Drosophila Temperature Preference Rhythms: An Innovative Model to Understand Body Temperature Rhythms

Abstract

Human body temperature increases during wakefulness and decreases during sleep. The body temperature rhythm (BTR) is a robust output of the circadian clock and is fundamental for maintaining homeostasis, such as generating metabolic energy and sleep, as well as entraining peripheral clocks in mammals. However, the mechanisms that regulate BTR are largely unknown. Drosophila are ectotherms, and their body temperatures are close to ambient temperature; therefore, flies select a preferred environmental temperature to set their body temperature. We identified a novel circadian output, the temperature preference rhythm (TPR), in which the preferred temperature in flies increases during the day and decreases at night. TPR, thereby, produces a daily BTR. We found that fly TPR shares many features with mammalian BTR. We demonstrated that diuretic hormone 31 receptor (DH31R) mediates Drosophila TPR and that the closest mouse homolog of DH31R, calcitonin receptor (Calcr), is essential for mice BTR. Importantly, both TPR and BTR are regulated in a distinct manner from locomotor activity rhythms, and neither DH31R nor Calcr regulates locomotor activity rhythms. Our findings suggest that DH31R/Calcr is an ancient and specific mediator of BTR. Thus, understanding fly TPR will provide fundamental insights into the molecular and neural mechanisms that control BTR in mammals.

Keywords: Calcitonin receptor; Calcr; DH31; DH31R; Drosophila; PDF; PDFR; body temperature rhythms; circadian rhythms; locomotor activity rhythms.

Figure 1

Comparison between the human body temperature rhythm (BTR) and the Drosophila temperature preference rhythm (TPR). The human BTR (A) (replotted the figure using an example form Duffy JF et al. 1998, Figure 3 [3]) and the Drosophila TPR (B) (modified from Kaneko et al. 2012, Figure 1 [18]). The shadow area in (A) shows the sleep period, and (B) shows the dark period. ZT: zeitgeber time. *: p < 0.05, ***: p < 0.001 (comparison to ZT 1–3). Given that the body temperature of Drosophila is close to their ambient temperature, the TPR results in fluctuations in body temperature. The TPR shows a rhythmic pattern similar to that of the human BTR.

Figure 2

Drosophila temperature preference behavioral assay. A temperature gradient from 18 to 32 °C is generated in a chamber made with a metal plate and a plexiglass cover. Flies are introduced into the chamber through the holes in the cover. Within 30 minutes, the flies settle in the locations with their preferred temperatures. Because their body temperatures are very close to the ambient temperature, their body temperatures can be determined by measuring the temperature in the place where they are located. The diagram is modified from Goda et al. 2014, Figure 3 [26].

Figure 3

The TPR is regulated by different regulatory mechanisms at different times of the day: daytime, night onset, and predawn. Daytime TPR: The increase in preferred temperature during the daytime (ZT 1–12: shown by the red rectangle). Night-onset TPR: The dramatic decrease in preferred temperature at the transition from day to night (ZT 10–15: shown by blue rectangle). Predawn TPR: The preferred temperature just before dawn is similar to that of early morning (ZT 22–24: shown by green rectangle). The TPR is regulated by different regulatory mechanisms in each of these periods. The graph is modified from Kaneko et al. 2012, Figure 1 [18]. *: p < 0.05, ***: p < 0.001 (comparison to ZT 1–3).

Figure 4

DH31R regulates the daytime TPR. Comparison of TPR between Dh31r mutant flies (Dh31r^1/Df, red line in (A–C) and heterozygous control flies (Dh31r^1/+, gray line in (A) or Dh31r^Df/+, gray line in (B)) or Dh31r genomic rescue flies (rescue, blue line in (C)). Dh31r mutant flies show an abnormal daytime TPR, in which they prefer a constant temperature of approximately 27°C during the daytime. The control flies exhibit a normal daytime TPR, in which their preferred temperature increases during the daytime (A,B). Genomic rescue of Dh31r flies restored normal daytime TPR (C). Dh31r¹ is a P-element insertion mutant (PBac {WH}Dh31-R^f05546), and Dh31r^Df is a deletion mutant [Df(2R) BSC273]. In the Dh31r genomic rescue fly, the Ch321-57F06 BAC clone, which includes the entire Dh31r gene region, is inserted into the genome. The graphs are modified from Goda et al. 2018, Figure 1 [23]. *: p < 0.05, **: p < 0.01 (comparison to ZT 1–3).

Figure 5

PDFR and DH31 regulate the night-onset TPR. Comparison of daytime TPR (A,C) or night-onset TPR (B,D) between w¹¹¹⁸ and Pdfr mutant (Pdfr⁵³⁰⁴) (A,B) or Dh31 mutant (Dh31^#51) (C,D) flies. Both Pdfr⁵³⁰⁴ and Dh31^#51 flies show significantly dampened night-onset TPR (B,D), while both exhibit robust increases in preferred temperature during the daytime (A,C). The data suggest that both PDFR and DH31 are responsible for the night-onset TPR. The graphs are modified from Goda et al. 2016, Figure 1 and Figure 3 [40]. t-test: **: p < 0.01, ****: p < 0.0001.

Figure 6

PDF regulates the predawn TPR. Comparison of TPRs between w¹¹¹⁸ and Pdf mutant (Pdf⁰¹) flies. Pdf⁰¹ flies exhibit robust daytime (ZT 1–12) and night-onset TPRs (ZT 10–15). However, their preferred temperatures continue decreasing during the night and are much lower before dawn (ZT 22–24) than that of w¹¹¹⁸ flies (predawn TPR). These data suggest that PDF is important for the predawn TPR. t-test between w¹¹¹⁸ and Pdf⁰¹ in each ZT: **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Figure 7

The Drosophila TPR is regulated by different mechanisms at different times of the day. Models for regulatory mechanisms of the TPR in each time range: daytime (ZT 1–12), night onset (ZT 10–15), and predawn (ZT 22–24). The daytime TPR: DN2s are the main clock neurons. DH31 acts on DH31R in a subset of clock neurons to regulate the daytime TPR. PDF is also involved in daytime TPR regulation. The night-onset TPR: DH31 acts on DN2s via PDFR to regulate the night-onset TPR. The predawn TPR: An AC-sLN-DN2 neural circuit regulates the proper setting of temperature preference before dawn.

Figure 8

Calcr contributes to body temperature regulation during the night (the active phase for mice). Mouse BTR (A) and locomotor activity rhythm (B) in control (Calcr^+/+: blue line) and Calcr knockout (Calcr^−/−: orange line) mice. The body temperatures of wild-type mice showed a deep trough at midnight, whereas the body temperatures of Calcr knockout mice lost the characteristic trough and remained relatively unchanged during the night (A). The locomotor activity rhythm in Calcr knockout did not show a significant difference from that of the control, suggesting that Calcr is responsible for mammalian body temperature regulation but not locomotor activity rhythm during the night (the active phase for mice). White and black bars on the graphs indicate the 12-h light and dark phases. The graphs are modified from Goda et al. 2018, Figure 6 [23]. *: p < 0.05, ***: p < 0.001.