Estimating the Intra-Puparial Period of Chrysomya nigripes Aubertin Using Morphology and Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) Spectroscopy

<i>Chrysomya nigripes</i> Aubertin, 1932, is a species of Calliphoridae widely distributed in Southeast Asia, with studies and case reports confirming the value of this species in estimating the minimum postmortem interval (PMI_min). However, data on the growth and development of this species' intra-puparial age are not yet complete. Here, we investigated the intra-puparial morphological changes of <i>C. nigripes</i> at seven temperatures, ranging from 16 °C to 34 °C. We also investigated the potential value of Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) coupled with chemometric methods for the intra-puparial age estimation of <i>C. nigripes</i> at 19 °C, 25 °C, and 31 °C. The spectral data within the wavenumber range of 1800-900 cm^-1, collected from the second thoracic segment of all puparia, were processed. Through this procedure, the mean values of ATR-FTIR spectra of <i>C. nigripes</i> of puparia at each intra-puparial age under various constant temperature conditions were obtained. The results showed that at 16 °C, <i>C. nigripes</i> could not complete its developmental process, while it could do so at the remaining six constant temperatures. With an increase in temperature, the average duration of the intra-puparial period was reduced from the longest at 19 °C of 192 ± 0 h to 77.3 ± 4.6 h at 34 °C. The intra-puparial morphological changes were divided into 12 sub-stages, and the development of the compound eyes, mouthparts, antennae, thorax, legs, wings, and abdomen were divided into 6-8 sub-stages. The Partial Least Squares Discriminant Analysis (PLS-DA) classification model predicted better results compared to the Random Forest (RF) classification model, with an accuracy of 58.3%, 77.8%, and 100% at 19 °C, 25 °C, and 31 °C, respectively. In this study, each sub-stage of the <i>C. nigripes</i> pupa and the time range of structure emergence were recorded, and it was concluded that the spectral trends were time-dependent. Thus, ATR-FTIR combined with chemometrics could also be used as a tool to assist in estimating the intra-puparial stage of <i>C. nigripes</i> and provide a reference value for PMI_min.