Natural alleles of the clock gene timeless differentially affect life-history traits in Drosophila

Abstract

Circadian clocks orchestrate a variety of physiological and behavioural functions within the 24-h day. These timekeeping systems have also been implicated in developmental and reproductive processes that span more (or less) than 24 h. Whether natural alleles of cardinal clock genes affect entire sets of life-history traits (i.e., reproductive arrest, developmental time, fecundity), thus providing a wider substrate for seasonal adaptation, remains unclear. Here we show that natural alleles of the timeless (tim) gene of Drosophila melanogaster, previously shown to modulate flies' propensity to enter reproductive dormancy, differentially affect correlated traits such as early-life fecundity and developmental time. Homozygous flies expressing the shorter TIM isoform (encoded by the s-tim allele) not only show a lower dormancy incidence compared to those homozygous for ls-tim (which produce both the short and an N-terminal additional 23-residues longer TIM isoform), but also higher fecundity in the first 12 days of adult life. Moreover, s-tim homozygous flies develop faster than ls-tim homozygous flies at both warm (25°C) and cold (15°C) temperatures, with the gap being larger at 15°C. In summary, this phenotypic analysis shows that natural variants of tim affect a set of life-history traits associated with reproductive dormancy in Drosophila. We speculate that this provides further adaptive advantage in temperate regions (with seasonal changes) and propose that the underlying mechanisms might not be exclusively dependent on photoperiod, as previously suggested.

Keywords: circadian clock; developmental time; early-life fecundity; photoperiodism; reproductive dormancy; seasonality; timeless.

FIGURE 1

ls-tim homozygous females show reduced early-life fecundity compared to their s-tim homozygous counterparts. The lines used in this study are called Houten (Hu) s-tim and ls-tim, from the location in the Netherlands where the natural population of origin was collected. (A) Proportions (±SEM) of s-tim (black) and ls-tim (red) homozygous females in reproductive dormancy after 11 days at 12°C under both short (LD 8:16) and long (LD 16:8) photoperiods. Statistical analysis was performed using one-way ANOVA (post hoc: Tukey test), df = 19, ****p < 0.0001. (B) Average number of eggs laid daily (±SEM) by s-tim and ls-tim homozygous females at 23°C (LD 12:12) during the first 12 days of adult life. Statistical analysis was performed using the Kolmogorov-Smirnov test, ****p < 0.0001. (C) Total number of eggs laid (±SEM) by s-tim and ls-tim homozygous females during the 12 days considered. Statistical analysis was performed by t test, df = 20, ****p < 0.0001.

FIGURE 2

ls-tim homozygous flies’ egg-to-adult development is delayed compared to that of s-tim homozygous flies at 25°C. (A) Pupariation curves of s-tim (black) and ls-tim (red) homozygous larvae, pupariating over time at 25°C (LD 12:12). The interval between the time of egg deposition and pupariation was used to quantify developmental time. Log-rank (Mantel-Cox) test, p < 0.0001. AED: After Eggs Deposition. The number of residual, non-pupariated larvae from each genotype at different timepoints is reported below the graph. (B) Average number (±SEM) of pupariating s-tim and ls-tim homozygous larvae at 25°C over-time. (C) Egg-to-adult developmental time of s-tim and ls-tim homozygous flies at 25°C. Statistical analysis of developmental time (hours after egg deposition, AED) was performed by Mann-Whitney U test, ****p < 0.0001. (D) % increase in the developmental time of ls-tim flies at 25°C compared to their s-tim counterpart used as reference (t test, df = 4, **p < 0.01).

FIGURE 3

Differences in developmental time between s-tim and ls-tim homozygous flies increase at lower temperature and lead to changes in the size of emerging flies. (A) Pupariation curves of s-tim (black) and ls-tim (red) homozygous larvae pupariating over time at 15°C (LD 12:12). The time spanning between egg deposition and pupariation was defined as developmental time. Log-rank (Mantel-Cox) test, p < 0.0001. AED: After Eggs Deposition. The number of residual, non-pupariated larvae from each genotype at different timepoints is reported below the graph. (B) Average number (±SEM) of pupariating s-tim and ls-tim homozygous larvae at 15°C over time. (C) Egg-to-adult developmental time of s-tim and ls-tim homozygous flies at 15°C. Statistical analysis of developmental time (hours after egg deposition, AED) was performed by Mann-Whitney U test, ****p < 0.0001. (D) % increase in the developmental time of ls-tim flies at 15°C compared to s-tim individuals used as reference (t test, df = 4, **p < 0.01. (E) Adult weight of s-tim and ls-tim homozygous males and females emerging in a 24 h window. For every biological replicate, flies were weighted in batches of 10, and the average weight (±SD) of each fly determined and plotted. Statistical analysis was performed by t test, df = 4, ***p < 0.001. (F) Increase in adult weight in ls-tim homozygous males and females expressed as % (±SD) of the weight of their s-tim counterparts. Statistical analysis was performed by t test, ***, df = 4, p < 0.001.