Erica L. Westerman

Mate preference learning, where individuals use past social experience to choose mates, is prevalent in many species. Yet its consequences on reproductive investment, and whether individuals differentially invest in reproduction based on their learned perception of the mates phenotype, are unknown. We addressed these questions using the butterfly Bicyclus anynana, where males acquire individual preferences for artificially painted 0-UV dorsal hindwing spotted (DHSN) females, who then lay more eggs. We measured the spermatophore proteins transferred by naive and experienced males to females manipulated to have preferred (0-DHSN) and unpreferred (2-DHSN) wing patterns. Using data independent acquisition (DIA), we identified 2144 proteins in the B. anynana spermatophore transferred to the female during mating. Experienced males that mated with preferred and unpreferred females had more differentially abundant (DA) proteins in their spermatophores that have functions in circadian rhythms, oogenesis, and neural signalling than naive males. Proteins associated with oogenesis were DA in spermatophores transferred to preferred females, which may contribute to why preferred females lay more eggs. Overall, our study provides evidence for the role of experience-induced behavioural plasticity in tailoring male ejaculates in butterflies and identifies proteins that influence female physiology and behaviours which directly affect the pairs overall reproductive fitness.

Reproductive isolation is a central driver of speciation and can be reinforced by both innate mate preferences and mate preference learning. Pigmentation genes are strong candidates for pleiotropic effects on these processes because they shape visual traits used in mate choice and may also influence neural function, yet their role in learning is poorly understood. Here, we test whether the pigmentation gene yellow affects innate mate preference and aversive mate preference learning in female Bicyclus anynana , and whether these effects are associated with changes in brain dopamine levels. Using CRISPR-Cas9, we knocked out the yellow gene, developed a mutant line, and tested the mate preferences and mate preference learning ability of mutant females compared to wild-type (WT) females. We find that loss of yellow does not alter assortative mating based on pigmentation or disrupt innate visual or olfactory preferences. In contrast, loss of yellow does influence learning ability, as mutant females failed to modify mate preference following aversive premating experience, indicating an impairment in aversive learning. Despite yellow ’s role in the melanin biosynthesis pathway, brain dopamine levels remain unchanged in mutant females relative to WT females. These findings identify yellow as a pleiotropic gene influencing both pigmentation and aversive mate preference learning, providing evidence that pigmentation genes can shape behavioral processes important for reproductive isolation and speciation. Significance statement: Pigmentation genes are best known for controlling color patterns, but they may also influence behavior through shared neural pathways. Here, we show that the pigmentation gene yellow is required for aversive mate preference learning in the butterfly Bicyclus anynana . Females lacking yellow retain normal innate visual and olfactory mate preferences but fail to learn to avoid previously unattractive mates. Surprisingly, this learning deficit is not associated with altered dopamine levels, suggesting that downstream neural signaling pathways are involved instead. These findings identify a gene that influences both pigmentation and learned mate preference, providing evidence that pigmentation genes can shape behavioral processes important for reproductive isolation and speciation.

Many animals change their behavior in response to social experiences by learning. Although social learning is adaptive, not all individuals learn. In Heliconius melpomene, H. m. malleti males respond to 2-day prior failed copulation experience by decreasing courtship, whereas H. m. rosina males do not. Here, we explore the transcriptomic differences in both the neural (brain) and sensory (eyes, antennae) tissues underlying this natural diversity in male aversive mate-preference learning. While the transcriptomic profiles of the two subspecies are inherently different across all three tissues, we found the greatest difference between the good ( H. m. malleti ), and bad ( H. m. rosina ) mate-preference learners in the brain, followed by the sensory tissues. Known learning genes and Gene Ontology terms were associated with differences in mate-preference learning, suggesting conserved learning pathways across animals. Genes within putative magic loci associated with colors, odors, and locomotion, were also differentially expressed between H. m. malleti and H. m. rosina, suggesting multimodal sensory processing may drive behavioral variance in these two subspecies. Overall, our study identifies genetic underpinnings for differences in preference learning, both in neural processing and sensory tissues. Selection on these genes/networks could result in preference learning-induced reinforcement, leading to reproductive isolation and speciation.

Signal efficacy is crucial to communicative behaviors. To be effective, a signal once broadcast must be capable of being received and interpreted by a receiver or receivers. In addition to maximizing the amount that a signal stimulates receptive organs, the timing of when a signal is broadcast would optimally synchronize with a receiver's peak receptivity. Here, we investigated whether peak timing of male courtship, which includes release of a pheromone required for copulation to occur, is synchronized with female pheromone sensitivity in the butterfly Bicyclus anynana (Lepidoptera: Nymphalidae). To do this, we performed behavioral, pheromone, and electrophysiological assays at dawn, noon and dusk. We also investigated the diel rhythmicity of antennae gene expression by performing RNA sequencing on antennal tissues collected at the same three time points. We found that although males court most often near dusk and produce pheromones at constant levels throughout the day, females are more sensitive to odorants in the morning. Furthermore, female antennae exhibited differential and diurnally rhythmic expression of olfactory receptors and genes associated with learning and sexual receptivity, as well as gene ontology (GO) terms related to fatty acid metabolism and trehalose biosynthesis, supporting our diel electrophysiological results. Previous literature indicates that females form both positive and negative mate preference associations with male pheromones in the morning, regardless of whether males court females. Thus, this asynchrony may enable female B. anynana to learn mate preferences before they are courted by conspecific (or potentially heterospecific) males.

Mate preference learning, where individuals learn to prefer or avoid specific phenotypes during mate selection, is pervasive across animal taxa and influences reproductive isolation and trait evolution. Despite its significance, the genetic basis underlying the associated valence attribution (whether to prefer or avoid) remains largely unclear. Both in terms of what genes are associated with attributing valence, and whether valence attribution is associated with transcriptional changes in both sensory tissues and neural circuits in the brain, or is restricted to the brain. Here, we investigate the neurogenomic basis of positive and negative mate preference learning using Bicyclus anynana butterflies. We put females in positive or negative training scenarios, as well as a naïve control, and compared their transcriptomic profiles across three tissues: antennae, eyes, and brain using RNA-sequencing. Our results reveal tissue-specific transcriptional responses, with the antennae showing higher transcriptomic changes during negative training and the eyes showing higher transcriptomic changes during positive training, relative to the naïve control, indicating that valence attribution during learning may not be restricted to transcriptional changes in the brain. We identified a subset of genes in each tissue whose expression patterns changed with valence, as well as genes previously linked to classical conditioning pathways, supporting the hypothesis that imprinting-like learning and classical conditioning share molecular mechanisms. Our findings suggest that valence attribution in mate preference learning involves tissue-specific transcriptional responses in sensory and brain tissues, emphasising the role of peripheral sensory systems in modulating learned mate preferences.

Synopsis: Plant–pollinator interactions have persisted and evolved over millions of years. These interactions are shaped by environmental factors. However, global environmental changes are disturbing these interactions in the Anthropocene. One way both plants and pollinators can respond (and potentially adapt) to these changing environments is through phenotypic plasticity mediated by epigenetic modifications and non-genetic inheritance. Yet, research on how, and to what extent, epigenetic modifications and non-genetic inheritance shape plant–pollinator dynamics is rare. In this forward-looking perspective, we discuss different ways in which the environment mediates epigenetic marks and non-genetic inheritance into the subsequent generation. By taking a broader perspective, we discuss four mechanisms of epigenetic modification and non-genetic inheritance in both plants and pollinator systems: epigenetic modifications, inter-generational non-genetic inheritance, transgenerational non-genetic inheritance, and cultural transmission. We discuss the roles of various epigenetic marks and the transfer of molecules that cause epigenetic changes and non-genetic inheritance in plants and pollinators, which either directly or indirectly affect the outcome of plant–pollinator interactions. We provide a framework for the ecological and evolutionary implications for inheritance of acquired traits driving plant–pollinator interactions and discuss its importance in a rapidly changing environment. Lastly, we suggest ways to experimentally test the role of epigenetic marks and non-genetic inheritance, and how to integrate such mechanisms into long-term studies on plant–pollinator interactions during the Anthropocene.

Synopsis: Animal-mediated pollination is one of the most ecologically and economically important mutualisms and serves as a remarkable example of cross-kingdom communication and coevolution. Unfortunately, pollinators, plants, and the interactions between them are threatened in the Anthropocene. While pollination emerges from interactions across biological scales, existing research and expertise have developed in distinct silos reflecting traditional fields of study such as ecology, plant physiology, neuroethology, etc. This forward-looking review and perspective is a culmination of the “Plant-pollinator interactions in the Anthropocene” symposium at the 2025 Society for Integrative and Comparative Biology meeting, which collected expertise across these disciplinary silos to identify pressing questions our community needs to tackle in the next decade. In this perspective piece, we argue that an integrative, organismally informed systems approach is critical to unraveling the complexity of how plant-pollinator relationships are impacted by dynamic anthropogenic stressors. Specifically, this calls for an intentional and iterative integration of holistic modeling studies with empirical studies. Modeling the emergent properties driven by organismal interactions in pollination systems can identify impactful variables; this in turn should drive design of empirical studies that elucidate how organisms respond to changing environments in the context of those impactful variables, feeding back into improved models. Repetition of this process will allow better predictive power over pollination stability in changing landscapes. Finally, we consider both existing barriers to this integration, as well as emerging opportunities (such as new technologies) that can help bridge across traditional fields.

Synopsis: Floral color plays a critical role in shaping plant–pollinator interactions, yet the extent to which lepidopteran flower visitation is color-dependent remains underexplored. Using over 8000 community-science observations from the southwestern Ozarks, central USA, from 2002 to 2024, we assessed whether butterflies and moths visit flowers randomly or according to color preferences, and how these preferences vary across taxa. Our results reveal that flower visitation by Lepidoptera is both flower color-dependent and Lepidoptera taxon-specific. Magenta flowers stood out as the most distinct in terms of their visitors. Although broad color preferences emerged at the family level, species-level analyses uncovered even greater complexity, with unique, non-random combinations of preferred flower colors. Lycaenidae exhibited the narrowest color spans, frequently visiting white and beige flowers. In contrast, monarchs and fritillaries (both from Nymphalidae), swallowtails (Papilionidae), and many skippers (Hesperiidae) visited flowers of nearly all available hues at similar rates. Some observed patterns were consistent with findings from other geographic regions, such as a strong preference for magenta and lavender in diurnal hawkmoths (Sphingidae). However, others were not, as many butterfly lineages associated with yellow flowers in other geographic regions instead showed a pronounced preference for red, crimson, and blush flowers in our study area. Our findings highlight the potential of community-science data for studying pollinator behavior at an unprecedented spatial and temporal scale, while also demonstrating diversity in Lepidoptera floral preferences and the potential for geographic variation in floral preferences within species.

Signal efficacy is crucial to communicative behaviours. To be effective, a signal once broadcast must be capable of being received and interpreted by a receiver or receivers. In addition to maximizing the amount that a signal stimulates receptive organs, optimally the timing of when a signal is broadcast would synchronize with a receiver’s peak receptivity. Here, we investigated whether peak timing of male courtship, which includes release of a pheromone required for copulation to occur, is synchronized with female pheromone sensitivity in the butterfly Bicyclus anynana (Lepidoptera: Nymphalidae). To do this, we performed behavioural and electrophysiological assays at dawn, noon, and dusk. We also investigated the circadian rhythmicity of antennae gene expression by performing RNA-Sequencing on antennal tissues collected at dawn, noon and dusk. We found that although males court most often near dusk, females are more sensitive to odorants in the morning. Furthermore, antennae exhibited differential and diurnally rhythmic expression of odorant receptors and genes associated with learning and sexual receptivity, as well as gene ontology (GO) terms related to fatty acid metabolism and trehalose biosynthesis, supporting our circadian electrophysiological results. Previous literature indicates females form both positive and negative mate preference associations with male pheromones in the morning, regardless of whether males court females. Thus, this asynchrony may enable female B. anynana to learn mate preferences before they are courted by conspecific (or potentially heterospecific) males. Summary: Male Bicyclus anynana butterflies court more at dusk while females are more sensitive to odorants in the morning. Patterns of antenna gene expression also suggest circadian rhythmicity of odorant sensitivity.