Hygienic Bees Research Articles

Varroa destructor mite population dynamics in africanized honeybee (Apis mellifera) colonies in a semi-arid region.

Most published data on mite infestation rates in semi-arid regions have been collected over only 3 or 4 months during a specific period of the year. Therefore, the aim of this study was to observe parasite-host dynamics of hygienic and non-hygienic Africanized bee colonies considering environmental factors that may influence Varroa destructor mite infestation rates in a semi-arid region. To this end, the brood puncture method was applied to 37 colonies, forming two groups, namely G1, encompassing 16 hygienic colonies, and G2, comprising 21 non-hygienic colonies. After forming the groups, 300 worker bees from each colony were examined monthly for mite infestations and the data were correlated with climatological records. The monthly infestation average was considered low, below 10%, except in November, when it reached 12.19% ± 6.45. No statistically significant difference was observed for inter-group infestation rates (P > 0.05). When mite infestation rates were associated with climatic variables, they were linked to colony losses (32%) due to swarming. No significant correlations between hygienic behaviour and parasite infestation rates were noted. Nonetheless, these results support the idea that there is no need to apply acaricides for V. destructor control in Brazil.

Differential hygienic behavior of Apis cerana F. and Apis mellifera L. to Sacbrood virus infection

• Differences of hygienic removal of pin-killed pupae and SBV-killed larvae between workers. • mellifera workers removed more SBV-p.i larvae at the four- and five-day old. • SBV-dead broods remained in cells of A. cerana colonies was higher than A. mellifera. • SBV-inoculated A. mellifera colonies were higher than that of A. cerana. Beekeeping with Apis cerana of Korean apiculture is facing with serious colony collapse caused by invasive Sacbrood virus (SBV) disease. This fatal brood disease was the main reason of more than 90% colony lost in Korea leading almost the extinct crisis. Sacbrood virus can infect either larvae or adult honeybees, with a higher sensibility of larvae to the infection. Since SBV has spread to all over the country, efforts have been made to treat and prevent this devastating disease although no effective results have so far been obtained. Several studies have demonstrated that Apis mellifera bee colonies that express an efficient hygienic behavior exhibit a higher resistance to the brood disease. In this study we demonstrated that the differences of hygienic behavior between A. cerana and A. mellifera . A. cerana more efficiently removed the pin-killed brood than A. mellifera . On the other hand, A. mellifera more efficiently removed SBV-infected larvae and SBV-dead brood than A. cerana . However, it remains unclear whether the advantage of hygienic bee could have efficacy against Sacbrood disease on A. cerana colonies.

Retraction: Evaluation of queen cell acceptance and royal jelly production between hygienic and non-hygienic honey bee (Apis mellifera) colonies.

Retraction: Evaluation of queen cell acceptance and royal jelly production between hygienic and non-hygienic honey bee (Apis mellifera) colonies.

Multiple benefits of breeding honey bees for hygienic behavior

Honey bee colonies are prone to invasion by pests and pathogens. The combination of the parasitic mite Varroa destructor (Varroa) and the multiple viruses it vectors, is a major driver of colony losses. Breeding for hygienic behavior to reduce Varroa populations is considered a sustainable way to reduce the impact of Varroa on honey bee health. However, hygienic behavior may have a cost to the health of individual bees, both in terms of viral infection risk and immune function. To determine whether selection for hygienic behavior at the colony level is associated with trade-offs in honey bee viral infection and immune function, we compared Varroa populations, viral loads, and individual immune function between honey bee colonies that were bred for high and low hygienic behavior. Specifically, we measured Varroa infestation, Deformed wing virus DWV-A, DWV-B, Acute bee paralysis virus (ABPV), and Israeli acute paralysis virus IAPV viral genome levels in bee samples from artificially inseminated queens in our bi-directional selection program for hygienic behavior in Israel. In addition, we evaluated the expression of 12 genes from the Jak-STAT, Toll, IMD and RNAi immune pathways. We found significantly lower Varroa infestation and DWV loads in highly hygienic colonies than in colonies exhibiting low hygienic behavior. In addition, workers of the hygienic colonies had significantly higher expression of the immune genes PGRP-S2 and hymenoptaecin compared to workers from low hygienic colonies. These results indicate no trade-offs in breeding for hygienic behavior. Hygienic honey bees were associated with reduced Varroa populations and reduced DWV prevalence or load at the colony level. Individual immunity of hygienic bees was increased, which could also contribute to lower virus levels, although lower Varroa levels due to social immunity presumably contributed as well. In sum, we demonstrate multiple health benefits of breeding for honey bee hygiene.

Characteristics of some chitin body parts in worker bees (Apis mellifera L.) from hygienic and non-hygienic bee colonies

Abstract. In the conducted study the level of manifestation of hygienic behavior of 25 bee colonies was tested from the honey bee (Apis mellifera L.) in Bulgaria. During the testing the method used for killing the brood was with an entomological pin. Based on the results two types of colonies were determined – hygienic (where 48 h after killing the brood the percentage of cleaned cells with dead brood was over 95%) and non-hygienic (where 48 h after killing the brood the percentage of cleaned cells with dead brood was lower than 95%). From each bee colony young non-flying worker bees were taken and the morphometric measurements of their chitin body parts were made. In the study 18 morphological traits were tested and 2 indexes were calculated. High degree of reliability (p≤0.001) between hygienic and non-hygienic bee colonies was determined for 2 morphological traits - Total length of fore wing and Length of proboscis. For the traits Length of tibia and Width of Іst tarsus of fore leg and Width and Surface of the left wax mirror of IIIrd sternit medium degree of reliability (p≤0.01) was determined. In 3 other traits - Length of femur of fore leg, Length of IIIrd sternit and Length of IInd part of the cubital cell of the fore wing low reliability (p≤0.05) was established. The results obtained from the present study indicate the existence of statistically significant differences between the morphometric characteristics of worker bees from hygienic and non-hygienic bee colonies. These traits are related to the productivity of worker bees.

Evaluation of queen cell acceptance and royal jelly production between hygienic and non-hygienic honey bee (Apis mellifera) colonies.

Honey bees are crucial for pollination services globally and produce important hive products including honey, royal jelly, pollen, and propolis that are being used commercially in food, cosmetics, and alternative medicinal purposes. Among the bee products, royal jelly (RJ) has long attracted scientists' interest because of its importance in honey caste differentiation. The present research was carried out to determine the acceptance rate of queen cells, and RJ production between the hygienic and non-hygienic lines. Further, this study unveils the effect of pollen substitute diets on the queen cell acceptance rate and RJ yields between both bee stocks. Results showed that the uncapped brood cells and dead brood's removal percentage was significantly more in hygienic bee colonies in comparison to non-hygienic bee colonies (p < 0.05). The average percentage of larval acceptance was statistically higher in hygienic lines (64.33 ± 2.91%) compared to non-hygienic lines (29.67 ± 1.20%). Similarly, the RJ mean weight per colony differed statistically between both bee stocks (p<0.001), which were 12.23 ± 0.52 g and 6.72 ± 0.33 g, respectively. Moreover, our results demonstrated that a significant difference was observed in larval acceptance rate, RJ yields (per colony and per cup) between both bee stocks those fed on various diets. However, no significant difference was recorded in RJ yields (per colony and per cup) between both bee stock that feeds on either commercially available pollen or pollen substitute. This study may provide future applications in helping bee breeders to choose the bees that carry a higher level of hygienic behavior with high RJ production traits.

Hygienic behavior and fat body development in worker bees (Apis mellifera L.)

Abstract. Testing for the level of manifestation of the hygienic behavior of 32 bee colonies from the local honey bee (Apis mellifera L.) from three apiaries has been carried out. Depending on the level of manifestation of the cleansing instinct, two groups of bee colonies have been formed – hygienic (these clean over 95% of the cells with dead brood up to the 48th hour after killing the brood) and non-hygienic (these clean less than 95% of the cells with dead brood up to the 48th hour). Worker bee samples have been taken from the tested bee colonies to determine the level of fat body development. The fat body is important for the bee organism, since in addition to being a fat and carbohydrate storage depot, it is also considered to be the center of metabolism in insects. The analysis of the results from the present study revealed that the mean value of the level of fat body development in worker bees from the group of hygienic bee colonies was 2.76±0.038. In the group of non-hygienic colonies this value was 7.61% lower (2.55±0.079). This difference is statistically significant at p≤0.05. Significant differences have been found in the development of the fat body in worker bees from apiaries at a distance of 100 km without regard to hygienic behavior and according to hygienic behavior. The results obtained suggest that there is a correlation between the level of fat body development and the hygienic behavior in bees.

Semi-automatic detection of honeybee brood hygiene-an example of artificial learning to facilitate ethological studies on social insects.

Machine-learning techniques are shifting the boundaries of feasibility in many fields of ethological research. Here, we describe an application of machine learning to the detection/measurement of hygienic behaviour, an important breeding trait in the honey bee (Apis mellifera). Hygienic worker bees are able to detect and destroy diseased brood, thereby reducing the reproduction of economically important pathogens and parasites such as the Varroa mite (Varroa destructor). Video observation of this behaviour on infested combs has many advantages over other methods of measurement, but analysing the recorded material is extremely time-consuming. We approached this problem by combining automatic tracking of bees in the video recordings, extracting relevant features, and training a multi-layer discriminator on positive and negative examples of the behaviour of interest. Including expert knowledge into the design of the features lead to an efficient model for identifying the uninteresting parts of the video which can be safely skipped. This algorithm was then used to semiautomatically identify individual worker bees involved in the behaviour. Application of the machine-learning method allowed to save 70% of the time required for manual analysis, and substantially increased the number of cell openings correctly identified. It thereby turns video-observation of individual cell opening events into an economically competitive method for selecting potentially resistant bees. This method presents an example of how machine learning can be used to boost ethological research, and how it can generate new knowledge by explaining the learned decision rule in form of meaningful parameters.

Reproduction of Varroa destructor does not elicit varroa sensitive hygiene (VSH) or recapping behaviour in honey bee colonies (Apis mellifera)

Suppressed mite reproduction (SMR) is an important trait for the selection of Varroa resistant honey bee colonies. It has repeatedly been assumed that SMR is an effect of varroa sensitive hygiene (VSH) when hygienic bees preferably remove those brood cells where the mite has reproduced. We here compare the VSH behaviour of honey bees toward brood cells artificial infested with a varroa mite. By infesting half of the brood cells directly after the cell capping and the other half only 24 h later, we established two groups with high (> 75%) and low (< 2%) mite reproduction. After 8 days, about 40% of the infested brood cells were removed, however without any difference between both groups. Likewise, no group differences were recorded in the percentage of recapped brood cells. This strongly indicates that the presence of mite offspring is not a crucial trigger for the VSH behaviour. SMR data like the percentage of non-reproducing mites are therefore not the optimal measure for the selection of colonies with high VSH.

Temporal changes in volatile profiles of Varroa destructor‐infested brood may trigger hygienic behavior in Apis mellifera

AbstractVarroa destructor Anderson &amp; Trueman (Acari: Varroidae) is one of the major contributors to the significant losses of western honey bee, Apis mellifera L. (Hymenoptera: Apidae), colonies worldwide. Hygienic behavior, in which individual workers detect, uncap, and remove unhealthy brood, is a type of social immunity that reduces pathogen and parasite loads in the colony. Previous evidence suggests that hygienic worker bees identify diseased brood through olfactory cues. The aims of the present work were (1) to study the hygienic behavior of worker bees toward V. destructor‐infested cells at different stages of brood development, (2) to explore changes in brood volatile profiles associated with the progression of mite infestation, and (3) to analyze the role of specific volatile compounds in triggering the hygienic behavior. Results showed that the removal rate of infested brood changed along its development and the progression of mite reproduction. Two compounds, ethyl hexanoate and α‐pinene, were present in volatile collections from mite‐infested pupae but absent from uninfested pupae. Field bioassays showed that these volatiles are relevant to elicit the hygienic behavior. A third compound, β‐ocimene, was present in infested and uninfested brood but its abundance pattern varied according to the infestation status throughout brood development. Specifically, for uninfested brood, the abundance of β‐ocimene showed a reduction in black‐eyed pupae, whereas for infested brood, its abundance decreased drastically in light‐pink‐eyed pupae and remained constant in black‐eyed pupae. Our results revealed that olfactory signals associated with V. destructor infestation change as the reproductive cycle of V. destructor progresses inside the cell. These changes can be mimicked to some extent by adding specific volatile compounds to the cell to induce hygienic removal. These findings shed light on the chemical basis of hygienic behavior against V. destructor and could facilitate the development of improved hygienic selection tools to breed mite‐resistant honey bee colonies.

Changes in chemical cues of Melissococcus plutonius infected honey bee larvae

European foulbrood (EFB), caused by Melissococcus plutonius, is a globally distributed bacterial brood disease affecting Apis mellifera larvae. There is some evidence, even if under debate, that spreading of the disease within the colony is prevented by worker bees performing hygienic behaviour, including detection and removal of infected larvae. Olfactory cues (brood pheromones, signature mixtures, diagnostic substances) emitted by infected individuals may play a central role for hygienic bees to initiate the disease-specific behaviour. However, the mechanisms of cue detection and brood removal, causing hygienic behaviour in EFB affected colonies, are poorly understood. Here, coupled gas chromatography-mass spectrometry (GC–MS) was used to detect disease-specific substances, changes in cuticular hydrocarbon (CHC) profiles, and brood ester pheromones (BEPs) of honey bee larvae artificially infected with M. plutonius. Although no diagnostic substances were found in significant quantities, discriminant analysis revealed specific differences in CHC and BEP profiles of infected and healthy larvae. β-Ocimene, a volatile brood pheromone related to starvation and hygienic behaviour, was present in all larvae with highest quantities in healthy young larvae; whereas oleic acid, a non-volatile necromone, was present only in old infected larvae. Furthermore, γ-octalactone (newly discovered in A. mellifera in this study) was detectable in trace amounts only in infected larvae. We propose that the deviation from the olfactory profile of healthy brood is supposed to trigger hygienic behaviour in worker bees. To confirm the relevance of change in the chemical bouquet (CHCs, BEPs, γ-octalactone, etc.), a field colony bioassay is needed, using healthy brood and hygienic bees to determine if bouquet changes elicit hygienic behaviour.

Time-accuracy trade-off and task partitioning of hygienic behavior among honey bee (Apis mellifera) workers

Behavioral specialization and cooperation are fundamental in the organization and success of social groups. Honey bee workers display hygienic behavior, defined as the detection, uncapping, and removal of unhealthy brood. We present detailed analyses of behavioral specialization and task partitioning among hygienic worker bees, focusing on uncapping of brood cells and removal of the cells’ content in freeze-killed brood assays. We demonstrate specialization of hygienic workers on either uncapping or removal and task partitioning among multiple individuals. Speed of hygiene decreases with the number of behavioral instances, suggesting a time cost for cooperation of multiple individuals. Additional analyses of an individual agent-based simulation of hygienic removal of Varroa-mite-infested brood demonstrate that erroneous removal of healthy brood can be reduced by task partitioning due to collective decision-making. Combined, our results indicate a speed-accuracy trade-off in the collective performance of hygienic behavior: Hygienic behavior may take longer when many individuals contribute and specialize on different tasks, but this organization of work also can prevent costly mistakes. This trade-off may explain the observed combination of elite workers and numerous other workers that contribute only little. Honey bees and other social animals form successful groups that are characterized by individual specialization on different tasks. Hygienic behavior of honey bees removes unhealthy brood from the nest and is important for the health of honey bee colonies. Here, we observed that individual worker bees specialize on either of the two main tasks involved in hygienic behavior: uncapping cells or removing brood. We also found that a few individuals perform the majority of the work and that the speed of hygienic behavior is higher when one individual works continuously instead of multiple individuals. Additionally, we show in computer simulations that the number of erroneous brood removals is decreased when separate workers contribute to the different aspects of hygienic behavior. Thus, our study indicates that a speed-accuracy trade-off might drive the evolution of task partitioning in hygienic behavior.

Influencias climáticas, regionales y cantidad de cría en el comportamiento higiénico de Apis mellifera

The hygienic behavior of honeybees (Apis mellífera) is a mechanism highly influenced by genetic effects that confers resistance against diseases and parasites. The objective of this study was to determine whether climatic, regional and brood quantity influences are related to hygienic behavior in beehives in the state of Jalisco, Mexico. A total of 142 colonies were evaluated by freezing pupae with liquid nitrogen in two climatic zones (mountain and valley). Colonies located in the municipalities of the low zone removed significantly more frozen brood than the colonies located in the municipalities of the higher zone (p 80%). This degree of cleanliness is acceptable for initiating selective breeding projects for highly hygienic bees to improve colony health, which would benefit the beekeeping industry.

Cuticular pheromones stimulate hygienic behavior in the honey bee (Apis mellifera)

The health of western honey bee (Apis mellifera) colonies is challenged by the parasitic mite Varroa destructor and the numerous harmful pathogens it vectors. Selective breeding for the naturally occurring social immune trait “hygienic behavior” has emerged as one sustainable approach to reducing the mites’ impact on honey bees. To expand our understanding of hygienic triggers and improve hygienic selection tools, we tested the hypothesis that the cuticular compounds (Z)-10-tritriacontene and (Z)-6-pentadecene, previously associated with unhealthy honey bee brood and/or brood targeted for hygiene, are triggers of honey bee hygienic behavior independent of brood health. In support of our hypothesis, application of synthetic (Z)-10-tritriacontene and (Z)-6-pentadecene onto brood and brood cell caps significantly increased hygienic behavior compared to application of similarly structured hydrocarbon controls (Z)-16-dotriacontene and (Z)-7-pentadecene. Furthermore, we demonstrate a significant positive correlation between colony-level hygienic responses to (Z)-10-tritriacontene and the traditional freeze-killed brood assay for selection of hygienic honey bee stocks. These results confirm biological activity of (Z)-6-pentadecene and reveal (Z)-10-tritriacontene as a novel hygiene trigger. They also support development of improved tools for honey bee colony monitoring and hygienic selection, and thus may accelerate development of honey bee stocks with greater resistance to Varroa and associated pathogens.

Stock-specific chemical brood signals are induced by Varroa and Deformed Wing Virus, and elicit hygienic response in the honey bee

The health of the honey bee Apis mellifera is challenged by the ectoparasitic mite Varroa destructor, and the numerous harmful pathogens it vectors. Existing pesticide-based Varroa controls are not sustainable. In contrast, one promising approach for improved honey bee health is the breeding of hygienic bees, capable of detecting and removing brood that is parasitized or diseased. In three experiments we find evidence to support the hypothesis that stock-specific chemical brood signals are induced by Varroa and Deformed Wing Virus, and elicit hygienic response in the honey bee. By collecting, analyzing, and running bioassays involving mite-infested and control brood extracts from three honey bee breeding stocks we: 1) found evidence that a transferrable chemical signal for hygienic behavior is present in Varroa-infested brood extracts, 2) identified ten stock-specific hydrocarbons as candidates of hygienic signaling, and 3) found that two of these hydrocarbons linked to Varroa and DWV were also elevated in brood targeted for hygienic behavior. These findings expand our understanding of honey bee chemical communication, and facilitate the development of improved hygienic selection tools to breed honey bees with greater resistance to Varroa and associated pathogens.

Brood Affects Hygienic Behavior in the Honey Bee (Hymenoptera: Apidae).

Despite receiving much attention, the ectoparasitic mite Varroa destructor (Anderson and Trueman) and the pathogens it vectors remain critical threats to the health of the honey bee Apis mellifera (Linnaeus) (Hymenoptera: Apidae). One promising intervention approach is the breeding of hygienic honey bees, which have an improved ability to detect and remove unhealthy brood from the colony, and are thus more resistant to Varroa. While much hygienic behavior-related research has focused on enhanced adult honey bee olfaction, less attention has been paid to the olfactory signals that originate inside the brood cell, triggering hygienic removal. Here, we hypothesized that selection for hygienic behavior in honey bees has influenced brood signaling, predicting that: 1) in a common social environment, removal rates differ among brood with different selective breeding histories, and 2) the removal rates of brood positively correlate to the hygiene level of the brood's colony of origin. To test these predictions, we cross-fostered brood subjected to control, wound, or Varroa treatment in unselected (UNS), Minnesota Hygienic (HYG), and Varroa-Sensitive Hygienic (VSH) colonies, and monitored individual brood cells for hygienic removal. Results confirmed both predictions, as brood from hygienic colonies was more likely to be removed than brood from UNS colonies, regardless of where the brood was fostered. These findings suggest that hygiene-related brood signals complement previously identified characteristics of hygienic adults, constituting an important mechanism of social immunity in honey bees. Thus, selective breeding for honey bee hygienic behavior may be improved through the utilization of field assays containing compounds related to larval signaling.

Varroa-specific hygienic behavior of Apis mellifera scutellata in Kenya

Varroa-specific hygienic behavior is a hereditary trait of honey bee (Apis mellifera), which supports resistance to Varroa destructor. This study investigated the response of Apis mellifera scutellata to Varroa-infested worker brood cells in Kenya, East Africa. Uncapping, removal of the brood, and disappearance of the introduced mite were recorded in a total of 690 cells into which live mites were introduced. We recorded a high proportion of untouched cells in controls (median, 80%) compared to manipulated cells in which mites had been introduced (median, 12.5%) with a significant difference (GLMM, p < 0.001). Mites were removed and cells were recapped in about 26% of the artificially infested brood cells. When ten, eight, and five mites were singly introduced in closely neighboring brood cells, hygienic bees were more responsive in the high mite density regime of eight and ten mites, an indication of a possibility that chemicals play a role in identification of Varroa-infested brood cells.

Expression analysis of genes putatively associated with hygienic behavior in selected stocks of Apis mellifera L. from Argentina

Hygienic behavior is an economically beneficial, heritable trait, which has evolved to limit the impact of honeybee pathogens. Selecting and breeding colonies with high levels of hygienic behavior has become a feasible and environmentally friendly strategy to control brood diseases in honeybee colonies worldwide. The identification of genes involved in the expression of this character may not only unravel molecular and biochemical pathways underlying hygienic behavior, but also serve as a practical approach to select disease resistance biomarkers useful for honeybee breeding programs. In the present work, we evaluated, at genetic level, Apis mellifera stocks selected for hygienic behavior, widely used for commercial apiculture in Argentina. We analyzed the expression profiles of five genes previously identified as candidates associated with hygienic behavior both in QTL and global gene expression studies in honeybees, more precisely, involved in perception and processing of olfactory information. We validated the differential expression of these genes as potentially responsible for behavioral differences in our selected stocks. Our results indicate that four of them (octopamine receptor, smell-impaired, odorant-binding protein 3, and odorant-binding protein 4) were differentially expressed between hygienic and non-hygienic bees within our highly hygienic colonies. The present findings improve our understanding of the molecular mechanisms underlying the differentiation of middle-age worker bees in their genetic propensity to perform hygienic behavior. This progress towards the genetic characterization of highly hygienic colonies that are commercially used in Argentine apiculture lays the groundwork for future development of targets for marker-assisted selection of disease-resistant honeybee stocks.

Odorant cues linked to social immunity induce lateralized antenna stimulation in honey bees (Apis mellifera L.)

Hygienic behaviour (HB) is a social immunity trait in honey bees (Apis mellifera L.) whereby workers detect, uncap and remove unhealthy brood, improving disease resistance in the colony. This is clearly economically valuable; however, the molecular mechanism behind it is not well understood. The freeze-killed brood (FKB) assay is the conventional method of HB selection, so we compared odour profiles of FKB and live brood to find candidate HB-inducing odours. Surprisingly, we found that significantly more brood pheromone (β-ocimene) was released from FKB. β-ocimene abundance also positively correlated with HB, suggesting there could be a brood effect contributing to overall hygiene. Furthermore, we found that β-ocimene stimulated worker antennae in a dose-dependent manner, with the left antennae responding significantly stronger than right antennae in hygienic bees, but not in non-hygienic bees. Five other unidentifiable compounds were differentially emitted from FKB which could also be important for HB. We also compared odour profiles of Varroa-infested brood to healthy brood and found an overall interactive effect between developmental stage and infestation, but specific odours did not drive these differences. Overall, the data we present here is an important foundation on which to build our understanding the molecular mechanism behind this complex behaviour.

Monitoring of Nosema Infections Levels During Hygienic Honey Bee Breeding Programs in Turkey

Monitoring of Nosema Infections Levels During Hygienic Honey Bee Breeding Programs in Turkey