Figure 2. A volcano plot showing the significance level (Y-axis, - logarithmic p-value) and logarithmic fold change in activity (X-axis) in the comparison between treatment groups for the analysis of H3K27ac. Non-significant loci are indicated with grey dots and loci with significantly (adjusted for multiple testing) different acetylation activity with red dots.
We proceeded and intersected the signals from the two different histone marks. This analysis unveiled that six of the 9 regions with significantly different activity signals in H3K27ac also had considerable activity peaks for H3K4me3, and an additional proximal differentially activated peak close to the gene SLC which has been inferred to be involved in carbohydrate metabolism.
Discussion
Here we used an experimental set-up where female painted lady butterflies were exposed to environments that varied in host plant abundance. We combined this with analysing regulatory element activity to get insights into the pathways that were differentially regulated between painted lady individuals exposed to different environmental conditions. Besides giving information about differences in transcriptional activity between female butterflies exposed to different environmental settings, this is one of the first attempts to characterise the genome wide distribution of H3K27ac and H3K4me3 in butterflies in detail.
We found that females that had access to host plants had laid significantly more eggs than females in cages without host plants. This is in line with the observations that female butterflies can sense both relative abundance of host plants and presence of conspecifics (Mugrabi-Oliveira & Moreira, 1996), and the amount of secondary compounds for oviposition selection (Reudler Talsma et al., 2008). The higher egg laying propensity in the treatments where females had access to host plants can obviously just be a consequence of availability of host plant substrate, and not indicate a delayed investment in reproduction. However, the observation supports previous results that indicate a higher frequency of reproductively active females in areas where host plants are abundant (Stefanescu et al., 2021).
In order to investigate how the differences in environment affected the activity of regulatory elements, we harvested the females from the different experimental cohorts at day five after eclosure and analysed regulatory element activity using ChIP-Seq. Information about the genome-wide regulatory landscape in Lepidoptera is limited to a few species. As a first step, we therefore identified genome-wide activity peaks for both H3K27ac and H3K4me3. Previous studies in Lepidoptera have shown that histone tail modifications (and regulatory activity) can vary across tissues and between developmental and metabolic stages (Cheng et al., 2018; Lewis et al., 2016). In the painted lady butterfly, 4,744 of the H3K4me3 peaks were located in proximal regions of genes potentially representing promoters and proximal regulatory elements. This is approximately in the same range as what has previously been observed in both B. mori (n = 5,599 ‘proximal elements’) and Heliconius erato (n = 5,399) (Cheng et al., 2018; Lewis et al., 2016). The observed agreement in both absolute numbers and the spatial distribution of activity peaks suggests that the genome wide distribution of these active histone marks has been accurately characterized in the painted lady butterfly.
The core question in this study was to investigate potential differences in regulatory element activity between treatment groups that could inform on how environmental differences affect gene regulation and, ultimately, the behaviour of individual butterflies. Only H3K27ac showed loci with significant differential activity between the treatment groups and 9 of those were in the vicinity of coding genes. When overlaid with information from the analysis of H3K4me3, we found that seven regions with differential activity coincided with H3K4me3 modifications. Hence, it is likely that the chromatin in the regions of these particular genes was accessible, since H3K4me3 activity was present in both treatments. In addition, H3K27ac and H3K4me3 have been shown to interact to enhance the transcriptional activity (Zhao et al., 2021) and it is therefore plausible that differential acetylation corresponds to significant differences in activity between individuals exposed to different environmental conditions (i.e. different host plant densities).
The potential functions of the significantly differentially activated genes were assessed and we found gene ontology information for eight of the candidate genes. This set included two genes associated with the mini-chromosome maintenance (MCM ) – the genes MCM6 andGEMININ - which have been shown to be involved in the regulation of MCM6 (Kushwaha et al., 2016). The MCM gene family consists of several gene copies that jointly affect chromatin unwinding and the complex has been shown to be involved in DNA-replication (Tsuruga et al., 2016). Interestingly, GEMININ , which showed a significant activity peak in individuals exposed to high host plant density, has been shown to be involved in DNA-replication control inB. mori (Tang et al., 2017) and expressed in insect ovaries during oogenesis (Quinn et al., 2001). It is hence tempting to speculate that the higher activity is associated with the more advanced reproductive mode in this treatment group. We also found that the genesTTN (titin-like) and SLC (solute carrier family 2) showed higher activity in the treatment where females had access to host plants. SLC is a member of a large family of transmembrane genes where the gene product forms a solute carrier that mediates transport of, for example, glucose and amino acids across cell membranes (Hediger et al., 2004). The titin-like gene product is a structural protein involved in muscle formation and function (https://flybase.org/reports/FBgn0086906.html; accessed 2022-12-09) and a different activity of regulatory regions of TTN in the treatment group with host plants is in line with the predictions of the oogenesis-flight syndrome, since flight muscle activity should be reduced when reproductive mode is more advanced compared to when individuals are in the migratory phase. In agreement with this, we found that an activity dependent transporter gene (ARC1 ) had significantly higher acetylation levels in the second intron in the group without host plant. In Drosophila , ARC1 mediates transfer of mRNA from motor neurons to the muscle tissue (Ashley et al., 2018) and it has also been linked body fat accumulation (Mosher et al., 2015). Both functions can be directly linked to migratory behaviour since motor neuron activity and efficient fat storage are key components of long-distance flight. The gene SPR (sex peptide receptor) also showed differential regulatory element activity between treatment groups, with a higher activity in the individuals exposed to an environment without host plants. SPR is expressed in the central nervous system and the reproductive tract in Drosophila melanogaster females where it has been shown to regulate post-mating responses, for example sperm release, egg-laying capacity and reduced receptivity (Avila et al., 2015; Haussmann et al., 2013). It seems plausible that this pattern reflects delayed female receptivity in female painted ladies when no host plants are available for egg laying. Another interesting gene that showed differential regulatory activity was the odorant binding protein, OBP . OBP expression is associated with pheromone binding activity and perception of smell (Hekmat-Scafe et al., 2002). Since pheromone signalling likely is a key component for courtship and mating acceptance in many lepidopterans, the difference in activity could be a result of differences in reproductive status between treatment groups. Finally, the butterflies with access to host plants showed a higher acetylation peak in the second intron of a juvenile hormone esterase (JHE) . JHE is involved in degradation of juvenile hormone (JH) , a key hormone in female reproductive maturation (Herman & Dallman, 1981) and associated with dispersal versus reproduction decisions in many insects (Ramaswamy et al., 1997). The increased activation of JHE in the butterflies with access to host plants is perhaps counterintuitive. However, since we harvested females five days after eclosure, it is possible, or even likely, that the differences in regulatory element activity we detected result from cascading effects that were initiated at an earlier time point. This does obviously not only concern the JHE / JHregulation, but all other differentially activated regions. Future efforts to characterize the genetic / regulatory underpinnings of the oogenesis-flight syndrome might benefit from sampling across multiple time points to cover the temporal dynamics of regulatory cascades.
Our results provide a starting point to investigate how regulation of the identified candidate genes affect individual behavioural strategies in painted lady butterflies and could also be relevant for future assessments of the generality of specific pathways underlying the oogenesis-flight syndrome in insects at a larger scale.
Acknowledgements
We would like to acknowledge members of the Backström lab, especially Orazioluca Paternò, for their work in the butterfly lab and Patrick Nylund at the Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, for providing the sonicator. This work was funded by the Swedish Research Council FORMAS (FORMAS research grant #2019-00670 to N.B.). The authors acknowledge support from the National Genomics Infrastructure in Stockholm funded by Science for Life Laboratory, the Knut and Alice Wallenberg Foundation and the Swedish Research Council, and SNIC/Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure. This work was also supported by NBIS/SciLifeLab long-term bioinformatics support (WABI). R.V. was supported by project PID2019-107078GB-I00/MCIN/AEI/10.13039/501100011033. G.T. was supported by the grant PID2020-117739GA-I00 MCIN / AEI / 10.13039/501100011033; all authors were also supported by the grant LINKA20399 from the CSIC iLink program.
Data availability
The raw sequence data is deposited in the European Nucleotide Archive (ENA) with accession number PRJEB59028. Scripts for the analyses are available in GitHub (https://github.com/EBC-butterfly-genomics-team).
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Supplementary information
Supplementary Table 1. Annotation information, genomic position and adjusted p-values for the significantly differentially activated genes identified in the comparison between treatment groups.