Transferrin-mediated iron sequestration suggests a novel therapeutic strategy for controlling Nosema disease in the honey bee, Apis mellifera.

Nosemosis C, a Nosema disease caused by microsporidia parasite Nosema ceranae, is a significant disease burden of the European honey bee Apis mellifera which is one of the most economically important insect pollinators. Nevertheless, there is no effective treatment currently available for Nosema disease and the disease mechanisms underlying the pathological effects of N. ceranae infection in honey bees are poorly understood. Iron is an essential nutrient for growth and survival of hosts and pathogens alike. The iron tug-of-war between host and pathogen is a central battlefield at the host-pathogen interface which determines the outcome of an infection, however, has not been explored in honey bees. To fill the gap, we conducted a study to investigate the impact of N. ceranae infection on iron homeostasis in honey bees. The expression of transferrin, an iron binding and transporting protein that is one of the key players of iron homeostasis, in response to N. ceranae infection was analysed. Furthermore, the functional roles of transferrin in iron homeostasis and honey bee host immunity were characterized using an RNA interference (RNAi)-based method. The results showed that N. ceranae infection causes iron deficiency and upregulation of the A. mellifera transferrin (AmTsf) mRNA in honey bees, implying that higher expression of AmTsf allows N. ceranae to scavenge more iron from the host for its proliferation and survival. The suppressed expression levels of AmTsf via RNAi could lead to reduced N. ceranae transcription activity, alleviated iron loss, enhanced immunity, and improved survival of the infected bees. The intriguing multifunctionality of transferrin illustrated in this study is a significant contribution to the existing body of literature concerning iron homeostasis in insects. The uncovered functional role of transferrin on iron homeostasis, pathogen growth and honey bee's ability to mount immune responses may hold the key for the development of novel strategies to treat or prevent diseases in honey bees.

Impacts of seasonality and parasitism on honey bee population dynamics.

The honeybee plays an extremely important role in ecosystem stability and diversity and in the production of bee pollinated crops. Honey bees and other pollinators are under threat from the combined effects of nutritional stress, parasitism, pesticides, and climate change that impact the timing, duration, and variability of seasonal events. To understand how parasitism and seasonality influence honey bee colonies separately and interactively, we developed a non-autonomous nonlinear honeybee-parasite interaction differential equation model that incorporates seasonality into the egg-laying rate of the queen. Our theoretical results show that parasitism negatively impacts the honey bee population either by decreasing colony size or destabilizing population dynamics through supercritical or subcritical Hopf-bifurcations depending on conditions. Our bifurcation analysis and simulations suggest that seasonality alone may have positive or negative impacts on the survival of honey bee colonies. More specifically, our study indicates that (1) the timing of the maximum egg-laying rate seems to determine when seasonality has positive or negative impacts; and (2) when the period of seasonality is large it can lead to the colony collapsing. Our study further suggests that the synergistic influences of parasitism and seasonality can lead to complicated dynamics that may positively and negatively impact the honey bee colony's survival. Our work partially uncovers the intrinsic effects of climate change and parasites, which potentially provide essential insights into how best to maintain or improve a honey bee colony's health.

Inferring pesticide toxicity to honey bees from a field-based feeding study using a colony model and Bayesian inference.

Honey bees are crucial pollinators for agricultural crops but are threatened by a multitude of stressors including exposure to pesticides. Linking our understanding of how pesticides affect individual bees to colony-level responses is challenging because colonies show emergent properties based on complex internal processes and interactions among individual bees. Agent-based models that simulate honey bee colony dynamics may be a tool for scaling between individual and colony effects of a pesticide. The U.S. Environmental Protection Agency (USEPA) and U.S. Department of Agriculture (USDA) are developing the VarroaPop + Pesticide model, which simulates the dynamics of honey bee colonies and how they respond to multiple stressors, including weather, Varroa mites, and pesticides. To evaluate this model, we used Approximate Bayesian Computation to fit field data from an empirical study where honey bee colonies were fed the insecticide clothianidin. This allowed us to reproduce colony feeding study data by simulating colony demography and mortality from ingestion of contaminated food. We found that VarroaPop + Pesticide was able to fit general trends in colony population size and structure and reproduce colony declines from increasing clothianidin exposure. The model underestimated adverse effects at low exposure (36 µg/kg), however, and overestimated recovery at the highest exposure level (140 µg/kg), for the adult and pupa endpoints, suggesting that mechanisms besides oral toxicity-induced mortality may have played a role in colony declines. The VarroaPop + Pesticide model estimates an adult oral LD50 of 18.9 ng/bee (95% CI 10.1-32.6) based on the simulated feeding study data, which falls just above the 95% confidence intervals of values observed in laboratory toxicology studies on individual bees. Overall, our results demonstrate a novel method for analyzing colony-level data on pesticide effects on bees and making inferences on pesticide toxicity to individual bees.

The active ingredients of a mitotoxic fungicide negatively affect pollen consumption and worker survival in laboratory-reared honey bees (Apis mellifera).

Recent observations of many sublethal effects of pesticides on pollinators have raised questions about whether standard short-term laboratory tests of pesticide effects on survival are sufficient for pollinator protection. The fungicide Pristine® and its active ingredients (25.2% boscalid, 12.8% pyraclostrobin) have been reported to have low acute toxicity to caged honey bee workers, but many sublethal effects at field-relevant doses have been reported and Pristine® was recently found to increase worker pollen consumption, reduce worker longevity and colony populations at field relevant concentrations (Fisher et al. 2021). To directly compare these whole-colony field results to more standard laboratory toxicology tests, the effects of Pristine®, at a range of field-relevant concentrations, were assessed on the survival and pollen consumption of honey bee workers 0-14 days of age. Also, to separate the effects of the inert and two active ingredients, bees were fed pollen containing boscalid, pyraclostrobin, or pyraclostrobin plus boscalid, at concentrations matching those in the Pristine® treatments. Pyraclostrobin significantly reduced pollen consumption across the duration of the experiment, and dose-dependently reduced pollen consumption on days 12-14. Pristine® and boscalid significantly reduced pollen feeding rate on days 12-14. Boscalid reduced survival in a dose-dependent manner. Consumption of Pristine® or pyraclostrobin plus boscalid did not affect survival, providing evidence against strong negative effects of the inert ingredients in Pristine® and against negative synergistic effects of boscalid and pyraclostrobin. The stronger toxic effects of Pristine® observed in field colonies compared to this laboratory test, and the opposite responses of pollen consumption in the laboratory and field to Pristine®, show that standard laboratory toxicology tests can fail to predict responses of pollinators to pesticides and to provide protection.

Field cross-fostering and in vitro rearing demonstrate negative effects of both larval and adult exposure to a widely used fungicide in honey bees (Apis mellifera).

Pollinators and other insects are experiencing an ongoing worldwide decline. While various environmental stressors have been implicated, including pesticide exposure, the causes of these declines are complex and highly debated. Fungicides may constitute a particularly prevalent threat to pollinator health due to their application on many crops during bloom, and because pollinators such as bees may consume fungicide-tainted pollen or nectar. In a previous study, consumption of pollen containing the fungicide Pristine® at field-relevant concentrations by honey bee colonies increased pollen foraging, caused earlier foraging, lowered worker survival, and reduced colony population size. Because most pollen is consumed by young adults, we hypothesized that Pristine® (25.2% boscalid, 12.8% pyraclostrobin) in pollen exerts its negative effects on honey bee colonies primarily on the adult stage. To rigorously test this hypothesis, we used a cross-fostering experimental design, with bees reared in colonies provided Pristine® incorporated into pollen patties at a supra-field concentration (230 mg/kg), only in the larvae, only in the adult, or both stages. In contrast to our predictions, exposure to Pristine® in either the larval or adult stage reduced survival relative to control bees not exposed to Pristine®, and exposure to the fungicide at both larval and adult stages further reduced survival. Adult exposure caused precocious foraging, while larval exposure increased the tendency to forage for pollen. These results demonstrate that pollen containing Pristine® can induce significant negative effects on both larvae and adults in a hive, though the magnitude of such effects may be smaller at field-realistic doses. To further test the potential negative effects of direct consumption of Pristine® on larvae, we reared them in vitro on food containing Pristine® at a range of concentrations. Consumption of Pristine® reduced survival rates of larvae at all concentrations tested. Larval and adult weights were only reduced at a supra-field concentration. We conclude that consumption of pollen containing Pristine® by field honey bee colonies likely exerts impacts on colony population size and foraging behavior by affecting both larvae and adults.

Can supplementary pollen feeding reduce varroa mite and virus levels and improve honey bee colony survival?

Varroa destructor is an ectoparasitic mite of immature and adult honey bees that can transmit several single-stranded RNA viruses to its host. Varroa reproduce in brood cells, and mite populations increase as colonies produce brood in spring and summer. Mite numbers also can sharply rise, particularly in the fall, by the migration of varroa into hives on foragers. Colonies with high levels of varroa and viruses often die over the winter. Feeding colonies pollen might keep virus levels low and improve survival because of the positive effects of pollen on immunity and colony growth. We compared varroa and virus levels and overwinter survival in colonies with (fed) and without (unfed) supplemental pollen. We also measured the frequency of capturing foragers with mites (FWM) at colony entrances to determine its relationship to varroa and virus levels. Colonies fed supplemental pollen were larger than unfed colonies and survived longer. Varroa populations and levels of Deformed wing virus (DWV) rose throughout the season, and were similar between fed and unfed colonies. The growth of varroa populations was correlated with FWM in fed and unfed colonies, and significantly affected DWV levels. Increasing frequencies of FWM and the effects on varroa populations might reduce the positive influence of supplemental pollen on immune function. However, pollen feeding can stimulate colony growth and this can improve colony survival.

Altered trajectories of neurodevelopment and behavior in mouse models of Rett syndrome.

Rett Syndrome (RTT) is a genetic disorder that is caused by mutations in the x-linked gene coding for methyl-CpG-biding-protein 2 (MECP2) and that mainly affects females. Male and female transgenic mouse models of RTT have been studied extensively, and we have learned a great deal regarding RTT neuropathology and how MeCP2 deficiency may be influencing brain function and maturation. In this manuscript we review what is known concerning structural and coinciding functional and behavioral deficits in RTT and in mouse models of MeCP2 deficiency. We also introduce our own corroborating data regarding behavioral phenotype and morphological alterations in volume of the cortex and striatum and the density of neurons, aberrations in experience-dependent plasticity within the barrel cortex and the impact of MeCP2 loss on glial structure. We conclude that regional structural changes in genetic models of RTT show great similarity to the alterations in brain structure of patients with RTT. These region-specific modifications often coincide with phenotype onset and contribute to larger issues of circuit connectivity, progression, and severity. Although the alterations seen in mouse models of RTT appear to be primarily due to cell-autonomous effects, there are also non-cell autonomous mechanisms including those caused by MeCP2-deficient glia that negatively impact healthy neuronal function. Collectively, this body of work has provided a solid foundation on which to continue to build our understanding of the role of MeCP2 on neuronal and glial structure and function, its greater impact on neural development, and potential new therapeutic avenues.

Pollen reverses decreased lifespan, altered nutritional metabolism and suppressed immunity in honey bees (Apis mellifera) treated with antibiotics.

Nutrition is involved in regulating multiple aspects of honey bee biology such as caste, immunity, lifespan, growth and behavioral development. Deformed wing virus (DWV) is a major pathogenic factor which threatens honey bee populations, and its replication is regulated by the nutrition status and immune response of honey bees. The alimentary canal of the honey bee is home to a diverse microbial community that provides essential nutrients and serves to bolster immune responses. However, to what extent gut bacteria affect honey bee nutrition metabolism and immunity with respect to DWV has not been investigated fully. In this study, newly emerged worker bees were subjected to four diets that contained (1) pollen, (2) pollen and antibiotics, (3) neither pollen nor antibiotics or (4) antibiotics alone. The expression level of two nutrition genes target of rapamycin (tor) and insulin like peptide (ilp1), one nutritional marker gene vitellogenin (vg), five major royal jellyprotein genes (mrjp1-5), one antimicrobial peptide regulating gene relish (rel), and DWV virus titer and its replication intermediate, negative RNA strand, were determined by qRT-PCR from the honey bees at 7 days post-antibiotic treatment. Additionally, honey bee head mass and survival rate were measured. We observed that antibiotics decreased the expression of tor and rel, and increased DWV titer and its replication activity. Expression of ilp1, mrjp1-5 and vg, and honey bee head mass were also reduced compared with bees on a pollen diet. Antibiotics also caused a significant drop in survivorship, which could be rescued by addition of pollen to the diet. Of importance, pollen could partially rescue the loss of vg and mrjp2 while also increasing the head mass of antibiotic-treated bees. Our results illuminate the roles of bacteria in honey bee nutrition, metabolism and immunity, which confer the ability to inhibit virus replication, extend honey bee lifespan and improve overall health.

Transcriptional response of honey bee (Apis mellifera) to differential nutritional status and Nosema infection.

BACKGROUND: Bees are confronting several environmental challenges, including the intermingled effects of malnutrition and disease. Intuitively, pollen is the healthiest nutritional choice, however, commercial substitutes, such as Bee-Pro and MegaBee, are widely used. Herein we examined how feeding natural and artificial diets shapes transcription in the abdomen of the honey bee, and how transcription shifts in combination with Nosema parasitism. RESULTS: Gene ontology enrichment revealed that, compared with poor diet (carbohydrates [C]), bees fed pollen (P > C), Bee-Pro (B > C), and MegaBee (M > C) showed a broad upregulation of metabolic processes, especially lipids; however, pollen feeding promoted more functions, and superior proteolysis. The superiority of the pollen diet was also evident through the remarkable overexpression of vitellogenin in bees fed pollen instead of MegaBee or Bee-Pro. Upregulation of bioprocesses under carbohydrates feeding compared to pollen (C > P) provided a clear poor nutritional status, uncovering stark expression changes that were slight or absent relatively to Bee-Pro (C > B) or MegaBee (C > M). Poor diet feeding (C > P) induced starvation response genes and hippo signaling pathway, while it repressed growth through different mechanisms. Carbohydrate feeding (C > P) also elicited 'adult behavior', and developmental processes suggesting transition to foraging. Finally, it altered the 'circadian rhythm', reflecting the role of this mechanism in the adaptation to nutritional stress in mammals. Nosema-infected bees fed pollen compared to carbohydrates (PN > CN) upheld certain bioprocesses of uninfected bees (P > C). Poor nutritional status was more apparent against pollen (CN > PN) than Bee-Pro (CN > BN) or MegaBee (CN > MN). Nosema accentuated the effects of malnutrition since more starvation-response genes and stress response mechanisms were upregulated in CN > PN compared to C > P. The bioprocess 'Macromolecular complex assembly' was also enriched in CN > PN, and involved genes associated with human HIV and/or influenza, thus providing potential candidates for bee-Nosema interactions. Finally, the enzyme Duox emerged as essential for guts defense in bees, similarly to Drosophila. CONCLUSIONS: These results provide evidence of the superior nutritional status of bees fed pollen instead of artificial substitutes in terms of overall health, even in the presence of a pathogen.

Impairments in the reproductive axis of female mice lacking estrogen receptor ß in GnRH neurons.

The effect of estrogen on the differentiation and maintenance of reproductive tissues is mediated by two nuclear estrogen receptors (ERs), ERα, and ERß. Lack of functional ERα and ERß genes in vivo significantly affects reproductive function; however, the target tissues and signaling pathways in the hypothalamus are not clearly defined. Here, we describe the generation and reproductive characterization of a complete-ERß KO (CERßKO) and a GnRH neuron-specific ERßKO (GERßKO) mouse models. Both ERßKO mouse models displayed a delay in vaginal opening and first estrus. Hypothalamic gonadotropin-releasing hormone (GnRH) mRNA expression levels in both ERßKO mice were similar to control mice; however female CERßKO and GERßKO mice had lower basal and surge serum gonadotropin levels. Although a GnRH stimulation test in both female ERßKO models showed preserved gonadotropic function in the same animals, a kisspeptin stimulation test revealed an attenuated response by GnRH neurons, suggesting a role for ERß in normal GnRH neuron function. No alteration in estrogen-negative feedback was observed in either ERßKO mouse models after ovariectomy and estrogen replacement. Further, abnormal development of ovarian follicles with low serum estradiol levels and impairment of fertility were observed in both ERßKO mouse models. In male ERßKO mice, no differences in the timing of pubertal onset or serum luteinizing hormone and follicle-stimulating hormone levels were observed as compared with controls. Taken together, these data provide in vivo evidence for a role of ERß in GnRH neurons in modulating puberty and reproduction, specifically through kisspeptin responsiveness in the female hypothalamic-pituitary-gonadal axis.

Nosema ceranae parasitism impacts olfactory learning and memory and neurochemistry in honey bees (Apis mellifera).

Nosema sp. is an internal parasite of the honey bee, Apis mellifera, and one of the leading contributors to colony losses worldwide. This parasite is found in the honey bee midgut and has profound consequences for the host's physiology. Nosema sp. impairs foraging performance in honey bees, yet, it is unclear whether this parasite affects the bee's neurobiology. In this study, we examined whether Nosema sp. affects odor learning and memory and whether the brains of parasitized bees show differences in amino acids and biogenic amines. We took newly emerged bees and fed them with Nosema ceranae At approximate nurse and forager ages, we employed an odor-associative conditioning assay using the proboscis extension reflex and two bioanalytical techniques to measure changes in brain chemistry. We found that nurse-aged bees infected with N. ceranae significantly outperformed controls in odor learning and memory, suggestive of precocious foraging, but by forager age, infected bees showed deficits in learning and memory. We also detected significant differences in amino acid concentrations, some of which were age specific, as well as altered serotonin, octopamine, dopamine and l-dopa concentrations in the brains of parasitized bees. These findings suggest that N. ceranae infection affects honey bee neurobiology and may compromise behavioral tasks. These results yield new insight into the host-parasite dynamic of honey bees and N. ceranae, as well as the neurochemistry of odor learning and memory under normal and parasitic conditions.

Smoke Conditions Affect the Release of the Venom Droplet Accompanying Sting Extension in Honey Bees (Hymenoptera: Apidae).

Honey bees (Apis mellifera) (Hymenoptera: Apidae) are social insects that have evolved a coordinated defensive response to ensure colony survival. Their nests may contain valuable resources such as pollen and nectar that are attractive to a range of insect and mammalian intruders and need protecting. With sufficient provocation, honey bees will mobilize and sting intruders, who are likely to incur additional stings. To inspect and manage their colonies, beekeepers apply smoke to decrease the likelihood of being stung. The use of smoke is a ubiquitous beekeeping practice, but the reasons behind its efficacy remain unknown. In this study, we examined the effects of smoke on honey bee defensive behavior by assessing individual sting extension responses under smoke conditions. We applied a brief voltage to the bee, ranging from a mild to a strong perturbation, and assessed four components of the sting extension reflex using two types of smoke. We found that smoke did not influence the probability of sting extension, but it did affect whether a venom droplet was released with the stinger. The venom droplet was more likely to be released at higher voltage levels, but this effect was significantly reduced under smoke conditions. Based on these results, we propose that the venom droplet coincides with greater agitation in individual bees; and smoke reduces the probability of its release. We speculate that the venom droplet serves to amplify the sting alarm pheromone, and smoke, in its ability to reduce droplet formation, may indicate that less alarm pheromone is released.

Sensitivity analyses for simulating pesticide impacts on honey bee colonies.

We employ Monte Carlo simulation and sensitivity analysis techniques to describe the population dynamics of pesticide exposure to a honey bee colony using the VarroaPop+Pesticide model. Simulations are performed of hive population trajectories with and without pesticide exposure to determine the effects of weather, queen strength, foraging activity, colony resources, and Varroa populations on colony growth and survival. The daily resolution of the model allows us to conditionally identify sensitivity metrics. Simulations indicate queen strength and forager lifespan are consistent, critical inputs for colony dynamics in both the control and exposed conditions. Adult contact toxicity, application rate and nectar load become critical parameters for colony dynamics within exposed simulations. Daily sensitivity analysis also reveals that the relative importance of these parameters fluctuates throughout the simulation period according to the status of other inputs.

Interlamellar CA1 network in the hippocampus.

To understand the cellular basis of learning and memory, the neurophysiology of the hippocampus has been largely examined in thin transverse slice preparations. However, the synaptic architecture along the longitudinal septo-temporal axis perpendicular to the transverse projections in CA1 is largely unknown, despite its potential significance for understanding the information processing carried out by the hippocampus. Here, using a battery of powerful techniques, including 3D digital holography and focal glutamate uncaging, voltage-sensitive dye, two-photon imaging, electrophysiology, and immunohistochemistry, we show that CA1 pyramidal neurons are connected to one another in an associational and well-organized fashion along the longitudinal axis of the hippocampus. Such CA1 longitudinal connections mediate reliable signal transfer among the pyramidal cells and express significant synaptic plasticity. These results illustrate a need to reconceptualize hippocampal CA1 network function to include not only processing in the transverse plane, but also operations made possible by the longitudinal network. Our data will thus provide an essential basis for future computational modeling studies on information processing operations carried out in the full 3D hippocampal network that underlies its complex cognitive functions.

Granulosa cell and oocyte mitochondrial abnormalities in a mouse model of fragile X primary ovarian insufficiency.

STUDY HYPOTHESIS: We hypothesized that the mitochondria of granulosa cells (GC) and/or oocytes might be abnormal in a mouse model of fragile X premutation (FXPM). STUDY FINDING: Mice heterozygous and homozygous for the FXPM have increased death (atresia) of large ovarian follicles, fewer corpora lutea with a gene dosage effect manifesting in decreased litter size(s). Furthermore, granulosa cells (GC) and oocytes of FXPM mice have decreased mitochondrial content, structurally abnormal mitochondria, and reduced expression of critical mitochondrial genes. Because this mouse allele produces the mutant Fragile X mental retardation 1 (Fmr1) transcript and reduced levels of wild-type (WT) Fmr1 protein (FMRP), but does not produce a Repeat Associated Non-ATG Translation (RAN)-translation product, our data lend support to the idea that Fmr1 mRNA with large numbers of CGG-repeats is intrinsically deleterious in the ovary. WHAT IS KNOWN ALREADY: Mitochondrial dysfunction has been detected in somatic cells of human and mouse FX PM carriers and mitochondria are essential for oogenesis and ovarian follicle development, FX-associated primary ovarian insufficiency (FXPOI) is seen in women with FXPM alleles. These alleles have 55-200 CGG repeats in the 5' UTR of an X-linked gene known as FMR1. The molecular basis of the pathology seen in this disorder is unclear but is thought to involve either some deleterious consequence of overexpression of RNA with long CGG-repeat tracts or of the generation of a repeat-associated non-AUG translation (RAN translation) product that is toxic. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Analysis of ovarian function in a knock-in FXPM mouse model carrying 130 CGG repeats was performed as follows on WT, PM/+, and PM/PM genotypes. Histomorphometric assessment of follicle and corpora lutea numbers in ovaries from 8-month-old mice was executed, along with litter size analysis. Mitochondrial DNA copy number was quantified in oocytes and GC using quantitative PCR, and cumulus granulosa mitochondrial content was measured by flow cytometric analysis after staining of cells with Mitotracker dye. Transmission electron micrographs were prepared of GC within small growing follicles and mitochondrial architecture was compared. Quantitative RT-PCR analysis of key genes involved in mitochondrial structure and recycling was performed. MAIN RESULTS AND THE ROLE OF CHANCE: A defect was found in follicle survival at the large antral stage in PM/+ and PM/PM mice. Litter size was significantly decreased in PM/PM mice, and corpora lutea were significantly reduced in mice of both mutant genotypes. Mitochondrial DNA copy number was significantly decreased in GC and metaphase II eggs in mutants. Flow cytometric analysis revealed that PM/+ and PM/PM animals lack the cumulus GC that harbor the greatest mitochondrial content as found in wild-type animals. Electron microscopic evaluation of GC of small growing follicles revealed mitochondrial structural abnormalities, including disorganized and vacuolar cristae. Finally, aberrant mitochondrial gene expression was detected. Mitofusin 2 (Mfn2) and Optic atrophy 1 (Opa1), genes involved in mitochondrial fusion and structure, respectively, were significantly decreased in whole ovaries of both mutant genotypes. Mitochondrial fission factor 1 (Mff1) was significantly decreased in PM/+ and PM/PM GC and eggs compared with wild-type controls. LIMITATIONS, REASONS FOR CAUTION: Data from the mouse model used for these studies should be viewed with some caution when considering parallels to the human FXPOI condition. WIDER IMPLICATIONS OF THE FINDINGS: Our data lend support to the idea that Fmr1 mRNA with large numbers of CGG-repeats is intrinsically deleterious in the ovary. FXPM disease states, including FXPOI, may share mitochondrial dysfunction as a common underlying mechanism. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: Studies were supported by NIH R21 071873 (J.J./G.H), The Albert McKern Fund for Perinatal Research (J.J.), NIH Intramural Funds (K.U.), and a TUBITAK Research Fellowship Award (B.U.). No conflict(s) of interest or competing interest(s) are noted.

Israeli acute paralysis virus: epidemiology, pathogenesis and implications for honey bee health.

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.

Population growth of Varroa destructor (Acari: Varroidae) in honey bee colonies is affected by the number of foragers with mites.

Varroa mites are a serious pest of honey bees and the leading cause of colony losses. Varroa have relatively low reproductive rates, so populations should not increase rapidly, but often they do. Other factors might contribute to the growth of varroa populations including mite migration into colonies on foragers from other hives. We measured the proportion of foragers carrying mites on their bodies while entering and leaving hives, and determined its relationship to the growth of varroa populations in those hives at two apiary sites. We also compared the estimates of mite population growth with predictions from a varroa population dynamics model that generates estimates of mite population growth based on mite reproduction. Samples of capped brood and adult bees indicated that the proportion of brood cells infested with mites and adult bees with phoretic mites was low through the summer but increased sharply in the fall especially at site 1. The frequency of capturing foragers with mites on their bodies while entering or leaving hives also increased in the fall. The growth of varroa populations at both sites was not significantly related to our colony estimates of successful mite reproduction, but instead to the total number of foragers with mites (entering and leaving the colony). There were more foragers with mites at site 1 than site 2, and mite populations at site 1 were larger especially in the fall. The model accurately estimated phoretic mite populations and infested brood cells until November when predictions were much lower than those measured in colonies. The rapid growth of mite populations particularly in the fall being a product of mite migration rather than mite reproduction only is discussed.

Mixed infections reveal virulence differences between host-specific bee pathogens.

Dynamics of host-pathogen interactions are complex, often influencing the ecology, evolution and behavior of both the host and pathogen. In the natural world, infections with multiple pathogens are common, yet due to their complexity, interactions can be difficult to predict and study. Mathematical models help facilitate our understanding of these evolutionary processes, but empirical data are needed to test model assumptions and predictions. We used two common theoretical models regarding mixed infections (superinfection and co-infection) to determine which model assumptions best described a group of fungal pathogens closely associated with bees. We tested three fungal species, Ascosphaera apis, Ascosphaera aggregata and Ascosphaera larvis, in two bee hosts (Apis mellifera and Megachile rotundata). Bee survival was not significantly different in mixed infections vs. solo infections with the most virulent pathogen for either host, but fungal growth within the host was significantly altered by mixed infections. In the host A. mellifera, only the most virulent pathogen was present in the host post-infection (indicating superinfective properties). In M. rotundata, the most virulent pathogen co-existed with the lesser-virulent one (indicating co-infective properties). We demonstrated that the competitive outcomes of mixed infections were host-specific, indicating strong host specificity among these fungal bee pathogens.

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

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.

Effects of Oral Exposure to Fungicides on Honey Bee Nutrition and Virus Levels.

Sublethal exposure to fungicides can affect honey bees (Apis mellifera L.) in ways that resemble malnutrition. These include reduced brood rearing, queen loss, and increased pathogen levels. We examined the effects of oral exposure to the fungicides boscalid and pyraclostrobin on factors affecting colony nutrition and immune function including pollen consumption, protein digestion, hemolymph protein titers, and changes in virus levels. Because the fungicides are respiratory inhibitors, we also measured ATP concentrations in flight muscle. The effects were evaluated in 3- and 7-d-old worker bees at high fungicide concentrations in cage studies, and at field-relevant concentrations in colony studies. Though fungicide levels differed greatly between the cage and colony studies, similar effects were observed. Hemolymph protein concentrations were comparable between bees feeding on pollen with and without added fungicides. However, in both cage and colony studies, bees consumed less pollen containing fungicides and digested less of the protein. Bees fed fungicide-treated pollen also had lower ATP concentrations and higher virus titers. The combination of effects we detected could produce symptoms that are similar to those from poor nutrition and weaken colonies making them more vulnerable to loss from additional stressors such as parasites and pathogens.