DOI: 10.5061/dryad.pk0p2ngxz
title: Data for: Field evidence for the role of plant volatiles induced by caterpillar oral secretion in prey localization by predatory social wasps

description: One assumed function of herbivore-induced plant volatiles (HIPVs) is to
 attract natural enemies of the inducing herbivores. Field evidence for
 this is scarce. Also, the assumption that elicitors in oral secretions
 that trigger the volatile emissions are essential for the attraction of
 natural enemies has not yet been demonstrated under field conditions.
 After observing predatory social wasps removing caterpillars from maize
 plants, we hypothesized that these wasps use HIPVs to locate their prey.
 To test this, we conducted an experiment that simultaneously explored the
 importance of caterpillar oral secretions in the interaction. Spodoptera
 caterpillars pinned onto mechanically damaged plants treated with oral
 secretion were more likely to be attacked by wasps compared to
 caterpillars on plants that were only mechanically wounded. Both of the
 latter treatments were considerably more attractive than plants only
 treated with oral secretion or left untreated. Subsequent analyses of
 headspace volatiles confirmed differences in emitted volatiles that likely
 account for the differential predation across treatments. These findings
 highlight the importance of HIPVs in prey localization by social wasps,
 hitherto underappreciated potential biocontrol agents, and provide
 evidence for the role that elicitors play in inducing attractive odor
 blends.
description: <em>Field Trials: </em>Pots containing two maize
 plants were randomly assigned to one of four treatments: unmanipulated
 control (CN), oral secretion only (OS), mechanical damage only (MD), and
 mechanical damage plus oral secretion (MD+OS). A pilot study on 1 January
 2022 (n = 6 per treatment) did not contain the OS treatment but was
 included in the analysis. Two trials were conducted the following winter
 on 13 (n = 11) and 21 (n = 12) December 2022.  In total, 220 plants in 110
 pots were used (treatment, n: CN, 29; OS, 23; MD, 29; MD+OS,
 29). To maximize emissions of HIPVs, we damaged the
 plants twice, once the evening before (17:00 h) and a second time the
 morning of the trial (7:00 h). We did this because, in response to
 specific elicitors in caterpillar oral secretion, plants typically
 initiate the <em>de novo</em> synthesis and release of various
 volatiles (Alborn et al. 1998; Schmelz et al., 2006). In maize, this takes
 about 8 hours and can be reinforced with repeated treatments (Turlings et
 al., 1993). The MD treatment consisted of using sandpaper to abrade about
 2 cm<sup>2 </sup>of the adaxial side of the two youngest
 developed leaves, avoiding the midvein. The OS treatment consisted of
 applying FAW regurgitant containing gut and<em> </em>salivary
 gland contents on the two youngest leaves (also on about 2
 cm<sup>2</sup>) by gently squeezing the head of one
 caterpillar until it regurgitated. One caterpillar was used as a source of
 oral secretion for each pot. For the MD+OS treatment, the plants were
 mechanically damaged and immediately after, the oral secretion was applied
 directly to the wounded tissue. After imposing the second damage
 treatment, approximately 14 hours after the initial treatment, one
 randomly selected<em> </em>live,
 3<sup>rd</sup><em> </em>FAW<em>
 </em>or VAW<em> </em>caterpillar was pinned to the
 center of each leaf. This timing coincides with when the wasps most
 actively forage (Prezoto et al., 2019). Live caterpillars (as opposed to
 freeze-killed) were used to ensure that wasps were acting as predators and
 not scavengers. At 9:00 h, pots containing induced
 plants with pinned caterpillars were arranged in spatial blocks containing
 one treatment set (CN, OS, MS, MS+OS, where 1 treatment set = 1 n) in
 maize plots within the experimental field. Each pot was approximately 30
 cm apart, forming a square with a random arrangement of treatments. Blocks
 were visited continuously over 75 minutes and the order in which sentinel
 caterpillars from the different treatments were removed in each block was
 recorded. <em>Volatile Collection: </em>We
 collected volatiles as described in Turlings et al (1998). Volatiles were
 trapped in filters (Porapak Q adsorbent, 25mg, 80-100, Merck, Darmstadt,
 DE) connected to the port of each glass bottle containing the odour source
 for 2 hours at a flowrate of 0.4 L/min. Each filter was then eluted with
 150 μL of dichloromethane (Suprasolv, GC-grade; Merck, Darmstadt, DE) into
 glass vials to which 10 μL of internal standards ([20ng/μL] of n-octane
 and n-nonyl acetate) were added. Samples were then kept at -80°C until
 further analyses. Volatile analyses were carried out
 using a gas chromatograph (Agilent 7890B) coupled to a mass spectrometer
 (Agilent 5977B) with Flame Ionization Detector technology (GC-MS / FID). A
 2 μL aliquot of each sample was injected in pulsed splitless mode onto a
 non-polar capillary column (Agilent HP-1; 30m length x 0.25mm ø x 0.25 μm
 thickness). After the injection, the temperature program maintained the
 temperature at 40°C for 3 minutes before increasing it progressively at a
 rate of 8°C min<sup>−1</sup> until reaching 100°C; it thence
 increased it at 5°C min<sup>−1</sup> until 200°C and
 subsequently rose it at 250°C for a 3 minutes post-run. Volatiles were
 identified using the NIST mass spectral library. Volatile emissions (ng
 hr<sup>-1</sup>) were calculated based on the peak areas of
 internal standards.
description: # Data for: Field evidence for the role of plant volatiles induced by
 caterpillar oral secretion in prey localization by predatory social wasps
 [https://doi.org/10.5061/dryad.pk0p2ngxz](https://doi.org/10.5061/dryad.pk0p2ngxz) ## Description of the data and file structure for field trials Data describes a sentinel caterpillar removal experiment where caterpillars were pinned to maize plants treated with 1 of 4 treatments (TRT), undamaged (C), oral secretion (S), mechanical damage (M), and the combination of M and S (M&S). We recorded the (Date), order of caterpillar removal (order_removed), individual plant id (individual) first caterpillar removed (first_removed) spatial block id (Spatial_plot).'NA' means that the caterpillar was not removed. Only the first caterpillar removed was used in our associated publication (first_removed). ## Code/Software for field trials R version 4.2.1; R Core Team 2022. data=read.csv("sentinel_wasp_BioLet_2.csv", stringsAsFactors = TRUE) lilibrary(emmeans) library(glmmTMB) library(plyr) data$TRT &lt;- factor(data$TRT, levels=c("C", "S", "M", "M&S")) data$TRT&lt;-mapvalues(data$TRT, from = c( "C", "S", "M", "M&S"), to = c("CN", "OS","MD", "MD+OS")) m1=glmmTMB(binom~TRT+Date+(1|Plot), family=binomial, data=data) emmeans(m1, pairwise ~ TRT, adjust = "fdr") ### HIPVs analyses The column headers for the HIPV analyses are sample id (sample), Treatment (TRT) (undamaged (C), oral secretion (S), mechanical damage (M), and the combination of M and S (MS). and the rest are the name of volatile compounds if known. We used standard parametric analyses for the HIPVs data as described in the manuscript. Units for HIPVs were (ng/h).