At first we decided to solve the HLB plague problem because it is affecting our country and our people. We knew we are not the first ones to make research about this disease and it´s characteristics. Before us, HLB has been addressed by using RNAi technologies that were to be directly applied to the targets which made them inconvenient for their use in the real natural environment.
We looked up for dsRNA technologies but they were nonspecific because it creates aleatory siRNA. So, we relied in the most certain path: siRNA technology, which assure specificity and results.

Taking this into account, we began the siRNA creation process by choosing what our main target would be: the infected plant or the disease´s vector, Diaphorina citri. We got to the conclusion that silencing genes in the psyllid will be the right solution to the problem as after doing an intensive research we found out that the disease was only transmitted through the psyllid (Diaphorina citri). Inside it´s genome there were very specific genes that helped the psyllid carry the disease and spread it. After a very substantial elimination, we got to choose four genes: Awd, RacI, SOD and Wnt. These will block the first steps of the infection and therefore end with the problem. It seems simple isn´t it?

Well, after we reached this, we were facing another problem. How were we going to apply our siRNA? We have seen that the existing delivery methods of the RNAi technologies were very complex and did not fulfilled their function. Therefore we decided to simplify the selection and application of RNAi technologies by creating a production mechanism, named BSLA which stands for Blue chromoprotein, sense, loop, antisense. BSLA will work as a universal siRNA reporter cassette that will carry our specific siRNA and assure it´s correct application.

Theoretically our siRNA works, but has it faced its real enemy? Our answer back then was: no. However we had a plan for that. Now that our new friends arrived, we gave them a really nice home, where they could get all the food, heat and love they needed. It was very important for us to give the Diaphorina a nice place to live, so they could settle there and form new families. Luckily for us, the insects began reproducing really fast and very soon, we found ourselves surrounded by an enormous psyllid community.

Now that we had a large psyllid population, it was the moment of testing if our designed siRNA were effective and recognized their corresponding target site in the genome of Diaphorina citri. The first thing we did was to extract the RNA from 20 individuals, making sure they were not in the reproductive stage. Since we care a lot about animal suffering, we performed this procedure making sure the psyllids were totally frozen in liquid nitrogen. Using the RNA, we performed a two-step RT-PCR using specific primers for the four genes and when we saw the gel, we were really excited to see that three of the four genes amplified the expected products. However, the RacI reaction showed a different product. Since the reported sequence states “like-sequence” it is possible that there are mismatches between the reported sequence and the real one. There are other reported sequences for this gene, but they are not specific for the designed primer either. We designed different siRNA but they were not available in time for us to continue.

Another important step to replace the number of psyllids that we were using was the establishment of the cell culture. This was presumably the most difficult step for us, since it was almost impossible to find the components for the medium reported by Hunter and Hert, beginning with the Schneider's insect medium. For this reason, we prepared two alternative mediums. We used 50 individuals for the cell extraction, once again, making sure of minimizing the suffering. The cells were cultured in our new mediums but they were not able to survive, due to the lack of key reagents, the short lifespan of the cells and the almost immediate contamination with fungi. Because of this, we had to forfeit the establishment of the cell culture and the following steps such as the transfection with siRNA and flow cytometry analysis.

For this reason, we were forced to make the in vivo studies via soaking. We took the psyllids and placed them near a droplet of siRNA for it to soak through the exoskeleton. Because of the delivery method, we observed little to no mortality from the soaking. Again, we performed the RNA extraction, cDNA synthesis and a real time PCR to know about the effects of the siRNA in the psyllids’ genome. In addition, we prepared some samples to test our mathematical model and our proposed delivery method with different chitosan molecules. This step was very intriguing for us, not only because the project was on the line, but because the thermal cycler stopped working and we needed to find another one of the same model.

It appeared like we already have it all figured out, but our siRNA had to survive terrible conditions to which it wasn't even prepared. So, we decided to look for one something that protected and maintained it free from any type of danger so that it could go and save the citrics. Our solution was the encapsulation. We discovered that we could create nanoparticles out of Chitosan, a biodegradable polysaccharide which can help siRNA molecules to bind and avoid its degradation by nucleases inside the D. citri´s cells and at the same time allow the super powerful siRNA to release RNA material.