Bioassays
Residual contact toxicity
The Log-rank test results showed that the treatments have significantly different residual contact toxicity to newly emerged bees (χ2= 394.1; df= 3; P< 0.0001) (Fig. 1A) and foragers (χ2= 249.6; df= 3; P< 0.0001) (Fig. 1B). The median survival time (MST) for newly emerged bees after contact with the residue of Proclaim Fit® and dimethoate occurred at 14 and 12 h, respectively, while the MST for the solvent and negative control bees was undefined (the curve never crossed the 50% survival probability line) (Fig. 1A). The MST for forager bees treated with the residue of Proclaim Fit® and dimethoate achieved after 14 and 6 h exposure, respectively (Fig. 1B).
Oral toxicity
The Log-rank test results showed that the treatments have significantly different oral toxicity to newly emerged bees (χ2= 396.1; df= 3; P< 0.0001) (Fig. 2A) and foragers (χ2= 208.8; df= 3; P< 0.0001) (Fig. 2B). The median survival time (MST) for newly emerged bees after oral exposure to Proclaim Fit® and dimethoate occurred at 12 and 16 h, respectively, while the MST for the solvent and negative control bees was undefined (Fig. 2A). The MST for forager bees orally exposed to Proclaim Fit® and dimethoate achieved after 17 and 4 h exposure, respectively (Fig. 2B).
Spray toxicity
The Log-rank test results showed that the treatments have significantly different spray toxicity to newly emerged bees (χ2= 353.6; df= 3; P< 0.0001) (Fig. 3A) and foragers (χ2= 170.5; df= 3; P< 0.0001) (Fig. 3B). The median survival time (MST) for newly emerged bees exposed to Proclaim Fit® and dimethoate as spray occurred at 4 h, while the MST for the solvent and negative control bees was undefined (Fig. 3A). The MST for forager bees exposed to Proclaim Fit® and dimethoate as spray achieved after 5 and 4 h exposure, respectively (Fig. 3B).
Susceptibility of newly emerged bees versus foragers to Proclaim Fit®
Based on estimated hazard ratio, newly emerged bees were 1.47 (95% CI= 1.02-2.12) and 2.78 (95% CI= 1.90-4.05) times more susceptible than foragers to Proclaim Fit® applied as residual contact (χ2= 6.76; df= 1; P< 0.0093) and oral (χ2= 35.86; df= 1; P< 0.0001), respectively. Spray toxicity of this insecticide for newly emerged bees was 1.08 (95% CI= 0.74-1.57) times of that for forages which was not significantly different (χ2= 0.06; df= 1; P< 0.8023).
Changes in the detoxifying enzymes activities
α-esterase activity
The interaction effects of the treatments, the exposure routes, and age of the bees on α-esterase activity was significant (F= 41.42; df t,e= 5,44; P˂ 0.001). Contact exposure to the residue of Proclaim Fit® and dimethoate significantly decreased the activity of α-esterase in newly emerged bees rather than the solvent and negative controls (F= 20.75; df t,e= 3,8; P< 0.001) (Fig. 4A). The activity of this enzyme in newly emerged bees orally exposed to Proclaim Fit® was not different from that to solvent control, but was significantly higher than that to dimethoate (F= 43.85; df t,e= 2,6; P< 0.001) (Fig. 4B). Proclaim Fit® significantly increased α-esterase activity in newly emerged bees in comparison with the rest of the treatments when applied via spray (F= 56.69; df t,e= 3,8; P< 0.001) (Fig. 4C). This enzyme showed highest activity when forager bees were exposed to Proclaim Fit® than the rest of the treatments via residual contact (F= 65.60; df t,e= 3,8; P< 0.001) and oral (F= 5.63; df t,e= 2,6; P< 0.042) (Fig. 4A and B). The activity of α-esterase in forager bees spray-exposed to Proclaim Fit® was not different from that to the solvent and negative controls, but was significantly lower than that to dimethoate (F= 13.05; df t,e= 2,6; P< 0.002) (Fig. 4C). The comparison of α-esterase activity in newly emerged versus forager bees after being exposed to the test treatments via different routes of exposure is presented in Fig 4.
β-esterase activity
The activity of β-esterase was significantly influenced by the interaction effects of the treatments, the exposure routes, and age of the bees (F= 3.54; df t,e= 5,44; P˂ 0.009). The activity of this enzyme in newly emerged bees exposed to the residue of Proclaim Fit® did not differ from that of controls, but was significantly higher than that of dimethoate (F= 9.43; df t,e= 3,8; P< 0.005) (Fig. 5A). This enzyme showed similar activities when newly emerged bees were exposed to the test treatments orally (F= 0.14; df t,e= 2,6; P< 0.870) and via spray (F= 2.00; df t,e= 3,8; P< 0.193) (Fig. 5B and C). In foragers, β-esterase was more active when the bees were exposed to the residue of Proclaim Fit® than other treatments (F= 4.90; df t,e= 3,8; P< 0.032) (Fig. 5A). The activity of this enzyme in the forager bees orally exposed to Proclaim Fit® was statistically equal to that to the dimethoate, but was significantly higher than that to the solvent (F= 9.50; df t,e= 2,6; P< 0.014) (Fig. 5B). The activity of β-esterase in forager bees spray-exposed to Proclaim Fit® was not different from that to the rest of the treatments (F= 2.83; df t,e= 3,8; P< 0.106) (Fig. 5C). The comparison of β-esterase activity in newly emerged versus forager bees after exposure to the test treatments via different routes of exposure is presented in Fig 5.
GST activity
The activity of GST was significantly affected by the interaction effects of the treatments, the exposure routes, and age of the bees (F= 49.77; df t,e= 5,44; P˂ 0.001). Proclaim Fit® significantly induced GST activity in newly emerged bees in comparison with the rest of the treatments when applied as residual contact (F= 72.49; df t,e= 3,8; P< 0.001) and spray (F= 35.08; df t,e= 3,8; P< 0.001) (Fig. 6A and C). This enzyme showed the lowest activity when newly emerged bees were orally exposed to Proclaim Fit® than dimethoate and solvent control (F= 35.15; df t,e= 2,6; P< 0.001) (Fig. 6B). In case of foragers, the activity of GST in Proclaim Fit®-treated bees was significantly higher than that in control bees when exposed as residual contact (F= 45.40; df t,e= 3,8; P< 0.001) and oral (F= 43.55; df t,e= 2,6; P< 0.001) (Fig. 6A and B). The activity of this enzyme in the forager bees exposed to Proclaim Fit® via spray did not differ from that to dimethoate, but was significantly lower than that in control bees (F= 12.53; df t,e= 3,8; P< 0.002) (Fig. 6C). Fig 6. also shows the comparison of GST activity between newly emerged and forager bees after exposure to the test treatments via different routes of exposure.
Since the interactions of exposure route, bee age, and type of enzyme were significant (F= 39.12; df t,e= 4, 36; P˂ 0.001), determining which enzyme is more involved in detoxification of Proclaim Fit® depends on how and at what age the bees are exposed to this insecticide. Nonetheless, it seems that GST and α-esterase are more likely to be induced than β-esterase when Proclaim Fit® molecules entered into the bees’ body.
Toxicity to worker bee larvae
The acute toxicity of the field-realistic dose of the treatments on honey bee larvae results clearly indicate that Proclaim Fit® (86.25% mortality) and dimethoate (93.75% mortality) are highly toxic to the honey bee larvae compared to the controls (F= 130.00, df t,e= 3,15, P< 0.001) (Fig. 7).