diagnostics Review Time Flies—Age Grading of Adult Flies for the Estimation of the Post-Mortem Interval Jens Amendt 1,*, Valentina Bugelli 2 and Victoria Bernhardt 1 1 Institute of Legal Medicine, University Hospital Frankfurt/Main, Goethe-University, 60323 Frankfurt/Main, Germany; [email protected] 2 Dipartimento di Scienze della Salute, Sezione di Scienze Medico Forensi, Università di Firenze, 50134 Firenze, Italy; valentina.bugelli@unifi.it * Correspondence: [email protected]; Tel.: +49-69-63017571 Abstract: The estimation of the minimum time since death is one of the main applications of forensic entomology. This can be done by calculating the age of the immature stage of necrophagous flies developing on the corpse, which is confined to approximately 2–4 weeks, depending on temperature and species of the first colonizing wave of flies. Adding the age of the adult flies developed on the dead body could extend this time frame up to several weeks when the body is in a building or closed premise. However, the techniques for accurately estimating the age of adult flies are still in their beginning stages or not sufficiently validated. Here we review the current state of the art of analysing the aging of flies by evaluating the ovarian development, the amount of pteridine in the eyes, the degree of wing damage, the modification of their cuticular hydrocarbon patterns, and the increasing number of growth layers in the cuticula. New approaches, including the use of age specific molecular profiles based on the levels of gene and protein expression and the application of near infrared spectroscopy, are introduced, and the forensic relevance of these methods is discussed. Citation: Amendt, J.; Bugelli, V.; Keywords: forensic entomology; pteridine; cuticular hydrocarbons; NIRS; gene expression; proteomics Bernhardt, V. Time Flies—Age Grading of Adult Flies for the Estimation of the Post-Mortem Interval. Diagnostics 2021, 11, 152. 1. Introduction https://doi.org/10.3390/diagnostics The age estimation of necrophagous blow fly larvae and pupae as a function of 11020152 ambient temperature and species is the key task of forensic entomology when it comes to the identification of the minimum post-mortem interval (PMImin), which is the time Academic Editor: Marco Di Paolo between the first insect colonization of a body and its discovery [1,2]. As adult blow flies are Received: 11 December 2020 able to colonize a corpse within minutes after death, age estimation of their juvenile stages Accepted: 19 January 2021 may yield “on-the-day” data for the PMI even several weeks post mortem. They are Published: 21 January 2021 min therefore the most important diagnostic tool in the scientific estimation of time since death in forensic medicine. An even greater extension of this period “several weeks post-mortem” Publisher’s Note: MDPI stays neutral would be nevertheless a great success. with regard to jurisdictional claims in published maps and institutional affil- Quantifying the weathering and chemical degradation of empty fly puparia (the iations. shells, in which the transformation of the maggot via the pupal stage into the adult fly takes place) discovered at a scene of a crime could be one opportunity, but relies on Gas Chromatography—Mass Spectrometry methods and equipment, and is still tentative, as the weathering varies among species. Moreover, puparia might be overlooked due to the tendency of maggots to seek sheltered places to pupate and therefore not be collected by an Copyright: © 2021 by the authors. untrained investigator. Last but not least, a variety of different methods should always be Licensee MDPI, Basel, Switzerland. considered in order to minimize the problem of variability and natural noise of biological This article is an open access article distributed under the terms and systems or living animals. Just as different methods of age determination of juvenile conditions of the Creative Commons necrophagous insects (further breeding to the adult stage, length measurements of larvae, Attribution (CC BY) license (https:// etc.) improve the accuracy of forensic entomological expertise, different techniques can also creativecommons.org/licenses/by/ usefully complement each other in the analysis of the remains of the necrophagous fauna. 4.0/). Adult flies at the scene offer an important complement and support here [3–5]. Senescence Diagnostics 2021, 11, 152. https://doi.org/10.3390/diagnostics11020152 https://www.mdpi.com/journal/diagnostics Diagnostics 2021, 11, x FOR PEER REVIEW 2 of 13 Diagnostics 2021, 11, 152 2 of 13 also usefully complement each other in the analysis of the remains of the necrophagous fauna. Adult flies at the scene offer an important complement and support here [3–5]. orSenescence aging is theor aging progressive is the progressive deterioration deterioration in the physiological in the physiological state of an state organism of an organ- over timeism over that culminatestime that culminates in death [6 in,7 ].death In the [6,7]. adult In fly,theit adult is linked fly, it with is linked various with morphological various mor- andphological molecular and variations, molecular andvariations, by using and these by variationsusing these for variations the aging for specimens, the aging which speci- remainmens, which on the remain scene ofon the the body scene after of the finishing body after their finishing development, their development, could open a could new dooropen to a new the estimation door to the of estimation the PMImin of. Thisthe PMI appliesmin. This especially applies to especially indoor scenes, to indoor where scenes, the majoritywhere the of majority routine cases of routine of insect cases infested of insect bodies infested are located bodies [8 are], and located where [8], hatched and where flies havehatched difficulty flies have leaving difficulty the room leaving due tothe closing room due conditions. to closing While conditions. under field While conditions, under field a distinctionconditions, between a distinction flies just between arriving flies and just those arriving which developedand those onwhich the bodydeveloped is difficult on the to achieve,body is difficult at indoor to scenes achieve, many at indoor of the fliesscenes originating many of fromthe flies the originating cadaver may from bestill the presentcadaver atmay the be scene still present for several at the weeks scene [ 9for], andseveral demonstrated weeks [9], and that demonstrated it is possible that to establish it is possible the originto establish of a population the origin of of such a population flies at a crime of such scene flies by at means a crime of a scene morphology by means examination. of a mor- Asphology adult fliesexamination. might reach As agesadult of flies up tomight 68 days reach [10 ages], adding of up their to 68 age days to the[10], time adding required their forage larval to the development time required and for metamorphosislarval development could and extend metamorphosis the calculable could period extend by severalthe cal- weeksculable (Figure period1). by The several present weeks review (Figure summarizes 1). The thepresent current review state summarizes of the art of estimatingthe current thestate age of ofthe adult art of flies estimating and evaluates the age possible of adult problems flies and and evaluates pitfalls possible regarding problems techniques and andpitfalls case regarding work. techniques and case work. Figure 1. An exemplary PMImin determination using a fictitious case. The dead body was discov- Figure 1. An exemplary PMImin determination using a fictitious case. The dead body was discovered inered an in apartment an apartment (20 ◦C (20 ambient °C ambient temperature), temperature), and onlyand only larvae, larv pupae,ae, pupae, and and empty empty puparia puparia of one of one single species, the blow fly Calliphora vicina, were found on the body and its surroundings. In single species, the blow fly Calliphora vicina, were found on the body and its surroundings. In order order to determine the PMImin, the most advanced stage of its development will be of relevance. In to determine the PMI , the most advanced stage of its development will be of relevance. In the the present case, thesemin are empty puparia. Since there is no current method to narrow this down presentwith respect case, theseto the aretime empty of hatching, puparia. the Since entomolo theregist is no will current assume method that the to narrowflies hatched this down immedi- with respectately before to the the time body of was hatching, found the in order entomologist to determine will assumea reliable that PMI themin. flies There hatched is a risk immediately that this beforemay lead the to body an underestimation was found in order of the to time determine period. a According reliable PMI to,min e.g.,. There Marchenko is a risk [11], that C. thisvicina may leadneeds to about an underestimation 21 days to complete of the a time complete period. deve Accordinglopment to,cycle e.g., at Marchenkoa temperature [11 of], C.20 vicina°C. Theseneeds 21 ◦ aboutdays would 21 days be to the complete PMImin ain complete the present development case, based cyclejust on at the a temperature immature specimens of 20 C. These (larvae, 21 pu- days pae) and remains (empty puparia). However, there are hundreds of adult C. vicina in the apart- would be the PMImin in the present case, based just on the immature specimens (larvae, pupae) and remainsment. Thirty (empty to puparia).forty specimens However, are therecaptured, are hundreds and by using of adult variousC. vicina techniquesin the apartment. (see below), Thirty their to age is estimated to be 15–17 days. Based on the assumption that the animals have developed on forty specimens are captured, and by using various techniques (see below), their age is estimated to the cadaver, this now means that we are giving a PMImin of 36 days in the entomological report (21 be 15–17 days.