Fig 1.
Anatomy and histology of G. mellonella larvae with emphasis on the digestive tract.
(A) Underside of a G. mellonella larva. The foregut, the midgut, and the hindgut are indicated by arrows. (B) Dissected digestive tract after instillation with methylene blue. (C) Schematic drawing of the digestive tract. The stomadeal valve (SV) separates foregut lined by cuticular epithelium from midgut lined by glandular epithelium. The proctodeal valve (PV) is located between midgut and hindgut. The distinct epithelium cranial to the PV is labeled in green. The ingesta (I) in the midgut is covered by the peritrophic membrane (PM) and separated from the mucosa by the ectoperitrophic space. The crop and both valves are surrounded by a thick layer of musculature (red). (D) Longitudinal and sagittal histological section along the middle through G. mellonella. (E) Magnification of mouth, esophagus, and crop lined by the cuticular epithelium and surrounded by muscle cells. (F) Magnification of the SV between crop and midgut. (G) Magnification of the midgut lined by glandular epithelium. The ingesta is surrounded by the peritrophic matrix (PM) and separated from mucosa by the ectoperitrophic space. (H) Magnification of the PV between midgut and hindgut lined by cuticular epithelium. Vacuolated columnar epithelial cells line the midgut cranial to the PV. (D)-(H) are paraffin sections stained by hematoxylin and eosin. Sections are indicated by numbers: 1 = mouth, 2 = esophagus, 3 = crop, 4 = glandular intestine, 5 = transition zone, 6 = cuticular intestine, 7 = rectum, 8 = anus. Photos of representative preparations are shown; the scales are indicated.
Fig 2.
Role of TcaA, HE, and TccC in insecticidal activity of W22703 towards G. mellonella.
Larvae were orally infected with W22703, its mutants lacking tcaA, HE, and tccC, and with mutants carrying the plasmids pACYC-HE, pACYC-tcaA, and pBAD-tccC. Application of LB medium served as control. Life span assays were performed for nine days, and the viability of the larvae was monitored each day to determine the survival rate of the larvae. The raw data were plotted by the Kaplan-Meier method. The Kaplan-Meier-plot is based on triplicates with 36 larvae in total per strain. The curves were compared to each other using the log-rank test, which generates a p value testing the null hypothesis that the survival curves are identical. Data were fit to exponential distribution. p values of 0.05 or less were considered significantly different from the null hypothesis (p value W22703 ΔHE/pACYC-HE = 0.0194; p value W22703 ΔtcaA/pACYC-tcaA = 0.0369; p value W22703 ΔtccC/pACYC-tccC = 0.0251). All graphs start at 100%.
Fig 3.
External morphology of larvae following oral infection with W22703 and its mutants.
The photos illustrate the outcome of the experiment shown in Fig 2. Black animals were dead, anthracite ones still alive. The numbers in the upper right angle of each photo indicates dead animals at the respective time-point. Infections with W22703 ΔtccC/pBAD-tccC were not documented by photos.
Fig 4.
Proliferation numbers of Y. enterocolitica W22703 strains in G. mellonella larvae.
Larvae were orally infected with W22703, W22703 ΔtcaA, W22703 ΔtcaA/pACAC-tcaA, W22703 ΔHE, W22703 ΔHE/pACYC-HE, W22703 ΔtccC, and W22703 ΔtccC/pBAD-tccC. Six animals per time-point were used, and the experiments were performed as triplicates, resulting in a total of 18 larvae per time-point per strain. Larvae fed with LB were used as a negative control. Standard deviations are indicated as error bars.
Fig 5.
Time course of G. mellonella infection by Y. enterocolitica W22703.
The tissue sections monitored by fluorescence microscopy show antibody-stained Y. enterocolitica cells in the (A) gut or (B) hemolymph of G. mellonella 4 h, 6 h, 12 h, 18 h, and 24 h after infection. (C) The controls depict the gut area of G. mellonella that were fed with LB (left) or infected with E. coli (middle) 24 h ago. The tissue sections were stained with a Yersinia-specific or an E. coli-specific antibody. Functionality of the anti-E. coli antibody was demonstrated by the application of E. coli into muscle tissue of chicken (right). Cyan-coloured areas in the gut area of G. mellonella are unspecific bonds of the anti-E. coli antibody. Representative preparations are shown; the scale is indicated. 1 = intestinal epithelium, 2 = intestinal lumen, 3 = fat tissue, 4 = hemolymph, 5 = appendix, 6 = Malpighian vessels, 7 = muscle cells, 8 = E. coli, 9 = antibody cross reactions.
Fig 6.
Detection of W22703 and its derivatives in the hemolymph of G. mellonella.
All photos show the hemolymph area of G. mellonella. (A) G. mellonella tissue sections 24 h p.i. with W22703 and its mutants. Staining was conducted using the anti-Yersinia antibody. (B) Tissue sections of larvae 24 h p.i. with W22703 tcaA::rfp, W22703 ΔHE tcaA::rfp, W22703 PHE::rfp, and W22703 ΔHE tcaA::rfp/pACYC HE. Staining was performed with the Yersinia-specific antibody (top row) or the anti-RFP antibody (bottom row), which was applied to the same tissue sections, to detect TcaA production as indicated exemplarily by arrows. Preparations of ten infected animals per strain were carried out. Photos of representative preparations are shown; the scales are indicated. 1 = exoskeleton, 2 = musculature, 3 = fat tissue, 4 = hemolymph.
Fig 7.
(A) Hemolymph cell preparations from G. mellonella orally infected with 6.1 × 105 CFU of W22703 or treated with LB medium as control. (B) Hemocytes of larvae after application of four W22703 mutants, showing cell morphology similar to those of the controls. Hemocyte aggregation and deformation was visible only upon infection with W22703. Hemolymph preparations were fixed with methanol and stained by Giemsa solution. Photos of representative preparations are shown; cells vary in size, the scale is indicated. An Olympus BX53 microscope (Olympus Europa, Hamburg, Germany) was used.
Fig 8.
COG categories of differentially expressed W22703 genes upon G. mellonella infection.
The numbers of down-regulated (light grey) and up-regulated (dark grey) genes in each COG category are indicated. The COG categories are ordered by descending ratio of up-to down-regulated genes. The data were taken from S1 Table.
Table 1.
Selection of down (log2 FC < -1.5, red)- and up (log2 FC > -1.5, green)-regulated Y. enterocolitica genes 12 h and 24 h p.i.
AMP, antimicrobial peptide; P, phosphate; PTS, phosphotransferase system; BCAA, branched-chain amino acid; dep., dependent; n.s., not significant.