Applied Design

The brainstorm

We initially decided to solve the HLB plague problem because it affects our country and our people. We know we are not the first ones to research about this disease and its characteristics. 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 environment.

Additionally, silencing technologies use dsRNA, which is nonspecific because it can be cut randomly through the cell’s Dicer mechanism, creating siRNA sequences that may hybridize with non-target genes. So, using siRNA directly, we are able to surpass this mechanisms and ensure specificity.

Starting the action

Taking this into account, we began the siRNA creation process by choosing what our main target would be: the infected plant or the disease vector, Diaphorina citri. We reached 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. Inside its genome there were very specific genes that helped the psyllid carry the disease and spread it. After a very substantial elimination, we chose four targets: Awd, RacI, SOD and Wnt. These will block the first steps of the infection, prevent the psyllid from harboring the disease and increase mortality, and therefore end with the problem. It seems simple, doesn’t it?

The Eureka! moment

Well, after we reached this, we faced 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 fulfill 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 ensure its correct application.

Taking care of our new buddies

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, Diaphorina citri specimens, 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 psyllids 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.

Testing our creation AKA the most difficult part of science

Now that we had a large psyllid population, it was the moment to test if our designed siRNA were effective and recognized their corresponding target site in the genome of Diaphorina citri. The first thing we did was extract the RNA from 20 individuals, previously 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 immediately 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, 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 (2009). For this reason, we prepared two alternative mediums. We used 50 individuals for the cell extraction, and 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.

Wait a minute... How do we take our teeny tiny solution to the big outside world?

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 citrus. 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.

Is this the best solution?

In Mexico there are 30 allowed pesticides which are forbidden in other countries. Approximately 4.55 tons were used for every 1000 hectares of pesticides (fungicides, herbicides and insecticides) between the year 2009 and 2010. In 2013, 37,455 tons of insecticides were used; 31,195 tons of herbicides and 42,223 tons of fungicide (Arellano-Aguilar & Rendón, 2017).

The benefits of having an increased fauna consequently improves the efficacy of pollination, and biological control agents that help suppress a broad range of pests. Organic grower’s communities, support the technology because it is a truly natural, innovative breakthrough, to manage many of the pests and pathogens which plague their industries (Andrade, 2016).

The parasitoid Tamarixia radiata was released in previous years and has established, but is not a significant source of mortality for D. citri at present (Michaud, 2002). This method of biological control has also been used in our home country, in fact, the research center located in Mérida, Yucatán is focused in the massive reproduction of this parasitoid for its release in infected plantations. During this visit, it was mentioned to us that this biological control is efficient but the T. radiata population is still insufficient to significantly control the reproduction of D. citri and consequently, the spread of Huanglongbing. Even the presence of natural predators of the psyllid such as Coccinellidae (ladybugs, beetles), ants, and spiders is not enough, as the psyllids reproduce much faster.

The importance of developing genomic methods for agricultural management comes from the need of a specific target solution to avoid the negative effects on other species, as the technology provides greater specificity in pest targeting, while reducing the potential negative effects on ecosystems and leaving beneficial insects and other organisms unharmed in crop ecosystems. Because all living things have evolved to break down dsRNA and use the nucleic acids as cellular nutrients, this technology will be safe for those who work in the fields or eat the produce.

References

Marutani-Hert, M., Hunter, W. & Hall, D. (2009). Establishment of Asian citrus psyllid (Diaphorina citri) primary cultures. In Vitro Cell.Dev.Biol.— Animal (2009) 45:317–320. Doi: 10.1007/s11626-009-9188-3

Arellano-Aguilar, O. & Rendón, J. (2017). La huella de los plaguicidas en México. Greenpeace. Retrieved from: http://m.greenpeace.org/mexico/Global/mexico/Graficos/2016/comida-sana/Plaguicidas_en_agua_ok_EM.pdf

Andrade, E. (2016). RNA Interference – Natural Gene-Based Technology for Highly Specific Pest Control (HiSPeC). InTech. Doi: http://dx.doi.org/10.5772/61612

Michaud, J. (2002). Biological control of Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae) in Florida: A preliminary report. Retrieved from: https://www.researchgate.net/publication/266214464_Biological_control_of_Asian_citrus_psyllid_Diaphorina_citri_Hemiptera_Psyllidae_in_Florida_A_preliminary_report