Multiple environmental stressors induce complex transcriptomic responses indicative of phenotypic outcomes in Western fence lizard

Abstract

Background: The health and resilience of species in natural environments is 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 lizards (WFL, Sceloporus occidentalis) using custom WFL transcriptome-based microarrays.

Results: Computational analysis including annotation enrichment and correlation analysis identified putative functional mechanisms linking transcript expression and toxicological phenotypes. 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 and adverse decreases in lizard growth at high doses. Functional enrichment results were indicative of inhibited potential for lipid mobilization and catabolism in TNT exposures which corresponded with increased inguinal fat weights and was suggestive of a decreased overall energy budget. Malaria infection elicited enriched 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 responses consistent with a decrease in WBC levels.

Conclusions: 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 bio-energetic constraints. Interestingly, both TNT and malaria individually increased transcriptional expression of immune-related genes and increased 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.

Keywords: Climate change; Food limitation; Genomics; Immune response; Malaria; Multiple stressors; Munitions; Reptiles; TNT.

Fig. 1

Experimental designs for microarray experiments across the three single/combined-stressor exposures in the Western fence lizard (Sceloporus occidentalis). Numbers within each cell represent the number of biological replicate microarray hybridizations contributing to the overall microarray analysis in the exposure matrix. Cells highlighted in gray represent microarrays hybridized using the same biological replicates for corresponding treatments across Exposure 2 and Exposure 3. Exp1, Exp2 and Exp3 represent Exposure 1, Exposure 2 and Exposure 3, respectively

Fig. 2

Summary results for microarray analyses. Venn diagrams in panel (a) display the differentially expressed transcripts found in common among treatments based on matching RefSeq Accession IDs. Hierarchical clustering analyses (b) identifed relationships based on significant differential transcript expression among treatment combinations for each exposure. Transcripts differentially expressed in common among repeated experimental exposures (c) and correlations in expression among these transcripts (d) provided opportunities for assessing reproducibility of experimental results. Note that no correlation was calculated for Diet given that only two targets were found in common. Key for Venn Diagrams: Exp = Exposures 1–3, Diet = food level, and Mal = malaria treatment. For hierarchical clustering analysis: infected = malaria infection, restricted = restricted diet and the values 0, 5, 10 and 20 = TNT dosing levels in mg/kg-d

Fig. 3

Transcript expression profiles for the significantly enriched annotation clusters (i.e. C1-C5). Significant clusters are mapped to each treatment for all exposures (See Fig. 1 for design). Clusters can be cross referenced against Table 1 for general functions and against Additional file 1: Table S6 for specific gene identities and expression values

Fig. 4

Relationships between differentially expressed gene transcripts identified within significant annotation clusters and toxicological phenotypes in the Western fence lizard. The plots represent selected relationships identified by correlation analysis (Additional file 1: Table S8) paired against expression of transcripts identified within significant annotation clusters (Additional file 1: Table S6). Toxicological phenotype data represented herein were significantly affected by the experimental exposures as described in McFarland et al. (2012) [1]