Volpe, Gennaro (2023) Rnai-Mediated immunosuppression for the development of new insect control technologies. [Tesi di dottorato]

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The need to develop new pest management strategies which are effective and ecologically sustainable has stimulated significant research efforts on molecules of natural origin and on gene regulation strategies that mimic and/or enhance mechanisms of pest suppression by insect antagonists. This area of research can allow the exploitation of a nearly untapped source of genes and molecules with potential bioinsecticide activity. Besides the direct use of virulence factors as physiological disruptors of pest insects, it is possible to target the molecular targets of these factors and alter vital functions of pests. This latter approach is made possible by genetic tools now available, such RNA interference (RNAi), which can be used to suppress the expression of host genes targeted by virulence factors, reproducing the negative/lethal effects that the natural antagonists have on their hosts. In this context, the present PhD thesis aims to develop (1) new solutions for insect pest control using RNAi mediated suppression of gene expression, and (2) sustainable and safe field delivery systems of dsRNA. The first part of the thesis reports the expression analysis of the immune gene Sl102, that is suppressed in parasitized noctuid moth larvae, during the embryogenesis of Spodoptera littoralis (Lepidoptera: Noctuidae). The functional role was analyzed by RNAi and by transmission electron microscopy (TEM) observations. The very high mortality observed upon gene silencing was associated with morphological alterations associated with a general delay in the embryonic development, suggesting a possible role of this gene in tissue organization and differentiation during embryogenesis. This embryonic mortality is of particular interest from an applied point of view, since it prevents the damage caused by the larval feeding stages. The second part concerns the study of a gene encoding a protein of the REPAT (REsponse to PAThogens) family, which is known to be involved in the response to entomopathogens infection. Here, the Slrepat1 gene in S. littoralis larvae has been identified and functionally characterized. This gene is mostly expressed in the midgut, and its dsRNA-mediated silencing determines an immunosuppressed phenotype that makes larvae more susceptible to the entomopathogen Bacillus thuringiensis. Therefore, the combined administration of the dsRNA targeting this gene and of B. thuringiensis would enhances the killing activity of the entomopathogen in an integrated pest management strategy. The third part is focused on the development of a new delivery and protection system for dsRNA molecules, which is based on the use of chitosan polymers and humic substances for the synthesis of a nanoconjugate. The results in terms of synthesis efficiency, physicochemical characterization, and in vivo administration to S.littoralis larvae indicate that the Chi/Hum-dsRNA nanoconjugate can be a valuable and a sustainable tool for the delivery of dsRNAs. The results of this PhD thesis pave the way towards the development of novel strategies of pest control which are not only based on direct pest suppression, but also on the enhancement of natural biocontrol agents, reinforcing the ecosystem services they provide.

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