Cell Tissue Res (2003) 312:197–202 DOI 10.1007/s00441-003-0723-3 REGULAR ARTICLE Harry M. Murray · Jeff W. Gallant · Susan E. Douglas Cellular localization of pleurocidin gene expression and synthesis in winter flounder gill using immunohistochemistry and in situ hybridization Received: 10 December 2002 / Accepted: 19 March 2003 / Published online: 17 April 2003  Springer-Verlag 2003 Abstract Pleurocidin is an antimicrobial peptide isolated from winter flounder and has been previously localized to mucous cells of the skin epidermis and the intestine. The present study was designed to determine the cell type involved in pleurocidin gene expression and protein synthesis in gills from the same species. Whole-mount in situ hybridization with a pleurocidin-specific RNA probe and whole-mount immunohistochemistry with an anti- pleurocidin antibody localized the expression of this gene and the synthesis of its corresponding protein in a population of cells primarily isolated to the non-lamellar portion of the gill filament. Histological techniques allowed the presumptive identification of these cells as eosinophilic granular cells. These observations suggest that pleurocidin is expressed in not one but two distinct populations of cells within the winter flounder, one being an important group of inflammatory cells, the eosinophil- ic granular cells. Keywords Immunohistochemistry · Pleurocidin · Gene expression · Gill · Winter flounder · Pseudopleuronectes americanus (Teleostei) Introduction Cationic antimicrobial peptides are small proteins that are becoming recognized as important players in the innate immune response of many species (Hancock and Scott 2000). The genes encoding these peptides are expressed in numerous tissue and cell types from a wide variety of different species including mammals, amphibians, insects, and fish (Hancock and Chapple 1999). In vertebrates, many of these peptides have been found to be associated with epithelial layers (Hancock and Scott 2000) or are synthesized and secreted by circulating and/or tissue granulocytes, i.e., leukocytes, neutrophils, and eosino- philic granular cells (Hancock and Chapple 1999; Levy 2000; Silphaduang and Noga 2001). The detection of the peptide pleurocidin in mucous cells of skin (Cole et al. 1997, 2000) and intestine (Cole et al. 2000) from winter flounder (Pseudopleuronectes americanus) has suggested that this protein might be important as an antimicrobial constituent of mucus found in association with specific epithelial cell layers. Our work with the reverse transcription/polymerase chain reaction technique indicates that the pleurocidin gene is also expressed in gill tissue from this species (unpub- lished); however, no information is available showing the specific cell types that could be involved. Since pleuro- cidin is known to be produced in other epithelial tissues, i.e., skin and intestine, this peptide might also be expressed in mucous cells distributed through the gill epithelium and co-secreted with mucus onto the epithelial surface, thus providing a barrier to microbial invasion at the interface of the delicate gill epithelium and the external environment. The purpose of the present study is to localize and identify the specific cell types involved in the synthesis of pleurocidin in winter flounder gill by using whole-mount in situ hybridization and immunohis- tochemistry. Materials and methods Tissue sampling All animals were maintained and sampled according to the guidelines set by the Canadian Council of Animal Care (Olfert et al. 1993). Juvenile winter flounder were held in tanks at the This project was partially funded by the National Research Council of Canada and AquaNet H. M. Murray AquaNet, Memorial University of Newfoundland, St. John’s, Newfoundland, A1C 5S7, Canada H. M. Murray ( ) ) · J. W. Gallant · S. E. Douglas Institute for Marine Biosciences, National Research Council, 1411 Oxford Street, Halifax, Nova Scotia, B3H 3Z1, Canada e-mail: Harry.Murray@nrc.ca Tel.: +1-902-4264319 Fax: +1-902-4269413