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><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 ...

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Research

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Biology

984 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 

A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or concentration-response curve, where mortality increases as the dose or concentration of an insecticide is increased. Standard lethal concentration (LC50) and lethal dose (LD50) tests that result in 50% mortality of a test population can be challenging for evaluating toxicity of insecticides against non-adult insect life stages, such as eggs and early instar or nymphal stages. However, this information is essential for understanding insecticide efficacy in all bed bug life stages, which affects control and treatment efforts. This protocol uses a standard dipping bioassay modified for bed bug eggs and a contact insecticidal assay for treating nymphal first instars. These assays produce a concentration-response curve to further quantify LC50 values for insecticide evaluations.

A Method for Evaluating Insecticide Efficacy against Bed Bug,  Cimex lectularius, Eggs and First Instars

Insecticide evaluations are often targeted against adult insects, rather than immature stages. Here, we present a protocol for evaluating insecticides against bed bug eggs with a comparison to the first nymphal bed bug stage. These protocols could be adjusted for other insects to evaluate insecticide efficacy in non-adult life stages.

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or ...

Environment

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were found up-regulated in the resistant house fly strain, whereas their roles in conferring insecticide resistance remained to be explored. Here, we designed a protocol for the functional characterization of carboxylesterases. Three example experiments are presented: (1) expression and isolation of carboxylesterase proteins through a baculovirus-mediated insect Spodoptera frugiperda (Sf9) cell expression system; (2) a cell-based MTT (3-[4, 5-dimethykthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) cytotoxicity assay to measure the tolerance of insect cells to different permethrin treatments; and (3) in vitro metabolic studies to explore the metabolic capabilities of carboxylesterases toward permethrin. The carboxylesterase gene Md&#945;E7 was cloned from a resistant house fly strain ALHF and used to construct a recombinant baculovirus for Sf9 cells infection. The cell viabilities against different permethrin treatments were measured with the MTT assay. The enhanced cell tolerance of the experimental group (Md&#945;E7-recombinant baculovirus infected cells) compared with those of the control groups (CAT-recombinant baculovirus infected cells and GFP-recombinant baculovirus infected cells) to permethrin treatments suggested the capabilities of Md&#945;E7 in metabolizing insecticides, thereby protecting cells from chemical damages. Besides that, carboxylesterase proteins were expressed in insect Sf9 cells and isolated to conduct an in vitro metabolic study. Our results indicated a significant in vitro metabolic efficiency of Md&#945;E7 toward permethrin, directly indicating the involvement of carboxylesterases in metabolizing insecticides and thus conferring insecticide resistance in house flies.

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Here, we present a protocol to produce house fly carboxylesterase proteins in vitro with a baculovirus mediated insect cell expression system and later functionally characterize their roles in metabolizing permethrin, thereby, conferring pyrethroid resistance by conducting cell-based MTT assay and in vitro metabolic studies.

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were ...

Biochemistry

Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei

Prior to recommending insecticides to treat the coffee berry borer (CBB) Hypothenemus hampei, it is valuable to know the mortality and repellency of these insecticides against adult insects or their impact on reproductive output. However, currently available methods assess adult mortality only, limiting the selection of novel insecticides with a different mode of action. In this work, different experimental methods were examined to identify the diverse effects on the CBB under laboratory conditions. For this, green coffee fruits (GFs) were collected and disinfected by immersion in sodium hypochlorite solution followed by UV light irradiation. In parallel, CBB adults from a colony were disinfected by immersion in sodium hypochlorite solution. To assess fruit protection (preinfestation), the fruits were placed in plastic boxes, and the insecticides were applied. Then, the CBB adults were released at a rate of two CBBs per GF. The GFs were left under controlled conditions to evaluate CBB infestation and survival after 1, 7, 15, and 21 days. To evaluate insecticide efficacy after CBB infestation (postinfestation), CBB adults were released to the GFs in a 2:1 ratio for 3 h at 21 &#176;C. Infested fruits showing CBB adults with their abdomens partially exposed were selected and placed in 96-well racks, and the CBBs boring into the fruits were treated directly. After 20 days, the fruits were dissected, and the CBB biological stages inside each fruit were recorded. The GFs served as substrates that mimic natural conditions to evaluate toxic, chemical, and biological insecticides against the CBB.

Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei

A method using green coffee fruits (GFs) was developed to test the toxicity of insecticides against the coffee berry borer (CBB). Insecticides or toxic substances were applied to disinfected GFs before or after CBB infestation. Insect mortality, repellency, and reproductive capacity, in addition to other parameters, were evaluated.

Prior to recommending insecticides to treat the coffee berry borer (CBB) Hypothenemus hampei, it is valuable to know the mortality and repellency of these ...

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (H&#252;bner)

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional genomics, and behavioral studies has made H. armigera a model species in Lepidoptera Noctuidae. To study the in vivo functions of and interactions between different genes, clustered regularly interspaced short palindromic repeats (CRISPR)/ associated protein 9 (Cas9) genome editing technology is a convenient and effective method used for performing functional genomic studies. In this study, we provide a step-by-step systematic method to complete gene knockout in H. armigera using the CRISPR/Cas9 system. The design and synthesis of guide RNA (gRNA) are described in detail. Then, the subsequent steps consisting of gene-specific primer design for guide RNA (gRNA) creation, embryo collection, microinjection, insect rearing, and mutant detection are summarized. Finally, troubleshooting advice and notes are provided to improve the efficiency of gene editing. Our method will serve as a reference for the application of CRISPR/Cas9 genome editing in H. armigera as well as other Lepidopteran moths.

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera H&#252;bner

Presented here is a protocol of Helicoverpa armigera (H&#252;bner) embryo microinjection and knockout mutant identification created by CRISPR/Cas9 genome editing. Mutant insects enable further research of gene function and interaction among different genes in vivo.

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional ...

Behavior

Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ingestion of provisions contaminated with pesticides may increase the mortality rate of the larvae, decrease the collection rate and the population of adult solitary bees in the next year from a demographic perspective. But there are limited studies on the effects of pesticides on the larvae of solitary bees. Therefore, understanding how pesticides influence the larvae of solitary bees should be considered an integral part of pesticide ecological risk assessment. This study presents a method to expose the larvae of solitary bee, Osmia excavata, to lethal or sublethal doses of pesticide, tracking larval weight gain, developmental duration, eclosion ability, and food consumption efficiency conversion of ingested food. To demonstrate the effectiveness of this method, the larvae of O. excavata were fed with provisions containing acute lethal and sublethal doses of chlorpyrifos. Then, the above indexes of the treated larvae were investigated. This technique helps to predict and mitigate the risk of pesticides to pollinators.

The present protocol explains a method to feed pesticide-contaminated provisions to the larvae of the solitary bees, Osmia excavata. The procedure examines the ecotoxicity of the pesticide to the larvae of the solitary bees.

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ...

Bioassays for Monitoring Insecticide Resistance

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.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with&#160;the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along&#160;a range of insecticide concentrations.
This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. ...

Optimized Bioassay for Entomopathogenic Fungi Against Mustard Aphid

Assessment of Aphidicidal Effect of Entomopathogenic Fungi against Parthenogenetic Insect, Mustard Aphid, Lipaphis erysimi (Kalt.)

The mustard aphid (L. erysimi) is a pest that infests various cruciferous crops and transmits plant viruses. To achieve eco-friendly pest management, entomopathogenic fungi (EPF) are potential microbial control agents for controlling this pest. Therefore, virulence screening of EPF isolates under Petri dish conditions is necessary before field application. However, the mustard aphid is a parthenogenetic insect, making it difficult to record data during Petri dish experiments. A modified system for detached-leaf bioassays was developed to address this issue, using a micro-sprayer to inoculate conidia onto aphids and prevent drowning by facilitating air-drying after spore suspension. The system maintained high relative humidity throughout the observation period, and the leaf disc remained fresh for over ten days, allowing parthenogenetic reproduction of the aphids. To prevent offspring buildup, a process of daily removal using a painting brush was implemented. This protocol demonstrates a stable system for evaluating the virulence of EPF isolates against mustard aphids or other aphids, enabling the selection of potential isolates for aphid control.

Author Spotlight: Eco-Friendly Pest Management Using Entomopathogenic Fungi Against Mustard Aphids 

This protocol presents an optimized detached-leaf bioassay system for evaluating the effectiveness of entomopathogenic fungi (EPF) against the mustard aphid (Lipaphis erysimi (Kalt.)), a parthenogenetic insect. The method outlines the data collection process during Petri dish experiments, enabling researchers to consistently measure the virulence of EPF against mustard aphids and other parthenogenetic insects.

The mustard aphid (L. erysimi) is a pest that infests various cruciferous crops and transmits plant viruses. To achieve eco-friendly pest management, ...

Larval Contact Assay: A Procedure to Evaluate Mosquito Larval Mortality following Direct Contact with Test Larvicidal Compound

Source: Brito-Sierra, C. A., et al. <a href="https://www.jove.com/v/57768">Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes</a> J. Vis. Exp. (2019)
In this video, we describe the larval contact assay to evaluate the toxicity of the dopamine receptor antagonist amitriptyline on the third instar stage larvae of mosquitoes, known vectors of several human infectious diseases. Assessing the larval mortality upon incubation with different amitriptyline concentrations over different time intervals helps determine its lethal concentration 50.

Source: Brito-Sierra, C. A., et al. Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes J. Vis. Exp. (2019)
In this video, ...

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

Summary

Explore More Videos

Chapters in this video

A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (Hübner)

Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees

Bioassays for Monitoring Insecticide Resistance

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

Assessment of Aphidicidal Effect of Entomopathogenic Fungi against Parthenogenetic Insect, Mustard Aphid, Lipaphis erysimi (Kalt.)

Larval Contact Assay: A Procedure to Evaluate Mosquito Larval Mortality following Direct Contact with Test Larvicidal Compound