الفهرس 
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Abstract
The present study aimed to investigate the effect of the natural additives (mint oil, sesame oil, chamomile oil and green tea oil) and the chemical additives (zinc sulfate, sodium carbonate, magnesium sulfate and citric acid) on the potency of the two entomopathogenic bacterial formulations; Spinosad and Bacillus thuringiensis (B.t) subsp. kurstaki represented in the commercial products; Tracer and Bay-8, respectively against the 2nd instar larvae of the cotton leaf worm, Spodoptera littoralis (Boisd.), (Lepidoptera : Noctuidae).
The obtained results could be summarized as follow:
1. Physico-chemical properties:
Suspensability test was evaluated in order to investigate the compatibility of the tested additive materials with the tested bio-insecticides solutions. The obtained results showed that all the tested additives were compatible with the tested bio-insecticides.
The obtained results indicated that the viscosity of Spinosad and B.t solutions increased after addition of the tested natural oil additives with the exception of green tea oil. In case of Spinosad solution, addition of green tea oil slightly reduced its viscosity. On the other hand, the viscosity values were the same in both B.t solution and B.t-green tea oil mixture.
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The current data cleared that the pH value of Spinosad and B.t solutions increased after addition of the natural oil additives (mint, sesame, chamomile and green tea oils). In addition, the tested chemical additives; ZnSO4, Na2CO3 and MgSO4 also increased the pH value of the resulting solutions to be more alkaline, while the additive citric acid reduced the pH value of the resulting solution to be acidic.
2. Toxicological and biological studies:
2.1. Effect of the tested additives on the larval mortality and some biological aspects of S. littoralis:
The effect of each tested additive material on the 2nd instar larvae of S. littoralis was evaluated. The obtained results revealed that the tested natural oil additives at concentration 0.3% and the chemical additives at concentration 0.1% had very low toxicity against the target pest as compared with the untreated larvae. In case of treatments with the natural additives, the corrected accumulated larval mortality percentages were 3.46, 3.46, 6.90 and 0.00% for mint, sesame, chamomile and green tea oils, respectively. In case of treatment with the chemical additives (ZnSO4, Na2CO3, MgSO4 and citric acid), they were 10.34, 0.00, 6.90 and 3.46%, respectively compared to those in the untreated larvae that recorded 3.33% larval mortality. Therefore, these additives could be considered nontoxic at the tested concentrations.
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2.2. Effect of Spinosad and B.t separately on the larval mortality and some biological aspects of S. littoralis:
Bioassay studies showed that, the LC50 value of the bio-insecticide Spinosad after 72h of treatment of S. littoralis 2nd instar larvae recorded 20.482ppm. On the other hand, the LC50 value of B.t was 7.079ml/L. The data also indicated that the accumulated larval mortality percentages increased with increasing the concentrations of the tested bio-insecticides. Furthermore, both weights of the 6th instar larvae and pupae were lower than that of the untreated ones. Moreover, the percentages of pupation and adult emergence were reduced with increasing the concentration of the tested bio-insecticide.
2.3. Effect of the tested additives on the toxicity of Spinosad and B.t against S. littoralis 2nd instar larvae:
Addition of each of the natural or chemical additives greatly enhanced or synergized Spinosad toxicity against S. littoralis 2nd instar larvae with the exceptions of Spinosad-green tea oil and Spinosad-Na2CO3 mixtures, where they had antagonistic effect on Spinosad toxicity. The LC50 value of Spinosad after 72h of treatment was reduced after addition of 0.3% (mint, sesame and chamomile oils) from 20.482ppm to 8.068, 11.603 and 14.394ppm, respectively. In contrast, addition of 0.3% green tea oil increased the LC50 value of Spinosad to 106.612ppm. On the
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other hand, the addition of the chemical additives (ZnSO4, citric acid and MgSO4) also reduced Spinosad LC50 value to be 12.805, 17.719 and 18.048ppm, respectively. Whereas, addition of 0.1% Na2CO3 increased Spinosad LC50 value to be 47.317ppm after 72h of treatment.
On the other hand, in case of B.t treatment addition of 0.3% mint, sesame and chamomile oils synergized the toxicity of B.t and reduced its LC50 value after 72h of treatment from 7.079ml/L to 3.549, 4.388 and 5.568ml/L, respectively. In contrast, addition of 0.3% green tea oil antagonized B.t toxicity and increased its LC50 value to be 11.245ml/L at the respective time post treatment. All the tested chemical additives (Na2CO3, ZnSO4, citric acid and MgSO4) synergized B.t toxicity and reduced its LC50 value to be 3.972, 4.193, 5.268 and 5.885ml/L, respectively.
The data revealed that 0.3% mint oil and 0.1% ZnSO4 were the most efficient additives in enhancing Spinosad toxicity against the treated larvae. On the other hand, 0.3% mint oil also and 0.1% Na2CO3 were the most efficient additives in enhancing B.t toxicity. Therefore, these combinations were subjected to further biochemical studies on the treated larvae.
Biochemical studies:
This part dealing with evaluation the effect of the LC50 values of Spinosad and B.t (only or in combination with the most efficient additives) on some biochemical parameters of S. littoralis larval body homogenate after 120h of treating the 2nd instar larvae. Total protein content and the activity of the enzymes acetylcholinesterase (AChE), α- and β-esterases, alkaline and acid phosphatases (ALP and ACP), glutathione-S-transferase (GST) and phenoloxidase (PO) were determined.
3.1. Total protein content:
Data indicated that there was a significant reduction in the total protein content in the treated larvae with Spinosad as well as its mixture with mint oil or ZnSO4 by percentages of change -14.027, - 37.544 and - 19.855, respectively.
On the other hand, treatment with B.t and its mixtures with mint oil or Na2CO3 showed also significant reduction in total protein content by percentages of change - 11.655, - 24.446 and - 20.835, respectively compared with the untreated one.
3.2. Acetylcholinesterase (AChE) activity:
The results recorded a significant reduction in AChE activity in the treated 2nd instar larvae with Spinosad (only or in combination with the additives) compared to the untreated larvae. There was also a significant decrease in the enzymatic activity
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when Spinosad combined with additives compared to treatment with Spinosad only. The percentages of change recorded (- 9.444, - 55.556 and - 31.667) for Spinosad, Spinosad-mint oil and Spinosad-ZnSO4 mixtures, respectively.
3.3. α- and β-esterases activity:
In Spinosad treatments α-esterase activity showed significant reduction with percentages of change (- 9.273, - 8.698 and - 21.852) for Spinosad, Spinosad-mint oil and Spinosad-ZnSO4 mixtures, respectively compared with the untreated larvae. In contrast, in case of B.t treatment the activity of α-esterase showed a significant increase with percentage of change 10.812. The two mixtures, B.t-mint oil and B.t-Na2CO3 induced significant reduction in α-esterase activity with percentages of change - 6.918 and - 19.831, respectively.
On the other hand, β-esterase activity showed a significant reduction in the treated larvae with Spinosad, Spinosad-mint oil and Spinosad-ZnSO4 mixtures by percentages of change - 26.698, - 34.887 and - 15.482, respectively. Inversely, β-esterase activity showed a significant increase in the treated larvae with (B.t and B.t-Na2CO3 mixture) by percentages of change 16.114 and 1.553, respectively. In case of B.t–mint oil mixture, β-esterase activity showed a significant decrease with percentage of change -18.299.
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3.4. Alkaline and acid phosphatases (ALP and ACP) activity:
The results cleared that there was a significant reduction in ALP activity with percentages of change (- 42.450, - 25.938 and - 17.438) for Spinosad, Spinosad-mint oil and Spinosad-ZnSO4 mixtures, respectively compared with the untreated larvae. In contrast, in case of B.t treatment the activity of ALP showed a significant increase with change percentage 2.949. B.t-mint oil mixture had a significant decrease in the enzyme activity with percentage of change - 10.086. The mixture of B.t-Na2CO3 showed a non-significant decrease in ALP activity with percentage of change - 0.163.
On the other hand, ACP activity indicated a significant reduction in the treated larvae with Spinosad, Spinosad-mint oil and Spinosad-ZnSO4 mixtures by percentages of change - 11.827, - 5.061 and - 8.365, respectively compared to the untreated larvae. Inversely, ACP activity showed a significant increase in the treated larvae with (B.t and B.t-Na2CO3 mixture) by percentages of change 42.941 and 23.228, respectively. In case of treatment with B.t–mint oil mixture ACP activity showed a non-significant increase with change percentage 0.959 compared with the untreated larvae.
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3.5. Glutathion S-transferase (GST) activity:
The present work reflected that there was a significant reduction in GST activity in the treated larvae with (Spinosad, Spinosad-mint oil and Spinosad-ZnSO4) by percentages of change - 9.178, - 15.942 and - 14.010, respectively compared with the untreated larvae.
On the other hand, treatment with (B.t and B.t-Na2CO3 mixture) induced significant increase in GST activity of the treated larvae by percentages of change 36.232 and 19.323, respectively compared with the untreated larvae. Meanwhile, B.t-mint oil mixture treatment indicated a non-significant increase in GST activity by percentage of change 1.449.
3.6. Phenoloxidase (PO) activity:
The results showed significant increase in the activity of PO by percentages of change 66.661 and 35.016 for treatments with Spinosad and Spinosad-ZnSO4 mixture, respectively. Whereas, phenoloxidase activity showed a non-significant increase in the treatment with Spinosad-mint oil mixture with small percentage of change 4.952 compared to the untreated larvae.
On the other hand, treatments with (B.t, B.t-mint oil and B.t-Na2CO3 mixtures) showed significant increase in the activity of phenoloxidase with percentages of change 42.405, 33.972 and 16.566, respectively.