In vitro evaluation of the effects of some plant essential oils on Ascosphaera apis,the causative agent of Chalkbrood disease

Ascosphaera apis is one of the major fungal pathogens of honey bee broods and the causative agent of Chalkbrood disease. The factors responsible for the pathogenesis of Chalkbrood disease are still not fully understood, and the increasing resistance of A. apis to commonly used antifungal agents necessitates a search for new agents to control this disease. The in vitro antifungal activities of 27 plant essential oils against two isolates of A. apis (Aksu-4 and Aksu-9) were evaluated. Out of the 27 plant essential oils tested, 21 were found to be effective in killing both isolates of A. apis. Based on their minimum fungicidal concentration (MFC) values, the effective oils were grouped into three categories: highly effective, moderately effective and minimally effective. Mountain pepper oil, Kala Bhangra oil, spearmint oil, babuna oil, betel leaf oil, carrot seed oil, cumin seed oil and clove bud oil were highly effective, with MBC values between 50.0 μg/mL and 600.0 μg/mL. Mountain pepper was the most effective essential oil, with an MBC value of 50.0 μg/mL. Citral and caryophyllene containing oils were the most effective with MIC 50 ppm. The essential oils tested exhibited significant antimicrobial activities against both strains of A. apis, and they may contain compounds that could play an important role in the treatment or prevention of Chalkbrood disease of honeybee.     

A reappraisal of orders and families within the subclass Chaetothyriomycetidae (Eurotiomycetes,Ascomycota)

The subclass Chaetothyriomycetidae (Eurotiomycetes, Ascomycota) is an assemblage of ecologically diverse species, ranging from mutualistic lichenised fungi to human opportunistic pathogens. Recent contributions from molecular studies have changed our understanding of the composition of this subclass. Among others, ant-associated fungi, deep-sea fungi and bryophilous fungi were also shown to belong to this group of ascomycetes. The delimitation of orders and families within this subclass has not previously been re-assessed using a broad phylogenetic study and the phylogenetic position of some taxa such as the lichenised family Celotheliaceae or the Chaetothyrialean bryophilous fungi is still unclear. In our study, we assemble new and published sequences from 132 taxa and reconstruct phylogenetic relationships using four markers (nuLSU, nuSSU, mtSSU and RPB1). Results highlight several shortfalls in the current classification of this subclass, mainly due to un-assigned paraphyletic taxa. The family Epibryaceae is therefore described to circumscribe a previously un-assigned lineage. Celotheliales ad int. is suggested for the lineage including the lichen genus Celothelium and various plant pathogens. The delimitation of the family Trichomeriaceae is also broadened to include the genus Knufia and some anamorphic taxa. As defined here, Chaetothyriomycetidae includes four orders (Celotheliales ad int., Chaetothyriales, Pyrenulales, and Verrucariales) and ten families (Adelococcaceae, Celotheliaceae, Chaetothyriaceae, Cyphellophoraceae, Epibryaceae fam. nov., Herpotrichiellaceae, Pyrenulaceae, Requienellaceae, Trichomeriaceae, and Verrucariaceae).     

Bumble Bees (Bombus spp) along a Gradient of Increasing Urbanization

Background

Bumble bees and other wild bees are important pollinators of wild flowers and several cultivated crop plants, and have declined in diversity and abundance during the last decades. The main cause of the decline is believed to be habitat destruction and fragmentation associated with urbanization and agricultural intensification. Urbanization is a process that involves dramatic and persistent changes of the landscape, increasing the amount of built-up areas while decreasing the amount of green areas. However, urban green areas can also provide suitable alternative habitats for wild bees.

Methodology/Principal Findings

We studied bumble bees in allotment gardens, i.e. intensively managed flower rich green areas, along a gradient of urbanization from the inner city of Stockholm towards more rural (periurban) areas. Keeping habitat quality similar along the urbanization gradient allowed us to separate the effect of landscape change (e.g. proportion impervious surface) from variation in habitat quality. Bumble bee diversity (after rarefaction to 25 individuals) decreased with increasing urbanization, from around eight species on sites in more rural areas to between five and six species in urban allotment gardens. Bumble bee abundance and species composition were most affected by qualities related to the management of the allotment areas, such as local flower abundance. The variability in bumble bee visits between allotment gardens was higher in an urban than in a periurban context, particularly among small and long-tongued bumble bee species.

Conclusions/Significance

Our results suggest that allotment gardens and other urban green areas can serve as important alternatives to natural habitats for many bumble bee species, but that the surrounding urban landscape influences how many species that will be present. The higher variability in abundance of certain species in the most urban areas may indicate a weaker reliability of the ecosystem service pollination in areas strongly influenced by human activity.     

Testing Dose-Dependent Effects of the Nectar Alkaloid Anabasine on Trypanosome Parasite Loads in Adult Bumble Bees

The impact of consuming biologically active compounds is often dose-dependent, where small quantities can be medicinal while larger doses are toxic. The consumption of plant secondary compounds can be toxic to herbivores in large doses, but can also improve survival in parasitized herbivores. In addition, recent studies have found that consuming nectar secondary compounds may decrease parasite loads in pollinators. However, the effect of compound dose on bee survival and parasite loads has not been assessed. To determine how secondary compound consumption affects survival and pathogen load in Bombus impatiens, we manipulated the presence of a common gut parasite, Crithidia bombi, and dietary concentration of anabasine, a nectar alkaloid produced by Nicotiana spp. using four concentrations naturally observed in floral nectar. We hypothesized that increased consumption of secondary compounds at concentrations found in nature would decrease survival of uninfected bees, but improve survival and ameliorate parasite loads in infected bees. We found medicinal effects of anabasine in infected bees; the high-anabasine diet decreased parasite loads and increased the probability of clearing the infection entirely. However, survival time was not affected by any level of anabasine concentration, or by interactive effects of anabasine concentration and infection. Crithidia infection reduced survival time by more than two days, but this effect was not significant. Our results support a medicinal role for anabasine at the highest concentration; moreover, we found no evidence for a survival-related cost of anabasine consumption across the concentration range found in nectar. Our results suggest that consuming anabasine at the higher levels of the natural range could reduce or clear pathogen loads without incurring costs for healthy bees.     

In vitro activity of 15-azasterol (A25822B) against chalkbrood pathogen Ascosphaera apis in the honey bee

The antifungal agent 15-azasterol A25822B was examined for effects on the growth and development of Ascosphaera apis. The minimum inhibition concentration (MIC) of azasterol against A. apis was 1 m. Growth and development of A. apis was completely controlled at this concentration. At a concentration of 0.01 m growth of A. apis was retarded and although sporocysts were formed developing spores were not be able to reach maturation. A major effect of azasterol at this low concentration was the accumulation of lipid in the hyphae, sporocysts and immature spores. In addition it caused a conformational change in mitochondria and damage to the spore membrane structure. On the basis of these results, further investigations of azasterol for the treatment of chalkbrood disease in the honey bee are warranted.Work was performed during sabbatical leave at the University of California, Davis.     

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.     

Ultrastructural changes in the spore and mycelia of Ascosphaera apis after treatment with benomyl (Benlate 50 W)

In Ascosphaera apis, after 8 days growth in darkness at 28° C, numerous sporocysts were observed, within which mature spores were seen aggregated into a spore ball. The mature spore of A. apis had a thick spore wall with an electron-opaque outer layer, a spore membrane with many depressions, and sporoplasm containing numerous ribosomes and mitochondria. In the cytoplasm of the mycelium, mitochondria with well-defined cristae and numerous ribosomes were observed. At a concentration of 1 g/ml of culture medium, benomyl appeared to inhibit colony growth of A. apis, but some sporocysts containing deformed spores were found. Deformed spores possessed a thick spore wall with a grainy matrix, and depressions were no longer detected in the spore membrane. Ribosomes were lacking in the sporoplasm and mitochondria appeared degenerate. The mycelium from the treated culture contained mitochondria with an electron-lucid matrix and no well defined cristae, while ribosomes were completely depleted. The significance of these observations in relation to the use of benomyl to control chalkbrood disease in the honey bee is discussed.     

Thresholds of Response in Nest Thermoregulation by Worker Bumble Bees, Bombus bifarius nearcticus (Hymenoptera: Apidae)

Regulation of nest temperature is important to the fitness of eusocial insect colonies. To maintain appropriate conditions for the developing brood, workers must exhibit thermoregulatory responses to ambient temperature. Because nest-mate workers differ in task performance, thermoregulatory behavior provides an opportunity to test threshold of response models for the regulation of division of labor. We found that worker bumble bees ( Bombus bifarius nearcticus ) responded to changes in ambient temperature by altering their rates of performing two tasks – wing fanning and brood cell incubation. At the colony level, the rate of incubating decreased, and the rate of fanning increased, with increasing temperature. Changes in the number of workers performing these tasks were more important to the colony response than changes in workers' task performance rates. At the individual level, workers' lifetime rates of incubation and fanning were positively correlated, and most individuals did not specialize exclusively on either of these temperature-sensitive tasks. However, workers differed in the maximum temperature at which they incubated and in the minimum temperature at which they fanned. More individuals fanned at high and incubated at low temperatures. Most of the workers that began incubating at higher temperatures continued performing this task at lower temperatures, when additional nest-mates became active. The converse was true for fanning behavior. These data are consistent with a threshold of response model for thermoregulatory behavior of B. bifarius workers.     

The Abundance and Pollen Foraging Behaviour of Bumble Bees in Relation to Population Size of Whortleberry (Vaccinium uliginosum)

Habitat fragmentation can have severe effects on plant pollinator interactions, for example changing the foraging behaviour of pollinators. To date, the impact of plant population size on pollen collection by pollinators has not yet been investigated. From 2008 to 2010, we monitored nine bumble bee species (Bombus campestris, Bombus hortorum s.l., Bombus hypnorum, Bombus lapidarius, Bombus pascuorum, Bombus pratorum, Bombus soroensis, Bombus terrestris s.l., Bombus vestalis s.l.) on Vaccinium uliginosum (Ericaceae) in up to nine populations in Belgium ranging in size from 80 m² to over 3.1 ha. Bumble bee abundance declined with decreasing plant population size, and especially the proportion of individuals of large bumble bee species diminished in smaller populations. The most remarkable and novel observation was that bumble bees seemed to switch foraging behaviour according to population size: while they collected both pollen and nectar in large populations, they largely neglected pollen collection in small populations. This pattern was due to large bumble bee species, which seem thus to be more likely to suffer from pollen shortages in smaller habitat fragments. Comparing pollen loads of bumble bees we found that fidelity to V. uliginosum pollen did not depend on plant population size but rather on the extent shrub cover and/or openness of the site. Bumble bees collected pollen only from three plant species (V. uliginosum, Sorbus aucuparia and Cytisus scoparius). We also did not discover any pollination limitation of V. uliginosum in small populations. We conclude that habitat fragmentation might not immediately threaten the pollination of V. uliginosum, nevertheless, it provides important nectar and pollen resources for bumble bees and declining populations of this plant could have negative effects for its pollinators. The finding that large bumble bee species abandon pollen collection when plant populations become small is of interest when considering plant and bumble bee conservation.     

Studies on the Erythrocytes of Larval and Adult Lampreys (Lampetra fluviatilis)

The erythrocytes of larval and adult Lampetra fluviatilis were examined in suspensions of whole blood, solutions of various ionic strengths, scanning electron photomicrographs and air dried smears. Although a wide range of shapes were observed in hyper- and hypotonic solutions, the cells in whole blood and isotonic solutions were predominantly biconcave with an eccentric nucleus. The biconcave shape was also seen in scanning electron photomicrographs of erythrocytes fixed with glutaraldehyde in lamprey Ringer. The mean diameter of larval and adult cells in whole blood suspensions was 11.2 and 11.6 μm respectively. Although in air dried smears the areas of the larval and adult erythrocytes were virtually identical, the erythrocyte nuclear area was markedly higher in ammocoetes with only a small amount of overlap between the values obtained for the two stages in the life cycle.     

Behavior of Adult and Larval Platysoma cylindrica (Coleoptera: Histeridae) and Larval Medetera bistriata (Diptera: Dolichopodidae) During Subcortical Predation of Ips pini (Coleoptera: Scolytidae)

The most abundant predators of the pine engraver, Ips pini (Say), include Platysoma cylindrica (Paykull) and Medetera bistriata Parent. We amended bark sandwiches with pine engravers and adult P. cylindrica and larval M. bistriata, and report observations on these endophytic predators. Adult P. cylindrica preferred adult prey, and appeared to be facultative egg predators, which contrasts with other Histeridae. The larvae of P. cylindrica fed on pine engraver larvae. Protection from predators is generally assumed to be an advantage of an endophytic life history, but we found that this predator exploited the herbivore's gallery architecture to facilitate prey capture. Larval M. bistriata also fed on pine engraver larvae. Immediate paralysis or death of the prey followed a strike with their tentorial rods. Such rapid immobilization, in conjuction with the glandular histology of Medetera spp., suggests that they use a toxin to arrest prey.     

First and southernmost records of Hirsutella (Ascomycota: Hypocreales) and Pandora (Zygomycota: Entomophthorales) species infecting Dermaptera and Psocodea

We present the first and southernmost records of the fungi Hirsutella strigosa Petch, H. citriformis Speare (Ascomycota: Hypocreales), and Pandora nouryi (Remaudière & Hennebert) Humber (Zygomycota: Entomophthorales) infecting Doru lineare (Eschscholtz) (Dermaptera: Forficulidae), Ectopsocus californicus (Banks) (Psocodea: Ectopsocidae), and Heterocaecilius sp. (Psocodea: Pseudocaeciliidae), respectively. This is the first report of naturally occurring pathogenic fungi infecting Psocoptera, and it is also the first report of P. nouryi from any host outside of the Aphididae. The three fungal species were morphologically described from their host insects and from microscopic preparations. Attempts to obtain pure fungal isolates were unsuccessful but slides and photographs of these fungi were preserved and deposited in mycological collections as herbarium material.     

First record of Erysiphe quercicola (Ascomycota: Erysiphales) on species of Quercus subgenus Cyclobalanopsis (evergreen oaks,Fagaceae)

During the DNA sequence analyses of specimens on Quercus subgenus Cyclobalanopsis deposited as Erysiphe gracilis or Cystotheca wrightii, some specimens exhibited DNA sequences that are different from the sequences of the two species. Based on BLAST search and morphological observations, the true causal agent was identified as E. quercicola. This is the first record of E. quercicola on species of subgenus Cyclobalanopsis. Previous studies revealed that E. quercicola is plurivorous infecting a wide range of tropical fruit trees and woody plants. Further studies are needed to address the question as to why only E. quercicola infect species of subgenus Cyclobalanopsis.     

Molecular phylogeny of the Laboulbeniomycetes (Ascomycota)

A first molecular-based phylogeny is presented for the Laboulbeniomycetes, a group of ascomycete fungi that utilize arthropods for nutrition and/or dispersal. Morphological diversification and life-history evolution has made it difficult to resolve relationships within the group, and to identify close relatives. Here, we infer a preliminary phylogeny based on acquisition of 51 new SSU rDNA sequences, representing a total of 65 taxa. The results of this study demonstrate that Laboulbeniomycetes is monophyletic, and related to Sordariomycetes. The class could be divided into at least 4 or 5 orders, though we refrain from formally giving names to these at this stage. Further evidence for the occurrence of asexuality within the class is provided by the inclusion of the genera Chantransiopsis and Tetrameronycha, both known only as asexual taxa with thalli consisting of linearly superposed cells. The precise placement of the genus Herpomyces (Herpomycetaceae), on cockroaches, remains unresolved in our analysis, but lies outside of the main clade of sexually reproducing Laboulbeniales. There is good support for this latter grouping, comprising taxa that are found on both aquatic and terrestrial hosts. Within this large assemblage, we recognize 5 distinct clades (clades E, F, G, H, I). Relationships among the so-called “aquatic genera” (≡ Ceratomycetaceae + some Euceratomycetaceae and Zodiomyces) are poorly resolved in our analyses, accounting for 3 of these clades (E, F, G), with the remainder of the taxa (largely equivalent to Laboulbeniaceae) split into two major groupings (clades H, I). Across all taxa, antheridial characteristics, features on which the earliest classifications were based, are shown to be homoplastic. On the other hand, features of perithecial development show an overall trend towards reduction, and appear to be phylogenetically informative. Morphological characters are identified that support the dichotomy in the Laboulbeniaceae and subclades within the two major groupings are discussed further in light of information on thallus morphology, development, and host relationships.     

中华蜜蜂6日龄幼虫肠道响应球囊菌胁迫的差异表达基因分析

蜜蜂球囊菌特异性侵染蜜蜂幼虫而导致白垩病,严重危害养蜂生产。本研究利用RNA-seq技术对健康及球囊菌胁迫的中蜂幼虫肠道进行深度测序,进而对宿主的差异表达基因进行深入分析。本研究中,幼虫肠道样品的RNA-seq共得到191167730条原始读段(raw reads),经过滤得到186284296条有效读段(clean reads),差异表达基因(DEG)分析结果显示上调与下调基因的数量分别为4513和385个。Gene ontology(GO)富集分析结果显示,上调基因富集在45个GO条目(term),富集基因数最多的是细胞进程、代谢进程及催化活性,下调基因富集在32个GO term,富集基因数最多的是代谢进程、单组织进程及催化活性,KEGG代谢通路(pathway)富集分析结果显示上调基因富集在193个pathway,其中富集基因数最多的是核糖体、氨基酸的合成、碳代谢。下调基因富集在59个pathway,其中富集基因数最多的是甘氨酸、碳代谢以及二羧酸代谢。深入分析发现宿主的细胞免疫被显著激活,体液免疫中的Toll-like与Jak-STAT信号通路也被球囊菌所激活。研究结果为揭示中蜂幼虫在球囊菌入侵后期的胁迫应答机制提供了重要的信息,也为解析中蜂幼虫的球囊菌抗性机制奠定了基础。     

Mating Behavior in Halictine Bees (Hymenoptera: Halictidae)

Studied two types of mate-orientation behavior in ♂♂ of the sweat bee, Lasioglossum rohweri: microterritoriality and patrolling. Observations were made in both the laboratory and the field. These ♂♂ behave uniquely for Hymenoptera in that 2–5 of them establish microterritories immediately around the same nest entrance. Some ♂♂ are exclusively patrollers, flying about flowers and nest sites. Rendezvous places (locations where animals are likely to find mates) in bees are: flowers, nesting sites, vegetative parts of plants, nest entrances, air, and nests. Territoriality in male bees may have arisen independently in 7 of the 9 families of bees.     

First record of Clonostachys rosea (Ascomycota: Hypocreales) as an entomopathogenic fungus of Oncometopia tucumana and Sonesimia grossa (Hemiptera: Cicadellidae) in Argentina

Clonostachys rosea (Link: Fries) Schroers, Samuels, Seifert, and Gams (Ascomycota: Hypocreales) has been reported as a mycoparasite of fungi and nematodes and as saprobe in soils, but this fungus has not been reported previously to be entomopathogenic. Many species of cicadellid leafhoppers cause economic damage to crops as vectors of plant pathogens. In the present work, we report the first record of C. rosea as an entomopathogenic fungus of two leafhoppers pest, Oncometopia tucumana and Sonesimia grossa (Hemiptera: Cicadellidae), in Argentina and evaluate the pathogenicity of C. rosea against them.