## 3 Results and Discussion
### Principal Component Analysis  
Figures 2-4 show the PCA score plots obtained from the analysis of the NIR spectra of the fifth instars, sixth instar larvae, and pre-pupae reared on soy waste and spent brewer's grains. Figure 2 displays the PCA score plot obtained for analysis of larvae sourced from two different feeds. PC1 explains 71% of the variation in the samples, while 26% of the variation was accounted for by PC2. It can be observed that the fifth instar larvae and the sixth instar pre-pupae tend to cluster together and are clearly separated from one another. This suggests that the information collected in the NIR spectra is capable of differentiating the fifth instar BSFL larvae from the sixth instar pre-pupae irrespective of the feed source used to rear them. NIR spectroscopy has demonstrated its ability to identify the age of several insect species, including _Musca domestic_ (common house flies), _M. autumnalis_ (flies), _Stonoxys calcirrans_ (flies), _Culicoides sonorensis_ (midges), _Anopheles gambiae_ (mosquito), and _Anopheles arabiensis_ (mosquito) [55; 56; 57; 58]. However, no reports were found on the use of NIR spectroscopy to distinguish BSFL samples based on their larval morphology. It can be interpreted from this PCA score plot (Figure 2) that the physiology of the larvae might play an important role in determining the traceability of BSFL. However, some fifth instar larvae were noted to overlap with the sixth instar pre-pupae samples--a plausible explanation is that some of the harvested fifth instar samples might be approaching the next morphological stage, which is the pre-pupae or the sixth instar. It was mentioned in the methods section that ten thousand 5-DOL (chronological age) larvae were introduced into the trays. However, there is a probability that certain eggs could have hatched earlier, resulting in the introduction of slightly older larvae than the intended (>5-DOL) larvae.  
Figure 1: Second derivative NIR spectra obtained from 5 th instar larvae and 6 th instar pre-pupae reared on soy waste and spent brewer’s grains.  
Figure 4: Principal component score plot of 6 th instar pre-pupae reared on soy waste and spent brewers’ grains and analysed using near infrared spectroscopy.  
Figure 3: Principal component score plot of 5 th instar larvae reared on soy waste and spent brewer’s grains and analysed using near infrared spectroscopy.  
Figure 2: Principal component score plot of 5 th instar larvae and 6 th instar pre-pupae reared on soy waste and spent brewer’s grains analysed using near infrared spectroscopy.  
Figure 3 displays the PCA score plot of the fifth instar BSFL larvae reared on two different waste streams. PC1 explained 86% of the variation, while PC2 explained 11% of the variation in the samples analysed. The fifth instar BSFL reared on soy waste were found to form distinct clusters from those reared on spent brewer's grain. The PCA score plot for the sixth instar pre-pupae samples reared using two different waste streams is presented in Figure 4, where PC1 explained 75% of the variation, and PC2 accounted for 23% of the variation in the samples analysed. Figure 5 shows the loadings derived from the PCA analysis. It was observed that the PCA loadings were different for each of the PCA models developed. The highest PCA loadings were observed in most of the wavenumbers/wavelengths described in the above section.  
The results obtained in this study indicated that the sixth instar pre-pupae and fifth instar larvae tend to cluster as a result of the feed source used to rear them. Similar results were reported where powdered _Bombyx mori_ was found to be separated into different groups based on the substrate used for rearing [59]. Similar trends were also observed when powdered crickets and buffalo worms obtained from two different commercial suppliers were analysed using mid-infrared spectroscopy [60]. Insects from the same species were separated into two different clusters due to the difference in their processing conditions [60].