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.
23 Related JoVE Articles!
Technique for Studying Arthropod and Microbial Communities within Tree Tissues
Institutions: Northern Arizona University, Acoustic Ecology Institute.
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.
Environmental Sciences, Issue 93, phloem sandwich, pine, bark beetles, mites, acoustics, phloem
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
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
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
Assessment of Age-related Changes in Cognitive Functions Using EmoCogMeter, a Novel Tablet-computer Based Approach
Institutions: Freie Universität Berlin, Charité Berlin, Freie Universität Berlin, Psychiatric University Hospital Zurich.
The main goal of this study was to assess the usability of a tablet-computer-based application (EmoCogMeter) in investigating the effects of age on cognitive functions across the lifespan in a sample of 378 healthy subjects (age range 18-89 years). Consistent with previous findings we found an age-related cognitive decline across a wide range of neuropsychological domains (memory, attention, executive functions), thereby proving the usability of our tablet-based application. Regardless of prior computer experience, subjects of all age groups were able to perform the tasks without instruction or feedback from an experimenter. Increased motivation and compliance proved to be beneficial for task performance, thereby potentially increasing the validity of the results. Our promising findings underline the great clinical and practical potential of a tablet-based application for detection and monitoring of cognitive dysfunction.
Behavior, Issue 84, Neuropsychological Testing, cognitive decline, age, tablet-computer, memory, attention, executive functions
In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae
Institutions: VU University Amsterdam.
In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo
, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871
, who showed that it was possible to reconstitute these complexes in vitro
starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro
reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g.,
pigment binding sites) or protein domain (e.g.,
protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro
currently used in our laboratory,
and examples describing applications of the method are provided.
Biochemistry, Issue 92, Reconstitution, Photosynthesis, Chlorophyll, Carotenoids, Light Harvesting Protein, Chlamydomonas reinhardtii, Arabidopsis thaliana
Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters
Institutions: The University of Memphis.
In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2
). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2
. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4
, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7
. Compared to traditional studies of locomotor activity in vivo
and SCN explants ex vivo
, cell-based in vitro
assays allow for discovery of cell-autonomous circadian defects5,8
. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase
as a reporter has become a common technique for studying circadian rhythms in mammals14,15
, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17
or stable transduction5,10,18,19
. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20
. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2
reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.
Genetics, Issue 67, Molecular Biology, Cellular Biology, Chemical Biology, Circadian clock, firefly luciferase, real-time bioluminescence technology, cell-autonomous model, lentiviral vector, RNA interference (RNAi), high-throughput screening (HTS)
Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example
Institutions: Jewish General Hospital, McGill University.
RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron regulatory proteins
, IRP1 and IRP2. They bind to iron responsive elements (IREs) within the untranslated regions (UTRs) of several target mRNAs, thereby controlling the mRNAs translation or stability. IRE/IRP interactions have been widely studied by EMSA. Here, we describe the EMSA protocol for analyzing the IRE-binding activity of IRP1 and IRP2, which can be generalized to assess the activity of other RNA-binding proteins as well. A crude protein lysate containing an RNA-binding protein, or a purified preparation of this protein, is incubated with an excess of32
P-labeled RNA probe, allowing for complex formation. Heparin is added to preclude non-specific protein to probe binding. Subsequently, the mixture is analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe migrates fast, while the RNA/protein complex exhibits retarded mobility; hence, the procedure is also called “gel retardation” or “bandshift” assay. After completion of the electrophoresis, the gel is dried and RNA/protein complexes, as well as free probe, are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE/IRP and other RNA/protein interactions. Moreover, EMSA can also be used to determine specificity, binding affinity, and stoichiometry of the RNA/protein interaction under investigation.
Biochemistry, Issue 94, RNA metabolism, mRNA translation, post-transcriptional gene regulation, mRNA stability, IRE, IRP1, IRP2, iron metabolism, ferritin, transferrin receptor
Using Luciferase to Image Bacterial Infections in Mice
Institutions: Texas A&M Health Science Center.
Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2
. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4
. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6
. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9
. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10
. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.
Infection, Issue 48, Bioluminescence, whole animal imaging, pathogenesis, click-beetle, in vivo imaging, firefly
An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus
Institutions: Georgetown University, The Ohio State University.
Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus
, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus.
Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus
due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.
Genetics, Issue 93, Aedes albopictus Asian tiger mosquito, photoperiodic diapause, RNA-Seq de novo transcriptome assembly, mosquito husbandry
Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity
Institutions: Case Western Reserve University.
Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.
Environmental Sciences, Issue 85, Coexistence, community assembly, environmental drivers, plant-soil feedback, soil heterogeneity, soil microbial communities, soil patch
Reconstitution Of β-catenin Degradation In Xenopus Egg Extract
Institutions: Vanderbilt University Medical Center, Cincinnati Children's Hospital Medical Center, Vanderbilt University School of Medicine.
egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus
egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3
. Herein, a method is described for isolating Xenopus
egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, β-catenin. Two different methods are described to assess β-catenin protein degradation in Xenopus
egg extract. One method is visually informative ([35
S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess β-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus
egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of β-catenin degradation.
Molecular Biology, Issue 88, Xenopus laevis, Xenopus egg extracts, protein degradation, radiolabel, luciferase, autoradiography, high-throughput screening
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (https://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
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
Using Coculture to Detect Chemically Mediated Interspecies Interactions
Institutions: University of North Carolina at Chapel Hill .
In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e.
biofilm formation, sporulation, virulence factor production, etc
.) Screening is performed under growth conditions where this phenotype is not
expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis
; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.
Microbiology, Issue 80, High-Throughput Screening Assays, Genes, Reporter, Microbial Interactions, Soil Microbiology, Coculture, microbial interactions, screen, fluorescent transcriptional reporters, Bacillus subtilis
Design and Construction of an Urban Runoff Research Facility
Institutions: Texas A&M University, The Scotts Miracle-Gro Company.
As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use due to increased demand for landscape irrigation. Improper irrigation practices and large rainfall events can result in runoff from urban landscapes which has potential to carry nutrients and sediments into local streams and lakes where they may contribute to eutrophication. A 1,000 m2
facility was constructed which consists of 24 individual 33.6 m2
field plots, each equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated urban landscapes. Runoff volumes from the first and second trials had coefficient of variability (CV) values of 38.2 and 28.7%, respectively. CV values for runoff pH, EC, and Na concentration for both trials were all under 10%. Concentrations of DOC, TDN, DON, PO4
, and Ca2+
had CV values less than 50% in both trials. Overall, the results of testing performed after sod installation at the facility indicated good uniformity between plots for runoff volumes and chemical constituents. The large plot size is sufficient to include much of the natural variability and therefore provides better simulation of urban landscape ecosystems.
Environmental Sciences, Issue 90, urban runoff, landscapes, home lawns, turfgrass, St. Augustinegrass, carbon, nitrogen, phosphorus, sodium
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
Obtaining Eggs from Xenopus laevis Females
Institutions: Emory University.
The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.
Basic Protocols, Issue 18, Current Protocols Wiley, Eggs, Xenopus laevis
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
Windowing Chicken Eggs for Developmental Studies
Institutions: University of California, Irvine (UCI).
The study of development has been greatly aided by the use of the chick embryo as an experimental model. The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation. These experiments have yielded important data on the development of the central and peripheral nervous systems. For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth. The embryo can be examined at successive developmental stages by re-opening the eggshell. While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times. In this method, the egg rests on its side and a small window is cut in the shell. After the experimental procedure, the shell is used to cover the egg for the duration of its development. Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period. This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.
Developmental Biology, Issue 8, Neuroscience, Chicken, Embryos, Electroporation, In ovo
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
Preparation and Fractionation of Xenopus laevis Egg Extracts
Institutions: Emory University.
Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.
Cellular Biology, Issue 18, Current Protocols Wiley, Xenopus laevis, Egg Extracts, Density Gradient Centrifugation, Light Membrane Fraction, Nuclear Fraction
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