Confirmation of QTL effects and evidence of genetic dominance of honeybee defensive behavior: results of colony and individual behavioral assays

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 fast-stinging 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.     

Genotype,Task Specialization,and Nest Environment Influence the Stinging Response Thresholds of Individual Africanized and European Honeybees to Electrical Stimulation

This study was conducted to analyze the stinging response thresholds of individual European and Africanized worker honeybees (Apis mellifera L.) to electrical stimulation. Newly emerged workers were identified, and either were placed into an incubator, into their natal colonies, or cross-fostered in common colonies of European or Africanized ancestry. Nest and guard bees of each type were collected and exposed to an electric stimulus of 0.5 mA, and the time they took to sting a leather substrate was recorded. Africanized bees consistently had significant lower thresholds of defensive response than European bees across all of the environments tested. Guards were faster to sting than nest bees only for the Africanized genotype, suggesting that alleles of African origin have pleiotropic effects on guarding and stinging. This is the first study that shows that single individuals specialized in guarding also may have a lower response threshold for stinging. Environmental effects were also evident. In all cases, bees responded faster to the electrical stimulation after being kept in environments other than their natal nest. Moreover, significant genotype by environment and genotype by task specialization interactions were found. Our results fit a model of division of labor based on differences in response thresholds to stimuli among workers of different genotypes and task groups that result in non-additive effects on colony behavior. Edited by Yong-Kyu Kim.     

A genetic analysis of the stinging and guarding behaviors of the honey bee

In order to identify genes that are influencing defensive behaviors, we have taken a new approach by dissecting colony-level defensive behavior into individual behavioral measurements using two families containing backcross workers from matings involving European and Africanized bees. We removed the social context from stinging behavior by using a laboratory assay to measure the stinging response of individual bees. A mild shock was given to bees using a constant-current stimulator. The time it took bees to sting in response to this stimulus was recorded. In addition, bees that were seen performing guard behaviors at the hive entrance were collected. We performed QTL mapping in two backcross families with SNP probes within genes and identified two new QTL regions for stinging behavior and another QTL region for guarding behavior. We also identified several candidate genes involved in neural signaling, neural development and muscle development that may be influencing stinging and guarding behaviors. The lack of overlap between these regions and previous defensive behavior QTL underscores the complexity of this behavior and increases our understanding of its genetic architecture.     

The behavioral genetics of colony defense in honeybees: Genetic variability for guarding behavior

Guard honeybees stand at the entrance of colonies and facilitate the exclusion of nonnestmates from the colony. In this study, we examined the hypothesis that genetic variability among individuals in colonies might explain variability in guarding activity. To do this, we cross-fostered honey bees between colonies with high-defensive responses and colonies with low-defensive responses in alarm pheromone tests. Individuals from high-defensive colonies were more likely to guard in their own colonies (controls) than cross-fostered bees from low-defensive colonies. Cross-fostered high-defensive bees also were more likely to guard in low-defense colonies. These results support the hypothesis that interindividual differences in guarding behavior are at least partially under genetic control. A positive correlation between number of guards and response to alarm pheromone demonstrates a link between behaviorally separated components of the overall defensive response.This work was supported by NSF Grant BNS 8605604.     

Olfactory stimulation of Africanized honey bee (Hymenoptera: Apidae) attacks by insect repellents

Three common insect repellents (N,N-diethyl-m-toluamide [DEET], Pyranha, and Repel X) were tested to determine whether they affected Africanized honey bee attack behavior. Eight Africanized honey bee (Apis mellifera L.) colonies were exposed in an alternating series to the test repellents or blank controls delivered in a stream of air directed toward the colony entrances. The response generated by the repellents and the controls was measured as the number of attacking honey bees recorded with an electronic temper tester. Neither a citronella-based repellent (Pyranha) nor DEET had any effect on colony behavior; however, Repel X consistently caused a greater attack response after exposure.     

Biochemical variability of venoms from individual European and Africanized honeybees (Apis mellifera).

To study biochemical differences between venom from individual honeybees, venom sacs from 103 European (EU) bees and 92 Africanized bees representing 12 different colonies were dissected, and the dry weight (DW) of venom from each bee was determined. Venom from each of these bees was studied with isoelectric focusing and functional assays for phospholipase A2 and melittin. Phospholipase concentrations in individual EU bee venoms varied between 1.8% and 27.4% (wt/wt). The melittin concentration in EU bee venom varied less and, on the average, was found to be much lower than previously reported. There was an eightfold to ninefold difference between lowest and highest venom sac DW contents, suggesting the possibility of highly variable venom delivery from bee stings. One EU bee contained greater than 300 micrograms of venom, three times the recommended maintenance dose for venom immunotherapy. Isoelectric focusing also demonstrated large differences between individual bees, with respect to major and minor components of their venoms. Africanized bees contained significantly less venom but more phospholipase than did EU bees. Bee venoms from different colonies differed in their DW content and in their concentrations of phospholipase and melittin. The results are relevant to the uncertainty of responses from sting challenges and field stings in allergic patients and massive stinging attacks on normal subjects.     

Genetic study of the aggressiveness of two subspecies ofApis mellifera in Bazil. IV. Number of stings in the gloves of the observer

Data are analyzed on an aspect of aggressiveness in workers from colonies of Africanized bees (Apis mellifera adansonii), Italian bees (Apis mellifera ligustica), their F ₁ hybrids, and backcrosses of the F ₁ to the parental stocks (Rothenbuhler method). The segregation values (3:1) in the backcrosses to the Africanized stock and nonsegregation in the backcrosses to the Italian stock suggest the existence of two pairs of genes (F₁/F₁;F₂/F₂ in the Italian bees and f₁/f₁;f₂/f₂ in the Africanized bees) which control a character defined as the number of stings in the gloves of the observer.This work was carried out in the Department of Genetics, Faculdade de Medicina de Ribeirão Preto, and in the Laboratory of Biology, Faculdade de Filosofia, Ciências, e Letras de Araraquara, with the financial assistance of Fundação de Amparo à Pesquisa do Estado de São Paulo. This paper is part of the author's doctoral dissertation.     

Phylogenetic analysis of the RNA-dependent RNA polymerase (RdRp) and a predicted structural protein (pSP) of the Chronic bee paralysis virus (CBPV) isolated from various geographic regions

Chronic bee paralysis virus (CBPV) is responsible for chronic paralysis, an infectious and contagious disease of adult honey bees (Apis mellifera L.). The full-length nucleotide sequences of the two major RNAs of CBPV have previously been characterized. The Orf3 of RNA1 has shown significant similarities to the RNA-dependent RNA polymerase (RdRp) of positive single-stranded RNA viruses, whereas the Orf3 of RNA2 encodes a putative structural protein (pSP). In the present study, honey bees originating from 9 different countries (Austria, Poland, Hungary, Spain, Belgium, Denmark, Switzerland, Uruguay and France) were analysed for the presence of CBPV genome. The complete genomic nucleotide sequence of the RdRp (1947 bp) and of the pSP (543 bp) from 24 honey bee positive samples was determined and the phylogenetic relationship among isolates was investigated. Four distinct genotypes of CBPV were observed.     

Double-stranded RNA reduces growth rates of the gut parasite Crithidia mellificae

Parasites of managed bees can disrupt the colony success of the host, but also influence local bee-parasite dynamics, which is regarded as a threat for wild bees. Therapeutic measures have been suggested to improve the health of managed bees, for instance, exploiting the bees’ RNA interference (RNAi) pathway to treat against viral pathogens. Gut trypanosomes are an important group of bee parasites in at least two common managed bee species, i.e., managed Apis mellifera and reared Bombus terrestris. In several trypanosomes, RNAi activity is present, while in other associated genes of RNAi, such as Dicer-like (DCL) and Argonaute (AGO), it is lost. Up to date, the ability to exploit the RNAi of gut trypanosomes of bees has remained unexplored. Here, we screened parasite genomes of two honey bee protozoa (Crithidia mellificae and Lotmaria passim) and two bumble bee protozoa (Crithidia bombi and Crithidia expoeki) for the presence of DCL and AGO proteins. For C. mellificae, we constructed a double-stranded RNA (dsRNA) targeting kinetoplastid membrane protein-11 (KMP-11) to test the RNAi potential to kill this parasite. Transfection with KMP-11 dsRNA, but also adding it to the growth medium resulted in small growth reduction of the trypanosome C. mellificae, thereby showing the limited potential to apply dsRNA therapeutics to control trypanosome infection in managed honey bee species. Within bumble bees, there seems to be no application potentials against C. bombi, as we could only retrieve non-functional DCL- and AGO-related genes within the genome of this bumble bee parasite.

     

Critical aspects of the Nosema spp. diagnostic sampling in honey bee (Apis mellifera L.) colonies

Nosemosis is one of the most widespread of the adult honey bee diseases and causes major economic losses to beekeepers. Two microsporidia have been described infecting honey bees worldwide, Nosema apis and Nosema ceranae, whose seasonality and pathology differ markedly. An increasing prevalence of microsporidian infections in honey bees has been observed worldwide during the last years. Because nosemosis has detrimental effects on both strength and productivity of the infected colonies, an accurate and reliable method to evaluate the presence of Nosema in honey bee colonies is needed. In this study a high degree of variability in the detection of microsporidia depending on the random subsample analyzed was found, suggesting that both sample size and the time of collection (month and day of sampling) notably affect the diagnosis.     

Intergenerational transfers may have decoupled physiological and chronological age in a eusocial insect

Life-history theory generally predicts that there should be no selection for longevity beyond the limit of reproductive capacity. However, the capacity to increase fitness may not end when individuals reach a state of functional sterility. Recent studies show that intergenerational transfers of resources from post-reproductive parents can increase the offspring's fitness, and analytical theory shows that age-trajectories of transfers may shape the course of senescence in social organisms. In eusocial insects, female roles are partitioned so that one phenotype or "caste" reproduces while another is responsible for resource transfers: the reproductive "queens" are arrested in a continuous reproductive mode, while transfer-activities such as hygienic behaviors, guarding, foraging and further food processing ("nursing") that increases the nutritional value of provisions are conducted by sterile "workers". Worker honey bees normally perform these tasks in a sequence so that nursing inside the protected nest is conducted prior to more risky exterior hive activities such as guarding and foraging. However, foragers may revert to nurse-activity in response to demographic changes, and worker bees can also develop into a stress resistant survival form with a 10-fold increase in lifespan. This elastic division of parental functions is believed to increase colony fitness. Further, it generates a stage-dependent trajectory of senescence that is difficult to address with established theories of aging. In the following, we show how a recent theory that includes resource transfers can be used to elucidate patterns of senescence in eusocial, non-reproducing individuals like the honey bee worker.     

Telomerase activity in mouse bone marrow and subpopulations of spleen colony-forming units

Telomerase activity is studied in mouse bone marrow and in splenic colonies produced by individual CFU characterized by different proliferative potential and time of colony formation (7–14 days). It is found that splenic CFU forming colonies on day 9 in culture exhibit maximum telomerase activity that 5-fold surpassed telomerase activity in the original bone marrow. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 3, pp. 311–313, March, 1999     

Deformities in the spore ofNosema apis as induced by itraconazole

Ultrastructural changes in the spores ofNosema apis were observed after infected honey bees were treated with itraconazole.Nosema spores from untreated honey bees developed normally, whereas those from treated bees exhibited many deformities. One particular feature noted was the absence of the polar filament. Lipid droplets were observed in the sporoplasm of treated spores. The significance of these observations in relation to the effectiveness of itraconazole againstN. apis is discussed.     

A Genetic Analysis of Avian Personality Traits: Correlated,Response to Artificial Selection

Individuals in a range of species consistently differ in their behavior towards mild challenges, over age and time. Differences have been found for several personality traits in a range of species. In great tits these traits have a genetic basis and are phenotypically correlated. Estimates of genetic correlations are, however, fundamental to understanding the evolution of consistent individual differences in behavior. This study analyzed two selection experiments on two avian personality traits, early exploratory behavior and risk-taking behavior. The selection lines used were both started using wild great tits (Parus major) from two natural populations. Genetic correlations were calculated using the response and the correlated response to artificial selection. We found genetic correlations ranging from 0.51 to 0.66, based on individual values, and from 0.84 to 1.00 based on nest means. Genetic correlations can be due to pleiotropic effects or to linkage disequilibrium. The different behavioral traits might therefore have a common genetic basis, possibly constraining independent evolution of personality traits in natural populations. These results are discussed in relation to domain generality and domain specificity of personalities.     

The ontogeny of kin discrimination cues in the honey bee,Apis mellifera

Cues used in the discrimination of relatives from nonrelatives by the honey bee reflect both genetic and environmental differences between groups. Discrimination is behaviorally expressed by acceptance into or agonistic rejection from the social group. We examine the development of these cues in field colonies and in controlled laboratory settings. Newly emerged worker honey bees are accepted by honey bee social groups at a high frequency. When bees are kept in a controlled laboratory environment for 5 days, acceptability into laboratory groups is determined largely by relatedness. Cues indicating relatedness develop in the laboratory within 12 h after the adult bee emerges. Bees older than 12 h are not accepted by field colonies regardless of relatedness. Bees maintained in a hive until 5 days after emergence are not accepted by related or unrelated laboratory groups (this is termed the hive effect). Bees maintained in hives for times as short as 5 h acquired the hive effect. In a cross-fostering experiment, the hive effect completely masked genetic differences.This work was supported by NSF Grants BNS 82-16787 and BNS 86-05604.     

Sexual and aggressive interactions in a visible burrow system with provisioned burrows

The visible burrow system (VBS) is a habitat providing burrows and an open area for mixed-set rat colonies. Provisioning of food and water in the burrows makes it unnecessary for potentially defensive animals to leave the burrows to eat/drink on the surface, and enables evaluation of new types of agonistic interactions that may emerge when this necessity is removed. In such colonies, subordinate males showed high magnitude tunnel guarding behavior, occupying a tunnel opening onto the surface and confronting the dominant. Dominants, in response, made lunges into the tunnels, but quickly retreated without gaining entry, apparently stopped by contact with the defender's vibrissae. Dominants also made and continued to make lateral attacks to the wall adjacent to the tunnels guarded by subordinates, although these were useless in terms of affording contact with the subordinate. Dominant-female agonistic interactions were more frequent than those of dominants and subordinates. These were largely initiated by the male, and involved female defensive behavior. Nonetheless, females, unlike subordinates, failed to show tunnel guarding and continued to utilize the surface freely. They also spent more time in the vicinity of the dominant over days of colony formation. This apparent paradox may reflect that females were seldom wounded, and that the initial site of male contact with females was the female's anogenital area, findings suggesting that interactions of males and females often reflect male sexual advances, countered by female defenses that effectively protect nonestrus females from mounting and copulation.     

Evaluation of the impact of Exomite Pro™ on Varroa mite (Varroa destructor) populations and honeybee (Apis mellifera) colonies: efficacy,side effects and residues

In this research, we examined the application of thymol-based powder, directly over the top of the brood frames in colonies with different population in a 2-year study. The efficacy against mites, the side effects on bees and the contamination of honey were studied comparably to thymol-based gel treatment. In one-store colonies, thymol-based powder treatment gave average efficacy 64.5 % and did not differ significantly from thymol-based gel treatment (65.4 %). The natural mortality in control colonies was 41.4 % and the corrected efficacy (E _T) during 2 years was 39.4 and 40.9 %, respectively. In two-store bee colonies, the application of thymol-based powder on top of each hive body gave higher E _T (45,4 %) than on top of the double body hive (40.4 %), without statistically significant differences. The average concentration of thymol residues in honey 24 days after the application was 368 and 1,119 μg kg^?1 for the honey of colonies treated with thymol-based powder and thymol-base gel, respectively.     

Positive and negative geotaxis: Sex-linked traits inDrosophila pseudoobscura

Using a Hirsch classification maze, selection was made for positive and negative geotactic behaviors in three different strains ofDrosophila pseudoobscura. Hybridization studies were then carried out with flies from the diverged strains. The geotactic scores of the parents and F₁ flies indicate that both negative and positive geotactic behaviors in these strains are strongly influenced by genes in the X chromosome. Additional hybridization studies using flies from strains with similar phenotypes suggest that the diverged strains contain similar alleles and the number of loci in the X chromosomes responsible for these types of behavior is limited. The loci may be highly organized in the X chromosome.This investigation was supported by NIH Research Grant GM 19583.     

Estimation of heritability of learning behavior in honeybees (Apis mellifera capensis)

The learning behavior of Cape honeybees (Apis mellifera capensis) is examined using conditioning of the proboscis extension reflex. The bees are tested cumulatively with several learning tests. The genetic influence on differences in the learning scores is calculated by means of heritability (h ²) estimations obtained by different methods. Theh ² values of the sum of the trials derived by each method range between 0.39 and 0.54. This shows that the proboscis extension reflex paradigm is suitable for the characterization and selection of genetic differences in learning behavior. Heritabilities in the narrow sense and in the broad sense are in the same range. The small differences obtained are discussed with respect to the type of selection existing in natural populations.This study was supported by the Deutsche Forschungsgemeinschaft (Br 827/1-2).