Final Research Report to The National Honey Board. Ramesh Sagili. Department of Horticulture, Oregon State University, Corvallis, OR

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1 Final Research Report to The National Honey Board Ramesh Sagili Department of Horticulture, Oregon State University, Corvallis, OR Project title: Comprehensive evaluation of role of nutrition in honey bee colony losses In the wake of deteriorating honey bee health, bee nutrition has attained greater importance than ever. Nutritional stress as a result of habitat loss might be significantly contributing to the recent rapid honey bee colony declines (Naug 2009). Nutrition is key in dealing with most of the stress factors that ultimately compromise the immune systems of honey bees. Anecdotal evidence suggests that beekeepers following a timely and consistent feeding regimen (both protein and carbohydrate sources) had significantly less colony losses compared to those that did not. Nutrition is one among several factors that have been cited or speculated as probable causes of Colony Collapse Disorder (CCD), but has not received adequate attention and has not been studied extensively. Honey bee colonies that are poorly nourished are more susceptible to the gut parasite Nosema ceranae when compared to the colonies that receive adequate nutrition (Eischen and Graham 2008). Various measures have been proposed to enhance pollen diversity such as planting hedge rows near the fields and cultivation of few acres of different crops in proximity of monocultures (Schmidt et al 1995). Pollen and nectar are the two primary resources for which bees forage. Pollen is consumed by nurse bees which biosynthesize proteinaceous glandular secretions that are progressively provisioned to developing larvae (Crailsheim et al. 1992, Winston 1987). Pollen is the sole source of protein for honey bees and is vital for their development and survival (Schmidt and Buchmann 1992). Large monocultures have restricted choice of pollen diet in honey bees (Schmidt et al. 1995).

2 Nutrition is not only important for colony growth and worker longevity, but also plays a vital role against defending pathogens (Ritz and Gardner 2006, Rowley and Powell 2007). Protein deficiencies might compromise immune responses of an organism such as encapsulation (Siva- Jothy et al 2005). Beekeepers provide protein/pollen supplement to colonies during pollen dearth to stimulate colony growth (Saffari et al. 2004, van der Steen 2007). Here we propose to conduct a comprehensive evaluation of role of nutrition in honey bee colony losses. The specific objectives of this proposal are: 1) Determine effects of nutrition on longevity, physiology and colony growth in honey bees 2) Investigate affects of nutrition on incidence of parasites and diseases and immunocompetence in honey bee colonies and 3) Evaluate effects of antibiotics (Terramycin and Fumagillin) used prophylactically by beekeepers on beneficial gut microorganisms of honey bees that aid in nutrient assimilation and gut proteolytic enzyme activity. Hypothesis 1: Poor nutrition significantly effects physiology, longevity, and colony growth in honey bees. Hypothesis 2: Poor nutrition leads to higher incidence of parasites and diseases and negatively impacts immunocompetence (immunity) in honey bee colonies. Materials and Methods Pollen collection Pollen loads were collected from foragers by fitting standard pollen traps onto hives. Almond, cherry, meadowfoam, and blueberry were the targeted crops. Pollen traps were installed on the hives when the bloom was 70 to 100 percent on target crops. Anthers were also collected from several individual plants of each species that were in bloom in each orchard where pollen traps were placed. Pollen and anther samples were sealed in plastic bags and immediately stored at - 20 C. Once all pollen was collected, pollen within each crop was mixed thoroughly. Pollen diet treatments for this study were comprised as follows:

3 Diet Pollen Composition % Crude Protein Monofloral 100% Almond 30 Monofloral 100% Meadowfoam 24 Polyfloral Polyfloral 50% Almond 50% Meadowfoam 25% Almond 25% Meadowfoam 25% Cherry 25% Blueberry Pollen analysis (in collaboration with Dr. Gretchen Jones of the USDA-ARS Texas Lab) Composite samples of each pollen diet treatment were analyzed for pollen species quantification. The composite samples were sorted by color of the corbicular pellets. Each color group was then dried and weighed in order to determine the quantity of each pollen specimen within the sample. Each color of each pellet sample was prepared and examined by light microscopy to identify any discrepancies between different pellet samples that are of the same colors. Each pellet was then acetolyzed following the method of Erdtman (1936). This removes the outer shell of the pollen grains and gives us a more accurate picture to identify. Various specimens of each pellet sample were mounted and preserved on slides. Ten to fifteen micrographs of each pellet sample were taken with a scanning electron microscope. Pollen will be identified to the lowest possible taxonomic ranking. Experimental Design Colonies were established in 5-frame nucleus boxes with naturally-mated sister queens and equal numbers of adult bees and stores. The nucleus colonies were placed in flight cage. Top feeders with 50% sucrose solution and external water feeders were provided for all colonies. One frame in each colony was empty built-comb; pollen treatments were inserted into these frames by distributing the raw pollen into the cells followed by a spray of 50% sucrose solution. This process simulates workers to pack pollen into the cells thus initiating the pollen processing procedure (Dreller and Tarpy, 2000). Bee-Pro, MegaBee, and Global Patties were three

4 commonly used artificial protein supplements served as three treatments groups for artificial diets. No pollen was present in these treatments hives. The pre-made patties were placed inside the hive, and replaced every week. Consumption was measured in patty weight consumed per week. Five replicates of each pollen and artificial treatment diet were performed. Nurse bee selection Bees were sampled under two different methods: 1) paint marked bees and 2) nurse bee behavior. For painted selection, brood frames from 4 source colonies, equipped with sister queens, were incubated for 24 hours at 33 C with 55% relative humidity. Newly emerged bees from those frames were painted with enamel paint on their thorax and released into the experimental colonies. Painted bees were selected within the nurse bee age range, which is between 4-14 days old. The selection process for bees exhibiting nurse behavior follows their examined characteristics in Schmickl et al. (2003) and Lindauer (1952). Total midgut proteolytic enzyme activity Ten 9-day-old bees and behaviorally-selected nurse bees were collected from the brood nest area for estimating total gut proteolytic enzyme activity each week. Gut proteolytic enzyme activity, was analyzed following the procedure of Sagili et al. (2005) and Michaud et al.(1995). Hypopharyngeal gland protein quantification Ten 9-day-old bees and behaviorally-selected nurse bees were collected from the brood nest area each week for estimating hypopharyngeal gland protein content. Bees weree cold euthanized, their hypopharyngeal glands dissected and stored in Tris buffer at -20 C for further analysis. Protein content of hypopharyngeal glands was determined using Bradford assay as per Sagili et al. (2005). Immunocompetence Ten 8-day-old bees and behaviorally-selected nurse bees were collected from the brood nest area each week for analysis of various immune response levels. Phenoloxidase (PO) and prophenoloxidase (ProPO) enzymes, present in the hemolymph, are apart of humoral immune

5 responses that are present in the hemolymph of honey bees. PO and ProPO are associated with wound healing response and immunity. Hemolymph was extracted with micro capillaries from the second abdominal tergite of the individual bee (Alaux et al. 2010). PO and ProPO analysis were prepared following the method of Laughton & Siva-Jothy (2010). 2-3ul of hemolymph was extracted from each bee and pooled for a total of 25ul of hemolymph preserved in sodium cacodylate buffer solution (NaCac; M sodium cacodylate, M calcium chloride, ph 6.5). Samples were stored in -80 C until further analysis. Reaction assays for PO and ProPO involved the suspension of hemolymph sample in PBS buffer (phosphate buffered solution; 150 mm NaCl, 10 mm Na2HPO4, ph 6.5) with L-dopa as a reacting agent. The reaction assays were recorded by a microplate reader that took readings every 31 seconds for one hour to produce viable reaction curves at 490nm absorbance. ProPO reaction assay was modified with the addition of α-chymotripsin as a binding agent. Measurements were reported as optical density per minute and normalized by total protein (mg) using a standard BCA assay. GOX measurements are performed following the methods of Ohashi et al. (1999). Colony Growth A standardized grid was used to assess colony resources: open brood, capped brood, pollen stores, nectar/honey stores (Pankiw et al., 2004). Colony growth was evaluated every 7 days. Queen supersedure and egg laying was monitored as well. Hypothesis 3: Honey bee colonies treated with antibiotics Terramycin and Fumagillin will have negative impact on proteolytic enzyme activity and beneficial bacteria in the honey bee gut. Materials and Methods Experimental Design Colonies were established in 10-frame Langstroth hives with naturally-mated sister queens and equal numbers of adult bees and stores. Treatment replications were conducted in two separate apiaries. The selected in-hive chemicals were Tylan, which consists of the antibiotic, tylosin, and Fumagillin, which is an antimicrobial treatment are commonly used in-hive chemicals. Tylan is used to target American Foulbrood. Fumagillin is used to target Nosema

6 spores.treatments groups were as follows: 1) Tylan, 2) Fumagillin, 3) Tylan and Fumagillin, and 4) no treatment. All parameters were measured 2 weeks before treatment, and two weeks after treatments. Five replicates of each chemical treatment were performed. Microbial counts Lactobacillus and Bifidobacterium are bacteria that are speculated to aid in digestion and immunity. Ten midguts of 15-day-old bees were analyzed for the amount of Lactobacillus and Bifidobacterium species via CFU counts. These two genera of bacteria will be quantified on selective media (MRS agar with 0.5% L-cysteine, 2% Fructose). The midguts were extracted from the honey bee, homogenized and water bath sonicated, and plated in a three 10-fold dilution pattern on the selective media. Plates were incubated at 37 C for 72 hours and then colony counts were recorded (Rada, 1997). Genera identification will be identified via cdna sequencing as described in Olofssen and Vásquez (2008). Results Data collected from above experiments will be analyzed soon. Analysis of variance will be used to analyze midgut proteolytic enzyme activity and hypopharyngeal gland protein content. Repeated measures ANOVA will be used to analyze colony growth data. Publications and Presentations: Preliminary findings from this study were presented at Oregon State Beekeepers Association conference, Western Apicultural Society conference and Montana State Beekeepers Association annual meeting during After completion of data analysis we plan to publish the results in Journal of Apicultural Research and Apidologie.