Maternal glucocorticoid levels during incubation predict breeding success, but not reproductive investment, in a free-ranging bird

ABSTRACT The hormone corticosterone (CORT) has been hypothesized to be linked with fitness, but the directionality of the relationship is unclear. The ‘CORT-fitness hypothesis’ proposes that high levels of CORT arise from challenging environmental conditions, resulting in lower reproductive success (a negative relationship). In contrast, the CORT-adaptation hypothesis suggests that, during energetically demanding periods, CORT will mediate physiological or behavioral changes that result in increased reproductive investment and success (a positive relationship). During two breeding seasons, we experimentally manipulated circulating CORT levels in female tree swallows (Tachycineta bicolor) prior to egg laying, and measured subsequent reproductive effort, breeding success, and maternal survival. When females were recaptured during egg incubation and again during the nestling stage, the CORT levels were similar among individuals in each treatment group, and maternal treatment had no effect on indices of fitness. By considering variation among females, we found support for the CORT-adaptation hypothesis; there was a significant positive relationship between CORT levels during incubation and hatching and fledging success. During the nestling stage CORT levels were unrelated to any measure of investment or success. Within the environmental context of our study, relationships between maternal glucocorticoid levels and indices of fitness vary across reproductive stages.


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ABSTRACT 22 The hormone corticosterone (CORT)) has been hypothesized to be linked with fitness, but the 23 directionality of the relationship is unclear. The "CORT-fitness hypothesis" proposes that high 24 levels of CORT arise from challenging environmental conditions, resulting in lower reproductive 25 success (a negative relationship). In contrast, the "CORT-adaptation hypothesis" suggests that, 26 during energetically demanding periods, CORT will mediate physiological or behavioural 27 changes that result in increased reproductive investment and success (a positive relationship). 28 During two breeding seasons, we experimentally manipulated circulating CORT levels in female 29 tree swallows (Tachycineta bicolor) prior to egg laying, and measured subsequent reproductive 30 effort, breeding success, and maternal survival. When females were recaptured during egg 31 incubation and again during the nestling stage, the CORT levels were similar among individuals 32 in each treatment group, and maternal treatment had no effect on indices of fitness. By 33 considering variation among females, we found support for the "CORT-adaptation hypothesis"; 34 there was a significant positive relationship between CORT levels during incubation and 35 hatching and fledging success. During the nestling stage CORT levels were unrelated to any 36 measure of investment or success. Within the environmental context of our study, relationships 37 between maternal glucocorticoid levels and indices fitness vary across reproductive stages.

INTRODUCTION 41
Within and among species individuals vary in the strategies used to maximise fitness, by 42 adjusting the relative effort put into current versus future reproductive events (Williams, 2005;43 Hansen et al., 2016). There is ample evidence that increased energy expenditure and effort 44 during one breeding bout results in decreased reproductive success, probability of re-nesting, or 45 survival in subsequent bouts (Nager, 2006;Crossin et al., 2013Crossin et al., , 2016Harms et al., 2014;Bleu, 46 Glucocorticoids (GCs) have been hypothesized to be a mediator of the trade-off between 48 current and future reproduction (Wingfield et al., 1998;Bleu, Gamelon & Saether, 2016;Hansen 49 et al., 2016). GCs are metabolic hormones that fluctuate daily with feeding and other activities, 50 and under resting conditions regulate energy balance (Landys, Ramenofsky & Wingfield, 2006;51 Wilcoxen et al., 2011;Hau & Goymann, 2015). In response to an environmental stressor, GC 52 levels increase rapidly, resulting in increased availability of metabolic substrates, and adjustment 53 of behaviors toward immediate survival (Wingfield & Sapolsky, 2003;Romero, 2004) while 54 inhibiting reproductive behaviour and physiology (Sapolsky et al., 2000;Dantzer et al., 2014), 55 i.e. the CORT-trade-off hypothesis (Patterson et al., 2014). 56 GCs are thought to play a role in translating environmental cues into adaptive 57 populations lay only one clutch of 5 or 6 eggs per season; the female then incubates the eggs for 111 about 14 days. Chicks are fed by both parents and fledge at approximately 21 days post-hatch. 112 We had two field sites near Peterborough Ontario, Canada ( boxes at the Sewage Lagoon was exposed soil and grasses; the greater surrounding area was 119 farmland consisting of both crop and pasture land. Nest boxes at the Sewage Lagoon were 5 to 120 10 metres from the water. 121 122 Experimental manipulation of maternal corticosterone levels 123 Nest boxes were monitored daily beginning 6 May in both years. When nests were about 75% 124 formed (when cup-shaped or when feathers were present), we captured females using cardboard 125 trapdoors over the nest box opening, or by surprising birds sitting in nest boxes. In 2015, seven 126 females were caught at night (between 2200 and 2400 hours) by surprising birds sitting in nest 127 7 2013). The design of our implants followed that of Ouyang et al. (2013), who used a 7 mm long 134 implant (ID 1.5 mm), sealed at both ends, and punctured with a single 0.3 mm hole. In great tits 135 (Parus major) this design increased corticosterone levels by ~2-fold above baseline for 136 approximately 30 days post-implantation (Table 1 in Ouyang et al. 2013). Sham treatment tree 137 swallows received sterilized empty implants. Empty implants weighed approximately 0.02g and 138 held an average of 0.007g ± 0.0007g of CORT. Once the implant was inserted, the incision in the 139 skin was sealed with a drop of 3M Vetbond (no. 1469SB). Each female was then aged as second 140 year, SY, or after-second year, ASY (Pyle et al., 1987). Flattened wing length was measured 141 with a standard ruler with a wing stop (±1mm), mass was measured with a Pesola spring scale 142 (±0.25g). All birds (including any males caught inadvertently) were banded with a federal 143 aluminum numbered leg band (Canadian Wildlife Service) and released. Birds were held for no 144 more than 10 minutes before release. In 2016 and 2017, any previously banded female from 145 2015 or 2016 was counted as a returned bird in the return rate analysis regardless of whether they 146 hatched a clutch that year. 147 We allocated females to the Control group if they were not caught prior to laying, either 148 because they did not enter the nest box while it had a trap, or because they began laying earlier 149 than we expected. Although these females did not receive an implant before egg laying they were 150 1 2 and because none of our response variables was correlated, we did not to use a post-hoc 249 correction for the number of tests performed (Perneger, 1998;Streiner, 2015). 250

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Morphological and hormonal measures of adult females 252 We ran preliminary tests to determine whether females that had been assigned to CORT or Sham 253 treatment groups differed in pre-implant body mass (measured at time of implant; females in the 254 Control group were not captured prior to incubation and thus there was no pre-implant mass 255 measurement). To test for possible differences in body size among treatments, we compared a 256 female's wing length (measured pre-laying in the CORT and Sham treatments, and during early 257 incubation in the Control females). Finally, we tested for differences in clutch initiation date in 258 Julian days among treatments. Separate linear models (LM) were run with female pre-laying 259 mass, wing length, and clutch initiation date as the response variable, and treatment (CORT,260 Sham, Control), site (Nature areas, Sewage Lagoon), year (2015,2016), and age (Second year, 261 SY; After second year, ASY) as the predictors. We did not include any interactions terms as they 262 were not of a priori interest. 263 To test whether implanted females differed in their probability of recapture depending on 264 treatment or year, we ran a generalized linear model (GLM) with binomial errors, with recapture 265 status (recaptured/non-recaptured) as the dependent variable, and treatment and year as the fixed 266 effects. To test whether the total number of individuals that retained their implants and 267 subsequently laid eggs differed between the CORT and Sham maternal treatment groups, we 268 used a chi-square test (because Control females were only captured post-egg laying, they were 269 not included in this analysis). 270 1 3

Maternal baseline corticosterone during incubation and nestling stages 272
To test whether treatment affected maternal CORT levels within each breeding stage (incubation 273 and nestling), we used linear models (LM) with either CORT during incubation (from hereafter 274 CORTinc) or CORT during the nestling stage (CORTnest) as the response variable and maternal 275 treatment, age, site, sample time (time from initial contact with bird to end of blood sample), and 276 clutch initiation date (in Julian days) as fixed effects. We had no a priori predictions regarding 277 interactions, so none was included in the models. 278 We analyzed CORTinc and CORTnest separately because CORTnest was only measured 279 in 2016. Baseline CORTinc measurements (N=59) had one suspected outlier (121.22 ng/ml) 280 removed prior to analysis. This value was > 3 standard deviations from the mean; considerably 281 higher than the 0.5 to 14 ng/ml range reported for previously (Franceschini et al., 2008;Ouyang 282 et al., 2011;Patterson et al., 2011;Madliger et al., 2015). Preliminary analyses were run with and 283 without this outlier, and although no difference was found in the pattern of significance of 284 To test for a relationship between CORTinc and indices of reproductive success, we used a 305 generalized linear mixed model (GLMM; glmer in R package lme4) with binomial errors, with 306 either hatching or fledging success as the response variable (0 or 1 for each chick) and CORTinc, 307 maternal treatment, age, site, and year as fixed effects, and nest ID as a random effect. To 308 explore the relationship between CORTnest and post-hatching reproductive success, we 309 examined individual nestling mass at day 14 post-hatch and fledging success as indices of 310 reproductive success. To test whether nestling mass differed with maternal CORT or treatment, 311 we used a LMM with nestling mass at day 14 as the response variable and CORTnest, maternal 312 treatment, maternal age, and site as fixed effects (year was not included because CORTnest was 313 measured in 2016 only), and Nest ID as a random effect. Finally, to test whether fledging success 314 differed with maternal CORT or treatment, fledging success (0 or 1 for each chick) was used as 315 the response variable in a GLMM with binomial errors with maternal treatment, maternal age, 1 5 site, and CORTnest as fixed effects, and Nest ID as a random effect. No interaction terms were 317 included in these analyses. 318 319

Measures of female survival 320
We estimated female survival by using the return rates of adult females to the study sites the 321 following spring and comparing this with CORTinc or CORTnest during the previous year in 322 separate models. Return rate (either 0 or 1) was the response variable in a general linear model 323 (GLM), with CORTinc (or CORTnest), treatment, year, age, site, and number of nestlings 324 fledged as main effects. In analyses of CORTnest, "year" was not included in the model because 325 CORTnest was only measured in a single year (2016). 326 327

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Morphology and hormonal measures of adult females 329 We implanted 45 females with corticosterone-filled implants (CORT), and 44 with sham 330 implants (Sham); an additional 23 females were captured for the first time during incubation and 331 were allocated to the Control treatment (Table 1). There was no difference in pre-egg laying 332 body mass between females allocated to the CORT and Sham groups (Table S1); females in the 333 Control group were not captured prior to egg laying, so there was no pre-egg laying mass. 334 Focusing on individuals that retained their implants, wing length and clutch initiation date did 335 not differ significantly among treatments (Table S1). There was no significant difference 336 between the Sham and CORT treatments in the percentage of females that retained their implants 337 and subsequently laid eggs (Sham: 45% (20 of 43), CORT: 31% (14 of 44); χ 1 6 There was no significant difference by year in the number of implanted females that were 341 recaptured during incubation (β = 0.800, SE = 0.509, z = 1.573, p = 0.116, N 2015 =34 recaptured 342 and 25 non-recaptured, N 2016 =22 recaptured and 8 non-recaptured. However, sham-implanted 343 individuals were more likely to be recaptured than CORT-implanted (β = 1.115, SE = 0.468, z = 344 2.381, p = 0.017, N Sham =33 recaptured and 11 non-recaptured, N CORT =23 recaptured and 22 non-345 recaptured). Control birds were not included in the recaptured/not recaptured analysis because 346 they were caught for the first time during incubation. 347 348 Implants failed to raise long-term maternal corticosterone levels 349 During incubation, females were recaptured on average 17.02 days (±0.63) after implantation 350 (range 7 to 26 days). Contrary to expectations, when females were recaptured there was no 351 difference in CORT levels among the 3 treatments (Table 2; Fig. 1A). Lay date (i.e., clutch 352 initiation date) was also not a significant predictor of CORTinc (Table 2). However, older 353 mothers (ASY) had higher CORTinc levels than SY mothers and levels differed between years 354 (Table 2). During nestling provisioning, maternal baseline CORT (CORTnest) did not differ 355 among treatments (Table 2, Fig. 1B), nor with any other fixed effects (Table 2). 356 357

Maternal corticosterone levels did not predict reproductive investment 358
Mean clutch size (±SE) of females was 5.3 eggs ± 0.1 (range = 3 to 7 eggs per nest, N=67 nests). 359 Reproductive investment during laying, measured as clutch mass, did not correlate with maternal 360 CORT levels during incubation (CORTinc) nor with maternal treatment, although older birds 361 had significantly heavier clutches (Table 3). Similarly, during the nestling stage, there was no 362 relationship between either maternal CORT (CORTnest) or treatment on the number of female 1 7 nest box visits (Table 3). Although maternal treatment did not influence nestling growth rate 364 between days 3 and 7, there was a marginally significant negative relationship between maternal 365 CORTnest and nestling growth rate (p = 0.092, Table 3). Maternal age influenced nestling 366 growth rates, with nestlings from SY mothers having higher growth rates than nestlings from 367 ASY mothers (Table 3). 368 369 Maternal corticosterone levels during incubation predicted reproductive success 370 As indices of reproductive success, we measured hatching success, nestling mass at day 14 post-371 hatch, and fledging success. Mothers with higher CORT during incubation (CORTinc) had 372 significantly higher hatching success (Table 4). Nestling mass at day 14 post-hatch was not 373 predicted by either CORTnest nor maternal treatment, although nestlings at the Nature Area site 374 tended to be heavier (Site: p = 0.064; Table 4). 375 The probability of a nestling fledging significantly increased with maternal corticosterone 376 levels measured during incubation (Fledging success A, Table 4). There was a marginally 377 significant negative relationship during the nestling phase (Fledging success B, p = 0.060), but 378 this was driven, at least in part, by a control female with the lowest hormone levels (2.34 ng/ml) 379 yet 100% fledging success. Maternal treatment had no effect on fledging success (Fledging 380 success A or B, Table 4). Fledging success tended to be higher at the Sewage Lagoon site 381 (Fledging success (A), Site: p=0.062; Table 4). 382 383 Maternal return rate was not significantly predicted by maternal corticosterone levels 384 Twenty-nine of 67 (43%) females (Sham, CORT or Control) returned in the year after they were 385 initially caught, and all returning females returned to the same breeding site where initially 1 8 caught. The number of females included in the analysis differs from totals in Table 1, because  387 only females with corticosterone measurements were included. The probability that a female 388 returned tended to increase with her incubation CORT levels (CORTinc, p = 0.054) and number 389 of fledglings in the previous year (Return rate (A): p=0.056, Table 5).There was no significant 390 effect of CORTnest or maternal treatment on the likelihood of a female returning to the nest sites 391 the following year (Table 5). 392

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DISCUSSION 394 Our data support the CORT-adaptation hypothesis. During egg incubation, corticosterone levels 395 of female tree swallows were positively related to two measures of reproductive success 396 (hatching and fledging success) and positively (albeit non-significantly) with female return rates. 397 During the nestling stage, there was no relationship between corticosterone and indices of either 398 reproductive investment or reproductive success. During neither period did we detect a 399 significant negative relationship between CORT and fitness, as predicted by the CORT-fitness 400 hypothesis. 401 402

Maternal corticosterone levels during incubation 403
We tested for a relationship between maternal corticosterone levels during incubation 404 (CORTinc) and clutch mass (as a single measure of reproductive investment), and hatching 405 success and survival to fledging as measures or reproductive success (Bonier et al., 2009b;406 Schoenle et al., 2017). We found no relation between CORTinc and clutch mass, however, 407 female tree swallows with higher CORTinc levels had greater hatching success and higher 408 fledging success. The positive relationship we detected with hatching success may be due to 409 During incubation, individuals may experience more unpredictable stressors than during the 431 nestling stage (Romero, 2002). For example, challenging environmental conditions such as lower 432 temperatures and scarcer food resources in early spring can cause a negative relationship 433 0 between both temperature and foraging success and baseline CORT levels, depending on the 434 fitness and environmental measure used (Angelier et al., 2007;Wingfield et al., 2010;Ouyang et 435 al., 2015). Because higher baseline levels may prime the body to perform better under stress, 436 females with higher baseline CORT during incubation in our study may have been better able to 437 meet these challenges (Romero, 2002). 438 439

Maternal corticosterone levels during chick rearing 440
We predicted that if there were a relationship between CORT and reproductive investment and 441 success, it would most likely emerge post-hatch, given the higher maternal energy expenditure 442 required during chick rearing than during incubation (Nilsson and Råberg, 2001;Humphreys et 443 al., 2006;Sakaluk et al., 2018) but see (Williams, 2018)  only when weather conditions were benign (Ouyang et al., 2015). Our inability to detect 476 relationships between maternal hormone levels during chick rearing and reproductive success 477 could be due to the influence of such factors as food availability or weather, both of which could 478 affect body condition and reproductive success of the mother (Schoech et al., 2007;Madliger and Love, 2016a). Maternal baseline CORT may also depend on the habitat type in which female tree 480 swallows were breeding (Madliger and Love, 2016b). While we found no significant difference 481 in reproductive investment between the two study sites, we did find that CORTinc and fledging 482 success tended to be higher at one of our sites (Sewage Lagoon). Reproductive success may 483 perhaps be mediated by a relationship between CORT and foraging conditions (Henderson et al., 484 2017), which could change from incubation to the nestling stage. 485 486

No relationship between corticosterone and return rates 487
We found a borderline (p = 0.054) positive relationship between CORTinc and the probability of 488 whether a female returned to the breeding sites the following year. One explanation for the lack 489 of significance is that the relationship between CORT levels and return rates may be non-linear. We implanted pre-egg laying females with corticosterone-filled silastic implants, but when 502 females were recaptured during early-to mid-incubation (mean ± SE: 17.0 days ± 0.6 after 503 implantation), the baseline CORT levels of implanted birds did not differ from unmanipulated 504 birds. However, CORT-implanted individuals had a lower probability of recapture during 505 incubation, consistent with a negative relationship between experimental CORT elevation and 506 survival (Schoenle et al., 2019 preprint). Across species, silastic implants have been successfully 507 used to raise CORT levels for anywhere from a few days (Astheimer et al., 2000;Hayward and 508 Wingfield, 2004;Criscuolo et al., 2005;Martin et al., 2005;Angelier et al., 2007) to three weeks 509 post-implantation in vivo (Ouyang et al., 2013) and in vitro (Newman et al., 2010). However, the 510 use of implants to raise CORT levels has not been consistently successful (Crossin et al., 2012;511 Ouyang et al., 2013;Hau and Goymann, 2015;Lattin et al., 2016, Torres-Medina et al., 2018. 512 Although the implants used in our study may have failed to release CORT, this seems unlikely 513 given that in vitro studies have shown that CORT continues to be released across the membrane 514 over 4 weeks (Newman et al., 2010). More likely, the implants resulted in decreased secretion of 515 endogenous CORT via negative feedback, or increased clearance of CORT from the blood via 516 increased excretory activity (Newman et al., 2010;Henriksen et al., 2011;Robertson et al., 517 2015). 518 Rather than experimentally manipulate CORT levels via implants, an alternative 519 approach may be to manipulate maternal condition, such as with feather clipping (Rivers et al., 520 2017), predator experiments (Clinchy et al., 2011Pitk et al., 2012), or density manipulations 521 (Bentz et al., 2013).Such an approach would encompass how maternal CORT levels change 522 based on how each female perceives her condition/ environment, how that is reflected in blood CORT levels, and how those levels might influence the next generation (Madliger and Love, 524 2016a;Rivers et al., 2017). 525

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Conclusions 527 The differing directionality of relationship between CORT and fitness among studies and species 528 raises the simple question: is there is a consistent relationship to be found among individuals 529 within a population? Many factors can affect both CORT and fitness, including condition (Love 530 et al., 2005), life-history stage (Romero, 2002), weather (Pakkala et al., 2016), habitat variability 531 (Madliger and Love, 2016b), and resource availability (Breuner and Burk 2019). If it can be 532 reasonably assumed that these will always differ among individuals, then perhaps there is no 533 consistent relationship, and any that may be detected will always be context-dependent 534 (Madliger and Love, 2016a). Recent meta-analyses that seek to understand relationships between 535 CORT and fitness across taxa, and studies that identify factors that contribute to context-536 dependence, are particularly valuable (Sorenson et al., 2017;Breuner and Burk 2019;Schoenle 537 et al., 2019 preprint;Bonier & Cox 2020). 538 The use of integrative measures of CORT may be an alternative way to improve our 539 understanding of the relationship between CORT and fitness. By measuring CORT deposited in 540 feathers during growth, or metabolites excreted in feces, it may be possible to infer CORT levels 541 over multiple days of the incubation or nestling stage (Lucas et al., 2006;Bortolotti et al., 2008;542 Romero and Fairhurst, 2016). For example, giant petrels that successfully bred had higher feather 543 CORT levels than failed breeders, but were less likely to breed the following year, a pattern 544 which was not observed using plasma CORT from these same individuals (Crossin et al., 2013). 545 Ideally, studies could be extended over the winter, as has been done recently in adult tree swallows (Vitousek et al., 2018). This would help elucidate the longer-term effects of maternal 547 CORT on offspring and maternal and fitness. 548 Integrated population models reveal local weather conditions are the key drivers of Table 1. Sample sizes of adult female tree swallows allocated to each maternal treatment group across two years.  Year was not included for CORTnest, because data were collected in a single year. Statistically significant main effects are in bold. CORTinc and CORTnest refer to maternal CORT levels during incubation and the nestling stage, respectively.
Year was not included in analyses of Nest box visits or Growth rate, because CORTnest was measured in a single year only. Adjusted R 2 values are provided for models without random effects; marginal (M) and conditional (C) R 2 values are provided for models with random effects. Statistically significant main effects are in bold. Each model includes Maternal ID as a random effect; marginal (M) and conditional (C) R 2 values are provided. There were two analyses of Return rate (A and B), with predictors including either CORTinc or CORTnest, respectively.
Year was not included in analyses of Return rate B, because CORTnest was measure in one year only. Statistically significant main effects are in bold.