Hypoxia in fish
Fish are exposed to large oxygen fluctuations in their aquatic environment since the inherent properties of water can result in marked spatial and temporal differences in the concentration of oxygen (see oxygenation and underwater). Fish respond to hypoxia with varied behavioral, physiological, and cellular responses in order to maintain homeostasis and organism function in an oxygen-depleted environment. The biggest challenge fish face when exposed to low oxygen conditions is maintaining metabolic energy balance, as 95% of the oxygen consumed by fish is used for ATP production through the electron transport chain. Therefore, hypoxia survival requires a coordinated response to secure more oxygen from the depleted environment and counteract the metabolic consequences of decreased ATP production at the mitochondria. This article is a review of the effects of hypoxia on all aspects of fish, ranging from behavior down to genes.
Hypoxia tolerance 
Hypoxia tolerance in fish can often be determined by assessing the value of critical O2 tension, often referred as Pcrit. Pcrit is a point where an oxy-regulator becomes an oxy-conformer. The oxygen consumption rate (MO2) of oxy-regulators are unaffected by the changes in environmental oxygen tension (PO2), whereas the MO2 of oxy-conformers are affected by the changes in PO2, showing decreased MO2 with decreasing PO2. Individuals with low Pcrit values are usually associated with high hypoxia tolerance while individuals with high Pcrit values tend to be more hypoxia sensitive. However, some species or individuals can be complete oxy-conformers and may not show distinctive Pcrit points. Therefore, experimentally defining hypoxia in such species can be difficult.
Oxygen sensing 
Oxygen sensing structures 
In mammals, several structures have been implicated as oxygen sensing structures; however, all of these structures are situated to detect aortic or internal hypoxia since mammals rarely run into environmental hypoxia. These structures include the type I cells of the carotid body, the neuroepithelial bodies of the lungs as well as some central and peripheral neurons and vascular smooth muscle cells. In fish, the neuroepithelial cells (NEC) have been implicated as the major oxygen sensing cells. NEC have been found in all teleost fish studied to date, and are likely a highly conserved structure within many taxa of fish. NEC are also found in all four gill arches within several different structures, such as along the filaments, at the ends of the gill rakers and throughout the lamellae. Two separate neural pathways have been identified within the zebrafish gill arches both the motor and sensory nerve fibre pathways. Since neuroepithelial cells are distributed throughout the gills, they are often ideally situated to detect both arterial as well as environmental oxygen.
Mechanisms of neurotransmitter release in neuroepithelial cells 
Neuroepithelial cells (NEC) are thought to be neuron-like chemoreceptor cells because they rely on membrane potential changes for the release of neurotransmitters and signal transmission onto nearby cells. Once NEC of the zebrafish gills come in contact with either environmental or aortic hypoxia, an outward K+ "leak" channel is inhibited. It remains unclear how these K+ channels are inhibited by a shortage of oxygen because there are yet to be any known direct binding sites for "a lack of oxygen", only whole cell and ion channel responses to hypoxia. K+ "leak" channels are two-pore-domain ion channels that are open at the resting membrane potential of the cell and play a major role in setting the equilibrium resting membrane potential of the cell. Once this "leak" channel is closed, the K+ is no longer able to freely flow out of the cell, and the membrane potential of the NEC increases; the cell becomes depolarized. This depolarization causes voltage-gated Ca2+ channels to open, and for extracellular Ca2+ to flow down its concentration gradient into the cell causing the intracellular Ca2+ concentration to greatly increase. Once the Ca2+ is inside the cell, it binds to the vesicle release machinery and facilitates binding of the t-snare complex on the vesicle to the s-snare complex on the NEC cell membrane which initiates the release of neurotransmitters into the synaptic cleft.
Signal transduction up to higher brain centres 
If the post-synaptic cell is a sensory neuron, then an increased firing rate in that neuron will transmit the signal to the central nervous system for integration. Whereas, if the post-synaptic cell is a connective pillar cell or a vascular smooth muscle cell, then the serotonin will cause vasoconstriction and previously unused lamellae will be recruited through recruitment of more capillary beds, and the total surface area for gas exchange per lamella will be increased.
In fish, the hypoxic signal is carried up to the brain for processing by the glossopharyngeal (cranial nerve IX) and vagus (cranial nerve X) nerves. The first branchial arch is innervated by the glossopharyngeal nerve (cranial nerve IX); however all four arches are innervated by the vagus nerve (cranial nerve X). Both the glossopharyngeal and vagus nerves carry sensory nerve fibres into the brain and central nervous system.
Locations of oxygen sensors 
Through studies using mammalian model organisms, there are two main hypotheses for the location of oxygen sensing in chemoreceptor cells: the membrane hypothesis and the mitochondrial hypothesis. The membrane hypothesis was proposed for the carotid body in mice, and it predicts that oxygen sensing is an ion balance initiated process. The mitochondrial hypothesis was also proposed for the carotid body of mice, but it relies on the levels of oxidative phosphorylation and/or reactive oxygen species (ROS) production as a cue for hypoxia. Specifically, the oxygen sensitive K+ currents are inhibited by H2O2 and NADPH oxidase activation. There is evidence for both of these hypotheses depending on the species used for the study. For the neuroepithelial cells in the zebrafish gills, there is strong evidence supporting the "membrane hypothesis" due to their capacity to respond to hypoxia after removal of the contents of the cell. However, there is no evidence against multiple sites for oxygen sensing in organisms.
Acute responses to hypoxia 
Many hypoxic environments never reach the level of anoxia and most fish are able to cope with this stress using different physiological and behavioural strategies. Fish that use air breathing organs (ABO) tend to live in environments with highly variable oxygen content and rely on aerial respiration during times when there is not enough oxygen to support water-breathing. Though all teleosts have some form of swim bladder, many of them are not capable of breathing air, and they rely on aquatic surface respiration as a supply of more oxygenated water at the surface of the water. However, many species of teleost fish are obligate water breathers and do not display either of these surface respiratory behaviours. Typically, acute hypoxia causes hyperventilation, bradycardia and an elevation in gill vascular resistance in teleosts. However, the benefit of these changes in blood pressure to oxygen uptake has not been supported in a recent study of the rainbow trout. It is possible that the acute hypoxia response is simply a stress response, and the advantages found in early studies may only result after acclimatization to the environment.
Aquatic surface respiration (ASR) 
In response to decreasing dissolved oxygen level in the environment, fish swim up to the surface of the water column and ventilate at the top layer of the water where it contains relatively higher level of dissolved oxygen. Most tropical and temperate fish species engage in ASR during hypoxia. ASR significantly affects survivability of fish during severe hypoxia. For example, a tropical fish, the shortfin molly survival rate was approximately four times higher when they were able to perform ASR than those which were not allowed to perform ASR during their exposure to extreme hypoxia.
Aerial respiration (air breathing) 
Aerial respiration is the ‘gulping’ of air at the surface of water to directly extract oxygen from the atmosphere. Aerial respiration evolved in fish that were exposed to more frequent hypoxia; also, species that engage in aerial respiration tend to be more hypoxia tolerant than those which do not air-breath during the hypoxia.
There are two main types of air breathing fish—facultative and non-facultative. Under normoxic conditions facultative fish can survive without having to breathe air from the surface of the water. However, non-facultative fish must respire at the surface even in normal dissolved oxygen levels because their gills cannot extract enough oxygen from the water.
Many air breathing freshwater teleosts use ABOs to effectively extract oxygen from air while maintaining functions of the gills. ABOs are modified gastrointestinal tracts, gas bladders, and labyrinth organs; they are highly vascularized and provide additional method of extracting oxygen from the air. Fish also use ABO for storing the retained oxygen.
Predation risk associated with ASR and aerial respiration 
Both ASR and aerial respiration require fish to travel to the top of water column and this behaviour increases the predation risks by aerial predators or other piscivores inhabiting near the surface of the water. In order to cope with the increased predation risk upon surfacing, some fish perform ASR or aerial respiration in schools in order to ‘dilute’ the predation risk. When fish can visually detect the presence of their aerial predators, they simply refrain from surfacing, or prefer to surface in areas where they can be detected less easily (i.e. turbid, shaded areas).
Gill remodelling in hypoxia 
Gill remodelling happens in only a few species of fish, and it involves the build up or removal of an inter-lamellar cell mass (ILCM). As a long term response to hypoxia, some fish are able to remodel their gills to increase the respiratory surface area. The increased respiratory surface area comes as a trade-off with increased metabolic costs because the gills are a very important site for many important processes including respiratory gas exchange, acid-base regulation, nitrogen excretion, osmoregulation, hormone regulation, metabolism, and environmental sensing.
The crucian carp is one species able to remodel its gill filaments in response to hypoxia. Their inter-lamellar cells have high rates of mitotic activity which are influenced by both hypoxia and temperature. In cold (15°C) water the crucian carp has more ILCM, but when the temperature is increased to 25°C the ILCM is removed, just as it would be in hypoxic conditions. This same transition in gill morphology occurs in the goldfish when the temperature was raised from 7.5°C to 15°C. This difference may be due to the temperature regimes that these fish are typically found in, or there could be an underlying protective mechanism to prevent a loss of ion balance in stressful temperatures. Temperature also affects the speed at which the gills can be remodelled: for example, at 20°C in hypoxia, the crucian carp can completely remove its ILCM in 6 hours, whereas at 8°C, the same process takes 3–7 days. The ILCM is likely removed by apoptosis, but it is possible that when the fish is faced with the double stress of hypoxia at high temperature, the lamellae may be lost by physical degradation. Covering the gill lamellae may protect species like the crucian carp from parasites and environmental toxins during normoxia by limiting their surface area for inward diffusion while still maintaining oxygen transport due to an extremely high hemoglobin oxygen binding affinity.
The naked carp, a closely related species native to the high-altitude Lake Qinghai, is also able to remodel their gills in response to hypoxic conditions. In response to oxygen levels 95% lower than normoxic conditions, apoptosis of ILCM increases lamellar surface area by up to 60% after just 24 hours. However, this comes at a significant osmoregulatory cost, reducing sodium and chloride levels in the cytoplasm by over 10%. The morphological response to hypoxia by scaleless carp is the fastest respiratory surface remodelling reported in vertebrates thus far.
Oxygen uptake 
Fish exhibit a wide range of tactics to counteract aquatic hypoxia, but when escape from the hypoxic stress is not possible, maintaining oxygen extraction and delivery becomes an essential component to survival. Except for the Antarctic ice fish that does not, most fish use hemoglobin (Hb) within their red blood cells to bind chemically and deliver 95% of the oxygen extracted from the environment to the working tissues. Maintaining oxygen extraction and delivery to the tissues allows continued activity under hypoxic stress and is in part determined by modifications in two different blood parameters: hematocrit and the binding properties of hemoglobin.
In general, hematocrit is the number of red blood cells (RBC) in circulation and is highly variable among fish species. Active fish, like the blue marlin, tend to have higher hematocrits, whereas less active fish, such as the starry flounder exhibit lower hematocrits. Hematocrit may be increased in response to both short-term (acute) or long-term (chronic) hypoxia exposure and results in an increase in the total amount of oxygen the blood can carry, also known as the oxygen carrying capacity of the blood. Acute changes in hematocrit are the result of circulating stress hormones (see - catecholamines) activating receptors on the spleen that cause the release of RBCs into circulation. During chronic hypoxia exposure, the mechanism used to increase hematocrit is independent of the spleen and results from hormonal stimulation of the kidney by erythropoetin (EPO). Increasing hematocrit in response to erythropoietin is observed after approximately one week and is therefore likely under genetic control of hypoxia inducible factor hypoxia inducible factor (HIF). While increasing hematocrit means that the blood can carry a larger total amount of oxygen, a possible advantage during hypoxia, increasing the number of RBCs in the blood can also lead to certain disadvantages. First, A higher hematocrit results in more viscous blood (especially in cold water) increasing the amount of energy the cardiac system requires to pump the blood through the system and secondly depending on the transit time of the blood across the branchial arch and the diffusion rate of oxygen, an increased hematocrit may result in less efficient transfer of oxygen from the environment to the blood.
Changing the binding affinity of hemoglobin 
An alternative mechanism to preserve O2 delivery in the face of low ambient oxygen is to increase the affinity of the blood. The oxygen content of the blood is related to PaO2 and is illustrated using an oxygen equilibrium curve (OEC). Fish hemoglobins, with the exception of the agnathans, are tetramers that exhibit cooperativity of O2 binding and have sigmoidal OECs.
The binding affinity of hemoglobin to oxygen is estimated using a measurement called P50 (the partial pressure of oxygen at which hemoglobin is 50% bound with oxygen) and can be extremely variable. If the hemoglobin has a weak affinity for oxygen, it is said to have a high P50 and therefore constrains the environment in which a fish can inhabit to those with relatively high environmental PO2. Conversely, fish hemoglobins with a low P50 bind strongly to oxygen and are then of obvious advantage when attempting to extract oxygen from hypoxic or variable PO2 environments. The use of high affinity (low P50) hemoglobins results in reduced ventillatory and therefore energetic requirements when facing hypoxic insult. The oxygen binding affinity of hemoglobin (Hb-O2) is regulated through a suite of allosteric modulators; the principal modulators used for controlling Hb-O2 affinity under hypoxic insult are:
- Increasing RBC pH
- Reducing Inorganic Phosphate Interactions
pH and inorganic phosphates (Pi) 
In rainbow trout as well as a variety of other teleosts, increased RBC pH stems from the activation of B-andrenergic Na+/H+ exchange protein (BNHE) on the RBC membrane via circulating catelcholamines. This process causes the internal pH of the RBC to increase through the outwards movement of H+ and inwards movement of Na+. The net consequence of alkalizing the RBC is an increase in Hb-O2 affinity via the Bohr effect. The net influx of Na+ ions and the compensatory activation of Na+/K+-ATPase to maintain ionic equilibrium within the RBC results in a steady decline in cellular ATP, also serving to increase Hb-O2 affinity. As a further result of inward Na+ movement, the osmolarity of the RBC increases causing osmotic influx of water and cell swelling. The dilution of the cell contents causes further spatial separation of hemoglobin from the inorganic phosphates and again serves to increase Hb-O2 affinity.
Changing Hb- isoforms 
Nearly all animals have more than one kind of Hb present in the RBC. Multiple Hb isoforms (see - isoforms) are particularly common in ectotherms, but especially in fish that are required to cope with both fluctutating temperature and oxygen availability. Hbs isolated from the European eel can be separated into anodic and cathodic isoforms. The anodic isoforms have low oxygen affinities (high P50) and marked Bohr effects, while the cathodic lack significant pH effects and are therefore thought to confer hypoxia tolerance. Several species of African cichlids raised from early stage development under either hypoxic or normoxic conditions were contrasted in an attempt to compare Hb isoforms. They demonstrated there were Hb isoforms specific to the hypoxia-raised individuals.
Metabolic challenge 
To deal with decreased ATP production through the electron transport chain, fish must activate anaerobic means of energy production (see anaerobic metabolism) while suppressing metabolic demands. The ability to decrease energy demand by metabolic suppression is essential to ensure hypoxic survival due to the limited efficiency of anaerobic ATP production.
Switch from aerobic to anaerobic metabolism 
Aerobic respiration, in which oxygen is used as the terminal electron acceptor, is crucial to all water-breathing fish. When fish are deprived of oxygen, they require other ways to produce ATP. Thus, a switch from aerobic metabolism to anaerobic metabolism occurs at the onset of hypoxia. Glycolysis and substrate-level phosphorylation are used as alternative pathways for ATP production. However, these pathways are much less efficient than aerobic metabolism. For example, when using the same substrate, the total yield of ATP in anaerobic metabolism is 15 times lower than in aerobic metabolism. This level of ATP production is not sufficient to maintain a high metabolic rate, therefore, the only survival strategy for fish is to alter their metabolic demands.
Metabolic suppression 
The most important factor of hypoxia tolerance is metabolic suppression. When oxygen levels decrease below Pcrit, fish are faced with the metabolic challenge of maintaining cellular energy balance through anaerobic metabolism. There are two ways fish can respond to this problem:
- Hypoxia-sensitive fish try to maintain a high metabolic rate
- Hypoxia-tolerant fish reduce their metabolic rate to conserve energy
The first response will shorten lifespan as there will be insufficient amounts of ATP to meet the high metabolic demands. The second response will prolong survival as they will be able to generate enough cellular energy to maintain a suppressed metabolic rate. As high as a 70% decrease in metabolic rate has been identified in some extremely hypoxia-tolerant fish such as goldfish, tilapia, and European eel. Although it is known that hypoxia-tolerant fish suppress their metabolism during hypoxia, the exact mechanisms to how this occurs is unclear. It is still unknown whether fish reduce ATP demand, increase ATP supply, or both. However, there is emerging evidence to support a decrease in ATP demand in response to hypoxia. The two most energetically expensive processes in a cell are protein turnover, which accounts for 55% of ATP utilization, and ion channel maintenance, which accounts for 25% of ATP utilization. In hypoxia-tolerant fish, the rate of protein synthesis is reduced in order to conserve ATP.
Energy conservation 
In addition to a reduction in the rate of protein synthesis, it appears that some species of hypoxia-tolerant fish conserve energy by employing Hochachka’s ion channel arrest hypothesis. This hypothesis makes two predictions:
- Hypoxia-tolerant animals naturally have low membrane permeabilities
- Membrane permeability decreases even more during hypoxic conditions (ion channel arrest)
The first prediction holds true. When membrane permeability to Na+ and K+ ions was compared between reptiles and mammals, reptile membranes were discovered to be five times less leaky. The second prediction has been more difficult to prove experimentally, however, indirect measures have showed a decrease in Na+/K+-ATPase activity in eel and trout hepatocytes during hypoxic conditions. Results seem to be tissue-specific, as crucian carp exposed to hypoxia do not undergo a reduction in Na+/K+ ATPase activity in their brain. Although evidence is limited, ion channel arrest enables organisms to maintain ion channel concentration gradients and membrane potentials without consuming large amounts of ATP.
Enhanced glycogen stores 
The limiting factor for fish undergoing hypoxia is the availability of fermentable substrate for anaerobic metabolism; once substrate runs out, ATP production ceases. Endogenous glycogen is present in tissue as a long term energy storage molecule. It can be converted into glucose and subsequently used as the starting material in glycolysis. A key adaptation to long-term survival during hypoxia is the ability of an organism to store large amounts of glycogen. Many hypoxia-tolerant species, such as carp, goldfish, killifish, and oscar contain the largest glycogen content (300-2000 µmol glocosyl units/g) in their tissue compared to hypoxia-sensitive fish, such as rainbow trout, which contain only 100 µmol glocosyl units/g. The more glycogen stored in a tissue indicates the capacity for that tissue to undergo glycolysis and produce ATP.
Tolerance of waste products 
When anaerobic pathways are turned on, glycogen stores are depleted and accumulation of acidic waste products occurs. This is known as a Pasteur effect. A challenge hypoxia-tolerant fish face is how to produce ATP anaerobically without creating a significant Pasteur effect. Along with a reduction in metabolism, some fish have adapted traits to avoid accumulation of lactate. For example the crucian carp, a highly hypoxia-tolerant fish, has evolved to survive months of anoxic waters. A key adaptation is the ability to convert lactate to ethanol in the muscle and excrete it out of their gills. Although this process is energetically costly is it crucial to their survival in hypoxic waters.
Gene expression changes 
DNA microarray studies done on different fish species exposed to low-oxygen conditions have shown that at the genetic level fish respond to hypoxia by changing the expression of genes involved in oxygen transport, ATP production, and protein synthesis. In the liver of mudsuckers exposed to hypoxia there were changes in the expression of genes involved in heme metabolism such as hemopexin, heme oxygenase 1, and ferritin. Changes in the sequestration and metabolism of iron may suggest hypoxia induced erythropoiesis and increased demand for hemoglobin synthesis, leading to increased oxygen uptake and transport. Increased expression of myoglobin, which is normally only found in muscle tissue, has also been observed after hypoxia exposure in the gills of zebrafish and in non-muscle tissue of the common carp suggesting increased oxygen transport throughout fish tissues.
Microarray studies done on fish species exposed to hypoxia typically show a metabolic switch, that is, a decrease in the expression of genes involved in aerobic metabolism and an increase in expression of genes involved in anaerobic metabolism. Zebrafish embryos exposed to hypoxia decreased expression of genes involved in the citric acid cycle including, succinate dehydrogenase, malate dehydrogenase, and citrate synthase, and increased expression of genes involved in glycolysis such as phosphoglycerate mutase, enolase, aldolase, and lactate dehydrogenase. A decrease in protein synthesis is an important response to hypoxia in order to decrease ATP demand for whole organism metabolic suppression. Decreases in the expression of genes involved in protein synthesis, such as elongation factor-2 and several ribosomal proteins, have been shown in the muscle of the mudsucker and gills of adult zebrafish after hypoxia exposure .
Research in mammals has implicated hypoxia inducible factor (HIF) as a key regulator of gene expression changes in response to hypoxia However, a direct link between fish HIFs and gene expression changes in response to hypoxia has yet to be found. Phylogenetic analysis of available fish, tetrapod, and bird HIF-α and -β sequences shows that the isoforms of both subunits present in mammals are also represented in fish Within fish, HIF sequences group close together and are distinct from tetrapod and bird sequences. As well, amino acid analysis of available fish HIF-α and -β sequences reveals that they contain all functional domains shown to be important for mammalian HIF function, including the basic helix-loop-helix (bHLH) domain, Per-ARNT-Sim (PAS) domain, and the oxygen-dependent degradation domain (ODD), which render the HIF-α subunit sensitive to oxygen levels. The evolutionary similarity between HIF sequences in fish, tetrapods and birds, as well as the conservation of important functional domains suggests that HIF function and regulation is similar between fish and mammalian species. There is also evidence of novel HIF mechanisms present in fish not found in mammals. In mammals, HIF-α protein is continuously synthesized and regulated post-translationally by changing oxygen conditions, but it has been shown in different fish species that HIF-α mRNA levels are also responsive to hypoxia. In the hypoxia tolerant grass carp, substantial increases in HIF-1α and HIF-3α mRNA were observed in all tissues after hypoxia exposure. Likewise, mRNA levels of HIF-1α and HIF-2α were hypoxia-responsive in the ovaries of the Atlantic croaker during both short and long term hypoxia.
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