Male reproductive gland proteins (mRGPs) impact the physiology and/or behavior of mated females in a broad range of organisms. We sought to identify mRGPs of the yellow fever mosquito, Aedes aegypti, the primary vector of dengue and yellow fever viruses. Earlier studies with Ae. aegypti demonstrated that "matrone" (a partially purified male reproductive accessory gland substance) or male accessory gland fluid injected into virgin female Ae. aegypti affect female sexual refractoriness, blood feeding and digestion, flight, ovarian development, and oviposition. Using bioinformatic comparisons to Drosophila melanogaster accessory gland proteins and mass spectrometry of proteins from Ae. aegypti male accessory glands and ejaculatory ducts (AG/ED) and female reproductive tracts, we identified 63 new putative Ae. aegypti mRGPs. Twenty-one of these proteins were found in the reproductive tract of mated females, but not of virgin females, suggesting that they are transferred from males to females during mating. Most of the putative mRGPs fall into the same protein classes as mRGPs in other organisms, although some appear to be evolving rapidly and lack identifiable homologs in Culex pipiens, Anopheles gambiae, and D. melanogaster. Our results identify candidate male-derived molecules that may have an important influence on behavior, survival and reproduction of female mosquitoes.

Cytoplasmic folate-mediated one carbon (1C) metabolism functions to carry and activate single carbons for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine. C1 tetrahydrofolate (THF) synthase, encoded by Mthfd1, is an entry point of 1Cs into folate metabolism through its formyl-THF synthetase (FTHFS) activity that catalyzes the ATP-dependent conversion of formate and THF to 10-formyl-THF. Disruption of FTHFS activity by the insertion of a gene trap vector into the Mthfd1 gene results in embryonic lethality in mice. Mthfd1 gt/؉ mice demonstrated lower hepatic adenosylmethionine levels, which is consistent with formate serving as a source of 1Cs for cellular methylation reactions. Surprisingly, Mthfd1 gt/؉ mice exhibited decreased levels of uracil in nuclear DNA, indicating enhanced de novo thymidylate synthesis, and suggesting that serine hydroxymethyltransferase and FTHFS compete for a limiting pool of unsubstituted THF. This study demonstrates the essentiality of the Mthfd1 gene and indicates that formate-derived 1Cs are utilized for de novo purine synthesis and the remethylation of homocysteine in liver. Further, the depletion of cytoplasmic FTHFS activity enhances thymidylate synthesis, affirming the competition between thymidylate synthesis and homocysteine remethylation for THF cofactors. Folate-mediated one-carbon (1C)3 metabolism is compartmentalized in the cytoplasm, mitochondria, and nucleus of mammalian cells (1). In the cytoplasm, 1C metabolism functions to carry and chemically activate single carbons for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine (2) (see Fig. 1). Methionine can be adenosylated to form S-adenosylmethionine (AdoMet), the major cellular methyl group donor required for the methylation of DNA, RNA, histones, small molecules, and lipids. Nuclear 1C metabolism functions to synthesize thymidylate from dUMP and serine during S phase through the small ubiquitin-like modifier-dependent translocation of cytoplasmic serine hydroxymethyltransferase (cSHMT), dihydrofolate reductase, and thymidylate synthase into the nucleus (3).Serine, through its conversion to glycine by SHMT, is a primary source of 1Cs for nucleotide and methionine synthesis (4). SHMT generates 1Cs in the cytoplasm, mitochondria, and nucleus, although the generation of 1Cs through SHMT activity in the cytoplasm is not essential in mice, indicating the essentiality of mitochondria-derived 1Cs for cytoplasmic 1C metabolism (5). In mitochondria, the hydroxymethyl group of serine and the C2 carbon of glycine are transferred to tetrahydrofolate (THF) to generate 5,10-methylene-THF by the mitochondrial isozyme of SHMT and the glycine cleavage system, respectively (6). The 1C carried by methylene-THF is oxidized and hydrolyzed to generate formate by the NAD-dependent methylene-