Development and Validation of Real-Time PCR Method to Estimate Stored Sperm in the Spermathecae of Ceratitis capitata (Diptera: Tephritidae).

The development of polymerase chain reaction (PCR) markers to identify theY chromosome of Ceratitis capitata Wiedemann has permitted the detection of sperm transferred to females during mating. However, a molecular technique to quantify the sperm transferred has not yet become available. The current method to quantify the amount of sperm has been the direct counting of sperm heads. Thus, the purpose of this research was to develop and validate an accurate molecular method of diagnosis based on the application of an absolute quantitative real-time PCR, which allows the assessment of the quantity of sperm stored in the spermathecae. For this, Y-specific sequences were used to re-design and test distinct sperm markers. From the amplification product of samples detected as strong positives in conventional PCR, a cloning process of the target sequence was carried out to build the required standard curve. A series of known dilutions of this standard material was prepared for the absolute quantification process. A Roche Lightcycler 480 Real-Time PCR System and SYBRGreen fluorescent dye were used to quantify the sperm contained in the spermathecae of 4-d-old mated females and virgins. Wild-type and Vienna-8 strain sterile males were used to quantify the sperm transferred at four mating durations (10, 30, 60, and 90 min) under laboratory conditions. To validate the reported quantitative method, our results were compared by counting sperm heads under a fluorescent microscope using the same experimental design. In addition, DNA samples were also evaluated and compared by conventional PCR.