Expression and function of <i>AmGR10</i> in honeybees.

<p>(A) Artificial expression of <i>AmGR10</i> in the nursing state. In October 2007, we collected nurse bees 0 and 7 days old (the latter from hives 1 and 2). In early November, we collected 29-day-old workers in an extended nursing state within the hive and foragers outside of the hive at the same time (hive 2). Twenty HPGs from each group were pooled for RT-PCR analyses. (B) Organ distribution of <i>AmGR10</i> expression in 20 HPGs, 150 ovaries, 150 mandibular glands, 150 salivary glands, 20 midguts, and 20 cerebral brains from 7-day-old nurse bees between July and October of 2007 and 2008 (hive 2). Organ preparations from each group were pooled for RT-PCR analyses. (C) Knockdown of <i>AmGR10</i> mRNA in the HPGs of 7-day-old nurse bees. One hundred bees in 4 independent experiments (25 bees × 4) injected with <i>ds AmGR10</i> (8.2 μg⁄2 μL, “RNAi”), 80 bees in 4 independent experiments (20 bees × 4) injected with nuclease-free water (2 μL, “MQ”), and 80 untreated bees in 4 independent experiments (20 bees × 4) (“Mock”) were placed back into their hives (hives 1, 2, and 3) immediately after treatment and were collected again 24 h later for qRT-PCR. For qRT-PCR analyses, 3 bees were used in 4 independent experiments (3 bees × 4) and the remaining bees were allowed to forage. All values are means ± SEM. *** <i>P</i> < 0.001 compared with each control. (D) Effect of RNAi on foraging behavior. We ascertained the age in each group from paint markings: 22 bees × 4 in “RNAi”, 17 bees × 4 in “MQ”, and 17 bees × 4 in “Mock”. We observed the bees over 3 consecutive days outside of the same hive (see RNAi section in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142917#sec002" target="_blank">Materials and Methods</a>) and their flights to ensure the onset of flight hazard were shown as cumulative numbers of bees initiating foraging, but thereafter we did not follow their survival or labors. All values are means ± SEM. *** <i>P</i> < 0.001 compared with each control.