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Isolation and characterization of human DNA from bed bug, Cimex lectularius L.,(Hemiptera: Cimicidae) blood meals

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The ability to identify individual human hosts based on analyses of blood recovered from hematophagous insects is beneficial for both medical and forensic entomology. Bed bugs, Cimex lectularius L. (Heteroptera: Cimicidae), may have several advantages over other blood-feeding arthropods for forensics because they do not remain on the host after their blood-feeding activity and remain in close proximity to a crime scene. Successful isolation, amplification, and sequencing of human DNA obtained from adult bed bugs is reported for the first time from this study. Engorged bed bugs were recovered from a human volunteer and from field collected samples from New York, New York, and Brazos County, Texas. Samples were preserved by drying, stored in 70% ethanol, or freezing at -20°C. DNA was extracted from individual insects, and polymerase chain reaction was conducted using short tandem repeat (STR), human mitochondrial DNA (mtDNA) hypervariable region (HVR1), and insect mtDNA 16S markers. Amplification of a STR marker used in forensic investigations, D18S51, a HVR1 marker, and an insect mtDNA 16S marker was successful. These results demonstrate that DNA isolated from bed bugs is qualitatively and quantitatively sufficient for DNA typing and could be helpful to identify individuals for forensic analysis.
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... Human DNA has also been isolated from common bed bugs (Cimex lectularius) after feeding, and the concentration of extracted DNA was adequate for DNA profiling [9,10]. Moreover, a complete human DNA profile has been generated from a DNA extract recovered up to 72 h after these bugs' post-blood meal [11]. ...
... Since this study's main aim was merely to detect human DNA traces within tropical bed bugs, only one common STR locus -the D18S51 marker (selected as it is easy to use in amplification), and one standard mitochondrial marker -HVR1, were used. The selection of markers was also adapted from the study by Szalanski et al. on the isolation and characterization of human DNA from common bed bugs (Cimex lectularius L.) [10]. ...
... PCR cycles for both HVR1 and D18S51 markers were adapted from Szalanski et al. [10]. For the HVR1 marker, PCR cycles consisted of 28 cycles of denaturing at 94ºC for 30 s, annealing at 60ºC for 30 s, an extension at 72ºC for 45 ssconds, and a final extension at 60ºC for 30 min. ...
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The ability to isolate and generate a DNA profle from human DNA recovered from tropical bed bugs (Cimex hemipterus) for identifying individuals can be useful for public health, forensic, and medical entomology. In this study, genomic DNA was recovered from both male and female bed bugs at every time interval tested (0, 1, 3, 5, 7, 14, 30, and 45 days post blood meal). The total DNA concentrations recovered from male bed bugs ranged from 12.93 to 65.97 ng/µL, while the total DNA concentrations from female bed bugs ranged from 8.93 to 44.53 ng/µL. However, based on the results from the BLAST search and PCR products, human DNA could be detected from female bed bugs at 0, 3, 5, 14, and 30 days post blood meal using the D18S51 marker. Concentrations of PCR products of the D18S51 locus from male bed bugs ranged from 4.20 to 35.50 ng/µL, whereas, for female bed bugs, concentrations ranged from 4.31 to 22.47 ng/µL. These were generally higher compared to the PCR products of the frst hypervariable part (HVR1) marker. The results indicate the HVR1 locus was less sensitive than the D18S51 locus
... However, human DNA isolated from an insect would have to be (1) stable and intact long enough to be useful in a forensic investigation and (2) unambiguously identifiable to an individual host. Researchers have successfully isolated, amplified, and profiled human mitochondrial DNA (mtDNA) from blood-feeding insects, including bed bugs [5] and human crab louse (Pthirus pubis) [6], and from maggots of the shiny blue bottle fly (Cynomyopsis [=Cynomya] cadaverina) feeding on human tissues [7,8]. Additional research has been conducted to isolate and profile human nuclear DNA from the blood meals of other insects such as human lice (Pediculus humanus capitis) [9] and mosquitoes (Culicidae) [10], and from maggots [11][12][13][14]. ...
... Szalanski et al. [5] demonstrated that DNA could be isolated from recently blood-fed bed bugs and it is qualitatively sufficient for DNA genotyping. However, to date, there are no documented reports about successful human blood identification and/or full human STR typing from a bed bug fed on human female, male, or pooled (female:male) blood. ...
... However, in 2018, a new potential use for Cimex detection dogs became apparent as bed bugs proved to be viable forensic physical evidence by yielding human DNA quantitation to help establish associations between perpetrators/victims to the scene of a crime [32]. Previous work had established that it was possible to extract human DNA from blood-sucking bed bugs [33]. ...
... A phylogenetic investigation based on the sequencing of mitochondrial genes is restricted to related species due to its high nucleotide substitution (Boore & Brown, 1998;Rawlings et al., 2001). In the present phylogeny, the partial mt 16S rRNA gene is used to strongly support the interrelationship between the recovered species with other hemipteran taxa at the generic level, this agreed with Hypša et al. (2002), Ribera et al. (2003), and Szalanski et al. (2006) Our findings confirmed that the order Hemiptera clustered four suborders, consistent with previous studies by Gordon et al. (2016) and Walker et al. (2016), who reported three lineages of herbivorous taxa of Sternorrhyncha, Auchenorrhyncha, and Coleorrhyncha were clustered by the species-rich insect order Hemiptera. ...
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Insecta is known to be the most diverse group of species, exhibiting numerous forms of endosymbiotic associations. Molecular techniques have provided significant indicators for insect–microbe interactions. The present study aimed to register one of the true bugs of pentatomomorpha and clarify its taxonomic position through phylogenetic analysis of the partial 16S rRNA gene region. A maximum likelihood analysis retrieved a generally well‐supported phylogeny based on Tamura 3‐parameter model. Based on the partial mitochondrial 16S rRNA gene sequences, a phylogenetic study of suborder Heteroptera relationships within Hemipteras' order was constructed. Sequences of 221 bases of the 3ʹ end of the gene from 28 species within 16 families were analyzed. This analysis and bootstrap confidence revealed two major clades comprising four suborders within Hemiptera, with a close relationship between Heteroptera + (Sternorrhyncha + (Auchenorrhycha + Coleorrhyncha)). Infraorder Pentatomomorpha is forming a sister group with a substantial bootstrap value to Cimicomorpha. Pyrrhocoroidea forms a sister relationship with Lygaeoidea + Coreoidea. There is a close relationship between Largidae and Pyrrhocoridae within Pyrrhocoroidea. The results show that the present species is firmly embedded in the genus Arhaphe with 94.35% sequence resemblance to its congeners. Besides, the recovered hemipteran species considered a potential model group for studying different symbionts. We propose both phylogenetic and ecological evolutionary developmental biology viewpoints for a more synthetic understanding of insect populations' molecular evolution. Highlights • • Our data highlighted the need of further taxonomic studies combining both morphological and molecular methods. • • Elevate the higher classification level of various species within different families.
... Research conducted in several countries shows that one in 14 women has been sexually abused and that there is a great under-reporting of this type of crime [7]. Pioneering studies in the isolation and amplification of human DNA using a forensic STR marker (D18S51) were performed on samples gathered from Cimex lectularius L bed bugs [8]. With the use of larvae of Calliphora vicina, which fed on human cadaver, it was possible to recover human autosomal DNA and Y chromosome [9]. ...
Article
The number of sexual crimes in Brazil, as in several other countries, is very high. In many of these crimes the women raped are murdered and their bodies are found days later, in an advanced state of decomposition, with intense cadaverous fauna. Forensic Entomology studies insects and other arthropods that can be used in the expert analysis of various types of crimes. Diptera, the order of insects that comprises the two-winged or true flies, represents one of the largest known groups of insects and is the principal source of cadaveric entomofauna. Members of its Calliphoridae family are observed in cadavers in all phases of decomposition. The retrieval and identification of human Y-STR DNA from the gastrointestinal tract of Calliphoridae species Chrysomya albiceps maggots and pupae can provide a good tool for the gathering of evidence in sexual crime investigations involving rape and death, in which the abandoned victim's body is found in a putrefied state. In this study, the animal model used was a female pig, Sus scrofa, which was sacrificed in a forested area with three shots from a .40 calibre Taurus pistol, and inoculated with semen to its anal and vaginal regions, simulating rape and homicide. During decomposition, 20 to 80 maggots were collected every 24 hours and preserved in 70% alcohol, totalling 289 maggots and 157 pupae (446 immatures) over a period of 14 days (336 hours) of decomposition. Each maggot was then dissected for removal of the digestive tract, which was placed in extraction buffer. The molecular phase proceeded with extraction, quantification, amplification and capillary electrophoresis of samples, testing 16 STR loci of the Y chromosome. It was possible to establish a partial Y-STR DNA profile, with the amplification of up to eight sites, by considering a combination of the samples taken at hours 144 h, 168 h, 192 h, 216 h, 240 h, 288 h, 312 h and 336 h.
... Forensic molecular entomology has proven to be a promising scientific tool; Szalanski et al. (2006) performed the first isolation and amplification of human DNA from the bedbugh Cimer lectularus (Hemiptera, Cimicidae) by utilization of the forensic STR marker D18S51. Similarly, human DNA was recovered from fly larvae that fed on a human corpse, obtaining a complete autosomic and Y-STR profile from various samples (Di Luise et al., 2008). ...
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Worldwide, several women become victims of rape every day. Many of those women are also murdered, with their bodies sometimes being found in an advanced state of decomposition, resulting in loss of evidence important to criminal investigations. Diptera is one of the main orders associated with human body decomposition. Fly species that belong to the family Calliphoridae are usually scavengers and are frequently found on decomposing bodies, thereby playing an important role in forensic ntomology. The recovery and genotyping of human Y-STR DNA from the gastrointestinal contents of the calliphorid Chrysomya albiceps larvae has promising applications in the investigation of sexual crimes, such as rape, and in cases of murder and abandonment of the victim’s body, which may be found in a state of decomposition. We studied this species of fly with the aim of supporting such investigations. After establishment of a colony, larvae were fed with decomposing human semen mixed in ground bovine meat (1 mL per 200 g beef). Larvae (10–15) were collected every 24 h and kept in 70% ethanol, to give a total of 96 larvae obtained after eight days of decomposition. The digestive system of each larva was resected. Molecular typing was conducted, which comprised sample extraction, quantification, amplification, and capillary electrophoresis with 16 STR loci from the Y chromosome. We succeeded in establishing a Y-STR DNA profile, with amplification of up to 11 loci, from individual samples, or up to 15 loci, when a combination of samples corresponding to the time-points 48, 72, 120, 144, and 192 h was used.
... The use of insects as forensic evidence is also important in order to detect nosocomial myiasis [12,13] or infection, whereby the insects can act as vectors of diseases, highlighting, sometimes, the state of neglect in which some elderly people live. In particular, entomological evidence can provide space-time information regarding events [14] thereby establishing a correlation between the insect, crime scene and victim [15], helping to identify the human remains [16][17][18][19][20][21] and investigating deaths caused by neglect. In some cases, fly species are difficult to distinguish morphologically, especially at the juvenile stage. ...
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This represents one of several sections of "A Bibliography Related to Crime Scene Interpretation with Emphases in Geotaphonomic and Forensic Archaeological Field Techniques, Nineteenth Edition" (The complete bibliography is also included at ResearchGate.net.). This is the most recent edition of a bibliography containing resources for multiple areas of crime scene, and particularly outdoor crime scene, investigations. It replaces the prior edition and contains approximately 10,000 additional citations. As an ongoing project, additional references, as encountered, will be added to future editions. The collection and analyses of insects, or invertebrates, from crime scenes is generally well known among homicide investigators and death scene investigators. It has been the compiler’s experience, however, that the actual practice of such collection, outside the presence of a forensic entomologist, is still overlooked or avoided. Often, an attitude prevails that that level of information is not necessary given the investigator’s knowledge of when an abduction took place, or a subject’s confession. In other situations, collections are not made simply because the investigators are not properly equipped with tools, chemicals, and packaging materials to collect and kill samples, or are not sure of what to do with, or how to store, live specimens. Entomological evidence is unique in that it is, in most criminal investigations, the only type of non-human evidence consisting of living, moving species. It is the hope of the compiler that this section will offer some answers toward appropriate collection procedures and equipment which are not expensive, do not involve a lot of time, or the need for additional manpower. The proper collection of entomological samples combined with accurate spatial, temporal, and environmental data, can yield valuable information toward determining postmortem intervals (Taphonomy - Decomposition and Time Since Death). Subject/Witness statements might be supported or disproved. Works such as Catts and Haskell (1990), and Lord and Burger (1983) have become standards in the field of forensic entomological procedures. In recent years compilations such as that by Byrd and Castner (2010) have demonstrated the increasing interest in forensic applications of a science which otherwise serves advancements in health and agriculture. Amendt, et al. (2007) offer "Standards and Guidelines" for this field of study. Entomology, as a means of determining post-mortem interval, continues to be scrutinized. This is not a bad thing. Any validation, clarification, or revocation of a forensic technique benefit crime scene interpretation. Many of the works below include laboratory analyses and research. That research goes beyond addressing the timing of a death or deposition (post-mortem interval) to toxicological determinations, interpretations of death scene versus depositional environments, et cetera. Like virtually every category in this bibliography, the study of entomology is contingent upon so many factors at a scene that an understanding of other disciplines is a neccessity. Obviously, taphonomy and pathology are directly related to insect activity on discovered remains. Environmental characteristics such as soils and plants, as well as body position either by accident or intentional, could influence the impact of insects in peri- and post-mortem activity. Again, the reader is refered to other categories such as Taphonomy, Geoarchaeology and Soil Science, and Criminal and Cultural Behavior. (2552 citations)
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The ability to identify individual human hosts based on analyses of blood recovered from the digestive tract of hematophagous arthropods has been a long-term pursuit in both medical and forensic entomology. Blood meal individualization techniques can bring important advancements to studies of vector-borne disease epidemiology. Forensically, these analyses may aid in assailant identification in violent crime cases where blood-feeding insects or their excreta are recovered from victims or at crime scenes. Successful isolation, amplification, and sequencing of human mitochondrial DNA obtained from adult human crab lice fed on human volunteers are reported. Adult lice were removed from recruited volunteers frequenting inner city health clinics. Live lice were killed by freezing and subsequently air dried at ambient temperature. A saliva sample was obtained from each volunteer and served as a DNA reference sample. Volunteers were afforded free, approved pediculosis treatment. Individual lice were subsequently processed using procedures developed for the extraction of mitochondrial DNA from human hair, teeth, and bone. The resulting DNA was amplified by the polymerase chain reaction and sequenced. Our results point to valuable avenues for future entomological research.
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The isolation, amplification, and characterization of human DNA from hematophagous (blood feeding) and necrophagous (carrion feeding) arthropods have been advanced significantly by the development of polymerase chain reaction (PCR) DNA sequencing methodologies. Historically, DNA technology has been successfully utilized to identify individual hosts upon which species of hematophagous arthropods have fed. The analysis of hematophagous insects' gut content blood meals has led to major advances in medical entomology and vector-borne disease epidemiology. In the forensic arena, the ability to apply similar techniques to insects recovered from badly decomposed remains has been greatly enhanced through the advent of mitochondrial DNA (mtDNA) techniques. Mitochondrial DNA analyses have been utilized to identify both the human remains upon which fly larvae (maggots) have fed and the species of the larvae themselves. The preliminary work detailed here demonstrates, for the first time, the successful application of mtDNA sequencing techniques to the analysis of necrophagous beetle larvae. A small sample of sap beetle larvae, Omosita spp. (Coleoptera: Nitidulidae), was collected from human skeletal remains during anthropological examination and analyzed for human DNA using mtDNA sequencing. The beetle larvae yielded mtDNA matching that of the host human bone. The results detailed here further demonstrate the robust nature of human mtDNA and the ability to recover valuable mtDNA evidence from forensically important, late decompositional stage insect species.
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In homicides with entomological evidence, it may be important to prove the presumed association of fly larvae to a corpse, especially if it is in doubt whether all maggots used for entomological expertise developed and fed on it. The present study demonstrates for the first time the possibility of analyzing human microsatellite DNA present in the digestive tract of necrophagous larvae that fed on decomposed bodies with a postmortem interval up to four months. The obtained human STR profiles support the association of a maggot to a specific corpse. In addition, the identification of the host species (e.g., animal source like pig) can be achieved by analysis of the cytochrome b gene. Maggots were collected from 13 corpses after various postmortem intervals and STR typing and HVR amplifications were performed using their crop contents. In seven cases, a complete STR profile was established, in two cases, an incomplete set of alleles was obtained, and in four cases, STR typing was not successful. HVR analysis was successful in all cases except one. The time of storage of the maggots and the length of the postmortem interval up to 16 weeks appeared to have no particular influence on the quality of the results.