Paola A. Magni

The Role of Forensic Botany in Crime Scene Investigation: Case Report and Review of Literature†

Management of a crime is the process of ensuring accurate and effective collection and preservation of physical evidence. Forensic botany can provide significant supporting evidences during criminal investigations. The aim of this study is to demonstrate the importance of forensic botany in the crime scene. We reported a case of a woman affected by dementia who had disappeared from nursing care and was found dead near the banks of a river that flowed under a railroad. Two possible ways of access to crime scene were identified and denominated “Path A” and “Path B.” Both types of soil and plants were identified. Botanical survey was performed. Some samples of Xanthium Orientalis subsp. Italicum were identified. The fall of woman resulted in external injuries and vertebral fracture at autopsy. The botanical evidence is important when crime scene and autopsy findings are not sufficient to define the dynamics and the modality of death.

Development and validation of a GC–MS method for nicotine detection in Calliphora vomitoria (L.) (Diptera: Calliphoridae)

Entomotoxicology is the application of toxicological methods and analytical procedures on necrophagous insects feeding on decomposing tissues to detect drugs and other chemical components, and their mechanisms affecting insect development and morphology and modifying the methodology for estimation of minimum time since death. Nicotine is a readily available potent poison. Because of its criminal use, a gas chromatography-mass spectrometry (GC-MS) method for the detection of nicotine in Calliphora vomitoria L. (Diptera: Calliphoridae) was developed and validated. Furthermore, the effect of nicotine on the development, growth rate, and survival of this blowfly was studied. Larvae were reared on liver substrates homogeneously spiked with measured amounts of nicotine (2, 4, and 6 ng/mg) based on concentrations that are lethal to humans. The results demonstrated that (a) the GC-MS method can detect both nicotine and its metabolite cotinine in immature C. vomitoria; (b) the presence of nicotine in the aforementioned three concentrations in food substrates did not modify the developmental time of C. vomitoria; (c) during the pupation period, larvae exposed to nicotine died depending on the concentration of nicotine in the substrate; and (d) the resultant lengths of larvae and pupae exposed to 4 and 6 ng/mg concentrations of nicotine were significantly shorter than those of the control.

Development of a GC-MS method for methamphetamine detection in Calliphora vomitoria L. (Diptera: Calliphoridae).

Entomotoxicology is the study of using insects for the detection of drugs and other chemical substances in decomposing tissues. One research aspect in particular is the effects of these substances on arthropod development and morphology, and their consequences on the post mortem interval estimation. Since methamphetamine (MA) is becoming commonplace as an illegal recreational drug, a GC-MS method for the detection of MA in Calliphora vomitoria L. (Diptera: Calliphoridae) was developed and validated. Furthermore, the effect of MA on the development, growth rates and survival of the blowfly was investigated. Larvae were reared on liver substrates homogeneously spiked with measured amounts of MA (5 ng/g and 10 ng/g) based on typical concentrations found in human tissue in cases of death caused by MA overdose. The experimental results demonstrated that (i) MA produced a significant increase in the developmental time from egg to adult in C. vomitoria, (ii) approximately 60% of larvae exposed to either dose of MA died during the pupation period and (iii) the resultant lengths of larvae and pupae were on average significantly larger than the controls.

Evaluation of the floating time of a corpse found in a marine environment using the barnacle Lepas anatifera L. (Crustacea: Cirripedia: Pedunculata).

Human activities involving water may result in a crime scene. Typically, death may be due to natural causes, homicide, or mass disasters. Decomposition in water is a complex process where many factors may interplay. Human remains in water are subject to many potential interactions, depending upon the remains themselves, the type of water and the characteristics of the water. A number of studies are focused on the decomposition process of the corpse in water, on the identification of the post mortem submersion interval (PMSI) and on the diagnosis of drowning, but very few studies consider the fate of floating remains in any aquatic environment. The following case describes a corpse found on a shore of the Tyrrhenian Sea (South West of Italy, Calabria Region). The corpse and the soles of his shoes were colonized by the barnacle Lepas anatifera L. (Crustacea: Cirripedia: Pedunculata). The analyses of the barnacles present on the corpse aided in the evaluation of the floating time of the corpse which assisted in estimating the minimum time since death.

Entomological evidence: Lessons to be learnt from a cold case review

Insects are known to be useful in estimating time since death, but this is only possible if samples are collected and preserved correctly according to best practices. This report describes a case where an 18-year old female was found dead and during the first medico-legal investigation which determined it was a homicide, entomological samples were collected but not considered. The case was then closed with no suspect. However, 9 years after the first investigation the courts decided that the case needed to be re-examined. In doing so the new review team decided that although the remaining entomological evidence was poorly preserved some extra information may be gained from its analyses. On inspection of the remaining samples of larvae no normal morphological analyses could be conducted. Molecular analyses were combined with an unorthodox morphological analysis to provide an estimate of the post-mortem interval based on insect evidence, indicating the value of multidisciplinary approaches to both cold and contemporary cases.

A Biological and Procedural Review of Forensically Significant Dermestes Species (Coleoptera: Dermestidae)

ABSTRACT The analyses of the insect species found on decomposing remains may provide useful information for the estimation of the minimum time elapsed since death and other parameters, such as causes and circumstances of death. The majority of research has focused on the early colonizing species, typically blowflies, while research concerning late colonizing insects is currently sparse. Dermestid beetles of the genus Dermestes L. (Coleoptera: Dermestidae) are one of the predominant insect species associated with decomposing remains during dry decay and skeletal stages of decomposition. In some dry environments, Dermestes species are likely to be the only necrophagous insects feeding on the decomposing remains. Furthermore, Dermestes species (immature and adults), their remains (cast skins and fecal material), and their artifacts (pupal chambers) are frequently found associated with ancient remains (e.g., mummies, fossils). Dermestes species have a worldwide distribution and are considered important in decomposition processes, forensic investigations, and economically as a known pest of stored products. Despite their recognized forensic importance, there is limited data documenting the ecology, biology, and the growth rates of the forensically relevant species. The aim of this review is to provide a comprehensive synopsis on the available literature concerning Dermestes species associated with forensic cases. In particular, aspects of colonization behavior, growth rates for forensic taxa and potential best practice guidelines for forensic casework encompassing late colonizing Dermestes species are discussed.

Effect of Continuous and Cyclic Exposure to a Cold Environment on the Development of Larvae of Lucilia sericata (Diptera: Calliphoridae) in Different Sized Larval Masses

Abstract Regulation of forensic practice in many countries prevents the pathologist performing an immediate autopsy. During the period prior to autopsy, the corpse and the insects possibly associated with it are stored in a mortuary with temperatures ∼4°C. When a corpse is found in a late stage of decay, fly immatures may be present as small or large larval masses. The purpose of refrigeration at 4°C is to slow down the decomposition of the corpse as well as the temporary disruption of the activity and development of the bacteria and the necrophagous insects associated with the corpse. The aim of this research is to investigate the growth and development of different larval masses of the blowfly Lucilia sericata (Meigen) (Diptera: Calliphoridae) when stored in a cold environment (4°C). The research was divided into experimental sessions comprising different storage conditions (continuous or cyclic exposure to a cold environment) for immature stages (second and third instar) and included four different sizes of larval mass (∼100, 500, 1,000, and 5,000 larvae) feeding on 4 kg of beef liver and replicated three times. Results show that if the larval mass has a size of ∼5,000 larvae, and the larvae have already reached third instar, then when they are exposed to a cold environment, their development continues. The storage condition at 4°C does not disrupt the development of such larvae. The number of larvae and their instar that make up the larval mass are essential data for the subsequent estimation of a correct minimum postmorteminterval.

Development and Validation of a Method for the Detection of α- and β-Endosulfan (Organochlorine Insecticide) in Calliphora vomitoria (Diptera: Calliphoridae)

Abstract Entomotoxicology studies employ analytical methods and instrumentation to detect chemical substances in carrion insects feeding from the decomposing tissues. The identification of such chemicals may determine the cause of death and may be used for the estimation of the minimum time since death. To date, the main focus of entomotoxicological studies has been the detection of drugs, whereas little information concerns the effects of pesticides on blowflies. Pesticides are generally freely available and more affordable than drugs but they can also be a home hazard and an accessible candidate poison at a crime scene. A QuEChERS extraction method followed by Gas chromatography–mass spectrometry (GC-MS) analysis was developed for the detection of α-and β-endosulfan (organochlorine insecticide and acaricide) in Calliphora vomitoria L. (Diptera: Calliphoridae) and validated. Furthermore, the effects of endosulfan on the morphology, development time and survival of the immature blowflies were investigated. Larvae were reared on liver substrates homogeneously spiked with aliquots of endosulfan corresponding to the concentrations found in body tissues of humans and animals involved in endosulfan poisoning. Results demonstrated that the combination of QuEChERS extraction and GC-MS provide an adequate methods to detect both α- and β-endosulfan in blowfly immatures. Furthermore, the presence of α- and β-endosulfan in the food source 1) prevented C. vomitoria immatures reaching the pupal instar and, therefore, the adult instar at high concentrations, 2) did not affect the developmental time of blowflies at low concentrations 3) affected the size of immatures only at high concentrations, resulting in significantly smaller larvae.

Development and validation of an HPLC-MS/MS method for the detection of ketamine in Calliphora vomitoria (L.) (Diptera: Calliphoridae)

Entomotoxicology is a branch of forensic entomology that studies the detection of drugs or other toxic substances from insects developing on the decomposing tissues of a human corpse or animal carcass. Entomotoxicology also investigates the effects of these substances on insect development, survival and morphology to provide an estimation of the minimum time since death. Ketamine is a medication mainly used for starting and maintaining anesthesia. In recent years ketamine has also been used as a recreational drug, and occasionally as a sedating drug to facilitate sexual assault. In both activities, it has resulted in several deaths. Furthermore, ketamine has been also implicated in suspicious deaths of animals. The present research describes for the first time the development and validation of an analytical method suited to detect ketamine in larvae, pupae, empty puparia, and adults of Calliphora vomitoria L. (Diptera: Calliphoridae), using liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). This research also considers the effects of ketamine on the survival, developmental rate and morphology (length and width of larvae and pupae) of C. vomitoria. The larvae were reared on liver substrates homogeneously spiked with ketamine concentrations consistent with those found in humans after recreational use (300 ng/mg) or allegedly indicated as capable of causing death in either humans or animals (600 ng/mg). The results demonstrated that (a) HPLC-MS/MS method is applicable to ketamine detection in C. vomitoria immatures, not adults; (b) the presence of ketamine at either concentration in the food substrate significantly delays the developmental time to pupal and adult instar; (d) the survival of C. vomitoria is negatively affected by the presence of ketamine in the substrate; (e) the length and width of larvae and pupae exposed to either ketamine concentration were significantly larger than the control samples.

The effect of antifreeze (ethylene glycol) on the survival and the life cycle of two species of necrophagous blowflies (Diptera: Calliphoridae)

Entomotoxicology involves the analysis of the presence and the effects of toxicological substances in necrophagous insects. Results obtained by entomotoxicological studies may assist in the investigation of both the causes and the time of death of humans and animals. Ethylene glycol (EG) is easy to purchase, sweet and extremely toxic. It may be consumed accidentally or purposefully, in an attempt to cause death for suicidal or homicidal intent. Several cases report fatalities of humans and animals. The present study is the first to examine the effects of EG on the survival, developmental rate and morphology of two blowfly species, (Diptera: Calliphoridae) typically found on corpses and carcasses: Lucilia sericata (Meigen) and L. cuprina (Wiedemann). Both species were reared on substrates (beef liver) spiked with three different concentrations of EG that could cause death in either a human or cat: 1/2LD50 (T1), LD50 (T2), 2LD50 (T3), in addition to a control treatment (C) with no EG. Results of this research show that: a) both species are unable to survive if reared on a food substrate spiked with the highest concentration of EG (T3), while lower and medium concentrations (T1, T2) affect, but not prevent, the survival and the completion of the life cycle of such species; b) adults of L. sericata eclose only in C and T1, while adults of L. cuprina in both C, T1, T2; however, c) the developmental time of both species reared in T1 and T2 is statistically slower than the control; d) the body length of the immatures of both of the species reared in T1 and T2 is statistically smaller than the control.