environmental modulation of the onset of air-breathing of
TRANSCRIPT
ENVIRONMENTAL MODULATION OF THE ONSET OF AIR-
BREATHING OF THE SIAMESE FIGHTING
FISH AND THE BLUE GOURAMI
JOSE FERNANDO MENDEZ SANCHEZ
DISSERTATION PREPARED FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF NORTH TEXAS
DECEMBER 2015
APPROVED:
Warren Burggren, Committee Chair
Dane Crossley, Committee Member
Ed Dzialowski, Committee Member
Ione Hunt von Herbing, Committee Member
Pamela Padilla, Committee Member
Arthur Goven, Chair of the Department of Biological Sciences
Costas Tsatsoulis, Acting Dean of the Toulose Graduate School
Mendez Sanchez, Jose Fernando. Environmental modulation of the onset of
air-breathing of the Siamese fighting fish and the blue gourami. Doctor of
Philosophy (Biology), December 2015, 149 pp., 28 figures, 1 table, references,
134 titles.
This study determined the effect of hypoxia on air-breathing onset and
physiological and morphological characters in larvae of the air breathing fishes
Trichopodus trichopterus and Betta splendens. Larvae were exposed intermittently
(12/12 h daily) to 20, 17, and 14 kPa of PO2 from 1 to 40 days post-fertilization.
Survival, onset of air breathing, wet body mass, ��𝑀O2, Pcrit were measured every 5
dpf. Hypoxia advanced by 4 days, and delayed by 9 days, the onset of air breathing
in Betta and Trichopodus, respectively. Hypoxia increased larval body length, wet
mass, and labyrinth organ respiratory surface of Betta, but did not affect these
factors in Trichopodus. Hypoxic exposure increased ��𝑀O2 by 50-100% at each day
throughout larval development in Betta, but had no effect on larval Trichopodus.
Hypoxia decreased Pcrit in Betta by 37%, but increased Pcrit in Trichopodus by 70%.
Larval Betta reared in hypoxia showed a modified heart rate:opercular rate ratio (3:1
to 2:1), but these changes did not occur in Trichopodus. Compared to Betta, the
blood of Trichopodus had a higher P50 and much smaller Bohr and Root effects.
These interspecific differences are likely due to ecophysiological differences: Betta is
a non- obligatory air-breather after 36 dpf with a slow lifestyle reflected in its low
metabolism, while Trichopodus is an obligatory air-breather past 32 dpf with an
athletic fast lifestyle and accompanying high metabolism.
ii
Copyright 2015
By
Jose Fernando Mendez Sanchez
iii
ACKNOWLEDGEMENTS
I would like to express my sincere appreciation and gratitude to
my advisor, Professor Warren Burggren, for his support during the six
years of teaching me the scientific lifestyle and proper English, for
introducing me to the fantastic world of the integrative biology, and for
all his patience. I’m very grateful with the members of my Committee -
Dane Crossley, Ed Dzialowski, Ione Hunt von Herbing, and Pamela
Padilla - for their contribution to my PhD formation in the classroom,
lab, fieldwork, and for their valuable comments and suggestions to
improve this thesis. I would also like to thank all of my lab mates - Kelly
Reyna, Francis Pan, Josele Flores, Silvia Branum, Josie Rositto, and
Mallory Burdick - for their supportive and inspiring fellowship. Thanks
especially to Monica and Delia; you brought me clarity and patience I
needed to actually finish my research. My participation at the Biological
Sciences PhD Program was possible with the support of NSF grants
IOS-1025823 and IOS-0942287. Also I would like to thank to Programa
de Mejoramiento del Profesorado (PROMEP México) for the grant
103.5/09/4407, to Universidad Autónoma del Estado de México grant
UAEMEX-127, and University of North Texas Toulouse Graduate
School Dissertation Fellowship.
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TABLE OF CONTENTS
CHAPTER 1 ........................................................................................1
INTRODUCTION TO HETEROKAIRY AND AIR-BREATHING FISH ....... 1
HYPOXIA AS A PROMOTER OF DEVELOPMENTAL PLASTICITY IN
FRESH WATER ....................................................................................... 5
FISH AIR-BREATHING AND LABYRINTH FISH ...................................... 8
INVESTIGATING HETEROKAIRY USING THE ONSET OF AIR-
BREATHING IN BIMODAL BREATHING FISHES ................................. 11
HYPOTHESES TO BE TESTED ....................................................... 14
AIMS IN AID OF TESTING THE HYPOTHESES ................................... 15
CHAPTER 2 ...................................................................................... 17
ENVIRONMENTAL MODULATION OF THE ONSET OF AIR-
BREATHING AND SURVIVAL OF BETTA SPLENDENS AND
TRICHOPODUS TRICHOPTERUS ........................................................ 17
INTRODUCTION .................................................................................... 17
MATERIALS AND METHODS ................................................................ 23
RESULTS .............................................................................................. 29
CHAPTER 3. ..................................................................................... 44
THE SUPPORTING LARVAL RESPIRATORY ANATOMY: HYPOXIA
MODULATION OF LARVAL GROWTH, GILL AND LABYRINTH ORGAN
MORPHOMETRICS AT THE ONSET OF AIR-BREATHING .................. 44
INTRODUCTION .................................................................................... 44
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MATERIALS AND METHODS ................................................................ 48
RESULTS .............................................................................................. 52
DISCUSSION ......................................................................................... 66
CHAPTER 4 ...................................................................................... 72
RESPIRATORY PHYSIOLOGICAL ADJUSTMENTS TO CHRONIC
HYPOXIA IN LARVAE OF THE AIR BREATHING ANABANTID FISHES
BETTA SPLENDENS AND TRICHOPODUS TRICHOPTERUS. ............ 72
INTRODUCTION .................................................................................... 72
METHODS ............................................................................................. 75
RESULTS .............................................................................................. 80
DISCUSSION ......................................................................................... 90
SUMMARY ............................................................................................. 99
CHAPTER 5 .................................................................................... 101
HEMOGLOBIN-OXYGEN EQUILIBRIUM CURVES, BOHR AND ROOT
EFFECTS COMPARISON IN TWO AIR-BREATHING FISH,
TRICHOPODUS TRICHOPTERUS AND BETTA SPLENDENS. .......... 101
INTRODUCTION .................................................................................. 101
METHODS ........................................................................................... 104
RESULTS ............................................................................................ 106
DISCUSSION ....................................................................................... 110
CHAPTER 6 .................................................................................... 116
CONCLUSIONS ................................................................................... 116
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REFERENCES ................................................................................ 127
vii
LIST OF ILLUSTRATIONS AND TABLES
Figure 1. Survival of B. splendens in three water PO2 treatments. a)
Continuous hypoxia. b) Intermittent hypoxia.▼=20 kPa, О=17kPa;
●=14kPa. Means and standard errors are plotted. ...................... 30
Figure 2. Survival of T. trichopterus at three water PO2 treatments. a)
Continuous hypoxia. b) Intermittent hypoxia.▼=20 kPa, О=17kPa;
●=14kPa. Means and standard errors are plotted. ...................... 31
Figure 3. a) Proportion of B. splendens larvae that are air-breathers,
plotted through development at different PO2 treatments. Means
and standard errors are plotted. ▼=20 kPa, О=17kPa; ●=14kPa.
b) Onset of air-breathing by PO2 treatment. 95% confidence
intervals are plotted and sample sizes are in parenthesis, *
represents significant differences (p<0.001). ................................ 33
Figure 4. a) Proportion of T. trichopterus larvae that are air-breathers,
plotted through development at different PO2 treatments. Means
and standard errors are plotted. ▼=20 kPa, О=17kPa; ●=14kPa.
b) Onset of air-breathing by PO2 treatment. 95% confidence
intervals are plotted and sample sizes are in parenthesis, *
represents significant differences (p<0.001). ................................ 35
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Figure 5. A) Larval body length of Betta through development in each
PO2 treatment. B) Effect of PO2 treatment on Betta larval body length
correcting for the effect of age. Means ± standard errors are
presented. * indicates a significant difference from control value
(p<0.05). ....................................................................................... 53
Figure 6. A) Trichopodus larval body length through development in
each PO2 treatment. B) Effect of PO2 treatment on Trichopodus
larval body length correcting for the effect of age. Means ± standard
error are presented. * indicates a significant difference from control
value (p<0.05). .............................................................................. 54
Figure 7. A) Betta larvae wet mass through development in each PO2
treatment. B) Effect of PO2 treatment on Betta larval wet mass
correcting for the effect of age. Means ± standard error are
presented. * indicates a significant difference from control value
(p<0.05). ....................................................................................... 55
Figure 8. A) Trichopodus larval wet mass through development in each
PO2 treatment. B) Effect of PO2 treatment on Trichopodus larval wet
mass correcting for the effect of age. Means ± standard errors are
presented. * indicates a significant difference from control value
(p<0.05). ....................................................................................... 56
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Figure 9. A) Effect of PO2 treatment on Betta larval condition factor (K)
correcting for the effect of age. B) Effect of PO2 treatment on
Trichopodus larval condition factor (K) correcting for the effect of
age. Means ± standard error are presented. * ............................ 57
Figure 10. A) Effect of PO2 treatment on Betta larval wet mass – body
length relationship. B) Effect of PO2 treatment on Trichopodus larval
wet mass – body length relationship. W = aLb, W = wet mass, L=
body. The values of a and b coefficients are showed for each PO2
group. The statistical significance and fit adjustment are also shown
with F, p, and r. The statistical difference within the b exponent of
the PO2 groups is presented with an ANOVA analysis. ................ 58
Figure 11. 4X compound microscope images of the dissected first
branchial arch from Betta (A) and Trichopodus (B) at 35 dpf. Closed
line shape and arrow show the forming labyrinth organ (lo) .......... 60
Figure 12. A) Variation of labyrinth organ respiratory surface (TSAlab)
at the onset of air-breathing in three PO2 levels and between the two
species. B) Lamellar respiratory surface (TSAlam) at the onset of air-
breathing in three PO2 levels and between the two species. Means
± standard errors are presented. Boxes enclose means not
significantly different from each other (p>0.05). ............................ 61
x
Figure 13. Variation of arch length (Larch) at the onset of air-breathing
in three PO2 levels and between the two species. Means ± standard
errors are presented. Boxes enclose means not significantly
different from each other (p>0.05) when the analysis was corrected
for body length. ............................................................................. 62
Figure 14. Variation of number of filaments per arch (NFarch) at the
onset of air-breathing in three PO2 levels and between the two
species. Means ± standard errors are presented. Boxes enclose
means not significantly different from each other (p>0.05). .......... 63
Figure 15. Variation of filament length (Lflm) at the onset of air-breathing
in three PO2 levels and between the two species. Means ± standard
errors are presented. Boxes enclose means not significantly
different from each other (p>0.05) when the analyses were corrected
for body length. ............................................................................. 64
Figure 16. Variation of number of lamellae per filament per arch (NLflm)
at the onset of air-breathing in three PO2 levels and between the two
species. Means ± standard errors are presented. Boxes enclose
means not significantly different from each other (p>0.05) when the
analyses was corrected for body length ........................................ 65
Figure 17. Variation of area of individual lamella per filament per arch
(SAlam) at the onset of air-breathing in three PO2 levels and between
xi
the two species. Means ± standard errors are presented. Boxes
enclose means not significantly different from each other (p>0.05).
...................................................................................................... 66
Figure 18. Oxygen consumption (MO2) of larval Betta splendens. A)
MO2 through 35 days of development reared in three levels of PO2.
B) MO2corrected for aged differences in the three larval populations.
Means ± SE are presented. N=9. An * indicates a significant
difference from control (20 kPa). ................................................... 81
Figure 19. Oxygen consumption (MO2) of larval Trichopodus
trichopterus. A) MO2 through 35 days of development reared in three
levels of PO2. B) MO2 corrected for aged differences in the three
larval populations. Means ± se are presented. N=9. An * indicates
a significant difference from control. ............................................. 82
Figure 20. Representative trace of 4 larval Trichopodus MO2. The fish
were grown at 14 kPa during 35 dpf; the average Pcrit is shown with
an arrow and it was estimated by the method of Muller and Seymour
(2011). Each symbol represents an individual analyzed in the Loligo
Systems respirometry system. ...................................................... 83
Figure 21. Comparison of Pcrit for larval Betta and Trichopodus reared
in different levels of PO2 at day 35 of development. Boxes enclose
xii
statistically identical means (p>0.05). N values for each group are in
parentheses. ................................................................................. 84
Figure 22. A comparison of heart rate (Hr) in larval Betta splendens (A)
and Trichopodus trichogaster (B) through 35 days of larval
development in three levels of PO2. Means ± SE are presented. N=9.
The horizontal lines represent the average Hr for all ages. ........... 85
Figure 23. Comparison of opercular rate (Or) through 35 days of larval
development in (A) Betta splendens and (B) Trichopodus
trichogaster reared in three different levels of PO2. Means ± SE are
presented. N=9. The horizontal lines represent the average Hr for
all ages.......................................................................................... 87
Figure 24. Representative traces taken from software analysis of video
images of Betta splendens (35 days post fertilization) raised in
hypoxia (17kPa). The dashed vertical lines towards the right of the
panels illustrate the ~ 2:1 ratio of heart beat to opercular movement
in this trace. See text for additional details. .................................. 88
Figure 25. The Hr:Or relationship in larvae of (A) Betta splendens and
(B) Trichopodus trichopterus from 5 to 35 dpf are presented. The
lines show the data for three groups reared in different levels of PO2.
N= 20 for each PO2 group of each species. In all cases r > 0.9 y
xiii
p<0.05 indicating significance of relationship. See text for statistical
analysis of differences between experimental groups ................... 89
Figure 26. Oxygen equilibrium curves for Betta splendens (A) and
Trichopodus trichogaster (B). Each line and symbol set represents
an individual. The sex is indicated on the legend. The mathematical
model that best fits the data set is presented along with the
associated mathematical and statistical values. .......................... 107
Figure 27. The Bohr effect on the whole blood oxygen equilibrium
curve of Betta splendens (A) and Trichopodus trichogaster (B).
Each line describes the fitted model created with 3 adult individuals
of each species ........................................................................... 108
Figure 28. Hill´s graph of Betta (A) and Trichopodus (B). Hill´s n values
are pointed with the arrows for each pH-CO2 value. ................... 110
Table 1. P50 and Φ values at different and CO2-manipulated pHs to
determine the magnitude of Bohr effect on the whole blood of both
fish species ................................................................................. 108
1
CHAPTER 1
INTRODUCTION TO HETEROKAIRY AND AIR-BREATHING FISH
Plasticity and Developmental Plasticity
The ability of an individual to express two or more genetically
controlled phenotypes in different environments is known as
phenotypic plasticity (Hill, et. al. 2008). When phenotypic plasticity is
studied in a development context this phenomenon is called
“developmental plasticity”, and refers to the fact that there are windows
of time during ontogeny when an organism is more prone to have an
altered developmental trajectory in response to stressors in the
external environment (Pigliucci, 1998; Burggren and Reyna, 2011).
During development these environmentally sensitive phenotypes, not
the genotype or the gene, are subject to selection, and they will
produce individuals with differential fitness (West-Eberhard, 2005).
Similarly, a physiological phenotype is a physiological feature of
an individual that is the result of specific genetic expression and/or non-
genetic regulation for a particular environment (Hochachka, et al.,
1998). When a physiological character is expressed during
development, it is termed a physiological developmental phenotype.
2
The varying of its form is also dependent on variation in the
environment, and this developmental physiological feature has an
impact on individual fitness (West-Eberhard, 2005). In other words,
developing individuals of the same species could present different
physiological phenotypes in different environments. How these
concepts fit into an evolutionary context will now be considered.
Heterokairy and Heterochrony
Heterochrony is the altered timing of the developmental events
as a mechanism of evolution (Gould, 1977). That is, heterochrony is
the interspecific variation in genetically altered timing of key
developmental landmarks (Spicer, et al., 2011). This concept can be
modified to consider intraspecific characters as well. Thus,
heterochrony is the alteration of the timing of the development of traits
(be they systems or components of that system) in one species relative
to an ancestral species (Spicer and Burggren, 2003; Spicer and
Rundle, 2006). Heterochrony is useful in understanding developmental
changes at the species level, but such changes also occur at the
population and individual level. Thus, heterokairy is the non-genetic
change in timing of the onset of physiological regulatory systems or the
plasticity in the timing of the onset of physiological regulatory systems
3
or their components below the level of the species, between-
populations and between individuals (Spicer and Burggren, 2003;
Spicer, et al., 2011).
A physiological regulatory system, just like morphological
characters, can be delayed or brought forward in both chronological
age and/or in a developmental sequence during an individual’s life.
Therefore, physiological heterokairy refers to plasticity in the timing of
the onset of physiological regulatory systems or their components
through development as a response to short term, acute environmental
stressors (Spicer and Burggren, 2003).
Physiological Heterokairy in Fish
Physiological heterokairy, though a relatively new concept begin
to be backed up by empirical evidence (Mueller et al., 2015). Moreover,
the concept has been gaining credibility from the reinterpretation of
previous data collected for other proposes. In an analysis by Spicer and
Rundle (2007), the concept of heterokairy was considered as the
alterations to developmental sequences at level of the individual, and
they summarized empirical evidence of this phenomenon. Below, are
described examples of heterokairy in fishes.
4
A clear example of heterokairy comes from the ontogeny of
salinity tolerance in salmonids during the transition from fresh water to
a marine existence, which was brought forward in development when
trout were pre-exposed to sea water (Hiroi and McCormik, 1995), were
administered cortisol or insulin (McCormic, 1995; McCormic et al.,
1991), or by temperature treatments (Handleland et al., 2004). As well,
thermal environment alters the ontogeny of muscle physiology of the
Atlantic Herring. The rostral-to-caudal progression of myofibril
synthesis begins earlier and slow muscle innervations appeared
earlier, as environmental temperature increased (Johnston et al.,
1997). Finally, a more recent heterokairy study of the California
Grunion showed plasticity in the hatching time. Embryos from the same
spawning may hatch at different times and different developmental
stages. They responded to unpredictable environment with diapause,
and then responded rapidly when the environment becomes favorable
with hatching (Moravek and Martin, 2011). Environmental stressors
drive Heterokairy. We will now consider the most common and
ecologically relevant environmental stressors.
5
HYPOXIA AS A PROMOTER OF DEVELOPMENTAL PLASTICITY IN FRESH WATER
Dissolved oxygen is the main limiting environmental factor for
life in fresh water (Diaz and Breitburg, 2009). Indeed, hypoxia-driven
natural selection is considered to be the single most important promoter
for evolution and diversification of air-breathing in fishes (Randall et al,
1881; Graham, 1997). Hypoxia has been defined in the ecological
literature as a shortage of O2, specifically dissolved O2 below 5-6 mg
O2/L in fresh water environments (Diaz and Breitburg, 2009; Verberk et
al., 2011). Clearly, defining a specific oxygen level as “hypoxia” is a
poor way to describe hypoxia, because what is functionally hypoxic for
one fish is certainly not functionally hypoxic for all fish (Farrell and
Richards, 2009).
Every response to hypoxia by a fish can be considered a
mechanism exposed to natural selection and therefore serves as a
source of plasticity (West-Eberhard, 2005). Fishes respond to hypoxia
on evolutionary time with a wide range of cellular-molecular,
physiological, anatomical and behavioral adaptations that varies
among species, life stages and habitats (Diaz and Breitburg, 2009).
6
At the molecular and cellular levels, when the circulatory and
breathing systems supplying O2 fail, the rate of mitochondrial O2
utilization declines, resulting in cellular hypoxia (Hill et al., 2008). The
response at this level is the increased expression of Hypoxia-Inducible
Factor 1 (HIF-1), which potentially affects the transcription of dozens or
hundreds of target genes. The target genes differ from tissue to tissue
within a single species and they also differ among species (Semenza,
2010). HIF-1 has an extremely broad range of potential actions. In
vertebrates, HIF-1 increases synthesis of glucose transporters,
enzymes of anaerobic glycolysis, and a form of mitochondrial
cytochrome oxidase that is particular efficient in using O2. HIF-1 also
promotes angiogenesis to create additional surface area of the same
diffusion distance (Hill et al., 2008; Semenza, 2010).
At the physiological level, the first step to maintaining oxygen
homeostasis in fish is the detection of hypoxia. The gills are the main
sites of oxygen chemoreception in developing aquatic vertebrates, and
oxygen-sensitive neuro-epithelial cells (NECs) are present in the gills
in larval stages of zebrafish (for reviews see Zaccone et al., 2008; Jonz
et al., 2015). In response to hypoxia, NECs undergo membrane
7
depolarization resulting from an inhibition of outwardly directed
potassium currents that ultimately elicit calcium entry and the release
of neurotransmitter (Perry et al., 2009). There is enough evidence to
consider nitric oxide (NO) as one of these neurotransmitters. Besides
having roles in regulating multiple biological functions (neurotransmitter
function, mediation of cell growth, apoptosis, platelet adhesion and
vasodilatation), NO is regarded as a regulator of oxygen-sensitive
phenotypes and a factor in the maintenance of oxygen homeostasis,
since low oxygen levels characterize the normal microenvironment of
cells in many tissues (Zaccone et al., 2008). Fish may exhibit multiple
additional sites of O2 chemoreception that show marked intraspecific
variability. However, little is known of the central pathways through
which information from peripheral O2 sensors is integrated to elicit
ventilatory chemoreflexes (Perry et al., 2009).
After the initial sensing of hypoxia, the most dominant response
to hypoxia in water-breathers is hyperventilation. In bimodal (water and
air) breathers the additional and often dominant response is the
increase in reliance upon air-breathing.
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These responses, collectively representing the hypoxic
ventilatory reflex, are frequently accompanied by increases in
hemoglobin oxygen binding affinity and blood O2 carrying capacity that
enhance O2 transfer and might therefore lower the ventilatory
convection requirement (Perry et al., 2009). At the same time the
cardiac response to acute hypoxia in fishes is typically reflex
bradycardia (a decrease in heart rate), but also an increase in stroke
volume and branchial vascular resistance (Gamperl and Driedzic,
2009).
FISH AIR-BREATHING AND LABYRINTH FISH
Air-breathing in fish involves the process of gas exchange, for
proposes of respiration, directly from the aerial environment. Air-
breathing is an adaptive response to habitats where O2 supplies may
be severely depleted. All air-breathing fish are also water-breathers;
that is, they ventilate their gills or other aquatic respiratory surfaces to
sustain adequate gas exchange (Burggren, 1979; Randall et al., 1982;
Graham, 1997). Phylogenetically speaking, air-breathing fishes are a
highly polyphyletic group, as air breathing has independently arisen in
49 known diverse fish families (Graham, 1997; Rüber et al., 2006).
9
The Labyrinth fishes (Perciformes: Anabantoidei) are a natural
assemblage of air-breathing fishes, and includes fishes such as the
gouramis, the Siamese fighting fish, and paradise fish. They possess
paired suprabranchial chambers, also called labyrinth organs, which
function in air-breathing. These are greatly modified epibranchial
structures housed, as the name suggests, in a cavity immediately
above the gill arches. Both the wall of the cavity and the modified
epibranchial are covered with a respiratory epithelium and assist in
accessory air-breathing. The “labyrinth” modifier derives from its
complex folding that greatly increases respiratory surface (Graham,
1997; Rüber et al., 2006).
Anabantid fishes are divided into two categories - facultative and
continuous air-breathers (Graham, 1997). Facultative air breathers do
not normally breathe air in normoxic water but need to adopt this mode
when exposed to conditions unfavorable for aquatic respiration
(hypoxia) or in response to increased O2 requirements (as a result of
changes in water temperature or activity). Continuous air-breathers
take air breaths at more less regular but not highly predictable intervals,
at all times, and under all aquatic conditions from hyperoxia to hypoxia.
Continuous air-breathers at the same time can be separated into two
10
types: obligatory and non-obligatory. Obligatory ones are not able to
survive on the quantity of O2 obtained by aquatic respiration, even in
normoxic water, and thus they always need supplemental aerial
oxygen. By contrast, non- obligatory air-breathers do not require air-
breathing to survive in normoxic water.
Developmentally, the site of the gas exchange as labyrinth
fishes begins with cutaneous exchange. Gills, and specifically
secondary lamellae, gradually displace the cutaneous exchange once
they have developed (Pelster, 2008). All anabantids are continuous
aquatic air breathers and they begin gulping air well before their ABO
is fully formed. The variation in ABO development is related to the
ambient conditions experienced by young fish during the 20 or so days
after hatching, while ABO development is taking place (Graham, 1997).
The development of an air ABO, the main gas exchanger of adults, is
accompanied by changes in the cardiovascular system necessary to
perfuse the gas exchange in the new organ, and then to separate
oxygenated blood in the ABO from systemic venous blood. As the
surface area available for aerial gas increases, the gills became
reduced in size and branchial oxygen uptake became an increasingly
smaller fraction of the total oxygen consumption. Branchial movements
11
were modified to alternate between pumping water past the gills and
pumping air into the ABO; water pumping could be eventually
abandoned (Smatresk, 1994; Graham, 2006).
INVESTIGATING HETEROKAIRY USING THE ONSET OF AIR-BREATHING IN
BIMODAL BREATHING FISHES
Air-breathing in bimodal breathing fishes is the culmination of
the maturation of an entire suite of structures and processes –
ventilatory control mechanisms, effective blood perfusion pathways,
specialized air-blood interfaces – that create a functional labyrinth
organ (Burggren and Warburton, 2007). Air-breathing is also a trait that
is likely to confer high fitness on aquatic larval fishes exposed to
aquatic hypoxia, and so hypoxia-driven natural selection of a
modifiable development plan for air-breathing might be anticipated.
Air-breathing behavior can be easily tracked because it requires
surfacing and stereotypic body movements resulting in the obvious
release of a gas bubble from the suprabranchial chamber containing
the labyrinth organ. The onset of air-breathing behavior in developing
fish is easy to follow by visual observation or with video devises (Blank,
2009). These characteristics make the onset of air-breathing an ideal
model for studying heterokairy, and the blue gourami and Siamese
12
fighting fish are particularly good model species for this purpose
(Randall et al., 1982). The Blue gourami Trichopodus trichopterus
(Osphronemidaea: Luciocephalinae) is an obligate air-breather
(Graham, 1997; Burggren, 1979; Blank, 2009; Burggren and Blank,
2009), while the Siamese fighting fish Betta splendens
(Osphronemidaea: Macropodinae), also commonly called “bettas” is a
non-obligatory air-breather (Graham, 1997). Both species are easily
reared and handled species of labyrinth fish that readily lend
themselves to the testing of the heterokairy hypothesis in the lab. They
are readily available from local pet stores, their development proceeds
rapidly, they can produce between 500 (Betta), and 1000-2000
(Gourami) eggs at a time, they hatch within 24-48 hours, and their fry
becomes free-swimming in 3 to 4 days (at 28oC) (Pollak, 1981).
The cardio-respiratory ontogeny and the transition to bimodal
respiration in the Blue gourami raised in normoxic and hypoxic
conditions has been described (Blank, 2009; Blank and Burggren,
2014). These developmental patterns are less known for Betta, but the
development could be very similar (Graham, 1997) and is an important
subject for additional observation. The low partial pressure of dissolved
oxygen in water triggers many aspects of air-breathing fish physiology
13
and development (Alton et al 2007; Blank, 2009; Burggren and
Haswell, 1979; Herbert and Wells, 2001; Hughes, 1973; Oliveira et al.,
2004; Smatresk , 1986; Warketin, 2007), and gouramis and bettas are
no exception. Blue gourami embryos and young larvae are entirely
aquatic and rely upon diffusion alone to meet their oxygen
requirements. As body size increases, the cardiovascular system
begins to contribute to oxygen distribution, and the later development
of the gills leads to active oxygen uptake via branchial respiration.
When the diffusion distances at the skin and gills increases to the point
that oxygen uptake across these surfaces is no longer adequate, the
developing suprabranchial chambers and labyrinth organs take over,
and the transition to air breathing commences. This transition usually
takes place at 21-22 days of post fertilization, when the animal has
reached a total body length of 10-12 mm (Blank, 2009).
In summary, the blue gourami and betta have labyrinth organs
that enable them to go through a transition from water-breathing to air-
breathing during their development. Important for the purposes of this
thesis, the onset of this transition is marked by a behavioral display that
is relatively easy to measure. These features make them suitable
examining heterokairy, making it possible to compare the timing of the
14
onset of the air breathing of larval fish raised under normoxic and
hypoxic environments and determine associated behavioral,
morphological and physiological mayor changes. Even more, it is
possible to compare between two air-breathing strategies (Graham,
1997), non-obligatory (Betta splendens) and obligatory (Trichopodus
trichopterus) air-breathers.
HYPOTHESES TO BE TESTED
This thesis will test three specific hypotheses related to the
onset of air-breathing in anabantid fishes:
Hypothesis #1: The onset of air breathing and supporting
anatomy and physiology of the Blue gourami and Siamese fighting fish,
exposed to hypoxia, will be delayed or brought forward in chronological
age.
Hypothesis #2: Survival of larvae of the Blue gourami and
Siamese fighting fish exposed to continuous hypoxia will be lower
compared with larvae of both species exposed to intermittent hypoxia.
15
Hypothesis #3: Hemoglobin of Siamese fighting fish will have
less O2 affinity than the blue gourami hemoglobin as a result of their
differences in air-breathing strategy and hypoxia tolerances.
AIMS IN AID OF TESTING THE HYPOTHESES
Testing the above-described hypothesis will emerge from
achieving for specific aims, as will now be described.
I. Comparing the effect of continuous and intermittent hypoxia on
survival of both species during the first 40 dpf
II. Evaluating the effect of intermittent hypoxia during the first 40
dpf of both species on: Survival and onset of air-breathing
a. Body growth (size and wet mass) and respiratory organs
size
b. Heart rate, ventilatory frequency, and mass specific ��O2
III. Determining the hemoglobin-oxygen dissociation curves for
both species
a. Evaluate the Borh shift of 0%, 1%, and 2.5% of CO2
b. Compare the P50 between both species and CO2 levels
These aims and hypothesis are laid out in the following four
chapters of this thesis. Chapter 2 introduces the heterokairy
16
hypothesis, and outlines how environmental modulation of the onset of
air breathing and survival will be used to test this hypothesis. Chapter
3 provides the basis quantitative morphology of the respiratory
structures of betta and gourami, and how these structures, larval
growth and the onset of air breathing are modulated by hypoxia.
Chapter 4 extends the observations of growth in normoxia and hypoxia
to mass-specific oxygen consumption, critical oxygen tension, and
ventilatory response to chronic hypoxia. Finally, Chapter 5 describes
the blood respiratory properties of these two species.
17
CHAPTER 2
ENVIRONMENTAL MODULATION OF THE ONSET OF AIR-BREATHING AND SURVIVAL OF BETTA SPLENDENS
AND TRICHOPODUS TRICHOPTERUS 1
INTRODUCTION
Phenotypic plasticity is the ability of an individual organism to
express two or more genetically controlled phenotypes in different
environments (Hill et al., 2008). In a developmental context, phenotypic
plasticity is evident in developmental plasticity, and refers to the fact
that there are some susceptible windows of time during ontogeny when
an organism’s developmental trajectory is altered in response to
environmental factors (Pigliucci, 1998; Burggren & Reyna, 2011).
During development this environmentally sensitive phenotype - not the
genotype or the gene – is subject to selection, and produces individuals
with differential fitness (West-Eberhard, 2005).
Phenotypes subject to modification include physiological
phenotypes, where a physiological feature of an individual results from
1 This chapter is presented in its entirety from Mendez-Sanchez, J. F. &
Burggren, W. W. (2014). Environmental modulation of the onset of air breathing and survival of Betta splendens and Trichopodus trichopterus. Journal of Fish Biology 84, 794–807 with permission of John Wiley and Sons, license number 3679110098467
18
specific gene expression and/or non-genetic regulation for a particular
environment (Hochachka et al., 1998). When a physiological feature is
expressed during development it is termed a physiological
developmental phenotype; variation in its expression is also dependent
on environmental variation and this developmental physiological
feature has an impact on individual fitness (West-Eberhard, 2005). In
other words, a single developing individual is likely to present different
physiological phenotypes in different environments – an example of
developmental plasticity.
Developmental plasticity can be interpreted in the framework of
two concepts: heterochrony and heterokairy. Heterochrony is the
altered timing of the developmental events as a mechanism of
evolution (Gould, 1977). Specifically, heterochrony is the genetically
based alterations in development (as evident from different times of
appearance of developmental landmarks) between different species
(Spicer, et al., 2011). Physiological heterochrony, then, is the alteration
of the timing of the development (first appearance or subsequent
maturation) of physiological traits in one species relative to an ancestral
species (Spicer & Rundle, 2006).
19
Heterochrony is useful in understanding developmental
changes at the species level but, importantly, such changes also occur
at the population and individual level and drive the composition of the
gene pool. Consequently, to understand developmental plasticity at
that level, the concept of “heterokairy” was introduced (Spicer and
Burggren, 2003; Spicer et al., 2011). Heterokairy is the non-genetic
change in timing of the onset of physiological regulatory systems, or
plasticity in the timing of the onset of physiological regulatory systems
or their components, below the level of the species – that is, between-
populations or individuals.
Physiological heterokairy is a relatively new concept, and as
such does not have a great deal of direct experimental support,
particular in vertebrates. However, the concept has been gaining
credibility based on the reinterpretation of previous data collected to
show developmental plasticity. In an analysis by Spicer and Rundle
(2007), heterokairy at the level of the individual was considered, and
they summarized empirical evidence of this phenomenon. For
example, the development of salinity tolerance in salmonids during the
transition from fresh water to a marine existence was brought forward
in development (that is, occurred earlier) when brook trout Salvelinus
20
fontinalis (Mitchill 1814) were pre-exposed to sea water (Hiroi &
McCormick, 1995), Coho salmon Oncorhynchus kisutch (Walbaum
1792) were administered cortisol or insulin (McCormick, 1995;
McCormick et al., 1991), or Atlantic salmon Salmo salar L. 1758 were
exposed to temperature treatments (Handlelanda et al., 2004). Other
examples involve the Atlantic herring Clupea harengus L.1758, where
the thermal environment alters the ontogeny of muscle physiology,
such that the rostral-to-caudal progression of myofibril synthesis as well
as slow fiber innervation appears earlier as environmental temperature
increased (Johnston et al., 1997).
Specific developmental markers for heterokairy were affected by
temperature inhaddock Melanogrammus aeglefinus L. 1758 (Martell, et
al., 2006). Blastopore closure, notochord vacuolation, retinal
pigmentation, the appearance of blood cells, and hatching all occurred
later in development at lower temperatures. Appearance of the optic
lumen, neural tube cavitation, and increased myofibril density per deep
cell all occurred earlier at lower temperatures. Notochord and eye
development were accelerated with increased embryonic temperature,
while myofibrillar genesis and neural tube development was similarly
slowed.
21
As a final example, California grunion Leuresthes tenius (Ayres
1860) provides evidence for heterokairy involving plasticity in the
hatching time. Embryos from the same spawning hatched at different
times and different developmental stages (Moravek & Martin, 2011).
They responded to unpredictable environment with diapause, and then
responded rapidly by hatching when the environment became
favorable.
Studies of heterokairy involving physiological processes such as
respiration and metabolism in vertebrates, including fishes, are lacking.
We elected to investigate the onset of aerial respiration in air-breathing
fishes in the context of heterokairy. Air-breathing in bimodal breathing
fishes and amphibians is the culmination of the maturation of an entire
suite of structures and processes – ventilatory control mechanisms,
effective blood perfusion pathways, specialized air-blood interfaces –
that create a functional air-breathing organ (Burggren & Warburton,
2007). Air-breathing is also a trait that is likely to confer high fitness on
aquatic larval fishes exposed to aquatic hypoxia, and so hypoxia-driven
natural selection of a development plan for air-breathing that is
modifiable might be anticipated. Air-breathing as a behavior in labyrinth
22
fishes can be easily tracked because it requires surfacing and highly
stereotypic body movements, and is further signaled by the obvious
release of a gas bubble from the suprabranchial chamber containing
the labyrinth organ. The onset of air-breathing behavior is also easy to
follow by visual observation or with video devices (Blank, 2009; Blank
& Burggren, 2013). These characteristics make the onset of air-
breathing an ideal model for studying heterokairy related to aerial gas
exchange.
The specific purpose of this study, then, was to determine if
chronic hypoxia could promote developmental plasticity in the onset of
air-breathing fish larvae, comprising the first vertebrate experiment
designed to test the heterokairy hypothesis with a discrete
physiologically-related developmental marker. Two easily reared and
handled species of labyrinth air-breathing fish readily lend themselves
to the testing of the heterokairy hypothesis in the laboratory. The three
spot gourami Trichopodus trichopterus (Pallas 1770), formerly
Trichogaster trichopterus, is an obligate air-breather (Burggren, 1979;
Graham, 1997; Herbert & Wells, 2001; Blank, 2009; Burggren & Blank,
2009). The Siamese fighting fish Betta splendens Regan 1910 is a
non-obligatory air-breather (Peters, 1978; Graham, 1997).
23
MATERIALS AND METHODS
Fish Species Used In Experimentation
Experiments were conducted on two different labyrinth fishes
from the Osphronemidae family – T. trichopterus subfamily
Luciocephalinae and B. splendens subfamily Macropodusinae (Froese
& Pauly, 2013). Both species develop rapidly and they produce large
numbers of eggs in each laying - 500 and 1000-2000, respectively -
and these eggs hatch within 24-48 h. Their fry become free-swimming
in 3 to 4 days (at 28oC) (Pollak, 1981).
Animal Maintenance And Breeding
Ten breeding pairs of the T. trichopterus were obtained from the
reproductive stock maintained in the Developmental Integrative Biology
Laboratory of the University of North Texas. Ten breeding pairs of B.
splendens were obtained from the Carolina Biological Supply
Company. Both species were maintained and conditioned for breeding
in 40 l aquariums equipped with under-gravel filters, held at 27°C and
a 12 light/12 dark photoperiod. Water chemistry was maintained as
follows: 6.5-7.5 pH, <40 ppm nitrate, <0.5 ppm nitrite, 50-120 ppm total
hardness, and 20 kPa of PO2. Adult fish and breeders were fed ad
24
libitum twice per day with a high protein diet (>40%) of dry flakes and
frozen brine shrimp and bloodworms.
Separate male and female groups comprising 4-6 adult T.
trichopterus were isolated for 2-3 weeks at 28°C. Once a male showed
darkened fins, reproduction was induced by moving into an aquarium
at 30-32°C for 1 week or until it started to build a visible bubble nest. A
female showing a bulky abdomen was then placed in with the primed
male and nocturnal spawning typically occurred within the next 2 days.
Two groups of adult B. splendens, one of 4 males and the other
of 4 females, were isolated for 2-3 weeks at 28°C. Individuals were
isolated in the same aquarium by a divider due to their aggressiveness.
Once a male showed bubble nest building behavior, a female showing
bulky abdomen and a white spot on the vent was placed next to the
male in a floating fine mesh cage. If the courtship dance occurred
between them, the female was released from the cage and nocturnal
spawning typically occurred within the next two days. After spawning
the female was taken out to avoid attacks from the male.
25
Larval Staging And Handling
All the eggs for the same experiment were taken from the same
clutch to avoid intra-clutch effects (see intra clutch variation in results
section). The chronological time was registered as standardized age
in days post fertilization (DPF). The fertilization day was considered
day 0, and for every cycle of 24 h a unit of day was added. The
experiment stopped at 50 days of post fertilization (DPF).
After 12 h of fertilization, eggs produced from the single clutch
were removed from the male’s bubble nest and placed in 20 l
experimental tanks. Each tank was equipped with a sponge bio-filter
and was maintained at 27°C with a 12 light/12 dark photoperiod. The
water quality for larvae was evaluated at shorter intervals than that of
water used for breeding, and was maintained as follows: 7.3-7.5 pH,
<10 ppm nitrate, <0.5 ppm nitrite, and 80-100 ppm total hardness.
The larvae of both species completely consumed their yolk
around 5 DPF. After that time, they were fed ad libitum with live
microworms from day 6 to day 10. Thereafter, they were fed with live
brine shrimp newborn nauplii. Prior to all measurements, larvae were
deprived of food for 12 h. They were then moved to experimental cages
26
in aquaria containing water with the same water quality and allowed to
acclimate for ~1.5 hours before experimentation. The experimental
cages consisted of floating plastic circular cages with a < 0.2 mm mesh
placed in 20 l aquaria maintained at a specific oxygen level. Oxygen
levels in the aquaria containing the experimental chamber were
regulated by bubbling into the water either room air (20 kPa) or a
mixture of room air and pure nitrogen, to achieve one of two desired
levels of hypoxia (17 kPa or 14 kPa). Each tank was filled with
conditioned water to 4/5 of its capacity, with the remaining 1/5 filled with
the gas that bubbled up through the aquarium. The tank was sealed
with a plexiglass cover with a valve to prevent atmospheric air from
leaking back into the tanks and to ensure that the gas and water phases
were in PO2 equilibrium. Water PO2 was monitored daily with an optical
oximeter probe ProODO (YSI Incorporated). The PO2 of the gas above
the water was measured and monitored daily with a ProOx 110 oxygen
sensor (Biospherix, Ltd). Having the experimental larvae exposed to
hypoxic air was not the natural condition for them, but it was deemed
necessary for this study to ensure that the larvae could not “escape”
internal tissue hypoxia through respiration with air.
27
Two types of hypoxic exposure – intermittent and continuous -
were created in different experiments. “Nocturnal intermittent”
exposure mimicked the dial cycle of hypoxia in tropical habitats were
these fish have evolved, namely 12 hours of normoxia during the day
and 12 hours of hypoxia during the night. That is, hypoxic exposure
was synchronized with the photoperiod employed.
Each PO2 population was created in triplicate, with 150 larvae
placed in three cages (50 on each) to develop and grow from 0 to 50
DPF. Water in the aquaria and the experimental cages within was
continuously circulated using a submersible pump (Marineland Maxy-
jet 400) to maintain homogeneous conditions of PO2 and water quality
within the cages.
Measurement of Larval Survival
Larval mortality for each day was registered for each of the 3
cages. The proportion of survivors (S) was determined as: S=(Ni –
Ncd)/Ni, where Ni=number initial of larvae in the cage (n=50), and
Ncd=cumulative number of dead larvae per day. Survival was compared
between the three PO2 levels and between continuous and intermittent
exposure.
28
Measurement of Onset of Air-Breathing
Air-breathing was measured in a group of 5 larvae from each
PO2 treatment with two replicate measurements for each group. A
different group of larvae were randomly selected daily. Fish that were
used for experimentation were removed from the general pool and not
used again. This group of larvae was exposed to extreme aquatic
hypoxia (PO2 3-5 kPa) for 10 min to see if air-breathing was stimulated.
A fish was considered to be “air-breathing” when it surfaced to inhale
air and subsequently released a bubble of gas through the opercular
opening. The percentage of air-breathers per day was recorded from
15 DPF until 100% of the sample had begun breathing air.
Statistical Analysis
The frequencies of survival and onset of air-breathing
independence of PO2 were tested by arranging contingency tables with
treatment in the columns and age in the rows and using an X2 analysis
(Zar, 2010). Additionally, a Kruskal-Wallis (H) comparison was
performed within the onset of air-breathing of each PO2 level. The
onset of air-breathing for each larva was computed in post-fertilization
29
days. Additional Spearman rank correlation (rs) was used to identify
patterns between the onset of air-breathing and levels of PO2.
RESULTS
Larval Survival
B. splendens in continuous normoxic conditions during the 40
DPF period had a survival rate of 65-75% through 50 DPF. The
corresponding value for T. trichopterus larvae was far lower, at 15-30
% (Figures 1a and 2a). For B. splendens, survival depended on the
PO2 treatment for both continuous exposure (X2=575, df=74, p<0.001;
Figure 1a) and nocturnal intermittent exposure (X2=207, df=74,
p<0.001; Figure 1b). The highest survival rate was in continuous
normoxia (>65%), while nocturnal intermittent hypoxia produced a low
survival rate (<25%; Figure1b). No larvae survived continuous hypoxia
beyond 9 DPF (Figure 1a). For T. trichopterus, survival depended on
the PO2 treatment for both continuous exposure (X2=2830, df=60,
p<0.001) and nocturnal intermittent exposure (X2=175, df=72, p<0.001;
Figures 2a and b). Survival rate in nocturnal intermittent hypoxia was
not significantly different from continuous normoxia (Figure 2b), but no
larvae survived continuous hypoxia beyond 9 DPF (Figure 2a).
30
Figure 1 Survival of B. splendens in three water PO2 treatments. a) Continuous hypoxia. b) Intermittent hypoxia.▼=20 kPa, О=17kPa; ●=14kPa. Means and standard errors are plotted.
31
Figure 2. Survival of T. trichopterus at three water PO2 treatments. a) Continuous
hypoxia. b) Intermittent hypoxia.▼=20 kPa, О=17kPa; ●=14kPa. Means and
standard errors are plotted.
32
Collectively, these initial findings of survival showed that
continuous hypoxia was deleterious for larvae through 9 DPF.
Consequently, the remainder of experiments were conducted either
under nocturnal intermittent hypoxia conditions or, for controls,
continuous normoxia.
Air-Breathing Onset
In a preliminary experiment with 3 normoxic groups of larval B.
splendens from different clutches and parents, the onset of air-
breathing was significantly different between clutches (H=74; n1=22
n2=31 n3=45; p<0.0001) with average and range values (in DPF) of 26
(25-27), 26 (23-29), and 40 (37-42). To avoid intra clutch variation, the
survival and onset of air-breathing experiments were therefore
conducted with larvae from the same clutch.
The air-breathing onset for B. splendens in continuous normoxic
exposure was an average of 39 DPF within a range of 37-42 DPF, with
50% of juveniles breathing air at 38 DPF (Figures 3a, 3b). In contrast,
the average onset of air-breathing at continuous normoxic exposure in
the T. trichopterus was 35 DPF with a range of (32-37), with 50% of
larvae breathing air at 34 DPF (Figures 4a, and 4b).
33
Figure 3. a) Proportion of B. splendens larvae that are air-breathers, plotted through development at different PO2 treatments. Means and standard errors are plotted.
▼=20 kPa, О=17kPa; ●=14kPa. b) Onset of air-breathing by PO2 treatment. 95%
confidence intervals are plotted and sample sizes are in parenthesis, * represents significant differences (p<0.001).
To test the hypothesis that the time of onset of air-breathing
could be modulated by hypoxia exposure in both B. splendens and T.
trichopterus, larvae of each species drawn from the same clutch were
exposed to one of continuous normoxia and two levels of nocturnal
34
intermittent hypoxia (17 kPa and 14 kPa). For B. splendens, the onset
of air-breathing depended on PO2 treatment (X2=56, df=24, p<0.001).
Nocturnal intermittent hypoxia at 17 kPa advanced the onset of air-
breathing by 4 days (Figure 3b). Moreover, the entire sample of the 14
kPa PO2 group began air-breathing 2 days before the normoxic group
(Fig. 3a). These results suggest that nocturnal hypoxia draws forward
the onset of air-breading (H=50, n21=45, n18=22, n15=20, p<0.0001;
Figure 3b).
In contrast to B. splendens, the onset of air-breathing in T.
trichopterus was independent of PO2 treatment (X2=9.3, df=26, p>0;
Figures 4a and 4b). However, in a non-parametric comparison the air-
breathing onset showed significant differences and the total sample of
hypoxic groups advanced to the air-breathing stage 8 days later than
the normoxic group (H=26.0, n21=40, n18=32, n15=25, p<0.0001). These
results suggest that intermittent, nocturnal hypoxia delays the onset of
air-breathing in at least 60% of the population (Figure 4b).
35
Figure 4 a) Proportion of T. trichopterus larvae that are air-breathers, plotted through development at different PO2 treatments. Means and standard errors are plotted.
▼=20 kPa, О=17kPa; ●=14kPa. b) Onset of air-breathing by PO2 treatment. 95%
confidence intervals are plotted and sample sizes are in parenthesis, * represents significant differences (p<0.001).
DISCUSSION
Larval Survival Rates
Survival of a fish under hypoxic conditions depends on several
basic capabilities. Of primary importance is the ability to increase
36
ventilation of the gas exchange organs, accompanied by appropriate
changes in blood perfusion of these organs (Burggren & Warburton,
2007). Failing this adjustment, important is the capacity to decrease
overall metabolic rate (metabolic depression), to tolerate increased
levels of metabolic by-products (particularly protons produced by
anaerobic metabolism and associated with lactic acid accumulation),
and to avoid and/or repair cellular injury following reoxygenation
(Bickler & Buck, 2007). Fish larvae are early in the process of
developing the systems necessary to respond or to tolerate hypoxia.
When combined with the fact that hypoxic stress alters the overall
developmental programming (Burggren & Bagatto, 2008), hypoxia can
generate mass larval fish mortalities in natural environments (Diaz &
Breitburg, 2009; Houde, 2002).
The present study revealed differences in larval survival
between continuous and intermittent hypoxia treatments for both B.
splendens and T. trichopterus. Continuous hypoxic stress likely
overwhelmed the response and/or tolerance capabilities of the larvae,
leading to their death. The result of this was complete population
mortality in continuous hypoxia by 9 DPF for both species. Nocturnal
intermittent hypoxia, on the other hand, likely gave larvae sufficient time
37
to recover and still continue subsequent development during alternate
episodes of stress, recovery and “normal development”. Interestingly,
the current study found that T. trichopterus was slightly more sensitive
to hypoxia in early larval life than reported for this species in an
independent study (Blank & Burggren, current issue). There are
numerous reasons for physiological variation between studies, and for
fish species the susceptibility of larvae to hypoxia can be altered by
epigenetic effects relating to maternal and paternal experiences (Ho &
Burggren, 2010; Burggren, 2013).
Natural initial mortality rate for freshwater larval fishes has been
estimated to be as high as 16 ± 4% per day (Fuiman, 2002; Houde,
2002). In the present experiment, the average mortality per day during
the experimental period (40 DPF) in the continuous normoxia (20 kPa)
was 1.4 ± 0.1%for B. splendensand 2.1 ± 0.1%for T. trichopterus; the
same parameter in continuous hypoxia was 2.7 ± 0.3% and 2.7 ± 0.2%,
and in nocturnal intermittent hypoxia was 2 ± 0.1% and 2.2 ± 0.06,
respectively, for each species. In this case the daily hypoxic-induced
mortality was less than 2.7% of the natural per day mortality in all
cases. This high survival rate can likely be explained by the absence
38
of predation, abundant food supply, and the maintenance of high water
quality in a laboratory setting.
The absolute expected mortality of larval freshwater fishes is
~95% in natural habitats (Fuiman, 2002; Houde, 2002). In the present
study mortality in response to continuous hypoxia was close to 100%
for both species. In contrast, mortality under intermittent, nocturnal
treatment was 74-76%for B. splendens and 81-85% for T. trichopterus.
Given these higher survival rates, intermittent hypoxia treatment was
subsequently employed to simulate the natural diel oxygen cycle where
these two species of air-breathing fishes evolved, namely tropical
freshwater ponds with high autotrophic oxygen production during the
day and high heterotrophic oxygen demand during the night (Graham,
1997; Farrell & Richards, 2009; Verberk et al., 2011). Though more
difficult to produce technically, clearly intermittent hypoxia creates a
more natural environmental stimulus, and is recommended for future
experimental exposure to hypoxia.
These results highlight dissolved oxygen as a limiting
environmental factor for larval fishes in fresh water (Diaz & Breitburg,
2009), and hypoxia as a strong natural selection force as in adult air-
39
breathing fish (Randall et al., 1881; Graham, 1997) with a high cost
evident in lowered larval survival. The current findings also showed that
the response of B. splendens to hypoxia certainly is not the same as in
T. trichopterus (Farrell & Richards, 2009). Possible reasons for these
differences are discussed below.
Hypoxic Manipulation of Air-Breathing Onset
The onset of air-breathing for Anabantid fishes has been
reported to take place over the broad species-specific range of 18-29
DPF (Prasad & Prasad, 1985; Graham, 1997), and specifically for T.
trichopterus at 20-25 DPF (Blank, 2009). Under normoxic conditions in
the present study, air-breathing onset occurred at 37-40 DPF for B.
splendens and 32-36 for T. trichopterus. The difference from previously
reported data for Anabantid fishes suggests a high variation of this
parameter as a result of heterochrony. In B. splendens there was
considerable inter clutch variation in the onset of air breathing.
Variation in the chronological age (timing) when normal air-breathing
onset occurs reflects developmental plasticity (West-Eberhard, 2005),
as well as different developmental trajectories for each species and
individuals within populations (Burggren & Reyna, 2011).
40
This study shows that hypoxic exposure can alter the timing of
onset of air-breathing onset within species. As such, these data
support the theory of heterokairy, in which developmental timing of
specific physiological process like aerial respiration has been moved
as a result of an environmental stressor like hypoxia (Spicer &
Burggren, 2003; Spicer et al., 2011). The results showed a particular
pattern for each species. In T. trichopterus, PO2s of both 17 and 14 kPa
delayed the onset of air breathing in an inversely correlated way (rs=-
1; df= 1, 2; p<0.0001). In contrast, intermittent hypoxia at a level of (17
kPa of PO2) brought air-breathing forward in the developmental plan
for B. splendens but, interestingly, hypoxia at the lower level of 14 kPa
had no significant effect. The reason for this differential response is not
evident, but the “dose-response curve” for the oxygen effect on the
developmental plan should not be assumed to be a simple linear
relationship. Indeed, at lower oxygen levels, larvae may simply not be
able to mount an adaptive response. The interspecific differences
between B. splendens and T. trichopterus in the “developmental
trajectory” (Burggren & Reyna, 2011) can be considered evidence of
heterochrony (Gould, 1977; Spicer, et al., 2011), because both species
diverge from the same phylogenetic family, Osphronemidae (Rüber, et
al., 2006; Froese & Pauly, 2012).
41
These interspecific differences in hypoxic effects may be related
to ecophysiological differences between species. Obligatory air-
breathing forms, like T. trichopterus, are unable to survive on the
quantity of O2 obtained by purely aquatic respiration (branchial plus
cutaneous), even in normoxic water, and thus they always need
supplemental aerial oxygen. By contrast, non-obligatory air-breathers
including B. splendens, do not require air-breathing to survive in
normoxic water, but can survive on continuous aquatic respiration (see
Lefevre et al., in current issue for additional discussion of facultative
and obligatory classifications of air-breathing fishes).
Based on the previous descriptions, the onset of air-breathing in
these Osphronemidae species proved to be a discrete developmental
marker for testing the hypothesis of heterokairy. The present
experiments show that B. splendens is a non-obligatory air-breather
after 36 DPF and hypoxia accelerates its air-breathing onset, while T.
trichopterus is an obligatory air-breather past 32 DPF and hypoxia
delays its onset of air-breathing. Both findings support the hypothesis
of physiological heterokairy.
42
Because the onset of air breathing can be accelerated or
delayed by hypoxia, the onset of air breathing – which involves
numerous physiological regulatory processes -represents a clear
example of the phenomenon of heterokairy (Spicer & Burggren, 2003).
Heterokairy serves as a form of compensation to environmental
stressors such as oxygen. Although examples within vertebrates of
heterokairy are as yet few in number, the movement in development of
the appearance of the ABO and its ventilation represents a case of
heterokairy that involves multiple levels of regulation. Modifications
must occur innumerous morphological structures and a whole series of
physiological regulatory systems that involve these structures (e.g.
respiratory, cardiovascular, musculo-skeletal, etc.). In the case of fish
exposed to hypoxia, known responses can include cellular-molecular,
physiological, anatomical and behavioral changes (Diaz & Breitburg,
2009), and one might reasonably anticipate that these responses can
additionally involve heterokairy.
Unknown at this time is the ultimate “cost” of responses
representing heterokairy in animals. By way of comparison, we know
that the “thrifty phenotype” sometimes evident in very low birth weight
mammal infants is helpful in infancy and in juveniles in regaining normal
43
body mass, but ultimately proves maladaptive later in later adult life
when “the metabolic syndrome” appears (Barker, 1997; see Isezuo,
2006 for review). Whether heterokairic responses in larvae that aid
larval survival ultimately prove maladaptive awaits further study.
44
CHAPTER 3.
THE SUPPORTING LARVAL RESPIRATORY ANATOMY: HYPOXIA MODULATION OF LARVAL GROWTH, GILL
AND LABYRINTH ORGAN MORPHOMETRICS AT THE ONSET OFAIR-BREATHING
INTRODUCTION
Hypoxia is a common environmental stressor in fishes, and has
been viewed as one of the primary factors in the evolution of air
breathing in fishes (Randall et al., 1982; Graham, 1997). Hypoxia has
many effects, including affecting food intake, rate of development, and
stimulation of ventilatory and cardiovascular reflexes (See Introduction,
Chapter 4).
Hypoxia in fishes limits growth primarily through a reduction in
appetite. This response and the associated growth retardation occurs
at relatively mild levels hypoxia (Wang, et al., 2009). As examples,
juveniles of turbot Scophthalmus maximus and sea bass Dicentrarchus
labrax were fed to satiation under hypoxia, and their growth and food
intake were depressed compared to normoxia (Pichavant, et al., 2001).
In juvenile catfish Silurus meridionalis, growth performance was also
impaired due to acclimation to dial cycling hypoxia (Yang, et al., 2013).
Juvenile piapara (Leporinus elongatus) exposed to severe hypoxia also
45
showed lower weight gain and feed conversion (the quantity of food
consumed divided by the weight gain over a given a time period) was
1.6 when compared to animals in moderate hypoxia (2.9) and normoxia
(1.8) (Filho, et al., 2005).
Chronic hypoxic exposure not only retards overall growth, but
also affects development of size and structure of fish respiratory organs
in aquatic species of fish. This has been observed in fully aquatic
fishes such as the Sailfin molly Poecilia latipinna, four species of knife
fishes Brachyhypopomus, the Nile perch Lates niloticus, the Crucian
carp Carassius carassius, and the goldfish Carassius auratus, all of
which increased gill surface area (lamellar) and the filament length in
response to chronic hypoxia (Timmerman & Chapman, 2004; Nilsson,
2007; Crampton, et al., 2008; Paterson, et al., 2010). However, the
effects of hypoxia on growth rate have only been studied in a few
species of air-breathing fishes, perhaps because researchers have
anticipated that obligate air-breathers are unlikely to be strongly
affected by aquatic hypoxia. On the other hand, facultative air-
breathers are negatively affected by aquatic hypoxia and the few
existing studies point to clear effects on growth (Wang, et al., 2009). In
the obligate air-breather striped snakehead Channa striata (synonym
46
of Ophicephalus striatus), the cost of compensating the low oxygen
availability by increasing surfacing and air-breathing frequency to
extract oxygen from the air actually exceeds the gain in food conversion
(Kramer & McClure, 1981). This situation is made more complicated
by the fact hypoxia reduced food consumption (Pandian &
Vivekanandan, 1976; Vivekanandan, 1977; Wang, et al., 2009).
Similarly, in the facultative air-breather the stinging catfish,
Heteropneustes fossilis, this compensation holds up to the point where
the cost of surfacing exceeds the gain in body mass. Beyond that point,
it is more efficient for the fish to rely solely on aquatic respiration
(Arunachalam, et al., 1976; Wang, et al., 2009).
Juvenile air-breathing fish tend to depend on aquatic respiration
until the air-breathing organ has sufficiently developed (Wang, et al.,
2009). For example, the South American arapaima (Arapaima gigas) is
entirely water-breathing up to 9 days post hatching, after which a
transition begins from gill lamellae to development of the air-breathing
organ. Hypoxia slows down this transition as well as growth itself
(Brauner, et al., 2004). The same pattern occurs in the paradisefish
Macropodus opercularis, in which the air-breathing organ develops
more slowly under hypoxic conditions (Ebeling & Alpert, 1966).
47
A final example comes from the obligate anabantid air breather,
the three spot gourami (Trichopodus trichopterus). Larvae exposed to
hypoxia (13% O2) showed no differences in total body length and total
body wet weight during development (1-90 days of post fertilization)
when compared with larvae reared in normoxic conditions (21% of O2)
(Blank, 2009). Interestingly these fish larvae showed hypoxia-induced
differences in the allometric relationships between the wet weight and
the branchial arches and the total labyrinth surface area.
Against this backdrop, the current study expands our knowledge
of phenotypical developmental plasticity in two species of air breathing
fishes. The goals of this study were to determine: 1) the effect of
intermittent nocturnal hypoxia on growth (size and weight) in pre air-
breathing larvae of the Siamese fighting fish and the three spot
gourami, and; 2) how hypoxia alters lamellar and labyrinth respiratory
surfaces in these species when their larvae first begin air-breathing.
We selected the three spot gourami Trichopodus trichopterus
(Pallas 1770) as an example of an obligate air breather (Burggren,
1979; Graham, 1997; Herbert & Wells, 2001; Blank, 2009; Burggren &
48
Blank, 2009) and the Siamese fighting fish Betta splendens Regan
1910, an example of a non-obligatory air breather (Peters, 1978;
Graham, 1997). Although both anabantids, the Siamese fighting fish
lives a more sedentary lifestyle in more hypoxic waters than the
gourami (Rainboth, 1996; Ruber et al., 2006; Monviseset al., 2009;
Froese & Pauly, 2015). Consequently, we anticipated that both species
would respond differently to hypoxic conditions. Specifically, we
hypothesized that the facultative air-breather (Betta) would exhibit
greater developmental plasticity, allowing it to be more tolerant to a
hypoxic environment, because Betta spends more time extracting
oxygen from the surrounding water and cannot avoid aquatic hypoxia
all the time. Alternatively, the obligate air-breather (Trichopodus) has
to extract oxygen from air as a mechanism to avoid aquatic hypoxia.
We additionally hypothesized that the larvae of these obligate and
facultative air breathing fish species would respond to hypoxia by
diminishing body size and increasing the absolute size of lamellar and
labyrinth respiratory surfaces.
MATERIALS AND METHODS
Larvae were maintained from 0 to 50 days post fertilization
(DPF) in three different PO2 levels: continuous normoxia (20 kPa) and
49
two intermittent nocturnal hypoxia groups (17 and 14 kPa). Details of
the complete experiment, water quality, and larvae maintenance were
described in the Methods section of Chapter 2.
Every five days post fertilization, five larvae of each treatment
were euthanized by moving them into buffered MS222 solution (pH =
7.0) until well after opercular movement stopped, indicating death.
Larvae were then fixed in a 10% neutral buffered formalin solution.
After 10 days of fixation, each individual was measured for length (mm)
and weight (mg). The gills and the labyrinth organ were dissected out,
and photographed with a 10 megapixel camera Leica model DFC450
under a compound microscope Leica model Wild M3Z. The pictures
were digitized, and counts made of lamellae and branchial filaments
and lengths and areas measured by using Image J 1.42 software
(http://rsbweb.nih.gov). All areas and lengths were calibrated in pixels
with the scale of a micrometer slide.
Two respiratory-related variables were calculated: total lamellar
respiratory surface (TSAlam) and total labyrinth organ respiratory
surface (TSAlab). TSAlam was obtained after the methods of Hughes
(1984) and Blank (2009). Average lamellar area ( X SAlam) was
50
determined using the mean of a medial lamella area of one proximal,
medial and distal gill filament for each of the four brachial arches. To
account for both lamella surfaces, each lamellar area was doubled. The
number of total lamella in each arch was multiplied by XSAlam to obtain
lamellar surface area per arch, designated as SAlamI, SAlamII, SAlamIII,
and SAlamIV. Finally, total lamellar surface area (TSAlamella) was
determined as the sum of SAlamI, SAlamII, SAlamIII, and SAlamIV multiplied
by two to account for both sides of the gills.
The total surface area of the labyrinth (TSAlab) was calculated
by first tracing the perimeter of the labyrinth organ, after Blank, (2009)
and Blank and Burggren (2014), and then determining the area with
Image J in a scaled picture of the I brachial arch. Each measured area
was doubled to account for both sides of the labyrinth respiratory
surface.
Gill arch length (Larch) of the four branchial arches (I, II, III, and
IV), as well as filament length (Lflm), were compared between the
species and the three PO2 levels. Also compared were the number of
filaments per ach (NFarch), number of lamellae per filament (NLflm) and
single lamella surface area (SAlam). For each branchial arch these
51
variables were measured on three filaments: anterior filament (A),
medial filament (M), and posterior filament (P), except on the IV arch
for which only A and P filaments were measured because of the small
size and number of filaments for this arch.
Summary of Measured Variables and Their Definitions
TSAlam = total lamellar respiratory surface
TSAlab = total labyrinth organ respiratory surface
XSAlam = average lamella area
SAlam = Arch I, ll, lll, and lV lamellar surface area
Larch = length of arches I, ll, lll, and lV
NFarch = number of filaments per arch I, II, II, and IV
Lflm = filament length
NLflm = number of lamellae per filament
SAlam = single lamella surface area
Statistical Analysis
ANOVA and MANCOVA comparisons were performed on wet
mass, body length, TSAlam, TSAlab, Larch, NFarch, Lflm, NLflm, and SAlamella
using species, PO2 level and, in some cases, age as factors. The
52
treatment groups were considered significantly different when the p
value was lower than 0.05.
RESULTS
Body Length
The average body length during the monitored 35 day
developmental period for larval Betta in continuous normoxic exposure
increased from 5.6 to 8.9 mm, while for Trichopodus in the same
condition average body length increased from 3.2 to 7.8 mm (Figures
5 and 6). For Betta, both age and PO2 treatment significantly affected
body length (Fage=13.8, df= 4, 64 and p<0.0001; FPO2=3.83, df= 4, 64
and p<0.05). The larval group exposed to a PO2 of 17 kPa of PO2 was
the largest, averaging 1.45 times longer than the normoxic group
(Figure 5A and 5B).
53
Figure 5. A) Larval body length of Betta through development in each PO2 treatment. B) Effect of PO2 treatment on Betta larval body length correcting for the effect of age. Means ± standard errors are presented. * indicates a significant difference from control value (p<0.05).
Trichopodus increased their body size during the monitored
developmental period (Fage = 51.92, df =7, 83, and p<0.0001), but P02
treatment did not affect body size (FPO2 = 1.96, df = 2, 83, and p>0.05)
(Figure 2A and 2B). These results for both species showed that hypoxia
at 17 kPa increased the body size by 14% in larval Betta, but hypoxia
did not significantly affect body length in Trichopodus.
54
Figure 6. A) Trichopodus larval body length through development in each PO2 treatment. B) Effect of PO2 treatment on Trichopodus larval body length correcting for the effect of age. Means ± standard error are presented. * indicates a significant difference from control value (p<0.05).
Body Wet Mass
The average body wet mass through 35 days of development
for larval Betta in continuous normoxic exposure was 2.26 to 5.5 mg,
while for Trichopodus in the same condition it was 0.26 to 4.36 mg
(Figures 7 and 8). For Betta, both age and PO2 treatment significantly
altered body wet mass (Fage=6.63, df = 4, 64, and p<0.001; FPO2= 3.53,
df = 2, 64, and p<0.05). At the onset of air-breathing (35 dpf), wet body
55
mass was 1.5 times greater in hypoxia at 17 kPa compared to the
normoxic Betta group (Figure 7A and 7B).
Figure 7. A) Betta larvae wet mass through development in each PO2 treatment. B) Effect of PO2 treatment on Betta larval wet mass correcting for the effect of age. Means ± standard error are presented. * indicates a significant difference from control value (p<0.05).
Trichopodus increased their body wet mass through
development (Fage=52, df = 7, 83, and p<0.0001), but PO2 treatment
did not affect this variable (FPO2=1.45, df=2, 83, and p>0.05) (Figure 8A
and 8B).
56
These results showed that, in Betta, only 17 kPa of PO2
increased body wet mass, but in the Trichopodus no hypoxic level
significantly affected body wet mass during development.
Figure 8 A) Trichopodus larval wet mass through development in each PO2 treatment. B) Effect of PO2 treatment on Trichopodus larval wet mass correcting for the effect of age. Means ± standard errors are presented. * indicates a significant difference from control value (p<0.05).
Condition Factor (K) and Wet Mas
- Body Length Relationship
The condition factor for Betta was significantly smaller in the
hypoxic group at 14 kPa (F=4.93, df=2, 64 and p<0.05) (Figure
9A). The b exponent of the body wet mass – body length relationship
57
of the two hypoxic groups was also smaller than the normoxic group
(F=6, df = 2, 50 and p<0.05) (Figure 10A).
Figure 9. A) Effect of PO2 treatment on Betta larval condition factor (K) correcting for the effect of age. B) Effect of PO2 treatment on Trichopodus larval condition factor (K) correcting for the effect of age. Means ± standard error are presented. *
For Trichopodus, the condition factor was not significantly
different between the PO2 treatment groups (F=2.9, df =2, 107 and
p>0.05) (Figure 5B). The b exponent for the body wet mass-body
length relationship showed no statistical differences (F=0.3, df= 2, 83
and p>0.05) (Figure 10B).
58
Figure 10. A) Effect of PO2 treatment on Betta larval wet mass – body length relationship. B) Effect of PO2 treatment on Trichopodus larval wet mass – body length relationship. W = aLb, W = wet mass, L= body. The values of a and b coefficients are showed for each PO2 group. The statistical significance and fit adjustment are also shown with F, p, and r. The statistical difference within the b exponent of the PO2 groups is presented with an ANOVA analysis.
Labyrinth Organ Respiratory Surface (TSAlab)
and Lamellar Respiratory Surface (TSAlam)
Average TSAlab for Betta was 0.24 mm2 at 35 post fertilization
days, and the corresponding value for Trichopodus was 0.13mm2
(figure 11). These values were significantly different from each other
(F = 69.79, df = 2, 17 and p<0.05). For Betta the P02 treatment at 17
kPa increased respiratory surface by 30% compared with the normoxic
59
group (F=11.14, df = 2, 8 and p<0.05), corrected for the effect of body
length (Figure 12B). However, in the Trichopodus PO2 did not
significantly affect TSAlab (F = 0.33, df = 2, 8 and p>0.05) (figure 12B).
TSAlam of Betta averaged 20.4 mm2; which was 3.3 times bigger
than for Trichopodus (6.06mm2). TSAlam significantly differed between
both species (F=69.8, df = 1, 17 and p<0. 05) when corrected for body
length (Figure 12A). The 14 kPa hypoxic group for Betta showed a
significant decrease in TSAlam (F=10.48, df = 2, 8 and p<0. 05).
However, in the Trichopodus hypoxic exposure did not significantly
affect this respiratory surface (F=0.52, df = 2, 8 and p>0.05).
60
Figure 11. Compound microscope images of the dissected first branchial arch from Betta (A) and Trichopodus (B) at 35 dpf. The forming labyrinth organ (lo) is indicated by the arrow and outlined by the solid line.
61
Figure 12. A) Variation of labyrinth organ respiratory surface (TSAlab) at the onset of air-breathing in three PO2 levels and between the two species. B) Lamellar respiratory surface (TSAlam) at the onset of air-breathing in three PO2 levels and between the two species. Means ± standard errors are presented. Boxes enclose means not significantly different from each other (p>0.05).
Gill Morphometrics
Arch length (Larch). When Larch was corrected for body length,
hypoxic treatment did not significantly affect Larch for either species
(F=0.41, df = 2, 71 and p>0.05). Betta had larger sizes on all the arches
than Trichopodus (F=7.31, df = 1, 71 and p<0. 05). The size of the
aches decreased significantly with increasing arch number for both
species (F=100, df = 1, 71 and p<0. 05) (Figure 13).
62
Figure 13 Variation of arch length (Larch) at the onset of air-breathing in three PO2
levels and between the two species. Means ± standard errors are presented. Boxes enclose means not significantly different from each other (p>0.05) when the analysis was corrected for body length.
Number of filaments per arch (NFarch). NFarch showed significant
differences between the two species (F=18.87, df = 1, 71 and p<0.05).
Betta had more filaments, particularly in the second, third, and fourth
arches where the labyrinth organ is not present. Hypoxia only affected
filament length in Betta, producing a significant decrease of filaments
of the third and fourth arches for the 14 kPa treatment (F=9.9, df= 2, 71
and p<0.05). Trichopodus showed no particular pattern of NFarch
related with PO2 treatment (Figure 14).
63
Figure 14 Variation of number of filaments per arch (NFarch) at the onset of air-breathing in three PO2 levels and between the two species. Means ± standard errors are presented. Boxes enclose means not significantly different from each other (p>0.05).
Filament length (Lflm). Gill filaments in Betta were significantly
longer than in the Trichopodus in all the branchial arches (F=7.3, df=
2, 71 and p<0.05), when Lflm was corrected for body length. Hypoxia
treatments did not induce any significant differences within the species
(F=0.13, df= 2, 71 and p>0.05) (Figure 15).
64
Figure 15. Variation of filament length (Lflm) at the onset of air-breathing in three PO2
levels and between the two species. Means ± standard errors are presented. Boxes enclose means not significantly different from each other (p>0.05) when the analyses were corrected for body length.
Number of lamellae per filament per arch (NLflm). Betta had
larger numbers of lamella per filament than Trichopodus (F=454, df =
1, 215 and p<0.05) on all gill arches. Hypoxia treatments produced a
slight but significant increase on NLflm on the medial and anterior
sections of the first filament in Betta (F=3.3, df = 2, 215 and p<0.05).
The Trichopodus showed no particular pattern of NLflm related with the
PO2 treatment (Figure 16).
65
Figure 16. Variation of number of lamellae per filament per arch (NLflm) at the onset of air-breathing in three PO2 levels and between the two species. Means ± standard errors are presented. Boxes enclose means not significantly different from each other (p>0.05) when the analyses was corrected for body length
Single lamella area per filament per arch (SAlam). SAlam was
significantly larger in Betta than Trichopodus (F=107, df = 2, 197 and
p<0.05), when SAlam was corrected for body length. SAlam decreased
significantly in Betta for the 14 kPa treatment (F=2.8, df = 2, 197 and
p<0.05). The Trichopodus larvae showed no particular pattern of SAlam
related to PO2 treatment (Figure 17).
66
Figure 17. Variation of area of individual lamella per filament per arch (SAlam) at the onset of air-breathing in three PO2 levels and between the two species. Means ± standard errors are presented. Boxes enclose means not significantly different from each other (p>0.05).
DISCUSSION
Larval Growth Patterns
One of the interesting findings of this study was the occurrence
of the hypothesized profound differences between larvae of Betta and
Trichopodus in their responses to hypoxic exposure. Larval Betta
showed a significant growth increase in body size and wet mass when
they were exposed to mild hypoxia (17 kPa). Additionally, severe
hypoxia (14 kPa of PO2) diminished the Betta condition factor and
67
showed a small weight at the same size on the body length - wet mass
relationship (14 and 17 kPa of PO2). In contrast, in larval Trichopodus
not a single growth variable was significantly altered by hypoxia, as
similar reported by Blank (2009). These data collectively showed the
higher sensitiveness of Betta to aquatic hypoxia and the absence of
response of growth variables of Trichopodus to the same
environmental factor.
The decreased fish growth pattern under hypoxic conditions
observed by Wang et al. (2009) and Filho et al. (2005) also occurred in
larval Betta as evident from the lower condition factor and wet mass
and body length relationship. However, when the data were analyzed
by the increase of weight and size as a factor of age, they showed that
mild hypoxia could enhance both growth variables. Interestingly,
Trichopodus larvae didn’t follow this predicted pattern. Perhaps the
growth in all treatments, including normoxia, is delayed relative to Betta
until they become air-breathers. Trichopodus´ strongest response at
this point of development is mortality, which is high in all the
experimental groups (Chapter 2). This higher mortality could be
compensated by the considerably higher number of eggs (2000-3000)
68
in a single clutch compared with Betta (300-500) (Clotfelter et al., 2006;
Monviseset al., 2009; Froese & Pauly, 2015)
Larval Betta larvae reared in hypoxia (17 kPa) for 35 days
showed a significant 30% increase on TSAlab, but in 14 kPa there was
a significant 50% decrease in TSAlam. The respiratory surfaces of
Trichopodus larvae did not show the same responses, but this might
be explained by the fact that larval Betta was 2-3 times larger in both
variables compared to Trichopodus larvae.
Branchial Plasticity
Larval Betta showed a direct relation between TSAlam and PO2,
which is opposite to that occurring in the cyprinids and some other fish,
where TSAlam increases as hypoxia decreases (Timmerman &
Chapman, 2004; Nilsson, 2007; Crampton et al., 2008; Paterson, et al.,
2010). All the fishes in the aforementioned studies were adult water-
breathers exposed to acute hypoxia treatment, with the same individual
being compared in a hypoxic and normoxic environment. This differs
from the current study, where the larvae were reared in chronic
hypoxia. Our results indicate that the decrease in TSAlam could be
compensated for an increase in TSAlab, possibly in preparation for a
69
future air-breathing way of life, as Blank (2009) suggested for older
Trichopodus.
In general, the branchial morphometric measurements did not
show differential responses related to PO2 treatments within species.
In contrast, the differences between the two fish species are statistically
significant in most of the gill morphometrics, with Betta tending to have
larger gills at equivalent developmental stages compared to the
Trichopodus.
A Species Comparison – Influence of Natural
Habitat on Respiratory Morphology.
We hypothesized that Betta would have different branchial
features from the Trichopodus related to their different natural histories.
The natural reproductive habitats of Betta are temporal isolated ponds
on standing waters of flood plains such as rice paddies. These habitats
are frequently hypoxic, even anoxic on the bottom, because of the high
temperatures and high organic content (Rainboth, 1996; Monviseset
al., 2009; Froese & Pauly, 2015). Betta is a bubble nest builder and
males provide intense care for eggs and early larva (Ruber et al., 2006;
Monviseset al., 2009). Young larval Betta are not able to escape these
70
hypoxic ecological conditions and natural selection may have led to a
greater tolerance of aquatic hypoxia. In this situation we predicted that
larval Betta could develop branchial features that facilitated its
tolerance of aquatic hypoxia, such as a higher TSAlam. TSAlam in Betta
was 5 times bigger than in Trichopodus, manifested in all features of
gill structure, and Betta also exhibited enhanced growth under mild
hypoxic conditions. These features could explain facultative air-
breathing condition of Betta (Peters, 1978; Graham, 1997) and its
ability to extract oxygen from the water at this larval stage (1-35 pfd).
In contrast to Betta, the Trichopodus’ natural reproductive
habitat occurs in lowland wetlands like marshes, swamps and canals
with seasonally floods that facilitate lateral migrations from the
mainstream, or other permanent water bodies, to flooded areas during
the flood season and allows their return to the permanent water bodies
at the onset of the dry season (Rainboth, 1996; Froese & Pauly, 2015).
Trichopodus are also bubble nest builder with male parental care
(Ruber et al., 2006; Monviseset al., 2009). Compared to Betta,
Trichopodus larvae are more active swimmers (Mendez-Sanchez, F.,
unpubl.). This feature favors the ability to escape these open habitats
and the hypoxia they present. For this possible reason, and in contrast
71
to Betta, Trichopodus did not exhibit phenotypic plastic responses to
aquatic hypoxia in either growth rates or respiratory surfaces.
Compared with larval Betta, larval Trichopodus have relatively small
respiratory surfaces. This leaves this species with air-breathing as the
mechanism to avoid aquatic hypoxia, and likely explains its obligate air-
breathing condition (Burggren, 1979; Graham, 1997; Herbert & Wells,
2001; Blank, 2009; Burggren & Blank, 2009).
72
CHAPTER 4
RESPIRATORY PHYSIOLOGICAL ADJUSTMENTS TO CHRONIC HYPOXIA IN LARVAE OF THE AIR- BREATHING
ANABANTID FISHES BETTA SPLENDENS AND TRICHOPODUS TRICHOPTERUS.
INTRODUCTION
Acute hypoxic exposure in aquatic fish triggers an assortment of
reflex responses aimed at maintaining homeostasis. Reflex
hyperventilation of the gills and the associated increase in branchial
irrigation is a common response to aquatic hypoxia, although this
stressor may additionally trigger aquatic surface respiration or actual
air-breathing in those species capable of aerial respiration (for reviews
see Johansen & Lefant, 1968; Burggren, 1982; Burggren & Johansen
1986; Perry, 2011; Milsom, 2012; Porteus et al., 2011; Martin, 2014;
Abdallah et al., 2015). Concurrent with hypoxia-induced increases in
gill ventilation is reflex bradycardia (a decrease in heart rate), as well
as an increase in stroke volume and branchial vascular resistance
(Pelster, 1999; Gamperl & Farrell, 2004; Farrell, 2007; Stecyk et al.,
2008; Gamperl and Driedzic, 2009; Tota et al 2011; Wilson et al.,
2014). In facultative air-breathing fish, aquatic hypoxia may also
promote an increased reliance on aerial O2 uptake and aquatic surface
respiration (Johansen & Lefant, 1968; Randall et al, 1981; Perry, 2009;
73
Gamperl and Driedzic, 2009; Perry, 2011). These physiological and
behavioral responses to aquatic hypoxia in fishes - collectively
representing the hypoxic ventilatory reflex - are often accompanied by
numerous other additional adjustments, including changes in
hemoglobin oxygen binding affinity, blood O2 carrying capacity, stroke
volume, and branchial vascular resistance, all of which contribute to
enhanced O2 transfer and potentially lowered ventilatory convection
requirement (Perry et al., 2009; Gamperl and Driedzic, 2009).
The hyperventilation reflex of fishes minimizes reductions in
arterial-blood PO2 associated with aquatic hypoxia, but may also be
metabolically expensive (Perry et al., 2009; Perry, 2011). Indeed, these
metabolic costs may be unsustainable - chronic hypoxia exposure of
anabantid fish larvae results in 85-100% mortality at day 9 post-
fertilization in the gourami (Trichopodus trichopterus) and the Siamese
fighting fish (Betta splendens) (Mendez-Sanchez and Burggren, 2014),
perhaps in part because of the high cost of gill ventilation with water or
other things like failure of adequate oxygen transport (Randall et al.,
1981; Graham, 1997; Diaz & Breitburg, 2009; Farrell & Richards,
2009).
74
Different fish species employ different strategies for dealing with
aquatic hypoxia. Hypoxia resistance, which can be defined as the
ability of a fish to actively maintain O2 extraction and thus routine
metabolic rate as O2 levels fall, allows animals to exploit more O2
variable environments (Mandic et al., 2013). The hyperventilation
reflex is critical for this strategy. However, some species exhibit
hypoxic tolerance, whereby as environmental PO2 falls, the fish no
longer actively maintains normal rates of aerobic O2 consumption. This
requires an ability to tolerate increasing levels of tissue hypoxia (Perry,
2011), and is often correlated with lower critical PO2 (Chapman et al.,
2002; Mandic et al., 2013).
Hypoxic resistance and tolerance has been extensively
investigated in adult aquatic fishes and, to a lesser extent, in adult air-
breathing fishes. However, the respiratory and metabolic responses of
larval fishes to hypoxia are only poorly understood, especially in air-
breathing species. The aim of this study, then, was to evaluate the
effect of chronic hypoxia on pre-air breathing larvae of the air-breathing
fishes the gourami (Trichopodus trichopterus) and the Siamese fighting
fish (Betta splendens). It was hypothesized that environmental hypoxia
increases heart rate and opercular rate and decreases ��𝑂2 and Pcrit in
75
both species, but that these responses would differ qualitatively and
quantitatively between Trichopodus and Betta, based on the findings
of Chapter 2 and 3 and the different habitats that these two species
inhabit.
METHODS
Rearing and Maintenance
Larval Trichopodus and Betta were maintained from day 0
(hatching) to 40 days post fertilization (DPF) in three different PO2
levels: continuous normoxia (20 kPa) and two intermittent nocturnal
hypoxia groups (17 and 14 kPa). Details of rearing protocol, water
quality, and larvae maintenance were described in the methods of
Chapter 2 and in Mendez Sanchez and Burggren (2014). Larvae were
then sampled for physiological variables until day 38 (Betta) or day 35
(Trichopodus).
Oxygen Consumption.
Every five days following fertilization, larvae from each treatment
were used to measure mass-specific O2 consumption ( ��𝑂2 ),
expressed as µmol O2/g/h. This variable was measured using
intermittent closed respirometry in normoxia (20kPa) using a Loligo
76
Systems respirometry system (Tjele, Denmark). Larvae were placed
in a 2.0 ml borosilicate glass microrespirometer chamber. Four
chambers were used simultaneously; alternately one of each was run
without fish and served as blank sample to determine the effects of
possible microbial respiration. In all cases the oxygen consumption of
microbial respiration was not detectable by the system’s sensors
(accurate to< < 0.0005 µmol O2/g/h). Water PO2 was measured in each
respirometer for 2-6 h, depending on the larva’s O2 consumption rate,
using temperature-compensated fiber-optic planar sensors attached to
an OXY-4 meter (PreSens). The signals from these probes were
displayed on a computer with the automated data acquisition system
DAQ-M. The AutoResp software (Loligo Systems ApS) calculated
mass-specific oxygen consumption from the rate of O2 decline in each
chamber over time, the elapsed time, the chamber volume, and the fish
mass.
Larvae of both species settled on the bottom of the chamber,
barely moving during the measurements. Consequently, the
��𝑂2 measured was considered as to be standard metabolic rate
(SMR). The potential oxygen gradient generated by larval respiration
in each chamber was avoided using a micro stir bar enclosed in a cage
77
to protect the larva from its rotatory movement.
We did not expect significant allometric influences on the
comparisons of ��𝑂2 by PO2 and DPF because of the relatively
increment of body mass at these early stages of these developing
animals. Additionally, the concept of “scaling” may simply not apply to
very young animals undergoing both organogenesis and systemic
growth (Burggren, 2005). However, to minimize any small scaling
effects we compare larvae on the same age at the same physiological
state and determined the allometric constant “b” for both species in the
relation Y = aXb, where Y was ��𝑂2, X is the body mass and b is the
allometric constant. Typically, the b value is 0.7 (Burggren; 2005). The
b value determined for Betta during this study was 0.018 (r2=0.207;
F=59; df=1,226; and p<0.0001) and for Trichopodus was 0.004
(r2=0.159; F=31; df=1,165; and p<0.0001). Thus, while the b was
significantly different from 0 for both species, not only was the R2 value
very low (indicating multiple contributors to the variance in these data),
but the actual exponent b in both species was so small as to be
biologically insignificant.
The critical oxygen tension (Pcrit, in kPa), defined as the oxygen
78
partial pressure (PO2) when ��𝑂2 can no longer be maintained at
normoxic levels with further decreases in PO2, was measured using the
same experimental design for ��𝑂2, but using closed as opposed to
flow-through respirometry. A two-phase linear regression model
(Yeager and Ultsch, 1989; Muller and Seymour, 2011) was applied to
the PO2 vs. ��𝑂2 data obtained during a 2-6 hour period of O2 decline
in the respirometer chamber. Due to the high natural mortality of the
young larvae (see Chapter 2) and to avoid intra-clutch variation, Pcrit
was only measured in six groups at the first day of air-breathing: three
groups of each PO2 treatment of each species with an age of 35 pfd
and from the same clutch.
Opercular Rate and Heart Rate
Opercular rate (Or, opercular beats*min-1) and heart rate (Hr,
beats*min-1) were measured every 5 days after larvae hatched. Larvae
(randomly selected and only used once) were placed in a 4.5 ml
transparent flow-through chamber for visual observation of opercular
and heart rates. Direct visualization of the heart of both species was
possible because the skin and muscles were transparent at these early
ages. Larvae were allowed to acclimate to the chambers for 1 h before
79
measurements were begun. Or and Hr was recorded for 60 sec using
a digital microscope (Celestron 44302-A) at a magnification of 150x.
A 10 second section of each video was analyzed with the open
source physics video analyzer Tracker 4.72 (Brown, 2012). This
software was used to automatically and simultaneously track heartbeat
and operculum movements using changes in “luma” (brightness in an
image = the "black-and-white" or achromatic portion of the image)
occurring through time in a selected area. Figure 1 shows
representative traces of the Tracker program output for opercular
movements and heart rate.
Statistical Analysis
MANOVA comparisons were performed on ��𝑂2, Pcrit, Hr, and Or
using PO2, age and, in some cases, species, as factors. MANCOVA
was used on ��𝑂2to correct for the effect of age using days of post-
fertilization as a covariate. To compare the effect of PO2 on the
relationship between Hr and Or, linear comparison of slopes and
intersections were also utilized. Treatment groups were considered
significantly different at P<0.05. All data are expressed as mean ± 1
standard error (SE). N values are indicated for individual means, and
80
total numbers of larvae used are indicated in the statistical descriptions.
RESULTS
Mass-Specific O2 Consumption
MO2 of Trichopodus was not significantly affected by
development, and was approximately 6.9 µmol O2/g/h throughout the
larval stages measured. This was approximately 60% higher than in
Betta at comparable stages (Figures 2A and 3A). Larval Betta, unlike
Trichopodus, showed a complex pattern of developmental change in
��𝑂2 under normoxic conditions, with significantly higher values at early
stages and declining values later in larval development (F=74, df=4,
228 and p<0.0001). The effects of hypoxic rearing were equally
complex in Betta. Mild chronic hypoxic exposure (17kPa) actually
stimulated ��𝑂2 above control (normoxic) levels, while more severe
chronic hypoxic exposure (14 kPa) depressed ��𝑂2 at all levels
(F=73.9, df = 2, 228 and p<0.0001) (Figure 18 A).
Analyzing the effect of hypoxia on ��𝑂2 corrected for age (i.e.
using age as a covariate) revealed a significant effect of hypoxia in the
mildly hypoxic 17 kPa group (F=18.2, df=2, 227, and p<0.0001) (Figure
18 B).
81
Figure 18. Oxygen consumption (��𝑂2) of larval Betta splendens. A) ��𝑂2 through 35
days of development reared in three levels of PO2. B) ��𝑂2 corrected for aged differences in the three larval populations. Means ± SE are presented. N=9. An * indicates a significant difference from control (20 kPa).
Larval Trichopodus similarly presented higher ��𝑂2 s during
earliest stages of development (F=137, df = 6, 167 and p<0.0001)
(Figure 19 A). In stark contrast to Betta, however, neither level of
chronic hypoxia induced significant changes in ��𝑂2 in the
Trichopodus. This finding was confirmed by analyzing the effect of
hypoxia corrected for age (F=0.2, df = 2, 167, and p>0.05) (Figure 19
B).
82
Figure 19. Oxygen consumption (��𝑂2) of larval Trichopodus trichopterus. A) ��𝑂2
through 35 days of development reared in three levels of po2. B) ��𝑂2 corrected for aged differences in the three larval populations. Means ± se are presented. N=9. An * indicates a significant difference from control.
Critical Oxygen Partial Pressure (Pcrit)
A representative graphic of the Pcrit determination is shown in
the figure 20. Betta and Trichopodus showed two different, opposing
patterns of Pcrit and how it was affected by rearing PO2 (Figure 21). In
larval Betta, Pcrit was positively correlated with rearing PO2 (F=5.5, df =
2, 21, and p<0.05), with Pcrit at 14 kPa decreased 37% from the value
in the normoxic population. In contrast, Pcrit in Trichopodus larvae
actually increased as rearing conditions grew more hypoxic (F=17, df
83
= 2, 19, and p<0.0001). Chronic rearing at a PO2 of 17 and 14 kPa PO2
increased Pcrit by 24% and 70%, respectively.
Figure 20. Representative traces of the ��𝑂2 decline with PO2 in four larval Trichopodus. The fish were grown at 14 kPa during 35 dpf; the average Pcrit is shown with an arrow and it was estimated by the method of Muller and Seymour (2011). Each symbol represents a single measurement of four different larvae.
84
Figure 21. Comparison of Pcrit for larval Betta and Trichopodus reared in different levels of PO2 at day 35 of development. Boxes enclose statistically identical means (p>0.05). N values for each group are in parentheses.
Heart Rate
Heart rate in larval Betta showed no significant changes as a
function of either development or with varying levels of chronic hypoxia
exposure (F=0.4, df=2,63 and p>0.05) (figure 22 A). The overall heart
rate for Betta, which averaged 156 ± 4 beats/min, was lower than in the
Trichopodus (212 ± 3 beats/min) at all comparable stages of
development. Unlike for Betta, however, Hr in Trichopodus decreased
slowly and significantly with development (F=29.1, df = 2, 60 and
p<0.0001). However, this decline occurred independently of hypoxia
85
(F=0.6, df = 2, 60 and p>0.05) (figure 22 B).
Figure 22. A comparison of heart rate (Hr) in larval Betta splendens (A) and Trichopodus trichogaster (B) through 35 days of larval development in three levels of PO2. Means ± SE are presented. N=9. The horizontal lines represent the average Hr for all ages.
Opercular Rate
Larval Betta were rapidly and extensively ventilating their gills
on Day 5, the first day of measurement. The overall Or average in larval
Betta was 77 ± 3 opercular beats/min and did not significantly vary
through development (F=0.4, df = 2, 63 and p>0.05 (Figure 23 A).
Rearing in chronic hypoxia also did not induce any significant changes
86
in gill ventilation measured in normoxia (F=7, df = 6, 63 and p>0.05)
(Figure 23 A).
Unlike larval Betta, larval Trichopodus reared in normoxia had
not yet established opercular beating on day 5 post-fertilization.
However, once opercular movements began on day 10, Trichopodus
Or average overall was 173 ± 6 beats/min. It was higher in Trichopodus
than in Betta at any given developmental stage. Interestingly, only the
larval group of Trichopodus chronically exposed to mild hypoxia (17
kPa of PO2) exhibited opercular beating on day 5. From 10 dpf onwards
there were no significant variation associated with hypoxia (F=0.6, df=
2, 60 and p>0.05) (Figure 23 B).
87
Figure 23. Comparison of opercular rate (Or) through 35 days of larval development in (A) Betta splendens and (B) Trichopodus trichogaster reared in three different levels of PO2. Means ± SE are presented. N=9. The horizontal lines represent the average Hr for all ages.
Heart Beat-Opercular Rate Relationship
Coupling of cardiac and respiratory activity has long been
appreciated, and gives insights into the maturity and complexity of
physiological regulatory systems (for reviews see Taylor et al., 2006;
Schulz et al., 2013; Dick et al., 2014). Figure 24 shows representative
traces of simultaneously recorded opercular rate and heart rate in a 35
day old Betta splendens. Analysis of the interaction of Hr and Or
showed no differences within ages in either species (other than 5 days
post-fertilization in Trichopodus), so the effect of this factor was not
88
considered from the perspective of their interaction. Thus, data for the
Hr:Or relationship was determined in individuals from 5 to 35 pfd.
However, the interaction of Hr and Or was affected by hypoxia rearing
level in both fish Betta and Trichopodus.
Figure 24. Representative traces taken from software analysis of video images of Betta splendens (35 days post fertilization) raised in hypoxia (17kPa). The dashed vertical lines towards the right of the panels illustrate the ~ 2:1 ratio of heart beat to opercular movement in this trace. See text for additional details.
Larval Betta reared in normoxia varied significantly from the line
of identity (1:1 ratio), showing an approximate timing of 3 heartbeats
per 1 opercular beat (Figure 25 A). Rearing in either level of chronic
hypoxia resulted in patterns of Hr:Or ratio of 2:1, that were significantly
different from each other, from the 1:1 ratio, and from the line
describing the normoxic ratio (Fslopes=2.57; df=3,7; p<0.05 and
Time (sec)
0 1 2 3 4 5 6 7
Ch
an
ge in L
um
a
at A
rea
of
Inte
rest
Operculum Movement
Heart Beat
Increase
Decrease
Increase
Decrease
89
Fintercepts=14.02; df=3,7; p<0.001). Essentially, hypoxic treatments
increased Or compared with the normoxic larva with the same Hr.
Hypoxic larvae also tended to have a Hr:Or with a slope more similar to
the 1:1 ratio than to the normoxic slope. For larval Trichopodus the
Hr:Or relationship was statistically identical to a 1:1 ratio (Figure 25 B),
and showed no significant differences between normoxic and hypoxic
larvae (Fslopes=1.62; df=3,7; p>0.05 and Fintercepts=0.83; df=3,7; p>0.05).
Figure 25. The Hr:Or relationship in larvae of (A) Betta splendens and (B) Trichopodus trichopterus from 5 to 35 dpf are presented. The lines show the data for three groups reared in different levels of PO2. N= 20 for each PO2 group of each species. In all cases r > 0.9 y p<0.05 indicating significance of relationship. See text for statistical analysis of differences between experimental groups.
90
DISCUSSION
Development in Normoxia
The mean ��𝑂2 of larval Betta, 4.8±0.34 µmol O2/g/h, decreased
significantly during the measured period of development. For
comparison, the larvae of the air-breather Hoplosternum littorale had a
��𝑂2 of 0.58-1.16 µmol O2/g/h (Sloman et al., 2009; Persaud et al.,
2006). Larvae of strictly water breathers tends to have higher ��𝑂2s:
the zebrafish Danio rerio, 8-38 µmol O2/g/h; the very active rainbow
trout Oncorhynchus mykiss, 236± 46.8 µmol O2/g/h and Atlantic
salmon Salmo salar, 2-13 µmol O2/g/h; Gilthead seabream Sparus
aurata and Senegalese sole Solea senegalensis, 10-30 µmol O2/g/h
(Wells & Pinder, 1996; Barrionuevo & Burggren, 1999; Rombough,
1998; Parra & Yufera, 2001; Barrionuevo et al., 2010). ��𝑂2 of larval
Betta was thus between the values of larval air breathers and water
breathers, which could be important for its facultative air-breathing
strategy (Peters, 1978; Graham, 1997). This intermediate ��𝑂2
provides larval Betta with the opportunity to behave like a water
breather or an air breather without drastically modifying its basic
metabolism. Indeed, being in the middle of both strategies facilitates a
broader possibility of maintaining appropriate metabolic levels.
91
Pcrit in larval Betta was 7.2 ± 0.4 kPa, compared with 4.6-8.1 kPa
for larval Hoplosternum littorale (Sloman et al., 2009), 9.3 ± 1.0 kPa in
the adult of the facultative air-breathing Amia calva (Porteus et al.,
2014), 7.3-9.9 kPa for larvae of the completely aquatic Danio rerio
(Barrionuevo & Burggren, 1999; Barrionuevo et al., 2010), 2- 6 kPa for
adults of Morguna adspersa, Melanotenia fluviatis and Hypseleotris sp.
exposed to natural hypoxia episodes as a consequence of droughts
(Stoffels, 2015). Considering Pcrit as measure of hypoxia tolerance
(Chapman et al., 2002; Mandic et al., 2013), this characteristic of Betta
is again in the middle of the aquatic and facultative air breathing fishes,
pointing to its facultative air-breathing habit. Having an intermediate
Pcrit makes easier the possibility to adjust its respiratory performance
to either a water or air breathing strategy (Robertson et al., 2015).
Hr and Or did not show important changes during development,
suggesting the lack of neural and hormonal control on both parameters
during the first 35 days of post fertilization (McKenzie et al., 2007;
Taylor et al., 2010). For similar temperatures larval Danio rerio Hr was
125-180 beats/min (Barrionuevo & Burggren, 1999), for the adult of the
facultative air-breathing Hoplerythrinus unitaeniatus Hr was 27 ± 2
92
beats/min (McKenzie et al., 2007), and the hypoxia tolerant adult
obligate gill breather the pacu Piaractus mesopotamicus 63 ± 6
beats/min (Leite et al., 2007;Taylor et al., 2009). Heart rate in larval
Betta and Trichopodus was higher (156 ± 4 and 212 ± 3 beats/min,
respectively) and aligned more with a strictly gill breathing fish.
The ventilatory frequency (Vf ) measured as Or of larval Betta
and Trichopodus in normoxia was 77 ± 3 and 173 ± 6 beats/min,
respectively. As examples of Or in adult gill breathing fish, rates in the
common dentex Dentex dentex and trout Oncorhynchus mykiss the Vf
were 71.3 ± 7 and 30 ±3 beats/min, respectively (Cerezo et al., 2006;
Porteus et al., 2011). The Vf of the hypoxia tolerant water breathers
the cachama Colossoma macropomum, the carp Cyprinus carpio, the
pacu Piaractus mesopotamicus was, 40 ± 4 ,17±1, and 90 ± 1
beats/min respectively (Kalinin et al., 2000; Porteus et al., 2011). In
adults of the facultative air-breathing Hoplerythrinus unitaeniatus Vf
was 74.5 ± 6.5 beats/min (McKenzie et al., 2007). The Or of Betta and
Trichopodus is over the range of adult gill breathers which is common
for aquatic larva with high metabolic rates; the aquatic larva of these
two fish behave like a strictly water breathers.
93
The Hr:Or relationship for larval Betta had an approximate timing
of 3 heartbeats for every 1 opercular beat. In the adults of another
facultative air-breathing fish, Lepisosteus osseus (Rahn et al., 1971),
the coupling between heart rate and gill ventilation ratio in normoxia
was approximately 1:1 (Smatresk, 1986). For adults of another
facultative air-breather Hoplerythrinus unitaeniatus (Oliverira et al.,
2004), the mean ventilatory frequency was slightly more than twice the
heart frequency, a typical relation for a fish with aquatic respiration
(McKenzie et al., 2007). For the hypoxia tolerant water breather
Piaractus mesopotamicus, the Hr:Or approximated 3:1(Leite et al.,
2007;Taylor et al., 2009). Clearly, larval Betta had less opercular
movements per heartbeat than the other two species of facultative air-
breathers employing aquatic respiration. This situation could be related
to the low level of physical activity of the larval Betta (Mendez-Sanchez,
F., unpubl.). Thus, in normoxic conditions the tissue O2 delivery
generated by the Hr:Or relationship could be adequate. Also, during the
early stages measured skin breathing is likely also directly involved in
providing gas exchange (Burggren & Blank, 2010). However, a
definitive answer will have to await additional data from other larval
fishes.
94
Larval Trichopodus had an MO2 of 9 ± 0.6 µmol O2/g/h, an
elevated value compared with larval Betta and the facultative air-
breather Hoplosternum littorale (Sloman et al., 2009; Persaud et al.,
2006). In fact, this value is in the range of active, aquatic fishes such
as Danio rerio and Oncorhynchus mykiss (Rombough, 1998;
Barrionuevo et al., 2010). Larval Trichopodus raised in normoxia are
active swimmers (Mendez-Sanchez, F., unpubl.) with a high oxygen
demand, yet were also more resistant to environmental hypoxia than
larval Betta. Indeed, Trichopodus showed the lowest Pcrit of the water
and air breathing species listed above. The Hr:Or ratio in Trichopodus
was ~1:1 - this synchrony between ventilation and heart rate in
normoxia may improve gas transport across the gills (Smatresk, 1986;
Rombough, 1999) by matching convective delivery of oxygen to the
gills with the ability of the perfusing blood to remove it, potentially
making this species more efficient in extracting O2 from the water.
Both species fit in the behavioral and locomotor gradient of fish
lifestyles described by Dwyer et al. (2014) and Stoffels (2015) at
different extreme points. On one hand, the slower lifestyle of Betta, an
ambush predator or ‘saltatory’ forager has mostly benthic habits while
ready for fast starts and powered turns for high-acceleration prey
95
capture. Low metabolic rates and high Pcrit characterize their
metabolism. On the other hand, Trichopodus fits to the faster lifestyle -
an active cruising and pursuit predator with endurance swimming and
sprints for sustained chases, patrolling, drift feeding, searching, etc.
These faster swimming fishes have higher metabolic rates and low Pcrit.
Development in Hypoxia
Betta and Trichopodus both responded physiologically to
chronic hypoxia, but with highly species-specific differences.
1) Betta: Rearing under mild hypoxia (PO2 =17kPa) increased
��𝑂2 in larval Betta at most stages of development, showing that these
larvae can regulate and increase aquatic oxygen consumption, even at
an early age. In this respect, larval Betta appears to be like the larvae
of aquatic species such as Danio rerio; (Barrionuevo et al., 2010)
whose oxygen uptake appears to be enhanced by mild hypoxia. Under
more severe oxygen stress (14 kPa), however, aquatic ��𝑂2 in Betta
was reduced compared to normoxia (figure 2). Physiological
adjustments to oxygen extraction from water to maintain homeostasis
are expensive (Perry et al., 2009; Perry, 2011) and the cost cannot
easily be sustained in severe hypoxia. In any event, the low
��𝑂2 presented by larval Betta at 14 kPa also reflects their physical
96
inability to maintain O2 uptake, which is reflected in low survival at this
PO2 in both continuous and intermittent hypoxia (Chapter 2).
Pcrit is a useful measure of hypoxia tolerance (Chapman et al.,
2002; Mandic et al., 2013). Larval Betta chronically reared under
hypoxic conditions showed a Pcrit that was 30% lower than when reared
in normoxia. A lower Pcrit reflects the ability of larval Betta to continue
extracting O2 to lower oxygen levels, essentially making them more
resistant to hypoxia being that this specie has evolved in more hypoxic
waters than Trichopodus (Rainboth, 1996; Monviseset al., 2009;
Froese & Pauly, 2015).
Facultative air-breathers evolved the air-breathing habit in
response to unpredictable environmental conditions, especially with
respect to ambient temperature and oxygen (Randal et al., 1981;
Graham, 1997; Brauner et al., 2004). To be a facultative air-breather
implies the ability to adjust physiological variables in the face of
unpredictable environment situations. Such ability was evident in larval
Betta when examining the Hr :Or relationship and the effect of hypoxic
rearing on this relationship. Chronic hypoxia induced higher Or s at the
same Hr, and the relationship between these two variables changed
97
from 3:1 in normoxia to 2:1. This relative increase in perfusion
compared to ventilation may also improve gas transport across the gills
(Smatresk, 1986), potentially making this species more efficient in
extracting O2 from the water under hypoxic conditions.
Collectively, these physiological responses of Betta can be
considered more plastic than Trichopodus (West-Eberhard, 2005; Hill
et al., 2008), allowing physiological compensation to environmental
hypoxia. Larval Betta was able to adjust ��𝑂2 , Pcrit, Hr and Or in
response to hypoxia to increase its hypoxia tolerance, supporting the
strategy of being a facultative air-breather (Graham, 1997). This larval
plasticity induced by hypoxia was similarly found in Danio rerio, in
which its increment in hypoxia tolerance (lower Pcrit) was associated
with the induction of HIF-1 during critical developmental windows
(Robertson et al., 2015).
2) Trichopodus: Larval Trichopodus, unlike larval Betta, showed
no significant responses to chronic aquatic hypoxia, a lack of
physiological plasticity potentially making this species less tolerant to
hypoxia (Chapman et al., 2002; Perry, 2011; Mandic et al., 2013). This
characteristic may also explain the low overall larval survival in hypoxia
98
(Mendez-Sanchez and Burggren, 2014). As an obligate air-breather,
the only mechanism that larval Trichopodus have to escape aquatic
hypoxia is to resort to air-breathing (Graham, 1997). The ��𝑂2 of
Trichopodus was ~ 60% higher than the ��𝑂2 of Betta at most days of
development. Not surprisingly, this was reflected in both higher Hr and
Or, likely required to provide for the higher oxygen demand of the
tissues of the Trichopodus larvae.
Rearing in chronic hypoxia had no significant effect on the ��𝑂2
of larval Trichopodus, but actually increased Pcrit. Thus, Trichopodus
showed less hypoxia tolerance when the environment in which they
were reared is more hypoxic. The inability of larval Trichopodus to
maintain aquatic oxygen consumption is likely strongly correlated with
fact that the juvenile and adult Trichopodus are obligate air-breathers
(Graham, 1997).
For larval Trichopodus the Hr:Or relationship was statistically
identical to a 1:1 ratio. There were no significant differences between
normoxic larvae and those chronically reared in hypoxia, showing a
lack of developmental phenotypic plasticity driven by oxygen
availability for these physiological features.
99
Respiratory Developmental Plasticity and
Physiological Heterokairy
Heterokairy is the non-genetic change in the timing of the onset
of physiological regulatory systems and their components, or plasticity
in the timing of the onset of physiological regulatory systems or their
components, below the level of the species, between populations or
individuals (Spicer & Burggren, 2003; Spicer et al., 2011; see
Discussion in Chapter 2). Interestingly, in larval Trichopodus (but not
Betta) chronic rearing in mild hypoxic actually advanced the onset of
opercular beating to before day 5, compared to day 10 in the normoxic
population. This response, apparently adaptive in that it would allow
earlier access to abundant aerial oxygen, provides another example of
heterokairy, along with the advancement of the onset of air-breathing
(Chapter 2, Mendez Sanchez and Burggren, 2014).
SUMMARY
In conclusion, these physiological data indicate that larval Betta
is in many ways better adapted to aquatic survival than larval
Trichopodus. This correlates well with the more hypoxic habitats
typically inhabited by larval Betta.
100
Chapter 5 will now explore the respiratory properties of the blood
of Betta splendens and Trichogaster trichopterus.
101
CHAPTER 5
HEMOGLOBIN-OXYGEN EQUILIBRIUM CURVES, BOHR AND ROOT EFFECTS COMPARISON IN TWO AIR
BREATHING FISH, TRICHOPODUS TRICHOPTERUSAND BETTA
SPLENDENS.
INTRODUCTION
The hemoglobin-oxygen equilibrium curve (OEC) is a tool for
understanding the function of respiratory pigments; it shows the
functional relationship between the percentage of binding sites that are
oxygenated and the ambient PO2. Specifically, the shape and position
of the curve reflects hemoglobin oxygen affinity and cooperativity, from
essentially hyperbolic to sigmoidal and quantified by Hill’s coefficient
“n”. The diagnostic parameter P50 indicates the PO2 at which half of
the hemoglobin is oxygenated (Nikinmaa, 2006; Wells, 2009; Hill et al.,
2012).
Hill’s equation describes the relationship between hemoglobin
oxygen saturation (SO2) and the oxygen partial pressure (PO2), thusly:
SO2=KPO2n/100+KPO2n.
If log SO2/100-SO2 is plotted against log PO2 for values of SO2
between 20% and 80%, then straight lines of slope “n” result (Eddy,
102
1973; Riggs, 1998; Kamshilov & Kamshilova, 2001; Hebert & Wells,
2001). Hill’s coefficient “n” typically has values from 1.0 where the
oxygen equilibrium curve is hyperbolic and hemoglobin exhibits less
cooperativity. When hemoglobin shows more cooperativity, n can
increase to 3.0 for a strongly sigmoidal curve (Eddy, 1973; Wells, 2009;
Mandic et al., 2009; Hill et al., 2012). Hill’s coefficient has been used
to measure hemoglobin cooperativity in the context of evolutionary
adaptation to hypoxia tolerance in sculpins (Scorpaeniformes:
Cottidae) (Mandic et al., 2009) and a physiological and ecological
response of the hemoglobin cooperativity as a mechanism to avoid
hypoxia by Pagrus auratus (Cook et al., 2013) and Pleuronectes
platessa (Wood et al., 1975).
Hemoglobin-oxygen binding is also strongly affected by proton
concentration. The so-called Bohr effect consists of a decrease in O2
affinity of the respiratory pigment due to a decrease in pH or an
increase in PCO2 partial pressure (Souza & Bonilla-Rodriguez, 2007;
Perry, 2011). The Bohr effect magnitude has been measured by the
Bohr factor expression Φ=Δlog P50/ΔpH, which reflects arteriovenous
pH gradient sustained by respiratory acidosis (C02)(Wells, 2009;
Hebert & Wells, 2001; Cook et al., 2013). The air-breather Trichopodus
103
trichopterus (Hebert & Wells, 2001) and the hypoxia tolerant Pagrus
auratus showed a modest Bohr effect induced by pH in hypoxia
acclimated fish (Cook et al., 2013).
Lowered pH also decreases the maximum possible oxygen
hemoglobin saturation even at high PO2s, a phenomenon known as the
Root effect (Nikinmaa, 2006; Wells, 2009; Hill et al., 2012; Souza &
Bonilla-Rodriguez, 2007;). This effect is activated by the addition of
tissue-specific lactic acid; among vertebrates, it is observed only in fish,
principally teleost fish (Wells, 2009; Hill et al., 2012). A classic example
in fish is the family of six oxygen equilibrium curves each with different
oxygen maximum saturation generated by six different pHs in the whole
blood of the eel Anguilla vulgaris at a constant temperature (Hill et al.,
2012). In contrast, there was no apparent Root effect in the blood of
the air-breather Trichopodus trichopterus even at the non-physiological
level of acidity of pH 6.6 (Hebert & Wells, 2001).
Hemoglobin-oxygen binding is also typically quite sensitive to
temperature, with increased temperature increasing the P50
(decreasing hemoglobin oxygen affinity). Fish hemoglobins typically
show significant temperature sensitivity as a way to compensate the
104
relation of aquatic hypoxia and temperature, for as temperature
increases, the obligatory decrease in oxygen solubility results in less
dissolved oxygen available for respiration (Nikinmaa, 2006; Wells,
2009; Hill et al., 2012).
Importantly, OECs as modified by the Bohr and Root effects and
the influence of temperature strongly reflect phenotypic plasticity,
especially in response to environmental stressors such as pH and
temperature. The relatively rapid turnover of red blood cells and the
hemoglobin they contain provides animals with the ability to quickly
modified blood oxygen transport characteristics in response to
changing environments (Nikinmaa, 2006; Wells, 2009; Hill et al., 2012).
The aims of this study were to 1) compare the parameters on
the hemoglobin-oxygen equilibrium curves between two air-breathing
fish species, Trichopodus trichopterus and Betta splendens, on a
gender-specific basis, and 2) determine the magnitude of the Bohr and
Root effects on both species. The parameters evaluated were partial
pressure at 50% of saturation (P50), oxygen maximal saturation
(SO2max), and Hill´s n value (n).
METHODS
105
Hemoglobin-oxygen dissociation curves
Ten Trichopodus trichopterus adults and ten Betta splendens
adults (5 females and 5 males) were obtained from the stock described
previously in Chapter 2. Because of the small size of these species,
blood sampling was done by cardiac puncture (Ostrander, 2000). Fish
were anesthetized using a dilution of MS222 in a separate container.
Once the fish lost equilibrium, it was positioned ventral side up. A 22-
gauge needle attached to a 1ml heparinized glass syringe, self-
prepared before sampling by aspiration and complete expulsion of 2ml
of standard heparin solution of 1000 U/ml (Chantler & Cox, 1999). It
was inserted into the heart and 50μl of blood were drawn. After the
syringe with the needle was removed, the fish was euthanized by
returning it to the buffered MS222 solution until all opercular movement
stops.
50 microliters of freshly drawn blood from one fish individual
were mixed with 5 ml of HEMOX-Solution, 20 microliters of Additive-A,
and 10 microliters of Anti-Foaming Agent (TCSSCI Scientific Supplies).
The mix was placed immediately in an automatic blood oxygen
dissociation analyzer Hemox Analyzer Model B (TCS Scientific Corp,
2007) at 27oC to determine the blood oxygen equilibrium curve.
106
Assessments of the relation between PO2 and SO2 were tested at CO2
concentrations of 0%, 1% and 2.5% to determine the Bohr and Root
effects.
Comparisons were made between species and sexes of the PO2
at which the hemoglobin was 50% saturated with oxygen (P50). Within
species and sexes comparisons were performed with Kruskal Wallis
(H) and U of Mann-Whithney (W) non-parametric analysis. P50,
SO2max and from the hemoglobin-oxygen dissociation curves were
compared between species and CO2 levels using an ANOVA or the
equivalent nonparametric H of Kruskal-Wallis. As the n coefficient was
a slope of the SO2/100-SO2 plotted against log PO2; this parameter was
compared within species with a multiple comparison of slopes. A
Φ=Δlog P50/ΔpH was estimated for each experimental individual and for
each 1% and 2% of CO2-pH level. An ANOVA was then also used to
compare between CO2-pH levels.
RESULTS
107
P50
The sigmoid curves for both adult Betta splendens and
Trichopodus trichogaster at 0% of CO2 are shown in figure 26 A. Whole
blood P50 was 0.38 ± 0.17 kPa in Betta but much higher in Trichopodus,
at 1.89 ± 0.58 kPa. These P50 values varied significantly between
species (W=36; n1=6, n2=6; p<0.01). The P50 distribution for males
and females for Betta was not significantly different for P50 (W=2, n♂
=3, n♀=3, and p>0.05), P80 (W=1, n♂=3, n♀=3, and p>0.05), and P90
(W=1, n♂=3, n♀=3, and p>0.05). Similarly, there were no significant
difference between males and females in Trichopodus on P50, P80, and
P90 (Ws=1, n♂=3, n♀=3, and ps>0.05) (figure 26 B).
Figure 26 Oxygen equilibrium curves for Betta splendens (A) and Trichopodus trichogaster (B). Each line and symbol set represents an individual. The sex is indicated on the legend. The mathematical model that best fits the data set is presented along with the associated mathematical and statistical values.
108
Bohr Shift
The pH of the whole blood-Hemox buffer mixture varied with %
CO2 as follows: 0% CO2, pH= 7.55; 1% CO2, pH= 7.44; 2.5% CO2 , pH=
7.25. Using these levels of pH, changes in blood pH produced
significant changes on the shape of the oxygen equilibrium curves for
both species, as shown on figure 27. P50 changes induce by pH, its
significance, and the magnitude of the Bohr effect expressed as Φ is
shown in table 1.
Figure 27 The Bohr effect on the whole blood oxygen equilibrium curve of Betta splendens (A) and Trichopodus trichogaster (B). Each line describes the fitted model created with 3 adult individuals of each species
Table 1. P50 and Φ values at different and CO2-manipulated pHs to determine the magnitude of Bohr effect on the whole blood of both fish species
P50 at designated Bohr Effect (Φ)
109
Species
pH (kPa) over designated pH intervals
pH 7.25
pH 7.44
pH 7.55
7.25-7.44
7.44-7.55
Mean
7.25-7.55
Betta splendens
2.1±1.07 *
0.4±0.01*
0.38±0.17
0.24
1.8
1.02
Trichopodus
trichogaster
3.36±0.96
0.4 ±0.01
1.89±0.58
0.43
0.16
0.29
An * indicates a significant difference (p<0.05) from control at pH 7.55
Root Effect
SO2 max for Betta change significantly with blood pH (F=29.93;
df=2,9; p<0.001), ranging from 100% at pH 7.55 to 84% ± 3.6 at pH
7.44 and 91.5% ±0.4 at pH 7.25 (Figure 27 A). The same variable for
Trichopodus did not change significantly with pH (F=1.6; df=2,11;
p>0.05), even though the figure 27 B shows the averages lines with
different SO2 max.
Hill’s Coefficient
Hill’s coefficient, n, for Betta at pH 7.55 (0% of CO2) was
2.17±0.6. This value was significantly different from that at pH 7.44,
which was 0.88±0.02, and from pH 7.25, which was 0.61±0.03 (F=
47.2, df=2, p<0.0001) (figure 28 A). For Trichopodus, n at pH 7.55 was
2.25±0.16. This value was significantly different from pH 7.44, which
110
was 1.7±0.007 and from pH 7.25 that was 1.48±0.01 (F= 48.2, df=2,
p<0.0001) (figure 28 B).
Figure 28. Hill´s graph of Betta (A) and Trichopodus (B). Hill´s n values are pointed with the arrows for each pH-CO2 value.
DISCUSSION
111
Overview
Some P50 values for teleost with different life styles at normoxia
and 0% of PCO2 are as follows: the freshwater breather rainbow trout
Oncorhynchus mykiss 2.8±0.13 kPa (Nikinmaa & Soivio, 1979); for the
amphibious mangrove rivulus Kryptolebias marmoratus 1.76±0.11 kPa,
an extremely hypoxia tolerant skin breather (PO2<2.5kPa) (Turco et
al., 2014); some cold-temperate marine fishes like the moderately
active benthopelagic Atlantic cod Gadus morhua 1.7 kPa; the active
benthopelagic Atlantic herring Clupea harengus 0.88 kPa; the inactive
demersal plaice Pleuronectes platessa 1.29 kPa; and the active
pelagic swimmer Atlantic mackerel Scomber scombrus 1.32 kPa
(Herbert et al., 2006); and the facultative air-breather armored catfish
Andstrus chagresi 2.63 kPa (Graham, 1983). On this study Betta
showed a P50 of 0.38±0.17 and Trichopodus of 1.89±0.58. The P50
value of Betta is the lowest of all the species above mentioned, it is
below of the hypoxia tolerant and benthic species. This feature is
associated with high oxygen affinity of its hemoglobin similar to the
environmental hypoxia tolerant fishes (Wells, 2009). Trichopodus,
differently, had an n value close to the active and pelagic fishes
showing less hemoglobin oxygen affinity than Betta. Those features
112
are similar to those active fish evolved to tolerate functional hypoxia
(Wells, 2009).
The hemoglobin cooperativity measured with the Hill’s
coefficient for different teleost is as follows: Antarctic cod Dissostichus
mawson, occurring at -1.9 °C, n=1.75 (Qvist et al., 1977); mangrove
rivulus Kryptolebias marmoratus n=1.35±0.03 (Turco et al., 2014); for
the cold-temperate marine fishes n=1.3-1.7 (Herbert et al., 2006);
rainbow trout Oncorhynchus mykiss n=1-2 (Weber et al., 1976); in
tench Tinca tinca blood n had values in the range of 0.84-1.75 (Eddy,
1973); the benthic cyprinid fish Pleuronectes platessa sowed n= 1.6-2
(Wood et al., 1975). Comparing these values with those obtained for
Betta and Trichopodus, 2.17±0.6 and 2.25±0.16 respectively, in fish n
the values are generally between 1.0 and 2.0 (Eddy, 1973). Both air-
breathing fish of this study showed slightly high values with a tendency
to be hyperbolic, resulting in high cooperativity compared to many
fishes. This cooperativity value dramatically decreased with decreasing
pH, in a statistically significant way for Betta for values below 1.0
indicating less cooperativity and possibly high oxygen affinity.
113
The Bohr shift magnitude for some teleost is enlisted as follows;
the highly hypoxia adapted New Zealand snapper Pagrus auratus
Φ=1.02±0.12 (Cook et al., 2013); Φ=0.64 - 0.75 in tench Tinca tinca
(Eddy, 1973); in the benthic fish Φ=0.06±0.003 Pleuronectes platessa
(Wood et al., 1975); in the Antarctic cod Dissostichus mawson Φ=0.48
(Qvist et al., 1977); and in the rainbow trout Oncorhynchus mykiss
Φ=1-1.7 (Nikinmaa & Soivio, 1979). In general, Hebert et al. (2006)
presented an integral analysis of Φ where the values for marine fishes
are between 0.9 and 1.06, and for freshwater fish species 0.6 and 0.98.
As it can be seen, the blood analysis of Betta in this study showed a
mean value of Φ of 1.02, which indicate a marked Bohr shift associated
with changes in blood pH. Contrarily, Trichopodus showed the smallest
mean Φ value of all fishes mentioned above. This reflects pH-induced
small effects on its hemoglobin structure (Souza & Bonilla-Rodriguez,
2007; Perry, 2011).
The reduced oxygen hemoglobin saturation produced by
decreased pH is known as the Root effect. In some marine teleost the
root effects was 12-19% reduction in total oxygen capacity (Hebert et
al., 2006); 12% on Pagrus auratus (Cook et al., 2013); and 10% on
Tinca tinca (Eddy, 1973). In contrast, there was no apparent Root effect
114
in the blood of the air-breather Trichopodus trichopterus, which confirm
the absence of Root effect in Trichopodus even at the non-
physiological level of acidity of pH 6.6 found by Hebert & Wells (2001)
Summary
Both fish species in this study are well framed within the gradient
of fish lifestyles proposed by Wells (2009); on one side are fish like
Betta. This species presents the smallest P50 values showing the
higher affinity of its hemoglobin, and also shows significant Bohr and
Root effects highlighted by signifficant change in the Hill’s n value.
Additionally, it showed the marked hyperbolic form (n closer to 3) of the
oxigen equilibrium curve showing low cooperativity. This low
cooperativity becomes dramaticaly higher with changes in pH. All of
these characteristics are typical of a environmental hypoxia tolerant
fish as decribed by Wells (2009). On the other hand, Trichopodus
trichpterus showed higher P50 values with lower hemoglobin-oxygen
affinity than Betta, characteristics of an active fish tolerant to funtional
hypoxia. This fish appears more adapted for an athletic lifestyle but also
preserves features of a fish adapted to environmental hypoxia with
small Bohr and Root effects .
115
Neither of the two species is strictly on the extreme of the
gradient functional hypoxia tolerant – environmental hypoxia tolerant,
but Betta showed characteristics such as the Bohr and root shifts that
favor physiological plasticy and explains its facultative air-breathing.
These characteristics give this species the option of behaving like a
strict environmental hypoxia-tolerant water breather. The hemoglobin
of Trichopodus did not show too many options for physiological
plasticity, leaving this species with only with the posibility to escape
hypoxic water by becoming an air-breather. This could explain its
obligate air-breather strategy and the high mortality of the pre air-
breathing larvae.
116
CHAPTER 6
CONCLUSIONS
In this concluding section, I provide answers to each single
implicit question associated with each set of hypothesis and aims. A
concrete interpretation is also provided, excerpted from the previous
discussions in the related chapters.
1. Did hypoxia induce high mortality on the pre air-breathing
larva of Betta and Trichopodus? Answer: Continuous
hypoxia (15%) killed 100% of the young larvae of both species,
and was deleterious beyond 9 DPF. Nocturnal intermittent
exposure to hypoxia produced a 75% of mortality on Betta larvae
and 80% on Trichopodus both beyond 35 DPF. Mortality of the
two species in normoxia (PO2 20kPa) was significantly less: 75%
for Betta and 85% for Trichopodus. Interpretation: Intermittent
hypoxia creates a more natural environmental stimulus, and is
recommended for future experimental exposure to hypoxia.
These results highlight dissolved oxygen as a limiting
environmental factor for larval fishes in fresh water (Diaz &
Breitburg, 2009), and hypoxia as a strong natural selection force
as in adult air-breathing fish (Randall et al., 1881; Graham,
117
1997) with a high cost evident in lowered larval survival. The
current findings also showed that the response of B. splendens
to hypoxia differs from T. trichopterus (Farrell & Richards, 2009).
2. Was there evidence for heterokairy on the onset of air-
breathing induced by hypoxic acclimation? Answer: Betta
larva advanced the onset of air-breathing. Nocturnal intermittent
hypoxia treatment at 17 kPa advanced the onset of air-breathing
by 4 days, and the group exposed to a PO2 of 14 kPa began air-
breathing 2 days before the normoxic group. In contrast,
Trichopodus delayed the onset of air-breathing by 8 DPF for
both levels of hypoxia. Interpretation: Variation in the
chronological age (timing) when normal air-breathing onset
occurs reflects developmental plasticity (West-Eberhard, 2005),
as well as different developmental trajectories for each species
and individuals within populations (Burggren & Reyna, 2011).
Hypoxic exposure can alter the timing of onset of air-breathing
onset within species. As such, these data support the theory of
heterokairy, in which developmental timing of specific
physiological process like aerial respiration has been moved as
a result of an environmental stressor like hypoxia (Spicer &
118
Burggren, 2003; Spicer et al., 2011). The results showed a
particular pattern for each species.
3. Did hypoxic acclimation affect larval growth? Answer:
Larval growth was affected for Betta but not for Trichopodus.
Mild hypoxia 17 kPa of PO2 increased 1.5 times body size and
wet mass, but more severe hypoxia conditions (14 kPa)
decreases the condition factor. Interpretation: These data
collectively showed the higher sensitivity of Betta to aquatic
hypoxia and the absence of a hypoxic response on growth
variables in Trichopodus. Interestingly, the decreased fish
growth pattern under hypoxic conditions observed by Wang et
al. (2009) and Filho et al. (2005) is not present in the larvae of
this two species.
4. Did hypoxic acclimation affect the size of the respiratory
surfaces at the onset of the air-breathing? Answer: Hypoxia
increased the respiratory surfaces for Betta but not for
Trichopodus. A PO2 of 17 kPa increased by 30% the labyrinth
respiratory surface of Betta. However, 14 kPa decreased the
lamellar respiratory surface. However, in the Trichopodus
119
hypoxic exposure did not significantly affect these respiratory
surfaces. Interpretation: Larval Betta could develop branchial
features that facilitated its tolerance of aquatic hypoxia, like the
enhanced growth and the increase size of TSAlab under mild
hypoxic conditions. These features could explain facultative air-
breathing condition of Betta (Peters, 1978; Graham, 1997) and
its ability to extract oxygen from the water at this larval stage (1-
35 pfd). In contrast, Trichopodus did not exhibit phenotypic
plastic responses to aquatic hypoxia in either growth rates or
respiratory surfaces; this leaves this species with air-breathing
as the mechanism to avoid aquatic hypoxia, and likely explains
its obligate air-breathing condition (Burggren, 1979; Graham,
1997; Herbert & Wells, 2001; Blank, 2009; Burggren & Blank,
2009).
5. Did hypoxic acclimation affect gill morphometrics?
Answer: Generally Betta had bigger gill structures than
Trichopodus. Hypoxia did not affect these structures in
Trichopodus; In contrast, in Betta this environmental factor
produced a significant decrease on the number of gill filaments
per arch and the area of a single lamella, but it produced a slight
120
significant increase on the number of lamella per filament.
Interpretation: In their natural habitat, young larval Betta are
not able to escape to hypoxic ecological conditions; natural
selection may have led to a greater tolerance of aquatic hypoxia.
In this situation we predicted that larval Betta could develop
branchial features that facilitated its tolerance of aquatic
hypoxia. Indeed, Betta showed plasticity on the respiratory
surfaces and the gill morphometrics. On the other hand, larval
Trichopodus are more active swimmers. This feature favors the
ability to escape these open habitats and the hypoxia they
present. Trichopodus did not exhibit phenotypic plastic
responses to aquatic hypoxia in either growth rates or
respiratory surfaces.
6. Did the routine metabolic rate, measured as mass-specific
oxygen consumption (��𝑶𝟐) , differ between species and
was it affected by hypoxic acclimation? Answer:
Trichopodus had a metabolic rate 60% higher than Betta at
comparable stage and weight. Mild chronic hypoxic exposure
(17kPa) actually stimulated the ��𝑂2 of Betta above control
121
(normoxic) levels, while more severe chronic hypoxic exposure
(14 kPa) depressed ��𝑂2. In stark contrast to Betta, however,
neither level of chronic hypoxia induced significant changes in
��𝑂2 in Trichopodus. Interpretation: ��𝑂2 of larval Betta was
between the values of larval air breathers and water breathers,
which could be important for its facultative air-breathing strategy
(Peters, 1978; Graham, 1997). This provides larval Betta with
the opportunity to behave like a water breather or an air breather
without drastically modifying its basic metabolism. On the
contrary, for Trichopodus this value is in the narrow range of
active, aquatic fishes with a high oxygen demand (Rombough,
1998; Barrionuevo et al., 2010), which means these fish larvae
will have higher mortality if they do not become air-breathers
because they are not able to restructure their anatomy to
increase their breathing.
7. Did Pcrit differ between species and was it affected by
hypoxia acclimation? Answer: The larvae of the two species
showed opposing patterns of the effects of hypoxic rearing on
Pcrit. Hypoxia decreased the Pcrit of Betta by 37%in contrast, the
Pcrit of Trichopodus increased by 70% as a result of growing in
122
chronic hypoxia. Interpretation: Considering Pcrit as measure of
hypoxia tolerance (Chapman et al., 2002; Mandic et al., 2013),
this characteristic of Betta is again in the middle of the aquatic
and facultative air breathing fishes, pointing to its facultative air-
breathing habit. Having an intermediate Pcrit makes easier the
possibility of adjusting its respiratory performance to either a
water or air breathing strategy (Robertson et al., 2015).
Trichopodus showed the lowest Pcrit of the water and air
breathing species (Barrionuevo & Burggren, 1999; Sloman et
al., 2009; Barrionuevo et al., 2010; Porteus et al., 2014; Stoffels,
2015). They also were more resistant to environmental hypoxia
than larval Betta. However, when hatched and grown in
hypoxia, the gourami’s tolerance to this factor decreases. In
contrast, Betta larvae become more tolerant when they born and
grow in hypoxic environments.
8. Did Hr and Or differ between species and were they affected
by hypoxic acclimation? Answer: The overall heart rate and
opercular rate for Betta were lower than in the Trichopodus at
all comparable stages of development. Hypoxia did not affect
the Hr and Or in larvae of both species. Interpretation: Hr and
123
Or did not show important changes during hypoxic treatments,
suggesting the lack of neural and hormonal control on both
parameters during the first 35 days of post fertilization
(McKenzie et al., 2007; Taylor et al., 2010). The Or of Betta and
Trichopodus is within the range of adult gill breathers which is
common for aquatic larva with high metabolic rates; the aquatic
larva of these two fish behave like a strictly water breathers.
9. How different was the relationship of Heart Beat:Opercular
Rate between species? And how did hypoxic acclimation
affect it? Answer: This relationship showed a ratio of 1:1 for
Trichopodus and 3:1 for Betta when the larvae were rear in
normoxia. Chronic hypoxia did not affect this ratio on
Trichopodus larvae, but for Betta it created a pattern of 2:1.
Interpretation: Clearly, larval Betta had less opercular
movements per heartbeat than species of facultative air-
breathers employing aquatic respiration (Leite et al.,
2007;Taylor et al., 2009). This situation could be related to the
low level of physical activity of the larval Betta. The Hr:Or ratio in
Trichopodus was ~1:1 - this synchrony between ventilation and
heart rate in normoxia may improve gas transport across the
124
gills (Smatresk, 1986; Rombough, 1999) by matching
convective delivery of oxygen to the gills with the ability of the
perfusing blood to remove it, potentially making this species
more efficient in extracting O2 from the water.
10. Did the hemoglobin oxygen equilibrium curves of adults
differ between species? Answer: Yes Betta presented a
marked hyperbolic curve with smaller P50 and higher
hemoglobin affinity than Trichopodus. This OEC was
significantly different from the sigmoid shape of Trichopodus.
Interpretation: Both air-breathing fish of this study showed
slightly high values with a tendency to be hyperbolic, resulting in
high cooperativity compared to many fishes. This cooperativity
value dramatically decreased with decreasing pH, in a
statistically significant way for Betta for values below 1.0
indicating less cooperativity and possibly high oxygen affinity.
The P50 value of Betta is the lowest of all the facultative air-
breathers, water breathers, amphibious, extremely hypoxia
tolerant, cold temperature marine fishes species, it is also below
of the hypoxia tolerant and benthic species (Nikinmaa & Soivio,
1979; Turco et al., 2014; Herbert et al., 2006; Graham, 1983).
125
This feature is associated with high oxygen affinity of its
hemoglobin similar to the environmental hypoxia tolerant fishes
(Wells, 2009). In contrast, Trichopodus had a hemoglobin
cooperativity value close to the active and pelagic fishes
showing less hemoglobin oxygen affinity than Betta. Those
features are similar to those active fish evolved to tolerate
functional hypoxia (Wells, 2009).
11. Did the hemoglobin of the both species show Bohr and
Root effects? Answer: Betta had significant Bohr and Root
effects highlighted by significant changes in the Hill’s n value.
On the other hand, the blood of Trichopodus showed much
lesser effects of increased proton concentration on hemoglobin-
oxygen affinity. Interpretation: Betta showed characteristics
such as the Bohr and root shifts that favor physiological plasticity
and explains its facultative air-breathing habit. These
characteristics give this species the option of behaving like a
strict water breather tolerant of environmental hypoxia. The
hemoglobin of Trichopodus did not show too many options for
physiological plasticity, leaving this species with only with the
possibility to escape hypoxic water by becoming an air-breather.
126
This could explain its obligate air-breather strategy and the high
mortality of the pre air-breathing larvae.
127
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