Polistes dominulus and Apis mellifera species were collected. The individuals were caught and subjected to a low-temperature, ice-controlled environment right away. To euthanize wasps and bees, freeze them for 5 hours at −20˚C.

Venom was obtained from poison glands of both social bees and wasps, and poisonous secretions were separated from venom glands using dissection instruments, a light microscope, were used to extract venoms from 208 wasp venom sacs, which were meticulously dissected from the wasps, macerated in a 1:1 (v:v) acetonitrile/water solution, and centrifuged at 5000 g for 5 minutes at room temperature and vacuum drying. The supernatant was collected, vacuum dried, weighed in a precision balance, and stored at 20˚C. Lipholization was used to obtain venom powder, vacuum drying the venom in silica gel.

A laboratory strain of S. littoralis was reared in the laboratory away from any insecticidal contamination at the Department of Cotton Leaf Worm, Branch of Plant Protection Research Institute at Zagazig, Sharqia Governorate under constant conditions 25˚C ± 2˚C and 60% ± 5% R.H. to provide insects used in the present investigation. Egg-masses were placed on leaves of castor bean oil, Ricinus communis in cylindrical glass jars (El-Defrawi et al., 1964).

After egg hatching, the newly hatched larvae were transferred into large rearing jars and provided with filter paper at the bottom of the jars to absorb excess moisture. Larvae were reared on fresh castor bean leaves until the end of larval stage. The formed pupae were collected and placed in clean jars until adult emergence. Each jar was provided with 10% honey solution soaked in cotton wool, which was renewed daily to avoid fermentation and growth of microorganisms. Fresh green castor leaves were introduced daily into clean jars.

Preliminary tests for each of the separate treatments were conducted using a variety of concentrations (in distilled water) for each of the venom concentrations (0.01, 0.02, 0.04, 0.08, 0.16 µl). Cotton worms were treated in glass jars with various concentrations of bee and wasp venoms then provided to early 4^th instar larvae to pupate on. According to [22], the offered treated larvae were in a wettable state, and the leaf-dipping strategy was used. Castor bean leaves (R. communities) were dipped in each concentration and dried at room temperature before being fed to newly moulted 4^th instar larvae. The larvae were allowed to feed for 24 hours before being fed new, untreated castor bean leaves until pupation. Controls for bee and wasp treatments were larvae that ate untreated castor bean leaves. For each concentration, eight replicates were carried out, with each replicate containing five larvae. The percentages of larval mortality and morphological deformity were calculated. The data were then subjected to probit analysis [23] to obtain the LC₅₀ values of both social bee and wasp venom as well as the concentration which causes 50% adult malformation (MC₅₀). Measuring the mortality and morphological changes through cotton worm pest treated with different concentrations of social wasp and bee venom through topical food, integument dropping, injection and spraying applications.

Injection: Cotton worms between 3^rd and 4^th instar (90 - 110 mg/individual) were used for injections. 0.1 mL of venom diluted in insect saline was injected into the metathoracic pleurite, and for all five concentration till 1.6 mL were injected. Injections were performed using a 29.5 gauge insulin syringe (B-D Ultra-Fine, Terumo Medical Corporation, Elkton, MD, USA). A cohort of ten insects was injected at each venom concentration, and a similar number of control insects were injected with insect saline.

Integument dropping: Lyophilized crude bee and wasp venoms were tested for biological activity by topically dropping µl of aqueous samples into newly eclosed fourth instar S. littoralis larvae (40 - 70 mg). For each concentration tested, 80 larvae were topically dropped on integument and toxic effects were monitored over 4 - 5 days.

Spraying: treatment doses are then harvested from the bees and wasps venom and formulated in acetone solvent for application using hand-held (panel d) or aerial-mounted sprayers.

Honey bee venom as well as yellow jacket the concentration which causes 50% larval malformations (MC₅₀) for cotton worm Spodoptera littoralis was measured. The sequential cumulative effect among treatments of cotton worm with either venoms were carried out by treatment of the 4^th instar larvae with the considered concentrations in order to obtain MC₅₀ effects of Apis mellifera and Polistes dominulus then, at the end of larval stage, the late 6^th instar larvae were allowed to pupate on castor bean treated with both venoms at which pupal MC₅₀ were estimated.

The effectiveness of various venom concentrations was tested on 4^th instar larvae by application, topical on food, spraying, topical on integument, and injection. Using distilled water, serial successive concentrations of each venom were created, starting with the recommended concentration. Castor bean leaf discs (9 cm in diameter) were dipped in the tested concentrations for 10 seconds, dried, and fed to larvae that had been starved for 4 - 6 hours before treatment [24]. The larvae were placed in 5 pound glass jars, and each treatment was repeated eight times (5 larvae per each). Only distilled water was used to dip the control discs. The larvae were given 48 hours to feed on the treated discs before being moved to the untreated ones. For everyone, mortality percentages were obtained after 24, 48, 72, and 96 hours. Probit analysis was used to statistically examine the dose toxicity regression lines [23]. Sun formulae were used to compute the Toxicity Index and Relative Potency [21]. At the end of each testing period, total mortalities were determined and corrected using Abbott's formula [25].

Samples of cotton leaves were picked up at random for each treatment at zero time directly after applying with four different treatment methods for cotton worm insect pest S. littoralis through spraying, topical on food application, dropping on integument and direct injection. The prepared samples concentrations were sealed in small flasks and transferred to the laboratory where they were offered to cotton leaf worm larvae (Aly, 1999). Five larvae were placed in each glass jar and allowed to feed on the treated leaves for 1 to 5 days then the survived larvae were transferred to other clean jars and supplied with fresh clean castor bean leaves for another 3 days. Eight replicates were used for each treatment. Cumulative mortalities were calculated at the end of each testing time [26].

The mortalities at the zero time were considered as initial kill; while the mean of the cumulative mortalities of the remaining tested times were considered as cumulative final effect.

Data were subjected to statistical analyses using a software package CoStat^® Statistical Software (2005) a product of Cohort Software. The significance of the main effects was determined by analysis of variance (ANOVA) and chi-square tests. The significance of various treatments was evaluated by Duncan’s multiple range tests (P < 0.05) [27].

Two bio pesticides from social bee and wasp venom [Hymenoptera] (used at MC₅₀, concentration caused more than 50% morphological malformation) were used for treatment of larvae of cotton leaf worm, Spodoptera littoralis. The obtained results revealed that wasp is a potent toxin (LC₅₀ ≤ 0.001 ppm) at initial time compared to bee venom (LC₅₀ = 0.49 ppm) when applied topically on food (Table 1 and Table 2).

On the other hand, the mode of action through morphological malformation, the malformation concentration fifty (MC₅₀ for cotton leaf worms treated by yellow jacket venom which was significantly higher at integument dropping and injection applications. All treatments caused higher significant morphological differences within integument, pores and dark bands when treated with Yellow jackets venom at final time interval.

The topical food application results revealed that wasp venom is a potent toxin causing morphological malformations that showed the highest effect on cotton leaf worm larvae while integument drooping highest effect was shown on the

second level at morphological deformations. MC₅₀ = 0.0013 ppm compared to bee venom MC₅₀ = 0.0157 ppm, while integument dropping application showed MC₅₀ = 0.0114 ppm) compared to bee venom MC₅₀ = 0.0303 ppm). The spraying application of wasp venom showed MC₅₀ = 0.014 ppm on treated cotton leaf worms compared to bee venom MC₅₀ = 0.139 ppm. Finally the injection application was MC₅₀ = 0.0412 ppm compared to bee venom (MC₅₀ = 0.792 ppm) (Figure 1 and Figure 2).

It was observed that venom applied by spraying has significant effect rather than by topical application. Spraying application of venom affect by black spots than topical application. Topical affect more by shrinkage of insect body while venom application by injection was observed to have the least morphological effect on cotton worm larvae.

According to LC₅₀ and LC₉₀ values, topical application of bee venom was the most effective method where the LC₅₀ and LC₉₀ values recorded 0.1, 0.809 ppm for 4^th instar treated with bee venom and 0.472 and 6.838 ppm for the 4^th instars larvae treated with wasp venom, respectively.

Meanwhile, injection application appeared to be the least effective against pest tested instars, where the LC₅₀ and LC₉₀ values against 4^th instar were 9.901 and 36.447 ppm for bee venom and the values against the 4^th one were 65.736 and 1000.775 ppm for wasp venom, respectively.

The rest venom application methods gave moderate effects against instars that manifested, the LC₅₀ and LC₉₀ levels were 0.204 and 2.311 ppm for bee venom, 0.255 and 5.484 ppm for wasp venom. 1.001, 12.34 ppm for topical on integument application with bee venom and 9.901, 36.447 ppm for topical on integument application with wasp venom, respectively for 4^th instar larvae (Table 1 and Table 2).

The social honey bee venoms killed cotton worms with LD₅₀ values in the range 69 - 126 mg/g, while the social Polistes sp. Wasp venom were slightly more potent, with LD₅₀ values of 46 - 48 mg/g (Figure 3). These values are comparable to the LD₅₀ value of 105 mg/g reported for injection of crude venom into 3^rd - 4^th instar Spodoptera littoralis. Thus, while there might be statistically significant

differences in potency between social bee and wasp venoms, it is remarkable that the LD₅₀ values of all of the venoms tested to date against cotton worm cluster in the range 69 - 242 mg/g.

Results indicated the important role of feeding period on treated leaves for 48 h followed by untreated leaves for 3 days. So, the evaluation was assessed using the cumulative mortalities. In this study mortality calculated after 72 hrs after each application of either social bee or wasp venom. The initial effect that (calculated as the cumulative mortalities at zero time) recorded 100%, 100%, 92%, 88% and

26% for spraying, topical on food, injection and on integument, respectively. Control recorded 1% mortality. Both topical application methods gave the highest significant mortalities compared to the control that manifested 0.76313 ± 0.43 and 0.921 ± 0.115 respectively for honey bee and 1.0169 ± 0.053 for wasp venoms (F_(4,389) = 6.56; P < 0.0001) (F_(4,389) = 35.65; P < 0.0001) (Table 3 and Table 4).

Cotton worm treatment with bee venom has accordingly reduced survival times at concentrations (0.04, 0.08, 0.16 ppm). Reduced survival times have reached 87% and 72% for A. mellifera while it was higher reached 98 and 100% for Polistes sp. (Table 4). For topical application on food and integument drop when compared with control. Caterpillars infected by spraying bee venom demonstrated significant survival reduction effects (F_(4,389) = 25.59; P < 0.00001) (F_(4,389) = 5.12; P < 0.0001) (Figure 3). It was low for both bee and wasp venoms effect on cotton worm mortality compared to infection by venom topical applications.

Topical application on food of bee venom indicated the mortality of S. littoralis was significantly different (χ² = 68.13; d.f. = 2; P < 0.0001) among applications with highest number on Polistes sp. than A. mellifera but lowest mortality on injection of both bee and wasp venoms respectively (Figures 4(a)-(d), Table 3 and Table 4).

Data represents the mean value ± S.E. from 25 cotton leaf worm/group with significance difference between the five different concentrations, using Chisquare test (χ² = 52.11, d.f. = 28, P ≤ 0.0037).

Data represents the mean value ± S.E. from 25 cotton leaf worm/group with significance difference between the five different concentrations, using Chisquare test (χ² = 47.43, d.f. = 28, P ≤ 0.01).

Different application methods of crude venom toxins on integument or into the hemolymph had not compromise the same mortality effect derived from species selectivity. Spraying and injection application of a high concentration (1 - 1.16 ml^–1) of bee venom to cotton leaf worm caused significant morphological differences. However, by passing the cuticle by topical dropping and topical food applications of either social bee or wasp venom into the hemolymph (1 - 1.16 ml^–1 per insect) caused 100% mortality in both species within 72 h. expressing different application effects to insect pests could be remedied by applying the venom in ways that targeted the pest.

Wasp venom applied on S. littoralis has the largest effect with (χ² = 53.10; d.f. = 2; P < 0.0001 for topical on food application and χ² = 12.18; d.f. = 2; P < 0.002 for integument application) which is higher than bee venom mortality and toxicological effects (Figures 5(a)-(d)).

Reduced cumulative survival by bee venom spraying caused 60% for bee and 75% for wasp (F_(4,389) = 9.98, P ≤ 0.004) mortality for the highest concentration 0.16 while reached 75% by spraying Polistes sp. Venom compared with infection by injection that has the lowest effect for bee causing 25% mortality and 83% for wasp venoms on Spodoptera littoralis (Figure 6 and Figure 8).

The mean of residual effect that calculated as the mean of cumulative mortalities from day 1 until day 5 after applying venom topically, injection and by spraying were manifested in (Table 1 and Table 2). Topical application on food and integument were detected the highest significant mortalities effect, whereas injection recorded the least significant mortality effect (18.40%) (18.40%) (F_(4,389) = 18.78; P < 0.001)

(F_(4,389) = 12.46; P < 0.0001) as compared to other venom application (Figure 7).

All treatments caused significant increases in total mortality and morphological changes during the larval until pupal stage and the sequential combined effect treatments had more decreasing effect than the individual treatments. According to the obtained result, bee and wasp venom could be considered as a biopesticide, and become more effective when used in sequential treatment (Table 2 and Figure 6).

Furthermore, we demonstrate that, despite significant differences in their venom most venom have strikingly similar insecticidal effectiveness. The fact that similar social bee and wasp venoms are not acquiring the same mechanism of action on lepidopteran insects suggests from results that bee venom toxins have a conserved mode of action. However, the precise role of their toxins identified as social insects still remains to be determined within our study.

Wasp venom applied on S. littoralis has the largest effect with (χ² = 53.10; d.f. = 2; P < 0.0001 for topical on food application and χ² = 12.18; d.f. = 2; P < 0.002 for integument application) which is higher than bee venom mortality and toxicological effects (Figure 7 and Figure 8).

All treatments caused significant increases in total mortality and morphological changes. According to the obtained result, bee and wasp venom could be considered as a biopesticide, and become more effective when used in sequential treatment.