Sperm storage patterns across Drosophila psuedoobscura populations

Research Team

Abstract/Introduction

Research Questions

Methods

Results/Findings and Discussion

Conclusions and Future Study

Acknowledgements

NACE Career Readiness Competencies

Research Team

Tessa Romah

Biomedical Engineering Pre-Medicine Co-Major

Dean Castillo

Department of Biological Sciences

Abstract/Introduction

Female mating decisions play a critical role in evolution, and these decisions extend beyond mate selection to include how females store and utilize sperm after mating. In Drosophila psuedoobscura, females store sperm in distinct organs, allowing for potential selection between sperm from multiple males. Previous work has shown that sperm competition contributes both to sexual selection and speciation, and that sperm competition dynamics differ across populations. Using females from these different geographies and males that had fluorescently labeled sperm, we examined how sperm is stored over time in female reproductive tracts. Our findings suggest that sperm storage is qualitatively the same across populations suggesting that initial storage would not contribute to sperm competition differences. This suggests that additional mechanisms might contribute to sperm storage when females mate with multiple males.

Females can store sperm in two distinct organs. Which organ sperm are stored determines which male sires the majority of the offspring.

In D. psuedoobscura females that occur in sympatry with the closely related D. persimili are able to reject heterospecific sperm. This does not occur in allopatric populations.

Research Questions

Will females from the different geographies will store sperm differently consistent with the differences in sperm rejection phenotypes?

Methods

I used a time series to determine sperm storage across different mating times. I used 4 populations from different geographic areas. Single pair matings were setup without anesthesia. After 1 day, 2 days, and 8 days female reproductive tracts were dissected and mounted on slides. I visualized sperm using a fluorescence microscope under 40X with contrast. I completed a minimum of 3 independent dissections for each genotype and time point combination. I used a male that had RFP molecule fused to protamine. This allowed me to visualize sperm in the reproductive tract since protamine binds to sperm tails and sperm heads.

This image depicts the mating setups and the different time points prior to dissecting the female reproductive tract. The tract was then placed on a slide and visualized under a microscope.

Results/Findings and Discussion

Sperm entered female storage organs quickly. In single pair matings, females had consistent patterns of storage regardless of their geographic origin. We did not see a difference between sympatric and allopatric females.

This image shows the female reproductive tract under a microscope without fluorescence, with the seminal receptacle and spermatheca labeled. Next to it are two images showing fluorescent sperm within both organs.

This table organizes the results of the experiment, showing a consistent pattern across all of the populations.

Conclusions and Future Study

Females did not show a difference in sperm storage between populations, but this occured after a single mating.

After a second mating sperm storage might shift so to better understand variation in female reproductive outcomes, future studies should examine how sperm from a second male is stored, as this may reveal distinct storage patterns. This could be achieved by incorporating a second male expressing GFP -labeled sperm to enable identification of the two different males. One of the initial observations that motivated my work was that sperm competition also contributes to reproductive isolation between species. In future experiments I could use the close relative D. persimilis that has labelled sperm and determine if there are sperm storage differences in this competitive context where females distinguish between sperm of different species

The following is an image of poster presented at the 2026 Undergraduate Research Forum.

Acknowledgements

I thank Yasir Ahmed-Braimah (Syracuse University) for the gift of the protamine-RFP stock. I also thank the Fernandes Lab (Miami University) for allowing us to use their fluorescent microscope. Finally, I would like to sincerely thank Dean for his constant support, guidance, and patience. His willingness to answer questions and teach me anything I needed to learn made this experience incredibly valuable.

NACE Career Readiness Competencies

Through my research experience, I developed strong critical thinking, communication, and teamwork skills. I enhanced my ability to analyze data, evaluate results, and adapt when challenges arose, while also improving how I present complex ideas clearly in both written and verbal formats. Working closely with mentors and peers allowed me to collaborate effectively, contribute to shared goals, and grow more confident in a team-based environment.

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