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.

Microbial ecology of the salmon necrobiome: evidence salmon carrion decomposition influences aquatic and terrestrial insect microbiomes.

Carrion decomposition is driven by complex relationships that affect necrobiome community (i.e. all organisms and their genes associated with a dead animal) interactions, such as insect species arrival time to carrion and microbial succession. Little is understood about how microbial communities interact with invertebrates at the aquatic-terrestrial habitat interface. The first objective of the study was to characterize internal microbial communities using high-throughput sequencing of 16S rRNA gene amplicons for aquatic insects (three mayfly species) in streams with salmon carcasses compared with those in streams without salmon carcasses. The second objective was to assess the epinecrotic microbial communities of decomposing salmon carcasses (Oncorhynchus keta) compared with those of terrestrial necrophagous insects (Calliphora terraenovae larvae and adults) associated with the carcasses. There was a significant difference in the internal microbiomes of mayflies collected in salmon carcass-bearing streams and in non-carcass streams, while the developmental stage of blow flies was the governing factor in structuring necrophagous insect internal microbiota. Furthermore, the necrophagous internal microbiome was influenced by the resource on which the larvae developed, and changes in the adult microbiome varied temporally. Overall, these carrion subsidy-driven networks respond to resource pulses with bottom-up effects on consumer microbial structure, as revealed by shifting communities over space and time.

Microbial Signatures of Cadaver Gravesoil During Decomposition.

Genomic studies have estimated there are approximately 10(3)-10(6) bacterial species per gram of soil. The microbial species found in soil associated with decomposing human remains (gravesoil) have been investigated and recognized as potential molecular determinants for estimates of time since death. The nascent era of high-throughput amplicon sequencing of the conserved 16S ribosomal RNA (rRNA) gene region of gravesoil microbes is allowing research to expand beyond more subjective empirical methods used in forensic microbiology. The goal of the present study was to evaluate microbial communities and identify taxonomic signatures associated with the gravesoil human cadavers. Using 16S rRNA gene amplicon-based sequencing, soil microbial communities were surveyed from 18 cadavers placed on the surface or buried that were allowed to decompose over a range of decomposition time periods (3-303 days). Surface soil microbial communities showed a decreasing trend in taxon richness, diversity, and evenness over decomposition, while buried cadaver-soil microbial communities demonstrated increasing taxon richness, consistent diversity, and decreasing evenness. The results show that ubiquitous Proteobacteria was confirmed as the most abundant phylum in all gravesoil samples. Surface cadaver-soil communities demonstrated a decrease in Acidobacteria and an increase in Firmicutes relative abundance over decomposition, while buried soil communities were consistent in their community composition throughout decomposition. Better understanding of microbial community structure and its shifts over time may be important for advancing general knowledge of decomposition soil ecology and its potential use during forensic investigations.

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

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.

The potential use of bacterial community succession in forensics as described by high throughput metagenomic sequencing.

Decomposition studies of vertebrate remains primarily focus on data that can be seen with the naked eye, such as arthropod or vertebrate scavenger activity, with little regard for what might be occurring with the microorganism community. Here, we discuss the necrobiome, or community of organisms associated with the decomposition of remains, specifically, the "epinecrotic" bacterial community succession throughout decomposition of vertebrate carrion. Pyrosequencing was used to (1) detect and identify bacterial community abundance patterns that described discrete time points of the decomposition process and (2) identify bacterial taxa important for estimating physiological time, a time-temperature metric that is often commensurate with minimum post-mortem interval estimates, via thermal summation models. There were significant bacterial community structure differences in taxon richness and relative abundance patterns through the decomposition process at both phylum and family taxonomic classification levels. We found a significant negative linear relationship for overall phylum and family taxon richness as decomposition progressed. Additionally, we developed a statistical model using high throughput sequencing data of epinecrotic bacterial communities on vertebrate remains that explained 94.4 % of the time since placement of remains in the field, which was within 2-3 h of death. These bacteria taxa are potentially useful for estimating the minimum post-mortem interval. Lastly, we provide a new framework and standard operating procedure of how this novel approach of using high throughput metagenomic sequencing has remarkable potential as a new forensic tool. Documenting and identifying differences in bacterial communities is key to advancing knowledge of the carrion necrobiome and its applicability in forensic science.

Microbial community succession of submerged bones in an aquatic habitat.

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.

A need for null models in understanding disease transmission: the example of Mycobacterium ulcerans (Buruli ulcer disease).

Understanding the interactions of ecosystems, humans and pathogens is important for disease risk estimation. This is particularly true for neglected and newly emerging diseases where modes and efficiencies of transmission leading to epidemics are not well understood. Using a model for other emerging diseases, the neglected tropical skin disease Buruli ulcer (BU), we systematically review the literature on transmission of the etiologic agent, Mycobacterium ulcerans (MU), within a One Health/EcoHealth framework and against Hill's nine criteria and Koch's postulates for making strong inference in disease systems. Using this strong inference approach, we advocate a null hypothesis for MU transmission and other understudied disease systems. The null should be tested against alternative vector or host roles in pathogen transmission to better inform disease management. We propose a re-evaluation of what is necessary to identify and confirm hosts, reservoirs and vectors associated with environmental pathogen replication, dispersal and transmission; critically review alternative environmental sources of MU that may be important for transmission, including invertebrate and vertebrate species, plants and biofilms on aquatic substrates; and conclude with placing BU within the context of other neglected and emerging infectious diseases with intricate ecological relationships that lead to disease in humans, wildlife and domestic animals.

Rehydration of forensically important larval Diptera specimens.

Established procedures for collecting and preserving evidence are essential for all forensic disciplines to be accepted in court and by the forensic community at large. Entomological evidence, such as Diptera larvae, are primarily preserved in ethanol, which can evaporate over time, resulting in the dehydration of specimens. In this study, methods used for rehydrating specimens were compared. The changes in larval specimens with respect to larval length and weight for three forensically important blow fly (Diptera: Calliphoridae) species in North America were quantified. Phormia regina (Meigen), Cochliomyia macellaria (F.), and Chrysomya rufifacies (Macquart) third-instar larvae were collected from various decomposing animals and preserved with three preservation methods (80% ethanol, 70% isopropyl alcohol, and hot-water kill then 80% ethanol). Preservative solutions were allowed to evaporate. Rehydration was attempted with either of the following: 80% ethanol, commercial trisodium phosphate substitute solution, or 0.5% trisodium phosphate solution. All three methods partially restored weight and length of specimens recorded before preservation. Analysis of variance results indicated that effects of preservation, rehydration treatment, and collection animal were different in each species. The interaction between preservative method and rehydration treatment had a significant effect on both P. regina and C. macellaria larval length and weight. In addition, there was a significant interaction effect of collection animal on larval C. macellaria measurements. No significant effect was observed in C. rufifacies larval length or weight among the preservatives or treatments. These methods could be used to establish a standard operating procedure for dealing with dehydrated larval specimens in forensic investigations.

Adult Blow Fly (Diptera: Calliphoridae) Community Structure Across Urban-Rural Landscapes in Michigan, United States.

Necrophagous insects play an important role in the decomposition of vertebrate carrion. The documented colonization, development, and succession of blow flies (Diptera: Calliphoridae) and other arthropods on decomposing carcasses make their communities relevant for use in decomposition ecology and forensic investigations. This relevance relies on the local pool of species available to colonize a carcass, but such community level survey data are not always available. The objective of this research was to conduct a baseline survey of adult Calliphoridae communities from urban-rural land use types in the Great Lakes region. To test how adult blow fly distribution varies with changing landscape in Mid-Michigan, sampling with baited jars and hanging traps was implemented over the summer months of June, July, and August 2017. To determine how blow fly communities differed in urban to rural land cover, seven cities were selected with site locations ranging from high intensity developed areas to cultivated crop fields. Over 97,000 individual flies were captured represented by 11 Calliphoridae species. The adult Calliphoridae communities were primarily structured by land use type and month of collection, with these two factors interacting, suggesting that the effect of location varied by time of year. The two most abundant species, Phormia regina (Meigen) and Lucilia sericata (Meigen), cumulatively comprised 88.5% adult flies from Mid-Michigan. These findings provide a baseline database of Great Lakes Calliphoridae, with potential use in forensic research and casework.

Dysbiosis in the Dead: Human Postmortem Microbiome Beta-Dispersion as an Indicator of Manner and Cause of Death.

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.

Characterizing the microbiome of ectoparasitic louse flies feeding on migratory raptors.

Louse flies (Diptera: Hippoboscidae) are obligate ectoparasites that often cause behavioral, pathogenic, and evolutionary effects on their hosts. Interactions between ectoparasites and avian hosts, especially migrating taxa, may influence avian pathogen spread in tropical and temperate ecosystems and affect long-term survival, fitness and reproductive success. The purpose of this study was to characterize the vector-associated microbiome of ectoparasitic louse flies feeding on migrating raptors over the fall migration period. Surveys for louse flies occurred during fall migration (2015-2016) at a banding station in Pennsylvania, United States; flies were collected from seven species of migrating raptors, and we sequenced their microbial (bacteria and archaea) composition using high-throughput targeted amplicon sequencing of the 16S rRNA gene (V4 region). All louse flies collected belonged to the same species, Icosta americana. Our analysis revealed no difference in bacterial communities of louse flies retrieved from different avian host species. The louse fly microbiome was dominated by a primary endosymbiont, suggesting that louse flies maintain a core microbial structure despite receiving blood meals from different host species. Thus, our findings highlight the importance of characterizing both beneficial and potentially pathogenic endosymbionts when interpreting how vector-associated microbiomes may impact insect vectors and their avian hosts.

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.

Detection of critical antibiotic resistance genes through routine microbiome surveillance.

Population-based public health data on antibiotic resistance gene carriage is poorly surveyed. Research of the human microbiome as an antibiotic resistance reservoir has primarily focused on gut associated microbial communities, but data have shown more widespread microbial colonization across organs than originally believed, with organs previously considered as sterile being colonized. Our study demonstrates the utility of postmortem microbiome sampling during routine autopsy as a method to survey antibiotic resistance carriage in a general population. Postmortem microbial sampling detected pathogens of public health concern including genes for multidrug efflux pumps, carbapenem, methicillin, vancomycin, and polymixin resistances. Results suggest that postmortem assessments of host-associated microbial communities are useful in acquiring community specific data while reducing selective-participant biases.

Machine learning performance in a microbial molecular autopsy context: A cross-sectional postmortem human population study.

BACKGROUND: The postmortem microbiome can provide valuable information to a death investigation and to the human health of the once living. Microbiome sequencing produces, in general, large multi-dimensional datasets that can be difficult to analyze and interpret. Machine learning methods can be useful in overcoming this analytical challenge. However, different methods employ distinct strategies to handle complex datasets. It is unclear whether one method is more appropriate than others for modeling postmortem microbiomes and their ability to predict attributes of interest in death investigations, which require understanding of how the microbial communities change after death and may represent those of the once living host. METHODS AND FINDINGS: Postmortem microbiomes were collected by swabbing five anatomical areas during routine death investigation, sequenced and analyzed from 188 death cases. Three machine learning methods (boosted algorithms, random forests, and neural networks) were compared with respect to their abilities to predict case attributes: postmortem interval (PMI), location of death, and manner of death. Accuracy depended on the method used, the numbers of anatomical areas analyzed, and the predicted attribute of death. CONCLUSIONS: All algorithms performed well but with distinct features to their performance. Xgboost often produced the most accurate predictions but may also be more prone to overfitting. Random forest was the most stable across predictions that included more anatomic areas. Analysis of postmortem microbiota from more than three anatomic areas appears to yield limited returns on accuracy, with the eyes and rectum providing the most useful information correlating with circumstances of death in most cases for this dataset.

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.

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.

Towards Quantifying Carrion Biomass in Ecosystems.

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.

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.

The potential use of cuticular hydrocarbons and multivariate analysis to age empty puparial cases of Calliphora vicina and Lucilia sericata.

Cuticular hydrocarbons (CHC) have been successfully used in the field of forensic entomology for identifying and ageing forensically important blowfly species, primarily in the larval stages. However in older scenes where all other entomological evidence is no longer present, Calliphoridae puparial cases can often be all that remains and therefore being able to establish the age could give an indication of the PMI. This paper examined the CHCs present in the lipid wax layer of insects, to determine the age of the cases over a period of nine months. The two forensically important species examined were Calliphora vicina and Lucilia sericata. The hydrocarbons were chemically extracted and analysed using Gas Chromatography - Mass Spectrometry. Statistical analysis was then applied in the form of non-metric multidimensional scaling analysis (NMDS), permutational multivariate analysis of variance (PERMANOVA) and random forest models. This study was successful in determining age differences within the empty cases, which to date, has not been establish by any other technique.

Frozen: Thawing and Its Effect on the Postmortem Microbiome in Two Pediatric Cases.

Previous postmortem microbiome studies have focused on characterizing taxa turnover during an undisturbed decomposition process. How coexisting conditions (e.g., frozen, buried, burned) affect the human microbiome at the time of discovery is less well understood. Microbiome data were collected from two pediatric cases at the Wayne County Medical Examiner in Michigan. The bodies were found frozen, hidden in a freezer for an extended time. Microbial communities were sampled from six external anatomic locations at three time points during the thawing process, prior to autopsy. The 16S rRNA V4 gene amplicon region was sequenced using high-throughput sequencing (Illumina MiSeq). Microbial diversity increased, and there was a distinct shift in microbial community structure and abundance throughout the thawing process. Overall, these data demonstrate that the postmortem human microbiome changes during the thawing process, and have important forensic implications when bodies have been substantially altered, modified, and concealed after death.