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.     

Microbiota Composition and Pulmonary Surfactant Protein Expression as Markers of Death by Drowning

Pathological diagnosis of drowning remains a challenge for forensic science, because of a lack of pathognomonic findings. We analyzed microbiota and surfactant protein in the lungs for a novel diagnosis of drowning. All rats were divided into drowning, postmortem submersion, and control groups. The water, lungs, closed organs (kidney and liver), and cardiac blood in rats were assayed by targeting 16S ribosomal RNA of Miseq sequencing. Lung samples were analyzed by immunohistochemical staining for surfactant protein A. The closed organs and cardiac blood of drowned group have a lot of aquatic microbes, which have not been detected in postmortem submersion group. Furthermore, intra‐alveolar granular staining of surfactant protein A (SP‐A) was severely observed in the drowned group than the postmortem submersion and control groups. The findings suggested that the presence of aquatic microbiota in the closed organs and increased expression of SP‐A could be markers for a diagnosis of drowning.     

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.     

The "coffin fly"Conicera tibialis (Diptera: Phoridae) breeding on buried human remains after a postmortem interval of 18 years

The "coffin fly,"Conicera tibialis Schmitz (Order: Diptera, Family: Phoridae), is well known for its frequent occurrence on buried corpses, in some cases after postmortem intervals of even 3-5 years. The present report describes the presence of a large amount of individuals of C. tibialis inside the coffin of a buried human corpse exhumed 18 years after death in central Spain. Adults, some of them newly emerged, and empty puparia were found in connection with the remains. Such postmortem interval is significantly longer than previously known for this species and raises the question on the current state of knowledge about the use of insects for estimating the postmortem interval in old, buried remains.     

Development of immersion skin in fresh and salt water at different water temperatures

The development of skin wrinkling was analysed using 48 hands from 24 adult corpses in systematic experiments involving submersion in fresh and salt water at varying water temperatures (10 degrees, 20 degrees, and 30 degrees C); the results were documented by photography. Skin wrinkling was observed after between 15 min and 72 h of submersion. These are the essential results of the study: skin wrinkling is highly dependent on temperature and begins after only 15 min of submersion. The development of wrinkling in salt water, especially after long periods (greater than 24 h), is different from that in fresh water. Washerwomen's hands are classified into four categories that allow practical, usable conclusions to be drawn about the duration of submersion. Previous opinions concerning the development and timing of washer-women's hands were verified by means of test photographs shown to 16 domestic and foreign experts on legal medicine. It became obvious that expertise in this area varies considerably. Concerning the forensic approach, these surveys again show that, even when many influencing factors are known (water temperature, osmolarity, etc.) and comprehensive documentation has been made, the length of submersion can only be very roughly assessed from macroscopical aspects.     

Algal colonization of submerged carcasses in a mid-order woodland stream

One of the primary goals of forensic pathology is the determination of time of death. In aquatic systems, one method to do this is to analyze the colonization of a corpse by algae. Algal communities typically follow a serial colonization pattern, therefore the taxa present at any given time may provide clues about postmortem submersion time. This study was undertaken to examine the algal colonization on rat carcasses in a medium-order woodland stream. Two habitats were studied: a low flow pool and a high flow riffle, with rats being removed from each site every 3 to 6 days over 31 days. The diversity of colonizing taxa increased at both sites as the study progressed, and after 17 days similar taxa were present (Sorensen's similarity index >60%) in each site. Some taxa, such as desmids (Chlorophyta), tended to increase in diversity throughout the study, making them possible indicators of submersion time. Diatoms were the most abundant taxa found in each site and accounted for 63 of the 92 total taxa identified. Due to their ubiquitous presence in nearly all streams, we suggest that diatoms may be the key organisms for the study of postmortem submersion in lotic systems.     

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.     

Chemical estimation of fresh water immersion intervals

Postmortem vitreous humor chemistry studies were reviewed in 25 cases in which death was followed by submersion in fresh water for known lengths of time. The potassium concentrations in cold temperature cases initially increased in a linear manner, but appeared to level off or decline after about 1 week. In hot weather cases, potassium levels rose rapidly during the first few days postmortem. The sodium and chloride levels decreased in a fairly regular fashion, with less temperature variation. Increasing variation in values began to occur at about 7 days. The data show that dilution of the contents of the eye by fresh water begins at about that time in cases of cold water submersion. A formula employing the weighted average of the potassium and chloride concentrations can be used to determine the duration of submersion in cold water over the range of 2-10 days.     

硅藻定量检验和种属鉴定的实验研究

本实验用家兔18只,分为溺死组、死后(抛尸)入水组及对照组,每组各6只。采用消化—光镜法作肺、肝、肾硅藻计数(定量)及种属鉴定检验,同时作水样检验及空白对照。结果:(一)进一步证实生前硅藻可经空气源性进入肺脏并存积于大循环内脏(肝、肾)的概念;(二)提示水中尸体肺脏如有大量硅藻检出,仅此即可作为诊断溺死的可靠依据;空气源性吸入及检验过程中的污染并不影响该检验结果对鉴定溺死的判断。种属分析溺死组肺与水样的主要硅藻种属相符,而肝、肾中硅藻计数及种属分析均无诊断意义。     

Stages of postmortem formation of washerwoman's skin--results of systematic qualitative and quantitative experimental studies

The development of washerwoman's hands depends upon the length of submersion. Until now there have been no results published on the qualitative and quantitative time-related changes with regard to washer-woman's hands. The fingertips of 50 hands from 35 corpses were qualitatively examined at early postmortem (1-4 h after death). The quantitative investigations were confined to the tips of the middle fingers. Submersion of the hands in water at 37 degrees C was interrupted at intervals of 10 min, 30 min, 60 min, 2 h, 3 h, 4 h, 6 h, 10 h and 24 h for a period of 30s-1 min. The width of three adjoining ridges of the skin were measured in three defined areas of the middle fingertips and photographs were taken. Quantitative results: No relevant differences were seen in the changes of the width of the skin ridges between the right and left hand after submersion in water; there was no specific difference between the sexes. During the soaking process the skin ridges influence each other. The age of the individual corpse has no influence on the change in width of the skin ridges. Concerning the qualitative test results, six time-limited stages could be classified: stage I: physiological dry state; stage II: softened state; stage III: swollen and compressed state; stage IV: partial wrinkle covering; stage V: epidermal lysis; stage VI: loss of relief outlines.     

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.     

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.     

PCR-DGGE法检测浮游生物16S rDNA在溺死鉴定中的应用

目的建立并评估PCR-DGGE法检测浮游生物对溺死鉴定的应用价值。方法大白兔30只随机分为3组:溺死组(n=12),死后抛尸组(n=12)和对照组(n=6);溺死组和死后抛尸组又分为2个亚组:东湖水域组(n=6)和墨水湖水域组(n=6)。死后提取心血和肺、肝、肾、脑等组织,匀浆后,采用Percoll密度梯度离心法分离浮游生物并提取其DNA,PCR扩增浮游生物特异的16S rDNA片段后分别用琼脂糖凝胶电泳及DGGE检测分析。2个溺死案例检材同法检验。结果溺死组各组织器官中浮游生物检测多呈阳性:肺(100%)、肝(83%)、肾(75%)、心血(83%)、脑(42%);死后抛尸组仅2例肺组织(16.7%)检出阳性;对照组全部阴性。溺尸肺组织DGGE分型图谱与相应溺死点水样分型图谱相似,而与非溺死点水样分型结果差异显著。2实际案例均呈阳性。结论本方法不仅有助于定性诊断溺死,而且通过比较产物的多样性可以推断溺死地点,在法医学溺死鉴定中具有较大实用价值。     

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.     

Metrological framework for selecting morphological characters to identify species and estimate developmental maturity of forensically significant insect specimens

Accurate age estimates of immature necrophagous insects associated with a human or animal body can provide evidence of how long the body has been dead. These estimates are based on species-specific details of the insects’ aging processes, and therefore require accurate species identification and developmental stage estimation. Many professionals who produce or use identified organisms as forensic evidence have little training in taxonomy or metrology, and appreciate the availability of formalized principles and standards for biological identification. Taxonomic identifications are usually most readily and economically made using categorical and qualitative morphological characters, but it may be necessary to use less convenient and potentially more ambiguous characters that are continuous and quantitative if two candidate species are closely related, or if identifying developmental stages within a species. Characters should be selected by criteria such as taxonomic specificity and metrological repeatability and relative error. We propose such a hierarchical framework, critique various measurements of immature insects, and suggest some standard approaches to determine the reliability of organismal identifications and measurements in estimating postmortem intervals. Relevant criteria for good characters include high repeatability (including low scope for ambiguity or parallax effects), pronounced discreteness, and small relative error in measurements. These same principles apply to individuation of unique objects in general.

Key points

• Metrological rigour can increase in forensic entomology by selecting measurements based on their metrological qualities.
• Selection of high-quality features for morphological identification of organisms should consider these criteria: (1) pronounced discreteness of features (minimising group overlap or maximizing interval); (2) high repeatability of assessment (such as symmetrical width rather than asymmetrical length); (3) small relative error in measurement (selecting the physically largest continuous rigid feature for measurement).
• These metrological principles also apply to individuation of unique objects in general.

     

Aquatic Crime: A New Horizon in Criminal Justice Research

This article focuses upon a relatively unexplored area of criminal activity, namely, aquatic or water based criminal activity. The specific concern is the need to focus upon the vast domain of water, engulfing over two-thirds of the earth's surface, in understanding and expaining many forms of intranational and international criminality. Emphasis is placed upon the need for comprehensive research in order to fill a void in criminological theory and to generate a better understanding of the potential of aquatic environments in promoting and concealing criminal activity. Moreover, comprehensive research is essential in order to promote a realistic assessment of the nature and extent of aquatic crime and to improve our ability to effectively combat such activity.     

Post-mortal lesions in freshwater environment

Postmortem animal feeding activity may cause considerable damage to bodies resulting in the modification of wounds, loss of identifying features and injury. Certain postmortem lesions may appear inflicted or non-inflicted antemortem injuries. At present, apart from cases in sea water, no data are available about post mortal lesions performed by aquatic organisms. This note that represents the first report concerning colonisation of a dead body by crustaceans a few hours after death, describes injuries caused by the amphipod Niphargus elegans on the face, and in particular on the eye region, of a young man dead by drowning. The lesions recorded in this case are comparable with the lesions caused by ants. The high plasticity in the food choice can allow Amphipoda to colonise drowning bodies in every moment after dead, however the benthonic behaviour of these animals suggests a more important role in the colonisation during post-mortem submersion periods.     

Detection of diverse aquatic microbes in blood and organs of drowning victims: first metagenomic approach using high-throughput 454-pyrosequencing

Current 454-pyrosequencing technology enables massive parallel sequencing. We used this technology to investigate the diversity of aquatic microbes in 14 specimens (blood and organs) of two drowning victims and in two water samples taken from the discovery sites. The 16S ribosomal RNA (rRNA) genes of microbes, which are often used to identify species (or genera), have nine highly variable regions (V1-V9), each of which is surrounded by conserved regions. Some parts within the conserved regions are common over domains of microbes, such as between bacteria and algae (16S rRNA genes on algal chloroplast genomes). We therefore simultaneously amplified the target regions (V7 and V8) of various microbes in the blood and organs of drowning victims using PCR with custom-designed primers that were based on the conserved regions. We then exhaustively analyzed the PCR products by pyrosequencing using the Genome Sequencer FLX Titanium system (Roche-454 Life Sciences). This approach identified a wide array of bacteria including cyanobacteria and algae including Bacillariophyceae (diatom), Cryptophyceae, Dictyochophyceae, Chrysophyceae and Trebouxiophyceae in the blood and organs of the victims and water at discovery sites. Our data further indicated that when conventional diatom testing of lungs yielded insufficient evidence of water aspiration, the detection of various exogenous microbes by 454-pyrosequencing is very useful to support a conclusion of death by drowning. To the best of our knowledge, this is the first attempt to use a new generation sequencer to investigate diverse aquatic microbes in the blood and closed organs of drowning victims.