# Aromatic plants, nest bacterial diversity, and nestling condition in Corsican blue tits

Hélène Dion-phénix, Gabrielle Gingras, Claire Doutrelant, Anne Charmantier,

Steven W. Kembel, Denis Réale

► To cite this version:

Hélène Dion-phénix, Gabrielle Gingras, Claire Doutrelant, Anne Charmantier, Steven W. Kembel, et al. Aromatic plants, nest bacterial diversity, and nestling condition in Corsican blue tits. Oikos, In press, 10.1002/oik.11620. hal-05401775

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Submitted on 6 Dec 2025

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OIKOS

Research Article

Aromatic plants, nest bacterial diversity, and nestling condition in
 Corsican blue tits

Hélène Dion-Phénix ID<fnref n="1" />, Gabrielle Gingras ID<fnref n="1" />, Claire Doutrelant ID<fnref n="2" />,
Anne Charmantier ID<fnref n="2" />, Steven W. Kembel ID<fnref n="1" /> and Denis Réale ID<fnref n="1" />

Correspondence: Hélène Dion-Phénix (dion-phenix.helene@uqam.ca)

Oikos

2025: e11620

doi: 10.1002/oik.11620

Subject Editor: Jakub Szymkowiak

Editor-in-Chief:

Paulo R. Guimaraes

Accepted 28 October 2025

<figure><img src="image_3.png" /></figure>

According to the ‘nest protection hypothesis’, some passerines incorporate fresh aro-
matic plants into their nests which reduce pathogens that can negatively affect nestlings.
We experimentally evaluated the effect of five aromatic plant species on the nest bacte-
rial microbiota of Corsican blue tits *Cyanistes caeruleus*. The experimental addition of
aromatic plants decreased bacterial diversity in nests collected post-hatching. We also
detected a weak effect of aromatic plants on nest bacterial composition. In the observa-
tional approach, we tested the effect of these plants on the bacterial microbiota diversity
and composition of eggshells and nests, and on nestling condition and behaviour. Bacterial
diversity decreased with the quantity of aromatic plants in nests containing nestlings and
on eggshells during incubation, but only in one of the three studied populations. Again,
there was a weak effect of aromatic plants on bacterial composition in nests and no effect
on eggshell bacterial composition. Finally, nests with a high quantity of aromatic plants
tended to have bigger and taller nestlings in two out of three populations. The quantity of
aromatic plants was not associated with speed of feather development and with docility.
Our results support the ‘nest protection hypothesis’, while highlighting differences in the
effect of aromatic plants among populations. To our knowledge, our study is the first to
reveal a correlation between the presence of aromatic plants and the bacterial diversity of
nests and eggshells in a natural bird population, and to demonstrate experimentally the
direct effect of five aromatic plant species on bacterial diversity in nest material.

Key words: aromatic plant, bacterial community, behaviour, blue tit, body condition, Cyanistes caeruleus, experiment, nest material, nest protection hypothesis, self-medication, potpourri hypothesis.

Introduction

Parasites, such as pathogenic bacteria or ectoparasites, play a major role in shaping
the selective environment of their host (Lymbery 2015). Animals can develop vari-
ous defense strategies against parasites (Laland et al. 1999), such as self-medication,

© 2025 The Author(s). Oikos published by John Wiley & Sons Ltd on behalf of Nordic Society Oikos

This is an open access article under the terms of the Creative Commons
Attribution License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited.

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<footnote n="1">¹Département des sciences biologiques, Université du Québec à Montréal, Montréal, QC, Canada</footnote>

<footnote n="2">²CEFE, Univ Montpellier, CNRS, EPHE, IRD, Montpellier, France</footnote>
where animal use substances produced by other species (often plants) (Clayton and Wolfe 1993). By doing so, animals influence selective pressures of the parasites and themselves. Depending on their specificity, substances may affect the entire arthropod or microbial community, including beneficial species. However, for the added substances to be considered as self-medication, they must be detrimental to at least one parasite (Abbott 2014). For example, chimpanzees *Pan troglodytes schweinfurthii* build sleeping platforms (night nests) using foliage and stems from trees with flying arthropod-repellent properties (Samson et al. 2013). Simone et al. (2009) found that honeybees *Apis mellifera* collect antimicrobial resin from certain plant species, reducing bacterial load and thus using less energy on immune function. Additionally, monarch butterflies *Danaus plexippus* lay their eggs on toxic plants to protect their offspring from parasites and disease (Lefèvre et al. 2010).

Some bird species incorporate different types of green materials rich in volatile compounds into their nests at different stages of the reproductive period. For instance, male European starlings *Sturnus vulgaris* and spotless starlings *Sturnus unicolor* add green plants, such as *Achillea millefolium* and *Salix alba*, until egg-laying begins (Gwinner 1997, Gwinner et al. 2000). In contrast, red-shouldered hawks *Buteo lineatus* use fresh branches from coniferous and deciduous trees, continuously renewing them throughout the incubation and nestling stages (Díaz-Lora et al. 2019). Similarly, female blue tits *Cyanistes caeruleus* incorporate various aromatic plant species into their nests, starting at the end of nest construction and continuing throughout the nesting period (Lambrechts and Santos 2000, Mennerat et al. 2008, 2009a 2009c, Díaz-Lora et al. 2019). Considering the energy and time invested in selecting, transporting, and renewing fresh plants in the nest (Mainwaring and Hartley 2013), this behaviour is likely costly, and this suggests that its maintenance in evolutionary time is due to the fitness impacts of the behaviour. Several hypotheses have proposed mechanisms by which this behaviour is advantageous. These hypotheses are not mutually exclusive and may result in similar outcomes.

The 'nest protection hypothesis' assumes that the secondary volatile compounds released by plants have a direct effect on microbial pathogens and ectoparasites affecting nestlings (Clark and Mason 1985, Clark 1991, Lafuma et al. 2001). Similarly, Lambrechts and Santos (2000) proposed the 'pot-pourri hypothesis', which suggests that incorporating multiple aromatic plant species creates a mixture of volatile compounds that enhances defense against pathogens. The 'drug hypothesis' posits that certain plant species positively affect nestling condition and development by stimulating the immune system of nestlings (Gwinner et al. 2000, Garrido-Bautista et al. 2023). The 'olfactory crypsis hypothesis' suggests that plant scents could act as a form of camouflage, reducing the risk of nest predation. By masking the scent of nestlings, these plants might make it harder for predators to locate the nest, thereby increasing chick survival rates (Grieves et al. 2022, Garrido-Bautista et al. 2023). Finally,

the 'courtship hypothesis' proposes that adding aromatic plants to the nest is related to mate choice. This behaviour may primarily signal parental investment to potential mates (Brouwer and Komdeur 2004, Dubiec et al. 2013).

Several studies have investigated the link between aromatic plants in nests and nestling development, morphology, physiology, microbiota, or abundance of parasites in the nests (Table 1). However, their conclusions often diverge according to the species or traits studied, and in some cases even for the same species–trait combinations. No study has tested the effect of aromatic plants on nestling behaviour even though olfactory signals in the environment can modulate various behaviours in birds (Caro et al. 2015).

Studies on the 'nest protection hypothesis' have mostly focused on the possible antiparasitic effects of aromatic plants. Garrido-Bautista et al. (2023), Lafuma et al. (2001) and Tomás et al. (2012) found a negative correlation between the abundance of aromatic plants and the presence of some parasites in the nests, while others reported no such association (Gwinner et al. 2000, Gwinner and Berger 2005, Mennerat et al. 2008, Tomás et al. 2012). Garrido-Bautista et al. (2023) even found a positive association between aromatic plants and the presence of blowflies and facultative parasitic mites. Fewer studies have focused on the potential effects of aromatic plants on the bacteria living in the nest environment or on nestlings. To our knowledge, no study has yet explored the impact of aromatic plants on the natural microbiota of nests (but see Clark and Mason 1985). One study investigated this effect on eggshell microbiota (Ruiz-Castellano et al. 2016), and two studies focused on the skin microbiota of nestlings (Gwinner and Berger 2005, Mennerat et al. 2009a). According to these studies, aromatic plants reduce bacterial richness and density on nestlings and reduce bacterial growth on eggshells under certain conditions. Birds engage in other behaviours that limit bacterial growth, such as removal of fecal sacs (Azcárate-García et al. 2019) and preening with uropygial gland secretions (Jacob et al. 2014). The latter is thought to reduce the risk of pathogenic infestation by lowering the overall bacterial load (Cook et al. 2005b, Mennerat et al. 2009a, Shawkey et al. 2009). Other behaviours, including incubation and feeding, may promote the transmission and establishment of beneficial bacteria (Shawkey et al. 2009, van Veelen et al. 2017, 2018, Maki et al. 2020). The bacteria present in the nest environment and on eggshells can colonize the nestling gut (Maki et al. 2020), and the gut microbiota can, in turn, influence the nestling body condition. Indeed, the gut microbiota can affect the immune system (Mazmanian et al. 2005), digestion (Martino et al. 2018) or may favor competitive exclusion of pathogenic bacteria by beneficial bacteria (Servin 2004). Thus, simultaneously lowering total bacterial load and diversity while enhancing the beneficial microbiota in the immediate environment or on the nestlings themselves could improve their condition. Jacob et al. (2014) observed an increase in tarsus length in great tit *Parus major* nestlings from nests that were vaporized with a solution that inhibited bacterial growth.
<table><caption>Table 1. Summary of key findings from previous studies on the relationship between aromatic plants (AP) in nests and nestling condition, bacterial microbiota, and parasite presence.</caption><thead><tr><th>Study</th><th>Species</th><th>Objective</th><th>Method</th><th>Effect of aromatic plants (AP)</th></tr></thead><tbody><tr><td>Clark and Mason 1985</td><td>European starling <i>Sturnus vulgaris</i></td><td>Test the nest protection hypothesis by evaluating the inhibitory effect of AP used by starlings on bacterial growth</td><td>Experimental addition of 17 fresh plant species in bacterial cultures of four laboratory strains of bacteria, and on bacteria found in starling nests.</td><td><strong>Bacterial microbiota.</strong> AP preferred by starlings control bacterial growth of three pure laboratory strains and inhibits the growth of bacteria found in starling nests.</td></tr><tr><td>Garrido-Bautista et al. 2023</td><td>Blue tit <i>Cyanistes caeruleus</i></td><td>Test both the drug and the nest protection hypothesis by evaluating the relationships between AP and nestling traits, as well as ectoparasite infestation</td><td>Observation of plant abundance in natural nests and cross-fostering experiments, along with the experimental addition of one aromatic plant species, <i>Mentha</i> sp., in natural nests.</td><td><strong>Nestling traits.</strong> AP was positively correlated to egg mass and the heterophil/lymphocyte ratio in nestlings in one of the two study years, but showed no correlation with nestling body mass, hatching or fledging success, hemoglobin levels, erythrocyte sedimentation rate, or other health metrics. The experimental addition of mint resulted in a negative effect on flange color saturation and brightness, but in a positive effect on color hue.<br><strong>Parasites.</strong> AP was positively correlated with blowflies and facultative parasitic mites and negatively correlated with obligatory parasitic mites. The experimental addition of AP led to a decrease in flea abundance.</td></tr><tr><td>Gładalski et al. 2020</td><td>Blue tit <i>Cyanistes caeruleus</i></td><td>Test the drug hypothesis by evaluating the effects of AP on nestling body condition</td><td>Experimental addition of three aromatic plant species: <i>Lavandula angustifolia</i>, <i>Achillea millefolium</i>, <i>Helichrysum italicum</i>, in natural nests.</td><td><strong>Nestling traits.</strong> AP resulted in increased hemoglobin concentration but had no effect on glucose concentration.</td></tr><tr><td>Gładalski et al. 2025</td><td>Great tit <i>Parus major</i></td><td>Test both the drug and the nest protection hypothesis by evaluating the effect of AP on nestling condition and ectoparasite infestation</td><td>Experimental addition of four aromatic plant species: <i>Lavandula angustifolia</i>, <i>Mentha sp.</i>, <i>Achillea millefolium</i>, <i>Helichrysum italicum</i>, in natural nests.</td><td><strong>Nestling traits.</strong> AP resulted in increased hemoglobin and hematocrit concentration but had no effect on glucose concentration, body mass and wing length.<br><strong>Parasites.</strong> AP had no effect on the total ectoparasite loads but led to a decrease of hard ticks (<i>Ixodidae</i>) abundance.</td></tr><tr><td>Gwinner et al. 2000</td><td>European starling <i>Sturnus vulgaris</i></td><td>Test both the drug and the nest protection hypothesis by evaluating the effect of green plants on nestling condition and ectoparasite infestation</td><td>Replacing natural nests with artificial ones that include six species of green plants: <i>Aegopodium podagraria</i>, <i>Heracleum sphondylium</i>, <i>Sambucus niger</i>, <i>Anthriscus sylvestris</i>, <i>Achillea millefolia</i>, <i>Salix alba</i></td><td><strong>Nestling traits.</strong> Green plants had no effect on fledging success but resulted in increased hematocrit levels and body mass (greater in nests with a high mite load).<br><strong>Parasites.</strong> Green plants had no effect on the abundance of ectoparasite (<i>Der manyssus gallinae</i>, <i>Ceratophyllus gallinae</i>).</td></tr><tr><td>Gwinner and Berger 2005</td><td>European starling <i>Sturnus vulgaris</i></td><td>Test both the drug and the nest protection hypothesis by evaluating the effect of green plants on ectoparasite and bacteria infestation, as well as on nestling condition</td><td>Replacing natural nests with artificial ones that include six species of green plants: <i>Aegopodium podagraria</i>, <i>Heracleum sphondylium</i>, <i>Sambucus niger</i>, <i>Anthriscus sylvestris</i>, <i>Achillea millefolia</i>, <i>Salix alba</i></td><td><strong>Nestling traits.</strong> Green plants resulted in increased fledgling body mass.<br><strong>Bacterial microbiota.</strong> Green plants resulted in decreased skin nestling bacterial load in the second week of life of nestlings, later in the season (June).<br><strong>Parasites.</strong> Green plants had no effect on ectoparasite (<i>Der manyssus gallinae</i>).</td></tr></tbody></table>

(continued)
<table><caption>Table 1. Continued.</caption><thead><tr><th>Study</th><th>Species</th><th>Objective</th><th>Method</th><th>Effect of aromatic plants (AP)</th></tr></thead><tbody><tr><td>Gwinner et al. 2018</td><td>European starling <i>Sturnus vulgaris</i></td><td>Test the effect of green plants on incubation behaviour and its subsequent impact on embryonic development and nestling condition</td><td>Replacing natural nests with artificial ones that include six species of green plants: <i>Aegopodium podagraria</i>, <i>Heracleum sphondylium</i>, <i>Sambucus niger</i>, <i>Anthriscus sylvestris</i>, <i>Achillea millefolia</i>, <i>Salix alba</i></td><td><strong>Nestling traits.</strong> Green plants resulted in increased embryonic development (through increased incubation time), in increased nestling body mass, and had no effect on hatching success.</td></tr><tr><td>Lafuma et al. 2001</td><td>Blue tit <i>Cyanistes caeruleus</i></td><td>Test the nest protection hypothesis by evaluating the effect of AP on ectoparasite</td><td>Experimental addition of one or four aromatic plant species: <i>Achillea ligustica</i>, <i>Helichrysum italicum</i>, <i>Lavandula stoechas</i> and <i>Cistus creticus</i>, in artificial nestboxes in laboratory conditions using domestic chicks</td><td><strong>Parasites.</strong> AP resulted in decreased mosquitoes (<i>Culex pipiens</i>) (more with plant mixture than mono-species treatment).</td></tr><tr><td>Mennerat et al. 2008</td><td>Blue tit <i>Cyanistes caeruleus caeruleus</i></td><td>Test the nest protection hypothesis by evaluating the effect of AP on ectoparasite infestation and its subsequent impact on nestling body condition</td><td>Observation of plant abundance in natural nests and experimental addition of two aromatic plant species: <i>Lavandula stoechas</i> and <i>Helichrysum italicum</i>, to natural nests</td><td><strong>Nestling traits.</strong> AP had no effect on nestling survival, body mass (9-11 days), and hematocrit level (14-15 days).<br><strong>Parasites.</strong> AP had no effect on the abundance of ectoparasite (<i>Protocalliphora</i> sp.) in natural non-manipulated nests.</td></tr><tr><td>Mennerat et al. 2009a</td><td>Blue tit <i>Cyanistes caeruleus caeruleus</i></td><td>Test the nest protection hypothesis by evaluating the effect of AP on bacterial load</td><td>Experimental addition of two aromatic plant species: <i>Lavandula stoechas</i> and <i>Helichrysum italicum</i>, in natural nests.</td><td><strong>Bacterial microbiota.</strong> AP resulted in decreased bacterial richness and density on nestlings, only under high blowfly (<i>Protocalliphora</i> sp.) infestation rate.</td></tr><tr><td>Mennerat et al. 2009b</td><td>Blue tit <i>Cyanistes caeruleus caeruleus</i></td><td>Test the drug hypothesis by evaluating the effect of AP on nestling development and condition</td><td>Experimental addition of five aromatic plant species: <i>Lavandula stoechas</i>, <i>Helichrysum italicum</i>, <i>Calamintha nepeta</i>, <i>Achillea ligustica</i>, <i>Pulicaria odora</i>, in natural nests and experimental brood enlargement</td><td><strong>Nestling traits.</strong> AP resulted in increased nestling body mass (in enlarged broods, but not in non-modified broods), in increased hematocrit level, and in faster feather development (only in one of the two years of the study).</td></tr><tr><td>Pires et al. 2020</td><td>Blue tit <i>Cyanistes caeruleus</i></td><td>Test the drug hypothesis by evaluating the effect of AP on reproductive performance and nestling development</td><td>Experimental addition of three aromatic plant species: <i>Lavandula dentata</i>, <i>Calamintha nepeta</i>, <i>Dittrichia viscosa</i>, in natural nests</td><td><strong>Nestling traits.</strong> AP resulted in increased tarsus length in large broods and had no effect on nestling survival rate and body mass.</td></tr><tr><td>Ruiz- Castellano et al. 2016</td><td>Spotless starling <i>Sturnus unicolor</i></td><td>Test the nest protection hypothesis by evaluating the effect of AP and feathers on eggshell bacterial load</td><td>Experimental addition of four aromatic plant species: <i>Marrubium vulgare</i>, <i>Artemisia barrelieri</i>, <i>Lamium amplexicaule</i>, <i>Anacyclus clavatus</i>, in natural and artificial nests without incubation, and with or without experimental contamination by bacterial strains known to be able to cross avian eggshells</td><td><strong>Bacterial microbiota.</strong> AP had no effect on the bacterial microbiota on eggshells in natural nests but resulted in decreased bacterial growth on eggshells from artificial experimentally contaminated nests.</td></tr></tbody></table>

(continued)
<table><caption>Table 1. Continued.</caption><thead><tr><th>Study</th><th>Species</th><th>Objective</th><th>Method</th><th>Effect of aromatic plants (AP)</th></tr></thead><tbody><tr><td>Tomás et al. 2012</td><td>Blue tit<br><i>Cyanistes caeruleus</i></td><td>Test both the drug and the nest protection hypothesis by evaluating the effect of AP on ectoparasites, as well as on nestling body condition and immune response</td><td>Experimental addition of two aromatic plant species: <i>Lavandula stoechas</i>, <i>Santolina chamaecyparissus</i>, in natural nests</td><td><b>Nestling traits.</b> AP had no effect on nestling body mass, tarsus length, wing length, phytohaemagglutinin, and hatching and fledging success.<br><b>Parasites.</b> AP resulted in decreased the abundance of fleas <i>Ceratophyllus gallinae</i> only in yearling female nests and had no effect on the abundance of mites <i>Der manyssus gallinoides</i>, blowfly <i>Protocalliphora azurea</i>, wasps <i>Nasonia vitripennis</i>, blood-sucking flies: blackflies (<i>Simuliidae</i>) and biting midges (<i>Ceratopogonidae</i>).</td></tr></tbody></table>

The aim of this research was to test the effect of aromatic plants on nest and eggshell bacterial microbiota, and on the body condition and behaviour of Corsican blue tit nestlings *Cyanistes caeruleus ogliastrae*. We employed an experimental approach to investigate the direct effects of five aromatic plant species commonly used by blue tits on the bacterial microbiota of the nest. While many studies have experimentally added fresh plants to nests occupied by birds, where many interactions are at play (i.e. incubation, feeding behaviour, nest sanitation), no study has yet tested the single and combined effect of the addition of aromatic plants in a semi-controlled environment using nests collected from a natural population. We also used an observational approach to study the effect of aromatic plants on nest and eggshell bacterial microbiota, and nestling body condition and behaviour, across three natural populations of Corsican blue tits.

Corsican blue tits use a variety of plant materials to build their nests. The nest foundation is mostly made of moss, small twigs and grass (Lambrechts and Santos 2000). They daily add aromatic plant fragments on top of the nest in quantities varying between 0.03 g and 0.31 g dry mass (Mennerat et al. 2008). The maximal quantity found in nest can reach up to 1.3 g dry mass (Mennerat et al. 2009c). The common aromatic plant species added by Corsican blue tits include *Lavandula stoechas*, *Mentha suaveolens insularis*, *Helichrysum italicum*, *Pulicaria odorata* and *Achillea ligustica* (Petit et al. 2002). These five genera are known for their antibacterial properties (*Achillea Bouteche* et al. 2024, *Helichrysum Viegas et al. 2014*, *Lavandula Zoubi* et al. 2020, *Mentha Afrokh* et al. 2024, *Pulicaria Ezoubeiri* et al. 2005). Some of these plants also have antifungal and anti-inflammatory properties (Lambrechts and Santos 2000, Gładalski et al. 2020). Female blue tits vary in their selection of aromatic plant species added to nests, suggesting individual preferences for certain aromatic plant species (Mennerat et al. 2009c). Such a repeatable choice in aromatic nest composition suggests that birds may select different plant species for distinct beneficial effects, a hypothesis that has rarely been tested (but see Lafuma et al. 2001).

Following the 'nest protection hypothesis', we predicted that the volatile compounds released by aromatic plants present in nests reduce the diversity and alter the composition

of the bacterial microbiota in blue tit nests. We collected abandoned nests to perform an experiment involving the addition of aromatic plants. We compared the diversity and composition of bacterial microbiota across nine plant treatments. Following the 'potpourri hypothesis', we predict that the addition of two aromatic plant species should decrease bacterial diversity and modify the composition of bacteria more than the addition of one aromatic species. We also predicted that the quantity of aromatic plants added by the parents during the breeding season would be negatively correlated with the diversity of the bacterial microbiota of nests and of eggshells and positively correlated with nestling body condition. To assess the nestling condition, we measured three traits (i.e. nestling body mass, tarsus length, and feather development). To explore the effect of aromatic plant on nestling behaviour, we measured docility in hand, a repeatable and heritable behaviour that represent reaction to predator (Brommer and Kluen 2012, Class et al. 2014, Dubuc-Messier 2017). The docility of nestlings is correlated with age and body mass (Dubuc-Messier 2017) and thus, is linked to nestling development.

Methodology

Experimental approach

We worked on three blue tit populations in Corsica during the spring of 2023 (EMuro, 42°35'N, 08°57'E; DMuro, 42°32'N, 08°55'E; EPirio, 42°34'N, 08°44'E). To assess the single and combined effects of different aromatic plants on nest microbiota, we collected 19 nests (EMuro; n=6; DMuro, n=6; EPirio, n=7) that had either experienced parental abandonment or clutch predation. Nests were abandoned at different stages of the breeding period and may differ in their initial bacterial microbiota. We thus classified the nests according to their breeding stages [pre-incubation (n=7), incubation (n=6) and post-hatching (n=6)]. We transferred the nests in sterile bags using sterile gloves and placed them in handmade sterilized nestboxes until the start of the experiment (mean: 10 days, min: 1 day, max: 22 days). We conducted the experiment in four-time blocks (first day of block 1: 2023-04-26, block 2: 2023-05-07, block 3: 2023-05-21, block 4: 2023-06-10).
## (A) Experimental Design

<figure><img src="image_5.png" /></figure>

### Treatments

#### Addition of one species

<figure><img src="image_6.png" /></figure>

Achillea
ligustica

Mentha
suaveolens

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

Lavandula
stoechas

<figure><img src="image_9.png" /></figure>

Pulicaria
odorata

<figure><img src="image_10.png" /></figure>

Helichrysum
italicum

#### Addition of two species

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

#### Control - no addition

<figure><img src="image_20.png" /></figure>

## (B) Observational Design

<figure><img src="image_21.png" /><figcaption>Figure 1. Summary of (A) experimental and (B) observational designs. In (B), the drawing shows how the observer hold the nestlings for the docility test.</figcaption></figure>

Each collected nest was weighed and homogenized, then divided into nine samples of equal mass, totaling 171 samples, that were subjected to nine treatments (Fig. 1A). The treatments corresponded to the addition of five single species (*Lavandula stoechas*, *Mentha suaveolens insularis*, *Helichrysum italicum*, *Pulicaria odorata* and *Achillea ligustica*), three

combinations of plant duos frequently observed in blue tit nests (*Lavandula stoechas* and *Mentha suaveolens insularis*, *Lavandula stoechas* and *Helichrysum italicum*, and *Mentha suaveolens insularis* and *Helichrysum italicum*), and one control sample with no plant addition. Each sample was then placed in plastic containers with a 26 mm hole covered with
a net to prevent birds from entering. These containers were designed to have a similar incubation chamber and hole entrance dimensions to the *Schwegler* artificial nestboxes used in our monitored populations to mimic their aeration conditions (Fig. 1A). Plastic material has been shown to have similar microclimate conditions to the commonly used wooden boxes (Scott-Baumann et al. 2022). These containers were hung outside over a one-week period. To induce an aromatic environment in the nestboxes, we added fresh aromatic plants every two days (day 1, 3 and 5), which was consistent with the observed behaviour of blue tits regularly renewing fresh plants in their nests (Mennerat et al. 2008).

We added aromatic plants to the top of the nest samples except for the control treatments, using a ratio of 1:3 (1 g of fresh aromatic plants for 3 g of nest sample). The mean mass of aromatic plant added in our samples was 0.56 g, corresponding to 0.14 g dry mass which is in the natural range of amounts of aromatic plants added by blue tits (0.03 g to 0.31 g). For the three duo treatments, we divided this value into two equal parts. To ensure freshness, we collected the five aromatic plant species on the same day they were added to the samples. To minimize variation in the microbiota of plant surfaces, we collected all plants of the same species from a single area of approximately 100 m². We collected *Lavandula stoechas* and *Mentha suaveolens insularis* near the EMuro site and *Achillea ligustica*, *Pulicaria odorata* and *Helichrysum italicum* near the DMuro site. We collected plants using gloves and scissors sterilized with 30% bleach followed by 70% ethanol and stored them in sterile bags at room temperature until being added to nest samples (within a maximum of four hours). To prevent cross-contamination between samples, we used new sterile gloves and disinfected surfaces and equipment with 30% bleach followed by 70% ethanol for each individual nest manipulation. On day 7, we removed aromatic plant fragments from each nest sample using sterile tweezers. We manipulated the control samples the same way as treatment samples (i.e. we opened the container to mimic plant addition at day 1, 3 and 5, and we manipulated the samples with tweezers on day 7). We also collected fresh plant leaves of the five species to compare their bacterial microbiota. We finally placed the samples in sterile bags and stored at -20°C until DNA extraction.

## Observational approach

In spring 2023, we followed the reproduction of the three populations as part of a long-term individual monitoring program (Charmantier et al. 2016, Blondel et al. 2006). These three populations occupy contrasting habitats and present morphological, physiological, behavioural, life-history and phenological differences. We characterized the nest composition of 212 first and second clutches in these populations (Mean laying date in DMuro: 11 April ± 16 days, EMuro: 14 April ± 10 days, EPirio: 4 May ± 9 days) during both the incubation and post-hatching stages every seven days or less for a total of 746 observations. Without disturbing the nest, we noted the percent cover of each of the same five aromatic plant species used in the experimental approach (*Lavandula*

*stoechas*, *Mentha suaveolens insularis*, *Helichrysum italicum*, *Pulicaria odorata* and *Achillea ligustica*). We estimated the percent cover of plants observed on top of the nest using Braun-Blanquet density scores (Braun-Blanquet 1932).

We captured nestlings at day 13–16 after hatching to measure indicators of their fitness and development. Most of the measures were made at 15 days (number of clutches: DMuro: 13 days: 2; 14 days: 7; 15 days: 72; 16 days: 1; EMuro: 13 days: 0; 14 days: 7; 15 days: 37; 16 days: 0; EPirio: 13 days: 12; 14 days: 16; 15 days: 23; 16 days: 1). Immediately after extracting a nestling from its nest, we measured docility by holding it upright with its head facing upward (Fig. 1B). We held each chick with one hand, positioning its head between our index and middle finger and its legs between our index and thumb to allow the bird to move its tail and wings freely. We counted the number of struggles within 10 seconds and calculated docility as the negative of this number (docility = -number of struggles) such that the most docile individuals had a value of 0. Docility showed a heritability of 0.40 but also increased with nestling age and mass (Dubuc-Messier 2017). Docility therefore represented individual differences in behaviour but may also be linked to development. Next, we weighted the nestling to the nearest 0.1 g using a portable digital scale (500 g/0.01 g), measured its tarsus length with a digital caliper to the nearest 0.01 mm, and scored its wing feather development based on an age table indicating typical feather development in blue tits (Supporting information). The measurement process for each chick took less than one minute. Body mass, tarsus length and feather development served as indicators of nestling conditions [DMuro (576 chicks, 82 clutches), EMuro (267 chicks, 43 clutches), EPirio (306 chicks, 51 clutches)].

We sampled nest material and eggshell swabs from 46 clutches, once during incubation and again during the post-hatching stage. For nest samples, we used sterile gloves to collect approximately 1 cm of the top layer from one side of the nest. During the incubation stage, we randomly selected either the right or left side for sampling, and during the post-hatching stage, we sampled the opposite side. Each nest sample was placed in a sterile bag. For eggshell swabs, we humidified a sterile swab with 1X phosphate-buffered saline (PBS 1X), gently swabbed the visible surfaces of the eggs during incubation and placed the tip of the swab in an Eppendorf tube filed with 200 µl of PBS 1X by breaking the wooden handle between the cap and the top of the tube. We stored all samples at -20°C until DNA extraction. We also collected field negative controls by opening sterile bags in the field and inserting our gloved hand inside, as done in the sampling procedure described above.

## DNA extraction of nest samples

DNA extraction was carried out in a DNA free room at the Centre d'Ecologie Fonctionnelle et Evolutive (CEFE-CNRS) in Montpellier, France. We washed the nest samples by adding 50 ml of PBS 1X solution and agitating for 5 minutes. We transferred 35 ml of the wash into a 50 ml Falcon tube. The tubes were then centrifuged at 4000 g for 20 minutes.
After removing the supernatant, the pellet was resuspended in 1 ml of PBS 1X. DNA extraction was performed using the PowerSoil Pro kit according to the manufacturer protocol, with the following modifications: mechanical lysis was extended to 20 minutes instead of 10 minutes, and 60 µl of elution solution was used instead of 200 µl. The extracted DNA was stored at -20°C until sequencing. As extraction negative controls, all DNA preparation and extraction steps were conducted using sterile bags without nest samples.

## DNA library preparation and sequencing

For each sample, we performed a PCR amplification of the V5–V6 region of the bacterial 16S rRNA gene using host DNA-excluding 799F/1115R primers (Chelius and Triplett 2001). The PCR was performed in a 25 µl mixture containing 1 µl of DNA extract, 0.2 µM of each primer, 0.5 U of Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific), 1X of Phusion HF Buffer, 0.2 mM of dNTPs, 3% DMSO and following this programme: initial denaturation at 98°C for 30 s, 35 cycles of denaturation at 98°C for 15 s, annealing at 64°C for 30 s and elongation at 72°C for 30 s and an extension step of 72°C for 10 min. For PCR negative controls, we replaced DNA with sterile water, and for PCR positive controls, we used ZymoBIOMICS Microbial Community DNA Standard (Zymo Research). We visualized all PCR products on 2% agarose gel, and we normalized them using Just-a-plate 96 PCR Purification and Normalization kit following the manufacturer's protocol (Charm Biotech).

We pooled the samples from the experimental study to form a first sequencing library, and the samples from the observational study in a second library. The pooled samples were purified with a ratio of 0.75 of NucleoMag using the manufacturer protocol (Macherey-Nagel). Quality control of the libraries was assessed as follows: libraries were quantified using the Qubit dsDNA HS Assay Kit (Invitrogen) and the NEBNext Library Quant Kit for Illumina (New England BioLabs), and the average fragment size was determined using a Bioanalyzer instrument (Agilent Technologies). Before sequencing, the PhiX control library (Illumina) was spiked into the amplicon pool to improve sequence yield. The sequencing was performed on an Illumina Miseq at the UQAM CERMO-FC Genomics Platform using the MiSeq reagent kit v3 (2 × 300 cycles; Illumina). The samples of the experimental study were sequenced in one run and the samples of the observational study in another run. Sequence data have been deposited in a public data repository (experimental study: https://doi.org/10.6084/m9.figshare.27953055 and observational study: https://doi.org/10.6084/m9.figshare.28640093).

## Data processing

We processed Illumina sequencing data using DADA2 ver. 1.12.1 (Callahan et al. 2016) to produce an error-corrected table of amplicon sequence variant (ASV) abundances in each sample for experimental samples, nest observational samples and eggshell swab samples separately. We used default parameters for all DADA2 analyses unless specified below.

Sequences were trimmed to remove forward and reverse primers and to a length of 160 nucleotides (forward reads) and 200 nucleotides (reverse reads) before ASV identification and merging of paired-end reads into a single ASV sequence.

We assigned a taxonomic annotation for all ASVs using the SILVA database ver. 138.1 (Quast et al. 2012) and removed all non-bacterial sequences (chloroplasts, mitochondria, eukaryotes, archaea; 0.09% of sequences for the experimental study, 0.007% for nest samples from the observational study, and 0.04% for eggshell swabs from the observational study) and sequences without a taxonomic phylum assigned (0.4% of sequences for the experimental study, 1.3% for nest samples from observational study, and 0.5% for eggshell swabs from observational study) before further data processing. We rarefied the ASV table to 14027 reads per sample for the experimental study, to 7363 for the nest samples from the observational study, and to 1464 for the eggshell swab samples from the observational study using the R package 'phyloseq' (McMurdie and Holmes 2013). We used these rarefaction thresholds to reach a plateau of ASV richness per sample, while allowing including all the samples except three eggshell swabs. All negative controls were excluded at the rarefaction step because they contained fewer sequences than the corresponding rarefaction threshold except for three eggshell swab negative controls. The composition of negative control and the remaining eggshell swab samples were different, and thus we removed these remaining negative control samples from further analysis. All PCR positive controls contained the ASVs expected from the known composition of the mock community and were removed from further analysis. Further details about rarefaction and control samples are provided in the Supporting information. The final dataset for the experimental study contained 169 nest bacterial microbiota samples (19 nests × 9 treatments – 2 discarded samples) and 15 plant bacterial microbiota samples (5 species × 3 samples). The final dataset for the observational study contained 92 nest bacterial microbiota samples (46 clutches × 2 breeding stages) and 43 eggshell swab samples (43 clutches during incubation).

## Statistical analysis

To test the effect of the addition of aromatic plant species on the bacterial diversity of nest samples, we ran a linear mixed model of the Shannon diversity in relation to the treatment, in interaction with the breeding stage of the nest at collection (pre-incubation, incubation, and post-hatching) and controlling for the nest of origin (before subdivision into 9 subsamples) as a random effect (n = 169) (model 1). We included the interaction with breeding stage because nest bacterial community and diversity could differ between the three stages and these communities may be affected differently by the treatments. We used a Levene's test to test for homogeneity of variances in Shannon diversity between breeding stages. We used a distance-based redundancy analysis with the Hellinger distance (dbRDA) to test if the addition of plant and the plant species, both in interaction with the breeding stages, and including the nest of origin as a cofactor, affected the
bacterial composition of the samples (n=169) (model 2). We also performed a PERMANOVA with the same model structure as for the dbRDA to calculate the adjusted proportion of variance explained by each term, and we tested the multivariate homogeneity of dispersion between the levels of each factor.

We used the quantity of aromatic plants as an explanatory variable for the observational study. We summed up the percent cover of the five studied aromatic species for each observation, using the median of each Braun-Blanquet class as an index of the quantity of plant material. We then calculated the mean of these values by clutch and by breeding stage. Finally, we applied a log-transformation to ensure that we had enough values along the axis of the explanatory variable. We used the term 'quantity of aromatic plants' in the text to refer to the log-transformed mean aromatic plant cover.

We used a linear mixed model to estimate the relationship between the bacterial Shannon diversity in the nests and the quantity of aromatic plants, categorised by population and by breeding stage (triple interaction). We also included the nest identity as a random effect (n=92) (model 3). We included the interaction with breeding stage for the same reasons as in the experimental approach, and the interaction with the population, since previous studies in our system have revealed that our populations often respond differently to the same environmental variables (Charmantier et al. 2016, Bonamour et al. 2019). We used a linear model to measure the relation between the bacterial Shannon diversity of eggshells (one sample per nest during incubation) and the quantity of aromatic plants by population (double interaction) (model 4). We also compared the quantity of aromatic plants between the three populations and the breeding stages (double interaction), and we included the nest identity as a random effect using a linear mixed model (n=746) (model 5). To estimate the among-nest variation in bacterial diversity in the experimental study (model 1) and in the observational study (model 3), and the among-female variation in the quantity of aromatic plants used as nest material (model 5), we calculated the adjusted repeatability (Eq. 1), and the unadjusted repeatability (Eq. 2) of the nest identity.

$$R_{adj}^2 = \sigma_{nest \diamond identity}^2 / \left( \sigma_{nest \diamond identity}^2 + \sigma_{residual}^2 \right) \quad (1)$$

$$R_{unadj}^2 = \sigma_{nest \diamond identity}^2 / \left( \sigma_{fixed}^2 + \sigma_{nest \diamond identity}^2 + \sigma_{residual}^2 \right) \quad (2)$$

We used a dbRDA with the Hellinger distance to measure the relation between the bacterial composition of nest samples and the quantity of aromatic plants for each population and breeding stage (triple interaction), and we included the nest identity in the model (n=92) (model 6). We finally used a dbRDA with the Hellinger distance to measure the relation between the bacterial composition of eggshells and the quantity of aromatic plants for each population (double interaction) (n=43) (model 7). We also performed a PERMANOVA for these last two models with the same model structure to

calculate the adjusted proportion of variance explained by each term, and we tested the multivariate homogeneity of dispersion between the levels of each factor.

To test the effect of the quantity of aromatic plants in the nest on nestling traits, we first performed a principal coordinate analysis (PCoA) on nestling traits using the Euclidean distance and used the scores of the first and second axis for each nestling in two subsequent models. In these models, we examined the relationship between the quantity of aromatic plants and the PCoA 1 score (model 8) and PCoA 2 score (model 9) controlling for the age of nestlings at the time of measurement and including the nest identity as a random effect (n=1149, 576 nestlings from DMuro, 267 nestlings from EMuro, and 267 nestlings from EPirio). We also ran a post hoc test on models 8 and 9 to determine whether the slope of the effect of the quantity of aromatic plants for each population differed from zero.

We used R to run all analyses and to produce the figures. We used the R package 'vegan' (Oksanen et al. 2024) for the dbRDA, the PERMANOVA, and the test of multivariate homogeneity of dispersion, and we used the function adonis_OmegaSq from the R package 'MicEco' (Russel and Oksanen 2025, https://github.com/Russel88/MicEco) to calculate an adjusted R² of each effect of the PERMANOVA. We used the R package 'lme4' to fit all the linear mixed models (Bates et al. 2015), 'lmerTest' to estimate p-values of t-tests of the estimates (Kuznetsova et al. 2017), and 'rptR' to estimate by parametric bootstrapping the 95% confidence interval and p-value associated with the adjusted and non-adjusted repeatability of the random effects in our models (Stoffel et al. 2017). We used the R package 'car' for the Levene's test (Fox and Weisberg 2019) and the package 'emmeans' (Lenth 2025) to test whether each individual slope in interaction terms differed from zero. Metadata and codes to reproduce all analysis and figures have been deposited in a GitHub public repository (https://github.com/HeleneDionPhenix/aromatic_plants).

# Results

## Experimental approach

### Bacterial diversity of nest samples

The addition of aromatic plant fragments in nest samples led to a decrease in Shannon diversity of nest bacterial communities for the eight treatments with plant addition compared to the control treatments, for the nests collected during the post-hatching stage but not for the two other stages (Fig. 2A). The bacterial diversity reduction effect of plant addition with two species combined did not differ from that of plant addition with a single species (Fig. 2A, control mean diversity=4.64, smallest differences with Mentha-Helichrysum treatment=-0.38, t=-2.13, p=0.035, statistical details in the Supporting information). We found no difference in Shannon diversity between the control samples from different breeding stages, although control samples from
<figure><img src="image_22.png" /><figcaption>Figure 2. Effect of the addition of aromatic plants at three breeding stages on the bacterial Shannon diversity estimated from a linear mixed model (model 1, n = 169). (A) Mean Shannon diversity of each treatment with 95% confidence intervals. (B) Shannon diversity of each sample; each line represents a nest (see the Supporting information for statistical details).</figcaption></figure>

incubation and pre-incubation nests tended to have a higher Shannon diversity than post-hatching nests (mean difference between post-hatching and incubation controls = 0.58, t = 1.53, p = 0.140; mean difference between post-hatching and pre-incubation controls = 0.52, t = 1.42, p = 0.170). There was a higher heterogeneity in Shannon diversity across post-hatching nests than across nests from incubation and

pre-incubation stages (Levene's test: F<sub>2,165</sub> = 25.8, p < 0.001, Fig. 2B). Samples from the same nest had similar Shannon diversities (nest adjusted repeatability = 80%, p < 0.001).

**Bacterial composition of nest samples**

Nest samples harbored a different bacterial composition according to the nest from which they came from and the
<table><caption>Table 2. Effect of plant addition (compared to control) and plant species at three breeding stages on the bacterial composition of the nest samples, accounting for the nest of origin as accessed by a dbRDA on the Hellinger distance (model 2, n=169).</caption><thead><tr><th>Source of variation</th><th>df</th><th>Sum of squares</th><th>Pseudo-F</th><th>Adjusted-R<sup>2</sup></th><th>p-value</th></tr></thead><tbody><tr><td>Plant addition</td><td>1</td><td>0.27</td><td>1.41</td><td>0.002</td><td>0.080</td></tr><tr><td>Plant species</td><td>7</td><td>1.34</td><td>1.01</td><td>&lt;0.001</td><td>0.446</td></tr><tr><td>Breeding stage</td><td>2</td><td>11.59</td><td>30.72</td><td>0.261</td><td>0.001</td></tr><tr><td>Nest</td><td>16</td><td>44.55</td><td>14.76</td><td>0.567</td><td>0.001</td></tr><tr><td>Plant addition:Breeding stage</td><td>2</td><td>0.48</td><td>1.28</td><td>0.003</td><td>0.099</td></tr><tr><td>Plant species:Breeding stage</td><td>14</td><td>2.55</td><td>0.97</td><td>&lt;0.001</td><td>0.637</td></tr><tr><td>Residual</td><td>125</td><td>23.58</td><td></td><td></td><td></td></tr><tr><td>Total</td><td>167</td><td>84.36</td><td></td><td></td><td></td></tr></tbody></table>

breeding stage when they were collected, with these factors explaining 57% and 26% of the total variation in composition among samples respectively (Table 2, Fig. 3). We found a weak tendency that plant addition affected nest bacterial composition regardless of the plant species, and a tendency that this effect varied between breeding stages (Table 2). Moreover, control samples (open squares in Fig. 3) were frequently associated with the highest values on the first axis in the ordination (RDA1) compared to the other samples from the same nest, especially so for post-hatching nests. For some nests, the plant addition samples had a similar bacterial composition, whatever the plant species added (full circles, close to each other), but differed from the control samples (Fig. 3B). As for Shannon diversity, the bacterial composition of samples from post-hatching nests was more heterogeneous than the bacterial composition of samples from nests in pre-incubation and incubation stages (multivariate homogeneity of group dispersions: $F_{2,166}=9.86, p < 0.001$; Fig. 3A).

## Observational approach

### Nest and eggshell bacterial diversities

We observed a negative effect of aromatic plants on the bacterial diversity of nests collected during post hatching but only in EPIrio ($-0.90 \pm 0.35, t=-2.57, p=0.01$, model 3, Fig. 4B) but observed no effect on nests during incubation (Fig. 4A). The Shannon diversity of bacteria on eggshells during incubation decreased with the quantity of aromatic plants added by the parent in EPIrio ($-0.52 \pm 0.26, t=-2.03, p=0.05$, model 4, Fig. 4C). For the populations of DMuro and EMuro, the Shannon diversity of nests (incubation and post-hatching) and eggshells (incubation) did not vary with the quantity of aromatic plants observed in the nests. For both breeding stages, the nests from the DMuro population contained more aromatic plants than nests from the two other populations (EPIrio-DMuro: $-0.69 \pm 0.14, t=-4.91, p < 0.001$; EMuro-DMuro: $-0.40 \pm 0.15, t=-2.64, p=0.009$), and there was no difference in the quantity of aromatic plants found in the nests during incubation and post-hatching for the three populations (model 5, Fig. 4D). For complete statistical information see the Supporting information.

### Nests bacterial compositions

The bacterial composition of nest material varied across the three populations, the breeding stages, and the nestboxes,

these three factors explaining respectively 7%, 4%, and 33% of the total variation among nest samples (model 6, Table 3). As shown by an adjusted-R² of 0.01, the quantity of aromatic plants observed in nests had a weak but significant effect on the bacterial composition of the nests (Table 3). Furthermore, there was no clear pattern of the effect of aromatic plant on the ordination Fig. (Fig. 5A). This effect did not vary across populations and stages (interactions in Table 3). We also noticed that the difference in the bacterial composition of nests during the post-hatching stage compared to the incubation stage was more important in the EPIrio population than in DMuro and EMuro populations (Fig. 5B).

### Eggshell microbiota composition

We found no significant effect of the quantity of aromatic plants found in the nest during incubation on eggshell bacterial composition, no matter the population, and no difference in eggshell bacterial composition across populations (model 7, Table 4).

### Nests repeatability

Across all models, the among-nest variation in bacterial microbiota diversity and composition was significant as was the among-nest variation in quantity of aromatic plants added by females during the reproductive period (Table 5). In the experimental study, after removing the variation explained by the plant addition treatment and by breeding stage, the variation in Shannon diversity among nests explained 80% of the residual variation, which indicated that the homogenization process before dividing the nest material among the nine samples was successful. The among-nest variation in the quantity of aromatic plants the parents added to the nest throughout the breeding season represented 30% of the total variation.

### Nestling condition

Nestling body mass and tarsus length were correlated at 99.9% on the first two axes of the ordination and respectively at 78.6% and 78.4% with the first axis of the ordination of nestling traits (Fig. 6A). Feather development and docility were correlated at -93.0% on the first two axes of the ordination and respectively at 56.6% and -78.2% with the second axis of the ordination (Fig. 6A). The PCoA1 scores (i.e. body mass and tarsus length) tended to increase with the
<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /><figcaption>Figure 3. Bacterial composition in relation with the treatment (plant addition and control treatments), for each breeding stage and considering the nest from which the samples came from as assessed by a distance-based redundancy analysis (dbRDA) on the Hellinger distance (model 2, n = 169). Open square dots represent control samples, round dots samples with plant addition, and the lines link samples from the same nests. All the figures present the results of the model 2, Table 2; (A) shows all the samples together, (B) presents the samples by breeding stage.</figcaption></figure>

quantity of aromatic plants in DMuro (0.02 ± 0.01, t=1.83, p=0.07) and EMuro (0.03 ± 0.02, t=1.81, p=0.07) populations, and there was no relation in EPIrio (0.00 ± 0.02, t=-0.03, p=0.98) (model 8, Fig. 6B). The quantity of aromatic plants was not correlated to PCoA2 score (i.e. feather development and inverse docility) in any of the three populations (model 9, Fig. 6C). For complete statistical information see the Supporting information.

## Discussion

Results from our experiment indicated that the five tested aromatic plant species reduced bacterial diversity in nests

that have contained nestlings (post-hatching), but not in nests collected during pre-incubation or incubation. Our observational study on wild blue tits revealed a negative relationship between aromatic plants and bacterial diversity in nests collected during post-hatching and on eggshells during incubation, but only in one of the three studied populations (EPIrio). No such relationship was observed in the other two populations (DMuro and EMuro). Aromatic plants had a weak effect on nest bacterial composition, with no variation among populations and breeding stages, and no effect on eggshell bacterial composition. Additionally, nests with a high quantity of aromatic plants tended to host heavier and taller nestlings in DMuro and EMuro populations and were not linked to docility and feather development.
<figure><img src="image_25.png" /><figcaption>Figure 4. Effect of the quantity of aromatic plants (log-transformed) on the bacterial Shannon diversity of (A) nests during incubation, (B) nests during post-hatching estimated with a linear mixed model (model 3, n = 92), and (C) eggshells during incubation estimated with a linear model (model 4, n = 46). (D) The mean differences of the quantity of aromatic plants in nests during incubation or post-hatching between the three populations, estimated with a linear mixed model (model 5, n = 746) (see the Supporting information for statistical details).</figcaption></figure>

Importance of phenology

The experimental addition of aromatic plants led to a decrease
in bacterial diversity in nests that were collected during the
post-hatching stage, but not in nests collected at an earlier
stage in the breeding period. The bacterial composition of
samples with and without the experimental addition of plants
was also slightly more contrasted at the post-hatching stage

than at the pre-incubation and incubation stages (Fig. 3).
Several nests collected during the experimental study already
contained aromatic plants before the treatment, which may
have influenced our results regarding bacterial diversity and
composition. The addition of plants to nests collected during
the post-hatching stage could have led to an accumulation of
volatile compounds from previously added plants. This may
<table><caption>Table 3. Bacterial composition of nest samples in relation to the quantity of aromatic plants (log-transformed) by population and by breeding stage, and accounting for the nest identity as accessed by a dbRDA on the Hellinger distance (model 6, n = 92).</caption><thead><tr><th>Source of variation</th><th>df</th><th>Sum of squares</th><th>Pseudo-F</th><th>Adjusted-R<sup>2</sup></th><th>p-value</th></tr></thead><tbody><tr><td>Aromatic plant quantity</td><td>1</td><td>0.87</td><td>2.27</td><td>0.014</td><td>0.001</td></tr><tr><td>Population</td><td>2</td><td>3.49</td><td>4.56</td><td>0.072</td><td>0.001</td></tr><tr><td>Breeding stage</td><td>1</td><td>1.93</td><td>5.03</td><td>0.042</td><td>0.001</td></tr><tr><td>Nest</td><td>43</td><td>33.87</td><td>2.06</td><td>0.330</td><td>0.001</td></tr><tr><td>Aromatic plant quantity:Population</td><td>2</td><td>0.71</td><td>0.93</td><td>&lt;0.001</td><td>0.653</td></tr><tr><td>Aromatic plant quantity:Breeding stage</td><td>1</td><td>0.41</td><td>1.07</td><td>&lt;0.001</td><td>0.300</td></tr><tr><td>Population:Breeding stage</td><td>2</td><td>1.68</td><td>2.20</td><td>0.025</td><td>0.001</td></tr><tr><td>Aromatic plant quantity:Population:Breeding stage</td><td>2</td><td>0.84</td><td>1.10</td><td>0.002</td><td>0.248</td></tr><tr><td>Residual</td><td>37</td><td>14.18</td><td></td><td></td><td></td></tr><tr><td>Total</td><td>91</td><td>57.98</td><td></td><td></td><td></td></tr></tbody></table>

be associated with a lower initial bacterial diversity compared to nests from earlier stages. However, we only observed a tendency for bacterial diversity to be lower in post-hatching controls than pre-incubation or incubation controls. Since heteroscedasticity can lead to decreased statistical power (Wilcox 1997), it is possible that this prevented us from detecting such differences. Gwinner and Berger (2005) found an increase in the amount of volatile compounds in starling nests in response to an increase in the quantity of fresh plants added to the nest. They also noted a decrease in bacteria load on nestlings in the last days of the breeding period (Gwinner and Berger 2005). This latter finding aligns with our results, which showed a decrease in bacterial diversity only in post-hatching nests from the experimental study and in post-hatching nests in the EPIrio population in the observational study.

Our observational study revealed a negative effect of aromatic plants on bacterial diversity in nests and on eggshells, but only in the EPIrio population, despite this population having a lower mean quantity of aromatic plants compared to the two other populations. These results highlight an intriguing variation between populations and breeding stages. In EPIrio, egg-laying occurs in early May, two weeks to one month later than both Muro populations (Charmantier et al. 2016). Consistent with our findings, Gwinner and Berger (2005) observed that the quantity and variety of volatile compounds in starling nests was higher later in the season. Several factors can influence the amount and the composition of volatile compounds released by aromatic plants, including plant development, the plant material (leaves or flowers), and environmental conditions such as temperature (Stefanakis et al. 2022). Some plant species release greater amounts of volatile compounds from mature flowers than from flower buds (Figueiredo et al. 2008), and the production of volatiles also increases later in the season (Stefanakis et al. 2022). Thus, the effect of aromatic plants on bacteria in EPIrio may have been stronger due to the plant phenological stage or the higher temperature during the breeding period in EPIrio compared to the Muro populations.

Additionally, many environmental differences between the three studied populations could affect the bacterial microbiota in the nest environment, many of which are explained by the later breeding phenology of blue tits in EPIrio. Infestation

by bloodsucking blowfly larvae *Protocalliphora spp.*, ectoparasites commonly found in blue tit nests, are more abundant in EPIrio than in the DMuro and EMuro populations (Tremblay et al. 2005). Mennerat et al. (2009a) found that bacterial density on nestlings was higher in nests infested by *Protocalliphora*, and that aromatic plants reduced bacterial density only in highly infested nests. Our results showed that the effect of breeding stages on nest bacterial composition varied among populations (Table 3, interaction), with a more significant difference between the incubation and post-hatching stages in the EPIrio population compared to the other two populations (Fig. 5B). We may have thus detected the effect of aromatic plants only in EPIrio partly due to the higher level of infestation in this population compared to the two others.

The decrease in bacterial diversity of nests in post-hatching and on the eggshells during incubation in the EPIrio population could potentially have implications for nestlings. A reduction in bacterial diversity on the eggshells could decrease the probability of pathogenic bacteria entering the shell, reducing the hatching probability (Cook et al. 2005a, Gantois et al. 2009). Moreover, the nest material and the eggshells are the first materials in direct contact with nestlings after hatching. The microbiota associated with these materials affects the intestinal and fecal microbiota in hens (Maki et al. 2020).

## Nest-specific signature of bacterial microbiota

Both experimental and observational nests varied strongly in their bacterial microbiota diversity and composition, as well as in the quantity of aromatic plants observed in the nests (Table 5). The among-nest variation in the quantity of aromatic plants added by females during the reproductive period represented one third of the total variation among observations. This indicates that females not only differed in their preference for plant species (Mennerat et al. 2009c), but also in the quantity they brought to their nest. The among-nest variation in bacterial microbiota diversity and composition reflects a nest-specific signature in the bacterial microbiota. In our populations, blue tits from the same nest share similar gut microbiota, including both breeding pairs and their nestlings (Dion-Phénix et al. 2021). Our results suggest that the nest environment also carries a microbiota signature, which
<figure><img src="image_26.png" /><figcaption>Figure 5. Bacterial composition of nest samples in relation to the quantity of aromatic plants (log-transformed) by population and by breeding stage. We accounted for nest identity and assessed the relation using a distance-based redundancy analysis (dbRDA) on the Hellinger distance (n=92). The lines link samples from the same nests. All the figures present the results of the model 6, Table 3: (A) all the samples together, (B) results by population.</figcaption></figure>

could be explained by bacterial transfer between the bird family and its nest environment.

Interestingly, in the experimental study the differences in bacterial composition between nests appears largely consistent, with nest identity explaining 57% of the total variation among samples. The important among-nest variation could partially explain the weak effect of aromatic plants on nest microbiota. Since the initial microbiota varied between nests, the aromatic plants may not have affected the same bacterial taxa across different nests. Furthermore, the bacterial nest signature became more pronounced as the season progressed.

Samples from the same nest consistently clustered together in the ordination at all breeding stages, but samples from different nests were more dispersed when collected during post-hatching, compared to those collected during pre-incubation or incubation.

## Differential effects of aromatic plants on nestlings across populations

Our results revealed a positive, albeit marginal, effect of aromatic plants on nestling body conditions in the two Muro populations. Nestlings from DMuro and EMuro tended
<table><caption>Table 4. Effect of the quantity of aromatic plants (log-transformed) by population on the bacterial composition of eggshell assessed by a dbRDA on the Hellinger distance among samples (model 7, n = 43).</caption><thead><tr><th>Source of variation</th><th>df</th><th>Sum of squares</th><th>Pseudo-F</th><th>Adjusted-R<sup>2</sup></th><th>p-value</th></tr></thead><tbody><tr><td>Aromatic plant</td><td>1</td><td>0.87</td><td>1.15</td><td>0.004</td><td>0.11</td></tr><tr><td>Population</td><td>2</td><td>1.74</td><td>1.15</td><td>0.007</td><td>0.06</td></tr><tr><td>Aromatic plant:Population</td><td>2</td><td>1.44</td><td>0.95</td><td>&lt;0.001</td><td>0.69</td></tr><tr><td>Residual</td><td>37</td><td>27.98</td><td></td><td></td><td></td></tr><tr><td>Total</td><td>42</td><td>32.04</td><td></td><td></td><td></td></tr></tbody></table>

to be heavier and to have longer tarsi in nests containing a greater quantity of aromatic plants. In contrast, the body mass and tarsus length of nestlings from EPIrIO were unaffected by the quantity of aromatic plants in the nest. Prey availability in the EPIrIO habitat is lower than in the Muro populations (Blondel et al. 2006, Dubuc-Messier et al. 2016) which may constrain nestling body mass and length. This reduces the beneficial effect of aromatic plants on these traits. No link was found between aromatic plants and docility or feather development in any populations, suggesting that volatile compounds had no effect on development or behaviour.

The differences observed among populations suggest that other biotic or abiotic factors may influence the effect of aromatic plants on nestling condition. Many environmental conditions experienced in early life, such as maternal immunisation or brood size, can influence morphological, physiological, or behavioural traits and these factors can interact with one another (Martyka et al. 2021). While adding aromatic plants to nests is part of the parents' rearing capability, other parental traits such as laying date and feeding rate also play major roles in nestling condition (Blondel et al. 2006, Grüebler and Naef-Daenzer 2010). Nestling traits may, therefore, be affected by more than just the quantity of aromatic plants if different parental care behaviours are correlated. It is generally assumed that early-laying parents are of better quality, invest more, and produce highly-quality offspring (Blondel et al. 1998). In the populations studied, females lay increasingly earlier in the first years of their life, and then later again during senescence (Auld and Charmantier 2011). Thus, females with poor rearing qualities who lay their eggs later may add fewer aromatic plants to the nest than higher-quality good rearing females. Our supplementary analysis revealed a negative association between aromatic plants and

hatching date (Supporting information). Therefore, we propose that the marginal correlation between aromatic plants and nestling traits may be indirectly related to hatching date and parental rearing capabilities.

The many differences in rearing conditions between the three populations could have interacted with the quantity of aromatic plants, resulting in various effects on nestling traits under study. Previous studies have observed an increase in body mass with the addition of aromatic plants only in experimentally enlarged broods (Mennerat et al. 2009b), a greater increase in body mass under higher ectoparasite infestations (Gwinner et al. 2000), a greater increase in tarsus length in large broods (Pires et al. 2020), and a more important increase in feather development in years of poor environmental conditions (Mennerat et al. 2009b). These studies suggest that the benefits of aromatic plants are only perceived when nestlings experience environmental constraints. However, in 2023, when our study was conducted, fledging success was higher than the average fledging success over the last twenty years for our populations, probably indicating a year with favourable environmental conditions that year (see complementary analysis in the Supporting information). Despite the generally favourable environmental conditions in 2023, we still detected a marginal correlation between aromatic plants and nestling traits in two out of the three populations. Using the axes of a PCoA on the four traits in our analysis provided a more comprehensive overview of nestling condition than considering each trait individually. Indeed, aromatic plants may affect multiple traits, so analysing them together in the same model may increase our power to detect biological relationships. Therefore, we recommend adopting a similar approach in other studies. Our results highlight that the effects of aromatic plants on nestlings can vary greatly,

<table><caption>Table 5. Nest repeatability (R²) of bacterial diversity and composition of the nest and in the quantity of aromatic plant added by the parents along the breeding season accessed by different models in the experimental and observational studies. Number of observations (n) and number of nests (j) are given for each model. The adjusted R² represents the proportion of the residual variation explained by the nest identities after accounting for the fixed effects, while the unadjusted R² represents the proportion of the total variation explained by nest identities.</caption><thead><tr><th rowspan="2">Model</th><th colspan="2">Adjusted</th><th colspan="2">Unadjusted</th><th rowspan="2">p-value</th></tr><tr><th>R<sup>2</sup></th><th>95% CI</th><th>R<sup>2</sup></th><th>95% CI</th></tr></thead><tbody><tr><td><strong>Experimental</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Nest bacterial diversity (model 1, n=169, j=19)</td><td>0.80</td><td>[0.68, 0.89]</td><td>0.52</td><td>[0.25, 0.73]</td><td>&lt;0.001</td></tr><tr><td>Nest bacterial composition (model 2, n=169, j=19)</td><td>—</td><td>—</td><td>0.57</td><td>—</td><td>0.001</td></tr><tr><td><strong>Observational</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Nest bacterial diversity (model 3, n=92, j=46)</td><td>0.23</td><td>[0.00, 0.50]</td><td>0.17</td><td>[0.00, 0.35]</td><td>0.051</td></tr><tr><td>Nest bacterial composition (model 6, n=92, j=46)</td><td>—</td><td>—</td><td>0.33</td><td>—</td><td>0.001</td></tr><tr><td>Quantity of aromatic plants (model 5, n=746, j=212)</td><td>0.33</td><td>[0.23, 0.40]</td><td>0.30</td><td>[0.22, 0.36]</td><td>&lt;0.001</td></tr></tbody></table>
<figure><img src="image_27.png" /><figcaption>Figure 6. Relationship between the nestling traits in three populations and the quantity of aromatic plants added to the nest by the parent. (A) The PCoA ordination shows the correlation between the four traits (body mass, tarsus length, feather development, and docility) in 1149 nestlings from the three populations (DMuro n = 576; EMuro n = 267; EPIrio n = 306). The relation between the quantity of aromatic plants and the position of nestlings on (B) the first axis of the PCoA (positively correlated with body mass and tarsus length), and (C) the second axis of the PCoA (positively correlated with feather development and negatively with docility) for the three populations, using two linear mixed models controlling for nestling age at measurement and with nest identity as a random effect (model 8 and 9, n = 1149). Long-dashed lines correspond to marginally significant relations and short-dashed lines correspond to insignificant relations in (B) and (C) (see the Supporting information for statistical details).</figcaption></figure>

even between populations of the same species living in close proximity.

## Support for the 'nest protection hypothesis'

Our results were mostly consistent with the 'nest protection hypothesis', suggesting that the aromatic compounds released by plants added to the nests had a direct antibacterial effect on both the nest and eggshell microbiota, which in turn affected nestling traits. Specifically, we found a correlation between aromatic plants and the bacterial diversity of nests and eggshells and a weak correlation with the composition of nest bacterial microbiota in the experimental study, and in one of the three studied populations, namely EPIrio. In the DMuro and EMuro populations, aromatic plants did not reduce bacterial diversity, but they did marginally improve nestling body condition. Therefore, in these populations, our results do not fully support the 'nest protection hypothesis' through bacterial effects. Aromatic plants could have increase body mass and tarsus length by directly stimulating

the immune system of nestlings, as suggested by the 'drug hypothesis'. An indirect effect of the rearing capabilities of the parents may also explained why aromatic plant quantity was positively correlated with nestling body mass and tarsus length if good quality parents bring more aromatic plants to the nest and produce bigger and taller nestlings than poor quality parents. Aromatic plants may influence ectoparasites in the nests, which could have potential consequences for the condition of nestlings that interact with reduced bacterial diversity. A subsequent study using a cross-fostering design, similar to the one conducted by Martyka et al. (2021) on great tits, could provide valuable additional insights into the effects of different early-life conditions.

We also tested the effect of adding two plant species together to the nest samples, and according to the 'potpourri hypothesis', we predicted that this combination would have stronger effect on bacterial diversity than adding only one species. Our result invalidated this prediction and thus does not support the 'potpourri hypothesis', which suggests that
each plant species acts differently on bacterial communities,
and that the addition of multiple species strengthen the anti-
bacterial effect (Lambrechts and Santos 2000). Indeed, we
found no difference in the effects of single plants and the
plant duos on the diversity and composition of the bacterial
microbiota in nest samples.

Conclusion

Our study is one of the first to explore the potential effect of
aromatic plants on the bacterial microbiota in the nest envi-
ronment, using advanced techniques such as next-generation
sequencing. The experimental approach allowed us to test
how the addition of aromatic plants affects the microbiota of
natural nests of Corsican blue tits. This innovative approach
allowed us to establish causal relationships while remaining
representative of real-world conditions. Our results suggested
that the phenology of reproduction is an important aspect
to consider when studying the effect of aromatic plants on
the nest microbiota. Moreover, the phenology of the aro-
matic plants themselves may also play an important role.
Our study proposed that blue tits may add aromatic plants to
their nests for multiple purposes, such as reducing bacteria,
which align with the ‘nest protection hypothesis’, or for their
benefits on nestling body condition independent of bacterial
effects in line with the ‘drug hypothesis’. Thus, we suggest
that additional studies are needed to assess how changes in
environmental conditions modulate the relationship between
aromatic plants and nestling conditions, as well as to inves-
tigate the potential cumulative effects of aromatic plants on
bacteria and nestling immune systems.

*Acknowledgements* – We thank all people involved in sample
collection, specifically Marilou Côté, Arlette Fauteux, and Victor
René de Cotret. We are grateful to the landowners for permission to
work on their property in Muro Valley and to the Office National
des Forêts and the Association Pour l'Etude Ecologique du Maquis
in the Fango Valley. We thank Christophe de Franceschi and people
from the Long-Term Projects platform (PLT CEFE-CNRS) for
help with the field work logistics. We thank Marie-Pierre Dubois
and people from the GEMEX platform (CEFE-CNRS) for the
help with DNA extraction. We thank Geneviève Bourret and the
CERMO platform from UQAM for the precious advice and help
at all laboratory steps. We thank Adèle Mennerat for the advice
and discussions. This document has been assisted by an AI tool,
specifically ChatGPT, to rephrase certain sections for clarity and
coherence.

*Funding* – This work was supported by the Fonds de recherche du Québec Nature et Technologies [grant numbers 282942, 305497, 342566], the Natural Sciences and Engineering Research Council of Canada [grant numbers RGPIN-2019-07079, RGPIN-2020-04745, BRPC-581630], the Observatoire des Sciences de l'Univers (OSU-OREME), and the long-term Studies in Ecology and Evolution (SEE-Life) program of the CNRS.

*Conflict of interest* – The authors declare no conflict of interest.

*Permits* – Blue tit captures and ringing were done under authorisations delivered by the Centre de Recherche sur la Biologie des Populations d’Oiseaux (CRBPO, Paris, permit for Anne

Charmantier no. 1907) and all animal experimentation procedures
were approved by the ethics committee for animal experimentation
of Languedoc Roussillon (APAFIS #8608-2017012011062214
authorized for five years on 2020-06-05).

Author contributions

**Hélène Dion-Phénix:** Conceptualization (equal); Data curation (equal); Formal analysis (lead); Funding acquisition (supporting); Investigation (equal); Methodology (equal); Visualization (lead); Writing – original draft (equal); Writing – review and editing (lead). **Gabrielle Gingras:** Conceptualization (equal); Data curation (equal); Formal analysis (supporting); Funding acquisition (supporting); Investigation (equal); Methodology (equal); Visualization (supporting); Writing – original draft (equal); Writing – review and editing (supporting). **Anne Charmantier:** Conceptualization (equal); Data curation (equal); Funding acquisition (equal); Methodology (equal); Project administration (equal); Resources (equal); Supervision (equal); Writing – original draft (supporting); Writing – review and editing (supporting). **Claire Doutrelant:** Conceptualization (equal); Data curation (equal); Funding acquisition (equal); Methodology (equal); Project administration (equal); Resources (equal); Supervision (equal); Writing – original draft (supporting); Writing – review and editing (supporting). **Steven W. Kembel:** Conceptualization (equal); Funding acquisition (equal); Methodology (equal); Project administration (equal); Resources (equal); Supervision (equal); Writing – original draft (supporting); Writing – review and editing (supporting). **Denis Reale:** Conceptualization (equal); Funding acquisition (equal); Methodology (equal); Project administration (equal); Resources (equal); Supervision (lead); Writing – original draft (supporting); Writing – review and editing (supporting).

Data availability statement

Data are available from the Figshare Repository for the
experimental study: https://10.6084/m9 figshare.27953055
(Dion-Phénix et al. 2025b) and for the observational study:
https://doi.org/10.6084/m9 figshare.28640093.v2 (Dion-
Phénix et al. 2025a).

Supporting information

The Supporting information associated with this article is
available with the online version.

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