RNAi of the circadian clock gene period disrupts the circadian rhythm but not the circatidal rhythm in the mangrove cricket

Abstract

The clock mechanism for circatidal rhythm has long been controversial, and its molecular basis is completely unknown. The mangrove cricket, Apteronemobius asahinai, shows two rhythms simultaneously in its locomotor activity: a circatidal rhythm producing active and inactive phases as well as a circadian rhythm modifying the activity intensity of circatidal active phases. The role of the clock gene period (per), one of the key components of the circadian clock in insects, was investigated in the circadian and circatidal rhythms of A. asahinai using RNAi. After injection of double-stranded RNA of per, most crickets did not show the circadian modulation of activity but the circatidal rhythm persisted without a significant difference in the period from controls. Thus, per is functionally involved in the circadian rhythm but plays no role, or a less important role, in the circatidal rhythm. We conclude that the circatidal rhythm in A. asahinai is controlled by a circatidal clock whose molecular mechanism is different from that of the circadian clock.

Figure 1.

Examples of the locomotor activity rhythm in male adults of Apteronemobius asahinai (a) anaesthetized (control), (b) injected with double-stranded RNA for β-lactamase, and (c) injected with double-stranded RNA for per and transferred from light–dark cycles (LD) to constant darkness (DD). Injection was performed on the sixth day (triangles). (i) Double-plotted actograms, (ii) chi-square periodograms of activities in subsequent DD, and (iii) histograms of activity levels counted for each circatidal period in DD are shown. Black and white bars above the actograms indicate light and dark phases, respectively. Grey boxes indicate no data. The oblique line in the periodogram indicates the significance level of α = 0.05 and a peak value above the line is designated as significant. Grey and white histograms represent odd and even circatidal cycles, respectively.

Figure 2.

The effects of per RNAi on circatidal and circadian rhythms in male adults of Apteronemobius asahinai under constant darkness subsequent to light–dark cycles. Crickets were anaesthetized (intact control), injected with double-stranded RNA for β-lactamase (dsbla), or injected with double-stranded RNA for per (dsper). The proportions with the same letters in each panel are not significantly different (Tukey-type multiple comparisons for proportions, p > 0.05). (a) The proportion of crickets with a circatidal rhythm and its period determined by a chi-square periodogram. The circatidal periods were not significantly different among the three groups (p > 0.05, ANOVA). (b) The proportion of crickets with a circadian rhythm among those with a circatidal rhythm determined by ANCOVA for the activity levels of even and odd circatidal cycles (electronic supplementary material, figure S1).