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\begin{document}

\title{Plasticity for the win: Flexible transcriptional response to host plant
switches in the comma butterfly (Polygonia c-album)}



\author[1]{Katharina Schneider}%
\author[1]{Rachel Steward}%
\author[1]{Maria de la Paz Celorio-Mancera}%
\author[1]{Niklas Janz}%
\author[1]{Dick Moberg}%
\author[1]{Christopher Wheat}%
\author[1]{Sören Nylin}%
\affil[1]{Stockholm University}%


\vspace{-1em}



  
  \date{May 22, 2024}


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\selectlanguage{english}
\begin{abstract}
Generalist butterflies are characterized by a broad host repertoire that
can comprise several families or even different orders of plants. The
genetic and physiological mechanisms underlying the use of such a wide
host range are still not fully understood. Earlier studies indicate that
the consumption of different host plants is associated with
host-specific gene expression profiles. It remained, however, unclear if
and how larvae can alter these profiles in the case of a changing host
environment. Using the polyphagous comma butterfly (Polygonia c-album)
we show that larvae can adjust their transcriptional profiles in
response to a new host plant. The switch to some of the host plants,
however, resulted in a larger transcriptional response and, thus, seems
to be more challenging. At a physiological level, no correspondence for
these patterns could be found in larval performance. This suggests that
a high transcriptional but also phenotypic flexibility are essential for
the use of a broad and diverse host range. We furthermore propose that
host switch tests in the laboratory followed by transcriptomic
investigations can be a valuable tool to examine not only plasticity in
host use but also subtle and/or transient trade-offs in the evolution of
host plant repertoires. key words: insect-plant association, host plant
adaptation, gene expression, phenotypic plasticity%
\end{abstract}%



\sloppy


\textbf{1.Introduction}

The interplay between plants and insects represents one of nature's most
common species interactions (Bernays and Chapman 1994; Stork 2018;
Jones\emph{et al.} 2022). Insects rely upon their host plants, as they
use them as a food source, for reproduction or as a shelter (Futuyma and
Moreno 1988; Kergoat \emph{et al.} 2017). When feeding on a particular
plant, herbivorous insects are exposed to a variety of different
structural and molecular components that can affect their cellular
homeostasis and in further consequence their fitness (for a summary of
different plant defense mechanisms see e.g. F\selectlanguage{ngerman}ürstenberg-Hägg \emph{et
al.} 2013). The efficient utilization of a host, thus, requires genetic
and physiological ``adjustments'' to the different properties of the
respective plant(s) (Ehrlich and Raven 1964; Després, David and Gallet
2007; Heidel-Fischer and Vogel 2015; Simon \emph{et al.} 2015; Xi, Guo
and Hu 2024). Within the order Lepidoptera, the majority of butterflies
(superfamily Papilionoidea) are purely herbivorous. The associations
with a host can be very exclusive, with a specialist insect species
using only one or a few host plant species (Bernays and Graham 1988; Via
1990; Bernays 1991; Forister \emph{et al.} 2015; Hardy \emph{et
al.}2020). In contrast, phytophagous generalists are characterized by a
broader host repertoire that can cover multiple plant families and
sometimes even different orders (polyphagy; Bernays and Graham 1988; Via
1990; Forister \emph{et al.} 2015; Hardy \emph{et al.} 2020). While
specialists should be able to develop strong adaptations to their hosts,
generalists encounter a much broader diversity of physically and
chemically different hosts and should require a more flexible set of
adaptations.

As suggested by Ehrlich and Raven (1964), the broad host repertoire of
polyphagous butterflies can be achieved by including plants that are
chemically similar. Chemical similarity in plants can either result from
shared ancestry or convergent evolution (Janz and Nylin 1998; Janz,
Nyblom and Nylin 2001; Kergoat \emph{et al.} 2005;
Heidel-Fischer\emph{et al.} 2009). Adaptations to one specific host
could, thus, also serve as preadaptations for another plant with a
similar chemical profile and allow an expansion of the host repertoire
(Janz and Nylin 2008; Agosta and Klemens 2008; Agosta \emph{et al.}
2010). This idea has received some support from simulation studies
(Araujo \emph{et al.}2015) and at the level of gene expression
(Heidel-Fischer \emph{et al.}2009; Celorio-Mancera \emph{et al.} 2023).
Using the polyphagous comma butterfly (\emph{Polygonia c-album} ),
Heidel-Fisher \emph{et al.} (2009) showed that similarities in the gene
expression profiles on different hosts could be explained by a close
phylogenetic relatedness or the same growth form of the plants. Despite
these similarities in the gene expression profiles, the overall patterns
indicated a very host-specific transcriptional response in the larval
gut (Heidel-Fisher \emph{et al.}2009). Such host-specific gene
expression profiles in \emph{P. c-album}were further supported by
Celorio-Mancera \emph{et al.} (2013). Based on this, it has been
suggested that adaptations to host plants can profitably be viewed as
``\emph{modules of co-expressed genes and their resulting phenotypic
appearance} '' (cf. Celorio-Mancera \emph{et al.}2023). The latter
study, which examined the overall gene expression patterns on different
plants in \emph{P. c-album} and related species, furthermore, showed
that these specific modules can be conserved over evolutionary time and
can show quite distinct gene expression profiles that differ in the
degree of overlap among plants (Celorio-Mancera\emph{et al.} 2023).
These similarities or differences could potentially be used as an
indicator for overlapping mechanisms to cope with a plant's properties.

Another important factor when describing environment-specific expression
patterns is how quickly insects can adapt their transcriptional response
to a new host. A high degree of plasticity and rapid responses to a host
switch can ensure that juvenile stages are able to react quickly to a
changing environment (cf. Xiao \emph{et al.} 2019). In many phytophagous
insects in which the host is consumed during their larval stages, the
actual host plant is determined by the mother (König \emph{et al.} 2016;
see also Nylin and Janz 1993; Gripenberg \emph{et al.} 2010; Refsnider
and Janzen 2010). It is still largely unknown if and to which extent
larvae of such species can leave their host plant and look for
alternatives, in case of deteriorating plant quality or oviposition
mistakes (but see Nylin et \emph{al.} 2000; Schäpers \emph{et al.}2016).
Although such scenarios are not well documented, larval movement within
plants should often be necessary for similar reasons. Plastic
adaptations to different plants and plant parts without a long temporal
lag could ensure that larvae survive and complete their development
without severe fitness costs. Thus, an experimental shift of host plant
during larval development should give some indication of how difficult
an evolutionary host plant shift would be for caterpillars. Furthermore,
previous studies have mainly focused on gene expression in larvae that
were reared on the same host plant during their entire development (for
a summary see Birnbaum and Abbot 2020). The transcriptional profiles
could, thus, not exclusively be associated with the direct responses to
a certain host but also were the product of different developmental
trajectories linked to a particular plant (cf. Celorio-Mancera et al.
2023). Experimental shifts could now help to further identify genes and
transcriptional adjustments that are crucial for the use of a specific
host plant.

\emph{Polygonia c-album} (Linnaeus, 1758), the comma butterfly, is a
polyphagous butterfly species in the family Nymphalidae that feeds on
trees, bushes and herbs from four different orders: Rosales, Fagales,
Malphigiales and Saxifragales (Nylin 1988). Within these orders the
repertoire of the comma comprises \emph{Urtica} , \emph{Humulus}
and\emph{Ulmus} (Urticaceae and other ``urticalean rosids''),
\emph{Betula}and \emph{Corylus} (Betulaceae) as well as \emph{Salix}
(Salicaceae) and\emph{Ribes} (Grossulariaceae) (Nylin 1988). For some of
these plants, quite characteristic chemical profiles have been
described. Urticaceae, for instance, are rich in alkaloids and phenols,
leaves of the Salicaea contain flavonoids and phenolic glycosides, while
the presence of cyanogenic compounds has been reported for the genus
Ribes (Hegnauer, 1973). \emph{Ribes} is particularly unusual as a host,
as its use is rather rare among butterflies. Within the family of
Nymphalidae (\textasciitilde{}6,100 species; Van Nieukerken \emph{et
al.} 2011), for instance, \emph{Ribes} is only consumed by a few species
of the genus\emph{Polygonia} (Gamberale-Stille \emph{et al.} 2019),
which together with the distinct gene expression patterns
(Celorio-Mancera \emph{et al.} 2013, 2023) indicates very specific and
exclusive adaptations to a likely chemical divergent host. Such
plant-specific profiles now represent a good prerequisite for
investigating the plasticity of adaptations.

In this study, the larvae's ability to adjust to a new host within one
generation was investigated. Special attention was paid to how, and how
fast, polyphagous butterfly larvae can react to a new plant in terms of
gene expression. Earlier studies already showed that larvae can be moved
to another host plant during their development without major effects on
their performance (Söderlind 2012). It is now assumed that also the
underlying transcriptional patterns can be adjusted very rapidly in
response to the new environment. Based on the patterns found in previous
studies, it was expected that diet shifts between hosts that strongly
overlap in their gene expression profiles should be easier (i.e. faster
and with fewer responding genes). In contrast, a slower and more drastic
adjustment is assumed for switches between plants with very opposing
patterns (e.g. \emph{Urtica} vs. \emph{Ribes} ; see
Celorio-Mancera\emph{et al.} 2023). To test this, two host switch
experiments were combined with a performance screening. It was predicted
that:

(1) There is a temporal course of transcriptional adjustments. Shortly
after the host switch, the gene expression profiles will still
correspond to the first host, but can change quickly to regain cellular
homeostasis on the new host plant. (2) There is a lower number of
transcriptional differences between hosts with similar chemical
properties. It is assumed that overlapping processes are involved when
feeding on host plants that show similarities in their chemical
composition, either due to a close phylogenetic relatedness or a shared
growth form (e.g. Switch A). These overlaps will result in a lower
number of differentially expressed genes. (3) Switching to chemically
more different hosts is more challenging. (in contrast to (2)) The use
of hosts with very distinct structural and chemical properties (e.g.
Switch B), often requires more transcriptional and physiological changes
to regain cellular homeostasis that will manifest in a higher number of
differentially expressed genes. (4) Differences in gene expression
profiles will have a physiological correspondence. Depending on the
extent of required adjustments, the switch to certain hosts can be
associated with higher costs, which can result in a reduced larval
performance.

\textbf{2.Material and Methods}

2.1. Host plants

This study is based on two switch experiments that differed in the host
plants involved. The host plants for the first experiments (Switch A)
comprised \emph{Urtica dioica} (stinging nettle), \emph{Salix
caprea}(sallow) and \emph{Ulmus glabra} (elm). For the second switch
experiment (Switch B) \emph{Urtica dioica} , \emph{Salix caprea} and
\emph{Ribes uva-crispa} (gooseberry) were used. Leaf material of the
host plants was collected in the close surrounding area of Stockholm
University, Stockholm, Sweden (N 59° 21' 47'', E 18° 03' 38'').

2.2. Butterfly rearing and host switch

Mated females of \emph{Polygonia c-album} were collected in Åkersberga
(N 59° 28' 56'', E 18° 21' 18'') and Hansta (N 59° 26' 11'', E 17° 54'
14'') near Stockholm. For oviposition, females were individually placed
into separate cages (50x50x40cm) with a transparent roof and a net in
the front. The cages contained a cut \emph{Urtica} branch and sugar
water for feeding. Moistened paper towels were distributed at the floor
of each oviposition cage. All cages were placed below a light source (LD
8:16). Eggs were collected every morning.

Immediately after hatching, larvae were placed into separate cups
(volume: 1.25 oz) containing one leaf of either \emph{Urtica, Salix}
or\emph{Ulmus} (Switch A), or \emph{Urtica, Salix} or \emph{Ribes}
(Switch B) respectively. To prevent the leaves from drying out quickly,
wet cotton was wrapped around their petioles. The quality of the leaves
was checked regularly. Leaf material that was too dry, or of too small
an amount was replaced. Larvae were kept at 18C (L:D 18:6) during their
development.

One day after molting into the fourth instar, larvae were switched to
another plant (Figure 1). For this, larvae were first allowed to feed
for one hour on the original host (Host 1) -- to ensure larval feeding
this was preceded by a 6-hour fasting phase. After feeding on Host 1,
larvae had to fasten again for one more hour before being transferred to
the new plant (Host 2; Figure 1b). Host 2 could either be the same plant
species (Control) or one of the alternative host plants (Switch). This
resulted in a total of 9 different host combinations per switch
experiment.

To measure the transcriptional response to these host switches, the
midguts of the larvae were dissected under phosphate saline solution (pH
7.4, 10 mM) 2 or 17 hours after they initiated feeding on the second
host (Host2). The gut was emptied and Malpighian tubules were removed.
Dissected gut tissue was snap frozen using liquid nitrogen and stored at
-80C. Sampling gut tissue at two different time points allowed us to
assess how quickly caterpillars can adjust to their new host
environment. Moreover, it also enabled a further distinction between
transcriptional changes due to the switch itself (i.e. possible stress
responses) and ``true'' host plant effects. In total, midguts were taken
from 72 caterpillars (1 larva x 4 families x 9 different host
combinations x 2 different time points per treatment). In the same way,
the second switch experiment was conducted using \emph{Urtica}
,\emph{Salix} and \emph{Ribes} . Here, midgut tissue from 72
caterpillars (2 larvae x 2 families x 9 different host combinations x 2
different time points per treatment) was collected for the differential
gene expression analysis. In both experiments, one sample had to be
discarded due to low quality.

The effect of host switches on the larval performance was investigated,
but only for Switch B. For this, 1-2 caterpillars from 5 different
families were reared at 18C (LD: 12:12) on either \emph{Urtica}
,\emph{Salix} or \emph{Ribes} (n=72)\emph{.} Larvae were moved to a new
plant (same or alternative host species) upon reaching the fifth instar.
Their weight was measured daily until pupation. The daily growth rate
was calculated as:

Growth rate =\(\frac{\log(\text{mass})}{\text{developmental}\ \text{time}}\)

2.3.RNA extraction

The frozen gut tissue was first transferred into 300\selectlanguage{greek}µ\selectlanguage{english}l Tri-reagent
(ZymoResearch) and homogenized with a clean pistil. The tissue continued
to be homogenized after adding 700\selectlanguage{greek}µ\selectlanguage{english}l of additional Tri-reagent and
samples were incubated for 5 minutes at room temperature. After the
incubation, 100\selectlanguage{greek}µ\selectlanguage{english}l of bromo-chloro propane (BCP) (Sigma-Aldrich) per
milliliter Tri-reagent were added. The samples were mixed thoroughly and
incubated for another 10 minutes. Subsequently, the samples were
centrifuged at 12,000g for 13 minutes at 4C. The aqueous supernatant was
then transferred into a fresh tube and 500\selectlanguage{greek}µ\selectlanguage{english}l isopropanol (Sigma-Aldrich)
per milliliter Tri-reagent were added. Samples were mixed and incubated
for 8 minutes at room temperature. After centrifuging for 8 minutes at
room temperature and 12,000g, the supernatant was discarded. 1
milliliter of 75\% Ethanol (KiiltoClean) was added per milliliter
Tri-reagent and samples were centrifuged at 7500g for 5 minutes at room
temperature. The ethanol was fully removed and the remaining pellets
were air dried. Dry pellets were subsequently dissolved in 90\selectlanguage{greek}µ\selectlanguage{english}l
pre-heated (55C) RNA-storage solution (Invitrogen). DNA was removed
using the DNA-\emph{free}  DNA removal Kit (Invitrogen) following the
manufacturer's protocol. The quality of the extracts was assessed using
Bioanalyzer (Agilent RNA 6000 Nano). RNA concentration was quantified
using a Qubit 2.0 fluorometer (Invitrogen). Library preparation (with
Poly-A-selection) and sequencing was done at the NGI SciLifelab in
Stockholm. RNA libraries were sequenced with 2x101bp paired-end
sequencing on an Illumina TrueSeq platform.

2.4. Analysis

2.4.1. Read alignment and quantification

The quality of the raw sequence data was checked using FastQC (version
0.11.9; Andrews 2010) and MultiQC (version 1.10; Ewels \emph{et a} l.
2016). Adapter and quality trimming was performed using Trimmomatic
(version 0.36; -phred 33, SLIDINGWINDOW:4:5, LEADING:5, TRAILING:5,
MINLEN25; Bolger \emph{et al.} 2014). The trimmed sequences were again
checked with FastQC and MultiQC. Trimmed reads were mapped against an
inhouse created reference genome of \emph{Polygonia
c-album}(Celorio-Mancera \emph{et al.} 2021) using a STAR 2-pass
approach (version 2.7.2b; Dobin \emph{et al.} 2013). Mapping success was
checked with samtools (version 1.12; Danecek \emph{et al.} 2021) and
MultiQC. Abundance estimations were done using featureCounts from
Subread (version 2.0.0; Liao \emph{et al.} 2014).

2.4.2. Differential gene expression

Differential gene expression analysis was performed using the R-package
edgeR (version 3.40.0; Robinson \emph{et} \emph{al.} 2010). For this,
the count matrix including 21,761 genes was first split into the two
time points (2 vs.17 hours) and separate gene expression lists were
created. Filtering was done using the filterByExpression-function of
edgeR (default setting) with information about the host combination as a
group-factor, which retained 13,126 (Switch A, 2hrs) 12,811 (Switch A,
17hrs), 12,622 (Switch B, 2hrs) and 12,910 (Switch B, 17hrs) genes.
Filtered data were then normalized using CalcNormFactors. Differential
gene expression was analysed using QLFit (robust=TRUE) and glmQLFTest
(FDR \textless{}0.05). Here, an ANOVA-like framework was used to
investigate the main effect of Host 1 and Host2, as well as their
interaction. We further used pairwise comparisons to assess the
differences between treatments. To account for potential differences due
to relatedness between samples, family was included in the models.
Significantly differentially expressed genes were identified as those
with an adjusted P-value (FDR, Benjamini-Hochberg correction) less than
0.05.

We generated a functional annotation using the web-based version of
eggNOG-emapper (Huerta-Cepas \emph{et al.} 2019; Cantalapiedra \emph{et
al.} 2021), which relies on HIMMER (Eddy 2011), DIAMOND
(Buchfink\emph{et al.} 2021) and MMSEQS2 (Steinegger and S\selectlanguage{ngerman}öding 2017)
when searching for sequence alignments. Briefly, genes were converted to
amino acid sequences using gffread (Pertea and Pertea 2020) and
submitted to eggNOG emapper using default parameters, including
automatic identification of taxonomic scope (i.e., functional
annotations were not limited to those confirmed in arthropods). We
obtained functional annotations for 12,469 of 17,606 genes in
the\emph{P. c-album} annotation, however only 8034 of these genes were
annotated with gene ontology (GO) terms. Enrichment of GO terms among
genes that differed significantly in their expression between the
treatments was tested using topGo (Alexa and Rahnenfuhrer 2016).
Significant enrichment was tested using Fisher's exact tests corrected
using topGO's parent-child algorithm. This algorithm evaluates
enrichment of a term in relation to its parent terms and so reduces
false positives due to the ``inheritance'' problem -- where more
specific terms are likely to be falsely enriched if their parent terms
are enriched (Grossmann et al. 2007). Here we focused on enrichment of
biological process terms and only considered GO-terms that were
annotated to at least five genes.

2.5. Congruence with earlier results

In order to investigate the repeatability of our observations, and
whether host-specific gene expression is primarily a direct response to
the host or a down-stream effect, we compared the set of differentially
expressed genes per contrast from this study to the sets of co-expressed
genes reported under similar host plant treatments but without a switch
(Celorio-Mancera \emph{et al.} 2023). In order to do this, it was
necessary to join the gene sets from two different annotation databases.
For this purpose, we used DIAMOND (\emph{blastp} flag, default
parameters, 16930 unique queries aligned) to annotate the predicted
protein set from the \emph{P. c-album} genome in the present study,
which relied on an ortholog \emph{Heliconius melpomene} database
generated and described by Celorio-Mancera \emph{et al.} (2023). The
output table of annotated sets of genes from \emph{P. c-album} was used
in turn to name the lists of differentially expressed genes obtained
from the different contrasts.

2.6 Performance

We tested the effect of host plant switches on larval performance using
linear mixed models (lme4, version 1.1.31, Bates \emph{et al.} 2015),
including ID of the mothers and individual-ID as random effects. Data
wrangling, analysis and visualization were performed in R with the
support of tidyverse packages (ggplot2, version 3.4.0, Wickham 2016).

\textbf{3.Results}

3.1. Data distribution

We compared gene expression patterns for both switch experiments using
principal component (PC) analysis. Overall, individuals did not cluster
by pre- or post-switch host at either time point. (Supplementary Figures
1 and 2). Rather, individuals that experienced specific transitions
between hosts stood out from the remaining larvae. For example, in
Switch A, individuals that were switched from \emph{Ulmus}
to\emph{Urtica} tended to cluster separately from other samples. This
pattern could be seen in the first two PC axes after 2 hours, and for
the second and third PC axes after 17 hours. In Switch B, individuals
that originally fed on \emph{Salix} clustered separately from the other
larvae after 2 hours, but this pattern was lost after17 hrs. In
contrast, individuals moved to \emph{Urtica} clustered only after 17
hours (PC axes 1 \& 2, Supplementary Figure 2e). While individuals did
not cluster by family on any of the first three PC axes in Switch A,
family clusters were clearly discernible in the Switch B PCA. This was
especially true for PC axis 3. Due to this, family structure was
accounted for in the downstream analyses.

3.2. Global assessment of gene expression in response to host switches

We assessed the influence of the two hosts involved in a host switch in
an ANOVA-like framework, testing for an overall effect of Host 1 on
expression, an overall effect of Host 2 on expression, or an interacting
effect of Host 1 and Host 2 at each timepoint. To do this, we combined
all of the different results across individual hosts, in order to get a
global assessment of host switching effects in each of our two
experiments. Overall, the total number of significantly differentially
expressed genes (FDR \textless{} 0.05) differed between the two switch
experiments (Figure 2). In Switch A, a total of 4.2\% (n = 550) and
1.9\% (n = 249) of genes were significantly differentially expressed
after 2 and 17 hours of feeding, respectively. At both time points, the
highest number of differentially expressed genes was associated with the
host they were feeding on before the switch (i.e. Host 1). This differed
considerably from Switch B, in which most differences in expression
between larvae that could be attributed to Host 1 and Host 2 shifted
over time. After 2 hours 32.9\% of genes (n = 4148) were significantly
differentially expressed, and the majority of these were associated with
the pre-switch plant (Host 1). After 17 hours 32.7\% (n = 4216) of genes
showed significantly different expression. Here, the highest number of
differentially expressed genes was driven by the new host (Host 2). This
suggests that the actual plants that are involved in a switch experiment
play an important role in the time course and extent of expression
differences.

3.3. Differentially expressed genes between switches

To further understand the transcriptional effects of switching between
two particular host plants, we compared differential gene expression
between caterpillars that were exclusively reared on the same host (i.e.
Control, e.g. \emph{UdUd} ) with individuals that were moved to an
alternative host (i.e. Switch, e.g. \emph{UdSc} ). In both switch
experiments, the host pairs that showed the highest number of
differentially expressed genes after 2 hours clearly differed from those
at the 17 hours after the switch (Figure 3).

In Switch A, the highest number of significantly differentially
expressed genes was generally found between comparisons in which larvae
were switched onto \emph{Urtica} (aka \emph{Ud} ; Figure 3a). After 2
hours, most differentially expressed genes were found in the switch
from\emph{Ulmus} to \emph{Urtica} (\emph{UgUg} - \emph{UgUd} ). In the
other comparisons, the number of significantly differentially expressed
genes was 6 or fewer. After 17 hours, the number of differentially
expressed genes was significantly lower. Here, a maximum of 3
significantly differentially expressed genes were found in the
comparison between the\emph{Urtica} and \emph{Salix} -control line
(\emph{UdUd} - \emph{ScSc} ).

Many more genes were found to be differentially expressed in Switch B
than in Switch A. Moreover, a more dynamic pattern was found when
comparing the two time points. After 2 hours, the highest number of
differentially expressed genes was measured in comparisons
between\emph{Urtica} and \emph{Salix} and between \emph{Salix}
and\emph{Ribes} . After 17 hours, overall higher numbers of
differentially expressed genes were found in switches that involved
\emph{Ribes.} By this time, most transcriptional differences were
detected in the pairwise comparison of the \emph{Urtica} control line
with the switch\emph{Urtica} to \emph{Ribes} (i.e. \emph{UdUd} -
\emph{UdRu} ). This further indicates that the same or similar processes
underlie the use of plants with similar chemical properties. In
contrast, the utilization of hosts that represent a chemically or
structurally more challenging environment requires more specific
mechanisms and, thus, a higher number of transcriptional changes.

Due to the low number of transcriptional differences after 17 hours in
Switch A, we focus subsequent analyses on Switch B. However, the
corresponding figures for Switch A can be found in the

supplementary material (Supplementary Figure 3, Supplementary Table 1).

3.4. Transcriptional adjustments to the host switch

Given that larvae in switch B showed distinct shifts in transcriptional
profiles between 2 and 17 hours after switching to a novel host, we
wanted to explore whether these larvae were recovering the expression
patterns of larvae reared continuously on the novel host (i.e, the
control lines). This would demonstrate extensive adaptive
transcriptional plasticity with an adjustment of a core set of genes
specific to a particular host plant. Alternatively, gene expression in
switched larvae may reflect stress responses caused by switching to any
novel host as well as transient adjustments to a specific host plant
(which would not be seen in control lines). To test these alternative
hypotheses, we evaluated the transcriptional differences between
switches and control lines against expectations from pairwise
comparisons with larvae exclusively feeding on one host (e.g,\emph{UdUd}
- \emph{UdRu} compared to \emph{UdUd} - \emph{RuRu} , i.e. Control).
This framework allowed for transcriptional profiles to be classified
into gene expression patterns that either matched or contradicted their
control line comparisons (Figure 4).

After 2 hours, gene expression profiles in larvae switched to a new host
mainly corresponded to those of their pre-switch host (Figure 5),
consistent with category 1 in Figure 4. This supported our previous
results (Figure 2), and suggests that after 2 hours larvae had not yet
adjusted their transcriptional response to the new plant. Overall, this
pattern was found for all combinations of host plants, and the direction
of a switch did not seem to play a crucial role. However, a slight
deviation from this broader pattern was found in the switch
from\emph{Salix} to \emph{Urtica} ; after 2 hours, six genes showed
early transcriptional adjustments to the new host (category 2, Figure
4). These genes were all involved in the ``regulation of
\emph{peptidoglycan recognition protein signaling pathway''}
(Supplementary Table 2). In the reciprocal switch from \emph{Urtica} to
\emph{Salix} , these differences were not found. In addition, one gene
that matched category 2 expectations was found 2 hours after individuals
were moved from\emph{Salix} to \emph{Ribes} . This gene showed a
significant enrichment for ``\emph{regulation of transcription involved
in G1/S transition of mitotic cell cycle} '' and
``\emph{biomineralization} ''. Again, the switch into the other
direction (\emph{Ribes} to \emph{Salix} ) did not show these
differences.

After 17 hours, the differences in the gene expression indicated
host-specific transcriptional plasticity, depending strongly on the
plants involved and the direction of the switch. Though few genes were
differentially expressed between \emph{Urtica} - \emph{Salix} switches
and the respective control lines after 17 hours (e.g., \emph{UdUd}
-\emph{UdSc} and \emph{ScSc} - \emph{ScUd} ; Figure 5), a large
proportion (51.4\% (n=38), 28\% (n=14) respectively) of these genes were
not also differentially expressed between the control lines (consistent
with category 3, Figure 4). This suggests these genes are not typically
involved in the more stable patterns of gene expression promoting the
ability to eat these different hosts (category 1), but instead reflect a
general stress response due to the switch between hosts, or a transient
response in order to regain homeostasis on a specific host. The
strongest transcriptional discrepancy was found in the switches
between\emph{Urtica} and \emph{Ribes} . In both directions a large
proportion of the genes showed an adjustment to the new host
(\emph{UdUd} -\emph{UdRu} : 9.5\% (n=123); \emph{RuRu} - \emph{RuUd} :
17.9\% (n=90)). Especially in the switch to \emph{Ribes,} most of these
genes were associated with functions directly involved in metabolic
processes (Supplementary Table 2). This pattern was also found in the
switch from\emph{Salix} to \emph{Ribes} (\emph{ScSc} - \emph{ScRu} ) but
not in the other direction, which further supports a crucial role of
these genes for the utilization of \emph{Ribes} . The majority of the
genes were, however, uniquely differentially expressed between switch
but not the control lines (\emph{UdUd} - \emph{UdRU} : 84.6\% (n=1096),
\emph{RuRu} -\emph{RuUd} : 60.4\% (n=304), \emph{ScSc} - \emph{ScRu} :
73\% (n=580)). Our hypothesis that these genes are associated with a
response to the experience of eating a novel host is supported by an
enrichment of genes that were involved in metabolic processes and genes
that could be associated with a stress response. These patterns are in
line with our expectations as they confirm that a switch to an
alternative host can not only represent a stressful situation but also
requires a specific regulation of molecular and physiological processes
to regain cellular homeostasis in the new environment. \emph{}

3.5. Congruence with previous studies

Switches between \emph{Urtica} and \emph{Ribes} are hypothesized to be
the most challenging for \emph{P. c-album} larvae
(Celorio-Mancera\emph{et al.} 2023). Our results support this
hypothesis, finding the largest expression differences associated with
switching between these two hosts (Figures 2, 5), and a large proportion
of differentially expressed genes that are unique to switches (i.e.
consistent with category 3, Figure 4). We thus aimed to evaluate the
extent to which our results overlap with these earlier results, in which
larvae were not switched between hosts but instead reared on the same
host until they were sampled (Celorio-Mancera \emph{et al.} 2023).
First, it can be noted that the results of the two studies are highly
congruent. In the present study, there were 199 genes differing
significantly in gene expression between \emph{Urtica} and \emph{Ribes}
controls. Of 113 genes upregulated on \emph{Ribes} , 91 could be matched
to genes in the earlier database, and 29 of these genes were also
significantly upregulated on\emph{Ribes} in the earlier study
(Celorio-Mancera \emph{et al.} 2023). 85 genes were upregulated on
\emph{Urtica} in our study, out of which 75 could be matched, and 20 of
these genes were also upregulated on\emph{Urtica} in the previous
study\emph{.} Only one gene was found to be expressed in the opposite
direction between the two studies (upregulated on \emph{Urtica} in our
study, downregulated in the previous). Moreover, genes differing most
strongly (highest fold-change) in both studies were most likely to
overlap. For instance, 7 of the genes with highest fold-change in the
present study were found among the 20 genes differing most strongly in
the earlier study (Celorio-Mancera\emph{et al} . 2023). In both studies,
a synaptic vesicle protein and an antibacterial peptide were strongly
upregulated on \emph{Urtica} . Similarly, a strong upregulation of an
aldo-keto reductase, a proline dehydrogenase, a spermine oxidase-like
and one non-annotated gene was found on \emph{Ribes} in both studies. We
further used an extensive functional annotation from
Celorio-Mancera\emph{et al} . (2023) to identify the putative function
of some of the genes that were highly up- or downregulated after a
switch from\emph{Urtica} to \emph{Ribes} or vice versa at 17 hours after
the switch. These genes are candidates for representing a direct
response to the new host plant rather than a downstream effect. In this
study, we could confirm the involvement of genes that had previously
been indicated by Celorio-Mancera \emph{et al.} (2023). Moreover, we
were also able to identify new candidates that appear to play an
important role in the direct response and, thus, the utilization of
\emph{Urtica}and \emph{Ribes} (Table 1).

3.6. Larval performance

In order to test whether switching to a new host also has consequences
at a phenotypic level, the performance of larvae was measured until they
reached the pupal stage. Despite the transcriptional responses found
here, the host switches had no significant effect (\selectlanguage{greek}χ\selectlanguage{english}2= 13.372, df= 8,
p=0.0997) on the larval growth rate (Figure 6). Even after excluding
pre-switch days from the analysis, no significant effect (\selectlanguage{greek}χ\selectlanguage{english}2= 3.1934,
df= 8, p=0.9216) on growth rate could be measured (Supplementary Figure
4). This suggests that the caterpillars of \emph{Polygonia c-album} can
adjust to a new host without major expenses for their development.

\textbf{4.Discussion}

Earlier results from \emph{Polygonia c-album} and related species have
suggested that adaptations to host plants can be understood as modules
of host-specific gene expression and the resulting phenotypic traits
(Celorio-Mancera \emph{et al.} 2023). In the present study, it could now
be shown that the larvae of such polyphagous butterflies can also
``switch'' between these modules during their individual development to
adjust their molecular and cellular processes to a new host plant. As
predicted, 2 hours after the switch, the larvae still showed gene
expression patterns that corresponded to Host 1 (i.e. category 2), while
Host 2-specific profiles, that were expected from the comparison of the
non-switched controls (i.e. category 1), were predominantly found after
17 hours (in support of Hypothesis 1 in the Introduction). The extent of
transcriptional adjustment depends strongly on the similarity of the
host plants (in support of Hypotheses 2 and 3). For some comparisons
even after 17 hours only very few differentially expressed genes were
found, that corresponded to expression profiles expected for the second
host. The overall low number of differentially expressed genes between
some plants may result from shared modules and, consequently, the use of
the same or similar downstream processes. This agrees with a high
similarity of the host plants used in Switch A. \emph{Urtica dioica}
and\emph{Ulmus glabra} both belong to the ``urticalean rosids'',
while\emph{Salix caprea} and \emph{Ulmus glabra} have the same growth
form. It was shown that both a close phylogenetic relationship, as well
as similar physical appearance can explain similarities in the chemical
properties and defense mechanisms (Feeny 1976) and, thus, suggest the
involvement of overlapping modules. Such similarities in the gene
expression response to these hosts were already indicated by
Heidel-Fischer \emph{et al.} (2009) and Celorio-Mancera \emph{et
al.}(2012, 2023). The comparatively small number of transcriptional
differences that were found in switches between U\emph{rtica}
and\emph{Salix} is moreover consistent with the overlapping gene
expression profiles that were previously shown by Celorio-Mancera
\emph{et al} . (2023).

In other switches, a high number of expression differences would, in
turn, indicate the involvement of more specific and exclusive gene
expression profiles and developmental pathways. The high number of
transcriptional differences that were found in switches that
involved\emph{Ribes} and especially between the phylogenetically and
chemically diverse \emph{Urtica} and \emph{Ribes} (Switch B), is
consistent with the overall opposing gene expression profiles between
these two hosts (Celorio-Mancera \emph{et al.} 2023).

Based on these transcription profiles, it could be assumed that switches
containing \emph{Ribes} indeed represent a more complex situation. The
differences between the different directions of the host switches,
further indicated that switches to \emph{Ribes} appear to be more
challenging than the reverse. This could be related to the special
chemical composition of \emph{Ribes} . The leaves of \emph{Ribes}
species are described to contain cyanogenic compounds (Hegnauer 1973),
which can act as a potent chemical defense against several herbivores
(Bellotti and Arias 1993; F\selectlanguage{ngerman}ürstenberg-Hägg \emph{et al} . 2013). The
gene expression patterns found here support this assumption. Most of the
genes with a significantly different expression on \emph{Ribes} were
mainly involved in metabolic processes (Table S1). This was further
confirmed by a closer examination of the upregulated genes
on\emph{Ribes} in comparison to \emph{Urtica} . In addition, an
increased expression was recorded in genes that were directly or
indirectly associated with metabolism and detoxification (e.g. Aldo-keto
reductase, Glutathione s-transferase 1-like).

In agreement with Celorio-Mancera \emph{et al} . (2023) an upregulation
of the proline dehydrogenase was found, which besides the metabolic
degradation of proline is also involved in energy recovery and response
to (micro)environmental stress (Phang, Liu and Zabirnyk 2010; Servet
2012). Furthermore, an upregulation of spermine oxidase like
on\emph{Ribes} could be shown again. Spermine oxidase is involved in the
metabolic processing of polyamines (Chang \emph{et al.} 2021). Moreover,
it was also described to play a role in the response to oxidative
stress, which, in addition to the upregulation of a Methionine sulfoxide
reductase (HMEL008564-PA) may indicate a role in the reaction to
cyanogenic glycosides found in \emph{Ribes} leaves.

An involvement of serine proteases could also be observed. Proteases
have been shown to play an important role in the breakdown of various
plant proteins and, thus, contribute to the digestion and nutrient
absorption of host plant material (Celorio-Mancera \emph{et al.} 2013;
Chikate \emph{et al.} 2013). Moreover, proteases are involved in
overcoming chemical plant defenses like protease inhibitors (Ryan 1990).
For this, larvae can not only express a wide range of different
proteases (e.g. serine proteases and serine-type endopeptidase) but also
regulate their expression according to a specific host plant
(Chougule\emph{et al.} 2005; Celorio-Mancera \emph{et al.} 2013; Chikate
\emph{et al.} 2013; Vogel \emph{et al.} 2014). Such host-specific
involvement of proteases has previously been shown for \emph{P.
c-album}(Celorio-Mancera \emph{et al} . 2013). Our results further
support these previous findings, and strengthen the assumption that
proteases are crucial candidates for the usability and processing of a
broad host repertoire.

Besides an upregulation of these genes, increased expression has also
been found in genes that are responsible for the development of the gut
peritrophic matrix, which are rather a physiological adaptation to the
usability of a host (Celorio-Mancera et al 2023; this study). The
peritrophic matrix is a membranous layer that encases the food bolus and
thus separates the ingested material from the gut epithelium (Lehane
1997). By this it represents an important protective barrier, as it
prevents plant toxins and other harmful substances from entering the
other body tissue (Puninean \emph{et al.} 2010, Celorio-Mancera \emph{et
al.} 2013). An upregulation of the peritrophic matrix development can
now either indicate a strengthening of the gut wall as a defensive
mechanism, or the initiation of processes that repair damages from
interactions with toxic components (see Celorio-Mancera \emph{et
al.}2013 for details; see also Pérez-Hedo \emph{et al.} 2012). Previous
studies already showed differential expression of genes involved in
structural constituents of the cuticle as a response to
\emph{Ribes}(Celorio-Mancera \emph{et al.} 2013). An increased
production of chitin to strengthen or repair the intestinal tissue,
thus, seems to be required for the usability of \emph{Ribes} after a
switch.

The downregulation of Sialin-like after a switch to \emph{Ribes} can be
interpreted as a transient response to a previous upregulation
on\emph{Urtica} . This is in agreement with its upregulation
on\emph{Urtica} shown in the earlier study by Celorio-Mancera \emph{et
al.} (2023). Sialin-like is likely involved in nitrate transport
and\emph{Urtica} is characterized by a high nitrate content (Hegnauer
1973; Taylor 2009). When switching larvae to \emph{Ribes} , they quickly
have to react to compensate for the lower nitrate concentration. This
also agrees with the upregulation of transmembrane proteins on
\emph{Urtica.}Transmembrane proteins are responsible for the exchange of
substances into and out of the cell and can, thus, play an important
role in the maintenance of cellular concentrations as well as signaling.

The ``unexpected'' differences after the switches (i.e. category 3),
were mostly involved in metabolism. However, a considerable proportion
also included genes that played a role in signaling and responses to
different (stress) stimuli, which further indicates a stress reaction to
the switch itself. The repeated observation of the upregulation of some
specific genes further implies their role in adaptation to a particular
host plant. In contrast to the present project, previous studies
measured the gene expression of larvae that were reared on the same host
plant during their entire development (Heidel-Fischer \emph{et al.}
2009; Celorio-Mancera \emph{et al.} 2013, 2023). Here, changes in the
transcriptional profiles were measured in short time intervals after the
switch to a new host. The up- or downregulation of a particular gene
can, thus, be interpreted as a direct response to a specific host,
further confirming its contribution to the digestibility and usability
of the plant. These genes now represent important candidates that could
be targeted during knockout experiments to further determine their role
in host plant adaptations.

The discrepancy in the gene expression between \emph{Urtica}
and\emph{Salix} in the two experiments may be due to annual variation in
host quality. The two switch experiments were carried out in different
years. Differences in temperature as well as in the amount of
precipitation are likely to influence the physical and chemical
properties of host plants (De Bruyn, Scheirs and Verhagen 2002; Scheirs
and de Bruyn 2005; Liu \emph{et al.} 2023). These variations could then
cause the differences in the transcriptional response between years.

Despite the transcriptional differences, host switches did not affect
the performance of the larvae (i.e. Hypothesis 4 was not supported).
Previous studies on sequential diet shifts in \emph{P. c-album} already
showed that caterpillars can switch easily between \emph{Urtica}
and\emph{Salix} without major costs for their performance (Söderlind
2012). The present study now also showed that in switches that were
assumed to be more challenging, larvae could still adjust to the new
host without significant physiological and developmental deviations.
Even when moved to \emph{Ribes} larval performance did not differ from
those in other treatments. This suggests that also at a phenotypic
level, larvae can flexibly adapt to the new host environment.

The ability to adjust the transcriptional and phenotypic response
demonstrates that the usability of a host is not determined by the
experience of larvae during their early development. This quick
adjustment of gene expression allows the caterpillar to flexibly switch
to an alternative host and, therefore, to compensate for poor host
quality due to, for instance, rapidly changing environmental conditions
or oviposition mistakes. The larvae of \emph{P. c-album} have been
described to be relatively mobile and can cover substantial distances
even as neonates (Nylin \emph{et al.} 2000, Schäpers \emph{et al.}2016).
It is, thus, possible that the caterpillars under some circumstances can
move to an alternative host plant if necessary (cf. Gamberale-Stille et
al 2014). Moreover, this plasticity can make caterpillars more resistant
to changes \emph{within} a host plant. Reduced host quality, seasonal
changes of the chemical composition, as well as an increase in potential
defensive chemicals (e.g. induced defenses) may require altered or
different cellular and physiological processes for the further use of a
plant.

The described plasticity in gene expression is, however, perhaps more
important in an evolutionary context. The findings support the
assumption that the colonization of new plants does not require entirely
new adaptations, but that existing adaptations to established hosts can
act as preadaptations and facilitate the colonization of novel plants
(ecological fitting; Janzen 1985; Agosta 2006; Agosta \emph{et
al.}2010). Nevertheless, the switch to some hosts still seems to be more
complicated than to others and it takes longer until the state of
homeostasis is reached when larvae are switched to such plants. Although
we did not find any significant performance costs of the hosts switches,
it should not be ruled out that physiological trade-offs were involved
in the evolution of the host repertoire of \emph{Polygonia} butterflies
at some point. In line with this interpretation, the challenging switch
to \emph{Ribes} in the present study seems to be reflected in
evolutionary patterns, as butterflies outside of \emph{Polygonia} rarely
have been able to colonize this host.

In conclusion, investigating the immediate response to a host switch
helps characterizing genetic modules (sensu Celorio-Mancera et al. 2023)
associated with host plant metabolism and detoxification, in that it
disentangles direct responses to the plant from overall downstream
effects. In addition, we propose that host switch experiments in the
laboratory followed by transcriptomic investigations can be a valuable
tool to examine not only plasticity in host use but also subtle and/or
transient trade-offs in the evolution of host plant repertoires.

\textbf{5. Acknowledgement}

The computations of this project were enabled by resources in the
project SNIC 2022/22-337 provided by the National Academic
Infrastructure for Supercomputing in Sweden (NAISS) at UPPMAX, funded by
the Swedish Research Council through grant agreement no. 2022-06725.
This research was supported by grants from the Swedish Research Council
to SN (2019-03441) and CWW (2017-04386). KS would like to acknowledge
Julie Lorent from the National Bioinformatics Infrastructure Sweden
(NBIS) for her advice in the course of the Swedish Bioinformatics
Advisory Program. We would also like to thank Estelle Cerisier for
providing illustrations of larvae and host plants.

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\textbf{Author Contributions}

This study was designed by SN, NJ, CWW and MC. Wild butterflies were
collected by SN, NJ. Larval rearing, switch experiment and gut sampling
was performed by MC. RNA-extractions were done by MC (Switch A) and KS
(Switch B). Data were analysed by KS, RAS, DM and SN. The manuscript was
written by KS with the help of all the authors.

\textbf{Tables and Figures}

Figures in higher resolution are also provided as individual PDFs.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1. Experimental setup.} The present study is based on two
switch experiments in which the larvae were moved to a different host
plant. during their development. (\textbf{a} ) The first switch
experiment (Switch A) included \emph{Urtica dioica} (\emph{Ud}
),\emph{Salix caprea} (\emph{Sc} ) and \emph{Ulmus glabra} (\emph{Ug} ).
For the second experiment (Switch B) \emph{Urtica dioica} , \emph{Salix
caprea} and \emph{Ribes uva-crispa} (\emph{Ru} ) were used. A summary of
all host combinations are given for each switch experiment. Here rows
correspond to the original host (Host 1) and columns to the host the
larvae were transferred to. (\textbf{b} ) In both experiments larvae
were first reared on one out of three established host plants (Host 1).
When reaching the fourth larval instar, caterpillars were moved to a new
host plant (Host 2). This new host could either be of the same (Control,
e.g\emph{UdUd} ) or one of the alternative plant species (Switch,
eg.\emph{UdSc} ). After the transfer larvae were allowed to feed for 2
respectively 17 hours on Host 2 before the midgut was collected for
RNA-extraction and sequencing. A summary of all host combinations per
sampling time point are given for each switch (\textbf{d} and\textbf{e}
). Here rows correspond to the original host, while columns represent
the host the larvae are transferred to. Switches to the same host
species (Control) are highlighted in grey.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2. Host effects on differential gene expression at the
two sampling time points} . The individual effect of the two host plants
as well as the effect of their interaction in the number of
differentially expressed genes (FDR \textless{} 0.05) was measured at
both sampling time points (2 hrs and 17 hrs). The effect of the hosts
differed between the experiments: In Switch A (top row) the highest
number of significantly differentially expressed genes was associated
with Host 1 (H1, blue) at both time points. In Switch B a stronger
effect of Host 2 (H2, yellow) was measured 17 hours after the host
switch. The interaction between the two hosts (H1xH2, grey) explained
only a small or no proportion of the significantly differentially
expressed genes. Percentages in parentheses represent the proportion of
the total number of significantly differentially expressed genes at a
given time point. The highest number of significantly differentially
expressed genes at a time point is shown in bold.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3. Host switches associated with the highest number of
differentially expressed genes varied between 2 and 17 hours.} The
number of significantly differentially expressed genes (FDR \textless{}
0.05) after a host switch was compared to the corresponding control line
(e.g. UdUd - UdSc). Here the comparisons with the highest numbers showed
strong differences between the sampling times. Vertical barplots show
the total number of significantly differentially expressed genes in each
pairwise comparison, horizontal barplots (set size) represent the
proportion of unique and shared genes. Overlaps in the differential gene
expression are shown by the dot-plots. Differences in Switch A are shown
on the left side (\textbf{a} -\textbf{c} ), Switch B is presented on the
right side (\textbf{d} -\textbf{f} ). Ud= \emph{Urtica dioica} ,
Sc=\emph{Salix caprea} , Ug= \emph{Ulmus glabra} , Ru=\emph{Ribes
uva-crispa.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4. Schematic of expected expression differences after the
host switch, when compared to differences between control lines using
the example switch \emph{Urtica} to \emph{Ribes}.} Here, we use the
differences between Host 1 control lines (\emph{UdUd} ) and either the
Host 2 control lines (\emph{RuRu} ) on the X axis or the switch from
Host 1 to Host 2 (\emph{UdRu} ) on the y-axis to identify 5 different
categories of expression change: (1) Differences in the gene expression
after the host switch correspond to differences found between the
controls, indicating an adjustment to the new host (green). (2) No
differences in gene expression after the switch despite differences
between the controls, corresponding to the gene expression profiles on
Host 1 (yellow). (3) Differences in gene expression after the switch but
no differences between the controls (blue). (4) differences in gene
expression after the switch that are opposite to differences between
controls (orange) were considered as ``mismatches''. (5) Genes that were
not significantly differentially expressed are not shown here. We
predict that 2 hours after a switch the majority of transcriptional
differences will match categories 2 and 3. After 17 hours, larvae should
predominantly show adjustments to the new host (category 1). Expression
changes of category 4 are not expected, and if seen may indicate
transcriptional dysregulation upon switch to the novel host.\emph{Ud}
=\emph{Urtica} \emph{dioica} , \emph{Ru} =\emph{Ribes uva-crispa.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5. Differences in the transcriptional profiles of larval
midgut genes} 2 hours (n=12,622) and 17 hours (n=12,910) after the host
switch. After two hours most of the differentially expressed genes still
showed expression profiles specific for a response to Host 1 (yellow).
After 17 hours these patterns had largely changed and host-specific
responses to the second plant (green) as well as expression differences
that were not expected from the differences between the controls (blue,
orange) were found. Ud= \emph{Urtica dioica} , Sc=\emph{Salix
caprea,}Ru=\emph{Ribes uva-crispa.}\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\textbf{Table 1. Candidate genes involved in a direct response to
\emph{Urtica} and \emph{Ribes.}} Genes with at least five-fold
differential expression between \emph{Urtica} and \emph{Ribes}
treatments 17 hours after a switch to Host 2 compared to controls
remaining on Host 1 (i.e., \emph{UdUd} - \emph{UdRu} or \emph{RuRu} -
\emph{RuUd}). Gene name and functional description are given for each
gene. Furthermore, names of orthologs genes in \emph{Heliconius
melpomene} and the corresponding gene description are given. Gene
function was determined based on the functional annotations of both
datasets. Moreover, the regulation of gene expression on Host 2 after
the switch is stated. Each gene was classified based on the categories
of transcriptional changes shown in figure 2. Note that an upregulation
on one host can be interpreted as a downregulation on the alternative
host. Genes in bold were highly upregulated on the same host also in the
earlier study (without a switch; Celorio-Mancera \emph{et al.} 2023) .
NA = not annotated. Each gene was classified based on the categories of
transcriptional changes shown in figure 2. \emph{Ud}=\emph{Urtica}
\emph{dioica}, \emph{Ru}=\emph{Ribes uva-crispa} & \textbf{Table 1.
Candidate genes involved in a direct response to \emph{Urtica} and
\emph{Ribes.}} Genes with at least five-fold differential expression
between \emph{Urtica} and \emph{Ribes} treatments 17 hours after a
switch to Host 2 compared to controls remaining on Host 1 (i.e.,
\emph{UdUd} - \emph{UdRu} or \emph{RuRu} - \emph{RuUd}). Gene name and
functional description are given for each gene. Furthermore, names of
orthologs genes in \emph{Heliconius melpomene} and the corresponding
gene description are given. Gene function was determined based on the
functional annotations of both datasets. Moreover, the regulation of
gene expression on Host 2 after the switch is stated. Each gene was
classified based on the categories of transcriptional changes shown in
figure 2. Note that an upregulation on one host can be interpreted as a
downregulation on the alternative host. Genes in bold were highly
upregulated on the same host also in the earlier study (without a
switch; Celorio-Mancera \emph{et al.} 2023) . NA = not annotated. Each
gene was classified based on the categories of transcriptional changes
shown in figure 2. \emph{Ud}=\emph{Urtica} \emph{dioica},
\emph{Ru}=\emph{Ribes uva-crispa} & \textbf{Table 1. Candidate genes
involved in a direct response to \emph{Urtica} and \emph{Ribes.}} Genes
with at least five-fold differential expression between \emph{Urtica}
and \emph{Ribes} treatments 17 hours after a switch to Host 2 compared
to controls remaining on Host 1 (i.e., \emph{UdUd} - \emph{UdRu} or
\emph{RuRu} - \emph{RuUd}). Gene name and functional description are
given for each gene. Furthermore, names of orthologs genes in
\emph{Heliconius melpomene} and the corresponding gene description are
given. Gene function was determined based on the functional annotations
of both datasets. Moreover, the regulation of gene expression on Host 2
after the switch is stated. Each gene was classified based on the
categories of transcriptional changes shown in figure 2. Note that an
upregulation on one host can be interpreted as a downregulation on the
alternative host. Genes in bold were highly upregulated on the same host
also in the earlier study (without a switch; Celorio-Mancera \emph{et
al.} 2023) . NA = not annotated. Each gene was classified based on the
categories of transcriptional changes shown in figure 2.
\emph{Ud}=\emph{Urtica} \emph{dioica}, \emph{Ru}=\emph{Ribes uva-crispa}
& \textbf{Table 1. Candidate genes involved in a direct response to
\emph{Urtica} and \emph{Ribes.}} Genes with at least five-fold
differential expression between \emph{Urtica} and \emph{Ribes}
treatments 17 hours after a switch to Host 2 compared to controls
remaining on Host 1 (i.e., \emph{UdUd} - \emph{UdRu} or \emph{RuRu} -
\emph{RuUd}). Gene name and functional description are given for each
gene. Furthermore, names of orthologs genes in \emph{Heliconius
melpomene} and the corresponding gene description are given. Gene
function was determined based on the functional annotations of both
datasets. Moreover, the regulation of gene expression on Host 2 after
the switch is stated. Each gene was classified based on the categories
of transcriptional changes shown in figure 2. Note that an upregulation
on one host can be interpreted as a downregulation on the alternative
host. Genes in bold were highly upregulated on the same host also in the
earlier study (without a switch; Celorio-Mancera \emph{et al.} 2023) .
NA = not annotated. Each gene was classified based on the categories of
transcriptional changes shown in figure 2. \emph{Ud}=\emph{Urtica}
\emph{dioica}, \emph{Ru}=\emph{Ribes uva-crispa} & \textbf{Table 1.
Candidate genes involved in a direct response to \emph{Urtica} and
\emph{Ribes.}} Genes with at least five-fold differential expression
between \emph{Urtica} and \emph{Ribes} treatments 17 hours after a
switch to Host 2 compared to controls remaining on Host 1 (i.e.,
\emph{UdUd} - \emph{UdRu} or \emph{RuRu} - \emph{RuUd}). Gene name and
functional description are given for each gene. Furthermore, names of
orthologs genes in \emph{Heliconius melpomene} and the corresponding
gene description are given. Gene function was determined based on the
functional annotations of both datasets. Moreover, the regulation of
gene expression on Host 2 after the switch is stated. Each gene was
classified based on the categories of transcriptional changes shown in
figure 2. Note that an upregulation on one host can be interpreted as a
downregulation on the alternative host. Genes in bold were highly
upregulated on the same host also in the earlier study (without a
switch; Celorio-Mancera \emph{et al.} 2023) . NA = not annotated. Each
gene was classified based on the categories of transcriptional changes
shown in figure 2. \emph{Ud}=\emph{Urtica} \emph{dioica},
\emph{Ru}=\emph{Ribes uva-crispa} & \textbf{Table 1. Candidate genes
involved in a direct response to \emph{Urtica} and \emph{Ribes.}} Genes
with at least five-fold differential expression between \emph{Urtica}
and \emph{Ribes} treatments 17 hours after a switch to Host 2 compared
to controls remaining on Host 1 (i.e., \emph{UdUd} - \emph{UdRu} or
\emph{RuRu} - \emph{RuUd}). Gene name and functional description are
given for each gene. Furthermore, names of orthologs genes in
\emph{Heliconius melpomene} and the corresponding gene description are
given. Gene function was determined based on the functional annotations
of both datasets. Moreover, the regulation of gene expression on Host 2
after the switch is stated. Each gene was classified based on the
categories of transcriptional changes shown in figure 2. Note that an
upregulation on one host can be interpreted as a downregulation on the
alternative host. Genes in bold were highly upregulated on the same host
also in the earlier study (without a switch; Celorio-Mancera \emph{et
al.} 2023) . NA = not annotated. Each gene was classified based on the
categories of transcriptional changes shown in figure 2.
\emph{Ud}=\emph{Urtica} \emph{dioica}, \emph{Ru}=\emph{Ribes uva-crispa}
& \textbf{Table 1. Candidate genes involved in a direct response to
\emph{Urtica} and \emph{Ribes.}} Genes with at least five-fold
differential expression between \emph{Urtica} and \emph{Ribes}
treatments 17 hours after a switch to Host 2 compared to controls
remaining on Host 1 (i.e., \emph{UdUd} - \emph{UdRu} or \emph{RuRu} -
\emph{RuUd}). Gene name and functional description are given for each
gene. Furthermore, names of orthologs genes in \emph{Heliconius
melpomene} and the corresponding gene description are given. Gene
function was determined based on the functional annotations of both
datasets. Moreover, the regulation of gene expression on Host 2 after
the switch is stated. Each gene was classified based on the categories
of transcriptional changes shown in figure 2. Note that an upregulation
on one host can be interpreted as a downregulation on the alternative
host. Genes in bold were highly upregulated on the same host also in the
earlier study (without a switch; Celorio-Mancera \emph{et al.} 2023) .
NA = not annotated. Each gene was classified based on the categories of
transcriptional changes shown in figure 2. \emph{Ud}=\emph{Urtica}
\emph{dioica}, \emph{Ru}=\emph{Ribes uva-crispa} & \textbf{Table 1.
Candidate genes involved in a direct response to \emph{Urtica} and
\emph{Ribes.}} Genes with at least five-fold differential expression
between \emph{Urtica} and \emph{Ribes} treatments 17 hours after a
switch to Host 2 compared to controls remaining on Host 1 (i.e.,
\emph{UdUd} - \emph{UdRu} or \emph{RuRu} - \emph{RuUd}). Gene name and
functional description are given for each gene. Furthermore, names of
orthologs genes in \emph{Heliconius melpomene} and the corresponding
gene description are given. Gene function was determined based on the
functional annotations of both datasets. Moreover, the regulation of
gene expression on Host 2 after the switch is stated. Each gene was
classified based on the categories of transcriptional changes shown in
figure 2. Note that an upregulation on one host can be interpreted as a
downregulation on the alternative host. Genes in bold were highly
upregulated on the same host also in the earlier study (without a
switch; Celorio-Mancera \emph{et al.} 2023) . NA = not annotated. Each
gene was classified based on the categories of transcriptional changes
shown in figure 2. \emph{Ud}=\emph{Urtica} \emph{dioica},
\emph{Ru}=\emph{Ribes uva-crispa}\tabularnewline
\textbf{Host pair comparisons} & \textbf{Gene name} &
\textbf{Description} & \emph{H. melpomene} \textbf{ortholog} &
\textbf{Description based on \emph{H. melpomene} ortholog} &
\textbf{Gene function} & \textbf{Regulation of gene expression on Host
2} & \textbf{category}\tabularnewline
\emph{UdUd} - \emph{UdRu} & g3036 & chitin binding; involved biological
process described with chitin metabolic process & HMEL002002-PA &
Peritrophic matrix 1-b precursor & Chitin binding, Gut peritrophic
matrix development & Upregulation on \emph{Ribes} & 3\tabularnewline
\emph{UdUd} - \emph{UdRu} & g94 & Proline dehydrogenase &
\textbf{HMEL003569-PA} & Sluggish isoform a-like; proline dehydrogenase
& Proline metabolism & Upregulation on \emph{Ribes} & 3\tabularnewline
\emph{UdUd} - \emph{UdRu} & g12792 & Trypsin-like serine protease &
HMEL003683-PA & Hemolymph proteinase 9 & Serine protease & Upregulation
on \emph{Ribes} & 3\tabularnewline
\emph{UdUd} - \emph{UdRu} & g11908 & NA & HMEL004395-PA & NA & NA &
Upregulation on \emph{Ribes} & 3\tabularnewline
\emph{UdUd} - \emph{UdRu} & g13507 & Glutathione S-transferase,
C-terminal domain & HMEL006527-PA & Glutathione s-transferase 1-like &
Detoxification & Upregulation on \emph{Ribes} & 3\tabularnewline
\emph{UdUd} - \emph{UdRu} & g1835 & Zinc finger, C2H2 type, GLIS2 &
HMEL006553-PA & Zinc finger protein gli1-like & Transcription factor &
Upregulation on \emph{Ribes} & 1\tabularnewline
\emph{UdUd} - \emph{UdRu} & g6954 & Peptide methionine sulfoxide
reductase & HMEL008564-PA & Methionine sulfoxide reductase & Response to
oxidative stress & Upregulation on \emph{Ribes} & 1\tabularnewline
\emph{UdUd} - \emph{UdRu} & g18164 g18165 & NA & \textbf{HMEL009345-PA}
& Aldo-keto reductase & Metabolism, detoxification & Upregulation on
\emph{Ribes} & 1\tabularnewline
\emph{UdUd} - \emph{UdRu} & g7930 & Immunoglobulin domain &
HMEL012261-PA & Lachesin-like & Neurogenesis & Upregulation on
\emph{Ribes} & 3\tabularnewline
\emph{UdUd} - \emph{UdRu} & g500 & NA & HMEL014522-PA &
Endonuclease-reverse transcriptase & DNA repair & Upregulation on
\emph{Ribes} & 3\tabularnewline
\emph{UdUd} - \emph{UdRu} & g8589 & NA & HMEL015934-PA & Lipase & Lipid
catabolism, Phagocytosis & Upregulation on \emph{Ribes} &
3\tabularnewline
\emph{UdUd} - \emph{UdRu} & g19077 g19078 & NA & \textbf{HMEL016210-PA}
& NA & NA & Upregulation on \emph{Ribes} & 1\tabularnewline
\emph{UdUd} - \emph{UdRu} & g12522 g12526 & NAD(P)-binding Rossmann-like
domain & \textbf{HMEL016306-PA} & Spermine oxidase-like & Polyamine
metabolism & Upregulation on \emph{Ribes} & 1\tabularnewline
\emph{UdUd} - \emph{UdRu} & g2848 & NA & NA & NA & NA & Upregulation on
\emph{Ribes} & 3\tabularnewline
\emph{UdUd} - \emph{UdRu} & g11512 & NA & NA & NA & NA & Upregulation on
\emph{Ribes} & 1\tabularnewline
\emph{UdUd} - \emph{UdRu} & g18835 & NA & NA & NA & NA & Upregulation on
\emph{Ribes} & 1\tabularnewline
\emph{UdUd} - \emph{UdRu} & g17488 & Major Facilitator Superfamily &
HMEL008930-PA & Sialin-like transporter & Transmembrane transport,
Sialic acid transport & Downregulation on \emph{Ribes} &
3\tabularnewline
\emph{UdUd} - \emph{UdRu} & g21009 & Chorion protein & HMEL011168-PA &
Chorion class cb protein m5h4-like & Structural protein & Downregulation
on \emph{Ribes} & 3\tabularnewline
\emph{UdUd} - \emph{UdRu} & g3540 g19683 & metallocarboxypeptidase
activity; involved in biological process described with proteolysis &
HMEL012401-PA & Carboxypeptidase-like & Protein metabolism &
Downregulation on \emph{Ribes} & 1\tabularnewline
\emph{UdUd} - \emph{UdRu} & g15631 g15249 & NA & \textbf{HMEL012909-PA}
& Synaptic vesicle glycoprotein-like & Transmembrane transport &
Downregulation on \emph{Ribes} & 1\tabularnewline
\emph{UdUd} - \emph{UdRu} & g15247 & synaptic vesicle &
\textbf{HMEL012912-PA} & Synaptic vesicle protein 2-like & Transmembrane
transport & Downregulation on \emph{Ribes} & 3\tabularnewline
\emph{UdUd} - \emph{UdRu} & g1204 & NA & NA & NA & NA & Downregulation
on \emph{Ribes} & 3\tabularnewline
\emph{UdUd} - \emph{UdRu} & g15555 & NA & NA & NA & NA & Downregulation
on \emph{Ribes} & 3\tabularnewline
\emph{RuRu} - \emph{RuUd} & g11903 & Trypsin-like serine protease &
HMEL004401-PA & serine protease easter-like; Clip domain serine protease
like 4 & Immune response & Upregulation on \emph{Urtica} &
3\tabularnewline
\emph{RuRu} - \emph{RuUd} & g12017 & NA & HMEL008841-PA & NA & NA &
Upregulation on \emph{Urtica} & 3\tabularnewline
\emph{RuRu} - \emph{RuUd} & g188 & N-acetylmuramoyl-L-alanine amidase &
HMEL009426-PA & peptidoglycan recognition protein-d & Innate immune
response & Upregulation on \emph{Urtica} & 3\tabularnewline
\emph{RuRu} - \emph{RuUd} & g1152 & NA & HMEL010472-PA & transcription
factor hnf-4 homolog & Involved in gut formation & Upregulation on
\emph{Urtica} & 3\tabularnewline
\emph{RuRu} - \emph{RuUd} & g2711 & Alkaline phosphatase & HMEL010183-PA
& alkaline phosphatase, tissue-nonspecific isozyme & Transmembrane
transport & Upregulation on \emph{Urtica} & 3\tabularnewline
\emph{RuRu} - \emph{RuUd} & g15249 & NA & \textbf{HMEL012909-PA} &
Synaptic vesicle glycoprotein-like & Transmembrane transport &
Upregulation on \emph{Urtica} & 1\tabularnewline
\emph{RuRu} - \emph{RuUd} & g7501 & Gloverin-like protein &
\textbf{HMEL017163-PA} & Gloverin; antibacterial peptide & Immune
response, Antimicrobial defense & Upregulation on \emph{Urtica} &
1\tabularnewline
\emph{RuRu} - \emph{RuUd} & g19716 & NA & HMEL017191-PA & unclassified
opsin & NA & Upregulation on \emph{Urtica} & 3\tabularnewline
\emph{RuRu} - \emph{RuUd} & g15789 & NA & NA & NA & NA & Upregulation on
\emph{Urtica} & 3\tabularnewline
\emph{RuRu} - \emph{RuUd} & g9519 & structural molecule activity &
HMEL002160-PA & NA & structural integrity of a complex & Downregulation
on \emph{Urtica} & 3\tabularnewline
\emph{RuRu} - \emph{RuUd} & g1835 & Zinc finger, C2H2 type, GLIS2 &
HMEL006553-PA & Zinc finger protein gli1-like & Transcription factor &
Downregulation on \emph{Urtica} & 1\tabularnewline
\emph{RuRu} - \emph{RuUd} & g354 & Carboxylesterase family, CES5A &
HMEL009236-PA & esterase fe4-like & Oxidative stress resistance &
Downregulation on \emph{Urtica} & 3\tabularnewline
\emph{RuRu} - \emph{RuUd} & g17913 & NA & HMEL014531-PA & NA & NA &
Downregulation on \emph{Urtica} & 3\tabularnewline
\emph{RuRu} - \emph{RuUd} & g10799 & Fungal protease inhibitor &
HMEL015606-PA & cationic peptide cp8 precursor & Immune response &
Downregulation on \emph{Urtica} & 3\tabularnewline
\emph{RuRu} - \emph{RuUd} & g19077 g19078 & NA & \textbf{HMEL016210-PA}
& NA & NA & Downregulation on \emph{Urtica} & 1\tabularnewline
\emph{RuRu} - \emph{RuUd} & g3815 & NA & NA & NA & NA & Downregulation
on \emph{Urtica} & 3\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6. Host switches did not affect larval
performance.}Larvae were reared on \emph{Urtica} , \emph{Salix} or
\emph{Ribes} and moved to a new host plant when reaching the 5th instar
(n=9-10 per treatment). To measure larval performance the daily growth
rate was calculated as log(mass in \selectlanguage{greek}µ\selectlanguage{english}g)/developmental time. No
significant differences in the growth rate were found between the
different treatments. The average age of larvae when they reached the
5th instar and were, thus, switched to a new host is indicated by the
vertical line. 95\% confidence interval is shown as shading for the
smoothed curves. The letters on top of each panel represent the plants
involved in the host switch. The first letter pair corresponds to Host
1, the second letter pair to Host 2. Ud=\emph{Urtica dioica} ,
Sc=\emph{Salix caprea} , Ru=\emph{Ribes uva-crispa} .

\selectlanguage{english}
\FloatBarrier
\end{document}

