Chen, S., Kaufman, M.G., Korir, M.L. and Walker, E.D.
Notes: Researchers were interested in quantitating the ingestion of Flavobacterium hibernum by eastern tree hole mosquito (Aedes triseriatus) larvae, and better understand the fate of the bacteria once ingested. The NanoLuc™ luciferase gene Nluc was inserted into a bacterial expression plasmid then introduced to F. hibernum by conjugation to create a bacterial strain expressing NanoLuc™ luciferase. The researchers determined the number of bacteria by diluting cultured F. hibernum, adding an equal volume of Nano-Glo® Luciferase Assay Reagent and measuring luminescence. Alternatively, the bacteria were lysed with Passive Lysis Buffer and lysozyme before assaying with an equal volume of Nano-Glo® Luciferase Assay Reagent. This method could determine the number of bacteria down to 700 cells/ml. To study the larval ingestion of the NanoLuc™-expressing F. hibernum, the bacteria were cultured, washed in PBS and resuspended to 7.3 × 105 cells/ml. Three milliliters of this feeding solution was inoculated with six A. triseriatus larvae of three different stages [3rd instar larvae (6 days past hatch), 4th instar larvae (9 days past hatch) and pupae (12 days past hatch)]. The number of bacterial cells remaining after a feeding interval was sampled and quantitated using the Nano-Glo® reagent as detailed earlier.
Third-instar larvae were starved for 2 hours in sterile water then 2.8 × 109 cells/ml of the NanoLuc™ expression bacteria were added and incubated with the larvae for 4 hours. After incubation, the mosquito larvae were rinsed well, and 2ml of larvae suspended in sterile water were transferred to a six-well plate to assess the rate of digestion. The amount of bacteria inside the larvae and the incubation solution were determined by sampling at 0, 0.5, 1, 1.5, 2, 2.5, 3 and 4 hours. To quantitate the internal bacteria, three larvae were pooled, homogenized, re-suspended in water and analyzed using the Nano-Glo® Luciferase Assay Reagent. To more closely mimic the tree hole environment in which the A. triseriatus larvae feed, six 2nd instar larvae were transferred to a 20ml microcosm of tree-hole water and beech leaf that was then inoculated with the NanoLuc™-expressing F. hibernum at 4.7 × 105 cells/ml. Bacterial cells in the microcosm were sampled at 0, 1, 2, 3 and 6 days, washed, resuspended in PBS and 50µl used to measure NanoLuc™ luciferase activity. (4441)