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SummaryGut microbes can play an important role in digestion, disease resistance, and the general health of animals, but little is known about the biology of gut symbionts in Apis mellifera. As part of the BEEBOOK series describing honey bee research methods, we provide standard protocols for studying gut symbionts. We describe non-culture-based approaches based on Next Generation Sequencing (NGS), methodology that has greatly improved our ability to identify the microbial communities associated with honey bees. We also describe Fluorescent In Situ Hybridization (FISH) microscopy, which allows a visual examination of the microenvironments where particular microbes occur. Culturing methods are also described, as they allow the researcher to isolate particular bacteria of interest for further study or gene identification, and enable the assignment of particular functions to particular gut community members. We hope these methods will help others advance the state of knowledge regarding bee gut symbionts and the role they play in honey bee health. Métodos estandar para investigar simbiontes intestinales de Apis mellifera ResumenLos microbios intestinales pueden jugar un papel importante en la digestión, la resistencia a las enfermedades, y la salud general de los animales, pero se conoce poco sobre la biología de los simbiontes intestinales en Apis mellifera. Como parte de la serie BEEBOOK que describe los métodos de investigación en la abeja, ofrecemos protocolos estándar para el estudio de simbiontes intestinales. Se describen métodos no basados en cultivos sino sobre la base de la secuenciación de nueva generación (NGS según sus siglas en inglés), metodología que ha mejorado en gran medida nuestra capacidad para identificar las comunidades microbianas asociadas con la abeja de la miel. También describimos la microscopía de hibridación in situ fluorescente (FISH), la cual permite un examen visual de los microambientes donde viven microbios particulares. También se describen métodos de cultivo, que permiten al investigador aislar bacterias de interés particular para posteriores estudios o para la identificación de genes, y permitir asignar funciones particulares a determinados miembros de la comunidad intestinal. Esperamos que estos métodos ayudarán a otros a avanzar en el estado del conocimiento sobre simbiontes intestinales de abejas y el papel que desempeñan en la salud de las abejas de la miel.

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BackgroundThe Hunt bumble bee (Bombus huntii Greene, Hymenoptera: Apidae) is a holometabolous, social insect important as a pollinator in natural and agricultural ecosystems in western North America. Bumble bees spend a significant amount of time foraging on a wide variety of flowering plants, and this activity exposes them to both plant toxins and pesticides, posing a threat to individual and colony survival. Little is known about what detoxification pathways are active in bumble bees, how the expression of detoxification genes changes across life stages, or how the number of detoxification genes expressed in B. huntii compares to other insects.ResultsWe found B. huntii expressed at least 584 genes associated with detoxification and stress responses. The expression levels of some of these genes, such as those encoding the cytochrome P450s, glutathione S-transferases (GSTs) and glycosidases, vary among different life stages to a greater extent than do other genes. We also found that the number of P450s, GSTs and esterase genes expressed by B. huntii is similar to the number of these genes found in the genomes of other bees, namely Bombus terrestris, Bombus impatiens, Apis mellifera and Megachile rotundata, but many fewer than are found in the fly Drosophila melanogaster.ConclusionsBombus huntii has transcripts for a large number of detoxification and stress related proteins, including oxidation and reduction enzymes, conjugation enzymes, hydrolytic enzymes, ABC transporters, cadherins, and heat shock proteins. The diversity of genes expressed within some detoxification pathways varies among the life stages and castes, and we typically identified more genes in the adult females than in larvae, pupae, or adult males, for most pathways. Meanwhile, we found the numbers of detoxification and stress genes expressed by B. huntii to be more similar to other bees than to the fruit fly. The low number of detoxification genes, first noted in the honey bee, appears to be a common phenomenon among bees, and perhaps results from their symbiotic relationship with plants. Many flowering plants benefit from pollinators, and thus offer these insects rewards (such as nectar) rather than defensive plant toxins.

The potential for Metarhizium anisopliae (Metschinkoff) to control the parasitic mite, Varroa destructor (Anderson and Trueman) in honey bee colonies was evaluated in field trials against the miticide, tau-fluvalinate (Apistan). Peak mortality of V. destructor occurred 3-4 d after the conidia were applied; however, the mites were still infected 42 d posttreatments. Two application methods were tested: dusts and strips coated with the fungal conidia, and both methods resulted in successful control of mite populations. The fungal treatments were as effective as the Apistan, at the end of the 42-d period of the experiment. The data suggested that optimum mite control could be achieved when no brood is being produced, or when brood production is low, such as in the early spring or late fall. M. anisopliae was harmless to the honey bees (adult bees, or brood) and colony development was not affected. Mite mortality was highly correlated with mycosis in dead mites collected from sticky traps, indicating that the fungus was infecting and killing the mites. Because workers and drones drift between hives, the adult bees were able to spread the fungus between honey bee colonies in the apiary, a situation that could be beneficial to beekeepers.

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We tested two genes together in hybrid poplars (genus Populus), CP4 and GOX, for imparting tolerance to glyphosate (the active ingredient in Roundup® herbicide). Using Agrobacterium-based transformation, 80 independent transgenic lines (i.e., products of asexual gene transfer) were produced in a variety of hybrid poplar clones (40 lines in Populus trichocarpa Torr. & Gray × Populus deltoides Bartr., 35 lines in Populus tremula L. × Populus alba L., and five lines in P. tremula × Populus tremuloides Michx.). We evaluated glyphosate tolerance over 2 years in field studies conducted in eastern and western Oregon. Ten percent of our transgenic lines showed no foliar damage or reduced growth after being sprayed with Roundup® at concentrations above normal commercial rates. Lack of damage was associated with expression of the CP4 gene but not of the GOX gene. It was suspected that GOX caused undesirable side effects, so we produced 12 lines into which only the CP4 gene was inserted. The performance of these newly regenerated lines was compared with an identical number of lines, produced in the same genotype, that had previously been engineered to contain both CP4 and GOX. Growth of the lines transformed with just CP4 was significantly better than those containing both genes and exhibited less damage in response to glyphosate treatment. This is the first report of transgenic poplars exhibiting high levels of glyphosate tolerance when grown under field conditions. With a modest transformation effort, it is possible to produce lines with commercially useful levels of glyphosate tolerance and little apparent collateral genetic damage.

Recent declines in bee populations coupled with advances in DNAsequencing technology have sparked a renaissance in studies of bee-associated microbes. Megachile rotundata is an important field crop pollinator, but is stricken by chalkbrood, a disease caused by the fungus Ascosphaera aggregata. To test the hypothesis that some gut microbes directly or indirectly affect the growth of others, we applied four treatments to the pollen provisions of M. rotundata eggs and young larvae: antibacterials, antifungals, A. aggregata spores and a no-treatment control. We allowed the larvae to develop, and then used 454 pyrosequencing and quantitative PCR (for A. aggregata) to investigate fungal and bacterial communities in the larval gut. Antifungals lowered A. aggregata abundance but increased the diversity of surviving fungi. This suggests that A. aggregata inhibits the growth of other fungi in the gut through chemical or competitive interaction. Bacterial richness decreased under the antifungal treatment, suggesting that changes in the fungal community caused changes in the bacterial community. We found no evidence that bacteria affect fungal communities. Lactobacillus kunkeei clade bacteria were common members of the larval gut microbiota and exhibited antibiotic resistance. Further research is needed to determine the effect of gut microbes on M. rotundata health.