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In the medico-legal application of forensic entomology, estimating the time of death is critical and traditionally relies on changes in observable traits of carrion feeding insect larvae. Traits such as size, weight, and morphology can be used to predict the insect specimen age and help define the minimum time since death. The blowfly Phormia regina Meigen (Diptera: Calliphoridae) is a key forensic insect, yet age estimation for older maggots in this and other carrion-feeding species is particularly challenging due to the limited morphological changes in the late-stage larvae. To enhance age-estimation precision, we employed transcriptomic profiling on blowfly maggots, aiming to identify genes as markers for time of death estimation. Our study characterized maggot development, reinforcing that weight and behavior cannot precisely determine age between 100 and 130 hours at 27.5 °C. We built a chromosomal scale annotated genome, establishing a reliable database for uncovering transcriptomic signatures during larval development. Applying differential gene expression analyses, weighted gene co-expression network analysis, and the generalized linear model, we identified nine candidate genes (y5078, y5076, agt2, ech1, dhb4, asm, gabd, acohc, ivd) that delineate the age of otherwise indeterminate maggots. This research introduces a molecular approach to address a longstanding problem in forensic entomology and promises to increase precision in determining the time of death at a crime scene.

A series of experiments were conducted on Phormia regina, a forensically important blow fly species, that met the requirements needed to create statistically valid development models. Experiments were conducted over 11 temperatures (7.5 to 32.5 °C, at 2.5 °C intervals) with a 16:8 L:D cycle. Experimental units contained 20 eggs, 10 g of beef liver, and 2.5 cm of sand. Each life stage (egg to adult) had five sampling times. Each sampling time was replicated four times for a total of 20 measurements per life stage. For each sampling time, the cups were pulled from the chambers, and the stage of each maggot was documented morphologically through posterior spiracular slits and cephalopharyngeal development. Data were normally distributed with the later larval (L3m) and pupation stages having the most variation within and transitioning between stages, particularly between 12.5 °C and 20.0 °C. The biological minimum was between 10.0 °C and 12.5 °C, with little egg development and no egg emergence at 7.5 °C and no maturation past L1 at 10.0 °C. Phormia regina did not display increased mortality associated with the upper temperature of 32.5 °C. The development data generated illustrate the advantages of large data sets in modeling blow fly development and the need for curvilinear models in describing development at environmental temperatures near the biological minima and maxima.

Puparial cases are common remnants of necrophagous flies in crime investigations. They usually represent the longest developmental time and, therefore, they can be very useful for the estimation of the post-mortem interval (PMI). However, before any PMI estimate, it is crucial to identify the species of fly eclosed from each puparium associated with the corpse. Morphological characteristics of the puparium are often distinctive enough to permit a species identification. But, even an accurate morphological analysis of empty puparia cannot discriminate among different species of closely related flies. Furthermore, morphological identification may be impossible if the fly puparia are poorly preserved or in fragments. This study explores the applicability of biomolecular techniques on empty puparia and their fragments for identification purposes. A total of 63 empty puparia of necrophagous Diptera resulting from forensic casework were examined. Samples were divided into three groups according to size, type and time of eclosion in order to verify whether the physical characteristics and puparia weathering can influence the amount of DNA extraction. The results suggest that a reliable genetic identification of forensically important flies may also be performed from empty puparia and/or their fragments. However, DNA degradation can deeply compromise the genetic analysis since the older the fly puparia, the smaller are the amplified fragments.

Common DNA-based species determination methods fail to distinguish some blow flies in the forensically and medically important genus Lucilia Robineau-Desvoidy. This is a practical problem, and it has also been interpreted as casting doubt on the validity of some morphologically defined species. An example is Lucilia illustris and L. caesar, which co-occur in Europe whilst only L. illustris has been collected in North America. Reports that these species shared both mitochondrial and nuclear gene sequences, along with claims that diagnostic morphological characters are difficult to interpret, were used to question their separate species status. We report here that amplified fragment length polymorphism profiles strongly support the validity of both species based on both assignment and phylogenetic analysis, and that traditional identification criteria based on male and female genital morphology are more reliable than has been claimed.

•We present the first probabilities for time since death based on insect succession.•Few of the 266 hexapod species observed could support a confidence interval.•Postmortem interval estimation was more precise using two instead of one species. Carrion insect succession patterns have long been used to estimate the postmortem interval (PMI) during a death investigation. However, no published carrion succession study included sufficient replication to calculate a confidence interval about a PMI estimate based on occurrence data. We exposed 53 pig carcasses (16±2.5 kg), near the likely minimum needed for such statistical analysis, at a site in north-central Indiana, USA, over three consecutive summer seasons. Insects and Collembola were sampled daily from each carcass for a total of 14 days, by this time each was skeletonized. The criteria for judging a life stage of a given species to be potentially useful for succession-based PMI estimation were (1) nonreoccurrence (observed during a single period of presence on a corpse), and (2) found in a sufficiently large proportion of carcasses to support a PMI confidence interval. For this data set that proportion threshold is 45/53. Of the 266 species collected and identified, none was nonreoccuring in that each showed at least a gap of one day on a single carcass. If the definition of nonreoccurrence is relaxed to include such a single one-day gap the larval forms of Necrophilaamericana, Fanniascalaris, Cochliomyia macellaria, Phormiaregina, and Luciliaillustris satisfied these two criteria. Adults of Creophilus maxillosus, Necrobiaruficollis, and Necrodessurinamensis were common and showed only a few, single-day gaps in occurrence. C.maxillosus, P.regina, and L.illustris displayed exceptional forensic utility in that they were observed on every carcass. Although these observations were made at a single site during one season of the year, the species we found to be useful have large geographic ranges. We suggest that future carrion insect succession research focus only on a limited set of species with high potential forensic utility so as to reduce sample effort per carcass and thereby enable increased experimental replication.

The accurate determination of time since death, or postmortem interval (PMI), can be critical in the investigation of suspicious deaths. Knowing when a suspicious death occurred can limit the number of potential suspects to those without a viable alibi for the time of the crime. The forensic techniques currently employed to determine PMI: pathology, entomology, and anthropology, are accurate over different time periods following death. A large gap in time exists between the capabilities of forensic entomology and traditional anthropology, leaving a period in which PMI is difficult to estimate. In this study, time-dependent differences in RNA decay rates were examined to extend the time frame over which early PMI estimates can be made. Comparing the decay rates of a large, labile segment of β-actin RNA and a smaller, more stable, non-overlapping segment of the same RNA from tooth pulp, we were able to estimate PMI values of pigs buried within a shallow grave for up to 84 days. This compares favorably to an estimate of PMI using insect data. Full skeletonization and loss of insect activity was observed by day 28 of our study. In addition to differences in RNA decay rates, morphological changes were observed in the pulp as it aged postmortem. To provide a quantitative measure of progressive color changes, analysis of digital photographs of each tooth's pulp were used to construct a simple colorimetric assay. This assay was then used to cluster ages of pulp samples by color. The two assays, used in combination with one another, can create a more precise estimate of PMI. The potential advantages of this molecular means of estimating PMI include extending the time frame for such estimates, is applicable to samples collected worldwide (no specialized knowledge of local insect fauna is required), is relatively fast, and inexpensive.

•First forensic entomology experiment in the United Arab Emirates.•Survey of the very early colonizers.•Flesh flies were dominant and the species Sarcophaga dux and Wohlfahrtia nuba were the most important taxa.•First record of feeding larvae of the flesh fly Wohlfahrtia indigens on carrion. The carrion insect species that most quickly deposit offspring on a corpse are, when available, likely to yield a more useful estimate of postmortem interval (PMI) compared to later arrivals. This is in part because the age of the oldest larva will be as close as possible to the PMI when doing a development analysis, and because the preappearance interval (PAI), the time the corpse was exposed before insect colonization, corresponds to the narrowest window of time since death for an insect-free corpse when doing a succession analysis. Given replicated training data a prediction of exposure time for a corpse can be in the form of a confidence set, and the maximum value of that set for an insect-free corpse is a probabilistic version of PAI. To discover the insect species likely to be useful in the early postmortem period in the United Arab Emirates we exposed 216 rat carcasses outdoors at two sites in Dubai over three-day periods during winter. Rats were sampled twice each day without replacement and kept in the lab to allow carrion insects to complete development to the adult stage. The fly species produced in this way were Sarcophaga dux, S. ruficornis, Wohlfahrtia nuba, W. indigens (Sarcophagidae), Chrysomya albiceps (Calliphoridae), and Musca domestica (Muscidae). To the best of our knowledge this is the first record of W. indigens larvae feeding on carrion. While adult C. albiceps and M. domestica were abundant on the carcasses, C. albiceps colonized too slowly to be useful for this type of succession analysis within this time frame, and M. domestica emerged from a single rat. The Sarcophagidae were rapid colonizers, and under these conditions the probability is>90% that a carcass would remain free of S. dux larvae not more than 57 h and free of W. nuba larvae for not more than 51 h.

Many authors produced carrion insect development data for predicting the age of an insect from a corpse. Under some circumstances, this age value is a minimum postmortem interval. There are no standard protocols for such experiments, and the literature includes a variety of sampling methods. To our knowledge, there has been no investigation of how the choice of sampling method can be expected to influence the performance of the resulting predictive model. We calculated 95 % inverse prediction confidence limits for growth curves of the forensically important carrion flies Chrysomya megacephala and Sarconesia chlorogaster (Calliphoridae) at a constant temperature. Confidence limits constructed on data for entire age cohorts were considered to be the most realistic and were used to judge the effect of various subsampling schemes from the literature. Random subsamples yielded predictive models very similar to those of the complete data. Because taking genuinely random subsamples would require a great deal of effort, we imagine that it would be worthwhile only if the larval measurement technique were especially slow and/or expensive. However, although some authors claimed to use random samples, their published methods suggest otherwise. Subsampling the largest larvae produced a predictive model that performed poorly, with confidence intervals about an estimate of age being unjustifiably narrow and unlikely to contain the true age. We believe these results indicate that most forensic insect development studies should involve the measurement of entire age cohorts rather than subsamples of one or more cohorts.

[...]we could not produce the coefficients and standard errors shown in MM09 Table 1. Because those two sets of numbers should be mathematically related, and are not, they cannot both be correct. [...]if three events are assumed to be independent then the probability of all three is the product of the probabilities.