feeding assay to evaluate the effects of quercetin on helicoverpa armigera growth and development

Feeding Assay를 통한 Helicoverpa armigera 에 대한 식물화학적 효과 평가

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.

저자 스포트라이트: 곤충 해충 관리를 위한 사료 분석법의 개발 및 과제 

Helicoverpa armigera에 대한 Phytochemicals의 살충 효과를 평가하기 위한 공급 분석 시스템 개발

This study is conducted to develop a simple and cost-effective feeding assay method for evaluating the effect of various natural molecules, including phytochemicals for their antifeedant and insecticidal properties against thieving insects, and identified potential molecules from these assay can be evaluated in field condition for pest management. Multiple assays have been developed to evaluate the effect of synthetic and natural molecules on insect feeding, growth, development, and behavior, further assays have been designed, such as dip case tool, diet covering assay, contact assay, droplet feeding assay, choice feeding assay, and lip dish test for insecticide evaluation, Maintaining uniform experimental conditions, addressing issues, like infection and cannibalism, ensuring even phytochemical distribution in larval diet, tracking, feeding behavior accurately, and determining optimal phytochemical concentrations post challenges, batch variation, and experimental design complexities must be managed. Additionally, transitioning from the lab assist to the field application, considering formulation and scalability is demanding.In the obligatory feeding assay, a large number of molecules can be tested simultaneously with a large number of insect sample sizes. The advantage of this feeding assay is that the test compound is mixed with an artificial diet, thus we have control over diet composition, which provides robust and reproducible results.

Watch this Scientific Journal Video about Feeding Assay to Evaluate the Effects of Quercetin on Helicoverpa armigera Growth and Development at JoVE.com

Feeding Assay to Evaluate the Effects of Quercetin on Helicoverpa armigera Growth and Development  

Helicoverpa armigera 성장 및 발달에 대한 퀘르세틴의 효과를 평가하기 위한 수유 분석

시작하려면 곤충 배양실의 모슬린 천으로 덮인 플라스틱 번식실에서 Helicoverpa armigera 신생아를 보관하십시오. 그런 다음 새로 나온 신생아를 성장을 위해 갓 준비한 병아리콩 기반 인공 식단에 가는 붓을 사용하여 부드럽게 옮깁니다. 먹이 분석을 위해 대조군 및 치료 세트를 위해 21초 인스타 유충을 수집합니다.미리 준비한 대조군과 케르세틴이 함유된 사료를 작은 조각으로 자르고 사료의 무게와 곤충의 몸무게를 기록합니다. 곤충을 배양 바이알에 조심스럽게 옮기고 각 식단을 바이알에 넣고 곤충이 먹이를 먹게 합니다. 둘째 날부터 10일째 되는 날까지 곤충 몸의 무게를 기록한 후 주어진 식단, 먹지 않은 식단 및 frass를 기록합니다.10일째에 살아 있는 유충과 죽은 유충과 번데기를 세십시오. 데이터를 기록하고 분석하기 위해 학생 t 검정을 사용하여 대조군과 치료군 간의 곤충 체중을 비교합니다. 살아있는 죽은 유충과 번데기 수를 사용하는 그래프 소프트웨어를 사용하여 생존율에 대한 Kaplan-Meier 곡선을 플로팅합니다. 그런 다음 번데기 비율을 계산하고 유충 발달 및 영양 지수를 비교합니다.본 연구에서 퀘르세틴이 함유된 사료를 먹인 곤충 유충은 대조군에 비해 현저히 감소하여 몸 크기가 줄어드는 결과를 보였다. 또한, 대조군에 비해 퀘르세틴을 먹인 유충의 먹이 비율이 눈에 띄게 감소하는 것이 관찰되었습니다. 또한, 퀘르세틴을 먹인 유충에서 번데기 비율이 현저히 감소하여 치료 시 발달 지연이 있음을 시사했습니다.퀘르세틴이 함유된 사료를 먹은 곤충의 경우, 섭취한 음식이 체물질로 전환되는 효율이 대조군에 비해 약 9% 감소했습니다. 소화된 음식의 전환 효율은 대조군에 비해 약 49% 감소했습니다. 퀘르세틴을 먹인 곤충의 대략적인 소화율은 대조군에 비해 5% 증가했습니다.

feeding-assay-to-evaluate-effects-quercetin-on-helicoverpa-armigera

Research

JoVE Journal

Biology

Feeding Assay to Evaluate the Effects of Quercetin on Helicoverpa armigera Growth and Development

71 조회수.  CSIR-National Chemical Laboratory. 시작하려면 곤충 배양실의 모슬린 천으로 덮인 플라스틱 번식실에서 Helicoverpa armigera 신생아를 보관하십시오. 그런 다음 새로 나온 신생아를 성장을 위해 갓 준비한 병아리콩 기반 인공 식단에 가는 붓을 사용하여 부드럽게 옮깁니다. 먹이 분석을 위해 대조군 및 치료 세트를 위해 21초 인스타 유충을 수집합니다.미리 준비한 대조군과 케르세틴이 함유된 사료를 작은 조?...

비디오: Feeding Assay to Evaluate the Effects of Quercetin on Helicoverpa armigera Growth and Development  

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.

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.

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

연구

생물학

Preparing a Quercetin-Containing Chickpea-Based Artificial Diet for Evaluating its Effect on Helicoverpa armigera Growth

Preparing a Quercetin-Containing Chickpea-Based Artificial Diet for Evaluating its Effect on Helicoverpa armigera Growth  

To begin, weigh the desired components of fractions A, B, and C separately in a beaker, and prepare a homogenous mixture using a spatula or magnetic stir. Boil fraction C at around 100 degrees Celsius in a microwave for five minutes. Then add it to fraction A and mix thoroughly.Once the mixture cools down, add fraction B and pour the prepared control diet mixture into a transparent polystyrene Petri dish. To prepare quercetin containing an artificial diet, dissolve quercetin hydrate and dimethyl sulfoxide, then add the dissolved quercetin into fraction B, followed by a mixture of fractions A and C.Add an equivalent dimethyl sulfoxide to the prepared controlled diet.

헬리코베파 아미게라 성장에 대한 효과를 평가하기 위한 퀘르세틴 함유 병아리콩 기반 인공 식단 준비

To begin, keep Helicoverpa armigera neonates in the plastic breeding chamber covered with a muslin cloth in the insect culture room. Then gently transfer newly emerged neonates using a fine paintbrush on a freshly prepared, chickpea based artificial diet for growth. For the feeding assay, collect 21 second instar larvae for the control and treatment set.Cut previously prepared control and quercetin containing diet into small pieces, and record the weight of the diet and the insect's body. Carefully transfer the insect into the culture vial, add the respective diet into the vial, and let the insect feed. From the second day till the 10th day, record the weight of the insect body followed by a given diet, uneaten diet, and frass.Count the live and dead larvae and pupae on day 10. To record and analyze the data, compare the insect body weight between the control and treatment groups using a student t test Plot a Kaplan-Meier curve for survival percentage using the graphing software using live dead larvae and pupae count. Then calculate the percent pupation and compare larval development and nutritional indices.In the present study, the insect larvae fed with quercetin containing diet showed a significant decrease compared to the control group, thereby resulting in reduced body size. Also, a notable reduction was observed in the feeding rate of quercetin fed larvae compared to the control. Furthermore, there was a significant decrease in percent pupation in quercetin fed larvae suggesting developmental retardation upon treatment.For insects fed on the quercetin containing diet, the efficiency of conversion of ingested food to body matter was reduced by approximately 9%compared to the control. The efficiency of conversion of digested food was reduced by approximately 49%compared to the control. The approximate digestibility in quercetin fed insects was increased by 5%compared to the control.