dTRPA1 Modulates Afternoon Peak of Activity of Fruit Flies Drosophila melanogaster

Abstract

Daily rhythms in Drosophila under semi-natural conditions (or SN) have received much recent attention. One of the striking differences in the behaviour of wild type flies under SN is the presence of an additional peak of activity in the middle of the day. This is referred to as the afternoon peak (A-peak) and is absent under standard laboratory regimes using gated light and temperature cues. Although previous reports identified the physical factors that contribute towards the A-peak there is no evidence for underlying molecular mechanisms or pathways that control A-peak. We report that the A-peak is mediated by thermosensitive dTRPA1 (drosophila Transient Receptor Potential- A1) ion channels as this peak is absent in dTRPA1 null mutants. Further, when natural cycles of light and temperature are simulated in the lab, we find that the amplitude of the A-peak is dTRPA1-dependent. Although a few circadian neurons express dTRPA1, we show that modulation of A-peak is primarily influenced by non-CRY dTRPA1 expressing neurons. Hence, we propose that A-peak of activity observed under SN is a temperature sensitive response in flies that is elicited through dTRPA1 receptor signalling.

Fig 1. Under semi-natural conditions the mid-day A-peak depends upon dTRPA1.

(A) Average activity profiles of wildtype (w ¹¹¹⁸) and two dTRPA1 null mutant lines (dTRPA1 ^ins and TRPA1 ^KI-GAL4) under SN conditions with different light intensities. Under low light and high temperature, w ¹¹¹⁸ show three peaks of activity with distinct A-peak (arrow) whereas mutant flies display bimodal activity lacking the A-peak. Under high light and high temperature, w ¹¹¹⁸ show two distinct peaks in the afternoon- one coinciding with L_max and another with T_max (arrows). Interestingly, dTRPA1 nulls also show an A-peak corresponding to L_max (arrow) but not with T_max. (B) In DD+SN also, w ¹¹¹⁸ depict distinct A-peak (arrow) whereas dTRPA1 null flies do not. Error bars are SEM. Three axes on the right represent environmental factors—light (L-lux), temperature (T-degree Celsius) and relative humidity (H-percentage). Arrows indicate A-peak displayed by more than 25% flies.

Fig 2. Simulating natural temperature profile in the laboratory generates dTRPA1-dependent A-peak.

(A, left) Average activity profiles of flies under L_r+T_r32 (L_max and T_max occur in phase) wherein wild type w ¹¹¹⁸ show a prominent A-peak (arrow) but dTRPA1 null flies (dTRPA1 ^ins- red and TRPA1 ^KI-GAL4-violet) do not show an A-peak. (A, left) Average activity profiles of flies under L_r+T_r32 (L_max and T_max occur in phase) wherein wild type w ¹¹¹⁸ show a prominent A-peak (arrow) but dTRPA1 null flies (dTRPA1 ^ins- red and TRPA1 ^KI-GAL4-violet) do not show an A-peak. (A, -middle) Flies under simulated light and temperature protocols with different temperature maxima (L_r+T_r28). Neither genotype exhibits an A-peak under L_r+T_r28 when T_max reached 28°C. (A, right) dTRPA1 ^oex flies (blue curves) show A-peak similar to their controls under L_r+T_r32. (B, left) Activity/rest profiles of w ¹¹¹⁸ depict a distinct A-peak corresponding to T_max but not to L_max under out of phase L_r+T_r32 whereas dTRPA1 ^ins flies do not show any peak. (B, right) dTRPA1 ^oex flies show an enhanced A-peak compared to controls and the peak coincides with T_max. All control flies showed an A-peak conjugated with T_max like w ¹¹¹⁸ flies. (C, left) Percentage activity during 1 hr of T_max (32°C) when light and temperature gradually changed (left) in phase and (right) out of phase. Under both regimes, dTRPA1 ^ins flies showed significantly reduced activity compared to w ¹¹¹⁸. dTRPA1 ^oex flies showed higher A-peak activity than controls under out of phase L_r+T_r32. All other experimental details are same as in Fig 1.

Fig 3. dTRPA1-dependent A-peak can be elicited by gradual temperature cycles even in absence of other time cues.

(A) Flies were subjected to simulated temperature protocol under constant darkness (DD+T_r32). All control genotypes show an A-peak. (Left) w ¹¹¹⁸ flies display A-peak coinciding with T_max 32°C whereas dTRPA1 ^ins flies do not, although the latter responded to temperature changes outside dTRPA1 activation range similar to w ¹¹¹⁸. (Middle) dTRPA1 ^oex flies show an enhanced A-peak compared to their respective controls. (Right) Heterozygous TrpA1 ^KI-GAL4 /+ flies (violet curve) show A-peak whereas homozygous TrpA1 ^KI-GAL4 null flies (grey curve) do not. (B, left) Comparison of percentage activity during 1 hr of T_max (32°C). dTRPA1 ^ins flies show significantly reduced activity compared to w ¹¹¹⁸ and dTRPA1 ^oex flies show higher A-peak activity than controls under DD+T_r32. (B, right) Heterozygous TrpA1 ^KI-GAL4 /+ flies show higher A-peak activity than TrpA1 ^KI-GAL4 null flies. All other experimental details are the same as in Figs 1 and 2.

Fig 4. dTRPA1-dependent A-peak can be elicited by gradual temperature cycles under constant light.

(A) Average activity profiles of flies in simulated natural temperature cycles under constant light (100 lux) (LL₁₀₀+T_r32). (Left) w ¹¹¹⁸ flies display an A-peak while dTRPA1 ^ins flies do not. (Right) dTRPA1 ^oex flies show an enhanced A-peak compared to their parental controls. (B) Percentage activity during 1 hr of T_max (32°C). dTRPA1 ^ins flies showed significantly reduced activity compared to w ¹¹¹⁸ and dTRPA1 ^oex flies show higher A-peak activity than controls under LL₁₀₀+T_r32. All other experimental details are the same as in Figs 1 and 2.

Fig 5. CRY-negative dTRPA1 neurons mediate the enhanced A-peak under gradual light and temperature cycles.

(A) Neuronal ablation or dTRPA1 over-expression either under (top) Pdf-GAL4 or (bottom) cry-GAL4 does not abolish occurrence of A-peak under L_r+T_r32. (B) Comparison of percentage activity during 1 hr of T_max (32°C). (B, top) Pdf-GAL4 mediated ablation or dTRPA1 over-expression elicits A-peak similar to the A-peak amplitude of their respective controls. (B, bottom) Ablation of CRY positive neurons leads to reduction in A-peak activity compared to controls whereas over-expression of dTRPA1 in CRY positive neurons has no effect on A-peak amplitude. (C) Over-expression of dTRPA1 only in non-CRY neurons targeted by dTRPA1 ^SH -GAL4 (dTRPA1 ^SH -GAL4 + cry-GAL80 < UAS-dTRPA1), leads to enhanced A-peak (blue curve, arrow) compared to controls. (D) Comparison of percentage activity also depicts that flies over-expressing dTRPA1 in CRY negative, dTRPA1⁺ neurons show enhanced A-peak compared to controls. All other experimental details same as Figs 1 and 2.

Fig 6. dTRPA1-A isoform rescues the mutant phenotype of dTRPA1 null flies under gradual light and temperature cycles.

(A) Overexpression of dTRPA1 isoforms dTRPA1-A or dTRPA1-D under dTRPA1 ^SH -GAL4 is able to elicit A-peak under L_r+T_r32. (B) Rescue of dTRPA1 function in dTRPA1 ^ins background only with dTRPA1-A but not with dTRPA1-D isoform. (B, left) dTRPA1-A which is known to be active 24–29°C elicits an A-peak similar to UAS-dTRPA1 mediated A-peak when driven under dTRPA1 ^SH -GAL4. (B, right) Restoring dTRPA1-D isoform expression in dTRPA1 ^ins null background induces an A-peak not different from the parental controls in null background. dTRPA1-D isoform, whose activation threshold is ~34°C, is not able to rescue occurrence of A-peak in mutant background. (C, left) Comparison of percentage activity show that over-expression of either dTRPA1-A or dTRPA1-D isoform does not alter A-peak amplitude compared to their respective controls during 1 hr of T_max. (C, right) Restoring expression of only dTRPA1-A isoform under dTRPA1 ^SH -GAL4 in dTRPA1 ^ins null background is able to elicit A-peak in flies compared to their parental controls in null background which have significantly low levels of A-peak activity. All other experimental details are same as in Figs 1 and 2.