Detection of Substances on Insects

Successful detection of substances has been accomplished by several extraction methods from maggots, pupae and adults of Diptera and even from the feces of beetles (Miller et al. 1994).

Insects may also be reliable animal models for immunohistochemical studies for detecting and surveying metabolism of some substances. Morphine was detected on third larval instar maggots from Calliphora vicina Linnaeus (Diptera: Calliphoridae) fed with an artificial diet mixed with the drug. Drug-positive maggots presented a characteristic haemolymph colour and intense immune reaction between the exocu-ticle and the endocuticle. These results indicated that morphine was stored inside the cuticle of the maggots during their development (Bourel et al. 2001a). Cocaine was detected on sections of second and third stage larvae by immunohistochemistry using the peroxidase-complex method. Positive specimens showed a more intense immunoreaction in an area located at the limit between exocuticle and endocuticle. These results constitute an evidence of cocaine accumulation in the cuticle of the larvae during their development. Neglecting the importance of this information may cause errors of up to12 h in estimating the PMI (Alves Jr et al. 2007).On the other hand, diethylpropion (Inibex®) was not detected in larvae of Chrysomya megacephala and Chrysomya putoria using this methodology, suggesting the rapid excretion of drug (Alves Jr et al. 2008).

Beyer et al. (1980) was the first to purpose analysing insects collected from decomposed bodies to detect substances. Ever since, there were researchers testing various drugs and techniques on maggots, frequently aimed at using them as toxi-cological samples for detecting substances like bromazepam, levomepromazine (Kintz 1990a), malathion (Gunatilake et al. 1989), fenobarbital, brazotam, oxazepam (Kintz 1990b), cocaine (Goff et al. 1989), morphine (Bourel et al. 2001b), diazepam (Carvalho et al. 2001), and such in cases of criminal nature like suicide, homicide, kidnapping, etc (Bourel et al. 1999; Gunatilake and Goff 1989; Goff et al. 1991, 1992; Sadler et al. 1997).

The insects most frequently used to estimate PMIs and as toxicological analysis samples belong to the families Calliphoridae (blowflies) and Sarcophagidae (flesh flies). These flies are expert agile flyers that feed on corpses, and are usually the first to arrive to the corpse or death scenario (Goff and Lord 1994).

Homogenized insect samples can be quite easily analyzed through toxicological procedures like radio-immune-analysis (RIA), gas chromatography (GC), thin layer chromatography (TLC), high pressure liquid chromatography (HPLC) and gas mass analysis (GC-MS). The most indicated procedure is fractioning the samples by chromatography followed by mass spectrometry analysis, because their combined precision permits separating, identifying and quantifying chemical substances and contaminants from illicit substances at minute amounts (Moore and Casale 1994; Collins et al. 1997). For example, chromatography is commonly applied to at least three types of investigation involving Cannabis (marijuana) (Gudzinowicz et al. 1980):

Identification of Cannabis in obtained plant samples

Determining the origin of the obtained Cannabis samples

Designing profiles of Cannabis samples according to their geographic origins

Gas chromatography is remarkable for its very high resolution and sensitiveness. With it, it is possible analyzing samples composed of multiple substances at minute quantities, a fact that would limit the use of other techniques. On the other hand, sample preparation must follow several steps and the results may take considerable time (Collins et al. 1997).

The advantages of using maggots instead of body tissues can be seen in chroma-tography. Chromatograms obtained from larval extracts present less endogenous contaminant peaks than chromatograms from human body extracts (Kintz et al. 1990b). This is particularly useful if the sampled material is decomposed, and insects are rather easily collected and reared in laboratory.

Larvae from flies (maggots) feeding from contaminated human tissues ingest drugs and toxins recently taken by the dead person when alive. These substances are transferred through the food chain, usually bioaccumulating in beetles that predate on blowfly larvae. Consequently, the beetles can also be used for toxicological analysis in some cases (Introna et al. 2001).

Nuorteva and Nuorteva (1982) detected mercury in larvae, pupae and adult insects reared on mercury-contaminated fish. Similar procedures were employed in a forensic investigation in where the body of a woman was found in putrefactive state and covered with maggots in a rural region of Finland. The distinctly low mercury levels present in the maggots indicated a probable geographic origin for the victim: an area almost free of mercury contamination. They also detected mercury in adult beetles of Creophilus maxillosus (Staphylinidae) and Tenebrio molitor (Tenebrionidae) that were feeding from necrophagous maggots reared on tissues containing mercury. No adverse effects on the staphilinids development were observed, whereas, adult tenebrionids displayed a significant reduction of their activities.

In the absence of maggots, especially in bodies on advanced putrefaction, necrophagous and some predator coleopterans that feed on necrophagous insects may be used as toxicological samples. Dermestes frischi Kugelann (Diptera:

Dermestidae) and Thanatophilus sinuatus Fabricius (Diptera: Silphidae) beetles were reared on food containing morphine and it was possible to detect the drug in all development stages of D. frischi and in the larval stages of T. sinuatus (Bourel et al. 2001c).

Nolte et al. (1992) recorded the first forensic case where it was possible detecting cocaine and benzoylecgonine in fly larvae collected from decomposing humans. The body was practically bones and no injuries were apparent. What could be the cause of death? The victim's girlfriend told the authorities he could have been on cocaine when he disappeared. Samples of muscles and insect larvae were submitted for toxicological analysis and results were positive to benzoylecgonine, a cocaine metabolite. Cocaine is toxic and can cause fatal effects in the heart, intestines, brain and lungs. In this investigation, the association of this knowledge with the absence of injuries and the drug metabolites on larvae and human muscle allowed the establishment of cocaine overdose as the cause of death.

In 1985 Leclercq and Brahy detected the presence of arsenic through toxicological analysis of species of flies of the families Piophilidae, Psychodidae and Fanniidae in a case of accidental death in France. Other researchers, Gunatilake and Goff (1989) detected traces of the organophosphate insecticide Malathion in calliphorid maggots from a suicidal, through GC analysis.

Parathion is an insecticide and acaricide widely used in agriculture. It is rapidly absorbed by the soil and it is degraded by photolysis. Cases of intoxication by inappropriate use, inadequate use of protection gear and suicides through direct ingestion are fairly frequent, especially in agricultural regions. Wolff et al. (2004) employed high performance liquid chromatography columns to separate and quantify para-thion in arthropods collected from decomposing drug-treated rabbits. It was detected in different development stages of ten arthropod species of the orders Diptera, Coleoptera, Hymenoptera, Isopoda and Acari, which were collected during all decomposition phases (from fresh stage to dry remains). Besides their small size, isopods and acarids accumulated parathion in quantities enough to be detected. The chitinous/proteic matrix of the cuticle contributes in fixing the drug onto the arthropods (Introna et al. 2001). The drug presented deterrent and insecticidal activity at the mouth entrance of the drug-treated rabbits. This is relevant since most necrophagous insects lay eggs on body cavities. No significant alterations on insect succession were observed, in contrast to results from a similar experiment using malathion (Gunatilake and Goff 1989), wherein a delay in insect oviposition and different taxa were found on carcasses containing (or not) insecticidal contaminants. Further research is warranted to understand the various effects of insecticides over carrion-feeding entomofauna.

Kintz et al. (1990c) successfully demonstrated the potentialities of entomotoxicology by describing many cases. An interesting case was that of a body found a couple of months after death. Liquid chromatography was employed to analyse tissue samples of his heart, liver, lung, spleen and kidneys as well as calliphorid maggots collected from his body. The results from both materials indicated the presence of triazolam, oxazepam, phenobarbitol, alimemazine and clomipramine.

Positive identification of morphine was achieved by Introna et al. (1996) from empty puparia of calliphorid flies reared on a diet containg the drug. Likewise, Goff et al. (1993) detected amitriptyline and nortriptyline in fly larvae developing on rabbit carcasses containing different concentrations of these substances. Drugs have also been detected in fly puparia and beetle exuvia found around the mummified body of dead person missing for about 2 years (Miller et al. 1994).

Manhoff et al. (1991) used gas chromatography and mass spectrometry to detect the presence of cocaine in mummified tissues, blood samples, maggots and beetle faeces from a corpse. Hair samples from a person can also be used to detect cocaine and other substances several years after his death (Kidwell 1993). Substances may deposit on puparia and drugs may be released from insect chitin.

Carvalho et al. (2001) reared C. albiceps and C. putoria (Diptera: Calliphoridae) larvae on livers from rabbits given twice the lethal dosage (LD) of diazepam. Drug was detected in all analysed tissues of rabbit and in almost all stages of development of both fly species showing insects can be used as an alternative sample for toxicological analysis (Table 9.1) although it was not observed a correlation between tissue and larvae concentrations.

Comparisons of drug concentrations between those in human tissues and blowfly larvae use to show different patterns of distribution that may be related to differences in species physiology as well to the chemical properties of the drugs (Campobasso et al. 2004). Several previous studies have confirmed the reliability of entomological specimens for qualitative analyses, although quantitative extrapolations are still unreliable. However, even if only as qualitative specimens, carrion feeding immature and adult stages of insects may serve as alternative toxicological evidence or sources of toxicological analysis, supporting the final diagnosis of narcotic intoxication (Campobasso et al. 2004).

Table 9.1 Diazepam quantification (mg/mg) in samples of rabbit tissues and fly specimens through of GC-MS analysis








Larvae of C.albiceps Larvae of C.putoria Puparia of C.albiceps Puparia of C.putoria Adult of C.albiceps Adult of C.putoria

9.531 (a) 5.943 (a) 3.228 (b) 3.341 (b) 4.029 (b) 0.342 (b) 0.479 (b) 0.233 (b) 3.022 (b) 0.051 (b) 0.000 (b)

Means in a column followed by the same letters are not significantly different (P < 0.05)

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