In recent decades many pollinators have declined in abundance or contracted their ranges, presenting a potential threat to agricultural productivity and biodiversity ( Biesmeijer et al., 2006 ; Potts et al., 2010 ). Insect pollinators are crucial for agricultural productivity providing pollination services to a wide variety of crops with an estimated global value of €153 billion per year ( Gallai et al., 2009 ; Potts et al., 2010 ). Moreover, pollinators have a key role in maintaining wild plant communities with over two thirds of flowering plants being dependent on pollinators to reproduce ( Ollerton, Winfree & Tarrant, 2011 ). Thus there is an urgent need to understand the underlying causes driving current pollinator declines.

Materials and methods

Bees and pathogen screening Fifteen nests of B. terrestris audax with approximately 60 workers were obtained from Biobest (Westerlo, Belgium) via Agralan Ltd (Swindon, UK). They were first screened microscopically and via PCR for common bumblebee and honeybee parasites (Nosema bombi, N. ceranae, Crithidia bombi, Apicystis bombi) by analysing a subset of workers (10% of workers) immediately upon arrival. A small sample of hind gut, mid gut and malpighian tubules was dissected out and homogenized in ddH 2 O. DNA was extracted with 10% Chelex (Bio-Rad, Hemel Hempstead, Hertfordshire, UK). PCR protocols and parasite-specific primers followed Graystock et al. (2013).

Microcolonies and treatments These 15 queenright nests were divided into 60 queenless microcolonies each consisting of 10 workers (4 microcolonies per queenright nest). The four microcolonies created from each queenright nest were randomly assigned to one of the four treatments: control, pesticide (clothianidin), parasite (N. ceranae) and exposure to both pesticide and parasite, thus we had a fully factorial design. Using microcolonies instead of whole nests enabled us to control for genetic background. Furthermore, the use of microcolonies is one of the methods recommended by EFSA for risk assessment studies (European Food Safety Authority, 2013). A small amount of brood and wax from the original queenright nest was given to each microcolony to stimulate nest building. Typically in a queenless microcolony, some workers will begin laying unfertilised eggs which develop into male bees. Bees resided in circular plastic containers (diameter 11 cm, height 9 cm) with an aluminium mesh cover to allow air ventilation, and were maintained in the dark at 26 °C (±1 °C) and 55% RH (±5%). Microcolonies assigned as pesticide or pesticide + parasite treatment were provided with an ad-lib supply of 60% sugar water solution contaminated with 1 ppb clothianidin (Sigma-Aldrich, Gillingham, UK) and microcolonies assigned as control or parasite treatments were given untreated food. Stock solutions were dissolved in acetone and dietary concentrations were made on the day of provisioning. Fresh sugar water solutions (contaminated with clothianidin or not) were renewed every third day, and the amount of sugar water solution collected was recorded. All microcolonies were provided with an ad-lib supply of untreated pollen (Biobest via Agralan Ltd, sterilised by gamma irradiation with a cobalt-60 source at dose rates between 25–45 kGy) renewed every third day. Three days after establishment of the microcolonies, 8 bees within each microcolony individually received either a meal of 4 μl of 30% sugar water (control and pesticide treatments) or 30% sugar water containing a controlled dose of circa 130,000 N. ceranae spores (parasite and pesticide + parasite treatments) (viability 98.6%, viability test with 0.4% Trypan blue. Viability of the spores was confirmed by infection of honeybees). Any remaining bees in the microcolony were discarded. The rationale for only treating 8 bees was that we expected a small percentage of workers to die prior to inoculation, so each microcolony contained 2 surplus bees to ensure that an equal number per microcolony would be available to be treated. The dose administered is typical of that used in honeybee studies (e.g. Alaux et al., 2010; Doublet et al., 2015) and dosages less than 100,000 spores have been found to infect bumblebees (Graystock et al., 2013). Bees were starved for 2 h before treatment. To facilitate the delivery of the meal, bees were first immobilized by placing them in a cooler bag with ice blocks for approximately 10–15 min. Recovering bees ingested the inoculum when their proboscis was touched with a droplet of the spore solution at the tip of a micropipette. A solution (in ddH 2 O) of freshly isolated N. ceranae spores (originating from a naturally infected honeybee hive from Spain) was obtained by homogenising abdomens of adult honeybees and purifying the homogenate by centrifugation in 95% Percoll® (Sigma-Aldrich). Identity of the parasite was confirmed by PCR. After parasite treatment, microcolonies were monitored for worker mortality and production of males every third day for 3 weeks, and then daily for 10 days. Newly emerged males were removed from the microcolonies. The number of eggs, larvae and pupae were counted at the end of the experiment. Fecundity of workers was measured as the total number of males, eggs, larvae and pupae produced during the experimental period. After the experiment, a subset of alive (n = 177) and dead bees (n = 57) were screened for N. ceranae infection (on average 5 bees per microcolony) as described above.

PER assays and memory retention Proboscis extension response (PER) assay is a standard assay of learning ability in bees, which tests their ability to learn an association between an odour (conditioned stimulus, CS) and sugar reward (unconditioned stimulus, US) (Bitterman et al., 1983; Laloi et al., 1999). PER learning assays began 7 days after the parasite treatment (10 days after the start of chronic pesticide exposure). The day before PER conditioning started, workers were placed in a cooler bag with ice blocks for approximately 10–15 min until immobilized, before being harnessed in plastic tubes modified from 1 ml pipette tips. The head of the bee was held in place using a “yoke” made from a paper clip (see Riveros & Gronenberg, 2009). Harnessed bees were fed to satiation with 60% sugar solution and then starved for 15 h. In total 3 workers per microcolony were harnessed. The following day bees were first allowed 20 min to acclimate to the conditions of the room in which the PER assays were conducted (mean temp 23 °C). Then one bee at a time was placed at 5 cm distance from a continuous air flow (circa 2 L min−1, aquarium pump, Hidom, Shenzhen, China) delivered by a silicon tube (diameter 4 mm) with a 1 ml pipette tip at the end. A floral odour (CS) linalool (Sigma-Aldrich) was then delivered by switching the air flow pass through a 20 ml syringe containing a 2 × 20 mm filter paper with 5 μl 2 M of the odorant in mineral oil. An extractor fan was placed behind the bee to allow extraction of any residual odour. One PER assay was composed of 10 CS-US trials with each trial conducted as follows: 15 s air flow, 3 s CS (air flow with odour only), 3 s both CS and US (unconditioned stimulus: touching the antennae and proboscis with a toothpick dipped in 60% sugar solution (not spiked with pesticide) and allowing the bee to lick), 1 s US only, 8 s air flow. The Inter-Trial Interval (ITI) was 10 min. Extension of the proboscis during each CS and US was recorded. After 10 CS-US trials, a final level of learning acquisition was assessed by recording whether a bee extended its proboscis when presented with only the conditioned stimulus without the sugar reward (US). To test whether bees remembered the learned association, a memory retention test was conducted 2 h after each PER assay, where again only the conditioned stimulus was presented, in the absence of the US. Ten minutes before and after each PER assay and after each memory retention test, bees were tested for motivation to respond to sugar stimulus by touching the antennae with a toothpick covered with 60% sugar solution and observing whether extension of proboscis occurred. Bees that showed a negative response to the sugar solution were excluded from the analyses. Also, bees that showed 4 or more sequential negative responses to US during PER assays were considered unmotivated and were excluded from analyses. After testing memory retention, bees were released from the harness, returned to their microcolony and their survival was observed.