Larval RNA Interference in Silkworm Bombyx mori through Chitosan/dsRNA Nanoparticle Delivery
Summary
Presented here is a protocol for chitosan/dsRNA nanoparticle delivery in silkworm Bombyx mori larvae to induce gene silencing through ingestion.
Abstract
The silkworm, Bombyx mori, is an important economic insect with thousands of years of history in China. Meanwhile, the silkworm is the model insect of Lepidoptera with a good accumulation of basic research. It is also the first insect in Lepidoptera with its complete genome sequenced and assembled, which provides a solid foundation for gene functional study. Although RNA interference (RNAi) is widely used in reverse gene functional study, it is refractory in silkworms and other Lepidopteran species. Previous successful RNAi-related research to deliver double-stranded RNA (dsRNA) was performed through injection only. Delivery of dsRNA through feeding is never reported. In this article, we describe step-by-step procedures to prepare the chitosan/dsRNA nanoparticles, which are fed to the silkworm larvae by ingestion. The protocol includes (i) selection of the proper stage of silkworm larvae, (ii) synthesis of dsRNA, (iii) preparation of the chitosan/dsRNA nanoparticles, and (iv) feeding the silkworm larvae with chitosan/dsRNA nanoparticles. Representative results, including gene transcript confirmation and phenotype observation, are presented. dsRNA feeding is a simple technique for RNAi in silkworm larvae. Since silkworm larvae are easy to rear and large enough to operate, it provides a good model to demonstrate larval RNAi in insects. In addition, the simplicity of this technique stimulates more student involvement in research, making silkworm larvae an ideal genetic system for use in a classroom setting.
Introduction
The silkworm, Bombyx mori, is an insect domesticated more than 5000 years ago in China. Due to its ability to produce silk, the silkworm is an important economic insect in Chinese agriculture and sericulture. The silkworm is second only to the fruit fly as the model insect. As a model insect in Lepidoptera, the silkworm is easy to rear, with a large body size and plenty of mutants. Meanwhile, the silkworm is the first Lepidopteran insect with its complete genome sequenced1. A lot of databases providing information for genome2, transcriptome3, expressed sequence tag (EST)4, non-coding RNA5, and microsatellite6 are also available to the public. The above facts make the silkworm a perfect model for genetic research.
RNA interference (RNAi) is a cellular process in which double-stranded RNA (dsRNA) molecules bind and slice the complementary messenger RNA (mRNA), thereby achieving the silencing effect of the target gene. This mechanism is naturally present in bacteria to defend against the invasion of viruses7. Later, it was found that RNAi is conserved in animals, plants, and microbes. Due to its powerful sequence-specific silencing effect, RNAi is used in fundamental research to manipulate gene expression and study gene function. RNAi is achieved through the delivery of dsRNA into cells.
In insects, there are three common ways to deliver dsRNA, which are microinjection, feeding, and soaking8. At the moment, successful RNAi reports in the silkworms through naked dsRNA delivery are conducted by dsRNA injection9. The advantages of microinjection are the immediate delivery of dsRNA into the hemolymph and precise dsRNA amount control. However, certain disadvantages of microinjection also exist. For example, it is time-consuming, and it requires delicate devices. It is also important to optimize the injection needles, injection volume, and dsRNA amount. Therefore, an alternative way to deliver dsRNA to silkworms becomes necessary. Because an insect's exoskeleton is a water-tight barrier that is made of chitin, soaking insect larvae to achieve RNAi is rarely reported, which is not a good option for RNAi in insects. Feeding of dsRNA is labor-saving, cost-effective, and easy to perform10. This method is also applicable for high-throughput gene screening11. However, it is found that a DNA/RNA non-specific nuclease, namely BmdsRNase, is present in the midgut and midgut juice of the silkworm larvae12. This nuclease is shown to digest dsRNA, preferably13. Therefore, feeding naked dsRNA to the silkworm to silence the gene expression seems to be difficult.
Recently, nanoparticle-shielded dsRNA is proved to be a good alternative to increase the RNAi efficiency by feeding14. Chitosan is an inexpensive, nontoxic, and biodegradable polymer, which can be prepared by deacetylation of chitin, a naturally occurring and the second most abundant biopolymer after cellulose15. Because the amino group in the chitosan is positively charged and the phosphate group on the backbone of the dsRNA is negatively charged, the chitosan/dsRNA nanoparticles could be formed by self-assembly of polycations16. Chitosan/dsRNA nanoparticles are effective in achieving RNAi through larval feeding in mosquitos Aedes aegypti and Anopheles gambiae17, cotton spotted bollworm Earias vittella18 and carmine spider mite Tetranychus cinnabarinus19.
In order to develop a methodology for dsRNA delivery by feeding in silkworms to gain successful RNAi efficiency, this report focuses on describing step-by-step procedures on how to prepare the chitosan/dsRNA nanoparticles and feed the nanoparticles to the silkworm larvae. This methodology is relatively inexpensive, labor-saving and easy to follow, which can be adapted for gene silencing studies in other insects. We aim to provide an easier protocol for the Lepidopteran dsRNA delivery method with higher RNAi efficiency.
Protocol
1. Silkworm species and rearing
- Rear at least 120 freshly hatched 1st instar silkworm larvae of B. mori P50 strain with fresh mulberry leaves at 25 ± 1 °C, photoperiod 12 h Light:12 h Dark, and 75% ± 5% relative humidity.
2. Selection of silkworm larvae
- Pick day 1 of the 5th instar larvae for the RNAi experiment; the silkworm larvae grow fast after day 3 of the 5th instar, which is ideal for comparing the differences in the larval appearance.
NOTE: Alternatively, younger stages, such as the 3rd or 4th instar, could be used for the RNAi experiment. However, it requires constant dsRNA treatment until the 5th instar, which is relatively expensive and material costing.
3. Synthesis of dsRNA
- Identifying dsRNA target fragment: Select the coding sequence of a target gene and align it with other homologous genes to determine the conserved region. Design gene-specific primers in the non-conserved region for subsequent dsRNA target fragment amplification. For RNAi experiments in insects, the typical dsRNA target fragment is generally 400-600 bp. The minimum size could be 200 bp.
NOTE: To improve the efficiency and successful rate of RNAi experiments, some web-based tool can be used to design the dsRNA target, such as dsRNA Engineer (https://dsrna-engineer.cn/). - Producing PCR product template: Add a T7 RNA polymerase promoter (5'-TAATACGACTCACTATAGG-3') to the 5'-end of either primer designed above. Perform standard PCR to amplify the dsRNA target fragment. Run a 1% agarose gel in 1x TAE to verify a single PCR product of the expected size. After that, purify the PCR product with a purification kit (Table of Materials) and use it for subsequent transcription.
NOTE: To store the target PCR product for long-term and obtain high yields, it is suggested to ligate the PCR product to a plasmid. The plasmid should be linearized before the PCR reaction. - Generation of dsRNA: Use a T7 RNAi System (Table of Materials) to generate the dsRNA. Set up the reaction by adding the components at room temperature according to the manufacturer's instructions. Mix the reaction by pipetting gently and incubate at 37 °C for 30 min.
NOTE: To maximize the yield, incubation at 37 °C can be extended up to 2-6 h. For templates containing a secondary structure or GC-rich, incubation can be performed at 42 °C instead to improve the yield of dsRNA. - Annealing to form dsRNA: Incubate the reaction at 70 °C for 10 min, then slowly cool to room temperature (~20 min). This will anneal the dsRNA.
- DNase and RNase treatment: Pipette 1 µL of the supplied RNase solution to 199 µL of nuclease-free water to make a freshly diluted RNase solution. Add 1 µL of freshly diluted RNase solution and 1 µL of the supplied RQ1 RNase-free DNase per 20 µL reaction volume, mix gently, and incubate at 37 °C for 30 min. The single-stranded RNA (ssRNA) and the template DNA in the reaction will be removed after this treatment.
- Alcohol precipitation: Add 1 volume of isopropanol or 2.5 volumes of 95% ethanol along with 0.1 volume of 3 M Sodium Acetate (pH 5.2) to the reaction. Mix the reaction by vortexing and incubate on ice for 5 min to form a cloudy mass. Spin at maximum speed in a centrifuge for 10 min to collect a white pellet at the bottom of the tube. Discard the supernatant and wash the pellet with 0.5 mL of cold 70% ethanol to remove the residual salt. Remove the ethanol after the wash. Allow the pellet to air dry at room temperature for 15 min. Resuspend the pellet with nuclease-free water in 2-5 times of the initial reaction volume. Store at -20 °C or -70 °C.
NOTE: The dsRNA pellet should not be overly dried, since this could make it difficult to fully resuspend. To ensure sufficient resuspension, a minimum of 2 volumes are needed. - Quantity and quality check of dsRNA: Dilute the dsRNA in nuclease-free water at a ratio of 1:100 to 1:300. Determine the concentration of dsRNA using a microvolume spectrophotometer (Table of Materials) according to the manufacturer's instructions. To quantify dsRNA, multiply the concentration by the volume. To assess the quality of dsRNA, use agarose gel electrophoresis. Prepare a 1% agarose gel in 1x TAE and dilute the dsRNA at 1:50 with nuclease-free water. Use 5 µL of diluted dsRNA per lane and stain the gel in 0.5 mg/mL ethidium bromide for at least 15 min for visualization.
NOTE: dsRNA migrates more slowly than dsDNA.
4. Preparation of the chitosan/dsRNA nanoparticles
- Make 100 mM sodium acetate (0.1 M NaC2H3O2 and 0.1 M acetic acid, pH 4.5 in deionized water) and 100 mM sodium sulfate (100 mM Na2SO4 in deionized water) buffer at room temperature.
- Dissolve commercialized chitosan (from shrimp shells, ≥75% deacetylated; Table of Materials) in 100 mM sodium acetate buffer to make a 0.02% (w/v) chitosan solution.
- Dissolve 20 µg of dsRNA in 50 µL of nuclease-free water and add it to 50 µL of 100 mM sodium sulfate buffer to make a 100 µL dsRNA solution.
- Add 100 µL of chitosan solution to 100 µL of dsRNA solution. Simultaneously, prepare a control by adding 100 µL of chitosan solution to 100 µL of 50 mM sodium sulfate. Mix and heat the mixtures at 55 °C for 1 min.
- Vortex the mixture immediately with a high-speed vortex for 30 s to allow the formation of the nanoparticles.
- Centrifuge the mixture at 13,000 x g at room temperature for 10 min to obtain a white pellet. Transfer the supernatant to a fresh 1.5 mL tube. Let the pellet air-dry at room temperature for 10 min.
- Determine the concentration of dsRNA in the supernatant by using a microvolume spectrophotometer (Table of Materials). Use the supernatant from the control as a blank. Multiply the concentration by volume to calculate the total amount of dsRNA remaining in the supernatant. Calculate the percentage of dsRNA encapsulated in the nanoparticles by the amount remaining in the supernatant divided by the starting amount of dsRNA.
NOTE: Even though the nanoparticles could be kept for 4-37 °C for at least 15 days before use14, it is suggested to use the nanoparticles as soon as possible.
5. Feeding the silkworm larvae with chitosan/dsRNA nanoparticles
- Pick freshly molted 5th instar silkworm larvae (i.e., Day 1 of the 5th instar) of the same size for the feeding experiment. Place the larvae individually into each well of a 6-well plate before closing the cover and starve for 24 h.
- Rinse fresh mulberry leaves with deionized water and dry them with clean kitchen paper. The mulberry leaves should be dry without water drops. Cut into 1 cm x 1 cm mulberry leaf discs.
- Dissolve chitosan/dsRNA nanoparticles into nuclease-free water at 500 ng/µL prior to use. Dilute the control chitosan nanoparticles (prepared in 4.4) with same volume of nuclease-free water. Prepare naked dsRNA to 500 ng/µL in nuclease-free water.
- Use the chitosan/dsRNA nanoparticles for RNAi knockdown experiment, the control chitosan nanoparticles as a blank/negative control. Use the naked dsRNA to compare the differences with chitosan/dsRNA nanoparticles.
- Coat 10 µL of chitosan/dsRNA nanoparticles, chitosan, and naked dsRNA on the surface of each leaf disc, respectively. Air-dry the nanoparticle solution and dsRNA solution on the leaf discs at room temperature for 5 min.
- Feed each larva with one nanoparticle-coated or dsRNA-coated leaf disc per day. Provide nanoparticle-coated or dsRNA-coated leaf discs to the larvae continuously for 5 days. Fresh mulberry leaves can be provided after the coated leaf disc is fully eaten by the larvae each day.
- On day 6, anesthetize the larvae on ice until they do not move and dissect the larvae for sampling. Cut off the thorax with scissors on a clean Petri dish. Pull out the midgut with a tweezer. Remove the content in the midgut and wash the midgut in another Petri dish filled with nuclease-free water. Store the midgut in a 1.5 mL tube and keep at -70 °C.
6. Confirmation of gene silencing
- Perform quantitative Real-time PCR (qRT-PCR) to calculate the relative transcript quantification. At least three biological replicates and three technical replicates for each biological replicate are required. At least two reference genes are recommended to normalize the relative transcript. A Bombyx Toll9-2 (BmToll9-2) gene is tested. Evaluate the gene silencing efficiency by comparing the mean value of the relative transcript to the control group and convert it to percentage.
NOTE: The qRT-PCR amplification condition, primer information, and relative transcript calculation can be found in our recent publication20. - Analyze the RNAi phenotype to evaluate the gene silencing effect. Observe the size of the larvae and cocoons. Take photos daily to record appearances.
NOTE: RNAi can affect morphology, metamorphosis, physiology, and behavior. The observation of RNAi-related phenotypes depends on the genes targeted.
Representative Results
To evaluate the RNAi efficiency, an immune gene targeting BmToll9-2 was chosen for analysis. BmToll9-2 gene is well characterized in the lab, and gene silencing by dsRNA injection results in lighter and smaller larvae in our recent publication20. To confirm the RNAi efficacy by ingestion through chitosan/dsRNA nanoparticles, chitosan nanoparticles were used as a control, and naked dsRNA was compared at the same time.
Compared with the control, chitosan nanoparticles without dsRNA, naked dsRNA targeting the BmToll9-2 gene shows no silencing effect. Feeding of chitosan/dsRNA nanoparticles in the silkworm significantly inhibits the transcript, with a 79% knockdown (Figure 1). The inhibition of the relative transcript indicates that the BmToll9-2 gene was successfully silenced through chitosan/dsRNA ingestion.
The knockdown of the BmToll9-2 gene significantly affects the growth of the silkworm. When observing the larvae, the BmToll9-2-silenced larvae are smaller than the control larvae (Figure 2A). When comparing the cocoons, the BmToll9-2-silenced cocoons are also smaller (Figure 2B). Chitosan/dsRNA nanoparticles show successful RNAi effects in both transcript and phenotype in the silkworm.
Figure 1: Relative transcript of BmToll9-2 after chitosan/dsBmToll9-2 nanoparticles ingestion in B. mori 5th instar larvae. The relative mRNA levels of BmToll9-2 in the larvae fed with chitosan nanoparticles as control, naked dsRNA, and chitosan/dsRNA nanoparticles. Data were represented as means ± standard deviation of three biological replications. For each biological replication, there were three technical replications. Different letters on the bars indicate significant differences based on one-way ANOVA analysis. Please click here to view a larger version of this figure.
Figure 2: Phenotype observation after feeding with chitosan/dsBmToll9-2 nanoparticles. Chitosan nanoparticles are used as a control. (A) The appearance of silkworm larvae after ingestion of nanoparticles. (B) The appearance of silkworm cocoons after ingestion of nanoparticles. Please click here to view a larger version of this figure.
Discussion
A proper stage is important for RNAi phenotype observation, depending on the genes targeted. Our preliminary results showed that Toll9-2 is involved in the growth of the silkworm. The size and weight of the silkworm larvae increase rapidly at the 5th instar21. Therefore, the 5th instar larvae are selected as the stage for the chitosan/dsRNA nanoparticles feeding experiment. It is also possible to select the 3rd or 4th instar larvae for feeding experiments and observe the developmental differences at the 5th instar. However, the nanoparticle feeding experiments require continuous feeding daily14. Feeding the larvae at a younger stage requires more dsRNA preparation, which is relatively more expensive. For the above reasons, we suggest selecting the freshly molting silkworm larvae of the 5th instar as the starting point of the nanoparticle feeding experiment.
Optimization of the nanoparticle feeding parameters is also important. A constant nanoparticle feeding for 5-7 days to achieve efficient RNAi is common in current studies14,18,22,23. Because dsRNA is the most expensive ingredient in RNAi experiments, nanoparticles could be fed to the larvae every other day, which may help to reduce half of the cost of dsRNA preparation. Another way to cut the cost is to optimize the amount of dsRNA used in feeding. In this protocol, 5 µg of chitosan/dsRNA nanoparticles are used for each larva, which is the universal dose for dsRNA injection in the lab. Considering 5 µg of dsRNA is a high dose and inhibition by nanoparticle feeding is significant. It is suggested to reduce the amount of dsRNA in the experiment. However, the impact of the above two parameters on the RNAi efficiency needs to be evaluated in future experiments.
The ratio of chitosan and dsRNA could be optimized. In this protocol, 20 µg of chitosan in 100 µL of sodium acetate is mixed with 20 µg of dsRNA in 100 µL of sodium sulfate. In the first chitosan/dsRNA nanoparticles report, 20 µg of chitosan in 100 µL of sodium acetate is mixed with 32 µg of dsRNA in 100 µL of sodium sulfate16. In some other reports, 1 µg of chitosan in 100 µL of sodium acetate is mixed with 1 µg of dsRNA in 100 µL of sodium sulfate18,23. Therefore, the amount of chitosan and dsRNA could be optimized to achieve the best loading efficiency of the nanoparticles and optimal RNAi silencing effect.
The storage of chitosan/dsRNA nanoparticles needs to be considered. The chitosan/dsRNA nanoparticles are prepared prior to the feeding experiment, and it is suggested that they be used immediately. A recent report showed that chitosan/dsRNA nanoparticles were stored at 4 °C, 24 °C and 37 °C for 15 days and were still stable19. Thus, the chitosan/dsRNA nanoparticles could be prepared in advance and stored at 4 °C in the fridge since dsRNA is more stable at low temperatures.
Besides dsRNA, some small RNA, such as small interfering RNA (siRNA) and short hairpin RNA (shRNA), can be used in nanoparticle preparation for RNAi experiments. For example, siRNA nanoparticles improve plant resistance to Pseudomonas syringae24. Delivery of shRNA nanoparticles caused successful RNAi effect in the neotropical brown stink bug Euschistus heros25. Therefore, nanoparticles can be applied to any RNA molecules used in the RNAi experiment.
The RNAi efficiency in Lepidoptera, including silkworms, is reported to be refractory26. Delivery of dsRNA is only through injection in silkworms, which is time-consuming and labor-costing. With this chitosan/dsRNA nanoparticle delivery method, a significant silencing effect is observed either in transcript or phenotype in the silkworm larvae and cocoons, possible due to the less degradation of dsRNA by the protection of chitosan19. This method is labor-saving and easy to perform, which can be adapted to other insects. The ease of this protocol can be applied not only in scientific research but also in classroom teaching demonstration and practice. The nanoparticle delivery method is a new direction for future biocontrol and pest management27.
Disclosures
The authors have nothing to disclose.
Acknowledgements
This study was funded by the National Natural Science Foundation of China (31501898), the Science and Technology Program of Guangzhou (202102010465), the Guangzhou Higher Education Teaching Quality and Teaching Reform Project (2022JXGG057), and the Research project of the Open Online Course Steering Committee of Guangdong Provincial Universities (2022ZXKC381).
Materials
| 1.5 mL centrifuge tube | Sangon | F601620 | for dsRNA or nanoparticles reaction |
| 10 μl pipette | Eppendorf | P13473G | to aspirate or resuspend liquid |
| 100 μl pipette | Eppendorf | Q12115G | to aspirate or resuspend liquid |
| 2.5 μl pipette | Eppendorf | P20777G | to aspirate or resuspend liquid |
| 20 μl pipette | Eppendorf | H19229E | to aspirate or resuspend liquid |
| 200 μl pipette | Eppendorf | H20588E | to aspirate or resuspend liquid |
| 6-well Clear TC-treated Multiple Well Plates | Costar | 3516 | for silkworm rearing individually |
| Acetic acid | Aladdin | A116165 | to make TAE |
| Agarose M | BBI Life Sciences | A610013 | for agarose gel electrophosis |
| Analytical balance | Sartorius | BSA224S | to weight ingredients |
| Centrifuge | Sartorius | Centrisart A-14C | to centrifuge to form dsRNA or nanoparticles |
| Chitosan | Sigma-Aldrich | C3646 | to combine with dsRNA for preparation of nanoparticles |
| EDTA | Sangon | A500895 | to make TAE |
| Ethanol | Aladdin | E130059 | to make TAE, or for dsRNA precipitation |
| Freezer | Siemens | iQ300 | to store dsRNA or nanoparticles |
| GoTaq Green Master Mix | Promega | M712 | for PCR reaction |
| GoTaq qPCR Master Mix | Promega | A6002 | for qRT-PCR reaction |
| Isopropanol | Aladdin | I112011 | for dsRNA precipitation |
| NanoDrop Microvolume UV-Vis Spectrophotometer | ThermoFisher | One | to determine the concentration of dsRNA |
| ph meter | Sartorius | PB-10 | to prepare buffers |
| SanPrep Column PCR Product Purification Kit | Sangon | B518141 | for PCR product purification |
| Sodium acetate | Sigma-Aldrich | S2889 | to make 100 mM sodium acetate buffer |
| Sodium sulfate | Sigma-Aldrich | 239313 | to make 100 mM sodium sulfate buffer |
| T7 RiboMAX Express RNAi System | Promega | P1700 | for dsRNA synthesis |
| ThermoMixer | Eppendorf | C | for dsRNA generation or nanoparticles heating |
| Tris | Sangon | A501492 | to make TAE |
| Vortex | IKA | Vortex 3 | to prepare chitosan/dsRNA nanoparticles |
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