Interest in the topic of local intra- and interfield adult WCR movement grew in advance of the registration and commercialization of the first transgenic maize variety for corn rootworm in 2003: YieldGard® Rootworm. This variety is active against root-feeding diabroticite beetle larvae, including the WCR (EPA Office of Pesticide Programs, 2003). The potential for development of resistance to a particular Bacillus thuringien- sis (Bt)-plant-incorporated protectant and Bt technology in general war- rants action to reduce the risk of Bt resistance and to preserve both the current and the future utility of Bt (EPA Office of Pesticide Programs, 2001). To this end, the US Environmental Protection Agency (EPA) has mandated insect resistance management (IRM) plans for all transgenic crop varieties with Bt-plant-incorporated protectants (EPA Office of Pesticide Programs, 2001). A key element of IRM plans for Bt crops is the planting of non-Bt refuges that provide a large reservoir of Bt-susceptible insects that will disperse and randomly mate with potentially Bt-resistant adults emerging from nearby Bt crops. The IRM plan for YieldGard® Rootworm includes a 20% structured non-Bt refuge (EPA Office of Pesticide Programs, 2003). How well a particular refuge design promotes adequate mating between rootworms from the Bt crop and the non-Bt refuge will depend on how well the relative size and placement of crop and refuge fields are matched to the movement capabilities of WCR adults.
The defining role that adult WCR movement plays in the design and success of refuge configurations has sparked a renewed interest in quanti- fying individual beetle behaviour and movement patterns in the field.
Detailed insect activity data are often obtained by directly observing insects or applying mark–release–recapture methods to quantify move- ment parameters. Many techniques have been used to mark and release individual insects so that they may be identified if later recaptured at another location (reviewed by Hagler and Jackson, 2001). Mass marking techniques have been used in WCR field and laboratory dispersal studies (Lance and Elliott, 1990; Naranjo, 1990; Oloumi-Sadeghi and Levine, 1990; Spencer et al., 1999a).
In 1999 and 2000, extensive observations of WCR movement were made in an east-central Illinois maize field using mark–release–recapture methods. During 1999 and 2000, 4726 and 13,090 WCR adults,
134 J.L. Spencer et al.
respectively, were captured in maize fields and marked with fluorescent powder before being released in the centre of a concentric circular array of vial traps positioned in a 4-acre maize field. Marked insect recovery rates were disappointingly low. In 1999, 21 marked males and three marked females were recaptured (0.51% recovery); in 2000, four marked males and four marked females were recaptured (0.06% recovery). Based on distance from release point and interval between release and recovery, we calculated movement rates for males and females. Male movement rates were 4.6 m/day in 1999 and 16.1 m/day in 2000. Female movement rates were 2.6 m/day in 1999 and 21.8 m/day in 2000. While these rates fall broadly within the Coats et al. (1987) estimate of < 30 m/day for movement by tethered females engaging in trivial flight, the low recovery rates were troublesome. These results suggested that monitoring beetle dispersal using mark–release–recapture methods was very challenging, if not impossible, given the extremely high WCR abundance in east-central Illinois. The logistics of collecting, handling and marking enough WCR to ensure that a reasonable number of marked insects were recovered led us to consider a novel alternative to standard mark–release–recapture methods (Spencer et al., 2003).
For several years, observation of maize and soybean tissues in the gut contents of dissected WCR that were collected in soybean and maize fields, respectively, had been used as a ‘marker’ to indicate recent inter- field movement. Knowledge of average gut passage time for various ingested plant tissues allowed scientists to estimate rates of interfield movement. Though useful in a general sense, because both tissues were common in the landscape, it was not possible to know with any certainty where a particular insect originated; furthermore, the process of dissec- tion and identification was slow. An ideal marker for monitoring WCR movement would be one the insects acquired during the course of normal behaviour and it would have an isolated, identifiable source and be unambiguously detectable.
During WCR studies in transgenic maize plots, it was discovered that the presence of tissue from YieldGard® Rootworm maize (expressing the Cry3Bb1 protein) could be detected in the bodies of adult WCR that had recently fed on transgenic plant material (there are no acute toxic effects of Cry3Bb1 protein on adult WCR) (Spencer et al., 2003). Ingested trans- genic tissue is detected using lateral flow test strips (Bt-Cry3Bb1 ImmunoStrips, AgDia, Elkhart, Indiana, USA) that react specifically to the presence of the Cry3Bb1 protein expressed in YieldGard® Rootworm corn. The strips indicate the presence (two lines appear) or absence (one line appears) of Cry3Bb1 protein when inserted into a buffer solution con- taining a pulverized WCR beetle (Fig. 6.4). In calibration tests, CryBb1 protein was detectable inside WCR beetles up to 24 h after they last con- sumed YieldGard® Rootworm tissue (50% were Cry3Bb1-positive after 12–16 h). Owing to the diel movement patterns of WCR adults, the 24 h detection interval for Cry3Bb1 protein using the AgDia ImmunoStrip is well suited to monitoring daily movement by WCR.
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Transgenic tissue detection (TTD) has several advantages over tradi- tional mark–release–recapture methods of monitoring insect movement.
There is no handling of the insects to apply a marker since WCR acquire the marker during feeding. Because the YieldGard® Rootworm maize is continually available to insects in the field, new insects are constantly
‘marked’ as they feed. The short detection interval ensures that any observed displacement must have occurred within 24 h. Lastly, the prob- ability of detecting marked insects is much higher using TTD because the pool of marked insects is higher (and constantly being renewed). By growing isolated blocks of transgenic maize that are contiguous with non- transgenic maize varieties or other crops in rotation with maize, it is pos- sible to measure rates of WCR movement out of transgenic maize. Since the distance from a source of Cry3Bb1-expressing maize is known and the detection interval is also known, we can calculate rates of movement for captured WCR between the transgenic maize and other locations. Over 3 years, we have found good coincidence between movement rate estimates derived from TTD and those generated from mark–recapture methods and have enjoyed a tremendous increase in the proportion of marked insects we recover. Nearly 15% (109/737) of WCR captured on 1 day in 2001
136 J.L. Spencer et al.
Fig. 6.4.Immunostrip test for detection of Cry3Bb1 protein (expressed in YieldGard® Rootworm maize; Monsanto Company, St Louis, Missouri, USA) contained in the gut contents of individual WCR beetles. Cry3Bb1-specific Immunostrips are available from AgDia, Inc., Elkart, Indiana (USA). The presence of two lines on an Immunostrip after development (10 min) in a processed WCR sample indicates that Cry3Bb1 protein was expressed in maize tissue ingested by the sample insect. The post-feeding detection interval for Cry3Bb1 protein in WCR gut contents is 24 h.
Pestles with abrasive
Tubes with buffer solution WCR Adult with transgenic
maize tissues (expressing Cry3Bb1) in gut contents
Cry3Bb1-specific
immunostrips Completed Cry3Bb1 detection tests
Cry3Bb1 (+)
Cry3Bb1 (–)
1 2
were Cry3Bb1-positive in plots surrounding YieldGard® Rootworm maize, compared to the 0.106% (9/8497) of fluorescent powder-marked WCR recovered within our trapping array in 1999. Within maize plots and between maize and soybean plots, c. 85–90% of male and female WCR moved ≤4.6–9.1 m/day (Spencer et al., 2003).