General introduction
Climate change poses a significant threat to aquatic animal life, particularly in the Mekong River Delta, where rising water temperatures and increased CO2 levels are critical concerns According to the International Panel on Climate Change (IPCC, 2014), temperatures in this region are expected to rise by 2.5-3.5°C over the next century, while atmospheric CO2 levels are projected to increase by 3% annually.
Projected increases in water temperature are expected to adversely affect the functions of marine and freshwater ecosystems, as highlighted by various studies (Roessig et al., 2004; Brander, 2007; Rijnsdorp et al., 2009; Pệrtner & Peck, 2010; Hofmann and Todghram, 2010; Madeira et al., 2012; Crozier &).
Hutchings, 2014; Lefevre et al., 2016) and fish populations through effects on fish physiology, respiration, metabolism, food ability, growth, behaviors, reproduction and/or mortality (Watts et al., 2001; Cnaani, 2006; Sigh et al.,
Temperature is a crucial physical factor influencing animal distribution, but rising environmental CO2 levels (hypercarbia) pose significant risks by causing acid-base imbalances, reducing water pH, and triggering cardioventilatory and respiratory changes.
Research on the physiological changes and adaptive capabilities of aquatic animals is crucial, particularly in light of projected increases in water temperature and hypercarbia Understanding these effects can provide valuable insights into how these species cope with changing environmental conditions.
The hypothesis of oxygen capacity limited thermal tolerance suggests that elevated temperatures adversely affect fish by compromising the oxygen delivery mechanism to their tissues This concept highlights the critical relationship between temperature stress and oxygen availability, as explored in various studies (Portner, 2001; Portner and Farrell, 2008; Munday et al., 2008; Munday et al., 2009; Nilsson et al.).
2009; Portner, 2010; Neuheimer et al., 2011) It is due to the dissolved oxygen level decreasing with progressive increases of the temperature whilst fish oxygen demand significantly increases with the elevated temperature
Elevated temperatures combined with hypoxia can severely impact the metabolism and overall performance of aquatic organisms (McBryan et al., 2013) Fish in tropical regions are particularly vulnerable, as they often exist close to their upper thermal limits, making even slight temperature increases detrimental to their health (Nelson et al., 2016; Tewksbury et al., 2008).
However, there is growing evidence of studies that do not conform that hypothesis (Clark et al., 2013; Norin et al., 2014; Wang et al., 2014; Lefevre,
Air-breathing fish species have developed hypoxic water tolerance, likely as an evolutionary response to historical conditions characterized by higher temperatures and lower atmospheric oxygen levels than those we experience today.
Investigating the effects of the elevated temperature and hypoxia on the metabolism of the air-breathing fish is important to assess the effects of climate change
Research indicates that one key adaptive mechanism in response to environmental factors is the ability to modify gill morphology Studies by Tuurala et al (1998), Sollid et al (2003, 2005), Sollid and Nilsson (2006), Ong et al (2007), Matey et al (2008), and Mitrovic and Perry highlight this important aspect of adaptability in aquatic organisms.
Intensive research on the respiratory adaptations of water-breathing fish, such as crucian carp, goldfish, and salmonids, has revealed that their gills can modify interlamellar cell mass (ILCM) to adjust respiratory surface area in response to environmental changes This remarkable ability raises intriguing questions among scientists about whether gill remodeling represents a contemporary adaptation or an ancient evolutionary trait.
Gill remodeling may be an ancient trait linked to evolutionary changes, as evidenced by air-breathing fish that adapt their gill morphology to environmental shifts This concept is particularly significant in the study of C ornata, a species that has existed for at least 300 million years, providing valuable insights that could inform predictions about other fish species.
The rising atmospheric CO2 levels, attributed to global warming, significantly impact aquatic ecosystems Dissolved CO2 is more soluble than oxygen, meaning even a slight increase in atmospheric CO2 can lead to a substantial rise in dissolved CO2 in water bodies In the Mekong River Delta, for instance, PCO2 levels range from 0.02% to 0.6%, adversely affecting fish populations (Li et al., 2013) Additionally, intensive aquaculture practices contribute to hypercarbic environments, further threatening aquatic life.
The study investigates the effects of hypercarbia and hypercapnia on clown knifefish (C ornata), a tropical facultative air-breathing species, by exposing them to extreme conditions such as elevated temperatures (33°C), hypoxia, and water hypercarbia It focuses on the cardioventilatory responses, blood gas and pH changes, and the location of CO2/H+-sensitive chemoreceptors, which are crucial for understanding how these fish adapt their respiratory metabolism and gill plasticity throughout their growth cycles The findings aim to clarify the equivocal responses of facultative air-breathing fish to hypercarbic exposure, as highlighted by previous research (Damsgaard et al., 2015; Deharai, 1962; Tuong et al., 2018b).
Research objectives
The main objectives of this dissertation were to evaluate the impacts of climate change (in case of theelevated temperature, hypoxia, elevated CO2)on
In specific, respiration of Clown knifefish at 27C and 33C in combination with normoxia and hypoxia evaluating through SMR, percentage of air- breathing, Q10 value, specific dynamic action (SDA) and growth
To comprehend how fish adapt their respiratory systems to elevated temperatures and low oxygen levels, researchers studied gill morphology, focusing on gill remodeling and the estimation of respiratory surface area and water-blood thickness Additionally, fish responses to hypercarbia and hypercapnia were assessed to evaluate cardioventilatory parameters.
Temperature and hypoxia
Temperature is a measure of the kinetic energy of a specific group of molecules, each possessing its own mass and velocity, as described by the equation Ek = (1/2)m.v² (Callen and Scott, 1998) Temperature is commonly measured using two scales: Celsius (ºC) and Kelvin (K).
The Celsius scale defines 0ºC as the freezing point of water and 100ºC as its boiling point, while the Kelvin scale is derived from Celsius by adding 273.15 (Erickson and Tiberghien, 1985) Through biological evolution, organisms, including fish, have adapted to temperature variations, categorized as poikilotherms and homeotherms (Schulte, 2011) Poikilotherms are organisms that adjust their body temperature according to the environmental temperature, whereas homeotherms maintain a constant internal temperature regardless of external conditions.
Endotherms and ectotherms differ in how they regulate body heat, with endotherms relying on internal metabolic processes while ectotherms depend on external environmental heat Most fish are poikilothermic ectotherms, meaning their body temperature fluctuates with ambient temperatures As the temperature of a system rises, so does its thermal energy, which accelerates chemical reaction rates due to increased molecular collisions and higher energy levels among molecules This increase in reaction rates is linked to a greater fraction of molecules surpassing the activation energy required for reactions Additionally, temperature changes can weaken essential molecular bonds, such as hydrogen bonds and ionic interactions, which are crucial for the structure and function of enzymes, proteins, nucleic acids, and membranes.
These changes influent the molecular levels therefore, not easy to be observed in short time scales
Hypoxia refers to low oxygen levels in aquatic environments, which can lead to anoxia, the complete absence of dissolved oxygen in water Key factors contribute to the development of these conditions, as highlighted by Diaz and others.
Breitburg, 2011).The concentration of dissolved oxygen in aqueous solution obeys Henry’s Law as well as other dissolved gases
Where C is the molar concentration of dissolved oxygen, α is the solubility coefficient and P is a partial pressure of oxygen (Rombough, 2011)
Dissolved oxygen concentration in aquatic environments is inversely affected by temperature and salinity Water's capacity to hold oxygen is significantly lower than that of air, with oxygen levels in the atmosphere at 20.95%, equating to 209.5 ml/L, while water at 27ºC contains only 11.13 ml/L of dissolved oxygen (Boyd, 1982).
Oxygen is essential for the survival of aquatic animals, and a decrease in dissolved oxygen (DO) levels can lead to hypoxia, which threatens their life This decline can be caused by two main factors: water column stratification that prevents oxygen-rich surface water from reaching deeper layers, and the decomposition of organic matter in the bottom water that consumes available oxygen Hypoxia can also occur in shallow water columns, particularly when aquatic plants and phytoplankton cease photosynthesis, leading to a dominance of respiration that further reduces oxygen levels This phenomenon, known as diel-cycling hypoxia, can be reversed when oxygen production resumes the following day Overall, hypoxia can arise naturally due to stratification, poor circulation, and organic decay, as well as through human activities in intensive aquaculture.
Temperature and hypoxia: their effects on metabolism of air-breathing
Temperature is a critical physical factor that significantly impacts ecosystems and determines animal distribution (Fry, 1971) It affects various organs and tissues differently (Schulte et al., 2011) and plays a vital role in the biological performance of organisms In particular, temperature is a key determinant of fish reproduction and growth, with extensive research conducted on its effects across marine and freshwater ecosystems (Cossins and Bowler, 1987; Roessig et al., 2004; Brander, 2007; John & David, 2009).
Rijnsdorp et al., 2009; P ệ rtner & Peck, 2010; Hofmann and Todghram, 2010;
The International Panel on Climate Change (IPCC) predicts that by 2100, coastal South-East Asia could experience temperature increases of 2.5-3.5°C, particularly under the most extreme scenario (RPC8.5) This rise in temperatures raises significant concerns regarding the adaptation of both terrestrial and aquatic animals to changing environmental conditions.
Tropical fish are believed to be more susceptible to temperature increases compared to temperate fish, as they already exist close to their optimal temperature ranges This vulnerability is particularly concerning with even slight rises in temperature (Hoegh-Guldberg et al., 2007; Deutsch et al., 2008; Munday et al., 2008; Tewksbury et al., 2008).
Understanding the ecological impacts of climate change on fish requires a focus on how oxygen supply affects their physiology and behavior Research by Nilsson et al (2009) and P ệ rtner (2010) emphasizes the importance of these factors in assessing the overall consequences for aquatic ecosystems.
As environmental temperatures rise, the levels of dissolved oxygen in water decline, while the metabolic rate of fish increases, creating a gap between the oxygen demand of fish and the available supply in their surroundings This phenomenon has been corroborated by numerous studies (Farrell et al., 2008; Munday et al., 2008, 2009).
Nilsson et al., 2009; Neuheimer et al., 2011) while other investigations have showed a different idea (Clark et al., 2013; Norin et al., 2014; Wang et al.,
The metabolic responses of air-breathing fishes to elevated temperatures remain a topic of debate (Lefevre, 2016) Regardless of varying results, understanding the oxygen cascade from the external environment into the fish's body is crucial, as it significantly impacts energy balance and overall fish performance.
The evolution of air-breathing fishes has been shaped by their adaptation to hypoxic environments and elevated temperatures, which presented challenges in meeting their oxygen demands compared to current conditions (Graham 1997).
Air-breathing fish species have successfully inherited traits from their ancestors that enable them to adapt to various environmental factors These fish are now widespread globally, with certain species like striped catfish and snakehead becoming dominant in aquaculture Farmers often culture these species in high densities within non-aerated ponds, where they may experience hypoxia or anoxia due to their air-breathing capabilities However, hypoxia may not be beneficial for these fish, as it requires more energy to surface for air and increases their vulnerability to predators The critical oxygen tension (Pcrit) serves as a key indicator of hypoxic tolerance, representing the oxygen pressure point where the resting metabolic rate intersects with the oxygen-dependent metabolic rate.
The determination of Pcrit in air-breathing fishes is not particularly useful, yet it indicates the minimum dissolved oxygen levels that trigger air-breathing behavior Fish oxygen demand, represented by Standard Metabolic Rate (SMR), is essential for maintaining vital functions such as breathing, circulation, and osmoregulation under specific temperature and environmental conditions Additionally, digestion plays a crucial role in providing energy and nutrients necessary for fish survival, growth, and reproduction Jobling (1981) noted that oxygen demand increases during digestion, a phenomenon known as Specific Dynamic Action (SDA), which varies with temperature and can be negatively impacted by hypoxia, leading to reduced appetite and growth (Jobling and Davies, 1980; Soofiani and Hawkins, 1982; Wang et al., 2009).
Investigating the growth rates of air-breathing fish species under elevated temperatures and hypoxia is crucial as aquaculture expands Increased water temperatures boost biochemical reaction rates, leading to enhanced growth in certain facultative air-breathing species, such as P hypophthamus and C ornata, particularly with a temperature rise of 6°C However, when combined with ambient hypoxia, growth rates decline despite the same temperature levels To understand the impact of temperature on fish metabolism, the temperature coefficient (Q10) is utilized, measuring the biological reaction rate changes with a 10°C temperature increase These indicators are essential for understanding fish responses and adaptations to climate change and environmental hypoxia.
Where T2 and T1 are temperature levels, and R1 and R2are biological process rates or reaction rates
Fig 2.1: Pcrit value of a C.ornata
Fish gills serve as a multipurpose organ essential for gas exchange, osmotic regulation, acid-base balance, and nitrogenous waste excretion, functioning similarly to kidneys (Evan et al., 2005; Diaz et al., 2009) While many water-breathing fish possess large gill surface areas, this can lead to increased energetic costs of osmoregulation (Nilsson, 1986; Gonzalez & McDonald, 1992), heightened susceptibility to toxins (Wood, 2017), greater risk of pathogen attachment, and an increased likelihood of injury (Sundin and Nilsson, 1998) Conversely, fish gills are believed to retain ancient adaptive traits that allow fish to respond to environmental changes through gill plasticity (Nilsson, 2007; Nilsson et al., 2012) The brachial epithelium of fish gills has demonstrated the ability to adapt to various environmental factors, including temperature, oxygen levels, toxins, parasites, and physical activity (Tuurala et al.).
Research has shown that fish gills undergo significant morphological transformations in response to environmental changes, particularly in interlamellar cell mass (ILCM) For instance, the crucian carp (Carassius carassius) exhibited a full ILCM when exposed to normoxia at 8°C, but the gills transformed to a normal appearance with bare lamellae under hypoxic conditions (6-8% air saturation) (Sollid et al., 2003) Similarly, goldfish (Carassius auratus) demonstrated gill morphology changes in response to both elevated temperatures (>25°C) and hypoxia (Sollid et al., 2005) These findings indicate that fish gills are highly sensitive to vital environmental factors such as temperature and dissolved oxygen levels, as well as to metabolic and osmoregulatory imbalances Additionally, exhaustive exercise has also been shown to induce gill morphological changes in both goldfish and crucian carp (Brauner et al.).
Research has demonstrated that aluminum affects salmonids (Nilsson et al., 2012) and influences acid-base regulation in Pangasius (Phuong, unpublished data) Additionally, a decrease in ILCM indicates that the surface area of fish gills increases as critical oxygen tension decreases, which is significant for enhancing oxygen uptake (Sollid et al., 2003; Fu et al., 2011).
Gill remodeling is not only observed in recently evolved water-breathing fish but also in air-breathing species Research has shown that gills can change morphology in anabantoid fish like the dwarf gourami and killifish when exposed to acidic water or when transitioning to land The obligate air-breathing fish Arapaima gigas exhibits a unique development where its gills transform from protruded lamellae in early life stages to a non-protruded form as it matures and begins using its air-breathing organ This change is irreversible Additionally, the tropical air-breathing fish Pangasianodon hypopthalmus can adjust its ILCM in response to high temperatures and low oxygen levels, indicating the presence of ancient gill remodeling traits across various fish species.
3 Gill remodeling
Hypercarbia and its effect on cardioventilatory responses
Concurrently, elevated temperature and rising CO2 are two severe issues consequently following the climate change Concentrations of atmospheric
CO2 had increased at rate of 1% per year in the 20 th century and have turned to
Recent studies indicate that rising CO2 concentrations, which have increased by approximately 3% per year (IPCC, 2014), are significantly altering ocean chemistry by lowering water pH and reducing carbonation (Cao et al., 2007) This acidification adversely affects a wide range of marine organisms, including fish, leading to various physiological challenges such as acid-base imbalance (Claiborn et al., 2002; Brauner and Baker, 2009) and impacting overall aquatic life (Talmage and Gobler, 2011; Nowicki et al., 2012; Munday et al., 2012).
PCO2 increase, respiratory difficulty, as well as circulation and metabolism effects (Ishimatsu et al., 2005), and mortality in adult fish (Ishimatsu et al.,
Exposure to hypercarbia and the resulting acidosis negatively impact feed intake and growth performance in fish within aquaculture systems.
In the Lower Mekong River Delta, the water PCO2 levels range from 0.02 to 0.6% and pH levels of 6.9–8.4 with monthly and spatial variations (Li et al.,
In tropical aquaculture systems, high stocking densities often lead to overfeeding and hypoxia, particularly in stagnant water bodies, which can result in severe hypercarbia in fish ponds For instance, Pangasius is commonly cultured in these challenging environments.
PCO levels exceeds 17 mmHg (~2.5%) towards the end of the growth cycle
(Damsgaard et al., 2015) while that levels can exceed 60 mmHg (~8%) have been reported in the natural (Furch and Junk, 1997)
Fish exhibit varied responses to different levels of water PCO2 (hypercarbia), particularly in their cardioventilatory functions Typically, when exposed to hypercarbia, fish increase the volume of water they take in with each breath and/or their breathing frequency Additionally, there is often an increase in air-breathing frequency alongside a decrease in heart rate, as noted in studies by Janssen and Randall (1975), Burleson and Smatresk (2000), Reid et al (2000), and Perry.
McKendry, 2001; McKendry and Perry, 2001; Gilmour, 2001; Milsom et al.,
Research indicates that fish species exhibit varying hypercarbic responses, with sensitivities differing significantly among them For instance, rainbow trout and zebrafish are highly sensitive to even slight increases in PCO2, while other species such as eel, carp, tambaqui, and traira require higher PCO2 levels (over 5%) to elicit a response Additionally, in air-breathing fish, exposure to hypercarbia leads to increased gill ventilation, although at elevated PCO2 levels, gill ventilation may be inhibited while air-breathing is stimulated.
1982; Sanchez and Glass, 2001; Sanchez et al., 2005; Boijink et al., 2010) It has been also found that there is no change of gill ventilation (Johansen, 1966;
Research by McMahon and Burggren (1987), Thomsen et al (2017), and Tuong et al (2018a) highlights the impact of hypercarbia on air-breathing fish, specifically regarding their air-breathing frequency For instance, the facultative air-breathing fish C ornata exhibited no change in gill ventilation but demonstrated a significant increase in air-breathing frequency during exposure to severe hypercarbia levels of approximately 5% (Tuong et al., 2018a).
The respiratory responses to CO2 and H+ levels are primarily mediated by sensitive chemoreceptors known as neuroepithelial cells (NECs) located throughout the gill arches Research by Milsom (2012) and Jonz et al (2015) has demonstrated that these CO2/H+ receptors are innervated by the glossopharyngeal nerve (IXth cranial nerve).
X th (vagus) cranial nerves Existent data to date have shown that CO2/H + chemoreceptors mainly monitor the changes of CO2 level in ambient water
Numerous studies have demonstrated that intra-arterial injections of CO2/H+ do not impact gill ventilation, as evidenced by the findings of Reid et al (2000), Sundin et al (2000), McKendry and Perry (2001), and Perry.
Research indicates that acetazolamide injections, which inhibit carbonic anhydrase to promote CO2 retention and blood acidification, do not lead to ventilatory changes (McKendry, 2001; Gilmour et al., 2005; Boijink et al., 2010) Conversely, Wood and Munger (1994) demonstrated that carbonic anhydrase injections can progressively reduce the increased ventilation following exhaustive exercise in rainbow trout, which is characterized by elevated PCO2 and [H+] Additionally, intra-arterial CO2/H+ injections have been shown to stimulate ventilation in rainbow trout (Janssen and Randall, 1975).
The orientation and location of chemoreceptors in fish, which monitor cardiorespiratory responses to hypercarbia and hypercapnia, are intriguing areas of study Research indicates that most fish species respond to external CO2 changes, with sensitive receptors primarily located at the gill sites and in the orobranchial cavity However, evidence regarding internal responses and the orientation of central chemoreceptors remains limited (Milsom, 2012) Additionally, Boijing et al (2010) conducted examinations in Jeju, contributing to this ongoing research.
Hoplerythrinus unitaeniatus, a facultative air-breathing fish, exhibits gill ventilatory responses to hypercarbic water, with CO2-sensitive receptors located on the first gill arch, while air-breathing responses are found across all gill arches In a study by Boijing et al (2010), total gill nerve denervation eliminated all gill ventilations, yet air-breathing responses persisted Similarly, C ornata demonstrated responsiveness to hypercarbia and hypercapnia, as evidenced by reactions to acetazolamide and CO2 injections in both intact and denervated fish, suggesting that the CO2-sensitive receptors in C ornata are likely of internal orientation and may function as central chemoreceptors (Tuong et al., 2018a).
In water-breathing fish, the role of central CO2/H+ chemoreceptors remains unclear; however, evidence suggests their presence in air-breathing fish, particularly in Sarcopterygian lungfish, as documented in studies by Smith (1930) and Delaney et al (1974, 1977), along with Babiker (1979).
Sanchez et al., 2001), and seems to be present in gar, Siamese fighting fish
(Wilson et al., 2000; Hedrick et al., 1991) and now in clown knifefish (Tuong et al., 2018a).
Air-breathing fish species
Many species in tropical regions possess air-breathing organs (ABO) that enable them to survive by absorbing oxygen from the air These species can be categorized as facultative or continuous air-breathers, including both obligate and non-obligate types Among the 400 known air-breathing fish species, only 25 have been successfully raised in aquaculture, as noted by Lefevre et al (2014) Notably, certain air-breathing fish, such as the Tra catfish (Pangasius hypophthamus) and clown knifefish, have thrived in intensive aquaculture systems due to their specialized respiratory adaptations.
The article provides detailed information on various air-breathing species, highlighting the specific types of air-breathing organs (ABOs) they possess Notable examples include the C ornata, the buccal pharyngeal epithelial surface in snakehead (Chana striata), swamp eels (Monopterus albus), and the brachial diverticulae, or labyrinth organ, found in climbing perch (Anabas testudineous) and walking catfish (Clarias batrachus) A comprehensive list of these species and their respective ABO types can be found in Table 2.1.
The evolution of air-breathing organs in fish is a response to hypoxic conditions typically found in tropical waters, with evidence suggesting that these adaptations originated in freshwater environments rather than marine settings (Graham, 1978) This evolutionary process has led to a diverse array of air-breathing mechanisms, which can be categorized based on their structural characteristics, anatomical locations, and levels of development (Graham, 1997) These mechanisms include respiratory gas bladders, head-related organs, structures derived from the digestive tract, and skin adaptations.
I Organs relates to head region:
- Ia Buccal cavity, pharyngeal, branchial, opercular chambers covered by respiratory epithelia and increasing surface area as well as volume of chambers
- Ib Pharyngeal and branhchial pouches, the gills, and gill derivatives
II Organs derived from digestive tract including: esophagus, pneumatic ducts, stomaches and intestines
III The skin plays an auxilary function in aerial-respiration in many kinds of fish
Species in group I and II are mainly aquatic air-breathing fish in freshwater
Groups Ib, II, and III enhance ABO modeling through evolutionary specialization, as air bladders did not fulfill recruitment under natural selection for ABO Notably, group III includes amphibious fish that can extract oxygen from the air while expelling carbon dioxide (CO2) through their gills or skin.
Variations in the gas bladder structures and functions of different Pangasius species have been documented, highlighting the unique characteristics of each species (Graham, 1997) For instance, the air bladder of Pangasius sutchu is a single chamber that is wide and bilobed at the anterior end, tapering to a smaller point at the rear (Browman and Kramer, 1985) The volume of the air bladder in this species ranges from approximately 2% to 8% of its body volume, with the surface area of the air bladder estimated to be less than 1%.
14%of body mass, and measurement of diffusion distance from air to blood are around 1-2 μm (Browman and Kramer, 1985)
Table 2.1.Air-breathing organ types (ABO) and names of common air- breathing fish species (Information is based on data of Graham 1997)
Genus species Common name ABO References
Arapaima gigas Arapaima Swim bladder (Greenwood and Liem, 1984)
Chitala ornata Clown knifefish Swim bladder (Dehadrai, 1962)
Pangasianodon hypophthalmus Stripped catfish Swim bladder
(Taylor, 1831),(Owen, 1846), (Day, 1877; Browman and Kramer, 1985)
Chana striata Snakehead fish Suprabranchial chamber
(Taylor, 1831),(Das, 1928), (Liem, 1980, 1984)(Ishimatsu and Itazawa, 1981, 1993)
Monopterus albus Swamp eels Skin, mouth, (Liem, 1980; Liem and Inger,
Clarias spp Walking catfish Suprabranchial chamber, skin
Anabas testudineus Climbing perch Labyrinth organ
(Taylor, 1831),(Day, 1868)(Das, 1928; Liem, 1963, 1980; Liem and Inger, 1987)
Osphronemus gourami Giant gourami Labyrinth organ (Day, 1877)
Trichopodus spp Gouramies Labyrinth organ (Herbert and Wells, 2001)
Helostoma temminckii Kissing gourami Labrynth organ (Liem, 1967)
Anguilla spp Eels Skin, Swim bladder
(Mott, 1950), (Fange, 1976), (Berg and Steen, 1965)
Hyposstomus plecostomus Sucker mouth catfish Stomach
(Carter and Beadle, 1930), (Gradwell, 1971), (Gee, 1976)(Graham, 1983)
Misgurnus anguillicaudatus Loach Intestine (McMahon and Burggren, 1987)
Hoplosternum littorale Tamuata Intestine (Carter and Beadle, 1930)
Gobiidae spp Mudskipper Skin, mouth (Graham, 1976)
Organisms with ABO (accessory breathing organs) located in or near the digestive tract are found between the branch point of the pneumatic duct and the sphincter that separates the esophagus from the stomach This area is characterized by a rich vascular network, resulting in a highly red appearance, and the diffusion distance for air and blood is less than 1μm Certain fish species, such as those in the Loricariid and Trichomycterid families, utilize their stomachs as ABOs, although research on their morphology and histology remains limited Additionally, other fish species, including Misgurnis, Cobitis, and Acanthophthalmus, possess gut structures that function as respiratory organs, while Monopterus albus, Monopterus cuchia, and Clarias batrachus are known to respire through their skin (Graham and Wegner, 2010).
Introduction
Temperature is a crucial physical factor influencing ecosystem function and animal distribution, as highlighted by Fry (1971) Its effects are hierarchical, impacting various organs and tissues at different rates (Schulte et al., 2011) Notably, the temperature niches for activity, reproduction, and growth are narrower than the critical temperature (Tcrit) at which movement ceases (Cossins and Bowler, 1987) The IPCC's most extreme climate model (RPC8.5) forecasts temperature increases of 2.5–3.5°C for coastal Southeast Asia by 2100, raising concerns about the ability of animals to adapt to these new temperature regimes (IPCC, 2014; Farrell, 1997; Heath and Hughes, 1973; Lee et al., 2003; McKenzie et al., 2016; Somero, 2011) Tropical fish, in particular, are already living near their thermal maximum, making them especially vulnerable to even slight temperature increases (Deutsch et al., 2008; Hoegh-Guldberg et al., 2007; Munday et al., 2017).
2008; Nilsson et al., 2009; Tewksbury et al., 2008)
Understanding how temperature impacts oxygen supply to tissues is crucial for predicting the ecological effects of global warming in aquatic systems (Purtner, 2010; Purtner and Knust, 2007) This concept has gained significant support in teleost fish (Farrell et al., 2008; Munday et al., 2008; Munday et al., 2009; Neuheimer et al., 2011; Nilsson et al., 2009), although it remains a topic of debate, as emerging evidence suggests that many species do not follow this trend (Clark et al., 2013; Lefevre, 2016; Norin et al.).
Tissue oxygen supply is crucial for animal energy balance and evolutionary fitness, as inadequate oxygen can hinder survival The evolution of air-breathing in vertebrates, particularly in air-breathing fish, was likely driven by historical challenges related to oxygen availability due to higher surface temperatures and lower atmospheric oxygen levels Although only about 450 of the nearly 30,000 known teleost species are capable of air-breathing, they play a significant role in aquaculture, surpassing global production levels of salmonids The clown knifefish (Chitala ornata) exemplifies an air-breathing fish with a unique body structure and gymnotiform locomotion, utilizing its modified swim bladder for oxygen intake This species is commonly found in Southeast Asia's shallow waters, including rivers, lakes, and floodplains, where it thrives as a nocturnal carnivore with a diverse diet of insects, worms, shrimp, crabs, and small fish.
Chitala, a species known for its preference to conceal itself in submerged wood, aquatic plants, and shrubs during the day, has gained significant commercial value in aquaculture Its production has seen a remarkable rise, currently reaching approximately 500 tons annually in the lower Mekong region.
The Mekong delta environment of Chitala is characterized by a mean water temperature ranging from 27 to 29°C, which is projected to rise due to climate change To assess the impact of this temperature increase on the species' aerobic metabolism, researchers evaluated the critical water PO2 and measured the standard and routine metabolic rates at 27 and 33°C in both normoxia and hypoxia Additionally, the metabolic response to digestion, known as specific dynamic action, was measured at both temperatures in normoxia, and fish growth was determined in both normoxia and hypoxia These measurements are crucial in understanding the overall energy budget and fitness of Chitala, particularly given its importance in human protein supply.
Materials and methods
Clown knifefish (C ornata) were sourced from a local farm and acclimated in well-aerated 1 m³ tanks at Can Tho University's College of Aquaculture and Fisheries in Ninh Kieu district, Can Tho city, Vietnam, for a duration of two weeks prior to experimentation For respirometry experiments, fish weighing between 60-100 g were selected, while growth experiments utilized individuals with an initial weight of 35-45 g The water temperature in the holding tanks was consistently maintained at 27°C, with regular water changes to ensure optimal conditions.
2 days to maintain good water quality (NO2 -< 0.5 mg L -1 , NO3 -< 90 mg L -1 ,
< 0.02 mg L -1 , dissolved oxygen >95%, pH≈7.6 and PCO2< 0.5 mg L -1 )
Fish were fed commercial floating pellets containing 43% protein (Stella S3,
In a study conducted by Nutreco Company in Ho Chi Minh City, Vietnam, fish were randomly selected and placed in holding tanks within a respirometry laboratory The temperature in the tanks was controlled at either 27°C or 33°C, and the fish were fasted for 48 hours prior to the respirometry measurements.
Oxygen uptake (ṀO2, mgO2kg -1 h -1 ) was measured using two-phase intermittent flow respirometry as described in Lefevre et al (2011, 2014a,
2014b, 2016) The fish chamber (2.4 L) was flushed after each closed phase
To maintain water PO2 levels above 17.3 kPa (130 mmHg), both the water and air phases were flushed for 15 minutes, with 12 minutes allocated to water and 3 minutes to air, ensuring the return to normoxic conditions At the end of each respirometry trial, bacterial oxygen consumption was measured, and fish ṀO2 was calculated by subtracting bacterial ṀO2 from the actual measured ṀO2 In the hypoxia treatment, an additional respirometry controller was utilized to regulate the PO2 in the water bath, ensuring that the incoming water during the flushing sequence achieved the desired PO2 levels Relays were employed to manage the dosing of nitrogen (N2) bubbled into the water bath, effectively lowering the PO2 as needed.
2.3 Experimental protocols for metabolic rate 2.3.1 Experiment 1.1: P crit and choice of hypoxia level
In our initial experiment, we established the optimal hypoxia level to encourage air-breathing in subsequent tests Fish were subjected to fasting tanks at temperatures of either 27°C or 33°C for a duration of 48 hours before measurements were taken.
In a controlled study, eight fish weighing an average of 97.3±6.3 g were placed in respirometers to measure their oxygen consumption (ṀO2) over a 19-hour period, assessing both water and air phases Following this measurement, the air phase was eliminated by flooding the chamber, and water circulation was halted The oxygen levels in the water were monitored until the fish lost their equilibrium, which occurred after approximately 3 to 4 hours, at which point the fish were returned to their holding facilities.
The critical oxygen partial pressure (Pcrit) for each fish was determined by intersecting the individual's standard metabolic rate (SMR), calculated using the R script from Chabot et al (2016) based on combined oxygen uptake from air and water, with the final closed period plot from Lefevre et al (2011) The hypoxia levels were established as the temperature-specific mean Pcrit minus one standard deviation, resulting in hypoxia levels of 4.7 kPa at 27°C and 6.0 kPa at 33°C.
Fig 3.1 Pcrit determination of C.ornata at 27°C and 33°C, mean±S.E.M, N=8
In later experiments, fish were acclimated to their measurement temperature for at least 30 days to mitigate the potential confounding effects of short-term temperature responses during metabolic rate assessments.
2.3.3 Experiment 1.2: SMR and RMR measurements
In a study measuring oxygen consumption, eight fish (69.6±2.9 g) were placed in a two-phase respirometer for 19 hours under normoxic conditions After this initial measurement, the fish were returned to their holding tank, and background oxygen consumption was recorded The respirometer system was then cleaned and equilibrated with nitrogen and oxygen to achieve the appropriate partial pressure of oxygen (PO2) before the fish were subjected to hypoxia measurements for another 19 hours Background oxygen consumption was assessed in both air and water phases, following the methodology outlined by Lefevre et al (2011), to account for oxygen diffusion between the two phases By subtracting the background oxygen consumption (which was less than 15% of the metabolic rate) from the water phase, the researchers were able to calculate the oxygen consumption from both water and air Standard metabolic rate (SMR) and routine metabolic rate (RMR) were then determined for each treatment by combining the measured oxygen consumption rates from both phases.
R script from Chabot et al (2016)
2.3.4 Experiment 2: metabolism and partitioning during digesting
Twelve fish, weighing approximately 60.7±1.6 g and 60.2±3.2 g at temperatures of 27°C and 33°C respectively, were fasted for 48 hours at their respective temperatures before individual oxygen consumption (ṀO2) was measured for 20 hours in normoxic conditions Following this, the fish were removed from the respirometer, and bacterial oxygen consumption was recorded for one hour After reweighing, the fish were force-fed 2% of their body mass with high-protein commercial pellets and then returned to the chamber for an additional 45 hours of ṀO2 measurement The standard metabolic rate (SMR) was determined from the initial measurement period, while the specific dynamic action (SDA) was calculated by subtracting the SMR from the postprandial ṀO2 The SDA area was computed using the trapezoid method, and the duration of SDA was assessed from the time the fish returned to the chamber until their ṀO2 levels returned to SMR plus 2 standard error of the mean (S.E.M.).
The Specific Dynamic Action (SDA) was estimated by calculating the area under the total ṀO2 and Standard Metabolic Rate (SMR) curves using the trapezoid method To convert the SDA into energy expenditure, the SDA coefficient was derived by dividing the energy expenditure by the energy consumed from the meal, expressed in kilojoules (kJ) using the oxygen coefficient of 13.56 kJ mgO2 -1 for carnivorous species, as established by Elliott and Davison in 1975.
2.3.5 Experiment 3: growth performance of Chitala ornata
In a study involving 120 fish (mean weight 41.2±0.3 g), the subjects were divided into four groups and tagged with FDX-B microchips They were raised in a recirculating aquaculture system at temperatures of 27±0.5°C or 33±0.5°C, under either normoxic or hypoxic conditions (5 kPa) in 2000 L tanks The water flow was maintained at 0.19 m³/h, with parameters kept within specific ranges: PCO295% air saturation in normoxia, pH≈7.6, NH3 +≈0.02 mg/L, and NO3 -≈90 mg/L Hypoxia was controlled by bubbling nitrogen into the tanks, while a floating bubble plastic cover allowed the fish to access air and feed The fish were fed commercial pellets with 43% protein, and their mass and length were recorded after 1, 2, and 3 months By the end of the experiment, the fish weights ranged from 150 to 220 g, with specific growth rates (SGR) calculated monthly based on the final fish mass.
Mi is the initial, and t is time (days) between measurements
Data analysis was conducted using Sigmaplot 12.5, with normality and variance homogeneity assessed through the Shapiro-Wilks test, leading to the arcsine transformation of % air uptake A two-way ANOVA evaluated the effects of temperature, oxygen levels, and their interaction on standard metabolic rate (SMR), resting metabolic rate (RMR), and % air uptake, while Holm-Sidak multiple comparison procedure was applied for within-treatment effects The Student's t-test compared mean parameters in digestion experiments, and growth rate along with specific growth rate (SGR) was analyzed using a one-way ANOVA with repeated measures Mean values of SMR and RMR were further compared using the Student's t-test, with results presented as mean ± S.E.M Statistical significance was determined at a probability (p) value of P < 0.05.
Results
3.1 The effects of temperature and hypoxia on oxygen uptake and partitioning
The critical partial pressure of oxygen (Pcrit) significantly increased with temperature, rising from 6.1±0.5 kPa at 27°C to 8.7±1.0 kPa at 33°C This trend indicates that as hypoxia intensifies, higher temperatures lead to an earlier dependence on air-breathing Importantly, the observed decrease in oxygen consumption (ṀO2) during the experiment does not suggest that the fish behaves as an oxy-conformer; instead, it reflects an initial disturbance caused by the reduction in water oxygen levels.
When the water circulation in the respirometer is halted for measurement, the impact of oxygen levels on metabolic rates becomes evident The findings indicate that oxygen concentration significantly influences partitioning at both temperatures, while temperature only affects partitioning under hypoxic conditions, not in normoxia Additionally, temperature significantly impacts both standard metabolic rate (SMR) and routine metabolic rate (RMR), with temperature coefficients (Q10) of 2.6 and 2.9 for SMR in normoxia and hypoxia, respectively, and coefficients of 2.5 and 2.3 for RMR.
Oxygen levels did not significantly affect resting metabolic rate (RMR), but hypoxia notably decreased standard metabolic rate (SMR) at 33°C from 112 to 95 mgO2 kg -1 h -1, with no similar effect observed at 27°C Time plots of metabolic rate illustrate that this species remains calm in normoxia at 27°C, but a reliable SMR estimate is only achievable after 12 hours In normoxic conditions at 33°C, air-breathing became increasingly important; however, it was initially suppressed at the start of measurements, stabilizing after 5 hours.
Table 3.1 presents the results of SMR (mgO2 kg -1 h -1), RMR (mgO2 kg -1 h -1), and the percentage of aerial oxygen uptake from air, alongside their associated p-values derived from a two-way ANOVA The data is expressed as mean ± S.E.M with a sample size of N=8 Significant effects are indicated by ‡ for oxygen levels at specific temperatures (P