The Summer Is Coming: nocte and timeless Genes Are Influenced by Temperature Cycles and May Affect Aedes aegypti Locomotor Activity

Abstract

Mosquitoes exhibit activity rhythms, crucial for the transmission of pathogens, under the control of a circadian clock. Aedes aegypti is one of the world's leading vectors. For decades, several studies have linked the rise in ambient temperature with the increase in their activity. Here, we identify candidate genes whose expression is influenced by temperature cycles and may affect Aedes locomotor activity. We observed that timeless completely lost its rhythmic expression in light/dark, with out-of-phase temperature cycles, and by RNAi mediated knockdown of nocte, an important gene for Drosophila circadian synchronization by temperature cycles. Thus, timeless and nocte are important genes for synchronization by temperature cycles in Aedes aegypti. To reinforce our findings, we simulated in the laboratory the gradual temperature fluctuations that were as close as possible to daily temperature variations in Brazil. We observed that the activity and the expression of the molecular circadian clock of Ae. aegypti differs significantly from that of mosquitoes subjected to constant or rectangular abrupt changes in temperature. We suggest that for understanding the circadian behavior of Aedes with possible implications for intervention strategies, the seminatural paradigm needs to replace the traditional laboratory study.

Keywords: Aedes aegypti; circadian gene expression; circadian rhythms; clock genes; seminatural cycles; temperature cycles.

FIGURE 1

Natural and simulated temperature cycles. (A) Natural temperature conditions during fall equinox (pink line), spring equinox (blue continuous line) or the average of equinoxes (orange line) in Rio de Janeiro, RJ, Brazil. (B) Gradual simulated temperature cycle (orange line). ZT, zeitgeber time (h); NT, natural time (h) in accordance with the official Brasília time (BRT, UTC-3).

FIGURE 2

Activity in light/dark cycles with simulated dawn and dusk or in constant darkness. (A) Average locomotor activity patterns of Aedes aegypti under gradual LD (yellow continuous line). The average pattern was obtained from the average activity from the 3rd to the 5th day in gradual LD. (B) Locomotor activity profiles of the first day in constant darkness (black continuous line). (C) Average actograms of Aedes aegypti. The mosquitoes were kept in gradual LD for 6 days, then light/dark cycles were interrupted (DD) for 10 days (n = 14). Temperature remained constant (25°C) every day. Area in actogram indicates light/dark conditions: lights on = white, lights off = gray. Horizontal bars above the average activity profiles lines and actogram indicate light/dark or temperature regimen: lights on = white, lights off = black, 25°C = orange. Dotted lines in average activities profiles indicate the beginning and the end of the photophase in LD or subjective photophase in DD. Error bars are not shown for clarity.

FIGURE 3

Activity profile of Ae. aegypti under seminatural or rectangular TC. (A,B) Average activity patterns of Aedes aegypti under seminatural TC (A, n = 51) or rectangular TC (B, n = 115) and constant darkness. Bars above the average activities profiles lines indicate light/dark or temperature regimen: lights off = black, 20°C = blue, 25°C = orange, 30°C = red. Dotted lines indicate the beginning and the end of the subjective photophase in DD. Arrows present the E peak. Error bars are not shown. (C) Analysis of E peak phase of each individual under seminatural TC (ZT 8.65 ± 2.16; lilac circles) or rectangular TC (ZT 6.03 ± 1.54; blue circles) with constant darkness. ***P < 0.001 in accordance with the Mann-Whitney U-test. The average activity patterns or the individual activity profiles used for the analysis of E peaks were both obtained from the average activity from the 4th to the 6th day in each condition.

FIGURE 4

Activity of Aedes aegypti over several days in seminatural or rectangular temperature cycles. (A,B) Double-plotted actograms of average activity in seminatural TC (A, n = 51) or rectangular TC (B, n = 115) with constant darkness. First, the mosquitoes were entrained by each TC for 6 days; then temperature cycles were phase-delayed by 6 h, and they were kept in that new condition for 7 days. (C,D) The graphics show the trajectories of the activity peaks of each individual before and after phase-shift of seminatural TC (C, n = 23) or rectangular TC (D, n = 30). (C) E peak is shown under seminatural TC (lilac dots). (D) In rectangular TC, we see the E peak (blue dots) and the peaks that happened just after the temperature rise (orange dots) or temperature drop (red dots). Black bars above the actograms indicate darkness. Areas in actograms represent the temperature cycles: 20°C = blue, 25°C = orange, 30°C = red.

FIGURE 5

Circadian expression of clock genes in Ae. aegypti under seminatural TC with constant darkness. The graphs show the expression of per, tim, cry2, cyc, Pdp1, vri, Clk, E75, and cwo genes in the head of Ae. aegypti under seminatural TC with constant darkness (lilac lines). The average was obtained from data of four independent experiments. The y-axis indicates the relative mRNA abundance and the x-axis represents the time points (ZT). Bars above the graphs indicate darkness or temperature regimen: lights off = black, 20°C = blue, 25°C = orange, 30°C = red. Rhythmic or arrhythmic genes are identified with “R∼” or “A-” symbols, respectively, according to One-way ANOVA. For more details, see Supplementary Table 2.

FIGURE 6

Activity in light/dark cycles with temperature cycles in natural phase or out of phase. (A) Average activity profile of Aedes aegypti under seminatural LD with in-phase (green continuous line) or out-of-phase (orange continuous line) TC. Dotted lines indicate the beginning and the end of the photophase. Error bars are not shown. M represents the Morning peak and E represents the Evening peak. (B) Double-plotted actograms of average activity when mosquitoes were kept in LD with in-phase TC for 6 days, and after phase-shifting by 12 h. The mosquitoes were kept in that new out-of-phase condition for 7 days (n = 74). Bars above the actogram or average activities profiles lines represent light/dark cycle: lights on = white, lights off = black. Temperature cycle is indicated by bars above the average activities profiles lines or area in actogram: 20°C = blue, 25°C = orange, 30°C = red. The average patterns were obtained from the activity data from the 3rd to the 5th day before or after phase-shift of seminatural TC.

FIGURE 7

Circadian expression of clock genes when Zeitgebers are in phase or conflicting. The graphs display the relative RNA abundance of per, tim, cry2, cyc, Pdp1, vri, Clk, E75, and cwo in head of Ae. aegypti under seminatural LD with in-phase (green lines) or out-of-phase (orange lines) TC. The average was obtained from data of four independent experiments. Bars above the graphs indicate light/dark or temperature regimen: lights on = white, lights off = black, 20°C = blue, 25°C = orange, 30°C = red. Arrhythmic or rhythmic genes are labeled with “A-” or “R∼” symbols, respectively, in accordance with the One-way ANOVA. The color of each symbol indicates the regimen: green = LD with TC in-phase, orange = LD with TC out-of-phase. For more details, see Supplementary Table 2.

FIGURE 8

Relative RNA abundance of nocte in Aedes aegypti. (A,B) Pattern of daily transcriptional expression of nocte in the head (A) or body (B) of mosquitoes under seminatural LD with out-of-phase TC (mosquitoes no injected with dsRNA). The average was obtained from data of four experiments. Bars above the graphs indicate light/dark or temperature regimen: lights on = white, lights off = black, 20°C = blue, 25°C = orange, 30°C = red. According to the One-way ANOVA, nocte was arrhythmic in the head and body (labeled with “A-” symbol, further details in the text). (C) Expression of nocte at ZT 21, in the head and body of Aedes aegypti injected with dsRNA of LacZ (blue) or nocte (red) and submitted to LD with out-of-phase TC. The analysis were performed on the fourth day after the injection with the dsRNAs. The experiments were repeated three times and the highest value between the two groups is applied as reference. A t-test was used to compare the groups in each tissue. Asterisks indicate when RNA abundance is significantly different among groups (P < 0.05). NS, non-significant different of RNA abundance between groups.

FIGURE 9

Activity of Ae. aegypti injected with dsRNA. The graph shows the activity profile of mosquitoes injected with dsRNA of LacZ (blue line) or nocte (red line). The average patterns were from the activity data from the 3rd to the 5th day in LD with out-of-phase TC. Bars above the graphs indicate light/dark or temperature regimen: lights on = white, lights off = black, 20°C = blue, 25°C = orange, 30°C = red. Error bars were omitted for clarity. Dotted lines indicate the beginning and the end of photophase.

FIGURE 10

Circadian expression of clock genes in Ae. aegypti injected with dsRNA. The graphs show the expression of per, tim, cry2, cyc, Pdp1, vri, Clk, E75, and cwo in the head of Ae. aegypti injected with dsRNA of LacZ (blue line) or nocte (red line). The mosquitoes were submitted to LD with out-of-phase TC. The average was obtained from data of three independent experiments. All analyzes were performed on the fourth day after the injection with the dsRNAs. Bars above the graphs indicate light/dark or temperature regimen: lights on = white, lights off = black, 20°C = blue, 25°C = orange, 30°C = red. Arrhythmic or rhythmic genes are labeled with “A-” or “R∼” symbols, respectively, in accordance with the One-way ANOVA. For more information, see Supplementary Table 2.