Green synthesis of AgNP–ligand complexes and their toxicological effects on Nilaparvata lugens

Background Despite developments in nanotechnology for use in the pharmaceutical field, there is still a need for implementation of this technology in agrochemistry. In this study, silver nanoparticles (AgNPs) were successfully prepared by a facile and an eco-friendly route using two different ligands, 2ʹ-amino-1,1ʹ:4ʹ,1″-terphenyl-3,3″,5,5″-tetracarboxylic acid (H4L) and 1,3,6,8-tetrakis (p-benzoic acid)-pyrene (TBAPy), as reducing agents. The physiochemical properties of the as-obtained AgNPs were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), X-ray diffraction (XRD) and transmission electron microscopy (TEM). The toxicity of H4L–AgNP and TBAPy–AgNP against the brown planthopper (BPH, Nilaparvata lugens) was also measured. Results SEM and TEM analyses demonstrated the formation of quasi-spherical AgNP structures in the presence of H4L and TBAPy. Insecticidal assays showed that TBAPy is less effective against N. lugens, with a median lethal concentration (LC50) of 810 mg/L, while the toxicity of H4L increased and their LC50 reached 786 mg/L 168 h posttreatment at a high concentration of 2000 mg/L. H4L–AgNPs were also highly toxic at a low concentration of 20 mg/L, with LC50 =  ~ 3.9 mg/L 168 h posttreatment, while TBAPy–AgNPs exhibited less toxicity at the same concentration, with LC50 =  ~ 4.6 mg/L. Conclusions These results suggest that the synthesized AgNPs using the two ligands may be a safe and cheaper method compared with chemical insecticides for protection of rice plants from pests and has potential as an effective insecticide in the N. lugens pest management program. Supplementary Information The online version contains supplementary material available at 10.1186/s12951-021-01068-z.

low risk on the environment [7,8]. In particular, AgNPs have attracted great attention in the field of biology because of their exceptional and tunable surface plasmon resonance (SPR) [9,10]. Therefore, AgNPs have promising potential in bioscience, originating from their low ecotoxicological characterization [11,12].
The brown planthopper (BPH) Nilaparvata lugens (Stål; Homoptera: Delphacidae) is an economically significant insect pest of rice plants in Asia [13]. BPH is distributed throughout many countries, such as Australia, China, Bangladesh, Cambodia, Fiji, India, Indonesia, and Japan. BPH causes damage to rice plants directly through feeding, and the plants turn yellow and rapidly dry up; this condition is called "hopper burn. " In addition, BPH causes extensive harm to rice plants by transmitting two viruses, rice ragged stunt virus and rice grassy stunt virus [14]. Because of its high adaptive ability to changing social practices, high reproductive potential, and long-distance migrating capability, insecticides are essential and applied widely to control this monophagous insect throughout Asia [15]. However, the extensive use of insecticides to control this pest over many years has led to resistance to most of classes of chemical insecticides (organochlorines, organophosphates, carbamates, and pyrethroids). In addition, BPH has distinct biological and behavioral attributes, for example, short up-growth time, high fertility, and dispersal ability [16][17][18]. The increase in insects resistant to chemical insecticides and toxicity concerns have motivated researchers toward the development of new insecticides based on AgNPs [11,19,20]. The organic ligand 2'-amino-1,1ʹ:4ʹ,1″-terphenyl-3,3″,5,5″tetracarboxylic acid (H 4 L) was first reported by Bharadwaj et al. [21] to synthesize non-interpenetrated porous metal-organic frameworks (PMOFs) containing paddle-wheel secondary bonding units (SBUs). Pyrene and its derivatives have been widely investigated as fluorescence probes in many applications. For example, pyrene-labeled oligonucleotides have been used to study DNA hybridization [22,23], and pyrene-labeled lipids have been developed to examine the depth-dependent quenching of fluorescence in lipid bilayers [24][25][26].
Recently, pyrene was used as a ligand to synthesize a mesoporous MOF and a catalytically active iridium pincer complex [27,28].
Exploring an effective and versatile synthesis route to fabricate noble metal NPs without any toxic chemicals is still a challenge but desirable. In this study, we developed an efficient liquid-phase reduction method to produce a Ag colloidal suspension using two organic compounds in the presence of silver nitrate (AgNO 3 ) as a metallic precursor. Such an approach could guarantee suspended AgNPs through the solution without any additives and toxic substances. Transmission electron microscopy (TEM) micrographs confirmed the formation of an Ag 0 nanocluster in the H 4 L and 1,3,6,8-tetrakis (p-benzoic acid)-pyrene (TBAPy) ligands. The prepared H 4 L-AgNPs and TBAPy-AgNPs exhibited remarkable performance as insecticidal agents against BPH. Furthermore, the effect of treatment on enzyme levels in the insect system was explored and the action mechanism addressed as well. To the best of our knowledge, this is the first report on the synthesis, characterization, and insecticidal potential of AgNPs against BPH using the two organic ligands H 4 L and TBAPy.

Synthesis of AgNPs with H 4 L and TBAPy
AgNPs were prepared by direct reduction of the Ag source (i.e., AgNO 3 ) in the presence of H 4 L and TBAPy. AgNO 3 solution (1, 3, and 5 mM) was prepared in 150 mL of Milli-Q water and kept at 60 °C in a water bath for 10 min. Then, a defined amount of H 4 L or TBAPy dissolved in 2 mL of dimethyl sulfoxide was mixed in with vigorous magnetic stirring at 70 °C for 2 h before cooling down to room temperature. The reaction between ligands and Ag persisted under stirring in the dark at room temperature, and the formation of a Ag 0 colloidal suspension was observed by optical color change (a reddish-brown color with H 4 L and a yellowish-black color with TBAPy). Additionally, the reduction of Ag ions to AgNPs was confirmed by sampling an aliquot of the reaction solution (4 mL) and analyzing it with a UV-Vis spectrophotometer. The H 4 L-and TBAPy-AgNPs were centrifuged at 10,000 rpm, and the colloidal suspension was collected and maintained in a vacuum oven at 120 °C for 2 h before further characterization.

UV-Vis spectral analysis of AgNPs
The UV-Vis patterns of the AgNP samples were recorded using 1 mL of AgNPs diluted with 3 mL of double-distilled water on a UV-2550 spectrophotometer (Shimadzu, Japan) at a wavelength of 200-800 nm with a resolution of 1 nm [29].

FT-IR analysis of AgNPs
Spectra of the dry powder of the AgNP samples were recorded under identical conditions at 500-4000 cm −1 with an amplitude of 4 cm −1 using Fourier transform infrared spectrometer (FT-IR, Vector 22; Bruker Corporation, Germany). Before measurements, 1 mg of the AgNP sample was mixed with 300 mg of KBr, cast into circular pellets, and stabilized under reactive humidity.

EDX analysis of AgNPs
The surface of the as-synthesized AgNP samples was viewed by an energy-dispersive X-ray spectroscopy (EDX, SU-8010; Hitachi, Japan). A thin film of the prepared AgNP samples was deposited onto a well-cleaned silicon substrate and gently dried. Next, the samples were attached to a circular metallic stud by carbon tape and inserted into the vacuum chamber of the microscope using a cylindrical lever. Analyses were performed at an accelerating voltage of 3 kV.

TEM and XRD analyses of AgNPs
The morphologies and microstructures of the obtained H 4 L-and TBAPy-AgNPs were observed by TEM (JEOL-JEM-1230; JEOL, Japan) operated at an accelerating voltage of 200 kV. The samples were mixed with ethanol under gentle sonication to disperse the powder into the solution well. A drop of the suspension was pipetted onto a copper grid and then dried at room temperature. The phase purity and crystalline structure of the as-synthesized compounds were measured by wide-angle X-ray diffraction (XRD) using a Siemens X-ray diffractometer (Siemens, Germany). The XRD patterns were collected with a monochromated CuKα radiation source at a potential of 40 kV and a current of 30 mA [30].

3Dynamic light scattering (DLS) and Zeta potential
The average particle size and size distribution of AgNPs were evaluated using the particle size analyzer system (Zeta sizer, Malvern Instruments Ltd., USA), with the same device, the zeta potential of AgNPs was measured to determine the stability of nanoparticles.

Insect rearing
BPH adults were initially gathered from rice fields in Hangzhou, Zhejiang Province, China, and reared on a susceptible variety of rice (TN1) in net cages at 26 ± 2 °C in a 14:10 h light:day (L:D) cycle at 70% − 80% relative humidity in the Laboratory of Urban Entomology, Institute of Insect Sciences, Zhejiang University, China. All tested insects were preserved on the same rice variety. Both sexes of BPH were collected from the population (20 individuals/replicate) and used in the experiment.

Toxicity tests of two AgNP-ligand complexes against the brown planthopper
The standard methods to determine the lethal concentration of H 4 L-and TBAPy-AgNPs on BPH adults were followed. Before defining the final doses of compounds, a preliminary test was performed to determine the dosage range of an insecticide with different levels of mortality before conducting toxicity tests. The concentrations of all samples were 500, 1000, 1500, and 2000 mg/L prepared in a 100 mL beaker Borosil (Mumbai, India) using deionized water as a solvent for the two ligands without Ag (positive control). For toxicity tests, H 4 L-and TBAPy-AgNP colloids were diluted using double-distilled water as a solvent to the desired concentrations (5, 10, 15, and 20 mg/L). Dimethyl sulfoxide was used as a negative control. Next, 20 BPH adults per replicate were released on rice plants in plastic cups (500 mL) and covered with water-impregnated cotton. After spraying the prepared compounds, all cups were covered with muslin cloth held in place with rubber bands to prevent the insects from escaping during the experiment. Each toxicity test, including a set of the control group (distilled water), was tested with four replicates of four concentrations of each compound. The plastic cups with treated insects were put into an incubator at 26 ± 1 °C in a 14:10 h L:D photoperiod. Mortality was recorded at 24 h intervals up to 10 days after spraying. Moribund insects were also considered for determining mortality after gentle prodding with a fine brush.

Mechanisms of action of AgNPs on N. lugens Preparation of whole-body homogenates
The treatment and control BPH groups (15 adult individuals per group) were pooled and homogenized in Eppendorf tubes (held in crushed ice) using a Teflon hand homogenizer in 1 mL of 0.9% saline for ultimate estimation of total proteins, acetylcholine, esterases, and phosphatases action. The whole-body BPH homogenates were centrifuged at 12,000 × g and 4 °C for 10 min, and the clear supernatants were used for biochemical examination. The solution for homogenization and glassware were kept at 4 °C before use, and the homogenates were hung on ice before further investigation.

Determination of protein concentration
The proteins in BPH whole-body homogenates were first precipitated by 80% ethanol [31], and the protein concentration was estimated using the Lowry method [32].

Acetylcholinesterase assays
With slight modifications, acetylcholinesterase activity in the BPH whole-body homogenates was spectrophotometrically measured using acetylcholine iodide as a substrate [33]. Each aliquot of the homogenate (200 µL) was mixed successively with 200 µL of sodium phosphate buffer (100 mM, pH 7.5), 50 µL of 10 mM 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) and 50 µL of 12.5 mM acetylcholine iodide. After incubation for 5 min at room temperature, the optical density of the samples was tested at 400 nm against a suitable reagent blank.

Esterase assay
Carboxylesterase activity in the whole-body BPH homogenates was determined using the Van Asperen method [34]. Briefly, 200 μL of control and treatment BPH homogenates was mixed with 2 mL of the α-or β-naphthyl acetate solution, and the reaction was extended for 30 min at room temperature. After incubation, 500 μL of fast blue sodium dodecyl sulfate (SDS) reagent was added (22.5 mg OF fast blue salt in 2.25 mL OF distilled water and 5% w/v SDS in 0.2 M phosphate buffer; pH 7.2), and the color was allowed to develop for 15 min at room temperature. The optical density of the samples was measured at 588 nm using a spectrophotometer against the respective reagent blank.

Phosphatase assay
The levels of acid and alkaline phosphatases in the whole-body BPH homogenates were measured using the Asakura method with slight modifications [35]. Acid phosphatase activity was estimated by mixing 100 μL of insect homogenate with 400 μL of 50 mM sodium acetate buffer (pH 4.0) and 500 μL of 15 mM β-nitrophenyl phosphate. Alkaline phosphatase activity was estimated by mixing 100 μL of BPH homogenate with 400 μL of 50 mM Tris-HCl buffer (pH 8.0) and 500 μL of 15 mM p-nitrophenyl phosphate. After incubation for 15 min at 37 °C in a water bath, the enzymatic reaction was stopped by adding 100 μL of 0.5 N NaOH solution and centrifuged at 4000 × g for 5 min. Absorbance of the resulting clear supernatant from each sample was checked at 440 nm against the appropriate reagent blank.

Determination of AgNP content in the brown planthopper by TEM
The AgNP accumulation in the adult BPH body was evaluated by TEM [36]. Briefly, midgut extracts of AgNP treatment (LC 30 ) and control groups were collected, anesthetized, and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 48 h at 4 °C. The samples were washed several times with phosphate-buffered saline (PBS) for 15-20 min each time, then postfixed in 2% aqueous osmium tetroxide for 1-2 h, washed again with distilled water four to six times for 30-45 min each time, dehydrated in a series of alcohols, and embedded in spur resin. The samples were incubated at 80 °C overnight for complete polymerization. Ultrathin Sects. (50-70 nm) were cut with a glass knife on an ultramicrotome (LEICA EM UC7 ultratome) and then mounted on copper grids. The sections were stained with uranyl acetate and alkaline lead citrate for 5-10 min, and the samples were characterized with TEM (Model H-7650 TEM; Hitachi-Japan).

Histological analysis
Histological analysis of the BPH digestive system was performed using adult insects (treatment and control groups). Briefly, 20 BPH adults were exposed to a median lethal concentration (LC 50 ) of H 4 L, TBAPy, H 4 L-AgNPs, and TBAPy-AgNPs for 5 days, as previously described [37] with a small modification. Briefly, the insects were fixed in 10% buffered formaldehyde for 48 h, dehydrated using a graded series of ethanol, and cleared with xylene solution. Next, they were embedded in paraffin blocks using melted paraffin at the embedding station. The paraffin blocks were cut into 5-µm-thick sections using a rotary microtome and stained with hematoxylin and eosin. The glass slides were examined for abnormalities under a light microscope.

Data analysis
The mortality (%) of BPH treated with H 4 L-and TBAPy-AgNPs were subjected to probit analysis for calculating LC 50 and LC 90 with 95% confidence limits of lower and upper values [38]. For evaluating the biological variables observed in the experiments, one-way analysis of variance (ANOVA) and Duncan's test were performed to assess the variation between both treatments. Chi-square values and all analyses were analyzed using CoStat ver. 6.311 (CoHort Software, Monterey, USA), and SPSS Statistics ver. 16.0 (SPSS Inc., Chicago, IL, USA).

Synthesis of H 4 L and TBAPy
H 4 L and TBAPy are two organic ligands formed during the synthesis of the frameworks L (Cu) and NU-1000 [39,40]. However, this study is the first attempt to prepare a Ag colloidal suspension as an insecticide using these ligands. H 4 L was obtained from the chemical reaction of 3,5-dimethylphenylboronic acid to form benzene-3,5-dicarboxyelester-boronic acid [41], and the final product was obtained according as previously described with slight modifications [40]. The reaction by-products and compounds were checked by elemental analysis, such as 1 H NMR (Additional file 1: Scheme S1). TBAPy was fabricated via the initial bromination of pyrene, followed by the reaction of tetrabromopyrene with 4-methoxycaronylphenyl boronic acid as previously described with some modifications (Additional file 1: Scheme S2; Figures S1, S2, and S3).

Formation mechanism of H 4 L-and TBAPy-AgNPs
H 4 L-and TBAPy-AgNPs were constructed by the direct recombination of Ag cations and ligand anions throughout the electrostatic attraction. The anchoring of Ag + into the organic ligands to form H 4 L-and TBAPy-AgNPs via a simple liquid-phase reduction process was confirmed by different analyses, such as FT-IR, UV-Vis spectroscopy, XRD, TEM, and EDX, as described later. We proposed that the functional groups induced onto the surfaces of ligands facilitate the nucleation of Ag seeds at the start of the reaction and the primarily grown seeds tend to aggregate and recrystallize to form quasi-spherical AgNPs after a prolonged period of growth (Fig. 1). Furthermore, the induced functional groups might improve the binding between the metallic cations and the ligand species in the resulting complexes.

UV-Vis spectroscopy
UV-Vis spectroscopy is a technique for verifying NP development in a solution. In this study, the integrated H 4 L-and TBAPy-AgNO 3 systems underwent a chemical reduction process, and the change in color to a reddish-brown for H 4 L or yellowish-black for TBAPy indicated the formation of a Ag colloidal suspension ( Fig. 2A, B). The obtained complexes were primarily observed by a UV spectrophotometer, and the recorded signals displayed major absorption peaks centered at 425 and 430 nm for H 4 L-and TBAPy-AgNPs, respectively, which is mainly indexed to generate AgNPs (Fig. 2C). Such characteristics match well with those observed for a single SPR at 405, 420, 448, and 450 nm [10,11,42]. The deviation of the SPR shoulder of anisotropic particles might be attributable to their shape and size [43,44]. However, the control samples prepared without AgNO 3 salt depicted an SPR peak at 230 nm originated from the organic matrices (Fig. 2C).

FT-IR analysis
FT-IR measurements were performed to obtain detailed information about the formation mechanism of AgNPs and functional groups. The responses of the as-designated H 4 L-and TBAPy-AgNPs were collected in the range from 500 to 4000 cm −1 (Fig. 3). The absorption bands of H 4 L-AgNPs were detected at 3384, 1698, 1380, 1013, and 522 cm −1 owing to the vibration of the chemical particles of H 4 L. For TBAPy-AgNPs, the characteristic bands were located at 3397, 1687, 1413, 1017, and 521 cm −1 within the same wavenumber window. The broad absorption peaks at 3384 and 3397 cm −1 for H 4 L-and TBAPy-AgNPs, respectively, can be attributed to the strong vibration modes of O-H stretching and bending, while the sharp characteristics peaks at 1698 and 1687 cm −1 for H 4 L-and TBAPy-AgNPs, respectively, are associated with the stretching vibration of carboxylic acid and some variable stretching and bending peaks that would be involved in the growth process of AgNPs [41]. In addition, the peak shift from 1380 to 1013 cm −1 for H 4 L-AgNPs and from 1413 to 1017 cm −1 for TBAPy-AgNPs is largely ascribed to the contribution of the -C-H and O-H bending vibration of aliphatic and aromatic C-H plane distortion vibrations of methyl, methylene, and methoxy groups during reduction. Such characteristic modes demonstrate the extended vibrational bands in charge of compounds such as flavonoids and terpenoids [44][45][46]. Fouad et al. J Nanobiotechnol (2021) 19:318

TEM, XRD, and EDX analyses of AgNP-based complexes
The microstructure and surface morphologies of the as-synthesized AgNPs were analyzed by TEM (Fig. 4). The TEM images of both H 4 L-and TBAPy-AgNPs at different concentrations of AgNO 3 showed more microscopic structures of Ag 0 -intercalated organic ligands. The organic frameworks displayed thin films of biomolecules that are beneficial for attaining highly stable nanocrystals for prolonged durations. The samples exhibited quasi-spherical AgNPs anchored into the entire film of the underlying substrates etched into a porous structure. These AgNPs were firmly interconnected to the transparent networks, which is probably due to the electrostatic interaction between the metallic cations and the surface functional groups of the organic matrices, as well as interfacial interaction among NPs (Fig. 4). The dispersion of the Ag 0 nanocluster formed by H 4 L was better than that TBAPy, suggesting a relatively low growth rate of NPs formed by H 4 L (Fig. 4C). The NPs formed by TBAPy revealed a lumpy morphology made up of agglomerated NPs (Fig. 4D-F). The Ag 0 particles produced by both ligands appeared thicker with increasing AgNO 3 concentration to 3 and 5 mM because of their high tendency to aggregate into larger clusters ( Fig. 4 and Additional file 1: Figure S4), which is a result expected due to ultrafast dissociation of AgNO 3 into water and thereby rapid growth kinetics. The darker areas are mainly associated with the agglomeration and condensed overlapping of the adjacent NPs because of thermodynamic kinetic changes. It seems reasonable that the reduction mode and growth mechanism of NPs evolves with the nature of the reducing agent and is strongly correlated with reaction conditions, such as time, temperature, and reactant concentration.
Accordingly, the reduction rate of AgNPs was fast at the initial stage of nucleation and crystallization. The freshly formed nanocrystals are thermodynamically unstable, owing to their high surface energies. Subsequently, these nanoclusters were guided to drive the following stage of self-assembly with extra generated nanocrystals into larger NPs by changing the corresponding interfacial energies. Moreover, the heating of the reaction solution with strong reflux may facilitate AgNP formation. We proposed that the variation in nanocrystal free energies might probably play a role in influencing the reaction kinetics and nucleation mechanism. It is worth mentioning that the architecture and geometry of the constructed NPs could significantly influence biological systems, particularly pest administration programs [47]. Accordingly, the obtained AgNPs are expected to play an important role in contributing to insecticidal assays.
The crystalline structure of the obtained AgNPs was studied by XRD analysis, as illustrated in Fig. 5. The H 4 L-AgNP spectrum (Fig. 5A)  diffractions of the cubic Ag phase, respectively. The slight change in the diffraction positions is probably due to the change in the chemical microstructure of the ligands. We believe that the induced strain can affect the character of nanocrystallites in the crystal structure. These results are consistent with those obtained for AgNPs prepared by using the cell-free supernatants of two bacteria Bacillus amyloliquefaciens and B. subtilis [11]. The composition and elemental analysis of both H 4 Land TBAPy-AgNPs are illustrated Additional file 1: Figure S5. The mapping profiles of both complexes demonstrated that the bulk Ag represents the major component. The spectra of both H 4 L-and TBAPy-AgNPs (Additional file 1: Figures S6A and S6B) displayed a strong peak at 3 keV attributed to AgNPs and demonstrated the reduction of Ag + to Ag 0 with the assistance of H 4 L and TBAPy capping salts. Besides, Al, S, Si, Cl, and O probably originated from the silicon substrate used to prepare the samples prior to EDX analysis and the organic precursors used to prepare the two ligands. The corresponding cross-sectional compositional line profiles displaced that the Ag intensity is much stronger than that of other elements detected, which evidences the predominance of Ag in the NPs. These findings agree well with the reported EDX profiles and existing results in the literature [43,48] and indicate the effectiveness of our ligands as environmentally friendly compounds to create Ag 0 -based complexes. Additionally, this may be an efficient strategy to prepare other noble metal NPs.

Nanoparticle size and stability of the nanoparticles
The average size of H 4 L-AgNP was found to be 7 nm to 75 nm, while the particle size of TBAPy-AgNP was found to be 10 nm to 85 nm (Additional file 1: Figure S7A, B). The zeta potential is usually utilized to know the constancy of colloidal systems. The zeta potential of nanoparticles was evaluated in methanol as dispersant. In this report, zeta potentials of H 4 L-and TBAPy-AgNPs were − 28.1 mV and − 27.2 mV, which denoted the stability of silver nanoparticles suspensions (Additional file 1: Figure S7C, D). Mostly, a suspension that displays an absolute zeta potential less than 20 mV is considered unstable and will cause precipitation of particles from solution, while the absolute zeta potential higher than 20 mV is stable [49,50]. Guilger-Casagrande et al. reported that all the samples of nanoparticles presented negative values with the AgNP-TS sample showing the highest electronegativity − 33.3 mV [51].

Toxicity of AgNPs toward unsexed brown planthopper adults
Immediate toxicity bioassay demonstrated that H 4 Land TBAPy-AgNPs are toxic to unsexed adult BPH. The concentration of our applied materials and the posttreatment time significantly affected the mortality for all tested adult insects. At high concentrations (1500 and 2000 mg/L), the toxicity of H 4 L (71.25% and 78.50%, respectively) was higher than that of TBAPy (64.50% and 70.50%, respectively) 10 days posttreatment (Additional file 1: Figure S8A). Similarly, at low concentrations (15 and 20 mg/L), H 4 L-AgNPs caused higher mortality (92% and 98%, respectively) compared with TBAPy-AgNPs (70% and 79% mortality, respectively) in BPH adults 10 days posttreatment (Additional file 1Figure S8B). Moreover, TBAPy were less effective against BPH at a high concentration, with an LC 50 and LC 90 of 810 and 1982 mg/L, respectively, 168 h posttreatment. However, the toxicity of H 4 L against BPH increased, with an LC 50 and LC 90 of 786 and 1919 mg/L, respectively, 168 h posttreatment (Table 1). Furthermore, at a similar concentration of dimethyl sulfoxide as a solvent for the two ligands (negative control), there was no obvious effect on adult BPH 168 h posttreatment. However, H 4 L-AgNPs were highly toxic to BPH, with an LC 50 and LC 90 of 3.9 and  14.2 mg/L, respectively, after 168 h posttreatment, while TBAPy-AgNPs were less toxic at the same concentration, with an LC 50 and LC 90 of 4.6 and 16.1 mg/L, respectively ( Table 2). The regression equation suggested a linear relationship between the rate of mortality and small doses of AgNPs, with a positive relationship between doses, AgNP shapes, and the insect body's absorption of high quantities of AgNPs that caused their death in the end. Over 15 days of careful observation, 100% mortality was seen in BPH adult at all concentrations of H 4 L-and TBAPy-AgNPs (Fig. 6), indicating that insect mortality might largely be due to the toxic inner effects of the AgNPs inside the cuticle. On the basis of our findings, the performance of AgNPs is strongly correlated with the concentration of AgNO 3 , with the highest activity being achieved with 1 mM for both ligands. In summary, the toxicity and insecticidal activity decreased in the order of 1 mM > 3 mM > 5 mM, which might be due to a decrease in reaction exposure. TEM analysis showed that the agglomeration rate increased with increasing concentration of the Ag precursor (Additional file 1: Figure S4). It is noteworthy that the efficiency of NPs can be negatively affected by such aggregation where specific NPs can participate in the biological reaction, while the remaining become inactive because the reaction takes place on the top layer of the Table 2 Insecticidal effects of synthesized H 4 L-AgNPs and TBAPy-AgNPs against brown planthopper N. lugens adult LC 50 lethal concentration that kills 50% of the exposed insects, LC 90 lethal concentration that kills 90% of the exposed insects, LCL lower confidence limit, UCL upper confidence limit, χ 2 chi-square test, df degrees of freedom, P-value probability value, R-value correlation coefficient  6 BPH adults dead after treatment of exposure to H 4 L-AgNPs and TBAPy-AgNPs exposed NPs. In contrast, a well-dispersed nanocluster can guarantee full use of the exposed area to the biological process and all the NPs are efficiently involved (Additional file 1: Figure S4). The cross-activity of NPs driving an individual cell can prevent molting and other physiological procedures that accelerate mortality [11,52] [50].

Inhibitory activity of H 4 L, TBAPy, and synthesized AgNP-based complexes on brown planthopper enzymes
Considering the insecticidal activity of H 4 L-and TBAPy-AgNPs, it is also necessary to know their mechanism of action. We measured the effects of AgNPs and ligands on the biochemical constituents of BPH. In general, the tested samples caused variation in the normal biochemical constituents of BPH, with either an increase or a decrease in their activity compared to the controls (Fig. 7). We studied on the effects of H 4 L-AgNPs, TBAPy-AgNPs, and pure H 4 L and TBAPy on different enzymes involved in biological processes in BPH adults and estimated total protein levels, acetylcholinesterase activity, α-and β-carboxylesterase activity, and acid and alkaline phosphatase activity. At 72 h posttreatment, H 4 L-and TBAPy-AgNPs significantly decreased the total protein level from the control value of 2.45 to 1.42 and 1.16 mg protein/mL of homogenate (p > 0.05), respectively (Fig. 7A); significantly decreased acetylcholinestrase activity from the control value of 0.072 to 0.018 and 0.01 μM ACT/mg/min (p > 0.05), respectively (Fig. 7B); significantly decreased α-carboxylesterase Esterases are the primary enzymes involved in the development of resistance mechanisms to chemical insecticides by splitting carboxyl ester and phosphodiester bonds [53]. We found that H 4 L-and TBAPy-AgNPs are able to highly reduce α-and β-carboxylesterase activity, and the level of detoxifying enzymes is significantly down regulated during BPH development. Hassan et al. treated mosquitoes with salicylic acid (SA), 3,5-dinitrosalicylic acid (DNS), and AgNPs and found reduced levels of acetylcholinesterase after treatment with SA-and DNS-AgNPs compared with the controls. They also found that α-and β-carboxylesterase activity was highly inhibited after treatment with SA-and DNS-AgNPs to 0.33 and 0.30 µM α,β-naphthol/min/mg protein, respectively, compared with the controls [54]. At 72 h posttreatment, H 4 L-and TBAPy-AgNPs significantly increased acid phosphatase activity from the control value of 0.224 to 0.869 and 1.035 mM/mg/min (p > 0.05), respectively (Fig. 7E), while they decreased alkaline phosphatase activity from the control value of 1.751 to 0.596 and 0.426 mM/mg/min (p > 0.05), respectively (Fig. 7F). Acid and alkaline phosphatases play an important role in the hydrolytic cleavage of phosphoric acid esters and regulate the acid-alkali balance [55]. In addition, these enzymes are important for many significant physiological processes, such as metabolism and cellular signaling [56].

AgNP accumulation in brown planthopper by TEM
The LC 30 exposure and control of BPH were used for TEM analysis to observe AgNP accumulation in the midgut cells of BPH adults. Figure 8A shows the microvilli structures in the midgut region of the control group. H 4 L-and TBAPy-AgNP-treated BPH showed accumulation of Ag particles in the midgut cells as dark spots, indicated by yellow arrows and circles, respectively. We observed distinct localization and accumulation of AgNPs in the rough endoplasmic reticulum (ER), in addition to beneficial bacteria (Figs. 8B, C). Our results are in agreement with the findings of Yasur et al. [36], who observed the accumulation of AgNPs in the rough ER and some other cell organelles of the midgut cells of AgNP-treated Achaea janata larvae. Similarly, gold (Au) NPs accumulated in the rough ER and cell vesicles of AuNP-treated Drosophila melanogaster [57]. TEM insect images showed that the AgNPs absorbed by midgut cells localize and accumulate in the cells' organelles, which could lead to detrimental effects, including DNA damage, genotoxicity, mitochondrial dysfunction, altered cell morphology, and subsequent necrosis or apoptosis [58].

Histological studies
The LC 50 exposure and control of BPH for 72 h were used for histological analysis. We found histological structure alterations in the midgut epithelial cells in treated insects (Fig. 9). The midgut area, including epithelial cells and the brush border, were significantly affected by H 4 L-and TBAPy-AgNPs compared with the controls. The midgut epithelium of the controls exhibited normal cytoplasmic characteristics with a regular microvilli lining (Fig. 9A) at 40 × magnification. H 4 L, TBAPy, H 4 L-AgNPs, and TBAPy-AgNPs caused histological alterations in the midgut, such as elongation of epithelial cells protruding into the lumen and disintegration of the brush border (Figs. 9B, C). TBAPy-AgNPs partially destroyed the epithelial cells in the posterior region of the midgut (Fig. 9D). Notably, H 4 L-AgNPs caused severe damage in the midgut epithelial cells, with cytopathological variations, such as destruction of epithelial cells and degradation of nuclei (Fig. 9E). Severe lesions were also seen in the midgut epithelial cells, including broken membranes, brush border damage, and vacuolization (Fig. 9D, E).

Conclusions
This paper highlights AgNP synthesis via a liquid-phase reduction approach using two new alternative organic ligands H 4 L and TBAPy as reducing agents, obtained by different chemical techniques. These two ligands can directly transform dissolved Ag ions into quasi-spherical architectures. H 4 L-and TBAPy-AgNPs were used as insecticides to control the BPH population. At the same concentration, H 4 L-AgNPs are more toxic than TBAPy-AgNPs after 10 days of direct contact with BPH. Furthermore, the toxicity increases with increasing doses. In addition, H 4 L-and TBAPy-AgNPs significantly affect the midgut of BPH compared with H 4 L and TBAPy alone and cause cytopathological variations, such as destruction of epithelial cells and degradation of nuclei. Our findings provide insights into the mechanism of action of AgNPs, which operate by affecting enzymatic pathways. AgNP-ligand formulations produce a synergetic effect to combat the adverse effects of bulk insecticide on the environment. AgNP-intercalated organic ligands present a new and potential tool for protecting rice plants from pests and may be used as an eco-friendly insecticide at low doses to reduce the BPH population in rice fields.