Female acoustic signaling of sexual immaturity depresses male courtship in Drosophila
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eLife Assessment
This important study characterizes wing flicking in sexually immature Drosophila melanogaster females as a rejection response behavior that produces patterned acoustic signals and suppresses male courtship. The authors combine behavioral and acoustic measurements with manipulations of sensory input and targeted neuronal populations to provide solid evidence for how sexual maturation is signaled. With some clarification, additional information, and a more cautious interpretation of the findings in the discussion, this study will be of interest to neurobiologists and ethologists studying sexual maturation and behavior and will open up opportunities to address questions across several distinct areas, including sexual dimorphism, sexual maturation, and even the evolution of sexual signals.
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Abstract
In an early phase of life, most animals are behaviorally and physiologically not yet able to reproduce but show adaptations specific to a juvenile state. In Drosophila females, sexual maturation after metamorphosis is achieved by acquiring receptivity to male courtship and completing oogenesis, a transition that is under hormonal control and requires coordinates changes in the nervous system. Here, we show that immature females display a transient signaling behavior during the time window of asexuality by flicking their wings in response to and dependent on male courtship stimuli. Immature wing flicks require the activity of Doublesex (Dsx) expressing central brain pC1a neurons that mediate receptivity in mature virgins. Immature wing flicks generate patterned sound pulse trains that differ from other intraspecific acoustic signals, but resemble pulses produced during mature male agonistic interactions. Courting males exposed to immature flicks shorten courtship singing and abstain from copulation attempts, indicating that immature wing flicks serve as an effective rejection signal of asexual females to minimize futile male mating pursuits.
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eLife Assessment
This important study characterizes wing flicking in sexually immature Drosophila melanogaster females as a rejection response behavior that produces patterned acoustic signals and suppresses male courtship. The authors combine behavioral and acoustic measurements with manipulations of sensory input and targeted neuronal populations to provide solid evidence for how sexual maturation is signaled. With some clarification, additional information, and a more cautious interpretation of the findings in the discussion, this study will be of interest to neurobiologists and ethologists studying sexual maturation and behavior and will open up opportunities to address questions across several distinct areas, including sexual dimorphism, sexual maturation, and even the evolution of sexual signals.
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Reviewer #1 (Public review):
The authors investigate a previously described behavior of sexually immature female Drosophila melanogaster, wherein immature females flick their wings in response to male courtship. They record, analyze, and characterize the auditory signals generated by these wing-flicks and show that these sounds reduce male courtship song and copulation attempts. The study combines several techniques including behavioral observations, acoustic analysis, developmental experiments, sensory manipulations, and neuronal manipulations to investigate the stimulus dependence and neural basis of this behavior. Amidst all the literature on sexual communication, this study uniquely addresses an interesting question on communication during sexual maturation, providing valuable insights on why and how sexual maturation could be …
Reviewer #1 (Public review):
The authors investigate a previously described behavior of sexually immature female Drosophila melanogaster, wherein immature females flick their wings in response to male courtship. They record, analyze, and characterize the auditory signals generated by these wing-flicks and show that these sounds reduce male courtship song and copulation attempts. The study combines several techniques including behavioral observations, acoustic analysis, developmental experiments, sensory manipulations, and neuronal manipulations to investigate the stimulus dependence and neural basis of this behavior. Amidst all the literature on sexual communication, this study uniquely addresses an interesting question on communication during sexual maturation, providing valuable insights on why and how sexual maturation could be signaled.
A major strength of the study is its behavioral and acoustic measurements with manipulations of sensory input and identified neurons. Comparative analysis of immature wing-flick, male-male aggression, and male courtship song was equally interesting. The developmental analysis also provides a clear temporal relationship between female maturation, wing flicking, and mating receptivity. Together, these experiments provide compelling evidence that immature female wing flicking is a reproducible behavior in response to male courtship, and that it significantly reduces male copulation attempts. Identifying a defined population of neurons that coordinate this response behavior further supports this study.
However, it must be pointed out that some of the broader conclusions extend beyond what is directly demonstrated. In particular, while the evidence supports the interpretation that wing flicking functions as a rejection or deterrence signal from sexually immature females, the designation of this behavior as a signal of "asexuality" and the proposed evolutionary relationship between female wing flicking and male agonistic wing flicking are less firmly established. Furthermore, I think the similarity in acoustic structure and the involvement of related neuronal populations is not sufficient to conclude a) that the two behaviors share an evolutionary origin or b) that one behavior evolved through modification of the other. Similarly, proposed explanations concerning energetic costs, predation risk, and adaptive benefits should be presented as hypotheses rather than conclusions unless directly tested. These interesting points can be included in a subsection "Ideas and Speculation", to clearly distinguish directly demonstrated effects and evolutionary or mechanistic interpretations.
In conclusion, the study contributes to our understanding on communication during sexual maturation.
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Reviewer #2 (Public review):
Summary:
This study characterizes wing flicking in sexually immature female Drosophila as a transient behavior that produces patterned acoustic signals and suppresses male courtship. The authors show that wing flicking is restricted to a short developmental window, is influenced by multiple components of male courtship and juvenile hormone-dependent maturation, and depends on Dsx+ pC1 neurons. They further show that wing flicking shortens male song bouts and reduces copulation attempts, and propose that it functions as a rejection signal associated with sexual immaturity. Overall, this is an interesting and valuable study that provides new insight into a little-characterized stage of female sexual maturation. The core characterizations are compelling, and some additional clarification and refinement of the …
Reviewer #2 (Public review):
Summary:
This study characterizes wing flicking in sexually immature female Drosophila as a transient behavior that produces patterned acoustic signals and suppresses male courtship. The authors show that wing flicking is restricted to a short developmental window, is influenced by multiple components of male courtship and juvenile hormone-dependent maturation, and depends on Dsx+ pC1 neurons. They further show that wing flicking shortens male song bouts and reduces copulation attempts, and propose that it functions as a rejection signal associated with sexual immaturity. Overall, this is an interesting and valuable study that provides new insight into a little-characterized stage of female sexual maturation. The core characterizations are compelling, and some additional clarification and refinement of the mechanistic interpretations would further strengthen the manuscript.
Strengths:
The work expands our understanding of female sexual behavior and highlights sexual maturation as an active transition between distinct behavioral states rather than simply the acquisition of receptivity. The comparison with the post-mating unreceptive state is especially informative. It also brings richness to the understanding by combining behavioral, developmental, and neural analyses and interpretation. The data showing how male behaviors are influenced by female wing flicks are particularly compelling and represent a critical aspect of behavioral characterization that is often overlooked.
Weaknesses:
Event-level analysis of behaviors such as singing, chasing, tapping, licking, or attempted copulation would help clarify the sensory and social context of female wing flicks. The neural conclusion also requires some caution. The split-GAL4 reagent used to assign the phenotype to pC1a labels two pC1 neurons per hemisphere in this study, whereas pC1a is generally described as a single bilateral pair. Given the functional heterogeneity of pC1 subtypes, the current data do not fully exclude a contribution from another pC1 subtype. While the comparison with male agonistic wing signals is informative and interesting, the rejective wing flicks of immature females may be more directly comparable to the previously reported wing flicks and song produced by young and mature males in response to male courtship, which are not mentioned or explored in this study.
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Reviewer #3 (Public review):
Summary:
Female receptivity to male courtship is regulated by many factors, including development and internal status. In mature, mated females, male courtship triggers ovipositor extrusion, which prevents intromission. In immature flies, however, rejection occurs through another behavior: immature wing flicking. How this behavior is engaged and regulated is unclear. Here, Bernet & Philipsborn advanced in this matter by defining behavioral and neuronal keys related to this phenotype. The authors show that immature wing flicking depends on pheromones and acoustic cues from the males to be triggered. Additionally, the authors identify the pC1a cluster as key for this behavior.
Strengths:
(1) In contrast to the regulation of ovipositor extrusion, immature wing flicker, while observed, has not been fully …
Reviewer #3 (Public review):
Summary:
Female receptivity to male courtship is regulated by many factors, including development and internal status. In mature, mated females, male courtship triggers ovipositor extrusion, which prevents intromission. In immature flies, however, rejection occurs through another behavior: immature wing flicking. How this behavior is engaged and regulated is unclear. Here, Bernet & Philipsborn advanced in this matter by defining behavioral and neuronal keys related to this phenotype. The authors show that immature wing flicking depends on pheromones and acoustic cues from the males to be triggered. Additionally, the authors identify the pC1a cluster as key for this behavior.
Strengths:
(1) In contrast to the regulation of ovipositor extrusion, immature wing flicker, while observed, has not been fully characterized. This is a significant step forward in our understanding of the ethology around female receptivity.
(2) The behavioral approaches that the authors used to determine the nature and correlation of immature wing flicker with male courtship are very convincing.
Weaknesses:
While the manuscript has clear strengths, particularly in describing immature wing flicking behavior, several analyses make some conclusions difficult to assess. This is especially troublesome since some of these assertions are the main points the authors highlight as significant findings. In particular:
(1) The authors describe immature female wing flicking as resembling that produced by mature males during agonistic encounters. While I see some similarities, this assessment is subjective. In Figure 1D, the authors present comparative metrics without statistics; thus, it is difficult to assess the validity of the results. Implications, however, can be direct: do females produce acoustic waves to mimic males and deter mating partners (or vice versa)?
(2) From Figure 3F, it is difficult to conclude that the arborization pattern does not differ between mature and immature flies (here, referred to as a 5h vs 10d difference). This may be an artifact of the representative images, but the arborization at 10d appears less dense, particularly around the PI area. If this difference is consistent, this could also reflect neuronal maturation contributing to this phenotype, which is particularly relevant to what the authors discuss in lines 309-339. Differences in synaptic partners might potentially explain the contrasting action of pC1 neurons in immature vs mature females (PMCID: PMC11386958).
(3) The experiments presented do not exclude the possibility that other pC1 neurons, in addition to pC1a neurons, affect the behavior. As written (line 363), the statement indicating that pC1a is the only one affecting immature female flicking is then incorrect.
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