Seasonality in circadian locomotor activity and serum testosterone level in the subtropical tree sparrow (Passer montanus)

    Seasonality in circadian locomotor activity and serum testosterone level in the subtropical tree sparrow (Passer montanus) Anand S. Dixit, Namram S. Singh PII: DOI: Reference:

S1011-1344(16)30108-7 doi: 10.1016/j.jphotobiol.2016.02.014 JPB 10251

To appear in: Received date: Revised date: Accepted date:

6 August 2015 17 February 2016 22 February 2016

Please cite this article as: Anand S. Dixit, Namram S. Singh, Seasonality in circadian locomotor activity and serum testosterone level in the subtropical tree sparrow (Passer montanus), (2016), doi: 10.1016/j.jphotobiol.2016.02.014

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ACCEPTED MANUSCRIPT Seasonality in circadian locomotor activity and serum testosterone level in the

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subtropical tree sparrow (Passer montanus)

Anand S. Dixit*1 and Namram S. Singh2 Department of Zoology, North- Eastern Hill University, Shillong-793022, Meghalaya, India 2

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Department of Zoology, Cotton College State University, Guwahati-781001, India

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E mail: *[email protected] and [email protected]

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Running title: Seasonality in circadian activity and testosterone

Corresponding address: *1

Anand S. Dixit

Department of Zoology, North-Eastern Hill University, Shillong-793022, Meghalaya E mail: [email protected] 0364-2722309,-2722311 FAX: +91-364-2550108

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ACCEPTED MANUSCRIPT Abstract Seasonality in daily locomotor activity pattern was investigated in the subtropical tree

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sparrow by exposing a group of birds to natural day lengths (NDL) for 30 days and another

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group to 12L/12D for 14 days followed by transfer to constant dim light (LLdim) for another

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15 days in four different seasons of the year. Serum testosterone levels were also measured during different seasons. Sparrows, under NDL, exhibited distinct circadian rhythmicity in their locomotor activity with almost similar general pattern in different seasons that restricted

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mainly to the light hours. However, they showed season-dependent differences in the

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characteristics of circadian locomotor activity rhythm. Birds, when exposed to 12L/12D, showed entrainment of their locomotor activity rhythm with the activity confined mainly

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during the light phase. Though, tau (τ) under free run conditions did not show any significant

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difference, the activity period varied significantly in different seasons. The highest level of testosterone was recorded in the spring season that corresponded with the maximum

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locomotor activity in spring months. The seasonality in daily locomotor activity correlates with the seasonal changes in testosterone levels suggesting the influence of gonadal steroids

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on endogenous circadian system which is indicative of adaptation of tree sparrow to local photoperiodic conditions. Keywords: locomotor activity, circadian, photoperiod, testosterone, tree sparrow

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1. Introduction

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Majority of behavioural and physiological functions of the organism show rhythmic variation

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with a daily repetition. Most daily rhythms are endogenous, and are merely synchronized by

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the environment [1, 2]. Variable environmental factors (zeitgeber) like light [3, 4], temperature [5, 6], food availability [4, 7, 8,], and social cues [9, 10] oscillate around a 24h period and play a role in this synchronisation [11]. Birds periodically measure some of these

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environmental factors and synchronize their internal rhythms with external periodicity under

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natural conditions [12]. Thus, rhythmic breeding and other physiological processes in birds have evolved through the development of timing mechanisms governed by the oscillatory

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systems. Of all the zeitgebers that may act as entraining agents to circadian rhythms, the most

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prominent are the daily changes in day and night [3, 13]. The synchronization of circadian rhythms is achieved by zeitgeber induced phase shifts which are possible in two directions:

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phase advance (the onset of circadian rhythm is earlier than the onset of the zeitgeber) and phase delay (the onset of the zeitgeber leads to the onset of circadian rhythm) [14, 15]. Phase

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angle difference (ψ) is time difference between zeitgeber and the displayed internal rhythm. Testosterone (T) regulates behaviours such as song production and territorial aggression during breeding seasons in most avian species [16]. Increased plasma levels of T in the spring months induce expression of secondary sexual characters, sperm production, and sexual behaviour in male birds [17]. Increase in T level has also been shown to modulate locomotor activity rhythm in some avian species [18, 19, 20, 21]. The possibility that testosterone may affect the circadian system is of considerable interest not only because such effect could explain some of the variations in activity period (α) and phase angle difference (ψ); hormonal changes may, in addition, be the basis of the dramatic seasonal changes in the

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ACCEPTED MANUSCRIPT circadian pattern of locomotor activity in migratory birds [21]. There may be similar effect in seasonally breeding non migratory birds that needs investigation.

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Among vertebrates, birds show well defined circadian rhythmicity [22, 23] in distinct

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physiological and behaviour features that change with the change of season [3]. Seasonal

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changes in day and night cycle influence daily change in pattern of short term behavioural functions such as locomotor activity in many avian species [24, 25, 26]. Most of these studies focus on birds of temperate region and especially on migratory species. Investigations at

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relatively lower latitudes are fewer in view of large number of avian inhabitants at these

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latitudes. As various characteristics of natural photoperiod (amplitude, length and duration) vary systematically not only with the change of seasons at a given latitude but they also vary

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with the change of latitudes. Therefore, critical laboratory experiments are required on birds

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inhabiting low latitudes also to observe similarity/differences in their responses with greater attention on local non-migratory species. Therefore, we carried out present study at Shillong

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(25°34’N, 91° 53’E) on the tree sparrows (Passer montanus), a resident species, to study their locomotor activity pattern and also to test whether they show seasonality in their circadian

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clock characteristics. The correlation between seasonal variations in T concentration and circadian characteristics was also examined to understand the adaptive strategies of sparrows in the changing photoperiod. 2. Materials and Methods The study was done during four different seasons of a year (winterDecember/January, spring- March/April, summer-June/July and autumn-September/October) with the experiments beginning at the times of longest (June), shortest (December) and two equinoxes (March and September) photoperiods. Male tree sparrows were captured from the wild and acclimatized to captive conditions in an outdoor aviary for 7 days before bringing them indoors and subjecting them to various experiments. Locomotor activity of the birds 4

ACCEPTED MANUSCRIPT under experimentation was measured by keeping birds singly in activity cages (60 x 45 x 35 cm) furnished with two perches and mounted with a Napoleon miniature passive infrared

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detector (Maximum electronic Ltd., Israel) with a range of 16m and wide angle (100°) field

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of view to detect movement of the bird in the cage. Each sensor was connected to separate

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channel of a window XP-compatible computer and the recording and analyses of the activity data (actogram) were done by using the Chronobiology Kit (Release Version 1c, © 19982004) software program of the Stanford Software Systems (Stanford, USA). Food and water

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were provided ad libitum, and replenished twice a week. In photoperiodic experiments, the

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activity cages carrying birds were kept in light proof wooden chambers (size: 90 x 75 x 75 cm) illuminated by light available from compact fluorescent tubes (CFL, Phillips). Light on

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and off were controlled by automatic digital time switches (Crono Digital Time Switches,

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Larsen and Toubro LTD., India). The photoperiodic chambers were well aerated through inlets and outlets connected to air circulators.

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2.1. Locomotor activity under natural day length: This experiment was carried out during four different seasons over the year i.e.

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winter, spring, summer and autumn and began in the middle of December, March, June and September, respectively. Acclimatized birds (n=5) were kept individually in activity cages receiving natural photoperiods to record their locomotor activity for 30 days. Here, they were visually separated by wooden plank partitioning between the cages. The times of sunrise and sunset were recorded from the local meteorological station as published daily in “Shillong Times” newspaper. 2.2. Locomotor activity under artificial day length: This experiment was also performed at four different times in a year as mentioned above. Acclimatized birds (n = 5) were housed individually in activity cages and exposed to a

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ACCEPTED MANUSCRIPT constant photoperiod of 12L/12D with the light intensity of ~400 lux in the light phase in a photoperiodic chamber for 14-days. Birds were then released in LLdim with light intensity of

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<1 Lux for another 15 days and their locomotor activity was recorded.

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2.3. Testosterone assay:

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Blood samples were collected at the beginning of the experiments in four different season of the year. Briefly, the blood samples were collected in a small volume of about 100-

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150µl with the help of a capillary tube by puncturing the wing vein using a 26 gauge needle. This type of blood sampling is almost non-invasive and causes no risk to bird health. The

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serum was collected and frozen at - 40oC until used for the hormone assay. A highly sensitive and specific commercially available ELISA kit (Dia Metra,Italy) was used for the estimation

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of testosterone (product no. DK0002) in blood serum following the protocol supplied with the

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kit. This assay has been validated and used for the measurement of testosterone in other avian

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and fish species [27, 28]. However, a validation test of this assay was not performed for our species. The absorbance was recorded at 450 nm with an automatic microplate reader (BioRad iMarkTM Microplate reader). Serum testosterone concentration was expressed as

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ng/ml. The lower detection limit of the assay for testosterone was 0.075ng/ml. The intra- and inter-assay variations were 4.6% and 7.5%, respectively. 2.4. Statistical analyses: The data from different experiments are presented as mean ± S.E.M. Analyses of period and activity distributions were done using the activity recorded in the first 10 days. The data were analyzed using a two-way ANOVA followed by post hoc Bonferroni test for group comparisons. A one way ANOVA was also used when only one factor was involved followed by Newman-Keul΄s Multiple range‘t’test, if ANOVA indicated a significant

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ACCEPTED MANUSCRIPT difference. Significance was taken at 95% confidence limit. Cosiner analysis was done as per Singh et al. [29]. No mortality was recorded during the course of experiment.

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3. Results

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3.1. Locomotor activity under natural day length:

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A distinct rhythmicity was observed in the locomotor activity of tree sparrow maintained under natural photoperiods in all four seasons over the year (Fig. 1 a-d). They

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showed similar general patterns of activity in different seasons. However, birds exhibited season-dependent differences in the characteristics of activity rest cycle (Fig.1 a-d). The

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cosiner regression curve of circadian activity exhibited a distinct sinusoidal rhythmicity in all the four season (Fig. 2 a-d). The circadian activity was found significantly different among 3, 198

=52.25, P<0.0001; hours: F23,

198=46.93,

P<0.0001 and

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the four seasons (season: F

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interaction: F69, 198=3.65, P<0.001; two way ANOVA: Fig. 2 a-d). There was gradual increase

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in average activity per hour from winter leading to its peak in spring, declining slightly thereafter in summer and reaching to its minimal value in autumn (Fig. 2 a-d). The average activity per hour was significantly higher (P<0.001) in the spring when compared to those of

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other three seasons over the year (Fig. 2 a-b). Further, the average activity recorded in summer was found significantly higher (P<0.001) than those observed in winter and autumn (Fig. 2 a, c and d). The activity in autumn was significantly (P<0.05) lower as compared to activity in winter (Fig. 2 a and d). Various characteristics of the activity rhythm in tree sparrow were affected by seasons and the bird showed peak activity much earlier in the spring season (Acrophase: 4.62±0.25h; Mesor: 210.5±13.71 and Amplitude: 296.3±19.38; r2=0.7719) followed by the winter (Acrophase: 4.98±0.28h; Mesor: 134.1±10.21 and Amplitude: 194.1±14.44; r2=0.7237), then summer (Acrophase: 5.07±0.31h; Mesor: 206.3.03±13.37 and Amplitude: 257.1.±18.90; r2=0.7284) and finally autumn (Acrophase: 5.40±0.21h, Mesor: 82.5±4.68 and Amplitude: 7

ACCEPTED MANUSCRIPT 116.5±6.62; r2=0.8176) seasons (Fig. 3). A compression of phase difference (ψ) between activity on and sun rise for first ten days of the study revealed significant seasonal variations 80

=9.188, P<0.0001; days: F9,

80=1.493,

P=0.164; interaction: F27,

80=1.576,

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(season: F3,

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P=0.0617; two way ANOVA; Fig. 4 a). Similar results were obtained when seasonal phase

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differences between activity off and sunset were compared (season: F3, 80 = 8.449, P< 0.0001; days: F9, 80=1.115, P=0.3621; interaction: F27, 80=1.032, P=0.4390; two way ANOVA: Fig. 4 a). ψ between onset of activity and sunrise was recorded maximum in spring (+0.94±0.07h)

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and minimum in autumn (+0.69±0.05 h). However, the ψ between offset of activity and

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sunset was maximum in spring (+1.08±0.08h) but minimum in winter months (+0.78±0.03h). Significant variation in the α (seasons: F3, 80 = 172.9, P< 0.0001; days: F9, 80=0.7558, P = 0.6670 and interaction: F27, 80=1.322, P=0.1698; two way ANOVA) was observed among the

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birds of different seasons. The α increased gradually from winter through spring and attained maximum in summer (June/July: 13.18±0.11h) that declined thereafter in autumn (Fig. 4 b).

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Further, two way ANOVA showed significant difference in activity movement per day among the birds of four different seasons (seasons: F3, 240 = 88. 37, P<0.0001) but not among

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days (F29, 240 =0.7264, P=0.8477) and interaction (F87, 240 =0.6017, P=0.9967). The activity was significantly higher in birds of winter, spring and summer (P<0.001, Fig. 5) when compared to autumn birds. 3.2. Locomotor activity under artificial day length: There was entrainment of activity rhythm under light dark cycle of 12L/12D with activity confined mainly in the light phase (Fig. 6 a-d). Two way ANOVA revealed significant variation in the circadian activity in different seasons (season: F3,

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=3.598,

P<0.0001; days: F23, 198 =55.75, P<0.0001 and interaction: F69, 198 =3.598, P<0.0001; Fig. 7 ad). The circadian activity movement was significantly higher in winter months when compared to the spring (P<0.001), summer (P<0.05) and autumn (P<0.001) seasons. Further, 8

ACCEPTED MANUSCRIPT the movement was significantly more (P<0.001) in spring and summer months when compared to autumn months. There was no significant difference (P>0.05) between the

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movement in summer and spring months. Analysis using cosiner regression curve based on

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activity movement revealed sinusoidal rhythmicity in all the four seasons (Fig. 7 a-d.). The α

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was observed earlier in summer (Acrophase: 3.68±0.28 h; Mesor: 173.8±12.48 and Amplitude: 237.7±17.66; r2=0.7242) followed by spring (Acrophase: 4.48±0.27h; Mesor: 175.4±11.79 and Amplitude: 235.7±16.68; r2=0.7433), winter (Acrophase: 4.49±0.21h;

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Mesor: 227.8±10.88 and Amplitude: 293.9±15.39; r2=0.8410) and autumn (Acrophase:

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6.16±0.36h; Mesor: 61.79±5.35 and Amplitude: 80.82 ±7.56; r2=0.6231) seasons (Fig. 8). Significant differences in onset (season: F3, 80=0.6699,

=13.54, P<0.0001; days: F9,

P=0.879) and offset (season: F

80=0.6353,

3, 80

=11.76,

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P=0.7636 and interaction: F27,

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P=0.0045; days: F9, 80=0.6353, P=0.7636 and interaction: F27,80=0.7933, P=0.7470; two way ANOVA) of activities were observed among different seasons when the differences between

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light ON and start of the activity and between light OFF and the end of the activity were compared for the first 10 days (Fig. 9 a). Two way ANOVA revealed significant difference

80 =1.185,

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among activity periods (α) during different seasons (season: F3, 80 =6.872, P=0.0004; days: F9, P=0.3162 and interaction: F27, 80 =1.180, P=0.2802; Fig. 9 b). The α during winter

(12.74±0.10 h) was significantly longer when compared with that of autumn (12.32±0.09 h: P<0.05). Further, the α was significantly more in spring when compared to summer (P<0.01) under 12L/12D. The birds with entrained activity under12L/12D started showing free running activity pattern when transferred to LLdim, (Fig. 6a-d). The τ, calculated from the activity records of 14 days in the free running condition, did not show any significant difference among different seasons (F 3, 11 =0.1148, P=0.9466: one way ANOVA; Fig. 10 a). However, significant variation in α, under free running condition, was noticed among four seasons (seasons: F

3, 80

=20.31, P<0.0001; days: F

9, 80

=1.006, P=0.4421 and interaction: F

27, 80

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ACCEPTED MANUSCRIPT =1.020, P=0.4544; two way ANOVA). α was much higher in spring when compared with those in winter, summer (P<0.001) and autumn (P<0.01) months (Fig. 10 b). Mean

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activity/day for total duration of the experiment (period of exposure to 12L/12D and LLdim)

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differed significantly among the seasons (seasons: F 3, 240 =59.77, P<0.0001; days: F 29, 240 =

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8.181, P<0.0001 and interaction: F 29, 240 =8.181, P<0.0001; two way ANOVA; Fig. 11). Mean activity count was significantly higher (P<0.01) in winter, spring and summer seasons

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when compared to that of autumn season. 3.3. Seasonal variation in testosterone levels:

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Significant variation in the serum levels of testosterone (F3, 19=17.60, P<0.0001; one way ANOVA: Fig. 12) was observed in the blood samples collected during four seasons of

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the year. The peak testosterone concentration was recorded in spring and was significantly

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higher (0.856±0.06 ng/ml) when compared with those of summer (P<0.01), autumn

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(P<0.001) and winter (P<0.001) seasons. Though the testosterone concentration declined in summer birds, it was significantly higher than autumn (P<0.05) birds. The minimal level of

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testosterone was recorded in autumn. 4. Discussion

The tree sparrow displays a circadian pattern in its locomotor activity with a strong preference of restricting activity during the light phase and arresting it in the dark phase as is true for a day active bird. The preference of an organism in being active during the day or night has an evolutionary significance since it enables organism to perform certain activities at the time of the day or the year when it is most advantageous. The cosiner regression curve of circadian activity exhibited a distinct sinusoidal rhythmicity in all the four seasons (Fig. 2 a-d). Further, the daily activity rhythm of sparrows gets entrained to daily changing photoperiod; this is done on a day to day basis. The adaptive value of a circadian rhythm lies

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ACCEPTED MANUSCRIPT in its being rightly phased in relation to the external environment. This is achieved by the synchronization of the circadian oscillators with zeitgebers. Though the birds exhibit almost

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similar general pattern of activity among four different seasons over a year, they show

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season-dependent differences in the characteristics of activity rest cycle (Figs. 2-5). The

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entrainment of locomotor activity to a specific phase of the day may be a result of physiological or morphological constraint which commonly reflects the time most advantageous to the sparrow, that is, when food resources are most abundant and attainable or

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when predators are least active [30, 31]. The above observations are consistent with those

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obtained on other birds studied under changing natural photoperiod such as house swift, Apus affinis [32]; great tits, Parus major [33]; white crowned sparrows, Zonotrichia leucophrys

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gambelii [34]; starlings, Sturnus vulgaris [35]; house sparrow, Passer domesticus [36, 37]

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and blackheaded bunting, Emberiza melanocephala [38]. Our birds displayed higher activity in morning than in the evening hours (Fig. 2 a-d)

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and this was more distinct in spring and summer when compared with the other two seasons (winter and autumn). A study on house sparrow by Trivadi et al. [37] revealed a bimodal

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activity which was considered as a reflection of the two oscillatory components (morning and the evening) of the circadian system. The bimodality was imprecise in May, June, and October. Almost similar bimodal activity pattern, as observed in house sparrow, was also noticed in the European starling [18] and the blackheaded bunting [38]. However, such bimodality was not observed in our study birds (Figs. 1 and 2). The α in tree sparrow increases with increase in day length in spring attaining maximum in summer and declining thereafter in autumn reaching to minimum in winter season under NDL (Fig. 4 b). This is supported by the study of Binkley and Mosher [39] who observed that the α was much longer in the house sparrow held under 16L/8D than under 8L/16D. A study on temperate population of house sparrows reported shortening of the α to 9.6 h in the month of December

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ACCEPTED MANUSCRIPT when natural days were only 9.35 h long, and lengthening of the α to 15 h in June when natural days were 15.02 h long [36]. It was further observed in the tree sparrow that the

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changes in day length during different seasons change the characteristics of the daily activity

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rhythm. Significant differences in activity onset (P<0.01) and offset (P<0.05) when

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compared with the light ON and OFF, respectively, were observed in four different seasons. There were phase advances in activity onsets and offsets which were recorded maximum in spring season (onset: +0.94±0.07h and offset: +1.08±0.08 h) in our study bird (Fig. 4 a). A

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study on house sparrow also revealed changes in the activity onsets, ends, and activity

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duration with shortening and lengthening day lengths. There was phase lead (+ψ) of activity onsets by about 0.2h, with reference to the sunrise, when days were lengthening, and of

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activity ends by about 0.2 h, with reference to the sunset, when days were shortening [36].

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Photoperiod has been reported to alter the phase relationship between onset of activity and the time of light on (ψ) in some avian species [13, 39, 40]. Further, the daily activity began

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earlier in relation to sunrise during spring in the breeding season than on similar day lengths during autumn in the non-breeding season in our study bird. Similar observations were also

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reported in some free living birds [25]. This early onset of locomotor activity during spring months may be advantageous to birds in territorial defence and in other activities scheduled to occur in the early morning hours indicating the influence of seasons on circadian system in relation to annual cycle of reproduction. There was entrainment of activity rhythm of sparrow under 12L/12D with activity confined mainly in the light phase. Birds showed continued activity throughout the 12h light period that subsided at light off under 12L/12D. Similarly, house sparrows also exhibit entrainment of their activity rhythm under 16L/8D and 8L/16D [31]. Tree sparrows, when transferred from 12L/12D to LLdim (constant condition), revealed their endogenous rhythm of locomotor activity which free-ran in the absence of a zeitgeber (LD cycle). The starting point

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ACCEPTED MANUSCRIPT of the free-run coincided with the phase angle of entrainment during the 12L/12D cycle (Fig. 6 a-d). This suggests that the light-dark cycle was most likely the principle entraining agent

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rather than the rhythm being the consequence of masking [41]. Though the τ for the free

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running activity rhythm did not differ significantly under different seasons in tree sparrow,

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the α varied significantly with maximum in spring season (Fig. 10 a and b). In our study, phase setting was observed in LLdim (constant condition) following the transfer of sparrow form 12L/12D. Birds when transferred from 12L/12D to LLdim, began their activity

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11.25±0.47h (winter), 13.33±0.52h (spring), 11.507±0.355h (summer) and 12.32±0.45h

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(autumn) from the last light dark cycle in which α were 12.74±0.15 h, 12.53±0.15h, 12.525±0.13h and 12.31±0.21h, respectively. In a different study on house sparrow, a similar

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result was obtained when the birds were transferred form 16L/8D and 8L/16D to DD [39].

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However, in a study on the house sparrow by Trivedi et al. [37], conducted at around the times of two equinoxes (i.e., March and September), the activity rhythms were synchronized

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to the time of sunrise with a positive phase angle difference (+ψ) of 0.41 ± 0.05 h (March) or 0.31 ± 0.06 h (September). When deprived of daylight hours from sunrise and sunset,

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sparrows remained synchronized with a similar phase angle difference: +ψ= 0.80 ± 0.24 h (March) or 0.37 ± 0.06 h (September). Study on mean activity count per day in tree sparrow under natural (NDL) and artificial (12L/12D and LLdim) photoperiodic conditions revealed high activity in spring and summer seasons as compared to autumn. (Figs. 5 and 11). These responses correlate well with the activity of birds in nature during spring and summer months when they remain actively engaged in finding preferred mate, nest building, rearing of the young ones, searching foods and territorial defence. Thus, the tree sparrows exhibited seasonal trend in the characteristics of their circadian activity without significantly altering the τ of their endogenous circadian system (Fig. 10 a). Further, the seasonal changes in activity correspond to the changes in the

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ACCEPTED MANUSCRIPT natural photoperiodic cycles showing adaptation of endogenous circadian system to their photoperiodic conditions.

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Serum levels of testosterone were found to be maximum in the spring and minimum in

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autumn (Fig. 12) in nature in our birds. The activity movement per day under NDL (Fig. 5)

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followed the trend observed in testosterone levels and was recorded greater in spring and summer seasons. Similarly, the activity movement per day was significantly higher in spring and summer seasons as compared to the autumn even under the entrained (12L/12D) and free

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running (LLdim) conditions (Fig. 11) in our birds. The birds transferred to LLdim during the

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reproductive season with significantly higher testosterone levels showed a longer α than the birds moved to constant condition during the non-breeding season having lower testosterone

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levels (Figs. 10 b, 12). Further, maximum phase advance in activity onsets was recorded

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during spring in our study bird (Fig. 4 a). These observations indicate that the testosterone level is somehow correlated with the duration of activity (α) and the activity movement per

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day in the tree sparrow. However, further investigation on the effect of testosterone treatment on locomotion is required to reach to any conclusion. Similar results were observed in some

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other birds when transferred to LLdim during the breeding season [21, 35]. Furthermore, while α increases steadily in the non-castrated birds whose testes grew in LL, it remains the same in the castrated birds [21]. Finally, injection of testosterone in castrated birds induces clear increases in α. These reports on the effect of testosterone on α on some birds together with the results obtained in our experiments on tree sparrow clearly indicate that the daily activity time of free-living male birds is considerably longer and often starts considerably earlier during the breeding season than on the days with similar photoperiods in autumn in the nonbreeding season. An earlier onset of activity may be of adaptive significance in these birds when they establish or defend a territory which are the activities known to be concentrated to the early morning hours. The experiments on male tree sparrow indicate that

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ACCEPTED MANUSCRIPT the variations in the levels of testosterone have no effect on the period of circadian activity rhythms (τ) over the seasons under artificial photoperiods (Fig. 10 a). This is in conformity

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with the report on some male birds that the testosterone has only little or no effect on the

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period of circadian activity rhythm [42, 43, 44, 45]. Still, there is some limited evidence

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suggesting that in the Common redpoll (Carduelis flammea) and in the European starling (Sturnus vulgaris), τ measured in LL is shorter during the breeding season that at other times of the year [20, 21]. However, other studies have failed to find such a relationship [35, 46].

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Moreover, in starling, maintained in LL, neither castration nor testosterone treatment of

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castrated male changes the period of circadian activity rhythm [21]. Thus, our findings on tree sparrow suggest that although the testosterone has no effect on τ, it does have effects on

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α and some other circadian characteristics. The mechanism by which these effects take place

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is not known. It has been pointed out that there are a variety of direct and indirect ways steroid hormones could influence the circadian system [42, 43, 44, 45]. A direct action of sex

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steroid on the clock system itself has been questioned, but not ruled out, since the suprachiasmatic nuclei (SCN) is not a major tissue for the uptake or binding of sex steroids

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[45]. A variety of indirect influences have also been proposed, including the possibility that steroid hormones act on steroid sensitive neurons which have input to the circadian pacemaker. In addition, due to the many hormonal changes which occur in response to alterations in circulating steroid levels (eg. change in gonadotropins and gonadotropinreleasing hormone activity), it may be that other hormones mediate the effects of steroids in circadian rhythmicity [47]. The production of testosterone (T) in male tree sparrows facilitates reproductive physiology and behaviors needed to compete for mates and defend territories during the breeding season. This is supported by a study on the Lapland longspurs (Calcarius lapponicus) that indicated possible correlation between photoentrainment and the elevated concentration of sex steroids. The temporal changes in circulating levels of

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ACCEPTED MANUSCRIPT testosterone may be the result of cost-benefit trade-off i.e., high level of testosterone is advantageous for successful territorial and mate acquisition [48]. Once territorial boundaries

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are established, testosterone levels decline as male – male interaction decrease and parental

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care take over [49].

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5. Conclusions

Seasonality in daily activity rest cycle in tree sparrow correlates with the seasonal reproductive cycle whereby hormonal changes corresponding to reproduction influence the

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endogenous circadian system to regulate the activity. Tree sparrows show circadian

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characteristics that are almost similar to those of its temperate populations with minor differences, which might be indicative of the local adaptations. Although there are noticeable

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seasonal differences in certain characteristics of the circadian activity rhythm, which tend to

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indicate that hormonal changes might be involved but all that is purely correlative at this stage. It seems most likely that the processes involved in control of certain features of the

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circadian rhythms such as the activity duration show seasonal fluctuations without affecting more fundamental features of the circadian rhythms such as the circadian period, which

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appears to be the characteristic of the species. It may be concluded on the basis of the present study that the subtropical population of tree sparrow possesses distinct seasonality in the circadian activity rhythm that correlates with seasonal fluctuations in reproductive functions indicating their adaptation to local photoperiodic conditions. Conflict of Interest: The author(s) declared no conflicts of interest to authorship, research or publication of this article. Acknowledgements: Financial support through DST grant to A.S.D. from the Department of Science and Technology of the Government of India, New Delhi is gratefully acknowledged. References

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Figures

Figure 1: Activity rest cycles in the tree sparrow under natural day length (NDL) during four

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seasons of the year.

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Figure 2: Distribution of activity-1h over 24 h under NDLduring four seasons of the year.

daily rhythms.

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Dotted lines represent the cosiner regression curve drawn through the time points to deduce

year.

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Figure 3: The time of peak activity (acrophase) under NDL in four different seasons of the

Figure 4: a) Phase angle differences upon comparing activity onset with light ON and activity offset with light OFF and b) activity period for the first 10 days during four different seasons of the year under NDL. Figure 5: Activity movement per day in four different seasons of the year. Figure 6: Representative actograms of tree sparrow exposed to 12L/12D and then released into LL dim in four different seasons over the year.

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ACCEPTED MANUSCRIPT Figure 7: Distribution of activity-1 h over 24 h under 12L/12Dduring four seasons of the year. Dotted lines represent the cosiner regression curve drawn through the time points to

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deduce daily rhythms.

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Figure 8: The time of peak activity (acrophase) under 12L/12D in four different seasons of

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the year.

Figure 9: a) Phase angle differences upon comparing activity onset with light ON and activity

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offset with light OFF and b) activity period for the first 10 days under 12L/12D in four

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different seasons of the year.

Figure 10: (a) tau and (b) activity period for the first 10 days under LL dim in four different

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seasons of the year.

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Figure 11: Plot of activity movement per day over the duration of the experiment under

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12L/12D and LL dim in four different seasons of the year. Figure 12: Variation in circulating testosterone levels in blood serum during four different

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seasons of the year.

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ACCEPTED MANUSCRIPT Highlights Tree sparrow exhibits circadian rhythmicity in activity that confine to light periods.

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They show season-dependent differences in characteristics of circadian activity rhythm.

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Entrained activity rhythm in 12L/12D free runs under LLdim without seasonal change in τ.

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Seasonal changes in activity followed testosterone levels with peaks in spring.

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Testosterone influence circadian system indicating adaptation to local photoperiod. .

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