DOI: 10.5061/dryad.ncjsxkt3b
title: Agricultural intensification impairs behavioral abilities and the expression of genes associated with social responsiveness in honey bees

description: The honey bee Apis mellifera is one of the main pollinators in
 agroecosystems, and consequently, its colonies are exposed to agricultural
 intensification. These practices lead to increased use of agrochemicals,
 resulting in fragmented and more homogeneous habitats. In an agricultural
 setting located in the region of the Argentine Pampas, behavioral and
 molecular approaches were performed in honey bee colonies to compare their
 global state at different times of crop management. Our results show that
 foraging honey bees captured in these beehives impaired their gustatory
 sensitivity, cognitive abilities, and the brain’s expression of several
 genes related to metabolic, immune and neuronal processes associated with
 social behavior in bees after pesticide application and the flowering of
 the surrounding crops. To our knowledge, no previous study has reported
 the impaired effects of agricultural intensification on insect pollinators
 from an integrative neurobiological perspective under realistic
 conditions.
description: Methods <strong>Colony
 activity</strong> We evaluated as a colony
 activity indicator, the number of incoming bees (incoming rate) at the
 entrance of the hives at each sampling moment. In each apiary, we randomly
 chose eight hives. The same eight hives were measured in the two sampling
 moments. We counted the arrivals in the hives chosen for 1 min, twice a
 day for three consecutive days, once in the morning and once in the
 afternoon. According to the presence or absence of pollen loads on their
 hind legs, incoming bees were recorded as pollen or non-pollen (nectar)
 bees, respectively. Plot, Moment and year were taken as fixed variables,
 while hive was taken as a random variable in a Negative binomial
 model. <strong>Pollen
 traps</strong> Pollen traps were placed in three
 randomly selected hives (different from the eight hives used for colony
 activity) for about 6 h and for three consecutive days. The weight of
 pollen loads collected in each pollen trap was measured. Color-classified
 pollen loads of each trap were weighed separately. Then, pollen grains
 were identified under the microscope to the lowest taxonomic level
 possible using a palynological database. Once color-classified pollen
 loads were identified, the total weight and the weight of pollen collected
 from the crop and the surrounding wild flora were calculated. Plot, Moment
 and year were taken as fixed variables, while hive was taken as a random
 variable in a Normal model. To analyze the proportion of pollen collected
 from the crop and wild flora, a Non-parametrical model was used.
 <strong>Gustatory responsiveness</strong>
 To evaluate gustatory responsiveness at the apiary level, we
 captured incoming honeybees at the hive entrances from different colonies
 and for three consecutive days. This methodology was carried out in the
 three apiaries and both sampling moments, before and after crop flowering
 and pesticide application, and in two years. Each forager bee was exposed
 to a sucrose solution of a specific concentration chosen in a
 pseudorandomized way (0.1%, 0.3%, 1%, 3%, 10%, 30%, or 50% w/w). At the
 end of the experiment, a gustatory response proportion was obtained for
 each concentration of sucrose solution, based on the number of responsive
 bees over the total evaluated bees. As we obtained a response ratio for
 each sucrose solution concentration, the statistics analyses were
 independent among the different solution concentrations tested. Plot,
 Moment and year were taken as fixed variables in a Binomial
 model. <strong>Learning score
 </strong> To evaluate the association between a
 pure odor with a sucrose reward via classical olfactory conditioning by
 using the proboscis extension response procedure, we captured incoming
 honeybees at the hive entrances from different colonies and for three
 consecutive days. This methodology was carried out in the three apiaries
 and both sampling moments, before and after crop flowering and pesticide
 application, and in two years. At the end of the experiment, a score
 defined as the sum of PERs throughout the procedure was obtained for each
 bee (learning score). Plot, Moment and year were taken as fixed variables
 in a Poisson model. As we obtained a score of responses for each forager
 bee evaluated, individual bees were not taken as a random
 variable. <strong>Gene
 expression</strong> The expression of genes
 associated with social responsiveness in honeybees was measured to assess
 the impact of pesticide application and the changes in the food resources’
 availability at the molecular level. In each apiary, we randomly chose
 four hives, and 10 brains of forager bees were pooled. Thus, we utilized
 four biological replicates per apiary. Plot, Moment and year were taken as
 fixed variables, while hive was taken as a random variable in a Gamma
 model.