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

1. Rear at least 120 freshly hatched 1^st 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

1. Pick day 1 of the 5^th instar larvae for the RNAi experiment; the silkworm larvae grow fast after day 3 of the 5^th instar, which is ideal for comparing the differences in the larval appearance.
   NOTE: Alternatively, younger stages, such as the 3^rd or 4^th instar, could be used for the RNAi experiment. However, it requires constant dsRNA treatment until the 5^th instar, which is relatively expensive and material costing.

3. Synthesis of dsRNA

1. 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/).
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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

1. 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.
2. 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.
3. 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.
4. 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.
5. Vortex the mixture immediately with a high-speed vortex for 30 s to allow the formation of the nanoparticles.
6. 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.
7. 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

1. Pick freshly molted 5^th instar silkworm larvae (i.e., Day 1 of the 5^th 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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

1. 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.
2. 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.

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...

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 5^th instar²¹. Therefore, the 5^th instar larvae are selected as the stage for the chitosan/dsRNA nanoparticles feeding experiment. It is also possible to select the 3^rd or 4^th instar larvae for feeding e...

Materials

Name	Company	Catalog Number	Comments
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