Div. of Biological Sciences
Non Technical Summary
Honey bees are responsible for the annual pollination of at least $17 billion worth of US crops, but face severe health challenges. Nosema ceranaeis a fungus thatcauses a common and widespread infection that is associated with poor colony health and Colony Collapse Disorder. Currently, Nosema diseaseis primarily treated with a single antibiotic. Because of the potential for antibiotic resistance and the transfer of resistant genes in humans, it is desirable to find an alternative treatment, one that relies upon the natural honey bee immune system. Based upon data from bees reared in the lab, we found thatfeeding honey bee larvae heat-killed N. ceranaspores can activate honey bee immunity and substantially reduces infection levels when these "immunized" bees are exposed to Nosema as adult bees. The proposed research will test if this "immune-priming", a vaccine-like treatment can also work (1) in naturally-reared honey bee larvae, (2)in beesimmunized as adults, and (3) in field tests of colonies. To test the effectiveness of our treatments, we will measureindividual bee infection level, the number of infected bees per treatment, bee immune gene activation, and effects on bee longevity.
Animal Health Component
Research Effort Categories
Goals / Objectives
Honey bees are responsible for the annual pollination of at least $17 billion worth of US crops, but face severe health challenges. Nosema ceranaecauses a common and widespread infection that reduces honey bee health and is primarily treated with a single antibiotic. Activating insect immunity against fungal infectionsprovides a natural countermeasure that should be difficult for pathogens to evolve resistance against. This has been demonstrated with heat-killed bacteria, but it remains unclear if this strategy would work with a heat-killed fungal pathogen like Nosema cerana.In our preliminary data, we show that autoclaved N. ceranaespores fed to A. melliferalarvae reared in vitro (immune priming) and subsequently fed live Nosemaspores as adults have infection levels reduced by 85% upon adult death. The proposed research would test the efficacy of this immune priming treatmentin vivo and with field colonies. If successful, we will have developed a new treatment against N. ceranaeand created a potentallytransformative approach to treating honey bee fungal diseases. Specifically, we will determine if immune priming conveys protection against infection when administered in vivo to (1) larvae or (2) adults upon emergence. Based upon these results, we will test if (3) immune primingeither larvae or young adults in field colonies boosts colony immunity against Nosema infection. We will measure individual bee infection level (midgut spores/bee), number of infected bees per treatment, bee immune gene activation, and effects on bee longevity.Overall goal:We will test if immune priming activates honey bee immunity againstN. ceranaeand therefore decreases subsequent infection when bees are exposed toN. ceranae.Ourspecific objectivesare as follows. We will determine if immune priming conveys protection against infection when administeredin vivoto (1) larvae or (2) adults upon emergence. Based upon these results, we will test if (3) immune priming either larvae or young adults in field colonies boosts colony immunity againstNosemainfection. We will measure pathogen prevalence (proportion of infected bees per treatment), abundance (spore counts /bee), immune gene activation, and effects on longevity.
Obj. 1 (Exp. 1) Testing effects of immune priming treatment on larvae naturally reared in vivo (Nieh, UCSD)This experiment tests the efficacy of vaccinating larvae reared in natural colonies. To obtain larvae of the same age, we will place an empty worker brood comb frame in the colony, remove the frame after 6 days, and visually determine that the larvae, based upon size, are three days old. We will select brood patch with approximately 300 three-day old larvae and divide them into two sections. A clear plastic sheet will be placed over the brood patch and marked to indicate larvae locations. Each section will be randomly assigned to one of the two treatments. One group will receive only 50% sterile sucrose syrup (control) and another group will receive 40k autoclaved spores per bee (vaccinated). Each larva (n=150 larvae per treatment) will be fed 2 μl of 50% syrup with a pipette. A previous study showed that nurses did not remove much of such food, because at least 80% of such provided food was consumed by larvae (Hanley et al., 2003). The comb will then be returned to the colony for 5 days and then moved to an incubator at 50% humidity and 34.5 °C (Milbrath et al., 2013). Cages made of hardware cloth will be placed over each treatment area so that newly emerged bees from the two groups do not mix (Hanley et al., 2003). We will have four treatments (Table 1): two treatments at larval stage (vaccinated or not) and two treatments at adult stage (inoculated with live spores or not). Inoculation will be during the first day of worker emergence, with 2 μl of syrup (with or without spores). Bees will then be isolated for 30 min to reduce spore transmission. Inoculating 1-day old bees resulted in higher Nosema spore loads than inoculating 5-day old bees (Milbrath et al., 2013). Each custom-made plastic cage (Eiri et al., 2015) will have 25 workers and sterilized pollen (bee-collected pollen irradiated to kill potential pathogens mixed with 50% sucrose solution) and 50% sterile sucrose solution in a gravity feeder (5 ml syringe). The pollen and sugar solution will be replaced each 5 days. Five bees per treatment will be sampled at age 1 (day of emergence), 6 and 14 to determine how long gene activation from the immune priming treatment lasts. These bees will be recorded as "censored" during survival analysis. Remaining bees will be observed for mortality. Dead bees will be removed and frozen for midgut spore counts (Cantwell, 1970). In total, we plan to run 25 bees per treatment per colony, for a total of 2,000 bees.Immune gene analysis (Huang, MSU): We will measure expression of four immune genes, abaecin, defensin, apidaecin, and hymenoptacin (Chaimanee et al., 2012a) and two reference genes, actin (Chaimanee et al, 2012) and GAPDH ((Scharlaken et al., 2008)). Samples will be shipped from UCSD to MSU using RNAlater. Equal numbers of treated and control bees are run on each 96-well plate with all 6 genes. Each gene per sample will be run in triplicate using standard methods.Obj. 2 (Exp. 2) Testing adult immune priming treatment (Nieh, UCSD)This experiment tests the effect of vaccinating newly emerged adult bees. A frame of capped brood will be removed from each colony, placed inside a nuc box and incubated as in Exp. 1. Vaccination (Table 1) will be administered to newly emerged adult bees, which will be isolated, treated (2 µl dose), and caged as in Exp.1. Sample sizes will be the same as Exp. 1. At 6 days of age, bees will be cooled and placed into individual vials and receive the inoculation as in Exp. 1 (Table 1). Half of the bees will receive live N. ceranae spores, and the other half sterile sucrose only. Bees will then be treated as in Exp. 1. The immune gene analysis (Huang, MSU) will be identical to Exp. 1.Obj. 3 (Exp. 3): Testing vaccination with field tests using full-sized colonies. (Huang, MSU) This experiment tests the effect of vaccinating bees (either in brood or young adults, depending upon the results of Exps. 1 & 2) in field colonies. Bees will be obtained and vaccinated with 40k Nosema spores per bee, as described in Exp 1. Unlike the first two experiments, we will only have two treatments: (t2) un-vaccinated and (t4) vaccinated bees so that we can increase the number of marked bees per treatment. Each of the two treated areas will be covered with a cage made with hardware cloth, allowing us to separate the two bee groups. For each treatment, 100 bees will be individually marked with queen bee tags and 200 bees marked with the same color paint mark. In the inoculation phase, we will capture these bees individually in vials, feed them 40K spores per bee in 2 µl of sugar syrup, or syrup only (Milbrath et al., 2013) and release them after 1 hr to the natal colony. Tagged bees will be tracked for survival by checking for their presence once every 5 days. If we vaccinate in adults, then the two groups of bees will be paint-marked (N=400 per group) in two different colors, vaccinated individually or fed syrup; and returned to their natal colony. On day 5, we will recover 300 bees from each group, cool them and inoculate both groups with live spores as each bee wakes up. One subset (n=100 per group) will be tagged and one (N=200 per group) painted with an additional paint (i.e. they become "double-painted"). Any bees with only one paint would be not used for the following observations. Double-paint-marked bees will be sampled for immune gene expression (N=10 bees) on day 6 and for Nosema spore counts (day 10 and day 20). Age of first foraging will be determined by blocking the colony entrance for 30 min in the morning and in the afternoon and recording the tag ID of the returning bees. We will measure the age of first foraging to assay the effects of Nosema infection and of our immune priming treatment. N. ceranae infection decreases the age of first foraging (Goblirsch et al., 2013), similar to N. apis (Huang, 2011). This experiment will be repeated in at least four 10-comb colonies (600 bees per colony, 2,400 bees total). To limit potential Nosema spread, all colonies will be in an isolated apiary. Based upon our preliminary qPCR results, and immunes genes identified in the literature, we will perform qPCR to analyze gene activation in bees that receive our field treatments.Table 1.Treatments and expected results for Exps. 1 & 2. The immune priming treatment consists of 40,000 (40K) autoclaved spores in 50% sucrose and the control consists of sterile 50% sucrose with no spores. Gene activation will be measured with separate groups of bees sampled at ages described in the experiments. Total midgut spore counts will be measured upon adult death.Overall treatmentVaccinationExp. 1: larvae (3-day old)Exp. 2: adults (1-day old)InoculationExp. 1: adults (1-day old)Exp. 2: adults (6-day old)Expected result1) 0K/0Ksterile sucrosesterile sucroseNo gene activation, no infection2) 0K/40Ksterile sucrose40K live sporesGene activation from infection alone, high infection3) immune priming treatment/0Kimmune priming treatmentsterile sucroseGene activation from immune priming treatment alone, no infection4) immune priming treatment/40Kimmune priming treatment40K live sporesGene activation from immune priming treatment & infection, no or low infection