Expression of aquaporin 3 in gills of the Atlantic killifish (Fundulus heteroclitus): Effects of seawater acclimation

Abstract

Estuarine fish, such as the Atlantic killifish (Fundulus heteroclitus), are constantly and rapidly exposed to changes in salinity. Although ion transport in killifish gills during acclimation to increased salinity has been studied extensively, no studies have examined the role of aquaglyceroporin 3 (AQP3), a water, glycerol, urea, and ammonia transporter, during acclimation to increased salinity in this sentinel environmental model organism. The goal of this study was to test the hypothesis that transfer from freshwater to seawater decreases AQP3 gene and protein expression in the gill of killifish. Transfer from freshwater to seawater decreased AQP3 mRNA in the gill after 1 day, but had no effect on total gill AQP3 protein abundance as determined by western blot. Quantitative confocal immunocytochemistry confirmed western blot studies that transfer from freshwater to seawater did not change total AQP3 abundance in the gill; however, immunocytochemistry revealed that the amount of AQP3 in pillar cells of secondary lamellae decreased in seawater fish, whereas the amount of AQP3 in mitochondrion rich cells (MRC) in primary filaments of the gill increased in seawater fish. This response of AQP3 expression is unique to killifish compared to other teleosts. Although the role of AQP3 in the gill of killifish has not been completely elucidated, these results suggest that AQP3 may play an important role in the ability of killifish to acclimate to increased salinity.

Figure 1. kfAQP3 mRNA expression during acclimation to seawater

Expression of kfAQP3 mRNA was determined by Q-PCR. Freshwater acclimated fish (FW) were moved to seawater (SW) and kfAQP3 mRNA was measured in gills harvested at 1h, 1 day, 2 days, 7 days, and 14 days after transfer. Data expressed as mean ± standard error of means. * p < 0.05 compared to freshwater (FW). N= 5 or 6 per group.

Figure 2. Verification of the kfAQP3 antibody

A, B: To verify the specificity of the kfAQP3 antibody, HEK293T were transfected with kfAQP3a cDNA at the concentrations indicated and kfAQP3 abundance was determined by western blot analysis. (A) Representative experiment. (B) Summary of three experiments. None of the non-specific bands were different among the different samples. * p < 0.01 compared to control. N=3 per group. C: In addition, cells were transfected with 1.0 μg of either kfAQP3, kfAQP7, or kfAQP9 and processed for western blot analysis. A protein of the right size was only identified in cells transfected with kfAQP3.

Figure 3. kfAQP3 protein abundance during acclimation to seawater

Killifish acclimated to freshwater (FW) were moved to seawater (SW) and kfAQP3 protein abundance in the gill was measured by western blot at 1h, 1 day, 2 days, 7 days, and 14 days post-transfer (a). There was no significant difference among the different treatment groups (p = 0.25). N= 8 per time point. A representative blot of kfAQP3 and β-actin, as loading control, is shown in b.

Figure 4. kfAQP3 localization in gills of fish acclimated to freshwater or seawater

Gills from killifish acclimated to either freshwater (FW) or seawater (SW) were isolated and fixed and probed with an anti-kfAQP3 antibody (green) and subsequently viewed by immunofluorescence confocal microscopy. Tissue was also labeled with an anti-Na⁺-K⁺ ATPase antibody (red) to identify MRC. (A) Representative images. Background refers to tissue probed with antibody pre-incubated with immunizing peptide. Arrowheads indicate MRC in the primary filament and arrow indicates secondary lamellae. Scale bar is equal to 50 μm. (B) Quantification of the mean fluorescence intensity of the gill tissue in FW and SW fish. (C, D) Single cell quantification of kfAQP3 in primary filament (C) and secondary lamellae (D). Data are presented as mean ± SEM of the fluorescence intensity (arbitrary units). N = 4 per treatment. * p < 0.001