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).
References
Ackery, P. R. (1988). Hostplants and classification: A review of
nymphalid butterflies. Biological Journal of the Linnean Society ,33 , 95–203.
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J.
(1990). Basic local alignment search tool. Journal of Molecular
Biology , 215 , 403–410.
Ashley, J., Cordy, B., Lucia, D., Fradkin, L. G., Budnik, V., &
Thomson, T. (2018). Retrovirus-like Gag protein Arc1 binds RNA and
traffics across synaptic boutons. Cell , 11 , 262–274.
Avila, F. W., Mattei, A. L., & Wolfner, M. F. (2015). Sex peptide
receptor is required for the release of stored sperm by matedDrosophila melanogaster females. Journal of Insect
Physiology , 76 , 1–6.
Beacon, T. H., Delcuve, G. P., López, C., Nardocci, G., Kovalchuk, I.,
van Wijnen, A. J., & Davie, J. R. (2021). The dynamic broad epigenetic
(H3K4me3, H3K27ac) domain as a mark of essential genes. Clinical
Epigenetics , 13 , 138.
Bhaumik, V., & Kunte, K. (2018). Female butterflies modulate investment
in reproduction and flight response to monsoon-driven migrations.Oikos , 127 , 285–296.
Chapman, J. W., & Drake, V. A. (2019). Insect migration. In In
Encyclopedia of animal behavior. (M.D. Breed, J. Moore). Academic
Press.
Chapman, J. W., Reynolds, D. R., & Wilson, K. (2015). Long-range
seasonal migration in insects: Mechanisms, evolutionary drivers and
ecological consequences. Ecology Letters , 18 , 287–302.
Cheng, D., Cheng, T., Yang, X., Zhang, Q., Fu, J., Feng, T., Gong, J.,
& Xia, Q. (2018). The genome-wide transcriptional regulatory landscape
of ecdysone in the silkworm. Epigenetics and Chromatin ,11 , 48.
Dingle, H. (2014). Migration. The biology of life on the move.Oxford University Press.
Dingle, H., & Drake, V. A. (2007). What is migration?Bioscience , 57 , 113–121.
Ewels, P. A., Peltzer, A., Fillinger, S., Patel, H., Alneberg, J., Wilm,
A., Garcia, M. U., Di Tommaso, P., & Nahnsen, S. (2020). The nf-core
framework for community-curated bioinformatics pipelines. Nature
Biotechnology , 38 , 3.
Haussmann, I. U., Hemani, Y., Wijesekera, T., Dauwalder, B., & Soller,
M. (2013). Multiple pathways mediate the sex-peptide-regulated switch in
female Drosophila reproductive behaviours. Proceedings of
the Royal Society B: Biological Sciences , 280 , 20131938.
Hediger, M. A., Romero, M. F., Peng, J. B., Rolfs, A., Takanaga, H., &
Bruford, E. A. (2004). The ABCs of solute carriers: Physiological,
pathological and therapeutic implications of human membrane transport
proteins. Pflügers Archiv , 447 , 465–468.
Hekmat-Scafe, D. S., Scafe, C. R., McKinney, A. J., & Tanouye, M. A.
(2002). Genome-wide analysis of the odorant-binding protein gene family
in Drosophila melanogaster . Genome Research , 12 ,
1357–1369.
Herman, W. S., & Dallman, S. H. (1981). Endocrine biology of the
painted lady butterfly Vanessa cardui . Journal of Insect
Physiology , 27 , 163–168.
Howe, F. S., Fischl, H., Murray, S. C., & Mellor, J. (2017). Is H3K4me3
instructive for transcription activation? Françoise S. Howe, Harry
Fischl, Struan C. Murray, Jane Mellor. BioEssays , 39 ,
1–12.
Jiang, X. F., Luo, L. Z., & Sappington, T. W. (2010). Relationship of
flight and reproduction in beet armyworm, Spodoptera exigua(Lepidoptera: Noctuidae), a migrant lacking the oogenesis-flight
syndrome. Journal of Insect Physiology , 56 , 1631–1637.
Johnson, C. G. (1969). Migration and dispersal of insects by
flight. Methuen.
Kushwaha, P. P., Rapalli, K. C., & Kumar, S. (2016). Geminin a multi
task protein involved in cancer pathophysiology and developmental
process: A review. Biochimie , 131 , 115–127.
Law, C. W., Chen, Y., Shi, W., & Smyth, G. K. (2014). voom: Precision
weights unlock linear model analysis tools for RNA-seq read counts.Genome Biology , 15 , R29.
Lewis, J. J., van der Burg, K. R. L., Mazo-Vargas, A., & Reed, R. D.
(2016). ChIP-Seq-annotated Heliconius erato genome highlights
patterns of cis-regulatory evolution in Lepidoptera. Cell
Reports , 16 , 2855–2863.
Li, H. (2013). Aligning sequence reads, clone sequences and assembly
contigs with BWA-MEM. ArXiv , 00 , 1–3.
Lohse, K., Wright, C., Talavera, G., Garcia-Berro, A., & Darwin Tree of
Life Consortium. (2021). The genome sequence of the painted lady,Vanessa cardui , Linnaeus 1758. Wellcome Open Research ,6 , 324.
Menchetti, M., Guéguen, M., & Talavera, G. (2019). Spatio-temporal
ecological niche modelling of multigenerational insect migrations.Proceedings of the Royal Society Series B: Biological Sciences ,286 , 20191583.
Mosher, J., Zhang, W., Blumhagen, R. Z., D’Alessandro, A., Nemkov, T.,
Hansen, K. C., Hesselberth, J. R., & Reis, T. (2015). Coordination
between Drosophila Arc1 and a specific population of brain
neurons regulates organismal fat. Developmental Biology ,405 , 280–290.
Mugrabi-Oliveira, E., & Moreira, G. R. P. (1996). Conspecific mimics
and low host plant availability reduce egg laying by Heliconius
erato phyllis (Fabricius) (Lepidoptera, Nymphalidae). Revista
Brasileira de Zoologia , 13 , 929–937.
Patel, H., Wang, C., Ewels, P., Silva, T. C., Peltzer, A., Behrens, D.,
Garcia, M., mashehu, Rotholandus, Haglund, S., & Kretzschmar, W.
(2021). nf-core/chipseq: Nf-core/chipseq v1.2.2 - Rusty Mole .
Zenodo.
Quinn, L. M., Herr, A., McGarry, T. J., & Richardson, H. (2001). TheDrosophila Geminin homolog: Roles for Geminin in limiting DNA
replication, in anaphase and in neurogenesis. Genes and
Development , 15 , 2741–2754.
Rada-Iglesias, A., Bajpai, R., Swigut, T., Brugmann, S. A., Flynn, R.
A., & Wysocka, J. (2011). A unique chromatin signature uncovers early
developmental enhancers in humans. Nature , 470 , 279–283.
Ramaswamy, S. B., Shu, S., Park, Y. I., & Zeng, F. (1997). Dynamics of
juvenile hormone-mediated gonadotropism in the Lepidoptera. Insect
Biochemistry and Physiology , 35 , 539–558.
Reudler Talsma, J. H., Biere, A., Harvey, J. A., & van Nouhuys, S.
(2008). Oviposition cues for a specialist butterfly: Plant chemistry and
size. Journal of Chemical Ecology , 34 (9), 1202–1212.
Ritchie, M. E., Phipson, B., Wu, D., Hu, Y., Law, C. W., Shi, W., &
Smyth, G. K. (2015). Limma powers differential expression analyses for
RNA-sequencing and microarray studies. Nucleic Acids Research ,43 , e47.
Ruiz Vargas, N., Rowe, L., Stevens, J., Armagost, J. E., Johnson, A. C.,
& Malcolm, S. B. (2018). Sequential partial migration across monarch
generations in Michigan. Animal Migration , 5 , 104–114.
Santos-Rosa, H., Schneider, R., Bannister, A. J., Sherriff, J.,
Bernstein, B. E., Emre, N. C. T., Schreiber, S. L., Mellor, J., &
Kouzarides, T. (2002). Active genes are tri-methylated at K4 of histone
H3. Nature , 419 , 407-411.
Shipilina, D., Näsvall, K., Höök, L., Vila, R., Talavera, G., &
Backström, N. (2022). Linkage mapping and genome annotation give novel
insights into gene family expansions and regional recombination rate
variation in the painted lady (Vanessa cardui ) butterfly.Genomics , 114 , 110481.
Stefanescu, C., Páramo, F., Åkesson, S., Alarcón, M., Ávila, A.,
Brereton, T., Carnicer, J., Cassar, L. F., Fox, R., Heliölä, J., Hill,
J. K., Hirneisen, N., Kjellén, N., Kühn, E., Kuussaari, M., Leskinen,
M., Liechti, F., Musche, M., Regan, E. C., … Chapman, J. W.
(2013). Multi-generational long-distance migration of insects: Studying
the painted lady butterfly in the Western Palaearctic. Ecography ,36 (4), 474–486.
Stefanescu, C., Ubach, A., & Wiklund, C. (2021). Timing of mating,
reproductive status and resource availability in relation to migration
in the painted lady butterfly. Animal Behaviour, 172 ,
145–153.
Talavera, G., Bataille, C., Benyamini, D., Gascoigne-Pees, M., & Vila,
R. (2018). Round-trip across the Sahara: Afrotropical painted lady
butterflies recolonize the Mediterranean in early spring. Biology
Letters , 14, 20180274 .
Talavera, G., & Vila, R. (2016). Discovery of mass migration and
breeding of the painted lady butterfly Vanessa cardui in the
Sub-Sahara: The Europe–Africa migration revisited. Biological
Journal of the Linnean Society , 120 , 274–285.
Tang, X. F., Chen, X. Y., Zhang, C. D., Li, Y. F., Liu, T. H., Zhou, X.
L., Wang, L., Zhang, Q., Chen, P., Lu, C., & Pan, M. H. (2017). Two
Geminin homologs regulate DNA replication in silkworm, Bombyx
mori . Cell Cycle , 16 , 830–840.
Tigreros, N., & Davidowitz, G. (2019). Flight-fecundity trade-offs in
wing-monomorphic insects. Advances in Insect Physiology ,56 , 1–41.
Tsuruga, H., Yabuta, N., Hosoya, S., Tamura, K., Endo, Y., & Nojima, H.
(2016). HsMCM6: A new member of the human MCM/P1 family encodes a
protein homologous to fission yeast Mis5. Genes to Cells ,2 , 381–399.
Wegener, G. (1996). Flying insects: Model systems in exercise
physiology. Experientia , 52 , 404–412.
Wiklund, C., & Friberg, M. (2022). Testing the migration syndrome:
Comparative fecundity of migratory and non-migratory nymphaline
butterflies. Ecological Entomology , 2022 , 1–7.
Zhang, Y., Liu, T., Meyer, C. A., Eeckhoute, J., Johnson, D. S.,
Bernstein, B. E., Nusbaum, C., Myers, R. M., Li, W., & Liu, S. (2008).
Model-based analysis of ChIP-Seq (MACS). Genome Biology ,9 , R137.
Zhao, W., Xu, Y., Wang, Y., Gao, D., King, J., Xu, Y., & Liang, F. S.
(2021). Investigating crosstalk between H3K27 acetylation and H3K4
trimethylation in CRISPR/dCas-based epigenome editing and gene
activation. Scientific Reports , 11 , 15912.
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.