Black/White Preference and Novel Tank Test to ...

Trends in Biosciences 11(14), Print : ISSN 0974-8431, 2493-2498, 2018

Black/White Preference and Novel Tank Test to Evaluate Anxiety in Adult Goldfish, Carassius auratus FARAH BANO, PRAGYA GUPTA, PRIYANKA AGARWAL AND MOHAMMAD SERAJUDDIN* Fish Biology Research Lab Department of Zoology, University of Lucknow, Lucknow, Uttar Pradesh * email: [email protected]

ABSTRACT Two types of preference tests (black/white and upper/lower tests) were used for measuring anxiety-like behaviour in the goldfish Carassius auratus. The fish preferred a black to a white zone, or the lower to the upper zone of the experimental tank. The fish spent significantly less time and displayed strong white-avoidance with less locomotors activities. The shuttling events were not significantly different between the two compartments of the testing apparatus. The locomotors activity of the fish and time spent in lower zone was significantly different from the upper zone in the novel test. The number of erratic movements and freezing bouts were not significantly different in the novel tank test. The relative efficiency of two complementary tests is supposed to be considerable evidence signifying the preference and aversion in goldfish. Key words

aquarium chamber, goldfish, novel-tank test, preference test

Anxiety is an emotion associated with risk assessment-like behaviour toward potential threats through exposure to a new environment or a potential adverse stimulus in the environment (Graeff and Zangrossi Junior, 2010). Fishes, including Goldfish are considered to be relevant as an experimental model in anxiety therapy (Kah and Chambolle, 1983; Yoshida, 1983; Bonn, 1987). The dark/ light preference test was carried out as an exploration model to measure locomotors activity in dark/light environments to assess the index of anxiety (Belung and Griebel, 2001; Hascoett et al. 2001; Prut and Belzung, 2003). Recent reports, on dark/light preference test was carried out by many workers particularly on the goldfish Carassius auratus (Yoshida et al., 2005; Maximino et al., 2007; Gazolla, 2008) to evaluate the anxiety. Besides these, role of several neuropeptides such as n-octanoylated ghrelin, octadecanneuropeptide, orexin, neuromedin U (NMU) regulating locomotor activity of the goldfish have been reported by Matsuda et al. (2006b, 2011); Nakamachi et al. (2006); Maruyama et al. (2008). The novel-tank test was carried out to develop the natural geotaxic instinct where the fish initially swim to the bottom of a novel experimental tank with gradual increase in vertical activity to explore the environment (Egan et al., 2009; Blaser and Rosemberg, 2012; Stewart et al., 2012; Kalueff et al., 2013). The preference (zebrafish) to the bottom of the novel tank was compared to thigmotaxis of rodents (Levin et al., 2007; Rosemberg, 2011) and the degree of ‘bottom dwelling’ was interpreted as an index of anxiety.

The light/dark preference test is a simple and painless that does not require conditioning because it assesses the natural tendencies of fishes as pointed by Ali et al. (2011). Maximino et al. (2010b, 2011) validated the test for the assessment of behaviour and pointed out the advantages of light/dark preference over other behavioural tests such as shyness-boldness, activity-exploration, approachavoidance and fear-avoidance test. The novel-tank test is also too easy to be employed in the laboratory without any critical laboratory equipments. The different behavioural tests evoke different response and may stimulate different neurological pathways and no single behavioural testing paradigm for anxiety like behaviour adequately captures the necessary information to interpret the results (Blaser and Rosemberg, 2012; Maximino, 2012). Therefore, keeping the above point in mind the present study was an attempt to integrate and consolidate the two different behavioural tests. The work carried out by Nakamachi (2014) based on the influence of hormone ‘Orexin A’ on the locomotor and anxiogenic-like activities in the goldfish, Carassius auratus. However, the main aim of conducting the experiment was to estimate the integrated view of natural preference or aversion in the goldfish with respect to black/white and upper/lower preference without any external hormonal treatment.

MATERIALS AND METHODS BEHAVIOURAL TESTING Adult goldfish (Carassius auratus, 12-18 g body weight) of both the sexes were purchased commercially and acclimated under controlled light-dark conditions (12 h light/12 h dark) with the room temperature regulated to 20-25 o C for 3 weeks before use. The experiments were performed between August and November 2016. Fish were fed with artificially prepared food comprised of 35% fish meal, 28% mustard oil cake, 28% rice barn, 2% sunflower and cod liver oils each, 5% carboxy methyl cellulose and multivitamin and multimineral tablets (‘Becozyme Forte’ Glaxo India Ltd, 25 tablets/kg foods) throughout the investigation once a day at 10.00 am. The faecal matter was removed before feeding the fish. The amount of food offered to the fish in each aquarium was kept constant (5% of body weight of stocked fish). A total of 6 test apparatus (75 cm long, 30 cm wide and 30 cm high) was used for the behavioural study of the fish. The 60 individuals were used for both the test (three replicates of thirty fish each) which was carried out for a month. Prior to each observation the observer sat in front of the tank for 10 min to allow the ûsh to acclimate to the observer’s presence and was immediately followed by the recording of their behavioural activities.

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Fig. 1. Test apparatus for the Black/White Preference test (75 × 30 × 30) cm. The tank was divided in half vertically. One exterior half was covered with white paper and the other half with black paper. Each behavioural activity was observed individually in a single session for 5 minutes.

BLACK/WHITE PREFERENCE TEST The tank was divided in two equal sized compartments and the sides of the compartments were made white and black using white and black paper except bottom. There was no physical barrier between the two compartments and no lid was placed above the compartments (Fig.1). The individuals of the fish were placed

in the centre of the white and dark zones of the testing apparatus and behaviour was measured are (1) total distance (cm) moved by fish over time for each zone (light/ dark) zones of test apparatus to evaluate the general locomotors activity and the the preference was depicted by position/place of the fish in either compartments at the end of each 1 min, (2) total time spent within each compartment to assess zone preference/avoidance, (3) the total number of shuttling events were counted as done by Blaser and Rosemberg (2012), Maximino et al.

Fig. 2. Test apparatus for the Novel Tank Test (75×30×30) cm. The tank was divided in half horizontally by using a black line drawn on the tank exterior.

BANO et al., Black/White Preference and Novel Tank Test to Evaluate Anxiety in Adult Goldfish, Carassius auratus

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(2012). Shuttling events were defined as short, less than 1 sec crossings into the white compartment (Kalueff et al., 2013).

NOVEL-TANK TEST

3(A)

The experimental tanks were divided horizontally into two equal compartments by using a straight black line drawn on exterior to mark upper (shallow) and lower (bottom) zones of the tank and provided with the small gravels and pebbles in the lower zone (Fig. 2). The individuals of the fish were placed in the mid of both the zones of the testing apparatus and behaviour was measured are (1) the number of transitions to the upper/lower zone and the preference was depicted by position/place of the fish in either compartments at the end of each 1 min (2) total time spent in the upper/lower half of the tank, (3) duration of freezing bouts and (4) number of erratic movements as per Blaser et al. (2010), Kistler et al. (2011) and Blaser and Rosemberg (2012). Freezing bouts were defined as periods of no body movement for at least 1 sec on the bottom of the tank. Erratic movements were defined as sharp, rapid changes of direction or darting.

STATISTICAL ANALYSIS Data are presented as mean±SEM and a probability level of 5 % was used as the minimal criterion of significance. Paired-sample t-tests (two tailed) were applied to all the behaviour measured for the comparison between the respective zones. A statistical analysis was carried out through Graph Pad Prism software (version 5.01).

RESULTS AND DISCUSSION BLACK/WHITE PREFERENCE TEST

3(B)

Student t-test revealed that the total distance moved in two compartments (black/dark) of the test apparatus was significantly different (t=8.820; p=0.0001; Fig. 3A). The time spent by the fish was significantly different between both the compartments (t=6.018; p=0.0002; Fig. 3B). Besides these, no differences in the shuttling events (t=1.496; p=0.1519; Fig. 3C) were recorded.

NOVEL-TANK TEST The t-test showed a significant difference between the movements in upper and lower zones (t=3.434, p = 0.00306; Fig. 4A), but the total time spent in the two zones of the test apparatus in the present study was significantly different (t=2.8485, p=0.007331; Fig. 4B). No significant differences were found in the number of freezing bouts (t=0.4874, p=0.6319; Fig. 4C) and the erratic movements (t=0.8076, p=0.4299; Fig. 4D).

3(C) Fig. 3. Behavioural testing of the light/dark preference test. 3(A) Total distance moved over time, 3(B) Time spent per zone and 3(C) Number of shuttling events

The black/white preference tests and novel-tank customized for adult goldfish in the present study showed a pattern of anxiety like behaviour towards white and upper zones of the testing apparatus. The total distance moved and time spent in white and black compartments was significantly different and more in dark zone indicated the probable preference of black zone in gold fish. It was found to be in the agreement of Gouveia et al. (2005) who pointed out that it may be the tendency of the fish to protect against predation (from birds and other predators) or to explore the new environment for food and sexual partners. The present

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4(A)

4(B)

4(C)

4(D)

Fig. 4. Behavioural testing of the novel tank test. 4(A) Number of transitions, 4(B) Time spent per zone, 4(C) Number of freezing bouts and 4(D) Number of erratic movements finding is also similar to that of Mathur and Guo (2010) and Maximino et al. (2010b) who also reported the preference for black (dark) environment over white in adult zebra fish and concluded that the possible reason of it as age-factor (Miklósi and Andrews, 2006) and the maturation of melanophores (Langsdale 1993; Mc Clure, 1999). The numbers of shuttling events of the gold fish were not significantly different between the two compartments of the light/dark testing apparatus. The similar result was reported by Collymore et al. (2015) in zebrafish. The goldfish was lingered, preferred and showed the more locomotors activities in lower half of the tank in novel test pointing out the original natural benthic feeding habits of the fish. The goldfish showed fewer entries in the upper zone of the testing apparatus indicated that the goldfish appears to be anticipating for the feed. The fish spent significantly more time in the lower zone, due to the nibbling behavior which suggested that the fish mainly prefer to take food from benthic environment (Gupta and Banerjee, 2009). Goldfish being benthic omnivore feeder (Moyle, 2002)

and feed particularly on preferred chironomids (benthic) than mosquito larvae (surface breather) (Gupta and Banerjee, 2009). Thus the lower zone (bottom zone of the testing apparatus) is considered to be the ideal habitat for the adult goldfish. Increased frequency of erratic movements and freezing bouts in the upper half of the compartment has been associated with greater anxiety-like behaviors (Egan et al., 2009; Blaser et al., 2010 and Stewart et al., 2012). The simple tests as novel tank and light–dark tests have emerged as potentially useful behavioral measures used to evaluate psychomotor activities notably, anxietylike behavior of fish which allow the experimenters to assess that how well fish adapt to various stressor conditions (Grossman et al., 2010; Sackerman et al., 2010; Wong et al., 2010, 2012; Blaser and Penulosa, 2011; Maximino et al., 2010 a, b and 2012; Parker et al., 2012). The integrated view is supposed to be considerable evidence suggesting the compatability of both the tests about the preference and aversion in goldfish.

BANO et al., Black/White Preference and Novel Tank Test to Evaluate Anxiety in Adult Goldfish, Carassius auratus

ACKNOWLEDGEMENT The authors thank the Head, Department of Zoology, University of Lucknow for providing facility and administrative support. One of the authors is thankful to the University Grants Commission (UGC), New Delhi for the JRF fellowship [Ref No-19/06/2016 (i) EU-V], which is given to undertake the advanced research.

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Received on 16-03-2018

Accepted on 19-03-2018