The utility of blow fly (Diptera: Calliphoridae) evidence from burned human remains.

Burning of human remains is a common method to conceal or destroy evidence associated with homicides and illegal activities. However, data regarding blow fly colonization of burned remains are scarce, with all previously published empirical studies focusing only on non-human animals. It is critically important to obtain basic data on blow fly colonization patterns of burned human remains as such evidence may represent the only feasible method for PMI estimation in cases of burning. In this study, we thermally altered six human donors to a Crow-Glassman Scale Level 3 (CGS-3) and placed them at the Anthropology Research Facility at the University of Tennessee in Summer 2021, Spring 2022, and Summer 2022. Six unburned human donors were used as controls. Observations for insect activity began within 24 h of placement and continued twice weekly through decomposition. Age estimations were performed with immature blow flies to estimate the time of colonization (TOC), and accuracy was assessed against the time of placement for each donor. All burned donors examined in this study were colonized by blow flies. No significant difference in species composition was determined between treatments, though TOC estimations from burned donors were slightly (but significantly) less accurate than TOC estimations from unburned donors (80% vs. 83% accuracy; χ2 = 0.041, df = 1, P = 0.840). These results indicate that blow flies can successfully colonize human remains burned to CGS-3 and that accurate TOC estimations can still be generated from larval specimens. Though several limitations to this study exist (e.g., inconsistent donor BMI, lack of donor temperature data), our results underscore the utility of entomological evidence in cases of burned human remains.

Temporal population genetic structure of Phormia regina (Diptera: Calliphoridae).

The genetic structure of forensically important blow fly (Brauer & Bergenstamm) (Diptera: Calliphoridae) populations has remained elusive despite high relatedness within wild-caught samples. This research aimed to determine if the implementation of a high-resolution spatiotemporal sampling design would reveal latent genetic structure among blow fly populations and to elucidate any environmental impacts on observed patterns of genetic structure. Adult females of the black blow fly, Phormia regina (Meigen) (Diptera: Calliphoridae), were collected from 9 urban parks in Indiana, USA over 3 yr and genotyped at 6 polymorphic microsatellite loci. The data analysis involved 3 clustering methods: principal coordinate analysis (PCoA), discriminant analysis of principal components (DAPC), and STRUCTURE. While the PCoA did not uncover any discernible clustering patterns, the DAPC and STRUCTURE analyses yielded significant results, with 9 and 4 genetic clusters, respectively. Visualization of the STRUCTURE bar plot revealed N = 11 temporal demarcations indicating barriers to gene flow. An analysis of molecular variance of these STRUCTURE-inferred populations supported strong temporally driven genetic differentiation (FST = 0.048, F'ST = 0.664) relative to geographic differentiation (FST = 0.009, F'ST = 0.241). Integrated Nested Laplace Approximation and Boosted Regression Tree analyses revealed that collection timepoint and 4 main abiotic factors (temperature, humidity, precipitation, and wind speed) were associated with the genetic subdivisions observed for P. regina. A complex interplay between environmental conditions, the unique reproductive strategies of the blow fly, and the extensive dispersal abilities of these organisms likely drives the strong genetic structure of P. regina in the Midwestern US.

Not by the Book: Observations of Delayed Oviposition and Re-Colonization of Human Remains by Blow Flies.

Postmortem interval estimations can be complicated by the inter-individual variation present in human decomposition. Forensic entomologists may especially face challenges interpreting arthropod evidence in scenarios that are not "by the book", or that vary in unexpected ways. Therefore, it is important to report instances where blow fly colonization does not align with expected soft tissue decomposition as blow fly larvae are often used to produce a time of colonization (TOC) estimation to infer a minimum PMI. We followed the decomposition and blow fly activity of three human donors at the Anthropology Research Facility (University of Tennessee). Delayed oviposition occurred on one donor 115 d post-placement, whereas two donors experienced blow fly re-colonization after cessation of the consumption phase, one 22 d and one more than 200 d after blow fly larvae were last observed. A null hypothesis model tested whether the entomological TOC and anthropological total body score (TBS) estimations encompassed the time of placement (TOP) for each donor. While the null hypothesis was rejected for all TOC estimations, it could not be rejected for the TBS estimations. We discuss how the non-linear nature of human decomposition can pose challenges to interpreting blow fly evidence and suggest that forensic entomology practitioners should recognize these limitations in both research endeavors and applied casework.

Insects as Chemical Sensors: Detection of Chemical Warfare Agent Simulants and Hydrolysis Products in the Blow Fly Using LC-MS/MS.

In this work, blow flies were investigated as environmental chemical sample collectors following a chemical warfare attack (CWA). Blow flies sample the environment as they search for water and food sources and can be trapped from kilometers away using baited traps. Three species of blow flies were exposed to CWA simulants to determine the persistence and detectability of these compounds under varying environmental conditions. A liquid chromatography mass spectrometry (LC-MS/MS) method was developed to detect CWA simulants and hydrolysis products from fly guts. Flies were exposed to the CWA simulants dimethyl methylphosphonate and diethyl phosphoramidate as well as the pesticide dichlorvos, followed by treatment-dependent temperature and humidity conditions. Flies were sacrificed at intervals within a 14 day postexposure period. Fly guts were extracted and analyzed with the LC-MS/MS method. The amount of CWA simulant in fly guts decreased with time following exposure but were detectable 14 days following exposure, giving a long window of detectability. In addition to the analysis of CWA simulants, isopropyl methylphosphonic acid, the hydrolysis product of sarin, was also detected in blow flies 14 days post exposure. This work demonstrates the potential to obtain valuable samples from remote or access-restricted areas without risking lives.

Blow fly stable isotopes reveal larval diet: A case study in community level anthropogenic effects.

Response to human impacts on the environment are typically initiated too late to remediate negative consequences. We present the novel use of stable isotope analysis (SIA) of blow flies to determine human influences on vertebrate communities in a range of human-inhabited environments, from a pristine national park to a dense metropolitan area. The refrain "you are what you eat" applies to the dietary isotope record of all living organisms, and for carrion-breeding blow flies, this translates to the type of carcasses present in an environment. Specifically, we show that carnivore carcasses make up a large proportion of the adult fly's prior larval diet, which contrasts to what has been reportedly previously for the wild adult fly diet (which consists of mostly herbivore resources). Additionally, we reveal the potential impact of human food on carcasses that were fed on by blow flies, underscoring the human influences on wild animal populations. Our results demonstrate that using SIA in conjunction with other methods (e.g., DNA analysis of flies) can reveal a comprehensive snapshot of the vertebrate community in a terrestrial ecosystem.

First record of the oriental latrine fly, Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae), in Tennessee, USA.

PURPOSE: Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) is a medically and forensically important blow fly species that invaded the United States three decades ago and has continued to expand its distribution across the country ever since. Unlike many other blow fly species, larvae of C. megacephala can develop in feces, particularly from humans. Additionally, C. megacephala is known to carry antibiotic-resistant bacteria in even greater quantities than house flies in tropical areas. This behavior, along with its dissemination into and prevalence within human inhabited environments (e.g., outdoor markets, urban neighborhoods), makes C. megacephala a potential threat to human health. This short communication serves as the first record of C. megacephala in Tennessee, USA. METHODS: Collections of adult and larval blow flies were made from two sets of human remains decomposing at the Anthropology Research Facility (ARF) at the University of Tennessee. Specimens were confirmed by an expert to be C. megacephala. RESULTS: A total of seven individual specimens (four adults and three larvae) of C. megacephala were collected from human bodies at the ARF. These results indicate that C. megacephala is not just dispersing into this environment as adults, but actively colonizing human remains in this semi-urban area. CONCLUSION: These observations support the previously described behavior and habitat of this filth-breeding fly in many Asian countries where it is considered medically and forensically important. Therefore, continuously updated distribution records, like this one, are critical for tracking the movement of C. megacephala across the United States.

The oncometabolite L-2-hydroxyglutarate is a common product of dipteran larval development.

The oncometabolite L-2-hydroxyglutarate (L-2HG) is considered an abnormal product of central carbon metabolism that is capable of disrupting chromatin architecture, mitochondrial metabolism, and cellular differentiation. Under most circumstances, mammalian tissues readily dispose of this compound, as aberrant L-2HG accumulation induces neurometabolic disorders and promotes renal cell carcinomas. Intriguingly, Drosophila melanogaster larvae were recently found to accumulate high L-2HG levels under normal growth conditions, raising the possibility that L-2HG plays a unique role in insect metabolism. Here we explore this hypothesis by analyzing L-2HG levels in 18 insect species. While L-2HG was present at low-to-moderate levels in most of these species (<100 pmol/mg; comparable to mouse liver), dipteran larvae exhibited a tendency to accumulate high L-2HG concentrations (>100 pmol/mg), with the mosquito Aedes aegypti, the blow fly Phormia regina, and three representative Drosophila species harboring concentrations that exceed 1 nmol/mg - levels comparable to those measured in mutant mice that are unable to degrade L-2HG. Overall, our findings suggest that one of the largest groups of animals on earth commonly generate high concentrations of an oncometabolite during juvenile growth, hint at a role for L-2HG in the evolution of dipteran development, and raise the possibility that L-2HG metabolism could be targeted to restrict the growth of key disease vectors and agricultural pests.

Female Blow Flies As Vertebrate Resource Indicators.

Rapid vertebrate diversity evaluation is invaluable for monitoring changing ecosystems worldwide. Wild blow flies naturally recover DNA and chemical signatures from animal carcasses and feces. We demonstrate the power of blow flies as biodiversity monitors through sampling of flies in three environments with varying human influences: Indianapolis, IN and two national parks (the Great Smoky Mountains and Yellowstone). Dissected fly guts underwent vertebrate DNA sequencing (12S and 16S rRNA genes) and fecal metabolite screening. Integrated Nested Laplace Approximation (INLA) was used to determine the most important abiotic factor influencing fly-derived vertebrate richness. In 720 min total sampling time, 28 vertebrate species were identified, with 42% of flies containing vertebrate resources: 23% DNA, 5% feces, and 14% contained both. The species of blow fly used was not important for vertebrate DNA recovery, however the use of female flies versus male flies directly influenced DNA detection. Temperature was statistically relevant across environments in maximizing vertebrate detection (mean = 0.098, sd = 0.048). This method will empower ecologists to test vertebrate community ecology theories previously out of reach due practical challenges associated with traditional sampling.

New Distribution Record for Lucilia cuprina (Diptera: Calliphoridae) in Indiana, United States.

Determining range expansion for insect species is vital in order to evaluate their impact on new ecosystems and communities. This is particularly important for species which could be potentially harmful to humans or domestic animals. Lucilia cuprina Wiedemann (Diptera: Calliphoridae) can act as a facultative ectoparasite and has an extensive history as the primary inducer of sheep-strike in Australia, New Zealand, and Africa. We present here the first record of this species in Indiana, United States. Lucilia cuprina's range expansion northward in the United States may be indicative of changing environmental conditions conducive to the proliferation of this species into historically cooler climates. The presence of this species could significantly impact forensic death investigations utilizing dipteran larvae to estimate a minimum postmortem interval. If range expansion of this species is not taken into account by a forensic entomologist (especially if L. cuprina is not known previously in their region), an inaccurate minimum postmortem interval (PMIMIN) estimation may be made, given the differences in development times for both species. Therefore, the range expansion of this fly could have large impacts for many different entomological disciplines.

Chemical Assay for the Detection of Vertebrate Fecal Metabolites in Adult Blow Flies (Diptera: Calliphoridae).

Filth flies are commonly implicated in pathogen transmission routes due to their affinity for vertebrate waste and their synanthropic associations. However, solidifying the link between flies and infected feces in the wild can be difficult, as interpretations made solely from microbial culturing or sequencing methods may represent an incomplete picture of pathogen acquisition. We present an analytical assay using high performance liquid chromatography tandem mass spectrometry (HPLC MS/MS) to detect vertebrate fecal metabolites (urobilinoids) in adult blow fly guts. Proof of concept experiments consisted of controlled feeding in which flies were grouped into three treatments (unfed, exposure to beef liver tissue, and exposure to canine feces; N = 20/treatment) using the black blow fly Phormia regina Meigen (Diptera: Calliphoridae). It was revealed that only feces-related samples exhibited peaks with an m/z of 591 and MS/MS spectra consistent with urobilinoids. These peaks were not seen for beef liver tissue, flies exposed to beef liver tissue, or unfed flies. Samples taken directly from beef liver tissue and from feces of several animals were also tested. To test this assay in wild flies, 216 flies were additionally analyzed to determine whether they had ingested vertebrate feces. About 13% of the wild flies exhibited these same peaks, providing a baseline measure of blow flies collected in urban and residential areas consuming feces from the environment. Overall, this assay can be used for P. regina collected in an applied setting and its integration with microbial culturing and sequencing methods will help to improve its use.

Redescription of Myianoetus muscarum (Acari: Histiostomatidae) Associated with Human Remains in Texas, USA, with Designation of a Neotype from Western Europe.

Mites are receiving renewed interest in forensic entomology, but the identification of many species associated with carrion and human remains is problematic. We review the taxonomy of the mite species currently known as Myianoetus muscarum (L., 1758) and designate a neotype from the collection of Jean Cooreman in Brussels, Belgium. The species is redescribed based on Cooreman's specimens, specimens previously reported from California and Ontario, Canada, and a large series of new specimens collected from a human corpse in Texas. The species is compared with and distinguished from other species of Myianoetus.

Developmental variation among Cochliomyia macellaria Fabricius (Diptera: Calliphoridae) populations from three ecoregions of Texas, USA.

Forensic entomologists rely on published developmental datasets to estimate the age of insects developing on human remains. Currently, these datasets only represent populations of targeted insects from specific locations. However, recent data indicate that populations can exhibit genetic variation in their development, including signatures of local adaptation demonstrated by regionally distinct plastic responses to their environments. In this study, three geographically distinct populations of the secondary screwworm, Cochliomyia macellaria Fabricius (Diptera: Calliphoridae; College Station, Longview, and San Marcos, TX, USA), a common blow fly collected from human remains in the southern USA, were reared in two distinct environments (cool 21 °C, 65 % relative humidity (RH); and warm 31 °C, 70 % RH) over 2 years (2011 and 2012) in order to determine differences in development time and mass. Significant differences in immature and pupal development time, as well as pupal mass, were shown to exist among strains derived from different populations and years. For immature development times, there was evidence of only an environmental effect on phenotype, while genotype by environment interactions was observed in pupal development times and pupal mass. College Station and San Marcos populations exhibited faster pupal development and smaller pupal sizes in the cooler environment relative to the Longview population, but showed an opposite trend in the warm environment. Rank order for College Station and Longview populations was reversed across years. Failure to take genetic variation into consideration when making such estimates can lead to unanticipated error and bias. These results indicate that genetics will have little impact on error when working with Texas genotypes of C. macellaria at ~30 °C and 70 % RH, but will have a more meaningful impact on error in postmortem interval estimates with this species in cooler, drier environments.