# A Laser Irradiation Method for Controlling _Pieris rapae_ Larvae
## 3 Results
### Experiment 4: Verification Test with 1 st- to 5 th-Instar Pieris rapae Larvae  
In the field environment, 1 st- to 5 th-instr _P. rapae_ larvae existed simultaneously. Therefore, to verify the universality of the working parameters in controlling the _P. rapae_ larvae under field situations, the optimal combination of parameters was used to irradiate 1 st- to 5 th-instrs. The larvae were collected in the Hunan Province Key Laboratory of Intelligent Agricultural Machinery and Equipment, Changsha, Hunan Province, China, from 13 July to 18 July 2020. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline \multirow{2}{*}{**Value**} & \multicolumn{3}{c}{**Optimal Working Parameters 1**} & \multicolumn{3}{c}{**Mean (\%) 24 h**} & \multicolumn{3}{c}{**Mean (\%) 24 h**} \\  & \(A\) & \(B^{2}\) & \(C\) & \(D\) & **Antifeedant Percentage** & **Mortality Rate** & **Comprehensive Score 3** & **Desirability 4** \\ \hline Predicted 5 & 5.222 & 7.070 & 1.585 & 1.000 & 91.01 & 100.00 & 0.979 & 0.980 \\ Actual 6 & 5.222 & 7.070 & 1.585 & 1.000 & 98.33 \(\pm\) 0.41a & 96.67 \(\pm\) 3.33a & & \\ Bias 7 (\%) & & & & & 7.44 & –3.45 & & \\ \hline Predicted 5 & 7.500 & 6.189 & 1.177 & 1.000 & 88.50 & 100.50 & 0.922 & 0.952 \\ Actual 6 & 7.500 & 6.158 & 1.177 & 1.000 & 98.49 \(\pm\) 0.60a & 100 \(\pm\) 0.00a & & \\ Bias 7 (\%) & & & & & 10.14 & –0.5 & & \\ \hline Predicted 5 & 7.500 & 4.961 & 1.539 & 1.000 & 86.76 & 100.00 & 0.891 & 0.918 \\ Actual 6 & 7.500 & 4.908 & 1.539 & 1.000 & 97.91 \(\pm\) 0.72a & 100 \(\pm\) 0.00a & & \\ Bias 7 (\%) & & & & & 11.39 & 0.00 & & \\ \hline \hline \end{tabular}  
* \(A\) is the laser power (W); \(B\) is the irradiation area (mm\({}^{2}\)); \(C\) is the laser opening time (s); and coded value 1.000 for \(D\) (irradiation position) corresponds to the actual parameter of the middle of the abdomen. The cross-validating model used 3 groups of optimized samples with estimated responses. 2 The irradiation area (\(B\)) of 7.07 mm\({}^{2}\), 6.158 mm\({}^{2}\) and 4.908 mm\({}^{2}\) indicates that the diameters are 3 mm, 2.8 mm and 2.5 mm, respectively. 3 Comprehensive score was calculated according to the mean antifeedant percentage and the mean mortality rate within 24 h posttreatment (Equation (2)). Calculating comprehensive score requires more than three groups of data; therefore, there is no comprehensive score in the actual. 4 Desirability represents satisfaction of the composition of the optimized sample with estimated responses. 5 The response values and reliability of the optimized sample combination for prediction. 6 Actual values are mean \(\pm\) SES of three biological replicates. Mean values followed by the same letter in the column are not different by the [PERSON]’s honestly significant difference test (\(p\) > 0.05). 7 Bias (\(\%\)) = 100 \(\times\) (Actual – Predicted)/Actual. The negative sign in front of the biases indicates an inverse deviation between the value of actual and predicted, while vice versa applies for the positive sign.  
\end{table} 
Table 4: Composition of optimized samples with estimated responses and the cross-validation of the model. As shown in Figure 4, the 1 st- to 3 rd-instar larvae showed no significant differences in the antifeedant percentages (24 h, \(F\) = 8.00, df = 4, 10, \(p\) = 0.004; 48 h, \(F\) = 6.01, df = 4, 10, \(p\) = 0.010) or mortality rates (12 h, \(F\) = 16.50, df = 4, 10, \(p\) = 0.0002; 24 h, same, all 100%). The antifeedant percentages and mortality rates of the 5 th-instar larvae were lower than those of the 4 th-instar larvae, but no markedly significant difference was found between the 4 th- and 5 th-instar larvae (the antifeedant percentage: 24 h, \(F\) = 1.78, df = 1, 4, \(p\) = 0.254; 48 h, \(F\) = 0.32, df = 1, 4, \(p\) = 0.603; mortality rate, 12 h, \(F\) = 1.00, df = 1, 4, \(p\) = 0.374; 24 h, 100 \(\pm\) 0.00%, 96.67 \(\pm\) 3.33%). Moreover, the mortality rate of the 4 th- and 5 th-instar larvae at 36 h was 100%.