DOI: 10.5061/dryad.n02v6wxb4
title: Hairy roadblock: Stem trichomes impede herbivore movement in a species and instar-dependent manner

description: Plants have evolved to deploy a vast set of defense mechanisms against
 insect herbivores, including physical barriers like leaf trichomes with
 pre and post ingestive effects. Yet, the role of stem trichomes in
 plant-herbivore interactions, particularly mobility dynamics, is poorly
 understood. To test this, we quantified the climbing speed of third and
 fifth instar soybean looper (SBL, Chrysodeixis includens) and fall
 armyworm (FAW, Spodoptera frugiperda) in the presence (intact) and absence
 (shaved off) of soybean (Glycine max) stem trichomes. The results show
 that SBL was significantly slowed down due to stem trichomes, but FAW was
 not affected. When compared between instars, fifth instar larvae were
 twice as fast as the third instar of both herbivores. Interestingly,
 significantly more fifth instar SBL successfully climbed to the top of the
 plant, compared to fifth instar FAW. Anecdotal observations also revealed
 a unique behavior of FAW and SBL larvae where they “girdle” the stems to
 remove the stem trichomes as they climb up. Our findings clearly
 demonstrate that stem trichomes are an effective barrier that slows down
 the herbivores by restricting their mobility. However, they are not as
 effective against a generalist herbivore like FAW, and the effects are
 larval stage dependent.
description: <strong>2. Materials and Methods</strong>
 <strong>(a) Plant materials</strong>
 Soybean plants were grown in Milo J. Shult Agricultural Research
 & Extension Center (SAREC), Arkansas Agricultural Experiment
 Station, University of Arkansas. Magellan cultivar was used for this study
 known for its drought tolerance, but no insect resistance has been
 discovered (See [16] for more details on Magellan). Plants were grown in
 potting soil (Farmers Co-Op, Van Buren, Arkansas, USA) in standard
 greenhouse conditions (16:8 light: dark cycle, ~70% relative humidity
 (RH), and a temperature of 28°C–30°C).
 <strong>(b) Insects</strong>
 Soybean looper and fall armyworm eggs were were obtained from
 Benzon Research Inc., Pennsylvania, USA and Frontier Agricultural
 Services, Calexico, CA, USA respectively. See Gautam et al., (2024) for
 more details regarding SBL and FAW rearing in lab.  Both SBL and FAW were
 reared in laboratory at University of Arkansas at 25°C, 70% RH until
 3<sup>rd</sup> and 5<sup>th</sup> instar
 stage. <strong>(c) Shaving the stem
 trichomes</strong> The stem trichomes and extra
 branches were removed with the help of sharp razor blades, but carefully,
 to avoid damage to the stem epidermis. The removal of stem trichomes was
 confirmed by observing the presence of trichomes under scanning electron
 microscopy (SEM), DSEM (DSEM; SNE-4500 Plus Tabletop Nanoimages LL,
 Pleasanton, CA, USA). A random section of the mid portion of the stem was
 excised (1cm × 1 cm) with the help of a sharp razor to obtain bark from
 both unshaved and shaved stems. This bark was observed under SEM at 190×
 and 170× magnification for unshaved and shaved stems respectively to
 confirm the removal of trichomes from shaved stems (Figure 1a-d). The
 overall experiment was conducted in batch of 16 plants at a time (for
 shaved and unshaved), and the larvae were replaced after completion of
 each experiment. Few leaves were left at the apical part of each plant to
 attract SBL and FAW into climbing the stem (Figure 1e).
 <strong>(d) Time to reach the
 target</strong> The larvae reared in the lab were
 starved for 3 hours before the experiment was conducted. Starvation makes
 larvae more sensitive to food during experiments such as time to initiate
 feeding, and choice assays. Third-instar SBL and FAW (N=16 each) were
 released on shaved and unshaved soybean plants and allowed them to climb
 up the stem to reach the leaves on the top. The larvae of both SBL and FAW
 are comparable in size as they are reported to reach 34-36 mm during their
 final instars. A 20 cm mark was made from the base of the plant to track
 the distance travelled by each caterpillar. The time taken by the larvae
 to reach the mark was also recorded with a stopwatch to calculate speed of
 the larvae. The speed with which the larvae travel along the shaved and
 unshaved stems was calculated by using the following equation.
 If the larvae reached the 20 cm mark, the total distance travelled was noted as 20 cm and the respective time taken was recorded. Only those larvae were included in the analysis which moved from the spot they were released at and the ones which did not move at all were labelled as 0 for the distance travelled. After the experiment, each caterpillar was removed from the plant and allowed to feed on artificial diet. The experiment was repeated for fifth instar larvae of SBL and FAW. <strong>(e) Statistical analysis</strong> The average speed of larvae was analyzed via full factorial analysis with speed as a response variable and herbivore species, shaving treatment, and larval instars as factors. ANOVA was conducted followed by Tukey HSD for post hoc analysis and appropriate transformations were carried out to meet the assumptions of normality. Further analyses were conducted separately for SBL and FAW to explore the effects of shaving treatment, larval instars and their interaction on the speed with which the larvae travelled across the soybean stems. The larvae’ success in reaching the marked region was compared by using chi-square test and compared across shaved vs unshaved and third instar vs fifth instar for both SBL and FAW. Similar chi-square tests were carried out to compare SBL and FAW across shaved vs unshaved, and third vs fifth instar. The statistical analyses were conducted using data.table package in R.
description: # Hairy roadblock: Stem trichomes impede herbivore movement in a species
 and instar-dependent manner Dataset DOI:
 [10.5061/dryad.n02v6wxb4](https://doi.org/10.5061/dryad.n02v6wxb4) ##
 Description of the data and file structure ### Files and variables ####
 File: Supplementary_data.xlsx **Description:** The file contains the
 consolidated data obtained during the experiment.  Sheet 1 - Data Sheet 2
 - Metadata and definition of variables.  ##### Variables * Speed (cm/min):
 Average speed of the larvae to climb the stem in the given time  *
 Completed: Measure of successful climbing * distance_cm: total distance
 travelled by the larvae within the given timeframe. (in centimeters). 
 "0" distance was assigned for the larvae that did not move from
 their initial position at all. Hence they correspond to "not
 reached". ## Code/software The data was analyzed using R studio. 
 Data.table package was used to handle the dataset.  ANOVA was conducted
 followed by Tukey HSD for post hoc analysis and appropriate
 transformations were carried out to meet the assumptions of normality. The
 larvae’ success in reaching the marked region was compared by using
 chi-square test and compared across shaved vs unshaved and third instar vs
 fifth instar for both SBL and FAW. ## Access information Other publicly
 accessible locations of the data: * NA Data was derived from the following
 sources: * NA