The starting point of our project consisted on designing our own siRNA sequences, to target specific gene expression in Diaphorina citri . For this method to be effective and applicable to a real-life environment, it was of utmost importance ensuring the sequences were specific for the vector and wouldn’t target other specimens, resulting in unplanned modifications to the environment.

Several BLASTs were performed during the design of each of our four siRNA sequences.

For the Awd (Abnormal wing disc) gene target sequence, the BLAST analysis showed the closest match at 79%, which provided enough variation to ensure safety.

For the WNT (Wingless) gene target sequence, the BLAST analysis showed a complete match with Anticrates phaedima. However, this corresponds to a species of moth found only in Australia, which doesn’t inhabit the same ecosystems as Diaphorina citri , and is not found on citrus plantations.

For the SOD (Superoxide dismutase 1) gene target sequence, the BLAST analysis showed no matches, being Crassostrea madrasensis the closest at a 92% match. This corresponds to a species of mollusk, and, due to the propensity of RNA to degrade, it is impossible for it to affect the ecosystem this species inhabits.

For the Rac I gene target sequence, the BLAST analysis showed correspondence with the Diaphorina citri genome exclusively, making it completely specific.

An integral part of our project required the use of live specimens of Diaphorina citri . These were obtained from SENASICA, a government institution dedicated to agroalimentary sanity investigation. To ensure the psyllids were comfortable and able to live and reproduce, special rearing cages were built, based on the work from Skelley and Hoy.

Cages were constructed from a PVC structure and consisted of a small enclosed cage inside a larger, open cage, double lined with fine white polyester mesh to prevent psyllids from escaping. The mesh was tied into a knot on one side and secured with an elastic band, allowing us to reach inside the cages for placement or recollection of the insects. Light bulbs were incorporated at the top to control daily light exposure and provide some heat. Psyllids were exposed to 18 hours of light and 6 hours of darkness daily.

In addition, our team leader received special training about the handling of the psyllids at the Regional massive reproduction laboratory of Tamarixia radiata (Laboratorio Regional de reproducción masiva de Tamarixia radiata), located in the city of Mérida, Yucatán. In this laboratory, Diaphorina citri is the study subject in the attempt of controlling HLB disease using natural methods.

The host plants used were orange jessamines (Murraya paniculata), which is a natural host to the psyllids. The plants were washed with a 3% acetic acid solution and then with detergent to kill any pests that could be present and then were washed thoroughly with water. After inspecting for any remaining insects they were cut to induce the production of new shoots where Diaphorina citri females are able to oviposit.

Temperature was controlled using fan heaters kept at a distance of 1 meter from the cages and running 24 hours. The temperature reached 21ºC inside the cages, which allowed for slow reproduction. Humidity was measured using a reptile hygrometer, and cotton balls soaked in water were placed at the base of the plants to provide extra humidity inside the artificial environment. Humidity was kept at around 55%.

RNA Extraction and cell culture

Since our sequences were designed to be specific and effective in a real-life environment, we had to perform two protocols involving live insects: total RNA extraction and cell extraction for culture. While both of these required live psyllids, we took measures to ensure the insects would not suffer, get contaminated, or cause harm.

Prior to these protocols (and all handling), psyllids were placed inside ventilated jars at -4ºC for several minutes, which caused them to become drowsy. This prevented the insects both from flying and possibly escaping the laboratory, and made it easy for us to handle them for the experiments. Additionally, this provided a painless experience for them, since the dissections were made immediately and quickly. For the RNA extraction, psyllids were first placed on a mortar containing liquid nitrogen, which quickly and painlessly killed them.

In addition to this precaution, a previous cell quantification was performed using three specimens, with the objective of measuring the amount of insects that would be needed for cell culturing, and avoiding killing a large quantity of psyllids unnecessarily.

The following document was expended by the Regional massive reproduction laboratory of Tamarixia radiata certifiying that our team leader asisted to the technical route in the installations of the laboratory, and was instructed on the handling of Diaphorina citri .