Transepithelial delivery of macromolecular biopesticides

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

Enhancement of Bacillus thuringiensis toxicity by feeding Spodoptera littoralis larvae with bacteria expressing immune suppressive dsRNA

RNAi interference (RNAi) for insect pest control is often used to silence genes controlling vital functions, thus generating lethal phenotypes. Here, we propose a novel approach, based on the knockout of an immune gene by dsRNA-expressing bacteria as a strategy to enhance the impact of spray applications of the entomopathogen Bacillus thuringiensis ( Bt). The target gene, Sl 102, controls the encapsulation and nodulation responses in the noctuid moth Spodoptera littoralis (Lepidop- tera, Noctuidae). To deliver Sl 102 dsRNA, we have developed a bacterial expression system, using HT115 Escherichia coli. This allows a much cheaper production of dsRNA and its protection against degradation. Transformed bacteria (dsRNA- Bac) administered through artificial diet proved to be more effective than dsRNA synthesized in vitro, both in terms of gene silencing and immunosuppression. This is a likely consequence of reduced dsRNA environmental degradation and of its protected release in the harsh conditions of the gut. The combined oral administration with artificial diet of dsRNA-Bac and of a Bt-based biopesticide (Xentari™) resulted in a remarkable enhancement of Bt killing activity, both on 4th and 5th instar larvae of S. littoralis, either when the two components were simultaneously administered or when gene silencing was obtained before Bt exposure. These results pave the way toward the development of novel Bt spray formulations containing killed dsRNA-Bac, which synergize Bt toxins by suppressing the insect immune response. This strategy will preserve the long-term efficacy of Bt-based products and can, in principle, enhance the ecological services provided by insect natural antagonists

Evolution of an insect immune barrier through horizontal gene transfer mediated by a parasitic wasp.

Genome sequencing data have recently demonstrated that eukaryote evolution has been remarkably influenced by the acquisition of a large number of genes by horizontal gene transfer (HGT) across different kingdoms. However, in depth-studies on the physiological traits conferred by these accidental DNA acquisitions are largely lacking. Here we elucidate the functional role of Sl gasmin, a gene of a symbiotic virus of a parasitic wasp that has been transferred to an ancestor of the moth species Spodoptera littoralis and domesticated. This gene is highly expressed in circulating immune cells (haemocytes) of larval stages, where its transcription is rapidly boosted by injection of microorganisms into the body cavity. RNAi silencing of Sl gasmin generates a phenotype characterized by a precocious suppression of phagocytic activity by haemocytes, which is rescued when these immune cells are incubated in plasma samples of control larvae, containing high levels of the encoded protein. Proteomic analysis demonstrates that the protein Sl gasmin is released by haemocytes into the haemolymph, where it opsonizes the invading bacteria to promote their phagocytosis, both in vitro and in vivo. Our results show that important physiological traits do not necessarily originate from evolution of pre-existing genes, but can be acquired by HGT events, through unique pathways of symbiotic evolution. These findings indicate that insects can paradoxically acquire selective advantages with the help of their natural enemies

Modelli e nuove tecnologie per il trasporto merci e la logistica

Dottorato di Ricerca in: Tecnologie e pianificazione ambientale, Ciclo XXII, a.a. 2008-2009Università degli Studi della Calabri

An Innovative Method to Produce Metal Foam Using Cold Gas Dynamic Spray Process Assisted by Fluidized Bed Mixing of Precursors

Metallic foams are known for their interesting physical and mechanical properties such as high stiffness, very low specific weight, high compression strength, unusual acoustic and thermal properties and good energy absorption characteristics. These materials are currently manufactured by means of several different processes. The limit of these conventional technologies is the impossibility to produce foams with complex geometry. This paper deals with the study of an innovative method to produce complex shaped precursors for aluminum foams through cold gas dynamic spray deposition process (CGDS). Aluminum alloy (Al-Si) fine powders were previously mixed with the blowing agent (titanium-hydride, TiH2). The mixing process was carried out by means of sound-assisted fluidized bed apparatus that allows to obtain an homogeneous mixture of the two elements. The mixed particles were then sprayed by means of the CGDS process on a stainless steel sheet that simulates the real component to be reinforced with the foam. Subsequently the obtained precursor was heated up in a furnace The produced metal foam was characterized using optical and electron microscopy in order to study the cell morphology and distribution. The obtained results showed the effectiveness of this method to produce aluminum foams with complex shap

Aluminum foam made via a new method based on cold gas dynamic sprayed powders mixed through sound assisted fluidization technique

Metal foams are an interesting class of materials with very low specific weight and unusual physical, mechanical and acoustic properties due to the porous structure. In recent years several manufacturing techniques were developed. The limit of these techniques is that it is difficult, even if impossible, to manufacture precursors and then foams able to reinforce complex shaped components; this drawback, to date, limits the application of metal foams. This proof of concept paper is focused on the study of an innovative manufacturing technique able to produce complex shaped precursors. The key idea is to spray a powder mixture (made of both aluminum alloy powders as metal matrix and titanium hydride particles as foaming agent) through the cold gas dynamic spray on a free shape metallic substrate and then carry out the foaming process. A preliminary granulometric analysis was carried out to estimate the particles mean size and then sound assisted (140dB–80 Hz) fluidization process was used to achieve a homogenous and deep mixing between the fine metal powders and the blowing agent ones. In particular, two different types of mixtures with 1 wt% and 2.5 wt% of TiH2 were investigated; moreover, air compressed as well as helium were used as CGDS carrier gas in order to ensure a higher impact velocity and a better compacting of the powders. Finally, the cross sections of manufactured solid foams were observed by means of a SEM microscope for having information about internal metallurgical phenomena as well as the distribution and morphology of foam cells. Macrographs of created porous structures showed the effectiveness of the developed innovative manufacturing process

Data from: Evolution of an insect immune barrier through horizontal gene transfer mediated by a parasitic wasp

Genome sequencing data have recently demonstrated that eukaryote evolution has been remarkably influenced by the acquisition of a large number of genes by horizontal gene transfer (HGT) across different kingdoms. However, in depth-studies on the physiological traits conferred by these accidental DNA acquisitions are largely lacking. Here we elucidate the functional role of Sl gasmin, a gene of a symbiotic virus of a parasitic wasp that has been transferred to an ancestor of the moth species Spodoptera littoralis and domesticated. This gene is highly expressed in circulating immune cells (haemocytes) of larval stages, where its transcription is rapidly boosted by injection of microorganisms into the body cavity. RNAi silencing of Sl gasmin generated a phenotype characterized by a precocious suppression of phagocytic activity by haemocytes, which was rescued when these immune cells were incubated in plasma samples of control larvae, containing high levels of the encoded protein. Proteomic analysis demonstrated that the protein Sl gasmin is released by haemocytes into the haemolymph, where it opsonizes the invading bacteria to promote their phagocytosis, both in vitro and in vivo. Our results show that important physiological traits do not necessarily originate from evolution of pre-existing genes, but can be acquired by HGT events, through unique pathways of symbiotic evolution. These findings indicate that insects can paradoxically acquire selective advantages with the help of their natural enemies

Raw data Di Lelio et al., 2019

Numerical data that underlies tables and graphs (figures 1-7, 9, 10; table 1) and statistics of the PLOS Genetics article from di Lelio et al., 2019