Silicon Fertilisation Affects Morphological and Immune Defences of an Insect Pest and Enhances Plant Compensatory Growth

doi:10.21203/rs.3.rs-773578/v1

Insect herbivores employ various defences, including morphological, behavioural, and immune responses against their natural enemies (e.g., predators, parasitoids) which can make biocontrol of herbivorous pests challenging. Silicon (Si) accumulation in plants is a potent physical defence against herbivores. However, it remains uncertain how Si affects pest defences to their enemies and plant defences following herbivore attack. We grew the model grass, Brachypodium distachyon, hydroponically with (+Si) or without (–Si) Si and investigated the impacts of Si on morphological (integument resistance and thickness), behavioural (flee, headrear, thrash, and regurgitation), and immune defences of the cotton bollworm, Helicoverpa armigera. We further examined the effects of Si on plant compensatory growth and leaf trichome production. Larval growth, leaf consumption, and integument resistance were lower when feeding on +Si plants compared to when feeding on –Si plants. Larval integument thickness, defensive behaviours, hemocyte density and lysozyme-like activity in the hemolymph were unaffected by Si. Larvae fed on +Si plants had higher hemolymph phenoloxidase (PO) and total-PO activities than larvae fed on –Si plants, although this did not enhance larval melanisation response. Furthermore, Si supply increased plant compensatory growth and constitutive trichome production whereas herbivory induced trichome production only on –Si plants. We provide the first evidence that Si fertilisation affects insect defences in addition to reducing their growth and feeding. Lower integument resistance might enhance larval vulnerability to parasitoids and pathogens and higher PO activities could impose fitness costs (e.g., delayed development), potentially increasing overall insect susceptibility to enemies.

Environmental Engineering

silicon defence

Helicoverpa armigera

plant compensatory growth

insect morphological defence

insect defensive behaviours

insect immune defence

The nutritional quality and physical and chemical defences of plants can substantially impact insect herbivore fitness (i.e., bottom-up effects) (Singer and Stireman 2005; Vidal and Murphy 2018). This can produce cascading effects on natural enemies of insect herbivores such as predators and parasitoids (top-down forces) via changes in host insect defences and vulnerability to attack (Forkner and Hunter 2000; Pekas and Wäckers 2020). For example, feeding on high-quality host plants can enhance insect immune defences such as encapsulation and melanisation of invaders (i.e., parasitoid eggs) (Diamond and Kingsolver 2011; Gherlenda et al. 2016). Moreover, insect herbivores can exploit plant defence chemicals (e.g. secondary metabolites) to self-medicate (Garvey et al. 2021) or to defend against their enemies via sequestration (Winde and Wittstock 2011).

Among insect pests, larval Lepidoptera encounter extensive top-down pressures (Bernays 1997) and are often managed with biological control (Stiling and Cornelissen 2005). As counteradaptations, larvae have evolved an arsenal of defences against enemies, including morphological, behavioural, chemical, and immune defences (Greeney et al. 2012). For instance, thicker integuments or larger body spines can function as morphological defences against parasitoids (Gross 1993). Moreover, larvae can show evasive behaviours including thrashing, twisting, or dropping to avoid enemies, and aggressive behaviours including headrearing, regurgitating, or biting while encountering enemies (Greeney et al. 2012; Gross 1993). However, after successful attacks by pathogens or parasitoids, larvae mostly rely on immune defence, driven by cellular and humoral mechanisms. Hemocytes, the circulating hemolymph cells in larvae, can cause phagocytosis of microorganisms and encapsulation of foreign entities such as parasitoid eggs or larvae (Lavine and Strand 2002). Humoral responses include the actions of phenoloxidase (PO), a key enzyme that regulates melanisation of invaders and wound healing (Eleftherianos and Revenis 2011), and lysozyme activity that degrades bacteria and fungi (Moreno-García et al. 2013).

Plant defence against herbivores comprises resistance (e.g., physical and chemical barriers to herbivory) and tolerance to attack, including compensatory growth following herbivory (Núñez-Farfán et al. 2007). The physical and chemical defences in plants can be expressed constitutively or induced when challenged by herbivores (Gatehouse 2002). Silicon (Si) accumulation in plants is recognized as an effective physical defence against chewing herbivores (Massey and Hartley 2009; Reynolds et al. 2016), particularly in grasses including cereal crops, as they possess limited secondary metabolite defences and can accumulate relatively higher amounts (up to 10% of dry weight) of Si (Moore and Johnson 2016; Vicari and Bazely 1993). Plants deposit silica (SiO₂) in tissues, including physical defence structures (e.g., trichomes), following active uptake or passive absorption of aqueous orthosilicic acid via roots and transportation via xylem (Ma and Yamaji 2006; Mandlik et al. 2020). Silicification makes plant tissue tougher and abrasive, and hence, less masticable and digestible for chewers, often causing mandibular wear while feeding (Massey and Hartley 2009) and gut damage while passing silicified trichomes through the digestive tract (Andama et al. 2020). Insect herbivores generally feed less on silicified plants and have retarded growth because of reduced nutrient assimilation (Massey and Hartley 2009). However, it remains uncertain whether malnourishment caused by siliceous plants makes insects more vulnerable to their enemies via compromised defences. This question is of interest as Si accumulation by plants has recently been shown to promote the attraction of natural enemies of herbivores (Islam et al. 2021; Liu et al. 2017) and to impact tri-trophic interactions by changing host insects’ body size (Hall et al. 2021).

In addition to reducing insect attack, either via direct physical defences or via compromising the defences of insect pests against their own enemies, Si has the potential to improve defences of previously attacked plants against future attack (Johnson et al. 2019). Research on Si-based plant defence against herbivores has mostly focused on plant resistance, with the role of Si in tolerance relatively neglected. Furthermore, herbivory often induces increased Si accumulation in plants (Islam et al. 2020; Massey et al. 2007), however, how Si induction impacts the production of other inducible defence structures such as trichomes remain understudied, though there are reports that Si can enhance constitutive trichome density (Biru et al. 2021; Johnson et al. 2021).

To the best of our knowledge, the impacts of Si on insect defences against their natural enemies have yet to be investigated. We grew the model grass, Brachypodium distachyon, hydroponically with (+ Si) or without (− Si) Si and investigated the impacts of Si supply on defences of the cotton bollworm, Helicoverpa armigera and plant defences following herbivory. Specifically, our objectives were (i) to elucidate the effects of Si supplementation of plants on larval growth and feeding and consequently on larval morphological (integument resistance and thickness), behavioural (in the absence or presence of a stimulus) and immune (under immunologically naïve and challenged conditions) defences; and (ii) to determine the impacts of Si supply on plant compensatory growth following herbivory, and constitutive and induced trichome production. Brachypodium distachyon is a temperate grass, widely studied because of its genetically tractable genome, short life cycle, and phylogenetic connection to important cereal crops (Scholthof et al. 2018). Helicoverpa armigera is a global pest of many high-value agricultural and horticultural crops, costing over US$7 billion annually due to crop losses and management expenses (Jones et al. 2019). We hypothesized that Si negatively affects larval feeding and growth, and thus attenuates larval defences by limiting physiological resources.

Plants and insect herbivores

Brachypodium distachyon (accession Bd21-3) seeds were obtained from the French National Institute for Agricultural Research (INRA, Versailles, France). Seeds were softened by soaking in water for 2 hrs and dehusked manually using forceps. Seeds were then sterilised in a solution of 0.9% NaOCl and 0.1% Triton-X for 30 min and subsequently, washed in water several times. Sterilised seeds were sown in wet perlite in germination trays and kept refrigerated at 4°C for 7 days for cold stratification and further grown for 12 days in a naturally lit glasshouse at 22/18°C day/night temperatures on a cycle of 14L:10D and 50% (± 6%) relative humidity. Uniform seedlings were transplanted to non-aerated hydroponic vessels, each comprised of two nested disposable plastic cups (480 ml) with a fitted foam disc at the top as per Hall et al. (2020). Foam discs were slot cut to accommodate a seedling in each vessel. Cups were weekly filled with 370 ml of freshly prepared nutrient solution with (+ Si) or without (− Si) Si supplementation as per Hall et al. (2020). Plants (N = 150) were grown in the same glasshouse environment and cups were rotated weekly to avoid any position bias. Helicoverpa armigera (Lepidoptera: Noctuidae) larvae were supplied by CSIRO Agriculture and Food, Narrabri, NSW, Australia and were reared on an artificial diet modified from Teakle and Jensen (1985) before inoculating on plants.

Experimental design and treatments

Liquid potassium silicate (K₂SiO₃) (21% K₂O and 32% SiO₂, Agsil32, PQ Australia, SA, Australia) was added to the nutrient solution to create + Si treatment. The concentration of silicic acid was maintained at 2 mM (SiO₂ equivalent) in + Si solution as polymerisation occurs above this concentration (Ma and Yamaji 2006). The added K⁺ ions in the + Si treatment were balanced in the control treatment (–Si) by adding KCl (Sigma-Aldrich, MO, USA). Both + Si and –Si solutions were adjusted to pH 5.5 with HCl. Plants were assigned to herbivore treatments (insect or no insect) after being grown hydroponically for five weeks. Eighty plants (40 + Si and 40 –Si) were randomly exposed to a second instar H. armigera larva for seven days. Larvae were starved for 24 hrs in Petri dishes before being weighed and placed onto plants. The rest of the plants were kept insect-free. All plants were kept caged for seven days in transparent acrylic cylinders (see Johnson et al. (2020) for cage specifications). Larvae were starved for 24 hrs following removal from plants to allow all frass to be expelled, and subsequently weighed to estimate larval relative growth rates (RGRs). Insect RGR is a combined estimate for plant antixenotic (i.e. effects on growth due to starvation) and antibiotic effects and was calculated as larval mass gain relative to initial body mass per unit of time [mass gained (mg)/initial mass (mg)/time (days)] (Massey and Hartley 2009). We collected larval frass randomly from 40 hydroponic vessels (20 holding + Si plants and 20 holding –Si plants) and matching Petri dishes following larval starvation and oven-dried it at 60°C for three days before weighing (i.e., as a proxy for larval leaf consumption (Murray et al. 2013)). Further, 40 larvae (20 fed on + Si plants and 20 fed on –Si plants) were used for assessing behavioural and morphological defences and the remaining 40 larvae were used for measuring immune defence. Thirty insect-fed plants (15 of each Si treatment) were randomly harvested immediately after insect herbivory, as were 30 insect-free plants (15 of each Si treatment). Plants were oven-dried at 60°C for seven days before measuring shoot dry biomass to assess insect damage on + Si and –Si plants. Eighty other plants (N = 20 of each treatment) were grown for a further two weeks to elucidate the effects of Si supply on plant compensatory growth and leaf trichome density.

Measurements of larval behavioural and morphological defences

We measured larval escape response (i.e., flee) in the absence of any stimulus using 40 larvae as per Vogelweith et al. (2014). Each larva was placed singly on a gridded plastic sheet (86 × 112 cm) and acclimated for 20 s under a glass jar. Following the jar removal, the number of squares crossed by a larva within 60 s (minimum time to exit the sheet as observed in the preliminary experiment) was recorded. As no stimulus was involved, some larvae (seven fed on + Si plants and four fed on − Si plants) did not make any movements and were omitted from the analysis. Following flee measurements, each larva was placed on a white filter paper, acclimated for 30 s under a glass jar and then gently pinched three times at 20 s intervals using bracket-placing tweezers at the abdominal end to imitate a predator attack (Cornell et al. 1987). The behaviours of larvae were video recorded (Logitech c270 webcam) and three distinct larval behaviours were characterized and logged for each larva: (a) ‘headrearing’ as characterized by the backward movement of the head and the anterior body portion to the posterior part, (b) ‘thrashing’ as characterized by the side-to-side swing movement of the head and the anterior body portion, and (c) ‘regurgitating’ on the filter paper (Cornell et al. 1987).

We further measured larval integument resistance and integument thickness following the protocol of Vogelweith et al. (2014) with minor modifications. Larvae were placed at − 20°C for 20 min and then thawed for 10 min before starting measurements. Integument resistance was measured by using a drill press equipped with a hypodermic needle (Terumo, 0.5 × 16 mm). A precision scale (± 0.1 mg) was positioned on the drill press table and each larva mounted on a cardboard sheet was placed over the scale. The needle was slowly lowered down by rotating the lever until it breached the dorsal integument. The scale reading (mg) when the needle breached the integument was recorded. Two measurements were taken from each larva, one on the thoracic region and another on the abdomen, and the average value was counted. Larvae were further dissected and all internal organs and fat bodies were removed before measuring integument thickness using a thickness gauge (Teclock SM-112, Japan, precision ± 0.01 mm) (Iltis et al. 2018).

Measurements of larval immune defences

We measured larval innate immunity (10 larvae fed on + Si plants and 10 larvae fed on − Si plants) in terms of hemocyte density, phenoloxidase (PO) activity, total-PO activity (combined estimate of PO and its precursor, prophenoloxidase), and lysozyme-like enzyme activity in the hemolymph. For this, immunologically naïve larvae were chilled on ice for 20 min and a proleg was removed from each larva using a sterile micro scissor and 3 µl of hemolymph was collected immediately using a sterile micropipette. Of this, 2 µl was flushed into a pre-chilled microcentrifuge tube containing 20 µl of cold phosphate buffer saline (PBS, pH 7.4), and 10 µl of this hemolymph-PBS mixture was immediately spread over a Neubauer Improved Haemocytometer and hemocytes were counted under a phase-contrast microscope at 400x magnification (Axio Vert.A1, Zeiss, Australia). We measured total hemocyte density as well as the density of individual hemocytes, classified according to Vogelweith et al. (2016) and Ribeiro and Brehélin (2006) into prohemocytes, granulocytes, plasmatocytes, spherulocytes, and oenocytoids. The rest of the hemolymph-PBS mixtures was stored at − 20°C for the later measurements of PO and total-PO activities following the protocol of Vogelweith et al. (2011). Briefly, samples were thawed slowly on ice and centrifuged (3000 g, 4ºC) for 15 min. Further, 5 µl of supernatant from each sample was transferred to a microplate well containing 20 µl of PBS with either 140 µl of distilled water for measuring PO activity or 140 µl of chymotrypsin solution (Sigma-Aldrich, 0.98 mg/14 ml of distilled water) for measuring total-PO activity. Finally, 20 µl of L-DOPA solution (Sigma-Aldrich, 8 mg/2 ml of distilled water) was added as a substrate to each microplate well and the absorbance readings were taken for samples and negative controls at 490 nm in a microplate reader (CLARIOstar Plus, BMG Labtech) for 40 min at 60 s intervals. The enzyme activity was calculated from the slope of the reaction curve (i.e., change in optical density per min) at the linear phase and are reported as the activity per microliter of pure hemolymph.

An additional 1 µl of pure hemolymph was flushed into a pre-chilled microcentrifuge tube containing 10 µl of reaction buffer (pH 6.24, Sigma-Aldrich) and stored at − 20°C for the subsequent measurements of lysozyme-like activity. Lysozyme-like activity was measured by a turbidity assay using Micrococcus lysodeikticus (Sigma-Aldrich) as a substrate following the method modified from Adamo et al. (2016). In short, 10 µl of hemolymph-PBS mixture from each sample was transferred to a microplate well containing 180 µl of a suspension of M. lysodeikticus cells (1 mg/10 ml) in reaction buffer (pH 6.24, Sigma-Aldrich). Lysozyme standards (Sigma-Aldrich) within the linear range of assays were run concurrently as positive controls along with the test samples and negative controls, and the kinetic decrease in absorbance was recorded in the microplate reader at 450 nm wavelength for 10 min at 50 s intervals. Lysozyme-like activities are presented in ‘unit’ where one unit represents 0.001 change in optical density per minute.

Furthermore, we measured encapsulation and melanisation responses of larvae (10 larvae fed on + Si plants and 10 larvae fed on − Si plants) when immunologically challenged, simulating a solitary endoparasitoid attack (Moreno-García et al. 2013). For this, a nylon monofilament (0.2 mm diameter) was implanted 2 mm deep in the hemocoel through the dorsal abdominal part of each larva according to Gherlenda et al. (2016). After 24 hrs, we collected 3 µl of hemolymph from each larva and measured hemolymph PO, total-PO and lysozyme-like activities as per the procedures described previously. The nylon implants were removed and photographed using a stereo microscope (Olympus, SZ61) mounted camera (Infinity1, Teledyne Lumenera) along with a control filament (i.e., not implanted in hemocoel). The photos were further processed in ImageJ (National Institutes of Health, Maryland; Version 1.52) and the mean grey values were estimated on a scale of 0 (light) to 255 (dark). The melanisation scores were calculated using the formula: 1 – (mean grey value of the treatment filament/mean grey value of the control filament) (Garvey et al. 2021).

Measurements of leaf trichome density, plant biomass, and Si concentrations

We measured the density of non-glandular trichomes (or macrohairs) on leaves of all 80 plants (N = 20 of each treatment) grown for an additional two weeks following herbivory or no herbivory. Non-glandular trichomes can defend plants against insect herbivores, often more effectively than plant secondary metabolites (Carmona et al. 2011), and herbivory can induce increased trichome density on newly emerged leaves (Dalin et al. 2008). We sampled the newly emerged, fully expanded leaves from plants and counted trichomes on abaxial and adaxial surfaces (4 mm × 4 mm area in the middle) under a stereo microscope (Olympus, SZX10). Plants were further harvested, oven-dried at 60ºC for 7 days and shoot and root biomass were recorded. Of these plants, we measured Si concentrations (% dry mass) in leaves and roots of all + Si plants (20 insect-fed plants and 20 insect-free plants). For this, ca. 80 mg oven-dried, ball-milled samples were analysed in an X-ray fluorescence spectrometer (Epsilon 3^x; PANalytical, EA Almelo, The Netherlands) according to Reidinger et al. (2012). Measurements were standardized using a certified plant material of known Si concentration (NCS ZC73018 Citrus leaves, China National Institute for Iron and Steel).

Statistical analyses

All data were analysed in the statistical software environment R, version 3.6.1 (R Core Team 2019). The distributions of dependent and independent variable datasets were compared using quantile-quantile plots and homogeneity of variance was assessed using ‘residuals versus fits’ plots. Larval frass and morphological and behavioural defence parameters were analysed using Wilcoxon’s rank-sum tests as the assumptions of normality of residuals or homoscedasticity were violated. Larval RGR, hemocyte density, PO, total-PO, lysozyme-like activity, and leaf and root Si concentrations were analysed using Student’s t-tests. The effects of Si on larval integument resistance and PO activity were further analysed using multiple linear regressions, incorporating larval weight gain or final weight as a covariate. The relationships between larval integument resistance and weight gain and final weight were explored using Spearman’s correlations. Leaf trichome density and plant shoot and root biomass were analysed using two-way analysis of variance (ANOVA) tests, considering Si and insect herbivore as fixed factors. In case of significant effects, differences between group means were determined by Tukey’s HSD tests.

Insect growth, feeding, and defensive traits

Larval RGR and frass production were significantly lower (− 300% and − 85%, respectively) when feeding on + Si plants compared to when feeding on –Si plants (Fig. 1a-1b; Table 1). However, none of the larval defensive behaviours (flee, headrear, thrash, and regurgitation) were affected by Si (Table S1). Larvae fed on + Si plants had significantly lower integument resistance than larvae fed on –Si plants (Fig. 2a; Table 1) despite no changes in integument thickness (Fig. 2b; Table 1). Integument resistance was strongly and positively correlated with larval weight gain (ρ = 0.83, p < 0.001) and final weight (ρ = 0.98, p < 0.001) (Fig. S1). Multiple linear regression analyses show that Si had a significant effect on integument resistance after adjusting for the effect of larval weight gain as a covariate (Table 3). However, Si had no significant effect on integument resistance when the effect of larval final weight was controlled (Table 3). The density of individual and total hemocytes in larval hemolymph was unaffected by Si (Fig. S2; Table S2). Larvae fed on + Si plants had significantly higher PO and total-PO activities in the hemolymph compared to larvae fed on –Si plants under both immunologically naïve (unchallenged) (+ 71% and + 86%, respectively) and challenged conditions (+ 33% for both) (Fig. 3a-3b; Table 1). Accordingly, Si had a significant effect on hemolymph PO activity after controlling for the effect of larval weight gain or final weight as a covariate (Table 3). However, the melanisation responses of larvae fed on + Si or –Si plants were similar (Fig. 4, Table 1). Lysozyme-like activity in the hemolymph was unaffected by Si, regardless of immunity challenge (Fig. S3; Table S2).

Leaf trichomes, plant compensatory growth, and silicon concentrations

Silicon in interaction with herbivory significantly affected leaf trichome density, whereby + Si plants had higher (+ 51%) trichome density on abaxial leaf surfaces than − Si plants in the absence of insect herbivory (Fig. 5a; Table 2). However, following herbivory, trichome density on abaxial leaf surfaces of − Si plants increased by 46% and became statistically similar to insect-fed + Si plants. Likewise, trichome density on adaxial leaf surfaces of − Si plants significantly increased (+ 62%) following herbivory (Fig. 5b; Table 2). In terms of plant biomass, insect herbivory significantly reduced shoot biomass of both − Si and + Si plants (− 16% and − 14%, respectively) harvested immediately after herbivory (Fig S4, Table S3). There was a significant interaction between Si and herbivory on shoot biomass of plants harvested two weeks afterwards, whereby insect-fed + Si plants produced similar shoot biomass to insect-free, +Si or –Si plants (Fig. 6a; Table 2), indicating a higher compensatory growth with the provision of Si. However, root biomass was unaffected by Si or herbivory (Fig. 6b; Table 2). Besides, herbivory significantly increased leaf Si accumulation by ca. 29% but had no effect on root Si concentrations (Fig. 7; Table 1).

We report for the first time, to our knowledge, how Si supplementation of plants impacts the morphological and behavioural defences of an insect herbivore. Silicon reduced larval growth and feeding, thereby lowering integument resistance which was associated with compensatory production of immunity proteins in the hemolymph. Moreover, Si supply enhanced constitutive trichome production on leaves and augmented plant compensatory growth whereas insect herbivory induced trichome production on plants that had no access to Si.

Silicon weakened larval morphological defences but did not affect defensive behaviours

Larvae fed less on + Si plants (i.e., as evident from the lower levels of frass production) and displayed retarded growth rates which are in line with the existing literature on the effects of Si against chewing herbivores, including folivores (Biru et al. 2021; Islam et al. 2020; Johnson et al. 2020), borers (Kvedaras et al. 2009; Nikpay et al. 2015) and root feeders (Frew et al. 2017). The three-fold decrease in larval growth rates observed here due to malnutrition could potentially impact biocontrol by natural enemies. For example, Remmel et al. (2011) estimated that a two-fold rise in the linear size of folivorous insect larvae increases 3.6-fold avian predation rates while reducing 4.9-fold arthropod predation rates.

We found a strong positive correlation between larval final weight and integument resistance. In accordance with this, Iltis et al. (2018) reported a positive correlation between larval body size and integument resistance of the lepidopteran grape pest, Lobesia botrana, despite no changes in integument thickness. Although it is not evident how larger larvae showed higher integument resistance despite the same integument thickness, we speculate that there might have some variations in the integument ultrastructures of larvae fed on –Si plants compared to larvae fed on + Si plants, including the extent of sclerotization and tanning. This finding suggests that feeding on + Si plants could make larvae more vulnerable to parasitoid attacks as parasitoid oviposition is more successful in smaller larvae with a lower integument resistance (Beckage and Riddiford 1978; Gross 1993). We observed consistent defensive behaviours in H. armigera larvae, irrespective of lower leaf consumption and growth rates. This concurs well with the findings of Zhou et al. (2017) who found that evasive (escape) and aggressive (thrashing and dropping) behaviours of the oriental armyworm, Mythimna separata, were unaffected by body size and weight.

Interactions between larval morphological and immune defences

Our results indicate a potential trade-off between insect morphological and immune defences; larvae fed on + Si plants had lower integument resistance and higher hemolymph PO and total-PO activities. Such trade-offs between different defensive traits have been demonstrated in other lepidopteran (Vogelweith et al. 2014) and hymenopteran (Boevé and Schaffner 2003) larvae, including trade-offs due to feeding on different host plants (Vogelweith et al. 2011). For instance, in several sawfly species, a lower integument resistance and propensity for bleeding have been linked to a higher hemolymph deterrence against predators such as ants and wasps (Boevé and Schaffner 2003). Our results differ from the previous study by Frew et al. (2017) who found no effects of Si supplementation of sugarcane on PO activity of the root-feeding grub, Dermolepida albohirtum. Even though the underlying mechanisms of how Si enhanced PO activity in the hemolymph are not clear, previous research has shown that consumption of plant toxins (e.g., nicotine) or starvation could be immunotherapeutic for insects and could enhance PO activity (Garvey et al. 2021; Yang et al. 2007). We suggest that Si accumulation in plants forced larvae into starvation and consumption of silicified tissues and trichomes might cause larval gut damage and exert physiological stress on them, contributing to higher hemolymph PO activities. This high level of PO can impose fitness costs on larvae as production and maintenance of PO is very costly (González-Santoyo and Córdoba-Aguilar 2012) and resource allocation to PO can constrain resources for other physiological functions, including larval development (Cotter et al. 2008) and expression of sexual traits (Siva–Jothy 2000).

Higher PO activity did not enhance melanisation

Notably, we found that higher PO activities in the hemolymph did not enhance the melanisation response when larvae were immunologically challenged. Since PO functions against pathogens and parasitoids via melanogenesis (i.e., a process whereby PO oxidises phenols to quinones which further polymerises to melanin) (Eleftherianos and Revenis 2011), this finding suggests that high PO activity does not imply high immune defences in larvae (González-Santoyo and Córdoba-Aguilar 2012). The possible explanations for similar melanisation responses in larvae are twofold. First, melanisation depends on both PO activity and hemocyte density as invaders need to be first coated (i.e., encapsulation) by adhesive hemocyte cells (Lavine and Strand 2002). Given that the density of hemocytes in larvae fed on + Si or –Si plants was similar, larvae might produce an identical encapsulation of the nylon implants. Second, both the activation of PO and the production of melanin (a nitrogen-rich compound) require substantial investments of nitrogen (González-Santoyo and Córdoba-Aguilar 2012), which can limit nitrogen for melanogenesis in larvae fed on + Si plants, especially considering the fact that Si and nitrogen accumulation in plants can interact antagonistically (Wu et al. 2017).

Post-attack plant defences

We found that Si increased constitutive trichome density on leaves, which substantiates previous studies on grasses (Biru et al. 2021; Johnson et al. 2021) and other crops (Acevedo et al. 2021). Interestingly, herbivory induced trichome production only on –Si plants, because + Si plants were already better defended with constitutively-produced silicified trichomes, which were found to be essential for defence against chewing herbivores in rice (Andama et al. 2020). We suggest that investment in induced defence by –Si plants might compromise, in part, their capacity for compensatory growth as there could be a potential trade-off between plant growth and defence (Herms and Mattson 1992; but see Koricheva 2002). Conversely, with the provision of Si, insect-fed plants produced similar biomass to insect-free plants within two weeks of the recovery period, despite initial biomass losses, substantiating the previous finding that Si can support plant compensatory growth (Johnson et al. 2019).

Our study establishes that Si supplementation of plants can affect defensive traits of insect herbivores, which might generate cascading effects on higher trophic levels. Moreover, Si supply can underpin constitutive trichome production on leaves and plant compensatory growth following herbivory. We found a potential trade-off between larval morphological and immune defences when fed on siliceous plants; a lower integument resistance was associated with higher PO and total-PO activities in the hemolymph although it did not enhance the melanisation response of larvae when challenged. We presume that the lower integument resistance of larvae could enhance their vulnerability to some natural enemies and higher PO activities could impose physiological costs, potentially impeding other physiological functions and possibly expanding overall insect susceptibility to enemies. Further research is needed to understand the consequences of such changes in host insect defences and nutritional quality for predators and parasitoids. Nonetheless, our study provides evidence for the bottom-up effects of siliceous host plants on insect defensive traits and presents a framework for studying the impacts of Si on top-down control via changes in host insect defences.

Contribution of authors

TI and SNJ conceived and designed the study. TI conducted experimental work, collected and analysed data, and drafted the manuscript. SNJ and BDM supervised TI. All authors critically revised the draft and approved the final manuscript for publication.

Acknowledgements

TI is the holder of a scholarship as part of an Australian Research Council Future Fellowship (FT170100342) awarded to SNJ. We would like to thank Mr Burhan Amiji and Dr Craig Barton for their technical supports to the work. We also thank Ximena Cibils-Stewart, Rocky Putra, Fikadu N. Biru and Jamie M. Waterman for their helpful edits to the manuscript.

Data availability

The data that supports the findings of this study are available in the figshare repository, https://doi.org/10.6084/m9.figshare.15085815.v1

Conflicts of interest

The authors declare that there is no conflict of interest.

Acevedo FE, Peiffer M, Ray S, Tan CW, Felton GW (2021) Silicon-mediated enhancement of herbivore resistance in agricultural crops. Front Plant Sci 12:631824. https://doi.org/10.3389/fpls.2021.631824

Adamo SA, Davies G, Easy R, Kovalko I, Turnbull KF (2016) Reconfiguration of the immune system network during food limitation in the caterpillar Manduca sexta. J Exp Biol 219:706-718. https://doi.org/10.1242/jeb.132936

Andama JB, Mujiono K, Hojo Y, Shinya T, Galis I (2020) Nonglandular silicified trichomes are essential for rice defense against chewing herbivores. Plant Cell Environ 43:2019-2032. https://doi.org/10.1111/pce.13775

Beckage NE, Riddiford LM (1978) Developmental interactions between the tobacco hornworm Manduca sexta and its braconid parasite Apanteles congregatus. Entomol Exp Appl 23:139-151. https://doi.org/10.1111/j.1570-7458.1978.tb03016.x

Bernays EA (1997) Feeding by lepidopteran larvae is dangerous. Ecol Entomol 22:121-123. https://doi.org/10.1046/j.1365-2311.1997.00042.x

Biru FN, Islam T, Cibils-Stewart X, Cazzonelli CI, Elbaum R, Johnson SN (2021) Anti-herbivore silicon defences in a model grass are greatest under Miocene levels of atmospheric CO₂. Global Change Biol 27:2959-2969. https://doi.org/10.1111/gcb.15619

Boevé J-L, Schaffner U (2003) Why does the larval integument of some sawfly species disrupt so easily? The harmful hemolymph hypothesis. Oecologia 134:104-111. https://doi.org/10.1007/s00442-002-1092-4

Carmona D, Lajeunesse MJ, Johnson MTJ (2011) Plant traits that predict resistance to herbivores. Funct Ecol 25:358-367. https://doi.org/10.1111/j.1365-2435.2010.01794.x

Cornell JC, Stamp NE, Bowers MD (1987) Developmental change in aggregation, defense and escape behavior of buckmoth caterpillars, Hemileuca lucina (Saturniidae). Behav Ecol Sociobiol 20:383-388. https://doi.org/10.1007/BF00302980

Cotter SC, Myatt JP, Benskin CM, Wilson K (2008) Selection for cuticular melanism reveals immune function and life-history trade-offs in Spodoptera littoralis. J Evol Biol 21:1744-1754. https://doi.org/10.1111/j.1420-9101.2008.01587.x

Dalin P, Ågren J, Björkman C, Huttunen P, Kärkkäinen K (2008) Leaf trichome formation and plant resistance to herbivory. In: Schaller A (ed) Induced Plant Resistance to Herbivory. Springer, Dordrecht, pp 89-105

Diamond SE, Kingsolver JG (2011) Host plant quality, selection and trade-offs shape the immunity of Manduca sexta. Proc R Soc B 278:289-297. https://doi.org/10.1098/rspb.2010.1137

Eleftherianos I, Revenis C (2011) Role and importance of phenoloxidase in insect hemostasis. J Innate Immun 3:28-33. https://doi.org/10.1159/000321931

Forkner RE, Hunter MD (2000) What goes up must come down? Nutrient addition and predation pressure on oak herbivores. Ecology 81:1588-1600. https://doi.org/10.1890/0012-9658(2000)081[1588:WGUMCD]2.0.CO;2

Frew A, Powell JR, Hiltpold I, Allsopp PG, Sallam N, Johnson SN (2017) Host plant colonisation by arbuscular mycorrhizal fungi stimulates immune function whereas high root silicon concentrations diminish growth in a soil-dwelling herbivore. Soil Biol Biochem 112:117-126. http://dx.doi.org/10.1016/j.soilbio.2017.05.008

Garvey M, Bredlau J, Kester K, Creighton C, Kaplan I (2021) Toxin or medication? Immunotherapeutic effects of nicotine on a specialist caterpillar. Funct Ecol 35:614-626. https://doi.org/10.1111/1365-2435.13743

Gatehouse JA (2002) Plant resistance towards insect herbivores: a dynamic interaction. New Phytol 156:145-169

Gherlenda AN, Haigh AM, Moore BD, Johnson SN, Riegler M (2016) Climate change, nutrition and immunity: Effects of elevated CO₂ and temperature on the immune function of an insect herbivore. J Insect Physiol 85:57-64. https://doi.org/10.1016/j.jinsphys.2015.12.002

González-Santoyo I, Córdoba-Aguilar A (2012) Phenoloxidase: a key component of the insect immune system. Entomol Exp Appl 142:1-16. https://doi.org/10.1111/j.1570-7458.2011.01187.x

Greeney HF, Dyer LA, Smilanich AM (2012) Feeding by lepidopteran larvae is dangerous: A review of caterpillars’ chemical, physiological, morphological, and behavioral defenses against natural enemies. Invertebr Surviv J 9:7-34

Gross P (1993) Insect behavioral and morphological defenses against parasitoids. Annu Rev Entomol 38:251-273. https://doi.org/10.1146/annurev.en.38.010193.001343

Hall CR, Mikhael M, Hartley SE, Johnson SN (2020) Elevated atmospheric CO₂ suppresses jasmonate and silicon‐based defences without affecting herbivores. Funct Ecol 34:993-1002. https://doi.org/10.1111/1365-2435.13549

Hall CR, Rowe RC, Mikhael M, Read E, Hartley SE, Johnson SN (2021) Plant silicon application alters leaf alkaloid concentrations and impacts parasitoids more adversely than their aphid hosts. Oecologia 196:145-154. https://doi.org/10.1007/s00442-021-04902-1

Herms DA, Mattson WJ (1992) The dilemma of plants: to grow or defend. Q Rev Biol 67:283-335

Iltis C, Martel G, Thiéry D, Moreau J, Louâpre P (2018) When warmer means weaker: high temperatures reduce behavioural and immune defences of the larvae of a major grapevine pest. J Pest Sci 91:1315-1326. https://doi.org/10.1007/s10340-018-0992-y

Islam T, Moore BD, Johnson SN (2020) Novel evidence for systemic induction of silicon defences in cucumber following attack by a global insect herbivore. Ecol Entomol 45:1373-1381. https://doi.org/10.1111/een.12922

Islam T, Moore BD, Johnson SN (2021) Silicon suppresses a ubiquitous mite herbivore and promotes natural enemy attraction by altering plant volatile blends. J Pest Sci. https://doi.org/10.1007/s10340-021-01384-1

Johnson SN, Hartley SE, Ryalls JMW, Frew A, Hall CR (2021) Targeted plant defense: silicon conserves hormonal defense signaling impacting chewing but not fluid-feeding herbivores. Ecology 102:e03250. https://doi.org/10.1002/ecy.3250

Johnson SN, Reynolds OL, Gurr GM, Esveld JL, Moore BD, Tory GJ, Gherlenda AN (2019) When resistance is futile, tolerate instead: silicon promotes plant compensatory growth when attacked by above- and belowground herbivores. Biol Lett 15:20190361. https://doi.org/10.1098/rsbl.2019.0361

Johnson SN, Rowe RC, Hall CR (2020) Silicon is an inducible and effective herbivore defence against Helicoverpa punctigera (Lepidoptera: Noctuidae) in soybean. Bull Entomol Res 110:417-422. https://doi.org/10.1017/S0007485319000798

Jones CM, Parry H, Tay WT, Reynolds DR, Chapman JW (2019) Movement ecology of pest Helicoverpa: implications for ongoing spread. Annu Rev Entomol 64:277-295. https://doi.org/10.1146/annurev-ento-011118-111959

Koricheva J (2002) Meta-analysis of sources of variation in fitness costs of plant antiherbivore defenses. Ecology 83:176-190. https://doi.org/10.1890/0012-9658(2002)083[0176:MAOSOV]2.0.CO;2

Kvedaras OL, Byrne MJ, Coombes NE, Keeping MG (2009) Influence of plant silicon and sugarcane cultivar on mandibular wear in the stalk borer Eldana saccharina. Agric For Entomol 11:301-306. https://doi.org/10.1111/j.1461-9563.2009.00430.x

Lavine MD, Strand MR (2002) Insect hemocytes and their role in immunity. Insect Biochem Mol Biol 32:1295-1309. https://doi.org/10.1016/s0965-1748(02)00092-9

Liu J, Zhu J, Zhang P et al. (2017) Silicon supplementation alters the composition of herbivore induced plant volatiles and enhances attraction of parasitoids to infested rice plants. Front Plant Sci 8:1265. https://doi.org/10.3389/fpls.2017.01265

Ma JF, Yamaji N (2006) Silicon uptake and accumulation in higher plants. Trends Plant Sci 11:392-397. https://doi.org/10.1016/j.tplants.2006.06.007

Mandlik R, Thakral V, Raturi G, Shinde S, Nikolić M, Tripathi DK, Sonah H, Deshmukh R (2020) Significance of silicon uptake, transport, and deposition in plants. J Exp Bot 71:6703–6718. https://doi.org/10.1093/jxb/eraa301

Massey FP, Ennos AR, Hartley SE (2007) Herbivore specific induction of silica-based plant defences. Oecologia 152:677-683. https://doi.org/10.1007/s00442-007-0703-5

Massey FP, Hartley SE (2009) Physical defences wear you down: progressive and irreversible impacts of silica on insect herbivores. J Anim Ecol 78:281-291. https://doi.org/10.1111/j.1365-2656.2008.01472.x

Moore BD, Johnson SN (2016) Get tough, get toxic, or get a bodyguard: Identifying candidate traits conferring belowground resistance to herbivores in grasses. Front Plant Sci 7:1925. https://doi.org/10.3389/fpls.2016.01925

Moreno-García M, Córdoba-Aguilar A, Condé R, Lanz-Mendoza H (2013) Current immunity markers in insect ecological immunology: assumed trade-offs and methodological issues. Bull Entomol Res 103:127-139. https://doi.org/10.1017/S000748531200048X

Murray TJ, Tissue DT, Ellsworth DS, Riegler M (2013) Interactive effects of pre-industrial, current and future [CO₂] and temperature on an insect herbivore of Eucalyptus. Oecologia 171:1025-1035. https://doi.org/10.1007/s00442-012-2467-9

Nikpay A, Soleyman-Nejadian E, Goldasteh S, Farazmand H (2015) Response of sugarcane and sugarcane stalk borers Sesamia spp. (Lepidoptera: Noctuidae) to calcium silicate fertilization. Neotrop Entomol 44:498-503. https://doi.org/10.1007/s13744-015-0298-1

Núñez-Farfán J, Fornoni J, Valverde PL (2007) The evolution of resistance and tolerance to herbivores. Annual Review of Ecology, Evolution, and Systematics 38:541-566. https://doi.org/10.1146/annurev.ecolsys.38.091206.095822

Pekas A, Wäckers FL (2020) Bottom-up effects on tri-trophic interactions: Plant fertilization enhances the fitness of a primary parasitoid mediated by its herbivore host. J Econ Entomol 113:2619-2626. https://doi.org/10.1093/jee/toaa204

R Core Team (2019) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria

Reidinger S, Ramsey MH, Hartley SE (2012) Rapid and accurate analyses of silicon and phosphorus in plants using a portable X‐ray fluorescence spectrometer. New Phytol 195:699-706. https://doi.org/10.1111/j.1469-8137.2012.04179.x

Remmel T, Davison J, Tammaru T (2011) Quantifying predation on folivorous insect larvae: the perspective of life-history evolution. Biol J Linn Soc 104:1-18. https://doi.org/10.1111/j.1095-8312.2011.01721.x

Reynolds OL, Padula MP, Zeng R, Gurr GM (2016) Silicon: potential to promote direct and indirect effects on plant defense against arthropod pests in agriculture. Front Plant Sci 7:744. https://doi.org/10.3389/fpls.2016.00744

Ribeiro C, Brehélin M (2006) Insect haemocytes: What type of cell is that? J Insect Physiol 52:417-429. https://doi.org/10.1016/j.jinsphys.2006.01.005

Scholthof K-BG, Irigoyen S, Catalan P, Mandadi KK (2018) Brachypodium: A monocot grass model genus for plant biology. Plant Cell 30:1673-1694. https://doi.org/10.1105/tpc.18.00083

Singer MS, Stireman JOI (2005) The tri-trophic niche concept and adaptive radiation of phytophagous insects. Ecol Lett 8:1247-1255. https://doi.org/10.1111/j.1461-0248.2005.00835.x

Siva–Jothy MT (2000) A mechanistic link between parasite resistance and expression of a sexually selected trait in a damselfly. Proc R Soc B 267:2523-2527. https://doi.org/doi:10.1098/rspb.2000.1315

Stiling P, Cornelissen T (2005) What makes a successful biocontrol agent? A meta-analysis of biological control agent performance. Biol Control 34:236-246. https://doi.org/10.1016/j.biocontrol.2005.02.017

Teakle RE, Jensen JM (1985) Heliothis punctigera vol 2. Handbook of insect rearing. Elsevier, Amsterdam,

Vicari M, Bazely DR (1993) Do grasses fight back? The case for antiherbivore defences. Trends Ecol Evol 8:137-141. https://doi.org/10.1016/0169-5347(93)90026-l

Vidal MC, Murphy SM (2018) Bottom-up vs. top-down effects on terrestrial insect herbivores: a meta-analysis. Ecol Lett 21:138-150. https://doi.org/10.1111/ele.12874

Vogelweith F, Moret Y, Monceau K, Thiéry D, Moreau J (2016) The relative abundance of hemocyte types in a polyphagous moth larva depends on diet. J Insect Physiol 88:33-39. https://doi.org/10.1016/j.jinsphys.2016.02.010

Vogelweith F, Thiéry D, Moret Y, Colin E, Motreuil S, Moreau J (2014) Defense strategies used by two sympatric vineyard moth pests. J Insect Physiol 64:54-61. https://doi.org/10.1016/j.jinsphys.2014.03.009

Vogelweith F, Thiéry D, Quaglietti B, Moret Y, Moreau J (2011) Host plant variation plastically impacts different traits of the immune system of a phytophagous insect. Funct Ecol 25:1241-1247. https://doi.org/10.1111/j.1365-2435.2011.01911.x

Winde I, Wittstock U (2011) Insect herbivore counteradaptations to the plant glucosinolate-myrosinase system. Phytochemistry 72:1566-1575. https://doi.org/10.1016/j.phytochem.2011.01.016

Wu X, Yu Y, Baerson SR, Song Y, Liang G, Ding C, Niu J, Pan Z, Zeng R (2017) Interactions between nitrogen and silicon in rice and their effects on resistance toward the brown planthopper Nilaparvata lugens. Front Plant Sci 8:28. https://doi.org/10.3389/fpls.2017.00028

Yang S, Ruuhola T, Rantala MJ (2007) Impact of starvation on immune defense and other lifehistory traits of an outbreaking geometrid, Epirrita autumnata: a possible causal trigger for the crash phase of population cycle. Ann Zool Fenn 44:89-96

Zhou J, Meng L, Li B (2017) Defensive behaviors of the Oriental armyworm Mythimna separata in response to different parasitoid species (Hymenoptera: Braconidae). PeerJ 5:e3690. https://doi.org/10.7717/peerj.3690

Table 1 Summary output of Student’s t-tests for comparing larval RGR, larval immune defence parameters, and plant Si concentrations and of Wilcoxon’s rank-sum tests for comparing larval frass weight and morphological defence parameters. Statistically significant effects (p < 0.05) are indicated in bold

Response variable	Fig.	Statistical analysis
Response variable	Fig.	df	Test statistic (t^a/W^b)	p value
Larval RGR ^a	1a	78	11.18	<0.001
Frass dry weight ^b	1b	-	381.5	<0.001
Larval morphological defences ^b
Integument resistance	2a	-	385.5	<0.001
Integument thickness	2b	-	227.5	0.442
Immune defences of naïve (unchallenged) larvae ^a
PO activity	3a	18	−2.94	0.009
Total-PO activity	3b	18	−2.74	0.014
Immune defences of challenged larvae ^a
PO activity	3a	18	−2.28	0.035
Total-PO activity	3b	18	−2.82	0.011
Melanisation score	4	18	1.27	0.222
Si concentrations in plants ^a
Leaf Si	5	38	−4.85	<0.001
Root Si	5	38	−0.06	0.949

^aAnalysed using a Student’s t-test.

^bAnalysed using a Wilcoxon’s rank-sum test.

Table 2 Summary output of two-way ANOVA tests for the effects of Si and insect herbivory on leaf trichome density and plant biomass. Statistically significant effects (p < 0.05) are indicated in bold

Response variable	Fig.	df	Si		Herbivore		Si × Herbivore
Response variable	Fig.	df	F	p value	F	p value	F	p value
Abaxial trichome density	6a	1,76	7.49	0.008	5.08	0.027	9.69	0.003
Adaxial trichome density	6b	1,76	0.02	0.888	24.62	<0.001	8.11	0.006
Shoot biomass	7a	1,76	63.13	<0.001	5.00	0.028	13.42	<0.001
Root biomass	7b	1,76	0.001	0.982	0.95	0.333	0.01	0.908

Table 3 Summary output of multiple linear regression analyses for the effects of Si on larval integument resistance and hemolymph PO activity after controlling for the effect of larval weight gain or final weight as a covariate. Statistically significant effects (p < 0.05) are indicated in bold

Predictor	Integument resistance				PO activity
Predictor	β	t	df	p value	β	t	df	p value
(Regression model 1)
Larval weight gain	11.09	1.37	37	0.179	−0.0003	−0.70	37		0.491
Si	−188.77	−2.25	37	0.031	0.03	2.96	37		0.005
(Regression model 2)
Larval final weight	2.19	19.50	37	<0.001	−0.0003	−0.80	37		0.431
Si	−15.98	−0.87	37	0.393	0.03	3.13	37		0.003

SupplementaryinformationIslametal2021.docx

Silicon Fertilisation Affects Morphological and Immune Defences of an Insect Pest and Enhances Plant Compensatory Growth

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Plants and insect herbivores

Experimental design and treatments

Measurements of larval behavioural and morphological defences

Measurements of larval immune defences

Measurements of leaf trichome density, plant biomass, and Si concentrations

Statistical analyses

Results

Insect growth, feeding, and defensive traits

Leaf trichomes, plant compensatory growth, and silicon concentrations

Discussion

Silicon weakened larval morphological defences but did not affect defensive behaviours

Interactions between larval morphological and immune defences

Higher PO activity did not enhance melanisation

Post-attack plant defences

Conclusions

Declarations

References

Tables

Supplementary Files

Status:

Journal Publication

Version 1