Co-localization and unique distributions of two clock proteins CYCLE and CLOCK in the cephalic ganglia of the ground cricket, Allonemobius allardi

Abstract

CYCLE (CYC) and CLOCK (CLK) are transcriptional activators of the circadian clock genes, period (per) and timeless (tim), binding at E-boxes of their upstream regulatory region in Drosophila. CYC-like and CLK-like immunohistochemical reactivities (CYC-ir and CLK-ir) were investigated in the ground cricket, Allonemobius allardi, in which immunohistochemical reactivities for three circadian clock proteins (PERIOD, Doubletime, and Cryptochrome), two neuropeptides (crustacean cardioactive peptide and diapause hormone), and arylalkylamine-N-acetyltransferase had previously been mapped in the brain-subesophageal ganglion (SOG) complex. CYC-ir and CLK-ir occurred predominantly in the cytoplasm of the neurons distributed mainly in the central brain, SOG, and corpora cardiaca. Double-labeling experiments showed that CYC-ir and CLK-ir were co-localized only in the mandibular and maxillary neuromeres of the SOG. The neuronal processes in the dorsolateral region of the protocerebrum partially shared the immunoreactivities, whereas most of the other immunoreactivities were unique. The optic lobe showed reactivity to anti-CYC at small proximal frontodorsal cells and to anti-CLK at small proximal frontoventral cells. The frontal ganglion exhibited CYC-ir in the cell bodies that lacked CLK-ir. No difference in their number, distribution, or staining intensity was found between sampling under light:dark regimes of 16:8 and 12:12. The levels of both CYC-ir and CLK-ir showed no oscillation throughout a 24-h period. The co-localization pattern suggests that the midline cells of the SOG share most of the circadian-related immunoreactivities, thus constituting the heart of the circadian clock in A. allardi.