BACKGROUND
chemical defenses of plants influence the host range of herbivorous insects  <cit> .
although by no means the only factor involved in shaping insect-host associations  <cit> , few researchers would argue against its general importance.
however, there is an ongoing argument over the reasons for similarities among plant defense chemicals.
there are two ways for evolution to achieve similarity: either through shared ancestry or through evolutionary convergence .
ehrlich and raven  <cit>  suggested that related insects tend to feed on related plants, and several other studies have continued to demonstrate a role of host plant phylogeny  on patterns of host plant use  <cit> .
hence, there is strong support for a historical component in patterns of host plant use.
on the other hand, several authors have also pointed out that plant chemistry does not always follow phylogeny  <cit> .
feeny  <cit>  suggested that plant defense strategies should differ depending on their "apparency"; plants that are long-lived and/or physically large will always be found by attacking insects and should possess constitutive chemical defenses such as tannins, terpenes, and flavanoids  that have a quantitative, dosage-dependent effect.
unapparent plants, with lower risk of detection by herbivores, should instead tend to utilize induced chemical defenses.
trees should be the most apparent of plants as they are both physically large and long-lived.
according to the apparency hypothesis, trees should then tend to have more convergent constitutive defenses than herbs, and as a consequence we should see more host shifts involving trees than herbs.
despite some criticism and uncertainty regarding the mechanisms involved, the apparency theory has remained a useful framework for thinking about the evolution of plant chemical defense in the context of plant community heterogeneity  <cit> .
in a phylogenetic reanalysis of ehrlich and raven's  <cit>  study on butterfly and plant coevolution, janz & nylin  <cit>  found strong effects of both plant phylogeny and growth form on patterns of host use among butterflies.
an overwhelming majority of host shifts occurred while feeding on trees, giving support for feeny's "plant apparency" hypothesis.
trees appeared to serve as a "bridge" that could facilitate host shifts between distantly related plants.
hence, there is support for both shared ancestry and convergent evolution in the large-scale chemical structuring of insect-host associations, but the mechanistic basis remains largely unknown.
however, recent years have seen great progress in understanding of the molecular mechanisms that enable insect to feed on certain host plants.
in general it is assumed that insects apply phase i and phase ii detoxifying enzymes to metabolize secondary plant compounds.
several studies have for example revealed the important role of the cytochrome p <dig> enzyme family for detoxification of plant secondary compounds as well as insecticides.
<cit> .
glutathione-s-transferases  have also been shown to be induced in generalist and specialist lepidopteran larvae upon feeding on their host plants  <cit> .
wittstock et al.
<cit>  identified the nitrile-specifier protein  in pieris rapae.
nsp redirects the glucosinolate hydrolysis and by that enables the pierinae butterflies to feed on the plant family brassicaceae.
further research has been done on the evolution of nsp showing its evolution by domain and gene duplication from a gene of unknown function that is widespread in insect species  <cit> .
plutella xylostella is also feeding on glucosinolate containing plants.
here the glucosinolate sulfatase  inhibits the hydrolysis of glucosinolates completely by forming a desulfo-glucosinolate.
in spite of progress in recent years, much is still to be discovered in the detoxification mechanisms of insects.
in the present study we aim to investigate the molecular basis of the diverse host plant range of the comma butterfly .
p. c-album is a widespread polyphagous butterfly species of the family nymphalidae.
it is found all over eurasia, from england to japan and from the central of sweden to the northern tip of africa.
the larvae can be found on host plants from several taxa: the "urticalean rosids" urtica, humulus and ulmus and the distantly related salix , ribes , betula and corylus   <cit> ; hence the species is at the extreme end of polyphagy among butterflies, although by no means an indiscriminate generalist  <cit> .
for this study, we used a test array with three host plants of p. c-album that are either closely related  or share the same growth form .
following feeny's "plant apparency" hypothesis and the phylogenetic patterns of host shifts found by janz & nylin  <cit>  and janz et al.
<cit> , we expected to find more similarities in the gene expression profiles of caterpillars that have been feeding on plants that either have a shared ancestry  or belong to the same growth form .
RESULTS
general patterns of gene expression
the pattern of expression in the midgut corresponded to the formed predictions that more similarities will be found in the gene expression profiles of caterpillars that have been feeding on plants that either have a shared ancestry  or belong to the same growth form  .
there were significantly more uniquely upregulated genes in the larval midgut on the urtica and salix diets than on the ulmus diet  =  <dig>   <dig>   <dig> , χ <dig> =  <dig> , p =  <dig> ).
pairwise similarities in upregulation were higher between plants that shared either growth form or were phylogenetically related  =  <dig>   <dig>   <dig> , χ <dig> =  <dig> , p =  <dig> ).
there were no corresponding patterns in the restbodies .
here, more genes were uniquely upregulated on urtica and ulmus than on salix  =  <dig>   <dig>   <dig> , χ <dig> =  <dig> , p =  <dig> ) and there were more similarities in upregulated genes between the "unrelated" plants than between the trees or urticalean rosid groups  =  <dig>   <dig>   <dig> , χ <dig> =  <dig> , p <  <dig> ).
function and patterns of differentially expressed genes
in total we identified  <dig> differentially expressed genes,  <dig> in the midgut of p. c-album and  <dig> in the restbody .
in the midgut six sequences gave no hit and in the restbody  <dig> sequences gave no hit in blast searches.
many of the differentially expressed genes in the midgut are likely to be involved in metabolism and digestion, ranging from protein degradation to starch and lipid breakdown for nutrient acquisition.
we could also identify eight ribosomal genes and seven genes that are involved in translation regulation and the dna structure in the cell nucleus.
furthermore, we found three genes involved in immunity and one transmembrane transport domain.
there were also differentially expressed proteases, but we did not identify any differentially expressed known detoxifying enzymes in the midgut.
four of the differentially expressed genes were unknown .
in the restbody of the comma butterfly larvae again, the majority of the gene products are most likely involved in the metabolism of the caterpillar or translate into ribosomal proteins.
seven of the sequenced gene fragments appear to have a structural role, and are mostly involved in chitin binding.
we could also identify translation regulation genes and some involved in cellular architecture .
one sequence showed moderate similarities  to potential detoxification genes, namely a cytochrome p <dig> of plutella xylostella .
confirmation of differentially expressed genes
of the total  <dig> genes originating from genefishing, we picked  <dig> genes  to confirm differential gene expression pattern with qrt-pcr.
we were able to see identical expression patterns in the genefishing and in the qrt-pcr results for  <dig> of the  <dig> genes .
partial similarity  in expression patterns between two independent methods could be observed in  <dig> genes  and  <dig> genes  behaved differently .
in the midgut, genes that showed similar expression patterns as in the genefishing experiment included proteins involved in digestion, namely chymotrypsinogen-like protein  <dig>  serine protease, chymotrypsin-like protease, alpha-amylase, trypsin-like protease, trypsin ia precursor, long-chain acyl-coa synthetase and short-chain dehydrogenease/reductase  <dig>  in addition, also a ribosomal protein s <dig> and the immune response related protein cobatoxin showed same expression patterns in the qrt-pcr and in the differential gene expression study.
in the restbodies alpha-amylase and the serine protease precursor were similarly expressed.
the stress related heat shock cognate  <dig> protein and a potential cytochrome p <dig> also showed similar expression pattern in both methods.
discussion
our expression data suggest a complex interaction between the comma butterfly and its host plants.
on the one hand, each plant species appears to require a very specific subset of genes to be regulated in the midgut upon feeding.
this involves digestion proteins, immunity related genes, general metabolism genes and ribosomal protein as well as translational and transport genes.
in the restbody we also find cell signaling related domains, hormone synthesis genes and genes involved in silk production, suggesting a gene regulation, tuned specifically for each plant species.
on the other hand, counting of upregulation of genes in the midgut shows a suggestive pattern .
both species of the urticales family , as well as both trees, have a higher agreement of gene regulation than do the stinging nettle and the great sallow.
especially the digestion and ribosomal genes show clear differences here .
this suggests that phylogenetic and/or growth form relatedness demand more similar expression profiles in the midgut of the caterpillars.
however, this pattern only holds true for the midgut of the comma butterfly; the restbody of the caterpillars show a completely different picture.
here the stinging nettle and great sallow diet in contrast share the highest similarity in gene expression, while the wych elm appears to require less gene upregulation in general .
the midgut is the place of contact with the food bolus, and the location where the first detoxifying and digestive actions will take place, whereas the restbody is only indirectly involved in this process by receiving the solubilized products and only sometimes toxic or toxin degradation products.
here the actual nutrient content is of more importance.
this might explain the converse picture we observe in those two tissues.
while the defense compounds of the phylogenetically or growth form-related plants might be more similar, our data suggests that the nutritional value does not follow this line.
previous data on larval performance show that caterpillars of p. c-album perform differently on the plant species used in this study, suggesting different levels of adaptation and/or different nutritional values .
the general pattern is that the plants in urticales  support the highest growth rates, while larvae grow slower on s. caprea .
these differences are also reflected in the preference hierarchy of the ovipositing females, which means that the preference-performance correlation is good on a population level  <cit>  .
oviposition preference and larval performance on u. glabra are more variable between years than the other plants, so that in some years it is considerably less preferred than u. dioica, with a correspondingly lower performance .
this could be caused by higher between-year variability in u. glabra compared to the other two hosts.
such unpredictable variation would make it more difficult to adapt to the host.
indeed, the lower amount of uniquely upregulated genes on ulmus found in this study would suggest such insufficient adaptation to this host.
in the midgut we could not detect any differentially expressed known detoxifying genes, such as cytochromes p450s or glutathione-s-transferases .
however, those enzymes are with high probability active in the gut of the comma butterfly larvae when encountering plant material.
the genefishing method identifies solely differentially expressed genes.
hence, p. c-album might very well express such phase i and phase ii detoxifying genes, but in a constant manner independent of the diet.
being a highly polyphagous insect species, the comma butterfly might be endued with very broad acting detoxifying enzymes that can cope with a wide variety of the compounds and hence are expressed continuously when feeding independent of the diet.
the lack of differentially expressed detoxifying enzymes could also be due to overlaps of toxic chemistry on the three host plants.
with the exception of cytochrome p450s and gsts not much is known about detoxifying enzymes applied by generalist insects.
hence such expressed enzymes could not be identified in databases.
in the midgut we identified  <dig> unknown differentially expressed genes, which could very well be unknown genes involved in detoxification.
we could, however, identify a differentially expressed gene homologous to a cytochrome p <dig> of p. xylostella in the restbody of p. c-album larvae.
cytochrome p450's are also involved in the metabolism of many endogenous compounds, hence this p <dig> expressed in the restbody is possibly not involved in the detoxification of plant defense compounds, but in metabolism.
while the detoxifying mechanisms appear not to be differentially expressed we see genes belonging to other classes differentially expressed.
short-chain dehydrogenases  for example are upregulated in the midguts of on urtica and ulmus feeding larvae.
scdh form a large protein family with highly different enzymes, which only share 15-30% identity among each other  <cit> .
they are present in all the life forms studied so far, have a very wide substrate spectrum and are generally involved in cellular differentiation and signaling  <cit> .
not surprisingly, also many digestive enzymes are differentially expressed in the midgut .
for example an alpha-amylase was down-regulated in both tree diets in comparison to the stinging nettle.
amylases are enzymes participating in carbohydrate digestion.
it is known that insects possess different amylases for starches  <cit> .
it is possible that the differential expression we observe is due to different starch contents in the stinging nettle.
we also found several serine proteases being differentially expressed upon feeding on the host plants, namely trypsins and chymotrypsins.
plants possess proteinase inhibitors , which are insect inducible peptidases that can suppress insect proteinases and by that reduce the digestibility  <cit> .
it has been shown that lepidopteran larvae adapt their proteinases expression profile to the pi content of their food plant, upregulating proteinases that are insensitive to the plant pis  <cit> .
our expression patterns suggest different proteinase requirements for the three host plants.
we also excised four bands that were identified as homologous to bmlipase-1; three of them were highly expressed in larvae feeding on urtica and one on salix.
insect midgut lipases have been studied in few insects so far and little to nothing is known on different expression of lipases in insects  <cit> .
they form in insects a gene family that underwent many duplication events  <cit>  with resulting diverse and overlapping function.
bmlipase- <dig> from bombyx mori shows a high antiviral activity against bombyx mori nucleopolyhedrovirus, although it is not inducible by viral infection.
its main function is probably as a digestive enzyme, as it is exclusively expressed in the gut tissue and has lipase activity  <cit> .
we also found many ribosomal proteins to be differentially regulated upon feeding on different host plants, namely seven in the midgut and twelve in the restbody of p. c-album.
ribosomal genes are considered to be stably expressed and have been suggested and used as housekeeping genes in expression analysis.
however, there are a number of ribosomal proteins that have been found to be differentially expressed upon different treatment and in different tissues  <cit> , suggesting that there are major differences in expression patterns between different ribosomal proteins.
the complex pattern found in this study should perhaps not be surprising, considering that secondary metabolites, leaf texture and nutrient contents vary greatly between different plant species, and also depending on the plant age  <cit> .
the three chosen p. c-album host plants have different leaf architecture, with the stinging nettle possessing thin hairy leaves, and both trees waxy leaves, that differ in shape.
besides the prominent stinging trichomes that only provide an effective defense against vertebrate herbivores  <cit> , stinging nettle leaves also contain the phenolic compound caffeic acid, tannins, nicotine in measurable amounts and flavonoid glucosides  <cit> .
salix leaves has low levels of toxins but more condensed tannins  <cit> , which is similar to the situation in elm  <cit> .
phenolic compounds, nicotine, tannins and flavonoids have been shown to affect insects feeding on them.
tannins for example are astringent, bitter plant polyphenols that either bind and precipitate or shrink proteins.
studies on the effect of tannins on the gut ph and redox potential of larvae found that many specialist and generalist insects have developed adaptations to cope with them  <cit> .
tannin metabolites are for example oxidized during the gut passage in the aquatic caterpillar acentria ephemerella.
resistance against polyphenols appears to be correlated in general with better repair mechanisms in the gut tissue that enables to cope with free radical stress occurring during oxidation better  <cit> .
nicotine acts as agonist of the postsynaptic nicotinic acetylcholine receptors of the insect central nervous system.
metabolism of nicotine has been attributed to the action of cytochrome p <dig>  <cit> .
flavonoids in contrast can be sequestered from the diet and used for protection and pigmentation.
in b. mori for example three flavonoid glycosides could be isolated from the cocoon shell  <cit> .
the role of these secondary plant compounds in the interaction between p. c-album and its host plant and the adaptations p. c-album developed to them however, is not known.
different plants also possess different microfloras  <cit> , which affect immune response-related gene expression in the midgut of lepidopteran larvae  <cit> .
we detected cobatoxin and gloverin homologous genes that are known to be inducible by bacterial challenge to be differentially expressed in the midgut tissue  <cit> .
we see gloverin expression only in urtica fed larvae and cobatoxin in salix fed larvae, suggesting different microbial environments on those two species.
in addition to differences in plant secondary metabolites p c.-album must therefore also face different bacterial quantities and qualities on its various host plants.
CONCLUSIONS
our data suggest a complex picture of gene expression in response to host plants, but also revealed some general patterns.
while each plant evidently requires a unique gene regulation in the caterpillar, both phylogenetic relatedness and host plant growth form appear to influence the expression profile of the polyphagous comma butterfly, in agreement with phylogenetic studies of host plant utilization in butterflies.

 chemical defenses of plants influence the host range of herbivorous insects. although by no means the only factor involved in shaping insect-host associations, few researchers would argue against its general importance. however, there is an ongoing argument over the reasons for similarities among plant defense chemicals. there are two ways for evolution to achieve similarity: either through shared ancestry or through evolutionary convergence. ehrlich and raven suggested that related insects tend to feed on related plants, and several other studies have continued to demonstrate a role of host plant phylogeny on patterns of host plant use. hence, there is strong support for a historical component in patterns of host plant use. on the other hand, several authors have also pointed out that plant chemistry does not always follow phylogeny. feeny suggested that plant defense strategies should differ depending on their "apparency"; plants that are long-lived and/or physically large will always be found by attacking insects and should possess constitutive chemical defenses such as tannins, terpenes, and flavanoids that have a quantitative, dosage-dependent effect. unapparent plants, with lower risk of detection by herbivores, should instead tend to utilize induced chemical defenses. trees should be the most apparent of plants as they are both physically large and long-lived. according to the apparency hypothesis, trees should then tend to have more convergent constitutive defenses than herbs, and as a consequence we should see more host shifts involving trees than herbs. despite some criticism and uncertainty regarding the mechanisms involved, the apparency theory has remained a useful framework for thinking about the evolution of plant chemical defense in the context of plant community heterogeneity. in a phylogenetic reanalysis of ehrlich and raven's study on butterfly and plant coevolution, janz & nylin found strong effects of both plant phylogeny and growth form on patterns of host use among butterflies. an overwhelming majority of host shifts occurred while feeding on trees, giving support for feeny's "plant apparency" hypothesis. trees appeared to serve as a "bridge" that could facilitate host shifts between distantly related plants. hence, there is support for both shared ancestry and convergent evolution in the large-scale chemical structuring of insect-host associations, but the mechanistic basis remains largely unknown. however, recent years have seen great progress in understanding of the molecular mechanisms that enable insect to feed on certain host plants. in general it is assumed that insects apply phase i and phase ii detoxifying enzymes to metabolize secondary plant compounds. several studies have for example revealed the important role of the cytochrome p enzyme family for detoxification of plant secondary compounds as well as insecticides.. glutathione-s-transferases have also been shown to be induced in generalist and specialist lepidopteran larvae upon feeding on their host plants. wittstock et al. identified the nitrile-specifier protein in pieris rapae. nsp redirects the glucosinolate hydrolysis and by that enables the pierinae butterflies to feed on the plant family brassicaceae. further research has been done on the evolution of nsp showing its evolution by domain and gene duplication from a gene of unknown function that is widespread in insect species. plutella xylostella is also feeding on glucosinolate containing plants. here the glucosinolate sulfatase inhibits the hydrolysis of glucosinolates completely by forming a desulfo-glucosinolate. in spite of progress in recent years, much is still to be discovered in the detoxification mechanisms of insects. in the present study we aim to investigate the molecular basis of the diverse host plant range of the comma butterfly. p. c-album is a widespread polyphagous butterfly species of the family nymphalidae. it is found all over eurasia, from england to japan and from the central of sweden to the northern tip of africa. the larvae can be found on host plants from several taxa: the "urticalean rosids" urtica, humulus and ulmus and the distantly related salix, ribes, betula and corylus; hence the species is at the extreme end of polyphagy among butterflies, although by no means an indiscriminate generalist. for this study, we used a test array with three host plants of p. c-album that are either closely related or share the same growth form. following feeny's "plant apparency" hypothesis and the phylogenetic patterns of host shifts found by janz & nylin and janz et al., we expected to find more similarities in the gene expression profiles of caterpillars that have been feeding on plants that either have a shared ancestry or belong to the same growth form.  general patterns of gene expression the pattern of expression in the midgut corresponded to the formed predictions that more similarities will be found in the gene expression profiles of caterpillars that have been feeding on plants that either have a shared ancestry or belong to the same growth form. there were significantly more uniquely upregulated genes in the larval midgut on the urtica and salix diets than on the ulmus diet =, χ =, p = ). pairwise similarities in upregulation were higher between plants that shared either growth form or were phylogenetically related =, χ =, p = ). there were no corresponding patterns in the restbodies. here, more genes were uniquely upregulated on urtica and ulmus than on salix =, χ =, p = ) and there were more similarities in upregulated genes between the "unrelated" plants than between the trees or urticalean rosid groups =, χ =, p < ). function and patterns of differentially expressed genes in total we identified differentially expressed genes, in the midgut of p. c-album and in the restbody. in the midgut six sequences gave no hit and in the restbody sequences gave no hit in blast searches. many of the differentially expressed genes in the midgut are likely to be involved in metabolism and digestion, ranging from protein degradation to starch and lipid breakdown for nutrient acquisition. we could also identify eight ribosomal genes and seven genes that are involved in translation regulation and the dna structure in the cell nucleus. furthermore, we found three genes involved in immunity and one transmembrane transport domain. there were also differentially expressed proteases, but we did not identify any differentially expressed known detoxifying enzymes in the midgut. four of the differentially expressed genes were unknown. in the restbody of the comma butterfly larvae again, the majority of the gene products are most likely involved in the metabolism of the caterpillar or translate into ribosomal proteins. seven of the sequenced gene fragments appear to have a structural role, and are mostly involved in chitin binding. we could also identify translation regulation genes and some involved in cellular architecture. one sequence showed moderate similarities to potential detoxification genes, namely a cytochrome p of plutella xylostella. confirmation of differentially expressed genes of the total genes originating from genefishing, we picked genes to confirm differential gene expression pattern with qrt-pcr. we were able to see identical expression patterns in the genefishing and in the qrt-pcr results for of the genes. partial similarity in expression patterns between two independent methods could be observed in genes and genes behaved differently. in the midgut, genes that showed similar expression patterns as in the genefishing experiment included proteins involved in digestion, namely chymotrypsinogen-like protein serine protease, chymotrypsin-like protease, alpha-amylase, trypsin-like protease, trypsin ia precursor, long-chain acyl-coa synthetase and short-chain dehydrogenease/reductase in addition, also a ribosomal protein s and the immune response related protein cobatoxin showed same expression patterns in the qrt-pcr and in the differential gene expression study. in the restbodies alpha-amylase and the serine protease precursor were similarly expressed. the stress related heat shock cognate protein and a potential cytochrome p also showed similar expression pattern in both methods. discussion our expression data suggest a complex interaction between the comma butterfly and its host plants. on the one hand, each plant species appears to require a very specific subset of genes to be regulated in the midgut upon feeding. this involves digestion proteins, immunity related genes, general metabolism genes and ribosomal protein as well as translational and transport genes. in the restbody we also find cell signaling related domains, hormone synthesis genes and genes involved in silk production, suggesting a gene regulation, tuned specifically for each plant species. on the other hand, counting of upregulation of genes in the midgut shows a suggestive pattern. both species of the urticales family, as well as both trees, have a higher agreement of gene regulation than do the stinging nettle and the great sallow. especially the digestion and ribosomal genes show clear differences here. this suggests that phylogenetic and/or growth form relatedness demand more similar expression profiles in the midgut of the caterpillars. however, this pattern only holds true for the midgut of the comma butterfly; the restbody of the caterpillars show a completely different picture. here the stinging nettle and great sallow diet in contrast share the highest similarity in gene expression, while the wych elm appears to require less gene upregulation in general. the midgut is the place of contact with the food bolus, and the location where the first detoxifying and digestive actions will take place, whereas the restbody is only indirectly involved in this process by receiving the solubilized products and only sometimes toxic or toxin degradation products. here the actual nutrient content is of more importance. this might explain the converse picture we observe in those two tissues. while the defense compounds of the phylogenetically or growth form-related plants might be more similar, our data suggests that the nutritional value does not follow this line. previous data on larval performance show that caterpillars of p. c-album perform differently on the plant species used in this study, suggesting different levels of adaptation and/or different nutritional values. the general pattern is that the plants in urticales support the highest growth rates, while larvae grow slower on s. caprea. these differences are also reflected in the preference hierarchy of the ovipositing females, which means that the preference-performance correlation is good on a population level. oviposition preference and larval performance on u. glabra are more variable between years than the other plants, so that in some years it is considerably less preferred than u. dioica, with a correspondingly lower performance. this could be caused by higher between-year variability in u. glabra compared to the other two hosts. such unpredictable variation would make it more difficult to adapt to the host. indeed, the lower amount of uniquely upregulated genes on ulmus found in this study would suggest such insufficient adaptation to this host. in the midgut we could not detect any differentially expressed known detoxifying genes, such as cytochromes p450s or glutathione-s-transferases. however, those enzymes are with high probability active in the gut of the comma butterfly larvae when encountering plant material. the genefishing method identifies solely differentially expressed genes. hence, p. c-album might very well express such phase i and phase ii detoxifying genes, but in a constant manner independent of the diet. being a highly polyphagous insect species, the comma butterfly might be endued with very broad acting detoxifying enzymes that can cope with a wide variety of the compounds and hence are expressed continuously when feeding independent of the diet. the lack of differentially expressed detoxifying enzymes could also be due to overlaps of toxic chemistry on the three host plants. with the exception of cytochrome p450s and gsts not much is known about detoxifying enzymes applied by generalist insects. hence such expressed enzymes could not be identified in databases. in the midgut we identified unknown differentially expressed genes, which could very well be unknown genes involved in detoxification. we could, however, identify a differentially expressed gene homologous to a cytochrome p of p. xylostella in the restbody of p. c-album larvae. cytochrome p450's are also involved in the metabolism of many endogenous compounds, hence this p expressed in the restbody is possibly not involved in the detoxification of plant defense compounds, but in metabolism. while the detoxifying mechanisms appear not to be differentially expressed we see genes belonging to other classes differentially expressed. short-chain dehydrogenases for example are upregulated in the midguts of on urtica and ulmus feeding larvae. scdh form a large protein family with highly different enzymes, which only share 15-30% identity among each other. they are present in all the life forms studied so far, have a very wide substrate spectrum and are generally involved in cellular differentiation and signaling. not surprisingly, also many digestive enzymes are differentially expressed in the midgut. for example an alpha-amylase was down-regulated in both tree diets in comparison to the stinging nettle. amylases are enzymes participating in carbohydrate digestion. it is known that insects possess different amylases for starches. it is possible that the differential expression we observe is due to different starch contents in the stinging nettle. we also found several serine proteases being differentially expressed upon feeding on the host plants, namely trypsins and chymotrypsins. plants possess proteinase inhibitors, which are insect inducible peptidases that can suppress insect proteinases and by that reduce the digestibility. it has been shown that lepidopteran larvae adapt their proteinases expression profile to the pi content of their food plant, upregulating proteinases that are insensitive to the plant pis. our expression patterns suggest different proteinase requirements for the three host plants. we also excised four bands that were identified as homologous to bmlipase-1; three of them were highly expressed in larvae feeding on urtica and one on salix. insect midgut lipases have been studied in few insects so far and little to nothing is known on different expression of lipases in insects. they form in insects a gene family that underwent many duplication events with resulting diverse and overlapping function. bmlipase- from bombyx mori shows a high antiviral activity against bombyx mori nucleopolyhedrovirus, although it is not inducible by viral infection. its main function is probably as a digestive enzyme, as it is exclusively expressed in the gut tissue and has lipase activity. we also found many ribosomal proteins to be differentially regulated upon feeding on different host plants, namely seven in the midgut and twelve in the restbody of p. c-album. ribosomal genes are considered to be stably expressed and have been suggested and used as housekeeping genes in expression analysis. however, there are a number of ribosomal proteins that have been found to be differentially expressed upon different treatment and in different tissues, suggesting that there are major differences in expression patterns between different ribosomal proteins. the complex pattern found in this study should perhaps not be surprising, considering that secondary metabolites, leaf texture and nutrient contents vary greatly between different plant species, and also depending on the plant age. the three chosen p. c-album host plants have different leaf architecture, with the stinging nettle possessing thin hairy leaves, and both trees waxy leaves, that differ in shape. besides the prominent stinging trichomes that only provide an effective defense against vertebrate herbivores, stinging nettle leaves also contain the phenolic compound caffeic acid, tannins, nicotine in measurable amounts and flavonoid glucosides. salix leaves has low levels of toxins but more condensed tannins, which is similar to the situation in elm. phenolic compounds, nicotine, tannins and flavonoids have been shown to affect insects feeding on them. tannins for example are astringent, bitter plant polyphenols that either bind and precipitate or shrink proteins. studies on the effect of tannins on the gut ph and redox potential of larvae found that many specialist and generalist insects have developed adaptations to cope with them. tannin metabolites are for example oxidized during the gut passage in the aquatic caterpillar acentria ephemerella. resistance against polyphenols appears to be correlated in general with better repair mechanisms in the gut tissue that enables to cope with free radical stress occurring during oxidation better. nicotine acts as agonist of the postsynaptic nicotinic acetylcholine receptors of the insect central nervous system. metabolism of nicotine has been attributed to the action of cytochrome p. flavonoids in contrast can be sequestered from the diet and used for protection and pigmentation. in b. mori for example three flavonoid glycosides could be isolated from the cocoon shell. the role of these secondary plant compounds in the interaction between p. c-album and its host plant and the adaptations p. c-album developed to them however, is not known. different plants also possess different microfloras, which affect immune response-related gene expression in the midgut of lepidopteran larvae. we detected cobatoxin and gloverin homologous genes that are known to be inducible by bacterial challenge to be differentially expressed in the midgut tissue. we see gloverin expression only in urtica fed larvae and cobatoxin in salix fed larvae, suggesting different microbial environments on those two species. in addition to differences in plant secondary metabolites p c.-album must therefore also face different bacterial quantities and qualities on its various host plants.  our data suggest a complex picture of gene expression in response to host plants, but also revealed some general patterns. while each plant evidently requires a unique gene regulation in the caterpillar, both phylogenetic relatedness and host plant growth form appear to influence the expression profile of the polyphagous comma butterfly, in agreement with phylogenetic studies of host plant utilization in butterflies. 
expression patterns could be confirmed with an independent method for of tested genes. while host plant chemistry is influenced by phylogeny, also the growth forms of plants appear to influence the plant defense strategies as first postulated by feeny. pairwise similarities in upregulation in the midgut of the caterpillars were higher between plants that shared either growth form or were phylogenetically related. while each plant requires a unique gene regulation in the caterpillar, both phylogenetic relatedness and host plant growth form appear to influence the expression profile of the polyphagous comma butterfly, in agreement with phylogenetic studies of host plant utilization in butterflies. in the present study we aim to investigate the molecular basis of the diverse host plant range of the comma butterfly by testing differential gene expression in the caterpillars on three host plants that are either closely related or share the same growth form. in total genes were identified to be differentially expressed in p. c-album after feeding on different host plants, of them in the midgut and in the restbody of the caterpillars. no known detoxifying enzymes were found to be differently regulated in the midgut after feeding on different host plants. nevertheless, it is well established that chemical defenses of plants influence the host range of herbivorous insects. our data suggest a complex picture of gene expression in response to host plant feeding. the mechanisms that shape the host plant range of herbivorous insect are to date not well understood but knowledge of these mechanisms and the selective forces that influence them can expand our understanding of the larger ecological interaction.
 the mechanisms that shape the host plant range of herbivorous insect are to date not well understood but knowledge of these mechanisms and the selective forces that influence them can expand our understanding of the larger ecological interaction. nevertheless, it is well established that chemical defenses of plants influence the host range of herbivorous insects. while host plant chemistry is influenced by phylogeny, also the growth forms of plants appear to influence the plant defense strategies as first postulated by feeny. in the present study we aim to investigate the molecular basis of the diverse host plant range of the comma butterfly by testing differential gene expression in the caterpillars on three host plants that are either closely related or share the same growth form.  in total genes were identified to be differentially expressed in p. c-album after feeding on different host plants, of them in the midgut and in the restbody of the caterpillars. expression patterns could be confirmed with an independent method for of tested genes. pairwise similarities in upregulation in the midgut of the caterpillars were higher between plants that shared either growth form or were phylogenetically related. no known detoxifying enzymes were found to be differently regulated in the midgut after feeding on different host plants.  our data suggest a complex picture of gene expression in response to host plant feeding. while each plant requires a unique gene regulation in the caterpillar, both phylogenetic relatedness and host plant growth form appear to influence the expression profile of the polyphagous comma butterfly, in agreement with phylogenetic studies of host plant utilization in butterflies. 