Signatures of correlated evolution predict new members of a protein network required for Drosophila female post-mating responses. Geoffrey D. Findlay¹, Nathaniel L. Clark^1,2, Jessica L. Sitnik¹, Charles F. Aquadro¹, Mariana F. Wolfner¹. 1) Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY; 2) Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA.

   Mating induces long-term changes in Drosophila melanogaster females, including decreased receptivity to courtship, increased egg production, and efficient sperm storage. These changes are caused primarily by a seminal fluid protein, sex peptide (SP), which females receive from the male and store for several days. Five additional seminal fluid proteins (Sfps) are required for SP to be stored in females and to act over the long term. To discover new members of this SP network, we used a new comparative genomic method to detect correlated changes in the rates of protein evolution across the Drosophila phylogeny. The logic was to discover new proteins within the SP network by virtue of their shared evolutionary selective pressures. We first confirmed that the known network proteins showed correlated evolutionary rates. We then used each member of the network to computationally query hundreds of male and female reproductive proteins for correlated evolution and used RNAi to functionally evaluate potential candidates. RNAi tests of 16 top candidates identified three male Sfps and two female-expressed proteins that are each required for the long-term effects of SP on fertility and female receptivity. Molecular genetic analysis showed the three new male proteins are required for the transfer of other network proteins and for SP storage in mated females. The two female proteins, in contrast, act downstream of SP storage. Our results provide the first demonstration that signatures of correlated evolution can be used prospectively to predict new members of a protein network. In addition, they have expanded our knowledge of the male-derived portion of the SP network and identified new female regulators of SP action.