1. Volume 29, Issue 22, Pages 3928-3936.e3 (November 2019)
Life at High Latitudes Does Not Require Circadian Behavioral Rhythmicity under Constant Darkness 
Enrico Bertolini, Frank K. Schubert, Damiano Zanini, Hana Sehadová, Charlotte Helfrich-Förster, Pamela Menegazzi 
Current Biology 
Volume 29, Issue 22, Pages e3 (November 2019)
DOI: /j.cub
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2. Current Biology 2019 29, 3928-3936.e3DOI: (10.1016/j.cub.2019.09.032)
Copyright © 2019 The Author(s) Terms and Conditions

3. Figure 1
C. costata, C. pararufithorax, and C. procnemis Possess the Ancestral Low-Latitude Clock Network Neuroarchitecture
(A) Schematic representation of the CRY and PDF neurochemistry within the LNvs of low- (left) and high-latitude (right) Drosophila species.
(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 [14]. The Chymomyza genus diverged circa 5 million years before the Drosophila radiation [9] and Chymomyza species are today found at both high and low latitudes [10, 11].
(C) PDP1 (green) and PDF (magenta) immunoreactive neurons in the brain of C. costata.
(D) PDP1 (green) and PDF (magenta) immunoreactive neurons in the brain of C. pararufithorax.
(E) PDP1 (green) and PDF (magenta) immunoreactive neurons in the brain of C. procnemis.
(F) PDP1 (green) and PDF (magenta) immunoreactive lateral neurons in C. costata.
(G) PDP1 (green) and PDF (magenta) immunoreactive lateral neurons in C. pararufithorax.
(H) PDP1 (green) and PDF (magenta) immunoreactive lateral neurons in C. procnemis. Scale bars: 100 μm (C–E) and 25 μm (F–H).
(I–K) CRY (yellow) and PDF (magenta) immunoreactive neurons in C. costata (I), C. pararufithorax (J), and C. procnemis (K). Scale bars: 50 μm.
DNs, dorsal neurons; l-LNvs, large ventrolateral neurons; LNds, dorsolateral neurons; ME, medulla; PDF−LNv, PDF negative ventrolateral neuron; POC, posterior optic commissure; s-LNvs, small ventrolateral neurons; SEZ, subesophageal zone.
See also Figure S1.
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4. Figure 2
C. costata, C. pararufithorax, and C. procnemis Show Low-Latitude Locomotor Activity under Light:Dark Cycles
(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 [34].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.
(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: Hf = 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).
See also Figure S2 and Tables S1 and S2.
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5. Figure 3
The High-Latitude C. costata and D. ezoana Are Arrhythmic in DD, whereas the Low-Latitude C. pararufithorax, C. procnemis, and D. melanogaster Are Rhythmic
In the upper panel are shown representative actograms of flies exposed to LD16:8 for 4 days and then released in DD. Below, for each species, the rhythmicity levels in DD are shown as barplots. All flies show entrainment during the LD regime, but only C. pararufithorax, C. procnemis, and D. melanogaster show rhythmic locomotor activity when in DD. In contrast, 89% of D. ezoana and 93% of C. costata scored as arrhythmic. The actograms are shown as double plots with the LD16:8 cycles during the first 4 days indicated by the black (night) and white (day) bar on top. The gray shaded area in the actogram indicates darkness. Rhythmicity levels are summarized in green and red for rhythmic and arrhythmic flies, respectively. The number of flies analyzed is indicated within each bar. The phylogenetic tree on top represents phylogenetic relationships among species according to Markow and O’Grady [34]. See also Figure S3 and Table S3.
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6. Figure 4
The Molecular Clock of D. ezoana Stops in DD, whereas that of C. costata Fails to Sustain a Rhythmic Output
(A) PDP1 oscillation within the lateral clock neurons (s-LNvs, PDF−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, 16]. We could not analyze data for the PDF−LNvs 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.
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