Fig 1.
Traditional diffusion hypothesis, ammonia transport mechanism, and structural and functional correspondences between protonephridial and metanephridial systems.
(a) Illustrated phylogenetic relationship between Nephrozoa, Xenacoelomorpha, and non-bilaterians [13]. Excretory organs or specialized excretory cells/tissues using active transport and ultrafiltration are so far only reported in the group of Nephrozoa. (b) Cartoon depiction of the structural components of metanephridia (podocyte, duct, tubule) in comparison to protonephridia (terminal cell, duct, tubule) and summary of the expression domains of orthologous selected genes in relation to their components. (c) NH3 cellular transport. NH3 is secreted into the lumen fluid via parallel H+ and NH3 transport. This involves passive diffusion through the cell membrane (dashed lines), facilitated diffusion via the Rh, active transport via the NKA, the hyperpolarization-activated cyclic nucleotide-gated HCN, and AQ as well as the generation of H+ gradient by a v-ATPase and the CA, which transforms CO2 into H+ and HCO3−. Vesicular ammonia-trapping mechanism is also illustrated. at, active transport; AQ, aquaporin transporter; CA, carbonic anhydrase; cd2ap, CD2-associated protein; HCN, K+[NH4+] channel; NH3, ammonia; NKA, Na+/K+[NH4+] ATPase; Rh, Rhesus glycoprotein; slc, solute carrier transporter; ul, ultrafiltration; v-ATPase, vacuolar H+-ATPase proton pump; zo1, zonula occludens 1. Animal depictions are from phylopic.org and are not copyright protected (Public Domain Mark 1.0 license).
Table 1.
Summary of expression patterns of excretion-related genes in I. pulchra and M. stichopi.
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.
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.
Fig 4.
Evolution of excretory mechanisms.
Illustration of the proposed direction of fluxes in Cnidaria and Xenacoelomorpha and evolution of active ammonia transport and ultrafiltration mechanisms. Cnidaria (e.g., N. vectensis) excrete across their intestinal epithelium (and probably across the epidermis too) via diffusion, whereas in xenacoelomorphs, excretion occurs both via diffusion across the epidermis and gut-associated tissues and via active transport across gut-associated tissues. Ultrafiltration mechanism originated within Nephrozoa. cu, cuticle; me, mesoglea.