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In the present study, it was examined if the locomotor activity of both male and female Indian freshwater crab, Barytelphusa cunicularis is rhythmic under LD 12:12 photo cycles and if this rhythm free runs following their transfer from LD to DD or LL. Further, it was investigated if B. cunicularis exhibits bimodal pattern of locomotor activity under LD, DD and LL schedules. The specimens were placed for recording the activity at each light regime in between 10-12 days for male crabs and 15 days for female crabs. The locomotor activity was recorded at three different light regimes; DD, LD (12:12h) and LL at 150 lux. Data were analyzed for documenting a circadian rhythm in locomotor-activity (ح = 24h) with the help of Cosinor rhythmometry. The locomotor activity rhythm in B. cunicularis, when exposed to LD the crab shows the bimodal and multimodal patterns of activity anticipating lights-on and lights-off condition. The free running rhythm under constant light and dark regimen are observed irrespective of gender. The 24-h average mean is more in male as compare to female. Amplitude is higher in DD and lower in LL conditions. It may be possible that, the crab possesses a functional oscillator, which regulates its overt circadian rhythm in locomotor activity. Circadian Locomotor Activity in The Freshwater Crab, Barytelphusa Cunicularis (Westwood, 1836): Under Artificial Illumination INTRODUCTION Among the crustaceans, the freshwater and marine decapods have been used as experimental models to study circadian organization in invertebrates (Warner, 1977; DeCoursey, 1983; Brown, 1983; Arechiga et al., 1993; Fuentes-Pardo and Hernandez-Falcon, 1993). Notably among those are: (1) American horseshoe crab, Limulus polyphemus (Chabot et al., 2007, 2004); (2) American freshwater crab, Pseudothelphusa americana (Vania et al., 2005); (3) Eastern Pacific fiddler crab, Uca princes Smith (Stillman and Barnwell, 2004); (4) the fiddler crab, Uca subcylindrica Stimpson (Thurman and Broghammer, 2001; Thurman, 1998); (5) shore crab, Carcinus maenas (Naylor, 1996; Reid and Naylor, 1990); (6) the East African fiddler crabs, Uca urvillei and Uca annulipes (Lehmann et al., 1974). They prominently exhibit bimodal activity pattern under LD schedules characterized by occurrence of peaks in activity corresponding to light-on and light-off timings. The locomotor activity rhythm has also been reported to free-run under DD and LL in freshwater crab, Pseudothelphusa americana (MirandaAnaya et al., 2003) and fiddler crab, Uca subcylindrica (Thurman and Broghammer, 2001; Thurman, 1998). Interestingly, the bimodality in the pattern of locomotor activity has been found to remain unaltered upon transfer from LD to DD, whereas following transfer to LL unimodal pattern in locomotor activity has been conspicuous (Miranda-Anaya et al., 2003). MATERIALS AND METHODS Procurement and recording of locomotor activity Live specimens of B. cunicularis were collected from local wetlands and freshwater ponds during rainy season and transferred to animal house. Male and female crabs were segregated and kept in separate stock aquaria containing tap water. The crabs were fed with boiled egg white on alternate day and fresh tap water was replenished a day after each feeding. They were acclimated to the laboratory conditions for 15 days. During the period of acclimation the crabs were exposed to LD 12:12 (Light onset: 06:00) photoperiod and relatively constant room temperature that varied between 22oC and 24oC. The light fraction of the LD had an intensity of 150 lux. Crabs were kept individually in a specially design transparent rectangular aquaria measuring 40 x 10 x 15 cm. The bottom of each aquarium consisted of two parts, such as aquatic zone and terrestrial zone. The former was filled with tap water up to 5 cm height. Each crab was provided with the option to select either of the zones. The rhythmic locomotor activity of the crabs were monitored and recorded by using IR sensors placed in the middle of the aquatic zone. IR beam interruptions caused by the movement of crabs were fed to a 20-channel Angus event recorder. Experimental protocol Experiment 1: Five LD-acclimated male crabs (average body weight = 58.59 g) were randomly selected and were exposed to DD for 10 days. Thereafter, they were exposed under LD 12:12 (Light onset: 06:00), DD, LD 12:12 (Light onset: 06:00), and LL in a sequential manner. In each schedule they had an experience of 10-12 days. Animal husbandry and maintenance activities were carried out under red dim light during the dark fraction of LD schedules and DD. Experiment 2: The experimental protocol was identical with that of the experiment 1, but the specimens were chosen female crabs (average body weight: 57.81 g) and that they were exposed under DD, LD, DD, LD, and LL sequentially for 15 days each. Construction of actogram and digitization of data Actogram was constructed day wise by placing locomotor activity record of each day, one below the other. Double plotting of activity was executed for the better visualization of rhythmic patterns. The qualitative data on the actogram were digitized, using the technique adopted by Thurman and Broghammer (2001). Each hour on the actogram was divided into six 10-minute bins and scored for activity. To represent the sum of the activities for the entire hour integers between 1 and 7 were used. The former digit indicated that the crab was dormant during the entire hour and the latter indicated that the crab was active at least once during all six 10-minute bins. The process of digitization was completed for each day and each time series. Statistical Analysis of Data Data were analyzed for documenting a circadian rhythm in locomotor activity (t = 24 h) with the help of Cosinor rhythmometry (Nelson et al., 1979). A rhythm was characterized by estimating three parameters, such as the Mesor (M, rhythm-adjusted mean), the amplitude (A, half of the difference between the highest and the lowest value of the rhythmic function) and the acrophase (O, timing of the highest value of the rhythmic function). A power spectrum method was also employed for detecting prominent period (t) in individual time series for locomotor activity (De Prins et al., 1986). RESULTS Both male and female crabs were more active during the onset of dark phase. The daily records of locomotor activity of one male and one female, chosen as representatives, are depicted (Fig. 1 & Fig. 2), respectively. A bout of increased activity was also noticeable corresponding to the timings of light onset, while crabs were under LD 12:12. This bimodality in locomotor activity of crabs disappeared, when they were subjected to DD or LL, irrespective of gender (Fig. 1 & Fig. 2). The observed bimodality was more distinct and precise in case of female crab (Fig. 2). However, in general the rhythm detection ratio was of lower order in the group of female crabs (Table 1) cosinor summary of the characteristics of locomotor activity rhythm in freshwater crab, B. cunicularis. Rhythm-adjusted mean, Mesor The male crabs were always more active as compared with the females, irrespective of the photo regimens. This was gauged |
