Experimental evaluation on the applicability of necrobiome analysis in forensic veterinary science

Despite the wide usage of animals as models in forensic studies, the investigations of fundamental legal questions involving domesticated and nondomesticated animals were always given marginal attention compared to "human forensic," and only recently the interest in the discipline is increasing. Our research focuses on the effect of the fur coat on the activity and development of microbial decomposers. In order to test this variable never assessed before, rabbit carcasses were used and results show that: (i) distinct and significant temporal changes in terms of metabolic activity and taxa distribution can be tracked over the decomposition process; (ii) the richness and the diversity of the bacterial communities does not significantly vary over time, but it does not mean that the species Operational Taxonomic Units (OTUs) do not change; (iii) the presence/absence of the fur on the carcasses does not significantly affect either the bacterial communities' functional activity or the diversity intra- and intercommunity, neither at phylum nor at family resolution; (iv) the functional activity and the ecological diversity of the bacterial communities are significantly affected by the body region, while the relative abundance is not. Obtained data confirm previous observations and provide new insight in the Forensic Veterinary field in terms of equally using them in order to derive a statistical model for the PMI estimation. As a future perspective, a contribution to the Forensic Entomology approach will be given in legal investigations when domestic or wild animals are involved, regardless of the presence of a hair layer

Do longer sequences improve the accuracy of identification of forensically important Calliphoridae species?

Species identification is a crucial step in forensic entomology. In several cases the calculation of the larval age allows the estimation of the minimum Post-Mortem Interval (mPMI). A correct identification of the species is the first step for a correct mPMI estimation. To overcome the difficulties due to the morphological identification especially of the immature stages, a molecular approach can be applied. However, difficulties in separation of closely related species are still an unsolved problem. Sequences of 4 different genes (COI, ND5, EF-1\u3b1, PER) of 13 different fly species collected during forensic experiments (Calliphora vicina, Calliphora vomitoria, Lucilia sericata, Lucilia illustris, Lucilia caesar, Chrysomya albiceps, Phormia regina, Cynomya mortuorum, Sarcophaga sp., Hydrotaea sp., Fannia scalaris, Piophila sp., Megaselia scalaris) were evaluated for their capability to identify correctly the species. Three concatenated sequences were obtained combining the four genes in order to verify if longer sequences increase the probability of a correct identification. The obtained results showed that this rule does not work for the species L. caesar and L. illustris. Future works on other DNA regions are suggested to solve this taxonomic issue

State of the art of the funerary archaeoentomological investigations in Italy

Archaeoentomology is a branch of environmental archaeology focusing on insects and other arthropods contributing, with other disciplines, to reconstruct a more complete view of past events, climate and landscape. Funerary archaeoentomology focuses on the arthropods associated with human remains or with elements part of funerary rituals such as offerings. In the last years, several papers have been published on Italian material covering around 5000 years. Sardinian necropolis, Egyptian mummies and offerings stored in public collections are among the oldest samples in which insects have been evidenced. Mummified and skeletonised bodies of kings, Saints and common people are another important source of insects in addition to remains of WWI soldiers. The arthropod remains found during these investigations belong mainly to Diptera (flies), Coleoptera (beetles) and Arachnida (mites). The aim of this paper is to summarise the Italian data about insects associated with human remains of archaeological interest in order to increase the interest and the utilisation of a discipline too often underestimated

Puparia cleaning techniques for forensic and archaeo-funerary studies

Diptera puparia may represent both in forensic and archaeo-funerary contexts the majority of the entomological evidence useful to reconstruct the peri and post-mortem events. Puparia identification is quite difficult due to the lack of identification keys and descriptions. In addition, external substances accumulated during the puparia permanence in the environment make the visualization of the few diagnostic characters difficult, resulting in a wrong identification. Six different techniques based on physical and chemical treatments have been tested for the removal of external substances from puparia to make identification at species level feasible. Furthermore, the effects of these methods on successful molecular analyses have also been tested as molecular identification is becoming an important tool to complement morphological identifications. The results of this study indicate that cleaning via warm water/soap, the sonication and treatment with a sodium hydroxide solution are the best methods to achieve a good quality of the samples

Record of Leptometopa latipes (Diptera: Milichiidae) from a human cadaver in the Mediterranean area

In forensic entomology, insects are used mainly to obtain information about the time since death. This information is obtained studying the developmental rate of the first colonizers, principally species in the families Calliphoridae, Sarcophagidae, Muscidae, Stratiomyidae and Phoridae. However, species belonging to other families can provide information about body transfer or the season of the death. Among them Milichiidae are flies rarely reported from human cases despite the larvae of some species are known as saprophagous feeding on plant and animal decomposing matter. A potential cause of the lack of records of these species from forensic cases can be related with the paucity of descriptions and illustrations of the immature stages. In this article, the entomological samples collected from a human body found inside an apartment in a Maghreb country, in Northern Africa, is reported and Leptometopa latipes (Diptera: Milichiidae) is described in detail. Molecular analysis is also reported to confirm the morphological analysis

DNA characterization from gut content of larvae of Megaselia scalaris (Diptera, Phoridae)

none6si---mixedMukherjee S.; Singh P.; Tuccia F.; Pradelli J.; Giordani G.; Vanin S.Mukherjee S.; Singh P.; Tuccia F.; Pradelli J.; Giordani G.; Vanin S

Environmental necrophagous fauna selection in a funerary hypogeal context: The putridarium of the Franciscan monastery of Azzio (northern Italy)

Insects play a fundamental role in the removal and recycling of the organic matter from human cadavers and animal carcasses. Several factors can affect both the cadaver decomposition and colonisation by insects and other animals. Accessibility to the body is one of these factors. The archaeological, anthropological and entomological investigations conducted inside the Crypt of the Franciscan Monastery in Azzio (Varese, northern Italy) allowed the reconstitution of the funerary practices used by a Franciscan community between the 17th and 18th centuries and provides a good example of necrophagous entomo-fauna selection. Human remains were found in the putridarium chamber, a semicircular room with sixteen narrow and semicircular niches, where bodies were located until the complete decomposition of the soft tissues. Numerous puparia were collected from the bones, the clothes and the niche walls. The majority belonged to Hydrotaea capensis (Diptera: Muscidae) and a few puparia of scuttle flies in the family Phoridae were detected among the studied material. Fragments of beetles in the families Staphylinidae and Cryptophagidae were also sampled. The entomological findings clearly support the hypothesis that the bodies were quickly transferred into the crypt after death and that the specific hypogeal (subterranean) environment selected the entomofauna community composition. The observations here reported with similar entomological findings cited in the literature support the hypothesis of a specific selectivity on insects' community by underground environments (e.g. graves, crypts). This conclusion can be applied to outline a common pattern in archaeological contexts and in indoor forensic cases

First report of Heleomyzidae (Diptera) recovered from the inner cavity of an intact human femur

One of the main characteristics of the Mafia of Gargano is their use of ritual murders: they shoot their victims in the face and then conceal the corpses in the numerous natural ravines present in the Gargano area. Skeletal remains are often recovered in a poor state of preservation under particular conditions related to the environmental situation. Humidity, temperature and environmental contaminants could be considered very important for forensic examinations and are strictly related to the bone preservation status. One of the most important analyses is the identification of the victim: the success rate is linked to the condition of the bones.During military investigations in the Gargano area, several bones were recovered and analyzed in a karst ravine about 30 m deep. The forensic examination highlighted the presence of fly puparia from an intact human femur. The colonization of the inner bone cavity by a species of the Heleomyzidae family is described for the first time. Puparia, despite not being identified at the species level, are described and illustrated and their potential role in the degradation of the victim's DNA is discussed. This work increases our knowledge about the effects of Diptera in the taphonomic process underlying the need of a multidisciplinary approach to skeletal investigations