Development and validation of forensically useful growth models for Central European population of Creophilus maxillosus L. (Coleoptera: Staphylinidae).
ABSTRACT: 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.
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: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: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:Common DNA-based species determination methods fail to distinguish some blow flies in the forensically and medically important genus Lucilia Robineau-Desvoidy. This is a practical problem, and it has also been interpreted as casting doubt on the validity of some morphologically defined species. An example is Lucilia illustris and L. caesar, which co-occur in Europe whilst only L. illustris has been collected in North America. Reports that these species shared both mitochondrial and nuclear gene sequences, along with claims that diagnostic morphological characters are difficult to interpret, were used to question their separate species status. We report here that amplified fragment length polymorphism profiles strongly support the validity of both species based on both assignment and phylogenetic analysis, and that traditional identification criteria based on male and female genital morphology are more reliable than has been claimed.
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: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 subfamily Luciliinae is diverse and geographically widespread. Its four currently recognised genera (Dyscritomyia Grimshaw, 1901, Hemipyrellia Townsend, 1918, Hypopygiopsis Townsend 1916 and Lucilia Robineau-Desvoidy, 1830) contain species that range from saprophages to obligate parasites, but their pattern of phylogenetic diversification is unclear. The 28S rRNA, COI and Period genes of 14 species of Lucilia and Hemipyrellia were partially sequenced and analysed together with sequences of 11 further species from public databases. The molecular data confirmed molecular paraphyly in three species-pairs in Lucilia that hamper barcode identifications of those six species. Lucilia sericata and Lucilia cuprina were confirmed as mutual sister species. The placements of Dyscritomyia and Hypopygiopsis were ambiguous, since both made Lucilia paraphyletic in some analyses. Recognising Hemipyrellia as a genus consistently left Lucilia s.l. paraphyletic, and the occasionally-recognised (sub)genus Phaenicia was consistently paraphyletic, so these taxa should be synonymised with Lucilia to maintain monophyly. Analysis of a matrix of 14 morphological characters scored for adults of all genera and for most of the species included in the molecular analysis confirmed several of these findings. The different degrees of parasitism were phylogenetically clustered within this genus but did not form a graded series of evolutionary stages, and there was no particular relationship between feeding habits and biogeography. Because of the ubiquity of hybridization, introgression and incomplete lineage sorting in blow flies, we recommend that using a combination of mitochondrial and nuclear markers should be a procedural standard for medico-criminal forensic identifications of insects.
Project description:Determining the age of juvenile blow flies is one of the key tasks of forensic entomology when providing evidence for the minimum post mortem interval. While the age determination of blow fly larvae is well established using morphological parameters, the current study focuses on molecular methods for estimating the age of blow flies during the metamorphosis in the pupal stage, which lasts about half the total juvenile development. It has already been demonstrated in several studies that the intraspecific variance in expression of so far used genes in blow flies is often too high to assign a certain expression level to a distinct age, leading to an inaccurate prediction. To overcome this problem, we previously identified new markers, which show a very sharp age dependent expression course during pupal development of the forensically-important blow fly Calliphora vicina Robineau-Desvoidy 1830 (Diptera: Calliphoridae) by analyzing massive parallel sequencing (MPS) generated transcriptome data. We initially designed and validated two quantitative polymerase chain reaction (qPCR) assays for each of 15 defined pupal ages representing a daily progress during the total pupal development if grown at 17 °C. We also investigated whether the performance of these assays is affected by the ambient temperature, when rearing pupae of C. vicina at three different constant temperatures-namely 17 °C, 20 °C and 25 °C. A temperature dependency of the performance could not be observed, except for one marker. Hence, for each of the defined development landmarks, we can present gene expression profiles of one to two markers defining the mentioned progress in development.
Project description:Histone deacetylase inhibitors (HDACi) are being investigated for the control of various human parasites. Here we investigate their potential as insecticides for the control of a major ecto-parasite of sheep, the Australian sheep blowfly, Lucilia cuprina. We assessed the ability of HDACi from various chemical classes to inhibit the development of blowfly larvae in vitro, and to inhibit HDAC activity in nuclear protein extracts prepared from blowfly eggs. The HDACi prodrug romidepsin, a cyclic depsipeptide that forms a thiolate, was the most potent inhibitor of larval growth, with equivalent or greater potency than three commercial blowfly insecticides. Other HDACi with potent activity were hydroxamic acids (trichostatin, CUDC-907, AR-42), a thioester (KD5170), a disulphide (Psammaplin A), and a cyclic tetrapeptide bearing a ketone (apicidin). On the other hand, no insecticidal activity was observed for certain other hydroxamic acids, fatty acids, and the sesquiterpene lactone parthenolide. The structural diversity of the 31 hydroxamic acids examined here revealed some structural requirements for insecticidal activity; for example, among compounds with flexible linear zinc-binding extensions, greater potency was observed in the presence of branched capping groups that likely make multiple interactions with the blowfly HDAC enzymes. The insecticidal activity correlated with inhibition of HDAC activity in blowfly nuclear protein extracts, indicating that the toxicity was most likely due to inhibition of HDAC enzymes in the blowfly larvae. The inhibitor potencies against blowfly larvae are different from inhibition of human HDACs, suggesting some selectivity for human over blowfly HDACs, and a potential for developing compounds with the inverse selectivity. In summary, these novel findings support blowfly HDAC enzymes as new targets for blowfly control, and point to development of HDAC inhibitors as a promising new class of insecticides.
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.