Performing Department
(N/A)
Non Technical Summary
Ammonia can be extremely toxic to fish if accumulated in the body. This event accounts for one of the main causes of substantial fish losses in aquaculture practices. Elevated ammonia and iron, the two fundamental and recurrent stress conditions often present in catfish aquaculture system, can inhibit the elimination of toxic ammonia from the fish. Therefore, under these scenarios, to prevent productivity loss, ammonia must be excreted efficiently to avoid reaching lethal/sub-lethal levels. Developing strategies to facilitate ammonia elimination by the fish gills typically under stressful environments is a major challenge but presents an opportunity for UAPB to advance aquaculture sustainability. The proposed project is aimed at developing a multi-faceted and interdisciplinary research approach (from whole-organismal to transcriptome) to identify and explore a set of Rhesus glycoproteins as an imperative ammonia excretory pathway in catfish. In addition, various factors (e.g., feeding rations, water pH, dietary cortisol) that can potentially trigger the functionality of Rhesus glycoproteins will be evaluated as potential tools to mitigate hyperammonia toxicosis induce by high environmental ammonia and iron. Key indicators of growth, ammonia-homeostasis and physio-chemical performance associated with Rhesus glycoproteins modulation under testing conditions will be determined. Laboratory experimentations will also be complemented with large-scale outdoor -ponds to obtain better insights into what might be expected in a commercial farm setting. This proposal will improve and strengthen the research capacity of the aquaculture centre at UAPB, and outcomes will have positive impacts on aquaculture production as well as on the environment.
Animal Health Component
100%
Research Effort Categories
Basic
65%
Applied
35%
Developmental
(N/A)
Goals / Objectives
The overall goal is to explore the potential strategies to enhance the functionality of Rh glycoproteins as a protective mechanism against the toxicity induced by excessive ammonia accumulation in catfish aquacultureSpecific objectives of this project are to, 1. Identify and characterize Rh glycoproteins isoforms, and subcellular localization in the gills.2. Assess the triggering effects of various factors (feeding rations, water pH and dietary cortisol) on Rh glycoprotein isoforms expression, and the consequence on ammonia handling when catfish are challenged with elevated water ammonia and iron.3. Determine the integrated performance of fish at growth, physio-biochemical and cellular level under the scenario of differentially expressed Rh glycoproteins (defined in objective #2)4. Determine the practical implication of feeding rations, water pH and dietary cortisol to mitigate endogenous ammonia toxicity under natural conditions in aquaculture ponds.
Project Methods
Objective 1: Identification, characterization and localization of Rh glycoprotein isoforms Determining sequence of Rh glycoproteins isoforms: RNA will be extracted from the gills of acclimated fish using RNeasy 96 QIAcube HT Kit (Qiagen). RNA samples will be shipped on dry ice to Novogene (Sacramento, CA) for downstream molecular analyses. Directional RNA-sequencing libraries will be prepared for Illumina NextSeq sequencing.Phylogenetic analysis for Rh homology: Raw sequencing data will be aligned to a reference transcriptome for channel catfish using the program Bowtie. Tests for differential expression will be completed using the edgeR Bioconductor package. Transcriptome sequences will also be subjected to HMMER based homology techniques to derive Rh homologues in our experimental fish species, and phylogenetic analysis will be carried out by neighbour joining method for our own sequence(s) and NCBI retrieved gene sequences. Thereafter, gene specific primers will be designed from the obtained sequences using Primer3 software.Detection of specific Rh proteins isoform in the gill homogenate: Heterologous fish antibodies will be utilized in the immunohistochemistry staining and Western blot analysis.Localization of the Rh glycoproteins in the gill: In situ hybridization will be carried out to determine the cellular location of Rh glycoproteins in gill tissue. Immunohistochemistry will be performed to determine the subcellular localization of these proteins.Objective 2: Assess the triggering effects of various factors on Rh glycoprotein isoforms expression.WP 2.1: Determine the lethal dose-response curve for the experimental conditions: We will determine the LC50 (lethal concentration) for the ammonia and iron in a time-resolved manner (10 days and 21 days). Following a range finding test, five concentrations of ammonia and iron will be selected based on 'concentration-response slopes' to determine the respective 10 and 21-day LC50 values. Exposures will occur in 40-L glass aquaria. Each concentration will be tested in triplicate (each with 10 fish) and will include parallel respective control groups. Stock solutions of ammonia and iron will be prepared with analytical grade salt of NH4HCO3 and FeCl3, respectively. For all experiments, fish mortality will be recorded at specific intervals up to 10 and 21 days. The 10- and 21-day LC50 values will be calculated by the Probit Analysis testWP 2.2: To examine the potential ability of different factors to increase Rh glycoproteins expression under challenging scenarios, and corresponding consequences on facilitating ammonia excretion and alleviating ammonia accumulation toxicity Effect of different feeding ration: Fish will be fed with three different rations per day, 0.5% body weight (low ration, will be designated as 'control' group), 1.5 % body weight (medium ration) and 2.5 % body weight (high ration). These feeding groups will be subsequently exposed to sub-lethal doses (25% 10-day LC50, comparable conditions occurring in intensive indoor fish culture systems; FAO 1989; Helfrich and Libey, 1991) of ammonia as well as iron for up to 45 days.Impact of elevated water pH: Juvenile catfish will be reared in three experimental water pH - 7.2 (control), 8.0 and 8.5. These groups will be subsequently exposed to sub-lethal doses (25% 10-day LC50) of ammonia as well as iron for up to 45 days. Elevated water pH will be maintained by addition of 0.1 M KOH.Modulation via dietary supplementation of cortisol: Experimental diets will be prepared to contain 0 (Control), 50, or 250 µg cortisol/g of food. The experimental diets will be prepared by spraying cortisol (hydrocortisone; Sigma Chemicals, USA) dissolved in 100% ethanol onto the surface of the food pellets to acquire the mentioned concentrations in feed (Bernier et al., 2004). Control diet will also be sprayed with 100% ethanol to maintain the homogeneity with cortisol supplemented diets. Catfish will be hand-fed the experimental diets to apparent satiation twice daily. The effects of each of these experimental diets will be tested against sub-lethal doses (25% 10-day LC50) of ammonia and iron for 45 days.Experimental set up and Sampling procedure: Each experiment will be performed in triplicate (n=25 per tank) in 1000 L tanks. For each group, fish (n=3 per tank) will be sampled at intervals of 15, 30 and 45 days.Analytical techniques:Gene expression: mRNA expression of Rh glycoprotein isoforms in the gill will be quantified by qRT-PCR by using the primers designed in objective #1.Protein expression: Western blots will be performed for detecting protein expression levels of Rh glycoproteins isoforms using protein isolated from the gills and primary antibodies raised in objective #1.Ammonia excretion rates (µmol/g/h): will be calculated as ([Amm]i- [Amm]f) × V/(t × M); where [Amm]i and [Amm]f are the initial and final concentrations of ammonia in water obtained from comparison to a standard curve. V indicates volume (L), t time (h), and M mass (g).Ammonia accumulation: Ammonia concentrations in plasma and collected tissues will be quantified using an enzymatic ammonia assay kit.Objective 3: To assess the accompanied effect of modulated Rh glycoprotein expression on key indices of performances at growth, physiological, biochemical and cellular levelFor all experimental groups and each sampling described in objective #2, the following indices will be investigated to determine the fish performance:Growth: Fish will be bulked weighed to determine various indices of growth performance including weight gain (%), specific growth rate (SGR), feed conversion rate (FCR) and survival (%).Metabolic efficiency: Metabolic expenditure will be evaluated by measuring indicators of tissue energy stores, protein, glycogen and lipid in fish hepatic tissues.Stress hormone and ion homeostasis: Cortisol hormone will be quantified through a commercially available ELISA kit (Enzo Life Sciences, US). Ion homeostasis in plasma will be examined by measuring electrolyte (Na+ K+, Cl-, Ca2+ etc.) status by AAS.Oxidative injury and anti-oxidant defense status: a wide array of oxidative stress indicators and anti-oxidative enzymes will be assessed.Objective 4: Validate the application of experimental factors (feeding rations, water pH and cortisol supplement) to alleviate endogenous ammonia toxicity under natural conditions Following the proof-of-principle experiment in laboratory conditions, a more ambitious experiment will be conducted in natural conditions in outdoor 0.1-acre (404 m2) earthen experimental ponds located at the aquaculture research station at UAPB. Based on the objective #2 and #3, the feeding ration, water pH and dietary cortisol supplementation that elicits the best outcome (highest criterion will be based on up-regulated Rh glycoproteins, enhanced ammonia excretion, reduced endogenous ammonia load, and higher fish growth performance) will be selected for the pond trial. Each of these selected treatments will be paired with their respective controls, and the experiment will be conducted in duplicate. The experimental ponds will be stocked with 250 catfish juveniles. The concentration of ammonia/iron spike will be 10% of the calculated 21-day LC50 value. Fish will be fed ad libitum two times daily. Ammonia/iron exposure conditions will be regularly maintained throughout the experiment and water will be renewed (if needed) to avoid build-up of waste products. Following 60 days of experimentation, ammonia content (in plasma, liver, muscle and brain) as well as growth rate performance (weight gain (%), SGR, FCR) will be determined at 21, 45 and 60 day intervals, and will be compared with the respective controls.