Project Summary/Abstract The male ejaculate of internally fertilizing organisms is a complex mixture of diverse components including sperm and numerous other macromolecules. Research on male contributions to fertility has mainly focused on sperm, and, more recently on proteins present in the seminal fluid. Much less attention has been directed toward understanding the role that other ejaculate molecules might play in fertilization success. The goal of the proposed research is to investigate the functional significance of ejaculate RNA, which is found in the ejaculates of diverse organisms ranging from humans to insects, though little is known about its potential function. Specifically, the proposed research involves three specific aims, which together evaluate whether coding and long non-coding RNA (lncRNA) in the ejaculate of male Drosophila arizonae has functional effects on fertility either by directing the production of key proteins or through regulatory effects within the female reproductive tract. Aim one combines ribosome profiling (ribo-seq) and proteomics to assess whether male mRNAs are translated within females. The challenge of differentiating male and female RNA and proteins within the female reproductive tract is overcome by exploiting natural genetic variation in the D. arizonae study system and by the adoption of recently developed methods for metabolically labeling the proteome of adult Drosophila using stable isotope labeled amino acids. Because lncRNAs often function through interactions with protein, aim two involves the identification of RNA-protein interactions using RNA Antisense Purification with Mass Spectrometry (RAP-MS). Interacting proteins with annotated functions will provide key insight into the mechanisms by which lncRNAs function. Aim three proposes to generate knockout male lines for a select number of RNAs using the CRISPR genome editing system. Reproductive output of crosses between knockout males and wild-type females will be compared to wild-type crosses, with the expectation that if a male RNA is involved in fertility, crosses involving knockout males will have lower reproductive output than crosses with wild-type males. Altogether the results of the proposed studies will provide novel insights into the molecular interactions between male and female molecules that ultimately determine fertilization success. Moreover, this research may have important implications for identifying the underlying causes of idiopathic infertility, the development of assistive reproductive techniques, and the design of new strategies for integrative pest management.