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Abstract
The present study was carried out to investigate the role of esterases as a resistance mechanism in the larvae of Cx. pipiens resistant to the pyrethroid insecticide cypermethrin. The results can be summarized as follows:
1. Larvicidal toxicity of certain insecticides against susceptible laboratory strain of mosquito Cx. pipiens (Diptera: Culicidae)
Nine insecticides representing different classes or types with different modes of action were chosen to evaluate their toxicity against the 4th instar larvae of laboratory strain (S-strain) of Cx. pipiens. The tested insecticides in the present study were, five pyrethroids (cypermethin, alpha-cypermethrin, lambda-cyhalothrin, deltamethrin, fenvalerate), one organophosphorus (chloropyrifos), one carbamate (methomyl), one neonicotinoid (imidacloprid) and one bioinsecticides )spinosad(. The larval-dip bioassay method was used to evaluate the toxicity of the tested insecticides and toxicity (mortality was recorded 24th after treatment) was expressed as LC50 in ug a.i./L (µg/L). The results indicated that the pyrethroid insecticide alpha-cypermethrin was the most toxic insecticide (LC50 = 0.14 µg/L) followed by cypermethrin (0.170), lambda-cyhalothrin (0.211), deltamethrin (0.447), chloropyrifos (1.591), spinosad (2.895), fenvalerate (2.912), imidacloprid (39.02) and methomyl (243.86). The difference in toxicity between the most toxic insecticide (alpha-cypermethrin) and the least toxic one (methomyl) was up to 1742 fold.
2- Development of cypermethrin resistance in Cx. pipiens larvae
Larvae of Cx. pipiens field strain were exposed to cypermethrin at an LC50 for ten successive generations. The results indicated that the resistance ratio (RR50) of the parent field strain (G0) gradually increased by different rate from generation to the next as a result of selection pressure with cypermethrin. The RR50 values compared to the susceptible strain were 4.38, 5.61, 7.26, 10.26, 17.45, 51.15, 105.25, 365.78, 599.59, 1157.52 and 1785.58-fold at G0, G1, G2, G3, G4, G5, G6, G7, G8, G9 and G10, respectively.
3- Cross-resistance spectrum in CYP-R-strain
To investigate the cross-resistance spectrum in CYP-R-strain, toxicity of alpha-cypermethrin, lambda-cyhalothrin, deltamethrin, fenvalerate, chloropyrifos, methomyl, imidacloprid and spinosad were tested against 4th instar larvae of CYP-R-strain. Results indicate that the CYP-R-strain of Cx. pipiens larvae exhibited different level of cross-resistance to the tested insecticides, i.e., fenvalerate (1831.42-fold), alpha-cypermethrin (1586.01-fold), deltamethrin (619.76-fold), lambda-cyhalothrin (366.66-fold), chloropyrifos (23.19-fold), methomyl (16.16-fold), imidacloprid (5.97-fold) and spinosad (17.33-fold).
4-The mechanistic role of esterases in resistance of Cx. pipiens larvae
The esterase inhibitor synergist triphenyl phosphate (TPP) was used to study the effect of inhibiting esterases on toxicity of the nine candidate insecticides against the S-strain and CYP-R- strain of Cx. pipins larvae. The synergistic ratio (SR) showed that very low level of synergism (SR ranged from 0.99 to 1.34) was obtained with all the insecticides tested. The response of the CYP-R-strain larvae to the tested insecticides along with the esterase inhibitor TPP was somewhat distinguished. TPP increased the effectiveness of cypermethrin (the pressuring insecticide) to a very high degree (SR 13.45). With other pyrethroids, TPP synergized alpha-cypermethrin to 42.1-fold, deltamethrin to 7.5 fold; lambda-cyhalothrin to 2.35 fold and fenvalerate to 1.19 fold. On the other hand, TPP slightly synergized chlorpyrifos (SR= 1.23); methomyl (SR= 1.55), imidacloprid (SR= 1.52) and spinosad (SR= 2.76). The effectiveness of the esterases inhibitor synergist (TPP) on the resistance level of the tested insecticides in CYP-R-strain was determined and the results obtained indicate the following two points:
(a) TPP reduced the resistance level toward cypermethrin from 1785.58 to 134.56-fold (92.46% reduction); toward alpha-cypermethrin from 1586.01 to 39.777-fold (97.49% reduction), toward lambda-cyhalothrin from 366.6 to 203.59-fold (44.47% reduction); toward fenvalerate from 1831.42 to 1737.02-fold (5.15% reduction). Except for fenvalerate, the tested pyrethroids appear to be highly sensitive to esterases as a resistance mechanism in CYP-R-strain of Cx. pipiens.
(b) For tested insecticides other than pyrethroids TPP decreased the RR50 values of chlorpyrifos from 23.19 to 20.06-fold (13.5% reduction); of methomyl from 16.16 to 14.01-fold (13.3% reduction); of imidacloprid from 5.97 to 3.91-fold (34.51% reduction); and of spinosad from 17.33 to 6.21-fold (64.1% reduction).
5- Kinetics and frequency distribution of larval esterases from susceptible and cypermethrin selected strains
Double reciprocal plots for esterase activity toward -naphthyl acetate in larval homogenates of Cx. pipiens mosquito from susceptible and cypermethrin selected generations [G0 (parent field strain), G3, G5, G7, G9 and G10] were examined. The Michaelis-Menten constants, Km values were 2.01, 1.9, 2.17, 1.9, 1.8, 1.93 and 1.96x10-4 M for S-strain and Cypermethrin parent strain (G0), selected generations (G3, G5, G7, G9 and G10) respectively. The corresponding maximum velocity (Vmax) were 11.05, 14.47, 23.87, 35.71, 72.99, 144.93 and 217.39 nmol substrate hydrolyzing/min /mg protein, respectively. The kinetic constants from the present study suggest that the non-specific esterases play an important role in resistance of Cx. pipiens larvae to cypermethrin. This mechanistic role is likely due to be an over-production of the same esterase(s).
The frequency distribution of the esterase activity in S-, field parent and CYP-R strains was measured in homogenate of individual larvae from each strain. It clear from the distribution curves that:
(a) The difference between the highest and lowest activity of the esterases of individual larvae were 8.0, 16.0 and 3.5-fold for susceptible, field parent and CYP-R-strain, respectively.
(b) The mean of non-specific esterase activity among the tested individuals representing the susceptible, field parent and CYP-R-strain were 17.66, 61.31 and 216.36 nmol substrate hydrolyzing/min/larva, respectively. These values seem to be concomitant with the degree of cypermethrin resistance in these strains.
In general, the bioassay and biochemical data of this study has proven the hypothesis of esterase-mediated insecticide resistance in field population, as well as in laboratory-induced cypermethrin resistance, of C. pipiens larvae.