Quantification of vitellogenin mRNA induction in mosquitofish (Gambusia affinis) by reverse transcription real-time polymerase chain reaction (RT-PCR)

Abstract

A method to quantify induction of vitellogenin (Vtg) mRNA in adult male mosquitofish was developed. Male mosquitofish were exposed to 0, 1, 20 and 250 ng l⁻¹ 17β-oestradiol (E₂) for 4 and 8 days in static exposures, and liver Vtg mRNA and 18S rRNA expression were quantified in duplex RT-PCR. Liver 18S rRNA expression was very consistent among individuals, and there was a highly significant increase in Vtg mRNA expression after exposure of mosquitofish for just 4 days at 250 ng l⁻¹ E₂. Lower doses did not induce Vtg mRNA expression even at 4 or 8 days. This method could be used as a rapid test to detect exposure of mosquitofish to oestrogenic chemicals. Further work is needed to determine if increased Vtg mRNA levels in male mosquitofish induce Vtg synthesis, and to determine the usefulness of the method in field sampling.

Keywords: :

• 18S rRNA
• LUX® primers
• one-step RT-PCR
• Vtg mRNA

Acknowledgements

Research was funded by an Australian Research Council (ARC) Strategic Partnership with Industry — Research and Training (SPIRT) grant in collaboration with CalAqua and a Foundation for Research, Science and Technology (FRST) Enterprise scholarship in collaboration with the New Zealand Water Environment Research Foundation (NZWERF) to F. L. Many thanks to J. Morton and D. Palmer for the use of their laboratory facilities, and to the iCycler at Lincoln University; to K. Gately for helpful advice, and to K. Trought at Landcare Research; to D. Gleeson and C. Eason for comments on earlier drafts; and to C. Bezar for editorial services.