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.

2. Materials and Methods

(a) Plant materials

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).

(b) Insects

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^rd and 5^th instar stage.

(c) Shaving the stem trichomes

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).

(d) Time to reach the target

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.

(e) Statistical analysis

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.