Are bumblebee queens exposed to neonicotinoids in the field?

Two visits were made to two winter OSR (B. napus) fields (variety PR46W21) at Shiplake Farm, Oxfordshire, United Kingdom (51° 30' 15.2" N, 0° 54' 00.7" W) during early April 2014 when the crop was in flower. Crop seeds had been treated with Modesto seed treatment (clothianidin and β-cyfluthrin; Bayer Crop Science) and planted the previous year. Transects around the edge of the fields (distance around each field: 2 km and 0.94 km) and through the centre of the crop (0.3 km and 0.4 km respectively) were walked between 11:00 and 15:00 on days when weather conditions were suitable (sunny and dry with minimal wind). Transects were walked once per visit at a steady pace (total walking time per visit: 3 h) and all bumblebee species within 2 m of the transect were recorded, along with the caste and activity of each bee. Queens of the B. lucorum complex (B. lucorum, Bombus cryptarum and Bombus magnus) cannot be reliably separated using morphological features alone69, and so these were recorded as B. lucorum complex.

Impacts of multiple stressors on B. terrestris queens

Colonies

Fifteen B. terrestris audax colonies were obtained from Koppert. Colonies were kept in the laboratory in darkness at 22 °C and a red light was used for colony manipulation. Colonies were fed ad libitum with 50% Ambrosia (EH Thorne), an inverted sugar syrup solution (herein referred to as syrup) and frozen honeybee-collected pollen pellets (Koppert). On arrival, 10% of the workers from each colony were dissected and screened microscopically for the parasites C. bombi (Trypanosomatidae), Nosema bombi (Microsporidia) and Apicystis bombi (Neogregarinida) using a Nikon eclipse (50i) compound microscope at 400× magnification. No parasite infections were detected at this stage.

Mating

Males and gynes (reproductive females) were removed from colonies as callows (newly emerged bees) and kept communally in single-sex wooden boxes (24 cm × 14 cm × 10.5 cm) with nest mates of the same age and fed ad libitum with pollen and syrup.

Four days after eclosion, gynes were mated with unrelated males of at least four days of age. Mating took place in a 60 cm × 50 cm × 50 cm wooden framed arena with plastic mesh sides under natural light at a temperature of 22 °C. Up to 25 males from a single colony were placed into the arena and left to acclimatize for 10 min. Unrelated gynes from another single colony and age group were then added to the arena. Mating pairs were removed from the arena immediately and the time, date, male and female colony, and age were recorded. Once mating was complete, the male was removed and frozen at −20 °C. The mated queen was kept in an individual plastic box (13 cm × 11 cm × 6.8 cm) containing a small amount of tissue paper to remove excess moisture and immediately provided with 100 µl of inoculum (see below for inoculum preparation). When this full amount had been consumed, the queen was provided with ad libitum food (pollen and syrup) for between two and four days after mating (depending on how quickly the inoculum was consumed), at which point it was weighed and placed into hibernation (see below). Queens that did not consume the full amount of inoculum within four days were excluded from the experiment.

Gynes that did not mate on the first attempt were kept in their communal boxes as described above, and further mating attempts (up to five attempts per gyne) were made (with different groups of males), until mating took place. Males were also kept until mating had occurred, and mating attempts continued until males were two weeks of age, at which point they were frozen at −20 °C.

Preparation and delivery of C. bombi inoculum

C. bombi was obtained from naturally infected wild B. terrestris queens collected from Windsor Great Park, Surrey, United Kingdom (51° 25' 05.9" N, 0° 36' 19.5" W) during the spring of 2013. Queens were also screened for N. bombi, S. bombi and A. bombi and any queens co-infected with these parasites were removed. Crithidia-infected queens were placed in the laboratory in Perspex queen-rearing boxes (13.3 cm × 8 cm × 5.6 cm) with ad libitum syrup and pollen, and kept in a dark room at a constant temperature of 28 °C and 50% humidity (conditions suitable for colony initiation). Eleven naturally infected queens (and their colonies in six cases) were available at the start of the experiment; 10 µl of faeces was collected from each of these, combined and used to infect 20 stock worker bees collected from each of the experimental colonies. This ensured that a wide range of naturally occurring strains of C. bombi was available for the infection of experimental queens. All collected faeces was combined and diluted with 0.9% Ringer’s solution to make 1 ml of solution. C. bombi cells were filtered using a modified protocol for purification23 originally developed by Cole70. This process was repeated using wild-caught queens from the same population that were not infected with C. bombi, A. bombi, N. bombi or S. bombi to provide a control.

The stock bees were taken from the experimental colonies to account for any filtering of the parasite strains by workers before infection of the experimental queens71. Workers were removed from each colony and starved for a period of four hours. Each stock bee was then individually fed a 10 µl drop of inoculum (containing 10,000 C. bombi cells) and observed until all the liquid had been consumed. These stock bees were then kept communally in wooden boxes with their nest mates and fed pollen and syrup ad libitum. The same process was repeated using faeces from the uninfected wild queens to create a control stock.

To make the inoculum for the experimental queens, an equal volume of faeces (10 µl) was collected from each box of stock bees on each day that inoculation took place. This was combined and purified as described above. The resulting solution was diluted with syrup and 100 µl of this inoculum (containing at least 20,000 C. bombi cells) was provided in a feeding tube for each queen. The same process was repeated using the C. bombi-free faeces from the control stock bees.

Hibernation

Mated queens (only those that had consumed the full volume of inoculum) were weighed, placed into 50 ml tubes (Falcon) with damp sterilized sand and kept in a dark incubator at a constant temperature of 4 °C for either 6 weeks or 12 weeks. After this hibernation period, the queens were removed from the tubes and re-weighed. Surviving queens were then placed into Perspex queen-rearing boxes (13.3 cm × 8 cm × 5.6 cm) with ad libitum syrup and pollen and kept in a dark room at a constant temperature of 28 °C and 50% humidity.

Pesticide exposure

A total of 319 mated queens were placed into hibernation. Of these, 20 died during hibernation and a further 68 were excluded from the final analysis. Exclusion was due to a lack of replication for their natal colony (as a result of nest mates being lost (n = 60)), accidental infection with C. bombi (n = 6) and accidental death (n = 2). The remaining 231 queens (from eight colonies) were allocated to either the pesticide or control treatment. The distribution of queens across the eight treatment groups is shown in Table 3.

Table 3 Summary of queen numbers allocated to the eight treatment groups in the experiment Full size table

Three days after emergence from hibernation, queens in the pesticide treatment group were provided with syrup containing 2.4 ppb thiamethoxam, which is equivalent to that found in stored nectar in bumblebee colonies foraging in agricultural environments in the UK72 and significantly below mean levels reported in stored pollen from bumblebee colonies foraging in agricultural environments in the UK73. As established in the field trial above, bumblebee queens forage on neonicotinoid-treated OSR crops and are therefore likely to be exposed to these pesticides as they establish a colony in the spring.

Analytical standard thiamethoxam (Pestanal; Sigma–Aldrich) was mixed with Acetone (Fluka; Sigma–Aldrich) to give a stock solution of 100 mg ml–1. Aliquots of this stock were diluted with syrup to give a final concentration of 2.4 ppb thiamethoxam. Acetone alone was diluted in the same way to provide a solvent control. Solution was freshly made on each day of the experiment. Samples of treated syrup from two dates in the experiment were collected and analysed for thiamethoxam residues using liquid chromatography–mass spectrometry (Food and Environment Research Agency). Average residues were found to be 2.5± 0.085 µg kg–1.

Queens were provided with the pesticide-treated syrup (or acetone-treated syrup in the case of the control group) for 14 days, and the amount consumed by the queen during this time was measured twice (once after 7 days, at which point the feeder was replenished with fresh treated syrup, and again after 14 days) using a 25 ml measuring cylinder to an accuracy of 0.25 ml. The average evaporation rate was measured by keeping feeders (n = 10) in empty rearing boxes for one week and calculating the volume lost during this time. Syrup consumption data were then corrected for evaporation. Queens were provided with ad libitum untreated syrup for the remainder of the experiment.

Post-hibernation monitoring

After hibernation, all queens were provided with a pollen ball (ground pollen pellets mixed with syrup to form a soft dough, shaped into a cylinder of approximately 1 cm in height and diameter) in which to lay their eggs and as a source of food. Unused pollen balls (which contained no eggs or brood) were changed twice a week to provide a source of fresh pollen for the queens. Pollen balls containing brood were left in the box and an additional pollen ball or dish of loose pollen was provided twice a week.

Queens were monitored daily for mortality and egg laying. All bees that died during the experiment were frozen at −20 °C on the day of death. The first date of egg laying (colony initiation) was recorded, as was the date that the first adult worker eclosed. Queens that had not initiated a colony ten weeks after emergence from hibernation were frozen at −20 °C. Queens that had a brood were kept for an additional four weeks to monitor the development of the brood into adult workers.

Each queen was checked for the presence of C. bombi (by microscopic examination of a fresh faecal sample) three times during the experiment. The first check occurred 4 days after the end of hibernation, the second 11 days after hibernation and the third 30 days after hibernation.

Dissection

All dissections were performed using a Nikon microscope (SM2800) at a magnification of ×10 to ×30. At the end of the experiment, all queens were dissected and checked microscopically for the presence of C. bombi (as described for the parasite screening above). Queens were also screened for N. bombi and A. bombi to verify the earlier colony-screening results. Neither of these parasites was found at this stage.

Analysis

Models were constructed for each analysis using some or all of the following factors: hibernation (short or long), pesticide (pesticide or control), parasite (exposed to the parasite or not exposed) and infection (infected or uninfected; this was assessed through the four parasite checks. If C. bombi was detected during any of these, the individual was considered to be infected). The following covariates were also considered: pre-weight (pre-hibernation weight), post-weight (post-hibernation weight), weight loss (proportion of weight lost during hibernation) and thorax (thorax width). The natal colony of the queen and of her mate (QColony and Mcolony) were considered as random factors in mixed models and compared with equivalent models without random factors. In the analysis of egg laying and colony development, all queens that died during the experiment were excluded, as they had not been present during the entire 10 (or 14) week observation period. The details of each analysis are summarized in Table 2.

All analyses were performed in R (version 3.1.1; ref. 74) using the packages lme475 and survival76.

Model selection

To select the optimal model for each analysis, Akaike information criterion (AIC)c values (AIC values corrected for small sample sizes) were compared for a set of candidate models. First, mixed models with one or both of the random factors Qcolony and Mcolony were compared with equivalent models with no random factors77. This was used to decide the random structure used in further model selection (one random factor, both random factors or no random factors). Candidate models were then constructed including biologically meaningful combinations of the fixed factors listed above. These were compared with the null model (no fixed factors) and full model (all fixed factors). Two random factors—queen colony and male colony—were included in the initial comparisons, but did not improve the fit of any of the models and so were not included. Two-way interactions between treatments were considered, but due to lack of coverage three-way interactions were not. Interactions between covariates and treatments were included if data visualisation indicated that this may be useful. The AICc values were used (these were chosen over AIC values due to the small sample sizes) and the optimal model (with the lowest AICc) was selected. When AICc values for different models were within two units of the lowest, model averaging was undertaken78 (except in cases for which the null model was among these, in which case the null was assumed to be optimal). Final models were verified graphically for fit and to ensure all assumptions had been met52,77. Interpretation of the importance of factors within the final models was based on the size of the estimate (the larger the estimate, the greater the effect size of that factor) and 95% CIs (those that did not cross zero were considered reliable and important to the model). Model selection tables are available in the Supplementary Tables 2–8.

Modelling methods

The colony capacity is the average number of colonies produced over one season. It is calculated as the product of the average number of gynes produced per colony taking a certain value, the probability of surviving six months in hibernation and consequently initiating a colony and the probability of successfully mating and finding a new nest site—the p nm variable. Although we do not know the value of any of the components of p nm with absolute certainty, several studies have quantified the success of various stages of the lifecycle. Baer and Schmid-Hempel51 studied the number of gynes produced by colonies under field conditions. They found that 18 colonies produced 155 gynes—an average of 8.61 gynes per colony. We assumed that the number of gynes produced follows a geometric distribution with mean m g (the mean number of gynes). The probability of surviving hibernation (which we called p h ) and the probability of being able to initiate a colony following survival (which we called p c ) were studied by Beekman et al.46. They found that out of 45 queens, 23 survived a 6 month hibernation period, from which 11 (of 23) initiated a colony. We assumed that both the number of survivors and the number of colony-initiating queens is binomially distributed. Here, we use this information to calculate the likelihood of the colony capacity taking a certain value.

Following emergence from the natal colony, gynes need to mate, survive hibernation and then find a nest site. p nm has not been studied quantitatively; therefore, no information on the value of this parameter exists. Figure 3a shows the probability of the colony capacity taking a certain value.

Next, we estimated the effect of pesticide exposure on the population. In this study, the effect of thiamethoxam on queen survival after three months of hibernation was tested experimentally using control queens and queens treated with pesticide. Assuming that the numbers surviving were binomially distributed, we established the likelihood of the multiplicative effect of pesticide exposure.

To do this, we conservatively assumed that the effect of thiamethoxam on colony initiation after hibernation is the same for six months as it is for three months (in reality it is likely to be higher) and inferred the likelihood of this effect. Subsequently, we normalized the likelihood before combining the results to establish the probability of the colony capacity after neonicotinoid exposure taking a certain value (Fig. 3b). For intermediate values of p nm , it can be seen that the colony capacity is likely to be reduced below 1 (Fig. 3d), which means populations will become extinct.

As B. terrestris populations have persisted in natural environments, we know the colony capacity of an extant population has to be at least 1. The expected number of colonies produced per colony can vary over the years and the information we have is restricted to an observation in a single year. However, even though we have no comparable data on the number of gynes produced, additional studies indicate that the productivity of B. terrestris in the same location was around 50% lower in a previous year51,79. This indicates that the data used here to estimate the colony capacity represent a relatively good year. We can thus reason that if the number of daughter colonies falls below one in a good year, a population cannot persist and, therefore, the persistence of B. terrestris in the wild tells us that our estimate of the colony capacity based on a good year has to be at least 1. We therefore conditioned the result on the colony capacity exceeding 1 by setting the probability of colony capacities below 1 to 0 and renormalizing (see Fig. 3c). We then took the effect of pesticide into account and calculated the probability of the colony capacity taking a certain value under pesticide exposure (Fig. 3d). The total probability of the colony capacity estimated to be to less than 1 was 28%. As we made several restrictive assumptions to reach this result it is very likely to be a conservative estimate.

Full details of these probability calculations are provided in the Supplementary Information.

Data availability

The data that support the findings of this study are available in Supplementary Data 1. The Mathematica notebook used to generate Fig. 3 is available from V.A.A.J. on request.