Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

Summary

This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes.

Abstract

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

Introduction

RNAi has been demonstrated as an effective method to knockdown gene expression through a mechanism of a post-transcriptional silencing pathway triggered by dsRNA molecules in many eukaryotes¹. Over the past decade of study, RNAi has become a useful tool to study the functions of genes from development to behavior by depleting the expression of specific genes via injection and/or feeding of dsRNA in various taxa of insects^2,3. Due to the specificity and robustness of the depleting effect, the application of RNAi is currently being considered as a potential strategy for pest control management^4,5. However, the efficiency of RNAi varies widely between insect species, depending on the different genes being targeted and the delivery methods. A growing body of evidence suggests that the instability of dsRNA, which is degraded by ribonucleases, is a critical factor in the limited efficacy of RNAi^5,6. For instance, the low RNAi sensitivity in Manduca sexta has been explained by the fact that the dsRNA mixed with hemolymph was quickly degraded within 1 hour⁷. Similarly, the presence of alkaline nucleases in the midgut, which efficiently degrade ingested dsRNA, is strongly correlated with low RNAi efficiency in different insect orders^8,9,10.

The oral delivery of dsRNA is particularly interesting for the application of RNAi in a pest control strategy, but a method to retard the degradation of dsRNA by the nucleases in the midgut has not yet been developed, which would have the potential to ensure effective RNAi through feeding. However, the unresponsiveness of RNAi to oral delivery of dsRNA has been reported by feeding large amount of dsRNA, e.g. 50 µg in Bombyx mori, or continuously feeding for 8 days (8 µg dsRNA in total) in the locust species. The German cockroach, Blattella germinica, is highly sensitive to RNAi by the injection of dsRNA^11,12,13,14, but is not responsive to dsRNA through feeding. Recently, Lin et al. (2017) have demonstrated that the dsRNA encapsulated with liposome carriers results in successful RNAi to knockdown the α-tubulin gene expression in the midgut and trigger significant mortality of the German cockroach¹⁵. As the degradation of dsRNA in the midgut is the limiting factor for oral RNAi, the liposome carriers serve as a vehicle to protect dsRNA from degradation, which is readily applicable in other insects with strong nuclease activities in the gut. Of note, the reason for choosing the particular transfection reagent (see Table of Materials) we used as liposome carrier in the current protocol is that it has been tested for insect cell line transfection with less toxicity, according to the manufacturer's instructions. According to the comparison of different liposome transfection systems in Gharavi et al. (2013)¹⁶, the efficiency of transfecting small interfering RNA (siRNA) is approximately the same between this and other commercially available systems that have been used for dsRNA delivery systems in other insects^17,18.Furthermore, our feeding method is careful enough to ensure the proper amount of dsRNA is ingested by each cockroach, and that the results are robust and confirmed. In summary, the present protocol and results demonstrate that using dsRNA lipoplexes improves dsRNA stability and opens the door to the design of the strategy oral delivery of RNAi, which is a promising approach for pest control in the future.

Protocol

1. Synthesis and Preparation of dsRNA

1. Identify the dsRNA target sites in the 3' untranslated region of the target genes. The dsTub is used for targeting the α-tubulin (tub) gene (GenBank accession number: KX228233), and dsEGFP as a negative dsRNA control is designed from the sequence of enhanced green fluorescence protein (EGFP; GenBank accession number: LC311024).
2. Perform standard PCR amplification to synthesize the dsRNA templates with gene-specific primers containing the T7 promoter sequence (5'-TAATACGACTCACTATAGGG-3'). The PCR amplification conditions and primer information used are those reported by Lin et al. (2017)¹² (see Table of Materials).
3. Proceed with dsRNA preparation using a T7 Transcription Kit (see Table of Materials), following the manufacturer's instructions.
   Note: To yield high quantity dsRNA, mix two reactions in one tube (in total 40 µL), and incubate at 37 °C overnight.
4. Add 2 µL of DNase (see Table of Materials) into dsRNA samples for 15 min at 37 °C to digest DNA templates.
5. Add 200 µL extraction reagent (see Table of Materials) and 40 µL chloroform, then vortex.
6. Centrifuge for 10 min at 13,000 x g at 4 °C, then collect supernatant (150 µL) in a new microcentrifuge tube.
7. Precipitate dsRNA by adding 150 µL isopropanol and incubating for 15 min on ice.
8. Centrifuge for 15 min at 13,000 x g at 4 °C, then remove supernatant.
9. Add 200 µL of the 70% ethanol to wash dsRNA pellets. Centrifuge for 10 min at 13,000 x g at 4 °C.
10. Remove the ethanol and repeat the ethanol wash steps.
11. Remove the ethanol and dry dsRNA pellet by centrifugal vacuum concentrators for 3 min, then resuspend dsRNA with RNase-free water.
12. Determine the quantity of the purified dsRNA using a micro-volume UV-Vis spectrophotometer (see Table of Materials) according to the manufacturer's instructions and adjust to the desired concentration (e.g., 0.25 µg/µL for preparation of dsRNA lipoplexes).
13. Store the dsRNA samples at -20 °C for up to 3 months.

2. Preparation of dsRNA Lipoplexes

1. Dilute 4 µL of the transfection reagent (see Table of Materials) by adding 4 µL of 5% glucose solution. Dilute 4 µL of the dsRNA (1 µg) by adding 4 µL of 5% glucose solution.
2. Add the diluted liposome reagent solution immediately into the diluted dsRNA solution all at once. Vortex briefly to mix them, then incubate for 15 min at 25 °C.
   Note: Do not mix the solutions in the reverse order.
3. The dsRNA lipoplexes are ready for use. Use within 1 hour of preparation, and discard after 1 hour.
   Note: According to the manufacturer's instructions, the lipoplexes should be used as soon as possible.

3. Collection of Extracellular Enzymes from Hemolymph and Midgut Juice

1. Insect saline buffer (10x stock)
   1. Mix 900 mL double distilled water with 109.3 g NaCl, 15.7 g KCl, 6.3 g CaCl₂, and 8.3 g MgCl₂·6H₂O.
   2. Adjust pH to 6.8, and fill up to 1,000 mL.
2. Hemolymph collection
   1. Anesthetize the cockroaches on ice until they do not move for 3 min.
   2. Hold the cockroach with two fingers (thumb and index finger), and turn the ventral side of the cockroach up.
   3. Use the dissecting scissor to cut off the tip of a coxa of a hind leg off.
      Note: Cut the connected intersection between the coxa and trochanter. The excision of the other part of coxa was less efficient to collect hemolymph.
   4. Squeeze the abdomen gently with middle finger, and collect the hemolymph bleeding from the incision with a 10 µL micropipette.
   5. Pool the hemolymph from several cockroaches (5 individuals) in a microcentrifuge tube.
      Note: Keep the microcentrifuge tubes on ice to prevent melanization. The average volume of hemolymph obtained from a male cockroach is 2 - 3 µL.
   6. Spin down the hemolymph briefly with a benchtop centrifuge for 10 s.
   7. Transfer desired volume of hemolymph (i.e., 10 µL), and mix it with 50 µL of 1x insect saline buffer.
   8. Centrifuge for 10 min at 1,000 x g at 4 °C to spin down hemocytes. Transfer the supernatant to a clean microcentrifuge tube.
   9. Quantify the total protein concentration using a micro-volume UV-Vis spectrophotometer by measuring A280¹⁵.
   10. Adjust each sample to have the same protein concentration (6 mg/µL of total protein).
3. Midgut juice collection
   1. Anesthetize the cockroaches on ice. Fix the cockroaches ventral side up on the dissection plate with cold 1x insect saline buffer using insect pins.
   2. Dissect the abdomen with fine tweezers. Remove the entire gut (including the front, mid, and hind gut) to a fresh dish with 1x insect saline buffer.
   3. Remove the front and hind gut, and quickly transfer the midgut to a microcentrifuge tube that contains 100 µL insect saline buffer.
   4. Pool the midguts from six cockroaches in the same tube, and vortex for 10 s.
   5. Centrifuge for 10 min at 1,000 x g at 4 °C to spin down the hemocytes and gut tissues. Transfer the supernatant to a clean microcentrifuge tube.
   6. Quantify the total protein concentration using a micro-volume UV-Vis spectrophotometer by measuring A280¹⁵.
   7. Adjust each sample to have the same protein concentration (6 mg/µL of total protein).

4. dsRNA Degradation Assay

1. Freshly prepare 4 µL of the naked dsRNA or dsRNA lipoplexes (1 µg).
2. Mix dsRNA solutions with 10 µL of insect saline buffer (control), extracted enzymes from hemolymph, or midgut juice.
3. Add 2 µL of either EGTA (20 mM) or RNase-free water to separate samples as controls for inhibition of enzyme activities.
4. Incubate for 1 hour or longer (6, 12, or 24 hours) at 25 °C.
5. Add 200 µL extraction reagent and 40 µL chloroform, then vortex.
6. Centrifuge for 10 min at 13,000 x g at 4 °C, then collect supernatant (150 µL) in a new microcentrifuge tube.
7. Precipitate dsRNA by adding 150 µL isopropanol and incubating for 15 min on ice.
8. Centrifuge for 15 min at 13,000 x g at 4 °C, then remove supernatant.
9. Add 200 µL of the 70% ethanol to wash dsRNA pellets. Centrifuge for 10 min at 13,000 x g at 4 °C.
10. Remove the ethanol and repeat the ethanol wash steps.
11. Remove the ethanol and dry dsRNA pellet by centrifugal vacuum concentrators for 3 min. Resuspend dsRNA with 10 µL RNase-free water.
12. Check the integrity of treated dsRNA via gel electrophoresis on a 1.5% agarose gel.

5. Oral Administration of dsRNA

1. Maintain cockroaches on an ad libitum diet of dry food (dog chow). Deprive water supply from the cockroaches one day before dsRNA ingestion experiments.
2. Hold a cockroach by grabbing its wings with the flexible forceps (Figure 1, step 5).
   Note: Do not grab the body parts of cockroaches.
3. Prepare the dsRNA lipoplexes, as well as naked dsRNA, for feeding, as described in section 2.
   Note: The dsRNA lipoplexes should be prepared freshly before feeding.
4. Feed 4 µL of dsRNA lipoplexes or naked dsRNA (250 ng) with a micropipette.
5. Slowly pipet the droplet of the dsRNA solution close to the mouthparts of cockroaches, and let the cockroaches ingest the droplet completely.
6. Perform the ingestion experiments twice a day (1 h after lights on and 1 h before lights off) for 8 days or 16 days continuously.
   Note: All cockroaches acquired water only through manual feeding with dsRNA solution or control reagents (e.g., nuclease free water, glucose solution, and liposome reagent) during ingestion experiments.
7. Provide water bottles to the cockroaches after 16 days of ingestion of dsRNA experiments.

6. Assess Knockdown

1. At chosen time points (2, 9, and 17 days after dsRNA ingestion assay), collect the dsEGFP- and dsTub-treated insects and dissect the chosen tissues (e.g., midgut).
2. Perform quantitative real-time PCR (qRT-PCR). The qRT-PCR amplification conditions and primer information as reported by Lin et al. (2017)¹².
3. Assess the control and dsRNA-treated insects daily to check mortality and remove the cockroaches that are no longer moving.

Results

A simplified scheme of the protocol for the oral delivery of dsRNA is presented in Figure 1, where the key steps for preparation of dsRNA lipoplexes are shown.

In order to investigate the protection given by liposome carriers upon dsRNA degradation in the midgut juice of B. germanica, an ex vivo assay where dsTub lipoplexes were incubated with midgut juice was conducted, and the in...

Discussion

This protocol presents a method for effective RNAi through oral delivery of dsRNA lipoplexes, involving protection against ribonuclease digestion in the midgut juice of the German cockroach. As shown in other studies in various insect species, the poor RNAi effect through oral delivery of dsRNA is mostly accounted for by the degradation of dsRNA^8,9,10. This protocol produces liposomes that serve as protective vehicles in oral de...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
GenJe Plus DNA in vitro Transfection reagent	SignaGen	SL100499	for lipoplexes preparation
Blend Taq plus	TOYOBO	BTQ-201	for PCR
Fast SYBR Green Master Mix	ABI	4385612	for qPCR
FirstChoice RLM-RACE Kit	Invitrogen	AM1700	for 3' UTR identification
MEGAscript T7 Transcription Kit	Invitrogen	AMB13345	for dsRNA synthesis
TURBO DNase	Invitrogen	AM2239	remove DNA template from dsRNA
TRIzol	Invitrogen	15596018	for dsRNA or total RNA extraction
RQ1 RNase-Free Dnase	Promega	M6101	remove DNA template from total RNA
chloroform	Sigma-Aldrich	C2432	for dsRNA or total RNA extraction
2-Propanol	Sigma-Aldrich	I9516	for dsRNA or total RNA extraction
ethanol	Sigma-Aldrich	24102	for dsRNA or total RNA extraction
Diethyl pyrocarbonate, DEPC	Sigma-Aldrich	D5758	for RNase free water preparation
glucose solution	Sigma-Aldrich	G3285	for lipoplexes preparation
Sodium chloride, NaCl	Sigma-Aldrich	S7653	insect saline buffer formula
Potassium chloride, KCl	Sigma-Aldrich	P9333	insect saline buffer formula
Calcium chloride, CaCl2	Sigma-Aldrich	C1016	insect saline buffer formula
Magnesium chloride hexahydrate, MgCl2.6H2O	Sigma-Aldrich	M2670	insect saline buffer formula
EGTA	Sigma-Aldrich	E3889	enzyme inhibitor
dissecting scissor	F.S.T.		cockroach dissection
fine tweezers	F.S.T.		cockroach dissection
flexible tweezer	F.S.T.		cockroach holding
pipetman	RAININ	P10	sample preparation
microcentrifuge tube	Axygen	MCT175C, PCR02C	sample preparation
pipette tip	Axygen		sample preparation
vortexter	Digisystem	vm1000	sample preparation
Minispin centrifuge	The Gruffin Group	GMC 206	for liquid spin down
Centrifuge	ALC	PK121R	sample preparation
pH meter	JENCO	6071	for pH adjust
micro-volume spectrophotometer	Quawell	Q3000	nucleic acid quantitative
PCR Thermal cycler	ABI	2720	for template PCR or  dsRNA synthesis incubation
quantitative real-time PCR	ABI	StepOne plus	gene expression quantitative
Centrifugal Vacuum Concentrators	eppendorf	5301	for dsRNA or total RNA extraction
Multipette	eppendorf	xstream	for real-time PCR sample loading
Agarose I	amresco	0710	for nucleic acid electrophoresis
tub gene specfifc forward preimer	tri-I biotech		GGG ACA AGC CGG AGT GCA GA
tub gene specfifc reverse preimer	tri-I biotech		TCC TGC TCC TGT CTC GCT GA
dsTub template forward primer	tri-I biotech		TAA TAC GAC TCA CTA TAG GGA CAA GCC GGA GTG CAG
dsTub template reverse primer	tri-I biotech		TAA TAC GAC TCA CTA TAG GGT CCT GCT CCT GTC TCG CTG
dsEGFP template forward preimer	tri-I biotech		TAA TAC GAC TCA CTA TAG GGT ATG GTG AGC AAG GGC GAG GAG
dsEGFP template reverse preimer	tri-I biotech		TAA TAC GAC TCA CTA TAG GGT GGC GGA TCT TGA AGT TCA CC
tub qPCR forward primer	tri-I biotech		GGA CCG CAT CAG GAA ACT GGC
tub qPCR reverse preimer	tri-I biotech		CCA CAG ACA GCC TCT CCA TGA GC
ef1 qPCR forward primer	tri-I biotech		CGC TTG AGG AAA TCA AGA AGG A
ef1 qPCRreverse preimer	tri-I biotech		CCT GCA GAG GAA GAC GAA G