Migration increases mitochondrial oxidative capacity without increasing reactive oxygen species emission in a songbird.

Birds remodel their flight muscle metabolism prior to migration to meet the physiological demands of migratory flight, including increases in both oxidative capacity and defence against reactive oxygen species. The degree of plasticity mediated by changes in these mitochondrial properties is poorly understood but may be explained by two non-mutually exclusive hypotheses: variation in mitochondrial quantity or in individual mitochondrial function. We tested these hypotheses using yellow-rumped warblers (Setophaga coronata), a Nearctic songbird which biannually migrates 2000-5000 km. We predicted higher flight muscle mitochondrial abundance and substrate oxidative capacity, and decreased reactive oxygen species emission in migratory warblers captured during autumn migration compared with a short-day photoperiod-induced non-migratory phenotype. We assessed mitochondrial abundance via citrate synthase activity and assessed isolated mitochondrial function using high-resolution fluororespirometry. We found 60% higher tissue citrate synthase activity in the migratory phenotype, indicating higher mitochondrial abundance. We also found 70% higher State 3 respiration (expressed per unit citrate synthase) in mitochondria from migratory warblers when oxidizing palmitoylcarnitine, but similar H2O2 emission rates between phenotypes. By contrast, non-phosphorylating respiration was higher and H2O2 emission rates were lower in the migratory phenotype. However, flux through electron transport system complexes I-IV, II-IV and IV was similar between phenotypes. In support of our hypotheses, these data suggest that flight muscle mitochondrial abundance and function are seasonally remodelled in migratory songbirds to increase tissue oxidative capacity without increasing reactive oxygen species formation.

Songbirds avoid the oxidative stress costs of high blood glucose levels: a comparative study.

Chronically high blood glucose levels (hyperglycaemia) can compromise healthy ageing and lifespan at the individual level. Elevated oxidative stress can play a central role in hyperglycaemia-induced pathologies. Nevertheless, the lifespan of birds shows no species-level association with blood glucose. This suggests that the potential pathologies of high blood glucose levels can be avoided by adaptations in oxidative physiology at the macroevolutionary scale. However, this hypothesis remains unexplored. Here, we examined this hypothesis using comparative analyses controlled for phylogeny, allometry and fecundity based on data from 51 songbird species (681 individuals with blood glucose data and 1021 individuals with oxidative state data). We measured blood glucose at baseline and after stress stimulus and computed glucose stress reactivity as the magnitude of change between the two time points. We also measured three parameters of non-enzymatic antioxidants (uric acid, total antioxidants and glutathione) and a marker of oxidative lipid damage (malondialdehyde). We found no clear evidence for blood glucose concentration being correlated with either antioxidant or lipid damage levels at the macroevolutionary scale, as opposed to the hypothesis postulating that high blood glucose levels entail oxidative costs. The only exception was the moderate evidence for species with a stronger stress-induced increase in blood glucose concentration evolving moderately lower investment into antioxidant defence (uric acid and glutathione). Neither baseline nor stress-induced glucose levels were associated with oxidative physiology. Our findings support the hypothesis that birds evolved adaptations preventing the (glyc)oxidative costs of high blood glucose observed at the within-species level. Such adaptations may explain the decoupled evolution of glycaemia and lifespan in birds and possibly the paradoxical combination of long lifespan and high blood glucose levels relative to mammals.

Sarcolipin relates to fattening, but not sarco/endoplasmic reticulum Ca2+-ATPase uncoupling, in captive migratory gray catbirds.

In order to complete their energetically demanding journeys, migratory birds undergo a suite of physiological changes to prepare for long-duration endurance flight, including hyperphagia, fat deposition, reliance on fat as a fuel source, and flight muscle hypertrophy. In mammalian muscle, SLN is a small regulatory protein which binds to sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and uncouples Ca2+ transport from ATP hydrolysis, increasing energy consumption, heat production, and cytosolic Ca2+ transients that signal for mitochondrial biogenesis, fatigue resistance and a shift to fatty acid oxidation. Using a photoperiod manipulation of captive gray catbirds (Dumetella carolinensis), we investigated whether SLN may play a role in coordinating the development of the migratory phenotype. In response to long-day photostimulation, catbirds demonstrated migratory restlessness and significant body fat stores, alongside higher SLN transcription while SERCA2 remained constant. SLN transcription was strongly correlated with h-FABP and PGC1α transcription, as well as fat mass. However, SLN was not significantly correlated with HOAD or CD36 transcripts or measurements of SERCA activity, SR membrane Ca2+ leak, Ca2+ uptake rates, pumping efficiency or mitochondrial biogenesis. Therefore, SLN may be involved in the process of storing fat and shifting to fat as a fuel, but the mechanism of its involvement remains unclear.

Dietary fatty acids and flight-training influence the expression of the eicosanoid hormone prostacyclin in songbirds.

Diet shifts can alter tissue fatty acid composition in birds, which is subsequently related to metabolic patterns. Eicosanoids, short-lived fatty acid-derived hormones, have been proposed to mediate these relationships but neither baseline concentrations nor the responses to diet and exercise have been measured in songbirds. We quantified a stable derivative of the vasodilatory eicosanoid prostacyclin in the plasma of male European Starlings (Sturnus vulgaris, N = 25) fed semisynthetic diets with either high (PUFA) or low (MUFA) amounts of n6 fatty acid precursors to prostacyclin. Plasma samples were taken from each bird before, immediately after, and two days following a 15-day flight-training regimen that a subset of birds (N = 17) underwent. We found elevated prostacyclin levels in flight-trained birds fed the PUFA diet compared to those fed the MUFA diet and a positive relationship between prostacyclin and body condition, indexed by fat score. Prostacyclin concentrations also significantly decreased at the final time point. These results are consistent with the proposed influences of precursor availability (i.e., dietary fatty acids) and regulatory feedback associated with exercise (i.e., fuel supply and inflammation), and suggest that prostacyclin may be an important mediator of dietary influence on songbird physiology.

Flight muscle size reductions and functional changes following long-distance flight under variable humidity conditions in a migratory warbler.

Bird flight muscle can lose as much as 20% of its mass during a migratory flight due to protein catabolism, and catabolism can be further exacerbated under dehydrating conditions. However, the functional consequences of exercise and environment induced protein catabolism on muscle has not been examined. We hypothesized that prolonged flight would cause a decline in muscle mass, aerobic capacity, and contractile performance. This decline would be heightened for birds placed under dehydrating environmental conditions, which typically increases lean mass losses. Yellow-rumped warblers (Setophaga coronata) were exposed to dry or humid (12 or 80% relative humidity at 18°C) conditions for up to 6 h while at rest or undergoing flight. The pectoralis muscle was sampled after flight/rest or after 24 h of recovery, and contractile properties and enzymatic capacity for aerobic metabolism was measured. There was no change in lipid catabolism or force generation of the muscle due to flight or humidity, despite reductions in pectoralis dry mass immediately post-flight. However, there was a slowing of myosin-actin crossbridge kinetics under dry compared to humid conditions. Aerobic and contractile function is largely preserved after 6 h of exercise, suggesting that migratory birds preserve energy pathways and function in the muscle.

Upper bound for broadband radiofrequency field disruption of magnetic compass orientation in night-migratory songbirds.

Night-migratory songbirds have a light-dependent magnetic compass sense, the mechanism of which is thought to depend on the photochemical formation of radical pairs in cryptochrome (Cry) proteins located in the retina. The finding that weak radiofrequency (RF) electromagnetic fields can prevent birds from orienting in the Earth's magnetic field has been regarded as a diagnostic test for this mechanism and as a potential source of information on the identities of the radicals. The maximum frequency that could cause such disorientation has been predicted to lie between 120 and 220 MHz for a flavin-tryptophan radical pair in Cry. Here we show that the magnetic orientation capabilities of Eurasian blackcaps (Sylvia atricapilla) are not affected by RF noise in the frequency bands 140 to 150 MHz and 235 to 245 MHz. From a consideration of its internal magnetic interactions, we argue that RF field effects on a flavin-containing radical-pair sensor should be approximately independent of frequency up to 116 MHz and that birds' sensitivity to RF disorientation should fall by about two orders of magnitude when the frequency exceeds 116 MHz. Taken together with our earlier finding that 75 to 85 MHz RF fields disrupt the magnetic orientation of blackcaps, these results provide compelling evidence that the magnetic compass of migratory birds operates by a radical pair mechanism.

The structure of songbird MHC class I reveals antigen binding that is flexible at the N-terminus and static at the C-terminus.

Long-distance migratory animals such as birds and bats have evolved to withstand selection imposed by pathogens across the globe, and pathogen richness is known to be particularly high in tropical regions. Immune genes, so-called Major Histocompatibility Complex (MHC) genes, are highly duplicated in songbirds compared to other vertebrates, and this high MHC diversity has been hypothesised to result in a unique adaptive immunity. To understand the rationale behind the evolution of the high MHC genetic diversity in songbirds, we determined the structural properties of an MHC class I protein, Acar3, from a long-distance migratory songbird, the great reed warbler Acrocephalus arundinaceus (in short: Acar). The structure of Acar3 was studied in complex with pathogen-derived antigens and shows an overall antigen presentation similar to human MHC class I. However, the peptides bound to Acar3 display an unusual conformation: Whereas the N-terminal ends of the peptides display enhanced flexibility, the conformation of their C-terminal halves is rather static. This uncommon peptide-binding mode in Acar3 is facilitated by a central Arg residue within the peptide-binding groove that fixes the backbone of the peptide at its central position, and potentially permits successful interactions between MHC class I and innate immune receptors. Our study highlights the importance of investigating the immune system of wild animals, such as birds and bats, to uncover unique immune mechanisms which may neither exist in humans nor in model organisms.

Dimerization of European Robin Cryptochrome 4a.

Homo-dimer formation is important for the function of many proteins. Although dimeric forms of cryptochromes (Cry) have been found by crystallography and were recently observed in vitro for European robin Cry4a, little is known about the dimerization of avian Crys and the role it could play in the mechanism of magnetic sensing in migratory birds. Here, we present a combined experimental and computational investigation of the dimerization of robin Cry4a resulting from covalent and non-covalent interactions. Experimental studies using native mass spectrometry, mass spectrometric analysis of disulfide bonds, chemical cross-linking, and photometric measurements show that disulfide-linked dimers are routinely formed, that their formation is promoted by exposure to blue light, and that the most likely cysteines are C317 and C412. Computational modeling and molecular dynamics simulations were used to generate and assess a number of possible dimer structures. The relevance of these findings to the proposed role of Cry4a in avian magnetoreception is discussed.

Transcriptomic effects of Perfluoralkyl acids on the adipose tissue of a songbird species at environmentally relevant concentrations.

Perfluoralkyl acids (PFAS) have been regarded as global pollutants for at least twenty years, with potentially negative physiological effects on multiple vertebrate species including humans. Here we analyze the effects of the administration of environmentally-relevant levels of PFAS on caged canaries (Serinus canaria) by using a combination of physiological, immunological, and transcriptomic analyses. This constitutes a completely new approach to understand the toxicity pathway of PFAS in birds. While we observed no effects on physiological and immunological parameters (e.g, body weight, fat index, cell-mediated immunity), the transcriptome of the pectoral fatty tissue showed changes compatible with the known effects of PFAS as obesogens in other vertebrates, particularly in mammals. First, transcripts related to the immunological response were affected (mainly enriched), including several key signaling pathways. Second, we found a repression of genes related to the peroxisome response and fatty acid metabolism. We interpret these results as indicative of the potential hazard of environmental concentrations of PFAS on the fat metabolism and the immunological system of birds, while exemplifying the ability of transcriptomic analyses of detecting early physiological responses to toxicants. As the potentially affected functions are essential for the survival of the animals during, for example, migration, our results underline the need for tight control of the exposure of natural populations of birds to these substances.

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.

Spatial and temporal specificity of neuroestradiol provision in the songbird.

Steroid hormones are often synthesized in multiple tissues, affect several different targets, and modulate numerous physiological endpoints. The mechanisms by which this modulation is achieved with temporal and spatial specificity remain unclear. 17ß-estradiol for example, is made in several peripheral tissues and in the brain, where it affects a diverse set of behaviors. How is estradiol delivered to the right target, at the right time, and at the right concentration? In the last two decades, we have learned that aromatase (estrogen-synthase) can be induced in astrocytes following damage to the brain and is expressed at central synapses. Both mechanisms of estrogen provision confer spatial and temporal specificity on a lipophilic neurohormone with potential access to all cells and tissues. In this review, I trace the progress in our understanding of astrocytic and synaptic aromatization. I discuss the incidence, regulation, and functions of neuroestradiol provision by aromatization, first in astrocytes and then at synapses. Finally, I focus on a relatively novel hypothesis about the role of neuroestradiol in the orchestration of species-specific behaviors.

Direct Interaction of Avian Cryptochrome 4 with a Cone Specific G-Protein.

BACKGROUND: Night-migratory birds sense the Earth's magnetic field by an unknown molecular mechanism. Theoretical and experimental evidence support the hypothesis that the light-induced formation of a radical-pair in European robin cryptochrome 4a (ErCry4a) is the primary signaling step in the retina of the bird. In the present work, we investigated a possible route of cryptochrome signaling involving the α-subunit of the cone-secific heterotrimeric G protein from European robin. METHODS: Protein-protein interaction studies include surface plasmon resonance, pulldown affinity binding and Förster resonance energy transfer. RESULTS: Surface plasmon resonance studies showed direct interaction, revealing high to moderate affinity for binding of non-myristoylated and myristoylated G protein to ErCry4a, respectively. Pulldown affinity experiments confirmed this complex formation in solution. We validated these in vitro data by monitoring the interaction between ErCry4a and G protein in a transiently transfected neuroretinal cell line using Förster resonance energy transfer. CONCLUSIONS: Our results suggest that ErCry4a and the G protein also interact in living cells and might constitute the first biochemical signaling step in radical-pair-based magnetoreception.

Computational Reconstruction and Analysis of Structural Models of Avian Cryptochrome 4.

A recent study by Xu et al. (Nature, 2021, 594, 535-540) provided strong evidence that cryptochrome 4 (Cry4) is a key protein to endow migratory birds with the magnetic compass sense. The investigation compared the magnetic field response of Cry4 from migratory and nonmigratory bird species and suggested that a difference in magnetic sensitivity could exist. This finding prompted an in-depth investigation into Cry4 protein differences on the structural and dynamic levels. In the present study, the pigeon Cry4 (ClCry4) crystal structure was used to reconstruct the missing avian Cry4 protein structures via homology modeling for carefully selected bird species. The reconstructed Cry4 structure from European robin, Eurasian blackcap, zebra finch, chicken, and pigeon were subsequently simulated dynamically and analyzed. The studied avian Cry4 structures show flexibility in analogous regions pointing to similar activation mechanisms and/or signaling interaction partners. It can be concluded that the experimentally recorded difference in the magnetic field sensitivity of Cry4 from different birds is unlikely to be due to solely intrinsic dynamics of the proteins but requires additional factors that have not yet been identified.

The biological effects of geomagnetic field exhibit an important potential for cancer and vascular diseases.

Xu et al. recently demonstrated that cryptochrome 4 (CRY4) protein, as a light-dependent magnetic receptor, can sense geomagnetic fields to guide night-migratory songbirds' navigation and evolution by the formation of composite radical pairs and electron transport. We aim to comment on CRY4 through radical pairs and electron transport for magnetic sensitive in night-migratory songbirds' migration and evolution. Additionally, we find that the role of magnetic fields is deeply concerning to the scientific community and very enlightening for the diagnosis and treatment of cancer and vascular disease. We believe that this commentary makes a significant contribution to the literature because it elaborates on the importance of the geomagnetic field to night-migratory songbirds and extends the diagnostic and therapeutic value to cancer and vascular disease.

Molecular parallelism in signaling function across different sexually selected ornaments in a warbler.

Extravagant ornaments are thought to signal male quality to females choosing mates, but the evidence linking ornament size to male quality is controversial, particularly in cases in which females prefer different ornaments in different populations. Here, we use whole-genome sequencing and transcriptomics to determine the genetic basis of ornament size in two populations of a widespread warbler, the common yellowthroat (Geothlypis trichas). Within a single subspecies, females in a Wisconsin population prefer males with larger black masks as mates, while females in a New York population prefer males with larger yellow bibs. Despite being produced by different pigments in different patches on the body, the size of the ornament preferred by females in each population was linked to numerous genes that function in many of the same core aspects of male quality (e.g., immunity and oxidative balance). These relationships confirm recent hypotheses linking the signaling function of ornaments to male quality. Furthermore, the parallelism in signaling function provides the flexibility for different types of ornaments to be used as signals of similar aspects of male quality. This could facilitate switches in female preference for different ornaments, a potentially important step in the early stages of divergence among populations.

Broadband 75-85 MHz radiofrequency fields disrupt magnetic compass orientation in night-migratory songbirds consistent with a flavin-based radical pair magnetoreceptor.

The light-dependent magnetic compass sense of night-migratory songbirds can be disrupted by weak radiofrequency fields. This finding supports a quantum mechanical, radical-pair-based mechanism of magnetoreception as observed for isolated cryptochrome 4, a protein found in birds' retinas. The exact identity of the magnetically sensitive radicals in cryptochrome is uncertain in vivo, but their formation seems to require a bound flavin adenine dinucleotide chromophore and a chain of four tryptophan residues within the protein. Resulting from the hyperfine interactions of nuclear spins with the unpaired electrons, the sensitivity of the radicals to radiofrequency magnetic fields depends strongly on the number of magnetic nuclei (hydrogen and nitrogen atoms) they contain. Quantum-chemical calculations suggested that electromagnetic noise in the frequency range 75-85 MHz could give information about the identity of the radicals involved. Here, we show that broadband 75-85 MHz radiofrequency fields prevent a night-migratory songbird from using its magnetic compass in behavioural experiments. These results indicate that at least one of the components of the radical pair involved in the sensory process of avian magnetoreception must contain a substantial number of strong hyperfine interactions as would be the case if a flavin-tryptophan radical pair were the magnetic sensor.

The form, function, and evolutionary significance of neural aromatization.

Songbirds have emerged as exceptional research subjects for helping us appreciate and understand estrogen synthesis and function in brain. In the context of recognizing the vertebrate-wide importance of brain aromatase expression, in this review we highlight where we believe studies of songbirds have provided clarification and conceptual insight. We follow by focusing on more recent studies of aromatase and neuroestrogen function in the hippocampus and the pallial auditory processing region NCM of songbirds. With perspectives drawn from this body of work, we speculate that the evolution of enhanced neural estrogen signaling, including in the mediation of social behaviors, may have given songbirds the resilience to radiate into one of the most successful vertebrate groups on the planet.

Analysis of avian Usutu virus infections in Germany from 2011 to 2018 with focus on dsRNA detection to demonstrate viral infections.

Usutu virus (USUV) is a zoonotic arbovirus causing avian mass mortalities. The first outbreak in North-Western Germany occurred in 2018. This retrospective analysis focused on combining virological and pathological findings in birds and immunohistochemistry. 25 common blackbirds, one great grey owl, and one kingfisher collected from 2011 to 2018 and positive for USUV by qRT-PCR were investigated. Macroscopically, most USUV infected birds showed splenomegaly and hepatomegaly. Histopathological lesions included necrosis and lymphohistiocytic inflammation within spleen, Bursa fabricii, liver, heart, brain, lung and intestine. Immunohistochemistry revealed USUV antigen positive cells in heart, spleen, pancreas, lung, brain, proventriculus/gizzard, Bursa fabricii, kidney, intestine, skeletal muscle, and liver. Analysis of viral genome allocated the virus to Europe 3 or Africa 2 lineage. This study investigated whether immunohistochemical detection of double-stranded ribonucleic acid (dsRNA) serves as an alternative tool to detect viral intermediates. Tissue samples of six animals with confirmed USUV infection by qRT-PCR but lacking viral antigen in liver and spleen, were further examined immunohistochemically. Two animals exhibited a positive signal for dsRNA. This could indicate either an early state of infection without sufficient formation of virus translation products, occurrence of another concurrent virus infection or endogenous dsRNA not related to infectious pathogens and should be investigated in more detail in future studies.

Early life neonicotinoid exposure results in proximal benefits and ultimate carryover effects.

Neonicotinoids are insecticides widely used as seed treatments that appear to have multiple negative effects on birds at a diversity of biological scales. Adult birds exposed to a low dose of imidacloprid, one of the most commonly used neonicotinoids, presented reduced fat stores, delayed migration and potentially altered orientation. However, little is known on the effect of imidacloprid on birds growth rate despite studies that have documented disruptive effects of low imidacloprid doses on thyroid gland communication. We performed a [Formula: see text] factorial design experiment in Zebra finches, in which nestling birds were exposed to a very low dose (0.205 mg kg body [Formula: see text]) of imidacloprid combined with food restriction during posthatch development. During the early developmental period, imidacloprid exposure resulted in an improvement of body condition index in treated nestlings relative to controls. Imidacloprid also led to compensatory growth in food restricted nestlings. This early life neonicotinoid exposure also carried over to adult age, with exposed birds showing higher lean mass and basal metabolic rate than controls at ages of 90-800 days. This study presents the first evidence that very low-dose neonicotinoid exposure during early life can permanently alter adult phenotype in birds.

Using breath δ13C analysis to determine the effects of dietary carbohydrate and protein on glucose and leucine oxidation at rest in the yellow-rumped warbler (Setophaga coronata).

Fat is the major fuel for migratory flight of birds, but protein is also catabolized. Flight range could be reduced if protein is used too quickly from muscles and organs, and it is important to understand factors that influence protein catabolism. Previous correlative studies suggested high protein diets may increase protein use in flight, although a wind tunnel study with yellow-rumped warblers (Setophaga coronata) did not support this relationship. We tested the hypothesis that diet composition affects nutrient oxidation in resting, fasted yellow-rumped warblers. For method development, we gavaged or subcutaneously injected warblers with 13C labelled glucose or leucine, and measured δ13C of breath CO2 in real time using infrared laser spectrometry. Regardless of route of administration, leucine had greater instantaneous and cumulative oxidation than glucose. Compared to subcutaneous injection, gavaged birds reached maximum oxidation rate faster for leucine and glucose, respectively, had a higher maximum oxidation rate, and reached final cumulative oxidation approximately faster for leucine or glucose, respectively, indicating immediate oxidation of the substrates by the digestive system. Warblers (N = 10 each) were fed isocaloric 60% carbohydrate or 60% protein diets for minimum 2 weeks, and subcutaneously injected with 13C labelled glucose or leucine. Diet composition had little effect on oxidation kinetics except that warblers fed high-carbohydrate reached final cumulative oxidation of leucine more quickly than those fed high-protein. The findings do not support the hypothesis that high protein diets increase the oxidation of protein during negative energy states in migratory birds, and provide methodology that could be applied to test it in flight.