Correction

ECOLOGYCorrection for “Omega-3 long-chain polyunsaturated fatty acidssupport aerial insectivore performance more than food quantity,”by Cornelia W. Twining, J. Thomas Brenna, Peter Lawrence,J. Ryan Shipley, Troy N. Tollefson, and David W. Winkler, whichappeared in issue 39, September 27, 2016, of Proc Natl Acad

Sci USA (113:10920–10925; first published September 16, 2016;10.1073/pnas.1603998113).The authors note that Fig. 3 appeared incorrectly. The cor-

rected figure and its legend appear below.

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Omega-3 long-chain polyunsaturated fatty acidssupport aerial insectivore performance more thanfood quantityCornelia W. Twininga,1, J. Thomas Brennab, Peter Lawrenceb, J. Ryan Shipleya, Troy N. Tollefsonc, and David W. Winklera

aDepartment of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY 14853; bDivision of Nutrition, Cornell University, Ithaca, NY 14853;and cResearch and Development, Mazuri Exotic Animal Nutrition, Purina Mills LLC, Gray Summit, MO 63039

Edited by Mary E. Power, University of California, Berkeley, CA, and approved August 2, 2016 (received for review March 9, 2016)

Once-abundant aerial insectivores, such as the Tree Swallow(Tachycineta bicolor), have declined steadily in the past severaldecades, making it imperative to understand all aspects of theirecology. Aerial insectivores forage on a mixture of aquatic andterrestrial insects that differ in fatty acid composition, specificallylong-chain omega-3 polyunsaturated fatty acid (LCPUFA) content.Aquatic insects contain high levels of both LCPUFA and their pre-cursor omega-3 PUFA, alpha-linolenic acid (ALA), whereas terres-trial insects contain much lower levels of both. We manipulatedboth the quantity and quality of food for Tree Swallow chicks in afull factorial design. Diets were either high-LCPUFA or low inLCPUFA but high in ALA, allowing us to separate the effects ofdirect LCPUFA in diet from the ability of Tree Swallows to converttheir precursor, ALA, into LCPUFA. We found that fatty acid com-position was more important for Tree Swallow chick performancethan food quantity. On high-LCPUFA diets, chicks grew faster, werein better condition, and had greater immunocompetence and lowerbasal metabolic rates compared with chicks on both low LCPUFAdiets. Increasing the quantity of high-LCPUFA diets resulted in im-provements to all metrics of performance while increasing the quan-tity of low-LCPUFA diets only resulted in greater immunocompetenceand lower metabolic rates. Chicks preferentially retained LCPUFA inbrain and muscle when both food quantity and LCPUFAwere limited.Our work suggests that fatty acid composition is an important di-mension of aerial insectivore nutritional ecology and reinforces theimportance of high-quality aquatic habitat for these declining birds.

aerial insectivores | omega-3 long-chain polyunsaturated fatty acids |nutritional ecology

Aerial insectivores, a paraphyletic group that includes theswallows, swifts, nightjars, and at least five different families

of flycatchers, were once abundant throughout both temperateand tropical regions. However, in the last half century, a numberof North American aerial insectivores across a diversity of fam-ilies and species, ranging from Common Nighthawks (Chordeilesminor) and Chimney Swifts (Chaetura pelagica) to Olive-sidedFlycatchers (Contopus cooperi) and Tree Swallows (Tachycinetabicolor), have undergone major declines (1, 2). For example,Tree Swallows, one of the best-studied model aerial insectivoretaxa in North America, have declined by 36% over the past 2 to 3decades (2). Experts have proposed several hypotheses, including(i) declines in aerial insects (3), (ii) habitat loss and degradation(3–5), (iii) environmental contaminants (6, 7), and (iv) climatechange and phenological mismatch (8, 9). Evidence exists tosupport all of these hypotheses, yet, at present, the exact causesof aerial insectivore declines remain unresolved, pointing to theneed for a more thorough understanding of all aspects of aerialinsectivore ecology.Past studies have documented the importance of food resources

for aerial insectivores, and numerous studies have suggested thataerial insectivore declines are linked to decreasing overall insectabundance (e.g., ref. 1). Research on Tree Swallows shows thatfood availability is linked with chick growth rates and fledging

success as well as egg size and composition (10, 11). Winkler et al.(11) found that environmental temperature had a strong effect onpatterns of Tree Swallow chick mortality, most likely through itseffect on insect activity levels. However, the sheer quantity of foodresources may not be the only important factor. Food quality andthe potential for mismatch between insect composition and thenutritional needs of aerial insectivores may also be importantdrivers of reproductive output and overall fitness for these birds(e.g., ref. 12).Food quality can be defined in many ways, including caloric

density, nutrient composition, and digestibility (13). Here, wefocus on differences in composition of macronutrients. Aerialinsectivores, like all animals, require organic compounds [e.g.,vitamins, amino acids, and fatty acids (FAs)] in addition to ele-mental nutrients (e.g., nitrogen, phosphorus, and calcium) togrow, develop, and complete their life cycles. Omega-3 long-chainpolyunsaturated FAs (LCPUFA), in particular the FAs docosa-hexaenoic acid (22:6n-3, DHA) and eicosapentaenoic acid (20:5n-3,EPA), are especially important organic compounds for mostanimals, affecting a range of important physiological processes,from immune function to vision and brain development (14).Birds and all other vertebrates must either consume EPA andDHA directly from diet or indirectly by consuming their molecularprecursor, the short-chain omega-3 PUFA, alpha linolenic acid(18:3n-3, ALA), and then converting ALA into EPA and DHA.The capability of any particular animal species to convert ALA tothe bioactive EPA and DHA depends on whether its diet containsEPA and DHA (14). Mammalian herbivores typically synthesize

Significance

Insect abundance is an important predictor of survival andperformance for many taxa of aerial insectivores, which forageon a mixture of aquatic and terrestrial insects that differ infatty acid composition, particularly omega-3 long-chain poly-unsaturated fatty acid (LCPUFA) content. We raised TreeSwallow (Tachycineta bicolor) chicks on either a high or lowquantity of feed with either high amounts of the LCPUFAfound in aquatic insects or an equivalent amount of the pre-cursor omega-3 PUFA, alpha-linolenic acid, but low LCPUFA.LCPUFA content was more important for Tree Swallow chickperformance than food quantity. Tree Swallows may be timingbreeding to coincide with the peak abundance of high-LCPUFAaquatic insects.

Author contributions: C.W.T. and D.W.W. designed research; C.W.T. and J.R.S. performedresearch; J.T.B., P.L., and T.N.T. contributed new reagents/analytic tools; C.W.T. and P.L.analyzed data; and C.W.T., J.T.B., P.L., J.R.S., T.N.T., and D.W.W. wrote the paper.

Conflict of interest statement: T.N.T. is employed at Purina Mills, LLC, which manufacturesthe product tested. The authors declare no other conflicts of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: cwt52@cornell.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1603998113/-/DCSupplemental.

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all EPA and DHA endogenously from ALA, whereas carnivoressuch as cats must obtain all of their DHA from diet (15). Theability of wild birds to synthesize DHA is not well characterized,but DHA concentrations are inversely related to mass (16). Forexample, DHA constitutes 12% of FAs in the muscles of HouseSparrows (Passer domesticus) (16), which are similar in size toTree Swallows, but can reach over 20% in Ruby-throated Hum-mingbird (Archilochus colubris) muscle (17).In the wild, aerial insectivores consume a combination of

terrestrial and aquatic insects (18), which differ in their FAcomposition (19). Aquatic insects contain much higher levels ofLCPUFA than do terrestrial insects, a difference driven by dif-ferences in the FA composition of aquatic and terrestrial primaryproducers (19, 20). Aquatic primary producers, such as diatomsand dinoflagellates, are rich in EPA and DHA (21), which can beincorporated into aquatic insect tissue (22). In contrast, vascularterrestrial plants contain little to no LCPUFA but do contain theirmolecular precursor ALA (14), which can be either incorporatedinto tissue or converted to LCPUFA to a minor degree by ter-restrial insects (19). As a consequence, from the perspective ofLCPUFA content, aquatic insects may constitute a higher-qualityfood for aerial insectivores than do terrestrial insects.However, because both aquatic and terrestrial insects contain

ALA, the relative value of aquatic insects depends on the ca-pacity of aerial insectivores to convert ALA into LCPUFA (19).The ability to elongate ALA into LCPUFA varies greatly acrosstaxa: Strict carnivores, such as cats (23, 24), and animals fromenvironments rich in LCPUFA, including most marine fish (25),have lost the ability to elongate ALA into LCPUFA and mustobtain them directly from diet. In contrast, terrestrial herbivoresappear to be relatively efficient at converting ALA to LCPUFA(26). The capacity of aerial insectivores to convert ALA toLCPUFA remains untested, but, as predators living around ri-parian areas with emergent aquatic insects rich in LCPUFA, theyappear likely to be limited by LCPUFA content in diet.The majority of past studies on avian FA requirements have

focused on domesticated herbivorous taxa, especially chickens(e.g., refs. 27 and 28). These studies found domestic hens to berelatively efficient at elongating ALA, EPA, and DHA (e.g., refs.27 and 28). Far fewer studies have experimentally manipulateddietary FA composition for wild birds (but see refs. 29–32).These studies have found that both dietary composition andelongation capacity of individual species affect avian FA com-position (32). However, with the exception of work by Pierce et al.(31) on Red-Eyed Vireos (Vireo olivaceus), these studies haveeither looked at seed- and fruit-eating passerines or fish-eatingseabirds. To our knowledge, no studies have explicitly examinedthe omega-3 FA requirements of any aerial insectivores. There-fore, we sought to understand the importance of food FA com-position for aerial insectivores by varying both food quality andquantity in a balanced factorial experimental design.In nature, the effects of food quality and quantity may be con-

founded because parents may provide chicks with an increasedquantity of food to make up for low-quality food. To address this,we experimentally manipulated both the quantity and FA com-position of food for wild-hatched nestling Tree Swallow chicks.Chicks were fed one of four diets: (i) a high-LCPUFA, high-quantity diet containing EPA and DHA (Hh); (ii) a low-LCPUFA,high-quantity diet containing high ALA and low omega-3LCPUFA (Lh); (iii) a high-LCPUFA, low-quantity diet (Hl); and(iv) a low-LCPUFA, low-quantity diet (Ll). We assessed size-specific growth rates, body condition, immunocompetence, andbasal metabolic rates (BMR) as metrics of performance. We alsodetermined the FA composition of brain and breast muscle tis-sue from a subset of chicks from each treatment group.

ResultsChicks on high-LCPUFA diets (Table S1) grew significantlymore rapidly than those on low-LCPUFA diets (Table S1) re-gardless of food quantity (ANOVA: treatment F3,128 = 59.889, P< 0.0001; Figs. 1 A and B and 2). Diet quality was more im-portant than quantity: Even Hl chicks grew significantly fasterthan did Lh chicks (Table S2 and Figs. 1B and 2). Among thehigh-LCPUFA groups, Hh chicks grew significantly faster thandid Hl chicks (Table S2 and Figs. 1B and 2). Among the low-LCPUFA groups, there were no significant differences betweenLl and Lh chicks (Table S2). There were no significant treatmentdifferences in head−bill or tarsus growth rates between treat-ments (ANOVA for head−bill: treatment F3,90 = 0.099, P = 0.96;ANOVA for tarsus: treatment F3,90 = 2.091, P = 0.107; Fig. 1C).Thus, the differences observed were in growth rates for mass, notstructural size.Chicks on high-LCPUFA diets were also in significantly better

condition (reflected by the ratio of mass to head−bill length andmass to tarsus length) than those on low-LCPUFA diets re-gardless of quantity (ANOVA for mass to tarsus: treatmentF3,115 = 225.673, P < 0.0001; ANOVA for mass to head−bill:treatment F3,115 = 276.462, P < 0.0001; Fig. 1D). Chicks on Hhwere in significantly better condition than were Hl chicks (TableS3), and Hl chicks were in significantly better condition than wereLh chicks (Table S3 and Fig. 1D). Among the low-LCPUFAgroups, chicks on Lh were in significantly better condition thanwere Ll chicks (Table S3).Chicks on high-LCPUFA diets had increased immunocompetence

compared with those on low-LCPUFA diets regardless of foodquantity (ANOVA: treatment F3,80 = 38.187, P < 0.0001; Fig. 1E).Even Lh chicks had significantly higher phytohemagglutanin(PHA) immune response ratios than did Ll chicks (Table S4 andFig. 1E). Among the high-LCPUFA groups, Hh chicks had sig-nificantly higher immune response ratios than Hl chicks (Table S4).Among the low-LCPUFA groups, there were no significant dif-ferences between Hl and Lh chicks (Table S4).Patterns in BMR were the reverse of those in immunocom-

petence (Kruskal−Wallis: χ2 = 7.941, df = 3, P < 0.047; Fig. 1 Eand F). Hh chicks had the lowest BMR whereas Ll chicks had thehighest BMR, and these differences were significant (Table S5and Fig. 1F). Hl chicks and Lh chicks had similar BMRs, whichwere not significantly different (Table S5 and Fig. 1F). We alsofound that Ll chicks had significantly higher BMR than Hlchicks, and Lh chicks had significantly higher BMR than Hhchicks (Table S5 and Fig. 1F).We found both treatment and tissue-based differences in FA

composition (Tables S6 and S7 and Fig. 3). Hh chicks had sig-nificantly higher percentages of EPA in brain, whereas chicks onboth high-LCPUFA diets had significantly higher percentages ofEPA in muscle (Kruskal−Wallis for brain EPA: χ2 = 7.567, df = 3,P = 0.056; Kruskal−Wallis for muscle EPA: χ2 = 9.088, df = 3,P = 0.028; Table S6 and Fig. 3). The percentage of DHA in brainwas significantly higher in Lh chicks compared with chicks oneither high-LCPUFA diet (Kruskal−Wallis: χ2 = 8.316, df = 3,P = 0.040; Table S6 and Fig. 3B). In contrast, the percentage ofDHA in muscle was highest in Hl chicks, but it was only signif-icantly higher than the percentage of DHA in muscle of Ll chicks(Kruskal−Wallis for muscle DHA: χ2 = 3.882, df = 3, P = 0.275;Table S6 and Fig. 3D). The percentage of total omega-3 FAs inbrain was also significantly higher in chicks on Lh diets comparedwith chicks on high-LCPUFA diets (Table S6). We found nosignificant differences in either the proportion of total omega-6FAs in either brain or muscle or the percentage of total omega-3FAs in muscle (Table S6). Muscle had significantly higher per-centages of EPA and total omega-6 FAs compared with brain,but it had similar percentages of total omega-3 FAs and signif-icantly less DHA (Table S6).

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DiscussionWe asked if food quality, in terms of LCPUFA, was as importantas food quantity for a model aerial insectivore species, the TreeSwallow. We manipulated food quantity and FA composition ina fully factorial design and assessed performance in Tree Swallowchicks by measuring changes in size (mass, head−bill length, andtarsus length), body condition, and differences in immunocom-petence and BMR at the conclusion of the experiment. Overall,we found strong evidence that LCPUFA content is as important, ifnot more important, than food quantity for aerial insectivores(Figs. 1 and 2). We also found significant differences in the FAcomposition of chicks on different diets, which suggested that thechicks preferentially retained EPA and DHA (Fig. 3).Hh chicks grew the fastest and were in the best condition,

whereas Ll chicks grew the slowest and were in the poorestoverall condition (Figs. 1 and 2). Interestingly, Hl chicks per-formed better than did Lh chicks (Figs. 1 and 2). Increasing thequantity of low-LCPUFA food had no effect on growth rates orcondition. Body mass and condition are two of the most im-portant predictors of Tree Swallow fledgling success and survivalin natural systems (33). Our results suggest that wild chicks withaccess to high-quality food resources, such as aquatic insects, arelikely to be in better condition than those with access to onlylower-quality terrestrial food resources. In addition, more high-quality food appears to further increase body mass and condi-tion, whereas more low-quality food does not.We found no significant differences in head−bill or tarsus growth

rates across treatments (Fig. 1C). This finding provides evidence thatthere is strong pressure to develop at a specific rate, even at the costof overall condition. Tree Swallows nestlings, like other passerines,suffer high mortality from predation (34), and, although bodymass and condition are strong predictors of survival after fledging,

there appears to be greater pressure to quickly reach fledgling sizeto be ready to fledge if threatened by predation (35, 36).We found that food quantity and quality had significant inter-

acting effects on immunocompetence, measured as PHA ratio,across treatments (Fig. 1E). In birds, PHA ratio is an indicator ofacquired T-cell proliferation and the ability to produce lymphocytesin response to pathogens (37). We found that Hh chicks had thehighest PHA ratios whereas Ll chicks had the lowest ratios and Hland Lh chicks had equivalent, intermediate PHA swelling responses.Our results suggest that wild Tree Swallow chicks with access tomore food, especially high-quality aquatic insects containing EPAand DHA, may be more likely to mount an effective immune re-sponse (3, 33). In addition to predators and food deprivation,pathogens are a significant source of early mortality in nestling TreeSwallows (38), and greater immunocompetence from higher foodquantity and quality likely increases Tree Swallow chick survival.Food quantity and quality also had significant interacting ef-

fects on BMR across treatments (Fig. 1F). Hh chicks had thelowest metabolic rates and Ll chicks had the highest BMR, eithermass-corrected or whole-organism (Table S5). Our low- andhigh-LCPUFA feeds had equal caloric content (Table S1), sodifferences in BMR are likely due to effects of feed FA compo-sition and total LCPUFA content, not total energy. The negativerelationship between total LCPUFA content of feed and BMRcould have resulted from costs of ALA elongation and desatura-tion to LCPUFA. For example, although feed for Hl and Hhchicks had the same FA composition, Hl chicks consumed lesstotal LCPUFA compared with Hh chicks, and thus may have re-quired additional energy to convert ALA into LCPUFA. Ourfindings agree with those of previous studies; for example, Pierceet al. (31) found that increasing unsaturated FAs in diet decreasedpeak metabolic rate for Red-Eyed Vireos. Across all treatments,our findings support the inverse relationship observed by Hulbert

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et al. (16) between avian body mass and both BMR and breastmuscle DHA across bird species.The FA composition of chicks provided evidence for both

dietary accumulation and preferential retention of the long-chain omega-3 FAs EPA and DHA (Fig. 3). In brain tissue, thepercentage of EPA was highest in Hh chicks, whose diet containedboth the highest percentage and the greatest total amount of EPA(Fig. 3A). Chicks on Hh and Hl diets had the highest percentagesof EPA in muscle tissue (Table S5 and Fig. 3C). The Lh and Lldiets may not have contained sufficient amounts of EPA to ac-cumulate dietary EPA, or Tree Swallows may be inefficient atconverting ALA to EP;. this suggests that EPA accumulation inthe Tree Swallow tissues may be based on dietary availability ofEPA, which is consistent with findings in other taxa (28).In brain tissue, the percentage of DHA was highest in Lh

chicks (Fig. 3B). This finding could have stemmed from eitherincreased elongation of ALA or preferential LCPUFA retentionin Lh chicks. We suggest a combination of elongation and pref-erential retention may have been at work: Lh chicks would havehad more energy to devote to elongation than did low-quantitychicks and would have had more non-LCPUFA FAs in diet topreferentially oxidize for fuel than did high-LCPUFA chicks. TreeSwallow muscle tissue had significantly less DHA than did brain(Table S6 and Fig. 3), potentially because phospholipid DHA is akey component of neural tissue (39). In muscle tissue, the pro-portion of DHA in muscle was highest in Hl chicks (Fig. 3D),which we suggest was due to preferential retention because Hlchicks were limited in energy but not in LCPUFA.Chicks on low-LCPUFA and/or low-quantity diets may have

either converted ALA to LCPUFA or preferentially retainedLCPUFA already present in tissue. Studies suggest that chickenembryos preferentially remove LCPUFA from yolk (27). How-ever, this does not appear to be the case with altricial chicks, suchas Barn Swallows (Hirundo rustica), which contain much less DHAat hatch than do precocial birds (40). We were unable to controlmaternal FA investment in eggs or parental feeding during the

chicks’ first few days of life, and all chicks originated from nestboxes on or near water. Thus, Hl and Lh chicks likely preferen-tially retained DHA from eggs and early life while oxidizing otherdietary fats for energy. In contrast, Hh chicks may paradoxicallyhave had lower tissue DHA concentrations precisely becauseDHA was abundant in diet, obviating the need to preferentiallyretain DHA.Past work on chickens found that higher levels of LCPUFA in

diet translated into increased proportions of LCPUFA in breastmuscle (e.g., ref. 28). We found that increasing the concentra-tions of LCPUFA in diet did not necessarily result in increasedLCPUFA content in Tree Swallow tissue. Instead, our findingson aerial insectivore chicks are closer to those of past studies onfreshwater zooplankton, which have found preferential retentionand biomagnification of LCPUFA compared with other FAsregardless of food quality (41). This suggests that there is strongpressure for aerial insectivores to obtain and retain LCPUFA inthe face of poor conditions. Our performance data suggest that,when food quantity or quality are low, saving LCPUFA for fu-ture use instead of burning them as fuel may result in lower bodymass and condition. Further studies using compound-specificstable isotope tracers (e.g., enriched δ13C) will be necessary todetermine if Tree Swallow chicks are able to convert ALA intoLCPUFA and thus whether LCPUFA are beneficial or abso-lutely essential components of diet.Previous work on Tree Swallows has attempted to link Tree

Swallow breeding season and nestling success with food avail-ability (9, 11). Our findings suggest that the abundance of high-quality aquatic insects relative to Tree Swallow phenology maybe a better predictor of breeding success than overall insectabundance. Aquatic insect abundance peaks earlier than ter-restrial insect abundance (42), and aquatic insects are often theonly available food early in the breeding season (43). Total insectabundance peaks later in the breeding season, yet Tree Swallowscomplete laying long before peak insect abundance, and theirbreeding success decreases with lay date (9). Ecologists havegenerally interpreted these findings to indicate that laying, al-though earlier than peak insect abundance, places chick rearing,thought to be the most energy-demanding phase of the breedingcycle, at a time of peak food availability (44). Our findings sug-gest an alternative interpretation, that Tree Swallows, and otherbirds, may be under selection to time their breeding seasonswhen insects high in LCPUFA are most available.Our findings have significant implications for aerial insectivore

conservation. Most North American aerial insectivores, includingthe Tree Swallow, are associated with aquatic or riparian habitats(43). We found evidence that feed containing LCPUFA repre-sentative of aquatic insects improves multiple metrics of TreeSwallow performance and that they preferentially retain thesehigh-quality fats. Our study suggests that large quantities of ter-restrial insects low in LCPUFA are, at best, no better than evensmall amounts of aquatic insects, even if they have high amounts ofthe LCPUFA precursor ALA. Land conservation is not enough foraerial insectivores to survive and thrive: Managers must conserveaquatic habitats that provide aerial insectivores with the highest-quality LCPUFA-rich aquatic insects.

MethodsWe collected 44wild Tree Swallow chicks from nest boxes around Ithaca, NewYork, from 29May 2015 to 7 June 2015. To prevent parental abandonment ofchicks, we removed all chicks from each nest box. All animal work was ap-proved under Cornell Institutional Animal Care and Use Committee protocol2001-0051, New York State Department of Environmental ConservationScientific Collection Permit 1477, and United States Fish and Wildlife ServiceMigratory Bird Scientific Collection Permit 757670.

Upon return to the laboratory, we weighed and sorted chicks into groupsof three to four birds to receive one of four feeding treatments: (i) Hh; (ii) Lh;(iii) Hl; and (iv) Ll. The high- and low-LCPUFA diets were not significantly dif-ferent in calories, moisture, crude protein, or crude fat (Table S1) and differed

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Fig. 2. Chick mass over time. Treatment means and SE bars are shown. Blackcircles represent our Hh treatment, gray circles represent our Hl treatment,black triangles represent our Lh treatment, and gray triangles represent ourLl treatment.

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only in FA composition (Table S1). All diets were based upon standard com-mercial Mazuri nestling feed (www.mazuri.com/mazurihandfeedingdiets-1.aspx). Standard nestling diets contained soybean oil as their principal fatsource. Our high-LCPUFA diets included a substitution of stabilized menhadenoil for soybean oil in a ratio of 7:3, and low-LCPUFA diets included a sub-stitution of flax oil for soybean oil in a ratio of 1:3. The resulting high-LCPUFAdiets contained ∼1.82% ALA, 3.74% EPA, and 3.44% DHA, and low-LCPUFAdiets contained ∼6.25% ALA, 1.47% EPA, and 1.42% DHA (Table S1). Low-quantity chicks were fed 4.5% of body mass per feeding session (the point atwhich begging still occurred at the end of the session), and high-quantity chickswere fed 6% of body mass per session (produced chick satiation at the end ofthe feeding session) of body mass per feeding. We grouped chicks of similarinitial sizes together in nest groups so as to avoid underfeeding or overfeedingindividual chicks, and we randomly split up chicks from the same clutch to avoidgenetic effects. There were multiple replicates of each food quality andquantity treatment that covered the full range of initial chick masses.Additional details on diets and care are described in SI Methods.

Each chick was weighed four times daily with an Ohaus Scout Pro bal-ance, and the average of that mass was used for calculations. We alsomeasured the head−bill and tarsus length of each chick to the nearest0.01 mm a minimum of two times over the course of the experiment, withMitutoyo Digimatic 500 calipers. Growth rates were calculated as [ln(massor length on day x) – ln(mass or length on day 0)]/(day x – day 0), and bodycondition was calculated as both the ratio of mass to head−bill length andthe ratio of mass to tarsus length. To measure immunocompetence, weused the simplified protocol described by Smits et al. (45) and detailed in SIMethods. To determine BMR, we used an open-flow pull-mode FoxBoxrespirometry setup coupled with a climate-controlled chamber at a flowrate of ∼490 mL/min following the methods of Lighton (46) and detailed inSI Methods.

We determined the whole-tissue FA composition of brain and pectoralmuscle for a subset of chicks from each treatment (n = 4). After euthanasia,we dissected and weighed out brain and pectoral muscle samples from fourchicks per treatment. FA methyl esters (FAMEs) were extracted from whole

tissues using a modified one-step method (47, 48) and quantified using a BPX-70 (SGE Inc.) column and an HP5890 series II gas chromatography flame ioni-zation detector. Chromatogram data were processed using PeakSimple. Responsefactors were calculated using the reference standard 462a (Nucheck prep).FAMEs were identified using a Varian Saturn 2000 ion trap with a Varian Star3400 gas chromatography mass spectrometer run in chemical ionization massspectrometry mode using Acteonitrile as reagent gas. FA composition dataare expressed as percent of total FA. We also calculated total omega-3 PUFAsand total omega-6 PUFAs.

We analyzed mass, size-specific growth rates for mass, tarsus length, size-specific growth rates for tarsus length, head−bill length, size-specific growthrates for head−bill length, the ratio of mass to tarsus length, the ratio ofmass to head−bill length, and PHA ratio through ANOVA, using treatmentgroup (the interaction of LCPUFA content and food quantity: Hh, Hl, Lh, andLl), nest, and individual as predictor variables. For all performance metricsexcept PHA ratio, which was only measured at the end of the experiment,we also ran analysis of covariance with experiment date as a covariate. Weused post hoc Tukey tests to interpret the direction and significance ofdifferences between treatment groups for variables that were significant asmain effects and assessed relative support between models using Aikaike’sInformation Criterion. To detect differences in our smaller datasets on BMRsand brain and muscle FA composition, we used nonparametric Kruskal−Wallis tests and performed Dunn tests to perform pairwise comparisonsbetween treatment groups (Hh, Hl, Lh, and Ll). We also compared differ-ences between brain and muscle fatty acid composition using Welch’s two-sample t tests. All statistical analyses were performed in R (version 3.2.2).

ACKNOWLEDGMENTS. The authors thank Sara Gonzalez for assistance withchick rearing and Rachel Corona and Vivien Ikwuozom for assistance withfatty acid analyses. D.W.W. acknowledges funding from National ScienceFoundation (NSF) Long Term Research in Environmental Biology Grant1242573, and C.W.T. acknowledges funding from the Cornell Lab of Ornithol-ogy Athena Fund, NSF Division of Environmental Biology Doctoral DissertationImprovement Grant 1500997, and fellowship support through the NSF GraduateResearch Fellowship Program.

0.15

0.20

0.25

0.30

Hh Hl Lh LlTreatments

Brain

EPA

(% to

tal f

atty

acid

s)

1.0

1.2

1.4

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Musc

le EP

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tota

l fat

ty ac

ids)

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High LowFood Quantity

Mass

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wth

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ay)

7

8

9

10

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Musc

le DH

A (%

tota

l fat

ty ac

ids)

A B

DC

Fig. 3. Fatty acid composition results for (A) brain EPA, (B) brain DHA, (C) muscle EPA, and (D) muscle DHA. Treatment means and SE bars are shown. Black circlesrepresent our Hh treatment, gray circles represent our Hl treatment, black triangles represent our Lh treatment, and gray triangles represent our Ll treatment.

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