Sealed Brood Cells Research Articles

Commercial probiotic formulas Bactocell and Levucell promote spring brood production in Apis mellifera L. (Hymenoptera: Apidae) colonies.

Honey bees are essential pollinators for several economically important crops. In temperate countries, honey bee colonies face multiple threats during the overwintering period, such as food availability, diseases, and confinement. Beekeepers commonly use chemicals to improve colony health during winter, but these products can have a negative impact on bee health and pathogens can develop resistance to them. Thus, there is a need for further development of alternative treatments. The aim of this study was to evaluate the impact of one endogenic bacterium (Bombella apis) and 2 commercial probiotic formulas (Bactocell and Levucell) on colony survival, spring development, and Vairimorpha (formerly Nosema) spp. spore count. Probiotic treatments were given in 1: 1 sugar syrup in October 2017 and April 2018, once a week for 2 wk. One experimental group was given Fumagilin-B, the only product approved in Canada to prevent nosemosis, once in October. The administration of 2 commercial probiotics, Bactocell (Pediococcus acidilactici) and Levucell (Saccharomyces cerevisiae boulardii), led to a significant increase in the number of sealed brood cells in spring. None of the probiotic treatments impacted the honey bee gut load of Vairimorpha spp. spores. The results suggest that beneficial microorganisms can improve spring development and performance of honey bee colonies.

Heritability of Apis mellifera recapping behavior and suppressed mite reproduction as resistance traits towards Varroa destructor.

The selection of honeybee strains resistant to the ectoparasitic mite Varroa destructor is generally considered as one of the most sustainable ways of coping with this major bee parasite. Thus, breeding efforts increasingly focus on resistance parameters in addition to common beekeeping traits like honey yield and gentleness. In every breeding effort, the success strongly depends on the quantifiability and heritability of the traits accounted. To find the most suitable traits among the manifold variants to assess Varroa resistance, it is necessary to evaluate how easily a trait can be measured (i.e., testing effort) in relation to the underlying heritability (i.e., expected transfer to the following generation). Various possible selection traits are described as beneficial for colony survival in the presence of Varroa destructor and therefore are measured in breeding stocks around the globe. Two of them in particular, suppressed mite reproduction (SMR, sensu lato any reproductive failure of mother mites) and recapping of already sealed brood cells have recently gained increasing attention among the breeders because they closely resemble resistance mechanisms of some Varroa-surviving honeybee populations. However, it was still unknown whether the genetic background of the trait is sufficient for targeted selection. We therefore investigated the heritabilities and genetic correlations for SMR and REC, distinguishing between recapping of infested cells (RECinf) and all cells (RECall), on an extensive dataset of Buckfast and Carniolan stock in Germany. With an accessible h² of 0.18 and 0.44 for SMR and an accessible h² of 0.44 and 0.40 for RECinf, both traits turned out to be very promising for further selection in the Buckfast and Carnica breeding population, respectively.

An investigation of the efficacy of hygienic behavior of various honey bee (Apis mellifera) races toward Varroa destructor (Acari: Varroidae) mite infestation

Hygienic behavior in honey bees reflects the social immunity against parasites and diseases and is considered one of the main factors of genetic resistance of the bee breeding program. Hygienic behavior refers to the uncapping and removal of diseased and dead larvae and pupae from sealed brood cells by the worker bees. Here, the hygienic behavior of Apis mellifera races was investigated in response to pin-killed assay and artificial Varroa mite-infested brood cells. Moreover, the reproduction behavior of Varroa mites was determined in various sizes of honey bee race comb cells. The results revealed that the percentage of uncapping and removal of dead broods were significantly higher in the Italian as compared to the Carniolan bee colonies (p < 0.05). Similarly, a significant difference was present in response to artificially infested brood cells with Varroa mites in Italian and Carniolan colonies over the day of inspection (p < 0.05). Overall, the width of cells of the two types of combs was significantly different. The smaller width of cells size combs reduces the Varroa mite reproduction behavior compared to larger cell size combs. More infestation of Varroa mite occurred in drone brood cells as compared to worker brood cells in both old and new comb types. This study contributed to understand the hygienic behavior of A. mellifera races in preparation for selecting and developing hygienic lines of bees given the important contribution of hygienic behavior to honey bee health.

Significant transcriptional changes in mature daughter Varroa destructor mites during infestation of different developmental stages of honeybees.

Varroa destructor is considered a major cause of honeybee (Apis mellifera) colony losses worldwide. Although V. destructor mites exhibit preference behavior for certain honeybee lifecycle stages, the mechanism underlying host finding and preference remains largely unknown. By using a de novo transcriptome assembly strategy, we sequenced the mature daughter V. destructor mite transcriptome during infestation of different stages of honeybees (brood cells, newly emerged bees and adult bees). A total of 132 779 unigenes were obtained with an average length of 2745 bp and N50 of 5706 bp. About 63.1% of the transcriptome could be annotated based on sequence homology to the predatory mite Metaseiulus occidentalis proteins. Expression analysis revealed that mature daughter mites had distinct transcriptome profiles after infestation of different honeybee stages, and that the majority of the differentially expressed genes (DEGs) of mite infesting adult honeybees were down-regulated compared to that infesting the sealed brood cells. Gene ontology and KEGG pathway enrichment analyses showed that a large number of DEGs were involved in cellular process and metabolic process, suggesting that Varroa mites undergo metabolic adjustment to accommodate the cellular, molecular and/or immune response of the honeybees. Interestingly, in adult honeybees, some mite DEGs involved in neurotransmitter biosynthesis and transport were identified and their levels of expression were validated by quantitative polymerase chain reaction (qPCR). These results provide evidence for transcriptional reprogramming in mature daughter Varroa mites during infestation of honeybees, which may be relevant to understanding the mechanism underpinning adaptation and preference behavior of these mites for honeybees. © 2020 Society of Chemical Industry.

Both hygienic and non-hygienic honeybee, Apis mellifera, colonies remove dead and diseased larvae from open brood cells.

Hygienic behaviour is a group defence in which dead or diseased individuals are excluded. In the honeybee, Apis mellifera, hygienic behaviour refers to uncapping and removing dead and diseased larvae and pupae from sealed brood cells. We quantified removal of freeze-killed and chalkbrood-infected larvae from open cells in 20 colonies. We also measured removal of freeze-killed brood from sealed cells. Study colonies ranged from non-hygienic to fully hygienic (52-100% removal within 2 days). All larvae killed in open cells were removed. This shows that all colonies, including those with low hygienic behaviour against dead brood in sealed cells, are highly hygienic against dead brood in open cells and suggests that low hygienic behaviour against dead brood in sealed cells is a trait in its own right. This may also contribute to understanding why hygienic behaviour is uncommon in A. mellifera, which is puzzling as it reduces several diseases without detrimental effects. In particular, the result provides indirect support for the hypothesis that there are two adaptive peaks conferring disease resistance: (i) high hygienic behaviour: diseased brood are removed quickly, in some cases before becoming infective; (ii) low hygienic behaviour: diseased brood remain isolated within sealed cells.This article is part of the Theo Murphy meeting issue 'Evolution of pathogen and parasite avoidance behaviours'.

Towards integrated control of varroa: 5) monitoring honey bee brood rearing in winter, and the proportion of varroa in small patches of sealed brood cells

Oxalic acid is used by beekeepers to control varroa mites, Varroa destructor. Oxalic acid is effective against mites phoretic on adult bees but not against mites in sealed brood cells. In Britain and other temperate areas, brood rearing diminishes in winter and may even fall to zero, providing a natural window of opportunity for varroa control with oxalic acid. We monitored brood rearing from September to March for four years in hives in Sussex, southern Britain. We also sampled phoretic mites on adult bees and in brood cells to determine the proportion protected from oxalic acid in sealed brood cells. Our results showed that December is consistently the month with the least sealed brood, with 17 times less than November or January, on average. However, winter reduction in brood rearing varied among years, and even in December some colonies still had sealed brood (range 9–52%, n = 4 winters). Although the amounts were low, even a small patch of c. 500–600 sealed cells (300 cm2) contains approximately 14% of the mites in a colony, enough to halve the duration of control provided by an oxalic acid treatment. Our results indicate that beekeepers should check their hives in winter if they plan to control varroa with oxalic acid. This will show which month(s) have the least capped brood, whether there is period when all colonies are broodless, and if not which hives have patches of capped brood to remove.

Transcriptome profiling of the honeybee parasite Varroa destructor provides new biological insights into the mite adult life cycle

BackgroundThe parasite Varroa destructor represents a significant threat to honeybee colonies. Indeed, development of Varroa infestation within colonies, if left untreated, often leads to the death of the colony. Although its impact on bees has been extensively studied, less is known about its biology and the functional processes governing its adult life cycle and adaptation to its host. We therefore developed a full life cycle transcriptomic catalogue in adult Varroa females and included pairwise comparisons with males, artificially-reared and non-reproducing females (10 life cycle stages and conditions in total).ResultsExtensive remodeling of the Varroa transcriptome was observed, with an upregulation of energetic and chitin metabolic processes during the initial and final phases of the life cycle (e.g. phoretic and post-oviposition stages), whereas during reproductive stages in brood cells genes showing functions related to transcriptional regulation were overexpressed. Several neurotransmitter and neuropeptide receptors involved in behavioural regulation, as well as active compounds of salivary glands, were also expressed at a higher level outside the reproductive stages. No difference was detected between artificially-reared phoretic females and their counterparts in colonies, or between females who failed to reproduce and females who successfully reproduced, indicating that phoretic individuals can be reared outside host colonies without impacting their physiology and that mechanisms underlying reproductive failure occur before oogenesis.ConclusionsWe discuss how these new findings reveal the remarkable adaptation of Varroa to its host biology and notably to the switch from living on adults to reproducing in sealed brood cells. By spanning the entire adult life cycle, our work captures the dynamic changes in the parasite gene expression and serves as a unique resource for deciphering Varroa biology and identifying new targets for mite control.

Spermatozoa production in male Varroa destructor and its impact on reproduction in worker brood of Apis mellifera.

Reproduction in Varroa destructor exclusively takes place within the sealed honey bee brood cell and is, therefore, limited by the duration of the postcapping period. Oogenesis, ontogenetic development and mating must be optimized to ensure the production of as many mated daughter mites as possible. One adult male mite has to mate with up to five sister mites and transfer 30-40 spermatozoa to each female. We analyzed the production and transfer of male spermatozoa during a reproductive cycle by counting all spermatozoa in the genital tracts of the male and daughter mites in 80 worker brood cells at defined times after cell capping. We could show that spermatozoa production in male mites is an ongoing process throughout their adult lifetime starting after the adult molt. The spermatozoa are transferred to the females in an early non-capacitated stage and require further maturation within the female's genital tract. Our study points out that a Varroa male has at any time in the brood cell enough spermatozoa to inseminate all daughter mites but does not waste energy in producing a big surplus. In total one male produced, on average, 125 spermatozoa during a reproductive cycle in worker brood which is sufficient for successful matings with at least three daughter mites. Spermiogenesis in Varroa males represents therefore a further adaptation to the limited time available for reproduction.

Infestation of Varroa destructor on Apis mellifera in Kashmir Himalaya

A study was conducted on the infestation of varroa mite Varroa destructor on Apis mellifera L. honey bee colonies in Kashmir Himalaya. It was through a survey covering 200 colonies from 10 locations at different altitudes during 2013. Relative adult bee infestation was observed to be between 9.12 to 11.33%, while in sealed worker brood cells it was 20.15 to 29.12%. Differences in infestation levels was found significant between locations, and ranged between 9.12 to 11.33% at Tangmarg and Medura Tral locations, due to altitude differences. In sealed worker brood cells infestation levels ranged between 20.15 to 29.12% at Deed Kote Kupwara and Penglina Pulwama.

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.

Spermatozoa capacitation in female Varroa destructor and its influence on the timing and success of female reproduction

Mating of Varroa destructor takes place inside the sealed honey bee brood cell. During copulation, male mites transfer the spermatozoa into the genital openings of the females. Before the fertilization of female germ cells, the transferred spermatozoa have to pass through a final maturation process inside the genital tract of the female, the so-called capacitation. We here describe for the first time the morphological changes and chronological sequence of spermatozoa capacitation within female V. destructor. We have defined seven distinct stages of spermatozoa during the process of capacitation and have shown that it takes about 5days from mating to the occurrence of spermatozoa ready for fertilization. This might explain the results of an additional experiment where we could show that freshly mated daughter mites need a phoretic phase on bees before their first reproduction cycle. The transfer of non-capacitated spermatozoa from male V. destructor and the resulting long capacitation period within the female mites seems to be a consequence of an adaptive pressure for the male mites to inseminate several daughter mites within the short time span inside the sealed honey bee brood cell.

Varroa mites, viruses and bacteria incidences in Kenyan domesticated honeybee colonies

ABSTRACTVarroa mite (Varroa destructor Anderson and Trueman) is a major global threat to the western honey bee (Apis mellifera L.). The ectoparasite has been implicated in the spread of honeybee viruses. Beekeeping plays a major role in transmission of the mite. The study aimed at assessing levels of Varroa infestation, bee viruses and bacteria incidences in domesticated honeybee colonies. Samples of adult honey bees, bee brood and Varroa mites were collected from Baringo, Narok, Kwale, Magarini, Voi, Ijara, Busia and Siaya in Kenya. Ten hives in each site were inspected for the presence of Varroa mites on adult bees using the icing sugar technique and forceps in sealed brood cells. The number of mites observed were recorded per site. Ribonucleic acid was extracted from the mites, brood and adult bees and a polymerase chain reaction was performed to detect the black queen cell virus. Pooled RNA samples of brood and adult bees were used in next generation sequencing on a 454 GS FLX platform to detect bee v...

Influence of different proteinaceous diets on consumption, brood rearing,and honey bee quality parameters under isolation conditions

This study was carried out to evaluate the efficacy of 5 proteinaceous diets on honey bees under isolation conditions. The diets were soybean meal, mesquite pod powder, date paste, Feedbee®, and corn gluten, along with control colonies (bees fed naturally on pollens). These 5 diets were supplied to honey bee colonies under isolation conditions, except for the control group. Food consumption amount, brood rearing activity, and some physiological parameters of the honey bees were measured. The best consumption amount was for date paste, followed by Feedbee, mesquite, and corn gluten, respectively. Concerning brood rearing activity, the highest mean of sealed brood cells was 174.7 cells/colony in colonies fed on Feedbee, while no brood was reared in colonies fed on soybean meal. The fresh weight, dry matter, and protein content of fully grown larvae and newly emerged workers were higher in colonies fed on bee bread than the colonies fed on the artificial diets. Total soluble solids %, total hemolymph count, and hemolymph protein % were varied among tested diets. Feedbee was able to enhance brood rearing, but Feedbee is expensive and not available in the local markets of Saudi Arabia. Hence, beekeepers are advised to use mesquite pod flour as a pollen substitute in dearth periods.

Mating disruption of the honeybee mite Varroa destructor under laboratory and field conditions

Mating in the mite Varroa destructor, an ectoparasite of the honeybee, takes place within the sealed brood cell of its host and is triggered by a female sex pheromone consisting of three fatty acids and their respective ethyl esters. In a laboratory bioassay, we observed interactions among offspring of a Varroa female at 11 days after host cell capping in the absence and in the presence of the sex pheromone and clearly demonstrated that male mites were not able to distinguish between receptive daughters and either older or immature and unreceptive females when exposed to the pheromone. In addition, mating attempts in the presence of the pheromone with otherwise receptive females were clearly of shorter duration and males often failed to select a receptive female. In order to evaluate the effect of pheromone exposure on successful copulations and the number of spermatozoa transferred under field conditions, we sprayed one of the pheromone components, oleic acid, on an empty brood comb before host egg-laying and Varroa infestation and counted the spermatozoa of daughter mites taken from this comb. We could show that the number of spermatozoa was indeed reduced, and 20 % of females lacked spermatozoa. Our results open up new possibilities and represent a promising step toward biological control of Varroa mites in beehives.

Colony development of two Carniolan genotypes (Apis mellifera carnica) in relation to environment

SummaryThe objective of this study was to compare the colony development cycle (unsealed and sealed worker brood, drone brood, pollen and colony strength) of two Apis mellifera carnica subpopulations in two distinct environments (alpine and continental). At each test location were two sub groups of 12 colonies headed by naturally mated sister queens from either the Institute of Apiculture Lunz am See, Austria (AT) or from the Faculty of Agriculture University of Zagreb, Croatia (HR). Colony development was monitored every 14 days. The HR genotype, adapted to a continental climate, had faster spring brood development in both environments. During spring and early summer the AT genotype maintained the number of sealed brood cells at a constant level in the more favourable conditions, although the amount of unsealed brood reached its maximum in early June. The environment influenced colony development, food stores and colony strength. Interaction between genotype and environment did not affect the number of unsealed brood cells, but the difference was statistically significant for the number of sealed brood cells. The study indicated the presence of a number of genotype and environment interactions between the two honey bee genotypes and their colony traits.

Influence of some plant extracts on Varroa mite and performance of honey bee Apis mellifera colonies

Nowadays the ectoparasite mite, Varroa jacobsoni still a serious problem affecting apiculture among Lybian beekeepers. Recently, the alternative control based on natural products extracted from medicinal and ornamental plants as control agents for the Varroa mite considered more save to human being and bees. plant extract of Thyme , mixture of Fenugreek & Santonica, Fenugreek alone and Santonica alone were evaluated against Varroa mite at the apiaries located at Sebha, Lybia during (March – April), 2012. The data clearly show that, an increased the efficiency of any extracts used for Varroa control with the superiority of Thyme followed by mixture of Fenugreek & Santonica, Fenugreek alone and finally Santonica alone. This extracts caused an obvious reduction of the rate of infestation either in the sealed brood or on the adult bees, moreover increase in the number of sealed worker brood cells and, lastly, an increase in the number of combs covered from both sides with adult worker bees (colony strength) in the treated colony.

The mating behavior of Varroa destructor is triggered by a female sex pheromone. Part 2: Identification and dose-dependent effects of components of the Varroa sex pheromone

Reproduction of female Varroa destructor happens within the sealed brood cell of the honeybee host. The mating represents the last step of the reproductive cycle and is usually performed between the mature male offspring and one or more daughter mites. By offering solvent extracts of freshly molted females to male Varroa mites in our mating bioassay, we could clearly confirm the presence of a volatile female sex pheromone. After separation of the extract into a polar and non-polar fraction, only the polar fraction elicited the typical mating behavior of male mites. GC-MS analysis of the active fraction revealed a pattern of three fatty acids as the main components and the respective ethyl esters. We could prove that all these substances stimulated the male mating behavior, and we present results on the dose-dependent reactions of the males toward these compounds. The identification of a Varroa sex pheromone might enable new options for a biological control of the parasite.

Activation and interruption of the reproduction of Varroa destructor is triggered by host signals (Apis mellifera)

The reproductive cycle of the parasitic mite Varroa destructor is closely linked to the development of the honey bee host larvae. Using a within colony approach we introduced phoretic Varroa females into brood cells of different age in order to analyze the capacity of certain stages of the honey bee larva to either activate or interrupt the reproduction of Varroa females. Only larvae within 18h (worker) and 36h (drones), respectively, after cell capping were able to stimulate the mite’s oogenesis. Therewith we could specify for the first time the short time window where honey bee larvae provide the signals for the activation of the Varroa reproduction. Stage specific volatiles of the larval cuticle are at least part of these activation signals. This is confirmed by the successful stimulation of presumably non-reproducing mites to oviposition by the application of a larval extract into the sealed brood cells. According to preliminary quantitative GC–MS analysis we suggest certain fatty acid ethyl esters as candidate compounds.If Varroa females that have just started with egg formation are transferred to brood cells containing host larvae of an elder stage two-thirds of these mites stopped their oogenesis. This confirms the presence of an additional signal in the host larvae allowing the reproducing mites to adjust their own reproductive cycle to the ontogenetic development of the host. From an adaptive point of view that sort of a stop signal enables the female mite to save resources for a next reproductive cycle if the own egg development is not sufficiently synchronized with the development of the host.The results presented here offer the opportunity to analyze exactly those host stages that have the capacity to activate or interrupt the Varroa reproduction in order to identify the crucial host signals.

Morphometry and number of spermatozoa in drone honeybees (Hymenoptera: Apidae) reared under different conditions

The effects of three factors operating during pre-emergence development period on some characteristics of drones were studied. Weight of newly emerged drones, length of forewing, length of tibia, length of femur, length and width of basitarsus and number of spermatozoa in drones from colonies in which the workers had access to drone brood (A), the size of brood cells differed (B) or the colony had a queen or was queenless (C), were determined. For this purpose, 9 colonies were chosen at random from the test apiary and prepared so that each contained one empty Langstroth frame with six small sub-frames containing drone combs, three of which were used to test the effect of one level and the other three the other level of each factor. The results showed that, colony status had a greater effect on the parameters measured than either the size of the brood cells or whether the workers had access to sealed brood cells. Most of the parameters were significantly different in C, whereas only a few in B and non in A were significantly different. The distance between discriminant scores in each experiment enhanced the previous results, as it was highest in C, followed by B and then A. Optimal drone characteristics may be recorded for colonies in which the queens were induced to lay unfertilized eggs in newly built drone combs, and then removed and the drone brood reared in a queen less colony.

How can cleptoparasitic drosophilid flies emerge from the closed brood cells of the red Mason bee?

Aculeate Hymenoptera provision their progeny with large amounts of food. To protect their investment against brood parasites, females of many bee and wasp species construct brood cells that are hard to penetrate when finally sealed. However, the sealed brood cells also pose a problem for parasites that oviposit in the brood cell during provisioning. Brood parasites are smaller than their host and may lack strong mandibles to break through the solid brood cell walls. Furthermore, in nests built in existing cavities, newly-eclosed brood parasites need information about the location of the nest entrance. In the present study, the mechanisms of emergence are investigated in Cacoxenus indagator Loew (Diptera, Drosophilidae), the major cleptoparasite in nests of the red Mason bee Osmia bicornis L. (Hymenoptera, Megachilidae). Larvae of C. indagator move to brood cells closer to the nest entrance and sometimes make small emergence holes in the final closure of the nest entrance. Nevertheless, approximately one-third of newly-eclosed flies orientate and break through at least one intact cell partition to emerge. Flies make most of their attempts to emerge at the correct side (i.e. the one pointing to the nest entrance, probably by using the shape of the cell partition as a cue). Newly-eclosed flies use their head blister (ptilinum) to exert hydraulic pressure on particles of the cell partitions and produce small holes. Thus, C. indagator exhibits a set of behavioural and physiological adaptations enabling them to successfully emerge even from closed brood cells of their host.