Abstract
RNA interference (RNAi) methods for insects are often limited by problems with double-stranded (ds) RNA delivery, which restricts reverse genetics studies and the development of RNAi-based biocides. We therefore delegated to insect symbiotic bacteria the task of: (i) constitutive dsRNA synthesis and (ii) trauma-free delivery. RNaseIII-deficient, dsRNA-expressing bacterial strains were created from the symbionts of two very diverse pest species: a long-lived blood-sucking bug, Rhodnius prolixus, and a short-lived globally invasive polyphagous agricultural pest, western flower thrips (Frankliniella occidentalis). When ingested, the manipulated bacteria colonized the insects, successfully competed with the wild-type microflora, and sustainably mediated systemic knockdown phenotypes that were horizontally transmissible. This represents a significant advance in the ability to deliver RNAi, potentially to a large range of non-model insects.
Highlights
Insects and arthropods are of enormous significance as agricultural or stored product pests and vectors of disease, as well as many species being beneficial such as pollinators
Insects at different developmental stages could be sustainably populated with the recombinant bacteria either by feeding on blood mixed with the bacteria or via coprophagy
The continual expression of dsRNA in the gut allows for sustained RNA interference (RNAi) so that phenotypic changes related to loss of gene function at each developmental stage can be assessed
Summary
Insects and arthropods are of enormous significance as agricultural or stored product pests and vectors of disease, as well as many species being beneficial such as pollinators. To highlight its broad applicability, we demonstrate the technology using two very different species of symbiont (one, a Gram-positive actinobacterium and the other, a Gram-negative gamma proteobacterium), in two contrasting insect species: the large, long-lived haematophagous insect R. prolixus (in which the dsRNA expression cassette is stably integrated into the chromosome of the symbiont, removing the requirement for constant antibiotic selection) and the much smaller, polyphagous Frankliniella occidentalis (western flower thrips) that has a short life cycle The latter species is a major global threat to agriculture that has extended its range from its native North America to all cultivated continents in the past 25 years, concomitantly developing considerable pesticide resistance [13].
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