Fungicides chlorothanolin, azoxystrobin and folpet induce transcriptional alterations in genes encoding enzymes involved in oxidative phosphorylation and metabolism in honey bees (Apis mellifera) at sublethal concentrations
Graphical abstract
Introduction
The widespread use of plant protection products (PPPs) in agriculture and private gardens may result in exposure of non-target organisms. Honey bees (Apis mellifera) are important pollinators in these areas and can encounter different PPPs. Exposure to PPPs is one among the reasons for population declines of honey and wild bees [1,2] and colony losses [3], often in combination with other factors such as Varroa destructor parasites. Bees are exposed while visiting flowers and they bring nectar and pollen to the hive. Thus, foragers may be affected by direct exposure but also the hive colony and larvae can be exposed by contaminated pollen and nectar. Pollen analyses in Europe and North America showed a high incidence of PPPs, and among them were often fungicides [4,5]. Experimental feeding of such common pollen-bound PPP-mixtures delayed foraging with perturbations of the energy metabolism of bees [6].
Thus, bees are often exposed to fungicides, which find widespread use in agriculture. For instance, in France, where about 67’000 tons of PPPs were sold annually between 2011–2015, over 40% were fungicides, of which copper and sulphur were most frequent, but synthetic compounds also find frequent application [7]. The French PPP reduction policy was not effective as PPPs application continues at considerable rate, particularly in grapevines, sugar beet, potatoes and apples, where average treatments per year may be over twenty times [7]. In some countries, including Switzerland, fungicides can also be sprayed when fruit trees are at blossom. This is because of the relatively low acute toxicity of fungicides, which are assumed to be without risks to pollinators. However, whether or not chronic or sublethal effects of fungicides in bees occur is very poorly known.
Fungicides may interact with other PPPs to produce synergistic effects [8]. This was described for combinations between neonicotinoid insecticides (clothianidin) and fungicides (i.e. propiconazole) [9,10], and effects included slow ovary maturation, decreased feeding and survival, and thus, a shortened nesting period in wild bees Osmia bicornis [11]. Fungicides that mainly interfere with the metabolism in fungi may also affect metabolism in insects. Triazole fungicides that are cytochrome P450-dependent monooxygenase (cyp) inhibitors, interfere with metabolism in bees and cause down-regulation of mitochondrion-related nuclear genes [12].
In our present study we focus on the transcriptional effects of three largely used fungicides to evaluate potential effects, which are currently unknown. Chlorothanolin is a broad-spectrum fungicide and has been found for instance in bee’s wax at concentrations of 47.38 ng/g [13]. Its mode of action in fungi is unclear, but it reduces the fungal intracellular glutathione level [14]. It is often applied to blooming crops when honey bees are present. It affected larval survival upon exposure via contaminated feed with 3.4 mg/L [15]. Other adverse effects on bees are unknown.
Azoxystrobin is a member of the strobilurin fungicides. It is frequently used and was the world’s biggest-selling fungicide in 1999 [16]. It inhibits mitochondrial respiration by binding at the Qo site of cytochrome b, which is part of the cytochrome bc1 complex in the inner mitochondrial membrane of fungi, but also of other eukaryotes [16]. Thus, it disrupts the energy cycle by halting ATP production. Azoxystrobin was found in poisoned bees in Germany [17], and in 17% of foraging bees collected in grassland in Colorado, USA, where up 25 ng/g tissue were detected [18]. Contaminated pollen was reported in Maine, USA, with 0.9 ppb per hive, and also royal jelly was found to contain azoxystrobin in Germany in concentrations of up to 0.91 ng/g [19]. The acute toxicity of azoxystrobin is higher than 200 μg/bee (https://www3.epa.gov/pesticides/chem_search/reg_actions/registration/fs_PC-128810_07-Feb-97.pdf), but very little is known about chronic effects of this important fungicide.
Folpet is a chloralkylthio-fungicide that acts by reacting with thiols, and thus alters proteins and enzymes in fungi [20]. By nonspecific reaction with thiols, folpet reacts with cysteine amino acids in proteins and glutathione, thus affecting the function of many proteins and enzymes. Folpet residues of 3.74 μg/g were detected in a propolis (resinous material produced by bees from plant exudates and buds mixed with bee saliva and wax) sample in Spain [21]. Currently, potential effects of this fungicide to pollinators are unknown.
Due to knowledge gaps in potential adverse effects of these frequently used fungicides and the lack of information on molecular effects, the aim of our study is to evaluate transcriptional responses in the brain of experimentally exposed honey bees. We focus on target genes that play an important role in the physiology of bees and which may be related to the mode of action of the fungicides, including interference with energy metabolism. We focus on the brain as it represents an important target organ for different PPPs. Our analysis at sublethal concentrations sheds new lights to potential adverse implications of bee’s exposures to fungicides.
Section snippets
Chemicals
Azoxystrobin, chlorothanolin and folpet (purities of all > 99%) were purchased from Sigma–Aldrich (Buchs, Switzerland). Stock solutions for each compound were prepared in DMSO and diluted into 20% sucrose-solution to a final concentration of 0.1% DMSO.
Experimental design of laboratory exposures
Adult forager honey bees (Apis mellifera carnica) of mixed age were obtained from frames from an outdoor colony placed at a location with no agricultural activity and pesticide use in the Black Forest (Germany, GPS: N 47.7667, E 7.8333) from end
Results
We exposed honey bees to the fungicides azoxystrobin, chlorothanolin and folpet at three different concentrations and at three different exposure times (24, 48 and 72 h) to explore and compare concentration-related and time-related molecular effects in the brain. To analyse for molecular effects, we assessed transcriptional alterations of selected genes including immune system regulating genes, genes involved in oxidative phosphorylation, genes encoding metabolism enzymes and genes linked to
Discussion
Here we show for the first time significant molecular effects of fungicides in the brain of honey bee workers. Transcripts of different important physiological pathways such as immune system, oxidative phosphorylation, detoxification and endocrine regulation were analysed. Of the three investigated fungicides, chlorothanolin showed strongest effects characterized by differential transcriptional expression of genes encoding enzymes and proteins related to oxidative phosphorylation,
Conclusion
Chlorothanolin showed strongest effects of the analysed fungicides. Marked down-regulation occurred for transcripts of genes linked to metabolism/detoxification, oxidative phosphorylation and hormone system. The observed effects were season-related; honey bees reacted often faster to chlorothanolin exposure in April than in June, probably due to different composition of the bees sampled (consisting of more nurse bees in April), and differences in temperature and flowering plants on which bees
Acknowledgments
We thank Eva Reinhard, Agroscope Liebefeld (Bern), for generous support. The study was supported by the Swiss Federal Office for Agriculture (BLW), Bern (contract no. REF-1062-22100, 627000648 to K.F.).
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