Assessing the impact of co-exposure to succinate dehydrogenase inhibitor (SDHI) fungicides and the intestinal parasite Nosema ceranae in the honey bee Apis mellifera

Highlights

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  The SDHI fluopyram at 5 mg/L and bixafen at 1 mg/L reduced A. mellifera survival
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  Fluopyram at 5 mg/L modified lipid reserves in A. mellifera
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  Fluopyram and fluxapyroxad had an impact on N. ceranae proliferation
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  Fluopyram and Nosema ceranae had an antagonistic effect on A. mellifera survival
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  Fluopyram at 1 mg/L masked N. ceranae effect on A. mellifera midgut transcriptome

Abstract

Over the past few decades, significant mortality rates have been reported in honey bee populations. The decline of these pollinators is thought to be linked to a combination of stressors, including both pathogens and pesticides. Here, we investigated the impact of chronic exposure of honey bees to a class of fungicides that inhibit succinate dehydrogenase (SDHI), in combination with the parasite Nosema ceranae. Bees were exposed under controlled laboratory conditions to N. ceranae and/or fed with two environmental concentrations of four different SDHIs (boscalid, bixafen, fluopyram, and fluxapyroxad). The bees were monitored for 21 days, during which several health parameters were evaluated, including survival, food consumption, parasitic load and lipid reserves. Additionally, a global RNA-Seq approach was used to analyze midgut transcriptional changes in non-infected and N. ceranae-infected bees treated with fluopyram. The results indicate complex and deleterious interactions of SDHI active substances, characterized by dose-response effects and non-monotonic reactions in uninfected bees. However, co-exposure to N. ceranae significantly modified these responses, with an antagonistic effect on survival and lipid reserves, which could be linked to mitochondrial disruption and activation of detoxification mechanisms. These results highlight the importance of considering bee co-exposure to multiple stressors over their lifespan.

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Introduction

Over recent decades, numerous studies have highlighted dramatic declines in pollinator populations, raising serious ecological and economic concerns (Goulson et al., 2015, Zattara and Aizen, 2021). These losses have profound implications for global food security and ecosystem stability, especially considering the vital role of animal-assisted pollination. Such pollination supports about 87.5% of flowering plant species (Ollerton et al., 2011), 75% of the world’s leading crop varieties (Klein et al., 2007), and contributes to approximately 9.5% of the total value of agricultural food production for human consumption (Gallai et al., 2009). In the global context of crop pollination, the Western honey bee (Apis mellifera) plays a crucial role (Hung et al., 2018). However, despite its importance, this species faces considerable threats (Potts et al., 2010, vanEngelsdorp et al., 2008), including the emergence of Colony Collapse Disorder (CCD) in the United States, characterized by substantial losses of overwintering colonies (Oldroyd, 2007).

Honey bees are impacted by multiple stressors that include biotic factors, such as parasites and pathogens, and abiotic factors, like climate change and agricultural practices (Belsky, 2019). While pollinating crops, forager bees can be exposed to various plant protection products either through direct contact during their application, or by interacting with contaminated substrates such as soil, water, or floral resources. Pesticides can exhibit systemic properties, either because they are included in seed coating or being absorbed by crops and transported to various parts of the plant, including pollen and nectar (Bonmatin et al., 2015). Consequently, contaminated bees may spread these chemicals throughout the hive, affecting its matrices and all individuals in the colony. Multiples studies have reported pesticide residues in different matrices, such as pollen, honey, bee bread, and wax (Böhme et al., 2018, Drummond et al., 2018, Mullin et al., 2010). Of these, fungicides are among the most frequently detected chemicals, likely due to their widespread use during flowering, which can expose bees during their pollination activities (Favaro et al., 2019). Recent reviews on pesticide exposure have documented a wide range of fungicide concentrations, with some levels exceeding thresholds of concern for chronic risks to bee populations (Rondeau and Raine, 2022, Végh et al., 2023).

Residues commonly found in beehive matrices include a new class of fungicides known as succinate dehydrogenase inhibitors (SDHI). SDHIs, used to control phytopathogenic fungi, inhibit the succinate dehydrogenase in the mitochondrial respiratory chain, a highly conserved enzyme across various living organisms (Duarte Hospital et al., 2023). Three SDHI molecules -boscalid, fluopyram, and fluxapyroxad- are particularly prevalent in beehive matrices. Boscalid, introduced to the market in 2002, has been widely used and frequently detected in analyses. Its concentrations in pollen have been reported to reach up to 512 ng/g (Simon-Delso et al., 2018), 962 ng/g (Mullin et al., 2010), 7,270 ng/g (Frazier et al., 2015), and even as high as 26,200 ng/g (Wallner, 2010). Boscalid has also been found in other bee-related matrices, including beebread (up to 1,300 ng/g) (Simon-Delso et al., 2014), wax (up to 388 ng/g) (Mullin et al., 2010), and honey bees themselves (up to 347 ng/g) (Frazier et al., 2015). The primary application methods for this fungicide are foliar spray during blooming and seed treatment. The high contamination levels observed for boscalid are not surprising, given that concentrations as high as 75,000 ng/g have been recorded in almond flowers in the USA (Frazier et al., 2015). Fluopyram and fluxapyroxad have also been detected in bee matrices, with concentrations reaching up to 4,050 ng/g and 353.6 ng/g in pollen, respectively (Friedle et al., 2021, McArt et al., 2017).

The widespread detection of SDHI fungicides in beehive matrices, coupled with a 2019 scientific alert regarding their possible risks, has raised significant concerns about the potential impact of these agrochemicals on bee populations (Bénit et al., 2019). Bénit et al. revealed that SDHIs inhibit succinate dehydrogenase activity not only in fungi but also in diverse non-target organisms, including earthworms, honey bees, and humans (Bénit et al., 2019). Consequently, studies have investigated the effects of these molecules on bee health, with a particular focus on boscalid, alone or mixed with other chemicals, including the formulation Pristine®. Such exposures have been linked to several adverse effects on bee physiology, including impaired digestion and intestinal integrity (Degrandi-Hoffman et al., 2015), altered gut microbiota (Dong et al., 2023), reduced longevity (Fisher et al., 2022, Glass et al., 2021), and behavioral changes (DesJardins et al., 2021).

Research on SDHI fungicides has primarily focused on their toxic effects on bees under single-stress conditions. However, bees in natural environments often face multiple stressors, leading to complex and diverse effects (Siviter et al., 2021). The combined impact of two stressors can be additive, synergistic, or antagonistic. Although synergistic interactions between pesticides have been extensively studied for their potential to intensify negative effects on bee health (Collison et al., 2016), recent research highlights the comparable importance of antagonistic interactions in determining the overall impact of pesticides on pollinators (Bird et al., 2021). These antagonistic interactions can occur when one stress attenuates the effects of another, potentially through the activation of defense mechanisms that indirectly reduce sensitivity to other stressors. Since bees are likely to be exposed to both pathogens and pesticides simultaneously in their environment, several studies have investigated the interactions between the most common microsporidian parasite in bees, Nosema ceranae, and pesticides, including insecticides, fungicides and/or herbicides (Aufauvre et al., 2014, Glavinic et al., 2019, Pettis et al., 2013, Tadei et al., 2020). Microsporidia are obligate intracellular parasites recognized for their limited metabolic capacities and the lack of ATP-producing mitochondria, making them highly dependent on the host cell’s energy (Han et al., 2020a, Han et al., 2020b). Given these parasitic traits, it is essential to investigate how these mitochondria-deficient parasites interact with SDHI pesticides, which inhibit the respiratory chain, to better understand their potential impact on bee health. One study has shown that the combination of boscalid and N. ceranae has synergistic negative effects on bee mortality and gut microbiota under laboratory conditions (Paris et al., 2020).

The present study aims to evaluate the effects of various SDHI fungicides on both uninfected and N. ceranae-infected honey bees under controlled laboratory conditions. Newly emerged worker bees were encaged and exposed to N. ceranae and/or fed with two environmental concentrations of four different SDHIs (boscalid, bixafen, fluopyram, and fluxapyroxad), according to the experimental design. The bees were monitored for twenty-one days to assess mortality rates. Additionally, we measured the parasite load in the digestive tract to evaluate the developmental success of N. ceranae, and the amount of lipid droplets in the fat body, a detoxification site reflecting the parasite’s energy dependence. To further explore the molecular responses of bees exposed to these combined stressors, a global RNA-Seq analysis was conducted to identify transcriptional changes in the midgut of bees infected with N. ceranae and/or exposed to fluopyram.