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

Michael B. Morgan, Charly C. Parker, Jerid W. Robinson, Emily M. Pierce

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

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 (20μg/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 800bp 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 20μg/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 20μg/l while other RDA products are most responsive at concentrations <20μg/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.

Original languageEnglish (US)
Pages (from-to)66-73
Number of pages8
JournalAquatic Toxicology
Volume110-111
DOIs
StatePublished - Apr 2012

Bibliographical note

Funding Information:
We acknowledge with appreciation the support of Berry College for the Faculty Development grant to M. Morgan as well as the Berry College/Sigma Xi grant to M. Morgan for the development of undergraduate research. We also wish to acknowledge the laboratory assistance of Michael Clemmons, Peggy Mahoney, Matt Moore, and Walt Wiley.

Copyright:
Copyright 2012 Elsevier B.V., All rights reserved.

Keywords

  • Biomarkers
  • Cnidarian
  • Expression profiles
  • Lindane
  • RDA
  • Stress response

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