Apoptosis In Gills Research Articles

MiR-8-3p regulates the antioxidant response and apoptosis in white shrimp, Litopenaeus vannamei under ammonia-N stress

Exposure to excess ammonia-N (NH3/NH4+) in aquaculture can disrupt physiological function in shrimp leading to enhanced oxidative stress and apoptosis, but little is known concerning the post-transcriptional regulation mechanism. In this study, the first miR-200 family member in crustacean was identified and characterized from Litopenaeus vannamei (designed as Lva-miR-8-3p). Lva-miR-8-3p was highly expressed in eyestalks, brainganglion, and gills. The expression of Lva-miR-8-3p in gills significantly decreased after ammonia-N stress, and Lva-miR-8-3p was confirmed to target IKKβ 3’UTR for negatively regulating IKKβ/NF-κB pathway. Overexpression of miR-8-3p promoted the hemolymph ammonia-N accumulation, total hemocyte count (THC) decrease, and gills tissue damage, thus resulting in a decreased survival rate of ammonia-exposed shrimp. Besides, Lva-miR-8-3p silencing could enhance the antioxidant enzymes activities and reduce the oxidative damage, whereas overexpression of Lva-miR-8-3p exerted the opposite effects. Furthermore, Lva-miR-8-3p overexpression was found to aggravate ammonia-N induced apoptosis in gills. In primarily cultured hemocytes, the cell viability decreased, the ROS content and caspase-3 activity increased after agomiR-8-3p transfection, while antagomiR-8-3p transfection caused the opposite change except the cell viability. These findings indicate that Lva-miR-8-3p acts as a post-transcriptional regulator in ammonia-N induced antioxidant response and apoptosis by negatively regulating IKKβ/NF-κB pathway.

NADK-mediated proline synthesis enhances high-salinity tolerance in the razor clam

Euryhaline organisms can accumulate organic osmolytes to maintain osmotic balance between their internal and external environments. Proline is a pivotal organic small molecule and plays an important role in osmoregulation that enables marine shellfish to tolerate high-salinity conditions. During high-salinity challenge, NAD kinase (NADK) is involved in de novo synthesis of NADP(H) in living organisms, which serves as a reducing agent for the biosynthetic reactions. However, the role of shellfish NADK in proline biosynthesis remains elusive. In this study, we show the modulation of NADK on proline synthesis in the razor clam (Sinonovacula constricta) in response to osmotic stress. Under acute hypersaline conditions, gill tissues exhibited a significant increase in the expression of ScNADK. To elucidate the role of ScNADK in proline biosynthesis, we performed dsRNA interference in the expression of ScNADK in gill tissues to assess proline content and the expression levels of key enzyme genes involved in proline biosynthesis. The results indicate that the knock-down of ScNADK led to a significant decrease in proline content (P<0.01), as well as the expression levels of two proline synthetase genes P5CS and P5CR involved in the glutamate pathway. Razor clams preferred to use ornithine as substrate for proline synthesis when the glutamate pathway is blocked. Exogenous administration of proline greatly improved cell viability and mitigated cell apoptosis in gills. In conclusion, our results demonstrate the important role of ScNADK in augmenting proline production under high-salinity stress, by which the razor clam is able to accommodate salinity variations in the ecological niche.

Effects of acute hypoxia and reoxygenation on histological structure, antioxidant response, and apoptosis in razor clam Sinonovacula constricta

Hypoxia is one of the major environmental problems limiting the healthy development of intensive aquaculture. Marine benthic shellfish are encountering heightened problems related to hypoxic stress as a result of ongoing human activities and aquaculture operations. Razor clam Sinonovacula constricta, a commercially valuable shellfish, has not yet been reported in studies on physiological changes caused by hypoxia and reoxygenation. To understand the negative effects of hypoxia and reoxygenation on the clams, we set up two low-oxygen concentration groups (DO 2.0 mg/L and DO 0.5 mg/L) and assessed multiple aspects of oxidative damage to their hepatopancreas and gills. After the hypoxic stress, the two tissues of the razor clam suffered varying degrees of damage, including cell degeneration and disruption of mitochondrial cristae. After reoxygenation, the 2.0 mg/L group recovered substantially, but the clams in the 0.5 mg/L group still unrecovered. The activities of antioxidant enzymes (MDA, T-AOC, SOD, GPX, and CAT) in clams were considerably altered by acute hypoxia and reoxygenation. Briefly, there was a growing and then declining trend in MDA, T-AOC, and SOD activities in the hepatopancreas, whereas GPX and CAT activities showed the converse trend. In the hepatopancreas and gills, the level of anti-apoptotic gene Bcl-2 transcripts gradually decreased with the duration of hypoxia and increased following reoxygenation. However, changes in the transcript level of the pro-apoptotic gene Bax were in contrast to that of Bcl-2. The TUNEL assay revealed that hypoxia caused apoptosis. Furthermore, at DO 0.5 mg/L, the degree of apoptosis was more significant than at DO 2.0 mg/L, and hepatopancreatic apoptosis was more severe than gill apoptosis. Collectively, our findings imply that hypoxia induces oxidative stress, histological damage, and apoptosis in razor clams in a concentration-dependent and tissue-specific manner. These consequences serve as a reminder that prolonged recovery periods may be required for razor clams to fully recover from oxidative damage resulting from hypoxia-reoxygenation episodes.

Effects of severe hypoxia and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) knock-down on its gene expression, activity, subcellular localization, and apoptosis in gills of the shrimp Penaeus vannamei

ABSTRACT Penaeus vannamei, experiences hypoxia in its natural habitat and in aquaculture. Under hypoxia, cells enhance anaerobic energy production through glycolysis dependent on the up-regulation of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in gills. In vertebrates, GAPDH translocates into the nucleus and displays “moonlighting” functions including apoptosis. These alternative localizations and functions have not been described in crustaceans. We examined the effect of severe hypoxia and GAPDH silencing by RNA interference (RNAi) on its mRNA expression localization and glycolytic activity in P. vannamei gills. Expression and cytosolic activity were up-regulated only in hypoxia-exposed shrimp, but not in hypoxia-silenced specimens. GAPDH was immunodetected in cytosol and nucleus regardless of oxygen conditions. Hypoxia and RNAi decreased activity in cytosol and nucleus without affecting protein abundance, which suggests that nuclear GAPDH may have non-glycolytic functions. Moreover, Caspase-3 (Casp-3) expression increased with GAPDH silencing, suggesting alternative roles for GAPDH in apoptosis evasion.

The physiological, biochemical and transcriptional responses to sulfamethoxazole in the Asian clam, Corbicula fluminea (O. F. Müller, 1774)

Sulfamethoxazole (SMX), a broad-spectrum antibiotic, has been widely used in the treatment and prevention of infection caused by bacteria in recent years. The present study was aimed to evaluate the response mechanisms to SMX stress in gills and digestive gland of Corbicula fluminea (O. F. Müller, 1774). To this end, clams were exposed to environmentally relevant concentrations of SMX (0, 1, 10 and 100 μg/L) for 7 and 28 days, and siphon behavior, tissue-specific enzymatic and transcriptional changes were assayed. Our results showed that exposure to SMX significantly suppressed filtration rate and acetylcholinesterase (AChE) activity, activated antioxidant defense system and elevated transcription of several genes related to cell apoptosis in gills and digestive gland of clams. In general, SMX at environmentally relevant concentrations exhibited a negative impact on siphon behavior and induced neurotoxicology, oxidative stress and cell apoptosis in C. fluminea. The current study will help broaden our understanding of the ecotoxicity of SMX on freshwater bivalves.

Responses of JNK signaling pathway to the toxic dinoflagellate Prorocentrum lima in the mussel Perna viridis

Diarrheic shellfish poisoning (DSP) toxins are widely distributed over the world, causing diarrhea, vomiting, and even tumor in human. However, bivalves, the main carrier of the DSP toxins, have some tolerant mechanisms to DSP toxins, though it remains unclear. In this study, we scrutinized the role of Jun N-terminal kinases (JNK) in tolerance of DSP toxins and the relationship between JNK, apoptosis and nuclear factor E2-related factor/antioxidant response element (Nrf2/ARE) pathways. We found that the phosphorylated level of JNK protein was significantly increased both in hemocytes (6 h) and gills (3 h) of the mussel Perna viridis after short-term exposure to DSP toxins-producing dinoflagellate Prorocentrum lima. Exposure of P. lima induced oxidative stress in mussels. Hemocytes and gills displayed different sensitivities to the cytotoxicity of DSP toxins. Exposure of P. lima activated caspase-3 and induced apoptosis in gills but did not induce caspase-3 and apoptosis in hemocytes. The short-term exposure of P. lima could activate Nrf2/ARE signaling pathway in hemocytes (6 h), while longer-term exposure could induce glutathione reductase (GR) expression in hemocytes (96 h) and glutathione-S-transferases (GST) in gills (96 h). Based on the phylogenetic tree of Nrf2, Nrf2 in P. viridis was closely related to that in other mussels, especially Mytilus coruscus, but far from that in Mus musculus. The most likely phosphorylated site of Nrf2 in the mussels P. viridis is threonine 504 for JNK, which is different from that in M. musculus. Taken all together, the tolerant mechanism of P. viridis to DSP toxins might be involved in JNK and Nrf2/ARE signaling pathways, and JNK play a key role in the mechanism. Our findings provide a new clue to further understand tolerant mechanisms of bivalves to DSP toxins.

Waterborne enrofloxacin exposure activated oxidative stress and MAPK pathway, induced apoptosis and resulted in immune dysfunction in the gills of yellow catfish Pelteobagrus fulvidraco

Enrofloxacin (ENR), one representative of fluoroquinolones widely used in husbandry, poses potential threats to aquatic organisms. However, its toxicological effect remained largely unknown. Here, we explored the influences of environmentally relevant concentration of ENR exposure on the gills of yellow catfish Pelteobagrus fulvidraco, a well-known freshwater teleost. After 10-weeks of waterborne ENR exposure at environmentally relevant concentrations, the gill tissues were collected and subsequently used for histopathology, enzyme activity, protein and mRNA expression analysis in yellow catfish. Our study revealed that waterborne ENR exposure reduced Na+/K+-ATPase activity, tight junction genes' expression and cytokines secretion, activated oxidative stress and induced apoptosis in gills. ENR exposure also activated MAPK signaling in a concentration-dependent manner. Thus, we demonstrated that environmentally relevant concentration of ENR exposure activated oxidative stress and MAPK pathway, resulted in immune dysfunction and induced intrinsic apoptosis. These results elucidated that ENR, as an environmental pollutant, had more complex toxicological effects on gills of fish than previously assessed, which provided novel insight into ENR toxicology and the assessment of its environmental risks.

Gill physiological and transcriptomic response of the threatened freshwater mussel Solenaia oleivora to salinity shift

Solenaia oleivora, a freshwater shellfish endemic to China, is becoming one of the most threatened freshwater mussels owing to water pollution, habitat fragmentation, and overfishing. Hence, exploring its response to different environmental factors is important for its conservation. In this work, we investigated the physiological and transcriptomic response of S. oleivora to increased salinity. We found that increased salinity caused the death of S. oleivora. High salinity caused shrinking and deformation of gill filaments, reduced gill cilia, and induced cell apoptosis in gills. The activities of superoxide dismutase (SOD), catalase (CAT), acid phosphatase (ACP), alkaline phosphatase (AKP), as well as glutathione (GSH) content were increased at the beginning of salinity stress (3−12 h), while SOD and ACP activities decreased at 48 h. Transcriptome data revealed that high salinity stress (48 h) induced 766 differentially expressed genes (DEGs). Among these DEGs, the majority of the stress response and ion transport-related genes were up-regulated, while most of the immune-related genes were down-regulated. In conclusion, these findings suggest that the antioxidant and immune functions of S. oleivora can be inhibited by high salinity, which may be one of the main reasons for its low survival rate under conditions of increasing salinity.

Elevated seasonal temperature disrupts prooxidant-antioxidant homeostasis and promotes cellular apoptosis in the American oyster, Crassostrea virginica, in the Gulf of Mexico: a field study.

One of the major impacts of climate change has been the marked rise in global temperature. Recently, we demonstrated that high temperatures (1-week exposure) disrupt prooxidant-antioxidant homeostasis and promote cellular apoptosis in the American oyster. In this study, we evaluated the effects of seasonal sea surface temperature (SST) on tissue morphology, extrapallial fluid (EPF) conditions, heat shock protein-70 (HSP70), dinitrophenyl protein (DNP, an indicator of reactive oxygen species, ROS), 3-nitrotyrosine protein (NTP, an indicator of RNS), catalase (CAT), superoxide dismutase (SOD) protein expressions, and cellular apoptosis in gills and digestive glands of oysters collected on the southern Texas coast during the winter (15°C), spring (24°C), summer (30°C), and fall (27°C). Histological observations of both tissues showed a notable increase in mucus production and an enlargement of the digestive gland lumen with seasonal temperature rise, whereas biochemical analyses exhibited a significant decrease in EPF pH and protein concentration. Immunohistochemical analyses showed higher expression of HSP70 along with the expression of DNP and NTP in oyster tissues during summer. Intriguingly, CAT and SOD protein expressions exhibited significant upregulation with rising seasonal temperatures (15 to 27°C), which decreased significantly in summer (30°C), leaving oysters vulnerable to oxidative and nitrative damage. qRT-PCR analysis revealed a significant increase in HSP70 mRNA levels in oyster tissues during the warmer seasons. In situ TUNNEL assay showed a significant increase in apoptotic cells in seasons with high temperature. These results suggest that elevated SST induces oxidative/nitrative stress through the overproduction of ROS/RNS and disrupts the antioxidant system which promotes cellular apoptosis in oysters.

Two-sided effects of prolonged hypoxia and sulfide exposure on juvenile ark shells (Anadara broughtonii)

Oxygen deficit and sulfide have been restrictive factors in mariculture zones. However, the adaptive mechanism in aquatic lives is still unclear. The commercial ark shells Anadara broughtonii were selected to test the tolerance and adaptive responses to prolonged and intermittent hypoxia with or without exogenous sulfide (mild, moderate, high) by evaluating their behavior, mortality, oxidative level, antioxidant responses, and the MAPK-mediated apoptosis in gills. The results indicated that the clams were tolerant to hypoxia and sulfide exposure but vulnerable during reoxygenation from the challenges. Even so, sulfide had remarkable effect on attenuating the accumulation of reactive oxygen species (ROS) and lipid peroxides caused by reoxygenation from prolonged hypoxia. The increase of glutathione level was probably as an early and primary protective response to prevent the expected reperfusion injury from reoxygenation. The challenges suppressed the oxidative level with a dose-dependent effect of sulfide, with an exception when exposed to mild sulfide. Synchronously, biphasic effects of exogenous sulfide on apoptotic cascade, which was induced by mild sulfide while it was inhibited by higher sulfide, were also detected in gills. The induced or inhibited apoptosis by hypoxia and sulfide kept to a typical ROS-MAPK-CASPASE cascade, desiderating further investigation.

Protective role of Angelica sinensis extract on trichlorfon-induced oxidative damage and apoptosis in gills and erythrocytes of fish

The study explored the effects of the extract of Angelica sinensis (EAs) on the apoptosis and oxidative damage parameters in gills and erythrocytes in trichlorfon-treated carp (Cyprinus carpio var. Jian) and of ligustilide on parameters above in trichlorfon-treated erythrocytes. At the end of a 60 days feeding trial, fish was exposed to 2.4 mg L−1 of trichlorfon (Based on animal model test) for 4 days to determine the effects of EAs on oxidative damage and apoptosis in gills and erythrocytes of carp. The results indicated that dietary EAs significantly increased the level of reduced glutathione (GSH) and the activities of glutamate-oxaloacetate transaminase (GOT), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reducase (GR) and glutathione-S-transferase (GST), decreased the levels of superoxide anion (O2·-) and malondialdehyde (MDA) and the activities of caspase-8 and caspase-9 and inhibited the trichlorfon-induced decreases in the level of GSH and the activities of Na+,K+-ATPase, GOT, glutamate-pyruvate transaminase (GPT), GPx and GST and increases in the levels of O2·-, hydroxyl radical, MDA and protein carbonyl and the activities of caspase-3, caspase-8, caspase-9, SOD and CAT in gills of carp. Meanwhile, dietary EAs effectively increased haematocrit (Hct), erythrocyte count (EC) and haemoglobin concentration (HbC) in the blood and the level of GSH and the activities of CAT, Na+,K+-ATPase and lactate dehydrogenase (LDH), and decreased the levels of met-haemoglobin (Met-Hb) and MDA and inhibited the trichlorfon-induced decreases in Hct, EC and HbC in the blood and the level of GSH and the activities of CAT, GPx, Na+,K+-ATPase and LDH and increases in the levels of PS exposure, O2·-, hydrogen peroxide (H2O2), Met-Hb and MDA in erythrocytes of carp. Moreover, the present study found that ligustilide, the most abundant active ingredient in Angelica sinensis, markedly suppressed the trichlorfon-induced PS exposure, hemolysis, increases in the levels of calcium ion, O2·-, H2O2 and Met-Hb and the activities of calpain and decreases in the activities of SOD, CAT and GPx in fish erythrocytes. These results demonstrated that dietary EAs could improve the function of erythrocytes and prevent the trichlorfon-induced oxidative damage and apoptosis in gills and erythrocytes of fish. The effects of EAs on the erythrocyte function may be closely associated with its inhibition on apoptosis and oxidative damage in fish. EAs could be used as an inhibitor of trichlorfon stress in fish.

Salmon Gill Poxvirus, the Deepest Representative of the Chordopoxvirinae.

ABSTRACTPoxviruses are large DNA viruses of vertebrates and insects causing disease in many animal species, including reptiles, birds, and mammals. Although poxvirus-like particles were detected in diseased farmed koi carp, ayu, and Atlantic salmon, their genetic relationships to poxviruses were not established. Here, we provide the first genome sequence of a fish poxvirus, which was isolated from farmed Atlantic salmon. In the present study, we used quantitative PCR and immunohistochemistry to determine aspects of salmon gill poxvirus disease, which are described here. The gill was the main target organ where immature and mature poxvirus particles were detected. The particles were detected in detaching, apoptotic respiratory epithelial cells preceding clinical disease in the form of lethargy, respiratory distress, and mortality. In moribund salmon, blocking of gas exchange would likely be caused by the adherence of respiratory lamellae and epithelial proliferation obstructing respiratory surfaces. The virus was not found in healthy salmon or in control fish with gill disease without apoptotic cells, although transmission remains to be demonstrated. PCR of archival tissue confirmed virus infection in 14 cases with gill apoptosis in Norway starting from 1995. Phylogenomic analyses showed that the fish poxvirus is the deepest available branch of chordopoxviruses. The virus genome encompasses most key chordopoxvirus genes that are required for genome replication and expression, although the gene order is substantially different from that in other chordopoxviruses. Nevertheless, many highly conserved chordopoxvirus genes involved in viral membrane biogenesis or virus-host interactions are missing. Instead, the salmon poxvirus carries numerous genes encoding unknown proteins, many of which have low sequence complexity and contain simple repeats suggestive of intrinsic disorder or distinct protein structures.IMPORTANCE Aquaculture is an increasingly important global source of high-quality food. To sustain the growth in aquaculture, disease control in fish farming is essential. Moreover, the spread of disease from farmed fish to wildlife is a concern. Serious poxviral diseases are emerging in aquaculture, but very little is known about the viruses and the diseases that they cause. There is a possibility that viruses with enhanced virulence may spread to new species, as has occurred with the myxoma poxvirus in rabbits. Provision of the first fish poxvirus genome sequence and specific diagnostics for the salmon gill poxvirus in Atlantic salmon may help curb this disease and provide comparative knowledge. Furthermore, because salmon gill poxvirus represents the deepest branch of chordopoxvirus so far discovered, the genome analysis provided substantial insight into the evolution of different functional modules in this important group of viruses.

Effects of sodium fluoride on MAPKs signaling pathway in the gills of a freshwater teleost, Cyprinus carpio

Exposure to elevated levels of fluoride can cause a variety of adverse effects in fish. Previously we showed that fluoride causes injuries and apoptosis in the gills of Cyprinus carpio. In this study, the effects of fluoride on caspase-3 activity and on accumulation of proteins in the MAPKs pathways were evaluated using Western blotting and immunohistochemistry methods in vivo and in vitro. In vivo experiments showed that the caspase-3 activity increased with fluoride exposure level in a dose-dependent pattern Western blotting and immunohistochemistry results indicated that ERK relative activation tended to decrease as a function of fluoride exposure concentration. In contrast, relative activation of JNK increased with fluoride exposure level. Fluoride exposure did not appear to affect p38 activation. Furthermore, pretreatment of branchial cells with MAPK-specific inhibitors effectively prevented JNK induction and ERK inhibition, respectively, as well as reversed caspase-3 activity in fluoride-treated branchial cells. Our results indicate that activation of JNK and inactivation of ERK were caused by increased ROS and decreased antioxidant capacity in the gills of chronically exposed C. carpio described previously, which eventually caused the observed apoptosis in the fluoride-exposed gills and cells in C. carpio. JNK activation and ERK inactivation mechanism play a crucial role in gill impairment induced by chronic fluorosis. These findings contribute to a better understanding of the initial molecular and cellular events in the gill of fish chronically exposed to fluoride.

Atlantic salmon (Salmo salar L.) smolts require more than two weeks to recover from acidic water and aluminium exposure

The detrimental effects of acid rain and aluminium (Al) on salmonids have been extensively studied, yet knowledge about the extent and rate of potential recovery after exposures to acid and Al episodes is limited. Atlantic salmon smolts in freshwater (FW) were exposed for 2 and 7-day episodes (ACID2 and ACID7, respectively) to low pH (5.7±0.2) and inorganic aluminium (Ali; 40±4μg) and then transferred to good water quality, control water (CW; pH 6.8±0.1; <14±2μg Ali). Al accumulation on gills after 2 and 7 days of acid/Al exposure was 35.3±14.1 and 26.6±1.8μgg−1 dry weight, respectively. These elevated levels decreased 2 days post transfer to CW and remained higher than in control (CON; 5–10μg Ali) for two weeks. Plasma Na+ levels in ACID2 and ACID7 smolts decreased to 141±0.8 and 138.6±1.4mM, respectively, and remained significantly lower than CON levels for two weeks post transfer to CW. Similarly, plasma Cl− levels in ACID7 smolts (124.3±2.8mM) were significantly lower than in CON, with Cl− levels remaining significantly lower in ACID7 (126.2±4.8mM) and ACID2 (127.6±3.7mM) than in CON following 9 and 14 days post-transfer to CW, respectively. ACID2 and ACID7 smolts sustained elevated plasma glucose levels post transfer to CW suggesting elevated stress for more than a week following exposure. While gill Na+, K+-ATPase (NKA) activity was only slightly affected in ACID2 and not in ACID7 smolts in FW, acid/Al exposure resulted in a transient decrease in NKA activity following SW exposure in both groups. Acid/Al episodes had limited impact on isoform specific NKA α-subunit mRNA during exposure. However, the transfer of ACID2 and ACID7 smolts to CW showed an increase in NKAα1a mRNA (the FW isoform) and inhibited the up-regulation of NKAα1b (the SW isoform), probably resulting in higher abundance of the enzyme favouring ion uptake. Gill caspase 3B gene transcription did not change in acid/Al treated smolts, indicating no increased apoptosis in gills. ACID2 and ACID7 treatments resulted in lower smolt-related gill transcription of the gene encoding the tight junction protein claudin 10e compared to CON, while the gene encoding claudin 30 showed lower mRNA expression only after 11 days SW exposure in ACID7 fish. Our data suggest that acid/Al conditions affect ion perturbations through a combination of alteration of the preparatory increase in paracellular permeability and negative impact on the SW type NKA α-subunit mRNA transcripts, and raise major concerns regarding the recovery of physiological disruption in smolts following acid/Al exposure. Smolts may require more than two weeks to fully recover from even short moderate episodes of acid/Al exposure. Acid/Al exposure thus probably has greater impact on salmon populations than previously acknowledged.

Physiological, molecular, and cellular mechanisms of impaired seawater tolerance following exposure of Atlantic salmon, Salmo salar, smolts to acid and aluminum

We examined the physiological, molecular, and cellular mechanisms of impaired ion regulation in Atlantic salmon, Salmo salar, smolts following acute acid and aluminum (Al) exposure. Smolts were exposed to: control (pH 6.5, 3.4 μg l −1 Al), acid and low Al (LAl: pH 5.4, 11 μg l −1 Al), acid and moderate Al (MAl: pH 5.3, 42 μg l −1 Al), and acid and high Al (HAl: pH 5.4, 56 μg l −1 Al) for two and six days. At each time-point, smolts were sampled directly from freshwater treatment tanks and after a 24 h seawater challenge. Exposure to acid/MAl and acid/HAl led to accumulation of gill Al, substantial alterations in gill morphology, reduced gill Na +/K +-ATPase (NKA) activity, and impaired ion regulation in both freshwater and seawater. Exposure to acid/MAl for six days also led to a decrease in gill mRNA expression of the apical Cl − channel (cystic fibrosis transmembrane conductance regulator I), increased apoptosis upon seawater exposure, an increase in the surface expression of mitochondria-rich cells (MRCs) within the filament epithelium of the gill, but reduced abundance of gill NKA-positive MRCs. By contrast, smolts exposed to acid and the lowest Al concentration exhibited minor gill Al accumulation, slight morphological modifications in the gill, and impaired seawater tolerance in the absence of a detectable effect on freshwater ion regulation. These impacts were accompanied by decreased cell proliferation, a slight increase in the surface expression of MRCs within the filament epithelium, but no impact on gill apoptosis or total MRC abundance was observed. However, MRCs in the gills of smolts exposed to acid/LAl exhibited morphological alterations including decreased size, staining intensity, and shape factor. We demonstrate that the seawater tolerance of Atlantic salmon smolts is extremely sensitive to acute exposure to acid and low levels of Al, and that the mechanisms underlying this depend on the time-course and severity of Al exposure. We propose that when smolts are exposed to acid and moderate to high Al concentrations, impaired seawater tolerance results from extensive gill Al accumulation, damage to the epithelium, reduced MRC and transport protein abundance, and a synergistic stimulation of apoptosis in the gill upon seawater exposure. When smolts are exposed to acid and low levels of Al, loss of seawater tolerance appears to be independent of these mechanisms and may result instead from a shift in the phenotype of MRCs present in the gill epithelium.