Larval RNA Interference in Silkworm Bombyx mori through Chitosan/dsRNA Nanoparticle Delivery
October 4th, 2024
Presented here is a protocol for chitosan/dsRNA nanoparticle delivery in silkworm Bombyx mori larvae to induce gene silencing through ingestion.
The scope of this research is to develop a methodology to feed the larvae of the silkworm Bombyx mori with dsRNA, resulting in gene silencing effect. This will help in gene regulation studies and high-throughput gene screening. Chitosan is an inexpensive, non-toxic, and biodegradable polymer.
Chitosan/dsRNA nanoparticles are found to be effective in causing the RNAi effect by feeding the mosquitoes. In the silkworm, RNAi is achieved only by injection of dsRNA so far. However, enough authenticated devices are required for injection.
This technique is also time-consuming. Feeding is a natural behavior in insects. Feeding the chitosan/dsRNA nanoparticles to silkworm to cause silencing effect has never been reported.
Additionally, this technique is labor-saving, cost-effective, and easy to perform. To begin, mix 0.1 molar sodium acetate and 0.1 molar acetic acid in deionized water at pH 4.5. Then, prepare 100 millimolar sodium sulfate buffer in deionized water at room temperature.
Weigh the commercialized chitosan to prepare 0.02%weight by volume solution and dissolve it in the prepared 100 millimolar sodium acetate buffer. Then, dissolve 20 micrograms of double-stranded RNA, or dsRNA, in 50 microliters of nuclease-free water. Add the solution to 50 microliters of the sodium sulfate buffer to make a 100 microliter dsRNA solution.
Next, add 100 microliters of the prepared chitosan solution to 100 microliters each of the dsRNA solution and 50 millimolar sodium sulfate. After mixing, heat the solutions at 55 degrees Celsius for one minute. Immediately vortex the mixture at high speed for 30 seconds to facilitate nanoparticle formation.
Centrifuge the mixture at 13, 000 G at room temperature for 10 minutes to obtain a white pellet. Transfer the supernatant to a fresh 1.5 milliliter tube and let the pellet air dry at room temperature for 10 minutes. Using a microvolume spectrophotometer, determine the concentration of dsRNA in the supernatant.
To calculate the percentage of dsRNA encapsulated, divide the amount remaining in the supernatant by the initial amount of dsRNA. To begin, prepare the chitosan double-stranded RNA nanoparticles for feeding the silkworm larvae. Then, pick freshly molted fifth instar silkworm larvae of similar size for the feeding experiment.
Place each larvae individually into a well of a six well plate. Cover the plate, and starve the larvae for 24 hours. Next, rinse fresh mulberry leaves with deionized water and dry them using clean kitchen paper.
Cut the fully dried leaves into one centimeter by one centimeter leaf discs. Dissolve the chitosan dsRNA nanoparticles in nuclease-free water at a concentration of 500 nanograms per microliter before use. Coat 10 microliters of chitosan/dsRNA nanoparticles, control chitosan nanoparticles, and naked dsRNA separately on the surface of each mulberry leaf disc.
Let the solutions air-dry on the leaf discs at room temperature for five minutes. Next, offer one nanoparticle-coated or dsRNA-coated leaf disc to each larvae every day. Once the coated leaf disc is fully consumed by the larvae each day, provide fresh mulberry leaves.
On day six, place the larvae on ice until they are immobile. To dissect the larvae, cut off the thorax with scissors on a clean Petri dish and use tweezers to pull out the midgut. After the removing the contents, wash the midgut in a Petri dish with nuclease-free water.
Freeze the midgut at minus 70 degrees Celsius in a 1.5 milliliter tube. Feeding chitosan/dsRNA nanoparticles in silkworms resulted in a 79%reduction in BmToll9-2 gene transcript levels compared to control treatments. Silkworm larvae treated with BmToll9-2-silencing chitosan/dsRNA nanoparticles were visibly smaller than the control larvae.
The cocoons from BmToll9-2-silenced larvae were also significantly smaller than those from the control group.
Overview
This study presents a novel protocol for the delivery of chitosan/dsRNA nanoparticles to silkworm larvae (Bombyx mori) via feeding, aimed at inducing gene silencing. The methodology is designed to facilitate gene regulation studies and high-throughput gene screening, showing significant silencing effects in targeted genes.
Key Study Components
Research Area
- Genetic regulation
- RNA interference (RNAi)
- Insect model systems
Background
- Chitosan is a biodegradable polymer that has potential in gene delivery systems.
- Traditional RNAi methods in silkworms required injection, which is labor-intensive.
- This feeding approach presents a cost-effective and easy alternative.
Methods Used
- Preparation of chitosan/dsRNA nanoparticles for oral delivery.
- Use of silkworm larvae (Bombyx mori) as a biological system.
- Application of centrifugation and spectrophotometric methods to assess dsRNA concentration.
Main Results
- Successful RNAi was achieved with a 79% reduction in BmToll9-2 gene transcripts.
- Silkworm larvae treated with dsRNA were visibly smaller than control groups.
- The cocoons from treated larvae were significantly smaller, demonstrating the effectiveness of the treatment.
Conclusions
- The study successfully demonstrates a new method for RNAi in silkworm larvae via feeding.
- This approach provides a labor-saving, efficient technique for genetic studies in insects.
Frequently Asked Questions
Executive Industry Relevance
Chitosan/dsRNA nanoparticle feeding in Bombyx mori enables scalable gene silencing for functional genomics and high-throughput screening in Lepidopteran models. This ingestion-based RNAi delivery overcomes injection bottlenecks, supporting efficient target validation and mechanistic de-risking in insect systems. The approach enhances predictive confidence for gene function studies, facilitating portfolio triage in agricultural and biotechnological R&D.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of gene function through targeted transcript knockdown in vivo.
- Supports biological de-risking by clarifying gene-phenotype relationships in a model organism.
- Facilitates predictive confidence in target selection for downstream screening.
Screening & Assay Development
- Prepares validated silkworm systems for high-throughput gene screening workflows.
- Standardizes RNAi delivery via feeding, improving reproducibility and scalability.
- Generates quantitative transcript reduction data for robust assay outputs.
Translational & Preclinical Research
- Aligns with disease-relevant insect models for translational gene function studies.
- Provides continuity from gene discovery to phenotype validation in preclinical settings.
- Reduces mechanistic ambiguity, supporting risk-adjusted advancement decisions.
Pipeline & Workflow Integration
This feeding-based RNAi method integrates into the early discovery-to-lead identification continuum for insect models, enabling functional genomics and target prioritization.
- Discovery Biology: Supports hypothesis testing and pathway clarification by enabling efficient gene silencing.
- Screening: Delivers reproducible, quantitative transcript knockdown for assay readiness.
- Analytics: Provides measurable gene expression reduction and phenotype outputs for comparative analysis.
- Translational Research: Connects gene function studies in silkworms to broader insect model applications.
- Enterprise Reuse: Establishes a scalable, labor-saving RNAi platform for repeated use in gene function pipelines.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in gene function studies.
- Operational Value: Streamlines workflows with standardized, scalable, and cost-effective RNAi delivery.
- Strategic Value: Enables better go/no-go decisions and capital efficiency in gene target pipelines.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of validated gene targets.
Implementation Considerations
- Requires expertise in nanoparticle preparation and insect handling.
- Needs access to spectrophotometric and centrifugation infrastructure for dsRNA quantification.
- Demands cross-team standardization of feeding and measurement protocols.
- Adaptation may be needed for other Lepidopteran or insect model systems.
- Practical limitations include species-specific RNAi responsiveness and nanoparticle uptake efficiency.
