Relative roles of temperature and photoperiod as drivers of metabolic flexibility in dark-eyed juncos.

Seasonal phenotypic flexibility in small birds produces a winter phenotype with elevated maximum cold-induced metabolic rates (=summit metabolism, Msum). Temperature and photoperiod are candidates for drivers of seasonal phenotypes, but their relative impacts on metabolic variation are unknown. We examined photoperiod and temperature effects on Msum, muscle masses and activities of key catabolic enzymes in winter dark-eyed juncos (Junco hyemalis). We randomly assigned birds to four treatment groups varying in temperature (cold=3°C; warm=24°C) and photoperiod [short day (SD)=8 h:16 h light:dark; long day (LD)=16 h:8 h light:dark] in a two-by-two design. We measured body mass (Mb), flight muscle width and Msum before and after 3 and 6 weeks of acclimation, and flight muscle and heart masses after 6 weeks. Msum increased for cold-exposed, but not for warm-exposed, birds. LD birds gained more Mb than SD birds, irrespective of temperature. Flight muscle size and mass did not differ significantly among groups, but heart mass was larger in cold-exposed birds. Citrate synthase, carnitine palmitoyl transferase and ß-hydroxyacyl Co-A dehydrogenase activities in the pectoralis were generally higher for LD and cold groups. The cold-induced changes in Msum and heart mass parallel winter changes for small birds, but the larger Mb and higher catabolic enzyme activities in LD birds suggest photoperiod-induced changes associated with migratory disposition. Temperature appears to be a primary driver of flexibility in Msum in juncos, but photoperiod-induced changes in Mb and catabolic enzyme activities, likely associated with migratory disposition, interact with temperature to contribute to seasonal phenotypes.

Activation of the immune system incurs energetic costs but has no effect on the thermogenic performance of house sparrows during acute cold challenge.

Trade-offs between the immune system and other condition-dependent life-history traits (reproduction, predator avoidance and somatic growth) have been well documented in both birds and mammals. However, no studies have examined the impact of immune activation on thermoregulatory performance during acute cold exposure. Because of their high surface-area-to-volume ratios, small birds incur high energetic costs associated with thermoregulation during cold exposure. Consequently, we predicted that the immune system and the thermoregulatory system would compete for energetic resources. To test this, we immunologically challenged adult house sparrows (Passer domesticus) with 5 mg kg(-1) of lipopolysaccharide (LPS) to induce an acute phase response and measured both resting (RMR; minimum metabolic rate) and summit ( ; maximal metabolic rate during cold exposure) metabolic rates. We found that birds injected with LPS had significantly higher RMR and than birds injected with phosphate-buffered saline, indicating that LPS-treated birds were able to support the cost of both immune activation and thermoregulation under conditions eliciting maximal thermogenic performance. These results suggest that, in the absence of a pathogen, birds that experience short-term activation of the immune system have higher energetic costs during cold exposure, but immune activation does not compromise maximum thermoregulatory performance.

Within-winter flexibility in muscle and heart lipid transport and catabolism in passerine birds.

Small birds in cold climates may show within-winter metabolic flexibility to match metabolic capacities to prevailing weather conditions. This flexibility may occur over periods of days to weeks, but the underlying mechanisms for such flexibility are not well understood. Because lipids are the primary fuel for sustained thermogenesis, we examined whether lipid transport and catabolism can mediate within-winter metabolic flexibility in two small temperate-zone wintering passerine birds, dark-eyed juncos (Junco hyemalis) and house sparrows (Passer domesticus). We used simple and multiple regression analyses to test for correlations of several lipid transporters in pectoralis muscle (plasma membrane-bound and cytosolic fatty acid-binding proteins, FABP; fatty acyl translocase, FAT/CD36) and regulatory enzymes (carnitine acyl transferase, CPT; ß-hydroxyacyl CoA dehydrogenase, HOAD) in pectoralis and heart with short-term (ST, 0-7 days), medium-term (MT, 14-30 days) and long-term (LT, 30-year mean daily and extreme minimum temperatures, day of winter season) temperature variables. We hypothesized negative correlations between these regulators and temperature variables. Juncos showed negative correlations for FABPs with ST or MT temperature variables, but other lipid transporters and regulatory enzymes showed positive correlations with ST or MT temperatures for juncos, suggesting no consistent pathway-wide response to within-winter temperatures. LT temperature variables showed several significant associations with lipid transporters and enzymes for juncos, but also not in consistent directions. House sparrows showed the expected negative correlations with LT temperatures for FABPpm, but positive correlations with temperature variables for FABPc, CPT and HOAD. Different species-specific patterns of variation and the absence of consistent pathway-wide responses to temperature suggest that the lipid transport and catabolism pathway is not a uniform mediator of within-winter metabolic flexibility among small birds.

Within-Winter Flexibility in Muscle Masses, Myostatin, and Cellular Aerobic Metabolic Intensity in Passerine Birds.

Metabolic rates of passerine birds are flexible traits that vary both seasonally and among and within winters. Seasonal variation in summit metabolic rates (Msum = maximum thermoregulatory metabolism) in birds is consistently correlated with changes in pectoralis muscle and heart masses and sometimes with variation in cellular aerobic metabolic intensity, so these traits might also be associated with shorter-term, within-winter variation in metabolic rates. To determine whether these mechanisms are associated with within-winter variation in Msum, we examined the effects of short-term (ST; 0-7 d), medium-term (MT; 14-30 d), and long-term (LT; 30-yr means) temperature variables on pectoralis muscle and heart masses, pectoralis expression of the muscle-growth inhibitor myostatin and its metalloproteinase activators TLL-1 and TLL-2, and pectoralis and heart citrate synthase (CS; an indicator of cellular aerobic metabolic intensity) activities for two temperate-zone resident passerines, house sparrows (Passer domesticus) and dark-eyed juncos (Junco hyemalis). For both species, pectoralis mass residuals were positively correlated with ST temperature variables, suggesting that cold temperatures resulted in increased turnover of pectoralis muscle, but heart mass showed little within-winter variation for either species. Pectoralis mRNA and protein expression of myostatin and the TLLs were only weakly correlated with ST and MT temperature variables, which is largely consistent with trends in muscle masses for both species. Pectoralis and heart CS activities showed weak and variable trends with ST temperature variables in both species, suggesting only minor effects of temperature variation on cellular aerobic metabolic intensity. Thus, neither muscle or heart masses, regulation by the myostatin system, nor cellular aerobic metabolic intensity varied consistently with winter temperature, suggesting that other factors regulate within-winter metabolic variation in these birds.

Summer-to-Winter Phenotypic Flexibility of Fatty Acid Transport and Catabolism in Skeletal Muscle and Heart of Small Birds.

Prolonged shivering in birds is mainly fueled by lipids. Consequently, lipid transport and catabolism are vital for thermogenic performance and could be upregulated along with thermogenic capacity as part of the winter phenotype. We investigated summer-to-winter variation in lipid transport and catabolism by measuring mRNA expression, protein levels, and enzyme activities for several key steps of lipid transport and catabolic pathways in pectoralis muscle and heart in two small temperate-zone resident birds, American goldfinches (Spinus tristis) and black-capped chickadees (Poecile atricapillus). Cytosolic fatty acid binding protein (FABPc; a key component of intramyocyte lipid transport) mRNA and/or protein levels were generally higher in winter for pectoralis muscle and heart for both species. However, seasonal variation in plasma membrane lipid transporters, fatty acyl translocase, and plasma membrane fatty acid binding protein in pectoralis and heart differed between the two species, with winter increases for chickadees and seasonal stability or summer increases for goldfinches. Catabolic enzyme activities generally showed limited seasonal differences for both tissues and both species. These data suggest that FABPc is an important target of upregulation for the winter phenotype in pectoralis and heart of both species. Plasma membrane lipid transporters and lipid catabolic capacity were also elevated in winter for chickadees but not for goldfinches. Because the two species show differential regulation of distinct aspects of lipid transport and catabolism, these data are consistent with other recent studies documenting that different bird species or populations employ a variety of strategies to promote elevated winter thermogenic capacity.

Migration-induced variation of fatty acid transporters and cellular metabolic intensity in passerine birds.

Because lipids are the main fuel supporting avian endurance activity, lipid transport and oxidation capacities may increase during migration. We measured enzyme activities, mRNA expression and protein levels in pectoralis and heart for several key steps of lipid transport and catabolism pathways to investigate whether these pathways were upregulated during migration. We used yellow-rumped (Setophaga coronata) and yellow (S. petechia) warblers and warbling vireos (Vireo gilvus) as study species because they all show migration-induced increases in organismal metabolic capacities. For yellow-rumped warblers, ß-hydroxyacyl CoA-dehydrogenase (HOAD) activities and fatty acid transporter mRNA and/or protein levels were higher during spring than fall in pectoralis and heart, except that fatty acid translocase (FAT/CD36) protein levels showed the opposite pattern in heart. Lipid transporter protein levels, but not mRNA expression, in pectoralis and heart of warbling vireos were higher either during spring or fall than summer, but this was not true for HOAD activities. For yellow warblers, pectoralis, but not heart, protein levels of lipid transporters were upregulated during migration relative to summer, but this pattern was not evident for mRNA expression or HOAD activity. Finally, muscle and heart citrate synthase and carnitine palmitoyl transferase activities showed little seasonal variation for any species. These data suggest that pectoralis and heart lipid transport and catabolism capacities are often, but not universally, important correlates of elevated organismal metabolic capacity during migration. In contrast, migration-induced variation in cellular metabolic intensity and mitochondrial membrane transport are apparently not common correlates of the migratory phenotype in passerines.

Phenotypic flexibility of skeletal muscle and heart masses and expression of myostatin and tolloid-like proteinases in migrating passerine birds.

Migrant birds require large flight muscles and hearts to enhance aerobic capacity and support sustained flight. A potential mechanism for increasing muscle and heart masses during migration in birds is the muscle growth inhibitor myostatin and its metalloproteinase activators, tolloid-like proteinases (TLL-1 and TLL-2). We hypothesized that myostatin, TLL-1 and TLL-2 are downregulated during migration in pectoralis and hearts of migratory passerines to promote hypertrophy. We measured seasonal variation of tissue masses, mRNA expression of myostatin, TLL-1, and TLL-2, and myostatin protein levels in pectoralis muscle and heart for yellow warblers (Setophaga petechia), warbling vireos (Vireo gilvus), and yellow-rumped warblers (Setophaga coronata). Pectoralis mass was greatest in spring for warbling vireos and yellow warblers, but was stable between spring and fall for yellow-rumped warblers. Heart mass was higher in spring than in fall for yellow-rumped warblers, lowest in fall for warbling vireos, and seasonally stable for yellow warblers. Pectoralis and heart mRNA expression of myostatin and the TLLs did not differ significantly for any of the three species, offering little support for our hypothesis for a prominent role for myostatin in regulating migration-induced variation in pectoralis and heart masses. In contrast, pectoralis myostatin protein levels were lowest in spring for all three species, consistent with our hypothesis. Myostatin protein levels in heart, however, were seasonally stable for warbling vireos and yellow warblers, and increased in spring relative to fall for yellow-rumped warblers. These data offer mixed support for our hypothesis for the pectoralis, but suggest that myostatin is not a prominent regulator of migration-induced heart hypertrophy. Moreover, the different seasonal patterns for pectoralis mRNA and protein expression suggest that post-transcriptional modification of myostatin may contribute to pectoralis mass regulation during migration.

Seasonal variation in pectoralis muscle and heart myostatin and tolloid-like proteinases in small birds: a regulatory role for seasonal phenotypic flexibility?

Seasonally variable environments produce seasonal phenotypes in small birds such that winter birds have higher thermogenic capacities and pectoralis and heart masses. One potential regulator of these seasonal phenotypes is myostatin, a muscle growth inhibitor, which may be downregulated under conditions promoting increased energy demand. We examined summer-to-winter variation in skeletal muscle and heart masses and used qPCR and Western blots to measure levels of myostatin and its metalloproteinase activators TLL-1 and TLL-2 for two small temperate-zone resident birds, American goldfinches (Spinus tristis) and black-capped chickadees (Poecile atricapillus). Winter pectoralis and heart masses were significantly greater than in summer for American goldfinches. Neither myostatin expression nor protein levels differed significantly between seasons for goldfinch pectoralis. However, myostatin levels in goldfinch heart were significantly greater in summer than in winter, although heart myostatin expression was seasonally stable. In addition, expression of both metalloproteinase activators was greater in summer than in winter goldfinches for both pectoralis and heart, significantly so except for heart TLL-2 (P = 0.083). Black-capped chickadees showed no significant seasonal variation in muscle or heart masses. Seasonal patterns of pectoralis and heart expression and/or protein levels for myostatin and its metalloproteinase activators in chickadees showed no consistent seasonal trends, which may help explain the absence of significant seasonal variation in muscle or heart masses for chickadees in this study. These data are partially consistent with a regulatory role for myostatin, and especially myostatin processing capacity, in mediating seasonal metabolic phenotypes of small birds.

Individual variation in thermogenic capacity is correlated with flight muscle size but not cellular metabolic capacity in American goldfinches (Spinus tristis).

Abstract Cold tolerance and overwinter survival are positively correlated with organismal thermogenic capacity (=summit metabolic rate [Msum]) in endotherms. Msum varies seasonally in small-bird populations and may be mechanistically associated with variation in flight muscle size or cellular metabolic capacity, but the relative roles of these traits as drivers of individual variation in thermogenic performance are poorly known. We measured flight muscle size by ultrasonography, pectoralis and supracoracoideus muscle masses, and muscular activities of key aerobic enzymes (citrate synthase, carnitine palmitoyl transferase, and ß-hydroxyacyl-CoA dehydrogenase) and correlated these measurements with Msum for individual American goldfinches (Spinus tristis) to test the hypotheses that muscle size and/or cellular metabolic capacity serve as prominent drivers of individual variation in organismal metabolic capacity. Ultrasonographic flight muscle size was weakly positively correlated with Msum ([Formula: see text]). Both log10-transformed Msum and flight muscle mass were significantly correlated with log10 body mass, so we calculated allometric residuals for log Msum and for log flight muscle mass to test their correlation independent of body mass. Flight muscle mass residuals were significantly positively correlated with Msum residuals, and this correlation was primarily driven by variation in pectoralis muscle mass. In contrast, none of the mass-specific activities of any enzyme in any muscle were significantly correlated with Msum. These data suggest that flight muscle size, not cellular metabolic capacity, is the primary driver of individual variation in thermogenic performance in goldfinches. This is consistent with the idea that phenotypic flexibility of flight muscle mass is a general mechanism mediating variation in metabolic performance in response to changing energy demands in birds.

Are maternal antibodies really that important? Patterns in the immunologic development of altricial passerine house sparrows (Passer domesticus).

BACKGROUND: Maternal antibodies are believed to play an integral role in protecting immunologically immature wild-passerines from environmental antigens. This study comprehensively examines the early development of the adaptive immune system in an altricial-developing wild passerine species, the house sparrow (Passer domestics), by characterizing the half-life of maternal antibodies in nestling plasma, the onset of de novo synthesis of endogenous antibodies by nestlings, and the timing of immunological independence, where nestlings rely entirely on their own antibodies for immunologic protection. METHODOLOGY/PRINCIPAL FINDINGS: In an aviary study we vaccinated females against a novel antigen that these birds would not otherwise encounter in their natural environment, and measured both antigen-specific and total antibody concentration in the plasma of females, yolks, and nestlings. We traced the transfer of maternal antibodies from females to nestlings through the yolk and measured catabolisation of maternal antigen-specific antibodies in nestlings during early development. By utilizing measurements of non-specific and specific antibody levels in nestling plasma we were able to calculate the half-life of maternal antibodies in nestling plasma and the time point at which nestling were capable of synthesizing antibodies themselves. Based on the short half-life of maternal antibodies, the rapid production of endogenous antibodies by nestlings and the relatively low transfer of maternal antibodies to nestlings, our findings suggest that altricial-developing sparrows achieve immunologic independence much earlier than precocial birds. CONCLUSIONS/SIGNIFICANCE: To our knowledge, this is the first in depth analyses performed on the adaptive immune system of a wild-passerine species. Our results suggest that maternal antibodies may not confer the immunologic protection or immune priming previously proposed in other passerine studies. Further research needs to be conducted on other altricial passerines to determine if the results of our study are a species-specific phenomenon or if they apply to all altricial-developing birds.

Ecoimmunity in Darwin's finches: invasive parasites trigger acquired immunity in the medium ground finch (Geospiza fortis).

BACKGROUND: Invasive parasites are a major threat to island populations of animals. Darwin's finches of the Galápagos Islands are under attack by introduced pox virus (Poxvirus avium) and nest flies (Philornis downsi). We developed assays for parasite-specific antibody responses in Darwin's finches (Geospiza fortis), to test for relationships between adaptive immune responses to novel parasites and spatial-temporal variation in the occurrence of parasite pressure among G. fortis populations. METHODOLOGY/PRINCIPAL FINDINGS: We developed enzyme-linked immunosorbent assays (ELISAs) for the presence of antibodies in the serum of Darwin's finches specific to pox virus or Philornis proteins. We compared antibody levels between bird populations with and without evidence of pox infection (visible lesions), and among birds sampled before nesting (prior to nest-fly exposure) versus during nesting (with fly exposure). Birds from the Pox-positive population had higher levels of pox-binding antibodies. Philornis-binding antibody levels were higher in birds sampled during nesting. Female birds, which occupy the nest, had higher Philornis-binding antibody levels than males. The study was limited by an inability to confirm pox exposure independent of obvious lesions. However, the lasting effects of pox infection (e.g., scarring and lost digits) were expected to be reliable indicators of prior pox infection. CONCLUSIONS/SIGNIFICANCE: This is the first demonstration, to our knowledge, of parasite-specific antibody responses to multiple classes of parasites in a wild population of birds. Darwin's finches initiated acquired immune responses to novel parasites. Our study has vital implications for invasion biology and ecological immunology. The adaptive immune response of Darwin's finches may help combat the negative effects of parasitism. Alternatively, the physiological cost of mounting such a response could outweigh any benefits, accelerating population decline. Tests of the fitness implications of parasite-specific immune responses in Darwin's finches are urgently needed.