Observations on the number of larvae presence on corpses found that its frequency was relatively low (Fig. 3) compared to the number of larvae that could be found on a corpse on land in the outdoor environment (Heo et al. 2007; Heo et al. 2008). Apart from the factors of the river current that may take away some of the entomological evidence, the reduced numbers of larvae on corpses may also be due to the technique of pulling the body ashore by the forensic officers which may cause most of the larvae or eggs on the body to be dislodged into the water. Therefore, training and improvements in the aspect of handling corpses found in aquatic environments should be conducted to preserve the entomological evidence as best as possible.
The presence of Ch. megacephala immature stages in all three cases strengthened the fact that it is the most forensically dominant fly species in Malaysia. This species can survive and compete successfully in various types of habitats under tropical climates (Lee et al. 2004; Syamsa et al. 2017), including aquatic environments as highlighted in this study. Similar observation was also reported by Sukontason et al. (2005) in Thailand where they recorded the presence of flies Ch. megacephala and Ch. rufifacies on corpses found in water reservoir areas. In Italy, among other flies recorded infesting bodies in aquatic environments were Ca. vicina and Ch. albiceps (Wiedemann) (Calliphoridae), Fannia sp. (Fannidae), Ophyra sp. (Muscidae), and Syritta pipiens Linnaeus (Syrphidae) (Magni et al. 2013).
One of the notable findings of this study was the presence of Eristalis spp. larvae in the second and third cases. The presence of Eristalis spp. of the Syrphidae family, also known as rattail maggot due to its unique morphology, indicates that this species is of forensic importance in Malaysia. This finding was also supported by Lee et al. (2004) and Ahmad et al. (2007), who also stated that this species could be an indicator of the death location as its immature stages require an aquatic environment to live and thrive. In the USA, Lindgren et al. (2015) conducted a year-long case study on simulated cadavers, observing the presence of Eristalis arbustorum larvae on one of the cadavers that were partially submerged in a grave filled with rainwater. Similarly, Archer and Ranson (2005) recorded the occurrence of this genus on decomposing bodies found in freshwater at Victoria, Australia. All of these studies agree that the preferences of this particular species on corpses associated with aquatic environments could be useful in forensic investigation, particularly to determine whether the body has been moved from one place to another.
Typically, corpses and carcasses in aquatic environments such as ponds or swamps will experience a series of submerging and floating phases (Mann et al. 1990; Heo et al. 2008; Magni et al. 2013; Ramos-Pastrana et al. 2019; Dalal et al. 2020). During this floating phase, forensically important flies will colonize the corpse and can be used for PMI estimation (Mann et al. 1990). However, the accuracy of PMI estimations is highly dependent on insect biology, including environmental preferences and constraints (Introna et al. 2011). Therefore, the PMI for all these cases could not be estimated due to the lack of information about the decomposition process for submerged bodies, especially in the aquatic environment in Malaysia. This is because most of the studies in the field of forensics emphasized on the terrestrial environment, with only 15% of research involving exposure to the aquatic environment (Merritt and Wallace 2010).
For case 1 of the current study, the body was in an active decomposition stage, while for both case 2 and case 3, the bodies were in the bloated stage of decomposition. The decomposition process may undergo modifications due to the corpse being exposed to low water temperature, which will slow down the decomposition process and affect the colonisation and the development of forensically important flies. Heo et al. (2008) conducted a study on the faunal distribution of pig carcasses placed in ponds. After a while, it was found that the pig carcass sank to the bottom of the pond before returning to float on the surface on the third day. Blowfly Ch. megacephala and Ch. rufifacies, on the other hand, were found to only start laying eggs on corpses on the fourth day. This indicates that the process of insect colonization of corpses found in aquatic environments can be delayed up to 4 days. More experimental studies should be conducted to ascertain the insect preference and behavior related to this specific condition. Heo et al. (2008) also observed that the activity of flies on pig carcasses in aquatic environments was not as active as the activity of flies observed on carcasses placed on land (Heo et al. 2007). Therefore, extra precaution is required when analyzing entomological evidence collected from an aquatic environment to avoid misinterpretation and errors in estimating PMI.
Haskell et al. (1989) stated that several factors influence the process of colonization of insects in watery areas including the size and position of the corpse, the depth of water, and the speed of water flow. According to Magni et al. (2013), the determination of PMI for submerged corpses should take into account several important parameters such as the process of limb disintegration experienced by corpses, adipocere formation, and collection techniques of entomological specimens in aquatic environments. In addition to the ambient temperature, the water temperature at the time of submergence plays a vital role in the development of fly larvae (Myskowiak and Doums 2002; Ames and Turner 2003). Hence, the knowledge of the biology of forensically important flies at low temperatures is critical to assist forensic entomologists in determining accurate PMI estimation. This requires expertise from various disciplines to ensure the reliability of the evidence and information obtained.
A laboratory study by Singh and Bala (2011) on larval Ch. megacephala and Ch. rufifacies found that the survival rates were inversely proportional to the period the larval had been immersed in the water. The stage of larvae found on the corpse when it underwent a sinking phase is also an important factor that determines the rate of larval survival. The lowest survival rate was observed in young 10-h-old larvae which were unable to withstand immersion periods of more than 2 h. For instar III larvae, the immersion period of more than 5 h was sufficient to provide 100% mortality on larval survival (Singh and Bala 2011). In addition to the larval stage, the immersion factors on pupa stage survival were also studied (Reigada et al. 2011). The pupa age factor plays an essential role in determining survival with longer soaking periods giving lower survival rates. Magni et al. (2021) studied the survival and eclosion of Ca. vomitoria (Linnaeus) (Calliphoridae) and L. sericata intra-puparial forms after submersion in various types of water. Both species were shown to have a higher survival rate in tap water than in river or salt water. Whereas the eclosion time after submersion was influenced by the age of intra-puparial forms when immersed, the types of water, and the duration of submersion. All of these studies are very useful in the investigation of cases of corpses found in the aquatic environment, especially for cases where the corpse undergoes a submerged phase after the larvae develop and enter the pupa stage.
