Entomotoxicology
The process of decomposition begins immediately after death and the process can be divided into five stages: fresh, bloated, decay, post-decay and skeletal. Therefore, the availability of tissues and blood samples for toxicological analysis is dependent on the state of decomposition. There are cases where blood and tissue samples are not available or suitable for analysis, the fly larvae found on the cadaver can be used as an alternative toxicological specimen. 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). Bourel et al. (2001) conducted a study which showed that morphine was detected on third larval instar maggots of Calliphora vicina Linnaeus (Diptera: Calliphoridae) fed with an artificial diet mixed with the drug. This shows that morphine was stored inside the cuticle of the maggots during their development. However, the detection of diethylpropion (Inebex) showed negative result in larvae of Chrysomya megacephala and Chrysomya putoria suggesting the rapid excretion of drugs (Alves et al.2008).

Insect succession
Insect succession is the wave or pattern of insects’ colonization on dead remains and is also affected by the surrounding environment. Invasion of a body by insects and other arthropods occurs soon after death (Anderson and Goff, 2000). They are capable to arrive and colonized within minutes of the death (Wells and Lamotte 2001). It is not unusual for human remains to be discovered in a highly decomposed or skeletonized state. Insect are the first organism to discover a body after death. They are capable of arriving and colonizing within minutes of the victim’s final breath.
The entomological basis for the postmortem interval estimation also is referred to as the period of insect activity (PIA) or the time of colonization (TOC). A variety of arthropods and their cast larval and puparial skins, however are commonly encountered on putrefied, mummified, and skeletonized remains. The use of necrophageous species as matrix for qualitative drugs detection is well documented and generally accepted by forensic toxicologist. Many compounds such as drugs, metals and pesticides have been detected in insects’ tissues in a forensic context (Augsburger et al, 2008)
The first insect that arrives on a dead body is the blow fly (Calliphoridae) and also flesh flies (Sarcophagidae). Therefore,blow fly, sarcophagid and muscid flies are the most important species that will provide information relating to the accurate estimation of the period of insect activity and thus accounting for a portion of the post mortem interval. The Calliphoridae are attracted to decomposing human tissue, animal carrion, some vegetative material and exploiting open wounds in living human and animals. For the flesh fly (Sarcophagidae), they feed on decomposing human and animals tissues as well as decomposing vegetation. The adults are often found on flowers because they are attracted on the nectar. In carrion, they also feed on excrement or exposed meats. They have been known to cause myiasis and may involve in the mechanical transmission of disease. They are attracted to carrion even under variety of condition including sun, shade, dry, wet, indoor, outdoor.
Life cycle of blowflies
Blow flies are among the first insects to detect and colonize human and animals’ remains. In experimental studies, calliphorid flies have been recorded arriving at the carcass within minutes of exposure. Blow flies locate human and animals remains in a two-step process that first consists of chemical detection via receptors on their antenna and a visual search. Once they locate the body, they will visually assess the size of the carcass and any sites of trauma.
Once the oviposition site is found, gravid female flies will begin to deposit their eggs. And then the cycle begins. Life cycle of blowflies is shown in Figure 2.1. The eggs hatched as soon as 24 hours and may also take up to 4 days to hatch into first instar larvae depending on the climate. The blowflies larvae feed on corpse and develop into second instar larva and then into third instar. In third instar stage, the larvae continue feeding then wanders away from the corpse either in clothes or in

Figure 2.1: The life cycle of blowflies

soil so that they can develop into pupate. After a number of days, adult flies emerge from pupa and the cycle begins again.
Both male and female adult Calliphoridae ranges from 6 to 14 mm in length. The adult size is dependent on species and food availability to the larval stages.
The mature larval which is the third instar of blow flies range from 8 to 23 mm in length and are white of cream in color. In this stage, it contains the posterior spiracles which are the primarily breathing apparatus of the larva. The posterior spiracles are important morphological indicators for species identification.
Beside blow flies and flesh flies, other insect such as beetles, bugs and ants may also appear. Large maggots masses form from fly larvae during the decay stage of decomposition. Many predaceous beetles will appear to feed off the plentiful maggots. At the post decay stage, various beetles that feed on the dry remains are predominant. By the final stage, where only consist of hair and bones, mainly mites as the useful indication of the PMI during this stage.
Factors that influence insect successions
The development of the insects is temperature dependent. The metabolic rate of the blowflies during carcass processing is increased with increased temperature. Necrophages are the newly hatched larvae and feed on corpses as they grow into pupae which take about 4 days. Then the pupae will burrows deep into the tissue and will fully developed into blowflies in a week. The development in each stage has their own period of time. Therefore the period of time depends on the food they ate and the temperature.
The burning and hanging of carcasses have also been studied to determine the effect on decomposition and arthropods succession. Wrapping a body in several layers of blankets also cause a delay up to several days in blowfly invasion of a corpse by reducing access to the body (Goff and Catts, 1992). The blow flies or fresh flies have shiny green, blue or black body and specific to certain regions and climates. Others insects such as Piophilidae or cheese skippers will arrive later, during protein fermentation. Some insects are not attracted to the dead body but are attracted to feed on other insects (Archer and Elgar, 2003).
During decay stage, Dipteran larvae forming large maggot’s masses are predominant. In addition, a large number of predaceous coleopterans begin to arrive. In post decay stage, beetles have a very strong mandible that enables them to utilize the tough remains such as cartilage and bones. Meanwhile, in skeletal stage, mites are the useful indicators of the PMI estimation (Early and Goff, 1999).
Larvae which feed on corpses may eat on drugs and toxicants which had been ingested by the deceased person (Goff and Lord 2001). Usually there are no biological sources such as urine, tissue or urine, therefore the analysis of insects encountered may enable toxicological assessment of the cause of death (Goff and Lord, 2001). Chemicals in or on the dead body might accompany drug overdose or suicide will effect on the insect succession. Insect-mediated decay process can be accelerated or decelerated depending on the substance concentration. It has been demonstrated that certain toxins such as illegal narcotics that present in the tissues can not only be detected in the larvae feeding on the corpse but also effect the rate of development (Gangliano-Candela and Aventaggiato 2001).
Effects of drugs on insects development
Goff (2001) observed that the presence of a ‘foreign’ substance in the body can alter the biological cycle of insects that are used in forensic investigations. It has been shown that larvae exposed to and that have fed on cocaine treated human tissues incorporate chemical substances from these substrates, including drugs that were taken by the person before death
A drugs or toxin can be detected in the larvae when its rate of absorption exceeds the rate of elimination. The effects of drugs and toxins on the rate of Diptera development is a paramount matter to solve before using maggots for post mortem intervals estimation. Study by Gunatilake and Goff, (1994) shows the development stages of C.megacephala were indicative of a minimum postmortem interval of 5 days whereas the victim had been seen alive 8 days prior to the discovery of the body. Verma et al (2013) observed the effects of drugs ethanol and cannabis on growth rates of the blowfly. It shows that control sample took an average of 4 days to grow from first instar to pupae stages, the samples grown in the presence of ethanol and cannabis showed a much faster growth rates. Study was conducted by Goff et al. (1991) using heroin on maggots. Heroin in tissue as morphine shows rapid development of maggots and produce a large numbers of maggots in all treated colonies until the maximum size was attained. Similar study was conducted on methamphetamine and it also shows increases in rates of development for the colonies fed on tissues containing lethal dosage of the drugs (Goff, 2001). Both median lethal and twice lethal dosage colonies failed to produce offspring by the second generation. While study on the effect of heroin on the development of Sarcophagidae (B.peregrina) fed on intoxicated rabbit tissues, Goff et al. (1991) observed that maggot grows at rates significantly faster from 18 to 96 hours when larvae reached their maximum length. The presence of morphine in the tissues demonstrated by Bourel et al. (2000) showed that there is slower rate in the development of larvae on rabbit carcass where it only received 50.0 mg/h morphine. Regarding narcotic intoxication, demonstrations with Radio Immune Analysis (RIA) to detect the presence of opiates(morphine) in larvae development on liver collected by bodies in which the cause of death was identified as opiates intoxication (Introna et al,2001).
Similar result on drugs was demonstrated by Goff et al. (1999) who administered varied dosage of cocaine and heroin to laboratory rabbits. The maggots developed more rapidly 36 hours after hatching if reared on liver or spleen on the rabbit with lethal dose of cocaine or twice the dose. The acceleration of larval development continued for 76 hours after hatching.
Similar study has been carried out by Bourel et al. (2001) on the effect of morphine on Diptera Calliphoridae. Morphine at different concentration of 0.5, 1.0 and 2.0 times the lethal dose slowed down the development growth rate of Calliphora vicina larvae during the first 60 hours of development. He also found that morphine slowed down the growth of Lucillia sericata in a dose-dependent manner at concentration approximately 5.8, 9.2 and 12.8 µg/g. Study has been carried out by Samuel et al.(2011) on effect of developmental stage and exposure to morphine in the diet on morphine secretion rate. The Malpighian tubules from third instar maggots (31.46 ± 0.70 mm) were longer than those of second instar maggots (18.73 ± 0.45 mm).
Similar study on effect of morphine hydrochloride on the larvae of Parasarcophaga crassipalpis showed that the larvae of Parasarcophaga crassipalpis grew faster with the increase of morphine hydrochloride concentration and the temperature (Zhang, et al., 2012).
Goff at al. (1994) reported that phencyclidine (PCP), a dissociative anesthetic which has the same pharmacological effects as ketamine, had no significant impact on larval development of Parasarcophaga ruficornis (Fabricius) (Diptera:Sarcophagidae). However, ketamine showed a promotion for the growth of L. sericata (Meigen) (Diptera: Calliphoridae) to some extent, especially at the first half of larval life (Zou, et al., 2013).
Calvalho (2012) shows the effects of the drug on C. albiceps and C. putoria may alter the time required for their development and generate error in PMI estimates obtained through entomological methods. Cocaine accelerated the larval and pupal development of both species. Thus, the lack of drug-related data in a postmortem estimate based on the normal development of C. albiceps and C. putoria could result in gross errors.
Opiates technology analysis for codeine and morphine yielded positive result on flesh fly developed on decomposed cadaver. Concentration of morphine in larvae reared on intoxicated rabbit carcass were 30-100 times lower than the concentrations found in the tissues based on the result illustrated by Hedouin et al (1999). Kharbouche (2008) showed the effect of Codeine accumulation and elimination in larvae, pupae, and imago of the blowfly Lucilia sericata and effects on its development. If codeine was present in the larvae substrate, 29-h interval bias on the evaluation of the larval stage duration calculated from the larvae weight. Similarly, a 21-h interval bias on the total duration of development, from egg to imago, has to be considered if codeine was present in the larvae substrate.