Ikeno et al. see parallels in their results that lead them to claim support for Bünning's hypothesis. They suggest that having targeted the principal positive regulator of the bean bug's clock (cyc) and a negative regulator (per), they have stopped the circadian clock at different phases of its oscillating cycle. It is unable to oscillate in response to the night-day cycle, but having become stuck in opposing phases, output signals are still delivered to give phenotypes that correspond, in the case of the cuticle, to those normally associated with opposite swings of the night-day pendulum. In the case of diapause, the differences in sensitivity to day length are interpreted as disruption of the photoperiodic timer, with the switch for diapause stuck in either one of two opposing positions. Thus, they see support for Bünning's proposition that the circadian clock mechanism lies at the heart of photoperiodicity.
These are, however, speculative suggestions only, venturing beyond the data, and in our view biased by the intrinsic appeal of connecting the circadian clock and photoperiodic time measurement. Based on the data, we would make the following points. First, both RNAi treatments lead to a reduction of per expression, so that any phenotypic differences observed must be attributed largely to the difference in cyc expression. Second, both treatments rendered the circadian clock dysfunctional. Third, all of the phenotypes that varied between the two RNAi treatments did so in the consistent absence of a functional circadian clock. If there is consistently no functional circadian clock in both RNAi treatments, then phenotypic differences between those treatments cannot be ascribed to the circadian clock. We would therefore conclude that the circadian clock as a functional unit (module) does not provide the essential clockworks for photoperiodic time measurement in R. pedestris.
Just because the circadian clock is dysfunctional does not mean that individual clock genes have no other pleiotropic effects [9]. The diapause response of bugs exposed to long and short days (Figure 4 in [6]) can be considered in terms of the presence of cyc expression (per RNAi treatment) or absence of cyc expression (cyc RNAi treatment). When cyc is expressed, there is an increase in ovarian development (non-diapause) compared to when cyc is not expressed, regardless of day length. The application of a juvenile hormone analog tells us that cyc is not acting at the level of the ovaries themselves. However, these results do not tell us where cyc is exerting its effect. This could be anywhere in the cascade of events (Figure 2), from the input of light [10] to the secretion of juvenile hormone in the corpora allata. Given that cyc encodes a positive transcriptional regulator, multiple pleiotropic effects are possible, and even likely.