Mediterranean songbirds show pronounced seasonal variation in thermoregulatory traits.

Addressing the patterns of variation in thermal traits is crucial to better predict the potential effects of climate change on organisms. Here, we assessed seasonal (winter vs summer) adjustments in key thermoregulatory traits in eight Mediterranean-resident songbirds. Overall, songbirds increased whole-animal (by 8%) and mass-adjusted (by 9%) basal metabolic rate and decreased (by 56%) thermal conductance below the thermoneutral zone during winter. The magnitude of these changes was within the lower values found in songbirds from northern temperate areas. Moreover, songbirds increased (by 11%) evaporative water loss within the thermoneutral zone during summer, while its rate of increase above the inflection point of evaporative water loss (i.e., the slope of evaporative water loss versus temperature) decreased by 35% during summer - a value well above that reported for other temperate and tropical songbirds. Finally, body mass increased by 5% during winter, a pattern similar to that found in many northern temperate species. Our findings support the idea that physiological adjustments might enhance the resilience of Mediterranean songbirds to environmental changes, with short-term benefits by saving energy and water under thermally stressful conditions. Nevertheless, not all species showed the same patterns, suggesting different strategies in their thermoregulatory adaptations to seasonal environments.

Heat tolerance limits of Mediterranean songbirds and their current and future vulnerabilities to temperature extremes.

Songbirds are one of the groups most vulnerable to extreme heat events. Although several recent studies have assessed their physiological responses to heat, most of them have focused solely on arid-zone species. We investigated thermoregulatory responses to heat in eight small-sized songbirds occurring in the Mediterranean Basin, where heatwaves are becoming more frequent and intense. Specifically, we determined their heat tolerance limits (HTLs) and evaporative cooling efficiency, and evaluated their current and future vulnerabilities to heat in southwestern Iberia, a Mediterranean climate warming hotspot. To do this, we exposed birds to an increasing profile of air temperatures (Ta) and measured resting metabolic rate (RMR), evaporative water loss (EWL), evaporative cooling efficiency (the ratio between evaporative heat loss and metabolic heat production) and body temperature (Tb). HTL ranged between 40 and 46°C across species, and all species showed rapid increases in RMR, EWL and Tb in response to increasing Ta. However, only the crested lark (Galerida cristata) achieved an evaporative cooling efficiency greater than 1. The studied songbirds currently experience summer Ta maxima that surpass the upper critical temperatures of their thermoneutral zone and even their HTL. Our estimates indicate that five of the eight species will experience moderate risk of lethal dehydration by the end of the century. We argue that the limited heat tolerance and evaporative cooling efficiency of small-sized Mediterranean songbirds make them particularly vulnerable to heatwaves, which will be exacerbated under future climate change scenarios.

The thermoregulatory role of relative bill and leg surface areas in a Mediterranean population of Great tit (Parus major).

There is growing evidence on the role of legs and bill as 'thermal windows' in birds coping with heat stress. However, there is a lack of empirical work examining the relationship between the relative bill and/or leg surface areas and key thermoregulatory traits such as the limits of the thermoneutral zone (TNZ) or the cooling efficiency at high temperatures. Here, we explored this relationship in a Mediterranean population of Great tit (Parus major) facing increasing thermal stress in its environment. The lower and upper critical limits of the TNZ were found to be 17.7 ± 1.6ºC and 34.5 ± 0.7°C, respectively, and the basal metabolic rate was 0.96 ± 0.12 ml O2 min-1 on average. The evaporative water loss (EWL) inflection point was established at 31.85 ± 0.27°C and was not significantly different from the value of the upper critical limit. No significant relationship was observed between the relative bill or tarsi size and TNZ critical limits, breadth, mass-independent VO2, or mass-independent EWL at any environmental temperature (from 10 to 40°C). However, Great tit males (but not females) with larger tarsi areas (a proxy of leg surface area) showed higher cooling efficiencies at 40°C. We found no support for the hypothesis that the bill surface area plays a significant role as a thermal window in Great tits, but the leg surface areas may play a role in males' physiological responses to high temperatures. On the one hand, we argue that the studied population occupies habitats with available microclimates and fresh water for drinking during summer, so active heat dissipation by EWL might be favored instead of dry heat loss through the bill surface. Conversely, male dominance behaviors could imply a greater dependence on cutaneous EWL through the upper leg surfaces as a consequence of higher exposure to harsh environmental conditions than faced by females.

Going to sleep with a full belly: Thermal substitution by specific dynamic action in shorebirds.

Many bird species occupy habitats where environmental temperatures fall well below their thermoneutral zone (TNZ), so they must deal with high energy costs of thermoregulation to keep in heat balance. In such circumstances, specific dynamic action (SDA) - also referred to as heat increment of feeding - could be used to substitute for these high thermoregulatory costs. If birds ingest food before going to roost in cold environments, the SDA will be beneficial as an energy-conserving mechanism by thermal substitution. We investigated the magnitude and duration of SDA in a small-sized shorebird, the dunlin Calidris alpina, while feeding on living prey. We simulated in the aviary the food availability of a semidiurnal tidal cycle, and calculated the thermal substitution by SDA below their TNZ at the beginning of the "high tide" (resting period), after feeding ad libitum during the "low tide" (feeding period). Within TNZ (25 °C), dunlins consumed 12% (2.15 kJ) of the gross energy intake in excess by the SDA, with a duration of ~95 min. At 10 °C, i.e. below the lower critical limit of TNZ, SDA magnitude and duration were reduced by 29% and 31%, respectively. The amount of food ingested significantly affected the duration and magnitude of SDA, as well as the dunlin's body temperature. Thermal substitution by SDA saved 11% of the dunlin's theoretical daily energy requirement during winter. This thermal substitution could be commonly used by birds going to roost in cold climates. Interacting with other different behavioral and/or physiological strategies would help to maintain lower energetic costs and enhance survival in cold environments.

Day and night use of habitats by northern pintails during winter in a primary rice-growing region of Iberia.

Loss of natural wetlands is a global phenomenon that has severe consequences for waterbird populations and their associated ecosystem services. Although agroecosystems can reduce the impact of natural habitat loss, drivers of use of such artificial habitats by waterbirds remain poorly understood. Using the cosmopolitan northern pintail Anas acuta as a model species, we monitored home-range and fine-scale resource selection across the agricultural landscape. Individuals were tracked using GPS-GSM transmitters, and a suite of environmental and landscape features were measured throughout the winter seasons. Spatial patterns of habitat use were analysed using generalized linear mixed effect models by integrating field-observations with GPS telemetry. All birds used rice fields as foraging grounds at night and commuted to an adjacent reservoir to roost during daylight. Home-ranges and maximum foraging distances of nocturnally foraging birds increased with decreasing availability of flooded fields, and were positively correlated with moonlight levels. Birds selected flooded rice paddies (water depth range: 9-21 cm) with standing stubble and substrate with pebbles smaller than 0.5 cm in diameter. Density of rice seeds, rice paddy size, and other environmental and landscape features did not emerge as significant predictors. Our findings indicate that nocturnal foraging of northern pintails within rice fields is driven primarily by straw manipulation, water level and substrate pebble size. Thus, the presence of standing stubble in flooded paddies with soft bottoms should be prioritized to improve foraging areas for dabbling ducks. These management procedures in themselves would not increase economic costs or affect rice production and could be applied for dabbling-duck conservation throughout the world.

Wetland salinity induces sex-dependent carry-over effects on the individual performance of a long-distance migrant.

Salinization is having a major impact on wetlands and its biota worldwide. Specifically, many migratory animals that rely on wetlands are increasingly exposed to elevated salinity on their nonbreeding grounds. Experimental evidence suggests that physiological challenges associated with increasing salinity may disrupt self-maintenance processes in these species. Nonetheless, the potential role of salinity as a driver of ecological carry-over effects remains unstudied. Here, we investigated the extent to which the use of saline wetlands during winter - inferred from feather stable isotope values - induces residual effects that carry over and influence physiological traits relevant to fitness in black-tailed godwits Limosa limosa limosa on their northward migration. Overwintering males and females were segregated by wetland salinity in West Africa, with females mostly occupying freshwater wetlands. The use of these wetlands along a gradient of salinities was associated with differences in immune responsiveness to phytohaemagglutinin and sized-corrected body mass in godwits staging in southern Europe during northward migration - 3,000 km from the nonbreeding grounds - but in males only. These findings provide a window onto the processes by which wetland salinity can induce carry-over effects and can help predict how migratory species should respond to future climate-induced increases in salinity.

Physiological, Morphological and Behavioural Responses of Self-Feeding Precocial Chicks Copying with Contrasting Levels of Water Salinity during Development.

Combined physiological and behavioural responses to salt loads during development have rarely been studied in air-breathing vertebrates able to inhabit hypersaline habitats, but they may be of particular importance in understanding, for example, the differences among species in patterns of habitat use or ontogenetic diet switches. Here, we compared the physiological and behavioural responses of self-feeding precocial chicks developed in contrasting levels of water salinity. The model species was the Black-winged Stilt (Himantopus himantopus) a precocial shorebird that breeds in a range of habitats from freshwater to hypersaline wetlands. Specifically, we compared resting metabolic rate (RMR), heat shock proteins (Hsp70), plasma ions, hematocrit, body mass, body size, growth rate and head-shaking behaviour of captive-reared Black-winged Stilt fledglings developed under fresh (0 ‰), saline (20 ‰), and hypersaline (60 ‰) water. Contrary to expectations, none of the physiological and morphological variables measured differed significantly among treatments. Likewise, the RMR of wild and captive-reared fledglings was similar. Surprisingly, the saltgland mass of wild fledglings from freshwater and those from hypersaline habitats was also similar. However, head-shaking, a behavioural response associated to minimize salt intake and to expel the secretions of salt glands, differed according to salinity source: head-shaking rate increased with increasing salinity. The results of this study support the key role of behavioural osmoregulation in avoiding salt stress during development.

Immunoreactive cortisone in droppings reflect stress levels, diet and growth rate of gull-billed tern chicks.

Blood levels of corticosterone have been traditionally analyzed to assess stress levels in birds; however, measuring steroid hormone metabolites in feces and droppings has gained much interest as a noninvasive technique successfully used for such purposed in vertebrates. Diet may affect these fecal metabolite levels (e.g., due to nutritional stress), however, this variable has not been taken into account in studies with chicks despite the great dietary flexibility of many avian species. In this study, we addressed for the first time this key issue and validated the technique in wild gull-billed tern chicks (Gelochelidon nilotica). Several enzyme immunoassays were used to determine the most appropriate test to measure the stress response. Subsequently, we performed an experiment in captivity to assess adrenocortical activity in gull-billed tern chicks fed with two diets: piscivorous vs. insectivorous. Finally, the relation between the chicks' growth rate and excreted immunoreactive glucocorticoid metabolites (EGMs) was also evaluated. We found the immunoreactive cortisone metabolites to be a good index of stress (as being an index of adrenocortical reactivity) in chicks of this species. Fish-fed chicks had higher levels of cortisone metabolites when comparing both concentration and total daily excreted metabolites. Within each treatment diet, cortisone metabolite levels and growth rates were negatively correlated. These findings suggest that the diet should be considered when using this technique for comparative purposes and highlight the trade-off between stress levels and chicks growth rates.

Unravelling trophic subsidies of agroecosystems for biodiversity conservation: food consumption and nutrient recycling by waterbirds in Mediterranean rice fields.

Waterbirds can reallocate a considerable amount of nutrients within agricultural fields and between agriculture sites and wetlands. However their effects on biogeochemical cycles have rarely been quantified. We estimated bird numbers, diet (from stable isotope analysis), food supply, and the food consumption on rice fields by overwintering waterbirds in one of the most important areas for rice production in southwestern Europe and a key area for various migrating and resident waterbird species. Herein, we modelled the nutrient (N and P) recycling in rice fields, and their transport to reservoirs. The energy consumption by waterbirds (96,605±18,311 individuals) on rice fields during winter averaged at 89.9±39.0 kJ·m(-2), with its majority (89.9%) belonging to foraging on rice seeds. Thus, the birds removed about 26% of rice seeds leftover after harvest (estimated in 932.5±504.7 seeds·m(-2) in early winter) wherein common cranes and dabbling ducks (four species) were the most important consumers. Waterbirds foraging and roosting in the rice fields recycled more than 24.1 (1.0 kg·ha(-1)) of N and an additional 5.0 tons (0.2 kg·ha(-1)) of P in the Extremadura's rice fields during winter. Additionally, we estimated that 2.3 tons of N and 550 kg of P were removed from rice fields and transported to reservoirs. The seasonal foraging of wildlife should result in a direct benefit for rice farmers by improving nutrient recycling through defecation by waterbirds with respect to artificial fertilisation. Additionally, rice fields located in the cranes' core wintering areas can provide sufficient food supply to induce habitat shift from their traditional wintering habitat in 'dehesas' to rice fields, which causes indirect socioeconomic benefit through reduced acorn consumption by cranes. Our modelling approach may thus be especially helpful for management decisions regarding rice agroecosystems in areas which are also important for the conservation of migratory waterbirds.

Time course and metabolic costs of a humoral immune response in the little ringed plover Charadrius dubius.

Despite host defense against parasites and pathogens being considered a costly life-history trait, relatively few studies have assessed the energetic cost of immune responsiveness. Knowledge of such energetic costs may help to understand the mechanisms by which trade-offs with other demanding activities occur. The time course and associated metabolic costs of mounting a primary and secondary humoral immune response was examined in little ringed plovers Charadrius dubius challenged with sheep red blood cells. As was expected, the injection with this antigen increased the production of specific antibodies significantly, with peaks 6 d postinjection in both primary and secondary responses. At the peak of secondary antibody response, the antibody production was 29% higher than that observed during the primary response, but the difference was nonsignificant. Mounting the primary response did not significantly increase the resting metabolic rate (RMR) of birds, whereas the secondary response did by 21%, suggesting that the latter was more costly in terms of RMR. In spite of the fact that the primary response did not involve an increase in RMR, birds significantly decreased their body mass. This could imply an internal energy reallocation strategy to cope with the induced immune challenge. Last, we found that RMR and antibody production peaks were not coupled, which could help to conciliate the variable results of previous studies. Collectively, the results of this study support the hypothesis that humoral immunity, especially the secondary response, entails energetic costs that may trade-off with other physiological activities.

Effects of salinity on the immune response of an 'osmotic generalist' bird.

Salt stress can suppress the immune function of fish and other aquatic animals, but such an effect has not yet been examined in air-breathing vertebrates that frequently cope with waters (and prey) of contrasting salinities. We investigated the effects of seawater salinity on the strength and cost of mounting an immune response in the dunlin Calidris alpina, a long-distance migratory shorebird that shifts seasonally from freshwater environments during the breeding season to marine environments during migration and the winter period. Phytohaemagglutinin (PHA)-induced skin swelling, basal metabolic rate (BMR), body mass, fat stores, and plasma ions were measured in dunlins acclimated to either freshwater or seawater (salinity: 0.3 and 35.0 ‰, respectively). Seawater-acclimated dunlins mounted a PHA-induced swelling response that was up to 56 % weaker than those held under freshwater conditions, despite ad libitum access to food. Freshwater-acclimated dunlins significantly increased their relative BMR 48 h after PHA injection, whereas seawater-acclimated dunlins did not. However, this differential immune and metabolic response between freshwater- and seawater-acclimated dunlins was not associated with significant changes in body mass, fat stores or plasma ions. Our results indicate that the strength of the immune response of this small-sized migratory shorebird was negatively influenced by the salinity of marine habitats. Further, these findings suggest that the reduced immune response observed under saline conditions might not be caused by an energy or nutrient limitation, and raise questions about the role of osmoregulatory hormones in the modulation of the immune system.

Avian BMR in marine and non-marine habitats: a test using shorebirds.

Basal metabolic rate (BMR) is closely linked to different habitats and way of life. In birds, some studies have noted that BMR is higher in marine species compared to those inhabiting terrestrial habitats. However, the extent of such metabolic dichotomy and its underlying mechanisms are largely unknown. Migratory shorebirds (Charadriiformes) offer a particularly interesting opportunity for testing this marine-non-marine difference as they are typically divided into two broad categories in terms of their habitat occupancy outside the breeding season: 'coastal' and 'inland' shorebirds. Here, we measured BMR for 12 species of migratory shorebirds wintering in temperate inland habitats and collected additional BMR values from the literature for coastal and inland shorebirds along their migratory route to make inter- and intraspecific comparisons. We also measured the BMR of inland and coastal dunlins Calidris alpina wintering at a similar latitude to facilitate a more direct intraspecific comparison. Our interspecific analyses showed that BMR was significantly lower in inland shorebirds than in coastal shorebirds after the effects of potentially confounding climatic (latitude, temperature, solar radiation, wind conditions) and organismal (body mass, migratory status, phylogeny) factors were accounted for. This indicates that part of the variation in basal metabolism might be attributed to genotypic divergence. Intraspecific comparisons showed that the mass-specific BMR of dunlins wintering in inland freshwater habitats was 15% lower than in coastal saline habitats, suggesting that phenotypic plasticity also plays an important role in generating these metabolic differences. We propose that the absence of tidally-induced food restrictions, low salinity, and less windy microclimates associated with inland freshwater habitats may reduce the levels of energy expenditure, and hence BMR. Further research including common-garden experiments that eliminate phenotypic plasticity as a source of phenotypic variation is needed to determine to what extent these general patterns are attributable to genotypic adaptation.

Plasma metabolite levels predict bird growth rates: A field test of model predictive ability.

Bird growth rates are usually derived from nonlinear relationships between age and some morphological structure, but this procedure may be limited by several factors. To date, nothing is known about the capacity of plasma metabolite profiling to predict chick growth rates. Based on laboratory-trials, we here develop predictive logistic models of body mass, and tarsus and wing length growth rates in Gull-billed Tern Gelochelidon nilotica chicks from measurements of plasma metabolite levels at different developmental stages. The predictive model obtained during the fastest growth period (at the age of 12 days) explained 65-68% of the chicks' growth rates, with fasting triglyceride level explaining most of the variation in growth rate. At the end of pre-fledging period, ß-hydroxybutyrate level was also a good predictor of growth rates. Finally, we carried out a field test to check the predictive capacity of the models in two colonies of wild Gull-billed Tern, comparing field-measured and model-predicted growth rates between groups. Both, measured and predicted growth rates, matched statistically. Plasma metabolite levels can thus be applied in comparative studies of chick growth rates when semi-precocial birds can be captured only once.

Understanding the energetic costs of living in saline environments: effects of salinity on basal metabolic rate, body mass and daily energy consumption of a long-distance migratory shorebird.

Many migratory vertebrates typically move between habitats with varying salinities during the annual cycle. These organisms clearly exhibit a remarkable phenotypic flexibility in their 'osmoregulatory machinery', but the metabolic consequences of salinity acclimatization are still not well understood. We investigated the effects of salinity on basal metabolic rate (BMR), body mass and daily energy consumption of a long-distance migratory shorebird, the dunlin (Calidris alpina), outside the breeding season. Mass-corrected BMR and daily energy consumption increased significantly by 17 and 20% between freshwater (0.3‰ NaCl) and saltwater (33.0-35.0‰ NaCl), respectively. Body mass in both captive and wild dunlins was lower (9-16%) in saline than in freshwater environments. These changes on BMR and body mass were quickly reversed by returning the birds to freshwater, suggesting that metabolic adjustment to saltwater and metabolic readjustment to freshwater are both processes that occur in a few days. Our findings support empirically that the processes of developing and maintaining an active osmoregulatory machinery are energetically expensive, and they could help to explain diet and/or habitat selection patterns along the flyway. Finally, we discuss whether body mass loss in saltwater may be a strategy to reduce maintenance cost in osmotically stressful conditions such as overwintering in marine habitats, and raise some methodological implications for studies of BMR-related outcomes using captive birds captured in saline environments.

Sex differences in digestive traits in sexually size-dimorphic birds: Insights from an assimilation efficiency experiment on black-tailed godwit.

Digestive assimilation efficiency is considered a trait with important implications for animal ecology. However, practically all studies have ignored the importance of sex differences in food assimilation efficiency (AE). Here, we investigated sex differences in dietary and physiological parameters in the Black-tailed Godwit Limosa limosa limosa feeding on rice seeds, a species with sexual dimorphism in body size and body mass. Gross daily food intake, gross energy intake, gross energy output and metabolizable energy intake did not vary significantly between sexes, but godwit females showed lower faeces energy density and higher AE than males. Mass-specific AE was similar in males and females, and the difference in AE could be attributed to the females' greater body mass. We suggest that a differential AE could play a role in explaining sex differences in habitat or micro-habitat selection during the non-breeding season in bird species with sexual dimorphism in size. Finally, we addressed the question about assimilation efficiency accuracy in models that estimate prey acquisitions by declining shorebirds as the Black-tailed Godwit.