DOI: 10.5061/dryad.ht76hdrh4
title: Honeybee optomotor behaviour is impaired by chronic exposure to insecticides

description: Honeybees use wide&[ndash]field visual motion information to
 calculate the distance they have flown from the hive, and this information
 is communicated to conspecifics during the waggle dance. Seed treatment
 insecticides, including neonicotinoids and novel insecticides like
 sulfoxaflor, display detrimental effects on wild and managed bees, even
 when present at sublethal quantities. These effects include deficits in
 flight navigation and homing ability, resulting in decreased survival of
 exposed worker bees. Neonicotinoid insecticides disrupt visual motion
 detection in the locust, resulting in impaired escape behaviours, but it
 had not previously been shown whether seed treatment insecticides disrupt
 wide&[ndash]field motion detection in the honeybee. Here, we show
 that sublethal exposure to two commonly used insecticides, imidacloprid (a
 neonicotinoid) and sulfoxaflor, results in impaired optomotor behaviour in
 the honeybee. This behavioural effect correlates with altered stress and
 detoxification gene expression in the brain. Exposure to sulfoxaflor led
 to sparse increases in neuronal apoptosis, localized primarily in the
 optic lobes, however there was no effect of imidacloprid. We propose that
 exposure to cholinergic insecticides disrupts the
 honeybee&[nprime]s ability to accurately encode
 wide&[ndash]field visual motion, resulting in impaired optomotor
 behaviours. These findings provide a novel explanation for previously
 described effects of neonicotinoid insecticides on navigation and link
 these effects to sulfoxaflor for which there is a gap in scientific
 knowledge. --
description: \subsection{Bees and insecticide exposures}<br>Worker bees
 (\textit{Apis mellifera}) were collected at the entrance to outdoor hives
 located at Falmouth Academy (Falmouth, MA) between July and August 2019.
 To reduce the inclusion of bees affected by age or illness, bees were
 discarded if they displayed low activity during the first hour after
 collection or appeared otherwise damaged. Bees were housed at the Marine
 Biological Laboratory (Woods Hole, MA) for 5 days in groups of 2-3 bees in
 5 cm\textsuperscript{3} acrylic boxes in a ventilated incubator at 32ºC
 and 55\% humidity, which is within the range of thermoregulation of
 honeybee hives \cite{Stabentheiner2010}. A 1.5 M sucrose solution was
 provided ad libitum to control bees (CTL), and treatment group bees were
 provided with the 1.5 M sucrose solution containing 50 ppb (195 nM)
 imidacloprid (IMD, Toronto Research Chemicals, Toronto, Canada), 50 ppb
 (180 nM) sulfoxaflor (SFX, Toronto Research Chemicals, Toronto, Canada),
 or a mixture of 25 ppb (97.5 nM) imidacloprid and 25 ppb (90 nM)
 sulfoxaflor (MIX). Treatments were sublethal during the 5 day exposure,
 and mortality of treated versus control bees did not vary over this
 period. <br>\subsection{Optomotor
 assay}<br>Using an open-loop, virtual reality arena that allows
 tethered honeybees to walk on an air-supported ball, we tested the effects
 of a 5 day chronic exposure to the neonicotinoid imidacloprid (IMD), novel
 insecticide sulfoxaflor (SFX) and a mixture of the two insecticides (MIX),
 versus a sucrose control on the optomotor response. The optomotor arena
 consisted of the air-supported ball, a USB camera module (ELP) positioned
 15 cm from the front of the ball, and two computer monitors (CUK Bionic
 B25GM, 24.5”, 240 Hz refresh rate) oriented at a right angle, such that
 the bee was facing the apex of the two monitors, 15 cm away from each
 screen (Figure 1A). Computer monitors displayed vertically-oriented
 translating black sine wave bars, to give the illusion of rotational optic
 flow. Wide-field visual motion elicited the optomotor response in tethered
 walking bees. The resulting rotation of the air-supported ball was video
 recorded and the fictive walking path was extrapolated using FicTrac
 \cite{Moore2014} (Figure 1B, Supplemental Information Video 1).
 We temperature-anesthetized the bees in a refrigerator at 4ºC
 until unresponsive (10-15 minutes). Once anesthetized, the dorsal side of
 the thorax was gently abraded with a blade to remove hairs, and a 1 ml
 pipette tip was affixed to the dorsal surface of the thorax using UV light
 cured dental glue (Prevest DenPro). Bees were then offered a drop of 1.5 M
 sucrose solution and allowed to recover in an incubator at 32ºC for 30
 minutes. After recovery, bees were positioned with a micromanipulator
 (World Precision Instruments) over the air supported ball in the optomotor
 arena. A terrarium lamp was positioned to warm the bee until it began to
 walk unprompted. Bees were excluded from the experiment if they did not
 start walking within 15 minutes. This accounted for approximately 15\% of
 the population, irrespective of treatment. In total, n=23 CTL, n=30 IMD,
 n=37 SFX, and n=25 MIX bees were included in the behavioural assay, and
 the response of all of these bees to the 0.0625 cpd, 16 Hz grating
 stimulus were recorded. For some stimuli, a smaller subset of bees were
 tested, to a minimum of n=13 CTL, n=11 IMD, n=15 SFX, and n=11 MIX for any
 given stimulus.  \subsection{Visual
 stimuli}<br>Visual stimuli were programmed in MATLAB R2019b
 (MathWorks, Natick, MA) by adapting code from the Psychophysics Toolbox
 Version 3 \cite{Brainard1997}. Translating vertical black and white
 sinusoidal bars were displayed in 10-second intervals, first leftwards on
 both screens then instantly changing direction to rightwards. This
 sequence was repeated 4 times, summing to a total of 80 seconds per
 stimulus. Eleven visual stimuli were tested, by varying either the spatial
 or temporal frequency, and were presented in a randomized order with a 3
 minute inter-stimulus interval. Stimuli covered spatial frequencies of
 0.5, 0.25, 0.125, 0.0625, and 0.0039 cycles per degree (cpd), where a
 smaller value represents wider gratings, which were displayed at a
 constant temporal frequency of 16 Hz. In addition, varying temporal
 frequencies of 4, 8, 16, 24, 32, 40, and 48 Hz were tested at a constant
 spatial frequency of 0.0625 cpd. The spatial frequencies were selected
 based on Ibbotson et al \cite{Ibbotson2017}, which demonstrated that
 descending neurons respond most robustly to spatial frequencies between
 0.03 and 0.05 cpd. A subset of animals were also tested with vertical
 black bars that were stationary (“no optic flow” stimulus) to measure
 differences in spontaneous behaviour between treatments. 
 \subsection{Fictrac video analysis} Optomotor
 behaviour tracking was performed online using FicTrac \citep{Moore2014}. A
 program was custom-written in Matlab to align the FicTrac data to the
 visual stimuli and extract the integrated yaw path (yaw radians over time)
 and total movement across all axes (Figure 1B). The integrated yaw was
 reset to 0 at the initiation of each stimulus and each stimulus direction
 switch (e.g., leftward to rightward) such that the output for leftward
 turns was positive, while rightward turns produced negative values (Figure
 1C).  We fit linear regressions to the 8 normalized
 response replicates (4 replicates per direction) for a given
 animal-stimulus pair. The slopes of the linear regressions for each of the
 four leftward and rightward stimuli were averaged to assess the symmetry
 of the behavioural responses. Absolute slopes of 0 represented perfectly
 symmetrical responses, while slopes of increasing magnitude signified
 greater response asymmetry. The R\textsuperscript{2} values for each
 linear regression were also averaged to produce a measure of path
 tortuosity. Responses that switched direction slowly or those that wavered
 from left to right produced lower R\textsuperscript{2} values than
 responses that quickly switched direction to follow the visual stimulus
 and maintained a consistent yaw for the duration of the stimulus. Movement
 distance (across all rotational axes) and diameter of the air supported
 ball were used to calculate the walking speed and this was averaged across
 the 4 leftward and 4 rightward stimulus presentations (80\,s of walking
 behaviour). To compare the magnitude of leftward and
 rightward optomotor responses, we changed the sign of the rightward
 responses so that both directions of responses were on the same scale
 (i.e., both positive slopes, Figure~1D). We calculated the response
 magnitude (cumulative yaw) as the maximum integrated yaw (\textit{ymax})
 averaged over stimulus presentations in both directions. Cumulative yaw
 was also compared between the leftward and rightward stimuli to determine
 if the responses were asymmetrical. Average \textit{ymax} values were
 calculated separately for leftward and rightward responses, and cumulative
 yaw asymmetry (\textit{y.sym}) was obtained by subtracting the smaller
 cumulative yaw value (\textit{ymax}\,b) from the larger value
 (\textit{ymax}\,a), and dividing by the larger value (\textit{ymax}\,a) to
 constrain measurements between 0 and 1. \textit{Y.sym} values close to
 zero represented symmetrical responses, while highly asymmetrical
 responses had \textit{y.sym} values closer to 1. Responses were labeled
 "biased" in the direction that elicited the larger response and
 "anti-biased" in the other direction. 
 \subsection{Relative quantification of gene
 expression}<br>A separate set of bees were chronically exposed to
 IMD, SFX, MIX, or sucrose (CTL) for 5 days, as described above (n = 25
 bees per treatment). Freshly dissected honeybee brains were snap frozen on
 dry ice and stored at -80ºC until further use (&lt;2 weeks). The
 material from 5 bee brains was pooled to one sample and 5 samples were
 generated per treatment group. Total RNA was isolated from each sample
 using the Maxwell® RSC miRNA Tissue Kit (Promega) and the Maxwell® RSC 48
 Instrument (Promega) according to the manufacturer’s instructions. RNA
 concentration was measured using the QuantiFluor® RNA System (Promega),
 and total RNA was quantified by Quantus Fluorometer (Promega) following
 manufacturer protocol, yielding between 120 - 210 ng/µl RNA and a total of
 7.2 – 12.6 µl.  Genes of interest were superoxide
 dismutase 1 (SOD1), catalase (CAT), cytochrome P450 enzymes (CYP9Q2 and
 CYP9Q3), and the reference gene GAPDH. Forward and reverse primer
 sequences and sequence accession numbers are described in Supplemental
 Information Figure 1A. Primers were either designed using NCBI Primer
 BLAST (NCBI) with 100\% specificity for the genes of interest or taken
 from previous studies \cite{Collins2004, Reim2013}. Quantitative reverse
 transcription PCR (RT-qPCR) was performed with a 1-step method combining
 reverse transcription and amplification in a single tube using the GoTaq
 1-Step RT-qPCR System (Promega). A 20 µl final volume contained 10 µl
 GoTaq qPCR Master Mix, 0.4 µl GoScript 1-step RT Master Mix, 0.5 uL of 10
 µM forward and reverse primer mix (Genewiz, New Jersey), 7.1 µl Nuclease
 Free Water, 0.8 µl MgCl\textsuperscript{2} and the RNA sample. Samples
 were loaded into a QuantStudio 5 (ThermoFisher Scientific) and run with
 the following cycling conditions: 45ºC for 15 minutes, 95ºC for 10
 minutes, 40 cycles of 95ºC for 15 seconds and 60ºC for 30 seconds (data
 collection step), and finally 72ºC for 60 seconds followed by the melt
 curve. RT-qPCR amplification was assessed with the normalized fluorescent
 signal (Delta Rn) from each sample (Supplemental Figure 1B). We used 1 –
 0.0001x dilution series of each RNA sample and the standard curve was used
 to demonstrate that the presence of PCR inhibitors was unlikely
 (Supplemental Figure 1C). A reaction without RT enzyme was used as
 reaction control for assessing the absence of DNA in all samples: no
 amplification could be detected in these samples.  We
 validated qPCR specificity using the melt curve analysis (Supplemental
 Figure 1D). Samples resulting in qPCR efficiency below 90\% were omitted
 from the analysis (3 of 20 samples). Data analysis was performed using
 QuantStudio5 (ThermoFisher Scientific) using the Cq threshold method
 \cite{Nolan2006, Pfaffl2001}. RNA amplification of genes of interest were
 normalized to the amplification of GAPDH for each sample (delta Ct), and
 then compared to the mean amplification of the control samples (delta
 delta Ct).  \subsection{TUNEL assay}<br>An
 additional cohort of honeybees (n = 4 per group) were treated with IMD,
 SFX or sucrose (CTL) for 5 days as described above. For tissue processing,
 cold-anesthetized honeybees were decapitated, the brain was dissected
 under cold PBS, pH 7.4 (as previously described by Groh and  Rössler
 \cite{Groh2011}) and immersion fixed in 4\% PFA/PBS, pH 7.4 at 4°C
 overnight. The tissue was washed in PBS for 12 hours at 4°C and incubated
 in 30\% sucrose/PBS at 4°C overnight. Prior to sectioning, tissue was
 immersed in Tissue-Tek O.C.T. compound (Sakura) and frozen on dry ice.
 Brains were sectioned at 12 µm using a cryostat, collected on SuperFrost
 Plus adhesion slides (Thermo Scientific), and stored at -20°C until
 further use. Two sections per brain (120 µm cutting distance) were mounted
 on each slide. For TUNEL assay, tissue was defrosted
 and washed 5 times with PBS, pH 7.4. Sections were permeabilized in 0.1\%
 Triton X-100, 0.1M sodium citrate pH 6.0 at RT for 30 minutes. After 4
 washes with PBS, sections were treated with TUNEL reagent (In situ cell
 death detection kit, fluorescein; Roche) in a dark, humidified 37°C
 chamber for 3 hours according to the manufacturer’s instructions. As
 negative control, no enzyme solution was applied during this step. As
 positive control, sections were treated with DNase I (1.5 U/µl) in 10 mM
 Tris-HCl, pH 7.4, 10 mM MgCl\textsuperscript{2}, 1 mg/ml BSA at 37°C for
 60 minutes prior to TUNEL development. All sections were washed 3 times
 with PBS and incubated with Hoechst 33342 (1:1000) in PBS for 10 minutes.
 After 2 washes in PBS, sections were mounted with Fluoromount aqueous
 mounting medium (Sigma-Aldrich). Images were acquired
 using an upright FV1000 confocal microscopy system (Olympus) equipped with
 a 20x/N.A. 0.85 oil immersion objective and using the laser lines 405 nm
 for Hoechst and 488 nm for TUNEL with standard filter sets. Image analysis
 was further performed using the open source software ImageJ/Fiji
 \cite{Schindelin2012}. We selected areas o interest (MB, La, Lo, Me) in
 the honeybee brain and quantified TUNEL+ cells only for areas rich in
 nuclei (Hoechst staining); hence, excluding the neuropil (Supplemental
 Figure 4A). Under blinded conditions, the cells within this area were
 defined for 2-4 sections per bee. Sections with poor Hoechst staining
 intensity were excluded from the analysis.
 \subsection{Consensus clustering and other statistical
 analyses}<br>We performed all statistical analyses in R version
 4.0.3 \cite{RCoreTeam2016}. Optomotor response types were clustered using
 a k-means algorithm to group the features described above measured from
 the behavioural responses of bees across treatments and visual stimuli.
 Prior to clustering, response features were z-scored. The silhouette
 method \cite{Rousseeuw1987}, predicted an optimal cluster count of 3
 resulting from the k-means algorithm. This cluster count was confirmed by
 a consensus clustering method \cite{Wilkerson2010}, an unsupervised
 learning technique that aggregates the results of multiple clustering
 runs. For each clustering iteration, we included 90\% of the total
 responses and 80\% of the features (4 of 5). The clustering algorithm was
 repeated for 2000 iterations, and the output from each clustering run was
 compared to define a consensus matrix. The proportion of behavioural
 responses assigned to each cluster was calculated for each treatment and
 visual stimulus. The features of the behavioural responses within each
 cluster were then compared to provide a quantitative illustration of the
 behavioural response types across the visual stimuli and
 treatments.  Data were tested for normality using the
 Shapiro-Wilk normality test and equal variance using Levene’s test from
 the car package \cite{Fox2019}. In the lmerTest package
 \cite{Kuznetsova2017} we used a linear mixed effects (LME) model to
 compare features of behavioural responses across treatments and stimuli,
 with animal as a random effect to account for repeated measures. Post hoc
 multiple comparisons were performed using the emmeans package
 \cite{Lenth2018} with the Kenward-Roger degrees-of-freedom method and
 Tukey P value adjustments. Relative gene expression, and spontaneous (no
 stimulus) behavioural responses were quantified with Kruskal-Wallis rank
 sum tests with Wilcoxon pairwise comparisons.  Statistical significance
 was assessed at p &lt; 0.05. In figures, asterisks denote statistical
 significance from post hoc multiple comparisons (&lt; 0.0001 = ‘***’;
  &lt; 0.001 = ‘**’; &lt; 0.05 = ‘*’), or letters show significant
 effects between groups.