The Pupal Age Estimation Of Sarcophaga Peregrina With Implications In Forensic Investigations

Background:Postmortem interval（PMI）estimation is crucial to investigation of cases.Forensic entomology has good prospects for PMI estimation of decomposed bodies,with necrophagous flies being the most common insect evidence in non-natural death cases.The unique advantages of necrophagous flies in PMI estimation have attracted widespread attention from forensic scientists.However,accurate and objective determination of developmental stages is a key step in PMI estimation based on insect evidence.The lack of basic developmental data on flies in different regions,objective indicators for estimating the age of necrophagous fly pupae,and the effects of temperature variation on fly growth and development seriously affect the accuracy and scientific validity of fly evidence,thus becoming a key problem that restricts the development of forensic entomology.Sarcophaga peregrina（Robineau-Desvoidy,1830）（Diptera:Sarcophagidae）is a common necrophagous fly on corpses and has significant forensic significance.Objective:（1）To gather fundamental growth and developmental data of S.peregrina under various constant temperature conditions with constant humidity.（2）To assess the potential of morphological techniques,including removing pupal cases for photography and HE staining scanning,chemical and spectral methods like cuticular hydrocarbons（CHCs）and attenuated total reflection Fourier-transform infrared spectroscopy（ATR-FTIR）,and molecular biology methods such as differentially expressed genes and metabolites in estimating the age of S.peregrina pupae.（3）To investigate the growth and developmental patterns of S.peregrina under temperature variation conditions and the methods of estimating the age of pupae.（4）To validate the practicality and precision of the age estimation technique for necrophagous fly pupae developed in this study with actual forensic entomology cases,with the ultimate goal of establishing a mature,stable,and scientific PMI estimation system.Through this research,the application value and scope of fly evidence in forensic science can be enhanced,providing a significant theoretical foundation for PMI estimation in cases.Methods:The S.peregrina adult flies were lured to rotten meat in the field,identified,and bred in the laboratory through successive generations.The study consisted of the following stages:（1）Basic growth and development data were collected for S.peregrina flies at constant temperatures of 15℃,20℃,25℃,and 30℃,with a humidity of 75%and a 12D:12L light cycle in an artificial climate chamber.Data collected included body length,developmental stage,developmental time,and accumulated temperature.（2）Age inference of S.peregrina pupae was investigated using various methods.Under constant temperatures of 15℃,20℃,25℃,and 30℃,samples of pupae were collected at different developmental stages.Morphological changes were analyzed by peeling off the pupal shell,taking photographs,and HE staining.Differentially expressed genes were screened by transcriptome sequencing,and the regulatory mechanisms of these genes were explored using RNAi and other techniques.Differentially expressed metabolites were identified using non-targeted metabolomics.CHCs and ATR-FTIR were used to analyze time-series changes in pupal tissues.（3）S.peregrina growth and developmental patterns were studied under variable temperature conditions（18℃-36℃and 22℃-30℃）,with a constant temperature of25℃as the control.Data was collected using various methods,and the application of CHCs,ATR-FTIR,and differentially expressed genes in pupal age inference under variable temperature conditions was explored.（4）Blind test experiments were conducted to verify the insect evidence-based PMI inference using the basic growth and development data and necrophilous fly pupal age inference methods obtained from the above research.Samples of necrophilous flies were collected from human corpses in actual cases.Results:The laboratory population of S.peregrina was established successfully.（1）the S.peregrina undergo development from larvae to adult emergence at temperatures ranging from 15℃to 30℃,with development times varying from 1090.3±30.6 hours to 280.3±4.5 hours.As expected,higher temperatures resulted in shorter development times.（2）Age determination of necrophagous fly pupae can be achieved using several methods.Morphological methods involve dividing the development of the pupal stage into 12 sub-stages and examining individual organ tissues.By means of HE staining scanning,the overall development of pupal stage of S.peregrina could be divided into 10sub-stages.Molecular biology methods involve analyzing differentially expressed genes,such as Sp Cht8,Sp Cht10,Sp TH,and Sp TPP,which play a crucial role in metamorphosis.Silencing these genes resulted in a range of developmental abnormalities,the results indicated that these candidate differentially expressed genes played an important regulatory role in the metamorphosis and development of S.peregrina.Establishing the fitting equation between these differentially expressed genes and pupal age could be used for the accurate inference of pupal age.Non-targeted metabolome sequencing showed that the metabolites were significantly different at different developmental stages of pupal stage.Chemical and spectroscopic methods:The infrared spectra with the wave length of 1000～1100 cm^-1 and the hydrocarbons such as C24 showed the potential to infer the age of pupal stage of S.peregrina by ATR-FTIR and GC-MS analysis.（3）Variable temperature conditions of 18°C～36°C and22°C～30°C have been shown to affect the development of S.peregrina.While the flies are capable of completing their entire metamorphosis,their body size,pupation rate,eclosion rate,and pupal weight are reduced.CHCs,ATR-FTIR,differentially expressed genes methods can also be used to predict the age of pupal stage in flies under variable temperature conditions.（4）Blind testing experiments have demonstrated that utilizing the growth and development data obtained in this study,in conjunction with the methods for estimating pupal age in necrophagous flies,can lead to more accurate and objective estimation of postmortem interval.Conclusion:（1）The research findings suggest that the S.peregrina fly is a significant necrophagous fly,and the developmental data gathered at constant temperatures ranging from 15°C to 30°C can serve as valuable reference material for forensic investigations.（2）The several methods such as morphological techniques involving pupal shell removal for photography and HE staining scanning,as well as chemical and spectroscopic approaches such as CHCs and ATR-FTIR,and molecular biology techniques like differential gene expression and differential metabolites,have the potential to estimate the age of pupae in necrophagous flies.（3）Although the basic developmental data of necrophagous flies vary under variable temperature conditions,the molecular biology,chemical and spectroscopic,and other pupal age estimation methods remain useful for such situations.