Using Representational Difference Analysis to detect changes in transcript expression of Aiptasia genes after laboratory exposure to lindane

Abstract

Molecular stress responses to pesticide exposures represent an understudied area of cnidarian transcriptome investigations. The organochlorine pesticide lindane is known to disrupt normal neuron function. Cnidarians with simple nervous systems are recognized as sensitive indicators of water quality, yet nothing is known about cnidarian responses to lindane. Sea anemones (Aiptasia pallida) were exposed for 4h to lindane (20 μg/l). Because anemones have neurons and lindane is known to target neurons, it is anticipated that cnidarian stress responses will include changes in transcription of genes associated with neurons. Representational Difference Analysis (RDA) was utilized to isolate differentially transcribed genes in the anemones exposed to the pesticide. After two rounds of RDA hybridizations, 148 amplified fragments ranging in size from 150 to 800 bp were cloned. Sequencing and bioinformatic analyses of 106 clones revealed 56 different gene fragments. Virtual Northern dot blots were used as a preliminary screening tool to identify the most responsive RDA products. To further characterize the specificity of response, additional anemones were exposed to a series of lindane concentrations (0, 0.2, 2.0, 10, and 20 μg/l). Northern dot blots were subsequently used to develop expression profiles for selected RDA products over the range of pesticide concentrations. The seven most responsive RDA products represent genes with products associated with neuron development, immune responses, and Ca(2+) binding/transport. The resulting expression profiles illustrate that these RDA products exhibit various degrees of concentration specificity with some RDA products being significantly up-regulated at 20 μg/l while other RDA products are most responsive at concentrations <20 μg/l. Results also demonstrate how RDA can be used to identify potentially important biomarkers of organochlorine exposure while generating new hypotheses about important phenomena such as endocrine disruption in cnidarians.