DOI: 10.5061/dryad.fttdz0952
title: Data from: Separate and synergistic anti-herbivore effects of non-glandular trichomes and leaf chemistry in a desert plant

description: Plant defense phenotypes commonly integrate physical and chemical traits
 that may act synergistically against herbivores, but empirical evidence
 for synergy as a defense strategy remains limited. We experimentally
 tested the separate and combined effects of two common plant defenses,
 non-glandular trichomes and leaf chemistry, on the feeding, behavior, and
 performance of a generalist herbivore. We conducted choice and no-choice
 assays that exposed beet armyworm caterpillars (Spodoptera exigua,
 Noctuidae) to desert stingbush (Eucnide urens, Loasaceae) leaves with
 manipulated trichome cover and raised caterpillars on a diet containing
 factorial combinations of E. urens trichomes and leaf extracts. We
 assessed differences in caterpillar behavior, performance, and survival
 across treatments. We found a significant interactive effect of trichomes
 and leaf chemistry that markedly decreased caterpillar growth, providing
 evidence for a defense synergism. Both defenses also reduced caterpillar
 performance individually. Leaf extracts contained a high proportion of
 putatively defensive compounds, including iridoid glycosides, which were
 associated with reduced caterpillar growth and high mortality before
 pupation. Barbed trichomes impeded caterpillar movement and, when
 consumed, caused mandible damage and reduced eclosion rates compared to
 controls. Our results indicate that two commonly co-expressed plant
 defenses can synergistically reduce herbivore performance. These findings
 provide a rare example of trait synergy as a defense strategy and
 represent a step forward in the effort to study plant defense
 holistically, as suites or syndromes of defenses, rather than isolated
 traits.
description: # Data from: Separate and synergistic anti-herbivore effects of
 non-glandular trichomes and leaf chemistry in a desert plant ##
 Description of the data and file structure These are the data and code to
 accompany the submitted manuscript entitled “Separate and Synergistic
 Anti-Herbivore Effects of Non-Glandular Trichomes and Leaf Chemistry in a
 Desert Plant”. Using a series of experiments with the desert stingbush
 (*Eucnide urens*: Loasaceae), we experimentally tested for synergisms
 between trichomes and chemistry on the feeding, behavior, and performance
 of a generalist herbivore. We conducted choice and no-choice assays that
 exposed beet armyworm caterpillars (*Spodoptera exigua* Hübner, Noctuidae)
 to *E.urens* leaves with manipulated trichome cover and raised
 caterpillars on a diet containing factorial combinations of trichomes and
 leaf metabolite extracts.  ### Files and variables #### File:
 choice_assay_caterpillar_data.csv **Description:** Data from an
 assay where *S. exigua* caterpillars were given a choice between *E.
 urens* leaf discs with different levels of trichome cover (shaved
 vs. unshaved). ##### Variables * dish_ID: Replicate ID * starved_mass_g:
 Mass, in grams, of caterpillars at the start of the assay. They were
 starved for 24 hours prior.  * loc_after_2hrs: Location of each
 caterpillar two hours into the assay (S = shaved leaf disc, U = unshaved
 leaf disc, NA = not on leaf).  * loc_after_18hrs: Location of each
 caterpillar 18 hours into the assay (S = shaved leaf disc, U = unshaved
 leaf disc, NA = not on leaf).  * trapped_after_18_hrs: Whether or not the
 caterpillar was trapped by trichomes on the leaf disc after 18 hours (Y =
 trapped, N = not trapped) * notes: notes about the location of trapped
 caterpillars #### File: choice_assay_herbivory_data.csv
 **Description:** Leaf herbivory data from an assay where *S. exigua*
 caterpillars were given a choice between *E. urens* leaf discs with
 different levels of trichome cover (shaved vs. unshaved). ##### Variables
 * dish_ID: Replicate ID * leaf_disc_treatment: Whether the herbivory data
 refer to the shaved or unshaved leaf disc in each replicate.  *
 proportion_consumed: Proportion of the leaf disc eaten by the
 caterpillars, estimated using the R package herbvar.  *
 leaf_area_consumed_mm2:  Area of the leaf disc, in square
 millimeters, eaten by the caterpillar, estimated using the R package
 herbvar.  * total_leaf_area_mm2: Total area of the leaf disc, in square
 millimeters, estimated using the R package herbvar.  #### File:
 factorial_diet_assay_data.csv **Description:** Data from an assay in which
 neonate *S. exigua* caterpillars were divided into four groups (n=35 per
 group) and raised to eclosion on one of four artificial diets: diet
 supplemented with trichomes isolated from *Eucnide urens* leaves, diet
 supplemented with *E. urens* leaf extract, a diet containing both
 trichomes and leaf extract, or a control diet with no trichomes or leaf
 extract added. ##### Variables * individual: individual caterpillar ID *
 treatment: whether the caterpillar was fed a control diet, diet with
 added trichomes, diet with added leaf extract, or diet with both trichomes
 and leaf extract.  * has_trichomes: binary variable indicating whether the
 caterpillar received a diet that contained trichomes (1 = yes, 0 = no) *
 has_metabolites: binary variable indicating whether the caterpillar
 received a diet that contained leaf metabolite extract (1 = yes, 0 = no) *
 tray_number: Tray in which the cup containing the caterpillar was
 placed during the assay. Cups were alternated by treatment within
 each tray, and trays were kept in a single layer within a growth chamber. 
 * cat_mass_MONTH_DAY: Mass, in grams, of the caterpillar at a given time
 point. Caterpillars were weighed every 48 hours. NA = individual was not
 weighed (typically because it had died).  * weighed_by_MONTH_DAY: letter
 (A or B) identifying which researcher performed the weighing. NA =
 individual was not weighed (typically because it had died).  *
 alive_MONTH_DAY: Whether or not the caterpillar was alive that day. Once a
 caterpillar died, we stopped weighing it.  * life_stage_MONTH_DAY:  Life
 stage of the insect, recorded once they started pupating. NA = individual
 died or pupated.  * Sex: Pupal sex, NA for individuals that did not
 survive to pupation.  * alive_after_30_days: whether an individual was
 alive after 30 days.  * Pupated: Whether an individual successfully
 pupated.  * pupal_mass: The mass, in grams, of each pupa.  * emerged:
 Whether a living adult moth emerged from the pupa (1 = yes, 0 = no) *
 notes: Various notes on caterpillar behavior, mortality, and appearance. 
 #### File: mandible_damage_measurements.csv **Description:** Damage to
 exuvial mandibles of *S. exigua* caterpillars fed control and
 trichome-augmented diet in the trichomes and metabolites assay. Damage was
 quantified by measuring (using the objectJ plugin in ImageJ) the length of
 the scars left by broken mandible teeth. ##### Variables * individual:
 caterpillar ID  * side: Whether measurements were taken from the left or
 right mandible.  * chip_length_um: The length, in um, of the scar left by
 a chipped mandible tooth.  #### File: no_choice_leaf_assay_data.csv
 **Description:** Data from an assay in which *S. exigua* larvae were
 raised on either shaved or unshaved *E. urens* leaves for 20 hours.  #####
 Variables * dish_ID: Replicate ID * treatment: Whether the caterpillar was
 given a shaved or unshaved leaf. * cat_starting_mass_g: The starting mass,
 in grams, of each caterpillar.  * cat_mass_g_20hrs: The mass, in grams, of
 each caterpillar at the conclusion of the assay.  * cat_status_20hrs:
 Description of whether the caterpillar was alive and if/where it was
 trapped at the conclusion of the assay.  * Trapped: Binary variable
 indicating whether the caterpillar was trapped (Y) or not trapped (N) on
 the leaf at the conclusion of the assay.  * proportion_consumed:
 Proportion of the leaf consumed by the end of the assay, estimated using
 the R package herbvar.  * total_leaf_area_mm2: Total leaf area, in mm^2,
 estimated using the R package herbvar.  * leaf_area_consumed_mm2: Area, in
 mm^2, of the leaf consumed by the end of the assay, estimated using the R
 package herbvar.  #### File: mzMine_steps_parameters.txt
 **Description:** Parameters used to group spectral features of *E. urens*
 extracts into compounds using mzMine #### File:
 SIRIUS_steps_parameters.txt **Description:** Parameters used for compound
 structure prediction using SIRIUS #### File: compound_feature_list.csv
 **Description:** Features of compound peaks identified from *E. urens*
 leaf extracts ##### Variables * rowID: compound number (unique identifier)
 * row m/z: mass to charge ratio of compound peak (positive ionization
 mode) * row retention time: elution time of compound peak in minutes *
 E120.mzXML Peak height: total ion current for compound in the pooled
 sample #### File: canopus_compound_summary.tsv **Description:** Summary of
 structure predictions and annotations from SIRIUS and CANOPUS  #####
 Variables * id: numerical order of compounds in the input file, followed
 by the input file name and the compound number * molecularFormula:
 molecular formula for MS peak predicted by SIRIUS * adduct: most likely
 adduct for predicted molecular formula (positive ionization) *
 precursorFormula: molecular formula of parent or precursor mass for MS2
 data predicted by SIRIUS * NPC#pathway: natural products pathway predicted
 by CANOPUS * NPC#pathway Probability: probability/confidence of CANOPUS
 natural products pathway prediction * NPC#superclass: natural products
 superclass predicted by CANOPUS * NPC#superclassProbability:
 probability/confidence of CANOPUS natural products superclass prediction *
 NPC#class: natural products class predicted by CANOPUS * NPC#class
 Probability: = probability/confidence of CANOPUS natural products class
 prediction * ClassyFire#most specific class: Classyfire most specific
 chemical class predicted by CANOPUS * ClassyFire#most specific class
 Probability: probability/confidence of CANOPUS Classyfire most specific
 chemical class prediction * ClassyFire#subclass: Classyfire chemical
 subclass predicted by CANOPUS * ClassyFire#subclass Probability:
 probability/confidence of CANOPUS Classyfire chemical subclass prediction
 * ClassyFire#class: Classyfire chemical class predicted by CANOPUS *
 ClassyFire#class Probability: probability/confidence of CANOPUS Classyfire
 chemical class prediction * ClassyFire#superclass: Classyfire chemical
 superclass predicted by CANOPUS * ClassyFire#superclass Probability:
 probability/confidence of CANOPUS Classyfire chemical superclass
 prediction * ClassyFire#all classifications: all Classyfire chemical
 classifications separated by ; * featureId: compound number (unique
 identifier) ## Code/software #### File:
 Synergistic_Effects_of_Trichomes_and_Chemistry_stats.R **Description:** R
 script used for statistical analysis and to generate manuscript figures.
 Statistical analyses
 (“Synergistic_Effects_of_Trichomes_and_Chemistry_stats.R”) were performed
 in RStudio using R version 4.4.2 and the following packages: tidyr - data
 wrangling plyr -  data wrangling dplyr - data wrangling ggplot2 - general
 plotting hrbrthemes - ggplot themes PairedData - to plot paired data in
 the leaf choice assay broom - summaries of statistical tests plotrix - 
 calculating standard error car - QQplot while testing ANOVA assumptions
 survival -  Kaplan-Meier survival analysis survminer - pairwise log-rank
 tests comparing Kaplan-Meier curves Effects - checking LMM model
 assumptions sjPlot - checking LMM model assumptions stringr - manipulating
 character strings during compound annotation