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.

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.

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 elements (iron, zinc and copper). Colonies were selected from four apiaries in the province of Bologna (Italy), located in different environmental and foraging conditions. Haemolymph samples were taken from June to October 2019. The Varroa infestation of the colonies was estimated by assessing the natural fall of the mites, while the bees were tested for Nosema spp. spores using microscopic method. Proteins were separated using SDS-PAGE and colony performance was assessed by determining adult bees, total brood, and 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 haemolymph proteins and colony performance measures. A positive correlation between pollen reserves and 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.

Although traditional methods of evaluating colony health provide a general overview, they are often subjective and imprecise. Therefore, a more integrative approach is needed. The aim of this study is to use a multi-omics strategy—combining proteomic and metallomic approaches—to evaluate the interactions between honeybees and their environment. Two apiaries were selected for the study: apiary A, which is located in an extensive agricultural landscape, and apiary M, which is located in a less anthropized environment. The distribution of honeybee soluble proteins and small biomolecules was analyzed using size-exclusion chromatography. These profiles varied significantly between sampling times and apiaries. During spring, a reduced high molecular mass protein peak and an elevated very low molecular mass molecules peak were observed in honeybees from apiary A. Hemolymph analysis corroborated these findings, showing a depletion of key nutritional proteins, such as vitellogenin and apolipophorin in this apiary during the same season. In addition to protein distribution, we studied also the speciation of zinc and copper using chromatographic and atomic absorption techniques. Our results indicate site and season-dependent variation in metal binding profiles, with evidence for the presence of metallothionein-like proteins capable of binding both copper and zinc. This study highlights the urgent need for integrated, multi-method research to understand the complex dynamics of honeybee health and emphasizes the fundamental role of agricultural practices in ensuring the health of honeybees.

Microalgae are used as dietary supplements for humans and animals, due to their excellent nutritional profile. This research aims to characterize Spirugrass^®, a co-product obtained after the extraction of phycocyanin from Limnospira platensis, and to evaluate whether a stabilization treatment involving high-pressure processing (HPP) affects its proteomic profile. The research also aims to evaluate the possible use of Spirugrass^® as feed integration for honeybee health. Proteins were extracted and fractionated using size exclusion chromatography (SEC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The iron content was measured using atomic absorption spectrometry (AAS). Samples of Spirugrass^® were subjected to HPP at 600 MPa for five minutes and the effect on the integrity of the protein profile was analyzed. Finally, nine groups of 30 newly emerged honeybees were supplemented with Spirugrass^® in a controlled laboratory experiment. SDS-PAGE and SEC enabled the assessment of the quality and integrity of the Spirugrass^® proteome, which contains 80% of the proteins found in the algal biomass, including phycocyanin. The phycocyanin purity indices were 2.07 ± 0.14 and 0.72 ± 0.13 for the L. platensis and Spirugrass^® extracts, respectively. Spirugrass^® maintains a consistent iron content of 261 ± 15 μg/g dry weight, equivalent to 74% of the iron present in the algal biomass. In both L. platensis and Spirugrass^®, iron was predominantly bound to high-molecular-mass proteins, including phycocyanin. Following HPP treatment, differences in the protein profiles were observed, which suggests partial protein degradation. Preliminary data obtained with honeybees are encouraging, as no mortality or adverse effects were observed and Spirugrass^® can be considered a promising candidate as feed supplement. Overall, the presence of consistent levels of soluble proteins, as well as protein-bound iron, suggests that Spirugrass^® could be used in animal nutrition.