Probiotic Prospecting for Alfalfa Leafcutting Bees - UNIVERSITY OF CALIFORNIA, RIVERSIDE

Goals / Objectives
Although chalkbrood can be a major source of mortality for the alfalfa leafcutting bee, no effective control strategy currently exists. In order to contribute to the availability of a healthy source of pollinators for alfalfa seed production, we have been studying interactions between bacteria and fungi that reside in the alfalfa leafcutting bee larval gut. The goal of this work is to discover microbes that hinder the growth of Ascosphaera aggregata, the fungus that causes chalkbrood. Previously, A. aggregata was found to inhibit the growth of other fungi in the larval gut, but no any single microbe was found that inhibits the growth of A. aggregata. In previous work, one hindrance to testing the effect of certain bacteria on A. aggregata was that these bacteria were resistant to the antibacterial treatments we used to remove bacteria. Additionally, it may be that combinations of bacteria work in concert to inhibit A. aggregata, which was impossible to tease out in our previous study. Therefore, the purpose of this project is to study how manipulated bacterial communities affect the growth of A. aggregata in the larval gut and subsequently, the incidence of the disease chalkbrood. Lactobacillus kunkeei, a bacterium that was found to be associated with alfalfa leafcutting bee larvae, is one of a mixture of lactic acid bacteria that protects honey bee larvae from pathogens. This suggests that these bacteria may act as probiotics (bacteria which, when ingested, increase the health of an organism), possibly by inhibiting A. aggregata. The first step in determining if these bacteria can act as probiotics for the alfalfa leafcutting bee is to experimentally determine if these bacteria affect bee health. Specific objectives are 1) To determine if Lactobacillus kunkeei can lower the abundance of A. aggregata in the larval gut and thereby lower incidence of chalkbrood and 2) to determine if combinations of bacteria can lower the abundance of A. aggregata in the larval gut and thereby lower incidence of chalkbrood. Year 2 Objectives: 1. Identify deleterious microbes in the ALCB larval gut. 2. Identify fungal or bacterial pathogens of parasitic wasps that may be leveraged to control parasitic wasps and not harm ALCBs.

Project Methods
(See original documents for Year 1 approach) Year 2 Approach: Objective 1: To identify microbes that compete with M. rotundata larvae for nutrients, larval nutrition will be measured while microbial communities associated with larvae are characterized. Fresh M. rotundata brood cells with eggs will be collected. Using female brood cells to control for size, rough measures of the amount of pollen each larvae eats will be made by weighing each cell before the egg hatches and again once lab-reared larvae have reached the last instar. Once larvae begin defecating, perform sterile dissection of each larva will be done, saving the gut for microbial sequencing and the rest of the larvae for nutritional analysis. Illumina sequencing of bacterial and fungal communities found in the larval guts will be used to characterize microbial communities. Established protocols will be used for sequencing bacterial and fungal communities. Nutrition will be assessed through quantification of the protein content of each larvae, using the bicinchoninic acid assay. The SparCC program will be employed to determine which microbes co-occur with lower or higher nutritional values. By culturing bacteria from larval guts, bacteria will be identified using standard molecular techniques, so that it can be determined if they also correlate with lower protein content in the above analysis. Objective 2: Microbial community analysis of hymenopteran parasites of M. rotundata also will be performed to characterize bacterial communities as well as eukaryotic microbes including fungi, trypanosomes, and neogregarines. Illumina sequencing will be used for analyses. To determine if there are putative biocontrol agents associated with wasps but not bees, microbiomes from bee larvae and the parasites will be compared. Known insect pathogens, such as Spiroplasma, Serratia, Wolbachia (reproductive parasite), nematodes, trypanosomes and neogregarine parasites will be targeted.