We have employed the proteomic approach in combination with mass spectrometry to study the immune response of honey bee workers at different developmental stages. Analysis of the hemolymph proteins of noninfected, mock-infected and immune-challenged individuals by polyacrylamide gel electrophoresis showed differences in the protein profiles. We present evidence that in vitro reared honey bee larvae respond with a prominent humoral reaction to aseptic and septic injury as documented by the transient synthesis of the three antimicrobial peptides (AMPs) hymenoptaecin, defensin1, and abaecin. In contrast, young adult worker bees react with a broader spectrum of immune reactions that include the activation of prophenoloxidase and humoral immune responses. At least seven proteins appeared consistently in the hemolymph of immune-challenged bees, three of which are identical to the AMPs induced also in larvae. The other four, i.e., phenoloxidase (PO), peptidoglycan recognition protein-S2, carboxylesterase (CE), and an Apis-specific protein not assigned to any function (HP30), are induced specifically in adult bees and, with the exception of PO, are not expressed after aseptic injury. Structural features of CE and HP30, such as classical leucine zipper motifs, together with their strong simultaneous induction upon challenge with bacteria suggest an important role of the two novel bee-specific immune proteins in response to microbial infections.

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SUMMARYDrones are haploid male individuals whose major social function in honey bee colonies is to produce sperm and mate with a queen. In spite of their limited tasks, the vitality of drones is of utmost importance for the next generation. The immune competence of drones -as compared to worker bees -is largely unexplored. Hence, we studied humoral and cellular immune reactions of in vitro reared drone larvae and adult drones of different age upon artificial bacterial infection. Haemolymph samples were collected after aseptic and septic injury and subsequently employed for (1) the identification of immune-responsive peptides and/or proteins by qualitative proteomic analyses in combination with mass spectrometry and (2) the detection of antimicrobial activity by inhibition-zone assays. Drone larvae and adult drones responded with a strong humoral immune reaction upon bacterial challenge, as validated by the expression of small antimicrobial peptides. Young adult drones exhibited a broader spectrum of defence reactions than drone larvae. Distinct polypeptides including peptidoglycan recognition protein-S2 and lysozyme 2 were upregulated in immunized adult drones. Moreover, a pronounced nodulation reaction was observed in young drones upon bacterial challenge. Prophenoloxidase zymogen is present at an almost constant level in non-infected adult drones throughout the entire lifespan. All observed immune reactions in drones were expressed in the absence of significant amounts of vitellogenin. We conclude that drones -like worker bees -have the potential to activate multiple elements of the innate immune response. Supplementary material available online at

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Ribonuclease P, the ubiquitous endonuclease required for generating mature tRNA 5' ends, is a ribonucleoprotein in most organisms and organelles, with the exception of mitochondria and chloroplasts of multicellular organisms. The cyanelle of the primitive alga Cyanophora paradoxa is the only photosynthetic organelle where the ribonucleoprotein nature of this enzyme has been functionally proven. tmRNA is another highly structured RNA: it can be aminoacylated with alanine, which is then incorporated into a tag peptide encoded on the same RNA molecule. This dual-function RNA has been found in bacteria, and its gene is also present in mitochondria and plastids from primitive organisms. Since nothing is known about the expression of this RNA in organelles, we have performed processing studies and determined the promoter of cyanelle pre-tmRNA. This RNA is transcribed as a precursor molecule in vivo. Synthetic transcripts of cyanelle pre-tmRNA, including or lacking the mature 3' CCA-end, are efficiently and correctly processed in vitro by bacterial RNase P ribo- and holoenzymes and by the homologous cyanelle RNase P. In addition to these experimental data, we propose a novel secondary structure model for this organellar tmRNA, which renders it more similar to its bacterial counterpart.