The Red Palm Weevil (RPW), Rhynchophorus ferrugineus (Olivier, 1970) (Coleoptera: Curculionidae) is an invasive exotic pest species of ornamental palms in Portugal. It has also been found affecting the banana production in the Madeira Islands. The concealed feeding habit of RPW borer is the main reason why spraying environmentally harmful chemical insecticides is not controlling the pest.

This three-year project's primary goals are to conduct the first survey of parasitic mites of RPW in Portugal and obtain recombinant anti-RPW endophytic bacteria for their eventual role in controlling this pest species. During the first year, we plan to diagnose parasitic mites in RPW adults and pupae collected from infested palm trees at three locations in Portugal (Porto metropolitan area, Algarve, and Madeira Islands) and infested banana plants at Madeira. We will determine mites' features –such as species composition and on-host distribution patterns– based on morphological and anatomical characters using a light microscope examination of dissected weevils. On-host distribution pattern and host sex preference behaviour will also be investigated. The prevalence (in %), mean abundance and mean intensity of parasitic infections will be determined to report the most frequent and abundant mite species. We also plan to search for fungi attached to mites' surface and investigate the effects of mites' phoresis on RPW eggs' number and viability, two essential insect fitness components. During the second year, we plan to isolate palm endophytic bacteria for their use as potential carriers of anti-RPW molecules. Endophytic genetically amenable Bacillus cereus (Bc)/B. thuringiensis (Bt) strains will be isolated from palm parts preferred by RPW larvae using a combination of microbial culturing, biochemical and genetic methods. Inoculation/re-isolation experiments will detect best palm seedlings' colonisers among true endophytes. Insect bioassays will be used to determine mortality (LC50) and moulting inhibition (MIC50) of anti-RPW molecules (i.e. Coleopteran-active Bt proteins and insecticidal RNAi). During the third year, we will construct recombinant endophytic bacteria and perform potted-plant bioassays. The DNA sequences encoding anti-RPW molecules will be inserted into the genome of endophytic Bc/Bt strains replacing the RNAse III locus, through a gene replacement system based on a thermosensitive-replication vector. Palm seedlings inoculated with recombinant anti-RPW endophytes will be evaluated for systemic control of RPW larvae under greenhouse conditions. We anticipate the first characterisation of mite species associated with RPW in Portugal, including the potential consequences of phoresis for RPW fitness, and the obtainment of recombinant anti-RPW microbial biopesticides for the RPW management toolkit.