Trevor Stamper

mtDNA-based identification of Lucilia cuprina (Wiedemann) and Lucilia sericata (Meigen) (Diptera: Calliphoridae) in the continental United States.

Existing data suggest that the forensically important dipteran species Lucilia cuprina (Wiedemann) and Lucilia sericata (Meigen) may be particularly difficult to discriminate using DNA sequence data. L. cuprina is paraphyletic with respect to L. sericata in mtDNA phylogenies, with some L. cuprina having mtDNA haplotypes that are very similar to those of L. sericata. We examine this problem by providing the first DNA data for L. cuprina from North America, including portions of both the mitochondrial COI gene and the nuclear 28S rRNA gene. With the new data, L. cuprina remains monophyletic for 28S but paraphyletic with respect to L. sericata for COI. However, we find that all flies that are identified as L. cuprina by morphology and have L. sericata-like mtDNA form a distinctly monophyletic mtDNA clade. This clade may possibly have originated by hybridization between L. cuprina and L. sericata, but its wide geographic distribution strongly suggests a singular origin as opposed to repeated incidents of hybridization. The phylogenetic results strongly support the hypothesis that L. cuprina and L. sericata can be discriminated using mtDNA sequence data. We find that a fragment of COI spanning approximately 1200 base pairs is sufficient to discriminate between the two species with greater than 95% bootstrap support.

DNA‐Based Identification of Forensically Important Lucilia (Diptera: Calliphoridae) in the Continental United States

Abstract:  Correct species identification is critical when dipteran larvae are used for inference of the postmortem interval. To facilitate DNA‐based identification of forensically important flies of the genus Lucilia in the continental United States, we develop a vouchered reference collection and DNA sequence database. A total of 122 specimens were collected for nine of the 10 species of Lucilia reported to occur in the continental United States. Using the polymerase chain reaction and DNA sequencing, data were obtained for an 1100‐bp region of the mitochondrial gene encoding cytochrome oxidase I (COI). We consider a species suitable for DNA‐based identification if it is exclusively monophyletic in >95% of bootstrap pseudoreplicate phylogenetic analyses. Seven of the nine species meet that criterion. Two species (Lucilia coeruleiviridis and Lucilia mexicana) share COI sequence and cannot be distinguished using our reference database. We conclude that DNA‐based identification is likely to be successful for the other seven species.

Phylogenetic relationships of flesh flies in the subfamily Sarcophaginae based on three mtDNA fragments (Diptera: Sarcophagidae)

In an effort to improve our knowledge of the phylogenetic relationships among species and genera of the subfamily Sarcophaginae, we analysed data from three mitochondrial gene fragments. Sequence data for portions of the genes cytochrome oxidase I (COI), cytochrome oxidase II (COII) and dehydrogenase subunit 4 (ND4) were obtained from 43 species of Sarcophagidae representing 15 genera. We used a Bayesian approach to simultaneously choose how best to partition the data and which substitution model to apply to each partition. Phylogenetic relationships were inferred using Bayesian Inference and Maximum Likelihood methods. Our results are consistent with monophyly of the subfamily Sarcophaginae (posterior probability 1; bootstrap support 93%), as well as with monophyly of several genera within the Sarcophaginae (including Sarcophaga s.l.; posterior probability 1; bootstrap support 97%). We found support for a sister‐group relationship between Ravinia Robineau‐Desvoidy and Oxysarcodexia Townsend, which has been hypothesised by past authors on the basis of morphological similarities, although this was supported only in the Bayesian analyses (posterior probability 0. 81–0. 98), and for some novel supra‐generic clades. Contrary to a recent morphological hypothesis, we do not find Helicobia Coquillett to be nested within Sarcophaga Meigen; our data suggest, but do not strongly support, a hypothesis that Peckia Robineau‐Desvoidy is the sister group to Sarcophaga.

The Nocturnal Ovipositing Behavior of Carrion Flies in Cincinnati, Ohio

Abstract:  The behavioral patterns of nocturnal oviposition represent a window of time that potentially has a large impact on postmortem interval estimations. We investigated the behavioral patterns of carrion flies at night by exposing euthanized rats between sunset and sunrise to see if carrion flies oviposited upon the carrion over two consecutive summers. We investigated urban and rural locations, in both lit and unlit conditions with n = 125. We found that nocturnal ovipositing did not occur in the Cincinnati metropolitan area. We conclude that nocturnal oviposition is an unlikely event in the Cincinnati metropolitan area.

The Nocturnal Oviposition Behavior of Carrion Flies in Rural and Urban Environments: Methodological Problems and Forensic Implications

ABSTRACT Unequivocal identification of the time at which larvae are deposited upon a carcass is essential for applying maggots as estimators of the post-mortem interval of that carcass. However, there is controversy as to whether flies oviposit at night or not. Nocturnal exposure of rat carcasses at three sites in the Cincinnati metropolitan region was used to examine this behavior for two field seasons, 2005 and 2006. In the 2005 field season, nocturnally exposed bait rats were observed to harbor live larvae following a maturation period, but all of the positive results were discovered to have resulted from post-exposure contamination. In the 2006 field season several methods were changed to correct for contamination and in that field season, nocturnal ovipositing was not found to occur.

An Efficient Technique for Primer Development and Application that Integrates Fluorescent Labeling and Multiplex PCR

Premise of the study: Development of genetic markers can be costly and time-consuming, especially when multiple primer pairs are fluorescently labeled. This step was streamlined by combining two techniques in the same PCR reaction: (1) custom-labeling of primers by the investigator and (2) multiplexing multiple primers together in the same reaction. Methods and Results: This technique was successfully used to develop microsatellite markers in several plant species. Microsatellites amplified with this multiplexing process were identical to those generated from PCR using individual primer pairs and with traditional methods using a priori labeled fluorescent primers. Tests of PCR cycling programs revealed that conditions recommended for the commercial kit generated stronger fragment peaks than the previously recommended cycling protocol. Conclusions: This technique is an efficient and economical way to fluorescently label multiple microsatellite primers in the same reaction. It is also applicable to other markers used in PCR amplification of genetic material.

Discordance between morphological species identification and mtDNA phylogeny in the flesh fly genus Ravinia (Diptera : Sarcophagidae)

Abstract. In order to better understand the phylogenetic relationships among species in the genus Ravinia Robineau-Desvoidy, 1863, we analysed data from two mitochondrial gene fragments: cytochrome oxidase I (COI) and cytochrome oxidase II (COII). We used Bayesian inference and maximum likelihood methods to infer phylogenetic relationships. Our results indicate that the genera Ravinia and Chaetoravinia, previously synonymised into the genus Ravinia (sensu lato) are each likely to be monophyletic (posterior probability 1; bootstrap support 85%). We found highly supported paraphyletic relationships among species of Ravinia, with relatively deep splits in the phylogeny. This conflict between the morphological species definitions and the mtDNA phylogeny could be indicative of the presence of cryptic species in Ravinia anxia (Walker, 1849), Ravinia floridensis (Aldrich, 1916), Ravinia lherminieri (Robineau-Desvoidy, 1830), and Ravinia querula (Walker, 1849).

Invading from the garden? A comparison of leaf herbivory for exotic and native plants in natural and ornamental settings

Abstract  The enemies release hypothesis proposes that exotic species can become invasive by escaping from predators and parasites in their novel environment. Agrawal et al. (Enemy release? An experiment with congeneric plant pairs and diverse above‐ and below‐ground enemies. Ecology, 86, 2979–2989) proposed that areas or times in which damage to introduced species is low provide opportunities for the invasion of native habitat. We tested whether ornamental settings may provide areas with low levels of herbivory for trees and shrubs, potentially facilitating invasion success. First, we compared levels of leaf herbivory among native and exotic species in ornamental and natural settings in Cincinnati, Ohio, United States. In the second study, we compared levels of herbivory for invasive and noninvasive exotic species between natural and ornamental settings. We found lower levels of leaf damage for exotic species than for native species; however, we found no differences in the amount of leaf damage suffered in ornamental or natural settings. Our results do not provide any evidence that ornamental settings afford additional release from herbivory for exotic plant species.

Post-Colonization Interval Estimates Using Multi-Species Calliphoridae Larval Masses and Spatially Distinct Temperature Data Sets: A Case Study

Common forensic entomology practice has been to collect the largest Diptera larvae from a scene and use published developmental data, with temperature data from the nearest weather station, to estimate larval development time and post-colonization intervals (PCIs). To evaluate the accuracy of PCI estimates among Calliphoridae species and spatially distinct temperature sources, larval communities and ambient air temperature were collected at replicate swine carcasses (N = 6) throughout decomposition. Expected accumulated degree hours (ADH) associated with Cochliomyia macellaria and Phormia regina third instars (presence and length) were calculated using published developmental data sets. Actual ADH ranges were calculated using temperatures recorded from multiple sources at varying distances (0.90 m–7.61 km) from the study carcasses: individual temperature loggers at each carcass, a local weather station, and a regional weather station. Third instars greatly varied in length and abundance. The expected ADH range for each species successfully encompassed the average actual ADH for each temperature source, but overall under-represented the range. For both calliphorid species, weather station data were associated with more accurate PCI estimates than temperature loggers associated with each carcass. These results provide an important step towards improving entomological evidence collection and analysis techniques, and developing forensic error rates.

Validating sonication as a DNA extraction method for use with carrion flies

Entomological evidence can be critical in establishing a postmortem interval estimate. DNA-based species identification can be an extremely valuable tool for forensic entomology. The problem of processing samples in a consistent, cost-effective manner that retains the morphological attributes of the specimen for vouchering has led us to investigate sonication as a primary means of non-destructive DNA extraction from carrion flies. We analyze the efficacy of this technique and compare it to an established DNA extraction technique - the Qiagen DNeasy tissue kit. Our results indicate that sonication produces a significant reduction in the sequence length and lower PHRED quality scores when compared to sequences using DNA obtained using the DNeasy kit, but species identification and phylogenetic inferences between sonication and DNeasy extractions are equivalent.