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

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. 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 nature^4,5. 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 imbalance^8,9. 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 phenolics^7,10. 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 pests^11,12. 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'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 assay^13,14. 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 assays^14,15.

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.

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, and prepare a homogenous mixture using a spatula/magnetic stirrer.
2. Boil Fraction C at around 100 °C using a microwave for 5 min, add to Fraction A, and mix it thoroughly.
3. After thoroughly mixing, let the mixed fraction cool down a little before adding Fraction B (Fraction B contains heat-labile components).
4. Pour into a transparent, polystyrene, 150 mm x 150 mm Petri dish.

2. Preparation of quercetin-containing artificial diet

1. 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.
2. 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).
3. Add an equal volume of organic solvent used for dissolving quercetin into the control diet.
   ​NOTE: Figure 1 shows the schematic representation of preparing artificial and quercetin-containing diets.

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 practices^16,17,18.

1. 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.
2. 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 moths^19,20.
   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.
3. Maintain the temperature at 25 ± 1 °C and relative humidity at 70% in the insect culture room, with a 16 h light:8 h dark photoperiod²¹.
4. Rear one generation of insects in the laboratory for homogeneity and then use it for feeding assay.
5. Optionally, increase the temperature of the insect culture room to 28 °C to speed up the growth of larvae and pupae²².

4. Setup for feeding assay

1. Collect 21 second instar larvae for each set (control and treatment) and keep them away from the diet, for approximately 1-3 h.
2. Cut the control and quercetin-containing diet into small pieces, record the weight of the diet given and the insect'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.
3. 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.
4. 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.
5. Freeze the insects on Day 10 if the study of developmental and morphological defects is not required.
   ​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.

5. Data recording and analysis

1. In GraphPad Prism software (see Table of Materials), choose an XY data table from the "Welcome or New Table" 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.
2. Compare the insect body weight between the control and treatment groups using a student t-test (α = 0.05).
3. Count the live and dead larvae and pupae on Day 10 to plot a Kaplan-Meier curve for survival percentage using the graphing software.
4. Count the number of pupae and calculate the percentage of pupation using the given formula:
5. Percentage of pupation (%) = (number of pupae formed/total number of larvae) x 100
6. Compare larval development in terms of nutritional indices²³ using 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

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...

Materials

Name	Company	Catalog Number	Comments
Agar Agar	Himedia	RM666	Solidifying agent
Ascorbic acid	Himedia	CMS1014	Vitamin C source
Bengal Gram	NA	NA	Protein and carbohydrate source
Casein	Sigma	C-5890	Protein source
Cholesterol	Sisco Research Laboratories	34811	Fatty acid source
Choline Chloride	Himedia	GRM6824	Ammonium salt
DMSO	Sigma	67-68-5	Solvent
GraphPad Prism v8.0			https://www.graphpad.com/guides/prism/latest/user-guide/using_choosing_an_analysis.htm
Methyl Paraben	Himedia	GRM1291	Antifungal agent
Multivitamin capsule	GalaxoSmithKline	NA	Vitamin source
Quercetin	Sigma	Q4951-10G	Phytochemical
Sorbic Acid	Himedia	M1880	Antimicrobail agent
Streptomycin	Himedia	CMS220	Antibiotic
Vitamin E capsule	Nukind Healthcare	NA	Vitamin E source
Yeast Extract	Himedia	RM027	Amino acid source