Elucidating biosynthesis of the Lutzomyia longipalpis sandfly pheromone for sustainable production and control of leishmaniasis

Leishmaniases are a group of neglected tropical diseases caused by a protozoan parasite. In Central and South America the disease is spread by the blood-feeding sand fly, Lutzomyia longipalpis and related species. The disease is a particular challenge in Brazil, which has the highest incidence of leishmaniasis. Population control of the Lutzomyia longipalpis fly is a recognised strategy for preventing leishmaniasis. Indiscriminate spraying of insecticide is, however, harmful to all insect fauna: including many beneficial insects, such as pollinators as well as prey for a diverse range of animals.

The use of pheromones to attract insects for population monitoring, mass trapping and mating disruption are all established approaches to pest control. Pheromones provide a level of specificity that allows targeted intervention and minimises negative effects on non-target species. Sand flies mate using a leking strategy, where large numbers of males and females accumulate on trees or patches on the ground. The pheromone will initiate and stabilise these assemblies for targeted insecticide treatment or netting. This approach has been investigated in mosquito control, and would be expected to be more effective with Lutzomyia due to their behaviour of leking close to the ground, rather than in airborne swarms

The sex and aggregation pheromone of Lutzomyia longipalpis is produced by the male and is attractive to both females and males of the species. Composition of the pheromone varies with geographical location, which results in a small number of 'chemotypes' of the insect. In Brazil, populations producing (S)-9-methylgermacrene-B ((S)-9MGB), (1S,3S,7R)-3-methyl-alpha-himachalene ((1S,3S,7R)-3MAH), or sobralene as the pheromone have been identified. Total chemical synthesis of these compounds is uneconomic, but by elucidating the biosynthesis and identifying the enzymes responsible for their synthesis in the insect, sustainable biocatalytic production can be realised. The potential future impact of this work in facilitating vector control, and thereby reducing cases of leishmaniasis, is therefore significant.

In this project, which comes under BBSRC's Bioscience for Health research heading, a series of six work packages will be used to elucidate the biosynthesis of the three pheromone compounds. We will exploit the existence of the different chemotypes for comparative transcriptome and proteome analysis, which - supported with existing genomic data - will point to the biosynthetic enzymes in each population. This parallel approach using transcriptomics, proteomics and genomic data will maximise the probability of identifying the pheromone biosynthetic machinery. Once identified, the enzymes will be expressed for functional characterisation and for sustainable production of the pheromone for mass trapping, and ultimately disease control.

This research project to elucidate the biosynthesis of the Lutzomyia pheromone is divided into 6 Work packages. In WP1, insects from the three chemotypes will be collected in Brazil, by Euzebio Sant'ana, our collaborator, who has considerable experience of collecting Lutzomyia in the field. This will aid in the identification of candidate genes/protein in WP3. In WP2, the pheromone composition of the collected chemotypes will be verified using GC-MS - in both Brazil and the UK.

Examination of the genomic data made available through Lutzomyia longipalpis sequencing programmes revealed 12 candidate IDSs/TPSs, which show homology with those from the flea beetle, Phyllotreta stridulata. This beetle uses a pheromone of related biosynthetic origin to (S)-9MGB, and is one of few insects for which the enzymes are functionally characterised. WP3 will be to construct cDNA libraries of different Lutzomyia longipalpis chemotypes. To assist identification of the IDS/TPS enzymes involved in production of pheromones, we will perform RNA-seq and proteomic analyses to screen transcription in pheromone glands of m/f of the chemotypes together. Male-specific enzymes that are unique to each chemotype will allow the narrowing of candidate enzymes.

In WP4, enzymes identified in WP3 will be expressed and purified for functional testing, biophysical characterisation (WP6), and substrate profiling. GC-MS will allow analysis of volatile products of terpene synthases (TPSs), and LC-MS will be used for detection of isopentenyl diphosphate intermediates by isopentenyl diphosphate synthases (IDSs). Whilst some precursor compounds for the enzyme assays are commercially available, a number will require chemical synthesis using established strategies (WP5). Once the enzymatic machinery to enable sustainable pheromone biosynthesis is fully identified, in the longer term we will explore means to produce the pheromone for insect control.