Molecular and epigenetic regulation of seasonal reproduction in Terai tree frog (Polypedates teraiensis).

Seasonal breeders predominantly use photoperiod as the predictable environmental cue to time their reproduction. Terai tree frogs are long-day seasonal breeders, but the molecular mechanism is unknown. We tested the role of different photoperiodic conditions on expression levels of candidate genes involved in seasonal reproduction and epigenetic regulation. Four experiments were performed. In experiment 1, frogs were exposed to long (LD: 16L:8D) or short photoperiod (SD: 8L:16D). In experiment 2, animals were procured at four different phases of breeding, i.e., during April (emergence just after hibernation), June (breeding phase), August (post-breeding), and October (just before hibernation). In experiments 3 and 4, frogs were exposed to equinox photoperiod but different (10, 100, or 500 lx) light intensities (exp. 3) or wavelength (red: 640 nm, green: 540 nm, blue: 450 nm or white; exp. 4). After 2 weeks, animals were euthanized, and their brain was harvested. mRNA levels of transcripts involved in photoperiodic transduction (Eya3 and Opn5), reproduction (Tshß, GnRH, Dio2, and Dio3), and epigenetics regulation (Dnmt1, Dnmt3a, Hdac1, Hdac3, and Tet2) were measured. Results show that LD promotes the upregulation of Eya3, Opn5, Tshß, GnRH, and Dio2. Differential expression of Opn5 during LD and SD suggests its involvement in light perception. Dio3 levels were upregulated in SD (exp.1) and during the post-breeding phase (exp. 2). These results employ the limited role of light intensity and spectrum in reproduction. This is the first study showing molecular machinery involved in the amphibian system's seasonal reproduction and epigenetic regulation.

Season dependent effects of urban environment on circadian clock of tree sparrow (Passer montanus).

Great efforts have been made recently to understand the effect(s) of urban environments on the circadian and seasonal physiology of wild animals, but the mechanisms involved remain largely unknown. Most laboratory studies and a few studies on animals in the wild suggest alterations occur in the physiological functions of organisms in urban habitats. Here, we addressed the effects of the interaction of seasons and urban environments on clock gene expression in three tissues of tree sparrows (Passer montanus). Tree sparrows (N = 30 per site per time of year) were procured from rural and urban habitats during periods corresponding to their three physiological states, i.e., June (longest photoperiod; reproductive phase), September (equinox photoperiod; refractory phase), and December (shortest photoperiod; sensitive phase). Birds (N = 5 per time per site per month) were sampled at six time points; ZT1, ZT5, ZT9, ZT13, ZT17, and ZT21 (ZT0 = sunrise time) and clock gene expression in the hypothalamus, pineal gland, and retina was studied. Our results show that there is persistence of the circadian clock in both rural and urban birds throughout the year. In urban birds Bmal1, Npas2, Per2, and Cry1 acrophases were advanced, compared to rural birds, while Clock acrophase was delayed, depending on the tissue and time of year. This difference could be because of changes in the availability, duration, and intensity of sunlight during different times of the year and/or differential photoreceptor sensitivities, differential physiological states, or a combination of all these factors. These important results reveal, for the first time in any species, season-dependent effects of an urban environment on the molecular machinery of the circadian clock.

Circadian timing in central and peripheral tissues in a migratory songbird: dependence on annual life-history states.

Predictable seasonal change in photoperiod triggers a sequential change in the daily activity-rest pattern, adaptive for migration in several bird species. The night-migratory black-headed bunting (Emberiza melanocephala) is day active under short photoperiods (8 h light:16 h dark, short day sensitive). Under long photoperiods (16 h light:8 h dark), the buntings are initially day active (long day premigratory) but subsequently become intensely night active (long day migratory) and after few weeks again return to a day active pattern (long day refractory). However, it is unclear how the daily expression of circadian genes changes during photoperiod-induced seasonal life-history states (LHSs). We measured period 2 (Per2), cryptochrome 1 (Cry1), brain and muscle arnt-like protein 1 (Bmal1), and circadian locomotor output cycles kaput (Clock) mRNA expressions in various neural and peripheral tissues of buntings in different LHSs and discovered differences of ∼2 to 6 h in the phase and 2- to 4-fold in amplitude of circadian oscillations of Per2, Cry1, and Bmal1 between photoperiod-induced LHSs. Phase relationship in mRNA oscillations was altered between oscillator components in the circadian pacemaker system (retina, pineal, hypothalamus) as well as in the peripheral (liver, muscle) tissues. These results show for the first time altered waveforms of clock gene expressions in all tissues in parallel with behavioral shifts and suggest the involvement of circadian system in photoperiod induction of seasonal LHSs in a migratory species.

Nocturnal melatonin levels decode daily light environment and reflect seasonal states in night-migratory blackheaded bunting (Emberiza melanocephala).

We proposed two perhaps overlapping hypotheses. Hypothesis 1 examined whether daily light information was transduced by a change in the pattern of daily melatonin secretion. Hypothesis 2 tested whether the melatonin amplitude peak was contingent upon seasonal states. To test these hypotheses, we performed three experiments on night migratory blackheaded buntings (Emberiza melanocephala). The first two experiments measured plasma melatonin levels in buntings exposed to light-dark (LD) cycles, with white and/or blue (450 nm) and red (640 nm) light periods differing by about 10-12 fold in the level of illuminance. In birds exposed to a 12 h day (white light) at 0.66 W m(-2) (dim) and 7.85 W m(-2) (bright) light intensities (experiment 1), night melatonin levels were significantly affected by the end of 8-week exposure in the dim, not bright, day with buntings showing a bimodal nocturnal melatonin peak. Similarly, in birds on 13 h days in white, blue and red light periods at 0.028 W m(-2) (dim) and 0.28 (bright) W m(-2) intensities (experiment 2), mid night melatonin levels were significantly higher in red than in the blue or white light periods after 4.5 weeks of exposure. The third experiment measured plasma melatonin levels in buntings that were held under natural light conditions (NDL, 27°N; experiment 3A), or exposed to LD cycles (experiment 3B). There were slightly more elevated melatonin levels early at night during the spring (photosensitive) than during the autumn (photorefractory) migration season, without a difference in nocturnal melatonin peak between these two times providing almost similar light hours. Similarly, there was no difference in mid night melatonin levels between photosensitive and photorefractory buntings subjected to a skeleton photoperiod (1L : 11D : 1L : 11D), although melatonin levels were higher in the first than in the second 11 h dark phase in photosensitive birds. Overall, these results show that (i) nocturnal melatonin levels decode the intensity and wavelength of the daily light environment, and (ii) the daily melatonin secretion pattern subtly reflects seasonal states in the migratory blackheaded bunting.

Adaptation of oxidative phosphorylation to photoperiod-induced seasonal metabolic states in migratory songbirds.

Eukaryotic cells produce chemical energy in the form of ATP by oxidative phosphorylation of metabolic fuels via a series of enzyme mediated biochemical reactions. We propose that the rates of these reactions are altered, as per energy needs of the seasonal metabolic states in avian migrants. To investigate this, blackheaded buntings were photoperiodically induced with non-migratory, premigratory, migratory and post-migratory phenotypes. High plasma levels of free fatty acids, citrate (an intermediate that begins the TCA cycle) and malate dehydrogenase (mdh, an enzyme involved at the end of the TCA cycle) confirmed increased availability of metabolic reserves and substrates to the TCA cycle during the premigratory and migratory states, respectively. Further, daily expression pattern of genes coding for enzymes involved in the oxidative decarboxylation of pyruvate to acetyl-CoA (pdc and pdk) and oxidative phosphorylation in the TCA cycle (cs, odgh, sdhd and mdh) was monitored in the hypothalamus and liver. Reciprocal relationship between pdc and pdk expressions conformed with the altered requirements of acetyl-CoA for the TCA cycle in different metabolic states. Except for pdk, all genes had a daily expression pattern, with high mRNA expression during the day in the premigratory/migratory phenotypes, and at night (cs, odhg, sdhd and mdh) in the nonmigratory phenotype. Differences in mRNA expression patterns of pdc, sdhd and mdh, but not of pdk, cs and odgh, between the hypothalamus and liver indicated a tissue dependent metabolism in buntings. These results suggest the adaptation of oxidative phosphorylation pathway(s) at gene levels to the seasonal alternations in metabolism in migratory songbirds.

Melatonin: an internal signal for daily and seasonal timing.

Melatonin is secreted only during night, irrespective of the habitat of an organism and the site of its synthesis and secretion, and hence known as "darkness hormone". Elevated melatonin levels reflect the nighttime. In vertebrates, the main site of melatonin production is the pineal gland. Species in which melatonin is also secreted from sources other than the pineal, as in some birds, relative contributions of different melatonin producing tissues to the blood melatonin level can vary from species to species. Melatonin acts through its receptors, which are members of the G protein-coupled (GPCR) superfamily. Three melatonin receptors subtypes MT1 (mella), MT2 (mellb), and MT3 (mellc) have been identified in different brain areas and other body organs of vertebrates. Melatonin synthesis and secretion are circadianly rhythmic. Changes and differences in specific features of melatonin signal can vary among species, and under a variety of natural environmental conditions. Two major physiological roles of melatonin are established in vertebrates. First, melatonin is involved in the circadian system regulated behavioural and physiological functions. Second, it is critical for the photoperiodic system. Besides, melatonin has been implicated in various ways both directly and indirectly to human health, including jet lag, sleep, immune system and cancer.

Daily behaviour can differ between colour morphs of the same species: a study on circadian activity behaviour of grey and pied zebra finches.

To investigate if the plumage colour mutation relates to circadian activity behaviour in the zebra finch, Taeniopygia guttata, wild type grey and pied mutant males were sequentially subjected for three weeks each to 12 h light:12 h darkness (12L:12D) and constant dim light (LL(dim)) condition. During the first 3 h of the 12 h day, pied finches were significantly greater active than grey finches. Also, as compared to grey, pied finches had longer activity duration in the day, with early activity onsets and late activity offsets. This was changed under free-running condition (LL(dim)), when the activity later in the subjective day (clock hour 9 and 11) was significantly greater in grey than in pied finches.Two colour morphs differed in daily activity profile, but not in the total daily activity or circadian rhythm period. Results suggest that greyzebra finches represent late chronotype, and could perhaps be better adapted to a seemingly stressful environment, such as low intensity LL(dim) in the present study.

Mikania micrantha silver nanoparticles exhibit anticancer activities against human lung adenocarcinoma via caspase-mediated apoptotic cell death.

Green-mediated synthesis of nanoparticles has earned a promising role in the area of nanotechnology due to their biomedical applications. This study describes the synthesis of silver nanoparticles (AgNPs) using Mikania micrantha leaf extract and its functional activities against cancer. The synthesis of AgNPs was confirmed using Ultraviolet-Visible (UV-Vis) spectrum that exhibited an absorption band at 459 nm. The bioactive compounds of M. micrantha leaf extract that functioned as reducing and capping agents were confirmed by a shift in the absorption bands in Fourier Transform Infra-red Spectroscopy (FT-IR). Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) studies validated the spherical shape and size of AgNPs, respectively. Energy Dispersive Spectroscopy (EDS) analysis revealed the presence of elemental silver. The crystalline nature of AgNPs was confirmed by the X-ray Diffraction Analysis (XRD). AgNPs effectively induced cytotoxicity and prevented A549 cell colony formation in a dose-dependent manner. Treatment of A549 cells with AgNPs also increased DNA damage, which was coupled with elevated lipid peroxidation and decreased antioxidant enzymes such as glutathione (GSH), glutathione-s-transferase (GST), and superoxide dismutase (SOD). Following AgNPs treatment, the mRNA expression levels of the pro-apoptotic genes as well as the activities of caspases were significantly elevated in A549 cells while the expression levels of anti-apoptotic genes were downregulated. Our study demonstrates the potential of the synthesised AgNPs for cancer therapy possibly targeting the apoptotic pathway.

Chronotype, sleep quality and academic performances among Mizo students.

Sleep is an essential component of the daily cycle. Age and puberty-related declines in sleep duration, delays in chronotype, and an increase in social jet lag have been consistently reported. This study examined chronotype and gender-based differences in adolescents' sleep quality, depression level, and academic achievements. Eight hundred fifteen school students (372 male and 443 female) aged 14 to 20 years voluntarily participated in this study. Horne and Östberg Morningness-Eveningness Questionnaire (MEQ) was used to measure their chronotype. Sleep duration, sleep quality, and daytime sleepiness were assessed by the Pediatric Daytime Sleepiness Scale (PDSS) and Epworth Sleepiness Scale (ESS). Pittsburgh's Sleep Quality Index (PSQI) was used to evaluate sleep habits. Cleveland Adolescent Sleepiness Questionnaire (CASQ) was used to measure the sleep pattern of the students. Zung Self-Rating Depression Scale (SDS) was used to assess the level of depression for the students. Our results show neither type of students dominated the population studied but interestingly, in comparison to evening type, morning type individuals were higher among the population. Chronotype-based sleep quality and academic performances were observed, and higher depression levels, poor sleep quality, poor academic performances were observed in evening type compared to neither type and morning type. In contrast to males, females had a poor sleep pattern (CASQ) and a higher depression level (SDS). Altogether, our study shows the effect of chronotype and gender on sleep quality and depression levels among school students.

Molecular expression of clock genes in central and peripheral tissues of white-rumped munia (Lonchura striata).

To synchronize with the fluctuating environment, organisms have evolved an endogenous time tracking mechanism referred to as the biological clock(s). This clock machinery has been identified in almost all cells of vertebrates and categorized as central and peripheral clocks. In birds, three independent circadian clocks have been identified in the hypothalamus, the pineal and the retina which interact and generate circadian time at a functional level. However, there is a limited knowledge of molecular clockwork and integration between central and peripheral clocks in birds. Therefore, we studied the daily expression of clock genes (Bmal1, Clock, Per2, Cry1, Npas2, Rev-Erbα, E4bp4, Pparα, Hlf and Tef) in three central circadian clocks (hypothalamus, pineal and retina), other brain areas (cerebellum, optic tectum and telencephalon) and in the peripheral tissues (liver, intestine, muscle and blood) of white-rumped munia. Adult birds were exposed to equinox photoperiod (12 L:12D) for 2 weeks and were then sampled (N = 5 per time point) at six-time points (ZT1, ZT5, ZT9, ZT13, ZT17 and ZT21). Daily expressions of clock genes were studied using qPCR. We observed daily variations and tissue-specific expression patterns for clock genes. These results are consistent with the autoregulatory circadian feedback loop proposed for the mammalian system and thus suggest a conserved tissue-level circadian time generation in white-rumped munia.

Seasonality in tropical birds.

The survival of offspring depends on environmental conditions. Many organisms have evolved with seasonality, characterized as initiation-termination-reinitiation of several physiological processes (i.e., body fattening, molt, plumage coloration, reproduction, etc). It is an adaptation for the survival of many species. Predominantly seasonal breeders use photoperiod as the most reliable environmental cue to adapt to seasonal changes but supplementary factors like temperature and food are synergistically involved in seasonal processes. Studies from diverse vertebrate systems have contributed to understanding the mechanism involved in seasonal reproduction at the molecular and endocrine levels. Long-day induced thyrotropin (thyroid-stimulating hormone) released from the pars tuberalis of the pituitary gland triggers local thyroid hormone activation within the mediobasal hypothalamus. This locally produced thyroid hormone, T3, regulates seasonal gonadotropin-releasing hormone secretion. Most of the bird species studied are seasonal in reproduction and linked behavior and, therefore, need to adjust reproductive decisions to environmental fluctuations. Reproductive strategies of the temperate zone breeders are well-documented, but less is known about tropical birds' reproduction and factors stimulating the annual breeding strategies. Here, we address seasonality in tropical birds with reference to seasonal reproduction and the various environmental factors influencing seasonal breeding.

Urban environment alter the timing of progression of testicular recrudescence in tree sparrow (Passer montanus).

Urbanization is a rapidly growing phenomenon that affects wildlife. Laboratory studies show the effects of night light on the physiology of the organisms. Limited studies have been conducted on birds in their natural habitat. Here, we studied the effects of the urban environment on reproduction-linked phenomenon and molecules involved in the regulation of seasonal breeding. Birds (N=5/time/site) were procured from urban and rural sites at specific times, i.e., in March (stimulatory phase), June (reproductive phase), September (refractory phase), and December (sensitive phase) of 2018. Immediately after procurement, birds were brought to the laboratory. Bodyweight, bill color, molt in body feathers, and testes size were recorded. The next day, all the birds were sacrificed in the middle of the day. Blood was collected and serum was used for ELISA of corticosterone, triiodothyronine (T3), and thyroxine (T4). mRNA levels of thyroid-stimulating hormone-ß (Tshß), type 2 deiodinase (Dio2), type 3 deiodinase (Dio3), gonadotropin-releasing hormone (GnRh), and gonadotropin inhibitory hormone (GnIh) were measured in hypothalamic tissue. Urban birds showed higher levels of corticosterone during the stimulatory phase. There was a delay in the initiation of testicular growth in urban birds and it was supported by reduced levels of T3 in blood plasma and relatively lower transcription of Dio2 and GnRH mRNA in urban birds. Our findings suggest that the urban environment delays the timing of reproduction in birds and could be the consequence of local environmental conditions.

Hypothalamus but not liver retains daily expression of clock genes during hibernation in terai tree frog (Polypedates teraiensis).

Circadian clock(s) allow an organism to be in synchrony with the surrounding environment and perform daily and seasonal physiological processes, including hibernation, migration, and reproduction. To cope with adverse environmental conditions, organisms have evolved various strategies. Insects undergo diapause, while some higher animals either migrate or hibernate/aestivate during unfavorable environmental conditions. Hibernation is an energy conservation strategy used to cope with adverse environmental conditions. Limited knowledge is available on the physiology of hibernation in non-mammalian vertebrates. Some studies suggest that metabolism is altered during amphibian hibernation, but nothing is known about the circadian clock. In the present study, we investigated daily oscillation of clock genes in the brain and liver of the terai tree frog (Polypedates teraiensis) during two annual phases of life: breeding and hibernation. Adult male terai tree frogs were procured from their natural habitat on the Mizoram University campus (23°N 92°E) during their breeding and hibernation phases. Body mass and testes weight were recorded. Animals were sacrificed at six time points: ZT1, ZT5, ZT9, ZT13, ZT17, and ZT21 (ZT0, zeitgeber time 0, indicates the sunrise time at the respective time of the year; N = 5 frogs per time point). Quantitative real-time polymerase chain reaction (qPCR) was performed for clock genes (Bmal1, Clock, Per2, and Cry2) in the hypothalamus and liver. Our results showed that body and testes weights decreased during hibernation. Further, the hypothalamus retained daily clock gene oscillations during breeding and hibernation. However, the liver lost this daily oscillation during hibernation. The maintained rhythm in hypothalamus in contrast to other hibernating animals might be the result of the fact that these animals hibernate at a higher temperature and might be more alert. As the animals have no food intake during their hibernation season which might be the reason the animals loose their rhythm in liver clock genes. These results suggest that retaining daily clock gene oscillations in the hypothalamic clock could be important for internal time tracking and post-hibernation emergence.

Effect of urban environment on pineal machinery and clock genes expression of tree sparrow (Passer montanus).

Increasing urbanisation is altering the physiology of wild animals and the mechanisms involved are largely unknown. We hypothesised that altering the physiology of urban organisms is due to the effect of extra light at night on the circadian clock by modulating the expression of pineal machinery and clock genes. Two experiments were performed. In Experiment 1, immediately after being procured from their respective sites (urban and rural sites), birds were released individually in LLdim light conditions. Circadian rhythm period, activity duration, and total activity count were calculated and did not differ between urban and rural birds. In Experiment 2, birds (from urban and rural habitats) were sampled at six time points at regular 4-h intervals, beginning 1 h after sunrise. We measured daily variations in plasma melatonin levels. We also analysed the expression levels of Aanat, Mel1A and Mel1B as an indicator of melatonin biosynthesis and action machinery. Clock and clock-controlled genes (Bmal1, Clock, Per2, Per3, Cry1 and Npas2) were studied in the hypothalamus, the pineal gland, and retina to investigate the effects of urban habitats on the circadian clock. Our results show that there is a lower expression of Aanat in the pineal gland and relatively low plasma melatonin levels in urban birds. Further, clock genes are also differentially expressed in all three central tissues of urban birds. We propose that alterations in the melatonin biosynthesis machinery and the expression of clock genes could result in miscalculations in the internal timing of the organism, with environmental timings leading to altered physiology in urban wild animals.

Temporal expression of genes coding for aryl-alkamine-N-acetyltransferase and melatonin receptors in circadian clock tissues: Circadian rhythm dependent role of melatonin in seasonal responses.

We investigated at the transcriptional level the role of daily rhythm in melatonin secretion in seasonal responses in the migratory blackheaded bunting (Emberiza melanocephala), which when exposed to short (SP) and long (LP) photoperiods exhibits distinct seasonal life-history states (LHSs). We reproduced the seasonal LHS by subjecting buntings to SP (8 h light: 16 h darkness, 8 L:16D), which maintained the nonmigratory/ nonbreeding phenotype, and to LP (16 L:8D), which induced the premigratory/ prebreeding, migratory/ breeding and nonmigratory/ postbreeding phenotypes. Plasma melatonin measured at 4 h intervals showed loss of the daily rhythm in the LP-induced premigratory/ prebreeding and migratory/ breeding LHSs. Subsequently, mRNA expression of genes coding for the aryl-alkamine-N-acetyltransferase (AANAT; the rate-liming enzyme of melatonin biosynthesis) and for the receptors for melatonin (Mel1A, Mel1B and Mel1C) was examined in the retina, pineal and hypothalamus; the interacting independent circadian clocks comprising the songbird circadian timing system. Except AANAT that was not amplified in the hypothalamus, we found significant alterations in both, the level and persistence of 24 h rhythm in mRNA expression of all genes, albeit with photoperiod and seasonal differences between three circadian clock tissues. Particularly, 24 h mRNA expression pattern of all genes, except retinal Mel1A, lacked a significant daily rhythm in the LP-induced migratory/ breeding LHS. These results underscore the overall importance of the circadian rhythm in the role of melatonin in photoperiodically-controlled seasonal responses in migratory songbirds.

Seasonality in terai tree frog (Polypedates teraiensis): Role of light and temperature in regulation of seasonal breeding.

Seasonality is represented as an initiation-termination-reinitiation of any physiological processes. Photoperiod is the most predictable environmental cue which organism use to time their daily physiology and seasonal functions. In natural light environmental conditions, day and night components change in terms of duration, intensity and spectrum of light available. In many vertebrate species, daytime light intensity and spectrum of light play a critical role in gonadal recrudescence-regression cycle. In tropical conditions, many amphibian species show a clear relationship between reproduction and seasonal distribution of rainfall. In temperate conditions, reproduction is usually centered during spring and summer seasons when environmental conditions are favorable. Poikilotherms are highly sensitive to change in environmental temperature and their physiology and metabolic activities depend on environmental temperature. How environmental factors (light and temperature) influence, the reproduction of terai tree frog (Polypedates teraiensis) is not known. We hypothesized that light acts as a proximate factor but the temperature is the ultimate factor for reproduction of terai tree frog. Three experiments were performed. In experiment one, we studied the annual reproductive cycle under natural environmental conditions. Beginning from the month of March till October 2016, we procured adult frogs (male and female) in the middle of each month. Monthly observations were recorded for body weight and gonadal weight and GSI was calculated. We found the annual change in body mass, gonadal weight and GSI in both male and female with high gonadal weight and GSI values from March to June. These results suggest that terai tree frog is seasonal breeders, and their breeding corresponds with long days. In experiment two we addressed the effects of light duration, light intensity and light spectrum on gonadal growth regression cycle. We observed that long days promote gonadal growth regression cycle. However, after achieving critical daylength there is no additive effect of light duration. Further, light intensity and spectrum have limited role in gonadal growth regression cycle of this species. In experiment three we tested the role of temperature on body weight and testicular growth under stimulatory photoperiod (12 L:12D). Group one was exposed to high temperature (34 ±â€¯2 °C), while group two was exposed to low temperature (22 ±â€¯2 °C). We found that low temperature promotes testicular recrudescence under laboratory conditions. Altogether our study suggests both photoperiod and temperature are involved in the regulation of seasonal breeding in tree frog. Findings from the above study could be used for captive breeding of amphibians and may be helpful in amphibian conservation programmes.

Daily expression of clock genes in central and peripheral tissues of tree sparrow (Passer montanus).

Almost all organisms live in a fluctuating environment. To achieve synchrony with the fluctuating environment, organisms have evolved with time-tracking mechanism commonly known as biological clocks. This circadian clock machinery has been identified in almost all cells of vertebrates and categorized as central and peripheral clocks. In birds, three independent circadian clocks reside within the nervous tissues in the hypothalamus, pineal and retina, which interact with each other and produce circadian time at a functional level. There is limited knowledge available of the molecular clockwork, and of integration between central and peripheral clocks in birds. Here, we studied daily expression of canonical clock genes (Bmal1, Clock, Per2, Per3, Cry1 and Cry2) and clock-controlled gene (Npas2) in all three central tissues (hypothalamus, pineal and retina) and in peripheral tissues (liver, intestine and muscle). Wild caught adult male tree sparrows were exposed to equinox photoperiod (12L:12D) for 2 weeks and after that birds were sacrificed (N = 5 per time point) at six time points (ZT1, ZT5, ZT9, ZT13, ZT17 and ZT21; ZT0 is lights on). Daily expression of clock genes was studied using qPCR. Bmal1, Clock, Per2, Per3, Cry1, Cry2 and Npas2 showed daily oscillation in all tissues except Cry2 in hypothalamus, pineal and intestine. We observed tissue-specific expression pattern for all clock and clock-controlled genes. Bmal1 transcripts expressed during early phase of night. Clock acrophase was observed during middle or late day time in the central clock while during early-to-middle phase of night in peripheral tissues. Npas2 expression pattern was similar to Bmal1. Per genes peaked either late at night or early during day time. However, Cry genes were peaked either at late day time (Cry1in retina, liver and intestine; Cry2 in liver and intestine) or at early night phase (Cry1 in hypothalamus, pineal and muscle; Cry2 in hypothalamus, pineal, retina and muscle). Our results are consistent with the autoregulatory circadian feedback loop, and suggest a conserved tissue-level circadian time generation in tree sparrows. Change in peak expression timing of these genes in different tissues implicates tissue-specific contribution of individual clock genes in the circadian time generation.

Temperature affects liver and muscle metabolism in photostimulated migratory redheaded buntings (Emberiza bruniceps).

We investigated the transcriptional effects of temperature on metabolism in the liver and flight muscle prior to the onset of spring migration in redheaded buntings. We measured changes in body mass, cellular lipid accumulation, and transcription of metabolic genes in the liver and flight muscle, during a week of exposure to 8-h short photoperiod (SP; 8L: 16D) and 13-h-long photoperiod (LP; 13L: 11D) at 22 °C to ascertain the responsiveness to LP, and during 2.5 weeks of LP at 22 and 38 °C to examine the effect of temperature on LP-induced metabolic response. Particularly, we measured mRNA expression of genes involved in the fatty acids and triglycerides biosynthesis (srebf1, elovl6, scd, fads2, and dgat2), glucose and lipid metabolism (sirt1, foxo1, ppara, pparg, hdac3, and hmgcr) in the liver, and fatty acid transport (cd36 and fabp3) and metabolism (myod1) in the flight muscle. Birds gained weight and had elevated lipid accumulation at 22 °C but not at 38 °C in the liver and muscle tissues. We also found significant differences in gene expression patterns during 2.5 weeks of LP exposure. For instance, as compared to week 1, there were decreased elovl6, dgat2, and srebf1 and increased foxo1 mRNA levels in the liver, and decreased cd36 and fabp3 mRNA levels in the muscle at 38 °C after 2.5 weeks of LP. It is suggested that a drastic change in temperature could alter the molecular metabolism in the liver and skeletal muscle with photoperiod-induced transition in the seasonal state in migratory songbirds.

Temperature alters the hypothalamic transcription of photoperiod responsive genes in induction of seasonal response in migratory redheaded buntings.

We investigated the temperature effects on hypothalamic transcription of genes involved in the induction of photoperiodic response in redheaded buntings. Birds were exposed at 22 and 38 °C to 13-h long photoperiods (LP), with controls at 22 °C on 8-h short photoperiods (SP). At 22 °C, compared to SP, we found higher tshb, eya3 and dio2 and low dio3 and gnih mRNA expressions after a week of LP; concomitant with testis recrudescence this confirmed buntings' responsiveness to LP-induced photostimulation. tshb, dio2 and gnrh mRNA levels were further increased by 2.5 weeks of LP at 38 °C. Temperature sensitive trpm8, but not trpv4, bdnf or adcyap1 also showed LP-induced expression at 22 °C. Concomitant changes in dnmt3b and tet2 mRNA expressions further suggested epigenetic modification of temperature influence on photoperiodic responses. These results demonstrate the role of temperature in hypothalamic molecular regulation of the photoperiodic gonadal response in seasonally breeding birds.

A robust and non-invertible fingerprint template for fingerprint matching system.

Fingerprint Recognition System is widely deployed in variety of application domain, ranging from forensic to mobile phones. Its widespread deployment in various applications were person authentication are required, has caused concern that a leaked fingerprint template may be used to reconstruct the original fingerprint and the reconstructed fingerprint can be used to circumvent all the applications the person is enrolled. In this paper, a non-invertible fingerprint template that stores only the relative geometric information about the minutiae points is proposed. The spatial location of the minutiae points in original fingerprint and its orientations are not available in the proposed template which makes it impossible to estimate the orientation of fingerprint from the template. The proposed template is invariant to rotation, translation and distortion and immune to reconstruction algorithm. The proposed system is experimented using standard FVC2000 database and yields better results in terms of EER and FMR as compared with latest techniques.