MATERIALS AND METHODS

Survey of rice weevils, darkling beetle, drugstore beetle, and cigarette beetle in Mecca regions, KSA

756 samples of stored products infected with rice weevils, darkling beetle, drugstore beetle, and cigarette beetle were collected from different 7 locations in Mecca regions. Per location was collected 108 samples were from 4 areas. Nine samples per one area and 3 replicates for all samples were collected.

Identification and the variation of morphological characters the rice weevils, darkling beetle, drugstore beetle, and cigarette beetle

Rice weevils and red flour beetle can be easily separated, and we can distinguish between cigarette and drugstore beetles by morphological and physical characters.

Effect of (Cypovirus1) infection on rice weevils, red flour beetle, drugstore beetle, and cigarette beetle larvae

Collection of infected samples

Samples of living and dead stem and cob borer larvae and pupae were collected from Maize and sorghum in farmers ‘fields in Ismailia governorates.

 Maize fields were selected based on plant growth stage and farmer's consent at intervals of approximately 5 Km along major roads within the field, plant will inspect at random for symptoms of stem borer damage. Further samples were collected by soil baiting [17] with stem borer larvae Sesamia calamistis was placed with the soil in plastic basins and held at ambient temperature after 1 week, live, dead and mycoses larvae were recovering and treated according to the following procedures.

Identification of pathogen

Samples containing suspected virus isolates (Cypovirus1) following examination of larval smears, were examined by symptoms appearance and Cypovirus1 particles were purified report to protocols of [18, 19] and showing inclusion bodies and viral particles by TEM and confirmed by Indirect- ELISA, according to Cherry et al. (1984).

Virus production

Suspensions of a virus isolate were prepared by triturating the original host larva in sterile distilled water to a volume of 1 ml and then filtering. Concentrations were defined as 1 larval equivalent (l.e.) ml−1. Suspensions were fed to 10 3rd instars larvae per isolate by applying 1 ml droplets via micropipette to small discs of artificial diet (15–20 mm3). Larvae were held in small glass tubes plugged with cotton wool. After 24 hours all larvae which had consumed the treated diet were supplied with a 0.5 cm3 plug of a clean diet and held until death or palpation.

Isolation of total genomic RNA

According to the manufacturer’s instructions, Genomic RNA was extracted from purified polyhedral by a standard guanidium isothiocyanate method after dissolving the purified polyhedral in alkali with triazole reagent (Gibco, BRL). RNA was then separated in 1% agarose gel in Tris-phosphate buffer. RNA segments were visualized on the ethidium bromide-stained (final concentration of 0.5 mg/ml) gel. Size markers (Bio Basic Inc. Canada) of 1,000 bp were used to determine segment sizes.

Preliminary assays with the propagated virus

Bio-component of (Cypovirus1) was prepared by mixing 1mg of powder infected larvae with 999mg inactivated integrate containing Carborandum and Talcum. Add 1gm from this component to 10 g, 100 g,1000 g, and 10000 g from stored products and mixture, put 60 larvae per each replicate and 180 larvae per each concentration, mortality was recorded daily until death or pupation, and all assays were replicated 3 times.

Effect of (Cypovirus1) on protein pattern of all larva stages

Protein extraction

The three larvae stage 3 stage, 6stage and 8 stages were collected from infected and healthy rice weevils, darkling beetle, drugstore beetle, and cigarette beetle larvae separately and ground (0.5g) to flour in a mortar by using liquid nitrogen in a mortar then proteins were extracted with 1ml extraction buffer. The sample will then transfer to a precool Eppendorf tube, vortex for 3 min, then store at 4°C for 30-60 min. The samples will centrifuge for 10 min at 500xg at 4°C and was transferred to a new Eppendorf and store at -20°C.

SDS-PAGE

SDS-PAGE was performed with a separating gel 12% (w/v) (pH8.8) and stacking gel 5% (w/v) (pH6.8) as described by [20]. Protein samples were mixed with an equal volume of protein sample buffer, denature at 80-90°C for 3-5 min. and immediately cool on ice and 25ml of each protein sample will load on each lane. Electrophoresis at 100 volts and 80 ma until the tracking dye reached the bottom of the gel. The gel was stain overnight in a protein staining solution and then wash twice with water. The gel was discolored in distaining solution several times. The protein molecular marker used was from Sigma (St. Louis, Mo, USA).

Statistical analyses

We were used ANOVA-type one-way to calculate the noteworthy diversity in the averages of the experimental treatments. A probability at a level of 0.05 or less will measure considerably [21].

RESULTS AND DISCUSSION

Survey of rice weevils, darkling beetle, drugstore beetle, and cigarette beetle in Mecca regions, KSA

From a total of 756 stored products, 608 (80.42%) Have been infested with at least one stored product beetles pests. Drugstore beetle was the most common stored product beetles with an infection rate of 67.20% (Table 1) and the incidence was particularly high in stored products (Ground anise, Ground cumin, all type of pasta, Nigella sativa, and Wheat flour these results were illustrated with (Figure 1), [4, 5] grown in Khulais (85.19%) and Taif, Rabigh, Jeddah, Bahra, Makkah, and Aljummum (74.10%, 71.30%, 71.29%, 67%, 54.63%, and 46.30%, respectively. Rice weevils ranked second (54.37%) and were mostly similarly distributed in all the regions (Khulais, Rabigh, Taif, Makkah, Jeddah, Bahra and Aljummum, 91.67%, 67.6%, 56.48%, 52.78%, 48.15&, 39.82% and 38.89%, respectively [1, 2]. In addition, the Darkling beetle recorded a high infection rate (50.93%) in the Khulais region, while, recorded a low infection rate in Aljummum (29.63%) [1]. On the other hand, the Cigarette beetle has recorded a low infection rate in all regions (36.38%), (Figure 1 and Table 1).

Table 1. Incidence of Rice weevils, Darkling beetle, Drugstore beetle, and Cigarette beetle infestation stored products samples from seven provinces in Mecca regions, KSA.

Effect of (Cypovirus1) infection on rice weevils, darkling beetle, drugstore beetle, and cigarette beetle larvae

Identification of pathogen

Samples containing suspected virus isolates (Cypovirus1) following examination of larval smears, were examined by symptoms appearance and Cypovirus1 is can be diagnosed from intact Drugstore beetle larvae host cells because the polyhedra are formed only in the cell cytoplasm. This was confirmed by transmission electron microscopy studies, which showed typical cytoplasmic polyhedral inclusion bodies (Figure 2) demonstrated that the occlusion bodies were of irregular shape and ranged from 2.2 to 4.9 µm in diameter [22]. whose dimension that the electron microscopy studies showed typical cytoplasmic polyhedral inclusion bodies that are icosahedral, and ranged from 2.4 to 5.3 µ m in diameter [23, 24]. The three-dimensional structures of full and empty Cypovirus1 by electron microscopy show identical outer shells but differ inside (Figure 2).

Cypovirus1 viral particles are icosahedral in shape and have 12 spikes or projections at each of the 12 vertices of the particles. Cypovirus1 has been isolated from Samples of living and dead stem and cob borer larvae and pupae. Unlike the multiple-shelled organization of other Reoviridae members, Cypovirus1 has a single-shelled capsid [13, 22] who mention that A cytoplasmic polyhedrosis virus (CPV) was isolated from the larvae of Thaumetopoea pityocampa and shown to cause an infection of midgut cells. On the other hand, this viral infection revealed several important diagnostic symptoms, including reduced feeding, discoloration of the posterior midgut, and extended development time of the larvae.

Electrophoretic analysis of dsRNA

An initial Cypovirus1 genome analysis by 1% agarose gel using a 14-cm gel at 75 V for 4 h generated seven RNA bands, some of which stained more intensely and appeared to contain more than one genome segment each (the first intense band contained segments 1, 2, and 3 and the fourth band contained segments 6 and 7). Analysis using a longer agarose gel with a lower voltage resolved the first band into three bands and the fourth band into two single bands, confirming that the genome contains a total of 10 segments. Approximate sizes of segments were estimated with size markers as follows: Seg-1, 3,846 bp; Seg-2, 3,612 bp; Seg-3, 3,431 bp; Seg-4, 3,100 bp; Seg- 5, 2,972 bp; Seg-6, 2,523 bp; Seg-7, 2,115 bp; Seg-8, 1,756 bp; Seg-9, 1,275 bp; Seg-10, 754 bp (Figure 3) [22].

Effect of (Cypovirus1) infection on stored products beetles

We are studying the effects of different concentrations of a new Bio - component of Cypovirus1 infection on the rice weevil adults, tobacco beetle, drugstore beetle, and darkling beetle larvae study their impact on the rates and severity of the infection. The highest percentage of mortality was recorded by concentrate 10/100 on tobacco beetle, drugstore beetle, darkling beetle larvae, and rice weevil adults (96.67%, 96.11%, 92.78%, and 90.00%) respectively, followed by concentrate 10/500 recorded 94.44% and concentrate 10/1000 recorded 70.56 %, on tobacco beetle larvae. On the other hand, a lower percentage of mortality was recorded by concentrate 1/25000 on the adult stage of rice weevil (33.33%), (Table 2), these results were illustrated with [14-16].

Table 2. Effect of (CPV) infection on four-stored product beetles pests.

Effect of (Cypovirus1) on protein pattern of all larva stages

Electrophoresis and densitometry analysis of protein bands by SDS-PAGE and illustrated in Figure 4. The results tabulated in Table 3 showed various distinguishable sole bands in both healthy and infected adult weevils Sitophilus oryzae, larvaes of Lasioderma serricorne, Stegobium paniceum, and Tribolium castaneum for which it could be used according to it is presented assign for the Cypovirus1 infection and effect of Cypovirus1 on protein pattern in infected weevils compared with healthy weevils. This results practically like be with [12].

Table 3. Hypothesized protein markers for the determined effect of different concentrations treatments from the Cypovirus1 on protein bands in infected Sitophilus orizae, Lasiderma serricorny, Stegobium paniceam, and Tribolium castaneum