The brain of larval Rhodnius prolixus releases neurohormones with a circadian rhythm, indicating that a clock system exists in the larval brain. Larvae also possess a circadian locomotor rhythm. The present paper is a detailed analysis of the distribution and axonal projections of circadian clock cells in the brain of the fifth larval instar. Clock cells are identified as neurons that exhibit circadian cycling of both PER and TIM proteins. A group of eight lateral clock neurons (LNs) in the proximal optic lobe also contain pigment-dispersing factor (PDF) throughout their axons, enabling their detailed projections to be traced. LNs project to the accessory medulla and thence laterally toward the compound eye and medially into a massive area of arborizations in the anterior protocerebrum. Fine branches radiate from this area to most of the protocerebrum. A second group of clock cells (dorsal neurons [DNs]), situated in the posterior dorsal protocerebrum, are devoid of PDF. The DNs receive two fine axons from the LNs, indicating that clock cells throughout the brain are integrated into a timing network. Two axons of the LNs cross the midline, presumably coordinating the clock networks of left and right sides. The neuroarchitecture of this timing system is much more elaborate than any previously described for a larval insect and is very similar to those described in adult insects. This is the first report that an insect timing system regulates rhythmicity in both the endocrine system and behavior, implying extensive functional parallels with the mammalian suprachiasmatic nucleus. J. Comp. Neurol. 518:1264 -1282, 2010. INDEXING TERMS: insect; clock; lateral neurons; PDF; neuroarchitecture; timing network; PERIOD; TIMELESS Circadian timing systems have been found in all organisms that have been studied, from bacteria to humans. They enable organisms to anticipate the arrival of favorable times of day (or seasons of the year) for execution of diverse rhythmic activities ranging from gene expression to hormone secretion, growth, reproduction, and behaviours. These circadian rhythms are driven by endogenous biological clocks centered primarily in groups of nerve cells. These cells generate rhythms with a periodicity of about 24 hours that become synchronized (entrained) to the precisely 24-hour external world by time signals (Zeitgebers) arising principally from components of the light/ dark cycle, such as dusk and dawn.The molecular machinery with which cells generate circadian rhythmicity was first elucidated in the fruit fly, Drosophila melanogaster. Several genes have been found that are central to the generation of circadian oscillations. These oscillations fundamentally comprise feedback loops between transcription of the genes and their protein products (reviewed by Hall, 2005;Taghert and Lin, 2005) and summarized by Nitabach and Taghert (2008) and Dubruille and Emery (2008). Rhythmic transcription of the canonical clock genes in Drosophila, period (per), and timeless (tim) leads to rhythmic formation of PE...

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The intermoult cycle of Oniscus has been divided into 15 stages, recognizable by changes in the appearance of the anterior sternites. The stages are related to concurrent microscopical changes in the integument. The normal stimulus which initiates premoult is followed by a period when no tissue changes are discernible. Apolysis occurs about 7 days after premoult initiation. Thus, use of apolysis to recognize premoult (as in many crustaceans) would result in 44% of premoult passing undetected. Storage of calcium in sternal deposits and the general cuticle of the anterior region during the latter half of premoult is associated with an arrest of cuticle secretion in this region; at the time of posterior ecdysis (Ep) the anterior region has no new exocuticle. The anterior region is thus functionally similar to the decapod gastrolith. Stored calcium is resorbed and the anterior exocuticle secreted during the 24 h following Ep, enabling ecdysis of the anterior region to follow. The accumulation of calcium in the anterior cuticle while the posterior cuticle is undergoing resorption and the secretion of exocuticle at different times in the two regions both imply that the integuments of anterior and posterior regions differ in their responses to the moulting hormone.

The calcium content of pieces of integument of standardized size from various regions of the body was measured at all 15 stages of the intermoult cycle. Calcium resorbed from the posterior integument during premoult is stored mainly in the sternal calcium deposits of the anterior region, which contain 20% of total body calcium in late premoult. Earlier arguments that the deposits are not a store of calcium are refuted. These deposits are similar to gastroliths in both structure and function. After posterior ecdysis, calcium in the deposits is resorbed rapidly and employed in calcification of the new posterior exocuticle. Calcium is then resorbed from the general anterior integument within 24 h and may accumulate transiently in the haemolymph. During this period resorption of integumentary calcium and calcification proceed simultaneously in the anterior and posterior regions of the animal. Ecdysis of the anterior region then occurs, and calcium from the haemolymph is employed in calcification of the new anterior exocuticle. Forty-eight percent of intermoult calcium content is conserved. However, both exuviae are eaten and it is argued that further calcium is conserved by resorption from the exuviae within the gut. It is suggested that this calcium, together with dietary calcium, is employed in calcification of the endocuticle after ecdysis. It is proposed that comparable events occur in all crustaceans that conserve calcium, but are rendered especially dramatic in isopods, owing to the unusual biphasic pattern of cuticle secretion.

This paper reports the localization in the Rhodnius prolixus brain of neurons producing the key neuropeptide that regulates insect development, prothoracicotropic hormone (PTTH) and describes intimate associations of the PTTH neurons with the brain circadian timekeeping system. Immunohistochemistry and confocal laser scanning microscopy revealed that the PTTH-positive neurons in larvae are located in a single group in the lateral protocerebrum. Their number increases from two in the last larval instar to five during larval-adult development. In adults, there are two distinct groups of these neurons composed of two cells each. A daily rhythm in content of PTTH-positive material occurs in both the somata and the axons in both larval and adult stages. These rhythms correlate with previous evidence of a circadian rhythm of PTTH release from brains in vitro. The key circadian clock cells of Rhodnius are eight neurons, which co-express pigment-dispersing factor (PDF) and the canonical clock proteins PER and TIM; PDF fills the axons. Equivalent cells control behavioral rhythms in other insects. Double labeling revealed intimate associations between axons of larval PTTH neurons and clock neurons, indicating a neuronal pathway from the brain timekeeping system for circadian control of PTTH release. Additional PDF neurons appear in the adult, associated with the second group of PTTH neurons. These findings provide the first direct evidence that neurons of the insect brain timekeeping system control hormone rhythms. The range of functions regulated by this timekeeping system is quite similar to those of the vertebrate suprachiasmatic nucleus, for which the insect system is a valuable model.