Microbial Biofilm Community Variation in Flowing Habitats: Potential Utility as Bioindicators of Postmortem Submersion Intervals

Biofilms are a ubiquitous formation of microbial communities found on surfaces in aqueous environments. These structures have been investigated as biomonitoring indicators for stream heath, and here were used for the potential use in forensic sciences. Biofilm successional development has been proposed as a method to determine the postmortem submersion interval (PMSI) of remains because there are no standard methods for estimating the PMSI and biofilms are ubiquitous in aquatic habitats. We sought to compare the development of epinecrotic (biofilms on Sus scrofa domesticus carcasses) and epilithic (biofilms on unglazed ceramic tiles) communities in two small streams using bacterial automated ribosomal intergenic spacer analysis. Epinecrotic communities were significantly different from epilithic communities even though environmental factors associated with each stream location also had a significant influence on biofilm structure. All communities at both locations exhibited significant succession suggesting that changing communities throughout time is a general characteristic of stream biofilm communities. The implications resulting from this work are that epinecrotic communities have distinctive shifts at the first and second weeks, and therefore the potential to be used in forensic applications by associating successional changes with submersion time to estimate a PMSI. The influence of environmental factors, however, indicates the lack of a successional pattern with the same organisms and a focus on functional diversity may be more applicable in a forensic context

Temporal and Spatial Impact of Human Cadaver Decomposition on Soil Bacterial and Arthropod Community Structure and Function

As vertebrate carrion decomposes, there is a release of nutrient-rich fluids into theunderlying soil, which can impact associated biological community structure andfunction. How these changes alter soil biogeochemical cycles is relatively unknown and may prove useful in the identification of carrion decomposition islands that have long lasting, focal ecological effects. This study investigated the spatial (0, 1, and 5 m) and temporal (3–732 days) dynamics of human cadaver decomposition on soil bacterial and arthropod community structure and microbial function. We observed strong evidence of a predictable response to cadaver decomposition that varies over space for soil bacterial and arthropod community structure, carbon (C) mineralization and microbial substrate utilization patterns. In the presence of a cadaver (i.e., 0 m samples), the relative abundance of Bacteroidetes and Firmicutes was greater, while the relative abundance of Acidobacteria, Chloroflexi, Gemmatimonadetes, and Verrucomicrobia was lower when compared to samples at 1 and 5 m. Micro-arthropods were more abundant (15 to 17-fold) in soils collected at 0 m compared to either 1 or 5 m, but overall, micro-arthropod community composition was unrelated to either bacterial community composition or function. Bacterial community structure and microbial function also exhibited temporal relationships, whereas arthropod community structure did not. Cumulative precipitation was more effective in predicting temporal variations in bacterial abundance and microbial activity than accumulated degree days. In the presence of the cadaver (i.e., 0 m samples), the relative abundance of Actinobacteria increased significantly with cumulative precipitation. Furthermore, soil bacterial communities and C mineralization were sensitive to the introduction of human cadavers as they diverged from baseline levels and did not recover completely in approximately 2 years. These data are valuable for understanding ecosystem function surrounding carrion decomposition islands and can be applicable to environmental bio-monitoring and forensic sciences

Evaluating Bioinformatic Pipeline Performance for Forensic Microbiome Analysis*,†,‡

Microbial communities have potential evidential utility for forensic applications. However, bioinformatic analysis of high‐throughput sequencing data varies widely among laboratories. These differences can potentially affect microbial community composition and downstream analyses. To illustrate the importance of standardizing methodology, we compared analyses of postmortem microbiome samples using several bioinformatic pipelines, varying minimum library size or minimum number of sequences per sample, and sample size. Using the same input sequence data, we found that three open‐source bioinformatic pipelines, MG‐RAST, mothur, and QIIME2, had significant differences in relative abundance, alpha‐diversity, and beta‐diversity, despite the same input data. Increasing minimum library size and sample size increased the number of low‐abundant and infrequent taxa detected. Our results show that bioinformatic pipeline and parameter choice affect results in important ways. Given the growing potential application of forensic microbiology to the criminal justice system, continued research on standardizing computational methodology will be important for downstream applications.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154468/1/jfo14213_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154468/2/jfo14213.pd

NECROBIOME FRAMEWORK FOR BRIDGING DECOMPOSITION ECOLOGY OF AUTOTROPHICALLY AND HETEROTROPHICALLY DERIVED ORGANIC MATTER

Life arises from death through species that decompose dead biomass or necromass. This paper provides a synthesis of the species responsible for dead plant and animal decomposition and describes a conceptual perspective—the “necrobiome”— that defines the diverse and complex communities that interact to recycle necromass. The concept brings unification to the previously disparate fields of plant and animal decomposition by discussing the universal processes occurring across all forms of necromass. It highlights the factors that make each form of dead biomass different in a way that defines how unique necrobiomes drive decomposition and ultimately shape ecosystem structure and function

Necrobiome framework for bridging decomposition ecology of autotrophically and heterotrophically derived organic matter

Decomposition contributes to global ecosystem function by contributing to nutrient recycling, energy flow, and limiting biomass accumulation. The decomposer organisms influencing this process form diverse, complex, and highly dynamic communities that often specialize on different plant or animal resources. Despite performing the same net role, there is a need to conceptually synthesize information on the structure and function of decomposer communities across the spectrum of dead plant and animal resources. A lack of synthesis has limited cross-disciplinary learning and research in important areas of ecosystem and community ecology. Here we expound on the “necrobiome” concept and develop a framework describing the decomposer communities and their interactions associated with plant and animal resource types within multiple ecosystems.We outline the biotic structure and ecological functions of the necrobiome, along with how the necrobiome fits into a broader landscape and ecosystem context. The expanded necrobiome model provides a set of perspectives on decomposer communities across resource types, and conceptually unifies plant and animal decomposer communities into the same framework, while acknowledging key differences in processes and mechanisms. This framework is intended to raise awareness among researchers, and advance the construction of explicit, mechanistic hypotheses that further our understanding of decomposer community contributions to biodiversity, the structure and function of ecosystems, global nutrient recycling and energy flow

Total RNA Analysis of Bacterial Community Structural and Functional Shifts Throughout Vertebrate Decomposition

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.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152033/1/jfo14083_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/152033/2/jfo14083.pd

Microbial Community Functional Change during Vertebrate Carrion Decomposition

Microorganisms play a critical role in the decomposition of organic matter, which contributes to energy and nutrient transformation in every ecosystem. Yet, little is known about the functional activity of epinecrotic microbial communities associated with carrion. The objective of this study was to provide a description of the carrion associated microbial community functional activity using differential carbon source use throughout decomposition over seasons, between years and when microbial communities were isolated from eukaryotic colonizers (e.g., necrophagous insects). Additionally, microbial communities were identified at the phyletic level using high throughput sequencing during a single study. We hypothesized that carrion microbial community functional profiles would change over the duration of decomposition, and that this change would depend on season, year and presence of necrophagous insect colonization. Biolog EcoPlates™ were used to measure the variation in epinecrotic microbial community function by the differential use of 29 carbon sources throughout vertebrate carrion decomposition. Pyrosequencing was used to describe the bacterial community composition in one experiment to identify key phyla associated with community functional changes. Overall, microbial functional activity increased throughout decomposition in spring, summer and winter while it decreased in autumn. Additionally, microbial functional activity was higher in 2011 when necrophagous arthropod colonizer effects were tested. There were inconsistent trends in the microbial function of communities isolated from remains colonized by necrophagous insects between 2010 and 2011, suggesting a greater need for a mechanistic understanding of the process. These data indicate that functional analyses can be implemented in carrion studies and will be important in understanding the influence of microbial communities on an essential ecosystem process, carrion decomposition

Bacterial Community Succession, Transmigration, and Differential Gene Transcription in a Controlled Vertebrate Decomposition Model

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

Hydrocarbon profiles throughout adult Calliphoridae aging: A promising tool for forensic entomology

Blow flies (Diptera: Calliphoridae) are typically the first insects to arrive at human remains and carrion. Predictable succession patterns and known larval development of necrophagous insects on vertebrate remains can assist a forensic entomologist with estimates of a minimum post-mortem interval (PMImin) range. However, adult blow flies are infrequently used to estimate the PMImin, but rather are used for a confirmation of larval species identification. Cuticular hydrocarbons have demonstrated potential for estimating adult blow fly age, as hydrocarbons are present throughout blow fly development, from egg to adult, and are stable structures. The goal of this study was to identify hydrocarbon profiles associated with the adults of a North American native blow fly species, Cochliomyia macellaria (Fabricius) and a North American invasive species, Chrysomya rufifacies (Macquart). Flies were reared at a constant temperature (25 °C), a photoperiod of 14:10 (L:D) (h), and were provided water, sugar and powdered milk ad libitum. Ten adult females from each species were collected at day 1, 5, 10, 20, and 30 post-emergence. Hydrocarbon compounds were extracted and then identified using gas chromatography–mass spectrometry (GC–MS) analysis. A total of 37 and 35 compounds were detected from C. macellaria and Ch. rufifacies, respectively. There were 24 and 23 n-alkene and methyl-branched alkane hydrocarbons from C. macellaria and Ch. rufifacies, respectively (10 compounds were shared between species), used for statistical analysis. Non-metric multidimensional scaling analysis and permutational multivariate analysis of variance were used to analyze the hydrocarbon profiles with significant differences (P < 0.001) detected among post-emergence age cohorts for each species, and unique hydrocarbon profiles detected as each adult blow fly species aged. This work provides empirical data that serve as a foundation for future research into improving PMImin estimates made by forensic practitioners and potentially increase the use of adult insects during death investigations

Necrophilous Insect Dynamics at Small Vertebrate Carrion in a Temperate Eucalypt Woodland

Insects associated with carrion are critical to the decomposition process and nutrient cycling in ecosystems. Yet the communities of insects associated with carrion vary between locations, and detailed case studies are necessary for identifying differences and similarities among contrasting habitats. In this study, we examined temporal changes in the crawling insect community collected from rabbit carcasses placed in contrasting grassland and tree habitats in southeastern Australia. We collected 18,400 adult insects, including 22 species of fly, 57 species of beetle, and 37 species of ant. We found significant effects of habitat type and time, but not their interaction, on the composition of the entire insect community. Several ant species showed early and rapid colonization and highest abundances during early stages of decay, including Iridomyrmex purpureus (Smith, 1858) under trees, and Iridomyrmex rufoniger (Lowne, 1865) and Rhytidoponera metallica (Smith, 1858) in grassland. We found that most fly species showed highest abundance during active decay, but Chrysomya varipes (Macquart 1851) was more abundant under trees than in grassland during this time. Beetles peaked during active or advanced decay stages, with Saprinus and Omorgus the most abundant genera. Our study demonstrates that strong replication of contrasting environmental treatments can reveal new information on habitat preferences of important carrion insect species. The numerical dominance of ants early in decomposition has implications for insect community structure via potential competitive interactions with flies, and should be more rigorously examined in future carrion studies.This project was funded by an Australian Research Council grant to Philip Barton (DE150100026)