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.

Analysis of cycle Gene Expression in Aedes aegypti Brains by In Situ Hybridization

“…However, since projections are observed from LA towards DA (see Figure 1), it seems a reasonable assumption, concerning to mushroom body, that cyc mRNA is more expressed in the calyx neuropil. This pattern of neuronal circuitry is consistent with that evinced in the cricket Allonemobius allard [39] and seems to be associated with a crucial role in olfactory learning in insects [24].…”

Circadian Rhythms and Period Expression in the Hawaiian Cricket Genus Laupala

“…It has been proposed that cycling of phosphorylation of clock proteins, as opposed to cycling of protein abundance, may be key to maintaining the circadian clock (Zheng and Sehgal 2008). Such a model may be consistent with the circadian clock of crickets, in which circadian clock protein levels are constant throughout the day (Shao et al 2008b; Lupien et al 2003; Shao et al 2006). In addition to the changes in putative phosphorylation sites, two non-conserved amino acid changes between L. cerasina and L. paranigra occurred within the PAS-B domain, a protein interaction domain.…”

“…However, even before the first clock mutant was reported in D. melanogaster , researchers had been investigating daily variation and circadian rhythms in crickets (Nowosielski and Patton 1963; Nowosielski and Patton 1964; Alexander and Meral 1967; McFarlane 1968). The circadian mechanism in crickets appears to differ from that of Drosophila , both in the molecular basis of the endogenous clock as well as the localization of the circadian pacemaker neurons (Sokolove and Loher 1975; Abe et al 1997; Shao et al 2008a; Shao et al 2008b; Abdelsalam et al 2008; Lupien et al 2003; Shao et al 2006; Shiga et al 1999). Understanding both the behavioral variation in circadian rhythms among crickets and mechanisms underlying cricket circadian clocks will provide insight into the evolution and plasticity of circadian rhythm generation, as well as further our understanding of the molecular basis of circadian timing.…”

“…Given the commonly observed daily cycling in cricket song, circadian rhythms of crickets have been a frequent subject of study, from early behavioral and physiological studies (Nowosielski and Patton 1963; Loher 1972; Loher 1974; Sokolove 1975; Sokolove and Loher 1975; Wiedenmann and Loher 1984; Abe et al 1997) to more recent molecular analyses (Hassaneen et al 2011; Shao et al 2008a; Shao et al 2008b; Moriyama et al 2008; Abdelsalam et al 2008; Lupien et al 2003; Shao et al 2006; Danbara et al 2010). Like other organisms, crickets possess an endogenous circadian clock (Loher 1972; Loher 1974; Abe et al 1997); however, there are physiological differences in the clock of crickets relative to Drosophila .…”

“…Like other organisms, crickets possess an endogenous circadian clock (Loher 1972; Loher 1974; Abe et al 1997); however, there are physiological differences in the clock of crickets relative to Drosophila . For example, the daily variation in expression of circadian clock proteins, including PER, CLK, TIM, which occurs in Drosophila (Dunlap 1999; Hardin 2005; Yu and Hardin 2006; Rosato et al 2006), appears to be lacking in crickets (Shao et al 2008a; Shao et al 2008b; Lupien et al 2003; Shao et al 2006). Additionally, CLK is the only one of these proteins which has been observed to translocate into the nucleus in crickets (Shao et al 2008a).…”