The Nanda–Hamner experiments yield neither a typically positive, nor a typically negative response in D. ezoana (Fig. 3). Therefore, newly developed T‐cycle experiments are used to test the involvement of a sustained or damped circadian clock in photoperiodic time measurement. The T‐cycle study shows that the photoperiodic clock of the northern fruit fly species D. ezoana measures an absolute night length of approximately 7 h to induce diapause, irrespective of the photoperiod and the period of T‐cycles (Fig. 5). The absolute night‐length measurement strongly suggests that the time measurement in D. ezoana is achieved by a simple hour‐glass clock. However, such apparently hour‐glass based night‐length measurement may also be achieved by a weak and highly Zeitgeber responsive oscillator based clock through an ‘external coincidence’ principle as proposed by Pittendrigh (1966). The circadian oscillators of such clocks are considered to be completely (or mostly) damped by the end of the naturally occurring photoperiods and to be reset to the same phase at the onset of the night, causing ϕ i to occur fixed hours afterwards. The night length is thus measured by sensing whether or not the ϕ i is exposed to light. The absolute night length measurement in T‐cycle experiments thus suggests that a weak circadian oscillator may underlie the photoperiodic clock in D. ezoana. The present T‐cycle experiments appear to decipher the type of clock involved in photoperiodic time measurement, even in cases in which the classical Nanda–Hamner or Bünsow experiments lead to ambiguous results. Interestingly, D. ezoana flies appear to also utilize a strongly damped circadian clock for controlling locomotor activity rhythms. This suggests that the same weak circadian clock may be involved in photoperiodic time measurement and behavioural rhythm control. These observations in D. ezoana lead to three questions: (i) is the clock that measures night length identical to the clock that controls activity rhythms in all species showing diapause; (ii) is a weak, highly damped circadian clock better suited for photoperiodic time measurement than a sustained robust circadian clock; and (iii) what are the limitations of the newly developed T‐cycle experiments?

Is the clock that measures night length identical to the clock that controls activity rhythms in all species showing diapause?

Circadian activity rhythms are a useful phase marker of the photoperiodic oscillator in many diapausing insects and their activity rhythms are therefore regarded as ‘hands of the photoperiodic clock’ (Kenny & Saunders, 1991), suggesting that the same clock controls activity rhythms and measures night length.

The results of the present study meet this expectation: D. ezoana flies appear to use a strongly damped oscillator for measuring night length and for controlling circadian activity rhythms. Nevertheless, there are superficial differences between the two clocks: the photoperiodic clock appears to damp within one cycle in DD, whereas the activity rhythm of D. ezoana flies free‐runs for a few cycles in DD. The reason for this difference may be a result of the different methods used in the diapause and activity rhythm assessment. The diapause assays measure the response of a population of flies, whereas locomotor activity recordings monitor the behaviour of single flies. Oscillator dampening is slightly different in individual flies. Some flies remain rhythmic for at least 5 days in DD, although the majority become arrhythmic after 2 days. As shown in Fig. 6(F), the average activity rhythm of the entire fly population, although still visible, is less clear than the rhythms of individual flies. Thus, the rhythmicity of a few flies disappears to some degree in the population. This may also explain the neither positive, nor negative Nanda–Hamner results. The clocks of the flies that remain rhythmic for a few days will respond approximately every 24 h to the light with diapause induction. However, the majority of the flies will barely respond. Because the measured diapause response is a mixture of the responses of the individual flies, results may be obtained that are neither positive, nor negative. The T‐cycle experiments are very different because they assess diapause induction not under free‐running conditions, as do the Nanda–Hamner experiments but under entrained conditions. As noted above, a highly damped oscillator or an hour‐glass clock will be reset every day at the onset of the night. Even weak circadian oscillators are highly responsive to LD cycles (Vitaterna et al., 2006; van der Leest et al., 2009). This may explain why even weak circadian clocks are reset by the light every day and thus measure night length. In summary, the present results are consistent with the hypothesis that the same clock governs diapause and rhythmic activity in D. ezoana.

Does this also hold true for species that exhibit ‘positive’ Nanda–Hamner responses and are thus expected to possess strong self‐sustained oscillator‐based photoperiodic clocks (Saunders & Lewis, 1987)? Diapausing insect species, such as the parasitic wasp Nasonia vitripenis, the flesh fly Sarcophaga argyrostoma and the spider mite Tetranychus urticae, are known to exhibit robust positive Nanda–Hamner responses (Saunders, 1973, 1974; Vaz Nunes & Veerman, 1986). Nasonia vitripennis additionally displays robust circadian activity rhythms (Bertossa et al., 2013), highlighting the assumption that the same robust circadian oscillator may control activity and measure photoperiod. If true, these insects should also show a prominent change in critical night length under the newly designed T‐cycle experiments described in the present study. To test this, the diapause incidence data are extracted from the published studies involving these species (Saunders, 1973, 1974; Vaz Nunes & Veerman, 1986) and re‐plotted as a function of day length (Fig. 7A, C, E) and night length (Figure 7B, D, F) for each T‐cycle separately, as carried out for D. ezoana in Fig. 4. In all three species, the diapause to nondiapause transition occurs at different day lengths under different T‐cycles (Figure 7A, C, E) but only in S. argyrostoma does it also occur at different night lengths (Fig. 7D). In N. vitripennis, the transition shows a tendency to occur at the same night length, irrespective of the T‐cycle period (Fig. 7B) and, in T. urticae, the curves at the four T‐cycles largely overlap (Fig. 7F). This suggests that at least T. urticae and perhaps also N. vitripennis may use a strongly damped circadian oscillator to measure night length despite the positive Nanda–Hamner responses. If true, this also implies that this oscillator does not coincide with the one controlling activity rhythms. It is nevertheless too early to draw any definitive conclusions because the extraction of the diapause incidence data from the published studies may still be imprecise. New studies with the three species in the newly designed T‐cycle experiments may help to solve the issue.

Figure 7 Open in figure viewer PowerPoint Nasonia vitripenis, Sarcophaga argyrostoma and Tetranychus urticae. The data reported were extracted from old Nanda–Hamner studies in the parasitic wasp N. vitripenis (Saunders, 1974 S. argyrostoma (Saunders, 1973 T. urticae (Vaz Nunes & Veerman, 1986 Diapause incidence in response to different day and night lengths under different T‐cycles inand. The data reported were extracted from old Nanda–Hamner studies in the parasitic wasp(Saunders,), the flesh fly(Saunders,) and the spider mite(Vaz Nunes & Veerman,). These studies conducted Nanda–Hamner experiments using a range of photoperiod conditions, thus allowing us to extract diapause incidence for more than one combination of photoperiods and night lengths for each LD periodicity. The extracted diapause incidence data were then plotted as a function of day length (A, C, E) and night length (B, D, F). Distinct line‐types and symbols in each plot represent different T‐cycle periods.

At present, it is not clear whether a positive Nanda–Hamner response is a proof for the involvement of a sustained circadian oscillator in photoperiodic time measurement or whether it is exclusively the result of a noncausal circadian influence on the diapause physiology, as suggested by Pittendrigh & Minis (1964) and Pittendrigh (1972). The T‐cycle experiments will help to answer this important question. Combined with activity rhythm recordings, these experiments will also help to clarify whether the clock controlling the behavioural rhythms is identical to the clock measuring night length for diapause induction.

There is a remarkable debate in the literature about this issue. Saunders et al. (1989) show that D. melanogaster flies remain capable of measuring day length in the absence of the canonical circadian clock gene period that is essential for normal activity rhythms, which suggests that the circadian clock is not involved in measuring day length. However, more recent studies indicate that the circadian clock gene timeless determines general diapause incidence (Tauber et al., 2007). The timeless gene is also shown to be involved in the photoperiodic response of the fly Chymomyza costata (Pavelka et al., 2003) and the period gene in the cricket Modicogryllus siamensis (Sakamoto et al., 2009). Furthermore, the clock genes period, cycle, mammalian type cryptochrome and clock are important for diapause in the bean bug Riptortus pedestris (Ikeno et al., 2010, 2011, 2013). Perhaps the most compelling evidence that not only the circadian clock genes, but also the circadian clock neurones driving activity rhythms are essential for photoperiodism comes from ablation experiments in the blow fly Protophormia terraenovae (Shiga & Numata, 2009). Protophormia terraenovae shows robust circadian rhythms and even the anatomical connections from certain circadian clock neurones to the diapause inducing neurosecretory cells in the brain are reported (Shiga & Numata, 2000; Hamanaka et al., 2005). So far, there are more studies in favour of the involvement of the same robust oscillator in circadian activity control and night length measurement than there are against it. The pitcher‐plant mosquito Wyeomyia smithii is perhaps the best example arguing against a role of the same oscillator in both processes. During the summer, the larvae of W. smithii live as top‐level predators in the water that is contained by the purple pitcher plant Sarracenia purpurea. In response to short‐day conditions, the third‐ or fourth‐instar W. smithii larvae enter diapause (Bradshaw & Lounibos, 1972). Formal, classical experiments show that a weak dampening oscillator participates in photoperiodic induction of diapause in W. smithii (Wegis et al., 1997; Bradshaw et al., 1998) and also that the influence of this oscillator decreases in populations living at high latitudes (Bradshaw & Holzapfel, 2001). Bradshaw & Holzapfel (2001) assume that the circadian clock of W. smithii is robust and argue that it would appear to be maladaptive to couple the robust circadian clock to the highly variable and rapidly evolving photoperiodic calendar. Instead, they propose that photoperiodic timing is a process separate from the circadian clock and capable of independent evolution without disrupting the temporal organization of daily events (Emerson et al., 2009). This hypothesis receives support from findings obtained in Drosophila littoralis flies, which show no apparent relationship between critical photoperiod and circadian gating of adult emergence over a latitudinal cline (Lankinen, 1986) and in which laboratory selection results in independent counter‐current ‘evolutions’ of critical photoperiod and circadian controlled adult emergence (Lankinen & Forsman, 2006). However, does the circadian clock necessarily determine the critical photoperiod or the critical night length? Perhaps the circadian clock only provides the necessary time reference to determine day or night length and it is environmental factors such as temperature that set the final critical photoperiod. Furthermore, is it true that all species with robust photoperiodic diapause also have robust circadian clocks? The circadian activity of W. smithii and T. urticae has not yet been investigated and several northern fly species with strong photoperiodism, such as D. ezoana (present study), D. littoralis (Riihimaa, 1996; Lankinen & Forsman, 2006) and Drosophila montana (Kauranen et al., 2012, 2016), have extremely weak circadian rhythms. Thus, the assumption that all species with robust diapause have robust circadian clocks may still be wrong, leaving the possibility open that the same weak circadian clock is involved in both processes.