J Forensic Sci, 2014 doi: 10.1111/1556-4029.12430 Available online at: onlinelibrary.wiley.com
PAPER PATHOLOGY/BIOLOGY
Zhou L€ u,1 M.S.; Xiandun Zhai,1 Ph.D.; Haimei Zhou,1 Ph.D.; Pu Li,1 B.Sc.; Jinqi Ma,1 B.Sc.; Ling Guan,1 M.S.; and Yaonan Mo,1 Ph.D.
Effects of Ketamine on the Development of forensically important Blowfly Chrysomya megacephala (F.) (Diptera: Calliphoridae) and its Forensic Relevance*
ABSTRACT: This study investigated effects of ketamine on the development of Chrysomya Megacephala (Diptera: Calliphoridae) at three
different temperatures. Larvae of the C. Megacephala were exposed to different concentrations of drugs and temperatures. The larval lengths, weights, and developmental durations of each stage were observed. This study demonstrated that ketamine, low temperature, and their synergistic action significantly suppressed the development of C. Megacephala (p < 0.001). The time that the larvae in all the treatments achieved the maximum length/weight was significantly delayed (p < 0.05), and that resulted in prolonged duration of larval and prepupal stages especially at low temperature. However, no linear correlations were discovered between ketamine concentration and growth rate of larval length/weight.
KEYWORDS: forensic science, forensic entomology, entomotoxicology, Chrysomya megacephala, drug abuse, Ketamine, developmental duration, postmortem interval
Insects associated with human remains are of fundamental importance in forensic entomology studies, as they can provide valuable information in criminal investigations. The use of insects as indicators to estimate postmortem interval (PMI) has been well documented and widely accepted in forensic practice. PMI estimation of corpse by the development of larvae of necrophagous flies is one of the main applications of forensic entomology in medico-legal death investigation (1–3). However, several factors such as environmental temperature fluctuation, photoperiod, maggot mass effect, nutritional status, hormonal regulation, and presence of chemicals in the corpse may affect the determination of the PMI, making the criminal investigation more difficult and, when not taken into consideration, leading to errors in the PMI estimation (3). It is of vital importance that errors in the PMI estimation are minimized as much as possible to analyze the factors. The presence of drugs and toxins in corpse tissues may affect the development rate of carrion-feeding insects there. Goff et al. (4) first found that cocaine and its metabolite benzoylecgonine could accelerate the development of Boettcherisca peregrina (Diptera: Sarcophagidae). As the developmental rate of larvae is one of the bases for PMI estimation in
1
Forensic Medicine Institute, Henan University of Science and Technology No. 31 Anhui Road, Jianxi District, Luoyang, Henan 471003, China. *Supported by the Basic & Frontier Study of Technology Project of Henan Province (Grant No. 112300410082), the Department of Education Project of Henan Province (Grant No. 200634004), and the Doctor Foundation of Henan University of Science and Technology (Grant No. 09001309). Received 27 Dec. 2012; and in revised form 23 May 2013; accepted 1 June 2013. © 2014 American Academy of Forensic Sciences
forensic entomology, toxicological analysis of insects is of great importance to correctly determine the PMI (5). Ketamine, first synthesized in 1962, is an NMDA receptor antagonist and a derivative of phencyclidine (PCP). Ketamine hydrochloride is used in intravenous anesthesia in clinical practice to replace PCP. Abuse of ketamine has recently gained popularity in China, and an increased trend of deaths due to ketamine intoxication has been noted during the past decade in the central China (6). The purpose of this study was to determine the effects of ketamine on the development of larvae of a forensically important blowfly Chrysomya Megacephala (Diptera: Calliphoridae), which is the dominant necrophagous species distributing area south of Yellow River in China in warm seasons. The larvae were reared in artificial diets containing different concentrations of ketamine. Interactions of ketamine concentration and temperature on the larval development were also observed. To the best of our knowledge, effects of ketamine on the development of C. Megacephala have not been reported. Materials and Methods Ketamine hydrochloride injections (2 mL: 1 g), produced by Jiangsu Hengrui Medicine Co., Ltd. (Lianyungang, China), were obtained from department of forensic toxicology of forensic medicine institute, Henan University of Science and Technology. The batch number of production of the injection is KH091204. Concentrations of all injections were determined by GC-MS method prior to use. Reference standard ketamine was obtained from the Institute of Forensic Science, Ministry of Public 1
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Security of China. Ethanol was purchased from Tianjin Damao Chemical reagent Factory, and xylene was purchased from Tianjin BASF Chemical Industry. Fresh pig meat and XX1-H8146BG agar powder (1200 g/cm2) produced in Japan was used to manufacture the artificial diets. Flies and larvae used in this study came from a stock colony of C. megacephala established from eggs, which were collected from fresh pig liver in the campus of Henan University of Science and Technology (N34°41′, E112°27′) in Luoyang city, PR China. The experimental colony was reared in an environmental chamber for three generations before use. Fresh liver was provided as an oviposition stimulus and site. Eggs were only collected when they were deposited in 1 h by 3–5 females. Egg batches from each single female were divided equally into four sections. These sections from different females were gathered into four colonies (c. 400 eggs each colony) and were laid on artificial diets in glass beakers. For the purpose of calculation, we defined 50 lg/g ketamine as the LD50 for human being as the oral ketamine LD50 of a 70 kg man is approximately 4.2 g (60 lg/g), and the intravenous LD50 is c. 2.8 g (40 lg/g) according to references (7,8). Four different doses of ketamine (the control, 0; 1/2LD50, 25 lg/g; LD50, 50 lg/g; 2LD50, 100 lg/g) were spiked into the artificial diets and fully mixed before solidification. The beakers were placed in transparent boxes (600 mm 9 440 mm 9 360 mm) containing sand and sawdust to prevent the postfeeding larvae from escape. All these boxes were deposited in environmental chamber at a constant temperature with 12:12 photoperiod and 75 10% humidity. The experiments were repeated at constant temperature of 32, 28, and 24°C. All the insects were observed every hour during larval stage, prepupal stage, and pupal stage. For each colony, 10 random larvae samples were collected every 12 h for physical measurements from the 16th hour since hatched until pupation. These samples of larvae were killed with a 50:50 v/v blend of ethanol and xylene and preserved in 75% alcohol. The samples were weighed with Mettler-Toledoâ (Zürich, Switzerland) AL104 electronic analytical balance (110 g 9 0.1 mg), produced in Switzerland. Lengths were measured with Shanghai Tool Worksâ outside
micrometer (25 mm 9 0.01 mm), produced in China. The weight and length data were recorded in form of mean SD. The maximum larval lengths, weights, the average growth rate of lengths, and weights were also calculated. The average growth rate of lengths (mm/h) = the maximum body length/developmental duration. The average growth rate of weights (mg/h) = the maximum body weight/developmental duration. One-way analysis of variance (ANOVA) was performed to investigate possible differences between the treatment colonies and the control. Duncan’s multiple comparisons test was used to compare the means for every factor. Kolmogorov–Smirnov test was performed to analyze the effects of ketamine concentration and temperature factors on the larval length, weight and to verify their interactions. These results were calculated and analyzed by the SPSS 15.0. A p < 0.05 was considered significant for all the analyses. Most figures were drawn and exported by the Origin Pro 8.0 (OriginLab, Northampton, MA).
Results Development of Larval Reared at Different Ketamine Concentrations At a constant temperature of 32°C, the average larval length in all treatment colonies was significantly less than the control from 16 h to 52 h after hatched (Table 1). The same period turned into 16–40 h at 28°C (Table 2) and 16 h–76 h at 24°C (Table 3). No correlation was observed between ketamine concentration and larval length in the treatment colonies. The growth rates of larval length of each colony at different temperatures are shown in Fig. 1. The growth rate of 1/2LD50 colony at 24°C was the most suppressed. However, no linear correlation was found between ketamine concentration and growth rate of larval length. Similar to the average larval length, the average weight of all treatment colonies was significantly smaller than the control from 16 h to 52 h after hatched at 32°C (Table 4), and from 16 h to 64 h (Tables 5 and 6) was also the same result at both 28°C and
TABLE 1––Mean lengths (mm) of larvae of C. megacephala at a constant temperature of 32°C. Time (h) 16 28 40 52 64 76
Control Mean (SD)
1/2LD50 Mean (SD)
LD50 Mean (SD)
2LD50 Mean (SD)
5.53 (0.18)a 10.68 (0.31)a 14.03 (0.38)a 17.79 (0.47)a 17.97 (0.55)a Pupation
4.48 (0.15)b 9.24 (0.32)b 12.55 (0.33)b 15.78 (0.52)b 16.38 (0.59)b Pupation
4.65 (0.17)c 8.97 (0.34)b 12.16 (0.31)c 16.58 (0.60)c 18.09 (0.42)a Pupation
4.36 (0.16)b 9.13 (0.32)b 13.03 (0.35)d 17.09 (0.55)d 18.18 (0.35)a Pupation
Mean in a line followed by the same letters are not significantly different.
TABLE 2––Mean lengths (mm) of larvae of C. megacephala at a constant temperature of 28°C. Time (h) 16 28 40 52 64 76 88 100
Control Mean (SD)
1/2LD50 Mean (SD)
LD50 Mean (SD)
2LD50 Mean (SD)
3.48 (0.27)a 8.88 (0.18)a 13.50 (0.46)a 17.88 (0.32)a 19.09 (0.37)a 17.90 (0.44)a Pupation
3.14 (0.21)b 7.35 (0.52)b 11.33 (0.64)b 16.87 (0.53)b 18.35 (0.51)b 18.90 (0.39)b 18.60 (0.53)a Pupation
3.16 (0.17)b 7.70 (0.42)b 13.17 (0.66)a 17.74 (0.44)ac 18.91 (0.38)ac 19.28 (0.46)c 17.83 (0.59)b Pupation
3.10 (0.28)b 7.73 (0.62)b 12.98 (0.64)a 17.36 (0.37)c 18.61 (0.54)bc 18.88 (0.31)b 18.52 (0.47)a Pupation
Mean in a line followed by the same letters are not significantly different.
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EFFECTS OF KETAMINE ON THE DEVELOPMENT OF C. MEGACEPHALA
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TABLE 3––Mean lengths (mm) of larvae of C. megacephala at a constant temperature of 24°C. Time (h) 16 28 40 52 64 76 88 100 112 124 136
Control Mean (SD)
1/2LD50 Mean (SD)
LD50 Mean (SD)
2LD50 Mean (SD)
3.79 (0.24)a 5.80 (0.44)a 8.35 (0.35)a 13.68 (0.55)a 16.52 (0.33)a 16.85 (0.38)a 17.30 (0.27)a 16.98 (0.49)a 16.97 (0.51)a Pupation
3.43 (0.19)b 5.21 (0.21)b 7.92 (0.31)b 12.30 (0.35)b 14.23 (0.54)b 16.17 (0.40)bc 16.17 (0.37)b 17.10 (0.46)a 17.54 (0.34)b 17.23 (0.49)a Pupation
3.47 (0.13)b 5.09 (0.30)b 7.44 (0.40)c 10.94 (0.44)c 14.53 (0.56)b 16.07 (0.41)b 17.16 (0.30)ac 17.64 (0.40)b 18.26 (0.32)c 17.79 (0.49)b Pupation
3.59 (0.21)b 5.04 (0.27)b 7.13 (0.33)c 11.22 (0.59)c 13.68 (0.54)c 16.46 (0.37)c 16.90 (0.33)c 17.75 (0.51)b 18.23 (0.51)c 17.81 (0.33)b Pupation
Mean in a line followed by the same letters are not significantly different.
length (60.17% of total effect) and weight (85.60%), followed by the variation of ketamine concentration (20.90% and 8.32%, respectively), and the synergistic action of both (18.93% and 6.08%, respectively). Effect on Developmental Duration of C. Megacephala The result of observation of effects on developmental duration (Figs. 5–7) showed that durations of larval stage of all the treatments were significantly prolonged (p < 0.05) compared to the control at different temperatures. Durations of prepupal stage of all the treatments were also prolonged (p < 0.05) except 32°C. However, durations of pupal stage of all the treatments were slightly shortened (p < 0.05) at 24°C. Discussion FIG. 1––Growth rates of larval body lengths (mm/h) of C. megacephala at constant temperature of 32, 28, and 24°C. All of the treatments grew slower than the control at 28 and 24°C.
24°C. No correlation between ketamine concentration and larval weight in the treatment colonies was observed, too. The growth rates of larval weight of each colony at different temperatures are shown in Fig. 2. The growth rate of 1/2LD50 colony at 24°C was the most suppressed, too. Similar to the result of larval length, no linear correlation was discovered between ketamine concentration and growth rate of larval weight. Interaction of Ketamine and Temperature on Larval Development There was a certain correlation of effects on the maximum larval length/weight between ketamine concentrations and environmental temperature (Figs. 3 and 4). This correlation showed that the maximum larval length/weight was the greatest at 28°C. No consistent changes in effect on larval length/weight were observed at different ketamine concentrations. The average growth rate of larval length/weight in all the treatments was more suppressed than the control at lower temperature, of which the most significant delay was the 1/2LD50 colony at lowest temperature. The results of Kolmogorov–Smirnov test indicated that the effects of ketamine concentration, temperature, and their synergistic action on the development of larvae were all statistically significant (p < 0.001). Of the three factors, temperature had the most significant effect on the growth rate of larval
Illicit drug abuse has risen to epidemic levels over the last decade in China because of the implementation of its open-door policies (9). The use of ketamine has recently gained popularity in China. An increased trend of illicit drug abuse deaths, mainly caused by heroin and ketamine during the past decade, has been reported in the central part of China (6). Previous studies have indicated that many drugs and their metabolites can either accelerate or delay the development of larvae of necrophagous flies feeding on cadavers. It has been reported that amitriptyline, hydrocortisone, sodium methohexital, butylscopolamine bromide, malathion, and ethanol delay the development of larval growth of necrophagous flies, while heroin, methamphetamine, cocaine, codeine, and diazepam accelerate the development of larvae (4,10–18). These effects can potentially cause misestimating of PMI by as much as 77 h in those drug-related deaths (10). Knowledge of the effects of different drugs on the growth rates of immature carrion-breeding insects could be useful in refining the estimation of PMI (1). Recently, Zou et al. (19) reported that ketamine could shorten the larval stage of Lucilia sericata (Diptera: Calliphoridae). In the present study, our result showed that ketamine can delay larval development of C. megacephala. The growth rates of larval length/weight of all treatment colonies of C. megacephala were significantly suppressed compared with the control. However, no significant dose-dependent effects of ketamine on the larval growth were observed. Interestingly, these results are completely opposite to the Zou’s. Bourel et al. (20) studied the effects of morphine on the development of L. sericata, and Tian et al. (21) studied the effects of morphine on the development of C. megacephala
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JOURNAL OF FORENSIC SCIENCES TABLE 4––Mean weights (mg) of larvae of C. megacephala at a constant temperature of 32°C.
Time (h) 16 28 40 52 64 76
Control Mean (SD)
1/2LD50 Mean (SD)
LD50 Mean (SD)
2LD50 Mean (SD)
2.6 (–) 26.9 (2.0)a 56.1 (4.7)a 108.4 (9.3)a 104.0 (9.5)a Pupation
1.8 (–) 20.2 (1.5)b 48.2 (3.5)b 92.9 (9.8)b 91.3 (10.0)b Pupation
2.0 (–) 19.8 (1.7)b 45.5 (3.1)b 94.4 (10.1)b 102.6 (7.5)ac Pupation
2.0 (–) 20.7 (1.7)b 47.4 (3.6)b 94.5 (9.4)b 95.9 (5.9)bc Pupation
Mean in a line followed by the same letters are not significantly different.
TABLE 5––Mean weights (mg) of larvae of C. megacephala at a constant temperature of 28°C. Time (h) 16 28 40 52 64 76 88 100
Control Mean (SD)
1/2LD50 Mean (SD)
LD50 Mean (SD)
2LD50 Mean (SD)
0.7 (–) 11.7 (1.2)a 30.0 (4.0)a 95.8 (5.3)a 116.4 (6.7)a 106.3 (4.9)a Pupation
0.5 (–) 7.4 (1.5)b 18.9 (3.8)b 69.1 (7.9)b 93.9 (8.5)b 108.2 (7.5)a 106.0 (9.2)ab Pupation
0.7 (–) 8.7 (0.9)c 26.7 (2.4)c 87.0 (5.7)c 108.7 (2.9)c 118.3 (5.3)b 100.4 (6.1)b Pupation
0.6 (–) 7.9 (1.1)bc 25.9 (2.4)c 79.4 (5.5)d 107.1 (5.0)c 103.8 (5.0)a 107.2 (3.9)a Pupation
Numbers in brackets are standard deviations. Mean in a line followed by the same letters are not significantly different.
TABLE 6––Mean weights (mg) of larvae of C. megacephala at a constant temperature of 24°C. Time (h) 16 28 40 52 64 76 88 100 112 124 136
Control Mean (SD)
1/2LD50 Mean (SD)
LD50 Mean (SD)
2LD50 Mean (SD)
0.3 ( ) 3.0 (0.7)a 9.5 (1.0)a 34.3 (2.5)a 65.0 (4.0)a 74.3 (5.8)a 82.0 (5.3)a 74.4 (9.7)ab 71.1 (9.3)a Pupation
0.2 ( ) 2.3(0.3)b 7.4 (1.3)b 24.6 (2.8)b 29.8 (3.1)b 52.1 (2.1)b 58.6 (2.0)b 70.6 (4.2)b 75.1 (4.4)a 63.7 (4.7)a Pupation
0.3 ( ) 1.7 (0.3)c 5.6 (0.6)c 16.5 (1.5)c 36.7(4.2)c 51.9 (5.9)b 64.8 (4.3)c 76.1 (3.7)ab 81.5 (4.7)b 79.5 (7.2)b Pupation
0.3 ( ) 1.8 (0.3)c 5.3 (0.4)c 19.3 (0.5)d 27.1 (3.9)b 60.5 (5.0)c 66.9 (4.1)c 80.9 (8.7)a 88.0 (7.7)c 74.5 (4.3)b Pupation
Numbers in brackets are standard deviations. Mean in a line followed by the same letters are not significantly different.
FIG. 2––Growth rates of larval body weights (mg/h) of C. megacephala at constant temperature of 32, 28, and 24°C. All of the treatments gained slower than the control at all three temperatures. This result indicates that the growth of larval length and weight of C. megacephala are not absolutely parallel.
FIG. 3––Variation trend of ketamine concentration—the maximum larval length. The maximum larval length/weight was the greatest at 28°C. No correlation was observed between ketamine concentration and maximum larval length.
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EFFECTS OF KETAMINE ON THE DEVELOPMENT OF C. MEGACEPHALA
FIG. 4––Variation trend of ketamine concentration—the maximum larval weight. The maximum larval length/weight was the greatest at 28°C. No correlation was observed between ketamine concentration and maximum larval weight.
FIG. 5––Developmental durations of C. megacephala at constant temperature of 32°C. The durations of larval stage of all the treatments were prolonged slightly.
FIG. 6––Developmental durations of C. megacephala at constant temperature of 28°C. The durations of larval stage of all the treatments were prolonged significantly
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FIG. 7––Developmental durations of C. megacephala at constant temperature of 24°C. The durations of larval and prepupal stages of all the treatments were prolonged significantly while the durations of pupal stage were slightly shortened.
using the same method of Bourel’s. Their results showed that morphine could suppress the development of L. sericata during the larval stage, but accelerate it of C. megacephala. It appears to indicate that different species of Luciliinae and Chrysomyinae may response differently to the same drug. Comparing with the results of Zou’s, our study confirmed this phenomenon. In the deaths of suspected ketamine intoxication, it should take the delayed growth effect into account when using regression against larval weight or length to estimate PMI. The effect of temperature on the insect larval development has also been studied extensively (22–24). The durations of larval stage increase with a decline in temperature. Our study showed that ketamine, low temperature, and their synergistic action significantly suppressed the development of C. Megacephala and prolonged its developmental durations. For the tested range of temperatures of 32, 28, and 24°C, the total effect could prolong the duration of larval and prepupal stages, while it slightly shorten the duration of pupal stage at low temperature. The total duration of the treatment colony was prolonged 33 h at most (24°C, 2LD50). The effects of ketamine on the growth rate of larval length, weight, and developmental duration, nevertheless, were not consistent. This phenomenon indicates that the growth of larval length, weight, and duration of larval stage of C. megacephala is not absolutely parallel. The present study showed ketamine only delayed the larval growth but not completely stop the development, and no linear correlation between the degree of inhibition and ketamine concentration was observed. Compared with single temperature-repeated experiment, our study explored the difference in toxicological effect of drug on the development of necrophagous fly at different temperatures for the first time and found the above-mentioned synergistic action of drug concentration and temperature. In addition, the fact should be noticed that the larvae were reared in an artificial diet in this study. Compared with administering the drug via ear vein of animals (20) or gavage method (13), the drug used in this study was not metabolized by a living system. We chose the artificial diet containing drug as the model of acute poisoning other than animal experiment for two reasons. First, ketamine will be metabolized and converted to norketamine in 2–3 h, and the effects from ketamine will last for 1–5 h in human body (25). As victims died quickly in most of cases
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relevant to ketamine overdose, there was still a mass of ketamine remained in cadavers (8). The second, the metabolic processes of the same drug are not necessarily the same in different species (26,27). Therefore, animal models of acute poisoning are not exactly as same as human in all situations. In view of these two reasons above, we performed the experiment based on O’Brien and Turner (28) and Oliveira et al. (12) methods and modified several details. In conclusion, ketamine can suppress the development of C. megacephala, and there is no linear dose-dependent relationship between ketamine concentration and the degree of inhibition of larval development. Knowledge of the effects of ketamine on the development of C. megacephala can be useful in estimation of PMI in suspected ketamine-related deaths. As different species of the Diptera may have different responses to the same drug, further research is needed to observe the effects of ketamine on the development of more species of necrophagous flies. Acknowledgments We thank Professor Liu Min of College of basic medicine and forensic medicine of Sichuan University for providing the craftsmanship of artificial diet support, and Professor Wang Jiangfeng of Guangdong Police College for some suggestions on flies feeding. We also thank Professor Li Ling of Chief Medical Examiner Office of Maryland for her help in revising this article. References 1. Catts EP, Haskell NH. Entomology and death: a procedure guide. Clemson, SC: Forensic Entomology Specialties, Joyce’s Print Shop, 1990. 2. Catts EP, Goff ML. Forensic entomology in criminal investigations. Annu Rev Entomol 1992;37:253–72. 3. Byrd JH, Castner JL. Forensic entomology: the utility of arthropods in legal investigations, 2nd edn. Boca Raton: CRC Press, 2010. 4. Goff ML, Omori A, Goodbrod J. Effect of cocaine in tissues on the rate of development of Boettcherisca peregrina (Diptera: Sarcophagidae). J Med Entomol 1989;26:91–3. 5. Gosselin M, Wille SM, Fernandez MM, Di FV, Samyn N, De Boeck G, et al. Entomotoxicology, experimental set-up and interpretation for forensic toxicologists. Forensic Sci Int 2011;208(1):1–9. 6. Zhou L, Liu L, Chang L, Li L. Poisoning deaths in central China (Hubei): a 10-year retrospective study of forensic autopsy cases. J Forensic Sci 2011;56:S234–7. 7. Bruce DL, Capan L. Antidepressants do not increase the lethality of Ketamine in mice. Br J Anaesth 1983;55(5):457–9. 8. Gable RS. Acute toxic effects of club drugs. J Psychoactive Drugs 2004;36(1):303–13. 9. Liu J, Si XM, Li HY. An investigation and analysis on addicts in city Dalian. Chin J Drug Abuse Prev Treatment 2004;10(1):24–5. 10. Goff ML, Brown WA, Omori AI, LaPointe DA. Preliminary observations of the effects of amitriptyline in decomposing tissues on the development of Parasarcophaga ruficornis (Diptera: Sarcophagidae) and implications of this effect to estimation of postmortem interval. J Forensic Sci 1993;38(2):316–22. 11. Musvasva E, Williams KA, Muller WJ, Villet MH. Preliminary observations on the effects of hydrocortisone and sodium methohexital on development of Sarcophaga (Curranea) tibialis Macquart (Diptera: Sarcophagidae), and implications for estimating post mortem interval. Forensic Sci Int 2001;120:37–41. 12. Oliveira HG, Gomes G, Morlin JJ Jr, Von Zuben CJ, Linhares AX. The effect of Buscopan on the development of the blow fly Chrysomya
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megacephala (F.) (Diptera: Calliphoridae). J Forensic Sci 2009;54(1):202– 6. Liu XS, Shi YW, Wang HY, Zhang RJ. Determination of Malathion levels and its effect on the development of Chrysomya megacephala (Fabricius) in South China. J Forensic Sci 2009;192(1):14–8. Tabor KL, Fell RD, Brewster CC, Pelzer K, Behonick GS. Effects of antemortem ingestion of ethanol on insect successional patterns and development of Phormia regina (Diptera: Calliphoridae). J Med Entomol 2005;42(3):481–9. Goff ML, Brown WA, Hewadikaram KA, Omori AI. Effect of heroin in decomposing tissues on the development rate of Boettcherisca peregrina (Diptera: Sarcophagidae) and implications of this effect on estimation of postmortem intervals using arthropod development patterns. J Forensic Sci 1991;36(2):537–42. Goff ML, Brown WA, Omori AI. Preliminary observations of the effect of methamphetamine in decomposing tissues on the development rate of Parasarcophaga ruficornis (Diptera: Sarcophagidae) and implications of this effect on the estimations of postmortem intervals. J Forensic Sci 1992;37(3):867–72. Kharbouche H, Augsburger M, Cherix D, Sporkert F, Giroud C, Wyss C, et al. Codeine accumulation and elimination in larvae, pupae, and imago of the blowfly Lucilia sericata and effects on its development. Int J Legal Med 2008;122(3):205–11. Carvalho LM, Linhares AX, Trigo JR. Determination of drug levels and the effect of diazepam on the growth of necrophagous flies of forensic importance in southeastern Brazil. Forensic Sci Int 2001;120:140–4. Zou Y, Huang M, Huang R, Wu X, You Z, Lin J, et al. Effect of Ketamine on the development of Lucilia sericata (Meigen) (Diptera: Calliphoridae) and preliminary pathological observation of larvae. Forensic Sci Int 2013;226:273–81. Bourel B, Hedouin V, Martin BL, Becart A, Tournel G, Deveaux M, et al. Effects of morphine in decomposing bodies on the development of Lucilia sericata (Diptera: Calliphoridae). J Forensic Sci 1999;44(2):354– 8. Tian J, Zhang MY, He B, Li ZM, Wang BX. Effect of morphine in rabbit tissues on growth of Chrysomya megacephala (Diptera: Calliphoridae) and its implication for estimation of postmortem intervals. Acta Entomol Sin 2004;47(6):715–8. Gomes L, Gomes G, Von Zuben CJ. The influence of temperature on the behavior of burrowing in larvae of the blowflies, Chrysomya albiceps and Lucilia cuprina, under controlled conditions. J Insect Sci 2009;9:14. Ames C, Turner B. Low temperature episodes in development of blowflies: implications for postmortem interval estimation. Med Vet Entomol 2003;17(2):178–86. Eliopoulos PA, Kontodimas DC, Stathas GJ. Temperature-dependent development of Chilocorus bipustulatus (Coleoptera: Coccinellidae). Environ Entomol 2010;39(4):1352–8. Mozayani A. Ketamine - effects on human performance and behavior. Forensic Sci Rev 2002;14(1–2):123–31. Campobasso CP, Gherardi M, Caligara M, Sironi L, Introna F. Drug analysis in blowfly larvae and in human tissues: a comparative study. Int J Legal Med 2004;118(4):210–4. Tominaga T, Negishi T, Hirooka H, Miyachi A, Inoue A, Hayasaka I, et al. Toxicokinetics of bisphenol A in rats, monkeys and chimpanzees by the LC–MS/MS method. Toxicology 2006;226(2–3):208–17. O’Brien C, Turner B. Effect of paracetamol on Calliphora vicina larval development. Int J Legal Med 2004;118(4):188–9.
Additional information and reprint requests: Zhou L€u, M.S. Forensic Medicine Institute Henan University of Science and Technology No. 31 Anhui Road Jianxi District, Luoyang Henan 471003 China E-mail: [email protected]
PAPER PATHOLOGY/BIOLOGY
Zhou L€ u,1 M.S.; Xiandun Zhai,1 Ph.D.; Haimei Zhou,1 Ph.D.; Pu Li,1 B.Sc.; Jinqi Ma,1 B.Sc.; Ling Guan,1 M.S.; and Yaonan Mo,1 Ph.D.
Effects of Ketamine on the Development of forensically important Blowfly Chrysomya megacephala (F.) (Diptera: Calliphoridae) and its Forensic Relevance*
ABSTRACT: This study investigated effects of ketamine on the development of Chrysomya Megacephala (Diptera: Calliphoridae) at three
different temperatures. Larvae of the C. Megacephala were exposed to different concentrations of drugs and temperatures. The larval lengths, weights, and developmental durations of each stage were observed. This study demonstrated that ketamine, low temperature, and their synergistic action significantly suppressed the development of C. Megacephala (p < 0.001). The time that the larvae in all the treatments achieved the maximum length/weight was significantly delayed (p < 0.05), and that resulted in prolonged duration of larval and prepupal stages especially at low temperature. However, no linear correlations were discovered between ketamine concentration and growth rate of larval length/weight.
KEYWORDS: forensic science, forensic entomology, entomotoxicology, Chrysomya megacephala, drug abuse, Ketamine, developmental duration, postmortem interval
Insects associated with human remains are of fundamental importance in forensic entomology studies, as they can provide valuable information in criminal investigations. The use of insects as indicators to estimate postmortem interval (PMI) has been well documented and widely accepted in forensic practice. PMI estimation of corpse by the development of larvae of necrophagous flies is one of the main applications of forensic entomology in medico-legal death investigation (1–3). However, several factors such as environmental temperature fluctuation, photoperiod, maggot mass effect, nutritional status, hormonal regulation, and presence of chemicals in the corpse may affect the determination of the PMI, making the criminal investigation more difficult and, when not taken into consideration, leading to errors in the PMI estimation (3). It is of vital importance that errors in the PMI estimation are minimized as much as possible to analyze the factors. The presence of drugs and toxins in corpse tissues may affect the development rate of carrion-feeding insects there. Goff et al. (4) first found that cocaine and its metabolite benzoylecgonine could accelerate the development of Boettcherisca peregrina (Diptera: Sarcophagidae). As the developmental rate of larvae is one of the bases for PMI estimation in
1
Forensic Medicine Institute, Henan University of Science and Technology No. 31 Anhui Road, Jianxi District, Luoyang, Henan 471003, China. *Supported by the Basic & Frontier Study of Technology Project of Henan Province (Grant No. 112300410082), the Department of Education Project of Henan Province (Grant No. 200634004), and the Doctor Foundation of Henan University of Science and Technology (Grant No. 09001309). Received 27 Dec. 2012; and in revised form 23 May 2013; accepted 1 June 2013. © 2014 American Academy of Forensic Sciences
forensic entomology, toxicological analysis of insects is of great importance to correctly determine the PMI (5). Ketamine, first synthesized in 1962, is an NMDA receptor antagonist and a derivative of phencyclidine (PCP). Ketamine hydrochloride is used in intravenous anesthesia in clinical practice to replace PCP. Abuse of ketamine has recently gained popularity in China, and an increased trend of deaths due to ketamine intoxication has been noted during the past decade in the central China (6). The purpose of this study was to determine the effects of ketamine on the development of larvae of a forensically important blowfly Chrysomya Megacephala (Diptera: Calliphoridae), which is the dominant necrophagous species distributing area south of Yellow River in China in warm seasons. The larvae were reared in artificial diets containing different concentrations of ketamine. Interactions of ketamine concentration and temperature on the larval development were also observed. To the best of our knowledge, effects of ketamine on the development of C. Megacephala have not been reported. Materials and Methods Ketamine hydrochloride injections (2 mL: 1 g), produced by Jiangsu Hengrui Medicine Co., Ltd. (Lianyungang, China), were obtained from department of forensic toxicology of forensic medicine institute, Henan University of Science and Technology. The batch number of production of the injection is KH091204. Concentrations of all injections were determined by GC-MS method prior to use. Reference standard ketamine was obtained from the Institute of Forensic Science, Ministry of Public 1
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Security of China. Ethanol was purchased from Tianjin Damao Chemical reagent Factory, and xylene was purchased from Tianjin BASF Chemical Industry. Fresh pig meat and XX1-H8146BG agar powder (1200 g/cm2) produced in Japan was used to manufacture the artificial diets. Flies and larvae used in this study came from a stock colony of C. megacephala established from eggs, which were collected from fresh pig liver in the campus of Henan University of Science and Technology (N34°41′, E112°27′) in Luoyang city, PR China. The experimental colony was reared in an environmental chamber for three generations before use. Fresh liver was provided as an oviposition stimulus and site. Eggs were only collected when they were deposited in 1 h by 3–5 females. Egg batches from each single female were divided equally into four sections. These sections from different females were gathered into four colonies (c. 400 eggs each colony) and were laid on artificial diets in glass beakers. For the purpose of calculation, we defined 50 lg/g ketamine as the LD50 for human being as the oral ketamine LD50 of a 70 kg man is approximately 4.2 g (60 lg/g), and the intravenous LD50 is c. 2.8 g (40 lg/g) according to references (7,8). Four different doses of ketamine (the control, 0; 1/2LD50, 25 lg/g; LD50, 50 lg/g; 2LD50, 100 lg/g) were spiked into the artificial diets and fully mixed before solidification. The beakers were placed in transparent boxes (600 mm 9 440 mm 9 360 mm) containing sand and sawdust to prevent the postfeeding larvae from escape. All these boxes were deposited in environmental chamber at a constant temperature with 12:12 photoperiod and 75 10% humidity. The experiments were repeated at constant temperature of 32, 28, and 24°C. All the insects were observed every hour during larval stage, prepupal stage, and pupal stage. For each colony, 10 random larvae samples were collected every 12 h for physical measurements from the 16th hour since hatched until pupation. These samples of larvae were killed with a 50:50 v/v blend of ethanol and xylene and preserved in 75% alcohol. The samples were weighed with Mettler-Toledoâ (Zürich, Switzerland) AL104 electronic analytical balance (110 g 9 0.1 mg), produced in Switzerland. Lengths were measured with Shanghai Tool Worksâ outside
micrometer (25 mm 9 0.01 mm), produced in China. The weight and length data were recorded in form of mean SD. The maximum larval lengths, weights, the average growth rate of lengths, and weights were also calculated. The average growth rate of lengths (mm/h) = the maximum body length/developmental duration. The average growth rate of weights (mg/h) = the maximum body weight/developmental duration. One-way analysis of variance (ANOVA) was performed to investigate possible differences between the treatment colonies and the control. Duncan’s multiple comparisons test was used to compare the means for every factor. Kolmogorov–Smirnov test was performed to analyze the effects of ketamine concentration and temperature factors on the larval length, weight and to verify their interactions. These results were calculated and analyzed by the SPSS 15.0. A p < 0.05 was considered significant for all the analyses. Most figures were drawn and exported by the Origin Pro 8.0 (OriginLab, Northampton, MA).
Results Development of Larval Reared at Different Ketamine Concentrations At a constant temperature of 32°C, the average larval length in all treatment colonies was significantly less than the control from 16 h to 52 h after hatched (Table 1). The same period turned into 16–40 h at 28°C (Table 2) and 16 h–76 h at 24°C (Table 3). No correlation was observed between ketamine concentration and larval length in the treatment colonies. The growth rates of larval length of each colony at different temperatures are shown in Fig. 1. The growth rate of 1/2LD50 colony at 24°C was the most suppressed. However, no linear correlation was found between ketamine concentration and growth rate of larval length. Similar to the average larval length, the average weight of all treatment colonies was significantly smaller than the control from 16 h to 52 h after hatched at 32°C (Table 4), and from 16 h to 64 h (Tables 5 and 6) was also the same result at both 28°C and
TABLE 1––Mean lengths (mm) of larvae of C. megacephala at a constant temperature of 32°C. Time (h) 16 28 40 52 64 76
Control Mean (SD)
1/2LD50 Mean (SD)
LD50 Mean (SD)
2LD50 Mean (SD)
5.53 (0.18)a 10.68 (0.31)a 14.03 (0.38)a 17.79 (0.47)a 17.97 (0.55)a Pupation
4.48 (0.15)b 9.24 (0.32)b 12.55 (0.33)b 15.78 (0.52)b 16.38 (0.59)b Pupation
4.65 (0.17)c 8.97 (0.34)b 12.16 (0.31)c 16.58 (0.60)c 18.09 (0.42)a Pupation
4.36 (0.16)b 9.13 (0.32)b 13.03 (0.35)d 17.09 (0.55)d 18.18 (0.35)a Pupation
Mean in a line followed by the same letters are not significantly different.
TABLE 2––Mean lengths (mm) of larvae of C. megacephala at a constant temperature of 28°C. Time (h) 16 28 40 52 64 76 88 100
Control Mean (SD)
1/2LD50 Mean (SD)
LD50 Mean (SD)
2LD50 Mean (SD)
3.48 (0.27)a 8.88 (0.18)a 13.50 (0.46)a 17.88 (0.32)a 19.09 (0.37)a 17.90 (0.44)a Pupation
3.14 (0.21)b 7.35 (0.52)b 11.33 (0.64)b 16.87 (0.53)b 18.35 (0.51)b 18.90 (0.39)b 18.60 (0.53)a Pupation
3.16 (0.17)b 7.70 (0.42)b 13.17 (0.66)a 17.74 (0.44)ac 18.91 (0.38)ac 19.28 (0.46)c 17.83 (0.59)b Pupation
3.10 (0.28)b 7.73 (0.62)b 12.98 (0.64)a 17.36 (0.37)c 18.61 (0.54)bc 18.88 (0.31)b 18.52 (0.47)a Pupation
Mean in a line followed by the same letters are not significantly different.
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EFFECTS OF KETAMINE ON THE DEVELOPMENT OF C. MEGACEPHALA
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TABLE 3––Mean lengths (mm) of larvae of C. megacephala at a constant temperature of 24°C. Time (h) 16 28 40 52 64 76 88 100 112 124 136
Control Mean (SD)
1/2LD50 Mean (SD)
LD50 Mean (SD)
2LD50 Mean (SD)
3.79 (0.24)a 5.80 (0.44)a 8.35 (0.35)a 13.68 (0.55)a 16.52 (0.33)a 16.85 (0.38)a 17.30 (0.27)a 16.98 (0.49)a 16.97 (0.51)a Pupation
3.43 (0.19)b 5.21 (0.21)b 7.92 (0.31)b 12.30 (0.35)b 14.23 (0.54)b 16.17 (0.40)bc 16.17 (0.37)b 17.10 (0.46)a 17.54 (0.34)b 17.23 (0.49)a Pupation
3.47 (0.13)b 5.09 (0.30)b 7.44 (0.40)c 10.94 (0.44)c 14.53 (0.56)b 16.07 (0.41)b 17.16 (0.30)ac 17.64 (0.40)b 18.26 (0.32)c 17.79 (0.49)b Pupation
3.59 (0.21)b 5.04 (0.27)b 7.13 (0.33)c 11.22 (0.59)c 13.68 (0.54)c 16.46 (0.37)c 16.90 (0.33)c 17.75 (0.51)b 18.23 (0.51)c 17.81 (0.33)b Pupation
Mean in a line followed by the same letters are not significantly different.
length (60.17% of total effect) and weight (85.60%), followed by the variation of ketamine concentration (20.90% and 8.32%, respectively), and the synergistic action of both (18.93% and 6.08%, respectively). Effect on Developmental Duration of C. Megacephala The result of observation of effects on developmental duration (Figs. 5–7) showed that durations of larval stage of all the treatments were significantly prolonged (p < 0.05) compared to the control at different temperatures. Durations of prepupal stage of all the treatments were also prolonged (p < 0.05) except 32°C. However, durations of pupal stage of all the treatments were slightly shortened (p < 0.05) at 24°C. Discussion FIG. 1––Growth rates of larval body lengths (mm/h) of C. megacephala at constant temperature of 32, 28, and 24°C. All of the treatments grew slower than the control at 28 and 24°C.
24°C. No correlation between ketamine concentration and larval weight in the treatment colonies was observed, too. The growth rates of larval weight of each colony at different temperatures are shown in Fig. 2. The growth rate of 1/2LD50 colony at 24°C was the most suppressed, too. Similar to the result of larval length, no linear correlation was discovered between ketamine concentration and growth rate of larval weight. Interaction of Ketamine and Temperature on Larval Development There was a certain correlation of effects on the maximum larval length/weight between ketamine concentrations and environmental temperature (Figs. 3 and 4). This correlation showed that the maximum larval length/weight was the greatest at 28°C. No consistent changes in effect on larval length/weight were observed at different ketamine concentrations. The average growth rate of larval length/weight in all the treatments was more suppressed than the control at lower temperature, of which the most significant delay was the 1/2LD50 colony at lowest temperature. The results of Kolmogorov–Smirnov test indicated that the effects of ketamine concentration, temperature, and their synergistic action on the development of larvae were all statistically significant (p < 0.001). Of the three factors, temperature had the most significant effect on the growth rate of larval
Illicit drug abuse has risen to epidemic levels over the last decade in China because of the implementation of its open-door policies (9). The use of ketamine has recently gained popularity in China. An increased trend of illicit drug abuse deaths, mainly caused by heroin and ketamine during the past decade, has been reported in the central part of China (6). Previous studies have indicated that many drugs and their metabolites can either accelerate or delay the development of larvae of necrophagous flies feeding on cadavers. It has been reported that amitriptyline, hydrocortisone, sodium methohexital, butylscopolamine bromide, malathion, and ethanol delay the development of larval growth of necrophagous flies, while heroin, methamphetamine, cocaine, codeine, and diazepam accelerate the development of larvae (4,10–18). These effects can potentially cause misestimating of PMI by as much as 77 h in those drug-related deaths (10). Knowledge of the effects of different drugs on the growth rates of immature carrion-breeding insects could be useful in refining the estimation of PMI (1). Recently, Zou et al. (19) reported that ketamine could shorten the larval stage of Lucilia sericata (Diptera: Calliphoridae). In the present study, our result showed that ketamine can delay larval development of C. megacephala. The growth rates of larval length/weight of all treatment colonies of C. megacephala were significantly suppressed compared with the control. However, no significant dose-dependent effects of ketamine on the larval growth were observed. Interestingly, these results are completely opposite to the Zou’s. Bourel et al. (20) studied the effects of morphine on the development of L. sericata, and Tian et al. (21) studied the effects of morphine on the development of C. megacephala
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JOURNAL OF FORENSIC SCIENCES TABLE 4––Mean weights (mg) of larvae of C. megacephala at a constant temperature of 32°C.
Time (h) 16 28 40 52 64 76
Control Mean (SD)
1/2LD50 Mean (SD)
LD50 Mean (SD)
2LD50 Mean (SD)
2.6 (–) 26.9 (2.0)a 56.1 (4.7)a 108.4 (9.3)a 104.0 (9.5)a Pupation
1.8 (–) 20.2 (1.5)b 48.2 (3.5)b 92.9 (9.8)b 91.3 (10.0)b Pupation
2.0 (–) 19.8 (1.7)b 45.5 (3.1)b 94.4 (10.1)b 102.6 (7.5)ac Pupation
2.0 (–) 20.7 (1.7)b 47.4 (3.6)b 94.5 (9.4)b 95.9 (5.9)bc Pupation
Mean in a line followed by the same letters are not significantly different.
TABLE 5––Mean weights (mg) of larvae of C. megacephala at a constant temperature of 28°C. Time (h) 16 28 40 52 64 76 88 100
Control Mean (SD)
1/2LD50 Mean (SD)
LD50 Mean (SD)
2LD50 Mean (SD)
0.7 (–) 11.7 (1.2)a 30.0 (4.0)a 95.8 (5.3)a 116.4 (6.7)a 106.3 (4.9)a Pupation
0.5 (–) 7.4 (1.5)b 18.9 (3.8)b 69.1 (7.9)b 93.9 (8.5)b 108.2 (7.5)a 106.0 (9.2)ab Pupation
0.7 (–) 8.7 (0.9)c 26.7 (2.4)c 87.0 (5.7)c 108.7 (2.9)c 118.3 (5.3)b 100.4 (6.1)b Pupation
0.6 (–) 7.9 (1.1)bc 25.9 (2.4)c 79.4 (5.5)d 107.1 (5.0)c 103.8 (5.0)a 107.2 (3.9)a Pupation
Numbers in brackets are standard deviations. Mean in a line followed by the same letters are not significantly different.
TABLE 6––Mean weights (mg) of larvae of C. megacephala at a constant temperature of 24°C. Time (h) 16 28 40 52 64 76 88 100 112 124 136
Control Mean (SD)
1/2LD50 Mean (SD)
LD50 Mean (SD)
2LD50 Mean (SD)
0.3 ( ) 3.0 (0.7)a 9.5 (1.0)a 34.3 (2.5)a 65.0 (4.0)a 74.3 (5.8)a 82.0 (5.3)a 74.4 (9.7)ab 71.1 (9.3)a Pupation
0.2 ( ) 2.3(0.3)b 7.4 (1.3)b 24.6 (2.8)b 29.8 (3.1)b 52.1 (2.1)b 58.6 (2.0)b 70.6 (4.2)b 75.1 (4.4)a 63.7 (4.7)a Pupation
0.3 ( ) 1.7 (0.3)c 5.6 (0.6)c 16.5 (1.5)c 36.7(4.2)c 51.9 (5.9)b 64.8 (4.3)c 76.1 (3.7)ab 81.5 (4.7)b 79.5 (7.2)b Pupation
0.3 ( ) 1.8 (0.3)c 5.3 (0.4)c 19.3 (0.5)d 27.1 (3.9)b 60.5 (5.0)c 66.9 (4.1)c 80.9 (8.7)a 88.0 (7.7)c 74.5 (4.3)b Pupation
Numbers in brackets are standard deviations. Mean in a line followed by the same letters are not significantly different.
FIG. 2––Growth rates of larval body weights (mg/h) of C. megacephala at constant temperature of 32, 28, and 24°C. All of the treatments gained slower than the control at all three temperatures. This result indicates that the growth of larval length and weight of C. megacephala are not absolutely parallel.
FIG. 3––Variation trend of ketamine concentration—the maximum larval length. The maximum larval length/weight was the greatest at 28°C. No correlation was observed between ketamine concentration and maximum larval length.
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EFFECTS OF KETAMINE ON THE DEVELOPMENT OF C. MEGACEPHALA
FIG. 4––Variation trend of ketamine concentration—the maximum larval weight. The maximum larval length/weight was the greatest at 28°C. No correlation was observed between ketamine concentration and maximum larval weight.
FIG. 5––Developmental durations of C. megacephala at constant temperature of 32°C. The durations of larval stage of all the treatments were prolonged slightly.
FIG. 6––Developmental durations of C. megacephala at constant temperature of 28°C. The durations of larval stage of all the treatments were prolonged significantly
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FIG. 7––Developmental durations of C. megacephala at constant temperature of 24°C. The durations of larval and prepupal stages of all the treatments were prolonged significantly while the durations of pupal stage were slightly shortened.
using the same method of Bourel’s. Their results showed that morphine could suppress the development of L. sericata during the larval stage, but accelerate it of C. megacephala. It appears to indicate that different species of Luciliinae and Chrysomyinae may response differently to the same drug. Comparing with the results of Zou’s, our study confirmed this phenomenon. In the deaths of suspected ketamine intoxication, it should take the delayed growth effect into account when using regression against larval weight or length to estimate PMI. The effect of temperature on the insect larval development has also been studied extensively (22–24). The durations of larval stage increase with a decline in temperature. Our study showed that ketamine, low temperature, and their synergistic action significantly suppressed the development of C. Megacephala and prolonged its developmental durations. For the tested range of temperatures of 32, 28, and 24°C, the total effect could prolong the duration of larval and prepupal stages, while it slightly shorten the duration of pupal stage at low temperature. The total duration of the treatment colony was prolonged 33 h at most (24°C, 2LD50). The effects of ketamine on the growth rate of larval length, weight, and developmental duration, nevertheless, were not consistent. This phenomenon indicates that the growth of larval length, weight, and duration of larval stage of C. megacephala is not absolutely parallel. The present study showed ketamine only delayed the larval growth but not completely stop the development, and no linear correlation between the degree of inhibition and ketamine concentration was observed. Compared with single temperature-repeated experiment, our study explored the difference in toxicological effect of drug on the development of necrophagous fly at different temperatures for the first time and found the above-mentioned synergistic action of drug concentration and temperature. In addition, the fact should be noticed that the larvae were reared in an artificial diet in this study. Compared with administering the drug via ear vein of animals (20) or gavage method (13), the drug used in this study was not metabolized by a living system. We chose the artificial diet containing drug as the model of acute poisoning other than animal experiment for two reasons. First, ketamine will be metabolized and converted to norketamine in 2–3 h, and the effects from ketamine will last for 1–5 h in human body (25). As victims died quickly in most of cases
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relevant to ketamine overdose, there was still a mass of ketamine remained in cadavers (8). The second, the metabolic processes of the same drug are not necessarily the same in different species (26,27). Therefore, animal models of acute poisoning are not exactly as same as human in all situations. In view of these two reasons above, we performed the experiment based on O’Brien and Turner (28) and Oliveira et al. (12) methods and modified several details. In conclusion, ketamine can suppress the development of C. megacephala, and there is no linear dose-dependent relationship between ketamine concentration and the degree of inhibition of larval development. Knowledge of the effects of ketamine on the development of C. megacephala can be useful in estimation of PMI in suspected ketamine-related deaths. As different species of the Diptera may have different responses to the same drug, further research is needed to observe the effects of ketamine on the development of more species of necrophagous flies. Acknowledgments We thank Professor Liu Min of College of basic medicine and forensic medicine of Sichuan University for providing the craftsmanship of artificial diet support, and Professor Wang Jiangfeng of Guangdong Police College for some suggestions on flies feeding. We also thank Professor Li Ling of Chief Medical Examiner Office of Maryland for her help in revising this article. References 1. Catts EP, Haskell NH. Entomology and death: a procedure guide. Clemson, SC: Forensic Entomology Specialties, Joyce’s Print Shop, 1990. 2. Catts EP, Goff ML. Forensic entomology in criminal investigations. Annu Rev Entomol 1992;37:253–72. 3. Byrd JH, Castner JL. Forensic entomology: the utility of arthropods in legal investigations, 2nd edn. Boca Raton: CRC Press, 2010. 4. Goff ML, Omori A, Goodbrod J. Effect of cocaine in tissues on the rate of development of Boettcherisca peregrina (Diptera: Sarcophagidae). J Med Entomol 1989;26:91–3. 5. Gosselin M, Wille SM, Fernandez MM, Di FV, Samyn N, De Boeck G, et al. Entomotoxicology, experimental set-up and interpretation for forensic toxicologists. Forensic Sci Int 2011;208(1):1–9. 6. Zhou L, Liu L, Chang L, Li L. Poisoning deaths in central China (Hubei): a 10-year retrospective study of forensic autopsy cases. J Forensic Sci 2011;56:S234–7. 7. Bruce DL, Capan L. Antidepressants do not increase the lethality of Ketamine in mice. Br J Anaesth 1983;55(5):457–9. 8. Gable RS. Acute toxic effects of club drugs. J Psychoactive Drugs 2004;36(1):303–13. 9. Liu J, Si XM, Li HY. An investigation and analysis on addicts in city Dalian. Chin J Drug Abuse Prev Treatment 2004;10(1):24–5. 10. Goff ML, Brown WA, Omori AI, LaPointe DA. Preliminary observations of the effects of amitriptyline in decomposing tissues on the development of Parasarcophaga ruficornis (Diptera: Sarcophagidae) and implications of this effect to estimation of postmortem interval. J Forensic Sci 1993;38(2):316–22. 11. Musvasva E, Williams KA, Muller WJ, Villet MH. Preliminary observations on the effects of hydrocortisone and sodium methohexital on development of Sarcophaga (Curranea) tibialis Macquart (Diptera: Sarcophagidae), and implications for estimating post mortem interval. Forensic Sci Int 2001;120:37–41. 12. Oliveira HG, Gomes G, Morlin JJ Jr, Von Zuben CJ, Linhares AX. The effect of Buscopan on the development of the blow fly Chrysomya
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Additional information and reprint requests: Zhou L€u, M.S. Forensic Medicine Institute Henan University of Science and Technology No. 31 Anhui Road Jianxi District, Luoyang Henan 471003 China E-mail: [email protected]
