Active mode of excretion across digestive tissues predates the origin of excretory organs
Fig 3
(a) Ammonia excretion rates of N. vectensis before (Ctrl) and after exposure for 2 hours to 50, 100, 200, and 500 μM and after exposure for 7 days in 1mM NH4Cl (boxplot). Excretion was measured over 2 hours following the HEA treatments in at least three independent biological replicates, each divided into two separate samples (six measurements in total). Bold horizontal bars in boxes indicate the median; lower and upper box borders indicate lower and upper quartile; and whiskers indicate minimum and maximum. Asterisks label significant changes. Significance, p < 0.02 (unpaired t test with unequal variance). (b) Quantitative relative expression of rhesus, nka, v-ATPase B, amts, and ca after exposure for 7 days in HEA (1 mM NH4Cl). Each circle represents the average of five independent biological replicates, each with three technical replicates. A 1-fold change represents no change; ≥2 indicates increased expression level significantly; ≤0.5 indicates decreased expression level significantly (red labels). (c) Effects of different inhibitors on ammonia excretion rates in N. vectensis (boxplot, with illustration and replicates similar to Fig 2d). The concentrations used were 5–15 μM Con-C as a V-ATPase A/B inhibitor, 1–3 mM azetazolamide as an inhibitor of the CA, 1–5 mM quabain as an NKA inhibitor, and 2–10 mM colchicine for inhibiting the microtubule network. Quabain was diluted in 0.5% DMSO, for which we used an appropriate Ctrl with 0.5% DMSO. N = 3 for all treatments. (d) Whole-mount in situ hybridization of rh 1, rh 2, rh 3, v-ATPase, and amt1/4b in feeding primary polyps. Anterior is to the top. (e) Protein localization of Rh and v-ATPase in N. vectensis early-juvenile polyps. The muscle filaments are labeled green with phalloidin, and the nervous system is stained cyan with tyr tubulin. Every picture is a full projection of merged confocal stacks. Nuclei are stained blue with DAPI. The regions shown are indicated with dashed boxes in the illustrated animal. Values underlying panels a and c are provided in S6 Table, and values underlying panel b are provided in S4 Table. amt, ammonia transporter; CA, carbonic anhydrase; Con-C, concanamycin C; Ctrl, control; DAPI, 4',6-diamidino-2-phenylindole; ebw, endodermal body wall; HEA, high environmental ammonia; mes, mesenteries; nka, Na+/K+[NH4+] ATPase; ph, pharynx; rh, Rhesus glycoprotein; sf, septal filament; ten, tentacles; tyr, tyrosinated; v-ATPase, vacuolar H+-ATPase proton pump.
doi: https://doi.org/10.1371/journal.pbio.3000408.g003