Immigration of Lygus spp. (Hemiptera: Miridae) and predaceous natural enemies to trap-cropped organic strawberry.

Lygus spp. are polyphagous pests that overwinter in weedy vegetation. In the spring on the central coast of California, Lygus spp. emigrate from weeds into strawberry fields. Subsequent feeding on strawberry flowers causes fruit deformation that precludes sale on the fresh market. Use of alfalfa (Medicago sativa L.) (Fabales: Fabaceae) as a trap crop has been hypothesized to prevent Lygus spp. colonization in strawberries. We examined the movement of Lygus spp. and associated predators from weeds to strawberry fields with alfalfa trap crops using a protein mark-capture technique. Insects and spiders were collected from weeds, strawberry, and alfalfa 1 day, 2 days, and ~2 wk after an albumin protein mark was applied to weeds bordering strawberry fields. For marked Lygus spp. that emigrated from weeds, the majority (79%) of adults were recovered from alfalfa trap crops; however, all nymphs immigrated to strawberry. Most protein-marked predators immigrated to strawberry, rather than trap crops, resulting in a marked predator-to-Lygus spp. ratio of 5:1. Trap cropping effectively reduced the colonization of Lygus adults in strawberry. Converting weedy areas to native perennial plantings could further mitigate the risk of pest migration, while simultaneously conserving beneficial insects.

A simple computerized Arduino-based control system for insect rotary flight mills.

Flight mills are widely used to investigate insect flight behavior. As technology advances, the means to build a computerized control system for a flight mill has become more accessible in terms of both price and availability of components. However, the specialized electronics and programming knowledge required to build such a system can still present an obstacle to interested parties. Here, we describe a simple and inexpensive flight mill control system that can be easily assembled and operated without specialized experience. The hardware and software components are built around an Arduino single-board microcontroller, which outputs raw data in the form of timestamped detections of rotations of the flight mill arm. This control system is suitable both as the basis for new flight mills and for replacing outdated computer controls on existing flight mills. Additionally, it can be used with any rotary flight mill design that uses an electronic sensor to count rotations.

Use of a Fluorophore to Tag Arthropods for Mark-Release-Recapture Type Research.

We examined the feasibility of externally marking insects with the liquid fluorescent forensic theft deterrent, SmartWater (SmartWater CSI, LLC.). We sprayed captive Lygus hesperus (Knight) (Hemiptera: Miridae), Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), and Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae) with SmartWater fluorophore, and then qualitatively examined them for fluorescence by visual inspection under ultraviolet (UV) light and quantitatively measured them with a multiwavelength microplate fluorometer. The results indicate that this product has enormous potential as a taggant for L. hesperus and B. tabaci. However, the marking efficiency for H. convergens was only adequate. The advantages and limitations of using SmartWater as a biological marker for arthropod mark-release-recapture research are discussed.

Arthropod Demography, Distribution, and Dispersion in a Novel Trap-Cropped Cotton Agroecosystem.

Vernonia [Vernonia galamensis (Cass.) Less.] (Asterales: Asteraceae) was examined as a potential trap crop for the cotton (Gossypium hirsutum L., Malvales: Malvaceae) arthropod complex. Four rows of vernonia were embedded within a 96-row cotton field. The abundance of true bug pests, true bug predators, and spiders were determined by whole-plant and sweep net sampling procedures during the early, middle, and late phases of the cotton-growing season. The census data showed that the arthropods had a strong preference for the vernonia trap crop throughout the cotton-growing season. The movement of the arthropods from the trap crop into cotton was also measured using the protein immunomarking technique as a mark-capture procedure. The arthropods inhabiting the vernonia trap crop were marked directly in the field with a broadcast spray application of egg albumin (protein) during each phase of the study. In turn, the captured specimens were examined for the presence of the mark by an egg albumin-specific enzyme-linked immunosorbent assay. Very few marked specimens were captured beyond the vernonia trap crop 1, 3, and 6 d after each marking event. The arthropods' strong attraction and fidelity to vernonia indicate that it could serve as a trap crop for cotton pests and a refuge for natural enemies.

Methyl Salicylate Fails to Enhance Arthropod Predator Abundance or Predator to Pest Ratios in Cotton.

Conservation biological control is a fundamental tactic in integrated pest management (IPM). Greater biological control services can be achieved by enhancing agroecosystems to be more favorable to the presence, survival, and growth of natural enemy populations. One approach that has been tested in numerous agricultural systems is the deployment of synthetic chemicals that mimic those produced by the plant when under attack by pests. These signals may attract arthropod natural enemies to crop habitats and thus potentially improve biological control activity locally. A 2-yr field study was conducted in the cotton agroecosystem to evaluate the potential of synthetic methyl salicylate (MeSA) to attract native arthropod natural enemies and to enhance biological control services on two key pests. Slow-release packets of MeSA were deployed in replicated cotton plots season long. The abundance of multiple taxa of natural enemies and two major pests were monitored weekly by several sampling methods. The deployment of MeSA failed to increase natural enemy abundance and pest densities did not decline. Predator to prey ratios, used as a proxy to estimate biological control function, also largely failed to increase with MeSA deployment. One exception was a season-long increase in the ratio of Orius tristicolor (White) (Hemiptera: Anthocoridae) to Bemisia argentifolii Bellows and Perring (= Bemisia tabaci MEAM1) (Hemiptera: Aleyrodidae) adults within the context of biological control informed action thresholds. Overall results suggest that MeSA would not likely enhance conservation biological control by the natural enemy community typical of U.S. western cotton production systems.

A Molecular Approach for Detecting Stage-Specific Predation on Lygus hesperus (Hemiptera: Miridae).

A molecular gut analysis technique is described to identify predators of Lygus hesperus (Knight), a significant pest of many crops. The technique is unique because it can pinpoint which life stage of the pest was consumed. Sentinel egg masses designed to mimic the endophytic egg-laying behavior of L. hesperus were marked with rabbit serum, while third instar and adult L. hesperus were marked with chicken and rat sera, respectively. Then, the variously labeled L. hesperus life stages were introduced into field cages that enclosed the native arthropod population inhabiting an individual cotton plant. After a 6-h exposure period, the predator assemblage, including the introduced and native L. hesperus population, in each cage were counted and had their gut contents examined for the presence of the variously marked L. hesperus life stages by a suite of serum-specific enzyme-linked immunosorbent assays (ELISA). The whole-plant sampling scheme revealed that Geocoris punticpes (Say) and Geocoris pallens Stal (Hemiptera: Geocoridae) and members of the spider complex were the numerically dominant predator taxa in the cotton field. The gut content analyses also showed that these two taxa appeared to be the most prolific predators of the L. hesperus nymph stage. Other key findings include that Collops vittatus (Say) (Coleoptera: Melyridae) and Solenopsis xyloni McCook (Hymenoptera: Formicidae) appear to be adept at finding and feeding on the cryptic L. hesperus egg stage, and that L. hesperus, albeit at low frequencies, engaged in cannibalism. The methods described here could be adapted for studying life stage-specific feeding preferences for a wide variety of arthropod taxa.

Refinement of the Protein Immunomarking Technique for Mark-Capture Research.

A follow-up study was conducted to further evaluate the marking efficiency of broadcast spray applications of egg albumin (from chicken egg whites) on Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae) in alfalfa. A previous study recorded exceptional marking efficiency (e.g., >95% of the population) on H. convergens when using relatively high concentrations (10 to 50%) of chicken egg whites. The present study examines marking efficiency of egg whites using lower concentrations of 2.5, 5.0, and 10.0%. We used cadaver and free-roaming beetles to measure protein mark acquisition (and retention) of each protein concentration by direct contact with the spray application and incidental contact with protein residue on the plant tissue, respectively. The vertical distribution of the protein mark was also determined by sampling the upper and lower portions of the alfalfa canopy. The data indicate, regardless of the egg white treatment, that the backpack sprayer provided uniform coverage of egg albumin on the alfalfa plants and cadaver beetles. Also, almost every free-roaming beetle acquired a mark within 24 h after contact exposure to protein marked plants. This study shows that a very low concentration of egg albumin is sufficient for marking arthropods directly in the field.

Laboratory Evaluation of Egg White and Milk External Biomarkers for Wasmannia auropunctata (Hymenoptera: Formicidae).

Acquisition and retention of two protein markers were tested on little fire ants, Wasmannia auropunctata Roger. Pure (100%) cow's milk and a dilution (10%) of chicken egg whites were applied to W. auropunctata directly by contact spray plus residue or indirectly via residual contact only with protein-marked plant debris. Protein-marked ants were held in plastic shoe-box-sized containers, collected at 0, 24, and 48 h after exposure to their respective marks, and then examined for the presence of the marks by a chicken egg albumin and milk casein-specific enzyme-linked immunosorbent assay. Cross-contamination rates were assessed by allowing ants marked with egg whites to interact with an equal number marked milk for 24 and 48 h, and then collected either individually or in bulk. Results indicated that the egg white biomarker was retained longer than milk and that more ants were successfully marked when the direct spray application method was employed. Cross-contamination rates were highest among bulk-collected ants and lowest among ants collected individually after 24 h. However, the rates of cross-contamination among individually collected ants increased and were similar to that of bulk-collected ants after 48 h. On the basis of our results, external protein marking may not be suitable if mass trapping is required or if the study extends beyond 24 h due to high cross-contamination rates among specimens collected in bulk and reduced marker detection rates.

A Gut Analysis Technique for Pinpointing Egg-Specific Predation Events.

A universal food immunomarking technique (UFIT) is described for postmortem gut analysis detection of predation on the egg stage of Lygus hesperus Knight (Hemiptera: Miridae). Collops vittatus Say (Coleoptera: Melyridae) and Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae) were fed a single L. hesperus egg that was marked with rabbit and chicken sera proteins. The protein-marked egg remnants were detectable in the guts of the majority of the predators by each sera-specific enzyme-linked immunosorbent assay (ELISA) for 3 to 6 h after a feeding event. A novel technique was then developed to expose protein-marked eggs to predators that simulated the L. hesperus endophytic oviposition behavior. The procedure entailed embedding L. hesperus eggs in an artificial substrate that mimicked the stem of a plant. A predator feeding choice study was then conducted in cages that contained a cotton plant and artificial stems containing endophytic (concealed) and exophytic (exposed) egg patches. The endophytic and exophytic egg treatments were marked with chicken and rabbit protein, respectively. The gut analyses revealed that higher proportions of both predator populations contained remnants of the exophytic egg treatment and L. hesperus eggs were more vulnerable to C. vittatus than H. convergens. This study shows how the UFIT can be used to pinpoint stage-specific feeding activity on two distinct egg exposure treatments (endophytic and exophytic) of the same species.

Dynamics of Predation on Lygus hesperus (Hemiptera: Miridae) in Alfalfa Trap-Cropped Organic Strawberry.

Alfalfa (Medicago sativa L.) (Fabales: Fabaceae) can be strategically planted as a trap crop for Lygus spp. in California's organic strawberry fields. Alfalfa has been shown to attract both Lygus spp. and, in turn, a Lygus-specific parasitoid, Peristenus relictus (Ruthe) (Hymenoptera: Braconidae). However, the impact of alfalfa trap-cropped strawberries on the Lygus spp. predator complex is unknown. Here we identify key predators of Lygus spp. found in organic strawberry. First, a general survey was conducted at an organic, non-trap cropped strawberry farm, to quantify predator abundance and to qualitatively assess their feeding activity on Lygus spp. We identified the 11 most abundant predator taxa present and, by using a Lygus-specific PCR assay, determined that about 18% of the insects and spiders contained Lygus spp. remains in their guts. We then conducted a study to examine alfalfa's role in conserving the most relevant predators in trap-cropped organic strawberries. Specifically, we quantified predator abundance and qualitatively measured predator feeding activity (by gut analysis) on Lygus spp. collected in strawberry plots either lacking or containing an alfalfa trap crop. Data revealed that some predator taxa, including the numerically dominant predator, Orius tristicolor (White) (Hemiptera: Anthocoridae), aggregated in alfalfa trap crops. The gut content analyses revealed that insect and spider predators collected from the alfalfa trap crop had a significantly higher proportion of their population containing Lygus spp. remains than those collected from nearby rows of strawberries. These results suggest that alfalfa trap cropping might be a useful tactic for conserving the biological control services of generalist predators in organically grown strawberries in California.

A Nonlethal Method to Examine Non-Apis Bees for Mark-Capture Research.

Studies of bee movement and activities across a landscape are important for developing an understanding of their behavior and their ability to withstand environmental stress. Recent research has shown that proteins, such as egg albumin, are effective for mass-marking bees. However, current protein mass-marking techniques require sacrificing individual bees during the data collection process. A nonlethal sampling method for protein mark-capture research is sorely needed, particularly for vulnerable, sensitive, or economically valuable species. This study describes a nonlethal sampling method, in which three non-Apis bee species (Bombus bifarius Cresson [Hymenoptera: Apidae], Osmia lignaria Say [Hymenoptera: Megachilidae], and Megachile rotundata Fabricius [Hymenoptera: Megachilidae]) were tested for a unique protein marker by immersing them momentarily in saline buffer and releasing them. Results showed that an egg albumin-specific enzyme-linked immunosorbent assay was 100% effective at detecting the protein on bees that were sampled nonlethally. Furthermore, this sampling method did not have an impact on bee survivorship, suggesting that immersing bees in buffer is a reliable and valid surrogate to traditional, destructive sampling methods for mark-capture bee studies.

Use of Body-Mounted Cameras to Enhance Data Collection: An Evaluation of Two Arthropod Sampling Techniques.

A study was conducted that compared the effectiveness of a sweepnet versus a vacuum suction device for collecting arthropods in cotton. The study differs from previous research in that body-mounted action cameras (B-MACs) were used to record the activity of the person conducting the arthropod collections. The videos produced by the B-MACs were then analyzed with behavioral event recording software to quantify various aspects of the sampling process. The sampler's speed and the number of sampling sweeps or vacuum suctions taken over a fixed distance (12.2 m) of cotton were two of the more significant sampling characteristics quantified for each method. The arthropod counts obtained, combined with the analyses of the videos, enabled us to estimate arthropod sampling efficiency for each technique based on fixed distance, time, and sample unit measurements. Data revealed that the vacuuming was the most precise method for collecting arthropods in the relatively small cotton research plots. However, data also indicates that the sweepnet method would be more efficient for collecting most of the cotton-dwelling arthropod taxa, especially if the sampler could continuously sweep for at least 1 min or ≥80 m (e.g., in larger research plots). The B-MACs are inexpensive and non-cumbersome, the video images generated are outstanding, and they can be archived to provide permanent documentation of a research project. The methods described here could be useful for other types of field-based research to enhance data collection efficiency.

Foraging Distance of the Argentine Ant in California Vineyards.

Argentine ants, Linepithema humile (Mayr) (Hymenoptera: Formicidae), form mutualisms with hemipteran pests in crop systems. In vineyards, they feed on honeydew produced by mealybugs and soft scales, which they tend and protect from natural enemies. Few options for controlling Argentine ants are available; one of the more effective approaches is to use liquid baits containing a low dose of an insecticide. Knowledge of ant foraging patterns is required to estimate how many bait stations to deploy per unit area. To measure how far ants move liquid bait in vineyards, we placed bait stations containing sugar water and a protein marker in plots for 6 d, and then collected ants along transects extending away from bait stations. The ants moved an average of 16.08 m and 12.21 m from bait stations in the first and second years of the study, respectively. Marked ants were found up to 63 m from bait stations; however, proportions of marked ants decreased exponentially as distance from the bait station increased. Results indicate that Argentine ants generally forage at distances <36 m in California vineyards, thus suggesting that insecticide bait stations must be deployed at intervals of 36 m or less to control ants. We found no effect of insecticide on distances that ants moved the liquid bait, but this may have been because bait station densities were too low to affect the high numbers of Argentine ants that were present at the study sites.

Protein Self-Marking by Ectoparasites: A Case Study Using Bed Bugs (Hemiptera: Cimicidae).

The ability to mark individuals is a critical feature of many entomological investigations, including dispersal studies. Insect dispersal is generally investigated using mark-release-recapture techniques, whereby marked individuals are released at a known location and then captured at a measured distance. Ectoparasite dispersal has historically been challenging to study, in part because of the ethical concerns associated with releasing marked individuals. Here, we introduce the protein self-marking technique, whereby ectoparasites mark themselves in the field by feeding on the blood of an introduced host. We demonstrate the potential of this technique using laboratory-reared bed bugs (Cimex lectularius L.) that marked themselves by feeding on either rabbit or chicken blood. We then used enzyme-linked immunosorbent assays to detect host-specific blood serum proteins in bed bugs. We assessed these protein markers' ability to 1) distinctively identify marked individuals, 2) persist following multiple feedings on an alternate diet, 3) persist over time across a range of temperatures, and 4) transfer from marked to unmarked individuals. Protein markers were detectable in bed bugs before and after molting, remained detectible after multiple feedings on an alternate diet, persisted regardless of whether an individual was starved or fed on an alternate diet following original mark acquisition, and did not transfer between individuals. The duration of detectability depended on temperature. Our results suggest that protein self-marking is an effective technique for marking bed bugs and holds promise for use in dispersal studies of ectoparasitic insects.

Administering and Detecting Protein Marks on Arthropods for Dispersal Research.

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ecological interactions. Arthropods are usually tracked in nature by tagging them with a unique mark and then re-collecting them over time and space to determine their dispersal capabilities. In addition to actual physical tags, such as colored dust or paint, various types of proteins have proven very effective for marking arthropods for ecological research. Proteins can be administered internally and/or externally. The proteins can then be detected on recaptured arthropods with a protein-specific enzyme-linked immunosorbent assay (ELISA). Here we describe protocols for externally and internally tagging arthropods with protein. Two simple experimental examples are demonstrated: (1) an internal protein mark introduced to an insect by providing a protein-enriched diet and (2) an external protein mark topically applied to an insect using a medical nebulizer. We then relate a step-by-step guide of the sandwich and indirect ELISA methods used to detect protein marks on the insects. In this demonstration, various aspects of the acquisition and detection of protein markers on arthropods for mark-release-recapture, mark-capture, and self-mark-capture types of research are discussed, along with the various ways that the immunomarking procedure has been adapted to suit a wide variety of research objectives.

Spatial density and movement of the Lygus spp. parasitoid Peristenus relictus (Hymenoptera: Braconidae) in organic strawberries with alfalfa trap crops.

Alfalfa trap crops are currently used to manage Lygus spp. in organic strawberry fields on the California Central Coast. The retention of Lygus spp. in alfalfa creates aggregated distributions that provide improved opportunities for biological control by the introduced parasitoid Peristenus relictus (Ruthe). The abundance and distribution of P. relictus between two trap crops separated by 50 strawberry rows were analyzed in 2008 and 2010. Parasitism of Lygus spp. nymphs by P. relictus (measured by larval abundance and % parasitism) was greatest in alfalfa trap crops compared with strawberry rows. A significantly positive correlation between host nymphs and P. relictus larvae in and between trap crops was found. Movement of P. relictus adults from a marked alfalfa trap crop into adjacent strawberry rows or trap crops was also studied in 2008 and 2009 using a chicken egg-albumin enzyme-linked immunosorbent assay mark-capture technique. In 2008 and 2009, 85 and 49% of protein-marked wasps were captured from central trap crops, respectively, indicating that alfalfa trap crops act as a concentrated "host-density anchor" in organic strawberry fields.

Dispersion, distribution, and movement of Lygus spp. (Hemiptera: Miridae) in trap-cropped organic strawberries.

Alfalfa (Medicago sativa L.) is a highly attractive plant host to Lygus spp. and is used as a trap crop in California organic strawberries to influence the dispersion and dispersal of these pests, particularly Lygus hesperus Knight. The abundance and distribution of Lygus spp. nymphs between two trap crops separated by 50 strawberry rows was analyzed in 2008 and 2010. Nymphs demonstrated a bimodal distribution in strawberries between trap crops, where nymphs were most abundant and aggregated in alfalfa, when compared with interior strawberry rows, where nymphs were less abundant. The majority of nymphs were concentrated in trap crops and nymphal densities in interior strawberry rows were well below economic thresholds. The movement of Lygus spp. from a marked alfalfa trap crop into adjacent strawberry rows or trap crops was also studied in 2008 and 2009 using a chicken egg albumin enzyme-linked immunosorbent assay mark-capture technique. The majority of marked-captured L. hesperus adults and Lygus spp. nymphs remained in alfalfa trap crops, rather than dispersing out into strawberry rows at 24 h, 48 h, and 2 wk, postprotein application. The attenuation of Lygus spp. movement in alfalfa associated with organic strawberries is a key component of successful trap cropping. A small percentage of marked adults and nymphs were captured in neighboring alfalfa trap crops, located 62 m from the point of protein application, highlighting the dispersal capacity of this key pest.

Plant water stress effects on the net dispersal rate of the insect vector Homalodisca vitripennis (Hemiptera: Cicadellidae) and movement of its egg parasitoid, Gonatocerus ashmeadi (Hymenoptera: Mymaridae).

Homalodisca vitripennis (Germar), a vector of Xylella fastidiosa, is associated with citrus plantings in California. Infested citrus orchards act as a source of vectors to adjacent vineyards where X. fastidiosa causes Pierce's disease. An analysis of the pattern and rate of movement of H. vitripennis and its egg parasitoid, Gonatocerus ashmeadi Girault, was conducted in a citrus orchard by using a protein mark-capture technique to quantify movement and net dispersal rates in the experimental areas. Treatments included irrigation at 100% of the crop evapotranspiration rate (ET(c)), 80, and 60% ET(c). Sex-specific net dispersal rates showed that H. vitripennis males and females moved consistently and contributed equally to the level of population change within treated areas. Trees irrigated at 60% ET(c) were the least preferred by H. vitripennis. Among all protein-marked individuals captured in the 60% ET(c) treatment, ≈ 75 and 88% in 2005 and 2006, respectively, were inflow individuals. Movement toward less preferable plants indicates that in agricultural landscapes dominated by perennial monocultures, there is a random component to H. vitripennis movement, which may result from the inability of H. vitripennis to use plant visual cues, olfactory cues, or both to make well-informed long-range decisions. The 80% ET(c) areas were a significant source of adult H. vitripennis and G. ashmeadi compared with the other treatments. Colonization rates by parasitoids were synchronized with the spatiotemporal distribution of H. vitripennis eggs. Results suggest that H. vitripennis movement from citrus into adjacent vineyards could be a result of random dispersal rather than oriented movement in response to host-plant characteristics.

Employing immunomarkers to track dispersal and trophic relationships of a piercing-sucking predator, Podisus maculiventris (Hemiptera: Pentatomidae).

Immunoproteins are markers that are useful for monitoring dispersal and/or pest consumption, but current application techniques are less effective for the large guild of piercing-sucking predators important in biocontrol. We quantified the use of protein immunomarks in tracking emigration of spined soldier bug, Podisus maculiventris Say (Hemiptera: Pentatomidae) and predation on the hornworm caterpillar, Manduca sexta L. (Lepidoptera: Sphingidae). An external protein mark was topically applied to adult P. maculiventris to assess persistence under field conditions for >2 wk. Internal marks were incorporated into the artificial diet of M. sexta to test retention of the internal mark in the prey and uptake of the mark by predators. External marks remained detectable in 100% of individuals after 3 d and >50% still tested positive at 12 d after application in the field. Internal diet-based marking was also effective in tracking feeding by P. maculiventris on M. sexta, especially using rabbit IgG that was far more persistent than chicken IgY. Nearly 90% of stink bugs fed caterpillars previously reared on protein-enriched diet retained their mark for 24 h. Surprisingly, diet concentration and time reared on diet had comparatively little impact on mark retention. Development on unmarked tomato leaves clearly diluted the initial diet mark, but plant-reared individuals that were marked were still successfully detected in 35 and 20% of the predators.

Foraging range of honey bees, Apis mellifera, in alfalfa seed production fields.

A study was conducted in 2006 and 2007 designed to examine the foraging range of honey bees, Apis mellifera (Hymenoptera: Apidae), in a 15.2 km(2) area dominated by a 128.9 ha glyphosate-resistant Roundup Ready® alfalfa seed production field and several non-Roundup Ready alfalfa seed production fields (totaling 120.2 ha). Each year, honey bee self-marking devices were placed on 112 selected honey bee colonies originating from nine different apiary locations. The foraging bees exiting each apiary location were uniquely marked so that the apiary of origin and the distance traveled by the marked (field-collected) bees into each of the alfalfa fields could be pinpointed. Honey bee self-marking devices were installed on 14.4 and 11.2% of the total hives located within the research area in 2006 and 2007, respectively. The frequency of field-collected bees possessing a distinct mark was similar, averaging 14.0% in 2006 and 12.6% in 2007. A grand total of 12,266 bees were collected from the various alfalfa fields on seven sampling dates over the course of the study. The distances traveled by marked bees ranged from a minimum of 45 m to a maximum of 5983 m. On average, marked bees were recovered ~ 800 m from their apiary of origin and the recovery rate of marked bees decreased exponentially as the distance from the apiary of origin increased. Ultimately, these data will be used to identify the extent of pollen-mediated gene flow from Roundup Ready to conventional alfalfa.