2026-03-26
Marcel Kaarow, Leonie Graser, Eileen Knorr, Anton Windfelder, Pascal Geisler, Frank Steiniger, Markus Oberpaul, Andreas Vilcinskas, Christoph Hellmann
The fall armyworm (FAW, Spodoptera frugiperda) is an invasive lepidopteran pest of staple crops. Its broad host range, ability to spread rapidly, and increasing resistance to pesticides pose a major threat to global food security. RNA interference (RNAi) offers a sustainable and targeted alternative to broad-spectrum chemical pesticides, but its efficacy is limited in lepidopterans primarily by the rapid degradation of double-stranded RNA (dsRNA) in the midgut and poor epithelial uptake. Here, we investigated lipid nanoparticles (LNPs) as a delivery strategy to enhance dsRNA stability and uptake in FAW larvae. LNP–dsRNA complexes (40–50 nm, +39 to +56 mV) were generated by the microfluidic mixing of a ternary lipid blend. Encapsulation protected dsRNA from degradation by gut enzyme extracts for up to 1 h, even under highly alkaline conditions (pH 11.5). The analysis of larvae exposed to Cy3-labeled dsRNA by fluorescence microscopy demonstrated that LNPs improved internal distribution beyond the gut lumen, whereas unformulated dsRNA mainly accumulated at the peritrophic membrane. These results indicate that LNPs resist the gut environment and overcome limited systemic uptake, the two major physiological barriers to RNAi in lepidopterans, enabling the more efficient delivery of dsRNA. This study establishes a lipid nanoparticle-based dsRNA delivery platform that overcomes key physiological barriers in FAW, providing a prerequisite for future in vivo gene knockdown and efficacy studies.
DOI: 10.3389/finsc.2026.17700552026-02-11
Sâmara V. Rocha, Chiara Bernardini, Poulami Sarkar, Chun-Yi Lin, Karina Fan, João Paulo R. Marques, Juan C. Cifuentes-Arenas, Maria Cristina S. Pranchevicius, Nelson A. Wulff, Fabrício J. Jaciani, Marcelo B. Cioffi, Daniel L. Stanton, Amit Levy, Andrea Soares-Costa
Huanglongbing (HLB) is considered the most serious Citrus disease for which there are currently no effective control methods. The putative agents of HLB, Candidatus Liberibacter asiaticus (CLas), and the vector Diaphorina citri, are widespread in citrus regions, causing losses in citrus cultivation worldwide. Studying the interactions between D. citri and CLas can elucidate disease epidemiology and determine specific targets for HLB control. This work aimed to improve our understanding of the host–bacteria (D. citri–CLas) relationship, focusing on cysteine peptidase (DcCathL) and its inhibition by citrus cystatin (CsinCPI-2). In this study, a combination of qPCR, FISH, and immunolocalization techniques was employed to detect DcCathL genes or proteins in CLas-free or -infected D. citri samples. To verify the effect of DcCathL inhibition on insect survival, an artificial diet assay containing recombinant CsinCPI-2 was performed with infected and healthy D. citri nymphs and adults. Accordingly, a relative expression of DcCathL 1.23 times higher and an approximately 3.3 times greater DcCathL transcripts in gut tissue were demonstrated in CLas-infected compared to CLas-free insects. Furthermore, the presence of DcCathL protein was detected in the gut, ovary, and salivary glands of D. citri, concentrated at the peripheral regions of the cells. The fluorescence signal associated with DcCathL indicates that the gut of infected D. citri contains 4.81 times more protein than the gut of a healthy insect. Similarly, the protein levels in the salivary glands and ovaries of infected insects were 1.38 and 1.1 times higher, respectively, compared to those of CLas-free insects. The efficacy of the artificial diet in delivering the recombinant protein to the insect was demonstrated by the detection of green fluorescent protein (GFP) in the intestinal tract of nymphs and adults. Finally, the CsinCPI-2 demonstrated a substantial increase in mortality among CLas-free nymphs (df = 1, p < 0.0001) and CLas-free or CLas-infected adults (df = 1, p = 0.0001). Thus, the development of inhibitors that can disrupt the interactions between bacteria and vectors by blocking DcCathL activity may represent a promising strategy to prevent the spread of HLB disease.
DOI: 10.3389/finsc.2026.1700002