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.

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.

Influence of female cuticular hydrocarbon (CHC) profile on male courtship behavior in two hybridizing field crickets Gryllus firmus and Gryllus pennsylvanicus

Background The hybridizing field crickets, Gryllus firmus and Gryllus pennsylvanicus have several barriers that prevent gene flow between species. The behavioral pre-zygotic mating barrier, where males court conspecifics more intensely than heterospecifics, is important because by acting earlier in the life cycle it has the potential to prevent a larger fraction of hybridization. The mechanism behind such male mate preference is unknown. Here we investigate if the female cuticular hydrocarbon (CHC) profile could be the signal behind male courtship. Results While males of the two species display nearly identical CHC profiles, females have different, albeit overlapping profiles and some females (between 15 and 45%) of both species display a male-like profile distinct from profiles of typical females. We classified CHC females profile into three categories: G. firmus-like (F; including mainly G. firmus females), G. pennsylvanicus-like (P; including mainly G. pennsylvanicus females), and male-like (ML; including females of both species). Gryllus firmus males courted ML and F females more often and faster than they courted P females (p < 0.05). Gryllus pennsylvanicus males were slower to court than G. firmus males, but courted ML females more often (p < 0.05) than their own conspecific P females (no difference between P and F). Both males courted heterospecific ML females more often than other heterospecific females (p < 0.05, significant only for G. firmus males). Conclusions Our results suggest that male mate preference is at least partially informed by female CHC profile and that ML females elicit high courtship behavior in both species. Since ML females exist in both species and are preferred over other heterospecific females, it is likely that this female type is responsible for most hybrid offspring production.

Male courtship behavior is triggered by female chemical cues in the scorpion Tityus pusillus (Scorpiones: Buthidae)

Recognizing conspecific individuals from other members of the community is important for many interactive behaviors, especially those involved in mate selection. We investigated whether male courtship behavior is triggered by chemical cues left by females on the substrate using the sedentary litter-dwelling scorpion Tityus pusillus Pocock, 1893, which is a small and common species distributed throughout the northeast Atlantic Forest in Brazil. In experiments using 50 pairs, we tested whether males recognize females by detecting sex-specific chemicals on the substrate. All males changed their behavior, performing pre-courtship acts when exposed to female-specific chemicals on the substrate, but they did not change their behavior when exposed to a clean substrate lacking female-specific chemicals. These results show that the male T. pusillus alters its behavior in the presence of female chemical cues, suggesting that males recognize females by detecting compounds left on the substrate and that the presence of these chemicals trigger the courtship behavior of the male T. pusillus.

Genetic architecture of male courtship behavior differences in the parasitoid wasp genus Nasonia (Hymenoptera; Pteromalidae)

Very little is known about the genetic basis of behavioral variation in courtship behavior, which can contribute to speciation by prezygotic isolation of closely related species. Here, we analyze the genetic basis and architecture of species differences in the male courtship behavior of two closely related parasitoid wasps Nasonia vitripennis and N. longicornis. Both species occur microsympatrically in parts of their ranges and have been found in the same host pupae. Despite strong postzygotic isolation mechanisms between these two Nasonia species, viable hybrid females can be produced in the laboratory if both species are cured of their Wolbachia endosymbionts. We used haploid F2 hybrid males derived from virgin F1 hybrid females of two independent mapping populations to study the genetic architecture of five quantitative and two qualitative components of their courtship behavior. A total of 14 independent Quantitative Trait Loci (QTL) were found in the first mapping population (320 males), which explained 4-25% of the observed phenotypic variance. Ten of these QTL were confirmed by a second independent mapping population (112 males) and no additional ones were found. A genome-wide scan for two-loci interactions revealed many unique but mostly additive interactions explaining an additional proportion of the observed phenotypic variance. Courtship QTL were found on all five chromosomes and four loci were associated with more than one QTL, indicating either possible pleiotropic effects of individual QTL or individual loci contributing to multiple courtship components. Our results indicate that these two evolutionary young species have rapidly evolved multiple significant phenotypic differences in their courtship behavior that have a polygenic and highly interactive genetic architecture. Based on the location of the QTL and the published Nasonia genome sequence we were able to identify a series of candidate genes for further study.

Dopaminergic neurons mediate male Drosophila courtship motivational state

Motivational states are important determinants of behavior. In Drosophila melanogaster, courtship behavior is robust and crucial for species continuation. However, the motivation of courtship behavior remains unexplored. We first find the phenomenon that courtship behavior is modulated by motivational state. A male fly courts another male fly when it first courts a decapitated female fly however, male– male courtship behavior rarely occurs under normal conditions. Male flies that have satisfied the need for sexual behavior show a decreased male–female sex drive. Therefore, in this phenomenon, the male fly’s courtship motivational state is induced by its exposure to female flies. Blocking dopaminergic neurons by expressing TNTe decreases motivational state-induced male–male courtship behavior without affecting male–female courtship behavior. Vision cues are another key component in sexually driven male–female courtship behavior. Here, we identify a base theory that the inner motivational state could eventually decide fly behavior.

The Role of miRNAs in Drosophila melanogaster Male Courtship Behavior

Drosophila melanogaster courtship, although stereotypical, continually changes based on cues received from the courtship subject. Such adaptive responses are mediated via rapid and widespread transcriptomic reprogramming, a characteristic now widely attributed to microRNAs (miRNAs), along with other players. Here, we conducted a large-scale miRNA knockout screen to identify miRNAs that affect various parameters of male courtship behavior. Apart from identifying miRNAs that impact male–female courtship, we observed that miR-957 mutants performed significantly increased male–male courtship and “chaining” behavior, whereby groups of males court one another. We tested the effect of miR-957 reduction in specific neuronal cell clusters, identifying miR-957 activity in Doublesex (DSX)-expressing and mushroom body clusters as an important regulator of male–male courtship interactions. We further characterized the behavior of miR-957 mutants and found that these males court male subjects vigorously, but do not elicit courtship. Moreover, they fail to lower courtship efforts toward females with higher levels of antiaphrodisiac pheromones. At the level of individual pheromones, miR-957 males show a reduced inhibitory response to both 7-Tricosene (7-T) and cis-vaccenyl acetate, with the effect being more pronounced in the case of 7-T. Overall, our results indicate that a single miRNA can contribute to the regulation of complex behaviors, including detection or processing of chemicals that control important survival strategies such as chemical mate-guarding, and the maintenance of sex- and species-specific courtship barriers.