Varroa destructor weakens honey bee external immunity by impairing melittin production

Varroa destructor weakens honey bee external immunity by impairing melittin production | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Varroa destructor weakens honey bee external immunity by impairing melittin production Michelina PUSCEDDU, Simon Tragust, Panagiotis Theodorou, Irene Ciabattini Bolla, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6238801/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Social insects employ venom as an external immune defence against pathogens and parasites. Like other Hymenoptera, the venom gland of honey bee serves as a reservoir of antimicrobial substances, primarily melittin. This study investigates the role of venom associated with grooming behaviour as an external immune defence in Apis mellifera workers infested by Varroa destructor . Using a multi-step approach, we first confirmed the presence of venom on bees' bodies using melittin as a marker. We then examined how grooming facilitates the distribution of venom on the bee's body. Further assays compared melittin levels on the bodies of Varroa -free and Varroa -infested workers and assessed the effects of bee-venom on mite activity. Our findings confirmed the occurrence of "venom bathing" in A. mellifera , whereby bees coat their bodies with antimicrobial substances through selfgrooming. excluding social components or environmental contamination. Infested bees spread larger amounts of venom on their bodies compared to uninfested bees and bee-venom significantly also reduced mite activity, suggesting venom functions as an external defence. However, Varroa negatively impacts melittin production. Our study reveals a previously unknown negative effect of V. destructor : impairment of honey bees' external immune defence through reduced melittin production. Biological sciences/Ecology Biological sciences/Zoology Health sciences/Diseases Apis mellifera mite ectoparasite bee venom grooming Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Bullet point Honey bees employ "venom bathing" via selfgrooming to distribute antimicrobial melittin on body surfaces. Varroa -infested bees spread proportionally more venom on their bodies whilst having reduced melittin production. Bee venom significantly reduces mite activity, suggesting its role as an external immune defence. Varroa destructor impairs honey bees' external immune defence through reduced melittin production. Introduction Collaboration among individuals in social insects leads to important benefits in brood care, foraging, antipredator defence, and other colony survival activities (Krause et al. 2002 ). However, the close and frequent interactions among genetically homogeneous nestmates, the large amounts of food stored in the nest and the relatively stable conditions in the nest-environment may also promote the spread of parasites and pathogens within the colony (Schmid-Hempel 1998 ). To counteract the risk of disease contraction and transmission within their societies, apart from their body innate immune system, social insects possess a diversity of physiological, behavioural and organizational adaptations, collectively often called social immunity (Cremer et al. 2007 , 2018 ; Wilson-Rich et al. 2009 ; Evans and Spivak 2010 ). As many of these adaptations act in the environment of a social insect society, they can also be conceptualized as external immune defence, i.e. traits acting outside the individual and either improving the protection from pathogens and parasites or manipulating the composition of the microbial community in favour of the host (Otti et al. 2014 ). For example, ants and honey bees incorporate tree resin with antimicrobial properties in their nests, thus modifying the microbial community of the nest environment (Chapuisat et al. 2007 ; Simone-Finstrom et al. 2010). In the case of ants, resin collection seems to be a mechanism implemented to prevent disease (Castella et al. 2008 ), whereas, in the case of honey bees, this behaviour can also have curative purposes, as suggested for the fungus Ascosphaera apis (Simone-Finstrom and Spivak 2012 ) and the mite Varroa destructor (Pusceddu et al. 2018 , 2019 , 2021a ). In addition to environment-derived bioactive substances (e.g., resins), self-produced substances, especially from exocrine glands such as the venom gland, can play a key role in external immunity (Tragust 2016 ; Baracchi and Tragust 2017 ). In many species of aculeate Hymenoptera, the sting apparatus and venom originally evolved as tools to kill prey but became a means of defence against predators, mainly vertebrates (Schmidt 1982 ). However, the venom of aculeate Hymenoptera is also a source of antimicrobial substances (Kuhn-Nentwig 2003 ; Moreau 2013 ), suggesting its potential use as an immune defence trait. The adaptive role of venom to sanitize oneself as well as the nest, other nest members and food is well known in ants (reviewed in (Tragust 2016 ; Koch et al. 2025 )), which can apply and spread their antimicrobial venom during grooming behaviour (e.g., (Tragust et al. 2013a )). Whether honey bees, or bees in general, also use their venom for sanitary purposes as ants do is less clear. Like ant venoms, bee venoms, especially the honey bee venom, are pharmacologically active products, which consist of a complex mix of biogenic amines, proteins, peptides, phospholipids, sugars, and volatile components (Dotimas and Hider 1987 ; Carpena et al. 2020 ; Guido-Patiño and Plisson 2022 ; Koludarov et al. 2023 ). Among species in the genus Apis , venom production can be influenced by seasonality (Ferreira et al. 2010 ), age and caste (Ali 2012 ), and species body size (Schmidt 1995 ). However, also in species with similar body size, differences in venom amount and composition occur. For instance, although the Asian honey bee A. cerana produces only half as much venom as A. mellifera , its venom contains a higher proportion of the peptide melittin (Schmidt 1995 ). Melittin is the main component of honey bee venom, accounting for 50% of the venom dry weight (Owen and Pfaff 1995 ; De Lima and Brocchetto-Braga 2003). Melittin has antiseptic (Kuhn-Nentwig 2003 ), strong antiviral (Kontogiannis et al. 2022 ), antifungal and antimicrobial properties (Habermann 1972 ). Apart from melittin, antimicrobial activity has also been suggested for other bee venom peptides such as apamin and MCD (Froy and Gurevitz 1998 ). In honey bees, the possible use of venom as an external immune defence trait in a social immunity context was, to our knowledge, first hypothesized by Baracchi and Turillazzi ( 2010 ), who found traces of venom compounds on the body of adult honey bees and on the surface of honeycombs. Later, the same authors found that although melittin was present in the venom of both open-nesting and cavity-nesting bees, it was detectable on the cuticle and combs only of the latter (Baracchi et al. 2011 ), thus suggesting that nesting ecology and environment shape the deposition of venom, potentially due to different associated disease pressures. Finally, with respect to the overall amount of venom peptides both in the venom and on the cuticle, the same authors (Baracchi et al. 2011 ) suggested that A. cerana performs a higher level of “venom bathing” compared to A. mellifera . This latter result is in accordance with a higher rate of grooming activity shown by A. cerana compared to A. mellifera , with grooming being one of the main mechanisms of resistance against parasitic mites in the Asian honey bee (Pritchard 2016 ). A relationship between grooming and venom is also suggested by the complete absence of venom peptides on the cuticle of freshly emerged bees, which probably cannot produce the venom yet, and adult drones in both bee species (Baracchi and Turillazzi 2010 ; Baracchi et al. 2011 ). Considering that previous research suggested that the role of venom in honey bees is well beyond the classical defence activity against predators (Baracchi et al. 2011 ), in our study we investigate the role of grooming in the distribution of venom compounds on the honey bee's body, as an external immune defence trait, upon challenge of A. mellifera by the parasitic mite Varroa destructor as well as the venom effects on the parasite activity. It is important to highlight that V. destructor was originally confined to the Asian honey bee, A. cerana (Rosenkranz et al. 2010 ). After the shift to the new host A. mellifera , this parasite spread worldwide and is currently the most serious pest of honey bees (Rosenkranz et al. 2010 ; Nazzi and Le Conte 2016 ). To conduct our study, we first performed chemical analyses to confirm the presence of venom on the bodies of worker bees (both nurses and foragers), using melittin as a marker. To exclude the possibility that the detected melittin originated from environmental contamination within the nest, we also quantified the amount of melittin on the bodies of drones and freshly emerged workers. Additionally, to ascertain that the melittin detected on the worker bees' bodies originated from venom produced by the bees themselves and smeared on the cuticle through grooming, we quantified the amount of venom on bees with either blocked or unblocked stingers. These bees were kept together in groups or separately. We then investigated if venom transfer to the body surface of honey bees is influenced by parasite presence. To do this, we quantified the amount of venom (using melittin as a marker) on the bodies of infested (both at the pupal and adult stage) and uninfested worker bees for comparison. To ensure that bees in the three experimental groups had an equal supply of venom and could thus equally utilize this defence mechanism, we also quantified the amount ofmelittin inside their venom sacs. In addition, since previous studies have suggested that grooming in A. mellifera is not effective in counteracting the mite (Büchler et al. 1992 ; Fries et al. 1996 ; Pettis and Pankiw 1998 ; Boecking and Spivak 1999 ; Pritchard 2016 ; Invernizzi et al. 2022 ), but it may play an additional role in relation to venom-spread, we determined the frequency and efficiency of selfgrooming and allogrooming through behavioural observations comparing infested and uninfested group of honey bee workers. Finally, through toxicological laboratory assays, we evaluated whether a biological amount of bee venom could induce lethal or narcoleptic effects on the ectoparasite V. destructor . Materials and methods Chemical analysis Standards and reagents. Melittin was purchased as certified analytical standard (purity ≥ 85%) by Sigma Aldrich (Milan, Italy), whereas acetonitrile (ACN), formic acid and HNO 3 (67–69%) were LC/MS grade (Sigma Aldrich, Milan, Italy), reagent grade (> 95%, Honeywell, Sigma Aldrich, Milan, Aldrich) and ultra-pure grade (Romil Spa, Cambridge, England) solvent, respectively. MilliQ water (18.25 MΩ × cm) was obtained from an integrated Millipore purification system (MilliQ, Merck, Milan, Italy). A melittin stock solution (1000 µg mL − 1 ) was prepared by solubilization in a 0.1% aqueous formic acid solution and stored at 4°C until use. The working solution (200 µg mL − 1 ) was freshly prepared each day by diluting the stock solution with a 0.1% aqueous formic acid solution. The five-point calibration curve was obtained by diluting the working solution with a 0.1% formic acid aqueous solution at concentrations of 0.1, 0.25, 0.50, 1, and 1.5 µg mL − 1 . Extraction of melittin from bees and the venom sac . Individual bees and venom sacs were placed into 1.5 mL Eppendorf tubes to extract melittin. An amount of 1 mL of 0.1% aqueous formic acid solution was added to each tube. Then, the tubes were vortexed for 1 min and for other 15 min on a rotary shaker (Reax Top, Heidolph, Germany). Finally, the solution was transferred to vials and analysed by LC-MS/MS. LC-MS/MS analysis. Analytical determinations of the solution from extracted bees were performed using an Agilent 1290 Infinity II UHPLC coupled to an Agilent 6470 Triple Quad LC-MS/MS mass detector paired with a MassHunter ChemStation. The column was a ZORBAX Eclipse Plus C18 (2.1 × 150 mm, 1.8 µm). A binary gradient, H 2 O + 0.1% formic acid (A) and ACN + 0.1% formic acid (B) was set as follows: T = 0 at 95% A, T = 4.30 min at 15% A, T = 5.80 min at 15% A, T = 7.70 min at 95% A, and 2 min post-run at 95%. The flow rate was 0.2 mL min − 1 , with 5 µl sample volume injected in positive mode. The mass detector gas and the sheet gas were set at 300°C and 250°C, with flow rates 5 L min − 1 and 11 L min − 1 , respectively. The nebulizer was at 30 psi and the capillary voltage was 4000 V. Melittin has a molecular weight (MW) of 2646.46 Da. Ionization in the positive ESI mode produces a stable MH + 4 ion with a MW of 712.44 (S1 Table). Analyses were carried out in dynamic MRM mode (S2 Table). Analytical method validation. To validate our analytical method, we followed the SANTE guidelines by evaluating linearity, selectivity, precision, limits of quantification of the method (LOQ), accuracy in terms of recovery, uncertainty, and matrix effect (SANTE/11312/2021). Six control bees were fortified by depositing an aliquot of solution of the analytical standard at 1 µg mL − 1 , left to rest for 24 h, and extracted as reported above. Each sample belonged to an independent experiment. Six solutions of the analytical standard at 0.1 µg mL − 1 (LOQ) and 1 µg mL − 1 (10 × LOQ) were analysed within one day to verify their repeatability (RSDr, intraday). Reproducibility (RSDwR) was calculated by analysing two analytical standard solutions over six days. Recovery results were analysed using matrix control standard calibration curves. The matrix effect was evaluated by comparing the analytical response of melittin in H 2 O + 0.1% formic acid with solutions prepared with extracts from unfortified control (blank) bees. Linearity was assessed by analysing standard calibration curves performed on five different days and was considered acceptable when the coefficient of determination was greater than 0.990. Selectivity was assessed by comparing the extracts of unfortified control bees with those spiked with the standard. The absence of chromatographic peaks at melittin retention times was a criterion for the selectivity of the confirmatory method. The expanded measurement uncertainty (U), a quantitative parameter of the reliability of the analytical method, was calculated by multiplying the combined uncertainty (u′) by a coverage factor k = 2, to obtain a 95% confidence level, using the following equation: u′= √𝑢′(𝑏𝑖𝑎𝑠)2+𝑢′(𝑝𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛)2; U = k × u′ The instrumental LOQ was calculated as the lowest fortification level that meets the method identification and performance criteria regarding recovery and precision. Experimental apiary The study was performed from April 2023 to November 2023 in an experimental apiary of the Department of Agricultural Sciences of the University of Sassari located in Ottava, Sardinia (Italy; latitude 40°46′23″, longitude 8°29′34″). The apiary consisted of 12 colonies of Apis mellifera maintained in Dadan-Blatt hives containing 10 combs each. During this period, the colonies were monitored every 2 weeks to check for the presence of the queen and food stores and to evaluate the sanitary status (disease symptoms and varroosis) of the bees. Before selecting the colonies to be used in the experiment, the V. destructor infestation level (%) of each colony was assessed following standard method (Pappas and Thrasyvoulou 1988). The infested colonies were used as a source of V. destructor mites, whereas the uninfested ones (infestation level < 1%) were used as a source of honeybee adults. Detection of melittin on the body of adult worker bees and drones We sampled 10 individuals per category: freshly emerged, nurse, forager bees and drones from three different colonies (totalling 30 individuals per category-group). Each sampled bee was immediately placed individually inside a 1.5 ml Eppendorf, sacrificed on dry ice, and stored at − 20°C without its abdomen until chemical analyses were performed by following the procedure described above. The abdomen was detached to prevent contamination of the collected bees' bodies during subsequent manipulations necessary for chemical analyses. Freshly emerged bees were directly sampled upon their emergence. To collect nurse bees, freshly emerged bees were marked on the thorax with a non-toxic colour upon emergence, returned immediately to their original hives and, seven days later, sampled from the hives. Forager bees with pollen load were captured at the hive entrance. Drones were sampled from inside the colonies. Origin of the melittin detected on the worker's body To establish the origin of melittin on the bee body surface, we quantified the amount of melittin present on the bodies of bees belonging to the following experimental groups: 1) 90 bees with blocked stingers, 2) a mix of 90 bees with blocked (45) and unblocked stingers (45), and 3) 90 bees with unblocked stingers (control group). The stinger was blocked using a stick to apply a droplet of non-toxic glue onto the tip of the abdomen. All bees were marked on the thorax with a non-toxic colour identifying the treatment. To exclude the family effect, experimental groups were replicated in three independent metal cages (10 cm × 10 cm × 5 cm), each containing a mix of 30 emerging bees each from three Varroa -free colonies (10 bees per colony per cage), on the same day. The larger inner side of the cages was covered by a sheet of bee-wax (9 cm × 9 cm), whereas the opposite side was closed by a glass window (10 cm × 10 cm). All cages were kept in an incubator (+ 31.5°C, 70% R.H., dark) with 50% (w/v) sucrose solution administered ad libitum with a graduated syringe (Williams et al. 2013 ). On the seventh day, all bees were sacrificed on dry ice and stored without abdomen at − 20°C until chemical analyses. Detection of melittin on the body of infested and uninfested workers and in the venom sac To determine if venom, using melittin as a proxy, varied quantitatively on the body surface of Varroa infested and uninfested bees, we set up the following experimental groups: 1) 90 workers parasitized during the adult stage, 2) 90 workers parasitized during the pupal stage, and 3) 90 uninfested workers (control). To obtain workers parasitized during the adult stage (phoretic infestation) and uninfested workers from the Varroa -free colonies, we used the method described in (Pusceddu et al. 2018 ). To obtain workers parasitized during the pupal stage (cell infestation) from the Varroa -infested colonies, we followed the protocol described in (Pusceddu et al. 2021a ). All groups were replicated in three independent metal cages (30 bees per cage), being set up on the same day and kept in an incubator under the same conditions described above (Williams et al. 2013 ). On the fifth day, each bee had its venom gland removed. Two sample types were obtained for each bee, as follows: 1) intact venom sac (source) and 2) body of the individual without abdomen. All samples were stored at − 20°C until chemical analyses. Behavioural observations To investigate whether grooming (selfgrooming and allogrooming) and the likely consequent application of venom to the body surface is influenced by Varroa infestation, we determined the frequency (e.g., number of events) of both grooming types by using the "all occurrences sampling" method (Altmann 1974 ). In addition, to evaluate the effectiveness of grooming in counteracting Varroa parasitism, the grooming behaviour was divided into three ethogram entries, as follows: 1) cleaning body with legs or mandibles + shaking the abdomen , 2) removing mite using bee’s legs or mandibles , and 3) damaging the mite using the mandibles . The frequency of each event entry was recorded. Thirty emerging bees from three Varroa -free colonies were mixed to prevent any family effects (10 bees from each colony) and placed in a metal cage as described above. To obtain workers parasitized during the adult stage (phoretic infestation) and uninfested workers from the Varroa -free colonies, we used the method described in (Pusceddu et al. 2018 ). The behavioural observations were repeated for three consecutive days during which phoretic infested and uninfested bee groups were compared. Selfgrooming and allogrooming occurrences were counted every day, at three different time slots (9:00–9:30; 13:30 − 14:00; 18:00–18:30), with 30-min observation sessions, corresponding to a total of 1 h and 30 min per day per cage. The cages were kept in an incubator under the same conditions as described above (Williams et al. 2013 ). All treatments were replicated using three independent cages (30 bees per cage) and were set up at the same time. Toxicology bioassay To evaluate the effect of bee venom on Varroa activity, five mites were placed in a 90-mm diameter Petri dish containing an absorbent paper 67 g/mq (APTACA SRL, Canelli, Italy) (Pusceddu et al. 2018 ). Each mite was wetted with 1 µl of a water solution containing 0.2 µl of bee venom extract (CITEQ, Groningen, The Netherlands) or 1 µl of water solution (control group). Mites were obtained from brood cells capped in the preceding 15 h, following the procedure described by (Nazzi et al. 2012 ). A total of 90 mites from six Varroa -infested colonies were tested, corresponding to 45 mites per treatment (control vs. venom). Mite activity was observed under a stereo microscope every 15 min for the first hour, every 20 min for the second hour, and every 30 min for the next six hours. Mites were considered inactive when they showed no response to contact stimulus using a stick (Milani 1995 ). This bioassay was replicated twice (180 mites in total). Both experiments were conducted under artificial light and at temperature of 32 ± 1°C. Statistical analysis We used a linear mixed model (LMM), followed by a Tukey post-hoc test, to investigate differences in melittin concentration (mg/L) on the bodies of worker bees (freshly emerged, nurse and forager) and drones. Colony was included as a random factor. Due to the absence of melittin on the bodies of freshly emerged worker bees and drones, statistical comparisons are reported only between nurse and forager worker bees. We used an LMM, followed by a Tukey post-hoc test, to examine the effect of treatment (control vs. bee blocked sting vs. mix) on melittin concentration (mg/L) on the bees’ bodies. Cage was included as a random factor. Due to the absence of melittin on the bodies of stinger blocked bees, statistical comparisons are reported only between control bees in single and mixed cages. We used LMMs, followed by a Tukey post-hoc test, to examine the effect of treatment (cell infestation, phoretic infestation, and Varroa -free) on melittin concentration (mg/L) on the bodies and venom gland of bees. In addition, we used an LMM, followed by a Tukey post-hoc test, to examine the effect of treatment (cell infestation, phoretic infestation and Varroa- free) on the proportion of melittin on the bodies of bees compared to the total amount of melittin (body + venom sac). Cage was included as a random factor in all models. To examine whether selfgrooming or allogrooming behaviour differed between groups of infested vs. uninfested bees, general linear mixed models (GLMMs) with negative binomial error structure were used. Experimental group (infested vs. uninfested bees) was used as a fixed factor and time slot, day of experiment and cage were used as random factors. To examine the effect of venom treatment (control treatment vs. bee venom treatment) on Varroa mite activity, a GLMM with binomial error structure was used. Treatment (control treatment vs. bee venom treatment), time of observation and their interaction were used as fixed factors and Petri dish was used as a random factor. All analyses were performed in R v.3.5.2 (R Core Team 2018 ). Mixed effects models were conducted using the R package lme4 (Bates et al. 2015 ). Model assumptions were checked visually and found to meet expectations, including homogeneity of variance, normality of residuals, and linearity. Due to lack of homogeneity of variance and normality, melittin concentration (mg/L) was square root-transformed in all models. Results LC-MS/MS method validation The correlation coefficient of the calibration lines (r 2 ) showed values oscillating between 0.992 and 0.999. The linearity was, therefore, above the condition set for the validation of the method. The accuracy data provided by the recovery experiments obtained from 6 replicates for the two concentrations tested ranged from 87.1 to 93.7% at the LOQ level and from 81.7 to 90.3% at the 10×LOQ (S3 Table). The values obtained showed a good extraction capacity of the proposed method, which was, therefore, assessed as suitable for the melittin analysis in the bee samples. The repeatability (RSDr) and reproducibility (RSDwR) showed values lower than 20%, thus showing good repeatability of the analytical method. The chromatograms of the control and standards did not show the presence of interfering peaks, thus indicating a good selectivity of the method (S1 Figure). The results obtained from the validation tests are consistent with the validation parameters of the SANTE/12682 /2019 guidelines. Detection of melittin on the body of adult worker bees and drones We found a detectable amount of melittin only in nurse and forager bees (Fig. 1 ). No melittin was observed in drones and freshly emerged bees (Fig. 1 ). Interestingly, the amount of melittin detected on nurses' bodies was significantly lower than that on foragers' bodies (LMM; Tukey post-hoc test; Z = 3.165, P = 0.001; Fig. 1 ). Origin of the melittin detected on the worker's body The experiment conducted to determine the amount of venom on the bodies of bees with blocked or unblocked stingers, whether housed individually or in groups, clearly demostrated that the melittin present on workers' bodies originates from the venom gland (Fig. 2 ). Specifically, no melittin was detected on the bodies of bees with blocked stingers, regardless of whether they were kept alone or alongside bees with unblocked stingers (Fig. 2 ). Detection of venom on the bodies of infested and uninfested workers and in the venom sac The amount of melittin detected on the cuticle of worker bees parasitized during the pupal stage (cell infestation) was significantly lower than in those parasitized as adults (phoretic infestation) (LMM; Tukey post hoc test; Z = 3.415, P = 0.001, Fig. 3 a) and in uninfested control bees (LMM; Tukey post hoc test; Z = 2.275, P = 0.034, Fig. 3 a). In contrast, there was no significant difference in the amount of melittin present on the bodies of adult-parasitized bees compared to the control bees (LMM; Tukey post hoc test; Z = 1.291, P = 0.196, Fig. 3 a). Furthermore, the amount of melittin detected in the venom sac was significantly lower in adult worker bees parasitized during the pupal stage than in control bees (LMM; Tukey post hoc test; Z = 2.442, P = 0.043, Fig. 3 b). Similarly, in adult workers parasitized as adults, the amount of melittin in the venom sac was lower than in control bees (approximately half on average), although this difference was not statistically significant (LMM; Tukey post hoc test; Z = 1.434, P = 0.227, Fig. 3 b). However, calculating the proportion of melittin found on the bodies of worker bees relative to total melittin amount (e.g., the sum of melittin present on the body and in the venom sac), showed that this proportion significantly increased in parasitized bees (both as adults and during the pupal stage) compared to unparasitized ones (LMM; Tukey post hoc test; Z = 3.308, P = 0.002; Z = 2.591, P = 0.014, respectively, Fig. 3 c). Behavioural observations We observed a total of 4841 selfgrooming events in which bees were cleaning their body using the legs + shaking the abdomen. Out of these, 3147 were observed in the phoretic infested group and 1694 in the uninfested group (65% vs. 35%). This difference was statistically significant (GLMM; χ 2 = 27.973, P < 0.001; Fig. 4 a). However, removing mites using bee’s legs was observed only 12 times out of the 3147 selfgrooming events observed in the Varroa -infested group (0.38%). Damaging the mite using bee’s mandibles did not occur in our study. We observed a total of 74 allogrooming events by cleaning nestmate body with mandibles. Out of these, 41 were observed in the phoretic infested group and 33 in the uninfested group (55% vs. 45%). This difference was not statistically significant (GLMM; χ 2 = 0.090, P = 0.763; Fig. 4 b). Removing mites from other bees using mandibles and damaging them did not occur in our study. Toxicology bioassay Exposure of the mites to honey bee venom resulted in a highly significant increase in the percentage of inactive mites compared to the control (approximately 7% vs 0.9%, respectively; GLMM; χ 2 = 12.191, P < 0.001; Fig. 5 a) Proportion of inactive mites also increased with time of observation (GLMM; χ 2 = 19.382, P < 0.001; Fig. 5 b). The interaction between treatment and time was not statistically significant (GLMM; χ 2 = 1.620, P = 0.203). Discussion The aims of this study were to investigate the role of grooming (selfgrooming and allogrooming) in the distribution of venom compounds on the honey bees’ body as an external immune defence trait under the challenge of the parasitic mite V. destructor , as well as to evaluate the effects of venom on the parasite’s activity. We first confirmed the results of earlier studies (Baracchi and Turillazzi 2010 ; Baracchi et al. 2011 ) by showing that melittin, a venom compound, is absent on the body surface of drones and freshly emerged bees but it is present on nurse and forager workers. We then found that melittin was absent on the body of workers that had access to their stingers blocked, thus indicating that venom is transferred from the stinger to the body surface, likely during selfgrooming, and is not picked up from other sources in the hive environment. We then found that venom transfer to the body surface was influenced by the presence of Varroa , with parasite presence leading, on one hand, to a proportional higher amount of melittin on the body surface relative to the total melittin (the sum of melittin present on the body and in the venom sac), and on the other hand, to a lower overall amount of melittin production. In addition, we also found that Varroa infested bees groomed themselves more than control bees. This supports the hypothesis that selfgrooming is responsible for the venom transfer to the body surface of honey bees and agrees with the proportionally higher amount of melittin on the body surface of Varroa infested bees. Lastly, we found that venom negatively influenced parasitic mite activity, thus indicating that venom deposition on the body surface of bees may serve as an external immune defence trait against parasites and/or pathogens. Our results exclude the possibility of environmental contamination or acquisition and indicate the presence of venom on the bee body surface through selfgrooming behaviour. As expected, bees with their stingers blocked, and therefore unable to release venom, did not show melittin on their bodies. This result remained consistent also when bees were reared in the same cage as control bees capable of releasing venom. These findings align with those reported by Baracchi and Turillazzi ( 2010 ) and are further supported by our results that showed the absence of venom on the bodies of drones sampled from the hive and the presence of venom on the bodies of nurse and forager bees. Notably, drones are also groomed by workers, although this behaviour occurs less frequently than among workers (Stout et al. 2011 ; Goins and Schneider 2011). If allogrooming behaviour had been responsible for venom spread, drones or bees with blocked stings would have had melittin on their bodies. In fact, our results clearly show not only that venom on the body surface of worker bees is not just an artefact, but also that it is likely used only as an external immune defence for personal hygiene rather than for sanitation of other adult nest members. This is because it is not spread via allogrooming to drones or bees with blocked stingers. In contrast, in ants, venom is used not only for personal sanitation via selfgrooming (e.g., (Tranter and Hughes 2015 )) but also for the sanitation of developing brood [21] and adult nest members via allogrooming (Beydizada et al. 2024 ). Interestingly, our research provides clear evidence that parasitisation leads to a lower amount of melittin in the venom. In fact, our study revealed that when bees were parasitized by Varroa during the pupal stage, the external immune system of forager bees was weakened, as evidenced by the lower levels of melittin found on their cuticles and venom sacs compared to healthy bees. This negative effect was more pronounced when bees were parasitized during the pupal stage rather than as adults. To our knowledge, this result had never been reported before and we believe that it can be explained by the high energy cost of venom production in aculeate Hymenoptera (Baracchi and Tragust 2017 ; Baumann 2018 ) and the physiological impairments caused by Varroa on honey bees (Rosenkranz et al. 2010 ; Nazzi and Le Conte 2016 ). Thus, assuming that bees cannot selectively choose which venom components to apply on their bodies and considering that parasitized bees use a greater proportion of the total melittin content from the venom sac compared to unparasitized bees, it is likely that parasitized worker bees spread larger amounts of venom on their bodies. However, this does not necessarily imply that the distribution of venom across the body represents a curative defence mechanism adopted by bees against V. destructor . In fact, the following question remains open: do bees use more venom (1) in response to Varroa attacks or (2) simply maintain a minimum level of antimicrobial substances on their bodies for the control of opportunistic or pathogenic microbes? A finding supporting the second hypothesis was the significantly higher amounts of melittin found on the bodies of foragers compared to nurses in our trial. It is well known that foragers face higher pathogen exposure (Pusceddu et al. 2021b ) and thus require additional protection compared to nurse bees. Therefore, it is debatable that bees seem capable of regulating the dosage of antimicrobial substances on their bodies according to their perceived level of protection needed. However, Baracchi and Turillazzi ( 2010 ) found that nurses' venom contains higher percentages of apamine and lower percentages of melittin compared to the venom of older nestmates (guards and foragers). Therefore, the difference we observed between the two cohorts of bees (nurses vs. foragers) likely resulted from variations in the composition of the venom distributed on the body rather than the quantity applied by the different bee cohorts. As expected, a significant increase in selfgrooming was observed in the group challenged with the ectoparasite Varroa . The behaviour of cleaning the body with legs and shaking the abdomen is likely involved in spreading of antimicrobial substances across the bee's body. Like in previous studies conducted on ants (Tragust 2016 ; Koch et al. 2025 ), our results suggest that selfgrooming in A. mellifera involves venom spread. However, our findings confirm the poor efficacy of selfgrooming in combating Varroa in A. mellifera . Indeed, the observed percentage of removal of the parasite during selfgrooming events was very low (only 0.38% of the cases), which was very similar to that observed by Peng et al. (Peng et al. 1987 ). Moreover, in accordance with Büchler et al. ( 1992 ), we never observed a bee damaging the mite with its mandibles. In accordance with our findings on absence of melittin on the body of drones and blocked stinger bees, an increase in allogrooming was not observed in these bees. This is consistent with the results of previous studies that have demonstrated that this behaviour is rare and occurs mainly during the nursing period (Cini et al. 2020 ; Pusceddu et al. 2021b ). In addition, we never observed the removal of parasite or bee damaging the mite with its mandibles during allogrooming behaviour. Our study also showed that bee venom has a detrimental effect on Varroa mites. However, the observed narcoleptic effect of the venom was relatively mild (approximately 7% of inactive mites) in comparison to other hive products such as raw propolis, which has an effect of 19–22% (Pusceddu et al. 2018 ), or ethanol propolis extracts, which have an effect of 45–90% (Garedew et al. 2002 ; Pusceddu et al. 2021a ). It could be assumed that venom on the body surface might provide protection against other honeybee parasites and/or pathogens (Fernández et al. 2014 ; Sani et al. 2022 ). For example, it is known that some viruses, namely Chronic bee paralysis virus and Israeli acute paralysis virus can be transmitted by topical application (Yañez et al. 2020 ) and contact with a virus-contaminated environment or infected bees (Bailey et al. 1983 ; Coulon et al. 2018 ; Amiri et al. 2014 , 2019 ). Therefore, given the strong antiviral effect of melittin (Kontogiannis et al. 2022 ), venom on the bee body surface might also help against virus transmission and infection. In support of this hypothesis, previous work has shown that bee venom, as a dietary supplement, increased the expression levels of immune genes encoding the antimicrobial peptides Abaecin, Defensin 2 and Hymenoptaecin as well as stimulated the production of juvenile hormone and vitellogenin secretion, and decreased Varroa infestation level within the colony (Seyam et al. 2022 ). Recently, Mahmoud et al. ( 2024 ) also showed an immunostimulatory as well as a beneficial role of venom in honeybees challenged by Vairimorpha ( Nosema ) ceranae. In addition, Hejníková et al. ( 2024 ) found that melittin is also synthesized in the fat body, which is apparently tolerated by bee cells and most likely protects the bee from infection. A hypothesis that would be interesting to test in future studies is if the narcoleptic effect induced by the venom on mite could facilitate its detachment from the bee's body during selfgrooming. Given the differences in venom composition and venom deposition among Apis species (35), it would be interesting to study venom bathing behaviour in relation to parasite efficacy control, both in A. mellifera and A. cerana , with the latter being the original host of Varroa destructor . This deserves further attention as it might explain some of the variations in mite resistant traits between species, e.g., mite infertility (Grindrod and Martin 2023 ). Another aspect that is important to consider is that the hive is a complex system where antimicrobial venom (Baracchi and Turillazzi 2010 ) coexists with other bioactive matrices such as propolis (Pusceddu et al. 2018 ), honey (Floris et al. 2021 ), wax (Felicioli et al. 2019 ), pollen (Didaras et al. 2020 ), and royal jelly (Alreshoodi and Sultanbawa 2015 ). The combination of these matrices with their bioactive properties could result in a synergistic effect against pests and pathogens (Erler and Moritz 2016 ; Erler et al. 2024 ). A synergistic effect of different antimicrobial substances was found in the study of Brütsch et al. ( 2017 ), who observed that wood ants can produce a potent antimicrobial cocktail by combining formic acid with tree resins. In conclusion, our results strongly suggest that, like ants, honeybees rely on a combination of selfgrooming behaviour and venom application for external immunity, which likely counteract opportunistic and pathogenic microorganisms. However, this defence system can be compromised by Varroa destructor , whose parasitizing activity on pupae leads to reduced melittin production in adult bees. This discovery reveals, to our knowledge, another previously unknown negative side effect caused by this mite. Declarations Ethics approval No ethical approval is required for this study. Funding This research was funded by: Fondazione di Sardegna, year 2016 and by Regione Autonoma della Sardegna (Italy), L.R. 7/2007, year 2016, project: ‘Self-medication in the hive: propolis and venom against the honeybee ectoparasite Varroa destructor ’. Author Contribution AS, IF, MP and ST conceived and designed research. ICB, JSN and MP conducted experiments. AA, AA and FC made the chemical analysis. PT analysed data. MP and AS wrote the manuscript. All authors read, reviewed and approved the manuscript. Acknowledgments The authors thank Dr. Ana Helena Dias Francesconi from the University of Sassari for revising the manuscript. Data Availability Data is provided within the supplementary information files References Ali, M. A. A. S. M. Studies on bee venom and its medical uses. Int. J. Adv. Res. Technol. 1 , 69–83 (2012). Alreshoodi, M. F. & Sultanbawa, Y. Antimicrobial activity of royal jelly. Anti-Infective Agents . 13 , 50–59. 10.2174/2211352513666150318234430 (2015). 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Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jorge","middleName":"Sanchez","lastName":"Navarro","suffix":""},{"id":446153498,"identity":"1b0a2933-a3d2-4bfc-919b-5ac6819cc2c6","order_by":5,"name":"Francesco Corrias","email":"","orcid":"","institution":"University of Cagliari, Department of Life and Environmental Sciences","correspondingAuthor":false,"prefix":"","firstName":"Francesco","middleName":"","lastName":"Corrias","suffix":""},{"id":446153499,"identity":"bc11a57d-4f69-4689-9e37-87f3f243ec09","order_by":6,"name":"Alessandro Atzei","email":"","orcid":"","institution":"University of Cagliari, Department of Life and Environmental Sciences","correspondingAuthor":false,"prefix":"","firstName":"Alessandro","middleName":"","lastName":"Atzei","suffix":""},{"id":446153500,"identity":"b415a95c-df1f-4601-8e94-b1bcc6ad0a26","order_by":7,"name":"Alberto Angioni","email":"","orcid":"","institution":"University of Cagliari, Department of Life and Environmental Sciences","correspondingAuthor":false,"prefix":"","firstName":"Alberto","middleName":"","lastName":"Angioni","suffix":""},{"id":446153501,"identity":"6cd51802-7d7e-4d41-9b6f-8b14c7f59be0","order_by":8,"name":"Ignazio Floris","email":"","orcid":"","institution":"University of Sassari, Department of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ignazio","middleName":"","lastName":"Floris","suffix":""},{"id":446153502,"identity":"e104a4bc-23b5-4cde-b382-91b26a460eb3","order_by":9,"name":"Alberto Satta","email":"","orcid":"","institution":"University of Sassari, Department of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Alberto","middleName":"","lastName":"Satta","suffix":""}],"badges":[],"createdAt":"2025-03-16 17:08:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6238801/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6238801/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-13440-2","type":"published","date":"2025-08-20T16:29:43+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81412987,"identity":"1ebb4892-d0c1-45fb-ba45-75c871169fb1","added_by":"auto","created_at":"2025-04-25 21:41:32","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":253355,"visible":true,"origin":"","legend":"\u003cp\u003eMelittin\u003cstrong\u003e \u003c/strong\u003eon the body (mg/L): drones \u003cem\u003evs\u003c/em\u003e. freshly emerged bees \u003cem\u003evs\u003c/em\u003e. nurses \u003cem\u003evs\u003c/em\u003e. foragers. Means ± SE are shown. Different letters indicate significant differences between groups (LMM; Tukey post-hoc test; P ≤ 0.05)\u003c/p\u003e","description":"","filename":"fig1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6238801/v1/76dc3d6708c943b707643615.jpeg"},{"id":81413411,"identity":"226dc2e7-d4a4-4555-a3c2-accd37526d55","added_by":"auto","created_at":"2025-04-25 21:49:32","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":387676,"visible":true,"origin":"","legend":"\u003cp\u003eMelittin\u003cstrong\u003e \u003c/strong\u003eon the body (mg/L): control (unblocked stinger) \u003cem\u003evs\u003c/em\u003e. stinger blocked \u003cem\u003evs.\u003c/em\u003e mixed cages. Means ± SE are shown. Different letters indicate significant differences between groups (LMM; Tukey post-hoc test; P ≤ 0.05)\u003c/p\u003e","description":"","filename":"fig2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6238801/v1/0837809131373424fee5de68.jpeg"},{"id":81412992,"identity":"34cdc607-a93b-4735-a901-54ec289fe2f1","added_by":"auto","created_at":"2025-04-25 21:41:32","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1016356,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Melittin\u003cstrong\u003e \u003c/strong\u003eon the body (mg/L): workers infested inside the bee cells \u003cem\u003evs\u003c/em\u003e. phoretic infested workers \u003cem\u003evs\u003c/em\u003e. control. (b)\u003cstrong\u003e \u003c/strong\u003eMelittin\u003cstrong\u003e \u003c/strong\u003ein the venom sac (mg/L): workers infested inside the bee cells \u003cem\u003evs\u003c/em\u003e. phoretic infested workers \u003cem\u003evs\u003c/em\u003e. control. (c) Proportional melittin on the body: workers infested inside the bee cells \u003cem\u003evs\u003c/em\u003e. phoretic infested workers \u003cem\u003evs\u003c/em\u003e. control. Means ± SE are shown. Different letters indicate significant differences between groups (LMM; Tukey post-hoc test; P ≤ 0.05)\u003c/p\u003e","description":"","filename":"fig3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6238801/v1/26aa65018c3172dffde7866b.jpeg"},{"id":81413415,"identity":"cf84024d-778d-4896-b349-45509e3ef622","added_by":"auto","created_at":"2025-04-25 21:49:32","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":440265,"visible":true,"origin":"","legend":"\u003cp\u003e(a)\u003cstrong\u003e \u003c/strong\u003eSelfgrooming behaviour in \u003cem\u003eVarroa-\u003c/em\u003efree \u003cem\u003evs.\u003c/em\u003e \u003cem\u003eVarroa\u003c/em\u003e-infested bees (phoretic infestation). Means and ± SE are shown. *** P \u0026lt; 0.001. (b) Allogrooming behaviour in \u003cem\u003eVarroa\u003c/em\u003e-free \u003cem\u003evs\u003c/em\u003e. \u003cem\u003eVarroa\u003c/em\u003e-infested bees (phoretic infestation). Means and ± SE are shown. ns P \u0026gt; 0.05\u003c/p\u003e","description":"","filename":"fig4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6238801/v1/2505837fae22aaa32f17ec8e.jpeg"},{"id":81412998,"identity":"fd7abbdb-0335-4c25-b393-0a09e8096ce0","added_by":"auto","created_at":"2025-04-25 21:41:32","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":566762,"visible":true,"origin":"","legend":"\u003cp\u003e(a)\u003cstrong\u003e \u003c/strong\u003eProportion of non-mobile (inactive) \u003cem\u003eVarroa\u003c/em\u003emites in the control (water solution) \u003cem\u003evs\u003c/em\u003e. the bee venom treatment. (b) Proportion of non-mobile (inactive) mites overtime of observation. Means and ± SE are shown.Plotted line show predicted relationship and the shaded area indicate the 95% confidence intervals. *** P\u0026lt;0.001\u003c/p\u003e","description":"","filename":"fig5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6238801/v1/8a9a9f7442fc5ec08513a222.jpeg"},{"id":89847856,"identity":"5b1ad784-a9fe-4498-b357-cdf5afcd01fc","added_by":"auto","created_at":"2025-08-25 16:44:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3737687,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6238801/v1/c857993a-f3d5-4342-9228-f2de759bb043.pdf"},{"id":81413412,"identity":"87b9c134-278e-4095-93ae-3273f0ea5b7e","added_by":"auto","created_at":"2025-04-25 21:49:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":366833,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6238801/v1/e6c621fc3471e77047f823e6.docx"},{"id":81412989,"identity":"b7444f5a-4062-49a3-b29d-2b54c491d808","added_by":"auto","created_at":"2025-04-25 21:41:32","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":36674,"visible":true,"origin":"","legend":"","description":"","filename":"DATAFILEtableS4S5S6S7.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6238801/v1/a498350b646f617fb9966e60.xlsx"},{"id":81412995,"identity":"4685c0da-ac32-45cf-adbd-f71698151e5c","added_by":"auto","created_at":"2025-04-25 21:41:32","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":323537,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6238801/v1/72d9bffa481b8275efcd2ffd.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Varroa destructor weakens honey bee external immunity by impairing melittin production","fulltext":[{"header":"Bullet point","content":"\u003col\u003e\n \u003cli\u003eHoney bees employ \u0026quot;venom bathing\u0026quot; via selfgrooming to distribute antimicrobial melittin on body surfaces.\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eVarroa\u003c/em\u003e-infested bees spread proportionally more venom on their bodies whilst having reduced melittin production.\u003c/li\u003e\n \u003cli\u003eBee venom significantly reduces mite activity, suggesting its role as an external immune defence.\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eVarroa destructor\u003c/em\u003e impairs honey bees\u0026apos; external immune defence through reduced melittin production.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Introduction","content":"\u003cp\u003eCollaboration among individuals in social insects leads to important benefits in brood care, foraging, antipredator defence, and other colony survival activities (Krause et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). However, the close and frequent interactions among genetically homogeneous nestmates, the large amounts of food stored in the nest and the relatively stable conditions in the nest-environment may also promote the spread of parasites and pathogens within the colony (Schmid-Hempel \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). To counteract the risk of disease contraction and transmission within their societies, apart from their body innate immune system, social insects possess a diversity of physiological, behavioural and organizational adaptations, collectively often called social immunity (Cremer et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wilson-Rich et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Evans and Spivak \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). As many of these adaptations act in the environment of a social insect society, they can also be conceptualized as external immune defence, i.e. traits acting outside the individual and either improving the protection from pathogens and parasites or manipulating the composition of the microbial community in favour of the host (Otti et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). For example, ants and honey bees incorporate tree resin with antimicrobial properties in their nests, thus modifying the microbial community of the nest environment (Chapuisat et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Simone-Finstrom et al. 2010). In the case of ants, resin collection seems to be a mechanism implemented to prevent disease (Castella et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), whereas, in the case of honey bees, this behaviour can also have curative purposes, as suggested for the fungus \u003cem\u003eAscosphaera apis\u003c/em\u003e (Simone-Finstrom and Spivak \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and the mite \u003cem\u003eVarroa destructor\u003c/em\u003e (Pusceddu et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). In addition to environment-derived bioactive substances (e.g., resins), self-produced substances, especially from exocrine glands such as the venom gland, can play a key role in external immunity (Tragust \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Baracchi and Tragust \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In many species of aculeate Hymenoptera, the sting apparatus and venom originally evolved as tools to kill prey but became a means of defence against predators, mainly vertebrates (Schmidt \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). However, the venom of aculeate Hymenoptera is also a source of antimicrobial substances (Kuhn-Nentwig \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Moreau \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), suggesting its potential use as an immune defence trait. The adaptive role of venom to sanitize oneself as well as the nest, other nest members and food is well known in ants (reviewed in (Tragust \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Koch et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e)), which can apply and spread their antimicrobial venom during grooming behaviour (e.g., (Tragust et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2013a\u003c/span\u003e)). Whether honey bees, or bees in general, also use their venom for sanitary purposes as ants do is less clear.\u003c/p\u003e \u003cp\u003eLike ant venoms, bee venoms, especially the honey bee venom, are pharmacologically active products, which consist of a complex mix of biogenic amines, proteins, peptides, phospholipids, sugars, and volatile components (Dotimas and Hider \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Carpena et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Guido-Pati\u0026ntilde;o and Plisson \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Koludarov et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among species in the genus \u003cem\u003eApis\u003c/em\u003e, venom production can be influenced by seasonality (Ferreira et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), age and caste (Ali \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and species body size (Schmidt \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). However, also in species with similar body size, differences in venom amount and composition occur. For instance, although the Asian honey bee \u003cem\u003eA. cerana\u003c/em\u003e produces only half as much venom as \u003cem\u003eA. mellifera\u003c/em\u003e, its venom contains a higher proportion of the peptide melittin (Schmidt \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Melittin is the main component of honey bee venom, accounting for 50% of the venom dry weight (Owen and Pfaff \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; De Lima and Brocchetto-Braga 2003). Melittin has antiseptic (Kuhn-Nentwig \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), strong antiviral (Kontogiannis et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), antifungal and antimicrobial properties (Habermann \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1972\u003c/span\u003e). Apart from melittin, antimicrobial activity has also been suggested for other bee venom peptides such as apamin and MCD (Froy and Gurevitz \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1998\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn honey bees, the possible use of venom as an external immune defence trait in a social immunity context was, to our knowledge, first hypothesized by Baracchi and Turillazzi (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), who found traces of venom compounds on the body of adult honey bees and on the surface of honeycombs. Later, the same authors found that although melittin was present in the venom of both open-nesting and cavity-nesting bees, it was detectable on the cuticle and combs only of the latter (Baracchi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), thus suggesting that nesting ecology and environment shape the deposition of venom, potentially due to different associated disease pressures. Finally, with respect to the overall amount of venom peptides both in the venom and on the cuticle, the same authors (Baracchi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) suggested that \u003cem\u003eA. cerana\u003c/em\u003e performs a higher level of \u003cem\u003e\u0026ldquo;venom bathing\u0026rdquo;\u003c/em\u003e compared to \u003cem\u003eA. mellifera\u003c/em\u003e. This latter result is in accordance with a higher rate of grooming activity shown by \u003cem\u003eA. cerana\u003c/em\u003e compared to \u003cem\u003eA. mellifera\u003c/em\u003e, with grooming being one of the main mechanisms of resistance against parasitic mites in the Asian honey bee (Pritchard \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A relationship between grooming and venom is also suggested by the complete absence of venom peptides on the cuticle of freshly emerged bees, which probably cannot produce the venom yet, and adult drones in both bee species (Baracchi and Turillazzi \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Baracchi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConsidering that previous research suggested that the role of venom in honey bees is well beyond the classical defence activity against predators (Baracchi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), in our study we investigate the role of grooming in the distribution of venom compounds on the honey bee's body, as an external immune defence trait, upon challenge of \u003cem\u003eA. mellifera\u003c/em\u003e by the parasitic mite \u003cem\u003eVarroa destructor\u003c/em\u003e as well as the venom effects on the parasite activity. It is important to highlight that \u003cem\u003eV. destructor\u003c/em\u003e was originally confined to the Asian honey bee, \u003cem\u003eA. cerana\u003c/em\u003e (Rosenkranz et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). After the shift to the new host \u003cem\u003eA. mellifera\u003c/em\u003e, this parasite spread worldwide and is currently the most serious pest of honey bees (Rosenkranz et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Nazzi and Le Conte \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo conduct our study, we first performed chemical analyses to confirm the presence of venom on the bodies of worker bees (both nurses and foragers), using melittin as a marker. To exclude the possibility that the detected melittin originated from environmental contamination within the nest, we also quantified the amount of melittin on the bodies of drones and freshly emerged workers. Additionally, to ascertain that the melittin detected on the worker bees' bodies originated from venom produced by the bees themselves and smeared on the cuticle through grooming, we quantified the amount of venom on bees with either blocked or unblocked stingers. These bees were kept together in groups or separately. We then investigated if venom transfer to the body surface of honey bees is influenced by parasite presence. To do this, we quantified the amount of venom (using melittin as a marker) on the bodies of infested (both at the pupal and adult stage) and uninfested worker bees for comparison. To ensure that bees in the three experimental groups had an equal supply of venom and could thus equally utilize this defence mechanism, we also quantified the amount ofmelittin inside their venom sacs.\u003c/p\u003e \u003cp\u003eIn addition, since previous studies have suggested that grooming in \u003cem\u003eA. mellifera\u003c/em\u003e is not effective in counteracting the mite (B\u0026uuml;chler et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Fries et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Pettis and Pankiw \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Boecking and Spivak \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Pritchard \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Invernizzi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), but it may play an additional role in relation to venom-spread, we determined the frequency and efficiency of selfgrooming and allogrooming through behavioural observations comparing infested and uninfested group of honey bee workers.\u003c/p\u003e \u003cp\u003eFinally, through toxicological laboratory assays, we evaluated whether a biological amount of bee venom could induce lethal or narcoleptic effects on the ectoparasite \u003cem\u003eV. destructor\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemical analysis\u003c/h2\u003e \u003cp\u003e \u003cb\u003eStandards and reagents.\u003c/b\u003e Melittin was purchased as certified analytical standard (purity\u0026thinsp;\u0026ge;\u0026thinsp;85%) by Sigma Aldrich (Milan, Italy), whereas acetonitrile (ACN), formic acid and HNO\u003csub\u003e3\u003c/sub\u003e (67\u0026ndash;69%) were LC/MS grade (Sigma Aldrich, Milan, Italy), reagent grade (\u0026gt;\u0026thinsp;95%, Honeywell, Sigma Aldrich, Milan, Aldrich) and ultra-pure grade (Romil Spa, Cambridge, England) solvent, respectively. MilliQ water (18.25 MΩ\u0026thinsp;\u0026times;\u0026thinsp;cm) was obtained from an integrated Millipore purification system (MilliQ, Merck, Milan, Italy). A melittin stock solution (1000 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was prepared by solubilization in a 0.1% aqueous formic acid solution and stored at 4\u0026deg;C until use. The working solution (200 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was freshly prepared each day by diluting the stock solution with a 0.1% aqueous formic acid solution. The five-point calibration curve was obtained by diluting the working solution with a 0.1% formic acid aqueous solution at concentrations of 0.1, 0.25, 0.50, 1, and 1.5 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExtraction of melittin from bees and the venom sac\u003c/b\u003e. Individual bees and venom sacs were placed into 1.5 mL Eppendorf tubes to extract melittin. An amount of 1 mL of 0.1% aqueous formic acid solution was added to each tube. Then, the tubes were vortexed for 1 min and for other 15 min on a rotary shaker (Reax Top, Heidolph, Germany). Finally, the solution was transferred to vials and analysed by LC-MS/MS.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLC-MS/MS analysis.\u003c/em\u003e Analytical determinations of the solution from extracted bees were performed using an Agilent 1290 Infinity II UHPLC coupled to an Agilent 6470 Triple Quad LC-MS/MS mass detector paired with a MassHunter ChemStation. The column was a ZORBAX Eclipse Plus C18 (2.1 \u0026times; 150 mm, 1.8 \u0026micro;m). A binary gradient, H\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;0.1% formic acid (A) and ACN\u0026thinsp;+\u0026thinsp;0.1% formic acid (B) was set as follows: T\u0026thinsp;=\u0026thinsp;0 at 95% A, T\u0026thinsp;=\u0026thinsp;4.30 min at 15% A, T\u0026thinsp;=\u0026thinsp;5.80 min at 15% A, T\u0026thinsp;=\u0026thinsp;7.70 min at 95% A, and 2 min post-run at 95%. The flow rate was 0.2 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with 5 \u0026micro;l sample volume injected in positive mode. The mass detector gas and the sheet gas were set at 300\u0026deg;C and 250\u0026deg;C, with flow rates 5 L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 11 L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. The nebulizer was at 30 psi and the capillary voltage was 4000 V. Melittin has a molecular weight (MW) of 2646.46 Da. Ionization in the positive ESI mode produces a stable MH\u0026thinsp;+\u0026thinsp;4 ion with a MW of 712.44 (S1 Table). Analyses were carried out in dynamic MRM mode (S2 Table).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalytical method validation.\u003c/b\u003e To validate our analytical method, we followed the SANTE guidelines by evaluating linearity, selectivity, precision, limits of quantification of the method (LOQ), accuracy in terms of recovery, uncertainty, and matrix effect (SANTE/11312/2021). Six control bees were fortified by depositing an aliquot of solution of the analytical standard at 1 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, left to rest for 24 h, and extracted as reported above. Each sample belonged to an independent experiment. Six solutions of the analytical standard at 0.1 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (LOQ) and 1 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (10 \u0026times; LOQ) were analysed within one day to verify their repeatability (RSDr, intraday). Reproducibility (RSDwR) was calculated by analysing two analytical standard solutions over six days. Recovery results were analysed using matrix control standard calibration curves. The matrix effect was evaluated by comparing the analytical response of melittin in H\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;0.1% formic acid with solutions prepared with extracts from unfortified control (blank) bees. Linearity was assessed by analysing standard calibration curves performed on five different days and was considered acceptable when the coefficient of determination was greater than 0.990. Selectivity was assessed by comparing the extracts of unfortified control bees with those spiked with the standard. The absence of chromatographic peaks at melittin retention times was a criterion for the selectivity of the confirmatory method. The expanded measurement uncertainty (U), a quantitative parameter of the reliability of the analytical method, was calculated by multiplying the combined uncertainty (u\u0026prime;) by a coverage factor k\u0026thinsp;=\u0026thinsp;2, to obtain a 95% confidence level, using the following equation:\u003c/p\u003e \u003cp\u003eu\u0026prime;= \u0026radic;\u0026#119906;\u0026prime;(\u0026#119887;\u0026#119894;\u0026#119886;\u0026#119904;)2+\u0026#119906;\u0026prime;(\u0026#119901;\u0026#119903;\u0026#119890;\u0026#119888;\u0026#119894;\u0026#119904;\u0026#119894;\u0026#119900;\u0026#119899;)2; U = k \u0026times; u\u0026prime;\u003c/p\u003e \u003cp\u003eThe instrumental LOQ was calculated as the lowest fortification level that meets the method identification and performance criteria regarding recovery and precision.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental apiary\u003c/h3\u003e\n\u003cp\u003eThe study was performed from April 2023 to November 2023 in an experimental apiary of the Department of Agricultural Sciences of the University of Sassari located in Ottava, Sardinia (Italy; latitude 40\u0026deg;46\u0026prime;23\u0026Prime;, longitude 8\u0026deg;29\u0026prime;34\u0026Prime;). The apiary consisted of 12 colonies of \u003cem\u003eApis mellifera\u003c/em\u003e maintained in Dadan-Blatt hives containing 10 combs each. During this period, the colonies were monitored every 2 weeks to check for the presence of the queen and food stores and to evaluate the sanitary status (disease symptoms and varroosis) of the bees. Before selecting the colonies to be used in the experiment, the \u003cem\u003eV. destructor\u003c/em\u003e infestation level (%) of each colony was assessed following standard method (Pappas and Thrasyvoulou 1988). The infested colonies were used as a source of \u003cem\u003eV. destructor\u003c/em\u003e mites, whereas the uninfested ones (infestation level\u0026thinsp;\u0026lt;\u0026thinsp;1%) were used as a source of honeybee adults.\u003c/p\u003e\n\u003ch3\u003eDetection of melittin on the body of adult worker bees and drones\u003c/h3\u003e\n\u003cp\u003eWe sampled 10 individuals per category: freshly emerged, nurse, forager bees and drones from three different colonies (totalling 30 individuals per category-group). Each sampled bee was immediately placed individually inside a 1.5 ml Eppendorf, sacrificed on dry ice, and stored at \u0026minus;\u0026thinsp;20\u0026deg;C without its abdomen until chemical analyses were performed by following the procedure described above. The abdomen was detached to prevent contamination of the collected bees' bodies during subsequent manipulations necessary for chemical analyses.\u003c/p\u003e \u003cp\u003eFreshly emerged bees were directly sampled upon their emergence. To collect nurse bees, freshly emerged bees were marked on the thorax with a non-toxic colour upon emergence, returned immediately to their original hives and, seven days later, sampled from the hives. Forager bees with pollen load were captured at the hive entrance. Drones were sampled from inside the colonies.\u003c/p\u003e\n\u003ch3\u003eOrigin of the melittin detected on the worker's body\u003c/h3\u003e\n\u003cp\u003eTo establish the origin of melittin on the bee body surface, we quantified the amount of melittin present on the bodies of bees belonging to the following experimental groups: 1) 90 bees with blocked stingers, 2) a mix of 90 bees with blocked (45) and unblocked stingers (45), and 3) 90 bees with unblocked stingers (control group). The stinger was blocked using a stick to apply a droplet of non-toxic glue onto the tip of the abdomen. All bees were marked on the thorax with a non-toxic colour identifying the treatment. To exclude the family effect, experimental groups were replicated in three independent metal cages (10 cm \u0026times; 10 cm \u0026times; 5 cm), each containing a mix of 30 emerging bees each from three \u003cem\u003eVarroa\u003c/em\u003e-free colonies (10 bees per colony per cage), on the same day. The larger inner side of the cages was covered by a sheet of bee-wax (9 cm \u0026times; 9 cm), whereas the opposite side was closed by a glass window (10 cm \u0026times; 10 cm). All cages were kept in an incubator (+\u0026thinsp;31.5\u0026deg;C, 70% R.H., dark) with 50% (w/v) sucrose solution administered \u003cem\u003ead libitum\u003c/em\u003e with a graduated syringe (Williams et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). On the seventh day, all bees were sacrificed on dry ice and stored without abdomen at \u0026minus;\u0026thinsp;20\u0026deg;C until chemical analyses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of melittin on the body of infested and uninfested workers and in the venom sac\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine if venom, using melittin as a proxy, varied quantitatively on the body surface of \u003cem\u003eVarroa\u003c/em\u003e infested and uninfested bees, we set up the following experimental groups: 1) 90 workers parasitized during the adult stage, 2) 90 workers parasitized during the pupal stage, and 3) 90 uninfested workers (control). To obtain workers parasitized during the adult stage (phoretic infestation) and uninfested workers from the \u003cem\u003eVarroa\u003c/em\u003e-free colonies, we used the method described in (Pusceddu et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). To obtain workers parasitized during the pupal stage (cell infestation) from the \u003cem\u003eVarroa\u003c/em\u003e-infested colonies, we followed the protocol described in (Pusceddu et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). All groups were replicated in three independent metal cages (30 bees per cage), being set up on the same day and kept in an incubator under the same conditions described above (Williams et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). On the fifth day, each bee had its venom gland removed. Two sample types were obtained for each bee, as follows: 1) intact venom sac (source) and 2) body of the individual without abdomen. All samples were stored at \u0026minus;\u0026thinsp;20\u0026deg;C until chemical analyses.\u003c/p\u003e\n\u003ch3\u003eBehavioural observations\u003c/h3\u003e\n\u003cp\u003eTo investigate whether grooming (selfgrooming and allogrooming) and the likely consequent application of venom to the body surface is influenced by \u003cem\u003eVarroa\u003c/em\u003e infestation, we determined the frequency (e.g., number of events) of both grooming types by using the \"all occurrences sampling\" method (Altmann \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1974\u003c/span\u003e). In addition, to evaluate the effectiveness of grooming in counteracting \u003cem\u003eVarroa\u003c/em\u003e parasitism, the grooming behaviour was divided into three ethogram entries, as follows: 1) \u003cem\u003ecleaning body with legs or mandibles\u0026thinsp;+\u0026thinsp;shaking the abdomen\u003c/em\u003e, 2) \u003cem\u003eremoving mite using bee\u0026rsquo;s legs or mandibles\u003c/em\u003e, and 3) \u003cem\u003edamaging the mite using the mandibles\u003c/em\u003e. The frequency of each event entry was recorded. Thirty emerging bees from three \u003cem\u003eVarroa\u003c/em\u003e-free colonies were mixed to prevent any family effects (10 bees from each colony) and placed in a metal cage as described above. To obtain workers parasitized during the adult stage (phoretic infestation) and uninfested workers from the \u003cem\u003eVarroa\u003c/em\u003e-free colonies, we used the method described in (Pusceddu et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The behavioural observations were repeated for three consecutive days during which phoretic infested and uninfested bee groups were compared. Selfgrooming and allogrooming occurrences were counted every day, at three different time slots (9:00\u0026ndash;9:30; 13:30\u0026thinsp;\u0026minus;\u0026thinsp;14:00; 18:00\u0026ndash;18:30), with 30-min observation sessions, corresponding to a total of 1 h and 30 min per day per cage. The cages were kept in an incubator under the same conditions as described above (Williams et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). All treatments were replicated using three independent cages (30 bees per cage) and were set up at the same time.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eToxicology bioassay\u003c/h2\u003e \u003cp\u003eTo evaluate the effect of bee venom on \u003cem\u003eVarroa\u003c/em\u003e activity, five mites were placed in a 90-mm diameter Petri dish containing an absorbent paper 67 g/mq (APTACA SRL, Canelli, Italy) (Pusceddu et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Each mite was wetted with 1 \u0026micro;l of a water solution containing 0.2 \u0026micro;l of bee venom extract (CITEQ, Groningen, The Netherlands) or 1 \u0026micro;l of water solution (control group). Mites were obtained from brood cells capped in the preceding 15 h, following the procedure described by (Nazzi et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). A total of 90 mites from six \u003cem\u003eVarroa\u003c/em\u003e-infested colonies were tested, corresponding to 45 mites per treatment (control \u003cem\u003evs.\u003c/em\u003e venom). Mite activity was observed under a stereo microscope every 15 min for the first hour, every 20 min for the second hour, and every 30 min for the next six hours. Mites were considered inactive when they showed no response to contact stimulus using a stick (Milani \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). This bioassay was replicated twice (180 mites in total). Both experiments were conducted under artificial light and at temperature of 32\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eWe used a linear mixed model (LMM), followed by a Tukey post-hoc test, to investigate differences in melittin concentration (mg/L) on the bodies of worker bees (freshly emerged, nurse and forager) and drones. Colony was included as a random factor. Due to the absence of melittin on the bodies of freshly emerged worker bees and drones, statistical comparisons are reported only between nurse and forager worker bees.\u003c/p\u003e \u003cp\u003eWe used an LMM, followed by a Tukey post-hoc test, to examine the effect of treatment (control \u003cem\u003evs.\u003c/em\u003e bee blocked sting \u003cem\u003evs.\u003c/em\u003e mix) on melittin concentration (mg/L) on the bees\u0026rsquo; bodies. Cage was included as a random factor. Due to the absence of melittin on the bodies of stinger blocked bees, statistical comparisons are reported only between control bees in single and mixed cages.\u003c/p\u003e \u003cp\u003eWe used LMMs, followed by a Tukey post-hoc test, to examine the effect of treatment (cell infestation, phoretic infestation, and \u003cem\u003eVarroa\u003c/em\u003e-free) on melittin concentration (mg/L) on the bodies and venom gland of bees. In addition, we used an LMM, followed by a Tukey post-hoc test, to examine the effect of treatment (cell infestation, phoretic infestation and \u003cem\u003eVarroa-\u003c/em\u003efree) on the proportion of melittin on the bodies of bees compared to the total amount of melittin (body\u0026thinsp;+\u0026thinsp;venom sac). Cage was included as a random factor in all models.\u003c/p\u003e \u003cp\u003eTo examine whether selfgrooming or allogrooming behaviour differed between groups of infested \u003cem\u003evs.\u003c/em\u003e uninfested bees, general linear mixed models (GLMMs) with negative binomial error structure were used. Experimental group (infested \u003cem\u003evs.\u003c/em\u003e uninfested bees) was used as a fixed factor and time slot, day of experiment and cage were used as random factors.\u003c/p\u003e \u003cp\u003eTo examine the effect of venom treatment (control treatment \u003cem\u003evs.\u003c/em\u003e bee venom treatment) on \u003cem\u003eVarroa\u003c/em\u003e mite activity, a GLMM with binomial error structure was used. Treatment (control treatment \u003cem\u003evs.\u003c/em\u003e bee venom treatment), time of observation and their interaction were used as fixed factors and Petri dish was used as a random factor.\u003c/p\u003e \u003cp\u003eAll analyses were performed in R v.3.5.2 (R Core Team \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Mixed effects models were conducted using the R package \u003cem\u003elme4\u003c/em\u003e (Bates et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Model assumptions were checked visually and found to meet expectations, including homogeneity of variance, normality of residuals, and linearity. Due to lack of homogeneity of variance and normality, melittin concentration (mg/L) was square root-transformed in all models.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS/MS method validation\u003c/h2\u003e \u003cp\u003eThe correlation coefficient of the calibration lines (r\u003csup\u003e2\u003c/sup\u003e) showed values oscillating between 0.992 and 0.999. The linearity was, therefore, above the condition set for the validation of the method. The accuracy data provided by the recovery experiments obtained from 6 replicates for the two concentrations tested ranged from 87.1 to 93.7% at the LOQ level and from 81.7 to 90.3% at the 10\u0026times;LOQ (S3 Table). The values obtained showed a good extraction capacity of the proposed method, which was, therefore, assessed as suitable for the melittin analysis in the bee samples. The repeatability (RSDr) and reproducibility (RSDwR) showed values lower than 20%, thus showing good repeatability of the analytical method. The chromatograms of the control and standards did not show the presence of interfering peaks, thus indicating a good selectivity of the method (S1 Figure). The results obtained from the validation tests are consistent with the validation parameters of the SANTE/12682 /2019 guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetection of melittin on the body of adult worker bees and drones\u003c/h2\u003e \u003cp\u003eWe found a detectable amount of melittin only in nurse and forager bees (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). No melittin was observed in drones and freshly emerged bees (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Interestingly, the amount of melittin detected on nurses' bodies was significantly lower than that on foragers' bodies (LMM; Tukey post-hoc test; Z\u0026thinsp;=\u0026thinsp;3.165, P\u0026thinsp;=\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOrigin of the melittin detected on the worker's body\u003c/h2\u003e \u003cp\u003eThe experiment conducted to determine the amount of venom on the bodies of bees with blocked or unblocked stingers, whether housed individually or in groups, clearly demostrated that the melittin present on workers' bodies originates from the venom gland (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Specifically, no melittin was detected on the bodies of bees with blocked stingers, regardless of whether they were kept alone or alongside bees with unblocked stingers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of venom on the bodies of infested and uninfested workers and in the venom sac\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe amount of melittin detected on the cuticle of worker bees parasitized during the pupal stage (cell infestation) was significantly lower than in those parasitized as adults (phoretic infestation) (LMM; Tukey post hoc test; Z\u0026thinsp;=\u0026thinsp;3.415, P\u0026thinsp;=\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) and in uninfested control bees (LMM; Tukey post hoc test; Z\u0026thinsp;=\u0026thinsp;2.275, P\u0026thinsp;=\u0026thinsp;0.034, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In contrast, there was no significant difference in the amount of melittin present on the bodies of adult-parasitized bees compared to the control bees (LMM; Tukey post hoc test; Z\u0026thinsp;=\u0026thinsp;1.291, P\u0026thinsp;=\u0026thinsp;0.196, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, the amount of melittin detected in the venom sac was significantly lower in adult worker bees parasitized during the pupal stage than in control bees (LMM; Tukey post hoc test; Z\u0026thinsp;=\u0026thinsp;2.442, P\u0026thinsp;=\u0026thinsp;0.043, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Similarly, in adult workers parasitized as adults, the amount of melittin in the venom sac was lower than in control bees (approximately half on average), although this difference was not statistically significant (LMM; Tukey post hoc test; Z\u0026thinsp;=\u0026thinsp;1.434, P\u0026thinsp;=\u0026thinsp;0.227, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). However, calculating the proportion of melittin found on the bodies of worker bees relative to total melittin amount (e.g., the sum of melittin present on the body and in the venom sac), showed that this proportion significantly increased in parasitized bees (both as adults and during the pupal stage) compared to unparasitized ones (LMM; Tukey post hoc test; Z\u0026thinsp;=\u0026thinsp;3.308, P\u0026thinsp;=\u0026thinsp;0.002; Z\u0026thinsp;=\u0026thinsp;2.591, P\u0026thinsp;=\u0026thinsp;0.014, respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBehavioural observations\u003c/h2\u003e \u003cp\u003eWe observed a total of 4841 selfgrooming events in which bees were cleaning their body using the legs\u0026thinsp;+\u0026thinsp;shaking the abdomen. Out of these, 3147 were observed in the phoretic infested group and 1694 in the uninfested group (65% \u003cem\u003evs.\u003c/em\u003e 35%). This difference was statistically significant (GLMM; χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;27.973, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). However, removing mites using bee\u0026rsquo;s legs was observed only 12 times out of the 3147 selfgrooming events observed in the \u003cem\u003eVarroa\u003c/em\u003e-infested group (0.38%). Damaging the mite using bee\u0026rsquo;s mandibles did not occur in our study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe observed a total of 74 allogrooming events by cleaning nestmate body with mandibles. Out of these, 41 were observed in the phoretic infested group and 33 in the uninfested group (55% \u003cem\u003evs.\u003c/em\u003e 45%). This difference was not statistically significant (GLMM; χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.090, P\u0026thinsp;=\u0026thinsp;0.763; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Removing mites from other bees using mandibles and damaging them did not occur in our study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eToxicology bioassay\u003c/h2\u003e \u003cp\u003eExposure of the mites to honey bee venom resulted in a highly significant increase in the percentage of inactive mites compared to the control (approximately 7% \u003cem\u003evs\u003c/em\u003e 0.9%, respectively; GLMM; χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;12.191, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) Proportion of inactive mites also increased with time of observation (GLMM; χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;19.382, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The interaction between treatment and time was not statistically significant (GLMM; χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;1.620, P\u0026thinsp;=\u0026thinsp;0.203).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe aims of this study were to investigate the role of grooming (selfgrooming and allogrooming) in the distribution of venom compounds on the honey bees\u0026rsquo; body as an external immune defence trait under the challenge of the parasitic mite \u003cem\u003eV. destructor\u003c/em\u003e, as well as to evaluate the effects of venom on the parasite\u0026rsquo;s activity. We first confirmed the results of earlier studies (Baracchi and Turillazzi \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Baracchi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) by showing that melittin, a venom compound, is absent on the body surface of drones and freshly emerged bees but it is present on nurse and forager workers. We then found that melittin was absent on the body of workers that had access to their stingers blocked, thus indicating that venom is transferred from the stinger to the body surface, likely during selfgrooming, and is not picked up from other sources in the hive environment. We then found that venom transfer to the body surface was influenced by the presence of \u003cem\u003eVarroa\u003c/em\u003e, with parasite presence leading, on one hand, to a proportional higher amount of melittin on the body surface relative to the total melittin (the sum of melittin present on the body and in the venom sac), and on the other hand, to a lower overall amount of melittin production. In addition, we also found that \u003cem\u003eVarroa\u003c/em\u003e infested bees groomed themselves more than control bees. This supports the hypothesis that selfgrooming is responsible for the venom transfer to the body surface of honey bees and agrees with the proportionally higher amount of melittin on the body surface of \u003cem\u003eVarroa\u003c/em\u003e infested bees. Lastly, we found that venom negatively influenced parasitic mite activity, thus indicating that venom deposition on the body surface of bees may serve as an external immune defence trait against parasites and/or pathogens.\u003c/p\u003e \u003cp\u003eOur results exclude the possibility of environmental contamination or acquisition and indicate the presence of venom on the bee body surface through selfgrooming behaviour. As expected, bees with their stingers blocked, and therefore unable to release venom, did not show melittin on their bodies. This result remained consistent also when bees were reared in the same cage as control bees capable of releasing venom. These findings align with those reported by Baracchi and Turillazzi (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and are further supported by our results that showed the absence of venom on the bodies of drones sampled from the hive and the presence of venom on the bodies of nurse and forager bees. Notably, drones are also groomed by workers, although this behaviour occurs less frequently than among workers (Stout et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Goins and Schneider 2011). If allogrooming behaviour had been responsible for venom spread, drones or bees with blocked stings would have had melittin on their bodies. In fact, our results clearly show not only that venom on the body surface of worker bees is not just an artefact, but also that it is likely used only as an external immune defence for personal hygiene rather than for sanitation of other adult nest members. This is because it is not spread via allogrooming to drones or bees with blocked stingers. In contrast, in ants, venom is used not only for personal sanitation via selfgrooming (e.g., (Tranter and Hughes \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)) but also for the sanitation of developing brood [21] and adult nest members via allogrooming (Beydizada et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInterestingly, our research provides clear evidence that parasitisation leads to a lower amount of melittin in the venom. In fact, our study revealed that when bees were parasitized by \u003cem\u003eVarroa\u003c/em\u003e during the pupal stage, the external immune system of forager bees was weakened, as evidenced by the lower levels of melittin found on their cuticles and venom sacs compared to healthy bees. This negative effect was more pronounced when bees were parasitized during the pupal stage rather than as adults. To our knowledge, this result had never been reported before and we believe that it can be explained by the high energy cost of venom production in aculeate Hymenoptera (Baracchi and Tragust \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Baumann \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and the physiological impairments caused by \u003cem\u003eVarroa\u003c/em\u003e on honey bees (Rosenkranz et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Nazzi and Le Conte \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThus, assuming that bees cannot selectively choose which venom components to apply on their bodies and considering that parasitized bees use a greater proportion of the total melittin content from the venom sac compared to unparasitized bees, it is likely that parasitized worker bees spread larger amounts of venom on their bodies. However, this does not necessarily imply that the distribution of venom across the body represents a curative defence mechanism adopted by bees against \u003cem\u003eV. destructor\u003c/em\u003e. In fact, the following question remains open: do bees use more venom (1) in response to \u003cem\u003eVarroa\u003c/em\u003e attacks or (2) simply maintain a minimum level of antimicrobial substances on their bodies for the control of opportunistic or pathogenic microbes? A finding supporting the second hypothesis was the significantly higher amounts of melittin found on the bodies of foragers compared to nurses in our trial. It is well known that foragers face higher pathogen exposure (Pusceddu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e) and thus require additional protection compared to nurse bees. Therefore, it is debatable that bees seem capable of regulating the dosage of antimicrobial substances on their bodies according to their perceived level of protection needed. However, Baracchi and Turillazzi (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) found that nurses' venom contains higher percentages of apamine and lower percentages of melittin compared to the venom of older nestmates (guards and foragers). Therefore, the difference we observed between the two cohorts of bees (nurses \u003cem\u003evs.\u003c/em\u003e foragers) likely resulted from variations in the composition of the venom distributed on the body rather than the quantity applied by the different bee cohorts.\u003c/p\u003e \u003cp\u003eAs expected, a significant increase in selfgrooming was observed in the group challenged with the ectoparasite \u003cem\u003eVarroa\u003c/em\u003e. The behaviour of cleaning the body with legs and shaking the abdomen is likely involved in spreading of antimicrobial substances across the bee's body. Like in previous studies conducted on ants (Tragust \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Koch et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), our results suggest that selfgrooming in \u003cem\u003eA. mellifera\u003c/em\u003e involves venom spread. However, our findings confirm the poor efficacy of selfgrooming in combating \u003cem\u003eVarroa\u003c/em\u003e in \u003cem\u003eA. mellifera\u003c/em\u003e. Indeed, the observed percentage of removal of the parasite during selfgrooming events was very low (only 0.38% of the cases), which was very similar to that observed by Peng et al. (Peng et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Moreover, in accordance with B\u0026uuml;chler et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), we never observed a bee damaging the mite with its mandibles. In accordance with our findings on absence of melittin on the body of drones and blocked stinger bees, an increase in allogrooming was not observed in these bees. This is consistent with the results of previous studies that have demonstrated that this behaviour is rare and occurs mainly during the nursing period (Cini et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pusceddu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). In addition, we never observed the removal of parasite or bee damaging the mite with its mandibles during allogrooming behaviour.\u003c/p\u003e \u003cp\u003eOur study also showed that bee venom has a detrimental effect on \u003cem\u003eVarroa\u003c/em\u003e mites. However, the observed narcoleptic effect of the venom was relatively mild (approximately 7% of inactive mites) in comparison to other hive products such as raw propolis, which has an effect of 19\u0026ndash;22% (Pusceddu et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), or ethanol propolis extracts, which have an effect of 45\u0026ndash;90% (Garedew et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Pusceddu et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt could be assumed that venom on the body surface might provide protection against other honeybee parasites and/or pathogens (Fern\u0026aacute;ndez et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Sani et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For example, it is known that some viruses, namely Chronic bee paralysis virus and Israeli acute paralysis virus can be transmitted by topical application (Ya\u0026ntilde;ez et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and contact with a virus-contaminated environment or infected bees (Bailey et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Coulon et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Amiri et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, given the strong antiviral effect of melittin (Kontogiannis et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), venom on the bee body surface might also help against virus transmission and infection. In support of this hypothesis, previous work has shown that bee venom, as a dietary supplement, increased the expression levels of immune genes encoding the antimicrobial peptides Abaecin, Defensin 2 and Hymenoptaecin as well as stimulated the production of juvenile hormone and vitellogenin secretion, and decreased \u003cem\u003eVarroa\u003c/em\u003e infestation level within the colony (Seyam et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Recently, Mahmoud et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) also showed an immunostimulatory as well as a beneficial role of venom in honeybees challenged by \u003cem\u003eVairimorpha\u003c/em\u003e (\u003cem\u003eNosema\u003c/em\u003e) \u003cem\u003eceranae.\u003c/em\u003e In addition, Hejn\u0026iacute;kov\u0026aacute; et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) found that melittin is also synthesized in the fat body, which is apparently tolerated by bee cells and most likely protects the bee from infection.\u003c/p\u003e \u003cp\u003eA hypothesis that would be interesting to test in future studies is if the narcoleptic effect induced by the venom on mite could facilitate its detachment from the bee's body during selfgrooming. Given the differences in venom composition and venom deposition among \u003cem\u003eApis\u003c/em\u003e species (35), it would be interesting to study \u003cem\u003evenom bathing\u003c/em\u003e behaviour in relation to parasite efficacy control, both in \u003cem\u003eA. mellifera\u003c/em\u003e and \u003cem\u003eA. cerana\u003c/em\u003e, with the latter being the original host of \u003cem\u003eVarroa destructor\u003c/em\u003e. This deserves further attention as it might explain some of the variations in mite resistant traits between species, e.g., mite infertility (Grindrod and Martin \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnother aspect that is important to consider is that the hive is a complex system where antimicrobial venom (Baracchi and Turillazzi \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) coexists with other bioactive matrices such as propolis (Pusceddu et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), honey (Floris et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), wax (Felicioli et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), pollen (Didaras et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and royal jelly (Alreshoodi and Sultanbawa \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The combination of these matrices with their bioactive properties could result in a synergistic effect against pests and pathogens (Erler and Moritz \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Erler et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A synergistic effect of different antimicrobial substances was found in the study of Br\u0026uuml;tsch et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), who observed that wood ants can produce a potent antimicrobial cocktail by combining formic acid with tree resins.\u003c/p\u003e \u003cp\u003eIn conclusion, our results strongly suggest that, like ants, honeybees rely on a combination of selfgrooming behaviour and venom application for external immunity, which likely counteract opportunistic and pathogenic microorganisms. However, this defence system can be compromised by \u003cem\u003eVarroa destructor\u003c/em\u003e, whose parasitizing activity on pupae leads to reduced melittin production in adult bees. This discovery reveals, to our knowledge, another previously unknown negative side effect caused by this mite.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003eNo ethical approval is required for this study.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by: Fondazione di Sardegna, year 2016 and by Regione Autonoma della Sardegna (Italy), L.R. 7/2007, year 2016, project: \u0026lsquo;Self-medication in the hive: propolis and venom against the honeybee ectoparasite \u003cem\u003eVarroa destructor\u003c/em\u003e\u0026rsquo;.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAS, IF, MP and ST conceived and designed research. ICB, JSN and MP conducted experiments. AA, AA and FC made the chemical analysis. PT analysed data. MP and AS wrote the manuscript. All authors read, reviewed and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe authors thank Dr. Ana Helena Dias Francesconi from the University of Sassari for revising the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the supplementary information files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAli, M. A. A. S. M. Studies on bee venom and its medical uses. \u003cem\u003eInt. J. Adv. Res. Technol.\u003c/em\u003e \u003cb\u003e1\u003c/b\u003e, 69\u0026ndash;83 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlreshoodi, M. F. \u0026amp; Sultanbawa, Y. 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Microbiol.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 943. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmicb.2020.00943\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2020.00943\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Apis mellifera, mite, ectoparasite, bee venom, grooming","lastPublishedDoi":"10.21203/rs.3.rs-6238801/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6238801/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSocial insects employ venom as an external immune defence against pathogens and parasites. Like other Hymenoptera, the venom gland of honey bee serves as a reservoir of antimicrobial substances, primarily melittin. This study investigates the role of venom associated with grooming behaviour as an external immune defence in \u003cem\u003eApis mellifera\u003c/em\u003e workers infested by \u003cem\u003eVarroa destructor\u003c/em\u003e. Using a multi-step approach, we first confirmed the presence of venom on bees' bodies using melittin as a marker. We then examined how grooming facilitates the distribution of venom on the bee's body. Further assays compared melittin levels on the bodies of \u003cem\u003eVarroa\u003c/em\u003e-free and \u003cem\u003eVarroa\u003c/em\u003e-infested workers and assessed the effects of bee-venom on mite activity. Our findings confirmed the occurrence of \"venom bathing\" in \u003cem\u003eA. mellifera\u003c/em\u003e, whereby bees coat their bodies with antimicrobial substances through selfgrooming. excluding social components or environmental contamination. Infested bees spread larger amounts of venom on their bodies compared to uninfested bees and bee-venom significantly also reduced mite activity, suggesting venom functions as an external defence. However, \u003cem\u003eVarroa\u003c/em\u003e negatively impacts melittin production. Our study reveals a previously unknown negative effect of \u003cem\u003eV. destructor\u003c/em\u003e: impairment of honey bees' external immune defence through reduced melittin production.\u003c/p\u003e","manuscriptTitle":"Varroa destructor weakens honey bee external immunity by impairing melittin production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-25 21:41:27","doi":"10.21203/rs.3.rs-6238801/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-22T07:29:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-21T21:43:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-20T10:04:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-15T20:36:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"48331739422775275961380520514190756724","date":"2025-04-04T13:15:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223469901058314789500804241034585441880","date":"2025-04-03T00:13:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69876648502755622515074341376845655279","date":"2025-04-02T20:09:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-02T11:38:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-27T15:02:41+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-27T14:58:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-26T17:44:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-16T16:52:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b597d6e3-8efa-430e-9525-fbc7340651c3","owner":[],"postedDate":"April 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47487864,"name":"Biological sciences/Ecology"},{"id":47487865,"name":"Biological sciences/Zoology"},{"id":47487866,"name":"Health sciences/Diseases"}],"tags":[],"updatedAt":"2025-08-25T16:40:50+00:00","versionOfRecord":{"articleIdentity":"rs-6238801","link":"https://doi.org/10.1038/s41598-025-13440-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-08-20 16:29:43","publishedOnDateReadable":"August 20th, 2025"},"versionCreatedAt":"2025-04-25 21:41:27","video":"","vorDoi":"10.1038/s41598-025-13440-2","vorDoiUrl":"https://doi.org/10.1038/s41598-025-13440-2","workflowStages":[]},"version":"v1","identity":"rs-6238801","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6238801","identity":"rs-6238801","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}