Use of DNA in forensic entomology

Forensic entomology contains three aspects: medicocriminal entomology, urban entomology, and stored product entomology. This article focuses more on the medicocriminal aspect and how DNA is analyzed with various blood feeding insects.

Blood meal extraction

In order to extract a blood meal from the abdomen of an insect so that it can be used to isolate and analyze DNA, the insect must first be killed by placing it in 96% ethanol. The killed insect can be stored at -20°C until analysis. When it is time for analysis the DNA first has to be extracted by dissecting the posterior end of the abdomen and collecting 25mg of tissue. The cut in the abdomen should be made with a razor blade as close to the posterior as possible to avoid the stomach. Using a DNA extraction kit, the DNA is extracted from the tissue. If the DNA is mixed with samples from more than one individual, it is separated using a species specific primer. Once extracted and isolated, the DNA sample goes through a polymerase chain reaction(PCR), is amplified and identified.

PCR works by analyzing species specific mitochondrial DNA. It is currently the most commonly used method to for species identification because not only is it very sensitive in that it requires only a small amount of biological material, but it also doesn’t require that material to be particularly fresh. The sample can be frozen and stored and still good for PCR later.

One hour is required for host DNA to reach the abdomen of an insect so DNA can be amplified one to forty-four hours after an insect feeds and some research suggests that the source of a blood meal can be determined up to two months post feeding.

In order to amplify DNA, it first has to be denatured. This is done by one minute exposure of the DNA to 95°C followed by thirty cycles of thirty second exposure to 95oC. The denatured DNA is then mixed with a specific primer. A chromatograph is then conducted on 2% agarose gel, stained, and viewed with UV fluorescence. The DNA is identified by looking for genome specific repetitive elements and by comparing it with known examples.

Haematophagous insects of forensic importance

Humans are constantly fed on by haematophagous (blood feeding) insects. The ingested blood can be recovered and used to identify the person from which it was taken. Bite marks and reactions to bites can be used to place a person in an area where those insects are found.

Order Diptera

• Mosquitoes, Family Culicidae

Due to erratic feeding habits, mosquitoes could potentially provide DNA evidence to many people in one area at a certain time. PCR analysis shows that identification of bitten individuals is possible with a low error rate, although multiple mosquitoes would be needed. The insects would need to be collected as soon as possible due to the insect’s high mobility and constant feeding. Research is centered on the mosquito due its widespread presence and affinity for feeding on humans.

• Biting midges, Family Ceratopogonidae
• Tsetse flies, Family Glossinidae
• Sheep keds, Family Hippoboscidae
• Stable and horn flies, Family Muscidae
• Sand flies, Family Phlebotominae
• Moth flies, Family Psychodidae
• Snipe flies, Family Rhagionidae
• Black flies, Family Simuliidae
• Horse flies, Family Tabanidae

Order Siphonaptera

Listed here are fleas commonly encountered by humans that could potentially be used for DNA identification.

• Sticktight and chigoe flea, Family Hectopsyllidae (formerly Tungidae)
• Cat flea (Ctenocephalides felis)
• Northern rat flea (Nosopsyllus fasciatus)
• Human flea (Pulex irritans)
• Oriental rat flea (Xenopsylla cheopis)

Order Hemiptera

• Bedbug (Cimex lectularius)

Cimex lectularius is an obligate parasite of humans. Testing a sample of a residence's bed bug population and screening for bites could reveal possible recent visitors to the structure, as they have been observed to feed approximately once a week in temperate conditions. A recent re-emergence of bedbug populations in North America as well as growing interest in the field of forensics may prove bedbugs to be useful investigative tools.

• Assassin bugs, Family Reduviidae

Order Phthiraptera

Lice can be indicators of contact with another person. Many species closely associated with humans can be easily transferred between individuals. DNA identification of multiple individuals using blood meals from body and head lice has been demonstrated in laboratory settings.

Suborder Anoplura

• Head louse (Pediculus humanus capitis)
• Body louse (Pediculus humanus humanus)
• Pubic louse (Phthirus pubis)

Other Arthropods

Order Ixodida

Due to the low probability of a tick detaching and falling to the ground at the scene of the crime, these may not be highly useful regardless of the large amount of blood and lymph they ingest. However, should an engorged tick be found in an area of interest, it would likely contain sufficient genetic material for identification.

Analysis of collected DNA

DNA identification of species can be a very useful tool in forensic entomology. Although it does not replace conventional identification of species through visual identification, it can be used to differentiate between two species of very similar or identical physical and behavioral characteristics. A thorough identification of the species through conventional methods is needed before an attempt at DNA analysis. This DNA can be obtained from practically any part of the insect like the body, leg, setae, antennae, etc. There are about one million species described in the world and many more that still have not been identified. A project termed "the barcode of life" was launched by Dr. Paul Herbert where he identified a gene that is used in cell respiration by all species, but is different in every species. This difference in sequence can help entomologists easily identify two similar species.

DNA sequencing is basically done in three steps: polymerase chain reaction (PCR), followed by a sequencing reaction, then gel electrophoresis. PCR is a step that cleaves the long chain of chromosomes into much shorter and workable pieces. Theses pieces are used as patterns to create a set of fragments. These fragments are different in length from each other by one base which is helpful in identification. Those sets of fragments are then separated by gel electrophoresis. This process uses electricity to separate DNA fragments by size as they move through a gel matrix. With the presence of an electric current the negative DNA strand marches toward the positive pole of the current. The smaller DNA fragments move through the gel pores much more easily/faster than larger molecules. At the bottom of the gel the fragments go through a laser beam that emits a distinct color according to the base that passes through.

Case studies

Research has already demonstrated the link between Forensic entomology and society by amplifying human DNA from blood meals through various methods. A case in Italy involving the murder of a woman utilized this technique in order to link to a suspect.

The woman’s body was discovered partially covered by sand on a beach in Sicily. Investigators suspected a prominent businessman whose car was observed in the area the night of the murder. During investigation of the suspect’s home no substantial evidence was collected apart from a mosquito blood meal stain on the wall. The stain was absorbed on wet filter paper and then scratched to recover the rest of the blood material off the wall. The insect’s remains were also collected in a tube for species identification. DNA was extracted from the collected blood sample and a PCR was performed. The DNA was identified as the victim’s, therefore at least placing the victim in the vicinity, if not in, the suspect’s home. The mosquito was identified as C. pipiens, a species that is not known to travel far distances, such as the distance between the suspect’s home and the beach where the victim was found. This evidence along with grains of sand and leaf fragments found on the suspect’s clothing that matched that found at the beach helped to convict the suspect of second degree murder. This also relates Entomology and the law.

Conclusion

Forensic entomology is a very important aspect for law enforcement. With the magnitude of information that can be gathered, investigators can more accurately determine time of death, location, how long a body has been in a specific area, if it has been moved, and other important factors. Forensic entomology is an extremely significant side for law enforcement officials to aid in numerous cases. As this branch of entomology progresses it will become a key facet in all investigations as it is steadily rising in popularity and usefulness.