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 (Lepidoptera, 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. Key message• RNAi for insect control requires the development of effective delivery strategies of dsRNA. • Bacteria expressing a dsRNA targeting an immune gene induce its silencing when ingested by larvae of the noctuid moth Spodoptera littoralis. • The resulting immunosuppression enhances the killing activity of a Bt-based biopesticide.• These immune suppressive bacteria can be used as synergistic factors to develop more effective Bt sprays, and to preserve Bt efficacy.Communicated by E. Roditakis.* S. Caccia

Genome sequencing data have recently demonstrated that eukaryote evolution has been remarkably influenced by the acquisition of a large number of genes by horizontal gene transfer (HGT) across different kingdoms. However, in depth-studies on the physiological traits conferred by these accidental DNA acquisitions are largely lacking. Here we elucidate the functional role of Sl gasmin, a gene of a symbiotic virus of a parasitic wasp that has been transferred to an ancestor of the moth species Spodoptera littoralis and domesticated. This gene is highly expressed in circulating immune cells (haemocytes) of larval stages, where its transcription is rapidly boosted by injection of microorganisms into the body cavity. RNAi silencing of Sl gasmin generates a phenotype characterized by a precocious suppression of phagocytic activity by haemocytes, which is rescued when these immune cells are incubated in plasma samples of control larvae, containing high levels of the encoded protein. Proteomic analysis demonstrates that the protein Sl gasmin is released by haemocytes into the haemolymph, where it opsonizes the invading bacteria to promote their phagocytosis, both in vitro and in vivo. Our results show that important physiological traits do not necessarily originate from evolution of pre-existing genes, but can be acquired by HGT events, through unique pathways of symbiotic evolution. These findings indicate that insects can paradoxically acquire selective advantages with the help of their natural enemies.

Lo scopo dello studio è stato quello di valutare i benefici della ricostruzione con lembo di SMAS nei pazienti affetti da neoplasie benigne della parotide e sottoposti a parotidectomia superficiale. Abbiamo condotto uno studio retrospettivo su 123 pazienti affetti da neoplasie benigne della ghiandola parotide ricoverati presso il Nostro istituto tra il Marzo 1997 e Marzo 2010. Tutti i pazienti arruolati sono stati sottoposti a parotidectomia superficiale. Il Nostro campione è stato diviso in due gruppi in base alla esecuzione (Gruppo 2) o no (Gruppo 1) di ricostruzione con lembo di SMAS dopo la parotidectomia superficiale. La ricostruzione con lembo di SMAS è stata eseguita in 64 pazienti. Un test chi quadro è stato utilizzato per valutare le differenze statistiche tra i due gruppi. Il livello di significatività statistica scelto è stato di p<0,05. Non è stata rilevata differenza statisticamente significativa tra i 2 gruppi per quanto riguarda l’ insorgenza di ematoma, infezione della ferita e paralisi del faciale (3,38 vs 1,56% [P > 0,05] , 8,47% vs 4,68% [P > 0,05, 5,08% vs 0,00%). La paralisi transitoria del facciale, la fistola, la depressione della cute e la sindrome di Frey sono significativamente più frequenti nei pazienti non ricostruiti con lembo di SMAS (10,16% vs 3,125% [P < 0,05], 13,55% vs 3,125% [P < 0,05] , 13,55% vs 3,125% [P < 0,05], 20,33% vs 0% [P < 0,05], rispettivamente). Il lembo di SMAS è capace di ridurre le complicanze funzionali ed estetiche che si verificano dopo la rimozione di un tumore benigno della parotide mediante parotidectomia superficiale, tra queste, riduce il verificarsi della sindrome di Frey .

BACKGROUND Potassium ion channels play a critical role in the generation of electrical signals and thus provide potential targets for control of insect pests by RNA interference. RESULTS Genes encoding the small conductance calcium‐activated potassium channel (SK) and the voltage‐gated potassium channel (SH) were knocked down in Tribolium castaneum by injection and oral delivery of dsRNA (dsTcSK and dsTcSH, respectively). Irrespective of the delivery mechanism a dose‐dependent effect was observed for knockdown (KD) of gene expression and insect mortality for both genes. Larvae fed a 400 ng dsRNA mg–1 diet showed significant gene (P < 0.05) knockdown (98% and 83%) for SK and SH, respectively, with corresponding mortalities of 100% and 98% after 7 days. When injected (248.4 ng larva–1), gene KD was 99% and 98% for SK and SH, causing 100% and 73.4% mortality, respectively. All developmental stages tested (larvae, early‐ and late‐stage pupae and adults) showed an RNAi‐sensitive response for both genes. LC50 values were lower for SK than SH, irrespective of delivery method, demonstrating that the knockdown of SK had a greater effect on larval mortality. Biosafety studies using adult honeybee Apis mellifera showed that there were no significant differences either in expression levels or mortality of honeybees orally dosed with dsTcSK and dsTcSH compared to control‐fed bees. Similarly, there was no significant difference in the titre of deformed wing virus, used as a measure of immune suppression, between experimental and control bees. CONCLUSION This study demonstrates the potential of using RNAi targeting neural receptors as a technology for the control of T. castaneum. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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