Correlation Between Increased Homing Flight Duration and Altered Gene Expression in the Brain of Honey Bee Foragers After Acute Oral Exposure to Thiacloprid and Thiamethoxam

Abstract

Neonicotinoids as thiamethoxam and thiacloprid are suspected to be implicated in the decline of honey bee populations. As nicotinic acetylcholine receptor agonists, they disturb acetylcholine receptor signaling in insects, leading to neurotoxicity and are therefore globally used as insecticides. Several behavioral studies have shown links between neonicotinoid exposure of bees and adverse effects on foraging activity, homing flight performance and reproduction, but the molecular aspects underlying these effects are not well-understood. In the last years, several studies through us and others showed the effects of exposure to neonicotinoids on gene expression in the brain of honey bees. Transcripts of acetylcholine receptors, hormonal regulation, stress markers, detoxification enzymes, immune system related genes and transcripts of the energy metabolism were altered after neonicotinoid exposure. To elucidate the link between homing flight performance and shifts in gene expression in the brain of honey bees after neonicotinoid exposure, we combined homing flight activity experiments applying RFID technology and gene expression analysis. We analyzed the expression of endocrine factors, stress genes, detoxification enzymes and genes linked to energy metabolism in forager bees after homing flight experiments. Three different experiments (experiment I: pilot study; experiment II: “worst-case” study and experiment III: laboratory study) were performed. In a pilot study, we wanted to investigate if we could see differences in gene expression between controls and exposed bees (experiment I). This first study was followed by a so-called “worst-case” study (experiment II), where we investigated mainly differences in the expression of transcripts linked to energy metabolism between fast and slow returning foragers. We found a correlation between homing flight duration and the expression of cytochrome c oxidase subunit 5A, one transcript linked to oxidative phosphorylation. In the third experiment (experiment III), foragers were exposed in the laboratory to 1 ng/bee thiamethoxam and 8 ng/bee thiacloprid followed by gene expression analysis without a subsequent flight experiment. We could partially confirm the induction of cytochrome c oxidase subunit 5A, which we detected in experiment II. In addition, we analyzed the effect of the feeding mode (group feeding vs. single bee feeding) on data scattering and demonstrated that single bee feeding is superior to group feeding as it significantly reduces variability in gene expression. Based on the data, we thus hypothesize that the disruption of energy metabolism may be one reason for a prolongation of homing flight duration in neonicotinoid treated bees.

Different effects of pesticides on transcripts of the endocrine regulation and energy metabolism in honeybee foragers from different colonies

“…Moreover, phytochemicals in nectar and pollen can also negatively interact with honeybee energy metabolism 28 . Changed expression of cox5a , cox5b and cox17 in mixed-aged foragers after exposure to spinosad also shows the possible effects of pesticides on energy balance in bees 24 . In addition, exposure of mixed-aged foragers to the fungicide chlorothanolin changed the expression of cox5a , cox5b and cox17 25 .…”

“…However, ilp-1 level can drop while foraging continuous, making the link to behavior ambiguous 53 , 54 . Various pesticides altered the expression of ilp-1 in the brain of foragers among them thiacloprid, thiamethoxam, cypermethrin, spinosad and chlorothanolin 24 – 27 . In contrast to the data presented here, which show a downregulation of ilp-1 after exposure to 0.06 ng/bee CPF, a study by Fent and colleagues showed an induction of ilp-1 after exposure to 0.6 ng/bee CPF.…”

“…For this purpose, foragers of the same age were exposed to sublethal concentrations of CPF and PS for 24 h followed by expression analysis of transcripts involved in oxidative phosphorylation ( cox5a , cox5b and cox17 ) as well as transcripts involved in endocrine functions ( buffy , hbg-3 , ilp-1 , vitellogenin , mrjp1 , mrjp2 and mrjp3 ) by qPCR. The selection of transcripts is because they regulate very important biological processes such as energy metabolism and conversion of nurse bees to foragers and that previous studies with other pesticides have already shown effects on these transcripts 21 – 24 . Oxidative phosphorylation plays an important role in energy metabolism and consists of various complexes such as NADH dehydrogenase.…”

“…For the exposure experiments, foragers of the same age were used and a single bee feeding approach was applied because it was shown in a previous study that the data generated in experiments with single bee feeding was less scattered than in experiments with group feeding 24 . Furthermore, foragers of the same age were used to have bees in the same physiological condition and thus to have the most controlled conditions possible.…”

Oral exposure to thiacloprid-based pesticide (Calypso SC480) causes physical poisoning symptoms and impairs the cognitive abilities of bumble bees

“…Furthermore, recent research has suggested that thiacloprid causes transcriptional changes in gene associated with mitochondria, which in turn affects metabolism and energy allocation in the bee brain [ 16 ]. The alterations in the energy metabolism in the brain can translate to various physiological and behavioural changes [ 9 , 16 , 27 ] and may be one reason for the reduced homing capacity, as well as for the reduced learning performance, after the lower exposure.…”