DOI: 10.5061/dryad.8sf7m0cmn
title: Colony fitness increases in the honey bee at queen mating frequencies higher than genetic diversity asymptote

description: Abstract Across the eusocial Hymenoptera, a queen’s mating frequency is
 positively associated with her workers’ genetic diversity and colony’s
 fitness. Over 90% of a colony’s diversity potential is achieved by its
 mother’s tenth effective mating (me); however, many females mate at levels
 of me &gt; 10, a zone we here call hyperpolyandry. We compared honey
 bee colony fitness at mating levels near and above this genetic diversity
 asymptote. We were interested in how hyperpolyandry affects colony
 phenotypes arising from both common tasks (brood care) and rare
 specialized tasks (parasite resistance). We used an unselected wild line
 of bees and a Varroa Sensitive Hygiene (VSH) line selected to resist the
 parasite Varroa destructor. Virgin queens were instrumentally inseminated
 to replicate the following queen/colony conditions: (1) VSH semen/low
 polyandry (observed mating number = mo = 9), (2) VSH semen/high polyandry
 (mo = 54), (3) wild type semen/low polyandry, or (4) wild semen/high
 polyandry. There was a positive effect of polyandry on brood survival, an
 outcome of common tasks, with highest values at mo = 54. There was an
 interaction between polyandry and genetics such that differences between
 genetic lines expressed only at mo = 54, with fewer mites in VSH colonies.
 These results are consistent with two hypotheses for the evolution of
 mating levels in excess of the genetic diversity asymptote: hyperpolyandry
 improves colony fitness by (1) optimizing genotype compositions for common
 tasks and (2) by capturing rare specialist allele combinations, resisting
 cliff-edge ecological catastrophes. Significance statement Polyandry is a
 female’s practice of mating with several males, storing their sperm, and
 using it to produce one or more clutches of genetically diverse offspring.
 In the social Hymenoptera, polyandry increases the genetic diversity and
 task efficiency of workers, leading to improved colony fitness. Over 90%
 of the increase in a colony’s diversity potential is achieved by its
 mother’s tenth mating; however, many females practice hyperpolyandry, a
 term we reserve here for mating levels above this genetic diversity
 asymptote. We show that a token of colony fitness arising from common
 tasks, brood survival, improves universally as one moves from sub- to
 hyperpolyandrous mating levels. However, a colony phenotype arising from a
 rare parasite resistance task is only expressed in the presence of the
 controlling alleles and under conditions of hyperpolyandry. These results
 suggest adaptive mechanisms by which hyperpolyandry could evolve.
description: Queens and inseminations<br> We performed an experiment
 near Athens, Georgia consisting of four treatments in a 2x2 factorial
 arrangement with two levels of genetic Varroa resistance (semen from
 VSH-selected drones or non-selected wild type drones) and two levels of
 polyandry (high or low). There was one dedicated research apiary in which
 all treatments were represented. Each colony consisted of a 5-frame
 Langstroth nucleus hive and was started with one 2-lb (0.9-kg) package of
 worker bees from one supplier practicing a common V. destructor treatment
 regimen, thus normalizing incipient parasite levels across the experiment.
 Colonies were arranged in one large circle at the apiary to minimize
 effects of bee and parasite drift (Dynes et al. 2019a). The experiment was
 set up in Apr 2018 and ran until Nov 2018. The year before, we made up two
 sets of dedicated drone-source colonies for future inseminations, one set
 headed by wild type unselected queens and the other headed by queens from
 the VSH line. In spring of 2018, we reared one wild
 type virgin queen and inseminated her with one wild type male. This
 single-drone inseminated queen was used to rear supersister virgin
 daughters who were subsequently inseminated and used in the experiment.
 This procedure minimizes maternal variation in experimental colonies
 (Harbo 1985). Supersister virgins were each randomly assigned one of four
 treatments in the 2x2 design: (1) VSH semen/low polyandry, (2) VSH
 semen/high polyandry, (3) wild type semen/low polyandry, or (4) wild type
 semen/high polyandry. We had nine strong VSH
 drone-source colonies come out of winter, a number that dictated the
 multiples for our polyandry ranges. For our low polyandry treatment, we
 inseminated each supersister virgin with one male from each of the VSH
 drone source colonies (mo=9), and for our high polyandry treatment each
 queen was inseminated with 6 males from each VSH drone source colony
 (mo=54). Supersister virgins in the wild type treatments similarly
 received one drone from each of 9 wild type drone source colonies (mo=9)
 or 6 from each wild type source colony (mo=54). Each
 virgin queen was emerged and housed in her 5-frame nucleus colony before
 and after the artificial insemination (AI) procedure, following methods of
 Cobey et al. (2013). Semen from 9 or 54 males, as per protocol, was
 collected into one common capillary tube. A different tube was used for
 each prescribed polyandry/genetic line; no contamination across treatments
 was possible. However, more than one queen was inseminated from any given
 tube. The volume of semen in each tube was increased by 15% with
 physiological saline (recipe 2.2.1 in Cobey et al. (2013)), thoroughly
 mixed in an Eppendorf tube to encourage a homogeneous drone representation
 per batch, re-drawn into the capillary tube, then used to inseminate 2-20
 queens at an average dose of 4 μL mixed semen each, thus removing effects
 of semen volume (Niño et al. 2012). Queens were
 narcotized three times with CO2 – once on the day of insemination and once
 on each of two successive days thereafter; CO2 narcosis stimulates egg
 laying (Mackensen and Roberts 1948). Experimental queens were housed in
 their home 5-frame nuclei and observed until all competent egg layers were
 identified. We waited six weeks thereafter to allow worker populations to
 turn over to progeny of the experimental queens.
 Dependent variables<br> Colonies were sampled at each of
 four time points (Jul, Aug, Sep, Nov). We sampled colonies to determine
 brood production, brood survival, Varroa mite levels, and adult bee
 populations. Brood production and worker bee population
 were derived by visually summing proportions of whole deep frames covered
 by brood or workers (Delaplane et al. 2013), converting frames of brood to
 cm2 by the observation that one deep Langstroth comb (both sides) = 1760
 cm2, and converting frames of adult bees to bee populations with the
 regression model of Burgett and Burikam (1985). When necessary, we
 converted cm2 brood to cells of brood with the conversion of 3.8 cells per
 cm2 (Delaplane et al. 2013). Brood survival was
 measured by removing a comb of open brood, laying over it a sheet of
 transparent acetate, using a felt-tip permanent marker to mark the
 location of 100 brood cells each containing a 1st or 2nd instar larva,
 then returning the comb to the hive; three days later the comb was
 retrieved, the same acetate laid on top of it, and the number of surviving
 brood cells recorded. Relative Varroa mite numbers are
 derived by inserting sticky sampling sheets into bottom board hive inserts
 and recording the number of Varroa mites trapped after 24 hours (Dietemann
 et al. 2013). Patriline determination<br> As a
 check on our success at creating two discrete classes of polyandry with
 AI, we were able to genotype workers from 18 colonies to determine
 effective realized paternity (me) in both polyandry classes.
 A 50-worker sample was taken from each of 8 colonies in the mo=9
 polyandry class and 10 colonies in the mo=54 polyandry class. DNA was
 extracted from the right hind leg for all honey bee specimens using a
 Qiagen DNeasy Blood & Tissue Kit. Hind legs were thoroughly
 pulverized with micro-scissors and digested with proteinase K in a 56°C
 water bath for 3 hours with sample agitation every hour. Extracted DNA was
 stored at -80°C until microsatellite amplification. Ten variable
 microsatellite loci were screened for all samples using two multiplexes:
 Plex 1 and Plex 2. Plex 1 included loci A107, A113, AP043, A024, and A006;
 Plex 2 included loci A28, A88, AP66, AP81, and B124 (Shaibi et al. 2008;
 Delaney et al. 2009). Both plexes were amplified for one cycle at 95°C for
 7 min, 30 cycles of 95°C for 30 sec, 54°C for 30 sec, 72°C for 30 sec, and
 a final extension at 72°C for 60 min. A 10 μL final reaction volume
 containing 5 μL of PCR Master Mix (Promega, Madison, WI), 1.0-2.5 μL of
 fluorescent dye-labeled primer, 0.9 μL of nuclease-free water, and 2 μL of
 DNA extract per sample was analyzed at the University of Delaware
 Sequencing and Genotyping Center at the Delaware Biotechnology Institute
 with an Applied Biosystems 3130 XL Genetic Analyzer via capillary
 electrophoresis. Microsatellite repeat sizes were
 scored using Geneious Prime® 2020.0.5 software (Biomatters Ltd). The
 number (N0) and frequency (pi) of full sibling patrilines was determined
 using raw microsatellite data in the software program COLONY 2.0.6.5
 (Jones and Wang 2010). The observed patriline number (N0), proportion of
 each patriline within each sample (pi), and number of worker bees in each
 respective sample (n) were used to calculate me after (Nielsen et al.
 2003; Tarpy et al. 2015):
 m_e=((〖n-1)〗^2)/(∑_(i=1)^(N_0)▒〖p_i^2 (n+1)(n-2)+3-n〗)
  
description: Identifiers for columns of interest in filename "Delaplane
 polyandry asymptote data for Dryad:"  
 A, state, just GA a placekeeper
 B, “nuc” short for “nucleus” is identifier for individual hive,
 one experimental unit. Nucs were sampled repeatedly at different time
 trials (column M) E, genetic type
 G, poly type I, ERP or effective realized
 paternity. For 18 hives only (!) we genotyped workers “after the fact” to
 see what the “real” paternity was. You can see that AI is very
 inefficient. The poly=54 group especially was all over the map.
 Nevertheless, we thought this could be an informative
 covariate. K, semen batch. The semen is drawn into
 little capillary tubes and used to inseminate queens. Because of genetic
 recombination, none of these mixtures are clones; hence, semen batch is
 another source of random variation. Numbers apply within state.
 M, trial, months at which dependent variables were measured.
 With honey bees there is always a huge seasonal effect as the colony grows
 and declines. Note ahead that not all dependent variables were measure all
 months. W, beepop, adult honey bee population, along
 with X, sqrtbeepop, transformed via square
 root. AA, cm2 honey bee brood, along with
 AB, brood square root transformed AD,
 percentage brood survival, along with AC, survival
 arcsine transformed AE, parasitic mite counts, along
 with AG, mites square root transformed
 AH, percentage of mites reproducing (possibly an indicator of
 VSH effect), along with AK, reproduction arcsine
 transformed AL, cm2 comb constructed, along
 with AN, comb square root transformed
 2-7 are structural elements. 8-19 are dependent
 variables of interest. Relevant column identifiers for
 second dataset, "Delaplane colony survival data for
 Dryad:" B, nuc C,
 whether colony survived to last sampling episode E,
 genetic type H, poly type