Infected Bees Research Articles

Deformed wing virus infection affects the neurological function of Apis mellifera by altering extracellular adenosine signaling

Deformed wing virus (DWV) infection is believed to be closely associated with colony losses of honeybee (Apis mellifera) due to reduced learning and memory of infected bees. The adenosine (Ado) pathway is important for maintaining immunity and memory function in animals, and it enhances antivirus responses by regulating carbohydrate metabolism in insects. Nevertheless, its effect on the memory of invertebrates is not yet clear. This study investigated how the Ado pathway regulates energy metabolism and memory in honeybees following DWV infection. Decreased Ado receptor (Ado-R) expression in the brain of infected bees resulted in a carbohydrate imbalance as well as impairments of glutamate-glutamine (Glu-Gln) cycle and long-term memory. Dietary supplementation with Ado not only increased the brain energy metabolism but also rescued long-term memory loss by upregulating the expression of memory-related genes. The present study demonstrated the regulation of the Ado pathway upon DWV infection and provides insights into the mechanisms underlying energy regulation and the neurological function of honeybees.

Natural extracts as potential control agents for Nosema ceranae infection in honeybees, Apis mellifera

Nosema disease is one factor that can cause colony decline in honeybees (Apis mellifera L.) worldwide. Nosema ceranae has outcompeted Nosema apis in the Western honeybee (A. mellifera) which is its original host. Fumagilin is an effective antibiotic treatment to control Nosema infection but currently it is forbidden in many countries. In this study, 12 plant extracts were evaluated for their toxicity to adult bees and antimicrosporidian activity under laboratory and field conditions. N. ceranae-infected adult bees were fed ad libitum with 50% sucrose solution containing 1% and 5% (w/v) of each plant extract. Bee mortality in N. ceranae-infected groups fed with plant extracts was higher than that in the control group treated with fumagilin. The results demonstrated that 9 of 12 extracts had high antimicrosporidian activity against N. ceranae and their efficacies were comparable to fumagilin. Spore reduction in infected bees was 4–6 fold less after extract treatment. Following laboratory screening, Annona squamosa, Ocimum basilicum, Psidium guajava and Syzygium jambos were tested in honeybee colonies. Plant extracts of 2% concentration (w/v) inhibited the development of Nosema spores after 30 days of treatment. At the end of experiment (90 days), spores in the plant extract treated groups were lower than in group treated with fumagilin but there was no significant difference. Although, extracts tested in this study showed high toxicity to bee in laboratory cages, they did not show negative affects on bees under whole colony conditions. Therefore, the effectiveness of plant extracts tested in this study was notable and warrants further study as potential Nosema control agents in honey bees. Plant extracts would offer a non-antibiotic alternative for Nosema control and help reduce the overuse of antibiotics in livestock.

The pathological effects of a Nosema ceranae infection in the giant honey bee, Apis dorsata Fabricius, 1793

Nosema ceranae is an intracellular microsporidian pathogen that lives in the midgut ventricular cells of all known honey bee Apis species. We suspect that N. ceranae may also cause energetic stress in the giant honey bee because this parasite is known to disrupt nutrient absorption resulting in energetic stress in the honey bee species Apis mellifera. To understand how N. ceranae impacts the energetic stress of the giant honey bee, A. dorsata, we measured the hemolymph trehalose levels of experimentally infected giant honey bees on days three, five, seven, and fourteen post infection (p.i.). We also measured the hypopharyngeal gland protein content, the total midgut proteolytic enzyme activity, honey bee survival, infection ratio, and spore loads comparing infected and uninfected honey bees across the same time frame. Nosema ceranae-infected honey bees had significantly lowered survival, trehalose levels, hypopharyngeal gland protein content, and midgut proteolytic enzyme activity. We found an increasing level of parasitic loads and infection ratio of N. ceranae-infected bees after inoculation. Collectively, our results suggest that the giant honey bee suffers from energetic stress and limited nutrient absorption from a N. ceranae infection, which results in lowered survival in comparison to uninfected honey bees. Our findings highlight that other honey bee species besides A. mellifera are susceptible to microsporidian pathogens that they harbor, which results in negative effects on health and survival. Therefore, these pathogens might be transmitted at a community level, in the natural environment, resulting in negative health effects of multiple honey bee species.

Experimental Nosema ceranae infection is associated with microbiome changes in the midguts of four species of Apis (honey bees)

Nosema ceranae is a unicellular microsporidian causing ventricular cell lysis in host Apis species. We hypothesized that the gut microbiota of honey bees, A. cerana, A. dorsata, A. florea, and A. mellifera, is disrupted by N. ceranae infection. Correspondingly, we investigated the impact of N. ceranae infection on gut populations of Bifidobacterium sp. and Apilactobacillus kunkeei in the selected Apis species. The bacteria populations were compared using a culture-dependent method and employing 16S rRNA gene sequencing between bees infected with 2.0 × 105 N. ceranae spores and those remaining uninfected. The number of N. ceranae spores per bee and the ratio of infected gut cells to uninfected cells were measured at five and ten days post infection (p.i.). N. ceranae spore load at ten days p.i. was significantly greater than at five days p.i. and was varied with species’ body size. For each of the four Apis species, N. ceranae treated bees had lower survival than did the control bees. Moreover, A. florae had the largest infection ratio of all the species. Across all Apis species, N. ceranae infected individuals had significantly lower mean numbers of Bifidobacterium sp. and A. kunkeei than did uninfected ones. Among infected bees, the average number of both bacteria was significantly greatest in A. dorsata and lowest in A. cerana. Overall, N. ceranae infection was associated with histopathological damage and also with lower populations of Bifidobacterium sp. and A. kunkeei in the midguts of the tested honey bee species.

Mitigating Nosema ceranae infection in western honey bee (Apis mellifera) workers using propolis collected from honey bee and stingless bee (Tetrigona apicalis) hives

Beekeepers need sustainable control options to treat Nosema ceranae infection in colonies of western honey bees (Apis mellifera L.) they manage. Propolis is a natural product derived from plant resins and contains chemical compounds with potential antimicrobial activity against N. ceranae. Here, we determined the efficacy of propolis from A. mellifera (USA) and Tetrigona apicalis (stingless bees, Thailand) colonies as treatments for N. ceranae infection in honey bee workers. Newly emerged bees were individually fed 2 μL of 50% (w/v) sucrose solution containing 1 × 105N. ceranae spores. Following this, the infected bees were treated with 50% propolis extracted from A. mellifera or T. apicalis hives and fed in 50% sucrose solution (v/v). All bees were maintained at 34 ± 2 °C and 55 ± 5% RH. Dead bees were counted daily for 30 d to calculate survival. We also determined infection rate (# infected bees/100 bees), infectivity (number of spores per bee) and protein content in the hypopharyngeal glands and hemolymph on 7, 14, and 21 d post infection as measures of bee health. Propolis from both bee species significantly reduced bee mortality, infection rate and infectivity compared with those of untreated bees and led to significantly greater protein contents in hypopharyngeal glands and hemolymph in treated bees than in untreated ones (p < 0.0001). In conclusion, propolis from A. mellifera and T. apicalis colonies shows promise as a control against N. ceranae infection in honey bees.

Vairimorpha (Nosema) ceranae Infection Alters Honey Bee Microbiota Composition and Sustains the Survival of Adult Honey Bees.

Simple SummaryThe gut microbiota, in addition to the hosts and the pathogens, has become the third factor involved in gut disease developments, including honey bees. Interestingly, various studies reported positive associations between the gut bacteria and the most commonly found microsporidian pathogen instead of negative associations. To investigate the positive associations, a prebiotic that also exists in honey was added in the trials. Bees fed the prebiotics have slightly higher pathogen counts but lower mortalities. Microbiota analyses suggested that bees with the infection have a microbiota composition similar to that of bees with a longer lifespan, and the prebiotic seemed to enhance the similarities. Since microsporidia typically cause chronic infections, the positive associations may serve to sustain the host lifespans which is the optimal outcome for the pathogen that the survived bees can withstand pathogen proliferation and transmit the pathogens. Although the mechanisms underlying the associations were not revealed, this study indicated that nosema disease management in bees through changes in microbiota may shorten the lifespans or enhance both the infection and the bee population. Such results have appeared in recent field studies. More studies will be needed for the disease management using bee gut microbiota.Vairimorpha (Nosema) ceranae is the most common eukaryotic gut pathogen in honey bees. Infection is typically chronic but may result in mortality. Gut microbiota is a factor that was recently noted for gut infectious disease development. Interestingly, studies identified positive, instead of negative, associations between core bacteria of honey bee microbiota and V. ceranae infection. To investigate the effects of the positive associations, we added isomaltooligosaccharide (IMO), a prebiotic sugar also found in honey, to enhance the positive associations, and we then investigated the infection and the gut microbiota alterations using qPCR and 16S rRNA gene sequencing. We found that infected bees fed IMO had significantly higher V. ceranae spore counts but lower mortalities. In microbiota comparisons, V. ceranae infections alone significantly enhanced the overall microbiota population in the honey bee hindgut and feces; all monitored core bacteria significantly increased in the quantities but not all in the population ratios. The microbiota alterations caused by the infection were enhanced with IMO, and these alterations were similar to the differences found in bees that naturally have longer lifespans. Although our results did not clarify the causations of the positive associations between the infections and microbiota, the associations seemed to sustain the host survival and benefit the pathogen. Enhancing indigenous gut microbe to control nosema disease may result in an increment of bee populations but not the control of the pathogen. This interaction between the pathogen and microbiota potentially enhances disease transmission and avoids the social immune responses that diseased bees die prematurely to curb the disease from spreading within colonies.

Transcriptome-level assessment of the impact of deformed wing virus on honey bee larvae

Deformed wing virus (DWV) prevalence is high in honey bee (Apis mellifera) populations. The virus infects honey bees through vertical and horizontal transmission, leading to behavioural changes, wing deformity, and early mortality. To better understand the impacts of viral infection in the larval stage of honey bees, artificially reared honey bee larvae were infected with DWV (1.55 × 1010 copies/per larva). No significant mortality occurred in infected honey bee larvae, while the survival rates decreased significantly at the pupal stage. Examination of DWV replication revealed that viral replication began at 2 days post inoculation (d.p.i.), increased dramatically to 4 d.p.i., and then continuously increased in the pupal stage. To better understand the impact of DWV on the larval stage, DWV-infected and control groups were subjected to transcriptomic analysis at 4 d.p.i. Two hundred fifty-five differentially expressed genes (DEGs) (fold change ≥ 2 or ≤ -2) were identified. Of these DEGs, 168 genes were downregulated, and 87 genes were upregulated. Gene Ontology (GO) analysis showed that 141 DEGs (55.3%) were categorized into molecular functions, cellular components and biological processes. One hundred eleven genes (38 upregulated and 73 downregulated) were annotated by KO (KEGG Orthology) pathway mapping and involved metabolic pathways, biosynthesis of secondary metabolites and glycine, serine and threonine metabolism pathways. Validation of DEGs was performed, and the related gene expression levels showed a similar tendency to the DEG predictions at 4 d.p.i.; cell wall integrity and stress response component 1 (wsc1), cuticular protein and myo-inositol 2-dehydrogenase (iolG) were significantly upregulated, and small conductance calcium-activated potassium channel protein (SK) was significantly downregulated at 4 d.p.i. Related gene expression levels at different d.p.i. revealed that these DEGs were significantly regulated from the larval stage to the pupal stage, indicating the potential impacts of gene expression levels from the larval to the pupal stages. Taken together, DWV infection in the honey bee larval stage potentially influences the gene expression levels from larvae to pupae and reduces the survival rate of the pupal stage. This information emphasizes the consequences of DWV prevalence in honey bee larvae for apiculture.

Propolis Extract and Chitosan Improve Health of Nosema ceranae Infected Giant Honey Bees, Apis dorsata Fabricius, 1793.

Nosema ceranae is a large contributing factor to the most recent decline in honey bee health worldwide. Developing new alternative treatments against N. ceranae is particularly pressing because there are few treatment options available and therefore the risk of increased antibiotic resistance is quite high. Recently, natural products have demonstrated to be a promising avenue for finding new effective treatments against N. ceranae. We evaluated the effects of propolis extract of stingless bee, Tetrigona apicalis and chito-oligosaccharide (COS) on giant honey bees, Apis dorsata, experimentally infected with N. ceranae to determine if these treatments could improve the health of the infected individuals. Newly emerged Nosema-free bees were individually inoculated with 106N. ceranae spores per bee. We fed infected and control bees the following treatments consisting of 0%, 50%, propolis extracts, 0 ppm and 0.5 ppm COS in honey solution (w/v). Propolis extracts and COS caused a significant increase in trehalose levels in hemolymph, protein contents, survival rates and acini diameters of the hypopharyngeal glands in infected bees. Our results suggest that propolis and COS could improve the health of infected bees. Further research is needed to determine the underlying mechanisms responsible for the improved health of the infected bees.

Impact of Nosema Disease and American Foulbrood on Gut Bacterial Communities of Honeybees Apis mellifera.

Simple SummaryNosema ceranae and Paenibacillus larvae are virulent pathogenic microbes in honeybee adults and larvae, respectively. However, there is still a lack of data on their dysbiosis effect on gut bacteria in honeybees. The guts of control and infected bees of both diseases were analyzed for bacterial communities by next-generation sequencing. The statistical data showed that Nosema ceranae affected bacterial dysbiosis in the adult honeybees’ guts. The guts of Paenibacillus larvae-infected honeybee larvae did not differ from the guts of larvae not infected with P. larvae. These data could be applied to control pathogens in the apicultural industry. Honeybees, Apis mellifera, are important pollinators of many economically important crops. However, one of the reasons for their decline is pathogenic infection. Nosema disease and American foulbrood (AFB) disease are the most common bee pathogens that propagate in the gut of honeybees. This study investigated the impact of gut-propagating pathogens, including Nosema ceranae and Paenibacillus larvae, on bacterial communities in the gut of A. mellifera using 454-pyrosequencing. Pyrosequencing results showed that N. ceranae was implicated in the elimination of Serratia and the dramatic increase in Snodgrassella and Bartonella in adult bees’ guts, while bacterial communities of P. larvae-infected larvae were not affected by the infection. The results indicated that only N. ceranae had an impact on some core bacteria in the gut of A. mellifera through increasing core gut bacteria, therefore leading to the induction of dysbiosis in the bees’ gut.

Africanized honey bees in Colombia exhibit high prevalence but low level of infestation of Varroa mites and low prevalence of pathogenic viruses.

The global spread of the ectoparasitic mite Varroa destructor has promoted the spread and virulence of highly infectious honey bee viruses. This phenomenon is considered the leading cause for the increased number of colony losses experienced by the mite-susceptible European honey bee populations in the Northern hemisphere. Most of the honey bee populations in Central and South America are Africanized honey bees (AHBs), which are considered more resistant to Varroa compared to European honey bees. However, the relationship between Varroa levels and the spread of honey bee viruses in AHBs remains unknown. In this study, we determined Varroa prevalence and infestation levels as well as the prevalence of seven major honey bee viruses in AHBs from three regions of Colombia. We found that although Varroa exhibited high prevalence (92%), its infestation levels were low (4.5%) considering that these populations never received acaricide treatments. We also detected four viruses in the three regions analyzed, but all colonies were asymptomatic, and virus prevalence was considerably lower than those found in other countries with higher rates of mite-associated colony loss (DWV 19.88%, BQCV 17.39%, SBV 23.4%, ABPV 10.56%). Our findings indicate that AHBs possess a natural resistance to Varroa that does not prevent the spread of this parasite among their population, but restrains mite population growth and suppresses the prevalence and pathogenicity of mite-associated viruses.

The Effect of Covert and Overt Infections on Disease Dynamics in Honey-Bee Colonies

Viral diseases of honey bees are important economically and ecologically and have been widely modelled. The models reflect the fact that, in contrast to the typical case for vertebrates, invertebrates cannot acquire immunity to a viral disease, so they are of SIS or (more often) SI type. Very often, these diseases may be transmitted vertically as well as horizontally, by vectors as well as directly, and through the environment, although models do not generally reflect all these transmission mechanisms. Here, we shall consider an important additional complication the consequences of which have yet to be fully explored in a model, namely that both infected honey bees and their vectors may best be described using more than one infection class. For honey bees, we consider three infection classes. Covert infections occur when bees have the virus under control, such that they do not display symptoms of the disease, and are minimally or not at all affected by it. Acutely overtly infected bees often exhibit severe symptoms and have a greatly curtailed lifespan. Chronically overtly infected bees typically have milder symptoms and a moderately shortened lifespan. For the vector, we consider just two infection classes which are covert infected and overt infected as has been observed in deformed-wing virus (DWV) vectored by varroa mites. Using this structure, we explore the impact of spontaneous transition of both mites and bees from a covertly to an overtly infected state, which is also a novel element in modelling viral diseases of honey bees made possible by including the different infected classes. The dynamics of these diseases are unsurprisingly rather different from the dynamics of a standard SI or SIS disease. In this paper, we highlight how our compartmental structure for infection in honey bees and their vectors impact the disease dynamics observed, concentrating in particular on DWV vectored by varroa mites. If there is no spontaneous transition, then a basic reproduction number R_0 exists. We derive a condition for R_0>1 that reflects the complexities of the system, with components for vertical and for direct and vector-mediated horizontal transmission, using the directed graph of the next-generation matrix of the system. Such a condition has never previously been derived for a honey-bee–mite–virus system. When spontaneous transitions do occur, then R_0 no longer exists, but we introduce a modification of the analysis that allows us to determine whether (i) the disease remains largely covert or (ii) a substantial outbreak of overt disease occurs.

Characterizing the Honey Bee Interactome using Mass Spectrometry‐Based Proteomics

Western honey bees (Apis mellifera), as a superorganism, have an essential ecological and economic role as pollinators. However, various understudied pathogens infecting honey bees pose a significant threat to their health. One such pathogen is Nosema ceranae, a microsporidian parasite which rapidly proliferates in the honey bee ventriculus (midgut) cells, weakening their immune systems by competing for nutrients. Upon completing their lifecycle in the midgut cells, Nosema spores rupture host cells and spread into the rest of the gut and subsequently to other bees in the colony. The resulting symptoms of the diseased colony have been attributed to colony death. We hypothesize that host-pathogen interactions underpin Nosema infection in honey bees, and therefore understanding pathological protein interactions will suggest tools for combating Nosema. To this end, we used an optimized version of the mass spectrometry-based co-fractionation experiment described in Kristensen et al. (2012) This method uses size exclusion chromatography (SEC) to separate protein complexes into fractionated samples. After measuring protein amounts in each fraction through mass spectrometry, a machine learning pipeline detects proteins with similar separation profiles as protein interactions (Stacey et al., 2017). We can then assess the effects of Nosema infection. Using an in vivo approach, we first constructed a honey bee interactome from uninfected midgut cells. This is the first honey bee interactome to date. Validation experiments using co-immunoprecipitation coupled with mass spectrometry (IP-MS) demonstrate that this interactome is high quality and will be a valuable tool to future researchers. Using this interactome as a baseline, we can then compare interactomes derived from infected honey bee cells to identify the protein-based mechanisms of Nosema infection. Preliminary results from this highlight interactome changes in various processes such as peroxiredoxin activity and carbohydrate binding. This work presents the first honey bee interactome map. In addition, we are gaining novel insight into how protein interactions change upon infection.

Seed Meals from Brassica nigra and Eruca sativa Control Artificial Nosema ceranae Infections in Apis mellifera.

Nosema ceranae is a widespread parasite responsible for nosemosis Type C in Apis mellifera honey bees, reducing colony survival. The antibiotic fumagillin is the only commercial treatment available, but concerns are emerging about its persistence, safety, and pathogen resistance. The use of natural substances from Brassicaceae defatted seed meals (DSMs) with known antimicrobial and antioxidant properties was explored. Artificially infected bees were fed for 8 days with candies enriched with two concentrations, 2% and 4%, of two DSMs from Brassica nigra and Eruca sativa, containing a known amount of different glucosinolates (GSLs). The food palatability, GSL intake, bee survival, and treatment effects on N. ceranae spore counts were evaluated. Food consumption was higher for the two 2% DSM patties, for both B. nigra and E. sativa, but the GSL intake did not increase by increasing DSM to 4%, due to the resulting lower palatability. The 2% B. nigra patty decreased the bee mortality, while the higher concentration had a toxic effect. The N. ceranae control was significant for all formulates with respect to the untreated control (312,192.6 +/− 14,443.4 s.e.), and was higher for 4% B. nigra (120,366.3 +/− 13,307.1 s.e.). GSL hydrolysis products, the isothiocyanates, were detected and quantified in bee gut tissues. Brassicaceae DSMs showed promising results for their nutraceutical and protective effects on bees artificially infected with N. ceranae spores at the laboratory level. Trials in the field should confirm these results.

Antiviral Activities of a Medicinal Plant Extract Against Sacbrood Virus in Honeybees

BackgroundSacbrood is an infectious disease of the honey bee caused by Scbrood virus (SBV) which belongs to the family Iflaviridae and is especially lethal for Asian honeybee Apis cerana. Chinese Sacbrood virus (CSBV) is a geographic strain of SBV. Currently, there is a lack of an effective antiviral agent for controlling CSBV infection in honey bees.MethodsHere, we explored the antiviral effect of a Chinese medicinal herb Radix isatidis on CSBV infection in A. cerana by inoculating the 3rd instar larvae with purified CSBV and treating the infected bee larvae with R. isatidis extract at the same time. The growth, development, and survival of larvae between the control and treatment groups were compared. The CSBV copy number at the 4th instar, 5th instar, and 6th instar larvae was measured by the absolute quantification PCR method.ResultsBioassays revealed that R. isatidis extract significantly inhibited the replication of CSBV, mitigated the impacts of CSBV on larval growth and development, reduced the mortality of CSBV-infected A. cerana larvae, and modulated the expression of immune transcripts in infected bees.ConclusionAlthough the mechanism underlying the inhibition of CSBV replication by the medicine plant will require further investigation, this study demonstrated the antiviral activity of R. isatidis extract and provides a potential strategy for controlling SBV infection in honey bees.

A novel method for the detection and diagnosis of virus infections in honey bees

In terms of infectious diseases caused by a variety of microorganisms, the ability to promptly and accurately identify the causative agents is the first step on the path to all types of effective management of such infections. Among the various factors that are affecting global bee health, viruses have often been linked to honey bee colony losses and they pose a serious threat to the fraction of agriculture that depends on the service of pollinators. Over the past few decades, PCR-based molecular methods have provided powerful tools for rapid, specific, and sensitive detection and the quantification of difficult-to-grow pathogenic microorganisms such as viruses in honey bees. However, PCR-based methods require nucleic acid extraction and purification, which can be quite laborious and time-consuming and they involve the use of organic solvents and chaotropic agents like phenol and chloroform which are volatile and highly toxic.In response, we developed a novel and non-sacrificial method for detecting viral infections in honey bees. As little as 1 μl of hemolymph was collected from adult workers, larvae, and queens of bee colonies by puncturing the soft inter-tergal integument between the second and third dorsal tergum with a fine glass capillary. The hemolymph was then diluted and subjected to RT-PCR analysis directly. The puncture wound caused by the glass capillary was found to heal automatically and rapidly without any trouble and the lifespan of the experimental workers remained unaffected. Using this method, we detected multiple viruses including Deformed wing virus (DWV), Black queen cell virus (BQCV), and Sacbrood virus (SBV) in infected bees. Furthermore, expressed transcripts that indicate the induction of innate immune response to the virus infections were also detected in the hemolymph of infected bees. The simplicity and cost-effectiveness of this innovative approach will allow it to be a valuable, time-saving, safer, and more environmentally friendly contribution to bee disease management programs.

Visual Diagnosis of the Varroa Destructor Parasitic Mite in Honeybees Using Object Detector Techniques.

The Varroa destructor mite is one of the most dangerous Honey Bee (Apis mellifera) parasites worldwide and the bee colonies have to be regularly monitored in order to control its spread. In this paper we present an object detector based method for health state monitoring of bee colonies. This method has the potential for online measurement and processing. In our experiment, we compare the YOLO and SSD object detectors along with the Deep SVDD anomaly detector. Based on the custom dataset with 600 ground-truth images of healthy and infected bees in various scenes, the detectors reached the highest F1 score up to 0.874 in the infected bee detection and up to 0.714 in the detection of the Varroa destructor mite itself. The results demonstrate the potential of this approach, which will be later used in the real-time computer vision based honey bee inspection system. To the best of our knowledge, this study is the first one using object detectors for the Varroa destructor mite detection on a honey bee. We expect that performance of those object detectors will enable us to inspect the health status of the honey bee colonies in real time.

The use of propolis for preventing and treating Nosema ceranae infection in western honey bee (Apis mellifera Linnaeus, 1787) workers

The health of western honey bees (Apis mellifera L.) is constantly affected by Nosema ceranae, a microsporidian threat to colonies worldwide. We extracted propolis, a natural product exhibiting antimicrobial properties, from honey bee hives, fed it to worker bees before or after infection with N. ceranae, and determined its ability to protect bees from and treat them after infection. Protection from N. ceranae was tested using newly emerged bees that were group fed (50 bees/group) 50% propolis extract for 2, 4 and 8 d prior to infection with 1 × 105 spores per bee. Treatment of N. ceranae was tested on newly emerged bees that were individually force-fed with 2 µL of 50% (w/v) sucrose solution containing 1 × 105 spores per bee and then treated with 50% propolis in 50% sucrose solution (v/v) at 0, 2, 4 and 8 d post infection (p.i.). Positive (sugar water + N. ceranae), negative (sugar water only), and solvent (ethanol + sugar water) controls were included in both studies. Propolis fed to honey bees for 4 or 8 d before infection was associated with significantly reduced mortality, infectivity, and N. ceranae infection rates compared to the positive controls. Moreover, providing propolis to infected honey bees at d 0, 2, and 4 p.i. significantly reduced bee mortality, infection, and infectivity rates compared to the positive controls and bees treated 8 d p.i. Therefore, propolis extracted from honey bee hives may be a promising alternative approach to antibiotics for protecting colonies from Nosema disease.

Immunity and physiological changes in adult honey bees (Apis mellifera) infected with Nosema ceranae: The natural colony environment

Nosema ceranae is a microsporidium that infects Apis mellifera, causing diverse physiological and behavioral alterations. Given the existence of individual and social mechanisms to reduce infection and fungal spread in the colony, bees may respond differently to infection depending on their rearing conditions. In this study, we investigated the effect of N. ceranae in honey bee foragers naturally infected with different fungal loads in a tropical region. In addition, we explored the effects of N. ceranae artificially infected young bees placed in a healthy colony under field conditions. Honey bees naturally infected with higher loads of N. ceranae showed downregulation of genes from Toll and IMD immune pathways and antimicrobial peptide (AMP) genes, but hemolymph total protein amount and Vitellogenin (Vg) titers were not affected. Artificially infected bees spread N. ceranae to the controls in the colony, but fungal loads were generally lower than those observed in cages, probably because of social immunity. Although no significant changes in mRNA levels of AMP-encoding were observed, N. ceranae artificially infected bees showed downregulation of miR-989 (an immune-related microRNA), lower vitellogenin gene expression, and decreased hemolymph Vg titers. Our results demonstrate for the first time that natural infection by N. ceranae suppresses the immune system of honey bee foragers in the field. This parasite is detrimental to the immune system of young and old bees, and disease spread, mitigation and containment will depend on the colony environment.

Testing the multiple stressor hypothesis: chlorothalonil exposure alters transmission potential of a bumblebee pathogen but not individual host health.

Numerous threats are putting pollinator health and essential ecosystem pollination services in jeopardy. Although individual threats are widely studied, their co-occurrence may exacerbate negative effects, as posited by the multiple stressor hypothesis. A prominent branch of this hypothesis concerns pesticide-pathogen co-exposure. A landscape analysis demonstrated a positive association between local chlorothalonil fungicide use and microsporidian pathogen (Nosema bombi) prevalence in declining bumblebee species (Bombus spp.), suggesting an interaction deserving further investigation. We tested the multiple stressor hypothesis with field-realistic chlorothalonil and N. bombi exposures in worker-produced B. impatiens microcolonies. Chlorothalonil was not avoided in preference assays, setting the stage for pesticide-pathogen co-exposure. However, contrary to the multiple stressor hypothesis, co-exposure did not affect survival. Bees showed surprising tolerance to Nosema infection, which was also unaffected by chlorothalonil exposure. However, previously fungicide-exposed infected bees carried more transmission-ready spores. Our use of a non-declining bumblebee and potential higher chlorothalonil exposures under some scenarios could mean stronger individual or interactive effects in certain field settings. Yet, our results alone suggest consequences of pesticide co-exposure for pathogen dynamics in host communities. This underlies the importance of considering both within- and between-host processes when addressing the multiple stressor hypothesis in relation to pathogens.

Potential of Fumagillin and Agaricus blazei Mushroom Extract to Reduce Nosema ceranae in Honey Bees.

Simple SummaryNosemosis is a disease in bees that causes severe problems for their vitality, reproduction and productivity. Traditionally, the treatment involves the application of fumagillin, an antibiotic with proven effect. Recently, fumagillin production and registration has faced problems worldwide, leading to the absence of adequate treatment. Motivated by the reported health issues and the occurrence of residues after fumagillin application, scientists around the world have sought a medication or a supplement that could help beekeepers to control Nosema. Current trends include the search for alternative non-antibiotic treatments. In a laboratory (cage) experiment, we studied the effect of fumagillin and natural extract of mushroom Agaricus blazei on the survival of Nosema infected bees, Nosema spore loads and levels of immune-related gene expression and oxidative stress markers. The results undoubtedly confirmed the anti-Nosema effect of fumagillin, as seen in better bee survival and monitored parameters; its application without Nosema infection (preventive) caused disturbance in some of the parameters. The application of A. blazei extract, however, showed positive effects in both preventive and curative applications. These beneficial properties of A. blazei extract indicate a potential that needs to be further investigated.Depending on the infection level and colony strength, Nosema ceranae, a microsporidian endoparasite of the honey bee may have significant consequences on the health, reproduction and productivity of bee colonies. Despite exerting some side effects, fumagillin is most often used for Nosema control. In this study, in a cage experiment, N. ceranae infected bees were treated with fumagillin or the extract of Agaricus blazei mushroom, a possible alternative for Nosema control. Bee survival, Nosema spore loads, the expression levels of immune-related genes and parameters of oxidative stress were observed. Fumagillin treatment showed a negative effect on monitored parameters when applied preventively to non-infected bees, while a noticeable anti-Nosema effect and protection from Nosema-induced immunosuppression and oxidative stress were proven in Nosema-infected bees. However, a protective effect of the natural A. blazei extract was detected, without any side effects but with immunostimulatory activity in the preventive application. The results of this research suggest the potential of A. blazei extract for Nosema control, which needs to be further investigated.