2794 Introduction Orcokinins are a family of arthropod neuropeptides that have been identified in decapod crustaceans and, more recently, in two species of insects (Stangier et al., 1992; Bungart et al., 1995; Huybrechts et al., 2003; Pascual et al., 2004; Hofer et al., 2005). All orcokinins are highly conserved between species and, in the investigated crustaceans, occur in multiple isoforms within a given species (Bungart et al., 1995; Yasuda-Kamatani and Yasuda, 2000; Skiebe et al., 2002; Huybrechts et al., 2003; Fu et al., 2005). The insect orcokinins identified to date, the tetradecapeptide Scg-OK1 from the locust Schistocerca gregaria and the dodecapeptide Blatella-OKL from the cockroach Blatella germanica, are closely related to crustacean orcokinins by identical N-terminal NFDEIDRSGF-sequence with crustacean Asn 13 -, Val 13 - and Ala 13 -orcokinins. Immunocytochemistry with an antiserum against Asn 13 - orcokinin revealed widespread distribution of orcokinins in the brain of polyneopteran insects, such as the cockroach Leucophaea maderae and the locust Schistocerca gregaria, but complete lack of immunostaining in the brain of endopterygote insects (Hofer et al., 2005). Labeling of brain interneurons and processes in the neurohemal retrocerebral complex in the cockroach and locust suggests that orcokinin-related peptides function both as hormones and as neuromodulators in these insects, as described for crustaceans. In the present study, we have analyzed the role of orcokinin- related peptides in the circadian system of the cockroach Leucophaea maderae. As in the fruitfly Drosophila melanogaster, neurons with arborizations in the accessory medulla (AMe), a small neuropil at the anterior base of the medulla, constitute the master circadian clock in the brain of the cockroach (Reischig and Stengl, 2003a) (reviewed by Homberg et al., 2003; Helfrich-Förster, 2005). The cockroach AMe consists of a core of dense nodular neuropil, embedded in and surrounded by coarse neuropil (Petri et al., 1995; Reischig and Stengl, 1996; Reischig and Stengl, 2003b). Six groups of somata near the AMe send neurites into the AMe neuropil (Reischig and Stengl, 2003b). Neurons of the distal tract connect the medulla to the AMe (Reischig and Stengl, 1996; Reischig and Stengl, 2003b; Petri et al., 2002) and are candidates for mediating light entrainment of the clock through photoreceptors of the compound eye (Roberts, 1965; The accessory medulla (AMe), a small neuropil in the optic lobe, houses the master circadian clock in the brain of the cockroach Leucophaea maderae and controls circadian rhythms in locomotor activity. Recently, members of the orcokinin family of crustacean neuropeptides were identified in a cockroach and a locust and were shown by immunocytochemistry to be prominently present in the AMe. In the cockroach L. maderae, about 30 neurons in five of six established cell groups of the AMe showed orcokinin immunostaining. By means of tracer injections into one AMe and immunostaining with anti-orcokinin antiserum, we show here that one orcokinin-immunoreactive ventral neuron and three ventromedian neurons directly connect both AMae. To determine a possible circadian function of orcokinin in the cockroach, we injected 150·fmol Asn 13 - orcokinin into the vicinity of the AMe at different circadian times. These experiments resulted in stable phase-dependent phase shifts of circadian locomotor activity of the cockroach. The shape of the resulting phase-response curve closely matched the phase-shifting effects of light pulses, and its amplitude was dependent on the amount of the injected peptide. Together with the anatomical data, the results suggest that orcokinin-related peptides play an important role in light entrainment pathways to the circadian clock via the contralateral compound eye. This study, furthermore, provides the first evidence for a physiological role of an orcokinin-related peptide in insects. Key words: orcokinin, insect brain, circadian rhythms, accessory medulla, phase-response curve, light entrainment, cockroach, Leucophaea maderae. Summary The Journal of Experimental Biology 209, 2794-2803 Published by The Company of Biologists 2006 doi:10.1242/jeb.02307 Evidence for a role of orcokinin-related peptides in the circadian clock controlling locomotor activity of the cockroach Leucophaea maderae Sabine Hofer and Uwe Homberg* Fachbereich Biologie, Tierphysiologie, Philipps Universität Marburg, D-35032 Marburg, Germany *Author for correspondence (e-mail: homberg@staff.uni-marburg.de) Accepted 3 May 2006 THE JOURNAL OF EXPERIMENTAL BIOLOGY