MATERIALS AND METHODS

Insects
A susceptible laboratory strain was collected in 1997 and has remained in culture without any exposure to insecticides. A field strain was collected from Beheira governorate in July 2001. All insect strains were reared on cotton plants in the laboratory as described by Coudriet et al. (1985).

Insecticides and Synergists Used
Insecticides used in this study were: Thiamethoxam (Actara 25% WP), profenofos (Selecron 72% EC). Synergists used in this study were: DEF (S,S,S-tributylphosphorotrithioate), diethylmaleate (DM), Piperonyl-butoxide (PB) and tricresylphosphate (TCP). Test concentrations were prepared by diluting the insecticides in water.

Bioassay
The bioassay method for obtaining concentration-response was described by Prabhaker et al. (1985). Briefly, attached cotton leaves were dipped for 5 sec in 100 mL of the desired concentration of each insecticide. Twenty adult whiteflies were exposed to the treated leaves confined in small cages of polyurethane foam each concentration and replicated five times. Mortality was recorded 24 h after treatment and LC₅₀ values were determined on the bases of standard probit analysis.

A field strain was subjected to laboratory selection pressure with each insecticide i.e., profenofos or thiamthoxam at a level producing 50% mortality to the adult stage. The level of developing resistance was determined at generations 3, 5, 7, 9 and 11 for each resistant strain Resistance Ratio (RR) were determined by dividing the LC₅₀’s of the R-strain by the LC₅₀’s of the S-strain and relative resistance were determined by dividing the LC₅₀’s of the R-strain by the LC₅₀’s of the field-strain. The results of selection and development of resistance are present in Table 1 and 2.

The synergistic of DEF, PB, DM and TCP on resistance were evaluated in both profenfos and thiamethoxam strains because DEF is known to act by inhibiting esterases, PB inhibits the mixed function oxidases DM inhibits glutathione transferases and TCP inhibits non-specific esterases.

Table 1:	Development of resistance in the whitefly, Bemisia tabaci to profenofos
Resistance ratio = LC50 of R-strain/LC50 of S-strain

Table 2:	Development of resistance in the whitefly, Bemisia tabaci to thiamethoxam
Resistance ratio = LC50 of R-strain/LC50 of S-strain, **ppm calculated as active ingredient

Three levels 50, 25 and 10 ppm from each synergist were used to evaluate their synergistic combination with thiamethoxam or profenofos.

At least six concentrations were tested for each insecticide alone and in combination with synergists. The mortality for treatments was corrected for control mortality by using Abbott’s formula (Abbott, 1925) and the LC₅₀’s were estimated by probit analysis (Finney, 1971). Toxicity of the insecticides + synergist compared with the insecticide alone was expressed as Synergistic Ratio (SR) LC₅₀ of insecticide alone divided by LC₅₀ of insecticide + synergist. The high value of synergistic ratio indicates the presence and the relative importance of detoxication enzymes responsible for a resistance.

RESULTS AND DISCUSSION

Resistance ratios for white fly profenofos-resistant strain as set up in Table 1 (RR = 720.L1) after selection for eleven generations. However, in the case of thiamethoxam the RR was 188.91-fold. This reflects the acceleration of building up resistance to profenofos faster than in the case of thiamethoxam as neonicoticnoid insecticides. In addition this may be due to the intensive used by organophosphate insecticides especially profenofos against whitefly in Egypt. However, thiomethaxam is recently introduced to be one the non-conventional pest control agent against white fly.

Various factors cause resistance in insects, including oxidative degradation by Mixed Function Oxidases (MFOs), hydrolytic degradation by esterases or glutathione transrerances and target site insensitivity (Oppenoorth and Welling, 1976). Detoxication is considered one of the major mechanisms of insecticide resistance (Ishaaya, 2000). Therefore synergists, as inhibitors of metabolic processes, were used to clarify the mechanism of resistance in Bemesia tabaci.

The results in Table 3 and 4 showed the response of profenofos and thiamethoxam whitefly resistant strains to the insecticide alone or to the insecticide + synergists. Data in Table 3 clearly show that piperonyl butoxide PB (mfos inhibitor) enhanced the toxicity of profenofos against R-strain when co-administered with 50 ppm PB which exhibited SR = 24.08 and the level of resistance decreased to ca., 30 fold. In addition diethylmalate DM (glutathione transferases inhibitor) was moderately synergized profenofos (SR ca. 11). This finding indicated major contribution by either mixed function oxidases or glutathione transferases to profenofos resistance in B. tabaci. Segall and Casida (1982) found that profenofos is sterospecifically converted to more potent inhibitor of acetylcholinesterase by mouse liver microsomal mixed function oxidase system. The chiral (-) isomer became a 34 fold better inhibitor of acetylcholiesterase in vitro while the less toxic (+) isomer was deactivated by factor of 2. Recently, Lee et al. (2006) added that pyraclofos, prothiofos, sulphrofos and profenofos are organophosphorus (OP) insecticides exhibited an insecticidal mechanism distinctive compared with that of OP insecticides. It is widely accepted hat O-ethyl, S-n-propyl phosphorothiolaates are activated by oxidation of sulfur atom and their activated forms show very high activity, but are too unstable.

Table 3:	Synergism of profenofos by DEF, PB, DM and TCP in the adults of Bemisia tabaci profenfos resistant strain
aSR: Synergist Ratio (LC50 of unsergized/LC50 of synergized treatment), bRR: Resistance Ratio (LC50 of R strain/LC50 of S-strain, cRSR: Relative synergism (RR of unsynergized/RR of synergized treatment)

Table 4:	Synergism of thiamethoxam by DEF, PB, DM and TCP in the adults of Bemisia tabaci thiamethoxam resistant strain
aSR: Synergist ratio (LC50 of unsergized/LC50 of synergized treatment), bRR: Resistance Ratio (LC50 of R strain/LC50 of S-strain, cRSR: Relative synergism (RR of unsynergized/RR of synergized treatment)

However, DEF and TCP (esterases inhibitors) slightly synergized profenofos. This suggests that hydrolytic enzymes not play a significant role in the resistance of whitefly to profenofos.

The above mentioned may be explain why PB (mfo’s inhibitor) and DM (glutathione transferases inhibitor) enhanced the toxicity of profenofos to profenofos resistant strain. Therefore, in this study we can draw attention to the role of mfo and glutathione transeferases as a possible mechanism of resistance of whitefly to profenofos.

In synergism study of thiamethoxam by DEF, PB, DM and TCP the results in Table 4 showed that DEF was the most effective synergist. The toxicity of thiomethoxam to R-strain was increased with SR = 19.36 and resistance ratio decreased to about 9-fold. In addition TCP (nonspecific esterases inhibitor) was moderately synergized thiamethoxam. However, PB and DM have a limited synergism when mixed with thiamethoxam.

As well known thiamethoxam is a second-generation neonicotinoid compound with systemic, stomach and contact activity. It belongs to thianicotinyl subclass that interferes with the nicotinic acetylcholine receptors in the insect’s nervous system. To explore the role of detoxication enzyme in resistant mechanism, Rauch and Nauen (2003) carried out a biochemical studies on the impact of enzymes in neonicotinoid resistance. The results showed that monooxygenase activity increased 2-3 fold in white fly moderately resistant strains (RF approximately 30), even 5-6 fold in highly resistant strain (RF approximately 1000). Only monoxyenase activity correlated with imidacloprid, thiamethoxam and acetamprid resistance and therefore seems to be the enzyme system responsible for neonicotinoid resistance strains from Spain.

On the other hand and in agreement with our results Byrene et al. (2003) demonstrated a lack of any significant NADPH dependent metabolism of imidacloprid in relation to resistance in B-type B. tabaci originating from Guatemala.

Generally Ishaaya (2001) reported that the mechanisms of resistance to insecticides acting outside the nervous system (e.g., insect growth regulators in Bacillus thuringiensis or to more novel neurotoxin (e.g. neonicotinoids) are less understood, but it is likely that they are subjected to detoxification and/or target site alteration.

CONCLUSION

The present study has shown that synergists can be used to identify the role of metabolic enzymes in the mechanism of resistance in whitefly to thiamethoxam and profenofos insecticides. Piperonyl butoxide and diethylmaleate emphasized the role of mixed function oxidases and glutathione transferase as major factors in detoxication mechanism in profenofos resistant strain.

High synergistic ratio obtained from mixing profenofos and DEF, reflects the role of estrases in the detoxication mechanism found in thiamethoxam resistant strain.