Due to its multifunctional and complex role in the honey bee colony functioning and health (construction material allowing food storage, brood rearing, thermoregulation, mediation in chemical and mechanical communication, substrate for pathogens, toxins and waste), Apis mellifera beeswax has been widely studied over the last five decades. This is supported by a comprehensive set of scientific reports covering different aspects of beeswax research. In this article, we present an overview of the methods for studying chemical, biological, constructional, and quality aspects of beeswax. We provide a detailed description of the methods for investigating wax scales, comb construction and growth pattern, cell properties, chemical composition of beeswax using different analytical tools, as well as the analytical procedures for provenancing beeswax and beeswax-derived compounds based on the hydrogen isotope ratio (IRMS). Along with classical physico-chemical and sensory analysis, we describe more precise and accurate methods for detection of adulterants in beeswax (GC-MS and FTIR-ATR). Moreover, we present methods for studying the influence of beeswax (comb foundation) adulteration on comb construction. Analytical protocols for determining the pesticide residues using different chromatographic and spectroscopic techniques are also described. As beeswax is an agent of high risk for the transmission of bee diseases, we present methods for detection of pathogens in beeswax. To ensure the reproducibility of experiments and results, we present best practice approaches and detailed protocols for all methods described, as well as their advantages and disadvantages.

There is no systematic report about propolis chemical biodiversity from the Adriatic Sea islands affecting its antioxidant capacity. Therefore, the samples from the islands Krk, Rab, Pag, Biševo and Korčula were collected. Comprehensive methods were used to unlock their chemical biodiversity: headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD) followed by gas chromatography and mass spectrometry (GC-MS); Fourier transform mid-infrared spectroscopy (FT-MIR); ultra high performance liquid chromatography with diode array detector and quadrupole time-of-flight mass spectrometry (UHPLC-DAD-QqTOF-MS) and DPPH and FRAP assay. The volatiles variability enabled differentiation of the samples in 2 groups of Mediterranean propolis: non-poplar type (dominated by α-pinene) and polar type (characterized by cadinane type sesquiterpenes). Spectral variations (FT-MIR) associated with phenolics and other balsam-related components were significant among the samples. The UHPLC profiles allowed to track compounds related to the different botanical sources such as poplar (pinobanksin esters, esters and glycerides of phenolic acids, including prenyl derivatives), coniferous trees (labdane, abietane diterpenes) and Cistus spp. (clerodane and labdane diterpenes, methylated myricetin derivatives). The antioxidant potential determined by DPPH ranged 2.6–81.6 mg GAE/g and in FRAP assay 0.1–0.8 mmol Fe²⁺/g. The highest activity was observed for the samples of Populus spp. origin. The antioxidant potential and phenolic/flavonoid content was positively, significantly correlated.

Chemical characterization of bee pollen is of great importance for its quality estimation. Multifloral and unifloral bee pollen samples collected from continental, mountain and Adriatic regions of Croatia were analyzed by means of physico-chemical, chromatographic (GC-MS), and spectroscopic (FTIR-ATR) analytical tools, aiming to conduct a comprehensive characterization of bee pollen. The most distinctive unifloral bee pollen with regard to nutritional value was Aesculus hippocastanum (27.26% of proteins), Quercus spp. (52.58% of total sugars), Taraxacumofficinale (19.04% of total lipids), and Prunusavium (3.81% of ash). No statistically significant differences between multifloral and unifloral bee pollen from different regions were found for most of the physico-chemical measurement data, with an exception of melezitose (p = 0.04). Remarkable differences were found among the bee pollen HS VOCs. The major ones were lower aliphatic compounds, monoterpenes (mainly linalool derivatives, especially in Prunusmahaleb and P.avium bee pollen), and benzene derivatives (mainly benzaldehyde in T.officinale and Salix spp.). Aldehydes C₉ to C₁₇ were present in almost all samples. FTIR-ATR analysis revealed unique spectral profiles of analyzed bee pollen exhibiting its overall chemical composition arising from molecular vibrations related to major macromolecules—proteins, lipids, and carbohydrates (sugars).

Although beeswax adulteration represents one of the main beeswax quality issues, there are still no internationally standardised analytical methods for routine quality control. The objective of this study was to establish an analytical procedure suitable for routine detection of beeswax adulteration using FTIR-ATR spectroscopy. For the purpose of this study, reference IR spectra of virgin beeswax, paraffin, and their mixtures containing different proportions of paraffin (5 - 95%), were obtained. Mixtures were used for the establishment of calibration curves. To determine the prediction strength of IR spectral data for the share of paraffin in mixtures, the Partial Least Squares Regression method was used. The same procedure was conducted on beeswax-beef tallow mixtures. The model was validated using comb foundation samples of an unknown chemical background which had been collected from the international market (n = 56). Selected physico-chemical parameters were determined for comparison purposes. Results revealed a strong predictive power (R² = 0.999) of IR spectra for the paraffin and beef tallow share in beeswax. The results also revealed that the majority of the analysed samples (89%) were adulterated with paraffin; only 6 out of 56 (11%) samples were identified as virgin beeswax, 28% of the samples exhibited a higher level of paraffin adulteration (>46% of paraffin), while the majority of the analysed samples (50%) were found to be adulterated with 5 - 20% of paraffin. These results indicate an urgent need for routine beeswax authenticity control. In this study, we demonstrated that the analytical approach defining the standard curves for particular adulteration levels in beeswax, based on chemometric modelling of specific IR spectral region indicative for adulteration, enables reliable determination of the adulterant proportions in beeswax.

Neuronal activity is temperature sensitive and affects behavioral traits important for individual fitness, such as locomotion and courtship. Yet, we do not know enough about the evolutionary response of neuronal phenotypes in new temperature environments. Here, we use long-term experimental evolution of Drosophila simulans populations exposed to novel temperature regimes. Here, we demonstrate a direct relationship between thermal selective pressure and the evolution of neuronally expressed molecular and behavioral phenotypes. Several essential neuronal genes evolve lower expression at high temperatures and higher expression at low temperatures, with dopaminergic neurons standing out by displaying the most consistent expression change across independent replicates. We functionally validate the link between evolved gene expression and behavioral changes by pharmacological intervention in the experimentally evolved D. simulans populations as well as by genetically triggered expression changes of key genes in D. melanogaster. As natural temperature clines confirm our results for Drosophila and Anopheles populations, we conclude that neuronal dopamine evolution is a key factor for temperature adaptation.