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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.