Sexual conflict and sexual networks in bed bugs: the fitness cost of traumatic insemination, female avoidance and male mate choice.

Sexual conflict is prevalent among animals and is primarily caused by the fact that the optimal mating rates are often higher in males than in females. While there is a growing appreciation that females can also gain from multiple matings, we still know relatively little about which sex controls the observed mating rates and how close it is to the optimal female mating rates. To address this issue, we tracked female bed bugs (Cimex lectularius) inseminated daily versus weekly and found that weekly inseminated females lived longer and produced over 50% more offspring. In a follow-up experiment employing a social network framework, we placed 24 bed bugs into a semi-naturalistic arena and recorded all sexual interactions. While recently inseminated females did not avoid males more often, they were more frequently rejected by males. Finally, we tracked avoidance behaviour in a single cohort of female bed bugs as they received six successive daily inseminations. Avoidance rates increased and insemination durations decreased with increasing number of prior inseminations. Overall, our results indicate high costs of polyandry. Although females possess some plastic avoidance strategies, the observed rates of insemination fall closer to the male rather than female optimum.

Genetic correlation between aggressive signals and fighting.

Theoretical analyses indicate that aggressive signals should positively correlate with the signallers' willingness and abilities to fight. Few experimental studies, however, have tested this prediction. In two experiments employing distinct, ecologically realistic protocols, we quantified the association between aggressive signals and fighting in fruit fly genotypes and found high positive genetic correlations between threat and fighting (rG = 0.80 and 0.74). Our results add to the growing body of experimental work indicating that aggressive signals have relatively high informational value.

The genetic basis of variation in sexual aggression: Evolution versus social plasticity.

Male sexual aggression towards females is a form of sexual conflict that can result in increased fitness for males through forced copulations (FCs) or coercive matings at the cost of female lifetime fitness. We used male fruit flies (Drosophila melanogaster) as a model system to uncover the genomic contributions to variation in FC, both due to standing variation in a wild population, and due to plastic changes associated with variation in social experience. We used RNAseq to analyse whole-transcriptome differential expression (DE) in male head tissue associated with evolved changes in FC from lineages previously selected for high and low FC rate and in male flies with varying FC rates due to social experience. We identified hundreds of genes associated with evolved and plastic variation in FC, however only a small proportion (27 genes) showed consistent DE due to both modes of variation. We confirmed this trend of low concordance in gene expression effects across broader sets of genes significant in either the evolved or plastic analyses using multivariate approaches. The gene ontology terms neuropeptide hormone activity and serotonin receptor activity were significantly enriched in the set of significant genes. Of seven genes chosen for RNAi knockdown validation tests, knockdown of four genes showed the expected effect on FC behaviours. Taken together, our results provide important information about the apparently independent genetic architectures that underlie natural variation in sexual aggression due to evolution and plasticity.

Female mating experience and genetic background independently influence male mating success in fruit flies.

When the reproductive interests of males and females conflict, males can evolve traits that are harmful to females, and females can coevolve traits to resist this harm. In the fruit fly, Drosophila melanogaster, there is genetic variation in female resistance traits, which can affect the pre- and post-mating success of males that try to mate with them. However, it is not clear to what extent the expression of these phenotypes can be modified by environmental factors such as sociosexual experience. Here, we tested how the genetic background of a female and her previous mating experience interact to affect the mating success of focal males. In the experience phase, we placed females from 28 distinct genetic backgrounds individually either with a single male (low conflict) or with three males (high conflict) for 48 hr. In the subsequent test phase, we measured the mating and post-mating fertilization success of focal males paired individually with each female. We found that focal males paired with females from the high-conflict treatment were less successful at mating, took longer to mate when they were successful, and had a lower proportion of paternity share. Furthermore, we identified significant female genetic variation associated with male mating success. These results indicate that female experience, along with intrinsic genetic factors, can independently influence different fitness components of her subsequent mates and has implications for our understanding of plastic female mating strategies and the evolution of sexually antagonistic traits in males and females.

Abnormal Social Interactions in a Drosophila Mutant of an Autism Candidate Gene: Neuroligin 3.

Social interactions are typically impaired in neuropsychiatric disorders such as autism, for which the genetic underpinnings are very complex. Social interactions can be modeled by analysis of behaviors, including social spacing, sociability, and aggression, in simpler organisms such as Drosophila melanogaster. Here, we examined the effects of mutants of the autism-related gene neuroligin 3 (nlg3) on fly social and non-social behaviors. Startled-induced negative geotaxis is affected by a loss of function nlg3 mutation. Social space and aggression are also altered in a sex- and social-experience-specific manner in nlg3 mutant flies. In light of the conserved roles that neuroligins play in social behavior, our results offer insight into the regulation of social behavior in other organisms, including humans.

Winners have higher pre-copulatory mating success but losers have better post-copulatory outcomes.

In many animals, the outcomes of competitive interactions can have lasting effects that influence an individual's reproductive success and have important consequences for the strength and direction of evolution via sexual selection. In the fruit fly, Drosophila melanogaster, males that have won previous contests are more likely to win in subsequent conflicts and losers are more likely to lose (winner-loser effects), but the direct fitness consequences and genetic underpinnings of this plasticity are poorly understood. Here, we tested how male genotype and the outcomes of previous male-male conflicts influence male pre- and post-copulatory success. We quantified pre-copulatory success in a choice and no-choice context, and post-copulatory success by quantifying ejaculate offensive and defensive ability. We found that winners have higher reproductive success compared to losers in both pre-copulatory scenarios. However, losers consistently mated for a longer duration, boosted female fecundity and had an increased paternity share when they were the first males to mate, suggesting increased investment into post-copulatory mechanisms. Finally, by using clonal hybrids from the Drosophila Genetic Reference Panel, we documented that genetic variation explained a sizeable proportion of the observed differences between lines, and of the interaction between line and winner and loser effects. Our results place the behavioural data on winner-loser effects in an evolutionary context by documenting the potential fitness gain to males from altering their reproductive strategy based on fighting experience. Our data may also explain the presence and maintenance of trade-offs between different male reproductive strategies.

Sociability in Fruit Flies: Genetic Variation, Heritability and Plasticity.

Sociability, defined as individuals' propensity to participate in non-aggressive activities with conspecifics, is a fundamental feature of behavior in many animals including humans. However, we still have a limited knowledge of the mechanisms and evolutionary biology of sociability. To enhance our understanding, we developed a new protocol to quantify sociability in fruit flies (Drosophila melanogaster). In a series of experiments with 59 F1 hybrids derived from inbred lines, we documented, first, significant genetic variation in sociability in both males and females, with broad-sense heritabilities of 0.24 and 0.21 respectively. Second, we observed little genetic correlation in sociability between the sexes. Third, we found genetic variation in social plasticity among the hybrids, with a broad-sense heritability of ~0.24. That is, genotypes differed in the degree of sociability after experiencing the same relevant social experience. Our data pave the way for further research on the mechanisms that underlie sociability as well as its ecological and evolutionary consequences.

Adult fruit fly attraction to larvae biases experience and mediates social learning.

We investigated whether adult fruit flies (Drosophila melanogaster) use cues of larvae as social information in their food patch choice decisions. Adult male and female fruit flies showed attraction to odours emanating from foraging larvae, and females preferred to lay eggs on food patches occupied by larvae over similar unoccupied patches. Females learned and subsequently preferred to lay eggs at patches with novel flavours previously associated with feeding larvae over patches with novel flavours previously associated with no larvae. However, when we controlled for the duration of exposure to each flavoured patch, females no longer preferred the flavour previously associated with feeding larvae. This suggests that social learning in this context is indirect, as a result of strong social attraction biasing experience.

Social attraction mediated by fruit flies' microbiome.

Larval and adult fruit flies are attracted to volatiles emanating from food substrates that have been occupied by larvae. We tested whether such volatiles are emitted by the larval gut bacteria by conducting tests under bacteria-free (axenic) conditions. We also tested attraction to two bacteria species, Lactobacillus brevis, which we cultured from larvae in our lab, and L. plantarum, a common constituent of fruit flies' microbiome in other laboratory populations and in wild fruit flies. Neither larvae nor adults showed attraction to axenic food that had been occupied by axenic larvae, but both showed the previously reported attraction to standard food that had been occupied by larvae with an intact microbiome. Larvae also showed significant attraction to volatiles from axenic food and larvae to which we added only either L. brevis or L. plantarum, and volatiles from L. brevis reared on its optimal growth medium. Controlled learning experiments indicated that larvae experienced with both standard and axenic used food do not perceive either as superior, while focal larvae experienced with simulated used food, which contains burrows, perceive it as superior to unused food. Our results suggest that flies rely on microbiome-derived volatiles for long-distance attraction to suitable food patches. Under natural settings, fruits often contain harmful fungi and bacteria, and both L. brevis and L. plantarum produce compounds that suppress the growth of some antagonistic fungi and bacteria. The larval microbiome volatiles may therefore lead prospective fruit flies towards substrates with a hospitable microbial environment.

Food selection in larval fruit flies: dynamics and effects on larval development.

Selecting food items and attaining a nutritionally balanced diet is an important challenge for all animals including humans. We aimed to establish fruit fly larvae (Drosophila melanogaster) as a simple yet powerful model system for examining the mechanisms of specific hunger and diet selection. In two lab experiments with artificial diets, we found that larvae deprived of either sucrose or protein later selectively fed on a diet providing the missing nutrient. When allowed to freely move between two adjacent food patches, larvae surprisingly preferred to settle on one patch containing yeast and ignored the patch providing sucrose. Moreover, when allowed to move freely between three patches, which provided either yeast only, sucrose only or a balanced mixture of yeast and sucrose, the majority of larvae settled on the yeast-plus-sucrose patch and about one third chose to feed on the yeast only food. While protein (yeast) is essential for development, we also quantified larval success on diets with or without sucrose and show that larvae develop faster on diets containing sucrose. Our data suggest that fruit fly larvae can quickly assess major nutrients in food and seek a diet providing a missing nutrient. The larvae, however, probably prefer to quickly dig into a single food substrate for enhanced protection over achieving an optimal diet.

Evolutionary biology of social expertise.

There is increasing evidence that competent handling of social interactions among conspecifics has positive effects on individual fitness. While individual variation in social competence has been appreciated, the role of long-term experience in the acquisition of superior social skills has received less attention. With the goal of promoting further research, we integrate knowledge across disciplines to assess social expertise, defined as the characteristics, skills and knowledge allowing individuals with extensive social experience to perform significantly better than novices on a given social task. We focus on three categories of social behaviour. First, animals can gain from adjusting social behaviour towards individually recognised conspecifics that they interact with on a regular basis. For example, there is evidence that some territorial animals individually recognise their neighbours and modify their social interactions based on experience with each neighbour. Similarly, individuals in group-living species learn to associate with specific group members based on their expected benefits from such social connections. Individuals have also been found to devote considerable time and effort to learning about the spatial location and timing of sexual receptivity of opposite-sex neighbours to optimise reproduction. Second, signallers can enhance their signals, and receivers can refine their response to signals with experience. In many birds and insects, individuals can produce more consistent signals with experience, and females across a wide taxonomic range can adaptively adjust mating preferences after perceiving distinct male signals. Third, in many species, individuals that succeed in reproducing encounter the novel, complex task of caring for vulnerable offspring. Evidence from a few species of mammals indicates that mothers improve in providing for and protecting their young over successive broods. Finally, for social expertise to evolve, heritable variation in social expertise has to be positively associated with fitness. Heritable variation has been shown in traits contributing to social expertise including social attention, empathy, individual recognition and maternal care. There are currently limited data associating social expertise with fitness, most likely owing to sparse research effort. Exceptions include maternal care, signal refinement, and familiarity with neighbours and group members. Overall, there is evidence that individuals in many species keep refining their social skills with experience throughout life. Hence we propose promising lines of research that can quantify more thoroughly the development of social expertise and its effects on fitness.

Recovery from social isolation requires dopamine in males, but not the autism-related gene nlg3 in either sex.

Social isolation causes profound changes in social behaviour in a variety of species. However, the genetic and molecular mechanisms modulating behavioural responses to social isolation and social recovery remain to be elucidated. Here, we quantified the behavioural response of vinegar flies to social isolation using two distinct protocols (social space preference and sociability, the spontaneous tendencies to form groups). We found that social isolation increased social space and reduced sociability. These effects of social isolation were reversible and could be reduced after 3 days of group housing. Flies with a loss of function of neuroligin3 (orthologue of autism-related neuroligin genes) with known increased social space in a socially enriched environment were still able to recover from social isolation. We also show that dopamine (DA) is needed for a response to social isolation and recovery in males but not in females. Furthermore, only in males, DA levels are reduced after isolation and are not recovered after group housing. Finally, in socially enriched flies mutant for neuroligin3, DA levels are reduced in males, but not in females. We propose a model to explain how DA and neuroligin3 are involved in the behavioural response to social isolation and its recovery in a dynamic and sex-specific manner.

Sexual conflict and social networks in bed bugs: effects of social experience.

Living in groups can provide essential experience that improves sexual performance and reproductive success. While the effects of social experience have drawn considerable scientific interest, commonly used behavioral assays often do not capture the dynamic nature of interactions within a social group. Here, we conducted 3 experiments using a social network framework to test whether social experience during early adulthood improves the sexual competence of bed bugs (Cimex lectularius) when placed in a complex and competitive group environment. In each experiment, we observed replicate groups of bed bugs comprising previously socialized and previously isolated individuals of the same sex, along with an equal number of standardized individuals of the opposite sex. Regardless of whether we controlled for their insemination history, previously isolated males mounted and inseminated females at significantly higher rates than previously socialized males. However, we found no evidence of social experience influencing our other measures of sexual competence: proportion of mounts directed at females, ability to overcome female resistance, and strength of opposite-sex social associations. We similarly did not detect effects of social experience on our female sexual competence metrics: propensity to avoid mounts, rate of successfully avoiding mounts, opposite-sex social association strength, and rate of receiving inseminations. Our findings indicate that early social experience does not improve sexual competence in male and female bed bugs.

Attraction to and learning from social cues in fruitfly larvae.

We examined the use of social information in fruitfly larvae, which represent an ideal model system owing to their robust learning abilities, small number of neurons and well-studied neurogenetics. Focal larvae showed attraction to the distinct odour emanating from food occupied by other larvae. In controlled learning experiments, focal larvae preferred novel odours previously paired with food occupied by other larvae over novel odours previously paired with unoccupied food. When we gave groups of larvae a choice between food patches differing in quality, more larvae aggregated on the higher-quality food, suggesting that attraction to and learning about cues associated with other larvae can be beneficial. Furthermore, larvae were more likely to find the best available food patch in trials when that food patch was occupied by other larvae than in trials when that food patch was unoccupied. Our data suggest, however, that the benefits from joining others may be at least partially offset by the fitness costs of increased competition, because larvae reared in isolation did as well as or better than larvae reared in groups on three key fitness parameters: developmental rate, survival rate and adult dry body mass. Our work establishes fruitfly larvae as a highly tractable model species for further research on the mechanisms that modulate behaviour and learning in a social context.

Evolution of sociability by artificial selection.

There has been extensive research on the ecology and evolution of social life in animals that live in groups. Less attention, however, has been devoted to apparently solitary species, even though recent research indicates that they also possess complex social behaviors. To address this knowledge gap, we artificially selected on sociability, defined as the tendency to engage in nonaggressive activities with others, in fruit flies. Our goal was to quantify the factors that determine the level of sociability and the traits correlated with this feature. After 25 generations of selection, the high-sociability lineages showed sociability scores about 50% higher than did the low-sociability lineages. Experiments using the evolved lineages indicated that there were no differences in mating success between flies from the low and high lineages. Both males and females from the low lineages, however, were more aggressive than males and females from the high lineages. Finally, the evolved lineages maintained their sociability scores after 10 generations of relaxed selection, suggesting no costs to maintaining low and high sociability, at least under our settings. Sociability is a complex trait, which we currently assess through genomic work on the evolved lineages.

Natural history of social and sexual behavior in fruit flies.

The past 2 decades have seen fruit flies being widely adopted for research on social behavior and aggression. This fruitful research, however, has not been well tied to fruit flies' natural history. To address this knowledge gap, I conducted a field study. My goal was to inform future research conducted in artificial surroundings, and to inspire new investigations that can rely more heavily on fruit flies' actual natural behavior. My two main novel findings were first, that flies in the field showed significant sociability, as they formed social groups rather than dispersed randomly among fruits of similar quality. Second, males showed fair levels of aggression towards each other as indicated by a lunging rate of 17 per hour, and lower rates of wing threat and boxing. Courtship was the most prominent activity on fruits, with females rejecting almost all males' advances. This resulted in an estimated mating rate of 0.6 per female per day. Flies showed a striking peak of activity early in the mornings, even at cold temperatures, followed by inactivity for much of the day and night. Flies, however, handled well high temperatures approaching 40 °C by hiding away from fruit and concentrating activity in the cooler, early mornings. My field work highlights a few promising lines of future research informed by fruit flies' natural history. Most importantly, we do not understand the intriguing dynamics that generate significant sociability despite frequent aggressive interactions on fruits. Males' responses to female rejection signals varied widely, perhaps because the signals differed in information content perceived by flies but not humans. Finally, flies tolerated cold early mornings perhaps owing to fitness benefits associated with increased mating and feeding opportunities at this time. Flies were adept at handling very high temperatures under the natural daily temperature fluctuations and availability of shelters, and this can inform more realistic research on the effects of global warming on animals in their natural settings.

Plasticity in male mating behavior modulates female life history in fruit flies.

In many species, intense male-male competition for the opportunity to sire offspring has led to the evolution of selfish reproductive traits that are harmful to the females they mate with. In the fruit fly, Drosophila melanogaster, males modulate their reproductive behavior based on the perceived intensity of competition in their premating environment. Specifically, males housed with other males subsequently transfer a larger ejaculate during a longer mating compared to males housed alone. Although the potential fitness benefits to males from such plasticity are clear, its effects on females are mostly unknown. Hence, we tested the long-term consequences to females from mating with males with distinct social experiences. First, we verified that competitive experience influences male mating behavior and found that males housed with rivals subsequently have shorter mating latencies and longer mating durations. Then, we exposed females every other day for 20 days to males that were either housed alone or with rivals, and subsequently measured their fitness. We found that females mated to males housed with rivals produce more offspring early in life but fewer offspring later in life and have shorter lifespans but similar intrinsic population growth rates. These results indicate that plasticity in male mating behavior can influence female life histories by altering females' relative allocation to early versus late investment in reproduction and survival.

Artificial selection on sexual aggression: Correlated traits and possible trade-offs.

Forced copulation is an extreme form of sexual aggression that can affect the evolution of sex-specific anatomy, morphology, and behavior. To characterize mechanistic and evolutionary aspects of forced copulation, we artificially selected male fruit flies based on their ability to succeed in the naturally prevalent behavior of forced matings with newly eclosed (teneral) females. The low and high forced copulation lineages showed rapid divergence, with the high lineages ultimately showing twice the rates of forced copulation as the low lineages. While males from the high lineages spent more time aggressively pursuing and mounting teneral females, their behavior toward non-teneral and heterospecific females was similar to that of males from the low lineages. Males from the low and high lineages also showed similar levels of male-male aggression. This suggests little or no genetic correlations between sexual aggression and non-aggressive pursuit of females, and between male aggression toward females and males. Surprisingly however, males from the high lineages had twice as high mating success than males from the low lineages when allowed to compete for consensual mating with mature females. In further experiments, we found no evidence for trade-offs associated with high forced mating rates: males from the high lineages did not have lower longevity than males from the low lineages when housed with females, and four generations of relaxed selection did not lead to convergence in forced mating rates. Our data indicate complex interactions among forced copulation success and consensual mating behavior, which we hope to clarify in future genomic work.

Social learning about egg-laying substrates in fruitflies.

Social learning, defined as learning from other individuals, has had dramatic effects on some species, including humans, in whom it has generated a rich culture. As a first step in examining the evolution of and mechanisms underlying social learning in insects, we tested for social learning in fruitflies (Drosophila melanogaster). Focal females (observers) that experienced novel food together with mated females (models), who had laid eggs on that food, subsequently exhibited a stronger preference for laying eggs on that food over another novel food compared with focal females that experienced the food alone. We observed no social learning, however, when observers experienced food with potentially more ambiguous social information provided by the presence of either virgin models or aggregation pheromone. This first documentation of social learning about egg-laying substrates in fruitflies builds on recent data indicating intricate use of social information by fruitflies and opens up exciting avenues for research on the evolution and neurogenetics of social learning using biology's major model system.

Bayesian approximations and extensions: optimal decisions for small brains and possibly big ones too.

We compared the performance of Bayesian learning strategies and approximations to such strategies, which are far less computationally demanding, in a setting requiring individuals to make binary decisions based on experience. Extending Bayesian updating schemes, we compared the different strategies while allowing for various implementations of memory and knowledge about the environment. The dynamics of the observable variables was modeled through basic probability distributions and convolution. This theoretical framework was applied to the problem of male fruit flies who have to decide which females they should court. Computer simulations indicated that, for most parameter values, approximations to the Bayesian strategy performed as well as the full Bayesian one. The linear approximation, reminiscent of the linear operator, was notably successful, and, without innate knowledge, the only successful learning strategy. Besides being less demanding in computation and thus realistic for small brains, the linear approximation was also successful at limited memory, which would translate into robustness in rapidly changing environments. Knowledge about the environment boosted the performance of the various learning strategies with maximal performance at large utilization of memory. Only for limited memory capacities, intermediate knowledge was most successful. We conclude that many animals may rely on algorithms that involve approximations rather than full Bayesian calculations because such approximations achieve high levels of performance with only a fraction of the computational requirements, in particular for extensions of Bayesian updating schemes, which can represent universal and realistic environments.