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 Ulmus toUrtica 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 Salix clustered separately from the other
larvae after 2 hours, but this pattern was lost after17 hrs. In
contrast, individuals moved to 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 < 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. UdUd ) with individuals that were moved to an
alternative host (i.e. Switch, e.g. 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 Urtica (aka Ud ; Figure 3a). After 2
hours, most differentially expressed genes were found in the switch fromUlmus to Urtica (UgUg - 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 theUrtica and Salix -control line (UdUd - 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 betweenUrtica and Salix and between Salix andRibes . After 17 hours, overall higher numbers of differentially
expressed genes were found in switches that involved Ribes. By
this time, most transcriptional differences were detected in the
pairwise comparison of the Urtica control line with the switchUrtica to Ribes (i.e. UdUd - 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,UdUd - UdRu compared to UdUd - 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 fromSalix to 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 peptidoglycan
recognition protein signaling pathway” (Supplementary Table 2). In the
reciprocal switch from Urtica to Salix , these differences
were not found. In addition, one gene that matched category 2
expectations was found 2 hours after individuals were moved fromSalix to Ribes . This gene showed a significant enrichment
for “regulation of transcription involved in G1/S transition of
mitotic cell cycle ” and “biomineralization ”. Again, the
switch into the other direction (Ribes to 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 Urtica - Salix switches
and the respective control lines after 17 hours (e.g., UdUd -UdSc and ScSc - 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 betweenUrtica and Ribes . In both directions a large proportion of
the genes showed an adjustment to the new host (UdUd -UdRu : 9.5% (n=123); RuRu - RuUd : 17.9% (n=90)).
Especially in the switch to 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 fromSalix to Ribes (ScSc - ScRu ) but not in the
other direction, which further supports a crucial role of these genes
for the utilization of Ribes . The majority of the genes were,
however, uniquely differentially expressed between switch but not the
control lines (UdUd - UdRU : 84.6% (n=1096), RuRu -RuUd : 60.4% (n=304), ScSc - 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.
3.5. Congruence with previous studies
Switches between Urtica and Ribes are hypothesized to be
the most challenging for P. c-album larvae (Celorio-Manceraet 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 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 Urtica and Ribes controls. Of 113 genes
upregulated on Ribes , 91 could be matched to genes in the earlier
database, and 29 of these genes were also significantly upregulated onRibes in the earlier study (Celorio-Mancera et al. 2023).
85 genes were upregulated on Urtica in our study, out of which 75
could be matched, and 20 of these genes were also upregulated onUrtica in the previous study. Only one gene was found to
be expressed in the opposite direction between the two studies
(upregulated on 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-Manceraet al . 2023). In both studies, a synaptic vesicle protein and an
antibacterial peptide were strongly upregulated on 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 Ribes in both studies.
We further used an extensive functional annotation from Celorio-Manceraet al . (2023) to identify the putative function of some of the
genes that were highly up- or downregulated after a switch fromUrtica to 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 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 Urticaand 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 (χ2= 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 (χ2= 3.1934,
df= 8, p=0.9216) on growth rate could be measured (Supplementary Figure
4). This suggests that the caterpillars of Polygonia c-album can
adjust to a new host without major expenses for their development.