Male Courtship Behavior Research Articles

Multiple stressors lead to complex responses in reproductive behaviors in an African cichlid

Abstract Exposure to multiple environmental stressors is a common occurrence that can affect organisms in predictable or unpredictable ways. Hypoxia and turbidity in aquatic environments are 2 stressors that can affect reproductive behaviors by altering energy availability and the visual environment, respectively. Here we examine the relative effects of population and the rearing environment (oxygen concentration and turbidity) on reproductive behaviors. We reared cichlid fish (the Egyptian mouthbrooder, Pseudocrenilabrus multicolor) from 2 populations (a swamp and river) until sexual maturity, in a full factorial design (hypoxic/normoxic × clear/turbid) and then quantified male competitive and courtship behaviors and female preference under their respective rearing conditions. Overall, we found that the rearing environment was more important than population for determining behavior, indicating there were few heritable differences in reproductive behavior between the 2 populations. Unexpectedly, males in the hypoxic rearing treatment performed more competitive and courtship behaviors. Under turbid conditions, males performed fewer competitive and courtship behaviors. We predicted that females would prefer males from their own population. However, under the hypoxic and turbid combination females from both populations preferred males from the other population. Our results suggest that reproductive behaviors are affected by interactions among male traits, female preferences, and environmental conditions.

Role of Doublesex-Dependent Phosphodiesterase 1c Expression in Gustatory Receptor Neurons in Male Courtship Behavior of Drosophila Melanogaster

Role of Doublesex-Dependent Phosphodiesterase 1c Expression in Gustatory Receptor Neurons in Male Courtship Behavior of Drosophila Melanogaster

Identification and Functional Analysis of the fruitless Gene in a Hemimetabolous Insect, Nilaparvata lugens.

The fruitless (fru) gene functions as a crucial "tuner" in male insect courtship behavior through distinct expression patterns. In Nilaparvata lugens, our previous research showed doublesex (dsx) influencing male courtship songs, causing mating failures with virgin females. However, the impact of fru on N. lugens mating remains unexplored. In this study, the fru homolog (Nlfru) in N. lugens yielded four spliceosomes: Nlfru-374-a/b, Nlfru-377, and Nlfru-433, encoding proteins of 374aa, 377aa, and 433aa, respectively. Notably, only Nlfru-374b exhibited male bias, while the others were non-sex-specific. All NlFRU proteins featured the BTB conserved domain, with NlFRU-374 and NlFRU-377 possessing the ZnF domain with different sequences. RNAi-mediated Nlfru or its isoforms' knockdown in nymph stages blocked wing-flapping behavior in mating males, while embryonic knockdown via maternal RNAi resulted in over 80% of males losing wing-flapping ability, and female receptivity was reduced. Nlfru expression was Nldsx-regulated, and yet courtship signals and mating success were unaffected. Remarkably, RNAi-mediated Nlfru knockdown up-regulated the expression of flightin in macropterous males, which regulated muscle stiffness and delayed force response, suggesting Nlfru's involvement in muscle development regulation. Collectively, our results indicate that Nlfru functions in N. lugens exhibit a combination of conservation and species specificity, contributing insights into fru evolution, particularly in Hemiptera species.

The melanin pigment gene black mediates body pigmentation and courtship behaviour in the German cockroach Blattella germanica.

Genes involved in melanin production directly impact insect pigmentation and can affect diverse physiology and behaviours. The role these genes have on sex behaviour, however, is unclear. In the present study, the crucial melanin pigment gene black was functionally characterised in an urban pest, the German cockroach, Blattella germanica. RNAi knockdown of B. germanica black (Bgblack) had no effect on survival, but did result in black pigmentation of the thoraxes, abdomens, heads, wings, legs, antennae, and cerci due to cuticular accumulation of melanin. Sex-specific variation in the pigmentation pattern was apparent, with females exhibiting darker coloration on the abdomen and thorax than males. Bgblack knockdown also resulted in wing deformation and negatively impacted the contact sex pheromone-based courtship behaviour of males. This study provides evidence for black function in multiple aspects of B. germanica biology and opens new avenues of exploration for novel pest control strategies.

The role of fruitless in specifying courtship behaviors across divergent Drosophila species.

Sex-specific behaviors are critical for reproduction and species survival. The sex-specifically spliced transcription factor fruitless (fru) helps establish male courtship behaviors in invertebrates. Forcing male-specific fru (fruM) splicing in Drosophila melanogaster females produces male-typical behaviors while disrupting female-specific behaviors. However, whether fru's joint role in specifying male and inhibiting female behaviors is conserved across species is unknown. We used CRISPR-Cas9 to force FruM expression in female Drosophila virilis, a species in which males and females produce sex-specific songs. In contrast to D. melanogaster, in which one fruM allele is sufficient to generate male behaviors in females, two alleles are needed in D. virilis females. D. virilis females expressing FruM maintain the ability to sing female-typical song as well as lay eggs, whereas D. melanogaster FruM females cannot lay eggs. These results reveal potential differences in fru function between divergent species and underscore the importance of studying diverse behaviors and species for understanding the genetic basis of sex differences.

Sexual behaviour of Anastrepha obliqua (Macquart) (Diptera: Tephritidae): Do laboratory domestication conditions influence male courtship behaviour?

AbstractWe analysed the influence of laboratory domestication, under relaxed conditions, on the courtship behaviour of the fruit fly species Anastrepha obliqua, an important agricultural pest. We compared the temporal patterns of pheromone emission (Calling behaviour) and the frequencies and sequences of the courtship behavioural units of males of a laboratory lineage and a wild lineage. Our results indicated similarities in the temporal behavioural patterns of calling, the durations of their behavioural sequences, the final sequences of courtships resulting in copulation, of wild and laboratory males. Differences, however, were observed between the two populations in terms of the frequencies of the behavioural units executed and the initial sequence of courtship. Differences were noted in the presence or absence of some behavioural units within the courtship behavioural repertoires of the laboratory‐reared and wild. The wild males did not show units such as Alignment, Contact, Fighting and Marking Leaf that were observed in the laboratory males' courtship behaviour under laboratory conditions; on the other hand, laboratory males did not show the Abdominal movements and Oscillation observed in the courtship behaviour of wild males. The rearing of A. obliqua males under relaxed conditions in the laboratory provides an environment adequate for the preservation of behavioural characteristics relevant to the successful mating, such as Movement, Arrowhead 1, and Attempt, and in temporal patterns of pheromone emission.

Ionotropic receptors mediate olfactory learning and memory in Drosophila.

Phenylacetaldehyde (PAH), an aromatic compound, is present in a diverse range of fruits including overripe bananas and prickly pear cactus, the two major host fruits for Drosophila melanogaster. PAH acts as a potent ligand for the ionotropic receptor 84a (IR84a) in the adult fruit fly and it is detected by the IR84a/IR8a heterotetrameric complex. Its role in the male courtship behavior through IR84a as an environmental aphrodisiac is of additional importance. In D. melanogaster, two distinct kinds of olfactory receptors, that is, odorant receptors (ORs) and ionotropic receptors (IRs), perceive the odorant stimuli. They display unique structural, molecular, and functional characteristics in addition to having different evolutionary origins. Traditionally, olfactory cues detected by the ORs such as ethyl acetate, 1-butanol, isoamyl acetate, 1-octanol, 4-methylcyclohexanol, etc. classified as aliphatic esters and alcohols have been employed in olfactory classical conditioning using fruit flies. This underlines the participation of OR-activated olfactory pathways in learning and memory formation. Our study elucidates that likewise ethyl acetate (EA) (an OR-responsive odorant), PAH (an IR-responsive aromatic compound) too can form learning and memory when associated with an appetitive gustatory reinforcer. The association of PAH with sucrose (PAH/SUC) led to learning and formation of the long-term memory (LTM). Additionally, the Orco1, Ir84aMI00501, and Ir8a1 mutant flies were used to confirm the exclusive participation of the IR84a/IR8a complex in PAH/SUC olfactory associative conditioning. These results highlight the involvement of IRs via an IR-activated pathway in facilitating robust olfactory behavior.

N-octyl acrylate is a candidate sex pheromone component involved in courtship in parasitoid wasp Microplitis mediator.

Sex pheromones are considered to play critical roles in partner communication of most parasitic Hymenoptera. However, the identification of sex pheromone components remains limited to a few families of parasitoid wasps. In this study, we functionally characterized a candidate sex pheromone component in Microplitis mediator (Hymenoptera: Braconidae), a solitary parasitoid of Noctuidae insects. We found that the body surface extract from female wasps could significantly stimulate courtship behavior of males. Gas chromatography-electroantennographic detection (GC-EAD) analysis revealed that a candidate semiochemical from extract triggered significant electrophysiological response of antennae of males. By performing gas chromatography-mass spectrometer (GC-MS) measurement, GC-EAD active compound was identified as n-octyl acrylate, a candidate sex pheromone component in female M. mediator. In electroantennogram (EAG) tests, antennae of male wasps showed significantly higher electrophysiological responses to n-octyl acrylate than those of females. Y-tube olfactometer assays indicated that male wasps significantly chose n-octyl acrylate compared with the control. Furthermore, male wasps showed a remarkable preference for n-octyl acrylate in a simulated field condition behavioral trial; simultaneously, n-octyl acrylate standard could also trigger significant courtship behavior in males. We propose that n-octyl acrylate, as a candidate vital sex pheromone component, could be utilized to design behavioral regulators of M. mediator to implement the protection and utilization of natural enemies.

Sequential mountings of a possible immature male by an adult male in Victoria’s Riflebird (Ptiloris victoriae)

Sexual behaviour in the Birds-of-paradise (family Paradisaeidae) is poorly understood and few studies have offered detailed descriptions of copulatory behaviours. We recorded courtship behaviours of Victoria’s Riflebird Ptiloris victoriae in the Atherton Tablelands in Queensland, Australia using motion triggered cameras and report a rare case of three sequential mountings by an adult male. While the recipient of these mountings performed female-typical sexual behaviours, it also briefly performed a male courtship display behaviour, suggesting that it may be an immature male. This observation raises several questions about the courtship behaviour of this species. Females may, for instance, occasionally solicit multiple copulations from preferred males to maximize the amount of transferred sperm. Another intriguing possibility is that immature males tolerate being mounted and potentially even mimic female sexual behaviours when learning from adult male displays. We also describe a novel adult male courtship behaviour, namely ‘nape-pecking’.

Copulatory courtship, body temperature and infection in Tenebrio molitor.

Ectothermic animals can raise their body temperature under varying circumstances. Two such situations occur during sexual activity (as metabolic rate rises during copulatory movements) and during infection (to control pathogens more effectively). We have investigated these two situations using Tenebrio molitor males. We recorded the copulatory courtship behavior of sick (= infected with Metharizium robertsii fungus) vs healthy males and its link with body temperature. We predicted a positive relation between copulatory courtship (measured as antennal and leg contact behavior) and body temperature, especially in sick males. We found that the intensity of contacts correlated with increased body temperature in sick males. Previous studies in this species indicated that partner females laid fewer eggs after mating with sick males above a certain male body temperature threshold. Thus, our present findings suggest that females may detect male infection via intensity of antennal-mediated courtship, body temperature or their combination. If this is the case, females may assess male cues directly related to health status such as body temperature.

The effect of short-term exposure to high temperatures on male courtship behaviour and mating success in the fruit fly Drosophila virilis

Human-induced climate change is leading to higher average global temperatures and increasingly extreme weather events. High temperatures can have obvious effects on animal survival, particularly in ectotherms. However, the temperature at which organisms become sterile may be significantly lower than the temperature at which other biological functions are impaired. In the fruit fly Drosophila virilis, males are sterilized at temperatures above 34 °C, but are still active and able to mate normally. We investigated the male behavioural changes associated with high-temperature fertility loss. We exposed males to a warming treatment of 34.4 °C or 36.6 °C for 4 h, and then recorded their mating behaviour after being allowed to recover for 24 h. Previous work in this species suggests that males exposed to 34.4 °C lose the ability to produce new sperm, but can utilize mature sperm produced before the heat shock. We therefore predicted that these males would increases their courtship rate, and reduce their choosiness, in order to try to ensure a mating before their remaining mature sperm die. In contrast, over two-thirds of males exposed to 36.6 °C are completely sterile. In standard mating trials, earlier exposure to 34.4 °C or 36.6 °C did not affect male courtship behaviour when compared to control males kept at 23 °C. Exposure to high temperatures also did not alter the extent to which males directed courtship toward females of the same species. However, males exposed to 36.6 °C were significantly slower to mate, and had a reduced likelihood of mating, when compared to control males. Overall, exposure to high temperatures did not alter male courtship behaviour, but did lower their likelihood of mating. This suggests that females can distinguish between normal and heat-sterilized males before mating, and that female mate choice may at least partly mitigate the population-level consequences of high-temperature induced male sterility in this species.

Decoding the genetic and chemical basis of sexual attractiveness in parasitic wasps.

Attracting and securing potential mating partners is of fundamental importance for reproduction. Therefore, signaling sexual attractiveness is expected to be tightly coordinated in communication systems synchronizing senders and receivers. Chemical signaling has permeated through all taxa of life as the earliest and most widespread form of communication and is particularly prevalent in insects. However, it has been notoriously difficult to decipher how exactly information related to sexual signaling is encoded in complex chemical profiles. Similarly, our knowledge of the genetic basis of sexual signaling is very limited and usually restricted to a few case studies with comparably simple pheromonal communication mechanisms. The present study jointly addresses these two knowledge gaps by characterizing two fatty acid synthase genes, that most likely evolved by tandem gene duplication, simultaneously impacting sexual attractiveness and complex chemical surface profiles in parasitic wasps. Gene knock-down in female wasps dramatically reduces their sexual attractiveness coinciding with a drastic decrease in male courtship and copulation behavior. Concordantly, we found a striking shift of methyl-branching patterns in the female surface pheromonal compounds, which we subsequently demonstrate to be the main cause for the greatly reduced male response. Intriguingly, this suggests a potential coding mechanism for sexual attractiveness mediated by specific methyl-branching patterns in CHC profiles. So far, the genetic underpinnings of methyl-branched CHCs are not well understood despite their high potential for encoding information. Our study sheds light on how biologically relevant information can be encoded in complex chemical profiles and on the genetic basis of sexual attractiveness.

Loss of gdnfa disrupts spermiogenesis and male courtship behavior in zebrafish

Spermatogenesis is essential for establishment and maintenance of reproduction in male vertebrates. Spermatogenesis, which is mainly regulated by the combined action of hormones, growth factors, and epigenetic factors, is highly conserved. Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-β superfamily. In this study, global gdnfa knockout and Tg (gdnfa: mcherry) transgenic zebrafish lines were generated. Loss of gdnfa resulted in disorganized testes, decreased gonadosomatic index, and low percentage of mature spermatozoa. In the Tg (gdnfa: mcherry) zebrafish line, we found that gdnfa was expressed in Leydig cells. The mutation in gdnfa significantly decreased Leydig cell marker gene expression and androgen secretion in Leydig cells. In addition, courtship behavior was disrupted in the male mutants. We present in vivo data showing that global knockout of gdnfa disrupts spermiogenesis and male courtship behavior in zebrafish. The first viable vertebrate model with a global gdnfa knockout may be valuable for studying the role of GDNF in animal reproduction.

Female Sex Determination Factors in Ceratitis capitata: Molecular and Structural Basis of TRA and TRA2 Recognition.

In the model system for genetics, Drosophila melanogaster, sexual differentiation and male courtship behavior are controlled by sex-specific splicing of doublesex (dsx) and fruitless (fru). In vitro and in vivo studies showed that female-specific Transformer (TRA) and the non-sex-specific Transformer 2 (TRA2) splicing factors interact, forming a complex promoting dsx and fru female-specific splicing. TRA/TRA2 complex binds to 13 nt long sequence repeats in their pre-mRNAs. In the Mediterranean fruitfly Ceratitis capitata (Medfly), a major agricultural pest, which shares with Drosophila a ~120 million years old ancestor, Cctra and Cctra2 genes seem to promote female-specific splicing of Ccdsx and Ccfru, which contain conserved TRA/TRA2 binding repeats. Unlike Drosophila tra, Cctra autoregulates its female-specific splicing through these putative regulatory repeats. Here, a yeast two-hybrid assay shows that CcTRA interacts with CcTRA2, despite its high amino acid divergence compared to Drosophila TRA. Interestingly, CcTRA2 interacts with itself, as also observed for Drosophila TRA2. We also generated a three-dimensional model of the complex formed by CcTRA and CcTRA2 using predictive approaches based on Artificial Intelligence. This structure also identified an evolutionary and highly conserved putative TRA2 recognition motif in the TRA sequence. The Y2H approach, combined with powerful predictive tools of three-dimensional protein structures, could use helpful also in this and other insect species to understand the potential links between different upstream proteins acting as primary sex-determining signals and the conserved TRA and TRA2 transducers.

Intense light unleashes male-male courtship behaviour in wild-type Drosophila.

Drosophila courtship studies have elucidated several principles of the neurogenetic organization of complex behaviour. Through an integration across sensory modalities, males perform stereotypic patterns of chasing, courtship song production and copulation attempts. Here we report a serendipitous finding that intense light not only enhances courtship toward female targets but also triggers unexpected courtship behaviours among male flies. Strikingly, in wild-type male-only chambers, we observed extreme behavioural manifestations, such as 'chaining' and 'wheeling', resembling previously reported male-male courtship behaviours in fruitless mutants and in transformants with ectopic mini-white+ overexpression. This male-male courtship was greatly diminished in a variety of visual system mutants, including disrupted phototransduction (norpA), eliminated eye-colour screening pigments (white), or deletion of the R7 photoreceptor cells (sevenless). However, light-induced courtship was unhampered in wing-cut flies, despite their inability to produce courtship song, a major acoustic signal during courtship. Unexpectedly the olfactory mutants orco and sbl displayed unrestrained male-male courtship. Particularly, orco males attained maximum courtship scores under either dim or intense light conditions. Together, our observations support the notion that the innate male courtship behaviour is restrained by olfactory cues under normal conditions but can be unleashed by strong visual stimulation in Drosophila.

Social experience and pheromone receptor activity reprogram gene expression in sensory neurons.

Social experience and pheromone signaling in olfactory neurons affect neuronal responses and male courtship behaviors in Drosophila. We previously showed that social experience and pheromone signaling modulate chromatin around behavioral switch gene fruitless, which encodes a transcription factor necessary and sufficient for male sexual behaviors. Fruitless drives social experience-dependent modulation of courtship behaviors and physiological sensory neuron responses to pheromone; however, the molecular mechanisms underlying this modulation of neural responses remain less clear. To identify the molecular mechanisms driving social experience-dependent changes in neuronal responses, we performed RNA-seq from antennal samples of mutants in pheromone receptors and fruitless, as well as grouped or isolated wild-type males. Genes affecting neuronal physiology and function, such as neurotransmitter receptors, ion channels, ion and membrane transporters, and odorant binding proteins are differentially regulated by social context and pheromone signaling. While we found that loss of pheromone detection only has small effects on differential promoter and exon usage within fruitless gene, many of the differentially regulated genes have Fruitless-binding sites or are bound by Fruitless in the nervous system. Recent studies showed that social experience and juvenile hormone signaling co-regulate fruitless chromatin to modify pheromone responses in olfactory neurons. Interestingly, genes involved in juvenile hormone metabolism are also misregulated in different social contexts and mutant backgrounds. Our results suggest that modulation of neuronal activity and behaviors in response to social experience and pheromone signaling likely arise due to large-scale changes in transcriptional programs for neuronal function downstream of behavioral switch gene function.

Drosophila Free-Flight Odor Tracking is Altered in a Sex-Specific Manner By Preimaginal Sensory Exposure.

In insects such as Drosophila melanogaster, flight guidance is based on converging sensory information provided by several modalities, including chemoperception. Drosophila flies are particularly attracted by complex odors constituting volatile molecules from yeast, pheromones and microbe-metabolized food. Based on a recent study revealing that adult male courtship behavior can be affected by early preimaginal exposure to maternally transmitted egg factors, we wondered whether a similar exposure could affect free-flight odor tracking in flies of both sexes. Our main experiment consisted of testing flies differently conditioned during preimaginal development in a wind tunnel. Each fly was presented with a dual choice of food labeled by groups of each sex of D. melanogaster or D. simulans flies. The combined effect of food with the cis-vaccenyl acetate pheromone (cVA), which is involved in aggregation behavior, was also measured. Moreover, we used the headspace method to determine the "odorant" identity of the different labeled foods tested. We also measured the antennal electrophysiological response to cVA in females and males resulting from the different preimaginal conditioning procedures. Our data indicate that flies differentially modulated their flight response (take off, flight duration, food landing and preference) according to sex, conditioning and food choice. Our headspace analysis revealed that many food-derived volatile molecules diverged between sexes and species. Antennal responses to cVA showed clear sex-specific variation for conditioned flies but not for control flies. In summary, our study indicates that preimaginal conditioning can affect Drosophila free flight behavior in a sex-specific manner.

The Drosophila dopamine 2-like receptor D2R (Dop2R) is required in the blood brain barrier for male courtship.

The blood brain barrier (BBB) has the essential function to protect the brain from potentially hazardous molecules while also enabling controlled selective uptake. How these processes and signaling inside BBB cells control neuronal function is an intense area of interest. Signaling in the adult Drosophila BBB is required for normal male courtship behavior and relies on male-specific molecules in the BBB. Here we show that the dopamine receptor D2R is expressed in the BBB and is required in mature males for normal mating behavior. Conditional adult male knockdown of D2R in BBB cells causes courtship defects. The courtship defects observed in genetic D2R mutants can be rescued by expression of normal D2R specifically in the BBB of adult males. Drosophila BBB cells are glial cells. Our findings thus identify a specific glial function for the DR2 receptor and dopamine signaling in the regulation of a complex behavior.

A genetically encoded sensor measures temporal oxytocin release from different neuronal compartments.

Oxytocin (OT), a peptide hormone and neuromodulator, is involved in diverse physiological and pathophysiological processes in the central nervous system and the periphery. However, the regulation and functional sequences of spatial OT release in the brain remain poorly understood. We describe a genetically encoded G-protein-coupled receptor activation-based (GRAB) OT sensor called GRABOT1.0. In contrast to previous methods, GRABOT1.0 enables imaging of OT release ex vivo and in vivo with suitable sensitivity, specificity and spatiotemporal resolution. Using this sensor, we visualize stimulation-induced OT release from specific neuronal compartments in mouse brain slices and discover that N-type calcium channels predominantly mediate axonal OT release, whereas L-type calcium channels mediate somatodendritic OT release. We identify differences in the fusion machinery of OT release for axon terminals versus somata and dendrites. Finally, we measure OT dynamics in various brain regions in mice during male courtship behavior. Thus, GRABOT1.0 provides insights into the role of compartmental OT release in physiological and behavioral functions.

Patterns of Population Structure and Introgression Among Recently Differentiated Drosophila melanogaster Populations.

Despite a century of genetic analysis, the evolutionary processes that have generated the patterns of exceptional genetic and phenotypic variation in the model organism Drosophila melanogaster remains poorly understood. In particular, how genetic variation is partitioned within its putative ancestral range in Southern Africa remains unresolved. Here, we study patterns of population genetic structure, admixture, and the spatial structuring of candidate incompatibility alleles across a global sample, including 223 new accessions, predominantly from remote regions in Southern Africa. We identify nine major ancestries, six that primarily occur in Africa and one that has not been previously described. We find evidence for both contemporary and historical admixture between ancestries, with admixture rates varying both within and between continents. For example, while previous work has highlighted an admixture zone between broadly defined African and European ancestries in the Caribbean and southeastern USA, we identify West African ancestry as the most likely African contributor. Moreover, loci showing the strongest signal of introgression between West Africa and the Caribbean/southeastern USA include several genes relating to neurological development and male courtship behavior, in line with previous work showing shared mating behaviors between these regions. Finally, while we hypothesized that potential incompatibility loci may contribute to population genetic structure across the range of D. melanogaster; these loci are, on average, not highly differentiated between ancestries. This work contributes to our understanding of the evolutionary history of a key model system, and provides insight into the partitioning of diversity across its range.