Effects of Ag/SiO2 Nanoparticles on Gene Expression of Digestive a-Amylase in Colorado Potato Beetle

Research Article

Austin J Biotechnol Bioeng. 2022; 9(1): 1116.

Effects of Ag/SiO2 Nanoparticles on Gene Expression of Digestive a-Amylase in Colorado Potato Beetle

Ashouri S1, Dolatyari M2, Pourabad RF1, Rostami A2,3* and Mirtaghioglu H4

1Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, Iran

2SP-EPT Labs, ASEPE Company, Industrial Park of Advanced Technologies, Tabriz, Iran

3OIC Research Group, University of Tabriz, Tabriz, Iran

4Faculty of Science and Literature, Department of Statistics, University of Bitlis Eren, Bitlis, Turkey

*Corresponding author: Ali Rostami, SP-EPT Labs, ASEPE Company, Industrial Park of Advanced Technologies, Tabriz, Iran; OIC Research Group, University of Tabriz, Tabriz-5166614761, Iran

Received: March 31, 2022; Accepted: April 26, 2022; Published: May 03, 2022

Abstract

Insecticidal properties of nanoparticles (NPs) have been considered widely in different studies. In this study, synthesis of Ag/SiO2 NPs is reported and the effect of the nanoparticles on the digestive a-amylase gene expression in the Colorado potato beetle (CPB), Lepidoptera decemlineata say is investigated. For this purpose, the inhibition of fourth instar larval digestive a-amylase with NPs was analyzed by different concentrations, and the highest inhibitory effect was recorded (72%) at 1000 ppm of Ag/SiO2 NPs. Mortality for the reared larvae from the first instar on potato leaves coated with 1000 ppm Ag/SiO2 NPs was 65%, while it is 8% for the control trial. To investigate the process, we conjugated the nanoparticles with extracted RNA, and the photoluminescent (PL) spectra of the obtained materials were measured and investigated. The results indicate that the FRET phenomenon occurs when the RNA reacts with nanoparticles. These results are according to the PL spectra of RNAs extracted from the larval fed with nanoparticles. The structure is simulated and calculated by the DFT method. The obtained results confirm chemical interaction between nanoparticles and nitrogenous bases on the RNA which can decrease gene expression (0.47 fold) compared to control and causes inhibition of larvae’s growth.

Keywords: a-amylase; Ag/SiO2; Nanoparticles; Gene expression; RTqPCR; FRET; DFT

Introduction

Indiscriminate use of pesticides causes some adverse effects on the environment such as increased pest resistance, reduced soil biodiversity, reduced nitrogen fixation, and decreased pollinators [1]. Hence, it is necessary to introduce new innovative technologies and methods to overcome these problems [2]. Biocompatible, biodegradable, and intelligent materials are currently an emerging area of interest in the field of efficient and safe pesticide formulation [3]. The nanoscale sciences have great importance in the agricultural revolution and one of the key focus areas for nanotechnology agricultural research is nano pesticides [1]. Therefore, there is a need to investigate the effects of nanoparticles (NPs) on insect pest species. The employment of NPs obtained through various synthesis routes as novel pesticides have recently attracted high research attention [4]. A notable number of researches have been conducted to test their toxic potential against a wide number of insect pests and vectors; ZnO NPs causes mortality and reduction in wood-feeding of Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae) [5] and shows insecticidal activity on adults of Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) [6]; SiO2 NPs causes midgut epithelial injury in intoxicated workers of Bombus terrestris (L.) (Hymenoptera: Apidae) [7]; Ag NPs has acute and chronic toxic effects on Drosophila melanogaster (Meigen) (Diptera: Drosophilidae) [8] and induces midgut epithelial cell damage in Aedes aegypti (L.) (Diptera: Culicidae) [9]. Amylase, protease, lipase, and invertase activities of Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) decrease when exposed to Ag NPs [10]. Also, Ag Nps induces oxidative stress in the larvae of S. litura and Achaea Janata (L.) (Lepidoptera: Erebidae), which is countered by antioxidant enzymes [11]. Ingestion of Ag NPs in D. melanogaster during the adult stage for a short and long time significantly affects egg-laying capability along with impaired ovarian growth [12]. As recently pointed out, total protein levels, acetylcholinesterase, a, and Β carboxylesterase activities decrease in Aedes albopictus (Skuse) and Culex pipiens (L.) (Diptera: Culicidae) when exposes to Ag NPs [13]. In addition, Ag NPs induce a decrease in total proteins, esterase, and phosphatase enzymes in the fourth instar larvae of A. albapictus [14]. Studies on the insecticidal effects of chemical or biological insecticides and their nanoparticles have been conducted as the effects of imidacloprid and Ag-Zn NPs on Aphis nerii (Fonscolombe) (Hemiptera: Aphididae) [15], and the entomopathogenic bacterium, Bacillus thuringiensis kurstaki (Btk) synthesized Ag NPs on Trichoplusia ni (Hubner) [16]. It has been found out greater insecticidal effects for NPs. Larvicidal activity of the bio-synthesized Au NPs using Artemisia vulgaris (L.) leaf extract has been found against A. aegypti and shows that Au NPs cause damage to the midgut, epithelial cells, and cortex [17].

It is well known that due to their similar size to cellular proteins, NPs can cross some of the barriers of biological systems. Toxicity mechanisms of NPs include disruption of membranes, oxidation of proteins, interruption of energy transduction, the formation of reactive oxygen species, the release of toxic constituents, and genotoxicity [18]. Concerning the genotoxic activity of NPs; Ag NPs has genotoxicity in the fourth instar larvae of Chironomus riparius (Meigen) (Diptera: Chironomidae) [19]. Glutathione-Stransferase genes up-or down-regulated in C. riparius according to the tested concentration of Ag NPs and Cd NPs [20], also Ag NPs led to prominent induction of genes related to oxidative stress and detoxification in C. riparius [21]. Genotoxic activities of Ag NPs, Co NPs, and Ni NPs have been demonstrated on D. melanogaster [22-25]. The genotoxic effects of Ag NPs biosynthesized using neem cake against larvae and pupae of A. aegypti have been evaluated [26]. Bombyx mori (L.) (Lepidoptera: Bombycidae) some cells signaling pathway genes were up-regulated by TiO2 NPs [27]. However, physiological events within the cell caused by the NPs are a question about the genotoxic effects of NPs [18]. Metal nanoparticles can bind to sulfur from proteins or to phosphorus from nucleic acids which lead to rapid denaturation of organelles and enzymes [4]. There is no report on the genotoxic effect of NPs on CPB, especially on digestive a-amylase enzyme gene expression. Therefore in the current investigation, a qPCR-based assay for amylase expression in the fourth instar larvae of L. decemlineata in response to Ag/SiO2 nanoparticles was developed. Moreover, to further understand the mechanism of action, the interaction between the insect’s RNA and nanoparticles was screened using the fluorescence resonance energy transfer (FRET) technique. Fluorescence resonance energy transfer (FRET) is a nonradiative energy transfer process in which energy transfers from an excited state of a donor to an energy level in the structure of the acceptor through long-range dipole-dipole interactions [28-33]. In this way, the acceptor absorbs the energy of the donor at the emission wavelengths. The acceptor does not need to remit the energy using the fluorescent process. The rate of energy transfer depends on the content of spectral overlap, the transition dipoles orientations, and the gap between the donor and acceptor [28-40].

Materials and Methods

Materials required for synthesis Ag/SiO2 nanoparticles

Materials used and their suppliers are as follows: AgNO3 (99% Sigma-Aldrich), polyvinyl pyrrolidone (PPV) K17 (M. W. 8000) (99% Sigma-Aldrich), tetraethyl orthosilicate (TEOS, Sigma), 30% NH4OH (25% Merck), ethanol (96% Merck).

Synthesis of Ag nanoparticles

Ag nanoparticles were fabricated by reducing Ag ions from the AgNO3 solution with NaBH4 solution. Briefly, 20mL of AgNO3 (0.1M) was added to the 3% solution of polyvinyl pyrrolidone (PPV) and stirred for 15min. 40mL of the AgNO3 (0.1M) was added dropwise to the solution. The solution was allowed to age at room temperature, in the dark, for 1h. The nanoparticles were centrifuged three times, 30min each time with water and ethanol.

Synthesis of nanoparticles coated with silica

The Ag nanoparticles were coated with silica with the following procedure. 7mL of a concentrated Ag nanoparticle solution was mixed with 40mL of ethanol and 400μL of 30% NH4OH, and subsequently, 4μg of TEOS was quickly added. The reaction was allowed to proceed for 45min at room temperature under vigorous stirring and then was stored in the refrigerator at 4°C for 24h then the synthesized Ag/ SiO2 nanoparticles were centrifuged and re-suspended in 10mL of deionized water.

Conjugation of the RNA with the synthesized Ag/SiO2 NPs

Two mL of the synthesized nanoparticles with the concentration of 30 ppm were added on 10mL of the diluted RNA (30 ppm) and the mixture was stirred for 1h. The temperature was 0°C. The obtained solution was centrifuged and washed several times by water and redispersed in 10mL of water.

DFT calculations

The electronic band structure along with the density of states (DOS) of bare Ag/SiO2 QD and its conjugation with Cytosine were calculated using DFT. Calculations were done with the CASTEP code [41] and optimized using the BFGS (Broyden-Fletcher- Goldfarb-Shanno) geometry optimization method [41]. In the calculation, Generalized gradient approximation (GGA) and the non-local gradient-corrected exchange-correlation functional as parameterized by Perdew-Burke-Ernzerhof (PBE) used which applies a plane wave basis set for the valence electrons and normconserving pseudopotential (NCP) [42] for the core electrons. The number of plane waves included in the basis was determined from cut-off energy (Ec) of 500.0eV. The summation over the Brillouin zone was carried out with k point sampling using a Monkhorst-Pack grid [43]. Geometry optimization under applied hydrostatic pressure with parameters of 3×3×3 was used to determine the modulus of a material (B) and its pressure derivative, B’ = dB/dP [44].

Insect rearing

The colony of CPB was collected from Ajabshir province of East Azarbayjan, Iran; maintained on potato foliage cultivar Agria, which tubers were supplied from Potato Research Center of Ardabil, Ardabil, Iran, and planted at the University of Tabriz, Tabriz, Iran. The rearing condition was about 27 ± 1 °C, 60 ± 5% relative humidity, 16:8 h (L:D) photoperiod, and white fluorescent light. Insects were reared from egg hatch to adult in clear plastic dishes containing daily fresh potato leaves.

Preparation of enzyme source for activity assays

Ten guts from fourth instar larvae (L4) of CPB were isolated by dissection and homogenized in 500μL cold distilled water. The mixture was centrifuged at 10000 rpm for 30min at 4°C. The supernatant was stored as an enzyme source at -20°C before analysis. The protein concentration of the enzyme samples was adjusted to 3mg/ml.

Amylase inhibition assay

The Alpha-amylase activity was measured by the dinitrosalicylic acid (DNS) procedure [45], using a 1% soluble starch as previously described [46]. In the laboratory inhibition assays, the enzyme extract was pre-incubated with 10μL Ag/SiO2 NPs at different concentrations (125, 250, 500, 1000, 2000, and 4000 ppm) before the addition of substrate for 30min at 37°C. The percentage of inhibition was calculated compared to control.

Feeding trials

Twenty-five newly emerged CPB larvae were reared on excised “Agria” potato leaves which were placed in aerated plastic arenas. The leaves were painted with 1000 ppm Ag/SiO2 NPs and replaced daily throughout the experiment. In control, insects fed with potato leaves were painted with xH2O. After ten days, the mortality of larvae (up to reaching L4 instar) was recorded and survived L4s were used in RNA extraction.

Total RNA isolation

Total RNA was isolated from three individuals of L4 whole body which were crushed in liquid nitrogen, and with the addition of the RNX-PLUS Solution (EX6101, Cinnagen®, Tehran, Iran) according to the manufacturer’s instruction with some modifications as follows: 1mLice cold solution was immediately added to the homogenized sample and incubated at room temperature for 5min. Samples were centrifuged at 12000 rpm at 4°C for 15min for removing non-homogenized particles. Then, 300μL chloroform was added and after vigorous shaking incubated on ice for 15min. Samples were centrifuged at the same condition and RNA was precipitated by adding the same volume of isopropanol to the upper phase and incubated on ice for 15min. After centrifugation, the pellet containing RNA was dislodged with 1mL of 75% ethanol and then centrifuged. This washing process was repeated twice to remove all chemical contamination. The pellet was left to dry at room temperature for a few minutes and dissolved in 50μL DEPC treated water. The purified RNAs were immediately used in the cDNA synthesis or were stored at -80°C until subsequent analysis.

The total RNA concentration was determined with appropriate dilution (1:100) using a photometer (Eppendorf®, Hamburg, Germany) at wavelengths from 260 and 280 nm where only total RNAs with A260/280 ratio ranging between 1.8-2 were used for further application.

The integrity of RNA was verified in 1% agarose (Sigma®, St Louis, MO, USA) gel electrophoresis stained with ethidium bromide by using loading dye containing formamide which allows RNA fragments to be separated. RNA samples were heat-denatured before separation at 70°C for 5min. The agarose gels were visualized under ultraviolet light.

Reveres transcription-PCR (RT-PCR)

Synthesize of cDNA (179bp) from high integrity and purify total RNA was performed using cDNA synthesis kit (YT4500, YTA®, Tehran, Iran) with oligo (dT)18 primer and M-MLV reverse transcriptase, following the protocol recommended by the manufacturer, as follow; 2μL total RNA (1μg/mL) was combined with 1μL oligo (dT)18 primer (50μM) and DEPC treated water to a final volume of 13.4μL. RNA and primers were denatured at 7°C for 5min using Primus 25 advanced thermocycler (PEQLAB Biotechnologie GmbH, Erlangen, Germany) and then incubated on ice for 5min. cDNA synthesis mix contained 4μL first strand buffer, 1μL dNTP (10mM each), 0.5μL RNasin (40U/μL) and 1μL M-MLV reverse transcriptase was added and incubation was continued at 42°C for 60min. The reaction was terminated by heating at 70°C for 5min. The synthesized cDNAs were stored at -20°C until analysis.

The RT-PCR products were subjected to gradient PCR (48-58°C) using Taq DNA Polymerase 2x Master Mix Red (Ampliqon®, Bie & Berntsen, Herlev, Denmark) at 94°C for 5min, followed by 35 cycles of denaturing at 94°C for 60sec, annealing at gradient temperatures for 40sec, extending at 72°C for 60sec and a final elongation step at 72°C for 7min. After optimizing annealing temperature, the PCR was carried out at 50°C. Also, the No-RT control sample was prepared to detect any potential contamination with genomic DNA. Subsequently, the amplicon sizes were verified by electrophoresis of the PCR products through 4% acrylamide gel.

The primer of the L. decemlineata a-amylase gene (LdAmy) was designed using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/ primer-blast) after a search of the gene sequence (Acc. LOC111504815) at NCBI (NATIONAL CENTER FOR BIOTECHNOLOGY INFORMATION-NCBI, 2012). Primers were designed from exonexon junction and had a Tm of over 50°C and little likelihood of secondary structure. Leptinotarsa decemlineata ribosomal protein S18 (LdRP18) was used as the endogenous reference gene, as it was stably expressed at different tissues and development stages [47]. The nucleotide sequences of these primers are shown in Table 1.