A Genetic RNAi Screen for IP3/Ca2+ Coupled GPCRs in Drosophila Identifies the PdfR as a Regulator of Insect Flight

Abstract

Insect flight is regulated by various sensory inputs and neuromodulatory circuits which function in synchrony to control and fine-tune the final behavioral outcome. The cellular and molecular bases of flight neuromodulatory circuits are not well defined. In Drosophila melanogaster, it is known that neuronal IP3 receptor mediated Ca2+ signaling and store-operated Ca2+ entry (SOCE) are required for air-puff stimulated adult flight. However, G-protein coupled receptors (GPCRs) that activate intracellular Ca2+ signaling in the context of flight are unknown in Drosophila. We performed a genetic RNAi screen to identify GPCRs that regulate flight by activating the IP3 receptor. Among the 108 GPCRs screened, we discovered 5 IP3/Ca2+ linked GPCRs that are necessary for maintenance of air-puff stimulated flight. Analysis of their temporal requirement established that while some GPCRs are required only during flight circuit development, others are required both in pupal development as well as during adult flight. Interestingly, our study identified the Pigment Dispersing Factor Receptor (PdfR) as a regulator of flight circuit development and as a modulator of acute flight. From the analysis of PdfR expressing neurons relevant for flight and its well-defined roles in other behavioral paradigms, we propose that PdfR signaling functions systemically to integrate multiple sensory inputs and modulate downstream motor behavior.

Pigment-dispersing factor signaling and circadian rhythms in insect locomotor activity

“…Though the acute excitation of PDF neurons does not result in measureable Ca 2+ changes in PdfR-expressing LN d s [71], focal application of PDF does cause small Ca 2+ increases in a subset of the DN1 p class of clock neurons [69]. Furthermore, PdfR has recently been identified as an IP 3 /Ca 2+ coupled receptor required for the maintenance of flight [20]. Thus, it appears that PdfR signaling causes Ca 2+ increases at least in some PdfR-expressing neurons, including some clock neurons [69,72].…”

“…Though expressed in relatively few neurons in insect nervous systems [4–12], PDF plays many roles in the control of behavior and physiology, including circadian rhythmicity [13], geotaxis [14], sleep and arousal [15–18], copulation [19], flight [20], the modulation of visceral muscle contraction [21] and tracheal growth [22]. PDF’s role in circadian timekeeping is its bestunderstood function and the focus of this review.…”

Model Organisms in G Protein–Coupled Receptor Research

“…Zhang and Emery argue that GW182 is a novel lightdependent rheostat modulating the amount of PDF GPCR signaling. Using an RNA interference screen of GPCRs coupled to inositol phosphate-stimulated calcium elevation, PDFR appears to be G q coupled in some neuronal subsets (Agrawal et al, 2013). To measure cyclic nucleotide levels, a number of investigators have used a fluorescence resonance energy transfer (FRET) reporter built around the cAMP-binding domain of the molecule EPAC (de Rooij et al, 1998;Nikolaev et al, 2004).…”

The Current Status and Development of Insect-Resistant Genetically Engineered Poplar in China

“…The first step of using RNAi should be the selection of appropriate target genes, enzymes, or receptors which may cause abnormal growth, development, or reproductive inhibition of target pests. RNAi technology had been successfully applied in the study of various types of insects in recent years and many suitable target genes have emerged, such as chitin synthesis-related enzymes (chitin synthase and chitinase gene) (Arakane et al, 2005; Chen et al, 2008), hormones and receptors related to growth and development (epidermal hormones receptors EcR and juvenile hormones) (Huang et al, 2013), enzymes that modulate energy metabolism (proton metabolized V-ATPase and cytochrome P450 enzymes that metabolize toxic substances) (Tang et al, 2012; Kotwica-Rolinska et al, 2013), as well as neuromodulation pathways receptor and related enzymes (Agrawal et al, 2013). RNAi-mediated down-regulation of poplar had been studied and proved that RNAi is also functional in poplar (Mryer et al, 2004; Bi et al, 2015).…”