In all three species, the spatial ( Figures 1 C and 1D) and temporal ( Figure 4 ) expression patterns of PDP1, CRY, and PDF allowed us to identify as bona fide clock neurons the same main clusters known from D. melanogaster and other low-latitude species []. These were dorsal neurons (DNs) located in the dorsal protocerebrum and lateral neurons (LNs) in the lateral cell body rind ( Figures 1 C and 1E). We could further subdivide the LNs into subgroups according to their size, anatomical location, and neurochemistry ( Figures 1 F–1K and S1 ). As in D. melanogaster, s- and l-LNvs co-express PDF ( Figures 1 F–1H and S1 A) and CRY ( Figures 1 I–1K). The l-LNvs send PDF positive projections toward both the optic lobes, whereas the s-LNvs innervate the superior posterior protocerebrum ( Figures 1 C and 1D). One LN, located in close proximity to the s- and l-LNvs but intermediate in size between them ( Figure S1 A), is CRY positive but PDF negative and hereafter named PDFLNvs ( Figures 1 I–1K). We propose that this neuron corresponds to the 5s-LNv of D. melanogaster []. We could also identify one cluster of more dorsally located LNs, named dorsolateral neurons (LNds), which, as in D. melanogaster [], do not express PDF and are heterogeneous regarding CRY expression ( Figures 1 I–1K). In none of the Chymomyza species, we found PDF-expressing cells in the dorsal protocerebrum. Based on these results, the architecture of the putative master clock of the three Chymomyza species showed conspicuous similarities with that of D. melanogaster. This was also true during development [] ( Figures S1 B–S1K). The parallelisms of the clock neuronal network among D. melanogaster and the Chymomyza species confirmed the idea that the D. melanogaster-like clock neuroarchitecture is the ancestral one [] and was somewhat expected, given that we already described this pattern outside the Drosophilidae family (i.e., Bactrocera oleae) []. Based on CRY and PDF functions in D. melanogaster [], we proposed that the loss of PDF in the s-LNvs and CRY in the l-LNvs in the high-latitude species might allow them to better cope with the long days typical of polar summers []. In this scenario, C. costata becomes an exception: it colonized high latitudes but, unlike the high-latitude Drosophila species, it maintains the ancestral clock neuroarchitecture. This suggests C. costata might have evolved adaptations different from those observed within the Drosophila genus.

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.

A pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila.

The neuroarchitecture of the circadian clock in the brain of Drosophila melanogaster.

Spatial and temporal expression of the period and timeless genes in the developing nervous system of Drosophila: newly identified pacemaker candidates and novel features of clock gene product cycling.

Neuroanatomical details of the lateral neurons of Drosophila melanogaster support their functional role in the circadian system.

Cosmopolitan and low-latitude fly species (such as D. melanogaster, D. hydei, Z. indianus, and B. oleae) generally co-express the photopigment cryptochrome (CRY) and the neuropeptide pigment dispersing factor (PDF) in two clusters of ventrolateral clock neurons (i.e., small [s-LNvs] and large [l-LNvs]) []. In contrast, all high-latitude Drosophila species so far investigated do not express CRY in the l-LNvs and PDF in the s-LNvs but express PDF in some cells located in the dorsal brain [] ( Figure 1 A). To understand whether similar modifications occurred also in high-latitude species outside the Drosophila genus, we studied the clock neuronal network of Chymomyza costata from Finland and compared it to that of Chymomyza pararufithorax and procnemis from southern Japan ( Figure 1 B). To identify and characterize their clock neuron clusters, we used antibodies against D. melanogaster PAR domain protein 1 (PDP1), CRY, and PDF. All antibodies already proved to work reliably in several fly species [].

(I–K) CRY (yellow) and PDF (magenta) immunoreactive neurons in C. costata (I), C. pararufithorax (J), and C. procnemis (K). Scale bars: 50 μm.

(C) PDP1 (green) and PDF (magenta) immunoreactive neurons in the brain of C. costata.

(B) Collection sites of the fly strains considered in this work. C. costata (1) and D. ezoana (5) were collected at 65°57’N, near the Oulanka Research Station, Kuusamo, Finland. C. pararufithorax (2) and C. procnemis (3) were collected in Southern Japan, at 26°28’N (Okinawa) and 33°35’N (Fukuoka), respectively, and obtained by the National Drosophila Species Stock Center at Cornell University (Ithaca, New York). D. melanogaster flies (4) were obtained from a commonly used laboratory strain, derived from a wild-type line collected at 40°48’N []. The Chymomyza genus diverged circa 5 million years before the Drosophila radiation [] and Chymomyza species are today found at both high and low latitudes [].

The genus Chymomyza CZERNY (Diptera, Drosophilidae) from New Guinea, Bismark Archipelago and Southest Asia, with an ecological note.

On the biology and karyology of Chymomyza costata Zetterstedt, with reference to the taxonomy and distribution of various species of Chymomyza (Dipt. Drosophilidae).

(A) Schematic representation of the CRY and PDF neurochemistry within the LNvs of low- (left) and high-latitude (right) Drosophila species.

These findings confirm the idea that flies carrying the ancestral low-latitude clock can neither delay their evening activity peak under long photoperiods nor maintain some rhythmic locomotor activity in LL, implying that these traits might not be essential for life in the North. While the clock of D. ezoana was driving a rather flexible locomotor activity, adjusting to a broad range of day lengths, the clock of C. costata seemed less plastic, with a morning and an evening activity peak tightly coupled to each other in LD. This difference under entrained conditions might be important in light of the circannual behavior of each species: D. ezoana overwinters as adult [], whereas C. costata undergoes a developmental arrest at the larval stage []. It was proposed that a clock driving robust rhythms, or in other words less sensitive to day length changes, might be a disadvantage in animals with strong circannual cycles [], like D. ezoana but unlike C. costata adults.

Diapause induction as an interplay between seasonal token stimuli, and modifying and directly limiting factors: hibernation in C. costata.

Animals living in subarctic regions are not only exposed to extremely long photoperiods but also to days with continuous light (LL), which, most likely via persistent activation of the photopigment CRY [], drives behavioral arrhythmicity in all low-latitude fly species so far investigated []. In contrast, we found that high-latitude Drosophila species retain some rhythmicity in LL and showed that this is due to the lack of CRY expression within their l-LNvs []. In agreement with this, the three Chymomyza species considered here express CRY in their l-LNvs ( Figure 1 ) and are unable to maintain rhythmicity in LL ( Figure S2 ).

(B) Timing of the evening activity peak for each species across the different photoperiods. Lights off transitions are marked by dotted lines. In LD12:12 and LD16:8, all Chymomyza species showed advanced evening activity compared to the Drosophila species (LD12:12: H f = 34.49, p < 0.001; LD16:8: H (4) = 60.46, p < 0.001). In LD20:4, C. costata peaked earlier than D. melanogaster (F (4–110) = 42.27; p < 0.01), whereas D. ezoana showed a later evening activity peak compared to the other species (F (4–110) = 42.27; p < 0.001). Evening activity timings flanked with the same letter are not significantly different (p > 0.05).

(A) Activity profiles of C. costata, C. pararufithorax, C. procnemis, D. melanogaster, and D. ezoana under LD12:12, LD16:8, and LD20:4. The phylogenetic tree on top summarizes species relationships according to Markow and O’Grady [].Under LD12:12, the evening activity peak occurred close to lights-off time in all species, but only in D. ezoana, this correspondence was maintained under the longer photoperiods. The average activity profiles are shown with SEM, i.e., gray lines above and below the mean (black). Activity levels are normalized to the maximum activity for each condition. Light regimes are represented by the environmental bars (white for day; black for night) on top of each box and by shaded areas in the background (gray for night). The evening peak time for each species and condition is shown by the boxplot. The number of flies analyzed is indicated at the top right of each box.

Drosophila species carrying the high-latitude clock show a rather unimodal daily locomotor activity profile and adjust to very long days by delaying their evening activity bout []. Such a locomotor activity profile depends, at least partially, on the PDF-negative s-LNvs and CRY-negative l-LNvs that characterize the clock network of these species []. Indeed, in D. melanogaster, flies with no PDF (especially in the s-LNvs) lack the morning activity bout becoming unimodal [], whereas flies with no CRY (especially in the l-LNvs) delay their evening activity more than wild-types []. The Chymomyza species considered here, despite their latitude of origin, carry a low-latitude D. melanogaster-like clock neuronal network and should therefore show a D. melanogaster-like locomotor activity under LD, i.e., strong bimodality and inability to track dusk under long photoperiods. The locomotor activity of C. pararufithorax and C. procnemis was never analyzed before, whereas few studies reported on the locomotor activity of C. costata. In 1970, Nuorteva and Hackman inferred information based on malt fruit bait catches in the field (Inari, Finland, 68°N) []: C. costata showed one bout of activity concomitant with the time of maximum environmental temperature (i.e., circa 25°C). On the day of collection, the authors recorded a minimum temperature of circa 2°C, making it hard to determine whether the activity peak observed was the result of a unimodal activity profile or merely a consequence of the low temperature. Based on later studies [], we can assert that the most likely explanation is the latter. It is well known that cold exposure suppresses locomotor activity in insects (see [] for a review on the topic) and cool temperatures can induce a unimodal activity profile even in the strongly bimodal D. melanogaster [].

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.

A pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila.

D. ezoana’s and C. costata’s Arrhythmicity as an Example of Convergent Evolution

1 Bloch G.

Barnes B.M.

Gerkema M.P.

Helm B. Animal activity around the clock with no overt circadian rhythms: patterns, mechanisms and adaptive value. 45 Lankinen P.

Riihimaa A.J. Weak circadian eclosion rhythmicity in Chymomyza costata (Diptera; Drosophilidae), and its independence of diapause type. 46 Dissel S.

Hansen C.N.

Özkaya Ö.

Hemsley M.

Kyriacou C.P.

Rosato E. The logic of circadian organization in Drosophila. 5 Menegazzi P.

Dalla Benetta E.

Beauchamp M.

Schlichting M.

Steffan-Dewenter I.

Helfrich-Förster C. Adaptation of circadian neuronal network to photoperiod in high-latitude European drosophilids. 47 Lin Y.

Stormo G.D.

Taghert P.H. The neuropeptide pigment-dispersing factor coordinates pacemaker interactions in the Drosophila circadian system. 25 Renn S.C.

Park J.H.

Rosbash M.

Hall J.C.

Taghert P.H. A pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila. 48 Stengl M.

Homberg U. Pigment-dispersing hormone-immunoreactive neurons in the cockroach Leucophaea maderae share properties with circadian pacemaker neurons. 49 Petri B.

Stengl M. Pigment-dispersing hormone shifts the phase of the circadian pacemaker of the cockroach Leucophaea maderae. 50 Singaravel M.

Fujisawa Y.

Hisada M.

Saifullah A.S.

Tomioka K. Phase shifts of the circadian locomotor rhythm induced by pigment-dispersing factor in the cricket Gryllus bimaculatus. 51 Lee C.M.

Su M.T.

Lee H.J. Pigment dispersing factor: an output regulator of the circadian clock in the German cockroach. 52 Hassaneen E.

El-Din Sallam A.

Abo-Ghalia A.

Moriyama Y.

Karpova S.G.

Abdelsalam S.

Matsushima A.

Shimohigashi Y.

Tomioka K. Pigment-dispersing factor affects nocturnal activity rhythms, photic entrainment, and the free-running period of the circadian clock in the cricket gryllus bimaculatus. 53 Beer K.

Kolbe E.

Kahana N.B.

Yayon N.

Weiss R.

Menegazzi P.

Bloch G.

Helfrich-Förster C. Pigment-dispersing factor-expressing neurons convey circadian information in the honey bee brain. 54 Hermann-Luibl C.

Yoshii T.

Senthilan P.R.

Dircksen H.

Helfrich-Förster C. The ion transport peptide is a new functional clock neuropeptide in the fruit fly Drosophila melanogaster. 55 Park J.H.

Helfrich-Förster C.

Lee G.

Liu L.

Rosbash M.

Hall J.C. Differential regulation of circadian pacemaker output by separate clock genes in Drosophila. Figure 4 The Molecular Clock of D. ezoana Stops in DD, whereas that of C. costata Fails to Sustain a Rhythmic Output Show full caption −LNvs, and LNds) of C. costata, D. melanogaster, and D. ezoana under LD16:8. We found time-dependent PDP1 fluctuation in all clock neuron clusters considered (C. costata: s-LNvs [H (7) = 39.04; p < 0.001], PDF−LNvs [H (7) = 45.61; p < 0.001], LNds [H (7) = 52.81; p < 0.001]; D. melanogaster: s-LNvs [H (7) = 33.86; p < 0.001], PDF−LNvs [H (7) = 25.69; p < 0.001], LNds [H (7) = 35.99; p < 0.001]; D. ezoana: s-LNvs [H (7) = 53.28; p < 0.001], LNds [H (7) = 19.30; p = 0.007]). Maximum levels of PDP1 occurred ∼4 and 3 h earlier in C. costata than in D. melanogaster and D. ezoana, respectively (H (2) = 15.18; p < 0.001). In C. costata, PDP1 peaked earlier in the s-LNvs compared to the other clusters (H (2) = 8.92; p < 0.05). PDP1 expression levels (±SEM) over time are represented as solid lines (s-LNvs in red, PDF−LNvs in blue, and LNds in green). The light regime is represented by the environmental bars on top of each panel (white for day and black for night). Zeitgeber time is plotted starting from the lights-on transition (ZT0), and night hours are represented by the shaded area in the background. The boxplots in each panel represent the peak time of PDP1 within the clock neuron clusters. Among the LNs, the l-LNvs were not considered because immunocytochemical assays fail to show clock protein cycling within these cells in flies kept in DD [ 15 Veleri S.

Brandes C.

Helfrich-Förster C.

Hall J.C.

Stanewsky R. A self-sustaining, light-entrainable circadian oscillator in the Drosophila brain. 16 Roberts L.

Leise T.L.

Noguchi T.

Galschiodt A.M.

Houl J.H.

Welsh D.K.

Holmes T.C. Light evokes rapid circadian network oscillator desynchrony followed by gradual phase retuning of synchrony. −LNvs in D. ezoana because, in this species, PDF is expressed only within the l-LNvs. (A) PDP1 oscillation within the lateral clock neurons (s-LNvs, PDFLNvs, and LNds) of C. costata, D. melanogaster, and D. ezoana under LD16:8. We found time-dependent PDP1 fluctuation in all clock neuron clusters considered (C. costata: s-LNvs [H= 39.04; p < 0.001], PDFLNvs [H= 45.61; p < 0.001], LNds [H= 52.81; p < 0.001]; D. melanogaster: s-LNvs [H= 33.86; p < 0.001], PDFLNvs [H= 25.69; p < 0.001], LNds [H= 35.99; p < 0.001]; D. ezoana: s-LNvs [H= 53.28; p < 0.001], LNds [H= 19.30; p = 0.007]). Maximum levels of PDP1 occurred ∼4 and 3 h earlier in C. costata than in D. melanogaster and D. ezoana, respectively (H= 15.18; p < 0.001). In C. costata, PDP1 peaked earlier in the s-LNvs compared to the other clusters (H= 8.92; p < 0.05). PDP1 expression levels (±SEM) over time are represented as solid lines (s-LNvs in red, PDFLNvs in blue, and LNds in green). The light regime is represented by the environmental bars on top of each panel (white for day and black for night). Zeitgeber time is plotted starting from the lights-on transition (ZT0), and night hours are represented by the shaded area in the background. The boxplots in each panel represent the peak time of PDP1 within the clock neuron clusters. Among the LNs, the l-LNvs were not considered because immunocytochemical assays fail to show clock protein cycling within these cells in flies kept in DD []. We could not analyze data for the PDFLNvs in D. ezoana because, in this species, PDF is expressed only within the l-LNvs. (B) PDP1 oscillation within the lateral clock neurons (s-LNvs, PDF−LNvs, and LNds) of C. costata, D. melanogaster, and D. ezoana under the first day of constant darkness. We found time-dependent PDP1 fluctuation in all clock neuron clusters of D. melanogaster (s-LNvs [H (6) = 38.04; p < 0.001], PDF−LNvs [H (6) = 26.58; p < 0.001], LNds [H (6) = 43.7; p < 0.001]) and in the LNds of C. costata LNds (H (6) = 34.29; p < 0.001). Fluctuations of PDP1 in C. costata s-LNvs and PDF−LNvs were not significantly time dependent (s-LNvs [H (6) = 11.29; p = 0.08], PDF−LNvs [H (6) = 9.84; p = 0.13]). PDP1 cycling was lost in D. ezoana (s-LNvs [H (6) = 6.12; p = 0.41], LNds [H (6) = 4.09; p = 0.66]). In C. costata, PDP1 maximum occurred 20.6 ± 0.82 h after the beginning of DD, which is comparable to the peak time in LD that occurred 19.05 ± 0.79 h after lights-on (U = 191; p > 0.05). In D. melanogaster, PDP1 maximum occurred 20.8 ± 0.2 h after the beginning of DD, whereas in LD, it occurred later during the day, i.e., 23.75 h ± 0.86 (Z = 29; p < 0.01). This observation fits with the free running period for this wild-type strain of D. melanogaster, which is slightly lower than 24 h. PDP1 expression levels (±SEM) over time are represented as solid lines (s-LNvs in red, PDF−LNvs in blue, and LNds in green). Night and day (i.e., subjective night and subjective day) of the previous LD cycle are represented by the environmental bars on top of each panel (dark gray for night and light gray for day). Circadian time is plotted starting from the beginning of constant conditions (CT0); subjective day and subjective night are represented by the light- and dark-gray-shaded areas, respectively. The peak time of PDP1 within the clock neurons is represented as mean ± SEM. (C) PDF oscillation at the dorsal terminal of the s-LNvs of C. costata under LD (upper panel) and DD (lower panel). Strong time-dependent PDF fluctuations were found in LD (H (7) = 43.36; p < 0.001), but not in DD (H (8) = 7.42; p = 0.049). PDF levels (±SEM) over time are represented as solid lines. Zeitgeber time is plotted starting from the lights-on transition (ZT0), whereas circadian time is plotted from the beginning of constant conditions (CT0). The light regimes (upper panel) or subjective night and subjective day (lower panel) are represented by the environmental bars on top and by the shaded area in the background of each panel. See also Figures 1 and S4 and Table S4 A clock unable to sustain rigid rhythms, in addition to a pronounced sensitivity to temperature stimuli, might be of adaptive significance for northern species that might need to respond to unpredictable changes in the environment independently of their endogenous timing []. The absence of behavioral rhythmicity in DD in C. costata and D. ezoana might be the output of a quickly dampening clock or of a clock that is not self-sustained. To investigate the two possibilities, we looked at the oscillation of PDP1 within the clock neurons ( Figure 4 ). For comparison, we conducted this experiment also in D. melanogaster, where we expected rhythmic PDP1 expression []. PDP1 cycled in all three species when flies were exposed to LD cycles ( Figure 4 A). PDP1 oscillation persisted in DD in the rhythmic D. melanogaster but was lost in the arrhythmic D. ezoana ( Figure 4 B). The loss of behavioral rhythms in D. ezoana might therefore be explained by a loss of core clock protein cycling. This is likely a consequence of the absence of PDF in the s-LNvs. It is known from D. melanogaster that the clock neurons, and especially the s-LNvs, which function as a master pacemaker in DD driving locomotor activity rhythms [], rely on PDF signaling to maintain a strong oscillation in their molecular clock []. In C. costata, despite the arrhythmic locomotor activity, the s-LNvs are expressing PDF, and we could expect PDP1 cycling in DD. This is indeed the case: PDP1 cycles in a circadian manner under constant conditions ( Table S4 ). We wondered whether, in C. costata, the reason behind the arrhythmic locomotor activity might rather be in a loss of coupling between the master clock and its output. To address this issue, we quantified PDF staining intensity at the s-LNv terminals. The role of PDF as an output factor of the circadian clock is conserved among several insect species []. Moreover, in D. melanogaster, the neuropeptide is known to be rhythmically released at the s-LNv terminals of wild-type, but not of arrhythmic clock mutants [].

56 Fernández M.P.

Berni J.

Ceriani M.F. Circadian remodeling of neuronal circuits involved in rhythmic behavior. 5 Menegazzi P.

Dalla Benetta E.

Beauchamp M.

Schlichting M.

Steffan-Dewenter I.

Helfrich-Förster C. Adaptation of circadian neuronal network to photoperiod in high-latitude European drosophilids. 57 Helfrich-Förster C.

Täuber M.

Park J.H.

Mühlig-Versen M.

Schneuwly S.

Hofbauer A. Ectopic expression of the neuropeptide pigment-dispersing factor alters behavioral rhythms in Drosophila melanogaster. 58 Gunawardhana K.L.

Hardin P.E. VRILLE controls PDF neuropeptide accumulation and arborization rhythms in small ventrolateral neurons to drive rhythmic behavior in Drosophila. 47 Lin Y.

Stormo G.D.

Taghert P.H. The neuropeptide pigment-dispersing factor coordinates pacemaker interactions in the Drosophila circadian system. 59 Yoshii T.

Wülbeck C.

Sehadova H.

Veleri S.

Bichler D.

Stanewsky R.

Helfrich-Förster C. The neuropeptide pigment-dispersing factor adjusts period and phase of Drosophila’s clock. 45 Lankinen P.

Riihimaa A.J. Weak circadian eclosion rhythmicity in Chymomyza costata (Diptera; Drosophilidae), and its independence of diapause type. 60 Kostál V.

Shimada K. Malfunction of circadian clock in the non-photoperiodic-diapause mutants of the drosophilid fly, Chymomyza costata. In C. costata, we found PDF cycling in LD, where flies showed strong locomotor activity rhythms, but not in DD, where they became mostly arrhythmic ( Figures 4 C and S4 ). For comparison, we analyzed PDF staining intensity also in the rhythmic C. procnemis. In accordance with their locomotor activity, C. procnemis showed cycling in PDF intensity both in LD and DD ( Figure S4 ). In D. melanogaster, daily changes in PDF intensity correlate with daily reorganization of the s-LNv axonal morphology []. Furthermore, disruption of either PDF accumulation at the s-LNv terminals or s-LNv arborization rhythms leads to arrhythmic locomotor activity []. Based on these observations and our findings in C. costata and C. procnemis, we speculate that loss of PDF cycling at the s-LNv terminals could be the cause of C. costata’s arrhythmicity. We can hypothesize that absence of rhythmic PDF release would eventually translate into a stopped clock [] or into a lack of coupling between the clock and its output. This idea would fit with previous observations that found dampening rhythms in C. costata (i.e., eclosion rhythm and per mRNA cycling) [].

39 Lu W.

Meng Q.J.

Tyler N.J.

Stokkan K.A.

Loudon A.S. A circadian clock is not required in an arctic mammal. 61 Beale A.

Guibal C.

Tamai T.K.

Klotz L.

Cowen S.

Peyric E.

Reynoso V.H.

Yamamoto Y.

Whitmore D. Circadian rhythms in Mexican blind cavefish Astyanax mexicanus in the lab and in the field. Our study shows that arrhythmicity in high-latitude environments can be achieved via either loss of molecular oscillation within the master clock or lack of coupling between the master clock and its output. These results fit well with previous findings in other organisms and can be generalized to weakly rhythmic environments all over the world. Lu and coworkers [] showed that the molecular clock stops cycling in reindeer fibroblasts in DD, whereas Beale and coauthors [] reported that the circadian clock of the Mexican blind cavefish Astyanax mexicanus keeps ticking under constant conditions but fails to drive rhythmic behavior.