Search

Osmia lignaria Say is used in combination with Apis mellifera L. to pollinate U.S. orchard crops. The deployment of O. lignaria requires artificial warming to synchronize adult bee emergence with crop bloom. However, current methods for emerging bees are time consuming and inefficient; the Hivetop Incubator (HTI) invention creates a space atop an A. mellifera hive whose heat flows through the screened bottom of the HTI to incubate cocooned O. lignaria adults therein. In response to HTI heat, O. lignaria adults chew out of cocoons, find an exit hole, and fly away to nest in provided nesting sites and, thereby, pollinate a crop. Objectives for studies performed in Utah and Washington were to: 1) determine whether HTIs inhibit A. mellifera thermoregulation or colony growth, 2) compare O. lignaria emergence duration from an HTI with and without hive heat, and 3) assess whether O. lignaria females leave HTIs located at the orchard edge to nest throughout the orchard. We found no significant differences between the internal temperatures of A. mellifera colonies with and without HTIs and no impact on A. mellifera food storage or brood production. Osmia lignaria in hive-heated HTIs emerged 3× faster than bees in unheated HTIs. Heated HTIs were significantly cooler than hive temperatures but significantly warmer than HTIs atop empty hive boxes. Osmia lignaria nest distribution was not correlated to the location of HTIs at the orchard edge. Overall, HTIs were effective for timely, on-site emergence of O. lignaria for orchard pollination without negatively impacting A. mellifera colonies.

Comprehensive decisions on the management of commercially produced livestock, including solitary bees, depend largely on associated knowledge of genetic diversity. In this study, we present novel microsatellite markers to support the breeding, management, and conservation of the blue orchard bee, Osmia lignaria. Native to North America, O. lignaria has been trapped from wildlands and propagated on-crop and used to pollinate certain fruit, nut, and berry crops. Harnessing the O. lignaria genome assembly, we identified 59,632 candidate microsatellite loci in silico, of which 22 were tested using molecular techniques. Of the 22 loci, 14 loci were in Hardy-Weinberg equilibrium (HWE) and demonstrated no linkage disequilibrium (LD) in two Intermountain North American wild populations in Idaho and Utah. We found no difference in population genetic diversity between the two populations, but there was evidence for low but significant population differentiation. Also, to determine if these markers amplify in other Osmia, we assessed 23 species across the clades apicata, bicornis, emarginata, and ribifloris. Nine loci amplified in three species/subspecies of apicata, 22 loci amplified in 11 species/subspecies of bicornis, 11 loci amplified in seven species/subspecies of emarginata, and 22 loci amplified in two species/subspecies of ribifloris. Further testing is necessary to determine the capacity of these microsatellite loci to characterize genetic diversity and structure under the assumption of HWE and LD for species beyond O. lignaria. These markers will help to inform the conservation and commercial use of trapped and managed O. lignaria and other Osmia species for both agricultural and non-agricultural systems.

Honey bees are highly valued for their pollination services in agricultural settings, and recent declines in managed populations have caused concern. Colony losses following a major pollination event in the United States, almond pollination, have been characterized by brood mortality with specific symptoms, followed by eventual colony loss weeks later. In this study, we demonstrate that these symptoms can be produced by chronically exposing brood to both an organosilicone surfactant adjuvant (OSS) commonly used on many agricultural crops including wine grapes, tree nuts and tree fruits and exogenous viral pathogens by simulating a horizontal transmission event. Observed synergistic mortality occurred during the larval-pupal molt. Using q-PCR techniques to measure gene expression and viral levels in larvae taken prior to observed mortality at metamorphosis, we found that exposure to OSS and exogenous virus resulted in significantly heightened Black Queen Cell Virus (BQCV) titers and lower expression of a Toll 7-like-receptor associated with autophagic viral defense (Am18w). These results demonstrate that organosilicone spray adjuvants that are considered biologically inert potentiate viral pathogenicity in honey bee larvae, and guidelines for OSS use may be warranted.

Honey bee (Apis mellifera) colonies continue to experience high annual losses that remain poorly explained. Numerous interacting factors have been linked to colony declines. Understanding the pathways linking pathophysiology with symptoms is an important step in understanding the mechanisms of disease. In this study we examined the specific pathologies associated with honey bees collected from colonies suffering from Colony Collapse Disorder (CCD) and compared these with bees collected from apparently healthy colonies. We identified a set of pathological physical characteristics that occurred at different rates in CCD diagnosed colonies prior to their collapse: rectum distension, Malpighian tubule iridescence, fecal matter consistency, rectal enteroliths (hard concretions), and venom sac color. The multiple differences in rectum symptomology in bees from CCD apiaries and colonies suggest effected bees had trouble regulating water. To ensure that pathologies we found associated with CCD were indeed pathologies and not due to normal changes in physical appearances that occur as an adult bee ages (CCD colonies are assumed to be composed mostly of young bees), we documented the changes in bees of different ages taken from healthy colonies. We found that young bees had much greater incidences of white nodules than older cohorts. Prevalent in newly-emerged bees, these white nodules or cellular encapsulations indicate an active immune response. Comparing the two sets of characteristics, we determined a subset of pathologies that reliably predict CCD status rather than bee age (fecal matter consistency, rectal distension size, rectal enteroliths and Malpighian tubule iridescence) and that may serve as biomarkers for colony health. In addition, these pathologies suggest that CCD bees are experiencing disrupted excretory physiology. Our identification of these symptoms is an important first step in understanding the physiological pathways that underlie CCD and factors impacting bee health.

Colony Collapse Disorder (CCD), a special case of collapse of honey bee colonies, has resulted in significant losses for beekeepers. CCD-colonies show abundance of pathogens which suggests that they have a weakened immune system. Since honey bee viruses are major players in colony collapse and given the important role of viral RNA interference (RNAi) in combating viral infections we investigated if CCD-colonies elicit an RNAi response. Deep-sequencing analysis of samples from CCD-colonies from US and Israel revealed abundant small interfering RNAs (siRNA) of 21–22 nucleotides perfectly matching the Israeli acute paralysis virus (IAPV), Kashmir virus and Deformed wing virus genomes. Israeli colonies showed high titers of IAPV and a conserved RNAi-pattern of matching the viral genome. That was also observed in sample analysis from colonies experimentally infected with IAPV. Our results suggest that CCD-colonies set out a siRNA response that is specific against predominant viruses associated with colony losses.

There are a number of RNA virus pathogens that represent a serious threat to the health of managed honey bees (Apis mellifera). That some of these viruses are also found in the broader pollinator community suggests the wider environmental spread of these viruses, with the potential for a broader impact on ecosystems. Studies on the ecology and evolution of these viruses in the arthropod community as a whole may therefore provide important insights into these potential impacts. We examined managed A. mellifera colonies, nearby non-Apis hymenopteran pollinators, and other associated arthropods for the presence of five commonly occurring picorna-like RNA viruses of honey bees - black queen cell virus, deformed wing virus, Israeli acute paralysis virus, Kashmir bee virus and sacbrood virus. Notably, we observed their presence in several arthropod species. Additionally, detection of negative-strand RNA using strand-specific RT-PCR assays for deformed wing virus and Israeli acute paralysis virus suggests active replication of deformed wing virus in at least six non-Apis species and active replication of Israeli acute paralysis virus in one non-Apis species. Phylogenetic analysis of deformed wing virus also revealed that this virus is freely disseminating across the species sampled in this study. In sum, our study indicates that these viruses are not specific to the pollinator community and that other arthropod species have the potential to be involved in disease transmission in pollinator populations.

An understanding of the anatomy and functions of internal and external structures is fundamental to many studies on the honey bee Apis mellifera. Similarly, proficiency in dissection techniques is vital for many more complex procedures. In this paper, which is a prelude to the other papers of the COLOSS BEEBOOK, we outline basic honey bee anatomy and basic dissection techniques.

Sacbrood disease, an affliction of honey bees (Apis mellifera) characterized by brood that fails to pupate and subsequently dies, is an important threat to honey bee health. The disease is caused by the sacbrood virus (SBV), a positive-, single-stranded RNA virus in the order Picornavirales. Because of the economic importance of honey bees for both pollination and honey production, it is vital to understand and monitor the spread of viruses such as SBV. This virus has been found in many places across the globe, including recently in some South American countries, and it is likely that it will continue to spread. We performed a preliminary study to search for SBV in two apiaries of Africanized honey bees in the State of São Paulo, Brazil, using RT-PCR and Sanger sequencing and found the first evidence of SBV in honey bee colonies in Brazil. The virus was detected in larvae, foraging and nurse bees from two colonies, one of which had symptoms of sacbrood disease, at the beginning of the winter season in June 2011. No SBV was found in samples from nine other nearby colonies.

Colony collapse disorder (CCD), a syndrome whose defining trait is the rapid loss of adult worker honey bees, Apis mellifera L., is thought to be responsible for a minority of the large overwintering losses experienced by U.S. beekeepers since the winter 2006-2007. Using the same data set developed to perform a monofactorial analysis (PloS ONE 4: e6481, 2009), we conducted a classification and regression tree (CART) analysis in an attempt to better understand the relative importance and interrelations among different risk variables in explaining CCD. Fifty-five exploratory variables were used to construct two CART models: one model with and one model without a cost of misclassifying a CCD-diagnosed colony as a non-CCD colony. The resulting model tree that permitted for misclassification had a sensitivity and specificity of 85 and 74%, respectively. Although factors measuring colony stress (e.g., adult bee physiological measures, such as fluctuating asymmetry or mass of head) were important discriminating values, six of the 19 variables having the greatest discriminatory value were pesticide levels in different hive matrices. Notably, coumaphos levels in brood (a miticide commonly used by beekeepers) had the highest discriminatory value and were highest in control (healthy) colonies. Our CART analysis provides evidence that CCD is probably the result of several factors acting in concert, making afflicted colonies more susceptible to disease. This analysis highlights several areas that warrant further attention, including the effect of sublethal pesticide exposure on pathogen prevalence and the role of variability in bee tolerance to pesticides on colony survivorship.

The peritrophic matrix (PM) lines the midgut of most insects, providing protection to the midgut epithelial cells while permitting passage of nutrients and water. Herein, we provide evidence that plant-mediated alteration of the PM contributes to the well-documented inhibition of fatal infection by Autographa californica multiple nucleopolyhedrovirus (AcMNPV) of Heliothis virescens F. larvae fed cotton foliage. We examined the impact of the PM on pathogenesis using a viral construct expressing a reporter gene (AcMNPV-hsp70/lacZ) orally inoculated into larvae with either intact PMs or PMs disrupted by Trichoplusia ni granulovirus occlusion bodies containing enhancin, known to degrade insect intestinal mucin. Larvae possessing disrupted PMs displayed infection foci (lacZ signaling) earlier than those with intact PMs. We then examined PMs from larvae fed artificial diet or plant foliage using electron microscopy; foliage-fed larvae had significantly thicker PMs than diet-fed larvae. Moreover, mean PM width was inversely related to both the proportion of larvae with lacZ signaling at 18h post-inoculation and the final percentage mortality from virus. Thus, feeding on foliage altered PM structure, and these foliage-mediated changes reduced baculoviral efficacy. These data indicate that the PM is an important factor determining the success of an ingested pathogen in foliage-fed lepidopteran larvae.

Controlling populations of varroa mites is crucial for the survival of the beekeeping industry. Many treatments exist, and all are designed to kill mites on adult bees. Because the majority of mites are found under capped brood, most treatments are designed to deliver active ingredients over an extended period to control mites on adult bees, as developing bees and mites emerge. In this study, a 17-h application of 50% formic acid effectively killed mites in capped worker brood and on adult bees without harming queens or uncapped brood. Neither acetic acid nor a combined treatment of formic and acetic acids applied to the West Virginia formic acid fumigator was as effective as formic acid alone in controlling varroa mites. In addition, none of the treatments tested in late summer had an effect on the late-season prevalence of deformed wing virus. The short-term formic acid treatment killed >60% of varroa mites in capped worker brood; thus, it is a promising tool for beekeepers, especially when such treatments are necessary during the nectar flow.

Cereal yellow dwarf virus -RPV (CYDV-RPV) is transmitted specifically by the aphids Rhopalosiphum padi and Schizaphis graminum in a circulative nonpropagative manner. The high level of vector specificity results from the vector aphids having the functional components of the receptor-mediated endocytotic pathways to allow virus to transverse the gut and salivary tissues. Studies of F 2 progeny from crosses of vector and nonvector genotypes of S . graminum showed that virus transmission efficiency is a heritable trait regulated by multiple genes acting in an additive fashion and that gut- and salivary gland-associated factors are not genetically linked. Utilizing two-dimensional difference gel electrophoresis to compare the proteomes of vector and nonvector parental and F 2 genotypes, four aphid proteins (S4, S8, S29, and S405) were specifically associated with the ability of S . graminum to transmit CYDV-RPV. The four proteins were coimmunoprecipitated with purified RPV, indicating that the aphid proteins are capable of binding to virus. Analysis by mass spectrometry identified S4 as a luciferase and S29 as a cyclophilin, both of which have been implicated in macromolecular transport. Proteins S8 and S405 were not identified from available databases. Study of this unique genetic system coupled with proteomic analysis indicated that these four virus-binding aphid proteins were specifically inherited and conserved in different generations of vector genotypes and suggests that they play a major role in regulating polerovirus transmission.

In colony collapse disorder (CCD), honey bee colonies inexplicably lose their workers. CCD has resulted in a loss of 50 to 90% of colonies in beekeeping operations across the United States. The observation that irradiated combs from affected colonies can be repopulated with naive bees suggests that infection may contribute to CCD. We used an unbiased metagenomic approach to survey microflora in CCD hives, normal hives, and imported royal jelly. Candidate pathogens were screened for significance of association with CCD by the examination of samples collected from several sites over a period of 3 years. One organism, Israeli acute paralysis virus of bees, was strongly correlated with CCD.

Reproduction of entomopathogenic nematodes requires that they escape recognition by a host’s immune system or that they have mechanisms to escape encapsulation and melanization. We investigated the immune responses of larvae for the greater wax moth (Galleria mellonella), tobacco hornworm (Manduca sexta), Japanese beetle (Popillia japonica), northern masked chafer (Cyclocephala borealis), oriental beetle (Exomala orientalis) and adult house crickets (Acheta domesticus), challenged with infective juveniles from different species and strains of entomopathogenic nematodes. The in vivo immune responses of hosts were correlated with nematode specificity and survival found by infection assays. In P. japonica, 45% of injected infective juveniles from Steinernema glaseri NC strain survived; whereas the hemocytes from the beetle strongly encapsulated and melanized the Heterorhabditis bacteriophora HP88 strain, S. glaseri FL strain, Steinernema scarabaei and Steinernema feltiae. Overall, H. bacteriophora was intensively melanized in resistant insect species (E. orientalis, P. japonica and C. borealis) and had the least ability to escape the host immune response. Steinernema glaseri NC strain suppressed the immune responses in susceptible hosts (M. sexta, E. orientalis and P. japonica), whereas S. glaseri FL strain was less successful. Using an in vitro assay, we found that hemocytes from G. mellonella, P. japonica, M. sexta and A. domestica recognized both nematode species quickly. However, many S. glaseri in M. sexta and H. bacteriophora in G. mellonella escaped from hemocyte encapsulation by 24 h. These data indicate that, while host recognition underlies some of the differences between resistant and susceptible host species, escape from encapsulation following recognition can also allow successful infection. Co-injected surface-coat proteins from S. glaseri did not protect H. bacteriophora in M. sexta but did protect H. bacteriophora in E. orientalis larva; therefore, surface coat proteins do not universally convey host susceptibility. Comparisons of surface coat proteins by native and SDS–PAGE demonstrated different protein compositions between H. bacteriophora and S. glaseri and between the two strains of S. glaseri.

The cDNA sequence of a salivary lysozyme in Helicoverpa zea (Lepidoptera: Noctuidae) was determined. The full-length cDNA is 1,032 bp, and it encodes a protein of 142 amino acids. This lysozyme has 90% identity with Heliothis virescens lysozyme and 76% identity with Manduca sexta lysozyme. There is a signal peptide of 20 amino acids at the N-terminus. The mature protein is about 14.4 kDa without the signal peptide. The pI value is greater than 9.5 as determined by isoelectric focusing. From genomic DNA, two introns and three exons were within the open reading frame (ORF). Southern blot analysis indicated that it is a single-copy gene. A time-course study revealed that the H. zea lysozyme gene was differentially expressed in the labial glands during the development of fifth-instar larvae, with the peak level of lysozyme mRNA being detected on day 1. Dot blot analysis showed different levels of H. zea lysozyme expression when the caterpillars fed on different plants. Further, the H. zea lysozyme could be detected with antibodies raised against the M. sexta lysozyme, and it was one of the most abundant secreted proteins in saliva collected directly from the caterpillar's spinneret. The potential role of the lysozyme on host plants in mediating susceptibility to bacterial disease is discussed in the context of tritrophic interactions.

Honey bee (Apis mellifera) colony losses pose a severe risk to the food chain. The IAPV (Israeli acute paralysis virus) was correlated with CCD, a particular case of colony collapse. Honey bees severely infected with IAPV show shivering wings that progress to paralysis and subsequent death. Bee viruses, including IAPV, are widely present in honey bee colonies but often there are no pathological symptoms. Infestation of the beehive with Varroa mites or exposure to stress factors leads to significant increase in viral titers and fatal infections. We hypothesized that the honey bee is regulating/controlling IAPV and viral infections in asymptomatic infections and this control is broken through "stress" leading to acute infections and/or CCD. Our aims were: 1. To discover genetic changes in IAPV that may affect tissue tropism in the host, and/or virus infectivity and pathogenicity. 2. To elucidate mechanisms used by the host to regulate/ manage the IAPV-infection in vivo and in vitro. To achieve the above objectives we first studied stress-induced virus activation. Our data indicated that some pesticides, including myclobutanil, chlorothalonil and fluvalinate, result in amplified viral titers when bees are exposed at sub lethal levels by a single feeding. Analysis of the level of immune-related bee genes indicated that CCD-colonies exhibit altered and weaker immune responses than healthy colonies. Given the important role of viral RNA interference (RNAi) in combating viral infections we investigated if CCD-colonies were able to elicit this particular antiviral response. Deep-sequencing analysis of samples from CCD-colonies from US and Israel revealed high frequency of small interfering RNAs (siRNA) perfectly matching IAPV, Kashmir bee virus and Deformed wing virus genomes. Israeli colonies showed high titers of IAPV and a conserved RNAi pattern of targeting the viral genome .Our findings were further supported by analysis of samples from colonies experimentally infected with IAPV. Following for the first time the dynamics of IAPV infection in a group of CCD colonies that we rescued from collapse, we found that IAPV conserves its potential to act as one lethal, infectious factor and that its continuous replication in CCD colonies deeply affects their health and survival. Ours is the first report on the dominant role of IAPV in CCD-colonies outside from the US under natural conditions. We concluded that CCD-colonies do exhibit a regular siRNA response that is specific against predominant viruses associated with colony losses and other immune pathways may account for their weak immune response towards virus infection. Our findings: 1. Reveal that preventive measures should be taken by the beekeepers to avoid insecticide-based stress induction of viral infections as well as to manage CCD colonies as a source of highly infectious viruses such as IAPV. 2. Contribute to identify honey bee mechanisms involved in managing viral infections.

Anacardic acids, alkyl phenolic acids excreted by tall glandular trichomes of the garden geranium, Pelargonium xhortorum, confer small-pest resistance. Up to 90% of the trichome exudate from mite-resistant P. xhortorum inbreds consists of an unusual anacardic acid with an unsaturated omega-5 (ω5) alkyl chain. As fatty acids are biochemical precursors to anacardic acids, we examined by GC the fatty acid composition of leaves, pedicels, petals, sepals, mature seeds, and glandular trichomes from pest-resistant and pest-susceptible Pelargonium inbred lines to determine the localization of ω5-fatty acids within plant tissues. The fatty acid composition of lipid classes (galactolipids, phospholipids, and neutral lipids) extracted from glandular trichomes from mite-resistant pedicels were also examined. ω5-Fatty acids (16:1Δ11 and 18:1Δ13) were found only in the glandular trichomes from pest-resistant geraniums (27.2% in trichomes of pedicels) and were localized predominantly in the phospho- and galactolipids (phosphatidylinositol, 25.9%; phosphatidylcholine, 18.2%; monogalactosyldiglyceride, 15.5%; and diglactosyldiglyceride, 14.0%).

Diploid zonal geraniums (Pelargonium ×hortorum) are able to resist attack by small arthropod pests such as the two-spotted spider mite (Tetranychus urticae Koch) when exudate produced by tall glandular trichomes contains a high percentage of ω5-unsaturated anacardic acids. Trichomes of susceptible plants exude primarily saturated anacardic acids. Inbred mite-resistant and -susceptible geraniums were reciprocally crossed and the F1, F2, and backcross generations were examined for anacardic acid composition and trichome density. Selected F2 plants were bioassayed for resistance to two-spotted spider mites. High concentrations of ω5-unsaturated anacardic acids in resistant plants are conditioned by a single dominant allele. We propose that inheritance of tall glandular trichome density can be controlled by a small number of loci (possibly as few as one) exhibiting codominance. F2, with low densities of tall glandular trichomes and producing ω5-unsaturated anacardic acids, displayed effective resistance to two-spotted spider mites as measured by mite mortality and fecundity. A genetic model for the biosynthesis of anacardic acids is proposed.

The zonal geranium (Pelargonium xhortorum) possesses tall glandular trichomes that secrete anacardic acids, a viscous, sticky exudate which has been suggested as the primary mechanism in two-spotted spider mite (Tetranychus urticae Koch) resistance. A new bioassay was developed using small Plexiglas® cylinders as chambers for evaluating the resistance of geranium leaves to the two-spotted spider mite. This bioassay was easy to prepare, required only 24 hours to conduct, exhibited no problems with desiccation, condensation, or mite accountability, and yielded reproducible results. This bioassay was then used to study the regeneration of resistance of attached geranium leaves after they were made mite-susceptible by removing the excreted anacardic acids with water. Washed leaves regained full resistance after 14 days.

Anacardic acids, a class of secondary compounds derived from fatty acids, are found in a variety of dicotyledonous families. Pest resistance (e.g., spider mites and aphids) in Pelargonium xhortorum (geranium) is associated with high levels (approximately 81%) of unsaturated 22:1 omega 5 and 24:1 omega 5 anacardic acids in the glandular trichome exudate. A single dominant locus controls the production of these omega 5 anacardic acids, which arise from novel 16:1 delta 11 and 18:1 delta 13 fatty acids. We describe the isolation and characterization of a cDNA encoding a unique delta 9 14:0-acyl carrier protein fatty acid desaturase. Several lines of evidence indicated that expression of this desaturase leads to the production of the omega 5 anacardic acids involved in pest resistance. First, its expression was found in pest-resistant, but not suspectible, plants and its expression followed the production of the omega 5 anacardic acids in segregating populations. Second, its expression and the occurrence of the novel 16:1 delta 11 and 18:1 delta 13 fatty acids and the omega 5 anacardic acids were specific to tall glandular trichomes. Third, assays of the recombinant protein demonstrated that this desaturase produced the 14:1 delta 9 fatty acid precursor to the novel 16:1 delta 11 and 18:1 delta 13 fatty acids. Based on our genetic and biochemical studies, we conclude that expression of this delta 9 14:0-ACP desaturase gene is required for the production of omega 5 anacardic acids that have been shown to be necessary for pest resistance in geranium.

A standard buffer (5 mM phosphate at pH 7) which is used to extract protein from insect eggs provided complete protein solubility for eggs from three of four tree-feeding lepidopteran species: obliquebanded leaf roller (Choristoneura rosaceana), forest tent caterpillar (Malacosoma disstria), and the eastern tent caterpillar (Malacosoma americanum). Under the same extraction protocol, egg proteins from the gypsy moth (Lymantria dispar), remained nearly insoluble. An array of methods typically used to solubilize insect egg proteins were tried and all but the most denaturing (2% SDS) were ineffective. Extraction buffers with typically high pH values were then evaluated. The results indicated that 1) solubility of gypsy moth egg proteins was pH dependent, and full solubility of most egg proteins required the extraction buffer to have a pH of 12 or more prior to the addition of eggs. We also determined that 2) the gypsy moth egg has a buffering capacity which must be surpassed for complete protein extraction, 3) low salt/high pH buffers gave slightly higher total protein values than did high salt/high pH buffers, 4) parental nutritional history (host species utilized) and egg developmental state (pre-embryonatedvs postembryonated/pre-hatch) were unrelated to the requirements for complete egg protein solubilization, and 5) the presence of soluble phenolics, compounds that have the potential to bind to protein and cause insolubility, was confirmed for the gypsy moth egg with 2-D paper chromatography and several other tests. Based on these results, we present a hypothesis about the cause of egg protein insolubility in the gypsy moth.

Composition of anacardic acids (phenolic acids known to be associated with small pest resistance in Pelargonium ×hortorum) was examined in 13 diploid and 25 tetraploid cultivars by high-performance liquid chromatography (HPLC). The presence of an unusual desaturation (omega (ω)-5) in the alkyl tail of anacardic acids present only in glandular trichome exudate of pest-resistant diploid inbred lines had previously been associated with a sticky-trap pest-resistance phenomenon. In this study, we examine Pelargonium cultivars for variability in anacardic acid composition to assess the distribution of ω5 desaturation among commercial cultivars, to determine possible interactions between ω5 desaturation and other plant desaturation mechanisms, and to examine the possible impact of ploidy on ω5 desaturation. An unsaturation index (UI) is derived to compare exudates differing widely in composition yet which may provide a similarly effective sticky-trap pest-resistance mechanism based on exudate viscosity. ω-5 Anacardic acids were observed in the glandular trichome exudate of all 38 commercial cultivars examined. No diploid cultivar produced ω5- and ω9- anacardic acids, although the simultaneous production of ω5 and ω9- anacardic acids was observed in three tetraploid cultivars. Total ω5- anacardic acids comprised from 42.4% (tetraploid cultivar Perlenkette-syn. Snowhite, Weiss) to 86.8% (tetraploid cultivar Amanda). Commercial P. ×domesticum cultivars had no ω5 anacardic acids. UIs ranged from 60.9 (tetraploid cultivar Dixieland) to 103.4 (diploid cultivar Pinto White). In contrast, anacardic acids collected from a pest-susceptible inbred line contained no ω5- anacardic acids and had a UI of 38.7. No significant differences among ploidy levels were observed for UIs or for most specific anacardic acid components, with the exception of 24:1 ω5- anacardic acid, in which the mean diploid value (32.1%) was significantly higher than that of the mean tetraploid value (27.6%). We conclude that ω5- anacardic acid production occurs in all Pelargonium cultivars observed and that these cultivars are predicted to exhibit resistance to small arthropod pests. Significant genetic variability in specific anacardic acid composition appears to exist among Pelargonium cultivars, suggesting that breeding for pest resistance can be readily monitored by HPLC of anacardic acids.

Biochemical and morphological components of 16 Pelargonium species and the P. ×hortorum interspecific complex were examined. Inflorescences and leaves of each species were analyzed for anacardic acids and the presence of glandular trichomes. Three species of the section Ciconium, P. acetosum, P. frutetorum, and P. inquinans, produced anacardic acids in association with glandular trichomes. only P. inquinans and P. frutetorum contained ω5- anacardic acids. An evolutionary model for the origin of anacardic acids and ω5- desaturation is proposed.

Recent ecological research has revealed that environmental factors can strongly affect insect immunity and influence the outcome of host-parasite interactions. To date, however, most studies examining immune function in mosquitoes have ignored environmental variability. We argue that one such environmental variable, temperature, influences both vector immunity and the parasite itself. As temperatures in the field can vary greatly from the ambient temperature in the laboratory, it will be essential to take temperature into account when studying vector immunology.

The isolation of high-quality RNA from various tissues (leaves, pedicels, glandular trichomes) of garden geranium (Pelargonium xhortorum) using various published methods is difficult due to numerous oxidizing compounds. A new RNA extraction method was developed through the combination and modification of two separate procedures (Rochester et al., 1986; Manning 1991). In addition to geranium tissues, this method is successful when used with other recalcitrant tissues such as mature needles of white pine (Pinus strobus) and mature leaves of poinsettia (Euphorbia pulcherrima). RNA quality was judged by spectrophotometric readings, denaturing agarose gels, and successful reverse transcription.

FAD-Glucose dehydrogenase (GLD) (EC 1.1.99.10) was assayed during the encapsulation response against abiotic implants (sterile latex) in fourth instar Manduca sexta larvae. No GLD activity was detected in fresh hemolymph or hemocytes of control or sham-treated larvae. Two types of responses occurred at 0.5 and 3 h after implantation. In the majority of larvae, only the encapsulation had high levels of GLD specific activity. In the second response, low levels of GLD specific activity were detected in hemocytes and cell-free hemolymph but not in the encapsulation. By 24 h after implantation, GLD activity was detected in the encapsulation tissue of all larvae. Levels of GLD specific activity did not significantly differ over time (0.5–72 h) during encapsulation. Freezing cell-free hemolymph from control larvae activated otherwise inactive hemolymph stores of GLD. In vitro experiments and histochemical staining demonstrated that GLD activity was localized in granules of plasmatocytes adhered to coverslips. Granules of both plasmatocytes and granulocytes contained GLD protein as detected by immunohistochemistry. Based on previous characterization of GLD, we hypothesize that GLD participates in strengthening the encapsulation and in the killing reaction, via reaction with quinones generated by phenoloxidase and subsequent production of free radicals.

Colony collapse disorder (CCD) is characterized by the unexplained losses of large numbers of adult worker bees (Apis mellifera) from apparently healthy colonies. Although infections, toxins, and other stressors have been associated with the onset of CCD, the pathogenesis of this disorder remains obscure. Recently, a proteomics study implicated a double-stranded DNA virus, invertebrate iridescent virus (Family Iridoviridae) along with a microsporidium (Nosema sp.) as the cause of CCD. We tested the validity of this relationship using two independent methods: (i) we surveyed healthy and CCD colonies from the United States and Israel for the presence of members of the Iridovirus genus and (ii) we reanalyzed metagenomics data previously generated from RNA pools of CCD colonies for the presence of Iridovirus-like sequences. Neither analysis revealed any evidence to suggest the presence of an Iridovirus in healthy or CCD colonies.

Resources supplied by mothers to offspring through the egg are known to significantly influence offspring life history traits in the gypsy moth, Lynzuntriu dispur. The purpose of this research was to determine the relative contribution of genetics (based on familial contribution) and the nutritional environment of the parents to the mean and variance of resources supplied to the eggs. Vitellogin, the dominant egg storage protein in the gypsy moth. was selected as the focus of the study. The amount of vitellogin in individual eggs from 48 mothers reared on one of four host species, quaking aspen, chestnut oak. red oak, or pitch pine was quantified with an immunoassay. Results of a nested analysis of variance show that both genetics and parental nutritional experience make significant contributions to egg vitellogin levels. When parents were reared on quaking aspen, vitellogin levels were highest and the expression of familial variation was greatest. This study shows that polyphagy can amplify phenotypic variance in reproductive traits through the interaction between genotype and nutritional environment. To the extent that egg resources influence offspring vigor, the fitness of offspring can include a time-lagged component which arises from the interaction between the parental genotype and the parental environment. The time-lagged expression of such a maternal trait is capable of influencing the rate and direction of character evolution and the stability of local population dynamics.

The barley cultivar Barsoy (Hordeum vulgare L.) contains an unusual 13.1 kb cellular double-stranded ribonucleic acid (dsRNA). This non-viral dsRNA is efficiently transmitted by both egg and pollen in crosses with dsRNA-free cultivars. The pedigree of Barsoy was reconstructed and tested for dsRNA occurrence and sequence similarity in an effort to determine the origin and distribution of this molecule. The ancestors of Barsoy barley were tested for the presence of dsRNA by CF-11 cellulose chromatography and agarose electrophoresis. Of 31 ancestral cultivars tested, eight contained a dsRNA with electrophoretic properties similar to the one from Barsoy. A cDNA probe made with Barsoy dsRNA revealed that the dsRNAs isolated from related cultivars have similar nucleotide sequences. The dsRNA was found in four out of five of the oldest ancestors of Barsoy tested. One of these cultivars, Nakano Wase, was introduced to the US from Japan for breeding purposes in 1911. These results support a theory for vertical transmission of the dsRNA from generation to generation and suggest that the molecule has been maintained in barley cultivars for over 80 years.

Although overall pollinator populations have declined over the last couple of decades, the honey bee (Apis mellifera) malady, colony collapse disorder (CCD), has caused major concern in the agricultural community. Among honey bee pathogens, RNA viruses are emerging as a serious threat and are suspected as major contributors to CCD. Recent detection of these viral species in bumble bees suggests a possible wider environmental spread of these viruses with potential broader impact. It is therefore vital to study the ecology and epidemiology of these viruses in the hymenopteran pollinator community as a whole. We studied the viral distribution in honey bees, in their pollen loads, and in other non-Apis hymenopteran pollinators collected from flowering plants in Pennsylvania, New York, and Illinois in the United States. Viruses in the samples were detected using reverse transcriptase-PCR and confirmed by sequencing. For the first time, we report the molecular detection of picorna-like RNA viruses (deformed wing virus, sacbrood virus and black queen cell virus) in pollen pellets collected directly from forager bees. Pollen pellets from several uninfected forager bees were detected with virus, indicating that pollen itself may harbor viruses. The viruses in the pollen and honey stored in the hive were demonstrated to be infective, with the queen becoming infected and laying infected eggs after these virus-contaminated foods were given to virus-free colonies. These viruses were detected in eleven other non-Apis hymenopteran species, ranging from many solitary bees to bumble bees and wasps. This finding further expands the viral host range and implies a possible deeper impact on the health of our ecosystem. Phylogenetic analyses support that these viruses are disseminating freely among the pollinators via the flower pollen itself. Notably, in cases where honey bee apiaries affected by CCD harbored honey bees with Israeli Acute Paralysis virus (IAPV), nearby non-Apis hymenopteran pollinators also had IAPV, while those near apiaries without IAPV did not. In containment greenhouse experiments, IAPV moved from infected honey bees to bumble bees and from infected bumble bees to honey bees within a week, demonstrating that the viruses could be transmitted from one species to another. This study adds to our present understanding of virus epidemiology and may help explain bee disease patterns and pollinator population decline in general.

Lymantria dispar has a long historical association with the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV), which is one of the primary population regulators of L. dispar in the field. However, host larvae exhibit strong developmental resistance to fatal infection by LdMNPV; the LD50 in newly moulted fourth instars is 18-fold lower than in the middle of the instar (48–72 h post-moult). Using a recombinant of LdMNPV expressing lacZ, we examined the key steps of pathogenesis in the host to explore mechanisms of developmental resistance. At the midgut level, we observed reduced primary midgut infections in mid-fourth instars, indicating increased sloughing of infected cells. Additional barriers were observed as the virus escaped the midgut. Mid-fourth instars had higher numbers of melanized foci of infection associated with the midgut, apoptotic tracheal epidermal cells and haemocytes, and reduced numbers of infected haemocytes later in infection. Our results show that the co-evolutionary relationship between L. dispar and LdMNPV has resulted in both midgut-based and systemic antiviral defences and that these defences are age-dependent within the instar. This age-related susceptibility may contribute to how the virus is maintained in nature and could influence management of L. dispar by using the virus.

How the innate immune system functions to defend insects from viruses is an emerging field of study. We examined the impact of melanized encapsulation, a component of innate immunity that integrates both cellular and humoral immune responses, on the success of the baculovirus Lymantria dispar multiple nucleocapsid nucleopolyhedrovirus (LdMNPV) in its host L. dispar . L. dispar exhibits midgut-based and systemic, age-dependent resistance to LdMNPV within the fourth instar; the LD 50 in newly molted larvae is approximately 18-fold lower than in mid-instar larvae (48–72 h post-molt). We examined the role of the immune system in systemic resistance by measuring differences in hemocyte immunoresponsiveness to foreign targets, hemolymph phenoloxidase (PO) and FAD-glucose dehydrogenase (GLD) activities, and melanization of infected tissue culture cells. Mid-instar larvae showed a higher degree of hemocyte immunoresponsiveness, greater potential PO activity (pro-PO) at the time the virus is escaping the midgut to enter the hemocoel (72 h post-inoculation), greater GLD activity, and more targeted melanization of infected tissue, which correlate with reduced viral success in the host. These findings support the hypothesis that innate immune responses can play an important role in anti-viral defenses against baculoviruses and that the success of these defenses can be age-dependent.

A physical method is described for the rapid isolation of plant trichomes, with emphasis on stalked glandular types. The technique involved breaking frozen trichomes with powdered dry ice and collection of glandular heads by sieving from larger tissue fragments. This method was applied to several plants that bear similar stalked trichomes: geranium (Pelargonium), potato (Solanum tuberosum), tomato (Lycopersicon esculentum), squash (Cucurbita pepo), and velvetleaf (Abutilon theophrasti). The tissue preparation was of sufficient quality without further purification for biochemical and molecular studies. The preparation maintained the biochemical integrity of the trichomes for active enzymes and usable nucleic acids. A large quantity of tissue can be harvested; for example, 351 milligrams dry weight of glandular trichomes were harvested from geranium pedicels in 12 hours. The utility of the technique was demonstrated by examining the fatty acid composition of tall glandular trichomes of geraniums, Pelargonium xhortorum L.H. Bailey. These purified cells contained high concentrations of unusual omega5-unsaturated fatty acids, proportionally 23.4% of total fatty acids in the trichomes. When the trichomes were removed, the supporting tissue contained no omega5-fatty acids, thereby unequivocally localizing omega5-fatty acids to the trichomes. Because omega5-fatty acids are unique precursors for the biosynthesis of omega5-anacardic acids, we conclude that anacardic acid synthesis must occur in the glandular trichomes.

The article discusses colony collapse disorder, which has killed large numbers of honeybees in the U.S. as of 2009. The disappearance of honeybees as a result of the disorder is described, noting the potential for a significant impact upon around 100 crops grown in the U.S. that require pollination. Possible solutions are also discussed, including proper hive hygiene and the development of antiviral drugs. INSETS: A COUNTRYWIDE SCOURGE;Many Suspects, No Convictions Yet;A BEE MEDICINE?;A BEE MEDICINE?

Background: Over the last two winters, there have been large-scale, unexplained losses of managed honey bee (Apis mellifera L.) colonies in the United States. In the absence of a known cause, this syndrome was named Colony Collapse Disorder (CCD) because the main trait was a rapid loss of adult worker bees. We initiated a descriptive epizootiological study in order to better characterize CCD and compare risk factor exposure between populations afflicted by and not afflicted by CCD. Methods and Principal Findings: Of 61 quantified variables (including adult bee physiology, pathogen loads, and pesticide levels), no single measure emerged as a most-likely cause of CCD. Bees in CCD colonies had higher pathogen loads and were co-infected with a greater number of pathogens than control populations, suggesting either an increased exposure to pathogens or a reduced resistance of bees toward pathogens. Levels of the synthetic acaricide coumaphos (used by beekeepers to control the parasitic mite Varroa destructor) were higher in control colonies than CCD-affected colonies. Conclusions/Significance: This is the first comprehensive survey of CCD-affected bee populations that suggests CCD involves an interaction between pathogens and other stress factors. We present evidence that this condition is contagious or the result of exposure to a common risk factor. Potentially important areas for future hypothesis-driven research, including the possible legacy effect of mite parasitism and the role of honey bee resistance to pesticides, are highlighted.

C22:1 omega 5-anacardic acid was found to be a good inhibitor of both potato lipoxygenase and ovine prostaglandin endoperoxide synthase with approximate IC50's of 6 and 27 microM, respectively. Very similar inhibition was seen with the crude exudate, rich in omega 5-anacardic acids, from glandular trichomes of an arthropod-resistant strain of geranium, Pelargonium xhortorum. The saturated anacardic acid (C22:0 sat), abundant in the trichome exudate of susceptible strains, was nearly as inhibitory toward both prostaglandin endoperoxide synthase and lipoxygenase as the omega 5-unsaturated compound. However, the dimethyl derivative of C22:1 omega 5-anacardic acid was a poor inhibitor of prostaglandin endoperoxide synthase and caused only moderate (32%) inhibition of lipoxygenase even at 135 microM. The possible role of prostaglandin endoperoxide synthase and lipoxygenase inhibition in the enhanced pest resistance of geraniums which produce the omega 5-AnAs is discussed.

Surface coat proteins (SCPs) of entomopathogenic nematodes are implicated in the suppression/evasion of host immune responses, which is required for successful host colonization. Steinernema glaseri NC strain SCPs suppressed immune responses in oriental beetle larvae (Exomala orientalis), a susceptible host for S. glaseri, in a dosage-dependent manner, thus protecting Heterorhabditis bacteriophora from being killed in the same host. Melanization of H. bacteriophora decreased from 92 ± 5% in the untreated check to 1 ± 3% when protected by injection of 230 ng of S. glaseri SCPs. As the SCPs dosage increased, freely moving H. bacteriophora increased from 3 ± 4% in the untreated group to 57 ± 15% with an SCPs dose of 940 ng. At 2 h and in the absence of SCPs, 8% and 11% of hemocytes of E. orientalis were stained by propidium iodide and Hoechst, respectively. When exposed to 300 ng/l SCPs, 70% and 96% were stained, respectively. At 6 h, propidium iodide stained 37% and 92% of the hemocytes without and with SCPs, respectively. In contrast, more than 90% of the cells were stained by Hoechst with or without SCPs. As native proteins, two isolated S. glaseri SCPs had an immunosuppressive effect; they were each composed of 38 kDa (PI = 4.6) and 56 kDa (PI = 3.6) subunits. SCP peptides were sequenced using LC-MS/MS and the mass fingerprints obtained with MALDI-TOF-MS; there were no significant matches found in peptide databases, which suggests that the SCPs studied are novel proteins. Twelve cDNA sequences were derived based on short peptides and 7 of them had no significant match against the Caenorhabditis elegans protein database. One of the cDNA matched an unknown C. elegans protein and the remaining 4 cDNAs matched proteins of C. elegans and Brugia malayi.

The Gld gene of Drosophila melanogaster is transiently expressed during every stage of development. The temporal pattern of Gld expression is highly correlated with that of ecdysteroids. Exogeneous treatment of third instar larvae with 20-hydroxyecdysone induces the accumulation of Gld mRNA in the hypoderm and anterior spiracular gland cells. During metamorphosis Gld is expressed in a variety of tissues derived from the ectoderm. In the developing reproductive tract, Gld mRNA accumulates in the female spermathecae and oviduct and in the male ejaculatory duct and ejaculatory bulb. These four organs are derived from closely related cell lineages in the genital imaginal disc. Since the expression of Gld is not required for the development of these reproductive structures, this spatial pattern of expression is most likely a fortuitous consequence of a shared regulatory factor in this cell lineage. At the adult stage a high level of the Gld mRNA is only observed in the male ejaculatory duct.

Background The GMC oxidoreductases comprise a large family of diverse FAD enzymes that share a homologous backbone. The relationship and origin of the GMC oxidoreductase genes, however, was unknown. Recent sequencing of entire genomes has allowed for the evolutionary analysis of the GMC oxidoreductase family. Results Although genes that encode enzyme families are rarely linked in higher eukaryotes, we discovered that the majority of the GMC oxidoreductase genes in the fruit fly (D. melanogaster), mosquito (A. gambiae), honeybee (A. mellifera), and flour beetle (T. castaneum) are located in a highly conserved cluster contained within a large intron of the flotillin-2 (Flo-2) gene. In contrast, the genomes of vertebrates and the nematode C. elegans contain few GMC genes and lack a GMC cluster, suggesting that the GMC cluster and the function of its resident genes are unique to insects or arthropods. We found that the development patterns of expression of the GMC cluster genes are highly complex. Among the GMC oxidoreductases located outside of the GMC gene cluster, the identities of two related enzymes, glucose dehydrogenase (GLD) and glucose oxidase (GOX), are known, and they play major roles in development and immunity. We have discovered that several additional GLD and GOX homologues exist in insects but are remotely similar to fungal GOX. Conclusion We speculate that the GMC oxidoreductase cluster has been conserved to coordinately regulate these genes for a common developmental or physiological function related to ecdysteroid metabolism. Furthermore, we propose that the GMC gene cluster may be the birthplace of the insect GMC oxidoreductase genes. Through tandem duplication and divergence within the cluster, new GMC genes evolved. Some of the GMC genes have been retained in the cluster for hundreds of millions of years while others might have transposed to other regions of the genome. Consistent with this hypothesis, our analysis indicates that insect GOX and GLD arose from a different ancestral GMC gene than that of fungal GOX.

Varroa mites ( Varroa destructor ) are ectoparasites of honey bees ( Apis mellifera ) and cause serious damage to bee colonies. The mechanism of how varroa mites kill honey bees remains unclear. We have addressed the effects of the mites on bee immunity and the replication of a picorna-like virus, the deformed wing virus (DWV). The expression of genes encoding three antimicrobial peptides (abaecin, defensin, and hymenoptaecin) and four immunity-related enzymes (phenol oxidase, glucose dehydrogenase, glucose oxidase, and lysozyme) were used as markers to measure the difference in the immune response. We have demonstrated an example of an ectoparasite immunosuppressing its invertebrate host with the evidence that parasitization significantly suppressed expression of these immunity-related genes. Given that ticks immunosuppress their vertebrate hosts, our finding indicates that immunosuppression of hosts may be a common phenomenon in the interaction and coevolution between ectoparasites and their vertebrate and invertebrate hosts. DWV viral titers were significantly negatively correlated with the expression levels of the immunity-related enzymes. All bees had detectable DWV. Mite-infested pupae developed into adults with either normal or deformed wings. All of the deformed-wing bees were greatly infected by DWV (≈10 6 times higher than varroa-infested but normal-winged bees). Injection with heat-killed bacteria dramatically promoted DWV titers (10 5 times in 10 h) in the mite-infested, normal-winged bees to levels similar to those found in mite-infested, deformed-wing bees. Varroa mites may cause the serious demise of honey bees by suppressing bee immunity and by boosting the amplification of DWV in bees exposed to microbes.

To determine the roles of varroa mites in activating or vectoring viral infections, we performed quantitative comparison of viral infections between bees with and without mites by dot blot analysis and enzyme-linked immunosorbent assay (ELISA). Under natural and artificial mite infestations, bee pupae contained significantly higher levels of Kashmir bee virus (KBV) and deformed wing virus (DWV) RNAs and KBV structural proteins than mite-free pupae. Moreover, in mite-infested bee pupae, DWV had amplified to extremely high titers with viral genomic RNA being clearly visible after separation of total bee RNA in agarose gels. Linear regression analysis has shown a positive correlation between the number of mites introduced and the levels of viral RNAs. The detection of viral RNAs in the nymph and adult mites underline the possible role of varroa in virus transmission. However, most groups of virus-free adult mites (9/12) were associated with bee pupae heavily infected by viruses, suggesting that the elevated viral titers in mite-infested pupae more likely resulted from activated viral replication. Based on these observations and our concurrent research demonstrating suppressed immune responses in bees infested with mites, we propose that parasitization by varroa suppresses the immunity of honey bees, leading to activation of persistent, latent viral infection.

Polydnaviruses (PDV) are obligate mutualistic symbionts found in association with some groups of parasitic Hymenoptera. In these groups, they suppress the immune response of the parasitoid's host and are required for successful parasitoid reproduction. Several PDV effects have been described in different experimental systems, but no clear picture of PDV mode of immunosuppression has emerged. No study to date has directly tested if PDV modes of action are evolutionarily conserved or divergent among parasitoid taxa within the Ichneumonoidea. We hypothesize the divergence in PDV mode of immunosuppression can be detected by identifying points of divergence in the immune response of different host species to PDV from one parasitoid species. This study tests the effects of purified PDV from Cotesia congregata on the immune response of three larval lepidopteran species that naturally are hosts of parasitoid species that differ in taxonomic relatedness to C. congregata. Here we demonstrate that despite associations with distantly related parasitoids (Ichneumonidae and Braconidae), Manduca sexta and Heliothis virescens showed similar patterns of increased glucose dehydrogenase (GLD) activity, suppressed cellular encapsulation in vitro, and increased time to pupation. In contrast, Lymantria dispar showed no response to C. congregata PDV across any of the parameters measured, even though it has an evolutionary association with several parasitoids closely related to C. congregata and within the Microgastrinae. The PDV immunosuppression in H. virescens and M. sexta does not correlate with host molecular phylogeny either. The suborganismal effects shown in M. sexta and H. virescens translated into significantly reduced pupation success in M. sexta only. Results demonstrate that while some PDV modes of immunosuppression in hosts may be divergent, others may be conserved across broad host groups.

Two proteins (SaM35 and SaM50) isolated from head tissues of the aphid vector, Sitobion avenae, were identified as potential receptors for barley yellow dwarf virus MAV isolate (Luteoviridae) based on MAV virus overlay assays and immunoblots of urea SDS 2-D gels. An anti-idiotypic antibody (MAV4 anti-ID) that mimics an epitope on MAV virions and competes with MAV in antibody binding assays also bound to SaM50 and SaM35 and to six additional proteins including a GroEL homolog. No MAV-binding proteins were detected from the nonvector aphid, Rhopalosiphum maidis, although MAV4 anti-ID did react with four proteins from R. maidis. It is hypothesized that SaM35 and SaM50 may be MAV receptors involved in MAV transmission based on their high affinity for MAV and their unique association with the vector, S. avenae. The additional aphid proteins binding the MAV4 anti-ID may represent less specific virus-binding proteins facilitating transmission through different aphid tissues.

The importance of honey bees to the world economy far surpasses their contribution in terms of honey production; they are responsible for up to 30% of the world's food production through pollination of crops. Since fall 2006, honey bees in the U.S. have faced a serious population decline, due in part to a phenomenon called Colony Collapse Disorder (CCD), which is a disease syndrome that is likely caused by several factors. Data from an initial study in which investigators compared pathogens in honey bees affected by CCD suggested a putative role for Israeli Acute Paralysis Virus, IAPV. This is a single stranded RNA virus with no DNA stage placed taxonomically within the family Dicistroviridae. Although subsequent studies have failed to find IAPV in all CCD diagnosed colonies, IAPV has been shown to cause honey bee mortality. RNA interference technology (RNAi) has been used successfully to silence endogenous insect (including honey bee) genes both by injection and feeding. Moreover, RNAi was shown to prevent bees from succumbing to infection from IAPV under laboratory conditions. In the current study IAPV specific homologous dsRNA was used in the field, under natural beekeeping conditions in order to prevent mortality and improve the overall health of bees infected with IAPV. This controlled study included a total of 160 honey bee hives in two discrete climates, seasons and geographical locations (Florida and Pennsylvania). To our knowledge, this is the first successful large-scale real world use of RNAi for disease control., Author Summary High rates of honey bee mortality continue to threaten food security and apicultural industries worldwide. At least some of these losses are likely the result of viral infections. Application of RNAi technologies in the treatment and management of disease promises new solutions to disease problems through the naturally occurring biological processes of living organisms. This study applied a novel dsRNA product developed specifically with the aim of improving honey bee health. The results demonstrate the successful application of RNAi strategies to improve disease tolerance. Honey bees were fed a dsRNA product, Remebee-I, in the presence of the Israeli Acute Paralysis Virus. Treatment resulted in larger colony populations and thus increased honey production. We show that IAPV specific homologous dsRNA successfully curbed the negative effects of IAPV infection in 160 honey bee hives in two discrete climates, seasons and geographical locations (Florida and Pennsylvania). We provide the first successful demonstration of the use of RNAi as a preventative treatment for an insect disease on such a large scale.