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.