Jeffrey D. Wells

Phylogenetic analysis of forensically important Lucilia flies based on cytochrome oxidase I sequence: a cautionary tale for forensic species determination

Forensic scientists are increasingly using DNA to identify the species of a tissue sample. However, little attention has been paid to basic experimental design issues such as replication and the selection of taxa when designing a species diagnostic test. We present an example using the forensically important fly genus Lucilia in which an increasingly larger sample size revealed that species diagnosis based on the commonly used cytochrome oxidase I gene (COI) was less straightforward than we initially thought. This locus may still be useful for diagnosing Lucilia specimens, but additional knowledge other than the genotype will be required to reduce the list of candidate species to include only forms that can be distinguished by COI. We believe that these results illustrate the importance of study design and biological knowledge of the study species when proposing a DNA-based identification test for any taxonomic group.

Estimating Maggot Age from Weight Using Inverse Prediction

Forensic entomological evidence is most often used to estimate the postmortem interval (PMI). Satisfactory techniques have not been available to quantify the precision of such a PMI estimate. For Cochliomyia macellaria (F.) (Diptera: Calliphoridae), we describe construction of a confidence interval on age of a larva, given its weight. The method requires a controlled experiment by which weights of larvae are observed at ages spread over sufficient range to cover the time from egg hatch up to postfeeding stage. A statistical model relating distributions of weights to age is formulated and fit to these data. We assumed a simple model in which both means and variances of weight distributions are linearly interpolated between sampled ages. The weight of a larva of unknown age is then compared to the fitted model via inverse prediction to compute the confidence interval on age of the larva.

Validation of a DNA-based method for identifying Chrysomyinae (Diptera: Calliphoridae) used in a death investigation

Many authors have proposed DNA-based methods for identifying an insect specimen associated with human remains. However, almost no attempt has been made to validate these methods using additional observations. We tested a protocol for identifying insects in the blow fly subfamily Chrysomyinae (Diptera: Calliphoridae) often found to be associated with a human corpse in Canada or the USA. This method uses phylogenetic analysis of DNA sequence from a short segment of the mitochondrial gene for cytochrome oxidase one (COI). Test chrysomyine COI sequences were obtained from 245 newly sequenced specimens and 51 specimens from the published literature. Published sequences from representatives of nonchrysomyine genera were also included to check for the possibility of a false positive identification. All of the chrysomyine test haplotypes were correctly identified with strong statistical support, and there were no false positives. This method appears to be an accurate and robust technique for identifying chrysomyine species from a death investigation in this geographic region. The far northern species Protophormia atriceps was not evaluated; therefore, caution is required in applying this method at very high latitudes in North America.

Molecular Systematics of the Calliphoridae (Diptera: Oestroidea): Evidence From One Mitochondrial and Three Nuclear Genes

ABSTRACT 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.

Mitochondrial DNA and STR analyses of maggot crop contents: effect of specimen preservation technique.

DNA analysis of maggot crop contents can be used to identify a missing body or aid entomologists with interpreting evidence used for PMI estimations. Entomological evidence is often collected and preserved to keep identifiable external features intact. The preservation methods currently in use may not be suitable for preserving DNA in the maggot crop for later analysis. In this study, carrion maggots raised on human tissue were preserved under the following 8 preservation conditions: no fluid at -70 degrees C, no fluid at 4 degrees C, no fluid at 24 degrees C, 70% ethanol at 4 degrees C, 70% ethanol at 24 degrees C, 95% ethanol at 24 degrees C, Kahles solution at 24 degrees C and formaldehyde at 24 degrees C. Maggots were dissected following 2 weeks, 8 weeks and 6 months of preservation. The maggot crops were extracted, human DNA was quantitated, and an attempt was made at amplifying mitochondrial DNA (mtDNA) and short tandem repeat (STR) loci. Both mtDNA and STRs were successfully amplified from maggots stored in ethanol or without any preservation fluid. Formalin-containing preservation solutions reduced the recovery of DNA. The best results were observed from maggots stored without any preservation fluid at -70 degrees C.

Survey of the Genetic Diversity of Phormia regina (Diptera: Calliphoridae) Using Amplified Fragment Length Polymorphisms

ABSTRACT There is very little information concerning carrion fly population genetic structure. We generated amplified fragment length polymorphism (AFLP) profiles for the common blowfly, Phormia regina (Meigen), from sites spanning the contiguous United States. Analysis of molecular variance (AMOVA) based on 232 loci found significant variation (&PHgr;SC = 23%) among discrete samples (those collected at a bait in one location over a short period of time). Samples collected in the same location but at different times were also distinct. When samples were pooled into geographic regions (east, central, west), the variation was negligible (&PHgr;CT = 0%). A Mantel test found only a very weak correlation between individual genetic and geographic distances. Relative relatedness coefficients based on shared allele proportions indicated individual samples were likely to contain close relatives. P. regina arriving at an individual carcass typically represent a nonrandom sample of the population despite a lack of geographic structure. A female blow fly produces hundreds of offspring at one time; therefore, newly emerged siblings may respond in concert to an odor plume. These results may be of interest to forensic entomologists, many of whom use a laboratory colony founded from a small sample for the growth studies that support casework. Discrepancies between published growth curves may reflect such random differences in the founding individuals.

p-values for postmortem intervals from arthropod succession data.

Two approaches, one based on the likelihood-ratio statistic and the other based on unconditioning Fishers exact test, are examined for obtaining a p-value in the comparison of the combination of arthropod species present on a mystery carcass to the observed frequency distribution of species combinations on carcasses exposed to the elements for a

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.

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 β-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 tooths 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.

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.