Graduate Program

Biological Sciences

Degree Name

Master of Science (MS)

Semester of Degree Completion

2008

Thesis Director

Ann Fritz

Thesis Committee Member

Gary Fritz

Thesis Committee Member

Gary Bulla

Abstract

Anastrepha suspensa females have four different sites of sperm storage in their reproductive tract, may mate with more than one male, and store sperm in differing amounts in each organ after copulation. In order to study the dynamics of sperm storage patterns and subsequent paternity outcomes, a method was developed for both genotyping and quantifying sperm in the spermathecae. Spermathecae were isolated and sonicated to remove maternal cells, and subsequently crushed between two microslides to release sperm from the capsule. After DNA isolation, microsatellite loci could be amplified by PCR for fragment analysis. A newly developed method was then used to identify and quantify sperm from two different male genotypes that could then be identified and quantified by measuring peak height and peak area using a CEQ 8000 XL Beckman Coulter TM Sequencer. A standard curve of peak area and height for different quantities of DNA amplified by PCR for a given microsatellite locus was used to estimate unknowns. Preliminary data was presented on correlations of the duration of first copulations versus second copulations and on sperm storage patterns for doubly-mated females. An inverse relationship was observed between the time females spent in copulation with first versus second males by correlation analysis and was found to be highly significant (r = -0.37691, p = 0.00001, N = 93). The first copulation duration is a good predictor of the second copulation duration and could also be used to predict whether the first or the second male's sperm would be stored. Over 80% of doubly-mated females that had short first copulations (1-15 minutes) stored sperm from one male only. Alternatively, most females with long first copulations(> 15 minutes) stored sperm from both males. After short first copulations, females storing one male's sperm had that of the second male primarily. After long first copulations, females storing one male's sperm had that of the first male primarily. Single spermathecae were never observed to contain the sperm of more than one male and it was found that 94.12% of all females storing sperm from both males had the first male's sperm in one spermatheca of the doublet spermathecae and the second male's sperm in the other spermatheca of the doublet spermathecae. Thirty-nine percent of all females had sperm from both males in their ventral receptacle. These results suggest the potential for cryptic female choice in this species because spermatozoa from different males were stored separately amongst the spermathecae, providing the possibility of female control of different males' sperm. However, sperm competition may occur within the ventral receptacle, as different males' sperm were mixed within that organ. Approximately, 69% of the doubly-mated females that stored sperm from single male contained the second male's sperm in their ventral receptacle. These data strongly imply a mechanism for second male precedence, and suggest that the paternity patterns (P2) must be critically interpreted with initial sperm quantity obtained by females as an important factor. The techniques described in this study should be generally applicable to all studies examining post-copulatory sexual selection.

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