ORIGINAL ARTICLE/ORİJİNAL ÇALIŞMA FULL PAPER TAM MAKALE

BEHAVIORAL AND PHYSIOLOGICAL STRESS RESPONSES OF Java ( gonionotus) TO ENVIRONMENTAL SALINITY CHALLENGE

Fahima Binte AMIN, Tania FARHANA, Golam Mohammod MOSTAKIM, Md. Mahiuddin ZAHANGIR, Mahbuba Monir MISHU, M. Sadiqul ISLAM Department of Fisheries Biology & Genetics Bangladesh Agricultural University Mymensigh-2202, Bangladesh

Corresponding author: Received: 02.07.2015 M. Sadiqul ISLAM, Department of Fisheries Biology & Accepted: 15.12.2015 Genetics Bangladesh Agricultural University Mymensigh- Published online: 22.06.2016 2202, Bangladesh E-mail: [email protected] Abstract:

Salinity impose stress on the physiology of the ex-

posed freshwater fish population can modify their

structure. In this study some indicators of behavioral

and physiological stress responses were examined dur- ing exposure of (Barbonymus gonionotus) to different saline concentrations. Based on the result of

median lethal salinity level (LC50) (12.9ppt), one sub- lethal (12ppt) and two lethal salinity levels (14ppt and 16ppt) were selected to study the acute stress respons-

es of fish for a period of 72h. A number of physiologi- cal responses, such as disturbance in body fluid, alter- ations of blood biochemical and hematological param-

eters and behavioral responses were detected with in- creasing ambient salinity. Fish exposed to different sub-lethal and lethal salinities exhibited clinical signs

agitated behavior, respiratory distress, abnormal nerv- ous behavior and death were recorded. From the pre- sent investigation, it was observed that higher salinity may affect the physiological and behavioral stress re- sponses in freshwater fish. Keywords: Behavior, Freshwater fish, Physiology, Salinity, Stress

JOURNAL OF AQUACULTURE ENGINEERING AND FISHERIES RESEARCH E-ISSN 2149-0236 2(4): 176-184 (2016) doi: 10.3153/JAEFR16019 176 © 2015-2016 ScientificWebJournals (SWJ) Journal of Aquaculture Engineering and Fisheries Research Amin et al., 2(4): 176-184 (2016)

Journal abbreviation: J Aquacult Eng Fish Res

Introduction Environmental changes in organisms are always Mymensingh) and reared in the cemented rectan- associated with a number of physiological re- gular tank and allowed to acclimatize to the la- sponses. Theoretically these responses can be boratory conditions for 15 days to remove the measured in fish and in other vertebrates in the suspected unhealthy subjects (handling and form of changes in hormonal or substrate concen- transportation stress) at 24-25°C. A total of 280 tration in the plasma or alterations in the blood individuals were randomly assigned. Fishes were parameters (Donaldson, 1981). Salinity is an im- fed twice a day with zooplankton (enriched with portant factor in the environmental condition of Daphnia, Keratella, etc.) and commercial dry fish by influencing osmotic pressure and metabo- pellets (Krishibid Fish Feed Ltd.). The fishes lism (Mustafayev and Mekhtiev, 2008); causing were considered well adapted to laboratory con- changes physiological functions, habits, and sur- ditions when mortality was recorded less than 1% vival of fish (Wang and Zhu, 2002). during acclimatization period of 15 days. Fish of both sexes were used without discrimination. Fish which inhabits only the freshwater environments may be extinct in the coastal areas The different levels of salinity concentrations due to saltwater intrusion and increase of the sa- used were achieved by serial dilution of brine linity. As compared to terrestrial inhabitants, fish water (150 ppt) with dechlorinated tap water, to and other aquatic creatures are subject to a get the desired concentrations. Pre-acclimatized broader variety of stressors because their homeo- fish were directly exposed to the different salinity static mechanisms are highly dependent on pre- levels (0, 2, 4, 6, 8, 10, 11, 12, 13, 14, 15 and vailing conditions in their immediate surround- 16ppt) for range finding test and determination of ings. Examples of additional stressors for aquatic LC10-100 by static bioassay. The OECD Directive other than terrestrial animals include No. 203 (1992) ‘Fish, acute toxicity test’ was fol- fluctuations in water salinity, pH, hardness, alka- lowed for the toxicity assay. Aeration was ap- 3 linity, dissolved solids, water level or current, plied to the aquaria (45×30×30 cm ) in order to and exposure to waterborne pathogens or toxi- obtain a homogeneous concentration of salt and cants. Being an aquatic the fish have to to maintain standard DO concentration. Several face various stressors and have to cope with regu- inspections were made during the experimental lar and unexpected fluctuation of different water period and dead fish if any, were removed imme- parameters to survive, and adapt physiologically diately. Fish were considered as dead when res- with alteration and modification of different or- piratory movement of the opercula stopped and gans and its behavior (Jasmin, 2013). there was no response to touch. The salinity con- centrations below the median lethal concentration Java barb (Barbonymus gonionotus) a stenohaline (LC50) value and above the LC50 values were con- freshwater fish species which was native to Asia sidered as sub-lethal and lethal salinity concen- but now spread worldwide and colonized in vari- trations, respectively. ous environments was used in the experimental stages of the study. It can tolerate a range of dif- To observe the behavioral stress responses, fish ferent salinities from freshwater up to 13 ppt were exposed to tap water (dechlorinated) (0ppt) (BFRI, 1993), but the behavioral and physiologi- and considered as control, one sub-lethal concen- cal mechanisms that underlie the salinity toler- tration (12ppt) and two lethal concentrations ance of freshwater species are not well under- (14ppt and 16ppt) of salinity for 72h. The abnor- stood. The present study examines the possible mal stress behaviors were observed by visual as- effects that osmoregulatory processes can have sessment as suggested by Aysel and Ayhan on the behavioral and physiological responses in (2010). Behavioral responses of fish such as con- Java barb that are subjected to increasing levels vulsions, equilibrium status or imbalance, fin of environmental salinity. movement, hyperactivity and swimming rate were observed. Some other behavioral criteria Materials and Methods were also observed as stress responses such as Healthy and moderate sized (8.02 ±1.44cm and shoaling, body and eye color etc. Behavioral 5.51 ±1.16g) Java barb (B. gonionotus) were col- stress responses were observed at different time lected from the local fish farm (Digarkanda, intervals (2, 4, 6, 12, 24, 48, 72h) and scored as 0

177 Journal of Aquaculture Engineering and Fisheries Research Amin et al., 2(4): 176-184 (2016)

Journal abbreviation: J Aquacult Eng Fish Res

to 5 (very weak, weak, moderate, strong, and almost 100% mortality was observed at 16ppt in very strong) according to degree of responses. 72h exposure period. Based on range finding test the LC50 of salinity for Java barb was determined To obtain blood samples, fish were caught gently between 10ppt and 16ppt. At salinity concentra- in a small scoop net and then quickly taken out tion of 11ppt 20% mortality and at 13ppt 50% from the water and fish were anaesthetized by mortality was found, 100% mortality was oc- 5mg/l of clove oil to avoid any handling stress in curred at 16ppt. Probit analysis showed that the order to minimize an error in normal blood val- LC50 of the fishes was about 12.90ppt. ues. Blood was obtained from the renal artery by cutting off the tail peduncle. Blood glucose was Lethal and sub-lethal effects of salinity on measured by using a digital blood glucose kit. A behavioral stress responses drop of blood sample was placed on the strips The fish exhibited a normal behavioral activity connected to the GLUCOLABTM Auto-coding without mortality when exposed to 0ppt. At sub- blood glucose test meter and results were record- lethal concentration of salinity (12ppt) a low ed. The values were expressed in mmol/l. Hema- stress responses were noticed like aggression, tological analyses were carried out by standard stunned posture, erratic swimming etc. Stress re- methods suggested by Blaxhall and Daisley sponses like impatience or agitated activeness or (1973). To examine red blood cell (RBC) and erratic movement observed at lethal concentra- white blood cell (WBC) counts, collected blood tions (14ppt and 16ppt) (Table 1, Figure 1). At was gently pushed into sterilized eppendorf tubes the beginning of experiment (up to 3 hours) the containing anticoagulant (EDTA) to give a final fish showed frequent movement from surface to concentration of 5mg EDTA per cm3 blood. bottom, then aggression and sometime showed Blood samples were mixed gently and counts jumping activity at the lethal concentrations of were made using a Neubauer hemocytometer. salinity. They also expressed highly increased Dehydration of fish body was measured to ob- opercular movements accompanied by excessive serve the effect of the sub-lethal and lethal con- secretion of mucus which indicates the respirato- centrations of salinity. Weights were recorded ry distress. Fish were scattered here and there. from the beginning to end of the experiment at Then the fish became very weak with a fade and different time intervals (0, 1, 3, 6, 12, 24, 48, gloomy body and eye color, settled at the bottom 72h). and finally died.

Median lethal concentration (LC50) values were Effects of salinity on biochemical and calculated from the data obtained in acute toxici- hematological parameters ty bioassays, by Finney’s (1971) method of “pro- Variations on the biochemical parameter such as bit analysis”. The data were subjected to an anal- blood glucose levels at both treatments (6.5ppt, ysis of variance (ANOVA), followed by compar- 50% of LC50 and 9.68ppt, 75% of LC50) were ison of means using Tukey's HSD test to deter- found to be increased within an hour and highest mine significance of each data treatment. In addi- about from 6-12h. Then glucose level gradually tion, the significance of differences of the values decreased and got back to its original states with- of RBC and WBC were determined by using “t in 24h (Figure 2). A significant (P<0.05) de- test”. Significant differences were indicated by P- crease of RBC values were registered up to 24h values <0.05. All statistical analyses were per- in fish that were exposed to sub-lethal salinities formed using SPSS16.0. (6.5ppt and 9.68ppt) compared to control (0ppt) Results and Discussion group (Figure 3). On the other hand, the pattern was different for WBCs: values showed a signifi- Median concentration (LC50) cant increase up to 24h in a salinity of 9.68ppt A study was carried out to find the range of salin- (75% of LC50), but subsequently returned to nor- ity tolerance of Java barb from 0ppt to 16ppt. mal values within 72h as in the control group There was no mortality observed up to 10ppt but (Figure 4).

178 Journal of Aquaculture Engineering and Fisheries Research Amin et al., 2(4): 176-184 (2016)

Journal abbreviation: J Aquacult Eng Fish Res

Table 1. Behavioral and clinical signs of Java barb after exposure 72h to different salinities

Behavioral signs Clinical signs Salinity (ppt) 0 12 14 16 Aggression 0 1 1 5 Agitated behavior Jumping 0 0 3 5 SP 0 2 3 3 FSBM 0 0 2 4 ES 0 1 3 3 OM 0 0 2 4 Respiratory distress AG 0 0 4 5 VPES 0 0 3 5 EMS 0 0 3 5 SSM 0 1 2 4 Abnormal nervous behavior SM 0 2 4 4 SD 0 0 3 5 DP 0 0 4 3 Death 0 0 3 5 Air gulping (AG), erratic swimming (ES), frequent surface to bottom movements (FSBM), vertical posture with exposed snouts (VPES), excessive mucus secretion (EMS), sluggish and swirling movements (SSM), state of motionless (SM), sudden darts (SD), different postures (DP), opercula movement (OM), stunned posture (SP); none (0), very weak (1), weak (2), moderate (3), strong (4), very strong (5)

Lethal and sub-lethal effects of salinity on Behavioral endpoints serve as valuable tools to osmotic challenge distinguish and evaluate effects of exposure to environmental stressors, and fish behavioral al- In this study lethal and sub-lethal effect of salini- terations can provide important indices for eco- ty on the osmotic change was determined by ob- system assessment (Kane et al., 2005). Through- serving variations of body weight of exposed fish out the study period fish exhibited low behavioral to different salinity concentrations (Figure 5). At stress responses when exposed to sub-lethal con- 12ppt, a gradual and slow decrease of weight was centration of salinity (12ppt). Impatience or agi- noticed up to 12h of exposure and within 24h tated activeness or erratic movements were ob- body weight got back to its original state. On the served when exposed to lethal concentrations other hand, at lethal concentrations of a sudden (14ppt and 16ppt). From the beginning up to 3 decrease of average weight (body fluid reduced hours of the lethal salinity exposure fish showed up to 19%) of fish were recorded at the beginning frequent movement from surface to bottom, ag- hours of exposure. Then the weight decreased gression and sometime showed jumping activity. gradually and got back to its original state (Fig- In addition, fish expressed highly increased oper- ure 5a, b). cular movements accompanied by excessive se- The stress response of a freshwater Java barb to cretion of mucus which indicating respiratory different salinity concentrations was evaluated distress of fish. Lawson and Anetekhai (2011) using the levels of biochemical and hematologi- and Aysel and Ayhan (2010) also noted similar cal parameters of blood and behavior as indica- behavioral stress responses after exposing fish in tors. Aquaculture urges for more accurate infor- higher salinities. mation on stress control, in order to be assured of The osmotic gradient between the plasma and the good health status of fish, especially those trans- environment is reduced in freshwater fishes when ferred to a new environment. Environmental sa- exposed to increase salinity. Kilambi and Zdinak linity fluctuation can act a stressor and leads to (1980) stated decreasing survival rates with in- give information on behavioral and physiological creasing salinity is characteristics of freshwater responses. stenohaline fish, and it has been postulated that this is due to increasing osmotic pressures at

179 Journal of Aquaculture Engineering and Fisheries Research Amin et al., 2(4): 176-184 (2016)

Journal abbreviation: J Aquacult Eng Fish Res

higher salinities. In the present study a gradual ter fish in saltwater is the absence of efficient and slow weight loss was noticed within 1-3 h of branchial and renal mechanisms that can readily sub-lethal salinity exposure whereas at lethal adjust to hypertonic media. This inability to ad- concentrations a rapid decrease of weight was just to increase salinity results in increased elec- observed and then the weight increased gradual- trolyte concentrations and osmotic stress ly. Similar trend also reported by Augley et al. (Furspan et al., 1984) which causes an increase in (2008) and Maceina and Shireman (1979). Nor- osmotic pressure and deterioration of cell func- ton and Davis (1977) suggested that the major tion. limiting factor affecting the survival of freshwa-

Figure 1. Degree of behavioral stress responses of Java barb at different salinity levels (AB= agitated behavior, RD= respiratory distress and ANB= abnormal nervous behavior

Figure 2. Sub-lethal effects of salinity on blood glucose levels at different time intervals in Java barb. Values are means ± SD. Asterisk (*) indicates significant difference at P<0.05. N = 5 fish at each salinity

180 Journal of Aquaculture Engineering and Fisheries Research Amin et al., 2(4): 176-184 (2016)

Journal abbreviation: J Aquacult Eng Fish Res

Figure 3. Effects of sub-lethal exposure of salinity on RBC levels at different time intervals in Java barb. Values are means ± SD. Asterisk (*) indicates significantly different (one-way analy- sis of variance, P<0.05, N =5 fish at each salinity)

Figure 4. Effects of sub-lethal exposure of salinity on WBC levels at different time intervals in Java barb. Values are means ± SD. Asterisk (*) indicates significantly different (one-way analy- sis of variance, P<0.05, N =5 fish at each salinity)

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Journal abbreviation: J Aquacult Eng Fish Res

(a)

(b)

Figure 5. a) Hydration levels and b) percentage of reduced body fluid of fishes during exposure to sub-lethal and lethal concentrations of salinity for a period of 72h. Values are means ± SD. N = 5 fish at each salinity.

The significant decrease in RBC counts at sub- certain RBC cells as observed at sub-lethal treat- lethal concentration might be due to haemolysis ments, the reduced blood oxygen carrying capaci- and shrinkage of blood cells by the effects of sa- ty can be compensated either through increasing linity. The reduction in the RBC counts of Java oxygen affinity and capacity of hemoglobin barb following exposure to salinity indicated a and/or increase in RBC production (Das et al., reduced blood oxygen carrying capacity (Das et 2006). al., 2006). In the context of distortion and lysis of

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An increased WBC counts established leukocyto- Augley, J., Mark, H., Teresa, F.F. & Alastair, sis, which is considered to be an adaptive value R.L. (2008). The effect of salinity on growth for the tissue under environmental stress. The and weight loss of juvenile plaice (Pleu- increase in WBC count at sub-lethal concentrati- ronectes platessa, L): An experimental test. ons can be correlated with an increase in anti- Journal of Sea Research, 60(4), 292-296. body production which helps in survival and re- Aysel, C.K.B. & Ayhan, O. (2010). Acute toxici- covery of the fish exposed to toxicants (Joshi et ty and histopathological effects of sub-lethal al., 2002). This also helps in the removal of cel- fenitrothion on Nile , Oreochromis ni- lular debris of necrosed tissue at a faster rate loticus. Journal of Pesticide Biochemistry (John, 2007). As a protective response of the and Physiology, 97, 32-35. body during stress, WBC increases through sti- mulation of leukopoietic process and enhanced Bani, A. & Vayghan, A.H. (2011). Temporal var- release of leukocytes to the blood circulation. iation in haematological and biochemical in- The released catecholamines, adrenaline and nor- dices of the Caspian kutum, Rutilus frisii ku- adrenaline, increase the conversion of liver gly- tum. Ichthyological Research, 58, 126-133. cogen to blood glucose to satisfy the greater Begg, K. & Pankhurst, N.W. (2004). Endocrine energy demands of the body to stress (Begg and and metabolic responses to stress in a la- Pankhurst, 2004). The variation of the WBC and boratory population of the tropical damsel- increased blood glucose levels in the present fish Acanthochromis polyacanthus Journal study indicated elevated stress levels in the Java of Fish Biology, 64, 133-145. barb which were most likely due to the distur- bance in the acid-base balance, respiratory home- BFRI (1993). Research Progress Report 1986- ostasis and ionic regulation during exposure to 1993 edited by M. A. Mazid, M. J. Alam, M. sub-lethal salinity. Consistent supports to the E Hoq and R. Begum. 136 pp above with several results which showed a signi- Blaxhall, P.C. & Daisley, K.W. (1973). Routine ficant increase in the WBC (Akinrotimi et al., haematological methods for use with fish 2012; Far et al., 2012; Geetha, 2014). blood. Journal of Fish Biology, 5, 771-781. Conclusion Das, P.C., Ayyappan, S. & Jena, J. (2006). Hae- The present study which aimed to mimic of fluc- matological changes in the three Indian ma- tuating salinities, has shown that short-term ex- jor carps, Catla catla (Hamilton), Labeo ro- posure of fish to salinities as high as 14ppt led to hita (Hamilton) and Cirrhinus mrigala extreme physiological disturbances as revealed in (Hamilton) exposed to acidic and alkaline changes observed in their behavioral and physio- water pH. Aquaculture, 235(1-4), 633-644. logical characteristics. From the overall study the Donaldson, E.M. (1981). The pituitary–interrenal perceptible concern might be that the Java barb axis as an indicator of stress in fish. In Stress can tolerate a definite range of salinity level and Fish (ed. AD Pickering), pp. 11-41. without being affected significantly, but extreme New York, London: Academic Press changes in salinity may cause some effects on physiology and behavior of the fish. Far, M.S., Roodsari, H.V., Zamini, A., Mirra- sooli, E. & Kazemi, R. (2012). The effects References of diazinon on behavior and some hemato- Akinrotimi, O.A., Agokei, E.O. & Aranyo, A.A. logical parameters of fry Rainbow Trout (2012). Changes in blood parameters of Ti- (Oncorhynchus mykiss). World Journal of lapia Guineensis exposed to different salini- Fish and Marine Sciences, 4(4), 369-375. ty levels. Journal of Environmental Engine- Finney, D.J. (1971). Probit Analysis. Univ. Press, ering and Technology, 1(2), 4-12. Cambridge, p. 333. Atamanalp, M., Yanik, T., Haliloglu, H.I. & A- Furspan, P., Prange, H.D. & Greenwald, L. ras, M.S. (2002). Alterations in the hemato- (1984). energetics and osmoregulation in the logical parameters of rainbow trout, On- catfish Ictalurus nebulosus, I. punctatus. corhynchus mykiss, exposed to cyperme- Comparative Biochemistry and Physiology, thrin. Israeli Journal of Aquaculture, Ba- 77, 773-778. midgeh, 54, 99-103.

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