DOI: 10.5061/dryad.0cfxpnw83
title: Silicon and Epichloë-endophyte defences in a model temperate grass diminish feeding efficiency and immunity of an insect folivore

description: Plants deploy diverse anti-herbivore defences which reduce feeding and
 performance of herbivores. Temperate grasses use silicon (Si) accumulation
 and Epichloë-endophytes for physical and chemical (i.e.
 endophytic-alkaloids) defence against insect herbivores. Recent studies
 suggest that Epichloë-endophytes increase Si accumulation in their host
 grass. It is unknown, however, how this affects Si-deposition on the leaf
 surface, their impacts on insect herbivore feeding efficiency and their
 immunity to potential infection/parasitism. To address this knowledge gap,
 we grew tall fescue (Festuca arundinacea) hydroponically with and without
 Si, in the absence or presence of the novel AR584 Epichloë-strain. We
 exposed plants to Helicoverpa armigera (Lepidoptera: Noctuidae) in both
 in-situ (intact leaves) and ex-situ (excised leaves) feeding trials and
 determined the effects of Si and endophyte defences on herbivore feeding
 efficiency, growth rates and immunity against potential
 infection/parasitism. Endophytic plants supplied with Si showed 110% and
 143% increases in leaf silica density and leaf Si concentrations,
 respectively, when exposed to herbivory, compared to non-endophytic plants
 that were herbivore-free. Despite the endophyte-mediated increases in Si
 concentrations, H. armigera was only affected by Si supply;
 growth rates decreased by 87% and most feeding efficiency indices
 decreased by at least 30%. Si supply also increased mandibular wear by
 16%, which was negatively correlated with H. armigera growth rates.
 Cellular and humoral immunity of H. armigera were negatively affected by
 both Si and endophytes. Endophytic-loline alkaloid concentrations were
 unaffected by Si supply or herbivory, whereas herbivory increased peramine
 concentrations by 290%.  To our knowledge, this is the first
 report of Si defences and Epichloë-endophyte-derived alkaloids
 compromising insect immunity via reduced melanisation response. Using tall
 fescue and H. armigera, our study suggests that deploying both physical
 (i.e. Si accumulation) and chemical (i.e. endophytic-alkaloids) defences
 acting against multiple insect herbivore traits, including feeding
 efficiency, growth and immunity, may be a successful defence strategy in
 temperate grasses. This multi-faceted defence may be particularly
 difficult for insect herbivores to overcome.
description: The statistical software R
 (R Core Team,
 2015) was utilized for all
 statistical analyses. Assumptions of normality for residuals were verified
 for all models according to the inspection of quantile-quantile plots. All
 figures were produced using the ‘ggplot2’ package (Wickham, 2016). To address hypothesis one (H1) that states that
 increased Si-defences (e.g. increase silicified phytoliths in the leaf
 surface) have no impact on endophyte-specific alkaloid production, foliar
 Si concentration and resulting silica cell density (response variables) of
 +Si plants only were analysed using a two-way ANOVA (type III
 sum-of-squares) with endophyte and herbivory treatments as fixed effect
 factors. To address H2 states that Si supply has no impact on
 endophyte-specific alkaloid production, using only endophytic plants, a multivariate
 analysis of variance (permutational MANOVA) with Si and herbivory
 treatments as fixed effects was utilized to determine differences in
 overall alkaloid profiles (response variables) with the ‘vegan’ package
 (Oksanen et al.,
 2019). The MANOVA function
 further provided results for univariate ANOVAs for treatment effects on
 individual alkaloids. Moreover, to address H3 and H4 that states
 that only Si supply
 negatively affects feeding efficiency indices (H3), whereas both Si and
 endophyte have (H4) negative effects on insect
 immunity, all herbivore
 parameters including, relative growth rate, mandibular wear, food
 utilization indices, and larval cellular and humoral immunity (response
 variables), were explored using a two-way ANOVA (type III) with endophyte
 and Si treatment as fixed factors. Additionally, a Pearson rank
 correlation coefficient test was used to analyse the relationship between
 relative growth rate and mandibular wear (Knowles, Siegmund, & Zhang,
 1991). Finally, although
 not part of the hypothesis, for plant growth and primary chemistry, a
 three-way ANOVA (type III) with endophyte, Si supply and herbivory
 treatments as fixed effect factors was performed.
 For the data analysed, we
 employed Type III ANOVAs and reported all interaction terms in the tables;
 we did not conduct model simplification analyses to assess the impact of
 non-significant interaction terms on the overall model because removing
 non-significant interactions can lead to an increase in the apparent
 sample size and may affect the power to detect main effects. Note that
 some individuals escaped our in-situ trial and thus, the data for this is
 unbalanced.
description: # Silicon and <i>Epichloë</i>-endophyte defences in a model
 temperate grass diminish feeding efficiency and immunity of an insect
 folivore.
 [https://doi.org/10.5061/dryad.0cfxpnw83](https://doi.org/10.5061/dryad.0cfxpnw83) ## Description of the data and file structure Dataset description. The metadata was subsetted  in different files for  R analysis as described in the methods of the manuscript: All files contain the main factor columns: * **ENDO** corresponding to endophyte level: AR584 (with endophyte) or Nil (without endophyte) * **Si** corresponding to silicon level: supplied (+Si) or not supplied (-So) with Silicon in hydroponic solution * **INSECT** corresponding to herbivore level: infested (+Herb) or not (-Herb) with Helicoverpa armigera * **OU** experimental unit To address hypothesis one (H1) that states that increased Si-defences (e.g. increase silicified phytoliths in the leaf surface) have no impact on endophyte-specific alkaloid production, foliar Si concentration and resulting silica cell density (response variables) of +Si plants only were analysed using a two-way ANOVA (type III sum-of-squares) with endophyte and herbivory treatments as fixed effect factors. To address H2 states that Si supply has no impact on endophyte-specific alkaloid production, using only endophytic plants, a multivariate analysis of variance (permutational MANOVA) with Si and herbivory treatments as fixed effects was utilized to determine differences in overall alkaloid profiles (response variables) with the ‘vegan’ package (Oksanen et al., 2019). The MANOVA function further provided results for univariate ANOVAs for treatment effects on individual alkaloids. All this was analyzed using the **INSITU.cvs** metadata. Colum references: **-PER** corresponds to peramine alkaloids **-LOL** corresponds to loline alkaloids **-NAL** corresponds to *N*-acetylloline alkaloids **-NANL** corresponds to *N*-acetylnorloline alkaloids **-NFL** corresponds to *N*-formylloline alkaloids **-Ha.I** corresponds to *H. armigera* initial weight **-Ha.F** corresponds to *H. armigera* final weight **-RGR** corresponds to *H. armigera* relative growth rate **-SI** corresponds to foliar silicon **-B.FSC** corresponds to filled silica cells in abaxial side of leaf **-T.FSC** corresponds to filled silica cells in adaxial side of leaf Moreover, to address H3 and H4 that states that only Si supply negatively affects feeding efficiency indices (H3), whereas both Si and endophyte have (H4) negative effects on insect immunity, all herbivore parameters including, relative growth rate, mandibular wear, food utilization indices, and larval cellular and humoral immunity (response variables), were explored using a two-way ANOVA (type III) with endophyte and Si treatment as fixed factors. Additionally, a Pearson rank correlation coefficient test was used to analyse the relationship between relative growth rate and mandibular wear (Knowles, Siegmund, & Zhang, 1991). Feeding efficiency indices calculations were performed using the **EXSITU.cvs** Colum references *H. armigera*: **-RGR** corresponds to relative growth rate **-ECI** corresponds to efficiency of conversion of ingested food **-ECD** corresponds to efficiency of conversion of digested food **-AD** corresponds to approximate digestibility **-RC** corresponds to relative consumption **-PNA** corresponds to proportion of nitrogen absorbed Immunity calculations were performed using the **IMUNITY.cvs** **-TRT** corresponds to immunity treatments: (1) control, (2) sham injection (procedural control), and (3) immune-challenge **-PO** corresponds to phenoloxidase  activity **-Protein** corresponds to haemolymph protein in *H. armigera* haemocoel **-Pl** corresponds to number of plasmatocytes in *H. armigera* haemocoel **-Gr** corresponds to number of granulocytes in *H. armigera* haemocoel **-Sp** corresponds to number of spherulocytes in *H. armigera* haemocoel **-total** corresponds to number of haemocytes (pPl+Gr+Sp)in *H. armigera* haemocoel **-MEL** corresponds to melanisation response Finally, although not part of the hypothesis, for plant growth and primary chemistry, a three-way ANOVA (type III) with endophyte, Si supply and herbivory treatments as fixed effect factors was performed using the **1STHARV.cvs and 2NDTHARV.cvs** metadata. Colum references: **-TILLER** corresponds to the number of tillers **-TW** corresponds to tiller width **-SDM** corresponds to shoot dry mass **-RDM** corresponds to root dry mass **-RS** corresponds to shoot to root ratio **-N** corresponds to shoot nitrogen **-C** corresponds to shoot carbon **-C/N** corresponds carbon nitrogen ratio ## Sharing/Access information via Drylab or e-mail request xcibils@inia.org.uy ## Code/Software e-mail request: xcibils@inia.org.uy