DOI: 10.5061/dryad.wwpzgmsgp
title: Date from: Top-down effects from parasitoids may mediate plant defense and plant fitness

description: Plants face many environmental stresses that can impact their survival,
 development, and fitness. Insects are the most diverse, abundant and
 threatening herbivores in nature. As a consequence, plants produce direct
 chemical and physical defenses to reduce herbivory. They also release
 volatiles to recruit natural enemies that indirectly protect them from
 herbivory. The recruitment of parasitic wasps can benefit plant fitness
 because they ultimately kill their insect hosts. Recently, studies showed
 that parasitoids can indirectly mediate plant defenses by modulating
 herbivore oral secretions. In addition to the direct benefits of
 parasitoids in terms of reducing herbivore survival, we tested if the
 reduction in induced defenses by parasitized caterpillars compared to
 non-parasitized caterpillars may reduce the costs associated with defense
 expression. We provide evidence that tomato plants treated with saliva
 from parasitized caterpillars have significantly higher fitness parameters
 including increased flower numbers (16.3%) and heavier fruit weight (15%),
 compared to plants treated with saliva from non-parasitized caterpillars.
 Since plants were grown without actual herbivores, the higher values for
 these fitness parameters were due to lower costs of induced defenses and
 not due to reduced herbivory by parasitized caterpillars. Furthermore, the
 resulting seed germination time was shorter and the germination rate was
 higher when the maternal plants were previously exposed to parasitized
 herbivore treatment compared to control (non-treated) plants. Overall,
 application of saliva did not result in transgenerational priming of
 offspring defense responses. However, offspring of parents exposed to
 caterpillar saliva had lower constitutive levels and higher induced levels
 of trypsin inhibitor than offspring from unexposed parents. This study
 shows that the saliva of parasitized caterpillars can modulate plant
 defenses and further demonstrates that the lower induction of plant
 defenses is associated with elevated plant fitness in the absence of
 herbivore feeding, suggesting that induced plant defenses are
 costly. 
description: <b>Caterpillar glucose oxidase enzyme activities in labial
 glands</b>    GOX activities were assessed as
 described by Eichenseer et al. (1999). The changes in absorbance values
 were recorded at 460 nm in a plate reader. Protein in each sample was
 quantified by Bradford assays using BSA (bovine serum albumin, Fraction V,
 Omnipur) as the protein standard (Bradford 1976).
   <b>Plant defense
 response</b> Twenty-four hours
 after saliva application, plant tissues were collected from the treated
 leaflet. RNA extraction, cDNA synthesis and qRT-PCR analysis were
 processed as described (Tan et al., 2018). Forty-eight hours after saliva
 application, plant tissues were collected from the third terminal leaflet
 for peroxidase (POD), polyphenol oxidase (PPO) and trypsin inhibitor (TI)
 assays. PPO and POD assays were performed as described by Felton et al.
 (1989). For trypsin inhibitor (TI) activity assays, samples were prepared
 and the absorbance values were recorded at 247 nm in a plate reader.
 Protein concentration in each sample was quantified by using Bradford
 assays.   <b>Plant fitness
 </b> A
 serrated wounding tool was used to wound the third terminal leaflet of
 tomato plants and immediately 15 µl of insect saliva from parasitized or
 non-parasitized caterpillars was applied with a pipette. Two days later,
 the same process was repeated on two leaflets of the
 4<sup>th</sup> leaf. Four days after the first application,
 insect saliva was applied to two leaflets of the
 5<sup>th</sup> leaf. In total, insect saliva was applied three
 times to five leaflets to simulate caterpillar feeding. Plants were
 watered as needed and pots were rotated randomly every week. Testing
 plants were excluded from other biotic and/or abiotic factors which might
 have varied with natural conditions. The first flowering date, flower
 number, fruit weight, seed weight and seed number were recorded to
 represent plant fitness. The first flowering date is the number of days
 between the first saliva application and appearance of the first
 flower.    <b>Second
 generation performance</b> Thirty seeds from each maternal
 plant were sown in potting soil in the greenhouse and a total of 10
 maternal plants of each treatment were used. Pots were placed on trays and
 water was added to the tray to maintain soil moisture. Seeds were observed
 daily for germination time. Germination rates of seeds were calculated at
 11 days after sowing as follows: (germinated seed #/ total seed # (30
 seeds)) x 100.   In the offspring defense response
 assay, there were three maternal plant treatments (C, P, and NP) and two
 herbivore treatments (N: no herbivore treatment, and H: herbivore
 treatment). Seeds were planted as described above. Plants with 3 fully
 expanded leaves were used for the experiment. Labial glands were collected
 and homogenized from non-parasitized caterpillars. For the herbivore
 treatment (H), 15 µl (1 µg/µl protein) of insect saliva was applied after
 mechanical wounding of the third terminal leaflet (counting from the
 bottom) of tomato plants, while the other half plants were without
 herbivore (N) treatment.    Twenty-four and 48 hours after
 saliva application, plant tissues were collected from the third terminal
 leaflets for polyphenol oxidase (PPO), peroxidase (POD) and trypsin
 inhibitor (TI) assays as described above. Each plant was only used
 once.   <b>Statistical
 analyses</b> Data were transformed as needed
 to obtain a normal distribution and to address homogeneity of residual
 variances; SAS 9.4 (SAS Institute Inc) was used for data analyses. The GOX
 activities in labial glands were analyzed by Student’s t test. Plant
 fitness was analyzed by One-way ANOVA (Proc GLM), and plant defense enzyme
 activities and seed germination rate were analyzed by Two-way ANOVA (Proc
 GLM), followed by means comparisons using the Tukey’s Least Significant
 Difference (LSD) test (significance level,<i>
 P</i>&lt;0.05). Seed germination days were analyzed by
 Kruskal-Wallis tests, followed by pairwise multiple comparison (DSCF)
 tests (significance level,<i> P</i>&lt;0.05).