الفهرس 
ACKNOWLEDGEMENTOnly 14 pages are availabe for public view
 |  | from | 143 |  |  |
| | | from | 143 | | |
Abstract
IV.1. Development of fenitrothion resistance in Culex pipiens larvae
To induce fenitrothion resistance in Culex Pipiens larvae, about 10000 4th instar larvae of Cx. Pipiens were collected from the field and exposed to fenitrothion selection pressure for fifteen generations using dipping method. This study revealed that:
The resistance ratio (RR50) of the parent strain (G0) was 16.07 fold. This RR50 value increased gradually from each generation to the next one as a result of selection pressure with fenitrothion. The regression line for fenitrothion against the parent field strain showed slope value as 2.28 which indicate a moderate level of heterogeneity of the parent strain to fenitrothion. The first four generations during the course of fenitrothion selection the development of resistance (RR50) was increased gradually in high rate from one generation to the next (The RR50 increased from 16.07-fold in G0 to 63.29-fold resistance in G4). By the 5th generation, the rate of resistance development to fenitrothion was fairly rapid where; the RR50 to fenitrothion increased during the 5th to the 8th generations from 99.51-fold in G5 to 397.18-fold resistance in G8. With the beginning of the 9th generation, the development of resistance to fenitrothion was increased very slowly during the last seventh generation from 400.55-fold in G9 to 426.70-fold resistance in G15 (plateau phase). Finally the slope was a volatile until the ninth generations (2.26 – 4.57) and then relatively proven in the last five generations (3.46 – 4.18). This is evidence of increasing homogeneity of strain in the last five generations.
IV.2. Development of propoxur resistance in Culex pipiens larvae
To induce propoxur resistance in Cx. Pipiens larvae, about 10000 4th instar larvae of Cx. Pipiens were collected from the field and exposed to propoxur selection pressure for fifteen generations using dipping method. This study revealed that:
The resistance ratio (RR50) of the parent strain (G0) was 1.35 fold. This RR50 value increased gradually from one generation to the next one as a result of selection pressure with propoxur. The regression line for propoxur against the parent field strain showed slope value as 1.90 which indicates a moderate level of heterogeneity of the parent strain to propoxur. The first five generations during the course of propoxur selection, the development of resistance (RR50) increased gradually from one generation to the next (The RR50 increased from 1.351-fold in G0 to 265.802-fold resistance in G5). By the 6th generation, the rate of development of resistance to propoxur was fairly rapid where the RR50 to propoxur increased during the 6th to the 11th generations from 265.802-fold in G6 to 1494.40-fold resistance in G11. With the beginning of the 12th generation, the rate of development of resistance to propoxur was increased very slowly during the last four generations from 1584.38-fold in G12 to 1688.90-fold in G15 (plateau phase).
Finally the slope values were volatile until the tenth generations (1.90 – 4.19) and then relatively proven in the last six generations (2.99 – 4.28). This is considered evidence of increasing homogeneity of the selected strain in the last six generations.
IV.3. Cross-resistance spectrum in a fenitrothion-resistant strain (FEN-R-Strain)
To investigate the cross-resistance spectrum in FEN-R-strain (426.7-fold resistance to fenitrothion) of Cx. Pipiens larvae, toxicity of thirteen insecticides including organophosphate (chlorpyrifos, diazinon and malathion), pyrithroids (cypermthrin, deltamthrin, fenvalerate and permethrin), carbamate (carbaryl, methomyl and propoxur) bioinsecticids (avermectin and spinosad) and an insect growth inhibitor (pyriproxyfen) were examined against 4th instar larvae of FEN-R-strain and S-strain. Results indicate that:
The FEN-R-strain of Cx. Pipiens larvae exhibited different levels of cross-resistance to the tested insecticides. The cross-resistance to the tested in the present study could be ranked in descending order of resistance ratio (RR50) as follow:
The FEN-R-strain proved to have a very high degree of cross-resistance to all the tested organophosphates but in different levels. Cross-resistance to malathion (RR50 = 634.86-fold) was slightly higher than of fenitrothion (RR50 = 426.70-fold).
Larvae of the FEN-R-strain displayed slightly high cross-resistance to the pyrethroid insecticide deltamthrin (RR50 = 22.32-fold) moderate cross-resistance to cypermthrin (RR50 = 16.90-fold) and permthrin (RR50 = 12.64-fold) and the least cross-resistance was to fenvalerate (RR50 = 1.67-fold). As expected, cross-resistance to insecticides structurally unrelated to the selecting OP-insecticide (fenitrothion) was far less than that of other OP-insecticids.
Larvae of the FEN-R-strain displayed slightly high cross-resistance to the carbamate insecticide propoxure (RR50 = 10.18-fold), low cross-resistance or tolerance to carbaryl (RR50 = 4.13-fold) and very low tolerance to methomyl (RR50 = 1.96-fold).
Larvae of the FEN-R-strain displayed slightly high cross-resistance to the bioinsecticide spinosad (RR50 = 250.53-fold) and low cross-resistance or tolerance to avermectin (RR50 = 8.52-fold). Unexpected results for spinosad cross-resistance were found in larvae of the FEN-R-strain.
Larvae of the FEN-R-strain displayed slightly low tolerance to the insect growth inhibitor insecticide pyriproxyfen (RR50 = 10.30-fold).
IV.4. Cross-resistance spectrum in a propoxur-resistant strain (PRO-R-Strain)
To investigate the cross-resistance spectrum in PRO-R-strain (1688.91-fold resistance to propoxur) of Cx. Pipiens larvae, toxicity of thirteen insecticides including carbamates (carbaryl and methomyl) organophosphates (chlorpyrifos, diazinon, fenitrothion and malathion), pyrithroids (cypermthrin, deltamthrin, fenvalerate and permethrin), bioinsecticids (avermectin and spinosad) and an insect growth inhibitor (pyriproxyfen) were examined against 4th instar larvae of PRO-R-strain and S-strain.
Results indicate that:
The PRO-R-strain of Cx. Pipiens larvae exhibited different levels of cross-resistance to the tested insecticides. The cross-resistance to the tested insecticides in the present study could be ranked in descending order according to the resistance ratio (RR50) as follow:
The PRO-R-strain proved to have a very high degree of cross-resistance to carbaryl (RR50 = 378.28-fold). On the other hand, the resistant strain had an even lower degree of cross-resistance toward methomyl (RR50 were 6.16-fold).
Larvae of the PRO-R-strain displayed slightly high cross-resistance to the organophosphate insecticides malathion (RR50 = 84.83-fold), fenitrothion (RR50 = 79.98-fold), chlorpyrifos (RR50 = 75.75-fold) and diazinon (RR50 = 34.41-fold).
Larvae of the PRO-R-strain displayed slightly moderate cross-resistance to the pyrethroid insecticide deltamthrin (RR50 = 13.86-fold), low cross-resistance to permethrin (RR50 = 5.65-fold) and cypermthrin (RR50 = 5.16-fold), while the least cross-resistance was to fenvalerate (RR50 = 1.32-fold).
Larvae of the PRO-R-strain displayed slightly high cross-resistance to the bioinsecticide spinosad (RR50 = 138.35-fold) and moderate cross-resistance to avermectin (RR50 = 12.92-fold). Unexpected results for spinosad cross-resistance were found in larvae of the PRO-R-strain.
Larvae of the PRO-R-strain displayed relatively high cross-resistance to the insect growth inhibitor insecticide pyriproxyfen (RR50 = 26.91-fold). Unexpected results for pyriproxyfen cross-resistance were found in larvae of the PRO-R-strain.
IV.5. Investigation of insecticide resistance mechanism(s) in FEN-R-strain and PRO-R-strain
IV.5.1. Synergism of insecticidal action
Quiet apart from various practical importances in using synergists, the fundamental investigation of synergism has led to much better appreciation of the mechanism of detoxication in insects and the basic biochemical processes involved in insecticide resistance.
IV.5.1.1. Synergism of fenitrothion action in larvae of the susceptible strain (S- strain) of Cx. Pipiens
The present study showed that very low levels of synergism (SR ranged from 1.10 to 1.27) were detected with fenitrothion by either using each synergist alone or using the three synergists together (insecticide + PBO + TPP + DEM, named the four way treatment) against susceptible strain. These results suggested that larvae of the susceptible strain did not have enough detoxification power towards fenitrthion.
IV.5.1.2. Synergism of propoxur action in larvae of the susceptible strain (S- strain) of Cx. Pipiens
The present study showed that very low levels of synergism (SR ranged from 0.99 to 1.09) were detected with propoxur by either using each synergist alone or using the three synergists together (propoxur + PBO + TPP + DEM, named the four way treatment) against susceptible strain. These results suggested that larvae of the susceptible strain did not have enough detoxification power towards propoxur.
IV.5.1.3. Synergism of fenitrothion action in larvae of the fenitrothion resistant strain (FEN-R- strain) of Cx. Pipiens
from the above results, it can be concluded that:
(1) PBO, TPP and DEM moderately synergized fenitrothion (SR = 3.12, 2.97 and 2.59-fold, respectively). The four ways treatment had the highest synergistic effect on the toxicity of fenitrothion against larvae of FEN-R-strain to the highest degree (SR = 12.36).
(2) A comparison between the synergistic effect (SR) of fenitrothion in the susceptible and FEN-R-strains (Table 6) with each synergist alone and / or with the three synergists together (the four way treatment) revealed that the degree of synergism was higher in the FEN-R-strain than in the susceptible one.
(3) A comparison between the degrees of synergism of fenitrothion with each synergist alone (the two way treatment) revealed that the oxidase inhibitor PBO was more efficient than the estrase inhibitor, (TPP) and the transferase inhibitor (DEM) in synergizing fenitrothion.
(4) The degree of synergism by TPP or DEM was less than that obtained by PBO for fenitrothion. These data suggested that the estrase(s) and GSTs in the FEN-R-strain of Cx. pipiens larvae play an important role in the toxicity of fenitrothion, but not as high as the role of oxidase (s). The four way treatment had more synergistic effect for fenitrothion against the FEN-R-strain larvae than using each synergist alone (the two way treatments). These results suggested that three metabolic detoxification enzymes, oxidase(s), esterase(s) and (GSTs) are likely involved in resistance of the FEN-R-strain larvae of Cx. Pipiens.
(5) Synergism in most cases of the FEN-R-strain larvae of Cx.pipiens clearly showed that the two way treatments and the four way treatment increased the slope values of the LCp-line of fenitrothion compared with the slope values of the LCp–line for fenitrothion alone. This finding means that using synergist(s) alone or together with fenitrothion increased the homogenetiy of the FEN-R-strain larvae in their response to the toxic action of fenitrothion.
IV.5.1.4. Synergism of propoxur action in larvae of the propoxur resistant strain (PRO-R- strain) of Cx. Pipiens
from the above results, it can be concluded that:
(1) PBO, TPP and DEM highly synergized propoxur (SR = 8.42, 7.15 and 8.02, respectively). The four ways treatment (propoxur + PBO + TPP + DEM) had the highest synergistic effect on the toxicity of propoxur against larvae of the PRO-R-strain (SR = 21.32).
(2) A comparison between the synergistic effect (SR) of propoxur in the susceptible and PRO-R-strains with the three synergists together (the four way treatment) revealed that the degree of synergism was higher in the PRO-R-strain than in the susceptible one.
(3) A comparison between the levels of synergism of propoxur with each synergist alone (the two way treatment) revealed that the oxidase inhibitor PBO was more efficient than the esterase inhibitor, TPP and the transferase inhibitor DEM in synergizing propoxur.
(4) The level of synergism by TPP or DEM was less than that obtained by PBO with propoxur. These data suggested that the esterase (s) and GSTs in the PRO-R-strain of Cx. pipiens larvae play an important role in the toxicity of propoxur, but not as high as the role of oxidase (s). The four way treatment had more synergistic effect for propoxur against the PRO-R-strain larvae than using each synergist alone (the two way treatment). These results suggested that three metabolic detoxification enzymes, oxidase(s), esterase(s) and (GSTs) are likely involved in conferring resistance to the PRO-R-strain of Cx. Pipiens.
(5) Synergism in most cases of the PRO-R-strain larvae of Cx.pipiens clearly showed that the two way treatments and the four way treatment increased the slope values of the LCp-line of propoxur compared with the slope value of the LCp–line for propoxur alone. This finding means that using synergist(s) alone or together with propoxur increased the homogeneity of the PRO-R-strain in their response to the toxic action of propoxur.
IV.5.1.5. The possible role of metabolic detoxification in insecticide resistance of the resistant strains of Cx. pipiens larvae
(A) FEN-R-strain:
These results indicate that:
(1) The presence of the oxidase inhibitor synergist (PBO) the resistance level was reduced for fenitrothion from 426.70 to 168.31 fold. This means that the percentage reduction in resistance level for fenitrothion by the oxidase(s) inhibitor synergist (PBO) was 60.56%.
(2) The presence of the esterase inhibitor synergist, TPP reduced the resistance ratio for fenitrothion. TPP reduced the resistance ratios toward fenitrothion from 426.70 to 158.58 fold. The percentage reduction in resistance level to fenitrothion was 62.84%.
(3) DEM decreased the RR for fenitrothion from 426.70 to 188.57 fold. The percentage reduction in RR values in the presence of DEM was 55.81%.
(4) The four way treatment reduced the RR values for fenitrothion from 426.70 to 43.82 fold. The reduction percentage of RR was 89.73%.
(5) The reduction in resistance level (RR) for fenitrothion indicate that the resistance level (RR alone) did not suppress completely by using the four way treatment (PB + TPP + DEM + insecticide) where the % reduction in RR was 89.73%. The results suggest that some other resistance mechanism(s), in addition to metabolic resistance, might be involved in larvae of the FEN-R-strain of Cx. pipiens.
(B) PRO-R-strain:
These results indicate that:
(1) The presence of the oxidase inhibitor synergist (PBO) the resistance level was reduced for propoxur from 1688.902 to198.54 fold. This means that the percentage reduction in resistance level for propoxur by the oxidase(s) inhibitor synergist (PBO) was 88.24%.
(2) TPP reduced the resistance ratio for propoxur. TPP reduced the resistance ratio toward propoxur from 1688.902 to 252.30 fold. The percentage reduction in resistance level to propoxur was 85.06%.
(3) DEM decreased the RR for propoxur from 1688.902 to 212.96 fold. The percentage reductions in RR values in the presence of DEM were 87.39%.
(4) The four way treatment reduced the RR value for propoxur from 1688.902 to 86.75 fold. The reduction percentage of RR value was 94.86 %.
(5) The reduction in resistance level (RR) for propoxur indicate that the resistance level did not suppressed completely by using the four way treatment (PB + TPP + DEM + insecticide) where the % reduction in RR was 94.86%. The results suggest that some other resistance mechanism(s), in addition to metabolic resistance, might be involved in larvae of the PRO-R-strain of Cx. pipiens.
IV.6. Biochemical assays of metabolic detoxification enzymes from susceptible and resistant strains of Cx. pipiens larvae
from the above results, it can be concluded that:
(1) Comparing the esterase activity from the susceptible and the resistant strains toward the four tested substrates indicated that the resistant strains exhibited higher activity than the susceptible strain with all the tested substrates.
(2) The EEN-R- and PRO-R-strains possesses higher level of GSTs activity than that of susceptible strain by 2.67 and 2.34-fold, respectively.
(3) The monooxygenase was more active in FEN-R- and PRO-R-strains than in susceptible strain by 5.54 and 5.14-fold, respectevly.
Finally, from the synergism studies and enzyme assays in the present investigation, it is suggested that the metabolic detoxification processes via general esterases, P-450 monooxygenases and GSTs play a significant role in conferring fenitrothion and propoxur resistance in larvae of the FEN-R-strain and PRO-R-strain of Cx. pipiens, respectevly. The results also suggested that other mechanisms such as target site insensitivity may be involved especially in resistance of larvae of the PRO-R-strain to propoxur.