Regulation of Las transmission and microbial colonization by the Asian citrus psyllid immune system

Regulation of Las transmission and microbial colonization by the Asian citrus psyllid immune system

Report Date: 07/30/2016
Project: 15-021   Year: 2016
Category: ACP Vector
Author: Kirsten Pelz-Stelinski
Sponsor: Citrus Research and Development Foundation

To examine the general effects of double strand RNA (dsRNA) uptake on D. citri fitness and biology, we have conducting dsRNA feeding experiments on D. citri adults of a CLas-free laboratory colony. dsRNA of genes that exist (e.g., V-type proton ATPase [v-ATPase]) or do not exist in D. citri (a bacterial GFP gene) were used for this study. Briefly, primers targeting each DNA fragment were designed and synthesized with T7 promoters tagged (5� ends). PCR were carried out and purified amplicons were used for dsRNA synthesis using the MEGAscript RNAi kit (Ambion, Austin, TX). dsRNA treatments were conducted by feeding D. citri with 20% sucrose solutions supplemented with 50 ng/�l of dsRNA and 0.5% of green food coloring dye, using previously described methods (Wuriyanghan, Rosa, and Falk 2011). The insects were separated by gender and 25 to 30 insects were used for each Gender x Treatment. For the negative control, blank elution buffers were added to the 20% sucrose solution. To determine whether the insects were feeding, we included a treatment group in which the insects did not have access to any diet. All insects were kept at a growth chamber and their mortality was recorded daily. Our preliminary data showed that insects that did not receive any diet had the lowest survivorship, indicating that D. citri in the other treatments were feeding on the sucrose diets. In females, the survivorship of both the GFP-dsRNA and v-ATPase-dsRNA treatment was lower than the buffer-only group. No significant difference was detected between the survivorship of the two dsRNA treatments. In males, all treatments other than the no-diet treatment had similar survivorship. Overall, these findings suggest that male and female D. citri may respond differently to the same dsRNA treatments; whether such difference is related with their physiology or behavior deserves further investigation. The reason why GFP-dsRNA and v-ATPase-dsRNA treatment resulted in similar effect on D. citri survivorship (particularly in females) will also be studied further. In the future, we will also be testing how similar RNAi treatments may influence D. citri�s immune system and ability to acquire or transmit CLas.


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