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Caste-Specific Expression Patterns of Immune Response and Chemosensory Related Genes in the Leaf-Cutting Ant, Atta vollenweideri.
PUBLISHED: 01-01-2013
Leaf-cutting ants are evolutionary derived social insects with elaborated division of labor and tremendous colony sizes with millions of workers. Their social organization is mainly based on olfactory communication using different pheromones and is promoted by a pronounced size-polymorphism of workers that perform different tasks within the colony. The size polymorphism and associated behaviors are correlated to distinct antennal lobe (AL) phenotypes. Two worker phenotypes differ in number of olfactory glomeruli in the AL and the presence or absence of an extremely large glomerulus (macroglomerulus), involved in trail-pheromone reception. The males AL contains three macroglomeruli which are presumably involved in detection of sex-pheromone components. We investigated the antennal transcriptome data of all major castes (males, queens and workers) and two worker subcastes (large and tiny workers). In order to identify putative odorant receptor genes involved in pheromone detection, we identified differentially expressed odorant receptor genes (OR-genes) using custom microarrays. In total, we found 185 OR-gene fragments that are clearly related to ORs and we identified orthologs for 70 OR-genes. Among them one OR-gene differs in relative expression between the two worker subcastes by a factor of >3 and thus is a very promising candidate gene for the trail-pheromone receptor. Using the relative expression of OR-genes in males versus queens, we identified 2 candidates for sex-pheromone receptor genes in males. In addition, we identified genes from all other chemosensory related gene families (13 chemosensory protein genes, 8 odorant binding protein genes, 2 sensory-neuron membrane protein genes, 7 ionotropic receptor genes, 2 gustatory receptor genes), and we found ant-specific expansions in the chemosensory protein gene family. In addition, a large number of genes involved in immune defense exhibited differential expression across the three different castes, and some genes even between the two worker subcastes.
23 Related JoVE Articles!
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Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model
Authors: Daniel Münch, Nicholas Baker, Erik M.K. Rasmussen, Ashish K. Shah, Claus D. Kreibich, Lars E. Heidem, Gro V. Amdam.
Institutions: Norwegian University of Life Sciences, Arizona State University.
Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best established model to study how plasticity in lifespan and aging is explained by social factors. The worker caste of honey bees includes nurse bees, which tend the brood, and forager bees, which collect nectar and pollen. Previous work has shown that brain functions and flight performance senesce more rapidly in foragers than in nurses. However, brain functions can recover, when foragers revert back to nursing tasks. Such patterns of accelerated and reversed functional senescence are linked to changed metabolic resource levels, to alterations in protein abundance and to immune function. Vitellogenin, a yolk protein with adapted functions in hormonal control and cellular defense, may serve as a major regulatory element in a network that controls the different aging dynamics in workers. Here we describe how the emergence of nurses and foragers can be monitored, and manipulated, including the reversal from typically short-lived foragers into longer-lived nurses. Our representative results show how individuals with similar chronological age differentiate into foragers and nurse bees under experimental conditions. We exemplify how behavioral reversal from foragers back to nurses can be validated. Last, we show how different cellular senescence can be assessed by measuring the accumulation of lipofuscin, a universal biomarker of senescence. For studying mechanisms that may link social influences and aging plasticity, this protocol provides a standardized tool set to acquire relevant sample material, and to improve data comparability among future studies.
Developmental Biology, Issue 78, Insects, Microscopy, Confocal, Aging, Gerontology, Neurobiology, Insect, Invertebrate, Brain, Lipofuscin, Confocal Microscopy
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Testing Drosophila Olfaction with a Y-maze Assay
Authors: Mégane M. Simonnet, Martine Berthelot-Grosjean, Yael Grosjean.
Institutions: UMR-6265 CNRS, UMR-1324 INRA, Université de Bourgogne.
Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.
Neuroscience, Issue 88, environmental effects (biological, animal and plant), genetics (animal and plant), life sciences, animal biology, behavioral sciences, Y-maze, olfaction, adult, choice, behavior, Drosophila melanogaster
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Dissection of Oenocytes from Adult Drosophila melanogaster
Authors: Joshua J. Krupp, Joel D. Levine.
Institutions: University of Toronto.
In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.
Developmental Biology, Issue 41, Drosophila, oenocytes, metabolism, cuticular hydrocarbons, chemical senses, chemical communication, pheromones, adult
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Authors: Johannes Felix Buyel, Rainer Fischer.
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
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Institutions: Arizona State University .
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Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Institutions: Fraunhofer USA Center for Molecular Biotechnology.
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Strategies for Study of Neuroprotection from Cold-preconditioning
Institutions: The University of Chicago Medical Center.
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Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting
Authors: Jesper T. Grønlund, Alison Eyres, Sanjeev Kumar, Vicky Buchanan-Wollaston, Miriam L. Gifford.
Institutions: University of Warwick , University of Warwick .
After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated. To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6. FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.
Plant Biology, Issue 68, Cellular Biology, Molecular Biology, Leaf protoplasts, fluorescence activated cell sorting, FACS, green fluorescent protein, GFP, cell type specificity, developmental stage specificity
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Institutions: The Samuel Roberts Noble Foundation.
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Electrophysiological Measurements from a Moth Olfactory System
Authors: Zainulabeuddin Syed, Walter S. Leal.
Institutions: 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. These approaches have been exploited for getting a better understanding of insect chemical communication. The identified stimuli can then be used as either attractants or repellents in management strategies for insect pests.
Neuroscience, Issue 49, Insect Olfaction, Electroantennogram (EAG), Single Sensillum Recordings (SSR), navel orangeworm
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Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation
Authors: Julien Brechbühl, Gaëlle Luyet, Fabian Moine, Ivan Rodriguez, Marie-Christine Broillet.
Institutions: University of Lausanne, University of Geneva.
Peter Karlson and Martin Lüscher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs. Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex. Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.
Neuroscience, Issue 58, Vomeronasal organ, VNO, pheromone, calcium imaging, tissue slice preparation, floating immunohistochemistry, GFP
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Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples
Institutions: University of Manitoba, University of Manitoba.
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High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity
Authors: Casey Trimmer, Lindsey L. Snyder, Joel D. Mainland.
Institutions: Monell Chemical Senses Center.
Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors—an essential step toward a model of odor coding in the mammalian olfactory system.
Neuroscience, Issue 88, Firefly luciferase, Renilla Luciferase, Dual-Glo Luciferase Assay, olfaction, Olfactory receptor, Odorant, GPCR, High-throughput
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Using Coculture to Detect Chemically Mediated Interspecies Interactions
Authors: Elizabeth Anne Shank.
Institutions: University of North Carolina at Chapel Hill .
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Authors: Brian H. Smith, Christina M. Burden.
Institutions: Arizona State University.
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Institutions: University of Würzburg.
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Authors: George Dimopoulos.
Institutions: Johns Hopkins University.
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Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes
Authors: Viktor Martyanov, Robert H. Gross.
Institutions: Dartmouth College.
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Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor
Authors: Limei Ma, Sachiko Haga-Yamanaka, Qingfeng Elden Yu, Qiang Qiu, SangSeong Kim, C. Ron Yu.
Institutions: Stowers Institute for Medical Research, The University of Kansas School of Medicine.
The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses. The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification. Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.
Neuroscience, Issue 58, Vomeronasal organ, VNO, pheromone, urine, slice preparation, G-CaMP2, calcium imaging
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