Ingestibility,Digestibility, and Engineered Biological Control Potential of Flavobacterium hibernum,Isolated from Larval Mosquito Habitats

Flavobacterium hibernum, isolated from larval habitats of the eastern tree hole mosquito, A. triseriatus, remained suspended in the larval feeding zone much longer (8 days) than other bacteria. Autofluorescent protein markers were developed for the labeling of F. hibernum with a strong flavobacterial expression system. Green fluorescent protein (GFP)-tagged F. hibernum cells were quickly consumed by larval mosquitoes at an ingestion rate of 9.5 × 10⁴/larva/h. The ingested F. hibernum cells were observed mostly in the foregut and midgut and rarely in the hindgut, suggesting that cells were digested and did not pass the gut viably. The NanoLuc luciferase reporter system was validated for quantitative larval ingestion rate and bacterial fate analyses. Larvae digested 1.87 × 10⁵ cells/larva/h, and few F. hibernum cells were excreted intact. Expression of the GFP::Cry11A fusion protein with the P20 chaperone protein from Bacillus thuringiensis H-14 was successfully achieved in F. hibernum. Whole-cell bioassays of recombinant F. hibernum exhibited high larvicidal activity against A. triseriatus in microplates and in microcosms simulating tree holes. F. hibernum cells persisted in microcosms at 100, 59, 30, and 10% of the initial densities at days 1, 2, 3, and 6, respectively, when larvae were absent, while larvae consumed nearly all of the F. hibernum cells within 3 days of their addition to microcosms.