Marisa Nardiello

The multifunctional polydnavirus TnBVANK1 protein: impact on host apoptotic pathway

Toxoneuron nigriceps (Hymenoptera, Braconidae) is an endophagous parasitoid of the larval stages of the tobacco budworm, Heliothis virescens (Lepidoptera, Noctuidae). The bracovirus associated with this wasp (TnBV) is currently being studied. Several genes expressed in parasitised host larvae have been isolated and their possible roles partly elucidated. TnBVank1 encodes an ankyrin motif protein similar to insect and mammalian IκB, an inhibitor of the transcription nuclear factor κB (NF-κB). Here we show that, when TnBVank1 was stably expressed in polyclonal Drosophila S2 cells, apoptosis is induced. Furthermore, we observed the same effects in haemocytes of H. virescens larvae, after TnBVank1 in vivo transient transfection, and in haemocytes of parasitised larvae. Coimmunoprecipitation experiments showed that TnBVANK1 binds to ALG-2 interacting protein X (Alix/AIP1), an interactor of apoptosis-linked gene protein 2 (ALG-2). Using double-immunofluorescence labeling, we observed the potential colocalization of TnBVANK1 and Alix proteins in the cytoplasm of polyclonal S2 cells. When Alix was silenced by RNA interference, TnBVANK1 was no longer able to cause apoptosis in both S2 cells and H. virescens haemocytes. Collectively, these results indicate that TnBVANK1 induces apoptosis by interacting with Alix, suggesting a role of TnBVANK1 in the suppression of host immune response observed after parasitisation by T. nigriceps.

Ecdysteroidogenesis and development in Heliothis virescens (Lepidoptera: Noctuidae): Focus on PTTH-stimulated pathways

Post-embryonic development and molting in insects are regulated by endocrine changes, including prothoracicotropic hormone (PTTH)-stimulated ecdysone secretion by the prothoracic glands (PGs). In Lepidoptera, two pathways are potentially involved in PTTH-stimulated ecdysteroidogenesis, mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/protein kinase B/target of rapamycin (PI3K/Akt/TOR). We investigated the potential roles of both these pathways in Heliothis virescens ecdysteroidogenesis. We identified putative proteins belonging to MAPK and PI3K/Akt/TOR signaling cascades, using transcriptomic analyses of PGs from last (fifth) instar larvae. Using western blots, we measured the phosphorylation of 4E-BP and S6K proteins, the main targets of TOR, following the in vitro exposure of PGs to brain extract containing PTTH (hereafter referred to as PTTH) and/or the inhibitors of MAPK (U0126), PI3K (LY294002) or TOR (rapamycin). Next, we measured ecdysone production, under the same experimental conditions, by enzyme immunoassay (EIA). We found that in Heliothis virescens last instar larvae, both pathways modulated PTTH-stimulated ecdysteroidogenesis. Finally, we analyzed the post-embryonic development of third and fourth instar larvae fed on diet supplemented with rapamycin, in order to better understand the role of the TOR pathway in larval growth. When rapamycin was added to the diet of larvae, the onset of molting was delayed, the growth rate was reduced and abnormally small larvae/pupae with high mortality rates resulted. In larvae fed on diet supplemented with rapamycin, the growth of PGs was suppressed, and ecdysone production and secretion were inhibited. Overall, the in vivo and in vitro results demonstrated that, similarly to Bombyx mori, MAPK and PI3K/Akt/TOR pathways are involved in PTTH signaling-stimulated ecdysteroidogenesis, and indicated the important role of TOR protein in H. virescens systemic growth.