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After death, microbes (including bacteria and fungi) colonize carrion from a variety of sources during the decomposition process. The predictable succession of microbes could be useful for forensics, such as postmortem submersion interval estimation (PMSI) for aquatic deaths. However, gaps exist in our understanding of microbial succession on submerged bone, particularly regarding longer‐term decomposition (>1 year), fungal composition, and differences between internal and external microbial communities. To further explore this potential forensic tool, we described the postmortem microbial communities (bacteria and fungi) on and within submerged bones using targeted amplicon sequencing. We hypothesized predictable successional patterns of microbial colonization would be detected on the surface and within submerged bones, which would eventually converge to a similar microbial community. To best replicate forensic contexts, we sampled bones from replicate swine (Sus scrofa domesticus) carcasses submerged in a freshwater pond, every three months for nearly two years. Microbial bone (internal vs. external) community structure (taxa abundance and diversity) of bones differed for both bacteria and fungi, but internal and external communities did not converge to a similar structure. PMSI estimation models built with random forest regression of postmortem microbiomes were highly accurate (>80% variation explained in PMSI) and showed promise for forensic purposes. Overall, we provide further evidence that internal and external bone microbial communities submerged in an aquatic habitat are distinct and each community undergoes predictable succession, demonstrating potential utility in forensics for modeling PMSI in unattended deaths and/or cold cases. [ABSTRACT FROM AUTHOR]

The postmortem microbiome plays an important functional role in host decomposition after death. Postmortem microbiome community successional patterns are specific to body site, with a significant shift in composition 48 h after death. While the postmortem microbiome has important forensic applications for postmortem interval estimation, it also has the potential to aid in manner of death (MOD) and cause of death (COD) determination as a reflection of antemortem health status. To further explore this association, we tested beta-dispersion, or the variability of microbiomes within the context of the "Anna Karenina Principle" (AKP). The foundational principle of AKP is that stressors affect microbiomes in unpredictable ways, which increases community beta-dispersion. We hypothesized that cases with identified M/CODs would have differential community beta-dispersion that reflected antemortem conditions, specifically that cardiovascular disease and/or natural deaths would have higher beta-dispersion compared to other deaths (e.g., accidents, drug-related deaths). Using a published microbiome data set of 188 postmortem cases (five body sites per case) collected during routine autopsy in Wayne County (Detroit), MI, we modeled beta-dispersion to test for M/COD associations a priori. Logistic regression models of beta-dispersion and case demographic data were used to classify M/COD. We demonstrated that beta-dispersion, along with case demographic data, could distinguish among M/COD – especially cardiovascular disease and drug related deaths, which were correctly classified in 79% of cases. Binary logistic regression models had higher correct classifications than multinomial logistic regression models, but changing the defined microbial community (e.g., full vs. non-core communities) used to calculate beta-dispersion overall did not improve model classification or M/COD. Furthermore, we tested our analytic approach on a case study that predicted suicides from other deaths, as well as distinguishing MOD (e.g., homicides vs. suicides) within COD (e.g., gunshot wound). We propose an analytical workflow that combines postmortem microbiome indicator taxa, beta-dispersion, and case demographic data for predicting MOD and COD classifications. Overall, we provide further evidence the postmortem microbiome is linked to the host's antemortem health condition(s), while also demonstrating the potential utility of including beta-dispersion (a non-taxon dependent approach) coupled with case demographic data for death determination. [ABSTRACT FROM AUTHOR]

Carrion plays an essential role in shaping the structure and functioning of ecosystems and has far-reaching implications for biodiversity conservation. The change in availability and type of carcasses throughout ecosystems can involve negative effects for scavenging communities. To address this issue, there have been recent conservation management measures of carrion provision in natural systems. However, the optimal conditions under which exposing carcasses to optimize conservation outcomes are still limited. Here, we used camera traps throughout elevational and vegetational gradients to monitor the consumption of 48 deer carcasses over a study period of six years by evaluating 270,279 photographs resulting out of 15,373 trap nights. We detected 17 species visiting carcass deployments, including five endangered species. Our results show that large carcasses, the winter season, and a heterogeneous surrounding habitat enhanced the frequency of carcass visits and the species richness of scavenger assemblages. Contrary to our expectations, carcass species, condition (fresh/frozen), and provision schedule (continuous vs single exposure) did not influence scavenging frequency or diversity. The carcass visitation frequency increased with carcass mass and lower temperatures. The effect of large carcasses was especially pronounced for mesopredators and the Eurasian lynx (Lynx lynx). Lynx were not too influenced in its carrion acquisition by the season, but exclusively preferred remote habitats containing higher forest cover. Birds of prey, mesopredators, and top predators were also positively influenced by the visiting rate of ravens (Corvus corax), whereas no biotic or abiotic preferences were found for wild boars (Sus scrofa). This study provides evidence that any ungulate species of carrion, either in a fresh or in previously frozen condition, attracts a high diversity of scavengers especially during winter, thereby supporting earlier work that carcass provisions may support scavenger communities and endangered species. [ABSTRACT FROM AUTHOR]

Multiple methods have been proposed to provide accurate time since death estimations, and recently, the discovery of bacterial community turnover during decomposition has shown itself to have predictable patterns that may prove useful. In this study, we demonstrate the use of metatranscriptomics from the postmortem microbiome to simultaneously obtain community structure and functional data across postmortem intervals (PMIs). We found that bacterial succession patterns reveal similar trends as detected through DNA analysis, such as increasing Clostridiaceae as decomposition occurs, strengthening the reliability of total RNA community analyses. We also provide one of the first analyses of RNA transcripts to characterize bacterial metabolic pathways during decomposition. We found distinct pathways, such as amino acid metabolism, to be strongly up‐regulated with increasing PMIs. Elucidating the metabolic activity of postmortem microbial communities provides the first steps to discovering postmortem functional biomarkers since functional redundancy across bacteria may reduce host individual microbiome variability. [ABSTRACT FROM AUTHOR]

Decomposing remains are a nutrient-rich ecosystem undergoing constant change due to cell breakdown and abiotic fluxes, such as pH level and oxygen availability. These environmental fluxes affect bacterial communities who respond in a predictive manner associated with the time since organismal death, or the postmortem interval (PMI). Profiles of microbial taxonomic turnover and transmigration are currently being studied in decomposition ecology, and in the field of forensic microbiology as indicators of the PMI. We monitored bacterial community structural and functional changes taking place during decomposition of the intestines, bone marrow, lungs, and heart in a highly controlled murine model. We found that organs presumed to be sterile during life are colonized by Clostridium during later decomposition as the fluids from internal organs begin to emulsify within the body cavity. During colonization of previously sterile sites, gene transcripts for multiple metabolism pathways were highly abundant, while transcripts associated with stress response and dormancy increased as decomposition progressed. We found our model strengthens known bacterial taxonomic succession data after host death. This study is one of the first to provide data of expressed bacterial community genes, alongside transmigration and structural changes of microbial species during laboratory controlled vertebrate decomposition. This is an important dataset for studying the effects of the environment on bacterial communities in an effort to determine which bacterial species and which bacterial functional pathways, such as amino acid metabolism, provide key changes during stages of decomposition that relate to the PMI. Finding unique PMI species or functions can be useful for determining time since death in forensic investigations. [ABSTRACT FROM AUTHOR]

Common forensic entomology practice has been to collect the largest Diptera larvae from a scene and use published developmental data, with temperature data from the nearest weather station, to estimate larval development time and post-colonization intervals (PCIs). To evaluate the accuracy of PCI estimates among Calliphoridae species and spatially distinct temperature sources, larval communities and ambient air temperature were collected at replicate swine carcasses (N = 6) throughout decomposition. Expected accumulated degree hours (ADH) associated with Cochliomyia macellaria and Phormia regina third instars (presence and length) were calculated using published developmental data sets. Actual ADH ranges were calculated using temperatures recorded from multiple sources at varying distances (0.90 m–7.61 km) from the study carcasses: individual temperature loggers at each carcass, a local weather station, and a regional weather station. Third instars greatly varied in length and abundance. The expected ADH range for each species successfully encompassed the average actual ADH for each temperature source, but overall under-represented the range. For both calliphorid species, weather station data were associated with more accurate PCI estimates than temperature loggers associated with each carcass. These results provide an important step towards improving entomological evidence collection and analysis techniques, and developing forensic error rates. [ABSTRACT FROM AUTHOR]

Necrophagous insect studies have shown that decomposing vertebrate remains are an important ephemeral resource within an ecosystem. However, the microbes (e.g., bacteria and archaea) that were a part of the once living organism and the exogenous taxa that colonize this postmortem resource remain largely underexplored. Also, it is not well understood how these two kingdoms interact to recycle decaying biomass, an important mechanistic question for ecosystem function ecology. To better understand microbial community dynamics throughout decomposition, we used swine carcasses (A/=6) as models for mammalian postmortem decomposition to characterize epinecrotic microbial communities from: the abdominal skin of replicate carcasses; the internal microbiome of individual necrophagous dipteran larvae (maggots); and the microbiome of dipteran larval masses that had colonized the carcasses. Sampling occurred every 12 h for the duration of the decomposition process. We characterized these microbial communities over time using high-throughput 16S amplicon sequencing. The relative abundance of microbial taxa changed over decomposition as well as across sampling locations, suggesting significant interactions between the environment, microbes, and insect larvae. Maggot masses were represented by multiple blow fly species in each mass: Phormia regina (Meigen), Lucilia coerulei-viridis (Macquart), and Cochliomyia macellaria (F.). Relative abundance of these species within the mass also changed as decomposition progressed, suggesting the presence of certain Calliphoridae species within a mass may be associated with temporal shifts of the microbial communities. These results provide new insight into the community ecology of carrion decomposition by providing new data on interactions of microbes and dipteran larvae overtime. [ABSTRACT FROM AUTHOR]

Simple Summary: Whereas vertebrate scavengers have a higher diversity reported at randomly placed carcasses, the drivers of insect diversity on carrion, such as the exposure type (fixed versus random) or the carrion species, are still incompletely understood. We analyzed beetle diversity at differently exposed carcasses in the low-range mountain forest of the Bavarian Forest National Park in Germany. We tested if scavenging beetles, similarly to vertebrate scavengers, show a higher diversity at randomly placed carcasses compared to easily manageable fixed places. Ninety-two beetle species at 29 exposed wildlife carcasses (roe, red deer, and red foxes) were detected. Beetle diversity was higher at fixed locations possessing extended highly nutrient-rich cadaver decomposition islands as important refuges for threatened red-listed species, such as Necrobia violacea (Coleoptera: Cleridae). Particularly noticeable in our insect traps were the following two rare species, the "primitive" carrion beetle Necrophilus subterraneus (Coleoptera: Agyrtidae) and the false clown beetle Sphaerites glabratus (Coleoptera: Sphaeritidae). In Europe, only the species S. glabratus out of the genus Sphaerites is present. This emphasizes the importance of carrion for biodiversity conservation. We clearly show the relevance of leaving and additional providing wildlife carcasses in a dedicated place in protected forests for preserving very rare and threatened beetle species as essential members of the decomposing community. In contrast to other necromass, such as leaves, deadwood, or dung, the drivers of insect biodiversity on carcasses are still incompletely understood. For vertebrate scavengers, a richer community was shown for randomly placed carcasses, due to lower competition. Here we tested if scavenging beetles similarly show a higher diversity at randomly placed carcasses compared to easily manageable fixed places. We sampled 12,879 individuals and 92 species of scavenging beetles attracted to 17 randomly and 12 at fixed places exposed and decomposing carcasses of red deer, roe deer, and red foxes compared to control sites in a low range mountain forest. We used rarefaction-extrapolation curves along the Hill-series to weight diversity from rare to dominant species and indicator species analysis to identify differences between placement types, the decay stage, and carrion species. Beetle diversity decreased from fixed to random locations, becoming increasingly pronounced with weighting of dominant species. In addition, we found only two indicator species for exposure location type, both representative of fixed placement locations and both red listed species, namely Omosita depressa and Necrobia violacea. Furthermore, we identified three indicator species of Staphylinidae (Philonthus marginatus and Oxytelus laqueatus) and Scarabaeidae (Melinopterus prodromus) for larger carrion and one geotrupid species Anoplotrupes stercorosus for advanced decomposition stages. Our study shows that necrophilous insect diversity patterns on carcasses over decomposition follow different mechanisms than those of vertebrate scavengers with permanently established carrion islands as important habitats for a diverse and threatened insect fauna. [ABSTRACT FROM AUTHOR]

The decomposition of animal biomass (carrion) contributes to the recycling of energy and nutrients through ecosystems. Whereas the role of plant decomposition in ecosystems is broadly recognised, the significance of carrion to ecosystem functioning remains poorly understood. Quantitative data on carrion biomass are lacking and there is no clear pathway towards improved knowledge in this area. Here, we present a framework to show how quantities derived from individual carcasses can be scaled up using population metrics, allowing for comparisons among ecosystems and other forms of biomass. Our framework facilitates the generation of new data that is critical to building a quantitative understanding of the contribution of carrion to trophic processes and ecosystem stocks and flows. Knowledge of carrion biomass in ecosystems is lacking for most biomes and animal species. Quantities derived from individual carcasses can be scaled up using population- and community-level concepts, allowing for comparisons with other forms of biomass within and among ecosystems. New data on carrion biomass will be critical to building a more complete and mechanistic understanding of trophic processes, and general ecosystem stocks and flows. [ABSTRACT FROM AUTHOR]