Confirmation of QTL Effects and Evidence of Genetic Dominance of Honeybee Defensive Behavior: Results of Colony and Individual Behavioral Assays

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1 Behavior Genetics, Vol. 32, No. 2, March 2002 ( 2002) Confirmation of QTL Effects and Evidence of Genetic Dominance of Honeybee Defensive Behavior: Results of Colony and Individual Behavioral Assays Ernesto Guzmán-Novoa, 1,3 Greg J. Hunt, 2 José L. Uribe, 1 Christine Smith, 2 and Miguel E. Arechavaleta-Velasco 2 Received 23 Mar Final 28 Oct The stinging and guarding components of the defensive behavior of European, Africanized, hybrid, and backcross honeybees (Apis mellifera L.) were compared and analyzed at both colony and individual levels. Hybrid and Africanized backcross colonies stung as many times as Africanized ones. European backcross colonies stung more than European bees but not as many times as Africanized or Africanized backcross colonies. The degree of dominance for the number of times that worker bees stung a leather patch was estimated to be 84.3%, 200.8%, and 145.8% for hybrid, backcross European, and backcross Africanized colonies, respectively. Additionally, both guards at the colony entrance and fast-stinging workers of one European backcross colony had a significantly higher frequency of an Africanized DNA marker allele, located near sting1, a QTL previously implicated in stinging behavior at the colony level. However, guards and faststinging bees from a backcross to the Africanized parental colony did not differ from control bees in their frequency for the Africanized and European markers, as would be expected if large genetic dominance effects for sting1 exist. These results support the hypothesis that genetic dominance influences the defensive behavior of honeybees and confirm the effect of sting1 on the defensiveness of individual worker bees. KEY WORDS: Apis mellifera; defensive behavior; Africanized honeybees; genetic dominance; QTL. INTRODUCTION The evolution of complex social organizations is an important issue in biology. Insect colonies may serve as model of complex systems, and honeybee colonies stand as an example of social organization because of their large number of members and their well-known division of labor (Winston, 1987). Honeybee colonies are typically 1 CENIFMA-INIFAP, Santa Crúz 29-B, Las Haciendas, Metepéc, Edo. de Méx., Mexico. 2 Department of Entomology, Purdue University, West Lafayette, IN To whom all correspondence should be addressed. Tel: (011-52) Fax: guzmane@inifap. conacyt.mx 95 composed of one reproductive (queen) and thousands of nonreproductive females (workers). The workers perform most of the tasks upon which the fitness of the colony depends. Female honeybees are diploid, but the males (drones) are haploid because they develop from unfertilized eggs (reviewed by Winston, 1987). A queen bee mates while flying with up to 17 males (Adams et al., 1977) and stores the sperm in a spermatheca for the rest of her life (Page, 1986). As a consequence, honeybee colonies with open-mated queens contain many subfamilies of workers, with each worker in a subfamily inheriting the same genetic contribution from the father (all the sperm cells produced by an individual male carry identical genomes; Page and Laidlaw, 1988). Natural selection acting at the level of colonies has probably been responsible for many observed features /02/ / Plenum Publishing Corporation

2 96 Guzmán-Novoa, Hunt, Uribe, Smith, and Arechavaleta-Velasco of colony organization (Crozier and Consul, 1976; Owen, 1986; Seeley, 1989). Selection acts on colonies, but the genes that affect heritable differences in colony social structure reside in the colony residents. So, the genotypic structures of colonies must change with respect to heritable components of division of labor, where individuals repeatedly perform specific tasks. Therefore, it is important to identify and study traits of individual workers that have heritable variability expressed at the colony level on which natural selection can operate. Heritable differences in the likelihood that individual nestmate workers perform specific tasks have been demonstrated repeatedly (Hellmich et al., 1985; Moritz and Hillesheim, 1985; Robinson and Page, 1988, 1989; Calderone and Page, 1988, 1991, 1992; Hillesheim et al., 1989; Rothenbuhler and Page, 1989; Breed et al., 1990; Breed and Rogers, 1991; Robinson et al., 1990; Page and Robinson, 1991; Guzmán-Novoa and Gary, 1993; Guzmán-Novoa et al., 1994; Giray et al., 2000). However, there are not many genetic studies that establish the connection between individual behavior and colony behavior. Finding evidence that establishes a relationship between genetically influenced individual behavior and colony behavior is important to demonstrate how individuals with different likelihoods to perform a task interact to influence a colony phenotype. Recently, a few studies have identified QTLs with effects on behavior in honeybees based on whole-colony measurements. Several QTLs that influence pollen foraging were identified in crosses between strains of colonies that were selected for either large or small amounts of pollen stored in the combs. These QTLs were subsequently shown to influence individual foraging behavior and physiology, using several independent crosses (Hunt et al., 1995; Page et al., 1995, 2000). We identified QTLs that influenced the numbers of stings deposited in whole-colony defensive-behavior assays, in crosses derived from Africanized honeybees (Hunt et al., 1998). One QTL (sting1) controlled experiment-wise error at p (LOD 3.57). Several other QTLs were also identified for their effect on the production of alarm pheromone components found in the stinging apparatus of worker bees (Hunt et al., 1999). These QTLs were independent of the previously identified loci that influenced stinging behavior at the colony level. However, no relationship has yet been established between QTLs influencing colony-level traits and defensive behavior of individual worker bees. Africanized honeybees are hybrids of African (Apis mellifera scutellata) and European honeybee races (primarily A. mellifera ligustica). Africanized bees originated in Brazil in 1956 (Kerr, 1967) and have since spread throughout most of the Americas. They were detected in Mexico in 1986 (Moffett et al., 1987) and in the United States in 1990 (Sugden and Williams, 1991). Africanized honeybees appear to have retained their highly defensive behavior and predominantly African genotype (Hall and Muralidharan, 1989; Lobo et al., 1989; Smith et al., 1989; Hall, 1990; Muralidharan and Hall, 1990; Rinderer et al., 1991; Sheppard et al., 1991; Hall, 1992a,b; Moritz and Meusel, 1992; Quezada-Euán and Medina, 1998). Africanized bees also outcompete European races in warmer climates. In our study area in Mexico, the frequency of African mitochondrial type in feral swarms increased from 29% to 96% in five years time (Guzmán-Novoa and Page 1999). High defensiveness is the most noticeable characteristic of Africanized bees (Stort, 1975a c; Collins et al., 1982; Guzmán-Novoa and Page, 1993, 1994a). In Mexico, these bees have caused thousands of stinging incidents that resulted in the deaths of more than 300 people as well as in several thousand animal fatalities (Guzmán-Novoa and Page, 1994b; Cajero, 1995). In the United States, only eight people have died as a consequence of stinging incidents since the arrival of Africanized bees (E. H. Erickson, pers. com.), but the number of fatalities could increase in the future if these bees continue to spread. The defensive behavior of Africanized bees is heritable in the broad sense (Stort, 1975c; Collins et al., 1984; Guzmán-Novoa and Page, 1994a), but the mode of inheritance is unclear. Stort (1975c) proposed that the highly defensive behavior of Africanized bees showed dominance. However, Collins et al. (1988) found primarily additive effects. Later, several studies supported the dominance hypothesis for whole-colony measures of stinging behavior, but the degree of dominance remains unclear (Guzmán-Novoa and Page, 1993, 1994a; DeGrandi-Hoffman et al., 1998). The two major components of the defensive behavior of honeybees are the tasks of guarding and stinging. Guard bees patrol the entrance to their colony. They inspect incoming bees, exclude bees (or other invertebrates) that are foreign to their nest, and alert other colony workers about intruders. Stinger bees fly out of the colony and sting intruders. A few studies have shown evidence of a correlation between the level of guarding behavior and the level of colony defensiveness in European honeybees (Breed et al., 1988; Robinson and Page, 1988; Breed and Rogers, 1991), but no studies on this subject have

3 Genetics and Honeybee Defensive Behavior 97 been conducted with Africanized bees. In contrast, many studies have reported evidence of genetic effects on the stinging behavior of Africanized bee colonies (Stort, 1975a c; Collins et al., 1982, 1984; Collins, 1986; Villa, 1988; Guzmán-Novoa and Page, 1993, 1994a; DeGrandi-Hoffman et al., 1998; Hunt et al., 1998), primarily measured at the colony level. Whether the defensive behavior of honeybee colonies shows additive or dominant inheritance may have implications for the fitness of honeybee colonies, for human and animal health, and for honeybee breeding. If the defensive behavior of honeybees is inherited additively, colonies of hybrid origin would be expected to become less fit in environments in which benefits associated with defensiveness for reducing predation outweigh the negative aspects of defensiveness, such as loss of worker bees that sting and the disruption of colony activities. However, additive inheritance would also make it easier to breed gentler bees, and fewer stinging incidents would be expected to occur. If dominance effects influence this behavior, breeding gentler bees would be more difficult to accomplish and more human and animal lives could be at risk. We describe results from behavioral assays of individual bees and linkage disequilibrium with a QTL in samples of bees performing tasks related to colony defense. We also present a more thorough analysis of the genetic dominance of defensive behavior. MATERIAL AND METHODS Experimental Colonies The experiments were conducted in Ixtapan de la Sal, Mexico (19 N, 99 W). Hybrid European- Africanized colonies were produced, and from these colonies, other colonies containing backcross workers were generated. The European colonies used for the experiments were derived from queens that had been imported previously from the United States and that had been maintained with instrumental insemination by a local beekeeper. The Africanized source was derived from colonies obtained locally. Morphometric (Sylvester and Rinderer, 1987) and mitochondrial DNA (Hall and Smith, 1991; Nielsen et al., 2000) analyses of the queens progeny were used to confirm the strain origin of our source colonies, but it is possible that some European-strain DNA may have been introgressed into the Africanized line. The morphometric analyses were based on the smaller wing sizes of the African subspecies. The mitochondrial assays were based on diagnostic restriction sites that are present in European strains but not in the African subspecies. A single-drone instrumental insemination (one drone for each queen) was used in all the crosses. Twelve Africanized and 9 European bee colonies were produced. To produce the colonies, queens were reared from six (randomly selected) of the source colonies (three Africanized and three European). Each of these queens was inseminated with the semen of one drone obtained from a colony of her respective origin, to produce virgin daughter queens in families that were closely related because they shared the same haploid father. Inseminated queens were identified by colored, numbered plastic tags (Graze KG, Weinstadt, Germany) glued to their thoraces and their right wings were clipped. Nine hybrid colonies were derived from these colonies (containing F 1 workers) by using either Africanized or European colonies as the drone source for each F 1 cross. An additional generation of 40 backcross colonies was produced, using the same procedure. Nineteen F 1 queens were each mated to an Africanized drone (19 drones), and the other 21 were each mated to a European drone (21 drones). Therefore, we had five different sets of experimental colonies: Africanized, European, F 1, Africanized backcross, and European backcross colonies. The experimental colonies were located in five different apiaries approximately 500 m apart. We positioned hives at least 5 m apart to minimize interhive drifting of workers. Behavioral Assays Each experimental colony was tested on three occasions with a defensive behavior assay. Fourteen days before conducting the defensive trials, the populations of the colonies were equalized by removing bees and frames of brood from the most populous colonies. Each of the colonies contained about 4,000 cm 2 (four frames) with capped brood and five frames with adult bees after equalization. Colony equalization was necessary to control for differences in colony population that may affect defensive behavior tests. We used a modification of a previous behavioral assay (Villa, 1988) to test the stinging behavior of the experimental colonies (see Guzmán-Novoa et al., 1999, for more details about methods for testing the defensiveness of honeybee colonies). The assay consisted of a 10 cm 8 cm black suede patch (a flag ) suspended on a piece of white wood (0.7 cm 0.5 cm 100 cm). To provide a stimulus to trigger the defensive response

4 98 Guzmán-Novoa, Hunt, Uribe, Smith, and Arechavaleta-Velasco of colonies, a flag was rhythmically elevated (about 4 cm) and lowered (about 4 cm; two swings per second) approximately 5 to 10 cm in front of the entrance of each hive. We removed the flag after the first 5 to 10 bees had stung the leather patch. These bees were grabbed with forceps from the leather patch (to which they were attached by their stingers) and stored in 96% ethanol until subsequent DNA analyses. These fast-responding bees were classified as first stingers. Immediately after removing the flag with the first stingers, another flag was presented in front of the colony entrance by another manipulator, to permit the bees to continue stinging until a period of 60 seconds was completed. After each trial, we packed and sealed the leather patches (from the second flag) for subsequent sting counts. All colonies within an apiary were simultaneously tested. Two operators who did not know the genotype of the colonies tested each colony. In addition to these three trials, each of two backcross colonies, one European (colony 33) and one Africanized (colony 60), was tested on 14 to 15 additional occasions (as described previously) to obtain at least 100 first stingers. Guard Bees Samples of at least 100 guard bees were collected with a vacuum device (Gary and Lorenzen, 1987) from each of the same backcross colonies where 100 first stingers were obtained (colonies 33 and 60). Guard bees were detected by observing their typical behavior (Ribbands, 1954) at the hive entrance for a period of 5 minutes before taking the sample. Guard bees stand with their forelegs off the ground, with their antennae held forwards to touch and inspect incoming bees. Additionally, we obtained random samples of worker bees ( controls ) from each of these backcross colonies. Between 45 and 100 control bees were collected from the outer combs of their respective hives. We chose not to collect from inner brood combs because brood combs would be more likely to contain nurse bees that are younger than guards. Samples were stored at 20 C in 96% ethanol until subsequent DNA analyses. Genetic Models The actual values obtained from the defensive trials of the experimental colonies were compared with expected values for number of stings that were calculated using two models. Both models assume genotypic additivity, meaning that individuals of a particular genotype would exhibit the same degree of defensiveness, regardless of whether they were living in a colony containing individuals of the same genotype as themselves or one that contained bees of another genotype. It is possible that bees of different genotypes interact to alter response thresholds, in which case a more complex model would be needed to describe the system (see Page and Robinson, 1991), but without knowing the nature of the interaction, it is premature to construct and use such a model. The first model assumes complete genetic dominance for the Africanized trait of high defensiveness (Equation 1). The second model assumes genetic additivity (i.e., hybrids are intermediate between European and Africanized workers, Equation 2). The expected values for stings in patches (E) were obtained as follows: E d P H R A (P E R E ) (P A R A ) (1) E a P H (R A R E )/2 (P E R E ) (P A R A ) (2) Where P H is the proportion of expected hybrid genotypes in the progeny P E is the proportion of expected European genotypes in the progeny P A is the proportion of expected Africanized genotypes in the progeny R A is the mean Africanized response R E is the mean European response Additionally, we estimated the degree of dominance (% d ) of the defensive behavior of hybrid and backcross colonies as follows: % d (R p E a )/(E d E a ) 100 (3) Where R p is the mean progeny response DNA Analyses To estimate the proportion of bees carrying the European and/or the African alleles for sting1 (which presumably affects the stinging behavior of honeybees at the colony level (see Hunt et al., 1998), randomly collected subsamples of at least 90 guard, 93 first stinger, and 45 control bees per each of the two selected backcross colonies were analyzed using sequencetagged site (STS) primers in the polymerase chain reaction (PCR) to amplify a marker linked to sting1. DNA was extracted from individual workers (Hunt, 1997) and dissolved in 300 L of H 2 O. Two microliters were used in PCR with primers that amplify fragments linked to sting1. One set of primers amplified stsn The other set amplified a fragment, sts , that was previously mapped to 8.5 cm distal from the most likely location for sting1 (Hunt et al., 1998). The

5 Genetics and Honeybee Defensive Behavior 99 primer sequences for the stsn were 5 CAA ATT AAA GTC TAC ACT AAA AAA and GCG AAG GTT GGT AAA TGG AA. The primers used to amplify the sts locus were 5 GCA GCC CCA TAA CCA AAG AAA AAT and GCA GCC CCA TTC CAT TTT TAG T. The PCR product for stsn was digested with the restriction enzyme, FOK1. This resulted in codominant markers for both loci. The phase of the marker alleles was determined by comparing the markers of the workers (guards, first stingers, and controls) to those of the haploid father of the F 1 queen. Statistical Analyses Data from the three defensive trials for each colony were summed. Analysis of variance and regression analyses were performed on these data. The data from the genotypic frequency of individual guards, first stingers, and control bees from two backcross colonies (colonies 33 and 60) were subjected to chi square analyses. We used a goodness-of-fit test to compare data from different behavioral groups to the 1:1 segregation that we expect for a backcross colony. RESULTS Differences were found among treatment colonies for the number of stings deposited by the bees in leather patches (Fig. 1). Significant associations between the marker linked to sting1 and the likelihood of individuals to guard the colony entrance or to sting the patches were found among individuals of the European backcross genotype, but not among individuals of the Africanized backcross genotype (Table I). Stings in Patches The number of stings deposited by the bees in leather patches, varied significantly among treatments (F 4, , P ; test based on ln (x) transformed data; Fig. 1) from an average of 38 in European colonies to an average of 136 in Africanized backcross colonies. There were no significant differences among F 1, Africanized backcross, and Africanized colonies for numbers of stings. European backcross colonies were different from European but not different from F 1 colonies. However, the backcross European colonies were also different from Africanized and backcross Africanized colonies. We compared responses generated by the different treatments with the expected values of the two proposed models. Results more closely resembled a model Fig. 1. Number of stings per minute for European (P. EU), African- (B. AF), and Euro- ized (P. AF), F 1 hybrid, Africanized backcross pean backcross (B. EU) colonies. Expected values for the genetic ad- presented as thin lines. ditivity and genetic dominance models are Actual values are presented by open boxes and SE bars, connected with thick lines. Different letters indicate significant differences based on protected LSD tests of ln (x) transformed data. of genetic dominance (Equation 1, Fig. 1). Results differed from the linear, additive model, as demonstrated by a significant quadratic regression coefficient ( , P 0.005). Additionally, the expected values of the F 1 and backcross colonies clearly lie outside of the 95% confidence intervals. Degree of Dominance The degree of dominance for the number of stings left by the bees in the leather patches, calculated with Table I. Number of Homozygous European (E/E), Heterozygous (A/E), and Homozygous Africanized (A/A) First-stinger Bees, Guard Bees, and Control Bees for Marker N4-.245, from a European Backcross Colony (B. EU), and from an Africanized Backcross Colony (B. AF). Africanized Alleles were Significantly Overrepresented in bees from B. EU, but No Significant (ns) Overrepresentation was Recorded in Bees from B. AF or from Control Samples. Colony n A/A A/E E/E Chi-Square P B. EU (stingers) B. EU (guards) B. EU (control) ns B. AF (stingers) ns B. AF (guards) ns B. AF (control) ns

6 100 Guzmán-Novoa, Hunt, Uribe, Smith, and Arechavaleta-Velasco Equation 3, was estimated at 84.3%, 200.8%, and 145.8% for F 1, backcross European, and backcross Africanized colonies, respectively. Guard and First Stinger Bees The proportions of heterozygous (carrying both the Africanized and European parental alleles for marker N4-.245) versus homozygous (carrying only European parental alleles for marker N4-.245) guards and first stinger bees from the European backcross colony did not fit a 1:1 segregation (P 0.01 and P 0.05, respectively; Table I). Heterozygous bees from the European backcross colony were 21.3% and 27.7% more likely to be first stingers and guards, respectively, than bees of pure European genotype. But the proportions of heterozygous versus homozygous guard and first stinger bees from the Africanized backcross colony were not different from the 1:1 expected segregation (Table I). In the European backcross family, the map distance between stsn and the linked marker, sts , was 21.9 cm and 9.1 cm in the first stingers and guards, respectively. Previous estimates for map distance were about 8 cm. The increased distance in the first-stingers sample was probably caused by errors in scoring sts , because this marker was more difficult to read on gels. However, the linkage of the markers confirms that we detected the same loci in these new populations of bees. DISCUSSION Results from both behavioral assays and DNA analyses demonstrate dominance affecting the defensive behavior of honeybee colonies. Dominance for the highly defensive colony phenotype is inferred from the nearly identical responses of F 1, Africanized backcross, and Africanized colonies. Moreover, the responses of European backcross colonies were not different from those of F 1 colonies, even though they differed from the two parental types. The responses of the experimental colonies resembled much more the expected values of the dominance model than the expected values of the additive model. In fact, the responses of backcross colonies suggested a degree of overdominance. The backcross to the Africanized parental colony had a higher mean stinging value than predicted by the dominance model, even though this was not statistically significant. The nonlinear relationship between the colony treatments and their responses may have been due to both genetic dominance and genotypic interactions between individuals within colonies. Guzmán-Novoa and Page (1994a) showed that co-fostered F 1 bees and European bees interact to alter individual likelihood to sting. Their data did not show a nonadditive interaction between the two genotypes when looking at individual stinging behavior. However, interactions between individuals may result in nonadditive effects when measured as a colony-level response. Genetic nonadditivity is also supported by our results with individual bees, showing a higher likelihood for guarding and first stinging for workers carrying the Africanized allele for marker N in a European backcross colony. But in the Africanized backcross colony, heterozygous and homozygous Africanized genotypes for marker N did not differ in their likelihood for guarding and first stinging, as would be expected if large dominance effects for the locus sting1 exist. An alternative explanation for these results is that the two F 1 queens of these colonies had different genotypes for sting1. Although very unlikely, this situation could have occurred if the Africanized queen-mother used for the F 1 cross was not fixed for high-defensive alleles of sting1 and each F 1 daughter inherited a different allele. Other studies also support the hypothesis of genetic dominance for high stinging behavior (Stort, 1975c; Guzmán-Novoa and Page, 1993, 1994a; De- Grandi-Hoffman et al., 1998), whereas Collins et al. (1988) suggested that the defensive behavior of honeybees was additive in inheritance, based on their observations of intermediate response of colonies with European queens that had been openly mated in an Africanized region. The lack of agreement of their results with ours may be due to their lack of control over mating or to differences in the sources of European or Africanized bees. Guzmán-Novoa and Page (1994a) hypothesized that behavioral dominance would occur for speed of stinging. Behavioral dominance may occur if an individual that has a low threshold of response to a specific stimulus performs a behavior elicited by the stimulus, thereby altering the probability that less sensitive individuals would perform that behavior (Page and Robinson, 1991). Therefore, worker bees with lower thresholds of response (presumably those carrying the Africanized allele for sting1) to the defensive stimulus (moving leather patch) are expected to be more likely to be the first to sting, and their actions may influence the behavior of nestmates through visual and pheromonal stimuli. This study is the first to confirm the effect of the locus sting1 on the defensive behavior of honeybees.

7 Genetics and Honeybee Defensive Behavior 101 Hunt et al. (1998) identified this locus affecting the stinging behavior of honeybees at the colony level, but the present study indicates this QTL also influences individual defensive behavior. These results agree with studies that demonstrate a relationship between guarding behavior and the relative defensiveness of a colony (Moore et al., 1987; Breed et al., 1988, 1990; Breed and Rogers, 1991; Hunt, unpublished data). In addition, the data provide strong evidence for the existence of genetic dominance effects in the defensive behavior of honeybees at both the colony and the individual levels. These results suggest that the production of less defensive stocks through selection would be relatively slow. Artificial introgression of European genes into honeybee populations (by frequent requeening and importation of drone sources) or maintenance of low levels of Africanization through selective breeding and management may be necessary to reduce stinging incidents. Despite the stated difficulties, the potential success of such breeding and management techniques was demonstrated in a 5-year study conducted with more than 3,000 colonies of bees in an Africanized area (Guzmán-Novoa and Page, 1999). Colonies reduced their defensiveness by more than 50%, on average, in response to frequent requeening and selection for European morphometrics, although not to the levels of European bee colonies. Although these studies showed a connection between colony-level and individual behavioral phenotype and QTL genotype, the genetic dominance at the colony level remains unexplained. 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