To begin, prepare the chitosan double stranded RNA nanoparticles for feeding the silkworm larvae. Then pick freshly molted fifth instar silkworm larvae of similar size for the feeding experiment. Place each larvae individually into a well of a six-well plate.
Cover the plate and starve the larvae for 24 hours. Next, rinse fresh mulberry leaves with deionized water and dry them using clean kitchen paper. Cut the fully dried leaves into one centimeter by one centimeter leaf discs.
Dissolve the chitosan dsRNA nanoparticles in nuclease-free water at a concentration of 500 nanograms per microliter before use. Coat 10 microliters of chitosan dsRNA nanoparticles, control chitosan nanoparticles, and naked dsRNA separately on the surface of each mulberry leaf disc. Let the solutions air dry on the leaf discs at room temperature for five minutes.
Next, offer one nanoparticle-coated or dsRNA-coated leaf disc to each larvae every day. Once the coated leaf disc is fully consumed by the larvae, each day, provide fresh mulberry leaves. On day six, place the larvae on ice until they are immobile.
To dissect the larvae, cut off the thorax with scissors on a clean Petri dish and use tweezers to pull out the midgut. After removing the contents. wash the midgut in a Petri dish with nuclease-free water.
Freeze the midgut at minus 70 degrees Celsius in a 1.5 milliliter tube. Feeding chitosan dsRNA nanoparticles in silkworms resulted in a 79%reduction in BmToll9-2 transcript levels compared to control treatments. Silkworm larvae treated with BmToll9-2 silencing chitosan dsRNA nanoparticles were visibly smaller than the control larvae.
The cocoons from BmToll9-2 silenced larvae were also significantly smaller than those from the control group.
