ResearcH

Use of a New Immunomarking Method to Assess Movement by Generalist Predators BetweenDavid R. Horton,a Cover Vincent Crop P. Jones, and and Thomas Tree R. UnruhCanopy

Abstract. in a Pear Orchard

Poor understanding of natural enemy movement between cover crop and crop habitats has limited effective use of cover crops to enhance biological control of pest in agricultural systems. To address this problem, we used an egg-albumin immunomarker sprayed on a legume cover crop to monitor movement by generalist predators from the cover crop into the canopy of pear trees in a Central Washington State pear orchard. Generalist predators in four taxa (, , Chrysopidae, Araneae) were collected from both cover crop and pear tree canopy, and tested using enzyme-linked immunonoassay (ELISA) methods to determine if the marker was present. In the three years of study, > 90% of arthropods collected from the cover crop had acquired the marker, indicating that we had excellent coverage and marking of arthropods with the solution. Between 17 and 29% (depending upon the year of study) of the approximately 2000 specimens collected from the tree canopy were also found to be marked, which is evidence that a percentage of the generalist predator community in the tree canopy had originated from or had visited the legume cover crop. These specimens included spiders and immature , which presumably colonized the tree either by walking up the tree trunk or (for spiders) by ballooning into the canopy. Some care must be taken in applying this technology or in interpreting results, due to unknown effects of the marker on behavior, and to questions of whether the marker might transfer from marked to unmarked insects as they interact in the field. Despite these caveats, the technology described here provides a new tool for assessing the effects of habitat management on movement of generalist predators in crop systems.

onservation biological control can be defined as the use of and Waddington (1994), and includes studies in nut crops (e.g., Bugg C et al. 1991, Smith et al. 1996), stone fruits (e.g., Stephens et al. 1998), tactics that preserve natural enemies in crop systems (Barbosa 1998). These tactics include efforts to minimize the effects and pome fruits (e.g., Fye 1983, Haley and Hogue 1990). of pesticides on natural enemies, changes in cultivation or harvest Despite the extensive literature, there is a lack of consensus about methods, or manipulation of non-crop habitats to act as refuges for the actual value of using cover crops in orchards to enhance biological natural enemies within colonization distance of the crop. Habitat control. Numerous studies demonstrate that diversification of agri- manipulation to enhance biological control includes planting of cover cultural systems (as through cover cropping or intercropping) leads crops (Smith et al. 1996) and establishment of hedgerows or other to higher densities and diversities of generalist predators (reviews by refuges adjacent to the crop (Rieux et al. 1999). The term “ecological Sheehan 1986, Bugg and Waddington 1994, Landis et al. 2000, Langel- engineering” has been used to describe these tactics (Gurr et al. 2004). lotto and Denno 2004). However, it is often unclear whether higher There is interest in using habitat manipulation to enhance biological densities or diversities of natural enemies actually lead to improved control, as shown by the growth in numbers of books and reviews that pest control and protection of the crop (Bugg and Waddington 1994, address this issue (Barbosa 1998, Pickett and Bugg 1998, Gurr et al. Snyder et al. 2005). This uncertainty is in part due to methodological 2004, Snyder et al. 2005). problems in many studies, including lack of replication, insufficient Cover crops are used in fruit or nut orchards to reduce soil erosion, plot size, or lack of pest suppression data (Bugg and Waddington 1994, improve soil quality, and to help control weed and arthropod pests (Sus- StatementSnyder et al. of 2005). Research Question tainable Agriculture Network 1998). Interest in the use of cover crops in orchards to enhance biological control of arthropod pests is shown by the growing literature on this topic in technical journals or books In addition to the methodological shortcomings mentioned above, (Bugg and Waddington 1994, Pickett and Bugg 1998), in trade journals limitations in our understanding of natural enemy biology have slowed (Alway 1998), and on websites (U.C. Davis 2002). The intent is to use progress in using cover crops as a means to enhance biological control cover crop habitats to supplement natural enemies with resources not in orchards. In particular, we have a poor understanding of movement provided by the crop, with the expectation that the cover crop will then by natural enemies between the cover crop and trees. For example, if be a source of natural enemies moving into the tree. The literature on the cover crop is highly attractive to natural enemies, it may act to limit coverAmerican crops Entomologist and biological • Volume control 55, Numberin orchards 1 was reviewed in Bugg movement by natural enemies into the tree. Indeed, it is conceivable49 Table 1. Number of generalist predators collected in cover crop (by sweep net) and tree canopy (by beating tray), and apparent habitat that a cover crop could actually lead to a net loss of biological control preferences based upon numbers in each habitat if it is attractive enough to divert natural enemies away from the target No. collected pest (see Snyder et al. 2005). Many reviews of habitat manipulation ex- in plicitly state that poor understanding of movement by natural enemies Cover Tree CoverApparent Tree habitat between non-crop and crop habitats has hindered progress in using preference TAXON Generalist habitat manipulation to enhance biological control (Lavandero et al. crop crop 2004, Jervis et al. 2004, Snyder et al. 2005, Denno et al. 2005). Orius tristicolor X We have begun a project to assess whether a legume cover crop in GeocorisHETEROPTERA X pyri- Nabis 662* 24*8 X pear orchards leads to increased biological control of a major pest cola spp. brevis 329* 0 X in orchards of North America and Europe: pear ( Anthocoris sp. tomentosus 99* X (Förster); : ). Generalist predators, dominated 59* 1159* by the true bugs (Heteroptera), have long been implicated in pear psylla 10* 459* Chrysopa oculata X control (Madsen et al. 1963, McMullen and Jong 1967, Westigard et al. ChrysoperlaCHRYSOPIDAE plorabunda X 1968, Unruh et al.1995), and are key components of integrated pest Eremochrysa 194* 1113 X? management programs in North America and Europe. In the western Chrysopa coloradensis 132* 155* X? Anthocoris sp. U.S., these predators include mirid and anthocorid species such as Chrysopa nigricornis 33 X 13 5* (Uhler) and spp. (Westigard et al. 1968, 4 47* Unruh & Higbee 1994, Horton & Lewis 2000). Other generalist preda- Hippodamia convergens X tors, including lacewings (Neuroptera: Chrysopidae), ladybird beetles CoccinellaCOCCINELLIDAE septempunctata X 382* 15* (Coleoptera: Coccinellidae), and spiders (Araneae) may also be locally Coccinella transversoguttata X? Hyperaspis lateralis 127* 118*95 X important (Westigard et al. 1986, Miliczky and Calkins 2001, Horton et Harmonia axyridis 116* 36 X al. 2002). Unfortunately, we have little understanding about how cover 112 crops in orchards affect biology of these species, including whether 11* 159* *, asterisks indicate that the samples included immatures (identified to species by rear- these generalist predators move readily from cover crops into the tree ing in the laboratory, as necessary). canopy, where the target pest (pear psylla) could then be attacked. Inferences about whether a non-crop refuge acts as a source of a given predator species are often based upon abundances of the herbaceousAnthocoris vegetation tomentosus to arboreal and habitats Deraeocoris (Honěk brevis 1985), although our predator in the two habitats, where a significant presence in both is results suggested that this species was common in the pear tree canopy assumed to be evidence of movement between them (see Nguyen et (Table 1). appear to prefer al. 1984, Rieux et al. 1999, Scutareanu et al. 1999, Horton and Lewis shrubs and trees (Horton and Lewis 2000, ChrysopaMiliczky and oculata Horton 2005), 2000, Miliczky and Horton 2005 for examples in orchards). Table 1 but can also be collected from herbaceous vegetation (Tamaki and summarizes relative abundance data from 3 years of sampling for 3 Weeks 1968, Miliczky and Horton 2005). , which we taxa of generalist predators (Coccinellidae, Chrysopidae, Heteroptera) rarely collected in pear trees, has actually been discounted as a preda- collected from a legume cover crop (see below for details on cover tor of tree pestsChrysopa in orchards oculata just because of its apparent preference crop) and from the tree canopy at a pear orchard in Central Washing- for herbaceous vegetation (Putman 1932). However, others have sug- ton State. Arthropods were collected using sweep nets and beating gested that is an important predator of orchard pests trays. We list the five numerically mostOrius common tristicolor species in eachAnthocoris taxon, Hippodamia(including pear convergens psylla), and may exhibit high densities in pear trees tomentosusranked by numbers in theDeraeocoris cover crop brevis samples. The most abundant or other fruit trees (Briand 1931, Wilde and Watson 1963). Similarly, species were in the HeteropteraChrysopa ( coloradensis (White), Guérin-Méneville, which was very abundant Péricart, and ), while the least common in the cover crop, was uncommon in our tray samples. In other pear Chrysopaspecies was oculata a green lacewingOrius ( tristicolor Banks). Chrysopa The data orchards, this species has been shown to occur regularly in the tree nigricornissuggest that the predator communityAnthocoris included tomentosus habitat specialists (e.g., canopy (Westigard et al. 1968). In sum, it is not clear that the results Say and Chrysoperla plorabunda in the cover crop;Hyperaspis from our pilot study (Table 1) describe true habitat preferences and lateralis BurmeisterCoccinella and septempunctata in the treeCoccinella canopy) potential for inter-habitat movement. transversoguttataand habitat generalists (e.g., (Fitch), Inferences about habitat specificity arising from data in Table 1 Mulsant, L., and possibly would lead to mistakes in developing cover crop recommendations if Faldermann). FromChrysopa these data, oculata one would assume the data do not reflect actual patterns in habitat use and movement by that a legume cover cropChrysopa is unlikely nigricornis to be a direct source of pear psylla the predators. Mistakes in inferring habitat specificity could occur for biological control by species such as (found mostly several reasons. The simplest reason is that sampling efficiency may in the cover crop) orChrysoperla plorabunda (found. mostly in the tree differ among habitats or insect species. For instance, if some species canopy), but that it could be useful with respect to control of pear psylla listed in Table 1 were sampled efficiently by sweep nets but not by by a species such as beating trays, the abundance data would have overestimated use of Abundance data in theChrysopa literature nigricornis support someand Harmonia of our generaliza axyridis- the cover crop by those predators. Or, the data in Table 1 may indeed tions in Table 1 about habitat preferences, although there are also reflect actual distribution of species on those days or times of day that inconsistencies. Thus, H. axyridis the samples were taken, but may fail to describe predator behavior at (Pallas) appear to prefer arboreal habitats (LaMana and Miller 1996, other times when conditions are different. Certain predators listed in Hagen et al. 1999), as suggested alsoCoccinella by our septempunctata data, although Table 1 may be most active at night (Pfannenstiel and Yeargan 2002), may also be locally abundant in herbaceous vegetation (LaMana and whereas all of our sampling was done during the daytime. The habitat 50Miller 1996). The ladybird beetle may prefer specialists listed in Table 1 may beAmerican more generalized Entomologist than • implied if

Spring 2009 their abundances in a certain habitat were caused by availability of prey at the time of sampling. In sum, species that we categorized as specialists in Table 1 may actually have wider ranges in habitat use than suggested, and thus may show relatively extensive movement between habitats. If true, one practical consequence is that data such as those in Table 1 would not allow us to reliably predict the value of a cover Assessmentcrop as a source of Predatorof predators Movement moving into the tree. Methods.

There are a number of marking methods which can be used to study movement of insects (reviews in Hagler and Jackson 2001, Jones et al. 2006). Most of the methods are impractical on a large- scale basis (Hagler and Jackson 2001). Hagler et al. (1992) developed methods that allow detection of vertebrate protein markers (rabbit or chicken immunoglobulin G (IgG)) using enzyme-linked immunosorbent assays (ELISA). The high cost of the purified IgG makes that method Fig. 1. Cover crop in April (top left panel) and in May (bottom left panel), impractical for many purposes. A major extension of the protein mark- with photographs of flowering vetch and red clover. ing technique has now been developed that substitutes low-cost crude food proteins for the rabbit or chicken IgG (Jones et al. 2006). Jones et al. (2006) developed ELISA protocols for these markers, and field-tested soy protein (as soy milk or soy flour), chicken egg whites (as liquid or water hardness (Jones et al. 2006). The solution was applied using a 94- powdered egg whites), and bovine casein (as liquid or powdered milk). liter electric weed sprayer (Scorpion Sprayer, Union City, TN) mounted The assays detected these proteins to levels of < 30 ppb for casein and on a 4-wheel all-terrain vehicle (Fig. 2). The egg solution was sprayed < 8 ppb for the other markers. There was no cross-reaction between through a boom (3 m in length) having 7 flat fan-tip nozzles spaced markers, which allows testing of a single insect specimen for multiple at 0.5 meters and approximately 1 m above ground. The marker was markers and tracking of inter-habitat movements in two directions (by applied at one- to two-week intervals between early June and August, marking different habitats with different proteins). The markers are for a total of 17 applications (summed2 over the three years). We used mixed in water, applied using standard agricultural spray equipment, 20-25 liters of solution per 100 m of cover crop, applied at 30 psi. and are stable on foliage in the field for at least 2 wks. Insects become Irrigation was done on weekends (i.e., between consecutive trials), to marked either by direct contact with the spray at time of application, avoid washing the marker from the cover crop during any given trial. or by walking across dried residue. Arthropods were collected from both tree and cover crop at 24 and Our studies were done in a 1-ha Bartlett pear orchard composed of 96 hours following each application of the marker. Predators were col- 5-8 year old trees (2.4 – 3 m tall), located 15 km east of Moxee, WA, and lected from the tree by jarring limbs with a rubber hose (Fig. 3), and managed by USDA-ARS. Trees were planted with 4.9 x 4.9 m spacing. trapping the dislodged insects on a section of butcher’s paper that had A 2.4 m wide strip of soil beneath the trees was kept vegetation-free been coated with a thin layer of an adhesive (Tangletrap liquid insect using herbicides. Three2 cover crop plots, each 4 aisles wide x 40 m long trap coating, Tanglefoot Company, Grand Rapids, MI). The arthropods were removed from the adhesive in the field using wooden toothpicks (approximately 400 m of coverPisum crop sativum per plot) were arvense planted in autumn (a new toothpick used for each specimen), and placed singly into 1.5 ml preceding each of three growing seasons. ViciaThe cover villosa crop (Fig. 1) was microtubes (Fig. 3). Similar methods were used to obtain arthropods a mix of Austrian winter pea ( Trifolium incarnatumL. ssp. (L.) Poiret) planted at 80 kg of seed per ha, hairy vetch ( Roth) at 58 kg of from the cover crop, except that the vegetation was shaken or beat over seed per ha, and crimson clover ( Lolium L.) perenneat 13 kg of seed (plus rhizobacteriaFestuca inoculant)rubra per ha. The rest of the orchard floor was composed of an 80:20 mix of perennial ryegrass ( L.) and red fescue ( L.). Orchard practices (pruning, fertilization, weed control) in experimental blocks were similar to practices used in commercial orchards.Acyrthosiphon Arthropod pests pisum were not managed. Irrigation was done using under-tree sprinklers.Frankliniella Potential occidentalis prey in the cover crop included pea , Lygus(Harris) (Hemiptera: Aphidi- dae), western flower thrips, (Pergande) (Thy- sanoptera: Thripidae), and immature spp. (Hemiptera: ), all occurring at high numbers in sweep net samples. Egg white protein was used as a marker to assess movement by generalist predators from the legume cover crop into pear tree canopy Fig. 2. Undiluted egg white (Fig. 2). The trials were done between June and August of 2004-2006. (top right panel) The cover crop was sprayed with liquid egg whites (MGW Brand frozen and spray apparatus. pasteurized egg whites, M.G. Waldbaum, Wakefield, NE) diluted in water to 20% (2004) or 10% (2005 and 2006). Water softener (tetrasodium ethylenediaminetetraacetic acid [EDTA]; Raynox®, Pace International, Wapato, WA) at 40 ml per 25 liters of solution was added to reduce American Entomologist • Volume 55, Number 1 51 across the residue could have acquired the marker (V.P.J. unpublished data). However, frequency of the marker in arthropods collected from the tree canopy on that date was not higher than observed during the rest of the collections (see below), suggesting that the drift inferred from the 20 June 2006 leaf samples did not contribute to marking of tree-collected arthropods. Over 90% of arthropods collected from the cover crop carried the egg marker, irrespective of whether the marker was applied at 20% dilution (2004; Table 2) or 10% dilution (2005-06; Table 3). These results indicate that we had good coverage of the cover crop with the egg solution, and that arthropods readily acquired the marker. Of more interest, 17–29% of tree-collected specimens carried the marker (Tables 2-3), which suggests that some proportion of generalist preda- tors inhabiting the tree canopy had recently visited the cover crop or had originated there. For the 20 June 2006 sample (i.e., the date on which drift was observed), 4 of the 35 (11.4%) arthropods collected Figure 3. Method used to collect samples from tree canopy, with examples from the two plots in which drift had occurred were found to carry of green lacewing and ladybird beetle trapped in adhesive. Bottom right the marker. Control arthropods collected off-site were uniformly free panel shows technique used to move a specimen from the adhesive into a of the marker. microtube. Approximately 20% of tree-collected predatory Heteroptera carried the cover crop marker (Tables 2-3). Species of Heteroptera are thought to be important generalist predators of pear pests in pear growing the top of the adhesive. Tree samples were collected before the cover regions worldwideDeraeocoris (Westigard brevis et and al. Anthocoris1968, Solomon tomentosus et al. 2000). Two crop samples were obtained, to lessen chances for contamination of species that are known important predators of pear psylla in the Pacific the tree specimens. Collecting methods replaced standard techniques Northwest are (Westigard (sweeping of vegetation or aspirating of insects from beating trays), et al. 1968, Fields and Beirne 1973, Horton and Lewis 2000). Both to prevent specimens from coming into contact with one anotheroC until species occur commonly on deciduous trees and shrubs (Horton and and transferring marker. Microtubes were put on ice immediately Lewis 2000), and our sampling data suggested that both species had a for transport to the laboratory. Specimens were stored at -2 preference for the tree canopyAnthocoris (Table 1). tomentosus However, the and marking Deraeocoris trials they were assayed for presence of the marker. In 2004, our objectives actuallybrevis indicated that the cover crop had been visited by a fairly Oriuslarge were primarily to show that the marker technology was suitable for percentagetristicolor, Nabis of tree-collectedGeocoris our purposes, and we collected only limited taxonomic information for (15-31% of specimens). Sample sizes for tree-collected the Coccinellidae and Chrysopidae. In 2005 and 2006, we identifiedLygus sp., and spp. were relatively small, but results Coccinellidae and Chrysopidae to species. Spiders were monitored all indicated that a percentage of those Heteroptera collected from trees three years, but were not identified to species. One pest taxon ( also had visited the cover crop (Tables 2-3). werespp.) wasused common as negative in the controls. cover crop, and was also assayed. Insects and Tree-collected chrysopids often carried the cover crop marker (Ta- spiders collected from row crops at least 300 m from the study orchard bles 2-3), including 50% of total chrysopids assayed in the 2004 study. Thus, the data indicate that adult green lacewings did indeed move Microtubes containing the specimens were removed from storage in between cover cropChrysoperla and tree plorabunda habitats. In 2005-2006, when we had the freezer. Each specimen was washed gently for 3 minutes with 1 ml better taxonomic resolution for the chrysopids, we found that 12.3% of tris-buffered saline solution. The buffer wash was then subjected to of tree-collected , categorized as a habitat gen- indirect ELISA (Jones et al. 2006) for detection of egg marker proteins. eralist (Table 1), carried the marker (Table 3). Putman (1932) noted Samples were considered to have been marked if the ELISA optical that this species occupied both tree and ground vegetation in peach Table 2. Percentage of insects from cover crop and tree habitats density readings were four standard deviations above the mean of found to be carrying the marker, 2004 data unmarked control insects of the same species (Jones et al. 2006). Tree In 2005 and 2006, 10-20 pear leaves per plot were collected 24 h N N following each spray application and assayed for presence of the egg Cover crop 551 TAXON % positive % positive protein; these assays were done to confirm that the marker had not Anthocoris tomentosus drifted into the trees during application to the cover crop. Leaves HETEROPTERADeraeocoris brevis 1105 95.196.7 21.4 were collected from low-hanging limbs nearest to the cover crop, as Nabis 100 95.0 2855 21.1 123 258 21.7 we anticipated that the leaves on those limbs would be most likely to Orius tristicolor Geocoris sp. 125 100.0 20.0 carry the marker if drift had indeed occurred. Seven to eight leaves 621 97.4 3 33.3 98.5 per plot were then randomly selected from the 10-20 leaves that were spp. 136 92.6 0 -- Results and discussion 99.8 85 collected, and assayed for the marker using ELISA. CHRYSOPIDAE 67 44 50.0 Hyperaspis lateralis 98.5 . For the 11 dates in 2005 and 2006 in COCCINELLIDAE 415 68.2 TOTAL which pear leaves were collected, the leaves were found to be free 66 61 60.7 of the marker in all but two plots for the 20 June 2006 sample; 5 of 1587 97.5 680 29.1 14 assayed pear leaves from those two plots on that date had levels Tree-collected specimens limited to adult insects; cover crop specimens included some nymphs and larvae. N = numbers assayed. 52of marker sufficiently high that some percentage of insects walking American Entomologist •

Spring 2009 Table 3. Percentage of insects from cover crop and tree habitats found to be carrying the marker, 2005 and 2006 data combined Tree ally moving up the tree trunk into the canopy. Over 13% of immature N N chrysopids collected from the tree canopy carried the marker. We had Cover crop large sample numbers for spiders in all three years, and found that TAXON % positive % positive Anthocoris tomentosus 11 15.9-17.4% of these predators collectedLygus from the tree had visited the HETEROPTERADeraeocoris brevis 1287 93.6 465 19.6 cover crop, presumably colonizing the tree canopy either by ballooning Orius tristicolor 100.0 121 31.4 Lygus Nabis 5560 96.7 326 15.0 or by walking up tree trunks. Our numbers were low for the tree Geocoris . 867 95.0 10 20.0 samples, but suggested that a large percentage of collected from sp. 100.0 4 25.0 11.9 the trees had originated in the cover crop (Table 4). spp 294 87.1 4 25.0 Some Caveats Chrysoperla plorabunda 15 Unknown effects of marker on arthropod behavior. Care is CHRYSOPIDAEEremochrysa 31 90.3 109 Chrysopa nigricornis 93.3 57 12.3 Chrysopa oculatasp. 87 85.7 47 10.6 Chrysopa coloradensis 10 -- 3 33.3 required in applying this technology and in interpretation of results. 87.5 0 -- One concern includes unknown effects of the marker on arthropod 100.0 2 0.0 Hippodamia convergens 5 behavior, an issue common with all marking techniques. That is, if the COCCINELLIDAECoccinella transversoguttata 12015 99.2 83 8.4 egg solution happened to be either repellent or attractive to predators, Coccinella septempunctata 74 98.6 20.0 movement into the tree canopy from the sprayed cover crop may have lateralis 100.0 10 10.0 Harmonia axyridis 21 100.0 26 7.7 occurred at a different rate than what would occur naturally from an Hyperaspis 10 100.0 7 0.0 unsprayed cover crop. These behavioral effects would lead to bias in TOTAL 0 -- 35 8.6 setting.estimating actual rates of movement. It would be useful to examine 1438 93.9 657 16.9 predatorTransfer behavior of marker on sprayed between and unsprayed marked andfoliage unmarked in a laboratory indi- Adult insects only were assayed (tree and cover crop specimens); data for immature insects are provided in Table 4. N = numbers assayed. viduals. Chrysoperla plorabunda A second issue arises because of the sensitivity of the assay. The liquid egg whites can be detected at 7.8 ppb (Jones et al. 2006), orchards of Canada. is potentiallyEremochrysa an impor- tant source of biological control in orchard systems of North America meaning that even minute quantities of the marker can be detected by (Szentkirályi 2001). Almost 11% of tree-collected sp. also the ELISA. We used a conservative threshold (4 standard deviations above the control mean) in analyzing ELISA results, to provide protec- carried theEremochrysa cover crop marker (Table 3). We are uncertain about this species’ role in orchard systems. Despite the relatively large numbers tion against accepting lower optical density readings as evidence for marker presence (Jones et al. 2006). Assay sensitivity also forced us of adult Eremochrysa encountered duringEremochrysa sampling (Table 1), none of the chrysopid larvae that we reared to adulthood during the study to develop new sampling methods. Traditional methods of collecting proved to be . Specimens of collected in this orchard arthropods in large numbers (i.e., use of sweep nets or beating study could have been visitors from adjacent shrub-steppe rangeland, trays) were not suitable because of concerns that marked specimens might transfer the marker to unmarked specimens as they came into where these lacewingsHyperaspis may be lateraliscommon (Zack et al.Harmonia 1998). axyridis contact with one another in the sweep net or aspirator vial. Instead, Species of CoccinellaCoccinellidae septempunctata that were at relativelyHyperaspis high numbers lateralis in samples included (Table 2), we moved specimens individually from the adhesive-covered butcher’s (Table 3), and (Table 3). paper into microtubes for storage. These methods made it difficult to obtain large numbers of certain species. was common in both cover crop and). tree samples (Table 1). Tree-col- Assay sensitivity may lead to questions about marker transfer from lected specimens of this species in 2004 showedHyperaspis very high lateralis frequency of marker (>60% of specimens; Table 2 These observations suggest that prey to predator. Specifically, it is conceivable that some proportion of the cover crop was an important source of coloniz- tree-dwelling predators that were found to be marked had acquired ing the tree canopy. This result was somewhat unexpected, as species the marker by feeding on marked prey rather than by visiting the in this genus are best known as predators of mealybugs (Hemiptera: cover crop. These circumstances could lead us to overestimate rates Table 4. Percentage of immature insects (Heteroptera, Chrysopidae, Pseudococcidae) and scale insects (Hemiptera: Coccidae)Pseudococcus (Gordon Coccinellidae), spiders, and Lygus spp. (Miridae) from cover crop maritimus1985, Hagen et al. 1999). These families includeQuadraspidiotus pest species perniciosus that are and tree habitats found to be carrying the marker common in the canopy of pear treesHyperaspis (grape lateralis mealybug, Tree (Ehrhorn); and San Jose scale, N N (Comstock)). It is not clear what was feeding on in Cover crop Harmonia axyridis, Coccinella sep- TAXON % positive % positive tempunctatathe cover crop,, Hippodamia although Gordon convergens (1985), and notes Coccinella that species transversoguttata in this genus Anthocoris tomentosus also feed on . Tree-collected DeraeocorisIMMATURE INSECTSbrevis (2005-06) 5 11.5 2 50.0 63 12.7 Coccinellidae 100.0 87 also included marked individuals (Table 3). These species are known Chrysopidae 13 100.0 30 13.3 to feed extensively on aphids (Honěk 1985, Hagen et al. 1999, Koch 70 85.7 6 66.7 2003), which may explain their use of the legume cover crop. 15.9 Lygus SPIDERS 88.9

Table 4 summarizes results forAnthocoris immatures tomentosus of Heteroptera, and Deraeocoris Coccinel- 2004 509 95.7 227 lidae, and Chrysopidae, and for spiders and one pest taxon ( spp.). 2005-06 431 404 17.4 brevis Lygus 558 11 Approximately 12% of nymphal LygusPEST spp. (2004) 97.7 63.6 carried the marker, suggesting that some nymphs of these two Lygus Lygus elisus Lygus hesperus species colonized the tree canopy from the orchard floor, presumably spp. (2005-06) 525 92.8 10 70.0 spp. includes mix of (Van Duzee) and (Knight) byAmerican walking Entomologist from the cover • Volume crop 55,and Number across 1the herbicide strip, eventu- 53 of movement between cover crop and tree for those predator taxa. led to transfer of the marker from prey to predator (Table 5). This Studies in which rabbit IgG protein was topically and internally applied result was obtained despite high optical density readings (approach- to prey showed that the protein signal was easily detected in whole ing 4.0) in several of the psylla taken from the marked plants (Table 5: body homogenates of both chewing and sucking predators that had subsample). Optical density readings for spiders were uniformly very been allowed to feed on the marked prey (Hagler and Durand 1994, low, irrespective of treatment (Table 5). Thus, under these conditions Hagler 2006). It is not clear whether an external wash of the predators and for these taxa, there was no evidence of marker transfer. Whether (as used in our studies) would also have detected the protein. Nor is these results would be representative for other taxa in this system or it clear whether more lightly marked prey, as might be produced as for predators and prey in other crop systems needs further work. At arthropods walk across marker residues on foliage, would have readily the very least, researchers who use this technology to assess predator transferred the marker. In our study system, it is unlikely that there movement should be aware that marker sensitivity may affect inter- was much movement (if any) from the cover crop into the tree by the Conclusionspretation of results. most important tree-dwelling prey (nymphal pear psylla), thus preda- tor specimens that had fed mostly on this prey species and which were also shown to be carrying the marker almost certainly had acquired the Poor understanding of predator movement between non-crop and marker from the cover crop rather than from their feeding activities. crop habitats has slowed progress in using habitat manipulation to Of more concern would be marker results for generalist predators that enhance biological control (Lavandero et al. 2004, Jervis et al. 2004, are capable of capturing and killing highly mobile arthropods (i.e., prey Snyder et al. 2005, Denno et al. 2005). The technology used here pro- that move extensively between habitats). Of the generalist predators vides an inexpensive method for marking large areas of habitat and monitored in our system, spiders are probably the most likely to have large numbers of arthropods, and thus affords us the means to assess fed on prey having these characteristics. questions about movement between cover crop and crop habitats. We have completed a small study in which marked adult pear psylla Our studies showed that 17-29% of specimens in the Heteroptera, were fed to jumping spiders (Salticidae). We chose to use psylla in these Coccinellidae, and Chrysopidae collected from trees carried the cover assays, as these insects were readily available and are easily marked crop marker, which we interpret as evidenceAnthocoris that there tomentosus was movement and (Jones et al. 2006). Psylla were marked by caging them for 24 hr on Deraeocorisfrom cover cropbrevis into tree canopy by these predators. Observations pear seedlings that had been coated with a 10% egg white solution. The include marker frequencies of 15-31% for seedlings were treated either by misting the solution onto the plants , two important predators of pear psylla which are or, in a second assay,Salticus by dipping the seedlings in solution. Spiders were generally thought to prefer tree and shrub habitats to herbaceous collectedSassacus from, Pelegrina shrubs, Phidippus growing , off-farm,and Phanias and included mid-sized to vegetation. Tree-collected immature insects and spiders also carried large immatures of (65% of specimens) with lesser numbers the marker, indicating that arthropods incapable of flight nonetheless of . Three treatments were were able to move from the cover crop into the tree canopy. compared: (1) spider fed one marked psylla and frozen immediately; The technology is flexible enough that it should be adaptable to (2) spider fed one marked psylla and frozen 24 hr later; and (3) spider address other questions in this cover crop system or in other systems. fed one marked psylla and a second, unmarked psylla 24 hr later. A For example, by applying different proteins to the cover crop and tree subsample of psylla from the marked plants was analyzed with ELISA canopy, it would be possible to assess movement by predators in both to confirm that the insects were indeed marked. Control (unmarked) directions (Hagler and Naranjo 2004), although some care must be psylla were obtained from a laboratory colony. Control spiders were taken in interpreting relative rates of movement if the two markers fed unmarked psylla and frozen immediately. Again, we used a con- differ in how effectively they mark the insects (Jones et al. 2006). We servative threshold (4 SD above the control mean) in accepting ELISA can partially correct for differences in marking efficiency between two scores as evidence of marker transfer. markers by evaluating the percentage marking that occurs in arthro- There was no evidence that feeding on marked psylla by spiders pods collected from the marked habitats. For example, if the egg marker Table 5. Results from spider feeding trials, showing optical density readings for marked psylla and spiders fed marked psylla, threshold optical density, and percentage of specimens exceeding threshold values (=% marked)

Treatment N a Optical density reading % exceeding Range Mean Median Threshold threshold (1) PEAR PLANT MISTED WITH 10% EGG SOLUTION Marked psylla (subsample) 10 0.005-3.095 0.514 0.092 0.013 90.0 Spider fed marked psylla, frozen immediately 1112 0.000-0.008 0.002 0.001 0.012 0.0 Spider fed marked psylla, frozen 24 h later 12 0.000-0.005 0.001 0.000 0.012 0.0 Spider fed marked psylla, unmarked psylla 24 h later, 0.000-0.007 0.001 0.000 0.012 0.0 then frozen

(2) PEAR PLANT DIPPED IN 10% EGG SOLUTION Marked psylla (subsample) 1514 0.001-3.998 1.412 0.762 0.005 85.7 Spider fed marked psylla, frozen immediately 1513 0.000-0.008 0.003 0.003 0.019 0.0 Spider fed marked psylla, frozen 24 h later 0.000-0.007 0.003 0.003 0.019 0.0 Spider fed marked psylla, unmarked psyllaSalticus 24 h later, 0.000-0.009 0.002 0.001 0.019 0.0 thena frozen Spiders were a mix of Salticidae, dominated by species. Control spiders, N = 12 (assay 1) and 14 (assay 2). Threshold is the reading four standard deviations above the mean of the control (unmarked) specimens. An optical density above threshold value is evidence that specimen has acquired marker. 54 American Entomologist •

Spring 2009 In is applied to the cover crop, and 90% of a particular taxon collected G.M. Gurr, S.D. Wratten and M.A. Altieri (Eds.), Ecological engineering for pest management: advances in habitat manipulation for arthropods. in the cover crop carries the marker, then the percentage marked in Hagen, K. S., N. J. Mills, G. Gordh and J. A. McMurtry. 1999. Terrestrial ar CSIRO Publishing, Australia. In tree-collected specimens could be adjusted to account for the less-than- - perfect marking occurring in the area of origin. In this specific example, Hagler,thropod J. R., predators A. C. Cohen, of insect D. andBradley-Dunlop, mite pests, pp. 383-503.and F. J. Enriquez. T.S. Bellows 1992. and we could multiply the percentage estimate for the tree-collected speci- T.W. Fisher (Eds.), Handbook of biological control. Academic Press, NY. mens by 1.1, to account for the fact that less than 100% (i.e., 90%) of New approach to mark insects for feeding and dispersal studies. Environ. the cover crop arthropods actually carried the marker. Hagler, J. R. and C. M. Durand. 1994. Entomol. 21: 20-25. The markers could also be used to directly test whether a pertur- A new method for immunologically bation of the cover crop affects movement of predators. For instance, Hagler,marking J. R., preyand C.and G. itsJackson. use in 2001.predation studies. Entomophaga 39: 257- the cover crop could be made less attractive to predators by killing the 265. prey (e.g., by use of a selective insecticide) or by destroying the cover Hagler, J. R. and S. E. Naranjo. 2004. Methods for marking insects: current techniques and future prospects. Annu. Rev. Entomol. 46: 511-543. crop itself (e.g., by mowing or use of herbicide), with aims to prompt A multiple ELISA system for simul- movement by predators into the tree canopy where the target pest Hagler,taneously J. R. 2006. monitoring intercrop movement and feeding activity of mass- can then be attacked. The marker technology could then be used to released insect predators. Int. J. Pest Mgmt. 50: 199-207. directly test whether the perturbation does indeed prompt movement. Development of an immunological technique for identifying Lastly, we have studies ongoing to combine the marker technology with Haley,multiple S. and predator-prey E. J. Hogue. 1990. interactions in a complex arthropod assemblage.Aphis Ann. Appl. Biol. 149: 153-165. recently developed molecular techniques for assessing gut contents of pomi Ground cover influence on apple aphid, predators (Agustí et al. 2003; T.R.U. unpublished data). By simultane- Honěk, A. DeGeer 1985. (Homoptera: Aphididae), and its predators in a young apple ous use of the marker technology and gut contents analysis, we will orchard. Crop Prot. 9: 225-230. test specifically whether predators that switch habitats from the cover Horton, D. R. and T.Habitat M. Lewis. preferences 2000. of aphidophagous coccinellidsAnthocoris (Cole- crop to the tree canopy then switch diet to attack the target pest, pear optera).and Deraeocoris Entomophaga brevis 30: 253-264. Seasonal distribution of spp. psylla. This approach has been used to monitor movement and feeding (Heteroptera: Anthocoridae, Miridae) in orchard by predators in row crops (Hagler and Naranjo 2004). Acknowledgments Horton,and non-orchard D. R., D. A. Broers, habitats T. of Hinojosa, Central Washington. T. M. Lewis, Ann. E. R. Entomol. Miliczky Soc. and Am. R. R.93: Lewis. 476-485. 2002.

We thank Robert Bugg, Steve Arthurs, and James Hagler for re- Diversity and phenology of predatory arthropods over- wintering in cardboard bands placed in pear and apple orchards of central viewing an earlier version of this manuscript. We also acknowledge Jervis, M. A., J. C. Lee and G. E. Heimpel. 2004. Washington state. Ann. Entomol. Soc. Am. 95: 469-480. Robert Bugg for raising the issue of whether predators might acquire In Use of behavioural and life-his- the marker by feeding on marked prey, and James Hagler for provid- tory studies to understand the effects of habitat manipulation, pp. 65-100. ing literature references on this topic. Field and laboratory help were G. M. Gurr, S. D.Wratten and M. A. Altieri (Eds.), Ecological engineering provided by Deb Broers, Merilee Bayer, Lila Scaife, Tamera Lewis, Gene Jones,for V.pest P., J.management: R. Hagler, J. F. advances Brunner, in C. habitatC. Baker manipulation and T. D. Wilburn. for arthropods. 2006. 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Stephens,and pears M. J., in C. Northern M. France, and S. D.Central Wratten Europe and C.– Frampton.3. Predators. 1998. Biocont. Sci.- ([email protected]) and (thomas.un Tech. 10: 91-128. [email protected]) are research entomologists at the Tree Fruit and VegetableVin- Fagopyrum esculentum Enhanc centInsect Jones Research Laboratory, USDA-ARS, 5230 Konnowac Pass Road, Wapato, WA ing biological control of leafrollers (Lepidoptera: Tortricidae) by sowing 98951, with research interests in the biological control of orchard pests. Sustainablebuckwheat Agriculture ( Network. 1998.) in an orchard. Biocontrol Sci. Tech. ([email protected]) is professor of entomology at Washington State 8: 547-558. University, Tree Fruit Research and Extension Center, 1100 N. Western Avenue,7 Managing cover crops profitably. Wenatchee, WA 98801. His research interests include biological control, pest Sustainable Agriculture Network, Washington, D.C. management, and population ecology of insects associated with tree fruits.

56 American Entomologist • Spring 2009