Location: Carl Hayden Bee Research Center
Project Number: 2022-21000-021-021-A
Project Type: Cooperative Agreement
Start Date: May 24, 2021
End Date: Sep 1, 2024
Objective 1) Determine the microbiome associated with larval health and disease. Overview and rationale: Although limited in geographic scope, our preliminary microbiome data reveals a wealth of information including a hypothesis for a potential new disease state containing well-known opportunistic pathogens. The data also suggest microbial communities associated with disease causing organisms (M. plutonius) that may increase virulence and promote disease progression. However, these patterns require verification from controlled experiments and greater sampling breadth to validate their deterministic nature, prevalence and association with larval phenotype. Expected outcomes: The results will provide decisive information concerning the various types and causes of brood disease, and new insight into the role of the honey bee microbiome in disease progression. Sampling broadly within and around disease “hot-spots” and longitudinally with respect to landscape and colony will further define the microbiome associated with European foul brood (EFB)-specific larval disease and larval disease in general, and provide isolates used to further verify virulence in vitro and in vivo. Most importantly, and as detailed in figure 2, the discovery and verification of microbiome types associated with disease forms the basis for controlled experiments in Objective 2.
Prior to larval collection, collaborator will photograph both visibly diseased and “non-diseased” areas of the colony, constructing a detailed (numbered) spatial map of individual brood samples (marked with pins) from the frame including a disease description, noting diagnostic characteristics associated with the disease state and non-diseased state. We will import photographs into power point and label each cell such that the cell phenotype can later be tied to the microbial communities. Using sterile technique, (nitrile gloves and flame sterilized tools), we will collect from the diseased pocket on the frame, sampling by individual cell into 2ml screw top cap tubes containing Ribonucleic acid (RNA) later solution, and when collecting concurrent samples for culturing, samples will be collected into physiological saline and placed on wet ice. When sampling a newly hatched 1st instar larvae we will collect all of the royal jelly with the larva. We will process a targeted subset of existing samples collected in 2016-2019 (see below) according to an informational throughput that promotes disease discovery and diagnosis based on photographic evidence and preliminary data. Working with multiple groups from beekeepers to apiary inspector to extension agents we will acquire more and more detailed samples of brood disease hot-spots including detailed photographs.