DOI: 10.5061/dryad.jh9w0vt9v
title: Larval nutrition impacts the scaling of adult metabolic rate with body mass in honeybees

description: Resting metabolic rate (RMR) is a fundamental physiological measure linked
 to numerous aspects of organismal function, including lifespan. Although
 dietary restriction in insects during larval growth/development affects
 adult RMR, the impact of larval diet quality on adult RMR has not been
 studied. Using in vitro rearing to control larval diet quality, we
 determined the effect of dietary protein and carbohydrate on honeybee
 survival-to-adulthood, time-to-eclosion, body mass/size and adult RMR.
 High carbohydrate larval diets increased survival-to-adulthood and
 time-to-eclosion compared to both low carbohydrate and high protein diets.
 Upon emergence, bees reared on the high protein diet were smaller and
 lighter than those reared on other diets, whilst those raised on the high
 carbohydrate diet varied more in body mass. Newly-emerged adult bees’
 reared on the high carbohydrate diet showed a significantly steeper
 increase in allometric scaling of RMR compared to those reared on other
 diets. This suggests that diet quality influences survival-to-adulthood,
 time-to-eclosion, and the allometric scaling of RMR. Given that
 agricultural intensification and increasing urbanisation have led to a
 decrease in both forage availability and dietary diversity for bees, our
 results are critical to improving understanding of the impacts of poor
 developmental nutrition on bee growth/development and physiology.
description: Honeybee (<i>Apis
 mellifera</i> L.) larvae were obtained from full-sized colonies
 housed on the University of Sussex campus, and reared in the laboratory
 using the <i>in vitro</i> method described by Schmehl
 <i>et al. </i>(2016). Briefly, three-day old larvae were
 removed from the comb using a grafting tool, transferred to individual
 wells of a 48-well cell culture plate, and placed into an incubator fixed
 at 35°C, 94% relative humidity (RH). Larvae were fed once per day for five
 days, and upon pupation transferred to a fresh cell culture plate.
 Survival was monitored daily until adult emergence. <i>Diet
 manipulation</i> A standard <i>in vitro</i> rearing diet of yeast (Sigma-Aldrich UK), royal jelly (The Raw Honey Shop, Brighton) and sugars (glucose and fructose, Sigma-Aldrich UK) was manipulated to contain differing amounts of protein (using royal jelly as a proxy) and/or carbohydrate (glucose and fructose), following the methods of Helm <i>et al.</i> (2017). Larvae were reared on one of five diets where the amount of protein and carbohydrate was either increased or decreased relative to the diet described by Schmehl <i>et al.</i> (2016).<sup>. </sup>Royal jelly<sup> </sup>was stored frozen at -20°C in 50 mL aliquots. Diets were freshly made every two days and stored at 4°C. Larvae were fed once per day for five days, and the volume of food varied according to the day of the experiment (Days 1 and 2 = 10 μL; Day 3 = 20 μL; Day 4 = 30 μL; Day 5 = 40 μL; and Day 6 = 50 μL). Between 60 and 78 larvae were assigned to each treatment group (<i>N</i> = 371 larvae in total; D1=78; D2=60; D3=78, D4=78; D5=77). Bees were reared in two cohorts, grafted on 30/9/2019 and 20/10/2019. Royal jelly nutritional values (supplementary data) were obtained by the supplier (The Raw Honey Shop, Brighton) using the international standard for royal jelly (ISO 12824:2016). From these values we calculated the proportion and ratio of protein:carbohydrate (P:C) in each of the five diets (Table 1). <i>Measuring resting metabolic rates</i> To determine how larval diet affects adult metabolism, the RMR of adult bees was measured on the day of emergence (between 14-17 days from the day of grafting) using flow-through respirometry, with CO<sub>2</sub> production used as a measure of metabolic rate. Emerging adults were first individually weighed to the nearest mg using a precision balance (Mettler Toledo, UK). Bees were then restrained using a small cylinder of metal mesh to allow gas exchange, before being placed into a 2 mL plastic chamber. Air scrubbed of CO<sub>2</sub> and H<sub>2</sub>O was then pumped through the chamber at a consistent rate of 100 mL min<sup>-1</sup> via a mass flow controller (GFC17; Aalborg, NY, USA), before passing through an infrared CO<sub>2</sub>-H<sub>2</sub>O analyser (Li7000, Li-Cor) which captured data on CO<sub>2</sub> production, relative to an empty control chamber (Nicholls <i>et al.</i>, 2017; Perl and Niven, 2018). The temperature in the room was held constant at 25°C (± 2°C) and recordings lasted for 20 minutes per bee. The first five minutes of the recording were treated as a settling period for the bee to adjust to the experimental set up and were excluded from analysis. During recording the plastic chamber was covered to ensure it was dark, which reduced bee movement. The order in which bees from different diet treatment groups were measured was randomised. After recording, bees were frozen to immobilise them, and digital callipers were used to measure the intertegular span (defined as the distance between the points at which the wings attach to the thorax) in mm, a proxy measure for body size (Cane, 1987). <i>Data analysis</i> Respirometry data was analysed using OriginPro software (Origin 2016, OriginLab Corporation, Northampton, MA, USA). Volumes of CO<sub>2</sub> were baseline corrected and temperature normalised using the Q10 correction for temperature differences. To calculate the rate of CO<sub>2</sub> production per bee, the volume of CO<sub>2</sub> (ppm) was converted to CO<sub>2</sub> fraction and multiplied by the flow rate (100 mL min<sup>-1</sup>). The integral of CO2 min<sup>−1</sup> <i>versus</i> min was calculated for a stable 15-minute period of the recording, and divided by this time to give a rate of µl CO<sub>2</sub> h<sup>-1</sup>. All statistical analyses were conducted in R 3.6.2 (R Core Team, 2019. https://www.R-project.org). To examine how diet quality impacts larval survival, Kaplan-Meier survival analysis was performed using the survfit function from the ‘survival’ package. The log-rank test was used to test for differences in survival between diet treatments with a Bonferroni correction for multiple comparisons. Linear and mixed effect models were performed by restricted maximum likelihood (REML) estimation using the lmer and glmer function from the ‘lme4’ package to test the impact of diet treatment on the time to adult emergence (days), wet body mass (mg), body size (using intertegular distance as a proxy measure; mm), body condition (body mass/body size; mg/mm) and CO<sub>2</sub> production (µL CO<sub>2 </sub>h<sup>-1</sup>). The continuous variables body mass, body size, body condition and CO<sub>2</sub> production were log transformed. Date of grafting was included as a random effect. For all models, Diet 2 was used as the reference category because bees in this treatment had the best survival. Significances of the fixed effects were determined using Satterthwaite’s method for estimation of degrees of freedom by using the anova function from ‘lmerTest’. Estimated marginal means (emm) and pairwise comparisons were obtained using the ‘lsmeans’ package and the <i>p</i>-value adjusted with the Tukey method. To test for differences in variance, we used the Brown-Forsythe test for non-normal data. All plots were made using the ‘ggplot2’ package.
description: Variables: grafted: Date larvae grafted into
 cell culture plate diet: Diet treatment fed to larvae,
 varying in protein:carbohydrate content (P:C D1 = 1:2.3; D2 =1:3.0; D3 =
 1:1.5; D4 = 1:1.9; D5 = 1:2.9) bee_ID: Individual bee
 identity Temp: Room temperature during RMR
 recordings emerged: Number of days after grafting until
 bees emerged as adults weight: Weight of emerging bee
 in mg interwing: Intertegular distance in mm
 Condition: Weight divided by interwing
 Normalised Temp: Temperature to which measurements were
 normalised Area under curve: Integral of CO2 vs
 seconds Temp Normalised area under curve: Integral
 adjusted for standardised temperature of 25C ml CO2:
 Total ml of CO2 produced CO2 ml/hour: Rate of CO2
 produced CO2 ul/hour: Rate of CO2 produced
 CO2.h.temp: Temperature corrected RMR in µl
 CO<sub>2</sub> h<sup>-1</sup>
 stage.death: Developmental stage at which bee died
 time.death: Developmental stage at which bee died (1= larva,
 2=prepupa, 3=pupa, 4=survived to adulthood) emergence:
 Survival to adulthood (0= died, 1=emerged as adult)