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.     

The effect of ionizing gamma radiation on natural and synthetic fibers and its implications for the forensic examination of fiber evidence

Circumstances of criminal activities involving radioactive materials may mean fiber evidence recovered from a crime scene could have been exposed to materials emitting ionizing radiation. The consequences of radiation exposed fibers on the result of the forensic analysis and interpretation is explored. The effect of exposure to 1-1000 kGy radiation doses in natural and synthetic fibers was noticeable using comparative forensic examination methods, such as optical microscopy, microspectrophotometry, and thin-layer chromatography. Fourier transform infrared spectroscopy analysis showed no signs of radiation-induced chemical changes in any of the fiber structures. The outcome of the comparative methods highlights the risk of "false negatives" associated in comparing colors of recovered fibers that may have been exposed to unknown radiation doses. Consideration of such results supports the requirement to know the context, including the environmental conditions, as much as possible before undertaking a forensic fiber examination.     

First record of Hermetia illucens (Diptera: Stratiomyidae) on human corpses in Iberian Peninsula

This article presents the first record of Hermetia illucens larvae on a human corpse in Spain (the second case report in Europe). Prepupae of H. illucens, and other insects, were recovered from the dead body of a 72-year-old man in an advanced stage of decomposition. The body was located in Reus (NE Spain), in October 1998. This article provides additional biological data on experimental studies and an update on the geographic distribution of this species in the Iberian Peninsula.     

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.     

The "last vestiges of life"

On 4 June 2010, Deputy State Coroner Schapel of the Coroner's Court of South Australia ruled that he had jurisdiction to conduct an inquest into the death of Tate Spencer-Koch, who was declared deceased following complications during her home-birth. Coroner Schapel considered he had jurisdiction on the basis that Tate was a "person in the eyes of the law" under the born alive rule, as she had completely left her mother's body and she could be shown to have been alive after birth, evident from the PEA detected in her heart 10 minutes following her delivery. Close consideration of this ruling, however, reveals issues with the application of the born alive rule in the case.     

Analysis of 683 drug packages seized from "body stuffers"

The wrappings and contents of 683 drug containers (bubbles) seized from 154 street dealers have been analysed since 2001, when the law enforcement policy in Hamburg, Germany, allowed the application of emetics to be used by so-called body stuffers for the recovery of swallowed drug packages. Of the bubbles recovered 96% contained cocaine. Cases of intoxication have not yet been observed but a 20-year-old man with a previously unknown toxic cardiomyopathy died 3 days after a sudden cardiac arrest which followed the forced application of ipecacuanha via correctly placed nasogastral tube.     

Eristalinus arvorum (Fabricius, 1787) (Diptera: Syrphidae) in Human Skull: A New Fly Species of Forensic Importance

A body of an unknown adult female was found within a shallow burial ground in Malaysia whereas the skull was exposed and visible on the ground. During autopsy examination, nine insect larvae were recovered from the interior of the human skull and subsequently preserved in 70% ethanol. The larvae were greyish in appearance, each with a posterior elongated breathing tube. A week after the autopsy, more larvae were collected at the burial site, and some of them were reared into adults. Adult specimens and larvae from the skull and from the burial site were sequenced to obtain DNA barcodes. Results showed all adult flies reared from the burial site, as well as the larvae collected from the skull were identified as Eristalinus arvorum (Fabricius, 1787) (Diptera: Syrphidae). Here, we report the colonization of E. arvorum larvae on a human corpse for the first time.     

The significance of the legal term "corpse" in forensic medicine

The definition of the term "corpse" is relevant for medicine in general and most particularly so for forensic medicine, as it has direct consequences on practical work: legally, postmortems and autopsies may only be performed on corpses. With this in mind, the legal term "corpse" was examined on the basis of German State Law regulations and provisions. Approximately half the burial acts include a definition of "corpse", but even here there are certain variations. The definitions for "corpse" can be divided into four categories: 1) according to the lack of vital signs or sure signs of death 2) according to the degree of decomposition and/or cohesion of body parts 3) on the basis of severed body parts, and 4) terminological differentiation of stillbirths and neonatal fatalities from miscarriages. Laws vary greatly in their definition of the term "corpse". Therefore, the decision whether or not human remains or parts thereof qualify as a "corpse" should be left to medical discretion.     

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.     

Presence of fecal material in diapers as a potential source of error in estimations of postmortem interval using arthropod development rates.

Second instar larvae of the fly Chrysomya megacephala (Diptera, Calliphoridae) were recovered from the diapers of a 16-month-old child abandoned by her mother on Oahu, Hawaii. The development of these larvae indicated a minimum period of 23.5 h of exposure prior to discovery of the child. Larvae of this species of fly are not normally associated with living tissues in Hawaii, but rather with feces and remains during the early stages of decomposition. Had the child in this case died and data not been provided detailing the site of infestation, the postmortem interval estimated would have been significantly longer than was actually the case, because of the development of the larvae inside the diapers of the living child. The need for caution in cases involving deaths of infants, the elderly, and individuals not capable of caring for themselves is stressed.     

Insects found on a human cadaver in central Italy including the blowfly Calliphora loewi (Diptera, Calliphoridae), a new species of forensic interest

In the case of unidentified bodies the estimation of the period since death or of the season of death plays an important role to focus the attention on a reduced number of people among the ones reported missing. Forensic entomology can be one of the most important methods for these estimations, as occurred in this case. Flies are typically the first insects to colonize a dead body. The case reported here concerns the colonisation by insects of a male body in advanced decay found during the winter in Central Italy. This case is of particular interest as few data are available on the entomological evidence in the cold season. In particular, in this case we recovered Calliphora loewi (Calliphoridae), a species never collected before on dead bodies in Southern Europe. Larvae of the black soldier fly Hermetia illucens (Stratiomyidae), pupae and larvae belonging to genus Hydrothea (Muscidae), and Necrobia rufipes (Cleridae) specimens were also collected. The estimated PMI enabled identification of the cadaver, confirmed by DNA analysis.     

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.     

Vladimir Shapoval: "The CEC Is Destined to Be the Scapegoat"

"The law is harsh, but it is the law"—the well-known ancient Roman saying is entirely suitable as a brief synopsis of the Lb.ua interview with the chairman of the Central Election Commission (CEC), Vladimir Shapoval.

"Any electoral legislation, I emphasize, any, will always be 95 percent the product of political expediency. Always. This way gives them an advantage—so be it." This is how he calmly parried my emotional "How can the advantage of the strong be codified in the law (!), and the weak essentially have no chance? What can you do, how can it be?" "All this ‘whining and crying,’ say, the law is this and that in substance, I do not accept it. The laws were adopted—so we will follow them. You cannot get away from it," he added.

It might seem to someone who does not know Vladimir Nikolaevich that the chief vote-counting official is being clever, "covering up" the "distortions" of the authorities "at the local level." He is in fact speaking frankly. The chairman of the Central Election Commission formed on the eve of the 2007 preterm parliamentary elections by a "coalition"—a retired Constitutional Court of Ukraine (CCU) justice, and a doctor of legal sciences who is an active member of the High Council of Justice (HCJ), he can permit himself that. Shapoval contrives to call things by their names: "How the law is written is another matter. And its parts are written abominably," he states bluntly.

In view of this, my discussion with Shapoval—formally tied to the start of a local elections campaign—went far beyond the bounds of a discussion of the legislation on this topic, and even the specifics of the campaign. The more so as I had already discussed this in detail with CEC Deputy Chairman Andrei Magera. Vladimir Nikolaevich, without concealing his indignation, related just what the HCJ is really afraid of (and it turned out it was not at all, or more precisely not only, what they are portraying it to be). Relying on nine years of experience as a CCU justice, he elucidated the risks of the invalidation of the 2004 constitutional reform by the current Constitutional Court—"only those who have face can lose it." Sketching out the situation in the body politic, he summed up, "If the Party of Regions (PoR) does not win the local elections, many questions will arise."     

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.     

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.     

The cocaine "body stuffer" syndrome: a fatal case

Body stuffer, sometimes called "mini packer", is the definition of someone who admits to or is strongly suspected of ingesting illegal drugs in order to escape detection by authorities, and not for recreational purposes or to transport the drug across borders. Cocaine is the drug most commonly involved in the body stuffer syndrome. Reported cases of body stuffer deaths are rare, however a fatality related to the ingestion of a plastic bag containing cocaine is described regarding a 17-year-old dealer. The authors describe how the cocaine body stuffer syndrome differs from the usual body packer. Histological and toxicological findings are examined and discussed for a better definition of this unique syndrome.     

Influence of storage on larval length and age determination of the forensically important blow fly Lucilia sericata (Diptera: Calliphoridae)

One of the main tasks in forensic entomology is the determination of the minimum post-mortem interval (PMI_min) based on the age of the juvenile insects feeding and developing on the dead body. An important task is to store the evidence appropriately so that the evaluation and expert report can be used in court. However, existing recommendations can be contradictory or lacking scientific validation, e.g. by proposing various preservation liquids without knowing whether and to what extent the period of storage in such a liquid has an effect on the length of the preserved larvae. Storage time can be an issue since, due to technical and procedural circumstances, killed larvae may be stored for hours, days, weeks or even longer prior length measurement. A changed body length would have consequences for the entomological report, as the age of the larvae is usually derived from their length.This study investigates the effect of four differently concentrated ethanol solutions (70%, 80%, 90% and 96%) during a storage period of up to 196 days on the body length of stored larvae of the forensically important blow fly species L. sericata (Diptera: Calliphoridae). Larvae of different ages (24 h, 48 h and 72 h after hatching) were killed by immersion in hot, non-boiling water (≥80 °C) for at least 30 s. Their lengths were measured immediately. Subsequently samples were stored in ethanol of appropriate concentration at room temperature (approx. 22 °C). Further length measurements were made at 16 different storage intervals between 1 and 196 days.Many specimens showed a length decrease for most storage conditions and all larval ages. However, there was a tendency for 48 h- and 72 h-old larvae to increase in length after the first days of storage of up to 1.1 mm which may lead to an erroneous overestimation of the PMI_min using this kind of specimens. All changes in length within each cohort over total time were in the range of +7% to −9.1%. Significant differences in length changes within the first days of storage were found mainly in larvae stored in 70%- and 80%-ethanol, but larvae stored in 90%- and 96%-ethanol showed first significant differences on day 56 at the earliest.Our results lead to the recommendation that the measurements of fly larvae samples should be taken immediately after killing and before storage to avoid any effects. Ethanol ≥90% should be used for storage.     

The "utility" principle within the scope of forensic determination of blood kinship using serostatistics

A "realistic" prior probability is always based on case experience (Akten-a-priori). In serological opinions pertaining to parentage, the realistic prior probability is only one piece of information in the whole body of evidence before the judge and does not have any special significance per se. There is no such thing as a "neutral" prior probability. It either implies "ignorance," in which case it cannot be "information," or it must be taken in connection with the utility principle, in which case it is not a "probability." The utility principle is defined in law and cannot be expressed in figures. The utility principle takes effect only when the judge reaches a decision (on the basis of all the evidence before him). It determines the relative importance of the participant's objects of legal protection which are at issue in the case. The expert is bound to apply a neutral utility component, i.e., in a two-hypothesis case (the normal situation) the significance of both the null and the counter hypothesis must carry the same weight. A null and/or a counter hypothesis can combine several single hypotheses; the mean value of their frequencies is taken. As a rule, one should avoid using a "prior case probability" ("Akten-a-priori") when calculating a W value. An "expectation of error" should be as realistic as possible and hence be obtained using a "prior case probability."     

Recovery of human remains after shark attack

Two cases of fatal shark attack are reported where the only tissues recovered were fragments of lung. Case 1: An 18-year-old male who was in the sea behind a boat was observed by friends to be taken by a great white shark (Carcharodon carcharias). The shark dragged him under the water and then, with a second shark, dismembered the body. Witnesses noted a large amount of blood and unrecognizable body parts coming to the surface. The only tissues recovered despite an intensive beach and sea search were 2 fragments of lung. Case 2: A 19-year-old male was attacked by a great white shark while diving. A witness saw the shark swim away with the victim's body in its mouth. Again, despite intensive beach and sea searches, the only tissue recovered was a single piece of lung, along with pieces of wetsuit and diving equipment. These cases indicate that the only tissue to escape being consumed or lost in fatal shark attacks, where there is a significant attack with dismemberment and disruption of the integrity of the body, may be lung. The buoyancy of aerated pulmonary tissue ensures that it rises quickly to the surface, where it may be recovered by searchers soon after the attack. Aeration of the lung would be in keeping with death from trauma rather than from drowning and may be a useful marker in unwitnessed deaths to separate ante- from postmortem injury, using only relatively small amounts of tissues. Early organ recovery enhances the identification of human tissues as the extent of morphologic alterations by putrefactive processes and sea scavengers will have been minimized. DNA testing is also possible on such recovered fragments, enabling confirmation of the identity of the victim.