RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

1. RNA isolation and de novo transcriptome assembly

1. Perform total RNA isolation from late stage third instar diving beetle larval eye tubes and adult beetles using an RNA isolation kit (see Table of Materials) that is designed for lipid-rich tissue. Isolate total RNA from T. marmoratus and sequence it following previously described methods¹⁸.
2. De novo transcriptome assembly
   NOTE: To assemble the transcriptome de novo, various bioinformatics platforms can be used (see Table of Materials). These platforms are commercially available and, in some cases, exist as Unix-based command line scripts. Since the assembly is de novo, it is recommended to assemble the transcriptomes independently on two platforms and compare the results to exclude false positives or false negatives.
   1. To start assembling the transcriptomes, upload the raw reads on the respective bioinformatics platform.
      NOTE: These files are usually compressed and in the format FASTQSANGER.GZ. There is no need to decompress the files as this format is accepted in the next step.
   2. Using the Trimmomatic command line, trim the raw reads to remove adapter sequences or short reads that fall below the default threshold. Decompress the trimmed raw reads; the files will now be in the FASTQ format. Concatenate the FASTQ raw reads for each sample to obtain files that are ready for de novo assembly.
   3. Assemble the concatenated raw reads de novo using any command line script that can assemble transcriptomes de novo. Save the assembled transcriptome, which will now have a list of contigs with their respective sequences, as a FASTA file. To increase the coverage of the assembly, combine and assemble multiple transcriptomes for the same species.
      NOTE: This process increases the chances of generating more accurate contigs and is especially important if low copy number genes are of interest.
   4. Once assembled, assess the transcriptome for completeness by calculating coverage scores (coverage assessment software is freely available, see Table of Materials).
      NOTE: A transcriptome with a coverage score >75% is considered good. However, the relevance of this score depends on the reason for transcriptome generation. For example, if the transcriptome is being used to identify low copy number genes, then a score >85% is better, as it signifies greater coverage.
   5. Annotate the de novo assembled transcriptome using a basic local alignment search tool (BLAST; see Table of Materials).
      NOTE: For this experiment, the transcriptome was annotated primarily against a database of known Drosophila proteins and a database of known beetle proteins. The list of annotations can be downloaded as a spreadsheet file and contig/contigs that indicate sequence similarity to the proteins of other organisms can be downloaded as a FASTA file.
   6. Identify the contig number/numbers of the protein of interest and extract the nucleotide sequences. Use BLAST to identify if protein-specific conserved regions (such as homeobox domains) are present in the annotated nucleotide sequence of interest. Check other contigs with the same annotation for nucleotide sequence overlaps to generate the sequence of a gene-specific transcript.
      NOTE: Identifying contigs with sequence overlap is not usually possible for all the genes, especially for low copy number genes.
   7. If the full-length sequence of the gene is needed, start with the contig that has the highest sequence similarity as an initiation point for identifying the nucleotide sequence of the whole mature transcript using techniques such as 3′ and 5′ rapid amplification of cDNA ends (RACE¹⁹).
   8. Annotate the assembly obtained from the second platform following steps 1.2.4–1.2.7.
      NOTE: Ideally, the results should be similar for the proteins of interest; however, coverage can differ between platforms to some extent.
   9. Use the assembled transcriptome to synthesize species-specific dsRNA as outlined in section 2.

2. Cloning and gene-specific dsRNA synthesis

NOTE: Gene-specific cloning and dsRNA synthesis has been described in detail for T. castaneum^20,21,22. The following steps are a brief overview.

1. Cloning, bacterial transformation, and plasmid sequencing
   1. Isolate total RNA from the organism (as described in step 1.1) and create complementary DNA (cDNA) using a reverse transcription kit (see Table of Materials). Identify one or two 100–1000 bp nucleotide sequences from contigs that are specific for the genes of interest.
      1. Design primer pairs spanning the entire identified stretch of nucleotides and amplify the sequence through a polymerase chain reaction (PCR). Add an extra 30 min step at the end to create 3′ adenine overhangs in the amplified DNA product. Purify this product using a PCR purification kit (see Table of Materials).
   2. Clone the purified product into a pCR4-TOPO plasmid vector (see Table of Materials) following the vendor’s protocol provided with the cloning kit. Using heat-shock treatment transform the cloned plasmids into competent bacterial cells (strain: E.coli DH10B) following the vendor’s protocol for competent cells (see Table of Materials), and screen for successfully transformed cells.
      NOTE: Plates with colonies can be wrapped in paraffin film to retain moisture and stored at 4 °C for up to one month.
   3. Pick colonies of transformed cells using a 10 µL pipette tip and culture in lysogeny broth (LB) containing ampicillin (50 µg/mL) in a shaker-incubator at 37 °C and 200 rpm for 24 h.
      NOTE: For long-term storage, cultured cells can be diluted 1:1 with 100% glycerol and frozen at -80 °C as glycerol stocks.
   4. Isolate plasmids containing the gene of interest from this culture using a miniprep isolation kit (see Table of Materials). Store the isolated plasmids (minipreps) at -20 °C after assessing the yield spectrophotometrically. Specifically, measure the sample absorbance at 260 nm, 280 nm and 230 nm. Calculate the ratios A₂₆₀/A₂₈₀ for quantifying DNA, and A₂₆₀/A₂₃₀ for quantifying DNA purity.
      NOTE: A 260/280 ratio of 1.8 is generally accepted as sufficiently pure DNA, ratios lower than 1.5 indicate the presence of residual extraction reagents such as phenol. The 260/230 ratio should be greater than or equal to the 260/280 ratio. Lower ratios indicate residual reagent contamination. The yield typically ranges from 100 to 500 ng/µL.
   5. Sequence the isolated minipreps to confirm that the gene-specific fragment has been successfully integrated, being flanked between 5′ T7 and 3′ T3 promoter regions in the plasmid; this can be done using universal T7 and T3 sequencing primers²². Use the minipreps with the gene-specific sequence of interest for dsRNA preparation.
2. PCR amplification and in vitro dsRNA synthesis
   1. PCR amplification and product purification
      1. Design plasmid-specific or gene-specific primers that have the T7 promoter sequence on their 5’ sides (see reference²² for details) to amplify a linear fragment of the inserted gene. Optimize the yield by setting up at least 10 reactions of 20 µL each.
         NOTE: The resulting product is flanked by two 20 bp T7 polymerase binding sites.
      2. Purify the PCR product using a PCR product purification kit (see Table of Materials), following the vendor’s column-based elution protocol. To increase the yield, repeat the final elution step up to 3 times with the same eluent. Assess the yield spectrophotometrically.
         NOTE: The yield usually ranges from 100 to 700 ng/µL. This purified product can be stored at -20 °C until further use.
   2. In vitro dsRNA synthesis and purification
      1. Use the gene-specific PCR purified product to synthesize dsRNA in vitro according to the protocol of a dsRNA synthesis kit of choice. If necessary, extend the incubation time for increased dsRNA production.
      2. Assess the progression of the reaction based on the opacity of the reaction mixture (the greater the amount of the dsRNA product, the higher the turbidity). At the end of the incubation, stop the reaction using a DNase treatment. To do so add 1 µL from a stock of 2 U/ µL, to the reaction mixture. Extend this treatment to 1–2 h to ensure the optimum degradation of the template DNA.
      3. Purify the dsRNA with a purification kit or via the following steps.
         1. Dilute the resulting product with 115 µL of nuclease-free water and add 15 µL of 3 M sodium acetate. Add 2 volumes of ice-cold 100% ethanol to this reaction mixture and precipitate the dsRNA overnight at -20 °C.
            NOTE: At this point, samples can be stored safely without affecting the final yield.
         2. Centrifuge the precipitate at 0 °C for 20 min at 9000 x g and discard the supernatant. Wash the product once with ice-cold 70% ethanol and centrifuge for 15 min at 13000 x g.
         3. Remove the supernatant and air dry the pellet for 5–15 min. Resuspend the pellet in up to 40 µL of nuclease-free water (this volume can be adjusted based on the isolated pellet size) and assess the yield and purity spectrophotometrically, as described in 2.1.4.
         4. Store the purified dsRNA at -20 °C until further use. Use the purified dsRNA for injection at the desired developmental stage.

3. Collection and preparation of early stage T. marmoratus embryos for dsRNA injections

1. Prepare an agarose plate for the incubation of T. marmoratus embryos.
   1. First, dissolve 20 mg of low-melting agarose powder in 100 mL of autoclaved distilled water (2% agarose) and then boil this mixture in a microwave until a clear solution is obtained. Take care with the hot solution, as air bubbles can cause it to overflow.
   2. Dispense this solution into a small Petri dish. To make grooves (which will hold the embryos) on the surface of the agarose plate, place three 1–2 mm wide plastic tubes (such as the tips of plastic transfer pipettes) on the surface of the liquid agarose before it solidifies.
   3. Once the agarose solidifies, use a pair of blunt forceps to remove these tubes. Add 500 µL of autoclaved distilled water using a P1000 micropipette to cover these indentations in the agarose.
2. Using a fine natural hair paintbrush, collect T. marmoratus embryos that are 5–8 h old from the beetle nesting sites in a glass cavity dish filled with autoclaved distilled water. Monitor the nesting sites frequently to assure that the obtained eggs are of the proper age.
3. Dechorionate the embryos under a stereomicroscope using fine dissection forceps. To do so, visualize the chorion under the microscope (at the desired magnification) by positioning the light source at an appropriate angle, then grab the chorion from two sides with sharp forceps, rip it open, and gently slide the embryo out. As there are two layers, ensure that both are removed.
4. Using a fine natural hair paintbrush, carefully transfer the embryos from the glass cavity dish to the agarose plate and arrange them in the grooves under a stereomicroscope. Take great care during this process, as dechorionated embryos are very fragile. Use the prepared embryos for dsRNA injections.
   NOTE: The slightly thicker end is the side of the embryo in which the head will develop.

4. dsRNA microinjections in early stage T. marmoratus embryos

1. To inject early stage embryos with gene-specific dsRNA, prepare microinjection needles using a microinjection needle puller (see Table of Materials). While visualizing the needle tip under a stereomicroscope, use a pair of fine forceps to break the needle tip, creating a sharp edge. Ideally, break the needle tip diagonally so that a sharper edge remains on one side, which will allow it to enter the embryo while causing minimal injury.
2. Thaw the purified stock dsRNA solution on ice, add 1 µL of the 10x injection buffer and top it off with double distilled water, to make 10 µL of injection solution. For injections, a dsRNA concentration of 1 µg/µL usually is suitable but can be adjusted according to the phenotypic severity of the resulting animals).
   NOTE: Prepare 1 mL of 10x injection buffer²² by adding 10 µL of 0.1 M sodium phosphate buffer (made by mixing 8.5 mL of 1 M Na2HPO4 and 1.5 mL of 1 M NaH2PO4 adjusted to a pH 7.6 at 25 °C with 100 µL of 0.5 M KCl), 100 µL of food dye and 790 μL of double-distilled water.
   1. Using a P10 pipette, backfill the microinjection needle with the working dsRNA solution. Once the solution has filled the tip of the needle, attach it to a microneedle holder connected to a microinjection system that utilizes pressure ejection technology (see Table of Materials).
3. Turn on the microinjection system and the pressurized air supply. Using the microvalve on the microinjection system, adjust the injection pressure to 15 psi. Adjust the injection duration using the time adjustment knob (set it to “Seconds”).
   1. As the injection duration depends on the required injection volume, if necessary, adjust this parameter on the microinjection system before each injection. Use an injection volume of 1–2 nL for early stage T. marmoratus embryos.
      NOTE: Higher injection volumes tend to interfere with the embryo survival rate.
4. To measure the volume of injection fluid that is dispensed by the microinjection system, calibrate the injection needle by assessing the volume of the fluid bubble that is formed at the tip of the needle when injecting into air. For T. marmoratus embryos, use an optimal bubble size of 100–200 µm. The injection needle is of good quality if this can be achieved at 15 psi with an injection duration of ~3 s.
5. Align the needle and the agarose plate containing the embryos under a stereomicroscope, such that the needle approaches the embryos at an angle between 45° and 60°.
6. Using the micromanipulator, move the microneedle slowly while monitoring the progress through the stereomicroscope. Once the tip of the needle is touching the surface of an embryo, carefully move the needle forward until it pierces the surface of the embryo. Do not perforate the embryo deeply with the needle tip since this can affect the survival of the embryo. To reduce variability, deliver the injection in the middle of the embryo.
7. Once the needle is inside the embryo, carefully press the injection button of the micro-injector to deliver the dose of dsRNA to the embryo. After injecting 1–2 nL of dsRNA, slowly retract the needle from the embryo, such that it does not cause the embryo to rupture.
   1. Inject the other embryos in a similar fashion. If the microinjection needle becomes blocked during the procedure, unblock it by increasing the injection pressure and duration. Visually identify successfully injected embryos by the presence of a colored spot at the site of the injection (since the injection buffer contains food coloring).
8. Carefully place the dish with injected embryos in a humidity chamber.
   1. To construct such a chamber, take any plastic box with a lid, sterilize it with 70% ethanol, allow it to dry, and place tissue soaked in autoclaved water at the bottom to provide a humid environment. Place this humidity chamber in an incubator with an appropriate temperature (in this case, 25 °C) and light cycle of 14 h light and 10 h dark.
9. Allow injected embryos to develop into first instar larvae, which requires 4–5 days. Monitor the progress of embryo development daily using a stereomicroscope to identify morphologically visible knockdown phenotypes as depicted in Figure 2.
10. Document this progress by taking digital images using a high-resolution camera. Remove dead embryos and replenish the water on the agarose plate daily to avoid desiccation and the possible spread of microbial contamination to other surviving embryos during the incubation period.
11. Perform appropriate controls for dsRNA injections, including buffer injections, injections of dsRNA synthesized against the sense strand of the gene of interest, and injections of dsRNA synthesized against sequences that do not exist within the target organism. Confirm RNAi knockdown using additional molecular methods²².

5. Preparation of T. marmoratus larvae for dsRNA injections

NOTE: Unlike early stage embryos, T. marmoratus larvae are relatively sturdy and can be injected with larger volumes. For example, second instars can be injected with up to 2 µL of dsRNA working solution and third instars with at least 3 µL without noticeable negative effects on the survival rate. To inject dsRNA, it is helpful to immobilize larvae by embedding them in agarose.

1. Before collecting the larvae, melt 2% agarose and keep it in a water bath at 60–70 °C to maintain it in liquid form. Prepare an immobilization dish by pouring melted 2% agarose into a small Petri dish and then cooling until solidified. Use blunt forceps to make a shallow groove in the agarose plate (roughly the size of the arthropod to be embedded) for each animal that will be injected.
2. Collect young larvae at the appropriate developmental stage (one stage prior to the stage desired for analysis). To do this, carefully drain the water from the culture cups containing the larvae and follow the steps mentioned below.
3. Anaesthetize on ice (carefully pick the larva out from its water cup and place it gently on ice) until the larva shows no notable movements. Use blunt, soft-ended forceps to carefully lift the larva from the ice and place it on the immobilization stage with its neck and body in the groove but its tail located above the agarose surface so that it will not be covered, which allows the larva to continue breathing through its tail spiracles.
4. Cover the larva with a thick layer of agarose that is still liquid but not dangerously hot. If it was accidentally covered, use the forceps to free the tail and the two segments below it from the agarose. Once the agarose solidifies, use the larva for dsRNA injections.

6. dsRNA microinjections in T. marmoratus larvae

1. Prepare and backfill a microinjection needle with the desired amount of gene-specific dsRNA working solution (as described in steps 4.1–4.2). Attach the needle to a needle holder connected to a manually controlled microinjection syringe. Before attaching the needle, pull the syringe plunger all the way out to avoid running out of injection pressure mid-procedure.
2. Position the immobilized larva such that the injection site, which is located dorsally between the third and fourth body plate segments, is aligned with the trajectory of the microinjection needle at a relatively flat angle (nearly parallel to the stage).
   NOTE: Angling the needle and the larva in this position is important, as it provides the path of least resistance for the needle tip to perforate the larvae with minimum damage.
3. Once the needle and the larva have been positioned, carefully move the needle tip into the larva using the micromanipulator while monitoring the progress through a stereomicroscope.
4. After the tip perforates the tissue, slowly and carefully apply pressure to the syringe. Adjust the injection pressure by observing the movement of the colored injection fluid in the needle under the microscope.
5. Carefully retract the needle after the injection. Use soft forceps to peel away and hence free the larva from the agarose. Transfer the larva back into a container with water (at room temperature) and allow it to develop further in an incubator or culture room at 25‒28 °C and a light cycle of 14 h light and 10 h dark.
6. Employ appropriate control injections and knockdown quantifications, as outlined in step 4.10.