Hemolymph Ammonia Research Articles

Dietary N-carbamylglutamate (NCG) supplementation improves the survival, digestive and immunological parameters by promoting ammonia excretion in Chinese mitten crab (Eriocheir sinensis), under ammonia nitrogen stress

Ammonia nitrogen is one of the most common environmental stress factors in aquaculture. The aim of this experiment was to evaluate the effects of ammonia nitrogen stress on ammonia metabolism, digestion and immune function as well as the protective effects of dietary N-carbamylglutamate (NCG) in the Chinese mitten crab (Eriocheir sinensis). Two feeds were used, namely a control feed (regular commercial feed) and an NCG feed (NCG (302.96 ± 4.07) mg/kg added). Subsequently, animals fed by these two feeds were exposed to 30 mg/L or 60 mg/L ammonia nitrogen stress (named A30 or A60) for 21 days, respectively. The rate of hemolymph ammonia excretion was significantly increased in NCG group compared to C (control) group (P < 0.05). Hemolymph ammonia levels were significantly lower in NCG A30 (NCG feed and A30) and NCG A60 (NCG feed and A60) groups compared to A30 (control feed and A30) and A60 (control feed and A60) groups (P < 0.05). Glutamate dehydrogenase and glutamine synthetase activities in the gills and hepatopancreas of NCG A60 group were significantly increased compared with the A60 group, which promoted glutamine synthesis (P < 0.05). The cumulative mortality rate of Eriocheir sinensis in NCG A30 and NCG A60 groups were effectively reduced (P < 0.05). The arginine levels of hepatopancreas were significantly increased in NCG A60 group (P < 0.05). Under ammonia nitrogen stress, NCG significantly increased the activities of trypsin, pepsin, lipase and α-Amylase in NCG feed group, and promoted feeding (P < 0.05). In hemolymph, hepatopancreas and intestine, NCG significantly increased nitric oxide synthase activity, lysozyme and phenol oxidase activities compared to the ammonia nitrogen stress group (P < 0.05). The results indicated that, NCG added in feed could decelerate the toxic effects of ammonia on Eriocheir sinensis by promoting ammonia excretion and accelerating glutamine and endogenous arginine synthesis by metabolism. Moreover, the activity of digestive enzymes and immune-related enzymes is improved.

Mechanisms of ammonotelism, epithelium damage, cellular apoptosis, and proliferation in gill of Litopenaeus vannamei under NH4Cl exposure.

Excessive ammonia-N in coastal environment and aquaculture threatens the health of marine organisms. To explore the mechanism of gill damage induced by ammonia-N, transcriptome of Litopenaeus vannamei 's gill was carried out under 20 mg/L NH4Cl for 0, 6, and 48 h. K-means clustering analysis suggested that ammonia excretion and metabolism-related genes were elevated. GO and KEGG enrichment analysis suggested that glycosyltransferase activity and amino acid metabolism were affected by ammonia. Moreover, histological observation via three staining methods gave clues on the changes of gill after ammonia-N exposure. Increased mucus, hemocyte infiltration, and lifting of the lamellar epithelium suggested that gill epithelium was suffering damage under ammonia-N stress. Meanwhile, the composition of extracellular matrix (ECM) in connective tissue changed. Based on the findings of transcriptomic and histological analysis, we further investigated the molecular mechanism of gill damage under multiple concentrations of NH4Cl (0, 2, 10, 20 mg/L) for multiple timepoints (0, 3, 6, 12, 24, 48, 72 h). First, ammonia excretion was elevated via ion channel, transporter, and exocytosis pathways, but hemolymph ammonia still kept ata high level under 20 mg/L NH4Cl exposure. Second, we focused on glycosaminoglycan metabolism which was related to the dynamics of ECM. It turned out that the degradation and biosynthesis of chondroitin sulfate (CS) were elevated, suggesting that the structure of CS might be destructed under ammonia-N stress and CS played an important role in maintaining gill structure. It was enlightening that the destructions occurred in extracellular regions were vital to gill damage. Third, ammonia-N stress induced a series of cellular responses including enhanced apoptosis, active inflammation, and inhibited proliferation which were closely linked and jointly led to the impairment of gill. Our results provided some insights into the physiological changes induced by ammonia-N and enriched the understandings of gill damage under environmental stress.

Effects of Ambient Ammonia‐Nitrogen Exposure on Uric Acid and Urea Metabolic Pathways and Tissue Distribution in the Swimming Crab Portunus trituberculatus

AbstractAmmonia‐nitrogen (ammonia‐N) is an important threat factor to the development of the crustacean breeding industry; however, information on nitrogen metabolism remains limited. In this study, effects of ammonia‐N on the metabolic pathways and tissue distribution of nitrogenous wastes were investigated in swimming crabs Portunus trituberculatus. The results showed that ammonia content in the hemolymph of crabs exposed to 1‐ and 5‐mg/L NH4Cl (ammonia‐N concentrations were 1.36 and 5.31 mg/L, respectively) significantly (P &lt; 0.05) increased with time. The expression of xanthine oxidase was significantly up‐regulated in gills and muscles but was significantly down‐regulated in the hepatopancreas of crabs exposed to 1‐ and 5‐mg/L NH4Cl after 6–12 h. The expression of uricase was significantly up‐regulated in gills and muscles at 3 h, whereas it was significantly down‐regulated in hepatopancreas during 6–12 h but up‐regulated again after 24 h. Moreover, the expression levels of carbamoyl phosphate synthetase III and arginase were significantly up‐regulated in gills and muscles but significantly down‐regulated in the hepatopancreas of crabs exposed to 5‐mg/L NH4Cl after 6–12 h. Furthermore, under 5‐mg/L NH4Cl stress, trace amounts of uric acid were detected, which significantly increased in hepatopancreas and hemolymph at 3 h, in muscles at 6–12 h, and in gills at 6–24 h. In addition, similar to hemolymph ammonia, urea was also significantly increased after NH4Cl exposure. Moreover, uric acid was mainly stored in muscles and urea was primarily synthesized in the hepatopancreas. This study provides a theoretical basis for further study on the nitrogen metabolism mechanism in crustaceans.

Expression and Functional Analysis of AMT1 Gene Responding to High Ammonia Stress in Razor Clam (Sinonovacula constricta).

Ammonium transporter 1 (AMT1), a member of ammonia (NH3/NH4+) transport proteins, has been found to have ammonia transport activity in plants and microorganisms. However, the functional characteristics and molecular mechanisms of AMT1 in mollusks remain unclear. The razor clam (Sinonovacula constricta) is a suitable model species to explore the molecular mechanism of ammonia excretion because of the high concentration of ambient ammonia it is exposed to in the clam-fish-shrimp polyculture system. Here, the expression of AMT1 in S. constricta (Sc-AMT1) in response to high ammonia (12.85 mmol/L NH4Cl) stress was identified by real-time quantitative PCR (qRT-PCR), Western blotting, RNA interference, and immunofluorescence analysis. Additionally, the association between the SNP_g.15211125A > T linked with Sc-AMT1 and ammonia tolerance was validated by kompetitive allele-specific PCR (KASP). A significant upregulated expression of Sc-AMT1 was observed during ammonia exposure, and Sc-AMT1 was found to be localized in the flat cells of gill. Moreover, the interference with Sc-AMT1 significantly upregulated the hemolymph ammonia levels, accompanied by the increased mRNA expression of Rhesus glycoprotein (Rh). Taken together, our findings imply that AMT1 may be a primary contributor to ammonia excretion in S. constricta, which is the basis of their ability to inhabit benthic water with high ammonia levels.

Effects of metamifop on ammonia production and metabolism of Monopterus albus

The use of herbicides is believed to have an impact on the metabolism, physiology and biochemistry of fish. In this study, we studied the effects of metamifop on the production and metabolism of Monopterus. albus living in the water. According to the semi-lethal concentration of metamifop for 96 h, four MET concentration groups (0.2-, 0.4-, 0.6- and 0.8 mg L−1) were set up for 96 h exposure test. The ammonia discharge rate decreased, hemolymph ammonia content increased significantly, and hemolymph urea nitrogen content decreased at all time periods of metamifop exposure. In liver, the protein content decreased, the neutral protease content increased significantly (p < 0.01), amino acid content increased, and ATP content increased significantly (p < 0.01). In brain, the protein content increased, the activity of acid protease, neutral protease and alkaline protease all decreased, amino acid content decreased significantly (p < 0.01), and the content of ATP decreased. Glutamic-pyruvic transaminase (GPT) activity did not change in liver but decreased in brain. Glutamine synthetase (GS) activity decreased in liver and increased in brain. Glutaminase (GLS) activity decreased in liver and increased in brain. In conclusion, the liver and brain tissues of M. albus react differently to MET exposure. The liver mainly synthesizes energy through hydrolyzed protein, while the brain mainly synthesizes protein. Amino acids produced by protein hydrolysis cannot be converted to alanine for storage, and the degraded amino acids lead to the elevation of endogenous ammonia. MET inhibits the removal of ammonia from M. albus. Only liver tissue can detoxify the eel by converting ammonia into glutamine. Brain should have to tolerate high levels of endogenous ammonia.

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.

Effects of microbe-derived antioxidants on growth, digestive and aminotransferase activities, and antioxidant capacities in the hepatopancreas of Eriocheir sinensis under ammonia nitrogen stress

This study evaluated the effects of feeding a microbial-derived antioxidant diet (MA) for 56 days on the growth performance of Eriocheir sinensis, as well as on digestive and metabolic enzyme activity and antioxidant capacity under ammonia stress (400 mg/L ammonia nitrogen for 48 h). The weight gain at 28d and the specific growth rate at 56ds for crabs in the MA group were significantly higher than those in the control diet (C) group (p < 0.05). Compared with the C group, the color of the hepatopancreas in the MA group was significantly improved (p < 0.05). A significant decrease in the level of Hemolymph ammonia was found in the microbial antioxidant ammonia-N (MA_A) group (p < 0.05), which may be related to the decrease of transaminase (AST, ALT, GDH) activities caused by MA diet (p < 0.05). There is a significant increase in the digestive enzyme activities (Trypsin and Lipase), which reduced the accumulation of triglycerides in the hepatopancreas in the (MA_A) group (p < 0.05). In addition, the antioxidant damage of crabs in hepatopancreas caused by ammonia nitrogen toxicity could also significantly be improved by MA fed in the (MA_A) group (p < 0.05). microbe-derived antioxidants (supplemented with 0.2% in feeds) can protect the health of hepatopancreas, promote growth, and improve the ability to resist ammonia nitrogen stress for E.sinensis.

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.

Physiological and Molecular Responses in the Gill of the Swimming Crab Portunus trituberculatus During Long-Term Ammonia Stress

Ammonia is a common environmental stressor encountered during aquaculture, and is a significant concern due to its adverse biological effects on vertebrate and invertebrate including crustaceans. However, little information is available on physiological and molecular responses in crustaceans under long-term ammonia exposure, which often occurs in aquaculture practices. Here, we investigated temporal physiological and molecular responses in the gills, the main ammonia excretion organ, of the swimming crab Portunus trituberculatus following long-term (4 weeks) exposure to three different ammonia nitrogen concentrations (2, 4, and 8 mg l–1), in comparison to seawater (ammonia nitrogen below 0.03 mg l–1). The results revealed that after ammonia stress, the ammonia excretion and detoxification pathways were initially up-regulated. These processes appear compromised as the exposure duration extended, leading to accumulation of hemolymph ammonia, which coincided with the reduction of adenosine 5′-triphosphate (ATP) and adenylate energy charge (AEC). Considering that ammonia excretion and detoxification are highly energy-consuming, the depression of these pathways are, at least partly, associated with disruption of energy homeostasis in gills after prolonged ammonia exposure. Furthermore, our results indicated that long-term ammonia exposure can impair the antioxidant defense and result in increased lipid peroxidation, as well as induce endoplasmic reticulum stress, which in turn lead to apoptosis through p53-bax pathway in gills of the swimming crab. The findings of the present study further our understanding of adverse effects and underlying mechanisms of long-term ammonia in decapods, and provide valuable information for aquaculture management of P. trituberculatus.

Effects of emersion on acid-base regulation, osmoregulation, and nitrogen physiology in the semi-terrestrial mangrove crab, Helice formosensis.

Emersion limits water availability and impairs the gill function of water-breathing animals resulting in a reduced capacity to regulate respiratory gas exchange, acid-base balance, and nitrogenous waste excretion. Semi-terrestrial crustaceans such as Helice formosensis mitigate these physiological consequences by modifying and recycling urine and branchial water shifting some branchial workload to the antennal glands. To investigate how this process occurs, Helice formosensis were emersed for up to 160 h and their hemolymph and urinary acid-base, nitrogenous waste, free amino acids, and osmoregulatory parameters wereinvestigated. Upon emersion, crabs experienced a respiratory acidosis that is restored by bicarbonate accumulation and ammonia reduction within the hemolymph and urine after 24h. Prolonged emersion caused an overcompensatory metabolic alkalosis potentially limiting the crab's ability to remain emersed. During the alkalosis, hemolymph ammonia was maintained at control levels while urinary ammonia remained reduced by 60% of control values. During emersion, ammonia may be temporarily converted to alanine as part of the Cahill cycle until re-immersion where crabs can revert alanine to ammonia for excretion coinciding with the crabs' observed delayed ammonia excretion response. The presence of high hemolymph alanine concentrations even when immersed may indicate this cycle's use outside of emersion or in preparation for emersion. Furthermore, H. formosensis appears to be uniquely capable of actively suppressing its rate of desiccation in absence of behavioral changes, in part by creating hyperosmotic urine that mitigates evaporative water loss.

Crustacean hyperglycemic hormone (CHH) regulates the ammonia excretion and metabolism in white shrimp, Litopenaeus vannamei under ammonia-N stress

To understand the adaptation of Litopenaeus vannamei to high environmental ammonia-N, RNA interference was used to investigate the function of crustacean hyperglycemic hormone (CHH) in the physiological process of neuroendocrine signaling transduction, and ammonia excretion and metabolism. The shrimp were exposed to 25 mg/L NH4Cl and injected with 20 μg/shrimp CHH dsRNA for 72 h. The results showed that hemolymph ammonia content increased under ammonia-N stress and further increased after CHH knockdown, suggesting that CHH can promote ammonia excretion. Moreover, after CHH knockdown, the levels of CHH, DA, and Wnts decreased significantly, the expression of receptor GC, DA1R, Frizzled and LRP 5/6 also decreased, while DA4R increased remarkably. Moreover, PKA and PKG decreased, while PKC markedly increased, and nuclear transcription factors (CREB and TCF) as well as effector proteins (β-catenin, FXYD2, and 14–3-3) were significantly downregulated. Furthermore, ammonia transporters Na+/K+-ATPase (NKA), K+channel, Rh protein, AQP, V-ATPase, and VAMP decreased significantly, while Na+/H+ exchangers (NHE) and Na+/K+/2Cl− cotransporter (NKCC) increased significantly. These results suggest that CHH regulates ammonia excretion in three ways: 1) by mainly regulating ion channels via PKA, PKC, and PKG signaling pathways; 2) by activating related proteins primarily through Wnt signaling pathway; and 3) by exocytosis, mostly induced by the PKA signaling pathway. In addition, the levels of Gln, uric acid, and urea increased in accordance with the activities of GDH/GS, XDH, and arginase, respectively, suggesting that ammonia excretion was inhibited but ammonia metabolism was slightly enhanced. This study deepens our understanding of the mechanism by which crustaceans respond to high environmental ammonia-N.

Ammonia-N exposure alters neurohormone levels in the hemolymph and mRNA abundance of neurohormone receptors and associated downstream factors in the gills of Litopenaeus vannamei.

Effects of ammonia-N (0.05, 2, 10 and 20 mg l-1) on the neuroendocrine regulation of ammonia transport were investigated in Litopenaeus vannamei The results showed that corticotrophin-releasing hormone, adrenocorticotropic hormone, dopamine, noradrenaline and 5-hydroxytryptamine concentrations in all ammonia-N groups increased significantly between 3 and 12 h. Cortisol increased significantly between 3 and 24 h. All hormones except crustacean hyperglycemic hormone were reduced to control levels. mRNA abundance of guanylyl cyclase increased significantly during the experiment. Dopamine receptor D4 and α2 adrenergic receptor mRNA abundance in treatments decreased significantly at the beginning, and eventually returned to the control level, whereas mRNA abundance of the 5-HT7 receptor increased significantly only within the first 12 h. Changes in protein kinase (PKA, PKG) mRNA abundance were similar to the patterns of biogenic amines and crustacean hyperglycemic hormone, peaking at 6 and 12 h, respectively, whereas PKC mRNA abundance decreased within 24 h. 14-3-3 protein, FXYD2 and cAMP-response element binding protein mRNA abundance increased significantly and peaked at 6 h. β-catenin and T-cell factor mRNA abundance increased significantly throughout the experiment and peaked at 12 h. The upregulation of Rh protein, K+ channel, Na+/K+-ATPase, V-type H+-ATPase and vesicle associated membrane protein (VAMP) mRNA, together with downregulation of Na+/K+/2Cl- cotransporter mRNA, indicated an adjustment of general branchial ion-/ammonia-regulatory mechanisms. Meanwhile, hemolymph ammonia concentration was significantly increased in most ammonia-N exposure groups. Histological investigation revealed the hepatopancreatic damage caused by ammonia-N. Results suggest that hormones, biogenic amines and Wnt/β-catenin play a principal role in adapting to ammonia-N exposure and facilitating ammonia transport.

Evidence that dopamine is involved in neuroendocrine regulation, gill intracellular signaling pathways and ion regulation in Litopenaeus vannamei.

The transport of ions and ammonia in gills may be regulated by neuroendocrine factors. In order to explore the mechanism of dopamine (DA) regulation, we investigated hemolymph neuroendocrine hormones, gill intracellular signaling pathways, ion and ammonia transporters, hemolymph osmolality and ammonia concentration in Litopenaeus vannamei after injection of 10-7 and 10-6mol DA per shrimp. The data showed a significant increase in crustacean hyperglycemic hormone (CHH) concentration at 1-12 h and a transient significant decrease in corticotrophin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and cortisol concentration under DA stimulation. The up-regulation of guanylyl cyclase (GC) mRNA, cyclic guanosine monophosphate (cGMP) and protein kinase G (PKG) concentration, together with the down-regulation of DA receptor D4 mRNA and up-regulation of cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), diacylglycerol (DAG) and protein kinase C (PKC) concentration suggested the activation of complicated intracellular signaling pathways. The expression of cAMP response element-binding protein (CREB), FXYD2 and 14-3-3 protein mRNA was significantly increased by PKA regulation. The increase in Na+/K+-ATPase (NKA) activity and the stabilization of V-type H+-ATPase (V-rATPase) activity were accompanied by an up-regulation of K+ channel, Na+/K+/2Cl- cotransporter (NKCC), Rh protein and vesicle associated membrane protein (VAMP) mRNA, resulting in an increase in hemolymph osmolality and a decrease in hemolymph ammonia concentration. These results suggest that DA stimulates the secretion of CHH and inhibits the release of cortisol, which activates intracellular signaling factors to facilitate ion and ammonia transport across the gills, and may not affect intracellular acidification.

Levels of Metabolic Enzymes and Nitrogenous Compounds in the Swimming Crab Portunus trituberculatus Exposed to Elevated Ambient Ammonia-N

This study investigated the effects of ammonia-N (control and 1 and 5 mg L−1) on the changes of metabolic enzymes activities and nitrogenous compounds levels in the gills, hepatopancreas, muscles, and hemolymph of the swimming crab Portunus trituberculatus. Results showed that the levels of hemolymph ammonia increased significantly at 6 h and then decreased to control level at 12 h. The activities of key enzymes involved in biosynthesis pathways of glutamine (Gln) and urea, such as glutamate dehydrogenase (GDH), glutamine synthetase (GS), and arginase (ARG), increased remarkably in different tissues of P. trituberculatus. Gln level was the highest in muscles, while urea content was the largest in hepatopancreas. The mRNA expression level of xanthine dehydrogenase (XDH) was downregulated sharply in gills but upregulated in the hepatopancreas and muscles. A small amount of uric acid was detected in the hemolymph. This result suggests that Gln in muscles may be used as an intermediary nitrogen repository, while urea may be primarily synthesized in the hepatopancreas and then transferred to other tissues via the hemolymph. Uric acid could be synthesized by the anabolism of purine nucleotides, in additon to the inhibitory activities of gills. In addition, the levels of Gln, urea and uric acid exhibited dose- and time-dependent effects with ammonia content. Therefore, P. trituberculatus could possibly convert ammonia into Gln, urea, or uric acid to detoxify ammonia during high ambient ammonia-N stress.

Ammonia excretion in aquatic invertebrates: new insights and questions.

Invertebrates employ a variety of ammonia excretion strategies to facilitate their survival in diverse aquatic environments, including freshwater, seawater and the water film surrounding soil particles. Various environmental properties set innate challenges for an organism's ammonia excretory capacity. These include the availability of NaCl and the respective ion-permeability of the organism's transport epithelia, and the buffering capacity of their immediate surrounding medium. To this end, some transporters seem to be conserved in the excretory process. This includes the Na+/K+(NH4+)-ATPase (NKA), the NH3/CO2 dual gas-channel Rhesus (Rh)-proteins and novel ammonia transporters (AMTs), which have been identified in several invertebrates but appear to be absent from vertebrates. In addition, recent evidence strongly suggests that the hyperpolarization-activated cyclic nucleotide-gated K+ channel (HCN) plays a significant role in ammonia excretion and is highly conserved throughout the animal kingdom. Furthermore, microtubule-dependent vesicular excretion pathways have been found in marine and soil-dwelling species, where, unlike freshwater systems, acid-trapping of excreted ammonia is difficult or absent owing to the high environmental buffering capacity of the surroundings. Finally, although ammonia is known to be a toxic nitrogenous waste product, certain marine species readily maintain potentially toxic hemolymph ammonia as a sort of ammonia homeostasis, which suggests that ammonia is involved in physiological processes and does not exist simply for excretion. Such findings are discussed within this Commentary and are hypothesized to be involved in acid-base regulation. We also describe excretory organs and processes that are dependent on environmental constraints and indicate gaps in the current knowledge in these topics.

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 and urea excretion in the swimming crab Portunus trituberculatus exposed to elevated ambient ammonia-N

In the present study of the swimming crab Portunus trituberculatus exposed to 0, 1, and 5mgL−1 NH4Cl, the effects of ammonia exposure on ammonia and urea content in hemolymph; activity of H+-ATPase (subunit A) and Na+/K+-ATPase (α-subunit) (NKA) in gills; mRNA expression levels of the crustacean Rh-like ammonia transporter (Rh), K+ Channel, Na+/K+/2Cl− co-transporter (NKCC), Na+/H+-exchanger (NHE), urea transporter (UT) and vesicle associated membrane protein (VAMP) in gills were investigated. The ultrastructure of gills was also evaluated. All these results in this study showed a dose-dependent effect with ammonia exposure concentration. The data displayed a significant increase in hemolymph ammonia and urea concentrations under ammonia exposure. The up-regulation of Rh mRNA together with up-regulation of K+-channel mRNA, NKA activity, down-regulation of NKCC and NHE mRNA suggested a coordinated protective response to maintain a relatively low ammonia concentration in the body fluids during ambient ammonia exposure. The up-regulation of VAMP, H+-ATPase activity along with the ultrastructure of gills suggested a mechanism of exocytotic ammonia excretion that may exit in the gill of P. trituberculatus. An increased production of urea and the up-regulated expression of UT suggested that the crab can detoxify elevated ammonia levels in the body fluids into urea when pathways of ammonia excretion are decreased after long term ammonia exposure.

Effects of ammonia exposure on nitrogen metabolism in gills and hemolymph of the swimming crab Portunus trituberculatus

Abstract The effects of ammonia-N (control, 1 and 5 mg L − 1 ) on nitrogen metabolism in gills and hemolymph of the swimming crab Portunus trituberculatus were investigated. The results showed that mRNA expression levels of Rh protein in gills were induced significantly by ammonia-N exposure. The levels of glutamine (Gln) and urea increased significantly in gills and hemolymph of P. trituberculatus exposed to high ammonia-N. After high ambient ammonia-N exposure, hemolymph ammonia increased significantly at 6 h and declined to control level at 12 h. The levels of Gln and urea in gills and hemolymph were significantly higher than control group during the whole exposure period and showed dose and time dependent effects. In addition, the activities of key-related enzymes involved in biosynthetic pathways of Gln and urea such as glutamate dehydrogenase (GDH), glutamine synthetase (GS) and Arginase (ARG) increased remarkably in the present study. This suggests that P. trituberculatus possesses some ammonia-detoxification strategies to cope with high ambient ammonia, including an up-regulation of Rh protein in gills and conversion ammonia to Gln and urea. The levels of Gln and urea in hemolymph could be considered as suitable physical indexes to evaluate ammonia toxicity to serve anti-ammonia selective breeding in the long run.

Physiological responses to emersion in the intertidal green crab, Carcinus maenas (L.)

The green shore crab, Carcinus maenas, undergoes on average 6 h periods of emersion during each low-tide cycle during the summer months. Under those conditions, the crab is cut off from its normal water environment and is exposed to potential stress from a suite of environmental and physiological changes: dehydration, compromised gas exchange and resultant internal hypoxia and hypercapnia, thermal stress, and ammonia toxicity. This study examined the comprehensive responses of the green crab in water and to a 6 h emersion period laboratory simulation of a tidal cycle followed by a 1 h re-immersion period, measuring indicators of dehydration, hemolymph osmolality, oxygen uptake, hemolymph acid–base status, heart and ventilatory rate, and hemolymph ammonia and ammonia excretion. Green crabs showed physiological responses of varying magnitude to 6 h of emersion. Individuals were found in the field exclusively under rocks and large clumps of seaweed where temperatures were approximately half those of exposed surfaces and relative humidity was about twice as high as ambient air. During emersion, crabs lost less than 5% of their wet weight, and hemolymph osmolality did not increase significantly. Oxygen uptake continued in air at about 50% of the control, aquatic values; and the gills continued to be ventilated by the scaphognathite, albeit at lower rates. Hemolymph lactate concentrations increased, indicating a shift to a greater reliance on anaerobic metabolism to support energetic needs. A slight acidosis developed in the hemolymph after 1 h of emersion, but it did not increase thereafter. Ammonia concentrations in the hemolymph were unchanged, as ammonia was volatilized by the gills and excreted into the air as NH3 gas. These results show that the green crab copes with emersion by seeking refuge in microhabitats that mitigate the changes in the physical parameters of intertidal emersion. Physiologically, desiccation is avoided, cardio-respiratory processes are maintained at reduced levels, and hemolymph acid–base balance is minimally affected. Ammonia toxicity appears to be avoided by a shift to excreting NH3 gas directly or indirectly to air.

In vivo Metabolic and Physiological Variations at Different Time Intervals and Cyto – Architectural Analysis of WSSV Infected Litopenaeus vannamei

In vivo Metabolic and Physiological Variations at Different Time Intervals and Cyto – Architectural Analysis of WSSV Infected Litopenaeus vannamei The metabolic and physiological changes aggravated by White Spot Syndrome Virus (WSSV) in host; Litopenaeus vannamei were analyzed. A total of 19 parameters such as, total protein, total carbohydrate, total glucose, total free amino acid, total fatty acid, fructose 1, 6 diphosphatase, aldolase, glucose 6 phosphatase, glucose 6 phosphate dehydrogenase, total hemocyte count, clotting time, oxyhemocyanin, hemolymph pH, hemolymph ammonia, phenoloxidase activity, respiratory burst activity, superoxide dismutase, oxygen consumption and ammonia excretion were examined for healthy (NEG) and WSSV infected (POS) shrimps.