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.

Photoperiod-driven concurrent changes in hypothalamic and brainstem transcription of sleep and immune genes in migratory redheaded bunting.

The molecular regulation of sleep in avian migrants is still obscure. We thus investigated this in migratory redheaded buntings, where four life-history states (LHS; i.e. non-migratory, pre-migratory, migratory and refractory states) were induced. There was increased night-time activity (i.e. Zugunruhe) during the migratory state with reduced daytime activity. The recordings of the sleep-wake cycle in buntings showed increased night-time active wakefulness coupled with drastically reduced front and back sleep during migratory phase. Interestingly, we found the buntings to feed and drink even after lights-off during migration. Gene expression studies revealed increased hypothalamic expression of glucocorticoid receptor (nr3c1), and pro-inflammatory cytokines (il1b and il6) in pre-migratory and migratory states, respectively, whereas in brainstem Ca2+/calmodulin-dependent protein kinase 2 (camk2) was upregulated during the migratory state. This suggested a heightened pro-inflammatory state during migration which is a feature of chronic sleep loss, and a possible role of Ca2+ signalling in promoting wakefulness. In both the hypothalamus and brainstem, the expression of melatonin receptors (mel1a and mel1b) was increased in the pre-migratory state, and growth hormone-releasing hormone (ghrh, known to induce sleep) was reduced during the migratory state. The current results demonstrate key molecules involved in the regulation of sleep-wake cycle across LHS in migratory songbirds.

Photoperiodic modulation of melatonin receptor and immune genes in migratory redheaded bunting.

The transcriptional regulation of innate immune function across annual life history states (LHS) remains obscure in avian migrants. We, therefore, investigated this in a migratory passerine songbird, redheaded bunting (Emberiza bruniceps), which exhibits long-distance vernal migration from India to Central Asia. We exposed the birds (N = 10) to differential photoperiodic conditions to induce a non-migratory (NM), pre-migratory (PM), migratory (MIG), and refractory (REF) state, and performed gene expression assays of melatonin receptors (MEL1A and MEL1B), and innate immunity-linked genes (IL1B, IL6, TLR4, and NFKB) in spleen and blood. We found a significant reduction in splenic mass and volume, and a parallel increase in fat accumulation, and testicular growth in birds under migratory state. The gene expression assay revealed an upregulation of MEL1A and MEL1B mRNA levels in both the tissues in MIG. Additionally, we found a nocturnal increase of splenic IL1B expression, and IL1B, IL6, and TLR4 expression in the blood. The mRNA expression of melatonin receptors and proinflammatory cytokine showed a positive correlation. These results suggest that melatonin relays the photoperiodic signal to peripheral immune organs, which shows LHS-dependent changes in mRNA expression of immune genes.

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.

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.

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.

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.

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.