Ecdysteroidogenesis in Heliothis virescens (Lepidoptera: Noctuidae): Recombinant Prothoracicotropic Hormone and Brain Extract Show Comparable Effects.

Prothoracicotropic hormone (PTTH) is a neuropeptide that triggers a cascade of events within the prothoracic gland (PG) cells, leading to the activation of all the crucial enzymes involved in ecdysone biosynthesis, the main insect steroid hormone. Studies concerning ecdysteroidogenesis predicted PTTH action using brain extract (BE), consisting in a complex mixture in which some components positively or negatively interfere with PTTH-stimulated ecdysteroidogenesis. Consequently, the integration of these opposing factors in steroidogenic tissues leads to a complex secretory pattern. A recombinant form of prothoracicotropic hormone (rPTTH) from the tobacco budworm Heliothis virescens (F.) (Lepidoptera: Noctuidae) was expressed and purified to perform in vitro tests in a standard and repeatable manner. A characterization of rPTTH primary and secondary structures was performed. The ability of rPTTH and H. virescens BE to stimulate ecdysteroidogenesis was investigated on the third day of fifth larval stage. rPTTH activity was compared with the BE mixture by enzyme immunoassay and western blot, revealing that they equally stimulate the production of significant amount of ecdysone, through a transduction cascade that includes the TOR pathway, by the phosphorylation of 4E binding protein (4E-BP) and S6 kinase (S6K), the main targets of TOR protein. The results of these experiments suggest the importance of obtaining a functional pure hormone to perform further studies, not depending on the crude brain extract, composed by different elements and susceptible to different uncontrollable variables.

Novel Factors of Viral Origin Inhibit TOR Pathway Gene Expression.

Polydnaviruses (PDVs) are obligate symbionts of endoparasitoid wasps, which exclusively attack the larval stages of their lepidopteran hosts. The Polydnavirus is injected by the parasitoid female during oviposition to selectively infect host tissues by the expression of viral genes without undergoing replication. Toxoneuron nigriceps bracovirus (TnBV) is associated with Toxoneuron nigriceps (Hymenoptera: Braconidae) wasp, an endoparasitoid of the tobacco budworm larval stages, Heliothis virescens (Lepidoptera: Noctuidae). Previous studies showed that TnBV is responsible for alterations in host physiology. The arrest of ecdysteroidogenesis is the main alteration which occurs in last (fifth) instar larvae and, as a consequence, prevents pupation. TnBV induces the functional inactivation of H. virescens prothoracic glands (PGs), resulting in decreased protein synthesis and phosphorylation. Previous work showed the involvement of the PI3K/Akt/TOR pathway in H. virescens PG ecdysteroidogenesis. Here, we demonstrate that this cellular signaling is one of the targets of TnBV infection. Western blot analysis and enzyme immunoassay (EIA) showed that parasitism inhibits ecdysteroidogenesis and the phosphorylation of the two targets of TOR (4E-BP and S6K), despite the stimulation of PTTH contained in the brain extract. Using a transcriptomic approach, we identified viral genes selectively expressed in last instar H. virescens PGs, 48 h after parasitization, and evaluated expression levels of PI3K/Akt/TOR pathway genes in these tissues. The relative expression of selected genes belonging to the TOR pathway (tor, 4e-bp, and s6k) in PGs of parasitized larvae was further confirmed by qRT-PCR. The down-regulation of these genes in PGs of parasitized larvae supports the hypothesis of TnBV involvement in blocking ecdysteroidogenesis, through alterations of the PI3K/Akt/TOR pathway at the transcriptional level.

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.