Ambient temperature-mediated changes in hepatic gene expression of a mammalian herbivore (Neotoma lepida).

Herbivores regularly ingest natural toxins produced by plants as a defence against herbivory. Recent work suggests that compound toxicity is exacerbated at higher ambient temperatures. This phenomenon, known as temperature-dependent toxicity (TDT), is the likely result of decreased liver function at warmer temperatures; however, the underlying cause of TDT remains speculative. In the present study, we compared the effects of temperature and dietary plant toxins on differential gene expression in the liver of an herbivorous rodent (Neotoma lepida), using species-specific microarrays. Expression profiles revealed a greater number of differentially expressed genes at an ambient temperature below the thermal neutral zone for N. lepida (22°C) compared to one within (27°C). Genes and pathways upregulated at 22°C were related to growth and biosynthesis, whereas those upregulated at 27°C were associated with gluconeogenesis, apoptosis and protein misfolding, suggestive of a stressed state for the liver. Additionally, few genes associated with xenobiotic metabolism were induced when woodrats ingested plant toxins compared to nontoxic diets, regardless of temperature. Taken together, the results highlight the important role of ambient temperature on gene expression profiles in the desert woodrat. Temperatures just below the thermal neutral zone might be a favourable state for liver metabolism. Furthermore, the reduction in the number of genes expressed at a temperature within the thermal neutral zone indicates that liver function may be reduced at temperatures that are not typically considered as thermally stressful. Understanding how herbivorous mammals will respond to ambient temperature is imperative to accurately predict the impacts of climate change.

Evidence for functional convergence in genes upregulated by herbivores ingesting plant secondary compounds.

BACKGROUND: Nearly 40 years ago, Freeland and Janzen predicted that liver biotransformation enzymes dictated diet selection by herbivores. Despite decades of research on model species and humans, little is known about the biotransformation mechanisms used by mammalian herbivores to metabolize plant secondary compounds (PSCs). We investigated the independent evolution of PSC biotransformation mechanisms by capitalizing on a dramatic diet change event-the dietary inclusion of creosote bush (Larrea tridentata)-that occurred in the recent evolutionary history of two species of woodrats (Neotoma lepida and N. bryanti). RESULTS: By comparing gene expression profiles of two populations of woodrats with evolutionary experience to creosote and one population naïve to creosote, we identified genes either induced by a diet containing creosote PSCs or constitutively higher in populations with evolutionary experience of creosote. Although only one detoxification gene (an aldo-keto reductase) was induced by both experienced populations, these populations converged upon functionally equivalent strategies to biotransform the PSCs of creosote bush by constitutively expressing aldehyde and alcohol dehydrogenases, Cytochromes P450s, methyltransferases, glutathione S-transferases and sulfotransferases. The response of the naïve woodrat population to creosote bush was indicative of extreme physiological stress. CONCLUSIONS: The hepatic detoxification system of mammals is notoriously complex, with hundreds of known biotransformation enzymes. The comparison herein of woodrat taxa that differ in evolutionary and ecological experience with toxins in creosote bush reveals convergence in the overall strategies used by independent species after a historical shift in diet. In addition, remarkably few genes seemed to be important in this dietary shift. The research lays the requisite groundwork for future studies of specific biotransformation pathways used by woodrats to metabolize the toxins in creosote and the evolution of diet switching in woodrats. On a larger level, this work advances our understanding of the mechanisms used by mammalian herbivores to process toxic diets and illustrates the importance of the selective relationship of PSCs in shaping herbivore diversity.

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.

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 animal's attempting to mitigate physiological damage caused by novel PSCs.

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.

Efficacy of the LouseBuster, a new medical device for treating head lice (Anoplura:Pediculidae).

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 subject's 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.

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.

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.

Host defence mediates interspecific competition in ectoparasites.

1. Interspecific competition influences which, how many and where species coexist in biological communities. Interactions between species in different trophic levels can mediate interspecific competition; e.g. predators are known to reduce competition between prey species by suppressing their population sizes. A parallel phenomenon may take place in host-parasite systems, with host defence mediating competition between parasite species. 2. We experimentally investigated the impact of host defence (preening) on competitive interactions between two species of feather-feeding lice: 'wing' lice Columbicola columbae and 'body' lice Campanulotes compar. Both species are host-specific parasites that co-occur on rock pigeons Columba livia. 3. We show that wing lice and body lice compete and that host defence mediates the magnitude of this competitive interaction. 4. Competition is asymmetrical; wing louse populations are negatively impacted by body lice, but not vice versa. This competitive asymmetry is consistent with the fact that body lice predominate in microhabitats on the host's body that offer the most food and the most space. 5. Our results indicate that host-defence-mediated competition can influence the structure of parasite communities and may play a part in the evolution of parasite diversity.

COMPARATIVE TRANSMISSION DYNAMICS OF COMPETING PARASITE SPECIES.

Competition-colonization trade-off models explain the coexistence of competing species in terms of a trade-off between competitive ability and the ability to colonize competitor-free patches of habitat. A simple prediction of these models is that inferior competitors will be superior dispersers. This prediction has seldom been tested in natural populations because measuring dispersal is difficult. Host-parasite systems are promising in this regard, especially those involving "permanent" parasites that complete their entire life cycle on the body of the host. Because of this close association with the host, the dispersal, i.e., transmission, of these parasites can be monitored very accurately. We tested the dispersal prediction of the competition-colonization model by documenting the transmission dynamics of feather-feeding lice, which are permanent, relatively host-specific parasites of birds. We compared two groups known as "wing" lice and "body" lice that are common parasites of Rock Pigeons (Columba livia Gmelin). The two groups are ecologically similar, and they compete for resources on the host. Previous work shows that body lice are competitively superior to wing lice, leading us to predict that wing lice should be better than body lice at dispersing to new host individuals. We tested this prediction by comparing the ability of wing and body lice to disperse between hosts using vertical- and horizontal-transmission mechanisms, including phoretic hitchhiking on parasitic flies (Diptera: Hippoboscidae). A series of experiments with both captive and wild birds confirmed that wing lice are much better than body lice at colonizing new hosts. Wing lice showed significantly greater vertical transmission to nestlings, and they were quite capable of phoretic transmission to new hosts on flies. In contrast, body lice were not phoretic. These results provide the first rigorous demonstration of phoretic transmission in lice, and they underscore the importance of a community-level approach to understanding the ecology of parasite transmission dynamics.