Oxidation and allocation of nectar amino acids during butterfly flight

3 Flying animals face extreme energetic demands, relying mainly on carbohydrates and lipids, with 4 occasional contributions from proteins and amino acids. In nectar-feeding species like butterflies 5 and hummingbirds, sugars are the primary fuel, yet the extent to which other nectar-derived 6 nutrients, like amino acids, are used for flight or retained for other functions remains unclear. 7 Using 13C-labeled nectar, we tracked the metabolic fate of sugars and amino acids during flight 8 in Pieris rapae butterflies. We found that proline and glycine, two of the most abundant nectar 9 amino acids, were oxidized alongside sugars. Importantly, flight intensity modulated nutrient 10 allocation from nectar: high-flight females incorporated less glycine into tissues, implying 11 diversion toward flight, while threonine deposition in abdomens increased, reflecting 12 prioritization for reproduction and storage. These findings reveal the complex role of nectar-13 derived nutrients in supporting locomotion and reproduction, while showing how nectar use can 14 modulate trade-offs between flight and fecundity. 15 16

17 Powered flight is a key adaptation across animals, allowing insects, birds, and bats to forage, 18 escape predators, locate mates, and disperse over long distances (Anderson & Ruxton, 2020; 19 Dudley, 2002; Ruaux et al., 2020). Yet flight is also the most energetically costly mode of 20 locomotion, far exceeding the metabolic demands of running or swimming (Bale et al., 2014; 21 Schmidt-Nielsen, 1972). In insects, for example, flight can demand up to 100 times the resting 22 metabolic rate (Anderson & Ruxton, 2020; Dudley, 2002; Ruaux et al., 2020). Thus, meeting 23 these energetic demands requires efficient fuel use, with flying animals relying primarily on 24 carbohydrates and lipids, and, in some cases, proteins and amino acids.The relative contribution 25 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 1, 2025. ; https://doi.org/10.1101/2025.09.29.679119doi: bioRxiv preprint of each fuel depends on the species, flight intensity, ecological context, and resource availability 26 (Weber, 2011). 27 For nectar-feeding animals such as butterflies, bees, and hummingbirds, nectar sugars are the 28 dominant energy source during flight (O’Brien, 1999; Suarez et al., 2011). Yet, nectar is a 29 complex resource that also contains small but physiologically relevant amounts of amino acids 30 (Baker & Baker, 1986; Nicolson & Thornburg, 2007). While studies, especially in insects, 31 indicate that some amino acids are oxidized from internal stores during flight (Teulier et al., 32 2016; Stec et al., 2021; Weeda et al., 1979), it is still unclear whether amino acids obtained 33 directly from nectar are similarly used in flight. 34 Beyond serving as a fuel for flight, nectar can also contribute to reproduction and long-term 35 somatic functions. In many butterflies and moths, for example, nectar sugars as well as amino 36 acids are incorporated into proteins and directly support oogenesis, supplementing or in some 37 cases even replacing nutrient reserves acquired during larval development (Jervis & Boggs, 38 2005; Levin et al., 2017; Mevi‐Schütz & Erhardt, 2005). Similarly, in bird pollinators, especially 39 those that rely heavily on nectar, amino acids in nectar have been suggested to help supplement 40 their nitrogen requirements (Nicolson, 2007; Roguz et al., 2019). This dual role of nectar-derived 41 resources may accentuate tradeoffs between flight and reproduction, as amino acids oxidized to 42 sustain flight are no longer available for egg production. Thus, understanding whether nectar 43 amino acids are metabolized during flight, or instead retained for tissue incorporation, is critical 44 to link flight energetics with fitness and help clarify the physiological mechanisms underlying 45 frequently observed trade-offs between flight and reproduction (Schmidt-Wellenburg et al., 46 2008; Tigreros & Davidowitz, 2019). 47 Here, we investigate the metabolic fate of nectar-derived nutrients in Pieris rapae, a common 48 butterfly that relies on floral nectar throughout its adult life. Using 13C-labeled sugars and amino 49 acids (proline, glycine, and threonine), we examined nutrient oxidation during flight as well as 50 nutrient deposition in thoracic and abdominal tissues under varying flight conditions. By 51 distinguishing which nectar components serve as immediate fuels and which are retained for 52 other functions, we provide new insight into the role of nectar composition in supporting the 53 energetics of insect flight. 54 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 1, 2025. ; https://doi.org/10.1101/2025.09.29.679119doi: bioRxiv preprint 55

56 Study organism and Rearing 57 The cabbage white butterfly (Pieris rapae) is a widespread species whose adults feed on floral 58 nectar, serving as pollinators for a variety of plants while obtaining sugars and amino acids as 59 nutritional rewards (Alm et al., 1990; Corbera et al., 2018; Hongfang et al., 2010; Rader et al., 60 2009). Like other pollinators, female P . rapae can detect and preferentially consume nectars 61 enriched in amino acids (Alm et al., 1990). This species faces high energetic demands due to its 62 investment in both flight and reproduction: females fly an average of 0.7 km per day (Scott, 63 1987) and lay an average of 278 ± 172 eggs over a short adult lifespan of about 12.2 ± 4.0 days 64 (Kimura & Tsubaki, 1986). 65 For this study, P . rapae were obtained from a laboratory colony established from a wild 66 population in Utah, USA. Larvae were reared in a greenhouse on Brassica oleracea. Pupae and 67 adults were maintained in a walk-in environmental chamber at 22 °C, 50% relative humidity, and 68 a 16:8 h light–dark cycle. 69 Nectar Treatments 70 In this study, we used artificial nectars that replicated the sugar and amino acid composition of 71 Lantana camara (Verbenaceae), a high-quality nectar source frequently visited by diverse 72 butterfly species. The composition of L. camara nectar has been well characterized and is widely 73 used in studies of nectar preference in P. rapae (Alm et al., 1990) and other Lepidoptera (Erhardt 74 & Rusterholz, 1998; Jervis & Boggs, 2005; Mevi-Schütz & Erhardt, 2003). Natural L. camara 75 nectar typically contains ~1 M sugars and ~10 mM amino acids, yielding an approximate 1:100 76 amino acid-to-sugar ratio. Of the amino acid pool, roughly 6% comprises three essential amino 77 acids, with threonine being the most abundant, while the remaining 94% consists of eight 78 nonessential amino acids, dominated by proline and glycine. 79 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 1, 2025. ; https://doi.org/10.1101/2025.09.29.679119doi: bioRxiv preprint To track use of specific nectar’s nutrients by female P . rapae, we created five lantana-mimicking 80 nectar treatments. Four contained a single 13C-labeled compound—13C₁-glycine, 13C₁-proline, 81 13C₁-threonine, or 13C-sucrose (enriched with cane sugar)—while the fifth served as a control, 82 containing beet sugar and only unlabeled amino acids (δ13C ≈ –26.5‰). The labeled amino acids 83 were selected because they are abundant in L. camara nectar and in other butterfly-pollinated 84 flowers (Mevi-Schütz & Erhardt, 2003; Roguz et al., 2019). All isotope tracers were obtained 85 from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). 86 87 Experiment 1. Oxidation of nectar nutrients during flight 88 To assess oxidation of nectar nutrients during flight, females were fed 48 h post-emergence with 89 10 µl of either a Control or one of four ¹³C₁-labelled nectars: ¹³C₁-glycine, ¹³C₁-proline, ¹³C₁-90 threonine, or ¹³C-sucrose. Four hours after feeding, each female was placed in a respirometry 91 chamber consisting of a 500 ml syringe fitted with an injection port and adjusted to provide a 92 200 ml chamber volume. This volume was sufficient to allow flight while enabling CO₂ 93 accumulation to ≥300 ppm. The syringe air was replaced with dried, CO₂-free air, sealed, and 94 females were flown for 4 min under natural light, with gentle shaking to prevent resting. A 20 ml 95 breath sample was then analyzed for δ¹³CO₂ using a cavity ring-down spectrometer (G2201-i, 96 Picarro Inc., Sunnyvale, CA) coupled to a SSIM2 Small Sample Isotope Module (Picarro Inc., 97 Sunnyvale, CA). 98 99 Experiment 2. The effect of flight on nectar nutrients depositions 100 To determine where nectar-derived amino acids are deposited, we used a different group of adult 101 females (48 h post-emergence) which were randomly assigned to one of two flight treatments—102 Low or High flight intensity—and fed either a control nectar or one of four ¹³C₁-labelled nectars. 103 For the Low flight intensity group, females were kept in a 30 × 30 × 30 cm mesh cage lined with 104 paper towel inside a climate-controlled walk-in chamber (LD 16:8 h, 22°C at 50% RH). For the 105 High flight intensity group, females were kept under the same conditions but were additionally 106 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 1, 2025. ; https://doi.org/10.1101/2025.09.29.679119doi: bioRxiv preprint stimulated to fly once per day, for five continuous minutes, by gently touching them with a fine 107 paintbrush (Niitepõld & Boggs, 2015). Preliminary trials showed that longer flight durations 108 caused extreme exhaustion, with butterflies becoming unresponsive. All females were fed once 109 daily with 10 µl of their assigned nectar treatment. After three consecutive days of the flight and 110 nectar treatments, individuals were euthanized by freezing at −20 °C. Thoraces and abdomens 111 were dissected, dried at 50 °C for 48 h, and homogenized. δ¹³C values were then measured using 112 cavity ring-down spectroscopy (CRDS; Picarro stable isotope analyzer, G2201-i) coupled with 113 an A0201 combustion module and A0301 gas interface. 114 Statistical Analysis 115 To assess nectar-derived nutrient oxidation (Experiment 1) and tissue deposition (Experiment 2), 116 we used Dunnett’s test to compare the δ¹³C values of breath or tissue samples from females fed 117 each ¹³C-labeled nectars against those of control females fed unlabeled nectar. 118 Because δ13C values can naturally change with tissue type and treatment, we used δ13C of control 119 females to estimate atom % excess (APE), which is a unit of enrichment (McCue et al., 2011; 120 Slater et al., 2001). We used 13C concentrations of both control and labelled tissues expressed in 121 δ13CVPDB to calculate the atom percent (AP) and then Atom Percent Excess (APE), defined as 122 APE = AP(label) – AP(control) (Slater et al., 2001). 123 The effects of flight on nutrient allocation to thoracic and abdominal tissues were analyzed using 124 two-way ANOV As with flight treatment, nectar nutrient treatment (¹³C-labeled vs. control), and 125 their interaction as fixed factors. When significant interactions were detected, we estimated 126 predicted values and performed pairwise comparisons of simple effects using the emmeans 127 package in R, applying a Tukey adjustment for multiple testing. All statistical analyses were 128 conducted in R 3.6.0. and assumptions of normality and homogeneity of variance were verified 129 prior to analysis. 130 131

AND DISCUSSION 132 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 1, 2025. ; https://doi.org/10.1101/2025.09.29.679119doi: bioRxiv preprint Nectar is a critical resource for flying pollinators, providing the energy needed to support their 133 high metabolic demands during flight (Brien, 1999; Suarez, 2005; Welch et al., 2006). While it is 134 well established that nectar sugars serve as the primary fuel for flight, far less is known about the 135 roles of non-sugar components, like amino acids. Here we investigated the use of nectar-derived 136 nutrients, including essential and non-essential amino acids, as metabolic fuels during flight and 137 how flight activity may influences their allocation to different tissues in female Pieris rapae. 138 139 Oxidation of nectar nutrients during flight 140 Females that fed on nectar containing ¹³C-labeled sugars, proline, or glycine showed significant 141 ¹³CO₂ enrichment (more positive δ¹³C values) in breath compared to controls fed unlabeled 142 nectar (Fig. 1), indicating active oxidation of 143 both sugars and non-essential amino acids. 144 Although previous studies have suggested that 145 nectar amino acids help meet the high 146 metabolic demands of flying nectarivores 147 (Carter et al., 2006; Levin et al., 2017; Stec et 148 al., 2021), our results provide the first direct 149 evidence that nectar amino acids are actually 150 oxidized during flight. At the same time, no 151 significant oxidation of threonine by flying 152 females was observed (Fig. 1), suggesting that 153 essential amino acids may be less likely to 154 contribute to locomotion, at least under our 155 experimental conditions. These results add to 156 the growing body of evidence highlighting the 157 importance of nectar amino acids for flying 158 animals, and in particular for insect 159 pollinators. Proline and glycine are common 160 constituents of floral nectar (Baker & Baker, 161 Fig. 1 Boxplots show the δ13C values from exhaled breath during flight of P . rapae females. Significant differences between the δ13C from control (n = 9, blue boxplot) and females that consumed 13C-labelled nectars indicate that a nectar-derived nutrient was oxidized during flight. Traced nutrients included sugars (n = 13), two non-essential amino acids: glycine (n = 15) and proline (n = 14), and an essential amino acid, threonine (n = 13). .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 1, 2025. ; https://doi.org/10.1101/2025.09.29.679119doi: bioRxiv preprint 1973; Rusterholz & Erhardt, 1998) and can act as a phagostimulant in pollinator insects species 162 (Lim et al., 2019; Ruedenauer et al., 2019). Several studies have shown that proline, in particular, 163 is selectively oxidized in insect flight muscle, especially during the initial 30 seconds of flight 164 (Stec et al., 2021; Teulier et al., 2016; Weeda et al., 1979). Though glycine’s role in flight is less 165 defined, its consumption has been linked to neuromodulation and memory in insects (Parkinson 166 et al., 2025), suggesting a dual function: fuelling flight and supporting cognitive processes 167 essential for effective foraging. 168 169 The effect of flight on nectar nutrients depositions 170 Gravimetric measurements of the impact of flight on 171 thorax and abdomen resources did not indicate any 172 differences (both thorax and abdomen p > 0.05). In 173 contrast, isotopic analysis of control, unlabeled females, 174 revealed significant changes in tissue carbon 175 composition due to flight intensity. Although abdominal 176 tissue had higher δ¹³C values than thoracic tissue 177 (ANOVA: F₁,₂₀ = 8.1, P = 0.01), females subjected to 178 high-intensity flight exhibited overall ¹³C enrichment in 179 both thorax and abdomen compared to low-flight 180 individuals (F₁,₂₀ = 6.26, P = 0.02), consistent with 181 increased oxidation and depletion of lipid stores (Fig. 182 2).This is in line with previous studies showing that 183 butterflies rely on both carbohydrates and lipids to meet 184 the high energy costs of flight, and that sustained flight 185 can rapidly deplete internal energy, often leading to flight fecundity tradeoffs (Tigreros & 186 Davidowitz, 2019). 187 Previous studies have shown that nectar in general and its amino acids in particular can be 188 rapidly incorporated, within minutes, into Lepidoptera flight muscle and developing oocytes 189 Figure 2 Changes in δ¹³C values in thorax and abdomen tissues of Pieris rapae females subjected to Low versus High flight intensity. Elevated δ¹³C values indicate ¹³C enrichment, reflecting relatively lower lipid content in abdomen tissue (compared to thorax) and highly active individuals. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 1, 2025. ; https://doi.org/10.1101/2025.09.29.679119doi: bioRxiv preprint (DeFino & Davidowitz, 2024; Levin et al., 2017). Given that the thorax serves as the primary site 190 of fuel combustion during flight (Dudley, 2002; Treidel et al., 2024), it can be expected that 191 incoming amino acids and sugars are preferentially oxidized rather than retained. Here, we 192 demonstrate that flight activity significantly alters the allocation of nectar nutrients across female 193 tissues (F₃, ₁₀₂ = 5.33, P = 0.002). In the thorax, the presence of nectar-derived nutrients declined 194 with increasing flight intensity (F₁,₅₁ = 195 4.99, P = 0.03; Fig. 3), consistent with their 196 increased metabolic oxidation or 197 mobilization. Interestingly, while most 198 labelled amino acids were detected in both 199 thorax and abdomen tissues (Fig. S1), 200 proline was notably absent in the thorax 201 under high flight conditions, and it only 202 approached significance in low-flight males 203 (P = 0.09; Fig. S1). This supports the 204 hypothesis that proline is rapidly oxidized 205 during flight, leaving little available for 206 tissue incorporation. 207 Patterns of nutrient incorporation and use 208 were distinct in the abdomen, reflecting its 209 key roles in nutrient storage and 210 reproduction (Fig. 3). We found that the 211 incorporation of nectar nutrients varied by 212 type (F3,51 = 4.9, P = 0.01): glycine 213 decreased (p = 0.002), while threonine 214 increased (p = 0.03) with higher flight 215 activity (Fig. 3; Table S1). Interestingly, proline and sucrose incorporation remained unchanged 216 (Fig. 3; Table S1). As the primary site of nutrient storage and reproduction, the abdomen likely 217 channels nectar-derived nutrients toward oogenesis and long-term metabolic demands (Boggs, 218 2009; O’Brien et al., 2002). The decline in abdominal glycine, an amino acid significantly 219 Fig. 3 Effects of flight, high vs. low intensity, on the use of nectar -derived nutrients in abdomen (left panel) and thorax (right panel ) tissues of P . rapae females. The y-axis represents the estimated marginal means ± 95CI of the atom percent excess (APE 13C (%)), with h igher APE values indicat ing greater incorporation of nectar derived sucrose, non-essential amino acids (glycine and proline) and an essential amino acid (threonine). .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 1, 2025. ; https://doi.org/10.1101/2025.09.29.679119doi: bioRxiv preprint oxidized during flight (Fig. 1), suggests that this non-essential amino acid may be diverted to 220 meet immediate energy demands, possibly contributing to a trade-off with female fecundity. 221 Conversely, the increase in threonine, an essential amino acid, may reflect its selective 222 prioritization for egg provisioning. These findings support the idea that adult females use nectar 223 to buffer against nutrient limitations, whether from poor larval diets (Mevi-Schütz & Erhardt, 224 2005; O’Brien et al., 2000) or increased metabolic demands from flight. 225 Collectively, our findings demonstrate that a flying nectarivorous insect utilizes both sugars and 226 specific amino acids—particularly proline and glycine—as metabolic fuels during flight. 227 Moreover, intense flight alters the fate and allocation of nectar-derived nutrients in adult tissues, 228 likely reflecting a trade-off between immediate energy needs and resource storage. Although 229 direct evidence in nectarivorous vertebrates is scarce, the high concentrations of amino acids 230 (including proline and glycine) in nectar of some bird-pollinated plants suggest these compounds 231 may serve similar functions in these species (Nicolson, 2007; Roguz et al., 2019). Thus, our 232

provide broader insights into how nectar composition can influence locomotion and 233 fitness of nectar-feeding species, emphasizing the ecological significance of amino acids in adult 234 diets, especially under conditions of elevated energy expenditure. 235 236

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