Development of pesticide resistance hampers the pest control program. It is therefore, imperative to evaluate the level of resistance in insects to launch a successful pest control strategy. The present study is aimed at determining the level of insecticide resistance in the Pak strain larvae and adult beetles of stored grain pests, Tribolium castaneum using their established resistant (CTC-12) and susceptible (FSS-ll) strains as controls. The present study also describes the efficacy of pirimiphos-methyl, an organophosphate insecticide and Fury, a synthetic pyrethroid, on the pest with particular reference to variety of esterases such as acetylcholinesterase (AChE), arylesterase (AE), carboxylesterase (CE) and cholinesterase (ChE), which have been taken as parameters of insecticide resistance. The activities of esterases were detennined biochemically and analyzed by polyacrylamide gel electrophoresis.
LC50 of pirimiphos-methyl against the 4th, and 6th instar larvae, and newly emerged and 15 days old beetles were 681, 1753, 4508 and 3.2 ppm, respectively, for Pak strain, 88, 743, 2674 and 1.08 ppm, respectively, for FSS-II strain, and 662, 5446, 5821 and 81 ppm, respectively, for CTC-12. The 15 days old adults were the most sensitive developmental stage. All the developmental stages of FSS-II showed comparatively lower LCsos than the other two strains.
The LC50 of Fury against Pak and FSS-II strains followed the same pattern as of pirimiphos-methyl. It was 405, 648, 560 and 121 ppm, respectively, for Pak strain, whereas it was 856, 1088,555 and 58 ppm, respectively, for the FSS-II strain. In CTC-12 strain the 4th and 6th instar larvae, and newly emerged and 15 days old adults had 1442, 1258, 1420 and 671 ppm for LC50. The 15 days old beetles had much less LC50 as compared with the other developmental stages.
The total esterase (TE) activity of the three strains decreased during the developmental process. The Pak and FSS-ll strains showed approximately the same TE level, whereas CTC-12 strain had higher activity when compared with the FSS-ll strain. The TE activity was significantly inhibited in the four stages of the three strains (Pak 54, 48, 69 and 59%; FSS-ll 80, 69, 62 and 6%: CTC-12 73, 62, 67 and 71%) after pirimiphos-methyl treatment. The Fury treatment, on the other hand, caused decrease (15%) in the 4th instar larvae of Pak strain, and all stages of the FSS-ll strain (17, 26, 20 and 36%). In CTC-12 strain the TE activity of 15 days old beetles increased (24%) after Fury treatment, while the remaining stages showed no significant change.
The CE activity decreased with the increasing age in all the three strains, al}d the CTC 12 strain showed the highest activity in all of its developmental stages. The pirimiphos-methyl treatment significantly decreased the CE activity in all the developmental stages (4th and 6th instar larvae, newly emerged and 15 days old beetles) of Pak strain (56, 29, 76 and 66%), FSS-II strain (81, 75, 64 and 27%) and CTC-12 strain (61, 58, 51 and 48%). The Fury treatment however did not show any significant change in the CE activity of all the developmental stages of Pak strain. The 4th and 6th instar larvae of FSS-ll and 4th instar larvae of CTC-12 strain showed decreased CE activity, whereas all other developmental stages remained unaffected.
The AChE activity increased from 4th instar larvae to newly emerged adult stages in Pak and CTC-12 strain, whereas it decreased significantly in 15 days old adults of .both the strains. In FSS-II strain, the 4th, 6th instar larvae and 15 days old adult had the same level of AChE activity, while the newly emerged adults showed significantly higher activity when compared with 4th ins tar larvae. The 4th and the 6th instar larvae and newly emerged adults of Pak and CTC-12 strains had higher AChE activity when compared with the FSS-ll strain. The 15 days old adult beetles of the three strains had almost the same AChE activity. After the treatment with pirimiphos-methyl and Fury the AChE activity decreased significantly in all stages of the three strains.
The ChE activity in Pak and FSS-Il strains remained almost constant throughout the development. In CTC-12 the ChE activity remained the same except for 15 days old beetles, which showed significantly higher activity when compared to the 4th instar larvae. The Pak and CTC-12 strains have lower ChE activity in all the stages than FSS Il strain. After the treatment with pirimiphos-methyl, the ChE activity increased (65, 59 and 37%) only in the 4th and 6th instar larvae and 15 days old adult beetles of Pak strain, whereas it decreased (31 %) in the newly emerged adults. The various developmental stages in FSS-ll and CTC-12 strains however showed opposite response. ChE activity of 15 days old beetles increased (21 and 80%), white that of other stages decreased after the treatment with pirimiphos-methyl. The Fury treatment resulted in increased ChE activity in the larval stages of Pak and FSS-Il, white the adult stages were not affected. In CTC 12 strain the ChE activity decreased (46 and 67%) significantly in the 4th instar larvae and newly emerged adult after Fury treatment whereas in 15 days old adult the ChE activity increased (40%) significantly.
The AE activity in all the three strains decreased from 4th instar larvae to 15 days old adults. Pak strain showed the least and CrC-12 strain the highest AE activity in all the developmental stages when compared with the FSS-Il strain. The activity was significantly decreased after pirimiphos-methyl treatment. After Fury treatment, the 4th instar larvae of Pak strain exhibited a decrease (33%) and 6th instar larvae showed increased AE activity (38%) whereas other stages remained unaffected. The 15 days old beetles of FSS-ll and 4th and 6th instar larvae of CTC-12 strain showed significantly decreased AE activity, while the other stages showed non significant changes after treatment with Fury.
The total soluble protein contents of 4th instar larvae and newly emerged adults of the Pak strain were higher than in the other two stages. The 6th instar larvae of FSS-ll strains had higher soluble protein contents, whereas the newly emerged adults and the 15 day old beetles showed lower content when compared with the 4th instar larvae. In CTC 12 strains this content decreased during the development. The soluble protein contents of all the developmental stages of Pak and CTC-12 strains were higher when compared with the FSS-ll strain. After treatment with pirimiphos-methyl the soluble proteins of 4th instar larvae of Pak strain decreased (30%), while that of other strains increased. The other stages of the three strains did not show any significant change after insecticide treatment. The Fury treated 4th instar larvae showed significant decrease (30%), whereas newly emerged adults aild 15 days old beetles showed increased soluble protein contents (28 and 12%). The various developmental stages of FSS-ll and CTC-12 strains showed increase of total soluble protein contents after treatment with Fury.
The esterases extracted from Pak, FSS-ll and CTC-12 strains of T. castaneum, with and without treatment of insecticides, were run on the polyacrylamide gel and electrophoresed to obtain variety of esterases under different experimental conditions. Four inhibitors, diagnostic for particular classes of esterases, were tested for their ability to inhibit the activity of esterases: parachloromercurobenzoic acid was used as inhibitor of AE, eserine sulfate as inhibitor of AChE, paraoxon as inhibitor of ChE, and triphenyl . Phosphate as inhibitor of CE.
The control electrophorertic pattern of Pak and FSS-ll strains showed seven distinct esterases bands. The 4th and 6th instar larvae of both the strains showed CE, ChE, AE and AChE bands, whereas AChE (band No.6) was only visible in the FSS-ll strain. The newly emerged adults of FSS-ll had all the esterases, while the newly emerged adults of Pak strain had low level of CE, ChE and AE activity, whereas AChE was absent. In 15 days old beetles of Pak and FSS-II strains all the seven esterases bands were prominent. After pirimiphos-methyl treatment the ChE, AE and AChE of all the developmental stages in Pak and FSS-ll strains were completely inhibited. The higher molecular weight CE, however, was not much affected. The Fury treatment did not effect the esterase bands pattern, except for the AChE, which decreased in intensity in Pak and FSS-ll strains, whereas AE was at the verge of disappearance.
The CTC-12 control pattern comprises of eight esterase bands in newly emerged adults and 15 days old beetles. The 4th and 6th instar larvae had almost similar pattern except for the absence of AChE (band No.8). The pirimiphos-methyl treatment generally reduced the intensity of all the esterases. The AE and AChE (bands 5, 7, 8) were completely inhibited in the newly elilerged adults and 15 days old beetles after insecticide treatment, however, the one band of AChE (band 6) remained unaffected. The Fury treatment had little effect on all the esterases except the bands No. 7 and 8 (AChE) in newly emerged adults and 15 days old beetles.
The esterases of all the development stages of Pak and FSS-II strains were significantly affected after pirimiphos-methyl treatment, whereas fury treatment comparatively did not produce that drastic effect. In CTC-12 strain esterase bands were decreased in intensity after treatment with both the insecticides whereas in other strains these bands were completely in all the development states.