RESEARCH ARTICLE Circannual testis and moult cycles persist under photoperiods that disrupt circadian activity and clock gene cycles in spotted munia Neha Agarwal 1,2 , Ila Mishra 1 , Ruchi Komal 2 , Sangeeta Rani 2 and Vinod Kumar 1, * ABSTRACT We investigated whether circannual rhythms underlying annual testis maturation and moult cycles are independent of duration and frequency of the light period and circadian clock control in non- photoperiodic spotted munia. Birds were subjected to an aberrant light–dark (LD) cycle (3.5 h L:3.5 h D; T7, where T is the period length of the LD cycle) and continuous light (LL, 24 h L:0 h D), with controls on 12 h L:12 h D (T24, 24 h LD cycle). We measured the behavioural activity pattern of the birds and 24 h mRNA oscillations of circadian clock genes (bmal1, clock, per2, cry1, cry2) in the hypothalamus, the putative site of seasonal timing. Diurnal munia were rhythmic in behaviour with the period of the activity–rest cycle matched to T7 and T24, and became behaviourally arrhythmic with activity scattered throughout 24 h under LL. Similarly, exposure to 3.5 h L:3.5 h D and LL caused arrhythmicity in 24 h clock gene expression, suggesting disruption of internal circadian timing at the transcriptional level; a significant rhythm was found under 12 h L:12 h D. During an exposure of 80 weeks, munia showed two to three cycles of testis maturation and wing primaries moult under all photoperiods, although with a longer period under 12L:12D. Thus, the frequency of light period under 3.5 h L:3.5 h D or LL disrupted circadian clock gene cycles, but did not affect the generation of circannual testis and moult cycles. We conclude that the prevailing light environment and hypothalamic circadian gene cycles do not exert direct control on the timing of the annual reproductive cycle in spotted munia, suggesting independent generation of the circadian and circannual rhythms in seasonally breeding species. KEY WORDS: Circadian rhythm, Circannual rhythm, Light–dark cycle, Moult, Spotted munia, Lonchura punctulata, Testicular cycle INTRODUCTION Most bird species reproduce at the best-suited time of the year, since mistiming will have severe fitness consequences for both parents and offspring (Helm et al., 2009; Helm and Lincoln, 2017). Birds show seasonal gonadal growth–regression cycles in response to the external environment, prevalently to the annual photoperiod changes (Kumar et al., 2010). Thus, annual time-keeping appears to operate at two levels. A wealth of accumulated evidence suggests the involvement of circannual clocks in the timing of changes in physiology and behaviour within each year. Captive birds show repeated cycles of gonadal maturation and moult with a cycle length of about 1 year under constant ‘neutral’ photoperiods [e.g. 12 h light:12 h darkness (12L:12D) or 12.25L:11.75D; Gwinner, 1986, 1996; Budki et al., 2012, 2014] or continuous light (LL; Holberton and Able, 1992; Budki et al., 2012, 2014). Also, birds show an annual gonadal cycle under the prevailing natural photoperiods, i.e. the annual photoperiod cycle is used as a calendar to time the gonadal maturation–regression cycle (Gwinner, 1986; Dawson et al., 2001; Kumar et al., 2010). This underscores that autonomous molecular switches respond to the photoperiod change and control the seasonal physiological states that make up an annual cycle. Circannual and photoperiodic timing may not be mutually exclusive. For example, annual photoperiod variations can entrain circannual gonadal cycles to periods as short as 4 months and as long as 2 years (Gwinner, 1986). Contrary to this, many low- latitude and equatorial seasonally breeding birds show persistent circannual cycles in gonadal maturation–regression, and fail to show a typical short- or long-day response (Chandola et al., 1975; Thapliyal, 1981; Gwinner, 1996). Furthermore, following gonadal maturation, several photoperiodic seasonally breeding birds undergo regression and exhibit photorefractoriness, and they continue to remain gonadally regressed as long as held under stimulatory long days (Sansum and King, 1976; Malik et al., 2014). As yet, less is understood about how a circannual timer operates at the cellular or molecular level (Kumar et al., 2010). A few recent studies advocate the pars tuberalis (PT) of the pituitary gland as the site of the ʻcircannual timer’, based on the role of PT-derived thyroid-stimulating hormone (TSH) in driving the expression of genes encoding type 2 and 3 deiodinases (dio2, dio3) in the ependymal tanycytes (Hazlerigg and Loudon, 2008; Shinomiya et al., 2014). Intriguingly, much of the evidence for PT TSH- induced deiodinase-dependent control of gonadotrophin-releasing hormone (GnRH) release, and consequently the initiation– termination–reinitiation of the gonad development cycle, comes mainly from the photoperiodic species, irrespective of whether they breed in the summer or winter. Therefore, PT TSH could be a regulatory output, and is not necessarily an integral component of the circannual time generator. Clock genes are, by contrast, an integral component of circadian timing. Further, PT TSH expression is dictated by circadian clock- controlled night melatonin secretion in mammals (Dardente et al., 2010), and by direct light input from the hypothalamic photoreceptors in birds (Nakane et al., 2010). Avian hypothalamic photoreceptors may also be involved in the measurement of photoperiod length, as shown by changes in the expression of neuropsin and rhodopsin photopigments between short and long photoperiods (Majumdar et al., 2015; see also Nakane et al., 2010; Stevenson and Ball, 2012). Thus, we could envisage a role of the circadian pacemaker system (CPS) in the annual timing of seasonal events. In birds, CPS is composed of circadian clocks in the hypothalamus, pineal gland and retina (Cassone and Menaker, 1984; Kumar et al., 2004; Cassone and Yoshimura, 2015). Near- 24 h timing in these clocks is generated by an autoregulatory Received 7 August 2017; Accepted 12 September 2017 1 IndoUS Center for Biological Timing, Department of Zoology, University of Delhi, Delhi 110 007, India. 2 Department of Zoology, University of Lucknow, Lucknow 226 007, India. *Author for correspondence (drvkumar11@yahoo.com) V.K., 0000-0002-0523-8689 4162 © 2017. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2017) 220, 4162-4168 doi:10.1242/jeb.167809 Journal of Experimental Biology