Recipient Organization
KENTUCKY STATE UNIVERSITY
(N/A)
FRANKFORT,KY 40601
Performing Department
Agriculture & Environmental Science
Non Technical Summary
Honey bee colony survival depends on the availability of pollen as a source of proteins, lipids, and micronutrients. Not all pollen species have adequate nutritional value; hence monoculture can impose dietary constraints harmful to honey bee health. In fact, pollen intake influences lifespan and several health indicators, including physiological metabolism, immunocompetence, disease tolerance, and resistance to pesticides. To offset pollen shortages or to boost honey productivity, beekeepers feed readily available pollen substitutes although the nutritional value of these feeds have not been fully studied. In addition to these protein supplements, more recently a new type of products, known as probiotics, are available to enhance colony health. Probiotics contain microorganisms that are fed to bees to enhance their gut microbiome, possibly by boosting the most beneficial strains of bacteria. As a result, overall colony health should improve in colonies fed probiotics since gut bacteria are associated with better digestion, disease resistance, and may even produce critical nutrients. Similar to the pollen substitutes, there is paucity of studies that unequivocally confirm the benefits of probiotics in beekeeping. A previous project that used whole genome transcription to measure honey bee nutritional response to pollen and the pollen substitutes MegaBee and Bee-pro revealed key differences between the three diets. To build on these outcomes, we will conduct feeding trials using a probiotic in combination with pollen or pollen substitutes to determine whether the addition of a probiotic has significant effects on honey bee health and whether any such effects are influenced by the diet formulation. Using quantitative polymerase chain reaction (qPCR), we will measure a set of marker genes involved in growth as well as the content of important microbiota taxa. Moreover, we will gauge the ability of a commercial probiotic to restore the honey bee gut microbiota when disrupted by an antibiotic treatment.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
Objective 1: Assess the effect of diet enhancement with probiotics on honey bee growthThe insulin/insulin-like signaling (IIS) pathway, which is involved in growth in insects (Wu and Brown, 2006), was shown to be a regulator of nutrient homeostasis and behavior in honey bees (Corona et al., 2007) as well as being a promoter of weight gain. The honey bee egg yolk protein, vitellogenin (Vg), was also shown to interact with the IIS pathway to regulate bee nutritional status (Amdam et al., 2012). To uncover how the bee gut microbiota affects host physiology, Zheng et al. (2017) measured weight, sucrose sensitivity (appetite), expression of IIS underlying genes, and Vg in microbiota-depleted bees versus those with an intact gut community. The results showed that the microbiota stimulates insulin signaling and Vg, which in turn affects satiety and ultimately promotes host weight gain, suggesting a role of the microbiota in the regulation of growth via the insulin pathway (Zheng et al., 2017). Based on these findings, and since probiotics theoretically boost the microbiota, we will measure weight gain and the expression of Vg and IIS-related genes in bees fed diets with and without probiotics. This design will allow us to assess the effects of probiotics on bee growth and identify the diet/probiotic treatment with the most positive effects (if any).Objective 2: Assess the effect of diet enhancement with probiotics on the gut microbiotaTaking advantage of the knowledge acquired on bee gut microbiome in relation to health, probiotics supplements are being commercialized for beekeeping to boost and shift the gut microbiota to more beneficial communities that will optimize the use of undigestible nutrients. For example, SuperDMF (Strong Microbials Inc., Milwaukee, WI) is marketed as an all-natural probiotic supplement containing a wide array of lactic acid bacteria and enzymes to support bee gut microbiome and gut health as well as to strengthen immunity and provide digestive aid. To test this assumption, bees will be fed the same diets as in objective 1 in the presence or absence of a probiotic and their core microbiota (Firm-4, Firm-5, Bifido, Gamma-1, and Alpha-1) will be quantified.Objective 3:Assess the effect of probiotics on the gut microbiome restorationThe antibiotic property of killing bacteria can be used to investigate the effects of microbiota disruption on honey bee health. For example, the disturbance of the honey bee gut microbiome via antibiotics resulted in increased levels of Nosema infection, suggesting that eliminating gut bacteria weakens the immune function and makes bees more susceptible to pathogens (Li et al., 2017). In beekeeping, antibiotics are routinely used to prevent outbreaks of American or European Foulbrood. In the U.S., the use of tetracyclines over the years has caused some strains of bee gut bacterial species to acquire several resistance loci to this class of antibiotics (Tian et al., 2012), in addition to severe gut dysbiosis with drastic and persistent effects on microbiome size and composition (Raymann et al., 2017). The use of antibiotics was also shown to increase mortality, possibly because of greater susceptibility to opportunistic pathogens (Raymann et al., 2017).In this project, bees will be fed a protein-rich diet with and without SuperDMF or a poor sucrose-based diet with and without SuperDMF while exposed to antibiotics to assess the extent of dysbiosis in relation to the nutritional status. The capability of commercial probiotics to restore the honey bee gut microbiome will also be assessed by quantifying the core microbiota post-treatment with antibiotics.
Project Methods
PROCEDURESFeeding trials for objective 1 and 2Healthy honey bee colonies from the Kentucky State University apiary will be used to produce newly emerged honey bees. To do so, frames with sealed brood will be kept in the dark at 34°C under 50-70% humidified atmosphere. Honey bees emerging within 24 hours will be collected and distributed in groups of individuals into 24 cages, with each cage representing an experimental replicate. Starting from day-1 post-emergence, the cages will be separated into 8 groups of 3, with each group receiving one of the following diets: Megabee/SuperDMF, Bee-pro/SuperDMF, pollen/SuperDMF, carbohydrates/SuperDMF, Megabee, Bee-pro, pollen, or carbohydrates (0.5 M sucrose syrup). The SuperDMF will administered as recommended by the manufacturer by sprinkling it on a piece of wax in the cages on which the bees cluster. The diets will be replaced every other day and dead bees will be recorded and removed. Ten bee workers will be collected per treatment at days 5, 10, and 15 post-treatment. The bees will be immobilized at 4°C, the whole-body wet weight will be recorded, and then the bees will be stored at -80°C until RNA extraction.Dysbiosis and feeding trials for objective 3Healthy honey bee colonies from the Kentucky State University apiary will be used to produce newly emerged honey bees as described. The honey bees emerging within 24 hours will be collected and distributed in groups of individuals into 24 cages with 100 bees per cage; each cage will represent an experimental replicate.Starting from day-1 post-emergence, the cages will be separated into 8 groups and fed: proteins, sucrose, proteins/SuperDMF, sucrose/SuperDMF, sucrose/antibiotics, proteins/antibiotics, proteins/SuperDMF/antibiotics, and sucrose/SuperDMF/antibiotics. The groups will be fed ad libitum, and those receiving protein diets will also receive carbohydrates consisting of 0.5 M sucrose syrup. The SuperDMF will be administered as recommended by the manufacturer by sprinkling on a piece of wax on which the bees cluster in the cages. For the antibiotic treatments, tetracycline will be suspended in 0.5 M sucrose syrup at a concentration of 450 ug/ml (Raymann et al., 2017).For the groups challenged with dysbiosis, the antibiotics will be fed for 5 consecutive days before removal on day 6 to start the recovery. All the dysbiosis cages fed proteins or sucrose only will each receive 15 marked worker bees from the originating hives to expose the caged bees to normal bee gut microbiota as in normal beekeeping conditions. The diet regimens will be maintained throughout the study and will be replaced every other day. Dead bees found at the time of diet replacement will be recorded and removed.To determine how the antibiotic treatment impacts the gut microbiota and whether a protein diet with a probiotic enhances the recovery from dysbiosis, bees will be collected at three time points (day 5, day 7, and day 10) of the experiment and stored at -80°C until DNA extraction. The trial will be conducted until all bees are dead to determine survival rates.RNA extraction and quantitative PCR assay for objective 1The bee abdomens and heads will be separated, and mRNA will be extracted separately from both body parts. After tissue homogenization in Trizol, mRNA will be extracted using the RNeasy kit (Qiagen, Germantown, MD) then reverse-transcribed to obtain cDNA. The expression levels of the genes encoding insulin-like receptor (InR) and vitellogenin will be quantified via qPCR from the RNA of the bee abdomens while insulin-like peptide (ILP) will be quantified from RNA of the heads. Previously published primer sequences will be utilized for the afore mentioned genes. As for housekeeping genes, we will test RP49, RPS5, S8, GAPDH and Actin, which we previously used and pick the best three genes for these experiments.Quantification of microbiota taxa for objective 2 and 3Twelve bees per biological replicate (cage) will be randomly chosen and rinsed with 7% benzalkonium bromide for 2 min and then rinsed four times with sterilized water to minimize bacterial contamination from the body surface. The midgut and hindgut will be collected with tweezers by clamping the end of the abdomen. To extract the DNA of the microbiota, we will utilize the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA) according to the manufacturer recommendations. The midgut and hindgut will be transferred to sterile 96-well sample extraction plates; each well will contain two sterile 3.2-mm steel beads and ~50 μL of 0.1-mm glass beads. The samples will then be subjected to lysis by using 180 μL of tissue lysis buffer (buffer ATL) per well and physically disrupted by shaking for six minutes at 30 hertz with TissueLyser (Qiagen, Valencia, CA). For protein removal, each sample will be treated with 20 μL proteinase K and incubated at 56°C overnight; the DNA will be then stored at -20°C until use.The abundance of the core microbiota taxa Firm-4, Firm-5, Bifido, Gamma-1, and Alpha-1 will be measured as copies of the 16S rRNA gene via the absolute qPCR method following published protocols (Guo et al., 2015). Briefly, the initial concentrations of the DNA template will be normalized between samples and two housekeeping genes, actin and ribosomal protein 49 (Rp49), will be used as internal PCR controls. A total reaction volume of 25 μL will contain the following reagents: 9.5 μL ddH2O, 12.5 μL 2X SybrGreen qPCR Master Mix (ThermoFisher Scientific, Applied Biosystems brand, Foster City, CA), 0.5 μL of each primer (10 μM), and 2μL of DNA template. qPCR reactions will be set at the following cycle conditions: 95°C for 10 min, 40 cycles at 95°C for 15 s, 60°C for 1 min; a melting curve will be observed at the end of each run. Standards consisting of 1:10 dilution series of adult worker samples will be prepared for each of the bacterial primers and will be run with each qPCR to establish a standard curve. The cycle threshold value for each sample will be compared to the standard curve to determine the copy numbers for each bacterium for each sample (Brucker and Bordenstein, 2012). The 16S rRNA copy numbers estimated by qPCR will be compared across different time points using ANOVA with Tukey's multiple comparisons test.