Cytochrome P450 2B diversity and dietary novelty in the herbivorous, desert woodrat (Neotoma lepida)

Abstract

Detoxification enzymes play a key role in plant-herbivore interactions, contributing to the on-going evolution of ecosystem functional diversity. Mammalian detoxification systems have been well studied by the medical and pharmacological industries to understand human drug metabolism; however, little is known of the mechanisms employed by wild herbivores to metabolize toxic plant secondary compounds. Using a wild rodent herbivore, the desert woodrat (Neotoma lepida), we investigated genomic structural variation, sequence variability, and expression patterns in a multigene subfamily involved in xenobiotic metabolism, cytochrome P450 2B (CYP2B). We hypothesized that differences in CYP2B expression and sequence diversity could explain differential abilities of woodrat populations to consume native plant toxins. Woodrats from two distinct populations were fed diets supplemented with either juniper (Juniperus osteosperma) or creosote bush (Larrea tridentata), plants consumed by woodrats in their respective desert habitats. We used Southern blot and quantitative PCR to determine that the genomic copy number of CYP2B in both populations was equivalent, and similar in number to known rodent copy number. We compared CYP2B expression patterns and sequence diversity using cloned hepatic CYP2B cDNA. The resulting sequences were very diverse, and clustered into four major clades by amino acid similarity. Sequences from the experimental treatments were distributed non-randomly across a CYP2B tree, indicating unique expression patterns from woodrats on different diets and from different habitats. Furthermore, within each major CYP2B clade, sequences shared a unique combination of amino acid residues at 13 sites throughout the protein known to be important for CYP2B enzyme function, implying differences in the function of each major CYP2B variant. This work is the most comprehensive investigation of the genetic diversity of a detoxification enzyme subfamily in a wild mammalian herbivore, and contributes an initial genetic framework to our understanding of how a wild herbivore responds to critical changes in its diet.

Figure 1. Number of woodrat CYP2B gene copies from individuals of both populations using qPCR.

Copy number was determined by comparing CYP2B amplification to the amplification of the single copy gene, SOD-I.

Figure 2. Maximum likelihood tree of woodrat CYP2B amino acid sequences.

Branch length is scaled to the amount of differentiation (see scale bar). Bootstrap support of >50% is indicated on the nodes. Branch labels consist of an individual animal identification number followed by population and diet treatment from the feeding trial. The tree is rooted with human CYP2B6 (#AAF13602.1). Letters (A to L) designate individual sequences or clades that share the same combination of amino acids at the 13 sites known to be important for CYP2B function. Letters correspond to the amino acid characterization in Table 1. For the four major clades, tree topology and the unique combination of substrate recognition amino acids are correlated. The pie charts specify the distribution of experimental treatments comprising each unique combination of substrate recognition residues in the major clades (A, F, G, and H). Size is a relative indicator of the number of sequences represented in that pie chart. For comparison, the pie chart in the upper left corner shows the relative representation of experimental treatments in the whole cloned dataset.

Figure 3. Differential distribution of sequenced CYP2B clones across population by dietary treatment.

Colors represent groups of sequences with unique combinations of 13 substrate recognition amino acid residues important for CYP2B enzyme function. The smallest wedges in each pie are a unique combination of substrate recognition amino acids that occurs only one time in that treatment. Letters correspond to amino acid characterization in Table 3 and phylogenetic relationships described in Figure 3.