DOI: 10.5061/dryad.wpzgmsbs4
title: Seasonal variation in defense behavior in European and scutellata-hybrid honey bees (Apis mellifera) in southern California

description: Nest defense in the honey bee (Apis mellifera) is a complex collective
 behavior modulated by various interacting social, environmental, and
 genetic factors. Scutellata-hybrid (“Africanized”) honey bees are usually
 considered to be far more defensive than European honey bees which are
 therefore preferred for commercial and hobbyist beekeeping. In the most
 recent zone of scutellata hybridization, the southern USA, the degree to
 which this defensiveness differs among current strains, and the extent to
 which defensiveness varies across a season has not been measured. We
 quantified the levels of A. m. scutellata ancestry in colonies and
 conducted a seasonal assessment (May through November) of colony nest
 defensiveness in feral scutellata-hybrid and managed European honey bee
 (A. mellifera ligustica) hives at two apiaries in Southern California.
 Standard measures of defensiveness were low in both scutellata-hybrid and
 European colonies during May. Defensiveness increased during the later
 months of the study in scutellata-hybrid colonies. Most measures of
 defensiveness did not increase in managed colonies. Defensiveness in the
 scutellata-hybrids appears lower than what has been previously documented
 in Brazil and Mexico, possibly due to their lower proportion of A. m.
 scutellata ancestry.
description: All honey bee hives were kept in two apiaries in San Diego,
 California (U.S.A.). Managed EHB colonies were maintained at the
 Biological Field Station (BFS) on the University of California San Diego
 campus in La Jolla, CA and feral bees were kept at the Elliot Chaparral
 Reserve (ECR), a largely undisturbed coastal sage scrub habitat (Fig. S1).
 Although a common garden design with all colonies maintained in the same
 apiary is desirable to control for multiple environmental factors, we used
 separate apiaries for the following reasons. First, the presence of
 <em>scutellata</em>-hybrids with EHB colonies in the same
 apiary has been shown to increase the defensiveness of the EHB colonies.
 We wished to independently measure the defensiveness of feral and managed
 colonies over time. Using separate apiaries prevented this interaction
 problem. In addition, for multiple years prior to this study, we hosted
 both <em>scutellata</em>-hybrids and EHB at the BFS apiary,
 not by design, but because our EHB colonies inevitably requeened
 themselves and, given the density of feral
 <em>scutellata</em>-hybrids throughout the region, these new
 queens often mated with <em>scutellata</em>-hybrid drones.
 Over time, EHB colonies at the BFS often became notably more defensive.
 Using the standard black flag test (see below) to measure defensiveness,
 we found that nearly all colonies, including EHB (confirmed by the
 presence of marked EHB queens) had elevated levels of defensiveness,
 corroborating the observations of other researchers. In addition, when we
 placed <em>scutellata</em>-hybrid and EHB colonies within the
 same apiary, we observed robbing in October and November. As a result, a
 high proportion of EHB colonies were robbed out and died, and their
 defensiveness could not be measured. Robbing also leads to increased
 defensiveness in an apiary. The use of two separate apiaries therefore
 enabled us to conduct a study of defensiveness over an extended period of
 time. However, we recognize that multiple factors differed between these
 two sites and we therefore do not directly compare defensiveness between
 our <em>scutellata</em>-hybrid and EHB colonies. Instead, we
 focus on how defensiveness varied over an extended time period, the
 longest and most detailed assessment to date for studies of defensiveness
 by these two bee types. Our managed colonies (BFS
 apiary) were inspected monthly and managed following standard beekeeping
 practices. Managed colonies were founded as nucleus colonies purchased
 from northern California, outside of the current range of
 <em>scutellata</em>-hybrids. They were also annually requeened
 with Varroa Sensitive Hygiene (VSH) <em>A. m. ligustica
 </em>queens bred for gentleness and obtained from Wildflower
 Meadows, an apiary located in Vista, CA, with the most recent requeening
 occurring in July 2021. Any BFS colonies that naturally requeened had
 their queen removed and were requeened with these VSH queens. From
 August-March, managed colonies were provided with sucrose solution (50%
 w/w) ad libitum and protein patties (Ultra Bee High Protein Pollen
 Substitute, Mann Lake LTD). EHB colonies were also placed on hive stands
 that excluded ants. In contrast, feral honey bee
 colonies (ECR apiary) were unmanaged and originated from feral swarms
 captured throughout San Diego County, likely to have ancestry admixed with
 <em>A. m. scutellata</em>. Feral colonies were not requeened
 nor treated for Varroa or any other diseases. No sucrose or protein
 supplements were ever provided to these colonies, and they did not have
 ant excluders. At both apiaries, each colony was housed
 in a single standard 10-frame Langstroth hive. Ten colonies were chosen
 from each apiary (total n = 20), based on comparable sizes and similar
 honey stores and brood comb areas. When any of these colonies absconded or
 died, we chose another colony in order to maintain a sample size of 10
 colonies evaluated per site each month. In total, 13 colonies across both
 sites (BFS, n = 7; ECR, n = 6) remained throughout the entirety of the
 study while all other colonies included in the study were only assessed
 for a portion of the study duration (ranging from 2–4 of the five months
 in which measurements were made).To assess the genetic composition of
 honey bee colonies from each apiary, we collected three honey bee workers
 from each colony (total n = 60). To ensure that these bees were from the
 hives being studied and not non-nestmates (e.g. robbers), we selected
 young bees (bees that had less wing wear and prominent white thoracic
 hairs) on brood combs. Honey bees from the ECR apiary were sampled in
 mid-November 2021, following the last defensiveness assay. Honey bees from
 the BFS apiary were sampled in late August, approximately two months after
 the last requeening (in early July 2021). Honey bees were euthanized and
 preserved in 100% ethanol at -20 °C. We separately extracted DNA from the
 crushed head of each bee using the standard protocol of the Qiagen DNAeasy
 Blood & Tissue extraction kit (Catalog ID: 69504). The DNA was
 submitted for DNA KAPA library construction and whole-genome sequencing at
 the Institute for Genomic Medicine (IGM), UC San Diego. All 60 individuals
 were multiplexed and sequenced across three lanes of an Illumina NovaSeqS4
 platform to produce 150-bp paired-end reads at ~20X coverage.