A conserved interdomain microbial network underpins cadaver decomposition despite environmental variables.

Microbial breakdown of organic matter is one of the most important processes on Earth, yet the controls of decomposition are poorly understood. Here we track 36 terrestrial human cadavers in three locations and show that a phylogenetically distinct, interdomain microbial network assembles during decomposition despite selection effects of location, climate and season. We generated a metagenome-assembled genome library from cadaver-associated soils and integrated it with metabolomics data to identify links between taxonomy and function. This universal network of microbial decomposers is characterized by cross-feeding to metabolize labile decomposition products. The key bacterial and fungal decomposers are rare across non-decomposition environments and appear unique to the breakdown of terrestrial decaying flesh, including humans, swine, mice and cattle, with insects as likely important vectors for dispersal. The observed lockstep of microbial interactions further underlies a robust microbial forensic tool with the potential to aid predictions of the time since death.

The microbiome of fly organs and fly-human microbial transfer during decomposition.

During decomposition, flies interact with the remains to lay eggs and acquire nutrients, and in the process, they bring their microbes with them. While it is known that flies have their own unique core microbiome, it is not known if flies associated with human cadavers have a different core microbiome. Differences in the fly microbiome may influence the types of microbes transmitted from the flies to the cadaver, therefore potentially affecting assembly of the human decomposer microbiome. The first purpose of this study was to characterize the microbiome of flies associated with human cadavers by fly organ and season. This is because fly interactions with cadavers vary by season, and because it is likely that external fly organs [i.e., the labellum and tarsi] make more direct contact and are likely involved in increased mechanical transmission with the cadaver than internal organs such as the oocyte. The second purpose of this study was to determine if the fly microbes contribute to the human decomposer microbiome. To accomplish these aims, 10 human cadavers were placed outdoors across three seasons and allowed to decompose. A total of 40 flies that landed on the cadaver were collected and dissected by the labellum, tarsi, and oocyte. In addition to fly collections, samples from the cadavers were collected using a sterile swab at sites including the cheek of the face, inner cheek, bicep, torso, and anus. Overall, it was shown that flies associated with human cadavers have a similar microbiome to flies from previous studies that were not associated with human cadavers. However, there are differences in the microbiome between seasons and fly parts. We also show evidence that flies act as a microbial source to the human decomposer microbiome, which is important for understanding the ecological mechanisms of human cadaver microbial community assembly.

Comparison of DNA extraction techniques for the recovery of bovine DNA from fly larvae crops.

Forensic entomology aids investigations using insects and is primarily associated with the estimation of post-mortem interval (PMI). Studies have shown that human DNA can be recovered from the crops of fly larvae. While several factors regarding the recovery of human DNA from crops have been studied, DNA extraction methods have not been thoroughly assessed. Determining a method for optimal extraction could aid crime laboratories in implementing DNA extraction from larvae and streamlining future research. Bovine DNA was used as a substitute for human DNA to test several DNA extractions kits. Four DNA extraction kits (Chelex®, PDQeX forensicGEM, EZ1® DNA Investigator, DNeasy® Powersoil® Pro Kit) were evaluated based on the quantity and quality of bovine DNA extracted. Extractions were performed on whole fly larvae and dissected crops. Quantification was performed using real-time PCR (qPCR) on a StepOne™ Real-Time PCR System with SYBR® Green using bovine-specific cytochrome b primers. The quality of extracts was determined by checking for inhibition using commercial qPCR chemistries with an internal PCR control (IPC). When using whole fly larvae, Powersoil® Pro yielded the highest average DNA yield (n = 10, 0.668 ± 0.458 ng/µl), while EZ1® DNA Investigator yielded the highest average with crops (n = 10, 0.605 ± 0.403 ng/µl). Chelex and forensicGEM yielded low amounts of bovine DNA, and its extracts were inhibited, unlike EZ1® and Powersoil® Pro, which have purification steps. Therefore, it is recommended to use EZ1® DNA Investigator coupled with automation on EZ1® Advanced XL to recover DNA from fly larvae crops.

A Pilot Study of Microbial Succession in Human Rib Skeletal Remains during Terrestrial Decomposition.

The bones of decomposing vertebrates are colonized by a succession of diverse microbial communities. If this succession is similar across individuals, microbes may provide clues about the postmortem interval (PMI) during forensic investigations in which human skeletal remains are discovered. Here, we characterize the human bone microbial decomposer community to determine whether microbial succession is a marker for PMI. Six human donor subjects were placed outdoors to decompose on the soil surface at the Southeast Texas Applied Forensic Science facility. To also assess the effect of seasons, three decedents were placed each in the spring and summer. Once ribs were exposed through natural decomposition, a rib was collected from each body for eight time points at 3 weeks apart. We discovered a core bone decomposer microbiome dominated by taxa in the phylum Proteobacteria and evidence that these bone-invading microbes are likely sourced from the surrounding decomposition environment, including skin of the cadaver and soils. Additionally, we found significant overall differences in bone microbial community composition between seasons. Finally, we used the microbial community data to develop random forest models that predict PMI with an accuracy of approximately ±34 days over a 1- to 9-month time frame of decomposition. Typically, anthropologists provide PMI estimates based on qualitative information, giving PMI errors ranging from several months to years. Previous work has focused on only the characterization of the bone microbiome decomposer community, and this is the first known data-driven, quantitative PMI estimate of terrestrially decomposed human skeletal remains using microbial abundance information. IMPORTANCE Microbes are known to facilitate vertebrate decomposition, and they can do so in a repeatable, predictable manner. The succession of microbes in the skin and associated soil can be used to predict time since death during the first few weeks of decomposition. However, when remains are discovered after months or years, often the only evidence are skeletal remains. To determine if microbial succession in bone would be useful for estimating time since death after several months, human subjects were placed to decompose in the spring and summer seasons. Ribs were collected after 1 to 9 months of decomposition, and the bone microbial communities were characterized. Analysis revealed a core bone decomposer microbial community with some differences in microbial assembly occurring between seasons. These data provided time since death estimates of approximately ±34 days over 9 months. This may provide forensic investigators with a tool for estimating time since death of skeletal remains, for which there are few current methods.

Microbiome Data Accurately Predicts the Postmortem Interval Using Random Forest Regression Models.

Death investigations often include an effort to establish the postmortem interval (PMI) in cases in which the time of death is uncertain. The postmortem interval can lead to the identification of the deceased and the validation of witness statements and suspect alibis. Recent research has demonstrated that microbes provide an accurate clock that starts at death and relies on ecological change in the microbial communities that normally inhabit a body and its surrounding environment. Here, we explore how to build the most robust Random Forest regression models for prediction of PMI by testing models built on different sample types (gravesoil, skin of the torso, skin of the head), gene markers (16S ribosomal RNA (rRNA), 18S rRNA, internal transcribed spacer regions (ITS)), and taxonomic levels (sequence variants, species, genus, etc.). We also tested whether particular suites of indicator microbes were informative across different datasets. Generally, results indicate that the most accurate models for predicting PMI were built using gravesoil and skin data using the 16S rRNA genetic marker at the taxonomic level of phyla. Additionally, several phyla consistently contributed highly to model accuracy and may be candidate indicators of PMI.

Microbial community assembly and metabolic function during mammalian corpse decomposition.

Vertebrate corpse decomposition provides an important stage in nutrient cycling in most terrestrial habitats, yet microbially mediated processes are poorly understood. Here we combine deep microbial community characterization, community-level metabolic reconstruction, and soil biogeochemical assessment to understand the principles governing microbial community assembly during decomposition of mouse and human corpses on different soil substrates. We find a suite of bacterial and fungal groups that contribute to nitrogen cycling and a reproducible network of decomposers that emerge on predictable time scales. Our results show that this decomposer community is derived primarily from bulk soil, but key decomposers are ubiquitous in low abundance. Soil type was not a dominant factor driving community development, and the process of decomposition is sufficiently reproducible to offer new opportunities for forensic investigations.

Four Forensic Entomology Case Studies: Records and Behavioral Observations on Seldom Reported Cadaver Fauna With Notes on Relevant Previous Occurrences and Ecology.

A yearlong survey of insect taxa associated with human decomposition was conducted at the Southeast Texas Applied Forensic Science (STAFS) facility located in the Center for Biological Field Studies of Sam Houston State University in Huntsville, TX. During this study, four insect-cadaver interactions were observed that represent previously poorly documented yet forensically significant interactions: Syrphidae maggots colonized a corpse in an aquatic situation; Psychodidae adults mated and oviposited on an algal film that was present on a corpse that had been recently removed from water; several Panorpidae were the first insects to feed upon a freshly placed corpse in the autumn; and a noctuid caterpillar was found chewing and ingesting dried human skin. Baseline knowledge of insect-cadaver interactions is the foundation of forensic entomology, and unique observations have the potential to expand our understanding of decomposition ecology.

Initial insights into bacterial succession during human decomposition.

Decomposition is a dynamic ecological process dependent upon many factors such as environment, climate, and bacterial, insect, and vertebrate activity in addition to intrinsic properties inherent to individual cadavers. Although largely attributed to microbial metabolism, very little is known about the bacterial basis of human decomposition. To assess the change in bacterial community structure through time, bacterial samples were collected from several sites across two cadavers placed outdoors to decompose and analyzed through 454 pyrosequencing and analysis of variable regions 3-5 of the bacterial 16S ribosomal RNA (16S rRNA) gene. Each cadaver was characterized by a change in bacterial community structure for all sites sampled as time, and decomposition, progressed. Bacteria community structure is variable at placement and before purge for all body sites. At bloat and purge and until tissues began to dehydrate or were removed, bacteria associated with flies, such as Ignatzschineria and Wohlfahrtimonas, were common. After dehydration and skeletonization, bacteria associated with soil, such as Acinetobacter, were common at most body sites sampled. However, more cadavers sampled through multiple seasons are necessary to assess major trends in bacterial succession.

Terrestrial laser scanning and a degenerated cylinder model to determine gross morphological change of cadavers under conditions of natural decomposition.

Decomposition can be a highly variable process with stages that are difficult to quantify. Using high accuracy terrestrial laser scanning a repeated three-dimensional (3D) documentation of volumetric changes of a human body during early decomposition is recorded. To determine temporal volumetric variations as well as 3D distribution of the changed locations in the body over time, this paper introduces the use of multiple degenerated cylinder models to provide a reasonable approximation of body parts against which 3D change can be measured and visualized. An iterative closest point algorithm is used for 3D registration, and a method for determining volumetric change is presented. Comparison of the laser scanning estimates of volumetric change shows good agreement with repeated in-situ measurements of abdomen and limb circumference that were taken diurnally. The 3D visualizations of volumetric changes demonstrate that bloat is a process with a beginning, middle, and end rather than a state of presence or absence. Additionally, the 3D visualizations show conclusively that cadaver bloat is not isolated to the abdominal cavity, but also occurs in the limbs. Detailed quantification of the bloat stage of decay has the potential to alter how the beginning and end of bloat are determined by researchers and can provide further insight into the effects of the ecosystem on decomposition.

Terrestrial laser scanning to model sunlight irradiance on cadavers under conditions of natural decomposition.

Human decomposition is a dynamic process that is influenced by both abiotic and biotic factors. Measuring these influences, in particular abiotic factors, on the decomposition process is often a challenge for scientists. Recently, researchers have turned to the use of advanced remote sensing technologies in forensic investigations. In this study, a new methodology is described that utilizes precise 3D images captured using terrestrial laser scanning (TLS) to calculate total solar irradiance on a cadaver in a partially forested environment. To test this new measurement approach under actual field conditions, three cadavers were placed in an outdoor environment to decompose. Laser scans were taken the day of placement and used to calculate the total solar irradiance at time points of 24 h, 1 week, and 1 month from placement. The results show that as time progresses, different cadavers at the field site and different areas of the same cadaver receive varying amounts of solar irradiance. The modeling based on these laser scans can be used to create predictive images of solar irradiance that may provide researchers with a new tool to help quantitatively assess the effect of solar irradiance on a cadaver ecosystem.

The living dead: bacterial community structure of a cadaver at the onset and end of the bloat stage of decomposition.

Human decomposition is a mosaic system with an intimate association between biotic and abiotic factors. Despite the integral role of bacteria in the decomposition process, few studies have catalogued bacterial biodiversity for terrestrial scenarios. To explore the microbiome of decomposition, two cadavers were placed at the Southeast Texas Applied Forensic Science facility and allowed to decompose under natural conditions. The bloat stage of decomposition, a stage easily identified in taphonomy and readily attributed to microbial physiology, was targeted. Each cadaver was sampled at two time points, at the onset and end of the bloat stage, from various body sites including internal locations. Bacterial samples were analyzed by pyrosequencing of the 16S rRNA gene. Our data show a shift from aerobic bacteria to anaerobic bacteria in all body sites sampled and demonstrate variation in community structure between bodies, between sample sites within a body, and between initial and end points of the bloat stage within a sample site. These data are best not viewed as points of comparison but rather additive data sets. While some species recovered are the same as those observed in culture-based studies, many are novel. Our results are preliminary and add to a larger emerging data set; a more comprehensive study is needed to further dissect the role of bacteria in human decomposition.

Exclusion of forensically important flies due to burying behavior by the red imported fire ant (Solenopsis invicta) in southeast Texas.

On March 3, 2009, the remains of an adult male were partially buried at the Southeast Texas Applied Forensic Science (STAFS) Facility at the Center for Biological Field Studies (CBFS), Sam Houston State University, Texas. The individual was buried except for a small portion of the left abdominal region. A postmortem incised wound was created in the exposed area with the intention of attracting carrion flies. Worker ants of a colony of Solenopsis invicta Buren 1972 (red imported fire ant) filled in the wound with soil, thereby monopolizing the exposed area of the corpse and excluding expected carrion insects from the wound. During the bloating phase, approximately nine days after burial, normal decomposition processes of the gut created a sufficient disruption of the ants, such that flies oviposited and larvae were able to colonize the corpse. Estimation of the postmortem interval (PMI) based on the minimum period of fly activity would be severely skewed should the remains be discovered at this point and growth rate of Diptera larvae be used as the primary determinant for the PMI. While S. invicta is an expected member of a carrion ecosystem in southeastern Texas, and is known to distort the PMI estimation through larval and egg removal, the complete exclusion of flies from the wound by the burial behavior of S. invicta was an unexpected and until now an unpublished occurrence.

Necrophagous caterpillars provide human mtDNA evidence.

Decomposition of large mammalian carcasses is greatly accelerated through the action of insects. Specialized feeders capable of digesting keratin and collagen found in skin, hair, and tendons and ligaments are attracted to corpses in late stages of dry decomposition and include Tinea pellionella, the casemaking clothes moth, and Tineola bisselliella, the webbing clothes moth (Lepidoptera; Tineidae). Until now, details of the caterpillar behavior as necrophagous insects were vague. Here, we detail the behavior of each species and document the incorporation of human hair into the portable larval shelters constructed by the caterpillars of T. pellionella. Hair of the decedent used as building material for caterpillar shelters provided enough starting template to amplify and sequence the HVI and HVII sections of the control region (mtDNA) of the decedent.

Comparison of phylogenetic signal between male genitalia and non-genital characters in insect systematics.

It is generally accepted that male genitalia evolve more rapidly and divergently relative to non-genital traits due to sexual selection, but there is little quantitative comparison of the pattern of evolution between these character sets. Moreover, despite the fact that genitalia are still among the most widely used characters in insect systematics, there is an idea that the rate of evolution is too rapid for genital characters to be useful in forming clades. Based on standard measures of fit used in cladistic analyses, we compare levels of homoplasy and synapomorphy between genital and non-genital characters of published data sets and demonstrate that phylogenetic signal between these two character sets is statistically similar. This pattern is found consistently across different insect orders at different taxonomic hierarchical levels. We argue that the fact that male genitalia are under sexual selection and thus diverge rapidly does not necessarily equate with the lack of phylogenetic signal, because characters that evolve by descent with modification make appropriate characters for a phylogenetic analysis, regardless of the rate of evolution. We conclude that male genitalia are a composite character consisting of different components diverging separately, which make them ideal characters for phylogenetic analyses, providing information for resolving varying levels of hierarchy. © The Willi Hennig Society 2009.

Insect successional pattern of a corpse in cooler months of subtropical southeastern Texas.

Here, we characterize the cool weather insect fauna found associated with partially skeletonized and desiccated human remains recovered from an abandoned house in an urban area of subtropical, coastal Galveston County, Texas, and use the information to conclude an approximate postmortem interval of 7-10 months. The predominant factors that allow for a confident assessment of the postmortem interval include climatological data, entomological data, and anthropological data. The documented insect fauna represents a unique assemblage present in a particular environment (an urban abandoned house in coastal Texas) at a particular time of year (winter) and includes expected forensically significant insects such as calliphorid flies, muscid flies, and dermestid beetles but also includes less commonly encountered insects such as an unusually dense population of live case-making clothes moths.

Gelechioidea (Insecta: Lepidoptera) systematics: a reexamination using combined morphology and mitochondrial DNA data.

Phylogenies of Gelechioidea (Insecta: Lepidoptera) historically have been in disagreement and definitions vary at the family and subfamily levels. Addition of new taxa or new characters drastically changes relationships indicating that current phylogenetic schemes require more investigation. This study is the first phylogenetic analysis of Gelechioidea to include molecular data. Here we present a combined analysis using Maximum Parsimony to investigate sister-group relationships within Gelechioidea. The addition of Cytochrome oxidase I and II to revised published morphological matrices gives 453 parsimony informative characters for the 42 taxa for which we have sequence data. The combined analysis resulted in two trees with mostly novel sister-group relationships. These results challenge current concepts of Gelechioidea, suggesting that traditional morphological characters that have united taxa may not be homologous structures and are in need of further investigation. A combination of morphological data with new molecular data will be the most robust method of study for Gelechioidea phylogenetics.