TITLE: USDA LTAR Common Experiment measurement: Natural pest suppression
AUTHORS: Nate Haan

[DESCRIPTION]
Pest suppression is a key ecosystem service worth billions of dollars annually, and it can vary with local management practices and landscape context. We propose to measure biocontrol services by deploying plasticine caterpillar mimics that record imprints of attacks by other organisms. Plasticine caterpillars are an easy and standardizable method indicating the attack rates on herbivorous lepidoptera larvae (Low et al. 2014, Howe et al. 2009, Haan & Landis 2023). They have been used successfully to study high-profile predation and its variation (e.g., Roslin et al. 2017). They also help identify the attacking organisms; bird beaks, arthropod mandibles, parasitoid wasp ovipositors, and small mammal teeth leave unique signatures on the dummy caterpillars.

[BEFORE_START]
The measurement described in this protocol for the LTAR network is dependent on acquiring extramural funding. Contact Nate Haan (nate.haan@uky.edu) for clarification on the ability to proceed with this protocol and whether samples will be processed centrally or at individual sites. 

[STEPS]
SECTION: Overview
1. Green plasticine caterpillars (3 cm × 15 cm) will be prepared and mounted on small wooden stakes in advance at a centralized location and mailed to each LTAR Site. 
SECTION: Overview
2. At each site, several caterpillars will be placed in the alternative and prevailing treatments at the plot and field scales. 
SECTION: Overview
3. The particulars of how many caterpillars, their precise field locations, and the deployment timings will vary depending on the interests and context of each station.
SECTION: Overview
4. However, we ask participating members to deploy at least 48 caterpillars across plots and fields at their site once per growing season (see Figure 1).
 
 
SECTION: Overview
5. The caterpillars will be deployed for 2 days (48 hours), then collected and stored in protective sleeves for mailing back and completing damage assessment.
SECTION: Dummy caterpillar preparation
6. We will prepare dummy caterpillars at a centralized location in advance and mail them to each participating LTAR Site (i.e., participants do not need to complete this step themselves).
SECTION: Dummy caterpillar preparation
7. Caterpillar production will use green Plastalina modeling clay (manufactured by Van Aken International): 
https://www.vanaken.com/products/4-5-lb-plastalina%E2%84%A2-non-hardening-modeling-clay-various-colors?variant=34624843120808
SECTION: Dummy caterpillar preparation
8. Caterpillars production will use a sugar paste extruder with a 3 mm aperture; such as the Clay Extruder Clay Gun Tool with 20 Discs available at https://a.co/d/ejat6qb.
SECTION: Dummy caterpillar preparation
9. The plasticine-loaded extruder will extrude a strand that will be cut into 15 mm segments using a razor blade or scalpel.
SECTION: Dummy caterpillar preparation
10. After examining all segments, those with imperfections will be discarded. 
SECTION: Dummy caterpillar preparation
11. The segments will be attached to a ~15 cm bamboo kabob stake (available at most grocery stores) 3 cm from the blunt end, using a small amount of super glue. 

SECTION: Dummy caterpillar preparation
12. Each set of caterpillars will then be mounted in foam and placed in a protective container for shipping.
SECTION: Deploying and collecting dummy caterpillars in the field
13. Each LTAR Site will receive a box of ~200 mounted plasticine caterpillars, plus a few backups, for deployment at their site.
SECTION: Deploying and collecting dummy caterpillars in the field
14. The number of caterpillars sent is adjustable based on site-to-site differences; however, the goal is to deploy 10 caterpillars within each plot-scale replicate and 30 within each field (i.e., for sites with four plot reps and two field reps per treatment [10 caterpillars * 2 treatments * 4 plot reps] + [30 caterpillars * 2 treatments * 2 field reps] = 200).
SECTION: Deploying and collecting dummy caterpillars in the field
15. If a different number of caterpillars is desired, discuss the deployment with the central biocontrol measurement coordinator.

SECTION: Deploying and collecting dummy caterpillars in the field
16. All caterpillars should be deployed +/- simultaneously within a site for 48 hours +/- 6 hours is acceptable, but attempt to minimize this variation.

 

SECTION: Deploying and collecting dummy caterpillars in the field
17. Avoid placing the caterpillars during inclement weather, when arthropod activity could be reduced. In environments with open soil, avoid times with heavy rainfall, as the caterpillars will tend to accumulate soil particles. 
SECTION: Deploying and collecting dummy caterpillars in the field
18. Seasonal deployment timing will vary among sites, and managers can choose the most appropriate and interesting time of year to measure pest suppression (e.g., preceding the time of year when pest outbreaks are of the highest concern at that site).

 

SECTION: Deploying and collecting dummy caterpillars in the field
19. The precise locations of caterpillar deployment within each plot can vary among sites depending on the locations of pre-established sampling stations or landmarks and on specific questions of interest to personnel at that site (e.g., how predation rates vary between plot edges and the plot interior or between the ground level and crop canopy). 

SECTION: Deploying and collecting dummy caterpillars in the field
20. Regardless of the precise location, caterpillars are easy to lose following deployment, so they should be placed in the same positions within each plot or field and marked with a pin flag or tape as applicable.
SECTION: Deploying and collecting dummy caterpillars in the field
21. Place the marker ~30 cm from the caterpillar (or eye level on a plant stem directly above it) to minimize its potential effect on attack rates. 
SECTION: Deploying and collecting dummy caterpillars in the field
22. After deployment, take GPS coordinates for each caterpillar location. See Figure 1 for an example of how to deploy caterpillars throughout plots and fields.
SECTION: Deploying and collecting dummy caterpillars in the field
23. When transferring caterpillars to the field, remove the number needed for a given plot or plot set from their shipping package and stick them in Styrofoam or a foam gardening pad to carry several at once.

 

SECTION: Deploying and collecting dummy caterpillars in the field
24. Caterpillars are deployable at the ground level, the canopy level, or both, depending on relevance at a given site. 
SECTION: Deploying and collecting dummy caterpillars in the field
25. If caterpillars are to be deployed at ground level, insert the kabob stake into the soil until the caterpillar is ~3 cm from the soil surface. 
SECTION: Deploying and collecting dummy caterpillars in the field
26. Height can be adjusted upward if there is thatch or vegetation present that would touch the caterpillar. 
SECTION: Deploying and collecting dummy caterpillars in the field
27. Depending on the focal crop, caterpillars can be placed directly adjacent to the base of the stem, as in Figure 2a. 
 
 
SECTION: Deploying and collecting dummy caterpillars in the field
28. If caterpillars are to be deployed in the crop canopy, the precise placement method will depend on the crop’s architecture. For example, in large grasses (prairie grasses, corn, sorghum), anchor the kabob stake securely in the leaf sheath (Figure 2b).
SECTION: Deploying and collecting dummy caterpillars in the field
29. In other cases, use a small amount of masking tape or a twist tie to hold the stake in place (Figure 2c). The precise method can be flexible so long as it is consistent within a site.
SECTION: Deploying and collecting dummy caterpillars in the field
30. In all cases, handlers and vegetation must not touch the caterpillars. The caterpillars can record even small brushes with vegetation, clothing, and fingers, possibly making markings difficult to interpret later on. 
SECTION: Deploying and collecting dummy caterpillars in the field
31. When installing a caterpillar, ensure leaves will not brush against it in a breeze.
SECTION: Deploying and collecting dummy caterpillars in the field
32. If a caterpillar sustains damage before deployment, replace it with a backup. 

 

SECTION: Deploying and collecting dummy caterpillars in the field
33. Label dummy caterpillars while collecting them at the end of 2880 min.

SECTION: Deploying and collecting dummy caterpillars in the field
34. For efficiency, write plot codes in advance on pieces of labeling tape attached to a flat surface portable in the field. 
SECTION: Deploying and collecting dummy caterpillars in the field
35. Each label should contain the date, plot or field treatment, replicate number, and an identifier for the station or location within the plot where the caterpillar was deployed.
SECTION: Deploying and collecting dummy caterpillars in the field
36. This last piece of information will vary among sites, and metadata will need to be provided to understand these differences during analysis. 
SECTION: Deploying and collecting dummy caterpillars in the field
37. Once a kabob has been picked up, wrap the tape around it or skewer it to keep it attached.
SECTION: Deploying and collecting dummy caterpillars in the field
38. Carry a piece of Styrofoam to transport the kabobs as you pick them up, then transfer them to the larger piece of Styrofoam they were shipped on after completing caterpillar collecting from a given plot.
SECTION: Deploying and collecting dummy caterpillars in the field
39. Finally, once all caterpillars are collected and placed in their original package, they can be shipped back to the central location for evaluation. 

 

SECTION: Evaluation, analysis
40. Each plasticine caterpillar will undergo inspection using a dissecting microscope. Damage to each caterpillar will be recorded. 
SECTION: Evaluation, analysis
41. Specifically, each caterpillar will be scored with a 0 (no marks), 1 (<10% of caterpillar length damaged), 2 (11-50% damaged), or 3 (>50% damaged) for each of the following types of markings: small mammal teeth, bird beaks, arthropod mandibles, arthropod ovipositors, incidental contact with vegetation, and incidental contact with a human during installation.

 

SECTION: Evaluation, analysis
42. To minimize observer bias, the same individual should review all specimens.
SECTION: Evaluation, analysis
43. Alternatively, multiple technicians can assess each caterpillar, with discrepancies between two observers averaged or resolved by a senior team member.
SECTION: Evaluation, analysis
44. Differences in attack odds are assessable with binomial generalized linear mixed models, and differences in the community composition of the attacking organisms (i.e., types of bite marks) are detectable using multivariate techniques such as Permutational MANOVA.

SECTION: Archiving
48. Each site should submit metadata describing the labeling scheme for plasticine caterpillars and GPS coordinates for all deployment locations. Beyond the treatment and replicate IDs, this submission should include information about where caterpillars were deployed (at an edge or the middle of the plot/field and in the canopy or soils), the crop or land use type they were deployed in, etc.
SECTION: Recommendations for data collection
50. Table 1. Summary of recommendations for measuring natural pest suppression.

 

SECTION: Quality control
45. Double-check all plasticine caterpillars during their installment to ensure they have no accidental markings from handling.

SECTION: Quality control
47. During evaluation, the number of observers will be limited, or multiple technicians will evaluate each caterpillar, with discrepancies resolved by a senior team member. Training materials may be provided to ensure all observers follow the same standards.
SECTION: Quality control
46. Do not expose plasticine to high temperatures, as caterpillars will lose their form at 150 °F.
SECTION: Archiving
49. All caterpillars will be photographed before disposal so that images can be checked in the future as needed (retaining original samples is usually not feasible due to space constraints).