Elsevier

Journal of Hazardous Materials

Volume 377, 5 September 2019, Pages 215-226
Journal of Hazardous Materials

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

https://doi.org/10.1016/j.jhazmat.2019.05.056Get rights and content

Highlights

  • First assessment of molecular effects of frequently used fungicides in honey bees.

  • Chorothanolin caused transcriptional alteration of genes related to oxidative phosphorylation.

  • Chorothanolin caused transcriptional alteration of genes regulating transition of honey bees.

  • Honey bees are more sensitive to chlorothanolin in April than in June.

Abstract

Fungicides are highly used for plant protection but their molecular and chronic effects are poorly known. Here, we analyse transcriptional effects in the brain of honey bees of three frequently applied fungicides, azoxystrobin, chlorothanolin and folpet, after oral exposure for 24, 48 and 72 h. Among transcripts assessed were genes encoding proteins for immune and hormone system regulation, oxidative phosphorylation, metabolism, and acetylcholine receptor alpha 1. Azoxystrobin and folpet induced minor alterations, including down-regulation of hbg-3 by azoxystrobin and induction of ndufb-7 by folpet. Chlorothanolin induced strong transcriptional down-regulation of genes encoding enzymes related to oxidative phosphorylation and metabolism, including cyp9q1, cyp9q2 and cyp9q3, acetylcholine receptor alpha 1 and hbg-3 and ilp-1, which are linked to hormonal regulation and behavioural transition of honey bees. Exposures to chlorothanolin in different seasonal times showed different responsiveness; responses were faster and often stronger in April than in June. Chlorothanolin caused the strongest effects and affected transcriptional abundance of genes related to energy production, metabolism and the endocrine system. Disturbed energy production may reduce foraging activity and hormonal dysregulation, such as the transition of nurse bees to foragers. Further analyses are needed to further substantiate potential adverse effects of chlorothanolin in bees on the physiological level.

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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