Honeybee intestines retain low yeast titers, but no bacterial mutualists, at emergence.

Honeybee symbionts, predominantly bacteria, play important roles in honeybee health, nutrition, and pathogen protection, thereby supporting colony health. On the other hand, fungi are often considered indicators of poor bee health, and honeybee microbiome studies generally exclude fungi and yeasts. We hypothesized that yeasts may be an important aspect of early honeybee biology, and if yeasts provide a mutual benefit to their hosts, then honeybees could provide a refuge during metamorphosis to ensure the presence of yeasts at emergence. We surveyed for yeast and fungi during pupal development and metamorphosis in worker bees using fungal-specific quantitative polymerase chain reaction (qPCR), next-generation sequencing, and standard microbiological culturing. On the basis of yeast presence in three distinct apiaries and multiple developmental stages, we conclude that yeasts can survive through metamorphosis and in naïve worker bees, albeit at relatively low levels. In comparison, known bacterial mutualists, like Gilliamella and Snodgrassella, were generally not found in pre-eclosed adult bees. Whether yeasts are actively retained as an important part of the bee microbiota or are passively propagating in the colony remains unknown. Our demonstration of the constancy of yeasts throughout development provides a framework to further understand the honeybee microbiota.

Colony-Level Effects of Amygdalin on Honeybees and Their Microbes.

Amygdalin, a cyanogenic glycoside, is found in the nectar and pollen of almond trees, as well as in a variety of other crops, such as cherries, nectarines, apples and others. It is inevitable that western honeybees (Apis mellifera) consistently consume amygdalin during almond pollination season because almond crops are almost exclusively pollinated by honeybees. This study tests the effects of a field-relevant concentration of amygdalin on honeybee microbes and the activities of key honeybee genes. We executed a two-month field trial providing sucrose solutions with or without amygdalin ad libitum to free-flying honeybee colonies. We collected adult worker bees at four time points and used RNA sequencing technology and our HoloBee database to assess global changes in microbes and honeybee transcripts. Our hypothesis was that amygdalin will negatively affect bee microbes and possibly immune gene regulation. Using a log2 fold-change cutoff at two and intraday comparisons, we show no large change of bacterial counts, fungal counts or key bee immune gene transcripts, due to amygdalin treatment in relation to the control. However, relatively large titer decreases in the amygdalin treatment relative to the control were found for several viruses. Chronic bee paralysis virus levels had a sharp decrease (-14.4) with titers then remaining less than the control, Black queen cell virus titers were lower at three time points (<-2) and Deformed wing virus titers were lower at two time points (<-6) in amygdalin-fed compared to sucrose-fed colonies. Titers of Lotmaria passim were lower in the treatment group at three of the four dates (<-4). In contrast, Sacbrood virus had two dates with relative increases in its titers (>2). Overall, viral titers appeared to fluctuate more so than bacteria, as observed by highly inconstant patterns between treatment and control and throughout the season. Our results suggest that amygdalin consumption may reduce several honeybee viruses without affecting other microbes or colony-level expression of immune genes.

Natural Product Medicines for Honey Bees: Perspective and Protocols.

The western honey bee remains the most important pollinator for agricultural crops. Disease and stressors threaten honey bee populations and productivity during winter- and summertime, creating costs for beekeepers and negative impacts on agriculture. To combat diseases and improve overall bee health, researchers are constantly developing honey bee medicines using the tools of microbiology, molecular biology and chemistry. Below, we present a manifesto alongside standardized protocols that outline the development and a systematic approach to test natural products as 'bee medicines.' These will be accomplished in both artificial rearing conditions and in colonies situated in the field. Output will be scored by gene expression data of host immunity, bee survivorship, reduction in pathogen titers, and more subjective merits of the compound in question. Natural products, some of which are already encountered by bees in the form of plant resins and nectar compounds, provide promising low-cost candidates for safe prophylaxis or treatment of bee diseases.

Effects of a Resident Yeast from the Honeybee Gut on Immunity, Microbiota, and Nosema Disease.

The western honeybee (Apis mellifera) has a core bacterial microbiota that is well described and important for health. Honeybees also host a yeast community that is poorly understood with respect to host nutrition and immunity, and also the symbiotic bacterial microbiota. In this work, we present two studies focusing on the consequences of dysbiosis when honeybees were control-fed a yeast that was isolated from a honeybee midgut, Wickerhamomyces anomalus. Yeast augmentation for bees with developed microbiota appeared immunomodulatory (lowered immunity and hormone-related gene expression) and affected the microbial community, while yeast augmentation for newly emerged bees without an established bacterial background did not lead to decreased immunity- and hormone-related gene expression. In newly emerged bees that had a naturally occurring baseline level of W. anomalus, we observed that the addition of N. ceranae led to a decrease in yeast levels. Overall, we show that yeasts can affect the microbiome, immunity, and physiology.

Draft Genome Sequence of the Yeast Kodamaea ohmeri, a Symbiont of the Small Hive Beetle.

Kodamaea ohmeri is a symbiont of the small hive beetle (SHB), which is a scavenger of honey bee colonies. The SHB causes absconding of the economically important honey bee (Apis mellifera) and deposits K. ohmeri in the honeycomb. We describe long-read sequencing and further analyses of the K. ohmeri genome.

Analysis of basidiomycete pigments in situ by Raman spectroscopy.

Basidiomycetes, that is, mushroom-type fungi, are known to produce pigments in response to environmental impacts. As antioxidants with a high level of unsaturation, these compounds can neutralize highly oxidative species. In the event of close contact with other microbes, the enzymatically controlled pigment production is triggered and pigment secretion is generated at the interaction zone. The identification and analysis of these pigments is important to understand the defense mechanism of fungi, which is essential to counteract an uncontrolled spread of harmful species. Usually, a detailed analysis of the pigments is time consuming as it depends on laborious sample preparation and isolation procedures. Furthermore, the applied protocols often influence the chemical integrity of the compound of interest. A possibility to noninvasively investigate the pigmentation is Raman microspectroscopy. The methodology has the potential to analyze the chemical composition of the sample spatially resolved at the interaction zone. After the acquisition of a representative spectroscopic library, the pigment production by basidiomycetes was monitored for during response to different fungi and bacteria. The presented results describe a very efficient noninvasive way of pigment analysis which can be applied with minimal sample preparation.

Dissimilar pigment regulation in Serpula lacrymans and Paxillus involutus during inter-kingdom interactions.

Production of basidiomycete atromentin-derived pigments like variegatic acid (pulvinic acid-type) and involutin (diarylcyclopentenone) from the brown-rotter Serpula lacrymans and the ectomycorrhiza-forming Paxillus involutus, respectively, is induced by complex nutrition, and in the case of S. lacrymans, bacteria. Pigmentation in S. lacrymans was stimulated by 13 different bacteria and cell-wall-damaging enzymes (lytic enzymes and proteases), but not by lysozyme or mechanical damage. The use of protease inhibitors with Bacillus subtilis or heat-killed bacteria during co-culturing with S. lacrymans significantly reduced pigmentation indicating that enzymatic hyphal damage and/or released peptides, rather than mechanical injury, was the major cause of systemic pigment induction. Conversely, no significant pigmentation by bacteria was observed from P. involutus. We found additional putative transcriptional composite elements of atromentin synthetase genes in P. involutus and other ectomycorrhiza-forming species that were absent from S. lacrymans and other brown-rotters. Variegatic and its precursor xerocomic acid, but not involutin, in return inhibited swarming and colony biofilm spreading of Bacillus subtilis, but did not kill B. subtilis. We suggest that dissimilar pigment regulation by fungal lifestyle was a consequence of pigment bioactivity and additional promoter motifs. The focus on basidiomycete natural product gene induction and regulation will assist in future studies to determine global regulators, signalling pathways and associated transcription factors of basidiomycetes.

Bacteria induce pigment formation in the basidiomycete Serpula lacrymans.

Basidiomycete fungi are characterized ecologically for their vital functional role in ecosystem carbon recycling and chemically for their capacity to produce a diverse array of small molecules. Chromophoric natural products derived from the quinone precursor atromentin, such as variegatic acid and involutin, have been shown to function in redox cycling. Yet, in the context of an inter-kingdom natural system these pigments are still elusive. Here, we co-cultured the model saprotrophic basidiomycete Serpula lacrymans with an ubiquitous terrestrial bacterium, either Bacillus subtilis, Pseudomonas putida, or Streptomyces iranensis. For each, there was induction of the gene cluster encoding a non-ribosomal peptide synthetase-like enzyme (atromentin synthetase) and an aminotransferase which together produce atromentin. Correspondingly, during co-culturing there was an increase in secreted atromentin-derived pigments, i.e., variegatic, xerocomic, isoxerocomic, and atromentic acid. Bioinformatic analyses from 14 quinone synthetase genes, twelve of which are encoded in a cluster, identified a common promoter motif indicating a general regulatory mechanism for numerous basidiomycetes.

Honey bee colonies act as reservoirs for two Spiroplasma facultative symbionts and incur complex, multiyear infection dynamics.

Two species of Spiroplasma (Mollicutes) bacteria were isolated from and described as pathogens of the European honey bee, Apis mellifera, ~30 years ago but recent information on them is lacking despite global concern to understand bee population declines. Here we provide a comprehensive survey for the prevalence of these two Spiroplasma species in current populations of honey bees using improved molecular diagnostic techniques to assay multiyear colony samples from North America (U.S.A.) and South America (Brazil). Significant annual and seasonal fluctuations of Spiroplasma apis and Spiroplasma melliferum prevalence in colonies from the U.S.A. (n = 616) and Brazil (n = 139) occurred during surveys from 2011 through 2013. Overall, 33% of U.S.A. colonies and 54% of Brazil colonies were infected by Spiroplasma spp., where S. melliferum predominated over S. apis in both countries (25% vs. 14% and 44% vs. 38% frequency, respectively). Colonies were co-infected by both species more frequently than expected in both countries and at a much higher rate in Brazil (52%) compared to the U.S.A. (16.5%). U.S.A. samples showed that both species were prevalent not only during spring, as expected from prior research, but also during other seasons. These findings demonstrate that the model of honey bee spiroplasmas as springtime-restricted pathogens needs to be broadened and their role as occasional pathogens considered in current contexts.