Field Application of an Innovative Approach to Assess Honeybee Health and Nutritional Status.

Environment, forage quality, management practices, pathogens, and pesticides influence honeybee responses to stressors. This study proposes an innovative approach to assess colony health and performance using molecular diagnostic tools by correlating hemolymph proteins with common measures of colony strength, prevalent honeybee pathogens (Varroa destructor and Nosema spp.), and essential trace elements (iron, zinc and copper). Colonies were selected from four apiaries located in different environmental and foraging conditions in the province of Bologna (Italy). Hemolymph samples were taken from June to October 2019. The Varroa infestation of the colonies was estimated by assessing the natural mortality of the mites, while the bees were tested for Nosema spp. spores using a microscopic method. Hemolymph proteins were quantified and separated using SDS-PAGE, and colony performance was assessed by determining adult bees, total brood, honey, and pollen reserves. The biomarkers measured proved to be useful for monitoring changes in performance and trophic conditions during summer and early autumn. Significant correlations were found between hemolymph proteins and colony performance measures. A positive correlation between pollen reserves, vitellogenin, and hexamerin 70a highlights the importance of these proteins for successful overwintering. In October, Varroa infestation was negatively correlated with total proteins, vitellogenin, apolipophorin II, transferrin, and hexamerin 70a, with negative implications for overwintering; furthermore, Varroa infestation was also negatively correlated with iron content, potentially affecting iron homeostasis.

SDS-PAGE-Based Quantitative Assay of Hemolymph Proteins in Honeybees: Progress and Prospects for Field Application.

In human and veterinary medicine, serum proteins are considered to be useful biomarkers for assessing the health and nutritional status of the organism. Honeybee hemolymph has a unique proteome that could represent a source of valuable biomarkers. Therefore, the aims of this study were to separate and identify the most abundant proteins in the hemolymph of worker honeybees to suggest a panel of these proteins that could represent useful biomarkers for assessing the nutritional and health status of the colonies and, finally, to analyze them in different periods of the year. Four apiaries were selected in the province of Bologna, and the bees were analyzed in April, May, July, and November. Thirty specimens from three hives of each apiary were sampled and their hemolymph was collected. The most represented bands obtained after 1D sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were cut from the gel, and the proteins were identified using an LC-ESI-Q-MS/MS System. A total of twelve proteins were unmistakably identified; the two most abundant proteins were apolipophorin and vitellogenin, which are known biomarkers of bee trophic and health status. The two other proteins identified were transferrin and hexamerin 70a, the first being involved in iron homeostasis and the second being a storage protein. Most of these proteins showed an increase from April to November, mirroring the physiological changes of honeybees during the productive season. The current study suggests a panel of biomarkers from honeybee hemolymph worth testing under different physiological and pathological field conditions.

A Machine Learning Approach to Study Demographic Alterations in Honeybee Colonies Using SDS-PAGE Fingerprinting.

Honeybees, as social insects, live in highly organised colonies where tasks reflect the age of individuals. As is widely known, in this context, emergent properties arise from interactions between them. The accelerated maturation of nurses into foragers, stimulated by many negative factors, may disrupt this complex equilibrium. This complexity needs a paradigm shift: from the study of a single stressor to the study of the effects exerted by multiple stressors on colony homeostasis. The aim of this research is, therefore, to study colony population disturbances by discriminating overaged nurses from proper aged nurses and precocious foragers from proper aged foragers using SDS-PAGE patterns of haemolymph proteins and a machine-learning algorithm. The KNN (K Nearest Neighbours) model fitted on the forager dataset showed remarkably good performances (accuracy 0.93, sensitivity 0.88, specificity 1.00) in discriminating precocious foragers from proper aged ones. The main strength of this innovative approach lies in the possibility of it being deployed as a preventive tool. Depopulation is an elusive syndrome in bee pathology and early detection with the method described could shed more light on the phenomenon. In addition, it enables countermeasures to revert this vicious circle.

Essential (Mg, Fe, Zn and Cu) and Non-Essential (Cd and Pb) Elements in Predatory Insects (Vespa crabro and Vespa velutina): A Molecular Perspective.

The recent introduction of the Asian yellow-legged hornet, Vespa velutina, into Europe has raised concern regarding the threat to honeybees and the competition with the European hornet, Vespa crabro. The aim of this study was to investigated essential (Mg, Fe, Zn, Cu) and non-essential (Cd and Pb) elements in these two species. Element concentrations were determined in the whole body and separately in the head, thorax and abdomen using atomic absorption spectrometry (AAS). The changes in essential element concentration and speciation during metamorphosis were also studied using size exclusion chromatography followed by AAS and proteomic analysis. In both species, the essential elements were more concentrated in the abdomen due to the presence of fat bodies. Magnesium, Fe and Zn concentrations were significantly higher in V. crabro than in V. velutina and could have been related to the higher aerobic energy demand of the former species required to sustain foraging flight. Low concentrations of Cd and Pb were indicative of low environmental exposure. The concentration and speciation of essential elements, particularly Fe, varied among the developmental stages, indicating a modification of ligand preferences during metamorphosis. Overall, the results in the present study provide a better understanding of the hornet metal metabolism and a foundation for additional studies.

Shotgun sequencing of honey DNA can describe honey bee derived environmental signatures and the honey bee hologenome complexity.

Honey bees are large-scale monitoring tools due to their extensive environmental exploration. In their activities and from the hive ecosystem complex, they get in close contact with many organisms whose traces can be transferred into the honey, which can represent an interesting reservoir of environmental DNA (eDNA) signatures and information useful to analyse the honey bee hologenome complexity. In this study, we tested a deep shotgun sequencing approach of honey DNA coupled with a specifically adapted bioinformatic pipeline. This methodology was applied to a few honey samples pointing out DNA sequences from 191 organisms spanning different kingdoms or phyla (viruses, bacteria, plants, fungi, protozoans, arthropods, mammals). Bacteria included the largest number of species. These multi-kingdom signatures listed common hive and honey bee gut microorganisms, honey bee pathogens, parasites and pests, which resembled a complex interplay that might provide a general picture of the honey bee pathosphere. Based on the Apis mellifera filamentous virus genome diversity (the most abundant detected DNA source) we obtained information that could define the origin of the honey at the apiary level. Mining Apis mellifera sequences made it possible to identify the honey bee subspecies both at the mitochondrial and nuclear genome levels.

Combined exposure to sublethal concentrations of an insecticide and a fungicide affect feeding, ovary development and longevity in a solitary bee.

Pollinators in agroecosystems are often exposed to pesticide mixtures. Even at low concentrations, the effects of these mixtures on bee populations are difficult to predict due to potential synergistic interactions. In this paper, we orally exposed newly emerged females of the solitary bee Osmia bicornis to environmentally realistic levels of clothianidin (neonicotinoid insecticide) and propiconazole (fungicide), singly and in combination. The amount of feeding solution consumed was highest in bees exposed to the neonicotinoid, and lowest in bees exposed to the pesticide mixture. Ovary maturation and longevity of bees of the neonicotinoid and the fungicide treatments did not differ from those of control bees. By contrast, bees exposed to the pesticide mixture showed slow ovary maturation and decreased longevity. We found a synergistic interaction between the neonicotinoid and the fungicide on survival probability. We also found an interaction between treatment and emergence time (an indicator of physiological condition) on longevity. Longevity was negatively correlated to physiological condition only in the fungicide and the mixture treatments. Delayed ovary maturation and premature death imply a shortened nesting period (highly correlated to fecundity in Osmia). Our findings provide a mechanism to explain the observed dynamics of solitary bee populations exposed to multiple chemical residues in agricultural environments.

A novel TaqMan® assay for Nosema ceranae quantification in honey bee, based on the protein coding gene Hsp70.

Nosema ceranae is now a widespread honey bee pathogen with high incidence in apiculture. Rapid and reliable detection and quantification methods are a matter of concern for research community, nowadays mainly relying on the use of biomolecular techniques such as PCR, RT-PCR or HRMA. The aim of this technical paper is to provide a new qPCR assay, based on the highly-conserved protein coding gene Hsp70, to detect and quantify the microsporidian Nosema ceranae affecting the western honey bee Apis mellifera. The validation steps to assess efficiency, sensitivity, specificity and robustness of the assay are described also.

Neonicotinoid pesticides and nutritional stress synergistically reduce survival in honey bees.

The honey bee is a major pollinator whose health is of global concern. Declines in bee health are related to multiple factors, including resource quality and pesticide contamination. Intensive agricultural areas with crop monocultures potentially reduce the quality and quantity of available nutrients and expose bee foragers to pesticides. However, there is, to date, no evidence for synergistic effects between pesticides and nutritional stress in animals. The neonicotinoids clothianidin (CLO) and thiamethoxam (TMX) are common systemic pesticides that are used worldwide and found in nectar and pollen. We therefore tested if nutritional stress (limited access to nectar and access to nectar with low-sugar concentrations) and sublethal, field-realistic acute exposures to two neonicotinoids (CLO and TMX at 1/5 and 1/25 of LD50) could alter bee survival, food consumption and haemolymph sugar levels. Bee survival was synergistically reduced by the combination of poor nutrition and pesticide exposure (-50%). Nutritional and pesticide stressors reduced also food consumption (-48%) and haemolymph levels of glucose (-60%) and trehalose (-27%). Our results provide the first demonstration that field-realistic nutritional stress and pesticide exposure can synergistically interact and cause significant harm to animal survival. These findings have implications for current pesticide risk assessment and pollinator protection.