Abstract
Hygienic behavior of honeybee workers is determined by two behavioral components, the uncapping and removing of dead brood, that are betieved to be determined by one Mendelian segregating locus each. A reevaluation of the data upon which this hypothesis was based revealed, however, that the underlying genetic mechanism may be more complex. For removing behavior more than one tocus seems to deter. mine the expression of the phenotype. A three-locus model better fits the original data set however, other patterns of inheritance of hygienic behavior in honeybees cannot be excluded.
thor is affiliated with the Bayerische Landesan. r Bienenzucht and lectures on genetics and zort the University of Erlangen/Nürnberg, Federal lic of Cermany. He wishes to thank R. H. Crozier. rzel, and H.-C. Spatz for stimulating discussions -ongly motivated him to reanalyze available data inheritance of hygienic behavior. Address re3quests to Dr. Moritz, Privatdozent, Bayerische

Hygienic behavior of honeybee workers is determined by two behavioral components, the uncapping and removing of dead brood, that are betieved to be determined by one Mendelian segregating locus each. A reevaluation of the data upon which this hypothesis was based revealed, however, that the underlying genetic mechanism may be more complex. For removing behavior more than one tocus seems to deter. mine the expression of the phenotype. A three-locus model better fits the original data set however, other patterns of inheritance of hygienic behavior in honeybees cannot be excluded.
thor is affiliated with the Bayerische Landesan. r Bienenzucht and lectures on genetics and zort the University of Erlangen/Nürnberg, Federal lic of Cermany. He wishes to thank R. H. Crozier. rzel, and H.-C. Spatz for stimulating discussions -ongly motivated him to reanalyze available data inheritance of hygienic behavior. Address re3quests to Dr. Moritz, Privatdozent, Bayerische ianstalt für Bienenzucht, Burgbergstr. 70, g520 :n, Federal Republic ol Germany.
I of Heredity I 988;79:257 -262; 0022-1503/88/$2.00
Hygienic behavior of honeybee workers (Apis mellifera L.) is believed to be one of the maior factors of resistance to infections of Bacillus /aruoe White (American foul brood).12'ta Field observations as well as laboratory experimentss'G have shown that bees of certain lineages are able to detect infected and dead brood in the capped brood cell. Workers of hygienic colonies uncap brood cells and remove the dead larvae. This leads to a decreased incidence of the infection and is. in combination with unsusceptible larvae, the maior biological factor for resistance to American foul brood (AFB). '4 The genetics of this behavioral trait of honeybees has been a widely known example of single-gene eftects on complex behavior since Rothenbuhlerr6 claimed that uncapping and removing may be controlled by only two loci. Recessive alleles, r for removing and u for uncapping, were postulated that, in homozygous workers, should lead to individuals performing complete hygienic behavior (r/r, u/u). Workers, heterozygous orwild-type at both loci, should not express the hygienic phenotype. Homorygosity at one of the two loci should result in the expression of the corresponding phenotype (ry': removing; u/re uncapping). Rothenbuhler'sr6 study became a classic experiment in behavioral genetics and is cited as the basic example of inheritance of behavior in most textbooks on behavioral genetics.
Indeed, Rothenbuhler's16 study was remarkable in that it was the first to deal with the problem of inheritance of social behavior. In regard to other social behavior, hygienic behavior does not correspond to a phenotype of an isolated individual, The context of a group is a central issue for the expression of social behavior. This has been shown for a variet5r of social insects, such as ants,3'a termites,22 bees,re and wasps.r0 The interactions among the group members are crucial for the expression of a group phenotype. For example, the alarm behavior of honeybees depends on linear (i.e., additive) but also nonlinear worker interactions.e Rothenbuhler found in follow-up studiesrr'20'2r that nonlinear interactions strongly affect hygienic behavior. In cases of strong nonadditive interactions among workers, however, the identification of a true two-locus system for hygienic behavior is extremely difficult. Unpredictable worker interactions may override the gene expression in individual workers of the group, making it impossible to estimate actual gene effects in individual genotypes in the group. Trump et al.2r touched on this problem when they found that mixed groups of hygienic and nonhygienic worker bees always showed the hygienic phenotype. The subgroup of hygienic workers was apparently large enough to uncap and remove all the dead larvae. Therefore, a reevaluation of Rothenbuhler's classic paper is meaningful because the interpretation of the results, based on two-locus Mendelian segregations, may constitute a simplification leading to unjustified conclusions.
The central problem is to assign the behavioral phenotypes to distinct classes of individuals with corresponding genotypes. Genetically determined behavior is Col." Jalle l. Hyglenlc behavtor ln Apis mellifera recalculated from Rothenbuhler'a data on the backcroEs of F1 hybrldo to the hyglenlc parental straln workers were allowed to rear the larvae to imagoes, and the number of inoculated larvae that were removed was determined daily.
The performance of all colonies of the F' generation was very close to the behavior of the susceptible line. This result indicated that the genes for hygienic behavior are definitely not additive. The crucial data on the backcross oftspring, which were given in the original paper as percentage survival of the initially inoculated larval sample, are listed in Table L The actual numbers of dead, removed, and surviving larvae are recalculated. Also shown in Table I is the survival of the controls (larvae inoculated with plain water). The information available from these control experiments was not used in Rothenbuhler's original analysis,ro Not all removed larvae were diseased; noninoculated larvae also were removed in the control experiments. Therefore, the control data are important in estimating the number of actually diseased larvae following the inoculation treatments for each colony. The total number of diseased larvae in each test can be estimated by adding the number of total larvae removed and dead larvae left and subtracting the number estimated from the percentage of noninfected control larvae removed. The effects of these control experiments on the estimation of the actual number of diseased and removed larvae are in some cases substantial.
Rothenbuhlerr6 also performed experiments in which he presented artificially uncapped dead brood to the test colonies. In these studies he was able to show that some of the colonies that were unable to uncap were able to remove dead larvae from uncapped cells. The data in the last column of Table l, in which removing is considered separately from uncapping, refer to these experiments. In contrast to Rothenbuhler's analysis, the percentage of removed larvae of colonies that did remove but did not uncap is computed using the information available from both experimental approaches. The number of dead larvae removed after both uncapping by the workers and artificial uncapping was determined in order to estimate the actual potential for removing of each colony.
Results
Rothenbuhlerr6 provided a vast amount of information, but not all of it was used in his original analysis. Here, the focus will be only on the data as presented in Table   Ceno oCol. * = number ol experimental colony. ÖGenotype -worker genotype as classified by Rothenbuhlerr6 (lor data blocks between lines). 'lnit. inoc. = number of larvae initially inoculated with spores ol Bacillus laruae. cTotal removed : total removed diseased and nondiseased larvae. 'Control removed -nondiseased larvae removed in control experiment. tTotal dead.= estimated percentage of dead larvae at the end of the experiment including removed larvae and those left dead in cells among inöculated. ,Dead lelt: dead larvae, not removed at end of experiment t% left of total = number of larvae removed as percentage of total inoculated larvae. r% left of dead = number of rarvae removed as percentage of diseased larvae among inoculated.
/ % lett capped = number of capped cells with dead larvae at end of experiment as percentage of cells with diseased larvae. | % not removed -number of larvae not removed from uncapped cells (artifrciauy and by workers) as percentage of actually diseased larvae. rclass = new classification as shown in Figure 2 : t: completely hygienic; [: partially hygienic;. = non-hygienic. often subject to continuous variation, and and one extremely resistant line of honthe expression of a genotype may have a eybees had been selected over more than mean and a variance within a group of in-30 years at extreme selection intensities. dividuals' Rothenbuhler's original study Rothenbuhler crossed these lines and did not consider variation within behav-made F, backcrosses to the corresponding ioral groups. In this study the variance hygienic or nonhygienic parental line. among the tested colonies is analyzed in InRothenbuhtei'ibioassay,16freshlyraid more detail in order to minimize the probeggs from one colony were ptaced for lems resulting from subjective classifica-haiching and rearing in another colony tions.
whose behavior *u, to be studied. When the larvae were approximately one day Materials and Methods old,rs large numbers of worker larvae (24 to 286) were inoculated with a spore sus_ The aim of Rothenbuhler'srs study was to pension of B. laruae and then placed into clarify the mode of inheritance of hygienic test colonies. A smaller number of control behavior. For his studies he had unique tarvae were not infected but instead were stocks available. one highly susceptible ,,inoculated" with plain water only. The l, not on the additional information concerning the time course of larvae removal, which also has high intercolony variability. The raw data are summarized in Figure  l . In the parental generation, all pure hygienic crrlonies removed every infected larvae in Rothenbuhler's bioassay. Colonies from the nonhygienic parental line had a larger variation for this character, ranging from colonies that removed 5% of the initially inoculated (not necessarily diseased) larvae up to 30% removal. For the hygienic behavior of the F, colonies, only pooled data of a very srnall sample size of five colonies were given, and the actual distribution of colony phenotypes remains unknown. The distributions of tlre backcrosses, however, are documented in detail, as shown in Figure l .
Using these data, one might conclude that a quantitative genetic model, assuming a pleiotropic phenotype depending on a large number of additive gene eflects, would be sufficient to explain the phenomenon of hygienic behavior. The interdeciles with the highest probabilities agree with the arithmetic means of the pure lines, suggesting plain additive inheritance (expected means; F, x nonhygienic: * -11.35%; Fr x hygienic: x : 3.79%). However, to decide on the basis of this analysis the inheritance of hygienic behavior would be erroneous because at least two factors simultaneously are involved in the determination of this phenotype. One is hy. gienic behavior of the workers, and the other is the susceptibility of the larvae. lf, for example, the larvae were completely resistant to AFB, there would be no dead brood left in the cells at the end of the experiment regardless of whether they were reared by hygienic or nonhygienic workers. Larval susceptibility has been shown to be an important factor in AFB resistance and may be subject to genetic variation.rT Tr.r adjust for this component in the analysis of hvgienic behavior, it is necessary to corrsider only those larvae which were actually diseased after the initial inoculation with B. loruae.ln Figure 2 the susceptibility of the larvae is plotted against the percentage of dead larvae left in the cells at the end of the experiment. There are three well-separated clusters, and the residual variance among the nonhygienic colonies can be reduced significantly by partitioning the data into three subsam. ples instead of two (F': 2.69, P < 0.025). Hygienic colonies that remove more than 90% of the infected larvae and nonhygienic colonies that leave more than 85% of deacl larvae show no correlation to larval susceptibility. The remaining colonies display a highly significant correlation between susceptibility and the percentage of dead larvae left in the cells (r: 0.85, P < 0.01). With increasing hygienic behavior susceptibility decreases, which is surprising because the larvae used in the test were not from the tested colony. The extremely nonhygienic colonies, however, apparently were tested with larvae that had Table 2 . Chl'square goodness of fft analysis of the backcross colonles to a unlmodal or btmodal dlstrlbutlon.
Left dead infected larvae, used in one colony tested, were always from the same comb, according to Rothenbuhler.r6 In contrast to Rothenbuhler's analysis, Figure 2 clearly displays three clusters instead of two. It can be seen that although there are concise clusters, there is variation within these clusters. Therefore. a more careful discrimination among the test colonies may result in more detailed information concerning a potentially underlying genetic system. others. It is still uncertain whether the other data represent one group or two different groups.
If 'one assumes that each genotype has a mean phenotype and a variance, it should be possible to show whether the nonhygienic honeybees resemble a normal distribution with one mean or fit closer to a bimodal distribution. In order to normalize the data, the percentage values were arcsin-transformed. Chi-square analysis based on the data in Table 2 shows that the distribution does not deviate significantly from an expected combination of two normal distributions (hygienics and nonhygienics) with the corresponding means and averages.
When uncapping is considered separately, there seems to be no reason to assume that the data set is composed of three distributions (Figure 4) . The trimodality of the nontransformed data may result mainly from the truncated percentage distribution.
For the removal of dead larvae, horvever, the results are different. The variance of the nonremovers under the trvosample hypothesis is significantly larger than in the remover group (Fro.rg : 7,27, P < 0.01). Splitting the data into three subsets, as in Figure 4 , shows no significant difference among the intragroup variances. Chi-square analysis reveals that three separate distributions better fit the data than does a model based on a twosample hypothesis. There is as significant deviation from normality of the nonremovers in the two-sample hypothesis (X, : 3.88; P < 0.05, I df). Because the chi-square analysis for the three-sample hypothesis "The data were subdivided as-follows: Two-sample hypothesis: removers,0-33% (= percentage dead larvae lett in cells); partial removers,33-100%. Three-sample hypothesis: removers,0-33%;-partial r-emovers,33-62%; nonremovers,67-100%. The original observed data (obs.) are compared with the theoretically expected (exp.) after angle transformation of the original data by computing the expected frequencies from the mean and standärä deviation o[ the corresponding data subset. Classes with expected values <4 were pooled with the adjacent class. ln some cases this was not possible because the resulting degree of freedom was less than l For each estimated parameter (mean, variance-), I degree of freedom is lost in the 1, analysis (-4 df in two-and -6 df in three-sample models). Pooled classes are indicated by brackets. 0 Two-sample hypothesis. ' Three-sample hypothesis. tively. However, colonies with intermecliate phenotypes were found in ratios that fit the two-locus model poorly. The postulation of a two-locus model for hygienic behavior seems to be a simplification that is not strongly supported by the data. The critical point is the trimodal distribution of the separately treated removing behav. ior in Figure 4 , ln the case of a one-locus model for each behavior, one would expect a clear-cut bimodal distribution. A nonepistatic model with two recessive alleles for removing bel'ravior might ex. plain the phenomenon, but a more complex underlying genetic mechanism cannot be ruled out, Workers homozygous at Table 3 . Chl.square analysls of lhe cornblned u and rphenotypes" Two-locus model both loci would express complete removing behavior. Homozygosity at only one locus would lead to partial removing behavior, and heterozygosity at both loci would lead to nonremoving behavior. This hypothesis also holds for the data presented in Figure 3 . Assuming the completely hygienic workers to be hornozygous at the one uncapping and the two removing loci (u/u,r/r,,rr/r), one would expect a distribution that is not significantly diflerent from the observed data (x2 : 5.96, P = 0.2, Table 3 ). The data significantly deviate from an additive fourlocus model, assuming both uncapping and removing to be controlled by two major loci each (x2 : 22,15; P < 0.01). ln partitular, the high number of complete hygienic phenotypes observed makes this model unlikely.
The general problem here is that apparently more than one locus for, at least, removal exists. This means that the expression of a phenotype will be affected by interactions between these loci. These interactions may be additive as assumed above, but any kind of nonadditive interaction ls posslble (e.g., epistasls), which makes predictions of the actual number of genes involved in the phenotype expression very difficult. A clearer picture probably would have been available from a classical quantitative genetic approach estimating the additive, dominance, and epistatic efiects. For such an analysis, however, a much larger sample size and a sophisticated breeding programr3 are necessary. The small sample size also affects the chi-square estimates in the goodness of fit analysis, which sufler substantial standard errors. MilneT recently estimated several genetic variance components of removing and uncapping and found only very low additive eflects, which is in line with the wide range of phenotypic variation observed by Rothenbuhler in the F, backcrosses to the nonhygienic parental line. In that group he even found the completely hygienic phenotype, which is not NOT REMOVED is statistically inappropriate, some fields having expected frequencies of less than 4, this hypothesis cannot be rejected.rs
Discussion
The maior problem in the analysis of the data is the identification of genotypes corresponding to behavioral phenotypes, which in any case suffers from subjective classifications. The pure hygienic phenotype is easy to detect, but it does not seem useful to classify all others as nonhygienic. This hypothesis implies that the hygienic genotypes have virtually no environmental variaiion, whereas the genotype expression of the nonhygienics is subject to an extreme environmental variabilitv. This is an assumption that could be coirect but definitely would need a solid experimental background before basic conclusions should be drawn. Based on the data available, the classification in Figure  2 shows three distinct clusters of colony phenotypes. This still leaves unanswered the question of the underlying genetic mechanism.
Apparently there is a lack of genetic correlation between uncapping and removing. The original interpretation of the data set resulted in a two-locus model for hv- in accordance with the above Mendelian two-or three-locus models. Furthermore, environmental factors can be strong. The position of the test brood in relation to the center of the brood nest of the colony is an important factor for hygienic behavior. The farther the distance from the brood nest, the less hygienic the worker bees (personal observation).
The failure of present-day studies to find results similar to those of Rothenbuhler may reflect the fact that the original hygienic or nonhygienic genes are not very common in the other populations of honeybees tested. The original variability was a result of a long-term selection toward .8. lcruae-resistant honeybees.rs Rare alleles or allele combinations therefore may have been maintained and selected for.
It is unlikely that the genetics of complex social behavior should follow simple Mendelian segregations in natural populations. Griffingr'z showed that the excessive complexity of interactions among workers of a colony of social Hymenoptera makes the expression of a particular behavioral phenotype highly unpredictable. In spite of this complexity, there is a strong need to understand the mechanisms of the genetics of social behavior. For practical purposes simplified models8 may be helpful. For basic behavior genetics research, however, the analysis and the impact of genotype interactions on the expression of group phenotypes is essential. In this light hygienic behavior supplies a prime experimental system to resolve the problem of worker interactions in genetic analysis. Further studies, based on the classic experiments of Rothenbuhler and coworkers, should enable us to gain deeper insights into the complex network of the inheritance of group characters.

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