MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY LE THI HONG GAM EFFECTS OF NITRITE, TEMPERATURE AND HYPERCAPNIA ON PHYSIOLOGICAL PROCESSES AND GROWTH IN CLOWN KNIFEFISH (Chitala ornata, Gray 1831) DOCTORAL DISSERTATION OF AQUACULTURE Can Tho, 2018 MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY LE THI HONG GAM EFFECTS OF NITRITE, TEMPERATURE AND HYPERCAPNIA ON PHYSIOLOGICAL PROCESSES AND GROWTH IN CLOWN KNIFEFISH (Chitala ornata, Gray 1831) Major: Aquaculture Major code: 62 03 01 DOCTORAL DISSERTATION OF AQUACULTURE Supervisor Prof Dr NGUYEN THANH PHUONG Can Tho, 2018 Data sheet Title: Effects of nitrite, temperature and hypercapnia on physiological processes and growth in clown knifefish (Chitala ornata, Gray 1831) Subtitle: PhD Dissertation Author: Le Thi Hong Gam, PhD student code: P0613005 Major: Aquaculture, Major code: 62 62 03 01 Affiliation: Department of Nutrition and Aquatic Products Processing, College of Aquaculture and Fisheries, Can Tho University, Vietnam Publication year 2018 Cited as: Le Thi Hong Gam, 2018 Effects of nitrite, temperature and hypercapnia on physiological processes and growth in clown knifefish (Chitala ornata, Gray 1831) Doctoral Dissertation College of Aquaculture and Fisheries, Can Tho University, Vietnam Keywords: Climate change, air-breathing fish, clown knifefish, nitrite, temperature, hypercapnia, methaemoglobin reductase activity, acid-base balance, ion exchange Supervisors: Prof Dr Nguyen Thanh Phuong, College of Aquaculture and Fisheries, Can Tho University, Viet Nam Assoc Prof Dr Mark Bayley, Zoophysiology, Department of Bioscience, Aarhus University, Denmark Assoc Prof Dr Do Thi Thanh Huong, Department of Nutrition and Aquatic Products Processing, College of Aquaculture and Fisheries, Can Tho University, Viet Nam Assoc Prof Dr Frank Bo Jensen, Department of Biology, University of Southern Denmark, Odense, Denmark i Result commitment I commit that this dissertation was investigated based on all the results of my study All showed data and results in the dissertation were honest and have never been published before The iAQUA project can completely use these data and results Can Tho, 18th Nov, 2018 ii Acknowledgements Foremost, my sincere thanks go to my principal supervisors Nguyen Thanh Phuong and Do Thi Thanh Huong, who have given me the opportunity of studying, enthusiastic guidances and detailed revisions for my thesis as well as positive encouragements in any situation throughout my academic research process I would also like to express my deep gratitude to Mark Bayley, who has supported, inspired, built my passion in doing researches, also revised the manuscripts and shared the life experiences for me being more mature My deep thanks also give to Frank Bo Jensen, who has taught me the techniques related to my main research about nitrite toxicity and helped me in manuscript revisions as well as showed me Odense city in my trip to Denmark The invaluable supports from all of them from the first day I stepped into the iAQUA project have brought to what I have today Also, I would like to give my thanks to Tobias Wang, the staff and students in Zoophysiology Section, Department of Bioscience, Aarhus University for my stays in Denmark I sincerely thank for the positive supports from Christian Damsgaard, who showed me the knowledge related to acid-base regulation My thanks also go to Roy, John, Elin and Louise for showing Aarhus city and inviting me to their home My great thanks give to the staff and students in Department of Nutrition and Aquatic Products Processing, where my project was investigated I greatly thank to Nguyen Quoc Thinh, Le Thi Minh Thuy and Tran Minh Phu, who shared me their experiences about studying PhD, writing and publishing the articles I would like to give my thanks to my fellow friends in iAQUA project: Nguyen Thi Kim Ha, Le My Phuong, Phan Vinh Thinh, and Dang Diem Tuong for their friendships and what we enjoyed together from the working environment as well as all the funs outside the campus, especially Phan Vinh Thinh, who has cared me from meals and movements during my intensively experimental works and the trips in Denmark I also appreciate the positive co-operations from Master students such as Nguyen Thi Thuy Vu and Tran Trong Nhan; Bachelor students: Ma Thanh Quoc Tri, Pham Quoc Boong, Dinh Phuc Tai, Ly Thi Ngoc Huynh, Nguyen Ngoc Mai and Dao Dang Hoang Ngan during our academic activities iii I would like to thank my family and my friends for their love and spiritual supports during my study My special thanks give to my sister Le Thi Hong Dao, who has supported me about both financial and mental sides from Bachelor’s degree up to date She has always been my side for sharing happiness and sadness throughout my whole life Last but not least, thanks to all the sacrifired clown knifefish in my project iv Table of contents Data sheet i Result commitment ii Acknowledgements iii Table of contents v List of figures x List of tables xii List of abbreviation xiv Summary xvi Tóm tắt xviii Chapter INTRODUCTION 1.1 Introduction 1.2 The objectives of dissertation 1.3 The main projects of dissertation 1.4 The hypotheses of dissertation 1.5 New findings of the dissertation 1.6 Significant contributions of the dissertation References Chapter LITERATURE REVIEW 2.1 The status and importance of aquaculture and fisheries 2.2 Climate changes and impacts on aquaculture and fisheries 2.3 The status of farming clown knifefish (C ornata) in MD 10 2.4 Background about effects of some key environmental parameters on physiological processes and growth in aquaculture 11 2.4.1 Temperature 11 2.4.2 Nitrite (NO2-) 14 2.4.3 Hypercapnia (elevated level of carbon dioxide) and acid-base balance 18 References 20 Chapter (Paper 1) 29 v EXTREME NITRITE TOLERANCE IN THE CLOWN KNIFEFISH CHITALA ORNATA IS LINKED TO UP-REGULATION OF METHAEMOGLOBIN REDUCTASE ACTIVITY 29 3.1 Introduction 30 3.2 Materials and methods 32 3.2.1 Experimental animals 32 3.2.2 Determination of acute nitrite toxicity (96 h LC50) 32 3.2.3 Sub-lethal exposures and blood sampling 33 3.2.4 Analysis of haemoglobin derivatives 34 3.2.5 Plasma ion and protein analysis 34 3.2.6 Measurements of whole body water content 35 3.2.7 Methaemoglobin reductase activity 35 3.2.8 Statistics 36 3.3 Results 36 3.4 Discussion 45 3.4.1 Nitrite tolerance 45 3.4.2 MetHb reductase activity 46 3.4.3 Plasma ions 47 3.5 Conclusions 49 References 49 Chapter (PAPER 2) 54 EFFECTS OF NITRITE EXPOSURE ON HAEMATOLOGICAL PARAMETERS AND GROWTH IN CLOWN KNIFEFISH (Chitala ornata, GRAY 1831) 54 4.1 Introduction 55 4.2 Materials and methods 56 4.2.1 Effects of nitrite on haematological parameters in C ornata 56 4.2.2 Effects of nitrite on growth of C ornata 57 4.2.3 Data analysis 57 4.3 Results and discussion 58 4.3.1 Effects of nitrite on haematological paramters in C ornata 58 4.3.2 Effects of nitrite on growth parameters in clown knifefish C ornata 62 4.4 Conclusions 64 References 64 vi Chapter (PAPER 3) 69 THE EFFECTS OF ELEVATED ENVIRONMENTAL CO2 ON NITRITE UPTAKE IN THE AIR-BREATHING CLOWN KNIFEFISH CHITALA ORNATA 69 5.1 Introduction 71 5.2 Materials and methods 73 5.2.1 Animal holding 73 5.2.2 Experimental protocols 74 5.2.3 Analytical procedures 74 5.2.4 Statistics 76 5.3 Results 76 5.3.1 Acid-base parameters and plasma ions 76 5.3.2 Nitrite uptake and levels of Hb derivatives 81 5.4 Discussion 85 5.5 Conclusions 88 References 88 Chapter (Manuscript 1) 93 THE COMBINED EFFECTS OF NITRITE AND ELEVATED ENVIRONMENTAL CO2 ON HAEMATOLOGICAL PARAMETERS IN SMALL-SIZED CLOWN KNIFEFISH (CHITALA ORNATA) 93 6.1 Introduction 94 6.2 Materials and methods 95 6.2.1 Animal handling and experimental protocols 95 6.2.2 Statistics 96 6.3 Results 97 6.3.1 Combined effects of nitrite and carbon dioxide on haematological parameters in small-sized C ornata 97 6.3.2 Combined effects of nitrite and carbon dioxide on acid-base parameters and plasma ions in small-sized C ornata 103 6.4 Discussion 107 6.5 Conclusions 111 References 111 Chapter (Manuscript 2) 115 EFFECTS OF DIFFERENT TEMPERATURES ON HAEMATOLOGICAL PARAMETERS IN CLOWN KNIFEFISH (CHITALA ORNATA) 115 vii 7.1 Introduction 116 7.2 Materials and methods 117 7.2.1 Experimental animals 117 7.2.2 Determination of temperature limits in the clown knifefish 118 7.2.3 Effect of different levels of temperature on haematological parameters 118 7.3 Results 120 7.3.1 Temperature tolerance in C ornata 120 7.3.2 Effects of different temperatures on physiological parameters in small-sized C ornata 121 7.3.3 Effects of different temperatures on physiological parameters in large-sized C ornata 127 7.4 Discussion 134 7.5 Conclusions 136 References 136 Chapter (Manuscript 3) 141 EFFECTS OF NITRITE AT DIFFERENT TEMPERATURES ON HAEMATOLOGICAL PARAMETERS AND GROWTH IN CLOWN KNIFEFISH CHITALA ORNATA 141 8.1 Introduction 142 8.2 Materials and methods 143 8.2.1 Experimental animals and general experimental design 143 8.2.2 Determination of acute nitrite toxicity (96 h LC50) at 30ºC and 33ºC in C ornata 144 8.2.3 Sub-lethal nitrite exposures at different temperatures and blood sampling in C ornata 144 8.2.4 Analysis of haemoglobin derivatives 146 8.2.5 Effects of nitrite at different temperatures on growth and digestive enzyme activities in C ornata 146 8.2.6 Calculations 147 8.2.7 Statistics 147 8.3 Results 148 8.4 Discussion 159 8.4.1 Values of 96 h LC50 for nitrite at different temperatures in C ornata 159 8.4.2 Effects of nitrite at different temperatures in C ornata 161 viii Chapter 10 GENERAL DISCUSSIONS 10.1 Effects of nitrite exposure to physiological functions in C ornata In aquatic system, nitrite is produced from the nitrification and denitrification processes of bacteria The high load of nitrogenous materials, organic wastes can cause elevated nitrite concentration in aquaculture system and natural watercourse (Eddy and Williams, 1987; Hargreaves, 1998; Jensen, 2003), but normal concentrations in the water are relatively µM (Jensen, 2003) This study found that the nitrite levels in C ornata ponds were 0.0055–0.0075 mM (fish size of 450-500 g), 0.0042–0.006 mM (fish size of 200-250 g), and 0.002–0.0035 mM (fish size of 5-10 g) According to Boyd (1990), safe concentrations of nitrite in aquaculture ponds are below 0.3 mg/L (0.0065 mM) Therefore, it can be considered that certain nitrite levels in C ornata ponds cause no effects to physiological functions and growth for this species In addition, the new finding about 96 h LC50 of 7.82 mM nitrite indicated that the air-breather C ornata become one of the most nitrite tolerant species compared to other species such as the water-breather common carp (96 h LC50 of 2.9 mM; Lewis and Morris, 1986), the facultative air-breathing P hypophthalmus (96 h LC50 of 1.65 mM, Lefevre et al., 2011), or the obligate air-breathing C striata (Lefevre et al., 2012) In sublethal nitrite exposure, some previous studies in the air-breathing fish showed effective mechanism of denitrification converting nitrite to nitrate and maintained plasma nitrite below ambient nitrite concentration (Lefevre et al., 2011; 2012) Interesting, in line with this mechanism for the reduction of nitrite uptake, upregulation of metHb reductase enzyme in metHb reduction plays the key role for reducing nitrite uptake after week of nitrite exposure with the first rate constant rising from 0.01 in controls to 0.046 min-1 after days of nitrite exposure MetHb reductase is considered to be a member of the cytochrome family of enzymes coded in mammals by the CYB5R3 gene with two forms including a membranebound form and a soluble form, metHb reductase functions in metHb reduction in the erythrocyte cytoplasma of mammals with the soluble form (Hultquist and Passon, 1971; Jaffé, 1981) where this enzyme locates in nucleated fish RBCs with the membrane-bound form (Saleh and McConkey, 2012) The formation of metHb in human is resulted from the deficiencies in this gene (Percy and Lappin, 178 2008) Therefore, this is the first experimental evidence about up-regulation of this enzyme found in fish, showing an improvement in evolution of aquatic animals Although C ornata had high tolerance of nitrite by its internal mechanism for metHb reduction, there were some disturbances in some haematological parameters such as the significant decline in plasma osmolality induced by dilution of body fluids during nitrite exposure (Jensen et al., 1987; Jensen, 1990a, 1996; Harris and Coley, 1991; Grosell and Jensen, 2000) and a limitation of active ion uptake by electrolyte-water balance (Jensen, 1990b) The acid-base status in C ornata was affected by nitrite exposure via a significant respiratory acidosis during nitrite exposure This completely differs with a respiratory alkalosis caused by the hyperventilation from the reduced blood oxygen carrying capacity as elevations in metHb (Jensen et al., 1987; Aggergaard and Jensen, 2001; Hvas et al., 2016) However, a possibility is that the facultative air-breathing C ornata may change the mechanism of oxygen uptake in a waterborne toxicant by increasing air-breathing frequency and reducing gill ventilation (Lefevre et al., 2014) 10.2 Effects of nitrite exposure on growth in C ornata Growth parameters are in generally affected by the accumulation of organic matters, leading the formation of microbial metabolites including ammonia, nitrite and hydrogen sulfide into the water column inducing to chronic stress on fish in culturing process (Das et al., 2004) As a result, the stress may subsequently form the exhaustion, diseases and mortality in fish Similarly, C ornata at the present study had significant effects in nitrite exposure of mM nitrite with the lowest gain weight, specific growth rate, but the highest FCR compared to those in control, 0.2 mM and 0.4 mM Survival rate at this highest nitrite exposure reached below 60 %, where it was approximately 95 % at control, 90 % at 0.2 mM and 82% at 0.4 mM nitrite after months culturing In addition, there were different impacts in combination of nitrite exposure elevated temperatures on nitrite tolerance, physiological functions and growth parameters C ornata had rather high tolerance of temperature with upper and lower temperature limits (41ºC and 12ºC) Thus, the values of 96 h LC50 at different temperatures changed significantly with the highest tolerance for nitrite at 30ºC (96 h LC50 of 8.12 mM), where they were 7.82 mM and 6.75 mM at 27ºC and 33ºC, respectively As a result, there were negative effects to growth parameters Typically, the survival rate was lowest at 30ºC and 33ºC in mM nitrite exposure 179 (70-75%) For nitrite effect, C ornata can reduce nitrite uptake by denitrification converting nitrite to nitrate (Doblander and Lacker, 1997; Jensen, 2003; Lefevre et al., 2011; 2012; Gam et al., 2017) 10.3 Effects of elevated temperatures to physiogical parameters in C ornata In aquatic systems, temperature is a critical environmental factor affecting on physiological processes such a food digestion, growth, metabolism, immunity and locomotion (Zeng et al., 2009) Therefore, haematological parameters are considered to essential factors to access physiological and pathological changes in fish In the present study, there were significant increases in RBCs, WBCs and plasma glucose at the first days exposed at 33ºC and 36ºC This indicated higher oxygen requirement due to higher metabolic rates in the elevations of temperature in tropical fish (Engel and David, 1964) In addition, elevated temperature caused disturbance in acid-base status with significant decreases in pHe (Heisler et al., 1976; Boulitier et al., 1987; Damsgaard et al., 2018; Thinh et al., 2018) In exposures of high temperature, C ornata had the reduction in blood pH in line with the rise of PCO2 and unchanged value of plasma bicarbonate This is explained by the decrease of ventilation to metabolic CO2 production for transepithelial ion exchange (Rahn, 1966), and the elevation in PCO2 related to the rise of air-breathing frequency (Lefevre et al., 2016) This is similar to Thinh et al., 2018, where the air-breathing swamp eels had the same trend in acid-base status On contrast, P hypothalamus exposed to elevated temperature induced the rises in both PCO2 and plasma HCO3- (Damsgaard et al., 2018) 10.4 Combined effects of hypercapnia and nitrite on nitrite uptake and acidbase regulation in C ornata It is presented that the regulation of pHe in environmental hypercapnia impossibly happens in most air-breathing fish species due to structure of reduced gill surface area as well as the reduction in branchial irrigation resulting from the shift to airbreathing (Shartau and Brauner, 2014) However, C ornata in the present study had rather high capacity in acid-base regulation with 50% pHe compensation in 21 mmHg CO2 during 96 h via a reduction in HCO3-/Cl- Among tropical airbreathing fishes, C ornata only had lower capacity of acid-base status compared to the striped catfish P hypophthalmus with full compensation of extracellular pH in 34 mmHg CO2 during 72 h (Damsgaard et al., 2015) by rise of plasma HCO3- Addition, nitrite competes with chloride with the same uptake mechanism in the gills via Cl-/HCO3- exchange, where active Cl- uptake can be replaced by nitrite 180 uptake and passive Cl- loss leading to the efflux of Cl- (Jensen et al., 1987; Eddy and Williams, 1987; Jensen, 2003) Therefore, some previous studies tested the hypothesis that hypercapnia can limit the uptake of nitrite (Jensen et al., 2000; Hvas et al., 2016) with different findings Environmental hypercapnia in the crayfish Astacus astacus protected against nitrite toxicity by a reduced nitrite uptake (Jensen et al., 2000), where a respiratory acidosis only induced to the decline of nitrite uptake in the initial stage, but subsequently metHb and plasma increased causing the lowering of functional Hb (Hvas et al., 2016) When testing this hypothesis, C ornata showed the similar response to the crayfish (Jensen et al., 2000) Incomplete pH regulation after 96 h in acclimated hypercapnia reduced the nitrite uptake during next 96 h in the combined exposure of hypercapnia and nitrite via the significant rise of plasma HCO3- This is basically explained by the reduced branchial Cl-/HCO3- exchange activity, or the reduced branchial ventilation and metabolic rate leading a reduction in nitrite uptake Nevertheless, hypercapnia in air-breathing fish encourages the increase of gill ventilation, but in some other species, the rise of hypercapnic levels may cause an inhibition with a related increase in air-breathing (Boijink et al., 2010) The reductions in metHb and plasma nitrite were accompanied with the decreases in plasma ions (Na+, Cl-) and osmolality in combination of hypercapnia and nitrite, showing an inhibition of active transport for reducing nitrite uptake across the gills via chloridemediated acid-base regulation References Aggergaard, S., Jensen, F.B., 2001 Cardiovascular changes and physiological response during nitrite exposure in rainbow trout Journal of Fish Biology 59(1): 13–27 Boijink, C., Florindo, L.H., Leite, C.A.C., Kalinin, A.L., Milsom, W.K., Rantin, F.T., 2010 Hypercarbic cardiorespiratory reflexes in the facultative air-breathing fish jeju (Hoplerythrinus unitaeniatus): the role of branchial CO2 chemoreceptors Journal of Experimental Biology 213: 2797-2807 Boutilier, R.G., Glass M.L and Heisler, N., 1987 Blood gases and extracellular/intracellular acid-base status as a function of temperature in the anuran amphibians Xenopus laevis and Bufo marinus Journal of Experimental Biology 130: 13-25 Boyd, C E., 1990 Water Quality in Ponds for Aquaculture Birmingham Publishing Co Birmingham, Alabana 482 pages 181 Damsgaard, C., Gam, L.T.H., Dang, D.T., Van Thinh, P., Huong, D.T.T., Wang, T., Bayley, M., 2015 High capacity for extracellular acid-base regulation in the airbreathing fish Pangasianodon hypophthalmus Journal of Experimental Biology 1290–1294 Damsgaard, C., Thomsen M T., Bayley, M and Wang, T., 2018 Air-breathing changes the pattern for temperature-induced pH regulation in a bimodal breathing teleost Journal of Comparative Physiology Part B (in press) Das P.C., Ayyappan, S., Jena J.K., Das, B.K., 2004 Effect of sublethal nitrite on selected haematological parameters in fingerling, Catla catla (Hamilton) Aquaculture Research, 35(9): 874-880 Doblander, C., Lackner, R., 1997 Oxidation of nitrite to nitrate in isolated erythrocytes: a possible mechanism for adaptation to environmental nitrite Canadian Journal of Fisheries and Aquatic Science, 54: 157–161 Eddy, F.B., Williams, E.M., 1987 Nitrite and Freshwater Fish Journal of Chemical Ecology, 3: 1–38 Engel, D.M., Davis, E.M., 1964 Relationship between activity and blood composition in certain marine teleosts Copeia 3: 586–587 Gam, L.T.H., Jensen, F.B., Damsgaard, C., Huong, D.T.T., Phuong, N.T., Bayley, M., 2017 Extreme nitrite tolerance in the clown knifefish Chitala ornata is linked to up-regulation of methaemoglobin reductase activity Aquatic Toxicology, 187: 9– 17 Grosell, M., Jensen, F.B., 2000 Uptake and effects of nitrite in the marine teleost fish Platichthys flesus Aquatic Toxicology, 50: 97–107 Hargreaves, J.A., 1998 Nitrogen biogeochemistry of aquaculture ponds Aquaculture, 166: 181–212 Harris, R.R., Coley, S., 1991 The effects of nitrite on chloride regulation in the crayfish Pacifastacus leniusculus Dana (Crustacea: Decapoda) Comparative Biochemistry and Physiology B, 161: 199–206 Heisler, J N., Weitz, H and Weitz, A M., 1976 Extra and intracellular pH with changes of temperature in the dogfish Scryliorhinus stellaris Respiration Physiology, 26: 249-264 Hultquist, D.E., Passon, P.G., 1971 Catalysis of methaemoglobin reduction by erythrocyte cytochrome B5 and cytochrome B5 reductase Nature: New Biology 229, 252–254 Hvas, M., Damsgaard, C., Gam, L.T.H., Huong, D.T.T., Jensen, F.B., Bayley, M., 2016 182 The effect of environmental hypercapnia and size on nitrite toxicity in the striped catfish (Pangasianodon hypophthalmus) Aquatic Toxicology, 176: 151–160 Jaffé, E.R., 1981 Methaemoglobinaemia Clinical and Laboratory Haematology, 10: 99–122 Jensen, F.B., Andersen, N.A., Heisler, N., 1987 Effects of nitrite exposure on blood respiratory properties, acid-base and electrolyte regulation in the carp (Cyprinus carpio) Journal of Comparative Physiology B, 157: 533–541 Jensen, F.B., Koldkjaer, P., Bach, A., 2000 Anion uptake and acid-base and ionic effects during isolated and combined exposure to hypercapnia and nitrite in the freshwater crayfish, Astacus astacus Journal of Comparative Physiology, Part B, 170: 489–495 Jensen, F.B., 1990a Nitrite and red cell function in carp: control factors for nitrite entry, membrane potassium ion permeation, oxygen affinity and methaemoglobin formation Journal of Experimental Biology, 152: 149–166 Jensen, F.B., 1990b Sublethal physiological changes in freshwater crayfish, Astacus astacus, exposed to nitrite: haemolymph and muscle tissue electrolyte status, and haemolymph acid-base balance and gas transport Aquatic Toxicology, 18: 51–60 Jensen, F.B., 1996 Uptake, elimination and effects of nitrite and nitrate in freshwater crayfish Astacus astacus Aquatic Toxicology, 34: 95–104 Jensen, F.B., 2003 Nitrite disrupts multiple physiological functions in aquatic animals Comparative Biochemistry and Physiology A, 135: 9–24 Lefevre, S., Jensen, F.B., Huong, D.T.T., Wang, T., Phuong, N.T., Bayley, M., 2011 Effects of nitrite exposure on functional haemoglobin levels, bimodal respiration, and swimming performance in the facultative air-breathing fish Pangasianodon hypophthalmus Aquatic Toxicology, 104: 86–93 Lefevre, S., Jensen, F.B., Huong, D.T.T., Wang, T., Phuong, N.T., Bayley, M., 2012 Haematological and ion regulatory effects of nitrite in the air-breathing snakehead fish Channa striata Aquatic Toxicology, 118–119: 48–53 Lefevre, S., Domenici, P., McKenzie, D.J., 2014 Swimming in air-breathing fishes Journal of Fish Biology, 84: 661–681 Lefevre, S., Bayley, M., McKenzie, D.J., 2016 Measuring oxygen uptake in fishes with bimodal respiration Journal of Fish Biology, 88: 206–231 Lewis, W.M., Morris, D.P., 1986 Toxicity of Nitrite to Fish: A Review Transactions of the American Fisheries Society, 115: 183–195 183 Percy, M.J., Lappin, T.R., 2008 Recessive congenital methaemoglobinaemia: Cytochrome b5 reductase deficiency Br J Haematol 141, 298–308 Rahn, H., 1966 Aquatic gas exchange: theory Respiration Physiology 1: 1-12 Saleh, M.C., McConkey, S., 2012 NADH-dependent cytochrome b5 reductase and NADPH methemoglobin reductase activity in the erythrocytes of Oncorhynchus mykiss Fish Physiology and Biochemistry, 38: 1807–1813 Shartau, R.B., Brauner, C.J., 2014 Acid-base and ion balance in fishes with bimodal respiration Journal of Fish Biology, 84: 682–704 Thinh, P.V., Phuong, N.T., Brauner, C.J., Huong, D.T.T., Wood, A., Kwan, G., Conner, J., Bayley, M and Wang, T., 2018 Acid-base regulation in the air-breathing swamp eel (Monopterus albus) at different temperatures Journal of Experimental Biology (In press) Zeng, L.Q., Cao, Z.D., Fu, S.J., Peng, J.L., Wang, Y.X., 2009 Effect of temperature on swimming performance in juvenile southern catfish (Silurus meridionalis) Comparative biochemistry and physiology, Part A, 153: 125-130 184 Chapter 11 CONCLUSIONS AND RECOMMENDATIONS 11.1 Conclusions This thesis presents an incredible tolerance to nitrite in C ornata with maintainance of metHb levels at subcritical levels with denitrification process converting nitrite to nitrate and nitrite concentration in the plasma maintain lower than the ambient nitrite level during sub-lethal time Although there were the related changes in plasma osmolality, plasma ions, protein and whole body content in nitrite exposure, this is the first time for showing up-regulation of erythrocyte metHb reductase with rate constant for metHb reduction increased from 0.01 in controls to 0.046 min-1 after days of 2.5 mM nitrite exposure Growth rate of C ornata in chronic nitrite exposure of mM (50%*96 h LC50) was significantly lower than that in control, 0.2 mM and 0.4 mM nitrite with low survival rate (59%) and high FCR (4.5) after 90 days of culture There were negative effects to the number of blood cells; metHb, Hct and Hb concentration while MCHC had no effect by these nitrite exposures after 14 days This is the second time about acid-base regulation found in the air-breathing species C ornata in the MD Although pH regulation capacity in C ornata (50% of pH regulation after 96 h at 21 mmHg CO2) was lower than that in the facultative air-breathing P hypophthalmus (100% of pH compsentation in 72 h in 34 mmHg CO2), this capacity of pH regulation was significantly better than that in other water-breathing species In addition, acclimated hypercapnia caused a decreased nitrite uptake by an apparent reduced transport rate of the branchial HCO3-/Cl- exchanger C ornata had high tolerance of temperature with limitation of 50 % mortality (12ºC and 41ºC for lower and upper limit, respectively) due to their living habitat in the tropical area MD Therefore, there were no significant impacts in the changes of haematological parameters, but pHe and PCO2 significantly fluctuated in the temperature ranges from 24ºC - 33ºC in both juvenile and commercial fish size while the mortality after days exposed to 36ºC in commercial size induced by sharp decrease in pHe and Hb concentration C ornata had higher tolerance of nitrite at 30ºC compared to this at 27ºC and 33ºC In addition, the number of red blood cells was significantly influenced by 185 combination of nitrite and elevated temperature while metHb and plasma NO2only affected in the initial stage of exposure In general, growth parameters such as GW, SR had no negative effects by elevated temperature and the combined exposure of nitrite and elevated temperatures, but FCR reached the highest value of 1.95 in mM nitrite at 33ºC compared to that in 27ºC 11.2 Recommendations 11.2.1 Recommendations for intensive farming systems From the results of dissertation, some recommendations for optimizing water quality and minimizing impacts to physiological processes and growth of fish in aquaculture ponds under climate change are showed as below: 1) Reducing stocking density and changing water regularly for minimizing the accumulation of all waste products and generation of toxic gases and supplying sufficient levels of dissolved oxygen for the system 2) Designing ponds with optimal sizes, outlet and inlet cannas of water for conveniently for regularly removing the waste products and accumulated toxic sediments for good water quality 3) Supporting aeration systems in situations of elevated temperatures and high carbon dioxide levels for supplying sufficient dissolved oxygen in farming systems 11.2.2 Recommendations for further studies From the new findings that we found about the impacts of nitrite, temperature and hypercapnia on physiological responses and growth in C ornata in this dissertation, some studies are expected to conduct in the future as below: 1) Chronic effects of nitrite on growth parameters in C ornata after exposed to extremely high nitrite concentration in a short duration in C ornata 2) Combined effects of nitrite and hypoxia on haematological parameters, metabolic rate and growth in C ornata 3) Effects of hypercapnia at different temperatures on acid-base regulation and growth in C ornata 186 List of appendices Appendix 3.2.1 Information in the C ornata culture ponds 5-10 g/fish 200-250 g/fish 450-500 g/fish Area 800 m2 600 m2 900 m2 Depth 1.5 m 1.5 m 1.5 m 20,000 fish 4,700 fish 16,500 fish 25 fish/m2 7.8 fish/m2 18.75 fish/m2 twice a day (5 kg worm+5 kg trash fish+2 kg commercial pellet)/time twice a day 10 kg/time (pellet) Skretting, 40% protein once a day 62.5 kg/time Skretting, 40% protein Water exchanged daily by tide twice a month 25% of water once times/month 50% of water once Weekly, 2-3 kg CaCO3/100 m3 Weekly, 2-3 kg CaCO3/100 m3 Weekly, 2-3 kg CaCO3/100 m3 Twice a week, – g/kg feed Twice a week, – g/kg feed Twice a week, – g/kg feed Fish size Total stocked fish Stocking density Feeding Water exchange Lime adding Vitamin C adding 187 Appendix 9.3: Determing the values of 96h LC50 for nitrite at 27, 30 and 33ºC in C ornata (SPSS analysis) Confidence Limits At 27⁰C Probit 30⁰C 95% Confidence Limits for nitrite 33⁰C 95% Confidence Limits for nitrite 95% Confidence Limits for nitrite Lower Bound Upper Bound Estimate Lower Bound Upper Bound Estimate Lower Bound Upper Bound -1.8436 -6.07391 0.530851 1.185309 0.003457 2.098659 -0.33004 -1.59976 0.64797 0.02 -0.71076 -4.48589 1.425663 1.998829 0.938144 2.822752 0.500241 -0.64412 1.385601 0.03 0.007997 -3.48111 1.996168 2.514981 1.529944 3.283394 1.027026 -0.03888 1.85468 0.04 0.548688 -2.72708 2.427156 2.903262 1.974338 3.630712 1.423306 0.415732 2.20824 0.05 0.988497 -2.11512 2.779122 3.219099 2.335218 3.913829 1.745649 0.785005 2.496352 0.06 1.362845 -1.5954 3.079852 3.487925 2.641891 4.155298 2.020014 1.098891 2.742003 0.07 1.691074 -1.14071 3.344535 3.723634 2.91036 4.367442 2.260578 1.373746 2.957753 0.08 1.984964 -0.73449 3.582427 3.934683 3.150367 4.557767 2.475975 1.619527 3.151251 0.09 2.252245 -0.36587 3.799609 4.126623 3.3683 4.731204 2.671869 1.842763 3.32752 0.1 2.498278 -0.02734 4.000303 4.303305 3.56859 4.89117 2.85219 2.047984 3.490045 0.15 3.51692 1.364403 4.841097 5.034813 4.393912 5.557406 3.598768 2.894342 4.166253 0.2 4.326505 2.455063 5.524789 5.616193 5.043949 6.092812 4.192125 3.562057 4.708626 0.25 5.021056 3.374774 6.127313 6.114965 5.595893 6.557873 4.701172 4.130096 5.178733 0.3 5.644785 4.183205 6.685897 6.562878 6.085693 6.981374 5.158312 4.635259 5.605859 0.35 6.222762 4.912657 7.223188 6.977936 6.533424 7.379953 5.58192 5.098083 6.006939 0.4 6.771206 5.582601 7.755259 7.371785 6.951804 7.764639 5.983883 5.53153 6.393252 0.45 7.301832 6.206027 8.294797 7.752839 7.349815 8.143602 6.372787 5.944668 6.773241 0.5 7.824045 6.792854 8.852494 8.127851 7.734532 8.523542 6.755525 6.344535 7.153927 0.55 8.346258 7.352055 9.437818 8.502864 8.112194 8.910536 7.138263 6.737253 7.541762 0.6 8.876884 7.893005 10.05983 8.883917 8.488962 9.310743 7.527166 7.128844 7.943299 0.65 9.425328 8.426332 10.72852 9.277767 8.871602 9.731168 7.929129 7.525976 8.365927 0.7 10.00331 8.964695 11.4569 9.692825 9.268329 10.18075 8.352737 7.936863 8.818944 0.75 10.62703 9.524168 12.26444 10.14074 9.690188 10.67219 8.809877 8.372692 9.315403 0.8 11.32159 10.12742 13.18342 10.63951 10.15381 11.22558 9.318925 8.850428 9.875815 0.85 12.13117 10.81171 14.27349 11.22089 10.68795 11.87688 9.912281 9.399476 10.53685 0.9 13.14981 11.65301 15.66473 11.9524 11.35307 12.70332 10.65886 10.08161 11.37728 0.91 13.39584 11.85379 16.00317 12.12908 11.51282 12.90382 10.83918 10.24526 11.58138 0.92 13.66313 12.07106 16.37169 12.32102 11.68604 13.12197 11.03507 10.42265 11.8035 0.93 13.95702 12.30904 16.77783 12.53207 11.87616 13.36219 11.25047 10.61725 12.04818 0.94 14.28525 12.57382 17.23243 12.76778 12.08809 13.63087 11.49104 10.83411 12.32192 0.95 14.65959 12.87463 17.75206 13.0366 12.32935 13.93775 11.7654 11.08088 12.63469 0.96 15.0994 13.22669 18.36393 13.35244 12.61225 14.29885 12.08774 11.37015 13.00281 0.97 15.64009 13.65777 19.11787 13.74072 12.95934 14.74347 12.48402 11.72492 13.45621 0.98 16.35885 14.22837 20.12255 14.25687 13.41973 15.33553 13.01081 12.19532 14.06012 0.99 17.49169 15.1233 21.71045 15.07039 14.14351 16.27052 13.84109 12.93455 15.01416 Estimate 0.01 188 List of pictures about experimental setup, blood sampling and devices of analysis used in the studies 189 190 191 192 ...MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY LE THI HONG GAM EFFECTS OF NITRITE, TEMPERATURE AND HYPERCAPNIA ON PHYSIOLOGICAL PROCESSES AND GROWTH IN CLOWN KNIFEFISH (Chitala ornata,. .. 2018 Effects of nitrite, temperature and hypercapnia on physiological processes and growth in clown knifefish (Chitala ornata, Gray 1831) Doctoral Dissertation College of Aquaculture and Fisheries,... hypercapnia on physiological processes and growth in clown knifefish (Chitala ornata, Gray 1831) was necessarily conducted to have an understanding about effects and adaption mechanisms of this