Neither sulfoxaflor, Crithidia bombi, nor their combination impact bumble bee colony development or field bean pollination

Many pollinators, including bumble bees, are in decline. Such declines are known to be driven by a number of interacting factors. Decreases in bee populations may also negatively impact the key ecosystem service, pollination, that they provide. Pesticides and parasites are often cited as two of the drivers of bee declines, particularly as they have previously been found to interact with one another to the detriment of bee health. Here we test the effects of an insecticide, sulfoxaflor, and a highly prevalent bumble bee parasite, Crithidia bombi, on the bumble bee Bombus terrestris. After exposing colonies to realistic doses of either sulfoxaflor and/or Crithidia bombi in a fully crossed experiment, colonies were allowed to forage on field beans in outdoor exclusion cages. Foraging performance was monitored, and the impacts on fruit set were recorded. We found no effect of either stressor, or their interaction, on the pollination services they provide to field beans, either at an individual level or a whole colony level. Further, there was no impact of any treatment, in any metric, on colony development. Our results contrast with prior findings that similar insecticides (neonicotinoids) impact pollination services, and that sulfoxaflor impacts colony development, potentially suggesting that sulfoxaflor is a less harmful compound to bee health than neonicotinoids insecticides.


Screening
On day 1, under red light and using forceps, 20 bees were removed from each colony, induced to defecate, and returned to the colony.Their pooled faeces was screened for parasites 27 to ensure the colonies were uninfected by microparasites.

Colony standardisation
On day 2, all the bees were removed from the colony, except for the queen.Afterwards, 20 bees were returned to the colony, chosen haphazardly.The resulting colony was then weighed and returned to its feeder.Colonies were allocated to treatments using a weight rank allocation, rotated between experimental blocks.

Crithidia bombi inoculation
On day 3, between 30 to 40 workers were removed from two colonies deliberately infected with Crithidia bombi (these functioned as parasite reserve colonies and are distinct from the experimental colonies), originally sourced from infected wild queens caught in Windsor Great Park in 2021.The faeces from these bees was pooled and purified 54 .The inoculum concentration was quantified using a Neubauer haemocytometer and diluted in 1 mL water to a dose of 525,000 cells, equivalent to 25,000 cells per bee (20 workers and the queen), a dose which induces a realistic infection.Four mL of 40% w/w sucrose was added and the inoculum was vortexed.The inoculum was pipetted into a 35 mm petri dish and presented to the colonies for 24 h, or 48 h if not consumed within the first 24 h.Control bees were exposed to 4 ml 40% w/w sucrose and 1 mL water.Once the inoculum was visually verified as consumed, bees were returned to their standard feeder.Colonies were left to develop for a week, allowing the infection to take hold.

Validation of infection
On day 10, colonies were screened for the presence of C. bombi to ensure successful inoculation, and that the control colonies were not accidentally infected.To achieve this, 30 workers (or all workers if < 30 were present) were removed from a colony and induced to defecate.Their faeces was screened for C. bombi presence/absence.An arbitrary 25% prevalence rate was chosen as our threshold for a successful infection (50% mean average) with no colonies discarded.If a Control or Sulfoxaflor-only colony had even a single instance of C. bombi, the whole colony was discarded (n = 2).The following day (day 11), colonies were driven from Royal Holloway to the University of Reading, with a travel time of around an hour, during which their sucrose reservoirs were closed.On day 12, colonies were rested for a day to settle in a well-ventilated room with controlled temperature (24-26 °C) and humidity (50 ± 20%).

Colony observations
Colony activity was measured by filming (using a digital camera placed on a tripod) and later scoring the number of bees leaving and returning to the colony using the event-logging software 'BORIS' 60  www.nature.com/scientificreports/colony entrance and allowed 10 min of acclimatisation starting from the moment the first bee left the colony, then turned on the camera to record the entrance of the colony.The number of visits made by bees to three field bean plants was also recorded for five minutes per plant for a total of 15 min, before returning the bees to the colony and closing the entrance.The same three plants were exposed to the same colony throughout the three days of observations, and all plants in different cages were exposed to colonies for the same amount of time (25 min/day).

Individual observations
Two field bean plants were assigned to one colony for individual observations throughout the three-day observation period.The observer allowed one bee out of the colony at a time and recorded its behaviour for a maximum of 15 min starting from the moment it left the colony, aiming for three bees per colony.Recorded behaviours included latency (time taken to visit the first flower), overall duration of foraging trip, time spent on each flower, time between one flower visit and the next, and if pollen was collected or not.Again, observations of individual bees were carried out using 'BORIS' 60 .

Pollination service
The three plants used for colony-level observations were also employed as phytometer plants, defined here as plants grown under the same conditions and used to assess the level of pollination delivered by each colony.One stem of each phytometer plant was marked with cable ties above and below the two floral nodes with the freshest and most receptive flowers being visited by bumble bees during the colony observations.After performing colony observations, the plants were transferred to an insect-free flight cage where they continued to grow and ripen for 2 months.At harvest, the number of pods per node between cable ties and node location was recorded, and pods were then dried in the oven for 48 h at 80 °C, after which pod weight, number of beans per pod, and weight of individual beans was recorded.

Colony development
On day 16, following the final day of observations, colonies were returned to a temperature-controlled room, and left on ad libitum unspiked sucrose (50% w/w) and pollen (Agralan), topped up weekly.Colonies were kept for a total of six weeks after the pesticide exposure, and then frozen at -20 °C (day 58).Colonies were returned to Royal Holloway and dissected in three batches.The total number of workers, males, queens, gynes, larvae, and pupae were recorded per colony, alongside the weight of the workers, males, queens, gynes and larvae.

Blinding
The entire University of Reading team was blinded to the treatments until after the experiment had concluded.
To facilitate this, colonies and pesticide solutions were labelled using alphabetical codes corresponding to the treatment.

Semi-field experiment
A total of 88 colony observations, 149 individuals, and 106 plants were successfully analysed (Table 1).Due to heavy rain and low temperatures, four experimental blocks had to be excluded from all analyses.Further, as the poor weather did not allow us to perform all individual observations in block one, this additional block was discarded from the individual observation analysis.Moreover, 21 files with data on number of bees leaving and re-entering colonies were lost, and thus could not be included in the analysis.Such files were randomly distributed across plants and treatments.Finally, one of the phytometer plants died and was thus discarded.Data were checked for correlations using the Pearson Product-Moment test, and linear and generalised linear mixed-effect models were built in Genstat 21st Edition 61 to assess the impact of treatments on bee behaviour and plant yield.The models with the lowest AICc value and ΔAICc ≤ 2 was selected as the model with the most explanatory power 62,63 .Fisher's protected LSD post-hoc tests were planned in case observation day or treatment would have been significant.Parameter estimates and standard errors are presented in the supplementary materials for the semi-field experiment work.
Individual and colony observations.Response variables for individual observations included duration of foraging trip, foraging rate, and pollen collection, with visitation rate used as a dependent variable for colony-level Day + (1|block and colony ID + observer)) was used.Treatment, observation day, and interaction between the two were included as fixed terms, while 'experimental block and colony ID' and 'observer' were included as random factors.Data were analysed using Linear Mixed Models (LMMs).
Pollination services.Response variables for pollination services included average number and weight of pods between cable ties, and were analysed using LMMs.The model (Metric ~ Treatment + First node location + (1|block and colony ID/plant ID)) was used.Treatment and location of first node were included as fixed terms, while 'plant' nested within 'experimental block and colony ID' were used as random factors.'First node location' was treated as a categorical variable including early (1 to 5), middle (6 to 10), and late flowering nodes (11 to 16).

Colony development
Statistical analyses were carried out in 'R' programming software version 3.6.2 64.Worker weight, larval weight, and number of pupae were analysed using LMMs, utilising the package 'lme4' 65 .Model assumptions were tested and met.The model (Metric ~ Treatment + Mass of bees upon delivery + (1|block)) was used.Mass of bees upon delivery was included as a fixed factor to account for the variation in the size of the colonies, which reflected the eggs, larvae, and pupae they had after standardisation.Larval weight and drone weight were analysed using a Kruskal-Wallis model, because the data were non-normal.The model (Metric ~ Treatment) was used, with a Benjamini-Hochberg p-value correction applied to account for multiple testing.Post-hoc testing is presented in the supplementary materials.

Colony behaviour
There was no treatment effect on visitation rate of colonies to field beans (Fig. 2), but a significant effect of observation day was found (Table 2), with the lowest visitation rate on day 14 (see Supplementary Materials for further analyses).

Individual behaviour
No significant effect of treatment, observation day, or interaction between the two was found on any metrics related to individual behaviour during foraging (Table 3 and Fig. 3, see Supplementary Materials for further analyses).

Pollination services
No significant effect of treatment or location of first node was shown on field bean pollination variables (Table 4 and Fig. 4, see Supplementary Materials for further analyses).www.nature.com/scientificreports/

Colony development
No significant effect of treatment was found for any metric (weight of workers, weight of larvae, weight of drone or number of pupae) (Table 5 and Fig. 5).

Discussion
Using a well replicated methodology and tracking a number of different outcomes, we find no evidence for negative effects of sulfoxaflor, Crithidia bombi, or their interaction on our measures of bumble bee behaviour, bean pollination, or colony growth and rearing of sexuals.Our results indicate that sulfoxaflor may not be as harmful to bumble bee pollination provisioning as the neonicotinoids thiamethoxam and imidacloprid 42,66 , and that the conditions under which sulfoxaflor impacts reproduction may be narrower than previously expected 47 .
Further, the presence of a parasitic stressor does not necessarily lead to the emergence of agrochemical impacts.Our semi-field experiment found no significant impact of sulfoxaflor at a field-realistic level of exposure on either colony or individual behaviour of bumble bees.Exposure to neonicotinoids, a related group of systemic agrochemicals that act as selective agonists of nicotinic acetyl choline receptors, has been linked to a reduction in bee visitation rate and pollen collection 42,[67][68][69] , which translates into less efficient flower visiting behaviour resulting in a lower production of seeds 42 .In contrast, our results are in line with previous studies, where sulfoxaflor did not impair bee behaviour 50,70 or pollination by honeybees 71 , although impacts on bumble bees have been found 72 .This difference in behavioural impacts between neonicotinoids and sulfoxaflor may explain the lack of an effect of sulfoxaflor on the yield of field bean plants in our experiment.Indeed, directly comparable  www.nature.com/scientificreports/testing of a neonicotinoid and sulfoxaflor found sulfoxaflor to have weaker impacts on locomotive behaviour 66 .Alternatively, small differences in our experimental paradigm to that of Stanley et al. 42 , with different exposure regimes and subsequent behavioural monitoring, may explain the difference in our results.However, the complete lack of any trends in colony behaviour, individual behaviour, or pollination services with respect to experimental treatments suggests that differential agrochemical impacts is the most likely explanation.We also found no impact of C. bombi on bee behaviour and pollination, contrary to previous studies where an effect on visitation rate and time spent on artificial flowers was found, resulting in an impairment of foraging abilities 40,41 .Consequently, under our experimental conditions, these laboratory effects do not appear to extrapolate to real-world bee-flower interactions.Finally, the absence of a sulfoxaflor-C.bombi interaction effect on foraging behaviour or pollination, in this semi-field realistic experiment, mirrors the results of a recent laboratory study 70 , which found no interactive effect of sulfoxaflor and C. bombi on bumble bee olfactory learning.Together, this suggests that sulfoxaflor, either on its own or in combination with a highly prevalent parasite, may be a less harmful alternative to neonicotinoids.
In addition to measuring behaviour and pollination services, we also found that neither sulfoxaflor, C. bombi, nor their interaction, impacted B. terrestris colony development.No effects, or unsupported data trends, were seen regarding the production of any caste or development stage, giving confidence in the result.Most previous studies investigating the impact of sulfoxaflor or C. bombi on colony development exposed queens before hibernation or exposed very young colonies 35,36,47 .One study with a similar experimental design is Fauser-Misslin et al. 52 , where exposure to both C. bombi and the agrochemical occur at a later stage in the colony life cycle.In contrast to our results, they found significant impacts of the neonicotinoids thiamethoxam and clothianidin on colony growth.They also found an interactive effect of these agrochemicals and C. bombi on mother queen survival.Together, this suggests that exposure to sulfoxaflor later in the season is less likely to reduce bumble bee colony health, and that sulfoxaflor is also less toxic to bumble bees than either thiamethoxam or clothianidin.However, we note that our experimental colonies had access to ad libitum food and were largely kept in controlled environmental conditions, in contrast to Siviter et al. 47 , where colonies were exposed to naturally fluctuating climatic conditions and had to forage for food.Consequently, it is possible that the semi-field experimental setup could have contributed to decreasing the impact of sulfoxaflor on the colony compared to an uncontrolled environment, such as an open crop field.Again, however, in Fauser-Misslin et al. 52 neonicotinoids still had a negative impact despite such buffering, suggesting that neonicotinoids are indeed more toxic to bumble bees than sulfoxaflor.www.nature.com/scientificreports/There are, of course, further caveats to our conclusion.Firstly, we only exposed bees through sugar water, while pollen is known to have significantly higher contamination levels 73 , and thus may lead to greater exposure in the field.Secondly, we performed behavioural observations while bumble bees had ad libitum access to the spiked solutions, contrary to Stanley et al. 42 , who observed behavioural changes after the exposure period to thiamethoxam was over.This was due to the fact that sulfoxaflor concentrations decrease quickly 57,74 , and although this may not have impacted the delivery of pollination services to field beans, having ad libitum access to gravity feeders might have discouraged workers from exiting the colonies and start foraging, particularly with adverse weather conditions.Thirdly, our bees were not immediately exposed to the peak concentration of sulfoxaflor, instead we started our exposure one day after the application.This decision was made based on the common practice of spraying blooming crops at dusk, when bees are least active, assuming that bees would not be exposed immediately after spraying also in a fully field-realistic scenario.However, once our exposure began, the peak sulfoxaflor levels started to quickly tail off, dropping from 0.047 mg/kg to below 0.004 mg/kg in just over three days.Hence, it remains possible that longer-term exposure to a lower dose could be more harmful than a shorter-term, higher-level one.While in the residue study we modelled 55 , residue levels quickly dropped below even 5 µg/kg (2.8 µg/kg five days after application and 1.8 µg/kg six days after), other residue studies have found 5 µg/kg for 14 days, so both exposure regimes represent real world scenarios (especially as sulfoxaflor can be sprayed more than once per crop).It is noteworthy that the residue studies which support either exposure regime assume a worst-case scenario where the crop is sprayed during bloom, which is not legal in the EU for sulfoxaflor products, although this is legal for some crops outside the EU.Whether such residue levels still exist with appropriate mitigation measures is unknown, however it is likely that the mitigation measure would further reduce the residue levels.This makes the exposure we modelled conservative.
The lack of interactions between C. bombi and sulfoxaflor in any of our metrics adds to results from previous studies 70 , which similarly found no interaction between this parasite and a range of pesticides, including clothianidin, thiamethoxam 52 , and glyphosate 20,75 .While no study has yet tested the impacts of combined C. bombi and pesticide exposure on a hibernating queen, when C. bombi is most impactful, these combined results do suggest that C. bombi does not meaningfully interact with many pesticides.
There is no indication of power limitation in our colony development study, with 15 to 17 colonies per treatment, and no unsupported trends in the data.However, adverse weather conditions during the semi-field experiment did limit data collection and the production of a balanced dataset, particularly for individual-level observations (see Table 1), although again there were no obvious trends.As the treatment day effect was significant for the visitation rate metric, it is reasonable to assume that the experiment was sufficiently sensitive to detect treatment effect differences, if they had been present.
Nectar-robbing behaviour of bumble bees on field beans has often been reported (e.g. 76), and it was also noticed during our experiment.Since Bombus terrestris are shorter-tongued than some other bumble bee species, it is possible they are not always able to fully exploit the long-tubed V. faba flowers, reducing their foraging efficiency 77 and leading to nectar-robbing, which requires lower effort and provides a higher nectar reward than that obtained by legitimate visitations 78 .However, it has been shown that B. terrestris are effective pollinators of field beans even after only a few flower visits 31 , and that, although nectar robbing can make field bean pollination less effective, it can still lead to higher pod production than non-pollinated flowers 59 .The absence of a 'no-pollination' control group in our experimental set-up means we cannot test this directly, or test whether any other yield limiting factor was preventing us detecting an effect of pollination by bees.However, our experimental plants were grown under similar conditions to those of previous experiments, where other inputs (e.g., nutrients, water) were not limiting, and the plants were highly responsive to pollination treatments 31,79 .
After our experiment concluded, the European Commission implemented a ban on outdoor use of sulfoxaflor 80 because of its risks, particularly its risk to bees 81 , finding it appropriate to allow its use only in permanent greenhouses.However, sulfoxaflor is still widely authorised globally.Our results add clarity to the understanding of how sulfoxaflor impacts bumble bee pollination and colony development, however our testing uses only one species (B.terrestris), with just one exposure scenario, and it should not be used to generalise across species or exposures.Exposure at different life cycle stages, or to a longer-term, lower-level cumulative exposure, may still be harmful 47 .While our study contributes to addressing the data gap, considerably more research, in particular field and semi-field work, is needed for the risk to bumble bees to be sufficiently clarified.Additionally, caution should be taken in interpreting the lack of an interaction with the parasite, C. bombi.This result should not be generalised across all parasites, as there is considerable diversity between parasites and parasite-pesticide interactions 19,21 .
Extrapolating from our results requires caution.Bee species have frequently been shown to differ in sensitivity to pesticides [82][83][84][85] , and consequently in experiencing sub-lethal effects 47 .For example, Boff et al. 85 found that Osmia bicornis exposed to field-realistic doses of sulfoxaflor showed changes in foraging behaviour, including the number of flower visits and flight performance, suggesting that impacts of this agrochemical on pollination services supplied by different bee species may vary.Moreover, in conventional agriculture, it is common practice to use several agrochemicals to control pests and increase crop yield 86 , as well multiple sprays of the same chemical.Therefore, it cannot be excluded that sulfoxaflor might interact with other pesticides, affecting bee health or behaviour.For example, Azpiazu et al. 87 found that sulfoxaflor decreased the survival of both Apis mellifera and Osmia bicornis when in conjunction with the fungicide fluxapyroxad, despite not having an individual impact on B. terrestris.Finally, there is substantial variability in how pesticides interact with different parasites (reviewed in Yordanova et al. 88 ).For example, Siviter et al. 49 observed that simultaneous exposure to sulfoxaflor and Nosema bombi increased B. terrestris mortality, while exposure to either stressor in isolation did not lead to the same effect.Future studies utilising other bee species and additional pesticides and parasites are clearly needed.
In conclusion, we found no impacts of chronic sulfoxaflor exposure, C. bombi, or their combination on bumble bee colony foraging behaviour, individual bee foraging behaviour, the pollination services they provide,

Figure 1 .
Figure 1.Summary of behavioural observation procedures showing the number of plants in each cage, always containing one colony.

Figure 2 .
Figure 2. A boxplot with horizontally jittered datapoints showing mean flower visitation rate (visits per flower per minute) by treatment.Bold line is the median, the box is bounded by the first and third quartile of the data, and the whiskers are the 95% confidence intervals.

Figure 3 .
Figure 3.A boxplot with horizontally jittered datapoints showing the mean duration of a foraging trip (in minutes) per bee, by treatment.Bold line is the median, the box is bounded by the first and third quartile of the data, and the whiskers are the 95% confidence intervals.

Figure 4 .
Figure 4.A boxplot with horizontally jittered datapoints showing the mean pod weight of field beans (in grams) by treatment.Bold line is the median, the box is bounded by the first and third quartile of the data, and the whiskers are the 95% confidence intervals.

Figure 5 .
Figure 5.A panel of boxplots with horizontally jittered datapoints.Top left: Cumulative weight of workers in a colony (in grams); Top right: number of pupae in a colony, Bottom left: cumulative weight of males (drones) in a colony (in grams); Bottom right: cumulative weight of larvae in a colony (in grams).Each boxplot shows the metric by treatment.Bold line is the median, the box is bounded by the first and third quartile of the data, and the whiskers are the 95% confidence intervals.

Table 1 .
Details on sample sizes of colony observations (visitation rates and bees leaving/returning to the colony), individuals, and employed plants across colonies included in the analysis.Colony and individual observation data came from 34 and 27 colonies respectively.

Table 3 .
Analysis of individual behaviour metrics: foraging trip, foraging rate, and pollen collection.

Table 4 .
Analysis of pollination metrics: number of pods and pod weight.

Table 5 .
Analysis of colony development metrics: weight of workers, weight of larvae, weight of drones and number of pupae.