In July 2014, one or two frames of emerging brood were removed from 15 healthy colonies housed in the Iowa State University research apiary. The following day, newly emerged bees from all frames were shaken into a large tub and gently mixed to provide a homogenized mixture of bees from the 15 different colonies. Bees from this mixture were then counted out into clear acrylic cages (10.16 × 10.16 × 7.62 cm) in groups of 35 per cage. Cages were randomly assigned to treatments and kept in a single walk-in insect rearing room kept at 32–34°C and 50% relative humidity. Each cage received a virus treatment (control 30% sugar solution or the same sugar solution with virus inoculum added) and a pollen dietary treatment (no pollen, polyfloral pollen, Cistus sp. (rockrose) pollen, or Castanea sp. (chestnut) pollen).
For the virus treatment, cages were provided an open feeder containing either 0.6 ml 30% sucrose solution or the same solution containing a 1 : 1000 dilution of viral inoculum (described in detail in Carrillo-Tripp et al. [43 (link)]). This dose was chosen through the production of dose–response curves to identify an inoculum concentration that would result in intermediate levels of mortality, as described in Carrillo-Tripp et al. [43 (link)]. Bees had ad libitum access to these feeders for 12 h, after which all of the solution had been completely consumed, and the open feeders were removed. For the rest of the experiment, bees had ad libitum access to untreated 30% sucrose solution fed through a drip feeder at the top of the cage.
Concurrent with the introduction of the open feeder, each cage received a pollen treatment. The cage either had no pollen added, polyfloral pollen, Cistus pollen, or Castanea pollen introduced into the bottom of the cage. Cistus and Castanea pollen were purchased from Pollenergie® (France). These pollens have been nutritionally well characterized, with Cistus being of overall lower quality (lower protein, amino acid content) and Castanea being of high quality (higher protein, amino acid content, beneficial affects during Nosema challenge) [24 (link)]. The polyfloral blend, identical to that described in Dolezal et al. [47 (link)], contained more than five species of pollen, with the most abundant being dandelion (Taraxacum sp. L.) and willow (Salix sp. L.), each of which made up approximately 8% by mass of the total blend. To this mixture, Cistus and Castanea pollen were added to a total of 8% by mass for each. In all cases, pollen was bee-collected and received in corbicular pellets. Each pollen source was homogenized into a powder in a coffee grinder, weighed out into 0.2 g aliquots and added to the bottom of each cage. After 24 h, remaining pollen was removed and replaced with fresh. In all cases, bees did not consume all of the pollen in any given 24 h period.
Mortality was monitored in all cages every 12 h with dead bees removed at each interval. Previous experiments had shown that mortality occurs primarily between 36 and 48 h post-infection (hpi, [43 (link)]) with some cages devoid of live bees by 72 hpi. Therefore, to sample bees during the height of infection but before death, six live bees were removed from each cage at 36 hpi. Mortality effects were measured as a cumulative percentage at 72 hpi, after which the experiment was ended because some cages had too few bees for meaningful analysis, similar to previous results [43 (link)]. Collected samples were stored at −70°C until processing.
For the virus treatment, cages were provided an open feeder containing either 0.6 ml 30% sucrose solution or the same solution containing a 1 : 1000 dilution of viral inoculum (described in detail in Carrillo-Tripp et al. [43 (link)]). This dose was chosen through the production of dose–response curves to identify an inoculum concentration that would result in intermediate levels of mortality, as described in Carrillo-Tripp et al. [43 (link)]. Bees had ad libitum access to these feeders for 12 h, after which all of the solution had been completely consumed, and the open feeders were removed. For the rest of the experiment, bees had ad libitum access to untreated 30% sucrose solution fed through a drip feeder at the top of the cage.
Concurrent with the introduction of the open feeder, each cage received a pollen treatment. The cage either had no pollen added, polyfloral pollen, Cistus pollen, or Castanea pollen introduced into the bottom of the cage. Cistus and Castanea pollen were purchased from Pollenergie® (France). These pollens have been nutritionally well characterized, with Cistus being of overall lower quality (lower protein, amino acid content) and Castanea being of high quality (higher protein, amino acid content, beneficial affects during Nosema challenge) [24 (link)]. The polyfloral blend, identical to that described in Dolezal et al. [47 (link)], contained more than five species of pollen, with the most abundant being dandelion (Taraxacum sp. L.) and willow (Salix sp. L.), each of which made up approximately 8% by mass of the total blend. To this mixture, Cistus and Castanea pollen were added to a total of 8% by mass for each. In all cases, pollen was bee-collected and received in corbicular pellets. Each pollen source was homogenized into a powder in a coffee grinder, weighed out into 0.2 g aliquots and added to the bottom of each cage. After 24 h, remaining pollen was removed and replaced with fresh. In all cases, bees did not consume all of the pollen in any given 24 h period.
Mortality was monitored in all cages every 12 h with dead bees removed at each interval. Previous experiments had shown that mortality occurs primarily between 36 and 48 h post-infection (hpi, [43 (link)]) with some cages devoid of live bees by 72 hpi. Therefore, to sample bees during the height of infection but before death, six live bees were removed from each cage at 36 hpi. Mortality effects were measured as a cumulative percentage at 72 hpi, after which the experiment was ended because some cages had too few bees for meaningful analysis, similar to previous results [43 (link)]. Collected samples were stored at −70°C until processing.
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