RP7 Ecotoxicogenomics of Emerging Chemical Issues
Thursday, 17 November 2005: 8:00 AM - 6:30 PM in Exhibit Hall

(KNO-1117-822100) Development of a Smallmouth Bass Quantitative Real-Time PCR Assay to Measure VTG1 Gene Induction in Male Fish from the South Branch of the Potomac River.

Knoebl, I.¹, Iwanowicz, L.^{2, 4}, O'Bara, C.³, Blazer, V.⁴, ¹ U. S. EPA, Ecological Exposure Research Division, Cincinnati, OH, USA² University of Massachusetts at Amherst, Amherst, MA, USA⁴ USGS-Leetown Science Center, National Fish Health Research Laboratory, Kearneysville, WV, USA³ West Virginia Department of Natural Resources, Parkersburg, WV, USA

ABSTRACT- A high incidence of intersex bass, primarily male smallmouth bass with previtellogenic oocytes, exists in the south branch of the Potomac River. Exposures to endocrine disrupting chemicals (EDCs) may be the cause of these abnormalities. Potential sources of EDCs to the river are municipal sewage treatment plant discharges and poultry and cattle feedlots located in close proximity to the river. In an attempt to determine whether EDCs, particularly estrogenic chemicals, from these effluents and discharges may be contributing to the observed intersex in fish, we have developed a quantitative real-time (Q-PCR) assay for smallmouth bass to measure the induction of the vitellogenin (VTG) gene in male fish. We isolated a VTG1 gene sequence using degenerate primers in a PCR reaction. The PCR products were cloned using E. coli cells and the clones were isolated and sequenced to confirm the identity of the gene fragment. The clones provided a template to generate a standard curve for smallmouth bass VTG1 and the gene sequence was then used to design Q-PCR primers. The Q-PCR assay was optimized and then used to measure VTG levels in smallmouth bass collected from various locations on the south branch of the Potomac River. Although this work was reviewed by EPA and approved for publication, it may not necessarily reflect official Agency policy.

Key words: Quantitative real-time PCR, Vitellogenin, smallmouth bass, Potomac River