Continuous records of carcass temperature during decomposition have shown two obvious elevation points. The first elevation is due to internal bacterial activities during putrefaction while the second elevation is related to maggot aggregation. Temperature reading in the second elevation is more significant as it occurred in a longer duration, has higher temperature range, and carcass-dependent. It was evident that at the beginning of death, temperature change pattern is similar regardless of carcass size, but it was different after 50 h of decomposition. Temperature after this period significantly associated with the heat produced by maggot aggregation. It was mentioned that maggot mass temperatures were elevated independently, not influenced by ambient temperatures after 69 h of decomposition (Joy et al. 2002). In a forensic postmortem, estimation of PMI after 72 h is more challenging as many internal and external factors are being involved (Henssge et al. 1995).One known external factors is the presence of maggot aggregation. Therefore, PMI estimation is more difficult after this time when actual temperature history where larvae developed is fluctuated depending on mass volume and its thermal generation. Their temperature can be over 20 °C above ambient which could accelerate the growth rate of flies (Turner and Howard 1992; Catts and Goff 1992).
In this study, carcass temperatures were varied depending on the size of the carcass. Higher body temperature in macaque carcass could be due to the fact that aggregation temperature is a function of the volume and larva number (Johnson and Wallman 2014; Heaton et al. 2014). A bigger carcass could provide a large area for aggregation and support a higher number of larvae making more heat be produced. Besides generating higher temperature, the big carcass had also lost weight faster. This finding is similar to a pig decomposition study in Hawaii (Hewadikaram and Goff 1991).The study mentioned that decomposition occurs more quickly in more massive pig because it has attracted more flies and supports a more significant number of feeding larvae (Hewadikaram and Goff 1991).
The volume of aggregation was not found correlated to aggregation temperature. This result demonstrated a disagreement with laboratory maggot study from a cultured colony (Johnson and Wallman 2014; Charabidze et al. 2011). This inconsistency could be due to the dynamicity of larvae in the aggregation in the carcass that makes it difficult to be measured accurately. Unlike in a laboratory setup, maggot mass in the carcass is dynamic and possesses three-dimensional arrangements with various depth, width, and area. Therefore, accurate calculation of volume is critical. However, in an outdoor maggot study in pig carcass, a relationship of volume to aggregation temperature was found, but this is only valid to selected aggregations which were tightly packed and able to retain metabolic heat (Slone and Gruner 2007). Due to this limitation, computed tomography scanning was introduced as a new measurement method for volume and temperature on a deceased person (Johnson et al. 2012, 2013).
Researchers have shown that the temperature in maggot mass is a product of ambient temperature during the initial 24 h of larval development, but not when the maggot is in the second and third instar (Peters 2003; Joy et al. 2002). Larva at a later stage could generate sufficient heat through aggregation, therefore terminating its dependency to the ambient temperature. Aggregation temperature in our study showed no correlation with ambient temperature and relative humidity, therefore supporting the fact that larvae were able to regulate their temperature for survival. This microclimate can, however, increase metabolic rate and accelerate growth. Thus it should be, therefore, critical for minimum PMI estimation.
During feeding stage, larvae are more concentrated at the soft tissue of carcass primarily at the abdomen and genitalia. Various species of feeding blowfly larvae were found to share their food in the early stage of death as there are plenty of soft tissues and less competition. During this time, a clear boundary species-specific aggregation was observed. This behavior is not unusual, and it is believed can benefit larvae by sharing salivary enzymes, regulating developmental temperature and protection from predator and parasites. Species-specific aggregation was found in two blowfly species Chrysomya albiceps (Weidemann) and Chrysomya marginalis (Weidemann) (Richards et al. 2009). Although this behavior is not fully understood, we hypothesized that this could be related to larva ability to detect chemical signals or attractant of their species. The previous study noted that Lucilia sericata could identify the larva-crawled area and stay within the aggregation (Boulay et al. 2015). This unique aggregation behavior is not prolonged. After food sources become limited, species-specific aggregation of Ch. megacephala ceased, taken over by Ch. rufifacies. Predation of Ch. rufifacies larvae has frequently been reported in the literature (Chin et al. 2009; Goodbrod and Goff 1990).
Ch. rufifacies aggregation temperatures were found higher than Ch. megacephala in most measurements. This species, also noted, could generate higher temperature than Calliphora vicinal (Johnson et al. 2014). Variation temperature between species could support the theory that aggregation temperature is species-specific where similar species have been shown to aggregate at the same temperature (Aubernon et al. 2016). The previous study has noted that L. sericata has aggregation temperature of around 33.3 °C while Calliphora vomitoria and Calliphora vicina were around 29.6 °C and 22.4 °C, respectively (Aubernon et al. 2016). We hypothesized that temperature difference between Ch. rufifacies and Ch. megacephala could be due to a species-specific factor. Besides that, hairy morphology and larva size of Ch. rufifacies which is relatively larger than Ch. megacephala might be the second reason for the higher heat produced by Ch. rufifacies during aggregation in our study. The relationship between larval size and temperature generation was previously demonstrated by a comparative study between Sarcophaga bullata (Sarcophagidae) and Protophormia terraenovae (Calliphoridae) (Rivers et al. 2010). Larval size is not only correlated to aggregation temperature but also has been found to correlate with their food quality, growth quality, and survival rate (Aubernon et al. 2016).
In our study, the highest numbers of aggregation were located under macaque carcass which was composed of third instar Ch. rufifacies. Ch. rufifacies larvae tend to stay under carcass until emerging rather than migrating away from the carcass. Research has shown that movement of Ch. rufifacies is nonrandom as they select the hottest spot in aggregation (Johnson et al. 2014). This strategy is believed as an effective way to protect them from unfavorable surrounding temperature and predation.
Based on our findings, locations primarily under carcass is the most suitable area that can give protection to larvae from cold air temperature, direct sunlight, and rainfall. Therefore, in a PMI estimation, measurement of multiple locations in a dead body at a death scene is crucial, and temperature measurements under a dead body should be given a priority as it could contains more packed aggregations. Infrared imaging is a useful choice to perform this measurement as it can effectively locate larva aggregation and measures the temperature without disturbing a dead body.(Johnson and Wallman 2013).