Validation by qPCR of Reference Genes for Reproductive Studies in the Invasive Apple Snail Pomacea canaliculata

ABSTRACT The South American freshwater gastropod Pomacea canaliculata is a highly invasive species. In introduced areas, it is a serious crop pest, responsible for great economic loss and ecological damage. It is also a vector of the nematode Angiostrongylus cantonensis that causes human meningoencephalitis. Many aspects and particularly its reproduction have been extensively studied, but little research has been conducted on this species regarding gene expression. To meaningfully interpret quantitative PCR, a powerful technique to develop this kind of study, validation of reference genes is essential but until now has not been undertaken. We selected the female albumen gland for its major role in egg production in order to evaluate the expression stability of the candidate reference genes EF1-α, RPL7, His H3.3, TUBB, 18S RNA, ACTB and GAPDH. Stability was analyzed under different reproductive activity conditions and defined based on three approaches: geNorm, NormFinder and the comparative ΔCt method. NormFinder selected GAPDH and ACTB as the best option to be used for normalization, whereas geNorm and the comparative ΔCt method indicate RPL7, GAPDH, and 18S RNA as the most stable genes under the conditions studied. These results will facilitate reproductive studies, particularly those using qPCR to evaluate factors that may affect fecundity of this conspicuous invasive species.