Astarita, Federica (2017) Transepithelial delivery of macromolecular biopesticides. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Lingua: English
Title: Transepithelial delivery of macromolecular biopesticides
Creators:
CreatorsEmail
Astarita, Federicafedericaastarita@gmail.com
Date: 9 April 2017
Number of Pages: 94
Institution: Università degli Studi di Napoli Federico II
Department: Scienze Chimiche
Dottorato: Biotecnologie
Ciclo di dottorato: 29
Coordinatore del Corso di dottorato:
nomeemail
Sannia, Giovannisannia@unina.it
Tutor:
nomeemail
Pennacchio, FrancescoUNSPECIFIED
Caccia, SilviaUNSPECIFIED
Date: 9 April 2017
Number of Pages: 94
Uncontrolled Keywords: biopesticide; delivery
Settori scientifico-disciplinari del MIUR: Area 07 - Scienze agrarie e veterinarie > AGR/11 - Entomologia generale e applicata
Area 05 - Scienze biologiche > BIO/11 - Biologia molecolare
Date Deposited: 07 Mar 2018 09:12
Last Modified: 12 May 2018 01:00
URI: http://www.fedoa.unina.it/id/eprint/11935
DOI: 10.6093/UNINA/FEDOA/11935

Abstract

The need to reduce the use of chemical pesticides in agriculture and the decrease in number of available molecules has promoted intense research efforts towards the identification of new bioinsecticides of natural origin and of appropriate delivery strategies. This has allowed the isolation and characterization of a wealth of bioactive molecules, mainly peptides and proteins derived from different sources such as bacteria, viruses, insect predators/parasitoids, arthropods and plants, which are natural antagonists of insects. Moreover, the study of the molecular mechanisms mediated by these novel bioinsecticides and the identification of their cognate receptors offer the opportunity to develop bioinspired strategies mimicking the negative effects on host insects by natural antagonists through the use of “RNAi mediated crop protection” technologies. The high specificity of these antagonistic associations and the targeted gene silencing action of properly tailored dsRNAs confer to these novel tools and strategies for pest control a high degree of selectivity, that make them ideal candidates for inclusion in sustainable IPM plans. However, specific delivery strategies have to be developed in order to allow these molecules to resist the harsh physicochemical environment of the insect gut and to overcome the lining epithelial layer. The present work aims at providing new delivery strategies for proteinaceus and oligonucleotidic molecules to control phytophagous insect pests. We focused, first, on the use of XXX as delivery vector of a neurotoxin derived from the salivary glands of the spider Segestria florentina, SFl2.6, and, then, screened specific XXX domains to assess their capacity to mediate transepithelial delivery of fused toxins. SFI2.6/XXX, SFI2.6/XXX domain1, SFI2.6/XXX domain2, SFI2.6/XXX domain3 fusion proteins have been produced in yeast. Recombinant SFI2.6/XXX and SFI2.6/XXX domain1 have been produced in suitable amounts for their use in feeding bioassays on the pea aphid Acyrthosiphon pisum. The high mortality obtained with these fusion proteins suggests that XXX and domain1 are extremely efficient carriers for the oral delivery of toxins active in the body cavity. Future studies including the other two XXX domains may further contribute to the development of novel delivery strategies. To promote the feasibility of RNAi technology for pest control, we propose a new delivery strategy of dsRNAs, here used to target host immune genes, in order to induce an immunosuppressive status enhancing the impact of entomopathogens. Two immune genes of the noctuid moth Spodoptera littoralis have been considered (Sl 102 and Sl gasmin), one of which (Sl gasmin) has been isolated and characterized as part of this thesis work, showing its potent role as opsonizing factor mediating phagocytosis. Vectors for the expression in bacteria of Sl 102 and Sl gasmin dsRNA have been designed and recombinant bacteria producing Sl 102 dsRNA, when orally delivered to S. littoralis larvae were able to specifically reproduce the high level of gene silencing observed with dsRNA obtained in vitro, along with the immunosuppressive phenotype induced. In conclusion, the results obtained with both fusion proteins and dsRNA-expressing bacteria are very promising and contribute to inspire new ideas for the delivery of insecticidal molecules, in particular of macromolecules targeting haemocoelic receptors or silencing host immune genes, in order to enhance insect sensitivity to natural antagonists.

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