Aden Abdi, Fatima (2015) STUDIO DEL VELENO DEL PARASSITOIDE DI AFIDI APHIDIUS ERVI. [Tesi di dottorato]

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Aphidius ervi (Hymenoptera, Braconidae) is an endophagous parasitoid of the pea aphid Acyrthosiphon pisum (Homoptera, Aphididae) and of various cereal aphids. Parasitized aphids show a notable inhibition of reproductive activity and metabolic alterations, which are nutritionally relevant for the developing wasp juveniles. Host regulation factors produced by A. ervi and responsible of these alterations are both of maternal origin, injected at the oviposition along with the egg, and of embryonic origin, in particular released by cells derived from the dissociation of the serosal membrane of the parasitoid embryo, denoted as teratocytes. The venom, injected at the oviposition, is the major factor responsible for the castration of the hosts, which is caused by the induction of apoptosis in the germarial cells of the aphid ovarioles. This event is triggered by a γ-glutamyl transpeptidase (Ae-γ-GT), which may likely induce an oxidative stress in germarial cells. Here this hypothesis was firstly tested, assessing if the exposure to Ae-γ-GT induce a change of glutathione (GSH) titer in the host tissues. GSH, measured by high performance liquid chromatography, was found to be significantly reduced in parasitized aphids and this difference was largely due to GSH decrease in the ovaries. Due to GSH role in the protection of oxidative stress, its decrease in parasitized aphids corroborates the experimental hypothesis. Transcriptomic analysis of the venom gland by RNAseq Ae-γ-GT allowed the identification of two isoforms of Ae-γ-GT (Ae-γ-GT1 and Ae-γ-GT2), which showed 51% sequence identity. Quantitative studies of their expression profile in venom glands and in the rest of the body have been performed using quantitative Real Time PCR, assessing the changes of the transcription rates as affected by time and oviposition activity. These two isoforms of Ae-γ-GT were produced in vitro in insect cells by recombinant baculovirus to carry out a more comprehensive molecular and functional characterization, aiming to shed light on their role in the host regulation process. This objective was also tentatively pursued by developing a gene silencing approach by RNAi (RNA interference). Indeed, three administration methods for the delivery of dsRNA targeting the Ae-γ-GT1 have been designed and their efficiency comparatively assessed. The first experimental approach consisted of haemocoelic injections of dsRNA in A. ervi pupae, then the delivery through the host aphid was attempted, by injecting dsRNA in the haemocoel of parasitized host aphids, or by feeding these latter on a liquid diet containing the dsRNA. The obtained results did not allow to select any of the experimental approaches pursued, as the results were inconsistent and further research work is necessary to develop a reliable and effective protocol to silence Ae-γ-GT.

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