Abundance and successional patterns of mite groups during stages of decomposition
The mite fauna of soil changes in relation to different stages of decomposition (Bornemissza 1957). Fluctuations in mite populations and succession of species can be used as time proxies, in a similar manner to insects. This study is the first to show that buried cadavers cause a significant increase in mite abundance and diversity in the surrounding soil, attracting a unique composition of mites compared to the local (in situ) soil mite fauna, confirming that mites are an important part of the arthropod succession of buried cadavers. This is also the first study to show that there are successional patterns of mite fauna associated with each decomposition stage of buried cadavers highlighting their importance as forensic markers of the decay stages. Mesostigmata mites were the most abundant and diverse group of mites from cadaver soils followed by Oribatida, Prostigmata and Astigmata. This is not surprising as Mesostigmata mites are free-living and soil-dwelling mites, the majority of which are predaceous and play a fundamental role in decomposition processes of organic material such as carrion in forest soils (Koehler 1999; Krantz 1998). Many predatory Mesostigmata species are phoretic associates of necrophagous and necrophilous Diptera and Coleoptera and therefore an abundance on cadavers is expected throughout decay. Mesostigmatids were more dominant in cadaver soils compared to control soils and were absent in the fresh stages. The Mesostigmata collected from cadaver soils were mainly predatory species, either originating from different soil horizons or arriving via phoresy with necrophagous and necrophilous Diptera and Coleoptera, or with subterranean mammalian scavengers carrying their own phoretic mites (Perotti and Braig 2009).
As expected, the abundance of Mesostigmata mites increased as decomposition progressed. As a cadaver enters the bloated stage, volatile organic compounds (VOCs) are released into the atmosphere, and calliphorids (Diptera) detect and colonise the carcass. Many initial colonising flies will be carrying phoretic mites, which detach from their host organism soon after arrival to a cadaver and proceed to rapidly feed and reproduce (Perotti and Braig 2009; Perotti et al. 2009). This explains the presence of Mesostigmata mites during the bloated stages of all cadavers. As expected for buried cadavers, Diptera were seen ovipositing on the slightly exposed areas of the buried cadavers from the bloated stages onwards and the resulting larvae were seen rapidly moving deep, consuming the soft tissue. As this occurs, there is an increase in food availability resulting in an increased density of the cadaveric fauna (Carter et al. 2007; Goff 1993). Consequently, this attracts a myriad of predatory mite species from the surrounding soil and/or via phoresy explaining why mid-decomposition stages were associated with an overall highest abundance of mites.
In contrast to cadaver soils, Oribatida was the most abundant and diverse group of mites in control soils, followed by Mesostigmata, Prostigmata and Astigmata. Oribatida mites are mostly secondary decomposers, typically associated with soil litter and plant detritus and are expected to be the most abundant group of mites in most soil types as they are representatives of the ‘normal’ soil fauna (Norton and Ermilov 2014). The majority of Oribatida mites do not possess specialised dispersal stages such as those seen with Mesotigmata, Prostigmata and Astigmata and their dispersal abilities are limited (Krantz and Walter 2009). Whereas Oribatida mites were present throughout the decay of P1 and P2 they were always less abundant than Mesostigmata mites during bloated, active and advanced stages but more abundant during the fresh stage. Most do not have phoretic associations with necrophagous insects and, therefore, the oribatids associated with the cadavers were most likely to be those already present in the soil or opportunistic oribatids migrating from other soil horizons.
In cadavers, although predatory Mesostigmata mites dominate, soil-dwelling oribatids are thought to disappear due to predation or migration away from the cadaver in response to environmental changes in the soil—e.g., increased soil pH—soon after a cadaver is introduced (Bornemissza 1957). In contrast, the results of this study demonstrate that the Oribatida mites did not entirely disappear on introduction of a cadaver as there was a slight increase after fresh decay of P1 and P2 and they occurred, although in reduced numbers, throughout most of the stages. However, their abundance did fluctuate and their abundance was always lower compared to cadaver soils and they were entirely absent during bloated and active decay of P3, when Mesostigmata mites were the most abundant. Their lower abundances and temporal absence may have been because Oribatida mites are predated by Mesostigmata mites. Most Oribatida species are saprophages and mycophages, only a minority are opportunistic species and feed on nematodes and micro-organisms such as fungi and bacteria that occupy ephemeral habitats such as a decaying cadaver (Krantz 1978; Norton and Ermilov 2014), explaining why they were not entirely absent from cadaver soils. Interestingly, Oribatida mites increased in abundance during the late stages of decay. The decrease in Oribatida mites as well as other mites from the Astigmata and Prostigmata during the dry stage of P2 may be explained by the presence of a greater number of the predatory Mesostigmata species Macrocheles matrius during this stage, compared to the dry stage of cadaver 1 and 3.
Our results also demonstrate that soil pH had a significant and positive effect on the abundance of mites. Therefore, differences in soil pH from cadaver soils are likely to have contributed to the small variations in patterns of mite abundance between the pig cadavers, for example, mite abundance decreased during advanced decay in P1 and P3 when soil pH also decreased, whereas mite abundance increased in P2 during advanced decay when soil pH increased (alkaline). Certain mite groups are known to be sensitive to soil pH, with acidic conditions more favourable to Oribatida than Mesostigmata mites in forest soils, and Oribatida less abundant in alkaline soils (Maraun and Scheu 2000). This pattern was reflected in our results: in P1 and P3, as soil pH became less alkaline in advanced decay, the abundance of Mesostigmata mites decreased, whereas in P2 the alkalinity of the soil pH increased and an increase in Mesostigmata mites was recorded. The increment in Mesostigmata numbers in advanced decay of P2 was mainly a dominance of Parasitidae mites; many Mesostigmata families such as Parasitidae can thrive in moderately alkaline conditions (Manu et al. 2021). In P1 and P2, where the pH declined slightly, the abundance of Parasitidae from active to advanced remained the same. By contrast, during active and advanced decay of P1, soil pH was less alkaline than in P2 and P3 and interestingly, a slightly higher abundance of Oribatida mites was noted. This change may be the result of indirect effects on the density of food sources for Oribatida mites, as soil pH fluctuations influence densities of bacteria and fungi. Species of Oppiidae and Quadroppiidae, which feed on these micro-organisms, were present during late decomposition when the soil remained slightly alkaline for all cadavers.
Astigmata mites were recovered in relatively low abundance, being the least diverse mite group in cadaver and control soils. Their occurrence did not follow any successional pattern in response to decomposition. Prostigmata mites appeared to have some significant association with decay stages and were more abundant than in control soils. In terrestrial ecosystems, Prostigmata mites have diverse feeding methods in soils and are known to be phytophagous, saprophagous, parasitic, paraphagous with insects as well as predaceous. Prostigmata mites are not commonly thought to be significant members of the carrion fauna as only a small number of Prostigmata species is predaceous, whereas most are parasites and parasitoids of insects (Eickwort 1990). Prostigmata mites appeared to be mainly associated with bloated, active and advanced stages of decay. Their reduction/absence in the fresh stages and in control soils may suggest that they are just opportunistic. Some species of Prostigmata are phoretic on Phoridae, ‘coffin flies’ (Fain and Greenwood 1991; Perotti et al. 2009), flies that are common in graves (Martín‐Vega et al. 2011; Motter 1898) and may bring along these phoretic prostigmatids.
Biodiversity of mites during stages of decomposition
In soils, the structure and dynamics of mite groups change in response to food availability and changes in environmental conditions (Behan-Pelletier 1999). The impact of vertebrate graves on the temporal succession and diversity of mite species is poorly understood; but variations in arthropod species richness in response to decay is known to occur in terrestrial environments (Schoenly and Reid 1987). The present study demonstrates that the biodiversity of mites in cadaver soils was higher during each decay stage compared to the corresponding control soils, apart from during the fresh stage (according to Shannon diversity index). Mite richness, diversity and evenness also fluctuated in control soils; however, this was likely a result of variations in spatial distribution of mites during the study, perhaps responding to seasonal effects, or temperature changes throughout the year.
In this study, active decay was the most species-rich and diverse decomposition stage (Shannon diversity index), whereas fresh decay was the least species-rich and least diverse stage. Biodiversity began increasing during the bloated stage, coincidentally with abundance of VOCs, produced through putrefactive processes attracting insects. Calliphorid eggs are a source of food and attract an abundance of predatory soil mites (Braig and Perotti 2009). Bloated stages showed the second highest diversities, confirming that shallow concealment still allows an adequate released of volatiles. As Diptera larvae develop during active decay, soft tissue breakdown is optimal and more VOCs are produced (Carter et al. 2007). This creates a highly nutrient-rich environment with ample food for a variety of arthropods, therefore active decomposition is associated with the greatest diversity of arthropods (Early and Goff 1986). Although the abundance of soil mites during the advanced and dry stage was still relatively high, the richness and evenness of species declined during these two stages. As the cadaver enters the advanced and dry stage, the amount of soft tissue present and arthropod activity is minimal and the food supply is gradually depleted resulting in a decline in faunal diversity (Carter et al. 2007), which is in line with the results of this study.
Successional patterns of mite families and species during stages of decomposition
Mesostigmata families and species
In terrestrial environments Parasitidae, Macrochelidae, Ascidae and Uropodidae mites are the most common associates of necrophagous insects and cadaveric decay (Perez-Martinez et al. 2019; Perotti and Braig 2009). This partially agrees with the results of this study as Parasitidae and Macrochelidae were the most abundant and diverse mite families. Interestingly, Ascidae and Uropodidae mites were not significantly abundant in the grave environment. Non-phoretic and phoretic Parasitidae species, especially from the genera Parasitus and Gamasodes, associated with necrophagous insects have been recovered from various types of decomposition scenes and stages in terrestrial environments, mainly on the surface (González-Medina et al. 2013; Kamaruzaman et al. 2018; Perez-Martinez et al. 2019; Reed 1958; Saloña-Bordas et al. 2010; Saloña-Bordas and Perotti 2014).
In studies of graves, Parasitidae mites can colonise shallow as well as deep graves (Goff 1991; Rai et al. 2020; Vanlaerhoven and Anderson 1999). Parasitidae mites increased in abundance as the cadaver progressed from bloated, active and advanced decay and decreased during the dry stage. Their colonisation of cadavers during these stages is primarily due to the arrival of their phoretic hosts, as many deutonymphal Parasitidae are phoretic with necrophagous and necrophilous flies (Fain and Greenwood 1991; Hyatt 1980; Rai et al. 2020; Saloña-Bordas and Perotti 2019) and beetles (Costa 1963; González-Medina et al. 2013; Hyatt 1980; Schwarz and Walzl 1996; Schwarz and Müller 1992). This is reflected by the results of this study where they were significantly more abundant in cadaver soils, suggesting that most species associated with the shallow graves are phoretic Parasitidae.
Cornigamasus lunaris (Parasitidae, Mesostigmata) was significantly associated with cadaver soils, and was found to be an indicator species of the bloated stage. This species is known to rapidly inhabit ephemeral habitats such as decomposing plant matter, dung and compost across Europe (Witaliñski 2005). Phoretic deutonymphs have been recovered from necrophilous insects visiting carrion outdoors (Perez-Martinez et al. 2019) and from a human cadaver indoors during an unspecified stage of decay (Anderson 1995). In Europe, phoretic deutonymphs have been found on dung beetles (Kirk 1992) and residing in the nests and fur of subterranean rodents (Várfalvyová et al. 2011). Even though a few individuals were found in control soils, its significant association with cadaver soils may suggest that the prevalence of C. lunaris in graves is related to phoresy on coprophagous and coprophilous insects. This species is considered coprophilous, its arrival at the bloated stage may be due to phoresy on early-colonising insects, on visiting rodents attracted to the volatiles, or because the graves were near small mammal nests.
Gamasodes spiniger (Parasitidae, Mesostigmata) was not significant to any soil type, but was an indicator species of active decay. It is a phoretic of necrophagous Diptera and Coleoptera (Copris hispanus) (Costa 1963). Phoretic deutonymphs of G. spiniger were collected attached to lesser dung fly, the Spelobia fly (Sphaeroceridae) during active decay of two human corpses in a shallow grave concealed with manure (Rai et al. 2020). It is a known traveller on Sphaeroceridae flies (Lundqvist 1998, Samsinak 1989), its occurrence in graves is expected as Sphaeroceridae are extremely common due to their ability to access the cadaver via small crevices in the soil (Pastula and Merritt 2013). Gamasodes spiniger is coprophilous, its affinity to active decay might be a result of the purging of internal fluids occurring during this stage (which includes faecal material). Gamasodes spiniger has been documented from surface carrion during different stages of decomposition (Anderson and VanLaerhoven 1996; Lundqvist 1998), suggesting that in graves this species is a strong marker of active decay as a result of phoretic arrival with small lesser dung flies attracted to decay fluids containing faecal material. Its association with both cadaver and control soils (though in greater abundance in cadaver soils) suggests that it can colonise shallow graves from the surrounding soil as well as phoretically with Diptera.
Vulgarogamasus remberti (Parasitidae, Mesostigmata) was found to be significantly associated with cadaver soil, but no significant association was found with decay stage, body region or a pig cadaver subject. This species is especially common in the fur and nests of above- and below-ground animals such as birds and moles (Hyatt 1980; Mašán and Stanko 2005). There is only one study documenting its occurrence with surface decay, attributing its phoretic arrival by visiting shrews (Perez-Martinez et al. 2019). Cadavers buried in graves of less than 30 cm deep are not entirely protected from small mammalian scavengers such as rodents (Rodriguez and Bass 1985). Small rodents can access cadavers in shallow graves via the surface or through subterranean channels and bring along predatory phoretic mites associated with their fur or species that reside in their nests. The colonisation of V. remberti and other Vulgarogamasus species found in the cadaver soils (V. kraeplini and V. oudemansi) is also likely due to phoretic arrival with subterranean small mammals visiting the cadavers for food.
Parasitus evertsi (Parasitidae, Mesostigmata) was significantly associated with the posterior region, with no association with either soil type or stage of decomposition. In England P. evertsi inhabits caves, tree holes, fur of shrews and other small mammals, soil and fungi (Hyatt 1980). It has been recovered from Yew (Taxus bacata) in England (Skorupski and Luxton 1998). Its association with cadavers is likely to be incidental as this species is already native to forest soils where it colonises decaying vegetation rather than carrion (Skorupski and Luxton 1998).
Eugamasus sp. (Parasitidae, Mesostigmata) was a significant indicator of advanced decay and it was specifically associated with the advanced decomposition of P2. This genus has phoretic associations with beetles (Moser and Roton 1971), its absence from control soils suggests arrival to the cadaver via phoretic dispersal perhaps from beetles rather than from the surrounding soil. Eugamasus colonization may be concurrent with occurrence of nematodes, which are known to increase in density and diversity during active, advanced and dry stages (Szelecz et al. 2016). Well studied species as E. cavernicolus inhabit caves, tunnels and nests of subterraneous mammals (Fenďa and Lukáš 2014).
The second most abundant and species-rich mite family in cadaver soils was Macrochelidae. Macrochelidae species have been recovered from cadavers during all stages of decay, in exposed (Goff 1989; Kamaruzaman et al. 2018; Saloña-Bordas et al. 2010; Szelecz et al. 2018) as well as buried cadavers (Anderson and VanLaerhoven 1996; Goff 1991; Kamaruzaman et al. 2018; Rysavy and Goff 2015; VanLaerhoven and Anderson 1999). Their high abundance in cadaver soils is not surprising as Macrochelidae species are predatory and are involved in the decomposition processes of ephemeral organic matter such as carrion, dung, compost and other organic materials. They prey on successive waves of Diptera and Coleoptera eggs and larvae and nematodes as well as a variety of micro-arthropods including Collembola (springtails) and other mites (Krantz 1998).
Macrochelidae mites may visit cadavers throughout decomposition from the early to the dry stages as female Macrochelidae mites are phoretic with necrophagous flies and carrion beetles (Barton et al. 2014; Perotti and Braig 2009; Perotti et al. 2009; Saloña-Bordas and Perotti 2015). Female Macrochelidae mites are amongst the first mites to colonise exposed cadavers in early stages with necrophagous Diptera (Early and Goff 1986; Goff 1989; Perotti and Braig 2009; Perotti et al. 2009; Rysavy and Goff 2015). The time of cadaver colonisation by Macrochelidae mites is dependent on the species, as some species are phoretic on early-colonising Diptera and will colonise during the early stages, whereas other species are phoretic on late-colonising Coleoptera and may arrive at later stages and feed on the eggs of flies (Early and Goff 1986; Goff 1991; Leclercq and Verstraeten 1988; Reed 1958). Colonisation patterns of Macrocheles mites in particular have complemented information on the time of death in case studies (Goff 1991; Kamaruzaman et al. 2018; Szelecz et al. 2018). In this study, they colonised at the bloated stages, likely to have arrived with early-colonising insects and decline in numbers during active and advanced decay, perhaps due to competition with a greater abundance of predatory Parasitidae mites. Their re-appearance in abundance at dry stages concurs with a reduction in Parasitidae. This study demonstrates that in graves Macrochelidae mites appear to colonise mainly during later stages of decay rather than early stages such as seen on surface decomposition.
Macrocheles is the most diverse genus of this family and the majority of Macrocheles species colonise cadavers through phoresy by virgin females, attaching to necrophagous and necrophilous Diptera and Coleoptera (Kamaruzaman et al. 2018). Macrocheles matrius was the most abundant in the present study. It most commonly inhabits poultry litter and farms and granaries, and is thought to be exogenous to forest soils but may exist in some forest habitats. In this study, it was a marker of cadaver soil and of the dry/remains stage, and was associated with the posterior body region. Macrocheles matrius has been found in the soil up to 10 cm deep beneath the exposed and dry human bones in a forest, agreeing with its association with the dry stage (Szelecz et al. 2018). It is a species mainly phoretic on mammals (Krantz and Whitaker 1988) and has been recovered from the nests of mound-building mice (Mašán and Stanko 2005). It may utilise small mammals to locate and colonise cadavers. Importantly, M. matrius has already been found utilising cadaver-associated dung beetles such as Geotrupes silvaticus for phoretic dispersal (Hughes 1976) which locates the beetles and the mites in the posterior area of the body, near the anus. In crime scenes, access to specific body parts by insects carrying mites, and thus occurrence of species-specific phoretic mites, can add information on localization of wounds, helping describe the circumstances related to death. For example, if the victim was attacked and/or where and which of the injuries might have caused the death; or, what type of attack was performed, was the victim raped? The presence of this species in the cadaver soils and colonisation during the dry stage is likely to be in accordance with the presence of Diptera and Coleoptera eggs and larvae as well as other mites which this species predates on (Soliman et al. 1978). The association with the body posterior region may explain its phoretic arrival with dung beetles, attracted to the volatiles from the faecal matter released with decay fluids from the anal region during active decomposition (Kamaruzaman et al. 2018).
Other Mesostigmata families colonised the cadavers in lower numbers throughout decomposition, such as Diagamasellidae, Pachylaelapidae, Ascidae, Diathrophallidae, Uropodidae, and Protodinychidae. Digamasellidae species are mainly predatory and feed on cadaveric fauna such as Diptera eggs and early instars, nematodes, fungi and Collembola (Walter et al. 1988), and deutonymphs are phoretic with necrophagous flies such as Muscidae (Pereira Sato et al. 2018). Muscidae flies are common in graves due to their smaller size compared to larger calliphorids (Gaudry 2010). Pachylaelapidae have been collected from a human cadaver in a shallow soil grave undergoing skeletisation (Goff 1991). In our study, Pachylaelaps longisetis was not significantly associated with cadaver soils but was significantly associated with only C2 (in two samples). This species has a patchy distribution in forest soils and is an unlikely marker of the grave fauna. Members of Pachylaelaps may occur in shallow graves incidentally, as P. longisetis has been recovered from bare soil samples up to a depth of 5 cm from spruce forests (Skorupski et al. 2009) and patchy distributions of soil organisms can result from varying soil organic matter content (Fromm et al. 1993).
Ascidae mites occurred during every stage of decay in low numbers (> 5 individuals) except in the fresh stage, suggesting that Ascidae mites are attracted to decomposition in shallow graves. Ascidae mites occupy a wide range of microhabitats in forest soils from the subsoil to leaf litter, and in dry as well as wet environments (Kalúz and Fenďa 2005), explaining their occurrence during the bloated, active, advanced and the dry stages. These mites are phoretic with Diptera of forensic importance (Perotti and Braig 2009). Ascidae mites have been recovered from exposed cadavers in past studies in forest soils (Perez-Martinez et al. 2019; Saloña-Bordas et al. 2010). This is their first report from graves. Diarthrophallidae mites have an exclusive commensal association with Passalidae beetles (Krantz and Walter 2009). They were found in low numbers (< 5 individuals) during active and advanced decay and were absent in control soils suggesting that their carriers were attracted to decomposition.
In graves, Uropodidae mites appear to be common during the skeletonised stages. For example, mites from the infra-order Uropodina (includes the family Uropodidae) were collected from buried skeletonised human remains (Goff 1991; Mariani et al. 2014), whereas Uropoda depressa (Uropodidae) was amongst the most abundant species collected from human graves by Motter (1898). The results of this study showed only one Uropodidae mite found during the active stage and one during and advanced stage of P2. Likewise, only two individuals of Protodinychidae were recovered in the bloated stage of P1 and were absent in control soils. The reason of their absence during this study could be due to this study finishing at the beginning of skeletal remains. Thus, their absence may suggest association with later skeletal remains, rather than with early stages. We cannot ignore that there might be attraction of Uropodidae to human cadavers, as this experiment was conducted with pig-carcasses.
Oribatida families and species
After introduction of the cadavers, there was disappearance of some incidental Oribatida mites such as fungivorous families Damaeidae and Suctobelbidae (Miko and Mourek 2008). These oribatids are likely to have moved away from the cadaver soon after its introduction due to changes in soil pH that led to a more alkaline soil environment. Scheloribates laevigatus (Scheloribatidae) was an indicator of control soils. Scheloribates laevigatus are soil-dwelling mites which inhabit the surface of soils residing in moss, humus and decaying wood and are microbial feeders (Hughes 1976). Until now its association with cadavers was almost unnoticed, but they might feed on microbes associated with decay in graves (Anderson and VanLaerhoven 1996). Two species of Oribatida were found to be significantly associated with the dry stages of P1 only: Quadroppia michaeli (Quadroppidae) and Ramusella clavipectinata (Oppidae). Colonisation during the dry stages may be due to the soil conditions normalising towards the end stages (Mariani et al. 2014; Walter and Proctor 1999). Their low numbers or absence in all other samples might suggest a patchy spatial dispersal in forest soil. Oribatida mites are known to exhibit specific spatial distributions in soil habitats due to their limited ability to disperse and their association to soil parameters such as microbial biomass and organic matter content (Minor 2011). Quadroppia michaeli and R. clavipectinata are fungivores and association to dry stages may result by attraction to fungal growth on bones (Hawksworth and Wiltshire 2011). The Oppiidae family is one of the very few Oribatida groups that display phoresy with beetles (Norton 1980), and R. clavipectinata has been found to be phoretically associated with Curculionoidea beetles (Ermilov and Frolov 2019). This species may have arrived with beetles in the dry stage to exploit the characteristic fungal growth on the bones at this stage.
Prostigmata families and species
This is the first study to highlight the occurrence of Prostigmata mites in graves. Most Prostigmata were Tydeidae and they were mainly associated with advanced decay, represented by a single species, Lorryia reticulata. Lorryia reticulata was a marker of cadaver soils, of advanced decay and P3. Until now, its association with decay, especially in graves was unknown. It was also significantly abundant in C3, in the samples taken while P3 was bloated. This family of mites has extremely diverse feeding strategies although most feed on algae, fungi and pollen. Tydeids occupy a large variety of micro-habitats within forests from soil litter, plant detritus, bark and hollows of trees to bird nests (Kaźmierski et al. 2018). Phoresy is uncommon in this family (Treat 1975). The significant association with the advanced decay is likely to have been opportunistic, arriving from the surrounding soil layers in order to feed on associated mites and their eggs, nematodes and fungi.