Forensic Entomology: Effective Role of Insects in Death Investigation

Forensic Science deals with the evidence used in criminal investigations to aid in solving cases. Every evidence has some importance in the case and insects found on the dead body during decomposition is another important evidence to aid in the death investigation. The study of those insects and applying it in a court of law is known as Forensic Entomology.

Introduction to Forensic Entomology

Entomology is the study of insects and related arthropods (crustaceans, spiders, etc). When the study of entomology is used to aid in legal death investigations, it is called Forensic Entomology.

The most visible type of Forensic Entomology is used in the investigation of death, abuse, and neglect cases. The most important contribution of the study of entomology is the estimation of the post mortem interval of time since death (TSD). Different information can be extracted from the study of arthropods on dead body present at a crime scene. Suspects can be linked to a crime scene by the presence of arthropods.

The role of a forensic Entomologist in a death investigation can be a major one. His/her role is to collect and identify arthropod specimens and then interpret the findings of environmental variables.

Forensic Entomologists can provide an objective estimate of time since death (TSD) as well as other valuable information concerning the circumstances surrounding the victim’s demise, including the season of death, location of death, movement or storage of remains after death, specific sites of injury on the body, post mortem artefacts on the body, use of drugs, and can even provide information to link a suspect to the crime scene, to child neglect or sexual molestation case, as well as in the identification of suspects.

Calliphorids have been known to cause myiasis and may be involved in the mechanical transmission of diseases. Myiasis is generally produced by the deposition of eggs or larvae by the female flies on the bodies of persons or animals, usually in body openings or in sores or wounds.

Medico-legal Entomology deals with the use of insects associated with a corpse at a crime scene in a legal investigation to provide data by using the normal methods of classic pathology. Determination of the postmortem interval is a crucial and fundamental step in any death investigation when the death has not been witnessed.

Estimation of the postmortem interval (PMI) is defined as the length of time between death and corpse discovery. At the onset of death, the medical parameters to establish the cause, manner, and time since death begin to degrade. With the progression of time and soft tissue decomposition, a postmortem determination becomes more difficult and less accurate.

Forensic Entomology is now an integral part of a death investigation while estimating time since death (TSD) beyond 72 hours. Forensic entomology is considered the most accurate method for estimating the elapsed time since death, particularly when more than 3 days have elapsed. Forensic Entomology was successfully utilized in the most famous Buck Ruxton case in 1935 in the UK.

Since then, the importance of Forensic Entomology has increased dramatically. The credit for the first entomology case goes to the French doctor Bergeret, who applies the study of Forensic Entomology in estimating time since death in 1855. Probably the best study on insects (Entomology) and their relationships with decay rates were reported in 1958 by Reed.

The use of Forensic Entomology to investigate cases of wrongful deaths has increased dramatically in recent years. Blowflies are among the first insects to discover and colonize on human remains. The larvae of blowflies are also used extensively in forensic entomology, predominantly to establish the minimum time elapsed since death.

In medico-legal death investigations, one of the most critical questions is, “When did the death take place?” An accurate estimation of the post mortem interval has special relevance in a homicide case because this information can narrow the field of possible suspects in a crime.

The application of the Entomology requires extensive knowledge of the factors, which interfere with the processes of colonization, development time, and the decomposition of the corpses by insects. Knowledge of the distribution, biology, ecology, and behaviour of insects found at a crime scene can provide information on when, where, and how the crime was committed.

Collection and preservation of evidence

To take advantage of the potential forensic value of arthropods, the evidence must be systematically collected and preserved. In 1983, the first paper on proper collection and preservation of important forensic evidence was published. A basic understanding of insect biology and anatomy, especially concerning flies and beetles, shall facilitate search, recognition, and collection of insect specimens for evidence.

The collected eggs or larvae should be placed directly into a preservative solution. The suggested preservative fluid is KAA (95% ethanol 80–100 ml, 20 ml glacial acetic acid and 10 ml kerosene), but several different methods exist. An excellent technique for preservation is to blanch the larvae in hot (nearly boiling) water for 60–120 s, and then place the blanched larvae in 80% ethyl alcohol. It is important to understand that with soft-bodied insect larvae, simple placement of the insect directly in 80% ethyl alcohol is not an adequate method of preservation.

Forensic Entomology is recognized in many countries as an important tool in legal investigations. With the advancement in forensic Entomology, investigators have also considered the effects of complicating factors that have been discussed in this paper. Various methods to estimate the postmortem interval from flies (Diptera: Calliphoridae) and problems associated with it have been reviewed in the study.

Measures for estimation of the postmortem interval (PMI)

Stages of succession

This approach takes advantage of the succession of the arthropod species commonly observed on carrion. The succession of insect communities on carrion varies according to the geographical location. The successional analysis may be used to estimate both minimum and maximum postmortem intervals.

The species typically occur in succession and respond to progressive changes in the carcass decomposition stages. The postmortem interval estimation applies to the remains in the more advanced stages of decomposition.

Limitations: To apply entomological evidence to the estimation of the postmortem interval based on the stages of succession, it is essential to precisely identify the species of insects attracted to the remains. Secondly, the succession patterns are typical for seasonal periods.

Age-dependent changes in the intestinal contents

Insect life cycles act as precise clocks, which begins within minutes of the death. These six-legged insects are attracted by the odour of the decaying corpse. The calliphorids arrive within a few minutes and the egg hatches to form the first instar. It undergoes moulting to form the third instar, which then enters the wandering stage.

The radiological examinations of the feeding behaviour of the maggots reveal that maggots stop eating immediately upon attaining their maximum length. During their subsequent development, the anterior intestine always remains empty. It is reported that by studying the intestinal filling in blowfly maggots the postmortem interval can be estimated efficiently.

Limitations: Determination of changes in the intestinal contents of maggots using radiological techniques is difficult. Such a technique for the ageing blowfly larvae has not been reported for most of the species of Calliphoridae.

 Development pattern

This method involves applying blowfly larval development times to investigations. During the early decomposition, entomological estimates of the postmortem interval (PMI) depend upon the period required for the insect species represented to develop to the growth stage encountered at the death scene.

Most often the larvae of the flies belonging to the families Calliphoridae and Sarcophagidae predominate. These flies currently provide the most accurate estimate of time since death (TSD). The age of specimens collected from the victim may be estimated to provide a minimum period since the death.

This approach requires detailed knowledge of the fly species used and the conditions at the crime scene but is relatively conservative. It is mentioned that because of their relatively short developmental cycles, blowflies and flesh flies are typically useful only during the first 3-4 weeks after death.

The growth rate of larvae can be studied by rearing them in the laboratory and this can give a reliable definition of time since death. Temperature and humidity heavily influence insect activities, such as the rate of oviposition and maturity.

Limitations: Development information is necessary about different geographical areas, as the more similar climatic and microenvironmental conditions between the experiment and the real case, the more accurate postmortem interval can be obtained.

Maggots appear to lengthen continuously during growth, developing at a predictable, species-specific temperature mediated growth rate. Thus within limits, their age may be estimated from their length, thereby providing a minimum estimate of time since death. The fluctuating temperatures also affect the development rate of blowflies.

Fly eggs

Forensic Entomologists use their knowledge of insects and their life cycles and behaviours to give clues about a crime. Egg-laying of a fly may give clues to the investigator about the time since death. Calliphorid flies feed on a fresh corpse and may arrive within a few minutes.

By studying the time of hatching of blowfly eggs in the laboratory at constant temperatures the data obtained can be used in actual cases. The estimate resulting from the experiment and laboratory developmental data shows the time of egg-laying within a period of about 2 h, which determines the time since death, as the Calliphorid species lays eggs immediately after death under favourable conditions.

Laboratory rearing of eggs of flies found on a corpse and studying the time of egg hatching or the emergence of the first instar can contribute to the determination of a short post mortem interval.

However, immediate oviposition may not occur even with those species which prefer carrion in the early fresh phase but may undergo a pre ovipositional pause. They may also feed on pooled blood as a protein supplement and then wait for digestion and assimilation of the meal.

The gravid female flies can retain eggs for 1–2 weeks in the absence of a suitable oviposition medium. Life table studies may be very time consuming and blowfly studies under laboratory conditions may be of dubious value in evaluating their potential in the field because many biotic and abiotic factors influence their survival, developmental rates, and fecundity.

However, while this is in part true, life history studies made under laboratory conditions allow researchers to collect the data needed for life table analysis to estimate their biotic potential under specific conditions, and these data can be used as a basis for simulation models for field use that include other factors.

Limitations: The arrival time of flies depends on many environmental factors such as sun-exposed/shaded carcass, cloud, rain, and other unfavourable conditions. The time of hatching of eggs in the laboratory at a constant temperature must be used carefully while studying the hatching of fly eggs under variable conditions in the field.

Ageing blowfly eggs through gene expression

The blowflies lay their eggs at predictable times in the decay cycle of the corpse. The immatures are difficult to identify up to the species level and they have to be reared up to the adult stage for correct identification and correct post mortem interval estimations.

This causes a delay of several weeks before time since death can be estimated. In criminal cases there is a need for quick investigations, hence, the reliable method of age estimation could be studying the expression of three genes (BCD, sell, cs) present in the blowfly eggs as a means of predicting age.

These models show that estimating egg age through gene expression made predictions within 2 h of the true age when all expression data are available, while the presence/absence of cs transcripts identified two age classes, together indicating that gene expression can be used to more precisely predict blowfly age.

Limitations: Specimen collection, preservation, time of storage, and DNA extraction strategies need to be refined.

Larval dispersal

The blowfly life cycle begins with the oviposition of eggs into eyes, mouth, and nasal passages where moisture and protection from the sun are optimal. Within hours (dependent on species and ambient temperatures) the eggs hatch and produce the first instars. These moult into second and third instars.

Feeding ends when larvae acquire the fat needed for pupariation. These larvae migrate to find a suitable site for pupariation. The lighter larvae tend to move longer distances for pupariation. This dispersal process can be important while estimating the minimum postmortem interval.

The investigator should be careful while concluding the minimum postmortem interval based on the size of those larvae that have dispersed to considerably long distances. Some of them may also migrate in search of a new food source.

The time taken for such dispersal should also be taken into consideration if pupae found away from the corpse are used to determine the minimum time since death (TSD). The larvae may also migrate on the decaying corpse from the other nearby corpse. Hence the larval dispersal is also an important factor and it should not be underestimated while estimating the post mortem interval.

Limitations: The larvae may be at risk of predation, prioritization, and desiccation during the post-feeding stage. If one wave of blowfly infestation has occurred and the post-feeding larvae have dispersed and are overlooked, the postmortem interval will be incorrectly determined.

Length of larvae

The postmortem interval is directly correlated with the length of the larva. The postmortem interval can be determined from the larvae of blowflies using the growth parameter and larval length as a ‘biological clock’. The insect larvae found on a corpse need to be collected and transferred to the laboratory in living conditions for identification and estimation of the postmortem interval.

The growth parameter of the egg, first, second, and third larval instars, total larval period, pupal period and egg-to-adult period can be studied in the laboratory. It is believed that older the maggots the more time has elapsed since death and this growth depends on the temperature. Hence, the oldest (longest) larvae provide the minimum postmortem interval.

Limitations: Larval specimens placed alive in most preservatives such as 70%, 75%, 80%, 90% and 100% ethyl alcohol, Kahle’s solution, and 10% formalin show colour changes, desiccation, drunkenness and agglomeration, and head curling. Reaction to the preservative type might be species-specific and that different instars of some species might also react differently.

Pupae

In certain indoor entomology cases, the corpse may be discovered only after the maggots have finished feeding and left it to pupate.

In these cases, it is not possible to estimate the time of death by examination of larvae, but structural and morphological changes in pupae or pupal cases should, in principle, enable a useful estimate to be reached.

During the growth stage, several species of blowflies spend 50% of their juvenile development in the pupal stage. Therefore, methods that are suitable for age estimation within this period would be valuable in entomology postmortem interval estimates.

During pupal development or metamorphosis, changes occur in gene expression also. The gene expression patterns of transcripts, which are differentially expressed during pupal development, show an age-dependent differential gene expression.

Limitations: Preservation methods for the pupal stage are poorly defined and inappropriate methods may result in nucleic acid degradation. These studies have used preservation at −80 °C or fresh pupae only, followed by an analysis of differentially expressed genes to ascertain age.

This application imposes the greatest limitations on the choice of the preservative method, as RNA is an inherently unstable molecule. Although newer molecular techniques are quite powerful, the technology is still expensive and DNA sequences are available for only some carrion breeding species at present. Furthermore, specific gene sequences are not appropriate for all species.

Difficulty in estimating postmortem interval

While Forensic Entomology plays an important role in the estimation of time since death after the corpse has been formed, yet there are some downsides which can alter the examination of postmortem interval. Those include:

• Myiasis
• Premortem condition of the deceased
• Maggot mass effect
• Fly pupae and puparium as contaminants
• Nocturnal oviposition
• Maggot development during morgue storage
• Fluctuating temperatures
• Indoors Forensic Entomology
• Weather Conditions
• Dispersal Time

Myiasis

Myiasis is the feeding by maggots on living tissues or dead tissues associated with a wound. In a forensic entomology context, myiasis is most frequently associated with facultative parasites in the families Calliphoridae, Sarcophagidae, and Muscidae.

Myiasis occurs primarily in indoor cases because sufferers are generally helpless infants or elderly people and may set in hours to weeks before death. If not fully appreciated, Myiasis can be a significant point of confusion for the Forensic Entomologist, appearing to give an estimate of postmortem far longer than the actual time since death.

Premortem condition of the deceased

The maggots found on the corpse can give the clue as to what happened. The study of these insects (entomology) can reveal the antemortem condition of the deceased or the manner of death. The presence of drugs and toxins taken internally before death can influence the estimation of time since death (TSD).

The presence of drugs in tissues can lead to under or overestimation of the postmortem interval. There is a necessity of considering the possible effects of drugs in tissues on insect growth rates when estimating the postmortem interval using entomology techniques.

Maggot mass effect

There can be a variation in the corpse temperature and ambient temperature. This is due to the activity of gregarious fly maggots present on the corpse. The maggot activity can lead to an increase in temperature approximately 1–3° Celsius above the ambient temperature.

This increase in temperature can lead to an increase in the development of maggots. Therefore, it can have detrimental effects on the accuracy of post mortem interval estimates.

Fly pupae and puparia as contaminants

In Forensic Entomology investigations, immature stages of the fly (egg, larva, or puparium) are used as entomological evidence at death scenes. The puparium represents the longest developmental time, which makes them useful indicators of time since death.

However, post-feeding larvae, which have originated from other remains, may migrate and pupate on forensic entomology samples at death scenes. These contaminants may erroneously lengthen postmortem interval estimates if no pupae or puparium are genuinely associated with the body.

Nocturnal oviposition

In Forensic Entomology, it was believed earlier that Calliphorid flies do not oviposit at night. But the research of some workers revealed that the flies crawl towards the food source and the oviposition occurs at a reduced rate. This can lead to an error of around 12 hours in the estimation of time since death.

Maggot development during morgue storage

The effects of refrigeration on insect development must also be considered. The insects present on the corpse are collected, refrigerated, and transferred to a laboratory for further studies. Even when the body infested with maggots is stored at 4°C, before the autopsy, it is seen that the low temperatures disrupt the insect’s development.

Low temperatures can also be indicated by the presence of dead larvae on the corpse.  A debate exists as to whether or not refrigeration affects maggot development.

As long as temperatures are sufficient for maggot development (above minimum developmental thresholds), movement of the body and changes in ambient temperature do not seem to retard or alter maggot feeding.

If the assumption is made that no insect development takes place during preautopsy refrigeration (−1°C to +4°C), potential error rates in the PMI estimation of (8.6–12.8)% occur.

Fluctuating temperatures

In some insects, the rate of development is constant at fluctuating temperatures but in some insects, the fluctuating temperatures have a greater impact on development.

Some species develop more rapidly under fluctuating temperatures and some insects take a longer time to develop under fluctuating temperatures. Negligence of fluctuating temperatures in legal cases can lead to distinctly wrong estimates of the postmortem interval.

Indoors Forensic Entomology

The bodies of socially isolated people may remain undiscovered in their own houses for prolonged periods. Medico-legal investigations of these cases are extremely difficult. Entomological examinations of the insect remains may estimate the time since death.

But the oviposition of flies in bodies found inside the buildings shows a decreased rate of egg batches and there is delayed oviposition of about 24 h. The delayed oviposition must be considered before a postmortem interval estimation is done.

Weather conditions

The insect fauna varies with the geographical location. The first colonizers on the corpse are the blowflies and their life cycle is important for estimation of time since death. It is dependent on the temperature and environmental conditions of the scene. The crime scene temperature and meteorological station temperature are also considered.

Although the gross weather conditions may be quite similar, microhabitat conditions may differ considerably and may not be comparable. Vegetational cover, air drainage, and slope exposure all influence microhabitat conditions greatly and this may also affect the development of flies.

Dispersal time

The larvae of flies feed on the corpse and moult into three instars. There is a rapid loss in the corpse weight and the post-feeding maggots begin to leave the corpse. To find a suitable site for pupariation, they migrate some distance away from the corpse. This period is known as the wandering stage.

For pupariation, the larvae prefer an environment protected from light and predators and may have a longer dispersal time to reach an appropriate pupation site. Hence, the dispersal time can vary and may influence the total time of development which may lead to an erroneous calculation of the postmortem interval.

Discussion

Diptera insects are of greatest forensic interest. Flies are considered to be nothing more than ecological scavengers and a nuisance to man. However, they are the important indicators of the post mortem interval usually in the first few weeks after death.

The examination of insects at the crime scene can provide other useful information, such as linking a suspect to a crime scene, providing analysis of badly decomposed bodies, indicating movement of the body, a period of neglect, and time of day and weather conditions at the time of insect colonization.

Various problems encountered in postmortem interval estimations should be taken into account. These areas require immediate research. The erroneous estimations could impede justice. Due to ethical reasons, animal models are used for experimental studies. Care must be taken when applying data from one carcass type (non-human/animal) to estimate the postmortem interval to another type (human).

Maggots play an important role in the study of Forensic Entomology therefore, they must be collected and preserved very cautiously. The kind of solution in which the maggots are killed or preserved has significant effects on their length. Hence, standard techniques should be used for collecting maggots at the crime scene and at the time of autopsy.

Blowflies (Diptera: Calliphoridae) are the first organisms to arrive on a dead body. Their offspring can give a good estimate of the time a body has been exposed to insects.

The age of the maggots can be calculated based on measurements of these morphological characters. Following the feeding stage, larvae disperse from the food source to pupate. The pupal stage can last up to 50% of the whole juvenile development. During this stage, age estimation is increasingly difficult because length, for instance, does not significantly change during metamorphosis.

The colour change in the puparium is also not a reliable method for postmortem interval estimation. Several different measures have evolved to estimate age from pupae such as internal morphological analysis of pupae through histological techniques, hydrocarbon profiles, and through the study of gene expressions.

The gene expressions, like human DNA fingerprints, are unique and this information allows the forensic entomologists to correctly identify the insect species and hence the postmortem interval. India needs to develop its internal database for future reference.

Apart from the above-mentioned references, more publications are needed, particularly regarding Indian species so that the insect evidence can be used in medico-legal investigations. The application of Forensic Entomology to criminal cases has also increased but this science has not gained the required popularity in India.

To make pathologists, police officers, and law personnel aware of the importance of Entomology, a standard protocol is required for the collection and preservation of entomological specimens containing taxonomic keys for the identification of Indian species. Each geographic region is comprised of unique factors that affect carrion decomposition rates and arthropod succession patterns.

Arthropod succession has been studied in India in the state of Punjab. Such studies are also required for other geographic regions of India. Accurate developmental data are required for all carrion species.

Due to the recent advancement in the field of Forensic Entomology, there is a need to understand the importance of the rate of development of flies about temperature, detailed development data are needed for Indian species to allow more precise postmortem interval estimates.