Silicon and Epichloë-endophyte defences in a model temperate grass diminish feeding efficiency and immunity of an insect folivore
Data files
Sep 28, 2023 version files 27.44 KB
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1STHARV.csv
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2NDHARV.csv
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EXSITU.csv
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IMUNITY.csv
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INSITU.csv
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README.md
Abstract
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.
README: 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
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
Methods
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.