Isolation of the Male-Specific Transformer Exon as a Method for Immature Specimen Sex Identification in Chrysomya megacephala (Diptera: Calliphoridae).

Being able to efficiently differentiate between male and female individuals in the immature forms of insects allows for investigations into sexually dimorphic patterns of growth rates and gene expression. For species lacking sex-specific morphological characteristics during these periods, alternative methods must be devised. Commonly, isolation of sex determination genes reveals sex-specific band patterns and allows for markers that can be used in insect control. For blow flies, a family that includes flies of medical and forensic importance, sex has previously been identified in some members using the male-specific exon in the transformer gene. This gene is relatively conserved between members of the genera Cochliomyia and Lucilia (Diptera: Calliphoridae), and we isolated a portion of this gene in an additional forensically and medically important blow fly genus using the widespread Chrysomya megacephala (F.). We found a relatively high level of conservation between exons 1 and 2 of transformer and were able to amplify a region containing the male-specific exon in C. megacephala. A sex-specific molecular diagnostic test based on the presence of sexually dimorphic PCR product bands showed the expected genotype for adults and intrapuparial period specimens of known sex. The same result could be obtained from single third-instar larval specimens, opening up the possibility to not only determine if development rates are sex dependent, but also to investigate the development of sexually dimorphic traits of interest in C. megacephala.

Body Size, Rather Than Male Eye Allometry, Explains Chrysomya megacephala (Diptera: Calliphoridae) Activity in Low Light.

Male Chrysomya megacephala (F.) blow fly compound eyes contain an unusual area of enlarged dorsal facets believed to allow for increased light capture. This region is absent in females and has been hypothesized to aid in mate tracking in low light conditions or at greater distances. Many traits used in the attraction and capture of mates are allometric, growing at different rates relative to body size. Previous reports concerning C. megacephala eye properties did not include measurements of body size, making the relationship between the specialized eye region and body size unclear. We examined different morphological features of the eye among individuals of varying sizes. We found total eye size scaled proportionately to body size, but the number of enlarged dorsal facets increased as body size increased. This demonstrated that larger males have an eye that is morphologically different than smaller males. On the basis of external morphology, we hypothesized that since larger males have larger and a greater number of dorsally enlarged facets, and these facets are believed to allow for increased light capture, larger males would be active in lower light levels than smaller males and females of equal size. In a laboratory setting, larger males were observed to become active earlier in the morning than smaller males, although they did not remain active later in the evening. However, females followed the same pattern at similar light levels suggesting that overall body size rather than specialized male eye morphology is responsible for increased activity under low light conditions.

Amplified fragment length polymorphism confirms reciprocal monophyly in Chrysomya putoria and Chrysomya chloropyga: a correction of reported shared mtDNA haplotypes.

Reinvestigation of mitochondrial haplotypes previously reported to be shared between the Afrotropical blowflies Chrysomya putoria Weidemann and Chrysomya chloropyga Weidemann (Diptera: Calliphoridae) revealed an error resulting from the misidentification of specimens. Preliminary amplified fragment length polymorphism (AFLP) analysis of the original and additional individuals again failed to find reciprocal monophyly, leading to a re-examination of the specimens for diagnostic male genitalic characters that were first described following the earlier study. Four of the original study specimens were found to have been misidentified, and definitive analysis of both mtDNA and AFLP genotypes using phylogenetic analysis and genetic assignment showed that each species was indeed reciprocally monophyletic. In addition to correcting the earlier error, this study illustrates how AFLP analysis can be used for efficient and effective specimen identification through both phylogenetic analysis and genetic assignment, and suggests that the latter method has special advantages for identification when no conspecific specimens are represented in the reference database.

Molecular phylogeny of the blowfly genus Chrysomya.

Chrysomya Robineau-Desvoidy (Diptera: Calliphoridae) is a genus of blowfly commonly observed in tropical and subtropical countries of the Old World. Species in this genus are vectors of bacteria, protozoans and helminths, cause myiasis, are predators of other carrion insects, and are important forensic indicators. Hypotheses concerning the evolution of sex determination, larval anatomy and genome size in Chrysomya have been difficult to evaluate because a robust phylogeny of the genus was lacking. Similarly, the monophyly of subgenera was uncertain. The phylogeny of Chrysomya spp. was reconstructed based on 2386 bp of combined mitochondrial cytochrome oxidase subunit I (COI) and nuclear carbamoylphosphate synthetase (CPS) genes. Maximum parsimony (MP), maximum likelihood (ML) and Bayesian analysis (BA) differed only slightly in the resulting tree topology. Chrysomya was monophyletic. Monogenic reproduction is almost certainly derived rather than, as has been suggested, primitive within the genus, and tuberculate larvae probably evolved twice. Genome size is more likely to have decreased over evolutionary time rather than, as has been suggested, increased within the genus, but its correlation with developmental time was not observed. The subgenera Microcalliphora, Eucompsomyia and Achoetandrus were recovered as monophyletic.

Survey of the genetic diversity of Phormia regina (Diptera: Calliphoridae) using amplified fragment length polymorphisms.

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 (phi(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 (phi(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.

Influence of an injury reduction program on injury and fitness outcomes among soldiers.

OBJECTIVE: This study evaluated the influence of a multiple injury control intervention on injury and physical fitness outcomes among soldiers attending United States Army Ordnance School Advanced Individual Training. METHODS: The study design was quasiexperimental involving a historical control group (n = 2559) that was compared to a multiple intervention group (n = 1283). Interventions in the multiple intervention group included modified physical training, injury education, and a unit based injury surveillance system (UBISS). The management responsible for training independently formed an Injury Control Advisory Committee that examined surveillance reports from the UBISS and recommended changes to training. On arrival at school, individual soldiers completed a demographics and lifestyle questionnaire and took an army physical fitness test (APFT: push-ups, sit-ups, and two mile run). Injuries among soldiers were tracked by a clinic based injury surveillance system that was separate from the UBISS. Soldiers completed a final APFT eight weeks after arrival at school. RESULTS: Cox regression (survival analysis) was used to examine differences in time to the first injury while controlling for group differences in demographics, lifestyle characteristics, and physical fitness. The adjusted relative risk of a time loss injury was 1.5 (95% confidence interval 1.2 to 1.8) times higher in the historical control men and 1.8 (95% confidence interval 1.1 to 2.8) times higher in the historical control women compared with the multiple intervention men and women, respectively. After correcting for the lower initial fitness of the multiple intervention group, there were no significant differences between the multiple intervention and historical control groups in terms of improvements in push-ups, sit-ups, or two mile run performance. CONCLUSIONS: This multiple intervention program contributed to a reduction in injuries while improvements in physical fitness were similar to a traditional physical training program previously used at the school.

Recent African derivation of Chrysomya putoria from C. chloropyga and mitochondrial DNA paraphyly of cytochrome oxidase subunit one in blowflies of forensic importance.

Chrysomya chloropyga (Wiedemann) and C. putoria (Wiedemann) (Diptera: Calliphoridae) are closely related Afrotropical blowflies that breed in carrion and latrines, reaching high density in association with humans and spreading to other continents. In some cases of human death, Chyrsomya specimens provide forensic clues. Because the immature stages of such flies are often difficult to identify taxonomically, it is useful to develop DNA-based tests for specimen identification. Therefore we attempted to distinguish between C. chloropyga and C. putoria using mitochondrial DNA (mtDNA) sequence data from a 593-bp region of the gene for cytochrome oxidase subunit one (COI). Twelve specimens from each species yielded a total of five haplotypes, none being unique to C. putoria. Therefore it was not possible to distinguish between the two species using this locus. Maximum parsimony analysis indicated paraphyletic C. chloropyga mtDNA with C. putoria nested therein. Based on these and previously published data, we infer that C. putoria diverged very recently from C. chloropyga.

Paraphyly in Hawaiian hybrid blowfly populations and the evolutionary history of anthropophilic species.

Complementary nuclear (28S rRNA) and mitochondrial (COI + II) gene markers were sequenced from the blowflies, Lucilia cuprina and Lucilia sericata, from Europe, Africa, North America, Australasia and Hawaii. Populations of the two species were phylogenetically distinct at both genes, with one exception. Hawaiian L. cuprina possessed typical L. cuprina-type rRNA, but had L. sericata-type mitochondrial (COI + II) sequences. An explanation for this pattern is that Hawaiian flies are hybrids and comparison of observed levels of sequence divergence to possible introduction events, e.g. Polynesian colonization, suggests that Hawaiian L. cuprina may be evolving rapidly. Moreover, the monophyly of these flies also suggests that the L. sericata mtDNA haplotype was apparently fixed in Hawaiian L. cuprina by lineage sorting, indicating a population bottleneck in the evolutionary history of these island flies.

DNA-based identification and molecular systematics of forensically important Sarcophagidae (Diptera).

Sarcophagid flies have many characteristics that make them ideal forensic indicators. However, their utility is severely limited because it is difficult or impossible to determine the species of a sarcophagid larva, and in many instances an adult specimen, based on anatomy. We developed a database of mitochondrial DNA sequence data that makes it possible to identify all sarcophagid species likely to be found feeding on a human corpse at an urban location in Canada or the USA. Analyses were based on a 783 base pair region of the gene for cytochrome oxidase subunit one (COI). The species analyzed, including some of no forensic importance that were included for purposes of phylogenetic comparisons, were members of the genera Sarcophaga, Peckia, Blaesoxipha, Rovinia, Wohlfahrtia, Brachicoma (all Sarcophagidae), and Musca (Muscidae).

DNA-based identification of forensically important Chrysomyinae (Diptera: Calliphoridae).

Identifying an insect specimen is an important first step in a forensic-entomological analysis. However, diagnostic morphological criteria are lacking for many species and life stages. We demonstrate a method for using mitochondrial DNA sequence data and phylogenetic analysis to identify any specimen of the blow fly subfamily Chrysomyinae likely to be collected from a human corpse within Canada or the USA. The reliability of the method was illustrated by analyzing specimens designed to mimic the information likely to be obtained from highly degraded specimens as well as specimens collected from widely separated geographic locations. Our sequence database may be suitable for another country provided the investigator knows the local fly fauna well enough to narrow the choice of chrysomyine species to those used in this study.

Human and insect mitochondrial DNA analysis from maggots.

During the course of our forensic investigations, we have encountered situations where it would have been useful to have evidence, other than direct contact between the two, for concluding that a carrion-fly maggot developed on a particular human victim. If a maggot collected during a death investigation did not develop on the victim, then its age is not relevant to estimating the postmortem interval. In this study we demonstrate that mitochondrial DNA (mtDNA) sequence data can be obtained from the dissected gut of a maggot that had fed on human tissue. These data can be used to identify both the human corpse upon which the maggot had been feeding and the species of the maggot itself.

Porous calcium polyphosphate scaffolds for bone substitute applications -- in vitro characterization.

Porous structures were formed by gravity sintering calcium polyphosphate (CPP) particles of either 106-150 or 150-250 microm size to form samples with 30-45 vol% porosity with pore sizes in the range of 100 microm (40-140 microm). Tensile strength of the samples assessed by diametral compression testing indicated relatively high values for porous ceramics with a maximum strength of 24.1 MPa for samples made using the finer particles (106-150 microm). X-ray diffraction studies of the sintered samples indicated the formation of beta-CPP from the starting amorphous powders. In vitro aging in 0.1 M tris-buffered solution (pH 7.4) or 0.05 M potassium hydrogen phthalate buffered solution (pH 4.0) at 37 degreesC for periods up to 30d indicated an initial rapid loss of strength and P elution by 1 d followed by a more gradual continuing strength and P loss resulting in strengths at 30d equal to about one-third the initial value. The observed structures, strengths and in vitro degradation characteristics of the porous CPP samples suggested their potential usefulness as bone substitute materials pending subsequent in vivo behaviour assessment.

Key to third-instar chrysomyinae (Diptera: Calliphoridae) from carrion in the continental United States.

The introduction of 4 Chrysomya Robineau-Desvoidy spp. to the Americas has made obsolete previously published keys to Nearctic calliphorid larvae, particularly those covering the subfamily Chrysomyinae. To assist forensic entomologists, ecologists, and public health workers, we provide a key to 3rd instars of 8 chrysomyine species reported from or likely to occur in carrion within the continental United States. The rare (in the United States) species Cochliomyia aldrichi Del Ponte, C. minima Shannon, and Chloroprocta idioidea (Robineau-Desvoidy) are not included because specimens and suitable descriptions were unavailable.

Molecular phylogeny of Chrysomya albiceps and C. rufifacies (Diptera: Calliphoridae).

Mitochondrial DNA was used to infer the phylogeny and genetic divergences of Chrysomya albiceps (Wiedemann) and C. rufifacies (Maquart) specimens from widely separated localities in the Old and New World. Analyses based on a 2.3-kb region including the genes for cytochrome oxidase subunits I and II indicated that the 2 species were separate monophyletic lineages that have been separated for > 1 million years. Analysis of DNA, in the form of either sequence or restriction fragment-length polymorphism (RFLP) data, will permit the identification of problematic specimens.

Human neutrophils express the prostaglandin G/H synthase 2 gene when stimulated with bacterial lipopolysaccharide.

Human blood neutrophils (PMN) rapidly release arachidonic acid (AA) from cellular phospholipids when stimulated in vitro with a variety of inflammatory agonists. Free AA is then metabolized via 5'-lipoxygenase to produce bioactive mediators such as leukotriene B4 and 5-hydroxyeicosatetraenoate. Arachidonic acid can also be metabolized via the cyclooxygenase or prostaglandin G/H synthase (PGHS) pathway to form prostaglandins and thromboxane. We show here that human blood PMN express the PGHS 2 gene when stimulated with bacterial lipopolysaccharide (LPS). PGHS 2 mRNA increases within 30 min after LPS stimulation and PGHS 2 immunoreactive protein is detectable by 5 h. Although PGHS 1 mRNA is detectable in PMN, no immunoreactive protein is observed in either resting or LPS-stimulated cells. Following stimulation with LPS and expression of PGHS 2, PMN increase secretion of prostaglandin E2. This phenotypic change in PMN could be an important mechanism for regulating inflammation.

Forensic use of Megaselia abdita and M. scalaris (Phoridae: Diptera): case studies, development rates, and egg structure.

Megaselia abdita Schmitz is reported from human corpses in 2 forensic cases in Chicago, IL. This fly has not been recorded in the Nearctic region before these occurrences. Descriptions of the death scenes and the insect indicators are given, as are developmental schedules and descriptions of the eggs of M. abdita and Megaselia scalaris (Loew). The presence of either fly on the body, together with blowflies or other insects, could contribute to a determination of the postmortem interval. Given their predilection for older carrion, these flies would be relegated to a secondary forensic role. Phoridae have been shown to be primary flies in Okinawa and Panama, and they may occur elsewhere as the only insect evidence, especially in sealed apartments inaccessible to large insects.

Interleukin-1beta expression after inhibition of protein phosphatases in endotoxin-tolerant cells.

Endotoxin (lipopolysaccharide [LPS]) is a potent activator of a number of inflammatory genes in blood leukocytes, including interleukin-1 (IL-1). Blood leukocytes isolated from patients with septic shock fail to produce IL-1 in response to LPS, a phenomenon known as endotoxin tolerance. To study the regulation of IL-1 expression in endotoxin-tolerant cells, the protein phosphatase inhibitor okadaic acid was used to examine the effects of protein phosphorylation on IL-1beta gene expression. We found that endotoxin-tolerant cells produced normal levels of IL-1beta when protein phosphatases were inhibited. In the human pro-monocytic cell line THP-1, okadaic acid increased mRNA accumulation and synthesis of IL-1beta protein. Normal and endotoxin-tolerant THP-1 cells accumulated IL-1beta mRNA and protein with similar delayed kinetics. Okadaic acid stabilization of IL-1beta mRNA appears to be the primary mechanism through which endotoxin-tolerant cells accumulate IL-1beta mRNA and protein. Endotoxin-tolerant cells were unable to activate transcription in response to okadaic acid. However, the transcription factor NF-kappaB, which is known to be involved in IL-1beta expression, was translocated to the nucleus in both normal and endotoxin-tolerant cells after treatment with okadaic acid. These studies revealed that protein phosphorylation can affect gene expression on at least two distinct levels, transcription factor activation and mRNA stability. Endotoxin-tolerant cells have decreased transcription activation potential, while IL-1beta mRNA stability remains responsive to protein phosphorylation.

Ki-ras mutation and p53 overexpression predict the clinical behavior of colorectal cancer: a Southwest Oncology Group study.

We assessed Ki-ras mutations by single-strand conformation polymorphism followed by DNA sequencing, p53 expression by immunohistochemistry, ploidy status, and S-phase fraction in 66 stage II and 163 stage III colon cancer patients enrolled on a randomized trial of surgery followed by observation or adjuvant levamisole or 5-fluorouracil (5FU) plus levamisole (Intergroup Trial 0035) to see whether these factors were independently associated with survival or with differential effects of adjuvant therapy. A Cox proportional hazards survival model was used to describe marker effects and therapy by marker interactions, with adjustment for the clinical covariates affecting survival. A Bonferroni adjustment was used to account for multiple testing. Mutation of the Ki-ras gene was found in 41% of the cancers and was associated with a poor prognosis in stage II but not stage III. In stage II, 7-year survival was 86% versus 58% in those with wild type versus Ki-ras mutations. After adjustment for treatment and clinical variables, the hazard ratio (HR) for death was 4.5; 95% confidence interval (CI), 1.7-12.1 (P = 0.012). p53 overexpression was found in 63% of cancers and was associated with a favorable survival in stage III but not stage II. Seven-year survival in stage III was 56% with p53 overexpression versus 43% with no p53 expression (HR, 2.2; 95% CI, 1.3-3.6; P = 0.012). Aneuploidy was more common in stage III than in stage II (66 versus 47%; P = 0.009) but was not independently related to survival in either group. The proliferative rate was greater in aneuploid than in diploid cancers but was not related to survival. There was no benefit of adjuvant therapy in stage II nor in any of the stage II subgroups defined by mutational status. In stage III, adjuvant therapy with 5FU plus levamisole improved 7-year survival in patients with wild-type Ki-ras (76 versus 44%; HR, 0.4; 95% CI, 0.2-0.8) and in those without p53 overexpression (64 versus 26%; HR, 0.3; 95% CI, 0.1-0.7). Adjuvant therapy did not benefit those with Ki-ras mutations or p53 overexpression. The effects of adjuvant therapy did not differ according to ploidy status or proliferative rate. Ki-ras mutation is a significant risk factor for death in stage II, and the absence of p53 expression is a significant risk factor for death in stage III colon cancer after adjustment for treatment and clinical covariates. Exploratory analyses suggest that patients with stage III colon cancer with wild-type Ki-ras or no p53 expression benefit from adjuvant 5FU plus levamisole, whereas those with Ki-ras mutations or p53 overexpression do not. An independent study will be required to determine whether response to adjuvant therapy in colon cancer depends on mutational status.