SBIR-STTR Award

Situation or Problem The blue orchard bee (BOB), Osmia lignaria, is a solitary, native bee species that has been demonstrated to be an excellent pollinator of almonds, cherries, apples, pears and other crops, but has yet to become available on a large scale. The economics and scientific knowledge are now in place for the BOB to become a commercial pollinator, but only if large populations of the bees can be supplied. One obstacle in raising these bees is that, at a minimum, 25% of the female bees allowed to emerge from their nests in an orchard will disperse from the intended pollination site. Dispersal can be greater (over 50%) when bees emerge from cocoons that were removed from their nests. Purpose In 2008, AgPollen conducted two field trials that showed that a BOB nest component could attract bees to construct more nests at a given site. For this project, we will build on that information using field and lab experiments. In California we will conduct two studies, one on almonds and one during propagation on wildflowers. In Utah, a third field trial will be conducted in an apple orchard. The nest component volatile chemicals will be extracted and identified. This will allow us to test various concentrations, fractions, and individual chemicals in our field tests. Chemicals will also be tested using a technique that can detect whether or not the bee can sense a particular chemical with its antennae. This research will seek a chemical or mix of chemicals that could be developed into a lure that will make nests more attractive to female BOBs. OBJECTIVES: Almond production depends on bee pollination and is the US's largest specialty export crop. Honey bees are transported from all over the US each year for almond pollination. Honey beekeepers currently face numerous problems including Varroa mites, American foulbrood, the small hive beetle, Africanized bees and most recently Colony Collapse Disorder. These problems have led to a shorter supply of colonies and consequently higher rental prices in the US. The blue orchard bee (BOB) (Osmia lignaria) is a solitary, native bee that has been shown to be an excellent pollinator of several crops including almonds, cherries and apples. For the BOB to be commercially successful, bee populations will need to increase in managed settings, i.e. orchards or farmed propagation sites, rather than from conventional wild-trapping of the bees. Much of the information needed to manage the species is readily available. There are, however, a few obstacles to growing a large population. One of these obstacles is the fact that in the open field, it can be expected that, at a minimum, 25% of the female bees allowed to emerge from their natal nests in an orchard will disperse from the intended pollination site. Dispersal can be greater (over 50%) when bees emerge from cocoons that were removed from their nests ("loose cocoons") prior to being placed in the orchard. Recent evidence shows that even when BOBs emerged from natal nests, establishment of females averaged 59%. Managing BOBs as loose cocoons will likely be a key feature of scaling up production to commercial levels, despite the potential increase in dispersal. Our understanding of how a newly emerged bee goes about locating a suitable nesting site is limited. As bee producers, we strive to create an optimal environment that will minimize dispersal away from the emergence site. Odors are an obvious choice for attracting bees and evidence indicates that BOBs are attracted to certain odor cues at the nest site. In preliminary studies conducted in 2008, we discovered that an extract made from a bee product increased nesting in two separate field trials. The overall objective of this Phase I proposal is to build upon what we know about natural bee products as nesting attractants. Funding from this proposal will be used to field test various fractions and combinations of the chemicals found in attractive extracts and then identify the chemicals found to show the most biological activity. If AgPollen is successful, the commercial BOB populations will grow rapidly and almond pollination will be more secure as it will not be entirely dependent on the services of the honey bee. APPROACH: In 2008, AgPollen conducted two field trials that showed that a BOB nest component could attract bees to construct nests. For this project, we will build on that information using field and lab bioassays. The nest component volatile chemicals will be extracted using chloroform and solid phase micro-extraction and identified using gas chromatography mass spectrometry and GC-flame ionization detection. Chloroform extracts will be tested under field conditions during almond pollination in California. Various concentrations of the extract and the actual component will be compared to control nests. Next, the extract will be offered along with extracts of other nesting components in screenhouses in California at AgPollen's propagation site. A third field study in a Utah apple orchard will test the extract, actual nest component and at least two specific chemicals found in the extract, identified by GC-mass spectrometry. Purified fractions of extract, separated using column chromatography will also be tested there. Lastly, extracts and chemical components will be tested using electroantenogram to test whether or not BOB females can detect the component chemicals. Ultimately, this research will seek a chemical or mix of chemicals that could be developed into a lure that will make nests more attractive to female BOBs. This research will be a cooperative effort between AgPollen LLC and USDA-ARS. Karin Anderson, postdoctoral researcher, will conduct the research under the direction of James Buckner (USDA-ARS), Theresa Pitts-Singer (USDA-ARS), Stephen Peterson (AgPollen) and David Moreland (AgPollen). Chemical extractions and analysis will be conducted at the USDA-ARS Biosciences Research Laboratory in Fargo, North Dakota, and field bioassays will be conducted at the USDA-ARS Bee Biology and Systematics Laboratory in Logan, Utah and at AgPollen field sites in California. Jordi Bosch (Autonomous University of Barcelona), an expert on Osmia species, has agreed to provide consulting services

Modern agriculture has relied almost exclusively on the honey bee for managed pollination services. Honey beekeepers currently face numerous problems including Varroa mites, American foulbrood, Africanized bees and most recently colony collapse disorder. These problems have led to a shorter supply of colonies and consequently higher rental prices in the United States. It is sensible to have more than one pollinator available to growers in case shortages become worse in the future. The blue orchard bee, Osmia lignaria, is a solitary, native bee that has been shown to be an excellent pollinator of several crops including almonds, cherries and apples. Almonds are the United States? largest specialty export crop and their production depends on bee pollination. Domestication and large-scale management of blue orchard bee is still developing, but there are already several business entities working with this species as an alternative pollinator. One of the management difficulties experienced with this bee is that many will fly away from their emergence site, and not return. It is not uncommon to lose up to half of the emerging females to such dispersal. In cooperation with USDA-ARS, AgPollen LLC conducted three blue orchard bee nest attractant field studies in 2009 with Phase I SBIR funding. These trials demonstrated that extract of a bee nest product could increase nesting by up to 2.7-fold compared to the control. Fractionation of the extract showed that free fatty acids and hydrocarbons were the most active in attracting bees to nests. A test of several free fatty acids found in the extract showed that three of them could increase nesting, and one of them increased nesting 8-fold compared to the control. The research proposed here will expand upon these results to optimize which chemical or chemical blend can most improve nesting success. This research will be a cooperative effort between AgPollen LLC and USDA-ARS. Jordi Bosch (CREAF, Autonomous University of Barcelona), an expert on Osmia species, has agreed to provide consulting services. Karin Anderson, postdoctoral researcher (AgPollen), will conduct the research under the direction of James Buckner (USDA-ARS), Theresa Pitts-Singer (USDA-ARS), Stephen Peterson (AgPollen) and David Moreland (AgPollen). Chemical extractions and analysis will be conducted at the USDA-ARS Biosciences Research Laboratory in Fargo, North Dakota, and field bioassays will be conducted at the USDA-ARS Bee Biology and Systematics Laboratory in Logan, Utah and at AgPollen field sites in California. Suterra, a leader in pheromone technology will provide assistance in formulation and release technology under a subcontract to this project. Paramount Farming Company, has agreed to cooperate on this project. The product developed from this enabling research would greatly enhance the rate of commercialization of blue orchard bee in almond production systems. By multiplying blue orchard bee populations more rapidly, orchardists will benefit from an alternative pollinator sooner, which will in turn help to secure an important segment of United States agriculture. OBJECTIVES: This Phase II proposal is to expand upon our demonstrated success in using scents from old nests to attract cavity-seeking blue orchard bee females to commercially placed nesting structures. During our Phase I research, we identified active components in the nest attractant and used these attractants to increase nesting success. The objectives for this Phase II proposal are to complete the following set of studies: (1) Multiple fine-tuning field bioassays of the attractant, (2) Electroantenogram bioassays of nest extract chemicals, (3) Field bioassays of attractant release and application technologies and methods, (4) Attractant blend longevity under field conditions, and (5) Large-scale proof-of-concept demonstration of the attractant. APPROACH: Full Formula Performance Bioassay (2011): The synthetic blend will be tested for female preference. Four replicates of each treatment in a randomized complete block design will be positioned in an almond orchard, prior to bloom. Bees will be released from other sites within the orchard, at least 30 m from the experimental shelters. Shelters will be checked in the evenings and completed nests will be removed and replaced. Once a week, female counts will be made at night. Analysis of variance will be conducted on female establishment and completed nests. Delivery Method Assay (2011): Four different technologies, to release the attractant at nesting sites will be tested. The lures will include (1) a rubber septa containing attractant, (2) attractant applied to filter paper, (3) a Biolure (R) controlled release applicator, and (4) a control. Four replicates of each treatment will be positioned in a randomized complete block design and placed into the orchard just prior to bloom. Analysis of variance will be conducted on female establishment and completed nests. Attractant Longevity Tests (2012): Lures will be removed from the freezer (where they are stored) and exposed to ambient conditions for 0, 1, 3, and 6 weeks. The lures will then be extracted using chloroform and quantified by gas chromatography to determine the proportion of chemical remaining. A bioassay of the variously aged lures will also be conducted in field cages. Large Scale Almond Proof-of-Concept Demonstration (2012): A large scale almond demonstration will be conducted with two treatments: (1) shelters with attractant and (2) shelters without attractant. The demonstration will be set up in three large orchards. Two plots of 20 acres will be selected in each orchard with at least a 100 m buffer between the plots. Bees will be released at least 30 m from the experimental shelters. A paired t-test will be used to compare female establishment and nest production. Extraction and Identification of Nest Chemical Compounds: Old nest components will be extracted with chloroform, evaporated and stored frozen under argon. The extract will be used in both field and laboratory bioassays and analyzed by GC-Flame Ionization Detection (-FID) and GC-Mass Spectrometry (-MS). In addition, Solid Phase Microextraction (SPME) will be used to identify any volatile components that may be lost during the chloroform extraction and therefore undetected by GC analysis. Purified fractions and chemical compounds will be separated using column chromatography. Lipids will be separated on a silica gel column and purified by high performance thin layer chromatography (HPTLC). Laboratory Bioassays and Assessment of Individual Chemicals: Extracts and chemical components of nests will be tested using electroantenogram. Female antennae will be excised and connected to an electrode through saline contacts. The antennae will then be placed in an air stream, and individual chemicals from an extract will be introduced into the air stream so that the change in action potential can be measured