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MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY LE MY PHUONG THE EFFECTS OF ELEVATED TEMPERATURE AND HYPOXIA ON THE RESPIRATORY ORGANS OF Pangasianodon hypophthalmus PHD DISSERTATION MAJOR: AQUACULTURE MAJOR CODE: 62 03 01 2018 MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY LE MY PHUONG THE EFFECTS OF ELEVATED TEMPERATURE AND HYPOXIA ON THE RESPIRATORY ORGANS OF Pangasianodon hypophthalmus PHD DISSERTATION MAJOR: AQUACULTURE MAJOR CODE: 62 03 01 SUPERVISORS Supervisor: Assoc Prof Dr DO THI THANH HUONG Co-supervisor: Assoc Prof Dr MARK BAYLEY 2018 DECLARATION I declare that this thesis is a scientific work implemented by myself All the results presented in the thesis are not published in any previous thesis or papers Supervisor PhD student Associ Prof Do Thi Thanh Huong i Le My Phuong Data sheet Title: The effects of elevated temperature and hypoxia on the respiratory organs of Pangasianodon hypophthalmus Subtitle: PhD Dissertation Author: Le My Phuong Affiliation: College of Aquaculture & Fisheries, Can Tho University, Vietnam; Zoophysiology, Department of Bioscience, Aarhus University, Denmark Publication year: 2017 Cite as: Phuong L.M (2017) The effects of elevated temperature and hypoxia on the respiratory organs of Pangasianodon hypophthalmus PhD Dissertation College of Aquaculture & Fisheries, Can Tho University, Vietnam and Zoophysiology, Department of Bioscience, Aarhus University, Denmark Keywords: climate change, hypoxia, temperature, morphometric, swim bladder, gill remodelling, air-breathing fish, Pangasianodon hypophthalmus Supervisor: Associate Professor Do Thi Thanh Huong, Deparment of Aquatic Nutrition and Products Processing, College of Aquaculture and Fisheries, Can Tho University, Vietnam Co-supervisor: Associate Professor Mark Bayley, Zoophysiology, Deparment of Bioscience, Aarhus University, Denmark Co-supervisor: Professor Jens Randel Nyengaard, Core Center for Molecular Morphology, Section for Stereology and Microscopy, Centre for Stochastic Geometry and Advanced Bioimaging, Aarhus University, Denmark ii Table of Contents Data sheet ii Table of Contents iii SUMMARY Chapter 1: INTRODUCTION 1.1 Oxygen requirement and adaptive mechanisms of fish to hypoxia .3 1.2 Characteristics of respiratory organs in striped catfish (Pangasianodon hypophthalmus) and adaptations to hypoxia 1.3 General gill structure in teleosts 1.4 Osmo-respiratory compromise in fish gills 1.5 Gill plasticity and environmental factors causing gill plasticity 1.6 Methods applied in morphometric studies 11 1.7 Objectives of dissertation 13 REFERENCES 15 Chapter (PAPER 1) 24 Recovery of blood gases and haematological parameters upon anaesthesia with Benzocaine, MS-222 or Aqui-S in the air-breathing catfish (Pangasianodon hypophthalmus) 24 Chapter (PAPER 2) 45 Ontogeny and morphometric of the gill and swim bladder of air-breathing striped catfish Pangasianodon hypophthalmus 45 Chapter (PAPER 3) 72 Gill remodelling and growth rate of striped catfish (Pangasianodon hypophthalmus) under impacts of hypoxia and temperatures 72 Chapter (PAPER 4) 101 Gill remodelling does not affect the rate of acid-base regulation in the striped catfish Pangasianodon hypophthalmus 101 Chapter 6: General discussion 118 Chapter 7: Conclusions and Perspectives 129 iii ACKNOWLEDGEMENTS Foremost, I would also like to give my thanks to Assoc Prof Do Thi Thanh Huong and Prof Nguyen Thanh Phuong in Can Tho University for always supporting, encouraging, and watching my steps during my study I would also like to express my gratitude to Assoc Prof Mark Bayley who has found and built my passions and directions for science as who I am and what I have today His guidance and enthusiasm have inspired and supported me moving forward and working by my best during my research and writing this thesis I am also grateful to Prof Jens Randel Nyengaard for teaching me stereological methods and supporting me during my working in his laboratory My sincere thanks also go to Prof Tobias Wang, Prof Atsushi Ishimatsu, and Prof Hans Malte for giving me special advices and precious and initiative suggestions which contribute importantly in my thesis Also, I acknowledge Christian Damsgaard who greatly contributed in interpreting and discussing data of the last manuscript in this thesis I would like to thank the staffs at the College of Aquaculture and Fisheries, Can Tho University (Vietnam), at the Zoophysiology Section, Department of Biological Sciences, and at Core Center for Molecular Morphology, Section for Stereology and Microscopy, Centre for Stochastic Geometry and Advanced Bioimaging, Aarhus University, Denmark where my projects were carried out Especially, I would like to thank Maj-Britt Lundorf for teaching me with helpful skills in histology and Per Guldhammer Henriksen for helping me during my experiments in Aarhus University I would also like to thank my fellow friends in iAQUA project: Nguyen Thi Kim Ha, Le Thi Hong Gam, Dang Diem Tuong, and Phan Vinh Thinh for their friendship and all the funs we have during the time we worked together in the project Finally, I would like to thank my family and my friends for their love and spiritually supporting me throughout my research, my writing, and my whole life My project was funded by The Danish International Development Agency (DANIDA), Ministry Affairs of Foreign Denmark, iAQUA project iv List of Tables Page Table 1.1 Anatomic diffusion factors (ADF, cm2µm-1kg-1) of gills and air-breathing organs (ABO) of different fish species Table 2.1 Effect of isoprenaline (10-5 mol l-1) on blood O2 saturation, Hct and MCHC Table 3.1 Relationship between body mass (Mb, g) and morphometrics (Y) of respiratory organs of Pangasianodon hypophthalmus according to the allometric equation Y = aMbb Table 3.2 Respiratory surface areas of lamellae (actual and potential) and swim bladder (mm2 g1 ); harmonic mean water- or air-blood barrier thickness (µm); total volumes of gill filaments and of swim bladder (mm3 g-1); percentages of secondary lamellar volume and air volume in swim bladders at different developmental stages of Pangasianodon hypophthalmus 35 54 58 Table 3.3 Anatomic diffusion factor (ADF) of gills and ABO of different fish species at the same size (100 g) 59 Table 3.4 Results of regression analysis from dimensions of respiratory organs (Y) in relation to body mass (Mb) in different fish species 65 Table 4.1 Lamellae respiratory surface area (mm2 g-1), harmonic mean water-blood barrier thickness (µm), volumes of gill filaments (mm3 g-1), the percentages of secondary lamellar volume and lamellae structural component volumes (pillar cells, blood spaces, epithelium) of Pangasianodon hypophthalmus exposed to elevated temperature and/or hypoxia Table 4.2 Evaluation the significant effects of temperature and/or hypoxia and parasites on growth and gill parameters of striped catfish (Pangasianodon hypophthalmus) v 83 84 List of Figures Page Fig 1.1 Respiratory branchial surface area and water-blood diffusion distance of different fish species Figure is adapted from Paper Fig 1.2 Schematic drawing and light micrographs show the sampling method for stereological observation applying VUR design for gills and swim bladder of Pangasianodon hypophthalmus 13 Fig 2.1 Time recorded for duration of anesthesia to reach the surgical plane, cannulation, and post-operative recovery of Pangasianodon hypophthalmus with the three anesthetics 30 Fig 2.2 Hematological and biochemical parameters of arterial blood following anesthesia in Pangasianodon hypophthalmus 31 Fig 2.3 Acid-Base status and chloride ions of arterial blood following anaesthesia in Pangasianodon hypophthalmus 33 Fig 2.4 Davenport diagrams of (a) Aqui-S, (b) MS222, and (c) Benzocaine Fig 3.1 Swim bladder processed for stereology and histological sections of swim bladder of Pangasianodon hypophthalmus Fig 3.2 Relative mass of the gill arches expressed as percentage of total 34 53 55 Fig 3.3 Bilogarithmic plots of lamellar surface areas in relation to body mass of Pangasianodon hypophthalmus 56 Fig 3.4 Bilogarithmic plots of swim bladder dimensions in relation to body mass of Pangasianodon hypophthalmus 57 Fig 3.5 Histological cross sections of gill filaments of Pangasianodon hypophthalmus before and after swimming 60 Fig 4.1 Respiratory lamella surface area according to body mass of juvenile striped catfish (Pangasianodon hypophthalmus) reared in different temperatures and/or hypoxia 81 Fig 4.2 vi Light micrographs of Pangasianodon hypophthalmus gill filaments from fish in different temperature and oxygen levels 81 Fig 4.3 Growth performance of juvenile striped catfish (Pangasianodon hypophthalmus) reared in different temperature and oxygen levels 85 Fig 4.4 Periodic specific growth rate (SGR) of juvenile striped catfish (Pangasianodon hypophthalmus) reared in different temperature and oxygen levels 86 Fig 4.5 Respiratory gill surface area and harmonic mean barrier thickness of several fish species with different life-styles and habitats 88 Fig 5.1 Light micrographs showing crossed sections of gill filaments of Pangasianodon hypophthalmus acclimated in hypoxia and and hyperoxia at 30°C following the hypercapnic exposure Fig 5.2 Davenport diagram showing acid-base regulation during hypercapnic exposure in cannulated Pangasianodon hypophthalmus being acclimated in hypoxia and hyperoxia Fig 5.3 Arterial blood parameters during hypercapnic exposure in cannulated Pangasianodon hypophthalmus being acclimated in hypoxia and hyperoxia vii 107 110 111 Sollid, J., De Angelis, P., Gundersen, K and Nilsson, G.E., 2003 Hypoxia induces adaptive and reversible gross morphological changes in crucian carp gills J Exp Biol, 206(20): 36673673 Sollid, J., Weber, R.E and Nilsson, G.E., 2005 Temperature alters the respiratory surface area of crucian carp Carassius carassius and goldfish Carassius auratus J Exp Biol, 208(6): 11091116 Stewart, P.A., 1978 Independent and dependent variables of acid-base control Respir physiol, 33(1): 9-26 Wood, C.M and Randall, D.J., 1973 The influence of swimming activity on sodium balance in the rainbow trout (Salmo gairdneri) J Comp Physiol A: Neuroethol Sens Neural Behav Physiol, 82(3): 207-233 Wood, C.M and Randall, D.J., 1973 The influence of swimming activity on water balance in the rainbow trout (Salmo gairdneri) J Comp Physiol A: Neuroethol Sens Neural Behav Physiol, 82(3): 257-276 117 Chapter 6: General discussion Well-developed gills in air-breathing Pangasianodon hypophthalmus In the majority of fish species, the gills represent the primary organ for fish respiration and they are also central in ionic and osmotic regulation, acid-base regulation, and in nitrogenous excretion (Evans et al., 2005; Diaz et al., 2009) As such, many studies have focussed on gill morphology as well as on changes in gill ultrastructure for better understanding the physiological state of fish in different environments (e.g Hughes and Flos, 1978; Perry, 1997; Mazon et al., 1998; Moron et al.,2009; Diaz et al.,2009) The basic structures of P hypophthalmus gills observed in this study are generally similar to other teleosts (Wilson and Laurent, 2002; Evans et al., 2005; Fernandes et al., 2012) The present study shows that respiratory lamella surface area of the air breathing striped catfish of 212 mm2 g-1 is surprisingly similar to that of active waterbreathing fish species such as Hoplias malabaricus (240 mm2 g-1; Sakuragui et al., 2003), Oncorhynchus mykiss (240 mm2 g-1; Hughes, 1972) and Tinca tinca (250 mm2 g-1; Hughes, 1972); much higher than the areas noted for other air-breathing fish species such as Arapaima giras (78 mm2 g-1; Fernandes et al., 2012), Channa punctatus (72 mm2 g-1; Hughes and Munshi, 1973; Hakim et al.,1978), Clarias mossambicus (22 mm2 g-1; Maina and Maloiy, 1986) The water – blood diffusion barrier of P hypophthalmus (3.22 µm) was very thin even compared to some mentioned water-breathers but was thicker than facultative air-breather Boleophthalmus boddarti (1.43 µm) These metrics demonstrate that the gills of P hypophthalmus are indeed well – developed This finding contributes to an explanation for the findings of Lefevre et al (2011) that P hypophthalmus has high capacity for aquatic respiration which can maintain its standard metabolic rate during normoxic condition by taking oxygen throughout its large gill surface area like other water-breathers It would also support for the findings of Damsgaard et al.(2015) that P hypophthalmus has high capacity for extracellular acid-base regulation during hypercapnia thanks to its large gill surface area while other air-breathing fish are inefficient with this mechanism Relationships between morphometrics of respiratory organs and body size in Pangasianodon hypophthalmus 118 It has long been known that fish gill SA in relation to body mass show a good fit with the equation Y=aWb This relationship has been confirmed for numerous water-breathing fish and several air-breathing fish Much of the variation in the exponent (b values), has been argued to be correlated with the activity level of the species and its habitat choice (Gray, 1954; Hughes, 1966; Palzenberger and Pohla, 1992; Fernandes et al., 1994; Severi et al., 1997) In the present study, the slopes of the regression lines for the actual and potential respiratory lamellar area against body mass were 0.83 and 0.92, respectively These values are within the range of 0.8 to 0.9 found in active water-breathing fish species, but significantly greater than found in other air-breathing fish, such as Anabas testudineus (0.615), Saccobranchus fossilis (0.746), Channa punctata (0.592) The high value of the slopes in P hypophthalmus also correlates with the active habits of this species with its strenuous migratory pattern between Laos and the Mekong river delta, a distance of more than 2000 Km (Zalinge et al., 2002; So et al., 2006) The regression line from log-log plot of swim bladder SA against body mass gave a slope of 0.714, which is smaller than that for gill SA (0.83 and 0.92, respectively for actual and potential SA) The slope of the regression line from the swim bladder volume of P hypophthalmus and body mass was 1.357, and the air volume in swim bladder against body mass was 1.482 It has previously been pointed out that the scaling relationship between respiratory SA and body size was similar to the relationship between oxygen consumption rates and body size in a variety of fish species, which log-log plot slopes also in the range of 0.8 to 0.9 (reviewed in Schmidt – Nielsen, 1984) However, it has been noted that these two slopes not always match (Singh and Munshi, 1985) The argument for this mismatch has typically been that the functional SA changes dramatically with oxygen demand as a result of recruitment of lamellae (Jones and Randall, 1978; Booth, 1978; Farrell et al., 1980; Wood and Perry, 1985; Perry and Wood, 1989) High plastic responses of P hypophthalmus gills to the different temperatures, hypoxic levels, and swimming activities This study found that there was a remodelling in the gills of P hypophthalmus Particularly, after weeks of experiment, the fish at 27ᵒC groups in both normoxic and hypoxic conditions were found with lamellae embedded partly or completely by a cell mass (ILCM), making the respiratory lamella surface area of these fish very low In contrast, the lamellae of groups at 33ᵒC were found to remain devoid of ILCM and hence a much larger surface area Remodelling of brachial surfaces 119 has been shown previously in carp exposed to hypoxic water, e.g in crucian carp Carassius carassius (Sollid et al., 2003), goldfish Carassius auratus (Sollid et al., 2005), and scaleless carp Gymnocypris prezewalskii (Matey et al., 2008) Sollid et al (2005) also found the gill remodelling in C carassius and C auratus could be induced by temperature changes, where lamellae were embedded by ILCM at low temperature and protruded at higher temperature Ong et al (2007) found mangrove killifish Kryptolebias marmoratus remodelled its gills in response to air exposure and Brauner et al (2004) found such gill morphological changes in Arapaima giras during the developmental programme transiting from water-breathing in juveniles to air-breathing in adults Nilsson (2007) hypothesized that fish gill remodelling could be an ancient mechanism, which could be present in many other teleost species but which remains largely unexplored Sollid and Nilssons (2006) stated that although gill remodelling occured in the crucian carp, the lamellae were always intact and the surface area changes were due to the embedding of cell mass between lamellae The present study agrees with this statement The lamellar surface is the primary site for gas exchange (Evans et al., 2005) and there is a correlation between lamellar surface area and respiratory needs among various fish species (Chapman et al., 2002) As already mentioned, gills of crucian carp have an increase in respiratory surface areas during hypoxia to increase oxygen uptake capacity (Sollids et al., 2003) In this study, however, P hypophthalmus showed a significant reduction of respiratory lamellae surface area at 27ᵒC when growing in either normoxic or hypoxic water This would indicate that striped catfish respond to temperature induced changes in metabolic oxygen demand rather than hypoxia in the environment This may be adaptive in minimising the costs much energy for other mechanisms such as ion or acid-base regulation from the environment In addition to lamellar recruitment changing SA, it has also been shown that ILCM can change the functional SA Thus, crucian carp (Carassius carassius) exposed to hypoxia in a closed respirometer at 20ᵒC significantly reduced their ILCM within 6h (Sollid et al., 2005) This species also reduced ILCM volume by 65% within 8h of an increase in swimming speed (Brauner et al., 2011) Similarly, the closely related goldfish (C auratus) lost 18% of ILCM within 30 of exposure to hypoxia at 7ᵒC (Tzaneva et al., 2011), and Fu et al (2011) found that 48h exposure to hypoxia or sustained exercise (70% of Ucrit) induced a significant increase in lamellar SA Perry et al (2012) found that while the gills of goldfish acclimated to low temperature (7ᵒC) were filled by ILCM, the SA increased by 45% after approximately 3h of swimming by reducing the ILCM 120 Similarly, in the present study we found that P hypophthalmus at 27ᵒC & normoxia have their gills partly embedded by ILCM, making their respiratory gill SA less 30% of their maximal area Swimming the fish for 20h caused an increase in SA, with the effect most pronounced at 33ᵒC where oxygen demand is highest The mechanisms involved in these changes in ILCM are at present unclear but Nilsson at al (2012) suggested two possible routes of ILCM removal: a slow route through increased apoptosis and decreased mitosis (Sollid et al., 2003) and a rapid route through simple shedding of ILCM cells into the water Given the rapidity of the changes incurred during swimming in both P hypophthalmus and goldfish it is likely that simple shedding of cells occurs during these urgent increases on oxygen demand It is clear that the main mediator of gill ADF is the oxygen demand of the fish Swimming has a large effect on τh in our study, which might be expected through increased lamella perfusion causing distension of the lamellae (Sovio and Tuurala, 1981) The temperature change alone in our study had no effect on gill diffusion distance τh but did cause a minor significant increase in gill SA This is in line with our previous findings in this species where the same temperature increase caused only a minor effect on SA unless combined with hypoxia (Phuong et al., 2017) In the eel (Anguilla anguilla), a larger temperature change (7 to 25ᵒC) was associated with a halving of the diffusion distance τh (Tuurala et al., 1998) The combined oxygen demand caused by both swimming and elevated temperature then led to both distension and hence thinning of τh and a massive increase in SA as a result of shedding of the entire ILCM It would be interesting to further investigate time scale of modulation of ILCM to determine how it might match the dynamics of the shifting oxygen environment and energetic needs of the animal The role of ILCM in offsetting negative costs of osmo-respiratory compromise has been discussed on numerous occasions (Nilsson et al., 2012; Perry et al., 2012; Phuong et al., 2017), but the costs of building and shedding of cell mass are presently unknown The effects of oxygen and temperature on growth of striped catfish This study demonstrated the significant effects of warm temperature (33ᵒC) on growth performance of striped catfish Pangasianodon hypophthalmus Particularly, the fish grew very fast at this temperature level, and were nearly 8-fold bigger than the groups at 27ᵒC over a period of 16 weeks There have been many studies on the effects of temperature on growth rate of many fish 121 species (Imsland et al., 2006; Jonassen et al., 2000; Handeland et al., 2008; Fang et al., 2010; Guerreiro et al., 2012), which maintained that elevated temperatures could drive fish growth It has been found that there is a correlation between elevated temperatures and growth up to the optimum point above which thermal stress occurs (Elliott and Hurley, 1997; Baum et al., 2005), and thanks to such relationship, there have been growth models developed in aquaculture, which allows for theoretically predicting potential growth changes as fish growth rate and final body weight, where adjustments in temperature lead to considerable decreases in time to market (Elliott and Hurley, 1997; Reid et al., 2015) Besides that, Guerreiro et al (2012) also found that feed intake, and feed efficiency of the fish observed were higher at higher temperature even the fish used low-protein diets; or the digestibility of dry matter and protein of salmonid fishes found were positively correlated to elevated temperature (Nicieza et al.,1994; Azevedo et al.,1998) It has not been known yet the temperature optimum for growth of P hypophthalmus, however it has been stated that the optimal temperature for growth is usually higher than the temperature level which the animals meet in nature (Imsland et al., 1996) In nature, the water temperature in striped catfish ponds is around 27-30ᵒC, however, it can be seen from this study that at 27ᵒC, feed intake (personal observation) and growth rate of P hypophthalmus were suppressed as compared to those at 33ᵒC In addition, Lefevre et al (2011b) supposed that under hypoxia condition, it would cost P hypophthalmus more energy for air-breathing and it would be benefit on growth and production if supply more oxygen into the striped catfish ponds This study has demonstrated that ambient oxygen saturation significantly affected the growth performance of P hypophthalmus (P