LAUSR

Significance Ammonium transporters (AMT) have been implicated in facilitating nitrogen assimilation in plants and bacteria and ammonia (NH3/NH4+) transport for nitrogen regulation and excretion in animals. However, the involvement of AMT proteins in mediating reproductive processes remains unexplored. We report that an insect AMT, AeAmt1, is expressed in the flagellum of spermatozoa from the dengue vector mosquito Aedes aegypti during sperm development through to egg fertilization. We provide evidence that AeAmt1 expression in spermatozoa is critical for sperm survival and overall male fertility. This description of ammonia transporter (AMT and Rh families) expression in the sperm of an animal provides a striking role for these evolutionarily conserved protein families and contributes to our understanding of mosquito reproductive biology. The ammonium transporter (AMT)/methylammonium permease (MEP)/Rhesus glycoprotein (Rh) family of ammonia (NH3/NH4+) transporters has been identified in organisms from all domains of life. In animals, fundamental roles for AMT and Rh proteins in the specific transport of ammonia across biological membranes to mitigate ammonia toxicity and aid in osmoregulation, acid–base balance, and excretion have been well documented. Here, we observed enriched Amt (AeAmt1) mRNA levels within reproductive organs of the arboviral vector mosquito, Aedes aegypti, prompting us to explore the role of AMTs in reproduction. We show that AeAmt1 is localized to sperm flagella during all stages of spermiogenesis and spermatogenesis in male testes. AeAmt1 expression in sperm flagella persists in spermatozoa that navigate the female reproductive tract following insemination and are stored within the spermathecae, as well as throughout sperm migration along the spermathecal ducts during ovulation to fertilize the descending egg. We demonstrate that RNA interference (RNAi)-mediated AeAmt1 protein knockdown leads to significant reductions (∼40%) of spermatozoa stored in seminal vesicles of males, resulting in decreased egg viability when these males inseminate nonmated females. We suggest that AeAmt1 function in spermatozoa is to protect against ammonia toxicity based on our observations of high NH4+ levels in the densely packed spermathecae of mated females. The presence of AMT proteins, in addition to Rh proteins, across insect taxa may indicate a conserved function for AMTs in sperm viability and reproduction in general.