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 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.

Keywords: Culicidae; dengue; insect physiology; insect reproduction; spermatogenesis.

Fig. 1.

AeAmt1 protein localization in male testes and spermatozoa stored within the spermathecae of female Aedes aegypti. (A) AeAmt1 (red) immunolocalization in a longitudinal section of a testis counterstained for nuclei (blue; DAPI). Zones of different stages of spermatogenesis, from primary spermatogonia (germ cells) to mature spermatozoa, are indicated (white labels). (B) High-magnification image of a single spermatozoon showing the nucleus (blue; DAPI) and the mitochondrial derivatives along the length of the flagellum (green; MitoTracker). (C) High-magnification image of AeAmt1 (red) localization counterstained for nuclei (blue; DAPI) in mature spermatozoa isolated from the testis and (D) bright-field image to corresponding C indicating the flagellum (black arrow), nucleus (yellow arrow), and region of the centriole adjunct (red arrow). (E) AeAmt1 (red) localization in a transverse section of spermathecae from mated female A. aegypti counterstained for nuclei (blue; DAPI) and (F) higher-magnification image of AeAmt1 (red) and nuclei (blue; DAPI) immunolocalization in spermatozoa from a single spermatheca of mated female A. aegypti from E. (G) The bright-field image corresponding to E indicating the sperm-filled spermatheca (black arrow). (H) Control slide (anti-AeAmt1 preincubated with 20x molar excess of AeAmt1-specific peptide) and (I) bright-field image corresponding to H. (Scale bars, 50 µm, unless indicated otherwise.)

Fig. 2.

Absence of AeAmt1 protein expression and localization in nonmated females and ammonium (NH₄⁺) content in spermathecae of nonmated and mated female Aedes aegypti. (A) AeAmt1 (red) immunolocalization is absent in longitudinal sections of spermathecae (white outlines) from nonmated females counterstained for nuclei (blue; DAPI). (B) Bright-field image corresponding to A. (C) AeAmt1 protein expression (Top) in isolated spermathecae from mated (m) and nonmated (n) females and Coomassie total protein staining (Bottom) using Western blotting. (D) Diluted [NH₄⁺] of pooled spermathecae (3 per animal) punctured in a 0.5 µL saline droplet from nonmated and mated female A. aegypti (Mann–Whitney U test, **P = 0.0077). Data are shown as mean ± SEM. (Scale bars, 50 µm.)

Fig. 3.

AeAmt1 protein localization in migrating spermatozoa within spermathecal ducts from blood-fed (48 h post blood meal, pbm) adult female Aedes aegypti. (A) AeAmt1 (red) immunolocalization in a longitudinal section of spermathecae (white outlines) counterstained for nuclei (DAPI, blue), (B) bright-field image corresponding to A, and (C) AeAmt1 (red) immunolocalization within spermathecal ducts containing migrating spermatozoa (white arrow) counterstained for nuclei (DAPI, blue) in a blood-fed female A. aegypti. (Scale bars, 50 µm.)

Fig. 4.

Total spermatozoa in the seminal vesicle of male Aedes aegypti following RNAi (dsRNA)-mediated knockdown of AeAmt1. (A) AeAmt1 protein abundance in male A. aegypti at 24 and 36 h postinjection with AeAmt1 (n = 3) or control β-lactamase (β-lac; n = 3) dsRNA (*P = 0.0467 for 24 h, unpaired Student’s t test, two tailed; P = 0.100 for 36 h, Mann–Whitney U test). (B) Representative Western blot images corresponding to A demonstrating AeAmt1 protein abundance (Top) and Coomassie total protein staining (Bottom) at 24 and 36 h after AeAmt1 and β-lac dsRNA injection. (C) Total spermatozoa number within the seminal vesicle of males at 24 h after AeAmt1 and β-lac dsRNA injection (*P = 0.0073; Mann–Whitney U test; each point represents an individual replicate value). (D) Representative images of immunofluorescent nuclei (grayscale, DAPI) from fixed spermatozoa in a 1 µL droplet of PBS from the seminal vesicles of AeAmt1 and β-lac dsRNA-injected males at 24 h postinjection. Data are shown as mean ± SEM.

Fig. 5.

Effects of AeAmt1 protein knockdown in males on egg laying and larval hatching (egg viability) of mated female Aedes aegypti. (A) Number of eggs laid (****P < 0.0001; Mann–Whitney U test), (B) number of larvae hatched (****P < 0.0001; Mann–Whitney U test), and (C) percentage of larval hatching (***P = 0.0006; Mann–Whitney U test) by individual females mated with males that were injected with AeAmt1 and β-lac dsRNA (n = 45 for β-lac; n = 46 for AeAmt1). Data are shown as mean ± SEM (each point represents an individual replicate value).