Diel periodicity and visual cues guide oviposition behavior in Phlebotomus papatasi, vector of old-world cutaneous leishmaniasis

Background Phlebotomine sand flies are vectors of human leishmaniases, important neglected tropical diseases. In this study, we investigated diel patterns of oviposition behavior, effects of visual cues on oviposition-site selection, and whether these affect the attraction of gravid Phlebotomus papatasi (Scopoli), the vector of old-world cutaneous leishmaniasis, to olfactory cues from oviposition sites. Methodology/principal findings To evaluate these questions, we conducted a series of experiments using attraction and oviposition assays within free-flight test chambers containing gravid females entrained under a 14:10 hrs light:dark photoperiod. By replacing sticky-screens or moist filter papers every three hours, we showed that oviposition site search occurs mainly in the latest part of the night whereas peak oviposition occurs during the early part of the night. Behavioral responses to olfactory oviposition cues are regulated by time-of-day and can be disrupted by transient exposure to a constant darkness photoperiod. Gravid females, but not any other stage, age, or sex, were attracted to dark, round oviposition jars, possibly resembling rodent burrow openings. This visual attraction disappeared in the absence of an illumination source. Egg deposition rate was not affected by jar color. Olfactory cues had the strongest effect when the visual cues were minimal. Conclusion and significance Our study showed, for the first time, that visual cues in the form of oviposition-site color, lighting level, and photoperiod are important in guiding the oviposition behavior of phlebotomine sand flies. Furthermore, such visual cues could modify the flies’ sensitivity to olfactory oviposition cues. Our results suggest that chemosensory and visual cues are complementary, with visual cues used to orient gravid females towards oviposition sites, possibly at long- to medium-ranges during crepuscular periods, while olfactory cues are used to approach the burrow in darkness and assess its suitability at close-range. Implications to sand fly control are discussed.
Author summary Sand flies are vectors of human leishmaniases, an important neglected tropical disease. An alternative approach to the conventional delivery of an insecticide to the vector is to bring the vector to the insecticide using oviposition (egg-laying)-site attractants. Olfactory cues originating from organic matter have been identified as important sources of oviposition attractants. However, nothing is known regarding visual assessment of oviposition sites by sand flies. Also, little is understood about diel egg-laying patterns of sand flies. Finally, it is not known if visual cues and time-of-day may affect their sensitivity to olfactory cues. In this study, we investigated these questions in a series of lab experiments using free-flight cage arenas, with Phlebotomus papatasi (vector of cutaneous leishmaniasis). We showed that peak oviposition-site search and sensitivity to olfactory cues occurs mainly in the latest part of the night whereas peak oviposition occurs during the early part of the night. We demonstrated that only gravid females, but not any other stage or sex, were attracted to dark, round oviposition jars resembling burrow openings. Finally, we showed that sensitivity to olfactory cues is reduced in the presence of strong visual cues and in the absence of natural diel photoperiod.