Positive selection of digestive Cys proteases in herbivorous Coleoptera.
Positive selection is thought to contribute to the functional diversification of insect-inducible protease inhibitors in plants in response to selective pressures exerted by the digestive proteases of their herbivorous enemies. Here we assessed whether a reciprocal evolutionary process takes place on the insect side, and whether ingestion of a positively selected plant inhibitor may translate into a measurable rebalancing of midgut proteases in vivo. Midgut Cys proteases of herbivorous Coleoptera, including the major pest Colorado potato beetle (Leptinotarsa decemlineata), were first compared using a codon-based evolutionary model to look for the occurrence of hypervariable, positively selected amino acid sites among the tested sequences. Hypervariable sites were found, distributed within -or close to- amino acid regions interacting with Cys-type inhibitors of the plant cystatin protein family. A close examination of L. decemlineata sequences indicated a link between their assignment to protease functional families and amino acid identity at positively selected sites. A function-diversifying role for positive selection was further suggested empirically by in vitro protease assays and a shotgun proteomic analysis of L. decemlineata Cys proteases showing a differential rebalancing of protease functional family complements in larvae fed single variants of a model cystatin mutated at positively selected amino acid sites. These data confirm overall the occurrence of hypervariable, positively selected amino acid sites in herbivorous Coleoptera digestive Cys proteases. They also support the idea of an adaptive role for positive selection, useful to generate functionally diverse proteases in insect herbivores ingesting functionally diverse, rapidly evolving dietary cystatins.
Département de phytologie, CRIV-Biotechnologie, Université Laval, Québec, QC G1V 0A6, Canada; Department of Plant and Soil Science, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, South Africa.
Juan
J
Vorster
Département de phytologie, CRIV-Biotechnologie, Université Laval, Québec, QC G1V 0A6, Canada.
Asieh
A
Rasoolizadeh
Département de phytologie, CRIV-Biotechnologie, Université Laval, Québec, QC G1V 0A6, Canada.
Marie-Claire
MC
Goulet
Département de biologie, Université Laval, Québec, QC G1V 0A6, Canada.
Conrad
C
Cloutier
Département de phytologie, CRIV-Biotechnologie, Université Laval, Québec, QC G1V 0A6, Canada; The University of Queensland, Australian Institute for Bioengineering and Nanotechnology, Centre for Biomolecular Engineering, St. Lucia, Queensland 4072, Australia.
Frank
F
Sainsbury
Département de phytologie, CRIV-Biotechnologie, Université Laval, Québec, QC G1V 0A6, Canada. Electronic address: dominique.michaud@fsaa.ulaval.ca.
Dominique
D
Michaud
D000595:Amino Acid Sequence; D000818:Animals; D005075:Biological Evolution; D001517:Coleoptera / Q000201:enzymology* / Q000235:genetics; D015891:Cystatins / Q000378:metabolism; D004064:Digestive System / Q000201:enzymology; D060434:Herbivory; D007814:Larva / Q000201:enzymology / Q000235:genetics; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D012641:Selection, Genetic
D015891:Cystatins; D010940:Plant Proteins; D010447:Peptide Hydrolases
Coevolutionary arms race; Digestive cysteine proteases; Herbivorous Coleoptera; Leptinotarsa decemlineata Say; Plant cystatins; Plant–insect interactions; Positive selection