Active mode of excretion across digestive tissues predates the origin of excretory organs
Fig 2
(a) WMISH of rhesus, v-ATPase, nka, and hcn in I. pulchra and M. stichopi. (b) Ammonia excretion rates of I. pulchra before (Ctrl) and after exposure for 2 hours to 50, 100, 200, and 500 μM and after exposure for 7 days in 1 mM 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 (p < 0.02 in an unpaired, 2-tailed t test with unequal variance). (c) Quantitative relative expression of rhesus, nka, v-ATPase B, amts, aq, and ca after 7 days of exposure in HEA (1 mM NH4Cl). Each circle indicates the average of three independent biological replicates, each with four technical replicates. Error bars indicate minimum and maximum of the biological replicates (averaged technical replicates). A 1-fold change represents no change; ≥2 indicates significantly increased expression level; ≤0.5 indicates significantly decreased expression level (red labels). (d) Effects of different inhibitors on ammonia excretion rates in I. pulchra (boxplot, with illustration and replicates similar to Fig 2b). The concentrations used were 5 μM Con-C as a v-ATPase A/B inhibitor, 1 mM azetazolamide as an inhibitor of the CA, 1 mM quabain as an NKA inhibitor, and 2 mM colchicine for inhibiting the microtubule network. Con-C was diluted in 0.5% DMSO for which we used an appropriate Ctrl with 0.5% DMSO. (e) Protein localization of Rhesus in I. pulchra and M. stichopi. Syncytium and gut are indicated in gray, and the magenta staining of the lumen in M. stichopi is false-positive staining of the gut content. Fluorescent pictures are projections of merged confocal stacks. The nervous system is stained green with tyr tubulin. (f) Double fluorescent WMISH of v-ATPase and nka, aq c and nka, v-ATPase and aq b, and v-ATPase and rhesus in I. pulchra. White areas in the first panel are the result of merged stacks and not of overlapping expression. Nuclei are stained blue with DAPI. Anterior is to the left. Scale bars are 50 μm for I. pulchra and 100 μm for M. stichopi. Values underlying panels b and d are provided in S6 Table, and values underlying panel c are provided in S4 Table. amt, ammonia transporter; aq, aquaporin; CA, carbonic anhydrase; Con-C, concanamycin C; Ctrl, control; DAPI, 4',6-diamidino-2-phenylindole; ds, digestive syncytium; gwc, gut-wrapping cell; HCN, K+[NH4+] channel; HEA, high environmental ammonia; NKA, Na+/K+[NH4+] ATPase; Rh, Rhesus glycoprotein; slc, solute carrier transporter; tyr, tyrosinated; v-ATPase, vacuolar H+-ATPase proton pump; WMISH, whole-mount in situ hybridization.
doi: https://doi.org/10.1371/journal.pbio.3000408.g002