RESUMO
The response of insects to orally delivered double-stranded RNA ranges widely among taxa studied to date. Long dsRNA does elicit a response in stink bugs but the dose required to achieve an effect is relatively high compared to other insects such Colorado potato beetle or western corn rootworm. Improving the delivery of dsRNA to stink bugs will improve the likelihood of using RNA-based biocontrols for the management of these economically important pests. Short hairpin RNA (shRNA) is a useful molecule with which to test improvements in the delivery of double stranded RNA in the neotropical brown stink bug, Euschistus heros, since shRNA alone does not elicit a clear effect like that for long dsRNA. Here, we show for the first time the oral delivery of shRNA triggering RNA interference (RNAi) in E. heros using 4 nm cerium oxide nanoparticles (CeO2 NPs) coated with diethylamioethyl dextran (Dextran-DEAE) as a carrier. We identified particle properties (coating composition and degree of substitution, hydrodynamic diameter, and zeta potential) and shRNA loading rates (Ce:shRNA mass ratio) that resulted in successful transcript reduction or RNAi. When the Z-average diameter of CeO2 Dextran-DEAE-shRNA NP complex was less than 250 nm and the zeta potential was in the 15-25 mV range (Ce:shRNA mass ratio of 0.7:1), significant mortality attributed to RNAi was observed with a shRNA concentration in feeding solution of 250 ng/µl. The degradation of the targeted troponin transcript by NP-delivered shRNA was equivalent to that observed with long dsRNA, while naked shRNA transcript reduction was not statistically significant. Elemental mapping by synchrotron X-ray fluorescence microprobe confirmed uptake and distribution of Ce throughout the body with the highest concentrations found in gut tissue. Taken together, our results suggest that a nanoparticle delivery system can improve the delivery of RNA-based biocontrols to E. heros, and therefore its attractiveness as an application in the management of this important pest in soybean production.
Assuntos
Heterópteros , Nanoestruturas , Animais , Heterópteros/genética , Interferência de RNA , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/genéticaRESUMO
The locus coeruleus (LC) is a chemoreceptive brain stem region in anuran amphibians and contains neurons sensitive to physiological changes in CO2/pH. The ventilatory and central sensitivity to CO2/pH is proportional to the temperature in amphibians, i.e., sensitivity increases with increasing temperature. We hypothesized that LC neurons from bullfrogs, Lithobates catesbeianus, would increase CO2/pH sensitivity with increasing temperature and decrease CO2/pH sensitivity with decreasing temperature. Further, we hypothesized that cooling would decrease, while warming would increase, normocapnic firing rates of LC neurons. To test these hypotheses, we used whole cell patch-clamp electrophysiology to measure firing rate, membrane potential (V(m)), and input resistance (R(in)) in LC neurons in brain stem slices from adult bullfrogs over a physiological range of temperatures during normocapnia and hypercapnia. We found that cooling reduced chemosensitive responses of LC neurons as temperature decreased until elimination of CO2/pH sensitivity at 10°C. Chemosensitive responses increased at elevated temperatures. Surprisingly, chemosensitive LC neurons increased normocapnic firing rate and underwent membrane depolarization when cooled and decreased normocapnic firing rate and underwent membrane hyperpolarization when warmed. These responses to temperature were not observed in nonchemosensitive LC neurons or neurons in a brain stem slice 500 µm rostral to the LC. Our results indicate that modulation of cellular chemosensitivity within the LC during temperature changes may influence temperature-dependent respiratory drive during acid-base disturbances in amphibians. Additionally, cold-activated/warm-inhibited LC neurons introduce paradoxical temperature sensitivity in respiratory control neurons of amphibians.
