Use of DNA in forensic entomology
Encyclopedia
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.
, 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 must then 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
. 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 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.
(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.
) have been utilized:
Order Hemiptera
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 [feedings, hours, days?] after feeding, thus demonstrating the potential use of this insect in forensic entomology
they ingest. However, should an engorged tick be found in an area of interest, it would likely contain sufficient genetic material for identification.
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, 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
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. 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. 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.
The woman’s body was discovered partially covered by sand on a beach in Sicily. Investigators suspected a prominent businessman, whose car was seen in the area the night of the murder. A search of the suspect’s home produced no substantial evidence, apart from a mosquito blood meal stain on the wall. Technicians absorbed the stain onto wet filter paper
and then scratched the rest of the blood material off the wall. They also collected the insect’s remains in a tube for species identification. They extracted DNA from the blood sample and performed PCR. They identified the DNA as the victim’s, which placed the victim in the vicinity, if not in, the suspect’s home. Experts identified the mosquito as C. pipiens, a species not known to travel far distances, such as between the suspect’s home and the crime scene. This evidence, along with grains of sand and leaf fragments on the suspect’s clothing that matched samples from the beach, helped convict the suspect of second degree murder. This also relates forensic entomology and the law
.
worked on this specific case.
The evidence (no details were provided as to the circumstances surrounding their collection) was received as two shipping forms and 17 gelatin capsules, each of which contained a hand-written label and either a dried fly larva (maggot), what appeared to be a fragment of adult insect cuticle, or what appeared to be a fragment of insect pupal cuticle (puparium).
A DNA extraction procedure was performed on each specimen using the commercial kit sold by Qiagen
. Each sample was subjected to chemical digestion but no deliberate mechanical damage. The middle portion was cut out of the body of the larvae specimens for digestion and DNA extraction following the manufacturer's instructions. Any tissue from the middle section that remained following digestion, plus the unprocessed anterior and posterior sections of each larva have been stored with the original label in a screw-cap vial filled with 95% ethanol. All material from a non-larval specimen was digested. Any fragments that remained following the extraction procedure were stored in ethanol as above. The outside of each vial was labeled with the extraction number.
Mitochondrial DNA sequence analysis was attempted on each DNA extract as described in Wells and Sperling using PCR and sequencing primers C1-J-1751 and C1-N-2191.
The results from the PCR experiment showed that the DNA analysis was successful with eight larval specimens. Analysis on the insect cuticle proved unsuccessful as no mitochondrial DNA was able to be extracted. The portion of the mitochondrial DNA molecule characterized by these procedures corresponds to base positions 1752–2190 of the commonly used system first established for Drosophila yakuba. The sequences (also known as haplotypes) obtained ranged from 414 bases to 439 bases in length. Below is an example of a larva specimen DNA analysis.
Specimen 4
Species determination was done based on a mitochondrial DNA (mtDNA) haplotype. Three specimens produced the same haplotype, matching the previously published haplotype for the calliphorid fly species Cynomya cadaverina
and therefore being identified as such.
Several specimens produced three very closely related haplotypes, indicating that
they are all the same species. However, these haplotypes are not closely related to any previously published or unpublished data. Therefore these specimens cannot be identified
based solely on haplotype data, although they are definitely not members of the forensically important and extensively studied fly families Calliphoridae or Sarcophagidae. There are several common carrion-feeding species in the family Muscidae
that have not been investigated using DNA methods, and one of these species, Hydrotea dentipes, fits the general appearance of these specimens. Given the current state of the science these specimens cannot be identified using DNA analysis.
Forensic entomology
Forensic entomology is the application and study of insect and other arthropod biology to criminal matters. It is primarily associated with death investigations; however, it may also be used to detect drugs and poisons, determine the location of an incident, and find the presence and time of the...
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
To extract a blood meal from the abdomen of an insect to isolate and analyze DNADNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
, the insect must first be killed by placing it in 96% ethanol
Ethanol
Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as 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 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
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
(PCR), is amplified and identified.
PCR works by analyzing species specific mitochondrial DNA
Mitochondrial DNA
Mitochondrial DNA is the DNA located in organelles called mitochondria, structures within eukaryotic cells that convert the chemical energy from food into a form that cells can use, adenosine triphosphate...
. 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 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 haematophagousHematophagy
Hematophagy is the practice of certain animals of feeding on blood...
(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 (DipteraDiptera
Diptera , or true flies, is the order of insects possessing only a single pair of wings on the mesothorax; the metathorax bears a pair of drumstick like structures called the halteres, the remnants of the hind wings. It is a large order, containing an estimated 240,000 species, although under half...
) have been utilized:
- 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 CeratopogonidaeCeratopogonidaeCeratopogonidae, or biting midges , are a family of small flies in the order Diptera...
- Tsetse flies, Family Glossinidae
- Sheep keds, Family HippoboscidaeHippoboscidaeHippoboscidae, the louse flies or keds are obligate parasites of mammals and birds. In this family there are winged species which can fly at least reasonably well, as well as others with vestigial or no wings which are flightless and highly apomorphic...
- Stable and horn flies, Family MuscidaeMuscidaeMuscidae are a family of flies found in the superfamily Muscoidea. The apical segment of the antennae of Muscidae are plumose, and the basal portion is smooth....
- Sand flies, Family PsychodidaePsychodidaeThe nematoceran family Psychodidae are small true flies with short, hairy bodies and wings giving them a "furry" moth-like appearance...
, Subfamily PhlebotominaePhlebotominaeMembers of the subfamily Phlebotominae are known outside of the United States by the name sand fly. This subfamily includes numerous genera of blood-feeding flies, including the primary vectors of leishmaniasis, bartonellosis and pappataci fever... - Snipe flies, Family RhagionidaeRhagionidaeRhagionidae or snipe flies are a small family of flies containing 21 genera.-Description:Rhagionidae are medium-sized or large flies with slender bodies and stilt-like legs. The mouthparts are adapted for piercing and many species are haematophagous as adults, while others are predatory on other...
- 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 fleaChigoe fleaThe chigoe flea or jigger is a parasitic arthropod found in most tropical and sub-tropical climates, not to be confused with the larval form of Trombiculidae found in more temperate climates. In Brazil, the parasite is referred to as bicho-de-pé . At 1 mm long, the chigoe flea is the smallest...
, Family HectopsyllidaeHectopsyllidaeHectopsyllidae is a small family of fleas, containing only the chigoe fleas and the sticktight fleas. They were formerly known as Tungidae, and by authorities that demote the Pulicoidea to family rank they are treated as subfamily Hectopsyllinae...
(formerly Tungidae) - Cat fleaCat fleaThe cat flea, Ctenocephalides felis, is one of the most abundant and widespread species of flea on Earth.-Overview:The cat flea's primary host is the domestic cat, but this is also the primary flea infesting dogs in most of the world. The cat flea can also maintain its life cycle on other...
(Ctenocephalides felis) - Northern rat fleaNorthern rat fleaThe northern rat flea is a species of flea that is found on domestic rats and house mice. Northern rat fleas are external parasites, living by hematophagy off the blood of rodents...
(Nosopsyllus fasciatus) - Human fleaHuman fleaThe human flea, Pulex irritans, 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 confined to the Nearctic and Neotropical regions...
(Pulex irritans) - Oriental rat fleaRat fleaThe Oriental rat flea , also known as the tropical rat flea, is a parasite of rodents, primarily of the genus Rattus, and is a primary vector for bubonic plague and murine typhus. This occurs when the flea has fed on an infected rodent, and then bites a human...
(Xenopsylla cheopis)
Order HemipteraHemipteraHemiptera is an order of insects most often known as the true bugs , comprising around 50,000–80,000 species of cicadas, aphids, planthoppers, leafhoppers, shield bugs, and others...
- BedbugBedbugCimicidae are small parasitic insects. The most common type is Cimex lectularius. The term usually refers to species that prefer to feed on human blood...
(Cimex lectularius)
Cimex lectularius is an obligate parasite
Obligate parasite
An obligate parasite is a parasitic organism that cannot complete its life cycle without dependence on its host.-See also:*Obligate intracellular parasite*Parasitism*Parasitic plant*Facultative 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 [feedings, hours, days?] after feeding, thus demonstrating the potential use of this insect in forensic entomology
- Assassin bugs, Family ReduviidaeReduviidaeReduviidae is a large, cosmopolitan family of predatory insects in the suborder Heteroptera...
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 louseHead louseThe head louse is an obligate ectoparasite of humans. Head lice are wingless insects spending their entire life on human scalp and feeding exclusively on human blood...
(Pediculus humanus capitis) - Body louseBody louseThe body louse is a louse which infests humans. The condition of being infested with head lice, body lice, or pubic lice is known as pediculosis.-Origins:...
(Pediculus humanus humanus) - Pubic louse (Phthirus pubis)
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 lymphLymph
Lymph is considered a part of the interstitial fluid, the fluid which lies in the interstices of all body tissues. Interstitial fluid becomes lymph when it enters a lymph capillary...
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
Species
In biology, a species is one of the basic units of biological classification and a taxonomic rank. A species is often defined as a group of organisms capable of interbreeding and producing fertile offspring. While in many cases this definition is adequate, more precise or differing measures are...
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
Consortium for the Barcode of Life
Consortium for the Barcode of Life runs the International Barcode of Life project, a collaborative effort which aims to use DNA barcoding to generate a unique genetic barcode for every species of life on earth...
" was launched by Dr. Paul D. N. Hebert, where he identified a gene that is used in cellular respiration
Cellular respiration
Cellular respiration is the set of the metabolic reactions and processes that take place in the cells of organisms to convert biochemical energy from nutrients into adenosine triphosphate , and then release waste products. The reactions involved in respiration are catabolic reactions that involve...
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
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
(PCR), followed by a sequencing reaction, then gel electrophoresis
Electrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...
. PCR is a step that cleaves the long chain of chromosomes 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. 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 Study #1
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 used this technique to identify 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 seen in the area the night of the murder. A search of the suspect’s home produced no substantial evidence, apart from a mosquito blood meal stain on the wall. Technicians absorbed the stain onto wet filter paper
Filter paper
Filter paper is a semi-permeable paper barrier placed perpendicular to a liquid or air flow. It is used to separate fine solids from liquids or air.-Properties:Filter paper comes in various porosities and grades depending on the applications it is meant for...
and then scratched the rest of the blood material off the wall. They also collected the insect’s remains in a tube for species identification. They extracted DNA from the blood sample and performed PCR. They identified the DNA as the victim’s, which placed the victim in the vicinity, if not in, the suspect’s home. Experts identified the mosquito as C. pipiens, a species not known to travel far distances, such as between the suspect’s home and the crime scene. This evidence, along with grains of sand and leaf fragments on the suspect’s clothing that matched samples from the beach, helped convict the suspect of second degree murder. This also relates forensic entomology and the law
Forensic entomology and the law
Forensic entomology deals with the collection of arthropodic evidence and its application, and through a series of tests and previously set of rules, general admissibility of said evidence is determined. Ultimately, the admissibility of forensic evidence is left up to the judgement of the court...
.
Case Study #2
Forensic Entomologist Dr. Jeff Wells from West Virginia UniversityWest Virginia University
West Virginia University is a public research university in Morgantown, West Virginia, USA. Other campuses include: West Virginia University at Parkersburg in Parkersburg; West Virginia University Institute of Technology in Montgomery; Potomac State College of West Virginia University in Keyser;...
worked on this specific case.
The evidence (no details were provided as to the circumstances surrounding their collection) was received as two shipping forms and 17 gelatin capsules, each of which contained a hand-written label and either a dried fly larva (maggot), what appeared to be a fragment of adult insect cuticle, or what appeared to be a fragment of insect pupal cuticle (puparium).
A DNA extraction procedure was performed on each specimen using the commercial kit sold by Qiagen
Qiagen
Qiagen is a provider of sample and assay technologies for molecular diagnostics, applied testing, academic and pharmaceutical research. Consolidated under the Dutch holding Qiagen N.V., the company operates more than 30 subsidiaries in over 18 countries. Qiagen’s shares are listed at the...
. Each sample was subjected to chemical digestion but no deliberate mechanical damage. The middle portion was cut out of the body of the larvae specimens for digestion and DNA extraction following the manufacturer's instructions. Any tissue from the middle section that remained following digestion, plus the unprocessed anterior and posterior sections of each larva have been stored with the original label in a screw-cap vial filled with 95% ethanol. All material from a non-larval specimen was digested. Any fragments that remained following the extraction procedure were stored in ethanol as above. The outside of each vial was labeled with the extraction number.
Mitochondrial DNA sequence analysis was attempted on each DNA extract as described in Wells and Sperling using PCR and sequencing primers C1-J-1751 and C1-N-2191.
The results from the PCR experiment showed that the DNA analysis was successful with eight larval specimens. Analysis on the insect cuticle proved unsuccessful as no mitochondrial DNA was able to be extracted. The portion of the mitochondrial DNA molecule characterized by these procedures corresponds to base positions 1752–2190 of the commonly used system first established for Drosophila yakuba. The sequences (also known as haplotypes) obtained ranged from 414 bases to 439 bases in length. Below is an example of a larva specimen DNA analysis.
Specimen 4
Species determination was done based on a mitochondrial DNA (mtDNA) haplotype. Three specimens produced the same haplotype, matching the previously published haplotype for the calliphorid fly species Cynomya cadaverina
Cynomya cadaverina
Cynomya cadaverina, also known as the shiny blue bottle fly, is a member of the family Calliphoridae, which includes both blow flies, as well as bottle flies. In recent years, this family has become a forensically important facet in many medicocriminal investigations in the growing field of...
and therefore being identified as such.
Several specimens produced three very closely related haplotypes, indicating that
they are all the same species. However, these haplotypes are not closely related to any previously published or unpublished data. Therefore these specimens cannot be identified
based solely on haplotype data, although they are definitely not members of the forensically important and extensively studied fly families Calliphoridae or Sarcophagidae. There are several common carrion-feeding species in the family Muscidae
Muscidae
Muscidae are a family of flies found in the superfamily Muscoidea. The apical segment of the antennae of Muscidae are plumose, and the basal portion is smooth....
that have not been investigated using DNA methods, and one of these species, Hydrotea dentipes, fits the general appearance of these specimens. Given the current state of the science these specimens cannot be identified using DNA analysis.