Decomposition and insect colonization patterns of pig cadavers lying on forest soil and suspended above ground.

Hanging is one of the most common methods of suicide, and, although it is known that decomposition and patterns of insect fauna colonization of hanging cadavers differ from those of cadavers decomposing on the ground, these differences have only been sparsely studied in Europe so far. We studied the decomposition and insect colonization patterns of ten pig (Sus scrofa domestica) carcasses in a spruce forest near Neuchâtel, Switzerland, over a 32-day period in summer 2013 (July 1st - August 2nd). Five pig carcasses were suspended horizontally in metal cages one meter above the ground and five carcasses were placed directly on the ground. The species occurrence and abundance of Coleoptera and Diptera recovered from passive pitfall traps and collected manually on the carcasses were recorded. Indicator species of decomposition stages and suspended vs. ground cadavers were identified by Indicator Value (IndVal) analysis. The ground carcasses decomposed significantly faster than the suspended carcasses. Coleoptera and Diptera communities differed significantly over time, between treatment, and the treatment effect varied over time. Diptera were evenly distributed among the two treatments. Coleoptera were more abundant and less differentiated on the ground carcasses and represent better indicators of cadavers decomposing on the soil as well as decomposition stages. Our results suggest possible applications such as evaluating if a hanged cadaver has been dropped to the ground, or inversely if a cadaver first lying on the soil has later been hung to fake a suicide. However differences among studies suggest possible effects of meso-climate determined by habitat which should be explored further.

Response of forest soil euglyphid testate amoebae (Rhizaria: Cercozoa) to pig cadavers assessed by high-throughput sequencing.

Decomposing cadavers modify the soil environment, but the effect on soil organisms and especially on soil protists is still poorly documented. We conducted a 35-month experiment in a deciduous forest where soil samples were taken under pig cadavers, control plots and fake pigs (bags of similar volume as the pigs). We extracted total soil DNA, amplified the SSU ribosomal RNA (rRNA) gene V9 region and sequenced it by Illumina technology and analysed the data for euglyphid testate amoebae (Rhizaria: Euglyphida), a common group of protozoa known to respond to micro-environmental changes. We found 51 euglyphid operational taxonomic units (OTUs), 45 of which did not match any known sequence. Most OTUs decreased in abundance underneath cadavers between days 0 and 309, but some responded positively after a time lag. We sequenced the full-length SSU rRNA gene of two common OTUs that responded positively to cadavers; a phylogenetic analysis showed that they did not belong to any known euglyphid family. This study confirmed the existence of an unknown diversity of euglyphids and that they react to cadavers. Results suggest that metabarcoding of soil euglyphids could be used as a forensic tool to estimate the post-mortem interval (PMI) particularly for long-term (>2 months) PMI, for which no reliable tool exists.

Soil chemistry changes beneath decomposing cadavers over a one-year period.

Decomposing vertebrate cadavers release large, localized inputs of nutrients. These temporally limited resource patches affect nutrient cycling and soil organisms. The impact of decomposing cadavers on soil chemistry is relevant to soil biology, as a natural disturbance, and forensic science, to estimate the postmortem interval. However, cadaver impacts on soils are rarely studied, making it difficult to identify common patterns. We investigated the effects of decomposing pig cadavers (Sus scrofa domesticus) on soil chemistry (pH, ammonium, nitrate, nitrogen, phosphorous, potassium and carbon) over a one-year period in a spruce-dominant forest. Four treatments were applied, each with five replicates: two treatments including pig cadavers (placed on the ground and hung one metre above ground) and two controls (bare soil and bags filled with soil placed on the ground i.e. "fake pig" treatment). In the first two months (15-59 days after the start of the experiment), cadavers caused significant increases of ammonium, nitrogen, phosphorous and potassium (p<0.05) whereas nitrate significantly increased towards the end of the study (263-367 days; p<0.05). Soil pH increased significantly at first and then decreased significantly at the end of the experiment. After one year, some markers returned to basal levels (i.e. not significantly different from control plots), whereas others were still significantly different. Based on these response patterns and in comparison with previous studies, we define three categories of chemical markers that may have the potential to date the time since death: early peak markers (EPM), late peak markers (LPM) and late decrease markers (LDM). The marker categories will enhance our understanding of soil processes and can be highly useful when changes in soil chemistry are related to changes in the composition of soil organism communities. For actual casework further studies and more data are necessary to refine the marker categories along a more precise timeline and to develop a method that can be used in court.

Comparative analysis of bones, mites, soil chemistry, nematodes and soil micro-eukaryotes from a suspected homicide to estimate the post-mortem interval.

Criminal investigations of suspected murder cases require estimating the post-mortem interval (PMI, or time after death) which is challenging for long PMIs. Here we present the case of human remains found in a Swiss forest. We have used a multidisciplinary approach involving the analysis of bones and soil samples collected beneath the remains of the head, upper and lower body and "control" samples taken a few meters away. We analysed soil chemical characteristics, mites and nematodes (by microscopy) and micro-eukaryotes (by Illumina high throughput sequencing). The PMI estimate on hair 14C-data via bomb peak radiocarbon dating gave a time range of 1 to 3 years before the discovery of the remains. Cluster analyses for soil chemical constituents, nematodes, mites and micro-eukaryotes revealed two clusters 1) head and upper body and 2) lower body and controls. From mite evidence, we conclude that the body was probably brought to the site after death. However, chemical analyses, nematode community analyses and the analyses of micro-eukaryotes indicate that decomposition took place at least partly on site. This study illustrates the usefulness of combining several lines of evidence for the study of homicide cases to better calibrate PMI inference tools.

Can soil testate amoebae be used for estimating the time since death? A field experiment in a deciduous forest.

Estimation of the post-mortem interval (PMI, the time interval between death and recovery of a body) can be crucial in solving criminal cases. Today minimum PMI calculations rely mainly on medical and entomological evidence. However, beyond 4-6 weeks even entomological methods become less accurate. Thus additional tools are needed. Cadaveric fluids released by decomposing cadavers modify the soil environment and thus impact soil organisms, which may thus be used to estimate the PMI. Although the response of bacteria or fungi to the presence of a corpse has been studied, to the best of our knowledge nothing is known about other soil organisms. Testate amoebae, a group of shelled protozoa, are sensitive bioindicators of soil physico-chemical and micro-climatic conditions and are therefore good potential PMI indicators. We investigated the response of testate amoebae to three decomposing pig cadavers, and compared the pattern to two controls each, bare soils and fake cadavers, in a beach-oak forest near Neuchâtel, Switzerland. Forest litter samples collected in the three treatments over 10 months were analysed by microscopy. The pig treatment significantly impacted the testate amoeba community: after 22 and 33 days no living amoeba remained underneath the pig cadavers. Communities subsequently recovered but 10 months after the beginning of the experiment recovery was not complete. The fake cadavers also influenced the testate amoeba communities by altering the soil microclimate during a dry hot period, but less than the cadavers. These results confirm the sensitivity of soil testate amoebae to micro-climatic conditions and show that they respond fast to the presence of cadavers - and that this effect although decreasing over time lasts for months, possibly several years. This study therefore confirms that soil protozoa could potentially be useful as forensic indicators, especially in cases with a longer PMI.