Combining spectrum and machine learning algorithms to predict the weathering time of empty puparia of Sarcophaga peregrine (Diptera: Sarcophagidae).

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.

The gut bacterial composition across life stages of Sarcophaga peregrina (Diptera: Sarcophagidae) and the effects of amikacin on their development.

Insects and microorganisms, ubiquitous organisms in the natural world, have developed intricate relationships throughout their evolutionary histories. However, most studies have concentrated on specific time points or life stages, but some limited studies have investigated the dynamics of microbial diversity within insects across life stages. Here, 16S rDNA sequencing technology was used to investigate the gut bacterial community across the life stages of Sarcophaga peregrina (Robineau-Desvoidy) (Diptera: Sarcophagidae). The results revealed that the gut bacterial diversity of S. peregrina varied with life stage and showed similarity in the nearby life stages. Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes were the dominant phyla in S. peregrina. Genera such as Providencia, Ignatzschineria, and Myroides are implicated in potentially pivotal roles during the developmental processes of this flesh fly. Furthermore, the effects of amikacin on the growth and development of S. peregrina were not statistically significant. However, we did observe significant changes at the protein level, which suggests a close association between protein-level alterations and growth and development. Additionally, we speculate that S. peregrina regulates its nutritional status during nonfeeding stages to meet the demands of eclosion. This study represents the first comprehensive examination of the intestinal bacterial composition across various life stages of S. peregrina. Our findings deepen our understanding of the gut microbiota in this flesh fly and lay the groundwork for further exploration into the intricate interactions between microorganisms and insects.

Age Determination of Chrysomya megacephala Pupae through Reflectance and Machine Learning Analysis.

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.

Age determination of Chrysomya megacephala (Diptera: Calliphoridae) using lifespan patterns, gene expression, and pteridine concentration under constant and variable temperatures.

Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae), is a blowfly species widely studied in medical, veterinary, and entomological research. Our study examined the impact of constant (15, 20, 25, 30, and 35 °C) and variable (ranging from 21.0 to 25.4 °C, with an average of 23.31 °C) temperatures on the development and larval body length of C. megacephala. Additionally, we analyzed the age of the adult C. megacephala through pteridine content and related metabolic genes analysis. Our findings revealed three distinct growth patterns: isomorphen diagram, isomegalen diagram, and thermal accumulated models. At constant temperatures of 15, 20, 25, 30, and 35 °C, egg-hatching times were 44.5 ± 8.9, 26.7 ± 4.6, 12.6 ± 1.1, 11.0 ± 1.0, and 9.9 ± 1.9 h, respectively, while it was 15.3 ± 5.9 h at variable temperatures. The total development times from oviposition to adult eclosion in C. megacephala required 858.1 ± 69.2, 362.3 ± 5.9, 289.6 ± 17.8, 207.3 ± 9.3, and 184.7 ± 12.1 h at constant temperatures of 15, 20, 25, 30, and 35 °C, respectively. This duration was extended to 282.0 ± 64.1 h under variable temperatures. However, no significant differences were found in hatching times and the total developmental durations between 25 °C and variable temperatures. A developmental threshold temperature (D0) of 9.90 ± 0.77 °C and a thermal summation constant (K) of 4244.0 ± 347.0° hours were ascertained. Pteridine content patterns varied significantly across constant temperatures, but not between 25 °C and variable temperatures. Sex and temperature were identified as the primary factors influencing pteridine levels in the head of C. megacephala. Gene expression associated with pteridine metabolism decreased following adult eclosion, matching with increased pteridine concentration. Further investigations are needed to explore the use of pteridine cofactors for age-grading adult necrophagous flies. These findings provide valuable insights into the lifespan of C. megacephala, thereby offering valuable groundwork for forthcoming investigations and PMImin determination.

Effect of Ciprofloxacin on the Composition of Intestinal Microbiota in Sarcophaga peregrina (Diptera: Sarcophagidae).

The intestinal bacteria of insects are crucial to the growth and development of the host. It has been found that various physiological processes of insects, such as immune response, metabolism, reproductive ability, and growth and development, involve the gastrointestinal flora. However, many external factors affect the composition of insects' intestinal microorganisms, such as the type of dietary substrate. Sarcophaga peregrina (Robineau-Desvoidy, 1830) (Diptera: Sarcophagidae) is of great significance in medicine and forensic science. In this study, we investigated the effects of ciprofloxacin on the growth and gut microbiota of S. peregrina. The results demonstrated that the maximum body length of larvae was not affected by ciprofloxacin, while the growth rate of body length quickened as the concentration of the drug increased. The weight of the pupa and adult was reduced significantly due to the effect of ciprofloxacin. After analyzing the gut microbiota composition of S. peregrina in different drug groups, it was indicated that Ignatzschineria, Providencia, Wohlfahrtiimonas, Proteus, Myroides, and Bacteroides play important roles in the growth of S. peregrina. However, they still need to be further studied. In general, ciprofloxacin can affect the gut microbial community structure, which in turn affects the fitness of the host.

Analysis of the Influence of Changing and Fixed Temperatures on the Growth and Pteridine Content in the Head of Adults Sarcophaga crassipalpis (Diptera: Sarcophagidae).

Flesh flies (Diptera: Sarcophagidae) are regarded as significant in medical and veterinary entomology, and their development models can be utilized as considerable markers to ascertain the minimum postmortem interval (PMImin). In this research, we explored the growth cycle and larval body length of Sarcophaga crassipalpis Macquart 1839 (Diptera: Sarcophagidae) reared under variable temperatures ranging from 15.7 to 31.1 °C, with an average of 24.55 °C and relative humidity ranges from 31.4 to 82.8% and at six fixed temperatures of 15, 20, 25, 30, 32, and then 35 °C. Moreover, pteridine from the head was used to assess adult age grading. Our results allowed us to provide three development models: the isomorphen chart, the isomegalen chart, and the thermal summation models. The time taken for S. crassipalpis to complete its development from larviposition to adult emergence at constant temperatures of 15, 20, 25, 30, 32, and 35 °C was 1256.3 ± 124.2, 698.6 ± 15.1, 481.8 ± 35.7, 366.0 ± 13.5, and 295.8 ± 20.5 h, respectively, except 35 °C, where all pupae were unable to attain adulthood. They lasted 485.8 ± 5.4 h under variable temperatures. The minimum developmental limit (D0) temperature and the thermal summation constant (K) of S. crassipalpis were 9.31 ± 0.55 °C and 7290.0 ± 388.4 degree hours, respectively. The increase in pteridine content exhibited variations across different temperatures. There was quite a considerable distinction in the pteridine contents of male and female S. crassipalpis at 15 °C (p = 0.0075) and 25 °C (p = 0.0213). At 32 °C and variable temperatures, the pteridine content between female and male S. crassipalpis was not statistically divergent. However, temperature and gender remain the main factors influencing the pteridine content in the head of S. crassipalpis. We aim to provide detailed developmental data on S. crassipalpis that can be used as a valuable resource for future research and PMI estimation.

Development of Forensically Important Sarcophaga peregrina (Diptera: Sarcophagidae) and Intra-Puparial Age Estimation Utilizing Multiple Methods at Constant and Fluctuating Temperatures.

Sarcophaga peregrina (Robineau-Desvoidy, 1830) has the potential to estimate the minimum postmortem interval (PMImin). Development data and intra-puparial age estimation are significant for PMImin estimation. Previous research has focused on constant temperatures, although fluctuating temperatures are a more real scenario at a crime scene. The current study examined the growth patterns of S. peregrina under constant (25.75 °C) and fluctuating temperatures (18-36 °C; 22-30 °C). Furthermore, differentially expressed genes, attenuated total reflectance Fourier-transform infrared spectroscopy, and cuticular hydrocarbons of S. peregrina during the intra-puparial period were used to estimate age. The results indicated that S. peregrina at fluctuating temperatures took longer to develop and had a lower pupariation rate, eclosion rate, and pupal weight than the group at constant temperatures did. Moreover, we found that six DEG expression profiles and ATR-FTIR technology, CHCs detection methods, and chemometrics can potentially estimate the intra-puparial age of S. peregrina at both constant and fluctuating temperatures. The findings of the study support the use of S. peregrina for PMImin estimation and encourage the use of entomological evidence in forensic practice.

Predicting the Weathering Time by the Empty Puparium of Sarcophaga peregrina (Diptera: Sarcophagidae) with the ANN Models.

Empty puparium are frequently collected at crime scenes and may provide valuable evidence in cases with a long postmortem interval (PMI). Here, we collected the puparium of Sarcophaga peregrina (Diptera: Sarcophagidae) (Robineau-Desvoidy, 1830) for 120 days at three temperatures (10 °C, 25 °C, and 40 °C) with the aim to estimate the weathering time of empty puparium. The CHC profiles were analyzed by gas chromatography-mass spectrometry (GC-MS). The partial least squares (PLS), support vector regression (SVR), and artificial neural network (ANN) models were used to estimate the weathering time. This identified 49 CHCs with a carbon chain length between 10 and 33 in empty puparium. The three models demonstrate that the variation tendency of hydrocarbon could be used to estimate the weathering time, while the ANN models show the best predictive ability among these three models. This work indicated that puparial hydrocarbon weathering has certain regularity with weathering time and can gain insight into estimating PMI in forensic investigations.