Transcriptomic diagnostics for identifying interactive effects of pesticides, food deficiency, and heat stress on bee health.

Pollination by bees is essential for the survival of many wild plants and harvesting of many crops. Over recent decades, however, bees have been in decline worldwide. This creates a major risk and potentially a huge cost - the economic value of pollination is estimated to be £430 million per year in the UK alone.

Causes for the recent bee declines include habitat loss which reduces access to food, changing temperatures, and exposure to pesticides. Indeed, previously authorised pesticides in fact have unintended impacts on bees, reducing their abilities to forage for food, and ultimately their survival. Three of the most commonly used pesticides were thus suspended in 2013 and banned by the European Commission in 2018.

Does this mean that pesticides that remain authorised pesticides are safe for bees? Unfortunately, the manners by which the effects of pesticides are evaluated have been too crude to be certain. To obtain clarity, we will apply a type of molecular medicine approach that has over the past decade dramatically improved treatment and understanding of human diseases.

We will apply the new technology to understand the effects of one established neonicotinoid pesticide (acetamiprid), and two new pesticides that have (with some contention) been heralded as "bee-safe" alternatives to the banned neonicotinoids: sulfoxaflor and flupyradifurone. For this purpose, we will perform controlled experiments in which we expose bees to individual pesticides and pesticide combinations - as occurs when neighbouring farms apply different products. Furthermore, we will perform experiments to determine whether the effects of pesticides change when simulating a warmer climate or if food has low nutritional quality - as is common in landscapes dominated by monocultures. Our experiments will be on two common British bee species that are also extensively bred for agricultural pollination: the social large earth bumblebee Bombus terrestris and the solitary red mason bee Osmia bicornis.

The "transcriptomic diagnostics" we apply to the bees will provide tens of thousands of measurements per experiment - rather than the handful obtained through traditional approaches. Indeed, we will measure changes in activity levels for each of the 12,000 gene building blocks in brain, flight muscle and a detoxification tissue of bees. This approach is highly sensitive and able to directly detect even subtle changes in the bees' physiologies and metabolisms. Furthermore, because we will measure gene activity in a detoxification tissue as well as brain and muscle, we will be able to determine whether changes in activity level are helping the bees cope with pesticide exposure.

Our work will 1) provide urgently needed insight into the effects of these specific pesticides on bees; 2) likely reveal previously unknown effects of authorised pesticides; 3) identify potential differences between these pesticides which are thought to act in similar manners; 4) clarify whether there are "non-additive" interactions between pesticides or with other environmental stressors; 5) whether conclusions about toxicity for one species are representative of another; and 6) unambiguously demonstrate the power and sensitivity of applying the "transcriptomic diagnostics" approach for determining bee health.

The biological knowledge we gain through this project will inform regulatory decisions about the currently authorised pesticides. Furthermore, we expect that the approach we develop will fundamentally improve future evaluation of pesticides for research and regulation. Ultimately, both will improve the fates of Britain's bees.

Bees are essential for agricultural productivity and ecosystem stability, but pesticides contribute significantly to recent bee declines worldwide. Despite the importance of balancing the beneficial and detrimental impacts of pesticides, we have limited understanding of the mechanisms through which pesticides affect pollinators, how exposure to multiple environmental stressors may worsen their effects, or how effects differ between species.

Detailed insight is beyond the reach of traditional pesticide susceptibility evaluation approaches, because they only measure few organism- or colony-level effects. Instead, we will use whole-transcriptome gene expression profiling (RNA-seq) in brain, midgut, and flight muscle, providing thousands of measurements per sample.

We will first expose Bombus terrestris bumblebees to three individual pesticides (acetamiprid, sulfoxaflor, and flupyradifurone) and combinations of these compounds, simulating what occurs when neighbouring fields have different treatments. Second, we will test whether pesticide effects are exacerbated when simulating a warmer climate or if low quality food is available - as is common around monocultures. Finally, we will perform similar experiments in the solitary red mason bee Osmia bicornis. We hypothesise that we will detect previously unknown effects of the pesticides, and non-linear interactions that lead to substantial differences in qualities and intensities of effects between pesticides, combination treatments, and species.

Our work will provide detailed insight into the diverse mechanisms by which the pesticides affect bees, how interactions with other environmental stressors may worsen pesticide effects, and how effects and interactions may differ between species. This research will provide clarity about whether such factors must be considered during pesticide evaluation. Furthermore, our approach pioneers a powerful new toolkit for pesticide research and assessment.

Large-scale agriculture depends on insect pollination services valued at over £430 million annually in the UK; for example, over 60,000 commercial Bombus terrestris bumblebee colonies are annually imported into the UK. Further, insect pollinators contribute significantly to ecosystem stability, with 87% of flowering plants requiring pollination for reproduction.

We will provide high-resolution comparisons to help evaluate the toxicity of currently available pesticides that target nicotinic acetylcholine receptors (nAChRs) - the most widely used class of insectivited. Moreover, we will provide general insight into differences in effects across pesticides and bee species. Finally, our project will showcase the power of transcriptomic diagnostics for this field.

Our project fits fully under the overarching BBSRC strategic priority "Agriculture and Food Security". Indeed, it is relevant to the priorities of "sustainably enhancing agricultural production", improving "welfare of managed animals". The high sensitivity of our approach will support the priority of "replacement, refinement and reduction in research using animals". Furthermore, our application of transcriptomics and bioinformatics aligns with BBSRC's enabling theme of "exploiting new ways of working through "data driven biology" and "systems approaches to the biosciences".

Our Pathways to Impact and scientific advisory board will help to ensure our project impacts:

1. Policy Makers and Advisors in Government and Non-Governmental Organisations: We will provide key novel insight on the relative toxicities of the focal pesticides. Furthermore, we will demonstrate that the transcriptomic diagnostics approach we are developing is highly sensitive, and able to provide detailed quantitative insight for evaluating toxicity in a manner inaccessible to traditional approaches. We expect transcriptomic diagnostics to become a powerful and practical tool for evaluation and regulation.

2. Pesticide and Seed Producers: Recent restrictions of pesticides - and increased awareness of their unintended consequences - create difficult challenges. Crop chemical companies will be able to integrate the highly-sensitive approach we are advancing into their pesticide research. They will benefit from being able to obtain a sensitive overview of impacts using few experiments. Furthermore, our gene- and pathway-level analyses provide new knowledge on the intended and unintended effects of the pesticides we focus on.

3. Farmers: The improved pesticide evaluation approach we are leveraging, and the specific insight we provide regarding the focal pesticides will ultimately benefit farmers as it will lead to pesticides that are less harmful to beneficial insect species. In the short term, our comparisons of the toxicities of the three focal pesticides can inform farmers' crop protection decisions.

4. General Public: Our work will help improve sustainability of agricultural practices, increasing the stability, productivity and security of our food supply and the maintenance of biodiversity. Outreach initiatives we will highlight the importance of wild and managed pollinators for ecosystem stability and the production of a broad range of nutritious, high-value food products. We will emphasise how innovative BBSRC-funded research can help overcome the challenge our society faces in achieving both food security and biodiversity conservation. Finally, we will stress the tremendous power of modern molecular techniques and bioinformatics for improving our understanding of the natural world.

5. Researchers: Our work will be of interest to public, private, and third-sector researchers (see Academic Beneficiaries), by raising awareness of the power of genomic approaches for research on ecotoxicology and effects of environmental stress on bees. Project staff members will receive dedicated training to build their careers. All data and analysis code will be open to ensure broad impact.