Analysis of the Drosophila host defense in domino mutant larvae, which are devoid of hemocytes

Abstract

We have analyzed the Drosophila immune response in domino mutant larvae, which are devoid of blood cells. The domino mutants have a good larval viability, but they die as prepupae. We show that, on immune challenge, induction of the genes encoding antimicrobial peptides in the fat body is not affected significantly in the mutant larvae, indicating that hemocytes are not essential in this process. The hemocoele of domino larvae contains numerous live microorganisms, the presence of which induces a weak antimicrobial response in the fat body. A full response is observed only after septic injury. We propose that the fat body cells are activated both by the presence of microorganisms and by injury and that injury potentiates the effect of microorganisms. Survival experiments after an immune challenge showed that domino mutants devoid of blood cells maintain a wild-type resistance to septic injury. This resistance was also observed in mutant larvae in which the synthesis of antibacterial peptides is impaired (immune deficiency larvae) and in mutants that are deficient for humoral melanization (Black cells larvae). However, if domino was combined with either the immune deficiency or the Black cell mutation, the resistance to septic injury was reduced severely. These results establish the relevance of the three immune reactions: phagocytosis, synthesis of antibacterial peptides, and melanization. By working in synergy, they provide Drosophila a highly effective defense against injury and/or infection.

Figure 1

Blood cells and fat body in domino larvae. Whole mounts of integument inner wall from wild-type (A) and domino (B and C) third-instar larvae. Groups of sessile plasmatocytes are frequently observed in wild-type larvae (A). However, in domino, a few abnormal hemocytes are occasionally seen (B), and microorganisms (arrow) accumulate in the hemocoele (C). Histochemical analysis showed the presence of toluidine and eosin. (For A–C, bars = 20 μm.) (D) Transmission electron micrograph of a plasmatocyte that has engulfed several bacteria (arrows). The experimental procedure was performed as described (21). N identifies the nucleus. (Bar = 1 μm.) Expression of antimicrobial reporter genes (E, diptericin-lacZ and F, drosomycin-green fluorescent protein) in a domino fat body lobe (in the absence of immune challenge). (Bar = 400 μm.)

Figure 2

Induction of genes encoding antimicrobial peptides in third-instar larvae of different genotypes. Total RNA was extracted from heterozygous (dom/+) or homozygous domino (dom/dom) larvae that were submitted to various treatments, and 20 μg samples were analyzed by Northern blotting. Blots were hybridized successively with the following random-primed cDNA probes: Dipt, diptericin; Drom, drosomycin; CecA, cecropin A1; Metch, metchnikowin, and rp49. c: control unchallenged larvae. ci: larvae collected 6 h after clean injury. Ec + Ml, Ec, Ml, Bb: larvae collected 6 h after pricking with a needle coated with E. coli and M. luteus (Ec + Ml), E. coli alone (Ec), M. luteus alone (Ml), or spores of B. bassiana strain 80.2 (Bb). Bb nat: larvae collected 24 h after natural infection by spores of B. bassiana. This experiment was repeated three times and yielded similar results.

Figure 3

Melanization and melanotic tumors in domino larvae. (A) Staining obtained 5 min after depositing a droplet of hemolymph from domino, Oregon^R (WT), and Bc larvae on a filter paper impregnated with l-3,4-dihydroxyphenylalanine, the substrate for phenoloxidase. Free-floating melanotic masses in Toll^10B/+ single (B) and dom/dom; Toll^10B/+ double mutants (C). In single mutants, the melanotic tumors are surrounded by layers of lamellocytes, as shown by the expression of the lacZ reporter of the l(3)03349 fly line (21), whereas no blood cells are associated in double mutants. (Bars = 40 μm.) (D) hop^Tum-l; dom/dom double-mutant larva exhibiting both the domino phenotype with black lymph glands (arrow) and a melanotic mass in the posterior region. (E) Natural infection of a domino larva by B. bassiana spores. On the left, the arrow indicates the lymph glands, and the larva on the right is an Oregon^R larva. Brown spots form at the penetration points of the fungi.

Figure 4

Survival of third-instar larvae of various genotypes to immune challenge. The survival rates (in percentages) of dom/+ (equivalent to wild-type rates, not shown), dom/dom (dom), Bc/Bc (Bc), imd/imd (imd), Bc, imd/Bc, imd (Bc,imd), Bc, dom/Bc, dom (Bc,dom), imd, dom/imd, dom (imd,dom) and Bc, imd, dom/Bc, imd, dom (Bc,imd,dom) larvae were scored over a 48-h period at 29°C after immune challenge and are presented with their confidence interval (P < 0.05). The larvae were either untreated (control), pricked with a clean needle (clean injury), or pricked with a needle coated with E. coli, M. luteus, or B. bassiana spores. For each experiment, 20–70 animals (M. luteus and B. bassiana) or 70–300 animals (control, clean injury, and E. coli) were used. Asterisks indicate 0% survival. For Bc, dom, and imd/dom as well as Bc, imd, dom combinations, two or three different recombinant chromosomes were tested, and all values were cumulated.