Indian Journal of Experimental Biology Vol. 52, May 2014, pp. 521-526

Photoperiodic effects on activity behaviour in the spiny eel ( pancalus)

Malik Zahid, Shalie Malik & Sangeeta Rani* Department of Zoology, University of Lucknow, Lucknow 226 007, India

Received 24 April 2013; revised 24 July 2013

The study focused on the characteristics of circadian locomotor activity in the spiny eel, M. pancalus, kept under different photoperiodic conditions. Two experiments were conducted. Experiment 1 tested the light intensity dependent effect on circadian rhythmicity of the locomotor activity in spiny eel. Three groups of were entrained to 12L:12D conditions for 10 days. Thereafter, they were released to constant conditions for 15 days as indicated below: group 1-DD (0 lux), group 2- LLdim (~1 lux) and group 3-LLbright (~500 lux). The locomotor activity of the fish, housed singly in an aquarium, was recorded continuously with infrared sensors connected to a computer. More than 90% activity of the eels was confined to the dark hours suggesting nocturnal habit. Under constant conditions, the activity in 7/9 fish in group 1, 4/8 in group 2 and 3/8 in group 3, started free running with a mean circadian period of 24.48 ± 0.17 h, 23.21 ± 0.47 h and 25.54 ± 1.13 h in respective groups. Remaining fish in each group became arrhythmic. This suggests that spiny eel can be synchronised to LD cycle and under constant conditions they free run with a circadian period. However, their activity under LL is light intensity dependent; higher the intensity, more disruption in circadian locomotor activity. Experiment 2 was conducted to study the effect of decreasing night length (increasing photoperiod) on circadian locomotor activity. The fish were sequentially exposed to 16D (8L:16D), 12D (12L:12D), 8D (16L:8D), 4D (20L:4D) and 2D (22L:2D) for 10 days in each condition, thereafter, they were released in constant dark (DD= 0lux). The results showed that the duration of night length affects both, the amplitude and duration of locomotor activity. It can be concluded that the spiny eels are nocturnal and that their locomotor activity is under the circadian control and may be influenced by the photoperiod.

Keywords: Circadian locomotor activity, Light intensity, Phase relation, Spiny eel Light is the most important environmental cue that Plotosus lineatus17, channel catfish Ictalurus entrains circadian rhythms1. The anticipate punctatus18, sea catfish Arius felis19, Japanese catfish the changes in the prevailing environment, for Silurus asotus20, European catfish Silurus glanis21, example, light:dark (L:D) cycle to optimize their Brazilian cave catfish Taunayia bifasciata22 and biological processes2-5. The circadian rhythms are Indian catfish Clarias batrachus23. However, limited endogenously regulated under constant environmental information is available on the locomotor activity in conditions e.g. constant dark (DD) or constant light the bottom dwelling fish. Therefore, in the present (LL), they free run with a period (tau; τ) close to, but paper attempts have been made to characterize not exactly of 24 h6. In vertebrates, the fish are a most circadian locomotor activity in the bottom dweller diverse group and depending on their time of being spiny eel, . The spiny eel is maximally active they can be categorized into diurnal, fresh water, bottom dwelling fish which can serve as a nocturnal, crepuscular species and some even show good model to study the circadian systems in a combinations of these features7,8. Fish when kept in nocturnal species. It is widely distributed throughout constant conditions for a prolonged period eventually India, Pakistan, Bangladesh24 and Nepal25. In India showed damped rhythms9-13. Circadian locomotor this fish plays an important role in meeting the activity has been widely studied under different LD nutritional requirements and also has high ornamental cycles in many fish, e.g., zebrafish Danio rerio14, value26-28. The study will be helpful in understanding tench Tinca tinca15, tilapia Oreochromis niloticus16, the circadian behaviour and physiology of other goldfish Carassius auratus7, Japanese sea catfish bottom dwelling nocturnal animals including fish.

—————— Materials and Methods *Correspondent author Telephone: 0522- 2740423 and housing — Adult spiny eel E-mail: [email protected] M. pancalus of both sexes (mean body weight: 522 INDIAN J EXP BIOL, MAY 2014

6.94±0.15 g, body length: 10.6 ± .022 cm) were used. accordingly. The fish were initially exposed to 16D They were captured from river Gomti, a tributary of (8L:16D) followed by 12D (12L:12D), 8D (16L:8D), Ganga, by using the cast nets and brought to the 4D (20L:4D) and 2D (22L:2D) for 10 days in each laboratory in water filled buckets and maintained in condition. At the end of 2D (22L:2D) exposure they the aquarium (60 × 30 × 30 cm, n=10 fish/aquarium) were released to constant dark (DD = 0 lux) for under natural light:dark conditions for another 10 days. acclimatization. Each aquarium was equipped with Data analysis — The data were analysed using aerators and charcoal filters. As this fish is a bottom Chronobiology software (Stanford Software Systems, dweller, PVC plastic pipes and coral stone bedding Stanford, California, USA). In both the experiments, were also provided in the aquarium. In each aquarium the phase difference (Psi, ψ) between activity onset the light was provided by a compact fluorescent lamp and lights off and between activity offset and lights (CFL; 5-watt cool compact fluorescent lamp, model on was calculated for each fish from each day B22C; Philips India Ltd.). The CFL was about 16 cm of recording under LD. During constant conditions above the water surface and provided an average light (DD / LL), the period of activity rhythm (tau, τ) was intensity of ~500 lux at that level. The light intensity obtained using chi-square periodogram. An hourly was measured by using the digital luxmeter (EP 628 activity profile (for 24 h) of each individual was Eurisem TECHNICS, Taiwan). The photoperiod was calculated and mean (± SE) profile of the group controlled automatically by a programmable digital was plotted. The significance of difference between timer (TM-619-2, Frontier, Taiwan). Water groups was determined by the Student’s t-test, temperature was kept constant at 22±2 °C and was one-way ANOVA with and without repeated continuously monitored by HOBO data logger, USA. measures (RM). The statistical analysis was done The fish were fed dried blood meal worms and live using Graph Pad Prism Software (version 5.0, San Chironomous larvae. All experiments adhered to the Diego, USA). ethical standards for biological research as outlined by Portaluppi et al29. Results The locomotor activity was recorded in small aquarium (size 30 × 22 × 26 cm ) housing single fish, Experiment 1: Circadian characteristics of locomotor equipped with one infrared diffuse-reflective sensor activity rhythms (Omron, E3S-AD62, Japan), installed on the front Results from this experiment are shown in Fig. 1. wall of each aquarium, 5 cm above the bottom and The actograms (Fig.1a-c; upper panel) showed that the placed at the centre, which detected movement of the locomotor activity of spiny eel in all the three groups fish within the aquarium. Each sensor was connected under 12L:12D was nocturnal; active during the night to a separate channel, and the recording was done and taking rest during the day (Fig.1a-f). The activity using a software program (Stanford Software counts were significantly high during night in all Systems, Stanford, California, USA) run on an IBM- the groups under LD (group1: F8,23 = 14.35, P < compatible computer. 0.0001; group 2 : F7,23 = 17.59, P < 0.0001; group 3: F7,23 = 17.50, P < 0.0001, 1-way RM ANOVA; Experiment 1: Circadian characteristics of locomotor Newman-Keuls test; Fig. 1d-f). The activity onset activity rhythms showed significantly increased counts during the hour Three groups of spiny eel, M. pancalus (n=8-9 just before lights off (ZT 10-11 vs ZT 11-12; P < 0.05, each) were exposed to 12L:12D for 10 days. On day t-test), which decreased significantly during the hour 11, they were released in constant conditions, as before lights on (ZT 22-23 vs ZT 23-24; P < 0.05, t- follows: group 1: complete dark (DD = 0 lux), group test). The activity pattern was unimodel with consistent 2: dim light (LLdim = ~ 1 lux) and group 3: bright light bouts during active phase, at night (Fig. 1d-f). (LLbright= ~500 lux). The onset and end of the activity under LD in all

Experiment 2: Effect of increasing photoperiod on the groups showed advanced phase relation with the circadian locomotor activity lights off and on respectively. The activity onset (ψon) The fish (n=9) were exposed to sequentially was phase advanced by 0.4±0.05 h and the end (ψend) decreasing night lengths in a 24 hour day. While by 0.6 ± 0.19 h (Fig.1a-f). Under constant conditions, decreasing the night length, the onset of night was the activity in each group started free running, kept constant, the end of night was changed however there was differential response in groups. ZAHID et al.: PHOTOPERIODIC EFFECTS & ACTIVITY BEHAVIOUR IN SPINY EEL 523

Fig. 1 —Representative actograms showing activity of spiny eel under similar 12L:12D and different constant condition (a) DD (b) LLdim (~1 lux) and (c) LLbright (~500 lux). Lower panel (d-f) shows the daily activity counts per hour in LD and constant conditions of three groups. Bar on the top of each actogram shows the light and dark (LD) cycles. The bars above X-axis in the figures in lower panel indicate the LD cycle and the constant conditions. Asterisk (*) show significance between preceding and following hour (i.e., ZT 10-11 vs ZT 11-12 and ZT 22-23 vs ZT 23-24). Significance was taken at the level of P < 0.05. For example, 7/9 fish in group 1 under DD, 4/8 in spiny eel exposed sequentially to reducing dark hours group 2 under LLdim and 2/8 in group 3 under LLbright (from 16 to 2 h) 8L:16D, 12L:12D, 16L:8D, 20L:4D showed circadian rhythmicity. The mean circadian and 22L:2D, in a 24 hour day (F4,23 = 17.30, P < period (tau, τ) calculated for the fish in free run was 0.0001, one-way RM ANOVA; Fig. 2a). As in 24.46 ± 0.19 h (DD, group 1), 23.21 ± 0.47 h (LLdim, experiment 1, the fish showed only nocturnal activity. group 2) and 25.54 ± 1.13 h (LLbright, group 3). It was The change in the night length affected the duration longer in LLbright than in LLdim and DD (F2,12 = 5.968, and amplitude of activity. Under 8L:16D, the activity P = 0.0197, one-way ANOVA). was more intense in the first 12 hours (ZT 8-20) of When compared with the counts in LD, the total night than in remaining 4 hours (ZT 20-24) (P = counts in constant conditions did not vary in group 1 in 0.0020, paired t-test). In other photoperiodic DD (P = 0.0765, paired t-test), however the activity conditions (12L:12D,16L:8D, 20L:4D, 22L:2D), however, the amplitude of activity did not show such counts were significantly reduced in group 2 (LLdim: P variation in their respective night hours (Fig. 2a). < 0.0001, paired t-test) and group 3 (LLbright: P = 0.0005, paired t-test; Fig. 1a-f). Comparing all the The activity in all the photoperiods was synchronised constant conditions, the total counts in DD were and restricted to the respective dark periods and the significantly high than in LLdim and LLbright, however transfer from one photoperiod to the next did not show the latter two groups did not show any difference from any transient cycle (Fig. 2, left panel). each other (F2,27 = 28.84, P < 0.0001, one-way The activity onset in relation with lights offset ANOVA, Neuman-Keuls test; Fig. 1 d-f; lower panel). (ψonset) showed no difference among the photoperiodic Experiment 2: Effect of increasing photoperiod on conditions, however the activity end with light offset circadian locomotor activity (ψend) was significantly advanced in 16D as compared The actogram (Fig. 2, left panel) shows the effect to rest of the photoperiods (F4, 28= 5.928, P = 0.0014, of photoperiod on circadian locomotor activity of the one-way RM ANOVA). The activity counts in 524 INDIAN J EXP BIOL, MAY 2014

Fig. 2 —Representative actogram of the fish exposed to sequentially changing light and dark (LD) conditions. The LD cycles to which the fish were exposed varied in its night length from 16D (8L:16D), to 12D (12L:12D), 8D (16L:8D), 4D (20L:4D) and 2D (22L:2D). The counts calculated under each condition are shown in the right panel (a) counts/hour in five different light dark (LD) conditions, (b) total activity counts/day in five different light dark conditions, and (c) Total activity counts two hours after the lights off in all the light dark cycles. Similar alphabets on the bars show no difference and different alphabets show significant difference between conditions. Significance was taken at the level of P < 0.05. different photoperiodic conditions were dependent on Discussion night length. Longer the night length, more were the This is the first report on circadian characteristics of total counts, however, there was no difference in locomotor activity in the spiny eel. During light period counts between 4D and 2D (F4,35 = 79.68, P < 0.0001, the fish restricted themselves to the burrow inside the one-way ANOVA; Fig. 2b). The counts were coral stones or plastic pipes (personal observation). The maximum in 16D and minimum in 4D and 2D. Under synchronisation of activity with LD cycle depicts that constant DD, 2/9 fish were rhythmic and free ran with the photoperiod acts as entraining cue. The fish did a period 25.50 ± 0.25 h. The shortest photoperiod not show transient cycles when transferred to different which the fish experienced was two hours of darkness LD cycles (Fig. 2, left panel), this could be due to so we also calculated the total activity counts of the the masking effect or the plasticity of circadian first two hours after light offset in all the system1,30,31. The demonstration of circadian activity photoperiodic conditions. It was maximum under rhythms in constant conditions indicated the existence 16D and minimum in 2D, however no difference of internal timing mechanism in spiny eel, although the was observed in 8D vs 4D and 4D vs 2D (F4,35 = response varied under different light periods in 36.96, P < 0.0001, one-way ANOVA; Fig. 2c). constant conditions. Of the total, 77% fish showed free ZAHID et al.: PHOTOPERIODIC EFFECTS & ACTIVITY BEHAVIOUR IN SPINY EEL 525

running rhythms when placed in DD, 50% in LLdim eels were exposed to DD after 22L:2D (experiment 2) (~1 lux) and only 25% in LLbright (~500 lux), suggesting only 2/9 fish remained rhythmic, however, when they that photoperiodic conditions may affect the circadian were released to DD after 12L:12D (experiment 1) period in the spiny eel. 7/9 fish were rhythmic. It could be for the reason that In the study, individual variations were observed very short night of 2 h under 22L:2D just before their under constant conditions. Not all the individuals were release in DD, might be a stressing photoperiod for rhythmic and they had variable circadian periods. them which disrupted their circadian rhythmicity or Similar results were observed in other species also. The the period of exposure under constant conditions per cent fish showing free running rhythm with large (10 days) was not long enough to restore it. variability in their circadian period varied with species. A comparison of the circadian rhythms of the For example, in tench, Tinca tinca, 41% and 50% of freshwater teleosts with those in the marine fish fish showed circadian rhythmicity under DD and in reveals that the fresh water fish relatively have ultradian LD pulses respectively15. Similarly, in flexible circadian system. This may be attributed to another study on goldfish, Carassius auratus, 57% in the relative instability of their environment41. DD, 57% in LL, and 67% in LD pulses showed Interestingly, as per the available reports on spiny rhythmicity under free running conditions7, however, in eels, the dark hours are best suited for their foraging rainbow trout, Oncorhynchus mykiss, the circadian and escape behaviour. rhythmicity is best seen under LL (83.8%) as compared To conclude, the result of the present study 32 to that in ultradian pulses (66.7%) and LLdim (16.7%) . suggests that spiny eel have flexible circadian system In the sharpsnout sea bream, Diplodus puntazzo, only and their locomotor activity is under clock control. one third of the individuals in the experiment showed While the locomotor activity seems to depend on the circadian rhythmicity33. The present results on spiny photoperiod, the night length may determine the eel are in conformity with other fish species. In spiny duration and amplitude of activity. eel the circadian period was longer under LL than bright Acknowledgement in LL , the activity counts were reduced under LL dim dim Financial support through a DST research grant, and LL suggesting that eel being nocturnal, the bright (SR/SO/AS–21/2008) from the Department of constant light might be stressful or induced negative Science and Technology of the Government of India masking effect as has been demonstrated in locomotor 30 is acknowledged. and feeding rhythms of gold fish, Carassius auratus . 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