ELUCIDATING THE ROLE OF BETA DEFENSINS IN STRESS RESPONSE IN RAINBOW TROUT

Project Methods
Aim 1:1) Determine the bioactivity of trout beta defensins (omDBs) against pathogensFour trout beta defensin recombinant proteins GFP conjugated (pTurboGFP-N vector (Evrogen)) will be produced via electroporation using the Amaxa Nucleofector II transfection system (Lonza) in RTG-2 (Rainbow trout gonadal cell line) and the supernatant of each over-expressing cell lines (rec-omDB-RTG-2) will be tested against the fungi Saprolegnia parasitica and Mucor hiemalis, the protozoan parasite Ichthyophthirius multifiliis (Ich), the Myxozoan parasite Myxobolus cerebralis, the viral pathogens infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV). Assays will be performed in a 96-well plate using the supernatant from the rec-ptGFP-RTG-2 as negative control and cecropin A (Sigma) as a positive control. All pathogens have been kindly donated by Dr. I. Salinas, Dr. O. Sunyer and Dr. Cain. For fungal pathogens, antifungal activity will be measured using plate and broth growth assays. For Ich, 20 protomonts will be added into a 24-well plate 6 h before incubation with 5 different concentrations of rec-omDB-RTG-2 supernatant or the negative control. Protomonts viability will be determined after 22 h as number of theronts released into the well and visualized by microscopy (Å~40 magnification). Morphological changes will be also evaluated. For M. cerebralis we will assess the ability of the myxosporean spore to recognize and invade host cells. For that 10^6/ml spores will be incubated for 24 or 48h with the rec-omDB-RTG-2 or control. Cell viability will be tested by double staining with Hoechst33342 dye and propidium iodine staining via flow cytometry and to confirm the results, cells will be visualized with using the Nikon Ti fluorescent microscope (40X magnification). Cellular invasion will be visualized by microscopy after H&E staining, for each recombinant cell line, 10 images will be taken, and spores will be counted. For virus inhibition, we will quantify the number of plaques forming units (pfus) in the recomDB- RTG-2 cells. Six different dilutions of virus will be incubated in a plate containing a confluent layer of rec-omDB-RTG-2 cells and subsequently cover with carboxymethyl cellulose. If the virus infects and lyses the cells, it will create plaques that will be visualized by cresyl violet staining, under the microscope (bright field). The mean number of pfus per well will be quantified in each treatment using five technical replicates.2) Determine the bioactivity of trout beta defensins against trout microbiota.Skin-associated bacteria will be collected from control rainbow trout (N=6). Purified bacterial suspensions will be adjusted to an O.D of 0.01 and incubated them each beta defensin supernatant or the negative control. Growth will be measured in a plate reader every 3 h for 30 h.Aim2:1) Characterization of omDB expression patterns in different tissues and during development.Here, we will perform fluorescent in situ hybridization (FISH) in mucosal tissues as well as spleen, head-kidney and liver from healthy control trout at different developmental stages. This is critical since salmonids experience different types of stress during their life cycle. Salmonids, like other fish, are also more or less susceptible to different pathogens at different life stages. Thus, we will obtain 100 trout larvae at 120 DD (degree days) from Lisboa Spring Hatchery (Pecos, New Mexico) and then acclimatize them for a week at 16°C. After acclimation, 12 larvae will be sampled once a week, starting from 222 DD, for a total of 5 time points. Subsequently, sampling will occur every 2 weeks until they reach adulthood. A total of N=6 animals will be sampled per time point. Skin cryosections will be fixed and stained with omDB oligoprobes (Eurofins). We will perform single molecule FISH or multiplex-FISH in which each omDB will be labeled with a different fluorophore. Cell nuclei will be stained with DAPI. For each sample, cryosections will also be stained with hematoxylin and eosin to identify the morphology and structure of the tissue. Sections with be visualized using a Nikon Ti fluorescent microscope. Based on the cellular and nuclear morphology as well as they position within the epithelium, we will be able to assign whether defensins are expressed by epithelial cells, goblet cells or immune cells. Additionally, we will perform RT-qPCR to detect developmental changes in omDB gene expression in trout skin, liver, spleen and head-kidney at each of the time points.2) Determine the effects of chronic stress on skin omDB expression and microbiomeAll rainbow trout used for in vivo experiments will have a weight of 100 g and will be provided by Lisboa Springs (Pecos, New Mexico). For this experiment fifty ug cortisol/g body weight in coconut oil or other oil mixtures as vehicle will be delivered into the peritoneal cavity of rainbow trout. Once injected, the coconut oil/cortisol mixture solidifies allowing an extended slow release of cortisol into the circulation over time. Using this model, mRNA levels of all four defensins will be measured in skin, liver, spleen and head-kidney (N=6) using RT-qPCR at days 1, 5 and 30 days after cortisol delivery. Cortisol levels in plasma will be measured at each time point using RIA. We will also collect a piece of skin for DNA extraction in order to determine if chronic stress results in changes of the skin-associated bacterial community. Microbiome profiling will be done by next generation sequencing of the 16S rDNA V1-V3. Briefly, three independent PCRs will be performed using the 5PRIME hot start mix (Applied Biosystems) using 28F and 519R primers. PCR products of correct size will be merged and cleaned, followed by Illumina library preparation using Kapa HiFi mix (Roche). Libraries will be sequenced on an Illumina MiSeq platform available in Dr. D. Dinwiddie's lab at the Clinical Translational Science Center (University of New Mexico). We will also quantify total bacterial loads in the skin using qPCR. Cryoblocks will be prepared for localization of omDBs as well as bacteria localization (using the EUB338 probe) by FISH. Morphological changes in the skin will be evaluated using routine histology with H&E staining and PAS staining for goblet cells.3) Determine the effects of acute stress omDB expressionFor the transport stress experiment, we will use an N=10 pre-transport and post-transport in order to make sure we have enough statistical power for cortisol and glucose measurements. For the heat stress experiments, 10 control animals will be maintained at a temperature of 16°C, while 30 trout will be exposed to acute heat stress increasing in increments of 3°C per hour up to 25°C. Thus, a total of 40 rainbow trout will be divided into four groups using two tanks per condition and subjected to acute heat stress for 30 min, 1 h or 3 h, while 10 fish will be kept at regular tank temperature (16°C) as control. Cortisol and glucose levels in plasma and blood will be measured as described in Tacchi et al, 2015. We will collect skin, liver, spleen and head-kidney for RNA extraction to perform RT-qPCR. Additionally, another piece of the skin will be collected for DNA extraction and microbiome profiling. To evaluate changes in skin microbiome composition we will perform next generation sequencing of the 16S rDNA V1-V3. Additionally, total bacterial load will be determined via qPCR. Finally, a piece of skin tissue will be snap frozen in OCT and cryoblocks will be used for localization of omDBs by FISH as well as bacteria by FISH using the universal probe EUB338. Finally, a piece of skin will be fixed in formalin to perform routine histology with H&E staining and PAS staining for goblet cells.