Forensic entomology when the evidence is "no insect." Best carrion fly species for predicting maximum postmortem interval in the United Arab Emirates.

The carrion insect species that most quickly deposit offspring on a corpse are, when available, likely to yield a more useful estimate of postmortem interval (PMI) compared to later arrivals. This is in part because the age of the oldest larva will be as close as possible to the PMI when doing a development analysis, and because the preappearance interval (PAI), the time the corpse was exposed before insect colonization, corresponds to the narrowest window of time since death for an insect-free corpse when doing a succession analysis. Given replicated training data a prediction of exposure time for a corpse can be in the form of a confidence set, and the maximum value of that set for an insect-free corpse is a probabilistic version of PAI. To discover the insect species likely to be useful in the early postmortem period in the United Arab Emirates we exposed 216 rat carcasses outdoors at two sites in Dubai over three-day periods during winter. Rats were sampled twice each day without replacement and kept in the lab to allow carrion insects to complete development to the adult stage. The fly species produced in this way were Sarcophaga dux, S. ruficornis, Wohlfahrtia nuba, W. indigens (Sarcophagidae), Chrysomya albiceps (Calliphoridae), and Musca domestica (Muscidae). To the best of our knowledge this is the first record of W. indigens larvae feeding on carrion. While adult C. albiceps and M. domestica were abundant on the carcasses, C. albiceps colonized too slowly to be useful for this type of succession analysis within this time frame, and M. domestica emerged from a single rat. The Sarcophagidae were rapid colonizers, and under these conditions the probability is>90% that a carcass would remain free of S. dux larvae not more than 57 h and free of W. nuba larvae for not more than 51 h.

A Forensic Entomological Analysis Can Yield an Estimate of Postmortem Interval, and Not Just a Minimum Postmortem Interval: An Explanation and Illustration Using a Case.

Several authors claimed that a forensic entomological analysis can never be interpreted as the postmortem interval (PMI), but that it can be interpreted as a minimum postmortem interval (PMImin ), or dead for not less than X amount of time. Because in most instances, a PMI estimate should be a range, that is, the set of values from maximum postmortem interval (PMImax ) to PMImin , objections to PMI estimation are objections to PMImax estimation. The arguments for this position did not address the substantial body of literature describing estimation of both PMImax and PMImin using insect succession analysis. This report reviews the scientific issues and presents a recent homicide investigation in which several forensic entomology experts used the absence of carrion insects on the corpse, a kind of succession analysis, to estimate PMI or PMImax .

Amplified fragment length polymorphism analysis supports the valid separate species status of Lucilia caesar and L. illustris (Diptera: Calliphoridae).

Common DNA-based species determination methods fail to distinguish some blow flies in the forensically and medically important genus Lucilia Robineau-Desvoidy. This is a practical problem, and it has also been interpreted as casting doubt on the validity of some morphologically defined species. An example is Lucilia illustris and L. caesar, which co-occur in Europe whilst only L. illustris has been collected in North America. Reports that these species shared both mitochondrial and nuclear gene sequences, along with claims that diagnostic morphological characters are difficult to interpret, were used to question their separate species status. We report here that amplified fragment length polymorphism profiles strongly support the validity of both species based on both assignment and phylogenetic analysis, and that traditional identification criteria based on male and female genital morphology are more reliable than has been claimed.

The forensically important blow fly, Chrysomya megacephala (Diptera: Calliphoridae), is more likely to walk than fly to carrion at low light levels.

One factor that influences estimates of time since death using entomological evidence is whether or not blow flies nocturnally oviposit. Field studies focusing on egg laying have found it occurs on an inconsistent basis. A key but poorly understood factor in nocturnal oviposition is a blow fly's ability to locate carrion under low light levels. It has been speculated that blow flies are more likely to walk than fly to carrion during the night, but this has not been empirically tested. We directly compared guided walking versus flying using infrared sensors under low light levels in laboratory conditions for Chrysomya megacephala (F.) (Diptera: Calliphoridae), a blow fly previously described to be nocturnal. We found C. megacephala is more likely to walk than fly toward carrion under low light levels (p=0.016). We did not, however, find differences between males and females for walking (p=0.48) or flying (p=0.42) despite male C. megacephala possessing eyes better suited for increased light capture. These results demonstrate the need to better understand where blow flies go at night, as bodies found within a fly's walking distance are more likely to be colonized.

A 454 sequencing approach to dipteran mitochondrial genome research.

The availability of complete mitochondrial genome (mtgenome) data for Diptera, one of the largest metazoan orders, in public databases is limited. The advent of high throughput sequencing technology provides the potential to generate mtgenomes for many species affordably and quickly. However, these technologies need to be validated for dipterans as the members of this clade play important economic and research roles. Illumina and 454 sequencing platforms are widely used in genomic research involving non-model organisms. The Illumina platform has already been utilized for generating mitochondrial genomes without using conventional long range PCR for insects whereas the power of 454 sequencing for generating mitochondrial genome drafts without PCR has not yet been validated for insects. Thus, this study examines the utility of 454 sequencing approach for dipteran mtgenomic research. We generated complete or nearly complete mitochondrial genomes for Cochliomyia hominivorax, Haematobia irritans, Phormia regina and Sarcophaga crassipalpis using a 454 sequencing approach. Comparisons between newly obtained and existing assemblies for C. hominivorax and H. irritans revealed no major discrepancies and verified the utility of 454 sequencing for dipteran mitochondrial genomes. We also report the complete mitochondrial sequences for two forensically important flies, P. regina and S. crassipalpis, which could be used to provide useful information to legal personnel. Comparative analyses revealed that dipterans follow similar codon usage and nucleotide biases that could be due to mutational and selection pressures. This study illustrates the utility of 454 sequencing to obtain complete mitochondrial genomes for dipterans without the aid of conventional molecular techniques such as PCR and cloning and validates this method of mtgenome sequencing in arthropods.

An evaluation of sampling methods used to produce insect growth models for postmortem interval estimation.

Many authors produced carrion insect development data for predicting the age of an insect from a corpse. Under some circumstances, this age value is a minimum postmortem interval. There are no standard protocols for such experiments, and the literature includes a variety of sampling methods. To our knowledge, there has been no investigation of how the choice of sampling method can be expected to influence the performance of the resulting predictive model. We calculated 95 % inverse prediction confidence limits for growth curves of the forensically important carrion flies Chrysomya megacephala and Sarconesia chlorogaster (Calliphoridae) at a constant temperature. Confidence limits constructed on data for entire age cohorts were considered to be the most realistic and were used to judge the effect of various subsampling schemes from the literature. Random subsamples yielded predictive models very similar to those of the complete data. Because taking genuinely random subsamples would require a great deal of effort, we imagine that it would be worthwhile only if the larval measurement technique were especially slow and/or expensive. However, although some authors claimed to use random samples, their published methods suggest otherwise. Subsampling the largest larvae produced a predictive model that performed poorly, with confidence intervals about an estimate of age being unjustifiably narrow and unlikely to contain the true age. We believe these results indicate that most forensic insect development studies should involve the measurement of entire age cohorts rather than subsamples of one or more cohorts.

Verification of AFLP kinship methods of entomological evidence by sequencing.

Kinship analysis allows the determination of sibship based on the individuals' genetic profile. In a recent empirical study, amplified fragment length polymorphism (AFLP) analysis was proposed as a test to determine kinship between Phormia regina individuals useful in inferring postmortem transport of a corpse. In order to validate this technique, mitochondrial DNA gene cytochrome oxidase II was sequenced for all individuals used in the previous study. Then, the relatedness coefficient based on AFLP profiles was determined for the pairs of individuals that had different haplotypes, and thus could not be full siblings, to determine a conservative false positive error rate of this proposed test. A majority, 96%, of pair wise comparisons of individuals with different haplotypes had relatedness coefficients <0.41 supporting the conclusion that AFLP analysis for full sibship is a valid and robust technique and thus useful for the detection of postmortem movement of a corpse.

Population genetic structure of an invasive forensically important insect.

The forensic utility of an insect can depend in part on its population structure. Although some native North American species have been characterized in this fashion, information is lacking for species that were introduced from elsewhere and that might have lower genetic diversity and less geographic differentiation. We surveyed Chrysomya megacephala, an Asian fly present in the continental USA since the 1980s. Amplified Fragment Length Polymorphism profiles were generated from adult insects collected across Florida and in Mobile, Alabama. Analysis of Molecular Variance on 151 polymorphic loci found significant but very small variation among samples. STRUCTURE and principal coordinate analyses produced the same two clusters in the population, consistent with C. megacephala in Florida having originated from two separate source populations. A weak negative correlation between genetic and geographic distances probably reflected the geographic arrangement of the genetic clusters. A positive relative relatedness coefficient for each sample indicated that flies arriving at a bait within a short time were likely to be close relatives, consistent with the earlier results for native North American carrion flies. However, genetic diversity estimated for the introduced Florida C. megacephala was lower than for native species or for published data on Malaysian C. megacephala, perhaps reflecting the genetic effects of being introduced to a new geographic region. Genetic assignment, a method that has been proposed as a way to infer corpse postmortem relocation, was much less successful for C. megacephala compared to the native species, possibly reflecting a history of admixture.

Evaluating the utility of hexapod species for calculating a confidence interval about a succession based postmortem interval estimate.

Carrion insect succession patterns have long been used to estimate the postmortem interval (PMI) during a death investigation. However, no published carrion succession study included sufficient replication to calculate a confidence interval about a PMI estimate based on occurrence data. We exposed 53 pig carcasses (16±2.5 kg), near the likely minimum needed for such statistical analysis, at a site in north-central Indiana, USA, over three consecutive summer seasons. Insects and Collembola were sampled daily from each carcass for a total of 14 days, by this time each was skeletonized. The criteria for judging a life stage of a given species to be potentially useful for succession-based PMI estimation were (1) nonreoccurrence (observed during a single period of presence on a corpse), and (2) found in a sufficiently large proportion of carcasses to support a PMI confidence interval. For this data set that proportion threshold is 45/53. Of the 266 species collected and identified, none was nonreoccuring in that each showed at least a gap of one day on a single carcass. If the definition of nonreoccurrence is relaxed to include such a single one-day gap the larval forms of Necrophilaamericana, Fanniascalaris, Cochliomyia macellaria, Phormiaregina, and Luciliaillustris satisfied these two criteria. Adults of Creophilus maxillosus, Necrobiaruficollis, and Necrodessurinamensis were common and showed only a few, single-day gaps in occurrence. C.maxillosus, P.regina, and L.illustris displayed exceptional forensic utility in that they were observed on every carcass. Although these observations were made at a single site during one season of the year, the species we found to be useful have large geographic ranges. We suggest that future carrion insect succession research focus only on a limited set of species with high potential forensic utility so as to reduce sample effort per carcass and thereby enable increased experimental replication.

First report of Blaesoxipha plinthopyga (Diptera: Sarcophagidae) from a human corpse in the U.S.A. and a new state geographic record based on specimen genotype.

Carrion flies in the taxonomic family Sarcophagidae are often recovered from a human corpse. However, because such specimens are difficult to identify, the forensic literature on this taxon is quite limited compared with that of the commonly employed Calliphoridae. Faced with a sarcophagid larva that could not be identified microscopically from a death investigation in the state of Idaho, we generated cytochrome oxidase one DNA sequence data from the specimen. Comparison to a reference data set of forensically significant sarcophagids from Canada and the U.S.A. confirmed that this was the first discovery of Blaesoxipha plinthopyga in a human corpse in the U.S.A. and the first record of this species in Idaho. Because B. plinthopyga occurs from the Northern U.S.A. to the Neotropics, it is potentially useful for estimating time since death at many locations.

Estimating postmortem interval using RNA degradation and morphological changes in tooth pulp.

The accurate determination of time since death, or postmortem interval (PMI), can be critical in the investigation of suspicious deaths. Knowing when a suspicious death occurred can limit the number of potential suspects to those without a viable alibi for the time of the crime. The forensic techniques currently employed to determine PMI: pathology, entomology, and anthropology, are accurate over different time periods following death. A large gap in time exists between the capabilities of forensic entomology and traditional anthropology, leaving a period in which PMI is difficult to estimate. In this study, time-dependent differences in RNA decay rates were examined to extend the time frame over which early PMI estimates can be made. Comparing the decay rates of a large, labile segment of ß-actin RNA and a smaller, more stable, non-overlapping segment of the same RNA from tooth pulp, we were able to estimate PMI values of pigs buried within a shallow grave for up to 84 days. This compares favorably to an estimate of PMI using insect data. Full skeletonization and loss of insect activity was observed by day 28 of our study. In addition to differences in RNA decay rates, morphological changes were observed in the pulp as it aged postmortem. To provide a quantitative measure of progressive color changes, analysis of digital photographs of each tooth's pulp were used to construct a simple colorimetric assay. This assay was then used to cluster ages of pulp samples by color. The two assays, used in combination with one another, can create a more precise estimate of PMI. The potential advantages of this molecular means of estimating PMI include extending the time frame for such estimates, is applicable to samples collected worldwide (no specialized knowledge of local insect fauna is required), is relatively fast, and inexpensive.

Molecular systematics of the Calliphoridae (Diptera: Oestroidea): evidence from one mitochondrial and three nuclear genes.

Approximately 8% of calyptrate species diversity comes from the Calliphoridae, which includes flies of medical, veterinary, and forensic importance. The status of family Calliphoridae has for years been the central systematic problem of the superfamily Oestroidea, and phylogenetic relationships between the key groups of the Calliphoridae are unresolved and controversial. We reconstructed phylogenies of the Calliphoridae within the larger context of the other Oestroidea based on 5,189 bp of combined data from one mitochondrial (cytochrome oxidase subunit one) and three nuclear (carbamoylphosphate synthetase, elongation factor one alpha, and 28S ribosomal RNA) genes using maximum parsimony, maximum likelihood, and Bayesian methods. Trees obtained from the different phylogenetic methods were almost identical. Calliphoridae is polyphyletic, with the phylogenetic position of Mesembrinellinae still uncertain but clearly outside the lineage that includes other Calliphoridae and some noncalliphorids, and Polleniinae is the sister group of the family Tachinidae. Strong support for a sister group relationship between Rhiniinae and traditional calliphorid subfamilies conflicts with a recent proposal to give Rhiniinae family status. All calliphorid subfamilies (except Calliphorinae) for which we had more than one species were monophyletic. Melanomyinae was nested within Calliphorinae. Toxotarsinae was more closely related to Calliphorinae rather than, as indicated by morphology, to Chrysomyinae. Efforts to resolve the relationships of the Oestroid families were largely inconclusive, although the monophyly of the superfamily was strongly supported.

A test for carrion fly full siblings: a tool for detecting postmortem relocation of a corpse.

We propose a genetic test for full sibship for a pair of carrion flies that could reveal the postmortem relocation of a corpse. A carrion fly larva is sometimes left behind when a corpse is moved. The discovery of full sibling larvae of approximately the same developmental stage at two locations would strongly suggest that a corpse was moved between those two sites. Distributions of pairwise comparisons of relatedness (R) coefficients were generated using amplified fragment length polymorphism profiles for nine samples of laboratory-generated full siblings as well as for a reference sample of nonfull sibling Phormia regina (Diptera: Calliphoridae). The mean relative R coefficient, a pairwise measure of the proportion of shared alleles, was 0.479 (±0.289 SD) for full siblings, close to the theoretical expectation of 0.5. A likelihood ratio (LR) test was based on observed distributions of R. R >0.55 corresponded to an LR >1000 favoring full sibship for that pair of individuals.

The population genetic structure of North American Lucilia sericata (Diptera: Calliphoridae), and the utility of genetic assignment methods for reconstruction of postmortem corpse relocation.

Lucilia sericata (Diptera: Calliphoridae) is a common urban blowfly, with a worldwide distribution. It is among the most important forensic insects, and a major veterinary pest. A previous amplified fragment length polymorphism (AFLP) survey of Phormia regina, another blowfly, found that the North American population structure results from the fact that flies trapped together at a bait are predominantly comprised of related individuals. We report here a similar population genetic pattern for L. sericata in North America based on AFLP genotypes with 249 loci. A STRUCTURE analysis found no population structure on a geographic scale, and analysis of molecular variance found a moderate amount of variation attributed to samples (adults collected at the same bait at the same time, Phi(SC)=20%, P=0.001). A Mantel test found a negligible correlation between geographic and genetic distances (R(2)=0.0063, P=0.02). The mean relative relatedness coefficient for every sample was positive (mean R=0.2486+/-0.18). Gravid females in a sample, those likely to oviposit on the same corpse, showed a pattern of relatively high relatedness similar to the total survey. Therefore, this pattern of local relatedness is likely to occur with larvae in a corpse, and if so it might support a genetic test for inferring the postmortem relocation of a corpse. This is because a larva may fall from the body at the original scene as it is moved. Connecting such a "stray" larva to the larval population in a corpse would provide powerful evidence that the corpse had been at both locations. Assignment tests resulted in a 96% success rate of assigning L. sericata individuals to their samples of origin.

DNA degradation and genetic analysis of empty puparia: genetic identification limits in forensic entomology.

Puparial cases are common remnants of necrophagous flies in crime investigations. They usually represent the longest developmental time and, therefore, they can be very useful for the estimation of the post-mortem interval (PMI). However, before any PMI estimate, it is crucial to identify the species of fly eclosed from each puparium associated with the corpse. Morphological characteristics of the puparium are often distinctive enough to permit a species identification. But, even an accurate morphological analysis of empty puparia cannot discriminate among different species of closely related flies. Furthermore, morphological identification may be impossible if the fly puparia are poorly preserved or in fragments. This study explores the applicability of biomolecular techniques on empty puparia and their fragments for identification purposes. A total of 63 empty puparia of necrophagous Diptera resulting from forensic casework were examined. Samples were divided into three groups according to size, type and time of eclosion in order to verify whether the physical characteristics and puparia weathering can influence the amount of DNA extraction. The results suggest that a reliable genetic identification of forensically important flies may also be performed from empty puparia and/or their fragments. However, DNA degradation can deeply compromise the genetic analysis since the older the fly puparia, the smaller are the amplified fragments.