Comparative hypoxia responses and oxygen sensing in galaxiid fishes
Ulrich, C. (2017). Comparative hypoxia responses and oxygen sensing in galaxiid fishes (Thesis, Doctor of Philosophy (PhD)). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/11575
Permanent Research Commons link: https://hdl.handle.net/10289/11575
Aquatic ecosystems are inherently characterised by limited oxygen availability and fluctuations in dissolved oxygen concentration, and therefore, environmental hypoxia (i.e. low oxygen conditions) is common. However, in recent decades, the frequency and global expansion of hypoxic environments have increased significantly, owing to intensified anthropogenic impacts on the natural environment. Fish depend on environmental oxygen for oxidative metabolism. In the absence of oxygen, alternative anaerobic mechanisms are in place to maintain energy metabolism. Efficiency and productivity of anaerobic metabolism are, however, comparatively low and may become rapidly insufficient in light of high physiological and metabolic oxygen demand. Correspondingly, substrates used in the oxygen-independent metabolism often are limited, and are converted into metabolites detrimental for the organism. When anaerobic mechanisms fail to meet the energy demand and lethal levels of anaerobic by-products have accumulated, hypoxic death is initiated. In this context, a variety of adaptive hypoxia-response strategies and hypoxia-sensitivities are established in fish, in dependence on species-specific oxygen demand and life strategies, thereby potentially creating very specific environmental niches occupied by distinct species. Accordingly, fish may demonstrate distinct habitat requirements and preferences in relation to the oxygen environment, potentially reflecting species-specific hypoxia sensitivities and response capacities, that may ultimately initiate adaptive radiation. In this context, a range of studies has been undertaken previously in a variety of species, however these are difficult to evaluate in a comparative context due to pronounced taxonomic differences between study species. New Zealand’s galaxiids, however, include species which occupy a wide range of environments. They are closely related and may demonstrate adaptive radiation to particular environments. The three related galaxiid species inanga (Galaxias maculatus), banded kokopu (Galaxias fasciatus) and black mudfish (Neochanna diversus) demonstrate minimised phylogenetic distances, yet exhibit specialised habitat requirements with distinctly different oxygen environments. Thus, it was the overarching goal of this research to establish whether these differences are based upon species-specific oxygen sensitivities and unique hypoxia response strategies and mechanisms, by utilising a novel, comparative combination of behavioural, physiological, molecular and gill morphological studies. To study whether the three closely related galaxiid species exhibit unique behavioural responses upon encountering a hypoxic environment, the ability to sense progressive hypoxia, as well as behavioural responses and hypoxia avoidance thresholds were investigated in a hypoxia-normoxia choice chamber. Inanga demonstrated avoidance of mild hypoxia (< 5.9 mg L-1) and increased the frequency of visits into both the hypoxic and normoxic sides of the choice chamber in severe hypoxia (at dissolved oxygen concentration below 3.6 mg L-1 for hypoxic side and below 1.9 mg L-1 for normoxic side). Banded kokopu responded to progressive hypoxia primarily with an increased frequency of aquatic surface respiration and an elevated swimming speed (body lengths (BL) s-1). Banded kokopu was less averse to hypoxia than inanga, as they displayed a lower hypoxia avoidance threshold and demonstrated horizontal migration from severe hypoxia (< 2.5 mg L-1). By contrast, no avoidance of, or other distinct behavioural response to hypoxia was observed in black mudfish. In conclusion, the three species exhibit unique behavioural responses upon encountering a hypoxic environment which demonstrates not only distinct behavioural response strategies towards hypoxia, but also indicates species-specific hypoxia sensitivities. To investigate whether these distinct hypoxia sensitivities and behavioural responses are based on different metabolic oxygen demands and oxygen consumption profiles, intermittent-flow respirometry was utilised to investigate routine metabolic rates at normoxia, mild and severe hypoxia. Inanga demonstrated oxygen consumption rates similar to banded kokopu, while black mudfish was lower. Inanga and banded kokopu maintained normoxic oxygen consumption in mild hypoxia and exhibited distinct critical oxygen concentrations (Ccrit), below which oxygen consumption rates declined, identifying these species as oxyregulators. Black mudfish was revealed to be an oxyregulator as well, but no Ccrit could be ascertained in this study, possible due to insufficiently severe levels of hypoxia. Inanga displayed the greatest hypoxia sensitivity, reflected in a Ccrit of 5.0 ± 0.4 mg L-1, while banded kokopu was slightly more tolerant to hypoxia with a Ccrit of 4.3 ± 0.1 mg L-1. In conclusion, the three species exhibit distinct oxygen consumption profiles and different critical dissolved oxygen concentrations. To examine whether swimming speed, as a measure of metabolic oxygen demand, and gill morphology are affected by prolonged hypoxia and whether responses to hypoxia are mediated by oxygen sensing neuroepithelial cells (NECs) and by the transcription protein hypoxia inducible factor 1 (HIF-1), the effect of inescapable moderate hypoxia at 5 mg L-1 dissolved oxygen concentration (without access to the water surface) on these parameters was investigated. Swimming speed decreased significantly from 21.6 ± 2.6 to 7.4 ± 2.0 BL min-1 in inanga, while no change was seen in banded kokopu and black mudfish. All three species presented NECs in the gill epithelia; however, hypoxia exposure did not elicit gill morphological adaptations or changes in NEC- and HIF-1 alpha density in any species. Gill morphological traits in black mudfish (i.e. wider respiratory lamellae than in inanga and banded kokopu) indicated an adaptation to emersion and aestivation, frequently observed in this species. HIF-1 alpha protein stabilisation at normoxic conditions indicated distinct differences between mammalian and piscine HIF-1 alpha pathway and hypoxia-inducible gene transcription. In conclusion, the three species exhibit specific adjustments of their swimming speed as a measure of metabolic oxygen demand, however, they do not alter their gill morphology in response to prolonged moderate hypoxia. Furthermore, responses to hypoxia are not mediated by changes in the density of NECs or HIF-1-positive cells. Overall, the findings from this thesis demonstrate that all three species are capable of managing hypoxic environments. In this context, they utilise distinct behavioural response strategies in progressive, escapable hypoxia as well as in prolonged, inescapable hypoxia, and they exhibit different oxygen consumption profiles and hypoxia sensitivities, possibly owing to specific metabolic oxygen demands and distinct behavioural and physiological response capacities. Therefore, the distinct habitat preferences of these galaxiid species are potentially a result of adaptive radiation in the context of the oxygen environment. These observations are relevant in regard to the anthropogenically caused increased expansion of hypoxic environments, affecting the natural habitats of inanga and, potentially, banded kokopu.
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