Summary Chalkbrood and stonebrood are two fungal diseases associated with honey bee brood. Chalkbrood, caused by Ascosphaera apis, is a common and widespread disease that can result in severe reduction of emerging worker bees and thus overall colony productivity. Stonebrood is caused by Aspergillus spp. that are rarely observed, so the impact on colony health is not very well understood. A major concern with the presence of Aspergillus in honey bees is the production of airborne conidia, which can lead to allergic bronchopulmonary aspergillosis, pulmonary aspergilloma, or even invasive aspergillosis in lung tissues upon inhalation by humans. In the current chapter we describe the honey bee disease symptoms of these fungal pathogens. In addition, we provide research methodologies and protocols for isolating and culturing, in vivo and in vitro assays that are commonly used to study these host pathogen interactions. We give guidelines on the preferred methods used in current research and the application of molecular techniques. We have added photographs, drawings and illustrations to assist bee-extension personnel and bee scientists in the control of these two diseases.

Recent biological invasions offer ‘natural’ laboratories to understand the genetics and ecology of adaptation, hybridization, and range limits. One of the most impressive and well-documented biological invasions of the 20th century began in 1957 when Apis mellifera scutellata honey bees swarmed out of managed experimental colonies in Brazil. This newly-imported subspecies, native to southern and eastern Africa, both hybridized with and out-competed previously-introduced European honey bee subspecies. Populations of scutellata -European hybrid honey bees rapidly expanded and spread across much of the Americas in less than 50 years. We use broad geographic sampling and whole genome sequencing of over 300 bees to map the distribution of scutellata ancestry where the northern and southern invasions have presently stalled, forming replicated hybrid zones with European bee populations in California and Argentina. California is much farther from Brazil, yet these hybrid zones occur at very similar latitudes, consistent with the invasion having reached a climate barrier. At these range limits, we observe genome-wide clines for scutellata ancestry, and parallel clines for wing length that span hundreds of kilometers, supporting a smooth transition from climates favoring scutellata -European hybrid bees to climates where they cannot survive winter. We find no large effect loci maintaining exceptionally steep ancestry transitions. Instead, we find most individual loci have concordant ancestry clines across South America, with a build-up of somewhat steeper clines in regions of the genome with low recombination rates, consistent with many loci of small effect contributing to climate-associated fitness trade-offs. Additionally, we find no substantial reductions in genetic diversity associated with rapid expansions nor complete dropout of scutellata ancestry at any individual loci on either continent, which suggests that the competitive fitness advantage of scutellata ancestry at lower latitudes has a polygenic basis and that scutellata -European hybrid bees maintained large population sizes during their invasion. To test for parallel selection across continents, we develop a null model that accounts for drift in ancestry frequencies during the rapid expansion. We identify several peaks within a larger genomic region where selection has pushed scutellata ancestry to high frequency hundreds of kilometers past the present cline centers in both North and South America and that may underlie high-fitness traits driving the invasion.

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The Latin American subcontinent contains some of the world's major honey producing and exporting countries, but the status of bee health in this part of the world has not been clearly documented. There have been no reports of massive colony losses in Latin America, at least from the symptoms of CCD (colony collapse disorder) or in the proportion and extent of the situations in the US and Europe. We examine possible reasons for the difference, and develop hypotheses that this prevailing good bee health could be due to: (1) the management of generally unselected bees with a certain natural resistance to diseases (tropical regions) or the selection of disease resistant bees (temperate regions); (2) a lower proportion of cropland over the total land area, resulting in more abundant or higher-quality pollen resources for bees;(3) the generally small-scale, low-income and little subsidized agriculture, and concomitant lower use of insecticides compared to industrialized countries. These general parameters may act synergistically, resulting in a large number of configurations across the tremendous ecological, social and economic diversity of Latin America. We suggest that the health of honey bees in Latin America may be ultimately due to the practices of low-income agriculture and beekeeping in the region, leading to more sustainable conditions for the bees. However the increasing trend of land use intensification in some parts of Latin America could lead to declines in honey bee health and population size. honey bee health / colony losses / disease resistance / genetic diversity / pollen nutrition

Hygienic behavior of honeybees involves inspection, uncapping and removal of diseased and dead brood from the colony. The objective of this work was to study the activities involved in hygienic behavior of individually tagged bees from selected hygienic (H) and non-hygienic (NH) colonies in the presence of chalkbrood infected brood (Ascosphaera apis) or pin-killed brood. No significant difference was detected in the age of bees inspecting, uncapping or removing brood in H and NH colonies; the median age was 15 days for all activities. The percentage of bees that performed these activities was significantly higher in H colonies. In NH colonies the bees that performed this behavior were more persistent but bees in H colonies were more efficient in the removal of the chalkbrood mummies. H colonies began uncapping more rapidly in response to the stimulus of dead brood independent of the method used to kill it. H and NH bees took the same amount of time to remove the mummies once they initiated the uncapping process but NH colonies took longer to remove pin-killed brood. These findings confirm previous behavioral studies on the activities of hygienic and non-hygienic bees toward freeze-killed brood, but this is the first time the entire process from inspection to removal was focused on individual cells containing actual diseased brood.

AbstractRecent biological invasions offer ‘natural’ laboratories to understand the genetics and ecology of adaptation, hybridization, and range limits. One of the most impressive and well-documented biological invasions of the 20th century began in Brazil with the escape of introduced African honey bees (Apis mellifera scutellata) in 1957. In less than 50 years, populations of ‘Africanized’ honey bees spread across much of the Americas, hybridizing with and outcompeting resident European honey bees. We use broad geographic sampling and whole genome sequencing of over 300 bees to map the distribution of African ancestry where the northern and southern invasions have presently stalled, forming replicated hybrid zones between Africanized and European bees in California and Argentina. California is much farther from Brazil, yet these hybrid zones occur at very similar latitudes, consistent with the invasion having reached a climate barrier. At these range limits, we observe genome-wide clines for African ancestry, and parallel clines for wing length that span hundreds of kilometers, supporting a smooth transition from climates favoring Africanized bees to climates where they cannot survive winter. We find no large effect loci maintaining exceptionally steep ancestry transitions. Instead, we find most individual loci have concordant ancestry clines across South America, with a build-up of somewhat steeper clines in regions of the genome with low recombination rates, consistent with many loci of small effect contributing to climate-associated fitness trade-offs. Additionally, we find no substantial reductions in genetic diversity associated with rapid expansions nor complete dropout of African ancestry at any individual loci on either continent, which suggests that the competitive fitness advantage of African ancestry at lower latitudes has a polygenic basis and that Africanized bees maintained large population sizes during their invasion. To test for parallel selection across continents, we develop a null model that accounts for drift in ancestry frequencies during the rapid expansion. We identify several peaks within a larger genomic region where selection has pushed African ancestry to high frequency hundreds of kilometers past the present cline centers in both North and South America and that may underlie high-fitness African traits driving the invasion.Author SummaryCrop pollination around the world relies on local honey bee populations, which vary in their behaviors and climatic ranges. Africanized honey bees (‘killer bees’) have been some of the most widely successful; originating in a 1950s experimental breeding program in Brazil, they rapidly came to dominate across most of the Americas. As a recent genetic mixture of imported African bees and European subspecies, Africanized honey bees have a patchwork of ancestry across their genomes, which we leverage to identify loci with an excess of African or European ancestry due to selection. We additionally use the natural replication in this invasion to compare outcomes between North and South America (California and Argentina). We identify several genomic regions with exceptionally high African ancestry across continents and that may underlie favored Africanized bee traits (e.g. Varroa mite resistance). We find evidence that a climatic barrier has dramatically slowed the invasion at similar latitudes on both continents. African-to-European ancestry transition zones span hundreds of kilometers where bees have intermediate African ancestry proportions, which can be used to map the genetic basis of segregating traits (here, wing length), and calls into question the biological basis for binary Africanized vs. European bee classifications.

During a reproductive cycle, not all daughter mites of Varroa destructor mate and thus leave the brood cells as virgins. Here, we show that virgin mites are present within both the phoretic (10%) and reproductive (8%) mite population. Most (n = 29 of n = 33) of these encountered virgins laid unfertilized (= male) eggs, and some (n = 10) mated later on with their own son. These findings were verified by tests with artificially reared virgin mites. Obviously, mating is not a prerequisite for Varroa reproduction. However, due to the small number of reproductive cycles, the contribution of virgins to the Varroa population is regarded as low. This study also confirms conclusively that sex of V. destructor is determined via arrhenotokous parthenogenesis and not-as previously assumed-via pseudo-arrhenotoky. Furthermore, reproductive parameters of naturally invaded and artificially introduced Varroa females were compared, and artificial infestation was reconfirmed as a suitable method.