Understanding the role of temperature in seasonal timing: Effects on behavioural, physiological and molecular phenotypes.

Organisms adapt to daily and seasonal environmental changes to maximise their metabolic and reproductive fitness. For seasonally breeding animals, photoperiod is considered the most robust cue to drive these changes. It, however, does not explain the interannual variations in different seasonal phenotypes. Several studies have repeatedly shown the influence of ambient temperature on the timing of different seasonal physiologies including the timing of migration, reproduction and its associated behaviours, etc. In the present review, we have discussed the effects of changes in ambient temperature on different seasonal events in endotherms with a focus on migratory birds as they have evolved to draw benefits from distinct but largely predictable seasonal patterns of natural resources. We have further discussed the physiological and molecular mechanisms by which temperature affects seasonal timings. The primary brain area involved in detecting temperature changes is the hypothalamic preoptic area. This area receives thermal inputs via sensory neurons in the peripheral ganglia that measure changes in thermoregulatory tissues such as the skin and spinal cord. For the input signals, several thermal sensory TRP (transient receptor potential ion channels) channels have been identified across different classes of vertebrates. These channels are activated at specific thermal ranges. Once perceived, this information should activate an effector function. However, the link between temperature sensation and the effector pathways is not properly understood yet. Here, we have summarised the available information that may help us understand how temperature information is translated into seasonal timing.

Physiological effects of food availability times in higher vertebrates.

Food availability is a crucial ecological determinant of population size and community structure, and controls various life-history traits in most, if not all, species. Food availability is not constant; there are daily and seasonal differences in food abundance. When coupled to appetite (urge to eat), this is expressed as the eating schedule of a species. Food availability times affect daily and seasonal physiology and behaviour of organisms both directly (by affecting metabolic homeostasis) and indirectly (by altering synchronization of endogenous rhythms). Restricted food availability times may, for example, constrain reproductive output by limiting the number or quality of offspring or the number of reproductive attempts, as has been observed for nesting frequency in birds. Consuming food at the wrong time of day reduces the reproductive ability of a seasonal breeder, and can result in quality-quantity trade-offs of offspring. The food availability pattern serves as a conditioning environment, and can shape the activity of the genome by influencing chromatin activation/silencing; however, the functional linkage of food availability times with epigenetic control of physiology is only beginning to emerge. This Review gives insights into how food availability times, affected by changes in eating schedules and/or by alterations in feeding environment or lifestyle, could have hitherto unknown consequences on the physiology and reproductive fitness of seasonally breeding vertebrates and those that reproduce year round.

Molecular changes associated with migratory departure from wintering areas in obligate songbird migrants.

Day length regulates the development of spring migratory and subsequent reproductive phenotypes in avian migrants. This study used molecular approaches, and compared mRNA and proteome-wide expression in captive redheaded buntings that were photostimulated under long-day (LD) conditions for 4 days (early stimulated, LD-eS) or for ∼3 weeks until each bird had shown 4 successive nights of Zugunruhe (stimulated, LD-S); controls were maintained under short days. After ∼3 weeks of LD, photostimulated indices of the migratory preparedness (fattening, weight gain and Zugunruhe) were paralleled with upregulated expression of acc, dgat2 and apoa1 genes in the liver, and of cd36, fabp3 and cpt1 genes in the flight muscle, suggesting enhanced fatty acid (FA) synthesis and transport in the LD-S state. Concurrently, elevated expression of genes involved in the calcium ion signalling and transport (camk1 and atp2a2; camk2a in LD-eS), cellular stress (hspa8 and sod1, not nos2) and metabolic pathways (apoa1 and sirt1), but not of genes associated with migratory behaviour (adcyap1 and vps13a), were found in the mediobasal hypothalamus (MBH). Further, MBH-specific quantitative proteomics revealed that out of 503 annotated proteins, 28 were differentially expressed (LD-eS versus LD-S: 21 up-regulated and 7 down-regulated) and they enriched five physiological pathways that are associated with FA transport and metabolism. These first comprehensive results on gene and protein expression suggest that changes in molecular correlates of FA transport and metabolism may aid the decision for migratory departure from wintering areas in obligate songbird migrants.

Daytime light spectrum affects photoperiodic induction of vernal response in obligate spring migrants.

It is not well understood how the spectral composition (wavelength) of daylight that varies considerably during the day and seasons affects photoperiodic responses in a seasonal species. Here, we investigated the molecular underpinnings of wavelength-dependent photoperiodic induction in migratory redheaded buntings transferred to 13 h long days in neutral (white), 460 nm (blue), 500 nm (green) or 620 nm (red) wavelength that were compared with one another, and to short day controls for indices of the migratory (body fattening and weight gain, and Zugunruhe) and reproductive (testicular maturation) responses. Buntings showed wavelength-dependent photoperiodic response, with delayed Zugunruhe and slower testis maturation under 620 nm red light. Post-mortem comparison of gene expressions further revealed wavelength-dependence of the photoperiodic molecular response. Whereas there were higher retinal expressions of opn2 (rhodopsin) and opn5 (neuropsin) genes in red daylight, and of rhodopsin-like opsin (rh2) gene in green daylight, the hypothalamic opn2 mRNA levels were higher in blue daylight. Similarly, we found in birds under blue daylight an increased hypothalamic expression of genes involved in the photoperiodic induction (thyroid stimulating hormone subunit beta, tshb; eye absent 3, eya3; deiodinase type 2, dio2) and associated neural responses such as the calcium signaling (ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2, atp2a2), dopamine biosynthesis (tyrosine hydroxylase, th) and neurogenesis (brain-derived neurotrophic factor, bdnf). These results demonstrate transcriptional changes in parallel to responses associated with migration and reproduction in buntings, and suggest a role of daylight spectrum in photoperiodic induction of the vernal response in obligate spring avian migrants.

Ambient temperature affects multiple drivers of physiology and behaviour: adaptation for timely departure of obligate spring migrants.

We investigated the role of ambient temperature in departure from wintering areas of migratory black-headed buntings in spring. Birds transferred at 22 and 35°C to long days were compared with one another and with controls held on short days for indices of readiness to migrate (Zugunruhe, fattening, mass gain), levels of testosterone and gonadal recrudescence. Temperature affected the development of migratory behaviour and physiology: buntings under long days at 35°C, compared with those at 22°C, showed altered migratory behaviour (daily activity and Zugunruhe onset), and enhanced muscle growth and plasma testosterone levels, but showed no effect on testis growth. Temperature was perceived at both peripheral and central levels, and affected multiple molecular drivers culminating into the migratory phenotype. This was evidenced by post-mortem comparison of the expression of 13 genes with known functions in the skin (temperature-sensitive TRP channels: trpv4 and trpm8), hypothalamus and/or midbrain (migration-linked genes: th, ddc, adcyap1 and vps13a) and flight muscles (muscle growth associated genes: ar, srd5a3, pvalb, mtor, myod, mstn and hif1a). In photostimulated birds, the expression of trpv4 in skin, th in the hypothalamus and midbrain, and srd5a3, ar, pvalb and mtor in flight muscle, in parallel with testosterone levels, was greater at 35°C than at 22°C. These results demonstrate the role of ambient temperature in development of the spring migration phenotype, and suggest that transcriptional responsiveness to temperature is a component of the overall adaptive strategy in latitudinal songbird migrants for timely departure from wintering areas in spring.

Changes in the expression of genes involved in DNA methylation and histone modification in response to daily food availability times in zebra finches: epigenetic implications.

We hypothesised that daily food availability times serve as an 'epigenetic' factor and affect reproductive physiology in continuously reproducing species. This we tested by measuring mRNA expression of genes coding for enzymes involved in DNA methylation-demethylation (dnmt, tet) and histone modification (hat1, hdac) in the hypothalamus, liver and gonads of male and female zebra finches that were paired for a year under 12 h light:12 h dark conditions with food availability restricted to 4 h in the morning (morning FA group) or evening (evening FA group), with controls provided with food ad libitum The overall hypothalamic and hepatic expression patterns of hat1 and hdac were similar but those of dnmt and tet were different between males and females. Irrespective of the timing of food availability, both hat1 and hdac mRNA levels were increased in the hypothalamus, but not in the liver, in which hat1 mRNA levels were increased in the morning FA group. While hypothalamic tet levels were higher in evening FA males, hepatic tet levels were higher in morning FA birds (tet1, only males). Gonadal expression levels similarly varied and showed sex differences. Histone-modifying genes did not show food availability effects, except for elevated testicular hdac3 levels. Similarly, testicular dnmt3b and tet2 mRNA levels were increased and decreased in morning and evening FA groups, respectively, whereas ovarian dnmt1 and tet2 levels were reduced in morning FA and tet1 levels were reduced in evening FA groups. The present results suggest that an enforced daily feeding schedule in the long term could serve as a conditioning environment that shapes overall hypothalamic regulation, and liver and gonadal function at the epigenetic level in diurnal vertebrates.

Involvement of steroid and antioxidant pathways in spleen-mediated immunity in migratory birds.

The molecular underpinnings of the spleen-mediated immune functions during the period of heightened energetic needs in the year are not known in avian migrants. We investigated this, in Palearctic-Indian migratory male redheaded buntings, which exhibited vernal (spring) premigratory / early testicular maturation states under artificial long days. This was evidenced by increased dio2 and decreased dio3 mRNA expression in the hypothalamus, elevated levels of circulating corticosterone and testosterone, and enlarged testes in long-day-photostimulated birds, as compared to unstimulated controls under short days. The concomitant decrease in both mass and volume of the spleen, and increase in the heterophil/ lymphocyte ratio suggested the parallel innate immunity effects in photostimulated buntings. Importantly, we found increased mRNA expression of genes coding for the cytokines (il15 and il34), steroid receptors (nr3c2) and oxidative stress marker enzymes (gpx1 and sod1) in the spleen, suggesting the activation of both immune and antioxidant molecular pathways during the early photostimulated state. However, the splenic expressions of il1ß, il6, tgfß, ar and nos2 genes were not significantly different between long-day stimulated and short-day unstimulated birds. The negative correlation of plasma corticosterone levels with spleen mass further indicated a role of corticosterone in the modulation of the spleen function, probably via nr3c2 gene encoded mineralocorticoid receptors. These results suggest the activation of the spleen-mediated innate immunity in anticipation of the heightened energetic stress state of the photostimulated spring migratory/breeding period in migratory songbirds.

Transcriptome-wide changes in testes reveal molecular differences in photoperiod-induced seasonal reproductive life-history states in migratory songbirds.

We investigated the molecular basis of seasonal testicular states in black-headed buntings (Emberiza melanocephala), in which seasonal migrations limit reproduction to a narrow time-window during the year. We examined testicular gene expressions in buntings during short day-induced photosensitive nonreproductive state (SDSE), and during long day-induced early (LDES) and late (LDLS) gonadal maturation and regressed photorefractory (LDRF) seasonal states. Using Kallisto, we pseudoaligned the RNA-Seq transcripts and calculated the transcript abundance. We found 1,799 differentially expressed genes (DEGs) between the four photoperiod-induced seasonal states. Further pairwise comparison with SDSE revealed 1,187, 1,224, and 1,238 DEGs in LDES, LDLS, and LDRF, respectively; 852 genes were common to three comparisons. We then identified genetic pathways putatively involved in seasonal testicular cycle. DEGs that enriched calcium ion binding were highly expressed in testicular maturation states. Similarly, DEGs that enriched glycolytic pathway were highly expressed in LDES and LDRF states. More specifically, quantitative polymerase chain reaction showed significant differences between the photoperiod-induced states in testicular expression of genes (HOOK1, RGS2, PRDX4, BCL6, CYFIP2, PDCD4, P2RX4, and GABRA1) involved in gametogenesis and associated pathways. Overall, these results show significant transcriptional differences, and provide insights into the molecular basis of seasonal changes in the reproductive life-history states in a photoperiodic species.

Metabolic plasticity mediates differential responses to spring and autumn migrations: Evidence from gene expression patterns in migratory buntings.

NEW FINDINGS: What is the central question of this study? What are the molecular underpinnings of seasonal metabolic plasticity during spring and autumn migrations in songbirds? What is the main finding and its importance? We report differences in mRNA levels of genes involved in the regulation of glucose and fat metabolism between photoinduced non-migratory and migratory states and between the spring and autumn migratory states. Higher expression of genes associated with fat mobilization and energy generation in the spring than in the autumn migration suggests differential activation of the metabolic pathways or alteration in the efficiency of existing functional machinery during annual journeys between nearly fixed destinations. ABSTRACT: The molecular underpinnings of metabolic plasticity underlying differential responses to spring and autumn migrations are not well understood. We investigated this by examining the differences in mRNA levels of metabolic genes in the liver, muscle and adipose tissues of night-migratory red-headed buntings between photostimulated non-migratory and migratory states and between spring and autumn migratory states. Buntings accumulated more subcutaneous fat and hepatic lipid, had higher body mass, larger adipose cells and higher circulating triglyceride and free fatty acid levels and exhibited more intense Zugunruhe in the spring migratory state than in the autumn migratory state. More importantly, we found differences in the hepatic expression of pdc and pdk genes, indicating a differential acetyl-CoA requirement, and of the mdh and ogdh genes, suggesting differential oxidative phosphorylation between the non-migratory and migratory states and between the spring and autumn migratory states. Differences in fasn, bmal1 and glut1 mRNA levels were consistent with this and suggested seasonal differences in lipogenesis and/or glucose uptake. Likewise, differences in mRNA levels of genes coding for lipases (atgl and lpl) suggested that adipose triglycerides and free fatty acids serve largely as the metabolic substrate. Furthermore, changes in mRNA levels of genes coding for the fatty acid binding protein (fabp3) and fatty acid translocases (cd36) were consistent with differential fat fuel supply (via circulating free fatty acids) to aerobically exercising flight muscles between the spring and autumn migrations. These results show seasonal adaptation of genetic pathway(s) underlying seasonal metabolic plasticity that seems to mediate differential responses to spring and autumn migrations in latitudinal migratory songbirds.

Difference in control between spring and autumn migration in birds: insight from seasonal changes in hypothalamic gene expression in captive buntings.

We hypothesized differences in molecular strategies for similar journeys that migrants undertake to reproduce in spring and to overwinter in autumn. We tested this in redheaded buntings (Emberiza bruniceps) photoinduced into spring and autumn migratory states, with winter and summer non-migratory states as controls. Compared with controls, buntings fattened, gained weight and showed Zugunruhe (nocturnal migratory restlessness) in the migratory state. Spring migration was associated with greater fat and body mass, and higher intensity of Zugunruhe, compared with autumn migration. Circulating corticosterone levels were higher in spring, while T3 levels were higher in autumn. Hypothalamic expression of thyroid hormone-responsive (dio2, dio3), light-responsive (per2, cry1, adcyap1) and th (tyrosine hydroxylase, involved in dopamine biosynthesis) genes showed significant changes with transition from non-migratory to the migratory state. There were significantly higher mRNA expressions in autumn, except for higher th levels in the spring. Furthermore, the expression patterns of dnmt3a (not dnmt3b) and tet2 genes suggested an epigenetic difference between the non-migrant and migrant periods, and the spring and autumn migrant periods. These results demonstrate for the first time seasonal transition in hypothalamic gene expressions, and suggest differences in regulatory strategies at the transcriptional level for spring and autumn migrations in songbirds.

Hypothalamic and liver transcriptome from two crucial life-history stages in a migratory songbird.

NEW FINDINGS: What is the central question of this study? What are the molecular underpinnings of the seasonal adaptation in a latitudinal migratory songbird? What is the main finding and its importance? We found changes in mRNA levels after a photoperiod-induced alteration of seasonal state in a captive long-distance latitudinal avian migrant. The hypothalamus and liver transcriptomes revealed genes involved in the regulatory and functional pathways between non-migratory and migratory states. Our results provide insights into mechanisms underlying homeostasis during seasonal changes that are conserved across most species, including humans. ABSTRACT: Very little is understood about genetic mechanisms underlying the onset of spring migration in latitudinal avian migrants. To gain insight into the genetic architecture of the hypothalamus and liver tissues of a long-distance migrant, we examined and compared the transcriptome profile of captive night-migratory black-headed buntings (Emberiza melanocephala) between photoperiod-induced winter non-migratory (WnM) and spring migratory (SM) life-history states under short and long days, respectively. High-throughput 454 pyrosequenced transcripts were mapped initially with reference to the genome of two phylogenetically close species, Taeniopygia guttata and Ficedula albicollis. The F. albicollis genome gave higher annotation results and was used for further analysis. A total of 216 (78 in hypothalamus; 138 in liver) genes were found to be expressed differentially between the WnM and SM life-history states. These genes were enriched for physiological pathways that might be involved in the regulation of seasonal migrations in birds. For example, genes for the ATP binding pathway in the hypothalamus were expressed at a significantly higher level in SM than in the WnM life-history state. Likewise, upregulated genes associated with the myelin sheath and focal adhesion were enriched in the hypothalamus, and those with cell-to-cell junction, intracellular protein transport, calcium ion transport and small GTPase-mediated signal transduction were enriched in the liver. Many of these genes are a part of physiological pathways potentially involved in the regulation of seasonal migration in birds. These results show molecular changes at the regulatory and metabolic levels associated with seasonal transitions in a long-distance migrant and provide the basis for future studies aimed at unravelling the genetic control of migration in birds.

Anthropometry and blood biomarkers of diabetes and their possible association with obesity and metabolic syndrome.

Background: Diabetes, a rapidly increasing heterogeneous disorder, is closely linked to the epidemic of obesity and metabolic syndrome (MetS). At present, we do not understand completely the blood biomarkers, molecular aetiology, and role of lifestyle modification and interventions to combat diabetes hand in hand with obesity and the MetS epidemic. Methods: To measure different anthropometric and blood biomarkers in pre-diabetic and diabetic patients, we collected data and blood samples from patients in a hospital OPD. This was a cross-sectional study that included the identification of possible relationships between different parameters to predict early diagnostic markers of diabetes. Results: We found increased body mass index (BMI), fasting blood glucose, neck, waist, and hip circumference, sagittal abdominal diameter, and skin fold thickness in the diabetic as compared to the pre-diabetic group. Also, serum uric acid and insulin resistance (HOMA-IR) values were significantly increased in diabetic individuals. We found a significant positive correlation between serum uric acid and BMI, fasting blood glucose, serum insulin, and HOMA-IR values. Conclusions: Here, we found that pre-diabetic and diabetic patients have increased fasting glucose levels while we did not find any difference in insulin levels. Both pre-diabetic and diabetic patients show high serum uric acid, positively associated with a higher prevalence of diabetes and HOMA-IR. Uric acid may hence be an important parameter for early diagnostics. These findings may be used as a basis for future studies that aim to identify the mechanistic details of the association of uric acid with insulin signaling and hence better understanding of the phenomenon associated with diabetes. Supplementary Information: The online version contains supplementary material available at 10.1007/s40200-023-01276-4.

Hypothalamic molecular correlates of photoperiod-induced spring migration in intact and castrated male redheaded buntings.

This study investigated the molecular changes associated with neural plasticity in photoperiodic induction of spring migration in intact and castrated redheaded bunting, Emberiza bruniceps. We measured the hypothalamic mRNA expression of genes in birds that were photostimulated into winter non-migratory and spring (vernal) migratory phenotypes under short and long photoperiods, respectively. These included genes associated with the appetitive phase of reproduction (spring migration drive, th and ddc genes encoding for tyrosine hydroxylase and dopamine decarboxylase enzymes, respectively), sleep/awake state (pmch gene encoding for pro-melanin concentrating hormone; hcrt and hcrtr2 encoding for the hypocretin/orexin and its receptor, respectively) and neurogenesis (dcx and neuN coding for doublecortin and neuronal nuclear proteins, respectively). Higher th mRNA levels suggested an upregulated dopamine synthesis in the hypothalamus of spring migrants. Similarly, elevated hcrt and hcrtr2 mRNA levels suggested an increased wakefulness, and those of dcx and neuN genes suggested an enhanced neurogenesis during the spring migration state. Further, compared to intact birds, the lower th and pmch, and higher hcrtr2 and neuN mRNA levels in castrates suggested a role of testicular steroids in modulation of the appetitive phase of reproduction, sleep and awake states, and neurogenesis during spring migration period. These results provide insights into molecular changes linked with important hypothalamic molecular pathways and steroidal influence in the photoperiodic induction of spring migration in obligate migratory songbirds.

Genetic Control of Avian Migration: Insights from Studies in Latitudinal Passerine Migrants.

Twice-a-year, large-scale movement of billions of birds across latitudinal gradients is one of the most fascinating behavioral phenomena seen among animals. These seasonal voyages in autumn southwards and in spring northwards occur within a discrete time window and, as part of an overall annual itinerary, involve close interaction of the endogenous rhythm at several levels with prevailing photoperiod and temperature. The overall success of seasonal migrations thus depends on their close coupling with the other annual sub-cycles, namely those of the breeding, post-breeding recovery, molt and non-migratory periods. There are striking alterations in the daily behavior and physiology with the onset and end of the migratory period, as shown by the phase inversions in behavioral (a diurnal passerine bird becomes nocturnal and flies at night) and neural activities. Interestingly, there are also differences in the behavior, physiology and regulatory strategies between autumn and spring (vernal) migrations. Concurrent molecular changes occur in regulatory (brain) and metabolic (liver, flight muscle) tissues, as shown in the expression of genes particularly associated with 24 h timekeeping, fat accumulation and the overall metabolism. Here, we present insights into the genetic basis of migratory behavior based on studies using both candidate and global gene expression approaches in passerine migrants, with special reference to Palearctic-Indian migratory blackheaded and redheaded buntings.

Control and adaptability of seasonal changes in behavior and physiology of latitudinal avian migrants: Insights from laboratory studies in Palearctic-Indian migratory buntings.

Twice-a-year migrations, one in autumn and the other in spring, occur within a discrete time window with striking alterations in the behavior and physiology, as regulated by the interaction of endogenous rhythms with prevailing photoperiod. These seasonal voyages are not isolated events; rather, they are part of an overall annual itinerary and remain closely coupled to the other annual subcycles, called seasonal life history states (LHSs). The success of migration depends on appropriate timing of the initiation and termination of each LHS, for example, reproduction, molt, summer nonmigratory, preautumn migratory (fattening and weight gain), autumn migratory, winter nonmigratory (wnM), prevernal (spring) migratory (fattening and weight gain), and spring migratory LHSs. Migration-linked photoperiod-induced changes include the body fattening and weight gain, nocturnal Zugunruhe (migratory restlessness), elevated triglycerides and free fatty acids, triiodothyronine and corticosterone levels. Hypothalamic expression of the thyroid hormone-responsive dio2 and dio3, light-responsive per2, cry1, and adcyap1 and th (tyrosine hydroxylase, involved in dopamine biosynthesis) genes also show significant changes with transition from wnM to the vernal migratory LHS. Concurrent changes in the expression of genes associated with lipid metabolism and its transport also occur in the liver and flight muscles, respectively. Interestingly, there are clear differences in the behavioral and physiological phenotypes, and associated molecular changes, between the autumn and vernal migrations. In this review, we discuss seasonal changes in the behavior and physiology, and present molecular insights into the development of migratory phenotypes in latitudinal avian migrants, with special reference to Palearctic-Indian migratory buntings.

Comparative Evaluation of Marginal Integrity of Three Esthetic Restorative Materials - An In-vitro Study.

CONTEXT: Microleakage is the major cause for the failure of dental restorations, especially in Class V cavities, as margins of such restorations are generally located in dentin or cementum. Microleakage evaluation is necessary as a means of evaluation of the marginal integrity of restorative materials. This would assist in developing techniques and materials that would reduce damage caused by the failure of the restorative marginal seal. AIM: The aim of this study is to analyze and compare the marginal integrity among three esthetic restorative materials, namely GC Fuji II LC, GC G-Aenial anterior composite resin, and GC Equia forte fil. SETTING AND DESIGN: Sixty orthodontically extracted caries-free premolar teeth with Class V restorations were divided into three groups. Microleakage was measured using an ordinal scale of 0-4, as given by Khera and Chan, in increasing order of dye penetration, which was observed under a microscope. MATERIALS AND METHODS: Study was conducted in sound human extracted premolars in which Standardized Class V cavities were prepared. Teeth were randomly and equally assigned to three groups (GC Fuji II LC, GC G-Aenial anterior composite resin, and GC Equia forte fil). Teeth were sectioned longitudinally into two halves using diamond discs and the sectioned halves of the teeth were evaluated for dye penetration under stereomicroscope. STATISTICAL ANALYSIS USED: Intergroup comparison of mean dye penetration scores were compared using the Kruskal-Wallis test along with post hoc pairwise comparison by Mann Whitney U test. The level of statistical significance was set at 0.05. RESULTS AND CONCLUSION: All the three groups (GC Fuji II LC, GC G-Aenial anterior composite resin, and GC Equia forte fil) tested showed microleakage at the tooth restoration interface. It was evident that microleakage was found to be highest with the Fuji II LC, both at occlusal and cervical levels. GC Equia forte exhibited the best performance in limiting microleakage around the restoration margins.

Photoperiodically driven transcriptome-wide changes in the hypothalamus reveal transcriptional differences between physiologically contrasting seasonal life-history states in migratory songbirds.

We investigated time course of photoperiodically driven transcriptional responses in physiologically contrasting seasonal life-history states in migratory blackheaded buntings. Birds exhibiting unstimulated winter phenotype (photosensitive state; responsive to photostimulation) under 6-h short days, and regressed summer phenotype (photorefractory state; unresponsiveness to photostimulation) under 16-h long days, were released into an extended light period up to 22 h of the day. Increased tshß and dio2, and decreased dio3 mRNA levels in hypothalamus, and low prdx4 and high il1ß mRNA levels in blood confirmed photoperiodic induction by hour 18 in photosensitive birds. Further, at hours 10, 14, 18 and 22 of light exposure, the comparison of hypothalamus RNA-Seq results revealed transcriptional differences within and between states. Particularly, we found reduced expression at hour 14 of transthyretin and proopiomelanocortin receptor, and increased expression at hour 18 of apolipoprotein A1 and carbon metabolism related genes in the photosensitive state. Similarly, valine, leucine and isoleucine degradation pathway genes and superoxide dismutase 1 were upregulated, and cocaine- and amphetamine-regulated transcript and gastrin-releasing peptide were downregulated in the photosensitive state. These results show life-history-dependent activation of hypothalamic molecular pathways involved in initiation and maintenance of key biological processes as early as on the first long day.

Seasonal reproductive state determines gene expression in the hypothalamus of a latitudinal migratory songbird during the spring and autumn migration.

We investigated gonadal effects on hypothalamic transcription of genes in sham-operated and castrated redheaded buntings photostimulated into spring and autumn migratory states. RNA-Seq results showed testes-dependent differences between spring and autumn migratory states. In particular, differentially expressed genes enriched G-protein-coupled receptor and calcium-ion signaling pathways during spring and autumn states, respectively. qPCR assay showed attenuated gabra5, ttr, thra and thrb expressions, suggesting reduced GABA and thyroid hormone effects on photo-sexual response in spring. In spring castrates, reduced npy, tac1 and nrcam and increased ank3 expression suggested testicular effects on the appetite, prolactin release and neuronal functions, whereas in autumn castrates, reduced rasgrp1, grm5 and grin1, and increased mras expression suggested testicular effects on the ras, G-protein and glutamate signaling pathways. Castration-induced reciprocal switching of pomc and pdyn expressions suggested effects on the overall homeostasis in both seasons. These results demonstrate transcriptome-wide changes, with season-dependent roles of testes in songbird migration.

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.