Use of DNA in forensic entomology
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Forensic entomology Forensic entomology is the scientific study of the colonization of a dead body by arthropods. This includes the study of insect types commonly associated with cadavers, their respective life cycles, their ecological presences in a given environme ...
has three sub-fields:
urban Urban means "related to a city". In that sense, the term may refer to: * Urban area, geographical area distinct from rural areas * Urban culture, the culture of towns and cities Urban may also refer to: General * Urban (name), a list of people ...
, stored product and medico-criminal entomologies. This article focuses on medico-criminal entomology and how DNA is analyzed with various blood-feeding insects. Forensic entomology can be an 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.


Blood meal extraction

To extract a blood meal from the abdomen of an insect to isolate and analyze DNA, the insect must first be killed by placing it in 96%
ethanol Ethanol (abbr. EtOH; also called ethyl alcohol, grain alcohol, drinking alcohol, or simply alcohol) is an organic compound. It is an alcohol with the chemical formula . Its formula can be also written as or (an ethyl group linked to a ...
. The killed insect can be stored at -20 °C until analysis. When it is time for analysis, the DNA must then be extracted by dissecting the posterior end of the abdomen and collecting 25 mg 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 The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) ...
(PCR), is amplified and identified. PCR works by analyzing species specific mitochondrial DNA. PCR is currently the most commonly used method of species identification. This results from the fact that it is very sensitive in that it requires only a small amount of biological material, and can also utilize material that is not particularly fresh. The sample can be frozen and stored while still remaining usable for later PCR. DNA requires one hour to reach the abdomen of an insect, so DNA can be amplified one to forty-four hours after an insect feeds. Some research suggests that the source of a blood meal can be determined up to two months post feeding. To amplify DNA, it must first be denatured by exposing it to a 95 °C temperature for one minute, followed by thirty cycles of thirty-second 95 °C exposures. Then denatured DNA is mixed with a specific primer. A
chromatograph In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it through a system (a ...
is 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 Hematophagy (sometimes spelled haematophagy or hematophagia) is the practice by certain animals of feeding on blood (from the Greek words αἷμα ' "blood" and φαγεῖν ' "to eat"). Since blood is a fluid tissue rich in nutritious pro ...
(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

The following among the flies ( Diptera) have been utilized: * Mosquitoes, Family
Culicidae Mosquitoes (or mosquitos) are members of a group of almost 3,600 species of small flies within the family Culicidae (from the Latin ''culex'' meaning "gnat"). The word "mosquito" (formed by ''mosca'' and diminutive ''-ito'') is Spanish for "litt ...
*: Due to erratic feeding habits, mosquitoes can provide valuable DNA evidence. Multiplex PCR enables reliable identification of bitten individuals from just one mosquito, even few days after taking a blood-meal. The insects would need to be collected as soon as possible due to the insect's high mobility, digestion of consumed blood (degradation of DNA) and repeated feeding, although dead specimens are also potentially valuable source of evidence DNA. Research is centered on the mosquito due its widespread presence and affinity for feeding on humans. * Biting midges, Family
Ceratopogonidae Ceratopogonidae is a family of flies commonly known as no-see-ums, or biting midges, generally in length. The family includes more than 5,000 species, distributed worldwide, apart from the Antarctic and the Arctic. Ceratopogonidae are holomet ...
* Tsetse flies, Family Glossinidae * Sheep keds, Family
Hippoboscidae __NOTOC__ Hippoboscidae, the louse flies or keds, are obligate parasites of mammals and birds. In this family, the winged species can fly at least reasonably well, though others with vestigial or no wings are flightless and highly apomorphic. As ...
* Stable and horn flies, Family
Muscidae Muscidae are a family of flies found in the superfamily Muscoidea. Muscidae, some of which are commonly known as house flies or stable flies due to their synanthropy, are worldwide in distribution and contain almost 4,000 described species i ...
* Sand flies, Family
Psychodidae Psychodidae, called drain flies, sink flies, filter flies, sewer flies, or sewer gnats, is a family of Fly, true flies. Some genera have short, hairy bodies and wings giving them a "furry" moth-like appearance, hence one of their common names, m ...
, Subfamily
Phlebotominae The Phlebotominae are a subfamily of the family Psychodidae. In several countries, their common name is sandfly; but that name is also applied to other flies. The Phlebotominae include many genera of blood-feeding (hematophagous) flies, includi ...
* Snipe flies, Family
Rhagionidae Rhagionidae or snipe flies are a small family of flies. They get their name from the similarity of their often prominent proboscis that looks like the beak of a snipe. Description Rhagionidae are medium-sized to large flies with slender bodies a ...
* Black flies, Family Simuliidae * Horse flies, Family
Tabanidae Horse-flies or horseflies are true flies in the family Tabanidae in the insect order Diptera. They are often large and agile in flight, and only the female horseflies bite animals, including humans, to obtain blood. They prefer to fly in s ...


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 The cat flea (scientific name ''Ctenocephalides felis'') is an extremely common parasitic insect whose principal host is the domestic cat, although a high proportion of the fleas found on dogs also belong to this species. This is despite the wid ...
(''Ctenocephalides felis'') *
Northern rat flea ''Nosopsyllus fasciatus'', the northern rat flea, is a species of flea found on domestic rats and house mice. Northern rat fleas are external parasites, living by hematophagy off the blood of rodents. ''N. fasciatus'' can bite humans, but they a ...
(''Nosopsyllus fasciatus'') *
Human flea The human flea (''Pulex irritans'') – once also called the house flea – is a cosmopolitan flea species that has, in spite of the common name, a wide host spectrum. It is one of six species in the genus '' Pulex''; the other five are all confi ...
(''Pulex irritans'') * Oriental
rat flea A rat flea is a parasite of rats. There are at least four species: * Oriental rat flea (''Xenopsylla cheopis''), also known as the tropical rat flea, the primary vector for bubonic plague * Northern rat flea (''Nosopsyllus fasciatus''). Accordin ...
(''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. Recent studies have revealed that human DNA can be recovered from bed bugs for up to 60 days after feeding, thus demonstrating the potential use of this insect in forensic entomology *Assassin bugs, Family
Reduviidae The Reduviidae are a large cosmopolitan family of the order Hemiptera (true bugs). Among the Hemiptera and together with the Nabidae almost all species are terrestrial ambush predators: most other predatory Hemiptera are aquatic. The main exampl ...


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 The head louse (''Pediculus humanus capitis'') is an obligate ectoparasite of humans. Head lice are wingless insects that spend their entire lives on the human scalp and feeding exclusively on human blood. Humans are the only known hosts of ...
(''Pediculus humanus capitis'') *
Body louse The body louse (''Pediculus humanus humanus'', also known as ''Pediculus humanus corporis'') is a hematophagic ectoparasite louse that infests humans. It is one of three lice which infest humans, the other two being the head louse, and the cr ...
(''Pediculus humanus humanus'') *
Pubic louse The crab louse or pubic louse (''Pthirus pubis'') is an insect that is an obligate ectoparasite of humans, feeding exclusively on blood. The crab louse usually is found in the person's pubic hair. Although the louse cannot jump, it can also liv ...
(''Pthirus 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 Lymph (from Latin, , meaning "water") is the fluid that flows through the lymphatic system, a system composed of lymph vessels (channels) and intervening lymph nodes whose function, like the venous system, is to return fluid from the tissues ...
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 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 In biology, a species is the basic unit of classification and a taxonomic rank of an organism, as well as a unit of biodiversity. A species is often defined as the largest group of organisms in which any two individuals of the appropriate s ...
through conventional methods is needed before an attempt at DNA analysis. This DNA can be obtained from practically any part of the insect, including the body, leg, setae, antennae, etc. There are about one million species described in the world and many more that have still not been identified. A project termed " the barcode of life" was launched by Dr. Paul D. N. Hebert, where he identified a gene that is used in
cellular respiration Cellular respiration is the process by which biological fuels are oxidised in the presence of an inorganic electron acceptor such as oxygen to produce large amounts of energy, to drive the bulk production of ATP. Cellular respiration may be des ...
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 The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) ...
(PCR), followed by a sequencing reaction, then gel electrophoresis. PCR is a step that cleaves the long chain of
chromosomes A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...
into much shorter and workable pieces. These 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.Klug, William, and Michael Cummings. Essential of Genetics. 6th ed. Upper Saddle River, NJ: Prentice Hall, 2007. 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.


References

{{Reflist Forensic entomology
Forensic entomology Forensic entomology is the scientific study of the colonization of a dead body by arthropods. This includes the study of insect types commonly associated with cadavers, their respective life cycles, their ecological presences in a given environme ...