Odorant binding proteins fromHermetia illucens: potential sensing elements for detecting volatile aldehydes involved in early stages of organic decomposition.

Organic decomposition processes, involving the breakdown of complex molecules such as carbohydrates, proteins and fats, release small chemicals known as volatile organic compounds (VOCs), smelly even at very low concentrations, but not all readily detectable by vertebrates. Many of these compounds are instead detected by insects, mostly by saprophytic species, for which long-range orientation towards organic decomposition matter is crucial. In the present work the detection of aldehydes, as an important measure of lipid oxidation, has been possible exploiting the molecular machinery underlying odour recognition inHermetia illucens(Diptera: Stratiomyidae). This voracious scavenger insect is of interest due to its outstanding capacity in bioconversion of organic waste, colonizing very diverse environments due to the ability of sensing a wide range of chemical compounds that influence the choice of substrates for ovideposition. A variety of soluble odorant binding proteins (OBPs) that may function as carriers of hydrophobic molecules from the air-water interface in the antenna of the insect to the receptors were identified, characterised and expressed. An OBP-based nanobiosensor prototype was realized using selected OBPs as sensing layers for the development of an array of quartz crystal microbalances (QCMs) for vapour phase detection of selected compounds at room temperature. QCMs coated with four recombinantH. illucensOBPs (HillOBPs) were exposed to a wide range of VOCs indicative of organic decomposition, showing a high sensitivity for the detection of three chemical compounds belonging to the class of aldehydes and one short-chain fatty acid. The possibility of using biomolecules capable of binding small ligands as reversible gas sensors has been confirmed, greatly expanding the state-of the-art in gas sensing technology.

Hermetia illucens (L.) (Diptera: Stratiomyidae) Odorant Binding Proteins and Their Interactions with Selected Volatile Organic Compounds: An In Silico Approach.

The black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), has considerable global interest due to its outstanding capacity in bioconverting organic waste to insect biomass, which can be used for livestock, poultry, and aquaculture feed. Mass production of this insect in colonies requires the development of methods concentrating oviposition in specific collection devices, while the mass production of larvae and disposing of waste may require substrates that are more palatable and more attractive to the insects. In insects, chemoreception plays an essential role throughout their life cycle, responding to an array of chemical, biological and environmental signals to locate and select food, mates, oviposition sites and avoid predators. To interpret these signals, insects use an arsenal of molecular components, including small proteins called odorant binding proteins (OBPs). Next generation sequencing was used to identify genes involved in chemoreception during the larval and adult stage of BSF, with particular attention to OBPs. The analysis of the de novo adult and larval transcriptome led to the identification of 27 and 31 OBPs for adults and larvae, respectively. Among these OBPs, 15 were common in larval and adult transcriptomes and the tertiary structures of 8 selected OBPs were modelled. In silico docking of ligands confirms the potential interaction with VOCs of interest. Starting from the information about the growth performance of H. illucens on different organic substrates from the agri-food sector, the present work demonstrates a possible correlation between a pool of selected VOCs, emitted by those substrates that are attractive for H. illucens females when searching for oviposition sites, as well as phagostimulants for larvae. The binding affinities between OBPs and selected ligands calculated by in silico modelling may indicate a correlation among OBPs, VOCs and behavioural preferences that will be the basis for further analysis.

Solanum nigrum Extract and Solasonine Affected Hemolymph Metabolites and Ultrastructure of the Fat Body and the Midgut in Galleria mellonella.

Glycoalkaloids, secondary metabolites abundant in plants belonging to the Solanaceae family, may affect the physiology of insect pests. This paper presents original results dealing with the influence of a crude extract obtained from Solanum nigrum unripe berries and its main constituent, solasonine, on the physiology of Galleria mellonella (Lepidoptera) that can be used as an alternative bioinsecticide. G. mellonella IV instar larvae were treated with S. nigrum extract and solasonine at different concentrations. The effects of extract and solasonine were evaluated analyzing changes in carbohydrate and amino acid composition in hemolymph by RP-HPLC and in the ultrastructure of the fat body cells by TEM. Both extract and solasonine changed the level of hemolymph metabolites and the ultrastructure of the fat body and the midgut cells. In particular, the extract increased the erythritol level in the hemolymph compared to control, enlarged the intracellular space in fat body cells, and decreased cytoplasm and lipid droplets electron density. The solasonine, tested with three concentrations, caused the decrease of cytoplasm electron density in both fat body and midgut cells. Obtained results highlighted the disturbance of the midgut and the fat body due to glycoalkaloids and the potential role of hemolymph ingredients in its detoxification. These findings suggest a possible application of glycoalkaloids as a natural insecticide in the pest control of G. mellonella larvae.

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.

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.

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.