Identification of Human Remains by DNA Analysis of the Gastrointestinal Contents of Fly Larvae

Dipterous fly larvae (maggots) are frequently collected from a corpse during a criminal investigation. Previous studies showed that DNA analysis of the gastrointestinal contents of maggots might be used to reveal the identity of a victim. However, this approach has not been used to date in legal investigations, and thus its practical usefulness is unknown. A badly burned body was discovered with its face and neck colonized by fly larvae. Given the condition of the body, identification was not possible. Short tandem repeat (STR) typing was performed using the gastrointestinal contents of maggots collected from the victim and was compared to STR profiles obtained from the alleged father. The probability of paternity was 99.685%. Thus, this comparative DNA test enabled the conclusive identification of the remains. This is the first reported case of analysis of human DNA isolated from the gastrointestinal tract of maggots used to identify a victim in a criminal case.     

根据绿蝇属昆虫推断死亡时间的法医学研究进展

尸源性昆虫在某些刑事案件的侦破中占有重要地位,对于推断死者的死亡时间和推断尸体是否有转移有重要的作用。双翅目中的丽蝇科绿蝇属昆虫是尸体上常见的昆虫之一,通常最先到达尸体,而且最喜阳光,该特点以及在尸体上发现的绿蝇属昆虫幼虫的生长发育阶段,可以为刑事案件的侦破提供十分有力的证据。本文主要从生物形态学和分子生物学对绿蝇的种属鉴定以及绿蝇日龄的推断等方面,综述了根据绿蝇属昆虫推断死亡时间的法医学应用研究进展。     

STR and SNP analysis of human DNA from Lucilia sericata larvae's gut contents

Importance of forensic entomology becomes inevitable when come across some incident where corpse is unidentifiable and lot of maggots or other insects are present. The most common application of forensic entomology is to use insects for the identification of specimens or human remains. DNA analysis recovered from a larva's gut contents can be used to identify a missing body. The obtained human STR and SNP profile support the association of a maggot to a specific patients or corpse. Main aim of this research was the identification of human DNA from gut contents of third instar maggots (larvae of Lucilia sericata) placed on diabetic patient's wounds for treatment purpose. Maggots (8–15) were taken from each diabetic patients (no. of the patients 8) and DNA was extracted from the gut contents manually by using Qiagen tissue protocol. Agarose gel electrophoresis was performed and the total size of DNA was seen using UV transilluminator. PCR amplification, STRs and SNPs profiling was then performed using PCR 9700 and AmpFLSTR Identifiler and SnaPshot Multiplex Kit (Applied Biosystems) respectively. The results were analyzed on ABI 310. SNP profiles were good and identifiable compared to the STRs where amplification was poor and the peaks were low. This may be the fact of the enzymatic activity present in the gut of the larvae which cause tremendous reduction in DNA size and thus yield. The results of this study reveals that it is possible to obtain a complete human profile using STRs and SNPs even if DNA is recovered from gut contents of maggots.     

贵阳市郊嗜尸蝇类群落组成及生长观察

目的观察嗜尸蝇类种群落组成、季节变动及蛆虫生长长度与死亡时间的关系，为在死亡时间推断中应用提供基础数据。方法采用荷兰猪尸／兔尸／猪肺作诱饵，在海拔1115．2～1120．8m的自然环境中吸引蝇种。捕抓特定时间段内优势蝇种，每日定时观察记录其在营养基上产卵、幼虫孵化、成熟幼虫爬离的时间，测量其长度，采用Excel软件进行数据处理。结果获取了贵阳市郊宽丽蝇、巨尾阿丽蝇、紫绿蝇、丝光绿蝇、棕尾别麻蝇、肥躯金蝇、大头金蝇和绯颜裸金蝇等8种常见嗜尸蝇类群落组成、季节变动及蛆虫生长长度的规律性变化数据，并用于实际案例死亡时问的推断。结论嗜尸蝇类群落组成、季节变动及蛆虫生长长度在推测死亡时间上有重要意义。     

Growth of Chrysomya megacephala (Fabricius) Maggots in a Morgue Cooler*

Abstract: In estimating the postmortem interval (PMI) using maggots obtained during autopsy, the forensic entomologist makes decisions regarding the effects of low‐temperature storage of the body on the insects. In this case report, a corpse was found in an abandoned house in the residential area of Bukit Mertajam, Penang, Malaysia. The maggots were found to be alive inside the mouth of the deceased although the corpse had been in the morgue cooler for 12 days. The maggots were reared and identified as Chrysomya megacephala (Fabricius). The emerged adult flies were kept as a stock colony, and the duration of development under the indoor fluctuating temperature regime was studied. The total duration of developmental process of this species was 9.5 ± 0.5 days, and the PMI estimated was 3.2 ± 0.6 days. This case report demonstrates the survival of Ch. megacephala maggots for 12 days and their growth inside the morgue cooler.     

Forensic entomo-toxicology.

Drugs present in a decomposing corpse may be identified through analysis of maggots feeding off it. Case reports in forensic entomo-toxicology are sparse and the data base is unstructured. Drug concentrations should be measured in residual skeletal muscle, the principal food source for fly larvae, as well as in washed maggots, and the fly species should be determined. An untested possibility is the analysis of puparia or puparial cases which could extend the time frame for analysis into years or even into palaeopathology. In deaths indoors, the analysis of flies known to have emerged from the corpse is a theoretical possibility. To what extent drugs are retained in successive levels of the food chain is entirely unknown; drugs might be detectable in beetles feeding off fly larvae.     

Entomological and Cardiologic Evidence of Time Since Death in Short Postmortem Intervals

Estimating the time since death of the deceased is a main goal in forensic investigations, but this can be challenging due to contradictory results derived from different investigations at the scene of death. We present a case of a 78‐year‐old woman, found dead in a small forest with broad‐leaved trees, whose husband had a history of domestic violence. Routinely performed investigations, such as postmortem rectal temperature and lividity, yielded inconsistent time since death results between only a few and longer than 20 h. This difficulty was most likely caused by high ambient temperatures of up to 38.0°C, which negatively influenced the informative value of the applied nomogram method. Additionally, performed entomological investigation of fly maggots (Lucilia illustris) recovered from the corpse and the assessment of heart pacemaker data revealed consistent and incontrovertible results. The presented case highlights the benefits of information provided by entomological investigations and data evaluation of cardiac implantable electronic devices and the combined use of the two techniques.     

The effects of larval crowding and food type on the size and development of the blowfly, Calliphora vomitoria

The use of entomological evidence in the estimation of the post mortem interval (PMI) often depends on the size and developmental stage of blowfly larvae collected from a corpse. Therefore, factors which can have an effect on the larval size and growth rate can have implications for reliable PMI determinations. This study explores the competitive effects of larval overcrowding on Calliphora vomitoria reared on three different pig tissues--liver, brain and muscle. The competitive feeding environment within the more crowded larval cultures resulted in increased development rates and the production of undersized larvae and adults. Variation in the extent of these effects was observed on each of the three body tissues, highlighting the importance of documenting the positions from which entomological evidence is recovered from a corpse.     

Influence of substrate tissue type on larval growth in Calliphora augur and Lucilia cuprina (Diptera: Calliphoridae)

The size of fly larvae is an important variable in the use of these insects to estimate postmortem interval. Furthermore, the nutritional intake of larvae is likely to vary subject to the part of a corpse on which they are feeding. A study was therefore conducted to investigate the effect of type of food substrate on larval growth in two species of forensically important Australian blowflies. After collection on sheep's liver in the laboratory, different groups of larvae of Lucilia cuprina (Wiedemann) and Calliphora augur (Fabricius) were grown on sheep's liver, meat, and brains, and their body lengths compared. Results indicated that the development of larvae fed sheep's liver was adversely affected compared with larvae fed meat and brain; they moulted later, reached maximum length more slowly and sometimes produced significantly smaller pupae. These findings, similar to those of another recent study, have obvious implications for postmortem interval determinations. Estimates may be considerably skewed if the site of collection of larvae at a death scene contains tissue types different to those used in reference experiments. We therefore recommend caution in forensic analyses that interpret crime scene data using developmental studies performed with a single type of larval food substrate.     

Surface sterilization of a maggot using bleach does not interfere with mitochondrial DNA analysis of crop contents

Mitochondrial DNA analysis of a maggot crop can identify the corpse a maggot has been feeding on. Analysis of the crop could be useful in a criminal investigation if maggots are found at a suspected crime scene in the absence of a body, or if there is a question of whether a maggot used in postmortem interval estimations moved onto the corpse from another food source. Such analysis can also resolve a chain of custody dispute if it has been suggested that larval samples have been switched. When recovering DNA from a maggot crop, the analyst must be careful to avoid external contamination. We investigated the effects of three simple wash methods developed to reduce external contamination. Maggots raised on pig liver and intentionally contaminated with cow blood were washed using water, 20% bleach, or a solution containing the enzyme DNase. Only washing the maggots in 20% bleach reduced the amount of vertebrate DNA amplified from the maggots' exteriors. No wash method affected the ability to recover DNA from the maggot crops.     

Unilateral occurrence of blowfly maggots in the face of a decomposing body

The corpse of a 41-year-old medical doctor was found in his bed. The body was partially dried out; parts of the hip region were skeletonized due to maggot activity. In the facial region of the corpse, blowfly maggots (Lucilia (Phaenicia) sericata [Meigen]) were found exclusively in one eye socket. This is an unusual occurrence since on that side, a bedlight (40 W light bulb) had been burning during the seven week post mortem interval. All other lights in the apartment were switched off, and no direct sunlight could enter the space where the body was found (only a TV set had been running all the time, ca. 2 m away from the head at the foot end of the bed). Obviously, the maggots who usually flee light had used up the one eye that was further away from the bedlight as a feeding source. Since the continuing mummification of the corpse led to a substantial restriction of feeding material, the maggots finally switched to the eye that the light was shining on.     

Nocturnal Oviposition Behavior of Necrophagous Dipterans in Kelantan,Malaysia*

Abstract: The likelihood of dipteran maggots colonizing a corpse due to nocturnal oviposition can be used to challenge the postmortem interval (PMI) estimated assuming diurnal oviposition. Earlier experiments tested nocturnal oviposition behavior by exposing fresh baits once during a single night. In this pilot study, oviposition behavior was studied using beef baits, which, simulating the decay of the body seen in case situations, decomposed inside cages designed to open and close at scheduled intervals during consecutive night or twilight periods. Freshly hatched maggots from diurnally oviposited eggs emerged in control baits on the third day, while a limited number of maggots attributable to nocturnal or twilight oviposition were observed in experimental baits only on the fifth or sixth day, indicating a categorical delay. These results suggest that such delayed and limited nocturnal oviposition is not forensically significant since the larger maggots deriving from diurnal oviposition would be the ones considered when estimating PMI.     

Experimental validation of forensic evidence: a study of the decomposition of buried pigs in a heavy clay soil.

In a murder investigation, where the victim had been strangled and buried in a shallow grave, there were discrepancies between the post mortem interval (PMI) as estimated from entomological studies and estimations determined from other evidence. This inconsistency provided the impetus for examining the decay process using pig carcasses as analogues for the human cadaver. The pigs were buried in the immediate vicinity of the original burial site in December 1996, which was the month when the victim was purported to have been interred in the previous year. The buried pigs were then monitored for 5 months which, based on the evidence other than the entomological, was the period over which the corpse was thought to have lain in the ground. The pig corpses were disturbed by scavengers in mid April: this was the same time that the human corpse was discovered in the previous year by scavengers. Insects played no role in the decomposition process until the pig carcasses had been exposed by animals. Blowflies, notably Calliphora vomitoria, were attracted to the exposed tissues and laid eggs from which larvae developed. Calliphora vomitoria is a species often used to estimate PMI. This investigation has shown that soil conditions and low seasonal temperatures had preserved the pig carcasses for longer than might be expected. Using the blowfly larvae to estimate PMI would have produced erroneous results had not the burial environment and exhumation history been investigated.     

DNA typing of human remains found in damp environments

DNA typing is often used to determine identity from human remains. The environment to which the material has been exposed, however, is crucial for the success of the investigation. Damp conditions in particular can cause a rapid degradation of DNA, even in bone and teeth, and thus reduce the chances of successful typing. Here, we present the results of investigations performed on four skulls that had been lying in a damp environment for periods ranging from almost 1 year to about 45 years. In none of these cases was DNA typing successful on bone or, where present, on teeth. Where remnants of brain tissue were used, however, complete STR typing was possible in one case, partial STR typing in another, and mtDNA sequencing could be carried out in three cases. These findings suggest that brain tissue is relatively resistant to putrefaction in damp environments and, unlike bone, appears to exhibit a certain degree of protection against DNA degradation.     

A Forensic Entomological Analysis Can Yield an Estimate of Postmortem Interval,and Not Just a Minimum Postmortem Interval: An Explanation and Illustration Using a Case

Several authors claimed that a forensic entomological analysis can never be interpreted as the postmortem interval (PMI), but that it can be interpreted as a minimum postmortem interval (PMI_min), or dead for not less than X amount of time. Because in most instances, a PMI estimate should be a range, that is, the set of values from maximum postmortem interval (PMI_max) to PMI_min, objections to PMI estimation are objections to PMI_max estimation. The arguments for this position did not address the substantial body of literature describing estimation of both PMI_max and PMI_min using insect succession analysis. This report reviews the scientific issues and presents a recent homicide investigation in which several forensic entomology experts used the absence of carrion insects on the corpse, a kind of succession analysis, to estimate PMI or PMI_max.     

A Case of Insect Colonization Before the Death

Forensic entomology is a branch of forensic science in which insects are used as evidence in legal investigations relating to humans, domestic animals and wildlife. One of the theoretical pillars on which the discipline is based concerns the fact that flies colonize a body after death. However in cases of myiasis, maggots are present before death, with consequences in the correct estimation of the minimum postmortem interval (mPMI). We report here the case of a woman, largely colonized by fly larvae, who has lain alive in her garden for four days prior to being rescued. Larvae were found on the conjunctivae, the bronchi, the rectum and vagina. The woman's death, two months later, was caused by tetanus. The consequences of myiasis on mPMI estimation are here discussed. In fact, despite she was still alive larvae, indicated and estimated age of 1.5–2.5 days, based on environmental and body temperature.     

PRAXIS--combined mu-Raman and mu-XRF spectrometers in the examination of forensic samples

A major role of forensic entomology is to estimate the post-mortem interval. An entomologist's estimate of post-mortem interval is based on a series of generally valid assumptions, error in any of which can alter the accuracy of an estimate. The initial process of collecting and preserving maggots can itself lead to error, as can the method of killing and preservation. Since circumstances exist where it is not possible to rear maggots, methods of killing and preservation can be vital to preserving the integrity of entomological evidence. In this study, a number of preservation techniques used at crime scenes and in mortuaries were examined, and their effect on feeding third-instar larvae of Calliphora augur and Lucilia cuprina evaluated. The preservatives used were 70, 75, 80, 90 and 100% EtOH, Kahle's solution and 10% formalin. Each treatment was replicated three times. The effect of handling on first- and second-instar, feeding and post-feeding third-instar larvae of C. augur was also examined and compared to unhandled controls. Finally, the effects of preservatives were noted when larvae of C. augur and L. cuprina were placed into preservatives alive. It was found that continued handling is detrimental to specimens because preservative evaporates from both the vial and the specimens. No single preservative type was found to be entirely suitable for both species if DNA retrieval is desired. Specimens placed into most preservatives alive exhibited adverse colour changes, desiccation, sunkeness and agglomeration. It is concluded that the reaction to preservative type might be species specific and that different instars of the same species might also react differently.     

Determination of Malathion levels and its effect on the development of Chrysomya megacephala (Fabricius) in South China

The present study determines the concentration of Malathion in rabbit tissues and Dipteral larvae feeding on those tissues. Malathion was found in all muscle and liver samples of the test rabbit, as well as larvae fed on it. Samples from the control rabbit and pupae from all colonies were negative for Malathion. Correlations were found between administered dosage, tissue concentrations and younger or prepuparial larvae. Effects of Malathion on the development rate of Chrysomya megacephala (Fabricius) were also reported. C. megacephala is the most common fly species found on corpses in South China during the early stages of decomposition. Significant differences in larval growth rate were both observed among the colonies fed on muscle and liver. The presence of Malathion in both muscle and liver appears to retard the normal growth rate of C. megacephala in larval stage. Larvae from all colonies fed on tissues from rabbits treated with Malathion were smaller and attained maximum length later than those from the control colony. Duration of the larval and pupal stages was both significantly prolonged for larvae on tissues from rabbit receiving Malathion than those from the control colony. The difference of the duration of the larval and pupal stages together from the muscle colonies would alter the postmortem interval estimation by up to 36 h. As for liver colonies, it would alter the postmortem interval estimation by up to 28 h. A significantly different duration of the larval and pupal stages from the muscle colonies would alter a postmortem estimate by up to 28 h relative to the liver colonies.     

Factors affecting decomposition and Diptera colonization.

Understanding the process of corpse decomposition is basic to establishing the postmortem interval (PMI) in any death investigation even using insect evidence. The sequence of postmortem changes in soft tissues usually gives an idea of how long an individual has been dead. However, modification of the decomposition process can considerably alter the estimate of the time of death. A body after death is sometimes subject to depredation by various types of animals among which insects can have a predominant role in the breakdown of the corpse thus, accelerating the decomposition rate. The interference of the insect community in the decomposition process has been investigated by several experimental studies using animal models and very few contributions directly on cadavers. Several of the most frequent factors affecting PMI estimates such as temperature, burial depth and access of the body to insects are fully reviewed. On account of their activity and world wide distribution, Diptera are the insects of greatest forensic interest. The knowledge of factors inhibiting or favouring colonization and Diptera development is a necessary pre-requisite for estimating the PMI using entomological data.     

Forensic entomology in the Hawaiian Islands. Three case studies

Three instances of the use of entomological techniques for the estimation of postmortem interval in homicide cases on the island of Oahu, Hawaiian Islands, are presented. These cases represent differing stages of decomposition and range from 8 to 53 days postmortem.