To synchronize a network of pacemakers in the Drosophila brain, a neuropeptide receptor specifically associates with adenylate cyclase 3 to create a “circadian signalosome.”

Highlights d Human NPY receptor-targeting drugs affect Ae. aegypti mosquito attraction to hosts d Ae. aegypti NPY-like receptor 7 is the mosquito target of behaviorally active drugs d NPYLR7 mutant mosquitoes show abnormal host-seeking regulation d Small-molecule NPYLR7 agonists suppress mosquito hostseeking and biting behavior

The neuropeptide PDF promotes the normal sequencing of circadian behavioral rhythms in Drosophila, but its signaling mechanisms are not well understood. We report daily rhythmicity in responsiveness to PDF in critical pacemakers called small LNvs. There is a daily change in potency, as great as 10-fold higher, around dawn. The rhythm persists in constant darkness, does not require endogenous ligand (PDF) signaling, or rhythmic receptor gene transcription. Furthermore, rhythmic responsiveness reflects the properties of the pacemaker cell type, not the receptor. Dopamine responsiveness also cycles, in phase with that of PDF, in the same pacemakers, but does not cycle in large LNv. The activity of RalA GTPase in s-LNv regulates PDF responsiveness and behavioral locomotor rhythms. Additional, cell autonomous PDF signaling reversed the circadian behavioral effects of lowered RalA activity. Thus RalA activity confers high PDF responsiveness, providing a daily gate around the dawn hours to promote functional PDF signaling.

SUMMARY Female Aedes aegypti mosquitoes typically mate only once with one male in their lifetime, a behavior known as “monandry” [1]. This single mating event provisions the female with sufficient sperm to fertilize the >500 eggs she will produce during her ~4–6-week lifespan in the laboratory [2]. Successful mating induces lifetime refractoriness to subsequent insemination by other males, enforcing the paternity of the first male [3–5]. Ae. aegypti mate in flight near human hosts [6], and females become refractory to remating within seconds [1, 3, 4], suggesting the existence of a rapid mechanism to prevent female remating. In this study we implicate HP-I, an Aedes- and male-specific peptide transferred to females [7], and its cognate receptor in the female, NPYLR1 [8], in rapid enforcement of paternity. HP-I mutant males were ineffective in enforcing paternity when a second male was given access to the female within 1 hour. NPYLR1 mutant females produced mixed paternity offspring at high frequency, indicating acceptance of multiple mates. Synthetic HP-I injected into wild-type but not NPYLR1 mutant virgins reduced successful matings. Asian tiger mosquito (Ae. albopictus) HP-I peptides potently activated Ae. aegypti NPYLR1. Invasive Ae. albopictus males are known to copulate with and effectively sterilize Ae. aegypti females by causing them to reject future mates [9]. Cross-species transfer of sperm and active seminal fluid proteins including HP-I may contribute to this phenomenon. This signaling system promotes rapid paternity enforcement within Ae. aegypti, but may promote local extinction in areas where they compete with Ae. albopictus.

We used real-time imaging to detect cAMP levels in neurons of intact fly brains to study mechanisms of circadian pacemaker synchronization by the neuropeptide PDF in Drosophila. PDF receptor (PDF-R) is expressed by both M (sLNv) and E (LNd) pacemaker sub-classes and is coupled to Gsα in both cases. We previously reported that PDF-R in M pacemakers elevates cAMP levels by activating the ortholog of mammalian Adenylate Cyclase 3 (AC3), but that AC3 disruptions had no effect on E pacemaker sensitivity to PDF. Here we show that PDF-R in E pacemakers activates a different AC isoform, AC78C, an ortholog of mammalian AC8. Knockdown of AC78C by transgenic RNAi substantially reduces, but does not completely abrogate, PDF responses in these E pacemakers. The knockdown effect is intact when restricted to mature stages, suggesting a physiological and not a development role for AC78C in E pacemakers. The AC78C phenotype is rescued by over-expression of AC78C, but not by over-expression of the rutabaga AC. AC78C over-expression does not disrupt PDF responses in these E pacemakers, and neither AC78C knockdown nor its over-expression disrupted locomotor rhythms. Finally, knockdown of two AKAPs, nervy and AKAP 200 partially reduces LNd PDF responses. These findings begin to identify the components of E pacemaker PDF-R signalosomes and indicate they are distinct from PDF-R signalosomes in M pacemakers: we propose they contain AC78C and at least one other AC.