Fungus Ascosphaera Apis Research Articles

Chalkbrood Disease Caused by Ascosphaera apis in Honey Bees (Apis mellifera)-Morphological and Histological Changes in Infected Larvae.

Chalkbrood is a mycological brood disease of the Western honey bee (Apis mellifera), caused by the fungus Ascosphaera apis. The aim of this study was the investigation of the pathology of artificially reared Apis mellifera larvae, experimentally infected with A. apis spores (1.0 × 103 spores/larva). Non-infected larvae served as control. Five living larvae and every dead larva were collected daily (day 1-7 p.i.). All larvae were macroscopically measured, photographed, formalin-fixed, and histologically processed (hematoxylin-eosin stain, Grocott silvering). Histological sections were digitized, and the size of the larvae was measured (mouth-after length, area) and statistically analyzed. Twenty-six larvae from the collected larvae (n = 64; 23 dead, 3 alive) showed histological signs of infection from 3 d p.i. onwards. The dead larvae showed macroscopically white/brown deposits, indistinct segmentation, and a lack of body elongation. Infected larvae were significantly smaller than the controls on days 3 p.i. (p < 0.05), 4 p.i. (p < 0.001), and 6 p.i. (p < 0.05). The early time of death, the low number of transitional stages, and the strong penetration of the larval carcass with fungal mycelium indicate a rapid and fulminant infection process, which is probably relevant for spreading the disease within the colony.

Widespread distribution of honey bee-associated pathogens in native bees and wasps: Trends in pathogen prevalence and co-occurrence

Pollinators have experienced significant declines in the past decade, in part due to emerging infectious diseases. Historically, studies have primarily focused on pathogens in the Western honey bee, Apis mellifera. However, recent work has demonstrated that these pathogens are shared by other pollinators and can negatively affect their health. Here, we surveyed honey bees and 15 native bee and wasp species for 13 pathogens traditionally associated with honey bees. The native bee and wasp species included 11 species not previously screened for pathogens. We found at least one honey bee-associated pathogen in 53% of native bee and wasp samples. The most widely distributed and commonly detected pathogens were the microsporidian Nosema ceranae, the bacterium Melissococcus plutonius, and the viruses deformed wing virus and black queen cell virus. The prevalence of viruses was generally higher in honey bees than in native bees and wasps. However, the prevalence of M. plutonius and the brood fungus Ascosphaera apis was significantly higher in some native bee species than in honey bees. The data also reveal novel trends in the association between co-occurring pathogens in honey bees and native bees and wasps at the pathogen community level. These results can inform the assessment of risks that native pollinator species face from pathogen stress, and indicate that many non-viral pathogens, notably M. plutonius and N. ceranae, are far more widely distributed and commonly found in native bees and wasps than previously thought.

Pathogenicity bioassays of Ascosphaera apis strains from Spanish provinces in bee larvae from Northern Argentina

Beekeeping is one of the main economic activities in several countries, and Argentina has become one of the most important exporters of honey and other products from hives. Ascosferosis, an emerging disease caused by the fungus Ascosphaera apis, affects bee larvae, causing mortality in the colonies. While many studies have focused on the infections of European Apis mellifera bees, we studied hybrid Apis mellifera scutellata bees from northern Argentina and fungal strains isolated from commercial pollen from Spanish provinces, to determine the susceptibility of local bees to the entomopathogenic A. apis. Different groups of larvae were administered sugar syrup suspensions with ascospores at different concentrations: a lower concentration of 5 × 103 spores/mL was used to know the susceptibility of the larvae and a higher concentration of 5 × 106 spores/mL was used to determine the pathogenicity, resulting in an average infection of 30% of the mummified larvae. Besides, an intermediate concentration of 5 × 104 spores/mL was administered, which allowed us to identify the most pathogenic strain at this concentration. This work provides the first approximation to the pathogenic effect of Spanish fungal strains on larvae of bees from northern Argentina.

Genetic variation of Ascosphaera apis and colony attributes do not explain chalkbrood disease outbreaks in Australian honey bees

Chalkbrood infection caused by the fungus Ascosphaera apis currently has a significant impact on Australia’s apicultural industry. We investigated the genetic variation of A. apis and colony and apiary level conditions to determine if an emerging, more virulent strain or specific conditions were responsible for the prevalence of the disease. We identified six genetically distinct strains of A. apis, four have been reported elsewhere and two are unique to Australia. Colonies and individual larvae were found to be infected with multiple strains of A. apis, neither individual strains, combinations of strains, or obvious colony or apiary characteristics were found to be predictive of hive infection levels. These results suggest that host genotype plays an important role in colony level resistance to chalkbrood infection in Australia.

Strains of Lactobacillus spp. reduce chalkbrood in Apis mellifera

Beekeeping activities have increased recently in Argentina, a country that is a major consumer of honey and other products from hives. With the advancement of monoculture areas in Argentina and worldwide, beekeepers move from one area to another in search of floral resources, thus spreading diseases such as chalkbrood, caused by the fungus Ascosphaera apis. Although there are few effective antifungals for the control of chalkbrood, different natural products have been investigated in recent years. Current research is focusing on the intestinal microbiota for the prevention of different pathogens and parasites. In this work, we analyzed the in vivo probiotic effect of three lactic acid bacteria (genus Lactobacillus spp.) isolated from pollen bread from apiaries of Jujuy province on A. apis strains from Spanish and Argentine provinces. Special hives were made for the assays, and a protective effect was observed in larvae of bees fed lactic acid bacteria added to sugar syrup at 105 CFU/mL concentrations, administered from May to September in two consecutive years. The results showed that the three lactic acid bacteria reduced larval mummification by percentages greater than 80%. Therefore, this work brings a first approximation of the in vivo probiotic effect of lactic bacteria against A. apis.

Environmental gut bacteria in European honey bees (Apis mellifera) from Australia and their relationship to the chalkbrood disease.

We report on aerobic “environmental” bacteria isolated from European honey bees (Apis mellifera). We determined the number of culturable aerobic bacteria in the gut of nurse bees sampled from locations around Australia. Bees from healthy colonies had 107–108 aerobic bacteria per g of bee gut, while bees from colonies with chalkbrood consistently had significantly fewer bacteria (104–105 bacteria per g). When colonies recovered from chalkbrood, bacterial numbers returned to normal levels, suggesting that counting aerobic bacteria in the gut could be used to predict an outbreak of the disease. Furthermore, Western Australian bees from the “Better Bees” program (bred to promote hygienic behaviour) had significantly higher numbers of aerobic gut bacteria compared to regular bees from healthy colonies. Bacteria with the ability to inhibit the chalkbrood pathogen were found in most bees from regular colonies (> 60%) but only in a few “Better Bees” (10%). Phylogenetic analysis of aerobic bacterial isolates that inhibited the chalkbrood pathogen revealed a close relationship (>97% sequence identity) to the genera Bacillus, Klebsiella, Pantoea, Hafnia, and Enterobacter (bacteria that have previously been isolated from honey bees), but we also isolated Maccrococcus and Frigoribacterium species (bacteria that were not previously identified in bees). Finally, we investigated the ability of bacteria to inhibit the chalkbrood fungus Ascosphaera apis. Mass spectroscopy analysis revealed that the bee gut isolates Frigoribacterium sp. and Bacillus senegalensis produce gluconic acid. We further found that this simple sugar is involved in chalkbrood fungal hyphal lysis and cytoplasmic leakage. Our findings suggest that “environmental” gut bacteria may help bees to control the chalkbrood pathogen.

Changes in Antioxidant Enzymes Activity and Metabolomic Profiles in the Guts of Honey Bee (Apis mellifera) Larvae Infected with Ascosphaera apis.

The fungus Ascosphaera apis, an obligate fungal pathogen of honey bee brood, causes chalkbrood disease in honey bee larvae worldwide. Biological characteristics of the fungal pathogen and the molecular interactions between A. apis and honey bees have been studied extensively. However, little is known about the effects of A. apis infection on antioxidant enzyme activities and metabolic profiles of the gut of honey bee larvae. In this study, sandwich enzyme-linked immunosorbent assay and LC-MS based untargeted metabolomic analysis were employed to determine the changes in the specific activities of antioxidant enzymes and the metabolomic profiles in gut tissues of A. apis-infected larvae (105 A. apis spores per larva) and controls. Results showed that specific activities of superoxide dismutase, catalase and glutathione S-transferase were significantly higher in the guts of the control larvae than in the guts of the A. apis-infected larvae. The metabolomic data revealed that levels of 28 and 52 metabolites were significantly higher and lower, respectively, in the guts of A. apis-infected larvae than in the guts of control larvae. The 5-oxo-ETE level in the infected larvae was two times higher than that in the control larvae. Elevated 5-oxo-ETE levels may act as a potential metabolic biomarker for chalkbrood disease diagnosis, suggesting that A. apis infection induced obvious oxidative stress in the honey bee larvae. The levels of metabolites such as taurine, docosahexaenoic acid, and L-carnitine involved in combating oxidative stress were significantly decreased in the gut of A. apis-infected larvae. Overall, our results suggest that A. apis infection may compromise the ability of infected larvae to cope with oxidative stress, providing new insight into changing patterns of physiological responses to A. apis infection in honey bee larvae by concurrent use of conventional biochemical assays and untargeted metabolomics.

Etiology, symptoms and prevention of chalkbrood disease: a literature review

ABSTRACT The fungus Ascosphaera apis, responsible for causing the chalkbrood disease of honey bees, is widely present in temperate regions of the northern hemisphere, but has also spread to other regions of the world such as Brazil. Although it is not usually lethal for the colony, it can reduce its population, hampering its development. This study is a review on the disease that presents a broad overview of its development, identification methods as well as ways to control it. Research shows that chalkbrood is associated with several factors and is most frequently found in colonies of Apis bees during the spring, when there is excess humidity and sudden temperature changes in the hive. Other factors such as viral or bacterial infection, the presence of the ectoparasite Varroa destructor, pesticide poisoning and poor nutrition of nurse bees can also affect its incidence and severity. Field diagnosis is made based on the presence of hardened mummified brood in the pupal stage, of white or black color, in the cells and entrance. Affected cells show dead pupae covered with white mycelia, resembling cotton, or hardened, dry and brittle, resembling chalk pieces, which originated the name. To date, there are no efficient methods to reduce the damage caused by chalkbrood. Genetic selection of bees with higher hygienic behavior and disease resistance is recommended.

The presence of a larval honey bee parasite, Ascosphaera apis, on flowers reduces pollinator visitation to several plant species

Many floral traits have evolved through pollinator-mediated selection. In selecting flowers for forage, pollinators rely on multi-modal flower signals and innate preferences. However, pollinators are known for their flexible foraging behavior which varies with experience and numerous cues, such as the presence of competitors and floral resource availability. The shared use of flowers by many pollinators leads to high rate of parasite transmission among pollinators, and flowers can be seen as hot-spots of parasite dispersal. Throughout the animal kingdom, behavioral adaptations enable avoidance of parasite exposure. Bumblebees are known to avoid contaminated flowers, however nothing is known regarding the impact of parasite presence on overall pollinator visitation. In this study, we measured the effect of a larval honey bee parasite, the fungus Ascosphaera apis causing chalkbrood disease, on hymenopteran pollinator visitation of several plant species in a natural setting. Several contaminated flowering plant species suffered not only the subsequent reduction of visitation by honey bees but also the subsequent reduction of their overall pollinator visitation. Two plant species with similar flower architecture were the least impacted by the presence of the spores. This study highlights the negative impact of the presence of pollinator parasites for plants and its possible consequences for floral evolution.

Plant Essential Oils Used Against Some Bee Diseases

The most common honey bee diseases are American foulbrood (AFB) caused by the bacterium Paenibacillus larvae, Chalkbrood caused by fungus Ascosphaera apis and diseases caused by parasitic mites such as Acarapis woodi, Varroa destructor. These diseases and pests not only cause economic loss but also cause ecological problems related to the role of honey bees, as the most important pollinators on Earth. Synthetic acaricides and antibiotics are used to keep the diseases and mites in control. Use of the drugs lead to the development of drug-resistant organisms, detrimental effect on non-target organisms and the residue problem in bee products. For this reasons, the need for alternative control methods has become compulsory in recent years. It has been known that some plant oils used widely in perfumery and food industry for flavor and smell have been used as repellent to certain insects, bactericide and fungicide. Therefore, intensive studies have been carried out on plants with anti-mites, antibacterial and antifungal potentials and these studies are still going on. Recently, studies in this area have shown that essential oils of plants such as thyme, cloves, mint, lemon grass, cinnamon, grapefruit, rosemary, marigold, are lethal to some mites, bacteria and fungi. In addition, it has been reported that some components, isolated from these plants such as sanguinarine, thymoquinone, capsaicin, carvacrol, citral, eugenol, thymol, show these effects on the organisms. As a result, in countries rich in biodiversity due to endemic plant species, the essential oils used in control of these diseases should be favored instead of or in combination with conventional drugs in integrated the disease management programs because of the lack of harmful effects of essential oils on non-target organisms and environment.

Fine mapping identifies significantly associating markers for resistance to the honey bee brood fungal disease, Chalkbrood

SummaryChalkbrood infection of honey bee (Apis mellifera) brood by the fungus Ascosphaera apis results in fatal encapsulation of susceptible larvae with a mycelial coat. Recent QTL analysis indicates that some level of physiological resistance exists in individual larvae. We performed a fine mapping analysis to define the genetic interval that confers resistance in the larvae and identify the strongest association molecular markers that could by useful for marker assisted selection of the trait. Evaluation of the interval suggests that only two possible genes (single Ig IL-related receptor-like, XM_003251514.1 and juvenile hormone-binding protein, XM_391872.4) are likely to be responsible for the resistance. Both genes are strong physiological candidates and potentially function as modulators of the antifungal-specific innate immunity pathway in insects.

Transcriptome analysis of the honey bee fungal pathogen, Ascosphaera apis: implications for host pathogenesis

BackgroundWe present a comprehensive transcriptome analysis of the fungus Ascosphaera apis, an economically important pathogen of the Western honey bee (Apis mellifera) that causes chalkbrood disease. Our goals were to further annotate the A. apis reference genome and to identify genes that are candidates for being differentially expressed during host infection versus axenic culture.ResultsWe compared A. apis transcriptome sequence from mycelia grown on liquid or solid media with that dissected from host-infected tissue. 454 pyrosequencing provided 252 Mb of filtered sequence reads from both culture types that were assembled into 10,087 contigs. Transcript contigs, protein sequences from multiple fungal species, and ab initio gene predictions were included as evidence sources in the Maker gene prediction pipeline, resulting in 6,992 consensus gene models. A phylogeny based on 12 of these protein-coding loci further supported the taxonomic placement of Ascosphaera as sister to the core Onygenales. Several common protein domains were less abundant in A. apis compared with related ascomycete genomes, particularly cytochrome p450 and protein kinase domains. A novel gene family was identified that has expanded in some ascomycete lineages, but not others. We manually annotated genes with homologs in other fungal genomes that have known relevance to fungal virulence and life history. Functional categories of interest included genes involved in mating-type specification, intracellular signal transduction, and stress response. Computational and manual annotations have been made publicly available on the Bee Pests and Pathogens website.ConclusionsThis comprehensive transcriptome analysis substantially enhances our understanding of the A. apis genome and its expression during infection of honey bee larvae. It also provides resources for future molecular studies of chalkbrood disease and ultimately improved disease management.

Polymorphic DNA sequences of the fungal honey bee pathogen Ascosphaera apis

The pathogenic fungus Ascosphaera apis is ubiquitous in honey bee populations. We used the draft genome assembly of this pathogen to search for polymorphic intergenic loci that could be used to differentiate haplotypes. Primers were developed for five such loci, and the species specificities were verified using DNA from nine closely related species. The sequence variation was compared among 12 A.apis isolates at each of these loci, and two additional loci, the internal transcribed spacer of the ribosomal RNA (ITS) and a variable part of the elongation factor 1α (Ef1α). The degree of variation was then compared among the different loci, and three were found to have the greatest detection power for identifying A.apis haplotypes. The described loci can help to resolve strain differences and population genetic structures, to elucidate host-pathogen interaction and to test evolutionary hypotheses for the world's most important pollinator: the honey bee and one of its most common pathogens.

Resistance to Chalkbrood Disease in Apis mellifera L. (Hymenoptera: Apidae) Colonies with Different Hygienic Behaviour

Chalkbrood disease affects the larvae of honeybees Apis mellifera L. and is caused by the fungus Ascosphaera apis. Infected larvae die when they are stretched in the cap cell and suffer a gradual hardening that ends in a very hard structure (mummie). Several studies have demonstrated that colonies that express an efficient hygienic behaviour (uncapping of cell and subsequent removal of dead brood) exhibit a higher resistance to the disease. However, it remains unclear whether the advantage of hygienic colonies over less hygienic ones lies in the ability to remove mummies or in the early detection of infected larvae and its cannibalization before they harden. To elucidate this aspect, the hygienic behaviour of 24 colonies, which were subsequently provided with pollen cakes containig A. apis, was evaluated. The number of mummies and the number of partially cannibalized and whole larvae in uncapped cells were recorded. The most hygienic colonies controlled the disease better. These colonies also had a higher tendency to uncap cells that contained infected larvae and cannibalize them. The presence of A. apis in partially cannibalized and whole larvae in uncapped cells indicate that the advantage of hygienic colonies over less hygienic ones lies in the early detection of infected larvae death and their quick removal from the cell before they become mummies.

Intracolonial genetic variability in honeybee larval resistance to the chalkbrood and American foulbrood parasites

The origin of multiple mating of queens in social Hymenoptera is a widely debated topic in evolutionary biology. One of the hypotheses is that genetic variability would benefit the colony by increasing its resistance to parasites through various mechanisms. One among the predictions of this hypothesis is that the resistance of different patrilines within a colony to parasites of different species should be independent, as a result of independent gene-for-gene interactions with each parasite. To test this aspect of the hypothesis, two honeybee colonies (Apis mellifera) were infected with the fungus Ascosphaera apis and two colonies with both A. apis and the American foulbrood bacterium Paenibacillus larvae. Patrilines were found to vary in resistance of larvae to A. apis in all four colonies, but similar variation in resistance was not found to P. larvae. Common resistance to both pathogens was not detected. This study supports the hypothesis that polyandry in social insects could have originated as an adaptation to decrease the impact of diseases.

Microsatellite loci for the fungus Ascosphaera apis: cause of honey bee chalkbrood disease

The fungus Ascosphaera apis is a worldwide fungal pathogen of honey bees. To provide tools for understanding the dispersal history of this pathogen, strain differences in virulence, and host-pathogen interactions, we used the draft genome assembly of A. apis to develop microsatellite loci for this species. We present testing results for 25 scorable loci revealing two to eight alleles per locus in a survey of Maryland isolates of this fungus.

Neric acid derivatives produced by the honey bee fungal entomopathogen Ascosphaera apis

Two yet unknown from natural sources monoterpenoids, 1 and 2, were isolated from a culture of the honey bee entomopathogenic fungus Ascosphaera apis and their structures were determined by spectroscopic methods. These compounds resembled certain pheromones of honey bees. Compounds 1 and 2 showed weak antifungal and antioxidant properties and 1 exhibited antibiotic activity. The compound p-hydroxyphenylethanol, showing antifungal properties, was also isolated and identified.

Genome sequences of the honey bee pathogens Paenibacillus larvae and Ascosphaera apis.

Genome sequences offer a broad view of host–pathogen interactions at the systems biology level. With the completion of the sequence of the honey bee, interest in the relevant pathogens is heightened. Here we report the genome sequences of two of the major pathogens of honey bees, the bacterium Paenibacillus larvae (causative agent for American foulbrood disease) and the fungus Ascosphaera apis. (causative agent for chalkbrood disease). Ongoing efforts to characterize the genomes of these species can be used to understand and mitigate the effects of two important pathogens, and will provide a contrast with pathogenic, benign and freeliving relatives.

Multiple micro-organisms in chalkbrood mummies: evidence and implications

SUMMARYThe pathogenic fungus Ascosphaera apis is the causative agent of the brood disease chalkbrood in honey bee (Apis mellifera) larvae. Infected, hardened larvae—in a powdered form—have been used to inoculate experimental honey bee colonies. While attempting to grow a pure isolate of A. apis from chalkbrood infected larvae, we discovered (via Gram staining and sequencing of the 5.8S rDNA region) that the inoculum also harbours many species of bacteria and additional species of fungi. Some of these fungi have been identified previously as antagonists of A. apis. It is not surprising that bacteria and fungi grow on dead bees; however, our data indicate that previous experiments intended to study the effects of A. apis in honey bees have rather studied the response of honey bees to a cocktail of fungal and bacterial agents. Accordingly, our results indicate that previous research on the A. apis-honey bee disease system was not specific to A. apis and raise the possibility that the observed honey bee response may have been muted, magnified, or both.

In Vitro Fungistatic Effect of Essential Oils Against Ascosphaera apis

Different essential oils were tested against the fungus Ascosphaera apis, the causal agent of Chalkbrood Disease of honey bees. Essential oils from lavandin (Lavandula x intermedia Emeric ex Loiseleur), coriander (Coriandrum sativum L.), laurel leaf (Laurus nobilis L.), false camphor (Cinnamomum glandulifera Nees), basil (Ocimum basilicum L.), tagetes (Tagetes minuta L.), rosemary (Rosmarinus officinalis L.) and eucalyptus (Eucalyptus globulus Labill.) were assayed to prove their fungistatic activity at different concentrations: 700, 800 and 900 μ.L/L. Strains of Ascosphaera apis were collected from apiaries of different places of Buenos Aires province (Argentina). At all concentrations tested, coriander oil was the most effective fungistatic control while basil and tagetes oils were effective only at 800 μL/L. No differences in effectiveness of oils were observed against the different strains of Ascosphaera apis.