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.

Artificial Rearing

Apis mellifera 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 (K₂SO₄) (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.

Diet Treatments

The study consisted of 72 treatment groups: nine diet qualities (Table 1) combined with eight diet quantities in a factorial design. Additionally, an ad libitum 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^th 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 in vitro diets (33, 35). However, royal jelly is the only protein source for in vitro 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.

Diet Quantities

The lowest diet quantity (160 l) was adopted from previous in vitro 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 ad libitum 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^th 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 ad libitum treatment, larvae were fed 200 l per day until gut purge. Following gut purge, individuals were moved into pupations plates.

Morphometrics

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. In vitro 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.

Data analysis and presentation of data

Statistical analyses were performed using R version 3.1.3 (R Core Team) (38). Use of additional R packages are reported below where appropriate.

Principal Component Analysis

A Principal Component analysis (PCA) was used to categorize an individual as a queen, worker or intercastes by comparing morphometric measurements between in vitro reared individuals and reference workers and queens. The Principal Components were calculated from hive-reared workers and commercially-reared queens using the prcomp function in the stats package, and the predict function was used to produce principal components for the in vitro 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.

Clustering Analysis

Clustering is a statistical analysis for group classification and was used to determine if in vitro 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 hclust function in the stats package. The results were graphed using the ColorDendrogram function within the sparcl package (40). The optimal number of clusters was calculated using the K-means clustering method.

Measure of contribution of diet quantity and quality to Principal Component 1

The influence of diet quality and quantity on PC1 was compared for the 72 treatments, excluding the single ad libitum treatment. A generalized linear mixed model (GLMM) was performed using the lme4 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.

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.