RNA Interference in the Egg Parasitoid, Trichogramma dendrolimi Matsumura

Trichogramma wasps are a group of egg parasitoids used to control laboratory pest in agriculture. The intracellular bacteria Wolbachia induce cysts but not genesis in some trichogramma wasps. We are trying to answer the metabolic method of Wolbachia and sex determination systems in Trichogramma wasps.

We have developed the RNA Interference method in Trichogramma wasps. This method is the vital genetic tool kit for investigating the gene functions of Trichogramma. So in major stages of Trichogramma, offspring develop within the host egg.

The body size of Trichogramma is extremely small and is approximately 0.5 millimeter in adult lens. The manipulation of genetic tools such as RNA interference and genome editing, presents a formidable challenge for this tiny parasitoid wasps. For decades genome function within the entire Trichogramma genus have been rarely explored.

The present methodology provide a robust model for investigating general regulations during developmental and physiological activities in Trichogramma wasps. To begin, add a solution of gum Arabic powder and water to a 9 centimeter by 16 centimeter card. Then place approximately 5, 000 eggs of Corycephalotica onto the card.

Subject the host egg cards to 30 minutes of ultraviolet irradiation to prevent the hatching of eggs. Subsequently, cut the paper with inactivated eggs into egg cards each containing approximately 300 to 500 eggs. Next, introduce a cohort of 80 to 120 Trichogramma dendrolimi wasps into a glass tube.

Seal the tube with cotton. For parasitization, allow the wasps to deposit their eggs into the host eggs for six hours. Then, promptly remove the wasps.

Cultivate the parasitized host eggs for approximately eight days at 25 degrees Celsius. After five days, the parasitized host eggs turned black. Transfer the host egg card to a dissecting microscope.

Using a pair of tungsten needles, meticulously remove the chorion from the host eggs and retrieve the pupa from within. Next, prepare a plate with 1.5%agar as substrate. Using a disinfected graver, etch several grooves on the agar.

Now using a small brush, transplant a pupa from the dissected host egg into one of the grooves on the agar substrate. To begin, turn on the glass needle puller. Then set the heat to 280, the velocity to 170, the delay to 250 and the pole to 30.

Bevel the tip of the capillary glass needle. Then, using a micro injection pump load approximately two microliters of ferritin heavy chain homology double stranded RNA solution into the injection needle. Transfer the agar plate containing Trichogramma dendrolimi pupae onto the compound microscope stage.

Carefully insert the injection needle into the abdomen of a pupa at an approximately 30 degree angle and gradually inject five nanoliters of ferritin heavy chain homology double stranded RNA solution. Incubate approximately 700 pupae per treatment at 25 degrees Celsius for 24 or 48 hours. Extract RNA content from 100 pupae per replicate.

From approximately 50 pupae per treatment, observe the wasps emergence. Finally, record the wasps emergence rate and deformity rate. T.dendrolimi pupae injected with double stranded ferritin heavy chain homology exhibited a significantly lower emergence rate compared to those injected with double stranded GFP or without injection.

In emerged wasps, 51.85%of T.dendrolimi wasps subjected to double stranded ferritin heavy chain homology developed deformed small wings. This deformation was absent in wasps injected with double stranded GFP or those without any injection. Additionally, T.dendrolimi pupae injected with double stranded ferritin heavy chain homology showed melanism, indicative of abnormal iron metabolism.

The melanism was not observed in wasps injected with double stranded GFP or those without any injection.