This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.
24 Related JoVE Articles!
Layers of Symbiosis - Visualizing the Termite Hindgut Microbial Community
Institutions: California Institute of Technology - Caltech.
Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth. Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.
Microbiology, issue 4, microbial community, symbiosis, hindgut
Analyzing the Movement of the Nauplius 'Artemia salina' by Optical Tracking of Plasmonic Nanoparticles
We demonstrate how optical tweezers may provide a sensitive tool to analyze the fluidic vibrations generated by the movement of small aquatic organisms. A single gold nanoparticle held by an optical tweezer is used as a sensor to quantify the rhythmic motion of a Nauplius larva (Artemia salina
) in a water sample. This is achieved by monitoring the time dependent displacement of the trapped nanoparticle as a consequence of the Nauplius activity. A Fourier analysis of the nanoparticle's position then yields a frequency spectrum that is characteristic to the motion of the observed species. This experiment demonstrates the capability of this method to measure and characterize the activity of small aquatic larvae without the requirement to observe them directly and to gain information about the position of the larvae with respect to the trapped particle. Overall, this approach could give an insight on the vitality of certain species found in an aquatic ecosystem and could expand the range of conventional methods for analyzing water samples.
Biophysics, Issue 89, optical tweezers, particle tracking, plasmonic nanoparticles, Nauplius, bioindicator, water sample analysis
Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals
Institutions: University of Illinois at Urbana-Champaign, University of Illinois at Urbana-Champaign, University of Illinois at Urbana-Champaign.
An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis
and M. faveolata
. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae
endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis
and M. faveolata
contain similar types of chlorophyll and chromatophores. However, M. annularis
and M. faveolata
exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation, (2) superior optical sectioning ability, and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.
Environmental Sciences, Issue 91, Serial block face imaging, two-photon fluorescence microscopy, Montastraea annularis, Montastraea faveolata, 3D coral tissue morphology and structure, zooxanthellae, chromatophore, autofluorescence, light harvesting optimization, environmental change
A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats
Institutions: Concordia University.
Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.
Behavior, Issue 91, Behavioral neuroscience, alcoholism, relapse, addiction, Pavlovian conditioning, ethanol, reinstatement, discrimination, conditioned approach
Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat
Institutions: University of Wisconsin-Madison.
Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical components. Both anxiety and fear can be studied in the laboratory by measuring the potentiation of the startle reflex. The startle reflex is a defensive reflex that is potentiated when an organism is threatened and the need for defense is high. The startle reflex is assessed via electromyography (EMG) in the orbicularis oculi muscle elicited by brief, intense, bursts of acoustic white noise (i.e.
, “startle probes”). Startle potentiation is calculated as the increase in startle response magnitude during presentation of sets of visual threat cues that signal delivery of mild electric shock relative to sets of matched cues that signal the absence of shock (no-threat cues). In the Threat Probability Task, fear is measured via startle potentiation to high probability (100% cue-contingent shock; certain) threat cues whereas anxiety is measured via startle potentiation to low probability (20% cue-contingent shock; uncertain) threat cues. Measurement of startle potentiation during the Threat Probability Task provides an objective and easily implemented alternative to assessment of negative affect via self-report or other methods (e.g.
, neuroimaging) that may be inappropriate or impractical for some researchers. Startle potentiation has been studied rigorously in both animals (e.g
., rodents, non-human primates) and humans which facilitates animal-to-human translational research. Startle potentiation during certain and uncertain threat provides an objective measure of negative affective and distinct emotional states (fear, anxiety) to use in research on psychopathology, substance use/abuse and broadly in affective science. As such, it has been used extensively by clinical scientists interested in psychopathology etiology and by affective scientists interested in individual differences in emotion.
Behavior, Issue 91,
Startle; electromyography; shock; addiction; uncertainty; fear; anxiety; humans; psychophysiology; translational
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
Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology
Institutions: San Diego State University, University of California San Diego.
Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.
Environmental Sciences, Issue 93, dissolved organic carbon, particulate organic matter, nutrients, DAPI, SYBR, microbial metagenomics, viral metagenomics, marine environment
Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites
Institutions: Royal Military College of Canada, Queen's University.
The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g.
carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.
Environmental Sciences, Issue 93, biochar, characterization, carbon sequestration, remediation, International Biochar Initiative (IBI), soil amendment
Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats
Institutions: Massachusetts General Hospital, Harvard Medical School.
Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that neural mechanisms for certain behavioral tasks and response behaviors are different in females and males. Using females in research studies can represent a challenge because of the variation of gonadal hormones during their estrous cycle. This protocol describes well-established procedures that are useful in investigating the role of estrogen in fear extinction memory consolidation in female rats. Phase of the estrous cycle and exogenous estrogen administration prior to extinction training can influence extinction recall 24 hr later. The vaginal swabbing technique for estrous phase identification described here aids the examination and manipulation of naturally cycling gonadal hormones. The use of this basic rodent model may further delineate the mechanisms by which estrogen can modulate fear extinction memory in females.
Behavior, Issue 96, estrogen, fear extinction, sex differences, estradiol, proestrus, metestrus, female, PTSD, anxiety
Detection of Foodborne Bacterial Pathogens from Individual Filth Flies
Institutions: U.S. Food and Drug Administration.
There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter
spp., Salmonella enterica,
and Listeria monocytogenes
and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter
spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica
(7%) and L.monocytogenes
(4%). No false positives were observed when detecting S. enterica
and L. monocytogenes
using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter
colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae
present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.
Environmental Sciences, Issue 96, Synanthropy, filth flies, Cronobacter, Listeria monocytogenes, Salmonella, Escherichia coli O157:H7, shiga-toxigenic E. coli, STEC, PCR-based methods, foodborne illness, foodborne outbreak investigations.
Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies
Institutions: University of Minnesota, Northern State University, Minnesota Pollution Control Agency, RMB Environmental Laboratories, Inc..
Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water’s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.
Environmental Sciences, Issue 101, biological monitoring, aquatic systems, aquatic ecology, water quality, macroinvertebrates, midge, Chironomid Pupal Exuviae Technique, rapid bioassessment protocol
Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians
Institutions: James Cook University.
Amphibians are experiencing the greatest decline of any vertebrate class and a leading cause of these declines is a fungal pathogen, Batrachochytrium dendrobatidis
), which causes the disease chytridiomycosis. Captive assurance colonies are important worldwide for threatened amphibian species and may be the only lifeline for those in critical threat of extinction. Maintaining disease free colonies is a priority of captive managers, yet safe and effective treatments for all species and across life stages have not been identified. The most widely used chemotherapeutic treatment is itraconazole, although the dosage commonly used can be harmful to some individuals and species. We performed a clinical treatment trial to assess whether a lower and safer but effective dose of itraconazole could be found to cure Bd
infections. We found that by reducing the treatment concentration from 0.01-0.0025% and reducing the treatment duration from 11-6 days of 5 min baths, frogs could be cured of Bd
infection with fewer side effects and less treatment-associated mortality.
Immunology, Issue 85, Batrachochytrium dendrobatidis, itraconazole, chytridiomycosis, captive assurance colonies, amphibian conservation
Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis
Institutions: Harvard University.
The amphipod Parhyale hawaiensis
is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale
has emerged as a promising model organism for laboratory studies of development, providing a useful outgroup comparison to the well studied arthropod model organism Drosophila melanogaster
. In contrast to the syncytial cleavages of Drosophila
, the early cleavages of Parhyale
are holoblastic. Fate mapping using tracer dyes injected into early blastomeres have shown that all three germ layers and the germ line are established by the eight-cell stage. At this stage, three blastomeres are fated to give rise to the ectoderm, three are fated to give rise to the mesoderm, and the remaining two blastomeres are the precursors of the endoderm and germ line respectively. However, blastomere ablation experiments have shown that Parhyale
embryos also possess significant regulatory capabilities, such that the fates of blastomeres ablated at the eight-cell stage can be taken over by the descendants of some of the remaining blastomeres. Blastomere ablation has previously been described by one of two methods: injection and subsequent activation of phototoxic dyes or manual ablation. However, photoablation kills blastomeres but does not remove the dead cell body from the embryo. Complete physical removal of specific blastomeres may therefore be a preferred method of ablation for some applications. Here we present a protocol for manual removal of single blastomeres from the eight-cell stage of Parhyale
embryos, illustrating the instruments and manual procedures necessary for complete removal of the cell body while keeping the remaining blastomeres alive and intact. This protocol can be applied to any Parhyale
cell at the eight-cell stage, or to blastomeres of other early cleavage stages. In addition, in principle this protocol could be applicable to early cleavage stage embryos of other holoblastically cleaving marine invertebrates.
Developmental Biology, Issue 85, Amphipod, experimental embryology, micromere, germ line, ablation, developmental potential, vasa
Electroporation of Mycobacteria
Institutions: Barts and the London School of Medicine and Dentistry, Barts and the London School of Medicine and Dentistry.
High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.
Microbiology, Issue 15, Springer Protocols, Mycobacteria, Electroporation, Bacterial Transformation, Transformation Efficiency, Bacteria, Tuberculosis, M. Smegmatis, Springer Protocols
Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises
Institutions: University of Kentucky, University of Toronto.
The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+
on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.
Neuroscience, Issue 47, Invertebrate, Crayfish, neurophysiology, muscle, anatomy, electrophysiology
Swimming Performance Assessment in Fishes
Institutions: University of Alberta.
Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, pollution, hypoxia and temperature. This paper describes a flexible protocol to assess fish swimming performance using equipment in which water velocity can be controlled. The protocol involves one to several stepped increases in flow speed that are intended to cause fish to fatigue. Step speeds and their duration can be set to capture swimming abilities of different physiological and ecological relevance. Most frequently step size is set to determine critical swimming velocity (Ucrit
), which is intended to capture maximum sustained swimming ability. Traditionally this test has consisted of approximately ten steps each of 20 min duration. However, steps of shorter duration (e.g. 1 min) are increasingly being utilized to capture acceleration ability or burst swimming performance. Regardless of step size, swimming tests can be repeated over time to gauge individual variation and recovery ability. Endpoints related to swimming such as measures of metabolic rate, fin use, ventilation rate, and of behavior, such as the distance between schooling fish, are often included before, during and after swimming tests. Given the diversity of fish species, the number of unexplored research questions, and the importance of many species to global ecology and economic health, studies of fish swimming performance will remain popular and invaluable for the foreseeable future.
Physiology, Issue 51, fish, swimming, Ucrit, burst, sustained, prolonged, schooling performance
Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes
Institutions: Dartmouth College.
SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference1
. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data1
. In this article, we utilize a web version of SCOPE2
to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs3,4
and has been used in other studies5-8
The three algorithms that comprise SCOPE are BEAM9
, which finds non-degenerate motifs (ACCGGT), PRISM10
, which finds degenerate motifs (ASCGWT), and SPACER11
, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well.
Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor.
Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run.
Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail1,2,9-11
Genetics, Issue 51, gene regulation, computational biology, algorithm, promoter sequence motif
A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals
Institutions: University of British Columbia, Okanagan Campus.
Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1
. This approach has proven to be especially useful when dealing with rare or elusive species2
. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps
). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas’ habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.
Genetics, Issue 49, Conservation genetics, noninvasive genetic sampling, Hair snares, Microsatellites, AFLPs, American pika, Ochotona princeps
Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana
Institutions: Institute for Environmental Protection and Research, Regional Agency for Environmental Protection in Emilia-Romagna.
Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia
to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia
) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia
). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2
. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3
. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4
. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.
Chemistry, Issue 62, Artemia franciscana, bioassays, chemical substances, crustaceans, marine environment
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
Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Institutions: University of Maine.
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
Basic Protocol, Issue 82, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises
Institutions: University of Kentucky, University of Kentucky, University of Oregon.
The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.
Neuroscience, Issue 80, Crustacean, joint, Muscle, sensory, teaching, educational, neuroscience
A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research
Institutions: Arizona State University.
Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g.
, food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera
) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.
Neuroscience, Issue 91, PER, conditioning, honey bee, olfaction, olfactory processing, learning, memory, toxin assay
Quantifying Learning in Young Infants: Tracking Leg Actions During a Discovery-learning Task
Institutions: University of Southern California, Temple University, Niigata University of Health and Welfare.
Task-specific actions emerge from spontaneous movement during infancy. It has been proposed that task-specific actions emerge through a discovery-learning process. Here a method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process. This discovery-learning task uses an infant activated mobile that rotates and plays music based on specified leg action of infants. Supine infants activate the mobile by moving their feet vertically across a virtual threshold. This paradigm is unique in that as infants independently discover that their leg actions activate the mobile, the infants’ leg movements are tracked using a motion capture system allowing for the quantification of the learning process. Specifically, learning is quantified in terms of the duration of mobile activation, the position variance of the end effectors (feet) that activate the mobile, changes in hip-knee coordination patterns, and changes in hip and knee muscle torque. This information describes infant exploration and exploitation at the interplay of person and environmental constraints that support task-specific action. Subsequent research using this method can investigate how specific impairments of different populations of infants at risk for movement disorders influence the discovery-learning process for task-specific action.
Behavior, Issue 100, infant, discovery-learning, motor learning, motor control, kinematics, kinetics