The role of the antennal glands and gills in acid-base regulation and ammonia excretion of a marine osmoconforming brachyuran.

The excretory mechanisms of stenohaline marine osmoconforming crabs are often compared to those of the more extensively characterized euryhaline osmoregulating crabs. These comparisons may have limitations, given that unlike euryhaline brachyurans the gills of stenohaline marine osmoconformers possess ion-leaky paracellular pathways and lack the capacity to undergo ultrastructural changes that can promote ion-transport processes in dilute media. Furthermore, the antennal glands of stenohaline marine osmoconformers are poorly characterized making it difficult to determine what role urinary processes play in excretion. In the presented study, ammonia excretory processes as well as related acid-base equivalent transport rates and mechanisms were investigated in the Dungeness crab, Metacarcinus magister - an economically valuable stenohaline marine osmoconforming crab. Isolated and perfused gills were found to predominantly eliminate ammonia through a microtubule network-dependent active NH4+ transport mechanism that is likely performed by cells lining the arterial pockets of the gill lamella where critical Na+/K+-ATPase detection was observed. The V-type H+-ATPase - a vital component to transbranchial ammonia excretion mechanisms of euryhaline crabs - was not found to contribute significantly to ammonia excretion; however, this may be due to the transporter's unexpected apical localization. Although unconnected to ammonia excretion rates, a membrane-bound isoform of carbonic anhydrase was localized to the apical and basolateral membranes of lamella suited for respiration. Urine was found to contain significantly less ammonia as well as carbonate species than the hemolymph, indicating that unlike those of some euryhaline crabs the antennal glands of the Dungeness crab reabsorb these molecules rather than eliminate them for excretion.

Short-term exposure to high pCO2 leads to decreased branchial cytochrome C oxidase activity in the presence of octopamine in a decapod.

In a recent mechanistic study, octopamine was shown to promote proton transport over the branchial epithelium in green crabs, Carcinus maenas. Here, we follow up on this finding by investigating the involvement of octopamine in an environmental and physiological context that challenges acid-base homeostasis, the response to short-term high pCO2 exposure (400 Pa) in a brackish water environment. We show that hyperregulating green crabs experienced a respiratory acidosis as early as 6 h of exposure to hypercapnia, with a rise in hemolymph pCO2 accompanied by a simultaneous drop of hemolymph pH. The slightly delayed increase in hemolymph HCO3- observed after 24 h helped to restore hemolymph pH to initial values by 48 h. Circulating levels of the biogenic amine octopamine were significantly higher in short-term high pCO2 exposed crabs compared to control crabs after 48 h. Whole animal metabolic rates, intracellular levels of octopamine and cAMP, as well as branchial mitochondrial enzyme activities for complex I + III and citrate synthase were unchanged in posterior gill #7 after 48 h of hypercapnia. However, application of octopamine in gill respirometry experiments suppressed branchial metabolic rate in posterior gills of short-term high pCO2 exposed animals. Furthermore, branchial enzyme activity of cytochrome C oxidase decreased in high pCO2 exposed crabs after 48 h. Our results indicate that hyperregulating green crabs are capable of quickly counteracting a hypercapnia-induced respiratory acidosis. The role of octopamine in the acclimation of green crabs to short-term hypercapnia seems to entail the alteration of branchial metabolic pathways, possibly targeting mitochondrial cytochrome C in the gill. Our findings help advancing our current limited understanding of endocrine components in hypercapnia acclimation. SUMMARY STATEMENT: Acid-base compensation upon short-term high pCO2 exposure in hyperregulating green crabs started after 6 h and was accomplished by 48 h with the involvement of the biogenic amine octopamine, accumulation of hemolymph HCO3-, and regulation of mitochondrial complex IV (cytochrome C oxidase).

Branchial CO2 and ammonia excretion in crustaceans: Involvement of an apical Rhesus-like glycoprotein.

AIM: To determine whether the crustacean Rh1 protein functions as a dual CO2 /ammonia transporter and investigate its role in branchial ammonia excretion and acid-base regulation. METHODS: Sequence analysis of decapod Rh1 proteins was used to determine the conservation of amino acid residues putatively involved in ammonia transport and CO2 binding in human and bacterial Rh proteins. Using the Carcinus maenas Rh1 protein (CmRh1) as a representative of decapod Rh1 proteins, we test the ammonia and CO2 transport capabilities of CmRh1 through heterologous expression in yeast and Xenopus oocytes coupled with site-directed mutagenesis. Quantitative PCR was used to assess the distribution of CmRh1 mRNA in various tissues. Western blotting was used to assess CmRh1 protein expression changes in response to high environmental ammonia and CO2 . Further, immunohistochemistry was used to assess sub-cellular localization of CmRh1 and a membrane-bound carbonic anhydrase (CmCAg). RESULTS: Sequence analysis of decapod Rh proteins revealed high conservation of several amino acid residues putatively involved in conducting ammonia transport and CO2 binding. Expression of CmRh1 in Xenopus oocytes enhanced both ammonia and CO2 transport which was nullified in CmRh1 D180N mutant oocytes. Transport of the ammonia analog methylamine by CmRh1 is dependent on both ionized and un-ionized ammonia/methylamine species. CmRh1 was co-localized with CmCAg to the apical membrane of the crustacean gill and only experienced decreased protein expression in the anterior gills when exposed to high environmental ammonia. CONCLUSION: CmRh1 is the first identified apical transporter-mediated route for ammonia and CO2 excretion in the crustacean gill. Our findings shed further light on the potential universality of dual ammonia and CO2 transport capacity of Rhesus glycoproteins in both vertebrates and invertebrates.

The role of octopamine and crustacean hyperglycemic hormone (CHH) in branchial acid-base regulation in the European green crab, Carcinus maenas.

Crustaceans' endocrinology is a vastly understudied area of research. The major focus of the studies on this topic to date has been on the molting cycle (and in particular, the role of crustacean hyperglycemic hormone (CHH)), as well as the role of other hormones in facilitating physiological phenotypic adjustments to salinity changes. Additionally, while many recent studies have been conducted on the acclimation and adaptation capacity of crustaceans to a changing environment, only few have investigated internal hormonal balance especially with respect to an endocrine response to environmental challenges. Consequently, our study aimed to identify and characterize endocrine components of acid-base regulation in the European green crab, Carcinus maenas. We show that both the biogenic amine octopamine (OCT) and the CHH are regulatory components of branchial acid-base regulation. While OCT suppressed branchial proton excretion, CHH seemed to promote it. Both hormones were also capable of enhancing branchial ammonia excretion. Furthermore, mRNA abundance for branchial receptors (OCT-R), or G-protein receptor activated soluble guanylate cyclase (sGC1b), are affected by environmental change such as elevated pCO2 (hypercapnia) and high environmental ammonia (HEA). Our findings support a role for both OCT and CHH in the general maintenance of steady-state acid-base maintenance in the gill, as well as regulating the acid-base response to environmental challenges that C. maenas encounters on a regular basis in the habitats it dwells in and more so in the future ocean.

Hiat1 as a new transporter involved in ammonia regulation.

The orphan transporter hippocampus-abundant transcript 1 (Hiat1) was first identified in the mammalian brain. Its specific substrate specificity, however, has not been investigated to date. Here, we identified and analyzed Hiat1 in a crustacean, the green crab Carcinus maenas. Our phylogenetic analysis showed that Hiat1 protein is conserved at a considerable level between mammals and this invertebrate (ca. 78% identical and conserved amino acids). Functional expression of Carcinus maenas Hiat1 in Xenopus laevis oocytes demonstrated the capability to transport ammonia (likely NH4+) in a sodium-dependent manner. Furthermore, applying quantitative polymerase chain reaction, our results indicated a physiological role for Carcinus maenas Hiat1 in ammonia homeostasis, as mRNA abundance increased in posterior gills in response to elevated circulating hemolymph ammonia upon exposure to high environmental ammonia. Its ubiquitous mRNA expression pattern also suggests an essential role in general cellular detoxification of ammonia. Overall, our results introduce a new ubiquitously expressed ammonia transporter, consequently demanding revision of our understanding of ammonia handling in key model systems from mammalian kidneys to crustacean and fish gills.

Identification of different physiological functions within the gills and epipodites of the American lobster: Differences in metabolism, transbranchial transport, and mRNA expression.

Transbranchial transport processes are responsible for the homeostatic regulation of most essential physiological functions in aquatic crustaceans. Due to their widespread use as laboratory models, brachyuran crabs are commonly used to predict how other decapod crustaceans respond to environmental stressors including ocean acidification and warming waters. Non-brachyuran species such as the economically-valuable American lobster, Homarus americanus, possess trichobranchiate gills and epipodites that are known to be anatomically distinct from the phyllobranchiate gills of brachyurans; however, studies have yet to define their potential physiological differences. Our results indicate that the pleuro-, arthro-, and podobranch gills of the lobster are functionally homogenous and similar to the respiratory gills of brachyurans as indicated by equivalent rates of H+Eq., CO2, HCO3-, and ammonia transport and mRNA expression of related transporters and enzymes. The epipodites were found to be functionally distinct, being capable of greater individual rates of H+Eq., CO2, and ammonia transport despite mRNA transcript levels of related transporters and enzymes being only a fraction found in the gills. Collectively, mathematical estimates infer that the gills are responsible for 91% of the lobster's branchial HCO3- accumulation whereas the epipodites are responsible for 66% of branchial ammonia excretion suggesting different mechanisms exist in these tissues. Furthermore, the greater metabolic rate and amino acid catabolism in the epipodites suggest that the tissue much of the CO2 and ammonia excreted by this tissue originates intracellularly rather than systemically. These results provide evidence that non-brachyuran species must be carefully compared to brachyuran models.

Characterization of two novel ammonia transporters, HIAT1α and HIAT1ß, in the American Horseshoe Crab, Limulus polyphemus.

The American horseshoe crab, Limulus polyphemus, excretes nitrogenous waste in the form of toxic ammonia across their book gills. The mechanism of this branchial excretion is yet unknown. In the current study, two isoforms of a novel ammonia transporter, LpHIAT1α and LpHIAT1ß, have been identified in L. polyphemus. Both isoforms have 12 predicted transmembrane regions and share 82.7% of amino acid identity to each other, and 77-86% amino acid homology to HIAT1 found in fish and crustaceans. In L. polyphemus, both isoforms were expressed in the gills, coxal glands, and brain. Slightly higher mRNA expression levels of LpHIAT1α were observed in the peripheral mitochondria-poor region of the gill (PMPA), central mitochondria-rich region of the gill (CMRA), and brain compared to the LpHIAT1ß isoform. A functional expression analysis of LpHIAT1α and LpHIAT1ß in Xenopus laevis oocytes resulted in a significantly lower uptake of the radiolabeled ammonia analogue 3H-methylamine when compared to controls, indicating an ammonia excretory function of the proteins. Exposure to elevated environmental ammonia (HEA, 1 mmol l-1 NH4Cl) caused an increase in mRNA expression of LpHIAT1ß in the ion-conductive ventral gill half. High mRNA expression of both isoforms in the brain, and the observation that LpHIAT1α and LpHIAT1ß likely mediate cellular ammonia excretion, suggests that these highly conserved ammonia transporters have an important housekeeping function in cellular ammonia elimination.

A Rhesus channel in the coral symbiosome membrane suggests a novel mechanism to regulate NH3 and CO2 delivery to algal symbionts.

Reef-building corals maintain an intracellular photosymbiotic association with dinoflagellate algae. As the algae are hosted inside the symbiosome, all metabolic exchanges must take place across the symbiosome membrane. Using functional studies in Xenopus oocytes, immunolocalization, and confocal Airyscan microscopy, we established that Acropora yongei Rh (ayRhp1) facilitates transmembrane NH3 and CO2 diffusion and that it is present in the symbiosome membrane. Furthermore, ayRhp1 abundance in the symbiosome membrane was highest around midday and lowest around midnight. We conclude that ayRhp1 mediates a symbiosomal NH4+-trapping mechanism that promotes nitrogen delivery to algae during the day-necessary to sustain photosynthesis-and restricts nitrogen delivery at night-to keep algae under nitrogen limitation. The role of ayRhp1-facilitated CO2 diffusion is less clear, but it may have implications for metabolic dysregulation between symbiotic partners and bleaching. This previously unknown mechanism expands our understanding of symbioses at the immediate animal-microbe interface, the symbiosome.

Postprandial nitrogen and acid-base regulation in the seawater acclimated green crab, Carcinus maenas.

The effects of feeding (meal of 3% of body mass) on acid-base and nitrogen homeostasis were investigated in the seawater acclimated green shore crab, Carcinus maenas. Feeding did not change gastric fluid pH (~pH 6); however, feeding was associated with a respiratory acidosis. Hemolymph HCO3- did not increase during this acidosis, although titratable and net acid efflux changed from an uptake to an excretion. Feeding affected the crabs' nitrogen homeostasis causing a substantial increase in hemolymph ammonia and urea concentrations after six hours. At this point, hemolymph urea accounted for ~1/3 of nitrogenous waste accumulated within the hemolymph, suggesting a potential role in ammonia detoxification. The postprandial increase in hemolymph ammonia coincided with an 18-fold increase in ammonia excretion rates that accounted for the majority of net acid excreted by the crabs. Urea excretion rates did not increase after feeding; however, branchial urease activity increased, implying that the gills may possess a mechanism to form excretable ammonia through the catabolism of urea. Our results demonstrate that despite an acidic gastric compartment, C. maenas does not experience a postprandial alkaline tide and that any feeding related acid-base challenges are primarily derived from metabolic acid production. Our findings also indicate that unlike the bicarbonate buffering acid-base compensatory response induced by hypercapnia and emersion, acid-base challenges upon feeding are compensated through changes in the excretion of acid equivalents, mainly in the form of ammonia.

Effects of single and dual-stressor elevation of environmental temperature and PCO2 on metabolism and acid-base regulation in the Louisiana red swamp crayfish, Procambarus clarkii.

Elevation of temperature and CO2 levels within the world's aquatic environments is expected to cause numerous physiological challenges to their inhabitants. While effects on marine ecosystems have been well studied, freshwater ecosystems have rarely been examined using a dual-stressor approach leaving our understanding of its inhabitants upon these challenges unclear. We aimed to identify the affects of elevated temperature and hypercapnia in isolation and in combination on the metabolic and acid-base regulatory processes of a freshwater crayfish, Procambarus clarkii. Crayfish were exposed to freshwater conditions that may be prevalent by the year 2100 and metabolic responses were determined after 14-days of exposure. In addition, changes in branchial mRNA expression of acid-base linked transporters were investigated. Interactions between exposure conditions influenced extracellular pH as well as the nitrogen physiology and routine metabolic rate of the crayfish. Crayfish exposed to individual and combined elevations in temperature and/or hypercapnia maintained an extracellular pH similar to that of control crayfish. Dual-stressor exposed crayfish seem to elevate the importance of ammonium as an excretable acid-equivalent based on an overall increase in the branchial mRNA expression of transporters related to ammonia excretion including the Na+/K+-ATPase, Rhesus-protein, and the V-type H+-ATPase. Overall, hypercapnia and dual-stressor conditions caused a metabolic depression that may have long-lasting consequences such as limited locomotion, growth, and reproduction. Future generations of crayfish given the chance to adapt over several generations may ameliorate these consequences.

Influence of ontogenetic development, temperature, and pCO2 on otolith calcium carbonate polymorph composition in sturgeons.

Changes to calcium carbonate (CaCO3) biomineralization in aquatic organisms is among the many predicted effects of climate change. Because otolith (hearing/orientation structures in fish) CaCO3 precipitation and polymorph composition are controlled by genetic and environmental factors, climate change may be predicted to affect the phenotypic plasticity of otoliths. We examined precipitation of otolith polymorphs (aragonite, vaterite, calcite) during early life history in two species of sturgeon, Lake Sturgeon, (Acipenser fulvescens) and White Sturgeon (A. transmontanus), using quantitative X-ray microdiffraction. Both species showed similar fluctuations in otolith polymorphs with a significant shift in the proportions of vaterite and aragonite in sagittal otoliths coinciding with the transition to fully exogenous feeding. We also examined the effect of the environment on otolith morphology and polymorph composition during early life history in Lake Sturgeon larvae reared in varying temperature (16/22 °C) and pCO2 (1000/2500 µatm) environments for 5 months. Fish raised in elevated temperature had significantly increased otolith size and precipitation of large single calcite crystals. Interestingly, pCO2 had no statistically significant effect on size or polymorph composition of otoliths despite blood pH exhibiting a mild alkalosis, which is contrary to what has been observed in several studies on marine fishes. These results suggest climate change may influence otolith polymorph composition during early life history in Lake Sturgeon.

Specialized adaptations allow vent-endemic crabs (Xenograpsus testudinatus) to thrive under extreme environmental hypercapnia.

Shallow hydrothermal vent environments are typically very warm and acidic due to the mixing of ambient seawater with volcanic gasses (> 92% CO2) released through the seafloor making them potential 'natural laboratories' to study long-term adaptations to extreme hypercapnic conditions. Xenograpsus testudinatus, the shallow hydrothermal vent crab, is the sole metazoan inhabitant endemic to vents surrounding Kueishantao Island, Taiwan, where it inhabits waters that are generally pH 6.50 with maximum acidities reported as pH 5.50. This study assessed the acid-base regulatory capacity and the compensatory response of X. testudinatus to investigate its remarkable physiological adaptations. Hemolymph parameters (pH, [HCO3-], [Formula: see text], [NH4+], and major ion compositions) and the whole animal's rates of oxygen consumption and ammonia excretion were measured throughout a 14-day acclimation to pH 6.5 and 5.5. Data revealed that vent crabs are exceptionally strong acid-base regulators capable of maintaining homeostatic pH against extreme hypercapnia (pH 5.50, 24.6 kPa [Formula: see text]) via HCO3-/Cl- exchange, retention and utilization of extracellular ammonia. Intact crabs as well as their isolated perfused gills maintained [Formula: see text]tensions below environmental levels suggesting the gills can excrete CO2 against a hemolymph-directed [Formula: see text] gradient. These specialized physiological mechanisms may be amongst the adaptations required by vent-endemic animals surviving in extreme conditions.

Impact of climate change on the American lobster (Homarus americanus): Physiological responses to combined exposure of elevated temperature and pCO2.

The physiological consequences of exposing marine organisms to predicted future ocean scenarios, i.e. simultaneous increase in temperature and pCO2, have only recently begun to be investigated. Adult American lobster (Homarus americanus) were exposed to either current (16 °C, 47 Pa pCO2, pH 8.10) or predicted year 2300 (20 °C, 948 Pa pCO2, pH 7.10) ocean parameters for 14-16 days prior to assessing physiological changes in their hemolymph parameters as well as whole animal ammonia excretion and resting metabolic rate. Acclimation of lobster simultaneously to elevated pCO2 and temperature induced a prolonged respiratory acidosis that was only partially compensated for via accumulation of extracellular HCO3- and ammonia. Furthermore, acclimated animals possessed significantly higher ammonia excretion and oxygen consumption rates suggesting that future ocean scenarios may increase basal energetic demands on H. americanus. Enzyme activity related to protein metabolism (glutamine dehydrogenase, alanine aminotransferase, and aspartate aminotransferase) in hepatopancreas and muscle tissue were unaltered in future ocean scenario exposed animals; however, muscular citrate synthase activity was reduced suggesting that, while protein catabolism may be unchanged, the net energetic output of muscle may be compromised in future scenarios. Overall, H. americanus acclimated to ocean conditions predicted for the year 2300 appear to be incapable of fully compensating against climate change-related acid-base challenges and experience an increase in metabolic waste excretion and oxygen consumption. Combining our study with past literature on H. americanus suggests that the whole lifecycle from larvae to adult stages is at risk of severe growth, survival and reproductive consequences due to climate change.

Comparing Electron Leak in Vertebrate Muscle Mitochondria.

Mitochondrial electron transfer for oxidative ATP regeneration is linked to reactive oxygen species (ROS) production in aerobic eukaryotic cells. Because they can contribute to signaling as well as oxidative damage in cells, these ROS have profound impact for the physiology and survival of the organism. Although mitochondria have been recognized as a potential source for ROS for about 50 years, the mechanistic understanding on molecular sites and processes has advanced recently. Most experimental approaches neglect thermal variability among species although temperature impacts mitochondrial processes significantly. Here we delineate the importance of temperature by comparing muscle mitochondrial ROS formation across species. Measuring the thermal sensitivity of respiration, electron leak rate (ROS formation), and the antioxidant capacity (measured as H2O2 consumption) in intact mitochondria of representative ectothermic and endothermic vertebrate species, our results suggest that using a common assay temperature is inappropriate for comparisons of organisms with differing body temperatures. Moreover, we propose that measuring electron leak relative to the mitochondrial antioxidant capacity (the oxidant ratio) may be superior to normalizing relative to respiration rates or mitochondrial protein for comparisons of mitochondrial metabolism of ROS across species of varying mitochondrial respiratory capacities.

Ammonia excretion and acid-base regulation in the American horseshoe crab, Limulus polyphemus.

Many studies have investigated ammonia excretion and acid-base regulation in aquatic arthropods, yet current knowledge of marine chelicerates is non-existent. In American horseshoe crabs (Limulus polyphemus), book gills bear physiologically distinct regions: dorsal and ventral half-lamellae, a central mitochondria-rich area (CMRA) and peripheral mitochondria-poor areas (PMPAs). In the present study, the CMRA and ventral half-lamella exhibited characteristics important for ammonia excretion and/or acid-base regulation, as supported by high expression levels of Rhesus-protein 1 (LpRh-1), cytoplasmic carbonic anhydrase (CA-2) and hyperpolarization-activated cyclic nucleotide-gated K+ channel (HCN) compared with the PMPA and dorsal half-lamella. The half-lamellae displayed remarkable differences; the ventral epithelium was ion-leaky whereas the dorsal counterpart possessed an exceptionally tight epithelium. LpRh-1 was more abundant than Rhesus-protein 2 (LpRh-2) in all investigated tissues, but LpRh-2 was more prevalent in the PMPA than in the CMRA. Ammonia influx associated with high ambient ammonia (HAA) treatment was counteracted by intact animals and complemented by upregulation of branchial CA-2, V-type H+-ATPase (HAT), HCN and LpRh-1 mRNA expression. The dorsal epithelium demonstrated characteristics of active ammonia excretion. However, an influx was observed across the ventral epithelium as a result of the tissue's high ion conductance, although the influx rate was not proportionately high considering the ∼3-fold inwardly directed ammonia gradient. These novel findings suggest a role for the coxal gland in excretion and in the maintenance of hemolymph ammonia regulation under HAA. Hypercapnic exposure induced compensatory respiratory acidosis and partial metabolic depression. Functional differences between the two halves of a branchial lamella may be physiologically beneficial in reducing the backflow of waste products into adjacent lamellae, especially in fluctuating environments where ammonia levels can increase.

Ammonia-independent sodium uptake mediated by Na+ channels and NHEs in the freshwater ribbon leech Nephelopsis obscura.

Freshwater organisms actively take up ions from their environment to counter diffusive ion losses due to inhabiting hypo-osmotic environments. The mechanisms behind active Na+ uptake are quite well understood in freshwater teleosts; however, the mechanisms employed by invertebrates are not. Pharmacological and molecular approaches were used to investigate Na+ uptake mechanisms and their link to ammonia excretion in the ribbon leech Nephelopsis obscura At the molecular level, we identified a Na+ channel and a Na+/H+ exchanger (NHE) in the skin of N. obscura, where the NHE was up-regulated when acclimated to extremely low [Na+] (0.05 mmol l-1, pH 5) conditions. Additionally, we found that leeches in dilute freshwater environments use both a vacuolar-type H+-ATPase (VHA)-assisted uptake via a Na+ channel and a NHE-based mechanisms for Na+ uptake. Immunolocalization of VHA and Na+/K+-ATPase (NKA) indicated at least two cell types present within leech skin, VHA+ and VHA- cells, where the VHA+ cells are probably involved in Na+ uptake. NKA was present throughout the epithelium. We also found that increasing ammonia excretion by decreasing water pH, ammonia loading leeches or exposing leeches to high environmental ammonia does not affect Na+ uptake, providing indications that an NHE-Rh metabolon is not present and that ammonia excretion and Na+ uptake are not coupled in N. obscura To our knowledge, this is the first study showing the mechanisms of Na+ uptake and their links to ammonia excretion in a freshwater invertebrate, where results suggest an ammonia-independent Na+ uptake mechanism relying on both Na+ channels and NHEs.

Ammonia excretion in the marine polychaete Eurythoe complanata (Annelida).

Ammonia is a toxic waste product from protein metabolism and needs to be either converted into less toxic molecules or, in the case of fish and aquatic invertebrates, excreted directly as is. In contrast to fish, very little is known regarding the ammonia excretion mechanism and the participating excretory organs in marine invertebrates. In the current study, ammonia excretion in the marine burrowing polychaete Eurythoe complanata was investigated. As a potential site for excretion, the 100-200 µm long, 30-50 µm wide and up to 25 µm thick dentrically branched, well ventilated and vascularized branchiae (gills) were identified. In comparison to the main body, the branchiae showed considerably higher mRNA expression levels of Na+/K+-ATPase, V-type H+-ATPase, cytoplasmic carbonic anhydrase (CA-2), a Rhesus-like protein, and three different ammonia transporters (AMTs). Experiments on the intact organism revealed that ammonia excretion did not occur via apical ammonia trapping, but was regulated by a basolateral localized V-type H+-ATPase, carbonic anhydrase and intracellular cAMP levels. Interestingly, the V-type H+-ATPase seems to play a role in ammonia retention. A 1 week exposure to 1 mmol l-1 NH4Cl (HEA) did not cause a change in ammonia excretion rates, while the three branchial expressed AMTs showed a tendency to be down-regulated. This indicates a shift of function in the branchial ammonia excretion processes under these conditions.

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

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.