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1 THE I MPACT OF MANAGED EUROPEAN HONEY BEE COLONIES ON THE COMPOSITION OF DRONE CONGREGATION AREAS IN REGIONS WHERE AFRICAN HONEY BEES ARE PRESENT By ASHLEY NICOLE MORTENSEN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013
2 2013 Ashley Nicole Mortensen
3 To my loving family who has supported me through
4 ACKNOWLEDGMENTS I graciously thank Arthur Mathisen for coordination and use of experimental apiary s ites and numerous other locations in search of DCAs; Mario Jacob and the employees of D&J Apiary for providing and managing Euro pean derived honey bee colonies ; Niko and Gudrun Koeniger for their invaluable advice on drone congregation area identification and drone trapping technique s ; Norm Gary for his generous contribution of synthetic 9 oda; Mark Dykes, Katy Evans, Stephanie Lon g, and Teresa Pitts for field assistance; Emily Helton and Shiloh Ripley for preparation of morphometric samples ; employees of the Florida Department of Agricultural and Consumer Services including Jerry Hayes, David Westervelt, K aty Evans, and Kelly Roger s for conducting all morphometric analyses; Cindy Tannahill for helping optimize the molecular protocols necessary for this thesis ; and my advisor, Dr. Jamie Ellis, and by committee, Dr. Glenn Hall and Dr. Bill Kern for their guidance Funding for this pro ject was provided by the Florida Department of Agricultural and Consumer Services (FDACS) through guidance of the Honey Bee Technical Council and the North East Florida Honey B ee Association
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 LITERATURE REVIEW ................................ ................................ .......................... 12 African Honey Bees in the Americas ................................ ................................ ...... 12 Persistence of African Genetics ................................ ................................ ....... 13 Hybrid dysfunction ................................ ................................ ..................... 14 Increased developmental stability ................................ .............................. 14 Pairwise breeding ................................ ................................ ...................... 15 Temporal isolation ................................ ................................ ...................... 15 Drone production and drift ................................ ................................ ......... 16 Reproductive rate ................................ ................................ ....................... 17 African patriline advantage ................................ ................................ ......... 17 Hybridization Zones ................................ ................................ .......................... 18 Africanization ................................ ................................ ................................ .... 2 0 Honey Bee Mati ng ................................ ................................ ................................ .. 21 Drone Congregation Areas ................................ ................................ ............... 22 Mating Flights ................................ ................................ ................................ ... 23 Population Structure of a DCA ................................ ................................ ......... 24 Synopsis and Objectives ................................ ................................ ......................... 25 2 SC IENTIFIC NOTE ON A SINGLE USER METHOD FOR IDENTIFYING DRONE CONGREGATION AREAS ................................ ................................ ....... 32 3 A SCIENTIFIC NOTE ON THE PREVALENCE OF THE COR DOVAN PHENOTYPE IN THE AFRICAN DERIVED HONEY BEE POPULATION IN THE SOUTHEASTERN UNITED STATES ................................ ............................. 41 4 USURPATION OF MANAGED EUROPEAN DERIVED HONEY BEE COLONIES VIA AFRICAN MATRILINE SWARMS IN THE SOUTHEASTERN UNITED STATES ................................ ................................ ................................ ... 46 Introduction ................................ ................................ ................................ ............. 46 Materials and Methods ................................ ................................ ............................ 47
6 Results ................................ ................................ ................................ .................... 48 Discussion ................................ ................................ ................................ .............. 49 5 MITIGATING THE IMPACT OF AFRICAN HONEY BEES: DETERMINING HOW MANAGED EUROPEAN HONEY BEE COLONIES AFFECT THE PROPORTION OF AFRICAN DRONES AT CONGREGATION ARE AS ................ 53 Introduction ................................ ................................ ................................ ............. 53 Materials and Methods ................................ ................................ ............................ 55 Results ................................ ................................ ................................ .................... 56 Discussion ................................ ................................ ................................ .............. 57 6 CONCLUSION ................................ ................................ ................................ ........ 64 LIST OF REFERENCES ................................ ................................ ............................... 67 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 73
7 LIST OF TABLES Table page 1 1 Descriptions of the factors hypothesized to contribute to the preservation of African genetics in the feral population in the face of expansion into regions hosting established European honey bee populations. ................................ ...... 29 1 2 Descriptions of methods by which managed European honey bee colonies ................................ ................................ ......................... 31 3 1 The characteristics of drones trapped at DCAs sampling the feral population (> 2.8 km to cd colonies) and DCAs proximal to apiaries of 96 commercial colonies headed by cd queens (0.25 km to cd colonies). Total drone counts are presented for drones with the cd phenotype, an African matriline, and with cd phenotype and African matriline. The percentage of collected African matriline drones that were cd is presented in the % African cd colum n. ............. 44 4 1 Number of colonies that received a FABIS score of > 80% likely African and their designation as hybridized, usurped, or mixed based on mtDNA anaylsis by sample period. Colonies declared as hybridized, usurped, or mixed are only reported at their first discovery. ................................ ................................ ... 52 5 1 Mitochondrial results for drones trapped at DCAs ~ 0.25 km of managed European honey bees or > 2.8 km from any managed colonies, and relative proportions of African and European matriline drones present at each DCA location type are presented. Columnar data followed by different letters are significantly different at P 0.05 (Chi Square test). ................................ .............. 62
8 LIST OF FIGURES Figure page 2 1 Satellite image map demonstrating the distribution of balloon stations within a potential DCA ................................ ................................ ................................ ..... 35 2 2 Williams (1987) drone trap ................................ ................................ .................. 36 2 3 Drone trap elevated 10 m above the ground using a 1.2 m chloroprene balloon at a DCA ................................ ................................ ................................ 37 2 4 Balloon station diagram with parts labeled ................................ ......................... 38 2 5 Collecting trapped drones into a 50ml centrifuge tube of 95% ethanol ............... 39 2 6 Drone trap elevated in a DCA ................................ ................................ ............. 40 3 1 Feral drones trapped at DCAs in central Florida. ................................ ................ 45 5 1 Satellite image of DCA locations in Orange and Osceola counties Florida ........ 60 5 2 2% agarose gel of the PCR RFLP results of 8 drones collected at a DCA >2.8 km from managed colon ies ................................ ................................ ........ 61 5 3 Distribution of African and European matrilines ................................ .................. 63
9 LIST OF ABBREVIATIONS 9 ODA 9 oxo 2 decenoic acid cd Cordovan BP Base Pair DCA Drone Congregation Area DNA Deoxyribonucleic Acid FDACS Florida Department of Ag riculture and Consumer Services mt Mitochondrial PCR RFLP Polymerase Chain Reaction Restriction Fragment Length Polymorphism
10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Master of Science THE I MPACT OF MANAGED EUROPEAN HONEY BEE COLONIES ON THE COMPOSITION OF DRONE CONGREGATION AREAS IN REGIONS WHERE AFRICAN HONEY BEES ARE PRESENT By Ashley Nicole Mortensen August 2013 Ch air: Jamie Ellis Major: Entomology and Nematology Following introduction into Brazil, African honey bees ( Apis mellifera scutellata) rapidly spread through the Americas dramatically changing the South American beek eeping industry. I a im to determine if the management of European honey bees ( A. m. ssp .) can mitigate the impact of African honey bee hybrids in the southeastern United States I conducted a series of studies to access: (1) the frequency of European colony takeover (i.e. usurpation ) by African matrilines, (2) the reliability of a phenotypic mutation, cordovan, as a marker of European ancestry, and (3) the extent to which the management of European colonies can modify the proportion of African matriline drones present at nearby drone congregat io n areas (DCAs). These studies require that DCA s be identified Consequently, I developed an efficient me thod for locating DCAs. Results indicate that: (1) colony usurpation is a rare event and does no t significantly contribute to the Africanization of Eu ropean apiaries, (2) the cordovan phenotype is associated with bot h African and European mitotypes and therefore cannot be u s ed as a reliable indicator of genetic ancestry, and (3) the management of European honey bee colonies can dramatically decrease the proportion of drones with African matrilines at nearby
11 DCAs. Additionally, the results support my assumption that a hybridization zone has developed in central Florida. Further experimentation in this region promises to offer valuable insight into the dynamics of African bee expansion into areas where European bee populations predominate and possible mechanisms contributing to the successful preservation of African phenotypes and matrilines in the population
12 CHAPTER 1 LITERATURE REVIEW African Honey B ees in the Americas In the tropics European derived temperate race honey bees, A. m. ligustica Spinola, carnica Pollmann, mellifera Linnaeus, causcasia Pollmann, and iberiensis Engel (Sheppard 1989b, a) produce small honey yields. In an effort to optimize honey production in the tropics, African honey bees, A. m. scutellata Lepeletier (formerly adansonii (Ruttner 1976) ), were imported to Brazil from Pretoria, South Africa in 1956. In the following year a portion of the origi nal African queens were introduced into the feral honey bee population near Rio Claro, So Paulo, Brazil (Kerr 1967) Subsequently, African derived honey bees have rapidly colonized South, Central, and southern North America at an annual expansion rate of up to 300 400 km a year (Taylor 1977, Buchmann 1982, Hall and Muralidharan 1989) This rate dramatically declined when the bees arrived in the temperate regions of North America Beekeepers in the American tropics have adapt ed to the management of African derived honey bees due to the exceptional performance of th e bee. Subsequently, apiaries were forced into isolated, remote locations and hobbyist beekeeping became almost nonexistent (Michener 1975) However, European derived honey bees will remain the primary subspecies managed by beekeepers in the United States (Taylor 1985) The ability to limit hybridization of managed European derived honey bees with feral African derived honey bees is of tremendous public concern due to anim al and human safety issues. In the United States efforts to minimizing hybridization of managed European derived honey bees are very important
13 Although it is recognized that African derived honey bees in the A mericas are not genetically identical to those in their native range (Schneider et al. 2004c), for conciseness I will refer to both African and European derived honey bees in the Americas as African and European, respectively Persistence of African Genetics African honey bees promptly populate an area with densities of up to 120 feral colonies per square mile within 2 3 years of arrival in a new territory (Taylor 1985) Moreover, b ehavior al and molecular evidence suggest there has been little introgression of European genetics into the feral A frican honey bee population as the African population has expanded (Hall 1992a, Lobo and Krieger 1992, Schneider et al. 2004 c ) Typical African behavioral characteristics, such as aggressive behavior, increased swarm production, and propensity to abscond, are predominant in m anaged and feral colonies within 2 years of African bees reaching an area (Taylor 1985, Sheppard et al. 1991) Furthermore, sampled feral populations in Mexico had no European mitochondrial DNA ( mtDNA ; Hall and Muralidharan 1989 ; Smith et al 1989 ) after the arrival of African bees into the area The lack of European mtDNA suggests an African population that has expanded through unbroken African matrilines i ndicating that any introgression that has occurred is unidirectional, with the mating of virgin African queen s to European dro nes being the only hybrids contributing to the expansion of the feral African population ( Smith et al 1989 ; Hall 1990) Managed when European queens mat e with African drones H owever, monitoring managed apiaries most likely exaggerates the impact of hybridization on the total population (Hall and Muralidharan 1989) T hese crosses contribute little to the success of the African population and may
14 even be a dead end process (Hall 1992b) Several theories, including hybrid dysfunction, increased develo pmental stability, pairwise breeding, temporal isolation, increa sed drone production and drift, increased reproductive rate and African patriline advantage have been developed to explain the predominance of African traits as the population expands into new areas hosting large European bee populations (Table 1 1) Hybrid dysfunction The disparity in gene flow between African and European populations in the Americas could be partially explained by the ex istance of dysfunctional hybrid offpring. Harrison a nd Hall (1993) found that pure African workers had higher mass specific metabolic rates than pure European workers. F urthermore, hybrid offspring African queens mated to European drones and European queens m ated to African drones had lower mass specific metabolic rates that pure European crosses, with hybrids of the European matriline having the lowest mass specific metabolic rates of all crosses. This increase in metabolic rate s uggest s that pure African hon ey bees have a greater flight capacity than European or hybrid bees Which may play a role in the African honey enhanced colony defense, foraging, and dispersal. Moreover, hybrid s with a European matriline preformed worse than hybrids with an African matriline. Indicating that the European matriline is a selective disadvantage in the feral population (Harrison and Hall 1993). Increased developmental stability Variation in the developm e ntal stability of African, European, and hybrid individuals has als o been documented. Schneider et al. (2003) found evid ence of differential developmental stability between pure African, European, and hybrid workers in the form of reduced fluctuating asymmetry of wing shape. African workers had the
15 least amount of asymmetry when compared to pure Europeans and both forms of hybrid. U nlike the metabolic rate findings, hybrids in this study had intermediate levels of wing asymmetry compared to that of the pure African and European crosses. These data did not indicate the presence of hybrid dysfunction. However, African honey be es were found to have increased developmental stability where compared to European and hybrid bees, that may result in increased fitness in African individuals. Pairwise breeding In addition to hybrid dysfunction, pairwise breeding has been hypothesized t o contribute to the preservation of African genetics in the feral population. Kerr and Bueno (1970) discovered e vidence that African and European queens were more likely to mate with drones of their own subspecies. They sampled brood 25 30 days after queen mating and determined that African queens mated with Afri can drones 58% of the time w hile European queens mated with European drones 64% of the time. Moreover, Taylor (1999) noted that in areas with abundant European drones African queens mated primarily with African drones (96 100%). This tendency towards li ke species mating would further promote a feral African population with little introgression of European genetics. Pairwise breeding may be a direct preference or a result of other physiological factors. The only apparent difference in mating behavior Kerr and Bueno (1970) identified was that African drones h ave a faster ejaculation speed. They suggested this may desynchroniz e copulation between African and European reproductives by preventing European queen from receiving sperm from some African drones Temporal isolation Temporal isolation of African and European mating flights, in the form of time of year and/or time of day, may further increase the likelihood of a queen pairing with a
16 drone of her own subspecies. In subtropical Mexico differential dro ne concentrations at drone congregation areas (DCAs) were noted based on season (Quezada Euan and May Itza 2001) Higher frequencies of African drones were present in March and April May had almost equal African and European proportions and European drones were the predominant subspecies i n June and July. Moreover, Taylor and Spivak (1984) theorized that there might be partial time of day isolation between the subspecies. Although this has not been confirmed for reproductive castes, European workers in Braz il are most active from 8 30 10 30 whereas African worker activity peaks in the afternoon (Michener 1975) Drone production and drift Together with the propensity of queens to breed with drones of their corresponding subspecies and potential temporal isolation, African colonies produce high numbers of drones even when resources are limit ed (Rinderer et al. 1987) In contrast, drone production of European colonies is dependent on resource availability and v ery few drones are produced in times of resource sc arcity. Furthermore, African drones are prone to drift between colonies exhibiting a tendency to enter Europea n colonies more so than other African colonies (Rinderer et al. 1985) This is a reproductive advantage for African bees as t here is a linear relationship between the number of migratory drones a colony is hosting and the number of drones that colony will produce. E ach additional migratory drone gained by a colony suppresses drone production of the host colony by 0.65 drones (Rinderer et al. 1987) Conversely, the loss of a migratory drone by its parent colony increases the (losing a single migrating drone results in the production of 0.65 more drones in the parent colony and a reduction by 0.65
17 drones in the colony receiving the migrating drone thus favoring the African parent colony a the DCA by 1.3 drones). in times of resource scarcity combine d with the suppression of European drone production could limit European drone production so severely that a small number of African colonies could produce the majority of drones in the reproductive population (Rinderer et al. 1985, Rinderer et al. 1987) In fact Rinderer et al. (1987) found the majority of drones in European colonies to be African in Western Venezuela further supporting the drift theory Reproductive rate African colonies further dominate the reproductive population by generating more virgin queens than Eu ropean colonies. African honey bees swarm more frequently and over a longer season than European honey bees (Winston 1979) The typical European colony seldom swarms its first year and swarms only once i n the spring of each subsequent year, with an average annual production rate of 0.92 swarms per year (Taylor 1977) In contrast, African colonies have been known to create swarms throughout the year (Taylor 1977) generating smaller swarms every 50 90 days that travel great distances to new nest sites (Winston 1979) These swarms no t only expand the feral African population into new territory but also increase the regional population size further increasing the prevalence of African genetics in that area. Af rican patriline advantage Despite the factors detailed above, i n the event that a n European queen mates with both African and European drones there is a developmental advantage for her daughter queens that have an African patriline ( 1998) When honey bee colonies
18 produce queens, several daughter q ueens are reared to adulthood simultaneously. In general, the first queen to emerge will chew holes into the sides of the cells of the developing queens, prompting the workers to abort the queens. Daughter queens with African patrilines develop faster and emerge before those with European patrilines (Degrandi Hoffman et al. 1998 b ) thus g iving African patriline queens a clear competitive advantage. Moreover, African patriline virgins have increased fighting success within the colony and were observed to pipe more frequently and kill mor e rivals than virgins with a European patriline in the same colonies (Schneider and Degrandi Hoffman 2003) It is hyp othesized that African patriline superiority may be an integral component of the asymmetrical nature of gene flow between African and European populations ( Schneider et al. 2004 c ) Hybridization Zones Studies assessing the reproductive advantages of African Honey bees have occurred in tropical areas, such as in Brazil (Rinderer et al. 1985) and Southern Mexico (Hall and Muralidharan 1989) where European honey bee performance is suboptimal. However, a hybridization zone will develop as the northern and southern African migratory fronts approach more temperate climates where Eu ropean honey bees are better suited (Taylor 1977) European honey bees are adapted for temperate environments and survive long harsh winters by forming a thermoregulatory cluster and feeding on stored honey (Ratnieks 1991) Unlike European honey bees, African honey bees are adapted for semi arid, sub Saharan environments (Sheppard et al. 1991) and clustering has not been documented. Additionally, African colonies have small honey stores as a result of rapid reprodu ctive swarming, (Michener 1975) Rather than overwinter in an area with
19 little or no resource availability, colonies abscond until they find an acceptable site or die (Michener 1975) African colonies migrate beyond the ir overwintering limit during warmer periods but those colonies die or migrate back within their climactic limits during the colder months (Taylor 1985) African honey bees already have reached this limit at the southern edge of their distribu tion in Argentina and Uruguay. In the Argentinian hybrid zone a significant amount of introgression has occurred and both European and African mitotypes are associated with a wide range of genetic markers and morphological characteristics (Sheppard et al. 1991) Furthermore, the predominate mitotypes behind and beyond the African overwintering border have remained predominantly African and European respectively (Sheppard et al. 1991) demonstrating an area of European advan tage beyond the overwintering limit o f the African population. In South America African honey bees appear to be limited to ecotypes in which the average high temperature in January (Argentinian summer) is 19 C (Taylor 1985) By applying this temperature limit to the northern expansion it has be en s peculated that the migratory border, and therefore the hybrid zone in North America will develop around Clinton, North Carolina (Taylor and Spivak 1984) However the African population does not appear to be ad vancing as far into the United S tat es as predicted. Pinto et al. (2005) characterized the nuclear and mitochondrial frequencies of A frican and European honey bees over 11 years as African honey bees expanded int o Texas. Their results describe a hybridized population where both African and European subspecies have made substantial genetic contribution i ndicating the formation of the no rthern hybridization zone. Moreover, the African
20 expansion has been stable in the area of Orlando, Florida since 2005 ( personal comm. David Westervelt, Florida Department of Agricultur e and Consumer Services (FDACS) Apiary Division Assistant Chief ). Therefore, o ther climatic factors such as average rainfall, total available water, and periods of dearth may play a role in limiting the African population Africanization The dynamics of African and European subspecies in the feral population and in mana ged Euro pean honey bee apiaries ar e most easily conceptualized as two distinct events. As discussed above the feral African population has expanded throughout the Americas with little to no contribution by European queenlines However, when the feral Afric an population exists in regions where European queen lines are actively maintained in managed apiaries the r occur (Hall and Muralidharan 1989 ; Smith et al 1989 ) There are three mechanisms by which European apiaries become Africanized ; usurpation, beekeepers collecting feral swarms and colonies, and hybri dization of European queens (Table 1 2) In the first mechanism an African swarms, containing a mated queen, usurp s a European colony by killing the European queen and introducing their own African queen as the laying queen of the colony (Michener et al. 1972) Usurpation is not well understood and has received little study (Schneider et al. 2004c) Moreover, the recorded annual frequencies of usurpation vary from 0 30.3% (Camazine 1986; Danka and Rinderer 1988; Danka et al. 1992; Vergara et al. 1993; Clarke et al. 2002; Schneider et al. 2004a; Schneider et al. 2004c) U surpation occurs quickly and can be difficult to distinguish from a colony that has naturally reared a replacement queen (Taylor 1985) However, dramatic genetic change occurs in the event of colony
21 usurpation (Danka and Rinderer 1988) In fact in this scenario no hybridization occurs and the colony is still truly African Similarly, by the second mode, when beekeepers c ollect feral colonies and swarms in regions hosting an established feral African population, Afr ican colonies can be introduced into the European apiary (Michener 1975, Taylor 1985) The third mode by which European apiaries become Africanized is through virgin European queens mating with African drones, producing hybrid Africanized offspring (Taylor 1985) Although European mtDNA does not contribute to the expansion of the African population (Hall 1992b) pairings of European queen s with African drones do occur. In southern Mexico, managed European colonies wer e sampled fifteen months after the arrival of African Honey bees. Despite having European mtDNA, these colonies had a high frequency of African nuclear markers (Hall 1990) thus revealing that seve ral generations of European quee ns had mated to African drones. r African. Of the three mechanisms described above hybridization of European queens with African drones is the only produced. In regions of the United States where feral African populations exist the management of European honey bees should focus on methods to minimize the chances of European queen s mating with Afric an drones Honey Bee Mating Honey bee queens are polyandrous mating with multiple drones in flight, during a series of flights that occur in the first weeks of her adult life. N umerous experiments have been conducted to determine the typical number of dro nes with which a virgin queen breeds Estimates based on semen volume in the queen reproductive tract
22 ( Woyke 1960) and genetic analysis of offspring (Jensen et al. 2005) both suggest t hat queen most often mate with 7 to 20 drones, w ith 11.6 12.1 matings being the average (Tarpy et al. 2004). Drone Congregation Areas Mature drones make daily flights to reproductive sites called drone congregation areas (DCAs). These DCAs persist throughout the reproductive season in the same locations and recur for many years (Ruttner 1985) In fact, drones of colonies brought to an area from great distances away immediately locate the regional DCAs (Tr ibe 1982) Drone congregation areas were first observed in 1958 (Jean Prost 1958) and comparatively little is known about the determination of DCA loc ations considering our advances in knowledge of other aspects of honey bee biology since that time It generally is believed that drones leave their colony flying towards a depression in the horizon (Ruttner 1966) until they reach a vertical relief such as a tree line or building (Zmarlicki and Morse 1963, Strang 1970) Tribe (1982) suggested the locations of DCAs are based primarily on wind, stating that drones typically fly upwind until encountering a turbulent area that could be cause d by buildings or trees. Thus, DCAs typically occur at breaks in th e horizon like open ings in forest areas, and along tree lines in fields (Ruttner 1985) Formation of a DCA is not dependent on the drone population size or the presence of a virgin queen (Strang 1970) In fact drones are more att racted to the location of the DCA than the virgin queen herself. Drones start their reproductive f l ights and assemble at DCAs about an hour prior to virgin queens beginning their mating flights Moreover, Ruttner (1985) demonstrated that dro nes will stop following a tethered queen once she is moved beyond the boundary of a DCA.
23 Once a virgin queen enters a DCA a drone comet of 20 to 41 drones will form and peruse her about 15 to 30 m above the ground (Gary 1963) Queen mandibular pheromone, specifically 9 oxo 2 decenoic (9 ODA), stimulates initial orientation of drones to the vir gin (Gary 1962), but pursuit of the queen is primarily visual once she is within range of the drones (Gary 1963, Van Praagh et al. 1980, Gries and Koeniger 1996) In fact drones have been observed to form small comets and ch ase other insects and animals in the DCA when queen mandibul ar pheromone is present Surprisingly, there is very little physical contact between the drones in the pursuing comet (Gries and Koeniger 1996) and it is suspected that the queen mandibular pheromone may play a role in drones d istinguishing the queen from other drones (Van Praagh et al. 1980) Mating Flights Drones prefer DCA s closer to their home colony. Few fly distances greater than 1.5 2 km to a DCA (Tribe 1982, Rowell et al. 1992) and most common distance traveled is 0.5 km or less (Rowell et al. 1992) with a recorded m aximum flight distance of 7 km. Th colony genetics and may be relevant in a virgin queens strategy for DCA selection (Koeniger et al 2005) V irgin queens fly further to a DCA than drones from the same apiary to avoid sibling breeding events (Rowell et al. 1992) Due to the haplo diploid sex determination system in honey bees inbreeding results in fertiliz ed individuals that are homozygous at the sex loci. These homozygous individuals dramatically decrease the productivity of a colony by developing into nonviable drone brood rather worker brood (Mackensen 1951).
24 J ensen et al. (2005) determined queens rarel y mate with drones from the same apiary and half of the pairings occurred at mating distances (the cumulative distance > 2.5 km. Supporting observations made by Woyke (1960) in which queens housed in apiaries h osting large numbers of drones had no difference in mating efficiency when compared to queen s house in an apiary 3 km from any drone source. Population Structure of a DCA The drones present at a DCAs are representative of the regional reproductive population because colonies that do not produce drones are unlikely to swarm (Moritz et al. 2003, Kraus et al. 2005) To date reports of the number of colonies contributing drones to an individual DCA vary greatly. In Germany one DCA was found to represent 238 colonies alt hough the DCA was located in close proximity to a large, managed apiary (Baudry et al. 1998) T he estimated number of contributing colonies for each of seven DCAs in Brazil was between 8 and 58. Moreover, South African studies determined there to be betwee n 29 to 36 contributing colonies at both of two DCAs (Shaibi et al. 2008) and another identified 12 during the winter and 72 in the summer (Jaff et al. 2009) T he Brazilian and African DCAs w ere influenced more strongly by feral populations than managed colonies. Collet et al. (2009) observed DCAs in Brazil t o be distributed more continuously than in Europe, which may have led to a lower number of con tributing colonies per DCA. Presently it is unclear if the discrepancy between colony numbers is due to colony dispersal in the area of the DCA, difference in DC A formation by African and European honey bees or some combination of both.
2 5 Synopsis and Objec tives Northern expansion of the African honey bee has stopped in central Florida in the Orlando area ( personal comm. David Westervelt, FDACS Apiary Division Assistant Chief) I suspect the northern hybrid zone has developed in this region as did the southe rn hybrid zone in Argentina. Hybridization zones are particularly interesting because they provide natural laboratories where genetic admixture and selection can be observed (Hewitt 1988) and the Florida honey bee population is ideal location to manipulate the repr oductive population to ensure virgin queens are likely to mate with European drones. Beekeepers have suggested that the management of European colonies in an area will help to minimize the impact of the feral African bee population. The FDACS states that gentle European honey bees are the best defe nse against feral African honey bees and ha s established best management practices for maintaining European honey bee colonies based on this untested assertion (FDACS 2013). European honey bees perform well in high population densities where food is the limiting resource (Ratnieks 1991) whereas African honey bees tend to abscond to areas with plentiful resources (Michener 1975) Considering these differences in strategy it is plausible that large European honey bee po pulations could minimize the siz e of the regional feral African population. Moreover Pinto et al. (2005) noted that the portion of the population wi th more European ancestry was more successful than those with African ancestry in areas of Texas where competi tion for limited resources arose In addition to combating the size of the feral African population methods to minimize the introgression of Af rican genes into managed European colonies in the United States are necessary. P ast research indicates this is possible by increasing the
26 probability of European drones breeding with virgin queens (Loper and Fierro 1991) However, Loper and Fierro (1991) employed 3 drone manipulation s simultaneously and were therefore unable to determine which of the 3 had the most impact : (1) aggressive trapping of fe ral drones, (2) introduction of large numbers of European drones source colonies, and (3) strategic placement of drone source colonies. The overall objective of this thesis is to determine the extent to which the management of European honey bees can mitig ate the impact of African honey bees in the southeastern United States. I will address the overall objective with the following specific aims: 1. Develop an effective technique for locating DCAs 2. Determine if the use of a r ecessive phenotypic color mutation cordovan ( cd ) c an identify European honey bees reliably 3. Determine the frequency at which feral African honey bee swarms usurp managed European honey bee colonies. 4. Determine if the management of European honey bee colonies can increase the proportion of European drones present at nearby drone congregation areas. Due to the dynamics of the honey bee mating system DCAs are ideal locations for assessing the influence of managed colonies on the total regional reproductive population. However DCAs are not read ily ap parent or easily identified. My first aim is to develop a technique that will allow me and future investigators, to readily identify experim ental drone congregation areas. Half of the DCAs I will be sampling at are located close to 96 managed Europe an honey bee colonies and all of these colonies are headed by a cd queen. Cordovan is a recessive color mutation ( Mackensen 1951; Laidlaw et al. 1953 ) that has been used in previous studies as an indicator of European ancestry (Rinderer et al. 1987; Degrandi
27 Hoffman et al. 1998a; DeGrandi Hoffman et al. 1998b; Schneider and Degrandi Hoffman 2002; Schneide r and Degrandi Hoffman 2003; Schneider et al. 2003; Schneider et al. 2004a) However, I suspect that this mutation is also present in the African population and therefor is not a reliable indicator of ancestry. I aim to determine if drones with both African and European matrilines express the cd phenotype in central Florida. Within my overall goal of determining the impact of managed Europ ean honey bees on the African honey bee population it is important that I verify the regional frequency of usurpation events. As discussed above, the reported frequencies of usurpation are highly variable, and i f managed European colonies are frequently usurped by African queens the experimental apiaries could be contributing to the African population rather than minimizing its influence. Finally I will determine the maternal ancestry of drones collected at DCAs located near by these managed apiaries an d from DCA s distant to any managed colonies These results are expected to determine if the managemen t of European honey bees can significantly reduce the proportion of African drones presen t at regional DCA s Thereby the risk of hybridization of European queens would be decreased by increasing the likelihood that virgin queen s attending those DCA s will mate with European drones rather than African drones. Cumulatively, the work presented in this thesis will offer insight into the dynamics of the African an d European honey bee populations with in, what I suspect to be, the hybridization zone at the northern front of the African population in the southeastern United States. My results will provide a foundation from which management practices
28 may be developed t o efficiently utilize European honey bees to combat the introgression of African traits into managed European colonies.
29 Table 1 1. Descriptions of the factors hypothesized to contribute to the preservation of African g enetics in the feral population in the face of expansion into regions hosting established European honey bee populations. Factors involved in preserving African Genetics Description References Hybrid dysfunction Hybrid individuals have lower mass specific metabolic rates that either pure African or European honey bees suggesting some level of hybrid dysfunction Harrison and Hall (1993) Increased developmental stability Fluctuating wing asymmetries suggest that African honey bees have the highest developmental stability. While pure Europea ns have the least and hybrid individuals are intermediate Schneider et al. (2003) Tendency towards pairwise breeding African queens are more likely to mate with African drones even in regions that have an abundance of European drones Kerr and Bueno (1970); Taylor (1999) Temporal isolation of African and European reproductives In Mexico African drones were most abundant in March and April and European drones were predominant in June and July. Further isolation may occur in daily drone flight time by subspecies, as is seen in the worker caste in Brazil. Michener (1975); Taylor and Spivak (1984);Quezada Euan and May Itza (2001) Heightened drone drift and production African drones are prone to drift into non parent colonies, and they are more likely to drift into European colonies than African ones. When hosting drifting drones, a colonies own drive to produce drones is suppressed. Rinderer et al. (1985); Rinderer et al. (1987) A dvantages for virgin queens with an African patriline When rearing new virgin queens, those with African patrilines emerge earlier, pipe more frequently, and kill more rival virgins than queens with European patrilines. Degrandi Hoffman et al. (1998 b ); Schneider and Degrandi Hoffman (2003) Schneider et al. (2004c) High reproductive rate African colonies swarm year round, up to every 50 days. Allowing for increased production of colonies and virgin queens compared to European colonies. Michener (1975); Taylor (1977); Winston (1979)
30 Table 1 1. C ontinued. Factors involved in preserving African Genetics Description References Usurpation of European colonies African swarms (containing a mated queen) invade European colonies, kill the resident queen, and introduce their own queen who begins to lay eggs Michen er et al. (1972); Taylor 1985; Danka and Rinderer (1988)
31 Table 1 2. Descriptions of methods by which managed European honey bee colonies Methods of 'Africanized' Description References Usurpation of European colonies African swarms invade European colonies, kill the resident queen, and introduce their own queen who begins to lay eggs Michener et al. (1972); Taylor 1985; Danka and Rinderer (1988) European Queens mating with African drones Virgin European queens mate with Afric an drones. Over successive generation offspring can be produced that have a high level of African genetics despite European maternal ancestry. Taylor (1985); Hall and Muralidharan (1989); Hall (1990); Hall (1992b) Advantages for virgin queens with an African patriline Queens with African patrilines emerge earlier, pipe more frequently, and kill more rival virgins than queens with European patrilines in the same colony Degrandi Hoffman et al. (1998 b ); Schneider and Degrandi H offman (2003); Schneider et al. (2004c) Introduction of captured African swarms and colonies Beekeepers capture feral colonies and swarms and introducing them into their apiaries Michener (1975); Taylor (1985)
32 CHAPTER 2 SCIENTIFIC NOTE ON A SINGLE USER METHOD FOR IDENTIFYING DRONE CONGREGATION AREAS Throughout the breeding season, d rone s of Western honey bees, Apis mellifera congregate on calm, sunny days with little to no wind at DCAs ( Zmarlicki and Morse 1963; Tribe 1982) Drone congregation areas are specific locations that typically occur in a n open area, free of trees and/or buildings and protected from wind by a vertical relief such as a tree line (Zmarlicki and Morse 1963; Ruttner 1966; Tribe 1982) Moreover, DCAs occur in the same location from year to year and they are found b y successive generations of drones (Tribe 1982; Ruttner 1985) Drone congregation areas are extremely useful in numerous aspects of honey bee research. Identification of a DCA is a prerequisite for many reproductive stud ies of the honey bee, as this behavior cannot be induced in artificial conditions (Schmolke 1977) D rones typically attend the closest DCA to their parent colony ( Koeniger et al. 2005) resulting in a mixture of managed and wild (or feral) colonies at DCA s located close to managed apiaries The dr ones present at any DCA are representative of the regional population allowing for population level a nalysis (Baudry et al. 1998) However, the determining factors for DCA formation are still unclear ( Scheiner et al. 2013 ) Previously published methods for locating DCAs are often labor intensive, requir e multiple researchers, and are less effective in areas with low colony population densities (i.e. feral populations). These techniques include listening for the loud humming of drone flight (Ruttner 1985) monitoring tether ed queens or queen dummies (Ruttner 1985; Muerrle et al. 2007) an d radar monitoring (Loper et al. 1987) We e xpanded upon
33 these methods to develop an efficient technique in which a single investigator can locate a DCA regardless of the regional honey bee population density. Locations meeting the known attributes of DCAs are identified using satellite imaging (for example, Google Earth Pro 7.1) and on site inspection. Once probable locations are selected, up to 6 balloon stations (Fig. 2 1) are evenly distributed throughout the location (between 75 150 m apart) for the duration of the regional drone flight period (see Scheiner et al. 2013 for methods to determine regional drone flight time ) Each balloon station consists of a 1.2 m chloroprene balloon equipped with a Williams (1987) drone trap (baited by a queen lure containing 1 mg synthetic 9 oxo 2 decenoic (9 ODA; Gary 1962; Gries and Koeniger 1996) suspende d below the trap opening (Fig. 2 2). The drone trap is attached 5m below the balloon and the bottom of the trap is between 10 30 m above the ground (Fig. 2 3). The tether line is at tached to a cinder block (Fig. 2 4). A balloon height of 10 m is ideal for initial detection of congregation areas for several practical reasons: (1) drones may not fly at higher elevations in less than ideal weather conditions, (2) a trap at a height of 10 m can be determined visually as empty without lowering the trap, and (3) there is less opportunity for tangling 10 m of line opposed to 30 m when lowing the trap to collect drones. A single investigator can easily visit 6 stations within a single, 20 30 minute interval Durin g inspections, the traps are lowered and all drones present collected (Fig. 2 5) As areas of increased drone activity (more drones being trapped ) become apparent, the balloon stations are relocated towards those areas. Final designation as a DCA can be ma de when d rone comets are observed (Fig. 2 6), drones are present
34 immediately at balloon elevation and > 50 drones are collected in a 20 minute interval (Tribe 1982; Muerrle et al. 2007) The presented method proved to be effective for a single investigator to readily identify DCAs in regions with low population densities, hosting only feral honey b ee colonies, and near large managed apiaries. Further benefits of the current method are that (1) e ach station actively traps drones while unattended, (2) no modification of the balloon station is necessary to collect samples and (3) a single investigator may collect samples at multiple DCAs simultaneously
35 Figure 2 1 Satellite image map demonstrating the distribution of balloon stations within a pot ential DCA Little to no drone activity was observed at all balloon stations except for the station marked by the orange star. This location was determined to be a DCA due to drone comet formation and >50 drones being trapped in 30 minutes. Satellite imaging provided by Google Earth Pro 7.1.
36 Figure 2 2. Williams (1987) drone trap equipped with visual queen dummies (b.) and pheromone queen dummies (c). Pheromone queen dummies are suspended 20 and 30 cm below the middle of the trap opening to attract drones to the area of the trap. Visual dummies are attached throughout the interior of the trap to draw drones (a.) up to the top of the trap. Photograph by Ashley N. Mortensen
37 Figure 2 3. Drone trap elevated 10 m above the ground using a 1.2 m chloroprene balloon at a DCA Note that this DCA occurs along the edge of an open area, near the surrounding tree line. Photograph by Ashley N. Mortensen.
38 Figure 2 4 Balloon station diagram with parts labeled: (a) chloroprene balloon inflated with helium, (b) swivel ho ok attachment to kite line for balloon and trap, (c) Williams (1987) drone trap with queen pheromone dummy suspended below, (d) 22.7 kg kite line, (e) kite reel secured to a cinder (cement) block, weighing 15 kg, with 2 bungee tie cords
39 Figure 2 5. Collecting trapped drones into a 50ml centrifuge tube of 95% ethanol. Suspension of the trap by a helium balloon allows the trap to be quickly and easily lowered for collections. Photograph by Ashley N. Mortensen.
40 Figure 2 6. Drone trap eleva ted in a DCA A comet of drones can be seen entering the trap. Approximately 200 drones are trapped in the top of the trap. Photograph by Ashley N. Mortensen.
41 CHAPTER 3 A SCIENTIFIC NOTE ON THE PREVALENCE OF THE CORDOVAN PHENOTYPE IN THE AFRICAN DERIVED HONEY BEE POPULATION IN THE SOUTHEASTERN UNITED STATES Readily identifiable phenotypic markers, such as color, are useful research tools in an array of disciplines. In the Western honey bee, Apis mellifera Linnaeus, a reces sive phenotype know as cordovan ( cd, Mackensen 1951; Laidlaw et al. 1953) has been used in numerous genetic and behavior studies ranging from the discovery of haplodiploid sex determination to kinship recognition, and spatial dynamics of the mating system (Mackensen 1951; Rowell et al. 1992; Breed et al. 1994) Cordovan individuals express red to brown cuticular coloration in all areas that otherwise would be black (Mackensen 1951) There is considerable variation in the amount of black cuti cular color expressed ordinarily in all subspecies of the Western honey bee. However, even in the most yellow of wild type workers and drones, the sixth abdominal tergum is always black (Tucker 1986) Thus, cd mutants are easily differentiated from their wild type counterparts, allowing them to be informative in laboratory and field studies. With the introduction of African derived honey bees, A.m. scutellata Lepeletier into the Americas (Kerr 1967) investigators have used this marker in a variety of comparative studies on African and European honey bees (Rinderer et al. 1987; DeGrandi Hoffman et al. 1998a; DeGrandi Hoffman et al. 1998 b ; Schneider and Degrandi Hoffma n 2002; Schneider and Degrandi Hoffman 2003; Schneider et al. 2003; Schneider et al. 2004a) However, I suggest that the cd phenotype be used cautiously, particularly as an indicator of European decent, as African and European derived populations in the United States have become increasingly admixed,
42 In June and July of 2012 I conducted a survey in which 400 drones were sam pled from each of 6 drone congregation areas for a total of 2,400 drones collected in Orange and Osceola counties, Florida. Three of the DCAs were located within 0.25 km of 96, 10 frame, commercial European honey bee colonies headed by cd queens. The 3 rem aining DCAs were located > 2.8 km from any managed honey bee colonies. Since drones typically travel < 2.0 km to a DCA (Rowell et al. 1992) drones trapped > 2.8 km from managed colonies were considered representative of the feral population. Drones were trapped via an aerial Williams (1987) trap, baited with a synthetic 9 oxo 2 decenoic (9 ODA) queen lure ( Gary 1962) and suspended 10 30 m above the ground by a ch loroprene balloon (see Chapter 2 ). The y then were preserved in 95% ethanol for transport to the laboratory where they were preserved at 80 C until molecular processing. Total DNA was extracted from each drone with 10% Chelex extraction (Walsh et al. 1991) and maternal ancestry determined using a PCR RFLP technique for a mitochondrial Cytochrome b gene diagnostic marker (Crozier et al. 1991; Pinto et al. 2003) Additionally, drones were determined visually to be wild type or cd In total, 829 cd drones were collected.125 drones were trapped at DCAs >2.8 km from any ma naged honey bee colonies (Table 3 1). Fourteen of the 125 i ndividuals had African mtDNA (or 3.4%). Moreover, 7 of 22 (or 31.82%) of the African matriline drones trapped at DCAs near managed colonies (0.25 km) were cd These data indicate that the cd mutation has been integrated into the feral population. Furthermore, the data show that the cd phenotype is associated with both African and European matrilines (Fig. 3 1).
43 In the state Florida t he African honey bee population not expanded northward beyon d its current limit in the central region of the state since 2005 ( personal comm. David Westervelt, FDACS Apiary Division Assistant Chief ) I suspect the feral population is highly hybridized in this region, as seen at the southern front in Argentina (Taylor 1977; Sheppard et al. 1991) Moreover, cd queens are becoming more popular in commercial and hobbyist apiaries, cumulatively leading to the incorporation of the cd phenotype into the feral A frican population in the southeastern United States. Under carefully controlled experimental designs, the cd allele will continue to be an informative research tool. However, I advise that care be taken to ensure feral cd individuals are not misinterpret ed within experimental conditions in regions where beekeepers maintain cd queens and feral African colonies exist.
44 Table 3 1. The characteristics of drones trapped at DCAs sampling the feral population (> 2.8 km to cd colonies) and DCAs proximal to apiar ies of 96 commercial colonies headed by cd queens (0.25 km to cd colonies). Total drone counts are presented for drones with the cd phenotype, an African matriline, and with cd phenotype and African matriline. The percentage of collected African matriline drones that were cd is presented in the % African cd column. DCA Proximity to managed colonies no. drones collected Total no. collected that were cd Total no. collected having an African Matriline Total no. cd collected having an African Matriline % total no. having an African matriline collected that were cd (column 5/column 4) >2.8 km to cd colonies 1200 125 412 14 3.40% 0.25 km to cd colonies 1200 704 22 7 31.82% Overall Totals 2400 829 434 21 4.84%
45 Figure 3 1. F eral drones trapped at DCAs in central Florida. European (a., b., c., d.) and African (e., f., g., h.) drones demonstrating cd mutation (a., b., e., f.) and wildtype (c., d., g., h.) cuticular colorations. Photograph by Lyle Buss.
46 CHAPTER 4 USURPATION OF MANAGED EUROPEAN DERIVED HONEY BEE COLONIES VIA AFRICAN MATRILINE SWARMS IN THE SOUTHEASTERN UNITED STATES Introduction Since their introduction in 1957 (Kerr 1967) African honey bees have rapidly colonized the western hemisphere This expansion has occurred via unbroken African matrilines (Hall and Muralidharan 1989 ; Smith et al 1989 ) with African bees r eplacing feral European hon ey bees in many regions. Probable mechanisms contributing to the dominance of the African matriline are thought to be rapid colony growth, high swarming rates (Schneider et al. 2004b ) frequent absconding, long distance swam movement (Ratnieks 1991) hybrid dysfunction (Harrison and Hall 1993) queen developmental time (Taylor 1999) drone mating advantage ( Schneider et al. 2004b; Schneider et al. 2004c) and nest usurpation (Michener 1972; Taylor 1985) The latter occurs when an intruding swarm (containing a mated queen) enter s an occupied colony, kills the resident queen, and introduces their own queen who begins to lay eggs (Michener 1972) Usurpation is not well understood. There has been little systematic study of the behavior (Schneider et al. 2004c) and annual frequencies are reported to vary from 0 30.3% (Camazine 1986; Danka and Rin derer 1988; Danka et al. 1992; Vergara et al. 1993; Clarke et al. 2002; Schneider et al. 2004a; Schneider et al. 2004c) Moreover, usurpation events typically are subtle and diff icult to distinguish from hybridization of a (Taylor 1985) However, dramatic genetic change occurs in the event of colony usurpation opposed to hybridization of a naturally reared replacement queen (Danka and Rinderer 1988)
47 In the present study, we aim to determine the extent t o which usurpa tion of managed European colonies contributes to the perpetuation of African matrilines at the north ern front of the African honey bee expansion in Florida, USA. The African expansion has been stable in central Florida since 2005 ( personal co mm. David Westervelt, FDACS Apiary Division Assistant Chief), and mtDNA analysis has confirmed the African matriline in this region (unpubl. data). We expect that usurpa tion of managed European colonies by African matrilines occurs at low frequencies in co mmercially managed apiaries, and does not perpetuate African matrilines in this region significantly. Materials and Methods We utilized a combination of morphometric and mtDNA analysis (Danka et al. 1992) to monitor up to 288 commercial European honey bee colonies distributed between 5 apiaries in Orange and Osceola Countie s (two apiaries of 96 colonies each and 3 apiaries of 32 colonies each) from December 2011 December 2012. During this time period, all colonies were managed in accordance with FDACS best management practices for maintaining European honey bees ( FDACS 2013 ) This includes requeening colonies in instances of queen failure, excessive defensive behavior, poor colony productivity, etc. In December of 2011, approximately 50 workers were collected from the comb of the uppermost box (or super) of each colony. Sampl es were collected in 95% ethanol and transported to the laboratory where forewing lengths of 10 randomly selected individuals per colony were morphometrically analyzed via the Fast African Bee Identification System (FABIS, Sylvester and Rinderer 1987) Any colony that receiv ed a FABIS score of > 80% likely to be African was requeened to begin the experimental
48 period. In March, June, September, and December of 2012, all colonies were resampled and assigned a FABIS score as described above. No requeening was directed by 2012 FABIS results and apiary managers were blind to the results. Each colony receiving a FAB IS score of > 80% likely African was analyzed further for African matriline using a Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR RFLP) technique (Crozier et al. 1991; Pinto et al. 2003) Colonies u h European derived queens from the original colo nies mating with African drones. Finally, m inconclusive category). The numbers of usurpation and h ybridization events for each sample period were totaled and the frequency o f each calculated based on the total number of queenright. If a colony showed hybridization or usurpation in successive sampling periods, its status was counted at its first discovery. Results The following data are detailed in Table 4 1: (1) the total num ber of live, queenright colonies, (2) the number of those colonies that received a FABIS score of > 80% likely to be African, and (3) the mtDNA designation of the colonies in point 2 for each sampling period. All African derived colonies identified at the June, September, and December sampling periods (27 total colonies) were classified as hybridization events rather than usurpation by a swarm headed by a queen with an African matriline. In March, 1 colony had both African and European mtDNA present at the time of sampling. However, this colony received a FABIS score of 16.8% likely African at the next sample period in June (well below the experimental threshold of > 80%). The other
49 22 African colonies from the March sampling were classified as hybridized. H ybridization rates for each sample where as follows; March 7.67%, June 4.10%, September 3.96%, and December 1.99%. No colonies showed evidence of usurpation. Discussion Previous usurpation research has focused on areas with established African hone y bee populations ( Danka and Rinderer 1988, Danka et al. 1992, Vergara et al. 1993 Schneider et al. 2004a, Schneider et al. 2004c ). The role of usurpation in the of the r egional European population are not well understood. We found no evidence that usurpation plays a significant role in the perpetuation of African matrilines at the northern front of the African honey bee expansion in the Southeastern United States. In fact our study indicates that apiary management practices and the lack of usurpation events resulted in no perpetuation of the African matriline in our studied apiaries. Hybridization of European matrilines was identified at each sample period, but no clear e vidence of usurpation by an African matriline was found. It is possible that the m 4 1) could have represented a recently usurped colony that had not fully replaced the worker population at the time of sampling. Pote ntially, the colony was requeened by the commercial apiary managers in accordance with the FDACS best management practices as it began to express more phenotypically African behavior, i.e. heightened defensive behavior (Michener 1972) This would have resulted in the low FABIS score recorded at the next sample collection in June.
50 During the study period, the hybridization rate appears to follow the honey bee swarming season with the highest rate in March. Then the frequency of hybridized colonies declines through June, September, and December. European colonies typically swarm once a year in the spring (Taylor 1977). At which time the original colony produces a vigin qeen to mate with regional drones. As more virgin qeueens are likely to be reared during the swarming season it is likely that the instances of hybridization would also increase. Interestingly, Quezada Euan and May Itza (2001) found a similar temporal pattern in the abundance of African drones present at drone congregation areas in subtropical Mexico They found the ratio of African to European drones present a t drone congregation areas was highest in March and decreased over the swarming season until the end of their sampling period in July. It is possible that higher African drone abundance during the early months of the swarming season may have led to an incr eased rate of hybridization of virgin queens produced by swarming colonies in our apiaries. Further investigation, incorporating multiple swarming seasons, is needed to determine if seasonal variation in subspecies abundance exists at DCAs in the southeast ern United States as does in the Yucatan. In the present study, it is possible that usurpation by hybridized queens possessing a European matriline were classified as hybridized rather than usurped. However, the focus of our study was to determine the cont ribution of usurpation specifically to the perpetuation of the African matrilines. Further stud ies integrating queen marking and monitoring of s upers edure, swarming, and beekeeper mediated
51 requeening should be conducted to expand our understanding of usurp Africanization process. The central Florida honey bee population offers a unique opportunity for understanding the interactions of African and European honey bee populations and the Afri canization process. We suspect substantian h ybridization is occurring in this region comparable to the southern front in Argentina (Taylor 1977; Sheppard et al. 1991) Further experimentation promises to offer valuable insight into the dynamics of the African and European honey bee populations and possible mechanisms contributing to the successful preservation of African phenotypes and matrilines in the population.
52 Table 4 1. Number of colonies that received a FABIS score of > 80% likel y African and their designation as hybridized, usurped, or mixed based on mtDNA anaylsis by sample period. Colonies declared as hybridized, usurped, or mixed are only reported at their first discovery. Sample Date Colonies Sampled > 80% FABIS Hybridized Usurped Mixed % of colonies hybridized March 287 23 22 1 7.67 June 268 11 11 4.10 September 278 11 11 3.96 December 251 5 5 1.99
53 CHAPTER 5 MITIGATING THE IMPACT OF AFRICAN HONEY BEES: DETERMINING HOW MANAGED EUROPEAN HONEY BEE COLONIES AFFECT THE PROPORTION OF AFRICAN DRONES AT CONGREGATION AREAS Introduction Following their introduct ion into Brazil, African honey bees rapidly spread through the Americas, dramatically changing the South American beekeeping industry ( Michener 1975 ) I n South America, managed African honey bees outperform European honey bees and quickly became the predominate subspecies upon enter ing new territory that was suitable for their habitation (Michener 1975 Hall and Muralidharan 1989 ) African honey bees are better adapted to the tropical environment of s outh and central America than European honey bees Furthermore, they outperform European honey bees in terms of survivability and honey production (Michener and al. 1972) However, d ue to undesirable traits, like the Af behavior, hobbyist beekeeping became nonexistent and managed apiaries were relocated to isolated locations (Michener 1975) African genetics now predominate honey bee populations in the American tropics d espite the efforts of beekeepers and local agenc ies to conserve favorable European traits (Hall and Muralidharan 1989 ; Smith et al 1989 ) As seen in South America it is important to the United States beekeeping industry that the introgression of African traits into European populations be limited (Taylor 1985) In Argentina, at the southern edge of the African expansion, a hybridizatio n zone has formed In the Argentinian hybridization zone substantial admixture occurs between the African and European populations (Sheppard et al. 1991) The northern e xpansion of the African honey bee population has halted in central Florida and been stable in this
54 region since 2005 ( personal comm. David Westervelt, FDACS Apiary Division Assistant Chief ). I suspect a hybri dization zone, compa rable the one in South Amer ica (Taylor 1977, Sheppard et al. 1991) has developed in central Florida T echn iques to limit the introgression of African genetics into the managed European honey bee population in the United States sh ould be focused in this region As the African population expanded into Texas it was noted that when competition for limited resources arose the portion of the population with more European ancestry was favored over those with African ancestry ( Pinto et al 2005) European honey bees are ada pted to and perform well in high population densities where food is the limiting resource (Ratnieks 1991) while African honey bees tend to abscond to areas with plentiful resources (Michener 1975) It has therefore been hypothesized that the management of European honey bees may itself be a method of control for the African honey bee population in the United States. In addition to creating unfavorable envir onmental conditions for African colonies saturation of the enviro nment with European colonies may also modify the regional reproductive population so that European queens are most likely to mate with European drones rather than hybridizing with African drones. H ybridization of managed European queens is the primary source of African genetics in managed European apiaries (Hall and Muralidharan 1989) M apiaries s hould focus on techniques to modify the abundance of African drones present at DCAs t hereby increasing th e probability that virgin European queens will mate with European drones (Loper and Fierro 1991)
55 Loper and Fierro (1991) monitored drones attending DCAs in Chiapas, Mexico and determine that the simultaneous implementation of three dr one techniques effecti vely manipulate d the regional reproductive populatio n. In the Loper and Fierro (1991) study, they; (1) aggressive ly trapped and removed feral drones prior to introduction of European drone source (2) introduced a large number of European drones to flood the regional population and (3) strategic ally placed introduced drone source colonies. However, d ue to the experimental design they were unable to ascertain which factor if any was most impactful. In the present study I aim to determine if the management of European honey bees can increase the proportion of African drones present at nearby drone congregations areas dramatically I expect that the management of European colonies will result in significantly more European drones present at near by DCAs. Materials a nd Methods Drones were collected in June and July of 2012 from 6 DCAs in Orange and Osceola counties, Florida. Three DCAs were located within 0.25 km of 96 10 frame commercial European colonies that had been est ablished in March of 2011 and the 3 remaining DCAs were > 2.8 km from any managed colonies (Fig. 5 1) Since drones typically travel < 2.0 km to a DCA (Rowell et al. 1992) drones trapped > 2.8 km from managed colonies were cons idered representative of the feral population. Drone collections occurred through the duration of the flight time via a Williams (1987) drone trap equipped two queen lure s baited with 1 mg of synthetic 9 oxo 2 decenoic ( 9 oda Gary 1962, Gries and Koeniger 1996) The trap was suspended below a white 1.2 m helium inflated, chloroprene balloon and e levation was determined by the dist ance from the bottom of the trap opening to the ground (see Chapter 2 ) Four
56 hundred drones were collected from each of the 6 DCAs Wit hin each DCA drones were caught at 2 heights: 200 drones from 10 m and 200 from 30 m. Collected drones were preserved in 95% ethanol and transported to the laboratory for storage at 80 C Total DNA was extracted individually from a hind leg of each drone with 10 % Chelex technique (Walsh et al. 1991) and maternal ance stry determined by digesting an amplified p ortion of the mitochondrial c ytochrome b gene with a Bgl II restriction enzyme (Crozier et al. 1991, Pinto et al. 2003) In individuals of European de s cent the Bgl II digestion cleaves the amplified 485 BP (base pair) c ytochrome b fragment into two, 194 and 291 BP fragments. African matriline lindividials do not have a Bgl II restriction site in the c ytochrome b fragment resulting in an intact 485 BP fragment that is easily d iscriminated from the banding pattern of European individuals whe n visualized under UV light on an ethiduim bromide stained 2% agarose/Tris acetic acid EDTA gel (Fig. 5 2 ) The proportions of African and European drones collected were analyzed by proximity to managed colonies and height within the DCA using Chi Square Analysis Results Mitochondrial results indicate d that DCAs distant to managed European apiaries had significantly higher prop ortion (p<0.0001) of African matriline drones (34.33% of the collected individuals) than did D CAs located close to managed European apiaries (1.83% of the collected individuals; Table 5 1). However, there was not a detectible difference in the vertical di stribution of African or European drones within the DCA (Fig. 5 3 ).
57 Discussion The data suggest that the mana gement of European colonies can inf luence the proportion of drones at DCAs that have an African matriline This is most likely the result of managed European colonies producing large numbers of drones that flooded the proximal DCAs. However, it is possible that the management of large European apiaries sa turated the environment, encouraging feral African colonies to abscond to areas with more resource availability. The latter cann ot be determined by mtDNA alone However, f urther investigation inco rporating nuclear analysis of closely linked microsatellites will allow for quantification of the regional population (Kraus et al. 2005) This will b e useful for determining if there are fewer African colonies in the proximity of the manage d apiaries. Regardless of the unde rlying cause for the changes seen in DCA composition, the data suggest that the managemen t of European colonies can be considered a viable option for limiting the introgression of African genetics. P aternal gene flow by way of European queens mating with A frican drones is the form of Africanization in managed European populations (Hall and Muralidharan 1989) Increasing the proportion of European drones present at a DCA will increase the likelihood that virgin queens at that DCA will mate with European, rather than African, drones (Loper and Fierro 1991) It appears this can be accomplished managing European bees in an area. Virgin queens fly to more distant DCAs than do drones from the same a piary. Drones typically travel < 0.5 km to a DCA whereas queens are estimated to travel 2.0 km to a DCA (Rowell et al. 1992, Jensen et al. 2005) Further research is needed to determine the concentration and distribution of managed European colonies needed to
58 modify the regional reproductive population to the extent that virgin queens produced in apiaries would themselves atten d a DCA with a high proportion of European drones. Past studies have indicated the existence of a slight tendency towards like subspecies mating where African and European queen s mated with drones of their same subspecies 58% and 64% of the time respective ly (Kerr and Bueno 1970b) Several theories including partial physiological barriers and temporal isolation exist to explain their tendencies (Kerr and Bueno 1970a, Taylor and Spi vak 1984, Hall 1992, Quezada Euan and Jesus May Itza 2001) In the present study drones were collected from both 10 and 30 m elevations to determine if there is a vertical distribution of African and European subspecies within t he DCA. However, no detectable difference in vertical flight behavior of African and European drones was observed. Th e frequency of African mtDNA 34.3% (Table 5 1) observed in the feral population (drones collected at DCAs > 2.8 km from managed colonies) supports the belief that significant hybridization is occurring in central Florida. African honey bees were confirmed in Orange and Osceola counties in 2005 via morphometric analysis ( personal comm. David Westervelt, FDACS Api ary Division Assistant Chief ), but have not expanded northward since that time. Studies of African mtDNA frequencies in predominantly African regions have reported between 83 88% African mtDNA (Sheppard et al. 1991, Diniz et al. 2003) and 69% of samples from the southern edge of the hybridization zone in the southwestern United States had African mtDNA (Pinto et al. 2005) I suspect the southeastern hybridization zone may exist in central Florida However, since the African population in Florida is not a part of the continuous African expansion, further investigation is requ ired to dete rmine if this is truly a hybridization
59 zone or if south /central population. The central Florida honey bee population offers a unique opportunity for understanding the interactions of African and E uropean honey bee populations and the Africanization process. Furthermore, experimentation in this region promises to offer valuable insight on possible population manipulation techniques to limit the introgression of undesirable African genetics into the manage European honey bee population.
60 Figure 5 1. Satellite image of DCA locations in Orange and Osceola c ounties Florida. Satellite imaging provided by Google Earth Pro 7.1.
61 Figure 5 2 2% agarose gel of the PCR RFLP results of 8 drones collecte d at a DCA >2.8 km from managed colonies. A single band of approximately 485 BP indicates African mtDNA (lane numbers: 1., 4., 5.), and 2 bands of approximately 194 and 291 BP indicate European mtDNA (lane numbers: 2., 3., 6., 7., 8.). 100 BP size marker can be seen on the far right lane, and lane 9 represents a negative control
62 Table 5 1 Mitochondrial results for drones trapped at DCAs ~ 0.25 km of managed European honey bees or > 2.8 km from any managed colonies, and relative proporti ons of African and European matriline drones present at each DCA location type are presented. Columnar data followed by different letters are significantly different at P 0.05 (Chi Square test). DCA locations Total no. drones c ollected Total drones collected with European mtDNA Total drones collected with African mtDNA % of total drones collected with European mtDNA % of total drones collected with African mtDNA 0.25 km from apiary 1200 1178 22 98.17% 1.83% a >2.8 km from apiary 1200 788 412 65.67% 34.33% b
63 Figure 5 3 Distribution of African and European matrilines. Total number of drones expressing an African or European matriline are presented based on the proximity of the DCA to managed European colonies and vertical distribution within that DCA from which they were trapped 0 100 200 300 400 500 600 700 African mtDNA European mtDNA 0.25 km at 10 m 0.25 km at 30 m >2.8 km at 10 m > 2.8 km at 30 m Matriline distribution based on DCA proximity to managed European colonies and height within the DCA Number of Drones
64 CHAPTER 6 CONCLU SION Cumulatively the experiments presented in this thesis confirm morphometric indications that the feral African honey bee population is established as far north as Orange and Osceola counties in central Florida. Furthermore, the frequency of Af rican mtDNA in the feral population and the occurrence of the cd phenotype in individuals with African matrilines suggest high levels of hybridization in the feral population. This region promises to provide a natural laboratory in which introgression of t he African and European honey bee populations can be studied (Hewitt 1988) Moreover, DCAs provide distinct s ites where populatio n level samples can be collected and the methods described herein allow for relatively simple identification of these location s. The studies revealed that the maternal introgression of African genetics into managed European colonies, by way of usurpation, does not appear to contribute to the Africanization of managed European apiaries. It is more likely that African matrilines ar e incorporated into managed colonies by b eekeepers collecting feral colonies and swarms (Michener 1975, Taylor 1985) In the case that beekeep e rs do not introduce African queenline s into their apiaries, paternal gene flow is the primary source of African g enetics observed in managed European colonies (Hall and Muralidharan 1989) Therefore techniques to limit the probability of virgin European queens mating with African drones will be the most influential means of Europe an honey bees in the United States. Furthermore the data suggest that the management of European honey bees can decrease the proportion of African drones present at nearby DCAs. However the minimum density and most effective distribution of European colonies to duplicate these
65 results on a large scale are still unclear and may, in fact, be unrealistic Most reasonably European colonies could be used to mitigate the African population in areas that are most sensitive to the presence of African honey bees such as popular tourist destinations or areas in which honey bee pr oducers are rearing and breeding virgin queens, rather than as a first line of defense throug hout the state. At this point it is unclear what t he minimum density of colonies needed to alter the proportion of African drones present at nearby DCAs is. The la rge European apiaries in these studies were most likely saturating the environment However, seemingly high numbers of managed European colonies may be necessary to modify the proportion of African drones present a t near by DCAs due to the African honey bee drone production advantages: (1) African colonies begin producing drones earlier in the breeding season (Quezada Euan and May Itza 2001) (2) African drones have a higher tendency to drift to non parent al colonies, (3) African drones are most likely to dr ift into European colonies, and (4) hosting drifter drones suppresses a drive to produce their own drones ( Rinderer et al. 1985) These factors cumulatively create an environment where a small number o f African colonies could produce the majority of the regional drones and European colonies could host only African drones ( Rinderer et al. 1985, Rinderer et al. 1987 ) Moreover, further study is necessary to determine what this ideal distribution of these European apiaries would be. Ideally, apiaries should be established in a way to ensure that virgin queens produced in drone source apiaries would themselves attend a DCA with a high proportion of European drones. Previous research has demonstrated that the majority of drones fly less that 0.5 km from their colony to a DCA ( Rowell et al.
66 1992). It is likely that an appropriate distribution of colonies would be approximately 1 km between apiaries so the area of dispersal from one a piary would reach to the area of dispersal of the next. However, addition al investigation is n eeded to determine the actual area over which a European apiary can modify the reproductive population The high level of population manipulation this type of management would likely result could further decrease the influence of Afric an genet ics by increasing the probability of virgin African queens mating with European drones, and/or causing European genetics to be selected for in the population due to resource limitation (Pinto et al. 2005). I used a mitochondrial technique to diagno sis maternal ancestry in the present studies. Regrettably, mtDNA provides no information on th e paternal genetic contribution and no hybridization can be identified. Recognizing that these experiments occur in a hybridization zone, where one expect s to se e high levels of introgression, further analysis of the nuclear genome promises to offer more insight into the interactions of the regional African and European populations.
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73 BIOGRAPHICAL SKETCH Ashley Mortensen graduated from Texas A&M University with a B.S. in animal science. She worked in z oological facilities as a primate and carnivore keeper for several years. Later, Ashley conduct ed respiratory physiology research as a biological scientist in a lab oratory at the University of Florida She earned a M.S. in entomology and nematology from the University of Florida, and will be continuing her education with a PhD in entomology and nema tology from the University of Florida.