Silica nanoparticles control a generalist pest, American serpentine leaf miner Liriomyza trifolii

: Insects quickly develop their resistance to conventional synthetic chemical insecticides. Silica nanoparticles (SiO 2 NPs) is a new promising approach not only in decreasing insect pest numbers but also for its safety regarding agricultural organisms such as plants and beneficial natural enemies. Here, we investigate the safety of SiO 2 NPs on the faba bean Vicia faba (Fabaceae) as a model plant for toxicity studies and its control efficiency against the American serpentine leafminer Liriomyza trifolii (Diptera: Agromyzidae) as a major devastating insect pest against a wide range of crops. The concentrations of 50, 100, 200 and 400 mg/L of SiO 2 NPs (spherical shape, 15.3 nm) as well as distilled water as the control were used to investigate the effect of the nanoparticles. Early stages of development of V. faba were evaluated. The same concentrations of SiO 2 NPs were applied by spraying on two weeks old bean seedlings to evaluate their toxic effect on the feeding, survival and body mass of L. trifolii . A qRT-PCR analysis was conducted to assess oxidative stress on the gene expression level of the major antioxidative enzymes, catalase and superoxide dismutase, in L. trifolii . SiO 2 NPs inhibited root development only at a high concentration and did not affect the germination percentage, germination time, shoot length and vigor index compared to the control. The survival rate of larvae was lower than the control at the highest concentration and the larval feeding velocity and pupal weight decreased at high concentrations. Gene expression of both enzymes at the pupal stage were not affected by SiO 2 NPs at any concentrations. This study suggests that care must be taken to utilize SiO 2 NPs at a suitable concentration in controlling pests, avoiding negative effects on plant growth.


Introduction
To date, synthetic insecticides are the main controlling strategies against insect pest. Despite their efficacy, these chemical substances generate some concerns principally insect resistance increased through successive generations. Also, the massive and random use of such strategies imports negative impacts to non-target organisms in addition to threats concerning human health.
The American serpentine leafminer Liriomyza trifolii is a highly polyphagous species and one of the most-damaging leaf miners found in tropical and subtropical areas. Liriomyza trifolii larvae consume the leaf mesophyll tissue and this interrupts the photosynthesis process in addition to spreading diseases leading to decrease in crops production including common bean Phaseolus vulgaris (Ibrahim 2008;Yıldırım et al. 2010) and faba bean Vicia faba (Aamer and Hegazi 2014; Bassiony et al. 2017).
Silicon dioxide nanoparticles (SiO2NPs) is the most popular among numerous types of nanomaterials (less than 100 nm in size) which has a great advantage in insuring definite interactions between molecules through the mesoporous structure which has ability of carrying specific functional groups (Kaziem et al. 2017). Thus, such small particles overcome the problem of developing insect resistance mechanisms to reduce the high-volume application of traditional chemical insecticides (Rani et al. 2014). This gives SiO2NPs additional advantage as a stable and environmentally safe insecticide due to its great versatility not only as an insecticide but also for improving the activity of other chemical insecticides. For human health, the World Health Organization (WHO) and the US Department of Agriculture guaranteed its safety (El-Naggar et al. 2020).
In addition to scarce reports on harmful or negative side effects on non-target organisms principally plants on early development stages (Yang et (Pavitra et al. 2018). This study may be the first report for SiO2NPs entomotoxic effect on leaf miners.
SiO2NPs might be a viable alternative to conventional pesticides according to their application, size, and concentration. Understanding of such effect requires studying of the mechanisms of SiO2NPs affecting the behavior, life history traits and gene expression of the target organisms. SiO2NPs are believed to interrupt insect physiological functions through respiratory blockage, cuticular damage, desiccation and midgut epithelium injuries (Caceres et al. 2019) in addition to altering enzymatic protection from oxidative stress.
Here we investigated the following to study the benefits of using SiO2NPs; • effect of SiO2NPs on V. faba seedling growth • effect of SiO2NPs on the feeding velocity, survival and body mass of the leafminer L. trifolii • expected genotoxicity SiO2NPs can induce on L. trifolii gene expression (oxidative stress genes).

Seed material
The seeds of faba bean V. faba L. were used to test nanoparticle effect on early development of plants. They were obtained from Canada by Kokusaipet food (Kobe, Japan). Common bean P. vulgaris L. was used to test nanoparticle effect on an insect pest feeding on the plant. The P. vulgaris seeds were obtained from Hokkaido, Japan by Nakahara Seed Co., Ltd. (Fukuoka, Japan).

Silicon dioxide nanoparticles
A white powder of silicon dioxide (SiO2) nanoparticles (NPs) [99.5% purity, 19.6 ± 5.8 nm (mean ± SD) in size and spherical in shape] were obtained from the US Research Nanomaterials, Inc. (Houston, Texas, USA). A stock solution (1000 mg/L) of SiO2NPs was prepared by dissolving the powder of SiO2NPs in distilled water, then sonicated for 30 min and centrifuged (2000 rpm, 25°C) for another 30 min to participate the non-dispersed agglomerated particles which was filtered using filter paper No. 2 (90 mm, Advantec, Japan). Then, the four different concentrations; 50, 100, 200 and 400 mg/L, of SiO2NPs were prepared for investigating toxic and genotoxic effects against V. faba M1 plants as well as an insect pest L. trifolii. The size and shape of SiO2NPs were inspected on a high-resolution transmission electron microscope (TEM) (JEM-2100, JEOL Ltd.) at an accelerating voltage of 200 kV.

Toxicity to V. faba germination and seedling growth
Dry and healthy seeds of V. faba were chosen randomly, sanitized using diluted sodium hypochlorite solution for three min, and washed three to four times by distilled water to be soaked in distilled water for 3 h. Then, the seeds were immersed in tested concentrations separately (60 seeds for each) for 24 h. After the treatment, seeds were thoroughly washed by distilled water to remove the residual amounts of SiO2NPs. The treated and control seeds were sown in six replicates for each treatment in a randomized complete block design (RCBD) to investigate germination. Ten seeds per replication were allowed to germinate and grow in a 15 cm diameter Petri dish (six dishes/treatment) lined with cotton moistened with distilled water. Seeds were observed for germination when the radicle was at least 3 mm length and the germinated seeds were counted every morning. Final germination percentage (G%) and mean germination time (MGT) were assessed to detect the effect of SiO2NPs on seed germination. These measures were derived as described by Ranal and Santana (2006) and Ranal et al. (2009) as follows: where is the number of germinated seeds on day i and N is the total number of seeds in each experimental treatment and where is the time in days from seeding to germination on day i. In order to evaluate seedling growth; on the fifth day, seeds of each replicate were removed to plastic pots filled by vermiculite and irrigated by water to allow them to germinate under laboratory conditions (25 ± 1 °C and 8L:16D). Seedling growth, in terms of shoot and root lengths, were measured after two weeks as the mean of five seedlings per replicate (six replicates/treatment). The values of these two criteria were summed up to calculate the seedling vigor index (SVI) that reflects a collective measure of yield. The value of SVI was calculated according to Dahindwal

Mass rearing
A culture of L. trifolii was maintained under a constant laboratory condition (25 ± 3 °C, 50 ± 10% RH and a 12L:12D photoperiod). Insects were reared inside a transparent cuboid cages (70 × 50 × 50 cm) opened on both sides by a window (20 × 20 cm) and the back side by two windows (50 × 20 cm) and all windows covered by mesh to insure a good ventilation. Two trays containing about 40 plants of common bean Phaseolus vulgaris L. (as a preferable host) was daily introduced. Thus, the reared insect can perform a continuous oviposition which will provide pupae to be used subsequently. The common bean was planted weekly under a long-day condition (a 16L:8D photoperiod, 21-27°C).

Biological parameters
Two weeks old healthy plants were chosen to be sprayed by one of the tested SiO2NPs concentrations (5 ml/plant) or distilled water (0 mg/L) as a control treatment. Plants were sprayed using an ordinary drizzle (drop size of 1.05 ± 0.13 mm) 15 cm away from plants to insure a full coverage of both surfaces of leaves (two leaves/plant). They were then left to dry at a room temperature inside a transparent cylindrical cage (70 cm height × 25 cm diameter) covered with a fine mesh at the top in a RCBD with six replications. After one hour, 48 h old males and females were released into the previously described cages (six for each treatment) to feed and lay eggs for 24 h. Then, the flies were removed, and laid eggs were allowed to complete their life cycle. To evaluate the toxic effect of SiO2NPs, the three biological parameters; larval survival rate, larval feeding velocity and pupal weight, were recorded: • Larval survival rate The number of total mines at early larval stage was counted every day for four days from the release of the adults. The number of pupae was counted every day for three days after the first pupa emerged. The larval survival rate was derived by dividing the number of pupae by number of mines at the early larval stage.
• Larval feeding velocity To detect the larval feeding activity, one mine was chosen randomly for each replicate and two digital pictures of the mine on the 2 nd and 3 rd days were captured to measure the length of both mines by dividing each mine (winding feeding tunnel, Ayabe et al. 2008) into short straight lines (taking into account the reference scale of each picture). Then, the larval feeding velocity was calculated as a measure of larval activity for tested material as follows: , where L1 and L2 refer to the lengths of the mine on the 2 nd and 3 rd days, respectively, and ∆T is the time between the two shots. One mine per replicate and six replicates per treatment were measured. •

Pupal weight
Five pupae (24 h old) per replicate were selected randomly and weighed by a microbalance (AT-20, Mettler Toledo, Switzerland) in six replications per treatment in a RCBD. Then, the pupae were kept in a freezer (−20 °C) for posterior genetic analysis.

Quantitative RT-PCR analysis on gene expression
The expression level of major antioxidative enzymes, catalase (CAT) and superoxide dismutase (SOD), in L. trifolii as a response to SiO2NPs treatments was examined using qRT-PCR. The P. vulgaris leaves were treated with four different concentrations of SiO2NPs and 48 h old pupae were analyzed. Individual pupa was homogenized in the homogenizer (BHA-6, As One) with Isogen II (Nippon Gene) and zirconia beads (3,000 rpm, 1 min). Total RNA was extracted from the homogenate and used as a template for RT-PCR. The cDNAs were synthesized using SuperScript IV VILO Master Mix (Thermo Fisher Scientific) according to the manufacturer's instruction. The target genes were amplified using recombinant Taq DNA polymerase and the tracer EvaGreen (Biotium) with the gene-specific primer sets; forward (F) and reverse (R): catalase (CG6871); F: 5´-GATGCGGCTTCCAATCAGTTG-3´ and R: 5´-GCAGCAGGATAGGTCCTCG-3´, and superoxide dismutase 2 (CG8905); F: 5´-AAGTCGGGCAAACTGCAACT-3´ and R: 5´-GGACGCACGTTCTTGTACTG-3´ obtained from FlyPrimerBank (Hu et al. 2013). As a reference gene, β-actin was chosen and amplified with the following primer set; F: 5´-TTGTATTGGACTCTGGTGACGG-3´ and R: 5´-GATAGCGTGAGGCAAAGCATAA-3´ (Chang et al. 2017). Negative controls containing water instead of cDNA template were included for each primer set. The amplification plots were analyzed in StepOnePlus real-time PCR system (Applied Biosystems), with the following cycling conditions: 94 °C for 3 min followed by 40 cycles consisting of 94 °C for 10 s, 60 °C for 15 s, and 72 °C for 15 s. Fluorescence readings were taken at the end of each cycle. The melting curve protocol contained 1 cycle at 95 °C for 15 s and 60 °C for 1 min. The temperature was increased from 60 °C to 95 °C at a rate of 0.3 °C s −1 . The gene expression level was normalized by dividing by a mean gene expression level of the control. Three pupae were analyzed per treatment.

Statistical analysis
The following tests were applied to test the effect of different concentrations of SiO2NPs on the plant and the insect. Data of plant parameters (G%, SVI as well as root and shoot lengths) and insect biological parameters (larval feeding velocity and pupal weight) were analyzed with ANOVAs after confirming the normality of data distributions. A posthoc Dunnett test was performed for multiple comparisons with the control. MGT was analyzed with a parametric survival test with the best fit Frechet distribution. Larval survival rate was analyzed with a logistic regression model with a logit link function, followed by a posthoc pairwise comparison with a Bonferroni correction. Kruskal-Wallis tests were performed on relative gene expression. All statistical tests were performed using JMP13.2.1. The results were presented as mean ± SE.

Larval survival rate
The larval survival rate was affected by applying SiO2NPs (likelihood ratio χ 2 4 = 25.25, P < 0.0001); it was lower than the control at 400 mg/L (Figure 2).

Pupal weight
The pupal weight (as a mean of five late pupae) showed significant difference among SiO2NPs concentrations (F4,25 = 8.38, P = 0.0002, Figure 4) and was lower than the control at 50,200 and 400 mg/L, partly reflecting LFV results; the higher feeding velocity the more pupal weight as feeding can affect the development of the later stages of insect life cycle.  .  Table A1. Effects of SiO2NPs on plant growth. NS, non-significant effect; +, higher than control; −, lower than control; @, at a concentration and/or a particle size; max, maximum; min, minimum.