The Auk 119(2):437-445, 2002 EXTRINSIC AND INTRINSIC SOURCES OF VARIATION IN PLASMA LIPID METABOLITES OF FREE-LIVING WESTERN SANDPIPERS {CALIDRIS MAURI) CHRISTOPHER G. GUGLIELMO/ PATRICK D. O'HARA, AND TONY D. WILLIAMS Centre for Wildlife Ecology, Behavioural Ecology Research Group, Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada ABSTRACT.?Plasma lipid metabolites may be useful indicators of mass changes in migra- tory birds. To test utility of plasma metabolites in field studies, we examined effects of sev- eral extrinsic (bleed time, time of day, location) and intrinsic (body mass, sex, age, migratory state) factors on plasma concentrations of triglyc?rides (TRIG), glycerol (GLYC), and B-OH- butyrate (BUTY) in free-living Western Sandpipers (Calidris mauri). TRIG and GLYC de- creased rapidly following capture (2-20 min), whereas BUTY did not change. GLYC and BUTY were negatively correlated to body mass. TRIG was positively correlated to body mass in migrant females, but not consistently in migrant males, or in females captured on the wintering grounds. Taking into account other sources of variation, the two measures of lipid utilization (GLYC and BUTY) varied little through the year. TRIG showed the greatest po- tential for use in field studies. TRIG was lowest during winter, when birds were leanest, and highest during spring and fall migration, when sandpipers were gaining mass rapidly at stopovers. TRIG differed between sandpipers refuelling a two stopover sites separated by 35 km, demonstrating that populations of birds can have characteristic lipid metabolite pro- files that may reflect local differences in fattening rate. Received 12 February 2001, accepted 12 December 2001. RESUMEN.?Los metabolitos lipid?eos del plasma pueden ser buenos indicadores de cam- bios en la masa corporal de las aves migratorias. Para poner a prueba su utilidad en estudios de campo, examinamos los efectos de varios factores extr?nsecos (tiempo de toma de la mues- tra de sangre, hora del d?a y lugar) e intr?nsecos (masa corporal, sexo, edad, estado migra- torio) sobre las concentraciones plasm?ticas de triglic?ridos (TRIG), glicerol (GLIC) y B-OH- butirato (BUTI) en individuos silvestres de Calidris mauri. TRIG y GLIC disminuyeron r?pidamente luego de la captura (2-20 min), mientras que BUTI no cambi?. GLIC y BUTI estuvieron correlacionados negativamente con la masa corporal. TRIG se correlacion? po- sitivamente con la masa corporal en hembras migrantes, pero esta relaci?n no fue consistente en machos migrantes ni en hembras capturadas en las ?reas de invernada. Teniendo en cuen- ta otras fuentes de variaci?n, las dos medidas de utilizaci?n de l?pidos (GLIC y BUTI) va- riaron poco a trav?s del a?o. TRIG mostr? el m?ximo potencial para uso en estudios de cam- po. TRIG fue m?s bajo durante el invierno cuando las aves estaban m?s livianas y m?s alto durante la primavera y el oto?o cuando ?stas estaban aumentando r?pidamente su masa en las ?reas de escala migratoria. TRIG mostr? diferencias entre aves que se estaban reaprovi- sionando en dos sitios de escala separados por 35 km, demostrando que las poblaciones pue- den tener perfiles de metabolitos lipid?eos caracter?sticos que podr?an reflejar diferencias locales en las tasas de engorde. VARIATION IN physiological state due to phocytes, immunoglobulins). Deciphering this anabolism, catabolism, or disease can result in information carried in the blood is essential to changes in circulating levels of hormones, met- medical and veterinary diagnostics (News- abolic fuels, electrolytes, albumin, nitrogenous holme and Leech 1985, Duncan et al. 1994, Ka- wastes, and immune system components (lym- neko et al. 1997, Lehmann 1998). The availabil- ity and low cost of analytical kits makes the analysis of blood samples to assess the physi- 1 Present address: Division of Biological Sciences, ological state (condition) of animals in nature University of Montana, Missoula, Montana 59812, very attractive (Mori and George 1978; Jenni- USA. E-mail: cgugliel@selway.umt.edu Eiermann and Jenni 1991, 1996; Andersson and 437 438 GUGLIELMO, O'HARA, AND WILLIAMS [Auk, Vol. 119 Gustafsson 1995; Brown 1996; Jenni and Jenni- Eiermann 1996; Dawson and Bertolotti 1997; Cannes 1999). Nevertheless, reliable interpre- tation of blood assays requires an understand- ing of how factors like stress, body mass, age, and sex affect baseline metabolite levels. Whereas these methods have been validated in humans and domestic animals, knowledge of the many possible sources of variation in wild animals remains rudimentary. Studies with captive birds indicate that plas- ma lipid metabolites can be used to measure mass change at the individual or population level. Mass gain from morning to mid-day re- lated positively to plasma triglyc?ride and neg- atively to B-OH-butyrate levels in Garden War- blers {Sylvia borin; Jenni-Eiermann and Jenni 1994). These same relationships were found in captive Western Sandpipers (Calidris niauri), and in addition, plasma glycerol was negative- ly correlated with rate of mass gain (Williams et al. 1999). At the individual level, determin- ing rate and direction of mass change may pro- vide a measure of physiological performance (fitness). At the population level, lipid metab- olite profiles could be used to compare rates of mass gain at different sites, and thus provide an index of habitat quality based on animal performance (Jenni and Jenni-Eiermann 1996, Williams et al. 1999). Field studies of day-feed- ing passerines seem to support the use of lipid metabolites in that manner (Jenni and Jenni- Eiermann 1996, Jenni-Eiermann and Jenni 1996, Schaub and Jenni 2001). It is unclear if the tech- nique can be applied easily to species like shorebirds, whose feeding may be related to both light and tide conditions. In this study, we examined effects of several extrinsic (bleed time, time of day, site) and in- trinsic (body mass, sex, age, migratory state) factors on plasma concentrations of triglyc?r- ides (TRIG), glycerol (GLYC), and B-OH-buty- rate (BUTY) in Western Sandpipers. This shore- bird migrates long distances between Arctic breeding areas and wintering grounds located mainly along the Pacific coast of the Americas (Wilson 1994). At some wintering areas (e.g. Panama), most first-year birds do not migrate north in the spring, whereas, simultaneously, premigratory adults fatten in preparation for their first northward flight (P. D. O'Hara un- publ. data, Guglielmo 1999). We measured me- tabolites in sandpipers during (1) winter resi- dency, when body mass is low and stable, (2) premigration, when adults gain mass at a low rate (0.09 g day-^; P. D. O'Hara unpubl. data), and (3) migratory stopover, when birds fatten at a high rate (0.4-1 g day-i; Butler et al. 1997). We hypothesized that TRIG, an indicator of mass gain, would be higher at migratory stop- over than during winter, and in Panama during premigration, TRIG would be higher in adults than yearlings. We expected GLYC and BUTY would follow a pattern opposite to TRIG. We also compared metabolites at two migratory stopover sites. METHODS Sample collection.?Wintering (nonmigratory) Western Sandpipers were sampled between 18 De- cember 1995 and 9 February 1996 at Chitr? on the Gulf of Panama (8?N, 79?W). Premigratory birds were sampled at Chitr? from 4 to 24 March 1996. Adult migrants were sampled during spring north- ward (30 April-7 May) and "fall" southward migra- tion (16-25 July), 1996 on the mudflats (25,000 ha) at Boundary Bay and Roberts Bank, British Columbia, Canada (49?10'N, 123?05'W). Juveniles were sampled on southward migration in 1996 at Boundary Bay (19-29 August) and at Sidney Island (8-28 August), a small stop-over site located 35 km southwest of Boundary Bay in the southern Strait of Georgia (Lis- simore et al. 1999). Due to permit limitations, only females were studied in Panama. We captured sandpipers with mist nets (Avinet, Dryden, New York) under permits from the Cana- dian Wildlife Service and INRENARE (Panama). Nets were in constant view, and blood sampling was timed from the moment of capture. In Panama, birds were caught on the falling tide returning from roost- ing and feeding at inland salt and shrimp ponds. At Boundary Bay and Sidney Island, birds were caught near the end of a high-low-high tide cycle to ensure that they had access to feeding areas for an extended period (6-10 h). About one-half of the samples were taken from the jugular vein of birds collected for body composition analysis (Guglielmo 1999). Ap- proximately 140 birds were sampled from the bra- chial vein (26 gauge needle, 200-300 (xL) with hep- arinized capillary tubes (VWR Scientific, Buffalo Grove, Illinois) at Chitr?, Boundary Bay, and Sidney Island. Whole blood was transferred to heparinized Eppendorf tubes (rinsed with 1,000 lU/mL porcine sodium heparin; SIGMA, USA), and stored above ice in a small cooler. Blood was centrifuged at 6,000 rpm (2,000 X g) for 10 min. Plasma was stored at -20?C until analysis. Animal handling protocols were ap- proved by the Simon Eraser University Animal Care April 2002] Plasma Lipid Metabolites 439 TABLE 1. Body mass (grams) and plasma lipid metabolite concentrations (mmol L"') of adult (A) and ju- venile (J) Western Sandpipers sampled at a variety of migratory stages and locations (within each sex, masses with shared superscripts are not significantly different; see text for metabolite statistics). Age/sex Mass Trig lyceride Glycerol B-OH -Butyrate Stage (Site) Mean SE/N Mean SE/N Mean SE/N Mean SE/N Winter (Panama) A/9 -??124.6 0.3/19 0.78 0.06/14 0.34 0.03/14 0.73 0.09/13 J/S ''23.3 0.3/16 0.70 0.09/14 0.42 0.05/14 1.08 0.16/9 Premigration (Panama) A/9 ??"27.9 0.8/15 1.61 0.18/15 0.30 0.02/15 0.51 0.10/13 J/S ''22.5 0.2/18 1.87 0.40/17 0.40 0.04/17 0.92 0.14/13 Spring (Boundary Bay) A/9 ?29.2 0.4/14 2.12 0.24/14 0.29 0.02/14 0.82 0.13/13 A/5 t25.6 0.6/14 1.75 0.21/14 0.33 0.03/14 ? ? Fall (Boundary Bay) A/9 ?29.0 0.9/11 2.47 0.27/11 0.26 0.03/11 0.50 0.14/11 A/ 0.15 in all cases). There was a consistent positive relationship between TRIG and body mass in females (P < 0.02), but not males (P > 0.14) except possibly for males at Sidney Island (P = 0.06). Thus, males and fe- males were analyzed separately because the ef- fect of body mass differed between them. Within females, there were no differences in residual TRIG (controlling for bleed-time and body mass) between adults and juveniles at any stage (P > 0.23), and there were no age by mass interactions (P > 0.29). Migratory stage and body mass interacted significantly in fe- males (P = 0.01), because there was no rela- tionship between body mass and TRIG in Pan- ama samples (Fig. 1). Within the Panama samples, premigratory birds of both ages had higher TRIG than winter birds (P = 0.0001; Fig. 1). In migrants, residual TRIG varied among stages (F = 17.3, df = 2 and 62, P = 0.0001), and was higher in the fall at Sidney Island than dur- ing spring or fall at Boundary Bay (P = 0.0001), which did not differ (P > 0.48; Fig. 1). In males, ages classes could not be combined because juveniles had higher TRIG than adults at Boundary Bay (evident in Table 1; P = 0.03). TRIG did not differ between spring and fall mi- grating adult males (P = 0.68). Male juveniles stopping at Sidney Island could not be com- pared directly to Boundary Bay juveniles be- cause of a significant site by mass interaction (P = 0.035). However, juvenile males and females could be combined at Sidney Island with no sex by mass interaction (P = 0.58). Doing so con- ta 0.2 OC O ? -c 9 lij -0.6 a. BODY MASS (g) FIG. 1. Plasma triglyc?ride concentrations of fe- male Western Sandpipers at various sites and sea- sons plotted against body mass. In nonmigrating birds sampled in Panama during the winter (open circles, thin solid line) and premigratory periods (open triangles, thick solid line), triglyc?ride levels were not related to body mass, but were higher in premigrants (P = 0.0001). Triglyc?ride was signifi- cantly related to body mass in spring and fall mi- grants sampled at Boundary Bay (closed circles, long dashed line), and fall migrants sampled at Sidney Is- land (closed triangles, short dashed line). Control- ling for body mass, triglyc?ride concentrations were higher at Sidney Island than Boundary Bay (P = 0.0001). firmed our previous finding in females alone; TRIG was higher at Sidney Island than at Boundary Bay (P = 0.0001). Glycerol.?There was a negative relationship between GLYC and bleed-time (logj^GLYC = -0.22[logi?bleed-time] - 0.28; F = 26.4, df = 1 and 187, R^ = 0.12, P = 0.0001), and a consistent negative relationship between GLYC and body mass controlling for bleed-time (logjgGLYC = -0.24[logiobleed-time] - 0.011[mass] + 0.017; F = 9.8, df = 1 and 186, P = 0.002). Residual GLYC declined significantly with time of day in fall migrants (F = 3.1, df = 1 and 70, R^ = 0.12, P = 0.003), and tended to do so in all other migra- tory stages. Thus, we controlled for bleed-time, mass, and time of day in all analyses. There were no sex effects (P > 0.11) or sex by mass interactions (P > 0.21) at any migratory stage, or for the data set as a whole. There were also no effects of age (P > 0.32) or age by mass interactions (P > 0.26). Compared to the other sites. Boundary Bay birds were generally caught later in the day, when time of day ap- peared to have the strongest negative effect on GLYC. That caused a significant stage by time April 2002] Plasma Lipid Metabolites 441 500 550 600 650 700 750 800 850 TIME OF DAY (MINUTES) FIG. 2. Residual plasma B-OH-butyrate concen- tration (controlling for body mass) in wintering and premigrant female Western Sandpipers versus time of day (R2 = 0.36, P = 0.0008). Time is minutes from midnight (sunrise approximately 0630 = 390 min; 0900 = 540 min; 1300 = 780 min). of day interaction (P = 0.009). Analysing sites separately, we found no difference in GLYC be- tween spring and fall migrants (P = 0.91), or between winter and premigrant birds (P = 0.70). Considering only the overlap in sampling times between Sidney Island and Boundary Bay (before 1600) eliminated the site by time of day interaction (P = 0.49), and indicated no dif- ference in residual GLYC between the two sites (P = 0.58). B-OH-Buti/rate.?BUTY was not significantly related to bleed-time overall (F = 2.3, df = 1 and 97, R^ = 0.01, P = 0.14), or in any stage (P > 0.35), but was consistently negatively related to body mass (logioBUTY = ?0.036[mass] + 0.72; F = 15.3, df = 1 and 97, R^ = 0.14, P = 0.0002). Controlling for body mass, BUTY tend- ed to decline with time of day at all stages, but not significantly (F = 1.8, df = 1 and 126, P = 0.18). However, in Panama, where morning sampling was possible, a decline was readily apparent in the first half of the day (08:00- 13:00; F = 14.5, df = 1 and 26, R^ = 0.36, P = 0.0008; Fig. 2). We controlled for body mass and time of day in subsequent analyses. Sexes were combined because they did not differ (P = 0.57) and there was no sex by mass interaction (P = 0.30). There was a significant age by mass interaction in fall migrants (P = 0.01), but not among Panama samples (P > 0.53). Within Panama samples, BUTY did not differ among winter adults, winter juveniles, and premigration season juveniles (P > 0.18), however all three had higher BUTY than pre- migratory adults (evident in Table 1; P < 0.05). To compare Panama to Boundary Bay, we an- alyzed ages separately. BUTY did not differ among winter, premigration season juveniles and fall migrant juveniles (F = 1.1, df = 1 and 44, P = 0.34). In adults, fall data could not be included in the analysis because of a significant stage by mass interaction (P = 0.04). However, BUTY in premigrant adults was lower than spring migrant (P = 0.03) and winter adults (P = 0.05), which did not differ (P = 0.52). DISCUSSION Bleed-time, body mass and time of day can affect plasma lipid metabolite levels to an ex- tent that could alter the interpretation of field data. Those covariates can be controlled statis- tically, however, so that hypotheses regarding sex, age, season, or site can be tested. Our most important findings were that (1) whereas in captivity TRIG, BUTY, and GLYC were related to rate of mass change (Williams et al. 1999), only TRIG varied in a predictable manner given the expected patterns of mass change in the field, and (2) TRIG can differ significantly be- tween stopover sites, indicating differences in the rate of mass gain (see also Ydenberg et al. 2002). Effects of covariates.?Lipid metabolite con- centrations were influenced by several vari- ables and interactions (Table 2). TRIG and GLYC declined following capture, most likely due to capture stress rather than short-term fasting, given the short bleed-times (5 min) of most of our samples. A similar rapid decline in TRIG after capture has been found in several passerine species (Jenni-Eiermann and Jenni 1991, 1996; Jenni and Jenni-Eiermann 1996). In passerine birds, longer fasts (30-120 min) are characterized by decreased TRIG and increased B-OH-butyrate (Jenni-Eiermann and Jenni 1991,1996, 1997). Body mass was a significant covariate for ev- ery metabolite. The two measures of lipid uti- lization (GLYC, BUTY) decreased with increas- ing body mass, whereas TRIG was positively related to body mass (but not under all condi- tions). Those relationships could result from (1) baseline metabolite levels changing inherently with body mass or fatness or (2) heavier birds having elevated fat deposition rates. In captive Western Sandpipers, absolute body mass ex- 442 GUGLIELMO, O'HARA, AND WILLIAMS [Auk, Vol. 119 TABLE 2. Summary of the effects of several covariates and class variables on plasma lipid metabolite con- centrations. (0 = no trend, ? = uncertain, Y = yes, N = no). Effect Triglyc?ride Glycerol B-OH-Butyrate Covariates Bleed-time Body mass Time of day Negative Positive/O 0 Negative Negative Negative Class Variables 0 Negative Negative/? Sex Sex X mass Age Age X mass Stage Stage X mass 7 Y ? = N, (? = Y N Y Y N N N N N Y N N Y Y/N Y Y plained, respectively, 64 and 21% of the varia- tion in TRIG and BUTY, regardless of whether birds were gaining or losing mass at the time (Williams et al. 1999). TRIG was also positively related to body mass in captive Red Knots {Ca- lidris canutus; L. Jenni pers. comm.). Based on that information, we treated body mass as a confounding factor to be controlled by AN- COVA. Nevertheless, heavier birds could fatten at higher rates if they gain better access to food through social dominance, or have enhanced physiological capacity for fattening (e.g. Car- penter et al. 1993, Piersma et al. 1999). Studies of captive passerines indicate that body mass is not a significant covariate of metabolite concen- trations (Jenni-Eiermann and Jenni 1994), yet in the field, metabolite levels in passerines have sometimes been found to be related to body mass (Gannes 1999, Schaub and Jenni 2001). That suggests that heavier birds may fatten at higher rates (Schaub and Jenni 2001), but net- ting studies suggest the opposite (Schaub and Jenni 2000). Clearly, experimental study of this complex issue is needed. Metabolite levels can vary with time of day mainly as a function of a bird's feeding behav- ior. In day-feeding passerines, BUTY or GLYC may be elevated, and TRIG may be low in the morning following the overnight fast (Jenni and Jenni-Eiermann 1996; Jenni-Eiermann and Jenni 1991,1997; Swain 1992; Gannes 1999). Af- ter feeding opportunities become available, metabolites reflecting fasting decrease, and TRIG increases rapidly, often stabilizing within a few hours of dawn (Jenni and Jenni-Eiermann 1996, but see Marsh 1983). Western Sandpipers are thought to feed mostly during daylight, and consistent with an overnight fast, GLYC and BUTY decreased through the day. In con- trast, TRIG was unaffected by time of day, pos- sibly due to two factors. First, we had few early morning captures, and may have missed a morning increase. Second, at each site we stan- dardized our netting effort to catch at a similar stage of the tide each day, a factor which may have an overriding influence on feeding behav- ior. Foraging by forest dwelling passerines is linked with hght availability, and one might ex- pect a similar pattern of metabolic changes each day (Jenni and Jenni-Eiermann 1996). Western Sandpiper feeding is probably related to both light and tide height. Our data suggest that sandpipers recover from overnight fasting in a similar way each day, regardless of tide conditions, possibly by finding terrestrial or lit- toral invertebrates. Rapid fattening, and asso- ciated high TRIG, may be more dependent on tide and the availability of high-quality feeding habitat (mudflats). Sex and age.?Sex was not generally an im- portant factor determining circulating levels of lipid metabolites. Raw TRIG values appear to be 13-27% lower in males than females (Table 1), but some of that difference can probably be attributed to the smaller body size of males (Wilson 1994). Comparison is complicated by the fact that in males, TRIG generally did not depend on body mass, whereas in females the relationship was consistently positive. Howev- er, in the one case where the relationship was positive in both males and females (Sidney Is- land), there was no difference in mass-correct- ed TRIG, indicating that fat deposition rate was similar in the two sexes. Bird age appeared to have little effect on lip- id metabolite concentrations, except for BUTY April 2002] Plasma Lipid Metabolites 443 during the premigration period, and for TRIG in fall migrant males at Boundary Bay. BUTY was lower in adult birds during premigration, but that can most likely be attributed to the fact that adults were undergoing fattening at that time, rather than to age per se (see below). Ju- venile males had higher TRIG than adults at Boundary Bay, but that was not true of females. Thus, based on TRIG of feeding sandpipers, ju- veniles making their first migration appear to have similar fattening performance to adults. Seasonal variation.?Plasma levels of metabo- lites related to lipid utilization (GLYC and BUTY) were relatively low throughout the year, and were similar to concentrations recorded in fed or short-term fasted birds (Jenni-Eiermann and Jenni 1991, Swain 1992, Gannes 1999). Stage by mass interactions were relatively com- mon, making direct comparisons among some stages impossible. GLYC did not differ among seasons. BUTY was low at all stages, as might be seen in fed birds, but was significantly re- duced in premigratory adults. During February and March in Panama, adult sandpipers deposit fat, but at a much slower rate (0.09 g day^^; P. D. O'Hara unpubl. data) than birds at migratory stopovers (0.4-1.0 g day^i; Butler et al. 1997). How premigratory adults adjust their physiology or time and en- ergy budgets during that period to allow for a net gain in body fat is unclear. Low BUTY could indicate reduced lipid use during premigration to spare stored lipids, as has been suggested in other studies (Suarez et al. 1990, Carpenter et al. 1993, Jenni and Jenni-Eiermann 1996, Jenni- Eiermann and Jenni 1996). BUTY influences protein metabolism (Robinson and Williamson 1980, Le Maho et al. 1981), and low BUTY could increase protein use from the diet, allowing in- gested lipids to be stored more efficiently. A re- duced contribution of lipid to energy metabo- lism could be responsible for the moderate rate of fat deposition (and lack of change in lean body mass; Guglielmo 1999) during premigra- tion in Panama. TRIG was the most informative of the lipid metabolites we measured. There was a pro- nounced seasonal difference in the relationship between TRIG and body mass that made inter- pretation difficult. However, the changes in concentration of this metabolite followed a pat- tern one might predict from expected changes in fattening rate through the year. TRIG was lowest, and was unrelated to body mass, dur- ing the winter when sandpipers were not de- positing fat. TRIG increased during premigra- tion, but contrary to our hypothesis, TRIG did not differ between juveniles and adults at that time. That may relate to the fact that birds were not caught during the height of feeding, or that rate of fattening in adults was too low to be de- tected. TRIG was highest, and was affected by body mass during spring and fall stopover when sandpipers increase body mass at a high rate (Lindstr?m 1991, Butler et al. 1997). TRIG was higher during migration than during the breeding season in wild passerine species (Bairlein and Totzke 1992, Jenni and Jenni-Eier- mann 1996), and TRIG cycled in relation to en- dogenous mass changes in captive Garden Warblers (Totzke and Bairlein 1998). Landscape-level variation in stopover quality.? Several studies suggest that plasma metabo- lites can be used to assess the relative quality of stopover sites for refuelling migrants (Jenni- Eiermann and Jenni 1994, Jenni and Jenni-Eier- mann 1996, Williams et al. 1999). Our results, and those of Schaub and Jenni (2001) demon- strate, in a natural situation, that birds at dif- ferent stopover sites can have distinctly differ- ent metabolite profiles, indicating differences in fattening rates. The mudflat at Sidney Island is much smaller than Boundary Bay, and con- sists of a tidal lagoon bordered by forest and dune shrubs which provide excellent attack cover for hunting raptors. Sandpipers of three species (Calidris maun, C. minutilla, and C. pu- silla) consistently weigh less at Sidney Island (Lissimore et al. 1999). Ydenberg et al. (2002) recently explored potential explanations for the use of Sidney Island by sandpipers of low body mass, and proposed a trade-off hypothesis, whereby higher pr?dation risk at Sidney Island is offset by better feeding conditions and more rapid fattening. At some point, pr?dation risk, due to increased body mass (wing loading), outweighs the feeding benefits and birds leave Sidney Island. The TRIG data supported the prediction of the trade-off hypothesis that fat- tening rates are greater at Sidney Island than Boundary Bay (Ydenberg et al. 2002). Most pro- grams for the conservation of migratory shore- birds (e.g. Western Hemisphere Shorebird Re- serve Network, Ramsar Convention on Wetlands) focus on the protection of major stopover sites, such as Boundary Bay. Our re- 444 GUGLIELMO, O'HARA, AND WILLIAMS [Auk, Vol. 119 suits indicate that small, high-quality stopover sites also may be important, especially for in- dividuals with low nutrient stores. Plasma metabolite analysis offers unique in- sight into the dynamics of physiological state of wild birds, and should be considered a stan- dard tool for the avian biologist. Future re- search should be directed at (1) demonstrating in the field that plasma metabolite levels cor- relate with the rate of mass gain estimated in- dependently (Winker et al. 1992, Cannes 1999, Dunn 2000), and (2) the effects of diet nutrient composition on metabolite to mass change relationships. ACKNOWLEDGMENTS Many thanks to all of those who helped in the field, especially C. Burgos, J. Christians, J. Dussureault, O. Egeler, J. Ferrigan, A. Lang, M. Lemon, D. Lissimore, J. Moran, Y. Morbey, E. Moreno, G. Reardon, E. Reid, G. Robertson, J. Roy, B. Sandercock, R Shepherd, M. Smyth, R. Stein, S. Tapia, J. Terris and C. Tucker. We are grateful to R. W. Butler, R. Einer, L. Z. Cannes, L. Jenni, S. Jenni-Eiermann, M. Klaassen, M. Landys, D. B. Lank, S. R. McWilliams, and R. C. 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