Molecular and Kinetic Properties of Two Acetylcholinesterases from the Western Honey Bee,Apis mellifera

We investigated the molecular and kinetic properties of two acetylcholinesterases (AmAChE1 and AmAChE2) from the Western honey bee, Apis mellifera. Western blot analysis revealed that AmAChE2 has most of catalytic activity rather than AmAChE1, further suggesting that AmAChE2 is responsible for synaptic transmission in A. mellifera, in contrast to most other insects. AmAChE2 was predominately expressed in the ganglia and head containing the central nervous system (CNS), while AmAChE1 was abundantly observed not only in the CNS but also in the peripheral nervous system/non-neuronal tissues. Both AmAChEs exist as homodimers; the monomers are covalently connected via a disulfide bond under native conditions. However, AmAChE2 was associated with the cell membrane via the glycophosphatidylinositol anchor, while AmAChE1 was present as a soluble form. The two AmAChEs were functionally expressed with a baculovirus system. Kinetic analysis revealed that AmAChE2 has approximately 2,500-fold greater catalytic efficiency toward acetylthiocholine and butyrylthiocholine than AmAChE1, supporting the synaptic function of AmAChE2. In addition, AmAChE2 likely serves as the main target of the organophosphate (OP) and carbamate (CB) insecticides as judged by the lower IC₅₀ values against AmAChE2 than against AmAChE1. When OP and CB insecticides were pre-incubated with a mixture of AmAChE1 and AmAChE2, a significant reduction in the inhibition of AmAChE2 was observed, suggesting a protective role of AmAChE1 against xenobiotics. Taken together, based on their tissue distribution pattern, molecular and kinetic properties, AmAChE2 plays a major role in synaptic transmission, while AmAChE1 has non-neuronal functions, including chemical defense.     

Genetics,Synergists, and Age Affect Insecticide Sensitivity of the Honey Bee,Apis mellifera

The number of honey bee colonies in the United States has declined to half of its peak level in the 1940s, and colonies lost over the winter have reached levels that are becoming economically unstable. While the causes of these losses are numerous and the interaction between them is very complex, the role of insecticides has garnered much attention. As a result, there is a need to better understand the risk of insecticides to bees, leading to more studies on both toxicity and exposure. While much research has been conducted on insecticides and bees, there have been very limited studies to elucidate the role that bee genotype and age has on the toxicity of these insecticides. The goal of this study was to determine if there are differences in insecticide sensitivity between honey bees of different genetic backgrounds (Carniolan, Italian, and Russian stocks) and assess if insecticide sensitivity varies with age. We found that Italian bees were the most sensitive of these stocks to insecticides, but variation was largely dependent on the class of insecticide tested. There were almost no differences in organophosphate bioassays between honey bee stocks (<1-fold), moderate differences in pyrethroid bioassays (1.5 to 3-fold), and dramatic differences in neonicotinoid bioassays (3.4 to 33.3-fold). Synergism bioassays with piperonyl butoxide, amitraz, and coumaphos showed increased phenothrin sensitivity in all stocks and also demonstrated further physiological differences between stocks. In addition, as bees aged, the sensitivity to phenothrin significantly decreased, but the sensitivity to naled significantly increased. These results demonstrate the variation arising from the genetic background and physiological transitions in honey bees as they age. This information can be used to determine risk assessment, as well as establishing baseline data for future comparisons to explain the variation in toxicity differences for honey bees reported in the literature.     

No Genetic Tradeoffs between Hygienic Behaviour and Individual Innate Immunity in the Honey Bee,Apis mellifera

Many animals have individual and social mechanisms for combating pathogens. Animals may exhibit short-term physiological tradeoffs between social and individual immunity because the latter is often energetically costly. Genetic tradeoffs between these two traits can also occur if mutations that enhance social immunity diminish individual immunity, or vice versa. Physiological tradeoffs between individual and social immunity have been previously documented in insects, but there has been no study of genetic tradeoffs involving these traits. There is strong evidence that some genes influence both innate immunity and behaviour in social insects – a prerequisite for genetic tradeoffs. Quantifying genetic tradeoffs is critical for understanding the evolution of immunity in social insects and for devising effective strategies for breeding disease-resistant pollinator populations. We conducted two experiments to test the hypothesis of a genetic tradeoff between social and individual immunity in the honey bee, Apis mellifera. First, we estimated the relative contribution of genetics to individual variation in innate immunity of honey bee workers, as only heritable traits can experience genetic tradeoffs. Second, we examined if worker bees with hygienic sisters have reduced individual innate immune response. We genotyped several hundred workers from two colonies and found that patriline genotype does not significantly influence the antimicrobial activity of a worker’s hemolymph. Further, we did not find a negative correlation between hygienic behaviour and the average antimicrobial activity of a worker’s hemolymph across 30 honey bee colonies. Taken together, our work indicates no genetic tradeoffs between hygienic behaviour and innate immunity in honey bees. Our work suggests that using artificial selection to increase hygienic behaviour of honey bee colonies is not expected to concurrently compromise individual innate immunity of worker bees.     

Extreme Recombination Frequencies Shape Genome Variation and Evolution in the Honeybee,Apis mellifera

Meiotic recombination is a fundamental cellular process, with important consequences for evolution and genome integrity. However, we know little about how recombination rates vary across the genomes of most species and the molecular and evolutionary determinants of this variation. The honeybee, Apis mellifera, has extremely high rates of meiotic recombination, although the evolutionary causes and consequences of this are unclear. Here we use patterns of linkage disequilibrium in whole genome resequencing data from 30 diploid honeybees to construct a fine-scale map of rates of crossing over in the genome. We find that, in contrast to vertebrate genomes, the recombination landscape is not strongly punctate. Crossover rates strongly correlate with levels of genetic variation, but not divergence, which indicates a pervasive impact of selection on the genome. Germ-line methylated genes have reduced crossover rate, which could indicate a role of methylation in suppressing recombination. Controlling for the effects of methylation, we do not infer a strong association between gene expression patterns and recombination. The site frequency spectrum is strongly skewed from neutral expectations in honeybees: rare variants are dominated by AT-biased mutations, whereas GC-biased mutations are found at higher frequencies, indicative of a major influence of GC-biased gene conversion (gBGC), which we infer to generate an allele fixation bias 5 – 50 times the genomic average estimated in humans. We uncover further evidence that this repair bias specifically affects transitions and favours fixation of CpG sites. Recombination, via gBGC, therefore appears to have profound consequences on genome evolution in honeybees and interferes with the process of natural selection. These findings have important implications for our understanding of the forces driving molecular evolution.     

In-depth Proteomics Characterization of Embryogenesis of the Honey Bee Worker (Apis mellifera ligustica)

Identifying proteome changes of honey bee embryogenesis is of prime importance for unraveling the molecular mechanisms that they underlie. However, many proteomic changes during the embryonic period are not well characterized. We analyzed the proteomic alterations over the complete time course of honey bee worker embryogenesis at 24, 48, and 72 h of age, using mass spectrometry-based proteomics, label-free quantitation, and bioinformatics. Of the 1460 proteins identified the embryo of all three ages, the core proteome (proteins shared by the embryos of all three ages, accounting for 40%) was mainly involved in protein synthesis, metabolic energy, development, and molecular transporter, which indicates their centrality in driving embryogenesis. However, embryos at different developmental stages have their own specific proteome and pathway signatures to coordinate and modulate developmental events. The young embryos (<24 h) stronger expression of proteins related to nutrition storage and nucleic acid metabolism may correlate with the cell proliferation occurring at this stage. The middle aged embryos (24–48 h) enhanced expression of proteins associated with cell cycle control, transporters, antioxidant activity, and the cytoskeleton suggest their roles to support rudimentary organogenesis. Among these proteins, the biological pathways of aminoacyl-tRNA biosynthesis, β-alanine metabolism, and protein export are intensively activated in the embryos of middle age. The old embryos (48–72 h) elevated expression of proteins implicated in fatty acid metabolism and morphogenesis indicate their functionality for the formation and development of organs and dorsal closure, in which the biological pathways of fatty acid metabolism and RNA transport are highly activated. These findings add novel understanding to the molecular details of honey bee embryogenesis, in which the programmed activation of the proteome matches with the physiological transition observed during embryogenesis. The identified biological pathways and key node proteins allow for further functional analysis and genetic manipulation for both the honey bee embryos and other eusocial insects.Embryogenesis is an important period during which the body plan of adult honey bees (Apis mellifera L.) is formed. This life stage, lasting 72 h, occurs during the egg laid by the queen before bees hatch as young larva. Worker bees are derived from fertilized eggs and develop through four distinct stages until the imago eventually emerges: egg, larva, pupa, and emerging adult (1–4). The worker is the dominate caste and engages in almost all aspects of social life: taking care of larvae, cleaning the hive, guarding the nest, and foraging for nectar and pollen for the colony. Understanding the developmental mechanism of embryogenesis of honey bee workers at the protein level is conducive to gaining a new insight into honey bee embryology, but information about the mechanisms of honey bee embryos at molecular level is still very limited.The embryo is recognized as an ideal model for genetic modification as compared with larva, pupa, and emerged adults (5). The environment for embryonic development requires a constant temperature of 34 °C and 80% relative humidity, which can easily be simulated under laboratory conditions. In contrast, rearing larvae or pupae is more challenging because they demand a specific temperature, humidity, and nutrition in the colony environment (1, 5). Furthermore, the honey bee has adapted an evolutionary strategy for better colony survival that makes it difficult to rear experimentally modified larvae and pupae within the colony (6, 7), nurse bees use acute judgment to identify and remove abnormal eggs or larvae (8). This adaptation makes raising experimentally treated bees, such as genetically manipulated eggs and larvae, very difficult in the honey bee colony (9–11). Because of totipotency and multiple differentiation potential, modified eggs could be hatched out normally and eventually some of them could be induced to morphologically and physiologically normal adult queens (12), increasing their usefulness as a model system. Moreover, the chorion of honey bee egg is more suitable for puncturing a hole for microinjection as it is much thinner than that of the fruit fly (Drosophila melonogastero) or the silk worm (Bombyx mori) (0.1–0.25 μm for honey bees compared with ∼17 μm for silk worm) (13, 14). These superiorities are quite promising for in vivo transgenic research on honey bee embryos.Until now, a number of genetic manipulations of the honey bee embryo have been developed. For example, embryonic cells in the pre-gastrula stage that have been transplanted with nuclear materials have developed into chimeric honey bee larvae (15). RNA interference (RNAi) has been used for honey bee embryos in vivo to characterize the functioning of specific genes (16) and for genetic effects on morphological differentiation (17, 18). Moreover, the cultivation of short-term (19–21), long-term (22), and immortalized cell lines (23), and the expression of non-Apis genes in cultured embryonic cells (24) have opened up a new era for genetic manipulation of honey bee embryos.Like Drosophila, Apis is a long germ insect in which segmentation occurs across the whole body (25). To date, although several studies have examined morphological change (2, 26, 27) and gene expression (25, 28, 29) during the period of embryogenesis in the honey bee, only a few works report on the preliminary results of the unraveling molecular underpinnings of worker (30) and drone (31) embryogenesis at the proteomic level, identifying only 107 proteins. MS-based proteomics is the primary technology that enables a system-wide view of proteomes and their changes. The development of MS with high resolution, high mass accuracy, and high sequencing speed now allows routine identification and quantification of proteins in a comprehensive and unbiased manner in biological samples with high confidence (32). These technological advances in LC-MS now allow the study of protein expression on a system-wide level (33). Therefore, an in-depth characterization of the proteome changes during the honey bee embryogenesis will provide greater understanding of the molecular mechanisms that underlie the process of embryogenesis in honey bee workers, and offers new insights into the embryology of other social insects.     

Nonsusceptibility of the Honey Bee,Apis mellifera (Hymenoptera: Apidae), to Steinernematid and Heterorhabditid Nematodes

We exposed honey bee workers and brood to four entomopathogenic nematode species under conditions normally encountered in the hive by spraying nematodes onto combs. Mortality of adult bees exposed to any of the nematode species was less than 10%, and there was no evidence of nematode infection when dead adults were dissected. To assess the impact of nematodes on brood, we used smaller-size honey combs placed in the second story (super) of a hive and large brood combs placed in the main section of the hive. Our results were inconsistent between these two experimental designs. The smaller honey combs sprayed with Steinernema carpocapsae contained the largest number of uncapped ceils, those sprayed with Heterorhabditis baeteriophora or S. riobravis contained an intermediate number of uncapped cells, and control combs and those sprayed with S. glaseri contained the fewest nmnber of uncapped cells. Large combs sprayed with S. riobravis contained more uncapped ceils than controls or those sprayed with S. carpocapsae, although the differences were not significant. Our results do not support the hypothesis that high-temperature-tolerant species of nematodes are necessarily more infective to honey bees.     

Linkage Map of the Honey Bee, Apis Mellifera, Based on Rapd Markers

A linkage map was constructed for the honey bee based on the segregation of 365 random amplified polymorphic DNA (RAPD) markers in haploid male progeny of a single female bee. The X locus for sex determination and genes for black body color and malate dehydrogenase were mapped to separate linkage groups. RAPD markers were very efficient for mapping, with an average of about 2.8 loci mapped for each 10-nucleotide primer that was used in polymerase chain reactions. The mean interval size between markers on the map was 9.1 cM. The map covered 3110 cM of linked markers on 26 linkage groups. We estimate the total genome size to be ~3450 cM. The size of the map indicated a very high recombination rate for the honey bee. The relationship of physical to genetic distance was estimated at 52 kb/cM, suggesting that map-based cloning of genes will be feasible for this species.     

Age and Behavior of Honey Bee Workers,Apis mellifera,that Interact with Drones

Colony reproduction in honey bees involves complex interactions between sterile workers and reproductive castes. Although worker–queen interactions have been studied in detail, worker–drone interactions are less well understood. We investigated caste interactions in honey bees by determining the age and behavior of workers that perform vibration signals, trophallaxis, and grooming with drones. Workers of all ages could engage in the different interactions monitored, although workers that performed vibration signals on drones were significantly older than those engaging in trophallaxis and grooming. Only 3–8% of workers engaged in the different behaviors were monitored. Compared with workers that performed vibration signals only on workers (‘worker vibrators’), those that performed signals on both workers and drones (‘drone vibrators’) had greater movement rates inside the nest, higher vibration signaling rates, and were more likely to have an immediate association with foraging. Both worker vibrators and drone vibrators contacted drones of all ages as they moved through the nest. However, drone vibrators contacted drones at higher rates, contacted slightly, but significantly younger drones, and were more likely to engage in trophallaxis and grooming with drones, in addition to vibrating them. Taken together, our results suggest that tiny proportions of workers belonging to separate, but overlapping age groups provide most of the care received by adult drones, and that drone vibrators comprise a subset of signalers within a colony that have an increased tendency to contact and interact with drones. Vibratory, tactile signals are involved in colony reproductive and movement decisions in a number of species of bees, wasps and ants, and may provide valuable tools for investigating caste interactions in many insect societies.     

Gregarines Found in the Honey Bee Apis mellifera Linnaeus in Venezuela.

SUMMARY. Gregarines were found for the first time in the honey bee Apis mellifera L. in Venezuela. The parasites attacked the inner wall of the ventriculus of the adult bees, causing heavy losses in apiculture in October 1954 and June 1955. The disease produced was called gregarina disease of the honey bee (in Spanish "gregarinosis de la abeja").     

The Bacterial Communities Associated with Honey Bee (Apis mellifera) Foragers

The honey bee is a key pollinator species in decline worldwide. As part of a commercial operation, bee colonies are exposed to a variety of agricultural ecosystems throughout the year and a multitude of environmental variables that may affect the microbial balance of individuals and the hive. While many recent studies support the idea of a core microbiota in guts of younger in-hive bees, it is unknown whether this core is present in forager bees or the pollen they carry back to the hive. Additionally, several studies hypothesize that the foregut (crop), a key interface between the pollination environment and hive food stores, contains a set of 13 lactic acid bacteria (LAB) that inoculate collected pollen and act in synergy to preserve pollen stores. Here, we used a combination of 454 based 16S rRNA gene sequencing of the microbial communities of forager guts, crops, and corbicular pollen and crop plate counts to show that (1) despite a very different diet, forager guts contain a core microbiota similar to that found in younger bees, (2) corbicular pollen contains a diverse community dominated by hive-specific, environmental or phyllosphere bacteria that are not prevalent in the gut or crop, and (3) the 13 LAB found in culture-based studies are not specific to the crop but are a small subset of midgut or hindgut specific bacteria identified in many recent 454 amplicon-based studies. The crop is dominated by Lactobacillus kunkeei, and Alpha 2.2 (Acetobacteraceae), highly osmotolerant and acid resistant bacteria found in stored pollen and honey. Crop taxa at low abundance include core hindgut bacteria in transit to their primary niche, and potential pathogens or food spoilage organisms seemingly vectored from the pollination environment. We conclude that the crop microbial environment is influenced by worker task, and may function in both decontamination and inoculation.     

Orientation-sensitive Neurons in the Brain of the Honey Bee (Apis mellifera)

Recent behavioural experiments have shown that bees are able to distinguish vertically presented patterns with orientation cues, although the locations of areas of black are randomized. To discriminate between two orientations, the bees must possess more than one orientation-sensitive neuron type. Therefore, the aim is to search for different types of orientation-sensitive cells of the honey bee, and measure their receptive field, velocity sensitivity and contrast sensitivity. Orientation-sensitive cells with two different types of orientation tuning-curves were recorded intracellularly in the mid-brain of the honey bee when the stimulus was a narrow bar (bar width = 5 degrees ). These cells are sensitive to bar movement within their large receptive field, which covers the visual field of one eye. They are quite distinct from the well-known directional motion detectors. The contrast sensitivity of the orientation-sensitive cells recorded in this study corresponds to results from behavioural experiments. The velocity-sensitivity curves of the orientation-sensitive cells differ from those of the direction-sensitive cells. Measurements of orientation sensitivity and contrast sensitivity when the stimulus is a wide bar (bar width = 10 degrees ), done in different eye regions, suggest that each orientation-sensitive cell receives visual signals from an array of orientational subunits within its receptive field. The correspondence between these physiological results and the results of recent behavioural experiments are discussed. Copyright 1997 Elsevier Science Ltd. All rights reserved     

Individual Learning Ability and Complex Odor Recognition in the Honey Bee, Apis mellifera L.

Individually restrained worker bees were trained to recognize complex odors in a conditioned proboscis extension assay. Three groups of bees were considered, based on the responses recorded during the experimental procedure: selective learners, nonselective learners, and nonlearners. For conditioning, three concentrations of two synthetic mixtures were used. The distribution of bees between groups was not significantly affected by the nature or by the concentration of the conditioning mixture. After conditioning, bees were tested with the individual compounds, and the responses were analyzed with respect to the three groups. Selective learners showed discriminative responses to a few key compounds, while nonselective learners responded to all the compounds, and nonlearners to none. These results showed that complex odor recognition is based on the recognition of key components and relies on the ability of bees to learn.     

Routes of Acquisition of the Gut Microbiota of the Honey Bee Apis mellifera

Studies of newly emerged Apis mellifera worker bees have demonstrated that their guts are colonized by a consistent core microbiota within several days of eclosure. We conducted experiments aimed at illuminating the transmission routes and spatiotemporal colonization dynamics of this microbiota. Experimental groups of newly emerged workers were maintained in cup cages and exposed to different potential transmission sources. Colonization patterns were evaluated using quantitative real-time PCR (qPCR) to assess community sizes and using deep sequencing of 16S rRNA gene amplicons to assess community composition. In addition, we monitored the establishment of the ileum and rectum communities within workers sampled over time from natural hive conditions. The study verified that workers initially lack gut bacteria and gain large characteristic communities in the ileum and rectum within 4 to 6 days within hives. Typical communities, resembling those of workers within hives, were established in the presence of nurse workers or nurse worker fecal material, and atypical communities of noncore or highly skewed compositions were established when workers were exposed only to oral trophallaxis or hive components (comb, honey, bee bread). The core species of Gram-negative bacteria, Snodgrassella alvi, Gilliamella apicola, and Frischella perrara, were dependent on the presence of nurses or hindgut material, whereas some Gram-positive species were more often transferred through exposure to hive components. These results indicate aspects of the colony life cycle and behavior that are key to the propagation of the characteristic honey bee gut microbiota.     

A SNP Based High-Density Linkage Map of Apis cerana Reveals a High Recombination Rate Similar to Apis mellifera

Background

The Eastern honey bee, Apis cerana Fabricius, is distributed in southern and eastern Asia, from India and China to Korea and Japan and southeast to the Moluccas. This species is also widely kept for honey production besides Apis mellifera. Apis cerana is also a model organism for studying social behavior, caste determination, mating biology, sexual selection, and host-parasite interactions. Few resources are available for molecular research in this species, and a linkage map was never constructed. A linkage map is a prerequisite for quantitative trait loci mapping and for analyzing genome structure. We used the Chinese honey bee, Apis cerana cerana to construct the first linkage map in the Eastern honey bee.

Results

F2 workers (N = 103) were genotyped for 126,990 single nucleotide polymorphisms (SNPs). After filtering low quality and those not passing the Mendel test, we obtained 3,000 SNPs, 1,535 of these were informative and used to construct a linkage map. The preliminary map contains 19 linkage groups, we then mapped the 19 linkage groups to 16 chromosomes by comparing the markers to the genome of A. mellfiera. The final map contains 16 linkage groups with a total of 1,535 markers. The total genetic distance is 3,942.7 centimorgans (cM) with the largest linkage group (180 loci) measuring 574.5 cM. Average marker interval for all markers across the 16 linkage groups is 2.6 cM.

Conclusion

We constructed a high density linkage map for A. c. cerana with 1,535 markers. Because the map is based on SNP markers, it will enable easier and faster genotyping assays than randomly amplified polymorphic DNA or microsatellite based maps used in A. mellifera.     

A Locomotor Deficit Induced by Sublethal Doses of Pyrethroid and Neonicotinoid Insecticides in the Honeybee Apis mellifera

The toxicity of pesticides used in agriculture towards non-targeted organisms and especially pollinators has recently drawn the attention from a broad scientific community. Increased honeybee mortality observed worldwide certainly contributes to this interest. The potential role of several neurotoxic insecticides in triggering or potentiating honeybee mortality was considered, in particular phenylpyrazoles and neonicotinoids, given that they are widely used and highly toxic for insects. Along with their ability to kill insects at lethal doses, they can compromise survival at sublethal doses by producing subtle deleterious effects. In this study, we compared the bee’s locomotor ability, which is crucial for many tasks within the hive (e.g. cleaning brood cells, feeding larvae…), before and after an acute sublethal exposure to one insecticide belonging to the two insecticide classes, fipronil and thiamethoxam. Additionally, we examined the locomotor ability after exposure to pyrethroids, an older chemical insecticide class still widely used and known to be highly toxic to bees as well. Our study focused on young bees (day 1 after emergence) since (i) few studies are available on locomotion at this stage and (ii) in recent years, pesticides have been reported to accumulate in different hive matrices, where young bees undergo their early development. At sublethal doses (SLD_48h, i.e. causing no mortality at 48h), three pyrethroids, namely cypermethrin (2.5 ng/bee), tetramethrin (70 ng/bee), tau-fluvalinate (33 ng/bee) and the neonicotinoid thiamethoxam (3.8 ng/bee) caused a locomotor deficit in honeybees. While the SLD_48h of fipronil (a phenylpyrazole, 0.5 ng/bee) had no measurable effect on locomotion, we observed high mortality several days after exposure, an effect that was not observed with the other insecticides. Although locomotor deficits observed in the sublethal range of pyrethroids and thiamethoxam would suggest deleterious effects in the field, the case of fipronil demonstrates that toxicity evaluation requires information on multiple endpoints (e.g. long term survival) to fully address pesticides risks for honeybees. Pyrethroid-induced locomotor deficits are discussed in light of recent advances regarding their mode of action on honeybee ion channels and current structure-function studies.