Recovery from cold-induced reproductive dormancy is regulated by temperature-dependent AstC signaling

Matthew R Meiselman; Michael H Alpert; Xinyue Cui; Jamien Shea; Ian Gregg; Marco Gallio; Nilay Yapici

doi:10.1016/j.cub.2022.01.061

. Author manuscript; available in PMC: 2023 Mar 28.

Published in final edited form as: Curr Biol. 2022 Feb 16;32(6):1362–1375.e8. doi: 10.1016/j.cub.2022.01.061

Recovery from cold-induced reproductive dormancy is regulated by temperature-dependent AstC signaling

Matthew R Meiselman ¹, Michael H Alpert ², Xinyue Cui ¹, Jamien Shea ¹, Ian Gregg ^1,³, Marco Gallio ², Nilay Yapici ^1,^*

PMCID: PMC8969192 NIHMSID: NIHMS1776772 PMID: 35176227

SUMMARY

Animals have evolved a variety of behaviors to cope with adverse environmental conditions. Similar to other insects, the fly, Drosophila melanogaster, responds to sustained cold by reducing its metabolic rate and arresting its reproduction. Here, we show that a subset of dorsal neurons (DN3s) that express the neuropeptide Allatostatin-C (AstC) facilitates recovery from cold-induced reproductive dormancy. The activity of AstC-expressing DN3s, as well as the AstC peptide levels, are suppressed by cold. Cold temperature also impacts AstC levels in other Drosophila species and mosquitoes, Ae. aegypti, and An. stephensi. The stimulatory effect of AstC on egg production is mediated by cholinergic AstC-R2 neurons. Our results demonstrate that DN3s coordinate female reproductive capacity with environmental temperature via AstC signaling. AstC/AstC-R2 is conserved across many insect species and their role in regulating female reproductive capacity makes them an ideal target for controlling the population of agricultural pests and human disease vectors.

In Brief

Many insects cease reproduction when faced with harsh environmental conditions. Meiselman et al. show cold-induced reproductive dormancy is regulated by circadian DN3s. DN3 activity and the expression of their peptide, AstC, is temperature-sensitive in flies and mosquitoes, suggesting a common means for coordinating reproduction and environment.

INTRODUCTION

Many animal species can undergo some type of programmed dormancy when exposed to prolonged adverse conditions such as intense summer heat or winter cold ^1-7. For example, most insects and some mammals respond to a sharp seasonal decrease in day length and/or temperature with an arrest in development and reproduction that protects them and their progeny from lethality ^1,3,8. Despite decades of research, how the nervous system evaluates changes in the environment, and accordingly enters or exits the dormant state, is poorly understood.

In Drosophila, reproductive dormancy is best understood in the female. Female flies arrest reproduction when exposed to persistent cold temperatures and short photoperiods ^9-14. Cold-induced dormancy involves a variety of changes in female behavior and physiology, but it is most often measured by the attenuation of the egg production ^2,6,9,15. In insects, ovary development is promoted by the circulating hormones ecdysone, juvenile hormone, and insulin-like peptides (ILPs), ^15-17. Previous studies suggest that circadian output or persistent cold temperatures can alter insulin signaling, which eventually changes the rate of egg production in females ^12,13,18,19. Recently, the cotranscription factor, EYES ABSENT (EYA), and the circadian clock protein, TIMELESS (TIM) have been shown to regulate reproductive dormancy ²⁰. However, little is known about how the fly nervous system senses persistent changes in temperature and accordingly adjusts the rate of egg production.

In this study, we investigated the impacts of photoperiod and environmental temperature on the reproductive capacity of Drosophila females and found that temperature, rather than photoperiod, determines the rate of egg production in virgin females. We performed a genetic screen to identify neurons that interrupt dormancy and stimulate egg production upon activation in cold. In addition to insulin-like peptide (ILP2) neurons, we unexpectedly found activation of groups of lateral and dorsal circadian neurons (E-cells and DN3s, respectively) was sufficient to stimulate egg production in dormancy-inducing conditions (~12°C, 8L:16D). DN3s make up the majority of the circadian clock neurons in the fly brain, however, their functions are poorly understood ^21,22. Here, we show that a subset of DN3s that express the neuropeptide Allatostatin-C (AstC) decrease their activity in response to cold. Manipulating the activity of AstC-expressing DN3s impacts proper execution of, and recovery from, cold-induced reproductive dormancy. Overexpression of AstC in DN3s accelerates egg production during recovery from cold temperatures and AstC signals through AstC-R2 in cholinergic neurons to modulate female reproduction. Furthermore, thermoregulation of AstC expression is conserved in multiple species of Drosophila and mosquitoes. Our results suggest that AstC release from DN3s is a critical factor that coordinates the reproductive capacity of females with persistent changes in environmental temperature.

RESULTS

Drosophila female reproductive capacity is altered by environmental temperatures

Drosophila oogenesis has a well-studied developmental program, which has 14 stages based on the morphology of the growing oocytes. Stage 14 oocytes are considered mature eggs ¹⁹ which are produced within the first 24-72 hours after eclosion in standard laboratory conditions ^19,23. To investigate how environmental conditions impact female reproductive capacity, we observed the effects of cold temperatures (12°C), and short photoperiod (8L:16D) on egg production. In these suboptimal conditions, females entered a reproductively dormant state, evident by the lack of mature eggs in their ovaries (Figure 1A, B) ^12,13,24. Next, we tested which environmental cue had the greatest impact on egg production. Using a custom-made chamber (Figure 1C), we housed virgin females in varying temperature and light-dark (LD) cycles and quantified the mature eggs they produced. While different LD cycles had no significant impact on egg production, reducing the temperature to 15°C, or below reduced egg production at all given LD cycles (Figure 1D).

Figure 1. — (A) Timeline for reproductive dormancy assessments.

(B) Ovaries of females kept at 12°C, 8L:16D (left) or 25°C, 12L:12D (right) (DAPI, scale bars=10μm).

(C) 3D model of the CHAPEL and its compartments.

(D) Egg quantification for females kept at varying temperature and light conditions (n=13-24, per condition).

(E) Egg quantification for intact and antennectomized females at 25°C and 12°C (n=16-17).

(F) Egg quantification for control and mutant females tested at indicated temperatures. (n=15-20).

(G) Egg quantification for females at indicated time points during recovery (18°C=blue, 25°C=red, 28.5°C= purple, n=10-25, two-way ANOVA with Tukey’s test).

(H) Egg quantification for antennectomized and intact females during recovery (n=8-25, two-way ANOVA with Bonferroni test).

(I) Egg quantification for females kept at 12L:12D (red) or total darkness (black) during recovery (n=9-25, two-way ANOVA with Bonferroni test).

See also Table S1.

To identify mechanisms that suppress egg production in the cold, we first investigated the role of known temperature sensors. In Drosophila, temperature is sensed mainly by the antenna. The terminal segment of the antenna, the arista, houses sensory neurons that respond to hot and cold temperatures ^25,26. Additional cold receptor neurons innervate the sacculus ^27-29. Neither surgical removal of the antenna including the sacculus (Figure 1E) nor known temperature sensor mutants ^30-32 impacted the suppression of egg production in cold (Figure 1F). Temperature was also the driving factor of egg production during recovery from cold (Figure 1G); neither antennectomy (Figure 1H) nor total darkness (Figure 1I) impacted the rate of egg production during recovery. Our results demonstrated cold temperature, via an antenna-independent pathway, is the main driver of reproductive dormancy in virgin females.

Activation of circadian neurons prevents proper execution of reproductive dormancy

To identify neurons that regulate reproductive dormancy, we carried out a genetic screen. We activated candidate neuronal populations under dormancy-inducing conditions using the GAL4/UAS system ³³ to drive the expression of a rat cold-activated cation channel, TRPM8 ³⁴. From 65 GAL4 lines tested, six GAL4 lines showed an increase in egg production upon neuronal activation consistently (Figure 2A, Figure S1A). From the six positives, Ilp2-GAL4 and Gr28B.B-GAL4, labeled insulin-like peptide producing neurons (IPCs) ^13,18 (Figure S1C). The remaining four positive lines labeled different classes of circadian neurons (Figure S1C). The results of the activation screen led us to focus our further investigation on the circadian neurons ^27,35-37. We screened selected GAL4 lines from the Janelia FlyLight collection ³⁸ and identified six GAL4 that labeled either dorsal-lateral neurons (LNds) or dorsal neurons (DNs) that consistently stimulated egg production upon activation in the cold (Figure 2B, C, Figure S1B, C). Next, using the enhancers from the screen positives, we generated four selective split GAL4 lines ^39,40. Split¹ labeled DN1p(s) and a few putative DN3s, but activation of these neurons did not strongly stimulate egg production in the cold (Figure 2D and 2H). Split² did not label any clock neurons and did not impact egg production (Figure 2E and 2I). Split³ and Split⁴ both labeled subsets of dorsal neurons (DNs) (Figure 2J, K) ⁴¹, and their activation promoted egg production (Figure 2F, G). Split⁴ (hereafter DN3-GAL4) specifically labeled ~14 DN3s that are TIM-positive (Figure S1D). We also observed a few scattered neurons in the Split⁴ ventral nerve cord (VNC) (Figure S1E), but these neurons were not critical for egg production in the cold (Figure S1F). Here, we focused on the role of DN3s in regulating egg production in virgin females.

Figure 2. — (A) Egg quantification for females kept at 10-12°C, 8L:16D for various GAL4s crossed to *UAS-TrpM8*. Positive GAL4s are colored blue for circadian neuron drivers and orange for lines that label IPCs (One-way ANOVA with Dunnett’s test, n=20-30).

(B) Egg quantification at 10-12°C, 8L:16D for GAL4 lines labeling circadian neurons crossed to *UAS-TrpM8*. Significant GAL4 lines are colored blue for dorsal neuron drivers and green for lines that label LNd neurons (One-way ANOVA with Dunnett’s test, n=12-30).

(C) Table showing positive GAL4s and circadian neurons they label.

(D-G) Egg quantification at 12°C, 12L:12D for split-GAL4 > *UAS-TrpM8* and genetic controls (n=30-60, One-way ANOVA with Tukey’s test).

(H-K). Images of fly brains (top panel) or lateral protocerebrum (bottom panel) of *split-GAL4>UAS-CD8-GFP* females (scale bars=50μm).

See also Figure S1 and Table S1.

Manipulation of a subset of DN3s perturbs thermal regulation of egg production

To further investigate how DN3s regulate egg production, we manipulated the activity of these neurons in different environmental conditions. DN3>TrpM8 females did not completely suppress egg production in the cold; they entered the reproductively dormant state upon cold exposure but were unable to maintain it (Figure 3A). Activation of DN3s with the bacterial voltage-gated sodium channel (NaChBac) ⁴² also stimulated egg production in the cold (Figure 3B) confirming increasing the activity of DN3s is sufficient to stimulate egg production in dormant females. Next, we investigated the effects of DN3 neuronal activation at warm temperatures. TRPA1³¹-mediated activation of DN3s after eclosion had no effect (Figure 3C), but the same manipulation significantly accelerated egg production during recovery from cold (Figure 3D). Ilp2, E-cell or DN1p activation also showed varying stimulatory effects on egg production, suggesting the activity of IPCs and multiple classes of circadian neurons are important during the recovery period (Figure S2B-D). Finally, we tested the impact of DN3 silencing on egg production by expressing the tetanus toxin light chain (TNT) ⁴³ in these neurons. At 25°C, DN3>Tnt females initially sped up egg production after eclosion and during recovery but subsequently slowed down the rate of egg synthesis (Figure 3E, F). When flies were moved from 25°C to 12°C, DN3>Tnt females were less responsive to cold – synthesizing more eggs than controls (Figure S2A). Based on our results, we concluded DN3 activity dynamics are critical to coordinate egg production with the environmental temperature.

Figure 3. — (A) Egg quantification for *DN3-GAL4>UAS-TrpM8* females and controls (12°C, 12L:12D) (n=16-18,). Two-way ANOVA with Tukey’s test is used to compare all time-course experiments and ovaries were assessed at indicated intervals unless otherwise stated.

(B) Egg quantification for *DN3-GAL4>UAS-NaChBac* females and controls that were moved from 25°C, 12°C. Ovaries were assessed 120 hours after eclosion (n=21, One-way ANOVA with Tukey’s test).

(C) Egg quantification for *DN3-GAL4>UAS-TrpA1* females and controls that were reared at 25°C and moved to 29°C at eclosion (n=14-18).

(D) Egg quantification for *DN3-GAL4>UAS-TrpA1* females and controls that were moved to 12°C then shifted to 29°C on the fourth day (n=15-20).

(E) Egg quantification for *DN3-GAL4>UAS-Tnt* females and genetic controls that were kept at baseline 25°C (n=18-25).

(F) Egg quantification for *DN3-GAL4>UAS-Tnt* females and genetic controls that were kept at cold 12°C for four days then moved 25°C (n=10-18).

See also Figure S2 and Table S1.

DN3 neural activity is temperature sensitive

To investigate how DN3s respond to changes in temperature, we performed whole-cell patch recordings of DN3s (Figure 4A). DN3s spontaneously fired action potentials at 25°C and exhibited both acute and persistent changes in their firing rate in response to temperature changes (Figure 4B). Specifically, we found that DN3s responded to cooling with an initial burst of action potential firing followed by a sustained decrease in firing rate (Figure 4C, white arrowhead). In contrast, heating led to a sustained rise in activity with a burst at temperature step offset (Figure 4D, white arrowhead). To test if temperature modulation of DN3s requires the antennae, we performed DN3 recordings in antennae-ablated flies. Upon acute ablation (by resection of the antennal nerves), cooling- or heating-induced transient changes in firing were abolished. However, changes in steady-state firing persisted in the absence of antennal input (Figure 4C, D, black arrowhead). Recordings done in other neurons (Figure S3A-C) did not show changes in firing rate in response to cooling, further confirming the specificity of DN3 responses to the changes in temperature (Figure 4E, F). We also measured DN3 activity in response to temperature changes using a transcriptional reporter of intracellular calcium (TRIC) ⁴⁴. TRIC signal in DN3s was reduced in flies that were kept at 12°C compared to 25°C (Figure 4G, H). In flies moved from 12°C to 25°C, the DN3 TRIC signal started to rise to levels seen in controls after six hours (Figure 4I). We did not see similar changes in the TRIC signal in response to cold temperatures in other neurons tested (Figure S3D-F).

In summary, DN3 firing rates and calcium levels are modulated by temperature. Importantly, consistent with their role in cold-induced reproductive dormancy, we observed a persistent temperature-dependent change in DN3 firing rate that is independent of antennal input (Figure 4C). Furthermore, DN3 firing rate declined with absolute temperature in the cold range (down to ~18°C, Figure 4F) suggesting progressive and long-lasting changes in membrane potential dynamics in DN3s in response to cooling.

DN3s regulate egg production through an insulin-independent pathway

How do DN3s regulate egg production? One potential target for DN3s is IPCs that are known regulators of egg production ^13,24. To elucidate whether IPCs may function (directly or indirectly) downstream of DN3s, we first used trans-Tango, a genetic method for trans-synaptic labeling of neural circuits in the fly brain ⁴⁵. We did not detect any IPCs in control or DN3>trans-Tango female brains (Figure 5A, C), while E-cell>trans-Tango clearly labeled IPCs (Figure 5B). We next investigated functional connectivity between DN3s and IPCs using two-photon calcium imaging coupled with optogenetic activation. We expressed GCaMP6s ⁴⁶ in IPCs and Chrimson ⁴⁷ in DN3s or, in E-cells (Figure 5D-F). While optogenetic activation of E-cells increased calcium levels in IPCs ⁴⁸, DN3 activation had no effect (Figure 5G-I). Our results suggest that DN3s might regulate egg production in the cold through means independent of the IPC pathway.

Figure 5. — (A-C) Confocal images of trans-Tango labeling of post-synaptic neurons for (A) control, (B) *E-cell,* and (C) *DN3s*. (B) E-cells and (C) DN3s are visualized by anti-GFP staining (green). Postsynaptic neurons for (B) E-cells and (C) DN3s are visualized by tdTomato expression and anti-Dsred staining (magenta) (scale bars=50μm).

(D-F) Confocal images of representative brains used in optogenetic stimulation experiments. (D) IPCs (green) express the genetically encoded calcium sensor, GCaMP6s. Chrimson (magenta) is expressed either in (E) E-cells or (F) DN3s (scale bars=50μm).

(G-I) *In vivo* optogenetic activation of (G) control, (H) E-cells, or (I) DN3s with Chrimson. Vertical shading indicates the activation period (10mA, 1s). Flies were kept at dormancy-inducing conditions (12°C and 12L:12D) for 48 hours before optogenetic stimulation (Error shading, mean ± SEM; n=5-6 per genotype, five stimulations per fly, Laser power=9.3mW, LED power=0.72mW).

See also Table S1.

Allatostatin-C in DN3s drives recovery from dormancy

Molecular architecture of DN3s has been recently described: Some of the DN3s are glutamatergic ⁴⁹ and the majority of these (~25 out of the ~40 DN3s) express the neuropeptide Allatostatin-C (Ast-C) ^50-54. The DN3-split-GAL4 used in this study labels 14 DN3s that are AstC-positive (Figure 6A). AstC is also expressed in some of the DN1s and PMP2 descending neurons (Figure 6B) ^51,55. Recently, AstC release by circadian DN1p neurons has been shown to regulate insulin signaling and alter the circadian rhythm of egg production ⁵⁶. To investigate whether AstC regulates egg production during cold-induced reproductive dormancy, we first checked if AstC expression is temperature-dependent. AstC immunoreactivity in the DN3s and PMP2 neurons was reduced at 12°C compared to 25°C (Figure 6B-D). AstC expression recovered to normal levels 12 hours after flies were moved to 25°C (Figure S4A-B). Other neuropeptides tested did not show similar temperature-dependent changes in expression (Figure S4C-H). Similar to D. melanogaster females, AstC expression was reduced in the cold across other Drosophilids (D. simulans, D. yakuba, D. virilis), and mosquitoes (Ae. aegypti, and An. stephensi) (Figure S5A-J).

Figure 6. — (A) Images of an example brain (left panel) or lateral protocerebrum (middle and right panel) of *DN3-GAL4>UAS-CD8-GFP* females co-stained for AstC (scale bars=50μm).

(B) Images of an example brain of control females kept at 25°C (left) or 12°C (right). Arrows indicate DN3s or PMP2s (scale bars=50μm).

(C-D) Quantification of AstC staining in (C) DN3s, (D) PMP2s (n=18, Welch’s t-test, ** p<0.01, *** p<0.001).

(E) Egg quantification for *AstC^−/−* and control females during recovery from cold exposure. Ovaries were assessed at indicated intervals (n=16-18, Two-way ANOVA with Bonferroni’s test).

(F-G) Overexpressing *AstC* in (F) DN3s facilitates egg production during recovery while overexpressing AstC in (G) PMP2s does not have an effect (n=10-20, Two-way ANOVA with Tukey’s test).

(H) Knocking down AstC in TIM neurons decreases the rate of egg production during recovery (n=12-16, Two-way ANOVA with Tukey’s test).

(I-J) Knocking down AstC in (I) DN3s or (J) PMP2s did not impact egg production during recovery (n=12-20, Two-way ANOVA with Tukey’s test).

(K) Activation of DN3s with NaChBac in the absence of AstC does not stimulate egg production in the cold (n=16-18, Two-way ANOVA with Tukey’s test).

(L) Activation of DN3s with TrpA1 in the absence of AstC does not stimulate egg production during recovery (n=15-19, Two-way ANOVA with Tukey’s test).

See also Figure S4, S5, and S6, and Table S1.

We next asked whether AstC peptide itself could modulate egg production in the cold or warm. Egg production was suppressed in AstC mutants (Figure S6A) in the cold similar to controls (Figure 6E, t=0 time point). However, during recovery, while controls rapidly resumed egg production, egg production in AstC mutants was significantly delayed (Figure 6E). As lack of AstC slowed down egg production during recovery from cold, we hypothesized that overexpression of AstC might accelerate it. To test our hypothesis, we first confirmed we can overexpress AstC mRNA and elevate the peptide levels in peptidergic neurons (Figure S6F-H). Indeed, overexpression of AstC in DN3s (Figure 6F), but not in PMP2s (Figure S6E, Figure 6G) elevated egg production during the recovery from cold.

Next, to determine which AstC neurons regulate egg production during recovery, we knocked down AstC in different populations of neurons. Knocking down AstC with Tim-GAL4 (Figure S6B) impaired egg production during recovery from cold (Figure 6H), while AstC knockdown with only DN3- or PMP2-splits did not suppress egg production during recovery (Figure 6I, J). This is likely due to partial knockdown of AstC with split-GAL4s, since DN3-split only labels ~50% of all AstC-expressing DN3s (Figure S6C, D). On the other hand, AstC knockdown with DN3-split was sufficient to attenuate elevated egg production caused by DN3 neuronal activation both in the cold and during recovery from cold (Figure 6K, L). Together, these results suggest that DN3 neural activity coupled with AstC release is necessary to coordinate egg production with environmental temperatures.

AstC regulates egg production through cholinergic AstC-R2 neurons

To further investigate how AstC regulates egg production, we injected the AstC peptide into females kept at different conditions. AstC injection (final hemolymph concentrations over 10nM) accelerated egg production during recovery from cold exposure (Figure 7A, Figure S7A, B), and slightly protected the rate of egg production in females that are moved to 12°C, 24-hours after eclosion (Figure S7D). We observed no effects of AstC injection on egg production in females that were constantly kept at 25°C (Figure S7C). Injection of AstC into hemolymph also rescued the egg production deficit seen in AstC mutants. In summary, circulating AstC is sufficient to stimulate egg production specifically after cold exposure similar to AstC overexpression in DN3s (Figure 6F).

Figure 7. — A) Egg quantification for control females injected with saline or 1 μM AstC and kept and assessed at indicated conditions (n=25-30 per time point, Two-way ANOVA with Tukey’s test). The same experimental regime was used in all injection/recovery experiments.

(B) 1μM AstC injection to *AstC^−/−* females rescues the egg production defect during recovery (n=16-19 per time point, Two-way ANOVA with Bonferroni’s test).

(C) Egg production is reduced during recovery in *AstC-R2^−/−* but not in *AstC-R1^−/−* females (n=16-19 per time point, Two-way ANOVA with Tukey’s test).

(D) 1μM AstC injection to *AstC-R2^−/−* females do not rescue reduced egg production during recovery (n=16-19 per time point, Two-way ANOVA with Bonferroni’s test).

(E) Images of an example brain (left panel) and ventral nerve cord (right) of *AstC-R2-GAL4>UAS-CD8-GFP* females (scale bars=50μm).

(F) Activation of AstC-R2 neurons inhibits egg production at (29°C, 12L:12D. Suppressing GAL4 expression with *ChAT-GAL80* abolishes the egg production defect caused by AstC-R2 activation (n=15-25, One-way ANOVA with Tukey’s test).

(G) Model for the temperature-dependent activity of DN3s and AstC/AstC-R2 regulation on egg production.

See also Figure S7 and Table S1.

AstC activates two G protein-coupled receptors (GPCRs), AstC-R1 and AstC-R2 ⁵⁷. To determine which AstC receptor is critical for regulating egg production in females, we tested the reproductive capacity of AstC-R1 and AstC-R2 mutants after cold exposure. AstC-R2 mutants showed significantly reduced egg production compared to controls, while AstC-R1 mutants showed no defect (Figure 7C). Furthermore, injection of AstC into AstC-R2 mutants did not stimulate egg production during recovery (Figure 7D) confirming AstC acts through AstC-R2 to modulate female reproductive capacity.

AstC-R2 is a G_i/o coupled receptor that is expected to inhibit neural activity upon AstC binding ^51,57. Hence, at 25°C since AstC levels are high, AstC-R2 neurons should be inhibited. Conversely, at 12°C since AstC levels are low, AstC-R2 neurons should be active. If this prediction is correct, artificial activation of AstC-R2 neurons (Figure 7E) should mimic the effects of cold and suppress egg production in warm temperatures. To test our hypothesis, we activated AstC-R2 neurons and found indeed AstC-R2>TrpA1 females synthesized almost no mature eggs (Figure 7F), similar to age-matched females in the cold. To identify which AstC-R2 neurons are responsible for the suppression seen in egg production, we inhibited the activity of GAL4 in selected cell types using the GAL80 repressor ⁵⁸. We ruled out a role for glial cells and motor neurons using Repo-GAL80 ⁵⁹, and Vglut-GAL80 ⁶⁰ respectively (Figure 7F, Figure S7E). Intriguingly, inhibiting GAL4 activity in cholinergic neurons using ChAT-GAL80 ⁶¹ completely abolished the reproductive suppression seen in AstC-R2>TrpA1 females (Figure 7F, Figure S7E). These results argue that cholinergic AstC-R2 neurons are a target for DN3/AstC, and their activation arrests egg production at warm temperatures.

DISCUSSION

Here, we investigated the impact of temperature and day length on the reproductive capacity of Drosophila females. We found that temperature is a critical factor that regulates egg production. We performed a neuronal activation screen and identified a subset of clock neurons, DN3s, whose activation disrupts cold-induced reproductive dormancy. The activity of DN3s is temperature-sensitive, and their activation stimulates egg production during and after cold exposure. DN3s utilize AstC as a signaling molecule to drive egg production, and AstC expression itself is temperature-dependent in multiple species of Drosophila and disease-causing mosquitoes. Finally, we found that DN3/AstC pathway acts through cholinergic neurons that express AstC-R2 to regulate egg production. Our data support a model in which DN3s alter their activity based on environmental temperature to speed up or slow down egg production in adult females (Figure 7G).

DN3s regulate recovery from cold-induced reproductive dormancy

Previous investigations mainly focused on the effects of photoperiod on female reproductive capacity ^12,13. In our study, we found that day length has a minimal impact on egg production. Our extensive analysis combining different LD cycles and temperature regimes showed that temperature is the dominant environmental factor regulating egg production in virgin females immediately after eclosion. It is possible LD cycles might have longer-term effects on egg production than we could monitor in the 4-to 5 days we examined in our experiments. Arresting reproduction when challenged by thermally adverse conditions might be a universal adaptive response to preserve resources that would otherwise be devoted to reproduction and protect future offspring until favorable environmental temperatures return. Here, we identified a group of temperature-sensitive circadian neurons, DN3s, that regulate egg production based on environmental temperatures. Activation of DN3s at cold temperatures or during recovery facilitates egg production, while inhibition of DN3s reduces egg production after eclosion and during recovery from cold. Interestingly, inhibition of DN3s in females that are moved from warm to cold also partially prevents the suppression of egg production. This could be related to chronic inhibition of DN3s which alters the dynamics of DN3 circuit activity in the female brain or due to the suppression of the DN3 responses to cooling (a rapid increase in firing which quickly adapts). By activating or silencing DN3 neurons artificially, we might be interrupting a dynamic neural signal. Circadian neurons are well known to form a highly interconnected network; thus, chronic activation and inhibition experiments might disturb this interconnected network activity and cause unexpected effects on fly physiology. Together, our results demonstrate that the proper functioning of DN3s is critical to coordinate egg production with environmental temperatures.

DN3 neural activity is temperature sensitive

Our measurements of neural activity using various methods showed that DN3 activity is persistently inhibited at cold temperatures and recovers when flies are moved to warm. Interestingly, the persistent modulation of DN3 activity was independent of the input provided by the temperature-sensing neurons in the fly antenna. We note that robust cold inhibition has been previously recorded for DN1a, a distinct group of dorsal neurons, in the context of temperature regulation of sleep-wake cycles ²⁷. Unlike DN3s, DN1a inhibition was nearly abolished by antennal resection. Interestingly, while antennal resection abolished temperature responses in several central nodes of the thermosensory circuit (e.g. TPN-IIs ²⁷, and other TPNs ⁶²), it did not eliminate temperature responses in DN1ps (which are activated, rather than inhibited, by cold temperature ³⁶). We also recorded the activity of olfactory projections neurons (GH146) that did not show any modulation in firing rate due to temperature decline. Together, these results suggest that the cold responses we observe in DN3s are a feature of this cell type. Currently, we do not know how DN3 neural activity is modulated by temperature. We speculate that DN3s either receive information from central brain neurons that are temperature-sensitive, or they sense temperature through a cell-autonomous pathway. Interestingly, similar to DN3s in the fly brain, in rats and hibernating ground squirrels, more than half of the neurons in the suprachiasmatic nucleus (SCN), a hypothalamic nucleus that regulates circadian rhythm, are active at warm temperatures, and rapidly reduce their firing rate during cooling ⁶³. Thus, we suggest, potent modulation of circadian neurons in response to temperature changes may be a conserved adaptation across insects and mammals.

AstC/AstC-R2 signaling regulates egg production in cold temperatures

Our data indicate that AstC is a critical factor that coordinates egg production with temperature changes. Decreased AstC expression in response to cold is conserved in multiple Drosophila species and mosquitoes. How does AstC release from DN3s regulate egg production? Recently, AstC release from DN1p(s) has been shown to change the egg production rhythm at warm temperatures by modulating the activity of IPCs ⁵⁶. In our functional imaging experiments, optogenetic activation of DN3s did not stimulate the activity of IPCs. The kinetics of the DN3/AstC effect on IPCs may be slow and occur over longer time frames. However, we also found that DN3 activation has a greater stimulatory effect on egg production compared to IPC activation. These results suggest that insulin signaling alone cannot explain the stimulatory effects of AstC/AstC-R2 on egg production. Furthermore, in our transsynaptic labeling experiments, we have identified neurons with projections in the superior medial protocerebrum (SMP) that are postsynaptic to DN3s. SMP is a major target of mushroom body output neurons, that regulate a variety of behaviors including foraging, feeding, and sleep ⁶⁴. Thus, it is possible that DN3/AstC signaling regulates egg production independent of the insulin pathway and by modulating the activity of neural circuits that directly regulate the physiological state of females.

CONCLUSION

Our identification of AstC/AstC-R2 signaling as a regulator of cold-induced reproductive dormancy is an entry point to neural circuits that regulate this physiological state in insects. Because regulation of AstC expression by temperature is conserved across Drosophila species and in mosquitoes, our results open the prospect of new strategies to control the reproduction of these insect vectors.

STAR METHODS

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Nilay Yapici (ny96@cornell.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The data that support the findings of this study are available from the lead contact upon reasonable request.
This study did not generate new code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon reasonable request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Flies

Drosophila melanogaster was maintained on conventional cornmeal-agar-molasses medium at 25°C and 60-70% relative humidity, under a 12hr light: 12hr dark cycle (lights on at 9 A.M.) unless otherwise stated. PMP2 split-GAL4 was identified using the electron microscopy volume reconstruction of a Hemi adult female brain ⁷⁹. These neurons have been also classified as DNP27 descending neurons in another study ⁸⁰. We backcrossed the AstC, AstC-R1, and AstC-R2 knock-out mutants for five generations to w¹¹¹⁸ background before testing their effects on egg production. Fly stocks and genotypes are provided in detail in the KEY RESOURCES TABLE and Table S1 respectively.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit polyclonal anti-AstC	Gift from J. Veenstra at Université de Bordeaux, France ⁶⁵	NA
Rat anti-TIM	Gift from Michael Young Rockefeller Institute ⁶⁶	NA
Mouse anti-AstA	Development Studies Hybridoma Bank	Cat# 5F10
Mouse anti-AstB (MIP)	Gift from Akira Mizoguchi ⁶⁷	NA
Mouse anti-PDF	Development Studies Hybridoma Bank	Cat# PDF C7
Rabbit polyclonal anti-dIlp2	Gift from J. Veenstra at Université de Bordeaux, France ⁶⁵	NA
Mouse monoclonal anti-DsRed	Takara Bio	Cat# 632392
Mouse monoclonal anti-GFP	Invitrogen	Cat# A-11120
Rabbit anti-GFP	Torrey Pines	Cat# TP401
Chicken polyclonal anti-GFP	Abcam	Cat# ab13970
Rabbit polyclonal anti-DsRed	Takara Bio	Cat# 632496
Mouse monoclonal anti-Bruchpilot	Development Studies Hybridoma Bank	Cat# Nc82
Alexa 488-conjugated goat anti-mouse IgG	Invitrogen	Cat# A-11001
Cy5 633-conjugated goat anti-mouse IgG	Invitrogen	Cat# A-10524
Alexa 488-conjugated goat anti-rabbit IgG	Invitrogen	Cat# 35552
Alexa 546-conjugated goat anti-rabbit IgG	Invitrogen	Cat# A-11035
Alexa 488-conjugated goat anti-chicken IgG	Invitrogen	Cat# A-11039
CF 568-conjugated goat anti-mouse IgG	Biotium	Cat# 20100
Alexa Fluor 594 Hydrazide	Invitrogen	Cat# A10438

Chemicals, Peptides, and Recombinant Proteins
All-trans retinal	Sigma	Cat#R2500
Allatostatin-C	Genscript	Custom
Vectashield Mounting Medium	Vector Laboratories	Cat# H-1200
Acetone	Sigma	Cat#179124-1L
Paraformaldehyde	Electron Microscopy Sciences	Cat# 15713
Normal Goat Serum	Sigma	Cat# G9023
Sodium Chloride	Sigma	Cat# S7653
Sodium Bicarbonate	Sigma	Cat# S5761
Potassium Chloride	Sigma	Cat# P9541
Calcium Chloride Dihydrate	Sigma	Cat#223506
Magnesium Chloride Hexahydrate	Sigma	Cat# M9272
HEPES	Sigma	Cat# H3375
D-(+)-Trehalose dihydrate	Sigma	Cat# T9531

Experimental Models: Organisms/Strains
Drosophila simulans	National Drosophila Species Stock Center (NDSSC)	#14021-0251.006
Drosophila virilis	National Drosophila Species Stock Center (NDSSC)	#15010-1051.00
Drosophila yakuba	National Drosophila Species Stock Center (NDSSC)	#14021-0261.00
Anopheles stephensi	Gift from Dr. Courtney Murdock, Cornell University	NA
Aedes aegypti	Gift from Dr. Laura Harrington, Cornell University	NA
Following strains are Drosophila melanogaster
w ^[1118]	Bloomington Stock Center	5905
Ir93a^−/−	Bloomington Stock Center	42090
Gr28b^−/−	Bloomington Stock Center	24190
TrpA1^−/−	Bloomington Stock Center	26504
11216-GAL4	Gift from Dr. Qiaoran Li ⁶⁸	NA
AkH-GAL4	Bloomington Stock Center	25683
Amn-GAL4	Korea Drosophila Resource Center	10010
AstA-GAL4	Bloomington Stock Center	51979
AstC-GAL4	Bloomington Stock Center	52017
Aug21-GAL4	Bloomington Stock Center	30137
Burs-GAL4	Bloomington Stock Center	51980
Capa-GAL4	Bloomington Stock Center	51969
CCAP-GAL4	Bloomington Stock Center	25685
CCHa1-GAL4	Korea Drosophila Resource Center	10021
CCHa2-GAL4	Bloomington Stock Center	84602
CG-GAL4	Bloomington Stock Center	7011
Clk-GAL4	Bloomington Stock Center	41810
CNMA-GAL4	Bloomington Stock Center	84619
Corazonin-GAL4	Bloomington Stock Center	51976
Cry-GAL4	Bloomington Stock Center	24514
Ddc-GAL4	Bloomington Stock Center	7010
Dh31-GAL4	Bloomington Stock Center	51988
Dh44-GAL4	Bloomington Stock Center	51987
Dimm-GAL4	Bloomington Stock Center	25373
Dsk-GAL4	Bloomington Stock Center	51981
dvPDF-GAL4	Gift from Dr. Michael Young ⁶⁹	NA
E-cell-GAL4 (DvPdf-GAL4, Pdf-GAL80)	Gift from Dr. Michael Young ⁷⁰	NA
Eh-GAL4	Bloomington Stock Center	6301
Eth-GAL4	Bloomington Stock Center	51982
FMRFamide-GAL4	Bloomington Stock Center	51990
Gr28B.a-GAL4	Bloomington Stock Center	57615
Gr28B.b-GAL4	Bloomington Stock Center	57616
Gr28B.c-GAL4	Bloomington Stock Center	57618
Gr28B.d-GAL4	Bloomington Stock Center	57620
Gr28B.e-GAL4	Bloomington Stock Center	57621
Hugin-GAL4	Bloomington Stock Center	58769
Ilp2-GAL4	Bloomington Stock Center	37516
Ilp3-GAL4	Bloomington Stock Center	52660
Ir25A-GAL4	Bloomington Stock Center	41728
Itp-GAL4	Bloomington Stock Center	84702
Lk-GAL4	Bloomington Stock Center	51993
Mip-GAL4	Bloomington Stock Center	51984
Myomodulin-GAL4	Bloomington Stock Center	30819
Myosuppressin-GAL4	Bloomington Stock Center	51986
NompC-GAL4	Bloomington Stock Center	36361
Npf-GAL4	Bloomington Stock Center	25681
Nplp1-GAL4	Korea Drosophila Resource Center	10008
Nplp2-GAL4	Korea Drosophila Resource Center	10023
Nplp4-GAL4	Korea Drosophila Resource Center	10022
Ntl-GAL4	Korea Drosophila Resource Center	10001
Ork-GAL4	Korea Drosophila Resource Center	10005
Pale-GAL4	Bloomington Stock Center	8848
Pdf-GAL4	Bloomington Stock Center	6899
Per-GAL4	Bloomington Stock Center	7127
Piezo-GAL4	Bloomington Stock Center	58771
Ppk-GAL4	Bloomington Stock Center	32079
Ppl-GAL4	Bloomington Stock Center	58768
Proc-GAL4	Bloomington Stock Center	51972
Ptth-GAL4	Gift from Dr. Naoki Yamanaka ⁷¹	NA
R61H02-GAL4	Bloomington Stock Center	39279
Rh1-GAL4	Bloomington Stock Center	8691
Rh3-GAL4	Bloomington Stock Center	7457
Rh4-GAL4	Bloomington Stock Center	8689
Rh5-GAL4	Bloomington Stock Center	7458
Rh6-GAL4	Bloomington Stock Center	7459
Rh7-GAL4 (PanR7-GAL4)	Bloomington Stock Center	8604
sNpf-GAL4	Bloomington Stock Center	51991
Tdc2-GAL4	Bloomington Stock Center	9313
Tim-GAL4	Gift from Dr. Michael Young ⁷²	NA
Tim(A3)-GAL4	Gift from Dr. Michael Young	NA
Trissin-GAL4	Bloomington Stock Center ⁷³	84695
TrpA1-GAL4	Bloomington Stock Center	76188
Yolk-GAL4	Bloomington Stock Center	58814
DN1-GAL4	FlyLight Split GAL4 Collection	LH2453
R10G01-GAL4	Bloomington Stock Center	48270
R14F03-GAL4	Bloomington Stock Center	48648
R16C05-GAL4	Bloomington Stock Center	48718
R16C09-GAL4	Bloomington Stock Center	48720
R18H11-GAL4	Bloomington Stock Center	48832
R19A11-GAL4	Bloomington Stock Center	47880
R19C05-GAL4	Bloomington Stock Center	48842
R19H06-GAL4	Bloomington Stock Center	49840
R20E08-GAL4	Bloomington Stock Center	49851
R31B01-GAL4	Bloomington Stock Center	49662
R43D05-GAL4	Bloomington Stock Center	41259
R51H05-GAL4	Bloomington Stock Center	41275
R54D11-GAL4	Bloomington Stock Center	41279
R57F07-GAL4	Bloomington Stock Center	46389
R61H08-GAL4	Bloomington Stock Center	39283
R65G02-GAL4	Bloomington Stock Center	39368
R66B05-GAL4	Bloomington Stock Center	39389
R67F03-GAL4	Bloomington Stock Center	39448
R70E02-GAL4	Bloomington Stock Center	39533
R70G01-GAL4	Bloomington Stock Center	39546
R73B06-GAL4	Bloomington Stock Center	42024
R75F10-GAL4	Bloomington Stock Center	46944
R77H08-GAL4	Bloomington Stock Center	39981
R77H09-GAL4	Bloomington Stock Center	39982
R78G01-GAL4	Bloomington Stock Center	40009
R80C12-GAL4	Bloomington Stock Center	47059
R80F05-GAL4	Bloomington Stock Center	48359
R85C03-GAL4	Bloomington Stock Center	47971
R85D05-GAL4	Bloomington Stock Center	40427
Split1-GAL4	Bloomington Stock Center	68852, 69456
Split2-GAL4	Bloomington Stock Center	70786, 69620
Split3-GAL4	Bloomington Stock Center	70786, 68520
Split4-GAL4 (DN3-GAL4)	Bloomington Stock Center	70786, 69630
Dilp2-LexA	Gift from Dr. Mark Wu ⁶⁸	NA
Vglut-GAL80	Bloomington Stock Center	58448
ChAT-GAL80	Yapici lab stock ⁶¹	NA
Repo-GAL80	Yapici lab stock ⁵⁹	NA
Tsh-GAL80	Yapici lab stock ⁷⁴	NA
10XUAS-Syn21-Chrimson88-tdT-3.1 (attP18), LexAop2-Syn21-opGCaMP6s (su(Hw)(attP8)	Gift from Dr. Michael Reiser ⁷⁵	NA
UAS-TrpM8	Gift from Dr. Ben White ⁷⁶	NA
10XUAS-IVS-mCD8-GFP (attP2)	Bloomington Stock Center	32185
10XUAS-IVS-mCD8-GFP (attP5)	Bloomington Stock Center	32188
UAS-AstC	Gift from Dr. Andreu Casali ⁷⁷	NA
UAS-TrpA1	Bloomington Stock Center	26263
UAS-Tnt-E2	Bloomington Stock Center	28837
UAS-NaChBac	Bloomington Stock Center	9466
UAS-AstC-RNAi	Bloomington Stock Center	25868
PMP2-GAL4	FlyLight Split GAL4 Driver Collection	SS00923
TRiC	Bloomington Stock Center	61679
trans-Tango	Bloomington Stock Center	77124
AstC−/−	Bloomington Stock Center	84453
AstC-R1−/−	Bloomington Stock Center	84454
AstC-R2−/−	Bloomington Stock Center	84455
AstC-R2-GAL4	Gift from Hiromu Tanimoto ⁷⁸	NA
GH146	Bloomington Stock Center	30026

Software and Algorithms
Adobe Illustrator 2021	Adobe	www.adobe.com
FIJI (ImageJ)	National Institutes of Health	https://fiji.sc/
GraphPad Prism 9.0.2.	GraphPad Software, La Jolla, CA	https://www.graphpad.com/scientific-software/prism/
ZEISS ZEN Imaging Software	Carl Zeiss, Oberkochen, Germany	www.zeiss.com
Pclamp (Clampex software v.9.2.1.9)	Axon Instruments/Molecular Devices	www.moleculardevices.com/www.moleculardevices.com/
Igor Pro v.6.37	Wavemetrics, Inc.	www.wavemetrics.com/www.wavemetrics.com/
Neuromatic v2.6i plug-in for Igor Pro	Rothman and Silver, 2018	www.neuromatic.thinkrandom.com/
Axograph v.1.70	Axograph	https://axograph.com/

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Mosquitoes

Ae. aegypti and An. stephensi were reared in the same walk-in incubator set to 28°C, 71.9 ± 9.5% humidity, and a light regime of 10hr light: 10hr dark and 2hrs each simulated dusk and dawn. Mosquito eggs were vacuum hatched and resulting larvae were allocated to rearing trays at a density of 200/1L dH₂O and fed with a fish food diet (Hikari Cichlid Gold, Hayward, CA, USA). Adult females were isolated in cardboard cups and kept at indicated temperatures with constant access to 10% sucrose until being sacrificed for brain dissections.

METHODS DETAILS

Mature Egg Quantification Assays

Virgin female progeny of the desired genotype was collected at eclosion and put in vials of no more than 8 females per vial. Upon dissection, whole ovaries were removed, fixed for 25 minutes in 4% paraformaldehyde at 23°C. Following fixing, ovaries were washed three times, 10 minutes each in PBST (PBS + 0.2% Triton X). PBST was then removed and replaced with VECTASHIELD® mounting medium with DAPI (Vector Laboratories, H1200) and mounted on glass slides. Ovaries were imaged on a Zeiss i880 confocal microscope using 405nm excitation and transmitted light. Z-stacks were used to count the number of mature eggs (stage 14 oocytes) in each ovary manually.

Egg production matrix.

w¹¹¹⁸ flies were reared at 25°C during development and virgin females were collected immediately after eclosion. Eight females were kept in food vials and moved into a container with eight light-controlled compartments each compartment had two vials. The entire enclosure was moved into an incubator that was set to the desired temperature. Females were kept in these conditions for four days. Ovaries were dissected, fixed, and stained according to the protocol described above.

Antennectomy testing.

Flies were reared at 25°C and moved to 12°C at eclosion. For antennectomized flies, flies were CO₂ anesthetized and antennae removed with sharp forceps before transfer. All flies were dissected, fixed, and stained after being kept in cold for four days.

TrpM8 screening.

Various GAL4 lines were crossed to UAS-TrpM8. Virgin female progeny were collected at eclosion and immediately moved to indicated cold temperatures. After five days, females were collected and ovaries were dissected, fixed, stained, and assessed. GAL4 lines that showed a statistically significant increase in egg production compared to controls (w¹¹¹⁸xUAS-TrpM8) during the activation screen were assessed again with their parental genetic controls.

Egg production assessment.

Virgin females were collected within two hours of eclosion and either kept at 25°C or moved to 29°C for TrpA1 experiments. Ovaries were dissected, fixed, stained, and assessed at indicated times.

Egg production assessment at cold.

Virgin females were collected within two hours of eclosion and either moved directly to 12°C or kept in 25°C for 24 hours first and then subsequently moved to 12°C. Ovaries were dissected, fixed, stained, and assessed at indicated times.

Recovery from cold-induced dormancy.

All data from cold-recovery experiments were taken from virgin females reared at 25°C (18°C for UAS-TrpA1 experiments), moved directly to 12°C for four days, and then returned to 25°C (29°C for UAS-TrpA 1 experiments) after the fourth day. Ovaries were dissected, fixed, stained, and assessed at indicated times.

Immunostaining and Confocal Microscopy

Flies were generally reared at 25°C or 12°C and dissected on day four after eclosion, except for trans-Tango experiments, in which flies were reared at 20°C and dissected/stained on day 20. Immunohistochemistry of different tissues was conducted as previously described ⁸¹. In brief, the organs were dissected in PBS and fixed in 4% paraformaldehyde (PFA)/PBS for 25 min at 23 °C. After washing in PBST (PBS + 0.2% Triton X-100) (5 times, 10 min each), the organs were blocked in 5% normal goat serum (NGS, Jackson Labs, 005-000-121) in PBST for 1 h at 23 °C, and then incubated with primary antibodies overnight in an orbital shaker at 23 °C. After washing in PBST (5 times, 10 min each), the sample organs were incubated with secondary antibodies overnight in an orbital shaker at 23 °C and washed again using PBST (5 times, 10 min each). Primary antibodies that were used: rabbit anti-GFP (1:500; Torrey Pines, TP401), chicken anti-GFP (1:500; Abcam, ab13970), mouse anti-nc82 (1:20; Development Studies Hybridoma Bank, DSHB, AB-2314866), rabbit anti-dsRed (1:500; Clontech, 632496), rabbit anti-TDC2 (1:250; Covalab, pAb0822-P), rabbit anti-Allatostatin-C (AstC) (1:500; a gift from J. Veenstra, Université de Bordeaux). Secondary antibodies that were used: Alexa Fluor 633 goat anti-rabbit IgG (1:500; Invitrogen, A-21052), Alexa Fluor 568 goat anti-mouse IgG (1:500; Biotium, 20101), Alexa Fluor 546 goat anti-rabbit IgG (1:500; Invitrogen, A27039), Alexa Fluor 488 goat anti-rabbit IgG (1:500; Dylight, 33552), Alexa Fluor 488 goat anti-chicken IgG (1:500; Invitrogen, A11039), anti-Horseradish Peroxiase-Cy3 conjugate (Jackson Immunoresearch, T123-165-021), and Alexa Fluor 405 Phalloidin (1:400; Invitrogen, A-30104). We used VECTASHIELD® (Vector Labs, H1000) for mounting the samples. All images were acquired using a Zeiss LSM 800 confocal microscope with 10XW, 20×, or 32XW lens at 1,024 × 1,024 resolution.

Whole-Cell Patch-Clamp Electrophysiology

Whole-cell patch-clamp electrophysiology experiments were performed on 2-3 days old flies. Females were anesthetized by brief cold exposure in an ice bath (~0°C) for ~1 min. Using a dissection microscope (Nikon SMZ1000), a small window in the head cuticle was opened and the underlying perineural sheath was gently removed using fine forceps (Moria Surgical). Brain tissue was exposed while maintaining connectivity with peripheral antennae and bathed in artificial hemolymph (AHL) solution containing the following: 103mM NaCl, 3mM KCl, 26mM NaHCO3, 1mM NaH2PO4, 8mM trehalose dihydrate, 10mM dextrose, 5mM TES, 4mM MgCl2, adjusted to 270-275mOSm. For experiments, 1.5mM CaCl2 was included, and the solution was continuously bubbled with 95% O2 5% CO2 to pH 7.3 and perfused over the brain at a flow rate of 1-2 mL/min. To target neurons for patching under the 2-photon microscope, DN3 split GAL4 neurons expressing GFP were excited at 840nm and detected using a photomultiplier tube (PMT) through a bandpass filter (490-560nm) using an Ultima 2-photon laser scanning microscope (Bruker, formerly Prairie Technologies). The microscope is equipped with galvanometers driving a Coherent Chameleon laser and a Dodt detector was used to visualize neural tissue/somata. Images were acquired with an upright Zeiss Examiner.Z1 microscope with a Zeiss W Plan-Apochromat 40×0.9 numerical aperture water immersion objective at 512 pixels × 512 pixels resolution using PrairieView software v. 5.2 (Bruker). Current clamp recordings were performed with pipettes pulled (Sutter P-97) using borosilicate capillary tubes (WPI Cat # 1B150F-4) with open tip resistances of 20 +/− 3 MΩ filled with an internal solution containing the following (in mM): 140 K-aspartate, 1 KCl, 1 EGTA, 10 HEPES, 4 Mg-ATP, 0.5 Na3-GTP, pH 7.3, 265 mOsm. To visualize the electrode and fill the cell after recording to confirm GFP colocalization, Alexa Fluor 594 Hydrazide (5 μM; Thermofisher Scientific Cat. # A10438) was added into the intracellular solution, excited using the 2-photon microscope at 840nm, and detected with a second PMT through a bandpass filter (580-630nm). Recordings were made using Axopatch 200B patch-clamp amplifier and CV203BU head stage (Axon Instruments), lowpass filtered at 2kHz, scaled to a 20x output gain, digitized with a Digidata 1320A, and acquired with Clampex software v.9.2.1.9 (Axon Instruments). Membrane potential recordings were made in current-clamp mode sampled at 10kHz.

Temperature stimulation.

For temperature stimulation, preparations were continuously perfused with Ca²⁺-containing AHL (as described above). AHL was gravity-fed through a 3-way valve (Lee company, part # LHDA1231315H) and the flow rate was adjusted through a flow regulator. Following the valve, the temperature was precisely regulated through 2 in-line solution heater/coolers (Warner, cat. # SC-20) in parallel with a dual-channel bipolar temperature controller (Warner Instruments, Cl-200A). Excess heat produced by each SC-20 Peltier was dissipated through a liquid cooling system (Koolance, Cat. # EXT-1055). To circumvent changes in resistivity and voltage offsets from changing the temperature of the bathing solution, the reference Ag-Cl pellet electrode was placed in an isolated well adjacent to the recording chamber (Warner Instruments, Cat. # RC-24N), filled with identical AHL and connected via a borosilicate capillary tube filled by 2% agar in 3M KCl. The bath temperature was precisely recorded using a custom Type T thermocouple with a 1cm exposed tip (Physitemp, Cat. # T-384A) connected to a thermometer (BAT-12, Physitemp) with an analog output connected to the digitizer and sampled at 10 kHz. The tip of the thermocouple was threaded through a borosilicate capillary tube and precisely placed near the antennae using a micromanipulator (MP-225, Sutter Instruments). In experiments in which single trials from individual cells were averaged, the line and shading indicate the mean temperature (°C) ± SD.

Transcriptional Reporter of Intracellular Ca²⁺ (TRIC) Analysis

Virgin females were collected at eclosion and moved to incubators set at either 25°C or 12°C, 12L:12D. On the fourth day, brains were dissected out and immediately imaged in PBS without fixing with a Zeiss i880 confocal microscope. In these experiments, to control for intraday variation all flies were assessed at Zeitgeber (ZT) 12.

Preparation of Flies for Optogenetics

Flies were transferred to a vial containing a mixture of fly food and 0.5mM ATR (all-trans-retinal, Sigma # R2500), on the second day after eclosion. After flies were transferred to ATR food, the whole vial was wrapped in aluminum foil. For the cold treated group, females were raised at 25°C but then kept at 12°C for 48 hours before the calcium imaging experiments. Optogenetic stimulation was performed using a 617nm LED that has been integrated into the light path of the two-photon microscope and LED light was delivered to the fly brain through the objective. LED light intensity was measured by an optical power meter (PM100D with S175C, Thorlabs) placed under the objective.

Brain Dissection for Two-Photon Calcium Imaging

Flies were first anesthetized by CO2, then placed on the sticky side of a short piece of transparent tape (about 2cm × 5cm, 3M™ Scotch® Transparent Film Tape 600), with fly’s leg facing up and wings attached to the tape. A short piece of hair (~2cm) was placed between the fly's head and thorax, pushing the fly's head toward the anterior side. Fly’s proboscis was manually extended using forceps until the labella stuck to the tape and stabilized in a fully extended position with UV curable resin (Bondic UV glue #SK8024). The tape was turned upside down and then attached to the 3D-printed fly holder. A small window was opened in the tape using forceps without injuring the fly, and the fly’s head was pushed head upward to make the head slightly past that window. The fly head was fixed in this position using UV curable resin. Once the fly’s head was fixed, physiological saline (108mM NaCl, 5mM KCl, 8.2mM MgCl₂·6H₂o, 2mM CaCl₂·2H₂O, 4mM NaHCO₃, 1mM NaH₂PO₄, 5mM Trehalose·2H₂0, 10mM Sucrose, 5mM HEPES, pH adjusted to 7.5, osmolarity measured to be 270mOsm) was applied to the fly head, and the head cuticle, air sacks and fat bodies were removed using forceps to gain optical access to fly brain. The holder carrying the fly was then placed under the two-photon microscope. During the calcium imaging experiments, the fly stood or walked on an air-suspended, spherical treadmill.

Two-Photon Calcium Imaging Coupled with Optogenetic Stimulation

The two-photon excitation source is a Ti:Sapphire laser centered at 920 nm (Coherent Chameleon Vision II, tuning range 680-1080). We used a two-photon microscope from Thorlabs (Bergamo II, Thorlabs) for calcium imaging equipped with a 16X Nikon CFI LWD Plan Fluor Objective Nikon (N16XLWD-PF). Laser power was measured using a power meter (PM100D with S175C, Thorlabs) at the objective side for all imaging experiments. A long-pass filter (FELH0600, Thorlabs) was used to reduce the background elevation caused by 617nm-LED stimulation used for optogenetic stimulation. Every fly brain was checked to confirm Chrimson-tomato and GCaMP6s expression before optogenetic stimulation. For IPC imaging experiments, we focused on the cell bodies of dIlp2 neurons and conducted volumetric time-lapse imaging upon 1s optogenetic stimulations. Z-planes were defined to encompass the entire volume of the IPCs. Usually, the distance between the starting and ending z-position is 70um. 8 planes were recorded, spaced ~10 μm apart and the entire volume was imaged at a rate of ~4.6 Hz. Fast-scanning volumetric imaging of the I PCs was necessary to minimize the effects of the fly’s movement on the recorded fluorescence signal. The calcium responses of IPCs reflected by GCaMP6s fluorescence intensity were recorded before and after optogenetic stimulation. At the end of each imaging experiment, the fly’s health was assessed by mechanical stimulation of the fly leg. If a fly did not respond to the mechanical stimulus, we considered that fly to be unhealthy and the data collected from that fly was not included in the final data analysis.

AstC peptide Injections

The AstC peptide (pGlu-VRYRQCYFNPISCF) was custom synthesized (GenScript, Piscataway, New Jersey). Flies were cold anesthetized and injected with either the AstC peptide or physiological saline using a Nanoject III, Programmable Nanoliter Injector (Drummond, # 3-000-207). The injection volume was set to 50nl. AstC peptide is dissolved in physiological saline (120mM NaCl, 3mM KCl, 1mM CaCl₂, 1mM MgCl₂, 5mM NaHCO₃, 5mM HEPES, 5mM Trehalose). The injection concentration range was 2nM to 100μM. Our calculations assume the total fly hemolymph volume is 1μl. Therefore, the final circulating concentration range after injections was 100pM to 5μM. After all members of a group were injected, animals were given 10 minutes to recover at room temperature before being moved to the indicated post-injection conditions.

QUANTIFICATION AND STATISTICAL ANALYSIS

Egg Number Quantification

Using the confocal stacks and the imaging software FIJI/ImageJ or Zen (Zeiss), we manually quantified the mature eggs from the DAPI stained ovary stacks. The mature eggs were identified by the presence of the dorsal appendages. The immature eggs lack dorsal appendages, and their size is smaller. In all panels, error bars indicate mean +/− SEM and groups labeled with different letters are significantly different unless stated otherwise (*p<0.05, **p<0.01, ***p<0.001).

AstC Expression Quantification

Using FIJI/ImageJ (NIH), AstC expression was quantified by measuring cell body brightness of DN3 and PMP2 neurons from females with indicated genotypes kept at 25oC or 12oC. AstC quantifications per genotype or condition were statistically compared using GraphPad Prism 9.0.2. The same procedure was used to quantify AstC expression for other Drosophilids and mosquito brains. The unit of all cell brightness in all the graphs is an arbitrary value calculated by quantifying the average pixel intensity in an ROI encircling the cell body of target neuron(s) and subtracting background (selected by encircling an unstained portion of the lateral horn). For DN3s, each point represents an average of cell bodies in each cluster. RNAi validation was done by measuring GFP and AstC staining intensity in DN3>AstC-RNAi;>GFP and DN3>GFP controls. Intensity was expressed as a ratio: GFP brightness-background/AstC brightness-background. For AstC staining during recovery, 6 neurons (3 per side) were averaged for each brain.

Transcriptional Reporter of Intracellular Ca²⁺ (TRIC) Quantification

Using FIJI/ImageJ (NIH), GFP and RFP cell body brightness of DN3s were quantified like above and used to create a ratio. The GFP/RFP ratios of brains kept at indicated conditions were statistically compared using GraphPad Prism 8.1.1.

Calcium Imaging Data Analysis

Two-photon volumetric imaging stacks were first projected in the z-axis and then registered with TurboReg (a FIJI plugin). Registration results were manually examined to avoid artifacts produced by incorrect registrations. ROI selection, average fluorescence computation, ΔF/F₀ calculation, and plotting were completed using a script written in MATLAB. Outlier stimulation segments (Max ΔF/F₀>10 or min ΔF/F₀ <10) were excluded from the dataset. For ROI selection, we draw a circle around all the cell bodies of IPCs labeled by Dilp2>GCaMP6s. We used binarization to get rid of dimmer pixels from the previously drawn ROI. The threshold for the binarization is manually selected to make sure the final ROI will include all the cell bodies while not including too much background. For ΔF/F₀ calculations, we first subtracted the background signal and the pre-stimulus GCaMP6s fluorescence (F₀=average of frames 5s before stimulus onset (t=−1s to −6s)) from the raw GCaMP6s fluorescence time series (F). The difference (ΔF) was then divided by the pre-stimulus GCaMP6s fluorescence (F₀) to obtain $Δ F ∕ F_{0} (\frac{Δ F}{F_{0}} = \frac{F - F_{0}}{F_{0}})$ . The data were aligned to the optogenetic stimulation (t=0 is when 617nm LED is turned on) and ΔF/F₀ values per genotype were plotted against time.

Whole-Cell Patch-Clamp Data Analysis

Whole-cell Patch Clamp recordings were analyzed offline using Axograph and Igor Pro. Action potentials were detected using custom scripts in Igor Pro using Neuromatic v2.6i plug-in ⁸². Peristimulus time histograms (PTH) of firing rate were made by binning detected spikes in 1-second bins, defining spikes/s (Hz). In experiments in which single trials from individual cells were averaged, the line and shading indicate the mean firing rate (Hz) ± SD. For experiments in which multiple sweeps were performed at a given stimulus temperature, average PTH was calculated per cell; the line and shading indicate the mean firing rate (Hz) ± SD. To test for a significant relationship between temperature and firing rate for DN3s, firing rates were fit to a linear model and significance (*) defined as p < 0.05.

Supplementary Material

NIHMS1776772-supplement-2.pdf^{(19.8MB, pdf)}

Table S1. Fly genotypes in detail. Related to Figures 1-7 and S1-S7.

NIHMS1776772-supplement-3.xlsx^{(18.2KB, xlsx)}

HIGHLIGHTS.

Reproductive dormancy in D. melanogaster is driven predominately by temperature.
Activation of DN3s interrupts reproductive dormancy.
DN3 activity is temperature-dependent, as is the expression of their peptide, AstC.
Activation of cholinergic AstC-R2 neurons induces reproductive dormancy.

ACKNOWLEDGEMENTS

We thank Dr. David Deitcher, Dr. Joe Fetcho, Dr. Mariana Wolfner, and the Yapici Lab for comments on the manuscript; Dr. Laura Harrington and Dr. Alexandra Amaro for their assistance in mosquito experiments; Dr. Qiaoran Li, Dr. Young-Joon Kim, Dr. Naoki Yamanaka, Dr. Michael Young, Dr. Benjamin White, Dr. Michael Reiser, and Dr. Andreu Casali for fly stocks, Dr. Laura Harrington for Ae. aegypti and Dr. Courtney Murdock for An. stephensi mosquito strains. We acknowledge Bloomington Stock Center (NIH P40OD018537) and the Developmental Studies Hybridoma Bank (NICHD of the NIH, University of Iowa) for reagents. Research in N.Y.’s laboratory is supported by NIH-R21 (R21AI149772) and NIH R35-MIRA (R35GM133698).

Footnotes

DECLARATION OF INTERESTS

The authors declare no competing interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS1776772-supplement-2.pdf^{(19.8MB, pdf)}

Table S1. Fly genotypes in detail. Related to Figures 1-7 and S1-S7.

NIHMS1776772-supplement-3.xlsx^{(18.2KB, xlsx)}

Data Availability Statement

The data that support the findings of this study are available from the lead contact upon reasonable request.
This study did not generate new code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon reasonable request.

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PERMALINK

Recovery from cold-induced reproductive dormancy is regulated by temperature-dependent AstC signaling

Matthew R Meiselman

Michael H Alpert

Xinyue Cui

Jamien Shea

Ian Gregg

Marco Gallio

Nilay Yapici

SUMMARY

In Brief

INTRODUCTION

RESULTS

Drosophila female reproductive capacity is altered by environmental temperatures

Figure 1. Egg production is strongly regulated by temperature in Drosophila melanogaster.

Activation of circadian neurons prevents proper execution of reproductive dormancy

Figure 2. Activation of specific clock neurons stimulates egg production in the cold.

Manipulation of a subset of DN3s perturbs thermal regulation of egg production

Figure 3. DN3s drive egg production in the context of cold temperatures.

DN3 neural activity is temperature sensitive

Figure 4. DN3s show persistent changes in their activity in response to temperature.

DN3s regulate egg production through an insulin-independent pathway

Figure 5. DN3s do not modulate the calcium activity of IPCs.

Allatostatin-C in DN3s drives recovery from dormancy

Figure 6. AstC release from DN3s stimulates egg production during and after cold exposure.

AstC regulates egg production through cholinergic AstC-R2 neurons

Figure 7. Cholinergic AstC-R2-expressing neurons regulate egg production.

DISCUSSION

DN3s regulate recovery from cold-induced reproductive dormancy

DN3 neural activity is temperature sensitive

AstC/AstC-R2 signaling regulates egg production in cold temperatures

CONCLUSION

STAR METHODS

RESOURCE AVAILABILITY

Lead contact

Materials availability

Data and code availability

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Flies

Mosquitoes

METHODS DETAILS

Mature Egg Quantification Assays

Egg production matrix.

Antennectomy testing.

TrpM8 screening.

Egg production assessment.

Egg production assessment at cold.

Recovery from cold-induced dormancy.

Immunostaining and Confocal Microscopy

Whole-Cell Patch-Clamp Electrophysiology

Temperature stimulation.

Transcriptional Reporter of Intracellular Ca2+ (TRIC) Analysis

Preparation of Flies for Optogenetics

Brain Dissection for Two-Photon Calcium Imaging

Two-Photon Calcium Imaging Coupled with Optogenetic Stimulation

AstC peptide Injections

QUANTIFICATION AND STATISTICAL ANALYSIS

Egg Number Quantification

AstC Expression Quantification

Transcriptional Reporter of Intracellular Ca2+ (TRIC) Quantification

Calcium Imaging Data Analysis

Whole-Cell Patch-Clamp Data Analysis

Supplementary Material

HIGHLIGHTS.

ACKNOWLEDGEMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Transcriptional Reporter of Intracellular Ca²⁺ (TRIC) Analysis

Transcriptional Reporter of Intracellular Ca²⁺ (TRIC) Quantification