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

Role of Ovarian Proteins Secreted by Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) in the Early Suppression of Host Immune Response

Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) is an endophagous parasitoid of the larval stages of the tobacco budworm, Heliothis virescens (Fabricius) (Lepidoptera, Noctuidae). During oviposition, T. nigriceps injects into the host body, along with the egg, the venom, the calyx fluid, which contains a Polydnavirus (T. nigriceps BracoVirus: TnBV), and the Ovarian Proteins (OPs). Although viral gene expression in the host reaches detectable levels after a few hours, a precocious disruption of the host metabolism and immune system is observed right after parasitization. This alteration appears to be induced by female secretions including TnBV venom and OPs. OPs, originating from the ovarian calyx cells, are involved in the induction of precocious symptoms in the host immune system alteration. It is known that OPs in braconid and ichneumonid wasps can interfere with the cellular immune response before Polydnavirus infects and expresses its genes in the host tissues. Here we show that T. nigriceps OPs induce several alterations on host haemocytes that trigger cell death. The OP injection induces an extensive oxidative stress and a disorganization of actin cytoskeleton and these alterations can explain the high-level of haemocyte mortality, the loss of haemocyte functionality, and so the reduction in encapsulation ability by the host

Phosphoproteomic Landscaping Identifies Non-canonical cKIT Signaling in Polycythemia Vera Erythroid Progenitors

Although stem cell factor (SCF)/cKIT interaction plays key functions in erythropoiesis, cKIT signaling in human erythroid cells is still poorly defined. To provide new insights into cKIT-mediated erythroid expansion in development and disease, we performed phosphoproteomic profiling of primary erythroid progenitors from adult blood (AB), cord blood (CB), and Polycythemia Vera (PV) at steady-state and upon SCF stimulation. While AB and CB, respectively, activated transient or sustained canonical cKIT-signaling, PV showed a non-canonical signaling including increased mTOR and ERK1 and decreased DEPTOR. Accordingly, screening of FDA-approved compounds showed increased PV sensitivity to JAK, cKIT, and MEK inhibitors. Moreover, differently from AB and CB, in PV the mature 145kDa-cKIT constitutively associated with the tetraspanin CD63 and was not endocytosed upon SCF stimulation, contributing to unrestrained cKIT signaling. These results identify a clinically exploitable variegation of cKIT signaling/metabolism that may contribute to the great erythroid output occurring during development and in PV

Synergistic Parasite-Pathogen Interactions Mediated by Host Immunity Can Drive the Collapse of Honeybee Colonies

The health of the honeybee and, indirectly, global crop production are threatened by several biotic and abiotic factors, which play a poorly defined role in the induction of widespread colony losses. Recent descriptive studies suggest that colony losses are often related to the interaction between pathogens and other stress factors, including parasites. Through an integrated analysis of the population and molecular changes associated with the collapse of honeybee colonies infested by the parasitic mite Varroa destructor, we show that this parasite can de-stabilise the within-host dynamics of Deformed wing virus (DWV), transforming a cryptic and vertically transmitted virus into a rapidly replicating killer, which attains lethal levels late in the season. The de-stabilisation of DWV infection is associated with an immunosuppression syndrome, characterized by a strong down-regulation of the transcription factor NF-κB. The centrality of NF-κB in host responses to a range of environmental challenges suggests that this transcription factor can act as a common currency underlying colony collapse that may be triggered by different causes. Our results offer an integrated account for the multifactorial origin of honeybee losses and a new framework for assessing, and possibly mitigating, the impact of environmental challenges on honeybee health

Protein tyrosine phosphatases of Toxoneuron nigriceps bracovirus as potential disrupters of host prothoracic gland function

The genomic sequence of the bracovirus associated with the wasp Toxoneuron nigriceps (Hymenoptera, Broconidae) (TnBV), an endophagous parasitoid of the tobacco budworm larvae, Heliothis virescens (Lepidoptera, Noctuidoe), contains a large gene family coding for protein tyrosine phosphatoses (PTPs). Here we report the characterization of cDNAs for two of the viral PTPs isolated by screening a cDNA library from haemocytes of parasitized host larvae. The two encoded proteins show 70% amino acid identity and are expressed in the fat body of parasitized hosts. In addition, one was expressed in inactivated prothoracic glands (PTGs), 24 h after parasitoid oviposition. The rapid block of ecdysteroidogenesis does not appear to be due to inhibition of general protein synthesis, as indirectly indicated by the unaltered S6 kinase activity in the cytosolic extracts of basal PTGs from parasitized host larvae. Rather, TnBV PTP over-expression in inactivated host PTGs suggests that gland function may be affected by the disruption of the phosphorylation balance of key proteins regulating points upstream from the ribosomal S6 phosphorylation in the PITH signaling cascade