## 3 Results and Discussion
### Effects of Abiotic Variables (P. regina)  
DA was carried out and confusion matrices were generated for the data from the studies of temperature, humidity, substrate, and diet. Although overall classification accuracies of 60% or more \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline  & \multicolumn{6}{c}{**G10 Generation**} & \multicolumn{3}{c}{**Total**} & \% **Correct** \\ \hline  & **From/To** & _C. macellaria_ & _L. cuprina_ & _L. sericata_ & _P. regina_ & & \\ \hline \multirow{4}{*}{G1 Generation} & _C. macellaria_ & 47 & 0 & 0 & 6 & 53 & 88.7 \\  & _L. cuprina_ & 2 & 49 & 2 & 1 & 54 & 90.7 \\  & _L. sericata_ & 0 & 0 & 52 & 2 & 54 & 93.0 \\  & _P. regina_ & 7 & 0 & 2 & 27 & 36 & 75.0 \\ \hline \hline \end{tabular}
\end{table} 
Table 6: Leave-one-out confusion matrix for the G1 generation chemical profiles. \begin{table}
\begin{tabular}{c l c c c c} \hline \hline  & \multicolumn{6}{c}{**G10 Generation**} & \multicolumn{3}{c}{**Total**} & \% **Correct** \\ \hline  & **From/to** & _C. macellaria_ & _L. cuprina_ & _L. sericata_ & _P. regina_ \\ \hline \multirow{4}{*}{G1 Generation} & _C. macellaria_ & 68.9\% (0.832) & & & \\  & _L. cuprina_ & & 73.5\% (0.839) & & \\  & _L. sericata_ & & & 58.9\% (0.648) & \\  & _P. regina_ & & & & 82.2\% (0.883) \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Classification accuracy and area under the curve (AUC) for the G1 and G10 pupae of each species. were achieved for all four experiments, in most cases the actual differences observed among the classes were marginal. For example, almost no difference in the lipid profile was manifest among specimens allowed to pupate in sawdust, in sand, or in vermiculite (data not shown). Classification accuracies were therefore poor, with a third of samples misclassified in the cases of pupae reared in sawdust and sand, and half of all samples in the case of vermiculite. In these cases, only the data in the experiment (for example, the pupation substrate) were used, therefore, it appeared that it did not matter what the pupation substrate was, there were no distinct classes formed. Results for pupae reared under different humidity conditions were almost as poor (data not shown), with discrimination only achieved for the sample set reared under normal 55% humidity conditions. This is perhaps a surprising result, as the cuticular hydrocarbons of insects are known to play a part in resistance to desiccation, and might be expected to vary in response to adverse environmental conditions such as low humidity. It should be noted that although discrimination could not be achieved in these cases, this is, in fact, beneficial. In the context of insect specimens recovered from a crime scene, it is to the advantage of an investigator if the chemical profiles do not vary significantly with temperature, humidity, and other uncontrollable factors. A reliable method for age and species determination that is not undermined or biased by biotic and abiotic variables is the gold standard for any analyst. Therefore, the lack of discrimination is, in this case, the desired result. Temperature appeared to have more of an impact on the lipid profiles, although its influence was still relatively small, as attested by the poor classification accuracies of 41.7% and 62.3% for pupae reared at 15 \({}^{\circ}\)C and 20 \({}^{\circ}\)C, respectively (data not shown). More substantial chemical differences were observed at 25 \({}^{\circ}\)C and 30 \({}^{\circ}\)C. In general, lauric acid, myristic acid, pentadecanoic acid, palmitic acid, and alkane G increased with temperature. Diet was the single-largest contributing factor to differences in the lipid profile (Figure 5). Classification accuracies (Table 7) were 100% for pupae reared on chicken liver and 87.5% for pupae reared on feeder rats. The statistical model was unsuccessful at distinguishing between pupae raised on high-fat ground beef and pupae fed on low-fat ground beef. Neglecting that minor distinction, pupae raised on ground beef were still correctly categorized 100.00% of the time.