Silicon and Epichloë-endophyte defences in a model temperate grass diminish feeding efficiency and immunity of an insect folivore

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 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

植物演化出多样的抗草食动物防御机制，以降低草食动物的取食行为与生存表现。温带禾本科植物通过硅（silicon, Si）积累与Epichloë内生真菌（Epichloë-endophytes），构建物理与化学（即内生生物碱）双重防御体系，抵御昆虫草食动物的侵害。已有研究表明，Epichloë内生真菌可提升宿主禾草的硅积累水平，但目前尚不明确这一效应如何影响叶片表面的硅沉积，以及其对昆虫草食动物取食效率、潜在感染/寄生免疫的作用。

为填补这一研究空白，本研究采用水培法培养高羊茅（Festuca arundinacea），设置添加硅与不添加硅、接种新型AR584菌株Epichloë内生真菌与不接种共四组处理。分别通过原位（完整叶片）与异位（离体叶片）取食试验，使植物暴露于棉铃虫（Helicoverpa armigera，鳞翅目：夜蛾科）的取食下，进而分析硅与内生真菌防御对草食动物取食效率、生长速率以及潜在感染/寄生免疫的影响。

与未接种内生真菌且无草食动物取食的植株相比，接种内生真菌且添加硅的植株在遭受草食动物取食时，叶片硅密度与叶片硅浓度分别提升110%与143%。尽管内生真菌可提升硅浓度，但棉铃虫的生长仅受硅供应的影响：其相对生长速率下降87%，多数取食效率指标至少降低30%。硅供应还使棉铃虫的上颚磨损程度提升16%，该指标与棉铃虫相对生长速率呈负相关。棉铃虫的细胞免疫与体液免疫同时受到硅与内生真菌的负向影响。内生洛林生物碱浓度不受硅供应或草食动物取食的影响，但草食动物取食可使佩拉姆碱浓度提升290%。

据我们所知，本研究首次报道硅防御与Epichloë内生真菌衍生的生物碱可通过降低黑化反应，削弱昆虫的免疫能力。本研究以高羊茅与棉铃虫为研究对象，结果表明，同时构建物理（即硅积累）与化学（即内生生物碱）双重防御体系，针对昆虫草食动物的取食效率、生长与免疫等多个性状发挥作用，可能是温带禾本科植物的高效防御策略。这种多维度防御体系对昆虫草食动物而言尤为难以突破。

# 数据集与文件结构说明
## 数据集描述
如论文方法部分所述，本研究将元数据拆分至不同文件，用于R语言分析：
所有文件均包含核心因子列：
- ENDO：内生真菌处理水平，分为AR584（接种内生真菌）与Nil（未接种内生真菌）
- Si：硅处理水平，分为水培液中添加硅（+Si）与不添加硅（原文标注为-So，疑似笔误）
- INSECT：草食动物处理水平，分为接种棉铃虫（+Herb）与未接种棉铃虫（-Herb）
- OU：实验单元

## 分析方法与数据文件
### 假说1（H1）验证
为验证假说1（H1）：增强的硅防御（例如叶片表面硅化植硅体的增加）对内生真菌特异性生物碱合成、叶片硅浓度及由此产生的硅细胞密度无影响。本研究仅针对添加硅的植株，采用以内生真菌与草食动物处理为固定效应因子的双因素方差分析（Type III平方和），分析上述响应变量。

### 假说2（H2）验证
为验证假说2（H2）：硅供应对内生真菌特异性生物碱合成无影响。本研究仅针对接种内生真菌的植株，采用以硅与草食动物处理为固定效应的置换多因素方差分析（permutational MANOVA），借助'vegan'包（Oksanen等，2019）分析生物碱整体谱（响应变量）的组间差异。该MANOVA函数还可输出针对单个生物碱的单因素方差分析结果，以解析处理对单一生物碱的效应。

以上分析均基于INSITU.csv元数据文件完成（原文标注为INSITU.cvs，疑似笔误）。

#### 列名说明
- PER：佩拉姆碱（peramine alkaloids）
- LOL：洛林生物碱（loline alkaloids）
- NAL：N-乙酰洛林碱（N-acetylloline alkaloids）
- NANL：N-乙酰去甲洛林碱（N-acetylnorloline alkaloids）
- NFL：N-甲酰洛林碱（N-formylloline alkaloids）
- Ha.I：棉铃虫初始体重
- Ha.F：棉铃虫终末体重
- RGR：棉铃虫相对生长速率
- SI：叶片硅含量
- B.FSC：叶片下表皮填充硅细胞数
- T.FSC：叶片上表皮填充硅细胞数

### 假说3（H3）与假说4（H4）验证
此外，为验证假说3（H3）：仅硅供应对取食效率指标存在负向影响，以及假说4（H4）：硅与内生真菌均对昆虫免疫存在负向影响，本研究采用以内生真菌与硅处理为固定因子的双因素方差分析（Type III），分析所有草食动物相关参数，包括相对生长速率、上颚磨损程度、食物利用指标以及幼虫细胞免疫与体液免疫（响应变量）。此外，采用皮尔逊秩相关系数检验，分析相对生长速率与上颚磨损程度之间的相关性（Knowles等，1991）。

取食效率指标的计算基于EXSITU.csv元数据文件完成（原文标注为EXSITU.cvs，疑似笔误）。
#### 棉铃虫相关列名说明
- RGR：相对生长速率
- ECI：摄入食物转化率（efficiency of conversion of ingested food）
- ECD：消化食物转化率（efficiency of conversion of digested food）
- AD：近似消化率（approximate digestibility）
- RC：相对取食量（relative consumption）
- PNA：氮吸收比例（proportion of nitrogen absorbed）

免疫相关指标的计算基于IMMUNITY.csv元数据文件完成（原文标注为IMUNITY.cvs，疑似笔误）。
#### 免疫相关列名说明
- TRT：免疫处理组：(1) 对照组，(2) 假注射组（程序性对照），(3) 免疫攻击组
- PO：酚氧化酶活性（phenoloxidase activity）
- Protein：棉铃虫血淋巴中血淋巴蛋白含量
- Pl：棉铃虫血淋巴中浆血细胞数量
- Gr：棉铃虫血淋巴中粒细胞数量
- Sp：棉铃虫血淋巴中小球细胞数量
- total：血淋巴细胞总数（Pl+Gr+Sp）
- MEL：黑化反应强度（melanisation response）

### 植物生长与基础化学组分分析
最后，尽管不属于本研究的假说范畴，针对植物生长与基础化学组分的分析，采用以内生真菌、硅供应与草食动物处理为固定效应因子的三因素方差分析（Type III），分析基于1STHARV.csv与2NDHARV.csv元数据文件完成（原文标注为2NDTHARV.cvs，疑似笔误）。
#### 列名说明
- TILLER：分蘖数
- TW：分蘖宽度
- SDM：地上部干重
- RDM：地下部干重
- RS：根冠比
- N：地上部氮含量
- C：地上部碳含量
- C/N：碳氮比