To fully understand the process of reproduction, it is essential to learn more about the many components of seminal fluid. These have been particularly well-studied in insects, where seminal fluid proteins (SFPs) have been demonstrated to be key regulators of the reproductive success of both males and females. This project will identify SFPs in the red flour beetle, Tribiolium castaneum, which is a worldwide, economically important pest of many different stored grains. Using a powerful combination of chemical and genetic techniques, the investigators will determine the specific effect these seminal proteins have on Tribolium reproductive success. One approach will be to use RNA interference (or RNAi), in which Tribolium will be injected with double stranded RNA that matches the genes responsible for producing those proteins. Using this technique, the investigators will be able to effectively halt the translation of the mRNA that contain the instructions to build each seminal protein. By then evaluating the reproductive performance of these knockdown males, the role these proteins play in determining the reproductive success of this species will be revealed. This project has both theoretical and practical applications. By understanding how seminal fluid proteins affect the interplay between males and females for control over the fertilization of a female's eggs, a greater understanding will be gained of the evolutionary forces at work in male and female co-evolution. Additionally, this research may reveal novel approaches to help control the reproductive output of this economically important agricultural pest. This project will likely provide training opportunities for several undergraduate students.
The purpose of this grant was to investigate and characterize specific molecules that are transferred in the ejaculate of male Tribolium castaneum beetles to females during mating. These molecules are known as seminal fluid proteins (referred to as SFPs hereafter) and serve a wide variety of functions. In insects, quite a bit is known about the role that SFPs play in the reproductive ecology of a given species. In addition to helping nourish and protect sperm once transferred, these molecules can initiate a series of behavioral and physiological shifts in females, causing mated females to reject other males and begin to produce and lay more eggs. Because these proteins are so important to reproduction, they have become a possible target for novel methods of biological control. For a stored products pest such as T. castaneum, which causes billions of dollars of economic damage a year, these sorts of biological controls are very much needed. Because of their ability to modulate reproductive fitness, a greater understanding of the myriad possible ways that SFPs can impact both males and females is an extremely important component of unraveling their true functional significance. To that end, T. castaneum is an optimal species. T. castaneum is a model system for the investigation of a mode of natural selection known as sexual selection, or the study of how evolution can shape how an individual can obtain or successfully copulate with a mate. In recent years sexual selection has been demonstrated to be episodic in nature, with different mechanisms relevant before, during and even after mating has already been completed. Therefore, in order to gain a better understanding of the totality of how sexual selection influences a given species reproductive fitness, it is necessary to have an experimental design inclusive of its episodic nature. Therefore this study had two goals: 1). Identify SFPs that are transferred from the male to the female. 2). Determine the functional role of these SFPs across multiple episodes of sexual selection This research project was the first to characterize and describe SFPs in this economically important species. Using a combination of proteomics and mass spectrometry, we identified 13 SFPs that males transfer to females in their ejaculate. These SFPs represent a broad category of protein types, including those that inhibit female reproductive proteins, regulate chemosensory functions and immune defense. This variety of functions likely reflects the multitude of tasks that these SFPs perform once transferred to the opposite sex. Additionally, we attempted to utilize a technique known as RNA interference (RNAi) to characterize the role that targeted SFPs play in the reproductive ecology of T. castaneum. RNAi is an experimental technique in which short pieces of RNA that are homologous to a gene producing a protein of interest (such as a SFP) is injected into an individual. The injection of this RNA should essentially halt or at least lessen the production of this protein. By comparing with a control that has not been injected with this RNA, this technique allows for a determination of the functionality of that targeted protein. Here we attempted to target the production of 3 different SFPs via RNAi. Injected individuals were then put through a variety of measurements of reproductive fitness and performance. These assays were designed to capture potential roles of these SFPs across the different episodes of sexual selection. Unfortunately, we were unable to detect any type of phenotype with our assays. However, the totality of this project establishes a solid foundation for future investigations of the role the SFPs play in both the reproductive ecology of T. castaneum and sexual selection in general. Thus, this project has advanced knowledge and understanding in multiple fields of study, a core value of NSF funded research
