Circadian rhythms are endogenous oscillations with a period of approximately 24 h (Saunders et al., 2002). These rhythms are generated by molecular oscillatory mechanisms consisting of transcriptional-translational feedback loops (Tataroglu & Emery, 2015). In the fruit fly Drosophila melanogaster, CLOCK (CLK)/CYCLE (CYC) heterodimers activate the transcription of period (per) and timeless (tim) from late day to early night, thereby increasing the protein products PER and TIM in the cytoplasm during the night (Hardin, 2009, Tataroglu and Emery, 2015). Late at night, they form a heterodimer, PER/TIM, which enters the nucleus and suppresses the transcriptional activity of CLK/CYC, their own transcription factor (Lee et al., 1998). This negative feedback reduces PER and TIM levels and releases CLK/CYC from inhibition by PER-TIM, leading to the next cycle (Allada et al., 1998; Rutila et al., 1998). However, other insects possess oscillatory mechanisms that differ from those of Drosophila (Tomioka and Matsumoto, 2019). In the silk moth Antheraea pernyi, the rhythmic expression of per is not controlled by the feedback of its product protein but by its antisense (Sauman and Reppert, 1996). In the honeybee Apis mellifera, tim is absent from the genome, and a light-insensitive, vertebrate-type cryptochrome (cry2) functions with per in transcriptional-translational feedback loops (Rubin et al., 2006, Yuan et al., 2007).

The most important role of circadian rhythms comprises synchronization with daily environmental cycles and the maintenance of an exact 24 h cycle by the synchronization, through which organisms can set the appropriate timing of daily behavioral and physiological events (Tomioka and Matsumoto, 2019). The main zeitgeber for the entrainment of circadian rhythms is the light–dark cycle. In D. melanogaster, blue light-sensitive CRY1, expressed in central clock neurons, functions as a circadian photoreceptor for photic entrainment (Emery et al., 2000, Stanewsky et al., 1998, Yoshii et al., 2008). Light-activated CRY1 resets molecular oscillations through the degradation of TIM (Ceriani et al., 1999, Emery et al., 1998, Lee et al., 1996). Degradation of TIM induces a delay in the clock during its increasing phase and an advance shift of the clock during its declining phase (Lee et al., 1996). A similar role for CRY1 has been suggested in the monarch butterfly, Danaus plexippus (Yuan et al., 2007, Zhu et al., 2008). Drosophila also uses rhodopsin for photic entrainment (Hanai et al., 2008, Hanai and Ishida, 2009, Saint-Charles et al., 2016).

However, some insects use photoreceptive systems other than CRY1 for photic entrainment (Helfrich-Förster, 2020). Many crickets and cockroaches use only compound eyes for photic entrainment because optic nerve severance results in free running, even under light–dark cycles (Nishiitsutsuji-Uwo and Pittendrigh, 1968, Tomioka and Chiba, 1984). In the cricket Gryllus bimaculatus, Opsin-long wavelength (OpLW) acts as the circadian photoreceptor (Komada et al., 2015). In bees and ants lacking CRY1, circadian photoreceptors are still unknown; however, the candidate might be a vertebrate opsin-3-like photoreceptor, pteropsin, which is expressed in several cerebral neurons located close to putative clock neurons (Fuchikawa et al., 2017, Velarde et al., 2005). These variations in circadian photoreceptors and oscillatory mechanisms of the clock, as mentioned above, suggest that insect clocks and their entrainment mechanisms are diverse, raising the question of what the prototype of insect clocks and its photic entrainment are. The solution to this question will lead to an understanding of insect clock diversity.

Firebrats Thermobia domestica forms a suitable template insect for this investigation because it is one of the most primitive. The experimental results thus far suggest that T. domestica has a clock with an oscillatory mechanism similar to that of crickets; tim and cyc are rhythmically expressed to peak at late day to early night and during the daytime, respectively (Kamae and Tomioka, 2012). The expression of tim is thought to be regulated by CLK/CYC and that of cyc by ecdysone-induced gene 75 (E75) and hormone receptor gene 3 (HR3) (Kamae et al., 2010, Kamae et al., 2014).

This study aimed to identify the circadian photoreceptors in firebrats T. domestica. The cry and opsin genes were focused on as candidate circadian photoreceptors. Sequences were searched using RNA-seq data (Misof et al., 2014) and identified the sequences of one cry gene and three opsin genes. Their possible roles as circadian photoreceptors were investigated using RNAi and behavioral analyses. The obtained results suggest that opLW2 serves as a circadian photoreceptor, involved in the photic entrainment of the circadian locomotor rhythm in firebrats T. domestica.