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: 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 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:.......................................................................................................................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


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.

iii


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.

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 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 CO 2/H
chemoreceptors existing in the facultative air-breathing, C. ornata.

+

Investigating cardioventilatory responses under the effects of CO 2 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 CO 2 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 (CO 2 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 CO 2 injection into ABO, and the
hypercarbia and hypercapnia. The results of CO 2 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 chemoreceptors and were indirectly
inferable to central chemoreceptors.

v


TÓM TẮT
Biến đổi khí hậu là một trong những vấn đề được quan tâm nhất trong nghiên

cứu 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 cứu
này để cung cấp các thông tin chuyên sâu về đánh giá tác động của sự tăng
nhiệt độ và nồng độ CO2. Các chỉ tiêu sinh lý hô hấp, các phản ứng hô hấp tim
mạch, sự thích nghi hình thái mang cá và tăng trưởng là các chỉ tiêu đã được
thực hiện để đánh giá ảnh hưởng của biến đổi khí hậu thông qua bốn nghiên
cứu.
Phản ứng hô hấp của cá theo sự tăng nhiệt độ và nồng độ oxy thấp được thực
hiện trong nghiên cứu thứ nhất. Ngưỡng oxy (P crit), trao đổi chất cơ bản
(SMR), tác động của tiêu hóa thức ăn lên hoạt động hô hấp (SDA) và tăng
trưởng của cá thát lát dưới ảnh hưởng của nhiệt độ dự báo (33°C) và nhiệt độ
trung bình hiện tại (27°C) kết hợp với hàm lượng oxy bão hòa và oxy thấp đã
được thực hiện. Kết quả cho thấy ở mức nhiệt độ dự báo cao nhất ở vùng đồng
bằng 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 đặc
điểm 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 cân
nhắc là việc dựa vào khả năng hô hấp khí trời có thể dẫn tới các hậu quả bất
lợi 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 dụng
phương pháp mô học lập thể được tiến hành trong thí nghiệm này. Kết quả cho
thấy cá có biến đổi hình thái mang bằng cách tăng sinh hoặc giảm sinh số
lượng các tế bào ở giữa lá mang thứ cấp (ILCM) trong môi trường oxy bão
hòa, và môi trường thiếu oxy và nhiệt độ cao. Diện tích bề mặt (SA) hô hấp
của mang bị ảnh hưởng nhiều bởi nhiệt độ và tình trạng thiếu oxy sau 30 ngày
nuôi. Khoảng cách khuếch tán giữa máu và nước giảm trong môi trường thiếu
oxy sau 30 ngày nuôi trong khi đó giá trị này giảm do nhiệt độ thể hiện rõ
sau60 ngày nuôi. Tính toán giá trị hệ số hô hấp (ADF) cho thấy chỉ số này cao

gấp 4 lần ở mức nhiệt độ 33°C trong môi trường oxy thấp so với ở mức 27°C
trong môi trường oxy bão hòa. Diện tích bề mặt của mang cá thát lát phù hợp
với các loài cá hô hấp khí trời. Các kết quả của nghiên cứu này củng cố cho
giả thuyết về sự tồn tại lâu dài của cơ chế biến đổi cấu trúc mang dưới tác
động 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 thụ
+

cảm CO2/H của cá đã được thực hiện trong nghiên cứu thứ ba. Cá nuôi trong
môi trường nước có tính a-xit (pH=6), môi trường CO 2 (tăng CO2 trong môi
trường nước, pH=6) và môi trường CO 2 cao trong máu (tiêm acetazolamide)
trong điều kiện oxy bão hòa. Thu mẫu phân tích các chỉ tiêu tần số hô hấp khí
trời, tần số hô hấp qua mang, nhịp tim, áp suất máu động mạch, và pH và
PCO2 máu. Trong môi trường a-xit (pH=6) thì sự thay đổi phản ứng của cá
không có ý nghĩa thống kê ở tất cả các chỉ tiêu khảo sát. Cá phản ứng lại môi
trường hypercarbia và môi trường hypercapnia qua sự tăng tần số hô hấp
nhưng 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 PCO2
trong máu cá tăng ở môi trường hypercarbia và môi trường hypercapnia (tiêm
acetazolamide). Kết quả của nghiên cứu cung cấp cơ sở về bằng chứng cho sự
+

hiện 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 bằng
cách tiêm CO2 vào cơ quan hô hấp khí trời (ABO) của cá không cắt dây thần
kinh, và ảnh hưởng của hypercarbia và hypercapnia lên cá cắt dây thần kinh
được thực hiện trong nghiên cứu thứ tư. Liều tiêm CO 2 vào bong bóng khí của
cá tăng dần theo nồng độ CO 2có trong hỗn hợp CO2 và không khí. Tiến hành cắt
th
th
các dây thần kinh thứ IX và X , và cho cá hồi phục trong 24 giờtrước khi
cho tiếp xúc với hypercarbia (tăng CO 2 ~ pH=6) và acetazolamide (tăng
+
[H ]và PCO2 trong máu). Kết quả cho thấy cả cá không cắt dây thần kinh
vàcắt dây thần kinh đều phản ứng tăng tần số hô hấp khí trờiđáng kể. Nhịp tim
chậm 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 nhận
được trong tất cả các mức nồng độ tiêm CO 2 vào ABO, và hypercarbia và
hypercapnia. Các kết quả tiêm CO2 vào ABO (không cắt dây thần kinh), và
hypercarbia và hypercapnia đã cung cấp thêm thông tin chứng minh sự tồn tại
+
của các thụ cảm CO2/H định hướng bên trong và gián tiếp cho thấy sự hiện
diện của cơ quan thụ cảm trung tâm.

vii


RESULT COMMITMENT
I commit that this dissertation was investigated based on all the results of my
studies. All the data and showed results in the dissertation were honest and
have never been published before. The iAQUA project can completely use
these data and results.

Can Tho, April ....., 2019
Supervisor

PhD student

Prof. Dr. Tran Ngoc Hai

Dang Diem Tuong

viii


LIST OF FIGURES
Chapter 1: Introduction

page
4

Fig.1.1: Diagram of research activities
Chapter 2: Literature review
Fig. 2.1: Pcrit value of a C. ornata.

14

Chapter 3:Clown knifefish (Chitala ornata) oxygen uptake and
its partitioning in present and future environments
determination of C.ornata at 27°C and
Fig. 3.1: Pcrit
33°C,mean±S.E.M, N=8.

42

Fig. 3.2: Oxygen partitioning in normoxia and hypoxia at 27°C and

46

33°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
mass 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 prefeeding 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
growth rate (%) (B) of C. ornata during 3 months at 27°C in

47
48

50

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
to different temperature and/or hypoxia.

69

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

70

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

70

Fig. 4.4: Calculated anatomic diffusion factor of C. ornata exposed
to different temperature and/or hypoxia.

72

ix


Fig. 4.5: One side gill arches of C. ornata showing five arches were
collected from a preserved sample. The fifth arch is reduced
without observing filaments.

75

Fig. 4.6: Gill filaments of C. ornata under light micrographs from
normoxia 27°C, hypoxia 33°C, at 0, 1 and 2 months. Fish gill
morphology was alike primary water breathing fish at preexperiment 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.

75

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

87

associated with type 1 and 2 air breaths.
Fig. 5.2: Air breathing frequency under control conditions and at
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.

89

Fig. 5.3: Buccal ventilation (N=8), opercular ventilatory frequency
(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.

90

Fig. 5.4: Heart rate and mean arterial pressure at 60 and 30 s before
and30, 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 StudentNewman-Keuls post hoc test; P<0.05; data are mean±S.E.M).

91

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

92

x


pH (left hand panels) or PCO 2 (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
in denervated fish
Fig. 6.1: The air breathing frequency of intact C. ornata injected
ascending percentage of CO2 air mixture into air bladder. Data are
presented as mean±S.E.M. The data were fit with regression
equations.

108

Fig. 6.2:Gill ventilation of intact C. ornata injected ascending

109

percentage of CO2 air mixture into air bladder. There is no
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

110

percentage 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
hypercarbia 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
mean±S.E.M.

112

Fig. 6.6: Gill ventilation of denervated C. ornata exposed to
hypercarbia and acetazolamide. (One way ANOVA for repeated

measurement followed by LSD post hoc test; P<0.05). Data were
presented as mean±S.E.M.

113

Fig. 6.7: Heart rates of denervated C. ornata exposed to
hypercarbia and acetazolamide. (One-way ANOVA for repeated
measurement followed by LSD post hoc test; P<0.05). Different

114

xi


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
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.

115

Fig. 6.9: Rate of air breathing presented as a function of arterial pH

116

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

20

Chapter 3: Clown knifefish (Chitala ornata) oxygen uptake and
its partitioning in present and future environments
Table 3.1: SMR (mgO2 kg h ), RMR (mgO2 kg h ) and
partitioning 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.

−1

−1

−1

−1

45

Table 3.2: SDA parameters prior and post feeding 2% of body

49

mass 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
different temperatures.

51

Table 3.4: The effect of temperature on SDA and SDA coefficient
in fish.

52

Chapter 4: Gill remodeling of Clown knifefish (Chitala ornata)
under impact of temperature and hypoxia
2 -1

Table 4.1: Lamellar surface area (mm g ), gill filament volume
3 -1

71

3 -1

(mm g ), lamellar volume (mm g ), harmonic mean (HM) water
2 -1

-1
blood thickness (µm) and anatomic diffusion factor (mm g µm )
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
hypoxic levels on gill parameters of C.ornata.

73

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

76

xiii


ABBREVIATIONS
[H+]:

Hydrogen ion concentration

AAS:

Apparent aerobic scope

ABO:

Air-breathing organ

ADF:

Anatomic diffusion factor

ATP:

Adenosine triphosphate

AZ:

Acetazolamide

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 air
Hypercapnia: High level of carbon dioxide in blood
I.D.:
ILCM:

Inner dimension
Inter lamellar cell mass

IPCC:

Intergovernmental Panel on Climate Change

ṀO2:

Oxygen uptake used in metabolism

NECs:

Neuroepithelial cells

NH3

Ammonia cation

+

:

NO2 −:
NO3 −

Nitrite ion

O.D.:
PBS:

Outer dimension
Phosphate buffer solution

:

Nitrate ion

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:

Specific dynamic action

SMR:

Standard metabolic rate

SDA coefficient: Specific dynamic action coefficient

xv


CHAPTER 1:
INTRODUCTION
1.1 General introduction
Climate change is dramatically challenging and threatening aquatic animal life
through rising water temperature and elevated CO 2 levels. It has been
predicted by international panel on climate change (IPCC, 2014) for Mekong

River Ddelta that the temperature will increase 2.5-3.5C in next 100 years;
and concurrently, the elevated atmospheric CO 2 levels will rise up at rate 3%
per year recently.
It has been indicated that projected the elevated water temperature may
negatively 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 animal
distribution, the rising environmental water CO 2 level (hypercarbia) is more
related 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 considered
intriguing targets to research under effects of projected elevated temperature
and hypercarbia in water.
A hypothesis of oxygen capacity limited thermal tolerance is proposed to
express negative impacts of the elevated temperature on the fish that is
underlying 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 fish
oxygen demand significantly increases with the elevated temperature.
Therefore, the elevated temperature integrating with hypoxia (the decrease of
the dissolved oxygen level) has been indicated that could result in a largely
severe 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 lived
near 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 hypoxic
water tolerance could be a result of an evolution under the effects of higher
temperatures and lower atmospheric oxygen pressure than the present.
Investigating the effects of the elevated temperature and hypoxia on the
metabolism of the air-breathing fish is important to assess the effects of
climate change.
Another aspect of the adaptive ability to environmental factors, it has been
exposed 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 waterbreathing fishes including crucian carp, goldfish and salmonids which their
gills have been found to increase or decrease interlamellar cell mass (ILCM) to
increase or decrease respiratory surface area with changes of the
environmental factors. This ability of fish is intriguing scientists that whether
the gill remodeling is a modern trend or an ancient trait (Nilsson, 2007;
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 the
air-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 CO 2level is rising year by year
consequently due to the global warming. The dissolved CO 2 level is more
dissoluble than the oxygen therefore, with a small increase of the CO 2 level in
the atmosphere, a largely significant increase of the dissolved CO 2 level can be
produced in the aqueous system. It has been reported that PCO 2 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 culture
systems 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 fish
+

species. Cardioventilatory responses derived from central CO 2/H -sensitive
chemoreceptors are still equivocal in the facultative air-breathing fish. The
effect of hypercarbia and hypercapnia on the cardioventilatory, and blood gas
+

and pH responses as well as CO 2/H -sensitive chemoreceptors location are
important to investigate.Clown knifefish (C.ornata), a tropical facultative airbreathing fish species (Deharai, 1962; Tuong et al., 2018b) was selected as a
model fish to challenge with a worst-case predicted temperature (33C),
hypoxia, water hypercarbia, internal hypercapnia to see how the respiratory
metabolism, gill plasticity responses and adaptation through the growth
performance,
orientation.

cardioventilation,

and

+

CO2/H -sensitive

chemoreceptors

1.2 Research objectives
The main objectives of this dissertation were to evaluate the impacts of climate
change (in case of theelevated temperature, hypoxia, elevated CO 2)on
C.ornata.
In specific, respiration of Clown knifefish at 27C and 33C in combination
with normoxia and hypoxia evaluating through SMR, percentage of airbreathing, Q10 value, specific dynamic action (SDA) and growth.
Consequently, to understand and explain how fish respiratory adaptation to the
elevated temperature and hypoxia, gill morphology was studied through ability
of gill remodeling and estimating respiratory surface area, water blood
thickness. 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 partial
pressure (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 the
branchial surface area, volume and water blood diffusion thickness under
the effects of the elevated temperature and hypoxia applying stereological
method.
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


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7