Caroline Marie Nieberding

There is broad evidence that the main driver of the ongoing biodiversity crisis is land-use change, which reduces and fragments habitats. The consequence of habitat fragmentation on behavioural responses of fitness-related traits in insects have been so far understudied. In herbivorous insects, oviposition-related behaviours determine access to larval food, and the fate of the next generation. We present a pilot study to assess differences in behaviours related to movement and oviposition in Limenitis camilla butterflies from Wallonia (Belgium), one of the most fragmented regions in Europe. We first quantified variation in functional habitat connectivity across Wallonia and found that fragmented habitats had more abundant, but less evenly distributed host plants of L. camilla. Secondly, we quantified the behaviours of field-caught L. camilla females originating from habitats with contrasted landscape connectivity in an outdoor experimental setting. We found differences in behaviours related to flight investment: butterflies from fragmented woodlands spent more time in departing flight, which we associated with dispersal, than butterflies from homogenous woodlands. Although results from this study should be interpreted with caution given the limited sample size, they provide valuable insights for the advancement of behavioural research that aims to assess the effects of global changes on insects.

For small ectotherm species, dealing with microclimatic variation during sensitive stages of their development is a critical component of their ecological interactions within and among different landscape settings. However, it is often unknown whether the use of habitats in different landscape settings may affect microclimatic conditions at the level of microhabitats (e.g., at host plants for herbivorous insects). As opposed to standard ecological monitoring experiments, we aim to identify the microclimatic variables that accurately represent the environment in which a small arthropod spends most of its life. As a case study, we selected grassy sites that corresponded to the microhabitat of grass‐feeding insects in general, and larvae of the butterfly Pararge aegeria in particular, as this insect recently expanded its habitat use from forest (edges) to agricultural and urban environments. We tested the extent to which local microclimates and microclimatic buffering capacity differed between tufts of grass in forest and in two anthropogenic (i.e., agricultural and urban) landscape settings by measuring microclimatic variables with sensors at the level of the grasses. We compared temperature, relative humidity, and vapor pressure deficit (VPD) during an exceptionally warm and dry summer period with parallel data from weather stations and tested for differences between microclimatic profiles among the three landscape settings. Microclimatic conditions correspond only partly to variables based on weather station data. Differences are particularly significant for relative humidity and VPD, but not for day and night temperatures. Effects of canopy openness on the variability of microclimatic variables are landscape‐specific. Host plants in agricultural settings experience strong microclimatic fluctuations and low short‐term variability in VPD. In urban settings, urban heat island effects increase night temperature but also show microclimatic buffering effects similar to the ones in forest settings. Our findings stress the functional implications of landscape‐specific microclimatic profiles at the appropriate organism‐centered scale. We discuss the significance of such microclimatic profiles for ecological studies dealing with ectotherms under climate and land use change.

Human induced environmental changes are accelerating at an unprecedented pace, forcing organisms to rapidly adjust their behaviours. There is broad evidence that the main driver of the ongoing biodiversity crisis is land-use change, that reduces and fragments natural habitats. However, the consequence of habitat fragmentation on behavioural responses of fitness-related traits such as oviposition site selection in insects, which represent about 50% of \ Earth’s species diversity, have been so far understudied. In herbivorous insects, oviposition-related behaviours determine larval food access, and thus the fate of the next generation. We present a pilot study to assess differences in oviposition-related behaviours in Limenitis camilla butterflies from Wallonia (Belgium), one of the most fragmented regions in Europe. We first quantified variation in functional habitat connectivity for L. camilla across Wallonia and found that fragmented habitats had more abundant, but less evenly distributed Lonicera periclymenum , the host plant of L. camilla . Secondly, we compared in a semi-natural experimental setting the behaviours of field-caught L. camilla females originating from habitats with contrasted landscape connectivity. We found differences in behaviours related to flight investment: butterflies from fragmented woodlands spent more time in non-compass orientation flight, which we associated with dispersal, than butterflies from homogenous woodlands, where L. periclymenum was less abundant and more evenly distributed. Although results from this study should be interpreted with caution given the limited sample size, they provide valuable insights for the advancement of behavioral research that aims to assess the effects of global changes on insects.

The impact of learning ability and sexual selection on the climate and biodiversity crisis is currently unclear. Using the African butterfly Bicyclus anynana, which shows strong phenotypic plasticity (i.e., polyphenism) in response to temperature, we tested whether learning affects mate preferences under climate warming. We first modelled climate warming scenarios and then showed experimentally that temperature becomes an unreliable cue to the onset of the dry season, adult butterflies displayed the wet season rather than the dry season form. Experienced females that were exposed to different male seasonal phenotypes during sexual maturation changed sexual preferences. Female fertilization success was reversed for naive compared with experienced females, likely reducing female fitness following climate warming. Our results emphasize the importance of sexual selection, learning, and their fitness consequences for understanding (mal)adaptation of natural populations to climate warming. Read the free Plain Language Summary for this article on the Journal blog.

Unraveling the origin of molecular pathways underlying the evolution of adaptive traits is essential for understanding how new lineages emerge, including the relative contribution of conserved ancestral traits and newly evolved derived traits. Here, we investigated the evolutionary divergence of sex pheromone communication from moths (mostly nocturnal) to butterflies (mostly diurnal) that occurred ~119 million years ago. In moths, it is the females that typically emit pheromones to attract male mates, but in butterflies males emit pheromones that are used by females for mate choice. The molecular bases of sex pheromone communication are well understood in moths, but they have remained relatively unexplored in butterflies. We used a combination of transcriptomics, real time qPCR, and phylogenetics to identify genes involved in the different steps (i.e., production, regulation, and reception) of sex pheromone communication of the butterfly Bicyclus anynana. Our results show that the biosynthesis and reception of sex pheromones relies both on moth-specific gene families (reductases) and on more ancestral insect gene families (desaturases, olfactory receptors, odorant binding proteins). Interestingly, B. anynana appears to use what was believed to be the moth-specific neuropeptide Pheromone Biosynthesis Activating Neuropeptide (PBAN) for regulating sex pheromone production. Altogether, our results suggest that a mosaic pattern best explains how sex pheromone communication evolved in butterflies, with some molecular components derived from moths, and others conserved from more ancient insect ancestors. This is the first large-scale investigation of the genetic pathways underlying sex pheromone communication in a butterfly.

Research on social learning has centered around vertebrates, but evidence is accumulating that small-brained, non-social arthropods also learn from others. Social learning can lead to social inheritance when socially acquired behaviors are transmitted to subsequent generations. Using oviposition site selection, a critical behavior for most arthropods, as an example, we first highlight the complementarities between social and classical genetic inheritance. We then discuss the relevance of studying social learning and transmission in non-social arthropods and document known cases in the literature, including examples of social learning from con- and hetero-specifics. We further highlight under which conditions social learning can be adaptive or not. We conclude that non-social arthropods and the study of oviposition behavior offer unparalleled opportunities to unravel the importance of social learning and inheritance for animal evolution.

Habitat fragmentation increases the isolation of natural populations resulting in reduced genetic variability and increased species extinction risk. Behavioral innovation through learning, i.e., the expression of a new learned behavior in a novel context, can help animals colonize new suitable and increasingly fragmented habitats. It has remained unclear, however, how reduced genetic variability affects learning for colonizing more or less suitable habitats. Here, we show that inbreeding in a subsocial invertebrate, the spider mite Tetranychus urticae , reduces novel host plant colonization and reproductive fitness. When provided with the possibility to learn from previous experience with a host plant species, outbred mites showed aversive learning ability, but inbred mites did not adapt their behavior. We further found a putative general cost of learning in both inbred and outbred mites. Our results reveal that inbreeding affects the learning component of behavioral innovation for host plant colonization.

The impact of learning ability and sexual selection on the climate and biodiversity crisis are currently unclear. Using the African butterfly Bicyclus anynana , which shows strong phenotypic plasticity (i.e., polyphenism) in response to temperature, we tested whether learning affects mate preferences under climate warming. We first modelled climate warming scenarios and then showed experimentally that as temperature becomes an unreliable cue to the onset of the dry season, adult butterflies displayed the wet season rather than the dry season form. Experienced females that were exposed to different male seasonal phenotypes during sexual maturation changed sexual preferences. Female fertilization success was reversed for naive compared to experienced females, likely reducing female fitness following climate warming. Our results emphasize the importance of sexual selection, learning, and their fitness consequences for understanding (mal)adaptation of natural populations to climate warming.

Unraveling the origin of molecular pathways underlying the evolution of adaptive traits is essential for understanding how new lineages emerge, including the relative contribution of conserved, ancestral traits, and newly evolved, derived traits. Here, we investigated the evolutionary divergence of sex pheromone communication from moths (mostly nocturnal) to butterflies (mostly diurnal) that occurred ~98 million years ago. In moths, females typically emit pheromones to attract male mates, but in butterflies pheromones and used by females for mate choice. The molecular bases of sex pheromone communication are well understood in moths, but have remained virtually unexplored in butterflies. We used a combination of transcriptomics, real time qPCR, and phylogenetics, to identify genes involved in different steps of sex pheromone communication in the butterfly Bicyclus anynana . Our results show that the biosynthesis and reception of sex pheromones relies both on moth-specific gene families (reductases) and on more ancestral insect gene families (desaturases, olfactory receptors, odorant binding proteins). Interestingly, B. anynana further appears to use what was believed to be the moth-specific neuropeptide Pheromone Biosynthesis Activating Neuropeptide (PBAN) for regulation of sex pheromone production. Altogether, our results suggest that a mosaic pattern best explains how sex pheromone communication evolved in butterflies, with some molecular components derived from moths, and others conserved from more ancient insect ancestors. This is the first large-scale analysis of the genetic pathways underlying sex pheromone communication in a butterfly.

Polyphenism is a type of phenotypic plasticity supposedly adaptive to drastic and recurrent changes in the environment such as seasonal alternation in temperate and tropical regions. The butterfly Bicyclus anynana shows polyphenism with well-described wet and dry seasonal forms in sub-Saharan Africa, displaying striking morphological, physiological and behavioural differences in response to higher or lower developmental temperatures. During the seasonal transition in the wild, the intermediate phenotype co-occurs with wet and dry phenotypes. In this study, we aimed to characterize the secondary sexually-selected wing traits of the intermediate form to infer its potential fitness compared to wet and dry phenotypes. Among the previously described wing morphological traits, we first showed that the area of the fifth eyespot on the ventral hindwing is the most discriminant trait to identify wet, dry and intermediate phenotypes in both sexes. Second, we characterized the intermediate form for two secondary sexually-selected wing traits: the area and UV reflectance of the dorsal forewing pupil and the composition of the male sex pheromone. We showed that values of these two traits are often between those of the wet and dry phenotypes. Third, we observed increasing male sex pheromone production in ageing dry and wet phenotypes. Our results contrast with previous reports of values for sexually-selected traits in wet and dry seasonal forms, which might be explained by differences in rearing conditions or sample size effects among studies. Wet, dry and intermediate phenotypes display redundant sexually dimorphic traits, including sexually-selected traits that can inform about their developmental temperature in sexual interactions.