The effects of elevated temperature and hypoxia on the respiratory organs of pangasianodon hypophthalmus

MINISTRY OF EDUCATION AND TRAINING 
CAN THO UNIVERSITY 
LE MY PHUONG 
THE EFFECTS OF ELEVATED TEMPERATURE AND 
HYPOXIA ON THE RESPIRATORY ORGANS OF 
Pangasianodon hypophthalmus 
PHD DISSERTATION 
MAJOR: AQUACULTURE 
MAJOR CODE: 62 62 03 01 
2017 
MINISTRY OF EDUCATION AND TRAINING 
CAN THO UNIVERSITY 
LE MY PHUONG 
THE EFFECTS OF ELEVATED TEMPERATURE AND 
HYPOXIA ON THE RESPIRATORY ORGANS OF 
Pangasianodon hypophthalmus 
PHD DISSERTATION 
MAJOR: AQUACULTURE 
MAJOR CODE: 62 62 03 01 
SUPERVISORS 
Supervisor: Assoc. Prof. Dr. DO THI THANH HUONG 
Co-supervisor: Assoc. Prof. Dr. MARK BAYLEY 
2017 
 1 
Data sheet 
Title: The effects of elevated temperature and hypoxia on the respiratory 
organs of Pangasianodon hypophthalmus 
Subtitle: PhD Dissertation 
Author: Le My Phuong 
Affiliation: College of Aquaculture & Fisheries, Can Tho University, 
Vietnam; Zoophysiology, Department of Bioscience, Aarhus 
University, Denmark. 
Publication year: 2017 
Cite as: Phuong L.M. (2017) The effects of elevated temperature and 
hypoxia on the respiratory organs of Pangasianodon 
hypophthalmus. PhD Dissertation. College of Aquaculture & 
Fisheries, Can Tho University, Vietnam and Zoophysiology, 
Department of Bioscience, Aarhus University, Denmark. 
Keywords: climate change, hypoxia, temperature, morphometric, swim 
bladder, gill remodelling, air-breathing fish, Pangasianodon 
hypophthalmus 
Supervisor: Associate Professor Do Thi Thanh Huong, Deparment of Aquatic 
Nutrition and Products Processing, College of Aquaculture and 
Fisheries, Can Tho University, Vietnam. 
Co-supervisor: Associate Professor Mark Bayley, Zoophysiology, Deparment of 
Bioscience, Aarhus University, Denmark. 
Co-supervisor: Professor Jens Randel Nyengaard, Core Center for Molecular 
Morphology, Section for Stereology and Microscopy, Centre for 
Stochastic Geometry and Advanced Bioimaging, Aarhus 
University, Denmark. 
 2 
Table of Contents 
Data sheet .......................................................................................................................... 1 
Table of Contents ............................................................................................................. 2 
ACKNOWLEDGEMENTS ............................................................................................ 3 
SUMMARY ...................................................................................................................... 4 
Chapter 1: INTRODUCTION ........................................................................................ 6 
1.1. Oxygen requirement and adaptive mechanisms of fish to hypoxia ........................ 6 
1.2. Striped catfish (Pangasianodon hypophthalmus) ................................................... 7 
1.3. General gill structure in teleosts .............................................................................. 8 
1.4. Osmo-respiratory compromise in fish gills ........................................................... 11 
1.5. Gill plasticity and environmental factors causing gill plasticity ........................... 12 
1.6. Methods applied in morphometric studies ............................................................ 14 
1.7. Objectives of dissertation......17 
REFERENCES ............................................................................................................. 17 
Chapter 2 (PAPER 1) .................................................................................................... 26 
Recovery of blood gases and haematological parameters upon anaesthesia with 
Benzocaine, MS-222 or Aqui-S in the air-breathing catfish (Pangasianodon 
hypophthalmus) ............................................................................................................... 26 
Chapter 3 (PAPER 2) .................................................................................................... 48 
Gill remodelling and growth rate of striped catfish Pangasianodon hypophthalmus 
under impacts of hypoxia and temperatures ............................................................... 48 
Chapter 4 (PAPER 3) .................................................................................................... 76 
Ontogeny and morphometric of the gill and swim bladder of air-breathing 
striped catfish Pangasianodon hypophthalmus ............................................................ 76 
Chapter 5 (PAPER 4) .................................................................................................. 104 
Gill remodelling does not affect the rate of acid-base regulation in the striped 
catfish Pangasianodon hypophthalmus ....................................................................... 104 
Chapter 6: Conclusions and Perspectives. ............................................................. 121 
APPENDICE.....125 
 3 
ACKNOWLEDGEMENTS 
Foremost, I would also like to give my thanks to Assoc. Prof. Do Thi Thanh Huong and 
Prof. Nguyen Thanh Phuong in Can Tho University for always supporting, encouraging, and 
watching my steps during my study. I would also like to express my gratitude to Assoc. Prof. 
Mark Bayley who has found and built my passions and directions for science as who I am and 
what I have today. His guidance and enthusiasm have inspired and supported me moving 
forward and working by my best during my research and writing this thesis. I am also grateful 
to Prof. Jens Randel Nyengaard for teaching me stereological methods and supporting me 
during my working in his laboratory. 
My sincere thanks also go to Prof. Tobias Wang, Prof. Atsushi Ishimatsu, and Prof. 
Hans Malte for giving me special advices and precious and initiative suggestions which 
contribute importantly in my thesis. Also, I acknowledge Christian Damsgaard who greatly 
contributed in interpreting and discussing data of the last manuscript in this thesis. 
I would like to thank the staffs at the College of Aquaculture and Fisheries, Can Tho 
University (Vietnam), at the Zoophysiology Section, Department of Biological Sciences, and 
at Core Center for Molecular Morphology, Section for Stereology and Microscopy, Centre for 
Stochastic Geometry and Advanced Bioimaging, Aarhus University, Denmark where my 
projects were carried out. Especially, I would like to thank Maj-Britt Lundorf for teaching me 
with helpful skills in histology and Per Guldhammer Henriksen for helping me during my 
experiments in Aarhus University. 
I would also like to thank my fellow friends in iAQUA project: Nguyen Thi Kim Ha, 
Le Thi Hong Gam, Dang Diem Tuong, and Phan Vinh Thinh for their friendship and all the 
funs we have during the time we worked together in the project. 
Finally, I would like to thank my family and my friends for their love and spiritually 
supporting me throughout my research, my writing, and my whole life. 
My project was funded by The Danish International Development Agency (DANIDA), 
Ministry Affairs of Foreign Denmark, iAQUA project. 
 4 
SUMMARY 
The striped catfish Pangasianodon hypophthalmus is one of the most important species 
in terms of both economy and physiology. The overall objective of this study is to provide 
input to the assessment of the effects of elevated temperature and/or hypoxia on the respiratory 
organs of air-breathing catfish Pangasionodon hypophthalmus by implementing stereological 
methods to reveal plasticity. This research will play a key role in a better understanding the 
capacity for adaptation of air-breathing fish to temperature increases. 
Four main projects were investigated in this study. In the first project, the disturbances 
in blood gasses and haematological parameters, caused by the three different anaesthetics, 
commonly used in aquaculture during transport as well as for surgical procedures, were 
investigated during recovery in Pangasianodon hypophthalmus. We found that these 
parameters were normalized within 24h and this was the first indication that P. hypophthalmus 
is unusual among air-breathing fish with its strong capacity for acid-base regulation. In 
addition, this study demonstrated that this species lacks the β-adrenergic swelling responses in 
red blood cells. 
In the second project, by applying vertical sections in stereology, gill morphometrics of 
P. hypophthalmus exposed to the average Mekong river present temperature (27°C) as well as 
to a constant 6°C elevation were investigated. These temperature treatments were combined 
with normoxic and hypoxic oxygen levels. We found strong plasticity in gills lamellar surface 
areas (SA), with highest SA under elevated temperature and hypoxia whereas almost 
eliminated. This plasticity was due to proliferation of cell mass between the secondary lamellae 
(ILCM), which was thus most developed in the normoxic low temperature group. Further, the 
diffusion distance from water to blood (HM) was thinnest (approximately 1.0µm) in the fish 
exposed to hypoxia and high temperature. At their largest, the gills of this species are on a 
weight specific basis similar to active water-breathers, such as trout, but with significantly 
shorter HM. This is the first documentation of ILCM in catfish and is similar to that found in 
cyprinids and seems to present support for the osmo-respiratory compromise phenomenon. In 
this study it was also demonstrated for the first time that this species grows much faster at 
higher than present temperatures, with an 8-fold higher growth rate at 33°C than 27°C. 
The third project, we examined the development of the gill and air-breathing organ 
(ABO) SA and HM with body size. By calculating the anatomical diffusion factor, it was 
possible to evaluate the importance of gills and ABO for oxygen uptake and to make 
 5 
interspecies comparisons. Here I found that the ADF of the gills is high (comparable to an 
active water-breather such as rainbow trout), even with fully developed ILCM. Further, when 
required, the gills can change rapidly (<20h) elevating ADF by a factor 3. In addition, the 
dimensions (respiratory SA, volume) of respiratory organs scaled with body mass, with the 
scaling slope for gills being in the range found for active water-breathing fish and with the 
swim bladder scaling as a mammalian lung. 
It has been argued that there is a compromise between gas exchange and ion regulation 
functions in fish gills. In the fourth project, we exposed the fish in two different oxygen levels 
(hypoxia and hyperoxia) to induce gill remodelling and to test whether such branchial 
remodelling affects the rate of acid/base regulation in response to aquatic hypercapnia in P. 
hypophthalmus. We found that there was no difference in the rate of acid-base regulation in 
these two groups, and suggest that despite its well-developed gills that there is a functional 
separation in this species occurring where the respiratory gill surfaces do not function in ion 
exchange. 
 6 
Chapter 1: INTRODUCTION 
1.1. Oxygen requirement and adaptive mechanisms of fish to hypoxia 
Climate change, hypoxia, and its effects 
It has been predicted that the environmental temperature is increasing as a result of 
global climate change (IPPC, 2014). It is clear that the projected increase in environmental 
temperature may lead to negative effects on fish populations as a result of disturbances on fish 
physiology, metabolism, immune functionality, reproductive capacity, behavior, growth 
performance, or mortality (Watts et al., 2001; Cnaani, 2006; Dalvi el al., 2009; khoshnevis 
Yazdi and Shakouri, 2010; Singh et al., 2013; Reid et al., 2015). Temperature elevation causes 
a reduction in oxygen concentration in water (hypoxia), and at the same time an increase in the 
metabolic demand for oxygen by living organisms, which has been argued to lead to aerobic 
scope reductions. These are the features underlying the oxygen- and capacity-limited thermal 
tolerance (OCLTT) hypothesis, which proposes that oxygen delivery is the key mechanism 
underlying the negative effects of increased temperatures in aquatic organisms (Pörtner, 2001; 
Pörtner and Farrell, 2008; Pörtner, 2010). It has also been noted that elevated temperature 
interacting with hypoxia could result in larger effects on organism performance (McBryan et 
al., 2013). Tropical regions are predicted to be seriously affected by the climate change. Indeed, 
there are more than 7000 freshwater fish species living in tropical regions, and it has been 
argued that since these animals are likely stenothermal and since they already live closer to 
their upper thermal limit, they may actually be the most vulnerable (Nelson, 1994; Tewksbury 
et al., 2008). Besides, tropical areas are known for significant fluctuations in oxygen content 
in the water, where water can be richly oxygenated in wet seasons and severely hypoxic or 
even anoxic in dry season (Welcomme, 1979; Lucas and Baras, 2008; Nguyễn Lâm Anh, 
2016). In addition, as a result of primary production in organically rich water with poor mixing, 
oxygen can fluctuate on a dual basis from deep hypoxia at night to hyperoxia during the day. 
Fish adaptive mechanisms to hypoxia 
Fish have developed a variety of adaptive mechanisms in their physiology, 
morphology, or behaviour to cope with hypoxia in the environment (Wu, 2002; McBryan et 
al., 2013). Firstly, fish have some biochemical and physiological responses to maintain oxygen 
delivery, such as increasing gill ventilation to increase water flow over the gills, increase gill 
perfusion (Randall, 1970, 1982; Wu, 2002), increase the amount of RBC (Randall, 1982; 
Soldatov, 1996; Paper 1), or increase affinity for O2 in haemoglobin (Randall, 1982; Val et al., 
 7 
1995; Damsgaard et al., 2015b). The fish may reduce their swimming activities to reduce 
oxygen demand and conserve energy expenditure under hypoxia (Schurmann and Steffensen, 
1994). Besides, numerous fish have been found with some mechanisms to increase capacity 
for oxygen uptake under hypoxic conditions by changing their respiratory structures (discussed 
in the following section). 
During long-term exposure to hypoxia, many fish species have formed an adaptive 
behaviour that they can migrate to more oxygenated waters or move to the surface of the water 
for aquatic surface respiration or aerial respiration when the water becomes hypoxic (Lewis, 
1970; Petersen and Petersen, 1990; Wannamaker and Rice, 2000; Kramer and Mehegan, 1981; 
Chapman et al., 1995). Air-breathing is one of the adaptive responses of fish to aquatic hypoxia 
(Johansen et al., 1970; Randall et al., 1981; Graham, 1997). Many species especially in tropical 
areas have evolved a transition from aquatic to aerial respiration (Graham, 1997). When the 
gills of fish fail to meet oxygen requirement for their metabolic  ... r-breathing fish have to date, been performed on Amazonian 
species from extremely ion-poor water (Shartau and Brauner, 2014). These species exhibit low 
capacities for pH regulation, and possibly the capacity for pH regulation was selected against due 
to the high metabolic cost of ionoregulation under these conditions (Heisler, 1982; Brauner et al., 
2004b; Harter et al., 2014). In contrast, air-breathing species inhabiting environments with higher 
ion levels, such as P. hypophthalmus in the Mekong Delta where ion levels such as calcium sodium 
and chloride are 10-50 times higher, may have retained the ability for pH regulation, as it is less 
metabolically costly to pH compensate in response to hypercapnia under these environmental 
conditions (Damsgaard et al., 2015; Gam et al., 2017; Thinh et al., unpublished data). 
Acknowledgements 
This study was funded by The Danish International Development Agency (DANIDA), 
Danish Ministry of Foreign Affairs, iAQUA project. 
 116 
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Chapter 6: Conclusions and Perspectives 
6.1 Conclusions 
In anaesthesia experiment with striped catfish, the all three anaesthetics effectively 
immobilized the fish and reduced responses to tactile stimulation to a level where transport or 
minor surgical procedures could be performed, but they also caused significant changes with 
similar patterns on gas and haematological parameters which were generally normalized within 24 
h. Especially, the main findings from this study showed that P. hypophthalmus is unusual among 
air-breathing fish with its strong capacity for acid-base regulation after a disturbance caused by 
the three anaesthetics. 
This dissertation also documents the high plastic responses of P. hypophthalmus gills to the 
different temperatures and hypoxic levels, with highest SA under high temperature and hypoxia 
whereas almost eliminated in lower temperature and normoxia due to the proliferation of cell mass 
between the secondary lamellae (ILCM). Moreover, the harmonic mean diffusion distance was 
found thinnest (around 1.0µm) in the fish exposed to hypoxia and high temperature. The findings 
of ILCM in P. hypophthalmus gills have revealed the first documentation of ILCM in catfish, 
which is similar to what have been found in cyprinids and seems to present support for the osmo-
respiratory compromise phenomenon. 
By measuring morphometrics (respiratory surface area and harmonic mean water- or air- 
blood diffusion distance) of respiratory organs of P. hypophthalmus, this thesis has 
morphologically demonstrated that this species has both well-developed gills and highly 
vascularized swim bladder indicating high capacity for both aquatic and aerial gas exchange. It 
was found that the ADF of the gills is high (comparable to an active water-breather such as rainbow 
trout), even with fully developed ILCM. Further, when required, the gills can change rapidly 
(<20h) elevating ADF by a factor 3. In addition, we have found there is a scaling relationship 
between these metrics with fish body mass, with the scaling slope for gills being in the range found 
for active water-breathing fish and with the swim bladder scaling as a mammalian lung. 
It was also demonstrated that the prolonged exposure to hypoxia has been found to cause 
a reduction in fish growth performance due to the energy cost for physiological and biochemical 
adjustments of the fish to these conditions. The results in this dissertation show that hypoxia 
 122 
negatively impacted on growth performance of P. hypophthalmus, possibly due to the extra 
respiratory cost for air-breathing of the fish; whereas, elevated temperature dramatically drive the 
growth of this species, with fish cultured at 33ᵒC growing approximately 8 fold faster compared 
to those at 27ᵒC. 
6.2 Perspectives 
The findings from growth performance of striped catfish under the effects of high 
temperature and/or hypoxia would illustrate that P. hypophthalmus still performs well under the 
effect of global warming when the predicted temperature to be increased 5ᵒC over the next century. 
In addition, the aquaculture production of P. hypophthalmus would be improved if improving the 
rearing conditions such as alleviating hypoxia by supplying oxygen or increasing water exchange 
in the culture system. 
From this thesis, moreover, the question remains as to why striped catfish reduces its 
branchial ADF in normoxic water. Reducing the respiratory SA by adding the ILCM barrier may 
help prevent ion loss or unwanted water uptake and therefore reduce ion-regulatory energy 
consumption. However, further research is needed to confirm this as we could see no evidence of 
mitochondrial rich cells on the secondary lamellae in the present study. 
Also, the mechanism as well as the speed with which ILCM can be increased or reduced 
should be examined and whether there are vascular shunts within the gills, or whether there is 
some other mechanism modulating the leakiness of lamellar epithelial membrane in this species. 
 123 
APPENDICE 
Fig. S1. Bilogarithmic plots of lamellar SA in relation to gill arch mass of Pangasianodon 
hypophthalmus. * indicates data from Phuong et al. (2017) of fish with no ILCM; # indicates 
potential lamella SA from first gill arch in this study. The equation for the regression line (Black 
line) for the whole data set is Y=0.834X +3.75 and R2=0.845. For the data from Phuong et al., 
(2017) the regression line (red dashed line) is given by Y=0.820X+3.71 and R2=0.705.