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The weathering time of empty puparia could be important in predicting the minimum postmortem interval (PMImin). As corpse decomposition progresses to the skeletal stage, empty puparia often remain the sole evidence of fly activity at the scene. In this study, we used empty puparia of Sarcophaga peregrina (Diptera: Sarcophagidae) collected at ten different time points between January 2019 and February 2023 as our samples. Initially, we used the scanning electron microscope (SEM) to observe the surface of the empty puparia, but it was challenging to identify significant markers to estimate weathering time. We then utilized attenuated total internal reflectance Fourier transform infrared spectroscopy (ATR-FTIR) to detect the puparia spectrogram. Absorption peaks were observed at 1064 cm -1 , 1236 cm -1 , 1381 cm -1 , 1538 cm -1 , 1636 cm -1 , 2852 cm -1 , 2920 cm -1 . Three machine learning models were used to regress the spectral data after dimensionality reduction using principal component analysis (PCA). Among them, eXtreme Gradient Boosting regression (XGBR) showed the best performance in the wavenumber range of 1800-600 cm -1 , with a mean absolute error (MAE) of 1.20. This study highlights the value of refining these techniques for forensic applications involving entomological specimens and underscores the considerable potential of combining FTIR and machine learning in forensic practice., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sarcophaga dux (Diptera: Sarcophagidae) is a necrophagous flesh fly species with potential forensic value for estimating minimum postmortem interval (PMI min ). The basic developmental data and precise age estimates of the pupae are significant for PMI min estimation in forensic investigations. In the present study, we investigated the development data of that species at seven constant temperatures varying from 16 °C to 34 °C, including body length changes of the larve, developmental duration and accumulated degree hours of the preadults. Several reference genes for relative quantification of the differentially expressed genes (DEGs) were firstly selected and evaluated in the pupae of different ages under different temperatures. The DEGs of the insects during the pupal period at different constant temperatures (34, 25 and 16 °C) were further analyzed for more precise age estimation. The results showed that the developmental durations of the preadults at 16, 19, 22, 25, 28, 31 and 34 °C were 1478.6 ± 18.3 h, 726.1 ± 15.8 h, 538.5 ± 0.9 h, 394.1 ± 9.5 h, 375.6 ± 10.8 h, 284.1 ± 7.3 h, and 252.5 ± 6.1 h, respectively. The developmental threshold temperature the flies was 12.27 ± 0.35 °C, and the thermal summation constant was 5341.71 ± 249.29° hours. The most reliable reference genes during the pupal period at different temperatures were found: GST1 and 18S rRNA for the 34 °C group, GST1 and RPL49 for 25 °C, and 18S rRNA and 28S rRNA for 16 °C. The four differential expression genes (Hsp60, A-alpha, ARP, and RPL8) have the potential to be used for more precise age estimation of pupal S. dux. This work provides important basic developmental data and a more precise age estimation method for pupal S. dux, and improves the value of this species for PMI min estimation in forensic investigations., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Simple Summary: The duration of pupa development on a cadaver holds the potential for estimating the time of colonization (TOC), which is often correlated with the postmortem interval (PMI) of decomposed bodies. Establishing an objective, precise, and efficient method for inferring pupa age has become paramount in forensic entomology. In this study, we observed temporal variations in the reflection spectrum of Chrysomya megacephala (Diptera: Calliphoridae) pupa, detectable through hyperspectral imaging (HSI). Additionally, we proposed that the eXtreme Gradient Boosting Regression (XGBR) model represents an optimal approach for estimating pupa development time based on HSI data. Estimating the age of pupa during the development time of the blow fly Chrysomya megacephala (Diptera: Calliphoridae) is of forensic significance as it assists in determining the time of colonization (TOC), which could help to determine the postmortem interval (PMI). However, establishing an objective, accurate, and efficient method for pupa age inference is still a leading matter of concern among forensic entomologists. In this study, we utilized hyperspectral imaging (HSI) technology to analyze the reflectance changes of pupa development under different temperatures (15 °C, 20 °C, 25 °C, and 30 °C). The spectrograms showed a downtrend under all temperatures. We used PCA to reduce the dimensionality of the spectral data, and then machine learning models (RF, SVR-RBF, SVR-POLY, XGBR, and Lasso) were built. RF, SVR with RBF kernel, and XGBR could show promise in accurate developmental time estimation using accumulated degree days. Among these, the XGBR model consistently exhibited the most minor errors, ranging between 3.9156 and 7.3951 (MAE). This study has identified the value of further refinement of HSI in forensic applications involving entomological specimens, and identified the considerable potential of HSI in forensic practice. [ABSTRACT FROM AUTHOR]

Simple Summary: The precise pupal age estimates of S. peregrina are significant for estimating the minimum postmortem interval (PMImin) in forensic investigations. In the present study, we first investigated the potential of attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and cuticular hydrocarbons (CHCs) for the age estimations of S. peregrina pupae at different constant temperatures (20 °C, 25 °C, and 30 °C). The orthogonal projections to latent structure discrimination analysis (OPLS-DA) and partial least squares (PLS) models were used to distinguish the pupae samples of different developmental ages. Sarcophaga peregrina (Robineau-Desvoidy, 1830) (Diptera: Sarcophagidae) is a forensically important flesh fly that has potential value in estimating the PMImin. The precise pupal age estimation has great implications for PMImin estimation. During larval development, the age determination is straightforward by the morphological changes and variation of length and weight, however, the pupal age estimation is more difficult due to anatomical and morphological changes not being visible. Thus, it is necessary to find new techniques and methods that can be implemented by standard experiments for accurate pupal age estimation. In this study, we first investigated the potential of attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and cuticular hydrocarbons (CHCs) for the age estimations of S. peregrina pupae at different constant temperatures (20 °C, 25 °C, and 30 °C). The orthogonal projections latent structure discrimination analysis (OPLS-DA) classification model was used to distinguish the pupae samples of different developmental ages. Then, a multivariate statistical regression model, partial least squares (PLS), was established with the spectroscopic and hydrocarbon data for pupal age estimations. We identified 37 CHCs with a carbon chain length between 11 and 35 in the pupae of S. peregrina. The results of the OPLS-DA model show a significant separation between different developmental ages of pupae (R2X > 0.928, R2Y > 0.899, Q2 > 0.863). The PLS model had a satisfactory prediction with a good fit between the actual and predicted ages of the pupae (R2 > 0.927, RMSECV < 1.268). The results demonstrate that the variation tendencies of spectroscopy and hydrocarbons were time-dependent, and ATR-FTIR and CHCs may be optimal for the age estimations of pupae of forensically important flies with implications for PMImin estimation in forensic practice. [ABSTRACT FROM AUTHOR]

Simple Summary: Forensic entomology plays a major role in postmortem interval (PMI) estimation, mainly based on morphological characteristics of larvae colonized on decomposed corpses. However, weight and length cannot be used as relevant parameters to calculate the age of wandering at the larval and pupal stages. In order to identify a specific indicator that may serve as a potential target, studies on the mechanism during postembryonic development may provide a foundation to establish a molecular age determination method. Sarcophaga peregrina (Robineau-Desvoidy, 1830) is a species of medical and forensic importance. In this study, transcriptome analysis was performed to investigate gene expression profiles from the larval to pupal stages of S. peregrina, which were used to identify differentially expressed genetic markers as candidates for molecular age estimation. This study explores molecular mechanisms during postembryonic development of S. peregrina. The developmental, stage-specific gene profiles suggest genetic markers for age prediction of forensically important flies. Sarcophaga peregrina (Robineau-Desvoidy, 1830) is a species of medical and forensic importance. In order to investigate the molecular mechanism during postembryonic development and identify specific genes that may serve as potential targets, transcriptome analysis was used to investigate its gene expression dynamics from the larval to pupal stages, based on our previous de novo-assembled genome of S. peregrina. Totals of 2457, 3656, 3764, and 2554 differentially expressed genes were identified. The specific genes encoding the structural constituent of cuticle were significantly differentially expressed, suggesting that degradation and synthesis of cuticle-related proteins might actively occur during metamorphosis. Molting (20-hydroxyecdysone, 20E) and juvenile (JH) hormone pathways were significantly enriched, and gene expression levels changed in a dynamic pattern during the developmental stages. In addition, the genes in the oxidative phosphorylation pathway were significantly expressed at a high level during the larval stage, and down-regulated from the wandering to pupal stages. Weighted gene co-expression correlation network analysis (WGCNA) further demonstrated the potential regulation mechanism of tyrosine metabolism in the process of puparium tanning. Moreover, 10 consistently up-regulated genes were further validated by qRT-PCR. The utility of the models was then examined in a blind study, indicating the ability to predict larval development. The developmental, stage-specific gene profiles suggest novel molecular markers for age prediction of forensically important flies. [ABSTRACT FROM AUTHOR]

Some representatives of flesh flies visiting/colonizing the decomposed remains demonstrated their values in estimating the minimal postmortem interval (PMImin) since death. However, the utility of sarcophagid flies has been seriously hampered by limited ecological, biological and taxonomic knowledge of them. Although mitochondrial genes have been proposed as a potential DNA barcode for the species-level identification of sarcophagids, some defects still remain such as the substantial memory and processing time taken for homologous comparisons online. Moreover, species identification is mainly achieved by Sanger sequencing based on PCR with genus-specific primers. In the present study we characterized 24 single nucleotide polymorphisms (SNPs) as robust markers of genetic variation for identifying different sarcophagids based on available cytochrome c oxidase I (COI) data and verified them through pyrosequencing (PSQ) technology to establish a SNP-based genotyping system. The system provides a preliminary foundation for developing a rapid, reliable, and high-throughput assay so as to efficiently and accurately identify the sarcophgid flies. Furthermore, the PSQ approach is proved to be faster, more cost-effective as well as more sensitive and specific than custom Sanger sequencing. [ABSTRACT FROM AUTHOR]