Silica and Selenium Nanoparticles Attract or Repel Scale Insects by Altering Physicochemical Leaf Traits

Although nanoparticles have gained attention as efficient alternatives to conventional agricultural chemicals, there is limited knowledge regarding their effects on herbivorous insect behavior and plant physicochemistry. Here, we investigated the effects of foliar applications of nano-silica (SiO2NPs) and nano-selenium (SeNPs), and bulk-size silica (SiO2) on the choice behavior of the arrowhead scale insect on mandarin orange plants. One leaf of a bifoliate pair was treated with one of the three chemicals, while the other was treated with water (control). The respective SiO2, SeO2, calcium (Ca), and carbon (C) content levels in the leaf epidermis and mesophyll were quantified using SEM–EDX (or SEM–EDS); leaf toughness and the arrowhead scale density and body size were measured. First-instar nymphs preferred silica-treated leaves and avoided SeNP-treated leaves. SiO2 content did not differ between control and SiO2NP-treated leaves, but was higher in bulk-size SiO2-treated leaves. The SiO2 level in the control leaves was higher in the SiO2NP treatment compared with that in the control leaves in the bulk-size SiO2 treatment. Silica-treated leaves increased in toughness, but SeNP-treated leaves did not; leaf toughness increased with mesophyllic SiO2 content. The insect density per leaf increased with leaf toughness, SiO2 content and, in the SiO2NP treatment, with epidermal C content. There was no correlation between SeO2 content and insect density. This study highlights the potential uses of SeNPs as an insect deterrent and of silica for enhancing leaf toughness and attracting scale insects.


Introduction
Citrus, recognized globally as a key fruit crop, offers various health benefits due to its richness in nutrients that reduce the risk of cardiovascular and liver deficiencies and cancers [1,2].Efficient and sustainable agriculture, characterized by a reduced reliance on chemical fertilizers, has prompted the exploration of nanofertilizers as promising alternatives for enhancing crop production.In recent decades, nanotechnology has emerged as a highly promising and progressive field, with numerous applications in applied science and technology [3]; nanoparticles (NPs) possess unique characteristics owing to their high surface reactivity and large surface area relative to volume [3].
After dispersing as crawlers (first-instar nymphs) from maternal scales and settling on nearby leaves, female arrowhead scale insects (Unaspis yanonensis) become sessile and remain in this location for the remainder of their life, including development and reproduction.The primary host plant for U. yanonensis is the Satsuma mandarin orange, Citrus unshiu, which is cultivated extensively in the southwestern part of Japan as well as in China, USA, Spain, Turkey, Croatia, South Korea, and Peru [4].The arrowhead scale typically goes through two to three generations each year in Japan [5].

Body Size of the Arrowhead Scale
The body size of female arrowhead scales was significantly reduced under th treatment (volume: p = 0.026, length: p = 0.048, width: p = 0.009; Table 2, Figure 2a) cating a negative effect on the development of the insects.By contrast, there were n cernible differences in scale size across all treatments (Table 2, Figure 2b).

Body Size of the Arrowhead Scale
The body size of female arrowhead scales was significantly reduced under the SiO 2 treatment (volume: p = 0.026, length: p = 0.048, width: p = 0.009; Table 2, Figure 2a), indicating a negative effect on the development of the insects.By contrast, there were no discernible differences in scale size across all treatments (Table 2, Figure 2b).

Leaf Toughness
Both SiO2 and SiO2NPs increased the toughness of the leaf, compared with the paired water-treated leaf (p < 0.001, p < 0.001, Table 3, Figure 3).By contrast, SeNPs did not affect the toughness of the leaf (p = 0.221, Figure 3).

Leaf Toughness
Both SiO 2 and SiO 2 NPs increased the toughness of the leaf, compared with the paired water-treated leaf (p < 0.001, p < 0.001, Table 3, Figure 3).By contrast, SeNPs did not affect the toughness of the leaf (p = 0.221, Figure 3).

Leaf Chemical Contents
In the case of SiO2 and SiO2NP treatments, there was a three-way interaction among the leaf tissue, treatment, and "control or treated leaf" factors (p = 0.016, Table 4, Figure 4): SiO2 content was higher in the mesophyll than in the epidermis (both adaxial and abaxial) in the SiO2 treatment (p < 0.001, Table 4) and in the treated leaves (p < 0.001, Table 4, Figure 4).When only water-treated leaves were compared, the levels of SiO2 content were different between the SiO2 and the SiO2NP treatments (F = 12.65, df1 = 1, df2 = 418, p < 0.001, Figure 4).In the case of SeNP treatment, SeO2 content was higher in treated leaves than in control leaves (p < 0.001, Table 5, Figure 5), with no difference observed between the epidermis and mesophyll.

Leaf Chemical Contents
In the case of SiO 2 and SiO 2 NP treatments, there was a three-way interaction among the leaf tissue, treatment, and "control or treated leaf" factors (p = 0.016, Table 4, Figure 4): SiO 2 content was higher in the mesophyll than in the epidermis (both adaxial and abaxial) in the SiO 2 treatment (p < 0.001, Table 4) and in the treated leaves (p < 0.001, Table 4, Figure 4).When only water-treated leaves were compared, the levels of SiO 2 content were different between the SiO 2 and the SiO 2 NP treatments (F = 12.65, df1 = 1, df2 = 418, p < 0.001, Figure 4).In the case of SeNP treatment, SeO 2 content was higher in treated leaves than in control leaves (p < 0.001, Table 5, Figure 5), with no difference observed between the epidermis and mesophyll.The cross-sectional images of leaves treated with SiO2 and SiO2NPs showed more densely and uniformly arranged mesophyll structure (Figure 6b,d) compared with watertreated leaves (Figure 6a,c).Conversely, there was no apparent difference in the leaf tissue structure between water-treated leaves and SeNP-treated leaves (Figure 6e,f).The cross-sectional images of leaves treated with SiO2 and SiO2NPs showed more densely and uniformly arranged mesophyll structure (Figure 6b,d) compared with watertreated leaves (Figure 6a,c).Conversely, there was no apparent difference in the leaf tissue structure between water-treated leaves and SeNP-treated leaves (Figure 6e,f).
In the SeNP treatment, no significant correlations were found between insect and plant traits (Table 7).
In the SeNP treatment, no significant correlations were found between insect and plant traits (Table 7).
Additionally, strongly negative correlations were consistently found between C and Ca content in both leaf tissues in all treatments (Table A1, Figure A2a, epidermis, ρ = −0.798,−0.826, and −0.769; p < 0.001, < 0.001, and < 0.001 for treatments with SiO2, SiO2NPs, Overall, in both silica treatments, increases in leaf SiO 2 content were associated with an increase in toughness and an increase in arrowhead scale density. In the SeNP treatment, no significant correlations were found between insect and plant traits (Table 7).

Discussion
We tested the hypotheses that SiO 2 and Se applied to C. unshiu will (1) affect the choice behavior of the arrowhead scale, U. yanonensis, and (2) increase leaf toughness, affecting scale density and body size.Scale insect nymphs were attracted to leaves treated with SiO 2 and SiO 2 NPs but avoided leaves treated with SeNPs.SiO 2 content did not differ between control (water-treated leaves) and SiO 2 NP-treated leaves but was higher in bulksize SiO2-treated leaves compared with water-treated leaves.SiO 2 and SiO 2 NPs increased the toughness of leaves, while SeNPs did not affect the toughness.There were positive correlations between leaf toughness and mesophyll SiO 2 content as well as between leaf toughness and insect density per leaf in both silica treatments.In the epidermis of leaves treated with SiO 2 NPs, increased C content-rather than SiO 2 content-was associated with increased leaf toughness.

Bulk SiO 2 and SiO 2 NPs
Irrespective of particle size, SiO 2 plays a crucial role in enhancing the toughness of plant tissues [26][27][28][29].This is also consistent with the results obtained in this study.Intriguingly, the application of lower concentrations of nano-silica was more efficient in influencing plants compared with its bulk counterpart.This result has also been demonstrated in cucumber, for which SiO 2 NP treatments increase hardness compared with an equivalent concentration (250 mg/L) of potassium silicate (K 2 SiO 3 ) [30].These findings extend to citrus plants, highlighting the importance of nano-silica in plant physiology.
While arrowhead scales exhibited a distinct preference for leaves treated with SiO 2 and SiO 2 NPs, the precise mechanism behind this attraction is unknown.It is plausible that the scales are drawn to treated leaves based on the emitted odors-a theory supported by earlier studies [17,27].Alternatively, the increased leaf toughness resulting from increased SiO 2 content may be a determining factor in the preference of arrowhead scale nymphs.This aligns with the observed feeding and oviposition preferences of other sucking hemipteran insects, such as whiteflies, which prefer thick leaves with compact vascular bundles [31].Since thick leaves with compact vascular bundles make them tougher [32], the density of whiteflies may increase with leaf toughness.The positive correlation between scale insect density and leaf toughness challenges conventional expectations of plant resistance against insect herbivores.While the expected negative relationship holds true for chewing insects, this positive correlation is a general trend in sucking insects with piercing-sucking mouth parts like whiteflies [32].For sessile sucking insects such as the arrowhead scale, this preference might suggest that tougher leaves provide a more secure anchoring site, thereby supporting insect survival.Although there was a strong positive correlation between leaf toughness and insect density, we could not determine whether it was tougher leaves that attracted the arrowhead scales or if the increased toughness was a result of insect feeding.
In the SiO 2 treatment but not in the SiO 2 NP treatment, insect body volume was reduced.Similarly, reduction (although non-significant) in dry body mass and body surface area was found in the sucking insect-the rice stalk stink bug, Tibraca limativentris-feeding on the rice treated with 1% potassium silicate solution (20 mL per pot), which seems to be due to a higher Si content in the rice [33].In our case, however, there was no correlation between leaf tissue Si content and scale body size, indicating that Si content in the plant is not responsible for the body size reduction.We hypothesize that more SiO 2 particles attached to the body surface as nymphs walked on the leaves treated with the high concentration of bulk SiO 2 , leading to a smaller size through physical dehydration.
Intraspecific competition is also not a causal factor of reduced body size because there was no negative correlation between scale density and body volume (Table 6).
We observed a smaller difference in silica content between nano-treated and watertreated leaves compared with the difference between bulk-size silica and water-treated leaves.This discrepancy may be attributed to the smaller particle size of nano-silica, potentially enhancing its mobility within the plant [34].The active or passive translocation mechanism responsible for this phenomenon remains a subject for future exploration.

SeNPs
This study demonstrated a strong repellent effect of Se or Se-treated plant against the arrowhead scale.This is consistent with the results of previous studies on other insects (e.g., the beet armyworm, Spodoptera exigua; the cabbage looper, Trichoplusia ni; the cabbage white, Pieris rapae; and the house cricket, Acheta domestica) [7].
Several studies have substantiated that Se can have repellent and toxic effects on various phytophagous insects and that, at the same time, both organic and inorganic Se compounds can exert toxic effects on insects to varying degrees [35].Previous studies consistently demonstrate that Se serves as an effective repellent for herbivorous insects, negatively impacting the feeding behavior of specific species [36][37][38].Crickets prefer to feed on leaves with low Se content [39].Similarly, a choice experiment showed that P. rapae larvae strongly preferred Se-absent leaves, exhibiting higher feeding rates compared with those of Se-present leaves [40].Laboratory studies showed that an Se-enriched diet acts as antifeedant for S. exigua larvae and influences their choice of plants and feeding site [41,42].At the same time, however, Se exerts a more pronounced negative impact on the natural enemies of herbivorous insects than on the herbivores themselves, which could be attributed to a less protected body morphology [43] or the biological transfer of Se from their herbivorous hosts [44].In recent studies, the application of specific concentrations of Se significantly influenced plant growth (Citrus reticulata at 150 mg/L [45]) and mitigated insect pest damage to a plant (Atractylodes macrocephala (Asteraceae) [46]).However, low concentrations of SeNPs (10-500 mg/L) can increase survival of pest insects (e.g., the azuki bean beetle, Callosobruchus chinensis, and the cowpea beetle, C. maculatus) [43,47,48], whereas higher concentrations of SeNPs or Se can inhibit the development and/or survival of both pest insects and their natural enemies (at 500-1000 mg/L, C. chinensis, C. maculatus, and the parasitoid-Anisopteromalus calandrae) [43,47,48] as well as plants such as A. macrocephala [46] and Citrus reticulata (at 200 mg/L) [45].Therefore, the application of Se within a reasonable stoichiometric range emerges as a crucial consideration for future research.
For example, smaller doses of SeNPs might be effectively used instead of selenium, resulting in a more positive influence on agricultural crops, attributed to the presumed biosafety and bioactivity of SeNPs [49].

Calcium (Ca) and Carbon (C)
Both scale density and leaf toughness increased with increasing epidermal C content in the SiO 2 NP treatment, whereas leaf toughness did not increase with increasing epidermal SiO 2 .There are experimental results that show that C or both Ca and C enhance leaf toughness [50][51][52][53].There is a negative correlation between the concentrations of Si and C in the aboveground tissues of grasses [50].Si enhances the accumulation of C in grasses [54].Si alone has been shown to be accumulated in the epidermis of the adaxial side of the citrus leaf, as a form of Si granules [55].Our SEM observation indicates a morphological change in the adaxial side of the mesophyll structure.In our study, Si and C content were independent of each other in the SiO 2 NP treatment.On the contrary, in this study, there was a significant negative correlation between mesophyllic C and epidermal SiO 2 in the SiO 2 treatment and a marginally significant negative correlation between mesophyllic Ca and SiO 2 in the SiO 2 NP treatment (Table A1).This might be partly due to a "dilution effect" in which an increase in C or Ca inevitably leads to a relative decline in SiO 2 [56].Therefore, Si and C may contribute in different ways (functional vs. structural) to increasing leaf structural toughness in C. unshiu.The relationship between Si and C needs further investigation.
In addition, the C and Ca content in leaves were negatively correlated in both the epidermis and mesophyll (Figure A2a,b).This is consistent with the findings in other woody plants [51].A wide range of insects tend to reject various forms of calcium (Ca) compounds present in crops, but insects with piercing-sucking mouthparts are less affected [57][58][59].Our present result is in line with this general trend.
This study marks a pioneering effort in comparing the impact of different particle sizes of silica on both a host plant and a pest insect.This is the first to show that silica-treated plants attract not only predators or parasitoids but also herbivores.Silica-treated plants might be used as a lure to trap scale nymphs.

Plant and Insect
The Satsuma mandarin orange, Citrus unshiu (Rutaceae), used in this study was the early ripening variety, Miyagawa-wase, which was cultivated and grown in a greenhouse.The environmental conditions were controlled at 25 ± 1 • C, 70% r.h., 450 ppm of carbon dioxide, and under natural sunlight.The potted soil was watered three times per week.
Twelve trees were planted in pots (volume: 12.8 L).The pots were filled with soil consisting of rice husk compost, coconut fiber, charcoal balls, perlite, effective microorganisms, and other components, with a pH range of 6.0-7.0.
To test preference by insects, a choice experiment was conducted as follows: Citrus leaves with female adults of the arrowhead scale, Unaspis yanonensis (Diaspididae), were collected from citrus trees in orchards located in Fukuoka Prefecture on 30 August 2022.

Reagents
We used bulk-size SiO 2 , SiO 2 NPs, and SeNPs, as well as distilled water as a control group.Each of the solutions was sonicated.The morphology of these particles was examined using a scanning electron microscope (SEM) (JSM-IT700HR, JEOL Ltd., Tokyo, Japan), operating at an accelerating voltage of 15 kV, and a transmission electron microscopy (TEM) (JEM2100HC, JEOL, Tokyo, Japan), operating at an accelerating voltage of 200 kV.

SiO 2 and SiO 2 NPs
The bulk-size SiO 2 (porous silica gel; Sieweves Co., Ltd., Aichi, Japan) was prepared at 0.16 mol/L (9.61 g/L) with distilled water.This preparation forms silicic acid Si(OH) 4 , which is water soluble upon contact with water.The SiO 2 used in the experiment had a particle size of 32.8 ± 8.7 µm (mean ± SE, n = 25, range: 3-93 µm), which was estimated from a SEM image.
SiO 2 NPs (US Research Nanomaterials, Inc., Houston, TX, USA) were prepared at 0.0016 mol/L (96.1 mg/L), which is 1 /100th of the concentration of the bulk SiO 2 .The SiO 2 NPs used in the experiment had a particle size of 13.0 ± 0.8 nm (mean ± SE, n = 15, range: 10-19 nm), which was estimated from a TEM image.

SeNPs
SeNPs were synthesized at room temperature through the reduction of sodium selenite (Na 2 SeO 3 ) with ascorbic acid (C 6 H 8 O 6 ), utilizing polysorbate 20 as a stabilizing agent [59].The SeNPs were stored at 4 • C and used within two months of synthesis.The SeNPs had a particle size of 48.3 ± 5.5 nm (mean ± SE, n = 13, range: 23-95 nm), which was estimated from a TEM image.The concentration of the SeNPs was adjusted to 0.0016 mol/L (126 mg/L), which is consistent with the concentration of the SiO 2 NPs.

Experiments Using Bifoliate Leaves
To control factors such as the morphology, physiology, and genetics of the leaves in our experiments, we used bifoliate new leaves (grown in 2022) for pairwise comparisons between water-treated control leaves and chemically treated leaves.SiO 2 , SiO 2 NPs, or SeNPs were applied as follows: We chose to use new leaves (current-year leaves) located in the upper canopy to ensure an even exposure of treated leaves to sunlight.We sprayed both adaxial and abaxial surfaces of one of the bifoliate leaves once with one of the solutions (0.74 ± 0.04 mL, mean ± SD, n = 5), totaling approximately 1.48 mL per leaf.The other leaves were sprayed likewise with distilled water.The treatment was conducted only once at the beginning of the experiment.Four bifoliates (i.e., eight leaves) per tree and three trees per treatment were used; hence, each treatment-control combination was replicated 12 times.
Plants 2024, 13, 952 13 of 18 4.3.1.Choice Experiment with the Arrowhead Scales On 30 August 2022, after the leaves has been sprayed, one leaf infested with one female adult scale collected from the orchard was placed at the point where the two leaves of a bifoliate branched, to allow the first-instar nymphs to choose between the two leaves.Forty-one days after the first appearance of the first-instar nymphs, the total number of arrowhead scales was recorded, followed by toughness measurements and EDX analyses.

Body Size of the Arrowhead Scale
We collected arrowhead scales from the choice experiments and calculated the body volume of adult females as well as the surface area of the scales to determine the effects of the different materials on insect development.We measured the length and width of bodies and scales to the precision of 0.001 mm with a microscope (VH-5500, Keyence, Osaka, Japan) for this purpose.Given that the bodies and scales of the arrowhead scales are approximately oval, we used Yanagi and Tuda's [60] formula for calculating volume: V = πLW 2 /12, which is half of an ellipsoid, where L is the main axis (i.e., length) and W is the minor axis (width) of the body or scale.The area of the scale was estimated using the formula S = πLW/4.

Leaf Toughness
The toughness (in Newtons, N) of 14 leaves from each treatment was measured using a rheometer (Compac-100, Sun Scientific Co., Tokyo, Japan) at a stress rate of 60 mm/min, at three different points.The mean toughness of the three points for each leaf was used in later statistical analysis.Measurement of leaf toughness was conducted 104 days after spraying.

Leaf Chemical Content
We obtained cross sections of leaves from the choice experiments using a razor blade, which was cleaned with ethanol before and after each use.Samples were fixed on an aluminum SEM mount covered with conductive carbon adhesive tape.The elemental composition of the samples was analyzed using a scanning electron microscope (SEM) (JSM-IT700HR) with an energy-dispersive X-ray spectrometer (EDX) (JED-2300 Analysis Station Plus, JEOL, Tokyo, Japan) at a low vacuum (30 Pa), 15 kV accelerating voltage, and 500× magnification.We measured three points within the epidermis of both the adaxial and abaxial surfaces and four points within the mesophyll.The SEM-EDX analysis was conducted on the same day as the toughness measurements.

Statistical Analyses
The number of arrowhead scales and the leaf toughness between bifoliate leaves were compared using paired t-tests for each treatment.The SiO 2 or SeO 2 content (mass %, mean per tissue per leaf) in leaves treated with SiO 2 , SiO 2 NPs, and SeNPs were arcsine square-root transformed and then analyzed using a general linear model; treatment (only for the two silica), leaf tissue, control or treated leaf, tree ID (nested within treatment), and leaf pair ID (nested within tree ID and treatment) were used as explanatory variables.Furthermore, SiO 2 content in water-treated leaves with their paired leaves treated with SiO 2 or SiO 2 NPs were compared between SiO 2 and SiO 2 NP treatments, using a subset of the general linear model.Multivariate correlations among scale density, body volume and scale area (both mean per leaf), leaf toughness (mean per leaf), and the content (mass %, mean per tissue per leaf) of treated elements (SiO 2 or SeO 2 ), C, and Ca in leaf epidermis and mesophyll were tested using nonparametric Spearman correlations.All statistical analyses were performed using JMP, version 13.0.

Figure 1 .
Figure 1.Proportion of arrowhead scales choosing a treated leaf over a paired control leaf t with water.Paired t-test results of the number of scales on each leaf of the leaf pairs are sho the right.***: p < 0.001.

Figure 1 .
Figure 1.Proportion of arrowhead scales choosing a treated leaf over a paired control leaf treated with water.Paired t-test results of the number of scales on each leaf of the leaf pairs are shown on the right.***: p < 0.001.

Figure 2 .
Figure 2. The effects of different treatments (SiO2, SiO2NPs, or SeNPs) on the size of the female arrowhead scales (mean ± SE).Shared letters above the bars indicate no significant differences.(a) Body volume (estimated), length, and width.(b) Scale area (estimated), length, and width.

Figure 2 .
Figure 2. The effects of different treatments (SiO 2 , SiO 2 NPs, or SeNPs) on the size of the female arrowhead scales (mean ± SE).Shared letters above the bars indicate no significant differences.(a) Body volume (estimated), length, and width.(b) Scale area (estimated), length, and width.

Figure 3 .
Figure 3. Difference in toughness between the treated leaf and the control leaf in the SiO2, SiO2NP, and SeNP treatments.***: p < 0.001 in paired t-tests.

Figure 3 .
Figure 3. Difference in toughness between the treated leaf and the control leaf in the SiO 2 , SiO 2 NP, and SeNP treatments.***: p < 0.001 in paired t-tests.

Figure 4 .
Figure 4. SiO2 content (mass %) in different leaf tissues (mean ± SE) of leaf pairs, where one leaf of the pairs was treated with SiO2 or SiO2NPs and the other leaf was treated with water.Shared letters above the bars indicate no significant differences.

Figure 5 .
Figure 5. SeO2 content (mass %, mean ± SE) of leaf pairs, where one leaf of the pairs was treated with SeNPs and the other leaf was treated with water.***: p < 0.001.

Figure 4 .
Figure 4. SiO 2 content (mass %) in different leaf tissues (mean ± SE) of leaf pairs, where one leaf of the pairs was treated with SiO 2 or SiO 2 NPs and the other leaf was treated with water.Shared letters above the bars indicate no significant differences.

Figure 4 .
Figure 4. SiO2 content (mass %) in different leaf tissues (mean ± SE) of leaf pairs, where one leaf of the pairs was treated with SiO2 or SiO2NPs and the other leaf was treated with water.Shared letters above the bars indicate no significant differences.

Figure 5 .
Figure 5. SeO2 content (mass %, mean ± SE) of leaf pairs, where one leaf of the pairs was treated with SeNPs and the other leaf was treated with water.***: p < 0.001.

Figure 5 .
Figure 5. SeO 2 content (mass %, mean ± SE) of leaf pairs, where one leaf of the pairs was treated with SeNPs and the other leaf was treated with water.***: p < 0.001.The cross-sectional images of leaves treated with SiO 2 and SiO 2 NPs showed more densely and uniformly arranged mesophyll structure (Figure6b,d) compared with watertreated leaves (Figure6a,c).Conversely, there was no apparent difference in the leaf tissue structure between water-treated leaves and SeNP-treated leaves (Figure6e,f).

Funding:
MT was supported by KAKENHI (19K06840) from JSPS (Japan Society for the Promotion of Science) and by the Initiative for Realizing Diversity in the Research Environment from MEXT (Ministry of Education, Science and Technology) to Kyushu University.

Figure A1 .
Figure A1.Correlations (a) between scale density and epidermal C content in the SiO 2 (p = 0.575) and SiO 2 NP (p = 0.005) treatments and (b) between leaf toughness and epidermal C content in the SiO 2 (p = 0.192) and SiO 2 NP (p = 0.049) treatments.

Figure A2 .
Figure A2.(a) Correlation between epidermal Ca and C content in all treatments.(b) Correlation between mesophyllic Ca and C content in all treatments.

Table 1 .
Paired t-test results on the number of arrowhead scales that chose either the control or the treated bifoliate leaf when one of the bifoliate leaves was treated with SiO 2 , SiO 2 NPs, or SeNPs and the other treated with water.
Plants 2024, 13, x FOR PEER REVIEW

Table 1 .
Paired t-test results on the number of arrowhead scales that chose either the control treated bifoliate leaf when one of the bifoliate leaves was treated with SiO2, SiO2NPs, or SeN the other treated with water.

Table 2 .
General linear model results of different treatments (SiO2, SiO2NPs, or SeNPs) on body volume and scale area of the arrowhead scale, Unaspis yanonensis.

Table 2 .
General linear model results of different treatments (SiO 2 , SiO 2 NPs, or SeNPs) on female body volume and scale area of the arrowhead scale, Unaspis yanonensis.

Table 3 .
Paired t-tests comparing the leaf toughness of paired leaves that were sprayed with either water or a chemical solution.

Table 3 .
Paired t-tests comparing the leaf toughness of paired leaves that were sprayed with either water or a chemical solution.

Table 4 .
General linear model analysis on the SiO2 concentration (mass %) in different leaf tissues (epidermis or mesophyll) after the foliar spray of SiO2 or SiO2NPs.Tree ID was nested within treatment, and leaf ID was nested within tree ID and treatment.

Table 5 .
General linear model analysis on the SeO2 content (mass %) in different leaf tissues (epidermis or mesophyll) after foliar spray of SeNPs.Leaf ID was nested within tree ID.

Table 4 .
General linear model analysis on the SiO 2 concentration (mass %) in different leaf tissues (epidermis or mesophyll) after the foliar spray of SiO 2 or SiO 2 NPs.Tree ID was nested within treatment, and leaf ID was nested within tree ID and treatment.

Table 5 .
General linear model analysis on the SeO 2 content (mass %) in different leaf tissues (epidermis or mesophyll) after foliar spray of SeNPs.Leaf ID was nested within tree ID.

Table 6 .
Multivariate correlation analysis on the leafwise parameters: SiO 2 content (mass %) in different leaf tissues (epidermis or mesophyll); scale body volume and density; and leaf toughness after the foliar spray of SiO 2 or SiO 2 NPs.Italic: 0.01 < p < 0.05, bold & italic: 0.001 < p < 0.01, and bold: p < 0.001.The number of pairs of samples is shown in parentheses.

Table 7 .
Multivariate correlation analysis on the leafwise parameters: SeO 2 content (mass %) in different leaf tissues (epidermis or mesophyll); scale body volume and density; and leaf toughness after foliar spray of SeNPs.Bold & italic: 0.001 < p < 0.01.The number of pairs of samples is shown in parentheses.

Table A2 .
Multivariate correlation analysis on the leafwise parameters: Ca and C content (mass %) in different leaf tissues (epidermis or mesophyll), scale insect body volume and scale area, insect density, and leaf toughness after foliar spray of SeNPs.Italic: 0.01 < p < 0.05, and bold: p < 0.001.The number of samples is shown in parentheses.