Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a ‘phloem sandwich’ and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.
18 Related JoVE Articles!
Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout (Salvelinus namaycush) from Its Prey
Institutions: U. S. Geological Survey, Grand Valley State University, Shedd Aquarium.
A technique for laboratory estimation of net trophic transfer efficiency (γ) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is described herein. During a 135-day laboratory experiment, we fed bloater (Coregonus hoyi
) that had been caught in Lake Michigan to lake trout (Salvelinus namaycush
) kept in eight laboratory tanks. Bloater is a natural prey for lake trout. In four of the tanks, a relatively high flow rate was used to ensure relatively high activity by the lake trout, whereas a low flow rate was used in the other four tanks, allowing for low lake trout activity. On a tank-by-tank basis, the amount of food eaten by the lake trout on each day of the experiment was recorded. Each lake trout was weighed at the start and end of the experiment. Four to nine lake trout from each of the eight tanks were sacrificed at the start of the experiment, and all 10 lake trout remaining in each of the tanks were euthanized at the end of the experiment. We determined concentrations of 75 PCB congeners in the lake trout at the start of the experiment, in the lake trout at the end of the experiment, and in bloaters fed to the lake trout during the experiment. Based on these measurements, γ was calculated for each of 75 PCB congeners in each of the eight tanks. Mean γ was calculated for each of the 75 PCB congeners for both active and inactive lake trout. Because the experiment was replicated in eight tanks, the standard error about mean γ could be estimated. Results from this type of experiment are useful in risk assessment models to predict future risk to humans and wildlife eating contaminated fish under various scenarios of environmental contamination.
Environmental Sciences, Issue 90, trophic transfer efficiency, polychlorinated biphenyl congeners, lake trout, activity, contaminants, accumulation, risk assessment, toxic equivalents
Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter
Institutions: Yale University, Virginia Tech, The Hebrew University of Jerusalem.
The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration1,2
. Plant litter comprises the majority of detritus3
, and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5
. However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6
. A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass7,8,9
because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism6
. Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption7
. Ultimately, prey stressed by predation risk increase their body C:N ratio7,10
, making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production6
. Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11
We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira
), a dominant grasshopper herbivore (Melanoplus femurrubrum
),and a variety of grass and forb plants9
Environmental Sciences, Issue 73, Microbiology, Plant Biology, Entomology, Organisms, Investigative Techniques, Biological Phenomena, Chemical Phenomena, Metabolic Phenomena, Microbiological Phenomena, Earth Resources and Remote Sensing, Life Sciences (General), Litter Decomposition, Ecological Stoichiometry, Physiological Stress and Ecosystem Function, Predation Risk, Soil Respiration, Carbon Sequestration, Soil Science, respiration, spider, grasshoper, model system
Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs
Institutions: VU University.
In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and activating sperm, the components in the seminal fluid can also influence female physiology to augment fertilization success of the sperm donor. While many studies have reported such effects in species with separate sexes, few studies have addressed this in simultaneously hermaphroditic animals. This video protocol presents a method to study effects of seminal fluid in gastropods, using a simultaneously hermaphroditic freshwater snail, the great pond snail Lymnaea stagnalis
, as model organism. While the procedure is shown using complete prostate gland extracts, individual components (i.e.
, proteins, peptides, and other compounds) of the seminal fluid can be tested in the same way. Effects of the receipt of ejaculate components on egg laying can be quantified in terms of frequency of egg laying and more subtle estimates of female reproductive performance such as egg numbers within each egg masses. Results show that seminal fluid proteins affect female reproductive output in this simultaneous hermaphrodite, highlighting their importance for sexual selection.
Physiology, Issue 88, Allohormone, Fresh-water snail, Gastropod, Lymnaea stagnalis, Mollusc, Pond snail, Prostate, Semen, Seminal fluid Sexual selection, Sperm
Measuring Glutathione-induced Feeding Response in Hydra
Institutions: India Institute of Science Education and Research, Pune.
Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.
Basic Protocols, Issue 93, Hydra, chemosensation, feeding response, feeding status, glutathione, prey, starvation
A Visual Assay to Monitor T6SS-mediated Bacterial Competition
Institutions: Imperial College London .
Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2
. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3
. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae
T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7
. Another striking example was recently documented using Pseudomonas aeruginosa
which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10
In the latter case,
three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1
). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10
. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa
, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14
, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli
DH5α) allowing a-complementation of the lacZ
gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.
Infection, Issue 73, Microbiology, Immunology, Infectious Diseases, Molecular Biology, Genetics, Biochemistry, Cellular Biology, Bacteriology, Bacteria, Type Six Secretion System, T6SS, Bacterial Competition, Killing Assay, Pseudomonas aeruginosa, E. coli, lacZ, CFU, bacterial screen, pathogens, assay
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
Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions
Institutions: University of Toronto, University of Toronto, University of Toronto.
The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae
as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub
) and the N-terminal half of the ubiquitin moiety (Nub
). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub
which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub
moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.
Cellular Biology, Issue 36, protein-protein interaction, membrane, split-ubiquitin, yeast, library screening, Y2H, yeast two-hybrid, MYTH
Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ
Institutions: College of William & Mary.
Studies performed in Drosophila melanogaster
embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila
larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila
immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ
morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.
Molecular Biology, Issue 90,
Drosophila, embryo, larvae, sonication, fixation, immunostain, immunofluorescence, organogenesis, development
Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes
Institutions: Université de Lorraine, Max Planck Institute for Chemical Ecology, aura optik.
Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.
Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthe
s, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.
In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes
digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes
traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.
Plant Biology, Issue 82, Plant, Plant Preparations, Plant Physiological Processes, Plant Pathology, micropreparation, mechanical dissection, glands, carnivory, Nepenthes, PCR, RNA
Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction
Institutions: Rutgers University, Koç University, New York University, Fairfield University.
We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the "cloud" and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in cabinet; two such cabinets (16 environments) may be controlled by one computer.
Behavior, Issue 84, genetics, cognitive mechanisms, behavioral screening, learning, memory, timing
Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish
Institutions: University of Ottawa, University of Ottawa, University of Ottawa.
Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals' access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal's body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal's body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.
Neuroscience, Issue 85, animal tracking, weakly electric fish, electric organ discharge, underwater infrared imaging, automated image tracking, sensory isolation chamber, exploratory behavior
Cortical Source Analysis of High-Density EEG Recordings in Children
Institutions: UCL Institute of Child Health, University College London.
EEG is traditionally described as a neuroimaging technique with high temporal and low spatial resolution. Recent advances in biophysical modelling and signal processing make it possible to exploit information from other imaging modalities like structural MRI that provide high spatial resolution to overcome this constraint1
. This is especially useful for investigations that require high resolution in the temporal as well as spatial domain. In addition, due to the easy application and low cost of EEG recordings, EEG is often the method of choice when working with populations, such as young children, that do not tolerate functional MRI scans well. However, in order to investigate which neural substrates are involved, anatomical information from structural MRI is still needed. Most EEG analysis packages work with standard head models that are based on adult anatomy. The accuracy of these models when used for children is limited2
, because the composition and spatial configuration of head tissues changes dramatically over development3
In the present paper, we provide an overview of our recent work in utilizing head models based on individual structural MRI scans or age specific head models to reconstruct the cortical generators of high density EEG. This article describes how EEG recordings are acquired, processed, and analyzed with pediatric populations at the London Baby Lab, including laboratory setup, task design, EEG preprocessing, MRI processing, and EEG channel level and source analysis.
Behavior, Issue 88, EEG, electroencephalogram, development, source analysis, pediatric, minimum-norm estimation, cognitive neuroscience, event-related potentials
Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions
Institutions: Wellcome Trust Sanger Institute.
Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1
, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives < 1 second) precluding their detection with many commonly-used high throughput methods2
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2
≤ 0.1 sec) with a low false positive rate3
. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4
, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (β-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3
Molecular Biology, Issue 61, Receptor-ligand pairs, Extracellular protein interactions, AVEXIS, Adhesion receptors, Transient/weak interactions, High throughput screening
Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum
Institutions: Miami University.
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.
Molecular Biology, Issue 92, RNA interference, RNAi, gene knockdown, red flour beetle, Tribolium castaneum, injection, double-stranded RNA, functional analysis, teaching laboratories
Barnes Maze Testing Strategies with Small and Large Rodent Models
Institutions: University of Missouri, Food and Drug Administration.
Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.
bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.
distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.
random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.
shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.
Behavior, Issue 84, spatial navigation, rats, Peromyscus, mice, intra- and extra-maze cues, learning, memory, latency, search strategy, escape motivation
In vitro Reconstitution of the Active T. castaneum Telomerase
Institutions: University of Pennsylvania.
Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (Tc
TERT) and the reconstitution of the active T. castaneum
telomerase ribonucleoprotein (RNP) complex in vitro
Telomerase is a specialized reverse transcriptase1
that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2
that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3
and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4
. Additionally, telomerase is dormant in most somatic cells5
in adults, but is active in cancer cells6
where it promotes cell immortality7
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9
. Prior to 2008, only structures for individual telomerase domains had been solved10,11
. A major breakthrough in this field came from the determination of the crystal structure of the active12
, catalytic subunit of T. castaneum
Here, we present methods for producing large quantities of the active, soluble Tc
TERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro
for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.
Molecular Biology, Issue 53, Telomerase, protein expression, purification, chromatography, RNA isolation, TRAP
In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces
Institutions: Johannes Kepler Universitat Linz.
Unraveling the interaction network of molecules in-vivo
is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al
., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo
. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.
Bioengineering, Issue 37, protein-protein interactions, quantification, in-vivo, micro-contact-printing, micro-patterned surfaces
Recordings of Neural Circuit Activation in Freely Behaving Animals
Institutions: University of Maryland.
The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. Traditional non-invasive methods require at least partially restrained research subjects, and they only allow identification of large numbers of simultaneously activated neurons. On the other hand, small ensembles of neurons or individual neurons can only be measured using single-cell recordings obtained from largely reduced preparations. Since the expression of natural behavior is limited in restrained and dissected animals, the underlying neural mechanisms that control such behavior are difficult to identify.
Here, I present a non-invasive physiological technique that allows measuring neural circuit activation in freely behaving animals. Using a pair of wire electrodes inside a water-filled chamber, the bath electrodes record neural and muscular field potentials generated by juvenile crayfish during natural or experimentally evoked escape responses. The primary escape responses of crayfish are mediated by three different types of tail-flips which move the animals away from the point of stimulation. Each type of tail-flip is controlled by its own neural circuit; the two fastest and most powerful escape responses require activation of different sets of large “command” neurons. In combination with behavioral observations, the bath electrode recordings allow unambiguous identification of these neurons and the associated neural circuits. Thus activity of neural circuitry underlying naturally occurring behavior can be measured in unrestrained animals and in different behavioral contexts.
Neuroscience, Issue 29, Electrophysiology, bath electrodes, neurons, behavior