Insect faunal succession on buried goat carcass in Aligarh Region of Uttar Pradesh, India, with implications in forensic entomology

Studies on the insect fauna of animal corpses, used as a vertebrate model, may help forensic investigation cases to estimate the post-mortem-interval (PMI), cause of death, and crime-scene location. Likewise, entomofauna of buried corpses can assist in determining the post-burial-interval (PBI), movement of the body or hiding of the crime. The bodies buried under the soil decompose at a slower rate than the body exposed. Also, there are fewer insects that can go underground to locate the corpse. Such types of studies on the insect fauna of buried carrion can help in forensic investigation cases. The current study aims to determine the succession of a goat carcass buried shallowly in an outdoor habitat of the Aligarh Region, North India. In the present study, we examined a goat carcass buried at a depth of nine inches to determine the type of insects capable of colonizing buried animal carcass in the study area of Aligarh, Uttar Pradesh, India. We have found five species of Diptera, three species of Coleoptera, one Hemipteran species, and one Hymenopteran species. Adult Dipterans found during different samplings were Megaselia scalaris (Loew 1866), Chrysomya megacephala (Fabricius 1794), and Calliphora vicina (Robineau-Desvoidy, 1830). Dipterans’ immature stages were found to be of Synthesiomyia nudiseta (van der Wulp, 1883) and Hydrotaea capensis (Wiedemann, 1830) Megaselia scalaris. Adults of Saprinus quadriguttatus (Fabricius, 1798), Saprinus splendens (Paykull, 1811), and Onthophagous quadridentatus (Fabricius, 1798) of order Coleoptera. Adult Cydnus species (Fabricius, 1803) of Hemiptera and Dorylus species (Fabricius, 1793) of Hymenoptera have also been recorded. During the sampling, the authors reported the stages of carcass disintegration and the insects associated with it. Flies, beetles, and ants have been recorded in association with the buried goat carcass, which may add to the knowledge of colonization of buried bodies in India and around the globe. S. nudiseta, H. capensis, and M. scalaris, which are found on the goat carrion in the current study, have also been reported on human corpses in the past. Furthermore, M. scalaris found consistently on two sampling dates beneath the ground on the goat carcass and was also seen running on the grave’s surface. In addition, authors have reported several species belonging to different order and families, corresponding to various stages of decomposition of corpse which were earlier not known from buried corpses from India. So, it is crucial and may have implications in medicolegal cases.


Background
The present study aims to analyze the insect succession and decomposition rate of a shallowly buried goat carcass in North Indian climatic conditions for two years. During putrefaction, corpses undergo considerable physical, chemical, and biological changes. Each decomposition stage is attractive to a group of arthropods, predominantly sarcosaprophagous insects such as Dipterans, Coleopterans, Hemipterans, Hymenopterans, and numerous others. Amongst these, Dipterans are the first ones to arrive on dead and decaying animal or human corpses, followed by Coleopterans, Hymenopterans, and Hemipterans.
Physical barriers such as soil cover or burial, body enclosed in containers/suitcases/drums, wrapped in clothing or sacs, and various others may hamper the insect activity. Time taken by insects to colonize the carrion placed above ground would be less than the carrion buried underground. Often, it has been seen that criminals bury the dead in shallow graves in haste, as digging requires much time and effort. Shallow grave entomofauna may also vary from the deep graves as some insects which can reach the thin soil cover may not locate the corpse buried deep (cited by Lundt, 1964).
Forensic entomology is a wide field of science that uses insect shreds of evidence in medicolegal investigations. The field has not only been confined to death investigations but also includes its broader aspects like in child/elderly neglect cases (Beneckea et al. 2009), unfit stored food products (Hagstrum and Athanassiou 2019), poor hygienic conditions of restaurants, and wildlife poaching cases (Anderson 1999). A thorough knowledge about insects can provide essential and valuable information regarding the cause and time of death. The diversity and succession of insects depend on several factors such as physical barriers, chemicals or toxins (Keshavarzi and Rassi, 2020), size, indoor/outdoor (Keshavarzi et al. 2019) and body mass of the corpse. Experiments conducted on flesh or animal tissue pieces may be misleading as the whole carcass has more surface area and body mass, resulting in a greater assemblage of insects than on an amount of animal tissue (LeBlanc et al., 2021). In addition, seasonal variations also influence the species richness and diversity of insects colonizing a corpse (Keshavarzi and Rassi 2020). In cases of pertinent death investigations, it requires the combined testimonies from various disciplines-from autopsy surgeon to forensic pathologist and other medical non-medical professionals. When a body is infested with insects, they must be collected in congruency with other biological evidence. These creatures could prove to be a vital parameter to many unanswered questions to the case mysteries.

Study site
The study was conducted in India, West of Uttar Pradesh, district Aligarh, microlocality Dhorra mafi. 27 o 54′ 41.96′′ N and 78 o 4′25.4′′ E, comprising a land area of 60' × 37' with vegetation of mulberries, brinjal, papaya, and mango on loamy soil. The average annual rainfall is 75.4 cm, and the average annual maximum and minimum temperatures are 31.30 o C and 17.90 o C, respectively. Relative Humidity (RH) values are about 61 to 84% in the morning and 46% to 75% in the afternoon during the southwest monsoon season (data retrieved from http://www.imdpune.gov.in/caui/smartcities/ALIGARH. pdf). It has three distinct seasons in terms of temperature conditions.

Study time
The period of the study is from early February 2018 to late March 2020.

Carrion
A fresh female, dead goat Capra hircus (Artiodactyla: Bovidae), was found on the 9th of February 2018. The death occurred due to the wound at its neck caused by the attack of some stray dogs. The goat age was about 3 months, the length was 2'2.5" from head to the start of tail, weight 8.6 kg, and body temperature 27 o C at the time of burial.

Examination procedure and taxonomic identification
The body was buried in a rectangular grave of dimensions 3'9" × 2'9" × 9" (Length × Breadth × Depth). The soil temperature of the lowermost depth of the grave was found to be 15 o C. The air temperature was 24 o C maximum and 8 o C minimum. The grave was in direct contact with the sunshine. The grave was opened and sampled every week from fresh to the late dry stage for collection and observation of insects. Afterward, it was sampled after 3 months, then followed by 6 months. Immature stages of insects were collected gently by using forceps and brushes and preserved in 70% ethanol. Adults were collected with the help of a small sweep net, particularly the Dipterans. Adult Coleopterans and Hemipterans were collected by handpicking with forceps and by sieving the soil. In addition, some of the immature stages of flies were reared in the laboratory at 27 ± 2 o C on goat meat up to the adult stage for identification purposes. The collected fauna was identified with the help of the taxonomic keys (Sawaby et al. 2018 andCarvalho, 2008) and later confirmed by the Zoological Survey of India, Kolkata.

Decomposition stages of goat carcass and insects associated
The carcass had undergone five stages of decomposition-fresh, bloated, active decay, post-decay, and dry stage. It took around 5 weeks from the fresh stage to reach to dry stage. The fresh phase lasted for about 1 week. Insects found to be associated with this stage during the first sampling (1 week after burial) were adults of Megaselia scalaris, Onthophagous quadridentatus, and Cydnus sp. During the second sampling (2 weeks after burial), the body was in the bloated stage. The fauna collected were adults of Megaselia scalaris, Saprinus quadriguttatus, and Cydnus sp. When the grave was opened and the carcass was exposed during sampling, Chrysomyia megacephala and Calliphora vicina were observed visiting the dead but could not oviposit. Immature stages of Synthesiomyia nudiseta (Muscidae) and larvae of Hydrotaea capensis (Muscidae) were found in large numbers in the deflated abdominal region, the active decay stage inside and outside of the abdomen within the grave. Furthermore, adults of Dorylus sp., Saprinus quadriguttatus, and Saprinus splendens were also collected during the third sampling (3 weeks after burial). In the active-decay stage, aggregation of ants (Dorylus sp.) was observed on the goat where maggots were found feeding. In the post-decay stage during the fourth sampling (4 weeks after burial), most of the body mass had been decomposed, larvae of H. capensis were still observed with cocoons of S. nudiseta inside the grave. From the fifth sampling onwards, the body was in the dry stage. Very few larvae of H. capensis and many cocoons of H. capensis and S. nudiseta were found during the fifth sampling. Live cocoons and empty puparial cases were collected during the sixth sampling. Empty puparia were found on seventh and eighth sampling. From the ninth sampling onwards, no fauna was detected till the last sampling (thirteenth).
The summary of results with the date of sampling, fauna collected, site of collection during sampling has been provided in Table 1. The images of stages of decomposition of goat carcass and insects collected during

Discussion
Insects are attractive to body fluids like urine, saliva, and fecal material oozing from natural openings and blood from wounds. However, the presence of larvae in these locales is a common phenomenon in exposed bodies lying above the ground (Beneckea et al. 2009). In the current study, it has been observed that no maggots were found on the wounded region of the neck and the other natural orifices of the goat (video clip 2 in supplementary files). This is probably due to the obliteration of bodily fluid and blood from wounds and natural orifices by the soil cover. The presence of maggots on the body beneath the ground is presumably due to the reasons that: 1. The flies oviposited on the grave's surface, and after hatching, larvae crawled and reached the target. 2. The adults had access to the corpse by burrowing through the soil. 3. The flies oviposited during brief exposure of goat carcass at the time of sampling.
Meanwhile, many calliphorids were found visiting the carcass during the sampling and exploring the resource but could not oviposit as no eggs or larvae were recorded on/in the body. It was also observed that at optimal temperature between 28 o C and 32 o C, the insect activity was maximum. As the temperature progressed, we found that immature stages have completed part of their life cycle.
The usual fly families that are considered as a typical carrion fauna are Calliphoridae, Sarcophagidae, and Muscidae. In the current study, Dipterans of Muscidae, Calliphoridae, and Phoridae families were collected. The larvae of two muscid species, i.e., Hydrotaea capensis and Synthesiomyia nudiseta, are recorded, dominant in the decay process. Megaselia scalaris, was found consistently on two sampling dates beneath the ground on the goat carcass and was also seen running on the grave's surface. M. scalaris, H. capensis, and S.nudiseta can be critical forensic indicators in the cases of buried bodies. The two species mentioned above have also been found inside the coffin and mummified bodies (Morrow et al. 2015).
Necrophagous beetles are either predatory on the larvae feeding on the carcasses or associated with the feeding on hairs and skin. Saprinus spp. of Histeridae, collected, is of forensic relevance and reported from most carcasses (Shayya et al. 2018). It has also been reported on the goat carcass above ground at Kullu, Himachal Pradesh, India (Singh and Bala 2019) and on a buried piece of pork at 60 cm in Punjab, India (Bala and Kaur, 2015). Dung beetles of the subfamily Scarabaeinae are a functionally important group of invertebrates that use both dung and carrion (Vernes et al. 2005). The scarabaeid beetle Onthophagous sp. reported from the current study has also been reported from other carrion resources such as the chicken liver and dead goat (Tshikae et al. 2008;Singh and Bala 2019). Hymenoptera, particularly ants, associated with exposed carrion and are generally represented by Pheidole megacephala or Dorylus species, although others may also occur (Ekanem and Dike 2010). In the current study, Dorylus sp. were found beneath the ground, feeding on the maggots and decaying flesh. This ant species has also been reported from a decaying rabbit at Patiala (Singh and Bharti, 2001). Ants also have a burying behavior of covering the carcass by soil with soil particles, preventing flies and beetles from colonizing the corpse (Singh et al. 2020).
Others have reported species from buried corpses such as Fannia canicularis, Muscina stabulans, Ophyra aenescens, and Megaselia scalaris on an exhumed infant skeleton in Argentina (Mariani et al. 2017). Calliphora vicina, C.vomitoria, Lucilia sericata, Muscina stabulans, and Muscina prolapsa have been reported to colonize buried remains at different depths (Gunn and Bird 2011). C. vicina and C. vomitoria were found to be attracted and oviposited through different zips of various gauge sizes of suitcases enclosing bait (Bhadra and Hart, 2014). Clothing also affects carcass decomposition and the pattern of insect succession. In a study in Western Australia, clothed pig carcass has more larval masses of L. sericata and Australophyra rostrata widely distributed than unclothed ones (Voss et al. 2011). In addition, the researches dealing with the seasonality of succession patterns in the same region reported that different dominant species and their arrival time vary in different seasons (Watson and Carlton 2005). There is little known about the buried corpse entomofauna from India. A study on a buried piece of pork from the state of Punjab is the only study reported from India, which reported only beetles (Bala and Kaur 2015). There are more studies on open lying carcasses than buried ones. There should be more exploration of buried corpses to get a more comprehensive data of insects that are able to colonize at different depths of burials and at different geographical regions of India.

Conclusion
Buried forensic entomology needs more exploration, especially in India, as the data is scanty. And more extensive studies need to be performed to develop a geographical database on arthropod succession from different habitats with applications in forensic and ecological fields. The current study provides basic information in the field of buried forensic entomology. However, we conclude that some insects can colonize buried carcasses, which requires more exploration and research in this direction.