Honeybee colonies compensate for pesticide-induced effects on royal jelly composition and brood survival with increased brood production

Clothianidin exposure in field experiments

The experimental bee yard was situated in a suburban area in Kirchhain (Germany) with sufficient natural pollen sources in late summer, where the honeybees were allowed to forage freely. The field experiment took place with 20 Apis mellifera carnica colonies in Kirchhainer mating nuclei (25.5 × 19.8 × 17 cm), established with sister queens (A. m. carnica breeding line, mother: 17:27:20:11) mated on an island mating station (Norderney) and 180 g of worker bees each. We tried to reduce the variance between colonies by using young sister queens from an island mating station. Furthermore, we used small hives to minimize food competition so that we were able to set up the colonies within a rather small area of 20 by 20 m. In detail, the worker bees originated from four healthy colonies of the institute, located at an apiary of the institute. In order to get a homogenous composition, worker bees of brood combs and honeycombs of two colonies were shaken into a box and mixed thoroughly, before they were uniformly distributed into 10 mating nuclei. This procedure was repeated to fill 20 mating nuclei. Subsequently, the mating boxes were placed in a cool room (12 °C) for three day, to acclimatize, before they were transported to the island mating station Norderney. The queens started to lay eggs at June 25, 2014. The clothianidin exposure started at July 28. Thus, the colonies containing freshly mated, egg-laying queens were allowed to establish and build up an intact brood nest for four weeks. Two days before clothianidin exposure (sampling day 0, S0; SFig. 1), the colony strength was assessed and the treatment groups were randomly assigned to the hives such that differences between treatment groups were minimized with respect to the strength of their colonies. Every week, from July 30 to September 10, each colony received 400 mL of Apiinvert (Südzucker AG, Mannheim, Germany) sugar syrup (39% w/v fructose, 31% w/v saccharose and 30% w/v glucose) spiked with clothianidin (1, 10 or 100 µg/L). Control colonies received sugar syrup containing the same concentration of the solvent (water) as the clothianidin-treated groups. The syrup was fed in zip-look bags placed inside the food chamber containing a climbing aid. After 1 week, the leftovers were removed and weighed to record food consumption. For all four experimental groups, the analyzed clothinidin levels were close to the target concentrations (Suppl. Table 1). Environmental data were recorded during the study period using a USB data logger (EL-USB-2, Lascar Electronics Ltd., temperature accuracy ± 0.5 °C, relative humidity accuracy ± 3%) located under the colonies.

Sampling

During weeks 3 and 7, random samples of worker bees, larvae and worker jelly were taken from each colony. In detail, 20 randomly chosen worker bees located on a brood comb and five larvae (larval stage: day 7 or 8 after egg laying) were immediately frozen for chemical analysis. To collect worker jelly, extra thick blotting paper (Protean, Xi size; Bio-Rad, Hercules, CA, USA) was cut into strips, cleaned in pure ethanol and acetone (Carl Roth, Karlsruhe, Germany) and dried in a heating cabinet at 80 °C. Each strip was inserted into five brood cells containing a small larva (developmental day 4–5) to suck up the worker jelly and the strips were immediately frozen.

To document brood development, each side of each comb was photographed within an empty hive box transformed into a photo box containing a digital camera (Canon PowerShot A1000 IS, Tokyo, Japan) and a ring-flash (Aputure Amaran AHL-C60 LED, Shenzhen, China). Brood documentation took place every week. The colonies were sampled starting with the control and then from the lowest to highest concentration of clothianidin to minimize the risk of carryover.

HPG size measurements

To obtain worker bees of a defined age, single frames of late-stage capped brood (Binder, Tuttlingen, Germany) were brought to the laboratory and incubated in the dark at 32 °C, with humidity provided by open water jars. The frames with worker brood were collected from two full-sized colonies, which were regularly inspected for symptoms of disease and tested for Chronic bee paralysis virus, Deformed wing virus, Acute bee paralysis virus, and Sac brood virus⁶⁵. Newly-emerged bees (≤ 24 h) were collected, color marked, and transferred to the experimental colonies on July 28 (15 marked bees per colony). During the second week of exposure, the marked bees (Suppl. Table 2) were removed from the colonies after 12 days in the hive and immobilized on ice. The HPGs were dissected in ice-cold phosphate-buffered saline (PBS, pH 7.4). The specimens were fixed in formaldehyde (4% in PBS, Carl Roth), rinsed three times in PBS, and mounted in Aquapolymount (Polysciences, Eppelheim, Germany). Three pictures of each gland (only one gland per bee) were photographed at 400x magnification using a phase contrast/fluorescence microscope (Leica DMIL, Leica camera DFC 420C) and LAS v4.4 image-capturing software (Leica Microsystems, Wetzlar, Germany). To measure the size of each gland, the diameters of 30 acini per bee were measured using ImageJ v1.49o (http://rsb.info.nih.gov/ij/index.html).

High-performance thin-layer chromatography

HPTLC was chosen as sensitive analytical method in order to detect qualitative and semi-quantitative differences in the composition of brood food and larvae of dosed hives. Individual larvae (one per colony) differing in their weights (200–500 mg/larva) were solely macerated and extracted in 1 mL n-hexane (> 99% pure, Rotisolv, Carl Roth) in an ultrasonic bath for 1 min and then vortexed for 1 min. For the analysis of worker jelly (from three cells per colony), adsorptive filter strips (Sugi strips, Kettenbach, Eschenburg, Germany) were cut in half and one part was dipped in brood combs until maximal absorption of the material, whereas the other strip was used as background control strip, and both strips were extracted as above. The supernatants of larvae and worker jelly were transferred to a fresh vial 1000 μL isopropylacetate/methanol (3/2, v/v). After maceration for 1 min, the mix was vortexed for 1 min and the supernatant was transferred to another vial. Between extractions, the samples were cooled on ice and stored at –20 °C.

The isopropylacetate/methanol extracts (20 µL/band for worker jelly and 7 µL/band for larvae) were sprayed onto the silica gel 60 F₂₅₄ HPTLC plate (Merck, Darmstadt, Germany) using an Automatic TLC Sampler 4. The plate of worker jelly was developed with a mobile phase consisting of chloroform/methanol/water/ammonia (30/17/2/1, v/v/v/v, all Carl Roth⁶⁶ and the plate of larvae with an 8-step gradient development based on methanol, chloroform, toluene and n-hexane^66,67. After drying in a stream of cold air for 2 min, the plate images were documented at UV 366 nm using the TLC Visualizer. For derivatisation, the chromatogram was dipped into the primuline solution (100 mg primuline in 200 mL acetone/water, 4/1 v/v, Sigma-Aldrich, Steinheim, Germany) at an immersion speed of 2.5 cm/s and an immersion time of 1 s, dried and derivatised as before. The data were processed using winCATS version 1.4.2.8121 (all instrumentation from CAMAG).

The bacterium Aliivibrio fischeri (NRRL-B11177, strain 7151), obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Leibniz Institute, Berlin, Germany), was used to assess a non-targeted, broad range of effective substances within the worker jelly. HPTLC plates were developed as described above, neutralized and immersed in a bacterial suspension, prepared according to DIN EN ISO 11,348–1 8⁴⁵ at an immersion speed of 3 cm/s and an immersion time of 2 s. The bioluminescence of the wet bioautogram was recorded in an interval of 3 min over 30 min using the BioLuminizer (CAMAG).

Brood assessment

The free extension of the open source program ImageJ, Fiji (http://fiji.sc/Fiji) was used to count all cells with eggs, larvae, or sealed brood, for every colony on every sampling day. The photos of a single comb from different sampling days were aligned in the program and all brood cells were counted. Because the colonies had no wax foundations, some cells on the edges of these naturally-built combs were at an unfavorable angle. Therefore, most but presumably not all cells with brood were visible in the photos. This uncertainty was similar in all hives. To estimate the brood survival, we first tracked the development of individual eggs, but found a high mortality rate even in control colonies. Therefore, we tracked the development of individual larvae (day 4–5 after egg laying) over 4 weeks (= sampling weeks). Brood survival was estimated for two periods during the experiment (weeks 1–4 and 3–7).

Modeling of demographic compensation

The aim of the model was to estimate the number of new larvae that must hatch each week in order to maintain a stable number of larvae up to 7 weeks of age given the survivorship schedule of larvae week by week. Let h denote the number of eggs that hatch into larvae each week, and let the probability that any individual larva dies in each successive week be m_i, where i takes values in the set {1, 2, …, 7} to indicate each of seven successive weeks, after which we assume that surviving larvae pupate. We used a demographic matrix model⁶⁸ to describe the state of the population of larvae each week as follows. Let l_x denote the number of larvae in the colony that are aged x weeks post-hatching and let m_x denote their per capita weekly mortality rate. The population of larvae is distributed into seven age classes and we also assign a class to queens, which give rise to larvae by producing eggs. Using the Lefcovitch matrix approach, the larval population of a colony can thus be viewed as a state vector n_t whose week-by-week change is the product of a matrix A and the population state vector n_t, as shown in Eq. (1):

The lowermost element of n_t is the number of queens (Q) which is here set to Q = 1 for all models, and the total number of larvae in the colony at any time is ({varvec{L}}_{{varvec{t}}} = mathop sum nolimits_{{varvec{x}}} {varvec{l}}_{{varvec{x}}}). Given the observed mortality schedule (m_x), we used the model to solve for the value of h that produces a stable value for L_t, as shown in Eq. (2):

We determined the mortality schedule by observing the survivorship of a larval cohort. For example, if a cohort of S_t larvae was originally marked at time t and, of these, S_t+1 survived until the following week, the per capita weekly mortality rate would be estimated as shown in Eq. (3):

BEEHAVE simulations

To predict the impact of clothianidin on colony development in standard hives we used BEEHAVE, a honeybee model that simulates colony dynamics and agent-based foraging in realistic landscapes⁴⁴(http://beehave-model.net/). Although BEEHAVE does not explicitly allow the incorporation of pesticides, the effect of pesticides on behavior and mortality can nevertheless be addressed^59,60. BEEHAVE simulates the development of a single honeybee colony, starting with 10,000 foragers on January 1. Colony dynamics are based on a daily egg laying rate, with the developmental stages eggs, larvae, pupae, and adults (in drones) or in-hive workers and foragers (in workers). The brood needs to be tended by in-hive bees, and the larvae additionally need to be fed with nectar and pollen. Foragers can scout for new food sources or collect nectar and pollen from sources already known. Successful foragers can recruit nestmates to the food source. Mortality rates depend on the developmental stage and the time spent on foraging. The colony dies if it either runs out of honey or if the colony size falls below 4000 bees at the end of the year. Swarming may take place when the brood nest grows to more than 17,000 bees before July 18. Under default conditions, two food sources are present at distances of 500 or 1,500 m. Daily foraging conditions are based on weather data from Rothamsted, UK.

We ran two sets of simulations: (A) We first set up BEEHAVE to mimic our experimental nucleus colonies. We then determined how the protein content of the jelly produced by nurses (ProteinFactorNurses) had to be modified to replicate the larval mortality we observed in our empirical data. (B) We then set up BEEHAVE under default conditions but modified ProteinFactorNurses according to the results from the previous simulations to assess the impact of clothianidin exposure under more realistic conditions.

To mimic the experimental nucleus colonies (simulation A), the maximum honey store (MAX_HONEY_STORE_kg) was reduced to 0.77 kg and the maximum size of the brood nest (MAX_BROODCELLS) was reduced to 250. Furthermore, HoneyIdeal was set to ‘true’, so that even though the honey store was small it was filled every day, reflecting the feeding of the experimental bees. In contrast, PollenIdeal was set to ‘false’, because the experimental colonies still had to forage for pollen. On day 209 (July 28), we set the number of pupae to 100 and the number of workers to 650, similar to the experimental colony sizes. During the exposure to clothianidin between days 211 (July 30) and 253 (September 10), the protein content of the jelly fed to the larvae (ProteinFactorNurses) was modified by the new variable ProteinNursesModifier_Exposed. We tested for ProteinNursesModifier_Exposed values from 0.6 to 1 in steps of 0.01. The main output of the simulation was the survival of the brood, calculated from the brood cohort sizes aged 19 days divided by the sizes of these cohorts when they were 3 days old. We calculated the mean brood survival over 30 replicates, using the last 10 cohorts only (i.e. those reaching the age of 19 days between September 1 and 10). Those parameter values for ProteinNursesModifier_Exposed resulting in brood survival most similar to the experimental brood survival were then chosen to represent the clothianidin concentrations of 100, 10 and 1 µg/L.

To assess the impact of clothianidin on standard colonies under more realistic conditions (simulation B), we ran BEEHAVE under default settings but reduced the protein content of the jelly (ProteinFactorNurses) during times of exposure. We assumed that colonies would be exposed when rapeseed plants are flowering, defined in the model as the period between days 95 (April 5) and 130 (May 10). ProteinNursesModifier_Exposed values representing the tested concentrations of clothianidin were derived from the previous set of simulations, and for the control we set ProteinNursesModifier_Exposed to 1 (i.e. no effect of clothianidin). Swarming was either prevented or allowed, in which case the simulation followed the colony remaining in the hive.

Statistical methods

Statistical analysis was carried out using R v3.4.2⁶⁹, including the add-on packages lme4⁷⁰ for linear mixed-effects models, pbkrtest⁷¹ for testing fixed effects in mixed-effects models, parallel⁶⁹ to increase computational power, RLRsim⁷² for testing random effects in mixed-effects model, multcomp⁷³ for multiple comparisons, and lattice⁷⁴ for various graphical displays. We used linear mixed-effects models for one- and two-factorial analysis of variance (ANOVA) or regressions as indicated and where necessary. In those models, the colonies were modeled as random effects to reflect the (longitudinal) grouping structure in the data. For the analysis of larval survival, we used a logarithmic transformation of the proportion of surviving larvae. When testing fixed effects in mixed-effects models, we used the Kenward-Roger method (and double-checked the results by comparing them with parametric bootstrap values). Where sufficient, we simplified the analysis using linear (fixed-effects) models. Model diagnostics were performed for all fitted models using qualitative tools such as normal q-q-plots for residuals and plots of residuals versus fitted values to assess the validity of model assumptions like homoscedastic normality. Dunnett’s test (or customized contrasts as appropriate) was used for multiple comparisons in post hoc analysis, and P values were appropriately adjusted for multiple testing within well-defined test families using the single-step or Westfall’s method. Model and analysis details, model diagnostic graphs, and further information are available in the supplemental statistical report.

Source: Ecology - nature.com