4.5. Insect Dissection and Collection of Samples

The cockroaches were subjected to CO₂ sedation and cleaned with 10% bleach, 70% ethanol and double washed with Type II water. They were fixed with entomological needles in supine position on a silicone plate, and the following tissues were extracted: salivary glands (pool of ten individuals, four replicates), foregut (pool of five individuals, four replicates), midgut (pool of five individuals, four replicates), hindgut (pool of five individuals, four replicates), Malpighian tubules (pool of six individuals, four replicates), and fat body (pool of three individuals, four replicates). Samples were collected in previously cooled Eppendorf tubes, treated overnight with RNAlater, frozen the next morning with liquid nitrogen and stored at −80 °C until RNA extraction.

For the collection of hemolymph samples (pool of forty individuals, three replicates), individuals were sedated with CO₂ and fixed with entomological needles in supine position on a silicone plate. Subsequently, one of the legs of the third pair was cut at the beginning of the second segment. Hemolymph was collected by capillarity by massaging the abdominal area. Hemolymph was collected directly in TRI Reagent (hemolymph to reagent ratio 1:3), and samples were frozen in liquid nitrogen and stored at −80 °C until RNA extraction.