From raw materials to validated system: the construction of a genomic library and microarray to interpret systemic perturbations in Northern bobwhite

Abstract

The limited availability of genomic tools and data for nonmodel species impedes computational and systems biology approaches in nonmodel organisms. Here we describe the development, functional annotation, and utilization of genomic tools for the avian wildlife species Northern bobwhite (Colinus virginianus) to determine the molecular impacts of exposure to 2,6-dinitrotoluene (2,6-DNT), a field contaminant of military concern. Massively parallel pyrosequencing of a normalized multitissue library of Northern bobwhite cDNAs yielded 71,384 unique transcripts that were annotated with gene ontology (GO), pathway information, and protein domain analysis. Comparative genome analyses with model organisms revealed functional homologies in 8,825 unique Northern bobwhite genes that are orthologous to 48% of Gallus gallus protein-coding genes. Pathway analysis and GO enrichment of genes differentially expressed in livers of birds exposed for 60 days (d) to 10 and 60 mg/kg/d 2,6-DNT revealed several impacts validated by RT-qPCR including: prostaglandin pathway-mediated inflammation, increased expression of a heme synthesis pathway in response to anemia, and a shift in energy metabolism toward protein catabolism via inhibition of control points for glucose and lipid metabolic pathways, PCK1 and PPARGC1, respectively. This research effort provides the first comprehensive annotated gene library for Northern bobwhite. Transcript expression analysis provided insights into the metabolic perturbations underlying several observed toxicological phenotypes in a 2,6-DNT exposure case study. Furthermore, the systemic impact of dinitrotoluenes on liver function appears conserved across species as PPAR signaling is similarly affected in fathead minnow liver tissue after exposure to 2,4-DNT.

Investigations of transcript expression in fathead minnow (Pimephales promelas) brain tissue reveal toxicological impacts of RDX exposure

“…was calculated around the mean relative expression for each RDX concentration. Confidence intervals that did not include unity were considered differentially expressed relative to controls as described in Rawat et al (2010).…”

Endocrinology: Advances through omics and related technologies

“…Aggressive behavior Homarus americanus, Astacus astacus, Drosophila Huber et al (1997), Kravitz and Huber (2003) and Nilsen et al (2004) Aging Bivalves, Drosophila, C. elegans, Macrostonum lignano (2009) minnows, quail and rat, supporting the suitability of Daphnia as an alternative model species (Deng et al, 2011;Rawat et al, 2010;Wintz et al, 2006). Further supporting the Daphnia model, researchers from Japan (Kato et al, 2011) used Daphnia to reveal how environmental sex determination is implemented by selective expression of a fundamental genetic component that is functionally conserved in animals using genetic sex determination, inferring that there is an ancient link between genetic and environmental sex determination.…”

Genomic investigation of year‐long and multigenerational exposures of fathead minnow to the munitions compound RDX

“…Genomics-based approaches, such as the use of DNA arrays to detect differentially expressed transcripts associated with contaminant exposure, have the potential to provide important insights into mechanisms of action [7][8][9][10][11], organism acclimation and adaptation [6], and development of meaningful biomarkers of exposure [12,13]. A fundamental understanding of genomic changes that occur during long-term and multigenerational exposure to low doses of a contaminant could significantly enhance the understanding of any chronic effects associated with the contaminant and also serve as a credible scientific basis for regulatory actions.…”