Performing Department
School of Agriculture, Fisheries and Human Science
Non Technical Summary
The catfish industry faces significant economic losses due to water-borne stressors, including elevated ammonia, hypoxic episodes, and iron load in aquaculture systems. These recurrent stress conditions pose a threat to the sustainable production of healthy aquatic food. To address this challenge, it is crucial to minimize and alleviate stress events in farmed organisms. In vertebrates including some fish, it is established that prior-exposure to stressors improves tolerance to subsequent threats by retaining the imprints of defensive strategies through the formation of 'stress-memory'. To our knowledge, potential consequences of early stress experiences remain unexplored in the aquaculture context. This project aims to fill this gap by investigating the innovative approach of 'developing stress memory' in catfish. The strategy involves artificially pre-exposing catfish to low levels of stressors, intending to mitigate the lethal and sub-lethal impacts of adverse conditions in culture systems. The project adopts an interdisciplinary approach, ranging from whole-organismal to transcriptome level responses, to evaluate the potential of 'stress memory' as a tool for enhancing the performance and production of catfish, a highly valued species in the US market. Laboratory experiments will be complemented by large-scale outdoor ponds to provide insights into real-world commercial farm settings. Project objectives address the area of advancing knowledge in water quality and sustainable agriculture. This endeavor aligns with the overarching goal of ensuring the productivity, sustainability, and viability of the catfish industry.
Animal Health Component
100%
Research Effort Categories
Basic
55%
Applied
45%
Developmental
(N/A)
Goals / Objectives
The overall goal is to establish a comprehensive strategy to improve the effectiveness and sustainability of management practices in catfish culture systems, specifically addressing issues arising from stressors. Our focus is on exploring the viability of boosting the growth and physiological resilience of fish facing severe or moderate stress conditions. We aim to achieve this enhancement by exposing the fish to mild stressors beforehand, laying the groundwork for enhanced stress tolerance.Specific objectives are to, 1. Investigate the stress memory or imprints in catfish for the experimental conditions of elevated ammonia level (hyperammonia), reduced oxygen content (hypoxia) and high iron in the water.2. Assess the potential effects of prior exposure to mild hyperammonia, hypoxia and high iron concentrations to prime catfish against the same future stress event via determining the growth, integrated physiological, metabolic and cellular response.3. Identify the genes and molecular pathways governing the coping ability of stress- primed fish by gene expression microarray.4. Determine the performance of hyperammonia, hypoxia as well as elevated iron-primed fish under natural conditions in aquaculture ponds.
Project Methods
Objective 1: Investigate stress memory or imprints in catfish for the experimental conditions of hyperammonia, high iron load and hypoxia.WP 1.1: Determine the lethal dose response curve for the experimental conditions: For catfish juveniles, we will determine the LC50 (lethal concentration) for the ammonia, hypoxia and iron in a time-resolved manner (10 days and 21 days).Experimental design and Bioassay LC50: Following a range finding test, five concentrations of ammonia, iron and hypoxia will be selected based on 'concentration-response slopes' to determine the respective 10 and 21-day LC50 values, by the Probit Analysis test.WP 1.2: Assessment of stress memory phenomena in response to the experimental conditions, and dose optimization for the induction of stress avoidance imprintsCatfish juveniles will be pre-exposed to mild doses of ammonia, iron or hypoxia in separate circular tanks (2000-L, in triplicate, with each tank containing 75 fish) for a period of 21 days, which is the pre-exposure timespan widely used in priming studies. The pre-exposure testing dose for each of these experimental factors will equal 5% and 10% of the respective calculated 10-day LC50 values. Following 21 days of pre-exposure, fish will undergo a 7-day recovery phase by placing them respectively in a separate series of 2000-L tanks filled with normal water (ammonia, iron or oxygen level equivalent to the parallel control tanks). To assess the occurrence of stress memory, each of the recovered fish groups and parallel naïve (control) groups will be subsequently exposed for 21 days to respective lethal (100% 10-day LC50) and sub-lethal doses (25% 10-day LC50, comparable conditions occurring in intensive indoor fish culture systems) in 160-L glass aquaria (in triplicate, 10 fish in each aquaria).Objective 2: Assess the potential effect of prior mild hyperammonia, iron exposure as well as hypoxia to protect catfish against future stress eventsExperimental design and sampling: based on the outcome of WP#1.2, the pre-exposure dose (5% or 10% 10-day LC50) for each treatment eliciting the best survival and growth performance will be selected for pre-exposing the fish in this experiment, and will be continued for 21 days (same as WP#1.2). Thereafter, pre-exposed and parallel control fish will be exposed to sub-lethal doses (25% 10-day LC50) of each treatment in 2000-L tanks (in triplicate) for up to 45 days. For each experimental group, fish will be sampled at intervals of 15, 30 and 45 days. A total of 9 fish will be randomly collected at each sampling point (15, 30 and 45 days) from three replicates (n=3 per tank) of each experimental group.Fish will be euthanized with an overdose of neutralized MS-222 and weighed. Subsequently, a blood sample will be collected using a heparinized syringe. Brain, gills and liver will be dissected and stored at −80°C for later physio-biochemical analysis. In addition, aliquots of brain tissue will be added to RNAlater and stored at -20°C for molecular analysis (objective #3).Analytical techniques: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: will be evaluated by measuring protein, glycogen and lipid content in fish hepatic tissues.Ammonia and iron bioaccumulation: Body burden of ammonia and iron in plasma, liver, gills and brain will be quantified using an enzymatic ammonia assay kit and ICP-OES, respectively.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+, Mg2+, Ca2+, Mn2+ etc.) status by ICP-OES.Oxidative injury and anti-oxidant defense status: we will determine the activity dynamics of a wide array of anti-oxidative enzymes and indices of oxidative stress.Objective 3: To identify genes and regulatory pathways underpinning the acclimation process in primed catfishTo get mechanistic insight into the regulatory mechanisms and identify genes involved in the acclimation process, we will perform microarray analysis that allows simultaneous investigation of expression levels of thousands of genes at once. Transcriptomic analysis using a channel catfish custom oligonucleotide microarray will be performed on the brain tissue (as it relates to memory imprints) obtained during objective #2. Samples for microarray analysis for each treatment (ammonia, iron and hypoxia) will be selected from the exposure time point (15, 30, or 45 days) at which the significant response occurred. The corresponding group that was not pre-exposed will be used for the comparative analysis.RNA isolation and quality control: Procedures will be performed per our previous protocol (Sinha et al., 2013, 2016).Microarray hybridization, data acquisition and analysis: Microarray experiments will be performed using a custom-designed, microarray platform with 4 × 44 K probes per slide. Four pools of RNA (from the 9 fish sampled in objective #2) will be produced. The microarray hybridization will be performed using a reference design, using a reference RNA sample, which is comprised of an equimolar mix of RNA extracted from all individual fish brain samples. Each experimental sample (labeled with Cy3TM) will be hybridized against this reference sample (labeled with Cy5TM) in a 2-color experiment. mRNA amplification, labeling and hybridization will be performed.Analysis of gene ontology: Gene ontology (GO) enrichment analysis for biological processes will be performed on all cDNA features that had GO identifiers associated using the Gene Ontology Enrichment Analysis Software Toolkit (GOEAST) program.Pathway analysis: Selected mRNA probes differentially expressed by pre-exposure and naïve groups will be used as inputs in the GeneMania pathway analysis tool (Montojo et al., 2010).Objective 4: Determine the performance of catfish pre-exposed to experimental scenarios (ammonia, iron and oxygen depletion) under natural conditions Following the proof-of-principle experiment in laboratory conditions, a more ambitious experiment will be conducted in natural conditions in outdoor 0.05-acre (202 m2) earthen experimental ponds.Exposure protocol: catfish juveniles will be pre-exposed to experimental treatments (ammonia, iron or oxygen depletion) for 21 days with the same dose used for objective #2. Similar to earlier experiments, pre-exposure to the stressors will be conducted in triplicate in indoor 2000-L tanks (75 fish/tank). Thereafter, pre-exposed and control fish will be stocked in ponds and immediately exposed to stressors at a concentration of 10% of the calculated 21-day LD50 value (comparable to conditions on aquaculture farms) for each of the treatments up to 60 days. Pre-exposed fish will be paired with a naïve (control) group (without pre-exposure) and the experiment will be conducted in duplicate. Each experimental pond will be stocked with 150 catfish juveniles. Fish will be fed ad libitum two times daily. Ammonia, iron and hypoxia exposure conditions will be regularly maintained throughout the experiment. Following 60 days of experimentation growth rate performance (weight gain (%), SGR, FCR) will be determined at 21, 45 and 60 day intervals, and will be compared with the respective non-pre-exposed (control) group.