A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.
In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae
This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.
1Oxitec Ltd, 2Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, 3Departamento de Epidemiologia, Universidade de São Paulo, 4Moscamed Brasil, 5Deptartment of Zoology, University of Oxford, 6Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM)
To achieve population suppression of Aedes aegypti using the RIDL® (Release of Insects carrying a Dominant Lethal) system, large numbers of male mosquitoes need to be released. This requires the use of mass rearing techniques and technology to provide reliable systems to obtain the maximum number of high quality male mosquitoes.
Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment
1Fachbereich Biologie, Entwicklungsbiologie, Philipps-Universität Marburg, 2Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg
This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.
1Department of Biology, Miami University
RNA interference (RNAi)-based gene knockdown techniques are at the core of Tribolium research. Here, we provide an overview of our larval RNAi technique in Tribolium castaneum. Larval RNAi is a simple, but powerful technique that provides quick access to loss-of-function phenotypes, allowing researchers to study gene functions in diverse contexts.
Published October 13, 2014. Keywords: Molecular Biology, RNA interference, RNAi, gene knockdown, red flour beetle, Tribolium castaneum, injection, double-stranded RNA, functional analysis, teaching laboratories
1Division of Biology, Kansas State University, 2Department of Medical and Molecular Genetics, Indiana University School of Medicine, 3Eck Institute for Global Health, University of Notre Dame, 4Department of Biological Sciences, University of Notre Dame, 5Department of Entomology, Kansas State University
Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.
Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.
1Biology Department, Wesleyan University
This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.
Published January 12, 2015. Keywords: Developmental Biology, Drosophila melanogaster, pupal eye, pattern formation, ommatidial development, live cell imaging, motion stabilization, fluorescence microscopy
1Department of Biology, University of Miami
This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.
This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.
An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus
1Department of Biology, Georgetown University, 2Insect Physiology Lab, EEOB, The Ohio State University
RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.
The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.
Published November 14, 2012. Keywords: Neuroscience, Anatomy, Physiology, Immunology, Developmental Biology, Drosophila, retina, photoreceptor, imaginal disc, larva, pupa, confocal microscopy, immunohistochemistry
1Department of Electrical and Computer Engineering, North Carolina State University
We present a novel surgical procedure to implant electrodes in Manduca sexta during its early metamorphic stages. This technique allows mechanically stable and electrically reliable coupling with the neuromuscular tissue to study flight neurophysiology dynamics. We also present a novel magnetic levitation platform for tethered studies of insect yaw.
Published July 12, 2014. Keywords: Behavior, Manduca sexta; telemetry; metamorphosis; bioelectronics; neurophysiology; electrophysiology; neuromuscular
Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain
A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo.
Published May 19, 2013. Keywords: Molecular Biology, Cellular Biology, Developmental Biology, Anatomy, Physiology, Genetics, Flow Cytometry, Cell Cycle, DNA Replication, Metamorphosis, Biological, drosophila, Gal4/UAS, insect metamorphosis, animal model
This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.
1Department of Biology, University of Rochester, 2W. M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges
Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.
1Department of Molecular, Cell and Developmental Biology (MCDB), University of California at Los Angeles (UCLA), 2Arizona College of Osteopathic Medicine (AZCOM), Midwestern University, 3MCDB, Broad Stem Cell Research Center, UCLA
The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.
1Center for Molecular Bacteriology and Infection, Imperial College London
The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.
Published November 22, 2013. Keywords: Infection, Bacterial Infections, Infection, Disease Models, Animal, Bacterial Infections and Mycoses, Galleria mellonella, Legionella pneumophila, insect model, bacterial infection, Legionnaires' disease, haemocytes
1Department of Biological Sciences, University of Toledo
Imaging of centrosomal proteins during Drosophila spermatogenesis is a powerful method to identify new proteins critical for centrosome biology as well as to elucidate the particular function of known players in this process.
Published September 20, 2013. Keywords: Developmental Biology, biology (general), genetics (animal and plant), animal biology, animal models, Life Sciences (General), Centrosome, Spermatogenesis, Spermiogenesis, Drosophila, Centriole, Cilium, Mitosis, Meiosis
Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.
This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.
Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.
Published December 11, 2012. Keywords: Infection, Microbiology, Immunology, Molecular Biology, Bacteriology, Entomology, Bacteria, Galleria mellonella, greater wax moth, insect larvae, intra haemocoelic injection, ingestion, animal model, host pathogen interactions
Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila
Morphological scaling relationships capture and describe organismal shape. We present a method to measure morphological scaling relationships across the natural range of body sizes in fully metamorphic insects. Using a simple diet manipulation we increase the distribution of trait sizes, permitting the accurate description of how shape and size co-vary.
1Laboratory of Molecular Biology of the Cell, Ecole Normale Supérieure de Lyon, 2INSERM U744, Institut Pasteur de Lille, Université Lille-Nord de France, 3Howard Hughes Medical Institute, Laboratory of Apoptosis and Cancer, The Rockefeller University
The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.
Published September 20, 2013. Keywords: Developmental Biology, Eye, Photoreceptor Cells, Genes, Developmental, neuron, visualization, degeneration, development, live imaging, Drosophila, photoreceptor, cornea neutralization, mitotic recombination
1Department of Computer Science and Engineering, University of California, Riverside, 2Department of Entomology, University of California, Riverside, 3Institute of Mathematics and Computer Sciences, University of São Paulo - USP, 4ISCA Technologies
We proposed a system that uses inexpensive, noninvasive pseudo-acoustic optical sensors to automatically and accurately detect, count, and classify flying insects based on their flying sound.
Published October 15, 2014. Keywords: Bioengineering, flying insect detection, automatic insect classification, pseudo-acoustic optical sensors, Bayesian classification framework, flight sound, circadian rhythm
1Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, 2Scholars Academy/MARC Scholar, University of Houston-Downtown, 3Genes and Development Graduate Program, University of Texas Graduate School of Biomedical Sciences, 4Neuroscience Graduate Program, University of Texas Graduate School of Biomedical Sciences
In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
Published May 18, 2012. Keywords: Neuroscience, Drosophila sensory neurons, thermal nociception, nociceptive sensitization, tissue damage, fly behavioral response, dendritic arborization neurons, allodynia, hyperalgesia, behavioral assay
A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.
Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes
1Department of Entomology, Virginia Tech
A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes. Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition.
1UMR 7503, Laboratoire Lorrain de Recherche en Informatique et ses Applications (LORIA), Centre National de la Recherche Scientifique (CNRS), 2UMR 1392 iEES-Paris, Institut d'Ecologie et des Sciences de l'Environnement de Paris, 3Physics of Biological Systems, Institut Pasteur
We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source.
Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.
Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species
Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.
Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti
1Agricultural Research Service, Henry A. Wallace Beltsville Agricultural Research Center, Plant Sciences Institute, Invasive Insect Biocontrol and Behavior Laboratory, United States Department of Agriculture
Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.
In order to examine gene expression in the pupal wing tissue of Bicyclus anynana, we present an optimized protocol for in situ hybridizations using riboprobes. We also provide guidelines for the further optimization of this protocol for use in pupal wings of other Lepidopteran species.
Measuring the yaw torque of tethered Drosophila with the torque meter allows the neuroscientist exquisite control of the stimulus situation of the experimental animal. Together with the unique genetic tools available in the fruit fly, this paradigm is used for a wide variety of neurobiological research.
1Center for Systems Biology, Massachusetts General Hospital, 2Institute for Biological and Medical Imaging (IBMI), Technical University of Munich and Helmholtz Center Munich, 3Department of Genetics, Harvard Medical School and Howard Hughes Medical Institute
Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.
Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.
Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster
Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.
Published December 19, 2011. Keywords: Developmental Biology, Cis-regulatory element, CRE, cis-regulatory module, enhancer, site-specific integration, reporter transgenes, confocal microscopy, regulatory logic, transcription factors, binding sites, Drosophila melanogaster, Drosophila