Cuticle deposition rhythm
Alternating bright and dark layers were observed in the endocuticle of the hind leg tibia of R. pedestris (Figure 1a). First, we confirmed that in R. pedestris the cuticle deposition rhythm free-ran under constant conditions (self-sustaining oscillation; Figure 1b), that the number of deposited cuticle layers varied with the given number of temperature cycles (entrainment to environmental cycles; Figure 1c) and that the Q10 value of the rhythm was close to 1.0 between 22.5°C and 27.5°C (temperature compensation; Figure 1d). Thus, the rhythm exhibited major properties of circadian rhythms, indicating that the cuticle deposition rhythm in R. pedestris is regulated by a circadian clock.
Gene silencing by double-stranded RNA (dsRNA) injection
Although gene suppression by dsRNA injection was not obvious in per at Zeitgeber time (ZT) 6 (6 h after light-on) and ZT18 on day 5 and in cyc at ZT6 on day 5, a clear suppression of target genes was detected in per at ZT8 on day 5 and day 20 and in cyc at ZT8 and ZT18 on day 5 and at ZT8 on day 20, indicating that RNAi by dsRNA injection is effective in R. pedestris (Figure 2a and b and Additional File 1). It should be noted that the expression level of per was also reduced in cyc RNAi insects (Figure 2c).
Effects of RNAi on cuticle deposition rhythm
In R. pedestris constantly maintained under short-day conditions, injection of 0.9% NaCl (saline) or control (β-lactamase: bla) dsRNA did not affect the alternating deposition of bright and dark layers, as in the case of intact insects (Figures 1a and 3b). In contrast, almost all insects injected with per dsRNA failed to deposit alternating cuticle layers, resulting in the production of a single thickened dark layer (Figure 3a left and 3b). Injection of cyc dsRNA also produced similar results, except that it generated a single bright layer (Figure 3a right and 3b). We also checked the cuticle deposition rhythm in all the individuals used in the following photoperiodic experiments and we obtained similar results irrespective of photoperiodic conditions (Additional File 2).
Effects of RNAi on diapause
We designed four experimental schedules with different combinations of short-day and long-day conditions before and after adult emergence and injected dsRNAs or saline into females on the day of adult emergence.
In the first experimental schedule, insects were continuously maintained under short-day conditions. Under these conditions, intact females did not develop ovaries and entered diapause in response to short days (Figure 4a) and most females injected with bla dsRNA or saline also entered diapause (Figure 4a). None of the individuals injected with cyc dsRNA showed any ovarian development (Figure 4a). In contrast, per RNAi induced ovarian development in approximately 50% of the females (Figure 4a).
In the second experimental schedule, insects reared under long-day conditions were transferred to short-day conditions on the day of adult emergence. Under these conditions, many intact females and females injected with bla dsRNA or saline suppressed ovarian development and entered diapause in response to short-day conditions (Figure 4b). Similarly, most females injected with cyc dsRNA suppressed ovarian development. However, mature ovaries were observed in all females injected with per dsRNA (Figure 4b).
In the third experimental schedule, insects were continuously maintained under long-day conditions. Under these conditions, all intact females and females injected with per dsRNA, bla dsRNA, or saline developed ovaries (Figure 4c). However, cyc RNAi suppressed ovarian development in approximately 80% of females (Figure 4c).
In the fourth experimental schedule, insects reared under short-day conditions were transferred to long-day conditions on the day of adult emergence. Under these conditions, most intact females and most females injected with bla dsRNA or saline developed ovaries (Figure 4d). Similarly, females injected with per dsRNA developed ovaries (Figure 4d). In contrast, no females matured their ovaries when cyc dsRNA was injected (Figure 4d).
The expression of juvenile hormone (JH)-regulated genes
In many insects, reproductive diapause is induced by the suppression of JH secretion [30]. Also, in R. pedestris, adult diapause is due to the cessation of JH secretion [31]. We were, however, unable to measure the JH concentration in the hemolymph of R. pedestris as hemipteran insects possess a novel type of JH [32]. A system that enables the quantification of JH concentrations in hemipterans has yet to be established. As an alternative approach, however, we could estimate the JH concentration by examining the expression of JH-regulated genes; expressions of cyanoprotein-α (CP-α) and vitellogenin (Vg) transcripts are induced by JH, whereas transferrin (Tf) transcript expression is suppressed, in R. pedestris [33, 34].
In this experiment, all insects were reared under short-day conditions. We then transferred some to long-day conditions on the day of adult emergence. CP-α and Vg transcripts were detected in reproductive females injected with saline and transferred to long-day conditions; they were also detected in reproductive females injected with per dsRNA under continuous short-day conditions. However, they were scarcely detectable in non-reproductive females injected with cyc dsRNA and transferred to long-day conditions or in non-reproductive females injected with saline under continuous short-day conditions (Figure 5 top and middle). Tf transcript was detected in diapause females injected with cyc dsRNA and transferred to long-day conditions and in diapause females injected with saline under continuous short-day conditions (Figure 5 bottom). In contrast, the transcript was undetectable in reproductive females injected with saline and transferred to long-day conditions and in reproductive females injected with per dsRNA under continuous short-day conditions (Figure 5 bottom). Thus, in reproductive females induced by per RNAi, CP-α and Vg transcripts were clearly expressed, whereas, in contrast, non-reproductive females induced by cyc RNAi expressed Tf transcript. These results indicate that JH secretion is suppressed in the cyc RNAi females, whereas it is induced in the per RNAi females.
JH analogue (JHA) application
JHA application induced reproduction in females injected with bla dsRNA or saline and even in females injected with cyc dsRNA. However, the application of a control (ethanol) had little or no effect on the ovarian status of these females (Figure 6). Thus, cyc RNAi did not disrupt the process directly involved in ovarian development but did affect some upstream cascade.