Introduction
Electroantennography (EAG) is a widely used technique to investigate the olfactory detection of arthropods to volatile organic compounds (Cork et al., Reference Cork, Beevor, Gough, Hall, McCaffery and Wilson1990; Piersanti et al., Reference Piersanti, Rebora, Marri and Salerno2024). However, the method of anaesthesia prior to antennal dissection can influence the integrity and functionality of sensory receptors, potentially affecting the results of such studies.
Cold anaesthesia, being a very economical and easy method, is commonly employed to temporarily immobilise invertebrates for experimental preparations, typically by placing the individual in a freezer until immobilisation occurs (Frost et al., Reference Frost, Shutler and Hillier2011). However, while cold anaesthesia is convenient for handling single individuals, it is less practical for immobilising groups, like honey bees, as they may cluster to generate and retain heat (Stabentheiner et al., Reference Stabentheiner, Kovac and Brodschneider2010). Moreover, cold anaesthesia has been shown to negatively affect bees’ behaviour and physiology, including impacts on short-term memory (Frost et al., Reference Frost, Shutler and Hillier2011), locomotion (Chen et al., Reference Chen, Fu, He and Wang2014), foraging (Poissonnier et al., Reference Poissonnier, Jackson and Tanner2015; Wilson et al., Reference Wilson, Holway and Nieh2006), and defensive behaviour (Groening et al., Reference Groening, Venini and Srinivasan2018). On the contrary, CO₂ exposure is a widely used method due to its simplicity and rapid immobilisation, making it ideal for experimental studies (Kohler et al., Reference Kohler, Meier, Busato, Neiger-Aeschbacher and Schatzmann1999). However, it is known to alter behaviour due to hypoxia-induced stress, as highlighted in some old studies (Ribbands, Reference Ribbands1950). It has also been shown to provoke changes in fecundity and longevity (Tasei, Reference Tasei1994) and in juvenile hormone titres (Bühler et al., Reference Bühler, Lanzrein and Wille1983). Diethyl ether, though less common, is effective for immobilising insects quickly, but its flammability and potential for adverse effects on both researchers and specimens make it less desirable for regular use (Arora and Gautam, Reference Arora, Gautam, Chawla, Singh and Kaushik2025; Cooper, Reference Cooper2001). While more costly, recent studies proposed isoflurane and sevoflurane as alternatives (Gooley and Gooley, Reference Gooley and Gooley2023). These volatile anaesthetics offer precise, reversible immobilisation with minimal impact on longevity and behaviour, making them particularly advantageous in experiments requiring repeated anaesthesia (Cooper, Reference Cooper2001, Reference Cooper2011; Rayl and Wratten, Reference Rayl and Wratten2016).
Eusocial insects use chemical communication to coordinate complex behaviours such as foraging, defence, and reproduction (Yew and Chung, Reference Yew and Chung2015). Among these chemical signals, alarm pheromones play a critical role in colony defence, triggering aggressive responses to perceived threats (Bruschini et al., Reference Bruschini, Cervo and Turillazzi2008).
In the Yellow-legged hornet, Vespa velutina nigrithorax, a species that has become an invasive pest in Europe (Darrouzet, Reference Darrouzet2024; Robinet et al., Reference Robinet, Darrouzet and Suppo2019) and Asia (Choi et al., Reference Choi, Martin and Lee2012; Takeuchi et al., Reference Takeuchi, Takahashi, Kiyoshi, Nakamura, Minoshima and Takahashi2017), understanding the function and detection of alarm pheromones is of particular ecological and applied interest (Berville et al., Reference Berville, Lucas, Haouzi, Khalil, Gévar, Bagnères and Darrouzet2023; Cheng et al., Reference Cheng, Wen, Dong, Tan and Nieh2017). This species has garnered increasing scientific interest as a study model due to its invasive nature and impact on native ecosystems and pollinator populations (Darrouzet, Reference Darrouzet2024). Its complex social behaviours (e.g., communication, foraging strategies, and colony organisation), along with its ecological and economic significance, make this species a valuable subject for research. The growing concern over its rapid spread and the threats it poses to biodiversity and apiculture (Carisio et al., Reference Carisio, Cerri, Lioy, Bianchi, Bertolino and Porporato2022; O’Shea-Wheller et al., Reference O’Shea-Wheller, Curtis, Kennedy, Groom, Poidatz, Raffle, Rojas-Nossa, Bartolomé, Dasilva-Martins, Maside, Mato and Osborne2023; Rojas-Nossa et al., Reference Rojas-Nossa, O’Shea-Wheller, Poidatz, Mato, Osborne and Garrido2023) has amplified the need for laboratory and field studies to acclimate, maintain, and manipulate live hornets. For example, this need is important to analyse pheromone production and perception by females (Berville et al., Reference Berville, Lucas, Haouzi, Khalil, Gévar, Bagnères and Darrouzet2023). In laboratory settings, anaesthesia is essential when handling individual hornets, as their venomous stings pose a risk of injury to researchers. Cold and CO2 narcosis are the most commonly used anaesthesia methods for hornets. While freezing is a common aesthetic in insect studies, its physiological effects on sensory perception are not fully investigated.
Consequently, prior to analysing the antennal responses of V. velutina nigrithorax to synthetic alarm pheromone components, in this study, we investigated which simple anaesthesia methods could be used, i.e. either CO₂ or freezing. By comparing the electroantennographic responses to key compounds, we aimed to assess the impact of anaesthesia method and to select the least detrimental one for this type of study.
Materials and methods
Insect sampling
Adult workers (foragers) of V. velutina nigrithorax were captured with a net at an apiary located within the experimental garden of the CEFE laboratory (Centre d’Écologie Fonctionnelle et Évolutive) in Montpellier (France). The individuals were housed separately in Faclon™ tubes (50 mL, 115 × 30 mm) before being tested. Experimental procedures were carried out at the CEFE from 25 November to 7 December 2024.
Aesthesia treatments
Hornets were anesthetised using either carbon dioxide (CO₂) exposure (n = 14) or cold treatment (n = 11). Individuals were placed under a CO2 flow for a few seconds until they stopped moving, or in a freezer (−20°C) for 2 min. Following anaesthesia, hornets were handled with forceps and one antenna (randomised between individuals) of each hornet was excised with microscissors, and prepared for EAG analyses.
Stimulation solutions
We prepared dilutions (0.01, 0.1, and 1 µg/µL) in paraffin of four synthetic compounds present in the alarm pheromone. These included 2-nonanone (Aldrich, CAS: 821-55-6), 2-undecanone (Aldrich, CAS: 112-12-9), and the two enantiomers (R and S) of 4,8-dimethyl-7-nonen-2-one (provided by the Scyll’Agro company) (Berville et al., Reference Berville, Lucas, Haouzi, Khalil, Gévar, Bagnères and Darrouzet2023). Ten microliters of each dilution was applied to filter paper and inserted into a glass pipette connected to an airflow delivery system.
EAG system
One of the two antennae (randomised) of each hornet was excised, and the distal tips were inserted into two glass capillaries filled with Ringer’s solution (NaCl/KCl/CaCl2/NaHCO3, Na+ 131 mmol/L, K+ 5 mmol/L, Cl− 111 mmol/L, C3H5O3 29 mmol/L). These capillaries were subsequently mounted onto silver electrodes of an EAG set-up Kombi Probe PRG-3 (SYNTECH®, Kirchzarten, Germany).
The antenna was placed within a continuous flow of purified and humidified air directed through a tube at a rate of 435 mL/min for stimulation. The tip of an odour cartridge, constructed from a Pasteur pipette, was inserted into a small opening in the airflow tube. Odour stimulation was delivered by a 0.5-s pulse of purified air through the cartridge, with a flow rate of 890 mL/min controlled by a CS-55 Stimulus Controller (Syntech, Kirchzarten, Germany). Electrophysiological responses were recorded using GcEad 2014 v1.2.5 software (Syntech, Kirchzarten, Germany). Each antenna was tested with three stimulus sequences, where each sequence included all four selected compounds at specific doses along with paraffin controls. The doses were presented in ascending order (0.01, 0.1, 1 µg/µL) for each sequence, with compounds applied in a randomised order. Paraffin controls were included at the beginning and end of each sequence. The EAG response amplitude was calculated by subtracting the average response to the paraffin for each sequence.
Analysis
Depolarisation amplitudes were quantified by subtracting the response to the solvent control from the response elicited by each test compound. Statistical analyses were performed using R (version 4.3.1, R Core Team). We performed t-tests to compare our two conditions for each concentration and compounds. Graphical representations were obtained with ggplot package.
Results and discussion
A significant difference was observed in antennal responses of V. velutina nigrithorax to synthetic alarm pheromone compounds tested depending on the method of anaesthesia. Hornets anesthetised with CO₂ exhibited robust electroantennographic responses to all tested compounds, while those anesthetised by cold showed no detectable response in any of the concentrations (fig. 1, table 1).
Comparison of the antennal depolarisation between cold- and CO2-anesthetised hornets for different concentrations of the selected pheromone compounds. (A) Concentration of 0.01 µg/µL. (B) Concentration of 0.1 µg/µL. (C) Concentration of 1 µg/µL.
T-test results of the antennal response between cold- and CO2-anasthetised hornets for the different synthetic alarm pheromone compounds at different concentrations (0.01, 0.1, and 1 µg/µL)
These results suggest that cold anaesthesia, achieved by placing the individual hornet in a freezer, may probably impair the functionality of antennal olfactory receptors. Freezing and thawing are known to cause cellular damage, potentially affecting the delicate structures of the antennal sensilla and their associated neurons. Additionally, rapid cooling may disrupt ion channels and synaptic transmission, leading to a loss of olfactory signal transduction (McGann et al., Reference McGann, Yang and Walterson1988).
In contrast, CO₂ anaesthesia gives interpretable results and appears to probably preserve the integrity of antennal receptors, allowing reliable detection of volatile compounds. This aligns with previous studies indicating that CO₂ has transient and reversible effects on insect physiology, minimising long-term impacts on sensory systems (Barie et al., Reference Barie, Levin and Amsalem2022; MacMillan et al., Reference MacMillan, Nørgård, MacLean, Overgaard and Williams2017). For instance, exposure to CO₂ anaesthesia has been shown to increase chill coma recovery time in Drosophila melanogaster, but this effect diminishes after a recovery period in air, suggesting minimal long-term physiological disruption (Nilson et al., Reference Nilson, Sinclair and Roberts2006). Poissonnier et al. (Reference Poissonnier, Jackson and Tanner2015) showed the bees were more active after a CO2 treatment compared to control and cold-treated bees, which could be beneficial for behavioural experiments.
The absence of response in cold-anesthetised hornets raises concerns about the reliability of freezing as a method of immobilisation in studies involving sensory physiology. While freezing is a convenient and commonly used technique, our results suggest it may introduce significant artefacts, particularly in chemical communication and behavioural ecological studies. A study by Wilson et al. (Reference Wilson, Holway and Nieh2006) on honeybees suggested that the cold-induced decrease in foraging could be due to impaired cognitive or sensory receptor abilities and therefore the lack of detection and response to recruitment stimuli. With our results, we could imagine that antennal receptors could be damaged, leading to the non-detection of pheromones and other molecules needed in this kind of behaviours.
Future research should explore the cellular and molecular impacts of freezing on insect sensory organs and evaluate alternative methods of immobilisation. In addition, further work is needed to determine whether the observed effects are consistent across other insect species and sensory modalities. By optimising experimental methodologies, we can improve the accuracy and reproducibility of studies in neurophysiology and chemical communication in arthropods.
Acknowledgements
We gratefully acknowledge C. Bresse and colleagues from the Scyll’Agro company for the financial help and scientific discussions. We thank E. Vandenbroucke for her help during the early phase of this project.
Author contributions
Dissections, data collection, and statistical analyses were carried out by C.M. EAG analyses were performed by C.M. and implemented in the analytic instrument by B.L. Writing and editing were carried out by all co-authors. D.E. managed the project and obtained the funds. All authors read and approved the final manuscript.
Financial support
The EAG system was funded by the Platform for Chemical Analysis in Ecology (PACE) at the CEFE (Centre of Functional and Evolutionary Ecology, Montpellier, France). C.M. was supported by a private fellowship from the Scyll’Agro company.
Competing interests
The authors declare no competing or financial interests.
Data availability
All relevant data and resources can be found within the article and its supplementary information.
Open access
