The blowflies of the Madeira Archipelago: species diversity, distribution and identification (Diptera, Calliphoridaes. l.).
ABSTRACT: 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: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: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: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: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: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: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:Defensins are the most widespread antimicrobial peptides characterised in insects. These cyclic peptides, 4-6 kDa in size, are folded into ?-helical/?-sheet mixed structures and have a common conserved motif of three intramolecular disulfide bridges with a Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6 connectivity. They have the ability to kill especially Gram-positive bacteria and some fungi, but Gram-negative bacteria are more resistant against them. Among them are the medicinally important compounds lucifensin and lucifensin II, which have recently been identified in the medicinal larvae of the blowflies Lucilia sericata and Lucilia cuprina, respectively. These defensins contribute to wound healing during a procedure known as maggot debridement therapy (MDT) which is routinely used at hospitals worldwide. Here we discuss the decades-long story of the effort to isolate and characterise these two defensins from the bodies of medicinal larvae or from their secretions/excretions. Furthermore, our previous studies showed that the free-range larvae of L. sericata acutely eliminated most of the Gram-positive strains of bacteria and some Gram-negative strains in patients with infected diabetic foot ulcers, but MDT was ineffective during the healing of wounds infected with Pseudomonas sp. and Acinetobacter sp. The bactericidal role of lucifensins secreted into the infected wound by larvae during MDT and its ability to enhance host immunity by functioning as immunomodulator is also discussed.
Project description:In this study, we present entomotoxicological data on the accumulation of cadmium and thallium in a forensically important blowfly, <i>Lucilia sericata</i>, and evaluate the reliability and utility of such information as toxicological evidence for poisoning as a cause of death. We observed that Cd and Tl content in different growing stages of <i>L. sericata</i> (larvae, puparial cases, and adults) was increasing with increasing metal concentration in the feeding substrate, namely metal-enriched liver. However, patterns of accumulation differed between the two metals investigated, showing a linear relationship for Cd and a saturable pattern for Tl. For cadmium, the highest bioaccumulation factor (BAF) was found in the larval stage (in the range of 0.20-0.25), while for thallium, puparial cases accumulated more metal than the other stages tested (BAF in the range of 0.24-0.42). Thallium was also observed to have a negative effect on larval growth, resulting in lower weight and smaller puparial size. With this study, we update the information on the bioaccumulation of cadmium in forensically important blowflies and provide the first report on the bioaccumulation of thallium as well as its developmental impact in blowflies. Specifically, our results suggest that analysis of puparial cases could yield useful information for entomotoxicological investigations. The content of Cd and Tl in larvae, puparial cases, and adults of <i>L. sericata</i> was determined by inductively coupled plasma mass spectrometry (ICP-MS). The validation parameters of the method such as sensitivity, detection limits, quantification limits, precision, and accuracy were evaluated. The method detection limit (MDL) for all types of samples was in the range of 1.6-3.4 ng g<sup>-1</sup> for Cd and 0.034-0.15 ng g<sup>-1</sup> for Tl, and the accuracy of the method was confirmed by a high recovery of metals from certified reference materials (91.3% for Cd and 94.3% for Tl).
Project description:Necrophagous Calliphoridae breed in vertebrate carrion. Their larvae aggregate and form large masses of individuals. These aggregated larvae can reach adulthood faster than scattered larvae, increasing their chances of survival. Furthermore, the gathering of larvae of different species suggests possible interspecific aggregation vectors. In this context, the effect of larval ground-left cues on larvae of Calliphora vomitoria and Lucilia sericata was studied. We used video tracking to follow larvae placed in binary choice tests. We observed (1) a preference of both species for a side marked by conspecific or heterospecific larvae compared to an unmarked side, (2) a preference of L. sericata larvae for a conspecific-marked side compared to a heterospecific-marked side but only at high concentration of cues and (3) a preference of both species for the side marked by the greater number of larvae. These results demonstrate that larvae leave a mark locally which is retentive, has an interspecific range, has an effect proportional to its intensity and whose strength varies depending on the emitting species. According to the self-organization theory, this mark could enhance larval gathering and promote interspecific aggregations. While not yet demonstrated, an interspecific Allee effect could explain the interspecific association of necrophagous calliphorid larvae.