Ảnh hưởng của nhiệt độ, điều kiện oxy thấp và CO2 cao lên hô hấp và sinh lý của cá thát lát còm chitala ornata (gray, 1831)

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY DANG DIEM TUONG THE EFFECTS OF TEMPERATURE, HYPOXIA ANDHYPERCARBIA ON RESPIRATION AND PHYSIOLOGY OF CLOWN KNIFEFISH CHITALA ORNATA (GRAY, 1831) DOCTORALDISSERTATION MAJOR: AQUACULTURE MAJOR CODE: 62 03 01 2018 MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY DANG DIEM TUONG THE EFFECTS OF TEMPERATURE, HYPOXIA AND HYPERCARBIA ON RESPIRATION AND PHYSIOLOGY OF CLOWN KNIFEFISH CHITALA ORNATA (GRAY, 1831) DOCTORALDISSERTATION MAJOR: AQUACULTURE MAJOR CODE: 62 03 01 Supervisors Prof Dr TRAN NGOC HAI 2018 Data sheet Title: The effects of temperature, hypoxia and hypercarbia on respiration and physiology of Clown knifefish Chitala ornata (Gray, 1831) Subtitle: PhD Dissertation Author: Dang Diem Tuong Affiliation: College of Aquaculture and Fisheries, Can Tho University, Vietnam Publisher: Can Tho University Publication year: Citation: Tuong, D D., 2018 The effects of temperature, hypoxia and hypercarbia on respiration and physiology of Clown knifefish Chitala ornata (Gray, 1831) PhD Dissertation, College of Aquaculture and Fisheries, Can Tho University, Vietnam Supervisors: Prof Dr Tran Ngoc Hai, College of Aquaculture and Fisheries, Can Tho University, Vietnam Co-supervisors: Assoc Prof Dr Do Thi Thanh Huong, Department of Nutrition and Aquatic Products Processing, College of Aquaculture and Fisheries, Can Tho University, Vietnam Assoc Prof Dr Mark Bayley, Zoophysiology, Department of Bioscience, Aarhus University, Denmark TABLE OF CONTENTS TABLE OF CONTENTS i ACKNOWLEDGEMENTS iii SUMMARY iv TÓM TẮT vi LIST OF FIGURES ix LIST OF TABLES .xiii ABBREVIATIONS xiv CHAPTER 1: INTRODUCTION 1.1 General introduction 1.2 Research objectives 1.3 Research contents/activities References CHAPTER 2: 10 LITTERATURE REVIEW 10 Temperature and hypoxia 10 Temperature and hypoxia: their effects on metabolism of air-breathing fishes 11 Gill remodeling 14 Hypercarbia and its effect on cardioventilatory responses 16 Air-breathing fish species 18 Clown knifefish (Chitala ornata) 21 References 23 CHAPTER 3: 38 CLOWN KNIFEFISH (CHITALA ORNATA) OXYGEN UPTAKE AND ITS PARTITIONING IN PRESENT AND FUTURE ENVIRONMENTS 38 Abstract 38 Introduction 39 Materials and methods 40 Results 44 Discussion 47 References 55 CHAPTER 4: 63 GILL REMODELING OF CLOWN KNIFEFISH (CHITALA ORNATA) UNDER IMPACT OF TEMPERATURE AND HYPOXIA 63 Abstract 63 Introduction 64 i Materials and methods 65 Results 69 Discussion 72 Conclusions 77 References 78 CHAPTER 5: 82 VENTILATORY RESPONSES OF THE CLOWN KNIFEFISH, CHITALA ORNATA, TO HYPERCARBIA AND HYPERCAPNIA 82 Abstract 82 Introduction 83 Materials and methods 85 Results 88 Discussion 91 References 96 CHAPTER 6: 102 VENTILATORY RESPONSES OF THE CLOWN KNIFEFISH, CHITALA ORNATA, TO AMBIENT WATER AND AIR HYPERCARBIA, AND HYPERCAPNIA IN DENERVATED FISH 102 Abstract 102 Introduction 103 Materials and methods 105 Results 108 Discussion 110 References 117 CHAPTER 7: 123 GENERAL DISCUSSIONS 123 CHAPTER 8: 132 CONCLUSIONS AND PERSPECTIVES 132 ii ACKNOWLEDGEMENTS First of all, I would like to give my deep appreciations to Assoc Prof Do Thi Thanh Huong, Prof Nguyen Thanh Phuong and Prof Tran Ngoc Hai of Can Tho University who supported, encouraged as well as straightened my direction during my study They have been great teachers who were willing to help me solving my troubles throughout the entire process and without them my PhD thesis would not have been finished My biggest thank go to Assoc Prof Mark Bayley who set a fire of passion on science and gave me a direction to become a real physiological scientist Moreover, he has made valuable connections between many famous scientists and young passionate scientists all over the world I myself felt like a real scientist among them that I have been highly inspired during my five-year PhD I would like to thank Prof William K Milsom who have taught me a lot about ventilation and chemoreceptors that contributed haft of my thesis contents Working with him was my biggest pleasure and fortune from the start to the end of this iAqua project that I have been luckily involved My thanks would like to go to Prof Tobias Wang at Aarhus University and Prof Jens Randel Nyegaardfrom Aarhus University Hospital who have supported and given me helpful advises and cares during time I have been in Denmark I would like to give my sincere thanks to Prof Atsushi Ishimatsu from Japan who was not only an expected teacher on cannulation in lab, but also a nice friend sharing many life experiences I also wish to thank staff members of the College of Aquaculture and Fisheries, Can Tho University, Vietnam; and of the Zoophysiology Section, the Department of Biological Science; the Stereology and Microscopy of AarhusUniversity Hospital, Denmark that have supported, and taught me laboratory skills during the time I have studied there I would like to give my thanks to my friends in iAQUA project, Nguyen Thi Kim Ha and Le My Phuong, who have shared interesting experiences and feeling during time we had been in Denmark Phan Vinh Thinh, Le Thi Hong Gam and Cristance Damgaard have supported my studies and brought me laughing moments to overcome stressful time I would like to thank all hard working students who have helped me in doing research works Finally, I would like to thank my family, my fiancée and my friends that always love and spiritually support me throughout my research on the way to achieve my PhD and all And thanks for all sacrificed fish! This thesis was included in iAQUA project funded by the Danish International Development Agency (DANIDA), Ministry Affairs of Foreign Denmark SUMMARY Climate change is one of the most concerns in scientific research regarding its effects on physiology, growth, adaption and/or extinction of aquatic animals Chitala ornata is an important species in aquaculture which has been investigated in this study to provide profound knowledge for assessment of elevated temperature and CO2 increase Respiratory physiology, cardiorespiratory responses, gill morphological adaptation and growth were target parameters to evaluate through four studies Respiratory responses to elevated temperature and hypoxia have been investigated in the first study Oxygen threshold (Pcrit), standard metabolic rate (SMR), specific dynamic action (SDA) and the growth under the effects of predicted elevated temperature (33°C) and average present temperature (27°C) in both normoxia (95% of oxygen saturation) and hypoxia (25% and 35% oxygen saturation) have been carried on C.ornata It has been found that at a worst-case model temperature for Mekong delta did not induce negative impact on respiratory physiology of C ornata Growth has actually been observed to increase at elevated temperature Air-breathing oxygen was an important ability of C ornata to diminish the effects of a severe hypoxic condition especially at the elevated temperature It is, however, important to consider that reliance on air-breathing may consequently bring disadvantages to fish such as energetic costs and fail of full oxygen saturation Ability of gill plasticity in C ornata under the effects of temperature (33 and 27°C) combining to normoxia (95% oxygen saturation)and hypoxia (25% and 35% of oxygen saturation) applying vertical sections in stereology were examined Results have shown that C ornata was able to transform the gill morphology which interlamellar cell mass (ILCM) was found to increase in the normoxia and decrease at the elevated temperature and in hypoxia Surface area (SA) of respiratory lamellae was significantly affected by the temperature and hypoxia after one month Harmonic mean water blood thickness significantly reduced by the hypoxia after one month while that reduction induced by the temperature took two months to have significant effects An anatomic diffusion factor (ADF) was found 4-fold higher at 33°C in hypoxia comparing to 27°C in normoxia The surface area of C ornata gills was consistent with those of air-breathing fish These results found in C ornata support the hypothesis of anciently long-term existence of the gill remodeling mechanism Hypercarbia and hypercapnia induced cardiorespiratory responses promoted + by CO2/H -sensitive chemoreceptors of C ornata have been investigated in the third study Intact C ornata has been exposed to acid water (pH=6), hypercarbic (CO2 increase, ~pH=6) and hypercapnic condition (injection of acetazolamide) at normocarbia We measured the changes of air-breathing frequency, gill ventilation frequency, heart rate, arterial blood pressure, and blood pH and plasma CO2 In acidosic condition, C ornata did not respond significant changes of any observed parameters It has been found that C ornata responded to the environmental hypercarbia and blood hypercapnia which dramatically increased air-breathing frequency but no significant changes of the gill ventilation, and revealed a modest bradycardia and fall in + the arterial blood pressure The blood [H ] and plasma PCO2 have been found to increase in both hypercarbia and acetazolamide The acetazolamide results provide an evidence of internally oriented cardiorespiratory CO2/H chemoreceptors existing in the facultative air-breathing, C ornata + Investigating cardioventilatory responses under the effects of CO2 injection into air-breathing organ (ABO) of intact C ornata, and the effects of the hypercarbia and hypercapnia on denervated C ornata were conducted in the last study The ascending CO2 percentages mixed with the air were injected th th into ABO Denervation of IX and X cranial nerves were performed in C ornata which were exposed to the hypercarbia (CO2 increase ~pH=6) and the + acetazolamide (internal [H ] and PCO2 increase) after 24h of recovery It has been found that both intact and denervated C ornata responded significant airbreathing frequencies Bradycardia and no significant changes of the gill + ventilations were also found in all treatments The increase of internal [H ]and PCO2 were found in all treatments of CO2 injection into ABO, and the hypercarbia and hypercapnia The results of CO2 injection into ABO of the intact fish, and the hypercarbia and hypercapnia in the denervated fish additionally gave documentation to confirm the existence of internally + oriented cardiorespiratory CO2/H -sensitive indirectly inferable to central chemoreceptors chemoreceptors and were TĨM TẮT Biến đổi khí hậu vấn đề quan tâm nghiên cứu khoa học phương diện ảnh hưởng lên sinh lý, tăng trưởng, thích nghi và/ diệt vong động vật thủy sản Cá thát lát còm (Chitala ornata) lồi quan trọng ni trồng thủy sản chọn nghiên cứu để cung cấp thông tin chuyên sâu đánh giá tác động tăng nhiệt độ nồng độ CO2 Các tiêu sinh lý hô hấp, phản ứng hô hấp tim mạch, thích nghi hình thái mang cá tăng trưởng tiêu thực để đánh giá ảnh hưởng biến đổi khí hậu thông qua bốn nghiên cứu Phản ứng hô hấp cá theo tăng nhiệt độ nồng độ oxy thấp thực nghiên cứu thứ Ngưỡng oxy (Pcrit), trao đổi chất (SMR), tác động tiêu hóa thức ăn lên hoạt động hơ hấp (SDA) tăng trưởng cá thát lát ảnh hưởng nhiệt độ dự báo (33°C) nhiệt độ trung bình (27°C) kết hợp với hàm lượng oxy bão hòa oxy thấp thực Kết cho thấy mức nhiệt độ dự báo cao vùng đồng sông Cửu Long không ảnh hưởng tiêu cực lên sinh lý hô hấp cá Tăng trưởng cá tăng mức nhiệt độ cao Khả hơ hấp khí trời đặc điểm quan trọng giúp cá giảm bớt ảnh hưởng hàm lượng oxy thấp, đặc biệt môi trường nhiệt độ tăng Tuy nhiên, điều quan trọng cần cân nhắc việc dựa vào khả hô hấp khí trời dẫn tới hậu bất lợi cho cá hao tốn lượng giảm khả bão hòa oxy máu Khảo sát khả biến đổi cấu trúc mang cá ảnh hưởng nhiệt độ (33 27°C) kết hợp với mơi trường oxy bảo hòa thiếu oxy áp dụng phương pháp mô học lập thể tiến hành thí nghiệm Kết cho thấy cá có biến đổi hình thái mang cách tăng sinh giảm sinh số lượng tế bào mang thứ cấp (ILCM) mơi trường oxy bão hòa, mơi trường thiếu oxy nhiệt độ cao Diện tích bề mặt (SA) hô hấp mang bị ảnh hưởng nhiều nhiệt độ tình trạng thiếu oxy sau 30 ngày nuôi Khoảng cách khuếch tán máu nước giảm môi trường thiếu oxy sau 30 ngày ni giá trị giảm nhiệt độ thể rõ sau60 ngày ni Tính tốn giá trị hệ số hô hấp (ADF) cho thấy số cao gấp lần mức nhiệt độ 33°C môi trường oxy thấp so với mức 27°C mơi trường oxy bão hòa Diện tích bề mặt mang cá thát lát phù hợp với loài cá hơ hấp khí trời Các kết nghiên cứu củng cố cho giả thuyết tồn lâu dài chế biến đổi cấu trúc mang tác động yếu tố môi trường Nghiên cứu nồng độ CO2 cao môi trường (hypercarbia) máu (hypercapnia) cá đến phản ứng hô hấp tim mạch thúc đẩy thụ + cảm CO2/H cá thực nghiên cứu thứ ba Cá ni mơi trường nước có tính a-xit (pH=6), mơi trường CO2 (tăng CO2 mơi trường nước, pH=6) môi trường CO2 cao máu (tiêm acetazolamide) điều kiện oxy bão hòa Thu mẫu phân tích tiêu tần số hơ hấp khí trời, tần số hô hấp qua mang, nhịp tim, áp suất máu động mạch, pH PCO2 máu Trong mơi trường a-xit (pH=6) thay đổi phản ứng cá khơng có ý nghĩa thống kê tất tiêu khảo sát Cá phản ứng lại môi trường hypercarbia môi trường hypercapnia qua tăng tần số hơ hấp thay đổi khơng có ý nghĩa so với tần số hô hấp qua mang; bên cạnh + phản ứng nhịp tim chậm giảm ấp suất máu cá Nồng độ [H ]và PCO2 máu cá tăng môi trường hypercarbia môi trường hypercapnia (tiêm acetazolamide) Kết nghiên cứu cung cấp sở chứng cho + diện thụ cảm CO2/H định hướng bên tồn lồi cá hơ hấp khí trời C ornata Khảo sát phản ứng hô hấp tim mạch ảnh hưởng hypercapnia cách tiêm CO2 vào quan hơ hấp khí trời (ABO) cá khơng cắt dây thần kinh, ảnh hưởng hypercarbia hypercapnia lên cá cắt dây thần kinh thực nghiên cứu thứ tư Liều tiêm CO2 vào bong bóng khí cá tăng dần theo nồng độ CO2có hỗn hợp CO2 khơng khí Tiến hành th th cắt dây thần kinh thứ IX X , cho cá hồi phục 24 giờtrước cho tiếp xúc với hypercarbia (tăng CO2 ~ pH=6) acetazolamide (tăng + [H ]và PCO2 máu) Kết cho thấy cá không cắt dây thần kinh vàcắt dây thần kinh phản ứng tăng tần số hô hấp khí trờiđáng kể Nhịp tim chậm khơng có thay đổi đáng kể tần số hô hấp qua mang thể tất + cá nghiệm thức Sự gia tăng [H ]và PCO2 máu ghi nhận tất mức nồng độ tiêm CO2 vào ABO, hypercarbia hypercapnia Các kết tiêm CO2 vào ABO (không cắt dây thần kinh), hypercarbia hypercapnia cung cấp thêm thông tin chứng minh tồn + thụ cảm CO2/H định hướng bên gián tiếp cho thấy diện quan thụ cảm trung tâm vii de Lima Boijink, C., Florindo, L H., Leite, C A., Kalinin, A L., Milsom, W K., & Rantin, F T (2010) Hypercarbic cardiorespiratory reflexes in the facultative air-breathing 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Abe, A S., & Crossley, D A (2014) The phylogeny and ontogeny of autonomic control of the heart and cardiorespiratory interactions in vertebrates Journal of Experimental Biology, 217(5), 690-703 Thomsen, M T., Wang, T., Milsom, W K., & Bayley, M (2017) Lactate provides a strong pH-independent ventilatory signal in the facultative airbreathing teleost Pangasianodon hypophthalmus Scientific Reports, 7(1), 6378 121 Tuong, D D., Borowiec, B., Clifford, A M., Filogonio, R., Somo, D., Phuong, N T., Milsom, W K (2018a) Ventilatory responses of the clown knifefish, Chitala ornata, to hypercarbia and hypercapnia Journal of Comparative Physiology B, 1-9 Tuong, D D., Ngoc, T B., Huynh, V T N., Phuong, N T., Hai, T N., Wang, T., & Bayley, M (2018b) Clown knifefish (Chitala ornata) oxygen uptake and its partitioning in present and future temperature environments Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 216, 52-59 Ultsch, G R (1987) The potential role of hypercarbia in the transition from water‐breathing to air‐breathing in vertebrates Evolution, 41(2), 442-445 Vidthayanon, C (2012) Chitala ornata The IUCN Red List of Threatened Species 2012 doi: 10.2305/IUCN.UK.2012-1.RLTS.T181056A1693604.en Viet, T V (2015) Applications of GIS for evaluation the current culture status of Clown knife fish (Chitala ornata) in Phung Hiep District, Hau Giang Province Journal of Can Tho University, 38, 109-115 Wilson, R., Harris, M., Remmers, J., & Perry, S (2000) Evolution of airbreathing and central CO(2)/H(+) respiratory chemosensitivity: new insights from an old fish? Journal of Experimental Biology, 203(22), 35053512 Wood, C., & Munger, R (1994) Carbonic anhydrase injection provides evidence for the role of blood acid-base status in stimulating ventilation after exhaustive exercise in rainbow trout Journal of Experimental Biology, 194(1), 225-253 Wood, C., Turner, J., Munger, R., & Graham, M (1990) Control of ventilation in the hypercapnic skate Raja ocellata: II Cerebrospinal fluid and intracellular pH in the brain and other tissues Respiration physiology, 80(2-3), 279-297 122 CHAPTER 7: GENERAL DISCUSSIONS Evaluation of methods used in the dissertation Methods and techniques used in researches were core important to explore many intriguing mysteries in science The method and technique decide validation, accuracy and precision of data and results Throughout this dissertation, the methods and techniques used were discussed and decided with professional advisors of fields In the first paper, bimodal intermittentclosed respirometry was a main method that was used recently (Lefevre et al., 2011, 2016) and developed at Zoophysiology section of Aarhus University This system of respirometry allows measuring both aerial and watering oxygen consumption of fish Oxygen electrode sensors (Visi Pro DO EX 120, Hamilton company)were calibrated to cope with area barometer pressure for an accurate and fast record of every that was important to have unbiased estimation of SMRs and Pcrits The intermittent closure of system and flush of water for 30 period to avoid accumulation of CO2, waste products and hypoxic water (Steffensen, 1989) reduced fish disturbance and stress In addition to measurement of SMRs, fish was placed into system in the morning and allowed to stable in a quiet and stable temperature room At the end of each fish measurement, all component of system were washed by chlorine water and chlorine free tap water to eliminate bacterial development (Lefevre et al., 2011) Growth and gill morphological studies under effect of temperature and hypoxia were performed in recirculating aquaculture system (RAS) which could maintain certain oxygen levels coinciding to each treatment by a control oxygard Pacific system (Oxyguard, Denmark) and other environmental parameters held within suitable ranges (Tuong et al., 2018b) In the second paper and first manuscript, C ornata was catheterized into dorsal aorta (Soivio et al., 1975) Fish was allowed to recover blood parameters to normal state for 24 h (Phuong et al., 2017a) Applying this technique in cardioventilatory study to take blood whilst holding fish in tank to continuously run experiment was significant important to have unstressed and reliable blood results at every sampling time In addition to blood gas analysis, an iSTAT was used to measure blood gas which was reported a potential temperature problem (Malte et al., 2014), and also pointed in trout for the accurate temperature of 10-20°C and the unreliable at low PCO2 (Harter et al., 2014) However, it was showed that PCO2 measured by iSTAT 123 at 30°C in tropical air-breathing fish was accurate when comparing with PCO2 results from tonometers (Damsgaard et al.,2015) Therefore, it is believed that the C ornata blood gas results from iSTAT were still reliable Another important method used in this dissertation was stereology which was applied to estimate respiratory surface area and water-blood diffusion distance This method was applied in human for more than 40 years (Weibel, 1963) while fish gill and organs were still estimated by many biased and potential errors methods (Price, 1931; Gray, 1954; Hughes, 1966, 1972; Hamkim et al., 1978; Mazon et al., 1998) The stereological method, using geometrical and statistical principles to estimate unbiased and quantitative target organ including surface area, volume and thickness, has been used for gill and gas exchange morphometric research recently including Lepidosiren paradoxa, Arapaima gigas, Pangasianodon hypophthalmus (de Moraes et al., 2005; da Costa et al., 2007; Fernandez et al., 2012; Phuong et al., 2017b, 2018) It has been also combined with random systematic sampling, cavalieri and vertical uniform random sections (VUR) allowing to significantly reduce the amount of tissues samples analysis Adaptive mechanism to climate change of clown knifefish Near et al (2012) has showed that the latest Actinterygian time-calibrated phylogeny in which C.ornata, a facultative air-breathing, an ancient fish species exists at least 300 million years ago During evolutionary pressure of long time of history, it is believed that C ornata is holding keys of many successful traits to survive up to date By exploring physiological aspects including respiratory metabolism, cardioventilation, gill morphological transformation and growth under three main effects of climate change including elevated temperature, hypoxia and elevated CO2 in the environment have revealed many dramatically important results that influences were broadly extensive in fish species In Vietnam, clown knifefish has been grown intensively in Mekong River Delta, especially in Hau Giang province (Viet, 2015) With a rapid development, C ornata is facing many consequences of environmental changes including hypoxia, elevated CO2 and a large daily change of temperature which are concerned and researched These issue impacts and C ornata responses studied in this dissertation in aspects of physiology including respiration, cardioventilation, gill morphological transformation and growth have been considered significant keys to investigate although climate change impacts are likely pervasive effects (Fry, 1971; Roessig et al., 2004; Brander, 124 2007; Rijnsdorp et al., 2009; PÖrtner and Peck, 2010; Hofmann and Todghram, 2010; Madeira et al., 2012; Crozier and Hutchings, 2014; Lefevre, 2016) The elevated temperature (6ºC above mean 27ºC in Mekong basin, worst-case of prediction), hypoxia (Pcrit levels where fish shifts from waterbreathing to air-breathing) and elevated CO2 induced air-breathing frequency increase in C ornata that mechanism has been normally found in air-breathing fish Nevertheless, hypoxia induced more oxygen ration of air-breathing than that of temperature whilst temperature induced increase of oxygen metabolic rate larger than in hypoxia (Tuong et al., 2018b) It can be likely that airbreathing mechanism is an adaptive behavior of air-breathing fish to hypoxia (Graham, 1997; Ishimatsu et al., 1998; Lefevre et al., 2011, 2012) However, increase of air-breathing rate meaning that fish is being exposed in risk of predators, imbalance of energetic cost and benefit income, and growth (Perry, 2011; Kramer, 1987; Lefevre et al., 2011b, 2012) In the other hand, it has been found that water-breathing is still an important mode of most facultative and obligate air-breathing fish In C ornata and P hypophthalmus, the results of Pcrits and SMRs showed their abilities to maintain SMR without increase of air-breathing rate and shift to air-breathing mainly when environmental oxygen level drop to their Pcrit values (Lefevre et al 2011; Tuong et al., 2018b) Lefevre et al (2012) has found that Chana striata, an obligate airbreathing, still relies on water-breathing of more than 30% and that ration is reduced when fish is exposed to hypoxia Therefore, fish its self still uses the water oxygen instead of the air oxygen because of the consequent disadvantages of the air-breathing In addition to hypoxic and elevated temperature adaptation, gill morphological transformation in C ornata was another significantly important adaptive mechanism The gill remodeling has been found initially in water-breathing crucian carp, gold fish and trout while air-breathing fish has been investigated later on Arapaima gigas, P hypophthalmus and present study C.ornata In the metabolic oxygen consumption study, C ornata was found to increase the SMR the most with elevated temperature (Tuong et al., 2018b) which also induced the most increase of respiratory surface area compares to 27ºC in normoxia Hypoxia was found to induce thinner water blood diffusion distance faster than that of temperature These result of metabolic oxygen demand and gill transformation showing that fish tend to adapt to uptake more water oxygen at both temperature and hypoxia instead of performing air-breathing The respiratory surface area responded to elevated temperature faster than to hypoxia while the water blood diffusion thickness responded to hypoxia faster 125 than to temperature after one month However, what is the highest dissolved oxygen level and lowest temperature that cause gill transformation was not determined in this dissertation Here we evaluated the gill morphological transformation at Pcrit levels that were estimated in the previous study of respiratory metabolism The gill transformation to increase SA at elevated can explain the increase water metabolic rate increase in respiratory experiment Therefore, it is likely that C ornata owns air breathing organ acting as instance support to secure the oxygen need and gill morphological transformation working as a way to optimal energy distribution and keep maintain of other function in balance + Another extraordinary finding of internal CO2/H sensitive chemoreceptors has been clearly found in C ornata (Tuong et al., 2018a) that indirectly + inferred to central chemoreceptors existence The central CO2/H have been supported in Sarcopterygian lungfish (Smith, 1930; Delaney et al.,1974, 1977; Babiker, 1979; Sanchez et al., 2001), in gar and Siamese fighting fish (Wilson et al., 2000; Hedrick et al., 1991) which showed the degree of evolution Obviously, higher evolutionary level serves more advantages to fish The + central chemoreceptors control internal CO2/H meaning balancing acid base + status of fish internally while external orientation of CO2/H chemoreceptors + only promote PCO2/[H ] ambient environment In the denervation experiment, it was important to note that the denervated C.ornata was less tolerant to exposures of the hypercarbia and hypercapnia (30 min) than the intact fish (60 th th min) Due to denervation of nerves IX and X performing, those denervated fish were checked ventilation, and heart rate and pressure if their parameters returned to normal ranges after surgery It is believed that all nerves were well cut as they were checked again after finishing the experiments, However, although the results of those experiments indirectly point out that C ornata + owns central chemoreceptors for CO2/H , there is not a direct experiment done + successfully Honestly, determination of central chemoreceptors of CO2/H in C ornata is a difficult experiment consuming lot of time for repeating and testing therefore, it is an excuse for weakness of this dissertation 126 References Babiker, M (1979) Respiratory behaviour, oxygen consumption and relative dependence on aerial respiration in the African lungfish (Protopterus annectens, owen) and an air-breathing teleost (Clarias lazera, C.) 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the air-breathing catfish Pangasianodon hypophthalmus J Comp Physiol B, 183(2), 215-221 Madeira, D., Narciso, L., Cabral, H N., & Vinagre, C (2012) Thermal tolerance and potential impacts of climate change on coastal and estuarine organisms Journal of Sea Research, 70, 32-41 Malte, C L., Jakobsen, S L., & Wang, T (2014) A critical evaluation of automated blood gas measurements in comparative respiratory physiology Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 178, 7-17 Mazon, A d F., Fernandes, M., Nolasco, M., & Severi, W (1998) Functional morphology of gills and respiratory area of two active rheophilic fish species, Plagioscion squamosissimus and Prochilodus scrofa Journal of fish biology, 52(1), 50-61 Near, T J., Eytan, R I., Dornburg, A., Kuhn, K L., Moore, J A., Davis, M P., Smith, W L (2012) Resolution of ray-finned fish phylogeny and timing of diversification Proceedings of the National Academy of Sciences, 109(34), 13698-13703 Perry (2011) 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and fisheries: towards a cause‐and‐effect understanding Journal of fish biology, 77(8), 1745-1779 Price, J W (1931) Growth and gill development in the small-mouthed black bass, Micropterus dolomieu Lacepède: Ohio State University Press Rijnsdorp, A D., Peck, M A., Engelhard, G H., Möllmann, C., & Pinnegar, J K (2009) Resolving the effect of climate change on fish populations ICES journal of marine science, 66(7), 1570-1583 Roessig, J M., Woodley, C M., Cech, J J., & Hansen, L J (2004) Effects of global climate change on marine and estuarine fishes and fisheries Reviews in fish biology and fisheries, 14(2), 251-275 Sanchez, A., Soncini, R., Wang, T., Koldkjaer, P., Taylor, E W., & Glass, M L (2001) The differential cardio-respiratory responses to ambient hypoxia and systemic hypoxaemia in the South American lungfish, Lepidosiren paradoxa Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 130(4), 677-687 Smith, H W (1930) Metabolism of the lung-fish, Protopterus aethiopicus Journal of Biological Chemistry, 88(1), 97-130 Soivio, A., Nynolm, K., & Westman, K (1975) A technique for repeated sampling of the blood of individual resting fish Journal of Experimental Biology, 63(1), 207-217 Steffensen, J F (1989) Some errors in respirometry of aquatic breathers: how to avoid and correct for them Fish physiology and biochemistry, 6(1), 4959 Tuong, D D., Borowiec, B., Clifford, A M., Filogonio, R., Somo, D., Phuong, N T., Milsom, W K (2018a) Ventilatory responses of the clown knifefish, Chitala ornata, to hypercarbia and hypercapnia Journal of Comparative Physiology B, 1-9 Tuong, D D., Ngoc, T B., Huynh, V T N., Phuong, N T., Hai, T N., Wang, T., & Bayley, M (2018b) Clown knifefish (Chitala ornata) oxygen uptake and its partitioning in present and future temperature environments 130 Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 216, 52-59 Viet, T V (2015) Applications of GIS for evaluation the current culture status of Clown knife fish (Chitala ornata) in Phung Hiep District, Hau Giang Province Journal of Can Tho University, 38, 109-115 Weibel, E R., Cournand, A F., & Richards, D W (1963) Morphometry of the human lung (Vol 1): Springer Wilson, R., Harris, M., Remmers, J., & Perry, S (2000) Evolution of airbreathing and central CO (2)/H (+) respiratory chemosensitivity: new insights from an old fish? Journal of Experimental Biology, 203(22), 35053512 131 CHAPTER 8: CONCLUSIONS AND PERSPECTIVES In the first experiment of respiratory metabolism, C ornata did not show any influent from 6°C elevated above average of27°C whereas growth performance was better in higher temperature The key success of C ornata under high temperature and hypoxia is ability of air-breathing that fish can fulfill the increase of oxygen demand Therefore, this finding can be suggested to farmers that when growing C ornata at high temperature, maintaining well aeration will bring better productions The ability of gill remodeling of C ornata allowing fish to adapt with environmental dissolved O2 and temperature changes has been indicated in the second research The elevated temperature induced the increase of SA faster than that of hypoxia whereas water blood diffusion thickness was driven thinner by hypoxia This finding in C ornata proves that the gill transformation is likely an ancient trait existed at least 300 million years ago The present study’s results are considered significantly important when it has been perspectively inferred that many extant fish species can also inherit the ability of gill plasticity to deal with the environmental changes In the third and last researches, we found that air-breathing response of C + ornata was driven by at least internally oriented CO2/H -sensitive chemoreceptors which were extraordinary finding in a facultative air-breathing + fish The internally oriented CO2/H -sensitive chemoreceptors control the acid-base status of arterial blood and cerebrospinal fluid whereas the + externally oriented CO2/H -sensitive chemoreceptors only monitor the + ambient CO2/H in the environment Therefore, these internal chemoreceptors represent the evolutionary levels of aquatic vertebrates, in fact, the existence + of central CO2/H -sensitive chemoreceptors in lungfish and tetrapods have been indicated in previous studies It is significantly important to investigate more studies to directly determine the central chemoreceptors on C ornata Therefore, climate change effects can likely be diminished for C ornata and maybe other fish species due to many evolutionary features helping them survival through many severe changes of environment in the past are still remained and used now However, it is necessary to investigate the integrating effect of complex changes of environmental factors and see how these combining impacts affect on growth of fish as well as other relative aspects of biology 132 ... (33°C) nhiệt độ trung bình (27°C) kết hợp với hàm lượng oxy bão hòa oxy thấp thực Kết cho thấy mức nhiệt độ dự báo cao vùng đồng sông Cửu Long không ảnh hưởng tiêu cực lên sinh lý hô hấp cá Tăng... CO2 Các tiêu sinh lý hô hấp, phản ứng hô hấp tim mạch, thích nghi hình thái mang cá tăng trưởng tiêu thực để đánh giá ảnh hưởng biến đổi khí hậu thông qua bốn nghiên cứu Phản ứng hô hấp cá theo... khí trời C ornata Khảo sát phản ứng hô hấp tim mạch ảnh hưởng hypercapnia cách tiêm CO2 vào quan hơ hấp khí trời (ABO) cá khơng cắt dây thần kinh, ảnh hưởng hypercarbia hypercapnia lên cá cắt dây