Combating insecticide resistance in the tomato leafminer, Tuta absoluta

Summary: The tomato leafminer, Tuta absoluta is an economically important insect pest of tomato production worldwide. This invasive species is a relatively recent introduction to the UK but since 2009 has become a growing problem to British tomato growers. Control of this species worldwide relies on the use of chemical insecticides and this is also true of the UK where two insecticides (spinosad and chlorantraniliprole) are used in combination with a biological control agent in an integrated pest management (IPM) programme. Recently, there have been reports of resistance development to both compounds in T. absoluta populations in Europe, and loss of efficacy reported to spinosad by certain growers in the UK. The aim of this proposed studentship is to build on this work and our previous research to understand the risk of resistance development in UK populations of T. absoluta and the mechanisms underlying resistance in UK and European populations. Insecticide bioassays and DNA-based diagnostics will be used to characterise the current status of resistance in the UK and the situation monitored over the life of the project. The molecular basis of resistance in European and UK populations will be characterised using a range of molecular approaches. Finally this knowledge will be translated into practical tools and solutions that can be used to prevent, slow, or overcome resistance. The project will be carried out in close collaboration with the British Tomato Growers Association who represent over 90% of the British Tomato industry.

Aims and Objectives:  

1) Resistance monitoring of UK populations of T. absoluta. The studentship will build on the 3 month monitoring project which commenced in August 2015 to monitor UK populations of T. absoluta for their susceptibility to three insecticide classes, spinosad, cholorantranilprole and indoxacarb in 2016, 2017 and 2018.  This will be carried out using A) discriminating dose insecticide bioassays which are relatively quick and easy to perform and will provide evidence of a shift in sensitivity compared to known susceptible standards, and B) in the case of spinosad (and in time to chlorantraniliprole, see 2 and 3 below) high-throughput DNA based diagnostic assays will be used to detect known resistance mechanisms (this allows resistance to be detected at very low frequency). This objective will retain flexibility during the life of the project to shift emphasis to (or include) other compounds as appropriate.

2) Identify the molecular basis of resistance of T. absoluta populations to chlorantraniliprole. Resistance to this compound is currently prevalent in Mediterranean populations of T. absoluta including in Spain (which is a major supplier of UK tomatoes). We have access to resistant populations from these regions and will use molecular approaches to characterise the basis of resistance. This objective is greatly facilitated by the fact that we recently created the first reference transcriptome for T. absoluta by Illumina sequencing of pooled RNA from all four life stages. From this transcriptome we have annotated the complete sequence of the chlorantraniliprole target-site (the ryanodine receptor) and the majority of the detoxification enzymes frequently involved in breaking down insecticides in resistant insects. The studentship project will exploit this transcriptomic resource to investigate target-site and metabolic resistance in Spanish populations (and UK populations if resistance is detected). In both cases we will use next-generation sequencing to rapidly characterise mutations and genes associated with resistance. A Spanish field resistant strain, a lab susceptible strain, and a chlorantraniliprole selected and parental unselected strain will be sequenced across two lanes of a HiSeq 2500. We have secured independent funding for this and samples will be sent for sequencing just prior to the project starting if this proposal is successful. The student will be shown how to map reads against our reference transcriptome using the Trinity software and, to investigate target-site resistance, reads encoding the ryanodine receptor will be compared between resistant (selected) and susceptible (unselected) to identify mutations that result in amino acid substitutions associated with resistance. The RyR is relatively highly expressed so we are confident we will obtain excellent coverage of the receptor. Analyses will focus on known resistance ‘hot-spots’ we have previously characterised in another Lepidopteran species (Plutella xylostella). In the case of metabolic resistance, after read mapping we will use EdgeR/DEseq to identify genes that are differentially expressed between susceptible and resistant strains of T. absoluta. Candidate genes putatively involved in metabolic detoxification showing differential expression will be further explored using quantitative PCR (qPCR). These analyses will identify genes encoding detoxification enzymes such as cytochrome P450s, glutathione-S-transferases and carboxylesterases that are overexpressed in resistant strains and may be involved in resistance. Because of the speed of the next-gen approach we envisage that the student will have time to carry out functional characterisation of select candidate mutations/genes using established methods in the Williamson and Bass labs [8].

3) Translation of fundamental research into tools or approaches to combat resistance. We envisage that the student may wish to explore three main areas under this objective. 1) The development of high-throughput diagnostics against resistance mechanisms identified in Objective 2 will allow UK populations to be screened for resistance to chlorantraniliprole. Such assays are not a substitute for insecticide bioassays but do allow resistance to be detected at an earlier stage (i.e. when present at very low frequency in a population). 2) If metabolic resistance is detected the ability of this to be overcome using an insecticide synergist such as piperonyl butoxide (PBO) will be explored. PBO is itself essentially non-toxic but inhibits the metabolic systems that resistance strains evolve to protect themselves and can therefore restore susceptibility to populations with this form of resistance. If this approach looks promising the effect of any co-application strategy will be explored in insecticide bioassays using bumblebees and the biological control agent Macrolophus pygmaeus to identify any off-target effects. Implementation of glasshouse trials would need to be explored in future projects in collaboration with the British Tomato Growers Association (BTGA). 3) If resistance to spinosad and/or chlorantraniliprole is identified in UK populations of T. absoluta, insecticide bioassays will be used to explore the efficacy of alternative insecticides against these strains (and also off-target organisms). The aim will be to identify a product that is not cross-resisted and is amenable to the current IPM strategy. Testing of any product at the glasshouse scale would again need to be explored in future projects in collaboration with the BTGA.