No abstract

In the temperate climates of middle Europe and North America, two distinct honeybee (Apis mellifera) populations are found in colonies: short-living summer bees emerge in spring and survive until summer, whereas long-living winter bees emerge in late August and overwinter. Besides the difference in their life spans, each of these populations fulfills a different role in the colonies and individual bees have distinct physiological and immunological adaptations depending on their roles. For instance, winter worker bees have higher vitellogenin levels and larger reserves of nutrients in the fat body than summer bees. The differences between the immune systems of both populations are well described at the constitutive level; however, our knowledge of its inducibility is still very limited. In this study, we focus on the response of 10-day-old honeybee workers to immune challenges triggered in vivo by injecting heat-killed bacteria, with particular focus on honeybees that emerge and live under hive conditions. Responses to bacterial injections differed between summer and winter bees. The latter induced more intense response, including higher expression of antimicrobial genes and antimicrobial activity, as well as a significant decrease in vitellogenin gene expression and its concentration in the hemolymph. The intense immune response observed in winter honeybees may contribute to our understanding of the relationships between colony fitness and infection with pathogens, as well as its association with successful overwintering.

European foulbrood (EFB) is an infectious disease of honey bees caused by the bacterium Melissococcus plutonius. A method for DNA isolation and conventional PCR diagnosis was developed using hive debris, which was non-invasively collected on paper sheets placed on the bottom boards of hives. Field trials utilized 23 honey bee colonies with clinically positive symptoms and 21 colonies without symptoms. Bayes statistics were applied to calculate the comparable parameters for EFB diagnostics when using honey, hive debris, or samples of adult bees. The reliability of the conventional PCR was 100% at 6.7 × 103 Colony Forming Unit of M. plutonius in 1 g of debris. The sensitivity of the method for the sampled honey, hive debris, and adult bees was 0.867, 0.714, and 1.000, respectively. The specificity for the tested matrices was 0.842, 0.800, and 0.833. The predictive values for the positive tests from selected populations with 52% prevalence were 0.813, 0.833, and 0.842, and the real accuracies were 0.853, 0.750, and 0.912, for the honey, hive debris, and adult bees, respectively. It was concluded that hive debris can effectively be utilized to non-invasively monitor EFB in honey bee colonies.

American foulbrood (AFB) is a dangerous disease of honeybees (Apis mellifera) caused by the spore-forming bacterium Paenibacillus larvae. According to the ERIC (enterobacterial repetitive intergenic consensus) classification, five genotypes are distinguished, i.e., I, II, III, IV, and V, which differ in their virulence and prevalence in colonies. In the Czech Republic, AFB prevalence is monitored by the State Veterinary Administration; however, the occurrence of specific P. larvae genotypes within the country remains unknown. In this study, our aim was to genotype field P. larvae strains collected in the Czech Republic according to the ERIC classification. In total, 102 field isolates from colonies with AFB clinical symptoms were collected from various locations in the Czech Republic, and the PCR genotypization was performed using ERIC primers. We confirmed the presence of both ERIC I and II genotypes, while ERIC III, IV, and V were not detected. The majority of samples (n = 82, 80.4%) were identified as ERIC II, while the ERIC I genotype was confirmed only in 20 samples (19.6%). In contrast to other European countries, the ERIC II genotype is predominant in Czech honeybee colonies. The ERIC I genotype was mostly detected in border regions close to Poland, Slovakia, and Austria.

Supplementary data S1 Qualitative and quantitative analyses of pigmentsQualitative analysis of pigments in the extracts was performed by reverse-phase HPLC-DAD according to a previously published protocol (Shukla et al., 2018). Quantitative analysis of chlorophyll a and b and lutein in C. vulgaris was performed by HPLC-APCI-HRMS using the analytical standard of the pigment for quantification. Solutions of chlorophyll a and b (Merck KGaA, Darmstadt, Germany) and lutein (Merck KGaA, Darmstadt, Germany) in methanol were prepared in the range of concentrations 0.1-100 µg/mL. The pigment standards together with individual samples were separated using Dionex UltiMate 3000 HPLC system (Thermo Scientific, Sunnyvale, CA, USA) coupled with a diode array detector (Shukla et al., 2018). The samples were loaded to a reverse-phase column (Phenomenex Kinetex C18 column, 150 × 4.6 mm, 2.6 μm) which was held at a constant temperature of 30°C. The chromatographic run was performed using as mobile phase the combination of 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B) using the following gradient: 0-1 min, 60% A; 1-20 min, 60%-0% A; 20-30 min, 0% A; 30-35 min, 0%-85% A, which was pumped at a constant flow rate of 0.6 mL/min. The Impact HD Mass Spectrometer was equipped with an atmospheric-pressure ionization source (APCI). The source parameters were as follows: the needle voltage was set at 4.0 kV, nitrogen was used both as the nebulizing gas (3 bar) and the drying gas (12 L/min), the drying heater was 200°C the APCI heater temperature was 450°C. The scanning range was 50-3,000 m/z operating in the positive ion mode. Concentrations of particular pigments in the extracts were calculated using linear regression from HPLC-APCI-HRMS signals. Results Qualitative and quantitative analyses of pigments present in extracts of C. vulgaris