Bee Tissues Research Articles

Systemic Israeli acute paralysis virus (IAPV) infection in bumblebees (Bombus terrestris) through feeding and injection.

Israeli acute paralysis virus (IAPV) can cause a systemic infection, resulting in mortality in both Apis and Bombus spp. bees. However, little is known about the virus infection dynamics within bee tissues. Here, we established systemic IAPV infections in reared bumblebee Bombus terrestris workers through feeding and injection and investigated the mortality, tissue tropism and viral localization. Injection of approximately 500 IAPV (IAPVinj stock) particles resulted in acute infection, viral loads within tissues that were relatively stable from bee to bee, and a distinctive tissue tropism, making this method suitable for studying systemic IAPV infection in bumblebees. Feeding with approximately 1 × 106 particles of the same virus stock did not result in systemic infection. A high-concentration stock of IAPV (IAPVfed stock) allowed us to feed bumblebees with approximately 1 × 109 viral particles, which induced both chronic and acute infection. We also observed a higher variability in viral titers within tissues and less clear tissue tropism during systemic infection, making feeding with IAPVfed stock less optimal for studying IAPV systemic infection. Strikingly, both infection methods and stocks with different viral loads gave a similar viral localization pattern in the brain and midgut of bumblebees with an acute infection. The implications of these findings in the study of the local immunity in bees and barriers to oral transmission are discussed. Our data provide useful information on the establishment of a systemic viral infection in bees.

Sample preservation, transport and processing strategies for honeybee RNA extraction: Influence on RNA yield, quality, target quantification and data normalization

Viral infections in managed honey bees are numerous, and most of them are caused by viruses with an RNA genome. Since RNA degrades rapidly, appropriate sample management and RNA extraction methods are imperative to get high quality RNA for downstream assays. This study evaluated the effect of various sampling-transport scenarios (combinations of temperature, RNA stabilizers, and duration) of transport on six RNA quality parameters; yield, purity, integrity, cDNA synthesis efficiency, target detection and quantification. The use of water and extraction buffer were also compared for a primary bee tissue homogenate prior to RNA extraction. The strategy least affected by time was preservation of samples at −80°C. All other regimens turned out to be poor alternatives unless the samples were frozen or processed within 24h. Chemical stabilizers have the greatest impact on RNA quality and adding an extra homogenization step (a QIAshredder™ homogenizer) to the extraction protocol significantly improves the RNA yield and chemical purity. This study confirms that RIN values (RNA Integrity Number), should be used cautiously with bee RNA. Using water for the primary homogenate has no negative effect on RNA quality as long as this step is no longer than 15min.

Quantifying exposure of wild bumblebees to mixtures of agrochemicals in agricultural and urban landscapes

The increased use of pesticides has caused concern over the possible direct association of exposure to combinations of these compounds with bee health problems. There is growing proof that bees are regularly exposed to mixtures of agrochemicals, but most research has been focused on managed bees living in farmland, whereas little is known about exposure of wild bees, both in farmland and urban habitats. To determine exposure of wild bumblebees to pesticides in agricultural and urban environments through the season, specimens of five different species were collected from farms and ornamental urban gardens in three sampling periods. Five neonicotinoid insecticides, thirteen fungicides and a pesticide synergist were analysed in each of the specimens collected. In total, 61% of the 150 individuals tested had detectable levels of at least one of the compounds, with boscalid being the most frequently detected (35%), followed by tebuconazole (27%), spiroxamine (19%), carbendazim (11%), epoxiconazole (8%), imidacloprid (7%), metconazole (7%) and thiamethoxam (6%). Quantifiable concentrations ranged from 0.17 to 54.4 ng/g (bee body weight) for individual pesticides. From all the bees where pesticides were detected, the majority (71%) had more than one compound, with a maximum of seven pesticides detected in one specimen. Concentrations and detection frequencies were higher in bees collected from farmland compared to urban sites, and pesticide concentrations decreased through the season. Overall, our results show that wild bumblebees are exposed to multiple pesticides when foraging in agricultural and urban landscapes. Such mixtures are detected in bee tissues not just during the crop flowering period, but also later in the season. Therefore, contact with these combinations of active compounds might be more prolonged in time and widespread in the environment than previously assumed. These findings may help to direct future research and pesticide regulation strategies to promote the conservation of wild bee populations.

Levels of Selected Persistent Organic Pollutants (PCB, PBDE) and Pesticides in Honey Bee Pollen Sampled in Poland.

Chemical plant protection is a commonly discussed factor potentially responsible for decline in pollinators and other beneficial insect populations. Various groups of chemicals including persistent organic pollutants could impact a bee colony’s welfare and are reported to be present in bee tissue and apiary products. The aim of this work was to evaluate the presence of selected persistent organic pollutant and pesticide residues in bee pollen originating from different geographical regions of Poland. Pesticide residues were identified in 60% of tested bee pollen samples. The compounds identified were mainly active ingredients of fungicide preparations. Insecticide active ingredients were up to 30% of the identified residues. The triazole fungicide tebuconazole and the neonicotinoid insecticide thiacloprid were the most frequently found pesticides in pollen. The highest pesticide concentration was determined for prothioconazole (356 μg kg-1). Mean concentrations of chlorinated biphenyls–EC6 and EC12 were 194 pg g-1 and 74 pg g-1, respectively. CB # 28 has the greatest share in the EC6 profile (mean 61 pg g−1, 31% contribution). Relatively high contributions were also observed for CBs # 101 (35 pg g−1, 18%), # 138 (36 pg g−1, 19%) and # 153 (33 pg g−1, 17%). CB # 114 and 118 have the highest share in the dioxin-like biphenyls fraction with mean concentrations of 17.6 and 37.6 pg g−1 (respectively 23 and 50%). Mean calculated concentrations of 39 polybrominated diphenyl ether congeners (Σ39 BDE) were 20 ± 27.7 pg g−1. High variability was observed between maximal and minimal determined concentration values. Individual BDEs were found at different frequencies and varying concentration levels. BDEs # 47, 75 and 99 dominated the profile with average concentrations of 3 pg g−1, 3.1 pg g−1, and 2.9 pg g−1, respectively.

The influence of sublethal doses of imidacloprid on protein content and proteolytic activity in honey bees (Apis mellifera L.)

The effect of sublethal doses of imidacloprid on protein content and activity of proteases on honey bees was analyzed. The study was conducted in three experimental groups: colonies from groups BE-5 and BE-200 were contaminated with 5 and 200 ppb of imidacloprid, respectively, via their food supply (syrup and pollen), while group BE was used as control (untreated). Bee samples were collected 3 and 10 weeks after feeding started. Protein concentration in bee tissue extracts was analyzed with reference: (a) to the dose of imidacloprid; and (b) duration of exposure to the chemical. The average quantity of protein content was significantly higher at the 3-week interval than in the 10-week interval and the bees from control colonies (BE) had significantly higher protein contents than contaminated bees (BE-5 and BE-200), even 3 weeks after feeding with imidacloprid started. Similarly, the activity of proteolytic enzymes (proteases) was found to be dependent on the dose of imidacloprid used, compared to bees from control colonies showing significantly higher activity.

A hybrid approach based on tissue P systems and artificial bee colony for IIR system identification

This paper presents a hybrid approach for infinite impulse response (IIR) system identification, called ABC-PS, that combines artificial bee colony (ABC) and tissue P systems. A tissue P system with fully connected structure of cells has been considered as its computing framework. A modification of ABC was developed as evolution rules for objects according to fully connected structure and communication mechanism. With the control of the object’s evolution-communication mechanism, the tissue P system designed can effectively and efficiently identify the optimal filter coefficients for an IIR system. The performance of ABC-PS was compared with artificial bee colony and several other evolutionary algorithms. Simulation results show that ABC-PS is superior or comparable to the other algorithms for the employed examples and can be efficiently used for IIR system identification.

Multi-Drug Resistance Transporters and a Mechanism-Based Strategy for Assessing Risks of Pesticide Combinations to Honey Bees.

Annual losses of honey bee colonies remain high and pesticide exposure is one possible cause. Dangerous combinations of pesticides, plant-produced compounds and antibiotics added to hives may cause or contribute to losses, but it is very difficult to test the many combinations of those compounds that bees encounter. We propose a mechanism-based strategy for simplifying the assessment of combinations of compounds, focusing here on compounds that interact with xenobiotic handling ABC transporters. We evaluate the use of ivermectin as a model substrate for these transporters. Compounds that increase sensitivity of bees to ivermectin may be inhibiting key transporters. We show that several compounds commonly encountered by honey bees (fumagillin, Pristine, quercetin) significantly increased honey bee mortality due to ivermectin and significantly reduced the LC50 of ivermectin suggesting that they may interfere with transporter function. These inhibitors also significantly increased honey bees sensitivity to the neonicotinoid insecticide acetamiprid. This mechanism-based strategy may dramatically reduce the number of tests needed to assess the possibility of adverse combinations among pesticides. We also demonstrate an in vivo transporter assay that provides physical evidence of transporter inhibition by tracking the dynamics of a fluorescent substrate of these transporters (Rhodamine B) in bee tissues. Significantly more Rhodamine B remains in the head and hemolymph of bees pretreated with higher concentrations of the transporter inhibitor verapamil. Mechanism-based strategies for simplifying the assessment of adverse chemical interactions such as described here could improve our ability to identify those combinations that pose significantly greater risk to bees and perhaps improve the risk assessment protocols for honey bees and similar sensitive species.

A fluorescent method for visualization of Nosema infection in whole-mount honey bee tissues

Honey bees are critical pollinators in both agricultural and ecological settings. The Nosema species, ceranae and apis, are microsporidian parasites that are pathogenic to honey bees. While current methods for detecting Nosema infection have key merits, additional techniques with novel properties for studying the cell biology of Nosema infection are highly desirable. We demonstrate that whole-mount staining of honey bee midgut tissue with chitin-binding agent Fluorescent Brightener 28 and DNA dye Propidium Iodide allows for observation of Nosema infection in structurally intact tissue, providing a new tool for increasing our understanding of Nosema infection at the cellular and tissue level.

A sensitive quantification of the peptide apidaecin 1 isoforms in single bee tissues using a weak cation exchange pre-separation and nanocapillary liquid chromatography coupled with mass spectrometry

Apidaecins represent an important group of antimicrobial peptides occurring in honey bee hemolymph, where they play an important role as key components of humoral immunity. The present study demonstrates the development of a highly sensitive assay for apidaecin 1 isoforms quantification in the hemolymph or body parts from honey bee individuals. The analytical protocol comprises apidaecins 1 purification and enrichment steps by weak cation-exchange chromatography (WCX) in laboratory-made WCX-Tip microcolumns combined with a desalting step on a reversed-phase sorbent (C8) carried in StageTips. Apidaecin-enriched fraction was analyzed by a reversed-phase based nanoliquid chromatography (C4) separation coupled with high-resolution mass spectrometry. The method performance was validated in its specificity, linearity (0–5pmol), recovery (∼45%), precision (<10% at 0.1pmol), limit of detection (∼50fmol), limit of quantification (0.1pmol) and sample stability. The method was successfully applied to analyze the content of apidaecin 1 isoforms in the following samples: hemolymph – 13.0ng/μL (95% confidence interval of 7.5–18.6ng/μL), thoraxes – 36.2ng/unit (95% CI of 18.9–53.6ng/unit) and heads – 12.9ng/unit (95% CI of 9.1–16.7ng/unit). Freshly emerged bees had apidaecin 1 isoforms levels below the limit of detection. Thus it was possible to use them as a competitive matrix for calibration standards to prevent losses of highly basic apidaecins. This new protocol for apidaecin 1 isoforms quantification represents a promising tool to study the role of apidaecins in honey bee immunity and can be considered as a proof-of-concept for the development of sensitive quantification methods for basic antimicrobial peptides in various organisms.

Distinguishing feral and managed honeybees (Apis mellifera) using stable carbon isotopes

The ability to distinguish feral and managed honeybees (Apis mellifera) has applications in studies of population genetics, parasite transmission, pollination, interspecific interactions, and bee breeding. We evaluated a diagnostic test based on theoretical differences in stable carbon isotope ratios generated by supplemental feeding. We evaluated (1) if carbon isotope ratios can distinguish feral and managed honeybees and (2) the temporal persistence of the signal after discontinuation of supplemental feeding. We compared carbon isotope ratios from four types of experimental colonies: feral, managed with and without supplemental feed, and managed with 13C-labeled glucose added to supplemental feed. There was a significant difference between the isotopic signatures of colonies receiving supplemental feed and unfed feral colonies. This difference, however, only persisted for a few weeks after supplemental feeding was discontinued, suggesting that this method may work best under a narrow range of conditions. This work highlights the potential for exploiting temporal turnover of carbon in bee tissues as a tool for studying nutrient flow in honeybee colonies.

A Cell Line Resource Derived from Honey Bee (Apis mellifera) Embryonic Tissues

A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz’s L15 medium and incubated at 32°C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10–14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.

Honey Bee tissues as a biomarker for Environmental pollution with heavy metals.

The purpose of this study is to verify the importance of the body surface and tissues of foraginghoney bee Apis mellifera as bioaccumulator of heavy metals that can therefore, be included inbiomonitoring programmes of heavy metals in terrestrial ecosystems. The presence of fiverepresentative heavy metals (copper, zinc, cadmium, lead and iron) was investigated in honeybee tissues (mid gut, hind gut and fat body), body surface and wings using proton induced X-rayemission method. X-ray spectrums of mid gut and fat body of forager bees demonstrated smallpeaks for Cd, Pb, Fe and Ni compared to the control. Moreover, slightly larger peaks for Cu andZn were noticed in the X-ray spectrums of whole honey bee mid gut, hind gut and fat body thanthose of the control. Forager honey bee body surface showed small peaks for Cu and Pbcompared to the control. Similarly, small peaks for Cd, Pb and Ni were noticed in the X-rayspectrums of wings and hind gut of the forager honey bee compared to control.

Rapid method for DNA extraction from the honey bee Apis mellifera and the parasitic bee mite Varroa destructor using lysis buffer and proteinase K

We developed a rapid method for extraction of DNA from honey bees, Apis mellifera, and from the parasitic bee mite, Varroa destructor. The advantages include fast processing and low toxicity of the substances that are utilized. We used lysis buffer with nonionic detergents to lyse cell walls and proteinase K to digest proteins. We tested whole thorax, thoracic muscle mass, legs, and antennae from individual bees; the mites were processed whole (1 mite/sample). Each thorax was incubated whole, without cutting, because exocuticle color pigment darkened the extraction solution, interfering with PCR results. The procedure was performed with autoclaved equipment and laboratory gloves. For each sample, we used 100 µL lysis buffer (2 mL stock solution of 0.5 M Tris/HCl, pH 8.5, 10 mL stock solution of 2 M KCl, 500 µL solution of 1 M MgCl2, 2 mL NP40, and 27.6 g sucrose, completed to 200 mL with bidistilled water and autoclaved) and 2 µL proteinase K (10 mg/mL in bidistilled water previously autoclaved, as proteinase K cannot be autoclaved). Tissues were incubated in the solutions for 1-2 h in a water bath (62°-68 °C) or overnight at 37 °C. After incubation, the tissues were removed from the extraction solution (lysis buffer + proteinase K) and the solution heated to 92 °C for 10 min, for proteinase K inactivation. Then, the solution with the extracted DNA was stored in a refrigerator (4°-8 °C) or a freezer (-20 °C). This method does not require centrifugation or phenol/chloroform extraction. The reduced number of steps allowed us to sample many individuals/day. Whole mites and bee antennae were the most rapidly processed. All bee tissues gave the same quality DNA. This method, even using a single bee antenna or a single mite, was adequate for extraction and analysis of bee genomic and mitochondrial DNA and mite genomic DNA.

EFFECT OF THE ACCESSORY PROTEIN FEEDING ON HONEY BEE WORKER TISSUES AND HYPOPHARYNGEAL GLANDS

The accessory protein diets major applied with most bee-keepers; dried medical yeast, baby milk powder and pollen grains were investigated in order to test their physiological reflections on worker honey bee tissues and glandular secretion of the royal jelly the final secretion of the hypopharyngeal glands which indi-cated privileged chemical properties with all tested foods particu-larly with the pollen grains caused increase the secretary globule densities, synchronism that with presence physiological active exceeding in the proliferating of the small digestive cells of the mid gut tissues and the fat bodies in the integument of the body cavity. The milk as supported protein let to higher synthesis of the connective tissues of the hypopharyngeal glands tissue fol-lowed with the medical dried yeast. The cytoplasmic albuminoidal granules of the fat cells and the Oenocytoids showed more densi-ties with all tested protein diets particularly with pollen grain treatment.

Sudden deaths and colony population decline in Greek honey bee colonies

During June and July of 2009, sudden deaths, tremulous movements and population declines of adult honey bees were reported by the beekeepers in the region of Peloponnesus (Mt. Mainalo), Greece. A preliminary study was carried out to investigate these unexplained phenomena in this region. In total, 37 bee samples, two brood frames containing honey bee brood of various ages, eight sugar samples and four sugar patties were collected from the affected colonies. The samples were tested for a range of pests, pathogens and pesticides. Symptomatic adult honey bees tested positive for Varroa destructor, Nosema ceranae, Chronic bee paralysis virus (CBPV), Acute paralysis virus (ABPV), Deformed wing virus (DWV), Sacbrood virus (SBV) and Black queen cell virus (BQCV), but negative for Acarapis woodi. American Foulbrood was absent from the brood samples. Chemical analysis revealed that amitraz, thiametoxan, clothianidin and acetamiprid were all absent from symptomatic adult bees, sugar and sugar patty samples. However, some bee samples, were contaminated with imidacloprid in concentrations between 14 ng/g and 39 ng/g tissue. We present: the infection of Greek honey bees by multiple viruses; the presence of N. ceranae in Greek honey bees and the first record of imidacloprid (neonicotonoid) residues in Greek honey bee tissues. The presence of multiple pathogens and pesticides made it difficult to associate a single specific cause to the depopulation phenomena observed in Greece, although we believe that viruses and N. ceranae synergistically played the most important role. A follow up in-depth survey across all Greek regions is required to provide context to these preliminary findings.

Detecting changes in habitat-scale bee foraging in a tropical fragmented landscape using stable isotopes

As the body of research on the ecosystem service of pollination grows, our ability to tackle a range of agricultural, conservation, and land management issues is limited by our understanding of pollinator foraging patterns and requirements. In particular, better knowledge of which habitats bees utilize for foraging over their lifetime would inform a range of applied and theoretical questions. Traditional methods of studying foraging are either impractical for insects (e.g. radio tracking) or else are limited spatially or temporally (e.g. observations by researchers). Here we describe a method for using stable isotopes of carbon and nitrogen from bee tissues to gain an integrated signal of which habitats a bee has foraged in over its lifetime, using three species of social stingless bee (Apidae: Meliponini) in a fragmented tropical forest landscape in southern Costa Rica as a test case. There were strongly significant differences in the carbon and nitrogen isotope signals of bees collected in the landscape extremes: the largest forests and largest open pastures in the region. We could not estimate proportions of dietary carbon and nitrogen derived from different habitats, however, due to the high sample variance. Still, isotope ratios reflect a signature of the habitat that a bee has foraged in, and we found significant relationships between foraging patterns and season, landscape context (proportion of forest surrounding sample sites) and local biotic context (bee and plant diversity and abundance). Landscape context was the most consistent factor among the analyses, in line with the idea that meliponines do not preferentially forage in deforested habitats. Bee and plant diversity and abundance showed various significant relationships with the isotopic values of the different species, but the patterns were inconsistent. These results are potentially consistent with habitat switching from pasture to forest in areas of greater competition, but the extreme spatiotemporal variability in resource density likely blurs these patterns.

β-Cyclodextrins as Carriers of Monoterpenes into the Hemolymph of the Honey Bee (Apis mellifera) for Integrated Pest Management

The Varroa mite ( Varroa destructor) is becoming ubiquitous worldwide and is a serious threat to honey bees. The cultivation of certain food crops are at risk. The most noted acaricides against Varroa mites are tau-fluvaninate and coumaphos, but the mites are showing resistance. Since these insecticides are used in the proximity of honey, it is desirable to use natural alternatives. Monoterpenoids such as thymol and carvacrol, that are constituents of oil of thyme and oil of origanum, show promise as acaricides against the Varroa mite ( Varroa destructor), but the delivery of these compounds remains a challenge due to the low water solubility and uncontrolled release into the colony. Beta-cyclodextrin (beta-CD) inclusion complexes of thymol, oil of origanum, and carvacrol were prepared on a preparative scale. Competitive binding was studied by fluorescence spectroscopy by using 6- p-toluidinylnaphthalene-2-sulfonate as a fluorescent probe. The complexes were characterized, and the competitive binding described by (1)H and (13)C NMR spectroscopy chemical shifts. The toxicity of beta-CD and the prepared complexes in enriched sucrose syrup was studied by conducting caged honey bee ( Apis mellifera) feeding trials. After the first and second weeks of feeding, hemolymph and gut tissue samples were acquired from the caged bee study. The levels of thymol and carvacrol were quantified by solid-phase microextraction gas chromatography mass spectroscopy, using an optimized procedure we developed. High (mM) levels of thymol and carvacrol were detected in bee tissues without any imposed toxicity to the bees, in an effort to deter Varroa mites from feeding on honey bee hemolymph.

Extreme thermotolerance and behavioral induction of 70-kDa heat shock proteins and their encoding genes in honey bees

Foraging honey bees frequently leave the hive to gather pollen and nectar for the colony. This period of their lives is marked by periodic extremes of body temperature, metabolic expenditure, and flight muscle activity. Following ecologically relevant episodes of hyperthermia between 33 degrees C and 50 degrees C, heat shock protein 70 (Hsp70) expression and hsp70/hsc70-4 activity in brains of nonflying laboratory-held bees increased by only two to three times baseline at temperatures 46-50 degrees C. Induction was undetectable in thoracic-flight muscles. Yet, thorax hsp70 mRNA (but not hsc70-4 mRNA) levels were up to ten times higher in flight-capable hive bees and foraging bees compared to 1-day-old, flight-incapable bees, while brain hsp70/hsc70-4 mRNA levels were low and varied little among behavioral groups. These data suggest honey bee tissues, especially flight muscles, are extremely thermotolerant. Furthermore, Hsp70 expression in the thoraces of flight-capable bees is probably flight-induced by oxidative and mechanical damage to flight muscle proteins rather than temperature.

Computational and transcriptional evidence for microRNAs in the honey bee genome

A total of 68 non-redundant candidate honey bee miRNAs were identified computationally; several of them appear to have previously unrecognized orthologs in the Drosophila genome. Several miRNAs showed caste- or age-related differences in transcript abundance and are likely to be involved in regulating honey bee development.

Evaluation of a real-time two-step RT-PCR assay for quantitation of Chronic bee paralysis virus (CBPV) genome in experimentally-infected bee tissues and in life stages of a symptomatic colony

A two-step real-time RT-PCR assay, based on TaqMan technology using a fluorescent probe (FAM-TAMRA) was developed to quantify Chronic bee paralysis virus (CBPV) genome in bee samples. Standard curves obtained from a CBPV control RNA and from a plasmid containing a partial sequence of CBPV showed that this assay provided linear detection over a 7-log range ( R 2 > 0.99) with a limit of detection of 100 copies, and reliable inter-assay and intra-assay reproducibility. Standardisation including RNA purification and cDNAs synthesis was also validated. The CBPV TaqMan methodology was first evaluated by quantifying the CBPV genomic load in bee samples from an experimental infection obtained by topical application. Up to 1.9 × 10 10 CBPV copies per segment of insect body (head, thorax and abdomen) were revealed whereas a lower CBPV genomic load was detected in dissected organs such as mandibular and hypopharyngeal glands, brain and alimentary canal (up to 7.2 × 10 6 CBPV copies). The CBPV genomic loads in different categories of bees from a hive presenting the trembling symptoms typical of Chronic paralysis were then quantified. Significantly higher CBPV loads were found in guard, symptomatic and dead bees (up to 1.9 × 10 13 CBPV copies) than in forager, drones and house bees (up to 3.4 × 10 6 CBPV copies). The results obtained for symptomatic or dead bees support the correlation between high CBPV genomic load and pathology expression. Moreover, the high CBPV genomic load revealed in guard bees highlights the possible pivotal role played by this category of bees in CBPV infection.