Short-term responses of spider mites inform mechanisms of maize resistance to a generalist herbivore

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.

Methods
Maize lines and plant maintenance

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.

Video tracking spider mite behavior on maize lines

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 cm2, 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 cm2 leaf-cutting from the middle section of the 8th 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 cm2 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).