Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

Artificial feeding and saliva collection provide a direct method for detecting effectors in insect saliva. However, this has not be described in detail before. This protocol is effective for saliva collection from saprophytic insects.

Additionally, it's a simple technique, as we use medium containing only sucrose as a artificial diet. Saprophytic insects'saliva may affect against the host's immune system to benefit feeding behavior and transmit pathogens. This medicine that can be used for insect behavior and the insect bone path research.

This is a simple method to perform. However, this suggest the mounting of clean experimental environment as saliva is being collected for further research. Begin by preparing a 5%sucrose solution for the artificial diet, and filter the solution through a 0.22 micron filter to remove bacterial contamination and impurities.

Starve approximately 200 small brown plant hopper larvae for three to five hours before introducing them into a feeding chamber. Next, to prepare the glass cylinders as feeding chambers, cover one open end of the chamber with a paraffin membrane before introducing the experimental insects, then transfer the insects into the glass cylinder and cover the other end of the chamber with stretched paraffin membrane. Add 200 microliters of artificial diet on the membrane, then cover the liquid with another layer of stretched paraffin membrane.

Finally, cover the chamber with aluminum foil, leaving the end with the artificial diet device exposed to the light. After 24 hours, collect the SBPH saliva by cooling the cylinder at four degrees Celsius to immobilize the insects, then uncover the outer film and collect the artificial diet into 1.5 milliliter sterile tubes using a sterile pipette. Keep the collected saliva at minus 80 degrees Celsius until analysis.

Next, rinse the inner membrane with 50 microliters of fresh artificial diet three times by pipetting softly, and collect the artificial washing diet into 1.5 milliliter sterile tubes. After the three rinses, add fresh artificial diet on the inner membrane and place a freshly stretched paraffin membrane on top. Finally, filter the collected samples through a 0.22 micron filter unit to remove microbes and other contaminants.

Transfer the collective saliva samples in a 500 microliter, 10 kilodalton centrifugal filter. Centrifuge the sample. Collect the supernatant and bring up the final volume to 100 microliters.

To measure the concentration of the collected saliva, turn on the UV visible spectrophotometer and wash the pedestals three times with double-distilled water. Select the options on the screen in the following order, proteins, protein A280. Select type and one absorbance unit equal to one milligram per milliliter.

Then check the box for baseline correction, 340 nanometers. Next, load two microliters of 5%sucrose aqueous solution as blank, then touch Blank at the bottom of the screen. Finally, after setting the standards, load two microliters of the collected saliva and record the protein concentration.

When feeding on 5%sucrose, more than 80%of SBPH survived for the first four days. However, from the fifth day onward, mortality increased quickly to 40%and on the seventh day, less than half of the SBPH survived. An SDS page analysis showed the concentrated saliva samples from RSV-infected SBPH saliva contained many more proteins than the 5%sucrose negative control.

In a Western blot analysis for detection of the RSV capsid protein, a non-infected and viruliferous insects. The RSV capsid protein was successfully detected in the viruliferous sample, an antibody designed against the LssgMP gene product detected a 78 kilodalton protein in non-infected and viruliferous samples, demonstrating that LssgMP is a saliva protein. A QRT PCR analysis of LssgMP transcript levels in different tissues showed that the Lssg transcript levels in the salivary gland was 20-fold higher than in the whole body and gut, confirming the specific expression of LssgMP in the salivary gland.

Starving the experimental insects before introducing them into chamber is necessary to ensure the efficiency of our saliva collection. Besides, an artificial medium should be collected and exchanged every 24 hours to reduce microbial contamination in the collected sample. This technique provides a direct method to study the function of saliva in transmitting pathogens.

Additionally, it can help study insect pathogen host interaction.