Insects:
The class Insecta, in the phylum Arthropoda, whose members are characterized by division into three parts: head, thorax, and abdomen. They are the dominant group of animals on earth; several hundred thousand different kinds having been described. Three orders, Hemiptera; Diptera; and Siphonaptera; are of medical interest in that they cause disease in humans and animals. (From Borror et al., An Introduction to the Study of Insects, 4th ed, p1)
JoVE Immunology and Infection
Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence
Department of Bacteriology, University of Wisconsin-Madison
The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.
JoVE General
Bioassays for Monitoring Insecticide Resistance
1Division of Plant Sciences, University of Missouri, Delta Research Center, 2Department of Entomology, Louisiana State University Agricultural Center
This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.
JoVE General
Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example
International Center for Tropical Agriculture, CIAT
This video explains mechanisms of host plant resistance to herbivory and demonstrates a no-choice test that estimates the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp.
JoVE General
A Simple Protocol for Extracting Hemocytes from Wild Caterpillars
Department of Biological Sciences, The George Washington University
Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.
JoVE Immunology and Infection
The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis
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.
JoVE Neuroscience
Vertical T-maze Choice Assay for Arthropod Response to Odorants
Entomology and Nematology Department, University of Florida
A vertical, T-maze olfactometer is described for assaying the behavioral response of arthropods. The olfactometer allows the experimenter to measure choices performed by test subjects when subjected to two potential odor fields. Both attraction to and repulsion from odorants can be measured with this device.
JoVE Neuroscience
Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe
Department of Biology, University of Washington
Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.
JoVE Neuroscience
Studying the Neural Basis of Adaptive Locomotor Behavior in Insects
Zoological Institute, University of Cologne
We describe a method to record motor activity, timed to the electrically recorded tarsal contact signal in a tethered insect, walking on a slippery surface. This is used to study the neural basis of adaptive behavior under reduced influence of mechanical interaction between legs through the substrate.
JoVE General
In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee
1Institut für Biologie - Neurobiologie, Freie Universität Berlin, 2Institut für Biologie - Neurobiologie, Free University Berlin - Freie Universitaet Berlin
Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.
JoVE Neuroscience
Electrophysiological Measurements from a Moth Olfactory System
Department of Entomology, University of California, Davis
Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.
JoVE General
RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes
1Biology Department, New Mexico State University, 2Molecular Biology Department, New Mexico State University
In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.
JoVE Neuroscience
Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae
Laboratory of Neurogenetics and Behavior, Rockefeller University
Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).
JoVE Immunology and Infection
A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti
Department of Biology, University of Richmond
A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.
JoVE General
Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles
Plant Mycotoxin Research, U.S. Department of Agriculture, Agricultural Research Service
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.
JoVE Editorial
December 2012: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Here are some highlights from the December 2012 Issue of Journal of Visualized Experiments (JoVE).
JoVE General
Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment
Boyce Thompson Institute for Plant Research, Cornell University
In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.
JoVE Editorial
April 2011: This Month in JoVE
Here are some highlights from the April 2011 Issue of Journal of Visualized Experiments (JoVE).
JoVE Immunology and Infection
Alphavirus Transducing System: Tools for Visualizing Infection in Mosquito Vectors
Microbiology, Immunology, and Pathology, Colorado State University
Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.
JoVE Immunology and Infection
Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes
1Department of Molecular Biology and Biochemistry, University of California, Irvine, 2Department of Microbiology and Molecular Genetics, University of California, Irvine
Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.
JoVE General
Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR
Department of Entomology, The Ohio State University
Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.
JoVE General
Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila
1Department of Biology & Biochemistry, University of Houston, 2Department of Zoology, Michigan State University
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.
JoVE General
Primary Neuronal Cultures from the Brains of Late Stage 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.
JoVE Neuroscience
Visualization of Proprioceptors in Drosophila Larvae and Pupae
A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.
JoVE General
Dissection of Oenocytes from Adult Drosophila melanogaster
Department of Biology, University of Toronto
In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.
JoVE Neuroscience
Electrophysiology of Scorpion Peg Sensilla
Department of Zoology, University of Oklahoma
This article describes an electrophysiological method for isolating chemical stimulation to individual sensilla via extracellular, tip-recordings under mineral oil.
JoVE General
Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection
1Plant Pathology, Cornell University, 2Boyce Thompson Institute for Plant Research, Cornell University
Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.
JoVE General
Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects
Boyce Thompson Institute for Plant Research, Cornell University
Plant resistance to chewing insect herbivores can be tested in several ways. Here, we demonstrate how to set-up a choice and a no-choice experiment with the model plant Arabidopsis thaliana to identify resistance against the pest species Pieris rapae.
JoVE Immunology and Infection
Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes
Department of Entomology, Virginia Tech
Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.
JoVE Neuroscience
Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits
Department of Biomedical Engineering, Washington University in St. Louis
We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.
JoVE Bioengineering
Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control
1Entomology, International Center for Tropical Agriculture (CIAT), Cali, Colombia, 2Sustainable Perennial Crops Laboratory, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland, USA
This protocol demonstrates two inoculation methods to introduce the fungal entomopathogen Beauveria bassiana as an endophyte in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control.
JoVE General
An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies
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.
JoVE General
Visually Mediated Odor Tracking During Flight in Drosophila
Department of Physiological Science, University of California, Los Angeles
Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.
JoVE General
A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila
Department of Physiological Science, University of California, Los Angeles
Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.
JoVE General
Herbivore-induced Blueberry Volatiles and Intra-plant Signaling
Department of Entomology, Rutgers University
A push-pull method for collecting plant volatiles is described. The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage. This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants.
JoVE Neuroscience
Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe
1Center for Integrative Genomics, University of Lausanne, 2Department of Biology, University of Konstanz
We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.
JoVE General
Visualizing Bacteria in Nematodes using Fluorescent Microscopy
Department of Bacteriology, University of Wisconsin-Madison
To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.
JoVE General
Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila
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.
JoVE Editorial
May 2012: This Month in JoVE
Here are some highlights from the May 2012 Issue of Journal of Visualized Experiments (JoVE).
JoVE General
Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro (Protozoa Culture) and in vivo (Microinjection into Termite Hindgut)
Department of Entomology, Louisiana State University Agricultural Center
We present procedures for demonstrating that ligands bind to the surface membrane of the cellulose-digesting protozoa in the gut of Formosan subterranean termites using fluorescent microscopy and that ligands coupled with lytic peptides kill these protozoa in vitro (anaerobic protozoa culture) and in vivo (injection into the termite hindgut).
JoVE General
Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies
Department of Biology, University of Kentucky, Lexington
We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.
JoVE Neuroscience
Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)
In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.
JoVE Neuroscience
Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique
1Monell Chemical Senses Center, 2Department of Physiology, Development & Neuroscience, Physiological Laboratory, University of Cambridge
Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.
JoVE Neuroscience
Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster
Department of Neurobiology, Stanford University
Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.
JoVE General
Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos
This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.
JoVE Immunology and Infection
RNA Interference in Ticks
1Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, 2(CSIC-UCLM-JCCM), Instituto de Investigación en Recursos Cinegéticos IREC
A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.
JoVE Neuroscience
Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas
Department of Biology, New York University
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.
JoVE Neuroscience
Ex vivo Culturing of Whole, Developing Drosophila Brains
1National Institute of Neurological Disorders and Stroke, 2National Human Genome Research Institute, National Institutes of Health, Bethesda, MD
This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.
JoVE Immunology and Infection
A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor
1Bioscience Medical Research Center, Niigata University Medical and Dental Hospital, 2First department of Internal Medicine, School of Medicine, Kyorin University, 3Neosilk Laboratory, Immuno Biological Laboratories Co., Ltd.
We designed a cell-free receptor binding assay in order to estimate the binding of granulocyte-macrophage colony-stimulating factor (GM-CSF) to the receptors. It enables us to evaluate competitive inhibition of biotinylated GM-CSF binding to soluble GM-CSF receptor alpha by GM-CSF autoantibody with excellent reproducibility.
JoVE Neuroscience
Appetitive Associative Olfactory Learning in Drosophila Larvae
1Department of Biology, University of Konstanz, 2Department of Biology, University of Fribourg
Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.
JoVE Immunology and Infection
An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response
1Biology Department, The City College of New York, CUNY, 2The Graduate Center, The City University of New York
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.
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