Drosophila Aversive Behavior toward Erwinia carotovora carotovora Is Mediated by Bitter Neurons and Leukokinin

Abstract

The phytopathogen Erwinia carotovora carotovora (Ecc) has been used successfully to decipher some of the mechanisms that regulate the interactions between Drosophila melanogaster and bacteria, mostly following forced association between the two species. How do Drosophila normally perceive and respond to the presence of Ecc is unknown. Using a fly feeding two-choice assay and video tracking, we show that Drosophila are first attracted but then repulsed by an Ecc-contaminated solution. The initial attractive phase is dependent on the olfactory Gr63a and Gaq proteins, whereas the second repulsive phase requires a functional gustatory system. Genetic manipulations and calcium imaging indicate that bitter neurons and gustatory receptors Gr66a and Gr33a are needed for the aversive phase and that the neuropeptide leukokinin is also involved. We also demonstrate that these behaviors are independent of the NF-kB cascade that controls some of the immune, metabolic, and behavioral responses to bacteria.

Protocol for a Binary Choice Feeding Assay Using Adult, Axenic Drosophila

“… CRITICAL: Do not exceed 4,000 × g during bacterial centrifugation, and do not centrifuge cells at 4°C but rather at 20°C–25°C. This will otherwise affect both the attractive and the aversive properties of Ecc-15 2141 for adult flies (for more explanation see Charroux et al., 2020 ). Remove the supernatant and wash the bacterial pellet with 20 mL of sterile 1× PBS.…”

“… Import the series of images into ImageJ as an “image sequence.” Save as a “AVI” selecting JPEG compression and 10 frames/s. Analyze the movie using Flybox as described in Charroux et al., 2020 . Note: The apparatus is placed on top of a LED panel (60 cm × 60 cm, 60 watts).…”

“…Non- starved flies, however, are expected to show no clear attraction phase to the Ecc-15 2141 contaminated solution and an attraction to sucrose that occurs slowly and progressively (See Figure 4 B and Methods Videos S1 , S2 , and S3 , right panels). See Charroux et al. 2020 for more explanations.…”

“…The bacterium used in this protocol, Erwinia carotovora carotovora-15 2141 ( Ecc-15 2141 ), is commonly used to decipher the mechanisms controlling host-pathogen interactions in the Drosophila model. For complete details on the use and execution of this protocol, please refer to Charroux et al. (2020) .…”

“…For complete details on the use and execution of this protocol, please refer to Charroux et al. (2020) .…”