The sandwich feeding assay for use with first instar nymphs of the Asian citrus psyllid, Diaphorina citri confirms the high susceptibility of this life stage to bacterial pesticidal proteins

Citrus greening or huanglongbing is the most important disease of citrus and threatens citrus production worldwide. As nymphs of Diaphorina citri play a crucial role in the acquisition and transmission of the citrus greening bacterium, suppression of this life stage is particularly important. However, the lack of a tractable feeding assay for use with first instar D. citri nymphs has impeded assessment of the toxicity of bioactives. Of several bacterial pesticidal proteins (BPP) that are toxic to D. citri adults, Mpp51Aa1 and Cry1Ba1, which have LC₅₀ values of 110 and 120 µg/mL respectively in adults, were fed to 1st instar nymphs in a newly developed assay. For this new sandwich feeding assay, parafilm layers containing feeding solution were placed on top of two 35 mm Petri dishes, with a concave surface created on each. Fifty nymphs were transferred to the membrane on one Petri dish, and the second Petri dish placed on the top to create a "sandwich" with the 1st instar nymphs in the middle. Nymphs were fed for four days and the LC₅₀ values for Mpp51Aa1 and Cry1Ba1 were calculated at 6.7 and 41.6 µg/mL respectively. Bioassays with bioengineered plants expressing Cry1Ba1 confirmed that the majority of D. citri mortality occurs during the 1st instar nymph stage, while egg laying adults are much less susceptible. Taken together, these results confirm that 1st instar D. citri nymphs are more susceptible to BPP than adults and demonstrate the utility of the sandwich feeding assay for effective screening of BPPs prior to investment into production of transgenic plants.