gariepinus were stocked under different densities (1, 5, or 10 fish/tank), and their locomotor activity recorded under light-dark (LD), constant light (LL), constant darkness (DD), and LD-reversed (DL) regimens. Under the LD cycle, catfish showed a crepuscular activity pattern, irrespective of stocking density, with most of the daily activity concentrated around the light-onset and light-offset times. When fish were subjected to DD, all 4 tanks with medium (5 fish) and high (10 fish) stocking densities showed circadian rhythmicity, with an average period (τ) of 23.3?±?0.5 and 24.6?±?0.5?h, respectively. In contrast, only 2 low (1 fish) density tanks showed free-running rhythms. Under LL, activity levels decreased significantly in comparison with levels observed under LD and DD. Moreover, fish of 1, 2, and 3 out of the 4 tanks with low, medium, and high densities, respectively, showed free-running rhythms under these conditions. When the photocycle was reversed (DL), fish of 3, 2, and 4 out of the 4 tanks with low, medium, and high stocking densities, respectively, showed gradual resynchronization to the new phase, and transient cycles of activity were observed. These results suggest that stocking density of fish affected the display of circadian rhythmicity and the intensity of activity levels. Thus, fish kept in higher densities showed more robust rhythmicity and higher levels of daily activity, indicating that social interactions may have an influence on behavioral patterns in the African catfish. (Author correspondence: lmvera@um.es)     

Light Cycles Given During Development Affect Freerunning Period of Circadian Locomotor Rhythm of period Mutants in Drosophila melanogaster

We reared wild type (Canton-S) and period mutant flies, i.e., per(S) and per(L), of Drosophila melanogaster in constant darkness, constant light or 24h light dark cycles with various light to dark ratios throughout the development from embryo to early adult. The locomotor activity rhythms of newly eclosed individuals were subsequently monitored in the lighting conditions, in which they had been reared, for several days and then in constant darkness. Circadian rhythms were clearly exhibited in constant darkness even in flies reared in constant light and constant darkness as well as flies reared in light-dark cycles, but the freerunning period differed among groups. The results suggest that the circadian clock is assembled without any cyclical photic information, and that the light influences the developing circadian clock of Drosophila to alter the freerunning period. The effects of light on the rhythm differed in some aspects between per(L) flies and the other two strains. Possible mechanisms through which light affects the developing circadian clock are discussed. Copyright 1997 Elsevier Science Ltd. All rights reserved     

Quantification of Locomotion and the Effect of Food Deprivation on Locomotor Activity in Drosophila

A new method to quantify locomotor behavior in Drosophila is presented, and compared with previous methods. It is based upon a radar wave, reflected by moving flies. A problem associated with the new apparatus is that its output is dependent on fly size. However, for the case the weight of the experimental flies has been determined, a correction is proposed. The method has been used by studying the effect of starvation upon locomotion in Drosophila melanogaster. It was found that starved flies are much more active than well fed flies. The importance of this effect under several conditions is discussed.     

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages ^1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) ³. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis ^4,5. Here, we have modified a previously described procedure for insulin injection ⁶ and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly''s ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.Download video file.^{(37M, mov)}     

Influence of Light Intensity on Plasma Melatonin and Locomotor Activity Rhythms in Tench

Melatonin production by the pineal organ is influenced by light intensity, as has been described in most vertebrate species, in which melatonin is considered a synchronizer of circadian rhythms. In tench, strict nocturnal activity rhythms have been described, although the role of melatonin has not been clarified. In this study we investigated daily activity and melatonin rhythms under 12∶12 light‐dark (LD) conditions with two different light intensities (58.6 and 1,091 µW/cm²), and the effect of 1 h broad spectrum white light pulses of different intensities (3.3, 5.3, 10.5, 1,091.4 µW/cm²) applied at middarkness (MD) on nocturnal circulating melatonin. The results showed that plasma melatonin in tench under LD 12∶12 and high light conditions displayed rhythmic variation, where values at MD (255.8±65.9 pg/ml) were higher than at midlight (ML) (70.7±31.9 pg/ml). Such a difference between MD and ML values was reduced in animals exposed to LD 12∶12 and low light intensity. The application of 1 h light pulses at MD lowered plasma melatonin to 111.6±3.2 pg/ml (in the 3.3–10.5 µW/cm² range) and to 61.8±18.3 pg/ml (with the 1,091.4 µW/cm² light pulse) and totally suppressed nocturnal locomotor activity. These results show that melatonin rhythms persisted in tench exposed to low light intensity although the amplitude of the rhythm is affected. In addition, it was observed that light pulses applied at MD affected plasma melatonin content and locomotor activity. Such a low threshold suggests that the melatonin system is capable of transducing light even under dim conditions, which may be used by this nocturnal fish to synchronize to weak night light signals (e.g., moonlight cycles).     

Time Course of Sensitivity of the Photoinducible Phase to Light in the Redheaded Bunting,Emberiza bruniceps

This study investigated the differential sensitivity of the photoinducible phase (Φi) to light in the redheaded bunting (Emberiza bruniceps). Using a skeleton paradigm, we assessed the rate and magnitude of testicular response as a function of the duration of an inducing light pulse and of the time of its introduction in Φi. For a period of 7 weeks, birds received at an intensity of ~100 lux, the same (6 h) entraining light stimulus with a varied inducing light pulse: 1, 2, 4 or 6 h beginning at zeitgeber time (zt) 11 (6L:5D:1L:12D, 6L:5D:2L:11D, 6L:5D:4L:9D or 6L:5D:6L:7D), or 1h pulses at zt 12 (6L:6D:1L:11D) or zt 16 (6L:10D:1L:7D). The testes were stimulated in all LD alternations, but duration- and time-dependent effects of the light pulse on the rate and magnitude of the testicular response were clearly evident. Illumination of a larger portion of Φi seems to result in higher rates of gonadal growth but there is a duration limit above which there will be no further increase of testicular response.     

Time Course of Sensitivity of the Photoinducible Phase to Light in the Redheaded Bunting, Emberiza bruniceps

This study investigated the differential sensitivity of the photoinducible phase (Φi) to light in the redheaded bunting (Emberiza bruniceps). Using a skeleton paradigm, we assessed the rate and magnitude of testicular response as a function of the duration of an inducing light pulse and of the time of its introduction in Φi. For a period of 7 weeks, birds received at an intensity of ~100 lux, the same (6 h) entraining light stimulus with a varied inducing light pulse: 1, 2, 4 or 6 h beginning at zeitgeber time (zt) 11 (6L:5D:1L:12D, 6L:5D:2L:11D, 6L:5D:4L:9D or 6L:5D:6L:7D), or 1h pulses at zt 12 (6L:6D:1L:11D) or zt 16 (6L:10D:1L:7D). The testes were stimulated in all LD alternations, but duration- and time-dependent effects of the light pulse on the rate and magnitude of the testicular response were clearly evident. Illumination of a larger portion of Φi seems to result in higher rates of gonadal growth but there is a duration limit above which there will be no further increase of testicular response.