Post-mating Refractoriness in Drosophila melanogaster Depends Upon Ecdysis Triggering Hormone Signaling

The decision to engage in courtship depends on external cues from potential mates and internal cues related to maturation, health, and experience. Hormones allow such information to be conveyed to distal tissues in a coordinated fashion. Here, we show Ecdysis-Triggering Hormone (ETH) is a regulator of male courtship in Drosophila melanogaster, and critical for mate choice and courtship inhibition after the completion of copulation. Preventing ETH release increases male-male courtship and decreases post-copulation courtship inhibition (PCCI). Such aberrant male courtship behavior in ETH-deficient males appears to be the consequence of inabilityto integrate pheromone cues into decision making. Silencing of ETH receptor (ETHR) in GR32A-expressing neurons leads to reduced ligand sensitivity and elevated male-male courtship. We find OR67D is critical for suppression of courtship after mating, and ETHR silencing in OR67D-expressing neurons, and GR32A-expressing neurons to a lesser degree, elevates post-copulation courtship. Finally, ETHR silencing in the corpus allatum increases post-copulation courtship; treatment of with juvenile hormone analog partially restores normal post-mating behavior. ETH, a stress-sensitive reproductive hormone, appears to coordinate multiple sensory modalities to guide Drosophila male courtship behaviors, especially after mating.

Male Barn Swallows Tolerate Nestling-Like Courtship Calls of Rival Males

Animals often exhibit conspicuous, and sometimes curious, courtship traits, such as nestling-like courtship display in birds, though modern studies of nestling-like courtship display (and calls) are virtually lacking. An exception is previous experiments on the barn swallow Hirundo rustica, demonstrating that females are equally attracted to playback of two structurally similar calls, nestling-like male courtship calls and nestling food-begging calls. The experiments support the sensory trap hypothesis, i.e., that male signals mimic nestling stimuli to exploit female parental care for nestlings. However, female attraction might not be the sole function of nestling-like traits, and males might also have a sensory bias toward nestling-like traits, in which males would be less aggressive toward characteristics typical of immature individuals. Here, I conducted playback experiments to study the function of nestling-like male courtship calls in the context of male–male interactions. Playback of male courtship songs induced frequent approaches by neighbouring males, while nestling-like male courtship calls or nestling food-begging calls induced fewer approaches, though male responses to the latter two vocalisations increased when approaching the nestling period. The observed pattern indicates that, by mimicking immature individuals, males attract intended signal receivers (i.e., females) while avoiding interference from eavesdroppers (i.e., neighbouring males). This unique function can explain why species with parental care exhibit immature-like behaviour.

A genetic screen for Drosophila social isolation mutants and analysis of sex pistol

Prolonged periods of forced social isolation is detrimental to well-being, yet we know little about which genes regulate susceptibility to its effects. In the fruit fly, Drosophila melanogaster, social isolation induces stark changes in behavior including increased aggression, locomotor activity, and resistance to ethanol sedation. To identify genes regulating sensitivity to isolation, I screened a collection of sixteen hundred P-element insertion lines for mutants with abnormal levels of all three isolation-induced behaviors. The screen identified three mutants whose affected genes are likely central to regulating the effects of isolation in flies. One mutant, sex pistol (sxp), became extremely aggressive and resistant to ethanol sedation when socially isolated. sxp also had a high level of male–male courtship. The mutation in sxp reduced the expression of two minor isoforms of the actin regulator hts (adducin), as well as mildly reducing expression of CalpA, a calcium-dependent protease. As a consequence, sxp also had increased expression of the insulin-like peptide, dILP5. Analysis of the social behavior of sxp suggests that these minor hts isoforms function to limit isolation-induced aggression, while chronically high levels of dILP5 increase male–male courtship.

Changes in splicing and neuromodulatory gene expression programs in sensory neurons with pheromone signaling and social experience

Social experience and pheromone signaling in ORNs affect pheromone responses and male courtship behaviors in Drosophila, however, the molecular mechanisms underlying this circuit-level neuromodulation remain less clear. Previous studies identified social experience and pheromone signaling-dependent modulation of chromatin around behavioral switch gene fruitless, which encodes a transcription factor necessary and sufficient for male behaviors. To identify the molecular mechanisms driving social experience-dependent neuromodulation, we performed RNA-seq from antennal samples of mutant fruit flies in pheromone receptors and fruitless, as well as grouped or isolated wild-type males. We found that loss of pheromone detection differentially alters the levels of fruitless exons suggesting changes in splicing patterns. In addition, many Fruitless target neuromodulatory genes, such as neurotransmitter receptors, ion channels, and ion transporters, are differentially regulated by social context and pheromone signaling. Our results suggest that modulation of circuit activity and behaviors in response to social experience and pheromone signaling arise due to changes in transcriptional programs for neuromodulators downstream of behavioral switch gene function.

Developmental plasticity in male courtship in Bicyclus anynana butterflies is driven by hormone regulation of the yellow gene

The organizational role for hormones in the regulation of sexual behavior is currently poorly explored. Previous work showed that seasonal variation in levels of the steroid hormone 20-hydroxyecdysone (20E) during pupal development regulates plasticity in male courtship behavior in Bicyclus anynana butterflies. Wet season (WS) males, reared at high temperature, have high levels of 20-hydroxyecdysone (20E) during pupation and become active courters. Dry season (DS) males, reared at low temperatures, have lower levels of 20E and lower courtship rates. Rescue of WS courtship rates can be achieved via injection of 20E into DS male pupae, but it is still unknown whether 20E alters gene expression in the pupal brain, and if so, the identity of those targets. Using transcriptomics, qPCR, and behavioral assays with a transgenic knockout, we show that higher expression levels of the yellow gene in DS male pupal brains, relative to WS brains, represses courtship in DS males. Furthermore, injecting DS males with 20E downregulates yellow to WS levels 4 hours post-injection, revealing a hormone sensitive window that determines courtship behavior. These findings are in striking contrast to Drosophila, where yellow is required for active male courtship behavior. We conclude that 20E plays an organizational role during pupal brain development by regulating the expression of yellow, which is a repressor of the neural circuity for male courtship behavior in B. anynana. This work shows that similar to vertebrates, hormones can also play an organizational role in insect brains, leading to permanent changes in adult sexual behavior.Significance StatementBehavioral plasticity in adult insects is known to be regulated by hormones, which activate neural circuits in response to environmental cues. Here, we show that hormones can also regulate adult behavioral plasticity by altering gene expression during brain development, adjusting the insect’s behavior to predictable seasonal environmental variation. We show that seasonal changes in the hormone 20E alters expression of the yellow gene in the developing pupal brain of Bicyclus anynana butterflies, which leads to differences in male courtship behavior between the dry and wet seasonal forms. This work provides one of the first examples of the organizational role of hormones in altering gene expression and adult sexual behavior in the developing insect brain.

Male courtship song drives escape responses that are suppressed for successful mating

Persuasion is a crucial component of the courtship ritual needed to overcome contact aversion. In fruit flies, it is well established that the male courtship song prompts receptivity in female flies, in part by causing sexually mature females to slow down and pause, allowing copulation. Whether the above receptivity behaviours require the suppression of contact avoidance or escape remains unknown. Here we show, through genetic manipulation of neurons we identified as required for female receptivity, that male song induces avoidance/escape responses that are suppressed in wild type flies. First, we show that silencing 70A09 neurons leads to an increase in escape, as females increase their walking speed during courtship together with an increase in jumping and a reduction in pausing. The increase in escape response is specific to courtship, as escape to a looming threat is not intensified. Activation of 70A09 neurons leads to pausing, confirming the role of these neurons in escape modulation. Finally, we show that the escape displays by the female result from the presence of a courting male and more specifically from the song produced by a courting male. Our results suggest that courtship song has a dual role, promoting both escape and pause in females and that escape is suppressed by the activity of 70A09 neurons, allowing mating to occur.

Transcriptome Analysis of NPFR Neurons Reveals a Connection Between Proteome Diversity and Social Behavior

Social behaviors are mediated by the activity of highly complex neuronal networks, the function of which is shaped by their transcriptomic and proteomic content. Contemporary advances in neurogenetics, genomics, and tools for automated behavior analysis make it possible to functionally connect the transcriptome profile of candidate neurons to their role in regulating behavior. In this study we used Drosophila melanogaster to explore the molecular signature of neurons expressing receptor for neuropeptide F (NPF), the fly homolog of neuropeptide Y (NPY). By comparing the transcription profile of NPFR neurons to those of nine other populations of neurons, we discovered that NPFR neurons exhibit a unique transcriptome, enriched with receptors for various neuropeptides and neuromodulators, as well as with genes known to regulate behavioral processes, such as learning and memory. By manipulating RNA editing and protein ubiquitination programs specifically in NPFR neurons, we demonstrate that the proper expression of their unique transcriptome and proteome is required to suppress male courtship and certain features of social group interaction. Our results highlight the importance of transcriptome and proteome diversity in the regulation of complex behaviors and pave the path for future dissection of the spatiotemporal regulation of genes within highly complex tissues, such as the brain.

The nuclear receptor DHR3/Hr46 is required in the blood brain barrier of mature males for courtship

The blood brain barrier (BBB) forms a stringent barrier that protects the brain from components in the circulation that could interfere with neuronal function. At the same time, the BBB enables selective transport of critical nutrients and other chemicals to the brain. Many of these processes are still poorly understood. Beyond these functions, another recently recognized function is even less characterized, specifically the role of the BBB in modulating behavior by affecting neuronal function in a sex dependent manner. Notably, signaling in the adult Drosophila BBB is required for normal male courtship behavior. Courtship regulation also relies on male-specific molecules in the BBB. Our previous studies have demonstrated that adult feminization of these cells in males significantly lowered courtship. Here, we conducted microarray analysis of BBB cells isolated from males and females. Findings revealed that these cells contain male- and female-enriched transcripts, respectively. Among these transcripts, nuclear receptor DHR3/Hr46 was identified as a male-enriched BBB transcript. DHR3/Hr46 is best known for its essential roles in the ecdysone response during development and metamorphosis. In this study, we demonstrate that DR3/Hr46 is specifically required in the BBB cells of mature males for courtship behavior. The protein is localized in the nuclei of sub-perineurial glial cells (SPG), indicating that it might act as a transcriptional regulator. These data provide a catalogue of sexually dimorphic BBB transcripts and demonstrate a physiological adult role for the nuclear receptor DH3/Hr46 in the regulation of male courtship, a novel function that is independent of its developmental role.