Introduction

The scientific study of human death and its effects on the environment is called forensic taphonomy which focuses on the decomposition process and the ecological influences that affect these changes1. This field examines the decomposition of cadavers in various environments, including different burial conditions, and the role of external factors like temperature, soil composition, burial depth, and insect activity2,3. In any homicide investigation, the precise estimation of postmortem interval (PMI) is very crucial. It refers to the period between death and subsequent retrieval of the dead body. There are numerous approaches for determining the postmortem interval that comprise early and late post-mortem changes. In cases where the bodies are retrieved in the advanced stages of decomposition wherein traditional methods fail in the assessment of PMI, forensic entomology can be very helpful in estimation of minimum postmortem interval (PMImin) on and under the ground4. The PMImin refers to the entomologically estimated time since death. It is based on the principle that insects typically associated with decomposing remains colonize a carcass just after death. Accordingly, assessing the age of the oldest immature insect specimen collected from the remains, in conjunction with the expected arrival time of adult females, provides an estimate of the minimum duration for which the remains have been available for insect colonization, thereby establishing the PMImin5.

Forensic entomology is the scientific discipline that focuses on the use of insects in criminal investigations6. Flies are known to arrive and colonize the cadaver shortly after death7. In the course of their work, forensic entomologists encounter bodies in a variety of conditions. These include bodies that are found on the surface, buried, floating, burned, indoors or outdoors, wrapped, located in high-rise buildings, as well as in a variety of other situations8. In each situation, access of insects to the body may differ, so various influencing conditions are required to be taken into consideration when arriving at a PMImin. Burial is one of the most used methods to conceal the body in homicidal cases. Perpetrators may choose burial as it can effectively hide evidence of the crime for an extended period, particularly if the grave is deep and well-hidden. Whereas shallow graves may be a practical choice in cases where time or resources are limited. Therefore, most clandestinely buried cadavers are found at various depths in a range of 30 to 90 cm9.

The decomposition process, rate, and insect access in buried environments are influenced by various factors, including soil type, soil pH, temperature, humidity, and burial depth10,11. Burial depth significantly impacts the accessibility of decomposing remains to insects; deeper burials may deter insect colonization, while shallow graves are more likely to be discovered by insects3,12. The decomposition stages in the buried environment are similar to those on the surface, but the decomposition rate of cadavers in the graves is slower13. Soil covers may restrict insect accessibility in burial environments and, consequently, decrease the decomposition rate3. It should however be noted that graves will not be devoid of insects, so the study of succession patterns of associated insects may be used in the investigation of a crime14. Blowflies (Calliphoridae) are the early colonizers on the exposed cadavers15, but in the graves especially deep burials, these species may not have access to the carcass16,17,18,19. In the buried environment the Phoridae, Sarcophagidae, and Muscidae are the most prominent ones followed by the coleopterans20.

By understanding the unique conditions and challenges posed by burial, forensic scientists can develop better tools and techniques to aid in legal investigations and contribute to the broader understanding of decomposition processes21. Although the forensic entomology field has seen significant advancements on the terrestrial surface, research in the context of buried environments remained relatively underdeveloped. Therefore, the purpose of the present study was to examine the decomposition pattern, insect access, and colonization on the buried carcasses at two different depths as well as on the terrestrial surface in the spring season. The frequent exhumation of buried carcasses also has a considerable effect on the decomposition rate and insect diversity, so, another focus of the present study was to compare the decomposition rate and entomofauna associated with undisturbed and frequently exhumed buried carcasses.

Methods

Experimental site and experiment design

The present study was conducted at the Entomological facility at Maharshi Dayanand University, Rohtak during the spring season for two consecutive years (March to May 2022 and March to May 2023). Rohtak is a semi-arid region in Haryana with coordinates 28.8955° N, 76.6066° E. This site was chosen for the study because it is free from any human activity, and this is a bushy area with plenty of insect activity.

Slaughtered pigs (Sus scrofa) of similar weight with an average of 40 kg were purchased from the local slaughterhouse. Pig carcasses were covered with a tarpaulin sheet and immediately transported to the study site to avoid any invasion of the insects before the experiment started.

A total of ten pig carcasses were used for the present study. Two pairs of pigs were buried at 40 cm and 80 cm depth respectively and one was placed above the ground during the period of March to May 2022. The remaining five pigs were used in the replicated study (March to May 2023). An excavator (Manufacturer JCB excavator Ltd.) was used to dig burial pits at a depth of 40 and 80 cm each (with measurements of 1 × 0.76 m (length and breadth)) with a 10 m distance between them to avoid the cross interference of the insects. The pits were excavated 30 min before the burial of the carcasses to prevent early colonization of the graves by terrestrial insects. The pig carcasses were placed in the cages (dimensions 90 × 60 × 40 cm with mess size 3 × 3 cm) and buried immediately after being placed in the burial pits. One pig carcass was exhumed frequently, whereas the other was left undisturbed, to compare their decomposition and the associated entomofauna at both the depths. The subaerially exposed carcasses were placed in cages with the same dimensions used for buried carcasses to avoid scavenger activity and allowed to decompose in the natural environment. To record the daily temperature and humidity data, a digital thermometer (Elitech-RC-4HC) was placed along with the pig carcasses on and under the ground. The experimental setup was the same for the replicated study. For understanding purposes, the pig carcasses have been coded as shown in Table 1.

Table 1 Shows the codes given to the pig carcasses.
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The sampling of the buried carcass (B40EF2022) was conducted on days 8, 15, 22, 30, 45, 60, 70, and 90 at a depth of 40 cm. Similarly, at 80 cm depth (B80EF2022 carcass) the sampling was carried out on days 8, 15, 22, 29, 35, 50, 65, 80, 110, 120, 150, 180, 210, and 240, and the decomposition stages were observed accordingly. Throughout the exhumation process, the grave soil was continuously hand-sorted for the presence of entomofauna. The insect activity around the graves was also observed for the carcasses which were kept undisturbed. The carcass (T2022) placed above the ground was monitored from morning to evening hours to observe the insect activity on and around the carcass. The adult insects were collected with the help of nets and forceps and their immature stages were collected by using the brushes and forceps and preserved in 70% ethanol for further identification. Morphological keys were used to identify the insects22,23,24,25 and were also confirmed by the Zoological Survey of India (ZSI), Kolkata, India. The standard error for the average temperature and humidity above and below the ground was calculated using the IBM SPSS 23 statistical software.

Results

The average temperature and humidity (± S. E) recorded in the spring season was 31.8 ˚C ± 0.50 and 40.6% ±1.16 respectively above the ground in both years. However, the average temperature in the graves was recorded as 22.8˚C ± 0.14 and 20˚C ± 0.16 respectively at 40 and 80 cm depth (Fig. 1). On the other hand, the average humidity in the buried environment was 40 ± 1.13 and 55% ± 0.64 respectively at both depths (Fig. 2). The average rainfall recorded was 41.6 and 190.8 mm during 2022 and 2023 respectively.

Fig. 1
Fig. 1
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Daily average temperature variations above and below the ground throughout the study period.

Fig. 2
Fig. 2
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Daily average humidity variations above and below the ground throughout the study period.

Fresh, bloated, active decay, advanced decay and dry stages of decomposition were observed for the carcasses above and below the ground. The stages of decomposition were similar at both the depths. Time taken by all the carcasses in each stage of decomposition is represented in the Fig. 3.

Fig. 3
Fig. 3
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Comparison of duration of stages of decomposition of carcasses placed above the ground and in the graves.

Approximately 1800 insect specimens from Diptera, Coleoptera and Hymenoptera orders were collected from the carcasses T2022 and T2023 above the ground. While approximately 950 insect specimens were collected from the buried carcasses. Insects belonging to orders Diptera, Coleoptera, and Isopoda were common on the carcasses (B40EF2022 and B80EF2022) buried at 40 and 80 cm depths respectively. Species belonging to order Hemiptera were found visiting the buried carcass B80EF2022 at 80 cm depth. The decomposition stages and entomofauna associated with the carcasses above the ground and in graves can be described as follows (Tables 2 and 3):

Table 2 Decomposition and insects associated with the pig carcasses on terrestrial surface.
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Table 3 Decomposition and insects associated with the pig carcasses buried at 40 and 80 cm depths.
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During the exhumation of undisturbed carcass buried at 40 cm depth (B40EO2022), it was observed that there was more tissue left as compared to the disturbed one (B40EF2022). During the exhumation only adults of beetle Saprinus sp. and Necrobia rufipes were found. Amongst the dipterans, pupal cases of Megaselia scalaris were encountered. The B80EO2022 carcass buried at 80 cm depth was found to be in the early dry stage and tissue loss was lesser as compared to B80EF2022 carcass. Undisturbed carcasses (B80EO2022 & B80EO2023) were found devoid of any insect activity at this depth. The number of insect species from different orders is shown in the Table 4.

Table 4 Number of insect species on the carcasses above the ground and in the buried environment.
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Discussion

A cadaver’s decomposition in a buried environment is a multifaceted process and can be affected by several variables such as soil composition, temperature, moisture, and burial depth. A lesser temperature difference was recorded in the buried carcasses at 40 and 80 cm depths as compared to that on the surface. The depth of burial could be the reason behind this because the carcasses in graves were protected by the soil coverings and weren’t exposed to direct heat. The rate of decomposition was also found to be slower for the carcasses (B40EF2022 and B80EF2022) buried at different depths as compared to the subaerially exposed carcass (T2022). Similar findings have also been observed in previous studies9,13,18,26,27,28,29 who noted that the decomposition gets prolonged due to reduced exposure to oxygen, temperature and access of insects to carcass.

It was interesting to observe that the rate of decomposition also varied at different burial depths. The decomposition rate was found to be much slower for the carcass (B80EF2022) buried at a depth of 80 cm as compared to the one buried at 40 cm depth (B40EF2022). The depth of burial is inversely proportional to the accessibility of the insects to the cadaver. Troutman et al.30 pointed out that the decomposition of the buried cadaver can take up to 8 times longer than the cadaver placed above the ground, which was also proven in the present study for the carcass buried at 40 cm depth. The B40EF2022 carcass took up to 7 times longer to decompose than the T2022 carcass. While at the 80 cm depth, this duration was even longer. A study at 75 cm depth conducted by Werner et al.13 too has reported that the decay rate of buried carcass can be 55 to 83% lesser than the surface remains which was found to be similar to the present study at a depth of 80 cm whereby the decay rate of B80EF2022 was 90% lesser than the carcass (T2022) on the terrestrial surface.

It was observed that frequent exhumation of a buried carcass significantly impacted the decomposition process and insect fauna. Each exhumation disturbs the burial environment, altering factors such as temperature, moisture levels and introduction to new insect species. The undisturbed carcass buried (B40EO2022) was found to retain more tissue mass as compared to the disturbed one (B40EF2022) during the exhumation which can be credited to the presence of diversity of insects on the disturbed one (B40EF2022). Although both carcasses were at the dry stage, a substantial amount of tissue mass remained underneath the dry skin in the B40EO2022 carcass owing to the presence of only two beetle species (Saprinus sp. and Necrobia rufipes) and one dipteran species (Megaselia scalaris). The replicated carcass B40EO2023 was also in the dry stage when exhumed and the tissue mass loss was almost same for both carcasses (B40EO2022 & B40EO2023). Bonacci et al.17 have also worked on the influence of frequent exhumation on the insect’s arrival on the buried carcasses. Though they found that the buried carcasses were in adipocere condition when exhumed only once with no colonization by the insects, they too have reported that frequently exhumed carcass was in the advanced decay stage at that time with a lot of activity of the insects. The present study is in congruence to Bonacci et al.18 for undisturbed carcasses (B80EO2022 & B80EO2023) buried at a depth of 80 cm wherein no insect activity was found. Undisturbed carcasses (B40EO2022 & B80EO2022) at 40 cm and 80 cm depths also showed significant differences in decomposition rates. The decay rate was found to be slower at 80 cm depth as compared to the 40 cm depth. It was found that there was more tissue on the carcass B80EO2022when it was exhumed only once as compared to the B40EO2022. These differences can be attributed to the depth of the burials. The forensic entomologists must carefully assess the burial site for signs of disturbance and consider these factors which might have altered the typical decomposition process. Frequent exhumation tends to lead to more rapid decomposition processes due to increased insect activity and variable microclimates (more exposure to the air). While undisturbed environments result in slower decomposition rate due to the lesser insect activity.

The succession pattern of entomofauna was the same for all the carcasses in both environments. Dipteran species were the early arrivers as well as the colonizers on the carcasses followed by the coleopterans. Variation in the arrival of insect fauna was observed above and below the ground in terms of the individual species. Blow flies (Calliphoridae) were the earliest visitors and colonizers followed by species belonging to Sarcophagidae and Muscidae families above the ground (T2022 and T2023) and this has been reported in the previous studies in different seasons28,31,32,33,34. According to Mabika et al.35 species belonging to Calliphoridae were also the early colonizers followed by Sarcophagidae. Musca domestica and Sarcophaga sp. have also been reported previously28,36,37. Piophilla casei species was also observed on the subaerially exposed carcasses (T2022 and T2023) during the present study and have also been reported by Bonnaci et al.18 and Thakur and Kumari36. Sacrophaga haemorrhoidalis, P. casei, and hister beetle have also been reported in India on the rabbit carcasses38. Dermestes maculatus and N. rufipes species were early colonizers amongst the coleopteran species followed by the Saprinus sp. These beetle species have also been observed in previous works32,36,39. As the decomposition process progresses, beetles are known to be of great forensic importance31. Adults and larvae of beetles are well recognized to consume dried tissue and remains40. Chrysomya megacephala, C. rufifacies, M. domestica, Dermestes maculatus, Necrobia rufipes, Histeridae, and Formicidae have also been mentioned in a previous study34.

Megaselia scalaris was found to be active on the buried carcasses (B40EF2022 and B80EF2022) at both depths in the current study. It was the only fly species which was able to access and reach the carcass (B80EF2022) buried at 80 cm depth during the present study. Due to its smaller size, M. scalaris is more easily able to make its way through the cracks in the soil in order to access the buried carcasses41. They prefer the darkness and narrow spaces and their ability to dig to various depths was confirmed in the present study because they were found at both the depths. Megaselia scalaris have also been reported in the previous studies at different depths9,17,18,29,42. Similarly, Pastula and Meritt9 reported the colonization by this fly at both 30 and 60 cm depth. They also reported the colonization by flesh flies (S. bullata) at 30 cm depth and of H. capensis at both 30 and 60 cm depth. These two species were not found during the current study. In the present study S. haemorrhoidalis colonized the buried carcass (B40EF2022) along with the M. scalaris. Megaselia scalaris has also been reported in India on the buried goat carcass15. Musca domestica was found to be active on the buried carcass (B40EF2022) at 40 cm depth which has also been recorded on the corpses buried at different depths28,43. Bourel et al.16 examined the coffins buried at 60 cm depth in Northern France. They found 3 species of Phoridae family, but they didn’t report the M. scalaris at this depth in any of the exhumations. But in the present research, M. scalaris was the dominant fly at both the depths. This can be credited to the change in the environmental parameters in both the regions and forensic entomologists should also take into consideration the influencing factors of the particular area.

Necrobia rufipes (Cleridae) and Syntomus foveatus (Carabidae) arrived on the carcasses (B40EF2022 and B80EF2022) at both depths. Necrobia rufipes species has been reported by Pastula and Merritt9 and Bala and Kaur44 at different depths and other species of Carabidae family have also been recorded in previous works18,44. Bala and Kaur44 only documented the presence of beetles on the buried pork liver and no flies were found on the porcine liver. On the contrary, during the present study the fly species were able to access and colonize the buried carcasses at both depths. In the present study, Anthelephila caeruleipennis were found at the 80 cm depth. Correa et al.26 too reported the Anthicidae species at the 30 cm depth, but they considered it as an incidental insect because of their low abundance but in the present study these insects were higher in numbers at 80 cm depth and were found on three different sampling occasions.

In forensic investigations, understanding the process of carcass decomposition at different depths can greatly improve the accuracy of estimation of PMI. The decomposition rate is impacted by various factors, including temperature, and moisture which vary with depth. By examining these variations, forensic scientists can develop more precise models to determine the time since death. This can be mainly critical in criminal cases where accurate PMI can impact the direction of investigations and the delivery of justice. The present study was the first one to study the effects of burial depths on the decomposition and associated entomofauna in a semiarid region of India. The results of the study showed differences in the decomposition rates and entomofauna on carcasses above and below the ground, signifying their importance to investigate cases with buried bodies in a similar condition. With the identification of these species, their temporal arrival and colonization, forensic entomologists can take one step closer to estimating the PMImin for buried cadavers. This study presents first-hand data on the arrival and colonization of the pig carcasses by the entomofauna in burial environment at two different depths. In addition to this, it also presents a comparison of the decomposition and insect fauna associated with the disturbed and undisturbed buried carcasses. The results point to the fact that the decomposition rate, insect arrival and colonization is greatly influenced by the frequent exhumation. Therefore, more research in this area is required to study the decomposition pattern and entomofauna associated with the undisturbed buried carcasses in real time.

Conclusion

The decomposition rate and insect fauna of carcasses exhibit significant differences in the burial environment and on the surface. The present study also concludes that environments significantly influenced the decomposition rate and insect fauna of carcasses. The carcasses decompose at the fastest rate on the surface due to direct exposure to environmental conditions which act as a driving force. Carcasses buried at a depth of 40 cm exhibited a slower decomposition rate compared to surface carcasses but decomposed more rapidly than those buried at 80 cm depth. The diversity and number of insect species associated with carcasses were found to be different at varying depths and on the surface. Surface carcasses attracted a wide range of insect species. Shallow-buried carcasses also attracted a variety of insects, although the diversity and abundance were lesser than those found on the surface. Deep-buried carcasses exhibited the least diversity and number of insect species, primarily involving the small-size soil-dwelling insects capable of penetrating the burial depth. Megaselia scalaris was the dominating species at both the depths and can play an important role in the estimation of post burial interval along with the help of beetle species found at these depths. These findings underscore the critical role of environmental exposure and burial depth in influencing both the decomposition process and the succession of insect fauna. This knowledge has practical applications in forensic science for estimating post-burial intervals and can enhance our understanding of ecological processes related to carrion decomposition in different environments. The absence of facilities to observe the decomposition and insect assemblages of the undisturbed buried carcasses remains a lacuna of the present study which can be attributed to the expenses involved. Nevertheless, this is also an opportunity for further research in this area.