New Insights into Geometric Morphometry Applied to Fish Scales for Species Identification.

The possibility of quick and cheap recognition of a fish species from a single dermal scale would be interesting in a wide range of contexts. The methods of geometric morphometry appear to be quite promising, although wide studies comparing different approaches are lacking. We aimed to apply two methods of geometric morphometry, landmark-based and outline-based, on a dataset of scales from five different teleost species: Danio rerio, Dicentrarchus labrax, Mullus surmuletus, Sardina pilchardus, and Sparus aurata. For the landmark-based method the R library "geomorph" was used. Some issues about landmark selection and positioning were addressed and, for the first time on fish scales, an approach with both landmarks and semilandmarks was set up. For the outline-based method the R library "Momocs" was used. Despite the relatively low number of scales analyzed (from 11 to 81 for each species), both methods achieved quite good clustering of all the species. In particular, the landmark-based method used here gave generally higher R2 values in testing species clustering than the outline-based method, but it failed to distinguish between a few couples of species; on the other hand, the outline-based method seemed to catch the differences among all the couples except one. Larger datasets have the potential to achieve better results with outline-based geometric morphometry. This latter method, being free from the problem of recognizing and positioning landmarks, is also the most suitable for being automatized in future applications.

[Diagnostic atypique d'une myiase]. / Atypical diagnosis of myiasis.

Muscoid larvae were observed on self-medicated dressing material loaded with purulent material taken from a 91-year-old hospital patient. These larvae were identified as Lucilia sericata. However, no larvae were found in the patient's tissues. The observation of larvae on dressings should not automatically lead to a diagnosis of cutaneous myiasis.

A New, Non-Invasive Methodology for the Molecular Identification of Adult Sarcophagidae from Collections.

Correct species identification is the cornerstone of all scientific studies that involve insects. Alongside traditional morphological identification techniques, molecular identification based on the characterization and analysis of specific mitochondrial or nuclear gene regions is becoming commonplace. Despite the good results that can be achieved, DNA extraction usually involves invasive techniques that lead to the partial or total destruction of specimens. In this work, a non-invasive DNA extraction technique is described. The technique was tested on the abdomens of dry-preserved Sarcophagidae (Diptera) specimens collected between 1889 and 2015. This allowed for the correct identification of species without impairing diagnostic morphological structures useful for further studies.

On-Site Inspection Form in Veterinary Cases: The Parma Veterinary Form.

The on-site inspection of the scene of an animal cadaver is crucial for a correct interpretation of the autopsy results, to determine the manner, method, and cause of death. This information plays a crucial role in the control of public health including the prevention of zoonoses. It is also fundamental for the recognition and the contrast of crimes against animals and to animal abuse phenomena, considered an alert sign of an anti-social or violent behavior of humans. Today the best veterinary procedure requires an accurate collection of the evidence at the scene that can be then handed to experts belonging to other forensic disciplines for further evaluation and data interpretation. In this paper authors suggest a form aiming to facilitate either the on-site and the autopsy activities, as a guarantee of the quality of the forensic process starting from the discovery scene up to the reconstruction of the case. Essential is training of non-medical personnel who often represent the first responder to be present on the scene. The form is inspired by the interdisciplinary form developed by the European Council of Legal Medicine and represents an initial tool to stimulate a multidisciplinary activity in close synergy with other forensic experts.

Morphological and molecular characterization of puparia of Piophilidae species of forensic relevance.

Piophilidae are a small family of Diptera with a worldwide distribution and which are historically associated with human activities. In addition to their economic importance, piophilid larvae can also be of medical and legal relevance. Within a medicolegal context, piophilids are frequently associated with cadavers in advanced stages of decomposition, thus being potentially useful forensic indicators and they have been reported also from archaeo-funerary contexts. An accurate species identification is therefore an essential prerequisite to ensure the reliable analysis of insect material in medical, forensic and archaeological investigations. Identification of the adult piophilid flies is possible because of the availability of identification keys, in contrast immature insects, especially puparia, have been poorly investigated and described. In this paper, puparia of 11 species of forensic interest (Piophila casei, Piophila megastigmata, Parapiophila atrifrons, Parapiophila flavipes, Parapiophila vulgaris, Protopiophila litigata, Liopiophila varipes, Prochyliza nigrimana, Prochyliza xanthosoma and Stearibia nigriceps in subtribe Piophilina and Centrophlebomyia furcata in subtribe Thyreophorina) are described and a molecular analysis, based on the COI sequencing, is presented to show the potential of the molecular approach in their identification.

Multidisciplinary Forensic Approach in "Complex" Bodies: Systematic Review and Procedural Proposal.

The recovery of severely altered cadavers (i.e., extensively decomposed, mummified, charred or dismembered) can be a challenge for forensic pathologists due to the difficulties in identification, PMI estimation and manner and cause of death determination. In such cases, integrating routine approaches (autopsy, histology, toxicology) to more specific forensic branches can be fundamental to improving the investigative process. In this paper a systematic review using PubMed, Scopus and Web of Science databases has been performed. The aim was to evaluate the forensic approaches implemented in the management of severely altered bodies due to decomposition, mummification, skeletonization, charring or dismemberment (to which we refer to as "complex"), and the role of each approach in the solution of a case. Then, the literature revision results were used to propose a schematic flowchart summarizing the post mortem activities that can be performed in forensic practice, adaptable in relation to each case.

Back to the Middle Ages: Entomological and Botanical Elements Reveal New Aspects of the Burial of Saint Davino of Armenia.

The natural mummy of Saint Davino Armeno (11th century) is preserved in the church of Saint Michele in Foro in the city of Lucca (Tuscany, Central Italy). The body of Davino is one of the oldest Italian mummies of a Saint, and his paleopathological study was performed in 2018. In the present research, we investigated the arthropod fragments and botanical remains collected from the body, coffin, and fabrics of Saint Davino. Entomological analyses outlined the presence of 192 arthropod fragments. Among these, Diptera, Muscidae (Hydrotaea capensis and Muscina sp.), and Phoridae (Conicera sp.) puparia were the most abundant. Regarding Coleoptera, Ptinidae (Anobium punctatum) were the most frequent, followed by Cleridae (Necrobia sp.), Trogidae (Trox scaber), Curculionidae (Sitophilus granarius), and Histeridae (Gnathoncus). Cocoons of Tineidae and Pyralidae moths were found, along with a propodeum joined to the petiole and a mesopleuron of an Ichneumoninae parasitoid. Numerous metamera of Julida and three scorpion fragments were also found. Botanical samples indicated the presence of a quite broad botanical community, including gramineous species, olives, evergreen oaks, and grapevine. Overall, entomological data allow us to argue that Saint Davino was first buried into the soil, probably in a wooden coffin, thus supporting the historical-hagiographic tradition according to which he was buried sub divo in the cemetery of Saint Michele. The preservation of the body as a natural mummy may have been facilitated by burial in a coffin that prevented direct contact of the corpse with the earth. Botanical remains offer confirmation of a late medieval urban environment rich in horticultural areas and trees, giving us a landscape that is very different from the current Tuscan city.

A review of morphological characters for the identification of three common European species of Sarcophaga s. str. (Diptera: Sarcophagidae), with an emphasis on female terminalia.

The subgenus Sarcophaga Meigen, 1824 (s. str.) currently comprises over 30 species distributed in the West Palearctic Region, the identification of which is normally based on characters of the male terminalia. Females of the three closely-related species Sarcophaga (Sarcophaga) carnaria (Linnaeus, 1758), S. (S.) subvicina Rohdendorf, 1937 and S. (S.) variegata (Scopoli, 1763), which are especially widespread and abundant in NW Europe, are considered morphologically indistinguishable by most authors. However, a few authors have proposed keys to separate females of these three species based on external and internal characters of the terminalia. Following a preliminary molecular identification using DNA barcode sequences (COI, cytochrome c oxidase subunit I), we herein revise the morphological characters used to differentiate female S. carnaria, S. subvicina and S. variegata in existing identification keys as well as search for additional diagnostic characters. Our results suggest that only one previously-proposed female character, namely the length to width ratio of abdominal sternite 7, can be used to separate S. subvicina from the other two species (Mann-Whitney U test: p < 0.0001), at least in a majority of cases. Other characters, such as the degree of sclerotisation and setation of tergite 8, show a high degree of overlap that does not allow to reliably separate females of these three species. Nevertheless, we propose a combination of characters that should allow the separation of female S. carnaria from female S. variegata in most cases. An additional analysis of males of the same species showed that the distribution of pruinosity and setation on syntergosternite 7+8, a character mentioned in a previously-published key, is also not reliable for identification.

Investigation of the skin lesions in lightning strike death.

Lightning strike-related deaths are unusual, and the victim bodies can present different lesions due to the different injury mechanisms associated with this event. Since the post mortem assessment can be challenging, the evaluation of the characteristics of the skin lesions becomes fundamental to reconstruct the event. Due to the paucity of literature on this topic, the authors report the case of a 59-year-old man found dead near his home after a thunderstorm. Initially considered a murder by gunshot, the autopsy revealed the typical lightning strike lesions, also known as Lichtenberg figures. The adequate interpretation of the autopsy data and the histological evidences allowed to reconstruct the death dynamic and to relate it to a lightning strike. The main lesions due to lightening are here reported and discussed in order to provide a workflow for the identification of lightening as cause of death in unwitnessed cases.

Forensic investigative issues in a fireworks production factory explosion.

Since their discovery in ancient China, fireworks rapidly spread throughout the world, where they have always been used to celebrate either popular or private events. Their use is nonetheless related to several risks, especially within production factories, since several injuries or even death can occur following an accidental ignition. In cases of major disasters related to fireworks explosions, stating the accidental or intentional nature of the event might prove challenging, thus raising the need of a multidisciplinary approach. In this regard, we here discuss the case of an accidental explosion that occurred in a fireworks production factory, accountable for five deaths and two hospitalisations.

Mentha pulegium L.: A Plant Underestimated for Its Toxicity to Be Recovered from the Perspective of the Circular Economy.

The aim of the study was to investigate the micromorphology of Mentha pulegium leaves and flowers harvested in three different Sicilian (Italy) areas with peculiar pedo-climatic conditions, and to characterize the phytochemical profile, the phytotoxic activity, and the eco-compatibility of their essential oils (EOs) for potential use as safe bioherbicides. Light microscopy (LM) and scanning electron microscopy (SEM) highlighted that M. pulegium indumentum consists of non-glandular and glandular trichomes of different types. Peltate trichomes of plants from the different sites showed few significant differences in dimension and abundance, but they were characterized by a surprisingly high number of secretory cells both in leaves and flowers. Phytochemical analyses showed that oxygenated monoterpenes were the most abundant class in all the EOs investigated (92.2-97.7%), but two different chemotypes, pulegone/isomenthone and piperitone/isomenthone, were found. The complex of morphological and phytochemical data indicates that soil salinity strongly affects the expression of the toxic metabolite pulegone, rather than the EO yield. Phytotoxicity tests showed a moderate activity of EOs against the selected species as confirmed by α-amylase assay. Moreover, the low toxicity on brine shrimp provided a rationale for the possible use of investigated EOs as eco-friendly herbicides.

Comparison of Accumulated Degree-Days and Entomological Approaches in Post Mortem Interval Estimation.

Establishing the post mortem interval (PMI) is a key component of every medicolegal death investigation. Several methods based on different approaches have been suggested to perform this estimation. Among them, two methods based their evaluation on the effect of the temperature and time on the considered parameters: total body score (TBS)/accumulated degree-days (ADDs) and insect development. In this work, the two methods were compared using the results of minPMI and PMI estimates of 30 forensic cases occurring in northern Italy. Species in the family Calliphoridae (Lucilia sericata, Calliphora vomitoria and Chrysomya albiceps) were considered in the analyses. The results highlighted the limits of the TBS/ADD method and the importance of the entomological approach, keeping in mind that the minPMI is evaluated. Due to the fact that the majority of the cases occurred in indoor conditions, further research must also be conducted on the different taxa to verify the possibility of increasing the accuracy of the minPIM estimation based on the entomological approach.

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.

Forensic Validity of the Third Molar Maturity Index (I 3M) for Age Estimation in a Russian Population.

The aim of this cross-sectional study was to test the accuracy of the third molar maturity index (I 3M) cut-off value (0.08) to distinguish between individuals above and below the adult age of legal responsibility (18 years) in a Russian population. A sample of 571 digital panoramic radiographs of healthy Russian minors and young adults (363 females and 208 males), aged between 14 and 24 years, was evaluated. The lower left third molars were analyzed by applying the cut-off value of 0.08 determined by Cameriere et al. (2008). Lin's concordance correlation coefficient (ρ c) and Cohen's kappa coefficient (κ) showed that repeatability and reproducibility are high for both intra- and interobserver errors. The I 3M value decreased with age in both sexes. Age distribution gradually decreases as I 3M increases in both girls and boys. In the male group, molar maturity stages 0-0.04, 0.04-0.08, 0.08-0.3, 0.3-0.5, and 0.9-1.4 were reached slightly earlier than in the female group. The results demonstrated that sensitivity is 0.96 in boys and 0.93 in girls; associated specificity values were both 0.98. The cut-off value of I 3M is statistically robust and thus valid for forensic application in a Russian population to determine whether or not a subject has reached 18 years of age. Finally, we compared our results with those of other studies in which the same I 3M cut-off value was tested on different populations. The method is novel as it is reliable and easily reproducible, thus ensuring a universal way of comparing the results obtained (based on a cut-off value) among many populations, in order to develop an ever-increasing database.

The Red Swamp Crayfish Procambarus Clarkii (the Louisiana Crayfish) as a Particular Scavenger on a Human Corpse.

An 85-year-old man was found deceased floating in an irrigation ditch 18 days after his disappearance. During crime scene investigation, specimens of the red swamp crayfish Procambarus clarkii were found in proximity of the body. The feeding activity of these nonendemic crayfishes contributed to the formation of specific injuries on the body and in the production of a large substance defects inside the corpse. The aim of this paper was to illustrate the scavenging activity of P. clarkii on a human body and highlight the potential postmortem artefacts caused by this species. This is the first report on a real case of postmortem injuries produced by P. clarkii crayfishes on a submerged human body. So far, crustaceans are not considered useful for the estimation of the minimum postmortem interval. However, the important modifications on the corpses deriving from the activity of these animals should be kept in consideration.

Potential treatment of leachate by Hermetia illucens (Diptera, Stratyomyidae) larvae: Performance under different feeding conditions.

In this study, the ability of H. illucens larvae (black soldier fly (BSF)) to metabolise different semisolid biowastes (e.g. kitchen waste, animal manure) has been applied to the treatment of landfill leachate. A testing programme has been developed by mixing leachate with three different solid supports: wheat bran, a biodegradable nutrient substrate, brewers' spent grain, a biodegradable nutrient residue from the brewery industry and sawdust, a low biodegradable residue from the wood industry. Larvae growth rate was monitored in terms of weight variation, mortality and time to reach the prepupal stage. Prepupal biomass composition was analysed in terms of crude protein, lipids and fatty acids. Substrates were monitored at the beginning and the end of tests for total solids (TS), total organic carbon (TOC), total Kjeldahl nitrogen (TKN), ammonia and (whenever significant) the 7-day Respirometric Index (RI7). The best performance was observed with wheat bran and brewers' spent grain, achieving an average larval weight ranging from 155.1 to 226.1 mg (w/w) with prepupation of more than 80% over 21 days. The initial TS, TOC and nitrogen content in feeding substrates had been metabolised (gasified and accumulated in prepupal biomass) by approximately 55%, 60% and 48%, respectively. Dry mass characterisation displayed a significant content of fats and proteins. The analysis demonstrated the suitability of BSF prepupal biomass for the production of biodiesel; however, the potential use of proteins as an animal feed needs further studies for assessing the presence of contaminants.

Insights on Funeral Practices and Insects Associated With the Tombs of King Ferrante II d'Aragona and Other Renaissance Nobles.

The impressive Sacristy of the Basilica of San Domenico Maggiore contains 38 wooden sarcophagi with the bodies of 10 Aragonese princes and other Neapolitan nobles, who died in the 15th and 16th centuries. To improve the knowledge about the entomofauna associated with bodies in archaeological contexts, herein we provide insights on the funerary practices and the insect community associated to Ferrante II King of Naples and other Italian Renaissance mummies of the Aragonese dynasty buried in the Basilica of St. Domenico Maggiore. We identified 842 insect specimens: 88% were Diptera (Muscidae, Fanniidae, and Phoridae), followed by 9% Lepidoptera (Tineidae) and 3% Coleoptera (Dermestidae and Ptinidae). Ninety-seven percent of the specimens were collected from the coffin of Francesco Ferdinando d'Avalos, which was the best preserved. A lack of fly species characterizing the first colonization waves of exposed bodies was noted. The most common fly was the later colonizing muscid Hydrotaea capensis (Wiedemann); only a few Fanniidae (Fannia spp.) were retrieved. The lack of blowflies, coupled with recording H. capensis as the dominant fly, supports our hypothesis that corpses have been kept indoors for a long time under confined environmental conditions. Other explanations include odorous oils/balms having been used in the embalming process, causing the delay or stopping the arrival of first colonizer flies. Hermetically sealing of the coffin with bitumen may also have played a role in preventing access to the corpses. This scenario describes a historical context characterized by a well-advanced knowledge of body preparation, with specific burial techniques adopted for nobles.

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.