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Patterns of oviposition and development of Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) and Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) on burned rabbit carcasses
Forensic Science International 260 (2016) 9–13
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Technical note
Patterns of oviposition and development of Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) and Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) on burned rabbit carcasses N.A. Mahat a,*, N.L. Zainol-Abidin b, N.H. Nordin a, R. Abdul-Wahab a, P.T. Jayaprakash c a b c
Faculty of Science, Universiti Teknologi Malaysia, 81310 UTM, Johor Bahru, Johor, Malaysia Department of Chemistry Malaysia, Jalan Sultan, 46661 Petaling Jaya, Selangor, Malaysia School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 July 2015 Received in revised form 28 December 2015 Accepted 29 December 2015 Available online 7 January 2016
Considering that crimes against animals such as illegal killing and cruelty have been alarmingly increasing and since burning is one of the common ways for disposing cadavers, ability to estimate minimum postmortem interval (PMI) using entomological data merits consideration. Chrysomya megacephala and Chrysomya rufifacies are common necrophagous species recovered from cadavers in many countries including Malaysia. Specific studies focusing on the oviposition and developmental patterns of both species on cadavers manifesting different levels of burn as described by the Crow– Glassman Scale (CGS) remain scarce. In four replicates, rabbit carcasses were burned to CGS levels #1, #2 and #3 by varying the amount of petrol used and duration of burning. Oviposition by C. megacephala and C. rufifacies was delayed by one day in the case of carcasses burned to the CGS level #3 (p < 0.05) when compared with that of controls. Such delay in oviposition was not observed in the CGS level #1 and #2 carcasses. No significant differences (p > 0.05) in the duration of development were found between control and burned carcasses. These findings deserve consideration while estimating minimum PMI since burning as a mean for disposing animal and human cadavers is gaining popularity. ß 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Postmortem interval (PMI) Wildlife forensics Burned carcasses Crow–Glassman Scale Chrysomya megacephala Chrysomya rufifacies
1. Introduction Forensic entomological data have been demonstrated to be useful for estimating the postmortem interval (PMI), especially for decomposing bodies discovered 72 h or more after death [1]. Similarly, forensic entomology has been utilized for investigating wildlife offences [2] viz. illegal killing and cruelty, trade and possession, as well as poaching [3,4]. Anderson [5] reported that wildlife animals are illegally killed for fur, meat and organs, and in this context, the ability to estimate PMI accurately would be extremely useful in refuting a suspect’s alibi. Chrysomya megacephala (Fabricius) remains the earliest necrophagous species to oviposit on decomposing corpses and animal models in Malaysia followed by Chrysomya rufifacies (Macquart) [6–10]. While climatological factors [8,11] and poisons [8] have been reported to
* Corresponding author. Tel.: +60 7 5534138; fax: +60 7 5566162. E-mail addresses: [email protected] (N.A. Mahat), [email protected] (N.L. Zainol-Abidin), [email protected] (N.H. Nordin), [email protected] (R. Abdul-Wahab), [email protected] (P.T. Jayaprakash). http://dx.doi.org/10.1016/j.forsciint.2015.12.047 0379-0738/ß 2016 Elsevier Ireland Ltd. All rights reserved.
influence oviposition and the development of necrophagous insects, entomological studies on the influence of burned carcasses/bodies appear to have been neglected [12]. Interestingly, Anderson [13] noted that ‘killers often try to dispose of a victim by burning the body’ and emphasized that ‘little research has been conducted on the effects of burning on insect succession’. For facilitating the uniform description of injuries to burned human bodies, Glassman and Crow [14] have described a graded scale (the Crow–Glassman Scale) that is divided into five levels in increasing order of destruction to the body. Unfortunately, a similar gradation for animals remains lacking. A previous study reported that initial oviposition occurred immediately on both the CGS level #2 burned and control pig carcasses, although the majority of the oviposition occurred the next day [15]. Heo et al. [16] studied partially burned pig carcasses in Malaysia and indicated no significant differences in the sequence of faunal succession or the rate of decomposition between the control and burned carcasses. However, these researchers [16] did not indicate the CGS level of burning, rendering it difficult to make the appropriate comparisons. Recognizing the applications of CGS levels for the estimation of
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PMI, Avila and Goff [15] recommended further investigations on the effects of different degrees of burning on arthropod succession patterns. In this context, it is pertinent to indicate that any factors that could influence initial oviposition or the duration of development of necrophagous insects may subsequently affect the accuracy of the PMI estimate [17]. Hence, the present study explores the patterns of oviposition and the development of C. megacephala and C. rufifacies across different CGS levels in burned rabbit carcasses and provides baseline data for investigating wildlife offences and human forensics.
2. Methods 2.1. Experimental design Using rabbits as animal models, this experiment (four replicates) was designed to investigate the possible influences of different levels of burn injury as prescribed in the Crow–Glassman Scale (CGS) [14] on the initial oviposition and completion of the first life cycle of C. megacephala and C. rufifacies. Male rabbits (Oryctolagous cuniculus) (4 for each replicate) (2.0–2.5 kg) sacrificed by slaughtering with the front part of the neck severed partially were purchased as dead carcasses from a rabbit meat seller in Kota Bharu between 7.00 and 7.30 am. The carcasses were transported individually to the decomposition site in separate sealed double plastic bags to prevent exposure to other arthropods prior to placement at the decomposition site. Among the four rabbit carcasses that formed a single replicate, one was used as the control, while the remaining three carcasses were burned to CGS level #1, CGS level #2 and CGS level #3, respectively. Glassman and Crow [14] describe the burn injuries of CGS level #1 as characteristic of typical smoke death (i.e., blistering on the epidermis, singeing of the head and facial hair), with the body remaining recognizable for identification. CGS Level #2 burns occur when the bodies are still recognizable with varying degrees of charring, and the destruction of the body remains limited to the absence of elements of limbs, genitalia and ears [14]. CGS Level #3 occurs when the body undergoes further destruction, leading to the absence of major portions of the limbs with unrecognizable identity, despite the presence of the head [14]. Because extremely extensive burning is required to obtain CGS levels #4 and #5, this study was restricted to CGS level #1, CGS level #2 and CGS level #3 only. For achieving the CGS level #1, CGS level #2 and CGS level #3 burn injuries; the carcasses were burned using 0.5 L of petrol (RON 95) for 1 min, 0.75 L of petrol (RON 95) for 5 min and 1.5 L of petrol (RON 95) for 33 min, respectively (Table 1). As much as possible, the petrol was poured evenly over each carcass. Repeating the above procedure, four replicates (i.e., 16 rabbit carcasses) were included in the present research. The decompositions for all of the four replicate experiments were conducted from the 1st–13th February 2013, 5th–17th February 2013, 9th–21st February 2013 and 27th March–8th April 2013, respectively. While each carcass was separated by a minimum distance of 20 m during every replicate, the decomposition sites for all of the different
Table 1 Detail of the four rabbit carcasses that formed one replicate and conditions of burning to achieve the required CGS level. Rabbits (n = 4)
Quantity of RON95 (L)
Duration of burning (min)
Control CGS level 1 CGS level 2 CGS level 3
Unburned intact carcass 0.5 0.75 1.5
0 1 5 33
replicate experiments were further separated by a minimum distance of 50 m to ensure the independence of these replicates. 2.2. Decomposition site and entomological observation The experiments (February to April 2013) were conducted in a sunlit habitat located within the Universiti Sains Malaysia Health Campus, Kubang Kerian, Kelantan, an eastern state of Peninsular Malaysia (606‘‘1’’N, 102017‘‘5’’E) at approximately 4.6 m above the sea level. The soil type at the decomposition site was loam, and human activity was minimal. Each carcass was placed individually with direct contact on bare soil and covered with a slotted plastic basket (basket length: 58 cm; basket width: 45 cm; basket height: 20 cm; slot length: 3 cm; slot width: 1 cm; mould width between slots: 0.5 cm) with two to three bricks on top to prevent scavengers, while allowing for the free access of flies. Entomological observations, as well as data on daily ambient temperature and rainfall, were recorded until the completion of the first developmental cycle of C. megacephala and C. rufifacies (i.e., evidence of the emergence of tenerals). Following the entomological observation methods prescribed by Mahat et al. [8], field observations, as well as collection, rearing and preservation of entomological specimens, were made. Taxonomic identification of larvae and tenerals was conducted based upon the morphological identification keys provided by previous researchers [18–20]. 2.3. Statistical analysis Data analysis was conducted using IBM SPSS version 20.0 software. The normality of the data used for statistical inference was tested using the Kolmogorov–Smirnov and Shapiro–Wilk tests. Considering the significance level of 0.05, the Kruskal–Wallis H with pairwise comparison using Mann–Whitney U test was used for comparing the differences in the oviposition and completion of life cycles for C. megacephala and C. rufifacies among the different groups. 3. Results and discussion Ambient temperatures remained similar for all four of the carcasses across all four replicates; the ambient temperature data recorded for the control carcasses alone are reported in Table 2. The means for the ambient temperatures and the total daily rainfall recorded during the four replicates ranged between 27.4 and 35.5 8C (Table 2) and 0.0–5.0 mm, respectively. In general, rain was not recorded during the first two days of decomposition in any of the four replicate experiments, except on the second day of decomposition in replicate 1, where drizzling was observed (0.4 mm). Neither incessant nor torrential rains were observed throughout the study. Results revealed that the times to initial oviposition by C. megacephala and C. rufifacies in the CGS level #1 and #2 burned carcasses were similar to that of control carcasses (p > 0.05, Tables 3 and 4), in concurrence with the findings reported by Avila and Goff [15]. It has been reported that blowflies are attracted by the odour emanating from decomposing corpses/carcasses [13], and burning may lead to the disintegration of charred soft tissues, which may be favourable for the colonization of insects [12]. Adding to the body of current knowledge, we found that initial oviposition by both necrophagous species was delayed by one day in the CGS level #3 burned carcasses (p < 0.05, Tables 3 and 4). The CGS Level #3 burned carcasses were relatively drier and had smaller amounts of seeping body fluids when compared with the controls and the CGS level #1 and #2 burned carcasses. Therefore, the delays in oviposition by C. megacephala and C. rufifacies observed in the CGS level #3 burned carcasses (p < 0.05,
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Table 2 Data on daily ambient temperature for the control carcasses during entomological observation. Day
1 2 3 4 5 6 7 8 9 10 11 12 13 *
Replicate 1
Replicate 2
Replicate 3
Replicate 4
Mean
Range
Mean
Range
Mean
Range
Mean
Range
30.0 29.3 27.9 28.1 28.3 – – – – – – – –
27.0–32.0 22.0–32.0 26.0–30.0 26.0–31.0 26.0–31.0 28.9* 30.1* 29.8* 31.5* 30.1* 31.1* 29.5* 30.0*
29.4 29.0 28.3 27.4 28.2 – – – – – – – –
27.0–32.0 22.0–32.0 23.0–32.0 25.0–31.0 22.0–32.0 30.1* 30.1* 29.8* 31.5* 30.1* 31.1* 29.5* 30.0*
31.7 28.2 27.8 27.4 28.2 – – – – – – – –
30.0–33.0 22.0–32.0 23.0–31.0 23.0–31.0 23.0–33.0 28.2* 28.8* 31.5* 30.2* 30.8* 29.8* 29.6* 29.1*
32.1 31.5 31.3 31.1 31.4 – – – – – – – –
26.0–35.0 26.6–35.0 26.0–34.0 26.0–35.0 27.0–34.0 35.5* 33.6* 33.8* 32.4* 32.5* 32.0* 29.6* 29.5*
The temperatures are the actual ambient temperature recorded since, during pupation, ambient temperature was measured once daily around noon.
Table 3 Initial oviposition and completion of the first developmental cycle of C. megacephala in all the four replicates of burned rabbit carcasses. Day and time for the first observation of Replicate
CGS level
Eggs
First instar
Second instar
Third instar
Prepupae
1
Control 1 2 3
Day-1/6 pm Day1/6 pm Day-1/6 pm Day-2/8 am*
Day-2/8 Day-2/8 Day-2/8 Day-2/6
am am am pm
Day-2/6 Day-2/6 Day-2/6 Day-3/8
Day-3/2 Day-3/2 Day-3/2 Day-4/8
Day-4/12 Day-4/12 Day-4/12 Day-5/10
noon noon noon am
Day-5/10 Day-5/10 Day-5/10 Day-6/10
am am am am
Day-9 Day-9 Day-9 Day-10
2
Control 1 2 3
Day-1/4 Day-1/6 Day-1/6 Day-2/8
Day-2/8 Day-2/8 Day-2/8 Day-2/6
am am am pm
Day-2/6 pm Day-2/4 pm Day-2/6 pm Day-3/10 am
Day-3/2 pm Day-3/2 pm Day-3/2 pm Day-4/10 am
Day-4/12 Day-4/12 Day-4/12 Day-5/12
noon noon noon noon
Day-5/10 Day-5/10 Day-5/10 Day-6/10
am am am am
Day-9 Day-9 Day-9 Day-10
3
Control 1 2 3
Day-1/4 pm Day-1/6 pm Day-1/6 pm Day-2/10 am*
Day-2/8 Day-2/8 Day-2/8 Day-2/6
am am am pm
Day-2/4 Day-2/4 Day-2/4 Day-3/8
Day-3/2 pm Day-3/2 pm Day-3/2 pm Day-4/10 am
Day-4/10 Day-4/10 Day-4/10 Day-5/10
am am am am
Day-5/10 Day-5/10 Day-5/10 Day-6/10
am am am am
Day-9 Day-9 Day-9 Day-10
4
Control 1 2 3
Day-1/6 Day-1/6 Day-1/6 Day-2/8
Day-2/8 Day-2/8 Day-2/8 Day-2/6
am am am pm
Day-2/6 pm Day-2/6 pm Day-2/6 pm Day-3/10 am
Day-3/2 pm Day-3/2 pm Day-3/2 pm Day-4/10 am
Day-4/12 Day-4/12 Day-4/12 Day-5/10
noon noon noon am
Day-5/10 Day-5/10 Day-5/10 Day-6/10
am am am am
Day-9 Day-9 Day-9 Day-10
pm pm pm am*
pm pm pm am*
pm pm pm am
pm pm pm am
pm pm pm am
Pupae
Tenerals
Kruskal–Wallis H with pairwise comparisons using the Mann–Whitney U test revealed a significant delay in oviposition for C. megacephala in CGS level 6¼3 carcasses (*) (p < 0.05) when compared with the control as well as CGS levels 6¼1 and 6¼2 carcasses. No significant differences (p > 0.05) in the durations for completing the life cycles for C. megacephala were found in control as well as CGS levels 6¼1-6¼3 carcasses. Significance level of 0.05 was used for determining the significant differences among groups. Field observations were made at every 2 h intervals from 8.00 am to 6.00 pm until the first observation of second instar larvae, then at every 4 h intervals until the first observation of pupae, beyond which the field observation was restricted to once daily around noon.
Tables 3 and 4) may be attributable to the greater charring of the tissues, resulting in a lessening of the decay odour emanating from the carcass after more extensive burning. In all control carcasses, the first, second and third instar larvae, as well as the prepupae of C. megacephala, were invariably observed at approximately 8 am and 4–6 pm on day-2, at approximately 2 pm on day-3 and 10 am to 12 noon on day-4, respectively (Table 3). The pupation period for C. megacephala that started on day-5 lasted for about four days, with the first emergence of tenerals on the 9th day (Table 3). In addition, the completion of the life cycle for C. rufifacies in the control as well as CGS level #1 and #2 burned carcasses was observed on day-11 of decomposition (Table 4). The emergence of tenerals of C. megacephala and C. rufifacies on days 9 and 11, respectively, was consistent with durations reported by previous studies conducted in Malaysia [8,10,16]. Because the initial oviposition of both C. megacephala and C. rufifacies was delayed by one day in CGS level #3 burned carcasses, the completions of their life cycles were subsequently delayed by one day compared to that of control carcasses (Tables 3 and 4). Considering that the durations for completing the life cycles for both C. megacephala and C. rufifacies across all the CGS levels (#1-3) were similar to that of control carcasses (p > 0.05, Tables 3 and 4), it can be
construed that burning carcasses up to CGS level #3 did not influence the durations required for necrophagous insects to complete their life cycles. The CGS levels prescribed by Glassman and Crow [14] provide a useful description of burn injuries to human bodies, and the same descriptions were extended for animal carcasses due to a lack of specific description of burns in animal models. Three different CGS levels produced using rabbits as animal models were used to study the influence of burn injuries on the initial oviposition and development of C. megacephala and C. rufifacies. Pertinently, Avila and Goff [15] utilized burned pig carcasses that corresponded to CGS level #2 in humans. Unlike other animals, pigs are considered to be an acceptable substitute for humans due to their similarities in body mass, skin structure, fat-to-muscle ratio and hair coverage [21], as well as their rates of decomposition and patterns of fly colonization [22]. Considering the morphological differences in aspect ratios, the presence of fur, as well as the weight of the rabbits used in this present study only ranged between 2.0 and 2.5 kg, while human body weights may be more than 20-fold higher, extrapolation of the data to represent burns in humans may not be appropriate. The greater than 20-fold differences in masses mean that rabbits have a far greater surface-area-to-volume ratio
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Table 4 Initial oviposition and completion of the first developmental cycle of C. rufifacies in all the four replicates of burned rabbit carcasses. Day and time for the first observation of Replicate
CGS level
Eggs
First instar
Second instar
Third instar
Prepupae
1
Control 1 2 3
Day-2/8 Day-2/8 Day-2/8 Day-3/8
am am am am*
Day-2/6 Day-2/6 Day-2/6 Day-3/6
pm pm pm pm
Day-3/12 Day-3/12 Day-3/12 Day-4/12
Day-4/2 Day-4/2 Day-4/2 Day-5/2
pm pm pm pm
Day-5/10 Day-5/10 Day-5/10 Day-6/10
am am am am
Day-6/8 Day-6/8 Day-6/8 Day-7/8
2
Control 1 2 3
Day-2/8 Day-2/8 Day-2/8 Day-3/8
am am am am*
Day-2/6 Day-2/6 Day-2/6 Day-3/6
pm pm pm pm
Day-3/2 Day-3/2 Day-3/2 Day-4/2
Day-4/4 Day-4/4 Day-4/4 Day-5/4
pm pm pm pm
Day-5/10 Day-5/10 Day-5/10 Day-6/10
am am am am
Day-6/10 Day-6/10 Day-6/10 Day-7/10
am am am am
Day-11 Day-11 Day-11 Day-12
3
Control 1 2 3
Day-2/8 Day-2/8 Day-2/8 Day-3/8
am am am am*
Day-2/6 Day-2/6 Day-2/6 Day-3/6
pm pm pm pm
Day-3/12 Day-3/12 Day-3/12 Day-4/12
Day-4/2 Day-4/2 Day-4/2 Day-5/2
pm pm pm pm
Day-5/10 Day-5/10 Day-5/10 Day-6/10
am am am am
Day-6/10 Day-6/10 Day-6/10 Day-7/10
am am am am
Day-11 Day-11 Day-11 Day-12
4
Control 1 2 3
Day-2/8 Day-2/8 Day-2/8 Day-3/8
am am am am*
Day-2/6 Day-2/6 Day-2/6 Day-3/6
pm pm pm pm
Day-3/2 Day-3/2 Day-3/2 Day-4/2
Day-4/4 Day-4/4 Day-4/4 Day-5/4
pm pm pm pm
Day-5/10 Day-5/10 Day-5/10 Day-6/10
am am am am
Day-6/8 Day-6/8 Day-6/8 Day-7/8
noon noon noon noon
pm pm pm pm noon noon noon noon
pm pm pm pm
Pupae
Tenerals am am am am
am am am am
Day-11 Day-11 Day-11 Day-12
Day-11 Day-11 Day-11 Day-12
Kruskal–Wallis H with pairwise comparisons using the Mann–Whitney U test revealed a significant delay in oviposition for C. rufifacies in CGS level 6¼3 carcasses (*) (p*) (p < 0.05) when compared with the control as well as CGS levels 6¼1 and 6¼2 carcasses. No significant differences (p > 0.05) in the durations for completing the life cycles for C. rufifacies were found in control as well as CGS levels 6¼1-6¼3 carcasses. Significance level of 0.05 was used for determining the significant differences among groups. Field observations were made at every 2 h intervals from 8.00 am to 6.00 pm until the first observation of second instar larvae, then at every 4 h intervals until the first observation of pupae, beyond which the field observation was restricted to once daily around noon.
than humans. Such aspects would result in greater dynamics of heat penetration into animal tissues than humans for any level of superficial burning as measured by the Crow–Glassman scale. At CGS level #3, the cracks in skin provide flies access to deeper tissues that are relatively undamaged by heat. In contrast, the deeper tissues of small animals, such as rabbits, would be likely to suffer much greater heat damage and become less attractive for egg-laying flies. Therefore, the delay in oviposition for the CGS level #3 rabbit carcasses observed in this study may not be likely to occur in much larger animals. However, the findings reported in this study may still be useful for wildlife forensic investigations involving animals similar to rabbits. In this research, petrol was used for maintaining the fire to achieve the intended CGS levels of burn injuries. Rumiza et al. [23] indicated that arthropods were not attracted to gasolineingested carcasses due to the strong odour of gasoline, which is known to repel insects. Because petrol is a volatile liquid, it is assumed that its odour as well as liquid traces would have evaporated during the fire and subsequent exposure of the carcasses to atmosphere. In certain arson cases where the burn injuries are caused by less volatile accelerants, the effects of residual accelerant may have to be considered. However, this limitation may not apply to dead bodies with burn injuries that are recovered in cases of structural fire, where contact with accelerants by the body would be limited. In conclusion, the initial oviposition and development of C. megacephala and C. rufifacies infesting CGS level #1 and #2 burned carcasses remained similar to that of controls. Significant delays of one day in the initial oviposition for both necrophagous species were observed in the CGS level #3 burned carcasses. Such findings would acquire forensic significance when a CGS-level #3 burned wildlife animal carcass with similar physical characteristics to a rabbit is found, and estimation of minimum PMI in these species is attempted for forensic wildlife investigation. Acknowledgements The authors are thankful to the Universiti Teknologi Malaysia for providing a Research University Grant (Q.J130000.2626.09J64) for conducting a research project in Forensic Entomology.
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Technical note
Patterns of oviposition and development of Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) and Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) on burned rabbit carcasses N.A. Mahat a,*, N.L. Zainol-Abidin b, N.H. Nordin a, R. Abdul-Wahab a, P.T. Jayaprakash c a b c
Faculty of Science, Universiti Teknologi Malaysia, 81310 UTM, Johor Bahru, Johor, Malaysia Department of Chemistry Malaysia, Jalan Sultan, 46661 Petaling Jaya, Selangor, Malaysia School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 July 2015 Received in revised form 28 December 2015 Accepted 29 December 2015 Available online 7 January 2016
Considering that crimes against animals such as illegal killing and cruelty have been alarmingly increasing and since burning is one of the common ways for disposing cadavers, ability to estimate minimum postmortem interval (PMI) using entomological data merits consideration. Chrysomya megacephala and Chrysomya rufifacies are common necrophagous species recovered from cadavers in many countries including Malaysia. Specific studies focusing on the oviposition and developmental patterns of both species on cadavers manifesting different levels of burn as described by the Crow– Glassman Scale (CGS) remain scarce. In four replicates, rabbit carcasses were burned to CGS levels #1, #2 and #3 by varying the amount of petrol used and duration of burning. Oviposition by C. megacephala and C. rufifacies was delayed by one day in the case of carcasses burned to the CGS level #3 (p < 0.05) when compared with that of controls. Such delay in oviposition was not observed in the CGS level #1 and #2 carcasses. No significant differences (p > 0.05) in the duration of development were found between control and burned carcasses. These findings deserve consideration while estimating minimum PMI since burning as a mean for disposing animal and human cadavers is gaining popularity. ß 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Postmortem interval (PMI) Wildlife forensics Burned carcasses Crow–Glassman Scale Chrysomya megacephala Chrysomya rufifacies
1. Introduction Forensic entomological data have been demonstrated to be useful for estimating the postmortem interval (PMI), especially for decomposing bodies discovered 72 h or more after death [1]. Similarly, forensic entomology has been utilized for investigating wildlife offences [2] viz. illegal killing and cruelty, trade and possession, as well as poaching [3,4]. Anderson [5] reported that wildlife animals are illegally killed for fur, meat and organs, and in this context, the ability to estimate PMI accurately would be extremely useful in refuting a suspect’s alibi. Chrysomya megacephala (Fabricius) remains the earliest necrophagous species to oviposit on decomposing corpses and animal models in Malaysia followed by Chrysomya rufifacies (Macquart) [6–10]. While climatological factors [8,11] and poisons [8] have been reported to
* Corresponding author. Tel.: +60 7 5534138; fax: +60 7 5566162. E-mail addresses: [email protected] (N.A. Mahat), [email protected] (N.L. Zainol-Abidin), [email protected] (N.H. Nordin), [email protected] (R. Abdul-Wahab), [email protected] (P.T. Jayaprakash). http://dx.doi.org/10.1016/j.forsciint.2015.12.047 0379-0738/ß 2016 Elsevier Ireland Ltd. All rights reserved.
influence oviposition and the development of necrophagous insects, entomological studies on the influence of burned carcasses/bodies appear to have been neglected [12]. Interestingly, Anderson [13] noted that ‘killers often try to dispose of a victim by burning the body’ and emphasized that ‘little research has been conducted on the effects of burning on insect succession’. For facilitating the uniform description of injuries to burned human bodies, Glassman and Crow [14] have described a graded scale (the Crow–Glassman Scale) that is divided into five levels in increasing order of destruction to the body. Unfortunately, a similar gradation for animals remains lacking. A previous study reported that initial oviposition occurred immediately on both the CGS level #2 burned and control pig carcasses, although the majority of the oviposition occurred the next day [15]. Heo et al. [16] studied partially burned pig carcasses in Malaysia and indicated no significant differences in the sequence of faunal succession or the rate of decomposition between the control and burned carcasses. However, these researchers [16] did not indicate the CGS level of burning, rendering it difficult to make the appropriate comparisons. Recognizing the applications of CGS levels for the estimation of
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PMI, Avila and Goff [15] recommended further investigations on the effects of different degrees of burning on arthropod succession patterns. In this context, it is pertinent to indicate that any factors that could influence initial oviposition or the duration of development of necrophagous insects may subsequently affect the accuracy of the PMI estimate [17]. Hence, the present study explores the patterns of oviposition and the development of C. megacephala and C. rufifacies across different CGS levels in burned rabbit carcasses and provides baseline data for investigating wildlife offences and human forensics.
2. Methods 2.1. Experimental design Using rabbits as animal models, this experiment (four replicates) was designed to investigate the possible influences of different levels of burn injury as prescribed in the Crow–Glassman Scale (CGS) [14] on the initial oviposition and completion of the first life cycle of C. megacephala and C. rufifacies. Male rabbits (Oryctolagous cuniculus) (4 for each replicate) (2.0–2.5 kg) sacrificed by slaughtering with the front part of the neck severed partially were purchased as dead carcasses from a rabbit meat seller in Kota Bharu between 7.00 and 7.30 am. The carcasses were transported individually to the decomposition site in separate sealed double plastic bags to prevent exposure to other arthropods prior to placement at the decomposition site. Among the four rabbit carcasses that formed a single replicate, one was used as the control, while the remaining three carcasses were burned to CGS level #1, CGS level #2 and CGS level #3, respectively. Glassman and Crow [14] describe the burn injuries of CGS level #1 as characteristic of typical smoke death (i.e., blistering on the epidermis, singeing of the head and facial hair), with the body remaining recognizable for identification. CGS Level #2 burns occur when the bodies are still recognizable with varying degrees of charring, and the destruction of the body remains limited to the absence of elements of limbs, genitalia and ears [14]. CGS Level #3 occurs when the body undergoes further destruction, leading to the absence of major portions of the limbs with unrecognizable identity, despite the presence of the head [14]. Because extremely extensive burning is required to obtain CGS levels #4 and #5, this study was restricted to CGS level #1, CGS level #2 and CGS level #3 only. For achieving the CGS level #1, CGS level #2 and CGS level #3 burn injuries; the carcasses were burned using 0.5 L of petrol (RON 95) for 1 min, 0.75 L of petrol (RON 95) for 5 min and 1.5 L of petrol (RON 95) for 33 min, respectively (Table 1). As much as possible, the petrol was poured evenly over each carcass. Repeating the above procedure, four replicates (i.e., 16 rabbit carcasses) were included in the present research. The decompositions for all of the four replicate experiments were conducted from the 1st–13th February 2013, 5th–17th February 2013, 9th–21st February 2013 and 27th March–8th April 2013, respectively. While each carcass was separated by a minimum distance of 20 m during every replicate, the decomposition sites for all of the different
Table 1 Detail of the four rabbit carcasses that formed one replicate and conditions of burning to achieve the required CGS level. Rabbits (n = 4)
Quantity of RON95 (L)
Duration of burning (min)
Control CGS level 1 CGS level 2 CGS level 3
Unburned intact carcass 0.5 0.75 1.5
0 1 5 33
replicate experiments were further separated by a minimum distance of 50 m to ensure the independence of these replicates. 2.2. Decomposition site and entomological observation The experiments (February to April 2013) were conducted in a sunlit habitat located within the Universiti Sains Malaysia Health Campus, Kubang Kerian, Kelantan, an eastern state of Peninsular Malaysia (606‘‘1’’N, 102017‘‘5’’E) at approximately 4.6 m above the sea level. The soil type at the decomposition site was loam, and human activity was minimal. Each carcass was placed individually with direct contact on bare soil and covered with a slotted plastic basket (basket length: 58 cm; basket width: 45 cm; basket height: 20 cm; slot length: 3 cm; slot width: 1 cm; mould width between slots: 0.5 cm) with two to three bricks on top to prevent scavengers, while allowing for the free access of flies. Entomological observations, as well as data on daily ambient temperature and rainfall, were recorded until the completion of the first developmental cycle of C. megacephala and C. rufifacies (i.e., evidence of the emergence of tenerals). Following the entomological observation methods prescribed by Mahat et al. [8], field observations, as well as collection, rearing and preservation of entomological specimens, were made. Taxonomic identification of larvae and tenerals was conducted based upon the morphological identification keys provided by previous researchers [18–20]. 2.3. Statistical analysis Data analysis was conducted using IBM SPSS version 20.0 software. The normality of the data used for statistical inference was tested using the Kolmogorov–Smirnov and Shapiro–Wilk tests. Considering the significance level of 0.05, the Kruskal–Wallis H with pairwise comparison using Mann–Whitney U test was used for comparing the differences in the oviposition and completion of life cycles for C. megacephala and C. rufifacies among the different groups. 3. Results and discussion Ambient temperatures remained similar for all four of the carcasses across all four replicates; the ambient temperature data recorded for the control carcasses alone are reported in Table 2. The means for the ambient temperatures and the total daily rainfall recorded during the four replicates ranged between 27.4 and 35.5 8C (Table 2) and 0.0–5.0 mm, respectively. In general, rain was not recorded during the first two days of decomposition in any of the four replicate experiments, except on the second day of decomposition in replicate 1, where drizzling was observed (0.4 mm). Neither incessant nor torrential rains were observed throughout the study. Results revealed that the times to initial oviposition by C. megacephala and C. rufifacies in the CGS level #1 and #2 burned carcasses were similar to that of control carcasses (p > 0.05, Tables 3 and 4), in concurrence with the findings reported by Avila and Goff [15]. It has been reported that blowflies are attracted by the odour emanating from decomposing corpses/carcasses [13], and burning may lead to the disintegration of charred soft tissues, which may be favourable for the colonization of insects [12]. Adding to the body of current knowledge, we found that initial oviposition by both necrophagous species was delayed by one day in the CGS level #3 burned carcasses (p < 0.05, Tables 3 and 4). The CGS Level #3 burned carcasses were relatively drier and had smaller amounts of seeping body fluids when compared with the controls and the CGS level #1 and #2 burned carcasses. Therefore, the delays in oviposition by C. megacephala and C. rufifacies observed in the CGS level #3 burned carcasses (p < 0.05,
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Table 2 Data on daily ambient temperature for the control carcasses during entomological observation. Day
1 2 3 4 5 6 7 8 9 10 11 12 13 *
Replicate 1
Replicate 2
Replicate 3
Replicate 4
Mean
Range
Mean
Range
Mean
Range
Mean
Range
30.0 29.3 27.9 28.1 28.3 – – – – – – – –
27.0–32.0 22.0–32.0 26.0–30.0 26.0–31.0 26.0–31.0 28.9* 30.1* 29.8* 31.5* 30.1* 31.1* 29.5* 30.0*
29.4 29.0 28.3 27.4 28.2 – – – – – – – –
27.0–32.0 22.0–32.0 23.0–32.0 25.0–31.0 22.0–32.0 30.1* 30.1* 29.8* 31.5* 30.1* 31.1* 29.5* 30.0*
31.7 28.2 27.8 27.4 28.2 – – – – – – – –
30.0–33.0 22.0–32.0 23.0–31.0 23.0–31.0 23.0–33.0 28.2* 28.8* 31.5* 30.2* 30.8* 29.8* 29.6* 29.1*
32.1 31.5 31.3 31.1 31.4 – – – – – – – –
26.0–35.0 26.6–35.0 26.0–34.0 26.0–35.0 27.0–34.0 35.5* 33.6* 33.8* 32.4* 32.5* 32.0* 29.6* 29.5*
The temperatures are the actual ambient temperature recorded since, during pupation, ambient temperature was measured once daily around noon.
Table 3 Initial oviposition and completion of the first developmental cycle of C. megacephala in all the four replicates of burned rabbit carcasses. Day and time for the first observation of Replicate
CGS level
Eggs
First instar
Second instar
Third instar
Prepupae
1
Control 1 2 3
Day-1/6 pm Day1/6 pm Day-1/6 pm Day-2/8 am*
Day-2/8 Day-2/8 Day-2/8 Day-2/6
am am am pm
Day-2/6 Day-2/6 Day-2/6 Day-3/8
Day-3/2 Day-3/2 Day-3/2 Day-4/8
Day-4/12 Day-4/12 Day-4/12 Day-5/10
noon noon noon am
Day-5/10 Day-5/10 Day-5/10 Day-6/10
am am am am
Day-9 Day-9 Day-9 Day-10
2
Control 1 2 3
Day-1/4 Day-1/6 Day-1/6 Day-2/8
Day-2/8 Day-2/8 Day-2/8 Day-2/6
am am am pm
Day-2/6 pm Day-2/4 pm Day-2/6 pm Day-3/10 am
Day-3/2 pm Day-3/2 pm Day-3/2 pm Day-4/10 am
Day-4/12 Day-4/12 Day-4/12 Day-5/12
noon noon noon noon
Day-5/10 Day-5/10 Day-5/10 Day-6/10
am am am am
Day-9 Day-9 Day-9 Day-10
3
Control 1 2 3
Day-1/4 pm Day-1/6 pm Day-1/6 pm Day-2/10 am*
Day-2/8 Day-2/8 Day-2/8 Day-2/6
am am am pm
Day-2/4 Day-2/4 Day-2/4 Day-3/8
Day-3/2 pm Day-3/2 pm Day-3/2 pm Day-4/10 am
Day-4/10 Day-4/10 Day-4/10 Day-5/10
am am am am
Day-5/10 Day-5/10 Day-5/10 Day-6/10
am am am am
Day-9 Day-9 Day-9 Day-10
4
Control 1 2 3
Day-1/6 Day-1/6 Day-1/6 Day-2/8
Day-2/8 Day-2/8 Day-2/8 Day-2/6
am am am pm
Day-2/6 pm Day-2/6 pm Day-2/6 pm Day-3/10 am
Day-3/2 pm Day-3/2 pm Day-3/2 pm Day-4/10 am
Day-4/12 Day-4/12 Day-4/12 Day-5/10
noon noon noon am
Day-5/10 Day-5/10 Day-5/10 Day-6/10
am am am am
Day-9 Day-9 Day-9 Day-10
pm pm pm am*
pm pm pm am*
pm pm pm am
pm pm pm am
pm pm pm am
Pupae
Tenerals
Kruskal–Wallis H with pairwise comparisons using the Mann–Whitney U test revealed a significant delay in oviposition for C. megacephala in CGS level 6¼3 carcasses (*) (p < 0.05) when compared with the control as well as CGS levels 6¼1 and 6¼2 carcasses. No significant differences (p > 0.05) in the durations for completing the life cycles for C. megacephala were found in control as well as CGS levels 6¼1-6¼3 carcasses. Significance level of 0.05 was used for determining the significant differences among groups. Field observations were made at every 2 h intervals from 8.00 am to 6.00 pm until the first observation of second instar larvae, then at every 4 h intervals until the first observation of pupae, beyond which the field observation was restricted to once daily around noon.
Tables 3 and 4) may be attributable to the greater charring of the tissues, resulting in a lessening of the decay odour emanating from the carcass after more extensive burning. In all control carcasses, the first, second and third instar larvae, as well as the prepupae of C. megacephala, were invariably observed at approximately 8 am and 4–6 pm on day-2, at approximately 2 pm on day-3 and 10 am to 12 noon on day-4, respectively (Table 3). The pupation period for C. megacephala that started on day-5 lasted for about four days, with the first emergence of tenerals on the 9th day (Table 3). In addition, the completion of the life cycle for C. rufifacies in the control as well as CGS level #1 and #2 burned carcasses was observed on day-11 of decomposition (Table 4). The emergence of tenerals of C. megacephala and C. rufifacies on days 9 and 11, respectively, was consistent with durations reported by previous studies conducted in Malaysia [8,10,16]. Because the initial oviposition of both C. megacephala and C. rufifacies was delayed by one day in CGS level #3 burned carcasses, the completions of their life cycles were subsequently delayed by one day compared to that of control carcasses (Tables 3 and 4). Considering that the durations for completing the life cycles for both C. megacephala and C. rufifacies across all the CGS levels (#1-3) were similar to that of control carcasses (p > 0.05, Tables 3 and 4), it can be
construed that burning carcasses up to CGS level #3 did not influence the durations required for necrophagous insects to complete their life cycles. The CGS levels prescribed by Glassman and Crow [14] provide a useful description of burn injuries to human bodies, and the same descriptions were extended for animal carcasses due to a lack of specific description of burns in animal models. Three different CGS levels produced using rabbits as animal models were used to study the influence of burn injuries on the initial oviposition and development of C. megacephala and C. rufifacies. Pertinently, Avila and Goff [15] utilized burned pig carcasses that corresponded to CGS level #2 in humans. Unlike other animals, pigs are considered to be an acceptable substitute for humans due to their similarities in body mass, skin structure, fat-to-muscle ratio and hair coverage [21], as well as their rates of decomposition and patterns of fly colonization [22]. Considering the morphological differences in aspect ratios, the presence of fur, as well as the weight of the rabbits used in this present study only ranged between 2.0 and 2.5 kg, while human body weights may be more than 20-fold higher, extrapolation of the data to represent burns in humans may not be appropriate. The greater than 20-fold differences in masses mean that rabbits have a far greater surface-area-to-volume ratio
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Table 4 Initial oviposition and completion of the first developmental cycle of C. rufifacies in all the four replicates of burned rabbit carcasses. Day and time for the first observation of Replicate
CGS level
Eggs
First instar
Second instar
Third instar
Prepupae
1
Control 1 2 3
Day-2/8 Day-2/8 Day-2/8 Day-3/8
am am am am*
Day-2/6 Day-2/6 Day-2/6 Day-3/6
pm pm pm pm
Day-3/12 Day-3/12 Day-3/12 Day-4/12
Day-4/2 Day-4/2 Day-4/2 Day-5/2
pm pm pm pm
Day-5/10 Day-5/10 Day-5/10 Day-6/10
am am am am
Day-6/8 Day-6/8 Day-6/8 Day-7/8
2
Control 1 2 3
Day-2/8 Day-2/8 Day-2/8 Day-3/8
am am am am*
Day-2/6 Day-2/6 Day-2/6 Day-3/6
pm pm pm pm
Day-3/2 Day-3/2 Day-3/2 Day-4/2
Day-4/4 Day-4/4 Day-4/4 Day-5/4
pm pm pm pm
Day-5/10 Day-5/10 Day-5/10 Day-6/10
am am am am
Day-6/10 Day-6/10 Day-6/10 Day-7/10
am am am am
Day-11 Day-11 Day-11 Day-12
3
Control 1 2 3
Day-2/8 Day-2/8 Day-2/8 Day-3/8
am am am am*
Day-2/6 Day-2/6 Day-2/6 Day-3/6
pm pm pm pm
Day-3/12 Day-3/12 Day-3/12 Day-4/12
Day-4/2 Day-4/2 Day-4/2 Day-5/2
pm pm pm pm
Day-5/10 Day-5/10 Day-5/10 Day-6/10
am am am am
Day-6/10 Day-6/10 Day-6/10 Day-7/10
am am am am
Day-11 Day-11 Day-11 Day-12
4
Control 1 2 3
Day-2/8 Day-2/8 Day-2/8 Day-3/8
am am am am*
Day-2/6 Day-2/6 Day-2/6 Day-3/6
pm pm pm pm
Day-3/2 Day-3/2 Day-3/2 Day-4/2
Day-4/4 Day-4/4 Day-4/4 Day-5/4
pm pm pm pm
Day-5/10 Day-5/10 Day-5/10 Day-6/10
am am am am
Day-6/8 Day-6/8 Day-6/8 Day-7/8
noon noon noon noon
pm pm pm pm noon noon noon noon
pm pm pm pm
Pupae
Tenerals am am am am
am am am am
Day-11 Day-11 Day-11 Day-12
Day-11 Day-11 Day-11 Day-12
Kruskal–Wallis H with pairwise comparisons using the Mann–Whitney U test revealed a significant delay in oviposition for C. rufifacies in CGS level 6¼3 carcasses (*) (p*) (p < 0.05) when compared with the control as well as CGS levels 6¼1 and 6¼2 carcasses. No significant differences (p > 0.05) in the durations for completing the life cycles for C. rufifacies were found in control as well as CGS levels 6¼1-6¼3 carcasses. Significance level of 0.05 was used for determining the significant differences among groups. Field observations were made at every 2 h intervals from 8.00 am to 6.00 pm until the first observation of second instar larvae, then at every 4 h intervals until the first observation of pupae, beyond which the field observation was restricted to once daily around noon.
than humans. Such aspects would result in greater dynamics of heat penetration into animal tissues than humans for any level of superficial burning as measured by the Crow–Glassman scale. At CGS level #3, the cracks in skin provide flies access to deeper tissues that are relatively undamaged by heat. In contrast, the deeper tissues of small animals, such as rabbits, would be likely to suffer much greater heat damage and become less attractive for egg-laying flies. Therefore, the delay in oviposition for the CGS level #3 rabbit carcasses observed in this study may not be likely to occur in much larger animals. However, the findings reported in this study may still be useful for wildlife forensic investigations involving animals similar to rabbits. In this research, petrol was used for maintaining the fire to achieve the intended CGS levels of burn injuries. Rumiza et al. [23] indicated that arthropods were not attracted to gasolineingested carcasses due to the strong odour of gasoline, which is known to repel insects. Because petrol is a volatile liquid, it is assumed that its odour as well as liquid traces would have evaporated during the fire and subsequent exposure of the carcasses to atmosphere. In certain arson cases where the burn injuries are caused by less volatile accelerants, the effects of residual accelerant may have to be considered. However, this limitation may not apply to dead bodies with burn injuries that are recovered in cases of structural fire, where contact with accelerants by the body would be limited. In conclusion, the initial oviposition and development of C. megacephala and C. rufifacies infesting CGS level #1 and #2 burned carcasses remained similar to that of controls. Significant delays of one day in the initial oviposition for both necrophagous species were observed in the CGS level #3 burned carcasses. Such findings would acquire forensic significance when a CGS-level #3 burned wildlife animal carcass with similar physical characteristics to a rabbit is found, and estimation of minimum PMI in these species is attempted for forensic wildlife investigation. Acknowledgements The authors are thankful to the Universiti Teknologi Malaysia for providing a Research University Grant (Q.J130000.2626.09J64) for conducting a research project in Forensic Entomology.
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