Hygienic Bees Research Articles

Hygienic behaviour in Brazilian stingless bees.

ABSTRACTSocial insects have many defence mechanisms against pests and pathogens. One of these is hygienic behaviour, which has been studied in detail in the honey bee, Apis mellifera. Hygienic honey bee workers remove dead and diseased larvae and pupae from sealed brood cells, thereby reducing disease transfer within the colony. Stingless bees, Meliponini, also rear broods in sealed cells. We investigated hygienic behaviour in three species of Brazilian stingless bees (Melipona scutellaris, Scaptotrigona depilis, Tetragonisca angustula) in response to freeze-killed brood. All three species had high mean levels of freeze-killed brood removal after 48 h ∼99% in M. scutellaris, 80% in S. depilis and 62% in T. angustula (N=8 colonies per species; three trials per colony). These levels are greater than in unselected honey bee populations, ∼46%. In S. depilis there was also considerable intercolony variation, ranging from 27% to 100% removal after 2 days. Interestingly, in the S. depilis colony with the slowest removal of freeze-killed brood, 15% of the adult bees emerging from their cells had shrivelled wings indicating a disease or disorder, which is as yet unidentified. Although the gross symptoms resembled the effects of deformed wing virus in the honey bee, this virus was not detected in the samples. When brood comb from the diseased colony was introduced to the other S. depilis colonies, there was a significant negative correlation between freeze-killed brood removal and the emergence of deformed worker bees (P=0.001), and a positive correlation with the cleaning out of brood cells (P=0.0008). This shows that the more hygienic colonies were detecting and removing unhealthy brood prior to adult emergence. Our results indicate that hygienic behaviour may play an important role in colony health in stingless bees. The low levels of disease normally seen in stingless bees may be because they have effective mechanisms of disease management, not because they lack diseases.

Responses of Varroa-resistant honey bees (Apis mellifera L.) to Deformed wingvirus

The negative impact of Deformed wing virus (DWV) on European honey bees Apis mellifera is magnified by Varroa destructor parasitism. This study compared the responses of two Varroa-resistant honey bee stocks, pure Russian honey bees (RHB) and out-crossed Varroa Sensitive Hygienic bees, Pol-line (POL) to DWV infection to that of Varroa- susceptible stock, Italian honey bees (IHB). Two-day-old larvae were fed with DWV lysate in different concentrations: undiluted DWV lysate (D1), D1:100, and D1:1000. The unfed larvae served as negative control. Combs containing test larvae were exposed to a common environment during their development using host colonies. Our results showed that only POL displayed variation in DWV levels when fed different DWV concentrations. POL fed highest concentration of DWV inoculum had the highest increase in DWV level than those fed low concentrations and unfed POL. This high increase in DWV level probably contributed to the decrease in the survival and median longevity (LT50) of D1-fed POL. Weights of newly eclosed D1-fed POL were similar to those of the two controls and DWV-fed bees. However, within IHB, D1-fed bees showed significant reductions in weight, days of survival and LT50. Regardless of the concentrations of DWV inoculum, the DWV levels were similarly low within RHB; adult bees had similar weights. Overall, larvae fed D1 had the highest rate of wing deformation. POL and RHB had numerically lower proportions of bees with deformed wings. This study suggests that RHB showed some degree of resistant to DWV as shown by no reduction on weight and numerically lower proportion of wing deformity when compared with the other bee stocks.

Individual precocity, temporal persistence, and task-specialization of hygienic bees from selected colonies of Apis mellifera

Abstract Hygienic behaviour is a complex trait that gives Apis mellifera L. resistance against brood diseases. Variability in the expression of hygienic behaviour is evidenced at the colony-level and is explained by the proportion and propensity of individual worker bees that engage in hygienic activities. We investigated the temporal performance and the dynamics of task-specialisation of individual bees over time, both in selected hygienic (H) and non-hygienic (NH) colonies. Then we evaluated the impact of these behavioural aspects on the colony performance. Bees that perform hygienic behaviour (hygienic bees) in our H colonies were more persistent in the hygienic activities throughout the days of the investigation. Such bees were more efficient in the removal of pin-killed brood than hygienic bees in the NH colonies. Hygienic bees in the H colonies were also specialist in the sub-tasks involved in the detection of odour stimulus from dead brood and continued to perform these activities throughout the days of the investigation (temporal persistence). Age-distribution of hygienic bees in the H colonies was asymmetrical, with a larger proportion of these bees performing hygienic activities early in life. At a colony-level, H showed higher efficiency compared to the NH colonies. The present results highlight the fact that individual behaviour may influence the collective dynamics of the hygienic behaviour in honeybee colonies. The results also note that the selection for highly hygienic colonies would result in changes in individual bees that improve the performance of the behaviour at the colony level. The relevance of task-partitioning and age-specialisation of hygienic bees on social immunity is discussed.

Hygienic Behaviour of Honeybee Colonies with Different Levels of Polyandry and Genotypic Composition

Abstract Honey bee queens were inseminated with diluted, homogenised semen collected from a few dozen drones. This procedure was carried out to increase the diversity of the queens’ offspring, which is in comparison to the offspring of queens inseminated with semen from only a few drones coming from one colony. Queens and drones were mated within carniolan bee (Apis mellifera carnica) subspecies, but 3 selected lines were used. Queens were reared from one line and drones from the same line, and two additional lines differing in hygienic behaviour wherein in one of them that trait was strongly evident. The aim of this study was to examine whether the level of enhanced genetic variability in colonies and simultaneously the participation of hygienic bees, would increase the performance of hygienic behaviour. Overall hygienic behaviour of colonies with a lower and greater genetic variability did not differ significantly and amounted to 52.1 and 47.0%, respectively. Colonies within the lower variability group, in which drones from line selected in hygienic behaviour performance were used for inseminating queens, had a significantly greater percent of cleaned pupae than other colonies (63.2%). Hygienic behaviour in other colonies was more dependent on the gene quotas of hygienic bees in the colonies rather than on the level of polyandry.

Differential viral levels and immune gene expression in three stocks of Apis mellifera induced by different numbers of Varroa destructor

The viral levels and immune responses of Italian honey bees (IHB), Russian honey bees (RHB) and an outcross of Varroa Sensitive Hygienic bees (POL) deliberately infested with one or two foundress Varroa were compared. We found that the Deformed wing virus (DWV) level in IHB inoculated with one or two foundress Varroa increased to about 103 or 105 fold the levels of their uninfested brood. In contrast, POL (102 or 104 fold) and RHB (102 or l04 fold) supported a lower increase in DWV levels. The feeding of different stages of Varroa nymphs did not increase DWV levels of their pupal hosts. Analyses of their corresponding Varroa mites showed the same trends: two foundress Varroa yielded higher DWV levels than one foundress, and the addition of nymphs did not increase viral levels. Using the same pupae examined for the presence of viruses, 16 out of 24 genes evaluated showed significant differential mRNA expression levels among the three honey bee stocks. However, only four genes (Defensin, Dscam, PPOact and spaetzle), which were expressed at similar levels in uninfested pupae, were altered by the number of feeding foundress Varroa and levels of DWV regardless of stocks. This research provides the first evidence that immune response profiles of different honey bee stocks are induced by Varroa parasitism.

First evidence of hygienic behaviour in the dwarf honey bee Apis florea

The hygienic behaviour of eight A. florea honey bee colonies was investigated in Thailand. Sealed brood was deep frozen or killed using entomological pins. The pins were 0.2 or 0.7 mm in diameter. The removal of killed brood by the bees was checked in intervals ranged from 24 to 60 h after it had been presented to the colonies. Almost eighty nine (89%) of the freeze-killed brood was removed by the bees within 24 h after being introduced into the combs, while 95% of the pin-killed brood was removed within 48 h after introduction. Thus, the A. florae colonies could be recognized as hygienic honey bees.

HYGIENIC BEES CLEANSE DAMAGED BROOD

It's a beekeeper's worse nightmare: to find their bee colonies decimated by Varroa mites. Hopping into brood cells just before the pupae are sealed in during metamorphosis, the mites reproduce and feed on the developing bees before hitching a lift on their emerging cellmates when development is complete. Initially a natural parasite of Asian honeybees, Varroa destructor began wreaking havoc among European bees during the last century. And the situation escalated when the parasites picked up naturally benign viruses, which had previously lived in harmony with their European hosts, transmitting them back in a sometimes-lethal form to the vulnerable bees. Fortunately though, all was not lost. Elke Genersch from the Institute for Bee Research, Germany, explains that nurse bees naturally weed out and dispose of damaged or deformed pupae while developing in the brood comb to protect the colony from infection.Yet, attentive nurse bees face a dilemma when presented with mite-infested brood. According to Genersch, honeybee viruses – such as deformed wing virus – mutate continually, so some mite populations carry virulent and crippling forms of the disease whereas the viruses transmitted by other mites are practically harmless. However, it was not clear whether the Varroa mites carrying harmless virus or no virus might also damage the developing pupae sufficiently to trigger removal. So, would bee nurses dispose of all developing pupae that are infected with Varroa mites, or dispose only of those pupae where the mites are carrying damaging forms of deformed wing virus (p. 264)?Teaming up with Sebastian Gisder, Genersch screened honeybee colonies infested with Varroa mites to identify colonies living with mites carrying virulent deformed wing virus and other colonies infested with mites carrying less-virulent – or even no – virus. Having isolated two mite-source colonies, the team then looked for colonies that were completely free of Varroa infestation – to provide clean brood – and identified hygienic colonies with good housekeepers, which had been selectively bred by Kaspar Bienefeld as part of a Varroa control strategy. Then Caspar Schöning and Ivonne Kretschmann tested how the good housekeepers would respond to virulent and non-virulent Varroa infestations to find out whether the bees respond to mite-induced damage or deformed wing disease.Taking clean brood combs from the uninfested colonies, the duo added virulent and less-virulent mites from the mite-source colonies to the comb's clean cells. Then, the team transferred the newly infested combs to the hygienic colonies and waited to see how the good housekeepers dealt with the contaminated combs. Would they dispose of all Varroa-infected pupae, regardless of deformed wing virus virulence, or would the nurses select out the pupae at risk from the most virulent virus, leaving the pupae infested with the less-virulent mites to develop?Amazingly, the bees homed in on the pupae that were infested with mites carrying the most virulent deformed wing virus, leaving the pupae that had been infested with less-virulent mites to develop to maturity. And when Sven Geiselhardt and Monika Hilker analysed the odours produced by pupae infested with virulent mites and pupae infested with the less-virulent mites, they found distinct differences between the odours emanating from both groups.‘The bees reacted towards the damage done to the brood by the virus rather than to the Varroa mite parasitisation per se’, says Genersch. She adds, ‘There is a certain combination of deformed wing virus and Varroa mite that is fatal for the bees and beekeepers need to treat against mites or breed for hygienic bees to get rid of this virulent combination.’

Social immunity in honeybees (Apis mellifera): transcriptome analysis of varroa-hygienic behaviour

Honeybees have evolved a social immunity consisting of the cooperation of individuals to decrease disease in the hive. We identified a set of genes involved in this social immunity by analysing the brain transcriptome of highly varroa-hygienic bees, who efficiently detect and remove brood infected with the Varroa destructor mite. The function of these candidate genes does not seem to support a higher olfactory sensitivity in hygienic bees, as previously hypothesized. However, comparing their genomic profile with those from other behaviours suggests a link with brood care and the highly varroa-hygienic Africanized honeybees. These results represent a first step toward the identification of genes involved in social immunity and thus provide first insights into the evolution of social immunity.

A scientific note about spectroscopic analysis of honey bee brood comb cappings in hygienic and non-hygienic honey bee colonies

Hygienic behavior (HB) involves the detectionof diseased, dead, and infected larvae or pupae andtheir selective removal from brood cells by workerbees (Arathi et al., 2000;ArathiandSpivak,2001).The efficiency of HB is affected by the ability ofbees to recognize infected or infested brood (bychemical and physical cues). Among the variousfactors that have been studied that could influencethe detection of cells with dead or diseased brood(Arathi et al., 2000), visual cues had not been inves-tigated. We examined the light transmittance char-acteristics of the cappings of brood combs to deter-mine if it would be possible for bees to detect ab-normal brood within capped cells using visual cues.Cell cappings from old and new brood combs(50 each) from each of three colonies of hygienicand non-hygienic bee lines were removed andtested. These previously selected hygienic colonieshad a mean of 99.8% HB, while the non-hygieniccolonies had a mean of 2.3% HB.The cappings were fixed in a support device(mask) that permitted light passage only throughthe cappings. Initially, the intensity of the light thatpassed through the mask was measured without acapping. This corresponds to the incident light in-tensity (

Hygienic behaviors of honey bees in response to brood experimentally pin-killed or infected withAscosphaera apis

Hygienic behavior of honeybees involves inspection, uncapping and removal of diseased and dead brood from the colony. The objective of this work was to study the activities involved in hygienic behavior of individually tagged bees from selected hygienic (H) and non-hygienic (NH) colonies in the presence of chalkbrood infected brood (Ascosphaera apis) or pin-killed brood. No significant difference was detected in the age of bees inspecting, uncapping or removing brood in H and NH colonies; the median age was 15 days for all activities. The percentage of bees that performed these activities was significantly higher in H colonies. In NH colonies the bees that performed this behavior were more persistent but bees in H colonies were more efficient in the removal of the chalkbrood mummies. H colonies began uncapping more rapidly in response to the stimulus of dead brood independent of the method used to kill it. H and NH bees took the same amount of time to remove the mummies once they initiated the uncapping process but NH colonies took longer to remove pin-killed brood. These findings confirm previous behavioral studies on the activities of hygienic and non-hygienic bees toward freeze-killed brood, but this is the first time the entire process from inspection to removal was focused on individual cells containing actual diseased brood.

Effect of Brood Type on Varroa-Sensitive Hygiene by Worker Honey Bees (Hymenoptera: Apidae)

AbstractHoney bees (Apis mellifera L.) (Hymenoptera: Apidae) have been selectively bred for varroa-sensitive hygiene (VSH), which is the removal of pupae that are infested by Varroa destructor Anderson & Trueman from capped brood cells. This hygienic behavior is a complex interaction of bees and brood in which brood cells are inspected, and then brood is either removed or recapped. Previous work has shown that VSH bees uncap and remove significantly more varroa-infested worker pupae than nonhygienic bees do, but nothing is known about the reactions of VSH bees to mite-infested drone brood. This study compared the reactions of VSH bees with mite-infested worker and drone brood in a laboratory test and a field test. VSH bees inspected brood cells containing mite-infested pupae of both types of brood, but they removed significantly fewer mite-infested drone pupae than mite-infested worker pupae after 1 wk. This result suggests that mite populations in VSH colonies could increase more rapidly when drone brood is available. Additionally, the percentages of uncapped pupae and uncapped mite-infested pupae were positively correlated to the natural infestation rate of brood after a 24-h exposure, but not after an exposure of 1 wk. This result suggests that the rate of uncapping brood by hygienic bees may depend on the infestation rate, which gradually decreases with longer exposures to bees that remove mite-infested pupae from capped brood.

Inefficient task partitioning among nonhygienic honeybees, Apis mellifera L., and implications for disease transmission

Hygienic behaviour in honeybee colonies involves the recognition and removal of diseased brood by worker bees. This task is further partitioned into subtasks of uncapping cells with diseased brood and removing the cell contents. Worker bees that express hygienic behaviour remove diseased brood from the colony before the pathogen reaches the infectious stage. Although all honeybees are capable of uncapping and removing diseased brood, only those colonies with hygienic bees, the genetic specialists, do so rapidly and hence efficiently limit disease transmission. Colonies with nonhygienic bees, however, uncap and remove diseased brood only after it becomes infectious, resulting in the handling and transmission of pathogens. To understand the behavioural repertoire of individual nonhygienic bees and how the performance of the behaviour is influenced by the presence of hygienic bees as nestmates, we observed nonhygienic bees that were maintained in colonies with varying proportions of hygienic and nonhygienic bees. Our results indicated that nonhygienic bees in mixed colonies were not stimulated to perform hygienic behaviour to a higher extent in the presence of hygienic bees. The rate, duration and probability of uncapping and removing dead brood by nonhygienic bees was significantly reduced in the presence of hygienic bees. In mixed colonies, as compared to a colony of hygienic bees, a higher proportion of uncapped cells were subsequently recapped, resulting in delayed removal of dead brood. This inefficient task partitioning would allow the pathogen to reach the infectious stage and increase the probability of disease transmission.

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.

Differences in olfactory sensitivity and behavioral responses among honey bees bred for hygienic behavior

Honey bees, Apis mellifera L., bred for hygienic behavior uncap and remove diseased and mite-infested brood. We hypothesized that within a colony bred for hygienic behavior, there would be differences in olfactory sensitivity among bees of the same age. We predicted that bees that initiate the behavior by perforating and uncapping brood would have greater olfactory sensitivity to the odor of the diseased brood, and would be better able to discriminate between odors of healthy and diseased brood, compared to bees that complete the behavior by removing the uncapped brood from the cells. Electroantennogram recordings of 15- to 21-day-old bees from three colonies demonstrated that bees collected while uncapping dead brood had significantly greater olfactory sensitivity to all concentrations of diseased brood odor compared to bees collected while removing brood. Proboscis-extension reflex discrimination conditioning demonstrated that 15- to 21-day-old bees collected while uncapping discriminated significantly better and generalized significantly less between the odors of diseased and healthy brood compared to bees collected while removing, when the odor of diseased brood was rewarded, but not when the odor of healthy brood was rewarded. Bees collected while uncapping brood that had been pierced with a pin had significantly less olfactory sensitivity than bees collected while uncapping freeze-killed brood, most likely because the pierced brood had greater stimulus intensity. Initiation of hygienic behavior depends on the olfactory sensitivity of the bee and stimulus intensity of the abnormal brood. Differential olfactory sensitivity and responsiveness among hygienic bees could lead to the apparent partitioning of the behavior into uncapping and removing components.

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.

Olfactory and behavioral response thresholds to odors of diseased blood differ between hygienic and non-hygienic honey bees (Apis mellifera L.).

Through the use of proboscis-extension reflex conditioning, we demonstrate that honey bees (Apis mellifera L.) bred for hygienic behavior (a behavioral mechanism of disease resistance) are able to discriminate between odors of healthy and diseased brood at a lower stimulus level than bees from a non-hygienic line. Electroantennogram recordings confirmed that hygienic bees exhibit increased olfactory sensitivity to low concentrations of the odor of chalkbrood infected pupae (a fungal disease caused by Ascosphaera apis). Three-week-old hygienic bees were able to discriminate between the brood odors significantly better than three-week old non-hygienic bees. However, the differential performance in brood odor discrimination was primarily genetically based, not a direct result of age, experience, or the temporary behavioral state of the bee. Lower stimulus thresholds for both the olfactory and behavioral responses of hygienic bees may facilitate their ability to detect, uncap and remove diseased brood rapidly from the nest. In contrast, non-hygienic bees, possessing higher response thresholds, may not be able to detect diseased brood as easily. Our results provide an example of how physiological and behavioral differences between the hygienic and non-hygienic honey bee lines, operating at the level of the individual, could produce colony-specific behavioral phenotypes.

Influence of colony genotypic composition on the performance of hygienic behaviour in the honeybee, Apis mellifera L.

Hygienic behaviour, an intranidal task performed by middle-aged worker bees is an important behavioural mechanism of resistance to disease and to attack by Varroa destructor, an ectoparasitic mite. We studied the effect of a colony's genotypic composition on the expression of this behaviour among worker bees by creating normal age-structured colonies with different proportions of bees belonging to hygienic and nonhygienic lines. We established four colonies with 0, 25, 50 or 100% of worker bees belonging to the hygienic line. Analyses of the behaviour of hygienic bees in these colonies indicated that the performance of hygienic behaviour depended on the proportion of hygienic bees in the colony. Hygienic bees in the 25% hygienic colony performed the behaviour well beyond middle age and were more persistent at the task compared with bees from the same genetic line in the other colonies. However, the colony with all worker bees from the hygienic line was more efficient in achieving the task despite a lack of persistence. We also observed that in the colony with 50 and 100% hygienic bees, the behaviour was partitioned into subtasks, and some bees performed the subtask of uncapping cells at higher frequencies than the subtask of removing cell contents. These results suggest that a colony's genotypic composition influences the performance and partitioning of hygienic behaviour. We propose that the performance of hygienic behaviour and its partitioning into subtasks could be determined by response thresholds of individual worker bees and that the rate of behavioural ontogeny may be controlled by the demand for specific tasks.

Field Assays for Hygienic Behavior in Honey Bees (Hymenoptera: Apidae)

Honey bee, Apis mellifera L., hygienic behavior is a mechanism of disease resistance and a mode of defense against the parasitic mite Varroa jacobsoni Oudemans. Hygienic bees uncap and remove diseased and parasitized brood from the nest. The propagation of colonies that demonstrate resistance to chalkbrood and American foulbrood and that remove pupae infested by Varroa mites is becoming increasingly important in apiculture. This study evaluates 2 commonly used field assays used to screen colonies for hygienic behavior: the freeze-killed brood and the pierced brood assays. Both involve determining the time required for worker bees to remove dead capped brood from a section of comb. Colonies in the experiment displayed a wide range of removal rates and were grouped as hygienic, nonhygienic, or intermediate. The results of experiments 1 and 2 indicated that neither the age nor the source of the frozen brood had a significant effect on the removal rate by hygienic colonies (i.e., those colonies that consistently uncapped and removed freeze-killed brood within 48 h). In experiment 3, only a weak correlation was found between the removal of young freeze-killed and pierced pupae, but a significant correlation existed between the removal of pre-eclosion freeze-killed and pierced pupae. Experiment 4 examined cues that elicit removal behavior by hygienic and non hygienic colonies. When pupae were pierced with an insect pin through the base of the cell (without piercing the wax cell capping), there was no difference in the number of pupae removed by the hygienic and nonhygienic colonies. On average, 30% of all pierced pupae survived the treatment, which considerably diminished the accuracy and reproducibility of the test. When pupae were treated with hemolymph extracted from either a live or freeze-killed pupa, there was also no difference in the rate of removal by hygienic and nonhygienic colonies. These results indicate that bees from nonhygienic lines can be induced to express hygienic behavior only if a sufficiently strong stimulus is present. Both hygienic and nonhygienic colonies removed significantly more pupae treated with hemolymph from a dead pupa than hemolymph from a live pupa, indicating that the cue that stimulates removal behavior is stronger in dead pupae. It is concluded that the freeze-killed brood assay is the most conservative and reliable screening procedure for hygienic behavior. The following procedures are recommended: Randomly selected comb sections (5 by 6 cm each) of capped brood should be cut from 1 healthy colony, frozen, and introduced into the test colonies. The assay should be repeated at least twice. Only colonies that remove >95% of freeze-killed brood within 48 h in both tests should be considered hygienic. When developing hygienic breeder stock, the hygienic colonies should be challenged with the American foulbrood or chalkbrood pathogen to ensure resistance.

Performance of hygienic honey bee colonies in a commercial apiary

Colonies with naturally mated queens from a hygienic line of Italian honey bees (Apis mellifera ligustica) were compared to colonies from a commercial line of Italian bees not selected for hygienic behavior. The following characteristics were compared: rate of removal of freeze-killed brood; amount of chalkbrood; incidence of American foulbrood; honey produc- tion; and the number of mites, Varroa jacobsoni, on adult bees. The hygienic colonies removed significantly more freeze-killed brood than the commercial colonies, had significantly less chalk- brood, had no American foulbrood, and produced significantly more honey than the commercial colonies. Estimates of the number of Varroa mites on adult bees indicated that the hygienic colonies had fewer mites than the commercial colonies in three of four apiaries. In previous stu- dies on the relation between hygienic behavior and resistance to diseases and mites, the test colonies contained instrumentally inseminated queens. This is the first study to evaluate hygienic stock in large field colonies with naturally mated queens. © Inra/DIB/AGIB/Elsevier, Paris