Ảnh hưởng của nhiệt độ, điều kiện oxy thấp và CO2 cao lên hô hấp và sinh lý của cá thát lát còm chitala ornata (gray, 1831) (tt)

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY DOCTORAL DISSERTATION SUMMARY MAJOR: AQUACULTURE MAJOR CODE: 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 THE 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 THE LIST OF PUBLISHED PAPERS 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 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 CHAPTER INTRODUCTION 1.1 Background Climate change is dramatically challenging and threatening aquatic animal life through rising water temperature and elevated CO2 levels It has been predicted by international panel on climate change (IPCC, 2014) for Mekong River delta that the temperature will increase 2.5-3.5C in next 100 years; and concurrently, the elevated atmospheric 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 pervasively determining 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 considered intriguing targets to research under effects of projected elevated temperature and hypercarbia in water A hypothesis of oxygen capacity limited thermal tolerance is proposed to express negative impacts of the elevated temperature on the fish that is underlying the oxygen delivery mechanism to tissues (Portner, 2001; Portner and Farrell, 2008; Munday et al., 2008; Munday et al., 2009; Nilsson et al., 2009; Portner, 2010; Neuheimer et al., 2011) It is due to the dissolved oxygen level decreasing with progressive increases of the temperature whilst fish oxygen demand significantly increases with the elevated temperature Therefore, the elevated temperature integrating with hypoxia (the decrease of the dissolved oxygen level) has been indicated that could result in largely 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 near their upper thermal limits and can be more vulnerable with a small increase of the temperature (Nelson et al., 2016; Tewksbury et al., 2008) However, there is growing evidence of studies that not conform that hypothesis (Clark et al., 2013; Norin et al., 2014; Wang et al., 2014; Lefevre, 2016) Indeed, it is argued that the air-breathing fish species which hypoxic water tolerance could be a result of an evolution under the effects of higher temperatures and lower atmospheric oxygen pressure than the present Investigating the effects of the elevated temperature and hypoxia on the metabolism of the air-breathing fish is important to assess the effects of climate change Another aspect of the adaptive ability to environmental factors, it has been exposed that one of adaptive mechanisms is ability of changing gill morphology (Tuurala et al., 1998, Sollid et al., 2003; Sollid et al., 2005; Sollid and Nilsson, 2006; Ong et al., 2007; Matey et al., 2008; Mitrovic and Perry, 2009) Intensive researches on this ability have been found in the water-breathing fishes including crucian carp, goldfish and salmonids which their gills have been found to increase or decrease interlamellar cell mass (ILCM) to increase or decrease respiratory surface area with changes of the environmental factors This ability of fish is intriguing scientists that whether the gill remodeling is a modern trend or an ancient trait (Nilsson, 2007; Nilsson et al., 2012) It has been proposed that gill remodeling can be an ancient trait induced by the evolutionary progress in the past when some of the air-breathing fishes has been found that are also able to transform their gill morphology to adapt to the environmental changes (Brauner et al., 2004; Ong et al., 2007; Huang and Lin, 2011; Phuong et al., 2017, 2018) This controversy is important to explore in C ornata because this species is an ancient fish existing at least 300 million years ago (Near et al., 2012) which will help to make an overview prediction for other fish species It is accounted that the atmospheric CO2 level is rising year by year consequently due to the global warming The dissolved CO2 level is more dissoluble than the oxygen therefore, with a small increase of the CO2 level in the atmosphere, a largely significant increase of the dissolved CO2 level can be produced in the aqueous system It has been reported that PCO2 level in Mekong River Delta ranging from 0.02-0.6% (Li et al., 2013) is affecting the fish animal life In aquaculture systems, intensive and super-intensive culture systems are practicing that lead to severe hypercarbic 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-breathing fish The effect of hypercarbia and hypercapnia on the cardioventilatory, and blood gas and pH responses as well as CO2/H+-sensitive chemoreceptors location are important to investigate.Clown knifefish (C.ornata), a tropical facultative airbreathing fish species (Deharai, 1962; Tuong et al., 2018b) was selected as a model fish to challenge with a worst-case predicted temperature (33C), hypoxia, water hypercarbia, internal hypercapnia to see how the respiratory metabolism, gill plasticity responses and adaptation through the growth performance, cardioventilation, and CO2/H+-sensitive chemoreceptors orientation 1.2 Research objectives The main objectives of this dissertation were to evaluate the impacts of climate change (in case of theelevated temperature, hypoxia, elevated CO2)on C.ornata In specific, respiration of Clown knifefish at 27C and 33C in combination with normoxia and hypoxia evaluating through SMR, percentage of air-breathing, Q10 value, specific dynamic action (SDA) and growth Consequently, to understand and explain how fish respiratory adaptation to the elevated temperature and hypoxia, gill morphology was studied through ability of gill remodeling and estimating respiratory surface area, water blood thickness Fish responses to the hypercarbia and hypercapnia were carried on to evaluate the cardioventilatory parameters as well as determinate CO2/H+ sensitive chemoreceptors 1.3 Research contents/activities This research includes main activities/studies: 1) Firstly, evaluating the effects of the elevated temperature on critical partial pressure (Pcrit), standard metabolism rate (SMR), specific dynamic action or digestion (SDA) (using a bimodal intermittent-closed respirometry); and on fish growth (in recirculating aquaculture system) 2) Secondly, investigating the gill morphology through estimating the branchial surface area, volume and water blood diffusion thickness under the effects of the elevated temperature and hypoxia applying stereological method 3) 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 CHAPTER 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: Pcrit 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 Eight fish from each temperature regime were placed in respirometers and ṀO2 measured for 19 h from water and air phases as described above Thereafter, the air phase was removed by flooding the chamber, circulation of water through the respirometer stopped, and water oxygen level monitored until the fish lost equilibrium at which time the fish were returned to holding facilities Loss of equilibrium occurred after 3-4 h The critical oxygen partial pressure (Pcrit) for each fish 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 oxygen consumption was measured for h Towards the end of this period, the fish was reweighed and carefully force fed with 2% of body mass using commercial pellets (43% protein, 13.39 kJ kg-1; Stella S3, Nutreco Company, Ho Chi Minh, Vietnam) The fish was then returned to the chamber and oxygen consumption measured for the next 45 h At this time, background ṀO2 was measured again The SMR was calculated from the first measurement period as described above and SDA (specific dynamic action) calculated by subtracting this SMR from this postprandial ṀO2 in the fed fish The area of the SDA was calculated using the trapezoid method as described in Lefevre et al (2012) Duration of specific dynamic action were calculated from the point that fish returned to the chamber until its total ṀO2 returned to SMR+2 S.E.M SDA were estimated by calculating area of total ṀO2 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 mgO2-1 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 groups After tagging with FDX-B microchips (Loligo systems, Denmark) and recording of body mass and length, were grown in a recirculating aquaculture system at 27±0.5 or 33±0.5°C in either normoxia or at a hypoxia (5 kPa) in 2000 L tanks Hypoxia was regulated the desired PO2 by bubbling nitrogen into the tanks under the control of oxyguard Pacific system (Oxyguard, Denmark) Fish mass and length were recorded after 1, and 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 was arcsine transformed A two-way ANOVA was used to test the effect of the temperatures, oxygen levels and interaction of temperature and oxygen levels on SMR, RMR and % air uptake and within treatment effects tested using Holm-Sidak multiple comparison procedure A student's t-test was used to compare mean parameters in digestion experiments Growth rate and specific growth rate (SGR) were tested using a one-way ANOVA with repeated measures (RM) Mean values of SMR and RMR were compared using Student t-test Data were presented by mean±S.E.M A probability (p value) of P