RESUMO
PA1b (Pea Albumin 1, subunit b) peptide is an entomotoxin, extracted from Legume seeds, with a lethal activity towards several insect pests, such as mosquitoes, some aphids and cereal weevils. This toxin acts by binding to the subunits c and e of the plasma membrane H+-ATPase (V-ATPase) in the insect midgut. In this study, two cereal weevils, the sensitive Sitophilus oryzae strain WAA42, the resistance Sitophilus oryzae strain ISOR3 and the insensitive red flour beetle Tribolium castaneum, were used in biochemical and histological experiments to demonstrate that a PA1b/V-ATPase interaction triggers the apoptosis mechanism, resulting in insect death. Upon intoxication with PA1b, apoptotic bodies are formed in the cells of the insect midgut. In addition, caspase-3 enzyme activity occurs in the midgut of sensitive weevils after intoxication with active PA1b, but not in the midgut of resistant weevils. These biochemical data were confirmed by immuno-histochemical detection of the caspase-3 active form in the midgut of sensitive weevils. Immuno-labelling experiments also revealed that the caspase-3 active form and V-ATPase are close-localized in the insect midgut. The results concerning this unique peptidic V-ATPase inhibitor pave the way for the utilization of PA1b as a promising, more selective and eco-friendly insecticide.
Assuntos
Proteínas de Insetos/genética , Inseticidas/toxicidade , Peptídeos/toxicidade , Pisum sativum/genética , Proteínas de Plantas/toxicidade , Toxinas Biológicas/toxicidade , ATPases Vacuolares Próton-Translocadoras/genética , Animais , Apoptose , Caspase 3/genética , Caspase 3/metabolismo , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Regulação da Expressão Gênica , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/metabolismo , Inseticidas/isolamento & purificação , Inseticidas/metabolismo , Pisum sativum/química , Pisum sativum/parasitologia , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Ligação Proteica , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Sementes/química , Sementes/genética , Sementes/parasitologia , Toxinas Biológicas/isolamento & purificação , Toxinas Biológicas/metabolismo , Tribolium/efeitos dos fármacos , Tribolium/metabolismo , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/metabolismo , Gorgulhos/efeitos dos fármacos , Gorgulhos/metabolismoRESUMO
Vertically transmitted endosymbionts persist for millions of years in invertebrates and play an important role in animal evolution. However, the functional basis underlying the maintenance of these long-term resident bacteria is unknown. We report that the weevil coleoptericin-A (ColA) antimicrobial peptide selectively targets endosymbionts within the bacteriocytes and regulates their growth through the inhibition of cell division. Silencing the colA gene with RNA interference resulted in a decrease in size of the giant filamentous endosymbionts, which escaped from the bacteriocytes and spread into insect tissues. Although this family of peptides is commonly linked with microbe clearance, this work shows that endosymbiosis benefits from ColA, suggesting that long-term host-symbiont coevolution might have shaped immune effectors for symbiont maintenance.