Characterizing the Circadian Locomotor Activity of Drosophila melanogaster yellow white Mutants under Different Temperatures

Citation: Egypt. Acad. J. Biolog. Sci. (A. Entomology) Vol. 10(2)pp: 2536 (2017) Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Entomology Journal publishes original research papers and reviews from any entomological discipline or from directly allied fields in ecology, behavioral biology, physiology, biochemistry, development, genetics, systematics, morphology, evolution, control of insects, arachnids, and general entomology. www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.


INTRODUCTION
Animal behavior and physiology is now known to be largely regulated by the circadian clock.An endogenous timing system composed of an oscillator that generates a 24-h rhythm, a photoreceptor that synchronizes the clock to the environmental light-dark (LD) cycles, and an output system that relays the timing information to overt output behaviors and physiological functions.They have been found to exist from bacteria to humans (Saunders, 2002;Dunlap et al., 2004;Tomioka and Matsumoto, 2010).Although light is the most important environmental cue to entrain the circadian clock in most animals, temperature fluctuations also play a major role in entrainment (Aschoff, 1981;Edmund, 1988;Tomioka and Yoshii, 2006).Temperature can change the free-running period, the phase of the rhythm, and is able to entrain the circadian rhythm in many animals such as lizards and insects (Lankinen and Riihimaa, 1997;Tomioka et al., 1998;Yoshii et al., 2002).
Circadian clocks help the organisms to anticipate the change in environmental conditions, thus increasing their ecological fitness.Temperature involvement in circadian regulation is especially important in maximizing the adaptability to selective pressures along latitudinal and altitudinal gradients (Hut et al., 2014).
Drosophila is the model of choice for circadian research as it has extensively been used in other behavioral studies.Analysis of Drosophila mutants made it possible to genetically dissect behaviors ranging from leg shaking, courtship, and associative learning to memory, including circadian regulation (Boynton and Tully, 1992;Allada and Chung, 2010).Screens for mutants with altered free-running periods, in constant darkness (DD), led to groundbreaking results of identifying the first clock gene, period (per) (Konopka and Benzer, 1971).Studying other clock mutants led to the discovery of other canonical clock genes including timeless (tim) (Sehgal et al., 1994), Clock (Clk) (Allada et al., 1998), cycle (cyc) (Rutila et al., 1998), and many others.
The yellow and white Drosophila mutation is widely used in genetic backgrounds in behavioral research.The yellow (y) gene, located on the X chromosome, affects adult melanisation that occurs at day 4 of puparium, resulting in an altered yellow pigmentation of the adult cuticle and its derivative structures.The mutant yellowish pigmentation is clearly distinguishable from the dark brown of wild type (Lindsley and Grell, 1972).The white (w) gene, on the other hand, encodes a transmembrane transporter protein involved in the uptake of essential precursor amino acids in the synthesis of eye pigments (Sullivan et al., 1974;Summers et al., 1982;Ewart and Howells, 1998).white mutation results in white-eyed flies with impaired vision and disturbed neurotransmitter levels (Borycz et al., 2008;Krstic et al., 2013).The altered pigmentation and neurophysiology in the y w mutants is expected to have a considerable effect on the light input pathways to the clock, which is the main clock-entraining environmental cue (Hassaneen, 2015;Yoshii et al., 2015).
The mutant y w D. melanogaster flies are used frequently in genetic and neurological studies of behavior to control for potential polygenic variability; however, very little is known about their circadian behavior.Therefore, this study aims to investigate the effect of temperature on their circadian clock and to determine their adaptability for living at different latitudes where different temperatures prevail.Results are also expected to expand our understanding of the clock in general and its role in different ecological niches.

Experimental Animals
Two fly lines were used in the experiments.The wild-type Canton S (CS) line, originally isolated from a wild strain near Canton, Ohio, in 1930 (Bridges and Brehme, 1944), was used as control.On the other hand, the mutant yellow white (y w) D. melanogaster flies were the experimental group.Both lines were obtained from the University of California, San Diego Drosophila Species Stock Center (DSSC).Adult males at the age of 4-7 days after eclosion were collected under CO 2 anesthesia and used in the experiments.All flies were reared on standard cornmeal/agar medium with yeast (0.85% agar, 2.2% sugar beet syrup, 8% malt extract, 3.3% yeast, 1% soy flour, 8% cornmeal, and 0.3% propionic acid) in conventional 2.8×9 cm food vials at 25°C and kept on a light-dark cycle of 12:12 hours (LD 12:12) in a humidity and temperature-controlled climate chamber (Schlichting et al., 2014).They were transferred to fresh food vials every week during light phase without anesthesia.

Locomotor Activity Recordings
Locomotor activity of male flies was recorded individually in glass tubes (5 mm in diameter × 65 mm long) with an air penetrable porous plug at one end and agar/sugar food (2% agar and 4% sucrose) on the other end for the Drosophila Activity Monitor (DAM2; TriKinetics Inc., Waltham, MA).This system consists of activity monitors that can simultaneously record the activity of 32 individual flies, an interface device, and a software for computerized data collections.An IR light beam crosses each tube and is detected by a photodetector on the other side.The software automatically generates text files in which the number of beam crosses, indicating the level of locomotor activity, is saved in a consecutive 1-min time span resolution for each individual fly.This assay is currently the most common behavioral assay in flies (Schlichting and Helfrich-Förster, 2015).The light intensity used in all experiments at the animal's level was 100 Lux (19 μW/cm 2 ).Lights-on and -off were controlled by a controller software (Trikinetics, Waltham, MA).Behaviors were recorded in a programmable incubator (MIR-154, Panasonic Biomedical, NL) at 10°, 18°, 20°, and 28°C under a light-dark cycle of 12 hours light and 12 hours dark (LD 12:12) for at least 9 days.Flies became adapted to the LD cycles in the recording monitors and their activity rhythm becomes stable within 1 day, therefore, the first two days of recordings were excluded to make sure that only data of stable rhythm is included in the analysis.Eight groups, 32 flies each, were used in the experiments.The actual number of flies survived and used in the analysis for each group is shown in (Fig. 1).

Data Analysis
Raw data collected in Microsoft Excel 2010 were used to draw double-plotted actograms using ActogramJ (http://actogramj.neurofly.de/)(Schmid et al., 2011), a plugin for ImageJ open-source software (http://rsb.info.nih.gov/ij), and a freely available software for data analysis in life sciences.Activity patterns of individual flies were analyzed by Chi-square periodogram analysis (Sokolove and Bushell, 1978) to determine whether the flies showed significant rhythmicity in their behavior under the experimental conditions at a significance level of 0.05.Average daily activity profiles of stably entrained flies of each line were calculated and plotted in 30-min blocks (not shown), each block represents the sum of activity in 30 minutes, then the phase of morning peak (M), siesta (S), and evening peak (E) was determined by checking individual activity profiles.This enabled reliable determination of peak onsets and offsets, because raw data at the high resolution of 1-min interval are often noisy.The M and E peaks of activity were considered to start when the activity increases gradually around the transition from light to dark and from dark to light, respectively, until reaching a peak then coming down again to a base line activity.
The siesta (S) is the steady activity at the base line in the middle of the light phase between the M and E peaks, which equals (E activity onset -M activity offset).To determine the timing of the peaks, the average day activity was smoothed by a moving average of 11 extending five time points before and after each time point.Consequently, randomly occurring spikes are reduced and the real maximum of the fly's activity can be determined.All data were analyzed and plotted using Microsoft Excel 2010 (Microsoft Corp., Redmond, WA) and SPSS Statistics for Windows, Version 22.0 (IBM Corp., Armonk, NY).The statistical tests used in data analysis were the Student t-test and the Analysis of Variance (ANOVA) followed by post hoc Tukey test.

Locomotor Activity Profiles under Different Temperatures
The general locomotor activity profile of the two fly lines in (LD12:12) under different temperatures showed many similarities (Figures 1 and 2).The y w flies expressed a crepuscular pattern with two peaks of activity around the transitions from dark to light and from light to dark, respectively, comparable to the morning (M) and evening (E) activity peaks known in D. melanogaster, with a resting mid-day siesta (S) in the middle.The activity reached a maximum once per activity peak.Circadian locomotor activity profiles calculated from the average locomotor activity per 1 min for 7 days is showed in (Fig. 1), while a representative actogram of a fly from each group is shown in (Fig. 2).
The average total daily locomotor activity of control CS flies increased significantly at 20°C and 28°C but was nearly stable at the low 10°C and 18°C° (ANOVA, F 3,121 = 59.4,p<0.0001).However, in the mutant y w flies, it increased significantly at 18°C then again at 20°C, but decreased significantly at 28°C (ANOVA, F 3,112 = 103.35,p<0.0001) (Fig. 3C).Comparing the two fly lines at each temperature step using t-test revealed that the low 10°C suppressed the total daily locomotor activity of y w flies more significantly than CS (t 55 =6.29, p<0.001), while the 20°C caused a significant increase in their activity compared to CS (t 61 =7.13, p<0.001).However at 18°C and the high 28°C, that activity level was nearly the same in the two fly lines (t 57 =0.85, NS) and (t 60 =1.89,NS), respectively (Fig. 3C).

Lights-on and Lights-off Locomotor Activity Peaks
Lights-on and lights-off peaks reflect the startle response of flies to the abrupt transition in the rectangular artificial light-dark cycle from dark to light and from light to dark, respectively (Fig 1).At the higher temperature of 28°C, The M peak is fused to the lights-on peak and the E peak is fused to the lights-off peak (Figs, 1A and  1E) in the two fly lines.As the temperature decreases, the M and E peaks migrate away from the lights-on and lights-off peak as in (Figs.1D and 1H).The lights-on activity peak of the CS flies increased significantly only at 20°C and 28°C, (ANOVA, F 3,121 = 25.72,p<0.0001), while the y w flies increased significantly at 18°C, 20°C, and 28°C (ANOVA, F 3,112 = 173.38,p<0.0001) (Fig. 4A).On the other hand, the lights-off activity peak increased in both CS and y w lines at 18°C, 20°C, and 28°C (ANOVA, F 3,121 = 172.97,p<0.0001) and (ANOVA, F 3,112 = 250, 70, p<0.0001), respectively (Fig. 4B).Comparing the two lines at every temperature using t-test revealed a pattern in lights-on peak similar to mean daytime activity (Figs.3A and  4A), also in lights-off peak and mean night-time activity (Figs.3B and 4B); however, that of y w flies was significantly suppressed only at 10°C and 18°C, but not at the higher temperatures.

Temperature-Adaptive Activity Timing
Although the general locomotor activity profile of the two fly lines under different temperatures showed many similarities (Figs. 1 and 2), differences can be found by analyzing profile segments.These differences were manifested in the timing of start and end of each activity peak, and consequently affected their duration (Fig. 5).
The start of the morning activity peak (M) in wildtype CS flies advanced significantly with every temperature increase (ANOVA, F 3,121 = 168.53,p<0.0001), while in y w flies it only advanced significantly at the highest temperature of 28°C (ANOVA, F 3,111 = 49.89,p<0.0001), but started at the same time at 10°C, 18°C, and 20°C.Comparing the two lines at each temperature step revealed that y w flies started their M peak significantly earlier than wildtype CS at 10°C (t 55 =3.63, p<0.001) and significantly later at 18°C (t 56 =5.28, p<0.001), 20°C (t 61 =9.73, p<0.001), and 28°C (t 60 =4.79, p<0.001).On the other hand, the end of M peak in wildtype CS flies delayed significantly only at the lowest temperature of 10°C (ANOVA, F 3,121 = 17.01, p<0.0001), but ended nearly at the same time at 18°C, 20°C, and 28°C.However, the y w files ended their M peak significantly earlier at 10°C then delayed significantly at 18°C (ANOVA, F 3,111 = 30.05,p<0.0001) with flies at 20°C and 28°C ending nearly at the same time.Comparing the end of M peak in the two lines at each temperature step revealed that y w flies ended their M peak significantly earlier than wildtype CS at 10°C (t 55 =5.97, p<0.001), significantly later at 18°C (t 56 =7.99, p<0.001), and 28°C (t 60 =3.51, p<0.001), but ending nearly at the same time at 20°C (t 61 =1.17, NS).The duration of M increased significantly with temperature in both wildtype CS (ANOVA, F 3,121 = 114.21,p<0.0001) and y w flies (ANOVA, F 3,111 = 65.81,p<0.0001) except for y w flies at 20°C (Figure 5).The length of the M peak in y w flies was shorter than CS at 10°C (t 55 =5.37, p<0.0001), 20°C (t 61 =6.88, p<0.001), and 28°C (t 60 =2.26, p<0.03), but they were similar at 18°C (t 56 =1.24, NS).The locomotor activity reached a maximum that usually lasts for about 15 minutes in average within the M peak (Fig. 1); from hereafter it will be denoted as MAX.The M peak's MAX of the wildtype CS flies was significantly delayed at 10°C (ANOVA, F 3,121 = 49.29,p<0.0001), while it occurred nearly at the same time, close to lights-on, at 18°C, 20°C, and 28°C.The M peak's MAX of the y w flies also occurred close to the lightson at 20°C and 28°C, but it was significantly delayed at 10°C and 18°C (ANOVA,F 3,111 = 14.56,p<0.0001).Comparing M's MAX between the two lines shoed that y w flies are significantly delayed compared to CS at 18°C (t 56 =5.80, p<0.001), 20°C (t 61 =2.66, p<0.01), and 28°C (t 60 =2.52, p<0.01), but significantly advanced at 10°C (t 55 =5.06, p<0.001) (Fig. 5).

DISCUSSION
The y w mutation in D. melanogaster flies is suggested to have neural implications and possible behavioral alterations.Mutant y males are at a mating disadvantage possibly through altered levels of neuroactive catecholamines that are synthesized via dopa-like melanin (Biessmann, 1985).This suggestion is supported by the fact that its transcript level undergoes significant changes during development, being highest in late embryos prior to hatching compared to much lower levels in larval instars and adults, even higher than in pupae when melanisation of the adult cuticle occurs.This prominent elevation at hatching cannot be explained solely via melanisation, since pigmentation is repeated at every molt (Biessmann, 1985).The white (w) gene, on the other hand, encodes a transmembrane ABC transporter protein involved in the uptake of guanine and tryptophan, which are indispensable precursors in the synthesis of red (drosopterins) and brown (ommochrome xanthommatin) Drosophila pigments (Sullivan et al., 1974;Summers et al., 1982;Ewart and Howells, 1998).The absence of pigments in the eye of flies without a functional w gene results in ommatidia without optical insulation.Hence, the vision of such whiteeyed flies is impaired, especially at high light intensities (Krstic et al., 2013).Their photoreceptors receive about 19 times more light than those of wild-type flies and their electroretinograms are abnormal (Wu and Wong, 1977).Since guanine is further required for the synthesis of dopamine and serotonin, also tryptophan is a precursor of serotonin (Coleman and Neckameyer, 2005), w mutants show much reduced levels and altered distributions of these neurotransmitters (Borycz et al., 2008).Accordingly, mutations affecting the w function have an impact on the neural control of various behaviors, independent of proper eyesight (Campbell and Nash, 2001).Regarding their circadian locomotion, y w mutants were found rhythmic in LD conditions and free-running in constant dark (DD) conditions with a shorter periodicity of (τ=23.82)and a preserved subjective morning and evening activity peaks (Hassaneen, 2015).
Results of this study showed that mutant y w D. melanogaster flies retained a functional circadian clock, since they expressed a crepuscular bimodal rhythm that preserved the general profile of the wildtype (Allada and Chung, 2010) even with reduced plasticity and robustness (Figures 1, 2, and 5).Total activity of y w flies increased significantly with temperature compared to wildtype CS during daytime (Light phase) (Figure 3(A)), while became more significantly suppressed during nighttime (dark phase) (Figure 3(B)).Both fly lines expressed the same total daily activity level at mid-range 18°C, but y w were significantly less active at the low 10°C and significantly more active at 20°C (Figure 3(C)).Thus, it seems that the y w mutation appears to impair the fly's ability to cope with changing temperature causing them to respond radically to its change, while the intact circadian clock of the wildtype seemed more efficient in buffering temperature effects within physiological ranges.The radical startle response to lights-on (Figure 4(A)) and lights-off (Figure 4(B)) in mutant y w flies might be another evidence on the circadian clock buffering effect on behavioral responses of wildtype flies to temperature fluctuations.
The circadian clock is adaptive to maximize ecological fitness of animals.In cold short days of winter, it is favorable for the animals to be active during the warmer parts of the day, while during the hot long summer days, it is better for them to shift their activity to the cooler morning and/or night (Yoshii et al., 2012).In this study, with increasing temperature, wildtype CS flies advanced their morning activity towards the dark phase, lengthened their mid-day siesta, and delayed their night activity (Figure 5).Although in this experiment, temperature was fixed in both lightand dark-phases, the flies preferred to avoid light in high-temperature, while seek light in low temperature.It makes sense because the flies primarily use light as an entrainment agent to the LD cycle, but also they seemed as if they are avoiding the damaging effect of high temperature naturally linked with sunny light phase, while in low temperature they delayed their activity as if they are seeking the warm temperature naturally linked to the light phase.y w mutation; however, seemed to impair this adaptive function and the mutants responded poorly to temperature fluctuations.The mutant y w flies significantly shifted their M peak only at the highest 28°C and they were mostly shorter and more delayed than wildtype CS.For the mid-day siesta, wildtype CS rested mostly longer with increased temperature.The E peak of y w flies was also much significantly advanced and longer compared to wildtype CS.Collectively, the behavioral changes in y w mutant made most of their locomotor activity in the light phase compared to the wildtype CS.In addition, their responses to light changes were radical at the low and high temperature extremes.Which suggest that the mutation exposed them to higher levels of environmental risk.
In conclusion, the y w mutation appeared to affect the circadian regulation of locomotor activity.Mutant y w flies appeared more vulnerable to environmental risk factors and less adaptive to temperature fluctuations compared to wildtype CS.The results provide new insights for better understanding of behaviors studied in the y w genetic background.Further studies are required to investigate the primary causes and mechanisms behind these findings.Impaired melanisation, eye pigmentation loss, disturbed neurotransmitter distributions, and levels in y w mutation could all be contributing to disturbed circadian control.

Fig. 1 :
Fig. 1: Average circadian locomotor activity profiles of control wild-type CS and y w mutant D. melanogaster flies under light-dark cycles of (LD 12:12) and temperatures of 10°C, 18°C, 20°C, and 28°C.Locomotor activity is represented on the yaxis as the mean infrared beam crossings per minute by a thick black line; the (Mean), with (Standard Error of Mean) represented by two thin lines above and below the mean.Zeitgeber time 0 (ZT0) indicates the beginning of the light phase and coincides with 6:00 am Egypt Standard Time.Black and white bars above the figures indicate dark and light phases of the circadian LD cycle, respectively.Grey shadings inside the graphs also represents dark phases of the LD cycle.Arrows indicate lights-on and lights-off activity peaks, while white and black arrowheads indicate the mathematically calculated maximum activity in the morning and evening activity peaks.n is the sample size.

Fig. 2 :
Fig. 2: Representative double-plotted actograms for the circadian locomotor activity rhythms of control wild-type CS and mutant y w D. melanogaster flies recorded under (LD 12:12) and 10°C, 18°C, 20°C, and 28°C for 7 days.Black and white bars above the figures indicate the dark and light phases, respectively.Shaded areas inside the figures also indicate dark phases.Zeitgeber times (ZT) are indicated on top of the figures, with ZT0 and ZT12 marking the beginning of the light and dark phase, respectively.

Figure 3 :
Figure 3: Effect of temperature on locomotor activity in daytime "light phase" (A), nighttime "dark phase" (B), and average total daily activity (C) of control wild-type CS and mutant y w D. melanogaster flies.Mean number of IR crossings/min across the 12 hours duration of daytime (A), nighttime (B), or the 24h (C) activity of 7 days is represented with standard error of mean.Sample size n for each fly group is as in Figure 1. a and b Indicate a significant difference between the locomotor activity level at a specific temperature step and the one before it within the CS or y w fly lines, respectively, using ANOVA, while *denotes a significant difference between the activity level of CS and y w flies at each temperature step using t-test, both at (p<0.05).

Fig. 4 :
Fig. 4: Effect of temperature on lights-on (A) and lights-off (B) locomotor activity peaks of control wild-type CS and mutant y w D. melanogaster flies under 10°C, 18°C, 20°C, and 28°C and (LD 12:12) for 7 days.Data represent the average activity of 15 minutes after lights-on and lightsoff (Mean ± standard error of mean).Sample size details are in Figure 1.* Indicates a significant difference between CS and y w flies at the same temperature using t-test, while a and b denotes a significant difference between the activity level at a temperature step and the step before it within the same fly line of CS and y w, respectively, using ANOVA, both at (p<0.05).

Fig. 5 :
Fig.5: Effect of temperature on timing of locomotor activity peaks of control wild-type CS and mutant y w D. melanogaster flies.Horizontal bars represent the average morning, mid-day siesta, and evening activity peaks for about 30 flies/group for 7 days.Start, end, and duration of these peaks has been compared within each group (Morning, mid-day siesta, and evening) using One-Way ANOVA followed by Tukey test.The "max" activity was also compared in morning and evening peaks.Significant differences between CS and y w within each group (Morning, siesta, and evening) at the same temperature is highlighted using (*) for the start, (a) for the maximum activity of 15 minutes, (b) for end, and (c) for the length of each activity peak.Data are represented as (Mean ± Standard error of mean).Error bars at the start and end of activity bars are for the duration of activity peaks.