SM, YP, and VN acknowledge the fellowship awarded by the University Grants Commission, Government of India, New Delhi. RJ acknowledges the Council of Scientific and Industrial Research (CSIR), India, and CSIR-National Chemical Laboratory, Pune, India, for financial support under project codes MLP036626, MLP101526, and YSA000826.

H. armigera larvae were acquired from ICAR-National Bureau of Agricultural Insect Resources (NBAIR), Bangalore, India. A total of 21 second instar larvae were used for the present study.
1.&#160;Preparation of chickpea-based artificial diet
NOTE: A list of ingredients required for preparing an artificial diet is mentioned in Table 1.
<ol>
	<li>Weigh all the fractions separately in a beaker, as listed in Table 1, and prepare a homogenous mixture using a spatula/magnetic stirrer.</li>
	<li>Boil Fraction C at around 100 &#176;C using a microwave for 5 min, add to Fraction A, and mix it thoroughly.</li>
	<li>After thoroughly mixing, let the mixed fraction cool down a little before adding Fraction B (Fraction B contains heat-labile components).</li>
	<li>Pour into a transparent, polystyrene, 150 mm x 150 mm Petri dish.</li>
</ol>
2. Preparation of quercetin-containing artificial diet
<ol>
	<li>Weigh the appropriate amount (1,000 ppm) of quercetin hydrate (see Table of Materials) and dissolve it properly into the minimum volume of organic solvents, such as ethanol (2 mg/mL), dimethyl sulfoxide (DMSO; 30 mg/mL), or dimethyl formamide (DMF). Here, DMSO is used for dissolving quercetin.</li>
	<li>Add dissolved quercetin into Fraction B, followed by addition into the mixture of Fractions A and C (the volume of water reduced from Fraction B equals the volume of DMSO added).</li>
	<li>Add an equal volume of organic solvent used for dissolving quercetin into the control diet. 
	&#8203;NOTE: Figure 1 shows the schematic representation of preparing artificial and quercetin-containing diets.</li>
</ol>
3. Rearing and maintenance of H. armigera culture
NOTE: Use appropriately cleaned and sterilized materials for insect rearing and maintenance. Handle the insects carefully by following all sterility and safety-related standard operating practices16,17,18.
<ol>
	<li>Keep H. armigera eggs in the breeding chamber (plastic jar covered with muslin cloth) with maintained conditions, as described in step 3.3. Then, gently transfer newly emerged neonates using a fine paintbrush on a freshly prepared chickpea-based artificial diet.</li>
	<li>Use an artificial diet for rearing the larvae, and 20% (w/v) sucrose solution with 1% (w/v) multi-vitamin (see Table of Materials) for adult moths19,20. 
	NOTE: As third and older instar larvae of H. armigera show a cannibalistic tendency, it is necessary to rear each larva in a separate vial.</li>
	<li>Maintain the temperature at 25 &#177; 1 &#176;C and relative humidity at 70% in the insect culture room, with a 16 h light:8 h dark photoperiod21.</li>
	<li>Rear one generation of insects in the laboratory for homogeneity and then use it for feeding assay.</li>
	<li>Optionally, increase the temperature of the insect culture room to 28 &#176;C to speed up the growth of larvae and pupae22.</li>
</ol>
4. Setup for feeding assay
<ol>
	<li>Collect 21 second instar larvae for each set (control and treatment) and keep them away from the diet, for approximately 1-3 h.</li>
	<li>Cut the control and quercetin-containing diet into small pieces, record the weight of the diet given and the insect&#39;s body, and carefully transfer the insects into culture vials. Allow the insects to feed on the respective diet. 
	NOTE: This should be considered as Day 0 of the feeding assay.</li>
	<li>Record the weight of the insect body, given diet, uneaten diet, and frass on alternate days (Days 2, 4, 6, 8, and 10) till the 10th day of assay.</li>
	<li>After Day 10, keep them feeding on their respective diet to observe further developmental and morphological changes. 
	NOTE: The developmental changes by means of: (1) larval-pupal intermediates, such as the posterior half body of pupae with larval cuticle patches, a head capsule, and thoracic legs; (2) prepupae with a completely blackened body; (3) undersized pupae with body shrinkage; (4) pupal-moth intermediates-moths with the old pupal skin. Morphological changes include malformed moth adults with abnormal bodies, twisted wings, and jointed legs. These changes are then compared with insects fed on the control diet.</li>
	<li>Freeze the insects on Day 10 if the study of developmental and morphological defects is not required. 
	&#8203;NOTE: Before freezing the larvae, they need to be kept deprived of the diet for at least 3 h to remove residual diet from the digestive tract.</li>
</ol>
5. Data recording and analysis
<ol>
	<li>In GraphPad Prism software (see Table of Materials), choose an XY data table from the &#34;Welcome or New Table&#34; dialog, and in that enter the number of insects replicate values side-by-side in the sub-columns. Then, give the title name to the X-axis as number of days, and in groups A and B, give the title name as control and quercetin treatment, respectively. Put the body weight of each insect under control and treatment to generate the body weight graph. 
	NOTE: Analysis in GraphPad may vary according to the sample size and the number of treatments.</li>
	<li>Compare the insect body weight between the control and treatment groups using a student t-test (&#945; = 0.05).</li>
	<li>Count the live and dead larvae and pupae on Day 10 to plot a Kaplan-Meier curve for survival percentage using the graphing software.</li>
	<li>Count the number of pupae and calculate the percentage of pupation using the given formula:</li>
	<li>Percentage of pupation (%) = (number of pupae formed/total number of larvae) x 100</li>
	<li>Compare larval development in terms of nutritional indices23&#160;using&#160;the following formulas, ECI (%) = (weight gain of larvae/weight of eaten feed) x 100 
	ECD (%) = (weight gain of larvae/[weight of eaten feed - weight of frass]) x 100 
	AD (%) = ([weight of eaten feed - weight of frass]/weight of eaten feed) x 100</li>
</ol>

Assessment of Phytochemical Effects on Helicoverpa armigera Through Feeding Assay

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The authors declared no conflict of interest.

Laboratory bioassays are useful to predict outcomes and produce comparative toxicity data on several compounds in a short period at a reasonable cost. The feeding bioassay helps to interpret the interactions between insect-insecticide and insect-plant-insecticides. It is an efficient method for measuring the toxicity of a variety of substances that significantly simplifies the process of establishing the lethal dose 50 (LD50), lethal concentration 50 (LC50), or any other lethal concentration or dose...

The biotic factors that affect crop productivity are mainly pathogenic agents and pests. Several insect pests cause 15% to 35% of agricultural crop loss and affect economic sustainability practices1. Insects belonging to the orders Coleoptera, Hemiptera, and Lepidoptera are the major orders of devastating pests. The highly adaptive nature of the environment has benefited lepidopterans in evolving several survival mechanisms. Amongst lepidopteran insects, Helicoverpa armigera (Cotton bollworm) can feed on around 180 different crops and cause significant damage to their reproductive tissues2. Worldwide, H. armigera infestation has resulted in a loss of around $5 billion3. Cotton, chickpeas, pigeon peas, tomatoes, sunflowers, and other crops are hosts for H. armigera. It completes its lifecycle on different parts of host plants. Eggs laid by female moths get hatched on the leaves, followed by their feeding on vegetative tissues during larval stages. The larval stage is the most destructive due to its voracious and highly adaptable nature4,5. H. armigera shows a global distribution and encroachment to new territories due to its remarkable attributes, such as polyphagy, excellent migratory abilities, higher fecundity, strong diapause, and the emergence of resistance to existing insect control strategies6.
Diverse chemical molecules from terpenes, flavonoids, alkaloids, polyphenols, cyanogenic glucosides, and many others are widely used for the control of H. armigera infestation7. However, frequent application of chemical molecules imparts adverse effects on the environment and human health due to the acquisition of their residues. Also, they show a detrimental effect on various pest predators, resulting in an ecological imbalance8,9. Therefore, there is a necessity to investigate safe and eco-friendly options for chemical molecules of pest control.
Natural insecticidal molecules produced by plants (phytochemicals) can be used as a promising alternative to chemical pesticides. These phytochemicals include various secondary metabolites belonging to the classes alkaloids, terpenoids, and phenolics7,10. Quercetin is one of the most abundant flavonoids (phenolic compound) present in various grains, vegetables, fruits, and leaves. It shows feeding deterrent and insecticidal activity against insects; also, it is not harmful to natural enemies of pests11,12. Thus, this protocol demonstrates the feeding assay using quercetin to assess its toxic effect on H. armigera.
Various bioassay methods have been developed to evaluate the effect of natural and synthetic molecules on an insect&#39;s feeding, growth, development, and behavioral patterns13. Commonly used methods include the leaf disk assay, choice feeding assay, droplet feeding assay, contact assay, diet covering assay, and obligate feeding assay13,14. These methods are classified based on how pesticides are applied to insects. The obligate feeding assay is one of the most commonly used, sensitive, simple, and adaptable methods to test probable insecticides and their lethal dose14. In an obligate feeding assay, the molecule of interest is mixed with an artificial diet. This provides consistency and control over the diet composition, generating robust and reproducible results. Important variables affecting feeding assays are the developmental stage of the insect, choice of insecticide, environmental factors, and sample size. The duration of the assay, interval between two data recordings, frequency and amount of diet fed, health of insects, and handling skill of operators can also influence the outcome of feeding assays14,15.
This study aims to demonstrate the obligate feeding assay to evaluate the effect of quercetin on H. armigera survival and fitness. Assessment of various parameters, such as insect body weight, mortality rate, and developmental defects, will provide insights into the insecticidal effects of quercetin. Meanwhile, measuring nutritional parameters, including the efficiency of conversion of ingested food (ECI), efficiency of conversion of digested food (ECD), and approximate digestibility (AD), will highlight the antifeedant attributes of quercetin.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar Agar</td>
			<td>Himedia</td>
			<td>RM666</td>
			<td>Solidifying agent</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Himedia</td>
			<td>CMS1014</td>
			<td>Vitamin C source</td>
		</tr>
		<tr>
			<td>Bengal Gram</td>
			<td>NA</td>
			<td>NA</td>
			<td>Protein and carbohydrate source</td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma</td>
			<td>C-5890</td>
			<td>Protein source</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sisco Research Laboratories</td>
			<td>34811</td>
			<td>Fatty acid source</td>
		</tr>
		<tr>
			<td>Choline Chloride</td>
			<td>Himedia</td>
			<td>GRM6824</td>
			<td>Ammonium salt</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>67-68-5</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>GraphPad Prism v8.0</td>
			<td></td>
			<td></td>
			<td>https://www.graphpad.com/guides/prism/latest/user-guide/using_choosing_an_analysis.htm</td>
		</tr>
		<tr>
			<td>Methyl Paraben</td>
			<td>Himedia</td>
			<td>GRM1291</td>
			<td>Antifungal agent</td>
		</tr>
		<tr>
			<td>Multivitamin capsule</td>
			<td>GalaxoSmithKline</td>
			<td>NA</td>
			<td>Vitamin source</td>
		</tr>
		<tr>
			<td>Quercetin</td>
			<td>Sigma</td>
			<td>Q4951-10G</td>
			<td>Phytochemical</td>
		</tr>
		<tr>
			<td>Sorbic Acid</td>
			<td>Himedia</td>
			<td>M1880</td>
			<td>Antimicrobail agent</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Himedia</td>
			<td>CMS220</td>
			<td>Antibiotic</td>
		</tr>
		<tr>
			<td>Vitamin E capsule</td>
			<td>Nukind Healthcare</td>
			<td>NA</td>
			<td>Vitamin E source</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Himedia</td>
			<td>RM027</td>
			<td>Amino acid source</td>
		</tr>
	</tbody>
</table>

developing a feeding assay system for evaluating the insecticidal effect of phytochemicals on helicoverpa armigera

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and agricultural productivity. In response, plants produce several phytochemicals that negatively impact the insect's growth and survival. This protocol demonstrates an obligate feeding assay method to evaluate the effect of a phytochemical (quercetin) on insect growth, development, and survival. Under controlled conditions, the neonates were maintained until the second instar on a pre-defined artificial diet. These second-instar larvae were allowed to feed on a control and quercetin-containing artificial diet for 10 days. The insects' body weight, developmental stage, frass weight, and mortality were recorded on alternate days. The change in body weight, the difference in feeding pattern, and developmental phenotypes were evaluated throughout the assay time. The described obligatory feeding assay simulates a natural mode of ingestion and can be scaled up to a large number of insects. It permits one to analyze phytochemicals' effect on the growth dynamics, developmental transition, and overall fitness of H. armigera. Furthermore, this setup can also be utilized to evaluate alterations in nutritional parameters and digestive physiology processes. This article provides a detailed methodology for feeding assay systems, which may have applications in toxicological studies, insecticidal molecule screening, and understanding chemical effects in plant-insect interactions.

Insect larvae fed on a diet containing 1,000 ppm quercetin showed a significant decrease in body weight of ~57% as compared to the control group (Figure 2A). The reduction in body weight resulted in a reduced body size of quercetin-treated larvae (Figure 2B). A notable reduction was observed in the feeding rate of quercetin-fed larvae as compared to the control (Figure 2C).
Also, larvae fed on quercetin s...

Watch this Scientific Journal Video about Development and Challenges of Feeding Assays for Insect Pest Management at JoVE.com

Author Spotlight: Development and Challenges of Feeding Assays for Insect Pest Management 

This protocol describes the obligate feeding assay to evaluate the potentially toxic effect of a phytochemical on the lepidopteran insect larvae. This is a highly scalable insect bioassay, easy to optimize the sublethal and lethal dose, deterrent activity, and physiological effect. This could be used for screening eco-friendly insecticides.

Developing a Feeding Assay System for Evaluating the Insecticidal Effect of Phytochemicals on Helicoverpa armigera

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and ...

developing-feeding-assay-system-for-evaluating-insecticidal-effect

Research

JoVE Journal

Biology

973 Views.  CSIR-National Chemical Laboratory. This protocol describes the obligate feeding assay to evaluate the potentially toxic effect of a phytochemical on the lepidopteran insect larvae. This is a highly scalable insect bioassay, easy to optimize the sublethal and lethal dose, deterrent activity, and physiological effect. This could be used for screening eco-friendly insecticides.

Video: Author Spotlight: Development and Challenges of Feeding Assays for Insect Pest Management 

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity greatly impacts cell behavior to regulate normal and pathologic processes including cell proliferation, differentiation, adhesion signaling and directional migration. In this regard, the innate ability of certain cell types to migrate towards a stiffer, or less compliant matrix substrate is referred to as durotaxis. This phenomenon plays an important role during embryonic development, wound repair and cancer cell invasion. Here, we describe a straightforward assay to study durotaxis, in vitro, using polydimethylsiloxane (PDMS) substrates. Preparation of the described durotaxis chambers creates a rigidity interface between the relatively soft PDMS gel and a rigid glass coverslip. In the example provided, we have used these durotaxis chambers to demonstrate a role for the cdc42/Rac1 GTPase activating protein, cdGAP, in mechanosensing and durotaxis regulation in human U2OS osteosarcoma cells. This assay is readily adaptable to other cell types and/or knockdown of other proteins of interests to explore their respective roles in mechanosignaling and durotaxis.

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential nutrient or produces antimicrobial compounds that its competitor is not resistant to. Here we describe a deferred growth inhibition assay, a method for assessing the ability of one bacterium to inhibit the growth of another through the production of antimicrobial compounds or through competition for nutrients. This technique has been used to investigate the correlation of nasal isolates with the exclusion of particular species from a community. This technique can also be used to screen for lantibiotic producers or potentially novel antimicrobials. The assay is performed by first culturing the test inhibitor-producing strain overnight on an agar plate, then spraying over the test competitor strain and incubating again. After incubation, the extent of inhibition can be measured quantitatively, through the size of the zone of clearing around the inhibitor-producing strain, and qualitatively, by assessing the clarity of the inhibition zone. Here we present the protocol for the deferred inhibition assay, describe ways to minimize variation between experiments, and define a clarity scale that can be used to qualitatively assess the degree of inhibition.

The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition.

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential ...

High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich meal, which consists of an emulsion of chicken egg yolk liposomes created by sonicating egg yolk in embryo media. Detailed instructions are provided to screen larvae for egg yolk consumption so that larvae that fail to feed will not confound experimental results. The chicken egg yolk liposomes can be spiked with fluorescent lipid analogs, including fatty acids and cholesterol, enabling both systemic and subcellular visualization of dietary lipid processing. Several methods are described to mount larvae that are conducive to short- and long-term live imaging with both upright and inverted objectives at high and low magnification. Additionally presented is an assay to quantify larval food intake by extracting the lipids of larvae fed fluorescent lipid analogs, spotting the lipids on a thin layer chromatography plate, and quantifying the fluorescence. Finally, critical aspects of the procedures, important controls, options for modifying the protocols to address specific experimental questions, and potential limitations are discussed. These techniques can be applied not only to focused, hypothesis driven inquiries, but also to a variety of screens and live imaging techniques to study dietary lipid metabolism and the control of food intake.

Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques.

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich ...

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby the permeability of the endothelium to blood cells, plasma macromolecules, and water can be adapted according to the physiological need. In certain diseases, cytokines and growth factors are released that target the endothelial barrier to transiently increase vascular permeability; however, their prolonged presence may cause chronic vascular hyperpermeability and thereby tissue-damaging edema. The Miles assay is an in vivo technique that allows researchers to study vascular hyperpermeability through the proxy measurement of vascular leakage. Here, we provide a detailed protocol on how to perform this procedure in the mouse, which is the most widely used model organism to study mammalian physiology and pathology. The procedure involves the intravenous injection of Evans blue dye to label the circulating albumin followed by multiple intradermal injections of permeability-inducing agents and vehicle control solutions into opposing flanks of the mouse. Consequently, Evans blue dye gradually leaks into the dermis, where it accumulates and can be extracted for quantification as leakage induced by the permeability-inducing agent relative to the vehicle. The Miles assay can be performed in wild type or genetically modified mouse models and may be combined with drug administration to study molecular mechanisms that regulate vascular permeability and identify agents/targets capable of inducing or blocking hyperpermeability.

Here, we present a protocol to measure the vascular leakage induced by intradermal administration of permeability promoting agents into the murine skin. This technique can be used to determine the ability of molecules to promote or inhibit vascular leakage or to study the molecular mechanisms that regulate vascular permeability.

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been conducted to investigate the effect of gut microbes as a whole; however, different microbes could exert distinct effects toward the host. This article provides the methodology to study the effect of each specific mosquito gut microbe and the potential mechanism.
This protocol contains two parts. The first part introduces how to dissect the mosquito midgut, isolate cultivable bacteria colonies, and identify bacteria species. The second part provides the procedure to generate antibiotic-treated mosquitoes and reintroduce one specific bacteria species.

This article presents a protocol to investigate the effect of individual mosquito gut bacteria, including isolation and identification of mosquito midgut cultivable microbes, antibiotic depletion of mosquito gut bacteria, and reintroduce one specific bacteria species.

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been ...