Confocal laser scanning microscopy as a valuable tool in Diptera larval morphology studies

Larval morphology of flies is traditionally studied using light microscopy, yet in the case of fine structures compound light microscopy is limited due to problems of resolution, illumination and depth of field, not allowing for precise recognition of sclerites’ edges and interactions. Using larval instars of cyclorrhaphan Diptera, we show the usefulness of confocal laser scanning microscopy (CLSM) for studying the morphological characters of immature stages by taking advantage of the autofluorescent properties of cephaloskeleton structures. We compare data obtained from killed but unprepared larvae with those from larvae prepared by clearing according to two commonly used methods, either with potassium hydroxide or with Hoyer’s medium. We also evaluated the CLSM application for examining already slide-mounted larvae stored in museum collections and those freshly prepared. Our results indicate that CLSM and 3D reconstruction are excellent for visualizing small, compound structures of cylrorrhaphan larvae cephaloskeleton, if appropriate clearing techniques, i.e. the application of KOH, are used. Maximum intensity projection of confocal data sets obtained from material freshly prepared and that stored in museum collection does not differ. Because of this and the fact that KOH is commonly used as a clearing method to examine the cephaloskeleton of Diptera larvae, it is possible, and highly recommended, to use slides already prepared with this method for re-examination by CLSM. We conclude that CLSM application can be an invaluable source of data for studies of larval morphology of Cyclorrhapha by way of taxonomic diagnoses, character identification and improvement in characters homologization.This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited

A Fly in the Ointment: Evaluation of Traditional Use of Plants to Repel and Kill Blowfly Larvae in Fermented Fish

Introduction: In rural areas in Laos, fly larvae infestations are common in fermenting fish. Blowflies (Chrysomyamegacephala, Diptera: Calliphoridae) are attracted to oviposit (and/or larviposit) onto fermenting fish which results ininfestations with fly larvae. Knowledge of traditional use of plants to repel larvae during the production of fermented fish iscommon and widespread in Lao PDR. Research Questions: How effective are the most salient species in repelling, and killing fly larvae in fermenting fish? Material and Methods: The three plant species most frequently reported to repel fly larvae during an ethnobotanical surveythroughout Lao PDR were tested for repellence and larvicidal activity of fly larvae infesting fermented fish. The lethality andrepellence of Tadehagi triquetrum (L.) H. Ohashi (Fabaceae), Uraria crinita (L.) Desv. ex DC. (Fabaceae) and Bambusa multiplex(Lour.) Raeusch. ex Schult. & Schult. f. (Poaceae) were tested in an experimental design using fermenting fish in Vientiane,Lao PDR. Results: The repellent effect of fresh material of T. triquetrum and U. crinita, and the larvicidal effect of fresh B. multiplex, issignificantly more effective than that of dried material of the same species, and the total effect (repellence and larvicidaleffect combined) for each of the three species was significantly more effective for fresh than for dry material. Fresh materialof T. triquetrum, U. crinita, or B. multiplex added on top of the fermenting fish repelled 50%, 54%, 37%, and killed 22%, 28%,and 40% of fly larvae. The total effect was not significantly different per species at 72%, 82%, and 77%, respectively. Discussion and Conclusions: The three most salient species are effective in repelling and killing fly larvae in the productionof fermented fish, and may be essential to augment food safety during traditional fermentation in open jars

The uses of Chrysomya megacephala (Fabricius, 1794)(Diptera: Calliphoridae) in forensic entomology:

Chrysomya megacephala (Fabricius, 1794) occurs on every continent and is closely associated with carrion and decaying material in human environments. Its abilities to find dead bodies and carry pathogens give it a prominence in human affairs that may involve prosecution or litigation, and therefore forensic entomologists. The identification, geographical distribution and biology of the species are reviewed to provide a background for approaches that four branches of forensic entomology (urban, stored-product, medico-criminal and environmental) might take to investigations involving this fly

A molecular, morphological, and physiological comparison of English and German populations of Calliphora vicina (Diptera: Calliphoridae).

The bluebottle blow fly Calliphora vicina is a common species distributed throughout Europe that can play an important role as forensic evidence in crime investigations. Developmental rates of C. vicina from distinct populations from Germany and England were compared under different temperature regimes to explore the use of growth data from different geographical regions for local case work. Wing morphometrics and molecular analysis between these populations were also studied as indicators for biological differences. One colony each of German and English C. vicina were cultured at the Institute of Legal Medicine in Frankfurt, Germany. Three different temperature regimes were applied, two constant (16°C & 25°C) and one variable (17-26°C, room temperature = RT). At seven time points (600, 850, 1200, 1450, 1800, 2050, and 2400 accumulated degree hours), larval lengths were measured; additionally, the durations of the post feeding stage and intrapuparial metamorphosis were recorded. For the morphometric and molecular study, 184 females and 133 males from each C. vicina population (Germany n = 3, England n = 4) were sampled. Right wings were measured based on 19 landmarks and analyzed using canonical variates analysis and discriminant function analysis. DNA was isolated from three legs per specimen (n = 61) using 5% chelex. A 784 bp long fragment of the mitochondrial cytochrome b gene was sequenced; sequences were aligned and phylogenetically analyzed. Similar larval growth rates of C. vicina were found from different geographic populations at different temperatures during the major part of development. Nevertheless, because minor differences were found a wider range of temperatures and sampling more time points should be analyzed to obtain more information relevant for forensic case work. Wing shape variation showed a difference between the German and English populations (P<0.0001). However, separation between the seven German and English populations at the smaller geographic scale remained ambiguous. Molecular phylogenetic analysis by maximum likelihood method could not unambiguously separate the different geographic populations at a national (Germany vs England) or local level.© 2018 Limsopatham et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The attached file is the published version of this article

A new PCR-based approach indicates the range of Clonorchis sinensis now extends to central Thailand

Differentiation of the fish-borne trematodes belonging to the Opisthorchiidae, Heterophyidae and Lecithodendriidae is important from a clinical and epidemiological perspective, yet it is impossible to do using conventional coprological techniques, as the eggs are morphologically similar. Epidemiological investigation therefore currently relies on morphological examination of adult worms following expulsion chemotherapy. A PCR test capable of amplifying a segment of the internal transcribed spacer region of ribosomal DNA for the opisthorchiid and heterophyid flukes eggs taken directly from faeces was developed and evaluated in a rural community in central Thailand. The lowest quantity of DNA that could be amplified from individual adults of Opisthorchis viverrini, Clonorchis sinensis and Haplorchis taichui was estimated at 0.6 pg, 0.8 pg and 3 pg, respectively. The PCR was capable of detecting mixed infection with the aforementioned species of flukes under experimental conditions. A total of 11.6% of individuals in rural communities in Sanamchaikaet district, central Thailand, were positive for ‘Opisthorchis-like’ eggs in their faeces using conventional parasitological detection techniques. In comparison to microscopy, the PCR yielded a sensitivity and specificity of 71.0% and 76.7%, respectively. Analysis of the microscopy-positive PCR products revealed 64% and 23% of individuals to be infected with O. viverrini and C. sinensis, respectively. The remaining 13% (three individuals) were identified as eggs of Didymozoidae, presumably being passed mechanically in the faeces following the ingestion of infected fishes. An immediate finding of this study is the identification and first report of a C. sinensis–endemic community in central Thailand. This extends the known range of this liver fluke in Southeast Asia. The PCR developed herein provides an important tool for the specific identification of liver and intestinal fluke species for future epidemiological surveys