The Potential of High-throughput Metagenomic Sequencing of Aquatic Bacterial Communities to Estimate the Postmortem Submersion Interval

Human remains can be discovered in freshwater or marine ecosystems, circumstances where insects and other invertebrates have infrequently been used for understanding the time of postmortem submersion. In this study, the identification and succession of epinecrotic bacterial communities on vertebrate remains were described during decomposition in a temperate headwater stream during two seasons (summer and winter). Bacterial communities were characterized with 454 pyrosequencing and analyzed at phyletic and generic taxonomic resolutions. There was a significant increase in genera richness over decomposition during both seasons. Additionally, multivariate statistical modeling revealed significant differences in bacterial communities between seasons at both taxonomic resolutions and siginificant genera differences among sampling days within each season, suggesting a succession of these communities. These data are the first to describe aquatic bacterial succession using high-throughput metagenomic sequencing on vertebrate remains submerged in a freshwater habitat, and provide initial evidence for their potential use in forensic investigations.     

The potential to determine a postmortem submersion interval based on algal/diatom diversity on decomposing mammalian carcasses in brackish ponds in Delaware

Recently botanical evidence has been studied to determine if it is useful in forensic investigations. This study was performed to examine stillborn piglet decomposition in a brackish water environment and to semi-quantitatively document stages of decomposition, degree day accumulation per stage as well as the algal/diatom diversity useful in determining a postmortem submersion interval (PMSI). Piglets and ceramic tiles were submerged in brackish ponds and sampled on a regular basis to document algal diversity and succession between substrates and stages of decomposition. Significantly greater weight was lost from piglet carcasses during the early floating and advanced floating decay stages. Seasonal effects were observed in degree-day accumulations. Diatom diversity was significantly greater on piglet carcasses compared to tile substrates. Algal diversity decreased over time on the piglet carcasses as well as the stage of decomposition. A significant relationship and strong correlation between algal diversity found on the piglet substrate with time was observed. Our results indicate that more research is needed to examine the potential to use diatoms in not only determining manner of death but also the duration of time (PMSI) a victim may have been immersed in an aquatic environment.     

Pig decomposition in lotic aquatic systems: the potential use of algal growth in establishing a postmortem submersion interval (PMSI)

While algal community composition has been examined as a qualitative indicator of postmortem submersion interval (PMSI), there have been no quantitative studies on using algal growth rates as PMSI estimators. The present study was undertaken to examine pig decomposition in streams and to develop a more quantitative approach to estimate a PMSI. Pigs and ceramic tiles were completely submerged and regularly sampled for periphyton growth. Five stages of decomposition were identified for the submerged pig carcasses according to physical characteristics. Algal growth rates, measured quantitatively as a function of chlorophyll-a concentration, were greater on pigs compared with tiles; however, microhabitat (pools versus riffles) did not significantly influence algal growth. Additionally, there was a strong correlation between algal growth rate and time on pigs and tile substrates. This strong correlation was observed after significant rain events. Our study documents for the first time a quantitative technique to determine the length of time a corpse has been submerged in water. We suggest that algal growth rates may be a useful quantitative indicator in criminal investigations involving corpses that are completely submerged in stream or riverine habitats.     

Comparison of Protocols for Measuring and Calculating Postmortem Submersion Intervals for Human Analogs in Fresh Water

Protocols for determining postmortem submersion interval (PMSI) have long been problematic for forensic investigators due to the wide variety of factors affecting the rate of decomposition of submerged carrion. Likewise, it has been equally problematic for researchers to develop standardized experimental protocols to monitor underwater decomposition without artificially affecting the decomposition rate. This study compares two experimental protocols: (i) underwater in situ evaluation with photographic documentation utilizing the Heaton et al. total aquatic decomposition (TAD) score and (ii) weighing the carrion before and after submersion. Complete forensic necropsies were performed as a control. Perinatal piglets were used as human analogs. The results of this study indicate that in order to objectively measure decomposition over time, the human analog should be examined at depth using the TAD scoring system rather than utilizing a carrion weight evaluation. The acquired TAD score can be used to calculate an approximate PMSI.     

Caddisflies assist with homicide case: determining a postmortem submersion interval using aquatic insects

Although few indicators of time since death for corpses found in aquatic ecosystems are comparable in precision to the insect indicators used in terrestrial cases, there are observations that can be useful in suggesting or ruling out an approximate PMSI (postmortem submersion interval). For example, the time intervals required for certain growth phases of aquatic insects, such as caddisflies, that may attach themselves to the submerged remains can be used to estimate a minimum PMSI. Approximately 8 of the 13 orders of insects containing species with aquatic or semi-aquatic stages are likely to be associated with carrion or corpses in aquatic habitats. We present a case study in which portions of a body from an adult male were discovered in a south central Michigan stream. The body was dismembered and portions were recovered from two bags floating and submerged in the stream. Insect specimens collected from mesh and plastic bags consisted of one fly larva belonging to the family Muscidae, and caddisfly larvae belonging to two families: the Limnephilidae. (case-makers) and the Hydropsychidae, (net spinners). We used unique case-building behaviors of the limnephilid caddisflies found on the remains to elucidate a PMSI range consistent with the disappearance of the victim. It is important for forensic investigators to understand that although some precision is lost in estimating a PMSI with aquatic insects, these organisms should not be ignored in gathering evidence from aquatic crime scenes, and in fact, they can provide valuable details in estimating a PMSI.     

Assessment of post-mortem submersion interval using total aquatic decomposition scores of drowned human cadavers

Investigating drowning-related deaths remains a significant problem for forensic personnel all over the world. The previously published decomposition scoring method like the total aquatic decomposition (TAD) score promises to estimate the correct post-mortem submersion interval (PMSI) in aquatic habitats through the assessment and calculation of the decomposition rate and accumulated degree days (ADD). The current study comprised of 53 drowned death cases belonging to various districts of Haryana from May 2016 to August 2017. The regression and Pearson's correlation indicated a significant correlation between the TAD scores and the actual ADD (calculated through water temperatures) (r² = 0.917) and between the actual and the estimated ADDs (calculated through TAD scores used by Heaton et al. [21]) (r = 0.9585). The results indicated that the estimated ADD tends to over predict the PMSI compared to the actual ADD. It is further confirmed by paired t-test, which showed the mean of actual ADD (mean = 349) to be significantly lower than the mean of estimated ADD (mean = 663). Moreover, these methods will help forensic investigators and researchers formulate region-specific regression equations for PMSI estimation.     

Variation in decomposition stages and carrion insect succession in a dry tropical climate and its effect on estimating postmortem interval

Insects have an important role in minimum postmortem interval (PMI_min) estimation. An accurate PMI_min estimation relies on a comprehensive study of the development and succession of local carrion insects. No published research on carrion insect succession exists for tropical north Queensland. To address this, we aimed to obtain preliminary observational data concerning the rate of decomposition and insect succession on pig carcasses in Townsville and compare these with other regions of Australia and overseas. Adult insects were collected daily from three pig carcasses for 30 d during summer and identified to family level. Observations of decomposition rate were made each day and progression through the stages of decomposition were recorded. Adult insects were identified to family and their presence/absence used as a proxy for arrival at/departure from the remains, respectively. These preliminary data highlight several interesting trends that may be informative for forensic PMI_min estimation. Decomposition was rapid: all carcasses were at the dry/remains stage by Day 5, which was substantially quicker than all other regions in the comparison. Differences were also observed in the presence/absence of insect families and their arrival and departure times. Given the rapid progression through early decomposition, we argue that later-arriving coleopteran taxa may be more forensically informative in tropical Australia, in contrast with temperate regions where Diptera appear most useful. This research contributes preliminary observational data to understanding insect succession patterns in tropical Australia and demonstrates the critical need for comprehensive local succession data for each climatic region of Australia to enable accurate PMI_min estimation. These data will inform future research targeted at gaining a more comprehensive understanding of insect succession in the Australian tropics.

Key points:

• We obtained preliminary observational data concerning the rate of decomposition and insect succession on pig carcasses in tropical Australia.
• Decomposition was rapid: all carcasses were at the dry/remains stage by Day 5.
• Coleopteran taxa may be more forensically informative in tropical Australia than dipterans.

     

Soft Tissue Decomposition of Submerged,Dismembered Pig Limbs Enclosed in Plastic Bags*

Abstract: This study examines underwater soft tissue decomposition of dismembered pig limbs deposited in polyethylene plastic bags. The research evaluates the level of influence that disposal method has on underwater decomposition processes and details observations specific to this scenario. To our knowledge, no other study has yet investigated decomposing, dismembered, and enclosed remains in water environments. The total sample size consisted of 120 dismembered pig limbs, divided into a subsample of 30 pig limbs per recovery period (34 and 71 days) for each treatment. The two treatments simulated non‐enclosed and plastic enclosed disposal methods in a water context. The remains were completely submerged in Lake Ontario for 34 and 71 days. In both recovery periods, the non‐enclosed samples lost soft tissue to a significantly greater extent than their plastic enclosed counterparts. Disposal of remains in plastic bags therefore results in preservation, most likely caused by bacterial inhibition and reduced oxygen levels.     

Potential Use of Bacterial Community Succession in Decaying Human Bone for Estimating Postmortem Interval,,

Bacteria are taphonomic agents of human decomposition, potentially useful for estimating postmortem interval (PMI) in late‐stage decomposition. Bone samples from 12 individuals and three soil samples were analyzed to assess the effects of decomposition and advancing time on bacterial communities. Results indicated that partially skeletonized remains maintained a presence of bacteria associated with the human gut, whereas bacterial composition of dry skeletal remains maintained a community profile similar to soil communities. Variation in the UniFrac distances was significantly greater between groups than within groups (p < 0.001) for the unweighted metric and not the weighted metric. The members of the bacterial communities were more similar within than between decomposition stages. The oligotrophic environment of bone relative to soft tissue and the physical protection of organic substrates may preclude bacterial blooms during the first years of skeletonization. Therefore, community membership (unweighted) may be better for estimating PMI from skeletonized remains than community structure (weighted).     

Detection of marine and freshwater bacterioplankton in immersed victims: Post-mortem bacterial invasion does not readily occur

We previously applied our method of detecting marine or freshwater bacterioplankton (bacteria) in the blood of immersed victims as a marker of drowning. However, we did not confirm the absence of post-mortem bacterial invasion during immersion. Here we examined the nature of bacterioplankton in blood samples from 21 immersed and 4 non-immersed cadavers. We found only freshwater bacterioplankton in the blood of two victims that were retrieved from the sea or an estuary inhabited by marine bacterioplankton even though one victim was highly putrefied. The results of diatom testing suggested that these two victims had drowned in fresh or brackish water with low salinity and then flowed out to the estuary or the sea. Two others were submerged in water, but representative bacterioplankton were undetectable in their blood although one victim was highly putrefied. Autopsy findings and the results of diatom tests did not indicate that the cause of death was drowning. As in previous studies, we identified freshwater bacterioplankton in the blood of seven other victims that had drowned in freshwater, marine bacterioplankton in the blood of four victims that had drowned in seawater and none in four victims found on land that had died by means other than drowning. Bacterioplankton in the blood of drowned victims appears to reflect the type of water aspirated and blood does not become easily contaminated with bacteria post-mortem even in decomposed bodies.     

论研究船舶污染事故中船员犯罪问题的意义

海洋环境污染已成为国际社会广泛关注的问题.基于海洋对于人类的重要性和海洋环境污染的严重程度,国际社会意识到要为海洋建立一种法律秩序,以便利国际交通和促进海洋的和平用途,海洋资源的公平而有效的利用,海洋生物资源的养护以及研究、保护和保全海洋环境. 这种法律秩序体系庞大,涉及到海洋开发、利用和保护的方方面面.海洋环境污染事故发生后有关行为人犯罪及刑事责任的问题,也当然被包含在这个法律秩序当中.分析研究船舶污染事故中船员犯罪问题的重要意义.     

A preliminary investigation of insect colonisation and succession on remains in New Zealand

A preliminary, systematic field study on the process of decomposition and associated insects was conducted, for the first time, in New Zealand. Using pig carcasses as an animal model for human decomposition, insect colonisation and succession was monitored in three different habitats in the Auckland region where remains are likely to be found. A significant difference in the rates of decomposition was found among the three different habitats of an open field, coastal sand dune area and native bush during the autumn/winter season. The primary colonisers of all carcasses were Calliphora stygia Fabricius (Calliphoridae), Chrysomya rufifacies Macquart (Calliphoridae) and Hydrotaea rostrata Robineau-Desvoidy (Muscidae). Two species were identified as possible representatives of the habitats in which they were found; Fannia sp. (Fanniidae) in the open field habitat and Calliphora hilli Patton (Calliphoridae) in the native bush habitat. Also identified was Sylvicola sp. (Anisopodidae) as a possible indicator of damp habitats as well as a likely indicator of a longer postmortem interval. This preliminary investigation presents a broad outline of the insects associated with remains and the order in which they appear in the Auckland region.     

The Marine Bill: An Overview and Some Thoughts

This paper examines the Marine and Coastal Access Bill as it was tabled on 8th December 2008. It explores the failure of the Bill to include marine protection of over 3,000,000 km² of the waters associated with overseas territories and Crown dependencies. It explains the necessity for close liaison with the European Commission and with the devolved administrations in Scotland, Northern Ireland and Wales, where responsibility for marine governance is not clear cut. It expresses some disappointment in the failure of the Bill to cover questions of ownership of marine resources in particular the right to fish, which remains held under ancient and flawed common law rules. The paper then investigates the content of the Bill, looking at the role and functions of the Marine Management Organisation including licensing of fishing vessels, wind farm consenting/creation of safety zones, consents under the Wildlife and Countryside Act, research, advice, assistance, training, and prosecutions. The Bill also establishes a framework for marine planning with potential for the creation of marine plans and marine policy statements covering the waters of England and Wales to the edge of the UK Exclusive Economic Zone. Such plans and statements will have persuasive effects on the organisations involved in marine management, but will not be completely binding. The Bill creates powers to licence numerous activities and if necessary delegate licensing function to the bodies such as the Marine Management Organisation. It also established new powers for the creation of Marine Conservation Zones. The paper explores the narrow purposes for which these Marine Conservation Zones and created, and contrasts these unfavourably with the draft Scottish Marine Bill, which permits Marine Conservation Zones for such purposes as community interest. The paper also acknowledges that no target has been set for the creation of Marine Conservation Zones, and raises some concerns over whether Marine Conservation Zones contain severe enough penalties, particularly in respect of commercial fishing where there is a specific exemption for fisheries damage. The paper explores the function of the new Inshore Fisheries Authorities which will replace existing Sea Fisheries Committees, but raises concerns that the new organisations will still remain vulnerable to conflicts of interests because of their membership.     

Succession pattern of carrion-feeding insects in Paramo, Colombia

The minimum postmortem interval can be estimated based on knowledge of the pattern of insect succession on a corpse. To use this approach requires that we take into account the rates of insect development associated with particular climatological conditions of the region. This study is the first to look at insect succession on decomposing carcasses in the high altitude plains (Paramo) in Colombia, at 3035 m above sea level. Five stages of decomposition were designated with indicator species identified for each stage: Callíphora nigribasis at the fresh stage; Compsomyiops verena at the bloated stage; Compsomyiops boliviana during active decay; Stearibia nigriceps and Hydrotaea sp. during advanced decay and Leptocera sp. for dry remains. A succession table is presented for carrion-associated species of the region, which can be used for estimating time since death in similar areas. Compsomyiops boliviana is reported for the first time in Colombia.     

An Aquatic Decomposition Scoring Method to Potentially Predict the Postmortem Submersion Interval of Bodies Recovered from the North Sea

This study aimed to develop an aquatic decomposition scoring (ADS) method and investigated the predictive value of this method in estimating the postmortem submersion interval (PMSI) of bodies recovered from the North Sea. This method, consisting of an ADS item list and a pictorial reference atlas, showed a high interobserver agreement (Krippendorff's alpha ≥ 0.93) and hence proved to be valid. This scoring method was applied to data, collected from closed cases—cases in which the postmortal submersion interval (PMSI) was known—concerning bodies recovered from the North Sea from 1990 to 2013. Thirty‐eight cases met the inclusion criteria and were scored by quantifying the observed total aquatic decomposition score (TADS). Statistical analysis demonstrated that TADS accurately predicts the PMSI (p < 0.001), confirming that the decomposition process in the North Sea is strongly correlated to time.     

Postmortem Intervals in Mice Submerged in Aqueous Environments at 20°C

Aqueous submersion can impede decay and produce decomposition stages not seen in terrestrial burials. To further understand the variances, fifty-four mice were submerged in freshwater, marine water, and a control environment at 20°C. The mice displayed sequential stages at differing rates over 6 weeks. Regression plots and comparative t-tests demonstrated that internal putrefaction, weight difference, and abdominal circumference of the aqueous environments varied significantly from the control group. The aqueous subjects did not vary significantly from each other quantitatively. The postmortem intervals were not consistent regardless of temperature or environment although a clear variance was noted between the control and the submerged groups.     

Initial study of arthropods succession and pig carrion decomposition in two freshwater ecosystems in the Colombian Andes

Entomological succession and trophic roles of arthropods associated with different stages of carcass decomposition were studied to estimate the post-mortem submersion interval in two freshwater ecosystems in the Colombian Andes, at an altitude of 2614 m. Pig carcasses were employed as models placed 68 m apart, one in a stream (lotic) and another in an artificial lake (lentic). Decomposition time to skeletal remains was 74 days in the lake and 80 days in the stream. Six phases of decomposition were established: submerged fresh, early floating, floating decay, bloated deterioration, floating remains and sunken remains. A total of 18,832 organisms associated with the carcasses were collected: 11,487 in the lake (four orders, 19 families and 33 species) and 7345 in the stream (eight orders, 15 families and 25 species). Organisms were classified in the following ecological categories: shredders, collectors, predators, necrophagous, sarcosaprophagous and opportunists. Physical and chemical properties of the habitats, such as water temperature, CO(2) and conductivity, varied according to rainfall. In the lake, shredders (Coleoptera: Tropisternus sp. and Berosus sp.) and collectors (Diptera: Chironomus sp.) were found to be associated with submerged phases. Predators (Odonata) were only present during the first phases. Coleoptera (Dytiscidae) were found during floating decay and bloated deterioration stages. In the stream, shredders (Hyalella sp.) and collectors (Simulium sp.) were found during all stages, whereas the predator Oxelytrum discicolle was found exclusively during the floating stages, during which body temperature increased in a fashion similar to active decay in terrestrial environments.     

Decomposition patterns in terrestrial and intertidal habitats on Oahu Island and Coconut Island, Hawaii

Decomposition studies were conducted at two sites on the Island of Oahu, Hawaii, to compare patterns of decomposition and arthropod invasion in intertidal and adjacent terrestrial habitats. The animal model used was the domestic pig. One site was on Coconut Island in Kaneohe Bay on the northeast side of Oahu, and the second was conducted in an anchialine pool located at Barber's Point Naval Air Station on the southwest shore of Oahu. At both sites, the terrestrial animal decomposed in a manner similar to what has been observed in previous studies in terrestrial habitats on the island of Oahu. Rate of biomass depletion was slower in both intertidal studies, and decomposition was primarily due to tide and wave activity and bacterial decomposition. No permanent colonization of carcasses by insects was seen for the intertidal carcass at Coconut Island. At the anchialine pool at Barber's Point Naval Air Station, Diptera larvae were responsible for biomass removal until the carcass was reduced below the water line and, from that point on, bacterial action was the means of decomposition. Marine and terrestrial scavengers were present at both sites although their impact on decomposition was negligible. Five stages of decomposition were recognized for the intertidal sites: fresh, buoyant/floating, deterioration/disintegration, buoyant remains, and scattered skeletal.