University of Groningen
Adaptation of Circadian Neuronal Network to Photoperiod in High-Latitude European
Drosophilids
Menegazzi, Pamela; Benetta, Elena Dalla; Beauchamp, Marta; Schlichting, Matthias;
Steffan-Dewenter, Ingolf; Helfrich-Foerster, Charlotte
Published in:
Current Biology
DOI:
10.1016/j.cub.2017.01.036
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Menegazzi, P., Benetta, E. D., Beauchamp, M., Schlichting, M., Steffan-Dewenter, I., & Helfrich-Foerster,
C. (2017). Adaptation of Circadian Neuronal Network to Photoperiod in High-Latitude European
Drosophilids. Current Biology, 27(6), 833-839. https://doi.org/10.1016/j.cub.2017.01.036
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Report
Adaptation of Circadian Neuronal Network to
Photoperiod in High-Latitude European Drosophilids
Graphical Abstract
Highlights
d
This is the first detailed analysis of fruit fly daily activity under
long days
d
D. ezoana and D. littoralis circadian clock differ from that of
D. melanogaster
d
In flies, CRY and PDF are essential for adapting to long days
dChanging CRY/PDF distribution in melanogaster mimics
northern species daily activity
Authors
Pamela Menegazzi,
Elena Dalla Benetta,
Marta Beauchamp,
Matthias Schlichting,
Ingolf Steffan-Dewenter,
Charlotte Helfrich-Fo¨rster
Correspondence
charlotte.foerster@biozentrum.
uni-wuerzburg.de
In Brief
The circadian clock modulates daily
activity. Menegazzi et al. found a
functional link between the clock
neurochemistry of fruit flies species and
their ability to adjust activity to long
summer days. The expression pattern of
clock components in northern species
might be of advantage for life in the north.
Menegazzi et al., 2017, Current Biology 27, 833–839
March 20, 2017ª 2017 The Author(s). Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.cub.2017.01.036
Current Biology
Report
Adaptation of Circadian Neuronal Network
to Photoperiod in High-Latitude
European Drosophilids
Pamela Menegazzi,1,3Elena Dalla Benetta,1,3,4Marta Beauchamp,1Matthias Schlichting,1,5Ingolf Steffan-Dewenter,2 and Charlotte Helfrich-Fo¨rster1,6,*
1Neurobiology and Genetics, Theodor Boveri Institute, Biocentre, University of Wu¨rzburg, 97074 Wu¨rzburg, Germany 2Department of Animal Ecology and Tropical Biology, Biocentre, University of Wu¨rzburg, 97074 Wu¨rzburg, Germany 3Co-first author
4Present address: Institute for Evolutionary Life Sciences, University of Groningen, 9700 Groningen, the Netherlands 5Present address: Department of Biology, Brandeis University, Waltham, MA 02453, USA
6Lead Contact
*Correspondence:charlotte.foerster@biozentrum.uni-wuerzburg.de http://dx.doi.org/10.1016/j.cub.2017.01.036
SUMMARY
The genus Drosophila contains over 2,000 species
that, stemming from a common ancestor in the Old
World Tropics, populate today very different
environ-ments [
1, 2
] (reviewed in [
3
]). We found significant
differences in the activity pattern of Drosophila
species belonging to the holarctic virilis group, i.e.,
D. ezoana and D. littoralis, collected in Northern
Europe, compared to that of the cosmopolitan
D. melanogaster, collected close to the equator.
These behavioral differences might have been of
adaptive significance for colonizing high-latitude
habitats and hence adjust to long photoperiods.
Most interestingly, the flies’ locomotor activity
corre-lates with the neurochemistry of their circadian
clock network, which differs between low and high
latitude for the expression pattern of the blue light
photopigment cryptochrome (CRY) and the
neuro-peptide Pigment-dispersing factor (PDF) [
4–6
]. In
D. melanogaster, CRY and PDF are known to
modu-late the timing of activity and to maintain robust
rhythmicity under constant conditions [
7–11
]. We
could partly simulate the rhythmic behavior of the
high-latitude virilis group species by mimicking their
CRY/PDF expression patterns in a laboratory strain
of D. melanogaster. We therefore suggest that these
alterations in the CRY/PDF clock neurochemistry
might have allowed the virilis group species to
colo-nize high-latitude environments.
RESULTS
The circadian clock network of D. ezoana and D. littoralis from Finland shows a different clock neurochemistry compared to that of D. melanogaster from Tanzania (Figures 1 and 2A). More precisely, high-latitude species show highly reduced
neu-ropeptide Pigment-dispersing factor (PDF) expression in the small ventrolateral clock neurons (s-LNvs) and lack the
photopig-ment cryptochrome (CRY) in the large ventrolateral clock neu-rons (l-LNvs). PDF fibers from the l-LNvs innervate instead the
central brain (Figure 2A). To identify the putative role of the pre-cise CRY and PDF expression pattern of the flies collected at high latitudes, we aimed to compare their locomotor activity to that of D. melanogaster under long photoperiods, i.e., light:dark cycles with 16 or 20 hr of light per day (LD 16:08 and LD 20:04, respectively) or constant environmental conditions. All flies en-trained to the LD cycles but D. ezoana and D. littoralis differed from D. melanogaster in their activity patterns (Figure 2B).
D. melanogaster showed a sharp morning activity peak, which
coincided with dawn simulation under both light regimes. This morning peak was followed by an evident siesta ending in the second half of the day, when the evening activity started. Whereas under LD 16:08 evening activity kept increasing until dusk, under LD 20:04 the evening activity maximum occurred 6 hr before dusk simulation (Figure 2B). Thus, under long photoperiods, D. melanogaster could only delay their evening activity to a limited extent. D. ezoana and D. littoralis showed more uniform activity levels throughout the light period compared to D. melanogaster (Figure 2B). They lacked the sharp morning peak and the prominent siesta. Moreover, the activity maxima of their broad evening activity occurred close to dusk even under LD 20:04,3 hr later than that of D. melanogaster (Figure 2B). Furthermore, the collected species differed in their ability to maintain locomotor activity rhythms under constant conditions (Figure 2C; Table S1). D. melanogaster remained rhythmic under constant darkness (DD) and lost rhythmicity un-der constant light (LL). In contrast, only 10% of D. littoralis and no
D. ezoana were rhythmic under DD (Figure 2C;Table S1). LL slightly improved rhythmicity in D. ezoana (Table S1). Taken together, these results indicate that the activity patterns previ-ously described for D. virilis (holarctic [5]) and D. montana (high latitude [4]) are not exceptional but hold true for D. ezoana and
D. littoralis. The peculiar PDF and CRY expression in the
ventro-lateral clock neurons and in the central brain of virilis group spe-cies therefore appears to correlate with their activity pattern. To clarify whether this correlation is causal, we aimed at stimulating
Current Biology 27, 833–839, March 20, 2017ª 2017 The Author(s). Published by Elsevier Ltd. 833 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
aspects of their rhythmic behavior under LD 20:04 by manipu-lating CRY and PDF expression in D. melanogaster.
The typical CRY expression pattern of the virilis group species (no CRY in l-LNvs as in D. ezoana and D. littoralis;Figure 2A) can
be mimicked in D. melanogaster (Figure 3) by downregulating CRY in all PDF-positive neurons (s-LNvs and l-LNvs) with help
of the Pdf-GAL4 driver [12] and in only the l-LNvs with the
c929-GAL4 driver [13]. CRY knockdown in the l-LNvs (or
s-LNvs plus l-LNvs) significantly reduced the morning peak and
delayed evening activity by40 min (Figure 3). In addition, it increased fly rhythmicity under LL (Figure 3): flies with no CRY in the l-LNvs (or s-LNvs plus l-LNvs) were significantly more
rhyth-mic (64% and 33%) than the controls (6%) (Figure 3;Table S2). There was no difference between the two strains, i.e., it is suffi-cient to knock down CRY in the l-LNvs to raise rhythmicity in LL.
In other words, mimicking virilis group species specific reduction of CRY expression in the l-LNvs of D. melanogaster is sufficient to
increase rhythmicity under LL. CRY expression did not affect rhythmicity in DD: all fly strains with manipulated CRY remained rhythmic under DD (data not shown).
To mimic PDF expression of D. ezoana and D. littoralis in
D. melanogaster, we used the c929>Pdf;Pdf01strain that
selec-tively limits PDF expression to the l-LNvs and to the central brain
(Figure 4A). We also limited the expression of PDF to the central brain in c929>Pdf;PdfG80;Pdf01flies. This enabled us to test whether PDF of non-clock cells can affect the activity rhythm of
D. melanogaster. Locomotor activity recordings of these two
strains were compared to Pdf>Pdf;Pdf01 flies, in which PDF
was rescued in a wild-type manner in the s-LNvs and l-LNvs,
and to R6>Pdf;Pdf01 flies that only express PDF within the s-LNvs. Similarly to the virilis group species, the flies with no
PDF in the s-LNvs, but expressing PDF in the l-LNvs and the
cen-tral brain or cencen-tral brain only, were arrhythmic under DD ( Fig-ure 4B;Table S3). Under LD conditions, all flies were entrained. In contrast to the flies with downregulated CRY, PDF-manipu-lated flies maintained the characteristic D. melanogaster activity pattern, with morning and evening peaks (Figure 4B). However, the maxima of evening activity occurred significantly later in flies expressing PDF ectopically in the central brain (with or without PDF-positive l-LNvs) as compared to controls (delay of1.6 hr;
Figure 4B). Thus, PDF expression in the l-LNvs and the central
brain, or even in the central brain alone, clearly has the potential to delay the evening peak and may account for the extended eve-ning activity of high-latitude species under long days.
To clarify how PDF signaling from the l-LNvs and in the central
brain affects the molecular clock, we compared cycling of the core clock protein PERIOD (PER) in all clock neurons between
Pdf01mutants and flies expressing PDF in l-LNvs and central
brain. As previously described for wild-type flies [14], PER was maximal around the transition from night to day in both fly strains, but clear differences in the amplitude and phase of PER cycling occurred in the two genotypes (Figures 3C and S1). PDF in the l-LNvs and in the central brain reduced PER
oscil-lation amplitude in all lateral clock neurons. Furthermore, it de-layed the PER increase and decrease in all clock neurons. This was most pronounced in clock neurons known to control eve-ning activity [15–17] (Figure S1). Thus, the delay of the evening peak seen in behavioral rhythmicity appears to be caused by a delay in the molecular oscillations in the evening clock neurons. In order to fully mimic the clock neurochemistry of the virilis group species in D. melanogaster, we simultaneously manipu-lated CRY and PDF by specifically downregulating CRY in the l-LNvs of flies that only express PDF in these clock neurons
and in the central brain. We found that these flies exhibited low morning activity (Figure 4D), significantly less compared to flies with the same PDF expression pattern but no CRY in l-LNvs (
Fig-ure 4B), and a broad evening activity bout (Figure 4D). In addi-tion, the former were more rhythmic than the latter in LL ( Fig-ure 4D;Table S2), showing that the manipulation of CRY and PDF is additive. Thus, their activity pattern resembled that of high-latitude species under long photoperiods.
DISCUSSION
Under long photoperiods and constant temperature in the lab, the high-latitude species D. ezoana and D. littoralis showed low morning activity and broad evening activity; they were also largely arrhythmic under constant conditions, with a tendency to a stronger rhythmicity under LL than under DD. The observed features are similar to those previously described for D. virilis and
D. montana [4, 5], suggesting that they are typical of the virilis group species. This may explain why this species group was able to colonize high-latitude environments, where it naturally experiences extremely long photoperiods. In contrast, species stemming from lower latitudes tend to show high morning and evening activity around dawn and dusk, with a siesta in between, and are robustly rhythmic under DD but largely arrhythmic under
Figure 1. Map with the Collection Sites of the Low- and High-Lati-tude Species
The collection site of the low-latitude D. melanogaster strain is in Tanzania (3S), the one of D. ezoana and D. littoralis in northern Finland (65N).
LL [18–21]. So far, only the rhythmic behavior of D. melanogaster from temperate zones and D. ananassae from India has been re-corded under artificial long days [22, 23]. Both studies showed that the evening peak cannot track dusk when the day gets too long. Here, with a D. melanogaster strain from close to the equa-tor (Tanzania; 3S), we observed an even more evident inability to track dusk. Thus, according to its activity rhythms, D.
mela-nogaster appears not to be suited for a life in the very north.
Indeed, the species D. melanogaster naturally inhabits equatorial to temperate regions [24, 25].
D. ezoana and D. littoralis, as well as other virilis group species,
lack CRY in the l-LNvs and PDF in the s-LNvs but additionally
ex-press PDF in the central brain [4–6]. We hypothesize that these species may have lost CRY and PDF in specific neuron subsets in the course of evolution. Unfortunately, little is known on CRY in other insects, but some information on PDF is available. PDF is present in neurons with small and large somata in several other Diptera and in a few polyneopteran insects [26–29]. In other spe-cies, such as kissing bugs and termites, PDF-expressing neu-rons could not be subdivided by size, but it seemed quite evident
Figure 2. CRY and PDF Expression in the Anterior Brain of D. melanogaster, D. ezoana, and D. littoralis as well as Rhythmic Behavior of the Three Fly Species under Long Photo-periods and Constant Conditions
D. melanogaster flies co-express CRY and PDF in the four s-LNvs and l-LNvs, respectively (A), they
exhibit morning (M) and evening (E) activity bouts under LD16:08 and LD20:4 (B), were rhythmic un-der constant darkness (DD) and arrhythmic unun-der constant light (LL) (C). D. ezoana and D. littoralis are devoid of CRY in the l-LNvs and of PDF in the
s-LNvs but have extra PDF in the central brain
(ar-rows in A). They exhibit reduced morning activity in comparison to D. melanogaster (F2,121= 11.58;
p < 0.0001; Tukey HSD D. melanogaster*D. ezoana: p < 0.05; D. melanogaster*D. littoralis: p < 0.0001), a broad evening activity bout (B), and are completely arrhythmic under DD but show some residual rhythmicity under LL (C). Percent-ages of rhythmic and arrhythmic flies are found in Table S1. Importantly, the evening activity bout occurs later in D. ezoana and D. littoralis than in D. melanogaster (F2,119= 122.79; p < 0.0001; Tukey
HSD D. melanogaster*D. ezoana: p < 0.0001; D. melanogaster*D. littoralis: p < 0.0001). The average evening peak timing is represented by the black dot (±SD) above the evening activity peak. The gray vertical bands in the average activity profiles (B) indicate dawn and dusk simulation; the numbers in the left upper corners are the number of flies recorded. The average activity profiles are shown with SEM (gray lines above and below the mean [black line]). The individual actograms (C) are shown as double plots with the LD 20:04 cycle during the first 5 days indicated as white (day), gray (dawn and dusk), and black (night) bars on top.
that they are composed by two subpopu-lations of neurons according to their pro-jection pattern [30, 31]. PDF might there-fore have been originally present in two groups of lateral neurons in insects (named s-LNvs and l-LNvs
in D. melanogaster) and may have subsequently disappeared from the s-LNvs of the Drosophila species, which inhabit
high-latitude environments. PDF-positive fibers from the l-LNvs might
have simultaneously started to innervate the central brain in these species.
CRY is regarded as the circadian photopigment of
D. melanogaster [10, 11, 32, 33]. cry mutants remain rhythmic un-der LL [10, 11, 32, 33]. In addition, cry mutants only exhibit low morning activity and can delay their evening activity peak under long photoperiods more than wild-type flies [8]. The latter fea-tures of locomotor activity are, to a certain extent, also present in high-latitude Drosophila species that lack CRY in the l-LNvs.
Thus, there is a causal relation between the presence of CRY in the l-LNvs and the rhythmic behavior of high-latitude
species. Nevertheless, downregulating CRY in the l-LNvs of
D. melanogaster, it is not sufficient to fully reproduce the behavior
of the virilis group species in terms of evening peak timing and LL rhythmicity. As previously proposed, it is likely that other factors, e.g., polymorphisms in the clock genes’ coding sequences
[34–38], might also play an important role. How CRY in the l-LNvs
of D. melanogaster can affect rhythmic behavior so drastically re-mains unclear. Nevertheless, the l-LNvs are also known to be
light-activated arousal neurons [39, 40], and without CRY, their excitability might be reduced [41].
The PDF-positive s-LNvs of D. melanogaster are crucial for
morning activity and circadian rhythmicity under DD: Pdf01
mu-tants, as well as flies in which PDF is specifically downregulated in the s-LNvs, lose both morning activity and the ability to sustain
robust free-running rhythms under DD [7, 42]. This correlates well with the here observed reduced morning activity and DD arrhythmicity of the virilis group species that lack PDF in the s-LNvs. PDF is also important for timing evening activity:
PDF-null mutants, as well as PDF receptor (PDFR)-PDF-null mutants exhibit early evening activity [7, 43, 44]. Here, we show for the first time that PDF release into the central lateral brain can delay PER cycling and evening activity under long photoperiods, the latter even independently of whether PDF is secreted from the l-LNvs or from PDF-positive cells in the central brain. This fits
to our recent observation that the PDF receptor is required in these evening neurons to delay the phase of evening activity un-der long photoperiods [44]. In addition, Cusumano et al. [45] showed that PDF from the l-LNvs is necessary to entrain the
eve-ning neurons (and eveeve-ning activity) via the visual system, in the absence of CRY. In accordance with these findings, flies ex-pressing PDF ectopically in the central brain or flies with misrouted l-LNvfibers show later evening activity [46–49]. This
effect seems not to depend on a rhythmic release of PDF, as the PDF-positive fibers originating from ectopic PDF-expressing
Figure 3. CRY Knockdown in the l-LNvs of
PDF>cryKDand c929>cryKDD. melanogaster
strains Reduces Morning Activity, Slightly Delays Evening Activity, and Increases Rhythmicity under LL
The average activity of the controls in the top-left panel is a pooled profile from both relevant up-stream activating sequence (UAS) and Gal4 con-trol lines. The actograms are representative ex-amples of individual flies. The black dot above the evening peak in the average activity profiles represents the average evening peak time (±SD). Activity levels during dawn simulation (arrows) are lower in PDF>cryKDand c929>cryKDcompared to the relevant Gal4 and UAS controls (F3,196= 21.7;
p < 0.0001; Tukey HSD pdf>cryKD *Gal4/UAS controls: p < 0.0001; c929>cryKD *Gal4/UAS con-trols: p < 0.0001). PDF>cryKD and c929>cryKD also show a later evening activity peak (F3,196= 28.47;
p < 0.0001; Tukey HSD pdf>cryKD
*Gal4/UAS controls: p < 0.0001; c929>cryKD
*Gal4/UAS controls: p < 0.0001). Labeling is as inFigure 2. Percentages of rhythmic and arrhythmic flies are found inTable S2.
neurons do not show a cycling in PDF im-munostaining [46]. Thus, it is likely that even PDF from non-clock might delay evening neurons of virilis group species. The only prerequisite would be for these fibers to be in the vicinity of the evening neurons, which seems to be the case here (seeFigures 2A and 4A; [6]; see also discussion in [46]).
Whereas the main function of PDF signals from the l-LNvs (and
other non-clock cells in the central brain) concerns the adjust-ment of evening activity to long days, CRY downregulation in the l-LNvs has the largest effects on the morning peak in LD
and on rhythmicity in LL. We were able to mimic certain features of the typical behavior of virilis group species in D. melanogaster by altering its PDF and CRY expression pattern, which might have been crucial in the adaptation of the virilis species to high-latitude environments. This theory also fits with the evolu-tionary history of the Drosophila genus, which originated from the Old World tropics. The virilis species group probably lost CRY in the l-LNvs as well as PDF expression in the s-LNvs but
gained PDF expression in the central brain and hence adjusted to environments subjected to major photoperiodic changes throughout the year. If our theory is valid, species phylogeneti-cally closer to the virilis group than D. melanogaster, but living in subtropical or temperate regions, should still show a
D. melanogaster-like CRY and PDF expression pattern. Future
experiments should therefore study the neuroanatomy and circadian behavior of flies that originate from the virilis-repleta ra-diation and inhabit tropical, subtropical, or temperate zones.
EXPERIMENTAL PROCEDURES Fly Strains and Rearing Conditions
Flies were reared on cornmeal/agar medium containing yeast at a constant temperature of 20C or 25C. D. melanogaster and D. littoralis flies were
Figure 4. PDF Restriction to the l-LNvs and the Central Brain Delays Evening Activity and PER Cycling in the Clock Neurons and Make
the Flies Arrhythmic under DD, whereas Simultaneous Manipulation of CRY and PDF Expression Makes the Rhythmic Activity Pattern of D. melanogaster Flies Similar to that of High-Latitude Species
(A) PDF expression under the control of c929 is driven in the l-LNvs and neurons close to them that arborize in the lateral central brain (arrow), not far from the
dorsolateral clock neurons that belong to the evening neurons (not stained).
(B) Average activity profiles show that PDF expression in the l-LNvs and central brain or central brain only is necessary and sufficient to delay the phase of the
evening peak, but (F3,108= 109.9; p < 0.0001; Tukey HSD c929>Pdf;Pdf01: p < 0.001; c929>Pdf;PdfG80;Pdf01: p < 0.001) not to sustain rhythmicity in DD.
Percentages of rhythmic and arrhythmic flies are found inTable S3.
(C) The mean peak time of PER oscillation within the clock neurons is delayed more than 1 hr in c929>Pdf;Pdf01
compared to Pdf01
mutants. Mean peak time was calculated from pooled data of all time points shown inFigure S1.
(D) D. melanogaster flies with PDF only in the l-LNvs plus central brain and without CRY in these cells behave similar to high-latitude species. The arrow in the
average activity profile points to the low morning activity (T6,59; p < 0.0001, compared to morning activity levels in c929>Pdf;Pdf01); the mean maximum of evening
activity (±SD) is indicated on top of the graph. Labeling is as inFigure 2. Percentages of rhythmic and arrhythmic flies are given inTable S2.
maintained under light:dark cycles of 12 hr (LD 12:12) and the strongly photo-periodic D. ezoana flies under constant light in order to avoid diapause induc-tion. Both D. littoralis and D. ezoana (1240J8) used in this study originated from wild populations collected in 2008 in northern Finland (Europe; 6596’N, 2918’E) and were kindly donated by A. Hoikkala, University of Jyv€askyl€a. The D. melanogaster strain was established from a population collected in Tanzania, Africa (3S) in 2014.
In order to manipulate PDF and/or CRY expression in the brain of D. melanogaster, we used three different GAL4 lines. The Pdf-GAL4 and R6-GAL4 drivers are expressed, respectively, in all ventrolateral clock neurons [12] or s-LNvs only [50]. The c929-GAL4 driver is expressed in the l-LNvs
and in neurosecretory cells in the central brain [13]. This driver was also used in combination with the GAL4 inhibitor GAL80, expressed under the con-trol of the Pdf promoter in order to block transgene expression in the l-LNvs
only [16]. For CRY downregulation (cryKD
), we used two UAS-cryRNAi lines (see theSupplemental Experimental Procedures), whereas to restrict PDF expression to subsets of the ventrolateral clock neurons, we combined the different GAL4 drivers with a UAS-Pdf construct in a Pdf01
background [7]. The following strains were used: (1) Pdf-GAL4;UAS-cryRNAi called pdf>cryKD
; (2) c929-GAL4;UAS-cryRNAi referred to as c929>cryKD
; (3) c929-GAL4 UAS-Pdf/+;Pdf01
, here referred to as c929>Pdf;Pdf01
; (4) c929-GAL4 UAS-Pdf/ Pdf-GAL80;Pdf01, here referred to as c929>Pdf;PdfG80;Pdf01; (5) Pdf-GAL4/ UAS-Pdf;Pdf01
, here called Pdf>Pdf;Pdf01
; and (6) UAS-Pdf/+;Pdf01
without any PDF expression, here called simply Pdf01
.
Locomotor Activity Recording and Analysis
Locomotor activity of individual 5- to 7-day-old male flies was recorded at 20C using the Drosophila Activity Monitor (DAM) system (Trikinetics) and analyzed as described previously [51]. For details, see theSupplemental Experimental Procedures.
For activity recordings under LD, all flies were exposed for 8 days to long photoperiods of LD 16:08 followed by 8 days of LD 20:04. For activity record-ings in constant conditions, after entrainment in LD 20:04, the flies were released either in constant darkness (DD) or constant light (LL) and recorded for 10 days.
Immunocytochemistry and Microscopy
For anatomical studies, flies were entrained for 5 days in LD 12:12 and collected at ZT23 (1 hr before lights on). For PER oscillation analysis, c929>Pdf;Pdf01and in the corresponding pdf01controls were entrained in LD 16:08 for 1 week, and on day 7, they were collected every 3 hr around the clock.
For each time point, 12 whole flies were fixed in 4% paraformaldehyde, 0.5% Triton X-100 dissolved in PBS (PBST). D. melanogaster flies were fixed for 2.5 hr, whereas D. ezoana and D. littoralis, due to their bigger size, were fixed for 3.5 hr. Staining and quantification of the signals followed the protocol described in [14]. For details, see theSupplemental Experimental Procedures.
Statistics
Statistical analysis (one- or two-way ANOVA analysis followed by Tukey HSD for normally distributed data; Mann-Whitney for non-normally distributed data) were performed using STATISTICA (StatSoft; DELL).
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures, one figure, and three tables and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2017.01.036.
AUTHOR CONTRIBUTIONS
C.H.-F. and P.M. conceived the study and wrote the paper. E.D.B. performed the experiments with the transgenic Drosophila flies and M.B. those with the Tanzania and Finland Drosophila species. M.S. helped with the crosses of the transgenic lines and brain dissection. P.M. supervised and analyzed all ex-periments. I.S.-D. collected the Tanzanian D. melanogaster strain, and both I.S.-D. and M.S. provided intellectual input.
ACKNOWLEDGMENTS
We thank Hannele Kauranen and Anneli Hoikkala for providing the northern Drosophila species; Heinrich Dircksen, Takeshi Todo, and Ralf Stanewsky for providing antibodies; and Franc¸ois Rouyer for transgenic fly lines. We are grateful to Enrico Bertolini for help with immunocytochemistry and to Sheeba Vasu, Pavitra Prakash, Sheetal Potdar, Koustubh Vaze, and Martijn Schenkel for comments on the manuscript. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) and the Vienna Drosophila Resource Center were used in this study. We thank the German Research Foundation (DFG; SFB1047, INST 93/784-1: projects A1 and C2) for funding and support. Received: October 25, 2016 Revised: December 14, 2016 Accepted: January 19, 2017 Published: March 2, 2017 REFERENCES
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