Functional circadian clock genes are essential for the overwintering diapause of the Northern house mosquito, Culex pipiens

Abstract

The short day lengths of late summer are used to program the overwintering adult diapause (dormancy) of the Northern house mosquito, Culex pipiens. Here, we investigated the role of clock genes in initiating this diapause and asked whether the circadian cycling of clock gene expression persists during diapause. We provide evidence that the major circadian clock genes continue to cycle throughout diapause and after diapause has been terminated. RNA interference (RNAi) was used to knock down the core circadian clock genes and to then assess the impact of the various clock genes on the ability of females to enter diapause. RNAi directed against negative circadian regulators (period, timeless and cryptochrome2) caused females that were reared under diapause-inducing, short day conditions to avert diapause. In contrast, knocking down the circadian-associated gene pigment dispersing factor caused females that were reared under diapause-averting, long day conditions to enter a diapause-like state. Our results implicate the circadian clock in the initiation of diapause in C. pipiens.

Keywords: Photoperiodism; RNA interference; cryptochrome 2; period; pigment dispersing factor; timeless.

Fig. 1.

Clock gene expression in non-diapausing and diapausing female Culex pipiens. (A) period (per), (B) timeless (tim), (C) Clock (Clk), (D) cycle (cyc), (E) cryptochrome1 (cry1) and (F) cryptochrome2 (cry2) relative mRNA expression measured by quantitative real-time PCR. Each point represents the average relative mRNA expression of 3–4 samples containing 10–20 female brains. One week old non-diapausing and post-diapause mosquitoes were exposed to 16 h of light and 8 h of darkness (16 h:8 h L:D), which is represented by the black open and filled bars below the x-axis (ZT, Zeitgeber time). One week old, 1 month old and 3 month old diapausing mosquitoes were exposed to 8 h:16 h L:D, which is shown by the gray open and filled bars below the x-axis. All mosquitoes were held at 18°C, except for post-diapause females, which were held at 25°C. Spline curves were fitted to the data using SigmaPlot. Standard error bars have been removed to enhance the clarity of the figure but are provided in supplementary material Figs 1–6.

Fig. 2.

Effects of per dsRNA injection on clock gene mRNA expression, ovarian development and lipid content. (A) Confirmation of per mRNA knockdown and the effects of per dsRNA on non-target circadian clock gene expression. Each bar represents the whole-body relative mRNA expression from mosquitoes that were collected at lights off 2 days after dsRNA injection. One week after dsRNA injection, the effects of per dsRNA on (B) average egg follicle length of 15 females and (C) average lipid content in 5 females that were reared under long (16 h:8 h L:D) or short day conditions (8 h:16 h L:D) at 18°C were measured. Effects of per dsRNA were compared with those observed in β-galactosidase (β-gal) dsRNA-injected (control) mosquitoes. Females were considered to be in diapause (percentage shown above bars) if the average egg follicle length was <80 μm and their lipid content was >30 μg mg⁻¹ mosquito. Bars represent s.e.m.; **P<0.01, ***P<0.001 (Student's t-test).

Fig. 3.

Effects of tim dsRNA injection on clock gene mRNA expression, ovarian development and lipid content. (A) Confirmation of tim mRNA knockdown and the effects of tim dsRNA on non-target circadian clock gene expression. Each bar represents the whole-body relative mRNA expression from mosquitoes that were collected at lights off 2 days after dsRNA injection. One week after dsRNA injection, the effects of tim dsRNA on (B) average egg follicle length of 15 females and (C) average lipid content in 5 females that were reared under long (16 h:8 h L:D) or short day conditions (8 h:16 h L:D) at 18°C were measured. Effects of tim dsRNA were compared with those observed in β-gal dsRNA-injected (control) mosquitoes. Females were considered to be in diapause (percentage shown above bars) if the average egg follicle length was <80 μm and their lipid content was >30 μg mg⁻¹ mosquito. Bars represent s.e.m.; *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).

Fig. 4.

Effects of cry2 dsRNA injection on clock gene mRNA expression, ovarian development and lipid content. (A) Confirmation of cry2 mRNA knockdown and the effects of cry2 dsRNA on non-target circadian clock gene expression. Each bar represents the whole-body relative mRNA expression from mosquitoes that were collected at lights off 2 days after dsRNA injection. One week after dsRNA injection, the effects of cry2 dsRNA on (B) average egg follicle length of 15 females and (C) average lipid content in 5 females that were reared under long (16 h:8 h L:D) or short day conditions (8 h:16 h L:D) at 18°C were measured. Effects of cry2 dsRNA were compared with those observed in β-gal dsRNA-injected (control) mosquitoes. Females were considered to be in diapause (percentage shown above bars) if the average egg follicle length was <80 μm and their lipid content was >30 μg mg⁻¹ mosquito. Bars represent s.e.m.; *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).

Fig. 5.

Effects of cyc dsRNA injection on clock gene mRNA expression, ovarian development and lipid content. (A) Confirmation of cyc mRNA knockdown and the effects of cyc dsRNA on non-target circadian clock gene expression. Each bar represents the whole-body relative mRNA expression from mosquitoes that were collected at lights off 2 days after dsRNA injection. One week after dsRNA injection, the effects of cyc dsRNA on (B) average egg follicle length of 15 females and (C) average lipid content in 5 females that were reared under long (16 h:8 h L:D) or short day conditions (8 h:16 h L:D) at 18°C were measured. Effects of cyc dsRNA were compared with those observed in β-gal dsRNA-injected (control) mosquitoes. Females were considered to be in diapause (percentage shown above bars) if the average egg follicle length was <80 μm and their lipid content was >30 μg mg⁻¹ mosquito. Bars represent s.e.m.; **P<0.01 (Student's t-test).

Fig. 6.

Effects of pigment dispersing factor (pdf) dsRNA injection on clock gene mRNA expression, ovarian development and lipid content. (A) Confirmation of pdf mRNA knockdown and the effects of pdf dsRNA on non-target circadian clock gene expression. Each bar represents the whole-body relative mRNA expression from mosquitoes that were collected at lights off 2 days after dsRNA injection. One week after dsRNA injection, the effects of pdf dsRNA on (B) average egg follicle length of 15 females and (C) average lipid content in 5 females that were reared under long (16 h:8 h L:D) or short day conditions (8 h:16 h L:D) at 18°C were measured. Effects of pdf dsRNA were compared with those observed in β-gal dsRNA-injected (control) mosquitoes. Females were considered to be in diapause (percentage shown above bars) if the average egg follicle length was <80 μm and their lipid content was >30 μg mg⁻¹ mosquito. Bars represent s.e.m.; *P<0.05, **P<0.01 (Student's t-test).