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.     

STR typing of human DNA from fly larvae fed on decomposing bodies

In homicides with entomological evidence, it may be important to prove the presumed association of fly larvae to a corpse, especially if it is in doubt whether all maggots used for entomological expertise developed and fed on it. The present study demonstrates for the first time the possibility of analyzing human microsatellite DNA present in the digestive tract of necrophagous larvae that fed on decomposed bodies with a postmortem interval up to four months. The obtained human STR profiles support the association of a maggot to a specific corpse. In addition, the identification of the host species (e.g., animal source like pig) can be achieved by analysis of the cytochrome b gene. Maggots were collected from 13 corpses after various postmortem intervals and STR typing and HVR amplifications were performed using their crop contents. In seven cases, a complete STR profile was established, in two cases, an incomplete set of alleles was obtained, and in four cases, STR typing was not successful. HVR analysis was successful in all cases except one. The time of storage of the maggots and the length of the postmortem interval up to 16 weeks appeared to have no particular influence on the quality of the results.     

Forensic genetic analysis of insect gut contents

Entomological evidence is most often used for estimating the postmortem interval, but fly larvae can also be a source of vertebrate DNA. Forensic analysis of DNA recovered from a larva's gut can be used to identify what the larva had been feeding on. During our previous research studies, we used the same DNA extraction for the dual purpose of identifying the insect species and associating a maggot with its last meal. In our experience, we have encountered several situations where this method for associating a maggot with a corpse would have been useful, such as removal of remains from a suspected crime scene, an alternative food source is nearby the scene or the body, and a chain-of-evidence dispute. However, since maggot gut content analysis is a quite brand-new area of study, many of the limitations of the technique have not yet been explored. The results of our most recent research studies suggest that third-instar larvae actively feeding on the corpse can be considered the best source of human DNA, better than postfeeding or starved larvae. In this paper, the state of the art of forensic genetic analysis of maggot gut contents is reviewed.     

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.     

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.     

Maggot-induced postmortem changes simulating gunshot wounds

In the warm season, human cadavers often are the breeding ground of fly larvae. In special conditions, the damage by maggots can take on the appearance of bullet holes. Preexisting injuries allow the maggots to penetrate the skin also in parts other than the body apertures commonly infested. Round tissue damage similar to gunshot-wounds are favored by the fact that the larvae of some types of flies bundle together to form feeding communities. The enzymes from the salivary glands of the fly larvae may in some rare instances even lead to perforation of bone lamellae (e.g. in the region of the orbital roofs). The case reports deal with 2 cases in which changes due to damage by maggots primarily gave the impression of gunshot-wounds.     

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.     

Forensic entomology exemplified by a homicide. A combined stain and postmortem time analysis]

The combined analysis of both ant and blow fly evidence recovered from a corpse, and from the boot of a suspect, suggested that an assumed scenario in a high profile murder case was likely to be true. The ants (Lasius fuliginous) were used as classical crime scene stains that linked the suspect to the scene. Blow fly maggots (Calliphora spec.) helped to determine the post mortem interval (PMI) with the calculated PMI overlapping with the assumed time of the killing. In the trial, the results of the medico-legal analysis of the insects was understood to be crucial scientific evidence, and the suspect was sentenced to 8 years in prison.     

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.     

Growth rates of the blowfly, Lucilia sericata, on different body tissues

Estimates of post-mortem interval based on forensic analysis of the age of blowfly larvae on a corpse, are generated from standardised larval development curves. These are derived from studies of the growth of larvae reared, usually, on liver and at a range of temperatures. However, it is possible that the species or organ on which the larvae feed, might significantly alter the rate of growth. The present study therefore compared the development of the blowfly Lucilia sericata (Meigen) (Diptera: Calliphoridae) fed on lung, liver and heart, from both cows and pigs. Half of the tissue samples used were liquidised while half were not, to assess whether structural differences in the food source were important. Larvae grew significantly faster and gave rise to larger adults when reared on pig compared to cow tissue and when reared on lung and heart compared to liver. Larvae completed feeding and wandered from the food source 31h earlier and grew 2mm longer when reared on lung compared to liver. Tissue structure had no or little effect on development. For the forensic entomologist, these results highlight the importance of recording the position of larvae removed from a body and the care that should be used when extrapolating development rates from standard curves based on larvae fed on only a single medium, particularly where that is liver.     

Some notes on the nocturnal larviposition by two species of Sarcophaga (Diptera: Sarcophagidae)

Experiments in the month of September 2005 were performed to study nocturnal larviposition by the flesh flies (Diptera: Sarcophagidae). Two species namely Sarcophaga albiceps Meigen and Sarcophaga hirtipes Wiedemann were observed to deposit their larvae during night time as well. Therefore, possibility of night larviposition by flesh flies should always be taken into consideration while determining age of maggots.     

Fly larvae and their relevance in forensic toxicology

Toxicological analyses on a putrefied cadaver are sometimes difficult to perform because of the absence of blood and urine. In this study, fly larvae, being living material, are proposed as a new medium of investigation in forensic toxicology. Bromazepam and levomepromazine were identified and assayed in the remains of cerebral tissue, in the clavicle of a putrefied cadaver, and in the fly larvae found on and in the corpse.     

Larder beetles (Coleoptera,Dermestidae) as an accelerating factor for decomposition of a human corpse

Larder beetles are known to feed directly upon decomposing carrion, with a preference for dried carrion. Under optimal environmental conditions (dry and warm), they can appear in large numbers. In our case, the mummified corpse of a human male was nearly skeletonized in less than 5 months in his apartment with windows closed and a room temperature between 25 degrees C (near the radiator) and 19.4 degrees C (near the body). There were very few empty fly pupae in the apartment, but a lot of adult hide beetles, their larvae and larval cast skins (exuviae) (Dermestes maculatus DEG.) belonging to the family of the larder beetles (Dermestidae). The beetles skeletonized the human corpse in such a short time because of ideal conditions for them in the apartment (dry and warm) making the body an optimal feeding ground because of his mummification.     

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.     

Toxicology and fly larvae on a putrefied cadaver

Toxicological analyses on a putrefied cadaver are sometimes difficult to achieve, due to the absence of blood and/or urine. In this study, morphine and phenobarbital were simultaneously identified and assayed in several tissues of a putrefied cadaver and in the fly larvae of Calliphoridae found on the corpse.     

Mitochondrial DNA sequencing of beetle larvae (Nitidulidae: Omosita) recovered from human bone

The isolation, amplification, and characterization of human DNA from hematophagous (blood feeding) and necrophagous (carrion feeding) arthropods have been advanced significantly by the development of polymerase chain reaction (PCR) DNA sequencing methodologies. Historically, DNA technology has been successfully utilized to identify individual hosts upon which species of hematophagous arthropods have fed. The analysis of hematophagous insects' gut content blood meals has led to major advances in medical entomology and vector-borne disease epidemiology. In the forensic arena, the ability to apply similar techniques to insects recovered from badly decomposed remains has been greatly enhanced through the advent of mitochondrial DNA (mtDNA) techniques. Mitochondrial DNA analyses have been utilized to identify both the human remains upon which fly larvae (maggots) have fed and the species of the larvae themselves. The preliminary work detailed here demonstrates, for the first time, the successful application of mtDNA sequencing techniques to the analysis of necrophagous beetle larvae. A small sample of sap beetle larvae, Omosita spp. (Coleoptera: Nitidulidae), was collected from human skeletal remains during anthropological examination and analyzed for human DNA using mtDNA sequencing. The beetle larvae yielded mtDNA matching that of the host human bone. The results detailed here further demonstrate the robust nature of human mtDNA and the ability to recover valuable mtDNA evidence from forensically important, late decompositional stage insect species.     

A model for the aging of fly larvae in forensic entomology

A method is described for determining the time of hatching of blowfly larvae on a corpse with respect to temperature. Temperature is known to have a profound effect on the rate of growth of fly larvae, and it is suggested that past assumptions that the growth of larvae found in a corpse has taken place at one approximated ambient temperature may have led to large overestimates of the development time of the larvae, and thus overestimates of time of death. Larvae of Lucilia cuprina, Calliphora vicina (= erythrocephala), Calliphora stygia and Calliphora hilli were cultured under constant temperatures, and their growth rates modeled with a logistic growth curve. Two programmes have been developed, one to compute the parameters for the logistic equations, the other is used to estimate the time elapsed from the time at which a sample is removed from a body to the estimated hatching time of the larvae comprising the sample, with temperatures and species as variables.     

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.     

Width as an alternative measurement to length for post-mortem interval estimations using Calliphora augur (Diptera: Calliphoridae) larvae

The length of fly larvae collected from corpses is often used to help provide an entomological estimate of time since death. However, 'head-curling' by larvae can affect the accuracy of length measurements. To investigate a possible resolution to this problem, larvae of Calliphora augur were grown on sheep's liver at two constant temperatures (20 and 25 degrees C). Replicate samples were collected at set time intervals until pupation. Body length and width were measured for individual larvae and examined as predictors of age. It was found that body width, as measured at the junction of the fifth and sixth abdominal segments, is comparable with body length for age prediction of maggots of C. augur grown at these temperatures. Furthermore, conversion of width to length can be done with 95% accuracy from a simple linear model.     

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.