Elsevier

Journal of Insect Physiology

Volume 121, February–March 2020, 104014
Journal of Insect Physiology

Impact of sublethal exposure to synthetic and natural acaricides on honey bee (Apis mellifera) memory and expression of genes related to memory

https://doi.org/10.1016/j.jinsphys.2020.104014Get rights and content

Highlights

  • Formic acid had the most and thymol had the least negative effect on bee memory.

  • Long-term memory PER at 48 hpt showed more effects of acaricides than at 24 hpt.

  • The most responsive memory-related gene tested to acaricides was defensin-1.

  • No direct relationship between defensin-1 expression and bee memory was observed.

  • Synthetic acaricides do not necessarily impact bee memory more than natural ones.

Abstract

Acaricides are used by beekeepers in honey bee (Apis mellifera L.) colonies to control parasitic mites, but may also have adverse effects to honey bees. In this study, five commonly used acaricides were tested for their sublethal effects on memory and expression of neural-related genes in honey bees. Memory measured with the proboscis extension reflex (PER) assay was significantly reduced by topical treatment of bees with a single LD05 dose of formic acid at 2 and 24 h post treatment (hpt). However, tau-fluvalinate, amitraz, coumaphos, and formic acid, but not thymol, resulted in memory loss at 48 hpt. The LD05 doses of the acraricides did not affect expression of neuroligin-1, related to memory, or expression of major royal jelly protein-1, related to both memory and development, although expression of both genes was affected at LD50 doses. The LD05 doses of thymol, formic acid, amitraz and coumaphos increased defensin-1 expression, which is related to both memory and immunity. The effect of thymol, however, may have been due to its impact on the immune response rather than memory. This study demonstrates that acaricides vary in their effects on bee’s memory, and that the widely used acaricide, formic acid, is particularly damaging.

Introduction

Honey bees (Apis mellifera L.) have sophisticated abilities to distinguish and respond to a wide range of stimuli in the environment, such as odor and color (Menzel et al., 2001, Hori et al., 2006), which are critical for the performance of foraging and nest behaviors such as hygienic behavior. Investigations on bee memory often rely on the use of reward learning studies, such as the proboscis extension reflex (PER) assay (Menzel, 2001), where bees are exposed to a conditioned stimulus (CS), such as an image or an odor, in relation to an unconditioned stimulus (US) or reward, such as food. The bees eventually establish an association between the two stimuli, which can be measured by a response, such as the extension of the proboscis from the mouth for feeding in the case of the PER assay (Erber et al., 1980, Bitterman et al., 1983, Menzel, 2012). As a result, the bee eventually reacts to both the CS and the US. Bee memory can then be evaluated by investigating how long information is retained based on the response. Memory may be affected by a variety of stresses, such as exposure to synthetic acaricides like coumaphos and tau-fluvalinate (Weick and Thorn, 2002, Frost et al., 2013, Williamson et al., 2013), insecticides (Decourtye et al., 2005), parasitic mites (Kralj et al., 2007) and viruses (Li et al., 2013).

Another means of assessing memory is by measuring expression of genes that have been associated with memory in bees. For example, Biswas et al. (2010) reported up-regulation of neuroligin-1 (NLG) in bees assessed by an olfactory PER assay, which may be due to the products of the post-synaptic NLG being critical for neuronal plasticity mechanisms, such as memory (Sudhof, 2008). Also, Wang et al. (2013) found that the gene for major royal jelly protein-1 (MRJP-1), which influences the synthesis of royal jelly in the hypopharyngeal glands, and the gene for defensin-1 (Def-1), which regulates the production of the antimicrobial peptide released in royal jelly, were among the 30 most up-regulated genes in bees trained with an olfactory PER assay. However, their relationship to memory is less clear, as MRJP-1 encodes a glycoprotein that affects development with larvae fed MRJP-1 developing into queens with a body almost two times larger and a lifespan almost ten times longer than larvae not fed MRJP-1 (Barchuk et al., 2007). Def-1 encodes an antimicrobial peptide released in royal jelly (Klaudiny et al., 2005) and honey (Kwakman et al., 2010). Expression of such genes in honey bees may be affected by a variety of stresses, such as sensory deprivation, which correlates with a decrease in NLG expression (Biswas et al., 2010), a sucrose only diet that correlates with decreased MRJP-1 expression (Hojo et al., 2010), and exposure to the neonicotinoid thiamethoxam, which correlates with decreased Def-1 expression (Tesovnik et al., 2017).

Acaricides are pesticides used to control parasitic mites, such as Varroa destructor, which is considered a major threat to honey bee populations worldwide (Rosenkranz et al., 2010). Acaricides are applied directly in the hive to control mites (Bonzini et al., 2011), however, they might cause damage to the bees. A synthetic acaricide commonly used in honey bee colonies is coumaphos, an organophosphate marketed as Checkmite® (Bayer Crop Science), which kills mites by inactivating acetylcholinesterase, thereby interfering with nerve signaling and function (Palmer et al., 2013). Topical application of coumaphos did not affect acetylcholinesterase activity in the bee brain and did not inhibit memory as measured by an olfactory PER assay (Weick and Thorn, 2002). In contrast, an acute higher coumaphos dose applied orally in sugar syrup increased olfactory memory as measured by PER (Williamson et al., 2013). Tau-fluvalinate, a pyrethroid marketed as Apistan® (Wellmark International), is a synthetic acaricide commonly used to control V. destructor that kills mites by blocking voltage-gated sodium and calcium channels in cells (Davies et al., 2007). Tau-fluvalinate at low doses negatively affected olfactory memory and at higher doses also reduced honey bee survival (Frost et al., 2013). However, Decourtye et al. (2005) showed that even higher oral dosages did not affect memory, which is surprising considering that Decourtye et al. (2005) used dosages of tau-fluvalinate up to eight times higher than Frost et al. (2013) and were administered for 11 consecutive days instead of once.

Acaricides have been reported to affect the expression of bee genes. For example, thymol and coumaphos caused a strong down-regulation of detoxification-related genes, such as cytochrome P450 306A1, while tau-fluvalinate and amitraz did not affect their expression (Boncristiani et al., 2012). Exposure of bees to amitraz, tau-fluvalinate or formic acid increased acetylcholinesterase expression, exposure to tau-fluvalinate, coumaphos, amitraz or formic acid increased vitellogenin expression, and exposure to tau-fluvalinate, coumaphos, amitraz, thymol or formic acid decreased cytochrome P450 9Q3 expression (Gashout et al., 2018). Thus far, studies of the effects of acaricides on honey bee memory measured by PER are limited and often contradictory, and there have been no studies yet to determine whether acaricides affect the expression of memory-associated genes.

Due to the importance of memory for bee behaviors, such as identifying flowers when foraging for pollen and nectar, as well as returning to hives during foraging trips, it is important to identify how acaricides differ in their potential detrimental effects on memory retention. Thus far, coumaphos, tau-fluvalinate and oxalic acid have been tested for their effects on memory (Weick and Thorn, 2002, Schneider et al., 2012, Frost et al., 2013), but there are no reports for other commonly used acaracides in hives, such as the synthetic acaricide, amitraz (ApiVar®; Mann Lake Ltd.), or the natural acaricides, thymol (Apiguard®; Vita Europe, Ltd.) and formic acid (Miteaway®; NOD Apiary Products, Ltd.). This study was designed to compare the effects of sublethal dosages of three synthetic acaricides, tau-fluvalinate, amitraz and coumaphos, as well as two natural acaricides, thymol and formic acid, on memory retention based on the PER assay and on the expression of related neural genes.

Section snippets

Chemicals

Technical grade (>98% purity) amitraz, coumaphos, tau-fluvalinate and formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Thymol was obtained from Fisher Scientific Ltd. (Ottawa, Ont., Canada).

Source of bees

Experiments were conducted at the Honey Bee Research Center of the University of Guelph, Ontario, Canada. Honey bee colonies containing naturally mated queens of the Buckfast strain were used as a source of workers. The colonies were treated against mites the previous fall and no Nosema

Memory retention

At 2 and 24 hpt, only formic acid significantly affected memory retention in the bees, compared to both controls (χ2 = 15.94, df = 1, P < 0.0001, χ2 = 17.41, df = 1, P < 0.0001, for 2 and 24 hpt, respectively; Table 2). Thus, formic acid reduced short-term memory retention, which declined to approx. half of that of the non-treated control by 24 hpt (Table 2). Long-term memory retention with LD05 doses of formic acid, tau-fluvalinate, amitraz and coumaphos was significantly reduced in the bees

Discussion

The PER assay is a very sensitive indicator of the effects of pesticides on learning ability and memory retention in bees (Pham-Delègue et al., 2002), but few studies have used it to analyze the impact of acaricides. In this study, the LD05 dose of formic acid was the most detrimental treatment being the only one to impair short and mid-term memory at 2 and 24 hpt. While formic acid treatment also resulted in a significantly lower PER for long-term memory, that effect was not significantly

Conclusion

In conclusion, this study is the first to compare LD05 doses between the most commonly used natural and synthetic acaricides on the PER responses of honey bees. The acaricides could be divided into three groups based on memory loss due to exposure to the LD05 doses. Formic acid was the most severe, followed by tau-fluvalinate, amitraz and coumaphos, and then thymol, which was the only acaricide not to affect PER compared to a non-treated control. Most studies of sublethal doses of acaricides on

Funding

This study was partially funded by a grant from the Ontario Ministry of Agriculture, Food and Rural Affairs to EG (OMAFRA-UoG research grant No. 0414-0317).

Acknowledgments

The following people contributed in different ways in the experiments conducted: Paul G. Kelly, Mollah Md. Hamiduzzaman, Abril Soria-Martínez, Nancy Bradbury, Mariana Guzman, Masha Burelo, Brooke Wallace and David Stotesbury.

References (55)

  • M.E. Bitterman et al.

    Classical conditioning of proboscis extension in honeybees (Apis mellifera)

    J. Comparat. Psycho1.

    (1983)
  • E. Bonnafé et al.

    Effect of a thymol application on olfactory memory and gene expression levels in the brain of the honeybee, Apis mellifera

    Environ. Sci. Pollut. Res.

    (2015)
  • S. Bonzini et al.

    Predicting pesticide fate in the hive (part 1): experimentally determined tau-fluvalinate residues in bees, honey and wax

    Apidologie

    (2011)
  • G.C. Brown

    Control of respiration and ATP synthesis in mammalian mitochondria and cells

    Biochem. J

    (1992)
  • J.E. Casida et al.

    Mechanisms of selective action of pyrethroid insecticides

    Annu. Rev. Pharmacol. Toxicol.

    (1983)
  • C.Y.J. Chen et al.

    An improved method for the isolation of total RNA from Malva pusilla tissues infected with Colletotrichum qloeosporioides

    J. Phytopathol.

    (2000)
  • T.G.E. Davies et al.

    DDT, pyrethrins, pyrethroids and insect sodium channels

    IUBMB Life

    (2007)
  • J.D. Dean et al.

    Comparison of relative RT-PCR and northern blot analyses to measure expression of β-1, 3- glucanase in Nicotiana benthamiana infected with Colltotrichum destructivum

    Plant Mol. Biol. Rep.

    (2002)
  • A. Decourtye et al.

    Comparative sublethal toxicity of nine pesticides on olfactory learning performances of the honeybee, Apis mellifera

    Arch. Environ. Contam. Toxicol.

    (2005)
  • J. Erber et al.

    Localization of short-term memory in the brain of the bee, Apis mellifera

    Physiol. Entomol.

    (1980)
  • T. Farooqui et al.

    Modulation of early olfactory processing by an octopaminergic reinforcement pathway in the honeybee

    J. Neurosci.

    (2003)
  • J. Felsenberg et al.

    Behavioural pharmacology in classical conditioning of the proboscis extension response in honeybees (Apis mellifera)

    J. Visual. Exp.

    (2011)
  • E.H. Frost et al.

    Effects of fluvalinate on honey bee learning, memory, responsiveness to sucrose, and survival

    J. Exp. Biol.

    (2013)
  • N.E. Gary et al.

    Vacuum device for collecting and dispensing honeybees (Hymenoptera: Apidae) and other insects into small cages

    Ann. Entomol. Soc. Am.

    (1987)
  • H.A. Gashout et al.

    Lethality of synthetic and natural acaricides to worker honey bees (Apis mellifera) and their impact on the expression of health and detoxification-related genes

    Environ. Sci. Pollut. Res.

    (2018)
  • M. Hojo et al.

    Reduced expression of major royal jelly protein 1 gene in the mushroom bodies of worker honeybees with reduced learning ability

    Apidologie

    (2010)
  • R.M. Hollingworth et al.

    Biological and neurotoxic effects of amidine pesticides

  • Cited by (23)

    • The adverse impact on lifespan, immunity, and forage behavior of worker bees (Apis mellifera Linnaeus 1758) after exposure to flumethrin

      2023, Science of the Total Environment
      Citation Excerpt :

      Honey bee loss adversely affects both natural plant biodiversity and crop production, which relies on insect pollination (Potts et al., 2010). Chemical drugs (pesticides and in particular insecticides/acaricides) are a significant stressor for honey bees, causing various physical and physiological effects on individual honey bees and colonies (Chmiel et al., 2020; Gashout et al., 2020; Olgun et al., 2020). There are two main categories of chemical drug residues in honey bee colonies: chemical drugs in the environment and veterinary drugs applied directly to the colony (Hristov et al., 2020).

    • Presence, persistence and distribution of thymol in honeybees and beehive compartments by high resolution mass spectrometry

      2021, Environmental Advances
      Citation Excerpt :

      It has been reported that thymol could induce brood removal or increase queen mortality (Floris et al., 2004; Glavan et al., 2020; Whittington, R., Winston, M.L., Melathopoulos, A.P., Higo, 2000) (Glavan et al., 2020; In- Floris et al. 2004; Whittington el al., 2000). Studies have also shown that this compound alter some metabolic responses such as detoxification gene expression pathways (Boncristiani et al., 2012), have a negative impact on the immune response of bees (Gashout et al., 2020) and can affect hygienic behavior of honeybees due to the interaction with olfactory receptors (Colin et al., 2019). After the administration of thymol treatment, residual contamination of beeswax, honey, beebread, bee brood or bees may occur (Tihelka, 2018; Bernal et al., 2020).

    • Effects of sequential exposures of sub-lethal doses of amitraz and thiacloprid on learning and memory of honey bee foragers, Apis mellifera

      2021, Journal of Asia-Pacific Entomology
      Citation Excerpt :

      Harnessed pollen foragers were fed 5 µl of (20 µg/ml = 0.1 µg/bee, 40 µg/ml = 0.2 µg/bee, 80 µg/ml = 0.4 µg/bee and 160 µg/ml = 0.8 µg/bee) of amitraz; (10 µg/ml = 0.05 µg/bee, 20 µg/ml = 0.1 µg/bee, 40 µg/ml = 0.2 µg/bee and 80 µg/ml = 0.4 µg/bee) of thiacloprid, and 50% sugar solution as a control. Individually harnessed bees were kept on the rack and fed 5 µl of 50% sucrose syrup three times a day using a micropipette (Gilson’s pipetman classicTM Korean analytical instruments, Korea) (Gashout et al., 2020). The number of bees surviving for 72 hrs was recorded (Fig. 3).

    View all citing articles on Scopus
    View full text