Formicidae Faunal Succession on Buried Rabbit Carcass: Implications for Forensic Entomology

Ants (Hymenoptera: Formicidae) are one of the most common insects in terrestrial habitats. They participate actively in the trophic system and are very helpful in the ecological decomposition process. They predate other arthropods that approach corpses (Catts, 1990) or eat directly on corpses (Bonacci et al., 2011; Ramón and Donoso, 2015).
       
Diptera are the most common order of insects discovered in a decomposing carcass and are used to estimate the minimum postmortem interval (PMImin) since they are the first insects to find and lay eggs on the cadaver (Smith, 1986). Coleoptera, the second-most common order in carcasses, are present during all stages of decomposition but particularly in the final stages (Mise et al., 2007). Hymenoptera is the third most abundant order of insects in corpses, primarily ants (Dos Santos, 2019), generally taking place during all stages of breakdown (Paula et al., 2016).
       
Physical barriers such as burial, bodies enclosed in containers, drums, or suitcases and various others may hamper insect activity. The time taken by insects to colonies the carrion placed above ground would be less than that of 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 deep graves, as some insects that can reach the thin soil cover may not locate the corpse buried deep (Sharif and Qamar, 2021).
       
Ants have a direct impact on the decomposition process, demonstrate that they can exploit animal carcasses as food sources and impede the activity of other insects that are crucial to forensic investigations, such as flies and beetles (Singh et al., 2020). On corpses, it can behave in a variety of ways. Necrophagous ants, like Camponotus sericeiventris, rip out pieces of the carcass and carry them to their nest, speeding up the breakdown process and causing injuries to the body that end up serving as a doorway for Diptera larvae to access wet areas, facilitating their feeding. Predator ant species Camponotus (Tanaemyrmex) sp. can delay the decomposition process because they carry a great number of eggs of flies to their nest for later feeding, thus interfering with decomposition (Paula et al., 2016). In any situation, ants are a crucial species for forensic professionals because they can hasten or slow down the process of decomposition and inflict injuries that may look like they were sustained before death, leading to false conclusions (Patel, 1994).
       
Newer studies have stated that forensic hints provided by ants affect the rate of remains decomposition (Campobasso et al., 2009; Chin et al., 2009), postmortem artifacts removal by ants (Heath and Byard, 2014) and ant species composition affect the position of carcasses (Chen et al., 2014).
       
To our knowledge, in the Kingdom of Saudi Arabia, no research was conducted in an attempt to identify the types of ants on buried corpses. In order to identify species and successions of ants, we carried out this investigation in the city of Riyad, Saudi Arabia, using rabbit carcasses as an animal model.

Study site
 
The research was done in Riyadh at King Saud University, 43°24 N, 46°36 E, on a 175 m by 250 m plot of ground containing acacia trees and several common wild herbs.
 
Study time
 
The study was conducted in two periods: the first period from late February to late May 2021 and the second period from September 5, 2021, to January 4, 2022.
 
Environment data
 
For the duration of the experiment, air temperature and relative humidity were recorded hourly using a Lascar EL-USB-2 data logger. and an EM50G data logger (Ecotone, Gdynia, Poland) was used for soil and was programmed to record the temperature and humidity once each hour throughout the experiment (Table 1).
 

 
Carrion
 
The Animal Use Committee of Al-Imam Muhammad Ibn Saud Islamic University in Riyadh, Saudi Arabia, approved the use of rabbits as the research animal for this study. Project number: 35-2021. 50 live, mature rabbits (Oryctolagus cuniculus L.) were obtained locally, weighing between 1400 - 2200 g. A day prior to the burial, a total of 24 holes measuring 60 × 30 cm were excavated using a short-handled spade. These holes were dug at depths of both 20 and 40 cm. The pits were positioned at least 10 meters apart from each other on all sides. On the morning of the burial, chloroform was employed to euthanize all the rabbits. The rabbits were positioned on a section of chicken wire measuring 30 × 60, which had holes with a diameter of 25 mm. This arrangement served the dual purpose of facilitating easier removal and preventing scavenging by other animals. 24 rabbits were buried in each study period and the exposed carcass was placed in robust steel 2-cm mesh cages with a layer of wire screening, each measuring 60 × 50 × 30 cm³.
 
Examination procedure and taxonomic identification
 
The grave was opened and sampled every 10 days for 120 days (four months) in both periods for the collection and observation of insects. Ants were collected from the exposed soil at the bottom of the hole and the layers of soil cover, in addition to the body of the rabbit, by handpicking with forceps. Then placed in Eppendorf tubes with 70% ethanol. The Formicidae fauna was identified at species level when possible with the aid of the identification keys of Collingwood (1985). Also, to confirm the identification of the species, we referred to experts in the Insect Museum, College of Food and Agricultural Sciences, King Saud University. Specialists were consulted and comparisons were made with the standards of the Entomological Reference Collection.

In total, eight types of ants were recorded in our study. In buried bodies, four types were recorded at a depth of 20 cm and two types only at a depth of 40 cm in the first period, while two types were found at a depth of 20 cm and one type only at 40 cm in the second period of the experiment. As for the exposed carcasses, seven types were recorded in the first period and six types in the second period (Table 2 and Fig 1).
 

 

       
These species follow three subfamilies: Myrmicinae, Formicinae and Ponerinae. Formicinae were prevalent in buried carcasses, while Myrmicinae were prevalent in exposed carcasses (Fig 2). Cataglyphis holgerseni (Formicinae) is the most abundant species at a depth of 20 cm and Cardiocondyla sp. (Myrmicinae) at a depth of 40 cm in the first period, while the numbers were very few in the second period of the experiment, but in the exposed carcasses, the species Messor ebeninus (Myrmicinae) was dominant in both study periods (Fig 3).
 

 

 
To the best of our knowledge, this is the first study of its kind to investigate ant fauna in buried carcasses and the resultant forensic implications. Previous work carried out in three different habitats (agricultural, desert and urban) in the city of Riyadh, Saudi Arabia, presented only Formicidae fauna associated with exposed carcasses (Mashaly et al., 2018) and two habitats that were ecologically differentiated (Al-Mekhlafi et al., 2021).  
       
Carcass decomposition time differed significantly between the two experimental periods in exposed bodies, the decomposition time of which was considerably extended to 30 days in the First period and 17 days in the second period, explaining the highest relative abundance and richness of ant species in those carcasses since these carcasses were exposed longer to the action of these insects. In the study of Paula et al., (2016), they discovered that pig carcasses exposed longer also attracted a greater variety of species. while bodies buried at both depths (20 and 40) reached the dry stage after 60 days of burial in the two study periods.
       
The first appearance of ants in buried corpses was in the stage of decay, as the species Camponotus aegyptiacus appeared in the second excavation (20 days), then the species Cataglyphis arenarius on the 30^th day and the species Cataglyphis holgerseni on the 40^th day. The dry phase began at the sixth excavation (60 days) and the species C. holgerseni and Cataglyphis arenarius were recorded on the 70^th day and the two species Messor ebeninus and Cardiocondyla sp. on the 90^th day. As for the second period, the first appearance was in the Bloated stage of the type C. arenarius, then the type C. aegyptiacus in the decay stage and finally, C. arenarius reappeared on the 40^th day and for the rest of the experiment period, no species was found (Table 3). As for the exposed carcasses, most of the species appeared in the three stages of decomposition (Bloated, decay and dry), except for C. aegyptiacus, which was found in the dry stage only and Brachyponera sennaarensis in the Bloated stage (Table 3).
 

       
M. niloticum appeared on exposed carcasses only and this species has not been recorded on cadavers before, but other species of the genus Monomorium have been recorded in previous studies; for example, M. floricola was found on rats in Malaysia (Singh et al., 2020), M. minimum on pigs in Mississippi, USA (Goddard et al., 2012) and in Egypt (Hamdy et al., 2022) recorded M. carbonarium, M. lipenyi, M. niloticus and M. salmonmis on the carcasses of rabbits and guinea pigs.
       
M. ebeninus was one of the most abundant species and was present at all stages in both periods of the experiment in both surface and buried carrions at a depth of 20 cm. The genus Messor was observed on buried carrion collected on pigs buried in Italy (Bonacci et al., 2021)  and South Africa (Botham, 2016) and on superficial carcasses in Saudi Arabia ((Al-Khalifa et al., 2021; Al-Qahtni et al., 2021).
       
The genus Cardiocondyla was collected only in the first period at a depth of 40 cm and on the exposed bodies at all stages of decomposition except the fresh and to our knowledge, this genus has not been recorded in the studies of forensic entomology in KSA, while many other studies have reported its association with buried (Botham, 2016) and surface carcasses (Chen et al., 2014; Leong et al., 2019; Souza, 2020).
       
C.  arenarius and C. holgerseni were collected in our current study in both periods and on Exposed and buried bodies in both depths. This genus was observed in Iran on a trap consisting of livestock, poultry and fish tissues (Tuzun et al., 2010); it was also recorded on the carcasses of rabbits buried in Iraq (Albushabaa, 2016); and in KSA  on human corpses (Al-Khalifa et al., 2021; Al-Qahtni et al., 2021). Diet flexibility is a feature many ant species possess and can lead to drastic changes such as those of B. sennaarensis, which has been documented as a granivore species and can shift to a carnivorous diet depending on the particular nature of environmental conditions (Lachaud and Déjean, 1994). This is confirmed by our current study, where this species was found on rabbit carcasses in the first period of the study. It was also collected from exposed carcasses of Impala in South Africa (Braack, 1986).   
       
Camponotus is a common ant genus found on corpses. It has been investigated by many authors and different species were associated with surface carcasses (Haddadi et al., 2019; Odo and Iloba, 2020; Souza, 2020) and buried remains (Bala and Kaur, 2015; Bonacci et al., 2021; Botham, 2016). This is consistent with our study, where the species C. fellah was found on superficial carrion only and the species C. aegyptiacus appeared on buried corpses at a depth of 20 cm and aboveground. (Viana et al., 2022) alone recorded 10 species of this genus on  the carcasses of exposed pigs in Brazil. Souza et al., (2008) documented C. rufipes preying on eggs and larvae of flies in the bulging stage, Camponotus sp. Was observed manifesting similar behaviour by predating on Calliphorid larvae in our current study (Fig 4). Silahuddin et al., (2015) also observed C. gigas as predators of adult Ch. megacephala.

More research is needed in buried forensic entomology, particularly in Saudi Arabia where there is a dearth of information. Additionally, more thorough research must be done to create a global database on the succession of insects from various ecosystems with applications in the forensic and ecological disciplines. In the area of buried forensic entomology, the current work offers fundamental knowledge. We do, however, draw the conclusion that some insects can colonize buried carcasses, which calls for additional investigation and study in this area.

Researchers Supporting Project number (RSP2023R112), King Saud University, Riyadh, Saudi Arabia.

O. Al-Zahrani and F.A. Al-Mekhlafi designed the study, F.A. Al-Mekhlafi, M.S. Al-Khalifa and M.S. Al-Khalifa conducted data analyses and wote the manuscript. OA performed field experiments. F.A. Al-Mekhlafi, A.H. Al-Qahtni and M.S. Al-Khalifa writing the manuscript and helped in conducted data analysis.

The conducted research is not related to either human or animal use.

All the data is available within the manuscript.

The authors declare no conflicts of interest.