Pervasive effects of temperature on the photoperiodic mechanism and the nature of the photoperiodic clock
In northern temperate species with an over-wintering diapause, temperature affects all levels of the photoperiodic response from photoreception to hormonal events triggering the diapause syndrome. Temperature signals include, inter alia, constant temperature, temperature cycles or thermoperiods, low and high temperature pulses, and temperature steps, up or down, all of them in conjunction with a daily photoperiod or in continuous darkness or continuous light. The effects of these temperature treatments on the photoperiodic induction of diapause have been considered in this review. Of particular importance, because they contribute to our understanding of the mechanism of the photoperiodic response itself, are daily thermoperiods in darkness and temperature pulses in conjunction with a light cycle.
Exposure of N. vitripennis and S. argyrostoma to daily thermoperiods in complete darkness (Saunders 1973b, 1984) suggested that light was essential for the photoperiodic response in Sarcophaga but not in Nasonia (Fig. 6) in accordance with the external coincidence model for the former and internal coincidence for the latter (Pittendrigh 1972). This conclusion was underlined in experiments using temperature pulses systematically interrupting the night of diapause inductive light cycles (Figs 7, 8), which again suggested external and internal coincidence for Sarcophaga and Nasonia, respectively. The external coincidence model for S. argyrostoma (Fig. 3) has been tested on a number of occasions (Saunders 2011) and its derivative, the damped circadian oscillator model of the clock-counter mechanism (Lewis & Saunders 1987; Saunders & Lewis 1987a,b) successfully accounts for the major features of photoperiodic time measurement in this species.
The internal coincidence model has been less well formulated than external coincidence and less completely characterized experimentally. Its origins lie in the numerous observations that the insect circadian system is of a multi-oscillator construction (Saunders 2002) with constituent oscillators varying both in period (τ) and their sensitivity to light, temperature and other entraining agents. In N. vitripennis, available evidence from Nanda–Hamner experiments, thermoperiod and low and high temperature pulses strongly indicate internal coincidence with separate, but interacting, “dawn” and “dusk” oscillators. Further elucidation of the photoperiodic mechanism in this species, however, must include more fundamental molecular and physiological investigations. These approaches are dealt with in the next section.
Evolution of insect photoperiodic clock mechanisms
Experiments involving daily thermoperiods in DD (Saunders 1973b, 1984) and low and high temperature pulses in light–dark cycles (Saunders 1984, 2002; this review) have indicated that the flesh fly S. argyrostoma and its parasitic wasp N. vitripennis measure seasonal changes in photoperiod with substantially different mechanisms: external coincidence in the former, but internal coincidence in the latter. In S. argyrostoma the external coincidence model has been extensively tested; the photoinducible phase (φi) has been located in the latter half of the night (at CT 21.5) by night interruptions and the so-called Lees experiment (see Lees 1973; Saunders 1979, 2012). The photoinducible phase has also been isolated from the effects of the “main” photophase in T experiments (Saunders 1979). On the other hand, in N. vitripennis, photoperiodic regulation of the diapause/nondiapause switch in development has been shown to operate in the absence of light, and temperature- and light-pulse experiments have indicated that: (i) more than one oscillation is involved; and (ii) a specific photoinducible phase is not necessary.
Further differences between external and internal coincidence, as shown in Sarcophaga spp. and N. vitripennis, have been revealed in spectral sensitivity and genomic studies. In spectral sensitivity studies using the flesh fly S. similis, Goto and Numata (2009) showed maximum sensitivity to UV and blue light (395–470 nm) when light fell early in the night, but a broader peak from 395 nm up through the blue and green (583 nm) into the red end of the spectrum for light falling in the second half of the night. This indicated that two photoreceptors were involved, one absorbing maximally in the blue and used for entrainment and the other, late in the night, absorbing at longer wavelengths and probably associated with diapause regulation at the photoinducible phase. In contrast, spectral sensitivity studies with N. vitripennis (Saunders 1975) showed that light falling both early and late in the night engendered identical action spectra with a broad peak at a maximum at 554–586 nm extending into much longer wavelengths up to 617 nm or more. This suggested that, although several photoreceptive molecules were involved, light had only a single role: that of entrainment. These data therefore supported the conclusion that Sarcophaga spp. and N. vitripennis measure photoperiod by substantially different mechanisms, seen here as external and internal coincidence, respectively.
The genome of a Sarcophaga sp. has yet to be sequenced but it may resemble that of Drosophila melanogaster Meigen, which includes timeless1 (tim1) and cryptochrome1 (cry1), but lacks cryptochrome2 (cry2) (Zhan et al. 2011). In Drosophila, the protein CRY1 is a dedicated blue-light photoreceptor responsible for entrainment (Emery et al. 1998; Stanewsky et al. 1998). In N. vitripennis, however, the genome lacks cry1 (and tim1) but contains the “mammalian-type” cry2 that encodes CRY2 acting not as a photoreceptor but as a transcriptional repressor in the auto-regulatory feedback loop itself (Schurko et al. 2010). In a number of other insects (Lepidoptera, mosquitoes and an aphid, for example) cry1, cry2 and tim1 are all represented (Zhan et al. 2011). Following earlier authors (Yuan et al. 2007; Sandrelli et al. 2008; Merlin & Reppert 2010), the form of the photoperiodic clock in these last species may be regarded as “ancestral”, whereas those in Sarcophaga (probably lacking cry2) and Nasonia (lacking tim1 and cry1) are probably “derived”.
Photoperiodically regulated diapause may occur at any stage of metamorphosis (embryo, larva, pupa or adult) but usually at a species-specific stage of development (Tauber et al. 1986). On the whole, there appears to be little phylogenetic relationship concerning the stage at which diapause may occur. Among the Culicidae, for example, different species may enter diapause as embryos, larvae or adults. These observations suggest that photoperiodic regulation and the type of diapause have evolved on numerous occasions as insects extended their distributions into higher latitudes or into areas with climates inimical to development and reproduction. However, many Sarcophaga spp. over-winter in a pupal diapause, most northern Drosophila spp. undergo an adult reproductive diapause and many Aëdes mosquitoes become dormant as embryos, suggesting some phylogenetic relationship in photoperiodic responses occurring between closely related species.
Given the increasing evidence that photoperiodic time measurement (even in some of those species presenting hourglass-like properties) is a function of the circadian system (Saunders 2002, 2010; Goto 2013) it is probable that insects have repeatedly “adopted” the pre-existing circadian system as the “clockwork” for photoperiodic time measurement. In larvae of Sarcophaga spp. (external coincidence) for example, the blue-light photoreceptor responsible for entrainment may be CRY1 and the longer wavelength photoreceptor(s) functioning at the photoinducible phase might be opsin-based (and probably brain-centered). In adults of N. vitripennis (internal coincidence), on the other hand (where CRY1 is absent) the photoperiodic photoreceptors in both the early and late subjective night might be opsin based and perhaps located in the compound eye. Recent data for the cricket Modicogryllus siamensis (Tamaki et al. 2013) also locate opsin-based photoreceptors in the compound eyes and gene silencing techniques (RNAi) for the genes encoding UV-, blue- and long-wavelength opsins result in a partial (although substantial) alteration of the crickets’ short-night responses (rapid nymphal development) to long-night responses (protracted nymphal development). Whether opsin-based photoreceptors are operating at the photoinducible phase in this species or merely for oscillator entrainment, however, is not clear.
The photoperiodic mechanisms in N. vitripennis and S. argyrostoma are clearly quite distinct and probably also derived from a more ancestral type of photoperiodic mechanism containing cry1 and cry2 as in various Lepidoptera, Homoptera, Heteroptera and Diptera–Nematocera, cited above. If that is the case these “ancestral” photoperiodic clocks might show characteristics of both external and internal coincidence. The latter, for instance, is indicated by the ability of several species of Lepidoptera to show diapause regulation by daily thermoperiod in the complete absence of light (Chippendale et al. 1976; Dumortier & Brunnarius 1977). Future progress in determining the nature of the photoperiodic mechanism in insects should involve a broad comparative approach including genomic and molecular studies on the one hand and “formal” and physiological studies on the other.