MICROALGAE AS A NOVEL PLATFORM TO IMPROVE HONEY BEE NUTRITION, MICROBIOME HEALTH, AND PATHOGEN RESISTANCE

<b>Project Methods</b><br> In general, measures of bee health and nutritional status will include spectrophotometry-based and qRT-PCR analyses.qRT-PCR using well-established protocols will be conducted to determine relative vitellogenin (Vg) expression (a nutritionally regulated storage lipoprotein), assess immune function (relative expression of key antimicrobial peptides and regulatory genes: abaecin, hymenoptaecin, defensin-1, relish, PPOact, and HSP90), and quantify viral loads using linearized plasmids (Deformed wing virus-A and B, Black queen cell virus, Chronic bee paralysis virus, Israeli-Acute-Kashmir bee virus complex, and Lake Sinai virus).An evaluation of oxidative stress will be conducted using the TBARS assay to determine lipid peroxidation using previously established methods. Statistical differences based on diet treatment in these health metrics will be identified by ANOVA including queen line as a random effect. For survival analyses, differences in lifespan will be compared across the treatment groups using the Kaplan-Meier survival analysis, followed by post-hoc pairwise analysis when a significant effect of treatment is determined. Protein mass spectrometry (LC-MS) will be performed by the Louisiana State University Proteomics Core Facility. We have optimized a novel method for absolute quantification of Vg using labeled peptides. Unique peptides in the honey bee proteome that correspond to Vg will be measured relative to total protein content per sample using the labeled peptides as internal standards.Queen sources: Queens used for experiments will be reared following routine practicesby "grafting" from 5 different queen lines and thus will result in all queens being tested as replicates of 5 different genotypes. All queens will be reared in a common environment to reduce effects of rearing environment on the subsequent measures. Queens will be instrumentally inseminated following routine practicesat our research unit with 7ul semen each from the same large pool of semen and housed in the same queen bank prior to the start of the experiment to control for rearing environment, mating experience, and adult environment as much as possible.Adult trials (workers and queens): For all adult trials involving queens and adult workers maintained in the laboratory, Plexiglass queen monitoring cages (QMCs) with a removable well plate where the queen can lay eggs will be used. Cages will be set up with 150 newly emerged workers and a single queen and be maintained at 34.5????C and 65% humidity following standard practices. Newly emerged bees (<12-hours old) will be sourced from the 5 colonies used as the source for the queen larvae at the USDA-ARS Honey Bee Lab. Cages will be provided ad libitum access to sucrose syrup and diet treatments in the form of a patty of pollen, commercial pollen substitute or microalgaecomprised of the dry protein source mixed with sucrose syrup and glycerol to maintain moisture and consistency as previously establishedLarval rearing: Larvae will be raised in the laboratory from 1st instar until emergence as an adult following previously used and well established protocols (Simone-Finstrom et al. 2016, Simone-Finstrom et al. 2018). The larval diet treatments will be a control diet containing a mixture of commercially produced royal jelly, yeast extract, glucose and fructose, or the control diet mixed with microalgae or pollen. Each larvae will be individually mass provisioned with 190mg of diet, an optimized amount for worker development. Larvae will be reared at 34.5°C and 95% humidity through pupation and then moved to 34.5°C and 80% humidity through eclosion following standard practices.Colony-level experiments:Working in collaboration with a commercial beekeeper, we will conduct a large-scale field trial using managed colonies involved in almond pollination. The trial will be comprised of three distinct apiaries within the operation. At each apiary, 16 colonies will serve as unfed controls, 16 colonies will be fed commercial pollen substitute,and 16 will be fed microalgae(48 colonies per apiary X 3 apiaries, N = 144 colonies). Colonies will be fed every two weeks. We will monitor colony brood production, adult bee populations, and survival from October through February to capture the most relevant time period related to supplemental feeding for almond pollination. We will also measure previously validated colony-level biomarkersto assess colony physiology and health. Targets of health used in Objective 1 will also be assessed including gut microbiota and pathogen abundance. These fine-scale measures will be determined for 6 colonies per treatment per apiary (N =54) for three different time points. Data will be analyzed as described above. The beekeeper involved in this objective will provide economic analyses based on the feeding trial outcomes in their specific operation. Economic analyses will take into consideration the cost of microalgae feed versus commercial pollen substitute feed and the effects on colony size, since the size of the colony going into almond pollination determines the pollination contract income. This will provide critical insights into the feasibility and profitability of implementing microalgae-based feed in commercial beekeeping operations and environments.