Vanessa cardui.
Running title:
Regulatory elements, oviposition and migratory behaviour in V. cardui .
Abstract
The oogenesis-flight syndrome reflects the temporal allocation of energy resources between dispersal / migration and reproduction and is a key concept in research on migratory behaviour in animals. In migratory butterflies, host plant abundance and quality may act as environmental cues to switch between the two states, but the mechanisms regulating this process are virtually unknown. Here, we used an experimental set-up to assess how variation in host plant abundance affected the activity of regulatory elements in the painted lady butterfly (Vanessa cardui ), a model species for insect migratory behaviour studies. Chromatin immunoprecipitation (ChIP-seq) was used to evaluate histone tail modifications of H3K27ac and H3K4me3, as a proxy for regulatory activity. The results indicate that recently eclosed females that had access to host plants invested in reproduction at an earlier stage and that variation in host plant abundance triggered significant differences in regulatory element activity via histone tail acetylation. The functions of genes in the vicinity of differentially activated regions were primarily associated with metabolism, egg shell formation, female receptivity, muscle activity, pheromone binding and chromosome maintenance. Our results provide a first glimpse into the regulatory underpinnings of how females perceive the environment and allocate resources for either migration or reproduction and a starting point for more detailed understanding of the links between environmental variation, gene regulation and behaviour in butterflies.
Introduction
Temporal heterogeneity in suitable habitat for reproduction is a main driver of insect adaptations. Some species cope with seasonally unfavourable conditions by entering a phase of dormancy, the diapause. Other species move between distinct geographic regions in more or less regular circuits to utilize temporally and spatially variable habitat patches. In insects, such migratory movements predominantly occur by flight and migration itself is obviously associated with individual costs (Dingle & Drake, 2007). Migratory flight can for example increase the risk for predation, and unpredictable weather conditions during migration can lead to severe deviations from the optimal flight direction and distance. In addition, migration may not necessarily lead to that the individual ends up in a more suitable habitat patch than the one it left. The energetic demands for migratory flight are also extremely high and a migrating individual has to allocate considerable resources to sustain the effort, resources that could otherwise be used for investment in reproduction (Wegener, 1996). Investment in reproduction can also come with a cost if conditions for egg laying are suboptimal and/or if host plant availability is limited. The evolution of migratory behaviour hence involves a cost-benefit trade-off, in essence reflecting the more short-term benefits of investing resources in reproduction in a potentially less suitable habitat patch versus the more long-term advantages of dispersing to, and colonizing, habitat patches with more suitable conditions for feeding and oviposition (Chapman et al., 2015).
The “oogenesis-flight syndrome” is a model for migratory insects which postulates that migratory and reproductive states are temporally separated during the life span of adult females (Chapman & Drake, 2019; Johnson, 1969). Although this generalisation does not apply to all migratory insects – observations suggest that some species are reproductively active also during the migratory phase (e.g. Jiang et al., 2010; Tigreros and Davidowitz, 2019) – there is a general consensus that the oogenesis-flight syndrome serves as a robust overarching model for insect migration studies (Bhaumik & Kunte, 2018; Chapman & Drake, 2019; Dingle, 2014). Two of the main model systems for butterfly migration are the monarch (Danaus chrysippus ) and the painted lady butterfly (Vanessa cardui ). In monarchs, females fulfil the oogenesis-flight syndrome during fall migration, but not in spring when they leave the overwintering areas and start migrating northwards (Ruiz Vargas et al., 2018). In the painted lady on the other hand, empirical data point towards lower mating propensity in females during the migratory phase indicating reduced investment in reproduction (Stefanescu et al., 2021). Besides that the oogenesis-flight syndrome potentially can be applied to the migrating female painted ladies, the species exhibits life-history characteristics that reflect long-distance migratory behaviour within different circuits that cover almost all continents – the best-studied circuit spans the range between sub-Saharan Africa and northern Europe (Menchetti et al., 2019; Stefanescu et al., 2013; Talavera et al., 2018; Talavera & Vila, 2016). In contrast to the monarch, painted ladies never enter diapause and the annual migratory circuit involves multiple generations (6-8, or even more) (Menchetti et al., 2019; Talavera & Vila, 2016). The species is highly polyphagous and can therefore utilize the wide range of host plants encountered during alternating seasons in different parts of the almost cosmopolitan distribution range (Ackery, 1988). The painted lady butterfly therefore constitutes an attractive model for investigating both the mechanistic basis of different aspects of migratory behaviour in general and the genetic underpinnings of the oogenesis-flight syndrome specifically.
The behavioural and physiological changes induced by environmental cues are mediated by the activation of different regulatory elements involved in transcriptional regulation. One way to characterise the genes and pathways involved is therefore to assess the chromatin state in regulatory elements. Chromatin accessibility and transcriptional activity is largely determined by histone tail modifications. For example, acetylation of the histone tail residue lysine 27 (H3K27ac) reduces the affinity of DNA to the histones. This leads to accessible chromatin around promoters and enhancers of actively transcribed genes (Rada-Iglesias et al., 2011). H3K4me3 on the other hand is predominantly positioned within promoters and proximal regulatory elements and has been associated with transcriptional activity via recruitment of transcription factors and histone modifiers (Santos-Rosa et al., 2002), although the precise molecular mechanisms here are not yet fully understood (Beacon et al., 2021; Howe et al., 2017). Chromatin immunoprecipitation followed by DNA-sequencing (ChIP-seq) is a method to identify DNA-sequences associated with attached proteins. By quantifying the enrichment of specific histone tail modifications, both the position and activity of regulatory elements and the adjacent genes can be characterized.
Here we used an experimental set-up where recently eclosed female painted lady butterflies were exposed to environments that differed in the availability of host plants. By quantifying differences in activity of regulatory elements between females from the treatment groups, we provide a first glimpse into how resource availability affects activation of different genetic pathways in a migratory butterfly.
Materials and methods