What tools do forensic entomologists use

Forensic entomology

Forensic entomology or the forensically applied entomology on corpses

Visit to the State Zoological Collection in Munich on June 28, 2017

A summary of various specialist lectures by Rosemarie Benke-Bursian

1) State Zoological Collection in Munich

The Zoological State Collection Munich (ZSM) is primarily a research station and is not only one of the largest zoological research museums in Germany, but is also one of the largest natural history research collections internationally. With around 11 million species, it also houses the largest collection of butterflies in the world.

The individual objects were brought together in the more than 200-year history of the house and the collection is still constantly being supplemented and expanded, be it through donations, inheritances or the purchase of collections from great naturalists and, last but not least, through research and collecting trips by scientific people and technical staff.

The information contained in these collections is systematically evaluated in the ZSM, with the research focus on taxonomy (classification scheme), phylogenetic systematics (biological systematics in the context of evolution), morphology (structure, shape, form, structure of living beings) and biodiversity (biological diversity) of the animal kingdom.

With the help of these collections, new animal species are described, relationships are clarified, the distribution of the individual species is documented and much more.

Since 2009 - as part of a special program - a molecular catalog of all approx. 35,000 Bavarian animal species has been created: the “Barcoding Fauna Bavarica”, so to speak a molecular “library of life”, which is attracting great attention worldwide. This catalog should also form the basis for cataloging throughout Germany.

With barcoding, the DNA of a marker gene - the mitochondrial cytochrome oxidase 1 = MT-CO1 or CO1 (an enzyme of the mitochondrial respiratory chain) - is sequenced and the respective sequence of the DNA base pairs (A, G, C, T) is added analogously to the barcode Associated with foods of the appropriate type.

The ZSM is not a museum with permanent exhibitions and therefore has no public opening times. Exceptions are special exhibitions and the open day, which takes place annually in October / November (2017 on Saturday, November 18).


2) Forensic entomology: definition and basic knowledge

Forensic entomology is forensic entomology and thus part of forensics (systematic investigation of criminal acts). Forensic entomology is used to clarify the time of death and possible circumstances of death based on the colonization of corpses by insects.
The focus is on all those insects that use a dead body as a source of food, to lay eggs (necrophageous insects) and those that are attracted by the "settlers" because they feed on them (necrophilic insects).

Forensic medicine has numerous options for determining the time of death in the first 48 to 72 hours. This includes the development of dead spots, body temperature, rigor mortis, electromuscular tests and various biochemical examinations. These values ​​are compared with the ambient temperature, the weight of the corpse, the type of clothing or covering of the body in order to then narrow down the time of death with the aid of various correction factors.
However, these classic methods can no longer be used after progressive tissue softening and structural dissolution (autolysis), which is usually the case after about 48 to 72 hours.
The colonization of the corpse by necrophageous insects, on the other hand, can make it possible to calculate the minimum lying time, the so-called post mortem interval (PMI), down to the day, even several weeks after the time of death. The type of insect also provides information on whether the location of the corpse corresponds to the crime scene or whether it was relocated.
In addition, the detection of drugs, poisons, medicaments or the identification of human DNA in these insects can provide information about the circumstances of death or be an approach for further criminal investigations.

A corpse that is accessible to insects is colonized by the first species within the first hours after death. These are mainly flies that use the corpse to lay eggs. The target locations for egg-laying are primarily the body cavities: mouth, nose, ears, eyes and the openings in the genital area, if they are exposed. The hatched larvae, which go through a total of three stages, use the corpse as food and only crawl away when they want to pupate.
In the meantime, more necrophageous insects have long been found on the corpse, because the different insect species colonize a corpse at different times of decomposition. All of these species have different development cycles.
The overall composition of the insect species, larvae and pupae provides information about how long the corpse has been lying down. The oldest stage of the first settlers is closest to the actual time of death. The age of the larvae or maggots is determined by their size.
Here, too, it is important to compare it with the other circumstances such as temperature, humidity, etc., because these not only influence the degree of decomposition of the corpse, but also the speed of development of the larvae and the cycle duration of the individual insects.

Insects are cold-blooded animals and therefore depend on their ambient temperature. Below a typical temperature threshold, they fall into hibernation or take a break in development, the maggot stops eating and only takes it up again when the temperature rises again. In cold seasons, this can lead to particularly long or interrupted cycle phases.
With the blow fly, for example, at a temperature of 30 ° C, it only takes seven days for the newly hatched maggot to pupate. If the temperature is around 12 ° C, however, it takes more than a month for the same development or the larva even dies completely. In severe winters, there is no insect colonization at all. Therefore, forensic entomology is mainly used in the warmer seasons.

Once all the data has been collected, the PMI, i.e. the length of time the corpse has been lying, can be read directly from a diagram in which the data is shown in relation to type, temperature and time.

The following insects are typically involved in a corpse colonization, with blowflies beginning the colonization:

1) Blowflies

2) ants

3) house flies

4) dung flies

5) wasps, bees

6) meat flies

7) raven beetle

8) cheese flies

9) humpback flies

10) colored beetles

11) Bacon beetle

12) earth beetles

While blowflies prefer a corpse that is just beginning to decompose, cheese flies do not lay their eggs until the decomposition process has progressed, and bacon beetles prefer a stage at which the corpse is already beginning to dry up again. With their powerful mouthparts, ants and beetles can also destroy hard parts or dried out corpses that are too hard for flies or make them accessible to flies.
Over 500 necrophage species have already been found on animal carcasses.



3) Forensic Entomology: Practical Process

After finding a corpse that is colonized with insects, a comprehensive photo documentation is created. The insects are then collected and killed so that they can be identified later.
The larvae, on the other hand, are more likely to be seized alive, because they can only be assigned to a certain species with difficulty and not clearly. They are further developed in the laboratory into the finished insect, on which the species can then be determined.

To determine the exact species, a determination under the microscope is necessary, because many species-specific features are reduced to the presence or absence of just a few bristles at a certain point and similar hard-to-see signs, which only specialized, experienced entomologists (entomologists) can clearly recognize can.
A determination of the time of death by means of insect colonization therefore takes an average of ten days, because even under optimal conditions it takes many days for the finished insect (imago) to hatch. In addition, there is the laborious determination of not just an insect, but an entire swarm.

Such a long time is of course not conducive to the police investigation.


4) DNA barcoding in combination with next-generation sequencing is revolutionizing forensic entomology

The work of the ZSM on "Barcoding Fauna Bavarica" ​​offers the opportunity to significantly accelerate species identification. With the help of this DNA barcoding, the insects are also cataloged via the species-specific DNA segment, the mitochondrial CO1 gene.
If you know the barcode of the CO1 gene of an insect, you can determine its species from the catalog - provided the insect has already been cataloged.
Since this method is also suitable for forensic entomology, the ZMS has been working for many years in cooperation with the Forensic Institute of the Bavarian State Criminal Police Office (BLKA) on a genetic reference database for forensically relevant insects. This database already includes a large number of typical necrophageous insects that originate from experiments with pig carcasses.

With the DNA barcoding, the rearing of the larvae and the morphological determination of species under the microscope can be omitted; it is sufficient to examine the mitochondrial DNA of the insect larvae. To do this, the DNA is isolated from the mitochondria and reproduced using a special method, the polymerase chain reaction (PCR) - this is necessary because many copies first result in so much material that the later genetic imprint can also be made visible. The DNA can then be sequenced, i.e. the base sequence in the DNA, i.e. the barcode of the CO1, can be read in the form of its initial letters (A, G, C, T) and then clearly assigned to an insect species.

For several years there has been an even faster method for sequencing: Next-Generation Sequencing (NGS). With this high-throughput sequencing, millions of DNA fragments can be sequenced in parallel.
For forensic entomology this means that it can sequence the entirety of all insects on a corpse in one fell swoop. There is no need to separate the individual insects and analyze them individually, you simply pour the entire mixture into the analyzer, which then spits out the barcodes of all the insects that have colonized the corpse, thereby speeding up the identification of species even further.
The forensic entomologists then only have to compare the barcodes with the genetic catalog for forensically relevant insects and can then use the species and other data - stage of development, temperature, etc. - to determine the PMI and thus the probable time of death.

Incidentally, DNA barcoding and NGS are currently not only revolutionizing forensics.