The impact of the decomposition process of shallow graves on soil mite abundance

Abstract Burial of a cadaver results in a slower decomposition rate, due to more stable below‐ground temperatures and restricted access to necrophagous insects. In such circumstances, analysis of the soil mesofauna, with emphasis on mites (Acari) may be more valuable in time‐of‐death estimations. The production of volatile organic compounds of cadaveric decay results in changes, especially in the soil pH, which in turn would affect the abundance and diversity of the associated mites. In general, the effects of decomposition and the consequently altered pH levels on the abundance of mites in shallow graves, as well as the effects of fluctuating above‐ground environmental parameters (temperature, relative humidity, and precipitation) remain unknown. Here, we found that the decay of three pig cadavers buried in shallow graves (<30 cm below) caused a significant increase in the soil pH throughout decomposition, from neutral to alkaline. Cadaver decay attracted an abundance of mites: with 300 mites collected from the three pig cadavers compared to 129 from the control soil samples at the same depth. Mites rapidly became more abundant in cadaver‐associated soils than in control soils after the fresh stage. Increasing soil pH had a positive impact on the abundance of mites in graves and there was a significant interaction between cadaver body temperature and soil pH. Above‐ground fluctuations in temperature, relative humidity, and precipitation were found to have no significant direct effect on mite abundance in grave or control soils.


| INTRODUC TI ON
Decomposition of exposed and buried cadavers is categorized into a series of stages of decay based on changes in physical characteristics [1][2][3][4][5]. A handful of studies have described the stages of decay of buried cadavers. Payne et al. [2] described five decay stages for buried pigs as fresh, inflated, deflation and decomposition, disintegration, and skeletonization. Vanlaerhoven and Anderson [5] also described five stages of decay of pigs buried at 30 cm: fresh, bloat, active, advanced, and dry/remains. Typically, these five stages are most widely described in vertebrate decomposition studies of both exposed and buried cadavers [8].
While the process of decomposition of buried and exposed cadavers is the same, the rate of decomposition of buried cadavers is much slower [5][6][7][8][9]. The biotic and abiotic parameters below ground are different to those above ground and the reduced insect activity in a grave environment means that buried bodies will decompose at an entirely different rate compared to an exposed cadaver in the same locality [10]. Temperature, access to insects, and the depth of burial are considered to be the three most influential factors [11].
Hence, decomposition of buried cadavers can be more difficult to characterize as the burial, depending on the depth of the grave, may significantly influence the temperature in the headspace of the corpse as well as the level of access to insects.
In terrestrial environments, a decomposing cadaver forms a nutrient-rich substrate for a variety of necrophagous, predatory/ parasitic, omnivorous, and adventive arthropods. Each decay stage attracts a unique assemblage of arthropods which arrive at the carcass in a series of waves, resulting in faunal succession occurring in a relatively predictable pattern [6,12]. The majority of existing research of the grave fauna is limited to insects, and that of other soil arthropods is largely unexamined. Several centimeters of soil covering the carcass may prevent access to a cadaver for major Calliphoridae species (Calliphora and Lucilia) [13][14][15]. Some insects can still gain access to an interred cadaver through macro-pores in the soil, though the access is restricted depending on the depth of burial and pore size [5,9,10,[15][16][17][18]. There is also limited knowledge on how far insects travel to colonize buried carcasses and only a handful of dipteran species (and some Coleoptera) are considered to have forensic importance in graves [15,[19][20][21]. For example, the Phoridae species Conicera tibialis [22][23][24][25] and Megaselia scalaris are frequently sampled from graves and utilized in PMI estimations [26,27].
The occurrence of insects in graves is somewhat random due to their limited access, so the use of insect succession in PMI estimations and trace evidence analysis of buried bodies can become unreliable. In circumstances, where activities and visitation patterns of insects are reduced or altered, analysis of the soil mesofauna, particularly the composition of the most predominant taxa, mites (Acari) inhabiting the surrounding soil may hold the forensic value. Several studies have demonstrated that mites are an important part of the cadaver fauna throughout decomposition [5,[28][29][30][31][32]. However, the variations in abundance and diversity of mites associated with each decomposing stage of buried cadavers in terrestrial habitats have not been explored. Mites are ubiquitous in natural and synanthropic habitats and are the most species-rich and abundant soil micro-arthropods in most soil types [33]. Mites have diverse feeding strategies and in soil, habitats are major contributors of organic decomposition of vertebrate tissue and dead plant matter. Mites, therefore, form a large part of the cadaveric fauna, especially in graves, where insect activity is absent or significantly reduced. More than 250,000 mites may be found in 10-cm depth in a square meter of forest soil and they are especially numerous in upper horizons where oribatids dominate, as compared to much greater depths, where mesostigmatids, astigmatids, and prostigmatids are predominant [34,35].
Mite population densities are affected by the fluctuations in the physical and chemical properties of soil [36]. During mammalian decomposition, the leakage of nutrient-rich cadaver fluids contributes to the alteration of the micro-environment and chemistry of the soil, in particular the soil pH, which in turn impacts the meso-faunal organisms. In several studies, cadaver decay has been found to cause an increase in the soil pH from the start to mid decomposition, to a more alkaline level, followed by a decrease toward the end stages of decay [37][38][39][40]. This is attributed mainly to the production and accumulation of ammonium ions (NH + 4 ) from the ammonification of proteins during autolysis and putrefaction occurring mainly during bloated and active decay [38,41]. The changes in soil pH are thought to directly affect the abundance of the meso-fauna throughout the decomposition, in particular the mites [42], however, the effect of the alteration in soil pH during decay on the abundance of the surrounding soil micro-arthropods such as mites is poorly understood.
The fauna of a cadaver is closely associated with the decomposition stages. However, the fauna may also be influenced by several extrinsic factors that are not directly linked to the cadaver or circumstances surrounding the death, such as the climatic conditions, ambient temperature, ambient humidity, rainfall, and snowfall.
These factors along with the intrinsic factors such as changes in the soil pH (as a result of decomposition) can significantly influence the fauna associated in terms of abundance and diversity. When a body is buried in a grave, it has greater protection from fluctuating environmental conditions such as rain, temperature, humidity, and snow, however, a grave of less than 20-cm depth is likely to be affected by the fluctuations in the above-ground environmental conditions [43].
The deeper the grave, the more protected the cadaver and its associated fauna are from environmental instabilities. Exposed cadavers, in terms of decomposition rate and the colonizing fauna, are more sensitive to such environmental parameters [5,10]. For example, parameters such as rain, snow, and ambient temperature can have a significant effect on the presence and abundance of fauna such as Diptera colonizing an exposed cadaver outdoors [44]. Calliphoridae activity is reduced during higher periods of rain and snowfall, and rainfall, in particular, can delay oviposition and pupation [26,45], while higher temperatures are more favorable for oviposition and development of some necrophagous flies and beetles [26]. It is not known if the mite fauna within a grave environment, where the cadaver is concealed by a thin layer of soil such as that in a shallow grave of less than 30 cm, will be affected by external factors such as temperature, humidity, rain, and snowfall in the same way that an exposed cadaver may experience.
Changes in the soil pH levels around a decaying cadaver have a major impact on the biotic elements, such as the population densities of bacteria, fungi, and mites [46,47]. For instance, neutral to slightly alkaline pH levels are favorable for soil bacteria, while acidic pH levels tend to be more favorable for fungal growth [46]. In turn, this impacts the composition of micro-arthropods such as mites that feed on these soil organisms [48]. The soil pH levels also influence abiotic parameters such as carbon content and nutrient availability, which fluctuate in response to cadaveric decay and directly influence the surrounding soil fauna [37,38]. However, the patterns of mite abundance associated with fluctuating soil pH during cadaveric decay are mostly unknown. Other extrinsic factors, such as seasonal fluctuations of temperature and rainfall, also impact mite densities [49,50], yet under cadaver decay circumstances, it is not known if these factors have a greater or a lesser effect on the abundance of mites that the effects of decomposition itself (food, shelter availability and soil pH).

| Aims and objectives
To use entomological and acarological evidence for time-of-death estimations, it is crucial to understand the stages and rates of decomposition that a cadaver will undergo in various scenarios and circumstances of death. The stage of decomposition will determine the abundance and diversity of mites in the surrounding soil, which is thought mainly to be an effect of food availability, though abiotic factors may also contribute. However, the extent to which fluctuations in soil pH, ambient temperature, humidity, and precipitation affect the mite abundances within a shallow grave environment remains unknown. Here, we examined the decomposition of pig cadavers in shallow graves to: (1) examine the patterns of total mite abundance associated with each decomposition stage, (2) investigate any links between the environmental factors and the decay of buried cadavers, and (3) analyze whether soil pH, ambient temperature, ambient relative humidity, and daily rainfall are associated with the abundance of mites throughout the decomposition process.

| Experimental design
The study was carried out over a period of 3 years (October 3, 2015 to September 30, 2018) within a temperate woodland located in the grounds of the Whiteknights Campus, University of Reading, Berkshire, UK (51°26′10.6″N 0°56′35.0″W). The relatively undisturbed area of deciduous trees and shrubbery measured approximately 80 m × 50 m. Although the main road is adjacent to the site, it is well concealed from the public view with the thick vegetation. The soil in this region is clay loam soil and slightly acidic with an average pH of 6.9.
Three freshly killed pig carcasses (Sus scrofa domesticus) (mean 33 kg) purchased from a slaughterhouse were used as temporal repeats (one per year), representing suitable proxies for the decay of human remains [12,51]. Each pig was killed on the day that each study commenced, every year. The total depth of the grave (10-

| Soil sampling and mite extraction
Digital images were taken to record the decomposition process of all three cadavers. The decay process of each carcass was grouped into five major decomposition stages, which were established by the observations of physical post-mortem changes to the cadavers.
'Cadaver soils' were collected as follows: A total of-300 ml volume of soil was sampled from immediately beneath each of the head, abdomen, and posterior regions of the pig carcass (total = 900 ml soil per sampling day) by lifting it at an angle with a large shovel and collecting soil from underneath with a hand shovel. Each carcass was inspected 3-5 times per week to record post-mortem changes (e.g., odors, maggot activity), and sampling days were selected corresponding to the five stages of decomposition (determined by the observation of the physical post-mortem changes). Sampling took place thrice within each stage. 'Control soils' were collected on the same day from the corresponding control plot, with three samples of 300-ml soil taken from the same depth as the cadaver soils.
Mite extraction was conducted for each sample over 7 days using manually constructed Berlese-Tullgren funnels placed underneath 40-watt incandescent light bulbs. Here, arthropods travel downward to avoid heat and light (Tullgren, 1918) where they are collected in vials of 70% (v/v) ethanol, preserving them prior to counting. Collection vial contents were transferred into petri dishes where a stereomicroscope was used to separate mites from all other arthropods, with any phoretic mites manually removed from each of these arthropods. Mites were then counted to generate the total for each soil sample (n = 90), corresponding to one of the five stages of decomposition for a cadaver soil (n = 45) or a corresponding control soil (n = 45).

| Collection of environmental data
Environmental data for each sampling day over the period The body surface temperature (°C) of each pig carcass was recorded using a handheld digital infra-red thermometer (Tekpower infrared DT8380 thermometer). Temperatures of the surface of the head, the torso, and the posterior region of the carcasses were taken on each day the soil was sampled and were averaged to give the mean body surface temperature. To obtain the pH of each sample of cadaver and control soil, 10 g of soil was mixed in 25 ml of deionized water following the soil pH measurement ratio of 1:2.5 (soil: solution) suggested by Aciego Pietri and Brookes [52].
After settling at room temperature for 30 min, a pen-type pH meter (Tekco plus Digital pH meter) was used to record the pH of the soil solution.

| Statistical analyses
For all the statistical tests, a p-value < 0.05 was accepted for rejecting the null hypothesis. Data on the duration of decomposition were tested for normality using the Anderson-Darling test. To examine the differences in the duration of the five major decomposition stages of the buried cadavers, a one-way analysis of variance (ANOVA) with post hoc Tukey honest significant difference (HSD) pairwise comparison was conducted with Minitab ® 19.2020.1.0.
Differences between cadaver body surface temperature and ambient air temperature were tested for significance using non- Statistical analyses of factors influencing mite abundance were conducted in R version 4.0.2 [53]. Generalized linear models with a Poisson distribution were initially used to examine the data. Due to the overdispersion of the data, generalized linear mixed-effects models (GLMM) with a negative binomial error structure and 'replicate' as a random factor were constructed to examine the changes in the mite abundance using the R package 'lme4' [54]. Models were fitted by restricted maximum likelihood. Interaction terms were examined for the variance-inflation due to multicollinearity using the vif function (VIF, variance-inflation factors) in R package 'car' [55].
Model fit for negative binomial GLMs was estimated using marginal (R 2 (m) ; variance explained by only the fixed effects) and conditional (R 2 (c) ; variance explained by the entire model, including both fixed and random effects) values with the trigamma method following [56].

| Timeline of cadaver decomposition
All three pig cadavers underwent the expected five major stages of decomposition for buried and exposed cadavers: fresh, bloated, active, advanced, and dry/remains (  Figure 1D). Larvae were mainly visible on exposed cadaver regions, for example, larval masses were prominent around the head and posterior regions of P3 from day 38.
Advanced decomposition was the longest decay stage for all three cadavers and was marked by the degradation of most of the soft tissue, with some dried soft tissue and remnants of decay fluids present, but mostly skin, cartilage, bone remaining, and weak decomposition odors ( Figure 1E). There was a reduction in decay flu- The duration data of all five decomposition stages (Table 1)   The abundance of mites was found to vary significantly with decomposition stage (fresh, bloated, active, advanced, dry), soil treatment (cadaver or control), and their interaction (Table 3, Figure 3, Table   S1). The estimated model fit (R 2 (m) = 0.425|R 2 (c) = 0.425) indicated that decomposition stage and soil treatment, rather than differences between replicates, were responsible for the observed variation in mite abundance. The overall pattern revealed a significant increase in mite abundance in cadaver soils during the bloated, active, advanced, and dry stages compared to the fresh stage (p < 0.001). Mite abundance in control soils was not significantly different from that of cadaver soils during the fresh (p = 0.210) or bloated (p = 0.077) stages, but was significantly lower in control compared to cadaver soils during the active, advanced, and dry stages (p < 0.05) (Table 3; Figure 3).

| Influence of environment on cadaver decay
The average body surface temperature (°C) reflected the average daily ambient air temperatures ( Figure 4A-C,

| Environmental impacts on mite abundance
Cadaver decay processes generated significant changes in soil pH ( Figure 5). Briefly, fresh cadaver soils and control soils generally exhibited neutral pH (~7), with cadaver soil rapidly becoming significantly more alkaline (~8-9) from the bloated stage onwards. The influence of environmental factors on mite abundance was investigated for cadaver and control soils separately. In cadaver soils, the abundance of mites was found to increase significantly with the increasing soil pH, and there was a significant interaction between the cadaver body temperature and soil pH ( Table 5, Table S2). Cadaver soil model fit (R 2 (m) = 0.359|R 2 (c) = 0.359) indicated that the measured environmental factors, rather than the differences between the replicate cadavers, were responsible for the observed variation in mite abundance.
In control soils, the abundance of mites was not significantly affected by any of the measured environmental variables (air temperature, rainfall, relative humidity and soil pH) ( Table 6). Control soil model fit (R 2 (m) = 0.033|R 2 (c) = 0.102) indicated that the differences between replicates, rather than measured environmental factors, were responsible for most of the observed variation in mite abundance.

| DISCUSS ION
This study demonstrated that cadaveric decay in shallow soil graves follows the same recognized physical postmortem changes associated with the five major stages of decay as reported by previous studies of buried cadavers [5,21,40] and surface cadavers [4,[57][58][59] in similar terrestrial environments and climatic conditions: fresh, bloated, active, advanced, and dry/remains. We found that the abundance of mites rapidly increased in cadaver soils, with significantly greater numbers of mites in bloated, active, advanced, and dry/remains stages compared to the fresh stage. Mite abundance in cadaver soils was significantly greater than in control soils during the active, advanced, and dry/remains stage, suggesting that the mite abundance alone can be a useful indicator of a shallow grave.
The soil pH and the interaction between pH and body temperature, influenced mite abundance in cadaver soils, but no significant association between the environmental factors and mite abundance were detected in control soils.

| Decomposition of vertebrate cadavers in shallow graves
Buried bodies are associated with significantly slower decomposition rates mainly due to the inhibited access for necrophagous insects, which along with the lowered temperature, is one of the major factors that results in delays to soft tissue breakdown [11,21,26,[60][61][62][63].
In our study, all three cadavers took up to or more than 12 months to transition into full-body skeletonization, which is similar to the decomposition rates seen in the previous studies of buried cadavers [8,10,21,64].
The decomposition rate of a buried body, especially in shallow graves (<30 cm), can be further influenced by environmental factors such as temperature, humidity, snowfall, and rainfall. Here, no significant differences were observed between the body surface temperatures of the pig cadavers and the daily average ambient temperature for any cadaver. This demonstrated that the temperatures within the graves were closely associated with the ambient temperatures and were directly proportional to the daily and seasonal fluctuations. Hence, slight variations in environmental parameters during each year of the study were likely to have had an influence

| Changes in mite abundance between decay stages
Mite abundance increased quickly and consistently following the addition of cadavers. Abundance during the fresh stage (~0-7 days) was comparable to the background levels exhibited by the control

| Effect of environmental parameters on decomposition
Cadaver body surface temperature throughout decomposition was directly associated with the ambient temperature, demonstrating that shallow burial appeared to have minimal effect on the grave temperature. Unlike with deeper graves (e.g., >60 cm), cadavers in shallow graves are less sheltered from above the ground environmental fluctuations [10,23,60,66]. Therefore, as with exposed cadav- Active decay is associated with an optimum larval activity and soft tissue breakdown, but low temperatures inhibit microbial

| Effect of environmental parameters on mite abundance
A total of 300 mites from cadaver soils and 129 from control soils were covered throughout the decomposition of all three cadavers.
The overall lower abundances compared to very high abundances reported earlier in forest soils may be explained by the location of the study site in an urban zone. Urban soils are affected in terms of biodiversity by pollution and environmental disturbances. This in turn can negatively impact the abundance and diversity of soil invertebrates such as mites in urban soils. The soil pH was the only measured environmental parameter that had a significant direct effect on the abundance of mites in cadaver soils. A positive relationship was found, with mite abundance increasing as the soil pH increased. An interactive effect was also observed between the body temperature and the soil pH: when both temperature and pH were low, mite abundance increased, and when both the temperature and the pH were high, mite abundance increased. By contrast, none of the other measured environmental parameters exhibited a significant influence on the mite abundance in control soils, demonstrating that the soil pH is one of the most important factors affecting mite densities in graves.
As decomposition enters bloated and active decay, putrefaction produces a myriad of carbon, nitrogen, and phosphorus-based volatile organic compounds (VOCs) as by-products. The mixture of decay fluids, fauna, flora, and highly nutritious VOCs influence the chemical properties of soil, in particular the pH [41]. Cadaver decomposition increases the soil pH because of ammonium (NH + 4 ) production, which is a by-product of the catabolism of amino acids [37,40].
Accumulation of NH + 4 occurs during mid-decomposition when the majority of soft tissue breakdown takes place; hence, explaining why there was an increase in the soil pH beneath all three cadavers from relatively neutral during fresh decay to moderately alkaline throughout bloated to advanced decay. A decline in the soil pH toward the very end of the decay (dry/remains stage) was observed from moderately to slightly alkaline. This can be attributed to a decrease in NH + 4 and other carbon and phosphorus-based compounds in the soil during the late stages of decomposition after completion of soft tissue decay during the active stage. The increase in the soil pH during early to intermediate stages of decay and a decline during the dry stages has been reported in other decomposition studies of exposed and buried cadavers in various soil types [8,10,37,39,60,64,68].
The soil pH is known to impact densities of bacteria and fungi [46], as well as soil meso-fauna such as collembola and nematodes, which in turn act as the main prey for predatory soil mites, such as Mesostigmata, which tend to increase in numbers as decomposition progresses [31,69]. This explains why mite occurrence and abundance were significantly affected by the soil pH during decomposition. Acidic conditions are known to be more favorable for certain mite groups, such as Oribatida mites, while alkaline conditions are less favorable for this group of mites [70].
While the cadaver body temperatures were closely related to the above-ground temperature fluctuations, the ambient temperature did not have a direct effect on the abundance of mites throughout the decomposition process, only displaying an interaction with cadaver soil pH. Similarly, while fluctuations in the ambient relative humidity, rainfall, and snowfall may have had some impact on delaying the rate of cadaver decay, they did not appear to significantly influence cadaver decay or the abundance of mites throughout the decomposition. This agrees with some previous studies showing that seasonal and environmental fluctuations, such as soil moisture content, did not have a significant effect on the abundance of soil mites [71,72], but contrasts with other studies that found a significant correlation between mite abundance and soil moisture content [73,74].
In conclusion, our study show that vertebrate cadavers buried in shallow graves (<30 cm) will undergo the same post-mortem changes and decomposition stages as previously described for exposed cadavers in terrestrial outdoor environments. However, the rate of decomposition is significantly slower and is influenced by a combination of reduced necrophagous dipteran activity and environmental fluctuations. Cadavers buried in shallow graves cause an increase in the soil pH. Previous studies have demonstrated that knowledge on the abundance of mites colonizing decomposing cadavers in relation to environmental conditions contributes with the information on the stage of decay and estimations of the post mortem interval [75][76][77]. This study has demonstrated that an abundance of mites colonizes shallowly buried cadavers, with total mite numbers rapidly increasing between fresh and later stages of decay. We have also shown that fluctuations in the above-ground temperature, humidity, rainfall, and snowfall impact the overall decomposition rate, while fluctuations of the pH of the grave soil significantly and directly influenced the abundance of mites colonizing each stage of the decay.
This last point demonstrates that the increase in soil pH due to decomposition is the major environmental parameter influencing the abundance of mites in shallow graves. The identification of the Acari from this study is being carried out [78], and the analysis of data at order, family, and species level, aims to compare diversity with stages of decomposition as well as body parts in the shallow grave environment.