DOI: 10.5061/dryad.h44j0zpgc
title: Diet quantity influences caste determination in honey bees (Apis mellifera)

description: In species that care for their young, provisioning has profound effects on
 offspring fitness. Provisioning is important in honey bees because
 nutritional cues determine whether a female becomes a reproductive queen
 or sterile worker. A qualitative difference between the larval diets of
 queens and workers is thought to drive this divergence; however, no single
 compound seems to be responsible. Diet quantity may have a role during
 honey bee caste determination yet has never been formally studied. Our
 goal was to determine the relative contributions of diet quantity and
 quality to queen development. Larvae were reared in vitro on nine diets
 varying in amount of royal jelly and sugars, which were fed to larvae in
 eight different quantities. Once adults eclosed, the queenliness was
 determined using Principal Component Analysis (PCA) on seven morphological
 measurements. We found that larvae fed the largest quantities of diet were
 indistinguishable from hive reared queens, independent of the proportion
 of protein and carbohydrate in the diet. Neither protein nor carbohydrate
 content had a significant influence on the first principle component
 (PC1), which explained 64.4% of the difference between queens and workers.
 Instead, the total quantity of diet explained a significant amount of the
 variation in PC1. Large amounts of diet in the final instar were capable
 of inducing queen traits, contrary to received wisdom that caste
 determination can only occur in the third instar. These results indicate
 that total diet quantity fed to larvae may regulate the difference between
 queen and worker castes in honey bees.
description: <i>Artificial Rearing</i><i>
 </i> <i>Apis mellifera </i>larvae
 were collected from nine hives near Fargo, Cass County, North Dakota
 during a three-week period in the summer of 2015. Hives were supplemented
 with pollen patties (Mann Lake, MN, USA) and a 1:1 sucrose-water solution
 (Brushy Mountain Bee Farm, NC, USA) during poor foraging conditions. First
 instar larvae (0-21 hours old) were transferred into 24-well cell culture
 plates (Falcon, Corning, Durham, NC) and placed onto 10 l of diet. The
 24-well plates were stored inside a modulator incubator chamber
 (Billups-Rothenberg, del Mar, CA, USA). Larvae were kept at a constant
 34C, darkness, and relative humidity of 96% using Potassium Sulfate
 (K<sub>2</sub>SO<sub>4</sub>) (33). Larvae were
 fed according to treatment in a factorial design of eight diet qualities
 and nine quantities, as described in the following sections. At the
 prepupal stage they were moved into 24-well cell culture plates containing
 Kimwipes (Kimtech Science, USA) sterilized in EtOH (34). Pupae were
 maintained at a constant 34C, darkness and 75% RH using NaCl until adult
 eclosion. Adults were stored at -20C.  
 <i>Diet Treatments</i> The study
 consisted of 72 treatment groups: nine diet qualities (Table 1) combined
 with eight diet quantities in a factorial design. Additionally, an
 <i>ad libitum </i>quantity treatment was added using the
 medium-protein medium-carbohydrate diet (Table 1). Each 24-well culture
 plate was randomly assigned to a diet quantity treatment. Within the
 plates, each row was assigned a diet quality treatment. Fresh diets were
 produced daily by homogenizing the ingredients for 10 minutes and warming
 in a 34C water bath for 10 minutes before feeding. The volume of diet
 produced each day depended on the number of larvae in the study that were
 still in the feeding stage. For all the treatments, larvae were fed the
 same amount until the 6<sup>th</sup> day of development, while
 diet quality remained the same throughout development.
   The reference diet (medium-protein
 medium-carbohydrate diet) was based on a previous study (35) that
 established the diet induced the development of worker adults. The other
 eight diets were produced by altering carbohydrates (glucose and fructose)
 and protein content (royal jelly: Pure Royal Jelly eBeeHoney.com, Ashland,
 OH, USA) in a full factorial design. Glucose and fructose have been
 previously used to alter carbohydrate content in <i>in
 vitro</i> diets (33, 35). However, royal jelly is the only protein
 source for <i>in vitro</i> diets because adding non-royal
 jelly proteins such as casein significantly decreases survival (36).
 Altering amounts of royal jelly to manipulate protein content also changes
 carbohydrate content because commercial royal jelly contains sugars in
 addition to proteins. Therefore, the protein, carbohydrate, and water
 content of the royal jelly was quantified using a Bradford assay and
 differential scanning calorimetry (see Supplemental Methods). The royal
 jelly contained 12.35% protein, 27% carbohydrates and 56% water. These
 values were used to calculate the percentage of macronutrients in each
 diet.   <i>Diet
 Quantities</i> The lowest diet quantity (160 l)
 was adopted from previous <i>in vitro </i>methods because this
 quantity produces workers (35). Quantity was increased by 30 l increments
 from 160 l to 370 l to produce the other treatments. There was an
 additional <i>ad libitum</i> treatment in which larvae were
 fed an excess of what they could consume. All larvae were fed the same
 amount during the first five days of development: day 1: 10 l, day 2: 10
 l, day 3: 20 l, day 4: 30 l, and day 5: 40 l, totaling 110 ul of diet
 over the five days. During the 6<sup>th</sup> day of
 development, larvae were fed different amounts depending upon the diet
 quantity treatment so that total diet quantity ranged from 160 l to 370
 l. In the <i>ad libitum </i>treatment, larvae were fed 200 l
 per day until gut purge. Following gut purge, individuals were moved into
 pupations plates.  
 <i>Morphometrics</i> Adult
 morphometrics can separate and classify castes, even when ovariole number
 and spermathecal size are excluded (37). The mandibles, basitarsus and
 head were dissected from adults (see Table S1 for sample sizes by
 treatment) and photographed. Morphometric measurements included total body
 wet weight, width and length of the basitarsus, width and length of the
 mandible, and width and length of the head (Fig. 1). Image J software was
 used for measurements. <i>In vitro</i> reared adults were
 compared to two reference populations: commercially-reared queens
 (Wildflower Meadows, Southern CA, USA) and hive-reared workers collected
 from the research hives in early spring.  
 <b><i>Data analysis and presentation of data
 </i></b> Statistical analyses were
 performed using R version 3.1.3 (R Core Team) (38). Use of additional R
 packages are reported below where appropriate.
   <i>Principal Component
 Analysis</i> A Principal Component analysis (PCA)
 was used to categorize an individual as a queen, worker or intercastes by
 comparing morphometric measurements between <i>in vitro
 </i>reared individuals and reference workers and queens. The
 Principal Components were calculated from hive-reared workers and
 commercially-reared queens using the <i>prcomp</i> function in
 the <i>stats</i> package, and the <i>predict
 </i>function was used to produce principal components for the
 <i>in vitro </i>reared individuals. The assumptions for
 sphericity, sample adequacy, and determinant of the matrix were tested and
 met. Principle Component 1 (PC1) was used for downstream
 analysis.   <i>Clustering
 Analysis </i> Clustering is a statistical
 analysis for group classification and was used to determine if <i>in
 vitro</i> reared individuals were grouped with reference queens or
 workers. Linkage distances were calculated using the complete method for
 hierarchical clustering (39). The cluster analysis was performed using the
 <i>hclust</i> function in the <i>stats</i>
 package. The results were graphed using the
 <i>ColorDendrogram</i> function within the <i>sparcl
 </i>package (40). The optimal number of clusters was calculated
 using the K-means clustering method.  
 <i>Measure of contribution of diet quantity and quality to
 Principal Component 1</i> The influence of diet
 quality and quantity on PC1 was compared for the 72 treatments, excluding
 the single <i>ad libitum</i> treatment. A generalized linear
 mixed model (GLMM) was performed using the <i>lme4</i>
 function (41). PC1 was the dependent variable and diet quantity and diet
 quality (protein, carbohydrate, and water proportion) were the independent
 variables (Table 1).  Hive was treated as a random effect because bees
 from different hives are expected to differ in size and shape because of
 parental genetics. The assumptions of collinearity, independence of data,
 and normality were calculated and met for the model.
description: The Word file "MainDataset_ColumnDescription" contains
 information on each column in the csv file called
 "QuantityVQuality_MainDataset.cvs."  The R files
 "MainAnalysis" and "Fig2_Graphs" contain the code used
 to do the data analysis.  The code is annotated to identify which commands
 go with each analysis.