May R. Berenbaum
Professor and Department Head
PhD, 1980, Cornell University
Berenbaum is interested in the chemical interactions between herbivorous insects and their hostplants, and the implications of such interactions on the organization of natural communities and the evolution of species. Her particular research interests focus on the secondary chemistry of the Umbelliferae (=Apiaceae) and the insect associates of these herbaceous plants. Current research approaches insect/plant coevolution at several levels. At the physiological level, her lab is investigating the modes of action and mechanisms of action of plant secondary metabolite defensive compounds in insect. Much of this work centers on phototoxic plant compounds, particularly the furanocoumarins, allelochemicals characteristic of the Apiaceae and Rutaceae. Sunlight, particularly in the near ultraviolet range (320-380 nm), is an abundant source of energy available to plants; absorption of a photon by a plant chemical can change its reactivity by altering its electron configuration and energy state. Many plant families have converged upon phototoxicity for resistance to pathogens, herbivores, and competitors. Although work in her laboratory is centered on the furanocoumarins, they have also investigated toxicity and mode of action of several biosynthetically distant chemical compounds in these same plant families, including furanoquinoline alkaloids, furanochromones, beta-carboline alkaloids, and polyacetylenes, with respect to their toxicity to insects and their mode of action. Two basic forms of phototoxicity exist. In type I reactions, the ground-state sensitizer forms an excited state, which following intersystem crossing goes to the more stable triplet state; this triplet state then by hydrogen ion or electron transfer reacts directly with target biomolecules (e.g., furanocoumarins and DNA). In type II reactions, the triplet sensitizer reacts with ground state molecular oxygen to form excited oxygen, which can proceed to react with target substrates. Resistance mechanisms to the two forms of phototoxicity may differ. Mechanisms under study in her lab are behavioral (e.g., leaf-rolling), physical (e.g., sequestration of carotenoid quenching pigments or production of melanin as a neutral density filter), and biochemical (e.g., antioxidant or detoxication enzymes such as catalase, superoxide dismutase, and glutathione reductase, as well as low molecular weight antioxidant compounds such as uric acid).
Biochemical resistance to furanocoumarins appears to involve cytochrome P450 mono-oxygenases in a variety of herbivorous insects; these detoxification enzymes provide another focus for our work. Approximately 75% of the species in the genus Papilio (Lepidoptera: Papilionidae) are associated with furanocoumarin-containing plants. In collaboration with Dr. Mary Schuler, of the Departments of Cell Biology and Plant Biology, they have been investigating the molecular basis of P-450-mediated metabolism of furanocoumarins, in order to understand its role in the evolution of specialization in this group. The black swallowtail butterfly Papilio polyxenes feeds almost exclusively on furanocoumarin-containing plants. Metabolism of furanocoumarins by these insects is consistent with cytochrome P450 mono-oxygenation; it is localized in midgut microsomes, it is inhibited by piperonyl butoxide and other cytochrome P450 inhibitors, and it is oxygen- and NADPH-dependent. Moreover, enzyme activity toward furanocoumarin substrates is induced by dietary furanocoumarins. This inducibility has provided a method for identifying and characterizing the gene or genes for furanocoumarin tolerance. Two cDNAs, CYP6B1v1 and CYP6B1v2, have been isolated from cDNA libraries made from midguts of P. polyxenes larvae induced by furanocoumarins. Northern analysis confirms that these P450 transcripts are inducible by hostplant linear furanocoumarins and to a lesser extent by angular furanocoumarins. That these cDNAs encode furanocoumarin-metabolizing P450 isozymes was demonstrated by baculovirus-mediated expression of the cDNAs. Another cDNA, CYP6B4v1, was isolated from cDNA libraries constructed from midgut RNA of induced Papilio glaucus, the tiger swallowtail. This more polyphagous swallowtail, with only a single host that contains furanocoumarins, possesses the ability to metabolize furanocoumarins; that CYP6B4 is responsible at least in part for this metabolism was confirmed by baculovirus-mediated expression. CYP6B4 metabolism of furanocoumarins resembles that of CYP6B1 in that linear furanocoumarins are metabolized with greater activity than are angular furanocoumarins. Isolation of the CYP6B1 and CYP6B4 genes (along with CYP6B3 from P. polyxenes and CYP6B5 from P. glaucus) demonstrated the presence of a highly conserved substrate recognition site (SRS1) as well as putative xanthotoxin-responsive elements in their promoter sequences. The presence of these conserved features is suggestive of a tight coevolutionary association with hostplant furanocoumarins in this group. Assessing the number and diversity of P450s in this family, in which the phylogeny is well established, can provide great insight into the genetic mechanisms underlying acquisition of and specialization on new host-plants. Recent isolation and characterization of furanocoumarin-inducible CYP6B8 cDNA in Helicoverpa zea (Lepidoptera: Noctuidae) provides an opportunity to compare allelochemical metabolism in swallowtails with metabolism by an extremely polyphagous species.
At the population level, they are using techniques of quantitative genetics to evaluate the likelihood of reciprocal genetic changes between interacting and ostensibly coevolving species. The parsnip webworm Depressaria pastinacella (Lepidoptera: Oecophoridae) and its primary host Pastinaca sativa provide an ideal system for examining reciprocal selective impact of host and insect; the plant, introduced into North America, has relatively few other insect associates and the insect feeds only on wild parsnip in many parts of its range. They have demonstrated that resistance in the wild parsnip is based on furanocoumarin characters that have heritabilities significantly different from zero; moreover, calculations of selection pressure exerted by the webworm indicate it is capable of inflicting sufficient damage to change the distribution of genetically based chemical traits within a plant population. By the same token, cytochrome P450-mediated metabolism of furanocoumarins by webworms is heritable and available for selection by hostplant chemistry. Moreover, significant metabolic costs have been demonstrated for both furanocoumarin production in the plant and furanocoumarin detoxification in the insect. The most dramatic evidence of reciprocal adaptive response in this system is the extraordinary matching of chemical phenotypes across a range of populations; in 3 of 4 populations examined, the profile of furanocoumarins produced by the plants corresponds precisely with the profile of furanocoumarin metabolic capabilities in the associated webworm populations. They are also investigating genetic variation in primary metabolites of parsnips and the impact of webworm herbivory on these chemical traits. Dissatisfaction with theories of chemical coevolution is due largely to the fact that reciprocal genetic changes are rarely demonstrated; their studies may provide the first quantitative data to allow a complete evaluation of the coevolutionary scenario described by Ehrlich and Raven (1964) and modified by Janzen (1980).
At the evolutionary level, the idea that the reciprocal genetic changes that take place between interacting populations can lead to speciation is an appealing but largely unsubstantiated one; the same gap that exists between micro- and macro- evolutionary changes also exists between micro- and macro-coevolutionary phenomena. The likelihood of coordinated reciprocal genetic changes between organisms differing in generation time, population structure and selective impact is difficult to evaluate a priori; however, assessments of host and insect phylogenies can provide an a posteriori test. The "reciprocal radiation" model accounts for a non-random fit to association by descent, with certain deviations from fit attributable to phytochemical divergence within taxa. Host transfers due to geographic proximity, ecological similarity or chemical convergence in contrast should generate noncongruent phylogenies. The lepidopteran family Oecophoridae (sensu lato) is an appropriate group in which to examine patterns of descent in host and herbivore. Representatives of the family feed on plants in virtually every order of angiosperm plants; relationships at the species, genus, and subfamily level, as determined by insect morphology, behavior and ecology, are being examined in the context of host phylogeny, ecology, geography and chemistry.
Representative and Recent Publications
- Berenbaum, M. R. and A. R. Zangerl 1992. Genetics of physiological and behavioral resistance to host furanocoumarins in the parsnip webworm. Evolution 46, 1373-1384.
- Cohen, M. B., M. A. Schuler and M. R. Berenbaum 1992. A host-inducible cytochrome P450 from a host-specific caterpillar: molecular cloning and evolution. Proc. Natl. Acad. Sci. USA 89, 10920-10924.
- Berenbaum, M. R. and A. R. Zangerl 1994. Costs of inducible defense in insects: effects of protein limitation on growth, silk production, and detoxification in parsnip webworms. Ecology 75, 2311-2317.
- Berenbaum, M. R. 1995. Chemical defense: theory and practice. Proc. Natl. Acad. Sci. USA 92, 2-8.
- Hung, C.-H., R. Holzmacher, E. Connolly, M. Berenbaum and M. Schuler 1996. Conserved promoter elements in the CYP6B genes suggest common ancestry for cytochrome P450 monooxygenases mediating furanocoumarin detoxification. Proc. Natl. Acad. Sci. USA 93: 12200-12205.
- Berenbaum, M. R., C. Favret and M. A. Schuler 1996. On defining "key innovations" in an adaptive radiation: cytochrome P450s and Papilionidae. Am. Nat. 148, S139-A155.
- Carroll, M., A. Hanlon, T. Hanlon, A. R. Zangerl and M. R. Berenbaum 1997. Behavioral effects of carotenoid sequestration by the parsnip webworm, Depressaria pastinacella. J. Chem. Ecol. 23, 2707-2719.
- Berenbaum, M. R. and A. R. Zangerl, 1998. Chemical phenotype matching between a plant and its insect herbivore. Proc. Natl. Acad. Sci. USA 95, 13743-13748.
May is also the author of several books, which are available here.