Progress in 2008-2009

Solter and Webster are amplifying Nosema apis and Nosema ceranae spores for a comparative mortality study among worker bees.  Resulting data is expected by late October 2009.
 

Objective 1.1C

Part 1, consisting of the cage experiments, ran from 28 October 2008 to 20 November 2008. Microarray analyses began 20 February 2009 and remain in progress. 
 

Objectives 1.2A-B and 1.2D-F

Solter has hired a postdoctoral research scientist, Dr. Wei-Fone Huang, to study progression of microsporidian disease in the honey bee host (with Z. Huang) and to conduct bioassays to evaluate temperature effects on bees that are multiply infected with microsporidia and viruses (with Cox-Foster and Ostiguy). She has determined that Nosema is present in the University of Illinois honey bee colonies and is currently sequencing the isolates to determine which species are present (N. apis and/or N. ceranae). In addition, Varroa mites and at least three virus species, most likely sacbrood, deformed wing virus and a paralysis virus, are present in the colonies. The identities of the viruses will be confirmed by the Cox-Foster lab at Penn State. Solter is building a large indoor screen cage to house a small honey bee colony (provided by Dr. Gene Robinson, Univ IL) for the remaining winter months. These bees have been removed from fumagillin-laced food sources and will be used to evaluate transmission of microsporidia in order to generate relatively Nosema-free bees for use in bioassays.  She plans to produce spores of N. apis and N. ceranae for use in experiments. 

Preliminary results of studies investigating the effects of pathogens on survivorship of eggs, larvae, pupae and adults indicate significant impacts on egg survivorship in infected colonies.  Complete results from 2009 work will be available by 12/31/09.
 

Objective 1.2C
Experiments have begun with newly emerged bees and also with 5-6 day old bees.  At two different doses, the virus was highly pathogenic to the newly emerged bees, and death was observed within 36-48 hours for the majority of these bees.  The day 5-6 old bees were more resistant.  With replicate experiments, using bees from different colonies for the replicates, we have used bees that were IAPV-free before feeding IAPV to them in sugar water.  We have also collected bees over time, after infection, to inquire as to whether the viruses has changed; these samples await RT-PCR. 

We are now extending our trials to inquire into dosage and how other ages of adult bees are affected.  In collaboration with Solter, we will also begin trials to inquire into the effect of Nosema on pathogenesis.  The revised completion dates for these studies are projected to be December 20, 2009 for IAPV and July 30, 2010 for DWV.
 

Objective 1.3

At proposal deadline, almost all apiaries are set up. J. Ellis (Florida), a late addition to our team, will be set up by mid-May 2009. The start dates for the rest are: Drummond 17 Apr, Spivak 7 Apr, Ostiguy 8 Apr, Aronstein 8 Apr, Visscher 11 Apr, and Sheppard 20 Apr. Cooperators are taking the initial bee samples for pathogen and pesticide analyses. 
 

Objective 1.4

Aronstien has refined two aims toward a Dip-Stick test for Nosema. In aim 1, she initiated development of the polyclonal antibodies against Nosema ceranae spore wall antigen SWP-32 (Aronstein, not published). Thus far, she has made a DNA construct for in vivo expression of SWP-32 fragment in rabbits and initiated injection protocol. Briefly, N. ceranae SWP-32 primers were designed to amplify 737 bp fragment of the 1025 bp gene. Gene analysis showed that this fragment is located down stream of the signal peptide (1 AA-22 AA). Primer sequences were designed as follows: SWP-32 F 5’- GAGCATTCTTTTAATTTA-3’, SWP-32R 5’-TTATTTTTCAAAACATCC-3’. Additional nested primer was designed at the 5’ of this fragment: 5’SWP-32 F 5’- ATGGATTTTTATTACCTT-3’. PCR amplification conditions were as follows: GoTaq polymerase activation step of 2 min at 95° C, followed by 35 cycles of 30 s at 95° C, 1 min at 44° C , and 1 min at 72° C, and a final extension step at 72° C for 10 min. PCR products were visualized by 0.8% agarose gel electrophoresis. PCR fragment amplified from N. cerane DNA was gel purified, cloned in pCR 2.1-TOPO plasmid vector (Invitronen) and sequenced insert (SeqWrite, Inc.). To determine the most immunogenic region of the translated sequence, the protein analysis was conducted using the on-line program Mobyle at (http://mobyle.pasteur.fr). A100 AA sequence was identified using this analysis and used for antibody production. 

A novel Genomic Antibody Technology (GAT) is being utilized to raise polyclonal antibody against N. ceranae SWP-32 protein (Strategic Diagnostics Inc., Newark, DE). Immunogens are expressed in vivo in animals, and therefore immediately presented to the immune system. GAT requires only the amino acid sequence of the target protein, eliminating the need for production and purification of the recombinant proteins. The plasmid expression vector (SDI. Inc) containing SWP-32 gene fragment was injected into rabbits following the company’s protocol. 

In aim 2, Aronstein will evaluate the performance characteristics of the SWP-32 antibody and design antibody-specific dipstick assay using a crude honey bee lysate. Future validation of this new assay will be conducted in partnership with a potential cooperator, a Biotech Company that will produce and market this device. At least two companies expressed interest in 2008-2009, and Aronstein is pursuing options for technology transfer with ARS administration. 
 

Objective 1.6

M. Ellis has hired Dr. Reed Johnson as a post-doctoral scientist to advance this objective. Previous work has shown that cytochrome P450 monooxygenase enzymes (P450s) and carboxylesterase enzymes are important for the detoxification of both tau-fluvalinate and coumaphos, both of which are currently registered as in-hive miticides for use against varroa mites. Deadly synergistic interactions have been observed in bees treated with sublethal doses of both coumaphos and tau-fluvalinate, probably due to the overloading of detoxicative enzyme pathway which both miticides share. Given bees' limited repertoire of detoxicative enzymes, the potential for similar synergistic interactions with newly registered miticides, or miticides used by beekeepers in the absence of registration, is high. 

Preliminary LD50 bioassays have shown that the newly registered miticide, fenpyroximate, present in Hivastan™ anti-mite patties, is also detoxified by P450s and carboxylesterases. Pre-treatment of bees with tau-fluvalinate or coumaphos synergistically increases the toxicity of this new miticide to bees. Preliminary bioassays using amitraz, which is not currently registered for use against varroa mites but was registered at one time, shows that this compound too is detoxified by P450s and carboxylesterases and is likely to also show synergism with the suite of other miticides. Bioassays to determine the relative importance of each detoxicative enzyme pathway, as well as tests for synergistic interactions, are also planned for thymol and oxalic acid, both of which are also in wide use against varroa mites. 

Synergism between pesticides and fungicides has also been a persistent concern for beekeepers, and it has been previously shown that erosterol-inhibiting fungicides, which work as P450 inhibitiors, can synergize the toxicity of pyrethroid pesticides applied to crops. We are also beginning to look for synergisms between the miticides and fungicides. Preliminary bioassays show that treatment of bees with P450-inhibiting fungicides increases the toxicity of both fenpyroximate and amitraz. 

The sublethal effects of pesticides, imidacloprid in particular, has been identified by many as a possible cause of CCD. However, the sublethal effects of in-hive miticides on bee behavior have largely gone unexamined. Using EthoVision tracking software we have begun examining the effects of sublethal doses of miticides on bee behavior. Rather than examining learning performance, as is commonly done through proboscis extension assays, EthoVision tracks individual bee movements inside a cage. Movement, if altered through pesticide exposure, could alter bees' ability to perform work in the hive, and, in the case of queens, could influence supersedure. Preliminary results show that sublethal doses of tau-fluvalinate can increase bee movement, while fenpyroximate decreases movement. Further work will focus on identifying the minimum dose of pesticide required to significantly alter bee behavior. 
 

Objective 2.2

Sheppard continues selecting and propagating WSU stocks underway since 2001 (originated as part of a grant to Cornell-WSU-USDA to establish an Africanized honey bee-free honey bee population from which to do selection). The starting place for these stocks was a large scale sampling of queen producers from across the US. Since 2005, this genetic material has been provided to beekeepers in the PNW via collaborative apiaries maintained by WSBA and local associations. Sheppard’s group provides queens annually to these apiaries across the state. Beekeepers subsequently graft from the WSU honey bee stocks and produce their own queens. As part of a long term effort, a collaborator and Sheppard brought in semen from A. m. ligustica and A. m. carnica under USDA-APHIS permit and inseminated virgin US production queens from CA bee breeders. The inseminated queens were kept at one of two quarantine apiaries established in the Palouse region of Washington. ARS tested the semen for the presence of viruses according to permit requirements, and inseminated queens from the carnica importation were released by USDA-APHIS. Following progeny testing of the Italian stocks, a small number of the ligustica inseminated queens were released. The permit is for a three year period (2008-2010) and is limited to sampling additional genetic material from the honey bee subspecies A. m. ligustica, A. m. caucasica and A. m. carnica for use in breeding efforts by the industry. 
 

Objective 3.4

Five studies were done investigating potential management and pollination efficacy of the commercial bumble bee, Bombus impatiens. 

Study 1 was designed to determine if bumble bees and honey bees select clones with more flowers over those with fewer flowers. The observations show that for bumble bees, honey bees and all species visiting blueberry during early bloom, the visitation increases with increased numbers of flowers in a clone. This suggests that one advantage to being one of the earliest clones to bloom is enhanced attraction of pollinators which should result in increased levels of fruit set. This study also suggests that early placement of hives in a blueberry field should not be detrimental to the bees as they will search out and recruit to clones that are in advanced stages of bloom. 

Study 2 was designed to determine if shade roofs and removal of pollen from bumble bee colonies affect the foraging intensity as measured by the number of foragers leaving and coming back to the colony. Contrary to our expectation that shading would lower quad internal temperature and stimulate foraging, we found that while shading did indeed lower temperature, foraging was positively related to increased temperatures. This suggests that shading could reduce foraging; however, it needs to be seen whether different temperature regimes that are common throughout a range of years in Maine during blueberry bloom (roughly mid-May to mid-June) will affect foraging in a similar way. The effects of pollen and nectar removal on foraging are not as clear. We plan on continuing this line of research in the near future as well as assessing other abiotic and biotic factors that might affect foraging. The goal of our study is to begin develop a better understanding of bumble bee foraging and pollination in lowbush blueberry with the aim at identifying factors that might result in better management of this pollinator resource. 

Study 3 was designed to determine if commercial bumble bees, Bombus impatiens, show preference to blueberry flower size and age. Single flower bioassays suggested that flower length was the only significant variable in determining flower choice by the foraging worker bumble bees in the flight cage. This relationship was positive, meaning that an increase in corolla length resulted in a greater probability that a bumble bee would choose the flower over a paired flower that was shorter in length. Flower width and age were not significant predictors of bumble bee preference, although corolla width and length were found to be moderately related. Therefore, both width and length measure a similar aspect of flower size, and length was a better predictor. Multiple flower bioassays suggested that flower density upon a stem did not enter into choice. The number of times that a flower had been previously visited showed a trend towards fewer visits to flowers that had been visited more times. The number of visits affecting choice might be due to the finding in some species of bumble bees that they mark flowers when foraging on them in order to reduce redundantly visiting previously visited flowers. 

Study 4 was designed to determine if bumble bees show preference to blueberry flowers compared to other flowering plant species commonly found in or adjacent to blueberry fields. Except for apple, Bombus impatiens foragers do appear to prefer blueberry to all co-flowering species both in percentage first visits and time spent foraging on flowers. 

Study 5 was designed to determine if bumble bees have potential to transmit the mummy berry fungus, Monilinia vaccinii-corymbosi. Our data suggest that the commercial bumble bee has a high likelihood of transmitting mummy berry spores to flowers resulting in secondary infection. In the future we plan to set up studies that quantify the ability of bumble bees to vector spores from lesions to flowers on healthy stems. 
 

Objective 3.5

Drummond is analyzing data from work done in 2005-2007 that bears on this objective. He compared efficacy of Bombus impatiens and the honey bee, Apis mellifera in the context of commercial lowbush blueberry pollination. Isolated fields were used as the unit of replication. Each field (range of 2-12 acres) was randomly assigned to a treatment group of stocking density. The stocking density treatments were either 4 hives / acre for the honey bee and 1, 2, 3, 4 and 5 colonies / acre. In addition, a control with no commercial pollinators was included. Replication of each stocking density ranged from 3-5 fields per stocking treatment. Drummond is assessing fruit set and yield as a function of stocking density and compiling economic data from the farmers involved in the study. Over the three-year period, fruit set and yield (wt / berry) were not significantly different just prior to harvest for pollinator species at the recommended stocking rates. 
 

Objective 4.1

The position for a dedicated technician to work under J. Skinner as the eXtension.org liaison has been filled by Mr. Michael Wilson. The Bee Health Community of Practice (CoP) application has been written, approved, and the community launched in fall 2008. The webpage is available to the public at http://www.extension.org/bee%20health. 43 people have joined the Bee Health community, linking the CAP team with other bee health experts. Community work pages have been populated with organizational information, website content outline, and 23 pages for the public site. Additional content has been obtained and is in preparation for upload, including bulletins in bee biology, best management practices, and diseases/pests. An online meeting protocol was established and we conducted meetings with the CoP, which included planning for the CAP team. In March 2009, the survey developed by the CAP survey committee (Objective 4.4) was converted and programmed by Michael Wilson to an online format using eXtension’s InstantSurvey web application. IRB approval for the online survey was successful through eXtension.org’s UNL, IRB protocol.
 

Objective 4.4

The survey instrument was written over the winter of 2008-2009. It was revised extensively following critiques from our Stakeholder Board. We have procured IRB approval through the University of Georgia, the lead institution in this CAP, and also through the resident provision available with eXtension.org via the University of Nebraska. The survey was launch in advance of our stated benchmark of 14 July 2009. In September 2009, participating beekeepers will receive from Nancy
Ostiguy a sampling kit and instructions for taking specimens of bees and wax for mite, disease, and pesticide residue analysis.