The paratransgenic sand fly: A platform for control of Leishmania transmission

Ivy Hurwitz,Heidi Hillesland,Ravi Durvasula,Annabeth Fieck,Pradeep Das

doi:10.1186/1756-3305-4-82

Abstract

BackgroundLeishmania donovani is transmitted by the bite of the sand fly, Phlebotomus argentipes. This parasite is the agent of visceral leishmaniasis (VL), an endemic disease in Bihar, India, where prevention has relied mainly on DDT spraying. Pesticide resistance in sand fly populations, environmental toxicity, and limited resources confound this approach. A novel paratransgenic strategy aimed at control of vectorial transmission of L. donovani is presented using Bacillus subtilis, a commensal bacterium isolated from the sand fly gut. In this work, B. subtilis expressing Green Fluorescent Protein (GFP) was added to sterilized larval chow. Control pots contained larval chow spiked either with untransformed B. subtilis or phosphate-buffered saline. Fourth-instar P. argentipes larvae were transferred into the media and allowed to mature. The number of bacterial colony forming units, relative abundance and the mean microbial load were determined per developmental stage.ResultsAddition of B. subtilis to larval chow did not affect sand fly emergence rates. B. cereus and Lys fusiformis were identified at each developmental stage, revealing transstadial passage of endogenous microbes. Larvae exposed to an exogenous bolus of B. subtilis harbored significantly larger numbers of bacteria. Bacterial load decreased to a range comparable to sand flies from control pots, suggesting an upper limit to the number of bacteria harbored. Emerging flies reared in larval chow containing transformed B. subtilis carried large numbers of these bacteria in their gut lumens. Strong GFP expression was detected in these paratransgenic flies with no spread of transformed bacteria to other compartments of the insects. This is the first demonstration of paratransgenic manipulation of P. argentipes.ConclusionsParatransgenic manipulation of P. argentipes appears feasible. Expression of leishmanicidal molecules via commensal bacteria commonly found at breeding sites of P. argentipes could render adult sand flies refractory to L. donovani infection.

Highlights

Leishmania donovani is transmitted by the bite of the sand fly, Phlebotomus argentipes
Bacterial analysis of larvae, pupae and emergent sand flies that developed in larval chow mixed with phosphate buffered saline (PBS) indicates that there is transstadial passage of indigenous bacterial flora (Table 1)
The use of an environmental commensal bacterium for delivery of foreign genes to developmental stages of the sand fly serves as a platform to consider paratransgenic approaches in field conditions as a tool to control vectorial transmission of L. donovani

Summary

Introduction

Leishmania donovani is transmitted by the bite of the sand fly, Phlebotomus argentipes This parasite is the agent of visceral leishmaniasis (VL), an endemic disease in Bihar, India, where prevention has relied mainly on DDT spraying. Our laboratory is developing paratransgenic strategies for control of Trypanosoma cruzi transmission by triatomine bugs as a method to reduce the human burden of Chagas disease in Latin America [2,3,4,5]. In this “Trojan Horse” approach, symbiotic gut-associated bacteria of the arthropod are transformed to express molecules with anti-parasite activity. It is our goal to develop a paratransgenic approach to control transmission of L. donovani by P. argentipes

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