Fig. 6 in Fig. 2 in Fig. 3 in Zavreliella shidai Cao & Tang, 2017, sp. n.

© 2017 Academia Sinica, Taiwan Open Access Total Evaporative Water Loss in Birds at Different Ambient Temperatures: Allometric and Stoichiometric Approaches Valery M. Gavrilov Department of Vertebrate Zoology and Zvenigorod Biological Station, M.V. Lomonosov Moscow State University, Moscow 119992, Russia (Received 28 March 2017; Accepted 14 November 2017; Published 14 December 2017; Communicated by Benny K.K. Chan) Valery M. Gavrilov (2017) Total evaporative water loss (TEWL) in Passeriformes and Non-Passeriformes was estimated by simultaneous measurements of energy expenditure and mass loss in resting birds. It was found that the percentage of heat dissipated by water evaporation depends on body size. Published data for 102 bird species were analyzed together with my own measurements for 157 bird species at thermally neutral temperatures (mostly 25°C) to establish the following relationship between TEWL and body mass: TEWL25°C Aves = 0.28 m0.701, R2 = 0.92, where TEWL is in g H2O/day and m is body mass (g). The scaling exponent 0.701 ± 0.007 is 0.05 greater than for the relationship of basal metabolic rate (BMR) to body mass. It was found that TEWL in passerines is higher than in non-passerines at all ambient temperatures by 50% at 25°C, 30% at 0°C, 39% at the lower critical temperature, and 59% at the upper critical temperature. The dependence of water loss on body mass at different ambient temperatures (TA) was found to vary in the same manner as evaporative heat loss. TEWL in Passeriformes is approximately 25-60% higher than in Non-Passeriformes (particularly at high TA), which is consistent with the ratio of their BMR levels. Within the thermoneutral zone, the proportion of heat dissipated by evaporation increases by approximately 2.6-fold in small passerines and by almost 4.1-fold in large passerines with the transition from the lower to upper critical temperature. In non-passerines, the proportion of evaporative heat losses increases by approximately 2.7 times within the thermoneutral zone in both large and small birds. The high basal metabolic rate in Passeriformes involves benefits like a higher maximum metabolic power and the ability to breed at lower ambient temperatures, but it comes with a cost: a significant expenditure of evaporative water. This cost is important because it is found to increase with body size in Passeriformes due to the forced evaporative heat loss, but it shows virtually no increase with body size in Non-Passeriformes. Thus, despite a high BMR significantly increasing ecological opportunities, this way of expanding the ecological niches is possible for the small size class only. These findings suggest that the high level of basal metabolic rate in Passeriformes in comparison to Non-Passeriformes determines the necessity for the former to utilize considerably larger amounts of water for evaporation to maintain the needed heat balance, especially at higher ambient temperatures and at larger body sizes. Key words: Water loss, Heat dissipation, Thermoregulation, Metabolic heat production, Energetic equivalent of the loss body mass, Birds. *Correspondence: Fax: + 74954211538. E-mail: [email protected] BACKGROUND Evaporation is a key component of an organism’s heat balance. It depends on metabolic rate, ambient temperature and relative humidity. Total evaporative water loss (TEWL) is a part of the animal’s heat balance that is especially important under warm conditions. Birds display a broad spectrum of morphological and functional adaptations aimed at improving the water balance. TEWL studies provide insight into those ecological and morphological properties that determine the geographic distribution of avian species. In individual animals of a given species, TEWL is Zoological Studies 56: 37 (2017) doi:10.6620/ZS.2017.56-37 1 © 2017 Academia Sinica, Taiwan influenced by water availability, feeding patterns and protein content of the diet (Bernstein 1971; Lasiewski et al. 1971; Marder and Ben-Asher 1983; Marder and Gavrieli-Levin 1987; Webster and Bernstein 1987; Wolf and Walsberg 1996; Arieli et al. 2002; Ophir et al. 2002; McKechnie and Wolf 2004; DeNardo et al. 2004; Hoffman et al. 2007). Several studies have shown that rates of evaporative heat loss are correlated with metabolic rates (Hoffman and Walsberg 1999; Marder et al. 1989; McKechnie and Wolf 2004; Webster and King 1987; Withers and Williams 1990; Clark and Dudley 2009, 2010). The rate of heat dissipation is proportional to TEWL and strongly influenced by acclimatization and, presumably, by natural selection; thus, it varies among species (Williams and Tieleman 2002). The need to maintain water balance restricts certain species to areas with sufficient rainfall, as demonstrated by studies of bird population density in Taiwan’s subtropical mountains (Walther et al. 2017). Rodríguez and Barba (2016) found that various physiological and behavioral adaptations related to water balance allow nestlings of small altricial birds to maintain a stable body temperature at high air temperatures during the breeding season. Basal metabolic rate (BMR) and TEWL are considered to be fundamental characteristics of the animal life history (Tieleman et al. 2006; Versteegh et al. 2008). It is known that Passeriformes and Non-Passeriformes differ in their BMR: Passeriformes’ BMR is about 1.5 times higher (McNab 2009, 2016). An important question is how this difference in BMR values relates to evaporative heat losses at rest and, especially, during flight. Does a high BMR imply that closely related species or individuals are characterized by a high peak power output and therefore high energy expenditures for ordinary existence and additional works (flight in particular)? Does BMR level determine the ratio of evaporative and nonevaporative heat loss? If BMR determines the other components of the energy budget, the energy expenditures for flight and the level of evaporative cooling in passerine birds should be higher than in non-passerines. In other words, if BMR determines the upper metabolic limit, then the ratio of BMR to the upper metabolic limit should be relatively constant. It could then be expected that the ratio of TEWL to oxygen consumption in passerines is be equal to those in non-passerines. Therefore, at the same ambient temperature, Passeriformes would have a higher TEWL than Non-Passeriformes. In this paper, heat dissipation in resting birds is analyzed at various ambient temperatures using the terms described below (the stoichiometric approach). A commonly cited allometric analysis of TEWL at thermally neutral temperatures is that of Crawford and Lasiewski (1968). They tabulated data for 42 species ranging in size from a 3-g hummingbird to a 100-kg ostrich (Struthio camelus). Williams (1996) noted that the allometric equation in Crawford and Lasiewski relating TEWL to body mass in birds was based on a relatively small sample size and constructed using procedures that might have biased the parameter estimation. In his report, Williams (1996) analyzed TEWL for 102 bird species ranging in size from hummingbirds to ostriches using both least-squares regression and phylogenetically independent contrasts. Using this approach, Williams (1996) established that (1) the slope of the allometric relationship between TEWL and body mass is higher than in the equation of Crawford and Lasiewski (1968), (2) birds from arid environments have lower TEWL than birds from more mesic environments, and (3) small and large birds have similar ratios of TEWL to oxygen consumption. The latter finding negates the idea that small desert birds replenish proportionately less of their TEWL with metabolic water than larger species do. Williams (1996) selected data taken at 25°C because this temperature is (1), at or near the lower critical temperature for many birds (2), not thermally stressful for most birds, and (3), the same temperature chosen by Crawford and Lasiewski (1968) for their analysis. In this paper we address the following questions: (1) How do TEWL estimates based on the energy equivalent of the body mass loss (q) correspond to TEWL estimates obtained by conventional methods reviewed by Williams (1996)? Since McNab (2009, 2016) convincingly demonstrated that Passeriformes and NonPasseriformes differ in their basal metabolic rate, we also address these questions: (2) What is the relationship between TEWL and body mass in Passeriformes and Non-Passeriformes birds at different ambient temperatures? And (3) How does the share of evaporative heat loss in Passeriformes and Non-Passeriformes vary from within the thermoneutral zone, i.e. from the lower critical temperature to the upper critical temperature? page 2 of 16Zoological Studies 56: 37 (2017) © 2017 Academia Sinica, Taiwan MATERIALS AND METHODS Bird sampling We analyzed more than 60 species of passerine birds representing the order’s full body size range-from the goldcrest (Regulus regulus, 5.5 g) to the Raven (Corvus corax 1,208 g) - and 30 species of non-passerine birds of a similar size range (25-4,000 g). All birds were kept in big aviaries at natural day lengths and temperatures. For migratory birds and subtropical species, the aviaries were heated in winter to maintain temperatures of 5-10°C. These conditions allow for an accurate analysis of seasonal acclimatization (Proser 1991). Measurements of energy values were made in winter (November-January, February) and summer (late May-June, late August-September) on non-molting birds. Studies of seasonal variation in energy expenditure, both at rest and at ordinary existence levels, were done at experimentally controlled temperatures, where TA varied from -28°C to + 40°C to construct a thermal energy profile for each species. Measurements of body mass variations in birds The following experiments were performed for a more precise determination of the rate at which food passes through the alimentary canal and the magnitude of mass loss at night. Groups of five chaffinches (Fringilla coelebs) and three wood or Carolina ducks (Aix sponsa) were placed immediately after evening feeding into a small light cage with a mesh floor. The excrements dropped sank in a cuvette with liquid mineral oil which prevented evaporation of water. The cage and the cuvette were connected to scale-levers to register body mass changes during the night. The rate of body mass loss after evening feeding stabilizes at different times in differentsized birds: on average, it occurs 2-4 h after feeding in small birds, and 6-8 h in large birds. Further loss proceeded at constant rates determined by water evaporation. Figure 1 exemplifies this trend, showing that the alimentary canal became empty five hours after feeding in Carolina ducks (body mass 470 g) and three hours after feeding in chaffinches (body mass 21 g). Based on our experiments, we assumed that 3 hours after the last feeding in small birds and 10-12 hours in large birds is sufficient for the alimentary canal to empty. All birds fasted for at least 3-12 h and were in the postabsorptive state during measurements. We thus assume that during measurements the body mass loss occurred at a constant rate. Metabolic rate measurements All oxygen consumption values were corrected to standard pressure and temperature according to the equations of Depocas and Hart (1957). Respirometry in our modification is based on measurements of air pressure and is therefore very sensitive to temperature changes. We carefully controlled the temperature in the laboratory and employed sealed chambers during metabolic rate measurements (Gavrilov 1997 2012a b 2014 2015). If temperature fluctuations in the sealed chamber within one hour of measurements exceeded 0.3°C, the corresponding data were not used. The average volume of consumed oxygen from the entire period of metabolic rate measurements was transformed into the volume at standard temperature and pressure and converted to kJ day-1 according to the equation 1 L of O2 = 15.97 + 5.16RQ (kJ) (Schmidt-Nielsen 1997). Oxygen consumption was measured at rest at different ambient temperatures in all species studied. Measurement of RQ Respiratory quotient (RQ) was determined by Haldane gas analyzer (Dolnik and Gavrilov 1973 1979; Gavrilov 1997 2015; Gavrilov et al. 2013). We measured energy expenditures at rest and the respiratory quotient in 26 species of passerine birds and 16 species of non-passerine birds in winter and summer. Measurements were made at different temperatures ranging from + 5°C to + 35°C. The stoichiometric approach for estimating total evaporative water loss This paper applied the stoichiometric approach for the determination of TEWL developed and described by Gavrilov (2014 2015). This method includes direct simultaneous measurements of the energetic equivalent of body weight loss (q) as the ratio between heat production (metabolic rate, MR, determined by the rate of oxygen consumption) and body weight loss (dm) at various TA: q = MR/dm page 3 of 16Zoological Studies 56: 37 (2017) © 2017 Academia Sinica, Taiwan Body weight loss of an animal in the postabsorptive state at a constant relative humidity is primarily determined by water evaporation. Therefore, the caloric equivalent is directly proportional to the specific caloric value of metabolically oxidized compounds and inversely proportional to the body weight loss dm. The latter is the sum of evaporative and cloacal water losses and is equal to the difference between the weights of consumed oxygen and liberated CO2, excreted products of nitrogen metabolism (protein oxidation), and excreted gastroliths. The complete algorithm used to calculate evaporative heat loss (He) from the energetic equivalent of body weight loss at rest for any combination of oxidized compounds was published elsewhere (Gavrilov 2015). In this work, we shall only give exponential equations for the relationships between evaporative heat loss as Fig. 1. Body mass loss per hour as a function of time after feeding for small (upper panel) and large (bottom panel) birds. Data for upper panel were slightly displaced horizontally to prevent overlapping. Body mass was measured every hour and thus body mass loss per hour is a mass difference between two successive measurements. page 4 of 16Zoological Studies 56: 37 (2017) © 2017 Academia Sinica, Taiwan a percentage of total heat loss (He, %) and the energetic equivalent of the body weight loss at rest. For purely lipid metabolism (lipid oxidation is the main source of energy for birds in winter) we have % He = 238.3q-0.98, where q is in kJ/g. Тhe oxidized substrate ratio is different in summer and the following coefficients were assumed: 0.7 for lipids, 0.1 for carbohydrates, and 0.2 for proteins (Dolnik and Gavrilov 1979; Gavrilov 2014 2015). Therefore, the energetic equivalents of body weight loss at rest were 0.7q for lipids, 0.lq for carbohydrates, and 0.2q for proteins. Тhе exponential equation for summer is % He = 239.3q-1.05, where q is in kJ/g. The evaporative heat loss (as a percentage of total heat loss) can be derived from the energetic equivalent of body weight loss at rest. From this relationship, we can determine the ratio between evaporative and non-evaporative heat losses at any ambient temperature. We used the above exponential equations to calculate the percentage of evaporative heat loss (He) from experimental measurements of q. Using the obtained percentage of evaporative heat loss and total heat production (SMR or BMR), we calculated the evaporative heat loss at various ambient temperatures: He0°С, HeTlc-the lower critical temperature, He25°С, and HeTuc-the upper critical temperature. Taking into account that the evaporative heat of 1 gram of water is equal to 2.4 kJ (Garai 2009), total evaporative water loss can be represented as TEWL = (BMR *% He /100)/2.4, where BMR is the total heat produced at any thermoneutral temperature TA and %He is the percentage of total heat loss through evaporation at this TA. Scaling analysis and statistic We performed a scaling analysis of evaporative water loss in Passeriformes and Non-Passeriformes birds at different ambient temperatures and seasons. First, we studied the relationship between TEWL and body mass by collating TEWL data at various ambient temperatures. We tested the ANOVA difference between the model in R statistic (R Development Core Team 2014) for log-transformed values of TEWL (g H2O/ day) and body mass (g). The study was performed according to the laws of the Russian Federation and Moscow State University regarding the capture and maintenance of wild animals. All birds were released after the experiment. RESULTS RQ measurements RQ values estimated in 26 species of passerine birds and 16 species of non-passerine birds at night in winter at ambient temperatures 5-35°C fell within the range 0.69-0.75 and averaged 0.72 ± 0.03 (n = 1024). These data confirmed that lipids were the primary source of energy expenditure in winter at night. Summer RQ values for the same species at night with ambient temperatures 5-35°C fell within the range 0.74-0.82 and averaged 0.77 ± 0.08 (n = 1024). Such RQs can be measured at the following ratios of oxidizable substrates: 0.7 for fat, 0.2 for carbohydrates and 0.1 for proteins, such that RQ = 0.7*0.7 + 0.2*1 + 0.1*0.82 = 0.77. Measurements of metabolic rate, energetic equivalent of body mass loss, and total evaporative water loss All the data obtained on heat loss at rest (BMR, SMR) and the associated values measured (Tlc, Tuc,q, and TEWL) for all bird species for two seasons are summarized in appendix 1. As an example, the energy metabolism values obtained in summer for one passerine species, the goal tit (Parus ater), are presented graphically in figure 2. The dependence on TA of the resting metabolic indices at night (Fig. 2 top) corresponds exactly to Scholander’s model: SMR decreases with rising TA and theoretically attains zero at a certain TA equal to body temperature (TB) under moderately cold stress, which corresponds to the beginning of the thermoneutral zone. Quantitatively, the metabolism or heat loss (SMR) is related to TA by a linear equation SMR = hl(TB-TA), where hl is the heat transfer coefficient or thermal conductance; it includes losses of heat by radiation, conduction and convection at low ambient temperatures where hl is minimal. The decrease in SMR discontinues at TA = Tlc, (Tlc page 5 of 16Zoological Studies 56: 37 (2017) © 2017 Academia Sinica, Taiwan is the lower critical temperature). At this point, SMR becomes equal to basal metabolic rate (BMR). With a further increase in TA, the energy expenditure remains unchanged, whereas the bird passes from the minimal wet thermal conductance (hl) to the maximal (hu) attained at TA = Tuc (Tuc is the upper critical temperature): hu = (BMR)/(TB-Tuc). Evaporative heat loss (He) dissipates 9.3% of heat at 0°C, 16.2-16.7% at Tlc and 35.160.2% at Tuc. At low TA, the role of heat loss via evaporation is minor: virtually all of the energy used in thermoregulation (SMR-BMR) is expended by conduction, convection, and radiation. Evaporative heat loss increases significantly in the thermoneutral zone, even though the birds increase thermal conductance. Fig. 2. (A) energy expenditure at rest (SMR, BMR, kJ per day, left scales) and the energy equivalent of lost body mass (q, kJ per g, right scales) as the functions of ambient temperature (TA, °C). Each symbol is a mean for several measurements in several birds at a given TA, vertical bars are SD. (B) Evaporative heat loss (He, kJ per day, right scale), non-evaporative heat loss (Hs, kJ per day, right scales) as the functions of ambient temperature (TA, °C) and percentage of heat loss through evaporation (He, %, left scales) as the functions of ambient temperature (TA, °C) in the Goal Tits (Parus ater) in winter. A B page 6 of 16Zoological Studies 56: 37 (2017) © 2017 Academia Sinica, Taiwan Allometric analysis Our data for 157 bird species (Appendix 1) with a thermally neutral temperature (25°C) indicate the following relationship between TEWL and body weight: TEWL at 25°C Aves = 0.27m0.71, r2 = 0.93, where the TEWL is expressed as g H2O g/day, and body weight is expressed as g (Fig. 3A). Evaporative water loss in Aves (based on combined data from this and Williams’ (1996) review) Comparison of our results for TEWL-obtained using measurements of the energy equivalent to body mass loss (q) and calculated for the same temperature (25°C)-with the results obtained by conventional methods (Williams 1996) revealed a good agreement between the two approaches, as shown in figure 3A: TEWL25°C (Willams), all = 0.29m0.68, r2 = 0.90; TEWL25°C (This study), Fig. 3. (A) total evaporative water loss (TEWL) at TA = 25°C as a function of body mass in all birds (this study and Willams, 1996). (B) total evaporative water loss (TEWL) at TA = 25°C as s function of body mass in Passeriformes and Non-Passeriformes (this study and Willams 1996). A B page 7 of 16Zoological Studies 56: 37 (2017) © 2017 Academia Sinica, Taiwan all = 0.27m0.71, r2 = 0.93. The differences are not significant (the difference in slope is ns F = 1.4578, p = 0.2284; the difference in the y-intercepts is ns F = 0.8987, p = 0.344). The data comparison demonstrates that determinations by the stoichiometric approach of total evaporative water loss yielded estimates that fit into the confidence intervals of all equations from the literature. After combining the data from table 1 of Williams (1996) for 102 species of birds with my data for 157 species of birds (Appendix 1) at a thermally neutral temperature (25°C), the resulting relationship between TEWL and body weight is: TEWL at 25°C Aves = 0.28m0.70, r2 = 0.92, where the TEWL is expressed as g H2O g/day, and m is body mass (g) (Fig. 3A). If the dichotomy between passerines and non-passerines is taken into account, we have TEWL at 25°C = 0.225m0.792 (n = 152 r2 = 0.874) SD = 0.025 for Passeriformes and TEWL at 25°C = 0.233m0.715 (n = 107 r2 = 0.929) SD = 0.019 for Non-Passeriformes. The differences are statistically significant for both the slope and y-intercept. On average, at TA = 25°C passerines expend 50% more water than non-passerines. The difference grows with increasing body size (Fig. 3B). Note that the highest slope of the regression line of the TEWL on body mass is in passerines at 25°C, because 25°C in some species is outside the thermoneutral zone. Total evaporative water loss in Passeriformes and non-Passeriformes at different ambient temperatures The established allometric relationships for three different ambient temperatures for passerines and non-passerine birds are shown in figures 4A, B, C. Table 1 shows the statistical significance of each pair of equations as the slope and y-intercept Table 1. Fitted curves (TEWL = amb, TEWL g/day, a - intersept, b - slope, m - body mass, g) for total evaporative water loss in various avian groups of species and comparison of TEWL in Passeriformes and non-Passeriformes birds at different ambient temperatures Groups of species Ambient temperatures TA NLim m, g a - intersept b - slope r2Differences between Passeriformes and non-Passeriformes Differences in slope Differences in intersepts Excess of TEWL in Passeriformes by Aves, N + D, S + W TA = 25°C 259 2.7-100000 0.28 0.70 0.917 Non-Passeriformes, N + D, S + W TA = 25°C 107 2.7-100000 0.23 0.747 0.874 t = 2.458 DF = 255 p = 0.01464 t = -2.846 DF = 255 p = 0.00479 50% Passeriformes, N + D, S + W TA = 25°C 152 5.5-1208 0.225 0.792 0.929 The slopes are DIFFERENT at p < 0.05 The intercepts are DIFFERENT at p < 0.05 Non-Passeriformes, N, S + W TA = 0°C 53 25.2-4010 0.21 0.710 0.970 t = 1.549 DF = 153 t = -38.336 DF = 153 30% Passeriformes, N, S + W TA = 0°C 103 5.5-1208 0.32 0.68 0.982 p = 0.12343 The slopes are NOT different at p < 0.05 p = 0.00000 The intercepts are DIFFERENT at i < 0.05 Non-Passeriformes, N, S + W TA = Tlc 53 25.2-4010 0.25 0.71 0.930 t = 1.264 t = -42.675 39% Passeriformes, N, S + W TA = Tlc 103 5.5-1208 0.16 0.74 0.910 DF = 153 p = 0.20804 The slopes are NOT different at p < 0.05 DF = 153 p = 0.00000 The intercepts are DIFFERENT at p < 0.05 Non-Passeriformes, N, S + W = Tuc TA = Tuc 53 25.2-4010 0.45 0.73 0.985 t = -1.178 t = -9.823 59% Passeriformes, N, S + W TA = Tuc 103 5.5-1208 0.56 0.78 0.969 DF = 153 p = 0.24052 The slopes are NOT different at p < 0.05 DF = 153 p = 0.00000 The intercepts are DIFFERENT at p < 0.05 D - Measurements were made during the active (daytime) phase of the avian circadian cycle; N - Measurements were made during the resting (nighttime) phase of the avian circadian cycle; W - Measurements were made during the nonproductive “winter” phase of the avian annual cycle; S - Measurements were made during the nonreproductive “summer” phase of the avian annual cycle. page 8 of 16Zoological Studies 56: 37 (2017) © 2017 Academia Sinica, Taiwan Fig. 4. (A) total evaporative water loss (TEWL) at TA = 0°C as function of body mass in Passeriformes and Non-Passeriformes (this study). (B) total evaporative water loss (TEWL) at TA TA = Tlc as function of body mass in Passeriformes and Non-Passeriformes (this study). (C) total evaporative water loss (TEWL) at TA TA = Tuc as function of body mass Passeriformes and Non-Passeriformes (this study). A B C page 9 of 16Zoological Studies 56: 37 (2017) © 2017 Academia Sinica, Taiwan Appendix 1. Thermoregulation energetics at rest at night, energetic equivalent of loss body mass at rest (q, kJ/g upon TA) and total evaporative water loss (TEWL, g/day) at different ambient temperatures in non-Passeriformes and Passeriformes in different seasons. (download) page 16 of 16Zoological Studies 56: 37 (2017)