الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Two novel nicotinoid insecticides (imidacloprid and acetamiprid) and two commercial bioinsecticides (viruset and profect) were investigated against one of the most serious insect pests in Egypt, the cotton leaf worm, S. littoralis. In order to diminish reliance on conventional insecticides, the interaction between them was studied. In order to study the insect immune response to these agents, biochemical and molecular studies on immune modulating enzymes and immune protein were also studied.
A. Bioassay studies:
1- Toxicity of the tested insecticides:
- Viruset significantly raised the mortality of the 4th instar larvae than Profect 48 hours post treatment.
- Imidacloprid was significantly more toxic on 2nd and 4th instar larvae than all tested compounds.
2- Successive application of tested compounds:
- All the combinations with lower concentrations (LC25) gave significantly more toxic effect and were superior in reducing larval population with enhanced effect than when they were applied individually.
- The successive application of LC25 of profect followed by treatment with LC25 of acetamiprid was promising in reducing the 2nd larval instar population.
- The successive application of LC25 of viruset followed by acetamiprid was promising in reducing the larval population.
B. Biochemical studies:
1- Change of total protein content of S. littoralis upon treatment with Neonicotinoids and Bioinsecticides.
- The neonicotinoid application raised the total protein content in contrast to bioinsecticides that caused a decrease in total protein content.
- Acetamiprid treatment significantly increased the total protein content 24 hours post treatment in comparison with both control and imidacloprid treatment.
- Viruset treatment significantly decreased the total protein content 48 hours post treatment in comparison with both control and profect treatment.
2- Change of total Soluble Protein Profile of S. littoralis upon Treatment with Neonicotinoids and Bioinsecticides.
- Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) showed that proteins of supernatant of untreated and treated 4th instar larvae were separated into 21 different bands by using COBB stain.
- The total number of bands in larvae of control and treatments with acetamiprid, imidacloprid, viruset and profect were 9,11,10,13 and 11 respectively.
- The molecular weight of bands in all samples ranged from11 to 255 KDa.
- Treatment with bioinsecticides led to the expression of 6 unique protein bands; three bands (No. 2, 13 and 20) appeared only with profect treatment with M.W. 148, 39 and 12 KDa. respectively.
- Three unique bands (No. 10, 16 and 17) were expressed only in case of viruset treatments with M.W. 47, 27 and 19 KDa. Respectively.
- Treatment with both acetamiprid and imidacloprid led to the expression of a polymorphic band (No. 19 with M.W. 16 KDa). In contrast, the same treatments led to the disappearance of band No. 18.
- 3- Enzyme assay:
3-1- Change of nonspecific esterase activities in the 4th instar larvae upon treatment with bioinsecticides.
- The α–esterase and β-esterase enzyme activity significantly increased in all treated larvae after 48 hours of exposure compared to the control. The highest activity is also observed in case of profect application 48 hours post treatment.
3-2-Change of Acid& Alkaline phosphatase activity in the 4th instar larvae upon treatment with bioinsecticides.
- The Alkaline phosphatase enzyme activity significantly increased in all treated larvae after 48 hours of exposure compared to the control. The highest activity is observed in case of viruset application 48 hours post treatment.
- The Acid phosphatase enzyme activity significantly increased in all treated larvae after 48 hours of exposure compared to the control with no significant difference between profect and viruset effects on the enzyme activity.
3-3- Change of Acetylcholinesterase (AChE) activity in the 4th instar larvae upon treatment with neonicotinoid insecticides.
- The enzyme activity increased significantly when using both imidacloprid and acetamiprid in comparison to control.
- Imidacloprid–treated larvae showed significantly higher enzyme activity by a magnitude of 2.0 than acetamiprid –treated ones.
3-4- Change of Glutathione S-transferase (GST) activity in the 4th instar larvae upon treatment with neonicotinoid insecticides.
- The enzyme activity significantly decreased 24 hours post treatment when using both imidacloprid and acetamiprid in comparison to control.
- The decrease of enzyme activity showed no significant difference between both treatments.
3-5- Change of cytochrome (P450) monooxygenase activity in the 4th instar larvae upon treatment with neonicotinoid insecticides.
- Imidacloprid caused insignificant decrease of the enzyme activity 24 hours post treatment.
- Acetamiprid caused insignificant increase of the enzyme activity 24 hours post treatment.
C. Molecular studies:
1- Detection of Transferrin transcript abundance in S. littoralis larvae upon infection with bioinsecticides.
- A fragment of 215 base pair (bp) corresponding to the transferrin transcript was detected in control and treated larvae.
- Transferrin (Tsf) transcript was detected in control and treated insect with LC25 and LC50 of both viruset and profect after 24 and 48 hours post infection.
1-1- Effect of viruset and profect on the relative expression of Tsf gene in S. littoralis.
- There was a significant decrease of Tsf gene expression level in S. littoralis post treatment with LC25 and LC50 of viruset compared to control groups and profect treatments. The effect was the same after 24 and 48 hours with different magnitude.
- There was a significant downregulation in Tsf gene expression in larvae treated with LC50 of viruset compared with that treated with LC25.
- There was a significant upregulation in Tsf gene expression in profect treated with LC25 and LC50 while, LC50 treatment showed two folds expression level more than LC25, as compared to control group, thus it gradually increased with increase the time of the exposure.
- By increasing the time from 24 to 48 hours, the expression level of Tsf transcript in larvae treated with viruset started to increase but did not reach the normal value as in control group.
1-2- characterization of S. littoralis transferrin (SpliTrf) partial cDNA and its position in a molecular phylogeny:
RT- PCR amplification approach using specific primers derived from conserved domains of insect transferrins permitted cloning of the isolated 215bp partial cDNA sequence for SpliTsf that encodes a deduced -65aa peptide with a molecular mass of approximately 7120.94 Da and an estimated pI value of 5.52.
Blastn comparisons and Clustal W alignment with other insect transferins further confirmed its identity as SpliTsf which is also supported by the protein molecular mass and isoelectric point calculations.
The deduced molecular phylogeny of insect Tsf was examined using nucleotide sequences of Tsf from a number of insects belonging to different families within Lepidoptera (Fig. 9). S. littoralis Tsf shows the highest identity to S. litura (88%), transferrin molecule (NCBI) data base (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi).