# Identification of five volatile organic compounds that trigger hygienic and recapping behaviours in the honey bee (Apis mellifera)

Amélie Noël, Charlène Dumas, Emilien Rottier, Dominique Beslay, Guy Costagliola, Christian Ginies, Florence Nicolè, Yves Le Conte, Fanny Mondet

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Amélie Noël, Charlène Dumas, Emilien Rottier, Dominique Beslay, Guy Costagliola, et al.. Identification of five volatile organic compounds that trigger hygienic and recapping behaviours in the honey bee (Apis mellifera). International Journal for Parasitology: Parasites and Wildlife, In press, 10.1016/j.ijpara.2025.01.009. hal-04999405v2

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# Identification of five volatile organic compounds that trigger hygienic and recapping behaviours in the honey bee (Apis mellifera)

Amélie Noël<sup>a,1</sup>, Charlène Dumas<sup>a</sup>, Emilien Rottier<sup>a</sup>, Dominique Beslay<sup>a</sup>, Guy Costagliola<sup>b</sup>, Christian Ginies<sup>c</sup>, Florence Nicolè<sup>d</sup>, Yves Le Conte<sup>a</sup>, Fanny Mondet<sup>a,*</sup>

## ARTICLE INFO

**Article history:**

Received 4 October 2024

Received in revised form 11 December 2024

Accepted 29 January 2025

Available online xxxx

**Keywords:**

Varroa destructor

Apis mellifera

Volatile organic compounds

VSH

Recapping

Chemical communication

Social immunity

## ABSTRACT

Varroa destructor, the main parasite of the honey bee (Apis mellifera), is having a devastating effect on beekeeping worldwide. The development of resistance traits in some colonies, linked with Varroa-sensitive hygiene (VSH) and recapping (REC) behaviours, provide an ideal avenue for long-term sustainable control of the parasite. The most important step in these behaviours is the detection of parasitized brood cells. Several semiochemicals released from Varroa-infested brood cells, targeted by VSH behaviour, trigger this behaviour. Most of these compounds are not very volatile. In the current work, we focus on the study of volatile organic compound (VOC) emissions from Varroa-infested cells. This study describes the emission of nine VOCs characteristic of Varroa parasitism, of which five could be identified and triggered hygienic and recapping behaviours. These five compounds were also tested with compounds already described in the literature, in relation to the volatile nature of the compounds. Using solutions containing 1–15 compounds, we looked at the cleaning and recapping behaviours of the workers. Behavioural results highlight the importance of the VOCs found in this study in the detection, opening and recapping of brood cells, while low volatile compounds seem to play a particularly key role in the sacrifice of pupae. Similar to the Varroa parasitization-specific (VPS) compounds, including the tetracosyl acetate alone, the cleaning of brood cells triggered by one of the compounds identified in this study, *n*-tetradecane, appears to be linked to the colony's ability to carry out VSH behaviour. This study opens new perspectives in the understanding of resistance behaviour of honey bees against their main parasite *Varroa destructor*.

© 2025 The Author(s). Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY license ([http://creativecommons.org/licenses/by/4.0/](http://creativecommons.org/licenses/by/4.0/)).

## 1. Introduction

The growing use of pesticides has led to resistance in pests and pathogens, affecting the quality and quantity of agricultural production worldwide (Traynor et al., 2020). Integrated pest management (IPM) aims to manage parasites and pathogens, reducing their impact on crops and limiting economic losses, while avoiding the use of pesticides (Karlsson Green et al., 2020). IPM is particularly relevant in beekeeping for controlling the main, globally distributed biotic stressor of the honey bee (Apis mellifera), the Varroa destructor mite. Varroa destructor is becoming increasingly resistant to synthetic acaricides, making its control increasingly complex (Martin, 2004; González-Cabrera et al., 2018; Mitton

et al., 2022). One promising option for mite control is the use of IPM techniques on Varroa-resistant honey bee populations. Such resistant honey bee colonies are able to survive by limiting the level of Varroa mites in the colony, thus reducing the need for acaricides. In several countries in Africa and Latin America, large honey bee populations do not rely on treatment for survival; several local untreated populations have also been identified in the US, South America and Europe (Locke, 2015; Moro et al., 2021). Resistance to Varroa can be explained by several traits, one of the most prominent being Varroa-sensitive hygiene (VSH) behaviour (Mondet et al., 2020). VSH is a hygienic behaviour specifically directed towards parasitized brood cells (Harbo and Harris, 2005) and consists of the detection, uncapping and cleaning of Varroa-infested brood cells by worker bees (Harbo and Harris, 2005). A variant of VSH behaviour is recapping behaviour where bees detect, uncap and recap the infested brood cell without touching its contents (Corrêa-Marques and De Jong, 1998; Boecking and Spivak, 1999; Aumeier et al., 2000). This recapping of the

<footnote><sup>a</sup>INRAE, UR 406 Abeilles et Environnement 84914 Avignon, France</footnote>

<footnote><sup>b</sup>INRAE, UR 1115 Plantes et Systèmes de culture Horticoles 84914 Avignon, France</footnote>

<footnote><sup>c</sup>INRAE, UMR 408 Sécurité et Qualité des Produits d'Origine Végétale 84914 Avignon, France</footnote>

<footnote><sup>d</sup>Université de Lyon, UJM-Saint-Étienne, CNRS, LBVpam 42100 Saint-Étienne, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: fanny.mondet@inrae.fr (F. Mondet).</footnote>

<footnote n="1">¹ Present address: Sveriges lantbruksuniversitet, Institutionen för Ekologi, 75007 Ulls väg 16, Uppsala, Sweden.</footnote>
parasitized brood cell could be sufficient to disrupt *Varroa* reproduction (Oddie et al., 2019). Choosing to use the natural resistance of colonies necessitates the identification of resistant colonies in order to select and breed for this trait. Currently, phenotyping colony resistance to *Varroa* is primarily carried out by measuring *Varroa* infestation, mite population growth and VSH behaviour, a process which is time consuming, and thus unsuitable for beekeepers managing both small and large number of colonies (Noël et al., 2020). Searching for chemical signals involved in the recognition of *Varroa*-infested brood cells is not only interesting for deciphering the mechanisms of this behaviour, but could be relevant as a tool used for phenotyping *Varroa*-resistant honey bee colonies.

The first step in the VSH and recapping behaviours relies on the ability of worker bees to specifically detect *Varroa*-infested brood cells. Chemical compounds are strongly involved in the recognition of disease and parasites among insects (Milutinović and Schmitt, 2022) and detection is mainly based on olfactory cues (Mondet et al., 2015; Nazzi and Le Conte, 2016). Currently eight low volatile compounds (LVCs) and two volatile organic compounds (VOCs) have been identified as triggering VSH behaviour (Nazzi et al., 2004; Wagoner et al., 2020; Liendo et al., 2021; Mondet et al., 2021). To date, it is not known whether a specific signal or blend of signals trigger VSH and recapping behaviour, or whether a common signal triggers the uncapping step followed by a specific signal triggering cleaning versus recapping of the brood cell. Compounds that trigger recapping behaviour rather than the sacrifice of the immature bee are still unknown; it may be the lack of specific cues emitted by the open brood cell that triggers recapping (Martin et al., 2020) or differences in sensibility between bees performing different hygienic tasks (Spivak et al., 2003). Compounds of both low and high volatility are thought to be highly involved in VSH and recapping behaviours. The detection step of VSH and recapping could occur in two steps. The first cue could be the emission of VOCs that would easily pass through the cap of the brood cell to signal the area where the parasitized cell is located (Kim et al., 2018). The second cue could involve LVCs that the worker would detect when it is close to the brood cell or when opening it. The recent identification of two VOCs (alpha-pinene and ethyl hexanoate, Liendo et al., 2021) from *Varroa*-parasitized brood cells, after the identification of LVCs, supports a similar signalling pathway. VOCs are highly likely to play a central role in the triggering of one of several steps of hygienic behaviours towards *Varroa*-infested brood cells.

The aim of this study was to improve our knowledge of the function of VOCs in the triggering of VSH and recapping behaviour, and to study the effect of the various compounds identified in the literature using a common standardised protocol. We carried out a comparative analysis of the VOCs emitted by *Varroa*-parasitized and non-parasitized brood. We have identified five VOCs characteristic of parasitized brood that trigger hygienic behaviour. The effects of the compounds on bees' behaviours was studied based on the volatility characteristics of the compounds (VOCs or LVCs). The hygienic behaviour was studied through the cleaning behaviour as well as the recapping behaviour of the treated brood cells, and with regard to the VSH status of the tested colonies.

## 2. Materials and methods

### 2.1. Identification of volatile compounds emitted by *Varroa*-parasitized brood

#### 2.1.1. *In vitro* rearing of *A. mellifera* brood

Rearing of the *A. mellifera* brood was conducted in vitro to avoid compounds present in the wax. Uncapped larvae in their last larval stage (L5) were reared in food-grade gelatin capsules (Ø 6.1 mm,

LGA, Laboratoire des Gélules et des Azymes, France). Capsules were pre-pierced, with nine holes at both ends and 27 holes along the body, with a sewing pin to favour gas exchange. The main part of the open capsules was placed on the brood cell to allow the larvae to enter freely into the capsules. Brood frames were then placed horizontally with the gelatin capsules facing downwards so that the larvae descended by themselves into the capsules to avoid excessive manipulation. After 3 h, the capsules were collected and closed by hand with the head of the gelatin capsule. This experiment was repeated on 10 different colonies. For each colony, one non-parasitized (NP) and one parasitized (P) group was created, composed of between 20 and 30 capsules each. For the P group, each capsule was infested with a *V. destructor* mite. *Varroa* originated from three donor colonies and adult mites were collected from workers using the icing sugar method (Dietemann et al., 2013). The capsules were placed horizontally at a slight angle in an incubator in order to mimic the orientation of the cells in a brood frame in the hive. After 12 h of rearing, due to shrinkage of the capsules due to the ambient humidity, the holes in the capsules were gently punched with a sewing needle so that the volatile gases could escape the cells, taking care not to injure the individual(s). The brood was reared in capsules for 8 days (black eyes and yellow thorax pupae) at 34 °C and 75% relative humidity.

#### 2.1.2. VOC capture and analysis

For the VOC captures, 10 capsules per colony for each group (NP and P) were placed in an open glass vial (10 mL, Sigma-Aldrich, France). VOCs were captured every 24 h from the same capsules. At the time of capture, the vial containing the 10 individuals was sealed with a polytetrafluoroethylene (PTFE) septum cap (Sigma-Aldrich, France). The septum was pierced in order to insert a polydimethylsiloxane divinylbenzene (PDMS-DVB) solid phase micro extraction (SPME) fibre (65 µm, manual holder, Sigma-Aldrich, France). For each capture, the vial and fibre were held horizontally and kept in an incubator (34 °C). Captures lasted for 20 min, after which the vials containing individuals were re-opened. Captures were carried out from day 1 to day 7 after capping, thus forming a total of 140 captures (10 for each group (NP and P) per day). Captures from empty capsules were made in the same way each day. Qualitative and quantitative analyses of VOCs were performed by gas chromatography coupled with mass spectrometry (GC-MS, GCMS-QP2010S, Shimadzu, France) equipped with a ZB 5 MS column (Zebron ZB 5 ms, 20 m × 0.18 mm, 0.18 µm thickness, Phenomenex, France). Desorption was realised in splitless mode, with the constant column flow at 0.82 mL/min; the carrier gas was helium. The injector temperature was set at 250 °C and the oven temperature program as follows: 45 °C isothermal held for 5 min, followed by temperature increases at a rate of 10 °C/min up to 250 °C. The oven was finally held at 250 °C for 4 min. The ion source was set at 200 °C. Identification of compounds of interest was performed using the NIST 2017 library and confirmed by injection of standards purchased from Sigma Aldrich.

### 2.2. Influence of compounds on VSH and recapping behaviours of brood cells

#### 2.2.1. Bioassay design for the evaluation of VSH and recapping behaviours

The bioassays tested the effect of different treatments containing compounds characteristic of *Varroa*-infested brood cells on VSH and recapping behaviours. To test the effects of the five VOCs identified, behavioural assays were conducted on 11 honey bee colonies, during July to August 2022, at Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAE) Research Centre of Avignon, France. To test the effects of mixtures of compounds specific to *Varroa* parasitism identified in
this work and those found in the scientific literature, behavioural assays were carried out on 31 honey bee colonies between September and October 2022. To cover the full spectrum of VSH scores, we used both treated (unselected for *Varroa* resistance) and untreated colonies (selected for high VSH performance). For each behavioural assay, homogeneous brood patches with brood cells containing white-eyed to pink-eyed pupae were injected with 1 µL of solution, using a Hamilton syringe (Sigma-Aldrich, France), just below the cap. In each brood frame, 15 to 30 cells per treatment were injected. The treated cells were organized in rows, taking care not to create patches. The status of the cells (emptied, uncapped) was observed at 6 h, 24 h and 48 h after injection. At 48 h, the recapping of the cells was checked by gently opening the remaining brood cells to look at the appearance of the underside of the cells caps (Harris et al., 2012).

### 2.2.2. Determination of the VSH score of the colonies

In each colony tested, one brood frame containing last instar larvae was collected, and cells undergoing capping were marked on a transparent plastic sheet and put back in the honey bee colony. After 6 h, these just-capped brood cells were artificially infested with *Varroa* mites, previously collected using the icing sugar method, from heavily infested colonies. Eight days later, cleaning of the artificially infested brood cells was checked. The VSH score was calculated as the number of cleaned brood cells (opened and fully cleaned) over the number of artificially infested brood cells (Tison et al., 2022).

### 2.2.3. Solution preparations of compounds

To test the effect of the five VOCs identified in this study, solutions containing only one of the five identified compounds and a solution containing all five compounds were prepared. For the single-compound solutions, the compound was diluted in isohexane (Sigma-Aldrich, France) to a concentration of 10% w/w. For the mixture, each compound was at 10% w/w in the final solution containing the five compounds and the solvent.

The sheer number of compounds identified in the scientific literature as triggering VSH behaviour did not allow testing of all possible mixtures. Two concentrations were tested, one at 10% w/w concentration (high concentration) (n = 16 colonies) with the aim of reproducing what had been reported in the scientific literature, and a 10-equivalent concentration (physiological concentration) (n = 15 colonies) to test a concentration closer to the biological conditions found in the brood cells. One equivalent corresponds to the amount of a given compound emitted by one parasitized brood cell. With the exception of the five VOCs identified in this study, the equivalents were calculated from the scientific literature (Salvy, M., 1999. Etude de la communication chimique intraspécifique chez *Varroa jacobsoni*, acarien parasite de l'abeille domestique, Apis mellifera. (Doctoral thesis), Université de Provence, France; Salvy et al., 2001; Nazzi et al., 2004; Liendo et al., 2021; Mondet et al., 2021). To observe potential synergy or additivity, compounds were mixed in order to obtain increasingly complex combinations until the final mixture containing all 15 compounds was tested (Fig. 1). Simple solutions were composed of the compounds identified together in the same publication (A: benzothiazole, dodecanoic acid, *n*-hexadecane, *n*-tetradecane and octanoic acid, B: alpha-pinene and ethyl hexanoate (Liendo et al., 2021), C: (Z)-6-pentadecene and (Z)-10-tritriacontene (Wagoner et al., 2020) and D: 2-tricosanone, 2-pentacosanone, tetracosyl acetate, 2-heptacosanone, hexacosyl acetate and 2-nonacosanone (Mondet et al., 2021), Fig. 1). *n*-Tetradecane was tested alone due to its rapid effect on hygienic behaviour in a previous bioassay (a, Fig. 1). Solutions containing all VOCs (A + B, Fig. 1) and LVCs (C + d and C + D, Fig. 1) were tested. The final solution was composed of VOCs and LVCs (A + B + C + D, Fig. 1). Due to solubility

problems at high concentrations, the mixture of the six *Varroa* parasitization-specific (VPS) compounds was replaced by tetracycl-syl acetate for the 10% concentration (Fig. 1). One colony was withdrawn from the analysis due to an excessive response to the solvent control (more than 40% of cleaning with the physiological concentration). Benzothiazole, dodecanoic acid, *n*-hexadecane, *n*-tetradecane, octanoic acid, alpha-pinene and ethyl hexanoate were purchased from Sigma Aldrich (France); (Z)-6-pentadecene, (Z)-10-tritriacontene and all VPS compounds were purchased after synthesis from Synthenova (France).

### 2.2.4. Determination of an equivalent of a parasitized brood cell for five of the VOCs

In order to determine the emission of each of the five VOCs emitted by a parasitized brood cell, calibration curves were performed for the quantification of these compounds. Each individual compound was diluted in isohexane with sequential dilutions ranging from $10^{-4}$ to $10^{-8}$ of pure compound and 10 µL were then placed in a sealed vial (10 mL). The septum was pierced to allow passage of the SPME fibre. Captures lasted 20 min. Quantitative analysis of the VOCs was carried out using GC-MS, following the same protocol described in section 2.1.2.

### 2.3. Statistical analyses

Statistical analyses were performed using R (v4.1.3) with R packages "mgcv" v1.8-41 (Wood, 2011), "RVAideMemoire" v0.9-83-7 (Hervé, M., 2023. "RVAideMemoire: Testing and plotting procedures for biostatistics. R package version 0.9-83-7", https://CRAN.R-project.org/package=RVAideMemoire), "vegan" v2.6-4 (Oksanen et al., 2022. "vegan: Community Ecology Package, https://vegan.r-forge.r-project.org/), "lme4" v1.1-28 (Bates et al., 2015), "car" v3.1-2 (Fox and Weisberg, 2019), "emmeans" v1.10.1 (Lenth, R.V., 2024. "emmeans: Estimated Marginal Means, aka Least-Squares Means", https://CRAN.R-project.org/package=emmeans) and "multcomp" v1.4-25 (Hothorn et al., 2008). *P*-values were considered significant at 0.05 or less.

For VOC captures, compounds detected less than six times out of the 10 replicates in each sample for each day were removed from the analysis. Day 1 was markedly different from the other days (early pre-pupae, capsules that shrunk, totally different VOC emissions), so it was removed from the rest of the analyses. To identify compounds characterising NP and P samples, a redundancy analysis (RDA) was performed from day 2 to day 7, with day and sample type (NP or P) and their interaction included as fixed factors. Linear mixed models were performed for each compound from day 2 to day 7, with day and sample type and their interaction included as fixed factors and vial ID as a random factor. The fit of the models was checked using the quality of the residues. Significance was checked with ANOVA and pairwise comparisons were performed on day and sample variables.

To study the effect of each solution on the number of cells visited (fully cleaned, uncapped and recapped), the data were analysed using a generalised linear mixed model (GLMM), with the number of brood cells visited compared with the remaining cells as a function of treatment (fixed factor) and colony (random factor). The effect of the solutions on the cleaning behaviour of the workers was analysed in the same manner. The effect of the solutions on the recapping behaviour of the workers was analysed with the number of brood cells visited compared with the remaining cells as a function of presence of compounds found in this study (fixed factor), presence of compounds found by other authors (fixed factors) and colony (random factor). Due to the non-linearity of the data, generalised additive mixed models (GAMMs) were fitted on the proportion of brood cells uncapped as a function of the VSH score of the colony, for each treatment. Most of the
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Scheme of the different mixtures used by injection in the brood cell to test the effects of compounds characteristic of *Varroa destructor*-infested brood on *Varroa* sensitive hygiene behaviour, at the two concentrations. Due to solubility issues at the high concentration, only one of the six *Varroa* parasitization-specific compounds (VPS) compounds was used for the higher concentration. High concentration: 10%w/w; physiological concentration: 10 equivalents. VOCs, volatile organic compounds; LVCs, low volatile compounds.</figcaption></figure>

curves were not monotonous and the inflection point was used to set a limit according to VSH performance (score of 47% of cleaning). Thus, colonies were separated into two groups (VSH+: good recognition and cleaning of *Varroa*-infested cells, score ranging from 48 –100%; VSH-: poor recognition and cleaning of *Varroa*-infested cells, score ranging from 0–46%). Colonies with a VSH score close to the threshold (± 1%) were removed from the analysis in order to have a net limit (one colony for the 10% concentration). To study whether VSH + colonies responded faster to compounds than VSH-colonies, generalized linear models were performed to analyse the effects of treatments on uncapping of injected cells by workers at 6 h, with the proportion of brood cells uncapped as a function of VSH groups (fixed factor).

## 3. Results

### 3.1. Identification of volatile organic compounds characteristic of *Varroa* parasitism

A total of 111 compounds were detected in the GC-MS profiles. None of these compounds were specific (presence/absence) to a sample group (NP/P). Comparison of total peak areas over time between the two groups showed no difference in total abundance (GAMM; NP: estimate = 2,911,042, P: estimate = 2,385,984, P = 0.94). The fluctuation of the two groups over time was also not significant (GAMM; NP over time: F = 0.379, P = 0.615; P over time: F = 0.051, P = 0.852). The first three components of the RDA explained 84.89% of the variance. Day, sample group and their interaction were significant (RDA; Day: F = 5.93, P = 0.001; Sample group: F

= 12.07, P = 0.001; Day:Sample group: F = 1.40, P = 0.026). The two sample groups were separated on component 2. To select the most characteristic compounds of the P group, we considered the results of the RDA, significant results of the ANOVA and at least three significant days in the pairwise comparisons (Figs. 2 and 3; Table 1). We selected nine compounds characterising parasitism (s43, s48, s54, s60, s67, s76, s81, s85, s86) (Fig. 3). The nine compounds were emitted in significantly greater quantities in the P group than in the NP group each day, except for compounds s43, s81 and s86 on the second, third and fourth days of rearing, and for compounds s54 and s60 on the fourth day of rearing (Fig. 3). Of the nine compounds, five were successfully identified (octanoic acid (s43), benzothiazole (s48), *n*-tetradecane (s67), dodecanoic acid (s81), *n*-hexadecane (s85)). For the four unidentified compounds, we were confident that three of them (s54, s60 and s76) belong to the alkane chemical family, but we had been unable to find the location of the unsaturation or methylation due to a lack of commercially available standards. We did not manage to find any information for the last one (s86).

### 3.2. Adult worker behaviour on newly identified VOC-treated brood cells

Brood cells treated with solutions containing one or more VOCs were more frequently visited than cells treated with the solvent control or those that received no treatment. There was a significant difference between treatments in brood cells visited (cleaned, uncapped or recapped) by workers ($\chi^2$ = 183.77, degrees of freedom (d.f.) = 7, P < 2.2e-16) (Fig. 4A). When visited, solvent and
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Redundancy Analysis (RDA) performed on chemical compounds captured on *Varroa destructor* parasitized (P) and non-parasitized (NP) honey bee brood reared in capsule, day 2 to day 7. For each day: (P) *n* = 10; (NP) *n* = 10. Each dot represents a sample of 10 pupae (P: orange (medium grey), NP: green (dark grey)). Compounds are represented by their IDs and grey arrows. The compounds characterising the P group and retained for the rest of the analysis are shown in bold. s43, octanoic acid; s48, benzothiazole; s54, unidentified; s60, unidentified; s67, *n*-tetradecane; s76, unidentified; s81, dodecanoic acid; s85, *n*-hexadecane; s86, unidentified.</figcaption></figure>

untouched controls were mainly recapped (Fig. 4A). Due to the small proportion of cleaned brood cells for the untreated brood cell controls, this control was not considered in subsequent analyses. There was a significant treatment effect in brood cell uncapping (uncapped and cleaned brood cells) behaviour (6 h: $\chi^2 = 99.161$, d.f. = 6, $P < 2.2e^{-16}$; 24 h: $\chi^2 = 124.38$, d.f. = 6, $P < 2.2e^{-16}$; 48 h: $\chi^2 = 143.2$, d.f. = 6, $P < 2.2e^{-16}$) (Fig. 4B). Each solution triggered a response with a different temporal dynamic. The response to the *n*-tetradecane solution was greater than for all other treatments 6 h after injection (Fig. 4B). At 24 h after injection, the response to the mixed solution was greater than for the other treatments, except for the *n*-tetradecane solution (Fig. 4B). At 48 h after injection, the responses to all of the treatments were greater than the control solvent (Fig. 4B).

### 3.3. Effects of compounds characteristic of *Varroa*-infested brood on brood cell visits by workers

For injections of compounds identified in the literature and the five VOCs, there was a significant difference between treatments for the two concentrations tested (10%: $\chi^2 = 789.71$, d.f. = 9, $P < 2.2e^{-16}$; 10 equivalents: $\chi^2 = 400.15$, d.f. = 10, $P < 2.2e^{-16}$) (Fig. 5A). Brood cells treated with solutions containing compounds characteristic of *Varroa*-parasitized brood cells were significantly more frequently visited (cleaned, uncapped and recapped) than solvent-treated or untreated brood cells (Fig. 5A). Due to the small proportion of cleaned brood cells, the untreated brood cell control

was not considered in the analysis of the cleaning behaviour but was still considered for the recapping analysis, as a significant basal level of recapping was detected in most colonies.

For the higher concentration (10% v/v), all treatments triggered more brood cell uncapping than the solvent control but the *n*-tetradecane solution (a), the mix of (Z)-10-tritriacontene & (Z)-6-pentadecene + tetracosyl acetate (C + d), and the mix of (Z)-10-tritriacontene and (Z)-6-pentadecene (C), triggered it more rapidly than others. There was a significant treatment effect (6 h: $\chi^2 = 629.21$, d.f. = 8, $P < 2.2e^{-16}$; 24 h: $\chi^2 = 651.35$, d.f. = 8, $P < 2.2e^{-16}$; 48 h: $\chi^2 = 633.83$, d.f. = 8, $P < 2.2e^{-16}$) (Fig. 5B). At 6 h and 24 h after injection, the highest uncapping rates were triggered by the mix of (Z)-10-tritriacontene and (Z)-6-pentadecene (C), the *n*-tetradecane solution (a), and the mix of (Z)-10-tritriacontene and (Z)-6-pentadecene + tetracosyl acetate (C + d) (Fig. 5B). At 24 h, the response to the *n*-tetradecane solution (a) and all the solutions containing the five VOCs in this study (A, A + B, A + B + C + d) exceeded 25% of the cleaning rate (Fig. 5B). At 48 h after injection, all solutions exceeded 50% of the cleaning rate, except for the solution of $\alpha$-pinene and ethyl hexanoate (B – Fig. 5B). The response triggered by the latter was still higher than the solvent control (Fig. 5B).

At physiological concentrations, the modalities triggered significantly different uncapping responses (6 h: $\chi^2 = 199.35$, d.f. = 9, $P < 2.2e^{-16}$; 24 h: $\chi^2 = 283$, d.f. = 9, $P < 2.2e^{-16}$; 48 h: $\chi^2 = 247.84$, d.f. = 9, $P < 2.2e^{-16}$) (Fig. 5B). The only solution that did not trigger a higher uncapping rate than the solvent control
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Adjusted means of areas (+/- SE) for the nine compounds of interest per 10 *Varroa destructor* parasitized (P) and non-parasitized (NP) honey bee brood samples reared in capsule, from day 2 to day 7. P: orange (medium grey), NP: green (light grey). For each day and each group, *n* = 10 colonies. Tukey-adjusted *P* values from pairwise comparisons of estimated marginal means. s43, octanoic acid; s48, benzothiazole; s54, unidentified; s60, unidentified; s67, *n*-tetradecane; s76, unidentified; s81, dodecanoic acid; s85, *n*-hexadecane; s86, unidentified. \* *P* < 0.05; \*\* *P* < 0.005; \*\*\* *P* < 0.001.</figcaption></figure>

<caption>**Table 1**</caption>

Results of the ANOVA showing the effects of sample groups on the compound area of the six compounds of interest found using gas chromatography coupled with mass spectrometry (GC-MS).

<table><thead><tr><th></th><th>s43</th><th>s48</th><th>s54</th><th>s60</th><th>s67</th><th>s76</th><th>s81</th><th>s85</th><th>s86</th></tr></thead><tbody><tr><th>χ<sup>2</sup></th><td>51.05</td><td>335.03</td><td>200.64</td><td>182.75</td><td>2372.90</td><td>177.34</td><td>39.30</td><td>166.18</td><td>60.46</td></tr><tr><th>P-value</th><td>9.006e<sup>-13</sup></td><td>&lt; 2.2e<sup>-16</sup></td><td>&lt; 2e<sup>-16</sup></td><td>&lt; 2e<sup>-16</sup></td><td>&lt; 2.2e<sup>-16</sup></td><td>&lt; 2e<sup>-16</sup></td><td>3.640e<sup>-10</sup></td><td>&lt; 2.2e<sup>-16</sup></td><td>7.528e<sup>-15</sup></td></tr></tbody></table>

For each group sample: *n* = 10.

s43, octanoic acid; s48, benzothiazole; s54, unidentified; s60, unidentified; s67, *n*-tetradecane; s76, unidentified; s81, dodecanoic acid; s85, *n*-hexadecane; s86, unidentified.

was the solution of α-pinene and ethyl hexanoate, 6 h and 24 h after injection (B – Fig. 5B). At 6 h, 24 h and 48 h after injections, the highest uncapping rates were triggered by all solutions containing *n*-tetradecane (a, A, A + B, A + B + C + d – Fig. 5B). At 48 h after injections, all solutions triggered at least a 25% cleaning rate, except for the solution of α-pinene and ethyl hexanoate (Fig. 5B).

In our study, almost 90% of the injected brood cells were employed by the workers for several treatments at the 10% concentration, leaving only a small proportion of cells available to measure recapping. The physiological concentration appeared to be more relevant to study this behaviour, as it triggered less brood cell cleaning (40% on average). Recapping of the remaining brood cells treated with the compounds from this study (Noël's VOCs), the other compounds from the scientific literature, and their interactions are significantly different from the control (Noël's VOCs: χ² = 59.34, d.f. = 1, P = 1.32e⁻¹⁴, Others: χ² = 21.87, d.f. = 1, P = 2.92e⁻¹⁴, Noël's VOCs: Others: χ² = 5.10, d.f. = 1, P = 0.02) (Fig. 6). Treatments containing the VOCs found in this study

triggered more recapping than treatments not containing them (Fig. 6).

### 3.4. Hygienic response of colonies to the treatments depending on the colony VSH scores

Colonies were divided into two groups according to their VSH score: VSH- colonies (6.66% < VSH- < 46.67%, average = 28.5%) and VSH+ colonies (50% < VSH+ < 90%, average = 68.8%). At the higher concentration, except for the solvent control, the five VOCs (A) solution, and the α-pinene and ethyl hexanoate (B), all solutions triggered a different response according to the ability of the colonies to perform VSH behaviour, with a higher response for VSH+ colonies than VSH- (Table 2, Fig. 7). The most pronounced differences between the two groups (VSH+/VSH-) were triggered by the *n*-tetradecane solution (a), the tetracosyl acetate solution (d), and the (Z)-10-tritriacontene and (Z)-6-pentadecene solution (C) (Fig. 7). For the physiological concentration, solutions containing tetracosyl acetate (d, D, C + D and A + B + C + D) and the *n*-te-
A

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

B

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

General legend

Unt: untreated cells

Iso: isohexane

s43: octanoic acid

s48: benzothiazole

s67: *n*-tetradecane

s81: dodecanoic acid

s85: *n*-hexadecane

Mix: all of the 5 VOCs

<caption>**Fig. 4.** Hygienic response of honey bee workers to brood cells treated with different treatments, 48 h after injection. (A) Proportion of brood cells in each status. (B) Adjusted uncapping rate means (+/- SE) of injected brood cells according to the different treatments 6 h, 24 h and 48 h after injections. For each treatment, *n* = 11 colonies. Intact, intact cells; recap, recapped cells; uncap, uncapped cells; empty, fully cleaned cells; VOCs, volatile organic compounds. Lowercase letters represent differences in significance.</caption>

brood cell cleaning depending on the VSH capacity (Table 2, Fig. 7).

4. Discussion

We analysed the VOC emissions of *V. destructor*-infested and non-infested honey bee brood (*A. mellifera*) reared in gelatin capsules, to identify candidates that could trigger VSH and/or recapping behaviours. To our knowledge, this is the first time that VOCs of *Varroa*-parasitized bee brood have been captured for several continuous days from the same individuals of brood reared in gelatin capsules. We have highlighted nine compounds characteristic of *Varroa*-infested brood cells, of which five were successfully identified and behaviourally tested in the field. These five VOCs triggered the cleaning of treated brood cells by workers and *n*-tetradecane triggered the fastest response of the compounds tested. We also compared the responses of workers to different mixtures of compounds described in the literature as specific to parasitized brood cells and triggering a hygienic response. The compounds used can be classified into two categories: VOCs with a high volatility (Liendo et al. (2021) and the five compounds identified in this study) and LVCs (Nazzi et al., 2004; Wagoner et al., 2020; Mondet et al., 2021). At physiological concentrations, we found the greatest effect on brood cell cleaning with the treatment containing all VOCs and LVCs. Our results showed that the VOCs found in this study are more likely to trigger recapping behaviour

than the other compounds from the scientific literature. Depending on the treatment, the cleaning behaviour differed according to the ability of the colony to perform the VSH behaviour.

Rearing bee brood in gelatin capsules makes it possible to be completely free of wax odours, while keeping the brood in an environment physically close to the wax cell (diameter, depth, volume available). This method allowed the capture of 111 compounds over the 7 days of experimentation, which illustrates the rich chemical emission landscape of the brood. Compounds found in the study of Noël et al. (2023) as being emitted in the pre-pupal and non-sclerotized pupal stages were all found here, except for hexenyl acetate. This rearing assay ensures that the vast majority of the compounds captured originate from the brood and/or the parasite. No difference was observed in the total amount of VOC emissions between parasitized and non-parasitized groups, suggesting a subtle modulation of brood emissions due to *Varroa* parasitism and confirming the ability of *Varroa* to mimic the olfactory profile of its host (Kather et al., 2015a, 2015b; Le Conte et al., 2015).

Amongst the compounds that were more abundant in the P group that in the NP group, we identified nine compounds that best characterized the parasitized state, in order to perform behavioural assays. The method used here allowed the capture of short carbon chain compounds such as compounds highlighted by Schöning et al. (2012) and Liendo et al. (2021). Schöning et al. (2012) analysed the odours of NP and P brood with high and low
<figure><img src="image_9.png" /><figcaption>B</figcaption></figure>

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

General legend

Unt: untreated
Iso: isohexane
a: n-tetradecane
A: five VOCs (n-tetradecane, n-hexadecane, benzothiazole, octanoic acid, dodecanoic acid)
B: alpha-pinene & ethyl hexanoate

LVCs
C: (Z)-10-tritracontene & (Z)-6-pentadecene
d: tetracosyl acetate
D: six VPS (2-tricosanone, 2-pentacosanone, tetracosyl acetate, 2-heptacosanone, hexacosyl acetate, 2-nonacosanone)

**Fig. 5.** Hygienic responses of honey bee workers to brood cells treated with different treatments, at two different concentrations, 48 h after injection. (A) Proportion of brood cells in each status (Intact: untouched cells, Recap: recapped cells, Uncap: uncapped cells, Empty: fully cleaned cells), at a high concentration and at physiological concentration. (B) Adjusted uncapping rate means (+/- SE) of injected brood cells according to the different treatments 6 h, 24 h and 48 h after injections. For each treatment, high concentration (10% w/w): *n* = 16 colonies, physiological concentration (10 equivalents): *n* = 15 colonies. VOCs, volatile organic compounds; LVCs, low volatile compounds. Unt, Iso: control; a, d: treatments with one compound; A, B, C, D: treatments containing compounds found in the same study; A = B, C + d, C + D: treatments containing all VOCs or all LVCs; A + B + C + d, A + B + c + d: treatment containing all of the compounds. Lowercase letters represent differences in significance.
<figure><img src="image_11.png" /><figcaption>**Fig. 6.** Adjusted recapping rate means (+/- SE) of remaining injected capped honey bee brood cells to physiological concentration treatments (10 equivalents). For each treatment, *n* = 15 colonies. VOCs, volatile organic compounds; Noël VOCs, treatments that included the five compounds found in this study (benzothiazole, dodecanoic acid, n-hexadecane, n-tetradecane and octanoic acid); Others: treatments that included compounds found in the scientific literature (alpha-pinene and ethyl hexanoate (Liendo et al., 2021), (Z)-6-pentadecene and (Z)-10-tritriacontene (Wagoner et al., 2020), 2-tricosanone, 2-pentacosanone, tetracosyl acetate, 2-heptacosanone, hexacosyl acetate and 2-nonacosanone (Mondet et al., 2021)). Mix: treatment containing all of the compounds. Lowercase letters represent differences in significance.</figcaption></figure>

deformed wing virus infection rates. They placed pupae of interest in a vial and performed compound captures using Tenax TA cartridges for 1 h at 34.5 ± 1°C (Schöning et al., 2012). Nine of the 39 compounds found by Schöning et al. (2012) in the headspace of the NP and P pupae were found in our captures (2-methylpropanoic acid, 2,3-butanediol (diastereomers), isoamyl acetate, benzaldehyde, (Z)-β-ocimene, (E)-β-ocimene, octanoic acid and methyl benzoate). Liendo et al. (2021) captured VOCs from the headspace of P (no Varroa present) and NP pupae at the pre-pupal (in our study: day 1), light pink-eyed pupae (in our study: day 4) and black-eyed pupae (in our study: day 6) stages in a glass aeration chamber. The captures were made for 24 h, at room temperature, with HayeSep Q cartridges. They found α-pinene and ethyl hexanoate to be specific to P brood (Liendo et al., 2021). We did not find ethyl hexanoate in our captures.α-pinene was present in trace amounts and could not be quantified. The differences in the

compounds found in these two studies and in our study may be due to the different properties of the adsorbents used, as each adsorbent has its own volatile affinity (Agelopoulos and Pickett, 1998). Differences may also be due to the duration of capture. In our study we used short captures whereas the studies cited used long captures. Their approaches could have the advantage of detecting and quantifying compounds that are emitted in small quantities by the brood. Differences in the time and temperature of capture, as well as the environment of the (pre-)pupa for capture, could also affect the compounds emitted and captured. Another contributing factor could be the presence (Schöning et al., 2012) or absence (Liendo et al., 2021) of mites and offspring in the samples during captures. These studies complement our understanding of the emissions from V. destructor-parasitized and non-parasitized brood. Our study presents the advantage of profiling the temporal evolution of emissions over seven continuous days.

Three of the compounds (dodecanoic acid, *n*-tetradecane and *n*-hexadecane) identified in this study had been found in trace amounts during the study of VOC capture on brood frames (Noël et al., 2023). Because SPME fibres have different affinities between volatile compounds (Roberts et al., 2000), the emission of wax compounds may have saturated the fibres, preventing the capture of compounds emitted in small quantities or with a lower affinity to the fibre. The capture of VOCs from the emissions of the brood reared in capsules provided the opportunity to avoid the interfering odours of the wax.

The five compounds identified in this study belong to three different chemical families: alkanes (*n*-tetradecane and *n*-hexadecane), carboxylic acids, in particular fatty acids (octanoic acid and dodecanoic acid), and benzothiazoles (benzothiazole). Alkanes are well known to be part of the chemical profile of bees, for both brood and adults (Salvy et al., 2000; Dani et al., 2005; Blomquist et al., 2010; Noël et al., 2023). *n*-Hexadecane was found to be emitted from dead brood extracts and triggered an increase in aggressive behaviour towards individuals treated with this compound (Breed and Stiller, 1992; Haber et al., 2019). The two alkanes identified in our study have already been found to be emitted by worker black-eyed pupae (Liendo et al., 2021). Changes in *n*-hexadecane and *n*-tetradecane emissions in pupae could influence the hygienic behaviour in workers by transmitting information about pupal health. Benzothiazoles have a broad spectrum of effects in pharmacology (Ali and Siddiqui, 2013). Their antimicrobial properties may play a major role in the defence against pathogens in the social spider *Stegodyphus dumicola* (Lammers et al., 2021). This class of compound could be involved in the pupal

<table><caption>**Table 2**

P-values of pairwise comparisons between Varroa-sensitive hygiene (VSH+) and VSH- colonies for each treatment.</caption><thead><tr><th>Solutions</th><th>ID</th><th>High concentration</th><th>Physiological concentration</th></tr></thead><tbody><tr><td>Isohexane</td><td>Iso</td><td>0.230</td><td>0.388</td></tr><tr><td>n-Tetradecane</td><td>a</td><td>8.17e<sup>-6</sup></td><td>0.015</td></tr><tr><td>Five VOCs</td><td>A</td><td>0.362</td><td>0.075</td></tr><tr><td>Alpha-pinene & ethyl hexanoate</td><td>B</td><td>0.097</td><td>0.995</td></tr><tr><td>Five VOCs + alpha-pinene & ethyl hexanoate</td><td>A + B</td><td>0.012</td><td>0.264</td></tr><tr><td>(Z)-10-tritriacontene & (Z)-6-pentadecene</td><td>C</td><td>3.7e<sup>-16</sup></td><td>0.075</td></tr><tr><td>Tetracosyl acetate</td><td>d</td><td>4.50e<sup>-5</sup></td><td>0.005</td></tr><tr><td>VPS</td><td>D</td><td>—</td><td>0.005</td></tr><tr><td>(Z)-10-tritriacontene & (Z)-6-pentadecene + tetracosyl acetate</td><td>C + d</td><td>2.07e<sup>-4</sup></td><td>—</td></tr><tr><td>(Z)-10-tritriacontene & (Z)-6-pentadecene + VPS</td><td>C + D</td><td>—</td><td>0.005</td></tr><tr><td>Final mix</td><td>A + B + C + d</td><td>0.012</td><td>—</td></tr><tr><td></td><td>A + B + C + D</td><td>—</td><td>9e<sup>-4</sup></td></tr></tbody></table>

VSH+, good recognition and cleaning of Varroa-infested cells; VSH-, poor recognition and cleaning of Varroa-infested cells; VOCs, volatile organic compounds; VPS, Varroa parasitization-specific compounds.
<figure><img src="image_12.png" /><figcaption>**Fig. 7.** Adjusted cleaning rate means (+/- SE) of injected honey bee brood cells according to the two *Varroa*-sensitive hygiene (VSH) groups (VSH+/VSH-) and the different treatments, 6 h after injections. For each treatment, for the high concentration (10% w/w), VSH+: *n* = 8 colonies, VSH-: *n* = 7 colonies; for the physiological concentration (10 equivalents), VSH+: *n* = 8 colonies, VSH-: *n* = 5 colonies. VSH-, VOCs, volatile organic compounds; LVCs, low volatile compounds. ISO: control; a, d: treatments with one compound; A, B, C, D: treatments containing compounds found in the same study; A + B, C + D: treatments containing all VOCs or all LVCs; A + B + C + D: treatment containing all of the compounds. \*P < 0.05; \*\*P < 0.005; \*\*\*P < 0.001.</figcaption></figure>

response to the mite or in the bees' immune response to the viruses transmitted by *Varroa*. Fatty acids are known to be a death signal for social insects (Wilson et al., 1958; Haskins and Haskins, 1974; Sun and Zhou, 2013; McAfee et al., 2018), and dodecanoic acid (also known as lauric acid) has been found to be emitted by liquid nitrogen-killed and freeze-killed pupae in the stingless bee (Neiva De Jesus et al., 2024). The two fatty acids found in this study could be also a signal of *Varroa* parasitism. Octanoic acid (also known as caprylic acid) and dodecanoic acid have been shown to be present in total lipids extracted from *Varroa* and *Varroa*-parasitized brood (Dmitryjuk et al., 2015). Dodecanoic acid was present on the cuticle of the *Varroa* foundress (Martin et al., 2002), and in greater quantities in total lipids extracts of parasitized drone pre-pupae than the *Varroa* mite (Dmitryjuk et al., 2015). Octanoic acid was only found in the total lipid extracts of parasitized pre-pupae drones (Dmitryjuk et al., 2015). With our capture system, it is not possible to distinguish between VOCs emitted by the brood or the mite; the *Varroa* mite could emit these five compounds in the reproductive phase, or the pupa could be attempting to disrupt the *Varroa* survival and/or reproduction, or signalling its own health status. Furthermore, two of those compounds (benzothiazole and *n*-tetradecane) triggered an electro-

physiological response from *Varroa* (Light et al., 2020) and octanoic acid has already been shown to be repellent to *Varroa* mites (Nazzi et al., 2004, Nazzi et al., 2009). This suggests that these compounds could have a physiological or behavioural effect on *Varroa*, thus affecting its life cycle.

The five VOCs triggered hygienic behaviour in the field bioassay. We expected that the mixture of the five compounds would trigger a faster and stronger response than the solutions containing a single compound. However, we observed a stronger response from the *n*-tetradecane compared with all other solutions tested, when measured 6 h post-treatment. Similarly, phenethyl acetate triggers greater hygienic behaviour than a mixture with two other compounds (Swanson et al., 2009); oleic acid triggers a cleaning rate equivalent to that obtained from the mixture of oleic acid and β-ocimene when applied to pupae inside uncapped brood cells (McAfee et al., 2018), and ethyl hexanoate triggers an equivalent cleaning rate alone or when mixed with α-pinene (Liendo et al., 2021). *n*-tetradecane is a potentially crucial signal in the detection process given the workers' rapid cleaning response to it and a potentially interesting target for studying the neural process triggered by *n*-tetradecane in individuals performing hygienic behaviour. Indeed, the rapid response of workers to cells injected
with *n*-tetradecane could be due to a specific neural pathway. For example, two odorant binding proteins that transport odorants to olfactory receptors facilitating their passage into the sensillary lymph, have been shown to be strongly involved in hygienic behaviour (Guarna et al., 2015; McAfee et al., 2018). Currently, only compounds with low affinity have been found for these two proteins (McAfee et al., 2018). *n*-Tetradecane could have a high affinity for one of them, enabling more rapid activation of the olfactory receptors.

Compounds inducing a behavioural response such as pheromones or other semiochemicals, are often optimal at a specific dose and no longer induce responses at lower or higher doses (Wyatt, 2014). Therefore, we decided to use two different concentrations to study behaviour induced by the compounds that are characteristic of parasitized brood: a physiological concentration (10 equivalents) and a high concentration (10% w/w). Behavioural studies commonly use a high concentration of compounds to test worker behaviour (Nazzi et al., 2004; Liendo et al., 2021; Wagoner et al., 2021). In parallel, the physiological dose approaches the natural emissions of *V. destructor*-parasitized brood cells. The dose of 10 equivalents was selected according to the results found by Mondet et al. (2021) that the response of workers to VPS treatment at 10 equivalents was low enough to observe potential synergistic or additive effects with treatments containing VPS. At this concentration, 48 h after injection, our results agree with those found by Mondet et al. (2021) (40% cleaning for Mondet et al., 2021; our study: 38% cleaning).

All compounds tested have been previously described as triggering hygienic behaviour (Nazzi et al., 2004; Liendo et al., 2021; Mondet et al., 2021; Wagoner et al., 2021), but the combination of these compounds in triggering hygienic behaviour had never before, to our knowledge, been tested. LVCs alone triggered a stronger response from workers at higher concentrations than at physiological concentrations. The lower response of workers to LVCs at the physiological concentration could reflect a lower perception of these compounds by the workers. Since these compounds are not very volatile, increased concentration may favour the passage of compounds through the waxy cap and promote perception of this chemical message by the workers. Another possibility could lie in differences in sensitivity to odorant between bees performing different tasks (Masterman et al., 2001; Spivak et al., 2003). Under such a hypothesis, recappers might exhibit a lower sensitivity to the LVCs than openers or cleaners. Also, at higher concentrations, a greater effect can be observed when the LVCs are all present, which was not observed at the physiological concentration. This effect can be seen as early as 6 h after injection. This suggests that a more complex message may have a greater impact on worker hygienic behaviour. The strong response of C and C + d mixtures may be linked to the presence of alkenes ((Z)-6-pentadecene and (Z)-10-tritriacontene). Alkenes play a key role in nestmate recognition in honey bees (Dani et al., 2005; Pradella et al., 2015). A change in the ratio can trigger a strong and rapid response from the workers. This strong responses of workers to LVCs confirmed the crucial roles of these compounds in hygienic behaviour (Nazzi et al., 2004; Mondet et al., 2021; Wagoner et al., 2021). In contrast to LVCs, increasing the complexity of the VOCs mixture did not necessarily increase the cell-cleaning rate. At the physiological dose, the mixture of all compounds triggered a greater cleaning response than the mixture of LVCs and the mixture of VOCs, and showed an additive effect of the compounds when used in blends. It has been suggested that highly volatile compounds coupled with less volatile compounds can be used as chemical cues by workers to identify brood cells and then to remove them (McAfee et al., 2018). The present study strongly suggests that workers use VOCs to target cells to be

uncapped. Uncapping would then allow the workers to access the LVCs and thus to trigger cleaning of the brood cell. Moreover at the higher concentration, LVCs seem to pass sufficiently through the cap to allow for identification of the cells to be cleaned, even before the uncapping event.

Recapping behaviour is difficult to study because the injection of compounds into the brood cells triggers hygienic behaviour, thereby reducing the number of cells available to be recapped. To truly measure recapping, the workers need to be inhibited from removing the pupae from brood cells. We thus decided to study the recapping behaviour of the remaining cells 48 h after injection for the lower concentration only, for which more uncleaned cells were present.

The brood cell is more likely to be recapped when the pupa is less likely to be damaged (Sprau et al., 2023). When the five VOCs found in this study were captured, the pupae did not appear to be in poor health. Furthermore, individuals that had completed their metamorphosis appeared to be in perfect health (normal size, no deformed wings). These VOCs could indicate parasitism without major varroosis symptoms. As the recapping rate of the total mixture was similar to the recapping rate of mixtures containing only the VOCs identified in this study (*n*-hexadecane, *n*-tetradecane, dodecanoic acid, octanoic acid and benzothiazole) and higher than that containing only compounds from previous studies, this suggests that our compounds are more likely to play a role in the recapping behaviour and may transmit that a pupa is "not so injured" (for example, with a low virus load). However, further studies are needed to confirm this hypothesis, in particular by comparing the health of pupae that undergo recapping with those that undergo hygienic behaviour.

This study investigated the impact of different compound mixtures on the VSH behaviour. Only the *n*-tetradecane, the tetracosyl acetate and the VPS solutions appeared to be linked to the ability of colonies to perform VSH behaviour at low concentrations. Bees selected for VSH behaviour are more sensitive to odour discrimination and can discriminate VOCs at very low concentrations (Ivanova and Bienefeld, 2023). For VPS compounds, only workers performing the VSH behaviour are able to discriminate the odours of healthy brood from the odours of healthy brood coupled with VPS at a low concentration (Mondet et al., 2021). These two compounds could play a key role in VSH behaviour.

The parasitism signal appears as a complex signal composed of several compounds, and the results of this study provide a better understanding of the impact of these signals on VSH and recapping behaviours. Nevertheless, it is important to remember that only five of the nine parasitism VOCs were successfully identified and therefore tested in behavioural tests. The four unknown VOCs, as well as less statically discriminating compounds, could be a missing piece in the puzzle of the volatile message of parasitism and could be involved in worker-brood communication as well as *Varroa*-brood communication.

The applied aim of characterising *Varroa* resistance traits in honey bees is to develop tools to enable beekeepers to fight the mite effectively. The discovery of new compounds with strong responses from *Varroa*-resistant colonies, that are linked to the colony's ability to perform VSH behaviour, could make it possible to develop an easy-to-use phenotype test for beekeepers to employ in *Varroa*-resistant honey bee selection programs. Our results confirm the effect of VPS compounds and show that only *n*-tetradecane, tetracosyl acetate and the VPS trigger greater hygienic behaviour in VSH+ than in VSH- colonies, at both high and physiological concentrations. Further studies are needed to fully understand the relationship between the individual behavioural response of workers towards compounds and the hygienic performance at the colony level.
CRediT authorship contribution statement

**Amélie Noël:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Charlène Dumas:** Writing – review & editing, Investigation, Data curation. **Emilien Rottier:** Writing – review & editing, Investigation, **Dominique Beslay:** Writing – review & editing, Investigation. **Guy Costagliola:** Writing – review & editing, Methodology, Data curation. **Christian Ginies:** Writing – review & editing, Methodology, Data curation. **Florence Nicolè:** Writing – review & editing, Data curation. **Yves Le Conte:** Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization. **Fanny Mondet:** Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Acknowledgements

This research was funded by a European Union H2020 Prima grant (#1812; Plant-B).

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