Circadian Eclosion Research Articles

Dependence of phase setting on the amplitude of square‐wave and pseudo‐sinusoidal temperature cycles in the circadian eclosion rhythm of the onion fly Delia antiqua

AbstractWith increasing soil depth, the diel temperature amplitude gradually decreases and the phase of the temperature cycle is delayed. The onion fly Delia antiqua (Meigen) (Diptera: Anthomyiidae), which pupates at a soil depth of 2–20 cm, advances the eclosion phase of its circadian rhythm as the temperature amplitude decreases. This ‘temperature amplitude response’ is considered to allow pupae located at any depth to eclose in the early morning. The present study examines the temperature amplitude response of D. antiqua under thermoperiods with amplitudes ranging between 1 and 20 °C. The thermoperiod to which pupae are exposed is either a square‐wave temperature cycle, which is generally used for laboratory experiments, or a pseudo‐sinusoidal temperature cycle, which more closely resembles natural daily temperature cycle, at an average temperature of either 25 or 20 °C. Bursts of eclosion invoked by the abrupt temperature increase in square‐wave cycles are apparently repressed in pseudo‐sinusoidal temperature cycles. Eclosion time shows a clear quantitative shift by 5.9–8.6 h in response to temperature amplitude regardless of the average temperature and the shape of the temperature cycle. The change in the eclosion time is clearly noticeable between amplitudes of 1 and 8 °C but is small between amplitudes of 8 °C or more. Delia antiqua may evolve to compensate for the depth‐dependent phase delay of temperature changes within the possible range of temperature amplitudes in actual soil environments.

The Drosophila apterous56f mutation impairs circadian locomotor activity

We investigated effects of apterous mutation ap56f on circadian locomotor activity, eclosion rhythms, and transcript levels of period and timeless in Drosophila. We investigated circadian locomotor activity and eclosion rhythms in ap56f and wild-type flies, their F1 and F2 offspring, and wingless vestigial mutants and show that ap56f disrupts circadian locomotor rhythms in a genetically recessive manner, that is not caused by the absence of wings. The apblt strain also showed impaired circadian activity rhythms, providing independent evidence for a significant role of apterous in circadian locomotor rhythm expression. The ap56f mutation did not disrupt a circadian eclosion rhythm or rhythmic expression of the period and timeless clock genes, indicating that apterous is not essential for circadian clock function, but is necessary for coupling locomotor activity to a circadian clock. Timeless transcription was reduced in ap56f flies in 12:12 LD, suggesting that apterous may modulate core clock gene expression.

The onion fly modulates the adult eclosion time in response to amplitude of temperature cycle

To confirm whether the amplitude of diel temperature cycles causes a phase shift of adult eclosion rhythm of the onion fly, Delia antiqua, the peak time (Ø(E)) of adult eclosion was determined under various thermoperiods with a fixed temperature either in the warm or cool phase and temperature differences ranging from 1°C to 4°C between the two phases. Irrespective of the temperature level during the warm or cool phase, Ø(E) occurred earlier with decreasing amplitude of the temperature cycle. The results strongly support the previous conclusion of Tanaka and Watari (Naturwissenschaften 90:76-79, 2003) that D. antiqua responds to the amplitude of temperature cycle as a cue for the circadian adult eclosion timing. The phase advance was larger in thermoperiods with a fixed warm-phase temperature than in those with a fixed cool-phase temperature. This might be ascribed to the interaction between the amplitude and level of temperature in the thermoperiodic regimes.

The LARK RNA-Binding Protein Selectively Regulates the Circadian Eclosion Rhythm by Controlling E74 Protein Expression

Despite substantial progress in defining central components of the circadian pacemaker, the output pathways coupling the clock to rhythmic physiological events remain elusive. We previously showed that LARK is a Drosophila RNA-binding protein which functions downstream of the clock to mediate behavioral outputs. To better understand the roles of LARK in the circadian system, we sought to identify RNA molecules associated with it, in vivo, using a three-part strategy to (1) capture RNA ligands by immunoprecipitation, (2) visualize the captured RNAs using whole-genome microarrays, and (3) identify functionally relevant targets through genetic screens. We found that LARK is associated with a large number of RNAs, in vivo, consistent with its broad expression pattern. Overexpression of LARK increases protein abundance for certain targets without affecting RNA level, suggesting a translational regulatory role for the RNA-binding protein. Phenotypic screens of target-gene mutants have identified several with rhythm-specific circadian defects, indicative of effects on clock output pathways. In particular, a hypomorphic mutation in the E74 gene, E74BG01805, was found to confer an early-eclosion phenotype reminiscent of that displayed by a mutant with decreased LARK gene dosage. Molecular analyses demonstrate that E74A protein shows diurnal changes in abundance, similar to LARK. In addition, the E74BG01805 allele enhances the lethal phenotype associated with a lark null mutation, whereas overexpression of LARK suppresses the early eclosion phenotype of E74BG01805, consistent with the idea that E74 is a target, in vivo. Our results suggest a model wherein LARK mediates the transfer of temporal information from the molecular oscillator to different output pathways by interacting with distinct RNA targets.

Independence of Genetic Geographical Variation between Photoperiodic Diapause, Circadian Eclosion Rhythm, and Thr-Gly Repeat Region of the Period Gene inDrosophila littoralis

Drosophila littoralis is a latitudinally widespread European species of the Drosophila virilis group. The species has ample genetic variation in photoperiodism (adult diapause) and circadian rhythmicity (pupal eclosion rhythm), with adaptive latitudinal clines in both of them. The possible common genetic basis between the variability of photoperiodism and circadian rhythms was studied by a long-term crossing experiment. A northern strain (65 degrees N) having long critical day length (CDL = 19.9 h) for diapause, early phase of the entrained rhythm in LD 3:21 (psi(LD3:21) = 12.3 h), and short period (tau= 18.8 h) of the free-running rhythm for the eclosion rhythm was crossed with a southern strain (42 degrees N) having short CDL (12.4 h), late eclosion phase (psi(LD3:21) = 20.2 h), and long period (tau= 22.8 h). After 54 generations, including free recombination, artificial selection, and genetic drift, a novel strain resulted, having even more "southern" diapause and more "northern" eclosion rhythm characteristics than found in any of the geographical strains. The observed complete separation of eclosion rhythm characteristics from photoperiodism is a new finding in D. littoralis; in earlier studies followed for 16 generations, the changes had been mostly parallel. Evidently, the genes controlling the variability of the eclosion rhythm and photoperiodism in D. littoralis are different but closely linked. To test for the possible gene loci underlying the observed geographical variability, the period gene was studied in 10 strains covering all the known clock variability in D. littoralis. The authors sequenced the most suspected Thr-Gly region, which is known to take part in the adaptive clock variability in Drosophila melanogaster. No coding differences were found in the strains, showing that this region is not included in the adaptive clock variability in D. littoralis.

Comparison of the circadian eclosion rhythm between non-diapause and diapause pupae in the onion fly, Delia antiqua: the change of rhythmicity

When pupae of Delia antiqua were transferred to constant darkness (DD) from light–dark (LD) cycles or constant light (LL), the sensitivity to light of the circadian clock controlling eclosion increased with age. The daily rhythm of eclosion appeared in both non-diapause and diapause pupae only when this transfer was made during late pharate adult development. When transferred from LL to DD in the early pupal stage, the adult eclosion was weakly rhythmic in non-diapause pupae but arrhythmic in diapause pupae. However, the sensitivity of the circadian clock to temperature cycles or steps was higher in diapause pupae than in non-diapause pupae; in the transfer to a constant 20 °C from a thermoperiod of 25 °C (12 h)/20 °C (12 h) on day 10 after pupation or from chilling (7.5 °C) in DD, the adult eclosion from diapause pupae was rhythmic but that from non-diapause pupae arrhythmic. In a transfer to 20 °C from the thermoperiod after the initiation of eclosion, rhythmicity was observed in both types of pupae. The larval stage was insensitive to the effect of LD cycle initiating the eclosion rhythm. In D. antiqua pupae in the soil under natural conditions, therefore, the thermoperiod in the late pupal stage would be the most important ‘Zeitgeber’ for the determination of eclosion timing.

Targeted ablation of CCAP neuropeptide-containing neurons of Drosophila causes specific defects in execution and circadian timing of ecdysis behavior.

Insect growth and metamorphosis is punctuated by molts, during which a new cuticle is produced. Every molt culminates in ecdysis, the shedding of the remains of the old cuticle. Both the timing of ecdysis relative to the molt and the actual execution of this vital insect behavior are under peptidergic neuronal control. Based on studies in the moth, Manduca sexta, it has been postulated that the neuropeptide Crustacean cardioactive peptide (CCAP) plays a key role in the initiation of the ecdysis motor program. We have used Drosophila bearing targeted ablations of CCAP neurons (CCAP KO animals) to investigate the role of CCAP in the execution and circadian regulation of ecdysis. CCAP KO animals showed specific defects at ecdysis, yet the severity and nature of the defects varied at different developmental stages. The majority of CCAP KO animals died at the pupal stage from the failure of pupal ecdysis, whereas larval ecdysis and adult eclosion behaviors showed only subtle defects. Interestingly, the most severe failure seen at eclosion appeared to be in a function required for abdominal inflation, which could be cardioactive in nature. Although CCAP KO populations exhibited circadian eclosion rhythms, the daily distribution of eclosion events (i.e., gating) was abnormal. Effects on the execution of ecdysis and its circadian regulation indicate that CCAP is a key regulator of the behavior. Nevertheless, an unexpected finding of this work is that the primary functions of CCAP as well as its importance in the control of ecdysis behaviors may change during the postembryonic development of Drosophila.

Comparison of the circadian eclosion rhythm between non-diapause and diapause pupae in the onion fly, Delia antiqua: the effect of thermoperiod

When non-diapause and diapause pupae of Delia antiqua were exposed to various thermoperiods where thermophase (T) was 25 °C and the cryophase (C) was 15 or 20 °C (TC 15 or TC 20) in constant darkness (DD), the majority of both types of flies emerged before the rise in temperature. Eclosion time was delayed at the lower cryophase temperature. Moreover, there was a significant difference in the time of adult eclosion between non-diapause and diapause pupae; diapause pupae eclosed earlier than non-diapause pupae. When the two types of pupae were transferred to a constant low temperature (15 or 20 °C) after having experienced TC 15 or TC 20 12:12 h, they showed circadian rhythmicity in eclosion. The free-running period ( τ) of the eclosion rhythm changed after transfer to constant low temperatures in both non-diapause and diapause pupae, suggesting that this change represents a transient cycle until the temperature-sensitive oscillator is coupled again to the temperature-insensitive pacemaker. However, diapause pupae tended to show a shorter τ than non-diapause pupae. This observation suggests that the difference in adult eclosion time under thermoperiodic conditions between non-diapause and diapause pupae is related to their different τ s.

Mutations for activity level in Drosophila jambulina perturbed its pacemaker that controls circadian eclosion rhythm.

Mutations for activity level, designated hpa and hra, in Drosophila jambulina altered properties of the pacemaker controlling eclosion rhythm. Entrainment of eclosion rhythm was studied in light-dark (LD) cycles of 12:12 h at 28 degrees C. The wild type strain entrained to LD cycles but the hpa or hra strain did not. When these strains were released from constant light (LL) to constant darkness (DD), the wild type strain free-ran while other two strains were arrhythmic. Temperature cycles entrained the wild type and hpa strain in DD and LL, and when transferred to constant temperature following thermoperiodic entrainment, the wild type strain free-ran in DD, while the hpa strain free-ran in LL as if the input of LL was essential for its free-running state. Temperature cycles entrained the hra strain in DD but not in LL, and free-running rhythmicities were never established, suggesting that the hra mutation has altered the basic properties of its pacemaker.

Comparison of the circadian eclosion rhythm between non-diapause and diapause pupae in the onion fly, Delia antiqua

The influence of pupal diapause on adult eclosion rhythm of Delia antiqua was investigated. When non-diapause and diapause pupae were exposed to various photoperiods at 15, 20 and 25 °C, both of them emerged as adults close to the light-on time, but the phase of eclosion varied with photoperiod and temperature. Moreover, there was a significant difference in the eclosion time between non-diapause and diapause pupae; the eclosion peak of diapause pupae was earlier than that of non-diapause pupae. When non-diapause and diapause pupae were transferred to constant darkness (DD) after having experienced LD 12:12 at 15, 20 and 25 °C, both showed circadian rhythmicity in eclosion. Although the free-running period ( τ) decreased slightly as temperature increased in both non-diapause and diapause pupae, the latter tended to show shorter τ than the former. This observation suggests that the observed difference in eclosion time in LD cycles between non-diapause and diapause pupae is due to differences in τ.

Persistence of eclosion rhythm in Drosophila melanogaster after 600 generations in an aperiodic environment.

The ubiquity of circadian rhythms suggests that they have an intrinsic adaptive value (Ouyang et al. 1998; Ronneberg and Foster 1997). Some experiments have shown that organisms have enhanced longevity, development time or growth rates when maintained in environments whose periodicity closely matches their endogenous period (Aschoff et al. 1971; Highkin and Hanson 1954; Hillman 1956; Pittendrigh and Minis 1972; Went 1960). So far there has been no experimental evidence to show that circadian rhythms per se (i.e. periodicity itself, as opposed to phasing properties of a rhythm) confer a fitness advantage. We show that the circadian eclosion rhythm persists in a population of the fruitfly Drosophila melanogaster maintained in constant conditions of light, temperature, and humidity for over 600 generations. The results suggest that even in the absence of any environmental cycle there exists some intrinsic fitness value of circadian rhythms.

Disruption of a Behavioral Sequence by Targeted Death of Peptidergic Neurons in Drosophila

The neuropeptide eclosion hormone (EH) is a key regulator of insect ecdysis. We tested the role of the two EH-producing neurons in Drosophila by using an EH cell-specific enhancer to activate cell death genes reaper and head involution defective to ablate the EH cells. In the EH cell knockout flies, larval and adult ecdyses were disrupted, yet a third of the knockouts emerged as adults, demonstrating that EH has a significant but nonessential role in ecdysis. The EH cell knockouts had discrete behavioral deficits, including slow, uncoordinated eclosion and an insensitivity to ecdysis-triggering hormone. The knockouts lacked the lights-on eclosion response despite having a normal circadian eclosion rhythm. This study represents a novel approach to the dissection of neuropeptide regulation of a complex behavioral program.

Timing of eclosion in marine insects on Mediterranean shores - studies on Clunio mediterraneus , C. ponticus and Thalassomyia frauenfeldi (Diptera: Chironomidae)

In three Clunio stocks, two from the Mediterranean Sea (Banyuls, Rovinj) and one from the Black Sea (Sosopol), an identical circasemilunar eclosion pattern in combination with a circadian eclosion time of day was evoked in breeding experiments under an artificial moonlight cycle (four nights of 0.3 lux every 30 d, 12 h light:12 h dark). The eclosion peaks occurred on days with artificial moonlight and approximately 2 weeks later, and with regard to the time of day between mid-night and lights-on. In spite of the weak tidal amplitudes on Mediterranean shores, extreme low water levels occur when a nocturnal low-water time coincides on days around full and new moon (spring-tide situation) with winds from land to sea. Field observations at Banyuls and Rovinj agree with the experimental results. In contrast to Clunio spp. adults (life span only a few hours), Thalassomyia frauenfeldi adults (life span up to several days in the laboratory) demonstrated an unsynchronized eclosion pattern in the experiments (no lunar concentration, wide diurnal eclosion gate). Although inhabiting the same intertidal area, T. frauenfeldi walking on exposed intertidal substrates can delay egg-deposition for days and can probably wait for favorable low water levels. The taxonomical status of European Clunio populations is reviewed; minute morphological distinctions are presented for C. mediterraneus in the otherwise morphologically uniform, but ecophysiologically differentiated genus.

North–south differences in circadian eclosion rhythm in European populations of Drosophila subobscura

North–south differences in circadian eclosion rhythm in European populations of Drosophila subobscura

Characterization oflinne, a new autosomal eclosion rhythm mutant inDrosophila subobscura

A new putative gene, named linne, affecting circadian eclosion rhythm was found in a Norwegian strain of Drosophila subobscura. The phenotype of linne may be novel among Drosophila clock mutants. Eclosion is practically arrhythmic in light-dark cycles, but diel temperature cycles of 4 degrees C amplitude effectively entrain eclosion in continuous light and in continuous darkness. After that, the rhythm continues in constant temperature and darkness, but unlike other strains, it continues in constant temperature and light as well. Thus in the linne strain the coupling between light signals and pacemaker is disrupted, not the pacemaker itself. Genetic effects of linne were tested in crosses with a wild strain. Arrhythmicity was recessive and autosomal and showed monogenic inheritance, suggesting that arrhythmicity in linne is based on mutation on a single locus.

Weak circadian eclosion rhythmicity in Chymomyza costata (Diptera: Drosophilidae), and its independence of diapause type

Pupal eclosion rhythms were studied in seven strains of C. costata originating between latitudes 43°N in Japan to 69°N in Finland. The strains represented the following types of diapause: larval obligatory, larval photoperiodic (critical daylengths from 14 h in the south to 21 h in the north), and no photoperiodic diapause. The eclosion rhythmicity of all strains was weak in all diel photoperiods. The free-running rhythms of eclosion were also of low amplitude. Three other species of Chymomyza were studied for comparison: Chymomyza distincta and C. fuscimana with a larval diapause and C. caudatula with an adult diapause. Only C. caudatula had the rhythmic eclosion that drosophilids have in general. We conclude that the exceptionally weak eclosion rhythmicity in C. costata is an adaptation for eclosion at any permissive time of day around midsummer. The weak rhythmicity of eclosion in C. costata would thus the phenotypic plasticity, an adaptation to the exceptional phenological timing of the species. There was no parallel variation between circadian eclosion rhythms and photoperiodism.

New short period mutations of the Drosophila clock gene per

Earlier work has indicated that the period length of Drosophila circadian behavioral rhythms is dependent on the abundance of the period ( per) gene product. Increased expression of this gene has been associated with period shortening for both the circadian eclosion (pupal hatching) rhythm and circadian locomotor activity rhythms of adult Drosophila. In this study it is shown that a wide variety of missense mutations, affecting a region of the per protein consisting of ∼ 20 aa, predominantly generate short period phenotypes. The prevalence of such mutations suggests that short period phenotypes may result from loss or depression of function in this domain of the per protein. Possibly mutations in the region eliminate a regulatory function provided by this segment, or substantially increase stability of the mutant protein.

Characterization ofAndante, A NewDrosophilaClock Mutant, and its Interactions with Other Clock Mutants

A new clock mutant, named Andante, has been identified on the X chromosome of Drosophila melanogaster. Andante lengthens the period of the circadian eclosion and locomotor activity rhythms by 1.5-2.0 hours. The phase response curves for the eclosion and activity rhythms, indicating light-induced phase shifts, show a similar degree of lengthening. Andante also lengthens the periods of other clock mutants, including Clock, and alleles of the period locus. Analysis of locomotor activity rhythms reveals that Andante is semi-dominant, and Andante rhythms are highly temperature compensated. The sine oculis mutation, which eliminates the outer visual system, has no effect on the period of Andante. Deficiency mapping indicates that Andante is located in the 1OE1-2 to 1OF1 region of the X chromosome, close to the miniature-dusky locus. Whereas Andante flies have a dusky wing phenotype, dusky flies do not have an Andante clock phenotype.

Genetic correlation between circadian eclosion rhythm and photoperiodic diapause in Drosophila littoralis.

Populations of Drosophila littoralis are known to be latitudinally highly variable in photoperiodic adult diapause and pupal eclosion rhythm. Phenotypic correlations between the two time-measuring systems among the strains from different latitudes are, however, weak. In the present study, two differing strains were crossed reciprocally in order to search for causal (genetic) correlations between the two traits in the strains. Segregation in the F2 generations showed that variation in each trait was based on a few variable loci only. In the F2, flies having different eclosion times also differed in their diapause. This association was not complete and could have been due to genetic linkage between the traits. For that reason, the hybrid generations were raised for eight generations more to allow recombination between the traits. In F8, selection against diapause was started in the lines by raising them in a light-dark cycle of 15:9, where only females of the southern type reproduce. After eight selected generations, the lines were studied for the traits. Diapause was completely of the southern type, and the eclosion rhythm had also changed in parallel. The change in the phase of the free-running rhythm was not complete. From the present experiment, and from earlier knowledge of the geographical variation in D. littoralis, I conclude that the same pacemaker that is seen in the eclosion rhythm could also participate in daylength measurement for diapause. However, there are also noncorrelated variable parts in the measuring systems of both traits, which may mask the correlated variation.

Geographical variation in circadian eclosion rhythm and photoperiodic adult diapause inDrosophila littoralis

The time measuring system ofDrosophila littoralis strains originating between 40–70° N was found to be highly variable and latitude dependent. The critical daylength for photoperiodic adult diapause varied from 12 h or no diapause response in the south to 20 h in north. The median timing of pupal eclosion rhythm varied correspondingly from 21 h to 12 h from lights off in LD 3∶21, and the period of free-running rhythm of eclosion from 24 h to 19 h. The phase of the free-running rhythm was also variable, and correlated with the phase of the entrained rhythm. Latitudinal variation in the entrained rhythm of eclosion and in diapause is adaptive, leading to eclosion early in the morning and to overwintering at the adult stage. In some strains with a late phase of eclosion, strong transient cycles were seen following the transition from LL to DD. A total damping of the free-running eclosion rhythm within 2–7 days was common to all strains. This damping was more pronounced in the northern strains. The phase and period of eclosion rhythms were statistically independent. Diapause was not correlated with any parameters of the eclosion rhythm in the analysis. Diapause may still be influenced by the period of the eclosion rhythm, even though its minor contribution may be masked by a more variable, eclosion rhythm independent system in the determination of diapause.