Effect of carcass model on maggot distribution and thermal generation of two forensically important blowfly species, Chrysomya megacephala (Fabricius) and Chrysomya rufifacies (Macquart)

The concept of accumulated degree hours (ADH) has been used to optimize the timing of insecticide applications to control agricultural pest which is based on the temperature-dependent rate of development of cold-blooded insects. In forensic entomology, this concept has been used to estimate the time of death by utilizing blowfly larva developmental records. In a laboratory, a total number of hours multiplied by temperature is used to determine the total ADH of a blowfly development. However, as ADH has been widely used, a problem arises due to the unknown thermal history of larva development. Researchers indicated that the ADH could be miscalculated when it is not referred to heat produced by the larvae within a maggot aggregation (Peters 2003; Slone and Gruner 2007). A previous finding showed that the actual temperature at which maggot develops is unknown, as maggots are continually moving down to the feeding site then back out to the exterior of the aggregation (Anderson and VanLaerhoven 1996). Moreover, the maggot aggregation temperature can remain high regardless of daily weather fluctuation. It was reported that maggot mass temperature could reach 16.7 °C above ambient temperature in a pig exposed to the sun in summer and slightly lower, 11.4 °C above ambient, in a pig in shade (Shean et al. 1993). A study also noted that heat can be higher, reaching 32 °C above ambient temperature (Turner and Howard 1992). Due to this thermal generation, a question has been raised whether postmortem interval (PMI) calculation based on surrounding temperature (usually obtained from the nearest weather station) is accurate and reliable. Therefore, a number of studies have emphasized on fly larval aggregation (Byrd and Butler 1998; Campobasso et al. 2001; Goff 2000; Greenberg 1991; Greenberg and Kunich 2002; Hewadikaram and Goff 1991; Joy et al. 2002). However, there are very few studies have accessed the larval aggregation formation in carcass exposed in a surrounding natural condition (Slone and Gruner 2007). Therefore, this research was carried out to extend knowledge on the distribution and thermal generation of blowfly aggregation in a surrounding natural environment using three common forensic entomology animal models which were laboratory rats, domestic rabbits, and long-tailed macaque. These models are standard and have been a surrogate to human cadaver in forensic entomology study in Malaysia (Azwandi and Abu Hassan 2009; Zuha et al. 2016; Rumiza et al. 2008).

In all carcass models, two blowfly species Ch. megacephala (Fabricius) and Ch. rufifacies (Macquart) were dominant, and their larva formed solid aggregations; therefore, making the examination of their aggregation parameters is possible. Adult Chrysomya nigripes (Aubertin), Chrysomya chani (Kurahashi), and Chrysomya villeneuvi (Patton) were also observed, but they did not form a clear aggregation and were unsuitable for examination. Thus, they were excluded from sampling and measurement. A total of 260 aggregations were included in the analyses which were obtained from those four rounds of experiment.

In the study, there was no difference in average temperature and relative humidity between each round of experiment, F (3,122) = 1.09, p = 0.357, ƞ² = 0.744. Based on continuous temperature measurement during decomposition, carcass temperature in the rectum started to elevate at two points, the first point at 20 h while the second point at 45 h (Fig. 1). The first elevation took place for only 10 h while the second elevation was longer and unique to carcass model. In the second elevation, rabbit and macaque carcasses were able to prolong temperature elevation until 83 h and 95 h respectively. Recorded maximum body temperature for rabbit was 37 °C while for macaque carcass was 40 °C. However, in rat carcass, the highest body temperature recorded was not more than 28 °C.

Our analysis indicated that rate of carcass weight loss after 6 days of decomposition was varied based on the animal model used F (2, 23) = 33.513, p = 0.001, ƞ² = 0.744. Multiple comparisons by Tukey HSD showed that weight loss in monkey carcasses (M = 0.789, SD = 0.130) was significantly higher than that in rat (M = 0.526, SD = 0.081) and rabbit carcasses (M = 0.589, SD = 0.069).

Distribution and frequency of each observed larval aggregation are shown in Table 2. Larval aggregation analysis indicated that Ch. megacephala was more associated with smaller carcasses. Twenty-three aggregations of this species were found in rat carcasses while only 7 and 14 in rabbit and monkey carcasses, respectively. Ch. rufifacies larvae dominated rabbit and monkey carcass with 85 and 113 aggregations, respectively. Records have shown that only 18 aggregations were found in rat carcasses. With regards to location, most of the aggregations of Ch. rufifacies were located below carcasses while Ch. megacephala aggregations were located mostly in the mouth and genitalia. The highest number of aggregations (42 aggregations) was by Ch. rufifacies which was located under macaque carcasses with a measured temperature of 44.1 °C. Most of the Ch. megacephala aggregation temperature was lower than that of Ch. rufifacies. In some carcass visits, we found that Ch. rufifacies and Ch. megacephala could form species-specific aggregation which was separated by a clear boundary (Fig. 2). Duration of this species-specific aggregation had taken place for only 1 day. It was then ceased followed by overwhelmed mass of Ch. rufifacies.

Carcass	Blowfly species	Mouth	Genitalia	Under carcass	Abdomen	Away from carcass	Other
Rat	Ch. megacephala	1 (29.9)	8 (28–31.1)	2 (31)	7 (27.9–33.4)	4 (29.1–31.7)	1 (28.4)
	Ch. rufifacies	3 (26.4–27.4)	2 (29.6–30.5)	2 (26.1–30.6)	0	11 (26.7–33.2)	0
Rabbit	Ch. megacephala	4 (30.5–31.1)	1 (31.9)	0	2 (34.5–34.5)	0	0
	Ch. rufifacies	9 (35.7–40.6)	18 (32.7–41.7)	34 (29.1–37.6)	11 (31–39.7)	13 (29.7–38.4)	0
Macaque	Ch. megacephala	8 (29–39)	2 (30.5–32.6)	0	0	2 (33.3–37.2)	2 (35.2–35.2)
	Ch. rufifacies	12 (32.4–40.4)	17 (32.6–41.6)	42 (26–44.1)	28 (29.1–41.9)	11 (32.3–40.8)	3 (40.1–40.1)
Total		37	48	80	48	41	6

Values in parenthesis are temperature range in °C

A strong correlation of aggregation temperature with carcass temperatures, r(188) = 0.65, p < 0.01 was found (Fig. 3). We also noted that the highest aggregation temperature occurred concurrently with the onset of third instar larvae and prolonged until the post-feeding larvae withdrawn from the carcass. Aggregation temperature was also found correlated to depth r(188) = 0.21, p < 0.05, but not to volume r(188) = 0.104, p = 0.28 (Fig. 3). No correlation was also observed between aggregation temperature and ambient temperature r(188) = 0.01, p = 0.885 and relative humidity r(188) = 0.07, p = 0.35 (Fig. 3).

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).

Although the findings obtained from this study were based on selected larval aggregations, the results have demonstrated the effect of corpse size, aggregation location, and species on the aggregation temperature of two well-known forensically important Malaysian blowflies. Based on the results of this study, larva aggregation location and its temperature in a corpse are critical in forensic entomology, and these aspects should be taken into consideration when handling a forensic investigation of decaying body. However, further fieldwork is needed to extend understanding of maggot mass thermoregulation and larva behavior in a dead body.