Animals

This research was conducted using honey bees (Apis mellifera L.) reared at National Taiwan University. Each bee colony tested was evaluated as stable and consisted of a normal egg-laying queen, larvae, pupae, honey and pollen. All colonies were monitored regularly to ensure that they were in optimum condition. If required, a 50% sucrose solution and artificial pollen were fed to a colony to maintain its status.

In our experiment, the honey bees were divided into two groups: the normal honey bees and the honey bees that were fed a sublethal dose of imidacloprid during their larval stage.

The normal honey bees were collected by choosing one comb with a large number of capped-brood cells. This comb was then placed in an incubator and the temperature was maintained at 33–34 °C. After the honey bees emerged, they were immediately marked with acrylic paint on their thoraxes for identification of their eclosion date and were then put back into their hive. Twenty honey bees of each age (1 day, 10 days and 20 days) were randomly collected. In addition, 20 foragers carrying pollen on their corbiculate legs were collected outside the hive. The collecting method for honey bees fed with imidacloprid is described below.

Preparation of the imidacloprid solution

Imidacloprid (95% TG, Bayer Cropscience AG, Monheim am Rhein, Germany) is a powder and was dissolved in dimethyl sulfoxide (DMSO, MP Biomedical). To reduce the effect of solvent on honey bees, the solvent used should not induce any abnormal behaviours in the honey bees. Previous studies have indicated that acetone significantly reduces the feeding activity of honey bees17, whereas DMSO does not. For this reason, DMSO was chosen as the solvent for imidacloprid. The amount of solvent remaining in the imidacloprid solution was trivial (below 0.0005%, v/v, which has been shown to be negligible in previous studies8,11,12,14,15,18).

Yang et al.23 demonstrated that a dose of imidacloprid above 0.04 ng would result in an apparent reduction in the bees’ olfactory associative ability after they matured into adults. Therefore, bees that received 0.04 or 0.4 ng of imidacloprid were deemed to have impaired olfactory learning ability and were designated as the experimental group. Bees receiving 0.004 ng of imidacloprid were designated as the group where the olfactory learning ability of the adult bee was not impaired. This was done to compare the density of synaptic units in the calyces of the mushroom bodies among the various groups. Yang et al.23 showed that capped-brood rate, pupation rate and eclosion rate were not affected by a dose of up to 200 ng. Therefore, in the experimental group, the comparatively higher dose of 2 ng imidacloprid, which did not affect the survival rate of the larvae, was added to test whether the development of neural connectivity in the brains of the worker bees was normal.

Thus, the concentrations of imidacloprid used in our experiment were: 1 μg/L (ppb), 10 μg/L, 100 μg/L and 500 μg/L, respectively. All concentrations used were diluted from the imidacloprid stock solution, which was prepared using a mixture of 0.5 mL distilled deionized water (DDW) and 0.5 mL DMSO to which 1 mg of imidacloprid powder was added, giving a concentration of 1 g/L. Then, 2 μL of the stock solution was added to 1998 μL of DDW, resulting in an intermediate concentration of 1000 μg/L. The test solutions in concentrations of 500 μg/L, 100 μg/L, 10 μg/L and 1 μg/L of imidacloprid were obtained by using 1000 μL, 200 μL, 20 μL and 2 μL of intermediate-concentration solution added to 1000 μL, 1800 μL, 1980 μL and 1998 μL of DDW, respectively.

Imidacloprid feeding process

After the appropriate colonies were selected, the queen of each colony was restricted to laying her eggs on one honeycomb within her hive so that the offspring’s age could easily be determined after they turned into larvae. The areas of the cells used for testing 1-day-old larvae were labelled by marking transparent slides placed on the honeycomb. The larvae were divided into five groups, one for the control group and the others for the experimental groups. It is typical for a normal egg-hatching queen to produce approximately 400 one-day-old larvae. These larvae were located on one side of the comb and were properly marked23.

After the marking procedure, the experimental groups were fed 1 μL of 1 μg/L, 10 μg/L, 100 μg/L and 500 μg/L concentration of imidacloprid, respectively, while the control group was fed a 0.0005% DMSO solution (the actual concentrations of DMSO fed to the larvae were 0.000001%, 0.00001%, 0.0001% and 0.0005%, respectively). As mentioned earlier, the negligible concentration of DMSO can be ignored and therefore the highest concentration of DMSO in the experimental group (0.0005%) was chosen to be fed to the control group. This solution was supplied to the larvae near the cell wall by means of a pipette. This feeding procedure was repeated daily for four days, resulting in a total intake of imidacloprid in the experimental groups of 0.004, 0.04, 0.4 and 2 ng/larva, respectively. We excluded those larvae that did not complete the four-day feeding process, either due to prior capping or death.

Usually, the cells are capped after six days and the larvae turn to pupae. To avoid any prior disturbance of the pupae and to ensure their stable state, we waited approximately seven days before transferring them into a 96-well plate and placing it in an incubator at 34 °C and a relative humidity of 70–80%. The pupae were placed in different 96-well plates that were separated in different compartments according to the concentration of imidacloprid with which they were fed, to avoid any mix-up after they reached the adult stage. Approximately six days after capping, the pupae became adults and emerged from their cells. After eclosion, the adult bees were marked on the thorax with acrylic paint of a different colour, depending on the treatment group to which they belonged and were then returned to their original hive. After excluding the dead, the average number of marked bees in each group was approximately fifty. Twenty adult bees from each group were then randomly collected on the 20th day after eclosion. These bees underwent an antibody staining procedure to reveal the density of their MG after being fed a sublethal dose of imidacloprid during their larval stage.

Immunocytochemistry

To analyse the effect of imidacloprid during the larval stage of honey bees on the MG in their calyces, we followed the protocol of Groh et al.47, but with some adjustments to be able to immunolabel the synaptic units in a whole-mount preparation. The tested bees were anaesthetized at 4 °C and their heads were fixed using beeswax. After removing the setae on the head capsule, the head was cut open along the edge and the glandular tissue, tracheae and membrane were exposed. The head was covered with cold physiological saline (130 mM NaCl, 5 mM KCl, 4 mM MgCl 2 , 5 mM CaCl 2 , 15 mM HEPES, 25 mM glucose, 160 mM sucrose; pH 7.2)47 and the glandular tissue, tracheae and membrane were removed, as well as the ocelli.

The head was then cut from the honey bee’s body and pre-fixed in 4% paraformaldehyde (Riedel-de Haën, 16005) at 4 °C in 0.01 M phosphate-buffered saline (PBS; 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.2) for 30 minutes52. The fixed brain was then dissected out from the head capsules under PBS. After a quick rinse in PBS, the brain was then placed in an Eppendorf tube and fixed in PFA for 20 hours at room temperature53.

The brains were washed in PBS five times (each time for 10 minutes) and were then permeabilized in 0.2% Triton X-100 (Tx) in PBS twice for 10 minutes each. They were then blocked in 2% normal goat serum (NGS; 005-000-121, Jackson ImmunoResearch Laboratories) in 0.2% PBS-Tx for 3 hours at 23 °C47.

For the synapse labelling process, the brains were incubated in the primary antibody SYNORF1 (monoclonal antibody against the Drosophila synaptic-vesicle-associated protein synapsin I (Erich Buchner, Wüerzburg, Germany)52,54), which was diluted 1:10 in 0.2% PBS-Tx with 2% NGS for four nights at 4 °C47. This antibody is commonly used in the synaptic immunostaining of Drosophila and honey bees37,40.

After being rinsed in PBS five times, for 10 minutes each time, the brains were incubated in goat anti-mouse DyLight 550 secondary antibody (Mouse IgG-heavy and light chain cross-adsorbed Antibody; Catalogue No. A90-516D3, Bethyl Laboratories, Inc.; diluted with 1:250 in PBS) for four nights at 4 °C47. Afterwards, they were washed in PBS five times, for 10 minutes each time and dehydrated in an ascending ethanol series (30%, 50%, 70%, 90%, 95%, 3X 100%). Finally, the brains were cleared in methyl-salicylate (M6752, Sigma-Aldrich, Germany) and mounted whole-mount in Permount (SP15-100; Fisher Scientific, Fair Lawn, NJ) on a modified concave slide (Marienfeld, Lauda-Königshofen, Germany). Finally, they were covered with cover glasses (thickness No. 1)52.

Confocal microscopy

The whole-mount preparations were scanned using a Laser Scanning Confocal Microscope (Leica TCS SP5 II, Germany) with a 20X objective lens (HCX PL APO CS 20X/0.70 DRY, WD = 0.59 mm), a 40X objective lens (HCX PL APO CS 40X/1.25 OIL, WD = 0.1 mm, UV/405 nm) and a 63X objective lens (HCX PL APO lambda blue 63X/1.40 OIL, WD = 0.1 mm, UV/405 nm). Synaptic units labelled with goat anti-mouse DyLight 550 secondary antibody were excited at 543 nm using a HeNe laser and emitted in the range of 560–580 nm. The scanning was performed at a resolution of 1024 × 1024 pixels at an acquisition speed of 100 Hz.

The calyces are located on the upper surface of the brain. This methodology allowed the scans to go through the entire structure of the calyces from top to bottom. Scans were taken at 5 μm intervals to measure the MG in the calyx region47. Each brain contains four calyces (Fig. 1). In the scanning process, the structure of each of the four calyces was scanned separately to obtain a scan of the complete structure. The total number of images captured for each calyx differed from brain to brain and depended on the depth of the calyx.

Image processing

The confocal images were processed by Imaris® (x64 version 7.6.5, Bitplane, Zürich, Switzerland). The images for the entire structure of the calyces were processed to obtain a 3D structure of each calyx formed by the many layers of images taken by confocal microscopy. By using this software, the 3D structural diagram could be framed and separated into different regions of interest by creating a new surface covering that specific part, such as the lips, collars and basal rings. The software then allowed us to view the 3D structural diagram of the original layers taken by the confocal microscope. Using these images, we started at the layer from which the boundaries of all the subparts in the calyces could be easily identified. The lip regions were the apparent ellipse on the two starting points of the U-shape. The boundary of the total collar region was determined by the line of the dense part in the collar because of its obvious borderlines. The rest was shown as the basal ring. Framing these subparts layer by layer, we reconstructed the 3D structural diagram of the calyces with different subparts shown with different surfaces. Using this software, we defined the lateral calyces with two lips, two collars and a basal ring (from left (l) to right (r): lip (l), collar (l), basal ring, collar (r) and lip (r)) and the medial calyces with two lips and a collar (lip (l), collar and lip (r)). Different treatments were randomly assigned as numbers without indicating the treatment group to which they belonged.

Data acquisition of MG counts

The MG counts for all the subparts of the calyces were determined by the Imaris® surface function. The surface function is created by framing each specific region layer by layer through the 3D structure. The diameter range of the MG was defined as being between 1.5 and 3.5 μm. This range was set by measurement of the MG through the Imaris® function. In addition, setting the diameter lower than 1.5 μm introduced background noise, while setting it to a value greater than 3.5 μm could cause miscalculation by overlapping parts that are not relevant for counting MG. We therefore chose this range of diameters to define the area for the MG count. The results—which processed the MG depicted as dots—were visually confirmed to ensure that all defined MGs in this diameter range were counted within the selected threshold range. Although previous studies have indicated that the human eye is the best tool for synapse identification because of the perception and expertise of the human viewer in defining the presence of labels55, the large amount of data in this study could not be processed entirely by the human eye.

We utilized the Imaris® software to calculate the enormous number of MG within each calyx using the filters to set the threshold of the MG-counting area. Through surface function framing of each specific subpart, the volume was calculated by analysing the entire surface region. The MG density was calculated by using the MG count and the calculated volume of each region.

Statistical analysis

Ten samples each were analysed from the group of normal bees and the experimental groups. To avoid bias caused by individual differences, the highest and lowest density values in each group were excluded from the statistical analysis.

The standard trends in all regions of the calyces for the group of normal bees were set by bees aged 1, 10 and 20 days after eclosion and foragers. The statistical values of spots, volumes and density were square-root transformed before analysis to conform to the assumption of a normal distribution when using Kolmogorov-Smirnov tests. To compare the differences in MG count, volumes of the different compartments and the density among bees of different ages, data were analysed using repeated-measures ANOVA when values fit normal distributions or GEE tests when they did not fit normal distributions. The comparisons between the control group and each imidacloprid-fed group in all regions were statistically analysed using the repeated-measures ANOVA when values fit normal distributions or the Mann-Whitney U test when they did not fit normal distributions.

Statistical analyses were performed with SPSS 21.0 software. For ease of interpretation, all figures and tables present raw data as the means ± standard deviation (SD). The brightness and contrast of the images presented in Figs 1 and 5 were slightly adjusted for better indication.