Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a ‘phloem sandwich’ and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.
23 Related JoVE Articles!
A Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)
Institutions: US Forest Service.
Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed. The disease is initiated by feeding activities of the invasive beech scale insect, Cryptococcus fagisuga
, which creates entry points for infection by one of the Neonectria
species of fungus. Without scale infestation, there is little opportunity for fungal infection. Using scale eggs to artificially infest healthy trees in heavily BBD impacted stands demonstrated that these trees were resistant to the scale insect portion of the disease complex1
. Here we present a protocol that we have developed, based on the artificial infestation technique by Houston2
, which can be used to screen for scale-resistant trees in the field and in smaller potted seedlings and grafts. The identification of scale-resistant trees is an important component of management of BBD through tree improvement programs and silvicultural manipulation.
Environmental Sciences, Issue 87, Forestry, Insects, Disease Resistance, American beech, Fagus grandifolia, beech scale, Cryptococcus fagisuga, resistance, screen, bioassay
A Venturi Effect Can Help Cure Our Trees
Institutions: Unversity of Padova.
In woody plants, xylem sap moves upwards through the vessels due to a decreasing gradient of water potential from the groundwater to the foliage. According to these factors and their dynamics, small amounts of sap-compatible liquids (i.e.
pesticides) can be injected into the xylem system, reaching their target from inside. This endotherapic method, called "trunk injection" or "trunk infusion" (depending on whether the user supplies an external pressure or not), confines the applied chemicals only within the target tree, thereby making it particularly useful in urban situations. The main factors limiting wider use of the traditional drilling methods are related to negative side effects of the holes that must be drilled around the trunk circumference in order to gain access to the xylem vessels beneath the bark.
The University of Padova (Italy) recently developed a manual, drill-free instrument with a small, perforated blade that enters the trunk by separating the woody fibers with minimal friction. Furthermore, the lenticular shaped blade reduces the vessels' cross section, increasing sap velocity and allowing the natural uptake of an external liquid up to the leaves, when transpiration rate is substantial. Ports partially close soon after the removal of the blade due to the natural elasticity and turgidity of the plant tissues, and the cambial activity completes the healing process in few weeks.
Environmental Sciences, Issue 80, Trunk injection, systemic injection, xylematic injection, endotherapy, sap flow, Bernoulli principle, plant diseases, pesticides, desiccants
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
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
Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies
Institutions: The University of Western Ontario, Instituto de Ciencias de la Vid y el Vino, Ghent University, University of Amsterdam.
The two-spotted spider mite, Tetranychus urticae
, is a ubiquitous polyphagous arthropod herbivore that feeds on a remarkably broad array of species, with more than 150 of economic value. It is a major pest of greenhouse crops, especially in Solanaceae
, tomatoes, eggplants, peppers, cucumbers, zucchini) and greenhouse ornamentals (e.g.
, roses, chrysanthemum, carnations), annual field crops (such as maize, cotton, soybean, and sugar beet), and in perennial cultures (alfalfa, strawberries, grapes, citruses, and plums)1,2
. In addition to the extreme polyphagy that makes it an important agricultural pest, T. urticae
has a tendency to develop resistance to a wide array of insecticides and acaricides that are used for its control3-7
is an excellent experimental organism, as it has a rapid life cycle (7 days at 27 °C) and can be easily maintained at high density in the laboratory. Methods to assay gene expression (including in situ
hybridization and antibody staining) and to inactivate expression of spider mite endogenous genes using RNA interference have been developed8-10
. Recently, the whole genome sequence of T. urticae
has been reported, creating an opportunity to develop this pest herbivore as a model organism with equivalent genomic resources that already exist in some of its host plants (Arabidopsis thaliana
and the tomato Solanum lycopersicum
. Together, these model organisms could provide insights into molecular bases of plant-pest interactions.
Here, an efficient method for quick and easy collection of a large number of adult female mites, their application on an experimental plant host, and the assessment of the plant damage due to spider mite feeding are described. The presented protocol enables fast and efficient collection of hundreds of individuals at any developmental stage (eggs, larvae, nymphs, adult males, and females) that can be used for subsequent experimental application.
Environmental Sciences, Issue 89, two-spotted spider mite, plant-herbivore interaction, Tetranychus urticae, Arabidopsis thaliana, plant damage analysis, herbivory, plant pests
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
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
A Technical Perspective in Modern Tree-ring Research - How to Overcome Dendroecological and Wood Anatomical Challenges
Institutions: Swiss Federal Research Institute WSL.
Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to environmental changes and to finally reconstruct such changes. This is done by analyzing growth variations back in time and correlating various plant-specific parameters to (for example) temperature records. Integrating wood anatomical parameters in these analyses would strengthen reconstructions, even down to intra-annual resolution. We therefore present a protocol on how to sample, prepare, and analyze wooden specimen for common macroscopic analyses, but also for subsequent microscopic analyses. Furthermore we introduce a potential solution for analyzing digital images generated from common small and large specimens to support time-series analyses. The protocol presents the basic steps as they currently can be used. Beyond this, there is an ongoing need for the improvement of existing techniques, and development of new techniques, to record and quantify past and ongoing environmental processes. Traditional wood anatomical research needs to be expanded to include ecological information to this field of research. This would support dendro-scientists who intend to analyze new parameters and develop new methodologies to understand the short and long term effects of specific environmental factors on the anatomy of woody plants.
Environmental Sciences, Issue 97, Cell parameters, dendroecology, image analysis, micro sectioning, microtomes, sample preparation, wood anatomy
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.
Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce (Lactuca sativa) Germplasm Collections
Institutions: United States Department of Agriculture.
This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa
L.) germplasm was screened for likely drought-tolerance traits. Fresh water availability for agricultural use is a growing concern across the United States as well as many regions of the world. Short-term drought events along with regulatory intervention in the regulation of water availability coupled with the looming threat of long-term climate shifts that may lead to reduced precipitation in many important agricultural regions has increased the need to hasten the development of crops adapted for improved water use efficiency in order to maintain or expand production in the coming years. This protocol is not meant as a step-by-step guide to identifying at either the physiological or molecular level drought-tolerance traits in lettuce, but rather is a method developed and refined through the screening of thousands of different lettuce varieties. The nature of this screen is based in part on the streamlined measurements focusing on only three water-stress indicators: leaf relative water content, wilt, and differential plant growth following drought-stress. The purpose of rapidly screening a large germplasm collection is to narrow the candidate pool to a point in which more intensive physiological, molecular, and genetic methods can be applied to identify specific drought-tolerant traits in either the lab or field. Candidates can also be directly incorporated into breeding programs as a source of drought-tolerance traits.
Environmental Sciences, Issue 98, Lettuce, Lactuca sativa, drought, water-stress, abiotic-stress, relative water content
Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects
Institutions: Cornell University.
Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which include many crops such as cabbage, broccoli, Brussel sprouts etc. Rearing of the insects takes place on cabbage plants in the greenhouse. At least two cages are needed for the rearing of Pieris rapae. One for the larvae and the other to contain the adults, the butterflies. In order to investigate the role of plant hormones and toxic plant chemicals in resistance to this insect pest, we demonstrate two experiments. First, determination of the role of jasmonic acid (JA - a plant hormone often indicated in resistance to insects) in resistance to the chewing insect Pieris rapae. Caterpillar growth can be compared on wild-type and mutant plants impaired in production of JA. This experiment is considered "No Choice", because larvae are forced to subsist on a single plant which synthesizes or is deficient in JA. Second, we demonstrate an experiment that investigates the role of glucosinolates, which are used as oviposition (egg-laying) signals. Here, we use WT and mutant Arabidopsis impaired in glucosinolate production in a "Choice" experiment in which female butterflies are allowed to choose to lay their eggs on plants of either genotype. This video demonstrates the experimental setup for both assays as well as representative results.
Plant Biology, Issue 15, Annual Review, Plant Resistance, Herbivory, Arabidopsis thaliana, Pieris rapae, Caterpillars, Butterflies, Jasmonic Acid, Glucosinolates
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
Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control
Institutions: International Center for Tropical Agriculture (CIAT), Cali, Colombia , United States Department of Agriculture, Beltsville, Maryland, USA.
is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana
may play a role in protecting plants from herbivory and disease. This protocol demonstrates two inoculation methods to establish B. bassiana
endophytically in the common bean (Phaseolus vulgaris
), in preparation for subsequent evaluations of endophytic biological control. Plants are grown from surface-sterilized seeds for two weeks before receiving a B. bassiana
treatment of 108
conidia/ml (or water) applied either as a foliar spray or a soil drench. Two weeks later, the plants are harvested and their leaves, stems and roots are sampled to evaluate endophytic fungal colonization. For this, samples are individually surface sterilized, cut into multiple sections, and incubated in potato dextrose agar media for 20 days. The media is inspected every 2-3 days to observe fungal growth associated with plant sections and record the occurrence of B. bassiana
to estimate the extent of its endophytic colonization. Analyses of inoculation success compare the occurrence of B. bassiana
within a given plant part (i.e.
leaves, stems or roots) across treatments and controls. In addition to the inoculation method, the specific outcome of the experiment may depend on the target crop species or variety, the fungal entomopathogen species strain or isolate used, and the plant's growing conditions.
Bioengineering, Issue 74, Plant Biology, Microbiology, Infection, Environmental Sciences, Molecular Biology, Mycology, Entomology, Botany, Pathology, Agriculture, Pest Control, Fungi, Entomopathogen, Endophyte, Pest, Pathogen, Phaseolus vulgaris, Beauveria bassiana, Sustainable Agriculture, hemocytometer, inoculation, fungus
Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment
Institutions: Cornell University.
Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.
Plant Biology, Issue 15, Annual Review, Nicotine, Aphids, Plant Feeding Resistance, Tobacco
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
Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species
Institutions: Uppsala University, Swedish University of Agricultural Sciences.
The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ
hybridization, a technique used to localize cell specific mRNA expression. The in situ
hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ
experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies
). Here we present a modified DIG in situ
hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies
. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana
and Brassica napus
. The protocol worked equally well for the species and genes studied. AtAP3
were observed in second and third whorl floral organs in A. thaliana
and B. napus
and DAL13 in microsporophylls of male cones from P. abies
. For P. abies
the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ
protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundström at Jens.Sundstrom@vbsg.slu.se
Plant Biology, Issue 26, RNA, expression analysis, Norway spruce, Arabidopsis, rapeseed, conifers
Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR
Institutions: The Ohio State University.
Emerald ash borer (EAB, Agrilus planipennis
) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus
spp) in North America.
EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1st
-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1
) is exemplified as the gene of interest and a ribosomal protein (AP-RP1
) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.
Cellular Biology, Issue 39, quantitative real time-PCR, peritrophin, emerald ash borer, gene expression
Bioassays for Monitoring Insecticide Resistance
Institutions: University of Missouri, Delta Research Center, Louisiana State University Agricultural Center.
Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50
) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50
can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50
using the same methodology.
Microbiology, Issue 46, Resistance monitoring, Insecticide Resistance, Pesticide Resistance, glass-vial bioassay
A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors
Institutions: Texas A&M University (TAMU), Texas A&M University (TAMU).
Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus
(Canestrini), and the mosquito Aedes aegypti
(Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al
., 2002 and Stables et al
., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.
Immunology, Issue 50, Aequorin calcium reporter, coelenterazine, G protein-coupled receptor (GPCR), CHO-K1 cells, mammalian cell culture, neuropeptide SAR studies (SAR= structure-activity relationships), receptor-neuropeptide interaction, bioluminescence, drug discovery, semi-throughput screening in plates
Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example
Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1
. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2
. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1
.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1
. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria
spp. Several species of African grasses of the genus Brachiaria
are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4
.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5
Plant Biology, Issue 52, host plant resistance, antibiosis, antixenosis, tolerance, Brachiaria, spittlebugs
Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles
Institutions: Agricultural Research Service.
Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2
When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3
Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5
by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella
) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8
Plant Biology, Issue 63, bioassay, chemoreception, electroantennography, electrophysiological response, high-throughput, host-plant volatiles, navel orangeworm, screening tool
Vertical T-maze Choice Assay for Arthropod Response to Odorants
Institutions: University of Florida .
Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, various technologies are being developed as control tools. A subset of these tools focuses on modifying the behavior of arthropods by attraction or repulsion. Therefore, arthropods are often the focus of behavioral investigations. Various tools have been developed to measure arthropod behavior, including wind tunnels, flight mills, servospheres, and various types of olfactometers. The purpose of these tools is to measure insect or arachnid response to visual or more often olfactory cues. The vertical T-maze oflactometer described here measures choices performed by insects in response to attractants or repellents. It is a high throughput assay device that takes advantage of the positive phototaxis (attraction to light) and negative geotaxis (tendency to walk or fly upward) exhibited by many arthropods. The olfactometer consists of a 30 cm glass tube that is divided in half with a Teflon strip forming a T-maze. Each half serves as an arm of the olfactometer enabling the test subjects to make a choice between two potential odor fields in assays involving attractants. In assays involving repellents, lack of normal response to known attractants can also be measured as a third variable.
Biochemistry, Issue 72, Molecular Biology, Basic Protocols, Entomology, Behavior, Eukaryota, Organic Chemicals, Chemical Actions and Uses, Life Sciences (General), Behavioral Sciences, Arthropod behavior, chemical ecology, olfactometer, chemotaxis, olfaction, attraction, repulsion, odorant, T-maze, psyllid, Diaphorina citri, insect, anthropod, insect model
Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae
Institutions: Kansas State University, Indiana University School of Medicine, University of Notre Dame, University of Notre Dame, Kansas State University.
Vector mosquitoes inflict more human suffering than any other organism—
and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae
and the dengue and yellow fever vector Aedes aegypti
. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae
and A. aegypti
Following larval feedings, knockdown, which is verified through qRT-PCR or in situ
can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.
Molecular Biology, Issue 97, vector biology, RNA interference, Anopheles gambiae, Aedes aegypti, dsRNA, siRNA, knockdown, ingestion, mosquito, larvae, development, disease