Fate of Ecc-15 bacteria in the larvae. The localization of Ecc-15 within larvae was determined by using a strain of Ecc-15 (Ecc-15-GFP) that expresses the GFP reporter gene. (A) In a larva naturally infected by Ecc-15-GFP, bacteria are clearly present in the pharynx and anterior midgut. (B) In a larva infected by Ecc-15-GFP, bacteria are present in the anterior spiracles. (C) Hemolymph collected from larvae infected for 16 h with Ecc-15 shows GFP-marked bacteria phagocytosed by hemocytes. P, pharynx; AM, anterior midgut.

Ecc-15 infects larvae through the digestive tract. dipt-lacZ larvae that were sealed at the mouth by ligaturing with a strand of human hair were naturally infected by Ecc-15 for 12 h. Each bar represents the level of β-galactosidase activity measured in one larva. Means and sd are indicated above. Ligatured larvae generally failed to express dipt-lacZ after exposure to Ecc-15, indicating that the digestive tract is the major route of infection by Ecc-15. Ligatured larvae still have an wild-type immune response after Ecc-15 injection, indicating that the ligature blocks only the route of infection and does not alter host defense response. Control, untreated larvae; Ecc-15, larvae infected by Ecc-15; ligatured Ecc-15, ligatured larvae infected by Ecc-15; ligatured injected, ligatured larvae collected 8 h after Ecc-15 injection.

Ecc-15 persists in developing larvae and pupae. Bacterial persistence was measured in wild-type (Or^R) and imd/imd mutant lines by plating appropriate dilutions of homogenates of five surface-sterilized larvae or pupae that were naturally infected with rifampicin-resistant strains of Ecc-15 (Ecc-15 rif^R) and Ecc-2046 (Ecc-2046 rif^R) and collected at different times after infection. Bacterial counts were obtained by plating the larval and pupal homogenates on LB medium containing rifampicin (100 μg/ml; ref. 14). The number of colony-forming units (cfu) per larva obtained at each time point after infection represents the mean of three independent measurements. L3, third instar larvae; P, pupae.

diptericin induction after natural infection by Ecc-15 requires hemocytes. (A) Quantitative measurements of β-galactosidase activity performed on five larvae collected 24 h after natural infection or bacterial injection by Ecc-15. Experiments were performed as described (3) on larvae carrying the dipt-lacZ reporter gene in either wild-type (WT) or l(3)hem/l(3)hem mutant backgrounds. C, control; NI, larvae incubated with Ecc-15 (natural infection); BI, third instar larvae injected with Ecc-15 (bacterial injection). Each bar represents an independent measurement. Means and sd are indicated above. (B) Histochemical staining of β-galactosidase activity is shown in the fat body of l(3)hem homozygous larvae carrying either the dipt-lacZ or drom-lacZ reporter genes. In contrast to injection, natural Ecc-15 infection failed to induce a strong expression of the dipt-lacZ reporter gene in the l(3)hem fat body. Larval fat bodies were isolated 24 h after infection and stained (5-bromo-4-chloro-3-indolyl β-d-galactoside) as described (11). C, unchallenged control larvae; Ecc-15 NI, natural infection by Ecc-15; Ecc-15 BI, bacterial injection of Ecc-15.

Induction of antimicrobial peptide genes after Ecc-15 infection. (A) Viable Ecc-15 is required to induce a larval immune response. Quantitative measurements of β-galactosidase activity in larvae carrying either the dipt-lacZ (Left) or the drom-lacZ (Right) reporter genes were performed on five larvae collected 24 h after various treatments. Each bar represents an independent measurement; mean and SD are indicated above. C, control larvae incubated in banana alone; 15 and 2046, larvae naturally infected with Ecc-15 and Ecc-2046, respectively; 15-HK, heat-inactivated Ecc-15 (40 min at 70°C); 15-BI, larvae injected with Ecc-15 (bacterial injection). (B) Northern blot analysis shows that Ecc-15 infection induces sustained diptericin expression in larvae and pupae collected at different time intervals after natural infection by Ecc-15. L3, third instar larvae; P, pupae; C, control. (C) Northern blot analysis shows that Ecc-15 infection of larvae induces the expression of all of the antimicrobial genes. Total RNA was collected 24 h after infection, and the blot was hybridized successively with the following cDNA probes: diptericin (dipt), cecropin A1 (cec A), attacin (att), drosomycin (drom), and ribosomal protein 49 (rp49) as an internal control. C, control larvae incubated with only banana; NI, larvae incubated with Ecc-15 (natural infection); BI, third instar larvae injected with Ecc-15 (bacterial injection). (D) Ecc-15 induces a systemic immune response. Histochemical staining of β-galactosidase activity is shown in the fat body of wild-type (Or^R) and imd homozygous larvae that were collected 24 h after infection and that carry the dipt-lacZ, cec-lacZ, or drom-lacZ reporter genes. Ecc-15 infection induces high expression of all three reporter genes in fat bodies from wild-type larvae. The absence of β-galactosidase activity in fat bodies isolated from infected imd larvae carrying the dipt-lacZ and cec-lacZ genes indicates that the induction of cec-lacZ and dipt-lacZ expression by Ecc-15 requires the imd gene product.