Clock genes period and timeless are rhythmically expressed in brains of newly hatched, photosensitive larvae of the fly, Sarcophaga crassipalpis Vladimı ´r Kos ˇta ´l a,b, *, Radka Za ´ vodska ´ a,c , David Denlinger d a Biology Centre of the Academy of Sciences of the Czech Republic, Institute of Entomology, Branisˇovska ´ 31, 370 05 C ˇ eske ´ Budeˇjovice, Czech Republic b Faculty of Science, University of South Bohemia, C ˇ eske ´ Budeˇjovice, Czech Republic c Pedagogical Faculty, University of South Bohemia, C ˇ eske ´ Budeˇjovice, Czech Republic d Department of Entomology, Ohio State University, Columbus, OH, USA 1. Introduction Period and Timeless proteins are the main players in the currently accepted molecular model of the central biological clock in Drosophila melanogaster. Two interconnecting transcription/ translation feedback loops, which spontaneously cycle with a period of approximately 24 h under constant darkness, are considered to constitute the core of the clock mechanism. In the core loop, Period and Timeless negatively regulate expression of their own genes by inhibiting activity of the transcription activator complex formed by the Clock and Cycle heterodimer. Binding of Clock/Cycle to enhancer E-box motifs activates transcription of per and tim and also of vri and pdp1, genes which form the second loop. The second loop is thought to confer stability and robustness to the core loop (for more detailed information see Hall, 2003; Hardin, 2005). While the roles of the clock proteins Per and Tim are relatively well understood in relation to the circadian clock (the central circadian oscillator driving daily rhythms in behavior), their role in photoperiodism (response to seasonal changes in day length) is less clear (Saunders et al., 2004; Bradshaw and Holzapfel, 2007). Photoperiodic sensitivity allows insects to monitor seasonal changes in day length (photoperiod) and thus properly time their development, reproduction, alteration of morphs, migration and diapause (Tauber et al., 1986; Danks, 1987; Kos ˇta ´ l, 2006). Formal analysis of the photoperiodic clock and counter mechanism suggests that unknown circadian oscillators participate in photo- periodic (seasonal) time measurement (for discussion see Saun- ders, 2002; Goldman et al., 2004). A logical question thus arises whether the well-known circadian elements of the biological clock are directly or indirectly involved in the photoperiodic time measurement system. This intuitive idea was directly tested for the first time when Saunders et al. (1989) showed that D. melanogaster females possessing two overlapping deletions of per were capable of discriminating between long and short days. Therefore, it was concluded that the per gene is not causally involved in photo- periodic induction of diapause. Despite such clearly negative results, two lines of reasoning still keep the original idea alive: (1) Journal of Insect Physiology 55 (2009) 408–414 ARTICLE INFO Article history: Received 7 October 2008 Received in revised form 16 December 2008 Accepted 16 December 2008 Keywords: Circadian clock Oscillator Photoperiodism Seasonal timer Diapause ABSTRACT While roles of the clock genes period (per) and timeless (tim) are relatively well understood in relation to circadian clocks, their potential roles in insect photoperiodism remain enigmatic. In this study, the expression of per and tim genes under two contrasting photoperiods is described in the central nervous system of photoperiodically sensitive, newly hatched first instar larvae of the flesh fly, Sarcophaga crassipalpis. Using qPCR, diel oscillations were observed in the mRNA levels of both genes under long-day (15 h light:9 h dark, promotes direct development) and short-day conditions (11 h light:13 h dark, induces pupal diapause). Peak per and tim mRNA oscillations were closely associated with the light/dark transition. The conspicuous difference between the two photoperiodic conditions was that the sharp increase in per and tim mRNA abundance occurred during the light phase under long days but during the dark phase under short days. The diel oscillations were, at least in part, driven by an endogenous component, as demonstrated by transferring larvae to continuous darkness. The cells displaying Tim- and Per-like immunoreactivities (Tim- and Per-LIRs) were localized using anti-Drosophila-Per and anti- Chymomyza-Tim antibodies. Per-LIR and Tim-LIR co-localized in three groups of cells in each brain hemisphere. Two other groups, one in the brain hemispheres and the other in the fused ventral nerve ganglion, expressed only the Per-LIR. ß 2009 Elsevier Ltd. All rights reserved. * Corresponding author at: Biology Centre of the Academy of Sciences of the Czech Republic, Institute of Entomology, Branis ˇovska ´ 31, 370 05 C ˇ eske ´ Bude ˇjovice, Czech Republic. Tel.: +420 387 775 324; fax: +420 385 310 354. E-mail address: kostal@entu.cas.cz (V. Kos ˇta ´ l). Contents lists available at ScienceDirect Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys 0022-1910/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2008.12.011