Ammonium transporter expression in sperm of the disease vector Aedes aegypti mosquito influences male fertility

Abstract

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 enrichedAmt(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.

lnc94638 is a testis‐specific long non‐coding RNA involved in spermatozoa formation in Zeugodacus cucurbitae (Coquillett)

“…These comparisons uncovered the unique temporal expression profiles of testis‐specific genes. Through FISH, lnc94638 was localized to the apical region of the Z. cucurbitae testis, a region characterized by the accumulation of mature spermatozoa, as has been previously shown for the mosquito testis (Durant and Donini, 2020). This distinctive distribution of lnc94638 suggested a potential function in spermatozoa formation of Z. cucurbitae .…”

Mutagenesis of the Ammonium TransporterAcAmtReveals a Reproductive Role and a Novel Ammonia-Sensing Mechanism in the Malaria Vector MosquitoAnopheles coluzzii

“…In addition to the peripheral olfactory responses to ammonia, ammonium transporters have recently been shown to be involved in other essential functions in the biology of insects including male fertility in Ae. aegypti (Durant and Donini 2020) and larval muscle control in Drosophila (Lecompte et al 2020). Here, CRISPR/Cas9-induced AcAmt -/- mutations similarly uncover several potentially non-olfactory phenotypes in An.…”

“…Even so, while significant fecundity deficits are reported here in AcAmt -/- mutants and AeAmt RNAi treatments in Ae. aegypti (Durant and Donini 2020), these phenotypes are likely to result from fundamentally different mechanisms working synergistically. In An.…”

“…coluzzii , the frequency of successful copulation (sperm delivery) is significantly reduced in both AcAmt -/- females and males, while the decrease of fecundity in Ae. aegypti appears to be due to a significant reduction in viable spermatozoa (Durant and Donini 2020). Importantly, this latter phenotype is specifically not observed in An.…”

The V-type H+-ATPase is targeted in anti-diuretic hormone control of the Malpighian ‘renal’ tubules

“…Molecular weight measurements were performed using Image Lab 5.0 software (Bio-Rad). PVDF membranes were then probed with Coomassie brilliant blue, since total protein normalization is now considered the benchmark method for quantitative analysis of western blot data (80, 81) and has been used in studies involving A. aegypti protein normalization (82). ImageJ software (NIH, USA) was used to quantify protein abundance.…”