Active mode of excretion across digestive tissues predates the origin of excretory organs

Most bilaterian animals excrete toxic metabolites through specialized organs, such as nephridia and kidneys, which share morphological and functional correspondences. In contrast, excretion in non-nephrozoans is largely unknown, and therefore the reconstruction of ancestral excretory mechanisms is problematic. Here, we investigated the excretory mode of members of the Xenacoelomorpha, the sister group to Nephrozoa, and Cnidaria, the sister group to Bilateria. By combining gene expression, inhibitor experiments, and exposure to varying environmental ammonia conditions, we show that both Xenacoelomorpha and Cnidaria are able to excrete across digestive-associated tissues. However, although the cnidarian Nematostella vectensis seems to use diffusion as its main excretory mode, the two xenacoelomorphs use both active transport and diffusion mechanisms. Based on these results, we propose that digestive-associated tissues functioned as excretory sites before the evolution of specialized organs in nephrozoans. We conclude that the emergence of a compact, multiple-layered bilaterian body plan necessitated the evolution of active transport mechanisms, which were later recruited into the specialized excretory organs.

The majority of genes encoding aquaporins were labeling digestive-associated domains and was expressed in the brain (Fig. 6A) and aq e was labeling parenchymal cells (Fig. 6G). In M. was expressed in cells of the anterior tip and the ventral epidermis (Fig. 7E).

3 6
The broad expression of the ammonia excretion-related genes and aquaporins shows that To reveal the excretory mechanism in Xenacoelomorphs, we conducted high environmental 2 6 2 ammonia (HEA) incubation experiments, as previously performed in a large array of animals 2 6 3 (summarized in 5,51 ), using I. pulchra because of its availability in sufficient numbers. We first 2 6 4 measured the pH of incubation mediums with different HEA concentrations (up to 1 mM) 2 6 5 and found no difference in pH, which could otherwise have influenced any excretion rates.

6 6
We then exposed animals to different HEA concentrations for a short period (2 hours) and 2 6 7 measured the ammonia excretion during the following two hours after bringing them back 2 6 8 into normal conditions, to test excretion via diffusion. The ammonia excretion rates of 2 6 9 exposed animals remained unchanged after exposure to 50 and 100 µM NH 4 Cl, compared to release in normal conditions. These results suggest that ammonia excretion is concentration- dependent, which is indicative of a diffusion mechanism. To test for a possible involvement of the excretion-related genes in the excretory mechanism 2 7 8 of xenacoelomorphs, we tested for alteration of mRNA expression levels in chronically HEA 2 7 9 exposed animals by quantitative relative expression experiments (qPCR) in I. pulchra. We 2 8 0 first exposed animals to 1mM HEA for 7 days, similar to conditions used in previous studies 2 8 1 (summarized in 5,51 ), and measured the ammonia excretion over 2 hours after bringing the 2 8 2 animals into normal conditions. As expected, the ammonia excretion rates were strongly putative role of these genes in ammonia excretion and suggest that acoels might not only excrete by diffusion and via passive transporters (rhesus, amts), but also by an alternative 2 9 0 active transport mechanism (nka). Inhibitor experiments support an active excretion mechanism via NKA transporter as well as a possible involvement of a vesicular transport mechanism, by conducting 2 9 6 pharmacological inhibitor assays in I. pulchra, as previously demonstrated in other animals 2 9 7 (summarized in 5,51 ). Inhibition of the CA by Azetazolamide, did not show any significant 2 9 8 change in ammonia excretion. Inhibition of the V-ATPase by Concanamycin C seemed to 2 9 9 lead to a decrease in ammonia excretion, although a 2-tailed t-test did not support a 3 0 0 significant change. In contrast, when perturbing the function of NKA with Quabain, the 3 0 1 ammonia excretion dropped significantly (Fig. 8c), which further support an active excretion 3 0 2 mechanism via NKA, similar to what is described for many nephrozoans [10][11][12][13]16,18,19,[24][25][26]30,52- Rhesus protein localization by immunohistochemistry (Fig. 8d). The protein localization (boxplot). Excretion was measured over two hours following the HEA treatments in at least three 3 2 7 independent biological replicates, each divided into two separate samples (six measurements in total). indicates the average of three independent biological replicates, each with four technical replicates.

3
Error bars indicate minimum and maximum of the biological replicates (averaged technical replicates). and nka, aquaporin c and nka, v-ATPase and aquaporin b and v-ATPase and rhesus in I. pulchra.

4 5
White areas in the first panel are the result of merged stacks and not of overlapping expression.

4 6
Nuclei are stained blue with 4',6-diamidino-2-phenylindole (DAPI). Anterior is to the left. Abbreviations: To obtain a better resolution and understanding of the relative topology of the differentially  gut-affiliated cells. Overall, these data revealed a similar spatial arrangement in gut-3 6 7 associated domains in both animals, which seems to be unrelated to the presence of an 3 6 8 epithelial gut or a syncytium.

6 9
Taken together, our findings suggest that I. pulchra uses different mechanisms for ammonia 3 7 0 excretion that are also known from nephrozoans; an active ammonia excretion mechanism 3 7 1 via NKA through the digestive system, as suggested by in situ hybridization, and a passive 3 7 2 vesicular transport mechanism likely mediated by Rhesus through digestive and likely also animals, these excretory mechanisms could be plesiomorphic for Xenacoelomorpha. across digestive tissues outside Nephrozoa, we also investigated a non-bilaterian species, the 3 8 0 cnidarian Nematostella vectensis ( Supplementary Fig. 1b), to test whether this excretion shown that the gastrodermis seems to be involved in osmoregulation 57 . Moreover, there is but localization studies that would suggest excretion sites are missing.
3 8 8 We first tested whether N. vectensis excretes via diffusion by exposing early juvenile animals 3 8 9 to HEA for 2 hours and measuring their ammonia excretion rates afterwards, similar to the 3 9 0 experiments performed with I. pulchra (Fig. 9a). We found that also in N. vectensis ammonia when the medium contained 500 µM NH 4 Cl. However, when we measured the excretion of 3 9 4 animals over 2 hours in a medium with an accordingly lowered pH we found that a difference 3 9 5 of 0.2 did not change the excretion rates (Supplementary Table 2). Therefore, the increase in concentration-dependent, supporting a diffusion mechanism also in N. vectensis. We then exposed animals to 1mM HEA for 7 days and tested the expression of the qPCR. As expected from the short-term HEA exposure experiment, specimens exposed for 7 4 0 4 days in HEA condition showed increased ammonia excretion rates (Fig. 9a). Also, the altered significantly (Fig. 9b). In contrast to I. pulchra, none of the two nka transporters Contrary to the results from acoels, we found that inhibition of vesicular transport did not 4 1 0 alter the ammonia excretion (Fig. 9e). We also inhibited the excretory function of v-ATPase 4 1 1 and CA proteins and found that none of them showed any significant change in ammonia 4 1 2 19 excretion rates (Fig. 9e). Finally, when we perturbed the function of NKA the ammonia 4 1 3 excretion rates did not alter (Fig. 9e), confirming the qPCR results and further supporting the 4 1 4 non-involvement of this transporter in excretion (Fig. 9a). These results suggest that the 4 1 5 ammonia excretion of N. vectensis is likely mediated via the passive Rhesus and AMT 4 1 6 transporters but neither relies on active transport mechanism mediated by NKA or on 4 1 7 vesicular ammonia-trapping excretion mode (Fig. 9e). To understand whether these genes were expressed in gastrodermal or epidermal cells, we  surface, which resembled gland cells 59 (Fig. 9d). The NKA antibody was localized in represents the average of five independent biological replicates, each with three technical replicates. replicates similar to Figure 5a). The concentrations used were 5-15 µM Concanamycin C as a v- ATPase A/B inhibitor, 1-3 mM Azetazolamide as an inhibitor of the CA, 1-5 mM Quabain as a NKA 4 5 8 inhibitor and 2-10 mM Colchicine for inhibiting the microtubule network. Quabain was diluted in 0,5% 4 5 9 DMSO for which we used an appropriate control with 0,5% DMSO. N=3 for all treatments. Abbreviations: ctrl, control; ebw, endodermal body wall; mes, mesenteries; ph, pharynx; sf, septal 4 6 1 filament; ten, tentacles. Overall, our findings show that acoelomorphs use, in addition to diffusion, also active 4 6 5 transport mechanisms, in contrast to what has been previously assumed for non- excretory sites but we were not able to detect any active transport mechanism. However, we mechanism likely reflects an ancient mechanism, before the evolution of specialized organs, apparatus' (Fig. 10). To conclude, our study shows that active transport mechanism and excretion through 4 8 9 digestive tissues predates the evolution of specialized excretory systems. If this is based on a 4 9 0 convergent recruitment, or if it reflects an ancestral state for Bilateria remains unclear.

9 1
However, if the latter is true, it correlates with the emergence of multilayered body plans and 4 9 2 solid internal parenchymes that separate the body wall from their digestive tract, as seen in 4 9 3 xenacoelomorphs and nephrozoans. We thus propose that diffusion mechanisms were the and via active transport across gut-associated tissues. Ultrafiltration mechanism originated within 5 0 5 Nephrozoa. Abbreviations: m, mesoglea; c, cuticle. Gene cloning and orthology assignment 5 1 5 Putative orthologous sequences of genes of interest were identified by tBLASTx search PCR using gene specific primers. PCR products were purified and cloned into a pGEM-T 5 3 8 Easy vector (Promega, USA) according to the manufacturer' s instruction and the identity of Adult specimens of Isodiametra pulchra (Smith & Bush, 1991), Meara stichopi Westblad, Animals were manually collected, fixed and processed for in situ hybridization as Situ Hybridization (WMISH) was performed according to the protocol outlined in 73 . For N.

7
vectensis, we followed the protocol described by 75 . Double Fluorescent In Situ Hybridization 5 5 8 (FISH) was performed as the colorimetric WMISH with the following modifications: After Animals were collected manually, fixed in 4% paraformaldehyde in SW for 50 minutes, washed 3 times in PBT and incubated in 4% sheep serum in PBT for 30 min. The animals