The health and resilience of species in natural environments are increasingly challenged by complex anthropogenic stressor combinations including climate change, habitat encroachment, and chemical contamination.
More...The health and resilience of species in natural environments are increasingly challenged by complex anthropogenic stressor combinations including climate change, habitat encroachment, and chemical contamination. To better understand impacts of these stressors, we examined the individual- and combined-stressor impacts of malaria infection, food limitation, and 2,4,6-trinitrotoluene (TNT) exposures on gene expression in livers of Western fence lizard (WFL, Sceloporus occidentalis) using custom WFL transcriptome-based microarrays. Computational analysis including annotation enrichment and correlation analysis identified putative functional mechanisms between transcript expression and toxicological phenotype. TNT exposure increased transcript expression for genes involved in erythropoiesis, potentially in response to TNT-induced anemia and/or methemoglobinemia, and caused dose-specific effects on genes involved in lipid and overall energy metabolism consistent with a hormesis response of growth stimulation at low doses contrasted with adverse effects on lizard growth at high doses. Functional enrichment and inguinal fat body weights suggest inhibition of lipid mobilization and catabolism by TNT coupled with a decreased overall energy budget. Malaria infection elicited enrichment of the expression of multiple immune-related functions likely corresponding to increased white blood cell (WBC) counts. Food limitation alone enriched functions related to cellular energy production and decreased expression of immune response consistent with a decrease in WBC levels. Despite these findings, the lizards demonstrated immune resilience to malaria infection under food limitation with transcriptional results indicating a fully competent immune response to malaria, even under bioenergetic constraints. Interestingly, each TNT and malaria individually tended to increase transcriptional expression of immune-related genes and increase overall WBC concentrations in blood; responses that were retained in the TNT x malaria combined exposure. The results demonstrate complex and sometimes unexpected responses to multiple stressors where the lizards displayed remarkable resiliency to the stressor combinations investigated.
Overall design: Three separate microarray analyses were conducted within this study. The three experiments investigated 3 pairwise stressor combinations, including: Exposure 1 - TNT x food limitation, Exposure 2 - food limitation x malaria infection, and Exposure 3 - TNT x malaria infection. All exposure details can be found in the companion paper, McFarland et al. (2012 Ecotoxicol. 21:2372–2390 DOI 10.1007/s10646-012-0993-1). This GEO entry represents Exposure 1 (TNT x food limitation): Briefly, the experiment was conducted as 30-day bioassay utilizing male Western fence lizards. Experimental treatments were deployed in a factorial treatment arrangement within a completely randomized experimental design. The Western fence lizards were dosed by daily gavage to TNT at 0 (control), 5, 10 and 20 mg/kg/d paired with either ad libitum feeding (10 crickets) or restricted feeding (2 crickets) treatments. All genomics investigations included at least 4 replicate liver samples collected from individual lizards per condition where most treatments included 6 replicates. At the completion of the exposures, animals were euthanized by CO2 asphyxia, dissected, and liver samples were fixed in RNA Later™ (Ambion, Austin, TX) following manufacturer's recommendations and stored at -80C until RNA extraction. Blood and various tissues were also collected for clinical endpoint analysis where individual and pair-wise stressor effects were tested for 29 toxicological endpoints (McFarland et al. 2012. Ecotoxicol. 21:2372–2390 DOI 10.1007/s10646-012-0993-1). All protocols were conducted consistent with Good Laboratory Practices and approved by the Institutional Animal Care and Use Committee at the U.S. Army Public Health Center.
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