Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best established model to study how plasticity in lifespan and aging is explained by social factors.
The worker caste of honey bees includes nurse bees, which tend the brood, and forager bees, which collect nectar and pollen. Previous work has shown that brain functions and flight performance senesce more rapidly in foragers than in nurses. However, brain functions can recover, when foragers revert back to nursing tasks. Such patterns of accelerated and reversed functional senescence are linked to changed metabolic resource levels, to alterations in protein abundance and to immune function. Vitellogenin, a yolk protein with adapted functions in hormonal control and cellular defense, may serve as a major regulatory element in a network that controls the different aging dynamics in workers.
Here we describe how the emergence of nurses and foragers can be monitored, and manipulated, including the reversal from typically short-lived foragers into longer-lived nurses. Our representative results show how individuals with similar chronological age differentiate into foragers and nurse bees under experimental conditions. We exemplify how behavioral reversal from foragers back to nurses can be validated. Last, we show how different cellular senescence can be assessed by measuring the accumulation of lipofuscin, a universal biomarker of senescence.
For studying mechanisms that may link social influences and aging plasticity, this protocol provides a standardized tool set to acquire relevant sample material, and to improve data comparability among future studies.
21 Related JoVE Articles!
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
RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)
Institutions: Arizona State University , Norwegian University of Life Sciences.
This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.
RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg
) and ultraspiracle (usp
), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.
The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.
Neuroscience, Issue 77, Genetics, Behavior, Neurobiology, Molecular Biology, Chemistry, Biochemistry, biology (general), genetics (animal and plant), animal biology, RNA interference, RNAi, double stranded RNA, dsRNA, double gene knockdown, vitellogenin gene, vg, ultraspiracle gene, usp, vitellogenin protein, Vg, ultraspiracle protein, USP, green fluorescence protein, GFP, gustatory perception, proboscis extension response, PER, honey bees, Apis mellifera, animal model, assay
Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)
Institutions: Bielefeld University.
Honey bees (Apis mellifera
L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2
. The worker bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g.
pollen or nectar) and brood3
. The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks4
, including hive building5
and pattern recognition6
. Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates7
. Bees use different floral signals like colors or odors7, 8
, but also tactile cues from the petal surface9
to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae10, 11, 12, 13
. This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees14
. The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern.
Neuroscience, Issue 70, Physiology, Anatomy, Entomology, Behavior, Sensilla, Bees, behavioral sciences, Sense Organs, Honey bee, Apis mellifera L., Insect antenna, Tactile sampling, conditioning, Proboscis extension response, Motion capture
C. elegans Tracking and Behavioral Measurement
Institutions: University of Toronto, Vrije Universiteit, Okinawa Institute of Science and Technology, University of Toronto.
We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans
as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans
is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2
. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3
, and general mutational phenotyping5
. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7
. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.
Neuroscience, Issue 69, Physics, Biophysics, Anatomy, Microscopy, Ethology, Behavior, Machine Vision, C. elegans, animal model
Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field
Institutions: USGS Western Ecological Research Center, University of California, Davis, University of California, Davis, University of Minnesota , Science Applications International Corporation.
Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri
) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.
Immunology, Issue 54, migratory birds, active surveillance, lyophilized reagents, avian influenza, H5N1
Flying Insect Detection and Classification with Inexpensive Sensors
Institutions: University of California, Riverside, University of California, Riverside, University of São Paulo - USP, ISCA Technologies.
An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow for the development of many useful applications in vector and pest control for both medical and agricultural entomology. Given this, the last sixty years have seen many research efforts devoted to this task. To date, however, none of this research has had a lasting impact. In this work, we show that pseudo-acoustic optical sensors can produce superior data; that additional features, both intrinsic and extrinsic to the insect’s flight behavior, can be exploited to improve insect classification; that a Bayesian classification approach allows to efficiently learn classification models that are very robust to over-fitting, and a general classification framework allows to easily incorporate arbitrary number of features. We demonstrate the findings with large-scale experiments that dwarf all previous works combined, as measured by the number of insects and the number of species considered.
Bioengineering, Issue 92, flying insect detection, automatic insect classification, pseudo-acoustic optical sensors, Bayesian classification framework, flight sound, circadian rhythm
Mouse Genome Engineering Using Designer Nucleases
Institutions: University of Zurich, University of Minnesota.
Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro
transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.
Genetics, Issue 86, Oocyte microinjection, Designer nucleases, ZFN, TALEN, Genome Engineering
Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches
Institutions: Centre National de la Recherche Scientifique (CNRS), Institut d'Ecologie et des Sciences de l'Environnement de Paris, Institut Pasteur.
Robots designed to track chemical leaks in hazardous industrial facilities1
or explosive traces in landmine fields2
face the same problem as insects foraging for food or searching for mates3
: the olfactory search is constrained by the physics of turbulent transport4
. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity5-6
, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones7
but also chemicals that are relevant to humans, e.g.
, volatile compounds emanating from cancer cells8
or toxic and illicit substances9-11
. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ
in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors12
. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies13
. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration14
or using nanostructured gas sensors that mimic insect antennae15
Neuroscience, Issue 90, robotics, electroantennogram, EAG, gas sensor, electronic nose, olfactory search, surge and casting, moth, insect, olfaction, neuron
A Video Demonstration of Preserved Piloting by Scent Tracking but Impaired Dead Reckoning After Fimbria-Fornix Lesions in the Rat
Institutions: Canadian Centre for Behavioural Neuroscience, University of Lethbridge.
Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; O’Keefe and Nadel, 1978; Mittelstaedt and Mittelstaedt, 1980; Landeau et al., 1984; Etienne, 1987; Gallistel, 1990;
Maurer and Séguinot, 1995). Piloting requires the use of the relationships between relatively stable external (visual, olfactory, auditory) cues, whereas dead reckoning requires the integration of cues generated by self-movement. Animals obtain self-movement information from vestibular receptors, and possibly muscle and joint receptors, and efference copy of commands that generate movement. An animal may also use the flows of visual, auditory, and olfactory stimuli caused by its movements. Using a piloting strategy an animal can use geometrical calculations to determine directions and distances to places in its environment, whereas using an dead reckoning strategy it can integrate cues generated by its previous movements to return to a just left location. Dead reckoning is colloquially called "sense of direction" and "sense of distance."
Although there is considerable evidence that the hippocampus is involved in piloting (O’Keefe and Nadel, 1978; O’Keefe and Speakman, 1987), there is also evidence from behavioral (Whishaw et al., 1997; Whishaw and Maaswinkel, 1998; Maaswinkel and Whishaw, 1999), modeling (Samsonovich and McNaughton, 1997), and electrophysiological (O’Mare et al., 1994; Sharp et al., 1995; Taube and Burton, 1995; Blair and Sharp, 1996; McNaughton et al., 1996; Wiener, 1996; Golob and Taube, 1997) studies that the hippocampal formation is involved in dead reckoning. The relative contribution of the hippocampus to the two forms of navigation is still uncertain, however. Ordinarily, it is difficult to be certain that an animal is using a piloting versus a dead reckoning strategy because animals are very flexible in their use of strategies and cues (Etienne et al., 1996; Dudchenko et al., 1997; Martin et al., 1997; Maaswinkel and Whishaw, 1999). The objective of the present video demonstrations was to solve the problem of cue specification in order to examine the relative contribution of the hippocampus in the use of these strategies. The rats were trained in a new task in which they followed linear or polygon scented trails to obtain a large food pellet hidden on an open field. Because rats have a proclivity to carry the food back to the refuge, accuracy and the cues used to return to the home base were dependent variables (Whishaw and Tomie, 1997). To force an animal to use a a dead reckoning strategy to reach its refuge with the food, the rats were tested when blindfolded or under infrared light, a spectral wavelength in which they cannot see, and in some experiments the scent trail was additionally removed once an animal reached the food. To examine the relative contribution of the hippocampus, fimbria–fornix (FF) lesions, which disrupt information flow in the hippocampal formation (Bland, 1986), impair memory (Gaffan and Gaffan, 1991), and produce spatial deficits (Whishaw and Jarrard, 1995), were used.
Neuroscience, Issue 26, Dead reckoning, fimbria-fornix, hippocampus, odor tracking, path integration, spatial learning, spatial navigation, piloting, rat, Canadian Centre for Behavioural Neuroscience
Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs
Institutions: University of Antioquia, University of Antioquia.
Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus
) were injected intradermally at the dorsal skin with 1 to 1.5 x l07
promastigotes of Leishmania
species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania
as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.
Immunology, Issue 62, Cutaneous leishmaniasis, hamster, Leishmania, antileishmanial drugs
A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos
Institutions: University of Notre Dame.
Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ
hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.
Developmental Biology, Issue 93, zebrafish, chemical genetics, chemical screen, in vivo small molecule screen, drug discovery, whole mount in situ hybridization (WISH), high-throughput screening (HTS), high-content screening (HCS)
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
Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers
Institutions: MIT - Massachusetts Institute of Technology.
The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and biogeochemical flux. However, to take advantage of nutrient patches in the ocean, swimming microbes must overcome the influences of physical forces including molecular diffusion and turbulent shear, which will limit the availability of patches and the ability of bacteria to locate them. Until recently, methodological limitations have precluded direct examinations of microbial behaviour within patchy habitats and realistic small-scale flow conditions. Hence, much of our current knowledge regarding microbial behaviour in the ocean has been procured from theoretical predictions. To obtain new information on microbial foraging behaviour in the ocean we have applied soft lithographic fabrication techniques to develop 2 microfluidic devices, which we have used to create (i) microscale nutrient patches with dimensions and diffusive characteristics relevant to oceanic processes and (ii) microscale vortices, with shear rates corresponding to those expected in the ocean. These microfluidic devices have permitted a first direct examination of microbial swimming and chemotactic behaviour within a heterogeneous and dynamic seascape. The combined use of epifluorescence and phase contrast microscopy allow direct examinations of the physical dimensions and diffusive characteristics of nutrient patches, while observing the population-level aggregative response, in addition to the swimming behaviour of individual microbes. These experiments have revealed that some species of phytoplankton, heterotrophic bacteria and phagotrophic protists are adept at locating and exploiting diffusing microscale resource patches within very short time frames. We have also shown that up to moderate shear rates, marine bacteria are able to fight the flow and swim through their environment at their own accord. However, beyond a threshold high shear level, bacteria are aligned in the shear flow and are less capable of swimming without disturbance from the flow. Microfluidics represents a novel and inexpensive approach for studying aquatic microbial ecology, and due to its suitability for accurately creating realistic flow fields and substrate gradients at the microscale, is ideally applicable to examinations of microbial behaviour at the smallest scales of interaction. We therefore suggest that microfluidics represents a valuable tool for obtaining a better understanding of the ecology of microorganisms in the ocean.
Microbiology, issue 4, microbial community, chemotaxis, microfluidics
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
Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae
Institutions: University of Michigan, University of Michigan, University of Michigan, University of Michigan, University of Michigan.
Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila
larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila
larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd
instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.
Bioengineering, Issue 84, Drosophila melanogaster, Live Imaging, Microfluidics, axonal injury, axonal degeneration, calcium imaging, photoconversion, laser microsurgery
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
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
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
● Perform plating procedures without contaminating media.
● Isolate single bacterial colonies by the streak-plating method.
● Use pour-plating and spread-plating methods to determine the concentration of bacteria.
● Perform soft agar overlays when working with phage.
● Transfer bacterial cells from one plate to another using the replica-plating procedure.
● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies
Institutions: US Pacific Basin Agricultural Research Center.
(Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1
. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus
has been a successfully used for biological control of fruit flies in multiple tropical regions2-4
. One impediment to the wide use of F. arisanus
for fruit fly control is that it is difficult to establish a stable laboratory colony5-9
. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12
. There is significant interest in F. arisanus
, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17
; interest is especially driven by the alarming spread of Bactrocera
fruit fly pests to new continents in the last decade18
. Further research on F. arisanus
and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus
based on a previously described approach12
. The method we describe here allows rearing of F. arisanus
in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.
Developmental Biology, Issue 53, Biological control, Tephritidae, parasitoid, French Polynesia, insectary
Spatial Multiobjective Optimization of Agricultural Conservation Practices using a SWAT Model and an Evolutionary Algorithm
Institutions: University of Washington, Iowa State University, North Carolina A&T University, Iowa Geological and Water Survey.
Finding the cost-efficient (i.e.
, lowest-cost) ways of targeting conservation practice investments for the achievement of specific water quality goals across the landscape is of primary importance in watershed management. Traditional economics methods of finding the lowest-cost solution in the watershed context (e.g.
) assume that off-site impacts can be accurately described as a proportion of on-site pollution generated. Such approaches are unlikely to be representative of the actual pollution process in a watershed, where the impacts of polluting sources are often determined by complex biophysical processes. The use of modern physically-based, spatially distributed hydrologic simulation models allows for a greater degree of realism in terms of process representation but requires a development of a simulation-optimization framework where the model becomes an integral part of optimization.
Evolutionary algorithms appear to be a particularly useful optimization tool, able to deal with the combinatorial nature of a watershed simulation-optimization problem and allowing the use of the full water quality model. Evolutionary algorithms treat a particular spatial allocation of conservation practices in a watershed as a candidate solution and utilize sets (populations) of candidate solutions iteratively applying stochastic operators of selection, recombination, and mutation to find improvements with respect to the optimization objectives. The optimization objectives in this case are to minimize nonpoint-source pollution in the watershed, simultaneously minimizing the cost of conservation practices. A recent and expanding set of research is attempting to use similar methods and integrates water quality models with broadly defined evolutionary optimization methods3,4,9,10,13-15,17-19,22,23,25
. In this application, we demonstrate a program which follows Rabotyagov et al.'s approach and integrates a modern and commonly used SWAT water quality model7
with a multiobjective evolutionary algorithm SPEA226
, and user-specified set of conservation practices and their costs to search for the complete tradeoff frontiers between costs of conservation practices and user-specified water quality objectives. The frontiers quantify the tradeoffs faced by the watershed managers by presenting the full range of costs associated with various water quality improvement goals. The program allows for a selection of watershed configurations achieving specified water quality improvement goals and a production of maps of optimized placement of conservation practices.
Environmental Sciences, Issue 70, Plant Biology, Civil Engineering, Forest Sciences, Water quality, multiobjective optimization, evolutionary algorithms, cost efficiency, agriculture, development
The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale
Institutions: University of Lethbridge.
Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation.
Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.
Neuroscience, Issue 18, rat skilled reaching, rat reaching scale, rat, rat movement element rating scale, reaching elements