Applications of RNA Interference in American Cockroach

Summary

The present protocol describes step-by-step guidelines for the RNAi operation techniques in P. americana.

Abstract

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous characteristics. Altogether with well-annotated genome sequences, these advantages have made American cockroach, Periplaneta americana, an important hemimetabolous insect model. Limited by the shortage of knockout strategy, effective RNA interference (RNAi)-based gene knockdown becomes an indispensable technique in functional gene research of P. americana. The present protocol describes the RNAi operation techniques in P. americana. The protocol includes (1) selection of the P. americana at proper developmental stages, (2) preparation for the injection setting, (3) dsRNA injection, and (4) gene knockdown efficiency detection. RNAi is a powerful reverse genetic tool in P. americana. The majority of P. americana tissues are sensitive to extracellular dsRNA. Its simplicity allows researchers to quickly obtain dysfunctional phenotypes under one or multiple targeting dsRNA injections, enabling researchers to better use the P. americana for developmental and metamorphic studies.

Introduction

RNA interference (RNAi), an evolutionarily conserved mechanism, gradually becomes an essential reverse-genetic tool to inhibit gene expression in many organisms¹, since Andrew Fire and Craig Mello² developed the double-stranded RNA (dsRNA) mediated gene silence strategy. dsRNA is cleaved into fragments of 21-23 nucleotides, small interfering RNAs (siRNAs), by the enzyme Dicer in cells to activate the RNAi pathway. Then siRNAs are incorporated into the RNA-induced silencing complex (RISC), which couples to the target mRNA, causes mRNA cleavage, and finally results in the loss of gene function^3,4,5. Among the insect species, many systemic RNAi experiments have so far been reported in lots of insect orders, such as Orthoptera, Isoptera, Hemiptera, Coleoptera, Neuroptera, Diptera, Hymenoptera, Lepidoptera, and Blattodea^5,6,7,8.

Cockroaches (Blattaria) are an essential insect family in developmental and metamorphic studies with their rapid growth cycles, strong adaptability to the environment, and high developmental plasticity⁹. Before discovering that RNAi was compatible with cockroaches, previous research only focused on cockroach prevention and control due to a scarcity of genetic manipulation techniques in cockroaches. The cockroach ootheca's unique structure made it challenging to perform embryo injection-based gene knockout with the CRISPR-Cas9 system. Besides, most tissues in cockroaches (such as P. americana) show robust systemic RNAi response, allowing for the rapid generation of dysfunctional phenotypes by injecting one or more targeting dsRNAs^9,10,11. These features made RNAi an indispensable technique in gene functional research in P. americana.

Even though the use of RNAi in functional gene research in P. americana has been reported, no detailed or step-by-step description was available. This report provides one step-by-step operational guideline for RNAi in P. americana, useful for gene function study in other cockroaches. Furthermore, this guide is not limited to Blattodea and can be applied to many other insects with minor modifications.

Protocol

The line of P. americana was initially provided by Dr. Huiling Hao. This species has been maintained with inbreeding for 30 years⁹.

1. Hatching and feeding of P. americana

1. Collect fresh oothecae (immediately post egg-laying) of P. americana and incubate in the dark incubator at 25 °C and 60% humidity for ~25 days. Then increase the temperature and humidity to 30 °C and 75% 3 days before hatching.
2. Use a sieve with 4 mm aperture to separate the hatched nymphs from oothecae.
3. Keep the nymphs in cylindrical containers (12 cm in diameter and 10 cm in height) in the dark at 28 °C, 70% humidity. Brush the inside edge of the containers with vaseline to prevent cockroaches from escaping. Provide rat food, water, and shelter (egg trays). Pay attention to the activity of the nymphs and regularly clean up the feces and debris.

2. Selection of the nymphs in proper instar

1. Use a glass tube to pick out the freshly molted P. americana (white in body color). Keep them in new containers and wait for the correct stage for treatment.
   ​NOTE: After ~19 days under the above feeding conditions, the nymphs in the 3^rd instars will be available for injection. The color of freshly molted cockroaches is white, and the instars are clarified by molting times.

3. Preparation of the target fragment with T7 promoters

1. Using the cDNA of P. americana as a template, design paired primers to perform PCR to obtain 300-800 bp DNA fragment of the target gene⁹. Then clone the PCR fragment into a pTOPO vector (see Table of Materials) for sequencing.
2. Using the target fragment DNA as a template, synthesize one new pair of primers with T7 promoter sequence (5'-GGATCCTAATACGACTCACTATAGG-3') at the 5' terminals and perform another round of PCR to obtain the target fragment with T7 promoters on each side⁹.

4. Transcription and synthesis of dsRNA in vitro

1. Add 10 µL of T7 2X Buffer, 2 µL of the Enzyme Mix, T7 Express (see Table of Materials), and 2 µg of PCR product (obtained in step 3.2) in the reaction system and add up the total volume to 20 µL with ddH₂O. Gently mix upside down manually and incubate at 37 °C for 30 min and 70 °C for 10 min, and then slowly cool to room temperature.
2. Dilute RNase A solution (4 µg/µL) with ddH₂O at a ratio of 1:200. Then add 1 µL of RQ1 RNase-Free DNase (1 U/µL) and 1 µL of diluted RNase A solution to the system (see Table of Materials).
   NOTE: Now, the volume of a single system is 22 µL.
3. Incubate at 37 °C for 30 min. Then add 10% of the total volume (when performing N reactions simultaneously, the total volume is N x 22 µL) of Sodium acetate and three volumes of the total volume of isopropanol.
4. Gently mix upside down manually, place on ice for 5 min, and centrifuge at 13,000 x g for 10 min at 4 °C.
5. Remove the supernatant with a pipette, wash the residue with 75% ethanol (with 25% diethyl pyrocarbonate (DEPC) treated water), and then centrifuge at 13,000 x g for 5 min at 4 °C.
6. Remove supernatant again. Air-dry the pellet for 15 min at room temperature. Use ~100 µL of DEPC water to dissolve dsRNA, then dilute the dsRNA to the final 2 µg/µL.
   ​NOTE: The dsRNA could be stored at -80 °C for 6 months.

5. Loading dsRNA solution into the syringe

1. Set up the program in the micro-injection pump (see Table of Materials) in advance to ensure that the volume of each injection is consistent. Before using the syringe, clean the syringe by filling it with DEPC water 8-10 times.
2. Install the 10 µL Syringe on the micro-injection pump, start the pump, and fill the syringe with 10 µL of dsRNA solution (prepared at step 4.6). Inject 1 µL of the dsRNA into a 3^rd instar nymph (see step 2) and ensure that it does not leak out to the body.

6. Injecting dsRNA to P. americana

1. Anesthetize the cockroaches with an increased concentration of CO₂ in a container until the cockroaches do not move anymore, and then proceed immediately with the injection.
2. Gently pick up the cockroach with tweezers, and deliver the cockroach toward the needle with hand. Next, insert the needle via the gap between two abdominal somites horizontally against the epidermis. About insert depth, the shallower, the better.
3. Then, inject the dsRNA solution into the cockroach. Finally, pull out the needle tip. Ensure that the needle tip is as close as possible to the epidermis to avoid damaging internal organs.
   NOTE: The injection should be made under a dissection microscope.
4. Put the injected cockroaches into clean bioassay containers. Wait for about 10-20 min to let them recover from the CO₂ effects. Label the containers with the date of injection, type and dose of dsRNA, and age of the P. americana.
5. Place the injected cockroaches in a dark environment at 28 °C, 70% humidity, provide water, feed, and shelter, and observe possible changes in the phenotype of the cockroaches regularly.

7. Knockdown confirmation and phenotypic analysis

1. Evaluate the efficiency of RNAi using any available molecular biology techniques such as quantitative Real-Time PCR (qRT-PCR) and Western blotting. For detailed qRT-PCR and Western blotting procedures, see References^1,12.
2. To observe and analyze RNAi-related phenotypes, use the microscope suitable for living animals.
   NOTE: RNAi may affect the morphology, behavior, molting, limb regeneration, and other physiological activities of cockroaches. The specific frequency of phenotype is calculated according to specific conditions.

Results

Figure 1 shows a successful injection. The microinjection syringe with a micro diameter needle should be horizontally placed on the booster (Figure 1A). The needle is inserted via the gap between two abdominal somites horizontally against the epidermis (Figure 1B). Ensure that the liquid goes into the P. americana abdomen. The too steep angle of the needle will damage the internal organs (Figur...

Discussion

This report described a methodological step-by-step RNAi strategy in P. americana; of note, it also can be applied to other cockroaches (Blattella germanica, for example) and many other insects with minor changes. However, the gene silencing efficiency of RNAi is not always high enough, with an obvious disadvantage compared with the gene knockout strategy¹³. The following residual effect of gene-level may interfere with the real phenotypes. To ensure the RNAi treatment is success...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
701 N 10 µL Syr (26s/51/2)	Hamilton	PN:80300	Injection
Incubator	Ningbo Jiangnan Instrument Factory	RXZ-380A-LED	For cockroaches hatching and feeding
Micro-injection pump	Alcott Biotechnology	ALC-IP600	Injection
pTOPO-Blunt Cloning Kit	Aidlab Biotechnology	CV16	For Gene clonging
quantitative Real-Time PCR Systems	Bio-Rad	CFX Connect	For qRT-PCR analysis
T7 RiboMAX Express RNAi System	Promega	P1700	For dsRNA synthesis, which contains Rnase A Solution (4 μg/μL), Sodium Acetate, 3.0M (pH 5.2), Enzyme Mix, T7 Express, Nuclease-Free water, Express T7 2x Buffer, RQ1 RNase-Free DNase
Thermal Cyclers	Bio-Rad	S1000	For DNA amplification