Molecular Biology of the Honeybee

Molecular Biology of the Honeybee Robin F.A. Moritz lnstitut fur Biologie, Technische Universitat Berlin, Franklinstr 28/29, 10587 Berlin, Germany

1 Introduction 105 2 Genes and sequences

107 2.1 Nuclear genes 107 3 Mitochondrial genome 114 3.1 Mitochondrial genes 116 3.2 Non-coding sequences and length variation 117 4 Gene activity in embryonic development 124 5 Population variability 125 5.1 Nuclear DNA markers 125 5.2 Mitochondrial DNA markers 129 6 Molecular evolution and biogeography 130 6.1 Molecular phylogeny of bees 130 6.2 Genetic variability among honeybee species 131 6.3 Molecular variability within species 133 7 Outlook 140 Acknowledgements 141 References 144

1 Introduction

The biological rules that govern the honeybee colony have fascinated scientists since Aristotle. The bustling activities of the thousands of workers are seemingly at random and yet in the end a unified and apparently coordinated system is achieved. The honeybee riddle of chaos on the one hand and yet pattern on the other was to occupy generations of scientists after Aristotle. It took a long time to grab some pieces of the great puzzle and understand the basics of communication in honeybee colonies. It was the great pioneers in the field of bee research who unravelled the mystery of identifying the dance language of the worker bee (von Frisch, 1965) and of queen control through pheromones (Butler, 1973). In the second half of this ADVANCES IN INSECI'PHYSIOLOGY VOL. 25 ISBN lL1?4l?1??5-7

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century bee research was dominated by studies focusing o n behavioural ecology and communication under the influence of Karl von Frisch's and Martin Lindauer's epochal work on honeybees. However. the focus on behavioural ecology was not always so strong. and in fact honeybees also entered the arena of modern research as a genetic model system. Mendel conducted experiments with bees but unfortunately he failed to achieve controlled matings in netted cages and could not produce any F2 offspring which he required to confirm his theories based on the pea experiments (Johannson, 1980). Real breakthroughs were achieved by Dzierzon (1845) who found that drones are parthenogenetically produced. Petrunkewitsch (1903) and Nachtsheim (1912, 1914) used honeybees in their studies to elucidate the mode of fertilization and chromosome duplication. Honeybees were among the prime genetic model systems used until Castle and Morgan introduced Drosoplzila to the field of genetics in the early 20th century. Yet after the wide acceptance of fruit flies as the genetic test organism, honeybee genetics became a very specialized field and even the introduction of artificial insemination by Laidlaw (1944) and Mackensen (1947) could not stop the decline of honeybee genetics. The honeybee system was difficult to handle, had a slow generation cycle and an annoyingly large number of chromosomes ( n = 16) which made linkage studies tedious. Controlled matings could only be achieved through artificial insemination, and the generation cycle was too long to generate a rapid series of high-quality publications, an important basis for successful research and the recruiting of funds. Maintenance costs were high and other organisms offered better, swifter, and less expensive ways to solve pending questions. One of the last strongholds of honeybee genetics was in behavioural genetics, but after the work of Rothenbuhler (1964) on the genetics of hygienic behaviour of worker honeybees, a depressing silence also broke out in international scientific journals in this field. Clearly. the molecular revolution of genetics took place leaving the honeybees aside. The molecular genetics of Drosophila melanogaster boomed while bee geneticists felt more attracted to applied breeding research. Only recently, after a surprisingly long abstinence of several decades, are geneticists slowly re-entering the scene in honeybee research, trying to catch up with modern techniques and addressing new and (more important) unique questions that can only be solved by using honeybees as a test system. Although honeybees as an animal model for basic genetics have their pitfalls, they are far from being useless in basic genetical research. In fact, they have several unique features that make them more attractive than any other genetical test system available in higher organisms. Particularly important are the following traits for genetical research: 1. Male haploidy allows the study of gene expression in haplotypes. This is important for both selection experiments and gene mapping studies.

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2. The rich behavioural repertoire and the social organization makes it a primc system in behavioural genetics. 3. The slow embryonic development offers plenty of opportunities to study gene regulation and expression during early development. These are just a few characteristics which make the honeybee profitable for basic genetical research. Since honeybees are of significant economical and ecological importance, there is obviously also a compelling need to understand their population and breeding genetics. Here also molecular techniques are aiding us in understanding processes in natural and artificial selection and the undcrlying genetic mechanisms. Today the stage is set for rapid progress in molecular honeybee genetics, since we can capitalize on the very detailed knowledgc obtained from Drosophila. Cloned DNA probes are available which enable us to rapidly map the honeybee genome; sequenced genes allow us a better understanding of gene regulation and evolution. In addition, mitochondria1 DNA is used in population genetic studies revealing the dynamics of natural selection in feral honeybee populations. Thus, molecular genetics of honeybees can both incrcase our understanding of basic genetic mechanisms and improve our knowledge of honeybee specific genetic problems. 2 Genes and sequences

2.1

NUCLEAR GENES

2.1.1 Elongation factor I The search for genes in honeybees has been limited and only a few genes have been isolated and sequenced so far. The most productive way of isolating genes has been through hybridization of honeybee DNA to known Drosophila genes. Walldorf and Hovemann (1990) studied a DNA coding for one of the three proteins ( a , p , y ) of the cytoplasmic elongation factor 1 (EF-1). EF-la catalyses the transport of the aminoacyl-tRNA to the 80s ribosome. In Drosophila melanogaster two independent genes (F1 and F2) code for EF-la (Hovemann et al., 1988). Walldorf and Hovemann (1990) found the EF-la gene of honeybees to be closely related to the corresponding coding region in Drosophila melanogaster. They cloned two fragments of 1.0kb and 1.1 kb. respectively, which revealed as much as 77% sequence homology to the EF-la F1 Drosophila reading frame. The same degree of homology was found for another elongation factor gene (EF-lcu, F2). Through this high sequence conservation Walldorf and Hovemann (1990) were able to locate the translation start and stop sequence in the gene (nucleotides 365 and 2121, respectively) and they also found two introns

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intron

FIG. 1 Putative structure of the E F - l a gene in Apis melliferu. The base pair numbering is adopted from Walldorf and Hovernann (1990).

similar to the Drosophila EF-la F2gene (Fig. 1). Walldorf and Hovemann (1990) suggested that the Apis EF-la gene evolved from a common ancestral =-type gene rather than the F1 gene which is free of introns. The isolated EF-la sequence of Apis codes for 461 amino acids with a calculated mass of 50.5 kDa. It is unclear whether Apis has more than one EF-la gene. In a southern hybridization of the Drosophila probe on an EcoRI digest of Apis mellifera DNA, Walldorf and Hovemann (1990) found a weak second signal at about 9.4 kb and they could not exclude the possibility of a second EF-la gene in honeybees. The high homologies between Drosophila and Apis are not necessarily surprising since the elongation factor is known to be encoded in an extremely conservative gene region with only little variance among different taxa. Only two more variable amino acid sites appear, at positions 186 and 315, respectively, by adding the Apis sequence to the already known sequence variability among eight different species (Walldorf and Hovemann, 1990). 2.1.2 Segmentation genes Fleig et a/. (1988) studied the homologies of several homeobox genes of Drosophila rnelanogaster and Apis rnellifera. They constructed a genomic library of Apis mellifera and identified a homeobox DNA sequence in a cloned 500 bp Cla-Sull fragment of Apis rnellifera showing 82% homology to the Drosophila gene Dfd, and coding for the identical amino acid sequence. They termed the honeybee gene H42 and found additional homologies that extended beyond the 5' and 3' ends of the box. They also argued that the position of the intron may be at an identical site in both honeybees and fruit flies. In further work, Walldorf et a/. (1989) compared other homeobox genes of Apis rnellifera and Drosophila rnelanogusfer in more detail. Drosophila DNA probes containing Antennupedia ( A n f p ) ,fushi tarazu ( f t z ) , sex combs

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TABLE 1 Sequenced homeobox genes in Apis mellifera

Drosophila probe abdominal-A Antennapedia Deformed fushi tarazu engrailed" invected"

muscle segment homeobox Sex combs reduced W-13

Honeybee clone

% amino acid similarity in the homeobox

E30 E6O E30 E60 H17

96.7 98.3 100 not detected 96.7 91.7 81.7 83.3 96.7 (with 5.4 kb intron)

H55 H40

98.3 54.2

H15 H90 H42 -

~~

T h e sequence similarity between eti and inv is smaller between than within species. It is therefore not possible to assign E30 and E60 to either gene.

reduced (Scr) and Deformed ( D f d ) hybridized to EcoRl digests of total genomic DNA of Apis rneffifera (Table 1). Although frz did yield hybridization signals in genomic honeybee DNA (Fleig et al., 1988), the authors were unable to detect a homologous conserved sequence in the honeybee. Screening their library with f t z , the only clones that were reisolated were those that had already been isolated with the Antp probe. Walldorf et al. (1989) concluded that, given the honeybee has an ftz homologous gene, it must have considerably diverged from the Drosophila gene. Alternatively, they suggest that the gene is completely lacking and other loci perform the ftz function. The genes of the engrailed class (en = engrailed and inv = invected) appeared to be conservative. Two clones, ,560 and E30, were highly homologous, yet it was not possible to assign the two Drosophila genes to the two honeybee clones because sequence divergence between the two genes within the species was less than between the species. The homology of the homeobox in clone H40 showed only 55.2% similarity to the Drosophila W-13 gene. No homeobox has been found in Drosophila with strong homologies to H40. Sommer et al. (1992) studied segmentation genes with the Cys2-His2 zinc-finger DNA binding motif. They found evolutionary conserved patterns between Drosophila and Apis for hunchback, Kriippel, and snail (Fig. 2). Particularly, Kriippel showed high similarities between both species with a 94.6% amino acid homology, and the authors suggest that it might be duplicated in the honeybee. It appeared that those amino acids which are believed to have a direct contact with the bases in the DNA are completely conserved. The escargot sequence from Drosophila had a higher homology

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hunchback

Kiiippel

snail

FIG. 2 Sequence alignment of finger fragments obtained from the genes of Drosophilu (top) and Apis (bottom). Identical amino acids are denoted with a dash, a deletion with a dot. The stars indicate those amino acids that are believed to be in direct contact with the bases in the DNA. For the snail gene (c) also the escargot sequence is plotted to reveal the sequence similarity to the Apis sequence.

to thc Apis fragment (91.4%) than had the paralog snail sequence (75.6%) which was initially used to isolate the Apis DNA (Sommer et ul., 1992).

2.1.3 Genes coding for honeybee venom compounds There are also few studies focusing on honeybee specific genes. These mainly include DNA regions coding for enzymes and peptides of bee venom (Table 2 ) which have been analysed because their composition is known from various detailed biochemical studies (Habermann and Jentsch, 1967; Shipolini er al., 1971; Bachmeyer et al., 1972; Habermann, 1972; Gauldie et ul., 1976, 1978; Suchanek et al., 1978; Kreil et al., 1980). Melittin is the main lytic peptide of the honeybee venom and is found in both queens and workers. Like most peptides it is initially synthesized as a larger precursor which is then proteolytically cleaved to the final product (Kreil, 1990). Typical of the melittin precursor is the pro-region which is composed of an amino acid sequence in which every other position is either alanine or

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TABLE 2 Composition ot t h e worker honeybee venom in 54 dry weight Enzymes Phospholipase A? Hyaluronidase Acid Phosphatase

l(klS% 2 (7,

Peptides Mclittin A pa mi n MCD-peptidc Secapin T e rt i a pi n

45-605? 2-3 %' 2 %' 1%

<1%

< 1%

proline. This region is removed by a stepwise cleavage of dipeptides through dipeptidylaminopeptidase (DPAP; Kreil et al., 1980). The end product is thus produced by removing 11 dipeptides of the sequence X-Ala or X-Pro, with X being alanine, glutamine or asparagine, by DPAP activity. Kreil et al. (1980) found that the end product, melittin, is only synthesized after the precursor has entered the venom sac. Vlasak et al. (1983) isolated mRNA from venom glands of queen bees and were able to synthesize a cDNA coding for prepromelittin; which is a transient precursor of melittin (Suchanek et ul., 1978). A clone with a 374 bp insert proved to contain an open reading frame which corresponded exactly to the prepromelittin amino acid sequence as determined by biochemical peptide analysis (Fig. 3 ) . Vlasak and Kreil (1984) isolated and sequenced a cDNA that coded for preprosecapin, which is a precursor for secapin, a minor compound in the venom glands of workers with an as-yet unknown function (Gauldie et al.,

ATG AAA 'ITC TTA GTC AAC GTT GCC CIT GTT TIT ATG GTC GTG TAC ATT TCT TAC ATC Met -Lys- Phe - Leu - Val -Asn - Val - M a -Leu - Val - Phe -Met - Val - Val - Tyr -1le -Ser-Tyr- IleTAT GCG GCC CCT GAA CCG GCA CCA GAG CCA GAG GCG GAG GCA GAC GCG GAG GCA Tyr - A l a - A la -P ro - G l u - P r o - A l a - P r o - G l u - P r o - G l u - Ala - G b - A l a - Asp- Ala-Glu - A h

0

GAT CCG GAA GCG GGA ATT GGA GC4 GTT CTG AAG GTA ?TA ACC ACA GGA TTG CCC Asp - Pro - Glu - Ala - Gly - Ile - Glv - Ala - Val - Leu - Lvs - Val -Leu - Thr - Thr - Glv -Leu - PrQ

GCC CTC ATA AGT TGG A'IT AAA CGT AAG AGG CAA CAG GGT TAG.. . . Ala - Leu - Ile - Ser - Tro - Ile - Lvs - Are - Lvs - ATP - Gln - G h - Gly

***

FIG. 3 DNA and amino acid sequence of prepromelittin and melittin (underlined). The putative peptide bond which is hydrolysed by the signal peptidase is marked by an arrow.

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ATG AAG AAC TAT TCA AAA AAT GCA ACA CAC 'ITA ATT ACG GTT (7IT CTA I T C AGC TlT Met - Lys - Asn - Tyr - Ser - Lys - Asn- Ala- Thr - His - Leu - Ile -Thr - Val - Leu - Leu - Phe - Ser - Phe,

GTT G'IT ATA CIT I T A ATT ATT CCA TCA AAA TGT GAA GCC G'IT AGC AAT GAT AGG Val - Val - Ile - Leu - Leu - Ile - Ile - Pro- Ser - Lys- Cys- Glu -Ala - Val - Ser - Asn -Asp - Arg-

0

CAA CCA 'ITG GAA GCA CGA TCT GCT GAT I T A GTC CCG GAA CCA AGA TAC ATT A l T Gln-Pro - L e u - G l u - A l a - Arg-Ser-Ala-Asp-Leu-Val -Pro - G l u - P r o - Arg-Tvr -1le - Ile GAT G'IT CCT CCT AGA TGT CCT CCA GGT TCT AAA TTC A'IT AAG AAC AGA TGT AGA ASR- Val - Pro - Pro - Ara - Cvs - Pro - Pro - Glv - Ser - Lvs - Phe - Ile - Lvs - Asn - A r p - Cvs - Are GTC ATA GTG CCT TAA Val - Ile - Val -Pro- **rt

.. . .

FIG. 4 DNA and amino acid sequence of preprosecapin and secapin (underlined). The putative end of the signal protein is marked by an arrow.

1976). Vlasak et al. (1986) argue that there is caste-specific variation for this compound, because the venom of queen honeybees has a higher secapin content than worker venom. The sequenced secapin precursor (Fig. 4) corresponds almost exactly to the secapin amino acid sequence as derived from biochemical analysis (Gauldie et al., 1978). A gene coding for the enzyme phospholipase A2 has been sequenced by Kuchler et al. (1989). Phospholipase A2 is the principal allergen and a major compound (up to 15% of the dry mass) of worker venom. Surprisingly this enzyme is caste specific and almost lacking in queen bee venom (Marz et al., 1981). Using polyclonal antibodies, Kuchler et al. (1989) prepared a cDNA coding for phospholipase A2 from honeybee venom glands. The isolated clone had a 540 bp insert with a deduced amino acid sequence that showed substantial homologies to phospholipase A2 from lizards and bovine pancreas. The similarities are particularly obvious in a three-dimensional analysis of the molecule structure. The critical residues that are essential for the catalytic activity are conserved in both the bovine and the honeybee venom enzyme. Using Northern blot technology, Marz et al. (1981) were able to confirm the apparent lack of an mRNA coding for phospholipase A2 in queens: they only found an mRNA of about 850 bp in worker bee venom RNA extracts but not in queen RNA. In a Southern blot with total honeybee DNA, the cloned cDNA probe hybridized to a single 1600 bp fragment and the authors concluded that only a single, rather small gene codes for phospholipase in the honeybee genome. Finally a DNA region coding for the enzyme hyaluronidase, which has been described as a 'spreading factor' (Habermann, 1972) to facilitate the rapid diffusion of the other venom compounds, has been sequenced by Gmachl and Kreil (1993).

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2.1.4 In situ hybridization Although several genes are now known and have been sequenced from the honeybee genome, we are still lacking any information on a genetical map. The production of a classical linkage map of the honeybee has proven difficult in the past. Only very few linkage groups are known (Tucker, 1986; Del Lama et al., 1985, 1993), and we have no information on which chromosomes these linkage groups are located. This is mainly due to the large number of chromosomes but also to the difficulties in identifying each of the 16 chromosomes with classical G or C banding techniques (Hoshiba and Kusangi, 1978; Hoshiba and Okada, 1986). Chromosome identification is primarily done by size, but since the size differences are small, it is usualiy only possible to identify the largest chromosome, numbered 1. A first step towards a better characterization of the chromosomal set can be achieved through in situ hybridization. Beye and Moritz (1993) recently adopted the fluorescence in situ hybridization technique (FISH) to the honeybee system. A DNA probe of Drosophila containing the repeat coding for the rRNA subunits 28S, 18S, 5.8s and 2s (Tautz et af., 1988), including the intergenic sequences, was used for hybridization to honeybee chromosomes prepared from larval drone testes. The tandem repeat gene occurs at about 250 copies on the X chromosome and 200 copies on the Y chromosome of Drosophila. Similar tandem repeat gene structures have been found in many other organisms (Spear, 1974). In the honeybee this probe hybridized at the telomeric position of two chromosomes (Fig. 5 ) . In subsequent studies (Beye and Moritz, 1994) probes with repetitive DNA cloned from honeybee

FIG. 5 In situ hybridization of an rDNA D. melunoguster probe to a haploid set of metaphase chromosomes of A. rnellifera (courtesy of M. Beye).

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FIG. 6 In situ hybridization of a repetitive DNA probe of A . meilifera to a haploid set of metaphase chromosomes isolated from drone testes. The probe is centromere specific and the two chromatids are clcarly visible (Beye and Moritz. 1994).

genomic DNA revealed chromosome-specific patterns and could be used to unambiguously identify the chromosomal set. Another probe proved to be ccntromere specific for 14 chromosomes (Fig. 6).

3 Mitochondria1 genome Thc mitochondria1 genome of the honeybee is much better known than its nuclear counterpart. With a length of 16300-17000 base pairs it has been shown to contain all those genes we know from Drosophila yakuba, currently the only other insect for which the complete sequence of mitochondria1 (mt) DNA is known (Clary and Wolstenholme, 1985). The study of honeybee mtDNA genes started with the work of Vlasak et al. (1987), who sequenced the region coding for the large ribosomal RNA. Crozier et al. (1989) sequenced a region of 2950 bp in which they recognized genes coding for four tRNAs (tryptophan, Icucine, aspartate and lysine) and two genes coding for two cytochrome oxidasc subunits (CO-1. CO-11). Cornuet and Garnery (1991) identitied another tRNA coding for tyrosine at

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FIG. 7 Map and gene order o f the circular mitochondrial genome of A . rnel/iferu (modified and redrawn from Crozier and Crozier, 1992) and D . yakubu (modified and redrawn from CIary and Wolstenholme, 1085). The dotted areas represent non-coding A +T-rich regions. The Bcll sites are given for A . rnellifera. The asterisks indicate differences in the codons of DNA sequences coding for tRNAs (hatched areas).

the beginning of this 2950 bp region. from position 56 to 124, being transcribed, however, in the opposite direction. Furthermore, they detected another tRNA-like sequence, with a GGG anticodon between the tRNA"y and tRNAtrPgenes. Meanwhile the complete sequence of mtDNA of Apis meflijera ligustica has been analysed by Crozier and Crozier (1992) yielding precise information on the genes located on the honeybee mtDNA. Much of the following section is based on their results (the sequence can be accessed through Genbank #L 06178). A map of the Apis mellifera mitochondrial genome is shown in Fig. 7 and compared with that of Drosophila yakuba, for which the mtDNA has also been sequenced in full (Clary and Wolstenholme, 1985). The similarities are striking with an almost identical gene order of the 37genes in both species. The high A + T content is typical for both mtDNAs. For example the CO-I region is composed of 75% and 80% A T in D. yakuba and A. rnellifera, respectively (Crozier et a[., 1989). A total of 84.9% of the entire mitochondrial genome is either A or T. In spite of these strong similarities there are some significant differences that need special consideration. For example guanine is the rarest nucleotide in A . mellifera (5.5%) whereas in D. yakuba cytosine is the least frequent. In its low guanine content A. melliferu mtDNA resembles that of the echinoderm Paracentrotus lividus (Crozier and Crozier, 1992). Also the total size of A. mellifera mtDNA can be substantially longer than that of D. yakuba, as will be seen later in a discussion of the non-coding regions in the honeybee mtDNA.

+

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F. A. MORlT2

MITOCHONDRIAL GENES

Although the genes encoding proteins, the control region and the rRNAs are in the same order as in D.yakuba, 11 of the 22 tRNA coding regions are in different positions. These position shifts are all located between the A+T-rich region and the ATPase8 gene (upper left quadrant in Fig. 7). The overlap of the ND2 and tRNACy" genes does not occur in D. yakuba, whereas the other overlap between ATPase8 and ATPase6 is found in both species. Crozier and Crozier (1992) suggest that the CO-I and CO-I1 genes do not overlap although no complete T A A stop codon is found. Citing Ojala et al. (1981), they argue that mRNAs from mtDNA can be polyadenylated at the 3' end, in this way completing the otherwise incomplete termination codon. In fact this phenomenon seems to be very common: Wolstenholme (1992) reports such incomplete termination codons as a general feature of those animal systems for which the complete mtDNA sequence is available. An exception is the cnidarian Metridiurn senile which Wolstenholme (1992) interprets as an indication for an early origin of the cleavage-polyadenylation mechanism. Initiation codons either code for methionine (three ATG and three ATA) or isoleucine (one ATC and six ATT) and no anomalous codons, like ATAA in D. yakuba (Clary and Wolstenholme, 1985), were found. All but the two stop codons for CO-I and CO-I1 (a single T) are TAA Surprisingly the A+T-rich region seems to lack the typical structure for the initiation of replication as found in vertebrates. Another A+T-rich duplicate region is found between the tRNA'"" and the CO-I1 region (Cornuet et af., 1991). However, it is as yet unclear whether this region can have replicate activity (see below), because some species and races of Apis seem to lack this duplication. The tRNA genes have striking similarities to the sequence of D. yakuba (Clary and Wolstenholme, 1985). The anticodons are identical but for the (TIT instead of CTT) and tRNAS" (TCT instead of GCT). tRNALayS Crozier and Crozier (1992) inferred the secondary structure of all tRNAs, and found a high A + T content (87.1%) which is higher than for the entire mitochondria1 genome (84.9%). Furthermore, they found various cases of base pair mismatch which has been shown to be a common phenomenon also in other insects (Clary et af., 1982, 1984; Clary and Wolstenholme, 1983a,b, 1984; DeBruijn, 1983; HsuChen and Dubin, 1984; Uhlenbusch et af., 1987). Four tRNAs coding for asparagine, leucine, threonine, and valine are characterized by an unusually long TUrCG stem with six nucleotide pairs (Fig. 8). Moreover, the TUrCG loop of the tRNATh' is surprisingly long (10 nucleotides). Typical, however, is the lack of the DHU arm in the tRNASer which is very likely to be universal (Wolstenholme, 1992). The genes coding for the large and small ribosomal unit and the 13 protein coding genes are listed in Table 3. Again we find strong similarities

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TABLE 3 The protein coding genes and the large and short rRNA coding regions of the Apis rnellifrra mitochondria1 genome. Nucleotide composition, sequence similarity to Drcxophila yakuba, and number of nucleotides Gene ATPase8 ATPase6 Cytochrome oxidase I Cytochrome oxidase I1 Cytochrome oxidase I11 Cytochrome b NADH dehydrogenase 1 NADH dehydrogenase 2 NADH dehydrogenase 3 NADH dehydrogenase 4 NADH dehydrogenase 4L NADH dehydrogenase 5 NADH dehydrogenase 6 Small rRNA Large rRNA

3' 6 A + T

% similarity to D. yakuba

Number of nucleotides

90 85 84 81 84 69 86 87 87 94 86 87 87 81 85

62 74 68 66 66 64 51 63 64 57 61 53 68 73"

64

156 678 1560 675 777 1149 915 999 35 1 1341 26 1 1662 501 786 1371

Data from Crozier and Crozier (1992). 'The similarity is slightly higher than given by Crozier and Crozier (1992) after improving the alignment of the D.yrkuba and A . meNiferu sequence.

to the D. yakuba genome. Similarities between D . yakuba and A . mellifera range from 51% to 75%. The G+C/A+T ratio is much higher in D. yakuba (0.43; Jukes and Bhushan, 1986) than in A . mellifera (0.18; Crozier and Crozier, 1992). This strong A + T bias in the honeybee mtDNA genome causes some differences in amino acid usage compared with Drosophila. For example alanine in Drusophila is replaced by serine in the honeybee at 43 sites (conserved at 42 sites). The most conserved amino acid appears to be arginine with 95% of the honeybee sites being conserved in Drosophila and 63% of the Drusophila sites being conserved in the honeybee.

3.2

NON-CODING S E Q U E N C ~ SA N D LENGTH VARIATION

Smith and Brown (1990) were the first to report on length variation and detected six locations with variable base pair numbers. Two sites showed small variability (20 bp) whereas the other four sites varied by about 100 bp. The small size variability is located within the C O - K O - I 1 region and can be easily detected o n Bcll digests. Cornuet and Garnery (1991) provided a plausible explanation for this length variation. Exactly 20 bp upstream of the Bcll site e (see Fig. 7) the following motif can be found: ..TGACCA ... With

A T A A A T T G A T A A C A T A

A A A A A A T

T

T T A G T T T G

T T T T T T A A

A A T C A

A

A

T T A A A T A A T T T A

A

T

C T

T A A

A A

T

G-T T A T A A T T A T A T A T T A T A A A A T T A A T A T A C G T T A A T T T T Q T G C A C T A A T T T A T A T A A T A T A T T A T G T A G

C

t RNA ASP

t RNA Leu Ja

7

D

A A T

G C C G

A A A A A T T T T G T A A A C A T A A

T

T A A A

A T T T

A T T A T A T A A T A A T T T A T

A A T A T A A T A T T A T A T A T G T

T

C

A A

T A

~

~

A G

T TA

A T T G T A A ?

A

T

A

T T

A A A A T T T

T T T C T

A T T T T A A A G A T A T T T T T T A A A A A A T T A A G A A A G T G

A

T A C

t RNAThr FIG. 8 Cloverleaf structures of four tRNAs with 6 bp T q CG stems. The T q C G loop of the tRNAThr(bottom left) is exceptionally long.

t RNAVal

I 0

z rn

<

m rn rn

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just a single transition at the third position of the GAC codon (coding for leucine) to G A T (also coding for leucine) to ..TGATCA ... a new Bcfl site is obtained. Combined with the loss of the Bcll site e, the gain of this new site could plausibly explain the two size variations without many presumptions. Other scenarios, however, are also possible and as long as the actual size variants have not been sequenced, the underlying mechanism will remain in the realm of speculation. The A . rnelfifera mitochondrial genome is 300-800 bp longer (depending on size variants) than that of D. yakuba, although the control region has been found to be 151 bp shorter. The larger total size is mainly due to non-coding intergenic sequences. As many as 618 non-coding nucleotides were found in the complete sequence data for the shortest Apis mitotype found in Apis mellifera ligustica (Crozier and Crozier, 1992) whereas only 183 were counted in D.yakuba (Clary and Wolstenholme, 1985). Particularly, the intergenic sequences (up to 831 bp; Cornuet and Garnery, 1991) between the tRNA"" and the CO-I1 genes are almost completely lacking in D.yakuba ( 5 bp only). Large mtDNA size variability is usually found near the control region of the mitochondrial genome (Moritz and Brown, 1987). According to Cornuet and Garnery (1991) two of the larger size variations found in honeybees are also located in the A+T-rich region which may function as the control region. Smith and Brown (1990) suggested that a variable number of tandem repeats between 80 and 100 bp causes this size variation. Yet again sequence data are lacking to support this hypothesis. The best studied size variability is between the (20-11 region and the tRNAl'" gene of the mitochondrial genome, and this has been found to be polymorphic in both European (Cornuet et a f . , 1991) and African subspecies (Garnery et al., 1992; Meusel and Moritz, 1992; Moritz et al., 1994). Cornuet et ul. (1991) analysed this region in detail and found repetitive sequences in the intergenic region between the tRNA"" and the CO-11 genes. They could identify two types of repeats which they named P and Q. Q is an obligatory sequence of 194-196 bp length found in all bees so far tested (Fig. 9). P with a length of 54 bp is facultative and is also present in a form Po (Cornuet and Garnery, 1991) with a 15 bp deletion which seems to be typical for the African subspecies of A . mellifera (Garnery et a / . , 1992) (Fig. 9). P is completely composed of A + T , whereas in the Q sequence 7.3% G + C is found. The size variation observed in several subspecies of honeybees is composed of various combinations of these two repeats. Whereas the Q repeat is variable within species, the P sequence has been found to be typical for various subspecies (Garnery et al., 1992). Table 4 lists all those types found to date. The repeat Q is composed of three subunits, Q,, Qz, Q3, which have conspicuous similarities with adjacent DNA regions. Cornuet and Garnery (1991) suggest that Q I might have diverged by duplication from the 3' end of

COI

I tRNAleuI

P

I

Q1 Q2 Q3

I COII 0 0

41-I

I

m

I

0

6 < W

rn rn

P

TTAATAAATTAATAT~TAT~T---------------TATATTTATTAAATTTAATTTATTAAA

******************* ******

...........................

TTAATAAATTAATAT~T~TAT~TATATTTATT~~TTAATT~ATTAAA Q3

FIG. 9 Gene order in the CO-I-CO-I1 region of the mitochondria1 D N A of A. meffifera.The intergenic region between tRNAL" and CO-I1 consists of a fragment P and one or various repeats of fragment Q which is composed of three subunits Q1, Qz, and Q3. The sequences of the intergenic fragments are shown below. P has a high similarity to Q3, and Q, closely matches the 3' end of CO-I. The similarity of Qz to tRNAL" is shown in Fig. 10.

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TABLE 4 Apis rnellifera subspecies and mitotypes Type of length variability

Subspecies

Reference

Q

carnica caucasica ligustica lamarc kii

Cornuet et al. (1991) Crozier and Crozier (1992) Crozier et al. (1989) Garnery et al. (1992) Smith (1991)

rnellifera iberica

Cornuet et al. (1991) Garnery et al. (1992)

scutellata capensis rnonticola unicolor

Cornuet et al. (1991) Hall and Smith (1991) Moritz and Meusel (1992) Smith (1991) Moritz et al. (1993)

scutellata

Moritz et al. (1993)

~~

X is a yet unknown sequence following the P,, fragment.

the CO-I gene, whereas Q2 bears strong similarities to the tRNA"" 3' end. In particular, the putative secondary structure of Q2 reveals striking similarities to the adjacent gene (Fig. 10). The aminoacyl arm, anticodon stem, and the T q C G stem are almost identical (16 out of 17 nucleotides). The three loops, however, differ substantially. The DHU loop of Q2 is twice as large in the corresponding tRNA'"" sequence, whereas the T K G loop lacks four nucleotides. Cornuet et af. (1991) suggest that the large DHU loop may impede the transcription of a functional tRNA. Q3 is very similar to Po and thus offers no such simple explanation for its evolutionary origin. Cornuet et af. (1991) argue that the entire region may function as an additional origin of replication finding support by the high A + T content (92.2%), and the secondary hairpin and cloverleaf structure. Such an additional origin of replication is surprising, and it is clearly lacking in D. yakuba, the closest species relative to honeybees for which complete sequence data are available. In fact a second origin of replication is uncommon in the animal systems studied so far (Harrison, 1989). Crozier and Crozier (1992) expressed doubts as to whether this highly variable region could function as an additional origin of replication. In attine ants the same region has been found to be extremely variable and sometimes completely lacking (Wetterer, cited in Crozier and Crozier, 1992). Yet, although this sequence may be lacking in ants, the Q sequence is present in one copy at least in all honeybees tested so far, thus leaving the issue still open.

T

A A G

A C G

G

T T T A A T A

A A A A T T A T

A A T T T T C A T A A A G T T

G C A T A T C A T G T A A

A

A T

T

T

T

T A

T A

A

A A A T A T T A T T A

T

T

T T T A A T T A

A A A A T T A A T

41 -I

A A T T T C T T A A A G A T A T T A T C A C T T A A

T T G A A T T T T A

t RNA Leu FIG. 10 Inferred cloverleaf structure of tRNALe"and the intergenic fragment Q2. Note the sequence identity in the aminoacyl arm, the anticodon stem and the W C G stem. Sixteen out of 17 nucleotide pairs are identical.

42

I rn

I

0 Z

rn

-c

m

rn

rn

124

R. F. A. MORlTZ

4 Gene activity in embryonic development

The segmentation of the embryo is one of the best understood steps in insect embryogenesis. In particular the findings in Drosophila rnelanogaster have given an important insight into gene regulation in early embryonic development (Niisslein-Vollhard and Wieschaus, 1980; Nusslein-Vollhard er ul., 1982). The pattern of gene activity corresponds to the external rnetameric subdivision in the larval body (Martinez-Arias and Lawrence, 1985; Akam, 1987; Lawrence et al., 1987 among others). The genetics of segmentation has also been studied in other insects, like the flour beetle, Tribolium custuneurn (Beeman et al., 1989), and the grasshopper (Patel et al., 1989).In honeybees Fleig et al. (1988) were the first to study gene expression of segmentation genes. The honeybee offers various attractive features as a model system to study embryogenesis. The potential banding and segmentation patterns can be more easily interpreted since the head of the honeybee embryo is not involuted as in Drosophilu. Furthermore, the gastrula of the honeybee is straight and not folded like in Drosophila. Gastrulation starts about 33 h after oviposition (Fleig and Sander, 1986). The first metameric units become visible at this very early developmental stage as slight transverse grooves in the gnathal and thoracic region. The most anterior groove is slightly angled and separates the antennae anlagen from the intercalary segments. By using an antibody staining technique, Fleig (1990) showed that the expression of the engrailed gene product directly correlates with the groove pattern in this early developmental stage. An alternating pattern of intense and weak stained stripes was found along the gastrulating embryo. The intensely stained mandibular, labial, and mesothoracic stripes were about two to three cell rows wide, whereas the faintly stained stripes of the maxillary and the prothoracic region consisted of a single irregular row of cells. As gastrulation continues, the engrailed stripes are seen along the whole body, appearing and vanishing one after the other in an antero-posterior sequence. The alternating pattern is replaced by a regular striped pattern of three cell rows wide coinciding with the rnetarneric groove pattern. The labial stripe, however, remains broader whereas the procephalic stripe is very weak and has only a few stained cells. After mid-gastrulation all nuclei in the pre-serosa (and later in the serosa) show engrailed activity, which has not been described for any other insect but the honeybee. During the germ band stage, and in prehatching larvae, engrailed activity persists. Activity is particularly strong in the four cell row just anterior to the bottom of each metameric groove. The general pattern of engrailed expression in Apis rnellifera is similar to that found during embryonic development of other arthropods (Kornberg el al., 1985: DiNardo et a!., 1985; DiNardo and O’Farrell, 1989; Patel et ul.,

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1989; Karr et af., 1989; Foe, 1989). The development of the stripe pattern and its topography on the embryo is somewhere between those found for Drosophila and Schistocerca. The honeybee has proved to be a most useful test system because of its size, its availability, and the straight structure (lack of head involution and no abdominal folding) of the embryo. In the future these features might even allow a direct analysis of the actual intercellular control processes that regulate the early metameric groove pattern in insects in general.

5 Population variability

Classically, genetic variability in populations has been analysed with isozyme polymorphisms. Although this technique has proved very powerful for the study of many insect populations, isozyme analysis in honeybees has often posed a problem. Honeybee population geneticists suffered for a long time from a notorious lack of suitable isozymes which revealed only a few polymorphisms (Sylvester, 1986). This was often attributed to male haploidy (Pamilo et af., 1978). However, Gan ef af. (1991) found 10 enzyme systems to be polymorphic in honeybees with an average degree of heterozygosity of 0.137. Thus it is also possible that the choice of gel running conditions was not always optimal in previous studies. The dynamics of gene frequency changes in honeybee populations can offer more ways of analysis than solitary insects can. Most interesting is the dual system of individual and colonial reproduction which results in potentially conflicting evolutionary strategies between workers, the sexual reproductives, and the colony as a whole. To examine the various individual and colonial fitness parameters one needs suitable genetic markers. This has proved to be a problem in the past when using allozyme markers. The progress in molecular analytical techniques, however, has allowed the direct utilization of DNA variability for population screening. Both nuclear and mitochondria1 DNA studies have been repeatedly conducted in the past few years. The analysis of exclusively queen-inherited markers, in combination with markers passed on by both sexes, can be studied by combining both nuclear and mitochondria1 DNA analysis. Such an approach is extremely helpful to dissect the selective importance of individual and colonial reproduction in honeybee populations. 5.1

NUCLEAR DNA MARKERS

Various nuclear DNA markers have been used to document genetic variability at the population and the colony level.

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R. F. A. MORlTZ

5.1.1 Variability at the population level Usually nuclear DNA variability is detected through restriction fragment length polymorphisms (RFLP) which are visualized by hybridization to a labelled DNA probe. Classically the DNA probe is isolated from a gene bank. Hall (1986, 1988) was the first to use random fragment probes of honeybee DNA cloned with the plasmid pBR322 in Escherichia coli. Hall (1990) found that 19 of his isolated probes were useful for discrimination between Africanized and European honeybees. He explicitly stressed the usefulness of three probes (P130, P138, P170) in this context. However, although Hall (1990) was able to reveal DNA variability among various subspecies, the discriminating power of the probes was limited. The frequency for the ‘typical’ European allele was either 0 or 1 for the probes P130 and P170, and probe P138 was only found at a frequency of 0.06 in Apis mellifera mellifera. This high degree of variability for a diagnostic allele was obtained from a small sample size of two to four colonies per European race. Even given that the only goal was to find discriminative alleles between the Africanized honeybee and US-American ‘European’ honeybees (a racial mix of honeybees imported from Europe), the discriminatory power was unsatisfactory. In honeybee populations sampled in Arizona and Florida (20 and 15 colonies, respectively), ‘European’ gene frequencies ranged from 0.70 to 0.83, indicating substantial polymorphisms in each of the samples. In statistical terms this allows for a one-sided- test only. The presence of a ‘European’ allele may qualify a honeybee as ‘European’; the lack of this allele, however, does not qualify a honeybee as ‘African’. Clearly a polymorphic locus has only limited power as a diagnostic test system to discriminate between two subspecies and further screening seems to be desirable to isolate DNA probes with better discrimination ability. Hall was well aware of this problem, and I fully agree with him that ‘future research to establish the nuclear DNA RFLP specificity among pure European races (including A . m. carnica and A . m. iberica) should prove valuable’ (Hall, 1990, p. 618). However, also in follow-up studies (Hall, 1992; Hall and McMichael, 1992), no unambiguous diagnostic and racespecific probes were found, although there were significant frequency differences in the different subspecies for various alleles. For example, allele 2A2-B was found at frequencies between 0.83 and 0.91 in African but only at 0.25 in west European bees. Nevertheless, such variability is inappropriately high for the use of these probes as diagnostic tools for the identification of unknown honeybees. Multivariate analysis based on allelic variability at several loci is likely to solve the problem but has not been utilized for molecular data so far. Genomic DNA probes, as found in Hall’s studies, are very powerful tools for studying the dynamics of population genetics, and they are very helpful for understanding the problem of the Africanized bee problem in the

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Americas, a subject to which I will return in detail later in this review. Currently the use of genomic DNA markers for the classification of racial types is premature. Successful and correct discrimination between European and African honeybees based on a few alleles only is very unlikely in the light of the vast number of highly diverse subspecies on each continent (Ruttner, 1988). Another approach to reveal intrapopulation variability was presented by Estoup et al. (1993). They isolated a series of microsatellites from a genomic library of the honeybee. One microsatellite which was composed of 13 CT tandem repeats associated with 9 G G T repeats proved to be highly variable in a population of A . mellifera. Using the flanking primers of the microsatellite, they found a strong length polymorphism in the PCR products yielding a total of 13 allelic forms. The same primers yielded considerably shorter PCR products in other honeybee species like A . cerana, A . florea and A . dorsata, which were not screened for intrapopulation polymorphism. 5.1.2 Variability within the colony Intracolonial genetic variability is high in honeybee colonies. This is due to the high degree of polyandry of the queen, with up to 20 drones mating a queen (Adams et al., 1977). As a consequence, up to 20 worker subfamilies or patrilines may coexist in the same colony. Since all workers are offspring from the same mother queen, workers with the same father are related by r = 0.75 (super-sisters), and those with different fathers are related by r = 0.25 (half-sisters). This intracolonial genetic variability is of particular interest for evolutionary and sociobiological aspects of honeybee biology. On the one hand the low genetic relatedness between patrilines might cause conflict in the colony, if workers recognize super-sisters and behave nepotistically. On the other hand genetic diversity in the colony may be advantageous (Moritz, 1989). For example genetic task specialization has been shown to be an important factor for division of labour in the colony (Page and Robinson, 1991) and genotypic intracolonial variability has been suggested to be an important fitness parameter for groups of bees and colonies (for recent reviews of these aspects of honeybee biology see Breed and Page, 1989; Moritz and Southwick, 1992). Until recently only isozyme or phenotypical mutant markers were used in studies to reveal patriline identity. This had many drawbacks because of the limited number of patrilines that can be tested with allozymes and the use of artificial insemination. Honeybee DNA probes have proved to be very useful in a study focusing on intracolonial variability. Various techniques have been tested and have shown the great potential of molecular tools to increase our understanding of the function of the honeybee colony as a whole. In a recent study by

128

R. F. A. MORITZ

Oldroyd et al. (1994), a DNA probe from a genomic library of the honeybee proved useful to identify different patrilines in a colony of A . florea, the Asian dwarf honeybee. They could identify as many as eight different RFLP patterns suggesting a mating with about eight drones. This is a surprisingly high number in the light of observations by Koeniger et al. (1989) who claimed that only a very limited number (less than five) of A . frorea drones mate with the queen, based on semen counts of queens returning from their mating flight. They found no semen in the oviducts and concluded that the A . frorea drones deposit their semen directly into the spermatheca with the complexly structured endophallus. The DNA data of Oldroyd et al. (1994) do not support this view, and the estimate of eight matings may be conservative since rare patrilines may not have been detected and some drones might have had the same RFLP type. Oldroyd et a f . (1994) could also confirm those observations made by Page and Robinson (1991) in A. meffifera, who found a variety of genetically determined specialists in the colony. Another approach using molecular tools for the identification of patrilines in a colony of honeybees has been DNA fingerprinting. Moritz et al. (1991) could discriminate between super- and half-sisters in a colony by using multilocus fingerprinting with the synthetic (GATA)4 oligonucleotide. Using the same oligonucleotide, Haberl and Moritz (1994) found 12 different subfamilies in a colony of A . rnellifera. A similar result was obtained by Blanchetot (1991) who used M13 as a probe for DNA fingerprinting. He was able to identify 11 patrilines in a single colony. Both figures are conservative in that patrilines may not have been identified if two drones had the same genotype and if the frequency of a patriline in the colony was very low. The genetic effective number of males was lower than the number of subfamilies found, because they were not equally represented. Twenty-five per cent of the workers belong to the most frequent subfamily, whereas rare subfamilies had frequencies of less than 2%. Using the estimator for the effective male number of Laidlaw and Page (1984), N , is between 6 and 7. Both empirical studies deviate substantially from the previous widely accepted estimate of an average of 17 effective matings per queen (Adams et a f . , 1977), which may be either an overestimate or a specific trait of the sampled Africanized honeybee population in South America. DNA amplification using random amplified polymorphic DNA technique (RAPD) has also been used to identify paternity in the honeybee colony. Hunt and Page (1993) and Fondrk et a f . (1993) used a 10 bp random primer with 50% GC content to screen a colony. They could clearly reidentify the four patrilines in a queen artificially inseminated with four drones. Some interpretational problems arose from non-paternal bands that resulted from chimaeric amplification products, known as PCR recombination. Nevertheless, Fondrk et al. (1993) could unambiguously classify each individual worker on the basis of 20 markers only.

MOLECULAR BIOLOGY OF THE HONEYBEE

5.2

129

MITOCHONDRIAL DNA MARKERS

Mitochondria1 DNA has been found to be inherited exclusively maternally in most animal systems (Moritz and Brown, 1987). In several cases, however, this rule might have been broken since heteroplasmic individuals have been found in various organisms. Brown el a f . (1992) found 42% heteroplasmy in sturgeon, and Hoeh et a f . (1991) observed 57% heteroplasmic individuals in Mytifus mussels. Kondo et a f . (1990) showed that in three out of 331 lineages paternal mtDNA replaced the original mtDNA in artificial crosses in Drosophifa. Gyllenstein et a f . (1991) reported on paternal leakage in mice. Looking at the special fertilization mechanism of the egg, honeybees seem to be another candidate for a significant paternal mtDNA inheritance. Apis meffifera has a polyspermic mode of fertilization, with many sperms entering the egg, including the mitochondria-rich tail (Blochmann, 1889; Petrunkewitsch 1901). Although many sperms have been found to enter the egg, only one of them fuses with the egg nucleus. The others remain as accessory sperms in the egg and usually disintegrate rapidly after the first cell divisions (Nachtsheim, 1912, 1914). Only rarely do they show mitotic activity yielding gynandromorph individuals (reviewed by Woyke and Hillesheim, 1990). Before an mtDNA marker can be used as an authentic maternal marker

30%

proportion paternal mtDNA I

20%

10%

0% 0

12

24

36

48 60 72 development (hrs)

84

96

108

120

FIG. 11 Decrease of paternal rntDNA after fertilization of the egg. When the larvae hatch after 96 h only traces of paternal rntDNA can be detected.

R. F. A. MORITZ

130

in honeybees, it seemed necessary to test whether any paternal leakage of mtDNA could contribute to mtDNA variability in the population. Meusel and Moritz (1993) pursued this in a study using hybrids of two races utilizing length polymorphism in the CO-I1 and tRNA"" region (see Section 2.3). They inseminated an A . meflifera capensis queen (0.4 kb insert) with A . meflifera carnica drones that did not have this insert and tested the progeny at various larval stages. Initially they found up to 23% paternal mtDNA in freshly laid eggs, but with ongoing development paternal mtDNA rapidly decreased (Fig. 11). In late larval stages only maternal mtDNA was found, indicating that in spite of the large initial paternal contribution only maternal mtDNA is genetically effective. The loss of paternal mitochondria has been reported for a variety of organisms. For example Anderson (1968) observed the degradation of paternal mitochondria in the sea urchin, Paracentrotus lividus, and even in isogamous fungi, in which mtDNA recombination can occur, the rapid disappearance of one parental type has been observed (Meland et al., 1991). In honeybees mtDNA seems to provide a safe tool to study maternal gene flow in populations. Several markers are available and particularly the size variants listed in Table 4 have been used in the past to characterize populations and racial types of the honeybee. 6 Molecular evolution and biogeography

6.1

MOLECULAR PHYLOGENY OF BEES

Besides studies at the population level molecular techniques are also helpful in phylogenetic studies. Similarities between different taxa can be evaluated by comparing RFLPs, restriction site maps or sequence data of specific DNA regions. The now classical techniques of using highly conserved ribosomal DNA have also been applied to honeybees, but with only limited success. Sheppard and McPheron (1991) report on a paucity of 18s rDNA variation in the genus Apis, not allowing for any phylogenetic analysis. Nevertheless, they did find sequence variability at the tribal level of the Apidae which could be used to produce a phylogenetic tree. Indeed, the need for a molecular approach to the phylogeny of the Apidae seems highly promising, because the classical phylogenies based on morphological or behavioural traits vary substantially among a variety of authors (see Fig. 12) The results from the 18s and 28s ribosomal DNA (Sheppard and McPheron, 1991) are based on seven informative sites and clearly more information is needed to get a more precise picture of the possible phylogenetic trees. Using the branch and bound routine of the PAUP 3.0 software package (Swofford, 1990) they found a parsimony tree as shown in Fig. 12 which differed from a similar tree produced by Cameron (1991; also using branch

131

MOLECULAR BIOLOGY OF THE HONEYBEE Euglossin i

Born bini Meliponini

Euglossini

Born bini Meliponini Apini

Melipon in i

<

Euglossini Meliponini

$

!!‘;::1

Born bini Apini

Euglossini Apjni

Meliponini

Meliponini

Born bini

FIG. 12 Alternative phylogenetic trees of the four tribes of the Apidae. (A) Michener (1944, 1990); (B) Michener (1974); (C) Winston and Michener (1977); (D) Plant and Paulus (1987); (E) Sheppard and McPheron (1991); (F) Cameron (1991). The phylogenetic trees E and F are based on sequence data from mtDNA (rRNA).

and bound of the PAUP 3.0 package) based on mitochondria1 DNA variability (Fig. 12). Although in both studies bumblebees and stingless bees are more similar than the Apini and Euglossini, the topologies of the two most parsimonious trees are different, indicating the need for complementary data in support of one or the other derived phylogenetic tree.

6.2

GENETIC VARIABILITY AMONG HONEYBEE SPECIES

Genetic variability within the genus can also be revealed using mtDNA variability. These studies are of particular interest because of the recent rediscovery of various ‘forgotten’ species of Apis. Maa (1953) recognized as many as 24 species divided into three genera: Micrapis (dwarf honeybees), Megapis (giant honeybees) and Apis (cave-breeding honeybees). Subsequent papers, however, ignored or criticized Maa’s system as unwarranted by the data set. For decades the genus Apis was composed of only four species (Ruttner, 1988), until the work on Asian honeybees was reintensified. Today Apis andreniformis (Wu and Kuang, 1987; Wongsiri et al., 1990) and Apis koschevnikovi (Koeniger et al., 1988; Tingek et al., 1988; Rinderer et al., 1989; Ruttner et al., 1989) are well established species, renewing the interest in the system originally developed by Maa. In spite of this interest, the molecular contribution towards a new systematics of the

132

R. F. A. MORITZ

Duplication Po

9 A. mellifera

Elongation

co

I1

A. c e r a n a

Regression

co

I

co

Regression

tRNA leu

A. d o r s a t a I1

A. florea

modification of leu

FIG. 13 Putative evolutionary patterns of the mtDNA CO-I-CO-I1 intergenic region in Apis (redrawn from Cornuet et al., 1991).

genus Apis has been scarce. Smith (1991) found that mtDNA of A . koschevnikovi and A. melliferu were both highly divergent from A . cerana samples, with more than 10% sequence divergence between each of the species. Garnery et ul. (1991) screened mtDNA of A. melliferu, A. cerana, A. floreu and A . dorsutu. They sequenced the intergenic region between the tRNA"" region and the CO-I1 gene, and found sequence divergence ranging from 7% to 11%. Based on the sequence data, Cornuet and Garnery (1991) present a most parsimonious phylogenetic tree, supporting the early divergence of A . floreu, and the close relationship of the two cave-breeding species A. melliferu and A . cerunu, which is in agreement with morphometrical, behavioural (Alexander, 1991) and allozyme data (Sheppard and Berlocher, 1989). Cornuet and Garnery (1991) developed a putative evolutionary pathway of the mtDNA region sequenced by Garnery et al. (1991), explaining the evolutionary changes through DNA duplication, elongation, and regression (Fig. 13). Cornuet and Garnery (1991) extended the sequence data 185 bp into the region of the large ribosomal unit yielding a total of 61 informative sites. Based on both a neighbour joining (Saitou and Nei, 1987) and a parsimony analysis (PAUP 3.0 software package) they obtained phylogenetic trees with Bombus lucorum as an outgroup, confirming the topology of the tree presented in Fig. 13. Cornuet and Garnery (1991) suggested that both cave-breeding species A. melliferu and A . ceruna diverged about 5.9 million years from their common ancestor. Willis et ul.

MOLECULAR BIOLOGY OF THE HONEYBEE

133

(1992) also presented a phylogenetic tree on the basis of mtDNA variability. They analysed the CO-I1 sequence of six different species, including A . koschevnikovi and A . andreniformis, using the CO-I1 sequence of the wasp (Excrisres roborator) as an outgroup. The most parsimonious tree derived in this study has a different topology. A. dorsara is considered as the most ancestral group which is in contradiction to Garnery’s results (Garnery er al., 1991; Cornuet and Garnery, 1991). The most surprising result is the placement of the cave-breeding A . koschevnikovi together with the two free breeding dwarf honeybees. Willis et a f . (1992) admit that this result is rather puzzling since the cave-breeding species are considered to be very similar, behaviourally , morphologically and ecologically (Smith, 1991). Also the low bootstrap value for the fork between the closely related A . meflifera and A . cerana (45.1%) may be an indication that additional data are required to obtain plausible phylogenetic trees. One would not necessarily need to share Willis er af.’s (1992) view, that because the oldest fossil bee is of a dorsara type (Culliney, 1983) therefore this supports A . dorsara as the most ancestral type. Also Ruttner (1988) found in a morphometrical study that the fossil Apis armbrusleri (10-12 m.y. b.p.) has a wing venation pattern very similar to that of A . dorsara. Although such evidence shows that dorsaru-type bees did exist in the lower Miocene, this does not mean that florea type bees did not exist. It could well be that an olderflorea-like fossil exists but has not been found or not been dated. The controversial results make it very clear that more molecular data are required to present a concise picture. Particularly in the light of the high A T content and the rapid evolution of honeybee mtDNA (Crozier er al., 1989), it seems wise to include other genes in a phylogenetic study as well and not only rely on a sequence analysis of a single mitochondria1 gene. 6.3

MOLECULAR VARIABILITY WITHIN SPECIES

The majority of mtDNA research has been used to study genetic variability within species. Mitochondria1 DNA has also been successfully used in a variety of studies characterizing subspecies and the biogeography of honeybee populations.

6.3.1 Apis cerana Smith (1991) constructed a distance tree based on RFLPs of various A . cerana populations (Fig. 14). Based on this information she suggested an evolutionary scenario in which the subspecies were connected during the late Pleistocene. At this time the sea level was about 120m lower and the Sunda Shelf, including Borneo, Sumatra and Java, was connected to the mainland explaining the close relationship (0-1.9% sequence divergence) among the populations found in Borneo, Malaysia, Japan, Thailand and

R. F. A. MORlTZ

134

M a 1a y si a

-

Ma1ays i a Borneo

India Thailand

A . florea

6

4

2

0

% sequence divergence

FIG. 14 UPGMA distance tree of Apis ceruna populations (redrawn from Smith, 1991).

India. Populations that were isolated on islands in that time period (Luzon, Andaman Islands) showed substantially higher sequence divergence from the other populations (2.9-5.6% divergence). Both other cave-breeding honeybees ( A . koschevnikovi and A . rnellifera) showed a sequence divergence of more than l o % , which is much higher than any intraspecific distance among subspecies.

6.3.2 Apis dorsata The systematics of the giant honeybees is controversial (as is systematics for most organisms). Maa (1953) recognized four different species which he grouped in a separate genus Megapis. Ruttner (1988) worked with only one species and gave the different types subspecies status. Sakagami et al. (1980) accepted Apis luboriosa of the Himalayan highlands as an individual species of giant honeybees in addition to Apis dorsata. Alexander (1991) and Otis (1991a) discussed the ambiguity of the dorsata group, forming either a genus-like group of four species or a single species with four subspecies. Smith (1991) favoured Ruttner’s (1988) nomenclature for her studies on mtDNA variability among the giant honeybees. Nevertheless, the sequence divergence she found between the different haplotypes was much larger than for any other honeybee species. She estimated as much as 12.23% sequence divergence between A . dorsata binghami and A . dorsata dorsata from the Asian mainland. This is a substantial difference for bee populations living in

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India India India India Borneo Ma lays i a Thailand Pakistan

I. d. dorsata

Malaysia Andaman Is.

A . d. b i n g h a m i

Sulawesi

1

10

8 6 4 2 % s e q u e n c e divergence

0

FIG. 15 UPGMA distance tree of the Apis dorsata group (redrawn from Smith, 1991).

the same geographic region. Apis mellifera from northern Europe has an estimated sequence divergence of 3% to A . m. capensis from South Africa (Smith, 1991). Although the large variability in the dorsata group does not in itself explain whether we are dealing with true species or subspecies, it clearly indicates that the giant honeybees are a much more diverse group than the cave breeding honeybees (Fig. 15).

6.3.3

Dwarf honeybees

Currently two species of dwarf honeybees are recognized, Apis florea and Apis andreniformis. Apis florea is found from the Persian gulf to Thailand whereas A. andreniformis is found in tropical Asia east of this distribution area. Smith (1991) screened samples from five different origins to determine sequence divergence (Fig. 16). Interestingly the distance between the two species is less than half of that of the giant honeybees! This certainly gives no support to the four subspecies classification of the dorsata group as presented by Ruttner (1988). This is particularly so because the dwarf bees are much poorer dispersers than are the giant honeybees. On the other hand, the data are still of very preliminary character, based on a scarce and limited sample set.

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Borneo Malaysia Japan Thailand India Luzon

I

Andaman Is.

1 5

4 3 2 1 % sequence divergence

0

FIG. 16 UPGMA distance tree of the dwarf honeybees (redrawn from Smith, 1991).

6.3.4 Apis melliferu

The western honeybee, A. melliferu, is certainly the best studied honeybee species concurring with its mtDNA variability. The most striking variability is the length variation in the CO-I-CO-I1 region which is composed of various numbers of tandem repeats as discussed above (Garnery et ul., 1992). There are three distinct haplotypes comprising a fragment P (lineage M), the African subspecies with a fragment Po which is 15 bp shorter (lineage A) and a lineage C lacking the P fragment altogether. Based on these data and additional restriction site and sequence data, Garnery et ul. (1992) developed a new phylogeographic tree for the species A. melliferu. Ruttner (1988) concluded from his biogeographic studies a Y-shaped branching in northern Africa. Two branches dispersed westward north and south of the Mediterranean, and one spread southward into central Africa. In this model west European A. melliferu melliferu were at the end of the branch along the North African coast, bridging to Europe at Gibraltar. East European A. m. curnicu were morphologically very different from A. m. melliferu and thought to be the end of the branch spreading at the south European Mediterranean coast, both subspecies divided by the Alps. Garnery et ul. (1992) constructed a triple-branched phylogeographic tree with the origin in Iran. They found that lineage A is more similar to C than A to M. Furthermore, C is less similar to M than to A (Fig. 17). This clearly

137

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3

I

FIG. 17 Biogeography of the lineages of Apis mellifera around the Mediterranean (redrawn from Garnery et al., 1992). Lineage M is the most northern branch leading to the A . mellifera subspecies of western Europe. Lineage C represents the races of the Balkan and the northern Mediterranean and lineage A comprises the African subspecies.

does not support Ruttner’s (1988) models which predict a higher similarity between the West European and African races. Garnery et al. (1992) plausibly explain the racial admixture in the Spanish population through secondary hybridization with African populations. Since various mitotypes have been suggested to be race typical (Cornuet et al., 1991; Cornuet and Garnery, 1991; Garnery et al., 1992; Meixner et al., 1993; Moritz et al., 1986; Sheppard et al., 1991a, b; Smith, 1988, 1991;

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Smith and Brown, 1990; Smith et al., 1989), they have been used to verify the racial origin of feral honeybee populations. For example Oldroyd et al. (1992) studied a honeybee population on Kangaroo island in southern Australia using restriction enzyme analysis on mtDNA in combination with isozyme variability. Interestingly they found that the isozyme data support the theory that no hybridization occurred after the introduction of Italian A. melfifera ligustica and the protection of this population by the Ligurian Bee Act of 1886. Yet surprisingly, mitochondria1 types proved to be those thought to be typical for A . mellifera mellifera (Sheppard et al, 1991a). Oldroyd et al. (1992) concluded that the degree of mtDNA variability found in honeybee populations may have been underestimated. The high variability of mitotypes is particularly apparent in the length variation between the CO-I and CO-I1 region (discussed above in detail). The variability for the number of repeats of the Q fragment is equal within and between races in southern Africa (Moritz et al., 1994). In a single race, A. mellifera mellifera, three different size types were found (Garnery et al., 1992) offering no obvious diagnostic power to discriminate among populations or subspecies.

6.3.5 The Africanized bee problem The problem of honeybee dispersal has become quite an important one during the past decades as the Africanized honeybee problem has developed into a serious issue of international scope. Among the various narratives about the outbreak of the problem, the following was favoured in a recent monograph on the problem (Spivak et a f . , 1991). Forty-seven or 48 queens (from a lot of 170 queens) from the Transvaal highveld (South Africa) and one queen from Tabora (Tanzania) were successfully introduced into colonies at Piracicaba (SP) in Brazil in 1956 to increase the productivity of the European honeybee stock which was believed to be poorly adapted to the tropical conditions of South America. In early 1957 queen excluders were removed from the flight entrances and 26 of these colonies swarmed. This is believed to be the beginning of the Africanized bee problem (Kerr, 1957). In addition to these accidentally escaped swarms it has been claimed that African honeybee queens have been systematically reared and distributed to beekeepers (Spivak el al., 1991). This clearly would be a much more plausible basis for the rapid spread of the African type honeybees. However, all of these claims are based on a questionable interpretation of data, unconfirmed personal communications and anecdotal material. The introduced stock, which was considered undesirable due to its strongly expressed stinging behaviour, spread rapidly throughout the continent, virtually eliminating the occurrence of European stock. Its current distribution ranges from Texas in the north to Argentina in the south. Because of its negative effects on the public, the ‘Africanized bee problem’

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is one of the better studied cases in population biology (for a comprehensive review, see Spivak et al., 1991). We know that the Africanized bees have a swarming rate about 13 times higher than European honeybees (Otis, 1982, 1991b). Furthermore, Africanized drones have been found to penetrate into European colonies and simply by their presence reduce the production of European drones (Rinderer et al., 1985). The phenotypical result of the Africanization process is quite clear. Feral honeybees in the Americas behave like true African bees and they phenotypically resemble the African rather than the European honeybee (Daly, 1991). In spite of the seemingly clear ecological data there is considerable controversy about the actual spread mechanisms of Africanized honeybees. Particularly, two testable hypotheses have been discussed to explain the process of Africanization. One model assumes the production of ‘hybrid’ populations and the spread of a phenotypically well adapted genotype through natural selection (Kerr and Bueno, 1970; Michener, 1975; Rinderer et al., 1985; Rinderer, 1986). The other model assumes the spread of the introduced queens as a pure African gene pool without hybridization, for example through hybrid inviability or pre-mating isolation (Taylor, 1985; Fletcher, 1991). Thus the problem is whether the Africanized bees spread exclusively through swarming or whether hybridization is also a significant factor in this process. In support of both hypotheses, a variety of studies focusing on mtDNA have been conducted, but unfortunately this has not yet solved the problem and still left space for contention. One set of studies on mtDNA (Hall and Muralidharan, 1989; Hall and Smith, 1991; Smith et al., 1989) supports the view that the African honeybees spread as pure maternal lineages. Theoretically mtDNA is a prime choice marker because it is inherited maternally (Meusel and Moritz, 1993). However, as soon as hybridization occurs between the two populations, which seems plausible in light of the increased fitness of the Africanized drones, European mitotypes are to be expected in the hybrid population. The higher the fitness of the Africanized drones, the higher the frequency of the European mitotypes. Moritz and Meusel (1992) modelled the spread of a neutral mitochondria1 marker which is initially linked to a fit nuclear genome. They could show that a determination of the cyto-nuclear disequilibrium in the hybrid zone would allow for an analysis of the significance of swarming and hybridization. Although a pure maternal spread through swarming might theoretically be possible in principle, Moritz and Meusel (1992) showed in a population genetic model that the spread of pure maternal lineages requires some extreme assumptions that may not be very likely in nature. Only strong assortative mating (as suggested by Taylor, 1985), a cyto-nuclear incompatibility (as suggested by Hall and Muralidharan, 1989) or functional hybrid sterility (as claimed by Fletcher, 1991) can explain the persistence of pure African mitotypes in a honeybee population with racial admixture. Although such mechanisms are possible

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and have been found in other organisms, there is as yet no such evidence for honeybee populations. Harrison and Hall (1993) found indications for a negative heterosis concerning the metabolic capacity of hybrid honeybees. The metabolic capacity (determined as watts/kg body weight) during agitated flight of a caged worker was about 15% less in the hybrid bees compared with pure African bees. It remains unclear however whether these differences in a rather artificial bioassay have any implications for the reproductive capacity of colonies. Rinderer et af. (1991) and Sheppard ef al. (1991a,b) found substantial hybridization in Africanized populations in Mexico, Brazil and Argentina, which is in line with the theoretical expectations as suggested by Moritz and Meusel (1992). Lob0 et af. (1989) also found racial admixture in feral honeybee populations in South America. These studies seem to contradict the view that the few imported African queens spread as pure maternal lineages in America. Most significant are those results in which a European mitotype is found with an African phenotype or nuclear genotype. These cases are in line with a view that hybrids do contribute to the gene pool and there are no biological barriers to hybridization. The issue may initially look academic but it is of substantial practical impact. It directly affects concepts for controlling the Africanized honeybee. If there is no hybridization between Africanized and European honeybees, any effort to interfere in the Africanization process through mating control and breeding would be void. In the light of the ongoing controversy it would certainly be premature to omit breeding control as a means to manage the Africanized honeybee. As long as contradicting empirical data are obtained all potentially possible control options have to be used. A shortcoming of all empirical studies on mtDNA variability is the lack of repeated sampling of the same transect in the hybrid zone between Africanized and European honeybees. It is impossible to determine a dynamic population process by taking a single ‘snap shot’ of gene and allele frequencies. It is only possible to determine nuclear and mitochondria1 gene flows through repeated sampling over several generations. Only then shall we be able to understand the genetic basis of the Africanized bee problem. 7 Outlook

Much of the cited work in this review reveals the major shortcomings of recent molecular research in honeybees. The field is trying hard to catch up in methodology with current standards in molecular research but it is in the early stages. Information is however rapidly accumulating. With the fast progress in molecular methodology and the increasing ease of performance of the various protocols, it is not difficult to predict that the boom of research in molecular honeybee research is yet to come. A typical example

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may be the analysis of the mitochondria1 genome of the honeybee, where within a few years the data accumulated from some patchy restriction fragment information (Moritz et af., 1986) to the complete sequence (Crozier and Crozier, 1992). The development of highly variable nuclear DNA markers will allow a further analysis of the intracolonial demography and the genetic basis for division of labour and colony organization. The analysis of structure and sequence of important genes will follow soon. Several groups are currently focusing on the analysis of the sex locus which indeed is one of the major interesting genes in Hymenoptera. The mapping of the genome will form the basis for such a study. The consequent use of the advantages offered by honeybees over other test systems will ensure a rapidly increasing body of most exciting studies, re-establishing its position as a most rewarding study organism in both physiological and genetical research. Acknowledgements

I wish to thank M. Beye, P. Kryger and H. G. Hall for their most helpful comments. I am grateful to J. M. Cornuet, R. H. Crozier, G. J . Hunt, B. Oldroyd, R. E. Page, T. E. Rinderer, and M. Winston, who provided preprints or submitted manuscripts which were most helpful to update this review.

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