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