Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

USDA-ARS

The African honey bee subspecies Apis mellifera scutellata has colonized much of the Americas in less than 50 years and has largely replaced European bees throughout its range in the New World. The African bee therefore provides an excellent opportunity to examine the factors that influence invasion success. We provide a synthesis of recent research on the African bee, concentrating on its ability to displace European honey bees. Specifically, we consider (a) the genetic composition of the expanding population and the symmetry of gene flow between African and European bees, (b) the mechanisms that favor the preservation of the African genome, and (c) the possible range and impact of the African bee in the United States.

The African honey bee: factors contributing to a successful biological invasion.

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.

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

Volatile agrochemicals such as 2-heptanone have potential in safely and effectively controlling important agricultural pests provided that they are properly delivered. The present study reports the permeability of starch gel matrices and various coatings, some of which are agricultural-based, that could be used in controlled release devices. Low-density, microcellular starch foam was made from wheat, Dent corn, and high amylose corn starches. The foam density ranged from 0.14 to 0.34 g/cm3, the pore volume ranged from 74 to 89%, and the loading capacity ranged from 2.3 to 7.2 times the foam weight. The compressive properties of the foam were not markedly affected by saturating the pore volume with silicone oil. The vapor transmission rate (VTR) and vapor permeability (VP) were measured in dry, porous starch foam and silicone-saturated starch gels. VTR values were highest in foam samples containing solvents with high vapor pressures. Silicone oil-saturated gels had lower VTR and VP values as compared to the dry foam. However, the silicone oil gel did not markedly reduce the VP for 2-heptanone and an additional vapor barrier or coating was needed to adequately reduce the evaporation rate. The VP of films of beeswax, paraffin, ethylene vinyl alcohol, a fruit film, and a laminate comprised of beeswax and fruit film was measured. The fruit film had a relatively high VP for polar solvents and a very low VP for nonpolar solvents. The laminate film provided a low VP for polar and nonpolar solvents. Perforating the fruit film portion of the laminate provided a method of attaining the target flux rate of 2-heptanone. The results demonstrate that the vapor flux rate of biologically active solvents can be controlled using agricultural materials.

Permeability of starch gel matrices and select films to solvent vapors.

Outcrossing of non-Bt cotton (Gossypium hirsutum (L.)) in refuges by transgenic Bt cultivars could reduce the efficacy of refuges for delaying resistance in seed-feeding pests. Based on reports that outcrossing decreased as distance from Bt cotton increased in small-scale studies, we hypothesized that increasing refuge width or distance from Bt fields would reduce outcrossing. In a large-scale study in Arizona, we quantified Bt seed in refuges of experimental and commercial fields, comparing outcrossing between in-field (narrow) and external (wide) refuges and among rows of refuges at various distances from Bt fields. Some refuges, including those in tightly controlled experimental plots, contained up to 8% adventitious Bt plants. Some, but not all, Bt plants likely resulted from Bt seed in the non-Bt seed bags. We did not detect a difference in outcrossing between in-field and external refuges. However, statistical power was low because outcrossing was low (< 0.4% of seeds) in both treatments. Higher outcrossing levels (< or = 4.6% of seeds) were observed in the studies measuring outcrossing at various distances from Bt fields, yet outcrossing did not decrease as the distance from Bt fields increased. We hypothesize that Bt plants in refuges cross-pollinated surrounding non-Bt plants, overshadowing the expected association between distance from Bt fields and outcrossing.

Outcrossed cottonseed and adventitious Bt plants in Arizona refuges.

A prominent theory states that animal phenotypes arise by evolutionary changes in gene regulation, but the extent to which this theory holds true for behavioral evolution is not known. Because "nature and nurture" are now understood to involve hereditary and environmental influences on gene expression, we studied whether environmental influences on a behavioral phenotype, i.e., aggression, could have evolved into inherited differences via changes in gene expression. Here, with microarray analysis of honey bees, we show that aggression-related genes with inherited patterns of brain expression are also environmentally regulated. There were expression differences in the brain for hundreds of genes between the highly aggressive Africanized honey bee compared with European honey bee (EHB) subspecies. Similar results were obtained for EHB in response to exposure to alarm pheromone (which provokes aggression) and when comparing old and young bees (aggressive tendencies increase with age). There was significant overlap of the gene lists generated from these three microarray experiments. Moreover, there was statistical enrichment of several of the same cis regulatory motifs in promoters of genes on all three gene lists. Aggression shows a remarkably robust brain molecular signature regardless of whether it occurs because of inherited, age-related, or environmental (social) factors. It appears that one element in the evolution of different degrees of aggressive behavior in honey bees involved changes in regulation of genes that mediate the response to alarm pheromone.

Honey bee aggression supports a link between gene regulation and behavioral evolution.

Does cognitive ability always correlate with a positive fitness consequence? Previous research in both vertebrates and invertebrates provides mixed results. Here, we compare the learning and memory abilities of Africanized honeybees (Apis mellifera scutellata hybrid) and European honeybees (Apis mellifera ligustica). The range of the Africanized honeybee continues to expand, superseding the European honeybee, which led us to hypothesize that they might possess greater cognitive capabilities as revealed by a classical conditioning assay. Surprisingly, we found that fewer Africanized honeybees learn to associate an odor with a reward. Additionally, fewer Africanized honeybees remembered the association a day later. While Africanized honeybees are replacing European honeybees, our results show that they do so despite displaying a relatively poorer performance on an associative learning paradigm.

Africanized honeybees are slower learners than their European counterparts.

Elucidating the mechanisms by which honey bees process pollen vs. protein supplements are important in the generation of artificial diets needed to sustain managed honeybees. We measured the effects of diet on protein concentration, hypopharyngeal gland development and virus titers in worker honey bees fed either pollen, a protein supplement (MegaBee), or a protein-free diet of sugar syrup. Workers consumed more pollen than protein supplement, but protein amounts and size of hypopharyngeal gland acini did not differ between the two feeding treatments. Bees fed sugar syrup alone had lower protein concentrations and smaller hypopharyngeal glands compared with the other feeding treatments especially as the bees aged. Deformed wing virus was detected in workers at the start of a trial. The virus concentrations increased as bees aged and were highest in those fed sugar syrup and lowest in bees fed pollen. Overall results suggest a connection between diet, protein levels and immune response and indicate that colony losses might be reduced by alleviating protein stress through supplemental feeding.

The effect of diet on protein concentration, hypopharyngeal gland development and virus load in worker honey bees (Apis mellifera L.).

The dynamics of viruses are critical to our understanding of disease pathogenesis. Using honey bee Deformed wing virus (DWV) as a model, we conducted field and laboratory studies to investigate the roles of abiotic and biotic stress factors as well as host health conditions in dynamics of virus replication in honey bees. The results showed that temperature decline could lead to not only significant decrease in the rate for pupae to emerge as adult bees, but also an increased severity of the virus infection in emerged bees, partly explaining the high levels of winter losses of managed honey bees, Apis mellifera, around the world. By experimentally exposing adult bees with variable levels of parasitic mite Varroa destructor, we showed that the severity of DWV infection was positively correlated with the density and time period of Varroa mite infestation, confirming the role of Varroa mites in virus transmission and activation in honey bees. Further, we showed that host conditions have a significant impact on the outcome of DWV infection as bees that originate from strong colonies resist DWV infection and replication significantly better than bee originating from weak colonies. The information obtained from this study has important implications for enhancing our understanding of host‑pathogen interactions and can be used to develop effective disease control strategies for honey bees.

Dynamics of persistent and acute deformed wing virus infections in honey bees, Apis mellifera.

A fundamental problem in meta-analysis is how to systematically combine information from multiple statistical tests to rigorously evaluate a single overarching hypothesis. This problem occurs in systems biology when attempting to map genomic attributes to complex phenotypes such as behavior. Behavior and other complex phenotypes are influenced by intrinsic and environmental determinants that act on the transcriptome, but little is known about how these determinants interact at the molecular level. We developed an informatic technique that identifies statistically significant meta-associations between gene expression patterns and transcription factor combinations. Deploying this technique for brain transcriptome profiles from ca. 400 individual bees, we show that diverse determinants of behavior rely on shared combinations of transcription factors. These relationships were revealed only when we considered complex and variable regulatory rules, suggesting that these shared transcription factors are used in distinct ways by different determinants. This regulatory code would have been missed by traditional gene coexpression or cis-regulatory analytic methods. We expect that our meta-analysis tools will be useful for a broad array of problems in systems biology and other fields.

New meta-analysis tools reveal common transcriptional regulatory basis for multiple determinants of behavior.

Un probl&egrave;me fondamental dans la m&eacute;ta-analyse est comment combiner syst&eacute;matiquement les informations de plusieurs tests statistiques pour &eacute;valuer rigoureusement une hypoth&egrave;se globale unique. En biologie des syst&egrave;mes, ce probl&egrave;me se produit lorsque vous essayez de mapper les attributs g&eacute;nomiques &agrave; des ph&eacute;notypes complexes tels que le comportement. Comportement et autres ph&eacute;notypes complexes sont influenc&eacute;es par des facteurs environnementaux et intrins&egrave;ques qui agissent sur le transcriptome, mais on sait peu sur la fa&ccedil;on dont ces d&eacute;terminants interagissent au niveau mol&eacute;culaire. Nous avons d&eacute;velopp&eacute; une technique informatique qui identifie les meta-corr&eacute;lations statistiquement significatives entre les profils d&#39;expression g&eacute;nique et les combinaisons de facteurs de transcription. Le d&eacute;ploiement de cette technique pour les profils de transcriptome de cerveau de ca. 400 abeilles individuels, nous montrons que divers facteurs d&eacute;terminants du comportement d&eacute;pendent partag&eacute;s combinaisons de facteurs de transcription. Ces relations sont r&eacute;v&eacute;l&eacute;es seulement quand nous avons consid&eacute;r&eacute;s comme complexes et des r&egrave;gles de r&eacute;glementation variables, ce qui sugg&egrave;re que ce partagent des facteurs de transcription sont utilis&eacute;s de mani&egrave;res distinctes de diff&eacute;rents d&eacute;terminants. Ce code de r&eacute;glementation aurait &eacute;t&eacute; manqu&eacute; par coexpression de g&egrave;ne traditionnel ou cis-r&eacute;gulation m&eacute;thodes analytiques. Nous pr&eacute;voyons que nos outils de m&eacute;ta-analyse sera utiles pour un vaste &eacute;ventail de probl&egrave;mes en biologie des syst&egrave;mes et d&#39;autres domaines.

Nouveaux outils de meta-analyse r&eacute;v&egrave;lent une base r&eacute;glementaire transcriptionnelle commune pour plusieurs d&eacute;terminants du comportement.

Un problema fondamentale nella meta-analisi &egrave; come combinare sistematicamente informazioni da molteplici test statistici per valutare rigorosamente una sola ipotesi overarching. Questo problema si verifica in biologia dei sistemi, quando si tenta di mapping degli attributi genomici di fenotipi complessi come il comportamento. Comportamento e altri fenotipi complessi sono influenzati da determinanti ambientali e intrinseche che agiscono su transcriptome, ma poco si sa su come questi fattori determinanti interagire a livello molecolare. Abbiamo sviluppato una tecnica informatica che identifica meta-associazioni statisticamente significative tra i modelli di espressione genica e combinazioni di fattore di trascrizione. Distribuzione di questa tecnica per profili transcriptome cervello da ca. 400 API individuali, mostriamo che diversi fattori determinanti del comportamento si basano su combinazioni condivise di fattori di trascrizione. Queste relazioni sono state rivelate solo quando abbiamo considerato complessi e variabile norme regolamentari, suggerendo che questi fattori di trascrizione in comune sono usati in modi diversi da diversi fattori determinanti. Questo codice normativo perso da coexpression gene tradizionale o cis-regolatori metodi analitici. Ci aspettiamo che gli strumenti di meta-analisi sar&agrave; utili per una vasta gamma di problemi in biologia dei sistemi e altri campi.

Nuovi strumenti di meta-analisi rivelano comuni transcriptional base normativa per pi&ugrave; determinanti del comportamento.

Hop (Humulus lupulus L.) beta acids (HBA) were tested for miticidal effects on varroa destructor Anderson and Trueman, a parasitic mite of the honey bee (Apis mellifera L.). When varroa were placed on bees that had topical applications of 1 % HBA, there was 100 % mite mortality. Bee mortality was unaffected. Cardboard strips saturated with HBA and placed in colonies resulted in mite drop that was significantly greater than in untreated hives. HBA was detected on about 60 % of the bees in colonies during the first 48 h after application. Mite drop in colonies lasted for about 7 days with the highest drop occurring in the first 2-3 days after treatment. There was a reduction in the percentages of bees with HBA and in the amounts on their bodies after 7 days. Bee and queen mortality in the colonies were not affected by HBA treatments. When cardboard strips saturated with HBA were put in packages of bees, more than 90 % of the mites were killed without an increase in bee mortality. HBA might have potential to control varroa when establishing colonies from packages or during broodless periods.

The effects of beta acids from hops (Humulus lupulus) on mortality of Varroa destructor (Acari: Varroidae).

Varroa (Varroa destuctor Anderson and Trueman) populations in honey bee (Apis mellifera L.) colonies might be kept at low levels by well-timed miticide applications. HopGuard(®) (HG) that contains beta plant acids as the active ingredient was used to reduce mite populations. Schedules for applications of the miticide that could maintain low mite levels were tested in hives started from either package bees or splits of larger colonies. The schedules were developed based on defined parameters for efficacy of the miticide and predictions of varroa population growth generated from a mathematical model of honey bee colony-varroa population dynamics. Colonies started from package bees and treated with HG in the package only or with subsequent HG treatments in the summer had 1.2-2.1 mites per 100 bees in August. Untreated controls averaged significantly more mites than treated colonies (3.3 mites per 100 bees). By October, mite populations ranged from 6.3 to 15.0 mites per 100 bees with the lowest mite numbers in colonies treated with HG in August. HG applications in colonies started from splits in April reduced mite populations to 0.12 mites per 100 bees. In September, the treated colonies had significantly fewer mites than the untreated controls. Subsequent HG applications in September that lasted for 3 weeks reduced mite populations to levels in November that were significantly lower than in colonies that were untreated or had an HG treatment that lasted for 1 week. The model accurately predicted colony population growth and varroa levels until the fall when varroa populations measured in colonies established from package bees or splits were much greater than predicted. Possible explanations for the differences between actual and predicted mite populations are discussed.

Population growth of Varroa destructor (Acari: Varroidae) in commercial honey bee colonies treated with beta plant acids.

Israeli acute paralysis virus (IAPV) is a widespread RNA virus of honey bees that has been linked with colony losses. Here we describe the transmission, prevalence, and genetic traits of this virus, along with host transcriptional responses to infections. Further, we present RNAi-based strategies for limiting an important mechanism used by IAPV to subvert host defenses. Our study shows that IAPV is established as a persistent infection in honey bee populations, likely enabled by both horizontal and vertical transmission pathways. The phenotypic differences in pathology among different strains of IAPV found globally may be due to high levels of standing genetic variation. Microarray profiles of host responses to IAPV infection revealed that mitochondrial function is the most significantly affected biological process, suggesting that viral infection causes significant disturbance in energy-related host processes. The expression of genes involved in immune pathways in adult bees indicates that IAPV infection triggers active immune responses. The evidence that silencing an IAPV-encoded putative suppressor of RNAi reduces IAPV replication suggests a functional assignment for a particular genomic region of IAPV and closely related viruses from the Family Dicistroviridae, and indicates a novel therapeutic strategy for limiting multiple honey bee viruses simultaneously and reducing colony losses due to viral diseases. We believe that the knowledge and insights gained from this study will provide a new platform for continuing studies of the IAPV-host interactions and have positive implications for disease management that will lead to mitigation of escalating honey bee colony losses worldwide.

Gloria Degrandi-Hoffman

Carl Hayden Bee Research Center

USDA-ARS

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Gloria Degrandi-Hoffman

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Carl Hayden Bee Research Center

USDA-ARS

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Carl Hayden Bee Research Center