Neuropeptide- and serotonin- cells in the brain of Rhodnius prolixus (Hemiptera) associated with the circadian clock

The neuronal pathways of the circadian clock in the brain of R. prolixus have been described in detail previously, but there is no information concerning the cells or their pathways which relay either inputs to the clock (e.g. for light entrainment), or outputs from it to driven rhythms. Here, we employ antisera to three neuropeptides (type A allatostatin-7, crustacean cardioactive peptide and FMRFamide), and serotonin in confocal laser scanning immunohistochemistry to analyze the distribution of cell bodies and their projections in relation to the principle circadian clock cells (lateral cells, LNs) for all four neuron types. LNs are revealed following labelling with anti- pigment dispersing factor in double labelled preparations. Regions of potential communication between ramifications of the LNs and each of the four other neuron types is described (identified by close superposition of their neurites in various brain regions), as is their detailed projections within the brain. Neuromodulation is sometimes suggested by close, but not intimate, proximity of varicosities of neurites. We infer that some neuron types comprise input pathways to the LNs, some are outputs to neuroendocrine or behavioral rhythms, and others participate in both input and output pathways, sometimes by the same neuron type but in different locations. For example, one retinula cell in each ommatidium is immunoreactive for allatostatin A; its axon projects to the medulla making superpositions with LNs, as do serotonin cells in the optic lobe, indicating roles of both neuron types in light input (entrainment) to the clock. But in other brain areas, these same types appear to mediate outputs from the clock. The accessory medulla has been widely reported as the principle center of integration in other insects; but we found sparse evidence of this in R. prolixus as it contains few neurites other than those from the clock cells. Rather, the importance of neural pathways involving the medulla and the superior protocerebrum is emphasized. We conclude that there is a vast and complex web of interactions in the brain with the LNs, which potentially receive multiple pathways of inputs and outputs that could drive rhythmicity in a multitude of downstream cells, rendering a host of output pathways rhythmic, notably hormone release from neurosecretory cells and behaviors.