Broadening the tools for studying sand fly breeding habitats: A novel molecular approach for the detection of phlebotomine larval DNA in soil substrates.

Phlebotomine sand flies constitute a group of haematophagous insects of great medical and veterinary importance. Despite the significant knowledge on the biology and behavior of adult sand flies in the wild, there is little information available on the ecology of their larval stages due to difficulties of detecting them in the soil. So far, investigations on sand fly breeding sites have been based on methods to catch emerging adults or on microscopic examination of the soil for the presence of immatures, neither of which is very precise or efficient for studying immatures in the field. Thus, there is a need for a better method to detect, identify and quantify immatures in soil samples. To address this issue we developed a novel molecular genetic approach for the detection of sand fly larval DNA within soil samples. Different numbers of live larvae of Phlebotomus papatasi and P. tobbi (alone or in combination), were mixed with soil and analyzed to identify and quantify the sand flies. By modifying already existing soil DNA extraction protocols in combination with a real-time PCR using species-specific primers, we successfully detected as little as one larva/40 ml of soil. We could also distinguish the two species of sand flies. This method should be very useful for increasing our knowledge of sand fly larval ecology in the field, and thus help develop more efficient, targeted control strategies against Phlebotomus sand flies.