The red flour beetle, Tribolium castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle’s body cavity.
In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.
22 Related JoVE Articles!
An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila
Institutions: University of Birmingham .
An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila
. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila
can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila
to investigate the molecular mechanisms underlying CNS regeneration and repair.
Neurobiology, Issue 73, Developmental Biology, Neuroscience, Molecular Biology, Cellular Biology, Anatomy, Physiology, Bioengineering, Central Nervous System, Neuroglia, Drosophila, fruit fly, animal models, Wounds and Injuries, Cell Physiological Phenomena, Genetic Phenomena, injury, repair, regeneration, central nervous system, ventral nerve cord, larva, live imaging, cell counting, Repo, GS2, glia, neurons, nerves, CNS, animal model
Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae
Institutions: University of Michigan , University of Michigan , University of Michigan , University of Michigan .
Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.
Developmental Biology, Issue 77, Anatomy, Physiology, Biophysics, Biomedical Engineering, Neurobiology, Neuroscience, Muscle, contraction, force, zebrafish, larvae, muscle function, muscle health, force generation, animal model
Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae
Institutions: University of Michigan, University of Michigan, University of Michigan, University of Michigan, University of Michigan.
Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila
larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila
larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd
instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.
Bioengineering, Issue 84, Drosophila melanogaster, Live Imaging, Microfluidics, axonal injury, axonal degeneration, calcium imaging, photoconversion, laser microsurgery
VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance
Institutions: The Samuel Roberts Noble Foundation.
Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus
(TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana
In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana
and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae
T1, P. syringae
, and X. campestris
. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.
Virology, Issue 78, Plant Biology, Infection, Genetics, Molecular Biology, Cellular Biology, Physiology, Genomics, Pathology, plants, Nonhost Resistance, Virus-induced gene silencing, VIGS, disease resistance, gene silencing, Pseudomonas, GFPuv, sequencing, virus, Nicotiana benthamiana, plant model
Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)
Institutions: University of Technology, Sydney.
Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques – stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.
Molecular Biology, Issue 91, super-resolution microscopy, fluorescence microscopy, OMX, 3D-SIM, Blaze, cell division, bacteria, Bacillus subtilis, Staphylococcus aureus, FtsZ, Z ring constriction
Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions
Institutions: Imperial College London, Institut Pasteur, Unité Macrophages et Développement de l'Immunité.
is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri
by compartmentalizing bacteria inside ‘septin cages’ and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro
using tissue culture cells and in vivo
using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.
Infection, Issue 91, ATG8/LC3, autophagy, cytoskeleton, HeLa cells, p62, septin, Shigella, zebrafish
From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data
Institutions: Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory.
Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g.
, signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation.
The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.
Bioengineering, Issue 90, 3D electron microscopy, feature extraction, segmentation, image analysis, reconstruction, manual tracing, thresholding
Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster
Institutions: Indiana University.
A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster
, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila
proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.
Cellular Biology, Issue 91, Drosophila, imaginal discs, eye, retina, dissection, developmental biology
Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment
Institutions: Philipps-Universität Marburg, Philipps-Universität Marburg.
During spermatogenesis in mammals and in Drosophila melanogaster,
male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin—the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster
offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo
in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.
Developmental Biology, Issue 91,
Ex vivo culture, testis, male germ-line cells, Drosophila, imaging, pharmacological assay
Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae
Institutions: The University of Texas MD Anderson Cancer Center, University of Houston-Downtown, University of Texas Graduate School of Biomedical Sciences, 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.
larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2
. The advantages of Drosophila
for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6
, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7
. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8
. In Drosophila
, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9
in addition to their recently discovered role as photoreceptors10
. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12
. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11
where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila
larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila
will likely inform our understanding of thermal nociception in other species, including vertebrates.
Neuroscience, Issue 63, Drosophila sensory neurons, thermal nociception, nociceptive sensitization, tissue damage, fly behavioral response, dendritic arborization neurons, allodynia, hyperalgesia, behavioral assay
An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response
Institutions: The City College of New York, CUNY, The City University of New York.
Most known parasitoid wasp species attack the larval or pupal stages of Drosophila
. While Trichopria drosophilae
infect the pupal stages of the host (Fig. 1A-C
), females of the genus Leptopilina
(Fig. 1D, 1F, 1G
) and Ganaspis
) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila
wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C
, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2
left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2
right). L. heterotoma
is one of the best-studied species of Drosophila
parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila
species as hosts1
. L. heterotoma
and L. victoriae
are sister species and they produce virus-like particles that actively interfere with the encapsulation response2
. Unlike L. heterotoma
, L. boulardi
is a specialist parasite and the range of Drosophila
species it utilizes is relatively limited1
. Strains of L. boulardi
also produce virus-like particles3
although they differ significantly in their ability to succeed on D. melanogaster1
. Some of these L. boulardi
strains are difficult to grow on D. melanogaster1
as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila
larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi
activates both immune arms1,4
. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A
). The fat body is a large multifunctional organ (Fig. 3B
). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4
. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5
. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F
). Some blood cells aggregate to form nodules (Fig. 5G-H
). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D
We present representative data with Toll signal transduction pathway components Dorsal and Spätzle (Figs. 4,5,7
), and its target Drosomycin
), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8
Immunology, Issue 63, Parasitoid wasps, innate immunity, encapsulation, hematopoiesis, insect, fat body, Toll-NF-kappaB, molecular biology
Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila
Institutions: University of Houston, Michigan State University.
The scaling of body parts is a central feature of animal morphology1-7
. Within species, morphological traits need to be correctly proportioned to the body for the organism to function; larger individuals typically have larger body parts and smaller individuals generally have smaller body parts, such that overall body shape is maintained across a range of adult body sizes. The requirement for correct proportions means that individuals within species usually exhibit low variation in relative trait size. In contrast, relative trait size can vary dramatically among species and is a primary mechanism by which morphological diversity is produced. Over a century of comparative work has established these intra- and interspecific patterns3,4
Perhaps the most widely used approach to describe this variation is to calculate the scaling relationship between the size of two morphological traits using the allometric equation y=bxα, where x and y are the size of the two traits, such as organ and body size8,9
. This equation describes the within-group (e.g., species, population) scaling relationship between two traits as both vary in size. Log-transformation of this equation produces a simple linear equation, log(y) = log(b) + αlog(x) and log-log plots of the size of different traits among individuals of the same species typically reveal linear scaling with an intercept of log(b) and a slope of α, called the 'allometric coefficient'9,10
. Morphological variation among groups is described by differences in scaling relationship intercepts or slopes for a given trait pair. Consequently, variation in the parameters of the allometric equation (b and α) elegantly describes the shape variation captured in the relationship between organ and body size within and among biological groups (see 11,12
Not all traits scale linearly with each other or with body size (e.g., 13,14
) Hence, morphological scaling relationships are most informative when the data are taken from the full range of trait sizes. Here we describe how simple experimental manipulation of diet can be used to produce the full range of body size in insects. This permits an estimation of the full scaling relationship for any given pair of traits, allowing a complete description of how shape covaries with size and a robust comparison of scaling relationship parameters among biological groups. Although we focus on Drosophila
, our methodology should be applicable to nearly any fully metamorphic insect.
Developmental Biology, Issue 56, Drosophila, allometry, morphology, body size, scaling, insect
Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises
Institutions: University of Kentucky, University of Toronto.
The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+
on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.
Neuroscience, Issue 47, Invertebrate, Crayfish, neurophysiology, muscle, anatomy, electrophysiology
In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution
Institutions: University of Tübingen, University of Tübingen.
Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila
larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3
. While other useful methods to anesthetize Drosophila melanogaster
larvae have been previously described4,5,6,7,8
, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1
, (2) a high survival rate of > 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2
and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1
in stable transgenic lines and the exon trap line FasII-GFP1
. (4) In contrast to other methods4,7
the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1
. The accompanying video details the function of individual parts of the in vivo
, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.
Basic Protocols, Issue 43, In vivo, Imaging, Drosophila, Neuromuscular, Synapse, Development, Microscopy, Anesthetization, Desflurane
Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae
Institutions: University of California, Irvine (UCI).
Developmental Biology, Issue 2, Drosophila, Imaginal Disks, Dissection Technique
Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects
Institutions: Cornell University.
Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which include many crops such as cabbage, broccoli, Brussel sprouts etc. Rearing of the insects takes place on cabbage plants in the greenhouse. At least two cages are needed for the rearing of Pieris rapae. One for the larvae and the other to contain the adults, the butterflies. In order to investigate the role of plant hormones and toxic plant chemicals in resistance to this insect pest, we demonstrate two experiments. First, determination of the role of jasmonic acid (JA - a plant hormone often indicated in resistance to insects) in resistance to the chewing insect Pieris rapae. Caterpillar growth can be compared on wild-type and mutant plants impaired in production of JA. This experiment is considered "No Choice", because larvae are forced to subsist on a single plant which synthesizes or is deficient in JA. Second, we demonstrate an experiment that investigates the role of glucosinolates, which are used as oviposition (egg-laying) signals. Here, we use WT and mutant Arabidopsis impaired in glucosinolate production in a "Choice" experiment in which female butterflies are allowed to choose to lay their eggs on plants of either genotype. This video demonstrates the experimental setup for both assays as well as representative results.
Plant Biology, Issue 15, Annual Review, Plant Resistance, Herbivory, Arabidopsis thaliana, Pieris rapae, Caterpillars, Butterflies, Jasmonic Acid, Glucosinolates
Protocols for Oral Infection of Lepidopteran Larvae with Baculovirus
Institutions: Iowa State University.
Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be infected with polyhedra by droplet feeding and diet plug-based bioassays. This accompanying Springer Protocols section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents, including discussion of the pros and cons for use of baculoviruses as insecticides, and progress made in genetic enhancement of baculoviruses for improved insecticidal efficacy.
Plant Biology, Issue 19, Springer Protocols, Baculovirus insecticides, recombinant baculovirus, insect pest management
Protocols for Microapplicator-assisted Infection of Lepidopteran Larvae with Baculovirus
Institutions: Iowa State University.
Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. . This video shows how lepidopteran larvae can be infected with microapplicator techniques in the gut with baculovirus polyhedra and in the hemolymph with budded virus. This accompanying Springer Protocols section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents. Formulation and application of baculoviruses for pest control purposes are described elsewhere.
Plant Biology, Issue 18, Springer Protocols, Baculovirus insecticides, recombinant baculovirus, insect pest management
Drosophila Larval NMJ Dissection
Institutions: Columbia University College of Physicians and Surgeons.
neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila
motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila
larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.
Developmental Biology, Issue 24, NMJ, Drosophila, Larvae, Immunohistochemistry, Neuroscienc
A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila
Institutions: King's College London.
eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila
eye to address fundamentally important biological questions.
Cellular Biology, Issue 34, fluorescence microscopy, Drosophila, eye, RFP, dissection, imaginal disc
Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations
Institutions: SUNY-University at Buffalo.
is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd
instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.
Developmental Biology, Issue 43, Fluorescence, Microscopy, Drosophila, 3rd instar larvae, larval segmental nerves, axonal transport
Dissection of Larval CNS in Drosophila Melanogaster
Institutions: Princeton University.
The central nervous system (CNS) of Drosophila larvae is complex and poorly understood. One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins. Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity. In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining. In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS. Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS. If the dissection is performed carefully the CNS will remain attached to the cuticle. We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides. We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS. The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.
Developmental Biology, Issue 1, Drosophila, fly, CNS, larvae