The genetic basis of diurnal preference in Drosophila melanogaster

- The genetic basis of diurnal preference in Drosophila melanogaster.
- Background: Most animals restrict their activity to a specific part of the day, being diurnal, nocturnal or crepuscular.
- The genetic basis underlying diurnal preference is largely unknown.
- Although time is one of the most important dimensions that define the species ecological niche, it is often a neglected research area [1].
- Most animal species exhibit locomotor activity that is restricted to a defined part of the day, and this preference constitutes the species- specific temporal niche.
- Selection for activity during a specific time of the day is driven by various factors, in- cluding preferred temperature or light intensity, food.
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- Full list of author information is available at the end of the article Pegoraro et al.
- In addition, experimental conditions in the laboratory set- ting (particularly light and temperature) often fail to simulate the high complexity that exists in natural con- ditions [1].
- In addition, a few studies in humans reported a significant association between polymorphisms in circa- dian clock genes and ‘morningness–eveningness’ chron- otypes, including a polymorphism in the promoter region of the period3 gene [7]..
- Sequence diver- gence in the period gene underlies the phase difference seen in locomotor and sexual rhythms between D.
- Flies also show nat- ural variation in the timing of adult emergence (eclosion), with a robust response to artificial selection.
- Further support for a genetic component to phase preference comes from our previous studies of allelic variation in the circadian-dedicated photoreceptor cryp- tochrome (CRY), where an association between a perva- sive replacement SNP (L232H) and the phases of locomotor activity and eclosion was revealed [15].
- Stud- ies of null mutants of the Clock gene (Clk jrk ) revealed that such flies became preferentially nocturnal [16], and that this phase switch is mediated by elevated CRY in a specific subset of clock neurons [17].
- Artificial selection for diurnal preference.
- As early as after 1 cycle of selection, the ND ratios of N and D flies were significantly different, relative to the original (control) population (Fig.
- The estimated heritability h 2 was higher for diurnality (37.1%) than for nocturnality, (8.4%) reflecting the asym- metric response of the two populations (Table S1)..
- The heritability value was slightly higher when ND ratios of mothers and daughters were regressed (Fig.
- We, therefore, tested whether our selection protocol asymmetrically affected the fitness of the N and D populations.
- After ~ 15 overlapping genera- tions from the end of the selection, in which selection has been relaxed, the ND ratios of N strain flies de- creased from 1.2 (C10) to 0.99, and those of D strain flies increased from 0.32 to 0.53.
- At this stage, we tested viability, fitness and egg- to-adult developmental time of the selection and control populations.
- Since the circadian system is a conceivable target for genetic adaptations that underlie diurnal preference, we tested whether the circadian clock of the N and D strains were affected by the Nocturnal/Diurnal artificial selection.
- Accordingly, we recorded the locomotor activ- ity of the selection lines following three generations after completion of the selection protocol, and measured vari- ous parameters of circadian rhythmicity..
- The phase of activity peak in the morning (MP) and in the evening (EP) differed between the populations (Figure S3A)..
- As expected, the MP of the N population was significantly advanced, as compared to that seen in both the C and D.
- Distribution of ND ratios of males from the starting population (C 0 , n = 176).
- Average ND ratios of males from selected populations per cycle of selection.
- Data points correspond to average ND ratios ± standard deviation (n .
- Grey points at the 4th and 10th cycle of selection correspond to ND ratios for the unselected control population (n = 19 – 78).
- The ND ratio of the original population is shown at C0.
- The EP of the N population was significantly delayed, as compared to that noted in the two other popula- tions.
- While D flies slept significantly more than C and N flies during the night, there was no dif- ference in the amount of sleep between N and C flies..
- The phases (φ) of the three populations did not differ significantly (Fig.
- We also tested the responses of the flies to an early night (ZT15) light stimulus and found no significant differences in their delay responses (Fig.
- The ND ratios of the isogenic lines resembled those of the progenitor selection lines (Figure S4A).
- The circadian behaviour of the isogenic lines differed, with the N* line having a longer FRP than both the D* and C* lines (Figure S5A).
- The locomotory acrophase of the N*.
- Since eclosion is regulated by the circadian clock [20, 21], we also compared the eclosion phase of the isogenic strains.
- Diurnal preference is partly driven by masking.
- We reasoned that light masking (i.e., the clock- independent inhibitory or stimulatory effect of light on.
- Congruently, when we ana- lysed the ND ratios of these flies in LD and DD conditions, we found that that both N* and C* flies be- came significantly more “diurnal” when released into constant conditions (N*, p <.
- Correlates of the molecular clock.
- To investigate whether differences in diurnal preference correlated with a similar shift in the molecular clock, we measured the intensity of nuclear PERIOD (PER) in key clock neurons (Fig.
- We also measured the expression of the Pigment Dis- persing Factor (PDF) in LNv projections (Figure S6-S7)..
- The signal measured in N* flies was lower than that measured in D* flies during the first part of the day (in particular at ZT3 and ZT7), yet increased during day- night transition at ZT11 and ZT13.
- There were no dif- ferences seen during the rest of the night..
- The predicted products of the DEGs were largely assigned to extracellular regions and pre- sented secretory pathway signal peptides.
- Acrophase angles of the free-run activity for N (black circles, n = 255), D (red circles, n = 159) and C (blue triangles, n = 65) populations.
- The free-run phases of the three populations did not differ (F p = 0.149, NS).
- flies, while genes involved in the immune response were up-regulated in N* flies.
- We investigated the contribution of various genes to nocturnal/diurnal behaviour using a modified version of the quantitative complementation test (QCT) [23]..
- Briefly, each of the natural alleles of a candidate gene is tested in association with a mutant allele of that gene, and their phenotypes are compared.
- We also tested the ion channel-encoding narrow abdo- men (na) gene, given its role in the circadian response to.
- The overall ANOVA between 4 days in LD and 4 days in DD conditions (starting from the second day in DD) indicates a significant effect of the light regime (i.e., LD vs.
- The ND ratios of D* flies did not significantly change in DD conditions (p = 0.94, NS).
- 6, Table S5), indicative of genetic variability in these genes contributing to the nocturnal/diurnal behaviour of the isogenic lines..
- Since switching from nocturnal to diurnal behaviour in mice has been shown to be associated with metabolic regu- lation [26], we also tested Insulin-like peptide 6 (Ilp6), and chico, both of which are involved in the Drosophila insulin pathway.
- This may indicate that different alleles and/or different genes were affected in the two.
- The appearance of the peak PER signal in ventral neurons (LNv: 5th-sLNv, sLNv, lLNv) was delayed in N* flies, as compared to D* flies (ZT1 vs ZT21 – 23), as indicated by significant time x genotype interactions: 5th-sLNv χ df = 11, p <.
- Selections for traits affect- ing fitness have been shown to have higher selection re- sponses in the direction of lower fitness [19].
- This phenomenon was observed both in the la- boratory and in the field [36], with light decreasing arousal in nocturnal animals and the opposite effect occurring in diurnal animals.
- This finding is reminiscent of the re- sults of our previous study in flies [37], where transcrip- tional differences between early and late chronotypes were present in genes up- and downstream of the clock but not in the clock itself.
- Most of the associations that were identified in each of the studies did not serve a clear circadian function, again underscoring the premise that diurnal preference is regulated by multiple loci both within and outside the circadian clock..
- 5 days old) from 272 isofemale lines from 33 regions in Europe and Africa (Table S2) in the same culture bottle containing standard sugar food.
- The progeny of this population was used in the artificial selection as generation 0 (C 0 .
- Using the R library GeneCycle and a custom- made script, we identified rhythmic flies and calculated their ND ratios.
- Correlation between ND ratios of fathers and sons ( n = 26 pairs).
- Selection population ND ratios after 5 months of selection relaxation.
- Distribution of ND ratios of males from the diurnal (D), nocturnal (N) and control (C) populations after 5 months.
- The ND ratio of the N population was significantly different from both that of the control and D populations (Kolmogorov-Smirnov (KS) t-test for N vs C D = 0.56, p <.
- The ND ratios of the D and C popula- tions were not significantly different (KS, D = 0.23, p = NS).
- Locomotor behaviour and sleep of the selection lines in LD condi- tions.
- ND ratios and sleep of isogenic strains.
- Distribution of ND ratios for males of the diurnal (D.
- Boxplots of free-running periods of the N* (n = 64), C* (n = 124) and D* (n = 157) isogenic lines.
- the bottom and upper ends of the box correspond to the upper and lower quartiles, respectively and the whiskers denote maximum and mini- mum values, excluding outliers.
- Acrophase angles of the free-running activity shown for the N* (black cir- cles, n = 64), D* (red circles, n = 157) and C* (blue triangles, n = 124) iso- genic lines.
- The phase in the N* line was delayed by 2.02 h, as compared to what was measured in the D* line, and by 1.38 h, as compared to what was measured in the C* line (F p <.
- Phase of eclosion in the N* (black circle, n = 75), D* (red circle, n = 111) and C*.
- There was no difference between N* and C* flies (F p = 0.78, NS).
- PDF staining in the N* (full lines) and D* (dashed lines) lines main- tained in a 12:12 LD cycle.
- 0.05 after Benjamini correc- tion, with the exception of the “ signal peptide ” term at ZT0, where p = 0.054.
- Artificial selection ND ratios and heritability.
- Vp is variance of the ND ratio per cycle of selection and Var(h 2 ) is the variance in h 2 due to gen- etic drift.
- KS D and pval are the results of the Kolmogorov-Smirnov test comparing ND ratios of two consecutive generations (n = 25 in all cases).
- Average ND ratios and standard deviations for each complementation test cross are shown.
- Compl indicates the result of the complementation test.
- Financial support has been provided by the Biotechnology and Biological Sciences Research Council (BBSRC, UK) grant BB/G02085X/1, which funded the postdoctoral fellowship of the lead author and supplies to conduct this research project.
- Polymorphism in the PER3 promoter associates with diurnal preference and delayed sleep phase disorder.
- Two clocks in the brain: an update of the morning and evening oscillator model in Drosophila.
- The molecular ticks of the Drosophila circadian clock..
- Analysis of temperature-sensitive mutants reveals new genes involved in the courtship song of Drosophila.
- Circadian rhythms of locomotor activity in the goldfish carassius auratus.
- Possible evidence for shift work schedules in the media workers of the ant species Camponotus compressus.
- Light masking in the field: an experiment with nocturnal and diurnal spiny mice under semi-natural field conditions.
- Period gene expression in the diurnal degu (Octodon degus) differs from the nocturnal laboratory rat (Rattus norvegicus).
- Period gene expression in the brain of a dual-phasing rodent, the octodon degus.
- Ectopic expression of ultraviolet-rhodopsins in the blue photoreceptor cells of drosophila: visual physiology and photochemistry of transgenic animals.
- A length polymorphism in the circadian clock gene Per3 is linked to delayed sleep phase syndrome and extreme diurnal preference

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