Sleep correlates with behavioral decision making critical for reproductive output in Drosophila melanogaster

Abstract

Balance between sleep, wakefulness and arousal is important for survival of organisms and species as a whole. While, the benefits of sleep both in terms of quantity and quality is widely recognized across species, sleep has a cost for organismal survival and reproduction. Here we focus on how sleep duration, sleep depth and sleep pressure affect the ability of animals to engage in courtship and egg-laying behaviors critical for reproductive success. Using isogenic lines from the Drosophila Genetic Reference Panel with variable sleep phenotypes we investigated the relationship between sleep and reproductive behaviors, courtship and oviposition. We found that three out of five lines with decreased sleep and increased arousal phenotypes, showed increased courtship and decreased latency to court as compared to normal and long sleeping lines. However, the male courtship phenotype is dependent on context and genotype as some but not all long sleeping-low courting lines elevate their courtship in the presence of short sleeping-high courting flies. We also find that unlike courtship, sleep phenotypes were less variable and minimally susceptible to social experience. In addition to male courtship, we also investigated egg-laying phenotype, a readout of female reproductive output and find oviposition to be less sensitive to sleep length and parameters that are indicative of switch between sleep and wake states. Taken together our extensive behavioral analysis here shows complex bidirectional interactions between genotype and environment and add to the growing evidence linking sleep duration and sleep-wake switch parameters to behavioral decision making critical to reproductive output.

Keywords: Behavior; Drosophila; Mating; Oviposition; Sleep.

Fig. 1.. Sleep phenotypes of long and short sleeper DGRP male flies.

(A) 3-day average of sleep duration in males during a 24-h period. (B) 3-day average of sleep bout length in males during a 24-h period. (C)3-day average of sleep bout number in dGRP males during a 24-h period. (D) 3-day average sleep (min/30 min) during a 24-h period. Sleep phenotype during daytime and nighttime of DGRP 38, 859 and 335 (each data point represents 30-min bin). ZT refers to zeitgeber time, ZT = 12 represents lights off. 21–63 flies of each genotype were assessed using Drosophila Activity Monitors with a 12:12 light/dark cycle for three days. Short sleepers (blue circles), normal sleepers (orange diamonds), and long sleepers (pink triangles). In this and all subsequent figures data represents mean and SEM *indicates p < 0.05, **indicates p < ***indicates p < 0.001. Number of male flies for each genotype were: Short sleepers (dGRP-38: 31, dGRP-310: 32, dGRP-365: 26, dGRP-808: 25, dGRP-832: 29), Normal sleepers (dGRP-21: 28, dGRP-301: 29, dGRP-307: 25, dGRP-859: 30), Long Sleepers (dGRP-235: 63, dGRP-313: 27, dGRP-335: 24, dGRP-338: 31, dGRP-379: 29).

Fig. 2.. Sleep phenotypes of long and short sleeper DGRP female flies.

(A) 3-day average of sleep duration in female flies during a 24-h period. (B) 3-day average of sleep bout length in female flies during a 24-h period. (C) 3-day average of sleep bout number in female flies during a 24-h period. (D) 3-day average sleep (min/30 min) during a 24-h period. Sleep phenotype during daytime and night-time of DGRP 38, 859 and 335 (each data point represents 30-min bin). ZT refers to zeitgeber time, ZT = 12 represents lights off. 24–31 flies of each genotype were assessed using Drosophila Activity Monitors and 12:12 light/dark cycle for three days. Short sleepers (blue circles), normal sleepers (orange diamonds), and long sleepers (pink triangles). Number of female flies for each genotype were: Short sleepers (dGRP-38: 29, dGRP-310: 30, dGRP-365: 31, dGRP-808: 28, dGRP-832: 31), Normal sleepers (dGRP-21: 23, dGRP-301: 29, dGRP-307: 25, dGRP-859: 27), Long Sleepers (dGRP-235: 31, dGRP-313: 30, dGRP-335: 29, dGRP-338: 28, dGRP-379: 24) Statistical analysis was by one-way ANOVA using Dunnett multiple comparisons with normal sleeper dGRP-859 as control. In this and all subsequent figures data represents mean and SEM *indicates p < 0.05, **indicates p < 0.01. ***indicates p < 0.001.

Fig. 3.. P(Wake), a measure of sleep depth and arousal in male and female DGRP lines.

Average P(Wake) or probability of sleep to wake transitions/min in (A) Male and (B) female flies. 21–63 flies of each genotype were assessed using Drosophila Activity Monitors with a 12:12 light/dark cycle for three days. Short sleepers (blue circles), normal sleepers (orange diamonds), and long sleepers (pink triangles). In this and all subsequent figures data represents mean and SEM *indicates p < 0.05, **indicates p < 0.01. ***indicates p < 0.001. Number of male flies for each genotype were: Short sleepers (dGRP-38: 31, dGRP-310: 32, dGRP-365: 26, dGRP-808: 25, dGRP-832: 29), Normal sleepers (dGRP-21: 28, dGRP-301: 29, dGRP-307: 25, dGRP-859: 30), Long Sleepers (dGRP-235: 63, dGRP-313: 27, dGRP-335: 24, dGRP-338: 31, dGRP-379: 29). Number of female flies for each genotype were: Short sleepers (dGRP-38: 29, dGRP-310: 30, dGRP-365: 31, dGRP-808: 28, dGRP-832: 31), Normal sleepers (dGRP-21: 23, dGRP-301: 29, dGRP-307: 25, dGRP-859: 27), Long Sleepers (dGRP-235: 31, dGRP-313: 30, dGRP-335: 29, dGRP-338: 28, dGRP-379: 24).

Fig. 4.. Sleep phenotypes of long and short sleeper DGRP male and female flies using video recordings and pixel movement-based measurement.

(A and B) Sleep duration (minutes) in male and female flies during a 24-h period. (C and D) Average P(Wake) or probability of sleep to wake transitions/ min in male and female flies. (E and F) Average P(Doze) or probability of wake to sleep transitions/ min in male and female flies. (G and H) Sleep phenotype during daytime and nighttime of DGRP 38 and 235 (each data point represents 30-min bins). ZT refers to zeitgeber time, ZT = 12 represents lights off. Short sleepers (blue circles), normal sleepers (orange diamonds), and long sleepers (pink triangles). In this and all subsequent figures data represents mean and SEM *indicates p < 0.05, **indicates p < 0.01. ***indicates p < 0.001, ***indicates p < 0.0001. Number of male flies for each genotype were: Short sleepers (dGRP-38: 16, dGRP-310: 22, dGRP-808: 39), Normal sleepers (dGRP-21: 39, dGRP-859: 30), Long Sleepers (dGRP-235: 23, dGRP-313: 23, dGRP-335: 39, dGRP-338: 17). Number of female flies for each genotype were: Short sleepers (dGRP-38: 17, dGRP-310: 24, dGRP-808: 45), Normal sleepers (dGRP-21: 40, dGRP-859: 36), Long Sleepers (dGRP-235: 21, dGRP-313: 23, dGRP-335: 42, dGRP-338: 20).

Fig. 5.. Courtship Index and latency in long-, short- and normal- sleeping DGRP males.

(A) Courtship wheel and arena used for assaying male courtship phenotypes. A pair of flies in an arena with male exhibiting unilateral wing extension. (B) Courtship index measured as time spent (in seconds) by a male fly exhibiting unilateral wing extension (a courtship specific behavior) during a 20-min trial. Courtship index of short sleepers (blue circles), normal sleepers (orange diamonds), and long sleepers (pink triangles). (C) Courtship latency measured as the time between start of the trial and initiation of the first bout of unilateral wing extension. Latency scores were coded such that 1 = 1–19 s 2 = 20–39 s, 3 = 40–59 s, 4 = 60–79 s, 5 = 80 s and above. (D) Percentage of flies that exhibited at least one courtship bout. 23–63 flies of each genotype were recorded in courtship wheel arenas and unilateral wing extensions were manually counted. Males were separated from beheaded females until video recording began. Latency was measured as time to first unilateral wing extension and data was binned. Short sleeping dGRP-38 were found to court significantly more than all other genotypes. Short sleepers (blue circles), normal sleepers (orange diamonds), and long sleepers (pink triangles). Data represents mean and SEM *indicates p < 0.05, **indicates p < 0.01. ***indicates p < 0.001, ***indicates p < 0.0001.

Fig. 6.. Correlational analysis within and between sleep and courtship parameters.

(A) Correlation analysis of P(Wake) and P(Doze) of male DGRP flies (short-, normal-, and long-sleeper lines). (B) Correlation analysis of courtship duration and latency of male DGRP flies (short-, normal-, and long-sleeper lines). (C and D) Correlation analysis of P(Doze) and P (Wake) vs courtship latency. (E and F) Correlation analysis of P(Doze) and P(Wake) vs courtship duration. We computed Spearman’s coefficient of correlation R between 2 groups at a time and two-tailed p values are indicated in all plots. Flies that did not court for the entirety of the 20-min duration are not included in the plots. For each of the genotypes we had 21–63 pairs. These genotypes included DGRP 38, 310, 365, 808, 832, 307, 859, 235, 335 and 338.

Fig. 7.. Social isolation does not affect sleep duration in short- and long-sleepers.

Sleep duration of socially isolated (I) and enriched (E) DGRP lines: short-sleepers with high courting phenotypes (38, 310 and 365), normal sleeper (859) and long sleepers with low courtship (235 and 335) in beam break-based DAM sleep assay. Isolated and enriched groups for each of the tested lines were compared using unpaired t-test with Welch’s correction. Data represents mean and SEM *indicates p < 0.05. Number of male flies for each genotype were between 18 and 26 and data were obtained from 2 independent trials.

Fig. 8.. Mate competition behavioral assays reveal that courtship phenotypes are plastic for longer sleeper DGRP 335 but not DGRP 235.

(A) Schematic of the mate competition assay showing a female CS (wild type fly) and two distinct DGRP males with variable sleep phenotype. One of the DGRP males is a short sleeper with high courtship phenotype. The other DGRP lines were long sleepers, low courters (DGRP 235 and 335) and normal sleeper (DGRP 859). (B, C and D) Courtship phenotype (time spent engaging in unilateral wing extension) of DGRP 38 (black bar) with long (335 and 235) or normal sleepers (859). Pink bars represent courtship phenotypes of long sleepers DGRP 235 and 335 (pink bar) and yellow bar indicates courtship phenotypes of normal sleeper DGRP 859. (E) Schematic of the mate competition assay showing a female CS (wild type fly) and two distinct DGRP males with variable sleep phenotype. One of the DGRP 38 male is a long sleeper with low courtship phenotype. The other DGRP lines were long sleepers-low courters (DGRP 235), short sleeper-high courter (DGRP 38), short sleeper-normal courter (DGRP 808) and moderate sleeper-normal courter (DGRP 859). (F) Courtship phenotype (time spent engaging in unilateral wing extension) of DGRP 335 in mate competition assay with DGRP 38 (black bar) and DGRP 235, 808 and 859 (grey bar). Data represents mean and SEM *indicates p < 0.05, **indicates p < 0.01. ***indicates p < 0.001, ***indicates p < 0.0001. Statistical analysis was conducted between two-samples by Unpaired t-test with Welch’s correction (B, C and D). For F, analysis was conducted between 4 samples by non-parametric Kruskal-Wallis test followed by Dunns’ correction.

Fig. 9.. Oviposition behavior in short-, normal- and long-sleepers.

(A) Number of eggs laid per chamber of a 24-chamber arena during a 3-day period. Each chamber housed two age-matched, mated female flies and eggs were counted manually. Short sleepers (blue circles), normal sleepers (orange diamonds), and long sleepers (pink triangles). (B) Single chamber of a 24 well plate arena containing apple juice agar media. Eggs were counted under the microscope. Data represents mean and SEM *indicates p < 0.5, **indicates p < 0.01. ***indicates p < 0.001, ***indicates p < 0.0001. We tested 48 flies/genotype (2 flies/chamber). Statistical analysis was by one-way ANOVA using Dunnett multiple comparisons with normal sleeper dGRP-859 as control. Data represents mean and SEM *indicates p < 0.5, **indicates p < 0.01. ***indicates p < 0.001.