Worker's genetic diversity has no relation to pollen diversity in a honey bee colony (Apis mellifera L.)

No differences were observed between the species diversity of pollen gathered by colonies with artificially reduced worker genetic diversity and those derived from naturally mated queens. Such a result does not support the hypothesis that polyandry increases colony fitness in which a genetic basis is created for using a wider range of food sources.     

Neuronal activity in a honey bee (Apis mellifera L.) with kynurenine deficiency

Neuronal activity of the antennal lobes, mushroom bodies, and cervical connective in wild-type honey bees and snowlaranija mutants was recorded at different stages of the ontogeny (on the 1st, 3rd, 7th, and 25th days). The mutation snowlaranija affects the structural gene of tryptophane oxygenase, the first key exzyme in the kynurenine pathway of tryptophane metabolism, and leads to a deficit of kynurenines. Changes in neuronal activity in nutant bees were most pronounced in the cervical connective. A significant decrease in the pulse rate was revealed only in homozygous but not in heterozygous individuals. This finding is in accordance with previously reported inhibitory effect of the mutation at the behavioral level. Less pronounced effects were obtained when the neuronal activity was recorded in the antennal lobes or mushroom bodies. This may be related to a complex character of biochemical changes in different parts of mutants brain.     

The impact of pollen consumption on honey bee (Apis mellifera) digestive physiology and carbohydrate metabolism

Carbohydrate‐active enzymes play an important role in the honey bee (Apis mellifera) due to its dietary specialization on plant‐based nutrition. Secretory glycoside hydrolases (GHs) produced in worker head glands aid in the processing of floral nectar into honey and are expressed in accordance with age‐based division of labor. Pollen utilization by the honey bee has been investigated in considerable detail, but little is known about the metabolic fate of indigestible carbohydrates and glycosides in pollen biomass. Here, we demonstrate that pollen consumption stimulates the hydrolysis of sugars that are toxic to the bee (xylose, arabinose, mannose). GHs produced in the head accumulate in the midgut and persist in the hindgut that harbors a core microbial community composed of approximately 10⁸ bacterial cells. Pollen consumption significantly impacted total and specific bacterial abundance in the digestive tract. Bacterial isolates representing major fermentative gut phylotypes exhibited primarily membrane‐bound GH activities that may function in tandem with soluble host enzymes retained in the hindgut. Additionally, we found that plant‐originating β‐galactosidase activity in pollen may be sufficient, in some cases, for probable physiological activity in the gut. These findings emphasize the potential relative contributions of host, bacteria, and pollen enzyme activities to carbohydrate breakdown, which may be tied to gut microbiome dynamics and associated host nutrition.     

Volatile compounds emitted by live European honey bee (Apis mellifera L.) queens

We used solid-phase microextraction (SPME, 65 microm PDMS-DVB fiber) to sample the volatile compounds emitted by live honey bee queens in several reproductive states (unmated queens, recently mated queens, and established mated queens), and compared them to the volatiles emitted by workers. We detected nine compounds that were present in at least 75% of the individuals in at least one type of bee, and which were not present in the sampling environment alone. Four of these compounds were present in queens but not in workers. One of these four compounds, identified as E-beta-ocimene, was expressed fully only in established mated queens and may be a signal of diploid egg-laying activity. The three remaining queen-specific compounds (including one identified as 2-phenylethanol) were associated with unmated queens and may mediate interactions between unmated queens and workers during queen elimination. The five common compounds that we detected in both queens and workers were hydrocarbons and may function as nestmate recognition cues. We consider these discoveries as a first step in determining the potentially important functions of volatile signals and cues within honey bee nests.     

Operant conditioning of antennal muscle activity in the honey bee (Apis mellifera L.)

Antennal movements of the honey bee can be conditioned operantly under laboratory conditions. Using this behavioural paradigm we have developed a preparation in which the activity of a single antennal muscle has been operantly conditioned. This muscle, the fast flagellum flexor muscle, is innervated by an identified motoneuron whose action potentials correlate 1:1 with the muscle potentials. The activity of the fast flagellum flexor muscle was recorded extracellularly from the scapus of the antenna. The animal was rewarded with a drop of sucrose solution whenever the muscle activity exceeded a defined reward threshold. The reward threshold was one standard deviation above the mean spontaneous frequency prior to conditioning. After ten conditioning trials, the frequency of the muscle potentials had increased significantly compared to the spontaneous frequency. The conditioned changes of frequency were observed for 30 min after conditioning. No significant changes of the frequency were found in the yoke control group. The firing pattern of the muscle potentials did not change significantly after conditioning or feeding. Fixing the antennal joints reduces or abolishes associative operant conditioning. The conditioned changes of the frequency of muscle potentials in the freely moving antenna are directly comparable to the behavioural changes during operant conditioning. Accepted: 29 March 2000     

Free amino acids in the haemolymph of honey bee queens (Apis mellifera L.)

In queen honey bees the free amino acid content in the haemolymph clearly depends on the physiological function and social environment of the individual. While in drones and workers the content of free amino acids increases after emergence until it reaches a peak in 5-day-old animals and decreases afterwards, the amino acid content in queens reaches its highest level (>60 nmol/ microl haemolymph) with the onset of egg laying (10 d of age). This level is about 2.5 times more than the highest level found in workers. Queens maintain this high level also when they are older (>30 d) and continue to lay eggs in average colonies. As in drones and workers, in queens the predominant amino acid is proline, which accounts for more than 50% of the total content of free amino acids in egg-laying individuals. When 10-day-old queens are prevented from mating and do not lay eggs, their amino acid content is significantly lower compared to laying queens of the same age. Also the social environment influences the contents of free amino acids in queens. When virgin queens were kept for 6 days with 20 worker bees and sufficient honey and pollen in an incubator, they had significantly lower concentrations of amino acids than virgin queens living for the same period with about 8000 workers in a colony. Most probably, the high amino acid concentration in the haemolymph is the basis for the high protein synthesis activity of laying queens.     

Selenium toxicity to honey bee (Apis mellifera L.) pollinators: effects on behaviors and survival

We know very little about how soil-borne pollutants such as selenium (Se) can impact pollinators, even though Se has contaminated soils and plants in areas where insect pollination can be critical to the functioning of both agricultural and natural ecosystems. Se can be biotransferred throughout the food web, but few studies have examined its effects on the insects that feed on Se-accumulating plants, particularly pollinators. In laboratory bioassays, we used proboscis extension reflex (PER) and taste perception to determine if the presence of Se affected the gustatory response of honey bee (Apis mellifera L., Hymenoptera: Apidae) foragers. Antennae and proboscises were stimulated with both organic (selenomethionine) and inorganic (selenate) forms of Se that commonly occur in Se-accumulating plants. Methionine was also tested. Each compound was dissolved in 1 M sucrose at 5 concentrations, with sucrose alone as a control. Antennal stimulation with selenomethionine and methionine reduced PER at higher concentrations. Selenate did not reduce gustatory behaviors. Two hours after being fed the treatments, bees were tested for sucrose response threshold. Bees fed selenate responded less to sucrose stimulation. Mortality was higher in bees chronically dosed with selenate compared with a single dose. Selenomethionine did not increase mortality except at the highest concentration. Methionine did not significantly impact survival. Our study has shown that bees fed selenate were less responsive to sucrose, which may lead to a reduction in incoming floral resources needed to support coworkers and larvae in the field. If honey bees forage on nectar containing Se (particularly selenate), reductions in population numbers may occur due to direct toxicity. Given that honey bees are willing to consume food resources containing Se and may not avoid Se compounds in the plant tissues on which they are foraging, they may suffer similar adverse effects as seen in other insect guilds.     

Antennal sucrose perception in the honey bee (Apis mellifera L.): behaviour and electrophysiology

Physiological mechanisms of antennal sucrose perception in the honey bee were analysed using behavioural and electrophysiological methods. Following sucrose stimulation of the tip of a freely moving antenna, the latency of proboscis extension was 320–340 ms, 80–100 ms after the first activity in muscle M17 controlling this response. When bees were allowed to actively touch a sucrose droplet with one antenna, contacts with the solution were frequent with durations of 10–20 ms and average intervals between contacts of approximately 40 ms. High sucrose concentrations led to short and frequent contacts. The proboscis response and M17 activity were largely independent of stimulus duration and temporal pattern. Taste hairs of the antennal tip displayed spike responses to sucrose concentrations down to at least 0.1%. The first 25 ms of the response were suitable for discrimination of sucrose concentrations. This time interval corresponds to the duration of naturally occurring gustatory stimuli. Sucrose responses between different hairs on the same antenna showed a high degree of variability, ranging from less than five to over 40 spikes per 0.5 s for a stimulus of 0.1% sucrose. This variability of receptor responses extends the dynamic range of sucrose perception over a large range of concentrations.     

Prevalence and infection intensity of Nosema in honey bee (Apis mellifera L.) colonies in Virginia

Nosema ceranae is a recently described pathogen of Apis mellifera and Apis cerana. Relatively little is known about the distribution or prevalence of N. ceranae in the United States. To determine the prevalence and potential impact of this new pathogen on honey bee colonies in Virginia, over 300 hives were sampled across the state. The samples were analyzed microscopically for Nosema spores and for the presence of the pathogen using real-time PCR. Our studies indicate that N. ceranae is the dominant species in Virginia with an estimated 69.3% of hives infected. Nosema apis infections were only observed at very low levels (2.7%), and occurred only as co-infections with N. ceranae. Traditional diagnoses based on spore counts alone do not provide an accurate indication of colony infections. We found that 51.1% of colonies that did not have spores present in the sample were infected with N. ceranae when analyzed by real-time PCR. In hives that tested positive for N. ceranae, average C_T values were used to diagnose a hive as having a low, moderate, or a heavy infection intensity. Most infected colonies had low-level infections (73%), but 11% of colonies had high levels of infection and 16% had moderate level infections. The prevalence and mean levels of infection were similar in different regions of the state.     

Mitochondrial structure and dynamics as critical factors in honey bee (Apis mellifera L.) caste development

The relationship between nutrition and phenotype is an especially challenging question in cases of facultative polyphenism, like the castes of social insects. In the honey bee, Apis mellifera, unexpected modifications in conserved signaling pathways revealed the hypoxia response as a possible mechanism underlying the regulation of body size and organ growth. Hence, the current study was designed to investigate possible causes of why the three hypoxia core genes are overexpressed in worker larvae. Parting from the hypothesis that this has an endogenous cause and is not due to differences in external oxygen levels we investigated mitochondrial numbers and distribution, as well as mitochondrial oxygen consumption rates in fat body cells of queen and worker larvae during the caste fate-critical larval stages. By immunofluorescence and electron microscopy we found higher densities of mitochondria in queen larval fat body, a finding further confirmed by a citrate synthase assay quantifying mitochondrial functional units. Oxygen consumption measurements by high-resolution respirometry revealed that queen larvae have higher maximum capacities of ATP production at lower physiological demand. Finally, the expression analysis of mitogenesis-related factors showed that the honey bee TFB1 and TFB2 homologs, and a nutritional regulator, ERR, are overexpressed in queen larvae. These results are strong evidence that the differential nutrition of queen and worker larvae by nurse bees affects mitochondrial dynamics and functionality in the fat body of these larvae, hence explaining their differential hypoxia response.     

Super and half-sister discrimination by honey bee workers (Apis mellifera L.) in a trophallactic bioassay

Summary Kin recognition and nepotism between honeybee workers (Apis mellifera L.) was analysed in a trophallactic bio-assay. Donor workers were fed dyed sugar syrup and introduced into a recipient group consisting of 12 to 15 workers of the same colony. After allowing for 1 hour of trophallaxis, the distribution of the dyed food was analysed with spectrophotometry. The subfamily composition in the recipient group was varied such that the donor bees had to discriminate between workers of 2 to 7 different patrilines. Donor bees preferentially fed super sisters if few patrilines were present in the recipient group. However, preferential feeding was not observed if the recipient group consisted of workers of more than three subfamilies. Since the natural degree of polyandry causes intracolonial genetic variance to exceed the genetic variability in the experiments, nepotistic behaviour among workers may not reveal intranidal subfamily recognition in honeybees.     

Myosins and DYNLL1/LC8 in the honey bee (Apis mellifera L.) brain

Honey bees have brain structures with specialized and developed systems of communication that account for memory, learning capacity and behavioral organization with a set of genes homologous to vertebrate genes. Many microtubule- and actin-based molecular motors are involved in axonal/dendritic transport. Myosin-Va is present in the honey bee Apis mellifera nervous system of the larvae and adult castes and subcastes. DYNLL1/LC8 and myosin-IIb, -VI and -IXb have also been detected in the adult brain. SNARE proteins, such as CaMKII, clathrin, syntaxin, SNAP25, munc18, synaptophysin and synaptotagmin, are also expressed in the honey bee brain. Honey bee myosin-Va displayed ATP-dependent solubility and was associated with DYNLL1/LC8 and SNARE proteins in the membrane vesicle-enriched fraction. Myosin-Va expression was also decreased after the intracerebral injection of melittin and NMDA. The immunolocalization of myosin-Va and -IV, DYNLL1/LC8, and synaptophysin in mushroom bodies, and optical and antennal lobes was compared with the brain morphology based on Neo-Timm histochemistry and revealed a distinct and punctate distribution. This result suggested that the pattern of localization is associated with neuron function. Therefore, our data indicated that the roles of myosins, DYNLL1/LC8, and SNARE proteins in the nervous and visual systems of honey bees should be further studied under different developmental, caste and behavioral conditions.     

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.     

Activity of protein kinase A and gustatory responsiveness in the honey bee (Apis mellifera L.)

The cAMP-dependent protein kinase A is involved in the induction of long-term memory and habituation in the bee. Gustatory responsiveness correlates strongly with associative and non-associative learning in bees. We tested whether protein kinase A activity in the antennal lobes correlates with gustatory responsiveness. Thirty minutes after feeding, bees with high gustatory responsiveness had a significantly higher protein kinase A activity than bees with low responsiveness. Ninety minutues after feeding, when gustatory responsiveness had increased in initially unresponsive bees, no changes in protein kinase A activity were found. We also tested time-dependent effects of protein kinase A activator and protein kinase A inhibitor on gustatory responsiveness. Injection of the protein kinase A activator adenosine 3'5'-cyclic monophosphate 8-bromo-sodium salt or of the protein kinase A inhibitor KT 5720 did not affect gustatory responsiveness within the first 4 h after treatment. Feeding of adenosine 3'5'-cyclic monophosphate 8-bromo-sodium salt over 4 days increased gustatory responsiveness in newly emerged bees and adult foragers. These results enable us to distinguish between two different forms of gustatory responsiveness: basal and transient gustatory responsiveness. Basal gustatory responsiveness correlates with protein kinase A activity and can only be modulated in the range of several days. Transient gustatory responsiveness appears to be independent of protein kinase A activity and can be modulated in the range of minutes to hours.     

Effects of honey bee (Apis mellifera L.) queen insemination volume on worker behavior and physiology

Honey bee colonies consist of tens of thousands of workers and a single reproductive queen that produces a pheromone blend which maintains colony organization. Previous studies indicated that the insemination quantity and volume alter queen mandibular pheromone profiles. In our 11-month long field study we show that workers are more attracted to high-volume versus low-volume inseminated queens, however, there were no significant differences between treatments in the number of queen cells built by workers in preparation for supersedure. Workers exposed to low-volume inseminated queens initiated production of queen-like esters in their Dufour's glands, but there were no significant difference in the amount of methyl farnesoate and juvenile hormone in worker hemolymph. Lastly, queen overwintering survival was unexpectedly lower in high-volume inseminated queens. Our results suggest that the queen insemination volume could ultimately affect colony health and productivity.