DOI: 10.5061/dryad.cc2fqz6g1
title: Data from: Individual and social heterosis act independently in honey bee (Apis mellifera) colonies

description: Heterosis occurs in individuals when genetic diversity, i.e.,
 heterozygosity, increases fitness. Many advanced eusocial insects evolved
 mating behaviors, including polyandry and polygyny, which increase
 inter-individual genetic diversity within colonies. The possibility of
 this structure of diversity to improve group fitness has been termed
 social heterosis. Neither the independence of individual and social
 heterosis nor their relative effect sizes have been explicitly measured.
 Through controlled breeding between pairs of Western honey bee queens
 (Apis mellifera L.; n=3 pairs) from two distinct populations, we created
 inbred colonies with low genetic diversity, hybrid colonies with high
 heterozygosity, and mixed colonies (combining inbred workers from each
 population) with low heterozygosity and high social diversity. We then
 quantified two independent traits in colonies: survival against bacterial
 challenge and maintenance of brood nest temperature. For both traits, we
 found hybrid and mixed colonies outperformed inbred colonies but did not
 perform differently from each other. During immune challenge assays,
 hybrid and mixed colonies experienced hazard ratios of 0.49 (95% CI [0.37,
 0.65]) and 0.69 (95% CI [0.50, 0.96]) compared to inbred colonies. For
 nest temperatures, hybrid and mixed colonies experienced 1.94±0.97°C and
 2.82±2.46°C less thermal error and 0.14±0.11°C2 and 0.16±0.06°C2 less
 thermal variance per hour than inbred lines. This suggests social and
 individual heterosis operate independently and may have similar effect
 sizes. These results highlight the importance of both inter- and
 intra-individual diversity to fitness, which may help explain the
 emergence of polyandry/polygyny in eusocial insects and inform breeding
 efforts in these systems.
description: Breeding Design Mated honey bee “founder”
 queens were sourced from the US states of Indiana (population A) and Texas
 (population B). On-going whole-genome scans indicate these populations
 represent the maximum possible genetic variance in naturalized honey bee
 populations in the United States (Ryals, Fikere et al. 2024, in prep.).
 All full-size colonies were kept at the Purdue University Research Apiary
 in West Lafayette, Indiana. We produced daughter queens and drones from a
 pair of founder queens A and B, one from each population. Using
 instrumental insemination, inbred crosses were performed for both A and B
 by mating daughter queens to a single drone from their own mother (AA and
 BB crosses). Accounting for haplodiploidy, their offspring have an
 expected inbreeding coefficient of 0.25. Reciprocal outbred crosses were
 also performed by mating a daughter queen of A to a single drone of B
 and<em> vice versa </em>(AB and BA crosses; Figure 1). Semen
 from a different, single drone was used in each cross to decrease unwanted
 genetic variation within worker groups and control for effects due to
 multiple mating. This design partitions the average
 genetic divergence existing between the chosen founder pair within
 individuals in the hybrid offspring of AB or BA and between individuals
 when combining inbred offspring of AA and BB in a single mixed colony.
 These groups were compared to the mean performance of inbred colonies to
 measure the effect of individual and social heterosis respectively.
 Hybrid, mixed<em>,</em> and<em>
 </em>inbred<em> </em>workers represent our three
 experimental groups. Because there is natural phenotypic variation in each
 population, the performance of all experimental groups and corresponding
 effect sizes of heterosis likely depend on the choice of founder queens.
 To achieve independent measurements of heterosis, the breeding design was
 repeated using three separate pairs of founder queens, resulting in 12
 instrumentally inseminated daughter queens and 15 worker groups.
 Apiculture To produce drones, uniquely
 paint-marked founder queens were introduced into separate queenless
 colonies and presented with a frame of wide-diameter “drone comb”,
 inducing them to lay unfertilized eggs that develop into drones. Each
 drone comb was cleared of bees and placed in a cage prior to drone
 eclosion. Newly emerged, caged drones were paint-marked according to
 parentage and returned to natal colonies. Daughter queens were produced
 following standard methods (Büchler et al., 2024). The presence of each
 founder queen was verified before grafting their larvae into paint-marked
 JZBZ cups (Mann Lake ltd. Z350). Grafting was scheduled so queens and
 drones would be ready for insemination around the same date. All grafted
 larvae were raised into queens in a single queenless “cell-building”
 colony. Prior to eclosion, all daughter queens were introduced into
 separate queenless, nucleus colonies sized for five standard Langstroth
 frames. Queen excluders were placed over entrances to prevent mating
 flights. Daughter queens in each replicate were
 instrumentally inseminated on the same day, 5-10 days after queen
 emergence and 10-20 days after drone emergence, following standard
 procedures (Büchler et al., 2024; Laidlaw, 1977). Daughter queens were
 captured from their nucleus colonies in small, marked cages and
 anesthetized with CO<sub>2</sub> gas. Marked drones were
 captured from natal colonies into large cages. Each queen was inseminated
 immediately after semen collection from the intended drone. All queens
 were marked with numbered disks. Queens were reintroduced to respective
 nucleus colonies (with excluders in place) and treated with
 CO<sub>2</sub> the following day to induce oviposition. All
 nucleus colonies were then fed one gallon sucrose solution to stimulate
 growth. After verifying the presense of capped worker brood (indicating
 successful insemination), colonies were expanded into 10-frame deep boxes
 and fed an additional gallon. Colonies were routinely inspected to remove
 “supersedure” queen cells and prevent replacement of daughter queens by
 workers. Immune Challenge Assay
 The immune challenge assay was performed in “micro-colonies,”
 each made from ~1.5 L plastic cups with mesh-covered windows for
 ventilation, a ~20 cm<sup>2</sup> piece of comb suspended from
 the top, absorptive paper towel covering the bottom, and two feeding tubes
 (15 ml Falcon tubes with 1 mm holes in the lids) entering from the top and
 in contact with the comb. Each micro-colony was filled with 20 marked,
 day-old worker offspring from a single experimental queen in hybrid and
 inbred trials and 10 from each queen in mixed trials. Because these groups
 are much smaller than natural colonies, more replication is allowed and
 bees likely experience increased stress leading to greater hazard. Taken
 together, this likely results in greater sensitivity to differences
 between treatments. A culture of <em>Serratia marcences</em>
 (Thomas Scientific laboratory stock) was combined with equal parts
 Pro-Sweet (Mann Lake ltd. Z320) and dispersed to micro-colonies in the
 treatment group. Bacterial stock was brought to a standard viscosity by
 achieving a reading at A600 nm of 0.11 on a NanoDrop spectrophotometer
 (Thermo Scientific ND-2000), corresponding to roughly
 3x10<sup>8</sup> cells per mL (estimated via hemocytometer),
 after incubating for 3 days at 23 °C in LB broth (Thermo Scientific
 H26760.36). The control group was fed equal parts Pro-Sweet and filtered
 water. Micro-colonies were kept in a dark incubator (Percival I-36NL) at
 34°C and checked at ~12-hr intervals to remove and record dead workers
 until all workers died. Within each replicate, mixed groups of all
 possible pairs of crosses were created, but for direct comparison to
 thermoregulation results, only the AA+BB mixed group was included in
 analysis (see supplemental figure 3 for summary of all mixed
 groups). Results from three micro-colonies were removed
 from analysis due to holes in cages allowing workers to escape or enter,
 potentially compromising their results. In total, 22 trials and 13
 controls were included for analysis. The survival of each micro-colony was
 estimated using the Kaplan-Meier survivor function (Kaplan &
 Meier, 1958) considering replicate as a stratification variable to allow
 for differences in baseline hazard function between family groups. Because
 stress likely increases as individuals are removed from trials,
 differences between treatments were tested using logrank tests (Peto
 & Peto, 1972) and estimated using a Cox proportional hazard model
 (Cox, 1972), neither of which assume a constant hazard function.
 Thermoregulation Assay The thermoregulation
 assay was performed in the field, following Jones et al., 2004, using
 newly-made queenless nucleus colonies sized for three langstroth frames.
 Within each replicate, five separate colonies were created using the
 offspring of experimental crosses: one for each inbred and outbred cross
 and one for the mixed group. Due to the premature death of an outbred
 queen (replicate 2), this gave a total of 14 colonies. After allowing
 inseminated queens to produce workers for at least 51 days to replace any
 unrelated workers in their (full-sized) home colonies, a total of 500 ml
 of workers were moved at night into each nucleus colony. For mixed nucleus
 colonies, one volume of 250 ml was added from each source, spraying bees
 with sugar water to minimize potential fighting during introduction. Each
 colony was provided a full frame of honey, a full frame of unrelated
 larvae, TempQueen pheromone (to simulate the presence of a queen; Mann
 Lake ltd. Z133), and a frame of empty comb. All nucleus colonies were
 moved to Purdue’s Lee Apiary in Americus, Indiana, roughly 20 km from
 natal colonies. They were placed randomly in full sun roughly 5 m apart
 with entrance directions varying randomly between SSE and SSW, and allowed
 to freely forage for seven days before temperature recordings
 began. To measure thermoregulation ability, iButton
 (Thermocron DS1921H) temperature sensors were placed in the brood nest
 recording every five minutes for four days between August 30th and
 September 3rd, 2023 (Figure 3A). An additional sensor was placed in a
 nucleus box without bees to record ambient temperature. At the end of the
 period, all sensors and data were recovered. For each colony, we subset
 data into one-hour windows (12 readings per window, 96 total windows) and
 calculated two response variables: thermal variance as the variance in
 temperature readings and thermal error as the absolute difference between
 recorded and optimal nest temperature (supplemental figure 1). To
 determine significance of individual and social heterosis, hybrid and
 mixed treatments were compared to the average of inbred controls within
 each replicate using a paired t-test for each thermal variable (for
 explicit paired data, see supplemental figure 2). All tests were single
 tailed because the directions of inequalities were explicitly predicted
 (see introduction).
description: # Data from: Individual and social heterosis act independently in honey
 bee (Apis mellifera) colonies data, analysis, and results for an
 experiment on social heterosis in honey bees ## Publication: *Individual
 and social heterosis act independently in honey bee (Apis mellifera)
 colonies* by: Ryals, D.K., Buschkoetter, A.C., J Krispn Given, and Harpur,
 B.A. Pre-Print:
 [https://doi.org/10.1093/jhered/esae043](https://doi.org/10.1093/jhered/esae043) ## Analysis * `therm_assay.Rmd`: Complete analysis for the thermoregulation assay * `immune_assay.Rmd`: Complete analysis for the immune challenge assay ## Data * `therm_data/sensors`: raw data uploads from iButton sensors * within each file, serial numbers are in cell C1 and data begin on row 22 with timestamps (UTC) and temperature (C) * `therm_data/ibkey.xlsx`: key to connect serial numbers for iButton sensors to experimental treatment and replication (see methods) * `immune_data/observations.xlsx`: log of all observations over the course of the immune challenge assay * sheet 1 `removed`: all removed bees (dead or censored) including datetime (EDT), cup (replicate) ID, paint color of individual, status of removed individual (1 = dead, 0 = censored), count (for identical observations), observer initial, and note. Empty cells are considered `NA` (Not Applicable) * sheet 2 `cups`: data for each replication or "cup", including start datetime (EDT), end datetime (controls only), the assay ID and paint color for each included genotype (two for mixed trials, one for single trials), control status (1 = control), categorical variable "run" for all trials begun at the same time, and additional notes. Empty cells are considered `NA` (Not Applicable) * `immune_data/id_key.xlsx`: key to link genotype ID's in `observations.xlsx` to replicate and treatment categories (see methods) # Note * The `figures` directory mentioned in code has been removed in accordance with publication copyright. Please see publication for complete figures.