ORIGINAL ARTICLE Pigment-dispersing factor affects circadian molecular oscillations in the cricket Gryllus bimaculatus Ehab HASSANEEN 1,2 , Alaa El-Din SALLAM 2 , Ahmad ABO-GHALIA 2 , Yoshiyuki MORIYAMA 1 and Kenji TOMIOKA 1 1 Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan; and 2 Zoology Department, Faculty of Science, Suez Canal University, Ismailia, Egypt Abstract Pigment-dispersing factor (PDF) is an important neurotransmitter in insect circadian systems. In the cricket Gryllus bimaculatus, it affects nocturnal activity, the free-running period and photic entrainment. In this study, to investigate whether these effects of PDF occur through a circadian molecular machinery, we measured mRNA levels of clock genes period (per) and timeless (tim) in crickets with pdf expression knocked-down by pdf RNAi. The pdf RNAi decreased per and tim mRNA levels during the night to reduce the amplitude of their oscillation. The phase of the rhythm advanced by about 4 h in terms of trough and/or peak phases. On the other hand, pdf mRNA levels were little affected by per and tim RNAi treatment. These results suggest that PDF affects the circadian rhythm at least in part through the circadian molecular oscillation while the circadian clock has little effect on the pdf expression. Key words: circadian clock, clock gene, PDF, RNAi. INTRODUCTION Circadian rhythms are biological oscillations recurring with a periodicity of about 24 h in a variety of physi- ological and behavioral phenomena from bacteria to humans (Dunlap et al. 2004). The rhythm is governed by an endogenous clock consisting of interacting tran- scriptional and translational molecular feedback loops (Dunlap 1999; Tomioka & Matsumoto 2010). In the cricket Gryllus bimaculatus De Geer (Orthoptera: Gryl- lidae), the major player of the clock is period (per) like in Drosophila melanogaster Meigen (Moriyama et al. 2008, 2009). Its mRNA levels show a clear circadian rhythm with a peak at early night together with timeless (tim), and knocking-down of its mRNA levels by RNAi leads to a loss of circadian locomotor rhythms (Moriyama et al. 2008, 2009). Like in some other insects (Renn et al. 1999; Lee et al. 2009), pigment dispersing factor (PDF) is an important neurotransmitter mediating the clock infor- mation to regulate circadian rhythms in the visual system and in the locomotor activity in the cricket (Saifullah & Tomioka 2003; Hassaneen et al. 2011). PDF is a octadeca neuropeptide expressed in three groups of neurons in the optic lobe located in the proximal medulla, in the dorsal and ventral region of the border between lamina and medulla neuropils (Okamoto et al. 2001; Abdelsalam et al. 2008). PDF shifts the phase of locomotor rhythms in a phase- dependent manner (Singaravel et al. 2003) and enhances the photoresponsiveness of the visual inter- neurons in the optic lobe (Saifullah & Tomioka 2003). With RNAi techniques, we have recently shown that knocking-down of pdf mRNA levels suppresses noctur- nal activity, shortens the free-running period in con- stant darkness, and enhances the photic entrainability to shifted light–dark cycles (LD) (Hassaneen et al. 2011). As to the enhancement of the photic entrainment in pdf RNAi crickets, there are two possible explanations. PDF may regulate the photic entrainment by affecting the responsiveness of visual interneurons and/or by regulating the property of the circadian clock. In the Correspondence: Kenji Tomioka, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-853, Japan. Email: tomioka@cc.okayama-u.ac.jp Received 20 November 2010; accepted 19 December 2010. Entomological Science (2011) 14, 278–282 doi:10.1111/j.1479-8298.2011.00445.x © 2011 The Entomological Society of Japan