Deformed Wing Virus-Induced Changes in Honey bee Reception and Preference for Pollen Scents

Deformed Wing Virus-Induced Changes in Honey bee Reception and Preference for Pollen Scents | 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 Research Article Deformed Wing Virus-Induced Changes in Honey bee Reception and Preference for Pollen Scents Diego Silva, Felipe Becerra, Sebastian Salazar, Nolberto Arismendi, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5582583/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Honey bees ( Apis mellifera ) play a crucial role in pollination, and their olfactory system is essential for food collection and source selection. This study evaluates how the Deformed Wing Virus (DWV), particularly its variant A, affects the olfactory sensitivity and behavioral responses of bees (10 to 20 days old) to volatile compounds from different pollen sources. We collected and analyzed the volatile fractions of three types of pollen (polyfloral and two monofloral) using dynamic HeadSpace and gas chromatography-mass spectrometry (GC-MS). The chemical analysis revealed differences in volatile compound profiles among the pollen types, including the presence of benzaldehyde, lilac alcohol, and 1–4 cineole, which are known to impact honey bee olfaction. Behavioral assays using a Y-olfactometer showed that while non-inoculated bees (N-DWV) responded to the aromas of all pollens in higher proportions, DWV-inoculated bees (I-DWV) exhibited significant reductions in behavioral responses. Specifically, I-DWV bees showed lower response rates, and a higher proportion of non-responding individuals compared to N-DWV bees. Notably, I-DWV bees were more attracted to the aroma of Eucryphia cordifolia when exposed simultaneously to the aroma of Rubus ulmifolius , possibly due to a self-medication behavior or a selective response to pollen with higher antiviral properties. Electrophysiological recordings indicated that DWV-A infection decreases olfactory sensitivity, particularly in response to complex odors. This decline in olfactory function and behavioral preferences could compromise foraging efficiency and overall colony health. These findings highlight the impact of DWV-A on honey bee sensory and behavioral processes, raising concerns about broader implications for colony survival and pollination services. Honey bees DWV-A Behaviors Figures Figure 1 Figure 2 Figure 3 I INTRODUCTION Pollination is crucial for ecosystems and the food industry, a behavior primarily carried out by honey bees ( Apis mellifera L.) (Potts et al., 2016). This species of pollinating insect is one of the most valuable animal species for large-scale food production systems, such as in canola and sunflower crops (Goodrich et al., 2018). It is predominantly used for pollinating monocultures over large areas, a global trend that has positioned A. mellifera as an economically significant organism for food production (Winfree et al., 2011; Goodrich et al., 2018). According to the Food and Agriculture Organization of the United Nations (FAO), out of the 100 crop species produced for human consumption, 71 are pollinated in agricultural systems by honeybees, thus supporting 90% of global food production (Klein et al., 2007). In Europe alone, out of the 264 crop species produced, 84% are exclusively pollinated by A. mellifera (UNEP, 2010). Similarly, 228 million tons of food produced in Latin America can be attributed directly to insect pollination, which represent US $ 77.82 billion (Basualdo et al., 2022). Thus, insect pollinators like honey bees provides direct and indirect benefits for human being by promoting biodiversity, food production and sociocultural utilities by goods and services (Etxegarai-Legarreta and Sanchez-Famoso, 2022). For bees, nectar is a source of energy, while pollen provides proteins, lipids, minerals, and vitamins (Yang et al., 2013). However, a diet lacking in nutritional content can reduce an ability of the bees to resist diseases (Alaux et al., 2010; Yang et al., 2013). Nurse honey bees are responsible for maintaining the nutritional strength of the colonies, as they are the ones who convert food into royal jelly, thus influencing the size of the colony, brood rearing, and overall strength, among other factors (Brodschneider & Crailsheim, 2010). The pollen consumption of each worker bee varies depending on their age and the tasks they perform within the colony. Honey bee larvae do not consume much pollen, while bees ingest large amounts (Babendreier et al., 2004). Older workers, often known as foragers, are unable to consume or digest pollen (Babendreier et al., 2004). Additionally, A. mellifera has been described as a polylectic pollinator, meaning that this insect does not have a specific, simple interaction with a single plant (Paoli and Galizia, 2021). Instead, it visits and recognizes multiple plant species solely to collect food sources like pollen and nectar (Paoli and Galizia, 2021). This action is performed with precise coordination of both memory processes and visual as well as olfactory recognition (Wright et al., 2002; Menzel et al., 2012). To recognize them, bees first need to detect the signals released by plants, which are sensed by their antennae, an organ primarily responsible for the sense of smell (Groot et al., 2016; Li et al., 2015; Paoli and Galizia, 2021). It is widely agreed that olfactory functioning and information processing are largely conserved among insects (Paoli and Galizia, 2021). This process begins in the antenna, where environmental compounds are intercepted and converted into an electrical signal, which is then sent to processing centers in the insect's brain (Paoli and Galizia, 2021, Ramirez et al., 2023). There, the information is received, interpreted, and analyzed through different memory mechanisms (Paoli and Galizia, 2021, Ramirez et al., 2023). This flow of information from the antenna to the brain is essential for the development of the insect’s complex behavioral tasks, whether within the hive or during foraging activities (Paoli and Galizia, 2021). However, the integrity of this insect’s olfactory system can be threatened by species-specific pathogens, such as the deformed wing virus (DWV). This pathogen belongs to the Iflaviridae family and is classified within Baltimore group IV. It has a positive-strand RNA genome organization like that of picornaviruses (Genersch and Aubert, 2010). With A. mellifera as its primary host, this virus has achieved global distribution, making it one of the most important pathogens on honey bees (Yue and Genersch, 2005; Sanpa, 2009; Williams et al., 2009; Mordecai et al., 2016). However, it has been shown that this pathogen can infect different species related to A. mellifera , including various bee species such as the Asian honey bee ( A. cerana F.) and the dwarf honey bee ( A. florea F.) (Allen & Ball, 1996; Ellis & Munn, 2005; Genersch & Aubert, 2010), as well as Bombus terrestris L. and B. pascuorum S., where symptoms of the disease have also been observed (Genersch et al., 2006). In fact, among the main symptoms of this pathogen are wing deformities, abdominal swelling, paralysis, and behavioral alterations. Furthermore, this pathogen can persist in bee colonies without presenting clinical symptoms, making early detection difficult (Benaet et al., 2017; Grozinger et al., 2019). It has been reported that this pathogen can replicate in different regions of the bee's brain, potentially compromising the normal functioning of the insect's neural network, with consequences for olfactory sensitivity and associated behaviors (Pizzorno et al., 2021). In general, DWV has been reported to be replicated in brain regions, including the neuropils responsible for vision and olfaction, which could interfere with various behavioral responses (Sha et al., 2009). The successful propagation of DWV has been attributed to its ability to inhabit different hosts, many of which can act as vectors, with the primary vector being the mite Varroa destructor A., which feeds on the fat bodies of the honey bees, facilitating virus transmission (Dainat et al., 2009; Forsgren et al., 2009). Other less efficient, unassisted transmission mechanisms are also known, such as trophallaxis, cannibalism, or vertical transovarial transfer (Amiri et al., 2018; Chen et al., 2006). To date, three epidemiologically significant variants of DWV (A, B, and C) have been identified; variant A is particularly associated with winter colony losses (Mordecai et al., 2016; Kevil et al., 2017). In Chile, specifically, the presence of variants A and B has been determined, with variant A being the most prevalent in the country's apiaries (Riveros et al., 2020). It has been specifically demonstrated that DWV-A can replicate in the epithelial cells of the antennae and that increased viral loads in the insect damage the cellular ultrastructure (Kim et al., 2019). This pathogen can affect olfactory sensitivity and reduce the gene expression of OBP-coding genes (Silva et al., 2021). It has been described that the increase in viral loads over time leads to the decrease of at least four out of the nine antenna-specific OBPs, notably the nurse bee activity regulators OBP5 and OBP11 (Silva et al., 2021). Additionally, recent studies have shown that high viral loads of DWV-A in both the head and antenna alter the behavioral response of nurse bees, and it has been determined that increased viral loads limit the insect's behavioral response (Silva et al., 2024). High viral loads of DWV-A in the head and antenna also affect the expression of genes coding for pre- and postsynaptic proteins, causing a reduction in the insects' behavioral responses (Silva et al., 2024). Also, it has been demonstrated that DWV-A can affect the physiology of the olfactory system of A. mellifera as well as its behavioral responses (Kim et al., 2019, Silva et al., 2021, Silva et al., 2024). However, how this pathogen impacts the physiological and behavioral consequences in relation to aromas associated with pollen from different plant species remain unknown. Therefore, the aim of this study was to evaluate the effects generated by the increase in viral loads in A. mellifera infected with the variant A of the deformed wing virus on olfactory reception and sensitivity in bees exposed to different aromas obtained from pollens of various species. III MATERIALS AND METHODS Preparation of the viral inoculum The viral inoculum used for all bioassays was prepared as described by Gusachenko et al. (2020) and Silva et al. (2024). The inoculum was obtained from colonies infected with the deformed wing virus (DWV), after confirming the presence of the Variant-A. A group of 20 symptomatic bees was homogenized in phosphate-buffered saline (1X PBS) for 90 seconds at high-speed using a Stomacher 80 laboratory blender (Seward, London, UK). The homogenate was then centrifuged twice: first at 1500× g for 10 minutes, followed by 10,000× g for 10 minutes, both at 4°C. The supernatant was purified by filtration through a 0.22 µM filter (PES, Merck Millipore, Darmstadt, Germany) and treated with RNase to destroy all unencapsidated RNA, as indicated by Skubnik et al. (2017). To determine the exact concentration of viral particles present in the inoculum, a 200 µL aliquot of the supernatant was collected for RNA extraction, cDNA synthesis, and subsequent quantification of the viral load via qPCR, following the methodology detailed in the RNA Extraction section. The remaining extracted inoculum was stored at − 80°C until further use. Viral Inoculation The adult A. mellifera used for all assays were obtained from the experimental apiary of the experimental center "El Nogal" (36°35′58.25″ S–72°04′51.77″ W), University of Concepción, Chillán, Chile. To standardize the age of the bees used in the various bioassays, brood frames were collected directly from hives and kept under control condition at 30°C ± 1; 60% ± 3 RH. Previously, bee colonies in the apiary were selected based on the levels of pathogens present, with the criterion that the donor hive of brood frames were free from other pathogens such as Nosema ceranae F. and Lotmaria passim S. Priority was also given to hives without or with low viral loads of acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV), and DWV. Newly emerged bees were then carefully collected and randomly confined in plastic cages (base = 8 cm diameter, top = 10 cm diameter, height = 15 cm). The experimental procedure was conducted similarly to what was previously published by Silva et al. (2021). In summary, newly emerged bees from the brood frames were inoculated with the viral suspension orally (Treatment I-DWV). A total of 5 µL of the viral suspension (1.0 × 10⁹ copy number per bee) in a 60% sucrose solution was used. All bees that did not consume viral suspension were excluded from the experiment. Non-inoculated bees with the viral suspension were used as the control group (Treatment N-DWV). In total, 1,700 worker bees were used for the subsequent bioassays. The bees were kept in 34 plastic cages, with 50 bees per cage (everyone was considered an independent replicate for future experiments), and the distribution was as follows: bees inoculated with the viral suspension, n = 850, distributed in 17 plastic cages with 50 bees per cage; non-inoculated bees, n = 850, distributed in 17 plastic cages with 50 bees per cage. Each plastic cage was provided with a supplement of 3 g of a pollen substitute (the commercial preparation included soybean, brewer’s yeast, corn starch, wheat flour, corbicular pollen, canola oil, and canola oil) and 60% sucrose syrup ad libitum, following the recommendations of Arismendi et al. (2020). Pollen collection, Chemical Characterization and Preparation of Stimuli For the preparation of the stimuli used in the various bioassays, four samples of corbicular pollen were collected from different experimental apiaries and locations: Pollen ( 1 ) from the University of Concepción, Chillán (36°35′58.25″ S–72°04′51.77″ W); Commercial pollen ( 2 ) from the Coihueco sector, Chillán (36°37′00″S-71°50′00″W) and Pollen ( 3 ) from the Río bueno sector, Los Ríos Region, Valdivia, Chile (40°16′40″S-72°33′47″W). The collections were carried out using pollen traps that were installed at the entrance of the bee hives, then the pollen samples were stored cold (-20) to avoid degradation of volatile compounds due to temperature. A sample (10 g) of each collected pollen was immediately sent for paleobotanical identification to Laboratory of Entomology, Universidad Austral de Chile. The paleobotanical identification was based on the Chilean pollen classification standard (NCh3255:2011). The samples were then categorized as follows: ( 1 ) Polyfloral pollen including pollen from Brassica campestris (32.77%), Trifolium pratense (30.40%), Rubus ulmifolius (14.18%), Lotus uliginosus (10.13%) and other plant species (12.52%); ( 2 ) monofloral pollen from Rubus ulmifolius (71%); and ( 3 ) monofloral native pollen from Eucryphia cordifolia (75%). The volatile fraction of the pollen samples was collected using the dynamic HeadSpace method. Fifty grams of pollen were encapsulated in a 450 mL Pyrex glass bell jar. The volatile fraction was collected using a 200 mg Porapak Q trap (80–100 mesh), previously cleaned with 1 mL of anhydrous ether and conditioned for 2 hours at 220°C under a constant flow of 60 mL min − 1 of nitrogen. The collection was performed over 24 hours using a positive/negative air pressure system, with an inlet flow of 0.7 L min − 1 and an outlet flow of 0.5 L min − 1 . The air entering the system was purified by an activated carbon filter. Volatiles were extracted from the Porapak Q trap by eluting with 1 mL of chromatographic-grade hexane. For relative quantification, 10 mg L − 1 of Tretalin was used as an internal standard. Characterization and relative quantification were performed using gas chromatography coupled with mass spectrometry (GC-MS) (GC-MS QP2010 Plus; Shimadzu), equipped with a capillary column (Rtx-5) 30 m x 0.25 mm ID x 0.25 µm; Restek Corporation). Helium was used as the carrier gas at a flow rate of 1.4 mL min − 1 . The oven was programmed to start at 40°C, held for 1 minute, with a temperature ramp of 8°C per minute until reaching 205°C, which was maintained for 3 minutes. The eluates were then stored in amber vials and kept at -40°C until use. Olfactory Reception Evaluation The technique of electroantennography (EAG) was used to determine the olfactory reception of A. mellifera adults inoculated and non-inoculate with the DWV-A, using the volatile fraction of the collected pollens as stimuli. The olfactory sensitivity evaluation was conducted at 5, 10, 15, and 20 days post viral inoculation, with five bees per treatment for each sampling point (n = 20 individuals per treatment, total experiment individuals n = 40). The antenna was used as the receptor organ, following the methodology described by Silva et al. (2021). One antenna was removed from the individual at the base of the scape with a scalpel, and then the first segment of the distal end was cut off using dissection scissors. The removed antenna was then mounted between two glass electrodes filled with a conductive saline solution of KCl (0.1 M) and 0.1% polyvinylpyrrolidone. After stimulation and data acquisition, the antenna was stored at -80°C along with the non-removed antenna used for EAG recording for later analysis. A volume of 10 µL of each stimulus was applied onto filter paper (Whatman No.1, Whatman®, Sigma-Aldrich, Darmstadt, Germany), and the stimulus was delivered to the antenna using a stimulus controller (CS-55, Syntech, Hilversum, Netherlands) with an airflow of 40 mL per s and a duration of 0.2 s, with a period of at least 30 s between each stimulation to allow for the recovery of the antennas basal signals. Each antenna was stimulated 3 times for each stimulus (volatile fraction); additionally, each antenna was stimulated with a blank (air) and a control (Hexane ≥ 99% GC, Sigma-Aldrich, Munich, Germany). Data were recorded using EAG 2014 software (Syntech, Hilversum, Netherlands) for the acquisition and analysis of the depolarizations obtained during the electroantennography. To correlate the DWV-A load with the electrophysiological response to the volatile stimuli, RNA extraction, subsequent cDNA synthesis, and viral load quantification by qPCR were performed on the antennas of bees from each treatment. RNA extraction, cDNA synthesis, and qPCR were completed according to Vargas et al. (2017). Behavioral Assays The behavioral response of A. mellifera adults to I-DWV and N-DWV treatments was evaluated using Y-tube olfactometry. Glass Y-olfactometers (21 cm long with an internal diameter of 3 cm) were designed (University of Concepción, Concepción, Chile), with glass odor chambers (15 cm long with an internal diameter of 3 cm) connected to the end of the central arm (Y-tube) to generate a filtered airflow at 280 mL min − 1 using a positive pressure air pump to deliver the stimulus. A filter paper (1 cm × 7.5 cm) was placed with 10 µL of each stimulus diluted in hexane (volatile fraction of collected pollens), then the paper strip was left to air dry for 30 s to evaporate the solvent and was subsequently placed inside one of the odor chambers, while only the solvent was left in the other odor chamber, considered as the control stimulus. Forty worker bees at 5, 10, 15, and 20 days post-inoculation (160 worker bees in total) per treatment (I-DWV or N-DWV, total n = 320) and per evaluated stimulus (n = 1280) were used. Before the test, each insect was acclimated inside the Y-tube by placing the individual in the testing area for 5 minutes, then carefully removed for 10 minutes, and the testing area was cleaned with 96% ethanol according to Arenas and Farina (2012). The bee was placed in the central arm and allowed to move in the olfactometer for 6 minutes; a choice was considered as the first movement towards any of the Y-tube arms within that time and where the insect remained for at least 30 s. The choice distribution was defined as follows: a positive choice towards the arm where the stimulus was applied, a negative choice towards the arm without the stimulus, and no choice when, at the end of the evaluation time, the insect did not choose either of the mentioned areas. A second olfactory assay was conducted, where bees from each treatment were exposed to choices between the volatile fractions of native pollen of E. cordifolia vs a no native pollen of R. ulmifolius (n = 160 bees per treatment, total n = 320). After each bee was tested, they were stored at -80°C until further analysis. RNA Extraction, cDNA synthesis and qPCR RNA extraction from each sample collected in the different bioassays; antennas and head in the olfactory reception evaluation and bees in the behavioral assays, were performed according to Kim et al. (2019), Mondet et al. (2018), and Silva et al. (2024) with minor modifications. The collected individuals and organs from each bioassay and sampling point were homogenized with 500 µL of Trizol™ (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) at maximum speed in a Retsch homogenizer. Subsequently, 5 µL of RNA Carrier was added, following the manufacturer's recommendations (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The homogenate was incubated on ice for 5 minutes, and 200 µL of chloroform was added, followed by incubation on ice for 3 minutes. The mixture was then centrifuged for 15 minutes at 10,000 g at 4°C, and the supernatant was collected for RNA extraction. RNA extraction was carried out according to the instructions provided by the E.Z.N.A. Total RNA I kit (Omega Bio-Tek, Norcross, GA, USA). RNA quality and yield were analyzed using a spectrophotometer (Infinite 200 PRO NanoQuant, Tecan Group, Männedorf, Switzerland) and visualized by agarose gel electrophoresis, and stored at -80°C. For cDNA synthesis, the high-capacity reverse transcription kit (Applied Biosystems) (Invitrogen, Life Technologies, Carlsbad, CA, USA) was used according to the manufacturer's instructions. Viral quantification was performed using the cDNA corresponding to the samples collected at each sampling point, with the β-Actin gene used as a normalization gene. qPCR was conducted using the commercial KAPA SYBR FAST Universal 2 qPCR MasterMix kit (KapaBiosystems, Wilmington, MA, USA), following the supplier’s instructions. Reactions were adjusted to a volume of 15 µL, including 20 ng of cDNA, 530 µM of each primer, and filtered sterile molecular-grade water to make up 15 µL. Thermal cycling conditions were adjusted to the requirements of each designed primer. Finally, qPCR analyses were performed using a Stratagene Mx3000P thermocycler (Agilent Technologies, Santa Clara, CA, USA) and data were analyzed using MxPro software (Stratagene, Agilent Technologies, Santa Clara, CA, USA). For viral load quantification, a standard curve was created using a purified PCR product (Wizard® VR SV Gel and PCR Clean-Up System, Promega, Madison, WI, USA). The purified amplicon was quantified using spectrophotometry (Epoch™ Microplate Spectrophotometer, BioTek, Winooski, VT, USA) and copy number calculations were performed according to Wu et al. (2017). Linear standard curves (95–100% efficiency) were generated using serial dilutions (1.0 × 10¹ to 1.0 × 10⁹) of purified cDNA viral copy numbers. Ct values were plotted against copy number values (log10). Thus, the sample copy number was estimated using Ct values and comparison with the linear equation of the standard curve and normalization values from β-actin gene calibration (Yang and Cox-Foster 2005). Finally, data were expressed as the number of DWV-A copies per bee, considering the dilutions performed during cDNA synthesis and qPCR. Data Analysis A nested analysis of variance (Nested ANOVA) was designed between treatments (I-DWV and N-DWV) and bee age (5, 10, 15, and 20 dpi) in the antennal electrical response (mV) to the volatile stimulus of the different pollens to be evaluated, followed by a Bonferroni test (p < 0.05) to separate the means at each evaluation point (5, 10, 15, and 20 dpi). A chi-square test (p < 0.05) was used to analyze the statistical difference in the responses (preference) of inoculated (I-DWV) and non-inoculated (N-DWV) honeybees at 5-, 10-, 15- and 20 days old in the olfactometric Y-tube test with different volatile fractions of pollens and R. ulmifolius vs E. cordifolia . Also, a binary (1 and 0) logistic regression was run to determine the likelihood of the preference of young bees for the volatile fraction of pollens in the Y-tube test as a function of the DWV-A load and bee age. The preference for the volatile fraction of pollens was marked as positive (yes = 1), and no preference or preference for the other compound was considered a negative response (no = 0). The data on all bees tested in the experiments (including the viral load of the head of inoculated and non-inoculated bees) were included in the logistic regression. IV RESULTS Chemical Characterization of the Volatile Fraction of Collected Pollens The chemical characterization of the volatile fraction of each species revealed differences in compound concentrations and types. The highest diversity of compounds and chemical groups was found in E. cordifolia pollen (Table 1 ), highlighting the presence of various terpenes such as menthol, camphene, 1,4-cineole, D-limonene, α-thujene, β-pinene, and α-pinene, with the latter two being the most abundant at 10.39 and 29.25 ppm, respectively. Terpenes were recorded in all pollens at concentrations of 0.9, 0.8 and 1.66 ppm of 1,4-cineole for Polyfloral pollen, R. ulmifolius and E. cordifolia , respectively. Additionally, all analyzed pollens contained benzaldehyde, an aromatic compound from stone fruit derivatives, at concentrations of 21.4, 11.7 and 1.32 ppm for Polyfloral pollen, monofloral pollen of R. ulmifolius and E. cordifolia , respectively. Table 1 Chemical composition of the volatile fraction of different curbicular pollens. RT Compound name Polyfloral R. ulmifolius E. cordifolia Concentration 1 (ppm) Concentration 1 (ppm) Concentration 1 (ppm) 6.283 Formic acid, hexyl ester - - 5.03 6.392 o-Xylene - - 3.73 6.450 1-Butanol, 3-methyl-, acetate - - 8.76 6.708 Pentanal, 2,4-dimethyl- - - 15.71 7.821 alpha.-Pinene - - 29.24 8.141 Camphene - - 2.09 8.290 Benzaldehyde 21.4 11.7 1.32 8.639 1-Butene, 4-isothiocyanato- 2.6 - - 8.744 beta.-Pinene - - 10.39 8.904 Caproic acid - - 3.27 9.165 n-Caprylaldehyde - - 2.6 9.329 alpha.-Thujene - - 0.58 9.719 Benzene, tert-butyl - - 5.3 9.825 D-Limonene - - 4.13 9.874 1,4-Cineol 0.9 0.8 1.66 10.434 p-Mentha-1,4-diene - - 11.96 11.859 Camphenol, 6- - - 0.83 12.085 Lilac aldehyde B 14.6 6.4 - 12.261 Lilac aldehyde C - 9.4 - 12.271 Lilac aldehyde A 20.7 - - 12.905 d-Menthol - - 0.97 13.293 Lilac alcohol B 7.7 2.9 - 13.490 Lilac alcohol A 2.5 4.2 - 13.771 Lilac alcohol D 6.1 0.9 - 17.458 alpha.-Ionone 0.2 - - 18.434 trans-.beta.-Ionone 0.5 - - 1 Concentration calculations were performed by comparing peaks with a standard curve, using tetralin. RT = Retention Time Olfactory Reception After evaluating peripheral perception or olfactory sensitivity in bees inoculated with the viral suspension (I-DWV) and non-inoculated (N-DWV) using electroantennography (EAG) assays, it was determined that the volatile fractions collected from all evaluated pollens were biologically active for bees response. Nested analysis of variance (Nested ANOVA) showed a significant interaction effect between deformed wing virus (DWV) infection treatment and bee age on the antennal electrical response (mV) to the volatile stimulus of polyfloral pollen (F ( 4 , 32 ) = 13.62 p = 0.0001) for R. ulmifolius pollen (F ( 4 , 32 ) = 16.566 p = 0.001) and for E. cordifolia pollen (F ( 4 , 32 ) = 25.012, p = 0.0001). It was observed that all bees inoculated with DWV-A (I-DWV) showed reduced electrophysiological responses compared to non-inoculated (N-DWV) at five days post-inoculation (dpi) (Fig. 1 ). Specifically, a 21%, 24%, 55%, and 40% reduction in EAG responses was observed in I-DWV bees compared to N-DWV bees when exposed to the polyfloral pollen aroma at 5, 10, 15, and 20 dpi, respectively. For R. ulmifolius pollen, the reduction was 16%, 55%, and 35% at 10, 15, and 20 dpi, respectively, and for E. cordifolia pollen, a reduction of 40%, 33%, and 55% was observed at 10, 15, and 20 dpi, respectively (Fig. 1 ). Olfactory Preference Subsequently, when evaluating the behavioral preferences using Y-tubes for bees inoculated with DWV-A (I-DWV) and non-inoculated bees (N-DWV), significant differences were observed in the behavioral responses of the insects at 10 days after inoculation. Inoculated bees (I-DWV) responded less frequently to each evaluated stimulus compared to non-inoculated (N-DWV). Specifically, when evaluating the behavioral preference between N-DWV and I-DWV bees exposed to the aroma of polyfloral pollen, we observed no difference between treatments at 5 dpi (X 2 = 0.072, p = 0.788). However, we observed significant differences at 10 dpi (X 2 = 8.142, p = 0.002), 15 dpi (X 2 = 9.796, p = 0.001), and 20 dpi (X 2 = 13.167, p = 0.001). For non-native species, R. ulmifolius pollen, we observed no difference between treatments at 5 dpi (X 2 = 0, p = 1). However, we observed significant differences at 10 dpi (X 2 = 16.038, p = 0.001), 15 dpi (X 2 = 13.941, p = 0.001), and 20 dpi (X 2 = 9.702, p = 0.007) and for native species, E. cordifolia pollen we observed no difference between treatments at 5 dpi (X 2 = 3.078, p = 0.079). However, we observed significant differences at 10 dpi (X 2 = 9.455, p = 0.002), 15 dpi (X 2 = 7.813, p = 0.005), and 20 dpi (X 2 = 11.028, p = 0.001). In summary, the proportion of I-DWV insects responding to the different stimuli was, on average, 30%, 37%, and 39% less than that of N-DWV bees for the volatile stimuli from polyfloral pollen, R. ulmifolius and E. cordifolia , respectively. Additionally, logistic regression analysis demonstrated the probability of preference for the different stimuli tested between DWV-A inoculated and non-inoculated bees (Fig. 3 ). For bees stimulated with the volatile fraction of polyfloral pollen, the viral load significantly influenced preference (Wald’s X² = 13.94, p = 0.0001, Odds ratio = 0.9), while bee age did not (Wald’s X² = 2.38, p = 0.12, Odds ratio = 1.03) (Fig. 3 A). Similarly, for bees stimulated with R. ulmifolius , a significant influence of viral load was observed (Wald’s X² = 30.04, p = 0.0001, Odds ratio = 0.86), whereas bee age did not show significance (Wald’s X² = 1.3, p = 0.25, Odds ratio = 1.02) (Fig. 3 B). Finally, for bees stimulated with E. cordifolia , a significant influence of viral load was reported (Wald’s X² = 29.39, p = 0.0001, Odds ratio = 0.84), while bee age did not have a significant influence (Wald’s X² = 0.23, p = 0.62, Odds ratio = 0.98) (Fig. 3 C). With the aim of determining whether bees were able to distinguish between one aromatic source and another based on the difference in the botanical origin of the pollens (non-native and native) We conducted a second behavioral bioassay, between bees inoculated and non-inoculated with DWV-A using a Y-tube olfactometer assay. In this essay, bees were exposed to the aromas of R. ulmifolius (non-native species) and E. cordifolia (native species) simultaneously to assess whether the bees showed a preference for one of the stimuli. It was observed that both inoculated (I-DWV) and non-inoculated (N-DWV) honey bees did not show a significant difference in preference towards the aroma of E. cordifolia , with preferences reported to be between 45–70% (Table 2 ). However, it was determined that at 15- and 20-days post-inoculation, significant differences existed in the behavioral response between the treatments towards the aroma of R. ulmifolius . Specifically, I-DWV bees responded in the range of 8–10% compared to N-DWV bees, where the attracted population ranged from 25–33%. Similarly, differences were observed in the proportion of insects that were not attracted to any stimulus. The proportion of bees in the I-DWV treatment that were not attracted ranged from 20–40%, while in the N-DWV treatment, it ranged from 5–15% throughout the duration of the assay. Additionally, we quantified the DWV-A viral load in bees from the N-DWV and I-DWV treatments using qPCR. For the N-DWV treatment, the observed viral load was 1 x 10⁴ copy number per bee at the beginning of the experiment and 1 x 10⁵ copy number per bee at 20 days post-viral inoculation. In contrast, for the I-DWV treatment, we detected a viral load of 1 x 10⁴ copy number per bee at the start of the experiment and 1 x 10¹⁵ copy number per bee at 20 days post-viral inoculation. V DISCUSSION The differences in chemical composition and abundance of compounds in the volatile fractions of the various collected pollens may be determined by their botanical origin, including both polyfloral and monofloral pollens (Prdun et al., 2021). Nonetheless, the presence of benzene-derived compounds such as benzaldehyde, which was found in all the pollens evaluated in this study, is a common compound in pollens from different botanical species, including both polyfloral (Starowicz et al., 2021) and monofloral sources such as Rosa spp. (Caser and Scariot, 2022), Lotus spp. (Ni et al., 2023), Taraxacum officinale , and Salix spp. (Prdun et al., 2021). It has been described that among the most prevalent chemical groups in volatile fractions of pollens are alkanes, aldehydes, acids, benzene derivatives, ketones, esters, sulfoxides, alcohols, pyrroles, furans, lactones, and terpenes (Starowicz et al., 2021). However, it has also been noted that the composition of these chemical groups can differ based on the botanical origin of the pollen (Da Silva et al., 2016; Lima et al., 2017). For instance, in Brazil, pollen was characterized by only two chemical groups: esters and alkanes (Carpes et al., 2013). Similarly, Lima et al. (2017) found no volatiles from the groups of alkanes, terpenes, disulfides, sulfoxides, benzene derivatives, acids, furans, lactones, and pyrroles in pollen samples of Mimosa caesalpiniifolia . This difference in composition, primarily due to the botanical origin of the species, could explain the abundance and richness of terpenes in the volatile fraction of E. cordifolia pollen (Table 1 ). Additionally, the presence of compounds such as lilac aldehyde and lilac alcohol in polyfloral and R. ulmifolius pollens may indicate the presence of other terpenes like linalool, from which a variety of compounds can be formed. For example, hydroxylation of monoterpenes can produce lilac aldehydes, while epoxidation of linalool produces lilac alcohols (Jerković et al., 2011). Moreover, differences in chemical compositions can also be attributed to the collection and storage methods; environmental factors such as temperature and relative humidity can initiate chemical degradation processes in pollens, altering their chemical profiles. Thus, better storage conditions would likely result in a more diverse chemical composition (Ni et al., 2023). On the other hand, studies conducted by Zhang et al. (2022) demonstrated that A. mellifera antennae are sensitive to benzaldehyde at different concentrations, ranging from 10–500 µg µL⁻¹. Although it was suggested that sensitivity was low, it remained between 0.5–2.1 mV without changes across different concentrations of the compound to which they were exposed. Similarly, the presence of lilac alcohol and benzaldehyde in the volatile fraction of Prunus sp. flowers was biologically active for honeybee in electroantennography (EAD) assays and elicited attraction in behavioral assays using Y-tubes (Su et al., 2022). Additionally, the presence of 1,4-cineole, a compound found in all the aromas characterized in this study and present in different concentrations, has biological activity in the olfactory system of A. mellifera by interacting with various olfactory system proteins in the insect (Briand et al., 2001). Studies by Liu et al. (2022) also showed that β-caryophyllene and β-pinene were biologically active and attractive in the olfactory system of A. mellifera . We can assume that the presence of these compounds in the volatile fractions of the tested pollens stimulates the antennae of honeybees and is responsible for the recorded olfactory sensitivity. In terms of sensitivity ranges, an interesting finding of this study was the recorded responses for polyfloral and monofloral native pollen from E. cordifolia , where sensitivity ranged between 0.4–0.6 mV, lower than those detected for R. ulmifolius pollen, with readings of 0.5–0.9 mV considering only non-inoculated honey bees. These changes in sensitivity may be associated with olfactory phenomena; models postulating ligand-receptor interactions suggest that complex aromas may saturate the olfactory system, which is associated with biologically active compounds in the chemical composition and high abundance. Studies by Zack et al. (2020) suggest that olfactory saturation can occur in simple aroma mixtures with as few as two biologically active compounds up to mixtures of twenty. While this olfactory saturation model was proposed in mice as a model organism, these postulates can be projected onto the olfactory system of A. mellifera due to the functional and evolutionary convergence of the olfactory nerve endings (Paoli and Galicia, 2021). This phenomenon may be responsible for the different readings recorded for each stimulus used in this assay; however, precise and specialized studies are needed to fully understand the functioning and regulatory mechanisms of the olfactory system of A. mellifera to reach a definitive conclusion. Additionally, as previously mentioned, many of the compounds found in the volatile fractions of the pollens in these studies are attractive to honey bees. However, the reductions in attraction detected for all stimuli had a significant contribution from the health condition of the bees, influenced by the viral load of DWV-A (Fig. 2 ). Previous studies have shown how the presence of this virus affects olfactory sensitivity, and some genes related to the detection of environmental chemical compounds, such as odorant-binding proteins (OBPs) (Silva et al., 2021). We previously described that adult A. mellifera antennae show reduced olfactory sensitivity to volatile fractions of essential oils, and the genes encoding OBPs 2, 5, 11, and 12 decrease in its expression when DWV-A load increases. Recently, we evaluated nurse bees with high viral loads (1 x 10 13 copy number per bee) change their behavioral preference, reducing their attraction when stimulated with benzyl alcohol, a component of the alarm pheromone emitted by larvae, which is related to OBPs 5 and 11 (Silva et al., 2024). Additionally, we demonstrated that genes involved in neuronal processes, such as presynaptic genes AmNrX-1 and postsynaptic gene AmNlG-1 , reduce their gene expression in relation to increasing viral loads (Silva et al., 2024). We assume that changes in sensitivity and behavioral responses in bees infected with DWV-A could be caused by the presence and replication of this virus on the head of nurse honeybees. Pizorno et al. (2021) determined through transcriptomic analyses of brains inoculated with DWV that downregulated genes involved biological processes encoding proteins related to cellular signaling, cell communication, and synaptic function in DWV-infected brains, suggesting that the presence and increase of viral loads could inhibit neuronal and brain physiology. Among the affected genes were G protein-coupled receptors (GPCRs) that have a broad function related to learning, memory, and behaviors associated with foraging (Schneider et al., 2006; Mustard et al., 2005; Schulz et al., 2002). Moreover, different GPCRs linked to various neuropeptides, based on functionality determined in Drosophila melanogaster M., could be involved in altering behavioral responses (Xu et al., 2010; Lee et al., 2013; Martelli et al., 2017). The hypothesis that the virus alters behavioral responses is supported by the numerous changes occurring in the insects’ brain as pathogen levels increase. Chen et al. (2021) demonstrated how increased viral load in the insects’ head affected long-term memory as a consequence of energy imbalance following disruption of the glutamate-glutamine (Glu-GLn) cycle and the decrease in the gene encoding the receptor (Ado-R) in the brains of infected bees, suggesting that DWV may have neurotoxic effects on A. mellifera . However, while the effects of brain physiology alterations have been evaluated in artificial infections with the deformed wing virus, it has been suggested that a sudden increase in viral loads in field conditions, due to stressors or attacks by pathogens such as V. destructor , can increase viral load, worsening symptoms and leading to neurodegenerative consequences, including changes in GABAergic regulation of the nervous system, compromising behavioral responses and memory processes in honey bees (Szymański et al., 2024). Finally, an interesting aspect to analyze was the differences in choices made by bees when exposed simultaneously to the aromas of R. ulmifolius and E. cordifolia . Although we previously reported that bees infected with the virus had difficulties in selecting aromas (Silva et al., 2024), we considered that much of this was due to the nature of the stimulus, as bees were exposed to a complex aroma obtained from a plant essential oil and a pure commercial compound. In this assay, both stimuli were obtained from a plants and corresponded to the aroma of pollen collected by the bees themselves. It was interesting to note that N-DWV bees did not show significant differences between one stimulus and another, whereas I-DWV bees were strongly attracted to the pollen aroma from E. cordifolia (Table 2 ). A possible explanation for this could be the phenomenon of self-medication described in bees, where they tend to select their food sources to improve their response to illness. Increases in resin collection by the colony in response to infections caused by Ascosphaera apis O. and increased populations of V. destructor have been reported (Simone-Finstrom & Spivak, 2012; Pusceddu et al., 2019). Similarly, infection caused by N. ceranae F. alters food source selection between infected and non-infected individuals, with high levels of the disease leading to a preference for honey with greater microbial activity (Gherman et al., 2014). Bees are also known to change their pollen selection to sources with higher nutritional quality, although this does not lead to an increase in foraging behavior (Ferguson et al., 2018). The possibility that bee’s behavioral responses and preferences are influenced by the immune benefits of pollen related to aroma could be determined by chemical characterization. The presence of chemical groups such as terpenes may indicate the interpretation of the aroma. For instance, terpenes derived from nectar and pollen of different species have antiviral effects on DWV (Palmer-Young et al., 2018). The terpene limonene, present in pollen from E. cordifolia , has antiviral effects against both variant A and B of the DWV (Boncristiani et al., 2021). However, to demonstrate this possibility, studies are needed to analyze the potential effect of these pollens on bee immunity and survival and to determine if this preliminary selection based on aroma and the health condition of bees leads to increased pollen consumption. The selection and discrimination of protein and food sources, such as pollen, is crucial to ensuring the survival of the hive. It has been shown that pollen differs in nutritional composition depending on its botanical origin (Di Pasquale et al. 2016). At the same time, this nutritional source will benefit the hive to a greater or lesser extent depending on its nutritional quality (Danihilk et al., 2018, Di Pasquale et al. 2016). Likewise, different botanical groups of pollen have a direct effect on the regulation of the immune system in bees (Bry´s et al., 2022, Danihilk et al., 2018, Di Pasquale et al. 2016). When observing the proportion of insects that did not participate in the final selection evaluation (Table 2 ), it was interesting to note that the proportion of insects unable to select any stimulus was higher in the population of bees with high viral loads (I-DWV), where we observed 30–40% of the insect population showing no preference for any stimulus, compared to honey bees non-inoculated with DWV-A, where only 3–10% showed no preference. This absence of choice supports the idea that the virus strongly affects bees' ability to respond behaviorally, even when they are exposed to attractive aromas. While many studies focus on neuronal changes as precursors to behavioral limitations, these studies are often centered on the deformed wing virus in general, but not specifically on master variants. A meta-analysis of transcriptomes generated by increasing viral loads of DWV-A highlighted that neuro-genomic changes in nurse bees would result in poor learning and early foraging behavior, leading to a higher incidence of infected bees mistakenly entering other colonies (Tranilleo et al., 2020). Similarly, a study reported by Kim et al. (2019) revealed structural damage caused by DWV-A, compromising all processes underlying peripheral perception. We have demonstrated here that DWV-A negatively and significantly affects honey bees (10–20 days old) in their olfactory sensitivity and perception, as well as their interpretation and selection of complex aromas derived from the volatile fractions emitted by different pollens. While we have shown what occurs from the perspective of peripheral perception and the underlying behavioral responses, we have not yet concluded the cause of these behavioral losses or how they might affect the integrity of the honey bee colonies into the hive. Furthermore, the question remains about what happens to foraging honey bees faced with complex and dynamic aromas in field conditions, where food source selection relies solely on peripheral perception. Therefore, more studies are required to answers these questions. VI. CONCLUSION In summary, the increase in DWV-A viral load negatively affects worker honeybees by reducing both their olfactory sensitivity and behavioral responses. This impact translates into a diminished ability to perceive and respond effectively to olfactory stimuli. Notably, honeybees with high viral loads exhibited a marked preference for native E. cordifolia pollen compared to other evaluated pollen types. This finding raises questions about whether such preference could represent an adaptive behavior aimed at mitigating the effects of pathogens. Further studies are required to explore this hypothesis and to determine the potential role of food selectivity as a resilience strategy against viral infections. Finally, the results indicate that heightened DWV-A infection can compromise honeybee selectivity and responses to food sources, directly affecting the functionality and integrity of the hive. References Alaux C, Ducloz F, Crauser D, Le Conte Y (2010) Diet effects on honeybee immunocompetence. Biol Lett 6:562–565. https://doi.org/10.1098/rsbl.2009.0986 Allen MF, Ball BV (1996) The incidence and world distribution of honeybee viruses. Bee World 77:141–162 Amiri E, Kryger P, Meixner MD, Strand MK, Tarpy D, Rueppell O (2018) Quantitative patterns of vertical transmission of deformed wing virus in honeybees. PLoS ONE 13(3):e0195283. https://doi.org/10.1371/journal.pone.0195283 Arenas A, Farina WM (2012) Learned olfactory cues affect pollen-foraging preferences in honeybees, Apis mellifera. Anim Behav 83(4):1023–1033 Arismendi N, Caro S, Castro MP, Vargas M, Riveros G, Venegas T (2020) Impact of mixed infections of gut parasites Lotmaria passim and Nosema ceranae on the lifespan and immune-related biomarkers in Apis mellifera . Insects, 11(7), 420 Babendreier D, Kalberer N, Romeis J, Fluri P, Bigler F (2004) Pollen consumption in honey bee larvae: a step forward in the risk assessment of transgenic plants. Apidologie 35:293–300. https://doi.org/10.1051/apido:2004016ï Basualdo, M., Cavigliasso, P., de Avila Jr, R. S., Aldea-Sánchez, P., Correa-Benítez,A., Harms, J. M., … Salvarrey, S. (2022). Status and economic value of insect-pollinated dependent crops in Latin America. Ecological Economics, 196, 107395. Benaets K, Van Geystelen A, Cardoen D, De Smet L, de Graaf DC, Schoofs L, Larmuseau MH, Brettell LE, Martin SJ, Wenseleers T (2017) Covert deformed wing virus infections have long-term deleterious effects on honeybee foraging and survival. Proc. Biol. Sci. 284 Boncristiani DL, Tauber JP, Palmer-Young EC, Cao L, Collins W et al (2021) Impacts of diverse natural products on honeybee viral loads and health. Appl Sci 11(22):10732 Briand L, Nespoulous C, Huet JC, Takahashi M, Pernollet JC (2001) Ligand binding and physico-chemical properties of ASP2, a recombinant odorant-binding protein from honey bee ( Apis mellifera L). Eur J Biochem 268:752–760. https://doi.org/10.1046/j.1432-1327.2001.01927.x Brodschneider R, Crailsheim K (2010) Nutrition and health in honeybees. Apidologie 41(3):278–294 Bryś MS, Skowronek P, Strachecka A (2021) Pollen Diet—Properties and Impact on a Bee Colony. Insects 12:798. https://doi.org/10.3390/insects12090798 Carpes ST, de Alencar SM, Cabral ISR, Oldoni TLC, Mourao GB, Haminiuk CWI, da Luz CFP, Masson ML (2013) Polyphenols and palynological origin of bee pollen of Apis mellifera L. from Brazil. Characterization of polyphenols of bee pollen. CYTA J Food 11:150–161 Caser M, Scariot V (2022) The Contribution of Volatile Organic Compounds (VOCs) Emitted by Petals and Pollen to the Scent of Garden Roses. Horticulturae 8:1049. https://doi.org/10.3390/horticulturae8111049 Chen P, Lu YH, Lin YH, Wu CP, Tang CK, Wei SC, Wu YL (2021) Deformed wing virus infection affects the neurological function of Apis mellifera by altering extracellular adenosine signaling. Insect Biochem Mol Biol 139:103674 Chen Y, Evans J, Feldlaufer M (2006) Horizontal and vertical transmission of viruses in the honey bee, Apis mellifera . J Invertebrate Pathol Soc Invertebrate Pathol 2006 Special Issue 92:152–159. https://doi.org/10.1016/j.jip.2006.03.010 Da Silva PM, Gauche C, Gonzaga LV, Costa ACO, Fett R (2016) Honey: Chemical composition, stability and authenticity. Food Chem 196:309–323 Dainat B, Ken T, Berthoud H, Neumann P (2009) The ectoparasitic mite Tropilaelaps mercedesae (Acari, Laelapidae) as a vector of honeybee viruses. Insectes Soc 56:40–43 Danihlík, J., Škrabišová, M., Lenobel, R., Šebela, M., Omar, E., Petřivalský, M.,… Brodschneider, R. (2018). Does the pollen diet influence the production and expression of antimicrobial peptides in individual honeybees? Insects, 9(3), 79. Di Pasquale, G., Salignon, M., Le Conte, Y., Belzunces, L. P., Decourtye, A., Kretzschmar,A., … Alaux, C. (2013). Influence of pollen nutrition on honey bee health: do pollen quality and diversity matter? PloS one, 8(8), e72016. Ellis JD, Munn PA (2005) The worldwide health status of honey bees. Bee World 86:88–101 Etxegarai-Legarret O, Sanchez-Famoso V (2022) The role of beekeeping in the generation of goods and services: The interrelation between environmental, socio-economic, and socio-cultural utilities. Agriculture 12:551 Evan C, Palmer-Young CÖ, Tozkar RS, Schwarz Y, Chen RE, Irwin LS, Adler JD, Evans (2017) Nectar and pollen phytochemicals stimulate honeybee (Hymenoptera: Apidae) immunity to viral infection. Journal of Economic Entomology Volume 110 Issue 5 October Pages 1959–1972 Ferguson JA, Northfield TD, Lach L (2018) Honey bee (Apis mellifera) pollen foraging reflects benefits dependent on individual infection status. Microb Ecol 76:482–491 Forsgren E, de Miranda JR, Isaksson M, Wei S, Fries I (2009) Deformed wing virus associated with Tropilaelaps mercedesae infesting European honey bees ( Apis mellifera ). Exp Appl Acarol 47:87–97 Genersch E, Aubert M (2010) Emerging and re-emerging viruses of the honey bee ( Apis mellifera L). Vet Res 41(6):54. 10.1051/vetres/2010027 Genersch E, Yue C, Fries I, de Miranda JR (2006) Detection of deformed wing virus, a honey bee viral pathogen, in bumble bees ( Bombus terrestris and Bombus pascuorum ) with wing deformities. J Invertebr Pathol 91:61–63 Gherman BI, Denner A, Bobiş O, Dezmirean DS et al (2014) Pathogen-associated self-medication behavior in the honeybee Apis mellifera. Behav Ecol Sociobiol 68:1777–1784 Goodrich BK (2019) Do more bees imply higher fees? Honeybee colony strength as a determinant of almond pollination fees. Food Policy 83:150–160 Groot AT, Dekker T, Heckel D (2016) The Genetic Basis of Pheromone Evolution in Moths. Annu Rev Entomol 61(1):99–117. 10.1146/annurev-ento-010715-023638 Grozinger CM, Flenniken ML (2019) Bee Viruses: Ecology, Pathogenicity, and Impacts. Annu Rev Entomol 64:205–226 Gusachenko ON, Woodford L, Balbirnie-Cumming K, Ryabov EV, Evans DJ (2020) Evidence for and against deformed wing virus spillover from honey bees to bumble bees: A reverse genetic analysis. Sci Rep 10:16847 Jerkovi´c I, Marijanovi´c Z, Malenica M (2011) Screening of Natural Organic Volatiles from Prunus mahaleb L. Honey: Coumarin and Vomifoliol as Nonspecific Biomarkers. Molecules 16:2507–2518 Kevill JL, Highfield A, Mordecai GJ, Martin SJ, Schroeder DC (2017) ABC Assay: Method development and application to quantify the role of three DWV master variants in overwinter colony losses of European honey bees. Viruses 9:314 Kim SH, Mercer A, Mitchell A, de Miranda JR, Ward V, Mondet F, Bostina M (2019) Viral infections alter antennal epithelium ultrastructure in honey bees. J Invertebr Pathol 168:107252 Kim SH, Mercer A, Mitchell A, de Miranda JR, Ward V, Mondet F, Bostina M (2019) Viral infections alter antennal epithelium ultrastructure in honey bees. J Invertebr Pathol 168:107252. 10.1016/j.jip.2019.107252 Klein AM, Vaissière BE, Cane JH, Steffan-Dewenter I, Cunningham SA, Kremen C, Tscharntke T (2007) Importance of pollinators in changing landscapes for world crops. Proc R Soc B Biol Sci 274:303–313 Lee G, Kikuno K, Bahn JH, Kim KM, Park JH (2013) Dopamine D2 receptor as a cellular component controlling nocturnal hyperactivities in Drosophila melanogaster. Chronobiol Int 30(4):443–459 Li Q, Liberles SD (2015) Aversion and attraction through olfaction. Curr Biol 25(3):120–129. https://doi.org/10.1016/j.cub.2014.11.044 Lima Neto JS, Lopes JAD, Moita Neto JM et al (2017) Volatile compounds and palynological analysis from pollen pots of stingless bees from the mid-north region of Brazil. Braz J Pharm Sci 53, e14093 Liu J, Zhang J, Shen J, Zhao H, Ma W, Jiang Y (2022) Differences in EAG Response and Behavioral Choices between Honey Bee and Bumble Bee to Tomato Flower Volatiles. Insects 13(11):987. https://doi.org/10.3390/insects13110987 Martelli C, Pech U, Kobbenbring S, Pauls D, Bahl B, Sommer MV et al (2017) SIFamide translates hunger signals into appetitive and feeding behavior in Drosophila. Cell Rep 20(2):464–478 Menzel R (2012) The honeybee as a model for understanding the basis of cognition. Nat Rev Neurosci 13(11):758–768 pmid:23080415 Mondet F, Rau A, Klopp C, Rohmer M, Severac D, Le Conte Y, Alaux C (2018) Transcriptome profiling of the honeybee parasite Varroa destructor provides new biological insights into the mite adult life cycle. BMC Genomics 19:1–19 Mordecai GJ, Wilfert L, Martin SJ, Jones IM, Schroeder DC (2016) Diversity in a honeybee pathogen: First report of a third master variant of the deformed Wing Virus quasispecies. ISME J 10:1264–1273 Mordecai GJ, Wilfert L, Martin SJ, Jones IM, Schroeder DC (2016) Diversity in a honeybee pathogen: First report of a third master variant of the deformed Wing Virus quasispecies. ISME J 10:1264–1273 Mustard JA, Kurshan PT, Hamilton IS, Blenau W, Mercer AR (2005) Developmental expression of a tyramine receptor gene in the brain of the honey bee, Apis mellifera. J Comp Neurol 483(1):66–75 Ni JB, Bi YX, Vidyarthi SK, Xiao HW et al (2023) Non-thermal electrohydrodynamic (EHD) drying improved the volatile organic compounds of lotus bee pollen via HS-GC-IMS and HS-SPME-GC-MS. Lwt 176:114480 Paoli M, Galizia GC (2021) Olfactory coding in honeybees. Cell Tissue Res 383:35–58. https://doi.org/10.1007/s00441-020-03385-5 Pizzorno MC, Field K, Kobokovich AL, Martin PL, Gupta RA, Mammone R et al (2021) Transcriptomic responses of the honey bee brain to infection with deformed wing virus. Viruses 13(2):287 Potts SG, Biesmeier JC, Kremen C, Neumann P, Schweiger O, Kunin WE (2010) Global pollinator declines: trends, impacts and drivers. Trend Ecol Evol 25:345–353 Prdun S, Sveˇcnjak L, Valenti´c M, Marijanovi´c Z, Jerkovi´c I (2021) Characterization of Bee Pollen: Physico-Chemical Properties, Headspace Composition and FTIR Spectral Profiles. Foods 10:2103. https://doi.org/10.3390/foods10092103 Pusceddu M, Piluzza G, Theodorou P, Buffa F, Ruiu L, Bullitta S et al (2019) Resin foraging dynamics in Varroa destructor-infested hives: a case of medication of kin? Insect Sci 26(2):297–310 Ramirez W, Thomas N, Muktar I, Riabinina O (2023) The neuroecology of olfaction in bees. Curr Opin Insect Sci 56:101018 Riveros G, Arismendi N, Zapata N et al (2020) Occurrence, prevalence and viral load of deformed wing virus variants in Apis mellifera colonies in Chile. J Apicult Res 59:63–68 Sanpa S, Chantawannakul P (2009) Survey of six bee viruses using RT-PCR in Northern Thailand. J Invertebr Pathol 100:116–119 Scheiner R, Bauman A, Blenau W (2006) Aminergic control and modulation of honeybee behaviour. Curr Neuropharmacol 4(4):259–276 Schulz DJ, Sullivan JP, Robinson GE (2002) Juvenile hormone and octopamine in the regulation of division of labor in honey bee colonies. Horm Behav 42(2):222–231 Shah KS, Evans EC, Pizzorno MC (2009) Localization of deformed wing virus (DWV) in the brains of the honeybee, Apis mellifera Linnaeus. Virol J 6:182 Silva D, Arismendi N, Alveal JP, Ceballos R, Zapata N, Vargas M (2024) Nursing Honeybee Behavior and Sensorial-Related Genes Are Altered by Deformed Wing Virus Variant A. Insects 15(2):80 Silva D, Ceballos R, Arismendi N, Dalmon A, Vargas M (2021) Variant A of the deformed wings virus alters the olfactory sensitivity and the expression of odorant binding proteins on antennas of Apis mellifera. Insects 12(10):895 Simone-Finstrom MD, Spivak M (2012) Increased resin collection after parasite challenge: a case of self-medication in honey bees? PLoS ONE, 7(3), e34601 Skubnik K, Novacek J, Fuzik T, Pridal A, Paxton RJ, Plevka P (2017) Structure of deformed wing virus, a major honey bee pathogen. Proc. Natl. Acad. Sci. USA. 114, 3210–3215 Starowicz M, Hanus P, Lamparski G, Sawicki T (2021) Characterizing the Volatile and Sensory Profiles, and Sugar Content of Beeswax, Beebread, Bee Pollen, and Honey. Molecules 26:3410. https://doi.org/10.3390/molecules26113410 Su W, Ma W, Zhang Q, Hu X, Ding G, Jiang Y, Huang J (2022) Honey Bee Foraging Decisions Influenced by Pear Volatiles. Agriculture 12:1074. https://doi.org/10.3390/agriculture12081074 Szymański S, Baracchi D, Dingle L, Bowman L, Manfredini F (2024) Learning performance and GABAergic pathway link to deformed wing virus in the mushroom bodies of naturally infected honey bees. J Exp Biol 227(13):jeb246766. https://doi.org/10.1242/jeb.246766 Traniello IM, Bukhari SA, Kevill J et al (2020) Meta-analysis of honey bee neurogenomic response links Deformed wing virus type A to precocious behavioral maturation. Sci Rep 10:3101. https://doi.org/10.1038/s41598-020-59808-4 UNEP Emerging Issues: Global Honeybee Colony Disorder and Other Threats to Insect Pollinators. United Nations Environment Programme (2010) Available online: https://www.unep.org/resources/report/unep-emerging-issues-global-honey-bee-colony-disorder-and-other-threats-insect (accessed on 22-08-2024) Vargas M, Arismendi N, Riveros G, Zapata N, Bruna A, Vidal M, Rodríguez M, Gerding M (2017) Viral and intestinal diseases detected in Apis mellifera in Central and Southern Chile. Chil J Agric Res 77:243–249 Williams GR, Rogers REL, Kalkstein AL, Taylor BA, Shutler D, Ostiguy N (2009) Deformed wing virus in western honey bees ( Apis mellifera ) from Atlantic Canada and the first description of an overtly-infected emerging queen. J Invertebr Pathol 101:77–79 Winfree R, Gross BJ, Kremen C (2011) Valuing pollination services to agriculture. Ecol Econ 71:80–88 Wright GA, Skinner BD, Smith BH (2002) Ability of honeybee, Apis mellifera , to detect and discriminate odors of varieties of canola ( Brassica rapa and brassica napus ) and snapdragon flowers ( Antirrhinum majus ). J Chem Ecol 28(4):721–740 pmid:12035922 Wu Y, Dong X, Kadowaki T (2017) Characterization of the copy number and variants of deformed wing virus (DWV) in the pairs of honey bee pupa and infesting Varroa destructor or Tropilaelaps mercedesae . Front Microbiol 8:1558 Xu J, Li M, Shen P (2010) A G-protein-coupled neuropeptide Y-like receptor suppresses behavioral and sensory response to multiple stressful stimuli in Drosophila. J Neurosci 30(7):2504–2512 Yang W, Kuang H, Wang S, Wang J, Liu W, Wu Z, Tian Y, Huang ZY, Miao X (2013) Comparative sucrose responsiveness in Apis mellifera and A. cerana Foragers. PLoS ONE 8. https://doi.org/10.1371/journal.pone.0079026 Yang X, Cox-Foster D (2005) Impact of an ectoparasite on the immunity and pathology of an invertebrate: Evidence for host immunosuppression and viral amplification. Proc Natl Acad Sci USA 102:7470–7475 Yue C, Genersch E (2005) RT-PCR analysis of Deformed wing virus in honeybees ( Apis mellifera ) and mites ( Varroa destructor ). J Gen Virol 86:3419–3424 Zak JD, Reddy G, Vergassola M et al (202) Antagonistic odor interactions in olfactory sensory neurons are widespread in freely breathing mice. Nat Commun 11, 3350. https://doi.org/10.1038/s41467-020-17124-5 Zhang J, Liu J, Gao F, Chen M, Jiang Y, Zhao H, Ma W (2022) Electrophysiological and Behavioral Responses of Apis mellifera and Bombus terrestris to Melon Flower Volatiles. Insects 13:973. https://doi.org/10.3390/insects13110973 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5582583","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":386385975,"identity":"57ed3535-ce02-45e5-b796-040ee0cba9e0","order_by":0,"name":"Diego Silva","email":"","orcid":"https://orcid.org/0009-0005-7265-0808","institution":"Universidad de Concepción","correspondingAuthor":false,"prefix":"","firstName":"Diego","middleName":"","lastName":"Silva","suffix":""},{"id":386387815,"identity":"fd89e9a8-f25a-4b6e-9868-def858f87220","order_by":1,"name":"Felipe Becerra","email":"","orcid":"","institution":"Universidad de Concepción","correspondingAuthor":false,"prefix":"","firstName":"Felipe","middleName":"","lastName":"Becerra","suffix":""},{"id":386387816,"identity":"e8fbf3f6-0f78-4986-b796-e0699dde8d80","order_by":2,"name":"Sebastian Salazar","email":"","orcid":"","institution":"Universidad de Concepción","correspondingAuthor":false,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Salazar","suffix":""},{"id":386387817,"identity":"ec5d7381-ccce-42b2-9346-a4e0cc43f5b5","order_by":3,"name":"Nolberto Arismendi","email":"","orcid":"","institution":"Universidad Santo Tomas","correspondingAuthor":false,"prefix":"","firstName":"Nolberto","middleName":"","lastName":"Arismendi","suffix":""},{"id":386387818,"identity":"d383117c-0296-4e1a-ba81-946385067435","order_by":4,"name":"Juan Alveal","email":"","orcid":"","institution":"Instituto de Investigaciones Agropecuarias","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Alveal","suffix":""},{"id":386387819,"identity":"2d06c24e-12e9-464f-a3bc-1447141381f0","order_by":5,"name":"Ricardo Ceballos","email":"","orcid":"","institution":"Instituto de Investigaciones Agropecuarias","correspondingAuthor":false,"prefix":"","firstName":"Ricardo","middleName":"","lastName":"Ceballos","suffix":""},{"id":386387820,"identity":"41685278-5359-484a-bbc8-70f53d049fc6","order_by":6,"name":"Nelson Zapata","email":"","orcid":"","institution":"Universidad de Concepción","correspondingAuthor":false,"prefix":"","firstName":"Nelson","middleName":"","lastName":"Zapata","suffix":""},{"id":386387821,"identity":"dc9c38e9-16ac-4dd6-9eef-3c60a196e0d5","order_by":7,"name":"Marisol Vargas","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYJCCAwwMFlBmBQNjA4h+QFiLBJR5BqolgbBFUC2MbURo0W0/+/DgjwqJxP7Zxy9+/DnvsGw/+wHGD/i0mJ1JNzjMc0Yicca5nGJp3m2HjWf2JDBL4NVyII3hMGObhDHDGZ4EacZthxM33GBgw+sws/PPGA7+BGqRP8OT/PPnnMOJ+wlquZHGcIC3TULO4Az7MQneBqAtEgS1PGMA+UXO8AwPmzXPsXTjGWcSm/H75Xwa88cfFTY8cmfYH9/8UWMt299++OCHD3i0IAEeAygDEjXEAPYHxKocBaNgFIyCEQYAi09RrJC4QHwAAAAASUVORK5CYII=","orcid":"","institution":"Universidad de Concepción","correspondingAuthor":true,"prefix":"","firstName":"Marisol","middleName":"","lastName":"Vargas","suffix":""}],"badges":[],"createdAt":"2024-12-04 22:50:24","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-5582583/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5582583/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70736241,"identity":"b76338f9-be86-4b80-87ba-989c06e2c61f","added_by":"auto","created_at":"2024-12-06 06:44:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":85491,"visible":true,"origin":"","legend":"\u003cp\u003eElectrophysiological response of adult \u003cem\u003eApis mellifera\u003c/em\u003e to different volatile fractions of pollen from various species. Asterisks above indicate significant differences according to the Bonferroni test (p\u0026lt;0.05) between the N-DWV and I-DWV treatments at each sampling point. Bars above the means at each sampling point indicate the standard error.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5582583/v1/0b47ffdc406ec7e1424c0ccf.png"},{"id":70736473,"identity":"84c8c54d-8487-4bbe-8fda-c828f7fffa6a","added_by":"auto","created_at":"2024-12-06 06:52:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":77897,"visible":true,"origin":"","legend":"\u003cp\u003eProportion of insects, inoculated (I-DWV) and non-inoculated (N-DWV) with a viral suspension of Deformed Wing Virus variant A (DWV-A), responding behaviorally to the volatile fraction of different collected pollens. P \u0026lt; 0.05, according to the Chi-Square analysis on the X-axis indicates significant differences between treatments for bees on the same day post viral inoculation. Bars above the means indicate 95% confidence intervals.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5582583/v1/285c25f967d7ca86f3cacb05.png"},{"id":70736243,"identity":"f201c932-0b65-4232-b2d0-adc9cdcb00c2","added_by":"auto","created_at":"2024-12-06 06:44:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":878133,"visible":true,"origin":"","legend":"\u003cp\u003eThree-dimensional logistic regression curve representing the probability of bee preference for different volatile pollen stimuli: polyfloral (A), \u003cem\u003eR. ulmifolius\u003c/em\u003e (B), and \u003cem\u003eE. cordifolia\u003c/em\u003e(C) based on DWV-A load and bee age in Y-tube assays. When bees were exposed to the multifloral pollen aroma, responding bees were marked as positive (yes = 1, n = 180), while those showing no preference were marked as negative (no = 0, n = 140). When exposed to the \u003cem\u003eR. ulmifolius\u003c/em\u003epollen aroma, responding bees were marked as positive (yes = 1, n = 162), and non-responding bees were marked as negative (no = 0, n = 158). When exposed to the E. cordifolia pollen aroma, responding bees were marked as positive (yes = 1, n = 230), and non-responding bees were marked as negative (no = 0, n = 90).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5582583/v1/66b42e99191ea7974f9a451d.png"},{"id":70737866,"identity":"6c20e6b7-2c8f-4c5b-bc9c-b5864059bc7e","added_by":"auto","created_at":"2024-12-06 07:08:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1637449,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5582583/v1/60f9db2d-89f9-4a0e-813c-c313abf3c42d.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eDeformed Wing Virus-Induced Changes in Honey bee Reception and Preference for Pollen Scents\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"I INTRODUCTION","content":"\u003cp\u003e Pollination is crucial for ecosystems and the food industry, a behavior primarily carried out by honey bees (\u003cem\u003eApis mellifera\u003c/em\u003e L.) (Potts et al., 2016). This species of pollinating insect is one of the most valuable animal species for large-scale food production systems, such as in canola and sunflower crops (Goodrich et al., 2018). It is predominantly used for pollinating monocultures over large areas, a global trend that has positioned \u003cem\u003eA. mellifera\u003c/em\u003e as an economically significant organism for food production (Winfree et al., 2011; Goodrich et al., 2018). According to the Food and Agriculture Organization of the United Nations (FAO), out of the 100 crop species produced for human consumption, 71 are pollinated in agricultural systems by honeybees, thus supporting 90% of global food production (Klein et al., 2007). In Europe alone, out of the 264 crop species produced, 84% are exclusively pollinated by \u003cem\u003eA. mellifera\u003c/em\u003e (UNEP, 2010). Similarly, 228\u0026nbsp;million tons of food produced in Latin America can be attributed directly to insect pollination, which represent US\u003cspan\u003e$\u003c/span\u003e 77.82\u0026nbsp;billion (Basualdo et al., 2022). Thus, insect pollinators like honey bees provides direct and indirect benefits for human being by promoting biodiversity, food production and sociocultural utilities by goods and services (Etxegarai-Legarreta and Sanchez-Famoso, 2022).\u003c/p\u003e \u003cp\u003eFor bees, nectar is a source of energy, while pollen provides proteins, lipids, minerals, and vitamins (Yang et al., 2013). However, a diet lacking in nutritional content can reduce an ability of the bees to resist diseases (Alaux et al., 2010; Yang et al., 2013). Nurse honey bees are responsible for maintaining the nutritional strength of the colonies, as they are the ones who convert food into royal jelly, thus influencing the size of the colony, brood rearing, and overall strength, among other factors (Brodschneider \u0026amp; Crailsheim, 2010). The pollen consumption of each worker bee varies depending on their age and the tasks they perform within the colony. Honey bee larvae do not consume much pollen, while bees ingest large amounts (Babendreier et al., 2004). Older workers, often known as foragers, are unable to consume or digest pollen (Babendreier et al., 2004). Additionally, \u003cem\u003eA. mellifera\u003c/em\u003e has been described as a polylectic pollinator, meaning that this insect does not have a specific, simple interaction with a single plant (Paoli and Galizia, 2021). Instead, it visits and recognizes multiple plant species solely to collect food sources like pollen and nectar (Paoli and Galizia, 2021). This action is performed with precise coordination of both memory processes and visual as well as olfactory recognition (Wright et al., 2002; Menzel et al., 2012). To recognize them, bees first need to detect the signals released by plants, which are sensed by their antennae, an organ primarily responsible for the sense of smell (Groot et al., 2016; Li et al., 2015; Paoli and Galizia, 2021).\u003c/p\u003e \u003cp\u003eIt is widely agreed that olfactory functioning and information processing are largely conserved among insects (Paoli and Galizia, 2021). This process begins in the antenna, where environmental compounds are intercepted and converted into an electrical signal, which is then sent to processing centers in the insect's brain (Paoli and Galizia, 2021, Ramirez et al., 2023). There, the information is received, interpreted, and analyzed through different memory mechanisms (Paoli and Galizia, 2021, Ramirez et al., 2023). This flow of information from the antenna to the brain is essential for the development of the insect\u0026rsquo;s complex behavioral tasks, whether within the hive or during foraging activities (Paoli and Galizia, 2021). However, the integrity of this insect\u0026rsquo;s olfactory system can be threatened by species-specific pathogens, such as the deformed wing virus (DWV). This pathogen belongs to the Iflaviridae family and is classified within Baltimore group IV. It has a positive-strand RNA genome organization like that of picornaviruses (Genersch and Aubert, 2010). With \u003cem\u003eA. mellifera\u003c/em\u003e as its primary host, this virus has achieved global distribution, making it one of the most important pathogens on honey bees (Yue and Genersch, 2005; Sanpa, 2009; Williams et al., 2009; Mordecai et al., 2016). However, it has been shown that this pathogen can infect different species related to \u003cem\u003eA. mellifera\u003c/em\u003e, including various bee species such as the Asian honey bee (\u003cem\u003eA. cerana\u003c/em\u003e F.) and the dwarf honey bee (\u003cem\u003eA. florea\u003c/em\u003e F.) (Allen \u0026amp; Ball, 1996; Ellis \u0026amp; Munn, 2005; Genersch \u0026amp; Aubert, 2010), as well as \u003cem\u003eBombus terrestris\u003c/em\u003e L. and \u003cem\u003eB. pascuorum\u003c/em\u003e S., where symptoms of the disease have also been observed (Genersch et al., 2006). In fact, among the main symptoms of this pathogen are wing deformities, abdominal swelling, paralysis, and behavioral alterations. Furthermore, this pathogen can persist in bee colonies without presenting clinical symptoms, making early detection difficult (Benaet et al., 2017; Grozinger et al., 2019). It has been reported that this pathogen can replicate in different regions of the bee's brain, potentially compromising the normal functioning of the insect's neural network, with consequences for olfactory sensitivity and associated behaviors (Pizzorno et al., 2021). In general, DWV has been reported to be replicated in brain regions, including the neuropils responsible for vision and olfaction, which could interfere with various behavioral responses (Sha et al., 2009).\u003c/p\u003e \u003cp\u003eThe successful propagation of DWV has been attributed to its ability to inhabit different hosts, many of which can act as vectors, with the primary vector being the mite \u003cem\u003eVarroa destructor\u003c/em\u003eA., which feeds on the fat bodies of the honey bees, facilitating virus transmission (Dainat et al., 2009; Forsgren et al., 2009). Other less efficient, unassisted transmission mechanisms are also known, such as trophallaxis, cannibalism, or vertical transovarial transfer (Amiri et al., 2018; Chen et al., 2006).\u003c/p\u003e \u003cp\u003eTo date, three epidemiologically significant variants of DWV (A, B, and C) have been identified; variant A is particularly associated with winter colony losses (Mordecai et al., 2016; Kevil et al., 2017). In Chile, specifically, the presence of variants A and B has been determined, with variant A being the most prevalent in the country's apiaries (Riveros et al., 2020). It has been specifically demonstrated that DWV-A can replicate in the epithelial cells of the antennae and that increased viral loads in the insect damage the cellular ultrastructure (Kim et al., 2019). This pathogen can affect olfactory sensitivity and reduce the gene expression of OBP-coding genes (Silva et al., 2021). It has been described that the increase in viral loads over time leads to the decrease of at least four out of the nine antenna-specific OBPs, notably the nurse bee activity regulators OBP5 and OBP11 (Silva et al., 2021). Additionally, recent studies have shown that high viral loads of DWV-A in both the head and antenna alter the behavioral response of nurse bees, and it has been determined that increased viral loads limit the insect's behavioral response (Silva et al., 2024). High viral loads of DWV-A in the head and antenna also affect the expression of genes coding for pre- and postsynaptic proteins, causing a reduction in the insects' behavioral responses (Silva et al., 2024). Also, it has been demonstrated that DWV-A can affect the physiology of the olfactory system of \u003cem\u003eA. mellifera\u003c/em\u003e as well as its behavioral responses (Kim et al., 2019, Silva et al., 2021, Silva et al., 2024). However, how this pathogen impacts the physiological and behavioral consequences in relation to aromas associated with pollen from different plant species remain unknown. Therefore, the aim of this study was to evaluate the effects generated by the increase in viral loads in \u003cem\u003eA. mellifera\u003c/em\u003e infected with the variant A of the deformed wing virus on olfactory reception and sensitivity in bees exposed to different aromas obtained from pollens of various species.\u003c/p\u003e"},{"header":"III MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of the viral inoculum\u003c/h2\u003e \u003cp\u003eThe viral inoculum used for all bioassays was prepared as described by Gusachenko et al. (2020) and Silva et al. (2024). The inoculum was obtained from colonies infected with the deformed wing virus (DWV), after confirming the presence of the Variant-A. A group of 20 symptomatic bees was homogenized in phosphate-buffered saline (1X PBS) for 90 seconds at high-speed using a Stomacher 80 laboratory blender (Seward, London, UK). The homogenate was then centrifuged twice: first at 1500\u0026times; g for 10 minutes, followed by 10,000\u0026times; g for 10 minutes, both at 4\u0026deg;C. The supernatant was purified by filtration through a 0.22 \u0026micro;M filter (PES, Merck Millipore, Darmstadt, Germany) and treated with RNase to destroy all unencapsidated RNA, as indicated by Skubnik et al. (2017). To determine the exact concentration of viral particles present in the inoculum, a 200 \u0026micro;L aliquot of the supernatant was collected for RNA extraction, cDNA synthesis, and subsequent quantification of the viral load via qPCR, following the methodology detailed in the RNA Extraction section. The remaining extracted inoculum was stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further use.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eViral Inoculation\u003c/h3\u003e\n\u003cp\u003eThe adult \u003cem\u003eA. mellifera\u003c/em\u003e used for all assays were obtained from the experimental apiary of the experimental center \"El Nogal\" (36\u0026deg;35\u0026prime;58.25\u0026Prime; S\u0026ndash;72\u0026deg;04\u0026prime;51.77\u0026Prime; W), University of Concepci\u0026oacute;n, Chill\u0026aacute;n, Chile. To standardize the age of the bees used in the various bioassays, brood frames were collected directly from hives and kept under control condition at 30\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1; 60% \u0026plusmn; 3 RH. Previously, bee colonies in the apiary were selected based on the levels of pathogens present, with the criterion that the donor hive of brood frames were free from other pathogens such as \u003cem\u003eNosema ceranae\u003c/em\u003e F. and \u003cem\u003eLotmaria passim\u003c/em\u003e S. Priority was also given to hives without or with low viral loads of acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV), and DWV. Newly emerged bees were then carefully collected and randomly confined in plastic cages (base\u0026thinsp;=\u0026thinsp;8 cm diameter, top\u0026thinsp;=\u0026thinsp;10 cm diameter, height\u0026thinsp;=\u0026thinsp;15 cm). The experimental procedure was conducted similarly to what was previously published by Silva et al. (2021). In summary, newly emerged bees from the brood frames were inoculated with the viral suspension orally (Treatment I-DWV). A total of 5 \u0026micro;L of the viral suspension (1.0 \u0026times; 10⁹ copy number per bee) in a 60% sucrose solution was used. All bees that did not consume viral suspension were excluded from the experiment. Non-inoculated bees with the viral suspension were used as the control group (Treatment N-DWV). In total, 1,700 worker bees were used for the subsequent bioassays. The bees were kept in 34 plastic cages, with 50 bees per cage (everyone was considered an independent replicate for future experiments), and the distribution was as follows: bees inoculated with the viral suspension, n\u0026thinsp;=\u0026thinsp;850, distributed in 17 plastic cages with 50 bees per cage; non-inoculated bees, n\u0026thinsp;=\u0026thinsp;850, distributed in 17 plastic cages with 50 bees per cage. Each plastic cage was provided with a supplement of 3 g of a pollen substitute (the commercial preparation included soybean, brewer\u0026rsquo;s yeast, corn starch, wheat flour, corbicular pollen, canola oil, and canola oil) and 60% sucrose syrup ad libitum, following the recommendations of Arismendi et al. (2020).\u003c/p\u003e\n\u003ch3\u003ePollen collection, Chemical Characterization and Preparation of Stimuli\u003c/h3\u003e\n\u003cp\u003eFor the preparation of the stimuli used in the various bioassays, four samples of corbicular pollen were collected from different experimental apiaries and locations: Pollen (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) from the University of Concepci\u0026oacute;n, Chill\u0026aacute;n (36\u0026deg;35\u0026prime;58.25\u0026Prime; S\u0026ndash;72\u0026deg;04\u0026prime;51.77\u0026Prime; W); Commercial pollen (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) from the Coihueco sector, Chill\u0026aacute;n (36\u0026deg;37\u0026prime;00\u0026Prime;S-71\u0026deg;50\u0026prime;00\u0026Prime;W) and Pollen (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) from the R\u0026iacute;o bueno sector, Los R\u0026iacute;os Region, Valdivia, Chile (40\u0026deg;16\u0026prime;40\u0026Prime;S-72\u0026deg;33\u0026prime;47\u0026Prime;W). The collections were carried out using pollen traps that were installed at the entrance of the bee hives, then the pollen samples were stored cold (-20) to avoid degradation of volatile compounds due to temperature. A sample (10 g) of each collected pollen was immediately sent for paleobotanical identification to Laboratory of Entomology, Universidad Austral de Chile. The paleobotanical identification was based on the Chilean pollen classification standard (NCh3255:2011). The samples were then categorized as follows: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Polyfloral pollen including pollen from \u003cem\u003eBrassica campestris\u003c/em\u003e (32.77%), \u003cem\u003eTrifolium pratense\u003c/em\u003e (30.40%), \u003cem\u003eRubus ulmifolius\u003c/em\u003e (14.18%), \u003cem\u003eLotus uliginosus\u003c/em\u003e (10.13%) and other plant species (12.52%); (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) monofloral pollen from \u003cem\u003eRubus ulmifolius\u003c/em\u003e (71%); and (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) monofloral native pollen from \u003cem\u003eEucryphia cordifolia\u003c/em\u003e (75%).\u003c/p\u003e \u003cp\u003eThe volatile fraction of the pollen samples was collected using the dynamic HeadSpace method. Fifty grams of pollen were encapsulated in a 450 mL Pyrex glass bell jar. The volatile fraction was collected using a 200 mg Porapak Q trap (80\u0026ndash;100 mesh), previously cleaned with 1 mL of anhydrous ether and conditioned for 2 hours at 220\u0026deg;C under a constant flow of 60 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of nitrogen. The collection was performed over 24 hours using a positive/negative air pressure system, with an inlet flow of 0.7 L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and an outlet flow of 0.5 L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The air entering the system was purified by an activated carbon filter. Volatiles were extracted from the Porapak Q trap by eluting with 1 mL of chromatographic-grade hexane. For relative quantification, 10 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of Tretalin was used as an internal standard. Characterization and relative quantification were performed using gas chromatography coupled with mass spectrometry (GC-MS) (GC-MS QP2010 Plus; Shimadzu), equipped with a capillary column (Rtx-5) 30 m x 0.25 mm ID x 0.25 \u0026micro;m; Restek Corporation). Helium was used as the carrier gas at a flow rate of 1.4 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The oven was programmed to start at 40\u0026deg;C, held for 1 minute, with a temperature ramp of 8\u0026deg;C per minute until reaching 205\u0026deg;C, which was maintained for 3 minutes. The eluates were then stored in amber vials and kept at -40\u0026deg;C until use.\u003c/p\u003e\n\u003ch3\u003eOlfactory Reception Evaluation\u003c/h3\u003e\n\u003cp\u003eThe technique of electroantennography (EAG) was used to determine the olfactory reception of \u003cem\u003eA. mellifera\u003c/em\u003e adults inoculated and non-inoculate with the DWV-A, using the volatile fraction of the collected pollens as stimuli. The olfactory sensitivity evaluation was conducted at 5, 10, 15, and 20 days post viral inoculation, with five bees per treatment for each sampling point (n\u0026thinsp;=\u0026thinsp;20 individuals per treatment, total experiment individuals n\u0026thinsp;=\u0026thinsp;40). The antenna was used as the receptor organ, following the methodology described by Silva et al. (2021). One antenna was removed from the individual at the base of the scape with a scalpel, and then the first segment of the distal end was cut off using dissection scissors. The removed antenna was then mounted between two glass electrodes filled with a conductive saline solution of KCl (0.1 M) and 0.1% polyvinylpyrrolidone. After stimulation and data acquisition, the antenna was stored at -80\u0026deg;C along with the non-removed antenna used for EAG recording for later analysis. A volume of 10 \u0026micro;L of each stimulus was applied onto filter paper (Whatman No.1, Whatman\u0026reg;, Sigma-Aldrich, Darmstadt, Germany), and the stimulus was delivered to the antenna using a stimulus controller (CS-55, Syntech, Hilversum, Netherlands) with an airflow of 40 mL per s and a duration of 0.2 s, with a period of at least 30 s between each stimulation to allow for the recovery of the antennas basal signals. Each antenna was stimulated 3 times for each stimulus (volatile fraction); additionally, each antenna was stimulated with a blank (air) and a control (Hexane\u0026thinsp;\u0026ge;\u0026thinsp;99% GC, Sigma-Aldrich, Munich, Germany). Data were recorded using EAG 2014 software (Syntech, Hilversum, Netherlands) for the acquisition and analysis of the depolarizations obtained during the electroantennography. To correlate the DWV-A load with the electrophysiological response to the volatile stimuli, RNA extraction, subsequent cDNA synthesis, and viral load quantification by qPCR were performed on the antennas of bees from each treatment. RNA extraction, cDNA synthesis, and qPCR were completed according to Vargas et al. (2017).\u003c/p\u003e\n\u003ch3\u003eBehavioral Assays\u003c/h3\u003e\n\u003cp\u003eThe behavioral response of \u003cem\u003eA. mellifera\u003c/em\u003e adults to I-DWV and N-DWV treatments was evaluated using Y-tube olfactometry. Glass Y-olfactometers (21 cm long with an internal diameter of 3 cm) were designed (University of Concepci\u0026oacute;n, Concepci\u0026oacute;n, Chile), with glass odor chambers (15 cm long with an internal diameter of 3 cm) connected to the end of the central arm (Y-tube) to generate a filtered airflow at 280 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e using a positive pressure air pump to deliver the stimulus. A filter paper (1 cm \u0026times; 7.5 cm) was placed with 10 \u0026micro;L of each stimulus diluted in hexane (volatile fraction of collected pollens), then the paper strip was left to air dry for 30 s to evaporate the solvent and was subsequently placed inside one of the odor chambers, while only the solvent was left in the other odor chamber, considered as the control stimulus. Forty worker bees at 5, 10, 15, and 20 days post-inoculation (160 worker bees in total) per treatment (I-DWV or N-DWV, total n\u0026thinsp;=\u0026thinsp;320) and per evaluated stimulus (n\u0026thinsp;=\u0026thinsp;1280) were used. Before the test, each insect was acclimated inside the Y-tube by placing the individual in the testing area for 5 minutes, then carefully removed for 10 minutes, and the testing area was cleaned with 96% ethanol according to Arenas and Farina (2012). The bee was placed in the central arm and allowed to move in the olfactometer for 6 minutes; a choice was considered as the first movement towards any of the Y-tube arms within that time and where the insect remained for at least 30 s. The choice distribution was defined as follows: a positive choice towards the arm where the stimulus was applied, a negative choice towards the arm without the stimulus, and no choice when, at the end of the evaluation time, the insect did not choose either of the mentioned areas. A second olfactory assay was conducted, where bees from each treatment were exposed to choices between the volatile fractions of native pollen of \u003cem\u003eE. cordifolia\u003c/em\u003e vs a no native pollen of \u003cem\u003eR. ulmifolius\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;160 bees per treatment, total n\u0026thinsp;=\u0026thinsp;320). After each bee was tested, they were stored at -80\u0026deg;C until further analysis.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA Extraction, cDNA synthesis and qPCR\u003c/h2\u003e \u003cp\u003eRNA extraction from each sample collected in the different bioassays; antennas and head in the olfactory reception evaluation and bees in the behavioral assays, were performed according to Kim et al. (2019), Mondet et al. (2018), and Silva et al. (2024) with minor modifications. The collected individuals and organs from each bioassay and sampling point were homogenized with 500 \u0026micro;L of Trizol\u0026trade; (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) at maximum speed in a Retsch homogenizer. Subsequently, 5 \u0026micro;L of RNA Carrier was added, following the manufacturer's recommendations (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The homogenate was incubated on ice for 5 minutes, and 200 \u0026micro;L of chloroform was added, followed by incubation on ice for 3 minutes. The mixture was then centrifuged for 15 minutes at 10,000 g at 4\u0026deg;C, and the supernatant was collected for RNA extraction. RNA extraction was carried out according to the instructions provided by the E.Z.N.A. Total RNA I kit (Omega Bio-Tek, Norcross, GA, USA). RNA quality and yield were analyzed using a spectrophotometer (Infinite 200 PRO NanoQuant, Tecan Group, M\u0026auml;nnedorf, Switzerland) and visualized by agarose gel electrophoresis, and stored at -80\u0026deg;C. For cDNA synthesis, the high-capacity reverse transcription kit (Applied Biosystems) (Invitrogen, Life Technologies, Carlsbad, CA, USA) was used according to the manufacturer's instructions. Viral quantification was performed using the cDNA corresponding to the samples collected at each sampling point, with the β-Actin gene used as a normalization gene. qPCR was conducted using the commercial KAPA SYBR FAST Universal 2 qPCR MasterMix kit (KapaBiosystems, Wilmington, MA, USA), following the supplier\u0026rsquo;s instructions. Reactions were adjusted to a volume of 15 \u0026micro;L, including 20 ng of cDNA, 530 \u0026micro;M of each primer, and filtered sterile molecular-grade water to make up 15 \u0026micro;L. Thermal cycling conditions were adjusted to the requirements of each designed primer. Finally, qPCR analyses were performed using a Stratagene Mx3000P thermocycler (Agilent Technologies, Santa Clara, CA, USA) and data were analyzed using MxPro software (Stratagene, Agilent Technologies, Santa Clara, CA, USA). For viral load quantification, a standard curve was created using a purified PCR product (Wizard\u0026reg; VR SV Gel and PCR Clean-Up System, Promega, Madison, WI, USA). The purified amplicon was quantified using spectrophotometry (Epoch\u0026trade; Microplate Spectrophotometer, BioTek, Winooski, VT, USA) and copy number calculations were performed according to Wu et al. (2017). Linear standard curves (95\u0026ndash;100% efficiency) were generated using serial dilutions (1.0 \u0026times; 10\u0026sup1; to 1.0 \u0026times; 10⁹) of purified cDNA viral copy numbers. Ct values were plotted against copy number values (log10). Thus, the sample copy number was estimated using Ct values and comparison with the linear equation of the standard curve and normalization values from β-actin gene calibration (Yang and Cox-Foster 2005). Finally, data were expressed as the number of DWV-A copies per bee, considering the dilutions performed during cDNA synthesis and qPCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eA nested analysis of variance (Nested ANOVA) was designed between treatments (I-DWV and N-DWV) and bee age (5, 10, 15, and 20 dpi) in the antennal electrical response (mV) to the volatile stimulus of the different pollens to be evaluated, followed by a Bonferroni test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) to separate the means at each evaluation point (5, 10, 15, and 20 dpi). A chi-square test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was used to analyze the statistical difference in the responses (preference) of inoculated (I-DWV) and non-inoculated (N-DWV) honeybees at 5-, 10-, 15- and 20 days old in the olfactometric Y-tube test with different volatile fractions of pollens and \u003cem\u003eR. ulmifolius\u003c/em\u003e vs \u003cem\u003eE. cordifolia\u003c/em\u003e. Also, a binary (1 and 0) logistic regression was run to determine the likelihood of the preference of young bees for the volatile fraction of pollens in the Y-tube test as a function of the DWV-A load and bee age. The preference for the volatile fraction of pollens was marked as positive (yes\u0026thinsp;=\u0026thinsp;1), and no preference or preference for the other compound was considered a negative response (no\u0026thinsp;=\u0026thinsp;0). The data on all bees tested in the experiments (including the viral load of the head of inoculated and non-inoculated bees) were included in the logistic regression.\u003c/p\u003e \u003c/div\u003e"},{"header":"IV RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eChemical Characterization of the Volatile Fraction of Collected Pollens\u003c/h2\u003e\n \u003cp\u003eThe chemical characterization of the volatile fraction of each species revealed differences in compound concentrations and types. The highest diversity of compounds and chemical groups was found in \u003cem\u003eE. cordifolia\u003c/em\u003e pollen (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), highlighting the presence of various terpenes such as menthol, camphene, 1,4-cineole, D-limonene, \u0026alpha;-thujene, \u0026beta;-pinene, and \u0026alpha;-pinene, with the latter two being the most abundant at 10.39 and 29.25 ppm, respectively. Terpenes were recorded in all pollens at concentrations of 0.9, 0.8 and 1.66 ppm of 1,4-cineole for Polyfloral pollen, \u003cem\u003eR. ulmifolius\u003c/em\u003e and \u003cem\u003eE. cordifolia\u003c/em\u003e, respectively. Additionally, all analyzed pollens contained benzaldehyde, an aromatic compound from stone fruit derivatives, at concentrations of 21.4, 11.7 and 1.32 ppm for Polyfloral pollen, monofloral pollen of \u003cem\u003eR. ulmifolius\u003c/em\u003e and \u003cem\u003eE. cordifolia\u003c/em\u003e, respectively.\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eChemical composition of the volatile fraction of different curbicular pollens.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003eCompound name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003ePolyfloral\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e\u003cem\u003eR. ulmifolius\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e\u003cem\u003eE. cordifolia\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003eConcentration\u003csup\u003e1\u003c/sup\u003e (ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003eConcentration\u003csup\u003e1\u003c/sup\u003e (ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003eConcentration\u003csup\u003e1\u003c/sup\u003e (ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e6.283\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003eFormic acid, hexyl ester\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e5.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e6.392\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003eo-Xylene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e3.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e6.450\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003e1-Butanol, 3-methyl-, acetate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e8.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e6.708\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003ePentanal, 2,4-dimethyl-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e15.71\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e7.821\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003ealpha.-Pinene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e29.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e8.141\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003eCamphene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e2.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e8.290\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003eBenzaldehyde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e21.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e11.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e1.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e8.639\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003e1-Butene, 4-isothiocyanato-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e8.744\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003ebeta.-Pinene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e10.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e8.904\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003eCaproic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e3.27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e9.165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003en-Caprylaldehyde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e9.329\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003ealpha.-Thujene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e9.719\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003eBenzene, tert-butyl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e5.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e9.825\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003eD-Limonene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e4.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e9.874\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003e1,4-Cineol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e1.66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e10.434\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003ep-Mentha-1,4-diene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e11.96\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e11.859\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003eCamphenol, 6-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e12.085\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003eLilac aldehyde B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e14.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e6.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e12.261\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003eLilac aldehyde C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e9.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e12.271\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003eLilac aldehyde A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e20.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e12.905\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003ed-Menthol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e13.293\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003eLilac alcohol B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e7.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e13.490\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003eLilac alcohol A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e13.771\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003eLilac alcohol D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e6.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e17.458\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003ealpha.-Ionone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 7.5011%;\"\u003e\n \u003cp\u003e18.434\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 21.2785%;\"\u003e\n \u003cp\u003etrans-.beta.-Ionone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.7477%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.5946%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 88.788%;\"\u003e\u003csup\u003e1\u003c/sup\u003eConcentration calculations were performed by comparing peaks with a standard curve, using tetralin. RT\u0026thinsp;=\u0026thinsp;Retention Time\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eOlfactory Reception\u003c/h2\u003e\n \u003cp\u003eAfter evaluating peripheral perception or olfactory sensitivity in bees inoculated with the viral suspension (I-DWV) and non-inoculated (N-DWV) using electroantennography (EAG) assays, it was determined that the volatile fractions collected from all evaluated pollens were biologically active for bees response. Nested analysis of variance (Nested ANOVA) showed a significant interaction effect between deformed wing virus (DWV) infection treatment and bee age on the antennal electrical response (mV) to the volatile stimulus of polyfloral pollen (F (\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;13.62 p\u0026thinsp;=\u0026thinsp;0.0001) for \u003cem\u003eR. ulmifolius\u003c/em\u003e pollen (F (\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;16.566 p\u0026thinsp;=\u0026thinsp;0.001) and for \u003cem\u003eE. cordifolia\u003c/em\u003e pollen (F (\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;25.012, p\u0026thinsp;=\u0026thinsp;0.0001).\u003c/p\u003e\n \u003cp\u003eIt was observed that all bees inoculated with DWV-A (I-DWV) showed reduced electrophysiological responses compared to non-inoculated (N-DWV) at five days post-inoculation (dpi) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Specifically, a 21%, 24%, 55%, and 40% reduction in EAG responses was observed in I-DWV bees compared to N-DWV bees when exposed to the polyfloral pollen aroma at 5, 10, 15, and 20 dpi, respectively. For \u003cem\u003eR. ulmifolius\u003c/em\u003e pollen, the reduction was 16%, 55%, and 35% at 10, 15, and 20 dpi, respectively, and for \u003cem\u003eE. cordifolia\u003c/em\u003e pollen, a reduction of 40%, 33%, and 55% was observed at 10, 15, and 20 dpi, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eOlfactory Preference\u003c/h2\u003e\n \u003cp\u003eSubsequently, when evaluating the behavioral preferences using Y-tubes for bees inoculated with DWV-A (I-DWV) and non-inoculated bees (N-DWV), significant differences were observed in the behavioral responses of the insects at 10 days after inoculation. Inoculated bees (I-DWV) responded less frequently to each evaluated stimulus compared to non-inoculated (N-DWV). Specifically, when evaluating the behavioral preference between N-DWV and I-DWV bees exposed to the aroma of polyfloral pollen, we observed no difference between treatments at 5 dpi (X\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.072, p\u0026thinsp;=\u0026thinsp;0.788). However, we observed significant differences at 10 dpi (X\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;8.142, p\u0026thinsp;=\u0026thinsp;0.002), 15 dpi (X\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;9.796, p\u0026thinsp;=\u0026thinsp;0.001), and 20 dpi (X\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;13.167, p\u0026thinsp;=\u0026thinsp;0.001). For non-native species, \u003cem\u003eR. ulmifolius\u003c/em\u003e pollen, we observed no difference between treatments at 5 dpi (X\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0, p\u0026thinsp;=\u0026thinsp;1). However, we observed significant differences at 10 dpi (X\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;16.038, p\u0026thinsp;=\u0026thinsp;0.001), 15 dpi (X\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;13.941, p\u0026thinsp;=\u0026thinsp;0.001), and 20 dpi (X\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;9.702, p\u0026thinsp;=\u0026thinsp;0.007) and for native species, \u003cem\u003eE. cordifolia\u003c/em\u003e pollen we observed no difference between treatments at 5 dpi (X\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;3.078, p\u0026thinsp;=\u0026thinsp;0.079). However, we observed significant differences at 10 dpi (X\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;9.455, p\u0026thinsp;=\u0026thinsp;0.002), 15 dpi (X\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;7.813, p\u0026thinsp;=\u0026thinsp;0.005), and 20 dpi (X\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;11.028, p\u0026thinsp;=\u0026thinsp;0.001). In summary, the proportion of I-DWV insects responding to the different stimuli was, on average, 30%, 37%, and 39% less than that of N-DWV bees for the volatile stimuli from polyfloral pollen, \u003cem\u003eR. ulmifolius\u003c/em\u003e and \u003cem\u003eE. cordifolia\u003c/em\u003e, respectively.\u003c/p\u003e\n \u003cp\u003eAdditionally, logistic regression analysis demonstrated the probability of preference for the different stimuli tested between DWV-A inoculated and non-inoculated bees (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). For bees stimulated with the volatile fraction of polyfloral pollen, the viral load significantly influenced preference (Wald\u0026rsquo;s X\u0026sup2; = 13.94, p\u0026thinsp;=\u0026thinsp;0.0001, Odds ratio\u0026thinsp;=\u0026thinsp;0.9), while bee age did not (Wald\u0026rsquo;s X\u0026sup2; = 2.38, p\u0026thinsp;=\u0026thinsp;0.12, Odds ratio\u0026thinsp;=\u0026thinsp;1.03) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Similarly, for bees stimulated with \u003cem\u003eR. ulmifolius\u003c/em\u003e, a significant influence of viral load was observed (Wald\u0026rsquo;s X\u0026sup2; = 30.04, p\u0026thinsp;=\u0026thinsp;0.0001, Odds ratio\u0026thinsp;=\u0026thinsp;0.86), whereas bee age did not show significance (Wald\u0026rsquo;s X\u0026sup2; = 1.3, p\u0026thinsp;=\u0026thinsp;0.25, Odds ratio\u0026thinsp;=\u0026thinsp;1.02) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Finally, for bees stimulated with \u003cem\u003eE. cordifolia\u003c/em\u003e, a significant influence of viral load was reported (Wald\u0026rsquo;s X\u0026sup2; = 29.39, p\u0026thinsp;=\u0026thinsp;0.0001, Odds ratio\u0026thinsp;=\u0026thinsp;0.84), while bee age did not have a significant influence (Wald\u0026rsquo;s X\u0026sup2; = 0.23, p\u0026thinsp;=\u0026thinsp;0.62, Odds ratio\u0026thinsp;=\u0026thinsp;0.98) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\n \u003cp\u003eWith the aim of determining whether bees were able to distinguish between one aromatic source and another based on the difference in the botanical origin of the pollens (non-native and native) We conducted a second behavioral bioassay, between bees inoculated and non-inoculated with DWV-A using a Y-tube olfactometer assay. In this essay, bees were exposed to the aromas of \u003cem\u003eR. ulmifolius\u003c/em\u003e (non-native species) and \u003cem\u003eE. cordifolia\u003c/em\u003e (native species) simultaneously to assess whether the bees showed a preference for one of the stimuli.\u003c/p\u003e\n \u003cp\u003eIt was observed that both inoculated (I-DWV) and non-inoculated (N-DWV) honey bees did not show a significant difference in preference towards the aroma of \u003cem\u003eE. cordifolia\u003c/em\u003e, with preferences reported to be between 45\u0026ndash;70% (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). However, it was determined that at 15- and 20-days post-inoculation, significant differences existed in the behavioral response between the treatments towards the aroma of \u003cem\u003eR. ulmifolius\u003c/em\u003e. Specifically, I-DWV bees responded in the range of 8\u0026ndash;10% compared to N-DWV bees, where the attracted population ranged from 25\u0026ndash;33%. Similarly, differences were observed in the proportion of insects that were not attracted to any stimulus. The proportion of bees in the I-DWV treatment that were not attracted ranged from 20\u0026ndash;40%, while in the N-DWV treatment, it ranged from 5\u0026ndash;15% throughout the duration of the assay.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cimg src=\"https://myfiles.space/user_files/58894_9946feeafa4c1df7/58894_custom_files/img1733466169.png\" width=\"643\" height=\"555\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eAdditionally, we quantified the DWV-A viral load in bees from the N-DWV and I-DWV treatments using qPCR. For the N-DWV treatment, the observed viral load was 1 x 10⁴ copy number per bee at the beginning of the experiment and 1 x 10⁵ copy number per bee at 20 days post-viral inoculation. In contrast, for the I-DWV treatment, we detected a viral load of 1 x 10⁴ copy number per bee at the start of the experiment and 1 x 10\u0026sup1;⁵ copy number per bee at 20 days post-viral inoculation.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"V DISCUSSION","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003cp\u003eThe differences in chemical composition and abundance of compounds in the volatile fractions of the various collected pollens may be determined by their botanical origin, including both polyfloral and monofloral pollens (Prdun et al., 2021). Nonetheless, the presence of benzene-derived compounds such as benzaldehyde, which was found in all the pollens evaluated in this study, is a common compound in pollens from different botanical species, including both polyfloral (Starowicz et al., 2021) and monofloral sources such as \u003cem\u003eRosa\u003c/em\u003e spp. (Caser and Scariot, 2022), \u003cem\u003eLotus\u003c/em\u003e spp. (Ni et al., 2023), \u003cem\u003eTaraxacum officinale\u003c/em\u003e, and \u003cem\u003eSalix\u003c/em\u003e spp. (Prdun et al., 2021). It has been described that among the most prevalent chemical groups in volatile fractions of pollens are alkanes, aldehydes, acids, benzene derivatives, ketones, esters, sulfoxides, alcohols, pyrroles, furans, lactones, and terpenes (Starowicz et al., 2021). However, it has also been noted that the composition of these chemical groups can differ based on the botanical origin of the pollen (Da Silva et al., 2016; Lima et al., 2017). For instance, in Brazil, pollen was characterized by only two chemical groups: esters and alkanes (Carpes et al., 2013). Similarly, Lima et al. (2017) found no volatiles from the groups of alkanes, terpenes, disulfides, sulfoxides, benzene derivatives, acids, furans, lactones, and pyrroles in pollen samples of \u003cem\u003eMimosa caesalpiniifolia\u003c/em\u003e. This difference in composition, primarily due to the botanical origin of the species, could explain the abundance and richness of terpenes in the volatile fraction of \u003cem\u003eE. cordifolia\u003c/em\u003e pollen (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Additionally, the presence of compounds such as lilac aldehyde and lilac alcohol in polyfloral and \u003cem\u003eR. ulmifolius\u003c/em\u003e pollens may indicate the presence of other terpenes like linalool, from which a variety of compounds can be formed. For example, hydroxylation of monoterpenes can produce lilac aldehydes, while epoxidation of linalool produces lilac alcohols (Jerković et al., 2011). Moreover, differences in chemical compositions can also be attributed to the collection and storage methods; environmental factors such as temperature and relative humidity can initiate chemical degradation processes in pollens, altering their chemical profiles. Thus, better storage conditions would likely result in a more diverse chemical composition (Ni et al., 2023).\u003c/p\u003e \u003cp\u003eOn the other hand, studies conducted by Zhang et al. (2022) demonstrated that \u003cem\u003eA. mellifera\u003c/em\u003e antennae are sensitive to benzaldehyde at different concentrations, ranging from 10\u0026ndash;500 \u0026micro;g \u0026micro;L⁻\u0026sup1;. Although it was suggested that sensitivity was low, it remained between 0.5\u0026ndash;2.1 mV without changes across different concentrations of the compound to which they were exposed. Similarly, the presence of lilac alcohol and benzaldehyde in the volatile fraction of \u003cem\u003ePrunus\u003c/em\u003e sp. flowers was biologically active for honeybee in electroantennography (EAD) assays and elicited attraction in behavioral assays using Y-tubes (Su et al., 2022). Additionally, the presence of 1,4-cineole, a compound found in all the aromas characterized in this study and present in different concentrations, has biological activity in the olfactory system of \u003cem\u003eA. mellifera\u003c/em\u003e by interacting with various olfactory system proteins in the insect (Briand et al., 2001). Studies by Liu et al. (2022) also showed that β-caryophyllene and β-pinene were biologically active and attractive in the olfactory system of \u003cem\u003eA. mellifera\u003c/em\u003e. We can assume that the presence of these compounds in the volatile fractions of the tested pollens stimulates the antennae of honeybees and is responsible for the recorded olfactory sensitivity.\u003c/p\u003e \u003cp\u003eIn terms of sensitivity ranges, an interesting finding of this study was the recorded responses for polyfloral and monofloral native pollen from \u003cem\u003eE. cordifolia\u003c/em\u003e, where sensitivity ranged between 0.4\u0026ndash;0.6 mV, lower than those detected for \u003cem\u003eR. ulmifolius\u003c/em\u003e pollen, with readings of 0.5\u0026ndash;0.9 mV considering only non-inoculated honey bees. These changes in sensitivity may be associated with olfactory phenomena; models postulating ligand-receptor interactions suggest that complex aromas may saturate the olfactory system, which is associated with biologically active compounds in the chemical composition and high abundance. Studies by Zack et al. (2020) suggest that olfactory saturation can occur in simple aroma mixtures with as few as two biologically active compounds up to mixtures of twenty. While this olfactory saturation model was proposed in mice as a model organism, these postulates can be projected onto the olfactory system of \u003cem\u003eA. mellifera\u003c/em\u003e due to the functional and evolutionary convergence of the olfactory nerve endings (Paoli and Galicia, 2021). This phenomenon may be responsible for the different readings recorded for each stimulus used in this assay; however, precise and specialized studies are needed to fully understand the functioning and regulatory mechanisms of the olfactory system of \u003cem\u003eA. mellifera\u003c/em\u003e to reach a definitive conclusion.\u003c/p\u003e \u003cp\u003eAdditionally, as previously mentioned, many of the compounds found in the volatile fractions of the pollens in these studies are attractive to honey bees. However, the reductions in attraction detected for all stimuli had a significant contribution from the health condition of the bees, influenced by the viral load of DWV-A (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Previous studies have shown how the presence of this virus affects olfactory sensitivity, and some genes related to the detection of environmental chemical compounds, such as odorant-binding proteins (OBPs) (Silva et al., 2021). We previously described that adult \u003cem\u003eA. mellifera\u003c/em\u003e antennae show reduced olfactory sensitivity to volatile fractions of essential oils, and the genes encoding OBPs 2, 5, 11, and 12 decrease in its expression when DWV-A load increases. Recently, we evaluated nurse bees with high viral loads (1 x 10\u003csup\u003e13\u003c/sup\u003e copy number per bee) change their behavioral preference, reducing their attraction when stimulated with benzyl alcohol, a component of the alarm pheromone emitted by larvae, which is related to OBPs 5 and 11 (Silva et al., 2024). Additionally, we demonstrated that genes involved in neuronal processes, such as presynaptic genes \u003cem\u003eAmNrX-1\u003c/em\u003e and postsynaptic gene \u003cem\u003eAmNlG-1\u003c/em\u003e, reduce their gene expression in relation to increasing viral loads (Silva et al., 2024). We assume that changes in sensitivity and behavioral responses in bees infected with DWV-A could be caused by the presence and replication of this virus on the head of nurse honeybees. Pizorno et al. (2021) determined through transcriptomic analyses of brains inoculated with DWV that downregulated genes involved biological processes encoding proteins related to cellular signaling, cell communication, and synaptic function in DWV-infected brains, suggesting that the presence and increase of viral loads could inhibit neuronal and brain physiology. Among the affected genes were G protein-coupled receptors (GPCRs) that have a broad function related to learning, memory, and behaviors associated with foraging (Schneider et al., 2006; Mustard et al., 2005; Schulz et al., 2002). Moreover, different GPCRs linked to various neuropeptides, based on functionality determined in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e M., could be involved in altering behavioral responses (Xu et al., 2010; Lee et al., 2013; Martelli et al., 2017). The hypothesis that the virus alters behavioral responses is supported by the numerous changes occurring in the insects\u0026rsquo; brain as pathogen levels increase. Chen et al. (2021) demonstrated how increased viral load in the insects\u0026rsquo; head affected long-term memory as a consequence of energy imbalance following disruption of the glutamate-glutamine (Glu-GLn) cycle and the decrease in the gene encoding the receptor (Ado-R) in the brains of infected bees, suggesting that DWV may have neurotoxic effects on \u003cem\u003eA. mellifera\u003c/em\u003e. However, while the effects of brain physiology alterations have been evaluated in artificial infections with the deformed wing virus, it has been suggested that a sudden increase in viral loads in field conditions, due to stressors or attacks by pathogens such as \u003cem\u003eV. destructor\u003c/em\u003e, can increase viral load, worsening symptoms and leading to neurodegenerative consequences, including changes in GABAergic regulation of the nervous system, compromising behavioral responses and memory processes in honey bees (Szymański et al., 2024).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, an interesting aspect to analyze was the differences in choices made by bees when exposed simultaneously to the aromas of \u003cem\u003eR. ulmifolius\u003c/em\u003e and \u003cem\u003eE. cordifolia\u003c/em\u003e. Although we previously reported that bees infected with the virus had difficulties in selecting aromas (Silva et al., 2024), we considered that much of this was due to the nature of the stimulus, as bees were exposed to a complex aroma obtained from a plant essential oil and a pure commercial compound. In this assay, both stimuli were obtained from a plants and corresponded to the aroma of pollen collected by the bees themselves. It was interesting to note that N-DWV bees did not show significant differences between one stimulus and another, whereas I-DWV bees were strongly attracted to the pollen aroma from \u003cem\u003eE. cordifolia\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A possible explanation for this could be the phenomenon of self-medication described in bees, where they tend to select their food sources to improve their response to illness. Increases in resin collection by the colony in response to infections caused by \u003cem\u003eAscosphaera apis\u003c/em\u003e O. and increased populations of \u003cem\u003eV. destructor\u003c/em\u003e have been reported (Simone-Finstrom \u0026amp; Spivak, 2012; Pusceddu et al., 2019). Similarly, infection caused by \u003cem\u003eN. ceranae\u003c/em\u003e F. alters food source selection between infected and non-infected individuals, with high levels of the disease leading to a preference for honey with greater microbial activity (Gherman et al., 2014). Bees are also known to change their pollen selection to sources with higher nutritional quality, although this does not lead to an increase in foraging behavior (Ferguson et al., 2018). The possibility that bee\u0026rsquo;s behavioral responses and preferences are influenced by the immune benefits of pollen related to aroma could be determined by chemical characterization. The presence of chemical groups such as terpenes may indicate the interpretation of the aroma. For instance, terpenes derived from nectar and pollen of different species have antiviral effects on DWV (Palmer-Young et al., 2018). The terpene limonene, present in pollen from \u003cem\u003eE. cordifolia\u003c/em\u003e, has antiviral effects against both variant A and B of the DWV (Boncristiani et al., 2021). However, to demonstrate this possibility, studies are needed to analyze the potential effect of these pollens on bee immunity and survival and to determine if this preliminary selection based on aroma and the health condition of bees leads to increased pollen consumption. The selection and discrimination of protein and food sources, such as pollen, is crucial to ensuring the survival of the hive. It has been shown that pollen differs in nutritional composition depending on its botanical origin (Di Pasquale et al. 2016). At the same time, this nutritional source will benefit the hive to a greater or lesser extent depending on its nutritional quality (Danihilk et al., 2018, Di Pasquale et al. 2016). Likewise, different botanical groups of pollen have a direct effect on the regulation of the immune system in bees (Bry\u0026acute;s et al., 2022, Danihilk et al., 2018, Di Pasquale et al. 2016).\u003c/p\u003e \u003cp\u003eWhen observing the proportion of insects that did not participate in the final selection evaluation (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), it was interesting to note that the proportion of insects unable to select any stimulus was higher in the population of bees with high viral loads (I-DWV), where we observed 30\u0026ndash;40% of the insect population showing no preference for any stimulus, compared to honey bees non-inoculated with DWV-A, where only 3\u0026ndash;10% showed no preference. This absence of choice supports the idea that the virus strongly affects bees' ability to respond behaviorally, even when they are exposed to attractive aromas. While many studies focus on neuronal changes as precursors to behavioral limitations, these studies are often centered on the deformed wing virus in general, but not specifically on master variants. A meta-analysis of transcriptomes generated by increasing viral loads of DWV-A highlighted that neuro-genomic changes in nurse bees would result in poor learning and early foraging behavior, leading to a higher incidence of infected bees mistakenly entering other colonies (Tranilleo et al., 2020). Similarly, a study reported by Kim et al. (2019) revealed structural damage caused by DWV-A, compromising all processes underlying peripheral perception.\u003c/p\u003e \u003cp\u003eWe have demonstrated here that DWV-A negatively and significantly affects honey bees (10\u0026ndash;20 days old) in their olfactory sensitivity and perception, as well as their interpretation and selection of complex aromas derived from the volatile fractions emitted by different pollens. While we have shown what occurs from the perspective of peripheral perception and the underlying behavioral responses, we have not yet concluded the cause of these behavioral losses or how they might affect the integrity of the honey bee colonies into the hive. Furthermore, the question remains about what happens to foraging honey bees faced with complex and dynamic aromas in field conditions, where food source selection relies solely on peripheral perception. Therefore, more studies are required to answers these questions.\u003c/p\u003e \u003c/div\u003e "},{"header":"VI. CONCLUSION","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003cp\u003eIn summary, the increase in DWV-A viral load negatively affects worker honeybees by reducing both their olfactory sensitivity and behavioral responses. This impact translates into a diminished ability to perceive and respond effectively to olfactory stimuli. Notably, honeybees with high viral loads exhibited a marked preference for native \u003cem\u003eE. cordifolia\u003c/em\u003e pollen compared to other evaluated pollen types. This finding raises questions about whether such preference could represent an adaptive behavior aimed at mitigating the effects of pathogens. Further studies are required to explore this hypothesis and to determine the potential role of food selectivity as a resilience strategy against viral infections. Finally, the results indicate that heightened DWV-A infection can compromise honeybee selectivity and responses to food sources, directly affecting the functionality and integrity of the hive.\u003c/p\u003e \u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlaux C, Ducloz F, Crauser D, Le Conte Y (2010) Diet effects on honeybee immunocompetence. Biol Lett 6:562\u0026ndash;565. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/rsbl.2009.0986\u003c/span\u003e\u003cspan address=\"10.1098/rsbl.2009.0986\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAllen MF, Ball BV (1996) The incidence and world distribution of honeybee viruses. Bee World 77:141\u0026ndash;162\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmiri E, Kryger P, Meixner MD, Strand MK, Tarpy D, Rueppell O (2018) Quantitative patterns of vertical transmission of deformed wing virus in honeybees. PLoS ONE 13(3):e0195283. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0195283\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0195283\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArenas A, Farina WM (2012) Learned olfactory cues affect pollen-foraging preferences in honeybees, Apis mellifera. Anim Behav 83(4):1023\u0026ndash;1033\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArismendi N, Caro S, Castro MP, Vargas M, Riveros G, Venegas T (2020) Impact of mixed infections of gut parasites \u003cem\u003eLotmaria passim\u003c/em\u003e and \u003cem\u003eNosema ceranae\u003c/em\u003e on the lifespan and immune-related biomarkers in \u003cem\u003eApis mellifera\u003c/em\u003e. Insects, 11(7), 420\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBabendreier D, Kalberer N, Romeis J, Fluri P, Bigler F (2004) Pollen consumption in honey bee larvae: a step forward in the risk assessment of transgenic plants. Apidologie 35:293\u0026ndash;300. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1051/apido:2004016\u0026iuml;\u003c/span\u003e\u003cspan address=\"10.1051/apido:2004016\u0026iuml;\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBasualdo, M., Cavigliasso, P., de Avila Jr, R. S., Aldea-S\u0026aacute;nchez, P., Correa-Ben\u0026iacute;tez,A., Harms, J. M., \u0026hellip; Salvarrey, S. (2022). Status and economic value of insect-pollinated dependent crops in Latin America. Ecological Economics, 196, 107395.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenaets K, Van Geystelen A, Cardoen D, De Smet L, de Graaf DC, Schoofs L, Larmuseau MH, Brettell LE, Martin SJ, Wenseleers T (2017) Covert deformed wing virus infections have long-term deleterious effects on honeybee foraging and survival. Proc. Biol. Sci. 284\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoncristiani DL, Tauber JP, Palmer-Young EC, Cao L, Collins W et al (2021) Impacts of diverse natural products on honeybee viral loads and health. Appl Sci 11(22):10732\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBriand L, Nespoulous C, Huet JC, Takahashi M, Pernollet JC (2001) Ligand binding and physico-chemical properties of ASP2, a recombinant odorant-binding protein from honey bee (\u003cem\u003eApis mellifera\u003c/em\u003e L). Eur J Biochem 268:752\u0026ndash;760. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1432-1327.2001.01927.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1432-1327.2001.01927.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrodschneider R, Crailsheim K (2010) Nutrition and health in honeybees. Apidologie 41(3):278\u0026ndash;294\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBryś MS, Skowronek P, Strachecka A (2021) Pollen Diet\u0026mdash;Properties and Impact on a Bee Colony. Insects 12:798. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/insects12090798\u003c/span\u003e\u003cspan address=\"10.3390/insects12090798\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarpes ST, de Alencar SM, Cabral ISR, Oldoni TLC, Mourao GB, Haminiuk CWI, da Luz CFP, Masson ML (2013) Polyphenols and palynological origin of bee pollen of \u003cem\u003eApis mellifera\u003c/em\u003e L. from Brazil. Characterization of polyphenols of bee pollen. CYTA J Food 11:150\u0026ndash;161\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaser M, Scariot V (2022) The Contribution of Volatile Organic Compounds (VOCs) Emitted by Petals and Pollen to the Scent of Garden Roses. Horticulturae 8:1049. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/horticulturae8111049\u003c/span\u003e\u003cspan address=\"10.3390/horticulturae8111049\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen P, Lu YH, Lin YH, Wu CP, Tang CK, Wei SC, Wu YL (2021) Deformed wing virus infection affects the neurological function of \u003cem\u003eApis mellifera\u003c/em\u003e by altering extracellular adenosine signaling. Insect Biochem Mol Biol 139:103674\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Evans J, Feldlaufer M (2006) Horizontal and vertical transmission of viruses in the honey bee, \u003cem\u003eApis mellifera\u003c/em\u003e. J Invertebrate Pathol Soc Invertebrate Pathol 2006 Special Issue 92:152\u0026ndash;159. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jip.2006.03.010\u003c/span\u003e\u003cspan address=\"10.1016/j.jip.2006.03.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDa Silva PM, Gauche C, Gonzaga LV, Costa ACO, Fett R (2016) Honey: Chemical composition, stability and authenticity. Food Chem 196:309\u0026ndash;323\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDainat B, Ken T, Berthoud H, Neumann P (2009) The ectoparasitic mite \u003cem\u003eTropilaelaps mercedesae\u003c/em\u003e (Acari, Laelapidae) as a vector of honeybee viruses. Insectes Soc 56:40\u0026ndash;43\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDanihl\u0026iacute;k, J., Škrabišov\u0026aacute;, M., Lenobel, R., Šebela, M., Omar, E., Petřivalsk\u0026yacute;, M.,\u0026hellip; Brodschneider, R. (2018). Does the pollen diet influence the production and expression of antimicrobial peptides in individual honeybees? Insects, 9(3), 79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDi Pasquale, G., Salignon, M., Le Conte, Y., Belzunces, L. P., Decourtye, A., Kretzschmar,A., \u0026hellip; Alaux, C. (2013). Influence of pollen nutrition on honey bee health: do pollen quality and diversity matter? PloS one, 8(8), e72016.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEllis JD, Munn PA (2005) The worldwide health status of honey bees. Bee World 86:88\u0026ndash;101\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEtxegarai-Legarret O, Sanchez-Famoso V (2022) The role of beekeeping in the generation of goods and services: The interrelation between environmental, socio-economic, and socio-cultural utilities. Agriculture 12:551\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEvan C, Palmer-Young C\u0026Ouml;, Tozkar RS, Schwarz Y, Chen RE, Irwin LS, Adler JD, Evans (2017) Nectar and pollen phytochemicals stimulate honeybee (Hymenoptera: Apidae) immunity to viral infection. Journal of Economic Entomology Volume 110 Issue 5 October Pages 1959\u0026ndash;1972\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerguson JA, Northfield TD, Lach L (2018) Honey bee (Apis mellifera) pollen foraging reflects benefits dependent on individual infection status. Microb Ecol 76:482\u0026ndash;491\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eForsgren E, de Miranda JR, Isaksson M, Wei S, Fries I (2009) Deformed wing virus associated with Tropilaelaps mercedesae infesting European honey bees (\u003cem\u003eApis mellifera\u003c/em\u003e). Exp Appl Acarol 47:87\u0026ndash;97\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGenersch E, Aubert M (2010) Emerging and re-emerging viruses of the honey bee (\u003cem\u003eApis mellifera\u003c/em\u003e L). Vet Res 41(6):54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1051/vetres/2010027\u003c/span\u003e\u003cspan address=\"10.1051/vetres/2010027\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGenersch E, Yue C, Fries I, de Miranda JR (2006) Detection of deformed wing virus, a honey bee viral pathogen, in bumble bees (\u003cem\u003eBombus terrestris\u003c/em\u003e and \u003cem\u003eBombus pascuorum\u003c/em\u003e) with wing deformities. J Invertebr Pathol 91:61\u0026ndash;63\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGherman BI, Denner A, Bobiş O, Dezmirean DS et al (2014) Pathogen-associated self-medication behavior in the honeybee Apis mellifera. Behav Ecol Sociobiol 68:1777\u0026ndash;1784\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoodrich BK (2019) Do more bees imply higher fees? Honeybee colony strength as a determinant of almond pollination fees. Food Policy 83:150\u0026ndash;160\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGroot AT, Dekker T, Heckel D (2016) The Genetic Basis of Pheromone Evolution in Moths. Annu Rev Entomol 61(1):99\u0026ndash;117. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1146/annurev-ento-010715-023638\u003c/span\u003e\u003cspan address=\"10.1146/annurev-ento-010715-023638\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrozinger CM, Flenniken ML (2019) Bee Viruses: Ecology, Pathogenicity, and Impacts. Annu Rev Entomol 64:205\u0026ndash;226\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGusachenko ON, Woodford L, Balbirnie-Cumming K, Ryabov EV, Evans DJ (2020) Evidence for and against deformed wing virus spillover from honey bees to bumble bees: A reverse genetic analysis. Sci Rep 10:16847\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJerkovi\u0026acute;c I, Marijanovi\u0026acute;c Z, Malenica M (2011) Screening of Natural Organic Volatiles from \u003cem\u003ePrunus mahaleb\u003c/em\u003e L. Honey: Coumarin and Vomifoliol as Nonspecific Biomarkers. Molecules 16:2507\u0026ndash;2518\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKevill JL, Highfield A, Mordecai GJ, Martin SJ, Schroeder DC (2017) ABC Assay: Method development and application to quantify the role of three DWV master variants in overwinter colony losses of European honey bees. Viruses 9:314\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SH, Mercer A, Mitchell A, de Miranda JR, Ward V, Mondet F, Bostina M (2019) Viral infections alter antennal epithelium ultrastructure in honey bees. J Invertebr Pathol 168:107252\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SH, Mercer A, Mitchell A, de Miranda JR, Ward V, Mondet F, Bostina M (2019) Viral infections alter antennal epithelium ultrastructure in honey bees. J Invertebr Pathol 168:107252. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jip.2019.107252\u003c/span\u003e\u003cspan address=\"10.1016/j.jip.2019.107252\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKlein AM, Vaissi\u0026egrave;re BE, Cane JH, Steffan-Dewenter I, Cunningham SA, Kremen C, Tscharntke T (2007) Importance of pollinators in changing landscapes for world crops. Proc R Soc B Biol Sci 274:303\u0026ndash;313\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee G, Kikuno K, Bahn JH, Kim KM, Park JH (2013) Dopamine D2 receptor as a cellular component controlling nocturnal hyperactivities in Drosophila melanogaster. Chronobiol Int 30(4):443\u0026ndash;459\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Q, Liberles SD (2015) Aversion and attraction through olfaction. Curr Biol 25(3):120\u0026ndash;129. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cub.2014.11.044\u003c/span\u003e\u003cspan address=\"10.1016/j.cub.2014.11.044\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLima Neto JS, Lopes JAD, Moita Neto JM et al (2017) Volatile compounds and palynological analysis from pollen pots of stingless bees from the mid-north region of Brazil. Braz J Pharm Sci 53, e14093\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Zhang J, Shen J, Zhao H, Ma W, Jiang Y (2022) Differences in EAG Response and Behavioral Choices between Honey Bee and Bumble Bee to Tomato Flower Volatiles. Insects 13(11):987. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/insects13110987\u003c/span\u003e\u003cspan address=\"10.3390/insects13110987\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartelli C, Pech U, Kobbenbring S, Pauls D, Bahl B, Sommer MV et al (2017) SIFamide translates hunger signals into appetitive and feeding behavior in Drosophila. Cell Rep 20(2):464\u0026ndash;478\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMenzel R (2012) The honeybee as a model for understanding the basis of cognition. Nat Rev Neurosci 13(11):758\u0026ndash;768 pmid:23080415\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMondet F, Rau A, Klopp C, Rohmer M, Severac D, Le Conte Y, Alaux C (2018) Transcriptome profiling of the honeybee parasite \u003cem\u003eVarroa destructor\u003c/em\u003e provides new biological insights into the mite adult life cycle. BMC Genomics 19:1\u0026ndash;19\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMordecai GJ, Wilfert L, Martin SJ, Jones IM, Schroeder DC (2016) Diversity in a honeybee pathogen: First report of a third master variant of the deformed Wing Virus quasispecies. ISME J 10:1264\u0026ndash;1273\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMordecai GJ, Wilfert L, Martin SJ, Jones IM, Schroeder DC (2016) Diversity in a honeybee pathogen: First report of a third master variant of the deformed Wing Virus quasispecies. ISME J 10:1264\u0026ndash;1273\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMustard JA, Kurshan PT, Hamilton IS, Blenau W, Mercer AR (2005) Developmental expression of a tyramine receptor gene in the brain of the honey bee, Apis mellifera. J Comp Neurol 483(1):66\u0026ndash;75\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNi JB, Bi YX, Vidyarthi SK, Xiao HW et al (2023) Non-thermal electrohydrodynamic (EHD) drying improved the volatile organic compounds of lotus bee pollen via HS-GC-IMS and HS-SPME-GC-MS. Lwt 176:114480\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaoli M, Galizia GC (2021) Olfactory coding in honeybees. Cell Tissue Res 383:35\u0026ndash;58. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00441-020-03385-5\u003c/span\u003e\u003cspan address=\"10.1007/s00441-020-03385-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePizzorno MC, Field K, Kobokovich AL, Martin PL, Gupta RA, Mammone R et al (2021) Transcriptomic responses of the honey bee brain to infection with deformed wing virus. Viruses 13(2):287\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePotts SG, Biesmeier JC, Kremen C, Neumann P, Schweiger O, Kunin WE (2010) Global pollinator declines: trends, impacts and drivers. Trend Ecol Evol 25:345\u0026ndash;353\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrdun S, Sveˇcnjak L, Valenti\u0026acute;c M, Marijanovi\u0026acute;c Z, Jerkovi\u0026acute;c I (2021) Characterization of Bee Pollen: Physico-Chemical Properties, Headspace Composition and FTIR Spectral Profiles. Foods 10:2103. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/foods10092103\u003c/span\u003e\u003cspan address=\"10.3390/foods10092103\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePusceddu M, Piluzza G, Theodorou P, Buffa F, Ruiu L, Bullitta S et al (2019) Resin foraging dynamics in Varroa destructor-infested hives: a case of medication of kin? Insect Sci 26(2):297\u0026ndash;310\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamirez W, Thomas N, Muktar I, Riabinina O (2023) The neuroecology of olfaction in bees. Curr Opin Insect Sci 56:101018\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiveros G, Arismendi N, Zapata N et al (2020) Occurrence, prevalence and viral load of deformed wing virus variants in Apis mellifera colonies in Chile. J Apicult Res 59:63\u0026ndash;68\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanpa S, Chantawannakul P (2009) Survey of six bee viruses using RT-PCR in Northern Thailand. J Invertebr Pathol 100:116\u0026ndash;119\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScheiner R, Bauman A, Blenau W (2006) Aminergic control and modulation of honeybee behaviour. Curr Neuropharmacol 4(4):259\u0026ndash;276\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchulz DJ, Sullivan JP, Robinson GE (2002) Juvenile hormone and octopamine in the regulation of division of labor in honey bee colonies. Horm Behav 42(2):222\u0026ndash;231\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShah KS, Evans EC, Pizzorno MC (2009) Localization of deformed wing virus (DWV) in the brains of the honeybee, \u003cem\u003eApis mellifera\u003c/em\u003e Linnaeus. Virol J 6:182\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSilva D, Arismendi N, Alveal JP, Ceballos R, Zapata N, Vargas M (2024) Nursing Honeybee Behavior and Sensorial-Related Genes Are Altered by Deformed Wing Virus Variant A. Insects 15(2):80\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSilva D, Ceballos R, Arismendi N, Dalmon A, Vargas M (2021) Variant A of the deformed wings virus alters the olfactory sensitivity and the expression of odorant binding proteins on antennas of Apis mellifera. Insects 12(10):895\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimone-Finstrom MD, Spivak M (2012) Increased resin collection after parasite challenge: a case of self-medication in honey bees? PLoS ONE, 7(3), e34601\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSkubnik K, Novacek J, Fuzik T, Pridal A, Paxton RJ, Plevka P (2017) Structure of deformed wing virus, a major honey bee pathogen. Proc. Natl. Acad. Sci. USA. 114, 3210\u0026ndash;3215\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStarowicz M, Hanus P, Lamparski G, Sawicki T (2021) Characterizing the Volatile and Sensory Profiles, and Sugar Content of Beeswax, Beebread, Bee Pollen, and Honey. Molecules 26:3410. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules26113410\u003c/span\u003e\u003cspan address=\"10.3390/molecules26113410\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu W, Ma W, Zhang Q, Hu X, Ding G, Jiang Y, Huang J (2022) Honey Bee Foraging Decisions Influenced by Pear Volatiles. Agriculture 12:1074. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/agriculture12081074\u003c/span\u003e\u003cspan address=\"10.3390/agriculture12081074\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzymański S, Baracchi D, Dingle L, Bowman L, Manfredini F (2024) Learning performance and GABAergic pathway link to deformed wing virus in the mushroom bodies of naturally infected honey bees. J Exp Biol 227(13):jeb246766. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/jeb.246766\u003c/span\u003e\u003cspan address=\"10.1242/jeb.246766\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTraniello IM, Bukhari SA, Kevill J et al (2020) Meta-analysis of honey bee neurogenomic response links Deformed wing virus type A to precocious behavioral maturation. Sci Rep 10:3101. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-020-59808-4\u003c/span\u003e\u003cspan address=\"10.1038/s41598-020-59808-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUNEP Emerging Issues: Global Honeybee Colony Disorder and Other Threats to Insect Pollinators. United Nations Environment Programme (2010) Available online: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.unep.org/resources/report/unep-emerging-issues-global-honey-bee-colony-disorder-and-other-threats-insect\u003c/span\u003e\u003cspan address=\"https://www.unep.org/resources/report/unep-emerging-issues-global-honey-bee-colony-disorder-and-other-threats-insect\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed on 22-08-2024)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVargas M, Arismendi N, Riveros G, Zapata N, Bruna A, Vidal M, Rodr\u0026iacute;guez M, Gerding M (2017) Viral and intestinal diseases detected in Apis mellifera in Central and Southern Chile. Chil J Agric Res 77:243\u0026ndash;249\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams GR, Rogers REL, Kalkstein AL, Taylor BA, Shutler D, Ostiguy N (2009) Deformed wing virus in western honey bees (\u003cem\u003eApis mellifera\u003c/em\u003e) from Atlantic Canada and the first description of an overtly-infected emerging queen. J Invertebr Pathol 101:77\u0026ndash;79\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWinfree R, Gross BJ, Kremen C (2011) Valuing pollination services to agriculture. Ecol Econ 71:80\u0026ndash;88\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWright GA, Skinner BD, Smith BH (2002) Ability of honeybee, \u003cem\u003eApis mellifera\u003c/em\u003e, to detect and discriminate odors of varieties of canola (\u003cem\u003eBrassica rapa\u003c/em\u003e and \u003cem\u003ebrassica napus\u003c/em\u003e) and snapdragon flowers (\u003cem\u003eAntirrhinum majus\u003c/em\u003e). J Chem Ecol 28(4):721\u0026ndash;740 pmid:12035922\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu Y, Dong X, Kadowaki T (2017) Characterization of the copy number and variants of deformed wing virus (DWV) in the pairs of honey bee pupa and infesting \u003cem\u003eVarroa destructor\u003c/em\u003e or \u003cem\u003eTropilaelaps mercedesae\u003c/em\u003e. Front Microbiol 8:1558\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu J, Li M, Shen P (2010) A G-protein-coupled neuropeptide Y-like receptor suppresses behavioral and sensory response to multiple stressful stimuli in Drosophila. J Neurosci 30(7):2504\u0026ndash;2512\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang W, Kuang H, Wang S, Wang J, Liu W, Wu Z, Tian Y, Huang ZY, Miao X (2013) Comparative sucrose responsiveness in \u003cem\u003eApis mellifera\u003c/em\u003e and \u003cem\u003eA. cerana\u003c/em\u003e Foragers. PLoS ONE 8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0079026\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0079026\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang X, Cox-Foster D (2005) Impact of an ectoparasite on the immunity and pathology of an invertebrate: Evidence for host immunosuppression and viral amplification. Proc Natl Acad Sci USA 102:7470\u0026ndash;7475\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYue C, Genersch E (2005) RT-PCR analysis of Deformed wing virus in honeybees (\u003cem\u003eApis mellifera\u003c/em\u003e) and mites (\u003cem\u003eVarroa destructor\u003c/em\u003e). J Gen Virol 86:3419\u0026ndash;3424\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZak JD, Reddy G, Vergassola M et al (202) Antagonistic odor interactions in olfactory sensory neurons are widespread in freely breathing mice. \u003cem\u003eNat Commun\u003c/em\u003e 11, 3350. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-020-17124-5\u003c/span\u003e\u003cspan address=\"10.1038/s41467-020-17124-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Liu J, Gao F, Chen M, Jiang Y, Zhao H, Ma W (2022) Electrophysiological and Behavioral Responses of \u003cem\u003eApis mellifera\u003c/em\u003e and \u003cem\u003eBombus terrestris\u003c/em\u003e to Melon Flower Volatiles. Insects 13:973. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/insects13110973\u003c/span\u003e\u003cspan address=\"10.3390/insects13110973\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"3e07dac7-5928-44a4-a1f9-e0420a9230f3","identifier":"10.13039/501100010751","name":"Fondo Nacional de Desarrollo Científico, Tecnológico y de Innovación Tecnológica","awardNumber":"1241994","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Universidad de Concepcion","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Honey bees, DWV-A, Behaviors","lastPublishedDoi":"10.21203/rs.3.rs-5582583/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5582583/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHoney bees (\u003cem\u003eApis mellifera\u003c/em\u003e) play a crucial role in pollination, and their olfactory system is essential for food collection and source selection. This study evaluates how the Deformed Wing Virus (DWV), particularly its variant A, affects the olfactory sensitivity and behavioral responses of bees (10 to 20 days old) to volatile compounds from different pollen sources. We collected and analyzed the volatile fractions of three types of pollen (polyfloral and two monofloral) using dynamic HeadSpace and gas chromatography-mass spectrometry (GC-MS). The chemical analysis revealed differences in volatile compound profiles among the pollen types, including the presence of benzaldehyde, lilac alcohol, and 1\u0026ndash;4 cineole, which are known to impact honey bee olfaction. Behavioral assays using a Y-olfactometer showed that while non-inoculated bees (N-DWV) responded to the aromas of all pollens in higher proportions, DWV-inoculated bees (I-DWV) exhibited significant reductions in behavioral responses. Specifically, I-DWV bees showed lower response rates, and a higher proportion of non-responding individuals compared to N-DWV bees. Notably, I-DWV bees were more attracted to the aroma of \u003cem\u003eEucryphia cordifolia\u003c/em\u003e when exposed simultaneously to the aroma of \u003cem\u003eRubus ulmifolius\u003c/em\u003e, possibly due to a self-medication behavior or a selective response to pollen with higher antiviral properties. Electrophysiological recordings indicated that DWV-A infection decreases olfactory sensitivity, particularly in response to complex odors. This decline in olfactory function and behavioral preferences could compromise foraging efficiency and overall colony health. These findings highlight the impact of DWV-A on honey bee sensory and behavioral processes, raising concerns about broader implications for colony survival and pollination services.\u003c/p\u003e","manuscriptTitle":"Deformed Wing Virus-Induced Changes in Honey bee Reception and Preference for Pollen Scents","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-06 06:43:57","doi":"10.21203/rs.3.rs-5582583/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"98be4d38-657c-4eef-b646-3698e6125316","owner":[],"postedDate":"December 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-06T06:43:57+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-06 06:43:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5582583","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5582583","identity":"rs-5582583","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}