الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
To date, Bacillus sphaericus is one of the most successful microbial larvicide for the control of Culex pipiens. Commercially available B. sphaericus biolarvicides are based on highly toxic bacterial strains characterized by their ability to express the binary (Bin) protoxin, a crystal protein produced in large amounts during sporulation. This heterodimer is formed by the Bin A (42-kDa) and Bin B (51-kDa) subunits that act in synergy to produce larvicidal activity upon Culex sp. larvae.
The Bin B subunit is responsible for the recognition and binding of the toxin to specific receptors on the midgut epithelium surface , while Bin A is primarily responsible for the toxic effects; but first the crystal has to be ingested by the larvae and the protoxin must be processed into toxin by the midgut enzymes.
The active insecticidal ingredients of Allium sativum is diallyl disulphide as organo-sulphers. Monoterpenes, including p-cymene, α-thujene, γ-terpinene, carvacrol, α-pinene and β-pinene, are the main constituents in the majority of Nigella sativa plant essential oils giving plants their unique odoriferous properties and bind to esterases inhibiting insect enzymes, interrupt insect protein configuration and inhibit cholinesterase. The natural pesticidal properties of organo-sulphers and monoterpenes make them useful as potential alternative pest control agents especially in their toxic effect on Culex pipiens.
1- Mosquito strain rearing:
Culex pipiens used in this study was collected from El- Gabal- El- Asfar in New Marge at (30°12’48”N 31°24’4”E). Field strains was used to start a self-perpetuating colony in Department of Entomology, Faculty of Science, Ain-Shams University under standard conditions of rearing, (27 2ᵒC), (70-80%) relative humidity and 16 hrs photoperiod.
2- Bioassay tests:
The larval bioassay tests were carried out according to the standard World Health Organization larval bioassay test method (WHO, 2005). Stock solution of Bacillus sphaericus 2362 and plant essential oils of garlic and black seed were prepared to obtain four concentrations of (0.1. 1, 10, 100 ppm.).
Observation on larval mortality was recorded till 48 hrs. Larvae were considered dead when they did not react to/with touching. Mortality data were subjected to probit analysis to analyze lethal Concentrations and lethal time (LC50, LC95 and LT50, LT95). Finally the data were corrected by Abbott’s formula
Results proved that Bacillus sphaericus was the most potent insecticide recording LC50 value as (0.25 ppm.) followed by Allium sativum and Nigella sativa recording (0.45 and 2.46 ppm.), respectively. The potentiation effect of B. sphaericus with Allium sativum and synergistic effect of B. sphaericus with Nigella sativa was investigated too.
Recording the time needed to induce mortality to Culex pipiens larvae, the data showed that 3 hrs. after application of Allium sativum started to induce kill ranging from 2% (at 0.1 ppm) to 46 % (at 100 ppm), followed by Nigella sativa recording mortality percentages ranging from (1%-28%) at the same concentrations. Bacillus sphaericus could not induce any mortality for 12 hrs. post application at low or high concentrations.
LT50 values at concentration (100 ppm.) showed that garlic oil needs 4 hrs. to kill half of the population, followed by black seed oil at 10.3 hrs., while Bacillus sphaericus need 38.7 hrs. to kill 50% of population. Statistical analysis proved a positive relationship between time intervals and mortality percentages in all treatments.
Data also showed that a combination between Bacillus sphaericus and Allium sativum at low concentration (0.1 ppm) can kill 4% of the insect larvae after 3 hrs. Bacillus sphaericus and Allium sativum 58% mortality was recorded at concentration 100 ppm after 6 hrs. While Bacillus sphaericus alone had no effect during the first 6 hrs. LT50 at the highest concentration for Bacillus sphaericus (100 ppm.) was (38.7 hrs.)
After mixing with Allium sativum LT50 decreased to (2.2 hrs.) which proved the potentiation effect between Bacillus sphaericus and Allium sativum. Mixing Bacillus sphaericus with Nigella sativa can induce mortality between (1%: 3%) at concentrations ranged between (0.1: 100 ppm.). By comparing LT50, the value decreased to be (8.3 hrs.) After mixing with Bacillus sphaericus, while Bacillus sphaericus recorded LT50 equal to (38.7 hrs.). Mixing of Nigella sativa with Bacillus sphaericus can kill 55% of the insect population after (12 hrs.) which indicate synergistic (additive) effect. While Bacillus sphaericus alone recorded 0% as mortality percentage after (12 hrs.) post treatment.
from laboratory evidence presented in this study and based on LC50 values, the results proved that most potent bio-insecticide applied alone was Bacillus sphaericus recording LC50 value equal (0.25 ppm.), followed by Allium sativum with LC50 equal (0.45 ppm.). The combination between B. sphaericus (microbial insecticide) and Allium sativum oil increased potency of the bio-insecticide. The Co-toxicity factor value of B. sphaericus and Allium sativum was 25 which indicate potentiation in using this combination. It also potentiates the insecticide activity (3 hrs.) after treatment which is not the case after using B. sphaericus alone. Moreover, the Co-toxicity factor of the combination between B. sphaericus and Nigella sativa show additive effect with value equal 16.6.
This is very important point, as one of the major obstacles of bacterial insecticides in Middle East zone is the long exposure to sun light and UV. radiation which inhibit spore germination during field application, and affect the bacterial cycle for toxin production. Decreasing the time needed to induce mortality from (38 hrs.) to (2 hrs.) will overcome the time of exposure of B. sphaericus to the day light and UV. increasing its efficiency.
3- Biochemical characterization of larval proteins:
One-dimensional gel electrophoresis was carried out in vertical polyacrylamide gels. Larval body proteins were analyzed by SDS-polyacrylamide gel. Electrophoresis conditions and procedures were as described according to SDS-molecular standard mixture of proteins; 200 kDa : 10 kDa (from Sigma) was used as a marker with a 4% stacking gel and a 10% separating gel, at 100 volts for (5 hrs.) at room temperature. After electrophoresis, proteins were stained for (2 hrs.) in Coomassie brilliant blue (CBB.R-250), then distained for (24 hrs.) till visualization of bands.