1. ESTABLISHMENT OF A STABLE CULTURE OF FISH ALARM CELLS HEATHER HINTZ AND WINNIFRED BRYANT, PH.D. DEPARTMENT OF BIOLOGY UNIVERSITY OF WISCONSIN-EAU CLAIRE Introduction The goal of this project was to establish a primary culture of alarm cells from the skin of Creek Chub (Semtilus atromaculatis). The skin (epithelium) is composed of different cell types. Included among them are cells that produce alarm substance. This compound is released following tissue damage, such as would occur during a predation event (Smith 1992, Canadian Journal of Zoology, 70(8): 1473-1476). When the alarm substance enters the water, anti- predatory behaviors are initiated by neighboring fish (e.g., darting, freezing, schooling). Alarm substance has not been isolated and commercial preparations are not available. Establishing a culture of fish epithelial cells will provide an immediate source of alarm substance that can be used in behavioral experiments with Creek Chub. To generate a viable culture of cells, we developed protocols for tissue collection and processing, and determined optimal media formulations and culture conditions. Developed Methods Anesthesia: Each fish was anesthetized by adding clove oil to the water for 2-3 minutes to induce a light plane of anesthesia to facilitate collection of cells. Upon being placed back in the water, the animal was immediately able to swim. Tissue Collection: The skin of the fish was lightly abraded with a scalpel to obtain epithelial cells. The cells were then placed in a conical vial containing phosphate-buffered saline (PBS) (minus Ca 2+ Mg 2+,ThermoScientific) supplemented with 1% penicillin/streptomycin (P/S, Gibco) and 1% Fungizone ® (Gibco). The cells were washed twice with buffer, centrifuging at room temperature for 5 minutes at 3000 rpm between each wash. Subsequent manipulations were performed under aseptic conditions. Media: After the final wash the pellet was added to 3 ml of L-15 Leibovitz culture medium supplemented with 20% fetal bovine serum (growth factor) and containing 1% P/S and 1% Fungizone ®. Physiological pH of Leibovitz L-15 is maintained through the salt, basic amino acid and galactose concentrations. In addition, Leibovitz L-15 is a CO 2 independent media so, unlike many cells cultures, ours did not require any additional supplementation of O 2 /CO 2. Cells were cultured in vented T25 culture flasks (Corning) at 20 o C (room temperature) on the benchtop. Bacterial contamination was an issue in some trials. P/S is usually effective at preventing bacterial contamination in most cell culture systems. Consequently, we isolated bacteria from our culture and screened them for antibiotic sensitivity. It was observed that the bacterial colonies could not grow on culture plates containing with tetracycline and kanomycin. Therefore we replaced P/S in our buffers and media with 1% tetracycline and 1% kanomycin. Fungal (yeast) contamination was also observed in some trials. To address this, we increased the concentration of Fungizone® to 1.5% in our buffers and media. Under these conditions we were able to maintain a stable cell culture for up to three weeks without contamination. Our current media formulation is: Leibovitz L-15 culture medium 20% fetal bovine serum 1% tetracycline 1% kanomycin 1.5% Fungizone ® Maintenance: The cells in our culture are not adherent. To maintain our suspension culture, every 48 hours cells and media were removed from the flask and placed in a conical vial. They were centrifuged at a 3000 rpm for 5 minutes and the supernatant was removed. The pellet was washed 3 times in PBS, centrifuging at 3000 rpm for 5 minutes at room temperature. After the final wash, the pellet was resuspended in culture media. Imaging: We monitored our culture daily using a phase contrast microscope fitted with a Moticam digital camera. Determination of Alarm Cell Viability: 3 ml of culture was removed from culture flask and centrifuged at 3000 rpm for 5 minutes. 1 ml of the supernatant was added to an aquarium containing Creek Chub. The fish exhibited anti-predatory behavior (data not shown). This indicates that cultured alarm cells are secreting alarm substance into the culture media and that the alarm substance is biologically active. Figure 2. Section of Creek Chub epithelium stained with haematoxylin-eosin and periodic acid-Schiff to indicate mucous cells (indicated by an asterisk) and alarm cells (indicated by an arrow). The tissue contains a mixed population of epithelial cells (Goulart and Young, 2013). Figure 1. Creek Chub (Semtilus atromaculatis) is the model organism for our study. Creek Chub are freshwater fish found in the Eastern US and Canada. Photo credit: http://gallery.nanfa.org/d/14373- 4/Semotilus+atromaculatus++Creek+Chub+2000.jpg Results Figure 3a. Suspension culture of fish epithelial cells. Alarm cells are indicated by arrow. Fibroblasts and mucus cells are also visible. Figure 3b. Alarm cells dividing on the fifth day of culture. Alarm cells comprise 10-20% of epithelial cells in our culture. Figure 3c. Alarm cells on second day of culture. Cells are approximately 20  m with a granular cytoplasm. Figure 3d. Alarm cells on fourth day of culture. Figure 3f. Alarm cells in second week of culture. Figure 3g. Alarm cells in the third week of culture. Cells are beginning to involute and appear apoptotic. Acknowledgements We would like to thank the Office of Research and Sponsored Programs for funding this project, Dr. Sasha Showsh for antibiotic screening of the cells, and Dr. David Lonzarich for technical assistance. Conclusions We successfully established and maintained a primary culture of alarm cells. This culture of cells is non-adherent and viable for approximately 3 weeks. We developed minimally invasive methods for tissue collection and optimized culture conditions for cells from organisms living in coldwater environments. There are no other reports in the literature of a fish epithelial cell culture containing alarm cells. Our method would allow further characterization of alarm cells and could be used as a source of alarm substance for use in bioassays. Future studies will focus on developing an enriched or purified culture of fish alarm cells and isolation/ chemical analysis of alarm substance.