Immunolocalization of clavanins in Styela clava hemocytes

Antimicrobial peptides play an important role in innate host defenses against infection. Clavanins are histidine-rich, amidated, 23-residue alpha-helical antimicrobial peptides that were isolated from a mixed population of Styela clava hemocytes. To learn which types of hemocytes contained clavanins, we raised a polyclonal antibody that recognized five different clavanins, and used it to localize these peptides by light and electron microscopy. Clavanins were present in the cytoplasmic granules and/or cytoplasm of five different types of granulocytes and they also occurred throughout the cytoplasm of macrophages. The orange G component of Mallory's trichrome stain had a high affinity for clavanins, and for the cytoplasmic granules of S. clava's hemocytes. Semiquantitative analysis of acid urea-PAGE gels suggested that clavanins and styelins comprised between 10 and 20% of the total cellular protein of eosinophilic granulocytes. Orange G and the century-old trichrome stain may provide simple screening tools for identifying cells that contain large amounts of antimicrobial peptides in mixed hemocyte populations.