Age estimation during the blow fly intra-puparial period: a qualitative and quantitative approach using micro-computed tomography.
ABSTRACT: Minimum post-mortem interval (minPMI) estimates often rely on the use of developmental data from blow flies (Diptera: Calliphoridae), which are generally the first colonisers of cadavers and, therefore, exemplar forensic indicators. Developmental data of the intra-puparial period are of particular importance, as it can account for more than half of the developmental duration of the blow fly life cycle. During this period, the insect undergoes metamorphosis inside the opaque, barrel-shaped puparium, formed by the hardening and darkening of the third instar larval cuticle, which shows virtually no external changes until adult emergence. Regrettably, estimates based on the intra-puparial period are severely limited due to the lack of reliable, non-destructive ageing methods and are frequently based solely on qualitative developmental markers. In this study, we use non-destructive micro-computed tomography (micro-CT) for (i) performing qualitative and quantitative analyses of the morphological changes taking place during the intra-puparial period of two forensically relevant blow fly species, Calliphora vicina and Lucilia sericata, and (ii) developing a novel and reliable method for estimating insect age in forensic practice. We show that micro-CT provides age-diagnostic qualitative characters for most 10% time intervals of the total intra-puparial period, which can be used over a range of temperatures and with a resolution comparable to more invasive and time-consuming traditional imaging techniques. Moreover, micro-CT can be used to yield a quantitative measure of the development of selected organ systems to be used in combination with qualitative markers. Our results confirm micro-CT as an emerging, powerful tool in medico-legal investigations.
Project description:Metamorphosis and, in particular, holometaboly, the development of organisms through a series of discrete stages (egg, larva, pupa, adult) that hardly resemble one another but are finely adapted to specific roles in the life cycle of the organism, has fascinated and mystified humans throughout history. However, it can be difficult to visualize the dramatic changes that occur during holometaboly without destructive sampling, traditionally through histology. However, advances in imaging technologies developed mainly for medical sciences have been applied to studies of insect metamorphosis over the past couple of decades. These include micro-computed tomography, magnetic resonance imaging and optical coherence tomography. A major advantage of these techniques is that they are rapid and non-destructive, enabling virtual dissection of an organism in any plane by anyone who has access to the image files and the necessary software. They can also be applied in some cases to visualize metamorphosis in vivo, including the periods of most rapid and dramatic morphological change. This review focusses on visualizing the intra-puparial holometabolous metamorphosis of cyclorraphous flies (Diptera), including the primary model organism for all genetic investigations, Drosophila melanogaster, and the blow flies of medical, veterinary and forensic importance, but also discusses similar studies on other insect orders. This article is part of the theme issue 'The evolution of complete metamorphosis'.
Project description:Despite widely accepted standards for sampling and preservation of insect evidence, unrepresentative samples or improperly preserved evidence are encountered frequently in forensic investigations. Here, we report the results of laboratory studies on the survival of Lucilia sericata and Calliphora vomitoria (Diptera: Calliphoridae) intra-puparial forms in hermetic containers, which were stimulated by a recent case. It is demonstrated that the survival of blowfly intra-puparial forms inside airtight containers is dependent on container volume, number of puparia inside, and their age. The survival in both species was found to increase with an increase in the volume of air per 1 mg of puparium per day of development in a hermetic container. Below 0.05 ml of air, no insect survived, and above 0.2 ml of air per 1 mg of puparium per day, survival reached its maximum. These results suggest that blowflies reveal a single, general pattern of survival under decreasing oxygen conditions and that this pattern is a product of number of developing insects, their age and the initial amount of available air. Implications for forensic entomology are discussed.
Project description:Blow flies are worldwide the most important insects from a forensic point of view. In Thailand, aside from the two most common species, Chrysomya megacephala (F.) and Chrysomya rufifacies (Macquart), Chrysomya chani Kurahashi was also found to be of forensic importance. We present a case of a human female cadaver in its bloated stage of decomposition, discovered at Pachangnoi Subdistrict, northern Thailand. Entomological sampling during the autopsy displayed an assemblage of numerous dipteran larvae. Macroscopic observations showed the coexistence of third instar larvae of the three blow flies C. megacephala, Chrysomya villeneuvi Patton, an unknown blow fly species and one muscid, Hydrotaea sp. The minimum post-mortem interval was estimated to be six days, based on the developmental rate of C. megacephala. The ID of the unknown larva, which is the focus of this report, was revealed later as C. chani by DNA sequencing, using a 1205 bp of cytochrome c oxidase subunit I (COI). The occurrence of C. chani on a human body revealed the need to analyse and describe the morphology of its immature stage, to enable forensic entomologists to identify this fly species in future cases. The morphological examination of the third instar was performed, revealing peculiar characteristics: protuberant tubercles encircling abdominal segments; 9-11 lobes on the anterior spiracle; six prominent pairs of tubercles along the peripheral rim of the eighth abdominal segment; a heavily sclerotized complete peritreme of the posterior spiracles. A key to differentiate the third instar of blow flies of forensic importance in Thailand is provided.
Project description:Blow flies are the first insect group to colonize on a dead body and thus correct species identification is a crucial step in forensic investigations for estimating the minimum postmortem interval, as developmental times are species-specific. Due to the difficulty of traditional morphology-based identification such as the morphological similarity of closely related species and uncovered taxonomic keys for all developmental stages, DNA-based identification has been increasing in interest, especially in high biodiversity areas such as Thailand. In this study, the effectiveness of long mitochondrial cytochrome c oxidase subunit I and II (COI and COII) sequences (1247 and 635 bp, respectively) in identifying 16 species of forensically relevant blow flies in Thailand (Chrysomya bezziana, Chrysomya chani, Chrysomya megacephala, Chrysomya nigripes, Chrysomya pinguis, Chrysomya rufifacies, Chrysomya thanomthini, Chrysomya villeneuvi, Lucilia cuprina, Lucilia papuensis, Lucilia porphyrina, Lucilia sinensis, Hemipyrellia ligurriens, Hemipyrellia pulchra, Hypopygiopsis infumata, and Hypopygiopsis tumrasvini) was assessed using distance-based (Kimura two-parameter distances based on Best Match, Best Close Match, and All Species Barcodes criteria) and tree-based (grouping taxa by sequence similarity in the neighbor-joining tree) methods. Analyses of the obtained sequence data demonstrated that COI and COII genes were effective markers for accurate species identification of the Thai blow flies. This study has not only demonstrated the genetic diversity of Thai blow flies, but also provided a reliable DNA reference database for further use in forensic entomology within the country and other regions where these species exist.
Project description:This paper introduces statistical shape modelling (SSM) for use in osteoarchaeology research. SSM is a full field, multi-material analytical technique, and is presented as a supplementary geometric morphometric (GM) tool. Lower mandibular canines from two archaeological populations and one modern population were sampled, digitised using micro-CT, aligned, registered to a baseline and statistically modelled using principal component analysis (PCA). Sample material properties were incorporated as a binary enamel/dentin parameter. Results were assessed qualitatively and quantitatively using anatomical landmarks. Finally, the technique's application was demonstrated for inter-sample comparison through analysis of the principal component (PC) weights. It was found that SSM could provide high detail qualitative and quantitative insight with respect to archaeological inter- and intra-sample variability. This technique has value for archaeological, biomechanical and forensic applications including identification, finite element analysis (FEA) and reconstruction from partial datasets.
Project description:The mechanical and structural properties of bone are known to change significantly with age. Within forensic and archaeological investigations, the medial end of the clavicle is typically used for estimating the age-at-death of an unknown individual. Although, this region of the skeleton is of interest to forensic and clinical domains, alterations beyond the macro-scale have not been fully explored. For this study, non-destructive micro-computed tomography (?-CT) was employed to characterize structural alterations to the cancellous bone of the medial clavicle. Fresh human cadaveric specimens (12-59 years) obtained at autopsy were utilized for this study, and were scanned with a voxel size of ~83 ?m. Morphometric properties were quantified and indicated that the bone volume, connectivity density, mineral density, and number of trabeculae decreased with age, while the spacing between the trabeculae increased with age. In contrast to other sub-regions of the skeleton, trabecular thickness, and degree of anisotropy did not correlate with age. Collectively, this could suggest that the network is becoming increasingly perforated with age rather than exhibiting trabecular thinning. These results are used in the context of deriving a potential protocol for forensic investigations by using this particular and largely unexplored region of the skeleton, and provide inspiration for future experiments concerning micro-architectural and small scale changes in other regions of the human skeleton.
Project description:The dramatic metamorphosis from larva to adult of insect orders such as Diptera cannot usually be witnessed because it occurs within an opaque structure. For the cyclorrhaphous dipterans, such as blow flies, this structure is the puparium, formed from the larval cuticle. Here, we reveal metamorphosis within the puparium of a blow fly at higher temporal resolution than previously possible with two-dimensional time-lapse videos created using the X-ray within a micro-computed tomography scanner, imaging development at 1?min and 2?min intervals. Our studies confirm that the most profound morphological changes occur during just 0.5% of the intrapuparial period (approx. equivalent to 1.25?h at 24°C) and demonstrate the significant potential of this technique to complement other methods for the study of developmental changes, such as hormone control and gene expression. We hope this will stimulate a renewed interest among students and researchers in the study of morphology and its astonishing transformation engendered by metamorphosis.
Project description:This is the first study to report Chrysomya pinguis (Walker) and Lucilia porphyrina (Walker) (Diptera: Calliphoridae) as forensically important blow fly species from human cadavers in Thailand, in addition to Chrysomya villeneuvi (Patton) already known in Thailand. In 2016, a fully decomposed body of an unknown adult male was discovered in a high mountainous forest during winter in Chiang Mai province. The remains were infested heavily with thousands of blow fly larvae feeding simultaneously on them. Morphological identification of adults reared from the larvae, and molecular analysis based on sequencing of 1,247 bp partial mitochondrial cytochrome c oxidase subunit 1 gene (CO1) of the larvae and puparia, confirmed the above mentioned 3 species. The approving forensic fly evidence by molecular approach was described for the first time in Thailand. Moreover, neighbor-joining phylogenetic analysis of the CO1 was performed to compare the relatedness of the species, thereby affirming the accuracy of identification. As species of entomofauna varies among cases in different geographic and climatic circumstances, C. pinguis and L. porphyrina were added to the list of Thai forensic entomology caseworks, including colonizers of human remains in open, high mountainous areas during winter. Further research should focus on these 3 species, for which no developmental data are currently available.
Project description:We present here the draft genome sequence of a Providencia stuartii strain, derived from the salivary glands of larval Lucilia sericata, a common blow fly important to forensic, medical, and veterinary science. The genome sequence will help dissect coinfections involving P. stuartii and Proteus mirabilis, as well as blow fly-bacteria interactions.
Project description:Laboratory x-ray micro-computed tomography (micro-CT) is a fast-growing method in scientific research applications that allows for non-destructive imaging of morphological structures. This paper provides an easily operated "how to" guide for new potential users and describes the various steps required for successful planning of research projects that involve micro-CT. Background information on micro-CT is provided, followed by relevant setup, scanning, reconstructing, and visualization methods and considerations. Throughout the guide, a Jackson's chameleon specimen, which was scanned at different settings, is used as an interactive example. The ultimate aim of this paper is make new users familiar with the concepts and applications of micro-CT in an attempt to promote its use in future scientific studies.