Effect of New Bio-component of Cytoplasmic Polyhedrosis Virus (Cypovirus1) with Silver Nanoparticles against Four Pests in Stored Food Products

This study was conducted to develop an approach for reducing severe damage to grains caused by various insect pests using the biological control agent Cypovirus1 and silver nanoparticles, both alone and in combination. Four types of beetles that infest stored products, including drugstore beetle ( Stegobium paniceum ), saw-toothed grain beetle ( Oryzaephilus surinamensis ), warehouse beetle ( Trogoderma variabile ), and Cowpea weevil ( Callosobruchus maculates ), were collected from seven different areas in the Kingdom of Saudi Arabia, specifically Mecca regions. We determined the morphological characteristics of the pests in various grains. Corn borer pupae and larvae of corn plants infected with Cypovirus1 were collected from the stems of live and dead maize plants in fields in Mecca regions to isolate Cypovirus1. Infected samples were examined using triple antibody sandwich-enzyme-linked immunosorbent assay (TAS-ELIZA) and reverse transcription-polymerase chain reaction (RT-PCR). Transmission electron microscopy (TEM) analysis revealed virus-containing occlusion bodies inside infected cells of corn borer larvae with irregular shapes and dimensions of 2.2–4.9 µm. The effects of different concentrations of the virus activated by synthetic spherical silver nanoparticles with an average diameter of 36.32 nm on the larva of grain pests taken were evaluated, and all treatments led to an increased mortality percentage after 72 h compared to at 48 h. The treatment mixture consisting of 10/100 viral and 400 µg/kg AgNPs led to the highest average death rates of the four insect larvae at 72 h after treatment. Protein bands that were present in the virus-infected larvae of the four pests were absent from healthy larvae, indicating viral infection.


INTRODUCTION
S t o re d g ra i n s a re a tta c ke d b y approximately 30 types of stored grain pests, causing serious economic damage to harvested grains, including the drugstore beetle, saw-grain beetle, khapra beetle, and cowpea weevil.
The drugstore beetle is a common pest in grain worldwide because of its ability to adapt to many different environments, 1 and can feed on various substances and grains.Because of these abilities, this beetle has been named as Stegobium paniceum. 2,3he saw-toothed grain beetle is distinguished by its dark brown color; its length ranges from 2.5 to 3.5 mm.This beetle belongs to the order Coleoptera and family Silvanidae.][6][7] The Khapra beetle, one of the five most important pests, is a serious exotic pest of grains and stored dry foodstuffs, and rankly highly as a target organism for exclusion by quarantine. 8,9Its distribution has been reviewed previously. 10The Khapra beetle belongs to the family Dermestidae. 9

The family of the Coleoptera
Chrysomelidae includes many pests of harvested grains, the most important of which is the cowpea weevil. 11This major pest damages cowpea grains, specifically by reducing the grain germination rate and reducing the grain quality by reducing its weight.After the cowpea weevil lays its eggs on the cereal surface, larvae feed on the cotyledons of the grain. 12any pesticides used to fumigate grain, including phosphine and methyl bromide, to combat grain pests have toxic effects on human health and negative impacts on the environment.Thus, in this study, we examined alternative approaches for grain pest control that are environmentally friendly with no effects on human health. 13We evaluated the effects of a bio-viral compound on the larvae of grain pests.][16][17][18][19] The larvae show symptoms of viral infection at four days after Cypovirus1 inoculation, including sudden destruction of the central gastric tissue, causing the larvae to stop feeding and develop vomiting and diarrhea.][21][22][23] Synthetic spherical silver nanoparticles (AgNPs) with an average diameter of 36.32 nm can easily diffuse through the plasma membranes of cells, as its size is similar to those of cellular proteins. 24,25AgNPs affect the permeability of the plasma membrane of treated larval cells, their respiration, and their ability to molt and develop.These alterations leading to toxicity are easily observable. 26he objectives of this study were to investigate the effect of different concentrations of Crypovirus1 and synthetic spherical AgNPs on four different types of beetle larvae that infest stored food products.

Survey of four stored product beetle pests in Mecca regions
Four types of grain pests were collected in seven different areas in Mecca, for a total of 756 samples of drugstore beetle, saw-toothed grain beetle, warehouse beetle, and cowpea weevil.We collected 108 samples per location from four areas representing nine samples (three replicates) per area.

Morphological characteristics of the pests
The drugstore beetle, 3 saw-toothed grain beetle, 9 warehouse beetle, 7 and cowpea weevil 12 were separated based on the morphological characteristics of the adult insects and larvae.

Infection of four pests in stored product beetles with Cypovirus1 Collection of infected samples
Pupae and larvae of maize infected with Cypovirus1 were collected from the stems of live and dead maize plants from fields in the Mecca regions.Other samples were collected from maize borer larvae Sesamia calamistis using bait comprised of molasses and yeast in the soil. 27

Pathogen identification
The collected corn borer larvae showed symptoms of infection with the virus such as very slow movement, abstinence from feeding, vomiting, and diarrhea, as evaluated serologically by indirect enzyme-linked immunosorbent assay as described previously. 28We confirmed the presence of viral particles and inclusion bodies in ultra-thin sections of the larvae using transmission electron microscopy and Cypovirus1 particles was purified using Larsen and Duffus protocol. 29,30

Virus production
Virus-infected corn borer larvae were ground in sterile distilled water, followed by filtration through filter paper to obtain the viral suspension, which was prepared at a concentration of 1 larva/mL.A viral dilution was prepared at a concentration of 10 3 from the prepared viral suspension, and 1 mL of the viral suspension was pipetted onto a small disc of artificial diet (15-20 mm 3 ) provided to the larvae to be infected with the virus.The prepared food tablets mixed with the viral suspension were introduced to the larvae at 24 h and placed in small glass tubes fitted with cotton wool stoppers.The discs were removed and replaced every 24 h, and symptoms of infection were monitored the larvae until death or pupation.

Isolation of total genomic RNA
We u s e d s t a n d a r d g u a n i d i u m isothiocyanate methods to purify the viral polyhedral structure and extract genomic RNA. 31 The dsRNA fragments were separated in a 1% agarose gel containing a 1-kbp marker and stained with ethidium-bromide.
Synthesis and collection of spherical AgNPs (avg.36.32 nm) using 8.0 mM of tri-sodium citrate dehydrate (C 6 H 5 O 7 Na 3 ) Sodium citrate (C 6 H 5 O 7 Na 3 solution; 8.0 M, 20 mL) heated at 60°C (as a reducing agent and surfactant) was added to 80 mL of AgNO 3 solution after heating at 60°C (as a raw material for manufacturing spherical AgNPs).][34][35] The reaction mechanism is expressed in the following equation 32 : The shape and size of the AgNPs were evaluated using electron microscopy.The spherical colloidal AgNPs were also characterized by radiation with 0.15405 nm Cu Kα radiation using X-ray diffraction (model D5000 Siemens, Munich, Germany).[34][35]

Preliminary assays with the propagated virus
Bio-component of (CPV) was prepared by mixture 1mg of powder infected larvae with 999mg inactivated integrate containing of carborandum and talcum.

Effect of Cypovirus1 activated by AgNPs on four beetle pests of stored products
The bio-viral component (10 mg) was added to 100 and 1000 mg of mixed stored products.Additionally, 200 and 400 µg AgNPs were added to 1 kg of mixed stored products.Additionally, 10 mg/1000 mg of virus solution was added to 400 µg of AgNPs.180 larvae per each concentration divided into 60 larvae were used (all assays were replicated three times).Mortality was recorded daily as a percentage.The mortality rates and severity of infection were determined at 48 and 72 h after treatment.
Symptoms of infestation of the medicinal beetle have been recorded on collected stored products samples including ground coffee beans, ground cumin, ground root of Curcuma longa, wormwood chamomile flowers, wheat flour, ground fennel seeds, meat spices (mixture of seven spices), cinnamon sticks crushed, rice grains, and ground fenugreek seeds.

Protein production pattern of all larva stages following treatment with Cypovirus1 activated with AgNPs Protein extraction
1 mL Extraction buffer (40ml SDS10%, 20ml Glycerol 15%, 8ml Tris 1M (pH=8.8),1ml EDTA 0.5M, H2O up to 100ml) added to the healthy and infected larvae at different life stages (0.5 g); these samples were ground in liquid nitrogen to extract

Statistical analysis
One-way analysis of variance (P ≤ 0.05) was used to calculate the remarkable assortment in the averages of the investigational treatments. 37

Incidence of pest infestation of stored products from seven provinces in Mecca regions
We found that 74.21% (561/756 samples) of stored products were infested with at least one pest.According to Figure 1, drugstore beetle showed the highest prevalence in stored products, with a mean infection rate of 64.29%.The highest rate of drugstore beetle infestation was observed in Jeddah (76.85%), whereas the lowest rate ).The infection rates of collected stored products samples including ground coffee beans, ground cumin, ground root of Curcuma longa, wormwood chamomile flowers, wheat flour, ground fennel seeds, meat spices (mixture of seven spices), cinnamon sticks crushed, rice grains, and ground fenugreek seeds are illustrated in Figure 2. 1,2 The second most prevalent pest was the saw-toothed grain beetle, with an infection rate of 54.89%; its highest infection rate was in Jeddah (66.67%) and its lowest infestation rate was in Rabigh (38.89%). 38,39In addition, warehouse beetle exhibited a low infection rate in Rabigh (30.56%). 38owpea weevil showed a low infection rate in all regions (35.05%) (Figure 1).

Effect of Cypovirus1 and AgNPs on beetle larvae
Table 1 shows the effect of viral activity on the larvae of drugstore beetle, saw-toothed grain beetle, and warehouse beetle, in addition to the effects on adult cowpea weevil by determining the infection rates and mortality percentage at 48 and 72 h after treatment.We treated insect larvae with different concentrations of each viral component (10/100 and 10/1000) and AgNPs (200 and 400 µg/kg) and in combination (10/100 viral component with 400 μg/kg AgNPs).All treatments   led to higher larvae mortality at 72 h than at 48 h.The treatment mixture consisting of 10/100 viral components and 400 µg/kg AgNPs led to the highest average death rates of larvae at 72 h (94.78%, 96.22%, 96.11%, and 92.78%) compared to those at 48 h (91.11%, 92.78%, 90.56%, and 86.67%) after treatment.Infection rates and the mortality percentage were increased at 72 h after treating the insect larvae with the viral component at concentrations of 10/100 (72.78%, 83.33%, 70.00%, and 83.89%) and 10/1000 (80.00%, 80.56%, 80.00%, and 71.67%) respectively compared with 48h, inactivated integrate and control treatments.This is because the virus has an incubation period inside the treated larvae, leading to increased infection rates and virus spread between infected larvae.In contrast, the mortality rates increased when the larvae were treated with 200 and 400 μg/kg AgNPs at 72 h after treatment (32.22%, 53.33%, 35.00%, and 56.67% for 200 μg/kg AgNPs; 83.89%, 93.89%, 82.22%, and 89.45% for 400 μg/kg AgNPs).This may be because AgNPs can enter cells by diffusing across the plasma membrane. 25AgNPs affect the plasma membrane permeability, respiration, and molting and development ability of larval cells, leading to toxicity. 26[22][23]

Protein pattern of all larva stages infested by Cypovirus1 activated with AgNPs
he presence of these protein bands is considered a means of detecting the virus in infected insect larvae (the presence of the virus was confirmed using TAS-ELIZA previously) and is distinguished from others in that it has high molecular weights, which have not appeared at all in healthy insect larvae.Synthesized protein bundles were present within the larvae treated with AgNPs, which were absent from untreated larvae including the protein band No. 6, 13, 24, 30, and 36 in treated larvae of a S. paniceum; 8, 15, 25, 31, and 38 in treated larvae of O. surinamensis; (1, 5, 12, 20, and 34 in treated larvae of T. variabile; and 4, 9, 19, 30, and 35 in treated adults of C. maculates, respectively.Thus, AgNPs can interfere in vital metabolic processes within larvae.These synthesized proteins influenced by AgNPs strongly impact insect antioxidant and detoxifying enzymes, leading to oxidative stress and cell death. 43he table 2 shows the effect of viral infection on proteins in infected larvae of the four types of pests. 16

CONCLUSION
From four types of pests of stored products (S.paniceum, O. surinamensis, T. variabile, and C. maculates) from seven different locations in the Mecca region.We found that 561/756 samples (74.21%) collected from the stored products were infested with at least one pest.Drugstore beetle showed the higher average infestation rate of stored products (64.29%).The second most prevalent pest in stored products was saw-toothed grain beetle (54.89%), with the highest infestation rate in Jeddah (66.67%) and lowest infestation rate in Rabigh (38.89%).The morphological characteristics of these insects were identified.The presence of distinctive occlusion bodies of the virus inside infected cells is considered conclusive evidence of a viral infection.In addition, Treating stored grain pest larvae under study with the viral compound mixture consisting of 10/100 viral and 400 µg/kg AgNPs led to the highest average mortality rates of the four insect larvae at 72 h after treatment, that attack stored grain materials and others in the warehouse.Therefore, we recommend in this study the use of this treatment to combat these pests, as it is considered a safe and environmentally friendly method that is not harmful to human health.
The effects of the results of electrophoresis of protein bands of larvae treated with the viral compound and silver nanoparticles show that viral infection of larvae in the presence of silver nanoparticles leads to the creation of protein bands indicating the presence of the viral infection and the concealment of other protein bands compared to untreated larvae.
The study also shows the effective combined effect of each of them in interfering with

Figure 1 .
Figure 1.Incidence of Drugstore beetle, Saw-toothed grain beetle, Ware house beetle, Cowpea weevil, and infestation stored products samples from seven provinces in Mecca regions, KSA

Figure 2 .
Figure 2. The domestic range is the most dangerous pest of stored products and grains, which is the drug beetle (a) Ground coffee beans infested with drugstore beetle, (b) Ground cumin infested with Drugstore beetle, (c) Ground cumin infested with Drugstore beetle, (d) Wormwood chamomile flowers infested with Drugstore beetle, (e) Wheat flour infested with drugstore beetle, (f) Ground fennel seeds infested with Drugstore beetle, (g) Meat spices (mixture of seven spices) infested with Drugstore beetle, (h) Cinnamon sticks crushed infested with Drugstore beetle, (i) rice grains infected with drugstore beetle, and (j) Ground fenugreek seeds infested with Drugstore beetle add enough Bio-Safe Coomassie Stain to completely cover the gel.Let stain for 1 hour on a shaker.If the protein signal is low, stain overnight.The protein molecular marker was from Sigma (St. Louis, MO, USA) and contained Albumin from Bovine serum (weight 66.0kDa), albumin from Egg (weight 45.0kDa), phosphorylase from Rabbit Muscle (weight 36 kDa) and carbonic Anhydrase from Bovine Erythrocytes (weight 29.0kDa).

Table 1 .
Effect of (CPV1) infection and AgNPs on four-stored product beetles pests