Temperature-Dependent Galleria mellonella Mortality as a Result of Yersinia entomophaga Infection

Abstract

The bacterium Yersinia entomophaga is pathogenic to a range of insect species, with death typically occurring within 2 to 5 days of ingestion. Per os challenge of larvae of the greater wax moth (Galleria mellonella) confirmed that Y. entomophaga was virulent when fed to larvae held at 25°C but was avirulent when fed to larvae maintained at 37°C. At 25°C, a dose of ~4 × 10(7) CFU per larva of a Y. entomophaga toxin complex (Yen-TC) deletion derivative, the Y. entomophaga ΔTC variant, resulted in 27% mortality. This low level of activity was restored to near-wild-type levels by augmentation of the diet with a sublethal dose of purified Yen-TC. Intrahemocoelic injection of ~3 Y. entomophaga or Y. entomophaga ΔTC cells per larva gave a 4-day median lethal dose, with similar levels of mortality observed at both 25 and 37°C. Following intrahemocoelic injection of a Yen-TC YenA1 green fluorescent protein fusion strain into larvae maintained at 25°C, the bacteria did not fluoresce until the population density reached 2 × 10(7) CFU ml(-1) of hemolymph. The observed cells also took an irregular form. When the larvae were maintained at 37°C, the cells were small and the observed fluorescence was sporadic and weak, being more consistent at a population density of ~3 × 10(9) CFU ml(-1) of hemolymph. These findings provide further understanding of the pathobiology of Y. entomophaga in insects, showing that the bacterium gains direct access to the hemocoelic cavity, from where it rapidly multiplies to cause disease.

FIG 1

Per os or intrahemocoelic assessment of Yersinia entomophaga or the Y. entomophaga ΔTC variant in Galleria mellonella larvae maintained at 25°C. (A) Cumulative mortality of G. mellonella larvae post-maximum per os challenge with Y. entomophaga or the Y. entomophaga ΔTC variant at 25°C. The mean ± SEM estimated LT₅₀ was 2.0 ± 0.1 days. The horizontal error bar denotes the 95% confidence interval (1.74 to 2.23 days) of the estimated LT₅₀ of 2 days. (B) Log dose of Y. entomophaga and mortality of G. mellonella larvae 5 days post per os challenge at 25°C. Shown is the fitted relationship using the probit model from the observed mortality of G. mellonella larvae challenged per os with various concentrations of Y. entomophaga. (C) Percentage of G. mellonella larval mortality (mean ± SEM) 5 days postaugmentation of the diet with sterile distilled water (control), wild-type Y. entomophaga (Y. ent) bacteria, the Y. entomophaga ΔTC variant, a sublethal dose of the Yen-TC, and a sublethal dose of Yen-TC in combination with the Y. entomophaga ΔTC variant (ΔTC + Yen-TC). (D) Fitted relationship using the probit model from the observed mortality of G. mellonella larvae hemocoelically injected with various numbers of cells of Y. entomophaga (solid line) or the Y. entomophaga ΔTC variant (broken line) 4 days postinjection.

FIG 2

Assessment of Galleria mellonella larval hemolymph postchallenge with Yersinia entomophaga(pGFPuvsp) in larvae maintained at 25°C, viewed using Olympus BX50 light microscope under fluorescent light at 400× magnification. (A) Hemolymph 24 h post-maximum per os challenge. Red arrows denote Y. entomophaga(pGFPuvsp) cells. (B) G. mellonella larval hemolymph 48 h post-larval death, containing ∼1.2 × 10⁹ CFU ml⁻¹ bacterial cells. (C) Atypical irregular partitioned bacteria (see the inset, where the scale bar is 10 μm). (D) Large (possibly septate) cells. Red arrows in panels B, C, and D denote lipid vesicles. Non-inset scale bars are 50 μm.

FIG 3

Assessment of Galleria mellonella larval hemocoelic fluid at various time points postinjection of Yersinia entomophaga YENA1GFP in larvae maintained at either 25 or 37°C viewed under white or fluorescent light at 400× magnification. (A) Hemocoelic fluid from larvae maintained at 25°C 36 h postinjection of 2 × 10⁷ CFU ml⁻¹. (B) The same sample viewed under UV fluorescent light with a red arrow denoting a fluorescing cell. (C) Hemocoelic fluid containing ∼5 × 10⁸ CFU ml⁻¹ ∼48 h postinjection. (D) The same sample viewed under UV fluorescent light, with >90% of the cells fluorescing, some elongated cells, and other cells in close association with the larval hemocytes (photo inset; scale bar, 10 μm). The micrographs are from hemocoelic fluid from larvae maintained at 37°C (E to F) at a cell density of ∼1.3 × 10⁹ CFU ml⁻¹ ∼48 h postinjection. Small cells were observed either singly or as aggregates; occasionally some of these cells were fluorescent (red arrows). Non-inset scale bars are 50 μm.

FIG 4

Yersinia entomophaga grown in LB broth at 37°C (A) and 25°C (B) at stationary phase showing flocking/aggregation of cells. Images were obtained by electron microscopy at 50,000× magnification of exponential-phase Y. entomophaga cells grown at 25°C (C) or 37°C (D and E), where panel D shows fimbriated (arrow) and nonfimbriated bacteria and panel E shows bacterial aggregates from which fimbriae can be visualized protruding from the leading edge (inset; scale bar, 0.5 μm) Non-inset scale bars are 1 μm.

FIG 5

Assessment of phenoloxidase (PO) activity in Galleria mellonella larvae maintained at 25°C that were intrahemocoelically injected with either Yersinia entomophaga, Escherichia coli, Yen-TC, or water. (A and B) Healthy G. mellonella larvae (A) and larvae 3 days postinjection of ∼1,000 Y. entomophaga cells (B). (C and D) Microcentrifuge tubes containing extracted hemolymph from Escherichia coli (C) and Y. entomophaga (D) 24 h postinjection, exhibiting melanization and an absence of melanization, respectively. (E) Assessment of PO activity of the hemocoelic fluid of insects injected with Y. entomophaga (Y. ent), purified Yen-TC, E. coli, or sterile distilled water at 5 or 24 h postinjection, compared with the noninjected control.

FIG 6

Confocal microscopy of Yersinia entomophaga(pGFPuvsp) associated with and within hemocytes from Galleria mellonella larvae maintained at 25°C. (A) Y. entomophaga(pGFPuvsp) inside a hemocyte. (B) Y. entomophaga(pGFPuvsp) within (arrows) and on hemocytes, stained with carboxy-SNARF-1 (for 3D depiction, see Movie S1 in the supplemental material). G. mellonella hemocytes were stained with SYTO 9-PI using a LIVE/DEAD BacLight kit and carboxy SNARF-1 (acetoxymethyl ester, acetate [Invitrogen]) 4 h postinjection of water (C) or 400 ng of Yen-TC (D). Scale bars are 10 μm.