Assessment of the ecotoxicological health status of Apis mellifera using a multi-tier approach based on biomarkers, proteomic analysis and quality and origin of bee products

Pollinating insects play a role of primary importance both in agriculture, ensuring the crops productivity, and in the conservation of plant biodiversity. Among pollinators, Apis mellifera L., 1958 (Hymenoptera: Apidae) is the most known and widespread species and the most valuable for its pollination service. This species is disappearing globally due to different reasons, such as climate change, the massive use of Plant Protection Products (PPPs) and other environmental contaminants diffusion, habitat fragmentation and parasites infections. The sub-lethal levels of pesticide residues and other anthropic contaminants, even not leading to the death of individuals, are able to cause problems in the development, behaviour and health of animals in the short and long term. Moreover, honey bees are exposed to mixtures of contaminants in the environment, that can cause different effects. However, there is a large gap in the assessment of the sub-lethal effects of these mixtures. Another gap in the research on the ecotoxicology of these animals is the assessment of the effects of commercial formulates instead of only using the active principles. Among the sub-lethal effects that were examined in the literature relating to pesticides exposure, rarely genotoxicity and immune system biomarkers were used. Anthropic activities could also be able to modify indirectly the quality and origin of bee products, since they can alter honey bees health and consequently their productivity. The use of an integrated approach to combine responses at different levels, could be a valid tool to evaluate the impact of contamination on these organisms. The goal of this thesis was to assess the health of honey bee colonies using a multi-tier methodology that included biomarker responses, proteomic analysis, and bee product quality and origin. This thesis was divided in two parts: ● A laboratory study, exposing Apis mellifera specimens to two commercial pesticides, the fungicide Sakura® and the herbicide Elegant 2FD, alone and in combination. The effects of these compounds were assessed integrating two methodologies, consisting in a set of biomarkers and a proteomics approach. Both pesticides modulated the detoxification process. The fungicide alone had also effects on the metabolism, while the herbicide demonstrated to be neurotoxic. The results from the mixture treatments demonstrated that the effects obtained were influenced mostly by the herbicide. The proteomic approach revealed that the two pesticides were able to affect the energy metabolism, the immune system and the protein synthesis. The proteomic approach should be improved to understand if and to what extent the above-mentioned post-translational changes happened, using specific antibodies to perform a more specific assessment. ● A two-year monitoring study, aiming to assess the ecotoxicological status of bees in natural environments. Apis mellifera specimens were sampled in 10 locations in Tuscany region characterised by varying contamination patterns. In this case, the used approach was made up of a set of biomarkers, used to assess the health status of honey bees, and the analyses of origin and quality of the honey, through melissopalynological and chemical-physical analyses. The biomarkers results obtained for the first year showed that the suburban area and the agricultural area were undergoing major stress but with different kinds of effects, probably because the contaminants were different in the various areas. In 2021 the specimens undergoing major stress were the ones coming from vineyards, that showed genotoxic effects, and clover field and wheat crops, showing alterations in nervous and immune systems. The comparison between the 2 years results showed that the organisms were undergoing major stress condition in 2021 compared to 2020. Bees from 2021 reported neurotoxic effects, the presence of oxidative stress and DNA damage. The different responses obtained could be due not only to contaminants but also to the changing of climatic conditions, such as differences in temperatures and rainfalls, which were also taken into consideration. The melissopalynological analysis showed that only in the clover field the pollen derived from the cultivation that we observed during the sampling. These findings suggest that the biomarker responses observed in A. mellifera specimens are probably not due to pollen contamination. In fact, organisms could come in contact with contaminants through other exposure routes. The carbohydrates, amino acids and humidity analysis showed that honey samples were not characterised by major differences, even if coming from different areas, except for the proportion of some amino acids, due to the presence of different pollens. Both the studies had also the goal to start filling a research gap regarding the assessment of effects on immune system and DNA damages, obtaining promising results. The integrated approaches that were used proved to be effective to observe the ecotoxicological health status of Apis mellifera from different points of view. The multi-trial approach would be a sensitive tool to measure sub-lethal effects, and not only lethal ones, of pesticide active principles and, more important, of pesticide commercial formulations. It would be helpful to improve the current risk assessment procedure for chemical registration and use, making the agricultural environment more pollinator-friendly.