DOI: 10.5061/dryad.gf1vhhmn2
title: Data from: Antibiotics in hives and their effects on honey bee physiology and behavioral development

description: Recurrent honey bee losses make it critical to understand the impact of
 human interventions, such as antibiotics use in apiculture. Antibiotics
 are used to prevent or treat bacterial infections in colonies. However,
 little is known about their effects on honey bee development. We studied
 the effect of two commercial beekeeping antibiotics on the bee physiology
 and behavior throughout development. Our results show that antibiotic
 treatments have an effect on amount of lipids and rate of behavioral
 development. Lipid amount in treated bees was higher than those not
 treated. Also, the timing of antibiotic treatment had distinct effects for
 the age of onset of behaviors starting with cleaning, then nursing and
 lastly foraging. Bees treated during larva-pupa stages demonstrated an
 accelerated behavioral development and loss of lipids, while bees treated
 from larva to adulthood had a delay in behavioral development and loss of
 lipids. The effects were shared across the two antibiotics tested,
 TerramycinR (oxytetracycline) and TylanR (tylosin tartrate). These results
 on effects of antibiotic treatments suggest a role of microbiota in the
 interaction between the fat body and brain that is important for honey bee
 behavioral development.
description: <strong>Antibiotic treatment</strong>
 A total of twelve (N=12) colonies of A. mellifera were selected
 for the experiment. The experiment was conducted during the summer seasons
 of 2013 (trial 1 n=6) and 2014 (trial 2 n=6) with different hives. The
 apiary is located at the University of Puerto Rico’s Agricultural
 Experimental Station in Gurabo, PR, (18°15'26.6"N
 65°59'11.5"W). Colonies were screened and health assessed.
 Colonies that had received previous treatment, sickly or weak were
 excluded. Colonies were paired based on hive population and composition.
 Selected control colonies were treated with powdered sugar, the vehicle of
 antibiotic. Experimental colonies were treated with the commercial
 Oxytetracycline “Terramycin” (Terra-Pro; Mann Lake Hackensack, MN, USA) or
 with Tylosin tartrate “Tylan” (Elanco, Greenfield, IN, USA), following the
 recommended commercial dose of the powdered antibiotic.
 <strong>Cross-fostering design</strong>
 Setup: To examine effects of exposure to antibiotics in
 physiology and behavioral development, we used twelve colonies (N=12) in a
 general cross-fostering design (Fig. 1) divided into two trials: trial 1
 treatment with Oxytetracycline (N=6 colonies, 3control:3antibiotic) and
 trial 2 treatment with Tylosin tartrate (N=6 colonies,
 3control:3antibiotic). Colonies were paired based on composition and
 randomly assigned a treatment; control or antibiotic. Initial treatment
 was performed in standard (40.6cm W x 50.4cm L x 24.4cm D) one story
 wooden hive boxes with eight frames. After three weeks of treatment, brood
 frames were collected from each pair and placed in an incubator (Percival,
 Perry, IA, USA) at 33°C for 24 hours. From each colony approximately 500
 emerging bees were marked on the thorax with numbered color tags (BioQuip,
 Compton, CA. USA) to indicate source colony and treatment (N=5,786 total
 bees from both trials; See Table 1). Marked bees were divided by equal
 numbers and introduced either to their original colony or the
 cross-fostered alternate colony background with continued treatment. Whole
 colonies content (including frames) were moved to observation hives. This
 approach resulted in four different treatment groups: (1) no exposure
 (-/-) bees raised in control colony, kept in control colony. (2)
 developmental exposure (+/-) bees raised in treatment colony, introduced
 into the control colony. (3) adult exposure (-/+) bees raised in control
 colony, introduced into the treatment colony. (4) developmental and adult
 exposure (+/+) bees raised in treatment colony and kept in treatment
 colony. Additionally, for further fat content analysis,
 twenty newly emerged bees (not numbered tagged) were collected at random
 from the control (n=10) and the antibiotic colonies (n=10) for each of the
 trials, prior cross-fostering. Also, an additional 100 bees from each
 colony were marked with paint of different colors in the thorax to
 indicate source colony and treatment and were cross-fostered; eg. 100 bees
 from C1 and 100 bees from A1 were marked with paint, 50 from C1 stayed in
 C1 the other 50 cross-fostered with A1 and same manner with the 100 bees
 from A1, to be later collected for the fat content analysis. This was
 performed for each pair of colonies. Colony forming
 units: We examined the efficacy of antibiotics by measuring colony forming
 units (CFU) (Fig. S1). Bees collected from the -/- and +/+ groups at one
 and seven days of age, were washed in 70% ethanol (Sigma-Aldrich, ST.
 Louis, MO, USA) and had their digestive tracts removed. The digestive
 tracts were homogenized by vortex mixer in 10mL of nutrient broth medium
 (Himedia, West Chester, PA, USA) followed by dilution plating on nutrient
 agar (Himedia) and incubated at 35°C under aerobic conditions for 48 hours
 (VWR, Radnor, PA, USA). Individual bacterial colonies were counted and
 multiplied by the degree of dilution to obtain CFU of the original sample
 (Olsen and Bakken, 1987). This CFU study is a control that demonstrates
 antibiotic applied to the colony reached bees and bacteria that can be
 cultured aerobically are reduced after antibiotic treatment.
 <strong>Dissections and fat measurement: extraction and
 weighing</strong> Sample collection: Since bees
 present a lipid profile that follows worker tasks and age stages (Toth and
 Robinson, 2005), previously painted marked bees were collected at 1
 (collected during cross-fostering setup), 7 and 14 days of age from each
 treatment group (10 bees per treatment group by age, n=100 from each
 trial). Of note, day 1 bees were collected only from the -/- and +/+
 groups. This is due to bees being newly emerged and thus not
 cross-fostered at this age. Age of collection was selected to match onset
 of behaviors as described by Seeley, 1982 and Moore et al., 1998.
 Dissections: Collected bees were dissected by separating the abdomen from
 the thorax. The entire digestive tract, along with the sting apparatus and
 any wax scales observed on the outside were removed from the abdomen.
 Weighing and lipid extraction: To measure lipid content of bees by age and
 treatment, we used the total body fat extraction method as described by
 O’Donnell and Jeanne, 1995. Briefly, fresh weight (weight of abdomen after
 dissection) was obtained using a Fisher 11 analytical balance (Fisher
 Scientific, Hampton, NH, USA) accurate to 0.1 mg. The abdomens were then
 placed in a drying oven at 70°C and dried for three days. Next after the
 abdomens were dried and weighed (dry weight), they were placed in 5mL of
 extraction solution (2:1 chloroform:methanol; Sigma-Aldrich) on a rotary
 shaker (Thomas Scientific, Swedesboro, NJ, USA). The solution was replaced
 every 24hrs for three days. After the three days of extraction period, the
 abdomens were placed in the drying oven at 70°C for two days. Abdomens
 were weighed after the second drying period (extracted weight). Abdomen
 water content was obtained by subtracting fresh weight and dry weight of
 each abdomen (data not shown). Total fat content was obtained by
 subtracting the dry weight and the extracted weight of each
 abdomen. <strong>Behavioral development
 assay</strong> To examine differences in rate of
 behavior development, the four treatment groups were followed and recorded
 in glass-walled observation hives with eight frames, in two daily scans of
 two hours each (at 10:00 and at 14:00 hrs.) until the beginning of
 foraging activities. We quantified cleaning behaviors (removing debris
 from honeycomb cells), brood cell visits (nursing), and foraging behavior
 during daily behavioral scan samples (Giray and Robinson, 1994; Moore et
 al., 1998). Age at onset of foraging was determined by two different
 criteria; bees observed bringing in pollen and time spent outside the
 hive. More than 10 minutes outside the hive was considered a foraging
 flight, and less than that time was considered as orientation flights and
 therefore not marked as foraging. These criteria are based on studies by
 Robinson 1987, Calderone and Page 1991, Winston and Katz 1992, and Moore
 et al. 1998. To measure rate of development, we focused
 on earliest performance of behaviors by focal group of individuals. Bees
 were divided by age groups, representative of the age cohorts by which
 tasks; cleaning, nursing, and foraging, should be performed or start at
 (Seeley, 1982): 3-&gt;6, 7-&gt;10, 11-&gt;14 days of age.
 Relative probability of task was examined as proportion of the number of
 bees performing cleaning, nursing or foraging task at the different age
 groups to the total number bees from each treatment group in the colonies.
 Although this proved to be an effective method, mean of onset of behaviors
 for all three tasks observed could not be determined, therefore we focused
 on comparative information based on proportions of task
 performed.  <strong>Statistical
 analysis</strong> CFU: We performed a
 Shapiro-Wilk test to determine if the distribution of the CFU counts was
 significantly different than normal. After confirming the data was not
 normal, we performed a Mann-Whitney U test to determine whether the CFU
 count was significantly different between -/-  and +/+ at day one and
 seven of age. Lipids: Since trial 1 (Oxytetracycline)
 and trial 2 (Tylosin tartrate) were conducted at two different summer
 seasons and with different colonies, data from both antibiotics were
 analyzed as independent factors. We performed a Shapiro-Wilk test to
 determine if the distribution of lipid content was significantly different
 than a normal distribution. After confirming the data did not showed a
 normal distribution, we performed a square-root transformation to ensure
 our data followed a normal distribution. After the assumption of normality
 was met, we performed a three-way ANOVA with type of treatment, age and
 antibiotic as independent factors to determine their interaction with
 lipid content. A Tukey test was used as a Post-Hoc analysis.
 Behavior counts: We determined that the behavior count data was
 over dispersed, therefore we used negative-binomial generalized-linear
 mixed model (GLMM) to determine if job count intercept and slope varied by
 hive. We determined that colony effects explained very little of the
 variation (only a 0.002%) for each of the worker counts. Therefore, we
 utilized a negative-binomial generalized-linear model (GLM) to examine the
 number of workers performing each task using type of treatment, age and
 type of antibiotic as independent factors. Data was
 analyzed using the statistical program R (R Core Team 2014) v. 3.5.2
 (2018-12-20). Packages: glmm (generalized-linear mixed models) v. 1.3.0,
 lme4 (lineal mixed-effects models) v. 1.1-20.