Mechanism of ammonia excretion in the freshwater leechNephelopsis obscura: characterization of a primitive Rh protein and effects of high environmental ammonia

Abstract

Remarkably little is known about nitrogenous excretion in freshwater invertebrates. In the current study, the nitrogen excretion mechanism in the carnivorous ribbon leech, Nephelopsis obscura, was investigated. Excretion experiments showed that the ribbon leech is ammonotelic, excreting 166.0 ± 8.6 nmol·grams fresh weight (gFW)−1·h−1 ammonia and 14.7 ± 1.9 nmol·gFW−1·h−1 urea. Exposure to high and low pH hampered and enhanced, respectively, ammonia excretion rates, indicating an acid-linked ammonia trapping mechanism across the skin epithelia. Accordingly, compared with body tissues, the skin exhibited elevated mRNA expression levels of a newly identified Rhesus protein and at least in tendency the Na+/K+-ATPase. Pharmacological experiments and enzyme assays suggested an ammonia excretion mechanism that involves the V-ATPase, Na+/K+-ATPase, and carbonic anhydrase, but not necessarily a functional microtubule system. Most importantly, functional expression studies of the identified Rh protein cloned from leech skin tissue revealed an ammonia transport capability of this protein when expressed in yeast. The leech Rh-ammonia transporter (NoRhp) is a member of the primitive Rh protein family, which is a sister group to the common ancestor of vertebrate ammonia-transporting Rh proteins. Exposure to high environmental ammonia (HEA) caused a new adjustment of body ammonia, accompanied with a decrease in NoRhp and Na+/K+-ATPase mRNA levels, but unaltered ammonia excretion rates. To our knowledge, this is only the second comprehensive study regarding the ammonia excretion mechanisms in a freshwater invertebrate, but our results show that basic processes of ammonia excretion appear to also be comparable to those found in freshwater fish, suggesting an early evolution of ionoregulatory mechanisms in freshwater organisms.

Ammonia excretion in the marine polychaeteEurythoe complanata(Annelida)

“…For this model, it is assumed that NH 4 + is transported from the blood into the branchial epithelial cells in an active manner by the Na + / K + (NH 4 + )-ATPase. While no direct evidence could be provided in this study because of the lethality of the NKA inhibitor ouabain to E. complanata, this mechanism has been shown in other marine polychaetes, namely Nereis succinea and Nereis virens, but also for the ammonia transporting epithelia of many other vertebrates and invertebrates (Evans et al, 1989;Larsen et al, 2014;Mangum, 1978;Quijada-Rodriguez et al, 2015;Weihrauch et al, 1998;Mangum, 1978). Further, movement of NH 4 + into cells may also be driven by the negative intracellular potential via a basolateral localized AMT, possibly EcAMT4.…”

“…If classic branchiae are absent, ammonia is excreted through other appendices such as the anal papillae found in some aquatic insect larvae (Donini and O'Donnell, 2005;Weihrauch et al, 2011). In addition, the skin (epidermal tissue) plays an import role in the excretion process when appendices are absent, as reported for some amphibians such as the African clawed frog Xenopus laevis and the neotenous newt Necturus maculosus (Cruz et al, 2013;Fanelli and Goldstein, 1964), but also for leeches (Quijada-Rodriguez et al, 2015), planarians (Weihrauch et al, 2012) and nematodes (Adlimoghaddam et al, 2015).…”

“…The actual transepithelial ammonia excretion mechanism varies between tissues and species but usually involves a basolateral Na + /K + -ATPase (NKA), which actively transports NH 4 + in exchange for Na + ions from the body fluids into the epithelial cell (Masui et al, 2002;Quijada-Rodriguez et al, 2015;Weihrauch et al, 1998), and a Rhesus-like (Rh)-protein which mediates basolateral ammonia transport (Adlimoghaddam et al, 2015;Nakada et al, 2007;Weihrauch et al, 2009). Further evidence is available suggesting that in freshwater organisms the exit of cellular ammonia occurs via apically localized Rh-proteins, a process energized by a co-localized V-type H + -ATPase (HAT) that acidifies the apical unstirred boundary layer, thereby generating an outwardly directed partial pressure gradient for NH 3 (ΔP NH3 ).…”

Aedes aegypti Rhesus glycoproteins contribute to ammonia excretion by larval anal papillae

“…For example, one of the more completely studied Rh-50 proteins, CeRhr-1 of the nematode, can mediate ammonia transport and is expressed at the transcript level predominantly in the hypodermis, where transcript abundance increases in response to high environmental ammonia (HEA), but has yet to be localized in the hypodermis (Adlimoghaddam et al, 2015;Ji et al, 2006). Similarly, a freshwater leech Rh protein, NoRhp, was shown to transport ammonia and alter transcript abundance in response to HEA; however, the protein has not been localized in tissues (Quijada-Rodriguez et al, 2015). Utilizing the basal and apical membrane markers, NKA and VA, respectively (see Patrick et al, 2006), we can conclude that the mosquito AeRh50s are localized on the apical and basal sides of the anal papillae epithelium; however, it remains unclear whether AeRh50-1 and AeRh50-2 are expressed on opposing membranes (apical versus basal) or within the same membranes (both apical and basal), because the antisera is expected to bind both AeRh50s.…”

Ammonia excretion in mytilid mussels is facilitated by ciliary beating

“…On the basolateral side, it has been hypothesized that TAM could be transported via NKA when K + is substituted by NH 4 + , a mechanism that has received biochemical support in bivalves as well (Pagliarani et al, 2008). For example, a recent study documented ouabain-sensitive enzymatic activity in freshwater leech skin extracts that was sustained when K + was substituted for NH 4 + in the assay, and ∼35% inhibition of TAM excretion rates across isolated leech skin (however, ouabain did not affect TAM excretion rates in experiments in live, whole animals) (Quijada-Rodriguez et al, 2015). Matching a previous report about low NKA activity gills (Melzner et al, 2009), neither plicate organ nor gill in Mytilus expresses abundant NKA, as no signal was detectable by immunofluorescence in the present study.…”