Host‐habitat Location by the Parasitoid,Nasonia vitripennis Walker (Hymenoptera: Pteromalidae)

This study investigated the role of odorant cues used during host‐habitat location by the generalist parasitoid, Nasonia vitripennis Walker. Nasonia vitripennis is a common parasitoid of Dipteran pupae found in association with decaying carrion. Behavioral assays were used to investigate the host‐habitat searching behavior under different scenarios. First, we demonstrated N. vitripennis to be significantly attracted toward odorant cues associated with decaying meat. The biological activity of nine of the volatile molecules constituting the odor of decaying meat was tested on the searching behavior of parasitoid females through two complementary chemoecological approaches: electroantennography (EAG) and olfactometry bioassays. Butanoic acid and butan‐1‐ol elicited high olfactory responses, but no attraction was induced by these two chemicals. Behavioral assays showed that, among the VOCs tested, methyldisulfanylmethane (DMDS) was the only volatile chemical to induce attraction in N. vitripennis.     

Conditioned Microplitis croceipes Cresson (Hymenoptera: Braconidae) detect and respond to 2,4-DNT: development of a biological sensor

We examined the ability of M. croceipes to learn, detect, and respond to 2,4-DNT, which is a volatile discriminator of trinitrotoluene (TNT). The percentage of conditioned wasps to detect and respond to the various concentrations of 2,4-DNT for > or = 15 sec was measured. Significantly more of the conditioned wasps responded to the concentration of 2,4-DNT used for conditioning than other concentrations examined. Accordingly, percent conditioned wasps to respond > or = 15 sec could be used as a suitable measure to screen air samples and distinguish between samples with or without the target odorant. The data recorded in this study indicate the measured behavior could be used to estimate the concentration of target odorants. Data in this study indicate M. croceipes can detect and respond to this compound, which provide further support for its development as a biological sensor.     

Electrophysiological and Behavioral Responses of Thanatophilus sinuatus Fabricius (Coleoptera: Silphidae) to Selected Cadaveric Volatile Organic Compounds

Soon after death, carcasses release volatile chemicals that attract carrion insects including Silphidae. Nevertheless, it is not known which chemical cues are involved in the attractiveness of the carcass. So far, little information is available on the chemical ecology of carrion beetles, particularly concerning the subfamily of Silphinae. The biological role of selected cadaveric volatile organic compounds including dimethyldisulfide (DMDS), butan‐1‐ol, n‐butanoic acid, indole, phenol, p‐cresol, putrescine, and cadaverine on the silphine species, Thanatophilus sinuatus Fabricius, was investigated using both electrophysiological and behavioral techniques. Among the tested cadaveric compounds, butan‐1‐ol and DMDS elicited the strongest electroantennography (EAG) from both T. sinuatus male and female antennae. In a two‐arm olfactometer, males and females were significantly attracted to DMDS for both tested doses, whereas only males were attracted to p‐cresol at 100 ng. Putrescine was repellent to males at the dose of 1 μg.     

Decaying Mouse Volatiles Perceived by Calliphora vicina Rob.‐Desv.

Volatiles emitted by decaying human remains are in the focus of recent research. The identification of core volatiles in this field is of high importance, because cadaveric volatiles generally show high variation. In this study, the volatile profiles of five mice (Myodes glareolus) were sampled with charcoal filter tubes from their time of death until advanced decay. Eleven compounds were quantitated by means of gas chromatography–mass spectrometry. Electroantennographic experiments with female Calliphora vicina antennae led to the identification of dimethyl trisulfide, dimethyl disulfide, nonanal, hexan‐1‐ol, 1‐octen‐3‐ol, 3‐methylbutan‐1‐ol, and heptanal as electrophysiologically active compounds. When these were compared, dimethyl trisulfide (17 ng/μL) and dimethyl disulfide (11 ng/μL) were found to be emitted in higher concentrations. The roles of these compounds and nonanal as core volatiles for cadaver detection or postmortem time determination and their correlation to the stages of decay and the accumulated degree days are discussed.     

Responses of Lucilia sericata Meigen (Diptera: Calliphoridae) to cadaveric volatile organic compounds

Flies of the Calliphoridae Family are the most forensically important insects because of their abundance on the decedent during the first minutes following death. Necrophagous insects are attracted at a distance by a decomposing body, through the use of volatile chemical cues. We tested the possible attractive role of some volatile organic chemicals (VOCs) released by decaying cadavers, on male and female of Lucilia sericata Meigen (Diptera: Calliphoridae). Two complementary approaches were used. Electroantennography (EAG) allowed identifying the semiochemicals that are detected by the olfactory system of L. sericata. Dose-response tests with EAG showed that dimethyl disulfide (DMDS) and butan-1-ol elicited the highest responses. Behavioral assays showed that, among the VOCs tested, DMDS and butan-1-ol are attractive for L. sericata, while the other VOCs are repulsive or do not cause any behavior. Our results may have potential implications in a better understanding of attractiveness of blowflies toward a corpse.     

The Effect of Flunitrazepam (Rohypnol®) on the Development of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) and its Implications for Forensic Entomology

This study investigated the potential effects of flunitrazepam (known as “date rape drug”) on the developmental cycle of Chrysomya megacephala, an important forensic species, and their possible implications for the calculation of the PMI. A 1050 C. megacephala eggs were divided into five groups with seven replications each. The eggs were placed on artificial diet prepared with four drug concentrations of flunitrazepam (4, 8, 16, and 32 ng/g), besides the control group (prepared with water). Were evaluated the potential effects on development time, weight gain, and mortality during the cycles. The drug had no significant effect on development time or mortality although it did affect the weight of the pupae and adults (Kruskal–Wallis, p < 0.05). The result can be deduced that the determination of the postmortem interval is not affected.     

Forensic Identification of CITES Protected Slimming Cactus (Hoodia) Using DNA Barcoding

Slimming cactus (Hoodia), found only in southwestern Africa, is a well‐known herbal product for losing weight. Consequently, Hoodia extracts are sought‐after worldwide despite a CITES Appendix II status. The failure to eradicate illegal trade is due to problems with detecting and identifying Hoodia using morphological and chemical characters. Our aim was to evaluate the potential of molecular identification of Hoodia based on DNA barcoding. Screening of nrITS1 and psbA‐trnH DNA sequences from 26 accessions of Ceropegieae resulted in successful identification, while conventional chemical profiling using DLI‐MS led to inaccurate detection and identification of Hoodia. The presence of Hoodia in herbal products was also successfully established using DNA sequences. A validation procedure of our DNA barcoding protocol demonstrated its robustness to changes in PCR conditions. We conclude that DNA barcoding is an effective tool for Hoodia detection and identification which can contribute to preventing illegal trade.     

Developmental Times of Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) at Constant Temperatures and Applications in Forensic Entomology

The characteristic life stages of infesting blowflies (Calliphoridae) such as Chrysomya megacephala (Fabricius) are powerful evidence for estimating the death time of a corpse, but an established reference of developmental times for local blowfly species is required. We determined the developmental rates of C. megacephala from southwest China at seven constant temperatures (16–34°C). Isomegalen and isomorphen diagrams were constructed based on the larval length and time for each developmental event (first ecdysis, second ecdysis, wandering, pupariation, and eclosion), at each temperature. A thermal summation model was constructed by estimating the developmental threshold temperature D₀ and the thermal summation constant K. The thermal summation model indicated that, for complete development from egg hatching to eclosion, D₀ = 9.07 ± 0.54°C and K = 3991.07 ± 187.26 h °C. This reference can increase the accuracy of estimations of postmortem intervals in China by predicting the growth of C. megacephala.     

The Effect of Soft Tissue on Temperature Estimation from Burnt Bone Using Fourier Transform Infrared Spectroscopy

This study investigated the effect of soft tissue and different exposure times on the prediction of burning temperatures of bone when using Fourier transform infrared spectroscopy (FTIR). Ovis aries rib bones were burnt at different temperatures and for varying time intervals. Results of a linear regression analysis indicated that burn temperatures can be predicted with a standard error of ±70°C from defleshed bone spectra. Exposure time does not have a significant impact on prediction accuracy. The presence of soft tissue has a significant impact on heat‐induced changes of the bone matrix in low (<300°C) as well as high temperatures (>800°C), slowing down combustion in the former and accelerating it in the latter (p < 0.05). At medium temperatures, no significant difference was noted. These results provide forensic investigators a new perspective with which to interpret the results of crystallinity measures derived from burnt bone.     

Decomposing Human Blood: Canine Detection Odor Signature and Volatile Organic Compounds

The admissibility of human “odor mortis” discrimination in courts depends on the lack of comprehension of volatile organic compounds (VOCs) during the human decay process and of the lack in standardized procedures in training cadaver dogs. Blood was collected from four young people who died from traffic accidents and analyzed using HS‐SPME/GC‐MS at different decompositional stages. Two dogs, professionally trained, were tested to exactly locate blood samples, for each time point of the experiment. We found a long list of VOCs which varied from fresh to decomposed blood samples, showing differences in specific compounds. Dog performance showed a positive predictive value between 98.96% and 100% for DOG A, and between 99.47% and 100% for DOG B. Our findings demonstrated that decomposing human blood is a good source of VOCs and a good target for canine training.     

The Effects of Soil Texture on the Ability of Human Remains Detection Dogs to Detect Buried Human Remains

Despite technological advances, human remains detection (HRD) dogs still remain one of the best tools for locating clandestine graves. However, soil texture may affect the escape of decomposition gases and therefore the effectiveness of HDR dogs. Six nationally credentialed HRD dogs (three HRD only and three cross‐trained) were evaluated on novel buried human remains in contrasting soils, a clayey and a sandy soil. Search time and accuracy were compared for the clayey soil and sandy soil to assess odor location difficulty. Sandy soil (p < 0.001) yielded significantly faster trained response times, but no significant differences were found in performance accuracy between soil textures or training method. Results indicate soil texture may be significant factor in odor detection difficulty. Prior knowledge of soil texture and moisture may be useful for search management and planning. Appropriate adjustments to search segment sizes, sweep widths and search time allotment depending on soil texture may optimize successful detection.     

SNP Miniplexes for Individual Identification of Random‐Bred Domestic Cats

Phenotypic and genotypic characteristics of the cat can be obtained from single nucleotide polymorphisms (SNPs) analyses of fur. This study developed miniplexes using SNPs with high discriminating power for random‐bred domestic cats, focusing on individual and phenotypic identification. Seventy‐eight SNPs were investigated using a multiplex PCR followed by a fluorescently labeled single base extension (SBE) technique (SNaPshot^®). The SNP miniplexes were evaluated for reliability, reproducibility, sensitivity, species specificity, detection limitations, and assignment accuracy. Six SNPplexes were developed containing 39 intergenic SNPs and 26 phenotypic SNPs, including a sex identification marker, ZFXY. The combined random match probability (cRMP) was 6.58 × 10^?19 across all Western cat populations and the likelihood ratio was 1.52 × 10¹⁸. These SNPplexes can distinguish individual cats and their phenotypic traits, which could provide insight into crime reconstructions. A SNP database of 237 cats from 13 worldwide populations is now available for forensic applications.     

The Morphology of Fecal and Regurgitation Artifacts Deposited by the Blow Fly Lucilia cuprina Fed a Diet of Human Blood

Fly feces and regurgitation deposits may be mistaken for bloodstain patterns at a crime scene, potentially compromising event reconstruction and/or misdirecting police resources. In some instances, these artifacts contain sufficient human biological material to generate a full DNA profile, sometimes 2 years after deposition. Clearly, it is important that investigators can make the distinction between artifacts and bloodstains. This study examined 6645 artifacts deposited on a smooth, nonporous surface after Lucilia cuprina were fed human blood. Artifacts were also compared with bloodstains on a variety of other surfaces. Both similarities and differences were found between artifacts and bloodstains, highlighting the need for an identification system to assist personnel with little training in bloodstain pattern analysis. The morphology of the artifacts has been described so that these deposits may be more clearly distinguished from bloodstains, targeted by crime scene personnel as potential sources of human DNA, and/or identified as potential evidence contaminants. Flowcharts have been devised to facilitate the analysis.     

A Novel Method for Identifying Shahtoosh

Shahtoosh, the down hair of the Tibetan antelope (Pantholops hodgsonii), is the noblest and most expensive wool in the world. The population of the animal has declined dramatically due to commercial poaching for the fiber. Traditional inspection for detection of shahtoosh has been performed by microscopic analysis. We developed a TaqMan real‐time PCR‐based DNA analysis method for identifying shahtoosh fibers. A set of probe and primers for the mitochondrial 12S ribosomal RNA gene that binds specifically to Tibetan antelope DNA was designed. A signal was detected with sensitivity to the 1:10,000 dilution of shahtoosh DNA. A fiber mixture of 1% of shahtoosh mixed with cashmere and even a single fiber can be detected with this method. The method is faster, more cost‐effective and more sensitive than other traditional sequencing methods and can be directly applied to identify shahtoosh and its processed products, which will be of value in illegal trade investigations.     

Temperature‐dependent Development of Parasarcophaga similis (Meade 1876) and its Significance in Estimating Postmortem Interval

Flesh flies are commonly found insects on decaying corpses that appears slightly later than blowflies, and their development patterns are significant indicators for minimum postmortem interval (PMI_min) estimation. In this study, the flesh fly Parasarcophaga similis (Meade 1876) was reared at nine constant temperatures ranging from 15°C to 35°C to examine indicators for estimating their age. We generated three development models, including isomorphen diagram, isomegalen diagram, and thermal summation model. Larval body length at different rearing temperatures was fit into an L = a + bT + cT² + dT³ equation with which the relationship between the larval body length (L) and the time after larviposition (T) was confirmed. The pupal stage was categorized into 13 substages according to intrapuparial morphological changes, and a detailed table was generated of the pupal developmental stages at five rearing temperatures, 15°C, 20°C, 25°C, 30°C, and 35°C. This study provides fundamental data in supporting P. similis as an indicator for PMI_min estimation.     

The Effects of Continuous Application of the TASER X26 Waveform on Sus scrofa,

This study investigated and evaluated the safety margins of the continuous long duration (up to 30 min) effect of the TASER X26 waveform, using a Sus scrofa model. Long duration continuous stimulus has not been evaluated on humans or human surrogates prior to this study. Swine were used as models due to similarities with humans in their skin and cardiovascular systems. Very long duration was used to determine both exposure dose and possible adverse physiological effects of dose. The trial began with an application of 10 min, and subsequent animals received increasing exposure time up to a survived maximum duration of 30 min. At the onset of this work, it was hypothesized that there would be a time limit after which most animals would not survive consistent with increased dose response. However, this hypothesis was not supported by the experimental results. All animals (10 of 10) survived up to 3 min. Seven of the 10 animals survived up to a 10‐min exposure and 3 of 5 animals with a 30‐min target exposure survived the full exposure. Surviving animals were recovered and observed for 24 h, with no postrecovery deaths. This suggests that swine (based on physiology) will not experience a fatal event when exposed to the TASER X26 for a continuous 3 min. Conclusions regarding longer duration (10–30 min) are not as certain due to the small sample sizes at these time intervals.     

Copycatting the smell of death: Deciphering the role of cadaveric scent components used by detection dogs to locate human remains

Human remains detection dogs (HRDD) are commonly used by law enforcement agencies to search for cadavers. Biological material is typically used as a training stimulus, also called aids, to train dogs to recognize the smell of cadavers. While HRDD training approaches have received extensive attention, information remains limited on the olfactory cues used to train them. Here, we aimed to decipher the chemical basis of detection dog olfaction. Five specific objectives were explored to precise whether the composition or the concentration of the training aids drives the HRDDs responses. We recorded the behavioral responses of four HRDDs exposed to different cadaveric-like smells. We found that HRDDs recognized a simplified synthetic aid composed of cadaveric compounds. The lowest concentration at which HRDDs continued to perceive the cadaveric smell was determined. HRDDs were not impacted by slight modifications to the chemical composition of a blend of odors that they have been trained with. HRDDs associated sulfur and nitrogen compounds as human cadaver. Our findings highlight a lack of specificity of HRDDs to cadaveric compounds, which could lead to error of detection. Moreover, all dogs did not positively respond to the same blends, despite being trained with the same aids and procedure. However, we confirmed that dogs could be trained with a simplified blend of molecules. The chemical composition of a training aid has, therefore, high consequences on the performance of the trained animal, and this conclusion opens additional questions regarding olfaction-based detection animals.     

An Experiment to Characterize the Decomposer Community Associated with Carcasses (Sus scrofa domesticus) on Oahu,Hawaii

Pig (Sus scrofa domesticus) carcasses were decomposed on the soil surface of a terrestrial habitat on the island of Oahu, Hawaii to begin characterizing the decomposer community. Results showed that carcasses can decompose rapidly on Oahu, primarily due to the activity of fly larvae, with ~80% of mass lost by 8 days (~220 ADD) postmortem. Scavenging was conducted exclusively by the small Indian mongoose (Herpestes javanicus), first feeding on larvae then feeding on the remains. Carcasses were habitats of warm temperature, little to no oxygen, slightly acidic/neutral pH, and high sodium concentration. Larval masses selected for a microbial community comprised of multiple bacterial taxa from phyla Firmicutes and Proteobacteria, particularly genera Clostridium, Proteus, and Providencia. These larval masses were well established from 3 to 8 days (~90 to ~220 ADD) postmortem. These data provide helpful, novel insight into the structure and activity of carcass decomposer communities on Oahu.     

Soilwater Conductivity Analysis to Date and Locate Clandestine Graves of Homicide Victims

In homicide investigations, it is critically important that postmortem interval and postburial interval (PBI) of buried victims are determined accurately. However, clandestine graves can be difficult to locate; and the detection rates for a variety of search methods (ranging from simple ground probing through to remote imaging and near‐surface geophysics) can be very low. In this study, simulated graves of homicide victims were emplaced in three sites with contrasting soil types, bedrock, and depositional environments. The long‐term monthly in situ monitoring of grave soil water revealed rapid increases in conductivity up to 2 years after burial, with the longest study evidencing declining values to background levels after 4.25 years. Results were corrected for site temperatures and rainfall to produce generic models of fluid conductivity as a function of time. The research suggests soilwater conductivity can give reliable PBI estimates for clandestine burials and therefore be used as a grave detection method.     

Detection of Anthrax and Other Pathogens Using a Unique Liquid Array Technology

A bead‐based liquid hybridization assay, Luminex^® 100?, was used to identify four pathogenic bacteria, Bacillus anthracis, Clostridium botulinum, Francisella tularensis subsp. tularensis, and Yersinia pestis, and several close relatives. Hybridization between PCR‐amplified target sequences and probe sequences (located within the 23S ribosomal RNA gene rrl and the genes related to the toxicity of each bacterium) was detected in single‐probe or multiple‐probe assays, depending on the organism. The lower limits of detection (LLDs) for the probes ranged from 0.1 to 10 ng. Sensitivity was improved using lambda exonuclease to digest the noncomplementary target strand. All contributors in 33 binary, ternary, and quaternary mixtures in which all components were present in a 1:1 ratio were identified with an 80% success rate. Twenty‐eight binary mixtures in which the two components were combined in various ratios were further studied. All target sequences were detected, even when the minor component was overshadowed by a tenfold excess of the major component.