High susceptibility of Diaphorina citri first instar nymphs to Bacillus thuringiensis-derived pesticidal proteins from multiple structural groups.

The Asian citrus psyllid, Diaphorina citri, is the primary vector of Candidatus Liberibacter asiaticus, the causative agent of citrus greening disease, which continues to threaten citrus production worldwide. Current management relies heavily on chemical insecticides, despite increasing concerns regarding resistance and environmental impacts. Because pathogen acquisition is most efficient during the nymphal stage, management strategies targeting immature psyllids may improve disease suppression. Although bacterial pesticidal proteins (BPP) have generally shown limited activity against D. citri, previous studies have focused on adults or late instar nymphs, potentially underestimating the susceptibility of earlier developmental stages. To address this gap, we conducted the largest screen to date of BPP against first instar D. citri. Thirteen proteins representing multiple structural classes were expressed in Bacillus thuringiensis or Escherichia coli, purified, and evaluated using sandwich feeding bioassays. Toxicity was assessed against first instar nymphs at a single dose, and LC50 values determined for proteins including Tpp78Aa1, Tpp78Ba1, Cry1Ca, Cry1Ea, that caused significant mortality relative to controls in the single-dose assays. Tpp78Aa1 and Cry1Ca were the most toxic, with LC50 values of 10.4 and 6.7 μg/mL, respectively. Marked disruption of the gut brush border and severe loss of microvilli was observed by transmission electron microscopy in fourth instar nymphs fed on Tpp78Aa1. These findings demonstrate the toxicity of Tpp78Ba1, Cry1Ca and Cry1Ea to D. citri for the first time and highlight the increased susceptibility of first instar nymphs to BPP relative to later developmental stages, supporting the targeting of this development stage in psyllid management programs.