Fig 1.
Comparative blockage and infection rates of O. montana and X. cheopis during a four-week period after a single infectious blood meal.
(A) Percentage of fleas that developed complete proventricular blockage. The mean and SD are indicated. (B) Mortality rate of uninfected (open symbols) and infected (closed symbols) O. montana (red symbols) and X. cheopis (blue symbols). (C) Percentage of O. montana (red symbols) and X. cheopis (blue symbols) still infected 7 and 28 days after an infectious blood meal. (D) Mean bacterial load in infected O. montana (blue symbols) and X. cheopis (red symbols) immediately after the infectious blood meal (day 0) and at 7 and 28 days after infection. The cumulative results of three independent experiments (n = 99 to 113 fleas each) are shown in (A, B); the results of each of the three experiments are plotted in (C, D). *P = 0.0003; **P < 0.0001 by Fisher’s exact test (two-tailed).
Fig 2.
Y. pestis causes complete proventricular blockage in O. montana fleas.
The left panels show the typical blockage phenotype in O. montana fleas immediately after a feeding attempt. Fresh red blood (arrows) that was unable to enter the midgut (MG) is seen in the esophagus (E). The fleas were infected with GFP-expressing Y. pestis. The middle and right panels are images of the dissected digestive tracts from the same fleas visualized by DIC and a combination of DIC + fluorescence microscopy, respectively, and confirm the presence of a dense Y. pestis biofilm that completely fills and blocks the proventriculus (PV). Scale bars = 100 μm.
Fig 3.
O. montana blocks earlier and survives longer after becoming blocked than X. cheopis.
Histograms of the temporal incidence of complete blockage in O. montana and X. cheopis (A, B) and their life span after becoming blocked (C). Dashed lines indicate the mean; curve fitting used a Gaussian model of the frequency distribution data. Blocked O. montana survived significantly longer than blocked X. cheopis; P < 0.0001 by log-rank (Mantel-Cox) test.
Fig 4.
Transmission dynamics of O. montana and X. cheopis fleas during a four-week period after a single infectious blood meal.
The number of Y. pestis CFU transmitted by O. montana (A) and X. cheopis (B) fleas by the early-phase (EPT, day 3) and proventricular biofilm-dependent (PBT) mechanisms in three independent experiments. See Tables 1 and 2 for details. (C) Number of Y. pestis CFU transmitted in the early phase (3 days after an infectious blood meal) by O. montana and X. cheopis in 8 independent experiments. Horizontal bars indicate the median. For these 16 EPT experiments, 103 to 202 fleas fed on day 3. The bacteremia level in the infectious blood meals ranged from 1.9 x 108 to 3.0 x109 CFU/ml.
Table 1.
O. montana transmission data summary.
Table 2.
X. cheopis transmission data summary.
Fig 5.
Proventricular blockage causes greater esophageal distension in O. montana than in X. cheopis.
(A-D) Representative images of foreguts from dissected uninfected and blocked O. montana and X. cheopis. Dashed red lines indicate the location of esophageal (E) and proventricular (PV) measurements used to calculate the E:PV ratio of uninfected fleas (open symbols; n = 20) and blocked fleas (closed symbols; n = 10 to 15) in (E); the mean and SD are indicated. *P < 0.05; **P < 0.01, ***P < 0.0001 by one-way ANOVA and Tukey’s multiple comparison test. Scale bars = 100 μm.