704

Seasonal Variation of Metabolic Thermogenesis in Eurasian Tree Sparrows (Passer montanus) over a Latitudinal Gradient Wei-Hong Zheng1 Ming Li2 Jin-Song Liu1,* Shu-Li Shao3 Xing-Jun Xu3 1 School of Life and Environmental Sciences, Wenzhou University, Wenzhou 325035, China; Key Laboratory for Water Environment and Marine Biological Resources Protection in Zhejiang Province, Wenzhou 325035, China; and Institute of Applied Ecology, Wenzhou University, Wenzhou 325035, China; 2Department of Biology, Daqing Normal College, Daqing 163712, China; 3College of Life Science and Engineering, Qiqihar University, Qiqihar 161006, China Accepted 3/30/2014; Electronically Published 8/19/2014

ABSTRACT Phenotypic flexibility of various morphological and physiological characters is widespread in animals. Resident endothermic animals of temperate climates provide a natural experiment in phenotypic flexibility. In this study, we took an integrative approach to assess seasonal and geographic influences on metabolism in Eurasian tree sparrows (Passer montanus). We measured resting metabolic rate (RMR), masses of internal organs, mitochondrial respiration capacities in liver and muscle, cytochrome C oxidase (COX) activities in liver and muscle, and circulating levels of plasma triiodothyronine (T3) and thyroxine (T4) in summer and winter sparrows at two sites from southeastern (Wenzhou) and northeastern (Qiqihar) China that differ in climate. Body masses of tree sparrows were significantly higher in winter than in summer at both sites but did not differ with latitude. RMRs of tree sparrows varied significantly with both latitude and season, with RMRs of Qiqihar birds being higher than those of Wenzhou birds and with RMRs being higher in winter than in summer. Consistently, dry masses of brain, lung, liver, gizzard, small intestine, rectum, and total digestive tract varied significantly with either latitude or season. State 4 respiration and COX activity in liver and muscle were remarkably higher in Qiqihar and increased significantly in winter. Circulating levels of plasma T3 also showed significant * Corresponding author; e-mail: [email protected].

Physiological and Biochemical Zoology 87(5):704–718. 2014. 䉷 2014 by The University of Chicago. All rights reserved. 1522-2152/2014/8705-2183$15.00. DOI: 10.1086/676832

seasonal and latitudinal variation and was higher in Qiqihar in winter than in other groups. These data suggest that tree sparrows mainly coped with cold by enhancing thermogenic capacities through heightened activity of respiratory enzymes and higher levels of plasma thyroid hormones (T3). These results are consistent with a pronounced seasonal and latitudinal phenotypic flexibility mediated through physiological and biochemical adjustments in Eurasian tree sparrows.

Introduction Over the last few decades, it has become apparent that phenotypic plasticity and phenotypic flexibility of various morphological and physiological characters are widespread in a variety of animals (Hammond et al. 2001; Piersma and Drent 2003; Starck and Rahmaan 2003). Phenotypic plasticity refers to variation in the phenotype generated by an environmental factor and defined by a reaction norm (Guglielmo and Williams 2003; McKechnie et al. 2006). In birds, phenotypic flexibility in metabolic power output is an important component of thermoregulatory responses to the elevated energy requirements experienced in seasonal environments. The study of phenotypic plasticity has become a central topic in evolutionary ecology (McKechnie et al. 2007). Resident endothermic animals of temperate climates provide a natural experiment in phenotypic flexibility (Liknes and Swanson 2011b). In birds, the ability to survive overwinter in seasonal environments requires efficient thermoregulatory mechanisms (Swanson 2001, 2010; Liu et al. 2008; Zheng et al. 2010; Liknes and Swanson 2011a, 2011b). The ecological significance of seasonal acclimatization for resident birds living in seasonally variable environments is obvious, and, consequently, knowledge of the flexibility of physiological parameters and the strategies employed to cope with extreme variability in temperature and food and water availability is important to understanding the survival of birds in these climates (Wiersma et al. 2007; Swanson and Palmer 2009). Winter is an energetically demanding period for small birds in temperate zones because thermoregulatory costs increase while food quality and availability are reduced (Yuni and Rose 2005). To cope with harsh environmental conditions, many small birds show seasonal variations in their morphology, physiology, and behavior (Swanson 1991a; Liknes and Swanson 1996; Yuni and Rose 2005; Zheng et al. 2008a, 2008b; Lindsay et al. 2009a, 2009c; Nzama et al. 2010). This results in the formation of what can be termed “seasonal phenotypes,” which

Thermogenesis in Small Birds at Different Latitudes 705 may involve a combination of phenotypically flexible responses to proximate (weather) factors (Swanson and Olmstead 1999; Liknes and Swanson 2011b). Heat production is commonly classified as either obligatory or facultative thermogenesis (Lowell and Spiegelman 2000; Silva 2006; Clapham 2012). Obligatory thermogenesis results from the widespread metabolic activity in many tissues and serves to maintain normal body temperature, which in homeotherms is usually higher than ambient temperature. This obligatory thermogenesis corresponds to the basal metabolic rate (BMR) or the resting metabolic rate (RMR; Enrique and Silva 2003; McNab 2009). BMR is the rate of energy transformation in a rested, awake, and fasted state in the absence of thermal stress and is the minimum metabolic rate of animals maintaining normal physiological function (McKechnie and Wolf 2004; Barcelo´ et al. 2009; McNab 2009; Swanson 2010). The use of BMR as an index of energy expenditure has received a great deal of attention from environmental physiologists, ecophysiologists, and comparative physiologists (Reynolds and Lee 1996; White et al. 2007; Zheng et al. 2008b; Lindsay et al. 2009b, 2009c; Nzama et al. 2010). Facultative thermogenesis is primarily produced in skeletal muscle, principally by shivering thermogenesis (Marsh and Dawson 1989; Swanson and Weinacht 1997; Swanson and Garland 2009; Swanson et al. 2012). Tropical birds typically have a lower BMR than high-latitude birds, which has been explained as an adaptation to avoid heat stress and to conserve water (Weathers 1997; Wiersma et al. 2007). In turn, the higher BMR of temperate and arctic birds has been explained as a direct or indirect result of the adaptation to a colder climate and a shorter breeding season, both presumably requiring higher metabolic capacities. The higher metabolic capacities in high-latitude species may involve a combination of genetic and phenotypically flexible responses to ultimate (climate) factors (Furness 2003; Wikelski et al. 2003; Swanson 2010; Liknes and Swanson 2011b). McKechnie (2008) and Swanson (2010) have identified three major physiological and morphological pathways whereby metabolic rates are up- or downregulated, namely, adjustments in organ masses, adjustments in the mass-specific metabolic intensities of specific organs, and adjustments in transport capacities for oxygen and metabolic substrates. Kersten and Piersma (1987) also hypothesized that natural selection adjusted the size of the internal organs to match energy requirements during parental care, the putative period of maximum energy expenditure, and that mass-independent variation in BMR reflects the relative size of such internal organs as the liver, kidney, and heart, which are thought to have high massspecific rates of oxygen consumption (Starck 1999; Williams and Tieleman 2000; Piersma and Drent 2003). Under basal metabolic conditions, the liver may contribute 25% of total heat production, and it is one of the largest and most metabolically active organs in endotherms (Swanson 1991b; Li et al. 2001; Villarin et al. 2003). Skeletal muscles have lower massspecific metabolic rates at rest than many central organs (Scott and Evans 1992), but due to their large total mass they may

contribute significantly to seasonal metabolic acclimatization (Chappell et al. 1999; Zheng et al. 2008a, 2010). Thyroid hormones (thyroxine [T4] and triiodothyronine [T3]) play pivotal roles in development and metabolism in homethermic animals (Yen 2001; Decuypere et al. 2005; Liu et al. 2006). In the complete absence of thyroid hormone, basal or resting energy expenditure can be reduced by 30% or more, a change associated with markedly reduced cold tolerance. This indicates that as much as 30% of obligatory thermogenesis depends on thyroid hormone and that this fraction of obligatory thermogenesis is essential for temperature homeostasis (Enrique and Silva 2003). Although seasonal changes in thermogenesis at the organismal level have been well documented in several wild species under laboratory or wild conditions (Zheng et al. 2008a; Swanson 2010; Liknes and Swanson 2011a, 2011b), studies of the intraspecific variability of thermal physiological responses and geographic variation in wild species have been relatively rare (Dawson et al. 1983; Swanson 1993; O’Connor 1996; Soobramoney et al. 2003; Olson et al. 2010). Few studies have investigated the physiological variation of birds with season or temperature across a latitudinal gradient (Weathers 1979; Furness 2003; Wikelski et al. 2003; Cavieres and Sabat 2008), and cellular evidence is lacking for birds that would allow understanding of the seasonal changes in thermogenesis at the cellular level in populations from different latitudes. In this study, we used an integrative approach to investigate whether seasonal phenotypic variation across a latitudinal gradient is associated with physiological, hormonal, or biochemical differences in Eurasian tree sparrows (Passer montanus). The Eurasian tree sparrow is a small granivorous passerine that inhabits vast areas of the continents of Europe and Asia (MacKinnon and Phillipps 2000). It has a high BMR and a wide zone of thermoneutrality (Deng and Zhang 1990). Tree sparrows cope with cold mainly through metabolic changes coupled with increased organ masses and heightened activities of respiratory enzymes (Liu and Li 2006; Liu et al. 2008; Zheng et al. 2008a; Li et al. 2010). Ours is the first study to assess seasonal variation in thermoregulatory mechanisms for tree sparrows over a latitudinal gradient. We hypothesized that physiological and biochemical metabolic characteristics would contribute to BMR variation. We predicted that winter tree sparrows from the northern area (Qiqihar) would have larger, metabolically expensive organs, higher activities of biochemical and cellular markers of metabolism, and higher plasma levels of thyroid hormones than their summer counterparts and than those birds from the southern area (Wenzhou). Material and Methods Animals This study was conducted in Qiqihar, Heihongjiang Province (47⬚33 N, 124⬚02E), and Wenzhou (27⬚29N, 120⬚51  E), Zhenjiang Province, China. In Qiqihar, the annual mean temperature is 3.4⬚C; mean daily maximum temperatures range from 38⬚C in July to ⫺33⬚C in January, and mean daily minimum temperatures range from 24⬚C in July to ⫺14⬚C in January. Mean

706 W.-H. Zhen, M. Li, J.-S. Liu, S.-L. Shao, and X.-J. Xu Table 1: Seasonal variations of liver and muscle thermogenesis in tree sparrows from two localities in China Wenzhou Category Sample size Body mass (g) Liver: Mitochondrial protein (mg g⫺1) State 4 respiration: nmol O2 min⫺1 mg mitochondrial protein⫺1 mmol O2 min⫺1 g tissue⫺1 Muscle: Mitochondrial protein (mg g⫺1) State 4 respiration: nmol O2 min⫺1 mg mitochondrial protein⫺1 mmol O2 min⫺1 g tissue⫺1

Qiqihar

Winter

Summer

Winter

Summer

10 20.5 Ⳳ .6

12 17.8 Ⳳ .4

12 19.6 Ⳳ .3

13 18.3 Ⳳ .2

19.74 Ⳳ .97

19.69 Ⳳ .74

19.00 Ⳳ .84

18.24 Ⳳ 1.05

23.15 Ⳳ 2.27 .47 Ⳳ .06

14.72 Ⳳ 2.47 .29 Ⳳ .05

34.71 Ⳳ 3.06 .68 Ⳳ .06

19.45 Ⳳ 2.92 .34 Ⳳ .05

6.70 Ⳳ .39

7.11 Ⳳ .52

7.34 Ⳳ .51

6.82 Ⳳ .60

42.36 Ⳳ 3.06 .29 Ⳳ .03

27.33 Ⳳ 1.89 .20 Ⳳ .02

43.95 Ⳳ 2.20 .32 Ⳳ .02

31.52 Ⳳ 2.04 .21 Ⳳ .03

Effects S***

S***, L** S***, L*

S*** S***

Note. Statistical significance was determined by two-way ANOVA; all results are expressed as means Ⳳ SEM. State 4 respiration of liver mitochondria varied significantly with latitude and season for both mitochondrial protein–specific and mass-specific bases. State 4 respiration of muscle mitochondria changed significantly with season for both mitochondrial protein–specific and mass-specific bases. S p season; L p latitude. *P ! 0.05. **P ! 0.01. ***P ! 0.001.

temperatures in winter (from December to February) and summer (from June to August) were ⫺14.7⬚C and 17⬚C, respectively, between 2007 and 2008 (Zheng et al. 2008a). In Wenzhou, the climate is warm-temperate, with an average annual rainfall of 1,700 mm spread across all months and slightly more precipitation during winter and spring. The mean annual temperature is 18⬚C; mean daily maximum temperatures range from 39⬚C in July to 8⬚C in January, and mean daily minimum temperatures range from 28⬚C in July to 3⬚C in January. Mean temperatures in winter (from December to February) and summer (from June to August) were 9.6⬚C and 29.0⬚C, respectively, between 2008 and 2009 (Wenzhou Bureau of Meteorology). In Qiqihar, we captured 10 birds in the winter (December) of 2007, 12 birds in the winter (from January to February) of 2008, and 25 birds in the summer (from June to August) of 2008. In Wenzhou, we captured 15 birds in the summer (from June to August) of 2008 and 16 birds in the winter (from January to February) of 2009. We tested for possible effects on data from the two latitudes. At capture, we weighed body mass (Mb) to the nearest 0.1 g with a Sartorius balance (model BT25S). Following capture, we transported birds to the laboratory and caged them for 1 or 2 d (50 # 30 # 20 cm3) outdoors under natural photoperiod and temperature before measurements. Birds were supplied food and water ad lib. The mean body masses of sparrows in winter and summer were 20.5 Ⳳ 0.6 and 17.8 Ⳳ 0.4 g in Wenzhou and 19.6 Ⳳ 0.3 and 18.3 Ⳳ 0.3 g in Qiqihar, respectively (table 1). All animal procedures were licensed under the Institutional Animal Care and Use Committee of the Institute of Zoology, Chinese Academy of Sciences.

Metabolic Trials We measured oxygen consumption using a closed-circuit respirometer containing 3.6-L animal chambers (Go´recki 1975) immersed in a water bath that maintained temperature at 25⬚ Ⳳ 0.5⬚C, which is near the thermal neutral zone for Eurasian tree sparrows (Deng and Zhang 1990). We obtained metabolic measurements at this temperature over a 60-min period that began after the birds had rested in their chambers for approximately an hour (Zheng et al. 2008a). RMR is the energy required to perform vital body functions while the body is at rest. Because it is doubtful that true BMRs can be achieved in the laboratory, the term RMR is often used to refer to such measurements, even when the standard conditions for BMR have been met (Swanson 2010). Before RMR measurement, we fasted birds for 4 h to ensure that they were in a postabsorptive state. H2O and CO2 were absorbed by silica gel and KOH, respectively. We discarded recordings of oxygen consumption when birds were active in the chamber from calculation of the metabolic rate for each individual. We made all measurements daily between 20:00 and 24: 00 and recorded oxygen consumption every 5 min over the test period. We used two consecutive, stable, and minimum recordings to calculate metabolic rates. We express metabolic rates as milliliters of O2 per hour after correcting all values to stpd conditions (Schmidt-Nielsen 1997). Measurements of Organ Masses After metabolic measurements, we sacrificed birds by decapitation and collected blood for plasma analyses. We removed the brain, heart, lungs, liver, kidneys, gizzard, small intestine, and rectum and weighed each organ to the nearest 0.1 mg. One

Thermogenesis in Small Birds at Different Latitudes 707

Figure 1. Seasonal variation in resting metabolic rate (RMR) in Eurasian tree sparrows from two localities in China. Results are expressed as means Ⳳ SEM; both whole and mass-independent RMRs are presented. Total (mL O2 h⫺1) and mass-independent (mL O2 g⫺1 h⫺1) RMR were significantly affected by latitude (L) and season (S). Three asterisks indicate P ! 0.001.

part of the liver was used for the collection of mitochondria. Internal organs, including the remaining part of the liver, were dried to constant mass over 2 d at 75⬚C and weighed to 0.1 mg (Williams and Tieleman 2000; Liu and Li 2006; Zhang et al. 2008).

1.96 mL of respiration medium with a Clark electrode (DW1; Hansatech Instruments, Norfolk, England), essentially as described by Estabrook (1967). We ran state 4 respiration measurements for 1 h under substrate-dependent conditions with succinate as the substrate (Liu et al. 2006; Zheng et al. 2008a, 2010).

Preparation of Mitochondria We quickly removed the liver and pectoral muscle from the sparrow carcass, placed them in ice-cold sucrose-buffered medium, removed any adhering tissue, blotted tissue dry, and weighed them to 0.1 mg. We chopped the liver samples coarsely with scissors, after which samples were rinsed and resuspended in 5 vol of ice-cold medium. We homogenized the samples using a Teflon/glass homogenizer. We minced the samples used for muscle mitochondrial preparations before treating samples with proteinase for 5–10 min, after which the proteinase was removed. We next resuspended muscle samples in 10 vol of ice-cold medium and homogenized them with a Teflon/glass homogenizer. We centrifuged the homogenate at 600 g for 10 min at 4⬚C in a Beckman centrifuge and discarded the pellets containing nuclei and cell debris. We centrifuged the supernatants at 12,000 g for 10 min at 4⬚C, resuspended the pellets, and then centrifuged the mixture again at 12,000 g, followed by resuspension in ice-cold medium (Rasmussen et al. 2004; Zheng et al. 2008a). We measured the protein content of mitochondria by the Folin phenol method with bovine serum albumin as the standard (Lowry et al. 1951). Mitochondrial Respiration We measured state 4 respiration (reflecting oxidative phosphorylation capacity) in liver and muscle mitochondria at 30⬚C in

Enzyme Activity and Hormone Concentration We measured the activities of cytochrome C oxidase (COX) in liver and muscle polarographically at 30⬚C using a Clark electrode according to Sundin et al. (1987). We expressed the activity of COX as nanomoles of O2 per minute per milligram of mitochondrial protein (Wiesinger et al. 1989; Zheng et al. 2008a, 2010). We determined the concentration of T3 and T4 in plasma by radioimmunoassay (RIA), using RIA kits for human use provided by the China Institute of Atomic Energy. We labeled antigen for T3 and T4 with 125NaI (Liu et al. 2006). Data Statistics We analyzed the data with SPSS for Windows (ver. 12.0). We examined the effect of season and latitude and their interaction with Mb, hormones, and enzymes using a two-way ANOVA. RMR (mL O2 g⫺1 h⫺1) was analyzed using a two-way ANCOVA (season # latitude) with Mb as the covariate, and organ dry mass data were analyzed with body dry mass minus organ dry mass (dry carcasses) as the covariate (Christians 1999). We expressed all results as means Ⳳ SEM and used Turkey’s HSD to identify differences among the four levers when the interaction (season # latitude) was significant. We accepted P ! 0.05 as a significant difference for all statistical comparisons.

Figure 2. Adjustment means of organ mass of tree sparrows in seasonal acclimatization from two localities in China. Results are expressed as means Ⳳ SEM; data are for dry masses. Brain, rectum, gizzard, and heart varied significantly with latitude (L); liver, rectum, small intestine, total digestive tract, and gizzard varied significantly with season (S); and rectum and lung varied significantly with the interaction between latitude and season (L # S). One asterisk indicates P ! 0.05, two asterisks indicate P ! 0.01, and three asterisks indicate P ! 0.001.

Thermogenesis in Small Birds at Different Latitudes 709

Figure 3. Seasonal variation in mitochondrial cytochrome C oxidase (COX) activity of liver in Eurasian tree sparrows from two localities in China. Results are expressed as means Ⳳ SEM. The activity of liver COX varied significantly with latitude (L) and season (S) on mitochondrial protein– specific (nmol O2 min⫺1 mg mitochondrial protein⫺1) and mass-specific (mmol O2 min⫺1 g tissue⫺1) bases. Two asterisks indicate P ! 0.01.

Results Mb and RMR Tree sparrows showed significant seasonal variation in Mb (ANOVA, F1, 74 p 36.925, P ! 0.001; table 1), with winter sparrows being heavier than summer sparrows. However, there was no significant difference in Mb between birds from the two latitudes (ANOVA, F1, 74 p 0.448, P 1 0.05) and no interaction between latitude and season (ANOVA, F1, 74 p 4.980, P 1 0.05; table 1). Total RMR (mL O2 h⫺1) was significantly affected by season (ANOVA, F1, 74 p 91.190, P ! 0.001) and latitude (ANOVA, F1, 74 p 40.647, P ! 0.001) but not by the interaction between latitude and season (ANOVA, F1, 74 p 0.052, P 1 0.05). Winter birds expressed higher RMR than summer birds, and those birds from Qiqihar had higher RMR than birds from Wenzhou. Similarly, mass-independent RMR (mL O2 g⫺1 h⫺1) was markedly affected by latitude (ANCOVA, F1, 73 p 44.599, P ! 0.001) and season (ANCOVA, F1, 73 p 40.781, P ! 0.001) but not by the interaction (ANCOVA, F1, 73 p 0.316, P 1 0.05; fig. 1). In addition, Mb had a significant effect on RMR (ANCOVA, F1, 73 p 4.830, P ! 0.05). Organ Masses There were no statistically significant effects of the covariate (body dry mass minus organ dry mass) on organs (brain, F1, 73 p 0.093, P 1 0.05; lung, F1, 73 p 0.477, P 1 0.05; liver, F1, 73 p 1.583, P 1 0.05; kidney, F1, 73 p 0.007, P 1 0.05; gizzard, F1, 73 p 0.612, P 1 0.05; small intestine, F1, 73 p 2.525, P 1 0.05; rectum, F1, 73 p 0.399, P 1 0.05; total digestive tract, F1, 73 p 2.916, P 1 0.05) except for heart (F1, 73 p 16.850, P ! 0.001). Brain dry mass varied significantly with latitude (ANCOVA, F1, 73 p 18.931, P ! 0.001) but not with season (ANCOVA,

F1, 73 p 0.179, P 1 0.05) or the interaction between latitude and season (ANCOVA, F1, 73 p 1.959, P 1 0.05), being higher in Qiqihar birds than in Wenzhou birds (fig. 2A). Liver dry mass was affected by season (ANCOVA, F1, 73 p 8.235, P ! 0.01), with winter sparrows having larger liver dry masses than their summer counterparts (fig. 2B). The effects of latitude (ANCOVA, F1, 73 p 0.089, P 1 0.05) and the interaction between latitude and season (ANCOVA, F1, 73 p 2.723, P 1 0.05) were not significant. Rectum dry mass was significantly affected by latitude (ANCOVA, F1, 73 p 6.988, P ! 0.05), season (ANCOVA, F1, 73 p 12.801, P ! 0.01), and the interaction between latitude and season (ANCOVA, F1, 73 p 10.660, P ! 0.01). Winter sparrows in Qiqihar had larger rectum dry masses than their summer counterparts, and tree sparrows from Wenzhou had larger rectum dry masses than Qiqihar birds in summer (post hoc, P ! 0.01; fig. 2C). Dry masses of the small intestine and total digestive tract varied significantly with season (ANCOVA, small intestine, F1, 73 p 111.484, P ! 0.001, fig. 2D; total digestive tract, F1, 73 p 164.764, P ! 0.001, fig. 2E) but not with latitude (ANCOVA, small intestine, F1, 73 p 2.281, P 1 0.05; total digestive tract, F1, 73 p 0.714, P 1 0.05) and the interaction between latitude and season (ANCOVA, small intestine, F1, 73 p 2.037, P 1 0.05; total digestive tract, F1, 73 p 1.646, P 1 0.05). Winter sparrows in winter had larger small intestine and total digestive tract dry masses than their summer counterparts. Gizzard dry mass varied significantly with latitude (ANCOVA, F1, 73 p 17.086, P ! 0.001) and season (ANCOVA, F1, 73 p 64.977, P ! 0.001) but not with the interaction between latitude and season (ANCOVA, F1, 73 p 0.535, P 1 0.05; fig. 2F), with tree sparrows from Qiqihar having larger gizzard dry mass than Wenzhou birds and winter birds having larger gizzards than their summer counterparts. Heart dry mass varied significantly

710 W.-H. Zhen, M. Li, J.-S. Liu, S.-L. Shao, and X.-J. Xu

Figure 4. Seasonal variation in mitochondrial cytochrome C oxidase (COX) activity of muscle in Eurasian tree sparrows from two localities in China. Results are expressed as means Ⳳ SEM. Muscle COX activity varied significantly with latitude (L) and season (S) on mitochondrial protein– specific (nmol O2 min⫺1 mg mitochondrial protein⫺1) and mass-specific (mmol O2 min⫺1 g tissue⫺1) bases. One asterisk indicates P ! 0.05, and three asterisks indicate P ! 0.001.

with latitude (ANCOVA, F1, 73 p 6.224, P ! 0.05) but not with season (ANCOVA, F1, 73 p 0.944, P 1 0.05) or the interaction between latitude and season (ANCOVA, F1, 73 p 1.542, P 1 0.05), with heart dry masses in Qiqihar birds being heavier than those in Wenzhou birds (fig. 2H). Lung dry mass was not affected by season (ANCOVA, F1, 73 p 0.256, P 1 0.05) or latitude (F1, 73 p 0.624, P 1 0.05) but was affected by the interaction between latitude and season (ANCOVA, F1, 73 p 10.251, P ! 0.01; fig. 2H). No significant variations with season, latitude, or the interaction between latitude and season occurred for kidney (ANCOVA, season, F1, 73 p 0.536, P 1 0.05; latitude, F1, 73 p 0.955, P 1 0.05; latitude and season, F1, 73 p 0.341, P 1 0.05; fig. 2I). Protein Content, Mitochondrial Respiration, and COX Activity in Liver Mass-specific mitochondrial protein content in liver tissue did not vary significantly with latitude (ANOVA, F1, 43 p 0.496, P 1 0.05), season (ANOVA, F1, 43 p 0.872, P 1 0.05), or the interaction between latitude and season (ANOVA, F1, 43 p 0.768, P 1 0.05; table 1). State 4 respiration of liver mitochondria varied significantly with latitude and season for both mitochondrial protein–specific (ANOVA, latitude, F1, 43 p 8.680, P ! 0.01; season, F1, 43 p 18.344, P ! 0.001; table 1) and massspecific (ANOVA, latitude, F1, 43 p 6.150, P ! 0.05; season, F1, 43 p 23.157, P ! 0.001; table 1) bases but not for the interaction between latitude and season (ANOVA, mitochondrial protein specific, F1, 43 p 1.521, P 1 0.05; mass specific, F1, 43 p 2.179, P 1 0.05; table 1). Mitochondrial respiration was higher in Qiqihar than in Wenzhou and was higher during winter than during summer. The activity of liver COX also varied signifi-

cantly with latitude and season on mitochondrial protein–specific (ANOVA, latitude, F1, 43 p 11.753, P ! 0.01; season, F1, 43 p 8.722, P ! 0.01; fig. 3) and mass-specific (ANOVA, latitude, F1, 43 p 8.955, P ! 0.01; season, F1, 43 p 13.794, P ! 0.01; fig. 3) bases but not with the interaction between latitude and season (ANOVA, mitochondrial protein specific, F1, 43 p 0.484, P 1 0.05; mass specific, F1, 43 p 0.001, P 1 0.05; fig. 3). Liver COX activity on a mitochondrial protein–specific basis was significantly higher in Qiqihar birds (winter: 27.53 Ⳳ 1.98 nmol O2 min⫺1 mg mitochondrial protein⫺1; summer: 23.09 Ⳳ 2.27 nmol O2 min⫺1 mg mitochondrial protein⫺1) than in Wenzhou birds (winter: 22.16 Ⳳ 1.81 nmol O2 min⫺1 mg mitochondrial protein⫺1; summer: 14.98 Ⳳ 1.57 nmol O2 min⫺1 mg mitochondrial protein⫺1). On a mass-specific basis, the activity of COX was also significantly higher in Qiqihar birds (winter: 0.55 Ⳳ 0.05 mmol O2 min⫺1 g tissue⫺1; summer: 0.41 Ⳳ 0.03 mmol O2 min⫺1 g tissue⫺1) than in Wenzhou birds (winter: 0.44 Ⳳ 0.04 mmol O2 min⫺1 g tissue⫺1; summer: 0.29 Ⳳ 0.03 mmol O2 min⫺1 g tissue⫺1). COX activity was significantly higher in winter than in summer for both Wenzhou and Qiqihar birds. Protein Content, Mitochondrial Respiration, and COX Activity in Muscle The mass-specific mitochondrial protein content of muscle did not vary significantly with latitude (ANOVA, F1, 43 p 0.104, P 1 0.05), season (ANOVA, F1, 43 p 0.010, P 1 0.05), or the interaction between latitude and season (ANOVA, F1, 43 p 0.769, P 1 0.05; table 1). State 4 respiration of muscle mitochondria changed significantly with season for both mitochondrial protein–specific (ANOVA, F1, 43 p 36.260, P ! 0.001; table 1) and mass-specific (ANOVA, F1, 43 p 17.860, P ! 0.001;

Thermogenesis in Small Birds at Different Latitudes 711

Figure 5. Relationship between resting metabolic rate (RMR) and cellular and biochemical metabolic makers of liver and muscle in Eurasian tree sparrows from two localities in China. Shown are linear regressions between RMR and cellular and biochemical metabolic makers. Correlation analysis demonstrated that liver mitochondrial state 4 respiration (A), liver mitochondria COX activity (B), muscle mitochondrial state 4 respiration (C), and muscle mitochondria COX activity (D) from the two localities were positively correlated with RMR. Filled triangles indicate winter in Wenzhou (WW), open triangles indicate summer in Wenzhou (WS), filled circles indicate winter in Qiqihar (QW), and open circles indicate summer in Qiqihar (QS).

table 1) bases, being higher in winter than in summer. However, state 4 respiration of muscle mitochondria did not vary significantly with latitude (ANOVA, mitochondrial protein specific, F1, 43 p 1.606, P 1 0.05; mass specific, F1, 43 p 1.115, P 1 0.05) or the interaction between latitude and season (ANOVA, mitochondrial protein specific, F1, 43 p 0.324, P 1 0.05; mass specific, F1, 43 p 0.160, P 1 0.05; table 1). Muscle COX activity also varied significantly with latitude and season on mitochondrial protein–specific (ANOVA, latitude, F1, 43 p 6.451, P ! 0.05; season, F1, 43 p 24.479, P ! 0.001; fig. 4) and massspecific (ANOVA, latitude, F1, 43 p 4.679, P ! 0.05; season, F1, 43 p 14.679, P ! 0.001; fig. 4) bases but not with the interaction between latitude and season (ANOVA, mitochondrial protein specific, F1, 43 p 0.210, P 1 0.05; mass specific, F1, 43 p 0.012, P 1 0.05; fig. 4). COX activity was significantly higher in winter than in summer for both Wenzhou and Qiqihar birds, and COX activity was significantly higher in Qiqihar than in

Wenzhou for both mitochondrial protein–specific and massspecific bases (fig. 4). There were significant positive relationships between massspecific mitochondrial state 4 respiration and RMR (r p 0.741, P ! 0.001; fig. 5A) and mass-specific mitochondrial COX activity and RMR (r p 0.691, P ! 0.001; fig. 5B) in liver and between mass-specific mitochondrial state 4 respiration and RMR (r p 0.744, P ! 0.001; fig. 5C) and mass-specific mitochondrial COX activity and RMR (r p 0.650, P ! 0.001; fig. 5D) in muscle. Plasma T3 and T4 Concentrations Plasma T3 levels were significantly affected by latitude (ANOVA, F1, 43 p 16.851, P ! 0.001) and season (ANOVA, F1, 43 p 37.268, P ! 0.001) but not by the interaction between latitude and season (ANOVA, F1, 43 p 1.733, P 1 0.05; fig. 6). T3 levels were

712 W.-H. Zhen, M. Li, J.-S. Liu, S.-L. Shao, and X.-J. Xu

Figure 6. Seasonal variation in plasma triiodothyronine (T3) and thyroxine (T4) in Eurasian tree sparrows from two localities in China. Results are expressed as means Ⳳ SEM. Plasma T3 levels were significantly affected by latitude (L) and season (S), and plasma T4 levels were also significantly affected by season. One asterisk indicates P ! 0.05, and three asterisks indicate P ! 0.001.

significantly higher for Qiqihar birds than for their counterparts in Wenzhou, and T3 levels were also significantly higher in winter than in summer for both populations. Significant seasonal variation also occurred for T4 levels (ANOVA, F1, 43 p 6.612, P ! 0.05; fig. 6), but T4 levels did not vary significantly with latitude (ANOVA, F1, 43 p 0.510, P 1 0.05) or with the interaction between latitude and season (ANOVA, F1, 43 p 1.475, P 1 0.05; fig. 6). T4 levels were significantly higher in winter than in summer. We documented significant positive relationships between T3 and RMR (r p 0.729, P ! 0.001; fig. 7), between T3 and mass-specific liver mitochondrial state 4 respiration (r p 0.556, P ! 0.001; fig. 8A), between T3 and mass-specific liver mitochondrial COX activity (r p 0.562, P ! 0.001; fig. 8B), between T3 and mass-specific muscle mitochondrial state 4 respiration (r p 0.455, P ! 0.01; fig. 8C), and between T3 and mass-specific muscle mitochondrial COX activity (r p 0.461, P ! 0.01; fig. 8D). Discussion Seasonal changes in temperature and photoperiod are important characteristics of temperate and high latitudes. Our results demonstrate that Eurasian tree sparrows show significant differences in Mb and physiological and biochemical characteristics between seasons and latitudes. Changes in Mb are an important adaptive strategy for many small birds, and those inhabiting seasonal environments maintain a stable Mb or show an increase in Mb when exposed to winter conditions (Liknes and Swanson 1996; Liu and Li 2006; Liu et al. 2008; Zheng et al. 2008a, 2008b). From the standpoint of causality, increased thermoregulatory heat production itself is a product of changes in these components, and an increase in Mb will decrease the surface-to-volume ratio, which can reduce heat loss and thereby also reduce living costs (Aschoff

1981; Schmidt-Nielsen 1997). Our present study indicates that tree sparrows showed small but significant seasonal variation in Mb. This pattern is similar to that noted in a congeneric species, the house sparrow (P. domesticus; Hart 1962; Liknes and Swanson 2011b; Swanson and Merkord 2013) and other small birds in temperate zones (Zheng et al. 2008a, 2008b; Chamane and Downs 2009). In addition to the seasonal variation in Mb, seasonal differences also occurred for some organ masses, such as liver, gizzard, small intestine, rectum, and total digestive tract, so variation in these factors also likely contributes to the seasonal differences in RMR. Thermoregulatory responses of small birds to cold have attracted considerable research attention due to their limited capacities for insulative acclimatization. They develop morphological, physiological, and behavioral adaptations that assist in coping with the various energy demands (Yuni and Rose 2005). These adaptations might include a reduction in the level of activity (Ambrose 1984), shifts in basal metabolism (Liknes et al. 2002; Liu and Li 2006), and variation in lipid content (Lill et al. 2006; Cooper 2007). An increased BMR, mediated through enhanced thermogenic properties, is an important factor for birds that live in cold environments (Schmidt-Nielsen 1997; Doucette and Geiser 2008; Zheng et al. 2008a). Our results indicate that RMRs of tree sparrows varied significantly with both latitude and season, with RMRs of Qiqihar birds higher than those of Wenzhou birds and RMRs higher in winter than in summer. Our results are also consistent with the hypothesis that tree sparrows living at different seasons (summer and winter) and latitudes (Wenzhou and Qiqihar) adjust their metabolic activity to the prevailing conditions (McKechnie 2008; McKechnie and Swanson 2010). Similar results have been reported for other bird species (Blem 1977; Bryant and Furness 1995; Wikelski et al. 2003; Wiersma et al. 2007). Weathers

Thermogenesis in Small Birds at Different Latitudes 713

Figure 7. Relationship between plasma triiodothyronine (T3) concentration and resting metabolic rate (RMR) in Eurasian tree sparrows from two localities in China. Shown is a linear regression between plasma T3 concentration and RMR. Correlation analysis demonstrated that RMRs from the two localities were positively correlated with plasma T3. Filled triangles indicate winter in Wenzhou (WW), open triangles indicate summer in Wenzhou (WS), filled circles indicate winter in Qiqihar (QW), and open circles indicate summer in Qiqihar (QS).

(1979) suggested that BMR of birds is correlated broadly with climate characteristics. A reduced level of endogenous heat production may thus have adaptive value in low-latitude species (Weathers 1977; Wikelski et al. 2003; Wiersma et al. 2007). Higher metabolic rates in midlatitude and high-latitude birds can contribute to improved cold tolerance and thus also have adaptive significance (Weathers 1979; Canterbury 2002). Kendeigh and Blem (1977) segregated species into “northern” (breeding distribution mainly north of 40⬚N) and “southern” (breeding distribution mainly south of 40⬚N) groups and compared regressions of BMR on Mb, finding that northern species had generally higher BMR than southern species mainly below 40⬚N. Tree sparrows in Wenzhou and Qiqihar fit nicely into the southern and northern categories, respectively, of Kendeigh and Blem (1977), and RMR in the two populations also fits the northern-southern dichotomy. Elevation of RMR in tree sparrows is presumably related to metabolic and/or morphological adjustments to meet the extra energy demands. Such organs as the gastrointestinal tract, liver, and kidneys have high mass-specific energy metabolism at rest and may contribute significantly to BMR (Daan et al. 1990; Piersma et al. 1996; Williams and Tieleman 2000; Liu and Li 2006; Zhang et al. 2008). Accordingly, visceral organs should be the primary determinants of BMR, and variation in BMR should be correlated with variation in the masses of these organs (Williams and Tieleman 2000; Hammond et al. 2001). What are the ecological implications of having larger guts for winteracclimatized birds? Birds in winter consumed more food, which apparently stimulated the enlargement of such organs as gizzard, small intestine, rectum, and total digestive tract. On the basis of histological measurements, possible cellular mechanisms of upregulating the digestive tract capacity are hyper-

plasia (production of more cells) and hypertrophy (increased cell size; Starck and Rahmaan 2003). Positive correlations of BMR with digestive organs have been documented for liver, gizzard, small intestine, and total digestive tract in some small birds (Starck and Rahmaan 2003; Tieleman et al. 2003; Zhang et al. 2008), so the effects of digestive organs on RMR for tree sparrows in this study is not without precedent. The selective pressures that influence metabolism may be complex and act through multiple avenues to influence metabolic rate. Two of these avenues are to alter the density of mitochondria and the concentration of enzymes in aerobic catabolic pathways and to alter the sizes of tissues/organs, such as liver or muscles (Li et al. 2001; Brand et al. 2003; Else et al. 2004; Zheng et al. 2008a, 2010; Liknes and Swanson 2011a). Early studies showed that cold acclimation and seasonal acclimatization can induce an increase in state 4 respiration and COX activity of the liver accompanied by enhanced RMR in small bird species, including Eurasian tree sparrows (Liu et al. 2008; Zheng et al. 2008a) and Chinese bulbuls (Zheng et al. 2010). Our data indicate that changes in liver cellular aerobic capacity paralleled changes in RMR, with both season and latitude (fig. 5A, 5B). Our data showed that liver mitochondrial state 4 respiration and COX activity increased significantly during winter in comparison to that during summer in both Wenzhou and Qiqihar, suggesting that tree sparrows increased the total respiratory capacity of the liver with colder climates in general. Thus, activation of liver mitochondrial respiration and elevations of COX activity appear to be one of the cellular mechanisms for elevating RMR. Skeletal muscle makes up nearly 40% of the Mb in many volant birds, and it is an important source of seasonal or coldinduced thermogenesis. Several studies have documented a pos-

714 W.-H. Zhen, M. Li, J.-S. Liu, S.-L. Shao, and X.-J. Xu

Figure 8. Relationships between plasma triiodothyronine (T3) concentration and cellular and biochemical metabolic makers of liver and muscle in Eurasian tree sparrows from two localities in China. Shown are linear regressions between plasma T3 concentration and cellular and biochemical metabolic makers. Correlation analysis demonstrated that liver mitochondrial state 4 respiration (A), liver mitochondria COX activity (B), muscle mitochondrial state 4 respiration (C), and muscle mitochondria COX activity (D) from the two localities were positively correlated with plasma T3. Filled triangles indicate winter in Wenzhou (WW), open triangles indicate summer in Wenzhou (WS), filled circles indicate winter in Qiqihar (QW), and open circles indicate summer in Qiqihar (QS).

itive seasonal or cold-induced correlation between thermogenic capacity and muscle mass, mitochondrial protein, state 4 respiration, and activities of citrate synthase and/or COX in small birds and mammals, including hoopoe larks (Williams and Tieleman 2000), Eurasian tree sparrows (Liu et al. 2008; Zheng et al. 2008a), Chinese bulbuls (Zhang et al. 2008; Zheng et al. 2010), and black-capped chickadees and house sparrows (Liknes and Swanson 2011a, 2011b). The present results support the notion that the mass-specific respiratory capacities of muscle are determined primarily by the amount of oxidase activity (Rasmussen et al. 2004). A significant increase in state 4 respiration and COX activity indicates an increase in cellular metabolic capacity and is associated with an increase in RMR. The state 4 respiration of tree sparrow flight muscle in both Wenzhou and Qiqihar increased during winter acclimatization. In addition, muscle COX activities of tree sparrows from Wenzhou and Qiqihar increased in winter by 62% and 38%, respectively,

on a specific-activity basis and increased 53% and 46%, respectively, on a mass-specific basis compared with their summer counterparts. These results demonstrate that muscle is an important contributor to basal thermogenesis in tree sparrows. Such a result has also been documented for congeneric house sparrows (Chappell et al. 1999). Thyroid hormone is the primary endocrine regulator of obligatory thermogenesis and can increase energy expenditure and stimulate basal thermogenesis by lowering metabolic efficiency (Yen 2001). Thyroid hormones are also important for facultative thermogenesis (Collin et al. 2003; Decuypere et al. 2005). Potential mechanisms for elevating facultative thermogenesis in birds include increasing muscle mass devoted to shivering thermogenesis, increasing muscle mass–specific aerobic enzyme capacity (Collin et al. 2003), and increasing heat production from mitochondrial respiration due to imperfect coupling to adenosine diphosphate phosphorylation (Dridi et

Thermogenesis in Small Birds at Different Latitudes 715 al. 2004). In addition, thyroid hormones can affect adaptive thermogenesis by influencing several aspects of energy metabolism, such as substrate cycling, ion cycling, and mitochondrial proton leakage (Yen 2001; Decuypere et al. 2005). Jenni-Eiermann et al. (2002) found that plasma T3 levels followed a unimodal pattern, with high values during the cold season in red knots. Li et al. (2010) and Zheng et al. (2010) showed that winter tree sparrows and Chinese bulbuls demonstrated significantly higher RMR than their summer counterparts, and winter birds also had a higher level of plasma T3 than summer birds. Liu et al. (2006) reported that little buntings eating thyroxine-laced poultry food showed increases in RMR, state 4 respiration, and COX activity in liver and muscle. This is consistent with a role for T3 in the adjustment of metabolic rate to acute energy demands. Our data indicate that plasma T3 levels varied with both latitude and season, and cellular properties of the liver and muscle were consistent with the changes in plasma T3 levels. Eurasian tree sparrows mainly coped with cold by enhancing thermogenic capacities through heightened activity of respiratory enzymes and an enhanced level of plasma thyroid hormones (T3). These results show that Eurasian tree sparrows exhibit a pronounced seasonal and latitudinal phenotypic flexibility with physiological and biochemical adjustments. Acknowledgments We are grateful to Professor De-Hua Wang and Qing-Fen Li for supplying a Kalabukhov respirometer for this study, and we especially thank Dr. David L. Swanson for providing several references, correcting the English, and giving some suggestions. This study was financially supported by the National Natural Science Foundation of China (30670324 and 31070366 to J.-S.L.). Literature Cited Ambrose S.J. 1984. The response of small birds to extreme heat. Emu 84:242–243. Aschoff J. 1981. Thermal conductance in mammals and birds: its dependence on body size and circadian phase. Comp Biochem Physiol A 69:611–619. Barcelo´ G., J. Salinas, G. Cavieres, M. Canals, and P. Sabat. 2009. Thermal history can affect the short-term thermal acclimation of basal metabolic rate in the passerine Zonotrichia capensis. J Therm Biol 34:415–419. Blem C. 1977. Reanalysis of geographic variation of house sparrow energetics. Auk 94:358–359. Brand M.D., N. Turner, A. Ocloo, P.L. Else, and A.J. Hulbert. 2003. Proton conductance and fatty acyl composition of liver mitochondria correlates with body mass in birds. Biochem J 376:741–748. Bryant D.M. and R.W. Furness. 1995. Basal metabolic rates of North Atlantic seabirds. Ibis 137:219–226. Canterbury G. 2002. Metabolic adaptation and climatic constraints on winter birds distribution. Ecology 83:946–957.

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