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

Chrysomya nigripes 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 C. nigripes 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 C. nigripes 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 C. nigripes of puparia at each intra-puparial age under various constant temperature conditions were obtained. The results showed that at 16 °C, C. nigripes 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 C. nigripes 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 C. nigripes and provide a reference value for PMI(min).