SUSTAINABLE SOLUTIONS TO PROBLEMS AFFECTING BEE HEALTH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1021437
• Multistate No.: NC-_old1173
• Project Start Date: Nov 21, 2019
• Project End Date: Sep 30, 2024
• Program Code: [(N/A)]- (N/A)

Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001

Performing Department
Entomology

Non Technical Summary
Honey bee colonies are constantly under threat from existing pest and disease complexes and also from other new species invasions. Recently, some honey bee colonies in the U.S have become infested with Nosema ceranae a gut parasite which is prevalent in the Asian honey bee. Nosema ceranae is a newly found fungal parasite that affects Apis mellifera worldwide (Evans and Schwartz, 2011; Higes et al., 2013). Currently little is known about biology and epidemiology of this relatively new species of Nosema and the effects of this species on honey bees at both the individual and colony level vary from study to study. Understanding the basis of this variation is important for recommending a course of action for beekeepers through BMPs. The many problems that currently face the U.S. honey bee population has underscored the need for sufficient genetic diversity at the colony, breeding, and population levels. Genetic diversity has been reduced by three distinct bottleneck events, namely the limited historical importation of subspecies and queens, the selection pressure of parasites and pathogens (particularly parasitic mites), and the consolidated commercial queen-production practices that have reduced the number of queen mothers in the breeding population. An additional goal of this research is to measure the genetic impact of stock importation and release on domestic stocks.Flowering plant species differ considerably in the nutritional content of their pollen, and this can have important ramifications for bee health (Levin and Haydak 1957, Standifer 1967). Nectar is also important for bee nutrition because, as the primary carbohydrate source for bees, sufficient quantities are essential for larval growth and meeting the energetic demands of bee activity (Brodschneider and Crailsheim 2010). The diversity and quality of floral resources at the landscape level may therefore have a significant impact on the nutrition of bees, particularly those with large foraging ranges such as honey bees. Bees are exposed to an array of xenobiotics in the course of foraging in a landscape natural plant-derived toxins, metals, pollutants, pesticides and spray adjuvants (Johnson et al., 2010). In high concentration these xenobiotics may have direct effects on bees, either through acute mortality at the larval or adult stage, or more subtly, through colony-level effects that harm a colonys chances of surviving over the course of a season. Combinations of xenobiotics may increase bees susceptibility to exposure to a particular pesticide, or the cocktail of xenobiotics may produce unexpected synergistic interactions (Glavan and Bozic, 2013). Further work is needed to determine relevant xenobiotic exposures in bees as well as establish exposure levels above which xenobiotic exposure directly harms bees ability to produce hive products and deliver pollination services. Insights gained can be provided to beekeepers, growers, manufacturers and regulators to mitigate any effect that xenobiotic exposure has on honey bee health and productivity.

Animal Health Component

60%

Research Effort Categories

Basic

20%

Applied

60%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
306	3010	1130	50%
305	3010	1130	50%

Knowledge Area
305 - Animal Physiological Processes; 306 - Environmental Stress in Animals;

Subject Of Investigation
3010 - Honey bees;

Field Of Science
1130 - Entomology and acarology;

Keywords

parasitic mites

parasites

microbes

pesticides

honey bee

pollinator health

Goals / Objectives
To evaluate the role, causative mechanisms, and interaction effects of biotic stressors (i.e. parasitic mites, pests, and pathogens) and abiotic stressors ((i.e. exposure to pesticides, poor habitat and nutrition, management practices) on the survival, health and productivity of honey bee colonies as well as within pollinator communities. To develop and recommend "best practices" for beekeepers, growers, land managers and homeowners to promote health of honey bees and pollinator communities.

Project Methods
Objective 1 -- To evaluate the role and causative mechanisms of parasitic mites, viruses, and microbes in pollinator abundance and honey bee colony success Rationale and Significance Honey bee colonies are constantly under threat from existing pest and disease complexes and also from other new species invasions. Within colony prevalence and intensity of Nosema ceranae and viral infection will be determined. Experimental colonies with four different age cohorts will be established and inoculated with known concentrations of Nosema spores. Two weeks after spore inoculation the experimental colonies will be killed by freezing and the prevalence and intensity of Nosema infection in each age cohort will be determined by light microscopy.Results from this study will provide insights on prevalence and intensity of Nosema ceranae. This information regarding prevalence and intensity will help better formulate Nosema sampling protocol that will help beekeepers assess realistic need for colony treatment. QT-PCR analysis will be used for determining infection levels of bees with IAPV, DWV, BQCV, SBV and other viral pathogens. Eggs, larvae, pupae or adults will be placed in a 1.5 ml tube containing RNALater and frozen at -80o C until analysis. Standard extraction protocols using RNAlater will be followed and QT-PCR using SYBR green will be performed using validated published primer sequences for bee pathogens (de Miranda et al., 2013; Evans et al., 2013). Individual-level consequences of pathogen infection: For cage studies, we use the following protocol to determine consequences of pathogen infection (Goblirsch et al., 2013): 1. Obtain newly emerged bees by incubating mature brood overnight inside cages at 35 degrees C and 50% RH. 2. Fresh Nosema spores (within 24 hrs) are obtained from live bees, cleaned of debris by centrifuging in water (5000xg, 10 ming), verified by PCR to be mono-specific. 3. Individual workers are starved for 2 hrs and then hand fed with a calibrated dose of Nosema spores in 2 microliter 50% sucrose syrup. Control bees are fed syrup only. 4. Workers are isolated in 20 ml glass scintillation vials for 30 min at 35C to reduce transfer of spores among bees. 5. Workers are then caged together and mortality observed daily. Sugar syrup (50%) and pollen arechanged every 5 days. 6. Pollen are frozen (-20C) and heated (60C) at 12 hr minimum for three cycles to inactivate potential spores of either N. ceranae or N. apis. 7. We then use survival analysis to compare different effects of different treatments, either using SAS or R package. For colony study, bees are paint-marked or tagged with numbers and inoculated with Nosema spores (or sugar only) and released to colonies. Survival is determined once every 5 days by noting the presence of numbered bees on each frame (2x). Age of first foraging is observed by recording the ID of returning bees at least 2 hours per day.Objective 2 -- To facilitate the development of honey bee stock selection, maintenance and production programs that promote genetic diversity and incorporate traits conferring resistance to parasites and pathogens. Analysis of diversity: We will use a meta-analysis approach to compare the pedigree relationships of honey bee reproductives (queens and their mates) across five different studies and to quantify the overall genetic diversity of breeding populations. We will compare the inferred genotypes of queens and their mates from microsatellite analyses of worker offspring from a feral Africanized honey bee population (which serves as a negative control for inbreeding), an experimentally derived population of sister queens (which serves as a positive control for inbreeding), and three separate commercially managed populations. We will also use microsatellite analysis to compare allelic diversity these New World populations of honey bees with populations of Old World bees where Apis mellifera is endemic. We will then compare the relatedness of all drones mated to each queen (mate-mate), all queens within each population (queen-queen), each queen with each of her mates (queen-mate), and all drones within each population (dronedrone).This will enable us to quantify the levels of genetic similarity among the managed honey bee populations compared to the two ends of that continuum. Preservation of favorable genetics and augmentation of diversity using imported honey bee semen. Since 2010 we have developed practical methods for the cryogenic storage and recovery of honey bee germplasm and maintained aliquots of imported material in a genetic repository. In 2013, we expanded this cryogenetic program to include conservation of top-tier genetics of US domestic stocks of honey bees. The goal of this program is to allow queen producers the future ability to breed through time via backcrossing to extant lines. Effective cyropreservation techniques have also made possible the importation of honey bee germplasm for evaluation and breeding purposes.Objective 3 -- To determine how land management practices affect pollinator nutrition and how nutrition affects honey bee colony productivity and success Rationale and Significance Flowering plant species differ considerably in the nutritional content of their pollen, and this can have important ramifications for bee health (Levin and Haydak 1957, Standifer 1967). Nectar is also important for bee nutrition because, as the primary carbohydrate source for bees, sufficient quantities are essential for larval growth and meeting the energetic demands of bee activity (Brodschneider and Crailsheim 2010). The diversity and quality of floral resources at the landscape level may therefore have a significant impact on the nutrition of bees, particularly those with large foraging ranges such as honey bees. We will investigate the role of nutrition on colony physiology, growth and immunocompetence, and recommend both bee and land management practices to improve bee nutrition, in several ways. These data can be incorporated into models used to support quantitative analyses and qualitative assessments for pollinator habitat enhancement, and to determine pollen preferences among Apis and non-Apis bees.Objective 4 -- To assess the effects of exposure to pesticides and other xenobiotics on the survival, health and productivity of honey bee colonies and pollinator abundance and diversity Rationale and significance Bees are exposed to an array of xenobiotics in the course of foraging in a landscape natural plant-derived toxins, metals, pollutants, pesticides and spray adjuvants (Johnson et al., 2010). In high concentration these xenobiotics may have direct effects on bees, either through acute mortality at the larval or adult stage, or more subtly, through colony-level effects that harm a colonys chances of surviving over the course of a season. Combinations of xenobiotics may increase bees susceptibility to exposure to a particular pesticide, or the cocktail of xenobiotics may produce unexpected synergistic interactions (Glavan and Bozic, 2013). Further work is needed to determine relevant xenobiotic exposures in bees as well as establish exposure levels above which xenobiotic exposure directly harms bees ability to produce hive products and deliver pollination services. Insights gained can be provided to beekeepers, growers, manufacturers and regulators to mitigate any effect that xenobiotic exposure has on honey bee health and productivity.

Progress 11/21/19 to 09/30/20

Outputs
Target Audience:Beekeepers, growers, land managers, homeowners, and other scientific professionals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Dr. Rangel hosted the 5th annual Art of Queen Rearing workshop, where she and her staff teach beekeepers about queen and drone production and management. [41] Preparing Winning Grants to USDA-NIFA. TAMU, 1500 Research Pkwy. 12 Sep. 2019. [40] Teaching with Technology Lunch Series II. TAMU, 601 Rudder. 30 November 2018. How have the results been disseminated to communities of interest?Dr. Rangel and/or her postdoctoral research fellow, graduate student(s) and undergraduate student(s) involved in these projects attended and presented oral talks or posters at at least one scientific conference per year, in one of the following conferences: • Annual National and Regional meetings of the Entomological Society of America (ESA), 2017, 2018, 2019 • American Bee Research Conference 2017, 2018, 2019 • Graduate Research Forum, Department of Entomology, Texas A&M University, 2017, 2018, 2019 • American Beekeeping Federation Annual meeting, 2017, 2018, 2019 • Texas Beekeepers Association annual convention, 2017, 2018, 2019 • Texas Beekeepers Association summer clinic, 2017, 2018, 2019 • Austin Area Beekeepers Association Bee School, 2017, 2018, 2019 • Central Texas Beekeepers Association Bee School, 2017, 2018, 2019 The PI, postdoctoral research fellow, graduate student(s) and undergraduate student(s) involved in these projects attended and presented oral talks or posters at least one beekeeping meeting per year to share our results with the beekeeping community, at: • Texas Beekeepers Association Summer Clinic or Annual Convention • American Beekeeping Federation • Local, county beekeepers association in TX • Heartland Beekeepers Association or any State or Regional Beekeeping conference The PI, postdoctoral research fellow, graduate student(s) and undergraduate student(s) involved in these projects also attendedand presented oral talks or posters at least one university-wide conference or meeting per year to share our results with the local academic/student community, which can be any combination of the following conferences: • Texas A&M University Department of Entomology's Annual Graduate Research Forum • Texas A&M University Department of Entomology's Seminar Series • Ecological Symposium, Texas A&M University • Student Research Week, Texas A&M University The PI, postdoctoral research fellow, graduate student(s) and undergraduate student(s) involved in these projects attended and presented in at least one community outreach event to share our results with the public and communicate our work through outreach activities to our local community, which can be any combination of the following conferences: • Brazos Valley Museum of Natural History's Boom Days • Texas A&M University's College of Agricultural and Life Sciences Annual Tailgate What do you plan to do during the next reporting period to accomplish the goals?We will continue to train undergraduate and graduate students, research staff and postdoctoral research associates to accomplish the research, service and outreach goals set by this Hatch Project, as we have been doing for the last eight years. 1. We will change knowledge of the students and postdocs involved in the projects through: • Development, execution, and delivery of research project • Completion of graduate level courses at Texas A&M University that will help increase the knowledge of students in the field of Entomology and Apiculture • Weekly laboratory meetings with all members of the Rangel Lab to assess progress in all objectives • Instruction of Honey Bee Biology, and Honey Bee Management courses at the undergraduate level at Texas A&M University • Development and execution of several scientific techniques/tools to accomplish research goals 2. We will evaluate the outputs involved in the projects by developing and executing the following experimental procedures: • Insect source. We will use Italian honey bee colonies and queens (Apis mellifera Ligustica) for all experiments, to be purchased from nearby producers in the city of Navasota, TX. We will label all queens to ensure they do not get inadvertently replaced by Africanized bee stocks. All queens heading source larvae for grafting will be super-sisters to each other. To create super-sister queens, all queens will be daughters of the same mother who will be single-drone inseminated (SDI), following the technique described by Laidlaw (1977), which has been used in similar studies • Grafting. Experimental queens will be raised by transferring young larvae from a colony to cells in another queenless colony to initiate queen-rearing behavior, a common procedure known as "grafting" • Observation colonies. We will create medium-sized honey bee colonies from larger colonies that live in nearby bee yards, all headed by artificially inseminated super-sister Italian queens. • Genotyping. To calculate queen mating frequency we will collect emerging brood from a queen's colony and extract DNA from their thoraces using DNeasyTM kits. The DNA samples will be screened at 6 highly polymorphic microsatellite loci • Spermatozoa viability analysis. We will analyze spermatozoa viability following a protocol similar to that described by Collins and Donoghue (1999). This will be done in collaboration with the Theriogenology Laboratory directors at Texas A&M University. • Statistical analysis. To test the effects of the queen-rearing environment on the morphological characteristics of queens, we will conduct non-parametric Kruskal-Wallis and ANOVA tests to analyze data • Drone rearing. We will use plastic frames sold commercially with hexagonal cell templates to rear drones in laboratory conditions. 3. We will evaluate our progress in the following general ways: We hope to be successful in grafting queens for all experiments. We also hope that drone rearing will occur early in each year's spring so that they can be produced early in the year, not only for drone quality measurements, but also to enable successful mating frequency of queens. We also hope that RNA can be extracted in large numbers to create the microRNA and mRNA libraries for the fertility fingerprint. 4. We will produce the following: • The PI, postdoctoral research fellow, graduate student(s) and undergraduate student(s) involved in these projects will produce at least two of the following tangible products per year to share our results with the public: • Scientific articles in peer-reviewed scientific journals • Articles in Trade magazines such as American Bee Journal, Bee Culture • Columns in the Texas Beekeepers Association Journal • Blogs and posts in social media sites such as the Facebook page of the TAMU honey bee lab, or its website • Oral presentations, posters, and outreach events outlined above

Impacts
What was accomplished under these goals? • Discovered that the prevalence of the levels of the microsporidian gut pathogens Nosema ceranae and N. apis have changed over the last 20 years in a feral honey bee population in South Texas, but have remained relatively at low numbers,and that N. apis disappeared from this population since 1998 • Discovered that the combination of miticides fluvalinate, amitraz and coumaphos in wax significantly decreases sperm numbers and viability in queens • Discovered that approximately 95% of the feral honey bees at Welder Wildlife Refuge is of Africanized maternal descent, and 60% of nuclear DNA contains alleles of African descent • Helped collect data for the national survey of colony losses led by the Bee Informed Partnership at the University of Maryland • Discovered that the chemical β-ocimene helps regulate foraging behavior of the honey bee • Discovered that endopolyploidy changes with age-related polyethism in honey bees • Discovered that exposure to in-hive miticides during queen development (used to control the ectoparasitic mite Varroa destructor) negatively affects queen retinue response, mating frequency, and ovary size • Showed that the miticides fluvalinate and coumaphos significantly decrease span of sexual maturity and lower sperm viability in drones • We mapped the location of feral honey bees at the Powdermill Nature Reserve in Pennsylvania to determine the mitochondrial DNA strains of these colonies, and their level of Nosema infections • We found severe negative synergistic effects of fungicides used in almond orchards during bloom on worker bee longevity • We discovered that honey bee associated viruses are found in ants that live in or around apiaries • We found that certain protein to lipid ratios in honey bee diets are preferred by bees, and these ratios help them combat viral disesases [in progress] • Dr. Rangel mentored 5 graduate students (two graduated with a Ph. D.) and over 10 undergraduate students and oversaw their research projects • Dr. Rangel and her students attended regional and national meetings of the Entomological Society of America • Dr. Rangel and her students attended and/or presented talks at the meetings of the Central Texas Beekeepers Association (Brenham, TX), the Austin Area Beekeepers Association (Austin, TX), the Texas Beekeepers Association (various locations and times), and the Brazos Valley Beekeepers Association (Bryan, TX) • Dr. Rangel has given or have been co-author in over 60 scientific talks, including eight international and several invited speakerpresentations at conference symposia • Served as Faculty Instructor in three Beekeeping Schools, where I showed a lot of my own research as part of the classes I instructed. Some of these included for 2019 are: [31] Brazos Valley Beekeepers Bee School. College Station, TX. 7 September 2019. [30] Keynote Speaker. Heartland Apicultural Society. Bowling Green, KY. 7-10 Jul 2019. [29] Keynote Speaker. Apiculture New Zealand Convention. Rotorua, NZ. 27-9 Jun 2019. [28] Long Island Beekeepers Association Meeting. Commack, NY. 22-24 March 2019. [27] Kansas Honey Producers Spring Conference. Lawrence, KS. 8-9 March 2019. [26] North East Kansas Beekeepers Association Conference. Lawrence, KS. 10 Mar 2019. [25] 8th Annual Austin Area Beekeepers Bee School. Round Rock, TX. 2 February 2019. [24] Louisiana Beekeepers Association Convention. Sulphur, LA. 6-8 December 2018. [23] Missouri Beekeepers Association Conference. Kirksville, MO. 17-19 Oct. 2018. [22] Georgia Beekeepers Association Conference. Lanier, GA. 27-29 September 2018. [21] Austin Area Beekeepers Association Monthly Meeting. Austin, TX. 17 Sept. 2018. [20] Brazos Valley Beekeepers Bee School. College Station, TX. 22 September 2018. [19] Keynote Speaker. Eastern Apicultural Society Meeting. Hampton, VA. 17 Aug. 2018. [18] Central Texas Beekeepers Association Bee School. Brenham, TX. 17 March 2018. [17] Ulster Bee Keepers Association Annual Conference. Greenmount College, Ulster, Northern Ireland. 9-11 March 2018. [16] 7th Annual Austin Area Beekeepers Bee School. Austin, TX. 27 January 2018.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Gonzalez AN#, Ing N, Rangel J (2017) Upregulation of antioxidant genes in the spermathecae of honey bee (Apis mellifera) queens after mating. Apidologie. 49(2): 224-234. DOI: 10.1007/s13592-017-0546-y.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Walsh E*, Rangel J (2018) Queen pheromones and mandibular gland dissection. Bee World. 95(1): 3-5. DOI: 10.1080/0005772X.2017.1373511.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Payne AN*, Rangel J (2018) The effect of queen insemination volume on honey bee (Apis mellifera) colony growth. Apidologie. 49(5): 594-605. DOI: 10.1007/s13592-018-0587-x.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Rangel J, Camp C**, Gonzalez A, Stoner** M, Hatter** A, Traver BE (2018) Genetic diversity and prevalence of Varroa destructor, Nosema apis and N. ceranae in managed honey bee (Apis mellifera) colonies in the Caribbean island of Dominica, West Indies. Journal of Apicultural Research. 57(4): 541-550. DOI: 10.1080/00218839.2018.1494892.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Powell JE, Eiri D, Moran NA, Rangel J (2018) Modulation of the honey bee queen microbiota: Effects of early social contact. PLoS ONE. 13(7): e0200527. DOI: 10.1371/journal.pone.0200527.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Corby-Harris V, Bowsher JH, Carr-Markell M, Carroll MJ, Centrella M, Cook SC, Couvillon M, DeGrandi-Hoffman G, Dolezal A, Jones JC, Mogren CL, Otto CRV, Lau P*, Rangel J, Schu?rch R, St. Clair A (2018): Emerging Themes from the ESA Symposium Entitled Pollinator Nutrition: Lessons from Bees at Individual to Landscape Levels. Bee World. DOI: 10.1080/0005772X.2018.1535951.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Fisher II A*, Rangel J (2018) Exposure to pesticides during development negatively affects honey bee (Apis mellifera) drone sperm viability. PLoS ONE. 13(12): e0208630. DOI: 10.1371/journal.pone.0208630.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Fisher II A*, Coleman C, Hoffmann C, Fritz B, Rangel J (2018) The effects of the insect growth regulators methoxyfenozide and pyriproxyfen and the acaricide bifenazate on honey bee (Hymenoptera: Apidae) forager survival. Journal of Economic Entomology. 111(2): 510516. DOI: 10.1093/jee/tox347.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ma R*, Villar G, Grozinger CM, Rangel J (2018) Larval pheromones act as colony-wide regulators of collective foraging behavior in honey bees. Behavioral Ecology. DOI: 10.1093/beheco/ary090.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Rangel J, Ward L (2018) Evaluation of the predatory mite Stratiolaelaps scimitus for biological control of the honey bee ectoparasitic mite Varroa destructor. Journal of Apicultural Research. 57(3): 425-432. DOI: 10.1080/00218839.2018.1457864.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Fisher II A*, Harrison K*, Love C, Varner D, Rangel J (2018) Spatio-temporal variation in honey bee (Apis mellifera) drone spermatozoa viability in central Texas apiaries. Southwestern Entomologist. 43(2): 343-356. DOI: 10.3958/059.043.0206.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Payne AN*, Walsh E*, Rangel J (2018) Initial exposure of wax foundation to agrochemicals causes negligible effects on the growth and winter survival of incipient honey bee (Apis mellifera) colonies. Insects. Jan 8;10(1). DOI: 10.3390/insects10010019.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Lau P*, Bryant V, Ellis JD, Huang ZY, Sullivan J, Schmehl DR, Cabrera AR, Rangel J (2019) Seasonal variation of pollen collected by honey bees (Apis mellifera) in developed areas across four regions in the United States. PLoS ONE 14(6): e0217294. DOI: 10.1371/journal.pone.0217294.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ma R, Rangel J, Grozinger CM (2019) Honey bee (Apis mellifera) larval pheromones may regulate gene expression related to foraging task specialization. BMC Genomics. DOI: 10.1186/s12864-019-5923-7.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Fisher II A*, Rangel J (2019) Environmental factors that affect the reproductive health of honey bee (Apis mellifera) drones - a review. Apidologie. 50: 759. DOI: 10.1007/s13592-019-00684-x
• Type: Journal Articles Status: Published Year Published: 2019 Citation: J Rangel. 2018. Texas Beekeepers Association Bimonthly Column. 6 columns per year.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: J Rangel. 2019. Texas Beekeepers Association Bimonthly Column. 6 columns per year.