DOI: 10.5061/dryad.h18931zrr
title: Short-term responses of spider mites inform mechanisms of maize resistance to a generalist herbivore

description: Plants are attacked by diverse herbivorous pests with different host
 specializations. While host plant resistance influences pest pressure, how
 resistance impacts the behaviors of generalist and specialist herbivores,
 and the relationship to resistance, is less well known. Here, we
 investigated the behavioral changes of a generalist herbivore, the
 two-spotted spider mite (TSM), and a specialist herbivore, the Banks grass
 mite (BGM), after introduction to no-choice Tanglefoot leaf-arenas (2×2
 cm2) of three maize inbred lines (B73, B75, and B96). The widely-used
 inbred line B73 is susceptible to spider mites, while B75 and B96 are
 known to be mite resistant, especially to TSM. Video tracking was used to
 record TSM and BGM walking, probing, feeding, resting, web-building and
 travel distance on arenas of each line. Mite oviposition was also recorded
 after 72 hours.  B75, a resistant line, decreased the feeding
 behavior of both mite species compared to B73 (susceptible control) and
 B96. Moreover, TSM appeared to be sensitive to both resistant lines (B75
 and B96) with reduced oviposition, and increased resting and web-building
 times compared to susceptible B73. In contrast, the specialist BGM showed
 no difference in oviposition, resting and web-building time across all
 maize inbred lines. Our study suggests that arthropod resistance traits in
 maize, as seen in B75 and B96, affect the generalist TSM behavior quite
 broadly, yet sensitivity to this resistance appears to be reduced as host
 specialization narrows. Therefore, other mechanisms of plant resistance
 may be needed for defense against a specialist like BGM.
description: <strong><em>Maize lines and plant maintenance
 </em></strong> Plants were grown at Utah
 State University’s Research Greenhouse and Laboratory, Logan, UT. Three
 maize inbred lines were selected for the study (B73, B75, and B96). B73 is
 susceptible to both BGM and TSM , while B75 and B96 have moderate- and
 high-level resistance to TSM, respectively. Two seeds
 per pot for each maize inbred line (B73, B75, and B96) were sown in 3.5 L
 pots filled with soil (Sunshine Mix #3, Sun Gro Horticulture, MA), 8 pots
 per line, distributed in a complete randomized design. Maize plants were
 grown under greenhouse-controlled conditions (25±2 °C, 60±5% RH, 16:8 hrs.
 (L:D) photoperiod) and fertigated at a rate of 4.8 kg/100L of 21N-5P-20K
 Peters Excel Water Soluble Fertilizer mixture (ICL Specialty Fertilizers,
 SC, USA) by using drip tape (DIG Corporation, CA, USA; 12.7 mm and 6.35 mm
 diameter tubing with 3.8 L/hr compensating emitters). At 8 weeks of age,
 plants were used to evaluate spider mite behavior.
 <strong><em>Video tracking spider mite behavior on
 maize lines</em></strong> We conducted a 3
 × 2 factorial design experiment using three levels of maize resistance
 (susceptible B73, and resistant lines B75 and B96) and two levels of mites
 (TSM and BGM). A rectangular plastic box (20 × 15
 cm<sup>2</sup>, Webstaurant Store, PA, USA) was used as an
 experimental unit and each treatment (3 maize inbred lines × 2 mite
 species) was replicated six times. A 3 × 3 cm<sup>2</sup>
 leaf-cutting from the middle section of the 8<sup>th</sup>
 leaf from each plant was collected, excluding the leaf midrib. We placed
 leaf-cuttings for each respective inbred line on a wet cotton pad on a
 plexiglass sheet fitted within the rectangular plastic box to prevent the
 leaf arena from desiccating. To ensure the cotton remained moist, boxes
 were filled halfway so the ends of the cotton pads were in contact with
 the water (the ends draped over the plexiglass sheet into the water below
 to allow wicking). A 2 × 2 cm<sup>2</sup> no-choice arena was
 created by placing Tanglefoot (The Scotts Miracle-Gro Company, OH, USA)
 non-phytotoxic wax barriers on the edges of each leaf-cutting to keep
 mites on the feeding site and prevent escape. BGM and TSM colonies used in
 the study were maintained in lab conditions [28±2 °C,
 50+5% RH, 16:8 hr (L:D) photoperiod] on B73 maize. One
 newly emerged adult female mite, mated and starved overnight, was
 introduced into the arena of each respective maize inbred line by using a
 fine paintbrush immediately after the leaf was cut.
 Using a Canon Eos 5D Mark III camera and 65mm MP-E lens, each
 female mite was recorded by video for 50 min following mite introduction
 to each respective arena. Each video was examined for six behaviors that
 included the total time that each mite spent 1) walking, 2) probing, 3)
 feeding, 4) resting, 5) web-building, and 6) traveling (total distance
 traveled in cm) for each mite in an arena using a behavior tracking
 software (OpenCV mite tracer, https://github.com/HMKRL/OpenCV-mitetrace). Briefly, videos were uploaded to the software, a tracer was placed on each spider mite, and the software tracked movement in the video and generated a path plot. The distance traveled by spider mites in arenas, and the times the mites moved and stopped were recorded. Manual visual inspection of each video was used to further assign times for specific behaviors (i.e., probing, feeding, resting, and web-building). Resting time was assessed as the time that mites were not moving, probing or feeding. Probing was apparent when a mite stopped its movement, short feeding events occurred in place, and forelegs showed a variety of small tactile movements. Web-building was assessed when mites swaying their forelegs in a side to side, a motion known to reflect connecting threads of silk. Finally, oviposition (number of eggs deposited) was recorded for each replicate 72 hours post mite introduction using a stereomicroscope (Leica S6 D Greenough, NJ, USA).
description: Data from mite oviposition on maize inbred lines were analyzed
 using a generalized linear model (Proc Glimmix; SAS 9.4 M4 University
 edition) within two-way ANOVA that included maize resistance (B73, B75,
 and B96) and mites (TSM and BGM) as fixed factors. Oviposition data were
 log transformed to conform to the assumptions of normality and
 heteroscedasticity. Video recordings (50 min each) were analyzed in 10 min
 intervals (0 to 10 min, 10 to 20 min, etc.). Proportions of time that each
 mite spent walking, probing, feeding, and resting within the 10 min
 intervals were analyzed using a generalized linear model within two-way
 ANOVA and repeated measures (5 time intervals) with a beta distribution.
 Data for web-building and travel distance were square-root transformed and
 analyzed using two-way ANOVA (maize inbred lines) with repeated measures
 (5 time intervals) using Proc Glimmix.
description: # Short-term responses of spider mites inform mechanisms of maize
 resistance to a generalist herbivore Author(s) Gunbharpur S. Gill1, Hsuan
 Bonny Lu2, Huyen Bui3, Richard M. Clark4,5, Ricardo A. Ramirez1*
 1Department of Biology, Utah State University, Logan, Utah 84322, USA.
 2Department of Kinesiology and Health Sciences, Utah State University,
 Logan, Utah 84322, USA. 3R&S Genetic, ARUP Laboratories, Salt Lake
 City, Utah 84108, USA. 4School of Biological Sciences, University of Utah,
 Salt Lake City, Utah 84112, USA. 5Henry Eyring Center for Cell and Genome
 Science, University of Utah, Salt Lake City, Utah 84112, USA. *Current
 address: Department of Entomology, Plant Pathology, & Weed Science,
 New Mexico State University, Las Cruces, New Mexico 88003, USA. **File
 list** Gill et al. 2023 mite behavior and eggs.xlsx This data set
 highlights the spider mite responses of twospotted spider mite and Banks
 grass mite to maize with varied host plant resistance in the lab. The
 behaviors investigated include time feeding, moving, probing, resting,
 web-building, and travel distance. Oviposition at the end of the trial was
 also recorded.