Ocean acidification alters foraging behaviour in Dungeness crab through impairment of the olfactory pathway.

Crustacean olfaction is fundamental to most aspects of living and communicating in aquatic environments and more broadly, for individual- and population-level success. Accelerated ocean acidification from elevated CO2 threatens the ability of crabs to detect and respond to important olfactory-related cues. Here, we demonstrate that the ecologically and economically important Dungeness crab (Metacarcinus magister) exhibits reduced olfactory-related antennular flicking responses to a food cue when exposed to near-future CO2 levels, adding to the growing body of evidence of impaired crab behaviour. Underlying this altered behaviour, we find that crabs have lower olfactory nerve sensitivities (twofold reduction in antennular nerve activity) in response to a food cue when exposed to elevated CO2 . This suggests that near-future CO2 levels will impact the threshold of detection of food by crabs. We also show that lower olfactory nerve sensitivity in elevated CO2 is accompanied by a decrease in the olfactory sensory neuron (OSN) expression of a principal chemosensory receptor protein, ionotropic receptor 25a (IR25a) which is fundamental for odorant coding and olfactory signalling cascades. The OSNs also exhibit morphological changes in the form of decreased surface areas of their somata. This study provides the first evidence of the effects of high CO2 levels at multiple levels of biological organization in marine crabs, linking physiological and cellular changes with whole animal behavioural responses.

Ammonia transport is independent of PNH3 gradients across the gastrointestinal epithelia of the rainbow trout: A role for the stomach.

Although the gastrointestinal tract (GIT) is an important site for nitrogen metabolism in teleosts, the mechanisms of ammonia absorption and transport remain to be elucidated. Both protein catabolism in the lumen and the metabolism of the GIT tissues produce ammonia which, in part, enters the portal blood through the anterior region of the GIT. The present study examined the possible roles of different GIT sections of rainbow trout (Oncorhynchus mykiss) in transporting ammonia in its unionized gas form-NH3 -by changing the PNH3 gradient across GIT epithelia using in vitro gut sac preparations. We also surveyed messenger RNA expression patterns of three of the identified Rh proteins (Rhbg, Rhcg1, and  Rhcg2) as potential NH3 transporters and NKCC as a potential ammonium ion (NH4 + ) transporter along the GIT of rainbow trout. We found that ammonia absorption is not dependent on the PNH3 gradient despite expression of Rhbg and Rhcg2 in the intestinal tissues, and Rhcg2 in the stomach. We detected no expression of Rhbg in the stomach and no expression of Rhcg1 in any GIT tissues. There was also a lack of correlation between ammonia transport and [NH4 + ] gradient despite NKCC expression in all GIT tissues. Regardless of PNH3 gradients, the stomach showed the greatest absorption and net tissue consumption of ammonia. Overall, our findings suggest nitrogen metabolism zonation of GIT, with stomach serving as an important site for the absorption, handling and transport of ammonia that is independent of the PNH3 gradient.

Impact of heatwaves and environmental ammonia on energy metabolism, nitrogen excretion, and mRNA expression of related genes in the indicator model system Daphnia magna.

Due to increasing anthropogenic impacts, heatwaves and prolonged exposure to elevated concentrations of ammonia (HEA) may occur in aquatic environments as a single stressor or a combination thereof, potentially impacting the physiology of exposed animals. In the current study, common water fleas Daphnia magna were exposed for one week to either a 5°C increase in temperature, an increase of 300 µmol l-1 total environmental ammonia, or to both of these stressors simultaneously. Exposure to elevated temperature caused a decrease in MO2, ammonia excretion rates, a downregulation of mRNA coding for key Krebs cycle enzymes and the energy consuming Na+/K+-ATPase and V-type H+-ATPase, as well as the energy distributing crustacean hyperglycemic hormone Rh-protein. High environmental ammonia inflicted a lesser inhibitory effect on the energy metabolism of Daphnia, but initiated ammonia detoxification processes via urea synthesis evident by elevated urea excretion rates and a mRNA upregulation of arginase. Effects observed under the combined stressors resembled largely the effects seen after acclimation to elevated temperature alone, potentially due to the animals' capability to efficiently detoxify critical ammonia loads. The observed physiological effects and potential threats of the environmental stressor are discussed in detail.

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.

Feeding through your gills and turning a toxicant into a resource: how the dogfish shark scavenges ammonia from its environment.

Nitrogen (N) appears to be a limiting dietary resource for elasmobranchs, required not only for protein growth but also for urea-based osmoregulation. Building on recent evidence that the toxicant ammonia can be taken up actively at the gills of the shark and made into the valuable osmolyte urea, we demonstrate that the uptake exhibits classic Michaelis-Menten saturation kinetics with an affinity constant (Km) of 379 µmol l-1, resulting in net N retention at environmentally realistic ammonia concentrations (100-400 µmol l-1) and net N loss through stimulated urea-N excretion at higher levels. Ammonia-N uptake rate increased or decreased with alterations in seawater pH, but the changes were much less than predicted by the associated changes in seawater PNH3 , and more closely paralleled changes in seawater NH4+ concentration. Ammonia-N uptake rate was insensitive to amiloride (0.1 mmol l-1) or to a 10-fold elevation in seawater K+ concentration (to 100 mmol l-1), suggesting that the mechanism does not directly involve Na+ or K+ transporters, but was inhibited by blockade of glutamine synthetase, the enzyme that traps ammonia-N to fuel the ornithine-urea cycle. High seawater ammonia inhibited uptake of the ammonia analogue [14C]methylamine. The results suggest that branchial ammonia-N uptake may significantly supplement dietary N intake, amounting to about 31% of the nitrogen acquired from the diet. They further indicate the involvement of Rh glycoproteins (ammonia channels), which are expressed in dogfish gills, in normal ammonia-N uptake and retention.

Ammonia excretion in Caenorhabditis elegans: Physiological and molecular characterization of the rhr-2 knock-out mutant.

Previous studies have shown the free living soil nematode Caenorhabditis elegans (N2 strain) to be ammonotelic. Ammonia excretion was suggested to take place partially via the hypodermis, involving the Na(+)/K(+)-ATPase (NKA), V-ATPase (VAT), carbonic anhydrase, NHX-3 and a functional microtubule network and at least one Rh-like ammonia transporter RHR-1. In the current study, we show that a second Rh-protein, RHR-2, is highly expressed in the hypodermis, here also in the apical membrane of that tissue. To further characterize the role of RHR-2 in ammonia excretion, a knock-out mutant rhr-2 (ok403), further referred to as ∆rhr-2, was employed. Compared to wild-type worms (N2), this mutant showed a lower rate of ammonia excretion and a lower hypodermal H(+) excretion rate. At the same time rhr-1, nka, vat, and nhx-3 showed higher mRNA expression levels when compared to N2. Also, in contrast to N2 worms, ∆rhr-2 did not show enhanced ammonia excretion rates when exposed to a low pH environment, suggesting that RHR-2 represents the apical NH3 pathway that allows ammonia trapping via the hypodermis in N2 worms. A hypothetical model for the mechanism of hypodermal ammonia excretion is proposed on the basis of data in this and previous investigations.