Standard methods for research onApis melliferagut symbionts

Abstract

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.

Interactions between Cooccurring Lactic Acid Bacteria in Honey Bee Hives

“…For example, Fructobacillus, a member of the Leuconostocaceae, may produce fermentative products, such as lactic acid, CO 2 , ethanol, or acetate (39); these products may selectively promote the growth of the honey beespecific microbial community. Indeed, selective culture of many of the "core" microbiome members requires elevated atmospheric CO 2 and acidic media such as MRS (40). Importantly, these effects do not require the presence of Fructobacillus in the bee gut but may be mediated by metabolic products ingested by the bee.…”

Genome-wide screen identifies host colonization determinants in a bacterial gut symbiont

“…S. alvi grows in a distinct layer adjacent to the gut epithelium, suggesting adherence (12,13) (Fig. 1).…”

“…An abundant constituent of the honey bee and bumble bee gut microbiota is Snodgrassella alvi (10), a member of Betaproteobacteria that is related to human commensals in the genus Neisseria (11). S. alvi colonizes the hindgut ileum, a region analogous to the proximal large intestine in humans and is normally found in contact with the intima lining, of the ileum (12,13).…”

Interactions between fungi and bacteria influence microbial community structure in the Megachile rotundata larval gut

“…rarefaction curves, the probability of an interspecific encounter (PIE, a diversity index) and Chao1-estimated OTU richness) and beta diversity (microbial diversity across individual guts, i.e. non-metric multi-dimensional scaling (NMDS) of Bray-Curtis dissimilarity matrices), again using the standard methods for honey bee gut symbionts [31] (see online electronic supplementary material for a detailed description). We additionally compared the effects of treatments on bacterial and fungal OTU richness, diversity (PIE), and Chao1-estimated richness (separately) using analysis of variance (ANOVA).…”

“…To utilize the UNITE ITS database [29] and to verify our 16S rDNA analyses, we conducted parallel analyses in the program QIIME [30]. For all analyses we followed the standard methods recommended for honey bee gut symbionts [31]. We analysed the 16S rDNA and ITS data separately, but used similar methods (see the electronic supplementary material for detailed descriptions of our analyses).…”