A Computational Error and Restricted Use of Time-series Analyses Underlie the Failure to Replicate period-Dependent Song Rhythms in Drosophila.

From 1980 to 1991, Kyriacou, Hall, and collaborators (K&H) reported that the Drosophila melanogaster courtship song has a 1-min cycle in the length of mean interpulse intervals (IPIs) that is modulated by circadian rhythm period mutations. In 2014, Stern failed to replicate these results using a fully automated method for detecting song pulses. Manual annotation of Stern's song records exposed a ~50% error rate in detection of IPIs, but the corrected data revealed period-dependent IPI cycles using a variety of statistical methods. In 2017, Stern et al. dismissed the sine/cosine method originally used by K&H to detect significant cycles, claiming that randomized songs showed as many significant values as real data using cosinor analysis. We first identify a simple mathematical error in Stern et al.'s cosinor implementation that invalidates their critique of the method. Stern et al. also concluded that although the manually corrected wild-type and perL mutant songs show similar periods to those observed by K&H, each song is usually not significantly rhythmic by the Lomb-Scargle (L-S) periodogram, so any genotypic effect simply reflects "noise." Here, we observe that L-S is extremely conservative compared with 3 other time-series analyses in assessing the significance of rhythmicity, both for conventional locomotor activity data collected in equally spaced time bins and for unequally spaced song records. Using randomization of locomotor and song data to generate confidence limits for L-S instead of the theoretically derived values, we find that L-S is now consistent with the other methods in determining significant rhythmicity in locomotor and song records and that it confirms period-dependent song cycles. We conclude that Stern and colleagues' failure to identify song cycles stems from the limitations of automated methods in accurately reflecting song parameters, combined with the use of an overly stringent method to discriminate rhythmicity in courtship songs.

Longitudinal assessment of health-span and pre-death morbidity in wild type Drosophila.

The increase in human life expectancy is accompanied by age-related cognitive and motor disability, thus raising the demand for strategies toward healthy aging. This requires understanding the biology of normal aging and late-life functional phenotypes. Genetic model organisms, such as Drosophila melanogaster, can help identifying evolutionary conserved mechanisms underlying aging. Longitudinal assessment of motor performance of more than 1000 individual flies revealed age-related motor performance decline and specific late-life motor disabilities. This allows defining heath- and ill-span and scoring late-life quality of individual flies. As in mammals, including humans, onset, duration, severity, and progression dynamics of decline are heterogenic and characterized by both, progressive worsening and sudden late-life events. Flies either become increasingly incapacitated by accumulating disability over multiple days prior to death, or they escape disability until few hours prior to death. Both late-life trajectories converge into a terminal stage characterized by stereotypical signs of functional collapse and death within 3 hours. Drosophila can now be used to evaluate life prolonging manipulations in the context of late-life quality. High sugar diet increases lifespan and late-life quality, whereas lifespan prolonging antioxidant supplementation has either no, or negative effects on late-life quality, depending on base diet and gender.

Failure to reproduce period-dependent song cycles in Drosophila is due to poor automated pulse-detection and low-intensity courtship.

Stern has criticized a body of work from several groups that have independently studied the so-called "Kyriacou and Hall" courtship song rhythms of male Drosophila melanogaster, claiming that these ultradian ∼60-s cycles in the interpulse interval (IPI) are statistical artifacts that are not modulated by mutations at the period (per) locus [Stern DL (2014) BMC Biol 12:38]. We have scrutinized Stern's raw data and observe that his automated song pulse-detection method identifies only ∼50% of the IPIs found by manual (visual and acoustic) monitoring. This critical error is further compounded by Stern's use of recordings with very little song, the large majority of which do not meet the minimal song intensity criteria which Kyriacou and Hall used in their studies. Consequently most of Stern's recordings only contribute noise to the analyses. Of the data presented by Stern, only perL and a small fraction of wild-type males sing vigorously, so we limited our reanalyses to these genotypes. We manually reexamined Stern's raw song recordings and analyzed IPI rhythms using several independent time-series analyses. We observe that perL songs show significantly longer song periods than wild-type songs, with values for both genotypes close to those found in previous studies. These per-dependent differences disappear when the song data are randomized. We conclude that Stern's negative findings are artifacts of his inadequate pulse-detection methodology coupled to his use of low-intensity courtship song records.

Melatonin increases the regularity of cardiac rhythmicity in the Drosophila heart in both wild-type and strains bearing pathogenic mutations.

Melatonin is a hormone that is critical for normal circadian and seasonal rhythmicity in a wide range of different animals. It is a powerful antioxidant commonly used to prevent reperfusion injury to the heart after infarction. We show here it has other more far-reaching effects on cardiac function. Using the Drosophila model, we show that injection of melatonin increases the regularity of heartbeat significantly and can rescue rhythmicity in flies bearing mutations that adversely affect cardiac function. Notably, melatonin increases cardiac regularity independent of alteration of heart rate. We provide compelling evidence that melatonin's action as an antioxidant is not the mechanism underlying improved cardiac performance. We have strong evidence that melatonin's action on the heart is mediated via a specific G-Protein-coupled receptor encoded by the CG 4313 gene that our results implicate as a candidate melatonin receptor. These results open a line of questioning about fundamental aspects of cardiac pacemaking.

Suppression of Tryptophan 2,3-Dioxygenase Produces a Slow Heartbeat Phenotype in Drosophila melanogaster.

The primary pathway utilizing tryptophan leads initially to kynurenine before branching. Products include nicotinamide adenine dinucleotide and important pigments in the eye. Products in this pathway have been linked to a number of pathologies. The gene encoding the first step in this pathway, tryptophan 2,3-dioxegenase, is encoded by the gene vermilion, initially discovered in Drosophila. In the fly, v is an important eye color marker, but is found to have multiple pleiotropic effects. We have uncovered significant effects of this mutation on the fly heart. The heart beats more slowly and more rhythmically in both males and females and in strains which we have outcrossed. In addition, the fly heart normally beats irregularly with multiple brief stoppages, and the time structure of these stoppages, as investigated by looking at interbeat intervals, is changed in flies bearing this mutation. Fewer flies bearing the v1 mutation show long hiatuses in beat compared to wild type, however, in some strains of the mutant animals that do, the number of stoppages in much greater and the mean duration is longer.

p38 MAP kinase regulates circadian rhythms in Drosophila.

The large repertoire of circadian rhythms in diverse organisms depends on oscillating central clock genes, input pathways for entrainment, and output pathways for controlling rhythmic behaviors. Stress-activated p38 MAP Kinases (p38K), although sparsely investigated in this context, show circadian rhythmicity in mammalian brains and are considered part of the circadian output machinery in Neurospora. We find that Drosophila p38Kb is expressed in clock neurons, and mutants in p38Kb either are arrhythmic or have a longer free-running periodicity, especially as they age. Paradoxically, similar phenotypes are observed through either transgenic inhibition or activation of p38Kb in clock neurons, suggesting a requirement for optimal p38Kb function for normal free-running circadian rhythms. We also find that p38Kb genetically interacts with multiple downstream targets to regulate circadian locomotor rhythms. More specifically, p38Kb interacts with the period gene to regulate period length and the strength of rhythmicity. In addition, we show that p38Kb suppresses the arrhythmic behavior associated with inhibition of a second p38Kb target, the transcription factor Mef2. Finally, we find that manipulating p38K signaling in free-running conditions alters the expression of another downstream target, MNK/Lk6, which has been shown to cycle with the clock and to play a role in regulating circadian rhythms. These data suggest that p38Kb may affect circadian locomotor rhythms through the regulation of multiple downstream pathways.

Maximum entropy spectral analysis for circadian rhythms: theory, history and practice.

There is an array of numerical techniques available to estimate the period of circadian and other biological rhythms. Criteria for choosing a method include accuracy of period measurement, resolution of signal embedded in noise or of multiple periodicities, and sensitivity to the presence of weak rhythms and robustness in the presence of stochastic noise. Maximum Entropy Spectral Analysis (MESA) has proven itself excellent in all regards. The MESA algorithm fits an autoregressive model to the data and extracts the spectrum from its coefficients. Entropy in this context refers to "ignorance" of the data and since this is formally maximized, no unwarranted assumptions are made. Computationally, the coefficients are calculated efficiently by solution of the Yule-Walker equations in an iterative algorithm. MESA is compared here to other common techniques. It is normal to remove high frequency noise from time series using digital filters before analysis. The Butterworth filter is demonstrated here and a danger inherent in multiple filtering passes is discussed.

Sleep-wake behavior in the rat: ultradian rhythms in a light-dark cycle and continuous bright light.

Ultradian rhythms are a prominent but little-studied feature of mammalian sleep-wake and rest-activity patterns. They are especially evident in long-term records of behavioral state in polyphasic animals such as rodents. However, few attempts have been made to incorporate ultradian rhythmicity into models of sleep-wake dynamics, and little is known about the physiological mechanisms that give rise to ultradian rhythms in sleep-wake state. This study investigated ultradian dynamics in sleep and wakefulness in rats entrained to a 12-h:12-h light-dark cycle (LD) and in rats whose circadian rhythms were suppressed and free-running following long-term exposure to uninterrupted bright light (LL). We recorded sleep-wake state continuously for 7 to 12 consecutive days and used time-series analysis to quantify the dynamics of net cumulative time in each state (wakefulness [WAKE], rapid eye movement sleep [REM], and non-REM sleep [NREM]) in each animal individually. Form estimates and autocorrelation confirmed the presence of significant ultradian and circadian rhythms; maximum entropy spectral analysis allowed high-resolution evaluation of multiple periods within the signal, and wave-by-wave analysis enabled a statistical evaluation of the instantaneous period, peak-trough range, and phase of each ultradian wave in the time series. Significant ultradian periodicities were present in all 3 states in all animals. In LD, ultradian range was approximately 28% of circadian range. In LL, ultradian range was slightly reduced relative to LD, and circadian range was strongly attenuated. Ultradian rhythms were found to be quasiperiodic in both LD and LL. That is, ultradian period varied randomly around a mean of approximately 4 h, with no relationship between ultradian period and time of day.

A survey of the protective effects of some commercially available antioxidant supplements in genetically and chemically induced models of oxidative stress in Drosophila melanogaster.

Oxidative stress remains one of the most well studied, albeit somewhat contentious, causes of age-related changes in humans. Consequently, a large number of putative antioxidant compounds are freely available in myriad formulations that are often not tested for their efficacy or regulated for quality control. Following the development of a Drosophila model of oxidative-stress dependent aging (p38 MAP K (p38K) mutants) in our laboratory, we attempted to test the protective effect of some of these commonly available formulations against oxidative stress, in the p38K model. As environmental exposure to oxidizing toxins has been linked to a variety of human diseases, we also tested the efficacy of these supplements on chemically-induced models of oxidative stress (paraquat and hydrogen peroxide exposure). Our results suggest that when added as a dietary supplement, some of these over-the-counter compounds, notably containing açai extracts, confer significant protection for both the p38K-dependent genetic model as well as the toxin-induced model. These products were also remarkably effective at dampening stress-induced expression of the detoxifying enzyme GSTD1 and eliminating paraquat induced circadian rhythm deficits. Overall, our results suggest potential benefits of dietary supplementation with some of these compounds, especially under conditions of elevated oxidative stress. These findings should be assessed in the context of other studies that seek to identify active principles in these extracts, determine their effective dosage for human consumption and evaluate the safety of long-term prophylactic applications.

Solitary and gregarious locusts differ in circadian rhythmicity of a visual output neuron.

Locusts demonstrate remarkable phenotypic plasticity driven by changes in population density. This density dependent phase polyphenism is associated with many physiological, behavioral, and morphological changes, including observations that cryptic solitarious (solitary-reared) individuals start to fly at dusk, whereas gregarious (crowd-reared) individuals are day-active. We have recorded for 24-36 h, from an identified visual output neuron, the descending contralateral movement detector (DCMD) of Schistocerca gregaria in solitarious and gregarious animals. DCMD signals impending collision and participates in flight avoidance maneuvers. The strength of DCMD's response to looming stimuli, characterized by the number of evoked spikes and peak firing rate, varies approximately sinusoidally with a period close to 24 h under constant light in solitarious locusts. In gregarious individuals the 24-h pattern is more complex, being modified by secondary ultradian rhythms. DCMD's strongest responses occur around expected dusk in solitarious locusts but up to 6 h earlier in gregarious locusts, matching the times of day at which locusts of each type are most active. We thus demonstrate a neuronal correlate of a temporal shift in behavior that is observed in gregarious locusts. Our ability to alter the nature of a circadian rhythm by manipulating the rearing density of locusts under identical light-dark cycles may provide important tools to investigate further the mechanisms underlying diurnal rhythmicity.

Ultradian components in the locomotor activity rhythms of the genetically normal mouse, Mus musculus.

Ultradian periodicities in physiological processes have been reported for a wide variety of organisms and may appear as bouts in locomotor activity. In some instances, this temporal organization can be related to some ethological strategy. In mice, however, ultradian rhythms have been reported largely in animals with circadian pacemakers disrupted either by genetic or surgical manipulation. Using analysis techniques capable of resolving periodicities in the ultradian range in the presence of strong diel periodicity, we found unequivocal evidence of ultradian rhythms in mice entrained to an light:dark cycle. We collected locomotor activity data of individuals from 11 genetically disparate strains of mice whose activity was recorded in 12 h:12 h L:D photoperiods for 3 days. Data were subjected to maximum entropy spectral analysis and autocorrelation, both before and after filtering to remove the 24-h periodicity. We found that every strain had a majority of individuals with strong ultradian rhythms ranging from ~3 to ~5 h. These periodicities were commonly visible in individual animals both in high-pass-filtered and in unfiltered data. Furthermore, when all raw data from a given strain were pooled to get a 24-h ensemble average across all animals and days, the rhythms continued to be discernable. We fitted Fourier series to these form estimates to model the frequency structure of each strain and found significant effects of strain and an interaction between period and strain indicating significant genetic variation for rhythmicity in the ultradian range. The techniques employed in this study should have wider use in a range of organisms and fields.

The relationship of heart function to temperature in Drosophila melanogaster and its heritability.

We measured heart rate and rhythmicity (regularity) of heartbeat in Drosophila melanogaster at five different temperatures (20, 25, 30, 35, and 37 degrees C) for a Florida population and estimated the narrow-sense heritability of both traits. Heritability of heart rate ranged from 0.17 to 0.24, but was statistically significant only at 20 degrees (h(2)=0.24) and at 30 degrees (h(2)=0.23). The heritability of heartbeat rhythmicity ranged from -0.034 to 0.11, and was not significant at any temperature. Heart rate increased linearly with increasing temperature; the temperature-dependence of heart rate was itself heritable (h(2)=0.29). Heart rhythmicity varied curvilinearly and was well-represented by a parabolic function, peaking at about 27 degrees which suggests a temperature optimum. The regularity of the heartbeat did not covary with heart rate except at 20 degrees . Neither heart rate nor regularity covaried with the change in heart rate with temperature. For this population of D. melanogaster, we conclude that there is substantial genetic variation for the mechanism whereby the cardiac pacemaker reacts to changes in temperature, but not for the cardiac pacemaker's rhythmicity. The small values of h(2) for temperature-specific heart rate and heartbeat rhythmicity suggest that these traits have been subjected to natural selection.

Analyses for physiological and behavioral rhythmicity.

Biological data that contain cycles require specialized statistical and analytical procedures. Techniques for analysis of time series from three types of systems are considered with the intent that the choice of examples is sufficiently broad that the processes described can be generalized to most other types of physiological or behavioral work. Behavioral circadian rhythms, acoustic signals in fly mating, and the Drosophila melanogaster cardiac system have been picked as typical in three broad areas. Worked examples from the fly cardiac system are studied in full detail throughout. The nature of the data streams and how they are acquired is first discussed with attention paid to ensuring satisfactory subsequent statistical treatment. Analysis in the time domain, namely simple and advanced plotting of data, autocorrelation analysis, and cross-correlation, is described. The search for periodicity is conducted through examples of analysis in the frequency domain, primarily spectral analysis. Nonstationary time series pose a particular problem, and wavelet analysis of Drosophila mating song is described in detail as an example. Conditioning of data to improve output with digital filters, Fourier filtering, and trend removal is described. Finally, two tests for noise levels and regularity are considered. All the nonproprietary software used throughout the work is available from the author free of charge and can be specifically tailored to the needs of individual systems.

Is vertical migration in Antarctic krill (Euphausia superba) influenced by an underlying circadian rhythm?

Antarctic krill (Euphausia superba) is a keystone species in the southern ocean ecosystem where it is the main consumer of phytoplankton and constitutes the main food item of many higher predators. Both food and predators are most abundant at the surface, thus krill hide in the depth of the ocean during the day and migrate to the upper layers at night, to feed at a time when the predatory risk is lowest. Although the functional significance of this diel vertical migration (DVM) is clear and its modulation by environmental factors has been described, the involvement of an endogenous circadian clock in this behaviour is as yet not fully resolved. We have analysed the circadian behaviour of Euphausia superba in a laboratory setting and here we present the first description of locomotor activity rhythms for this species. Our results are in agreement with the hypothesis that the circadian clock plays a key role in DVM. They also suggest that the interplay between food availability, social cues and the light:dark cycle acts as the predominant Zeitgeber for DVM in this species.

Statistical analysis of biological rhythm data.

The author has developed an ensemble of digital signal analysis techniques applicable to biological time series containing circadian and ultradian periodicities that is of very high resolution and functions well even in the presence of extreme noise and trend. A method for quantifying the significance, strength, and regularity of the rhythmic process is included. To illustrate these techniques, the author presents analyses of artificial periodic data containing varying amounts of noise, trend, and multiple periodicities. The periods and amplitudes of circadian and, where included, ultradian periodicities, and all other components of the test signals are known exactly. Analyses are illustrated in a step-by-step manner and the results are compared with the known input parameters. Trends are removed; spectra, autocorrelation functions, and rhythmicity indices are produced and discussed. References covering theory and details of all analyses are supplied. All programs employed are available from the author free of charge.

Conditional mutations in SERCA, the Sarco-endoplasmic reticulum Ca2+-ATPase, alter heart rate and rhythmicity in Drosophila.

To analyze the role of cytosolic calcium in regulating heart beat frequency and rhythm, we studied conditional mutations in Drosophila Sarco-endoplasmic reticulum Ca2+-ATPase, believed to be predominantly responsible for sequestering free cytosolic calcium. Abnormalities in the amount or structure of the SERCA protein have been linked to cardiac malfunction in mammals. Drosophila SERCA protein (dSERCA) is highly enriched in Drosophila larval heart with a distinct membrane distribution of SERCA at cardiac Z-lines, suggesting evolutionarily conserved zones for calcium uptake into the sarcoplasmic reticulum. Heart beat frequency is strikingly reduced in mutant animals following dSERCA inactivation, (achieved by a brief exposure of these conditional mutants to non-permissive temperature). Cardiac contractions also show abnormal rhythmicity and electrophysiological recordings from the heart muscle reveal dramatic alterations in electrical activity. Overall, these studies underscore the utility of the Drosophila heart to model SERCA dysfunction dependent cardiac disorders and constitute an initial step towards developing Drosophila as a viable genetic model system to study conserved molecular determinants of cardiac physiology.

Mutations in and deletions of the Ca2+ channel-encoding gene cacophony, which affect courtship song in Drosophila, have novel effects on heartbeating.

The cacophony (cac) locus of Drosophila melanogaster encodes the a-1 subunit of a voltage gated Ca(2+) channel, termed Dmca1A. A subset of mutations at this locus cause characteristic alterations in the male mating song, manifest as polycyclic pulses with higher than normal amplitude. This phenotype has been postulated to result from disruption of an oscillator involving the cac-encoded channel, nearly identical to a model proposed for the pacemaker of the Drosophila heart. We report here that flies bearing two intragenic mutations that affect song, cac(S) and cac(TS2), cause aberrant heartbeating. Hearts of both cac(S) and cac(TS2) mutants beat significantly more rapidly than wild type and the heartbeat is more regular across temperature. Deletions of the cac gene, heterozygous with cac(+), caused interestingly similar heartbeating anomalies. For the heart phenotypes, the mutations are dominant, unlike the effects of cac(S) on song. In sum, our results establish the hypothesis that the observed effects are a result of a reduced number of functional cac-encoded channels rather than any specific alteration in the protein, and that in addition to Dmca1A, a second Ca(2+) channel with different kinetics is also involved in pacemaking.

Resetting the circadian clock by social experience in Drosophila melanogaster.

Circadian clocks are influenced by social interactions in a variety of species, but little is known about the sensory mechanisms underlying these effects. We investigated whether social cues could reset circadian rhythms in Drosophila melanogaster by addressing two questions: Is there a social influence on circadian timing? If so, then how is that influence communicated? The experiments show that in a social context Drosophila transmit and receive cues that influence circadian time and that these cues are likely olfactory.

Advanced analysis of a cryptochrome mutation's effects on the robustness and phase of molecular cycles in isolated peripheral tissues of Drosophila.

BACKGROUND: Previously, we reported effects of the cry(b) mutation on circadian rhythms in period and timeless gene expression within isolated peripheral Drosophila tissues. We relied on luciferase activity driven by the respective regulatory genomic elements to provide real-time reporting of cycling gene expression. Subsequently, we developed a tool kit for the analysis of behavioral and molecular cycles. Here, we use these tools to analyze our earlier results as well as additional data obtained using the same experimental designs. RESULTS: Isolated antennal pairs, heads, bodies, wings and forelegs were evaluated under light-dark cycles. In these conditions, the cry(b) mutation significantly decreases the number of rhythmic specimens in each case except the wing. Moreover, among those specimens with detectable rhythmicity, mutant rhythms are significantly weaker than cry+ controls. In addition, cry(b) alters the phase of period gene expression in these tissues. Furthermore, peak phase of luciferase-reported period and timeless expression within cry+ samples is indistinguishable in some tissues, yet significantly different in others. We also analyze rhythms produced by antennal pairs in constant conditions. CONCLUSIONS: These analyses further show that circadian clock mechanisms in Drosophila may vary in a tissue-specific manner, including how the cry gene regulates circadian gene expression.

Signal analysis of behavioral and molecular cycles.

BACKGROUND: Circadian clocks are biological oscillators that regulate molecular, physiological, and behavioral rhythms in a wide variety of organisms. While behavioral rhythms are typically monitored over many cycles, a similar approach to molecular rhythms was not possible until recently; the advent of real-time analysis using transgenic reporters now permits the observations of molecular rhythms over many cycles as well. This development suggests that new details about the relationship between molecular and behavioral rhythms may be revealed. Even so, behavioral and molecular rhythmicity have been analyzed using different methods, making such comparisons difficult to achieve. To address this shortcoming, among others, we developed a set of integrated analytical tools to unify the analysis of biological rhythms across modalities. RESULTS: We demonstrate an adaptation of digital signal analysis that allows similar treatment of both behavioral and molecular data from our studies of Drosophila. For both types of data, we apply digital filters to extract and clarify details of interest; we employ methods of autocorrelation and spectral analysis to assess rhythmicity and estimate the period; we evaluate phase shifts using crosscorrelation; and we use circular statistics to extract information about phase. CONCLUSION: Using data generated by our investigation of rhythms in Drosophila we demonstrate how a unique aggregation of analytical tools may be used to analyze and compare behavioral and molecular rhythms. These methods are shown to be versatile and will also be adaptable to further experiments, owing in part to the non-proprietary nature of the code we have developed.