Jael R. Malenke

Competition promotes the evolution of host generalists in obligate parasites

Ecological theory traditionally predicts that interspecific competition selects for an increase in ecological specialization. Specialization, in turn, is often thought to be an evolutionary ‘dead end,’ with specialist lineages unlikely to evolve into generalist lineages. In host–parasite systems, this specialization can take the form of host specificity, with more specialized parasites using fewer hosts. We tested the hypothesis that specialists are evolutionarily more derived, and whether competition favours specialization, using the ectoparasitic feather lice of doves. Phylogenetic analyses revealed that complete host specificity is actually the ancestral condition, with generalists repeatedly evolving from specialist ancestors. These multiple origins of generalists are correlated with the presence of potentially competing species of the same genus. A competition experiment with captive doves and lice confirmed that congeneric species of lice do, in fact, have the potential to compete in ecological time. Taken together, these results suggest that interspecific competition can favour the evolution of host generalists, not specialists, over macroevolutionary time.

Expression of biotransformation genes in woodrat (Neotoma) herbivores on novel and ancestral diets: identification of candidate genes responsible for dietary shifts.

The ability of herbivores to switch diets is thought to be governed by biotransformation enzymes. To identify potential biotransformation enzymes, we conducted a large‐scale study on the expression of biotransformation enzymes in herbivorous woodrats (Neotoma lepida). We compared gene expression in a woodrat population from the Great Basin that feeds on the ancestral diet of juniper to one from the Mojave Desert that putatively switched from feeding on juniper to feeding on creosote. Juniper and creosote have notable differences in secondary chemistry, and thus, should require different biotransformation enzymes for detoxification. Individuals from each population were fed juniper and creosote diets separately. After the feeding trials, hepatic mRNA was extracted and hybridized to laboratory rat microarrays. Hybridization of woodrat samples to biotransformation probes on the array was 87%, resulting in a total of 224 biotransformation genes that met quality control standards. Overall, we found large differences in expression of biotransformation genes when woodrats were fed juniper vs. creosote. Mojave woodrats had greater expression of 10× as many biotransformation genes as did Great Basin woodrats on a creosote diet. We identified 24 candidate genes that may be critical in the biotransformation of creosote toxins. Superoxide dismutase, a free radical scavenger, was also expressed to a greater extent by the Mojave woodrats and may be important in controlling oxidative damage during biotransformation. The results are consistent with the hypothesis that biotransformation enzymes limit diet switching and that woodrats in the Mojave have evolved a unique strategy for the biotransformation of creosote toxins.

Host specialization differentiates cryptic species of feather-feeding lice.

Parasite species with differentiated host-specific populations provide a natural opportunity to explore factors involved in parasite diversification. Columbicola macrourae is a species of ectoparasitic feather louse currently recognized from 15 species of New World pigeons and doves. Mitochondrial sequences reveal five divergent haplotype clusters within C. macrourae, suggesting cryptic species. Each cluster is relatively host specific, with only one or a few hosts. We conducted a reciprocal transfer experiment with two of these lineages to test whether host use has an adaptive component. Our results demonstrate that the fitness of each lineage is considerably higher on its native host than on the novel host suggesting that one or more selective agents favor host specialization by the different lineages. In addition, we were able to morphologically separate individual lice from the two experimental lineages using discriminant function analysis. Furthermore, differences in the size of these louse lineages match differences in the size of their respective hosts, paralleling the strong correlation between parasite and host body size across the genus Columbicola. Together, these results suggest that selection in this cryptic species complex reflects selection across the whole genus, and that this selection, in part, contributes to the maintenance of host specialization.

Condition-Specific Competition Governs the Geographic Distribution and Diversity of Ectoparasites

Understanding how environmental parameters interact to govern species distributions is a shared goal of ecology and biogeography. Biotic and abiotic conditions can change distributions by affecting the nature of interspecific interactions. Although documented in free-living systems, this context dependency has been neglected in parasite interactions. We investigated the influence of condition-specific competition on the specificity of two species of feather lice (Phthiraptera: Ischnocera) that share a host, the mourning dove (Zenaida macroura). We show that relative humidity restricts the range of one species, Columbicola macrourae3 (i.e., the C. macrourae lineage found on mourning doves), to the more humid eastern United States. The second species, Columbicola baculoides, an arid-adapted species, is restricted to drier regions of the western United States by C. macrourae3, which outcompetes it in experiments. Thus, arid conditions in the West provide C. baculoides with a climatic refuge from the competitively superior C. macrourae3, effectively doubling parasite diversity on one host species. These results support the hypothesis that abiotic factors can determine species distributions on the stressful end of an environmental gradient while interspecific competition governs distributions at the benign end. The balance between these factors is subject to change as environmental conditions change, even if the host distribution remains unaffected.

Is the citrus-like plumage odorant of crested auklets (Aethia cristatella) a defense against lice?

Plumage odors may function as chemical defenses against ectoparasites in birds. We tested this hypothesis for crested auklets (Aethia cristatella), a species of colonial seabird that emits a very strong citrus-like odor from its plumage. This odorant contains known chemical repellents. We evaluated evidence for chemical defense in this species using two approaches. First, we exposed pigeon lice to the volatiles emitted by freshly plucked plumage and by whole specimens. Louse survivorship was compared between these treatments and two controls. Second, we compared louse abundance on crested auklets versus a closely related congener that nests in close association. Louse survivorship did not differ between crested auklet treatments and controls. Comparison of ectoparasite loads showed that crested auklets had significantly higher louse abundance than least auklets (A. pusilla), even after controlling for body size. Our results failed to support two expectations for chemical defense. Presence of the crested auklet plumage odorant in our experiments did not reduce louse life span. Presence of the aldehyde odorant in nature did not reduce louse abundance on crested auklets. Hence we conclude that the aldehyde odorant is not immediately lethal to lice at natural concentrations in plumage.

Cytochrome P450 2B Diversity and Dietary Novelty in the Herbivorous, Desert Woodrat (Neotoma lepida)

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.

Functional characterization of cytochromes P450 2B from the desert woodrat Neotoma lepida.

Mammalian detoxification processes have been the focus of intense research, but little is known about how wild herbivores process plant secondary compounds, many of which have medicinal value or are drugs. cDNA sequences that code for three enzymes of the cytochrome P450 (CYP) 2B subfamily, here termed 2B35, 2B36, and 2B37 have been recently identified from a wild rodent, the desert woodrat (Malenke et al., 2012). Two variant clones of each enzyme were engineered to increase protein solubility and to facilitate purification, as reported for CYP2B enzymes from multiple species. When expressed in Escherichia coli each of the woodrat proteins gave the characteristic maximum at 450nm in a reduced carbon monoxide difference spectrum but generally expressed at lower levels than rat CYP2B1. Two enzymes, 2B36 and 2B37, showed dealkylation activity with the model substrates 7-ethoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin, whereas 2B35 was inactive. Binding of the monoterpene (+)-α-pinene produced a Type I shift in the absorbance spectrum of each enzyme. Mutation of 2B37 at residues 114, 262, or 480, key residues governing ligand interactions with other CYP2B enzymes, did not significantly change expression levels or produce the expected functional changes. In summary, two catalytic and one ligand-binding assay are sufficient to distinguish among CYP2B35, 2B36, and 2B37. Differences in functional profiles between 2B36 and 2B37 are partially explained by changes in substrate recognition site residue 114, but not 480. The results advance our understanding of the mechanisms of detoxification in wild mammalian herbivores and highlight the complexity of this system.

Efficacy of the LouseBuster, a New Medical Device for Treating Head Lice (Anoplura: Pediculidae)

ABSTRACT Human head lice (Pediculus humanus capitis De Geer) occur worldwide and infest millions of children and adults every year. Head lice infestations, which are known as pediculosis capitis, are psychologically stressful, physically irritating, and are one of the leading causes of K-6 school absence. The prevalence of head lice in many countries is increasing rapidly because of resistance to chemicals used in many head lice treatments. We tested the efficacy of an alternative method for controlling head lice, the LouseBuster, a custom-built medical device designed to kill head lice and their eggs using controlled, heated air. A total of 56 infested subjects was treated with the LouseBuster, and the efficacy of the treatment was evaluated by comparing the viability of lice and eggs on randomly assigned pre- and posttreatment sides of each subjects scalp. We evaluate treatment efficacy in the hands of novice versus experienced operators. We also evaluate treatment efficacy on different hair types and at different ambient humidities. Overall mortality of lice and eggs was 94.8% after treatment by experienced operators. Novice operators also achieved good results after a short training session; their results did not differ significantly from those of experienced operators. No adverse events were associated with the LouseBuster treatment. The LouseBuster is efficacious for killing head lice and their eggs. The use of heated air is appealing because it is a fast, safe, nonchemical treatment. Head lice are also unlikely to evolve resistance to desiccation, which is the apparent mode of action.

An In Vivo Assay for Elucidating the Importance of Cytochromes P450 for the Ability of a Wild Mammalian Herbivore (Neotoma lepida) to Consume Toxic Plants

An in vivo assay using the cytochrome P450 (P450) suicide inhibitor 1-aminobenzotriazole (ABT) and 24-h food intake was developed to determine the relative importance of P450s in two populations of Neotoma lepida with respect to biotransformation of plant secondary compounds in the animals’ natural diets. The efficacy of ABT as a P450 inhibitor was first validated using hypnotic-state assays with and without pretreatment with ABT. Pretreatment with 100 mg/kg ABT by gavage increased hexobarbital sleep times 3–4-fold in both populations, showing effective inhibition of P450s in woodrats. Next, the Great Basin population was fed a terpene-rich juniper diet, and the Mojave population was fed a phenolic-rich creosote diet, with rabbit chow serving as the control diet in each group. Treatment with ABT inhibited food intake in the Great Basin population fed the juniper diet to a greater extent (35%) than the Great Basin population fed the control diet (19%) or the Mojave population fed the creosote diet (16%). The food intake of the Mojave population fed the control diet was not significantly inhibited by ABT. The findings suggest that the biotransformation of terpenes in juniper relies more heavily on P450s than that of phenolics in creosote. This assay provides an inexpensive and noninvasive method to explore the relative importance of P450s in the biotransformation strategies of wild mammalian herbivores.

Hepatic gene expression in herbivores on diets with natural and novel plant secondary compounds.

Herbivores are predicted to evolve appropriate mechanisms to process the plant secondary compounds (PSCs) in their diet, and these mechanisms are likely specific to particular suites of PSCs. Changes in diet composition over evolutionary time should select for appropriate alterations in metabolism of the more recent dietary components. We investigated differences in gene expression profiles in the liver with respect to prior ecological and evolutionary experience with PSCs in the desert woodrat, Neotoma lepida. This woodrat species has populations in the Mojave Desert that have switched from feeding on juniper to feeding on creosote at the end of the Holocene as well as populations in the Great Basin Desert that still feed on the ancestral diet of juniper and are naïve to creosote. Juniper and creosote have notable differences in secondary chemistry. Woodrats from the Mojave and Great Basin Deserts were subjected to a fully crossed feeding trial on diets of juniper and creosote after which their livers were analyzed for gene expression. Hybridization of hepatic mRNAs to laboratory rat microarrays resulted in a total of 20,031 genes that met quality control standards. We analyzed differences in large-scale patterns of liver gene expression with respect to GO term enrichment. Diet had a larger effect on gene expression than population membership. However, woodrats with no prior evolutionary experience to the diet upregulated a greater proportion of genes indicative of physiological stress compared with those on their natural diet. This pattern may be the result of a naïve animals attempting to mitigate physiological damage caused by novel PSCs.