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 TRAININGCAN THO UNIVERSITY DANG DIEM TUONG THE EFFECTS OF TEMPERATURE, HYPOXIA AND HYPERCARBIA ON RESPIRATION AND PHYSIOLOGY OF CLOWN KNIFEFISH CHITALA ORNATA G

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: 9 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: 9 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 ofNutrition and Aquatic Products Processing, College of Aquaculture andFisheries, 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: 1

INTRODUCTION 1

1.1 General introduction 1

1.2 Research objectives 3

1.3 Research contents/activities 3

References 5

CHAPTER 2: 10

LITTERATURE REVIEW 10

1 Temperature and hypoxia 10

2. Temperature and hypoxia: their effects on metabolism of air-breathing fishes 11 3 Gill remodeling 14

4 Hypercarbia and its effect on cardioventilatory responses 16

5 Air-breathing fish species 18

6 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

1 Introduction 39

2 Materials and methods 40

3 Results 44

4 Discussion 47

References 55

CHAPTER 4: 63

GILL REMODELING OF CLOWN KNIFEFISH ( CHITALA ORNATA ) UNDER IMPACT OF TEMPERATURE AND HYPOXIA 63

Abstract 63

1 Introduction 64

i

2 Materials and methods 65

3 Results 69

4 Discussion 72

5 Conclusions 77

References 78

CHAPTER 5: 82

VENTILATORY RESPONSES OF THE CLOWN KNIFEFISH, CHITALA ORNATA , TO HYPERCARBIA AND HYPERCAPNIA 82

Abstract 82

1 Introduction 83

2 Materials and methods 85

3 Results 88

4 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

1 Introduction 103

2 Materials and methods 105

3 Results 108

4 Discussion 110

References 117

CHAPTER 7: 123

GENERAL DISCUSSIONS 123

CHAPTER 8: 132

CONCLUSIONS AND PERSPECTIVES 132

ii

First of all, I would like to give my deep appreciations to Assoc Prof Do Thi ThanhHuong, Prof Nguyen Thanh Phuong and Prof Tran Ngoc Hai of Can Tho Universitywho supported, encouraged as well as straightened my direction during my study.They have been great teachers who were willing to help me solving my troublesthroughout the entire process and without them my PhD thesis would not have beenfinished

My biggest thank go to Assoc Prof Mark Bayley who set a fire of passion on scienceand gave me a direction to become a real physiological scientist Moreover, he hasmade valuable connections between many famous scientists and young passionatescientists all over the world I myself felt like a real scientist among them that I havebeen highly inspired during my five-year PhD

I would like to thank Prof William K Milsom who have taught me a lot aboutventilation and chemoreceptors that contributed haft of my thesis contents Workingwith him was my biggest pleasure and fortune from the start to the end of this iAquaproject that I have been luckily involved

My thanks would like to go to Prof Tobias Wang at Aarhus University and Prof JensRandel Nyegaardfrom Aarhus University Hospital who have supported and given mehelpful 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 anexpected teacher on cannulation in lab, but also a nice friend sharing many lifeexperiences

I also wish to thank staff members of the College of Aquaculture and Fisheries, CanTho University, Vietnam; and of the Zoophysiology Section, the Department ofBiological Science; the Stereology and Microscopy of AarhusUniversity Hospital,Denmark that have supported, and taught me laboratory skills during the time I havestudied there

I would like to give my thanks to my friends in iAQUA project, Nguyen Thi Kim Haand 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 CristanceDamgaard have supported my studies and brought me laughing moments toovercome stressful time I would like to thank all hard working students who havehelped me in doing research works

Finally, I would like to thank my family, my fiancée and my friends that always loveand spiritually support me throughout my research on the way to achieve my PhDand all And thanks for all sacrificed fish!

This thesis was included in iAQUA project funded by the Danish InternationalDevelopment Agency (DANIDA), Ministry Affairs of Foreign Denmark

iii

Climate change is one of the most concerns in scientific research regarding itseffects 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 ofelevated temperature and CO2 increase Respiratory physiology,cardiorespiratory responses, gill morphological adaptation and growth weretarget parameters to evaluate through four studies

Respiratory responses to elevated temperature and hypoxia have beeninvestigated in the first study Oxygen threshold (Pcrit), standard metabolic rate(SMR), specific dynamic action (SDA) and the growth under the effects ofpredicted 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 toconsider 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% and35% 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 inthe normoxia and decrease at the elevated temperature and in hypoxia Surfacearea (SA) of respiratory lamellae was significantly affected by the temperatureand hypoxia after one month Harmonic mean water blood thicknesssignificantly reduced by the hypoxia after one month while that reductioninduced by the temperature took two months to have significant effects Ananatomic 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 remodelingmechanism

iv

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 ofacetazolamide) at normocarbia We measured the changes of air-breathingfrequency, gill ventilation frequency, heart rate, arterial blood pressure, andblood 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 significantchanges of the gill ventilation, and revealed a modest bradycardia and fall inthe arterial blood pressure The blood [H+] and plasma PCO2 have been found

to increase in both hypercarbia and acetazolamide The acetazolamide resultsprovide 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 injectedinto ABO Denervation of IXth and Xth 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

air-breathing frequencies Bradycardia and no significant changes of the gillventilations were also found in all treatments The increase of internal [H+]andPCO2 were found in all treatments of CO2 injection into ABO, and thehypercarbia and hypercapnia The results of CO2 injection into ABO of theintact fish, and the hypercarbia and hypercapnia in the denervated fishadditionally gave documentation to confirm the existence of internally orientedcardiorespiratory CO2/H+-sensitive chemoreceptors and were indirectlyinferable to central chemoreceptors

v

TÓM TẮT

Biến đổi khí hậu là một trong những vấn đề được quan tâm nhất trong nghiêncứu khoa học về phương diện ảnh hưởng lên sinh lý, tăng trưởng, thích nghi

và/ hoặc diệt vong của động vật thủy sản Cá thát lát còm (Chitala ornata) là

một loài quan trọng trong nuôi trồng thủy sản đã được chọn trong nghiên cứunày để cung cấp các thông tin chuyên sâu về đánh giá tác động của sự tăngnhiệt độ và nồng độ CO2 Các chỉ tiêu sinh lý hô hấp, các phản ứng hô hấp timmạch, sự thích nghi hình thái mang cá và tăng trưởng là các chỉ tiêu đã đượcthực hiện để đánh giá ảnh hưởng của biến đổi khí hậu thông qua bốn nghiêncứu

Phản ứng hô hấp của cá theo sự tăng nhiệt độ và nồng độ oxy thấp được thựchiện trong nghiên cứu thứ nhất Ngưỡng oxy (Pcrit), trao đổi chất cơ bản(SMR), tác động của tiêu hóa thức ăn lên hoạt động hô hấp (SDA) và tăngtrưởng của cá thát lát dưới ảnh hưởng của nhiệt độ dự báo (33°C) và nhiệt độtrung bình hiện tại (27°C) kết hợp với hàm lượng oxy bão hòa và oxy thấp đãđược thực hiện Kết quả cho thấy ở mức nhiệt độ dự báo cao nhất ở vùng đồngbằng sông Cửu Long sẽ không ảnh hưởng tiêu cực lên sinh lý hô hấp của cá.Tăng trưởng của cá tăng ở mức nhiệt độ cao Khả năng hô hấp khí trời là đặcđiểm quan trọng giúp cá có thể giảm bớt ảnh hưởng của hàm lượng oxy thấp,đặc biệt trong môi trường nhiệt độ tăng Tuy nhiên, điều quan trọng cần cânnhắc là việc dựa vào khả năng hô hấp khí trời có thể dẫn tới các hậu quả bấtlợi cho cá như hao tốn năng lượng và giảm khả năng bão hòa oxy trong máu.Khảo sát khả năng biến đổi cấu trúc mang của cá dưới ảnh hưởng của nhiệt độ(33 và 27°C) kết hợp với môi trường oxy bảo hòa và thiếu oxy áp dụngphương pháp mô học lập thể được tiến hành trong thí nghiệm này Kết quả chothấy cá có biến đổi hình thái mang bằng cách tăng sinh hoặc giảm sinh sốlượng các tế bào ở giữa lá mang thứ cấp (ILCM) trong môi trường oxy bãohòa, và môi trường thiếu oxy và nhiệt độ cao Diện tích bề mặt (SA) hô hấpcủa mang bị ảnh hưởng nhiều bởi nhiệt độ và tình trạng thiếu oxy sau 30 ngàynuôi Khoảng cách khuếch tán giữa máu và nước giảm trong môi trường thiếuoxy sau 30 ngày nuôi trong khi đó giá trị này giảm do nhiệt độ thể hiện rõsau60 ngày nuôi Tính toán giá trị hệ số hô hấp (ADF) cho thấy chỉ số này caogấp 4 lần ở mức nhiệt độ 33°C trong môi trường oxy thấp so với ở mức 27°Ctrong môi trường oxy bão hòa Diện tích bề mặt của mang cá thát lát phù hợpvới các loài cá hô hấp khí trời Các kết quả của nghiên cứu này củng cố chogiả thuyết về sự tồn tại lâu dài của cơ chế biến đổi cấu trúc mang dưới tácđộng của các yếu tố môi trường

vi

Nghiên cứu nồng độ CO2 cao trong môi trường (hypercarbia) và trong máu(hypercapnia) cá đến phản ứng hô hấp tim mạch được thúc đẩy bởi các thụcảm CO2/H+ của cá đã được thực hiện trong nghiên cứu thứ ba Cá nuôi trongmôi trường nước có tính a-xit (pH=6), môi trường CO2 (tăng CO2 trong môitrường nước, pH=6) và môi trường CO2 cao trong máu (tiêm acetazolamide)trong điều kiện oxy bão hòa Thu mẫu phân tích các chỉ tiêu tần số hô hấp khítrời, tần số hô hấp qua mang, nhịp tim, áp suất máu động mạch, và pH vàPCO2 máu Trong môi trường a-xit (pH=6) thì sự thay đổi phản ứng của cákhông có ý nghĩa thống kê ở tất cả các chỉ tiêu khảo sát Cá phản ứng lại môitrường hypercarbia và môi trường hypercapnia qua sự tăng tần số hô hấpnhưng sự thay đổi không có ý nghĩa so với tần số hô hấp qua mang; bên cạnh

sự phản ứng nhịp tim chậm và giảm ấp suất máu cá Nồng độ [H+]và PCO2trong máu cá tăng ở môi trường hypercarbia và môi trường hypercapnia (tiêmacetazolamide) Kết quả của nghiên cứu cung cấp cơ sở về bằng chứng cho sựhiện diện của thụ cảm CO2/H+ định hướng bên trong tồn tại ở loài cá hô hấp

khí trời như C ornata.

Khảo sát phản ứng hô hấp tim mạch dưới ảnh hưởng của hypercapnia bằngcách tiêm CO2 vào cơ quan hô hấp khí trời (ABO) của cá không cắt dây thầnkinh, và ảnh hưởng của hypercarbia và hypercapnia lên cá cắt dây thần kinhđược thực hiện trong nghiên cứu thứ tư Liều tiêm CO2 vào bong bóng khí của

cá tăng dần theo nồng độ CO2có trong hỗn hợp CO2 và không khí Tiến hành cắtcác dây thần kinh thứ IXth và Xth, và cho cá hồi phục trong 24 giờtrước khi

cho tiếp xúc với hypercarbia (tăng CO2 ~ pH=6) và acetazolamide (tăng[H+]và PCO2 trong máu) Kết quả cho thấy cả cá không cắt dây thần kinhvàcắt dây thần kinh đều phản ứng tăng tần số hô hấp khí trờiđáng kể Nhịp timchậm và không có thay đổi đáng kể về tần số hô hấp qua mang thể hiện ở tất

cả cá của các nghiệm thức Sự gia tăng của [H+]và PCO2 trong máu ghi nhậnđược trong tất cả các mức nồng độ tiêm CO2 vào ABO, và hypercarbia vàhypercapnia Các kết quả tiêm CO2 vào ABO (không cắt dây thần kinh), vàhypercarbia và hypercapnia đã cung cấp thêm thông tin chứng minh sự tồn tạicủa các thụ cảm CO2/H+ định hướng bên trong và gián tiếp cho thấy sự hiệndiện của cơ quan thụ cảm trung tâm

vii

RESULT COMMITMENT

I commit that this dissertation was investigated based on all the results of mystudies All the data and showed results in the dissertation were honest andhave never been published before The iAQUA project can completely usethese data and results

Can Tho, April , 2019

viii

LIST OF FIGURES

Chapter 2: Literature review

Chapter 3:Clown knifefish (Chitala ornata) oxygen uptake and

its partitioning in present and future environments

Fig 3.1: Pcrit determination of C.ornata at 27°C and 4233°C,mean±S.E.M, N=8

Fig 3.2: Oxygen partitioning in normoxia and hypoxia at 27°C and 4633°C Hypoxic levels in experiment (PO2=4.7 and 6.0 kPa) at 27°C

and 33°C, respectively Mean±S.E.M, N=8

Fig 3.3: Total ṀO2pre-feeding and post-feeding with 2% of body 47mass in 27°C and 33°C(PO2=19–21 kPa), mean±S.E.M, N= 6

Fig 3.4: Partitioning of oxygen uptake of C ornata for 20 h pre- 48feeding to feeding and for 42 h after forced feeding of high protein

meal (2% of body mass) at 27 and 33°C Mean±S.E.M, N=6

Fig 3.5: Growth performance as weight gain (g) (A) and specific 50growth rate (%) (B) of C ornata during 3 months at 27°C in

normoxia (N27), 27°C in hypoxia (H27), 33°C in normoxia (N33)

and 33°C in hypoxia (H33), N=30, mean±S.E.M

Chapter 4: Gill remodeling of Clown knifefish (Chitala ornata)

under impact of temperature and hypoxia

Fig 4.1: Surface area of respiratory lamellae of C ornata exposed 69

to different temperature and/or hypoxia

Fig 4.2: Volume of respiratory lamellae of C ornata exposed to 70different temperature and/or hypoxia

Fig 4.3: Harmonic mean water-blood thickness of C ornata 70exposed to different temperature andor hypoxia

Fig 4.4: Calculated anatomic diffusion factor of C ornata exposed 72

to different temperature and/or hypoxia

ix

Fig 4.5: One side gill arches of C ornata showing five arches were 75collected from a preserved sample The fifth arch is reduced

without observing filaments

Fig 4.6: Gill filaments of C ornata under light micrographs from 75normoxia 27°C, hypoxia 33°C, at 0, 1 and 2 months Fish gill

morphology was alike primary water breathing fish at

pre-experiment and started to increase ILCM when fish was exposed to

normoxia 27°C after 1 and 2 months There was not ILCM

developing found in hypoxia at 33°C after 2 months

Chapter 5: Ventilatory responses of the Clown knifefish,

Chitala ornata, to hypercarbia and hypercapnia

Fig 5.1: Sample traces of buccal pressure and opercular impedance 87associated with type 1 and 2 air breaths

Fig 5.2: Air breathing frequency under control conditions and at 89

20, 40and 60 min into either acid (red symbols) or hypercarbia

(green symbols) exposure and following acetazolamide injection

(blue symbols) The * indicate differences between control and

exposure conditions (two-way ANOVA for repeated measures

followed by Holm-Sidak post hoc test; P < 0.05) Data are

mean±S.E.M

Fig 5.3: Buccal ventilation (N=8), opercular ventilatory frequency 90(N=8), heart rate (N=4) and mean arterial pressure (N=4) under

control conditions and at 20, 40 and 60 min into either acid (red

symbols) or hypercarbia (green symbols) exposure and following

acetazolamide injection (blue symbols) The * indicate differences

between control and exposure conditions (two-way ANOVA for

repeated measures followed by Holm-Sidak post hoc test; P<0.05)

Data are mean±S.E.M

Fig 5.4: Heart rate and mean arterial pressure at 60 and 30 s before 91and30, 60, 90 and 120 s after an air breathe White circles refer to

type 1 air breaths (N=4); black circles to type 2 air breaths (N=4)

Equal symbols indicate statistical differences for type 1 air breaths

(one-way ANOVA for repeated measures followed by

Student-Newman-Keuls post hoc test; P<0.05; data are mean±S.E.M)

Fig 5.5: Rate of air breathing expressed as a function of the arterial 92

x

pH (left hand panels) or PCO2 (right hand panels) Upper panels

show the mean data while the lower panels show all the data with

the regression equations that best fit each dataset

Chapter 6: Ventilatory responses of the Clown knifefish, Chitala

ornata, to ambient water and air hypercarbia, and hypercapnia

significant differences of gill ventilations between control and the

other injections (one way ANOVA for repeated measurement,

P<0.05) Data are presented as mean±S.E.M

Fig 6.3: Heart rates of intact C ornata injected ascending 110percentage of CO2 air mixture into air bladder There are no

significant differences of heart rates between control and the other

injections (one way ANOVA for repeated measurement, P<0.05)

Data are presented as mean±S.E.M

Fig 6.4: Rate of air breathing expressed as function of the arterial 111

pH (left hand panel) or PCO2 (right hand panel) The data were

showed as mean±S.E.M

Fig 6.5: Air-breathing frequency under controls and exposure of 112hypercarbia and acetazolamide Different alphabet indicates

significant differences (one-way ANOVA repeated measurement

followed by LSD post hoc test; P<0.05) Data are presented as

alphabet letters indicate significant differences between the control

and the other time points of exposure Data were presented as

mean±S.E.M

Fig 6.8: Heart rates at 60 and 30 s before and 30, 60, 90 and 120 s 115

after an air breathe of denervated C ornata (one-way ANOAVA

for repeated measurement followed by LSD post hoc test; P<0.05)

Data were presented as mean±S.E.M

Fig 6.9: Rate of air breathing presented as a function of arterial pH 116and PCO2 Two upper panels present mean values (mean±S.E.M)

Two below show all data of each single fish

xii

LIST OF TABLES

Chapter 2: Literature review

Table 2.1: Air-breathing organ types (ABO) and names of common 20air-breathing fish species Information is based on data of Graham

(1997)

Chapter 3: Clown knifefish (Chitala ornata) oxygen uptake and

its partitioning in present and future environments

Table 3.1: SMR (mgO2 kg−1 h−1), RMR (mgO2 kg−1 h−1) and 45partitioning as percentage of aerial oxygen uptake from air and

associated p-values for overall effects from two-way anova,

mean±S.E.M, N=8 Holm-Sidak post hoc multiple comparisons: ‡

indicates a significant effect (P<0.05) of oxygen level within a

given temperature, * indicates a significant effect of temperature

(P<0.05) within a given oxygen level n.s indicates non-significant

Table 3.2: SDA parameters prior and post feeding 2% of body 49mass in 27°C and 33°C (PO2=19-21kPa), mean±S.E.M, N=6

Table 3.3: Q10 values and % air uptake of air-breathing species at 51

different temperatures

Table 3.4: The effect of temperature on SDA and SDA coefficient 52

in fish

Chapter 4: Gill remodeling of Clown knifefish (Chitala ornata)

under impact of temperature and hypoxia

Table 4.1: Lamellar surface area (mm2g-1), gill filament volume 71(mm3g-1), lamellar volume (mm3g-1), harmonic mean (HM) water

blood thickness (µm) and anatomic diffusion factor (mm2g-1µm-1)

of C.ornata exposed to elevated temperature and/ or hypoxia Data

are presented as mean±S.E.M

Table 4.2: Comparison the significant effects of temperature and 73

hypoxic levels on gill parameters of C.ornata.

Table 4.3: Comparison of lamellar surface area, water-blood 76diffusion thickness and ADF of fish species

xiii

[H+]: Hydrogen ion concentration

AAS: Apparent aerobic scope

ABO: Air-breathing organ

ADF: Anatomic diffusion factor

ATP: Adenosine triphosphate

CO2/H+: Carbon dioxide/ hydrogen ion

CO2: Carbon dioxide

DMSO: Dimethyl sulfoxide

[H+]: Hydrogen ion concentration

HCO3-: Bicarbonate ion

Hypercarbia: High level of carbon dioxide in water or airHypercapnia: High level of carbon dioxide in bloodI.D.: Inner dimension

ILCM: Inter lamellar cell mass

IPCC: Intergovernmental Panel on Climate Change

ṀO2: Oxygen uptake used in metabolism

NECs: Neuroepithelial cells

NH3

+: Ammonia cation

NO2 −: Nitrite ion

NO3 −: Nitrate ion

O.D.: Outer dimension

PBS: Phosphate buffer solution

PCO2: Carbon dioxide pressure

Pcrit: Critical oxygen partial pressure

Q10: Temperature coefficient

RAS: Recirculating aquaculture system

RMR: Routine metabolic rate

xiv

SA: Surface area

SDA coefficient: Specific dynamic action coefficient

SDA: Specific dynamic actionSMR: Standard metabolic rate

xv

It has been indicated that projected the elevated water temperature maynegatively impact broadly marine and freshwater ecosystem functions

(Roessig et al., 2004; Brander, 2007; Rijnsdorp et al., 2009; PÖrtner & Peck, 2010; Hofmann and Todghram, 2010; Madeira et al., 2012; Crozier & Hutchings, 2014; Lefevre et al., 2016) and fish populations through effects on

fish physiology, respiration, metabolism, food ability, growth, behaviors,

reproduction and/or mortality (Watts et al., 2001; Cnaani, 2006; Sigh et al., 2013; Reid et al., 2015) While the temperature is considered as a key

importance of controlling physical factor pervasively determining animaldistribution, the rising environmental water CO2 level (hypercarbia) is morerelated to acid-base imbalance, water pH reduction and cardioventilatory as

well as respiratory changes (Gilmour, 2001; Claiborn et al., 2002; Ishimatsu et

al., 2005; Brauner and Baker, 2009; Talmage & Gobler., 2011; Nowicki et al.,

2012; Milson, 2012; Munday et al., 2012) Nevertheless, physiological

changes and adaptive ability of aquatic animals have been consideredintriguing targets to research under effects of projected elevated temperatureand hypercarbia in water

A hypothesis of oxygen capacity limited thermal tolerance is proposed toexpress negative impacts of the elevated temperature on the fish that isunderlying the oxygen delivery mechanism to tissues (Portner, 2001; Portner

and Farrell, 2008; Munday et al., 2008; Munday et al., 2009; Nilsson et al., 2009; Portner, 2010; Neuheimer et al., 2011) It is due to the dissolved oxygen

level decreasing with progressive increases of the temperature whilst fishoxygen demand significantly increases with the elevated temperature.Therefore, the elevated temperature integrating with hypoxia (the decrease ofthe dissolved oxygen level) has been indicated that could result in a largelysevere effects on aquatic organism in term of the metabolism and net result of

1

performance (McBryan et al., 2013).In addition, it has been argued that fish in

tropical areas can be affected severely because they have been already livednear their upper thermal limits and can be more vulnerable with a small

increase of the temperature (Nelson et al., 2016; Tewksbury et al., 2008).

However, there is growing evidence of studies that do not conform that

hypothesis (Clark et al., 2013; Norin et al., 2014; Wang et al., 2014; Lefevre,

2016) Indeed, it is argued that the air-breathing fish species which hypoxicwater tolerance could be a result of an evolution under the effects of highertemperatures and lower atmospheric oxygen pressure than the present.Investigating the effects of the elevated temperature and hypoxia on themetabolism of the air-breathing fish is important to assess the effects ofclimate change

Another aspect of the adaptive ability to environmental factors, it has beenexposed that one of adaptive mechanisms is ability of changing gill

morphology (Tuurala et al., 1998, Sollid et al.,2003; Sollid et al., 2005; Sollid and Nilsson, 2006; Ong et al., 2007; Matey et al., 2008; Mitrovic and Perry,

2009) Intensive researches on this ability have been found in the breathing fishes including crucian carp, goldfish and salmonids which theirgills have been found to increase or decrease interlamellar cell mass (ILCM) toincrease or decrease respiratory surface area with changes of theenvironmental factors This ability of fish is intriguing scientists that whetherthe gill remodeling is a modern trend or an ancient trait (Nilsson, 2007;

water-Nilsson et al., 2012) It has been proposed that gill remodeling can be an

ancient trait induced by the evolutionary progress in the past when some of theair-breathing fishes has been found that are also able to transform their gill

morphology to adapt to the environmental changes (Brauner et al., 2004; Ong

et al., 2007; Huang and Lin, 2011; Phuong et al., 2017,2018) This controversy

is important to explore in C ornata because this species is an ancient fish existing at least 300 million years ago (Near et al., 2012) which will help to

make an overview prediction for other fish species

It is accounted that the atmospheric CO2level is rising year by yearconsequently due to the global warming The dissolved CO2 level is moredissoluble than the oxygen therefore, with a small increase of the CO2 level inthe atmosphere, a largely significant increase of the dissolved CO2 level can beproduced in the aqueous system It has been reported that PCO2 level in

Mekong River Delta ranging from 0.02-0.6% (Li et al., 2013) is affecting the

fish animal life In aquaculture systems, intensive and super-intensive culturesystems are practicing that lead to severe hypercarbic environment of around

2

30mmHg toward the end of the growth cycles (Damsgaard et al., 2015) The

hypercarbic exposure normally induces different responses of specific fishspecies Cardioventilatory responses derived from central CO2/H+-sensitivechemoreceptors are still equivocal in the facultative air-breathing fish Theeffect of hypercarbia and hypercapnia on the cardioventilatory, and blood gasand pH responses as well as CO2/H+-sensitive chemoreceptors location are

important to investigate.Clown knifefish (C.ornata), a tropical facultative breathing fish species (Deharai, 1962; Tuong et al., 2018b) was selected as a

air-model fish to challenge with a worst-case predicted temperature (33°C),hypoxia, water hypercarbia, internal hypercapnia to see how the respiratorymetabolism, gill plasticity responses and adaptation through the growthperformance, cardioventilation, and CO2/H+-sensitive chemoreceptorsorientation

of gill remodeling and estimating respiratory surface area, water bloodthickness Fish responses to the hypercarbia and hypercapnia were carried on

to evaluate the cardioventilatory parameters as well as determinate

CO2/H+sensitive chemoreceptors

1.3 Research contents/activities

This research includes 4 main activities/studies:

1) Firstly, evaluating the effects of the elevated temperature on critical partialpressure (Pcrit), standard metabolism rate (SMR), specific dynamic action

or digestion (SDA) (using a bimodal intermittent-closed respirometry);and on fish growth (in recirculating aquaculture system)

2) Secondly, investigating the gill morphology through estimating thebranchial surface area, volume and water blood diffusion thickness underthe effects of the elevated temperature and hypoxia applying stereologicalmethod

3

3) Thirdly,cchallenging the cardioventilatory responses of C ornata to the

hypercarbia (high ambient water PCO2) and hypercapnia (high arterial[H+]/PCO2) as well as determiningthe locations of the cardioventilatory

CO2/H+ chemoreceptors in C ornata.

4) Finally, evaluating the effects of the hypercarbia and hypercapnia on the

cardioventilatory responses of denervated C ornata in processes of

continuously determining the location of CO2/H+-sensitive

chemoreceptors in C ornata.

Fig 1.1: Diagram of research activities

4

Brander, K M (2007) Global fish production and climate change

Proceedings of the National Academy of Sciences, 104(50), 19709-19714.

Brauner, C., & Baker, D (2009) Patterns of acid–base regulation during

exposure to hypercarbia in fishes Cardio-respiratory control in vertebrates

Claiborne, J B., Edwards, S L., & Morrison‐Shetlar, A I (2002) Acid–base regulation

in fishes: cellular and molecular mechanisms Journal of Experimental Zoology Part

A: Ecological Genetics and Physiology, 293(3), 302-319.

Clark, T D., Sandblom, E., & Jutfelt, F (2013) Aerobic scope measurements

of fishes in an era of climate change: respirometry, relevance and

recommendations Journal of Experimental Biology, 216(15), 2771-2782.

Cnaani, A (2006) Genetic perspective on stress response and diseaseresistance in aquaculture

Crozier, L G., & Hutchings, J A (2014) Plastic and evolutionary responses

to climate change in fish Evolutionary Applications, 7(1), 68-87.

Damsgaard, C., Tuong, D D., Thinh, P V., Wang, T., & Bayley, M (2015).High capacity for extracellular acid–base regulation in the air-breathing fish

Pangasianodon hypophthalmus Journal of Experimental Biology, 218(9),

1290-1294

Dehadrai, P V (1962) Respiratory function of the swimbladder of Notopterus

(Lacépède) Paper presented at the Proceedings of the Zoological Society of

London

Gilmour, K M (2001) The CO2/pH ventilatory drive in fish Comparative

Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 130(2), 219-240.

Hofmann, G E., & Todgham, A E (2010) Living in the now: physiological

mechanisms to tolerate a rapidly changing environment Annual review of

physiology, 72, 127-145.

5

Huang, C.-Y., & Lin, H.-C (2011) The effect of acidity on gill variations in

the aquatic air-breathing fish, Trichogaster lalius Comparative

Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 158(1), 61-71.

IPCC (2014) Climate Change 2014: Synthesis Report Contribution ofWorking Groups I, II and III to the Fifth Assessment Report of theIntergovernmental Panel on Climate Change [Core Writing Team, R.K.Pachauri and L.A Meyer (eds.)] IPCC, Geneva, Switzerland 151 pp.Ishimatsu, A., Hayashi, M., Lee, K S., Kikkawa, T., & Kita, J (2005) Physiological effects on fishes in a high‐CO 2 world Journal of geophysical research: Oceans, 110(C9).

Lefevre, S (2016) Are global warming and ocean acidification conspiringagainst marine ectotherms? A meta-analysis of the respiratory effects ofelevated temperature, high CO2and their interaction Conservation

physiology, 4(1), cow009.

Lefevre, S., Findorf, I., Bayley, M., Huong, D., & Wang, T (2016) Increased temperature tolerance of the air‐breathing Asian swamp eel Monopterus albus after high‐temperature acclimation is not explained

by improved cardiorespiratory performance Journal of fish biology, 88(1), 418-432.

Li, S., Lu, X., & Bush, R T (2013) CO 2 partial pressure and CO2 emission

in the Lower Mekong River Journal of Hydrology, 504, 40-56.

Madeira, D., Narciso, L., Cabral, H N., & Vinagre, C (2012) Thermaltolerance and potential impacts of climate change on coastal and estuarine

organisms Journal of Sea Research, 70, 32-41.

Matey, V., Richards, J G., Wang, Y., Wood, C M., Rogers, J., Davies, R., Brauner, C J (2008) The effect of hypoxia on gill morphology and

ionoregulatory status in the Lake Qinghai scaleless carp, Gymnocypris

przewalskii.Journal of Experimental Biology, 211(7), 1063-1074.

McBryan, T., Anttila, K., Healy, T., & Schulte, P (2013) Responses totemperature and hypoxia as interacting stressors in fish: implications for

adaptation to environmental change Integrative and comparative biology,

53(4), 648-659.

Milsom, W K (2012) New insights into gill chemoreception: receptor

distribution and roles in water and air breathing fish Respiratory

physiology & neurobiology, 184(3), 326-339.

6

Mitrovic, D., & Perry, S (2009) The effects of thermally induced gillremodeling on ionocyte distribution and branchial chloride fluxes in

goldfish (Carassius auratus) Journal of Experimental Biology, 212(6),

843-852

Munday, P., Kingsford, M., O’callaghan, M., & Donelson, J (2008) Elevated

temperature restricts growth potential of the coral reef fish Acanthochromis

polyacanthus Coral Reefs, 27(4), 927-931.

Munday, P L., Crawley, N E., & Nilsson, G E (2009) Interacting effects ofelevated temperature and ocean acidification on the aerobic performance of

coral reef fishes Marine Ecology Progress Series, 388, 235-242.

Munday, P L., McCormick, M I., & Nilsson, G E (2012) Impact of globalwarming and rising CO2 levels on coral reef fishes: what hope for the

future? J Exp Biol, 215(Pt 22), 3865-3873 doi: 10.1242/jeb.074765

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.

Nelson, J S., Grande, T C., & Wilson, M V (2016) Fishes of the World:

John Wiley & Sons

Neuheimer, A., Thresher, R., Lyle, J., & Semmens, J (2011) Tolerance limit

for fish growth exceeded by warming waters Nature Climate Change, 1(2),

110-113

Nilsson, G E (2007) Gill remodeling in fish–a new fashion or an ancient

secret? Journal of Experimental Biology, 210(14), 2403-2409.

Nilsson, G E., Crawley, N., Lunde, I G., & Munday, P L (2009) Elevated

temperature reduces the respiratory scope of coral reef fishes Global

Change Biology, 15(6), 1405-1412.

Nilsson, G E., Dymowska, A., & Stecyk, J A (2012) New insights into the

plasticity of gill structure Respiratory physiology & neurobiology, 184(3),

Nowicki, J P., Miller, G M., & Munday, P L (2012) Interactive effects ofelevated temperature and CO2 on foraging behavior of juvenile coral reef

fish Journal of Experimental Marine Biology and Ecology, 412, 46-51.

Ong, K., Stevens, E., & Wright, P (2007) Gill morphology of the mangrovekillifish (Kryptolebias marmoratus) is plastic and changes in response to

terrestrial air exposure Journal of Experimental Biology, 210(7),

Phuong, L M., Nyengaard, J R., & Bayley, M (2017) Gill remodelling and

growth rate of striped catfish Pangasianodon hypophthalmus under impacts

of hypoxia and temperature Comparative Biochemistry and Physiology

Part A: Molecular & Integrative Physiology, 203, 288-296.

Pörtner, H.-O (2010) Oxygen-and capacity-limitation of thermal tolerance: amatrix for integrating climate-related stressor effects in marine ecosystems

Journal of Experimental Biology, 213(6), 881-893.

Pörtner, H.-O., & Peck, M (2010) Climate change effects on fishes and fisheries: towards a cause‐and‐

effect understanding Journal of fish biology, 77(8), 1745-1779.

Pörtner, H (2001) Climate change and temperature-dependent biogeography:

oxygen limitation of thermal tolerance in animals Naturwissenschaften,

88(4), 137-146.

Pörtner, H O., & Farrell, A P (2008) Physiology and climate change

Science, 690-692.

Reid, G., Filgueira, R., & Garber, A (2015) Revisiting temperature effects on

aquaculture in light of pending climate change Aquaculture Canada 2014

Proceedings of Contributed Papers, 85.

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.

8

Singh, S., Sharma, J., Ahmad, T., & Chakrabarti, R (2013) Effect of water

temperature on the physiological responses of Asian catfish Clarias

batrachus (Linnaeus 1758) Asian Fish Sci, 26, 26-38.

Sollid, J., De Angelis, P., Gundersen, K., & Nilsson, G E (2003) Hypoxiainduces adaptive and reversible gross morphological changes in crucian

carp gills Journal of Experimental Biology, 206(20), 3667-3673.

Sollid, J., & Nilsson, G E (2006) Plasticity of respiratory structures—adaptive remodeling of fish gills induced by ambient oxygen and

temperature Respiratory physiology & neurobiology, 154(1), 241-251.

Sollid, J., Weber, R E., & Nilsson, G E (2005) Temperature alters therespiratory surface area of crucian carp Carassius carassius and goldfish

Carassius auratus Journal of Experimental Biology, 208(6), 1109-1116.

Talmage, S C., & Gobler, C J (2011) Effects of elevated temperature andcarbon dioxide on the growth and survival of larvae and juveniles of three

species of northwest Atlantic bivalves PLoS One, 6(10), e26941.

Tewksbury, J J., Huey, R B., & Deutsch, C A (2008) Putting the heat on

tropical animals SCIENCE-NEW YORK THEN WASHINGTON-,

320(5881), 1296.

Tuong, D D., Ngoc, T B., Huynh, V T N., Phuong, N T., Hai, T N., Wang,

T., & Bayley, M (2018) Clown knifefish (C.ornata) oxygen uptake and its partitioning in present and future temperature environments Comparative

Biochemistry and Physiology Part A: Molecular & Integrative Physiology,

216, 52-59.

Tuurala, H., Egginton, S., & Soivio, A (1998) Cold exposure increases

branchial water–blood barrier thickness in the eel Journal of fish biology,

53(2), 451-455.

Wang, T., Lefevre, S., Iversen, N K., Findorf, I., Buchanan, R., & McKenzie,

D J (2014) Anaemia only causes a small reduction in the upper criticaltemperature of sea bass: is oxygen delivery the limiting factor for tolerance

of acute warming in fishes? Journal of Experimental Biology, 217(24),

4275-4278

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Australian Veterinary Journal, 79(8), 570-574.

9

CHAPTER 2:

LITTERATURE REVIEW

1 Temperature and hypoxia

Temperature can be defined as a measurement of kinetic energy of specificgroup of molecules which have their own mass and velocity to perform kineticenergy (Ek)=(½)m.v (Callen and Scott, 1998) There are two unit scales oftemperature measurement including Celsius (ºC) and Kelvin scales (K).Celsius scale is defined as 0ºC at freezing point of water and 100ºC at boilingpoint of water, Kelvin scale is developed basing on Celsius scale which isconvertible exactly to kelvin scale by adding 273.15 to Celsius scale (Ericksonand Tiberghien, 1985) Through biological evolution, organisms or fish haveinvolved broadly to adapt to effects of temperature as known as poikilothermand homeotherm (Schulte, 2011) Poikilotherm is the term describingorganisms varying their body temperature to environmental temperature andvice a versa for homeotherm In homeothermic organism, there are terms of

―endotherm‖ and ―ectotherm‖ infering the source of heat affecting theanimals For endotherm, heat is maintained mainly by internally metabolicprocesses For ectotherm, organism body’s heat is base on the externally heatenergy of environment Most of fishes are poikiothermic ectotherm which theirtemperature much depend on ambient temperatures Therefore, increasing thetemperature of a system results in increase in thermal energy of the systemwhich is pointed to cause an increase in the rates of most chemical reaction.Increasing in rates of reaction are related to rising in collision betweenmolecules per unite time and increase the energy of molecules that collisionsoccur (increase fraction of molecules that exceed activation energy of thereaction) (Schulte, 2011) In addition to effects of the temperature on the rate

of processes, the temperature changes lead to weak bonds between themolecules such as hydrogen bonds, ionic bonds, hydrophobic interactionswhich are vital important to enzyme, protein and nucleic acids structures, andmenbrance functions (Hazel, 1995; Somero, 1995, 2004; Schulte, 2011) Thesechanges influent the molecular levels therefore, not easy to be observed inshort time scales

Hypoxia is a phenomenon of low pressures of oxygen in water environmentrelated to principal factors that lead to development of hypoxia or anoxia(phenomenon of complete absence of dissolved oxygen in water) (Diaz and

10

Breitburg, 2011).The concentration of dissolved oxygen in aqueous solutionobeys Henry’s Law as well as other dissolved gases.

Where C is the molar concentration of dissolved oxygen, α is the solubilitycoefficient and P is a partial pressure of oxygen (Rombough, 2011)

Dissolved oxygen concentration in the aquatic environment physicallydepends on internal temperature and salinity inversely In facts, the ability tohold the oxygen of aqueous solution is 20-30 times lower than the aerialsolution which contains 20.95% of oxygen per liter of air meaning 209.5 ml/L

of oxygen while only 11.13 ml/L at 27ºC in water environment (Boyd, 1982).Oxygen is vital to sustain the life of aquatic animals When the supply is cutoff or a phenomenon of the oxygen generation is exceeded by consumption,dissolved oxygen (DO) concentration can drops to below the level needed tosustain organism life that the low DO is called the hypoxia These factors can

be described as (1) water column stratification that separates bottom waterfrom exchange of the oxygen-rich in the surface water and (2) thedecomposition of organic matter in the bottom water that declines the oxygenlevel That two factors work in the deep water column, however, (3) in theshallow and smaller water column, the hypoxia can also occur When theaquatic plants and phytoplankton stop photosynthesizing and producingoxygen, respirations of these aquatic plants, phytoplankton and organismsstarts to dominate and reduce the oxygen levels (Conley et al., 2009) The state

of hypoxia disappears when oxygen producers start again on the next day Theform of this hypoxia is called diel-cycling hypoxia (Diaz and Rosenberg,2008; Diaz and Breitburg, 2011) In brief, hypoxia can occur naturally in watercolumn in related to the stratification, a poor circulation, the organic matterdecaying, and artificially in intensive and super intensive aquaculture (linked

to human activities) related to the diel-cycling hypoxia

2 Temperature and hypoxia: their effects on metabolism of

air-breathing fishes

Temperature has been considered as a controlling physical factor whichpervasively influences all levels of ecosystem and largely determines animaldistribution (Fry, 1971) It has been proposed that temperature affects different

levels of organs or tissues differently (Schulte et al., 2011) In aspect of

biological performance, temperature has been considered as a key factor that

11

influences reproduction and growth of fish (Cossins and Bowler, 1987) whichhave been vastly investigated on many fishes in marine and freshwater

ecosystem functions (Roessig et al., 2004; Brander, 2007; John& David, 2009; Rijnsdorp et al., 2009; PÖrtner & Peck, 2010; Hofmann and Todghram, 2010;

Madeira et al., 2012; Crozier & Hutchings, 2014; Lefevre et al., 2016) The

international panel on climate change (IPCC) predicting the increases of 3.5°C (the most extreme model (RPC8.5)) may occur in coastal South-EastAsia by 2100 (IPCC, 2014) The elevated temperature scenarios are raisingconcerns of adaptation of terrestrial and aquatic animals (Heath and Hughes,

2.5-1973; Farrell, 1997; Lee et al., 2003; Somero, 2011; McKenzie et al., 2016).

Tropical fishes have been proposed that they will be more vulnerable thantemperate fishes even small elevation of temperature because tropical fishes

have been already living near their optimum temperatures (Hoegh-Guldberg et

al., 2007; Deutsch et al., 2008; Munday et al., 2008; Tewksbury et al., 2008;

Nilsson et al., 2009) To reveal the ecological consequences of climate change

in fishes, it has been proposed that the key point is to understand the effects ofoxygen supply to animal bodies as well as their responses (PÖrtner, 2010;

PÖrtner and Knust, 2007) When the environmental temperature increases, thedissolved oxygen in the water decreases and the oxygen demand of fishmetabolic rate increases that subsequently lead to a fail to meet the oxygendemand of fish and oxygen supply of ambient environment Many studies have

supported this idea(Farrell et at., 2008; Munday et al., 2008, 2009; Nilsson et

al., 2009; Neuheimer et al., 2011) while other investigations have showed a

different idea (Clark et al., 2013; Norin et al., 2014; Wang et al., 2014;

Lefevre, 2016) Thus, mechanism of metabolic responses at elevatedtemperature in many air-breathing fishes are still a controversy However, nomatter a trend of results, the oxygen cascade from outside to inside of the fishbody under the effect of elevated temperatures still is an important aspect toinvestigate influencing the energy balance as well as fish performance

It has been emphasized that the evolution of air-breathing fishes has gonethrough hypoxic environment and insufficiently high demand oxygen underhigher levels of temperature comparing to present temperature (Graham 1997;Johasen, 1968) It seems to be that present air-breathing fish species inheritedmany good traits from their ancestors to survive and adapt with varying ofambient environmental factors successfully Air-breathing fishes now becomeextensive on over the world, some fish species also become dominant in worldaquaculture such as striped catfish and domestic aquaculture such assnakehead, walking catfish, climbing perch and clown knifefish Farmers

12

usually cultured these fish species at intensive density in non-aerated ponds,excess feeds in which they experience hypoxia or anoxia due to their air-breathing ability However, it has been argued that experiencing the hypoxiamay not benefit air-breathing fish species even in obligate air-breathing fishbecause they cost more energy for traveling to the surface to air breathe and

increase risk of predators (Lefevre et al., 2011b; Lefevre et al., 2012; Lefevre

et al., 2013) To present ability of hypoxic tolerance, Pcrit (a critical O2

tension) (Fig 2.1) is considered as a viable indicator which is defined as an

oxygen pressure point of an interaction of a resting or standard metabolic rate(SMR) line and O2pressure-dependent metabolic rate line (Mandic et al., 2009, Lefevre et al., 2011a) This value is different with temperature and specific

with species In air-breathing fishes, determination of Pcrit seems to be useless,

in the other hand, this value can represent the minimum dissolved O2 levelsthat stimulate fish to air breathe Oxygen demand of fish also is present asSMR which the minimum resting oxygen consumption is needed to maintainbasic fish body functions including breathing, circulation, excretion, proteinsynthesis, ion gradients, osmoregulation at a certain temperature and

environmental condition (Wang et al., 2009) In addition, digestion is also

considered an important physiological aspect which provided energy andnutrients for fish to survive, grow, and reproduce Jobling (1981) hadmentioned the increase of oxygen demand above the SMR during digestionstage is the specific dynamic action (SDA) which duration is temperaturedependent and is affected by hypoxia through reducing appetite and growth

(Jobling and Davies, 1980; Soofiani and Hawkins, 1982, Wang et al., 2009).

Growth rates under the effect of elevated temperature and hypoxia are aninteresting aspect desirable to investigate when aquaculture of air-breathingfish species are increasing.Elevated temperature rises the water temperature inthe environment leading to increase the biochemical reaction rate (Schulte et

al

., 2011) In some facultative air-breathing fish species, elevated temperature

of 6°C causes increases of growth rates on P hypophthamus and C ornata(Phuong et al 2017; Tuong et al 2018) However, in combination withambient hypoxia, growth rates are apparently reduced within the same levels

of temperature In order to present effect of temperature to biological reactionrates of fish, the temperature coefficient (Q10), a measure of the rate of change

of a biological reactions (here we evaluate the changes of metabolism) as aconsequence of increasing the temperature by 10°C, is used.These aboveindicators are significant to elucidate how fish response and adapt to climatechange and environmental hypoxia

13

Where T2 and T1 are temperature levels, and R1 and R2are biological process rates or reaction rates.

Fig 2.1: Pcrit value of a C.ornata

3 Gill remodeling

The fish gills are known as a multipurpose organ playing the vital roles inresponding to internal and external changes primarily responses as a media ofgas exchanges, osmotic regulation, acid-base regulation and nitrogenous

excretion as function of a kidney (Evan et al., 2005; Diaz et al., 2009).Most of

water-breathing fishes own large gill surface areas which can associated withmany problems such as increase of energetically cost of osmorespiration(Nilsson, 1986; Gonzalez &McDonald, 1992), increase of toxic entrance(Wood, 2017), increase of pathogen attachment and increase of possibility ofthreatening injury (Sundin and Nilsson, 1998) On the other hand, fish gills arethought to keep a secret key of ancient traits from ancestors that fish canbecome adaptive to changes of ambient environment by ability of gill

plasticity (Nilsson, 2007; Nilsson et al., 2012) The brachial epithelium of fish

gills has been showed that they are able to be transformed to changes of theenvironmental factors including temperature, oxygen level, toxic substances,

biotic factors (parasites), and animal state activity (exercise) (Tuurala et al.,

14

1998, Sollid et al., 2003; Sollid et al., 2005; Sollid and Nilsson, 2006; Ong et

al., 2007; Matey et al., 2008; Mitrovic and Perry, 2009) The transformation

was described as an increase or decrease of interlamellar cell mass (ILCM) of

fish gills (Sollid et al., 2003) A water-breathing fish, crucian carp (Carassius

carassius) has firstly been reported that there was a completely full of ILCM

in gill of fish exposed to 8°Cin normoxia The gills then transformed themorphometry into normal looking with bare lamellae when fish was exposed

to hypoxia (6-8% air saturation) (Sollid et al., 2003) Sollid et al (2005) latterly found that goldfish (Carassius auratus) was also able to transform gill

morphology responding to both warm water (>25°C) and hypoxia Fish gillsseem to be very sensitive and vulnerable to environmental and internalchanges when responding not only to vital environmental factors (temperatureand dissolved oxygen level) but also to unbalancing of metabolism andosmoregulation In fact, transformation of gill morphology has been also

induced by exhaustive exercise in goldfish and crucian carp (Brauner et al., 2011; Fu et al., 2011), aluminum in salmonids (Nilsson et al., 2012) and acid-

base regulation in Pangasius (Phuong unpublished data) In addition, thedecrease of ILCM is clearly shown that surface area of fish gills increased withreduce of critical oxygen tension (lower Pcrit value) having a functional

significance for oxygen uptake (Sollid et al., 2003; Fu et al., 2011).

It has been thought that gill remodeling can be a new fashion occurring inrecently evolutionary water-breathing fishes, however, in air-breathing fishspecies, it has been also emphasized that the gills are able to transform theirmorphology occurring in anabantoid (dwarf gourami) (Huang and Lin, 2011)

and killifish (Ong et al., 2007) when exposed to acid water and emerged from water to land, respectively Brauner et al (2004) has also found that an obligate air-breathing fish (Arapaima gigas) possesses protruded lamellar gills

at the early stage of its life as an exclusive water-breathing fish species andthen possesses gills without protruded lamellae when fish starts to use the air-breathing organ after getting 100g of body mass This phenomenon cannot bereversible found in this fish species Recently, a tropical air-breathing fish

species (Pangasianodon hypopthalmus) has been found that is able to increase

or reduce the ILCM under the effect of high temperature (33°C) and hypoxia

(35% air saturation) (Phuong et al., 2017; Phuong et al., 2018) These finding

on air-breathing fish may provide more evidences to support existence ofancient traits of gill remodeling that can be found in vastly extensive fishspecies

15

Gill remodeling was recorded in many fish species, however, estimatingsurface area and volume to present numerously these changes are rare The

publications of Phuong et al (2017, 2018) and da Costa et al (2007) applied

stereological method that bring us a precise estimation of filament, lamellar

surface area and volumes In the present study,C ornata was investigated on

metrical estimation of respiratory surface area and water blood diffusion

thickness under the effect of elevated temperature and hypoxia C ornata is

known as an ancient fish (discuss below) that is an important key to

investigate when C ornata was found to be able to transform its gill

morphology responding to both elevated temperature and hypoxia during 3

months of exposure (paper 4) This intriguing result in C ornataindicates that

the gill remodeling is likely an ancient characteristic kept through evolutionaryprocesses inherited by many fish species In addition, with the inherited trait,

at least, fish species are able to adapt and keep pace with the changesenvironment in progress of the climate change

4 Hypercarbia and its effect on cardioventilatory responses

Concurrently, elevated temperature and rising CO2 are two severe issuesconsequently following the climate change Concentrations of atmospheric

CO2 had increased at rate of 1% per year in the 20th century and have turned to

~3% per year recently (IPCC, 2014) Rising CO2 concentration willconsequently alter the ocean chemistry as well as lower pH of the water and

decline carbonation (Cao et al., 2007) and thus affects the aquatic life It has

been reported that rising CO2concentration influences wide range of livingorganism and fish in the ocean due to acidification of the ocean water

(Talmage and Gobler., 2011; Nowicki et al., 2012; Munday et al., 2012), base imbalance (Claiborn et al., 2002; Brauner and Baker, 2009), plasma

acid-PCO2 increase, respiratory difficulty, as well as circulation and metabolism

effects (Ishimatsu et al., 2005), and mortality in adult fish (Ishimatsu et al.,

2008) And as a result, exposure of hypercarbia and associated acidosis arelinked to reduce of feed intake and growth performance of fish in aspect of

aquaculture systems (Smart, 1981; Danley et al., 2005; Fivelstad et al., 2007).

In the Lower Mekong River Delta, the water PCO2 levels range from 0.02 to

0.6% and pH levels of 6.9–8.4 with monthly and spatial variations (Li et al.,

2013) However, in tropical aquaculture systems, where stagnant water bodyusually loads a high stocking density, overfeeding and hypoxia withoutaeration that subsequently cause severe hypercarbia in fish ponds (Damsgaard

et al., 2015) Pangasius, for example, is cultured in such environment that

PCO2 levels exceeds 17 mmHg (~2.5%) towards the end of the growth cycle

16

(Damsgaard et al., 2015) while that levels can exceed 60 mmHg (~8%) have

been reported in the natural (Furch and Junk, 1997)

Different levels of water PCO2 (hypercarbia) induce variable responses fromfish For cardioventilatory aspect, the responses of fish exposed to hypercarbiatypically are to increase the water volume of each breath and/or breathingfrequency, increase air-breathing frequency and fall in heart rate (Janssen and

Randall, 1975; Burleson and Smatresk, 2000; Reid et al., 2000; Perry and McKendry, 2001; McKendry and Perry, 2001; Gilmour, 2001; Milsom et al.,

2002; Perry and Reid, 2002) In addition, it has been recorded that hypercarbicresponses of fish species vary with a specific species, which sensitivities aretypically different Rainbow trout and zebrafish, for example, are verysensitive with small increase of PCO2 while the other species including eel,carp, tambaqui and traira need a higher PCO2 (>5%) to respond (Gilmour,2001; Milsom, 2012) In air-breathing fish species, it has been indicated thatgill ventilations increase when exposed to hypercarbia, and gill ventilations arealso inhibited while air-breathing is stimulated at a level of PCO2high enough

(Jesse et al.,1967; Johansen et al., 1967,1970; Graham and Baird, 1982; Sanchez and Glass, 2001; Sanchez et al., 2005; Boijink et al., 2010) It has

been also found that there is no change of gill ventilation (Johansen, 1966;

McMahon and Burggren, 1987; Thomsen et al., 2017, Tuong et al., 2018a)

and/or air-breathing frequency (Johansen and Lenfant, 1968; Lomholt and

Johansen, 1974) when air-breathing fish were exposed to hypercarbia C.

ornata, for example, is a facultative air-breathing fish that gill ventilations did

not change whereas air-breathing frequency showed a significant increase

during a severe hypercarbic exposure (~5%) (Tuong et al., 2018a).

The CO2/H+ respiratory responses are believed to arise primarily from theirsensitive chemoreceptors (neuroepithelial cells (NECs)) distributing

throughout the gill arches Milsom (2012) and Jonz et al (2015) have shown

that these CO2/H+ receptors are innervated by the IXth (glossopharyngeal) and

Xth (vagus) cranial nerves Existent data to date have shown that CO2/H+chemoreceptors mainly monitor the changes of CO2 level in ambient water.These results were indicated in many studies which obviously showed theintra-arterial injections of CO2/H+ without any effect on gill ventilation (Reid

et al., 2000; Sundin et al., 2000; McKendry and Perry, 2001; Perry and

McKendry, 2001; Gilmour et al., 2005; Boijink et al., 2010) as well as no

ventilatory change in acetazolamide injections (blocking carbonic anhydrase toincrease CO2 retention (hypercapnia) and blood acidification internally)

(Gilmour et al., 2005) In contrast, Wood and Munger (1994) showed the

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effect of carbonic anhydrase injections in reducing the increase ventilationprogressively of post exhaustive exercise of rainbow trout (increase PCO2 and[H+]following the exhaustive exercise) The injections of intra-arterial CO2/H+also stimulate the ventilation in rainbow trout (Janssen and Randall, 1975;

Aota et al., 1990).

It is intriguing to explore the orientation and the location of chemoreceptorsthat are responsible to monitor cardiorespiratory responses to hypercarbiaand/or hypercapnia of fish In most studied fish species, it has been reportedthat fish responds to external changes of CO2 and the sensitive receptors arefound to distribute at gill sites, and orobranchial cavity while internalresponses and central chemoreceptors orientation still receive modest

evidences (Milsom, 2012) Boijing et al (2010) examined in jeju (Hoplerythrinus unitaeniatus), a facultative air-breathing fish, exposing to

hypercarbic water induced the gill ventilatory responses which CO2 sensitivereceptors were located on the first gill arch, whereas that of air-breathingresponse were located on all the gill arches The author then decided todenervate the total gill nerves and subsequently eliminated all gill ventilationsbut the remaining of air-breathing responses were not completely eliminated

(Boijing et al., 2010) Extraordinarily, C ornata were found to respond both to

hypercarbia and hypercapnia (injection of acetazolamide and CO2 injectioninto air-breathing organs) for both intact and denervated fish that can beindirectly inferred that the CO2 sensitive receptors of C ornata at least is internal orientation and likely a central chemoreceptor (Tuong et al., 2018a).

In water-breathing fishes, central CO2/H+ chemoreceptor is equivocal, but inair-breathing fishes there are some evidences found in Sarcopterygian lungfish

(Smith, 1930; Delaney et al., 1974, Delaney et al., 1977; Babiker, 1979; Sanchez et al., 2001), and seems to be present in gar, Siamese fighting fish (Wilson et al., 2000; Hedrick et al., 1991) and now in clown knifefish (Tuong

et al., 2018a).

5 Air-breathing fish species

Most of species occuring in tropical zone own air-breathing organs (ABO)which help them being survival due to oxygen absortion from the air; some arefacultative, others are continous air-breathing species (obligate and non-obligate) Among 400 air-breathing fish species known, there are 25 species

have been raised in aquaculture as accounted of Lefevre et al (2014) Many

species of air-breathing fish have been cultured in intensive aquaculturesystem successfully because of their auxiliary ABO such as respiratory gas

bladder in Tra catfish (Pangasius hypophthamus) and clown knifefish

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(C.ornata), buccal pharyngeal epithelial surface (suprabranchial chamber) in snakehead (Chana striata), swamp eels (Monopterus albus), brachialdiverticulae (labrynth organ) in climping perch (Anabas testudineous) and walking catfish (Clarias batrachus) Names of air-breathing species, types

of air-breathing organ (ABO) and related references are listed in detail inTable2.1

Structure of air-breathing organs are results of species evolutions to survive inhypoxic conditions occurred usually in tropical water zone and there areevidences supporting that origion of evolution of air-breathing fish arose infreshwater instead of marine wateraccording to Graham (1978) Evolution ofair-breathing organs are diversible leading to many types of air-breathingmode as funtions of related remodelling organs They are listed basing onstructures, sites and degrees of development (Graham, 1997)includingrespiratory gas bladder, organs related to head region, organs derivedfrom digestive tube, and skin

I Organs relates to head region:

- Ia Buccal cavity, pharyngeal, branchial, opercular chambers covered

by respiratory epithelia and increasing surface area as well as volume ofchambers

- Ib Pharyngeal and branhchial pouches, the gills, and gill derivatives

II Organs derived from digestive tract including: esophagus, pneumaticducts, stomaches and intestines

III The skin plays an auxilary function in aerial-respiration in many kinds of fish

Species in group I and II are mainly aquatic air-breathing fish in freshwater.Group Ib, II and III perfect the ABO modelling through evolutionaryspecialization which air bladders were not fullfiled for recruiment undernatural selection for ABO Especially, in group III, amphibious fish can takeoxygen from the air and release cacbon dioxide (CO2) through gills or skin

Air bladders of some Pangasius species has been described that there are variations in gas bladder structures and functions of different Pangasius

species (Graham, 1997) It is described that air bladder of P.sutchu is a singlechamber, wide and bilobed anteriorly but smaller to a point at the end(Browman and Kramer, 1985) Volume of air bladder in this species can bearound 2-8% of body volume, ABO surface area is estimated less than 1-

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14%of body mass, and measurement of diffusion distance from air to blood

are around 1-2 μm (Browman and Kramer, 1985)

Table 2.1.Air-breathing organ types (ABO) and names of common

air-breathing fish species (Information is based on data of Graham 1997)

Arapaima Swim bladder (Greenwood and Liem, 1984)

Clown knifefish Swim bladder (Dehadrai, 1962)

(Taylor, 1831),(Owen, 1846),

Stripped catfish Swim bladder (Day, 1877; Browman and

Kramer, 1985)

Suprabranchial (Taylor, 1831),(Das, 1928),

Snakehead fish (Liem, 1980, 1984)(Ishimatsu

chamber

and Itazawa, 1981, 1993)

Swamp eels Skin, mouth, (Liem, 1980; Liem and Inger,

1987)

Walking catfish Suprabranchial (Das, 1928)(G Hughes and

chamber, skin Munshi, 1973)

(Taylor, 1831),(Day, 1868)(Das, Climbing perch Labyrinth organ 1928; Liem, 1963, 1980; Liem

and Inger, 1987)

Giant gourami Labyrinth organ (Day, 1877)

Gouramies Labyrinth organ (Herbert and Wells, 2001)

Kissing gourami Labrynth organ (Liem, 1967)

Eels Skin, Swim (Mott, 1950), (Fange, 1976),

bladder (Berg and Steen, 1965)

(Carter and Beadle, 1930),

Sucker mouth catfish Stomach (Gradwell, 1971), (Gee,

1976)(Graham, 1983)

Loach Intestine (McMahon and Burggren, 1987)

Tamuata Intestine (Carter and Beadle, 1930)

Mudskipper Skin, mouth (Graham, 1976)

Organisms with ABO in or adjacent to the digestive tract which occurs

between a branch point of pneumatic duct and sphincter dividing esophagus

and stomach In this zone, there are vastly large in vessels making highly redcolor, and air and blood diffusion distance is smaller than 1μm Species inLoricariid and Trichomycterids use stomachs as ABOs which studies are stilllimited on morphology and histology In addition, other fish species also own

the guts as respiratory organs consisting of Misgurnis, Cobitis,

Acanthophthalmus, and skin respiration including Monopterus albus, Monopterus cuchia and Clarias batrachus (Graham and Wegner, 2010).

6. Clown knifefish (Chitala ornata)

The clown knifefish, Chitala ornata (Order Oste-oglossiformes; Family,

Notopterus (Gray, 1831)) is a facultative air-breathing species (Dehadrai,

1962; Tuong et al., 2018) Their air-breathing organs are a unique shape which

functions are not only for air-breathing but reflecting both the exotic bodybuoyancy control, sound production and sound reception also (Dehadrai, 1962;Graham, 1997) Their extensive posterior projections between thepterygiophores are divided medially by a vertical septum While the anterior ofair-breathing organs is a wide connecting the dorsal left side of the esophagus

to the left-ventral side of the gas bladder through a short pneumatic duct It hasbeen reported that an internally single layer of the epithelium forms the aerialrespiratory surface which is observed to extend throughout posterior of organ(Dehadrai, 1962)

C ornatahabitats naturally in marsh-land, large water bodies and deep pools of

the mainstreams of the Mekong River including Thailand, Lao PDR,

Cambodia and Vietnam (Poulsen et al., 2004; Vidthayanon, 2012) They have

also been found in Myanmar and the Philippines as an introducing species(Vidthayanon, 2012) In Vietnam, they are grown intensively for aquacultureespecially in Hau Giang province with an estimated production of ~ 500 tons

per drop (Viet, 2015) C ornatahas been induced to breed artificially along

with producing artificial feeds for intensive production, these successful

achievements are playing an important role in domestic aquaculture (Lan et

al., 2013; Lan et al., 2015).

In aquaculture system, C ornata usually experiences many unfavorable

environmental conditions such as the hypoxia varying daily, accumulation ofexcess feeds and thus severe PCO2 and nitrite accumulation increasingly toward

the end of the growth cycles (environmental investigation at the cultured C ornataponds) C ornata is known as an air-breathing fish species that can sustain

severe hypoxia (Pcrit~6.1 to 8.7 kPa at 27 to 33°C, respectively (noting that this

Pcrit indicates the dissolved oxygen level that fish starts to shift

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from mainly water-breathing to totally air-breathing dependent)) (Tuong et al.,

2018b) In addition, ability of nitrite tolerance of Clown knifefish has beenindicated that is extremely high of 7.82 mM for 96h LC50 (Gam et al., 2017) Under combining effects of the nitrite and the hypercarbia, C.

ornatasurprisingly showed a significant decrease of the nitrite uptake in

hypercarbia which has been indicated recently (Gam et al., 2018).Surprisingly,

C ornatawere challenged to the elevated temperature and severe hypoxia

showing that the growth rates were actually better at higher temperature and at

normoxic condition (Tuong et al., 2018b).

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