Six quantitative trait loci influence task thresholds for hygienic behaviour in honeybees (Apis mellifera)

Honeybee hygienic behaviour provides colonies with protection from many pathogens and is an important model system of the genetics of a complex behaviour. It is a textbook example of complex behaviour under simple genetic control: hygienic behaviour consists of two components – uncapping a diseased brood cell, followed by removal of the contents – each of which are thought to be modulated independently by a few loci of medium to large effect. A worker’s genetic propensity to engage in hygienic tasks affects the intensity of the stimulus required before she initiates the behaviour. Genetic diversity within colonies leads to task specialization among workers, with a minority of workers performing the majority of nest‐cleaning tasks. We identify three quantitative trait loci that influence the likelihood that workers will engage in hygienic behaviour and account for up to 30% of the phenotypic variability in hygienic behaviour in our population. Furthermore, we identify two loci that influence the likelihood that a worker will perform uncapping behaviour only, and one locus that influences removal behaviour. We report the first candidate genes associated with engaging in hygienic behaviour, including four genes involved in olfaction, learning and social behaviour, and one gene involved in circadian locomotion. These candidates will allow molecular characterization of this distinctive behavioural mode of disease resistance, as well as providing the opportunity for marker‐assisted selection for this commercially significant trait.     

Evaluation of the time of uncapping and removing dead brood from cells by hygienic and non-hygienic honey bees

Most research on hygienic behavior has recorded the time taken by the colony to remove an experimental amount of dead brood, usually after one or two days. We evaluated the time that hygienic (H) and non-hygienic (NH) honey bees take to uncap and remove dead brood in observation hives after the brood was killed using the pin-killing assay. Four experimental colonies were selected as the extreme cases among 108 original colonies. Thirty brood cells were perforated with a pin in two H and two NH colonies and observations were made after 1, 2, 3, 4, 5, 6, and 24 h. Different stages of uncapping and removing were recorded. Differences in uncapping and removal between H and NH colonies were significant for all comparisons made at the different times after perforation. Using observation hives one obtains a better and faster discrimination between H and NH colonies than in full size colonies. It is possible to differentiate H and NH within a few hours after perforating the cells.     

Varroa destructor infestation in untreated honey bee (Hymenoptera: Apidae) colonies selected for hygienic behavior

Honey bee (Apis mellifera L.) colonies bred for hygienic behavior were tested in a large field trial to determine if they were able to resist the parasitic mite Varroa destructor better than unselected colonies of"Starline" stock. Colonies bred for hygienic behavior are able to detect, uncap, and remove experimentally infested brood from the nest, although the extent to which the behavior actually reduces the overall mite-load in untreated, naturally infested colonies needed further verification. The results indicate that hygienic colonies with queens mated naturally to unselected drones had significantly fewer mites on adult bees and within worker brood cells than Starline colonies for up to 1 yr without treatment in a commercial, migratory beekeeping operation. Hygienic colonies actively defended themselves against the mites when mite levels were relatively low. At high mite infestations (>15% of worker brood and of adult bees), the majority of hygienic colonies required treatment to prevent collapse. Overall, the hygienic colonies had similar adult populations and brood areas, produced as much honey, and had less brood disease than the Starline colonies. Thus, honey bees bred for hygienic behavior performed as well if not better than other commercial lines of bees and maintained lower mite loads for up to one year without treatment.     

Uncapping activity of Apis mellifera L. (Hymenoptera: Apidae) towards worker brood cells infested with the mite Varroa destructor Anderson & Treuman (Mesostigmata: Varroidae)

Varroosis, a disease caused by the mite Varroa destructor Anderson and Treuman has killed hundreds of thousands of Apis mellifera L. colonies in various parts of the world. Nevertheless, the damage caused by this mite varies with the type of bee and climate conditions. Varroa causes little damage to Africanized bee colonies in Brazil, as the infestation rates are relatively stable and low. We evaluated the hygienic behavior (uncapping and removal of brood) of highly hygienic Africanized bees using combs with worker brood cells infested (naturally) and no infested with V. destructor. The daily uncapping rate, measured in eight colonies during six days, was 3.5 fold higher in the combs infested with varroa compared to no infested combs. The results show that the Africanized bees are able to recognise and remove brood cells naturally infested with V. destructor what is an important mechanism for tolerance against varroa.     

Hygienic behavior of the honey bee (Apis mellifera) is independent of sucrose responsiveness and foraging ontogeny

Hygienic behavior in honey bees is a behavioral mechanism of disease resistance. Bees bred for hygienic behavior exhibit an increased olfactory sensitivity to odors of diseased brood, which is most likely differentially enhanced in the hygienic line by the modulatory effects of octopamine (OA), a noradrenaline-like neuromodulator. Here, we addressed whether the hygienic behavioral state is linked to other behavioral activities known to be modulated by OA. We specifically asked if, during learning trials, bees from hygienic colonies discriminate better between odors of diseased and healthy brood because of differences in sucrose (reward) response thresholds. This determination had to be tested because sucrose response thresholds are susceptible to OA modulation and may have influenced the honey bee's association of the conditioned stimulus (odor) with the unconditioned stimulus (i.e., the sucrose reward). Because the onset of first foraging is also modulated by OA, we also examined whether bees from hygienic colonies differentially forage at an earlier age compared to bees from non-hygienic colonies. Our study revealed that 1-day- and 15- to 20-day-old bees from the hygienic line do not have lower sucrose response thresholds compared to bees from the non-hygienic lines. In addition, hygienic bees did not forage at an earlier age or forage preferentially for pollen as compared to non-hygienic bees. These results support the idea that OA does not function in honey bees simply to enhance the detection of all chemical cues non-selectively or control related behaviors regardless of their environmental milieu. Our results indicate that the behavioral profile of the hygienic bee is sculpted by multiple factors including genetic, neural, social and environmental systems.     

Is the number of antennal plate organs (sensilla placodea) greater in hygienic than in non-hygienic Africanized honey bees?

Hygienic behavior is a desirable trait in honey bees (Apis mellifera L.), as hygienic bees quickly remove diseased brood, interrupting the infectious cycle. Hygienic lines of honey bees appear to be more sensitive to the odors of dead and diseased honey bee brood, and Africanized honey bees are generally more hygienic than are European honey bees. We compared the number of sensilla placodea, antennal sensory structures involved in the perception of odor, in 10 bees from each of six hygienic and four non-hygienic colonies of Africanized honey bees. The sensilla placodea of three of the terminal segments (flagellomeres) of the right antenna of each bee were counted with a scanning electron microscope. There were no significant differences in the mean numbers of sensilla placodea between the hygienic and non-hygienic bees, though the variance was higher in the hygienic group. Flagellomere 4 had significantly more sensilla placodea than flagellomeres 6 and 8. However, there was no significant difference between the other two flagellomeres. As hygienic bees are capable of identifying dead, injured, or infested brood inside a capped brood cell, sensilla placodea probably have an important role in enabling worker bees to sense sick brood. However, we did not find greater numbers of this sensory structure in the antennae of hygienic, compared to non-hygienic Africanized honey bees.     

Evaluations of the Removal of <Emphasis Type="Italic">Varroa destructor</Emphasis> in Russian Honey Bee Colonies that Display Different Levels of <Emphasis Type="Italic">Varroa</Emphasis> Sensitive Hygienic Activities

The removal of Varroa destructor was assessed in Russian honey bee (RHB) colonies with known levels of Varroa Sensitive Hygienic (VSH) and brood removal activities. The expression of grooming behaviour using individual bees was also measured using three groups of RHB displaying different VSH levels: low hygiene (RHB-LH, < 35% VSH), medium hygiene (RHB-MH, 35–70%) and high hygiene (RHB-HH, > 70%). Italian colonies (5.43–71.62% VSH) served as control. Our results demonstrated, for the first time, significant relationships between two hygienic responses (VSH activity measured as percent change in infestation and the actual brood removal of Varroa-infested donor comb) and two measurements of mite fall (trapped old mites/trapped mites or O/T and trapped young mites/trapped mites or Y/T). However, these relationships were only observed in RHB colonies. In addition, the RHB colonies that displayed the highest levels of hygiene (RHB-HH) also groomed longer in response to the presence of a V. destructor mite based on individual bee assays. The positive regressions between the two hygienic measurements and O/T and their negative regressions with Y/T suggest that the removal of infested brood prevented successful mite reproduction, ultimately suppressing V. destructor infestations in the RHB colonies. In addition, it is demonstrated that RHB resistance to V. destructor rests on both an increased hygienic response and the removal of phoretic mites, released by hygienic behaviour, through grooming. Both resistance traits are reflected in the O/T and Y/T ratios found in trapped mites from RHB colonies. None of the measurements involving mite injuries were associated with any measurements of hygiene and colony infestations.     

Phenotypic and Genetic Analyses of the Varroa Sensitive Hygienic Trait in Russian Honey Bee (Hymenoptera: Apidae) Colonies

Varroa destructor continues to threaten colonies of European honey bees. General hygiene, and more specific Varroa Sensitive Hygiene (VSH), provide resistance towards the Varroa mite in a number of stocks. In this study, 32 Russian (RHB) and 14 Italian honey bee colonies were assessed for the VSH trait using two different assays. Firstly, colonies were assessed using the standard VSH behavioural assay of the change in infestation of a highly infested donor comb after a one-week exposure. Secondly, the same colonies were assessed using an “actual brood removal assay” that measured the removal of brood in a section created within the donor combs as a potential alternative measure of hygiene towards Varroa-infested brood. All colonies were then analysed for the recently discovered VSH quantitative trait locus (QTL) to determine whether the genetic mechanisms were similar across different stocks. Based on the two assays, RHB colonies were consistently more hygienic toward Varroa-infested brood than Italian honey bee colonies. The actual number of brood cells removed in the defined section was negatively correlated with the Varroa infestations of the colonies (r² = 0.25). Only two (percentages of brood removed and reproductive foundress Varroa) out of nine phenotypic parameters showed significant associations with genotype distributions. However, the allele associated with each parameter was the opposite of that determined by VSH mapping. In this study, RHB colonies showed high levels of hygienic behaviour towards Varroa -infested brood. The genetic mechanisms are similar to those of the VSH stock, though the opposite allele associates in RHB, indicating a stable recombination event before the selection of the VSH stock. The measurement of brood removal is a simple, reliable alternative method of measuring hygienic behaviour towards Varroa mites, at least in RHB stock.     

Honey bee hygienic behaviour does not incur a cost via removal of healthy brood

In the honey bee, hygienic behaviour, the removal of dead or diseased brood from capped cells by workers, is a heritable trait that confers colony‐level resistance against brood diseases. This behaviour is quite rare. Only c. 10% of unselected colonies show high levels of hygiene. Previous studies suggested that hygiene might be rare because it also results in the removal of healthy brood, thereby imposing an ongoing cost even when brood diseases are absent. We tested this hypothesis by quantifying hygienic behaviour in 10 colonies using a standard technique, the freeze‐killed brood (FKB) bioassay. At the same time, we also quantified the removal of untreated brood. The study colonies showed a wide range in hygienic behaviour, removing 19.7–100% of the FKB. The removal of untreated brood ranged from 2% to 44.4%. However, there was no correlation between the two removal rates for any of the four age groups of untreated brood studied (eggs, young larvae, older larvae from uncapped cells and larvae/pupae from capped cells). These results do not support the cost‐to‐healthy‐brood hypothesis for the rarity of hygienic behaviour.     

Hygienic behavior in the honey bee (Apis mellifera L.) and the modulatory role of octopamine

Honey bees, Apis mellifera, which perform hygienic behavior, quickly detect, uncap and remove diseased brood from the nest. This behavior, performed by bees 15-20 days old and prior to foraging, is likely mediated by olfactory cues. Because the neuromodulator octopamine (OA) plays a pivotal role in olfactory-based behaviors of honey bees, we examined whether bees bred for hygienic and nonhygienic behavior differed with regard to their OA expression and physiology. We compared the staining intensity of octopamine-immunoreactive (OA-ir) neurons in the deutocerebral region of the brain, medial to the antennal lobes, between hygienic and nonhygienic bees (based on genotype and phenotype). We also tested how the olfactory responses of the two lines, based on electroantennograms (EAGs), were affected by oral administration of OA and of epinastine, a highly specific OA antagonist. Our results revealed that bees expressing hygienic behavior (irrespective of genotype) possessed OA-ir neurons that exhibited more intense labeling than same-aged bees not performing the behavior. In bees bred for nonhygienic behavior, OA significantly increased the EAG response to low concentrations of diseased brood odor. Conversely, in bees bred for hygienic behavior, epinastine significantly reduced the magnitude of the EAG response, a reduction not observed in nonhygienic bees. Our results provide two lines of evidence that OA has the potential to facilitate the detection and response of honey bees to diseased brood. We discuss the contributions of OA for behavioral shaping and its ability to bias the nervous system to express one form of behavior over another.     

Resistance of the honey bee, Apis mellifera to the acarian parasite Varroa destructor: behavioural and electroantennographic data

Abstract. One way in which Apis mellifera honey bees resist Varroa destructor is by detection and elimination of nestmates. This study uses behavioural tests and electroanntennography to assess the role of chemostimuli in recognition by honey bees of this acarian ectoparasite. Behavioural tests using living or dead parasites involved observation of honey bee grooming activity (antennation) under controlled conditions in Petri dishes, and removal behaviour (uncapping and elimination of parasitized and unparasitized control brood cells) under natural conditions. Some bees from colonies with both small and large parasite populations showed aggressive behaviour (biting). No difference was observed according to whether the mite was dead or alive. Under natural conditions, bees uncapped more parasitized cells than control cells. Electroantennographic tests were performed to measure sensitivity to various Varroa extracts at three concentrations (10, 20 and 30 Varroa Equivalents). Only 30 Varroa Equivalent methanol extracts made from Varroa collected from brood cells elicited significantly greater antennal response than controls (pure solvent). All three methanol extracts elicited significantly greater antennal response than controls. No response was observed using Varroa extracts made with acetone or hexane. These findings suggest that polar products may act as chemostimuli for recognition of V. destructor by honey bees. Further study will be necessary to determine which polar products are involved in this recognition and assess grooming and removal behaviour using these products.     

Rapid anti-pathogen response in ant societies relies on high genetic diversity

Social organisms are constantly exposed to infectious agents via physical contact with conspecifics. While previous work has shown that disease susceptibility at the individual and group level is influenced by genetic diversity within and between group members, it remains poorly understood how group-level resistance to pathogens relates directly to individual physiology, defence behaviour and social interactions. We investigated the effects of high versus low genetic diversity on both the individual and collective disease defences in the ant Cardiocondyla obscurior. We compared the antiseptic behaviours (grooming and hygienic behaviour) of workers from genetically homogeneous and diverse colonies after exposure of their brood to the entomopathogenic fungus Metarhizium anisopliae. While workers from diverse colonies performed intensive allogrooming and quickly removed larvae covered with live fungal spores from the nest, workers from homogeneous colonies only removed sick larvae late after infection. This difference was not caused by a reduced repertoire of antiseptic behaviours or a generally decreased brood care activity in ants from homogeneous colonies. Our data instead suggest that reduced genetic diversity compromises the ability of Cardiocondyla colonies to quickly detect or react to the presence of pathogenic fungal spores before an infection is established, thereby affecting the dynamics of social immunity in the colony.     

Task-specific expression of the foraging gene in harvester ants

In social insects, groups of workers perform various tasks such as brood care and foraging. Transitions in workers from one task to another are important in the organization and ecological success of colonies. Regulation of genetic pathways can lead to plasticity in social insect task behaviour. The colony organization of advanced eusocial insects evolved independently in ants, bees, and wasps and it is not known whether the genetic mechanisms that influence behavioural plasticity are conserved across species. Here we show that a gene associated with foraging behaviour is conserved across social insect species, but the expression patterns of this gene are not. We cloned the red harvester ant (Pogonomyrmex barbatus) ortholog (Pbfor) to foraging, one of few genes implicated in social organization, and found that foraging behaviour in harvester ants is associated with the expression of this gene; young (callow) worker brains have significantly higher levels of Pbfor mRNA than foragers. Levels of Pbfor mRNA in other worker task groups vary among harvester ant colonies. However, foragers always have the lowest expression levels compared to other task groups. The association between foraging behaviour and the foraging gene is conserved across social insects but ants and bees have an inverse relationship between foraging expression and behaviour.     

Resistance to Chalkbrood disease in Apis mellifera L. (Hymenoptera: Apidae) colonies with different hygienic behaviour

Chalkbrood disease affects the larvae of honeybees Apis mellifera L. and is caused by the fungus Ascosphaera apis. Infected larvae die when they are stretched in the cap cell and suffer a gradual hardening that ends in a very hard structure (mummie). Several studies have demonstrated that colonies that express an efficient hygienic behaviour (uncapping of cell and subsequent removal of dead brood) exhibit a higher resistance to the disease. However, it remains unclear whether the advantage of hygienic colonies over less hygienic ones lies in the ability to remove mummies or in the early detection of infected larvae and its cannibalization before they harden. To elucidate this aspect, the hygienic behaviour of 24 colonies, which were subsequently provided with pollen cakes containig A. apis, was evaluated. The number of mummies and the number of partially cannibalized and whole larvae in uncapped cells were recorded. The most hygienic colonies controlled the disease better. These colonies also had a higher tendency to uncap cells that contained infected larvae and cannibalize them. The presence of A. apis in partially cannibalized and whole larvae in uncapped cells indicate that the advantage of hygienic colonies over less hygienic ones lies in the early detection of infected larvae death and their quick removal from the cell before they become mummies.     

Brood Odor Discrimination Abilities in Hygienic Honey Bees (Apis mellifera L.) Using Proboscis Extension Reflex Conditioning

To understand the effect of abnormal brood odors on the initiation or control of hygienic behavior in honey bees, we employed the associative learning paradigm, proboscis extension reflex conditioning. Bees from two genetic lines(hygienic and non-hygienic) were able to discriminate between high concentrations of two floral odors equally well. Differential discrimination abilities were observed between the two lines when healthy and diseased brood odors were used, with the bees from the hygienic line discriminating between the pair of brood odors better than the non-hygienic bees. These results suggest that hygienic behavior in individual bees is associated with the bees' responses to olfactory stimuli emanating from diseased brood.     

Open-air-nesting honey bees Apis dorsata and Apis laboriosa differ from the cavity-nesting Apis mellifera and Apis cerana in brood hygiene behaviour

The cavity-nesting Apis mellifera and Apis cerana bees detect, uncap, and remove diseased brood. The hygiene behaviour of open-air-nesting bees Apis dorsata and Apis laboriosa was investigated in India and Nepal. Sealed A. dorsata pupae were pin-killed or deep-frozen. The workers removed 73 or 37% of damaged pin-killed pupae depending on the diameter of the pins, and only 7% of the frozen undamaged pupae. Migrating A. dorsata and A. laboriosa left unopened the sealed brood in deserted combs. Thus, A. dorsata and A. laboriosa do not open undamaged cells with dead brood. This behaviour is a more efficient mechanism in preventing the spread of diseases and parasitic mites than uncapping and removing dead pupae by A. mellifera and A. cerana. It may be beneficial for migrating A. dorsata and A. laboriosa to temporarily disuse part of the comb cells in exchange for arresting the mites there and thus reducing the increase of their population.     

Potential mechanism for detection by Apis mellifera of the parasitic mite Varroa destructor inside sealed brood cells

Abstract The parasitic mite Varroa destructor Anderson & Trueman is a major pest of the honeybee Apis mellifera L. throughout the world. Chemical agents currently used for mite control leave contaminating residues and promote pesticide resistance. As an alternative means of control, it would be useful to identify natural substances enabling bees to detect Varroa inside brood cells. These substances could then be used to trigger mite hygienic behaviour by bees.
In this study several techniques were used to screen substances that might allow detection of infested brood cells by bees. Gas chromatography-mass spectrometry analysis was performed on substances extracted in dichloromethane from the contents of brood cells. Solid phase microextraction and solid injection were performed on substances obtained from living and dead Varroa, respectively. Electroantennography was performed to assess the sensitivity of olfactory receptors in bee antennae to some of these substances.
Principal component analysis based on proportions of cuticular substances allowed discrimination between bees and other cell contents. Foundress Varroa exhibited the greatest dissimilarity to healthy pupae that were used as controls. Immature Varroa and faecal material were intermediate. High molecular weight compounds, mainly dimethylalkanes, were proportionally the most characteristic components of foundress Varroa . This finding suggests that these compounds would be the most apt to induce uncapping of cells infested by Varroa . Solid-phase microextraction and solid injection demonstrated the presence of aliphatic acids, esters, and one alcohol, eicosenol, in Varroa . Electroantennographic recordings showed that mite-resistant bees were more responsive to some acids and one ester. We speculate that these compounds may be involved in recognition of living Varroa by honeybees.     

Thresholds of Response in Nest Thermoregulation by Worker Bumble Bees, Bombus bifarius nearcticus (Hymenoptera: Apidae)

Regulation of nest temperature is important to the fitness of eusocial insect colonies. To maintain appropriate conditions for the developing brood, workers must exhibit thermoregulatory responses to ambient temperature. Because nest-mate workers differ in task performance, thermoregulatory behavior provides an opportunity to test threshold of response models for the regulation of division of labor. We found that worker bumble bees ( Bombus bifarius nearcticus ) responded to changes in ambient temperature by altering their rates of performing two tasks – wing fanning and brood cell incubation. At the colony level, the rate of incubating decreased, and the rate of fanning increased, with increasing temperature. Changes in the number of workers performing these tasks were more important to the colony response than changes in workers' task performance rates. At the individual level, workers' lifetime rates of incubation and fanning were positively correlated, and most individuals did not specialize exclusively on either of these temperature-sensitive tasks. However, workers differed in the maximum temperature at which they incubated and in the minimum temperature at which they fanned. More individuals fanned at high and incubated at low temperatures. Most of the workers that began incubating at higher temperatures continued performing this task at lower temperatures, when additional nest-mates became active. The converse was true for fanning behavior. These data are consistent with a threshold of response model for thermoregulatory behavior of B. bifarius workers.     

Dynamics of Collective Decision Making of Honeybees in Complex Temperature Fields

Endothermic heat production is a crucial evolutionary adaptation that is, amongst others, responsible for the great success of honeybees. Endothermy ensures the survival of the colonies in harsh environments and is involved in the maintenance of the brood nest temperature, which is fundamental for the breeding and further development of healthy individuals and thus the foraging and reproduction success of this species. Freshly emerged honeybees are not yet able to produce heat endothermically and thus developed behavioural patterns that result in the location of these young bees within the warm brood nest where they further develop and perform tasks for the colony. Previous studies showed that groups of young ectothermic honeybees exposed to a temperature gradient collectively aggregate at the optimal place with their preferred temperature of 36°C but most single bees do not locate themselves at the optimum. In this work we further investigate the behavioural patterns that lead to this collective thermotaxis. We tested single and groups of young bees concerning their ability to discriminate a local from a global temperature optimum and, for groups of bees, analysed the speed of the decision making process as well as density dependent effects by varying group sizes. We found that the majority of tested single bees do not locate themselves at the optimum whereas sufficiently large groups of bees are able to collectively discriminate a suboptimal temperature spot and aggregate at 36°C. Larger groups decide faster than smaller ones, but in larger groups a higher percentage of bees may switch to the sub-optimum due to crowding effects. We show that the collective thermotaxis is a simple but well evolved, scalable and robust social behaviour that enables the collective of bees to perform complex tasks despite the limited abilities of each individual.