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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

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.

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 NH 3 and CO 2 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 NH 4 + -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 CO 2 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.

The physiological consequences of exposing marine organisms to predicted future ocean scenarios, i.e. simultaneous increase in temperature and p CO 2 , have only recently begun to be investigated. Adult American lobster ( Homarus americanus ) were exposed to either current (16 °C, 47 Pa p CO 2 , pH 8.10) or predicted year 2300 (20 °C, 948 Pa p CO 2 , 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 p CO 2 and temperature induced a prolonged respiratory acidosis that was only partially compensated for via accumulation of extracellular HCO 3 – 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.

Dissolution of anthropogenic CO2 is chronically acidifying aquatic ecosystems. Studies indicate that ocean acidification will cause marine life, especially calcifying species, to suffer at the organism and ecosystem levels. In comparison, freshwater acidification has received less attention, rendering its consequences unclear. Here, juvenile Chinese mitten crabs, Eriocheir sinensis, were used as a crustacean model to investigate the impact of CO2-mediated freshwater acidification. Our integrative approach, investigating changes in the animal's acid–base homeostasis, metabolism, calcification, locomotory behaviour, and survival rate, indicates that this economically relevant crustacean will face energetic consequences from future freshwater acidification. These energetic trade-offs allow the animal to maintain its acid–base homeostasis at the cost of reduced metabolic activity, exoskeletal calcification, and locomotion, reducing the animal's overall fitness and increasing its mortality. Results indicate that present-day Chinese mitten crab could be heavily affected by freshwater acidification like their marine counterparts and emphasize the importance of understanding the long-term implications of freshwater acidification on species' fitness.

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 HCO

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−], $${\text{P}}_{{{\text{CO}}_{2} }}$$PCO2, [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 $${\text{P}}_{{{\text{CO}}_{2} }}$$PCO2) via HCO3−/Cl− exchange, retention and utilization of extracellular ammonia. Intact crabs as well as their isolated perfused gills maintained $${\text{P}}_{{{\text{CO}}_{2} }}$$PCO2tensions below environmental levels suggesting the gills can excrete CO2 against a hemolymph-directed $${\text{P}}_{{{\text{CO}}_{2} }}$$PCO2 gradient. These specialized physiological mechanisms may be amongst the adaptations required by vent-endemic animals surviving in extreme conditions.

Cation/proton exchangers of the cation proton antiporter 1 (CPA1) subfamily (NHEs, SLC 9) play an important role in many physiological processes, including cell volume regulation, acid–base homeostasis, and ammonia excretion. The soil nematode Caenorhabditis elegans (Maupas, 1900) (N2, 1968) expresses nine paralogues (NHX-1 to NHX-9). The current study was undertaken to investigate the role of the cation/proton exchanger in hypodermal Na+and H+fluxes, as well in ammonia excretion processes. Measurements using SIET (scanning ion-selective electrode technique) showed that the hypodermis promotes H+secretion and Na+uptake. Inhibitory effects on fluxes were observed upon application of amiloride but not EIPA, suggesting that NHXs are not involved in the transport processes. In response to stress induced by starvation or exposure to 1 mmol·L−1NH4Cl, pH 5.5, or pH 8.0, body pH stayed fairly constant, with changes in mRNA expression levels detected in intestinal NHX-2 and hypodermal NHX-3. In conclusion, the study suggest that hypodermal apically localized EIPA-sensitive Na+/H+exchangers do not likely play a role in ammonia excretion and Na+uptake in the hypodermis of C. elegans, whereas apical amiloride-sensitive Na+channels seem to be involved not just in hypodermal Na+uptake but indirectly also in NH4+and H+excretion.

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.

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.

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−1ammonia and 14.7 ± 1.9 nmol·gFW−1·h−1urea. 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.

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

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.

This chapter summarizes the current knowledge of processes involved in nitrogen excretion in aquatic crustaceans with focus on the species-rich (up to 10,000 species) infraorder Brachyura, the true crabs (Martin and Davis 2001). Besides the introduction that briefly covers pathways of the synthesis of nitrogenous waste products and the toxicity of ammonia, organs involved in the excretory processes will be introduced, such as the antennal gland and the gills. More emphasis will be given toward the gills and their capability to actively excrete ammonia in different haline species including the marine crabs Cancer pagurus and Metacarcinus magister, brackish water living Carcinus maenas, as well as freshwater dwelling Eriocheir sinensis. In more detail this chapter reviews the branchial ammonia excretion mechanisms in the green shore crab C. maenas, which is summarized in a working model at the end. Here the potential roles of the Na+/K+-ATPase, K+-channels, Rh-proteins, V-type H+-ATPase, the microtubule network, Na+/H+ exchangers (NHEs), ammonia transporters (AMTs), and aquaporins (AQPs) will be discussed. Besides cited literature, this chapter contains original data as well as cross-references to corresponding chapters within this book.

Aquatic invertebrates primarily excrete their nitrogenous waste in the form of ammonia, a highly toxic waste product of amino acid catabolism. However, very little is known about the mechanism and for that fact the tissues that are actually responsible for ammonia excretion in freshwater and soil-dwelling invertebrates. This chapter provides an overview of the current literature examining the ammonia excretion mechanisms in three phyla: Nematoda, Platyhelminthes, and Annelida. The introduction will briefly cover the challenges faced by freshwater and soil-dwelling invertebrates in terms of their ionoregulatory capabilities in addition to a general overview of nitrogenous waste excretion in aquatic invertebrates. Subsequently, nitrogenous waste excretion in the nematodes, annelids, and platyhelminthes will be discussed with an emphasis placed on the potential tissues involved in ammonia excretion, e.g., the nephridial system, epidermis, hypodermis, intestine, and the nematode-specific excretory system. This chapter will also provide an extensive review of the cutaneous ammonia excretion mechanism in two freshwater representatives of the phyla Annelida and Platyhelminthes, the ribbon leech (Nephelopsis obscura) and planarian (Schmidtea mediterranea). Additionally, the mechanisms of hypodermal ammonia excretion in the soil nematode (Caenorhabditis elegans) will be discussed in comparison with the mechanisms seen in N. obscura and S. mediterranea. Here a focus will be placed on the role of Na+/K+-ATPase, Na+/H+ exchangers (NHEs), H+-ATPase, Rhesus (Rh) proteins, and vesicular transport. With increasing studies on freshwater and soil-dwelling invertebrates, common mechanisms to that seen in more widely studied vertebrates are becoming more and more evident.

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 (