Extreme hyperthermia tolerance in the world’s most abundant wild bird

The thermal tolerances of vertebrates are generally restricted to body temperatures below 45–47 °C, and avian and mammalian critical thermal maxima seldom exceed 46 °C. We investigated thermoregulation at high air temperatures in the red-billed quelea (Quelea quelea), an African passerine bird that occurs in flocks sometimes numbering millions of individuals. Our data reveal this species can increase its body temperature to extremely high levels: queleas exposed to air temperature > 45 °C increased body temperature to 48.0 ± 0.7 °C without any apparent ill-effect, with individual values as high as 49.1 °C. These values exceed known avian lethal limits, with tolerance of body temperature > 48 °C unprecedented among birds and mammals.

the thermal tolerances of vertebrates are generally restricted to body temperatures below 45-47 °C, and avian and mammalian critical thermal maxima seldom exceed 46 °C. We investigated thermoregulation at high air temperatures in the red-billed quelea (Quelea quelea), an African passerine bird that occurs in flocks sometimes numbering millions of individuals. Our data reveal this species can increase its body temperature to extremely high levels: queleas exposed to air temperature > 45 °C increased body temperature to 48.0 ± 0.7 °C without any apparent ill-effect, with individual values as high as 49.1 °C. These values exceed known avian lethal limits, with tolerance of body temperature > 48 °C unprecedented among birds and mammals.
Survival and reproduction in hot environments are constrained by the upper limits to organisms' thermal tolerances. Under high environmental heat loads, the avoidance of lethal body temperature (T b ) drives fundamental behavioral trade-offs between thermoregulation and activities such as foraging 1,2 , and constraints on the evolution of upper thermal limits have important consequences for predicting responses to climate change 3 . Upper thermal limits also constrain performance under conditions of high metabolic heat production 4 in contexts that include livestock production and food security under hotter future conditions 5,6 .
Body temperatures (T b ) of vertebrates are thought to be limited to below 45-47 °C by the thermal sensitivity of cellular macromolecules 7-10 and oxygen supply limitation 11,12 . Among terrestrial vertebrates, critical thermal maxima for squamate reptiles, rodents and birds are usually below 46 °C [13][14][15][16] . The same is generally true of maximum T b values observed in birds, rodents and small bats in studies involving acute heat exposure but where critical thermal maxima were not quantified [17][18][19][20] . Typical lethal avian T b values are 46.2-47.7 °C in two species of towhees 21 and 46-47.8 °C in barred-rock chickens 16 , although the latter author reported lethal values as high as 48.8 °C associated with tracheal administration of 100% oxygen.
However, T b above the typical vertebrate range has occasionally been documented. Critical thermal maxima above 47 °C have been reported in a small number of desert lizards (reviewed by Clusella-Trullas et al. 13 ), with a value of 51.0 °C observed in ten adult Aspidoscelis sexlineata 22 . Among birds, three variable seed-eaters (Sporophila aurita), a passerine from the humid lowlands of Panama, survived T b = 46.8-47.0 °C without any apparent ill-effects 23 . In a pioneering study of the use of surgically-implanted transmitters to measure avian T b , Southwick 24 recorded T b = 47.7 °C in a single white-crowned sparrow (Zonotrichia leucophrys gambelli). However, cloacal T b measured simultaneously was 44.1 °C, and the 3.6 °C difference between this pair of measurements was the largest reported in the study 24 .
As part of a study of adaptive variation in avian heat tolerance, we investigated thermoregulation during acute heat exposure in the red-billed quelea (Quelea quelea). This small (18-g) African passerine is widely considered the most abundant non-domesticated bird on Earth, with post-breeding population estimates of ~ 1.5 billion individuals 25 . It is highly gregarious and forms huge flocks that may consist of several million individuals 26 . The peculiar natural history of this species led us to hypothesize that its thermal physiology differs from that of typical small songbirds. Red-billed queleas drink regularly 26 . However, the timing of flocks' visits to water sources is presumably determined by the average hydration status of large numbers of flock members rather than that of single individuals. Under conditions where hydration status potentially varies substantially across individuals within a vast flock, selection should favour the capacity for water conservation via facultative hyperthermia.

Gas exchange measurements.
An open flow-through respirometry system was used to measure evaporative water loss (EWL) and carbon dioxide production ( V CO 2 ) during measurements. Queleas were placed individually in 3-L (approximate dimensions 20 cm high × 15 cm wide × 10 cm deep) plastic chambers, previously shown to not absorb water vapour 27 , equipped with a mesh platform ~ 10 cm above a 1-cm layer of mineral oil into which excreta fell to prevent evaporation. The chambers were placed in a ~ 100 L ice chest modified such that temperature inside the chest was regulated using a Peltier device (AC-162 Thermoelectric Air Cooler, TE Technology, Traverse City MI, USA) controlled via a digital controller (TC-36-25-RS485 Temperature Controller, TE Technology, Traverse City MI, USA).
Atmospheric air supplied by an oil-free compressor was scrubbed of water vapour using a membrane dryer (Champion CMD3 air dryer and filter, Champion Pneumatic, Quincy IL, USA). The dried air was split into an experimental and baseline channel. A mass flow controller (Alicat Scientific Inc., Tuscon AZ, USA), calibrated using a soap-bubble flow meter (Gilibrator 2, Sensidyne, St Petersburg, FL, USA), regulated experimental flow rates to the animal chamber. The flow rate of the baseline channel was controlled using a needle valve (Swagelok, Solon, OH, USA). Within each chamber, the air inlet was positioned close to the lid with an elbow joint facing upwards (to minimize any potential convective cooling at higher flow rates) and the air outlet below the mesh platform to maximize air mixing. We used flow rates of 10.1-18.3 L min −1 , depending on air temperature and individual behaviour, with flow rate regularly adjusted during measurements to maintain chamber humidity below a dewpoint of − 7.7 °C.
A respirometry multiplexer (model MUX3-1,101-18 M, Sable Systems, Las Vegas, NV) in manual mode and an SS-3 Subsampler (Sable Systems) sequentially subsampled excurrent air from the chamber and baseline air. Subsampled air was pulled through a CO 2 /H 2 O analyzer (model LI-840A, LI-COR, Lincoln, NE, USA), which was regularly zeroed using nitrogen and spanned for CO 2 using a certified calibration gas with a known CO 2 concentration of 1900 ppm (AFROX, Johannesburg, South Africa). The H 2 O sensor of the Li-840A was regularly zeroed using nitrogen and spanned using a dewpoint generator (DG-4, Sable Systems, Las Vegas NV). Voltage outputs from the analyzers and thermistor probes were digitized using an analog-digital converter (model UI-3, Sable Systems) and recorded with a sampling interval of 5 s using Expedata software (Sable Systems). All tubing in the system was Bev-A-Line IV tubing (Thermoplastic Processes Inc., Warren, NJ, USA). experimental protocol. Measurements occurred during the day, and we quantified relationships between body temperature, metabolic heat production and evaporative heat dissipation over air temperatures of 28-52 °C by exposing birds to the same stepped air temperature profile involving 4-°C increments below 40 °C and 2-°C increments above 40 °C as used in previous studies [27][28][29][30] . Measurements commenced with a baseline air subsample until water and CO 2 readings were stable (5 min). Birds spent a minimum of 10 min at each air temperature, with stable average values over the last 5 min at each air temperature value included in subsequent analyses, followed by another 5 min baseline. This approach to quantifying physiological responses to heat exposure is functionally analogous to the sliding cold exposure protocol used to elicit maximum metabolic rates during cold exposure 32  www.nature.com/scientificreports/ During measurements, individuals were continuously monitored using a video camera with an infrared light source. Only data from birds that remained calm during measurements (i.e., no sign of agitation or sustained escape behavior) were included in analyses. Trials were terminated and individuals immediately removed from the chamber when a bird reached its thermal endpoint characterized by sustained escape behaviour (i.e., agitated jumping) or a loss of coordination or balance, often associated with a sudden decrease in EWL or resting metabolic rate. Individual critical thermal maximum was taken as the body temperature associated with the onset of loss of balance and or uncoordinated movement. Immediately after each bird was removed from the chamber, its belly feathers were dabbed with 80% ethanol to accelerate heat loss and it was placed in a recovery cage with ad libitum water and food. Each bird was later released at the site of capture. This experimental protocol has been used previously for multiple species and, in one study with opportunistic monitoring for several weeks post-release, no adverse effects were observed 33 . Data analysis. We corrected for analyzer drift and lag using the relevant algorithms in Expedata software (Sable Systems, Las Vegas NV, USA). Eqs. 9.5 and 9.6 from Lighton 34 were used to calculate V CO 2 and EWL from the lowest stable 5-min periods of CO 2 and water vapour at a given air temperature, assuming 0.803 mg H 2 O mL − 1 vapour. As individuals were likely post-absorptive, we estimated resting metabolic rate from V CO 2 assuming respiratory exchange ratio (RER) = 0.71 and converted rates of V CO 2 to metabolic rate (W) using 27.8 J ml −1 CO 2 35 . Rates of EWL were converted to rates of evaporative heat loss (EHL, W) assuming a latent heat of vaporization of water of 2.406 J mg −1 at 40 °C 36 . Body temperatures, rates of EWL and resting metabolic rates at thermoneutral air temperatures (Supplementary Fig. S1) were considered normothermic values.
All analyses were conducted in R 3.5.2 37 . Relationships between physiological response variables and air temperature as a predictor were analyzed using linear mixed-effects models ("lme" command) in the R package nlme 3.1-140 38 after using segmented 1.1-0 39 to identify inflection points. We accounted for pseudoreplication (multiple measurements per individual) by including individual identity as a predictor (random factor) in all analyses. We assessed significance at p < 0.05 and values are presented as mean ± s.d.

Results
The normothermic body temperature of queleas was 40.9 ± 0.9 °C (n = 20), a value typical for small passerines (Fig. 1). Above an inflection air temperature of 38.0 ± SE 0.6 °C, body temperature increased by 0.5 °C per 1 °C increase in air temperature. Body temperature reached an estimated critical thermal maximum (i.e., maximum values associated with a loss of coordination and motor function) of 48.0 ± 0.7 °C (n = 20) at an air temperature of 50.9 ± 1.5 °C. Individual maximum values were 46.4-49.1 °C, with 75% of individuals reaching body temperature ≥ 48.0 °C (Fig. 1). Concurrent measurements of metabolic heat production (MHP) and evaporative heat loss (EHL) (Supplementary Fig. S1) revealed that EHL/MHP reached a maximum value of 1.49 at air temperature > 46.9 ± SE 0.5 °C (Fig. 1), confirming the queleas' maximum evaporative cooling capacity had been attained.

Discussion
Patterns of T b during acute heat exposure supported our prediction that red-billed queleas have a pronounced capacity to tolerate hyperthermia. The species' critical thermal maximum is substantially higher than the known avian range (Fig. 2), exceeding by 2-3 °C the values associated with breakdown of respiratory function in poultry 15,16 and the body temperatures associated with loss of motor function in wild birds [27][28][29][30]40,41 . Moreover, the body temperature range tolerated by the queleas exceeds known avian lethal values for passerines 21 and domestic fowls 15,16 . Tolerance of body temperature > 48 °C is unprecedented among birds and mammals, with higher values having been reported only in ectothermic vertebrates 13 and invertebrates 42 .
The methods we used here to establish the upper limits of queleas' heat tolerance and evaporative cooling capacity are identical to those of recent studies involving ~ 55 bird species, including three arid-zone representatives of the Ploceidae 27 , the family to which Q. quelea belongs. Extreme hyperthermia tolerance comparable to that of the queleas appears to be absent among small passerines inhabiting arid regions where air temperature maxima may approach or exceed 50 °C 27,29,40 . That desert birds apparently lack the ability to tolerate comparably high body temperatures, despite strong selection for water conservation 43 , suggests there are substantial costs to such extreme hyperthermia tolerance. These costs could potentially be related the synthesis of heat shock proteins (HSPs) and interactions with stress responses via the modification of glucocorticoid receptor function 9,44 .
The capacity of queleas to dissipate evaporatively a maximum of ~ 150% of metabolic heat production is relatively modest for a passerine; among 30 species, maximum EHL/MHP was 1.75 ± 0.31 27,29,30,40,45 . Among aridzone passerines, regular-drinking species are capable of greater fractional increases in EWL and have higher heat tolerance limits compared to non-drinking species 45 . Our finding here of modest evaporative cooling capacity accompanied by extreme hyperthermia tolerance in a regularly-drinking species raises the possibility that coevolution of thermal physiology and water-dependence follows a different trajectory in species that form large flocks. Our hypothesis that avian social systems involving large flocks are associated with selection for pronounced hyperthermia tolerance could be tested further in gregarious species inhabiting hot, arid climates, particularly Australian species such as budgerigars (Melopsittacus undulatus) or cockatiels (Nymphicus hollandicus).
Our findings reveal it is possible for birds to evolve short-term tolerance of very high body temperature. Moreover, they identify red-billed queleas as a model for future studies of the physiological and molecular bases of extreme hyperthermia tolerance. We speculate that this species' ability to tolerate T b as high as 48-49 °C arises from an array of anatomical and molecular mechanisms, including a well-developed rete opthalmicum to maintain brain temperature well below core T b 46-48 and pronounced heat shock protein expression 44,49 . Understanding the processes underlying the queleas' ability to tolerate T b values lethal to other endotherms may, we suspect, prove useful for biotechnology aimed at developing greater heat tolerance in birds and other organisms.

Data availability
The data generated during this study are included in the Supplementary Information files accompanying this published article.