Electroporation-mediated RNA Interference Method in Odonata

This is a video protocol for electroporation-mediated RNA interference method in Odonata. Dragonflies and damselflies, members of the order Odonata, are among the most ancestral insects. Adult Odonata show a remarkable diversity in body colors and patterns, and many ecological and behavioral studies have been conducted.

However, molecular genetic studies on Odonata have been lacking, because gene functional analysis is very difficult. For example, the conventional RNAi method is not effective in Odonata species. Recently, we established an RNAi method in combination with in vivo electroporation.

Here we provide a video protocol for the electroporation-mediated RNAi in both dragonflies and damselflies. As our representative damselfly species, we use the blue-tailed damselfly, Ischnura senegalensis.

This is one of the most common damselfly species and often found in sunny ponds in Japan. First, we show the RNAi protocol targeting the abdomen of a damselfly, Ischnura senegalensis. After ice-cold anesthesia, attach two pins on both sides of the prothorax and fix the larva to a fixed stand.

Pull and stretch the inter-segmental membrane between the seventh and eighth abdominal segment using forceps. Keep the inter-segmental membrane stretched by hand. Insert the tip of the prepared capillary into the stretched inter-segmental membrane.

Inject 1 microliter of siRNA or dsRNA solution. Apply two droplets of ultrasound gel on the larval surface using forceps. Place electrodes on the ultrasound gel, with the positive electrode on the side of injection.

Generate 10-times electroporation pulses using an electroporator. Wipe off the remaining gel on the surface with a paper towel. Remove insect pins and keep the treated larvae on a wet paper towel for approximately one day for recovery.

Next, we show the RNAi protocol targeting the thorax of a damselfly, Ischnura senegalensis. After ice-cold anesthesia, attach two pins on both sides of the prothorax and fix the larva to a fixed stand. Pull and stretch the inter-segmental membrane between the prothorax and synthorax using hand.

Insert the tip of the prepared capillary into the stretched inter-segmental membrane. Inject one microliter of siRNA or dsRNA solution. Apply two droplets of ultrasound gel on the larval surface using forceps.

Place electrodes on the ultrasound gel with a positive electrode on the side of injection. Generate 10-times electroporation pulses using an electroporator. Wipe off the remaining gel on the surface with a paper towel.

Remove insect pins and keep the treated larvae on a wet paper towel for approximately one day for recovery. As a representative dragonfly species, we use the pied skimmer dragonfly, Pseudothemis zonata.

This is one of the most common dragonfly species and often found in shady ponds in Japan. Finally, we show the RNAi protocol targeting the abdomen of a dragonfly, Pseudothemis zonata. Stretch the inter-segmental membrane between the fourth and fifth abdominal segment.

Make a small hole with a fine needle between the fourth and fifth abdominal segment. Insert the tip of the prepared capillary into the prepared hole. Inject one microliter of siRNA or dsRNA solution.

Fix the larva to a fixed stand using pins. Apply two droplets of ultrasound gel on the larval surface using forceps. Place electrodes on the ultrasound gel with the positive electrode on the side of injection.

Generate 10-times electroporation pulses using an electroporator. Wipe off the remaining gel on the surface with a paper towel. Remove insect pins and keep the treated larvae on a wet paper towel for approximately one day for recovery.

Here we selected a melanin synthesis gene MCO2 as the target gene, and EGFP or bla as a negative control target. MCO2 is essential for the black color formation in various insects. First, we show the results in the abdomen of Ischnura senegalensis.

White arrowheads indicate the regions of suppressed black pigmentation where the positive electrode was placed upon electroporation. Inhibition of pigmentation was observed both by siRNA treatment and dsRNA treatment. The size and location of the RNAi phenotype varied considerably among individuals.

Without electroporation, no RNAi phenotypes were observed, indicating that electroporation is essential for RNAi in this species. Next, we show the results in the thorax of Ischnura senegalensis. Similarly, inhibition of black pigmentation was observed around the region where the positive electrode was placed upon electroporation.

Finally, we show the results in the abdomen of Pseudothemis zonata. As expected, the RNAi phenotype was detected around the region where the positive electrode was placed upon electroporation. We confirm that the electroporation-mediated RNAi method is very useful in Odonata.

It can induce local gene suppression almost 100% efficiency at least in epidermis, when we select appropriate developmental stages. This method has several advantages over the CRISPR/Cas9 method.

For example, the region where the RNAi phenotypes appear can be controlled by the position of the positive electrode upon electroporation, and the RNAi phenotypes can be easily compared with the control phenotypes side by side in the same individual. Moreover, this method does not require microinjection into tiny eggs, so it is much easier and faster to observe the phenotype than the CRISPR/Cas9 method.

Furthermore, this method can be applied to insects whose newly-laid eggs are difficult to collect. For example, females of Pseudothemis zonata lay eggs during flight, so it is very difficult to collect their newly-laid eggs.

We expect that this protocol may be generally applicable to non-model organisms when the conventional RNAi does not work efficiently.