Section 1: Light wavelength dependent effects on circadian behavior and physiology

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1 1 Section 1: Light wavelength dependent effects on circadian behavior and physiology Introduction Most circadian and photoperiodic studies have placed importance to the role of daylight in controlling daily and seasonal functions in vertebrates (Kumar et al., 2010). Across seasons, the day length changes in duration, intensity and spectrum. The spectral composition of daylight can affect the circadian and photoperiodic responses in vertebrates, including birds (Rani et al., 2002). Also there are changes in the nightillumination levels, which are perhaps buffered against a brighter day. In general, avian circadian and photoperiodic responses depend on the subjective interpretation of day and night illumination, hence the photophase contrast, rather than on the day light intensity alone as has been studied in different birds such as Japanese quail, Coturnix c. japonica, (Meyer et al., 1988); blackheaded bunting, Emberiza melanocephala (Kumar et al., 1992) and Indian weaver bird, Ploceus philippinus (Singh et al., 2012a). The activity-rest cycles (locomotor rhythms), are reliable and easily (noninvasively) measurable assay of the dynamics of clock system which can be studied both in the field and laboratory conditions. Using locomotor activity as phase marker, the characteristics of circadian system have been studied in birds (Subbaraj and Gwinner, 1985). Oscillations in spectral distribution of daily light available have been shown to synchronize avian circadian rhythm in field (Krull, 1976). Zebra finches, could entrain

2 2 their circadian activity rhythms under 12 short:12 long light wavelengths (3400:2900 K color temperatures) at equal irradiance (44 mw cm 2 ) (Demmelmeyer and Haarhaus, 1972). A similar light wavelength dependent effect on circadian activity rhythms has also been shown in other birds including brambling, Fringilla montifringilla common redpoll, Carduelis f. flammea (Pohl, 1999) and blackheaded bunting, Emberiza melanocephala (Malik et al., 2004). Besides the effects on circadian physiology, light spectrum has a massive impact on hormonal rhythms of different class of animals. The short light wavelengths ( nm; blue-green light) are shown to suppress night melatonin levels with delayed peak in several species, including fish, chicken, songbirds, rat and human (Malik et al., 2015). Most of the studies in fishes have convincingly shown that red light has a positive effect on their growth and metabolism whereas blue light induces stress (Karakatsouli et al., 2008). In frog, Rana cyanophlyctis, the ovarian follicular growth was maximum in red light, followed by yellow and green light (Joshi and Udaykumar, 1998). Recently, an interesting experiment assessed the impact of light wavelength on night melatonin levels in blackheaded buntings exposed to 13L:11D, where 13h day of white, blue or red lights with bright and dim intensities was given to birds, while 11h night was kept completely dark. Mid night melatonin levels were found significantly higher in red than in the blue or white light periods after 4.5 weeks of exposure, suggesting a complex interaction of light wavelengths and hormonal secretion in birds (Malik et al., 2015). Light wavelength have been shown to exert different effects on glucose, triglycerides and other metabolic

3 3 parameters with red light being more effective in inducing growth in other vertebrates like fish (Karakatsouli et al., 2008). Photoperiodic induction and other seasonally affected physiologies have also shown to be influenced by wavelength and intensity of light. In Syrian hamsters, 1 hour green, blue or near ultraviolet light, but not red or yellow light, presented during night induced the reproductive system. Also, at equal irradiances, different bandwidths of light have different effects on reproductive system of hamster (Brainard et al., 1983). The effect of light wavelength on seasonal reproductive phenomenon has been well documented in migratory blackheaded buntings (Emberiza melanocephala). When subjected to a stimulatory day length (13L:11D) in white, green (528nm) and red (654nm) colors at 100 lux intensity, photostimulation (weight gain and testis growth) occurred in all groups but the response was significantly greater in birds that were exposed to red light (Kumar and Rani, 1996). Further, this was demonstrated by comparing the gonadal growth in male buntings exposed to 14L:10D of blue light with those exposed to 12L:12D of red light with about 4 to 6 folds difference in the light intensity that long light wavelengths can mimic the stimulatory effects (compensation) of the duration and intensity of a longer photoperiod of short wavelengths (Kumar et al., 2000). Investigations on action spectra for photoperiodic responses showed that the responsiveness to long wavelength is many folds higher than short wavelength (Malik et al., 2002). Similar responses have also been reported to occur in several other bird species (for details see Kumar, 1997; Kumar and Rani, 1999; Rani et al., 2002). Thus, most of the studies have shown the effect of light wavelength on vertebrate s photoperiodic physiology. However, effect of wavelength transition on daily

4 4 rhythms and hormonal profile has not been studied. Also, effects of light wavelengths on avian circadian and photoperiodic responses, independent of duration and intensity of light period, have been scarcely investigated. Therefore in this section we have tried to demonstrate the effects of light wavelengths on the physiology and behavior of a nonphotoperiodic species; spotted munia (Lonchura punctulata; study 1), and photoperiodic species; migratory buntings (Emberiza melanocephala) and resident Indian weaver bird (Ploceus philippinus; study 2).

5 5 Section 1: Light wavelength dependent effects on circadian behavior and physiology Study 1: Effect of light wavelength on activity-rest behavior of spotted munia (Lonchura punctulata) 1.1. Abstract Spectrum of light continuously changes across the day, but more importantly during morning and evening civil twilight periods, which could be critical time of photoperiodic entrainment. We tested this hypothesis in spotted munia (Lonchura punctulata) exposed to different light wavelengths (blue: short wavelength; 450nm, red: long wavelength; 640nm) at equal energy (0.33 μm/m 2 /sec) levels. To test this hypothesis, two experiments were done. In experiment 1, effect of light wavelength was studied. Birds were entrained to 12L:12D for two weeks followed by continuous white light (LL) for another 2 weeks. After this birds were given 12L:12D again, but the light period was given either as white (group 1; control), blue (group 2) or red (group 3). Their locomotor activity was recorded throughout. Results showed that birds in all the groups shifted their activity to light phase regardless of the wavelength. In experiment 2, effect of transition of blue to red and vice-versa mimicking the spectral shift of natural dawn-dusk transition was studied. It had two parts; in part 1, the birds were first entrained to 12L:12D followed by LL for two weeks. Thereafter, they were given 1L:11D:1L:11D where both 1L pulses were replaced by white (group 1) or in combination of blue (B) and red (R) of 0.5 h each as B:R/R:B (group 2) or R:B/B:R (group 3). Part 2 of the experiment was similar to part 1 except that the skeleton photoperiod was 4L:8D:4L:8D and both 4L light pulses were replaced by white (group 1), or in combination of blue and red of 2h each as B:R/R:B (group 2) or R:B/B:R (group 3). The results showed that transition of light wavelengths can affect the locomotor activity pattern. Taken together, these results suggest that different monochromatic light at equal energy level when given with dark phase are perceived as light and that transition of blue to red and vice-versa during dawn and dusk affects locomotor activity pattern. The results invoke further study to understand the involvement of different photoreceptors, if any, in perceiving the photoperiodic information.

6 Introduction The spectral changes across the day can be important in entraining the circadian rhythms. Therefore, in the present study we have assessed the influence of two different light wavelengths on circadian locomotor activity of spotted munia given at equal energy levels. Spotted munia is a passerine finch inhabiting tropical and subtropical regions which unlike temperate regions, do not have much annual variation in the daylength and long twilight periods characterized by dramatic changes in spectral distribution. Thus, it is a good model system to study wavelength dependent effects on its circadian behavior. Therefore, in the present study, we tried to demonstrate light wavelength dependent effects on activity behavior in spotted munia (i) if presented with dark or (ii) as transition from blue to red and vice-versa during light onset (dawn) and offset (dusk) Materials and Methods Experiments Experiment 1: Wavelength dependent entrainment of locomotor activity rhythm Adult birds were captured from the wild, brought to the laboratory and kept in outdoor aviary for about a week for their acclimation. They were first entrained to 12h light: 12h darkness (12L: 12D); (L = 0.33 μm/m 2 /sec, D = 0 μm/m 2 /sec) for two weeks followed by continuous white light (LL) for another two weeks at identical light energy levels. Thereafter, birds were divided into 3 groups (n = 10 each) and were again exposed to 12L:12D where the light phase was given either white (12L:12D; group 1), blue (12B:12D; group 2) or red (12R:12D; group 3). The locomotor activity was monitored

7 7 throughout the experiment. Experimental conditions, methods of observation and statistical analyses were the same as described in the general materials and methods. We calculated total activity counts of each bird during day (ZT 0-12) and night (ZT 12-0) for entire duration of the experiment. Data was plotted as changes in activity counts throughout to clearly show trend of response as the experiment progressed. Experiment 2: Effect of light wavelength transitions at lights onset (dawn) and offset (dusk) This experiment was done in two parts. In part 1, the birds maintained in outdoor aviary were brought indoors and kept in 12L:12D condition in activity cages. After two weeks of entrainment they were released in LL (continuous light) for another two weeks. Thereafter, birds were divided into three groups (n = 6 each) and exposed to skeleton photoperiod (1L:11D:1L:11D). In the skeleton photoperiod both 1h light pulses were given either as white (group 1) or in combination of blue (B) and red (R) of 0.5h each as B:R/R:B (group 2) or R:B/B:R (group 3). Activity pattern was monitored in all the photoperiodic condition throughout the experiment. Part 2 of the experiment was same as part 1 except that the duration of light pulses was increased from 1h to 4h. Briefly, birds were kept in 12L:12D condition in activity cages for about two weeks. After that they were released in LL, for another two weeks. Thereafter, they were exposed to 4L:8D:4L:8D where 4 hours light pulses were given either as white (group 1) or in combination of (B) and (R) of 2h each as B:R/R:B (group 2) or R:B/B:R (group 3).

8 8 We calculated the activity of each 0.5h in 1h transition phase (part 1) and 2h (part 2) in 4h transition phase. Data from the experiment was plotted as changes in activity counts throughout the experiment to explain the response against light treatment Statistics The data were analyzed by using unpaired (between two groups) Student s t-test. Significance was considered at p < All statistical analyses were done by GraphPad prism software program version 5.0 (San Diego, USA) Results Experiment 1: Wavelength dependent entrainment of locomotor activity rhythm Figure 1, shows representative actograms of spotted munia exposed to different photoperiodic conditions. Birds in all the three groups were synchronized to 12L:12D with significantly more activity in 12L than in 12D (Group 1: p < , Group 2: p < , Group 3: p < ; Student s t-test, Figs. 2d, h, l) but when the birds were released in LL, their activity was disorganized after few cycles. On shifting to different wavelengths schedule their activity was restricted to light phase irrespective of the wavelength. Thus, this was similar to control group; white (Group 1: p < ; Student s t-test, Fig. 2d), blue light (Group 2: p < ; Student s t-test, Fig. 2h) and red light (Group 3: p < ; Student s t-test, Fig.2l). Experiment 2: Effect of light wavelength transitions at lights onset (dawn) and offset (dusk) Figure 3 shows the representative actograms of spotted munia exposed to different light schedule (part 1). All birds showed high activity in 12L than in 12D (Fig.

9 9 3), but in LL (9 out of 16) birds free ran, and remaining birds became arrhythmic after few cycles. During 1L:11D:1L:11D birds restricted their activity only during the light hours (dawn and dusk) regardless of the wavelength appeared (Fig. 3). When the activity in each 0.5h in 1h light pulse (both dawn and dusk) was calculated, no difference was observed in total activity counts in group 1 and group 2 [group 1: dawn (W/W); p = , dusk (W/W); p = , group 2: dawn (B/R); p = , dusk (R/B); p = ; Student s t-test, Figs. 5a, b)], by contrast, in group 3, significantly more counts in red light at dawn (R/B); p = and blue light at dusk (B/R); p = ; Student s t- test, Fig. 5c) were found. In group 2, few birds showed a free running rhythm in the wavelength treatment schedule along with consolidated bouts in that period which was otherwise the light phase in 12L, although this activity was absent in rest of the groups. Similarly, in part 2 of the experiment, birds were entrained to 12L:12D condition showing higher activity in 12L (Fig. 4). In LL (8 out of 13) birds free ran and remaining birds showed arrhythmic activity. In skeleton photoperiod (4L:8D:4L:8D), the birds restricted their activity towards light phase (4h) irrespective of the sequence of light wavelength presented (Fig. 4). When activity in each 2h in 4h light pulse (both dawn and dusk) was calculated, no difference was observed in activity counts in any of the groups (group 1: dawn (W/W); p = , dusk (W/W); p = ), group2: dawn (B/R); p = , dusk (R/B); p = and group 3: dawn (R/B); p = , dusk (B/R); p = Student s t-test, Figs. 5a, b, c ).

10 10 Section 1: Light wavelength dependent effects on circadian behavior and physiology Study 2: Effect of light wavelength on hormonal physiology of migratory bunting and non migratory Indian weaver bird 2.1. Abstract The present study investigated whether at identical duration and equal energy level (0.33 μm / m 2 / sec), birds presented with short (450 nm; blue, B) and long (640 nm; red, R) light wavelengths would differentially interpret them and exhibit wavelengthdependent circadian behavioral and physiological responses, regardless of the difference in their breeding latitudes. We compared synchronization of circadian behavioral and physiological responses in the temperate migratory blackheaded buntings and subtropical Indian weaver birds, presented with B:R and R:B spectral regimes at equal energy levels in a 12:12 paradigm, after they were arrhythmic under constant light, LL. Briefly, birds in two groups of each species (groups 1 and 2) entrained to 12h light: 12h darkness (12L:12D) were subjected to LL for two weeks, which rendered them arrhythmic in the activity pattern. Then, they were sequentially exposed for about two weeks each to 12B:12R and 12R:12B (group 1) or 12R:12B and 12B:12R (group 2). The activity pattern was monitored throughout, but melatonin and cortisol levels were measured once at the end of each light exposure, in middle of light and darkness in 12L:12D or at corresponding periods in LL and spectral light regimes. The blue and red light periods were interpreted as day and night, respectively, and showed activity and noactivity in non-migratory weaver birds or activity and intense activity (Zugunruhe, night time restlessness in caged migrants) in buntings. Plasma melatonin levels were low and high in blue and red light periods, respectively, in B:R, but not R:B regime, except when weaver birds presented with R:B regime in the first step (group 2) showed low amplitude elevated melatonin levels in the red light period. There was no effect of light wavelength was observed in the plasma cortisol, triglyceride, GOT and glucose levels. These results show wavelength-dependent synchronization of circadian activity and melatonin rhythms, and suggest that adaptation for synchronization of the circadian clock governed behavior and physiology to the photoperiodic environment is conserved.

11 Introduction In this study, it was hypothesized that birds presented with short and long light wavelengths at identical duration and equal irradiance (energy level) would respond similarly and exhibit wavelength-dependent circadian responses, regardless of the difference in their breeding latitudes. To investigate this, we compared synchronization of the circadian behavioral and physiological responses viz. melatonin secretion, physiological aspects of metabolism (blood glucose, triglycerides) and stress (cortisol, GOT, LDH) on two avian species, temperate migratory blackheaded bunting (Emberiza melanocephala) and sub tropical non-migratory Indian weaver bird (Ploceus philippinus) presented with B:R and R:B spectral regimes at equal energy levels in a 12:12 paradigm. Both these birds experience different lighting conditions in their annual life history stages and vastly differ in their physiologies even if both are long day breeders. Recently, it has been demonstrated that both these birds respond differently to photoperiod (stimulatory long days). Rate of induction of seasonal life history stages in the buntings was much faster than in Indian weaver birds even when the birds share the same lighting conditions for about 6 months (Malik et al., 2014). It has also been show recently that bunting and weaver birds differ in body weight gain response and metabolic processes including triglycerides in blood (Srivastava et al., 2014). Thus, it is quite possible that spectrum of light will have different effect on these two different bird species giving more insights to the circadian perception of different light colors with same energy levels and its effect on physiology. We used an experimental approach whereby the effects of short and long light wavelengths were tested in synchronization of the behavioral and physiological responses in circadianly arrhythmic birds. The prediction was that both species would

12 12 respond similarly to different light wavelengths, and interpret 12h periods in blue and red light as day and night, respectively, should the spectral sensitivity of photoreceptors mediating photoperiodism was a conserved trait among photoperiodic birds Materials and Methods Experiment This study included two photoperiodic songbirds, the blackheaded bunting (Emberiza melanocephala) and Indian weaver bird (Ploceus philippinus). Photosensitive birds (buntings: n = 14; weaverbirds: n = 18) were housed individually in activity cages providing programmed light cycles. Birds were initially kept on 8L:16D. On day 4, they were exposed to 12L:12D (L = 0.33 μm / m 2 / sec, D = 0 μm / m 2 / sec) by delaying the light offset by 4h. After about two weeks, birds were released in constant light (LL) at identical light energy levels for next two weeks to induce circadian arrhythmicity in the activity behavior. At this stage, they were distributed in two groups of each species (groups 1 and 2; n = 7 or 9 each) and subjected to spectral regimes (450 nm: blue, B; 640 nm: red, R) with identical light duration (12h) and energy level (0.33 μm/m 2 /sec). The 12L:12D was replaced with 12B:12R (group 1) or 12R:12B (group 2). After two weeks, the wavelength pattern was phase-inversed for another two weeks; group 1-12R:12B, group 2-12B:12R. This was done to remove bias, if any, in the interpretation of light wavelength by birds because of their order of presentation during the experiment. There was a difference of 2-3 days in the duration of exposure to a particular lighting regime between buntings and weaver birds, in order to stagger observation dates in two species for the sake of convenience.

13 Measurement of physiological parameters from blood Blood sample and plasma collection Blood samples were taken at hours 6 and 18 relative to lights on of the 12L:12D in each light condition; this was designated as ZT6 and ZT18 samples (ZT0, zeitgeber time = lights on). The same blood sampling times were used in LL, in the absence of another phase marker of daily oscillation. Samples of μl of blood were collected in heparinized capillary tubes by veni-puncture of the wing vein, and immediately centrifuged. The plasma was harvested and stored at -20 C until assayed for melatonin and cortisol by ELISA. All the spectrophotometric readings were taken at the specified wavelengths in Spectra Max M2e microplate reader, Molecular Devices LLC, USA Plasma melatonin assay Plasma melatonin concentration (pg/ml) was measured by ELISA using a specific melatonin kit (product no. RE54021, IBL International GmbH, Hamburg, Germany), previously standardized and used in our laboratory for the measurement of melatonin in weaver birds samples (Singh et al., 2012a). Melatonin was extracted and assayed from each plasma sample, as per manufacturer s protocol and instructions. Briefly, methanol extracted volumes of experimental samples, standards and controls were dried by vacuum concentrator. Obtained pellet was reconstituted in double distilled water, and 50 μl of each reconstituted sample volume and 50 μl melatonin anti- serum (rabbit, polyclonal) were incubated in a 96 well plate at 4 C for 20h. Thereafter, 150 μl of freshly prepared enzyme conjugate was added and incubated for 2h at room temperature with continuous shaking (500 rpm). Then, 200 μl of freshly prepared p-nitrophenyl phosphate (PNPP)

14 14 substrate solution was added to each well, and incubated for another 40 min. The reaction was stopped by adding 50 μl of PNPP stop solution to each well, and optical density (OD) was measured at 405 nm, using 650 nm wavelength as reference. OD of standard samples was plotted to make a standard curve, and the concentration of melatonin in experimental samples was calculated with reference to values in the standard curve. Individual values of each were used to calculate mean ± SEM melatonin levels (pg/ ml) for a group Plasma glucose and triglyceride measurements assay Glucose concentration in blood was measured using Accu-chek (Roche) active blood glucose monitoring system. Briefly, during blood sampling at respective time points and light regimes, one drop of blood was added to glucose strips which gave the glucose concentration (mg/ dl) for the respective samples. Values from the individual samples were then averaged for a particular group and shown as mean ± SEM. Blood triglyceride levels (which included free glycerol) was measured in the samples by Trinder-end point based ELISA kit (FAR diagnostics, Italy). The method has been used previously for buntings and weaver birds (Srivastava et al., 2014). Briefly, 10 μl standard, control or sample was mixed with 1ml of reagent supplied. After 10 min incubation at 37 ºC absorbance of standard and samples were taken against blank at 550 nm. The final concentration in a sample was calculated as triglycerides (mg/ dl) = abss/abs Std 200. Values of individual of a particular group was then averaged and plotted as mean ± SEM Plasma cortisol, GOT and LDH measurement assay

15 15 Plasma samples obtained were assayed for blood cortisol using solid phase enzyme-linked immunoabsorbent assay based competitive assay kit from IBL international, GmBH, Germany (product no. RE52611) as per the manufacturer s instructions. For this, 50 μl of standard, control and samples were used in duplicate and incubated with the supplied enzyme conjugate for 2h in room temperature with shaking ( rpm). After washing 4 times with supplied wash buffer 100 μl of TMB substrate was added into each well and incubated for 30 min at RT with shaking at rpm. 100 μl TMB stop solution was added after the incubation and optical density was recorded within 15 min at 450 nm (Reference wavelength of nm). The values for a group were averaged and plotted as mean ± SEM. Likewise, plasma lactate dehydrogenase (LDH) was measured using Infinite LDH kit (Accurex India; Cat no. L60713/L60813) as per manufacturer s protocol. Individual samples were mixed with working solution (800 μl R μl R2) and after initial incubation of 60 sec, three consecutive reading were taken at an interval of 30 sec at 340 nm. Mean change of absorbance was calculated per minute and results were calculated as activity of LDH (IU/ L) = Abs/ min Individual values were averaged for a particular group and reported as mean ± SEM. Glutamic oxaloacetic transaminase (GOT) was determined using diagnostic kit (Siemens Diagnostics, Vadodara, India, Product code 982). Individual samples were mixed with working reagent (Reagent 1 + Reagent 1A) and after initial incubation of 60 sec, 4 optical density reading at 60 sec interval between each was measured at 340 nm (Chakrabarti and Srivastava, 2012). Mean OD/ min multiplied with international unit

16 16 factor of 1746 gave the SGOT activity (IU/ L). Average of the individual values of a group was finally plotted as mean ± SEM Results Activity behavior Both species were synchronized to 12L:12D, with a diurnal activity pattern (Figs. 6a, f and 7a, f). Under 12L:12D, 2 or 3 buntings in both groups had developed the night activity, indicating the initiation of Zugunruhe. By the end of LL, all birds, except 3/14 buntings and 3/ 18 weaverbirds, were arrhythmic, with scattered activity throughout the 24h (Figs. 6b, g and 7b, g). On subsequent exposure to the B:R (group 1) and R:B (group 2) spectral regimes, birds were synchronized, as before, and exhibited response as if 12h blue was day and 12h red was night (Figs. 6c, h and 7c, h). The activity pattern was inversed in respective groups with the phase inversion of 12h light components in the subsequent (final) exposure (Figs. 6d, i and 7d, i). The restriction of Zugunruhe in the 12h red light further supported this component of an LD cycle which was interpreted as night (Figs. 6c, i). In synchronized states, the period of rhythm matched the external light regimes (tau, τ = T), i.e. in 12L:12D, 12B:12R and 12R:12B, regardless of the order of presentation of light wavelengths in groups 1 and 2 (Table 2). Consistent with this, total activity in the first and second 12h depended upon their interpretation as day and night. For instance, similar to significant difference in activity in light and dark periods of initial 12L:12D (p < 0.05, Figs. 6e, j and 7e, j), blue light period had significantly higher activity than the red light period in the spectral regimes, irrespective of the order of exposure. This was true for weaverbirds (p < 0.05, cf. Figs. 7c, h, d, i), but not for

17 17 buntings because of the development of Zugunruhe in night (red light period). Thus, buntings had significantly higher activity in red than in the blue light in B:R (p < 0.05, Figs. 6c, i), and only a relative difference between red and blue light components in the R:B condition (Figs. 6d, h) Plasma melatonin levels In both species, plasma melatonin levels were low during the day and significantly high at night, consistent with diurnal pattern of melatonin secretion (ZT6 vs. ZT18: bunting: p = ; Indian weaver bird: p = ; Student s t-test; Fig. 8a). This was lost in LL, with no difference between day and night melatonin levels (Fig. 8b). On exposure to spectral regimes, 12h red component had significantly higher melatonin levels than the 12h blue component, if light regimes were presented with B:R combination (p = 0.05, Student s t-test; Figs. 8c, d ). However, when presented as R:B combination, as was the case for group 1 in the second step and for group 2 in the first step, there was no difference in melatonin levels between two 12h halves, except a small albeit significant increased levels in red light in group 2 weaver birds (p < 0.05, Student s t-test; Figs. 8c, d) Plasma glucose and triglyceride levels Figure 9 compares the plasma glucose levels in two time points (ZT6 and ZT18) of migratory buntings and Indian weaver birds and in response to light spectrum. There was no difference observed in blood glucose level in any light schedule in both the species. Plasma triglyceride levels are depicted in fig. 10 of weaver birds and buntings in response to white, long and short wavelengths of light. Both (weaver bird: p = ,

18 18 buntings: p = , Student s t-test; Fig. 10a) showed significantly high triglyceride levels in (ZT6) of 12L:12D in comparison to dark phase (ZT18). In addition, buntings had higher triglycerides in ZT18 of LL group (p = , Student s t-test; Fig. 10b). But no significant difference was found in response to blue or red light in both the species Plasma cortisol, LDH and GOT levels Figure 11 shows the plasma cortisol levels of migratory buntings and Indian weaver birds in response to white, blue and red light. No difference in cortisol concentrations was found in blood plasma of both the species in response to 12L:12D, LL, 12B:12R, 12R:12B and the flip groups 12R:12B and 12B:12R. Fig. 12, compares the plasma lactate dehydrogenase (LDH) activity of migratory buntings and Indian weaver birds in response to different light schedules. Both weaver bird and buntings showed significantly high LDH activity at ZT6 of 12L:12D (baya: p = , bunting: p < ; Student s t-test; Fig. 12a). In LL, baya weaver showed a high LDH activity at ZT6 (p < , Student s t-test; Fig. 12b) whereas buntings showed no difference. In response to blue and red light, no significant difference was found in both species. Fig. 13, depicts the glutamic oxaloacetic transaminase (GOT) activity in Indian weaver bird and blackheaded buntings. Weaver birds did not show difference in the activity of GOT in any light schedule or in response to white, blue or red lights. Buntings on the other hand showed significantly high activity of GOT at ZT6 (12L:12D; p < and LL; p = , Student s t-test; Figs. 13a, b,) compared to ZT18. Moreover, significantly high GOT activity was found in blue light in both 12B:12R group (ZT6, p = , Student s t-test; Fig. 13c) and 12R:12B (ZT18, p = , Student s t-test; Fig. 13d). When light

19 19 treatment was phase inversed, although higher GOT activity was found in response to blue light treatment, but it was not statistically significant Correlation between activity and melatonin levels There was no significant correlation of activity with plasma melatonin levels in blackheaded buntings (group 1; melatonin: r = , r 2 = , p = ; Fig. 14a, group 2; melatonin: r = , r 2 = , p = , Fig. 14b), activity had significant negative correlation with plasma melatonin levels in Indian weaver birds (group 1; melatonin: r = , r 2 = , p = ; Fig. 14 c, group 2; melatonin: r = , r 2 = , p < ; Fig.14 d) Discussion The results obtained from these experiments showed that artificial light cycles of different monochromatic light (short, blue and long, red) at equal energy level may influence the circadian activity rhythms of spotted munia (study 1), Indian weaver bird and blackheaded bunting (study 2). This is supported by the experiments on bramblings in which the activity was restricted in blue light in 12B:12R (12h blue:12h red) cycles (Pohl, 1996). Again, common redpolls exposed to 16.5B:7.5R cycles with similar qualitative light parameters the rhythms were entrained and activity was mainly restricted to blue light phase (Pohl, 1999). These results further suggest that if birds were able to discriminate between light wavelengths, it may imply that different photoreceptors are involved in photoperiodic regulation of circadian system in birds. This is not surprising in view of the results from other species. In Gonyaulax, it has been shown that different photoreceptors (photopigments) and different pathways to the pacemaker are the basis for

20 20 entrainment of circadian rhythms by qualitative changes in illumination (Roenneberg and Hastings, 1991). In river lamprey, Lampetra japonica, neuronal activity is inhibited by short light wavelengths and excited by middle to long light wavelengths showing the maximum sensitivities of the inhibitory and excitatory responses at about 380 and 540 nm, respectively (Gordon and Brown, 1971). Since environmental light contains both inhibitory and excitatory components, neurons keep both sensitivities during daytime and could measure variation in spectral composition (Holzhausen and Roenneberg, 1991). In birds, spectral sensitivity of the photoreceptors mediating photic entrainment has also been suggested in domestic canary and brambling (Pohl, 1996, 1999). Interestingly, in present section (study 1; experiment 1), when munia were exposed to light and dark regime, in which light phase was replaced by white/blue/red, light wavelengths were able to entrain the locomotor activity showing that the circadian system in munia is responsive to light wavelength and thus any light is considered as subjective day in comparison to dark. This may mean that if not contrasted with other photoreceptors, any particular photoreceptor on its own can entrain the clock. Although the day light environment is always dominated by bright white light, there are very precise spectral changes during the day, especially during twilight periods. Spectral information, which is so unique in twilight times when there are large changes in irradiance and precise changes in the light spectrum, is utilized by fresh water alga Chlamydomonas (Kondo et al., 1991) and marine alga Gonyaulax (Roenneberg and Deng, 1997) to regulate their circadian timing system. But, there are less evidences for light-spectrum based regulation in the vertebrates. There are large changes in the amount

21 21 of light, its spectral composition and in the sun s position relative to the horizon which could be used by organisms for detecting the phase of twilight (Roenneberg and Foster, 1997). It has been demonstrated that twilight is primarily characterized by relative enrichment of the shorter wavelengths (<500 nm) compared to the mid-long wavelengths ( nm) (Peirson et al., 2009). Our results on twilight simulation suggest that transitions of light wavelengths are important and can influence the locomotor activity in spotted munia. Significant difference in total activity was found from red to blue at dawn and from blue to red at dusk (Nouber et al., 1983; Thorne et al., 2009). More activity in red light than in blue light at dusk (group 3) could be explained by higher degree of anticipation for blue light (cf. Fig. 5c). This is supported by other studies in mammals where the evening-active wild rabbits, light increments in blue light advanced the onset of activity than decrements in blue light (Nouber et al., 1983). Free running behavior in few birds at wavelength transition schedule could be explained by studies which showed that light information in eye is processed by two systems which works for image detection and regulation of biological clocks distinctly (i) image forming photoreceptor system, which involve in classical vision and (ii) nonimage-forming photoreceptor system, which detects changes in quantity and quality of light at twilight (Roenneberg and Foster, 1997). It warrants further 'tuned in' experiments to underline the importance of spectral transitions on circadian clock. Adding to this, effects of contrasting light wavelengths at equal energy levels were also performed in two photoperiodic bird species (study 2), which respond to long days positively but, differing in their physiology with blackheaded bunting being

22 22 migratory and Indian weaver birds being non-migratory. Both, buntings and weaverbirds interpreted short (blue) and long (red) light wavelengths applied at equal energy levels in a 12:12h cycle as day and night, respectively, with high activity during blue and low or no activity during red light period, except when buntings were in migratory state and exhibited Zugunruhe in red light night period (cf. Figs. 6 and 7). This similarity in wavelength-dependent response between species was in spite of difference in their annual itineraries and photoperiodic environments that they are exposed to in the wild, and regardless of the order of presentation of light wavelengths. Before used in this study, migratory buntings experienced temperate, consistently varying and subtropical photoperiodic environments during their breeding (~40 N), autumn migration (travel from ~40 N to ~25 N) and wintering periods, respectively, whilst weaver birds inhabited the subtropical (~27 N) photoperiodic environment (Ali and Ripley, 1974; Misra et al., 2004). These results are consistent with previous findings on blackheaded buntings in which in a skeleton photoperiod paradigm (6L:6D:1L:11D or 1L:11D: 1L:11D), B:R was more effective than R:B light regimes in synchronizing activity rhythms and inducing photoperiodic responses measured as fat deposition and weight gain, development of Zugunruhe and testicular recrudescence (Kumar and Rani, 1996; Malik et al., 2004). The response to light wavelength measured in melatonin secretion was not entirely consistent with activity behavior. There was a daily pattern of plasma melatonin with low levels during blue and high during the red light periods, when light wavelengths were presented as B:R but not as the R:B as a first step (in weaver birds only) in the order of presentation (cf. Fig. 8d). A precise reason why in R:B spectral regime, both blue and red light periods had a similar low daytime melatonin level is

23 23 unclear, but we would like to speculate it as if circadian oscillators governing the activity behavior and melatonin secretion respond differentially to spectral regimes. Dissociation between the activity behavior and melatonin secretion has been shown in weaver birds by presenting them to different light intensities in two halves of a 12:12 cycle (Singh et al., 2012a). Also, circadian oscillator(s) governing melatonin secretion may not change their phase, unlike the phase inversion in circadian oscillator(s) governing activity behavior with the onset of Zugunruhe in migratory buntings (Bartell and Gwinner, 2005; Rani et al., 2006). However, because of such a change in phase relationship between the rhythms in activity behavior and melatonin secretion with development of Zugunruhe, there may be an influence on night melatonin levels in migratory buntings (Gwinner et al., 1993), not in weaver birds (cf. Fig. 8). In fact, this is reflected in a significant negative correlation of 12h activity with corresponding melatonin levels in weaverbirds, and its absence (no correlation) in buntings (Fig. 14). Different light wavelength did not induce a differential metabolic reaction which was reflected in the more or less equal levels of triglycerides and blood glucose in both weaver birds and buntings. Again, contrary to our prediction, there was no diurnal pattern in the cortisol levels (Fig. 9). However, we would not rule out yet a daily (circadian) rhythm in cortisol secretion in view of the fact that blood samples twice-a-day in present study may have excluded those times at which cortisol levels were possibly low or high during the day. In mammals including humans, for instance, cortisol secretion with a daily rhythm peaks at the end of night, the sleep period (Weibel et al., 1995). Further, birds may use corticosterone without or with cortisol, as phase marker of their circadian activity (wake) and rest (sleep) cycles, but this is purely speculative at this time. LDH and GOT levels were also in general were not

24 24 different in blue or red light in both the birds which may be the effect of time point or may be spectral components of light induce equal amount of stress in these birds. The effects of spectral B:R and R:B regimes on activity behavior and physiology in the present study, may be due to activation of different groups of deep brain photoreceptor cells, with possibly different photopigment characteristics (Surbhi and Kumar, 2014) during the subjective times of day and night, as per initial 12L:12D regime (Kumar and Rani, 1996; Rani and Kumar, 2000). Consistent with photopigment based circadian entrainment in Gonyaulax (Roenneberg and Hastings, 1991) and mammals (Holzhausen and Roenneberg, 1991), our results may explain a possible difference in synchronization of circadian oscillators governing behavior and physiology in buntings and weaver birds between the B:R and R:B spectral regimes, presented beginning with blue and red light periods in the subjective morning, respectively. Finally, wavelength effects in buntings and weaver birds may not be dependent on light intensity and duration of exposure. A least suppressive long light wavelength (650 nm, red) applied at higher intensity and/ or for a longer duration, can depress night melatonin secretion (Zawilska et al., 1995). This seems to happen in natural photoperiodic environment, where daily alterations in duration, intensity and wavelength are not independent of each other. Furthermore, twilight periods of the natural light environment are rich in short wavelengths (< 500 nm), compared to long wavelengths during mid day (Roenneberg and Foster, 1997), which may explain why a diurnal species like buntings and weaver birds respond to blue light periods as day, of course for the duration that is sufficient to establish a stable synchronization.