Bacterial infection of fly ovaries reduces egg production and induces local hemocyte activation

Abstract

Morbidity, the state of being diseased, is an important aspect of pathogenesis that has gone relatively unstudied in fruit flies. Our interest is in characterizing how bacterial pathogenesis affects various physiologies of the fly. We chose to examine the fly ovary because we found bacterial infection had a striking effect on fly reproduction. We observed decreased egg laying after bacterial infection that correlated with increased bacterial virulence. We also found that bacteria colonized the ovary in a previously undescribed manner; bacteria were found in the posterior of the ovary, adjacent to the lateral oviduct. This local infection in the ovary resulted in melanization and activation of the cellular immune response at the site of infection.

Figure 1. Persistent bacterial infections reduce lifespan and reproduction

(a) Survival of female flies injected with bacteria or medium. n=90 flies. Symbols indicate mean. Bars indicate 95% confidence intervals. p<0.0001 when comparing S. typhimurium- or S. marcescens- or B. cenocepacia-injected flies to E. coli- or medium-injected flies by logrank test. (b) Bacterial growth in individual female flies. n=5 flies. The geometric mean and 95% confidence intervals are indicated. (c) Average eggs laid per female fly injected with bacteria or medium or left untouched. n=18. The geometric mean and 95% confidence intervals are indicated. Significance was determined by two-tailed unpaired t-test. p=.043 for E. coli- vs. medium-injected on day 10. See Supplemental Figure 1 for raw data and full time course. Note: There were no living flies on day 10 post-injection with B. cenocepacia or S. marcescens.

Figure 2. Bacterial virulence correlates with reduced egg production

(a) Survival of female flies injected with bacteria or medium. n=90 flies. Symbols indicate mean. Bars indicate 95% confidence intervals. p<0.0001 when comparing S. typhimurium- to SPI S. typhimurium-injected flies by logrank test. (b) Bacterial growth in individual female flies. n=5 flies. The geometric mean and 95% confidence intervals are indicated. (c) Average eggs laid per female fly injected with bacteria or medium, or left untouched. n=18 flies. The geometric mean and 95% confidence intervals are indicated. Significance was determined by two-tailed unpaired t-test. See Supplemental Figure 2 for raw data and full time course. (d) Diptericin RNA transcript levels in female flies injected with bacteria or medium. Values are normalized to day 0. n=3 RNA samples from 5 flies each. The mean and standard deviation are indicated. p<0.05 for wild type vs. SPI S. typhimurium at all time points as determined by two-tailed unpaired t-test.

Figure 3. Ovaries degenerate during infection

(a) DIC image of a single ovary dissected 7 days post-injection from a medium-injected female fly (left) and a S. typhimurium-injected female fly (right). All ovaries are shown posterior end (oviduct) up. (b) Percentage of dying egg chambers per ovary pair from female flies injected with S. typhimurium or medium. n=10 ovaries. The geometric mean and 95% confidence intervals are indicated. Significance was determined by two-tailed unpaired t-test.

Figure 4. Bacteria colonize the fly ovary

(a) Diagram describing the anatomy of the ovary. Each fly has two ovaries. The one on the left is shown whole while the one on the right is shown in an exploded view revealing three of the approximately 8 ovarioles that make up the ovary. Development proceeds from the anterior to the posterior end of the ovary, where eggs are laid through the common oviduct. The “debris zone” where bacteria and hemocytes are found is marked in green. (b) Bacterial growth in ovaries of female flies injected with bacteria. n=5 ovary pairs. The geometric mean and 95% confidence intervals are indicated. See Supplemental Figure 3 for raw data and full time course. (c) Ovaries dissected from a female fly 2 days post-injection with S. typhimurium pmig-1. DIC image (left), localization of green bacteria in the debris zone (center), overlay (right).

Figure 5. Fly hemocytes respond to ovary infection

(a) Ovary dissected from a hemolectin delta-Gal4, UAS-GFP female fly 7 days post-injection with medium. Note the green fluorescent hemocytes in the debris zone. (b–c) Ovaries dissected from hemolectin delta-Gal4, UAS-GFP female flies 7 days post-injection with S. typhimurium. The morphology of these cells differs from that seen in uninfected ovaries in (a) The white bar indicates 10 μm. (d) DIC image of ovaries from a female fly 10 days-post injection with S. typhimurium. Note the dark regions (marked with a triangle) that appear melanized. (e–f) Oviducts dissected from hemolectin delta-Gal4, UAS-GFP female flies 7 days post-injection with medium(e) or S. typhimurium(f). Note the diffence in the morphology of the labeled cells in uninfected versus infected ovaries. The white bar indicates 10 μm.