Determining low levels of africanization using three diagnostic techniques

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    DETERMINING LOW LEVELS OF AFRICANIZATION
    IN UNMANAGED HONEY BEE COLONIES
    USING THREE DIAGNOSTIC TECHNIQUES
    by
    Katherine Darger
    A thesis submitted to the Faculty of the University of Delaware in partial
    fulfillment of the requirements for the degree of Master of Science in Entomology
    Spring 2013
    Copyright 2013 Katherine Darger
    All Rights Reserved


    ABSTRACT
    “Killer bees†arrived in the United States in the year 1990. Questions have
    arisen regarding low levels of Africanization in regions bordering the locations with
    established, Africanized bees. Honey bees were collected and examined using three
    methods of testing to determine levels of Africanization. With morphometrics,
    mitochondrial DNA, and nuclear DNA tested with the use of microsatellites we found
    that the known Africanized bees collected by the Florida Department of Agriculture
    did not exhibit Africanization other than in the preliminary, morphometric test
    performed by the Department of Agriculture.

    Fast Africanized Bee Identification System (FABIS) is the field test for
    preliminary identification of suspected Africanized honey bees (Rinderer 1986).
    FABIS measures forewing length, fresh weight, dry weight, and femur length. This
    process takes approximately twenty minutes per colony (Sylvester and Rinderer 2009).
    Once the colony is identified with FABIS, the sample is analyzed with USDA-ID.
    Universal System for Detecting Africanization (USDA-ID) (Sanford 2006) is
    the test necessary to officially declare a case of Africanization in the Unites States.
    This process is laborious due to the obligation to accuracy. Twenty-five mounted
    parts of each specimen are needed to determine whether a colony is Africanized. This
    requires thorough training to insure accuracy, as it includes miniscule measurements,
    like the number of hamuli on the hindwing, the length and width of the basitarsus, and
    the distance between wax mirrors (Rinderer et al. 1993).
    Two newly established morphometric methods of analysis are less expensive
    and faster than the well-established methods. The Automatic Bee Identification
    System (ABIS) uses a comparison of plotted wing-vein junctions with a digital image
    of the forewing of the specimen. The process takes two minutes per sample and the
    accuracy rate is estimated to be 98.05% among bee species and 94% among honey bee
    subspecies (Francoy et al. 2008). Geometric morphometric analysis is used to
    delineate between honey bee subspecies. The test takes five minutes (Francoy et al.
    2006) and has a 99.2% estimated accuracy rate looking at the wing venation angles
    (Francoy et al. 2008, Francoy et al. 2009). Due to its precision and short preparation
    time, this test was used on the samples in this study.

    USDA-ARS Carl Hayden Honey Bee Research Center and the Florida Department of Agriculture and Consumer Services; Bureau of Plant and Apiary Inspection,
    respectively. The samples from the Florida Department of Agriculture were collected in Alabama, Georgia, and Florida but were grouped together for this study. The samples from Jerry Hayes, formerly of the Florida Department of Agriculture, were grouped together to increase the samples size. Known Africanized samples from South America as well as African samples from Pretoria and Kenya were provided by Dr. Walter S. Sheppard’s personal collection from Washington State University. Forty known Africanized honey bee samples were sent from Belize by Brenna Traver. Altogether, there are 82 samples of known African/Africanized honey bees.
    Morphometrics
    For our morphometric diagnoses, ten right forewings per colony from an
    assortment of colonies among our sampling collection were removed (Table 5). Our
    samples and the control samples were diagnosed in Brazil by Dr. David De Jong using
    the geometric morphometric technique of comparing wing venation (Francoy et al.
    2006, Francoy et al. 2008, Francoy et al. 2009). The results are displayed in Principle
    Component Analysis figures to exhibit the clustering of populations according to
    morphometric restrictions.
    Total DNA Extraction
    A hind leg from two bees per sample, totaling in 188 unmanaged bees and 81
    known Africanized bees, were cut into 4 to 6 pieces and placed in 150 μl of 10%

    Results
    The collection contained limited samples from certain states due to the time
    constraints of this project affecting the number of collecting seasons.
    Morphometrics
    Through the geometric wing analysis performed by Dr. David De Jong, several
    patterns emerged. The unmanaged and managed samples clustered together,
    separately from the representative type of each subspecies (A. m. liguctica, A. m.
    mellifera, A.m. carnica and A. m. caucasica) (Figure 2), showing that the bees in the
    US are a distinct hybrid. The known Africanized samples separated from each other, however the samples from Florida, Georgia, and Alabama all clustered together but outside the range of Africanized bees (Figure 3). The Africanized bees from Arizona clustered separately from the known Africanized bees from Florida as well as the bees from Africa and Brazil (Figure 3). Based on morphological characters, the bees from Florida may not be fully Africanized.


    Mitochondrial Testing
    The unmanaged and managed honey bee colonies were tested and found to
    have European mitotypes (Table 7). The Africanized samples from Florida and Belize
    contained European mitotypes. The Africanized samples from Arizona had a variety
    of mitotypes from the African lineage: A26, A26c, A1e predominantly, and A1. The
    Africanized samples from Belize had four European mitotypes that were C1 while the


    rest of the mitotypes were mostly A1 and A1e. The samples from Africa and Brazil
    had no European mitotypes. They had mitotypes of A4, A47, A26a, and A26c (Table
    1).
    T Tests were performed in Microsoft Excel to test for significant differences
    between the Africanized populations according to mitotypes. The p values for each
    comparison were well below the significance level of 0.05, suggesting that the
    Africanized samples from Belize, Arizona, Brazil, and Africa had significantly more
    mtDNA of African origin compared to the mitotypes of Africanized samples collected from Florida, Alabama, and Georgia. In the comparison of Africanized bees from Florida versus Arizona significant differences were found (p value less than 0.0001,60 degrees of freedom). The comparison of the bees from Florida versus Brazil and Africa was also significant (p value less than 0.0001, 31 degrees of freedom). The p
    value for the comparison of Florida versus Belize was 0.001 (96 degrees of freedom).
    Even the bees from Arizona, Brazil, Africa, and Belize were significantly different
    from each other based on mitotype. The p value for Arizona versus Brazilian and
    African samples was 0.000991 (53 degrees of freedom), while the comparison of
    Arizona versus Belize was 0.0075038 (118 degrees of freedom). The bees from Brazil
    and Africa were different from the bees from Belize (p value = 0.0000509, 89 degrees
    of freedom).


    Microsatellites
    The average number of alleles in each population tested ranged from 5.23
    ±2.12 in Africanized bees from Florida to 7.85 ± 3.25 in unmanaged populations
    (Table 2). Allelic richness was the lowest in the Africanized bees from Florida while the Africanized bees from Arizona had the highest allelic richness measured (Table 2).
    The expected heterozygosity was very similar with the largest range being between
    0.79±0.1 (in samples from Africa and Brazil) and 0.62±0.2 (from the Africanized in
    Florida/Alabama/Georgia and Managed populations) (Table 2).
    The STRUCTURE output consistently provided two populations: Unmanaged
    and managed clustering with the Africanized samples from Florida/Alabama/Georgia
    versus Africanized from Arizona, Africanized from Belize, and samples from Africa
    and Brazil (Figure 4). STRUCTURE is a program that uses multi-locus data to infer
    population structure, assign individuals to populations, and is often used to study
    hybrid areas.
    FST stands for fixation index and shows population differentiation due to
    structure variation as measured by Single Nucleotide Polymorphisms (SNPs) or
    microsatellites. According to the FST results (Table 3), there was a significant
    difference between the managed and unmanaged populations. There was also
    significance between the African and managed samples. Highly significant FST values
    were noted between Africanized bees from Arizona and managed bees and also
    between Africanized bees from Arizona and the unmanaged bees. Importantly, the
    Africanized samples were not significantly different from each other.
    Through the use of Genepop, Hardy Weinberg equilibrium was tested using
    Fis, or inbreeding coefficient, estimates to compare each locus for every population. In
    each instance, using the Fisher’s Method, the results were highly significant, thus the
    populations are not in equilibrium. Allele frequency data, which is the proportion of
    alleles compared to the number of genes, can be found in Table 6 in the Appendix.
    Discussion
    Currently, Africanized bees are officially diagnosed through morphometric
    methods only. The genetics of a honey bee are not often utilized in official
    determinations of Africanization. Should we combine morphometric and molecular
    data to identify the level of Africanization? Determining the process of Africanization
    as either incomplete or absolute can be done with the use of mitochondrial and nuclear
    DNA, as was performed in this study, and will serve as a good tool for tracking the
    introgression of Africanized genes into commercial and unmanaged honey bee
    populations in North America.
    Morphometrically, the data shows that there is an American bee, an amalgam
    that is distinctive from the originating, European subspecies (Figure 2). The GWV
    morphometric study further distinguishes between Africanized bees from Arizona and
    those from Africa and Brazil. Interestingly, the samples from
    Florida/Alabama/Georgia which were originally diagnosed as Africanized by the
    USDA-ID technique were found to have European ancestry using mtDNA markers,


    European morphology based on GWV and grouped with managed and unmanaged
    honey bee colonies in a population structure analysis using microsatellite markers.
    A panmictic population is one in which all forms of recombination are possible
    due to a lack of restriction caused by genetics and behavior, and all fertile individuals
    are potential partners. We hypothesized that the samples would separate into distinct
    populations delineated by lines of latitude. The samples tested in our study are not in
    Hardy Weinberg equilibrium, as shown through the use of the Fisher’s test in
    Genepop, which is indicative that they are not separate and distinct populations as
    would be expected based on geographic collecting locations. Essentially, our study
    indicates that bees on the east coast are not in distinct populations as would be
    surmised based on collection latitude. Northern bees were not differentiated from
    southern bees.
    Studies in Europe showed the M lineage, consisting of western and northern
    European bees, is genetically much more similar to A, or the African lineage than to C
    (eastern Europe) or O lineages (near East and central Asia) (Whitfield et al. 2006).
    Our data shows that two populations could be discerned based on microsatellite allele
    frequency. The Africanized samples from Arizona and Belize were genetically similar
    to the African bees from Africa and Brazil based on allele frequency data on the tested
    microsatellites. Furthermore, the unmanaged, managed, and Africanized bees from
    Florida/Alabama/Georgia shared enough alleles to be considered one population.
    These low levels of introgression can be seen in the Africanized samples collected
    from Florida/Alabama/Georgia (STRUCTURE output Figure 4) from the known


    African and Africanized populations from Arizona, Belize and Brazil. However, the
    Africanized samples from Florida/Alabama/Georgia still grouped with unmanaged and
    managed samples collected along the east coast. This could be due to the very recent
    Africanization of Florida (2010) or could be due to an incomplete Africanization of
    the entire east coast as supplanted by the migratory queen rearing and caged bee trade.
    High linkage disequilibrium in A. m. ligustica and A. m. mellifera accounts for the
    potential for high genetic variation and recombination (Whitfield et al. 2006). The
    opposite is true for A. m. scutellata and A. m, intermissa (Whitfield et al. 2006),
    accounting for infiltration of A. m. scutellata into European genetics and not the other
    way around.
    The mtDNA data supplants this conclusion, as the samples of Africanized
    bees from Florida/Alabama/Georgia did not exhibit Africanized mitotypes in the
    majority of the samples, rather they had European maternal influences, which were also found in the managed and unmanaged samples.
    According to Whitfield et al. (2006), New World bees in Brazil were highly
    Africanized, yet individuals had alleles from the M lineage. Our data shows bees from
    Florida had low levels of Africanization. Mid to late infiltration time showed
    substantially, but not exclusively, Africanized genes (Whitfield et al. 2006) which is
    substantiated by our data; the Africanized bees from Arizona, one of the earliest states
    declared Africanized, were determined to be Africanized based on morphometrics,
    mitochondrial DNA, and when tested with microsatellites.


    During the process of Africanization in Latin America, bees have historically
    had African nuclear and mitochondrial markers become the majority after the first five
    to ten years following the invasion (Schneider et al. 2004). This increase of African
    nuclear and mitochondrial genes over time is supported by our findings: the samples
    from Florida/Alabama/Georgia have European mtDNA and low levels of Africanized
    alleles while samples from Arizona consist of mostly African mitotypes and are
    accompanied by African nuclear alleles. The onset of the invasion into the
    southeastern U.S. as opposed to the desert southwest is supported by the observed
    differences in genetic composition between the two sets of samples and is due to the
    fact that Arizona was one of the earliest states colonized by Africanized bees due to
    the proximity to Central America. It stands to reason that this amount of time allowed
    for more complete introgression to occur, making Arizona further along in the
    Africanization process. All of the managed and unmanaged honey bees had European
    mitotypes (Table 7).
    In Texas, the first state to have Africanized bees, it was suggested that the rate
    of Africanization was enhanced by the decimation of European feral and managed
    colonies by the Varroa destructor mite (Pinto et al. 2005). The Varroa mite created a
    selection pressure as Africanized bees survive infestation. It was also suggested that
    Texas may be the northernmost range of Africanization possible (Pinto et al. 2005),
    while the most recent map of Africanization in the United States shows that as being a
    false prediction. This map, however, was constructed using USDA-ID as the
    diagnostic tool. If molecular tools were used diagnostically, as in our study, Florida
    would not be included. It is possible that the diagnostic techniques (USDA-ID) for
    determining Africanized swarms may be sensitive only to phenotype that is not yet
    linked to genotype and could find a diagnosis in bees with few genetically African
    indicators.
    Genetics should also not be used alone diagnostically. Through the use of
    mtDNA testing along with microsatellite data it was shown that the forefront of the
    Africanized bee movement is caused by Africanized drones mating with European
    queens (Pinto et al. 2005). The resulting hybrid drones were tested and found to be at
    a competitive disadvantage (Hall 1991) because of reduced flight speed, eventually
    leading to direct competition between European and Africanized queens (Pinto et al.
    2005). Our data shows that the Africanized bees from Florida/Alabama/Georgia were morphometrically Africanized only using USDA-ID not the GWV technique, and not necessarily genetically similar enough to the African bee to be considered an Africanized bee.
    The Africanized bees from Florida/Alabama/Georgia were different from the
    other Africanized samples from other locations using each of the three testing
    methods. Using GWV morphometric techniques, the Africanized samples from
    Florida/Alabama/Georgia clustered together as being identical to the managed and
    unmanaged samples from the north, central, and southern aspects of the eastern
    seaboard of the United States and not within the same populations as the Africanized
    bees from Arizona, Brazil, or Africa. No clinal gradient in the degree of
    Africanization was observed in the unmanaged samples collected along the eastern


    seaboard using microsatellite markers. Using mitochondrial testing, the mitotypes
    found in the Africanized bees from Florida/Alabama/Georgia proved to be of
    European ancestry which differed from the mostly pure African ancestry of the
    African and Africanized samples collected from Arizona, Belize, and Brazil. The
    difference in mitotypes found in the samples from Arizona, Belize and Africa warrant
    further research into possible routes of entry of Africanized colonies.
    Using microsatellite analysis to reconstruct actual population dynamics, the
    Africanized bees from Florida/Alabama/Georgia clustered into a population with the
    unmanaged and managed bees from the east coast while the populations from Arizona,
    Belize, Brazil, and Africa clustered together into another separate population. The
    lack of definitiveness in the three diagnostic tools leads us to the conclusion that
    sensitivity of genetic markers may not be the most useful factor in determining
    desirable stock for managing honey bees. Perhaps the best way to evaluate a hive is
    by phenotypic traits, such as aggressive tendencies, and other undesirable traits such
    as swarming and absconding, and not by genotypic traits. Containing a blend of
    markers denotes a level of Africanization but perhaps negative behavior should be the
    first line of diagnosis. Excessive stinging and swarming indicates a need for further
    tools to be used, but even without the negative diagnoses these traits should be
    discouraged.
    In conclusion, I hypothesized that unmanaged honey bee colonies have blends
    of genes from European and African influences and low levels of Africanization occur
    throughout the United States, and can be detected using microsatellite markers. This


    blend of genes was made evident by comparing morphometric and mitochondrial test
    results. The results from the USDA-ID technique conflicted with the results of the
    geometric wing venation, which depicted the Africanized samples from
    Florida/Alabama/Georgia as non-Africanized.
    Future Plans
    For future analysis of our data, more software using Bayesian analysis will
    further indicate population structure and degree of admixture. Bayesian Analysis of
    Population Structure (BAPS) is one program that shows population structure and
    information on introgression. Before processing the samples, we will prepare the data
    by forming “group-wise clustering†of the individuals from each population (Corander
    et al. 2009). All of the data will be pre-processed through the BAPS program prior to
    analysis of genetic information (Corander et al. 2009). Format is the key to successful
    analysis. In the future, the reasoning for the difference in sensitivity of the tests
    should be explored. Perhaps the diagnoses do not necessitate genotype, as
    morphologically and behaviorally the colonies are being confirmed to be Africanized.
    The USDA-ID test is performed after a colony is deemed overly aggressive in the
    field. With those two delineations, a need for genotypic examination may be
    unnecessary. Three of the tools used: geometric wing analysis, mtDNA testing, and microsatellites showed the Africanized bees from Florida, already confirmed
    Africanized by USDA-ID, to be incompletely or not at all Africanized.
    The probable Africanized drone front is a possibility in the Africanized
    counties in Florida, leading to the incomplete genetic introgression. The possibility of


    exchanging the morphometric program with a purely molecular diagnostic program is
    appealing in that it is more precise, less expensive, and less time consuming, but the
    sensitivity may under-estimate the aggressiveness of the hive due to incomplete
    introgression.


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