Genetic Criteria for Establishing Evolutionarily Significant Units in Cryan's Buckmoth

Buckmoths ( Hemileuca spp.) are day-flying saturniid moths with diverse ecologies and host plants. Populations that feed on Menyanthes trifoliata , known commonly as Cryan's buckmoths, have been found in only a few bogs and fens near eastern Lake Ontario in New York and near Ottawa in Ontario, Canada. Because of their unique ecological traits, geographic isolation from other Hemileuca populations, and the small number of sites they occupy, there is concern that the Cryan's buckmoth populations are phylogenetically distinct and should be protected. The Cryan's buckmoths have not yet been taxonomically described and do not appear to have clear distinguishing morphological characters. Both molecular genetic traits (allozymes and mitochondrial DNA sequences) and an ecologically based character (host performance) were investigated to determine whether these populations possess fixed diagnostic characters signifying genetic differentiation from other eastern Hemileuca populations. Such differences would merit separate conservation management as an evolutionarily significant unit. Our studies showed that the Cryan's buckmoths clearly belong to the Hemileuca maia species group, but they could not be readily distinguished from other members of that group by means of molecular genetic techniques. There were no fixed differences in alleles or haplotypes distinguishing any of the populations or species, suggesting recent divergence. Nonetheless, in the host-plant performance experiment only the Cryan's buckmoth larvae were able to develop on M. trifoliata , a significant difference from other Hemileuca larvae tested. The Cryan's buckmoth appears to be unique in host performance and warrants protection and management us an evolutionarily significant unit. In cases such as this where groups appear to have recently diverged, investigations into ecologically significant traits may provide indicators of conservation significance as reliable as molecular genetic markers.     

Xenobiotics: Substrates and inhibitors of the plant cytochrome P450

The ability of a plant cytochrome P450 to bind and metabolise plant endogenous molecules and xenobiotics was investigated. The work was performed on the yeast-expressed CYP73A1, a cinnamate 4-hydroxylase isolated fromHelianthus tuberosus. CYP73 controls the general phenylpropanoid pathway and is likely to be one of the most abundant sources of P450 in the biosphere. The enzyme shows a high selectivity toward plant secondary metabolites. Nevertheless, it oxygenates several small and planar xenobiotics with low efficiency, including an herbicide (chlorotoluron). One xenobiotic molecule, 2naphthoic acid, is hydroxylated with an efficiency comparable to that of the physiological substrate. This reaction was used to devise a fluorimetric test for the rapid measurement of enzyme activity. A series of herbicidal molecules (hydroxybenzonitriles) are shown to bind the active site without being metabolised. These molecules behave as strong competitive inhibitors of CYP73 with a K_i in the same micromolar range as the K_m for the physiological substrate. It is proposed that their inhibition of the phenylpropanoid pathway reinforces their other phytotoxic effects at the level of the chloroplasts. All our results indicate a strong reciprocal interaction between plant P450s and xenobiotics.