hormone nuclear receptor (EcR) exhibits circadian cycling in certain tissues, but not others, during development in Rhodnius prolixus (Hemiptera)

Abstract

The insect moulting hormones, viz. the ecdysteroids, regulate gene expression during development by binding to an intracellular protein, the ecdysteroid receptor (EcR). In the insect Rhodnius prolixus, circulating levels of ecdysteroids exhibit a robust circadian rhythm. This paper demonstrates associated circadian rhythms in the abundance and distribution of EcR in several major target tissues of ecdysteroids, but not in others. Quantitative analysis of immunofluorescence images obtained by confocal laser-scanning microscopy following the use of anti-EcR has revealed a marked daily rhythm in the nuclear abundance of EcR in cells of the abdominal epidermis, brain, fat body, oenocytes and rectal epithelium of Rhodnius. This EcR rhythm is synchronous with the rhythm of circulating hormone levels. It free-runs in continuous darkness for several cycles, showing that EcR nuclear abundance is under circadian control. Circadian control of a nuclear receptor has not been shown previously in any animal. We infer that the above cell types detect and respond to the temporal signals in the rhythmic ecdysteroid titre. In several cell types, the rhythm in cytoplasmic EcR peaks several hours prior to the EcR peak in the nucleus each day, thereby implying a daily migration of EcR from the cytoplasm to the nucleus. This finding shows that EcR is not a constitutive nuclear receptor, as has previously been assumed. In the brain, rhythmic nuclear EcR has been found in peptidergic neurosecretory cells, indicating a potential pathway for feedback regulation of the neuroendocrine system by ecdysteroids, and also in regions containing circadian clock neurons, suggesting that the circadian timing system in the brain is also sensitive to rhythmic ecdysteroid signals.

Ecdysteroid receptor (EcR) is associated with microtubules and with mitochondria in the cytoplasm of prothoracic gland cells of Rhodnius prolixus (Hemiptera)

“…The data from the Western blot studies above, IHC findings using various antibodies and fixation methods, together with previously published findings (Vafopoulou et al, 2005;Vafopoulou and Steel, 2006), collectively indicate that it is extremely probable that the antibodies recognize the native EcR of Rhodnius. For the experiments described below, the 9B9 antibody was used because it produced consistently intense immunofluorescence in both nucleus and cytoplasm.…”

“…EcR has been classically regarded as residing exclusively in the nucleus (reviewed by Riddiford et al, 2003), despite occasional reports of its presence in the cytoplasm (Lammerding-Köppel et al, 1998;Vafopoulou and Steel, 2006;Gwó$d$ et al, 2007). There are many examples of rapid actions of Ec that cannot be explained by genomic actions and indicate a number of potential non-genomic actions of Ec (reviewed by Schlattner et al, 2006).…”

Metamorphosis of a clock: Remodeling of the circadian timing system in the brain of Rhodnius prolixus (Hemiptera) during larval‐adult development

“…PTTH neurons are closely associated with axons of the circadian system in the brain (Vafopoulou et al, 2007). Circadian rhythms in release of PTTH (Vafopoulou and Steel, 1996a,b) and other hormones act as ''messengers of time'' to a diversity of target tissues (reviewed by Steel and Vafopoulou, 2006); they may function to entrain peripheral oscillators such as the prothoracic glands (Pelc and Steel, 1997;Vafopoulou and Steel, 1999) or to drive cycling of hormone receptors in target tissues Vafopoulou and Steel, 2006). These functions would orchestrate internal temporal order both within target cells of the hormone(s) and also within the organism as a whole.…”

Circadian rhythms in insect disease vectors

“…The rhythmic release of PTTH not only stimulates, but also entrains the release of ecdysteroids by PG, which also possess light-entrainable autonomous oscillators ( Steel & Vafopoulou 2006 ). The resulting rhythm in haemolymph ecdysteroid titre is read by the target tissue’s ecdysteroid nuclear receptors, which leads to internal tissue synchronisation and also triggers the rhythmic expression of downstream genes on different tissues such as fat body, rectal epithelium, oenocytes, neurosecretory cells and even the lateral clock neurons ( Vafopoulou & Steel 2006 ).…”