ABSTRACT

Food availability and quality are both critical for growing young animals. In nature, swallows (Tachycineta bicolor) and other aerial insectivores feed on both aquatic insects, which are rich in omega-3 highly unsaturated fatty acids (HUFAs), and terrestrial insects, which contain considerably lower amounts of omega-3 HUFAs. Carnivorous mammals and fishes must obtain omega-3 HUFAs from their diet, as they have lost the capacity to convert the precursor omega-3 α-linolenic acid (ALA) into omega-3 HUFAs. Thus, the relative value of aquatic versus terrestrial insects depends not only on the fatty acid composition of the prey but also on the capacity of consumers to convert ALA into omega-3 HUFAs. We used a combination of stable-isotope-labeled fatty acid tracers to ask whether, and how efficiently, tree swallows can deposit newly synthesized omega-3 HUFAs into tissue. Our data show for the first time that tree swallows can convert ALA into omega-3 HUFAs deposited in liver and skeletal muscle. However, high tree swallow demand for omega-3 HUFAs combined with low ALA availability in natural terrestrial foods may strain their modest conversion ability. This suggests that while tree swallows can synthesize omega-3 HUFAs de novo, omega-3 HUFAs are ecologically essential nutrients in natural systems. Our findings thus provide mechanistic support for our previous findings and the importance of omega-3 HUFA-rich aquatic insects for tree swallows and most likely other aerial insectivores with similar niches.

INTRODUCTION

Although dietary resources are crucial for animals throughout their life cycle, energy and nutrients are particularly critical during early life, especially for animals, like birds, that undergo rapid determinate growth. For wild birds with young that reach mature size rapidly, timing breeding phenology with food availability is essential for survival (Lyon et al., 2008). Climate change is already creating mismatches between the phenology of insect prey and the timing of breeding of insectivores like pied flycatchers (Ficedula hypoleuca; Both et al., 2006) and great tits (Parus major; Nussey et al., 2005). However, even if food resources are available during the breeding season, food availability in terms of energy alone may not be sufficient for successful breeding if available foods lack key nutrients. Recent studies suggest that there is also the potential for mismatches between the nutritional composition of available food resources and the complex nutritional needs of growing chicks (Twining et al., 2016a; C.W.T., J.R.S. and D.W.W., unpublished).

Food quality can be defined in many ways, including caloric density, nutrient composition and digestibility. Previous studies focusing on food quality in terms of fatty acids have found the ratio of omega-3:omega-6 fatty acids (e.g. Hulbert et al., 2005; Arnold et al., 2015) and omega-3 highly unsaturated fatty acids (HUFAs; Twining et al., 2016b) to be especially important drivers of animal performance and health. Here, we focused on food quality in terms of the availability of omega-3 HUFAs because we previously found it to be more important at low levels for insectivore chick growth performance than was food availability (Twining et al., 2016a). Omega-3 HUFAs, in particular the fatty acids DHA (docosahexaenoic acid, 22:6n-3) and EPA (eicosapentaenoic acid, 20:5n-3), are important organic compounds for most animals: they affect a range of important physiological processes from immune function through their role in eicosanoid and docosanoid signaling (Jump, 2002) to serving as the building blocks for vision and brain tissue (Brenna and Carlson, 2014). Dietary omega-3 and omega-6 fatty acids also influence cell membrane composition (Hulbert et al., 2005) with the potential to alter major thermoregulatory processes, such as hibernation (Arnold et al., 2015). Dietary omega-3 HUFAs may also be particularly important for the exercise performance of migratory birds by increasing the oxidative capacity of flight muscle (e.g. McWilliams et al., 2004; Guglielmo, 2010).

A major dichotomy in omega-3 HUFA availability exists between aquatic and terrestrial ecosystems: omega-3 HUFAs are abundant at the base of aquatic food webs, and aquatic insects incorporate omega-3 HUFAs from aquatic primary producers into tissues (Twining et al., 2016b; Torres-Ruiz et al., 2007). In contrast, terrestrial plants contain little to no omega-3 HUFAs, though they contain their molecular precursor ALA (Hixson et al., 2015; Twining et al., 2016b), which is either incorporated into tissue or to a minor degree converted to omega-3 HUFAs by terrestrial insects (Blomquist et al., 1991). Terrestrial seeds and the animals that consume them also have a higher proportion of their fatty acids as omega-6 fatty acids (Hixson et al., 2015). As a consequence of these differences at the base of food webs, aquatic insects are richer in omega-3 HUFAs than terrestrial insects and also have higher ratios of omega-3:omega-6 fatty acids compared with terrestrial insects (Hixson et al., 2015). In the wild, avian insectivores in riparian areas, such as tree swallows (Tachycineta bicolor), which are considered a model insectivore species, consume a mix of terrestrial and aquatic insect prey items (McCarty and Winkler, 1999; Winkler et al., 2013). Our recent studies show that differences in omega-3 HUFA availability found between aquatic and terrestrial insects (Hixson et al., 2015) can have strong consequences on tree swallow chick performance in the laboratory (Twining et al., 2016a) and that the composition of insects can have significant effects on tree swallow breeding success in the field (C.W.T., J.R.S. and D.W.W., unpublished).

Ultimately, the nutritional value of aquatic versus terrestrial insects depends not only on the fatty acid composition of these insects but also upon the capacity of tree swallows and other insectivores to convert ALA into omega-3 HUFAs. While strict carnivores, such as wild and domesticated cats, have lost the ability to elongate and desaturate sufficient amounts of ALA for their omega-3 HUFA needs and must obtain omega-3 HUFAs directly from their diet (e.g. Rivers et al., 1975; MacDonald et al., 1984; Davidson et al., 1986; Morris, 2002), omnivorous animals, from humans to many species of birds, can obtain omega-3 HUFAs through two pathways: either (1) directly by consuming food containing EPA and DHA, or (2) indirectly by consuming their molecular precursor, the shorter chain omega-3 polyunsaturated fatty acid (PUFA) α-linolenic acid (18:3n-3, ALA), and then converting ALA into omega-3 HUFAs through the biochemical processes of elongation and desaturation (Brenna and Carlson, 2014). For example, domestic chickens (Gallus domesticus), which are omnivorous but consume mainly terrestrial vascular plant-based foods in captivity, appear to be relatively efficient at ALA to omega-3 HUFA conversion (e.g. Cherian and Sim, 1991; Newman et al., 2002; Zdunczyk and Jankowski, 2013), with the capacity to survive and reproduce on omega-3 HUFA-free diets. In domestic chickens, tissue omega-3 HUFA content increases with increased dietary ALA (Linn et al., 1991). In addition, increasing ALA in maternal diet through foods such as flax seeds also increases omega-3 HUFA content in eggs, embryos and newly hatched chicks (Cherian and Sim, 1991).

Studies on wild birds have found that both nutritional requirements and foraging ecology affect avian tissue fatty acid composition (Pierce et al., 2005; Maillet and Weber, 2006; McCue et al., 2009). Most studies on wild birds (but see McWilliams et al., 2002; Pierce et al., 2005) have looked at either seed- and fruit-eating passerines (e.g. McCue et al., 2009), which consume a diet much lower in omega-3 HUFAs, or fish- and shellfish-eating shorebirds, which consume a diet much higher in omega-3 HUFAs than do riparian aerial insectivores (e.g. McWilliams et al., 2004). Furthermore, past studies on avian fatty acid requirements have made inferences about conversion efficiency indirectly based on the effects of dietary fatty acid content on exercise and/or developmental performance (e.g. Pierce et al., 2005; Twining et al., 2016b), and tissue omega-3 HUFA content (e.g. McWilliams et al., 2004; Guglielmo, 2010), but they have not definitively established whether the ALA to omega-3 HUFA conversion is metabolically feasible, unlike studies on humans and other mammals (Brenna et al., 2009).

Apart from obligate carnivores, animals generally retain the metabolic capacity to endogenously biosynthesize omega-3 HUFAs from precursors based in part on the omega-3 HUFA content of the diets that they have consumed over their evolutionary history. Thus, terrestrial herbivores consuming high ALA, low omega-3 HUFA terrestrial primary plants must endogenously synthesize effectively all of their omega-3 HUFAs, while carnivores can obtain all the omega-3 HUFAs in their diets by taking advantage of the ALA to omega-3 HUFA conversion performed at lower trophic levels (Castro et al., 2012; Brenna and Carlson, 2014). On this basis, tree swallows and other riparian aerial insectivores should have a limited capacity to convert ALA into the omega-3 HUFAs because they evolved with access to aquatic insects, obviating the need to maintain efficiency in this metabolic pathway. We previously found that omega-3 HUFAs have strong effects on tree swallow growth: chicks on higher relative omega-3 HUFAs to ALA feeds, designed to represent wild diets higher in aquatic insects, grew faster, were in better condition, and had increased immunocompetence and decreased metabolic rates compared with chicks on lower relative omega-3 HUFAs to ALA feeds designed to represent wild diets dominated by terrestrial insects (Twining et al., 2016a). In a long-term field study, we also that found aquatic insects, which, unlike terrestrial insects, are rich in omega-3 HUFAs, are a strong driver of long-term tree swallow fledging success in nature (C.W.T., J.R.S. and D.W.W., unpublished). Together, these findings suggest that the ALA to omega-3 HUFA conversion, if present, is likely to be inefficient in tree swallows.

To understand the physiological importance of omega-3 HUFAs for riparian aerial insectivores, we used rapidly growing tree swallow chicks as a model to ask: first, are birds that regularly consume high omega-3 HUFA dietary resources able to convert ALA into omega-3 HUFAs or are dietary omega-3 HUFAs strictly essential nutrients? Second, if birds with access to high omega-3 HUFA resources are able to convert ALA into omega-3 HUFAs, is their conversion and tissue deposition efficiency high enough to provide them with enough omega-3 HUFAs from dietary ALA in natural settings to avoid performance limitation or are dietary omega-3 HUFAs ecologically essential nutrients? We used enriched 13C stable isotope fatty acid tracers to trace the metabolic pathway of ALA through tree swallow chicks, adapting this method from studies on humans and small mammals, to directly quantify the ALA to omega-3 HUFA conversion for the first time in wild birds.

MATERIALS AND METHODS

We examined the ALA conversion capacity and efficiency in seven wild tree swallow chicks, Tachycineta bicolor (Vieillot 1808), from two sites near Ithaca, NY, USA (site 1: 42.515459°N, 76.335272°W, site 2: 42.504434°N, 76.465949°W). At both sites, we briefly removed chicks from the nest and fed them olive oil with or without dissolved δ13C-enriched ALA (Cambridge Isotope Laboratories, Cambridge, MA, USA) via syringe. Six chicks were dosed with a δ13C-enriched ALA tracer, serving as treatment chicks, and one was not dosed with a δ13C-enriched ALA tracer and served as a natural abundance δ13C control. At the time of dosing, all chicks were approximately 7 days old and had a mean (±s.d.) mass of 10.687±1.204 g. Chicks of this age can only be sexed with genetic tests. We dissolved 5 mg of δ13CALA in 2.5 ml of olive oil creating a 10 mg ml−1 solution of δ13CALA in olive oil. Each treatment chick received 0.25 ml of olive oil with dissolved δ13CALA followed by 0.25 ml of olive oil without tracer using the same syringe. The control chick received two 0.25 ml syringes of olive oil that did not come into contact with the tracer. The use of a single chick as a control is justified because of the low variability of natural isotope ratios (standard deviation of less than 1‰: Table S1) compared with the enrichment achieved by tracer administration of >100‰).

After dosing the chicks, we labeled them with non-toxic children's nail varnish before returning them to their nests for parental care and feeding. All chicks were killed at approximately 48 h post-dose as per United States Fish and Wildlife Service migratory bird scientific collection permit no. MB757670 and New York State Department of Environmental Conservation scientific collection permit no. 1477. All animal work was approved under Cornell Institutional Animal Care and Use Committee no. 2001-0051. Chicks were killed in the field by cervical dislocation; their livers and pectoral muscles were then removed for analysis.

Next, we performed compound-specific δ13C analysis and fatty acid composition analysis. Briefly, we extracted liver and pectoral muscle fatty acids and derivatized them to fatty acid methyl esters (FAMEs) using a modified one-step method (Garces and Mancha, 1993). We quantified fatty acid composition using a BPX-70 (SGE Inc., Troy, NY, USA) column and a HP5890 series II gas chromatograph-flame ionization detector (GC-FID). Chromatogram data were processed using PeakSimple. Response factors were calculated using the reference standard 462a (Nucheck prep). FAMEs were identified using a Varian Saturn 2000 ion trap with a Varian Star 3400 gas chromatography mass spectrometer run in chemical ionization mass spectrometry mode using acetonitrile as the reagent gas. We used gas chromatography combustion isotope ratio mass spectrometry (GCC-IRMS) to measure the δ13C signatures of ALA, EPA and DHA (Goodman and Brenna, 1992; Plourde et al., 2014). Briefly, an Agilent 6890 GC was interfaced to a Thermo Scientific 253 isotope-ratio mass spectrometer via a custom-built combustion interface. Peaks were confirmed to be baseline separated, and calibrated against working standards with isotope ratios traceable to international standards calibrated to VPDB (Caimi et al., 1994; Zhang et al., 2009).

Conversion from ALA to omega-3 HUFAs requires a complex series of biochemical reactions of varying efficiency in each organ, including transport across and deposition into membranes as well as transport between organs. From measurement of the amount of labeled omega-3 HUFAs in tissue, we derive an apparent conversion efficiency (CE) reflecting all processes leading to the deposition of newly synthesized omega-3 HUFAs. This parameter, derived from experimental measurements, can also be understood as net deposition of labeled omega-3 HUFAs in tissue, rather than a mechanistic conversion rate that would be measured for a single enzymatic reaction. In experimental animals, this process is sensitive in the short term to diet, with lower expression of genes involved in fatty acid conversion with omega-3 HUFA feeding, as well as subject to competition as a result of the various fatty acids in the diet. We assessed ALA to EPA and DHA conversion efficiency by calculating conversion levels according to Sheaff et al. (1995). Atom percentage excess (APE) was calculated as:
formula
(1)
where atom percentage is calculated directly from the measured δ13CX, X is a fatty acid (ALA, EPA, DPA, DHA), and tracer and control indicate chicks dosed or not dosed with tracer.
Total label, or the concentration of tracer per unit mass of tissue, was calculated as:
formula
(2)
Finally, we calculated a tissue-specific apparent CE for fatty acids as:
formula
(3)
where omega-3 HUFA refers to either DHA or EPA.

To understand how ALA-derived EPA and ALA-derived DHA accumulation varies between tissues where conversion activity occurs (i.e. liver) and those where it is expected to be lower (i.e. pectoral muscle), we divided liver CE by muscle CE for both EPA and DHA.

To understand the significance of our measured ALA CE on performance, we applied our calculations of apparent CE to the mean ALA content of nestling bird feeds and wild aquatic and terrestrial insects to estimate potential EPA and DHA synthesis. We calculated potential EPA and DHA (omega-3 HUFA) synthesis from ALA as:
formula
(4)
(where LC is long chain), and potential total EPA and DHA (omega-3 HUFA) as:
formula
(5)
We then calculated the fraction of EPA and DHA (omega-3 HUFA) from diet versus ALA conversion for each prey item as:
formula
(6)
formula
(7)

To understand how ALA-derived EPA and ALA-derived DHA accumulation varies between tissues where conversion activity occurs (i.e. liver) and those where it is minimal (i.e. pectoral muscle), we divided liver CE by muscle CE for both EPA and DHA.

To understand the potential impact of our measured ALA CE on growth performance, we applied our calculations of CE to the mean ALA content of nestling bird feeds and wild aquatic and terrestrial insects to estimate potential EPA and DHA synthesis. Insects were collected from eight sites around Ithaca, NY, USA, using a combination of emergence traps, pan traps and targeted hand-netting. We determined insect fatty acid composition following the same methods as described above for tree swallow tissue.

We calculated each of these measures for the following food items: (1) aquatic mayflies (Ephemeroptera, Heptageniidae), (2) aquatic stoneflies (Plecoptera, Perlidae), (3) aquatic dragonflies (Odonata, Anisoptera), (4) terrestrial beetles (Coleoptera), (5) terrestrial flies (Diptera, Brachycera), (6) terrestrial moths and butterflies (Lepidoptera), and (7) terrestrial bees (Hymenoptera, Apidae).

We compared stable isotope values between our treatment chicks (n=6) and control chick (n=1) using one-sample t-tests for both muscle and liver using control chick values as µ0. We used paired two-sample t-tests to compare CE and tissue deposition between liver (n=6) and muscle (n=6) for treatment chicks. To compare raw ALA and EPA, potential ALA-derived EPA and DHA, and total potential EPA and DHA between aquatic and terrestrial insects, we also used two-sample t-tests. We also used general linear models (GLM) to compare raw ALA and EPA, potential ALA-derived EPA and DHA, and total potential EPA and DHA between all insect groups using insect group as a factor. We performed post hoc Tukey contrasts on all GLMs to determine which insect groups were significantly different from each other. All statistical analyses were performed in R version 3.3.3 (R Core Team).

RESULTS

We first asked whether omega-3 HUFA synthesis supports our previous results showing that omega-3 HUFAs are key nutrients for tree swallows. We found evidence that tree swallow chicks can derive long chain PUFA from ALA: δ13CEPA and δ13CDHA values of liver and muscle from chicks fed δ13C-enriched ALA were significantly higher than controls (one-sample t-tests: t=4.62, d.f.=5, P<0.01 for liver; t=5.75, d.f.=5, P<0.01; Table 1). Although all chicks fed δ13C-enriched ALA showed evidence of ALA to omega-3 HUFA conversion and deposition of ALA-derived omega-3 HUFAs in tissues, we found substantial individual variation in CE between individuals across both field sites, especially for DHA (Table 1). We found that ALA-derived EPA and ALA-derived DHA were significantly higher in liver (ratio of 2.37 for EPA and 3.68 for DHA), where most conversion activity is likely to occur, than in pectoral muscle, where omega-3 HUFAs are deposited (two-sample t-test: t=6.80, d.f.=6.61, P<0.01 for EPA; t=2.55, d.f.=5.54, P<0.05 for DHA; Table 1).

Table 1.

Conversion efficiency of α-linolenic acid (ALA) to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in tree swallow chick liver and pectoral muscle

Conversion efficiency of α-linolenic acid (ALA) to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in tree swallow chick liver and pectoral muscle
Conversion efficiency of α-linolenic acid (ALA) to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in tree swallow chick liver and pectoral muscle

We next asked whether omega-3 HUFAs are ecologically essential nutrients for tree swallows based on measured CEs and tissue deposition levels combined with the fatty acid composition of potential insect prey (Table 2). Aquatic insects, especially mayflies and stoneflies, had much higher percentages of raw EPA than did terrestrial insects (two-sample t-test for aquatic versus terrestrial EPA: t=8.05, d.f.=13.96, P<0.01; Table 3), while terrestrial insects, especially terrestrial moths, butterflies and bees, had much higher percentages of ALA than did aquatic insects or terrestrial beetles and terrestrial flies (Fig. 1A; two-sample t-test for aquatic versus terrestrial ALA: t=−2.08, d.f.=42.81, P<0.05; Table 3).

Table 2.

Potential EPA from ALA, potential DHA from ALA, and potential total EPA at mean, maximum and minimum conversion efficiency

Potential EPA from ALA, potential DHA from ALA, and potential total EPA at mean, maximum and minimum conversion efficiency
Potential EPA from ALA, potential DHA from ALA, and potential total EPA at mean, maximum and minimum conversion efficiency
Table 3.

Generalized linear model (GLM) results for raw ALA and EPA in insects

Generalized linear model (GLM) results for raw ALA and EPA in insects
Generalized linear model (GLM) results for raw ALA and EPA in insects
Fig. 1.

Meanfatty acid (FA) contentand potential mean FA content from conversion of ALA to omega-3 HUFAs of different food sources. (A) α-Linolenic acid (ALA, 18:3n-3), docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3) content from food sources. (B) Potential EPA and DHA from conversion of ALA to omega-3 highly unsaturated fatty acids (HUFAs). (C) Potential total ALA, EPA and DHA content from food sources and conversion of ALA to omega-3 HUFAs. Values shown are means of pooled biological replicates: aquatic mayflies n=23, aquatic dragonflies and damselflies n=6, aquatic stoneflies n=11, terrestrial beetles n=20, terrestrial flies n=24, terrestrial bees n=22, terrestrial moths and butterflies n=24.

Fig. 1.

Meanfatty acid (FA) contentand potential mean FA content from conversion of ALA to omega-3 HUFAs of different food sources. (A) α-Linolenic acid (ALA, 18:3n-3), docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3) content from food sources. (B) Potential EPA and DHA from conversion of ALA to omega-3 highly unsaturated fatty acids (HUFAs). (C) Potential total ALA, EPA and DHA content from food sources and conversion of ALA to omega-3 HUFAs. Values shown are means of pooled biological replicates: aquatic mayflies n=23, aquatic dragonflies and damselflies n=6, aquatic stoneflies n=11, terrestrial beetles n=20, terrestrial flies n=24, terrestrial bees n=22, terrestrial moths and butterflies n=24.

We estimated that tree swallows could derive significantly more potential EPA and DHA from ALA in terrestrial moths, butterflies and bees than from other terrestrial or aquatic insects (Fig. 1B, Table 4). However, total EPA (raw EPA plus potential EPA from ALA) from aquatic insects was still significantly higher than the largely ALA-derived total EPA from terrestrial moths, butterflies and bees (two-sample t-test for aquatic versus terrestrial total EPA: t=8.08, d.f.=13.85, P<0.01; Fig. 1C, Table 5). Aquatic insect EPA was derived primarily from diet (Fig. 1) because aquatic insects had significantly higher raw EPA values than those of any terrestrial insects (Fig. 1). Unlike aquatic insects, terrestrial insects, especially terrestrial moths, butterflies and bees, had the potential to provide tree swallows with EPA primarily from ALA conversion (Fig. 1).

Table 4.

GLM results for potential ALA-derived EPA and DHA in insects

GLM results for potential ALA-derived EPA and DHA in insects
GLM results for potential ALA-derived EPA and DHA in insects
Table 5.

GLM results for potential total ALA and EPA in insects

GLM results for potential total ALA and EPA in insects
GLM results for potential total ALA and EPA in insects

In contrast to our findings for EPA, aquatic insects contained only trace amounts of DHA and no terrestrial insects contained any detectable DHA (Fig. 1A). Therefore, regardless of CE, over 95% of total DHA was derived from conversion, rather than diet, for all insect prey (Tables 1, 2). Because of their high ALA content, terrestrial moths, butterflies and bees had the potential to supply significantly more DHA than aquatic insects or other terrestrial insects (Fig. 1, Table 5). Our estimates for DHA from EPA-rich aquatic insects are likely conservative because our 13C-ALA tracer allowed us to measure ALA to DHA conversion as well as ALA-derived EPA conversion to DHA, but not direct EPA to DHA conversion (i.e. tree swallows could have converted additional unlabeled EPA to DHA).

DISCUSSION

Food quantity in terms of energy and food quality in terms of limiting nutrients are the major drivers of survival and performance in all living things everywhere, and especially in developing animals in the wild. We previously found that food containing omega-3 HUFAs reflective of aquatic insects improves multiple metrics of performance in tree swallow chicks in the laboratory (Twining et al., 2016a) and that the biomass of omega-3 HUFA-rich aquatic insects is a strong predictor of tree swallow chick fledgling production in nature (C.W.T., J.R.S. and D.W.W., unpublished). In the present study, we sought to understand these costs to chick performance further by determining whether and how tree swallow chicks can efficiently convert ALA into omega-3 HUFAs and deposit them into tissues. Our ALA tracer-based results reveal that tree swallow chicks are able to derive omega-3 HUFAs from ALA within liver and deposit ALA-derived omega-3 HUFAs into both liver and pectoral muscle. This evidence for ALA to omega-3 HUFA conversion and deposition of ALA-derived omega-3 HUFAs suggests that performance costs in chicks on low omega-3 HUFA diets (Twining et al., 2016a; C.W.T., J.R.S. and D.W.W., unpublished) may arise directly from the energetic cost of converting ALA to omega-3 HUFAs as well as indirectly from omega-3 HUFA limitation when ALA itself is in limited supply.

In our previous study, we found that tree swallow chicks suffered performance declines when fed on diets with 6.25% ALA, 1.47% EPA and 1.42% DHA compared with diets with 1.82% ALA, 3.74% EPA and 3.44% DHA (Twining et al., 2016a). While all insect taxa that we examined contained more than 1.82% ALA (the level of ALA in our higher performance dietary treatment), terrestrial Diptera provide less than the 3.74% EPA and 3.44% DHA levels in our high-performance dietary treatment (Fig. 1, Table 2), even at maximum measured deposition levels of 22.1% EPA and 34.1% DHA (Table 2). Terrestrial insects that did contribute more total EPA than that of our high-performance lab treatments provided substantially and significantly less EPA than aquatic insects (Fig. 1, Table 4). Only terrestrial butterflies and moths had the potential to provide more than sufficient total (raw plus potential from ALA) EPA and DHA (Fig. 1, Table 4). However, terrestrial butterflies and moths, beetles and bees are rare prey in tree swallow diets (McCarty and Winkler, 1999; D.W.W. and C.W.T., unpublished). Instead, true flies (Diptera), which are often highly abundant and relatively easy for tree swallows to catch compared with less-abundant and faster flying prey like butterflies and large odonates, dominate tree swallow diets (McCarty and Winkler, 1999). Thus, it is unlikely that the terrestrial insects that tree swallows actually consume can provide their chicks with sufficient ALA-derived omega-3 HUFAs in natural systems, making omega-3 HUFA-rich aquatic insects ecologically essential for tree swallow chicks.

Strictly essential nutrients are nutrients that animals are unable to synthesize from their molecular precursors in sufficient quantity to meet metabolic needs and must therefore be derived directly from the diet. We found that tree swallows can synthesize limited amounts of omega-3 HUFAs from precursors. However, omega-3 HUFA-rich prey like aquatic insects appear to be ecologically essential for tree swallows in natural systems because the ALA content of terrestrial insects and the ALA to omega-3 HUFA CE are insufficient to supply chicks with the omega-3 HUFAs that they require. In addition, ALA-derived omega-3 HUFA levels were much higher in liver, which is where most ALA to omega-3 HUFA conversion occurs, compared with pectoral (i.e. flight) muscle (Table 1), where omega-3 HUFAs are deposited and used by birds during energetically demanding and oxidatively stressful processes such as migration (e.g. McWilliams et al., 2004; Guglielmo, 2010), but are not synthesized. Our findings in tree swallows contrast with observations in chickens, which show increases in tissue omega-3 HUFAs with increased dietary omega-3 HUFAs, but are able to thrive even without dietary omega-3 HUFAs (Cherian and Sim, 1991).

Past studies directly measuring ALA to omega-3 HUFA CE have focused on humans and laboratory mammals, such as rats (Brenna et al., 2009). Human studies uniformly show that ALA conversion to EPA is significant and in the low percentage range, while conversion to DHA is barely above trace levels (Brenna et al., 2009). Our measured CE in tree swallow liver (Table 1), where presumably most omega-3 HUFA synthesis occurs, is broadly similar to that found in human blood (Burdge, 2004), where omega-3 HUFA synthesis is low or zero. In contrast, ALA-derived omega-3 HUFA levels in tree swallow pectoral muscle (Table 1), which is a more comparable tissue to blood in that omega-3 HUFA synthesis is also low or zero, were much lower than levels in human blood (Burdge, 2004). Studies in rat pups suggest that ALA to omega-3 HUFA conversion occurs at much higher efficiency than we found in tree swallows even when omega-3 HUFAs are present in the diet (Sheaff et al., 1995). However, although these studies suggest that ALA conversion is possible in humans and other mammals throughout their lifetime, most omega-3 HUFAs still come directly from dietary omega-3 HUFAs unless dietary sources are kept artificially low (Brenna et al., 2009).

Within the existing literature on ALA to omega-3 HUFA conversion in humans, it is well known that ontogeny, sex and reproductive status have important effects on CE. For example, human females of reproductive age are more efficient at ALA to omega-3 HUFA conversion than are human males of the same age (Burdge et al., 2002), which researchers have interpreted as an adaptation for provisioning human infants with omega-3 HUFAs (Brenna et al., 2009). Studies in humans also suggest that infants are more efficient at conversion than are adults or older children, although infant CE may be insufficient for optimal development in the absence of preformed dietary omega-3 HUFAs (Brenna et al., 2009). Even among human infants, those at earlier gestational ages appear to have higher omega-3 HUFA CE than do infants at later gestational ages (Carnielli et al., 2007).

Like human infants, the tree swallow nestlings that we studied are entirely reliant upon parental feeding. Nutritional demands are greatest for altricial temperate passerines like tree swallows during the nestling period when they undergo rapid growth, often doubling in mass every few days (Zach and Mayoh, 1982). Thus, the CE that we report for tree swallow nestlings is likely near maximum for the species under the diet conditions provided. Unlike human mothers, who provision their fetuses and nursing infants with a constant supply of their own digested nutrients including omega-3 HUFAs (Brenna et al., 2009), or even precocial species of birds like chickens, which provision embryos with high levels of ALA and omega-3 HUFAs in eggs (Lin et al., 1991; Speake and Wood, 2005), wild altricial birds like tree swallows invest little omega-3 HUFAs into eggs (Speake and Wood, 2005; C.W.T. and D.W.W., unpublished). Tree swallows also complete nearly all somatic growth, including growing the brain, eyes and other nervous tissues, within approximately 3 weeks. This means that nestling tree swallow chicks must acquire all of their omega-3 HUFAs within a very short time window, creating high selective pressure for high ALA to omega-3 HUFA CE and tissue deposition during the nestling period. As in humans, further studies on tree swallow adults are necessary to confirm how ALA to omega-3 HUFA CE varies throughout the life cycle.

Previous studies on birds and other animals in natural systems have not investigated ALA to omega-3 HUFA conversion mechanistically using stable isotope tracers. Thus, although it is clear that omega-3 HUFAs are crucial for many physiological processes at the molecular level (e.g. Hulbert et al., 2005; Arnold et al., 2015), unfortunately, it remains unclear whether omega-3 HUFAs are strictly or ecologically essential for many wild animals, including other birds. Most researchers have made inferences about CE based on data on the effects of dietary omega-3 HUFA content on performance or survival (e.g. Sargent et al., 1999) or tissue fatty acid composition (e.g. Cherian and Sim, 1991). However, when CE is low, costs to performance and survival may be due to either dietary omega-3 HUFA limitation or the costs associated with converting ALA to omega-3 HUFAs. In contrast, if CE is zero, then all costs to performance and survival must be due to direct limitation, making omega-3 HUFAs strictly essential. Direct measurements of CE are clearly necessary to distinguish between these possibilities.

Additional studies in our system and on other wild animals are also necessary to characterize the EPA to DHA conversion. In this study, we fed tree swallow chicks enriched ALA to characterize conversion of dietary ALA into both EPA and DHA. While many animals are also capable of converting dietary EPA into the DHA they need (Castro et al., 2012; Plourde et al., 2014), our estimates of potential total DHA do not include conversion of dietary EPA into DHA. Thus, our estimates likely underestimate the value of high-EPA aquatic insects as sources of DHA from dietary EPA. Future studies using multiple stable isotope tracers are needed to evaluate the relative value of both ALA and EPA as precursors for DHA.

Further studies are also needed to investigate selective transport of omega-3 HUFAs into tissues throughout the body in wild animals. Omega-3 HUFAs are used and may be selectively transported to and deposited in different metabolically active tissues throughout the body. We chose to examine synthesized omega-3 HUFAs in pectoral muscle as well as liver, finding similar levels of ALA-derived DHA and EPA within liver and within pectoral muscle (Table 1). However, based upon our current data, we cannot rule out the possibility that synthesized DHA could have been selectively transported and deposited at different rates from synthesized EPA in other tissues, such as brain. Future studies characterizing transport and deposition into tissues throughout the body are clearly necessary to fully capture all aspects of the ALA to omega-3 conversion and metabolism for animals in natural systems.

Understanding whether omega-3 HUFAs are strictly essential, ecologically essential or dispensable nutrients for birds and other wild animals is crucial for informing conservation efforts. In order to develop successful species-management plans, environmental managers must understand the full suite of food and habitat resources that wild animals require throughout their life cycle. For example, our findings in tree swallow chicks suggest omega-3 HUFA-rich aquatic insects and habitats are ecologically essential resources during a critical ontogenetic period. As a consequence, human activities that alter the composition and resulting nutritional quality of insect prey, such as land use change and pesticide use, as well as phenological shifts in insect emergence due to climate change have the potential to create nutritional mismatches for tree swallows (C.W.T., J.R.S. and D.W.W., unpublished). We hope that our field-based adaptation of an enriched stable isotope tracer method from human clinical studies is a starting point towards developing a general understanding of fatty acid nutritional ecology in wild animals.

Acknowledgements

We thank Jeremy Ryan Shipley for field assistance and tree swallow nestling feeding advice.

Footnotes

Author contributions

Conceptualization: C.W.T., P.L., D.W.W., A.S.F., J.T.B.; Methodology: C.W.T., P.L., J.T.B.; Formal analysis: C.W.T., P.L.; Investigation: C.W.T., P.L.; Resources: C.W.T., P.L., J.T.B.; Data curation: C.W.T.; Writing - original draft: C.W.T.; Writing - review & editing: D.W.W., A.S.F., J.T.B.; Supervision: D.W.W., A.S.F., J.T.B.; Funding acquisition: C.W.T., J.T.B.

Funding

C.W.T., P.L. and J.T.B. acknowledge funding from the U.S. Department of Agriculture (Hatch Grant NYC-399-7461); C.W.T. and A.S.F. acknowledge funding from the National Science Foundation (DDIG DEB-1838331); and C.W.T. acknowledges funding from a Cornell University College of Agriculture and Life Sciences Mellon Grant. C.W.T. was supported by the National Science Foundation Graduate Research Fellowship Program during the course of this project.

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Competing interests

The authors declare no competing or financial interests.

Supplementary information