ÆM-POLLY

D.D. del MUR n. 1409 del 14/09/2022

CUP: J53D23013930001

Coordinatore di progetto: Università degli Studi di Siena

Ruolo UNIBO: RL

Sostegno finanziario UE: 100.980,00 €

Ambiti di ricerca: Entomologia agraria

Responsabile Scientifico: Fabio Sgolastra

Durata: 30/11/2023 - 30/11/2025

Gruppo di ricerca: Fabio Sgolastra (DiSTAL), Marta Galloni (BiGeA).

Abstract

Bees and other insect pollinators, including wild bees, hoverflies, beetles, butterflies and moths, have been declining worldwide. In Europe, several initiatives have been adopted at European and national level to halt this trend, however, we still miss a reliable method to assess their efficacy. In this project we aim to develop and validate an integrated protocol to verify if the mitigation measures foreseen by the EU Green Deal are effective in halting and reversing the decline of wild pollinator. This monitoring protocol will integrate endpoints in terms of wild pollinator diversity with other endpoints able to assess the state of health at the sub-individual, individual and population level. These endpoints are selected and designed to diagnose biological alterations in wild pollinators due to different anthropogenic pressures. Using a set of biomarkers integrated with data from the monitoring activity we expect to obtain innovative results, covering several gaps on wild pollinator biodiversity, their health status and on the monitoring of the effectiveness of mitigation actions designed to halt and reverse biodiversity decline.

Results achieved

The monitoring protocol was implemented and applied in the Emilia-Romagna and Tuscany regions of Italy. For each region, two different macro-areas were selected. Each macro-area contained the three ecosystem types: (I) Natural ecosystems (N), (II) Agroecosystems with mitigation measures (A+) included practices aimed at improving pollinator communities, such as flower strips and reduced chemical pesticide use, (III) Agroecosystems without mitigation measures (A-). Within agroecosystems, orchards of the widespread apple crop were selected in the Emilia-Romagna region, and vineyards were selected in Tuscany. All sites were sampled during spring and summer in 2024.

The project results were innovative, addressing several knowledge gaps regarding wild pollinator biodiversity and health status, as well as the monitoring of the effectiveness of mitigation actions designed and recommended by the EU Green Deal to halt and reverse biodiversity decline. A monitoring protocol was developed, implemented, and applied across two different regions and cultivation types (orchards in Emilia-Romagna and vineyards in Tuscany). These sites were characterized by the presence (4 areas, A+) or absence (4 areas, A-) of mitigation measures, as well as by the inclusion of adjacent natural areas (4 areas, N). This monitoring protocol integrates ecological metrics—specifically the presence, abundance, and diversity of wild pollinator species—with endpoints designed to assess health status at the sub-individual, individual, and population levels. These endpoints were selected to diagnose biological alterations in wild pollinators resulting from various environmental pressures, including chemical stress from pesticides, climate change-related stressors, nutritional and water deficiencies, habitat loss, and diseases.

The integrated approach using a specific set of biomarkers was successfully applied to wild pollinator species for the first time. We established a comprehensive dataset of baseline physiological biomarkers for the three selected representative species: Apis mellifera, Bombus terrestris, and Sphaerophoria scripta. These baseline values were derived from the analysis of specimens collected across the four designated natural areas.

The second key result involves the assessment of the health status and biodiversity of wild pollinators monitored in agroecological sites, both in the presence and absence of mitigation measures. Detailed information was compiled and processed regarding the presence, abundance, and diversity of insect

pollinator species (bees, butterflies, and hoverflies), alongside data on morphometric and ecological traits across the various study areas. Comprehensive data regarding the applied biomarker set were produced and analyzed for Apis mellifera, Bombus terrestris, and Sphaerophoria scripta: immune system biomarkers: phenoloxidase (PO), prophenoloxidase (proPO) and differential haemocyte counts; nervous system enzyme activities: acetylcholinesterase (AChE) and carboxylesterases (CaE); biomarkers of oxidative stress and detoxification system: glutathione S-transferase (GST); biomarkers of metabolism: alkaline phosphatase (ALP); biomarkers of genotoxicity: Nuclear Abnormalities (NA) assay; biomarkers of energy mobilization: lipid, total protein, glucose and glycogen levels.

All bees and flowers samples collected in 2024 from 12 sites and two periods have been analyzed for pesticides, in total 56 different active ingredients (including insecticides, fungicides and herbicides) have been detected and about 50% of samples were found to be positive. The risk of agrochemical contamination for pollinators across ecosystems by assessing contamination levels in wild flowers and bee bodies was evaluated. The abundance models of the selected pollinator groups varied according to the group. Butterflies and wild bees varied significantly among ecosystems. Butterflies were significantly more abundant (p < 0.05) in natural ecosystems (N) and in agroecosystems with mitigation measures (A+) than in agroecosystems without mitigation measures (A-). Differences in contamination risk among ecosystems were more pronounced for honeybees than for wild bees. Natural areas showed similarly low risk levels to areas with mitigation measures and clearly differed from areas without mitigation measures. The Emilia-Romagna region exhibited the highest bee risk values. In general, when the contamination risk for wildflowers and bees increased, abundance decreased. Biomarker results demonstrate that mitigation measures (A+) in agroecosystems significantly bolster the resilience of pollinators compared to conventional (A-) or natural sites (N). Key benefits include: a) Physiological Protection: Bees in A+ sites exhibit lower neurotoxicity and reduced genotoxic stress. Conversely, A- sites trigger immune activation and detoxification enzymes (CaE, GST) as compensatory responses to pesticide exposure. b) Enhanced Nutrition: Mitigation practices like flower strips provide vital lipid and protein reserves for long-term survival, whereas bees in unmitigated areas rely on short-term carbohydrates. c) Metabolic and Gut Health: Elevated ALP activity in A+ bees indicates superior nutrient absorption and intestinal health.

The assessment of the health status and biodiversity of the wild pollinators addressed the subsequent indication regarding the effectiveness of the mitigation measures in the 2 types of cultivations (orchards and vineyards). The effectiveness of the measures is essential to reverse the trend of biodiversity loss in natural environments and agroecosystems. In this project, agroecological mitigation strategies were found to provide a resilient physiological barrier, effectively decoupling agricultural production from elevated agrochemical hazards and matching the chemical composition of managed sites with that of natural habitats. These practices were found both in orchards and vineyards to enhance pollinator’s health by stabilizing neurotoxic enzymatic pathways, mitigating genotoxic damage, and promoting the sequestration of high-quality macronutrients. In orchards, mitigation measures significantly increased butterfly abundance. A similar tendency was observed in vineyards, although the effect was not statistically significant. Mitigation measures also tended to increase honeybee and hoverfly abundance in both regions, but these effects did not reach statistical significance. In Tuscany, mitigation measures significantly increased the Faith’s phylogenetic diversity of butterflies and hoverflies while no significant tendencies were found in Emilia-Romagna. At the end, it can be assumed that, in evaluating the effectiveness of mitigation actions, biomarkers act promptly and are better able to highlight the effects of such measures, whereas ecological surveys require more time to achieve statistical power and may necessitate larger datasets. The Contamination Risk Index proved to be a valuable tool for the purposes of the ÆM-POLLY project.

Following the overall analysis of the results obtained, we carried out the fine-tuning and the definition of the final integrated monitoring protocol to assess the health status of wild pollinators and their biodiversity. This protocol is based on parameters, at various levels of biological organization, that proved to be the most sensitive and widely applicable for evaluating the effectiveness of mitigation measures.

After the elaboration and simplification of the integrated protocol, comprehensive, user-friendly guidelines to monitor the effectiveness of pollinator mitigation actions were produced in English and Italian language. These guidelines constitute a strategic tool for a wide range of stakeholders, including environmental and agricultural agencies, phytosanitary services, regulatory bodies, and policymakers, with farmers serving as the primary indirect beneficiaries.

Funding

Funded by the European Union - NextGenerationEU under the National Recovery and Resilience Plan (PNRR) - Mission 4 Education and research - Component 2 From research to business - Investment 1.1 Notice PRIN 2022 PNRR (DD N. 1409 del 14/09/2022), entitled "Assessing the effectiveness of mitigation measures on pollinator decline: an integrated multi-biomarker approach", proposal code P2022YWSSX - CUP J53D23013930001.