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Understanding interactions between RNAi and entomopathogens as a novel control strategy for insect pests

Principal Supervisor: Dr. Dave Chandler, School of Life Sciences

Co-supervisor: Dr. Graham Teakle, School of Life Sciences

PhD project title: Understanding interactions between RNAi and entomopathogens as a novel control strategy for insect pests

University of Registration: University of Warwick

Project outline:

Plant feeding insects are a major constraint on the production of food crops. At present, insect pest management is heavily reliant upon the use of synthetic chemical pesticides, but excessive often results in control failure through the evolution of resistance in target pest populations, while a large number of pesticides have been withdrawn in recent years because of government concerns about their safety to people and the environment. Therefore, there is an urgent need for alternative strategies for pest management. The project will focus on integrating novel biopesticide approaches for a more effective form of insect pest management.

At Warwick, we are experts in the development and use of microbial biopesticides based on entomopathogens. These plant protection tools are classified as low risk and the biopesticide market is currently undergoing rapid expansion. However, the efficacy of biopesticides on their own is lower than that of fully effective synthetic chemical pesticides and therefore they are best used in conjunction with other control tools as part of an Integrated Pest Management strategy.

For this project, we will investigate the interaction of entomopathogenic fungi with RNAI-mediated knock down of insect genes. A range of entomopathogenic fungi are being used as commercial biopesticides, but they are not suitable as a stand-alone treatment. There is also increasing interest in the use of RNAi as a novel pest management tool, either by the application of stabilised exogenous dsRNA to crops or through the production of GM varieties that express dsRNA in planta. RNAi generally does not provide total insect kill. In this project we will study the two methods as a combination treatment for insect control.

The research will be done initially with the model insect Tenebrio molitor (Coleoptera), which is amenable to RNAi-mediated knock down of gene expression. Basic principles and discoveries made with Tenebrio will then be applied to experiments with cabbage stem flea beetle, Psylliodes chrysocephala, a major pest of oilseed rape that is currently proving almost impossible to control using conventional chemical pesticides because of its evolved resistance to pyrethroids. The outline plan for the research is as follows:

  • Laboratory bioassays will be used to quantify the effects of selected strains of entomopathogenic fungi (Beauveria bassiana, Metarhizium brunneum) on the survival and longevity of larval T. molitor. 
  • Comparative genomics will be used to identify candidate genes for knock-down in T. molitor including genes encoding proteins with essential functions (regulation of the nervous system, the gut, and developmental pathways mediated by hormonal (ecdysteroid) and homeobox gene transcription factors) and genes associated with the inducible innate immune response (Toll pathway (MyD88), serine protease cascade, gram-negative binding proteins, antimicrobial peptides).
  • Candidate genes will be used as templates for dsRNA synthesis (RNA extraction, cDNA synthesis, primer design and optimisation, PCR and dsRNA synthesis). Dose response assays will then be done to quantify effects of selected dsRNAs on survival and longevity of T. molitor larvae. Gene expression methods will be used to confirm up-regulation of both Dicer2 and Argonaute in response to treatment with exogenous dsRNA.
  • Laboratory bioassays will be done to quantify the effects of co-applications of entomopathogenic fungi and dsRNAs on T. molitor larvae. This will involve applying co-agents at a range of doses, and a simple isobologram method will be used to determine the nature of the effect (synergism, antagonism or additive effect). Investigations of co-application of fungal biopesticides and dsRNA targeting the immune system will focus on determining whether dsRNA functions as a potentiator for improved fungal infection.
  • ‘Winning’ combinations of dsRNA and entomopathogenic fungi will be tested for their effects on larvae and adults of the cabbage stem flea beetle (CSFB), Psylliodes chrysocephala. The intention is to use pyrethroid-resistant strains of CSFB so that the effectiveness of the biopesticide strategy can be compared against a conventional pesticide spray programme.

BBSRC Strategic Research Priority: Food Security

Techniques that will be undertaken during the project:

  • Bioinformatics
  • Quantification and analysis of gene expression
  • PCR, insect rearing
  • insect microinjection and dissection
  • mycology and production of entomopathogenic fungi
  • Laboratory dose response bioassays of insect pathogens and insecticides
  • Statistical analysis and modelling (survival analysis, dose response analysis).

Contact: Dr. Dave Chandler, School of Life Sciences