Daniel Dezmirean

Pathogen-associated self-medication behavior in the honeybee Apis mellifera

Honeybees, Apis mellifera, have several prophylactic disease defense strategies, including the foraging of antibiotic, antifungal, and antiviral compounds of plant products. Hence, honey and pollen contain many compounds that prevent fungal and bacterial growth and inhibit viral replication. Since these compounds are also fed to the larvae by nurse bees, they play a central role for colony health inside the hive. Here, we show that honeybee nurse bees, infected with the microsporidian gut parasite Nosema ceranae, show different preferences for various types of honeys in a simultaneous choice test. Infected workers preferred honeys with a higher antibiotic activity that reduced the microsporidian infection after the consumption of the honey. Since nurse bees feed not only the larvae but also other colony members, this behavior might be a highly adaptive form of therapeutic medication at both the individual and the colony level.

Interactions among flavonoids of propolis affect antibacterial activity against the honeybee pathogen Paenibacillus larvae

Propolis is derived from plant resins, collected by honeybees (Apis mellifera) and renown for its antibacterial properties. Here we test the antibacterial effects of ethanolic extracts of propolis from different origins on Paenibacillus larvae, the bacterial pathogen that causes American Foulbrood, a larval disease that can kill the honeybee colony. All tested propolis samples inhibited significantly the growth of P. larvae tested in vitro. The extracts showed major differences in the content of total flavonoids (ranging from 2.4% to 16.4%) and the total polyphenols (ranging between 23.3% and 63.2%). We found that it is not only the content of compounds in propolis, which influences the strength of antimicrobial effects but there is also a significant interaction effect among flavonoids of the propolis extracts. We propose that interaction effects among the various chemical compounds in propolis should be taken into account when considering the antibacterial effects against honeybee pathogens.

Predominant and Secondary Pollen Botanical Origins Influence the Carotenoid and Fatty Acid Profile in Fresh Honeybee-Collected Pollen

Total and individual carotenoids, fatty acid composition of total lipids, and main lipid classes of 16 fresh bee-collected pollen samples from Romania were determined by high-performance liquid chromatography with photodiode array detection and capillary gas chromatography with mass detection. Analyzed samples were found rich in lutein, whereas β-criptoxanthin and β-carotene were present in a wide range of amounts correlated with predominant botanical origin of the samples. High amounts of lutein were correlated with the presence of Callendula officinalis, Taraxacum officinale and Anthylis sp. The highest amount of total lipids was found in samples where pollen from Brassica sp. was predominant. Lipid classes were dominated by polyunsaturated fatty acids. Saturated fatty acids were determined in variable amounts. Lipid and carotenoid contents present great variability, explained by the various botanical species present in the samples.

What Is the Main Driver of Ageing in Long-Lived Winter Honeybees: Antioxidant Enzymes, Innate Immunity, or Vitellogenin?

To date five different theories compete in explaining the biological mechanisms of senescence or ageing in invertebrates. Physiological, genetical, and environmental mechanisms form the image of ageing in individuals and groups. Social insects, especially the honeybee Apis mellifera, present exceptional model systems to study developmentally related ageing. The extremely high phenotypic plasticity for life expectancy resulting from the female caste system provides a most useful system to study open questions with respect to ageing. Here, we used long-lived winter worker honeybees and measured transcriptional changes of 14 antioxidative enzyme, immunity, and ageing-related (insulin/insulin-like growth factor signaling pathway) genes at two time points during hibernation. Additionally, worker bees were challenged with a bacterial infection to test ageing- and infection-associated immunity changes. Gene expression levels for each group of target genes revealed that ageing had a much higher impact than the bacterial challenge, notably for immunity-related genes. Antimicrobial peptide and antioxidative enzyme genes were significantly upregulated in aged worker honeybees independent of bacterial infections. The known ageing markers vitellogenin and IlP-1 were opposed regulated with decreasing vitellogenin levels during ageing. The increased antioxidative enzyme and antimicrobial peptide gene expression may contribute to a retardation of senescence in long-lived hibernating worker honeybees.

Botanical Origin Causes Changes in Nutritional Profile and Antioxidant Activity of Fermented Products Obtained from Honey

Honey as rich source of enzymatic and nonenzymatic antioxidants serves as health-promoting nutrient in the human body. Here, we present the first time a comparative study of nutritional profiles (e.g., acidities, sugar, organic acid profile, total polyphenolic, flavonoid content) for different unifloral, multifloral honeys and their fermented products, in correlation with their antioxidant activity. Additionally, an optimized method for HPLC separation of organic acids from honey was established. The total phenolic content of honey samples varied widely among the honey types compared to fermented products. High amounts of total flavonoids were quantified in heather honey, followed by raspberry, multifloral, black locust, and linden honey. A positive correlation between the content of polyphenols, flavonoids, and antioxidant activity was observed in honey samples. After fermentation, the flavonoid content of dark honey fermented products decreased significantly. Black locust and linden honeys are more suitable for fermentation because the decrease in antioxidant substances is less pronounced.

Climate rather than geography separates two European honeybee subspecies

Both climatic and geographical factors play an important role for the biogeographical distribution of species. The Carpathian mountain ridge has been suggested as a natural geographical divide between the two honeybee subspecies Apis mellifera carnica and A. m. macedonica. We sampled one worker from one colony each at 138 traditional apiaries located across the Carpathians spanning from the Hungarian plains to the Danube delta. All samples were sequenced at the mitochondrial tRNALeu‐cox2 intergenic region and genotyped at twelve microsatellite loci. The Carpathians had only limited impact on the biogeography because both subspecies were abundant on either side of the mountain ridge. In contrast, subspecies differentiation strongly correlated with the various temperature zones in Romania. A. m. carnica is more abundant in regions with the mean average temperature below 9 °C, whereas A. m. macedonica honeybees are more frequent in regions with mean temperatures above 9 °C. This range selection may have impact on the future biogeography in the light of anticipated global climatic changes.

Important Developments in Romanian Propolis Research

The most important developments in propolis analysis and pharmacological properties are discussed. In order to help in the Romanian propolis standardization, different methodologies for chemical composition analysis (UV-VIS, HP-TLC, and HPLC-DAD) are reviewed using new approaches and software (fuzzy divisive hierarchical clustering approach and ChromQuest software) and compared with international studies made until now in propolis research. Practical applications of Romanian propolis in medicinal therapy and cosmetics are reviewed, and quality criteria for further standardization are proposed.

Influence of phytochemical profile on antibacterial activity of different medicinal plants against gram-positive and gram-negative bacteria

Nettle (Urtica dioica), basil (Ocimum basilicum), thyme (Thymus serphyllium), costmary (Chrysanthemum balsamita) and yarrow (Achillea millefolium) are used worldwide as aromatic herbs and medicinal plants. The alcoholic macerates of these plants were prepared and their chemical composition was investigated by spectrophotometric methods and HPLC chromatography with photodiode array detection. Alcoholic extracts were tested also in vitro against 5 pathogenic bacterial strains: 2 Gram-positive (Staphyloccocus aureus and Bacillus cereus) and 3 Gram-negative (Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium). Minimum inhibitory concentrations for the extracts against all the microorganisms were determined by serial dilutions method. All the extracts demonstrated antibacterial activity against Gram-positive bacteria and partially against Gram-negative bacteria. In addition, nettle extract possessed the highest polyphenolic and flavonoid content. The results obtained indicated that these plants represent a potential source of natural antibacterial substances and can be used for the production of natural antibacterial formulations.

Medicinal Plants Based Products Tested on Pathogens Isolated from Mastitis Milk

Bovine mastitis a major disease that is commonly associated with bacterial infection. The common treatment is with antibiotics administered intramammary into infected quarters of the udder. The excessive use of antibiotics leads to multidrug resistance and associated risks for human health. In this context, the search for alternative drugs based on plants has become a priority in livestock medicine. These products have a low manufacturing cost and no reports of antimicrobial resistance to these have been documented. In this context, the main objective of this study was to determine the antimicrobial effect of extracts and products of several indigenous, or acclimatized plants on pathogens isolated from bovine mastitis. A total of eleven plant alcoholic extracts and eight plant-derived products were tested against 32 microorganisms isolated from milk. The obtained results have shown an inhibition of bacterial growth for all tested plants, with better results for Evernia prunastri, Artemisia absinthium, and Lavandula angustifolia. Moreover, E. prunastri, Populus nigra, and L. angustifolia presented small averages of minimum inhibitory and bactericidal concentrations. Among the plant-derived products, three out of eight have shown a strong anti-microbial effect comparable with the effect of florfenicol and enrofloxacin, and better than individual plant extracts possibly due to synergism. These results suggest an important anti-microbial effect of these products on pathogens isolated from bovine mastitis with a possible applicability in this disease.

Influence of geographic origin, plant source and polyphenolic substances on antimicrobial properties of propolis against human and honey bee pathogens

Natural honey bee products, especially propolis are well known for their biological properties. However, less well known are the impact of propolis’ single phenolic substances and variation across different landscapes, geographic locations and botanical origins. Here, we determined the chemical composition of propolis samples collected in different geographic areas of Romania with a continental climate. Main groups of polyphenols, as well as individual phenolics (phenolic acids and flavonoids), were identified and quantified. The antimicrobial activity of propolis ethanolic extracts and some of the most important propolis phenolics (caffeic and ferulic acid, pinocembrin, chrysin and galangin) were evaluated against different bacterial species. Analyzing the interaction of bacterial growth inhibition and different chemical parameters, revealed that flavone/flavonol, flavanone/dihydroflavonol, chrysin and galangin are largely responsible for the propolis’ antimicrobial activity against Gram-positive bacteria. Gram-negative bacteria were mostly inhibited by galangin, caffeic acid and naringenin. Chemical composition and antimicrobial activity of the propolis samples might be explained by the botanical sources present in the specific foraging areas. The highest amounts of polyphenols and flavonoids were measured in regions where deciduous forests were dominant to coniferous, or where large areas of willow could be observed.