Background

In the decomposition of human remains, every stage of decay is associated with specific insects, starting from flies to beetles, and finally moths. However, the most important insects in this process are flies from the family Calliphoridae, especially those from the subfamily Chrysomyinae, Calliphorinae, and Lucillinae. They are among the first insects to arrive on human remains for oviposition. After hatching, their offspring consume dead tissue (Wells et al. 2001a, 2001b). Fly offspring or larvae serve as a repository for drugs once they have consumed drug-contaminated tissues. As a result, they can be utilized in a toxicological analysis for drug detection, especially under conditions in which host tissue samples are not available or viable. Drug-contaminated tissues consumed by larvae are stored in the crop until they reach the pupal stage, so they serve as “living preservation capsules” of tissues from the deceased. However, when the pupal stage begins, digestion and metamorphosis occur, leading to a significant drop of drug concentration, meaning that the pupal stage has less forensic importance (Sadler et al. 1995).

It is well-documented that drugs accumulated in fly larvae could alter their normal life cycle duration (Goff et al. 1989; Bourel et al. 2001) as well as changing body weight and length (Oliveira et al. 2009). For example, morphine and heroin may accelerate larval development growth rate and slow development rate in the pupal stage (Carvalho et al. 2001). A study on Lucilia sericata (Meigen 1826) (Diptera: Calliphoridae) showed similar findings which heroin speed up their growth rate (Bourel et al. 2001). The pupal casing of L. sericata has been found to shed earlier at a higher concentration of morphine compared to a lower concentration (Bourel et al. 2001). Moreover, L. sericata larvae reared in rabbits treated with tramadol was noted reached maximum sizes earlier than in larvae reared on the control rabbit (El-Samad et al. 2011). Along with changes in developmental rates and variation in the size of larvae during their development, the presence of drugs or toxic substances in the insect food source can also be validated (Wells et al. 2001a, 2001b). Various drugs have been detected in different stages of fly development, such as amitriptyline (Sadler et al. 1997a; Wilson et al. 1993), phenobarbital (Beyer et al. 1980), lorazepam (Wood et al. 2003), amphetamine (Sadler et al. 1997b), cocaine (Nolte et al. 1992; Campobasso et al. 2004), codeine (Kharbouche et al. 2008), morphine (Hedouin et al. 1999), and methadone (Gosselin et al. 2010).

For drug detection, larval specimens in a forensic case are only useful if they are collected alive and properly preserved to ensure that minimal putrefactive fluids are produced from their own decomposition process. Forensic toxicological analysis of dead tissues from larvae would be misleading if these criteria were not met due to contamination of the sample with larvae putrefaction fluids (Levine et al. 2000; Kintz et al. 1990a, 1990b). Considering the impact of drugs on a normal fly life cycle and developmental rate, the identification of drug metabolites in each fly developmental stage is critical. Moreover, the pharmacokinetics of drugs in an insect is known to be influenced by the developmental stage, as well as species and feeding activity (Kharbouche et al. 2008). Therefore, we conducted a preliminary analysis with the objective of identifying both complete and non-complete heroin metabolites in each developmental stage of L. cuprina reared on heroin-treated meat prepared with five different concentrations. An unusual approach was used in this study in which heroin-treated meat was used, instead of an animal model. This considers a possibility that larvae could also have a chance to consume unmetabolized heroin in a forensic case. This study emphasizes detecting heroin metabolites in larvae consuming an unmetabolized drug; therefore, drug metabolism factors are excluded if a living animal model was used.

Methods

Maintaining stock culture for Lucilia cuprina

A sweep net was used to capture adult L. cuprina hovering or landing on bait prepared from fresh minced buffalo meat obtained from a local wet market. The captured flies were immediately transferred into an insect cage (30 cm x 30 cm x 30 cm) and brought back to the insectarium at the School of Biological Sciences, Universiti Sains Malaysia, for culturing at 29 ± 3 °C, under a photoperiod of 13 D: 11 L and RH of 75 ± 10%. The flies were fed two kinds of food: a mixture of 10% (w/v) sugar solution for adult and minced buffalo meat for fly oviposition. For the oviposition substrate, 500 g minced buffalo meat was placed in three petri dishes. The eggs observed from each petri dish were then transferred gently into a larva-rearing box containing 50 g minced buffalo meat. The larval development was monitored until pupation. When pupae were observed, they were transferred using forceps into a new container containing sterile fine sand; each container was then placed into an individual insect cage. Once the adult flies emerged, a mixture of 10% (w/v) sugar solution was provided as adult food. The first-generation adults were identified using a blowfly taxonomic identification key (Wallman 2001). Flies confirmed to be L. cuprina were reared until the third generation following the process described above. The eggs of that generation were then collected and used for subsequent experiments.

Application of heroin onto minced buffalo meat

Lyophilized heroin was prepared in saline to obtain concentrations of 500, 1000, 2500, 5000, and 10,000 ng/μl. Each heroin preparation was mixed with minced buffalo meat purchased from a wet market in a volume to weight ratio of 1:100 (I.E., 5 ml: 500 g) using a food processor to ensure a homogenous mixture. Minced buffalo meat that was not mixed with heroin (untreated) served as the control treatment. Each preparation of heroin-treated minced buffalo meat and untreated meat was then placed into half-filled sterile sand plastic containers and covered with a fabric mesh. A clump of L. cuprina eggs was placed onto each buffalo meat preparation, and the treatments were replicated five times. Every 8 h, 10 individual first instar larvae, 10 individual second instar larvae, and 10 migrating third instar larvae were harvested and cleaned, and their body length was measured. Larva instar stages were determined based on the characteristics of posterior spiracles. The larvae were then preserved for toxicological and molecular analysis following the method described below.

Dissecting the crop of Lucilia cuprina for toxicological analysis

Ten collected larvae from each harvesting were rinsed with distilled water to remove any foreign particles or other possible contamination that adhered to the larvae during previous procedures. The collected larvae were then labelled, preserved alive, and stored at -20 °C for toxicology analysis. For the first and the second instar larvae, the whole larvae tissue was used in the toxicology analysis because the crop was too small to be dissected by using a dissecting microscope (Carls Zeiss, United State). However, the third instar larvae were dissected to bring out the crop using a dissecting microscope. Before carrying out the dissection procedure, all larvae were washed with sterilized water to remove any possible contamination present on the external of the larval body. The cleaned larvae were placed under the dissecting microscope on a new sterile petri dish, and the instar of each larva was recorded based on the posterior spiracle’s characteristics. Only the third instar larvae of 10 mm in length were dissected for toxicology analysis purposes. After that, the posterior segment was cut using a sterilized scalpel, followed by a ventral incision to the anterior of the larvae. Later, each crop was removed using sterilized forceps and placed in a 1.5 mL Eppendorf tube for toxicological analysis. The working area was wiped with 70% alcohol before and after the procedure. The dissection was performed based on the standard method (Linville and Wells 2002).

Toxicological analysis

In the toxicological analysis, an alkali-acid base extraction was used, followed by gas chromatography mass spectrophotometry (GCMS) analysis (Agilent 6890 GC). The toxicological analysis adopted in this study adhered to an accredited test recommended by Jabatan Kimia Forensik Malaysia (The Forensic Chemistry Department of Malaysia) with identification number 0537 (JKMF0537). The GCMS was conducted with an Agilent GC instrument using a 5973-mass selective detector (MSD) and a non-polar column HP-5. Helium gas was applied as the carrier gas at a constant current rate of 2 ml/minutes, where 1 ul of the extract was injected in a split mode (40:1). The temperatures for the injector and the detector (transfer line) were set at 250 °C and 280 °C, respectively. The analysis was conducted at a temperature of 80 °C for 2 min; then increased to 150 °C at 20 °C/minute and held for 2 min; to 280 °C at 10 °C/minute and held for 5 min; and to 20 °C/min to 30 °C and held for 14 min. All the results obtained were analysed using the toxicology library available in the instrument.

Results

Overall ion chromatogram of tryptophan and morphine obtained from the third instar larvae are presented in Figs. 1 and 2, respectively. Meanwhile, information concerning mass spectral characteristics of tryptophan and morphine obtained from the third instar larvae are illustrated in Figs. 3 and 4, respectively. However, the mass spectral characteristics and the ion chromatogram could not be obtained for hydromorphone, because the metabolic yield was less than 50% than that of tryptophan and morphine.

Fig. 1
figure 1

Total ion chromatogram of Tryptophan 2,4-dimethoxy-5-ethoxyamphetamine (3.74 m/z) in the third instar larvae of L. cuprina after hydrolysis

Full size image
Fig. 2
figure 2

Total ion chromatogram of Morphine (4.94 m/z) in the third instar larvae of L. cuprina after hydrolysis

Full size image
Fig. 3
figure 3

Mass spectral characteristics of Tryptophan 2,4-dimethoxy-5-ethoxyamphetamine in the third instar larvae of L. cuprina after hydrolysis

Full size image
Fig. 4
figure 4

Mass spectral characteristics of Morphine in the third instar larvae of L. cuprina after hydrolysis

Full size image

Table 1 shows the mass to charge ratio and the quality of heroin metabolites measured in immature stages of L. cuprina reared on minced buffalo meat treated with different concentrations of heroin. At each sampling time, all analysed second and third instar larvae tested positive for heroin metabolites. In contrast, heroin metabolites were not detected in the first instar larvae and pupae. Tryptophan was detected in the second instar larvae reared on the 500, 1000, and 2500 ng/μl of heroin-treated meat, while morphine was detected in the second instar larvae reared on 10,000 and 5000 ng/μl of heroin-treated meat. Meanwhile, the metabolites detected in third instar larvae consisted of the following: hydromorphone for larvae reared on 500 and 1000 ng/μl of heroin-treated minced meat; tryptophan for larvae reared on 2500 ng/μl of heroin-treated meat; and morphine for larvae reared on the 5000 and 10,000 ng/μl of heroin-treated meat (Table 1).

Table 1 The total ion chromatogram (m/z) and quality (%) of heroin metabolite measured in larvae, pupae and adult stages of L. cuprina reared in meat containing different concentrations of heroin. m/z represents mass divided by change number and the horizontal axis in a mass spectrum is expressed in units of m/z
Full size table

The quality of tryptophan detected in the second instar larvae increased from 16% to 27% when heroin concentration increased from 500 ng/μl to 2500 ng/μl. On the other hand, the quality of morphine was detected to increase drastically from 11% to 95% in the second instar larvae that were collected from the 5000 ng/μl of heroin-treated meat compared to the 10,000 ng/μl of heroin-treated meat, respectively. However, the quality of hydromorphone in the third instar larvae reared on the 500 ng/μl of heroin-treated meat, and the 1000 ng/μl of heroin-treated meat was low at 1% and 9%, respectively.

Nonetheless, the highest quality of morphine at 96% was detected in the third instar larvae reared on the 5000 ng/μl of heroin-treated meat, whereas the highest quality of tryptophan at 53% was detected in the third instar larvae reared on the 2500 ng/μl of heroin-treated meat (Table 1). Moreover, the third instar larvae collected from 5000 and 10,000 ng/μl of heroin-treated meat produced positive results for morphine.

The toxicological analysis adopted for this study allowed for the rapid identification of heroin analytes, including tryptophan, hydromorphone, and morphine. The mass to charge ratio for tryptophan in the second instar larvae reared on the 500, 1000, and 2500 ng/μl of heroin-treated meat were 3.75, 4.78, and 3.76 m/z, respectively, while at the concentrations of 5000 and 10,000 ng/μl, the second instar larvae were found to have completely metabolized the heroin to morphine at a mass to charge ratio of 3.60 m/z.

The mass to charge ratio of hydromorphone in the third instar larvae reared on the 500 and the 1000 ng/μl of heroin-treated minced buffalo meat had 8.06 and 3.32 m/z, respectively. Other than that, the mass to charge ratio for tryptophan in the third instar larvae reared on the 2500 ng/μl of heroin-treated minced buffalo meat was 3.74 m/z; whereas in the 5000 and the 10,000 ng/μl of heroin-treated minced buffalo meat, the mass to charge ratio for morphine was 4.94 m/z (Table 1).

Discussions

In an entomotoxicology research set-up, drug-treated meat has one advantage compared to heroin-treated animal model, in that the concentration of heroin prior to feeding can be fixed. In an animal model, for instance drug-injected rats or rabbits, the rate of drug concentration to be fed on to larvae is uncontrolled and varies depending on current body metabolism and multiple other unknown internal and external factors. In contrast, however, in drug-prepared meat, the drug concentration before treatment is fixed; therefore, pre- and post-treatment results can be compared quantitatively using an adequate number of replicates. Even though living animals such as rats or rabbits are more suitable models and realistic to represent actual forensic scenario, reports have indicated that correlations between drugs inside human or animal tissue and feeding larvae have not been found due to unknown and complicating factors (Definis-gojanovic et al. 2007; Gosselin et al. 2011). Therefore, the effects of drug concentration on heroin metabolism before and after feeding are impossible to evaluate. In contrast, by using heroin-treated meat, this non-correlation issue is eliminated, and larval pre-feeding concentration can be controlled. Even though in reality, two metabolisms (in human body and fly larvae) are involved in routine forensic analysis and casework, the purpose to understand heroin metabolism in larvae in our case is achievable by using drug-treated meat.

We also consider the possibility that larvae could consume pure or unmetabolized heroin in a forensic case. If drug is taken by snorting powdered form, its residue on or around a dead body or clothing might also be consumed by fly larvae; therefore, first metabolism may begin in the feeding larvae. Although this is uncommon, as no such case has been reported, a record showed fatality rate, resulting from an overdose of heroin that involves routes of administration other than injection, is rather high (i.e. snorting or smoking) (Thiblin et al. 2004). Therefore, there is a chance that larvae could consume drug residue. If larvae had consumed drug residue, in this case, unmetabolized heroin, its metabolites in larvae would be important to be addressed. Moreover, some amount of unmetabolized heroin can remain in urine and therefore could be consumed by larvae.

Through a series of toxicological analysis on heroin-feeded L. cuprina, we have detected the three heroin metabolites of tryptophan, hydromorphone, and morphine. However, the metabolites were identified in the second and third instar larvae, but not in the first instar and pupa. In agreement with this finding, previous work has reported negative results for morphine in the pupae reared on a substrate spiked with high morphine concentration. They concluded that it was due to rapid elimination by the larval stages (Kharbouche et al. 2008). In contrast, different results were observed when morphine was detected in pupal cases and desiccated adults reared on minced-beef meat (Bourel et al. 2001). In addition to morphine, there are many other drugs detected in the puparium. For instance, cocaine was found in a pupal case of Calliphora vicina Robineau-Desvoidy, 1830 (Nolte et al. 1992), and amitriptyline was detected in pupal cases of Phoridae (Miller et al. 1994). Metabolites of methylenedioxyamphetamine (MDMA) were found in fly puparia, but not in the blood, liver, and larvae collected from tissues treated with 3,4-methylenedioxymethamphetamine, contradicting our study (Goff et al. 1997).

The absence of heroin metabolites at the pupal of L. cuprina showed an efficiency of metabolism and elimination of heroin during the larval stage. Similar findings were displayed for L. sericata pupae, suggesting that morphine has a rapid elimination rate (Kharbouche et al. 2008). Other factors might be associated with food storage location in the crop and rapid expansion and digestion of food during the post-feeding stage. These processes decrease the drug concentration in larvae, as well as the rate of drug elimination and absorption, resulting in a lack of heroin metabolites in both pupa and adult stages.

In our study, the third instar larvae showed high-quality morphine as compared to the second and the first instar larvae. This high-quality morphine detected from 5000 ng/μl and 10,000 ng/μl heroin concentrations justified the potential use of carrion-feeding insects for drug detection; therefore it can be an alternative toxicological specimen to that of direct testing of tissues from a corpse. The differences among stages might be due to the heterogeneous accumulation and elimination rates of morphine by specific developmental stage of fly, as pharmacokinetics of drugs is known as stage-dependence (Kharbouche et al. 2008). For example, active feeding larvae could have an absorption rate that exceeded the elimination rate. In contrast, elimination rate of non-feeding stage exceeded the absorption rate, therefore inducing a decrease in the opiate concentration in non-active larvae (Campobasso et al. 2004; Pien et al. 2004). Moreover, similar findings were present for opiate metabolism, as it has been observed that morphine has a rapid elimination rate (Kharbouche et al. 2008). The study had also found that codeine was not detected in the prepupa stage, unless they were reared in 30 mg/kg concentration, which was the highest level in the study. Therefore, undetected morphine during the pupa stage in our study is not unusual and has been explained by previous reports.

The high quality of morphine was detected in the third instar larvae in our study; it could also be related to the accumulation of morphine in adipocytes or integumentary cells. This built-up has been known for facilitating the excretion process by the Malpighian tubules in the third instar larvae (Bourel et al. 2001). Therefore, in toxicological analysis, the chance of tracing a high concentration of morphine is better at this stage. The ability of the third instar larvae to accumulate morphine is considered as an advantage and should be emphasized in the future study. Even though it was suggested that the third instar larvae are the most appropriate stage for toxicological analysis (Kharbouche et al. 2008; Sadler et al. 1995), the drug metabolism and pharmacokinetics in third instar larvae are still not adequately understood.

In a toxicological analysis of an advance decay dead body, putrefied tissue is not a recommended specimen for drug detection due to potential disturbance of extraction recoveries, chromatographic performances, as well as low separation and ionization efficiencies. These interruptions are making the drugs not turn up in the analysis (Skopp et al. 2001; Schloegl et al. 2006). With regard to this, fly larvae is a better candidate for drug analysis instead of putrefied tissue. In toxicological analysis, fly larvae have been reported to offer greater sensitivity compared to putrefied tissue or product from human remains (Kintz et al. 1990a, 1990b).

The data obtained in this study is preliminary; therefore it’s immature to directly be used as a reference or to apply in actual forensic case works. Nevertheless, it can be a subject for comparison to any similar future research when rats (i.e. liver) are chosen as fly rearing-food. Moreover, comparison of data from both settings is useful as Duke (2004) mentioned that comparisons of spiked foodstuff versus live-animal models should be carried out with a standardized experimental protocol for both experiments.

Despite the limited findings obtained in this study, we have pointed up a potential forensic use of L. cuprina. In a case where other biological samples, such as human tissues or blood are in an advanced state of decomposition or unavailable, the immature stages of fly become the best alternative substrate to be used for toxicological analysis. However, if drug metabolism in larvae is not fully understood, forensic examination might be compromised.

Conclusions

Even though heroin metabolites were detected at only two life stages of L. cuprina, the findings are still indicative of the potential of carrion-feeding insects as alternative toxicology specimens. Third instar larvae are excellent reliable specimens for toxicological analysis, as they can be used to detect the complete heroin metabolite before it is eliminated. However, the mechanism of how larvae metabolize drugs has not been extensively studied, and there is limited literature discussing the discovery of the metabolic products of drugs in fly larvae. Therefore, upcoming research may emphasize this aspect, as well as the pharmacokinetics of the drug in larvae of different species.