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 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'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.
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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.
- Weigh all the fractions separately in a beaker, as listed in Table 1, and prepare a homogenous mixture using a spatula/magnetic stirrer.
- Boil Fraction C at around 100 °C using a microwave for 5 min, add to Fraction A, and mix it thoroughly.
- After thoroughly mixing, let the mixed fraction cool down a little before adding Fraction B (Fraction B contains heat-labile components).
- Pour into a transparent, polystyrene, 150 mm x 150 mm Petri dish.
2. Preparation of quercetin-containing artificial diet
- 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.
- 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).
- 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 practices16,17,18.
- 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.
- 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. - 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.
- Rear one generation of insects in the laboratory for homogeneity and then use it for feeding assay.
- 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
- Collect 21 second instar larvae for each set (control and treatment) and keep them away from the diet, for approximately 1-3 h.
- 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. - 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.
- 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. - 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
- 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. - Compare the insect body weight between the control and treatment groups using a student t-test (α = 0.05).
- Count the live and dead larvae and pupae on Day 10 to plot a Kaplan-Meier curve for survival percentage using the graphing software.
- Count the number of pupae and calculate the percentage of pupation using the given formula:
- Percentage of pupation (%) = (number of pupae formed/total number of larvae) x 100
- 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
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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 showed a decrease in pupation rate by ~14% and delayed pupation, suggesting developmental retardation upon treatment (Figure 3A,B). Furthermore, ~77.65% of survival and lethal phenotypes were observed in insect larvae fed on a quercetin-containing diet (Figure 4A,B). The nutritional parameters were calculated for control and quercetin-fed larvae based on the consumption and utilization of food (Supplementary Table 1). The ECI to body matter and the ECD for insects fed on the 1,000 ppm quercetin-containing diet were reduced by ~9 % and ~49%, respectively. The decrease in ECD can be due to the lack of available metabolites in the insect body20. The AD of quercetin-fed insects was increased by ~5% compared to the control (Table 2). Overall, the obtained results indicate that quercetin has significant negative effects on insect growth and the development of H. armigera.
Figure 1: Schematic representation of the preparation of an artificial diet and quercetin-containing diet. Fractions A, B, and C are mixed to make an artificial and quercetin-containing diet. The larvae are fed on the respective diet for 10 days. Blue process arrows represent an artificial diet, while red process arrows represent the preparation of a quercetin-containing diet. Please click here to view a larger version of this figure.
Figure 2: Representative data from the quercetin feeding assay. (A) Body weight graph of H. armigera larvae following the feeding of 1,000 ppm quercetin compared to the control on Days 2, 4, 6, 8, and 10. The body weight of larvae is in milligrams (mg). (B) The average size of larvae is recorded on Day 10. Scale bar = 1 cm. (C) Average feeding rate recorded on Days 2, 4, 6, 8, and 10. The weight of the feed is in milligrams (mg). Blue circles and red squares represent the average data of the control and quercetin-treated insects on alternate days, respectively. Student t-test is used for comparison of the two groups (paired). Data represent mean ± SEM (n = 21 second instar larvae; *p < 0.05 indicates statistically significant). Please click here to view a larger version of this figure.
Figure 3: Representative data for pupation from the feeding assay. (A) Percentage of pupation graph. (B) Images of pupae (Day 15) showing a delayed and reduced pupation rate in quercetin treatment. Scale bar = 1 cm. Please click here to view a larger version of this figure.
Figure 4: Representative data of survival on Day 10 upon feeding of 1,000 ppm quercetin compared to control. (A) Kaplan-Meier survival graph for quercetin-fed insects indicates decreased survival rate. The control insects show a ~96% survival rate, and the quercetin-treated insects show a ~77.65% survival. (B) Images of lethal phenotypes of quercetin-fed larvae taken on Day 10. Scale bar = 1 cm. Please click here to view a larger version of this figure.
| Fraction A | ||
| 1 | Bengal Gram | 50 g |
| 2 | Yeast Extract | 12 g |
| 3 | Casein | 3.5 g |
| 4 | Sorbic Acid | 0.5 g |
| 5 | Methyl Paraben | 1 g |
| 6 | dH2O | 150 mL |
| Fraction B | ||
| 1 | Choline Chloride | 0.35 g |
| 2 | Streptomycin | 0.02 g |
| 3 | Ascorbic acid | 2 g |
| 4 | Cholesterol | 0.15 g |
| 5 | Multivitamin capsule | 1 |
| 6 | Vitamin E capsule | 1 |
| 7 | dH2O | 30 mL |
| Fraction C | ||
| 1 | Agar Agar | 6.5 g |
| 2 | dH2O | 180 mL |
Table 1: Composition of the artificial diet.
| Treatment (Quercentin concentration) | Nutritional indices (%) | ||
| ECI | ECD | AD | |
| 0 ppm | 73.044 | 208.148 | 35.092068 |
| 1000 ppm | 64.2771 | 159.871 | 40.2056684 |
Table 2: Effect of quercetin ingestion on the H. armigera feeding behavior and dietary utilization. Abbreviations: ECI = efficiency of conversion of ingested food; ECD = efficiency of conversion of digested food; AD = approximate digestibility.
Supplementary Table 1: Example of the data sheet for the quercetin feeding assay. Please click here to download this File.
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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 dose24,25. Various laboratory bioassays are used to determine insecticidal activity, insecticide resistance, and the toxicity of compounds, including diet covering, topical application, obligate feeding, the injection method, contact or residual, and the film method13,14. All of these methods can be used based on the aim of a particular study, however the ideal bioassay approach should be quick and effective26. Hence, the obligate feeding assay method discussed in this manuscript can be the bioassay of choice in several instances, except for sucking insects.
The obligate feeding assay described in this manuscript can be used to study the effect of any compound on the growth, development, feeding, and survival of insect larvae. In the representative results shown here, the insecticidal activity of quercetin was examined against H. armigera larvae, providing a rationale for further exploration. Significant reductions in body weight of ~57% (Figure 2A,B), changes in feeding rate (Figure 2C), and decreased survival rate of ~18% (Figure 4A,B) were observed in quercetin-fed larvae. Also, insects fed on a quercetin diet showed delayed and reduced pupation by ~14% (Figure 3A,B). A significant change was also observed in nutritional indices, including ECI, ECD, and AD (Table 2), compared to the control. Overall, these results indicate that quercetin has a deleterious effect on the growth, development, and survival of H. armigera larvae. All these observations follow the antibiosis effect of quercetin on Aedes aegypti27, Bactrocera cucurbitae Coquillett28, and Drosophila melanogaster29. Furthermore, these observations are found to be in accordance with increased lethality in Bombyx mori due to impairment of the immune system30, reduced larval weight, and fecundity in Spodoptera litura31, Hyphantria cunea12 and Eriosoma lanigerum32.
Taking precautions, such as uniformity in sample size, is crucial to reduce biological variance between experiments. To ensure reproducibility, the feeding assay must be carried out using insect larvae of the same instar in an insect culture room at consistent temperatures and humidity levels. While preparing an artificial diet, it must be assured that the phytochemical is uniformly mixed with the diet. To minimize the error due to phytochemical degradation over time, a freshly prepared diet is preferable for assay. The properties of phytochemicals, such as thermosensitivity, light sensitivity, solubility, etc., should be considered while preparing and storing the artificial diet. Diets that have been dried out over time may change in color and shrink, and they shouldn't be utilized for the feeding assay. The assay results must not be considered when the control's mortality rates are greater than 10%33. The materials, such as spatula, beakers, Petri dishes, etc., required for diet preparation and insect weighing should be separate for the control and treatment groups to avoid errors due to cross-contamination.
The insect feeding bioassay is highly specific and reproducible, but has some limitations. For example, when an insect attacks a plant, plant immunity produces structural or chemical traits to reduce herbivore feeding and thereby minimize herbivore damage34. However, these defensive traits and their effects are not observed during this assay. Another limitation is that the definite concentration of phytochemicals ingested by insects cannot be determined14. The stability of the nutritional content of the diet and used phytochemical is a major limiting factor that can influence its effect on insects.
In spite of the above-mentioned limitations, the obligate feeding assay is affordable and can test a large number of many insects simultaneously. Also, this assay can be adapted to screen several molecules to study their antifeedant and insecticidal properties against different classes of insects.
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Disclosures
The authors declared no conflict of interest.
Acknowledgments
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
| 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 |
References
- Popp, J., Pető, K., Nagy, J. Pesticide productivity and food security. A review. Agronomy for Sustainable Development. 33 (1), 243-255 (2013).
- da Silva, F. R., et al. Comparative toxicity of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) to selected insecticides. Insects. 11 (7), 431 (2020).
- Usman, A., Ali, M. I., Shah, M., e Amin, F., Sarwar, J. Comparative efficacy of indigenous plant extracts and a synthetic insecticide for the management of tomato fruit worm (Helicoverpa armigera Hub.) and their effect on natural enemies in tomato crop. Pure and Applied Biology. 7 (3), 1014-1020 (2018).
- Honnakerappa, S. B., Udikeri, S. S. Abundance of Helicoverpa armigera (Hubner) on different host crops. Journal of Farm Science. 31, 436-439 (2018).
- Edosa, T. T. Review on bio-intensive management of African bollworm, Helicoverpa armigera (Hub.): Botanicals and semiochemicals perspectives. African Journal of Agricultural Research. 14 (1), 1-9 (2019).
- Zhou, Y., et al. Migratory Helicoverpa armigera (Lepidoptera: Noctuidae) exhibits marked seasonal variation in morphology and fitness. Environmental Entomology. 48 (3), 755-763 (2019).
- Souto, A. L., et al. Plant-derived pesticides as an alternative to pest management and sustainable agricultural production: Prospects, applications and challenges. Molecules. 26 (16), 4835 (2021).
- Özkara, A., Akyıl, D., Konuk, M. Pesticides, environmental pollution, and health. Environmental Health Risk-Hazardous Factors to Living Species. , (2016).
- Alengebawy, A., Abdelkhalek, S. T., Qureshi, S. R., Wang, M. -Q. Heavy metals and pesticides toxicity in agricultural soil and plants: Ecological risks and human health implications. Toxics. 9 (3), 42 (2021).
- Tlak Gajger, I., Dar, S. A. Plant allelochemicals as sources of insecticides. Insects. 12 (3), 189 (2021).
- Riddick, E. W. Potential of quercetin to reduce herbivory without disrupting natural enemies and pollinators. Agriculture. 11 (6), 476 (2021).
- Gao, Y. -L., et al. The effect of quercetin on the growth, development, nutrition utilization, and detoxification enzymes in Hyphantria cunea Drury (Lepidoptera: Arctiidae). Forests. 13 (11), 1945 (2022).
- Durmuşoğlu, E., Hatipoğlu, A., Gürkan, M. O., Moores, G. Comparison of different bioassay methods for determining insecticide resistance in European Grapevine Moth, Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae). Turkish Journal of Entomology. 39 (3), 271-276 (2015).
- Paramasivam, M., Selvi, C. Laboratory bioassay methods to assess the insecticide toxicity against insect pests-A review. Journal of Entomology and Zoology Studies. 5 (3), 1441-1445 (2017).
- Clark, E. L., Isitt, R., Plettner, E., Fields, P. G., Huber, D. P. W. An inexpensive feeding bioassay technique for stored-product insects. Journal of Economic Entomology. 107 (1), 455-461 (2014).
- Waldbauer, G. P., Cohen, R. W., Friedman, S. An improved procedure for laboratory rearing of the corn earworm, Heliothis zea (Lepidoptera: Noctuidae). The Great Lakes Entomologist. 17 (2), 10 (2017).
- Friesen, K., Berkebile, D. R., Zhu, J. J., Taylor, D. B. Laboratory rearing of stable flies and other muscoid Diptera. JoVE. (138), e57341 (2018).
- Zheng, M. -L., Zhang, D. -J., Damiens, D. D., Lees, R. S., Gilles, J. R. L. Standard operating procedures for standardized mass rearing of the dengue and chikungunya vectors Aedes aegypti and Aedes albopictus (Diptera: Culicidae)-II-Egg storage and hatching. Parasites & Vectors. 8, 1-7 (2015).
- Nagarkatti, S., Prakash, S. Rearing Heliothis armigera (Hubn.) on an artificial diet. Technical Bulletin Commonwealth Institute of Biological Control. , (1974).
- Adhav, A. S., Kokane, S. R., Joshi, R. S. Functional characterization of Helicoverpa armigera trehalase and investigation of physiological effects caused due to its inhibition by Validamycin A formulation. International Journal of Biological Macromolecules. 112, 638-647 (2018).
- Abbasi, B. H., et al. Rearing the cotton bollworm, Helicoverpa armigera, on a tapioca-based artificial diet. Journal of Insect Science. 7 (1), 35 (2007).
- Armes, N. J., Jadhav, D. R., Bond, G. S., King, A. B. S. Insecticide resistance in Helicoverpa armigera in South India. Pesticide Science. 34 (4), 355-364 (1992).
- Waldbauer, G. P. The consumption and utilization of food by insects. Advances in Insect Physiology. 5, Academic Press. 229-288 (1968).
- Carpinella, M. C., Defago, M. T., Valladares, G., Palacios, S. M. Antifeedant and insecticide properties of a limonoid from Melia azedarach (Meliaceae) with potential use for pest management. Journal of Agricultural and Food Chemistry. 51 (2), 369-374 (2003).
- Diaz Napal, G. N., Palacios, S. M. Bioinsecticidal effect of the flavonoids pinocembrin and quercetin against Spodoptera frugiperda. Journal of Pest Science. 88, 629-635 (2015).
- ffrench-Constant, R. H., Roush, R. T. Resistance detection and documentation: the relative roles of pesticidal and biochemical assays. Pesticide Resistance in Arthropods. , 4-38 (1990).
- Gikonyo, N. K., Mwangi, R. W., Midiwo, J. O. Toxicity and growth-inhibitory activity of Polygonum senegalense (Meissn.) surface exudate against Aedes aegypti larvae. International Journal of Tropical Insect Science. 18 (3), 229-234 (1998).
- Sharma, R., Sohal, S. K. Bioefficacy of quercetin against melon fruit fly. Bulletin of Insectology. 66 (1), 79-83 (2013).
- Després, L., David, J. -P., Gallet, C. The evolutionary ecology of insect resistance to plant chemicals. Trends in Ecology & Evolution. 22 (6), 298-307 (2007).
- Shi, G., Kang, Z., Ren, F., Zhou, Y., Guo, P. Effects of quercetin on the growth and expression of immune-pathway-related genes in silkworm (Lepidoptera: Bombycidae). Journal of Insect Science. 20 (6), 23 (2020).
- Selin-Rani, S., et al. Toxicity and physiological effect of quercetin on generalist herbivore, Spodoptera litura Fab. and a non-target earthworm Eisenia fetida Savigny. Chemosphere. 165, 257-267 (2016).
- Ateyyat, M., Abu-Romman, S., Abu-Darwish, M., Ghabeish, I. Impact of flavonoids against woolly apple aphid, Eriosoma lanigerum (Hausmann) and its sole parasitoid, Aphelinus mali (Hald). Journal of Agricultural Science. 4 (2), 227 (2012).
- Brito-Sierra, C. A., Kaur, J., Hill, C. A. Protocols for testing the toxicity of novel insecticidal chemistries to mosquitoes. JoVE. (144), e57768 (2019).
- Mitchell, C., Brennan, R. M., Graham, J., Karley, A. J. Plant defense against herbivorous pests: exploiting resistance and tolerance traits for sustainable crop protection. Frontiers in Plant Science. 7, 1132 (2016).
