MINISTRY OF EDUCATION AND TRAININGCAN THO UNIVERSITY DOCTORAL DISSERTATION SUMMARY MAJOR: AQUACULTURE MAJOR CODE: 9 62 03 01 DANG DIEM TUONG THE EFFECTS OF TEMPERATURE, HYPOXIA AND HYPER
Trang 1MINISTRY OF EDUCATION AND TRAINING
CAN THO UNIVERSITY
DOCTORAL DISSERTATION SUMMARY
MAJOR: AQUACULTURE MAJOR CODE: 9 62 03 01
DANG DIEM TUONG
THE EFFECTS OF TEMPERATURE, HYPOXIA AND HYPERCARBIA ON RESPIRATION AND PHYSIOLOGY
OF CLOWN KNIFEFISH
CHITALA ORNATA (GRAY, 1831)
Can Tho, 2018
Trang 2THE RESEARCH WAS PERFORMED AND COMPLETED AT CAN THO UNIVERSITY
Supervisor: Prof Tran Ngoc Hai
Co-supervisor: Assoc Prof Mark Bayley
Assoc Prof Do Thi Thanh Huong
The dissertation will be defended at the Doctoral Dissertation
Assessment Committee at the Institute Level
At: ……….………
Time & Date:………
Reviewer 1: ………
Reviewer 2: ………
The dissertation can be found at:
The learning resource center, Can Tho University
The national library of Vietnam
Trang 3THE LIST OF PUBLISHED PAPERS
1 Tuong, D D., Borowiec, B., Clifford, A M., Filogonio, R., Somo, D.,
Phuong, N T., Milsom, W K (2018a) Ventilatory responses of the clown
knifefish, Chitala ornata, to hypercarbia and hypercapnia Journal of
Comparative Physiology B, 1-9.
2 Tuong, D D., Ngoc, T B., Huynh, V T N., Phuong, N T., Hai, T N., Wang,
T., & Bayley, M (2018b) Clown knifefish (Chitala ornata) oxygen uptake
and its partitioning in present and future temperature environments
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 216, 52-59.
Trang 4CHAPTER 1 INTRODUCTION1.1 Background
Climate change is dramatically challenging and threatening aquatic animal life
international panel on climate change (IPCC, 2014) for Mekong River delta that thetemperature will increase 2.5-3.5C in next 100 years; and concurrently, the elevatedatmospheric CO2 levels will rise up at rate 3% per year recently
It has been indicated that projected the elevated water temperature may negatively
impact broadly marine and freshwater ecosystem functions (Roessig et al., 2004; Brander, 2007; Rijnsdorp et al., 2009; PÖrtner & Peck, 2010; Hofmann and Todghram, 2010; Madeira et al., 2012; Crozier & Hutchings, 2014; Lefevre et al.,
2016) and fish populations through effects on fish physiology, respiration,
metabolism, food ability, growth, behaviors, reproduction and/or mortality (Watts et
al., 2001; Cnaani, 2006; Sigh et al., 2013; Reid et al., 2015) While the temperature
is considered as a key importance of controlling physical factor pervasivelydetermining animal distribution, the rising environmental water CO2 level(hypercarbia) is more related to acid-base imbalance, water pH reduction and
cardioventilatory as well as respiratory changes (Gilmour, 2001; Claiborn et al., 2002; Ishimatsu et al., 2005; Brauner and Baker, 2009; Talmage & Gobler., 2011; Nowicki et al., 2012; Milson, 2012; Munday et al., 2012) Nevertheless,
physiological changes and adaptive ability of aquatic animals have been consideredintriguing targets to research under effects of projected elevated temperature andhypercarbia in water
A hypothesis of oxygen capacity limited thermal tolerance is proposed to expressnegative impacts of the elevated temperature on the fish that is underlying the oxygen
delivery mechanism to tissues (Portner, 2001; Portner and Farrell, 2008; Munday et
al., 2008; Munday et al., 2009; Nilsson et al., 2009; Portner, 2010; Neuheimer et al.,
2011) It is due to the dissolved oxygen level decreasing with progressive increases
of the temperature whilst fish oxygen demand significantly increases with theelevated temperature Therefore, the elevated temperature integrating with hypoxia(the decrease of the dissolved oxygen level) has been indicated that could result inlargely severe effects on aquatic organism in term of the metabolism and net result of
performance (McBryan et al., 2013) In addition, it has been argued that fish in
tropical areas can be affected severely because they have been already lived neartheir upper thermal limits and can be more vulnerable with a small increase of the
temperature (Nelson et al., 2016; Tewksbury et al., 2008) However, there is growing evidence of studies that do not conform that hypothesis (Clark et al., 2013; Norin et
al., 2014; Wang et al., 2014; Lefevre, 2016) Indeed, it is
Trang 5argued that the air-breathing fish species which hypoxic water tolerance could be aresult of an evolution under the effects of higher temperatures and lower atmosphericoxygen pressure than the present Investigating the effects of the elevatedtemperature and hypoxia on the metabolism of the air-breathing fish is important toassess the effects of climate change.
Another aspect of the adaptive ability to environmental factors, it has been exposed
that one of adaptive mechanisms is ability of changing gill morphology (Tuurala et
al., 1998, Sollid et al., 2003; Sollid et al., 2005; Sollid and Nilsson, 2006; Ong et al.,
2007; Matey et al., 2008; Mitrovic and Perry, 2009) Intensive researches on this
ability have been found in the water-breathing fishes including crucian carp, goldfishand salmonids which their gills have been found to increase or decrease interlamellarcell mass (ILCM) to increase or decrease respiratory surface area with changes of theenvironmental factors This ability of fish is intriguing scientists that whether the gill
remodeling is a modern trend or an ancient trait (Nilsson, 2007; Nilsson et al., 2012).
It has been proposed that gill remodeling can be an ancient trait induced by theevolutionary progress in the past when some of the air-breathing fishes has beenfound that are also able to transform their gill morphology to adapt to the
environmental changes (Brauner et al., 2004; Ong et al., 2007; Huang and Lin, 2011; Phuong et al., 2017, 2018) This controversy is important to explore in C ornata because this species is an ancient fish existing at least 300 million years ago (Near et
al., 2012) which will help to make an overview prediction for other fish species.
It is accounted that the atmospheric CO2 level is rising year by year consequently due
therefore, with a small increase of the CO2 level in the atmosphere, a largelysignificant increase of the dissolved CO2 level can be produced in the aqueous
0.02-0.6% (Li et al., 2013) is affecting the fish animal life In aquaculture systems,
intensive and super-intensive culture systems are practicing that lead to severehypercarbic environment of around 30mmHg toward the end of the growth cycles
(Damsgaard et al., 2015) The hypercarbic exposure normally induces different
responses of specific fish species Cardioventilatory responses derived from central
CO2/H+-sensitive chemoreceptors are still equivocal in the facultative air-breathingfish The effect of hypercarbia and hypercapnia on the cardioventilatory, and bloodgas and pH responses as well as CO2/H+-sensitive chemoreceptors location are
important to investigate.Clown knifefish (C.ornata), a tropical facultative breathing fish species (Deharai, 1962; Tuong et al., 2018b) was selected as a model
air-fish to challenge with a worst-case predicted temperature (33C), hypoxia, waterhypercarbia, internal hypercapnia to see how the respiratory metabolism, gillplasticity responses and adaptation through the growth performance,cardioventilation, and CO2/H+-sensitive chemoreceptors orientation
1.2 Research objectives
Trang 6The main objectives of this dissertation were to evaluate the impacts of climatechange (in case of theelevated temperature, hypoxia, elevated CO2)on C.ornata.
In specific, respiration of Clown knifefish at 27C and 33C in combination with
value, specific dynamic action (SDA) and growth Consequently, to understand andexplain how fish respiratory adaptation to the elevated temperature and hypoxia, gillmorphology was studied through ability of gill remodeling and estimating respiratorysurface area, water blood thickness Fish responses to the hypercarbia andhypercapnia were carried on to evaluate the cardioventilatory parameters as well asdeterminate CO2/H+ sensitive chemoreceptors
1.3 Research contents/activities
This research includes 4 main activities/studies:
1) Firstly, evaluating the effects of the elevated temperature on critical partialpressure (Pcrit), standard metabolism rate (SMR), specific dynamic action ordigestion (SDA) (using a bimodal intermittent-closed respirometry); and on fishgrowth (in recirculating aquaculture system)
2) Secondly, investigating the gill morphology through estimating the branchialsurface area, volume and water blood diffusion thickness under the effects of theelevated temperature and hypoxia applying stereological method
3) Thirdly, challenging the cardioventilatory responses of C ornata to the
hypercarbia (high ambient water PCO2) and hypercapnia (high arterial[H+]/PCO2) as well as determiningthe locations of the cardioventilatory CO2/H+
chemoreceptors in C ornata.
4) Finally, evaluating the effects of the hypercarbia and hypercapnia on the
cardioventilatory responses of denervated C ornata in processes of continuously
determining the location of CO2/H+-sensitive chemoreceptors in C ornata.
Trang 7CHAPTER 2 METHODOLOGY
Study 1: Clown knifefish (Chitala ornata) oxygen uptake and its partitioning in
present and future environments
Fish: For respirometry experiments individuals of 60-100 g were chosen, whereas
for growth experiments a starting weight of 35-45 g was chosen
Respirometry: Oxygen uptake (ṀO2, mgO2kg-1 h-1) was measured using two-phase
intermittent flow respirometry as described in Lefevre et al (2011, 2014a, 2014b,
2016)
Experimental protocols:
P crit and choice of hypoxia: In an initial experiment, we determined an appropriate
hypoxia level to stimulate reliance on air-breathing in the remaining experiments.Fish were placed in fasting tanks at either 27 or 33°C for 48 h prior to measurement
measured for 19 h from water and air phases as described above Thereafter, the airphase was removed by flooding the chamber, circulation of water through therespirometer stopped, and water oxygen level monitored until the fish lostequilibrium at which time the fish were returned to holding facilities Loss ofequilibrium occurred after 3-4 h The critical oxygen partial pressure (Pcrit) for eachfish was calculated as the intersection of the individual's SMR, calculated using the R
script from Chabot et al (2016) on the combined oxygen uptake from air and water, and the plot from the final closed period (Lefevre et al., 2011) Accordingly, the
hypoxia levels subsequently used were 4.7 kPa at 27°C and 6.0 kPa at 33°C
Temperature acclimation: In subsequent experiments, fish were acclimated to their
measurement temperature for a minimum of 30 days to eliminate the possible confounding effects of short-term temperature responses occurring during the metabolic rate measurements.
Metabolism and partitioning during digesting: Twelve fish (N=6 at each
temperature) were fasted at their appropriate temperature for 48 h and individual
ṀO2 measured as described above for 20 h in normoxic water (~150 mmHg).Thereafter, fish were gently removed from the respirometer while bacterial oxygenconsumption was measured for 1 h Towards the end of this period, the fish wasreweighed and carefully force fed with 2% of body mass using commercial pellets
The fish was then returned to the chamber and oxygen consumption measured for thenext 45 h At this time, background ṀO2 was measured again The SMR wascalculated from the first measurement period as described above and SDA (specific
the fed fish The area of the SDA was calculated using the trapezoid method as
4
Trang 8described in Lefevre et al (2012) Duration of specific dynamic action were calculated from the
point that fish returned to the chamber until its total ṀO 2 returned to SMR+2 S.E.M SDA were estimated by calculating area of total ṀO 2 and SMR curves by trapezoid method The SDA were converted to energy equivalent as energy expended and SDA coefficient (SDA energy expenditure divided by meal energy consumption) converted to kJ using the oxygenated coefficient of 13.56 kJ
-1
for carnivores (Elliott and Davison, 1975)
Growth performance: One hundred and twenty fish (41.2±0.3 g, mean±S.E.M)
were randomly divided into 4 groups After tagging with FDX-B microchips (Loligosystems, Denmark) and recording of body mass and length, were grown in arecirculating aquaculture system at 27±0.5 or 33±0.5°C in either normoxia or at a
nitrogen into the tanks under the control of oxyguard Pacific system (Oxyguard,Denmark) Fish mass and length were recorded after 1, 2 and 3 months
Statistics: Data was analyzed using Sigmaplot 12.5 Data were tested for normality
and variance homogeneity using Shapiro-Wilks test and as a result, % air uptake wasarcsine transformed A two-way ANOVA was used to test the effect of thetemperatures, oxygen levels and interaction of temperature and oxygen levels onSMR, RMR and % air uptake and within treatment effects tested using Holm-Sidakmultiple comparison procedure A student's t-test was used to compare meanparameters in digestion experiments Growth rate and specific growth rate (SGR)were tested using a one-way ANOVA with repeated measures (RM) Mean values ofSMR and RMR were compared using Student t-test Data were presented bymean±S.E.M A probability (p value) of P<0.05 was considered a significant result
Study 2: Gill remodeling of Clown knifefish (Chitala ornata) under impact of
temperature and hypoxia
Fish: 120 Clown knifefish (33.1±1.13g)
Experimental protocol: Fish were randomly chosen and stocked into four tanks (2
pellet 43% of protein (Stella S3, Nutreco Company, Ho Chi Minh city, Vietnam)twice a day until satiation After 3 days in normoxia and at 27°C, six fish gills weresampled and stored in 10% of formalin- phosphate buffer solution (10% formalinPBS) for zero-day sample Then the temperature and the oxygen level were adjusted
to create four treatments of 27°C and normoxia (95% oxygen saturation), 27°C andhypoxia (25% oxygen saturation), 33°C and normoxia (95% oxygen saturation) and
33°C and hypoxia (30% oxygen saturation) (Tuong et al., 2018) Next fish gills were
sampled after one and two months (N=6 for each treatment) Fish body mass andlength were also recorded
Gill samples processing and stereological procedure: Methods of design of da
Costa et al (2007) to process, embed and section the gill samples was applied on C.
mgO 2
Trang 9ornata gills Cavalieri estimation and vertical uniform random sections –VUR was
used to section gill samples (Baddeley et al., 1986) Fish gills were randomly chosen
to take left or right gill side There are five gill arches in each side of C ornata gill,
while the fifth gill arch is completely reduced Therefore, two gill arches (the first orthe second gill arch and the third or the forth gill arch) were chosen and removed thecartilage and hard component carefully The remaining bare gill tissues wererecorded weight and subsequently dehydrated by graded alcohol series Thereafter,methyl methacrylate Technovit 7100 (Heraeus Kulzer, Germany) was used to embedgill tissues Each block of embedding contained four gill tissues from two fish thatthe horizontal plane was parallel with lateral opercular side of gill tissues and thevertical axis was perpendicular to this horizontal plane Each gill tissue wassubsequently rotated 40° from the previous one Each block was vertically sectioned
to take eight sections of 3 µm and distance from a chosen section to the other was the
same (da Costa et al., 2007) Hematoxylin and Eosin solutions were used to stain
these sections which were dry at 55°C for 24 h The samples then analyzed usingnewCast stereological software VIS (Visiopharm Integrator System, Olympus,Denmark) Reference volumes of gill component, surface areas of lamellae and waterblood barrier thickness were first estimated
Statistics: Data was analyzed using SPSS 18.0 A two-way ANOVA was used to test
the effects of the temperatures, oxygen levels and interaction of temperature andoxygen levels on lamellar surface area, gill filament volume, harmonic mean waterblood thickness and anatomic diffusion factor Data was presented as mean±S.E.M(Standard error mean) A probability (p value) of <0.05 was considered a significantvalue
Study 3:Ventilatory responses of the Clown knifefish, Chitala ornata, to hypercarbia
and hypercapnia
Fish: Clown knifefish (708±92 g)
Fish Preparation: Fish were first anaesthetized by immersion in benzocaine (0.35
aorta was catheterized with fine bore polyethylene tubing (PE 50; I.D 0.4 mm, O.D
0.8 mm) containing heparinized saline (50 IU/ ml) (Soivio et al., 1975) The cannula
was fed out of the buccal cavity through a hole in the rostrum and secured with nylonsuture A second polyethylene cannula (PE 100) for measuring buccal pressure wasintroduced into the buccal cavity through a hole in the skin between the corner of themandible and maxilla and also secured in place Finally, impedance electrodes werefitted to both opercula and sewn in place with nylon suture
Experimental protocol: Post-surgery, fish were allowed to recover for 24 h and on thesecond day, they were moved to the experimental tank The impedance leads wereconnected to an impedance convertor and the buccal catheter was connected to apressure transducer to measure gill ventilation and air-breathing frequency (breaths
Trang 10min-1) The dorsal aorta cannula was connected to a pressure transducer to record
blood gas analysis (PCO2 and pH) using an i-STAT VetScan Analyzer (Abaxis,Union City, CA, USA) It was also used to inject the carbonic anhydrase inhibitor,acetazolamide The pressure transducers were calibrated manometrically, connected
to pre amplifiers and all outputs monitored and recorded with a data acquisitionsystem (Dataq DI-720, DATAQ Instruments, Inc., Akron, OH, USA) recording at
125 Hz per channel
Fish (N=8) were first exposed to water of pH of 7.8 (the pH of the water in the tanks
was slowly added to lower water pH to 6.0 over the course of 15-30 min This acidic
pH was then maintained for 1 h, during which both temperature and pH weremeasured continuously The fish was then returned to the holding tank and allowed torecover for 24 h before being exposed to hypercarbia
The following day the fish (N=8) were exposed to hypercarbic water following asimilar procedure to that used for acid exposure A minimum of 30 min of control
the water pH reached a pH of 6.0, the same level as in the low pH trial The CO2 wasadjusted to maintain water pH constant at this level for 1 h
A separate group of fish (N=8) were used to study the effects of increasing blood
throughout this trial Following 30 min of control were recorded, Acetazolamide
the dorsal aorta catheter and measurements were obtained for 1 h beginning 15 minafter the injection to allow time for the acetazolamide to take effect
Blood samples: Arterial blood was withdrawn for blood gas analysis at the end of
control and treatment exposures for blood pH, PCO2 by iStat
Statistics: using a two-way repeated measure analysis of variance (ANOVA)
followed by a Holm-Sidak post hoc test to check the effects of hypercarbia and acidexposures and acetazolamide injections on the cardiorespiratory variables at control,
20, 40 and 60 min during exposure We used a one-way ANOVA for repeatedmeasures followed by a Student-Newman-Keuls post hoc test to verify changes inheart rate and mean arterial pressures before and after an air-breath
Study 4: Ventilatory responses of the Clown knifefish, Chitala ornata, to ambient
water and air hypercarbia, and hypercapnia in denervated fish
Fish: Clown knifefish (400-900 g)
Fish preparation: similar to study 3.
Trang 11For air-breathing organ cannulation, fish was catheterized with another polythenecannula (PE 100) into air-breathing organ through buccal cavity, esophagus andpneumatic duct.
For hypercapnic acetazolamide injection treatments, cranial nerves IXth and Xth atboth sides were dissected
Fish was returned to the resting tank to recover for 24 h before exposing totreatments
Experimental protocols:
air-breathing organ through air-breathing catheter, holding measurement for 15 min.The air above the experimental tank was held equivalent to the injection CO2
mixed with the air)
Fish (N=5) were get intra-arterial injections of CO2/H+ (both injection pH~2.5)through catheter at vena cava where the injection led to the heart and shortly came tothe gills to determine the location of the chemoreceptor at internally orientated gillsites through air-breathing responses
Six denervation fish (761.67±42.46 g) were exposed to normocapnic water pH=7.8
bubbled into experimental tank to make hypercarbic before running hypercarbiawhile pH was continuously monitoring until the water pH reached pH level=6.0.Thereafter, pH level was adjusted to stabilize ambient water pH at this level for 30min Finally, fish was moved back to the resting tank for 24 h recovery beforesubsequent acetazolamide injection
The following day six fish recovered from hypercarbia was gently moved toexperimental tank again to inject acetazolamide to study effects of increasing blood
the dorsal aorta catheter A measurement was then taken for minimum 30 min
Blood samples: similar to study 3
Trang 12CHAPTER 3:
RESULTS
Study 1: Clown knifefish (Chitala ornata) oxygen uptake and its partitioning in
present and future environments
The effects of temperature and hypoxia on oxygen uptake and partitioning
increased significantly with temperature from 6.1±0.5 kPa at 27°C to 8.7±1.0 kPa at 33°C, indicating that as hypoxia develops, increased temperature will cause an earlier reliance on air-breathing It should be noted here that the fall in ṀO2 in this experiment does not indicate that this fish is an oxy-conformer, rather that the fish is initially disturbed by the fall in water PO2 when the circulation of water in the respirometer is turned off for the measurement This was also reflected in the subsequent measurements of metabolic rate hypoxia such that oxygen level in the water had a significant effect on partitioning at both temperatures, whereas temperature only affected partitioning in hypoxia and not in normoxia (Table 1) Temperature had the expected significant effect on both the SMR and RMR (Table 1) with temperature coefficients (Q 10 ) for SMR of 2.6 and 2.9 in normoxia and hypoxia respectively, whereas the coefficients were 2.5 and 2.3 for RMR Oxygen level had no significant effect on RMR, but hypoxia significantly reduced the SMR at 33°C from 112 to 95 mgO 2 kg-1 h-1 This effect of oxygen level on SMR was not evident at 27°C These trends are also visible in the time plots of metabolic rate in Fig 1 In normoxia at 27°C it is evident that this species is generally calm in normoxia at 27°C but that a reliable estimate
of SMR is not available until 12 h have passed (Fig 1) In 33°C normoxic water, air-breathing was clearly more important, and it is evident from an examination of Fig 1 that air-breathing was suppressed at the beginning of the measurement, but stabilized after 5 h.
9
Pcrit