GENOMIC DNA RESTRICTION FRAGMENT LENGTH POLYMORPHISMS AT A HIGHLY POLYMORPHIC LOCUS DISTINGUISH OLD AND NEW WORLD SUBSPECIES OF THE HONEY BEE, Apis mellifera L. By MARGARET ANNE MCMICHAEL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1994 ACKNOWLEDGMENTS I am grateful for the efforts of so many people, and I am bothered by the omission of many names from these two pages. My thanks go to all who have helped me. First and foremost, Glenn Hall took a chance on me and has been patient and generous in providing the opportunity, direction, and freedom I have enjoyed in the course of obtaining my doctorate in his lab. The members of my advisory committee provided tremendous assistance; Andy Cockburn, Jim Maruniak, and Buffy Bondy. Don Campton generously donated his time in the initial stages of my research, and Jan Conn reviewed my manuscripts and postdoctoral research proposal. Chip Taylor provided a memorable week in Linnares, Mexico, and taught me the zen of smoker-lighting. Lois Lemmerman Myeroff, Case Western Reserve University, is responsible for teaching me all I knew about molecular biology when I was in the Mapstone/Goldthwait lab and as such was instrumental in my move to Florida. Aléjandra Garcia and Raquel McTeirnan have been immeasurably helpful to me here, and my way would have been rough without their tireless and unselfish assistance. Marjorie Hoy's lab members-particularly Owain Edwards (who also reviewed my manuscripts), Jim Presnail, Dr. Jey, and Greg ii McDermott-have been instrumental in advancing my research efforts. Reg Coler, Scott Yocom, and Owain Edwards helped me make slides for the meetings and for my departmental seminar. Dr. Strayer, Faith Oi, and Hugh Smith taught me a few things during one of my best experiences here; assisting in Principles of Entomology. Don Hall, Tom Dykstra, and Robin Goodson provided invaluable opportunities for me to lead their classes and tours of the Bee Lab. Scott Yocom recruited me for the Linnean Team, which was an exciting, not to mention a humbling, experience. Deserving additional, special mention are John Strayer and the great students he recruited for the department, with whom I have had the honor and pleasure to work. in TABLE OF CONTENTS ACKNOWLEDGMENTS ii ABSTRACT v INTRODUCTION 1 IDENTIFICATION AND GEOGRAPHICAL DISTRIBUTION OF ALLELES AT LOCUS 178 5 Introduction 5 Materials and Methods 6 Results 8 Discussion 30 ALLELE FREQUENCIES AT LOCUS 178 REVEAL HYBRIDIZATION OF EUROPEAN AND AFRICAN BEES IN THE NEOTROPICS 36 Introduction 36 Materials and Methods 36 Results 40 Discussion 58 LOCALIZATION OF VARIATION AT LOCUS 178 IN Apis mellifera (L.) BY RESTRICTION MAPPING 65 Introduction 65 Materials and Methods 67 Results 69 Discussion 93 CONCLUDING REMARKS 99 LITERATURE CITED 103 BIOGRAPHICAL SKETCH 111 iv Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy GENOMIC DNA RESTRICTION FRAGMENT LENGTH POLYMORPHISMS AT A HIGHLY POLYMORPHIC LOCUS DISTINGUISH OLD AND NEW WORLD SUBSPECIES OF THE HONEY BEE, Apis mellifera L. By Margaret Anne McMichael April 1994 Chairperson: H. G. Hall Major Department: Entomology and Nematology Honey bees (Apis mellifera L.) of African and European ancestry were distinguished by analysis of restriction fragment length polymorphisms (RFLPs) defined by two enzymes at a highly polymorphic locus corresponding to genomic probe pB178. Thirty-six Msp\ restriction fragment patterns, or variants, and thirty-three DdeI variants were identified. Sixty-five pairwise combinations of the Mspl and Dde I variants, referred to as alleles, were found among the individual drones tested. Variants and alleles were discontinuously distributed in USA and South African drones: only one Dde I variant and no Msp\ variant (hence no allele) were common to these two sample populations. The diversity in the variants and alleles found in the South African drones was v greater than in the USA drones. Msp\ variants were discontinuously distributed among workers bees of the subspecies A. m. mellifera (west European), A. m. Hgustica and A. m. caucásica (east European), and A. m. scutellata (South African). Ancestry in New World bees was inferred from variant and allele frequencies at this locus. In USA bees, variants characteristic of east and west European bees were found at frequencies consistent with previously identified nuclear and mitochondrial DNA markers. In neotropical drones, European and African origins were assumed for variants that were similar in fragment pattern to variants in the USA and South Africa, respectively. The alleles identified in the majority of neotropical colonies were African. There was little evidence of hybridization of African and east European bees in neotropical bees: variants specific to South African bees were detected at the highest frequencies, while east European-specific variants were absent or detected at very low frequencies. A variant that may be specific for A. m. mellifera was found throughout the neotropics at frequencies that varied from 19% to 33% but did not increase clinally in a northward direction. Determination of the allelic relationships between Msp\ fragments and between DdeI fragments was initiated by mapping these restriction sites within the probe, and correlating fragments on Southern blots to the locations of restriction sites on the resulting map. VI INTRODUCTION Descendants of ancestral honey bees migrated from the site of their origin, presumed to be northeast Africa or the Middle East, and became widely distributed in the Old World (Garnery, Cornuet & Solignac 1992). Subsequent adaptation to a wide variety of ecological conditions resulted in the evolution of subspecies of the honey bee, Apis mellifera L. (Ruttner 1988). These subspecies are distinguished on the basis of physical, behavioral, and ecological criteria and are defined quantitatively by discriminant analysis of morphological characters (Daly & Balling 1978; Ruttner 1988; Ruttner, Tassencourt & Louveaux 1978). Honey bees were first introduced to the New World by 17th and 18th century European settlers. For over two hundred years, the genetic diversity found in New World honey bees resulted from the importation of primarily four European subspecies or races: west and north European A. m. mellifera (referred to here as west European); north Mediterranean A m. ligustica Spinola and A. m. carnica Pollmann (which in this report will be called east European; Ruttner 1988); and the east European bee A m. caucásica Gorbachev (referred to here as east European) (Kent 1988; Kerr, DeLeon & Dardo 1982). 1 2 Despite abundant nectar sources in the neotropics, self-sustaining feral populations of European bees did not become established (Michener 1975). The poor performance of European bees in the tropics has been attributed to their failure to adapt to environmental conditions vastly different from those in which they evolved (Ruttner 1988). To improve commercial honey production, queens of a central and south African race, A. m. scutellata Lepeletier, were imported into Brazil in 1956 for experimental breeding with European bees. Swarms of African bees escaped into the wild (Kerr 1967) and subsequently proliferated to form large feral populations that spread through tropical South and Central America. African bees entered Texas in 1990 and Arizona in 1993. The release and spread of African bees has been disruptive to the behavior and ecology of the melliferous flora and fauna in the neotropics (Cantwell 1974; Michener 1975; Roubik 1980, 1989; Spivak, Fletcher & Breed 1991; Taylor 1977; Winston, Taylor & Otis 1983). Explanations for the migration pressure resulting from the introduction of the bees from Africa and for the rapid colonization of New World territories previously unoccupied by honey bees have focused on the extent to which neotropical bees represent an admixture of African and European subspecies (Hall 1990, 1991, 1992a; Hall & Muralidharan 1989; Rinderer 1986; Rinderer et at. 1985, 1991; Sheppard et al. 1991; Smith, Taylor & Brown 1989; Taylor 1985). The ability to distinguish honey bee subspecies is essential to ascertain the extent of hybridization. In turn, the degree of hybridization will influence the northern limit of African bee 3 introgression in the USA, about which there is considerable debate (Dietz 1986; Dietz, Krell & Pettis 1986; Rinderer 1986; Roubik 1986; Southwick, Roubik & Williams 1990; Taylor 1977; Taylor & Spivak 1984; Villa, Gentry & Taylor 1987; Villa, Rinderer & Collins 1993). The retention of African morphology and behavior in neotropical bees has stimulated the quest for the identification of their ancestry, which will aid in understanding the mechanisms contributing to their phenomenal success. Efforts to ascertain the extent of interaction between the extant European bees and the African bees in the neotropics have been hindered by a lack of genetic markers specific for each Old World subspecies introduced. Intermediate values of morphometric values and allozyme frequencies, taxonomic characters traditionally used to identify honey bee subspecies and subspecies groups, can only suggest that hybridization between races has occurred (Daly 1991; Del Lama et al. 1988; Nunamaker et at. 1984; Spivak et at. 1988; Sylvester 1982, 1986). Thus, supplemental genetic characters subject to little environmental modification have thus been sought. Mitochondrial DNA (mtDNA) has been used effectively to identify honey bee subspecies within populations in biogeographic studies (Arias, Soares & Nobrega 1990; Cornuet & Garnery 1991; Garnery, Cornuet & Solignac 1992; Hall & Smith 1991; Meixner, Sheppard & Poklukar 1993; Smith & Brown 1988, 1990; Smith 1991; Smith et al. 1991). Honey bee mtdna types are discontinuously distributed among subspecies, reflecting the sustained temporal 4 separation and independent evolution of temperate and tropical lineages (Avise et at. 1987; Cornuet & Garnery 1991). African mtDNA has been found in almost all feral neotropical colonies. The small proportion of European mtDNA that has persisted has remained largely confined to managed apiaries (Hall and Muralidharan 1989; Hall & Smith 1991; Smith, Taylor & Brown 1989). These findings supplied strong evidence that unbroken matrilines spreading as swarms have been primarily responsible for the expansion of the African bee population in the neotropics (Hall & Muralidharan 1989; Hall & Smith 1991; Smith, Taylor & Brown 1989). Yet the features of mtDNA that make these molecules good genetic markers (i.e. uniparental inheritance, no recombination) limit the information that can be obtained from their analysis. Genomic RFLPs at several loci have been identified that distinguish east European from west European and African bees, but not west European from African bees (Hall 1990, 1992b). Frequencies for the markers indicated limited hybridization of African with European bees, but it was not possible to assess hybridization with west European bees. Alleles at two loci, found at different frequencies in African and west European bees, were found at intermediate frequencies in neotropical bees, which could have resulted from hybridization between these two groups of subspecies (Hall 1992b). The objective of the research reported here was to identify genomic DNA (molecular) markers or characters that distinguish among the east European, west European, and African groups of subspecies. IDENTIFICATION AND GEOGRAPHICAL DISTRIBUTION OF ALLELES AT LOCUS 178 Introduction Nuclear DNA restriction fragment length polymorphisms (RFLPs) are biparentally inherited, codominantly expressed markers shown to be a valuable source of genetic variability in honey bees (Hall 1986, 1990, 1992b). Relative to the biochemical variability that can be discerned in allozyme frequencies, RFLPs are more abundant and do not require gene expression for their detection. East European- and African-specific RFLP markers have been identified, as well as markers common to African and a west European species (Hall 1986, 1990, 1992b). In neotropical colonies, the east European markers have been found at very low frequencies in areas where African bees have become established, indicating that there has been limited paternal introgression from European colonies into the African bee population (Hall 1990). One limitation to date in the use of RFLPs to study ancestry in neotropical bees has been the lack of additional markers specific for other subspecies, particularly A. m. mellifera. Described in this chapter are nuclear DNA RFLPs, detected with a single genomic probe, that distinguish bees of European and African ancestry. While 5 6 it is not possible to know the genotype of each queen bee introduced, nor many details of the events contributing to the establishment of New World honey bee populations, some reconstruction can be acheived by studying contemporary bees in the Old and New World. Evidence is provided that reflects the racial composition of Central and South American bees prior to the introduction of African bees, and indicates the occurrence of some, albeit limited, hybridization of European and African bees. Materials and Methods Sources of honey bees. Protocols described by Hall (1986, 1990) for the collection, caste determination, and preservation of bees were followed without modification. Drones were collected as larvae and pupae. South African samples, from four locations in the Transvaal, were collected in January 1990 by HGH. Brood samples from a colony in So Frango, Brasilia, were obtained in 1990 by J. Maruniak, University of Florida. Honduran samples included drones from feral colonies and from managed colonies established from feral swarms, collected in November 1989 by HGH and A. Suazo, Escuela Agricola Panamericana, Honduras. Samples from Tapachula, Mexico, obtained in January 1988 by HGH, were from feral swarms captured in bait hives maintained by the Mexican agency Secretariat of Agriculture and Hydrologic Resources (SARH), and from two managed apiaries. Sources of USA drones included a closed breeding population maintained in Arizona, 7 provided by J. Martin, G. Waller, G. Loper, and E. Erickson (Page, Erickson & Laidlaw 1982; Severson, Page & Erickson 1986); managed colonies in Kansas, provided by 0. Taylor, University of Kansas; the University of Florida apiaries; and the Kona Queen Company, Captain Hook, HI. Electrophoretic analysis of honey bee DNA. The cloned probe pB178 came from a library of Psfl-digested European honey bee DNA ligated into the plasmid vector pBR322 (Hall 1986). This clone was used as a radioactive probe for detecting RFLPs in honey bee DNA digested with restriction endonucleases and separated electrophoretically. Isolation of genomic DNA, restriction endonuclease digestions, electrophoresis, probe preparation and labeling, blotting, and hybridizations were conducted as previously described or cited (Hall 1986, 1990) without further modification. Initial detection of polymorphisms. In the initial search for polymorphisms, DNA was isolated from a pool of ten workers from a New World European (USA) colony and from a New World African (Costa Rica) colony (Hall 1986, 1990). DNA from each pooled sample was digested separately with nine restriction endonucleases, and separated in 2% agarose gels. The restriction fragment profiles for the European and African samples were compared following hybridization with cloned probe pB178. The sizes (in kilobase pairs, kb) of the restriction fragments were estimated by comparison to size standards using a HI-PAD digitizer (Houston Instruments). Polymorphisms were detected in the Msp\- and Datel-treated samples. 8 Additional individuals from many locations in the New and Old World were then analyzed to ascertain the subspecies and/or geographic distribution of the polymorphic fragments. The polymorphisms generated by MspI and DdeI are described here. For the identification of the Msp\ variants, 402 drone bees were analyzed. For the identification of the Dde I variants, 508 drones were analyzed, including 401 of those analyzed for the Msp\ variants. The pairwise combinations of the Msp\ and Dde I variants found in these 401 drones are referred to as alleles. Results Allelic nature of the polymorphisms. Initially, polymorphisms were detected with pB178 following Msp\ and Dde I digestion of DNA extracted from a sibling pool of New World European and African worker (female) honey bees. In the Msp\ digests, a 1.8kb fragment was detected only in the European sample, and a unique 1.1 kb fragment was detected only in the African sample. In the Dde I digests, the European sample contained a unique 1.25kb fragment, while the African sample contained unique 3.4kb and 1.0kb fragments. RFLPs are codominantly expressed in diploid workers, which can result in the comigration and masking of fragments. Therefore, further characterization of the Msp I and Dde I RFLPs was accomplished by analyzing drones (males), which are haploid offspring, parthenogenetically derived from the queen. 9 The correspondence of the region of the genome detected by probe pB178 to a contiguous, heritable segment of a chromosome, or locus (Fincham 1983; Hall 1990, 1992b), was demonstrated by analyzing at least six drone progeny from a single colony (queen). For each pB178/restriction enzyme combination, no more than two restriction fragment patterns were detected, which reflected the segregation of the queen's alleles (not shown). Allelic polymorphisms in individual drones were manifested as different restriction fragment patterns. The different patterns generated by a single restriction enzyme are referred to as variants. The pairwise combinations of the variants produced by the two enzymes in individual drones are referred to as alleles. Identification of MsoI variants. Most of the 36 Msp\ restriction patterns or variants identified are shown diagrammatically in Figure 1. Each variant was composed of approximately 8 to 12 restriction fragments between 2.0kb and 0.3kb. All of the variants included a 1.4kb fragment, one to four fragments between 1.3kb and 1.1 kb, two or three fragments of variable intensity between 0.7kb and 0.5kb, and one to four fragments smaller than 0.5kb (Figures 1, 2, & 3). MsdI variant pattern correlates with distribution. Variants with subsets of common fragments were consistently detected in individuals from populations shown previously to have similar genetic backgrounds (Hall & Muralidharan 1989; Hall & Smith 1991). To illustrate this Size, kb t-cnjoo *- cm ro^J-t-cN -- cn ro Ln ooo oo oooo ooooo cm cm n n n n *3 •cor^oocno«-cMco'*a-ir> OOOOOOOOO»— inininiAinioininifliniflinifiinin 222222222222222 1.8 1.4 1.2 1.1 0.85 0.75 0.6 0.4 Figure 1. Restriction fragments composing Msp\ variants detected at locus 178 in drones. The individual variants are identified above each lane. Numbers to left correspond to sizes of characteristic and distinguishing fragments. Figure 2. Msp\ restriction fragment patterns, or variants, detected in drone bees with genomic probe pB178. Individual variants are identified above each lane. Numbers to left correspond to sizes of fragments of molecular weight standards (see Materials and Methods): numbers to right correspond to sizes of honey bee DNA fragments. The M100 variants, indicative of European ancestry, are shown in the first panel (left). The M200 variants, also detected in bees of European ancestry, are shown in the second panel. The M300 variants, one of which was detected in each population sampled, are shown in the third panel. The M400 variants, detected in South African and neotropical bees, are shown in the fourth panel (right). CO T— CM CO T— CM T— CO CM r— CM CO LO o O o O O O o o o O o O o o o 1—1 1— T— T— T— T— CM CM CO CO CO CO "it ■tf CO 2 .60kb 1.8kb .85kb ,75kb .4 kb ro Figure 3. M500 group of Msp\ restriction fragment variants detected with pB178 in drone bees. These variants were detected in South African and neotropical bees. Left, molecular weight standard fragment sizes; right, sizes of honey bee DNA fragments. co T— CM 00 LO CO r- 00 cn o T— CM oo '3" o O o o o o o o o T— T— T— T— LJ L_ LO LO LO LO LO LO LO LO LO LO LO LO LO LO CO 2 l.5kb l.4kb 1.3kb 1.2kb .98kb *- ,85kb 15 correlation, variants consisting of patterns with common fragments and similar distributions have been grouped together. For each population sampled, a summary of the sample size, the number of variants in each Msp\ variant group, and the total number of variants detected are given in Table 1. Variants M101-M104 (Figure 2, first panel; see also Figure 1) were characterized by a pair of fragments of approximately 1.2kb and 0.85kb, and an intense signal at approximately 0.4kb. These variants differed from each other primarily in additional fragments, between 0.7kb and 0.5kb. Variants M103 and M104 were difficult to distinguish and will be referred to together as M103. Variants M201 and M202 (Figure 2, second panel) differed from M101 and M102 only in the fragments smaller than 0.5kb. Variants M201 and M202 were distinguished from each other by the fragments 0.7kb to 0.5kb in size. The M100 and M200 variants were found in drones from the USA, Mexico, and Honduras. The Msp\ variants M301-M304 (Figure 2, third panel; see also Figure 1) featured 1.8kb and 1.2kb fragments. Variants M301, M303, and M304 differed very slightly from each other in the size of the fragments at approximately 1.4kb and 1.2kb and in the fragments between 0.7kb and 0.5kb (fragments not clear in Figure 2 can be seen in Figure 1). Variant M302 was distinguished by a 0.75kb fragment and by fragments smaller than 0.5kb. At least one of the M300 variants was found in each population. 16 Table 1. Summary of populations examined for variants and alleles at locus 178. Sample origin South Africa Honduras Mexico USA Msp\ variant analysis: n: individuals 94 106 56 146 colonies 23 21 12 58 different variants: 19 18 11 8 M100 total8 0 1 2 3 M200 total 0 1 0 3 M300 total 2 1 2 2 M400 total 3 3 3 0 M500 total 14 12 4 0 variants unique to this population 10 2 0 3 Dde I variant analysis: n: individuals 124 145 75 164 colonies 24 26 13 59 different variants 19 17 15 8 D100 total8 0 0 2 2 D200 total 0 3 1 1 D300 total 1 4 4 3 D400 total 12 8 5 2 D500 total 6 2 3 0 variants unique to this population 10 3 1 1 Allele identification: n: individuals 93 106 56 146 colonies 23 21 12 58 alleles15 detected 28 25 14 14 alleles unique to this population 26 15 3 8 a Total number of different variants from each variant group detected in each sample population. b Pairwise combinations of Msp\ and DdeI variants detected with pB178 in individual drones. 17 Variants M401-M405 (Figure 2, fourth panel) were characterized by the presence of a 1.1 kb fragment and three fragments smaller than 0.5kb, and by the absence of the 0.85kb fragment common to nearly all other variants. The 1.1 kb fragment of the M400 group appeared to be allelic to the 0.85kb fragment (Figures 1 & 2). The M400 variants were distinguished from each other on the basis of restriction fragments between 1.4kb and 1.1 kb (fragments smaller than 0.7kb, not clear in Figure 2, can be seen in Figure 1). The M400 variants were found in drones from South Africa, Honduras, and Mexico. MspI variants M500 as a group (Figures 1 & 3)) were distinguished by the presence, absence, or intensity of one or more unique restriction fragments. For example, variant M501 contained a unique 1.5kb fragment and lacked the 0.85kb fragment. M502 contained one, and M510 and M511 contained two, restriction fragments at approximately 1.3kb; these three differed from each other in the restriction fragments smaller than 0.7kb. M503 contained two fragments near 1.4kb, similar to variant M403, but contained the 0.85kb fragment that M403 lacked. The M500 variants were found in drones from South Africa, Honduras, and Mexico. Identification of DdeI variants. All 33 Dde I restriction fragment variants identified are shown diagrammatically in Figure 4. Each pattern consisted of six or seven restriction fragments. Most variants exhibited a 0.75kb fragment and one to three Size kb / *- CM t-CMOO O O O O O «- i- CM CM CM Q Q Q Q Q >- CM 00 LO CO o o o o o o o 00 CO 00 00 CO CO Q Q Q Q Q Q Q CM CO ID CO r* 00 o o o o o o o Q Q Q Q Q Q Q OT O <- CM 00 <í IÍ) • 730bp 380bp 380bp 620bp 560bp 750bp 1250bp 420bp 350bp 1400bp 560bp 1300bp • p Pst\ i 730 1110 1220 1600 Msp I Msp I 2220 /Wspl 2636 2786 2880 Kpn\ 3630 3938 Hindill : M§0\ * 6000 /Wspl IÜ8 ,/Wspl * ■ Bgl\\ 6770 6020 Msp I /Wspl ?M8 7680 EcoRI : ®: 8140 /Wspl , /Wspl * Psrl2 Size, kb *— CM CO o o o «- CM o o CM CM CO t- CM O O O O CO CO CO CO <- CM CO LO o o o o o <-cMco''i-mcor''-ooOTO«r-cMco''tir> OOOOOOOOO»-.-.-*-*-*- ininininininiflifiinininiflininin 1.8 1.4 1.2 1.1 0.85 0.75 0.6 Figure 14. Hybridization of the 2.6kb Psfl, - KpnI region of pB178 (178P1K) to Southern blots containing honey bee genomic DNA digested with Msp\. Restriction fragments of each Msp\ variant (identified above each lane) to which 178P.,K hybridized are indicated. U1 Figure 1 5. Identification of Msp\ recognition sites in the region of the honey bee genome corresponding to the Pst\, - Kpn\ region of probe pB1 78. Locations of the Msp\ sites within 178?^, the 2.6kb Psil, - Kpn\ fragment of P178, were identified by digestion of 178?^ with Msp\ alone and paired with one restriction enzyme at a time (those shown in Figure 12 within the P.,K fragment). Additional MspI sites were hypothesized on the basis of fragments in other variants. The Msp I sites have been labeled a - k; Pand K represent the Psfl, and Kpn\ sites at the termini of the fragment, respectively. The Msp\ restriction fragment pattern of 178?^ corresponds to M101 or M103, with the restiction sites indicated. Restriction sites that could account for the patterns of several other Msp\ variants are also indicated. -420 — — Msp I-a M101/103: M201: -120 — 1 — — Msp l-b — P, b b 0.85kb 0.85kb 730 — — Msp l-c c c 0.38kb 0.38kb 1110 — — Msp\-d d d 1220 — — Msp l-e e e 0.38kb f 0.27kb 1600 — — Msp\-h h h 0.62kb 0.62kb 2200 — — Msp\-i i 0.56kb i 0.62kb 2636 — K 2786 — 2880 —* j k M300s: b 0.85kb c — 0.38kb d — e — f — 0.27kb h — 0.62kb 0.56kb j — M401: a M501: M503: b 1.1 kb 1.5kb 0.85kb c c c 0.38kb 0.38kb 0.49kb d d e e e f f f 0 0.27kb 0.27kb h h h 0.62kb 0.62kb 0.62kb i 0.56kb i 0.56kb i 0.56kb j j j *-c\in «-cm r-csjco^m <-c\icr)*tLOo.— cnco^ld Cj7p ooo oo oooo ooooo OOOOOOOOO»— «—*—«—»— *— oize, N cm n n n n tt lo lo lo m lo lo lo lo lo lo lo lo lo lo lo kb 222 22 2222 22222 222222222222222 1.8 1.4 1.2 1.1 0.85 0.75 0.6 0.4 M101/ M103: M102: 2200 2636 2786 2880 3630 3630 3700 Msp\ K Msp\ Msp\ H Msp\ Msp\ 0 66kb 0.62kl I (faint] 0.75kb 0.75kli Figure 16. Hybridization of the .9kb, KpnI - Hind\\\ region of probe pB178 (178KH) to Southern blots containing honey bee genomic DNA digested with Msp\. Restriction fragments of each Msp\ variant (identified above each lane) to which 178KH hybridized are shown. A faint hybridization signal at 0.56kb was seen in all variants except M102 and M201 (not shown). Msp\ sites that could account for the fragments detected in the variants are shown on the right. ^ CD 79 16, the loss of an Mspl site in this region (at approximately 2785bp) could explain the presence of the 0.62kb fragment in these two variants. Digestion of the 1.9kb region from Hind\\\ to Bgl\\ (178HB) with Msp\ produced fragments of approximately 1.3kb, 0.42kb, 100bp and 70bp. MspI sites were identified near the Hind III and Bgi\\ sites in subclone pG178HB (Figure 13). Mspl restriction fragments to which 178HB hybridized are shown in Figure 17. The Msp\ restriction fragment pattern of 178HB corresponded to variant M103: two site polymorphisms are indicated in Figure 17 that could account for the fragments detected by 178HB in two other variants. Digestion of the 2.0kb BgtII - EcoRI4 fragment (178BE4) with Msp\ produced fragments of approximately 1.4kb, 0.33kb, and 0.25kb (Figure 13). The Msp\ fragments to which 178BE4 hybridized in Southern blots are shown in Figure 18: these included combinations of approximately 1.8kb, 1.4kb, 1.25kb, 0.77kb, 0.56kb, and 0.35kb fragments. The loss of an Msp\ site in the 178HB region (Figures 13 & 17, at approximately 5420bp) could account for the 0.77kb fragment detected in variants M101 and M302, and for the detection of this fragment with both 178HB and 178BE4. Since 178BE4 hybridized to more Mspl fragments than had been found in the digestion of the insert and accounted for by overlapping with adjacent regions, the placement of Mspl sites within 178BE4 was based on double digests with Spel, Sph\, /Vs/I, and SaiI, and the results of the hybridizations of 178HB, 178BE4, and 178E4P2 (following). M103 M510 r- CM CO «-CM Size, 2 2 2 2 2 kb s 2 ^ 2 2 CO ^ «- CM o o o o CO CO CO CO «- CM CO ■'t IT) o o o o o ^ «tf- ^1- «t «-cMco'si-Lf)cor''COC)0«-cMcort-ir> ooooooooo«— «—«—«—«— «— LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO 3530 3630 3700 H Msp\ — Msp\ — M101 1.8 1.4 1.2 1.1 1.25kb 1.: 5kb 0.85 0.75 0.6 0.4 5000 5420 5440 Msp\ 0.42kb - Iglsp\ 5770 — Msp\ 0.’ 7kb Figure 17. Hybridization of the 1.9kb, Hiná\\\ - BglII region of probe pB178 (1 78HB) to Southern blots of honey bee genomic DNA digested with Msp\. Restriction fragments of each Msp\ variant (identified above each lane) to which 178HB hybridized are shown. Right: Msp\ restriction sites determined for the probe DNA (M103), and which could account for two other Msp\ variants. 00 o i— c\i co «- cm co ■'t cm cm co lo <-cNco*tLncor^oocT)OT-cMcO''tLn q;70 OOO OO OOOO OOOOO OOOOOOOOO--*-*-*-*-*- oize, <—>—*— cm cm cocococo loiciioininininiciininioinininm kb ^ ^ ^ ^ ^ ^5^52^2^255 1.8 1.4 1.2 1.1 0.85 0.75 0.6 0.4 Figure 1 8. Hybridization of the 2.0kb, BglII - £coRI4 region of probe pB1 78 (178BEJ to Southern blots containing honey bee genomic DNA digested with Msp\. Restriction fragments of each variant (identified above each lane) to which 178BE4 hybridized are shown. 00 Size, 22° ° ° kb ^ ^ 2 2 CO «— CNJ o o o o CO CO CO CO 2 2 2 2 «- cm co in o o o o o ■3- r)- 'Í 't >-cMco^tLncDr^ooo50T-c\ico^-ir) OOOOOOOOO»— »—»—*—«— «— inifiininiflinininininiriinininin 1.8 1.4 1.2 1.1 0.85 0.75 0.6 0.4 Figure 19. Hybridization of the 2.0kb, £coRI4 - Pst\2 region of probe pB178 (178E4P2) to Southern blots containing honey bee genomic DIMA digested with Msp\. Restriction fragments in each variant (identified above each lane) to which 178E4P2 hybridized are indicated. 00 ro 83 Digestion of the 2.0kb £coRI4 - Pst\2 fragment (178E4P2) with MspI produced fragments of approximately 1.3kb and 0.56kb. Two additional Msp I sites were found near the £coRI site in subclone pG178E4P2 (Figure 13). The fragments to which 178E4P2 hybridized, shown in Figure 19, appeared to include the 1.8kb, 1.25kb, and 0.56kb fragments to which 178BE4 hybridized. In the majority of variants, 178E4P2 hybridized to fragments of approximately 1.25kb and 0.56kb. The existence of the 1.8kb fragment in the M300 variants could be explained by the loss of the Msp\ site at 8140bp (Figure 13). Since it is likely that the duplication in the fragments to which 178BE4 and 178E4P2 hybridized resulted from contamination of 178BE4 with 178E4P2, the placement of the Msp\ sites shown in Figure 13 was based on the digestion of different aliquots of these two regions with Msp\, in the absence and presence of other restriction enzymes which have sites in each region. Identification of DdeI sites in 178. The approximate locations of Dde I sites identified in the honey bee DNA insert of pB178 are shown in Figure 20, in the same format as Figures 12 and 13. The results of Southern blot hybridizations are shown in Figures 21 - 25, in the same format as Figure 4. Digestion of 178?^ with Dde I produced fragments of approximately 1.3kb, 0.75kb, and 0.4kb. A single fragment of approximately 1.0kb was detected when 178?^, was digested with Dde I (Figure 20: refer to Figure 13 Figure 20. Map of DdeI restriction sites identified in honey bee genomic DNA insert of probe pB178. The Pst\ sites marking the ends of the insert were utilized for cloning in pBR322. Relative positions of sites were determined by digesting smaller regions of the insert (Psí^ - Kpn\, Kpn\ - H¡nú\\\, Hin6\\\ - Bg/\\, Bgl\\ - £coRI4, and £coRI4 - Pst\2) with Dde I in the absence and presence of restriction enzymes within each region, which were indicated in Figure 12 (with the exception of the region between the Kpn\ and Hincilll sites). The asterisks (*) indicate sites identified by sequencing the ends of the respective subclones. 1 Psri, Dde I 85 Pi 1300bp 750bp 2000bp 1250bp 1270bp 1200bp > 1150bp — P2 160 1460 2200 2636 3630 4280 6440 6630 6780 7060 7440 8300 Dde I Dde I Kpn\ Hinm Dde I m * Dde I Dde I EcoRI Dc/el Psrl2 9460 86 for 178?^, location and size). Analysis of the sequences of the ends of subclone pG^SP^ revealed a DdeI site at 150bp from the Pst\, site (Figures 20 & 21). The Dde I fragments to which 178P,K hybridized are shown in Figure 21. In the majority of variants, 178?^ hybridized to a fragment of approximately 0.75kb, and another fragment of approximately 1.3kb. In the D100 and D200 variants, a single fragment of approximately 2.1 kb was detected. The loss of the Dde I site at approximately 1450bp would account for the 2.1 kb fragment in the D100 and D200 variants. The variation in the size of the fragment of approximately 1.3kb may be the result of length polymorphism(s) closer to the site. There were no Dde I sites within 178KH. A single fragment in each variant, approximately 2.0kb, was detected when 178KH was used to probe this region, shown in Figure 22. On the basis of the Dde I sites identified in 178?^, it was concluded that the same 2.0kb fragment had been detected by 1 78PtK and 178KH, and would be detected by 178HB as well. The placement of the Dde I sites on either side of 178KH is shown in Figure 22. Digestion of 178HB with Dde I resulted in fragments of approximately 1.15kb and 0.75kb. The Dde I fragments to which 178HB hybridized are shown in Figure 23. In each variant, 178HB hybridized to the 2.0kb fragment detected by 1 78PtK and 178KH. One other fragment was detected by 178HB in each variant: approximately 1.0kb in the D100s and D400s, 1.25kb in the D200s and D300s, and 0.98kb in the D500s. Two possible locations for a site «- CM O O D301 t-cNco r-cMro^Lf>cD ^-cMn'tLncDr'OOOTO«-tNco'5í-Ln «- es co ■st ld co OOO OOOOOO OOOOOOOOO»— ooooooo _ es (N cm nnnnnco m lo m icnn m m Size, QQ QQQ QQQQQQ QQQQQQQQQQQQQQQ OOOOOOO kb 3.4 TPl 150 - - DdeI D101 2.3 2.0 1.3kb 1.4 1.25 1.1 1.0 2.1kb uso -- Dde I — 0.75 0.75kb Figure 21. Hybridization of the 2.6kb Psfl, - Kpn\ region of probe pB178 (178PK) to Southern blots containing honey bee genomic DNA digested with Dde I. Restriction fragments in each Dde I variant (idenntified above each lane) to which 178?^ hybridized are indicated. On the right is a map of the 178?^ region and the location of the Dde I sites determined for the probe (D301); the loss of the Dde I site at 1450bp accounts for the pattern detected in the D100 and D200 variants. 2200 --Dde I — 2635 -I- K 00 2200 -i— DdeI «- CM CM CD >- CM CO O- LO CD r-CMCOMj-LnCDr^COCDO^-CNCOO-Ln «- CM CD ID CO I** Size 00 000 000000 ooooooooo«-«-*-t-.-r- 0000000 ’ «— «— CM CM CM CO CO n CO CO (O ID ID ID LO ID ID If) kb QQ QQQ 0 0 0 0 0 0 OOOOOOOOOOOOOOO OOOQQOQ 3.4 2.3 2.0 2635 1.4 1.25 1.1 1.0 3530 0.75 4280 Figure 22. Hybridization of the .9kb Kpn\ - HindlW region of probe pB178 (178KH) to Southern blots containing honey bee DNA digested with Dde I. Restriction fragments in each Dde I variant to which 178KH hybridized are indicated. The map to the right indicates the location of the Dde I sites flanking 178KH which account for the fragment detected. The origin of the slight differences in the size of the fragment detected in each variant, from either a site(s) or length polymorphism(s), has not been identified. K 2.0kb H Dde\ 00 00 D201 D101 t-cm »- cm co «- cm oo lo co Size 00 000 000000 ' <- <- CM CM CNl n CO CO 00 CO CO kb QQ Odd dddddd r-CMCO^iflíDr^OOOSO^-CMC^^lO ooooooooo*-*-*-»-.-.- ddddddddddddddd i—cMoo^’Locor''- 0000000 LO LO LO LO LO LO LO d d d d d d d 2200 -i— Dde I 3.4 2.3 2.0 3530 — H 2.0kb 1.4 1.25 1.1 1.0 4280 - — Dde I H 1.25kb 0.75 1 .(kb Figure 23. Hybridization of the 1.9kb, Hind\\\ - Bgl\\ region of probe pB178 (178HB) to Southern blots containing honey bee DNA digested with Dde I. Restriction fragments to which 178HB hybridized are indicated. On the right is a map of the region encompassing 178HB and the Dde I sites defining the fragments to which 178HB hybridized. Restriction sites which could account for the fragment patterns in the D100 and D400 variants in this region are also indicated. The 2.0kb fragment was also detected with 178KH. 00 co 90 which could account for the 1 .Okb fragment seen in the D100 and D400 variants are shown in Figure 23. Digestion of 178BE4 with DdeI resulted in fragments of approximately 1.27kb, 0.39kb, and 0.25kb. The Dde I fragments to which 178BE4 hybridized are shown in Figure 24. In the majority of variants 178BE4 hybridized to fragments of approximately 2.1 kb, 1.3kb, and 1.05kb. A Dde I site was identified near the Bgl\\ site of subclone pG178BE4 (Figure 20). Since 178BE4 hybridized to more Dde I fragments than it appeared to contain, which also occurred when 178BE4 was used to probe Southern blots containing the Msp\ variants, the placement of Msp\ sites within 178BE4 was based on double digests with Spel, Sph\, /Vs/I, and SalI, and the results of the hybridizations of 178HB, 178BE4, and 178E4P2 (following). Digestion of 178E4P2 with Dde I produced fragments of approximately 1.2kb and 0.85kb (Figure 20). The fragments to which 178E4P2 hybridized are shown in Figure 25, and include what appeared to be the same 2.1 kb and 1.05kb fragments to which 178BE4 had hybridized. Sites corresponding to the fragments greater than 2.3kb in the D400 variants lie outside the probe region. The duplication in the Dde I fragments to which 178BE4 and 178E4P2 hybridized was concluded to be due to contamination of 178BE4 with 178E4P2. Therefore the placement of the Dde I sites shown in Figure 20 was based on the digestion of different aliquots of these two regions with Dde I, in the absence and presence of other restriction enzymes with sites in each region. Size kb «-CN t-cnco >- m (o ld cd '-c\irr>*tLncor^-cootel-4530/5280 M301/D200 — + — M301/D300 + + — M302/D100 — — + M303/D400 + + + M304/D500 + + + a ( + ), Site present; (—), site absent 98 geographically separated populations in the same species, and markers for following the movement of African bees in the USA. The sequences obtained for the pG178 subclones will be used to design primers which can be used to convert the RFLP analysis to a more expedient PCR format. It is predicted that by amplifying each region in the samples containing the different Msp\ and DdeI variants, the site and length polymophisms which define each variant can be identified. CONCLUDING REMARKS RFLPs at the polymorphic locus corresponding to probe pB178 represent the first nuclear DNA markers that distinguish east European or north Mediterranean (Garnery, Cornuet & Solignac 1992), west European, and South African bees. For this study, only workers from Europe were available, and the codominant expression and comigration of restriction fragments in diploid workers precluded the identification of individual variants in each of the European populations. Nonetheless, the detection of fragments characteristic of MspI and DdeI variant groups enabled the distribution of the Msp\ and DdeI variants in the European samples to be determined. A. m. mellifera was the first subspecies introduced to North America, in 1622 (Sheppard 1988), followed by A. m. Hgustica, A. m. carnica, and A. m. caucásica in the mid-to-late 1800s (Kent 1988; Oertel 1976; Pellet 1938; Sheppard 1989). A. m. mellifera overwintered well in colder climates, and tended to establish feral colonies upon swarming, but this race was defensive and had a greater susceptibility to brood diseases (Susceptibility 1982; Sheppard 1988). The east European bees, particularly A m. Hgustica, gained tremendous popularity for beekeeping due to their high productivity and the 99 100 facility of their management. A. m. Ugustica has dominated the hobby and commercial honey bee market in the USA for over a hundred years. It is likely that the importation of honey bees from Europe to the USA resulted in a reduction in variation, or genetic bottleneck, relative to the parent populations (Sheppard 1988). The identity and number of variants and alleles at locus 178 in the subspecies imported to the USA for beekeeping have not been determined but may be greater than those found in USA drones. Variants and alleles detected in USA drones likely represent a subset of the variability at this locus in European honey bee subspecies. The emphasis on vitality and productivity in beekeeping and queen-rearing practices has resulted in some degree of homogenization of European races in the USA. Documentation of the identity and numbers of European honey bee subspecies imported into the neotropics appears to be limited, and commercial and feral populations have not been thoroughly surveyed in the past (Kent 1988; Taylor 1977). It has been reported that the same races of European bees were introduced to North America and the neotropics, although the introductions occurred at different times (Goncalves 1974; Goncalves, Stort & DeJong 1991; Hellmich & Rinderer 1991; Kent 1988; Kerr, DeLeon & Dardo 1982; Lobo, Del Lama & Mestriner 1989; Rinderer & Hellmich 1991). The number of African queens imported and released in Brazil is not clear (Kerr 1967; Rinderer, Oldroyd & Sheppard 1993; Smith 1991) nor the number of drones with which each was mated, but the introduction probably resulted in 101 a reduction in variation relative to that of the parent South African population. Variants and alleles found in the South African samples may not have been represented in the individuals imported to the New World. Variants identified in the neotropical samples similar to but not detected in South Africa may reflect sampling error. Fletcher has provided a summary of past, repeated attempts to introduce A. m. Hgustica and A. m. caucásica to South Africa (Fletcher 1973, 1978). The European bees failed to become established, and apparently failed to interbreed with the indigenous subspecies, A. m. scutellata (Fletcher 1978). This conclusion may be supported by the detection of only one variant common (D404) to the USA/European and South African samples. The existence of this variant may represent a relic of a previously successful introduction of European genes into the South African honey bee gene pool, or it may represent an ancestral form. A more exhaustive survey, including the identification of variants and alleles present in the original temperate, European populations, may indicate how well the samples of the present study represent the variation throughout the parental and neotropical populations. While most of the variants and alleles detected in the neotropical bees were the same or similar in African bees, there is evidence that European markers have been retained by neotropical bees. In the region of Brazil into which the African bees were released there may have been a greater concentration of west European compared to east 102 European bees. The relatively constant frequency for the Msp\ variant M301 in neotropical bees suggests that it is a neutral marker, incorporated as a result of west European introgression into the African population as it was becoming established and has been carried along as the bees have migrated north (Lobo & Kreiger 1992; Smith 1991). The release and spread of African bees has been disruptive to the practice of beekeeping in the neotropics (Cantwell 1974; Goncalves, Stort & De Jong 1991; Michener 1975; Roubik 1980, 1989; Spivak, Fletcher & Breed 1991; Taylor 1977; Winston, Taylor & Otis 1983). 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Taylor OR (1985) African bees: potential impact in the United States. Bulletin of the Entomological Society of America, 31, 14-24. Taylor OR, Spivak M (1984) Climatic limits of tropical African honeybees in the Americas. Bee World, 65, 38-47. Villa JD, Gentry C, Taylor OR (1987) Preliminary observations on thermoregulation, clustering, and energy utilization in African and European honey bees. Journal of the Kansas Entomological Society, 60, 4-14. Villa JD, Rinderer TE, Collins AM (1993) "Overwintering" of Africanized, European, and hybrid honey bees (Hymenoptera: Apidae) in the Andes of Venezuela. Environmental Entomology, 22, 183-189. Winston ML, Taylor OR, Otis GW (1983) Some differences between temperate European and tropical African and South American honeybees. Bee World, 64, 12-21. BIOGRAPHICAL SKETCH I was born and raised in Des Moines, Iowa. I obtained a B.S. in chemistry, with concentrations in biology and math, from Clarke College, Dubuque, Iowa. My M.S. in biochemistry, investigating structural and kinetic properties of aldehyde dehydrogenase isozymes, was earned at Purdue University in the laboratory of Henry Weiner. I then worked at Rainbow Babies and Children's Hospital, Case Western Reserve University, Cleveland, Ohio, developing an animal model for studying pediatric giardiasis. When the grant money ran out, I was fortunate to obtain a position studying gene expression in central nervous system tumors with Tim Mapstone, M.D., Department of Neurosurgery, and Dave Goldthwait, M.D., Department of Biochemistry, CWRU. Recognizing that I was more interested in teaching and conducting entomological than medical research (and that I would rather be warm than cold), I called H. Glenn Hall, at the University of Florida, to inquire about the possibility of working toward my doctorate in his laboratory (/.e., I invited myself into his lab). I have met an outstanding group of people at UF, had terrific opportunities to travel and teach, and have enjoyed great freedom in conducting my research. 111 I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. // Atvwn Mad H. Glenn Hall, Chair Associate Professor of Entomology and Nematology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Jaraes E. Maruniak Associate Professor of Entomology and Nematology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Andrew F. Cockburn Assistant Professor of Entomology and Nematology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Bondu, Elizabeth ¿Bondy ^ Assistant Professorof Instruction and Curriculum This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. April 1994 Dean, College of Agriculture Dean, Graduate School LO I^So FLORIDA 3 i A V""L""11 »■' ■mil lili 1262 08556 8151