More Worker Capped Brood and Honey Bees with Less Varroa Load Are Simple Precursors of Colony Productivity at Beekeepers' Disposal: An Extensive Longitudinal Survey.

In response to the concerns of beekeepers on the decline of honey bee populations on lavender honey flow in the lavender fields of southeast France and the consequent decrease of honey production, our long-term survey (2009-2021) monitored the total weight gain collected by these colonies. This study shows the variations in the total weight gain according to regions, years, populations structure (bee number and quantity of capped brood) and Varroa load. Among these factors, years and regions support one third of the variations over this 13-year survey. At the beginning of the honey flow, capped brood is more important than the number of bees, whereas Varroa load severely limits the performance of the colonies. A threshold of 3 mites/100 bees seems to reflect the upper limit of the Varroa load below which the total weight gain is not affected. This survey provides useful information for the beekeepers to better prepare the colonies for this honey flow and allows them to compare their results obtained with our general description of the total weight gains by year.

To Treat or Not to Treat Bees? Handy VarLoad: A Predictive Model for Varroa destructor Load.

The parasitic Varroa destructor is considered a major pathogenic threat to honey bees and to beekeeping. Without regular treatment against this mite, honey bee colonies can collapse within a 2-3-year period in temperate climates. Beyond this dramatic scenario, Varroa induces reductions in colony performance, which can have significant economic impacts for beekeepers. Unfortunately, until now, it has not been possible to predict the summer Varroa population size from its initial load in early spring. Here, we present models that use the Varroa load observed in the spring to predict the Varroa load one or three months later by using easily and quickly measurable data: phoretic Varroa load and capped brood cell numbers. Built on 1030 commercial colonies located in three regions in the south of France and sampled over a three-year period, these predictive models are tools designed to help professional beekeepers' decision making regarding treatments against Varroa. Using these models, beekeepers will either be able to evaluate the risks and benefits of treating against Varroa or to anticipate the reduction in colony performance due to the mite during the beekeeping season.

ColEval: Honeybee COLony Structure EVALuation for Field Surveys.

Methods for the evaluation and comparison of the structure of numerous honeybee colonies are needed for the development of applied and fundamental field research, as well as to evaluate how the structure and activity of honeybee colonies evolve over time. ColEval complements existing methods, as it uses an online reference image bank for (human) learning and training purposes. ColEval is based on the evaluation of the surface area percentage occupied by different components of a honeybee colony: adult worker bees, open and capped brood, honey, nectar, and pollen. This method is an essential tool for the description of the evolution in the size of honeybee colonies. The procedure makes allowances for tendencies between different observers and uses them to calculate accurate measurements of honeybee colony evaluation. ColEval thus allows for a posteriori comparison of under- or over-evaluation made by different observers working on the same project; it is thus possible to eliminate observer bias in the measurements and to conduct large surveys.

Measuring biological age to assess colony demographics in honeybees.

Honeybee colonies are increasingly exposed to environmental stress factors, which can lead to their decline or failure. However, there are major gaps in stressor risk assessment due to the difficulty of assessing the honeybee colony state and detecting abnormal events. Since stress factors usually induce a demographic disturbance in the colony (e.g. loss of foragers, early transition from nurse to forager state), we suggest that disturbances could be revealed indirectly by measuring the age- and task-related physiological state of bees, which can be referred to as biological age (an indicator of the changes in physiological state that occur throughout an individual lifespan). We therefore estimated the biological age of bees from the relationship between age and biomarkers of task specialization (vitellogenin and the adipokinetic hormone receptor). This relationship was determined from a calibrated sample set of known-age bees and mathematically modelled for biological age prediction. Then, we determined throughout the foraging season the evolution of the biological age of bees from colonies with low (conventional apiary) or high Varroa destructor infestation rates (organic apiary). We found that the biological age of bees from the conventional apiary progressively decreased from the spring (17 days) to the fall (6 days). However, in colonies from the organic apiary, the population aged from spring (13 days) to summer (18.5 days) and then rejuvenated in the fall (13 days) after Varroa treatment. Biological age was positively correlated with the amount of brood (open and closed cells) in the apiary with low Varroa pressure, and negatively correlated with Varroa infestation level in the apiary with high Varroa pressure. Altogether, these results show that the estimation of biological age is a useful and effective method for assessing colony demographic state and likely detrimental effects of stress factors.

Combined neonicotinoid pesticide and parasite stress alter honeybee queens' physiology and survival.

Honeybee colony survival strongly relies on the queen to overcome worker losses exposed to combined stressors like pesticides and parasites. Queen's capacity to withstand these stressors is however very little known. The effects of the common neonicotinoid pesticide imidacloprid in a chronic and sublethal exposure together with the wide distributed parasite Nosema ceranae have therefore been investigated on queen's physiology and survivorship in laboratory and field conditions. Early physiological changes were observed on queens, particularly the increase of enzyme activities (catalase [CAT] and glutathione-S-transferase [GST] in the heads) related to protective responses to xenobiotics and oxidative stress against pesticide and parasite alone or combined. Stressors also alter the activity of two other enzymes (carboxylesterase alpha [CaE α] and carboxylesterase para [CaE p] in the midguts) involved in metabolic and detoxification functions. Furthermore, single and combined effects of pesticide and parasite decrease survivorship of queens introduced into mating hives for three months. Because colony demographic regulation relies on queen's fertility, the compromise of its physiology and life can seriously menace colony survival under pressure of combined stressors.

Neuronal plasticity in the mushroom body calyx during adult maturation in the honeybee and possible pheromonal influences.

Honeybee workers express a pronounced age-dependent polyethism switching from various indoor duties to foraging outside the hive. This transition is accompanied by tremendous changes in the sensory environment that sensory systems and higher brain centers have to cope with. Foraging and age have earlier been shown to be associated with volume changes in the mushroom bodies (MBs). Using age- and task-controlled bees this study provides a detailed framework of neuronal maturation processes in the MB calyx during the course of natural behavioral maturation. We show that the MB calyx volume already increases during the first week of adult life. This process is mainly driven by broadening of the Kenyon cell dendritic branching pattern and then followed by pruning of projection neuron axonal boutons during the actual transition from indoor to outdoor duties. To further investigate the flexible regulation of division of labor and its neuronal correlates in a honeybee colony, we studied the modulation of the nurse-forager transition via a chemical communication system, the primer pheromone ethyl oleate (EO). EO is found at high concentrations on foragers in contrast to nurse bees and was shown to delay the onset of foraging. In this study, EO effects on colony behavior were not as robust as expected, and we found no direct correlation between EO treatment and synaptic maturation in the MB calyx. In general, we assume that the primer pheromone EO rather acts in concert with other factors influencing the onset of foraging with its effect being highly adaptive.

Flight behavior and pheromone changes associated to Nosema ceranae infection of honey bee workers (Apis mellifera) in field conditions.

Parasites are known to cause the loss of individuals in social insects. In honey bee colonies the disappearance of foragers is a common factor of the wide extended colony losses. The emergent parasite of the European honey bee Nosema ceranae has been found to reduce homing and orientation skills and alter metabolism of forager bees. N. ceranae-infected bees also show changes in Ethyl Oleate (EO) levels, which is so far the only primer pheromone identified in workers that is involved in foraging behavior. Thus, we hypothesized that N. ceranae (i) modifies flight activity of honey bees and (ii) induces EO changes that can alter foraging behavior of nestmates. We compared flight activity of infected bees and non-infected bees in small colonies using an electronic optic bee counter during 28 days. We measured EO levels by gas chromatography-mass spectrometry and spore-counts. Bee mortality was estimated at the end of the experiment. Infected bees showed precocious and a higher flight activity than healthy bees, which agreed with the more elevated EO titers of infected bees and reduced lifespan. Our results suggest that the higher EO levels of infected bees might delay the behavioral maturation of same age healthy bees, which might explain their lower level of activity. We propose that delayed behavioral maturation of healthy bees might be a protective response to infection, as healthy bees would be performing less risky tasks inside the hive, thus extending their lifespan. We also discuss the potential of increased flight activity of infected bees to reduce pathogen transmission inside the hive. Further research is needed to understand the consequences of host behavioral changes on pathogen transmission. This knowledge may contribute to enhance natural colony defense behaviors through beekeeping practices to reduce probability of colony losses.

Seasonal variation in the titers and biosynthesis of the primer pheromone ethyl oleate in honey bees.

Honey bees allocate tasks along reproductive and non-reproductive lines: the queen mates and lays eggs, whereas the workers nurse the brood and forage for food. Among workers, tasks are distributed according to age: young workers nurse and old workers fly out and forage. This task distribution in the colony is further regulated by an increase in juvenile hormone III as workers age and by pheromones. One such compound is ethyl oleate (EO), a primer pheromone that delays the onset of foraging in young workers. EO is produced by foragers when they are exposed to ethanol (from fermented nectar) while gathering food. EO is perceived by younger bees via olfaction. We describe here the seasonal variation of EO production and the effects of Methoprene, a juvenile hormone analog. We found that honey bee workers biosynthesize more EO during the growing season than during the fall and winter months, reaching peak levels at late spring or summer. When caged workers were fed with syrup+d(6)-ethanol, labeled EO accumulated in the honey crop and large amounts exuded to the exoskeleton. Exuded levels were high for several hours after exposure to ethanol. Treatment with Methoprene increased the production of EO in worker bees, by speeding up its movement from biosynthetic sites to the exoskeleton, where EO evaporates. Crop fluid from bees collected monthly during the growing season showed a modest seasonal variation of in vitro EO biosynthetic activity that correlated with the dry and sunny periods during which bees could forage.

Biosynthesis of ethyl oleate, a primer pheromone, in the honey bee (Apis mellifera L.).

Honey bees undergo a physiological transition from nursing to foraging approximately 21 days after adult emergence. This transition is delayed by ethyl oleate (EO), a primer pheromone produced by foragers when exposed to ethanol from fermented nectar. We demonstrate here that two secreted α/ß-hydrolases (BeeBase ID: GB11403 and GB13365) are responsible for the reversible esterification of ethanol with oleic acid, giving EO. Expression of hydrolase GB11403 was shown to be significantly up-regulated in foragers, relative to nurses. Tissue perfusion experiments with labeled substrates consistently localized the highest level of EO production in the head, whereas in situ imaging revealed expression of relevant EO biosynthetic genes and enzymatic activity along the esophagus, the site of ethanol exposure during nectar intake. Both α/ß-hydrolases were expressed in Pichia pastoris, purified and were shown produce EO in vitro. Experiments with live bees fed ethanol demonstrated that EO formed in regurgitate accumulates in the honey crop and exudes to the exoskeleton, from where it exerts its primer effect on younger bees.

Sensory reception of the primer pheromone ethyl oleate.

Social work force distribution in honeybee colonies critically depends on subtle adjustments of an age-related polyethism. Pheromones play a crucial role in adjusting physiological and behavioral maturation of nurse bees to foragers. In addition to primer effects of brood pheromone and queen mandibular pheromone--both were shown to influence onset of foraging--direct worker-worker interactions influence adult behavioral maturation. These interactions were narrowed down to the primer pheromone ethyl oleate, which is present at high concentrations in foragers, almost absent in young bees and was shown to delay the onset of foraging. Based on chemical analyses, physiological recordings from the antenna (electroantennograms) and the antennal lobe (calcium imaging), and behavioral assays (associative conditioning of the proboscis extension response), we present evidence that ethyl oleate is most abundant on the cuticle, received by olfactory receptors on the antenna, processed in glomeruli of the antennal lobe, and learned in olfactory centers of the brain. The results are highly suggestive that the primer pheromone ethyl oleate is transmitted and perceived between individuals via olfaction at close range.

E-ß-ocimene, a volatile brood pheromone involved in social regulation in the honey bee colony (Apis mellifera).

BACKGROUND: In honey bee colony, the brood is able to manipulate and chemically control the workers in order to sustain their own development. A brood ester pheromone produced primarily by old larvae (4 and 5 days old larvae) was first identified as acting as a contact pheromone with specific effects on nurses in the colony. More recently a new volatile brood pheromone has been identified: E-ß-ocimene, which partially inhibits ovary development in workers. METHODOLOGY AND PRINCIPAL FINDING: Our analysis of E-ß-ocimene production revealed that young brood (newly hatched to 3 days old) produce the highest quantity of E-ß-ocimene relative to their body weight. By testing the potential action of this molecule as a non-specific larval signal, due to its high volatility in the colony, we demonstrated that in the presence of E-ß-ocimene nest workers start to forage earlier in life, as seen in the presence of real brood. CONCLUSIONS/SIGNIFICANCE: In this way, young larvae are able to assign precedence to the task of foraging by workers in order to increase food stores for their own development. Thus, in the complexity of honey bee chemical communication, E-ß-ocimene, a pheromone of young larvae, provides the brood with the means to express their nutritional needs to the workers.

New insights into honey bee (Apis mellifera) pheromone communication. Is the queen mandibular pheromone alone in colony regulation?

BACKGROUND: In social insects, the queen is essential to the functioning and homeostasis of the colony. This influence has been demonstrated to be mediated through pheromone communication. However, the only social insect for which any queen pheromone has been identified is the honey bee (Apis mellifera) with its well-known queen mandibular pheromone (QMP). Although pleiotropic effects on colony regulation are accredited to the QMP, this pheromone does not trigger the full behavioral and physiological response observed in the presence of the queen, suggesting the presence of additional compounds. We tested the hypothesis of a pheromone redundancy in honey bee queens by comparing the influence of queens with and without mandibular glands on worker behavior and physiology. RESULTS: Demandibulated queens had no detectable (E)-9-oxodec-2-enoic acid (9-ODA), the major compound in QMP, yet they controlled worker behavior (cell construction and queen retinue) and physiology (ovary inhibition) as efficiently as intact queens. CONCLUSIONS: We demonstrated that the queen uses other pheromones as powerful as QMP to control the colony. It follows that queens appear to have multiple active compounds with similar functions in the colony (pheromone redundancy). Our findings support two hypotheses in the biology of social insects: (1) that multiple semiochemicals with synonymous meaning exist in the honey bee, (2) that this extensive semiochemical vocabulary exists because it confers an evolutionary advantage to the colony.

Nosema spp. infection alters pheromone production in honey bees (Apis mellifera).

Pheromones in social insects play a key role in the regulation of group homoeostasis. It is well-established that parasites can modify hormone signaling of their host, but less is known about the effect of parasites on pheromone signaling in insect societies. We, thus, tested in honey bees (Apis mellifera) the effect of the widespread parasite Nosema spp. on the production of ethyl oleate (EO), the only identified primer pheromone in honey bee workers. Since environmental stressors like pesticides also can weaken honey bees, we also analyzed the effect of imidacloprid, a neonicotinoid widely used in agriculture, on EO production. We show that, contrary to imidacloprid, Nosema spp. significantly altered EO production. In addition, the level of Nosema infection was correlated positively with the level of EO production. Since EO is involved in the regulation of division of labor among workers, our result suggests that the changes in EO signaling induced by parasitism have the potential to disturb the colony homoeostasis.