Application of Chrysomya rufifacies（Diptera,Calliphoridae）to Postmortem Interval Estimation in Southwest China:A Case Report

LI Xue-bo,ZHAO Feng,DING Chun-li,LI Liang-liang,CHEN Lu-shi

1.Key Laboratory of Evidence Identification in Universities of Shandong Province,Shandong University of Political Science and Law,Jinan 250014,China;2.Institute of Forensic Science,Chongqing Municipal Public Security Bureau,Chongqing 400021,China;3.Guizhou Police College,Guiyang 550005,China

Abstract:Insect samples found on human corpses can provide the information important to estimating the minimum postmortem interval（PMImin）.A female cadaver,found in a deserted factory in Chongqing of China,was confirmed as a homicide case after the forensic investigation and autopsy.Determining the time of death was difficult due to the inconsistent degree of decomposition in different parts of the decedent.The insect specimens found on the cadaver were identified to be Chrysomya rufifacies（C.rufifacies,Macquart）by morphology and mitochondrial DNA sequence analysis.The PMImin was estimated to be 452 h,based on the developmental rate of C.rufifacies.The PMImin was estimated successfully to be almost precise,which provided an important entomological evidence for case investigation and suspect prosecution.In so doing,this highlights the usefulness of entomological evidence of specific species in the geographic area for PMI accurate estimation,especially in the case of advanced decomposed corpses.

Keywords:forensic entomology;postmortem interval; Chrysomya rufifacies;species identification

Introduction

Determining the time of death is one of the key issues in forensic investigation.Traditionally,estimation of the postmortem interval（PMI）focused on physical postmortem changes of the corpse in a crime scene.Many a factor that influences decomposition can even lead to wrongly determining the PMI[1].Sarcosaprophagous flies often play an essential role in the estimation of PMI when the information is not available concerning the postmortem phenomena.An accurate estimation often relies on the blowflies which first colonize the human cadaver,especially in the case of advanced decomposed stage[2-3].Calculating the developmental time of forensic insects on the corpse allows forensic scientists to estimate the time when the insects first colonize the cadaver,and this information can provide important clues for estimating the minimum postmortem interval（PMImin）[4-6].

Forensic entomologists estimate PMIminby examining the developmental stage of blowflies found on the corpse of the crime scene[7].It is critical,therefore,to identify the species of insect for the estimation of the PMImin,as there exists significant differences in the developmental rates among different species of blowflies[8-9].Efforts have been made to identify blowfly specimens by morphology[9-11]and mitochondrial DNA sequence analysis[4,12-14].Chrysomya rufifacies（C.rufifacies,Macquart）,commonly known as the hairy maggot blowflies,is one of the primary forensic species in most areas of China[15-17].In the current investigation,we presented a successful application in a criminal case ofC.rufifaciesspecimens,which were collected as corpse-based entomological evidence during the scene investigation.This case highlights the necessary combination of morphological and molecular biological methods for species identification,and suggests an urgent need of growing developmental data of different forensic blowflies in different regions,so that forensic entomological evidence can be better applied to practical cases.

Case history

Crime scene investigation

An unknown female corpse was found on November 16th of a year in the bushes of a deserted factory in Chongqing（N 29°59′23′′,E 106°54′26′′）,Southwest China.The remains were covered by a discarded plastic advertising banner（Fig.1A）.The truncus of the corpse wrapped with a jacket and shirt was highly decomposed,the head and both upper limbs becoming skeletonized remains.However,both lower limbs wore tight pants and stockings,beginning to decay slightly.The soft tissues of the chest and abdomen were extensively colonized with third instar maggots.In the hair were found lots of light brown to dark brown pupae,as well as several empty puparia（Fig.1B,C）on the corpse and jacket.Near the corpse there laid a pair of leather sandals and a handbag,in which were found the identification card and keys,indicating that the owner of these items was this decedent.The corpse was transported to the laboratory for further autopsy,medicolegal examination and identification.

Fig.1 The crime scene

To determine whether her death was caused by a crime,the scene was investigated extensively and carefully.Bloodstain was found at the scene with Bluestar®Forensic Latent bloodstain reagent（Monte-Carlo,USA）following the manufacturer’s instructions.DNA from the blood was amplified using AmpFℓSTR®Identifiler®Plus PCR Amplification Kit（Thermo Fisher Scientific,USA）according to the manufacturer’s instructions.DNA typing was analyzed using the GeneMapper®IDv3.2 software（Thermo Fisher Scientific,USA）and all the alleles of 16 STR loci matched with the decedent completely.

Autopsy findings

When a new crack was observed in the collar of the cadaver’s jacket,there appeared a wound that may have been caused by a sharp tool of a knife,on the surface of the third cervical vertebra.Moreover,fractures were discovered on the cadaver’s nasal bone and left temporal bone（Fig.2A,B）,while such common poisons as sedatives,rodenticides and organophosphorus pesticides were not detected in the corpse.

Fig.2 The skull

Apparently,the cause of death was homicide,which was decided based on the evidence collected during the scene investigation and systemic forensic examination.The evidence indicated that the victim was violently attacked before her death,and the site where the victim’s body was found was the crime scene.

Entomological examination

The insect samples,including larvae,pupae,and empty puparium,were collected from the corpse and the crime scene,before transferred to the laboratory in 70% alcohol for species identification.

The larvae,pupae,and empty puparia appeared hairy,having a distinct stout,and the elongated tubercles encircled the body segments.Each tubercle had a large base,with a gradually tapering tip having slender spines apically under the microscope equipped with LeicaM165C camera（Leica Microsystems,Switzerland;Fig.1C）.The morphological analysis of the specimens proved that all samples wereC.rufifaciesaccording to the published protocols[4,17-18].

From the corpse was extracted the mitochondrial DNA of the specimen.DNA amplification was performed according to the PCR protocol of the Phire Animal Tissue Direct PCR Kit（Thermo Fisher Scientific,USA）using published PCR primers[12],the cycling condition:95 ℃3 min;94 ℃3 s,45 ℃60 s,72 ℃ 60 s,35 cycles.Consequently,the PCR products were obtained to be purified through agar gel electrophoresis and sequenced on ABI PRISM®377 DNA Sequencer（Thermo Fisher Scientific,USA）.The sequences of 498 bp in cytochrome c oxidase subunit I（CO I）gene were acquired by multiplealignment DNAMAN version 6.0（Lynnon Biosoft Company,Canada）,and the sequence alignments were disposed.The obtained sequences were submitted to GenBank（http://www.ncbi.nlm.nih.Gov/equin/index.html）to be analyzed for sequence similarity to the published CO Ⅰsequences through Basic Local Alignment Search Tool（BLAST,https://blast.ncbi.nlm.nih.gov/Blast.cgi）analysis against the National Center for Biotechnology Information（NCBI）NT/NR database,hence the phylogeny tree constructed.Based on the BLAST,the specimen sequence from the corpse showed the highest consistency withC.rufifacies,with the max ident being 100%.This further verified that the insect specimens on the corpse wereC.rufifacies.

PMI estimation

The weather data of the crime scene in the past few weeks were obtained from the nearest meteorological station.In addition,its temperature and relative humidity were measured thrice a day to calibrate and improve the accuracy of the weather data.The corrected crime scene temperatures were calculated using a linear regression based on the temperature measured at the crime scene using an electronic thermohygrometer.

After the temperature measurement and calibration at the crime scene,it was found that the average temperature was 19.0 ℃ in the past 20 days according to the obtained weather data.The average temperature in the crime scene was higher than the threshold values forC.rufifacies[19].According to the developmental data from the previously published studies[19-20]and our previous experiments conducted under the relevant temperature in this region[21],we calculated that the duration of the oldest samples from egg to eclosion was 452 h at an average temperature of 19.0 ℃.Therefore,the entomological data indicated thatC.rufifaciescolonized the corpse for at least 452 h before the corpse was found.Based on such entomological evidence,we estimate the PMIminto be 452 h.

Suspect testimony

Thanks to this information,along with other physical evidence,the suspect was identified.The murderer confessed that he was resisted by the victim when he tried to rape the victim.In order not to expose his criminal act,he hit the victim’s head and face,causing her in a coma,before cutting her neck with a sharp knife,resulting in excessive blood loss and death.The actual time of the victim’s death occurred 456 h（19 days）ago before the corpse was found.

Discussion

The estimation of PMI is still remains one of the most challenging issues in forensic investigations,especially when the corpse is in a state of advanced decomposition.When investigating a corpse,the PMI can range from hours to days,months,or even years[22].The developmental stage or body size of the oldest immature insects becomes promising evidence in forensic investigations when the traditional physical postmortem changes cannot meet the requirement of PMI estimation.In the current case,the PMI was successfully estimated using the forensic entomological method,with only a 4-hour deviation,which illustrated the advantages and feasibility of this scientific approach.

Behind the success of this case existed three main reasons.The wounds on the head,face,and neck of the decedent,as well as the bloodstains on the scene,sent out attracting smell toC.rufifaciesto lay eggs quickly,causing a short time lag between death and the development ofC.rufifacies.Next,the discovery of a small number of empty puparium meant the oldest flies having just finished their pupa stage to become adults,which is well known to be an important critical time point for the estimation of PMI.Additionally,the development data ofC.rufifaciesobtained from the investigation in this area and the relevant reports in other areas[19-21]provided a scientific basis for this case.To the best of our knowledge,this is the first report of the successful application ofC.rufifaciesto a real case for estimating the PMI in Southwest China.

China has a long history of using insects as forensic evidence in criminal investigation[23],and the first documented forensic entomology case was reported in Song Dynasty[24].With a vast territory,China spans many latitudes and longitudes,the climate varying greatly from region to region.Its temperature and humidity can have an impact on the development rate of insects[25-26].As a result,the dominant species of sarcosaprophagous flies are quite different,and the developmental data of sarcosaprophagous flies are also not entirely consistent in different regions.The duration of each development milestone can be displayed in an isomorphen diagram,which serves as a good reference for the application of forensic entomology[19].It is necessary that forensic scientists investigate which species of sarcosaprophagous flies exist in some specific areas and the developmental data of various sarcosaprophagous flies in different regions[19,26].

As predominant blowfly species found in corpses across the world,C.rufifaciesserves as an important forensic evidence[15,17].In the current case,we calculated the PMIminaccording to the developmental stage of the empty puparia ofC.rufifaciesfound in the corpse,which suggested the forensic importance of collecting the puparium samples at a crime scene.In general,it is difficult to distinguish the species of closely related blowflies based on the morphology of the empty puparium[12].Furthermore,the poorly preserved or fragmented puparia are difficult for morphological identification.In the current case,the morphology of the larvae,pupae,and puparium was identified to beC.rufifaciesbased on its unique tubercles that are endowed with only sharp apical spines[4,18].Additionally,this conclusion was confirmed by the DNA bar coding analysis and phylogenetic tree,so that the fundamental clue was provided to estimate the time of death.

Due to the environmental differences where the corpses are hidden,the environmental temperature of the crime scene needs to be tested and calibrated[27-28].In this case,the average temperature of the scene was higher than that derived from the weather data by the nearest meteorological station,because the corpse was covered with a discarded advertising banner,which absorbed heat from the sun and prevented the entry of rainwater,without preventing the oviposition of the blowflies.Therefore,it is crucial to calibrate and improve the accuracy of weather data at the crime scene case by case.To our knowledge,there have been few related researches in this area;it is worth pursuing such forensic investigations in the future.

Acknowledgements

This study was supported by Natural Science Foundation of Shandong Province（ZR2020QH288）,Youth Innovation Team Development Project of Shandong Universities（2019KJE018）,Project of Shandong Province Higher Educational Science and Technology Program（J17KA240）,the Open Topic Project Foundation of Hubei Chongxin Forensic Appraisal Center（CXLL201803）,and Postgraduate Education Quality Improvement Program in Shandong Province（SDYAL18097）.