4.Discussion
Earlier results from 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 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. Urtica dioica andUlmus glabra both belong to the “urticalean rosids”, whileSalix caprea and 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 et al. (2009) and Celorio-Mancera et al.(2012, 2023). The comparatively small number of transcriptional
differences that were found in switches between Urtica andSalix is moreover consistent with the overlapping gene expression
profiles that were previously shown by Celorio-Mancera 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 involvedRibes and especially between the phylogenetically and chemically
diverse Urtica and Ribes (Switch B), is consistent with
the overall opposing gene expression profiles between these two hosts
(Celorio-Mancera et al. 2023).
Based on these transcription profiles, it could be assumed that switches
containing Ribes indeed represent a more complex situation. The
differences between the different directions of the host switches,
further indicated that switches to Ribes appear to be more
challenging than the reverse. This could be related to the special
chemical composition of Ribes . The leaves of 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; Fürstenberg-Hägg et al . 2013). The gene
expression patterns found here support this assumption. Most of the
genes with a significantly different expression on Ribes were
mainly involved in metabolic processes (Table S1). This was further
confirmed by a closer examination of the upregulated genes onRibes in comparison to 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 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 onRibes could be shown again. Spermine oxidase is involved in the
metabolic processing of polyamines (Chang 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 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 et al. 2013;
Chikate 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 (Chouguleet al. 2005; Celorio-Mancera et al. 2013; Chikate et
al. 2013; Vogel et al. 2014). Such host-specific involvement of
proteases has previously been shown for P. c-album(Celorio-Mancera 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 et al. 2010, Celorio-Mancera 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 et al.2013 for details; see also Pérez-Hedo et al. 2012). Previous
studies already showed differential expression of genes involved in
structural constituents of the cuticle as a response to Ribes(Celorio-Mancera et al. 2013). An increased production of chitin
to strengthen or repair the intestinal tissue, thus, seems to be
required for the usability of Ribes after a switch.
The downregulation of Sialin-like after a switch to Ribes can be
interpreted as a transient response to a previous upregulation onUrtica . This is in agreement with its upregulation onUrtica shown in the earlier study by Celorio-Mancera et
al. (2023). Sialin-like is likely involved in nitrate transport andUrtica is characterized by a high nitrate content (Hegnauer 1973;
Taylor 2009). When switching larvae to Ribes , they quickly have
to react to compensate for the lower nitrate concentration. This also
agrees with the upregulation of transmembrane proteins on 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 et al. 2009;
Celorio-Mancera 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 Urtica andSalix 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 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 P. c-album already
showed that caterpillars can switch easily between Urtica andSalix 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 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 P. c-album have been
described to be relatively mobile and can cover substantial distances
even as neonates (Nylin et al. 2000, Schäpers 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 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 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 Polygonia butterflies
at some point. In line with this interpretation, the challenging switch
to Ribes in the present study seems to be reflected in
evolutionary patterns, as butterflies outside of 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.