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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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Adaptation of Circadian N

euronal Network toPhotoperiod 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

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

Correspondencecharlotte.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.

.

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: 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 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

REFERENCES

1. Grimaldi, D.A. (1990). A phylogenetic, revised classification of the genera

in the Drosophilidae (Diptera). Bull. Am. Mus. Nat. Hist. 197, 1–139.

2. Throckmorton, L.H. (1975). The phylogeny, ecology, and geography of

Droop. In Handbook of Genetics, R.C. King, ed. (Plenum), pp. 421–459.

3. O’Grady, P.M., and Markow, T.A. (2009). Phylogenetic taxonomy in

Drosophila. Fly (Austin) 3, 10–14.

4. Kauranen, H., Menegazzi, P., Costa, R., Helfrich-Forster, C., Kankainen,

A., and Hoikkala, A. (2012). Flies in the north: locomotor behavior and

clock neuron organization of Drosophila montana. J. Biol. Rhythms 27,

377–387.

5. Bahn, J.H., Lee, G., and Park, J.H. (2009). Comparative analysis of

Pdf-mediated circadian behaviors between Drosophila melanogaster

and D. virilis. Genetics 181, 965–975.

6. Hermann, C., Saccon, R., Senthilan, P.R., Domnik, L., Dircksen, H., Yoshii,

T., and Helfrich-Forster, C. (2013). The circadian clock network in the brain

of different Drosophila species. J. Comp. Neurol. 521, 367–388.

7. Renn, S.C., Park, J.H., Rosbash, M., Hall, J.C., and Taghert, P.H. (1999). A

pdf neuropeptide gene mutation and ablation of PDF neurons each cause

severe abnormalities of behavioral circadian rhythms in Drosophila. Cell

99, 791–802.

8. Rieger, D., Stanewsky, R., and Helfrich-Forster, C. (2003). Cryptochrome,

compound eyes, Hofbauer-Buchner eyelets, and ocelli play different roles

in the entrainment and masking pathway of the locomotor activity rhythm

in the fruit fly Drosophila melanogaster. J. Biol. Rhythms 18, 377–391.

9. Dolezelova, E., Dolezel, D., and Hall, J.C. (2007). Rhythm defects caused

by newly engineered null mutations in Drosophila’s cryptochrome gene.

Genetics 177, 329–345.

10. Emery, P., So, W.V., Kaneko, M., Hall, J.C., and Rosbash, M. (1998). CRY,

a Drosophila clock and light-regulated cryptochrome, is a major contrib-

utor to circadian rhythm resetting and photosensitivity. Cell 95, 669–679.

11. Stanewsky, R., Kaneko, M., Emery, P., Beretta, B., Wager-Smith, K., Kay,

S.A., Rosbash, M., and Hall, J.C. (1998). The crybmutation identifies cryp-

tochrome as a circadian photoreceptor in Drosophila. Cell 95, 681–692.

12. Park, J.H., Helfrich-Forster, C., Lee, G., Liu, L., Rosbash, M., and Hall, J.C.

(2000). Differential regulation of circadian pacemaker output by separate

clock genes in Drosophila. Proc. Natl. Acad. Sci. USA 97, 3608–3613.

13. Hewes, R.S., Schaefer, A.M., and Taghert, P.H. (2000). The cryptocephal

gene (ATF4) encodes multiple basic-leucine zipper proteins controlling

molting and metamorphosis in Drosophila. Genetics 155, 1711–1723.

14. Menegazzi, P., Vanin, S., Yoshii, T., Rieger, D., Hermann, C., Dusik, V.,

Kyriacou, C.P., Helfrich-Forster, C., and Costa, R. (2013).Drosophila clock

neurons under natural conditions. J. Biol. Rhythms 28, 3–14.

15. Grima, B., Ch�elot, E., Xia, R., and Rouyer, F. (2004). Morning and evening

peaks of activity rely on different clock neurons of the Drosophila brain.

Nature 431, 869–873.

16. Stoleru, D., Peng, Y., Agosto, J., and Rosbash, M. (2004). Coupled oscil-

lators control morning and evening locomotor behaviour of Drosophila.

Nature 431, 862–868.

17. Rieger, D., Shafer, O.T., Tomioka, K., and Helfrich-Forster, C. (2006).

Functional analysis of circadian pacemaker neurons in Drosophila mela-

nogaster. J. Neurosci. 26, 2531–2543.

18. Helfrich-Forster, C. (2000). Differential control of morning and evening

components in the activity rhythm of Drosophila melanogaster–sex-spe-

cific differences suggest a different quality of activity. J. Biol. Rhythms

15, 135–154.

19. Konopka, R.J., Pittendrigh, C., and Orr, D. (1989). Reciprocal behaviour

associated with altered homeostasis and photosensitivity of Drosophila

clock mutants. J. Neurogenet. 6, 1–10.

20. Prabhakaran, P.M., and Sheeba, V. (2014). Temperature sensitivity of

circadian clocks is conserved across Drosophila species melanogaster,

malerkotliana and ananassae. Chronobiol. Int. 31, 1008–1016.

21. Vanlalhriatpuia, K., Chhakchhuak, V., Moses, S.K., Iyyer, S.B., Kasture,

M.S., Shivagaje, A.J., Rajneesh, B.J., and Joshi, D.S. (2007). Effects of alti-

tude on circadian rhythm of adult locomotor activity in Himalayan strains of

Drosophila helvetica. J. Circadian Rhythms 5, 1.

22. Rieger, D., Peschel, N., Dusik, V., Glotz, S., and Helfrich-Forster, C. (2012).

The ability to entrain to long photoperiods differs between 3 Drosophila

melanogaster wild-type strains and is modified by twilight simulation.

J. Biol. Rhythms 27, 37–47.

23. Prabhakaran, P.M., and Sheeba, V. (2012). Sympatric Drosophilid species

melanogaster and ananassae differ in temporal patterns of activity. J. Biol.

Rhythms 27, 365–376.

24. Demerec, M. (1950). Biology of Drosophila (John Wiley and Sons).

25. Patterson, J.T., and Stone, W.S. (1952). Evolution in the Genus Drosophila

(Macmillan).

26. N€assel, D.R., Shiga, S., Wikstrand, E.M., and Rao, K.R. (1991). Pigment-

dispersing hormone-immunoreactive neurons and their relation to seroto-

nergic neurons in the blowfly and cockroach visual system. Cell Tissue

Res. 266, 511–523.

27. Pyza, E., Siuta, T., and Tanimura, T. (2003). Development of PDF-immuno-

reactive cells, possible clock neurons, in the housefly Musca domestica.

Microsc. Res. Tech. 62, 103–113.

28. Pyza, E., and Meinertzhagen, I.A. (1997). Neurites of period-expressing

PDH cells in the fly’s optic lobe exhibit circadian oscillations in

morphology. Eur. J. Neurosci. 9, 1784–1788.

29. Sehadova, H., Sauman, I., and Sehnal, F. (2003). Immunocytochemical

distribution of pigment-dispersing hormone in the cephalic ganglia of pol-

yneopteran insects. Cell Tissue Res. 312, 113–125.

30. Vafopoulou, X., Terry, K.L., and Steel, C.G. (2010). The circadian timing

system in the brain of the fifth larval instar ofRhodnius prolixus (hemiptera).

J. Comp. Neurol. 518, 1264–1282.

31. Zavodska, R., Wen, C.J., Sehnal, F., Hrdy, I., Lee, H.J., and Sauman, I.

(2009). Corazonin- and PDF-immunoreactivities in the cephalic ganglia

of termites. J. Insect Physiol. 55, 441–449.

32. Emery, P., Stanewsky, R., Helfrich-Forster, C., Emery-Le, M., Hall, J.C.,

and Rosbash, M. (2000). Drosophila CRY is a deep brain circadian photo-

receptor. Neuron 26, 493–504.

33. Emery, P., Stanewsky, R., Hall, J.C., and Rosbash, M. (2000). A unique

circadian-rhythm photoreceptor. Nature 404, 456–457.

34. Tauber, E., Zordan,M., Sandrelli, F., Pegoraro, M., Osterwalder, N., Breda,

C., Daga, A., Selmin, A., Monger, K., Benna, C., et al. (2007). Natural selec-

tion favors a newly derived timeless allele in Drosophila melanogaster.

Science 316, 1895–1898.

35. Sandrelli, F., Tauber, E., Pegoraro, M., Mazzotta, G., Cisotto, P.,

Landskron, J., Stanewsky, R., Piccin, A., Rosato, E., Zordan, M., et al.

(2007). A molecular basis for natural selection at the timeless locus in

Drosophila melanogaster. Science 316, 1898–1900.

36. Petersen, G., Hall, J.C., and Rosbash, M. (1988). The period gene of

Drosophila carries species-specific behavioral instructions. EMBO J. 7,

3939–3947.

37. Hut, R.A., Paolucci, S., Dor, R., Kyriacou, C.P., and Daan, S. (2013).

Latitudinal clines: an evolutionary view on biological rhythms. Proc. Biol.

Sci. 280, 20130433.

38. Pegoraro, M., Noreen, S., Bhutani, S., Tsolou, A., Schmid, R., Kyriacou,

C.P., and Tauber, E. (2014). Molecular evolution of a pervasive natural

amino-acid substitution in Drosophila cryptochrome. PLoS ONE 9,

e86483.

39. Parisky, K.M., Agosto, J., Pulver, S.R., Shang, Y., Kuklin, E., Hodge, J.J.,

Kang, K., Liu, X., Garrity, P.A., Rosbash, M., and Griffith, L.C. (2008).

PDF cells are a GABA-responsive wake-promoting component of the

Drosophila sleep circuit. Neuron 60, 672–682.

40. Sheeba, V., Fogle, K.J., Kaneko, M., Rashid, S., Chou, Y.T., Sharma, V.K.,

and Holmes, T.C. (2008). Large ventral lateral neurons modulate arousal

and sleep in Drosophila. Curr. Biol. 18, 1537–1545.

41. Fogle, K.J., Parson, K.G., Dahm, N.A., and Holmes, T.C. (2011).

CRYPTOCHROME is a blue-light sensor that regulates neuronal firing

rate. Science 331, 1409–1413.

42. Shafer, O.T., and Taghert, P.H. (2009). RNA-interference knockdown of

Drosophila pigment dispersing factor in neuronal subsets: the anatomical

basis of a neuropeptide’s circadian functions. PLoS ONE 4, e8298.

43. Hyun, S., Lee, Y., Hong, S.T., Bang, S., Paik, D., Kang, J., Shin, J., Lee, J.,

Jeon, K., Hwang, S., et al. (2005). Drosophila GPCR Han is a receptor for

the circadian clock neuropeptide PDF. Neuron 48, 267–278.

44. Schlichting, M., Menegazzi, P., Lelito, K.R., Yao, Z., Buhl, E., Dalla

Benetta, E., Bahle, A., Denike, J., Hodge, J.J., Helfrich-Forster, C., and

Shafer, O.T. (2016). A neural network underlying circadian entrainment and

photoperiodic adjustment of sleep and activity in Drosophila. J. Neurosci.

36, 9084–9096.

45. Cusumano, P., Klarsfeld, A., Ch�elot, E., Picot, M., Richier, B., and Rouyer,

F. (2009). PDF-modulated visual inputs and cryptochrome define diurnal

behavior in Drosophila. Nat. Neurosci. 12, 1431–1437.

46. Helfrich-Forster, C., T€auber, M., Park, J.H., Muhlig-Versen, M.,

Schneuwly, S., and Hofbauer, A. (2000). Ectopic expression of the neuro-

peptide pigment-dispersing factor alters behavioral rhythms in Drosophila

melanogaster. J. Neurosci. 20, 3339–3353.

47. Wulbeck, C., Grieshaber, E., and Helfrich-Forster, C. (2008). Pigment-

dispersing factor (PDF) has different effects on Drosophila’s circadian

clocks in the accessory medulla and in the dorsal brain. J. Biol. Rhythms

23, 409–424.

48. Yoshii, T., Wulbeck, C., Sehadova, H., Veleri, S., Bichler, D., Stanewsky,

R., and Helfrich-Forster, C. (2009). The neuropeptide pigment-dispersing

factor adjusts period and phase of Drosophila’s clock. J. Neurosci. 29,

2597–2610.

49. Helfrich-Forster, C. (2014). From neurogenetic studies in the fly brain to a

concept in circadian biology. J. Neurogenet. 28, 329–347.

50. Helfrich-Forster, C., Shafer, O.T., Wulbeck, C., Grieshaber, E., Rieger, D.,

and Taghert, P. (2007). Development and morphology of the clock-gene-

expressing lateral neurons of Drosophila melanogaster. J. Comp. Neurol.

500, 47–70.

51. Schlichting, M., and Helfrich-Forster, C. (2015). Photic entrainment

in Drosophila assessed by locomotor activity recordings. Methods

Enzymol. 552, 105–123.

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