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Adaptation of Circadian Neuronal Network to Photoperiod in High-Latitude EuropeanDrosophilidsMenegazzi, Pamela; Benetta, Elena Dalla; Beauchamp, Marta; Schlichting, Matthias; Steffan-Dewenter, Ingolf; Helfrich-Foerster, CharlottePublished in:Current Biology
DOI:10.1016/j.cub.2017.01.036
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Report
Adaptation of Circadian N
euronal Network toPhotoperiod in High-Latitude European DrosophilidsGraphical 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
d Changing CRY/PDF distribution in melanogaster mimics
northern species daily activity
Menegazzi et al., 2017, Current Biology 27, 833–839March 20, 2017 ª 2017 The Author(s). Published by Elsevier Ltdhttp://dx.doi.org/10.1016/j.cub.2017.01.036
Authors
Pamela Menegazzi,
Elena Dalla Benetta,
Marta Beauchamp,
Matthias Schlichting,
Ingolf Steffan-Dewenter,
Charlotte Helfrich-Forster
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.
.
Current Biology
Report
Adaptation of Circadian Neuronal Networkto Photoperiod in High-LatitudeEuropean DrosophilidsPamela Menegazzi,1,3 Elena Dalla Benetta,1,3,4 Marta Beauchamp,1 Matthias Schlichting,1,5 Ingolf Steffan-Dewenter,2
and Charlotte Helfrich-Forster1,6,*1Neurobiology and Genetics, Theodor Boveri Institute, Biocentre, University of Wurzburg, 97074 Wurzburg, Germany2Department of Animal Ecology and Tropical Biology, Biocentre, University of Wurzburg, 97074 Wurzburg, Germany3Co-first author4Present address: Institute for Evolutionary Life Sciences, University of Groningen, 9700 Groningen, the Netherlands5Present address: Department of Biology, Brandeis University, Waltham, MA 02453, USA6Lead Contact*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.cub.2017.01.036
SUMMARY
The genus Drosophila contains over 2,000 speciesthat, stemming from a common ancestor in the OldWorld Tropics, populate today very different environ-ments [1, 2] (reviewed in [3]). We found significantdifferences in the activity pattern of Drosophilaspecies belonging to the holarctic virilis group, i.e.,D. ezoana and D. littoralis, collected in NorthernEurope, compared to that of the cosmopolitanD. melanogaster, collected close to the equator.These behavioral differences might have been ofadaptive significance for colonizing high-latitudehabitats and hence adjust to long photoperiods.Most interestingly, the flies’ locomotor activity corre-lates with the neurochemistry of their circadianclock network, which differs between low and highlatitude for the expression pattern of the blue lightphotopigment cryptochrome (CRY) and the neuro-peptide Pigment-dispersing factor (PDF) [4–6]. InD. melanogaster, CRY and PDF are known to modu-late the timing of activity and to maintain robustrhythmicity under constant conditions [7–11]. Wecould partly simulate the rhythmic behavior of thehigh-latitude virilis group species by mimicking theirCRY/PDF expression patterns in a laboratory strainof D. melanogaster. We therefore suggest that thesealterations in the CRY/PDF clock neurochemistrymight 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-
Current Biology 27, 833–839, MaThis is an open access article under the CC BY-N
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%ofD. 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
rch 20, 2017 ª 2017 The Author(s). Published by Elsevier Ltd. 833C-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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
(3�S), the one of D. ezoana and D. littoralis in northern Finland (65�N).
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 by �40 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 ofD.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;Pdf01 strain 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;Pdf01 flies. 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
834 Current Biology 27, 833–839, March 20, 2017
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 central 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 of �1.6 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 andmay 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
Pdf01 mutants 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 forD. 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 2. CRY and PDF Expression in the
AnteriorBrainofD.melanogaster,D.ezoana,
andD. littoralis aswell asRhythmicBehavior
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 under
constant light (LL) (C).D. ezoana andD. 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 aswhite (day), gray
(dawn and dusk), and black (night) bars on top.
LL [18–21]. So far, only the rhythmic behavior ofD.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; 3�S), 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 speciesD.melanogaster naturally inhabits equatorial
to temperate regions [24, 25].
D. ezoana andD. 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
Curren
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]. crymutants 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
t Biology 27, 833–839, March 20, 2017 835
Figure 3. CRY Knockdown in the l-LNvs of
PDF>cryKD and c929>cryKD D. 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>cryKD and c929>cryKD compared 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 in Figure 2.
Percentages of rhythmic and arrhythmic flies are
found in Table S2.
[34–38], might also play an important role. HowCRY in the l-LNvs
ofD.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)-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 evening 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-LNv fibers 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
836 Current Biology 27, 833–839, March 20, 2017
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 (see Figures 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 20�C or 25�C. 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 in Table S3.
(C) The mean peak time of PER oscillation within the clock neurons is delayedmore than 1 hr in c929>Pdf;Pdf01 compared to Pdf01mutants. Mean peak time was
calculated from pooled data of all time points shown in Figure 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 lowmorning activity (T6,59; p < 0.0001, compared tomorning activity levels in c929>Pdf;Pdf01); themeanmaximumof evening
activity (±SD) is indicated on top of the graph. Labeling is as in Figure 2. Percentages of rhythmic and arrhythmic flies are given in Table S2.
Current Biology 27, 833–839, March 20, 2017 837
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. BothD. littoralis andD. ezoana (1240J8) used in this study originated from
wild populations collected in 2008 in northern Finland (Europe; 65�96’N,29�18’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 (3�S) 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 the Supplemental 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
20�C using the Drosophila Activity Monitor (DAM) system (Trikinetics) and
analyzed as described previously [51]. For details, see the Supplemental
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;Pdf01 and in the corresponding pdf01 controls 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 the Supplemental 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.
838 Current Biology 27, 833–839, March 20, 2017
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 Francois 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
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