INTRODUCTION:

The potential use of insects for detecting drugs and other toxins in decomposing tissues has been widely demonstrated. In death investigations, Diptera and Arthropods can be reliable alternate specimens for toxicological analysis in the absence of tissues and fluids normally taken for such purposes. Entomotoxicology studies the application of toxicological analysis to carrion- feeding insects in order to evaluate drugs and toxins present on intoxicated tissue.

Entomotoxicology also investigates the effects caused by such substances on arthropods development in order to assist the forensic PMI estimates. At a crime scene, insects, particularly larvae, can be valuable as a source of information about the poisoning and/or drug consumption of a victim. This is especially true where the body is so badly decomposed that it is not possible to take samples of blood, urine, or the stomach contents. Immature insect stages, as well as adults, may retain or accumulate foreign chemicals present in a cadaver will vary. All these factors depends either how body is kept or which toxins was taken by the dead organism. For example, Grellner and Glenewinkel (1997) in their actual and desk-based study of exhumations, found indications of diazepam for a period of five years; mercury for one month (based on the literature) morphine up to 1.1 years(based on the literature). If these chemicals remain in a body for such a long duration then it became prior to determine their effect found at the time of development of the insects feeding on the carrions. It may helps in calculating the time since death or we called it postmortem interval (PMI). The ability of the insects to retain indications of such drugs means that they, themselves, can be a valuable source of information about the past behavior of the deceased [1], [3]. Forensic entomology is a branch of forensic science in which information about the insects is use to draw conclusion investigating legal cases relating to mammals (both human and wild life).insects can be used in the investigation of a crime scene on land and in water (Anderson, 1995; Erzinclioglu, 2000; keeper and casamatta, 2001).most of the entomological evidences discovered within a short time of being commited. Gaudry et al (2004) commented that in France 70% of cadavers were found outdoors and of these 60% were less than one month old.

Flies are the significant evidence found at the crime scene, by evaluating the lifecycle of the flies, we can calculate time since death. Age is derived by comparing the degree of development of maggots collected from the carrion with the maggots of the same species that were rared under constant known condition. The most measurable forensic entomological contemplates have focused on commonly recommended drugs. Insects specimen gathered from disintegrating bodies empower forensic entomologists to estimate the smaller then expected time after death interim (interval).The basic lifecycle of a normal fly is of 15-20 days in which they lay eggs that became larvae in about 7-8 days then after larval stage flies lifecycle cross the stage of pupa after which they became adult and ready to lay eggs. Over a period of a few days, flies are produced five or six batches of eggs. Females house flies favor damp, dark surfaces such as compost, manure and other decomposing organic material for egg laying. House fly eggs resemble individual grains of rice. Within a day, house fly eggs hatch into larvae, also known as maggots[3],[4]. Analysis of insects is quite easy after homogenization of the most representative specimen by common toxicological procedure such as gas chromatography(GC), Thin layer chromatography (TLC), High performance thin layer chromatography (HPTLC), High pressure liquid chromatography (HPLC-MS), Radio immune analysis(RIA), Gas mass analysis

(GC-MS)[2],[17].

History of research and development of forensic entomology:

Forensic entomology as it is today is derived from a long sporadic history of research dating back to 13th century China. Insects are known to have been used in the detection of crimes for a long time and number of researchers has written about the history of forensic entomology. During medieval times, the correlation between maggots on a corpse and the oviposition of adult flies was not recognized. However, the realistic and detailed illustration of corpses containing maggots was not unusual. The importance of insects in the decomposition of human bodies was documented, as early as the 19th century: the biologist Carl von Linné (Linnaeus) in 1767 said that “Three flies would destroy a horse as fast as a lion would”. In the early 20th century, entomological evidence has been used sporadically in several murder cases in Europe with increased success. In the second half of the 20th century there was been an increasing interest in forensic entomology and the topic was revived by many researchers. Forensic entomology continues to progress as a science, gaining credibility as research leads to a greater understanding of insects and refinement of methods. Employment of DNA analysis and use of scanning electron microscopy has helped improve accuracy in the classification and identification of insect species. Recent research has brought the possibility of extraction human DNA tissue and gunshot residue from the gut contents of feeding maggots. Entomotoxicology (detection of narcotics and toxins in the tissue of feeding insects on a decaying corpse) is an emerging field providing valuable tools in the investigation of homicides, suicides and other unattended deaths where chemicals are involved. Despite great strides having been made in fundamental and applied research, there are many questions yet to be answered. Forensic entomology is still a young discipline and there is still much room for progress. The scientific literature available on this topic, although constantly growing, remains small when compared to many other biological and legal subjects. Likewise, the number of qualified participating forensic entomologists capable of fully utilizing insect evidence is currently very small [11], [3].

MATERIALS AND METHODS:

Collection:

The carcass was obtained from the site of Arpa river, this was then transferred to the university for further studies. For the study of successional colonization all the important data was noted down on every day. A type of all flies which are spotted on dog, carcasses and environmental temperature was writing down. Two different types of larvae was found on the carcass of dog i.e., smooth larvae and hairy larvae. Sample of these larvae were collected which further reared in laboratory on beef liver sample which is collected from nearby the road. Growth and temperature are regularly noted down. The collected hairy larvae than reared into three different drug contained sample within about 1g/1kg of meat drug concentration. Three different kind of drugs i.e., paracetamol, caffeine and codeine are used for the determination of toxins.[3]

S. No.	No. of larvae	Content	Concentration
1	16	Paracetamol	1gm/1kg
2	20	Caffeine	1gm/1kg
3	22	Codeine	1gm/1kg
4	25	Controlled

We added equal amount of drugs (1gm/1kg) in three different sample of beef liver and keep them in same condition and same temperature. We noted down all records of its growth from ecolism to adult flies.

Insect sampling:

For performing insect sampling, we had just collected some maggots from the corpse. However, it is a factor leading to variability in drug detection. At first, during sampling of insects, insects (larvae or pupa) can be carried out, around or under the body or body discovery site. Before collecting the sample of insect’s investigator must be aware that the source of collection is only the decomposed corpse other than that of deceased. In addition several author demonstrate the importance of collecting at different body site, as inter-site sampling results in a high variation of drug concentration. This observation is logic as drugs are distributed in the body according to their physicochemical properties, leading to different drug concentrations in different organs and tissues, and thus also in insects reared on these different substrates. The best sampling site are the internal organs (e.g. liver) for the detection of toxin substance.

Sample preservation:

Once the specimens have been removed from the body, or the crime scene, they are washed with deionizer or tap water and the specimens are then frozen for storage at a temperature ranging from 20°C to 4°C until they are needed for analyses. Specimens are prepared for analysis in a variety of ways. They differ based upon the substance that is in question.

Inorganic substance:

For the analysis of inorganic substances, the arthropods are taken out of storage, washed, and then dried to insure the removal of any foreign human fluids. They are then crushed and stored in a porcelain crucible at a constant temperature of 650°C for 24 hours. The resulting ash has a high concentration of metals, which are then analyzed by acid digestion using 70% HNO3 (nitric acid).

Organic substances:

investigation starts up with washing and drying the specimens. 110 grams of larvae are finely cut and an internal standard solution is added. The specimens are then homogenized, in a 0.9% saline solution, followed by centrifuged. Strong acids or bases break down the chitinous exoskeleton to release any toxins present and the sample is allowed to extract overnight at a temperature of 65°C. The acid solution is then removed and the organic substances are available for further analyses. Pharmacokinetics of drugs in insects depends on the species, the developmental stage as well as on their feeding activity. Apart from necrophageous species, bioaccumulations can also occur in parasitoids, predators or omnivorous species. However, this drug bioaccumulation will not be similar as these species present different feeding behavior due to their diet or life history traits. For entomotoxicological investigations, use of necrophageous species belonging to Coleoptera and/or Diptera is recommended as they are the first to colonize the corpse. Necrophageous species are usually very common and abundantly present on the crime scene. Moreover, their biology and development are well-known, as they are already used in forensic entomology to estimate Postmortem Interval (PMI). Morphine and heroin were both believed to slow down the rate of fly development. However, closer examination of the effects of heroin on fly development has shown that it actually speeds up larval growth and then decreases the development rate of the pupal stage. This actually increases the overall timing of development from egg to adult. Cocaine and methamphetamine also accelerate the rate of fly development. Some effects of toxins on these arthropods depend on the concentration of the toxin while others simply depend on its presence. For example, cocaine (at the lethal dose) causes larvae to "develop more rapidly 36 (to 76) hours after hatching". The amount of growth depends on the concentration of cocaine in the area being fed upon. The amount of methamphetamine. [1], [6]-[9].

Arthropods studied:

Blowflies are the first insects that discover the corpse i.e., it comes first for feeding the carrions. Mainly blue and green bottle blowflies are abundant during summer months and over winter as larvae or pupae (Albert Cruz, 2006). A blowfly belongs to the family of Calliphoridae which includes green and blue bottle flies.

Life cycle of fly:

They have a complete life cycle which includes egg, larvae, pupa and adult stage:

1st stage- The adult flies lay eggs on the carcass, mainly on the area which have some injuries or wound or on the area which have some depths i.e. nose, eyes, ear, anus, etc

2nd stage- Eggs hatch into larvae (maggots) in about 12-24 hours.

3rd stage-The larvae continue to grow and shed their exoskeletons (molt) as they go through different instars stages.

· 1st instar- 5mm long after 1.8days.

· 2nd instar-10mm long after 2.5days.

· 3rd instar- 14-16mm long after 4-5 days.

· 4th instar- 17mm long, the larvae develops into pupa after burrowing in surrounding soil.

4th stage-Adults flies emerges from the pupa in about 6-8days. [3],[5],[7].

Calliphora vicina (Robineau-Desvoidy):

This is a large blowfly, 9–11mm in length. The front thoracic spiracle is orange in color (Smith, 1986). The head is black on top and the front half of the cheek (bucca) is reddish orange. The lower region of the face is black. There are black hairs on the jowls, irrespective of the jowl color. The thorax is black and the top of the thorax (the dorsum) is covered with a dense grayish shine (pubescence). There is a pair of strong bristles in a row in the centre of the thorax. These are called the acrostically bristles. Like other blowfly species, this species also has a fan of bristles, the hypo pleural bristles, on a plate above the coxa of each hind (third) leg, near the posterior spiracle. Look for this spiracle and you will spot them. The abdomen is blue with a silvery chequerboard effect (tessellation). The basicosta on the wing is yellowish in color, although this can fade to a yellowish-brown color.[1]

Blue bottle flies

Kingdom-Animilia

Phylum-Arthropods

Sub-Phylum-Hexapods

Class- Insecta

Order-Diptera

Family- Calliphoridae

Genus-Calliphorida Vicina

Lucilia sericata (Meigen):

This is commonly called a greenbottle because all the flies in this genus are a metallic green colour. In North America, Lucilia sericata is called Phaenicia sericata. Lucilia species are distinguished from other blowflies by having a ridge just above the squama,there are wing flap,whichhas tufts of hair on it. Lucilia sericata has a yellow-coloured basicosta. One of the differences between the larvae of Calliphora and Lucilia sericata is that the oral sclerite in the head skeleton (cephalopharyngeal skeleton) is transparent and so seems to be absent in larvae of Lucilia sericata. The identity of Lucilia sericata larvae can also be confirmed by looking at the rim of the final posterior segment of the larva. The protrusions found along the outer rim of the segment are called tubercles. They are named, from the top (12 noon position), the inner, median and outer (lower) tubercles. If the distance between the two inner tubercles is the same as the distance between the inner and the median tubercle, then this species can be identified as Lucilia sericata. This feature is characteristic of the third instar larvae. Erzinçlio˘glu (1987) found that around the posterior spiracles in first and second instar larvae of Calliphora and Lucilia there was a circle of hairs. In Calliphora species these hairs would be visible under low power, being very well-developed in Calliphora vomitoria, but would not be visible under low power in Lucilia species.

Green bottle flies

Kingdom-Animilia

Phylum-Arthropos

Sub-Phylum-Hexapods

Class- Insecta

Order-Diptera

Family- Calliphoridae

Genus-Calliphorida Lucilia

Sarcophagidae:

The common name for a member of this family of flies is the fleshfly. They are large and grayish in color and have a chequerboard (tessellated) abdomen which is silvery grey and a thorax with three stripes down. Colyer and Hammond (1951) considered this a difficult group from which to identify species with any degree of certainty, unless adult specimens are captured whilst mating (‘in cop’). Using the identity of the male, it is easier to confirm the identity of the female species. Help should be sought from taxonomists if these are the only family recovered from the body. The sarcophagid larvae are characterized by having a barrel-like shape with their posterior spiracles sunk into a hollow. The edge of the posterior segment has a large number of tubercles. This makes this family easy to distinguish as a larval stage. Some success has also been made in identification to species of larvae of the Sarcophagidae, using molecular methods (Zehner et al., 2004). [18], [19].

Sacrophaga (flesh fly)

Kingdom-Animalia

Phylum-Arthropods

Sub-phylum-Hexapod

Class-Insecta

Order- Diptera

Family-Sacrophagidae

Genus-Sacrophagidae

Effect of drugs on larvae:

For calculating the postmortem interval, particularly within the first 2-4 weeks of the decompositions, it is likely to be predicted that the insect will grow faster on the carrions or slower due to the presence of drugs on the carrion. (Byrd – Forensic Entomology) It has been known that heavy metals could be extracted from cadavers by using the larval stages of insects that feeds on carrion for a number of years. For example mercury has been extracted from calliphorid larvae, puparia, and adults feeding on fish which accumulate methylated – mercury (nuorteva and nuorteva, 1982). [1], [2].

The effects of drugs on larval lifecycle duration must be taken into account when calculating the time since death. sacrophagid species larvae fed on tissue which is highly intoxicated with drug that is cocaine, it metabolite at median lethal dose and twice the median lethal dose which show accelerated development time due to a reduced time in the larval stages, early pupation and adult eclosion (Goff, Omori and Goodbrod, 1989). Similarly, heroine speeds up the growth of larvae upto 29 hours (Goff et al, 1991). For the entomotoxicological study Goff in 1991 use peregrine maggots, which conclude that morphine hasten the growth of larvae [1]

Bourel et al, (1999) demonstrative that morphine reduce the speed of development in lucilia sericata and due to which investigator was not able to underestimate the time interval since death by 24 hours. Bourel et al (2001), using radio immune essay identify a concentration relationship between recovery of morphine and the original amount in the host tissue. They confirmed that lucilia sericata larvae were capable of excreting morphine during the post feeding stage and that tissue with concentration of 100-1000mg/kg [20]. Equally other drugs have little or no effect on development insect time. The effect of a particular drug or its metabolite (or both) on speed of insect development appears to be species specific, due to variation the intolerance levels for specific drugs. All these factors depends only whether the chemical sequestered in or excreted by the insects [1].

RESULTS AND DISCUSSION:

First step – Collection of larvae samples:

The larvae collected from the carcass are smooth larvae and hairy, from that four different flies are identified

1. Blue bottle flies ( calliphoridae )

2. Green bottle flies ( lucilia )

3. Sacrophagidae

4. Chrysome fly

From the smooth larvaes blue bottle flies, green bottle flies, sacrophagids developed and from hairy larvae the Chrysoma flies were developed.

Second step – Effects of drugs on lifecycle of blowfly

Liver of beef which is feed by flies

1st instar larvae

2nd instar larvae

3rd instar larvae

Microscopical examination of Spiracles of 3rd instar larvae

Drugs may have a major significance on insects over all development either its may hasten or slow down its development.There are certain drugs which do not effects its development such as paracetamol , caffeine effect has been observed in its development. Thus, the drugs can have a significant impact on insect growth.

Third step - Study of Successional colonization:

The stage of decomposing of corpse left on the soil surfaces or on the land. There are mainly six stage-

1. Fresh stage.

2. Bloated stage.

3. Decay stage.

4. Post-decay stage.

5. Skeleton stage.

6. Dry stage.

1. Fresh stage:

This stage starts just after the death and last for the moment when bloating starts . The first insects arrive on the dog carcass is blue bottle flies and green bottle flies. It is the first day of decomposing.

Fresh stage

2. Bloated stage:

3. Due to the bacterial activity continuous skin starts breaking or get putrefied. This is all because of the gases causing the dog carcass to bloat are generated by anaerobic bacteria metabolism nutrient. This is the perfect stage to distinguish. Whole body swells, starting with the abdomen, and became inflated air-balloon. Due to the foul smell more and more flies get attracted to the carcass.

Bloated stage

4. Decay stage:

5. After bloating stage , decaying of the carrion stars. It mainly starts when gases escape from the carrion and it became deflated. In this stage many beetles appeared to feed on the maggots. In this stage two types of larvae were found, one is smooth and other one is hairy larvae. 4-6 days were required for decaying.

Decay stage

6. Post Decay stage:

At this stage all the soft tissue has been removed , leaving most skin , cartilage and bones. In this stage, larvae were at its 3rd instar stage . on fifth day carrion goes in post-decaying stage. Most insects like calliphoridae and sarcophogidae departure from the remains because they can’t eat hard skin of the carcass. 7&8 days were required or arriving at the stage of post decay.

Post decay stage

7. Skeleton stage:

Only hairs and bones were left on the body, all tissues was eaten by the insects. In this stage only mites were found other than that flies and beetles. The 3rd instar larvae goes inside the soil and ready for the puparation stage after feeding sufficient to survive. Age of carcass at the skeleton stage is 7 days.

8. Dry stage:

In this stage the remains only have the dry bones. In this stage the larvae were found in dry stage, they were transformed into pupation. In pupation period, the color of pupa is changing from light brown to dark brown and then black with time. At the 8th day body completely decomposed and only dry bones were left.

LIMITATIONS:

As we all know that entomotoxicology is very much new branch of forensic entomology due to which there is lack of research in the emerging field. As an assessment to quantify the concentration of a drug in tissue using entomological evidence. The reason behind the less research article is drug only can be detected at the larval stage when the rate of absorption exceeds the rate of elimination. The sample of pupae and the third instars larvae does not contain any concentrations of drugs these indicate that drugs do not bioaccumulation in overall lifecycle of flies. This leads entomologists to theorize that toxins are eliminated / excreted from the larvae’s system over time, if they are not constantly receiving the toxins. This field of entomotoxicology in its infantile stage or in the beginning stage, hence lot of active research is needed in these field along with the new researcher with new ideas in these field.

CONCLUSION:

· Arthropods prove to be valuable tool in the investigation of homicides, suicides, and other unattended human deaths.

· Entomology can provide alternative specimens for drug detection in decomposed body.

· Paracetamol showed neutral effect on lifecycle of blowflies.

· Caffeine increased the time span of blowfly life cycle by 24 hours.

· Beef contain codeine does not show any further stages of larvae that means the larvae could not survive in codeine.

· Succession colonization took 29days for complete decomposition of carcass

· The flora of insects in succession were identified to be

1 Calliphora species

2 Lucilia species

3 Chrysoma species

4 Sarcophagids species

5 Mites

When the body has completely decomposed to a point where normal toxicological procedure are no longer valid, then insects themselves can reveal the identity of toxin.

Research has shown that the blowflies retain the toxin throughout the larval and puparial stages and eliminate it early in the adult stage.

REFERENCES:

1. Dorothy E. Gennard, University of Lincoln UK , Forensic entomology, An introduction, First edition, John Wiley & Sons Ltd publisher; 2007 pg. 24-26/pg.34-35.

2. Francesco Introna, Carlo Pietro Campobasso, Madison Lee Goff, Article in forensic science international 120, 2001 USA ,Entomotoxicology, : Elsevier Publication; page no. 42-47

3. Dr. C.R. Vasudeva Murthy, Miss. Manisa Mohanty, entomotoxicology;a review paper J Indian Acad Forensic Med, ISSN 0971-0973 page no. 82-84.

4. Jason Payne-Jame et al. Encyclopedia of Forensic Medicine and Legal Medicine. First edition. New York: Elsevier Publication; 2005.p 268-26

5. Miller, M. L, W. D. Lord, M. L. Goff, D. Donnelly, E. T. McDonough, and J. C. Alexis. 1994. Isolation of amitriptyline and nortriptyline from fly puparia (Phoridae) and beetle exuviae (Dermestidae) associated with mummified human remains. Journal of Forensic Sciences. 39:1305–13.

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12. Albert M. Cruz, 2006,crime scene intelligence,an experiment in forensic entomology.

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16. J.C. Beyer, W.F. Enos,M. Stajic,drug identificatiom through analysis of maggots , J.Forensic science 25 (1980) pg. 411-412.

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Received on 27.04.2017 Modified on 24.05.2017

Research J. Pharm. and Tech. 2018; 11(1): 65-72.

DOI: 10.5958/0974-360X.2018.00013.6