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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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. arm....

Protocol

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. Preparation of chickpea-based artificial diet

NOTE: A list of ingredients required for preparing an artificial diet is mentioned in Table 1.

  1. Weigh all the fractions separately in a beaker, as listed in Table 1

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
Agar AgarHimediaRM666Solidifying agent
Ascorbic acidHimediaCMS1014Vitamin C source
Bengal GramNANAProtein and carbohydrate source
CaseinSigmaC-5890Protein source
CholesterolSisco Research Laboratories34811Fatty acid source
Choline ChlorideHimediaGRM6824Ammonium salt
DMSOSigma67-68-5Solvent
GraphPad Prism v8.0https://www.graphpad.com/guides/prism/latest/user-guide/using_choosing_an_analysis.htm
Methyl ParabenHimediaGRM1291Antifungal agent
Multivitamin capsuleGalaxoSmithKlineNAVitamin source
QuercetinSigmaQ4951-10GPhytochemical
Sorbic AcidHimediaM1880Antimicrobail agent
StreptomycinHimediaCMS220Antibiotic
Vitamin E capsuleNukind HealthcareNAVitamin E source
Yeast ExtractHimediaRM027Amino acid source

References

  1. Popp, J., Pető, K., Nagy, J. Pesticide productivity and food security. A review. Agronomy for Sustainable Development. 33 (1), 243-255 (2013).
  2. da Silva, F. R., et al. Comparative tox....

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Feeding AssayPhytochemicalsInsecticidal EffectHelicoverpa ArmigeraObligate Feeding AssayQuercetinArtificial DietInsect GrowthInsect DevelopmentInsect Survival

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