MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY DOCTORAL DISSERTATION SUMMARY Major: Aquaculture Major code: 62 62 03 01 LE THI HONG GAM EFFECTS OF NITRITE, TEMPERATURE AND HYPERCAPNIA ON PHYSIOLOGICAL PROCESSES AND GROWTH IN CLOWN KNIFEFISH (Chitala ornata, Gray 1831) Can Tho, 2018 THE RESEARCH WAS PERFORMED AND COMPLETED AT CAN THO UNIVERSITY Supervisor: Prof Dr Nguyen Thanh Phuong Co-supervisor: Assoc Prof Dr Mark Bayley The dissertation will be defended at the Doctoral Dissertation Assessment Committee at the University 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 Gam, L.T.H., Jensen, F.B., Damsgaard, C., Huong, D.T.T., Phuong, N.T and Bayley, M., 2017 Extreme nitrite tolerance in the clown knifefish Chitala ornata is linked to up-regulation of methaemoglobin reductase activity Aquatic Toxicology 187: 9–17 Gam, L.T.H., Jensen, F.B., Huong, D.T.T., Phuong, N.T., and Bayley, M., 2018 The effects of elevated environmental CO2 on nitrite uptake in the air-breathing clown knifefish, Chitala ornata Aquatic Toxicology 196: 124-131 Gam, L.T.H., Vu, N.T.T., Nhu, P.N., Phuong, N.T and Huong, D.T.T., 2018 Effects of nitrite exposure on haematological parameters and growth in clown knifefish (Chitala ornata, Gray1831) Can Tho University Journal of Science 54(2): 1-8 CHAPTER INTRODUCTION 1.1 Background Climate change is defined as a change of climate that affected directly or indirectly human activity, replacing the composition of the global atmosphere, and natural climate change recorded over long-term comparable periods of time (UNFCCC, 1992) This change has been caused by the increases of toxic gases such as CO2, N20, CH4 and green house gas concentrations as well as a temperature rise of 2.5 degrees Fahrenheit (1-4 degrees Celsius) over the next century (IPCC, 2013) According to the evaluation of vulnerability, Vietnam had the 27 th rank among 132 countries over the world, which is under the impacts of climate change With topographic characteristics and natural geographical conditions, the Mekong Delta (MD) becomes one of the areas having the most impacts over the world The increases of temperature induce the rise of metabolism of organism and aquatic animals as well as decomposition of toxic compounds In the other hand, with the abundance of intensive culture system, overfeeding with waste products from excretion of aquatic animals has caused toxic gases such as: nitrite, carbon dioxide, ammonia, hydro sulfur…Especially, nitrite which is a product of nitrogen cycle, formed from ammonia in the condition of low dissolved oxygen level is well-documented toxin in aquatic system because it causes a lowering of blood oxygen with methaemoglobin formation (brown blood phenomenon), then leading a disturbance of respiration, physiological processes and growth (Kroupova et al., 2005) However, there have been a limited number of studies about effects of these environmental parameters to biological features, physiological processes in air-breathers, which may be seriously influenced by global climate change with their air-breathing activity To date only two studies exist in air-breathers in the striped catfish (Pangasionodon hypophthalmus) reported by Lefevre et al., 2011 and the snakehead (Channa striata) also reported by Lefevre et al., 2012 with typical results driven by high tolerance of nitrite in reducing nitrite uptake via gills and efficient denitrification mechanisms Besides, there have recently been several studies about effects of other environmental factors in air-breathing fish such as Damsgaard et al (2015) about effects of carbon dioxide on acid-base regulation in P hypophthalmus with high capacity of acid-base regulation compared to other airbreathing species Moreover, there is obviously not only one toxin existing in aquatic environment; the best assumption is that the combination of a variety of toxin may cause more bad effects by competition to uptake into fish blood The facultative air-breathing C ornata is an important species in aquaculture throughout South East Asia C ornata is not only of high commercial value as a source of protein for human consumption, but it is also a costly ornamental fish species in tropical aquaria Therefore, the present dissertation about “Effects of nitrite, temperature and hypercapnia on physiological processes and growth in clown knifefish (Chitala ornata, Gray 1831)” was necessarily conducted to have an understanding about effects and adaption mechanisms of this airbreathing fish under climate change 1.2 The objectives of dissertation The objectives of this dissertation were to investigate the effects of nitrite, high concentrations of carbon dioxide and elevated temperatures to physiological parameters and growth of the air-breathing C ornata during sub-lethal and chronic exposures of these factors in isolation and combination in order to provide a better physiological understanding, particularly recommendations and solutions for minimizing impacts of nitrite toxicity and its combination with other environmental elements in aquaculture ponds under global climate change 1.3 The main projects of dissertation a) Conducting a survey on some selected environmental parameters in C ornata ponds b) Determining the 96 h LC50 of nitrite and examining the effect of nitrite on haematological parameters and growth in C ornata c) Determining the activity of metHb reductase in metHb reduction in sub-lethal nitrite exposures in C ornata d) Investigating the combined effect of nitrite and hypercapnia (high concentration of carbon dioxide in the water) on haematological parameters in small-sized and large sized C ornata e) Determining the temperature tolerance and the effect of various levels of temperature on haematological parameters in small-sized and large sized C ornata f) Determining 96 h LC50 of nitrite at elevated temperatures and investigating the effects of nitrite at different temperature on haematological paramters in C ornata g) Examining the effects of nitrite at different temperatures on haematological parameters, growth and digestive enzyme activity in C ornata 1.4 The hypotheses of dissertation During nitrite exposure, C ornata reduce their branchial HCO3-/Clexchanging rate and/or increase the activity of erythrocyte NADH metHb reductase for metHb reduction and experience significant changes in exchanging rate of branchial ions for recovery b) pH regulation under a respiratory acidosis stimulate a reduction in branchial HCO3-/Cl- exchanger and thereby protect against nitrite toxicity in C ornata c) Chronic exposures of nitrite cause negative impacts to growth parameters such as low weight gain, low survival rate and high FCR in C ornata d) Elevated temperatures cause imbalance of acid-base status such as a reduction in pH and a rise of PCO2 , leading negative disturbances to blood cells, Hb and plasma ions in C ornata e) C ornata has low tolerance of nitrite in the elevation of temperature, leading to more significant effects to physiological parameters and growth compared to those in isolated exposure of nitrite or isolated elevated temperatures 1.5 Significant contributions and applicability of the dissertation The dissertation provides a better understanding about physiological knowledge of the air-breathing clown knifefish C ornata including recommendations and solutions for minimizing nitrite toxicity as well as its combination with other environmental elements in aquaculture ponds under global climate change With high tolerances of nitrite, temperature and hypercapnia in both sub-lethal and chronic levels, C ornata can properly adapt with extreme environmental changes such as temperature (24-33ºC), partial pressure of carbon dioxide (below 21 mmHg) and nitrite concentration (below 2.5 mM) contributing to the sustainable development of aquatic animals in the increases of temperature (1- 4ºC) in the next century and accumulation of toxic gases such as nitrite, carbon dioxide in intensive farming systems The results of dissertation will be reliable background for conducting deeper further studies about physiology in C ornata, other airbreathing species or comparing with physiological responses of this species to those in other aquatic animals under extreme environmental changes CHAPTER METHODOLOGY a) 2.1 Project 1: Extreme nitrite tolerance in the clown knifefish Chitala ornata is linked to up-regulation of methaemoglobin reductase activity Fish: C ornata (8-10 g; 28-31 ± 0.05 g); Chemical: NaNO2 (Merck) Determination of acute nitrite toxicity (96 h LC50): Fish (8-10g, n=576) were randomly distributed to 48 tanks (36-L water each) and 12 fish per tank The fish were fasted for days before exposure to 0, 2.6, 3.7, 4.8, 5.9; 7.0, 8.0, 9.1, 10.2, 11.3, 12.4 or 13.5 mM nitrite, with four replicate tanks for each concentration Sub-lethal exposures: Fish (n=300, body mass 31.8 ± 1.8g) were randomly taken from the 1m3 holding tank in a recirculation system with optimal water and placed in 200L experimental tanks with aerated water two days before experimentation Fish were fasted from this time until experiment termination Sub-lethal nitrite exposure concentrations were control, mM and 2.5 mM, with one tank per treatment (100 fish per tank) Extra nitrite was added during the experiment to maintain the chosen concentration Ten fish were sampled from each tank at days 0, 1, 2, 3, 4, 5, and - Blood sampling: Blood was sampled by caudal puncture The fish were placed on ice (which causes gentle initiation of a comatose state in this species) and 1mL of blood was withdrawn by from the caudal vein, using a heparinized syringe The fish were subsequently euthanized by severing the spine The blood was divided into two parts Half was used immediately for measurements of haemoglobin derivatives, haematocrit (Hct, ratio between volume of red blood cells), mean corpuscular haemoglobin concentration (MCHC, Huong and Tu, 2010) and extracellular pH (pHe), carbon dioxide tension (pCO2) and lactate, using the iSTAT analyzer (i-STAT Corporation, Princeton, USA) with CG4+ cartridges The remainder of the blood was centrifuged, and the plasma was stored at -80°C for subsequent analysis of ions and osmolality The values for pH and PCO2, HCO3- in the blood (Boutilier et al., 1985; Cameron, 1971) - Analysis procedures + Haemoglobin derivatives: The concentrations of oxyHb, deoxyHb, metHb, HbNO were calculated by spectral deconvolution, following the procedure described in Jensen (2007), Lefevre et al (2012) and Hvas et al (2016) using reference spectra prepared from C ornata blood + Plasma ions: Plasma was obtained by centrifuging blood at 6000g for to determine the osmolality and the concentrations of Na +, Cl-, NO2-, NO3- and protein Total osmolality was measured on a Fiske oneten osmometer (Fiske® Associates, Two Technology Way, Norwood, Massachusetts, USA) Plasma concentration of Na+ was measured using a flame photometer (Sherwood Model 420, Sherwood Scientific Ltd., Cambridge, UK) Plasma Cl- concentration was measured using a chloride titrator (Sherwood model 926S MK II Chloride analyzer) Plasma NO2- and NO3- was measured spectophotometrically using the Griess reaction (Miranda, 2001; Jensen, 2007; Lefevre et al., 2011) + Plasma protein and measurement of whole body water content: Plasma protein concentration was measured spectrophotometrically with Bio-rad protein assay (Bio-Rad Laboratory, Richmond, CA), using bovine serum albumin as standard (Bradford, 1976) Total body water was calculated from wet and dry weights of the fish The dry weight was determined by drying the fish at 60ºC until constant weight (for days) Methaemoglobin reductase activity: A series of fasted (2 days) fish (n=216, body mass of 28-30 g) were exposed to 0, and 2.5 mM nitrite, with each concentration replicated times (6 tanks per treatment) The fish were sampled for blood at times 0, and days In control tanks, fish were sampled at each sampling time, whereas three fish were sampled at each sampling in the nitrite tanks During the experiment, the water nitrite concentration was checked twice daily Blood (1.5 mL) was withdrawn from each fish of the exposure groups and washed four times (as above) with Ringer to obtain mL nitrite-cleaned RBC suspension This RBC suspension was equilibrated to 1% CO2 with 99% air, and the MetHb decay was followed (as described above), where after k in exposed fish was calculated Statistics: All figures were made in Sigma plot 12.5 All data were analyzed with PASW statistics (SPSS 18) Predicted mean, upper and lower 95% confidence intervals for the 96h LC50 were analyzed in JMP 9.0, using a logistic model A two- way ANOVA (the Holm-Sidak multiple comparison method, pair-wise comparison) was used to identify differences between treatments and sampling times for all parameters related to nitrite exposure (Hb derivatives, Hct, Hb, MCHC, plasma nitrite, plasma nitrate, plasma ions, plasma protein, osmolality and body water content) Normal distribution was tested using the Shapiro-Wilk test and where necessary data were log transformed to achieve normality A p value of less than 5% (p90%) for weeks before experimentation Fish were fed by commercial feed (shrimp feed with 38% protein, Tomboy Aquafeed company, Vietnam) Thirty percent of tank water was changed every second day to maintain optimal environmental condition (NO2- < µM, NO3- < 40 µM and NH3 < 40 µM) Feeding was stopped days before starting the experiment The experiment was performed in accordance with national guidelines on the protection and care of experimental in Vietnam A total of 24 fish were used - Fish cannulation: They were anaesthetized in 0.05 g L-1 benzocaine and a polyethylene PE40 catheter (Smiths Medical International Ldt., Kent, UK) was inserted into the dorsal aorta through the dorsal side of the mouth (Soivio et al., 1975), while the gills were irrigated with well-oxygenated water containing 0.025 g L-1 benzocaine Fish recovered in well-aerated water for 24 h before starting experimentation to allow post-operative normalization of blood gasses (Phuong et al., 2017a) - Blood sampling: The experimental set-up included a large 500-L tank from which water was re-circulated to smaller 120-L tanks with cannulated fish in each The water PCO2 was controlled with an Oxyguard Pacific system coupled with a G10ps CO2 probe and a K01svpld pH probe (Oxyguard International A/S, Farum, Denmark), which supplied CO2 to the water when pH changed above a value corresponding to the desired PCO2 in the water There were exposure groups: (i) normocapnia (PCO2 < 0.7 mmHg); (ii) hypercapnia (PCO2 = 21 mmHg); (iii) mM nitrite in normocapnic water and (iv) combined hypercapnia (acclimated hypercapnia) and 1mM nitrite In this combined group, the fish were cannulated then acclimated to hypercapnia (21 mmHg CO2) for 96 h before adding mM nitrite Water temperature was controlled at 27-28ºC throughout experiments and water PO2 was above 120 mmHg Nitrite was added as NaNO and tested after each sampling time During the exposures, a volume of 0.8 mL blood was withdrawn from the catherter at 0, 3, 6, 24, 48, 72 and 96 h The blood was divided into two parts Half was used immediately for measurements of haematological parameters including: Hct, pH e, PCO2, and Hb derivatives The remainder of the blood was centrifuged (6 at 6,000g), and the plasma was stored at -80°C for subsequent analysis 3; 4; 5; 6; 7; and 14 (3 fish/tank) After placing the fish on ice (which causes gentle initiation of a comatose state in this species), mL of blood was withdrawn by from the caudal vein, using a heparinized syringe The fish were subsequently euthanized by serving the spine for recognizing with other fish and maintaining the same stocking density - Analytical procedures: All haematological parameters chosen and their analyzing methods were similar as described in Project - Effects of nitrite at different temperatures on growth and digestive enzyme activities in C ornata: - Experimental method: Fish (8-10 g, n=900) were placed in 500-L experimental tanks with aerated water two days before experimentation The experiment was randomly conducted including treatments: 27ºC; 30ºC; 33ºC; mM nitrite at 27ºC; mM nitrite at 30ºC; and mM nitrite at 33ºC with tanks/treatment (50 fish per tank) After collecting growth samples at day 0, the temperatures were adjusted by heaters until reaching the chosen temperatures (2ºC per 12 h) Nitrite was subsequently added, and growth samples collected at day 30, 60, and 90 for measuring growth parameters such as DWG, SGR, SR, and FCR (thirty fish randomly sampled in each tank) Three fish per tank were collected intestine and stomach at day 90 for examining activities of digestive enzymes During experimentation, the fish were fed twice a day (the mixture of trashfish and commercial feed (Shrimp feed with 38% protein, Tomboy Aquafeed company) used in the first month, and then only commercial feed used until the end of growth experiment After 30 minutes feeding, uneaten feed was removed for counting the total number of pellets and weighed trashed fish used, then subtracting their humidity to obtain the actual weight of feed used Dead fish was removed every day, and thirty percent of the tank water was changed every third day to maintain optimal environmental conditions Nitrite concentration was checked before exchanging water for supplementing the lacked amount of nitrite - Analytical procedures: growth parameters were measured by similar methods as described in Project 1; digestive enzymes such as pepsine (Worthington, 1982), trypsine (Tseng et al., 1982), chymotrypsine (Worthington, 1982), α-Amylase (Bernfeld, 1951) - Statistics: Similar to Project However, a one-way ANOVA was used to identify differences between control treatment compared to other treatments for all growth parameters and digestive enzyme 2.7 Project 7: A survey on some environmental parameters in clown knifefish (Chitala ornata, Gray 1831) ponds 10 Sampling method: Examined environmental parameters were sampled at the C ornata ponds in Hau Giang province, Viet Nam, including six factors: temperature, pH, PCO2, PO2, [NO2-] and [NO3-] in the water at total ponds with different sizes of fish 5-10 g, 200-250 g, 500-700 g (3 ponds for each size of fish) The environmental parameters were measured and sampled at positions for each pond (2 corner positions and central position), layers for each position (surface 30 cm and bottom 1.2 m) in 24 h at specific time points: am, 12 pm, pm, pm, pm, 12 am, am, am Analytical method: Direct measurements on site such as temperature, pH, PCO2 and PO2 were showed immediately results by the devices The water samples for [NO2-] and [NO3-] measurements were transported to Can Tho University for measuring at the day after (Lefevre et al., 2011; Miranda et al., 2001) CHAPTER RESULTS AND DISCUSSION 3.1 Project 1: Extreme nitrite tolerance in the clown knifefish Chitala ornata is linked to up-regulation of methaemoglobin reductase activity 96 h LC50 for nitrite in C ornata: The 96 h LC50 for nitrite in this species was 7.82 mM (95% CI 6.79-8.85 mM), which make C ornata one of the most nitrite tolerant fish species studied (Fig 3.1.1) The reference spectra of Hb derivatives were obtained for C ornata (Fig 3.1.2A) C ornata shows very high tolerance compared to 1.65 mM NO2- in the facultative air-breathing P hypophthalmus (Lefevre et al., 2011) and 4.7mM NO2- in the obligate air-breathing C striata (Lefevre et al., 2012) This tolerance is significantly higher than the most nitriteresistant water breathers and more than twice that of the common carp (96h LC50 = 2.9 mM; Lewis and Morris, 1986) Fig 3.1.1 Mortality of C ornata (8-10 g) by a function of nitrite concentration Predicted mean, upper and lower values of LC50 96 h 11 Fig 3.1.2 (A) The reference spectra of Hb derivatives in C ornata at wavelengths from 480 to 700 nm (B) Spectrum of oxyHb from a fish exposed to mM nitrite for days and the fitted curve of oxyHb in reference spectra The rise in metHb to a maximum on day of nitrite exposure, then succeeded by a decrease in metHb during continued nitrite exposure (Fig 3.1.3), resembles the patterns seen in P hypophthalmus and C striata (Lefevre et al., 2011; 2012) This led these authors to suggest that the decline of meHb might result from up-regulation of metHb reductase, which is the enzyme responsible for reducing metHb to functional Hb Such up-regulation had also been suggested in carp recovering from nitrite exposure (Knudsen and Jensen, 1997) The present study confirms this hypothesis by showing that the rate constant for metHb reduction via erythrocyte metHb reductase is significantly elevated during nitrite exposure (Fig 3.1.4) Figure 3.1.3 Plasma NO2- (A), plasma NO3- (B), percentage metHb (C), percentage HbNO (D), functional Hb (E) and total plasma nitrite and nitrate after exposure to mM nitrite (controls, closed circles); mM nitrite (open circles) and 2.5 mM nitrite (closed triangles) in 0, 1, 2, 3, 4, 5, and days 12 Fig 3.1.4 (A) Rate constant (k, min-1) of metHb reductase in fish exposed to mM (controls, closed circles); mM (open circles) and 2.5 mM nitrite (closed triangles) in 0, and days (B) Examples of the decay of metHb in log(metHb) Examples of the decay in log(metHb) of a control fish and a fish exposed to 2.5 mM nitrite in days Acid-base status was significantly affected in the highest nitrite exposure group (Davenport diagram, Fig 3.1.5A) In this group, pHe fell from 7.73 to 7.56 at day (Fig 3.1.5C), where after it recovered by 0.1 unit Blood pCO2 rose from mmHg on day to 14 mmHg from day and onwards (Fig 3.1.5B) However, the significant respiratory acidosis during exposure was partially compensated by the associated increase in ion HCO3- This is quite contrary to the normal expectation in nitrite-exposed water-breathing animals, where a respiratory alkalosis is induced by the hyperventilation brought about by the reduced oxygen carrying capacity of the blood as methHb increases (Jensen et al., 1987; Aggergaard and Jensen, 2001; Hvas et al., 2016) Differently, the facultative C ornata has the opportunity to change the partitioning of oxygen uptake when confronted with a waterborne toxicant by increasing its reliance on air-breathing and reducing gill ventilation (Lefevre et al., 2014) Fig 3.1.5 Davenport diagram (A), blood PCO2(B), pHe (C) after exposure to mM (controls, closed circles); mM (open circles) and 2.5 mM nitrite (closed triangles) in 0, 1, and days 3.2 Project 2: Effects of nitrite exposure on haematological parameters and growth in clown knifefish (Chitala ornata, Gray 1831) In nitrite exposure, metHb significantly increased and reached the highest percentages on day (2.55±0.12, 4.30±0.32, and 29.54±0.72% at the treatments of 0.2, 0.4 and mM nitrite, respectively) (Fig 3.2.1A) MetHb formation is related to the formation of free peroxide and changes the properties of essential protein, including Hb and composition of erythrocyte membrane causing the reduction in Hb solubility, which damage erythrocyte structures and decompose them rapidly (Everse and Hsia, 1997; Bloom and Brandt, 2001) Hb and Hct 13 had decreasing trends during nitrite exposures; particularly at the highest level of nitrite exposure (Fig 3.2.1B,C) Hb concentration is converted to metHb and loses capacity with oxygen Higher concentrations of nitrite exposure cause higher concentrations of metHb generated and lower concentration of Hb (Kosaka and Tyuma, 1987; Jensen, 2009; Huong and Tu, 2010) The higher concentrations of nitrite were accompanied with the lower SR in all nitrite treatments Typically, the treatment of mM nitrite had the lowest survival rate (59%), which was significantly different from the treatments of control, 0.2 and 0.4 mM nitrite (95, 92 and 86%) (Fig 3.2.2A) FCR gradually increased from low to high nitrite levels being exposed (4.19±0.08 and 4.56±0.11 at the two highest nitrite concentrations (Fig 3.2.2B) This may be resulted from the metHb and HbNO formation, causing the low of oxygen capacity in the blood, subsequently affecting the fish growth during chronic nitrite exposures (Jensen, 2007) Fig 3.2.1 Haematological paramters in C ornata after 14 days exposed to nitrite (control, 0.2 mM, 0.4 mM, and mM) (A) MetHb), (B) Hb concentration, (C) Hct, and (D) MCHC Fig 3.2.2 Growth paramters in C ornata after 90 days exposed to nitrite: control, 0.2 mM, 0.4 mM and mM (A) SR and (B) FCR 14 3.3 Project 3: The effects of elevated environmental CO on nitrite uptake in the air-breathing clown knifefish Chitala ornata Nitrite exposure was associated with nitrite uptake in the plasma, but plasma NO2- increased significantly less during nitrite exposure in hypercapnia than in normocapnia (Fig 3.3.1A) The nitrite uptake induced a rise of blood metHb to 26% (nitrite group) and 14% (hypercapnia + nitrite) of total Hb after 48 h, whereupon metHb levels slowly decreased (Fig 3.3.1B) Despite lower maximal metHb levels during exposure to combined hypercapnia and nitrite than nitrite alone, the rate of metHb formation was highest during the initial hours of nitrite exposure in hypercapnia (Fig 3.3.1B) Plasma NO3- significantly increased, reaching 3.8 mM and 2.5 mM in 96 h in the nitrite and combined hypercapnia and nitrite groups, respectively (Fig 3.3.1E) The sum of plasma nitrite and nitrate (Fig 3.3.1F) is a good indicator of the total uptake of nitrite, as nitrate is formed by oxidation of nitrite (e.g in the reaction between nitrite and oxyHb) MetHb and HbNO levels were lower during exposure to combined hypercapnia and nitrite than during exposure to nitrite alone (Fig 3.3.1B,C), which is in line with the reduced nitrite uptake in the combined group It is notable that the initial increase in [metHb] is faster in the combined group than in the nitrite alone group (Fig 3.3.1) This is to be expected because of the lower initial pH in the combined group than in the nitrite alone group Hydrogen ions are required in the reactions between nitrite and Hb (both oxyHb and deoxyHb), and a reduced pH will therefore speed up the reaction rates (Jensen and Rohde, 2010) 15 Fig 3.3.1 Time-dependent changes in plasma NO2- (A), metHb percentage (B), HbNO percentage (C), functional Hb (D), plasma NO3- (E), and the sum of plasma nitrite and nitrate (F) during exposure to normocapnia (open circles), hypercapnia (21 mmHg CO 2, closed circles), mM nitrite (open triangles), and acclimated hypercapnia and nitrite (closed triangles) The changes in acid-base status in the different exposure groups are illustrated in a Davenport diagram (Fig 3.3.2) Hypercapnia led to an acute pH decrease along the buffer line followed by metabolic pH e compensation via HCO3- accumulation along the PCO2 ~ 21 mmHg isocline, reaching half-compensation by 96 h In the combined hypercapnia and nitrite group a further increase in bicarbonate occurred During exposure to nitrite alone there was a minor respiratory acidosis for some 24 h that subsequently became rectified by a small elevation of HCO3- (Fig 3.3.2) This study supports our hypothesis that environmental hypercapnia reduced branchial nitrite uptake via the branchial Cl-/HCO3 exchanger, since regulation of a respiratory acidosis causes a slowing of Cl- uptake via the exchanger and hence also reduces nitrite uptake Thus the response of C ornata to this combined exposure resembles that of the crayfish Astacus astacus (Jensen et al., 2000), but is different to that seen in the air-breathing teleost P hypothalamus, where nitrite uptake is only transiently decreased and subsequently increases (Hvas et al., 2016) Figure 3.3.2 Davenport diagram showing changes in acid-base status during exposure to normocapnia (open circles), hypercapnia (21 mmHg CO2, closed circles), mM nitrite (open triangles), and acclimated hypercapnia and nitrite (closed triangles) 3.4 Project 4: The combined effects of nitrite and elevated environmental CO2 on haematological parameters in small-sized clown knifefish (Chitala ornata) 16 The data showed that small-sized C ornata in general had the similar physiological responses to large-sized C ornata during combined exposures of hypercapnia and nitrite in isolation and combination via branchial chloride/bicarbonate exchanger Therefore, the results and discussion had similar trends to those in project 3.5 Project 5: Effects of different temperature on haematological parameters in clown knifefish (Chitala ornata) The upper and lower limits for temperature in C ornata were 41ºC and 12ºC, respectively This indicated that C ornata is one of the most temperature tolerant species in tropical area The physological responses described in the values of haematological parameters were similar between small-sized and large-sized C ornata in exposures of 24ºC, 27ºC, 30ºC and 33ºC It is considered that temperature is a critical environmental factor affecting on physiological processes such as food digestion, growth, metabolism, immunity, and locomotion (Zeng et al., 2009) In large-sized fish, plasma Na+ and plasma osmolality maintained unchanged in the groups of 24ºC, 27ºC, 30ºC while there were significant declines at the groups of 33ºC and 36ºC (Fig 3.5.1A,B) Stress indicators such as plasma glucose and plasma K + had significant increasing values by the elevated temperatures (Fig 3.5.1C, D) It is suggested that the levels of glucose in the blood is a stress indicator and a result of depletion in glycogen storage in the liver (Ojolick et al., 1995; Click and Engin, 2005) Fig 3.5.1 Plasma Na+ (A), plasma osmolality (B) plasma glucose (C), plasma K+ (D) in large-sized C ornata after exposed to five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC in 0, 1, 2, 3, 4, and days 17 Acid-base parameters were significantly affected by the elevation of temperature by the decrease of extracellular pH and the rise of PCO Plasma bicarbonate maintained constant in all temperatures while there was a rapid reduction in plasma Cl- in elevated temperatures (Fig 3.5.2) It is obvious that elevated temperatures cause the significant decreases in pHe (Heisler et al., 1976; Fobian et al., 2014; Damsgaard et al., 2018; Thinh et al., 2018) The increases in PCO2 in elevated temperatures may be explained by the increases of air-breathing frequency (Lefevre et al., 2016), subsequently the metabolic production of CO2 (Rahn, 1966) Fig 3.5.2 pHe (A), PCO2 (B) plasma HCO3- (C), plasma Cl- (D) in large-sized C ornata after exposed to five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC in 0, 1, 2, 3, 4, and days 3.6 Project 6: Effects of nitrite at different temperatures on haematological parameters and growth in clown knifefish Chitala ornata The values of 96 h LC50 for nitrite in C ornata were 8.12 mM at 30ºC, and 6.75 mM at 30ºC (Fig 3.6.1) while the 96 h LC50 for nitrite at 27ºC was 7.82 mM (Gam et al., 2017) 18 Fig 3.6.1 Mortality (96h LC50 for nitrite) of C ornata (8-10 g) at three different temperatures: 27ºC (A), 30ºC (B), and 33ºC (C) as a function of nitrite concentration Predicted mean, upper and lower 95% confidence intervals are presented as lines fitted to the data indicted as dots 96 h LC50 for nitrite at 27ºC is 7.82 mM (Gam et al., 2017) MetHb and plasma nitrite significantly peaked to the highest value at the highest temperature (36ºC) at day 2, but they decreased significantly at experimental termination (Fig 3.6.2A, B) This can be explained by the effective denitrification converting nitrite to nitrate and the effective activities of metHb reductase enzyme in metHb reduction (Doblander and Lackner, 1997; Jensen, 2003; Gam et al., 2017; 2018a,b) In addition, HbNO concentration increased during nitrite exposures at various temperatures in the present study (Fig 3.6.2C) The formation of HbNO is a result of the nitrite reduction to NO through the reaction between deoxygenated Hb and nitrite (Jensen, 2007) At the present study, there were significant declined in plasma ions such as Na+, Cl- and osmolality in elevated temperature(Fig 3.6.3), possibly resulting from the rise of nitrite absorption across the gills via Cl -/HCO3exchange (Evan et al., 2005; Jensen et al., 2000) A possibility is that dilution of body fluids are frequently seen during nitrite exposure (Jensen et al., 1987; Jensen, 1990a, 1996; Harris and Coley, 1991; Grosell and Jensen, 2000; Gam et al., 2017) 19 Fig 3.6.2 Plasma NO2- (A), metHb (B), HbNO (C), functional Hb (D), plasma NO3- (E), and total NO2- and NO3- (F) after exposed to nitrite at five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC in 0, 1, 2, 3, 4, and 14 days Fig 3.6.3 Plasma Na+ (A), plasma osmolality (B), plasma Cl- (C), plasma HCO3- (D) after exposed to nitrite at five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC in 0, 1, 2, 3, 4, and 14 days Davenport diagram (Fig 3.6.4) showed a significant respiratory acidosis during nitrite exposure at various levels of temperature, and pH compensated by the elevation of plasma HCO3- pHe significantly dropped with lower values in combination of nitrite and elevated temperatures compared to nitrite exposures alone (Gam et al., 2017; 2018a) Moreover, the air-breathing clown knifefish in this study can change the the portioning of oxygen uptake via air-breathing and a reduction in gill ventilation for avoiding environmental toxicants 20 Fig 3.6.4 Davenport diagram presenting the changes in acid-base status (A), blood PCO2 (B), and pHe (C) after exposed to nitrite at five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC in 0, 1, 2, 3, 4, and 14 days There were significant changes on growth parameters when C ornata were exposed to nitrite at various temperatures The results showed that SR decreased in nitrite exposure at elevated temperature (Fig 3.6.5), particularly the treatment of mM nitrite at 30 and 33ºC with significant difference compared to that at 27ºC In this study, mortality appeared in the initial culturing stage, subsequently decreasing during experiment because fish need a period of time to adapt, adjust for maintaining intercellular activities (Gerlach et al., 1990) Fig 3.6.5 Survival rate (SR) and feed conversion ratio (FCR) after 90 days exposed to nitrite at 27ºC (control); 30ºC; 33ºC; mM nitrite at 27ºC; mM nitrite at 30ºC; mM nitrite at 33ºC Different letters (a, b, c) in the same column show significant difference from control treatment Showed data are mean±SEM (n=30) 3.7 Project 7: A survey on some environmental parameters in clown knifefish (Chitala ornata, Gray 1831) ponds There were significant fluctuations in environmental parameters between daytime and nighttime (Fig 3.7 below) IPCC, 2013 predicts a temperature rise of 1- 4ºC over the next century Particularly, temperatures in C ornata ponds had the highest value at 12-3 pm (32.5ºC), but it slightly decreased at night time (30-31ºC at 12 – am) According to Boyd (1990), temperature in the ponds of tropical fish fluctuates in the range of 20-35ºC and optimal range of 28-32ºC Therefore, temperatures in C ornata ponds were considered to be safe for their physiological and growth processes Hypercapnic conditions appeared in the early morning with PCO2 of 14 mmHg and pH of 6.9 (450-500g) while there were fluctuations of PCO2 and pH around mmHg and 7.5 at am (5-10 g) The CO2 levels were higher in the ponds of large-sized fish compared to the ponds of small-sized fish Contrast to CO2 concentration, oxygen level reached saturated value with PO2 of 140 mmHg at pm while there was almost no oxygen at 36 am with pond depth of 1.5 m C ornata can normally grow in anoxic condition in the early morning thanks to their air-breathing assessory 21 organs used for taking oxygen directly from the air at hypoxic situations (Long, 2003) Reviewed by Diaz and Breitburg (2009), oxygen is commonly limited to the top m of the water column during daytime, whereas the water is completely anoxic at night in the conditions such as high pond temperatures (28-32ºC), high organic loading, low water exchange, and especially the absence of aeration According to Damsgaard et al., 2015, P hypophthalmus pond was completely anoxic at the water column of m with PCO2 of 28 mmHg in this depth The accumulations of nitrite and nitrate levels in the ponds of large sized fish were higher than those in the ponds of small-sized fish Particularly, the concentrations of nitrite and nitrate in the C ornata ponds were 0.0055-0.0075 mM and 0.058-0.068 mM (450-500 g/fish), whereas they were only 0.002 - 0.0035 mM (200-250 g/fish) and 0.025-0.035 mM (510 g/fish), respectively Nitrite and nitrate levels reached the highest values at pm in ponds of different fish sizes Boyd (1990) documented that safe concentrations of nitrogen products: nitrite below 0.3 mg/L (0.0065 mM in this study) and nitrate 0.2 – 10 mg/L (0.003-0.156 mM in this study) Therefore, levels of nitrite and nitrate in C ornata ponds changed in the optimal ranges 22 Figure 3.7 Temperature (A), pH (B), PCO2 (C), PO2 (D), [NO2-] (E), [NO3-](F) in the water at the C ornata ponds CHAPTER CONCLUSIONS AND RECOMMENDATIONS 4.1 Conclusions This thesis presents an incredible tolerance to nitrite in C ornata with maintenance of metHb levels at subcritical levels with denitrification process converting nitrite to nitrate and nitrite concentration in the plasma maintain lower than the ambient nitrite level during sub-lethal time Although there were the related changes in plasma osmolality, plasma ions, protein and whole body content in nitrite exposure, this is the first time for showing up-regulation of erythrocyte metHb reductase with rate constant for metHb reduction increased from 0.01 in controls to 0.046 min-1 after days of 2.5 mM nitrite exposure Growth rate of C ornata in chronic nitrite exposure of mM (50%*96 h LC50) was significantly lower than that in control, 0.2 mM and 0.4 mM nitrite with low survival rate (59%) and high FCR (4.5) after 90 days of culture There were negative effects to the number of blood cells; metHb, Hct and Hb concentration while MCHC had no effect by these nitrite exposures after 14 days This is the second time about acid-base regulation found in the airbreathing species C ornata in the MD Although pH regulation capacity in C ornata (50% of pH regulation after 96 h at 21 mmHg CO 2) was lower than that in the facultative air-breathing P hypophthalmus (100% of pH compsentation in 72 h in 34 mmHg CO2), this capacity of pH regulation was significantly better than that in other water-breathing species In addition, acclimated hypercapnia caused a decreased nitrite uptake by an apparent reduced transport rate of the branchial HCO 3-/Clexchanger C ornata had high tolerance of temperature with limitation of 50 % mortality (12ºC and 41ºC for lower and upper limit, respectively) due to their living habitat in the tropical area MD Therefore, there were no significant impacts in the changes of haematological parameters, but pHe and PCO2 significantly fluctuated in the temperature ranges from 24ºC - 33ºC in both juvenile and commercial fish size while the mortality after days exposed to 36ºC in commercial size induced by sharp decrease in pHe and Hb concentration C ornata had higher tolerance of nitrite at 30ºC compared to this at 27ºC and 33ºC In addition, the number of red blood cells was 23 significantly influenced by combination of nitrite and elevated temperature while metHb and plasma NO2- only affected in the initial stage of exposure In general, growth parameters such as GW, SR had no negative effects by temperature and the combined exposure of nitrite and temperatures, but FCR reached the highest value of 1.95 in mM nitrite at 33ºC compared to that in 27ºC 4.2 Recommendations For intensive farming systems: 1) Reducing stocking density and changing water regularly for minimizing the accumulation of all waste products and generation of toxic gases 2) Designing ponds with optimal sizes, outlet and inlet cannas of water for conveniently for regularly removing the waste products and accumulated toxic sediments regularly for good water quality 3) Supporting aeration systems in situations of elevated temperatures and high carbon dioxide levels for supplying sufficient dissolved oxygen in farming systems For further studies: 1) Chronic effects of nitrite on growth parameters in C ornata after exposed to extremely high nitrite concentration in a short duration 2) Combined effects of nitrite and hypoxia on haematological parameters, metabolic rate and growth in C ornata 3) Effects of hypercapnia at different temperatures on acid-base regulation and growth in C ornata 24 ... present dissertation about “Effects of nitrite, temperature and hypercapnia on physiological processes and growth in clown knifefish (Chitala ornata, Gray 1831) was necessarily conducted to have... Effects of nitrite exposure on haematological parameters and growth in clown knifefish (Chitala ornata, Gray1831) Can Tho University Journal of Science 54(2): 1-8 CHAPTER INTRODUCTION 1.1 Background... each The water PCO2 was controlled with an Oxyguard Pacific system coupled with a G10ps CO2 probe and a K01svpld pH probe (Oxyguard International A/S, Farum, Denmark), which supplied CO2 to the water