Jennifer L. Pechal

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.

Fluorescently labeled bacteria provide insight on post-mortem microbial transmigration

Microbially mediated mechanisms of human decomposition begin immediately after death, and are a driving force for the conversion of a once living organism to a resource of energy and nutrients. Little is known about post-mortem microbiology in cadavers, particularly the community structure of microflora residing within the cadaver and the dynamics of these communities during decomposition. Recent work suggests these bacterial communities undergo taxa turnover and shifts in community composition throughout the post-mortem interval. In this paper we describe how the microbiome of a living host changes and transmigrates within the body after death thus linking the microbiome of a living individual to post-mortem microbiome changes. These differences in the human post-mortem from the ante-mortem microbiome have demonstrated promise as evidence in death investigations. We investigated the post-mortem structure and function dynamics of Staphylococcus aureus and Clostridium perfringens after intranasal inoculation in the animal model Mus musculus L. (mouse) to identify how transmigration of bacterial species can potentially aid in post-mortem interval estimations. S. aureus was tracked using in vivo and in vitro imaging to determine colonization routes associated with different physiological events of host decomposition, while C. perfringens was tracked using culture-based techniques. Samples were collected at discrete time intervals associated with various physiological events and host decomposition beginning at 1h and ending at 60 days post-mortem. Results suggest that S. aureus reaches its highest concentration at 5-7 days post-mortem then begins to rapidly decrease and is undetectable by culture on day 30. The ability to track these organisms as they move in to once considered sterile space may be useful for sampling during autopsy to aid in determining post-mortem interval range estimations, cause of death, and origins associated with the geographic location of human remains during death investigations.

The Dynamic Maggot Mass Microbiome

Abstract 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 (N = 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 coeruleiviridis (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 over time.

Field Documentation of Unusual Post-Mortem Arthropod Activity on Human Remains.

ABSTRACT During a forensic investigation, the presence of physical marks on human remains can influence the interpretation of events related to the death of an individual. Some tissue injury on human remains can be misinterpreted as ante- or peri-mortem wounds by an investigator when in reality the markings resulted from post-mortem arthropod activity. Unusual entomological data were collected during a study examining the decomposition of a set of human remains in San Marcos, Texas. An adult female Pediodectes haldemani (Girard) (Orthoptera: Tettigoniidae) and an Armadillidium cf. vulgare (Isopoda: Armadilidiidae) were documented feeding on the remains. Both arthropods produced physical marks or artifacts on the remains that could be misinterpreted as attack, abuse, neglect, or torture. Additionally, red imported fire ants, Solenopsis invicta Buren (Hymenoptera: Formicidae), were observed constructing structures in the mark produced by the P. haldemani feeding. These observations provide insight into the potential of post-mortem arthropod damage to human remains, which previously had not been described for these taxa, and therefore, physical artifacts on any remains found in similar circumstances may result from arthropod activity and not ante- or peri-mortem wounds.

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

Abstract 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.

Post-Colonization Interval Estimates Using Multi-Species Calliphoridae Larval Masses and Spatially Distinct Temperature Data Sets: A Case Study

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.

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 the underlying soil, which can impact associated biological community structure and function. 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.

A large-scale survey of the postmortem human microbiome, and its potential to provide insight into the living health condition

The microbiome plays many roles in human health, often through the exclusive lens of clinical interest. The inevitable end point for all living hosts, death, has its own altered microbiome configurations. However, little is understood about the ecology and changes of microbial communities after death, or their potential utility for understanding the health condition of the recently living. Here we reveal distinct postmortem microbiomes of human hosts from a large-scale survey of death cases representing a predominantly urban population, and demonstrated these microbiomes reflected antemortem health conditions within 24–48 hours of death. Our results characterized microbial community structure and predicted function from 188 cases representing a cross-section of an industrial-urban population. We found strong niche differentiation of anatomic habitat and microbial community turnover based on topographical distribution. Microbial community stability was documented up to two days after death. Additionally, we observed a positive relationship between cell motility and time since host death. Interestingly, we discovered evidence that microbial biodiversity is a predictor of antemortem host health condition (e.g., heart disease). These findings improve the understanding of postmortem host microbiota dynamics, and provide a robust dataset to test the postmortem microbiome as a tool for assessing health conditions in living populations.

Interkingdom Community Interactions in Disease Ecology

A key tenet of community ecology is the interactions of individual organisms contribute to the ecological structure and function of ecosystems. Within these networks of interacting organisms are those taxa important for human and animal health: disease systems defined by combinations of host, pathogen, reservoir, and vector or a subset of these components. While the simplest disease system is that of the host and pathogen, more complex systems include the direct interactions of a pathogen with other hosts and the microbial communities of those hosts, reservoirs, and sometimes vectors. Each of these disease system components is made up of species that directly and indirectly interact with other species in ways that affect their individual fitness, population biology, and role in communities of the ecosystem. This chapter recognizes the direct interactions of those species that make up the primary components of disease systems; however, the focus and examples provided relate to the more indirect interkingdom (or domain) interactions that impact disease system components. The examples provided include how microbial communities mediate invertebrate and vertebrate fitness and behavior, often in systems where the hosts play important roles in pathogen transmission and disease emergence. The potential mechanisms of these interkingdom interactions are also developed in detail, as the mechanisms of such interactions are likely the target of future studies that could directly inform disease management strategies. Based on these examples and mechanisms, the existing literature suggests there are likely undiscovered and complex interactions of species within communities that affect disease systems.

Changes in Larval Mosquito Microbiota Reveal Non-target Effects of Insecticide Treatments in Hurricane-Created Habitats

Ephemeral aquatic habitats and their associated microbial communities (microbiomes) play important roles in the growth and development of numerous aquatic insects, including mosquitoes (Diptera). Biological control agents, such as Bacillus thuringiensis israelensis (Bti) or insect growth regulators (e.g., methoprene), are commonly used to control mosquitoes in these habitats. However, it is unknown how commonly used control compounds affect the mosquito internal microbiome and potentially alter their life history traits. The objectives of this study were threefold: characterize the internal microbiota of Aedes larvae (Culicidae) in ephemeral forested mosquito habitat using high-throughput amplicon based sequencing, assess how mosquito control treatments affect the internal microbial communities of larval mosquitoes, and determine if changes to the microbiome resulted from direct or indirect treatment effects. The larval microbiome varied in community composition and diversity with development stage and treatment, suggesting potential effects of control compounds on insect microbial ecology. While microbial community differences due to Bti treatment were a result of indirect effects on larval development, methoprene had significant impacts on bacterial and algal taxa that could not be explained by indirect treatment effects. These results provide new information on the interactions between pesticide treatments and insect microbial communities.