Molecular Adaptation of Chrysochus Leaf Beetles to Toxic Compounds in Their Food Plants
http://www.100md.com
分子生物学进展 2004年第2期
Institut für Zoologie, Universit?t Freiburg, Freiburg, Germany
E-mail: susanne.dobler@zoologie.uni-hamburg.de.
Abstract
Herbivores that feed on toxic plants must overcome plant defenses and occasionally may even benefit from them. The current challenge is to understand how herbivores evolve the necessary physiological adaptations and which changes at the molecular level are involved. In this context we studied the leaf beetles genus Chrysochus (Coleoptera, Chrysomelidae). Two species of this genus, C. auratus and C. cobaltinus, feed on plants that contain toxic cardenolides. These beetles not only avoid poisoning by the toxin but also use it for their own defense against predators. All other Chrysochus species feed on plants that are devoid of cardenolides. The most important active principle of cardenolides is their capacity to bind to and thereby block the ubiquitous Na+/K+-ATPase responsible for maintaining cellular potentials. By analyzing the DNA sequence of the putative ouabain-binding site of the -subunit of the Na+/K+-ATPase gene of Chrysochus and its close relatives feeding on plants with or without cardenolides, we here trace the evolution of cardenolide insensitivity in this group of beetles. The most interesting difference among the sequences involves the amino acid at position 122. Whereas all species that do not encounter cardenolides have an asparagine in this position, both Chrysochus species that feed on cardenolide plants have a histidine instead. This single amino acid substitution has already been shown to confer cardenolide insensitivity in the monarch butterfly. A mtDNA-based phylogeny corroborates the hypothesis that the asparagine at position 122 of the -subunit of the Na+/K+-ATPase gene as observed in Drosophila and other insects is the plesiomorphic condition in this group of leaf beetles. The later host-plant switch to cardenolide-containing plants in the common ancestor of C. auratus and C. cobaltinus coincides with the exchange of the asparagine for a histidine in the ouabain binding site.
Key Words: Chrysochus ? Na+/K+-ATPase -subunit ? cardenolide ? ouabain binding site
Introduction
Herbivorous insects face the challenge of tolerating or detoxifying the numerous noxious compounds that plants have evolved to ward off their insect enemies (c.f. Rosenthal and Berenbaum 1991). The adaptations that herbivores may evolve in their turn to overcome plant defenses and occasionally even benefit from them can be very intricate. This first became apparent in the case of the monarch butterfly (Lepidoptera, Nymphalidae, Danaus plexippus). Here, it was first proved that insects may not only adapt to toxic plant compounds and use a plant as food despite their presence but also accumulate these compounds in the body to deter their own predators (Brower, Brower, and Corvino 1967; von Euw et al. 1967). Such a sequestration of plant compounds has been described in numerous cases (Rowell-Rahier and Pasteels 1992; Nishida 1994/1995). The current challenge is to understand how herbivores evolve the necessary physiological adaptations and which changes at the molecular level are involved.
The specimens that we currently use to analyze the evolution of such adaptations at the molecular level are Chrysochus leaf beetles (Coleoptera, Chrysomelidae). Like the monarch butterfly, two species in this genus, C. auratus and C. cobaltinus, feed on plants with cardenolides (cardiac glycosides), in this case of the genera Asclepias (Apocynaceae, Asclepiadoideae) and Apocynum (Apocynaceae s.str.) (Dobler and Farrell 1999). All other members of the genus feed on plants belonging to the Apocynaceae/Asclepiadoideae (Vincetoxicum, Cynanchum and related genera), which are devoid of cardenolides (Hegnauer 1964). Chrysochus species have evolved spectacular adaptations to the presence of the toxins in their food plant. In C. auratus and C. cobaltinus the plant compounds are efficiently shuttled through the body into cuticular glands on pronotum and elytra. These glands then release upon disturbance defensive secretions containing cardenolides. In C. asclepiadeus, a species that feeds on plants devoid of cardenolides on the other hand, the secretions only contain amino acids and other simple autogenously produced substances (Dobler, Daloze, and Pasteels 1998).
Avoiding poisoning by the highly toxic cardenolides is the most obvious adaptation C. auratus and C. cobaltinus had to evolve to adopt sequestration of cardenolides. The most important active principle of cardenolides consists in their capacity to bind to and thereby block the ubiquitous Na+/K+-ATPase present in almost all animals and most tissues. This transmembrane carrier is responsible for the maintenance of cellular potentials and plays a decisive role in the formation of nervous action potentials. This also holds true for insects in which Na+/K+-ATPase is present in the nervous system and can usually be blocked by the cardenolide ouabain (Emery et al. 1998). Adaptation of insects feeding on cardenolide-containing plants should therefore involve some way of reducing the extent to which their Na+/K+-ATPase is affected by the toxins. Studies in the monarch butterfly and the milkweed bug Oncopeltus fasciatus showed, for example, that in these insects living on cardenolide plants, the sensitivity to ouabain is altered so that their Na+/K+-ATPase can no longer be inhibited by the presence of ouabain (Vaughan and Jungreis 1977; Moore and Scudder 1986). We here trace the evolution of cardenolide insensitivity in this group of beetles by analyzing the DNA sequence of the putative ouabain-binding site of the -subunit of the Na+/K+-ATPase gene of Chrysochus and its close relatives feeding on plants with or without cardenolides. To polarize character evolution more firmly, a representative of a closely related genus, Platycorynus sauteri, feeding on Tylophora species (Apocynaceae, Asclepiadoideae) devoid of cardenolides has been incorporated in these analyses. The interpretation is based on an mtDNA phylogeny of Chrysochus and its close relatives.
Materials and Methods
Genomic DNA of C. asclepiadeus (Cevio, Ticino, Switzerland), C. auratus (Boulder, Colorado, USA), C. chinensis (Flaming Cliffs, Mongolia, 1332m, leg. J. Carpenter), C. cobaltinus (Canby, California, USA), and Platycorynus sauteri (Coleoptera, Chrysomelidae; collected at Fengyuan, Taichung, Taiwan, leg. C.S. Lin) was extracted from thorax or legs of individual adult beetles using the Qiamp tissue kit (Quiagen, Hilden, Germany). In addition, adults of Creatonotus transiens (Lepidoptera, Arctiidae [analyzed previously by Holzinger and Wink 1996]) and a sample of 10 to 20 adults of Drosophila melanogaster were extracted according to the same procedure and used as positive control in the PCR. Primers for the amplification of the ouabain-binding site were constructed based on the coding sequences of the -subunit of the Na+/K+-ATPase gene of D. melanogaster (GenBank accession number AF044974) and the flea Ctenocephalides felis (GenBank accession number S66043): S458; 5'-GA(AG)TGGGT(AGCT)AA(AG)TT(CT)TG(CT)AA(AG)AA(CT)-3' and A906; 5'-CC(CT)TCIAC(AGCT)GC(AG)TTI(AG)T(AGCT)GA(AG)AA-3'. Numbers in primer names refer to the 3' end of the primer-binding site on the D. melanogaster sequence. PCR conditions consisted of 2 min denaturing at 95°C, 40 cycles of 95°C for 45 s, primer annealing at 47°C for 30 s, and primer extension at 72°C for 2 min plus a final extension step at 72°C for 2 min and refrigeration at 4°C until removal of the samples. A 550-bp fragment was amplified from all species. The PCR products were separated by electrophoresis in 1% agarose gels and individual bands purified with the NucleoSpin Extract Kit (Macherey-Nagel, Düren, Germany). Aliquots were ligated into the pCR2.1 vector of the TOPO TA Cloning Kit (Invitrogen, Paisley, UK). Escherichia coli were transformed with the respective plasmids by chemical transformation. Insert-containing clones were checked for insert length by colony PCR. Plasmid DNA was purified with the GKXTM Micro Plasmid Prep Kit (Amersham Pharmacia Biotech, Freiburg, Germany) and sequenced on a Licor 4000L sequencer (MWG Biotech, Ebersberg, Germany) using M13 and M13rev primers. The sequences were edited and aligned with Sequencher version 3.0 (Gene Corp., Ann Arbor, Michigan, USA). In addition, 1,241 bp of the mitochondrial cytochrome oxidase I and II and the tRNA leu were sequenced for Platycorynus sauteri following the procedures previously described to incorporate this representative of the sister genus into an existing mt DNA phylogeny of the genus Chrysochus (Dobler and Farrell 1999).
Results and Discussion
In all species sequenced here (GenBank accession numbers AJ617742–AJ617746), the -subunit of the Na+/K+-ATPase gene was found to contain an intron 54 to 55 bp in length after postion 603 or 606 of the D. melanogaster (accession number AF044974) coding sequence. The extracellular domain of 12 amino acids corresponding to the ouabain-binding site (residue 111 to 122) shows remarkable differences in the taxa sequenced here (fig. 1). Among the five leaf beetle species investigated, C. auratus and C. cobaltinus, which both live on cardenolide plants and use the compounds as antipredator defense, have an identical amino acid sequence. This sequence differs in four positions from the one exhibited by all three species that do not feed on cardenolide-containing plants. We then compared these sequences with the previously published sequences of D. melanogaster, Creatonotus transiens (accession number X70120), and the tobacco horn worm Manduca sexta (Lepidoptera, Sphingidae [accession number X70119]), all of which do not encounter cardenolides naturally, and by contrast to the sequence of the cardenolide tolerating and sequestering monarch butterfly Danaus plexippus (accession number X70118). Fifty percent of the amino acids are conserved among all species, whereas the rest of the sequences are more similar within each taxonomic group than among them. The most interesting difference among the sequences involves the amino acid at position 122. Whereas all species that do not encounter cardenolides have an asparagine in this postion, both Chrysochus species that feed on cardenolide plants and the monarch butterfly have a histidine instead. This single amino acid substitution has already been shown to confer cardenolide insensitivity in the monarch butterfly. Expression studies of the D. melanogaster Na+/K+-ATPase gene where the same amino acid substitution was introduced by site-directed mutagenesis demonstrated that the single mutation is sufficient to reduce drastically the binding capacity for ouabain (Holzinger and Wink 1996). Two other lepidopterans that feed on cardenolide plants, however, do not feature the same substitution but must have achieved tolerance to cardenolides by some other modification (Holzinger and Wink 1996). Alternative ways to achieve ouabain resistance have also been described in a line of canine kidney cells where an amino acid substitution in a transmembrane region of the enzyme alters the binding capacity for ouabain (Canessa et al. 1992). Although we do not know the effect of the other three amino acid substitutions that have occurred in the two Chrysochus species that feed on cardenolide plants, the mutagenesis and gene expression studies of the D. melanogaster Na+/K+-ATPase demonstrate unambiguously that the exchange of asparagine for histidine exhibited by the beetles is sufficient to reduce the sensitivity of the enzyme to ouabain dramatically.
FIG. 1. Nucleotide (a) and amino acid sequence (b) of the ouabain-binding site of the -subunit of Na+, K+-ATPase of Chrysochus spp. and Platycorynus sauteri (this study, in bold) as compared with Drosophila melanogaster, Creatonotus transiens, Manduca sexta, and Danaus plexippus. Sequences of species on cardenolide plants are in gray; numbers refer to amino acid position in the mature protein
To firmly interpret the direction of the changes observed, an mt DNA phylogeny of the genus Chrysochus (Dobler and Farrell 1999) was supplemented by the sequence of Platycorynus sauteri (fig. 2). In the single most-parsimonious tree, P. sauteri is placed as sister group to the genus Chrysochus while C. asclepiadeus and C. chinensis, on one hand, and C. auratus and C. cobaltinus, on the other hand, appear as sister taxa. This phylogeny corroborates the hypothesis that an asparagine at position 122 of the -subunit of the Na+/K+-ATPase gene as observed in Drosophila and other insects is the plesiomorphic condition in this group of leaf beetles. The later host-plant switch to cardenolide-containing plants in the common ancestor of C. auratus and C. cobaltinus nicely coincides with the exchange of the asparagine for a histidine. Although this substitution is obviously not the only way to achieve an improved resistance to ouabain (Holzinger and Wink 1996; Canessa et al. 1992), Chrysochus leaf beetles apparently evolved a very similar trick of preventing toxicity of the cardenolides in their food plants, as did the monarch butterfly.
FIG. 2. Single most-parsimonious tree (length = 635, CI = 0.824) of Chrysochus and its relatives based on mtDNA sequence data. The tree is rooted on Eumolpus species, a more distantly related member of the same tribe. Numbers in bold give bootstrap values. The gray bar indicates the host plant switch to cardenolide-containing plants in the ancestor of C. auratus and C. cobaltinus and coincides with the substitution of histidine for arginine in the ouabain-binding site of the Na+, K+-ATPase
Acknowledgements
We would like to thank Cheng-Shing Lin for sending the specimen of Platycorynus sauteri and Johannes von Lintig, Susanne Hessel, Cornelia Kiefer, and Johanna Lampert for helpful advice on the experiments. This work was partly supported by a Swiss National Science Foundation postdoctoral fellowship.
Literature Cited
Brower, L. P., J. V. Z. Brower, and J. M. Corvino. 1967. Plant poisons in a terrestrial food chain. Proc. Nat. Acad. Sci. USA 57:893-898.
Canessa, C. M., J. D. Horisberger, D. Louvard, and B. C. Rossier. 1992. Mutation of a cysteine in the first transmembrane segment of Na,K-ATPase -subunit confers ouabain resistance. EMBO 11:1687-1992.
Dobler S., D. Daloze, and J. M. Pasteels. 1998. Sequestration of plant compounds in a leaf beetle's secretion: cardenolides in Chrysochus. Chemoecology 8:111-118.
Dobler S., and B. D. Farrell. 1999. Host use evolution in Chrysochus milkweed beetles: evidence from behaviour, population genetics and phylogeny. Mol. Ecol. 8:1297-1307.
Emery A. M., P. F. Billingsley, P. D. Ready, and M. B. A Djamgoz. 1998. Insect Na+/K+ ATPase. J. Insect Physiol. 44:197-209.
Hegnauer R. 1964. Chemotaxonomie der Pflanzen. Dicotyledoneae: Acanthaceae–Cyrillaceae. Vol. 3. Birkh?user, Basel.
Holzinger F., and M. Wink. 1996. Mediation of cardiac glycoside insensitivity in the monarch butterfly (Danaus plexippus): role of an amino acid substitution in the ouabain binding site of Na+,K+-ATPase. J. Chem. Ecol. 22:1921-1937.
Moore L. V., and G. G. E. Scudder. 1986. Ouabain-resistant Na,K-ATPases and cardenolide tolerance in the large milkweed bug, Oncopeltus fasciatus. J. Insect. Physiol. 32:27-33.
Nishida R. 1994/1995. Sequestration of plant secondary compounds by butterflies and moths. Chemoecology 5/6:127-138.
Rosenthal G. A., and M. R. Berenbaum. 1991. Herbivores: their interactions with secondary plant metabolites. Academic Press, San Diego.
Rowell-Rahier M., and J. M. Pasteels. 1992. Third trophic level influences of plant allelochemicals. Pp. 243–277 in G. A. Rosenthal and M. R. Berenbaum, eds. Herbivores: their interactions with secondary plant metabolites. Academic Press, San Diego.
Vaughan G. L., and A. M. Jungreis. 1977. Insensitivity of lepidopteran tissues to ouabain: physiological mechanisms for protection from cardiac glycosides. J. Insect. Physiol. 23:585-589.
von Euw J., L. Fishelson, J. A. Parsons, T. Reichstein, and M. Rothschild. 1967. Cardenolides (heart poisons) in a grasshoper feeding on milkweeds. Nature 214:35-39.(Estelle Labeyrie and Susa)
E-mail: susanne.dobler@zoologie.uni-hamburg.de.
Abstract
Herbivores that feed on toxic plants must overcome plant defenses and occasionally may even benefit from them. The current challenge is to understand how herbivores evolve the necessary physiological adaptations and which changes at the molecular level are involved. In this context we studied the leaf beetles genus Chrysochus (Coleoptera, Chrysomelidae). Two species of this genus, C. auratus and C. cobaltinus, feed on plants that contain toxic cardenolides. These beetles not only avoid poisoning by the toxin but also use it for their own defense against predators. All other Chrysochus species feed on plants that are devoid of cardenolides. The most important active principle of cardenolides is their capacity to bind to and thereby block the ubiquitous Na+/K+-ATPase responsible for maintaining cellular potentials. By analyzing the DNA sequence of the putative ouabain-binding site of the -subunit of the Na+/K+-ATPase gene of Chrysochus and its close relatives feeding on plants with or without cardenolides, we here trace the evolution of cardenolide insensitivity in this group of beetles. The most interesting difference among the sequences involves the amino acid at position 122. Whereas all species that do not encounter cardenolides have an asparagine in this position, both Chrysochus species that feed on cardenolide plants have a histidine instead. This single amino acid substitution has already been shown to confer cardenolide insensitivity in the monarch butterfly. A mtDNA-based phylogeny corroborates the hypothesis that the asparagine at position 122 of the -subunit of the Na+/K+-ATPase gene as observed in Drosophila and other insects is the plesiomorphic condition in this group of leaf beetles. The later host-plant switch to cardenolide-containing plants in the common ancestor of C. auratus and C. cobaltinus coincides with the exchange of the asparagine for a histidine in the ouabain binding site.
Key Words: Chrysochus ? Na+/K+-ATPase -subunit ? cardenolide ? ouabain binding site
Introduction
Herbivorous insects face the challenge of tolerating or detoxifying the numerous noxious compounds that plants have evolved to ward off their insect enemies (c.f. Rosenthal and Berenbaum 1991). The adaptations that herbivores may evolve in their turn to overcome plant defenses and occasionally even benefit from them can be very intricate. This first became apparent in the case of the monarch butterfly (Lepidoptera, Nymphalidae, Danaus plexippus). Here, it was first proved that insects may not only adapt to toxic plant compounds and use a plant as food despite their presence but also accumulate these compounds in the body to deter their own predators (Brower, Brower, and Corvino 1967; von Euw et al. 1967). Such a sequestration of plant compounds has been described in numerous cases (Rowell-Rahier and Pasteels 1992; Nishida 1994/1995). The current challenge is to understand how herbivores evolve the necessary physiological adaptations and which changes at the molecular level are involved.
The specimens that we currently use to analyze the evolution of such adaptations at the molecular level are Chrysochus leaf beetles (Coleoptera, Chrysomelidae). Like the monarch butterfly, two species in this genus, C. auratus and C. cobaltinus, feed on plants with cardenolides (cardiac glycosides), in this case of the genera Asclepias (Apocynaceae, Asclepiadoideae) and Apocynum (Apocynaceae s.str.) (Dobler and Farrell 1999). All other members of the genus feed on plants belonging to the Apocynaceae/Asclepiadoideae (Vincetoxicum, Cynanchum and related genera), which are devoid of cardenolides (Hegnauer 1964). Chrysochus species have evolved spectacular adaptations to the presence of the toxins in their food plant. In C. auratus and C. cobaltinus the plant compounds are efficiently shuttled through the body into cuticular glands on pronotum and elytra. These glands then release upon disturbance defensive secretions containing cardenolides. In C. asclepiadeus, a species that feeds on plants devoid of cardenolides on the other hand, the secretions only contain amino acids and other simple autogenously produced substances (Dobler, Daloze, and Pasteels 1998).
Avoiding poisoning by the highly toxic cardenolides is the most obvious adaptation C. auratus and C. cobaltinus had to evolve to adopt sequestration of cardenolides. The most important active principle of cardenolides consists in their capacity to bind to and thereby block the ubiquitous Na+/K+-ATPase present in almost all animals and most tissues. This transmembrane carrier is responsible for the maintenance of cellular potentials and plays a decisive role in the formation of nervous action potentials. This also holds true for insects in which Na+/K+-ATPase is present in the nervous system and can usually be blocked by the cardenolide ouabain (Emery et al. 1998). Adaptation of insects feeding on cardenolide-containing plants should therefore involve some way of reducing the extent to which their Na+/K+-ATPase is affected by the toxins. Studies in the monarch butterfly and the milkweed bug Oncopeltus fasciatus showed, for example, that in these insects living on cardenolide plants, the sensitivity to ouabain is altered so that their Na+/K+-ATPase can no longer be inhibited by the presence of ouabain (Vaughan and Jungreis 1977; Moore and Scudder 1986). We here trace the evolution of cardenolide insensitivity in this group of beetles by analyzing the DNA sequence of the putative ouabain-binding site of the -subunit of the Na+/K+-ATPase gene of Chrysochus and its close relatives feeding on plants with or without cardenolides. To polarize character evolution more firmly, a representative of a closely related genus, Platycorynus sauteri, feeding on Tylophora species (Apocynaceae, Asclepiadoideae) devoid of cardenolides has been incorporated in these analyses. The interpretation is based on an mtDNA phylogeny of Chrysochus and its close relatives.
Materials and Methods
Genomic DNA of C. asclepiadeus (Cevio, Ticino, Switzerland), C. auratus (Boulder, Colorado, USA), C. chinensis (Flaming Cliffs, Mongolia, 1332m, leg. J. Carpenter), C. cobaltinus (Canby, California, USA), and Platycorynus sauteri (Coleoptera, Chrysomelidae; collected at Fengyuan, Taichung, Taiwan, leg. C.S. Lin) was extracted from thorax or legs of individual adult beetles using the Qiamp tissue kit (Quiagen, Hilden, Germany). In addition, adults of Creatonotus transiens (Lepidoptera, Arctiidae [analyzed previously by Holzinger and Wink 1996]) and a sample of 10 to 20 adults of Drosophila melanogaster were extracted according to the same procedure and used as positive control in the PCR. Primers for the amplification of the ouabain-binding site were constructed based on the coding sequences of the -subunit of the Na+/K+-ATPase gene of D. melanogaster (GenBank accession number AF044974) and the flea Ctenocephalides felis (GenBank accession number S66043): S458; 5'-GA(AG)TGGGT(AGCT)AA(AG)TT(CT)TG(CT)AA(AG)AA(CT)-3' and A906; 5'-CC(CT)TCIAC(AGCT)GC(AG)TTI(AG)T(AGCT)GA(AG)AA-3'. Numbers in primer names refer to the 3' end of the primer-binding site on the D. melanogaster sequence. PCR conditions consisted of 2 min denaturing at 95°C, 40 cycles of 95°C for 45 s, primer annealing at 47°C for 30 s, and primer extension at 72°C for 2 min plus a final extension step at 72°C for 2 min and refrigeration at 4°C until removal of the samples. A 550-bp fragment was amplified from all species. The PCR products were separated by electrophoresis in 1% agarose gels and individual bands purified with the NucleoSpin Extract Kit (Macherey-Nagel, Düren, Germany). Aliquots were ligated into the pCR2.1 vector of the TOPO TA Cloning Kit (Invitrogen, Paisley, UK). Escherichia coli were transformed with the respective plasmids by chemical transformation. Insert-containing clones were checked for insert length by colony PCR. Plasmid DNA was purified with the GKXTM Micro Plasmid Prep Kit (Amersham Pharmacia Biotech, Freiburg, Germany) and sequenced on a Licor 4000L sequencer (MWG Biotech, Ebersberg, Germany) using M13 and M13rev primers. The sequences were edited and aligned with Sequencher version 3.0 (Gene Corp., Ann Arbor, Michigan, USA). In addition, 1,241 bp of the mitochondrial cytochrome oxidase I and II and the tRNA leu were sequenced for Platycorynus sauteri following the procedures previously described to incorporate this representative of the sister genus into an existing mt DNA phylogeny of the genus Chrysochus (Dobler and Farrell 1999).
Results and Discussion
In all species sequenced here (GenBank accession numbers AJ617742–AJ617746), the -subunit of the Na+/K+-ATPase gene was found to contain an intron 54 to 55 bp in length after postion 603 or 606 of the D. melanogaster (accession number AF044974) coding sequence. The extracellular domain of 12 amino acids corresponding to the ouabain-binding site (residue 111 to 122) shows remarkable differences in the taxa sequenced here (fig. 1). Among the five leaf beetle species investigated, C. auratus and C. cobaltinus, which both live on cardenolide plants and use the compounds as antipredator defense, have an identical amino acid sequence. This sequence differs in four positions from the one exhibited by all three species that do not feed on cardenolide-containing plants. We then compared these sequences with the previously published sequences of D. melanogaster, Creatonotus transiens (accession number X70120), and the tobacco horn worm Manduca sexta (Lepidoptera, Sphingidae [accession number X70119]), all of which do not encounter cardenolides naturally, and by contrast to the sequence of the cardenolide tolerating and sequestering monarch butterfly Danaus plexippus (accession number X70118). Fifty percent of the amino acids are conserved among all species, whereas the rest of the sequences are more similar within each taxonomic group than among them. The most interesting difference among the sequences involves the amino acid at position 122. Whereas all species that do not encounter cardenolides have an asparagine in this postion, both Chrysochus species that feed on cardenolide plants and the monarch butterfly have a histidine instead. This single amino acid substitution has already been shown to confer cardenolide insensitivity in the monarch butterfly. Expression studies of the D. melanogaster Na+/K+-ATPase gene where the same amino acid substitution was introduced by site-directed mutagenesis demonstrated that the single mutation is sufficient to reduce drastically the binding capacity for ouabain (Holzinger and Wink 1996). Two other lepidopterans that feed on cardenolide plants, however, do not feature the same substitution but must have achieved tolerance to cardenolides by some other modification (Holzinger and Wink 1996). Alternative ways to achieve ouabain resistance have also been described in a line of canine kidney cells where an amino acid substitution in a transmembrane region of the enzyme alters the binding capacity for ouabain (Canessa et al. 1992). Although we do not know the effect of the other three amino acid substitutions that have occurred in the two Chrysochus species that feed on cardenolide plants, the mutagenesis and gene expression studies of the D. melanogaster Na+/K+-ATPase demonstrate unambiguously that the exchange of asparagine for histidine exhibited by the beetles is sufficient to reduce the sensitivity of the enzyme to ouabain dramatically.
FIG. 1. Nucleotide (a) and amino acid sequence (b) of the ouabain-binding site of the -subunit of Na+, K+-ATPase of Chrysochus spp. and Platycorynus sauteri (this study, in bold) as compared with Drosophila melanogaster, Creatonotus transiens, Manduca sexta, and Danaus plexippus. Sequences of species on cardenolide plants are in gray; numbers refer to amino acid position in the mature protein
To firmly interpret the direction of the changes observed, an mt DNA phylogeny of the genus Chrysochus (Dobler and Farrell 1999) was supplemented by the sequence of Platycorynus sauteri (fig. 2). In the single most-parsimonious tree, P. sauteri is placed as sister group to the genus Chrysochus while C. asclepiadeus and C. chinensis, on one hand, and C. auratus and C. cobaltinus, on the other hand, appear as sister taxa. This phylogeny corroborates the hypothesis that an asparagine at position 122 of the -subunit of the Na+/K+-ATPase gene as observed in Drosophila and other insects is the plesiomorphic condition in this group of leaf beetles. The later host-plant switch to cardenolide-containing plants in the common ancestor of C. auratus and C. cobaltinus nicely coincides with the exchange of the asparagine for a histidine. Although this substitution is obviously not the only way to achieve an improved resistance to ouabain (Holzinger and Wink 1996; Canessa et al. 1992), Chrysochus leaf beetles apparently evolved a very similar trick of preventing toxicity of the cardenolides in their food plants, as did the monarch butterfly.
FIG. 2. Single most-parsimonious tree (length = 635, CI = 0.824) of Chrysochus and its relatives based on mtDNA sequence data. The tree is rooted on Eumolpus species, a more distantly related member of the same tribe. Numbers in bold give bootstrap values. The gray bar indicates the host plant switch to cardenolide-containing plants in the ancestor of C. auratus and C. cobaltinus and coincides with the substitution of histidine for arginine in the ouabain-binding site of the Na+, K+-ATPase
Acknowledgements
We would like to thank Cheng-Shing Lin for sending the specimen of Platycorynus sauteri and Johannes von Lintig, Susanne Hessel, Cornelia Kiefer, and Johanna Lampert for helpful advice on the experiments. This work was partly supported by a Swiss National Science Foundation postdoctoral fellowship.
Literature Cited
Brower, L. P., J. V. Z. Brower, and J. M. Corvino. 1967. Plant poisons in a terrestrial food chain. Proc. Nat. Acad. Sci. USA 57:893-898.
Canessa, C. M., J. D. Horisberger, D. Louvard, and B. C. Rossier. 1992. Mutation of a cysteine in the first transmembrane segment of Na,K-ATPase -subunit confers ouabain resistance. EMBO 11:1687-1992.
Dobler S., D. Daloze, and J. M. Pasteels. 1998. Sequestration of plant compounds in a leaf beetle's secretion: cardenolides in Chrysochus. Chemoecology 8:111-118.
Dobler S., and B. D. Farrell. 1999. Host use evolution in Chrysochus milkweed beetles: evidence from behaviour, population genetics and phylogeny. Mol. Ecol. 8:1297-1307.
Emery A. M., P. F. Billingsley, P. D. Ready, and M. B. A Djamgoz. 1998. Insect Na+/K+ ATPase. J. Insect Physiol. 44:197-209.
Hegnauer R. 1964. Chemotaxonomie der Pflanzen. Dicotyledoneae: Acanthaceae–Cyrillaceae. Vol. 3. Birkh?user, Basel.
Holzinger F., and M. Wink. 1996. Mediation of cardiac glycoside insensitivity in the monarch butterfly (Danaus plexippus): role of an amino acid substitution in the ouabain binding site of Na+,K+-ATPase. J. Chem. Ecol. 22:1921-1937.
Moore L. V., and G. G. E. Scudder. 1986. Ouabain-resistant Na,K-ATPases and cardenolide tolerance in the large milkweed bug, Oncopeltus fasciatus. J. Insect. Physiol. 32:27-33.
Nishida R. 1994/1995. Sequestration of plant secondary compounds by butterflies and moths. Chemoecology 5/6:127-138.
Rosenthal G. A., and M. R. Berenbaum. 1991. Herbivores: their interactions with secondary plant metabolites. Academic Press, San Diego.
Rowell-Rahier M., and J. M. Pasteels. 1992. Third trophic level influences of plant allelochemicals. Pp. 243–277 in G. A. Rosenthal and M. R. Berenbaum, eds. Herbivores: their interactions with secondary plant metabolites. Academic Press, San Diego.
Vaughan G. L., and A. M. Jungreis. 1977. Insensitivity of lepidopteran tissues to ouabain: physiological mechanisms for protection from cardiac glycosides. J. Insect. Physiol. 23:585-589.
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