Seasonal variation in defense behavior in European and scutellata-hybrid honey bees (Apis mellifera) in southern California

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 scutellata-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 scutellata-hybrids and EHB at the BFS apiary, not by design, but because our EHB colonies inevitably requeened themselves and, given the density of feral scutellata-hybrids throughout the region, these new queens often mated with scutellata-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 scutellata-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 scutellata-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 scutellata-hybrids. They were also annually requeened with Varroa Sensitive Hygiene (VSH) A. m. ligustica 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 A. m. scutellata. 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.