ISOLATING, AMPLIFYING AND QUANTIFYING SPERM DNA IN ANASTREPHA SUSPENSA (DIPTERA: TEPHRITIDAE)

A major obstacle for many studies examining sperm competition and cryptic female choice in insects has been the identification and quantification of sperm stored in the sperm storage organs of females that have mated with two or more males. Historically, sexual selection studies have focused primarily on paternity outcomes for inferring potential underlying mechanisms (e.g., sperm competition and cryptic female choice). We describe a technique for isolating, genotyping and quantifying sperm in Anastrepha suspensa Loew, a species that has four sperm storage organs (three spermathecae and a ventral receptacle) that are minute (approximately 80 mu m) and exhibit complex interior structures restricting sperm recovery through simple dissection. With our protocol, we were able to isolate and amplify sperm DNA (PCR of microsatellite loci) without contamination from female cells, and quantify sperm contributed to a storage organ by one or more males. Briefly, sperm storage organs are dissected-out of the female abdomen, sonicated to remove female cells, rinsed in saline, crushed between micro-slides (1 x 2 mm), and placed in a microcentrifuge tube for DNA isolation in situ using a solution containing 10% chelex, proteinase-K and DTT. After boiling, the DNA is amplified by PCR. With this technique, we have successfully amplified microsatellite loci from as few as 10 +/- 3 sperm. Estimates of absolute numbers of sperm stored in sperm storage organs was accomplished by incorporating a reference amplicon standard in each sample during fragment analysis of microsatellite loci. The protocol described in this study enable the localization, identification and quantification of sperm from multiple males stored in female sperm storage organs and, therefore, generates data that can augment interpretations of paternity outcomes.