Su(H)S269A mutants are compromised in their response to parasitoid wasp infestation
(A) Quantification of melanized crystal cells from the last two segments of Su(H)gwtand Su(H)S269A larvae with and without wasp infestation as indicated. In the control, wasp parasitism causes crystal cell numbers to drop to a level of about 50%, whereas in Su(H)S269A mutants the number settles at the un-infested Su(H)gwt level. Each dot represents one analysed larva (n=70-100 as indicated). (B) Crystal cell index in larval lymph glands is given as ratio of Hnt-positive crystal cells per 1° lobe relative to the size of the lobe. Each point represents one analysed lobus (n=15). (A,B) Statistical analyses Kruskal-Wallis test, followed by Dunn’s test with *** p<0.001, ** p<0.01, * p<0.05, ns (not significant p≥0.05).
(C-F) Quantification of larval lamellocytes in the circulating hemolymph (C,D) or in lymph glands (E,F) before and after wasp infestation in Su(H)gwt versus Su(H)S269A. Lamellocytes were marked with either PPO3-Gal4::UAS-GFP (C,E) or atilla-GFP (D,F) as indicated. (C,D) The fraction of GFP-labelled lamellocytes of the total number of DAPI-labelled blood cells isolated from hemolymph is given; each dot represents ten pooled larvae (n=8-10 as shown). Representative image of labelled control hemolymph is shown above (DAPI-labelled nuclei in light blue, GFP in green). Scale bars 50 µm. (E,F) Lamellocyte index is given as number of GFP-labelled lamellocytes per area in the 1° lobe of the lymph gland. Each dot represents the lamellocyte index of one lobus (n=15). Representative Su(H)gwt lymph glands after wasp infestation are shown, co-stained for nuclear Pzg (in blue). Scale bars 100 µm. Statistical analyses with unpaired Student’s t-test; only significant differences are indicated (*** p<0.001). (A-F) Representative images for each genotype and condition are shown in supplemental Figure 2.
(G) qRT-PCR analyses measuring expression of NRE-GFP (left panel) and atilla (right panel). Transcript levels were quantified from hemolymph isolated from infested larvae at 0-6 h or 24-30 h post-infestation as indicated, relative to the untreated Su(H)gwtcontrol. Tbp and cyp33 served as reference genes. Shown data were gained from four biological and two technical replicates each. Left panel: Immediately after wasp infection, NRE-GFP expression dropped significantly in the Su(H)gwt control, and even further to about 30% 24-30 h post-infection, whereas it remained at 60%-70% in the infested Su(H)S269Amutants. Right panel: atilla transcripts remained stable at first in the Su(H)gwt control, to rise dramatically 24-30 h post-infection, in contrast to Su(H)S269A. Mini-max depicts 95% confidence, mean corresponds to expression ratio. Exact p-values are given in the raw data table. Significance was tested using PFRR from REST (*p<0.05).