Using the developmental gene bicoid to identify species of forensically important blowflies (Diptera: calliphoridae).
ABSTRACT: Identifying species of insects used to estimate postmortem interval (PMI) is a major subject in forensic entomology. Because forensic insect specimens are morphologically uniform and are obtained at various developmental stages, DNA markers are greatly needed. To develop new autosomal DNA markers to identify species, partial genomic sequences of the bicoid (bcd) genes, containing the homeobox and its flanking sequences, from 12 blowfly species (Aldrichina grahami, Calliphora vicina, Calliphora lata, Triceratopyga calliphoroides, Chrysomya megacephala, Chrysomya pinguis, Phormia regina, Lucilia ampullacea, Lucilia caesar, Lucilia illustris, Hemipyrellia ligurriens and Lucilia sericata; Calliphoridae: Diptera) were determined and analyzed. This study first sequenced the ten blowfly species other than C. vicina and L. sericata. Based on the bcd sequences of these 12 blowfly species, a phylogenetic tree was constructed that discriminates the subfamilies of Calliphoridae (Luciliinae, Chrysomyinae, and Calliphorinae) and most blowfly species. Even partial genomic sequences of about 500 bp can distinguish most blowfly species. The short intron 2 and coding sequences downstream of the bcd homeobox in exon 3 could be utilized to develop DNA markers for forensic applications. These gene sequences are important in the evolution of insect developmental biology and are potentially useful for identifying insect species in forensic science.
Project description:Knowledge on the taxonomic diversity and distribution of blowflies from the Madeira Archipelago is updated. New and interesting findings are reported for poorly studied islands and islets of this archipelago, together with a brief analysis of the diversity of Macaronesian Calliphoridae s. l. Seven blowfly species were collected during this study, including the first records of Calliphora vicina Robineau-Desvoidy, 1830, Chrysomya albiceps (Wiedemann, 1819), Lucilia sericata (Meigen, 1826), Pollenia rudis (Fabricius, 1794) and Stomorhina lunata (Fabricius, 1805) from Porto Santo, and of Calliphora vicina, Lucilia sericata and Stomorhina lunata from Desertas Islands. The presence of Calliphora loewi Enderlein, 1903 in Madeira Laurisilva forest is discussed and its first instar larva is redescribed, revealing important differences in relation to its original description. An identification key to the adult Madeiran blowflies is provided for the first time.
Project description:Species identification is a crucial step in forensic entomology. In several cases the calculation of the larval age allows the estimation of the minimum Post-Mortem Interval (mPMI). A correct identification of the species is the first step for a correct mPMI estimation. To overcome the difficulties due to the morphological identification especially of the immature stages, a molecular approach can be applied. However, difficulties in separation of closely related species are still an unsolved problem. Sequences of 4 different genes (COI, ND5, EF-1?, PER) of 13 different fly species collected during forensic experiments (Calliphora vicina, Calliphora vomitoria, Lucilia sericata, Lucilia illustris, Lucilia caesar, Chrysomya albiceps, Phormia regina, Cynomya mortuorum, Sarcophaga sp., Hydrotaea sp., Fannia scalaris, Piophila sp., Megaselia scalaris) were evaluated for their capability to identify correctly the species. Three concatenated sequences were obtained combining the four genes in order to verify if longer sequences increase the probability of a correct identification. The obtained results showed that this rule does not work for the species L. caesar and L. illustris. Future works on other DNA regions are suggested to solve this taxonomic issue.
Project description:Regional surveys were carried out in different parts of North West Pakistan among domestic animals (N=57,921) including pets and livestock identifying cases of traumatic myiasis (n=1037). A total of four surveys focused general livestock population during Eid ul Adha (Eid surveys; incidence=1.21%) while another four surveys (Miscellaneous surveys; incidence=7.34%) targeted animal population brought to veterinary hospitals and dispensaries. Timeframe spanned four years from 2012 to 2015. Maggots were sampled and location of the wound was recorded for each host. Taxonomic identification used light and electron microscopic techniques. Our dataset shows three species as principle agents of myiasis (n=882) including Chrysomya bezziana Villeneuve (n=394), Wohlfahrtia magnifica (n=244) and Lucilia cuprina Wiedemann (n=244). Others (n=155) including Chrysomya megacephala (Fabricius), Chrysomya rufifacies (Macquart), Lucilia sericata (Meigen), Lucilia illustris (Meigen), Lucilia porphyrina (Walker), Hemipyrellia ligguriens (Wiedemann), Calliphora vicina (Robineau-Desvoidy), Sarcophaga crassipalpalis (Macquart) and Sarcophaga species were identified as species of minor importance. The obligatory screwworm species W. magnifica is a first report from Pakistan. The results based on this dataset are presented in a recent publication "Distribution Modeling of three screwworm species in the ecologically diverse landscape of North West Pakistan" (Zaidi et al., 2016) .
Project description:The Bicoid morphogen evolved approximately 150 MYA from a Hox3 duplication and is only found in higher dipterans. A major difference between dipteran species, however, is the size of the embryo, which varies up to 5-fold. Although the expression of developmental factors scale with egg length, it remains unknown how this scaling is achieved. To test whether scaling is accounted for by the properties of Bicoid, we expressed eGFP fused to the coding region of bicoid from three dipteran species in transgenic Drosophila embryos using the Drosophila bicoid cis-regulatory and mRNA localization sequences. In such embryos, we find that Lucilia sericata and Calliphora vicina Bicoid produce gradients very similar to the endogenous Drosophila gradient and much shorter than what they would have produced in their own respective species. The common shape of the Drosophila, Lucilia and Calliphora Bicoid gradients appears to be a conserved feature of the Bicoid protein. Surprisingly, despite their similar distributions, we find that Bicoid from Lucilia and Calliphora do not rescue Drosophila bicoid mutants, suggesting that that Bicoid proteins have evolved species-specific functional amino acid differences. We also found that maternal expression and anteriorly localization of proteins other than Bcd does not necessarily give rise to a gradient; eGFP produced a uniform protein distribution. However, a shallow gradient was observed using eGFP-NLS, suggesting nuclear localization may be necessary but not sufficient for gradient formation.
Project description:Contemporary studies in forensic entomology exhaustively evaluate gene sequences because these constitute the fastest and most accurate method of species identification. For this purpose single gene segments, cytochrome oxidase subunit I (COI) in particular, are commonly used. However, the limitation of such sequences in identification, especially of closely related species and populations, demand a multi-gene approach. But this raises the question of which group of genes can best fulfill the identification task? In this context the utility of five gene segments was explored among blowfly species from two distinct geographic regions, China and Pakistan. COI, cytochrome b (CYTB), NADH dehydrogenase 5 (ND5), nuclear internal transcribed spacers (ITS1 and ITS2), were sequenced for eight blowfly species including Chrysomya megacephala F. (Diptera: Calliphoidae), Ch. pinguis Walker, Lucilia sericata Meigen L. porphyrina Walker, L. illustris Meigen Hemipyrellia ligurriens Wiedemann, Aldrichina grahami Aldrich, and the housefly, Musca domestica L. (Muscidae), from Hangzhou, China; while COI, CYTB, and ITS2 were sequenced for four species, i.e. Ch. megacephala, Ch. rufifacies, L. cuprina, and the flesh fly, Sarcophaga albiceps Meigen (Sarcophagidae), from Dera Ismail Khan Pakistan. The results demonstrate a universal utility of these gene segments in the molecular identification of flies of forensic importance.
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 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: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:The hairy rove beetle, Creophilus maxillosus (Linnaeus) (Staphylinidae), is recognized for its use in forensic entomology. However, insufficient developmental data exist for the Central European population of this species. Accordingly, we studied the development of C. maxillosus at ten constant temperatures (10-32.5 °C). Based on these results, linear and nonlinear developmental models were created and validated. We also studied the effect of different homogenous diets (third-instar larvae or puparia of Calliphora sp. Robineau-Desvoidy or Lucilia sp. Robineau-Desvoidy (Diptera: Calliphoridae) or mix of first- and second-instar larvae of Necrodes littoralis (Linnaeus) (Coleoptera: Silphidae)) on the development and mortality of C. maxillosus. Average total development times ranged between 122.21 days at 15 °C and 22.18 days at 30 °C. Beetles reached the adult stage in seven out of ten temperatures (15-30 °C). No beetles reached the adult stage when fed with larvae of N. littoralis; their development times at first and second larval stage were also significantly longer than in other food conditions. When C. maxillosus larvae were fed with blowfly larvae, the highest mortality was observed at the pupal stage, as compared when they were fed with blowfly puparia-at the first larval stage. While validating thermal summation models, the highest age estimation errors were found for beetles bred at 10 and 12.5 °C (between 21 and 43% for all developmental events). Age estimation errors were on average higher for pupation and eclosion than hatching and first and second ecdyses. While validating the models with specimens fed with different diets, the highest errors were recorded for beetles fed with N. littoralis larvae (22% for the first ecdysis and 33% for the second ecdysis) and Lucilia sp. puparia (32% for pupation and 22% for eclosion). Implications for C. maxillosus use in forensic entomology are discussed.