Honey Bee Breeding, Genetics, and Physiology Laboratory

BACKGROUND: Recent genomic analyses of arthropod defense mechanisms suggest conservation of key elements underlying responses to pathogens, parasites and stresses. At the center of pathogen-induced immune responses are signaling pathways triggered by the recognition of fungal, bacterial and viral signatures. These pathways result in the production of response molecules, such as antimicrobial peptides and lysozymes, which degrade or destroy invaders. Using the recently sequenced genome of the pea aphid (Acyrthosiphon pisum), we conducted the first extensive annotation of the immune and stress gene repertoire of a hemipterous insect, which is phylogenetically distantly related to previously characterized insects models. RESULTS: Strikingly, pea aphids appear to be missing genes present in insect genomes characterized to date and thought critical for recognition, signaling and killing of microbes. In line with results of gene annotation, experimental analyses designed to characterize immune response through the isolation of RNA transcripts and proteins from immune-challenged pea aphids uncovered few immune-related products. Gene expression studies, however, indicated some expression of immune and stress-related genes. CONCLUSIONS: The absence of genes suspected to be essential for the insect immune response suggests that the traditional view of insect immunity may not be as broadly applicable as once thought. The limitations of the aphid immune system may be representative of a broad range of insects, or may be aphid specific. We suggest that several aspects of the aphid life style, such as their association with microbial symbionts, could facilitate survival without strong immune protection.

Propolis is one of the most fascinating honey bee (Apis mellifera L.) products. It is a plant derived product that bees produce from resins that they collect from different plant organs and with which they mix beeswax. Propolis is a building material and a protective agent in the bee hive. It also plays an important role in honey bee social immunity, and is widely used by humans as an ingredient of nutraceuticals, over-the-counter preparations and cosmetics. Its chemical
\ncomposition varies by geographic location, climatic zone and local flora. The understanding of the chemical diversity of propolis is very important in propolis research. In this manuscript, we give an overview of the available methods for studying propolis in different aspects: propolis in the bee colony; chemical composition and plant sources of propolis; biological activity of propolis with respect to bees and humans; and approaches for standardization and quality control for the purposes of industrial application.

We have identified 44 serine protease (SP) and 13 serine protease homolog (SPH) genes in the genome of Apis mellifera. Most of these genes encode putative secreted proteins, but four SPs and three SPHs may associate with the plasma membrane via a transmembrane region. Clip domains represent the most abundant non-catalytic structural units in these SP-like proteins -12 SPs and six SPHs contain at least one clip domain. Some of the family members contain other modules for protein-protein interactions, including disulphide-stabilized structures (LDL(r)A, SRCR, frizzled, kringle, Sushi, Wonton and Pan/apple), carbohydrate-recognition domains (C-type lectin and chitin-binding), and other modules (such as zinc finger, CUB, coiled coil and Sina). Comparison of the sequences with those from Drosophila led to a proposed SP pathway for establishing the dorsoventral axis of honey bee embryos. Multiple sequence alignments revealed evolutionary relationships of honey bee SPs and SPHs with those in Drosophila melanogaster, Anopheles gambiae, and Manduca sexta. We identified homologs of D. melanogaster persephone, M. sexta HP14, PAP-1 and SPH-1. A. mellifera genome includes at least five genes for potential SP inhibitors (serpin-1 through -5) and three genes of SP putative substrates (prophenoloxidase, spätzle-1 and spätzle-2). Quantitative RT-PCR analyses showed an elevation in the mRNA levels of SP2, SP3, SP9, SP10, SPH41, SPH42, SP49, serpin-2, serpin-4, serpin-5, and spätzle-2 in adults after a microbial challenge. The SP41 and SP6 transcripts significantly increased after an injection of Paenibacillus larva, but there was no such increase after injection of saline or Escherichia coli. mRNA levels of most SPs and serpins significantly increased by 48 h after the pathogen infection in 1st instar larvae. On the contrary, SP1, SP3, SP19 and serpin-5 transcript levels reduced. These results, taken together, provide a framework for designing experimental studies of the roles of SPs and related proteins in embryonic development and immune responses of A. mellifera.

The parasitic mite Varroa destructor and the associated viruses it transmits are responsible for most instances of honey bee colony losses in the United States. As such, beekeepers utilize miticides to control Varroa populations. Widespread resistance has developed to the miticides fluvalinate and coumaphos. However, Varroa has largely maintained susceptibility to amitraz despite a long and extensive use history. Anecdotal reports of reduced amitraz effectiveness have been a widely discussed contemporary issue among commercial beekeepers. Amitraz resistance was measured by in vitro bioassays with technical amitraz as well as Apivar® efficacy tests. Amitraz resistance was evaluated in commercial beekeeping operations in Louisiana, New York, and South Dakota with a long history of amitraz use. This research shows that amitraz remains an effective Varroa control product in many operations. However, apiaries across operations displayed a wide range of amitraz resistance from no resistance to high resistance that resulted in Varroa control failure. The resistance ratios from in vitro amitraz bioassays were correlated with reduced Apivar® efficacy, demonstrating bona fide cases of Varroa control failures due to amitraz resistance. Therefore, amitraz resistance monitoring protocols need to be developed. A resistance monitoring network should be established to ensure the sustainability of miticide use for Varroa control.

BACKGROUND: Honey bees, Apis mellifera, face many parasites and pathogens and consequently rely on a diverse set of individual and group-level defenses to prevent disease. One route by which honey bees and other insects might combat disease is through the shielding effects of their microbial symbionts. Bees carry a diverse assemblage of bacteria, very few of which appear to be pathogenic. Here we explore the inhibitory effects of these resident bacteria against the primary bacterial pathogen of honey bees, Paenibacillus larvae. RESULTS: Here we isolate, culture, and describe by 16S rRNA and protein-coding gene sequences 61 bacterial isolates from honey bee larvae, reflecting a total of 43 distinct bacterial taxa. We culture these bacteria alongside the primary larval pathogen of honey bees, Paenibacillus larvae, and show that many of these isolates severely inhibit the growth of this pathogen. Accordingly, symbiotic bacteria including those described here are plausible natural antagonists toward this widespread pathogen. CONCLUSION: The results suggest a tradeoff in social insect colonies between the maintenance of potentially beneficial bacterial symbionts and deterrence at the individual and colony level of pathogenic species. They also provide a novel mechanism for recently described social components behind disease resistance in insect colonies, and point toward a potential control strategy for an important bee disease.

SUMMARYSuppressed mite reproduction (SMR) is a heritable trait of the honey bee (Apis mellifera) that can control the parasitic mite, Varroa destructor. The purpose of this study was to determine whether adult bees with the SMR trait affect mites in brood after cells are capped. Colonies with or without the SMR trait were each given a comb of newly-capped worker brood that was naturally infested with varroa. Each of 7 source colonies provided a comb of brood to at least one SMR (n = 9) and one control colony (n = 8). These combs were removed from their host colonies 8 days later and mite populations evaluated in cells with bee pupae that were >8 days post-capping. Colonies with SMR bees averaged 2.2% of their cells infested with mites; controls averaged 9.0%. Therefore, bees with the SMR trait apparently removed mites from capped cells. Of the mites that remained, the SMR colonies had a much lower rate of reproductive mites, 20% vs. 71%. This suggests that bees with the SMR trait removed reproductive mites more often than they removed non-reproductive mites. When comparing only the number of mites that produced no progeny, the groups were almost identical averaging 1.2 and 1.3 mites per 100 cells of brood. This suggests that the SMR bees did not remove mites from brood cells if the mites did not lay eggs. By targeting the reproductive mites, bees with the SMR trait give the illusion that nearly all of the mites are non-reproductive. Therefore, our selection for a low frequency of reproductive mites may have produced bees that remove reproductive mites from capped brood.Keywords: Apis melliferaVarroa destructorSMRhygienic behaviourparasitic miteshoney beesresistance

The ectoparasitic mite Varroa destructor is the most significant pathological threat to the western honey bee, Apis mellifera, leading to the death of most colonies if left untreated. An alternative approach to chemical treatments is to selectively enhance heritable honey bee traits of resistance or tolerance to the mite through breeding programs, or select for naturally surviving untreated colonies. We conducted a literature review of all studies documenting traits of A. mellifera populations either selectively bred or naturally selected for resistance and tolerance to mite parasitism. This allowed us to conduct an analysis of the diversity, distribution and importance of the traits in different honey bee populations that can survive V. destructor globally. In a second analysis, we investigated the genetic bases of these different phenotypes by comparing 'omics studies (genomics, transcriptomics, and proteomics) of A. mellifera resistance and tolerance to the parasite. Altogether, this review provides a detailed overview of the current state of the research projects and breeding efforts against the most devastating parasite of A. mellifera. By highlighting the most promising traits of Varroa-surviving bees and our current knowledge on their genetic bases, this work will help direct future research efforts and selection programs to control this pest. Additionally, by comparing the diverse populations of honey bees that exhibit those traits, this review highlights the consequences of anthropogenic and natural selection in the interactions between hosts and parasites.

Honey bees (Apis mellifera) are constantly dealing with threats from pathogens, pests, pesticides and poor nutrition. It is critically important to understand how honey bees’ natural immune responses (individual immunity) and collective behavioral defenses (social immunity) can improve bee health and productivity. One form of social immunity in honey bee colonies is the collection of antimicrobial plant resins and their use in the nest architecture as propolis. We review research on the constitutive benefits of propolis on the honey bee immune system, and its known therapeutic, colony-level effects against the pathogens Paenibacillus larvae and Ascosphaera apis. We also review the limited research on the effects of propolis against other pathogens, parasites and pests (Nosema, viruses, Varroa destructor, and hive beetles) and how propolis may enhance bee products such as royal jelly and honey. Although propolis may be a source of pesticide contamination, it also has the potential to be a detoxifying agent or primer of detoxification pathways, as well as increasing bee longevity via antioxidant-related pathways. Throughout this paper, we discuss opportunities for future research goals and present ways in which the beekeeping community can promote propolis use in standard colonies, as one way to improve and maintain colony health and resiliency.

Pollinators, including honey bees, are responsible for the successful reproduction of more than 87% of flowering plant species: they are thus vital to ecosystem health and agricultural services world-wide. To investigate honey bee exposure to pesticides, 168 pollen samples and 142 wax comb samples were collected from colonies within six stationary apiaries in six U.S. states. These samples were analyzed for evidence of pesticides. Samples were taken bi-weekly when each colony was active. Each apiary included thirty colonies, of which five randomly chosen colonies in each apiary were sampled for pollen. The pollen samples were separately pooled by apiary. There were a total of 714 detections in the collected pollen and 1008 detections in collected wax. A total of 91 different compounds were detected: of these, 79 different pesticides and metabolites were observed in the pollen and 56 were observed in the wax. In all years, insecticides were detected more frequently than were fungicides or herbicides: one third of the detected pesticides were found only in pollen. The mean (standard deviation (SD)) number of detections per pooled pollen sample varied by location from 1.1 (1.1) to 8.7 (2.1). Ten different modes of action were found across all four years and nine additional modes of action occurred in only one year. If synergy in toxicological response is a function of simultaneous occurrence of multiple distinct modes of action, then a high frequency of potential synergies was found in pollen and wax-comb samples. Because only pooled pollen samples were obtained from each apiary, and these from only five colonies per apiary per year, more data are needed to adequately evaluate the differences in pesticide exposure risk to honey bees among colonies in the same apiary and by year and location.

SummaryThe effect of population size on brood production, worker survival and gain or loss of honey was studied in colonies of honeybees (Apis mellifera) in Louisiana, USA. About 11 kg of bees were caged, stored for two days and subdivided into five populations numbering 2300, 4500, 9000, 17 000 and 35 000 bees. Each colony was started with a laying queen, no brood, and 230 bees per 1000 cm3 of hive space. The test ended 19 days after queen release, just before adult bees began to emerge. The test was conducted 10 times (two replicates being used in each of February, April, June, August and October). The two largest populations produced more honey per bee and in dearth times and winter consumed less honey per bee. Colonies of 4500 bees produced the most brood per bee; as population increased above that number brood production per bee decreased. However, during summer dearth, the colonies of 9000 bees produced the most brood per bee. Overall, the optimal colony size was 9000 bees; the rate of weight gain in colonies of this size was nearer to that of the two largest populations and the rate of brood production was nearer to that of the two smaller colonies.

Honey bee immune responses are composed of a complex suite of individual immune mechanisms and special types of behavioral adaptations. The main focus of this paper is innate immunity in the honey bee, and specifically, the role and function of antimicrobial peptides (AMPs). Insect innate immunity constitutes evolutionary conserved defense strategies that provide immediate responses against invading pathogens. It consists of the three levels of resistance: physical barriers as the first line of defense, cell-mediated immunity, and cell-free humoral immunity, a complex network of intracellular signaling pathways leading to activation of a variety of humoral factors. Among those, AMPs are recognized as key components of humoral immunity in many types of organisms. The two basic mechanisms of action are: (1) the generation of leaks into prokaryotic membranes; and (2) either inhibition of bacterial protein translation or folding. Recently, four families of AMPs (i.e., apidaecins, abaecin, hymenoptaecin and defensins) have been described in the honey bee. One of the defensins, defensin1, was originally isolated from royal jelly, and therefore named royalisin. In addition, several bioactive peptides (e.g., apamin and melittin) were identified in bee venom. The expression of bee AMPs is regulated mainly by two intracellular signaling pathways Toll and Imd/JNK. However, the extent of gene expression and peptides synthesis is affected by a number of different biotic and abiotic factors. In this review paper, we have attempted to discuss factors involved in activation of the honey bee AMPs and their role in bee resistance to microbial pathogens and environmental stress such as exposure to pesticides. We also discuss recent knowledge on the molecular regulation of bee AMPs. Although recent advances in genomics have produced a new understanding of bee immunity in general, the exact mechanisms of gene regulation within each of the immune signaling pathways and the complex network of these signaling pathways still await further investigations.

BACKGROUND: The ectoparasitic mite Varroa destructor has emerged as the primary pest of domestic honey bees (Apis mellifera). Here we present an initial survey of the V. destructor genome carried out to advance our understanding of Varroa biology and to identify new avenues for mite control. This sequence survey provides immediate resources for molecular and population-genetic analyses of Varroa-Apis interactions and defines the challenges ahead for a comprehensive Varroa genome project. RESULTS: The genome size was estimated by flow cytometry to be 565 Mbp, larger than most sequenced insects but modest relative to some other Acari. Genomic DNA pooled from ~1,000 mites was sequenced to 4.3× coverage with 454 pyrosequencing. The 2.4 Gbp of sequencing reads were assembled into 184,094 contigs with an N50 of 2,262 bp, totaling 294 Mbp of sequence after filtering. Genic sequences with homology to other eukaryotic genomes were identified on 13,031 of these contigs, totaling 31.3 Mbp. Alignment of protein sequence blocks conserved among V. destructor and four other arthropod genomes indicated a higher level of sequence divergence within this mite lineage relative to the tick Ixodes scapularis. A number of microbes potentially associated with V. destructor were identified in the sequence survey, including ~300 Kbp of sequence deriving from one or more bacterial species of the Actinomycetales. The presence of this bacterium was confirmed in individual mites by PCR assay, but varied significantly by age and sex of mites. Fragments of a novel virus related to the Baculoviridae were also identified in the survey. The rate of single nucleotide polymorphisms (SNPs) in the pooled mites was estimated to be 6.2 × 10-5 per bp, a low rate consistent with the historical demography and life history of the species. CONCLUSIONS: This survey has provided general tools for the research community and novel directions for investigating the biology and control of Varroa mites. Ongoing development of Varroa genomic resources will be a boon for comparative genomics of under-represented arthropods, and will further enhance the honey bee and its associated pathogens as a model system for studying host-pathogen interactions.

This study uses sibling analysis to measure the heritability in honey bees, Apis mellifera L., of characteristics that have been associated with resistance to the mite, Varroa jacobsoni Oudemans. Twenty-eight uniform colonies of bees were established on 13 May in Baton Rouge, LA, each with 1 kg of mite-infested bees and a queen. The 28 unrelated queens in these colonies were divided into 7 groups of 4 based on the insemination of 4 queens with the same mixture of semen from 1 of 7 sire colonies. After worker progeny from these queens had replaced the initial bee populations, a colony was related as a full sister to the other 3 colonies in its sire group and unrelated to the other 24 colonies. Heritability (h 2 ) was 1.24 for proportion of mites in brood, 0.65 for hygienic behavior, 0.89 for the duration of the capped period, 0.46 for suppression of mite reproduction, and 0.00 for physical damage to mites (measured by the presence of physically broken or dented mites on the bottom board). These results suggest that it should be possible to enhance the expression of 4 of these 5 characteristics with selective breeding of bees, thus reinforcing condence in our ability to breed honey bees for resistance to V. jacobsoni.

Early planted cotton ( Gossypium hirsutum L.) and varieties expressing the Bacillus thuringiensis (Bt) gene offer improved yield potential. It is not clear whether the current N recommendations remain appropriate for these new production options. The objectives were to determine how varying rates, application timing, and sources of N affected cotton dry matter partitioning, leaf chlorophyll (Chl) concentration, leaf Bt (Cry1Ac) endotoxin concentration, lint yield, and fiber quality. Four N treatments (112 kg N ha −1 anhydrous; 112 kg N ha −1 liquid; 112/56 kg N ha −1 split; 56/56 kg N ha −1 split) were applied to three cotton varieties (PM 1218BR, STV 4691B, and FM 832) planted on either an early or normal planting date from 2001 through 2004. The N response was consistent across planting dates and varieties for all data collected as shown by the lack of any interactions with these variables. Although N treatments had no effect on lint yield or any dry matter partitioning components, plants receiving the 112/56 kg N ha −1 split application treatment exhibited 14% greater leaf Cry1Ac concentration and a 3% greater leaf Chl concentration than the other N treatments. Early planted cotton had 5% greater leaf Chl concentration but a 12% lower leaf Cry1Ac concentration than normal planted cotton. Lower Cry1Ac levels may reflect enhanced remobilization of the leaf protein to feed the faster developing boll load of the early planted cotton. Early planted cotton yielded 10% more than normal planted cotton because it produced 9% more bolls. Current N recommendations for normal planted cotton also appear sufficient for early planted cotton.

The ectoparasitic mite, Varroa destructor, is the most destructive parasite of managed honeybee colonies worldwide. Since V. destructor transfers pathogens to honeybees, it may be adaptive for bees to respond to mite infestation by upregulating their immune responses. Mites, however, may overcome the host's immune responses by suppressing them, which could facilitate the mite's ability to feed on hemolymph. A humoral immune response of bees parasitized by V. destructor may be detected by studying the expression levels of antibacterial peptides, such as abaecin and defensin, known to be immune-responsive. Expression levels for these two antibacterial peptides changed non-linearly with respect to the number of mites parasitizing honeybee pupae. Bees exposed to low or moderate number of mites had fewer immune-related transcripts than pupae that were never parasitized or pupae with high mite loads. Although many of the pupae tested indicated the presence of bacteria, no correlation with mite numbers or immune-response levels existed. All bees tested negative for acute paralysis and Kashmir bee viruses known to be vectored by V. destructor.

Taxonomic identification of pollen has historically been accomplished via light microscopy but requires specialized knowledge and reference collections, particularly when identification to lower taxonomic levels is necessary. Recently, next-generation sequencing technology has been used as a cost-effective alternative for identifying bee-collected pollen; however, this novel approach has not been tested on a spatially or temporally robust number of pollen samples. Here, we compare pollen identification results derived from light microscopy and DNA sequencing techniques with samples collected from honey bee colonies embedded within a gradient of intensive agricultural landscapes in the Northern Great Plains throughout the 2010-2011 growing seasons. We demonstrate that at all taxonomic levels, DNA sequencing was able to discern a greater number of taxa, and was particularly useful for the identification of infrequently detected species. Importantly, substantial phenological overlap did occur for commonly detected taxa using either technique, suggesting that DNA sequencing is an appropriate, and enhancing, substitutive technique for accurately capturing the breadth of bee-collected species of pollen present across agricultural landscapes. We also show that honey bees located in high and low intensity agricultural settings forage on dissimilar plants, though with overlap of the most abundantly collected pollen taxa. We highlight practical applications of utilizing sequencing technology, including addressing ecological issues surrounding land use, climate change, importance of taxa relative to abundance, and evaluating the impact of conservation program habitat enhancement efforts.

Varroa mites (V. destructor) are a major threat to honey bees (Apis melilfera) and beekeeping worldwide and likely lead to colony decline if colonies are not treated. Most treatments involve chemical control of the mites; however, Varroa has evolved resistance to many of these miticides, leaving beekeepers with a limited number of alternatives. A non-chemical control method is highly desirable for numerous reasons including lack of chemical residues and decreased likelihood of resistance. Varroa sensitive hygiene behavior is one of two behaviors identified that are most important for controlling the growth of Varroa populations in bee hives. To identify genes influencing this trait, a study was conducted to map quantitative trait loci (QTL). Individual workers of a backcross family were observed and evaluated for their VSH behavior in a mite-infested observation hive. Bees that uncapped or removed pupae were identified. The genotypes for 1,340 informative single nucleotide polymorphisms were used to construct a high-resolution genetic map and interval mapping was used to analyze the association of the genotypes with the performance of Varroa sensitive hygiene. We identified one major QTL on chromosome 9 (LOD score = 3.21) and a suggestive QTL on chromosome 1 (LOD = 1.95). The QTL confidence interval on chromosome 9 contains the gene 'no receptor potential A' and a dopamine receptor. 'No receptor potential A' is involved in vision and olfaction in Drosophila, and dopamine signaling has been previously shown to be required for aversive olfactory learning in honey bees, which is probably necessary for identifying mites within brood cells. Further studies on these candidate genes may allow for breeding bees with this trait using marker-assisted selection.

BACKGROUND: Chronic infections can profoundly affect the physiology, behavior, fitness and longevity of individuals, and may alter the organization and demography of social groups. Nosema apis and Nosema ceranae are two microsporidian parasites which chronically infect the digestive tract of honey bees (Apis mellifera). These parasites, in addition to other stressors, have been linked to increased mortality of individual workers and colony losses in this key pollinator species. Physiologically, Nosema infection damages midgut tissue, is energetically expensive and alters expression of immune genes in worker honey bees. Infection also accelerates worker transition from nursing to foraging behavior (termed behavioral maturation). Here, using microarrays, we characterized global gene expression patterns in adult worker honey bee midgut and fat body tissue in response to Nosema infection. RESULTS: Our results indicate that N. apis infection in young workers (1 and 2 days old) disrupts midgut development. At 2 and 7 days post-infection in the fat body tissue, N. apis drives metabolic changes consistent with energetic costs of infection. A final experiment characterizing gene expression in the fat bodies of 14 day old workers parasitized with N. apis and N. ceranae demonstrated that Nosema co-infection specifically alters conserved nutritional, metabolic and hormonal pathways, including the insulin signaling pathway, which is also linked to behavioral maturation in workers. Interestingly, in all experiments, Nosema infection did not appear to significantly regulate overall expression of canonical immune response genes, but infection did alter expression of acute immune response genes identified in a previous study. Comparative analyses suggest that changes in nutritional/metabolic processes precede changes in behavioral maturation and immune processes. CONCLUSIONS: These genome-wide studies of expression patterns can help us disentangle the direct and indirect effects of chronic infection, and understand the molecular pathways that regulate disease symptoms.

To combat an increasing abundance of sucking insect pests, >40 pesticides are currently recommended and frequently used as foliar sprays on row crops, especially cotton. Foraging honey bees may be killed when they are directly exposed to foliar sprays, or they may take contaminated pollen back to hives that maybe toxic to other adult bees and larvae. To assess acute toxicity against the honey bee, we used a modified spray tower to simulate field spray conditions to include direct whole-body exposure, inhalation, and continuing tarsal contact and oral licking after a field spray. A total of 42 formulated pesticides, including one herbicide and one fungicide, were assayed for acute spray toxicity to 4-6-d-old workers. Results showed significantly variable toxicities among pesticides, with LC50s ranging from 25 to thousands of mg/liter. Further risk assessment using the field application concentration to LC1 or LC99 ratios revealed the risk potential of the 42 pesticides. Three pesticides killed less than 1% of the worker bees, including the herbicide, a miticide, and a neonicotinoid. Twenty-six insecticides killed more than 99% of the bees, including commonly used organophosphates and neonicotinoids. The remainder of the 13 chemicals killed from 1-99% of the bees at field application rates. This study reveals a realistic acute toxicity of 42 commonly used foliar pesticides. The information is valuable for guiding insecticide selection to minimize direct killing of foraging honey bees, while maintaining effective control of field crop pests.

Until recently, African and European subspecies of the honeybee (Apis mellifera L.) had been geographically separated for around 10,000 years. However, human-assisted introductions have caused the mixing of large populations of African and European subspecies in South and Central America, permitting an unprecedented opportunity to study a large-scale hybridization event using molecular analyses. We obtained reference populations from Europe, Africa, and South America and used these to provide baseline information for a microsatellite and mitochondrial analysis of the process of Africanization of the bees of the Yucatan Peninsula, Mexico. The genetic structure of the Yucatecan population has changed dramatically over time. The pre-Africanized Yucatecan population (1985) comprised bees that were most similar to samples from southeastern Europe and northern and western Europe. Three years after the arrival of Africanized bees (1989), substantial paternal gene flow had occurred from feral Africanized drones into the resident European population, but maternal gene flow from the invading Africanized population into the local population was negligible. However by 1998, there was a radical shift with both African nuclear alleles (65%) and African-derived mitochondria (61%) dominating the genomes of domestic colonies. We suggest that although European mitochondria may eventually be driven to extinction in the feral population, stable introgression of European nuclear alleles has occurred.