Hepatic transcriptome profiling of largemouth bass (Micropterus salmoides Lacépède) injected with Flavobacterium covae or lipopolysaccharide.

Flavobacterium covae (columnaris) is the most detrimental bacterial disease affecting the largemouth bass (Micropterus salmoides Lacépède) aquaculture industry. In the current study, fish received an intraperitoneal injection of either 1× PBS (100 µL), LPS in PBS (100 µL, 10 µg/mL), or F. covae (100 µL, 2.85 × 1011 CFU/mL) to simulate immunological challenges. After 24 h post-injection, liver tissue from the control and treated groups were then collected for transcriptome analysis. Results of the Gene Ontology (GO) and KEGG pathway analyses for the F. covae and LPS-injected groups found differentially expressed genes (DEGs) enriched primarily in toll-like receptors (TLRs), cytokine-cytokine receptors, complement and coagulation cascades, and the PPAR signalling pathways. This suggests that the liver immune system is enhanced by these five combined pathways. Additionally, the DEGs TLR5, MYD88, and IL-1 were significantly upregulated in F. covae and LPS-injected fish compared to the controls, whereas IL-8 was downregulated. The upregulation of TLR5 was unexpected as F. covae lacks flagellin, the protein that binds to TLR5. Additionally, it is unknown whether the TLR5 is upregulated by LPS. Further research into the upregulation of TLR5 is warranted. These results provide insight into immune responses and associated pathways contributing to the immune system in the liver during columnaris infection and induced response to LPS in largemouth bass.

Coinfection of channel catfish (Ictalurus punctatus) with virulent Aeromonas hydrophila and Flavobacterium covae exacerbates mortality.

Flavobacterium covae and virulent Aeromonas hydrophila are prevalent bacterial pathogens within the US catfish industry that can cause high mortality in production ponds. An assessment of in vivo bacterial coinfection with virulent A. hydrophila (ML09-119) and F. covae (ALG-00-530) was conducted in juvenile channel catfish (Ictalurus punctatus). Catfish were divided into seven treatments: (1) mock control; (2) and (3) high and low doses of virulent A. hydrophila; (4) and (5) high and low doses of F. covae; (6) and (7) simultaneous challenge with high and low doses of virulent A. hydrophila and F. covae. In addition to the mortality assessment, anterior kidney and spleen were collected to evaluate immune gene expression, as well as quantify bacterial load by qPCR. At 96 h post-challenge (hpc), the high dose of virulent A. hydrophila infection (immersed in 2.3 × 107 CFU mL-1 ) resulted in cumulative percent mortality (CPM) of 28.3 ± 9.5%, while the high dose of F. covae (immersed in 5.2 × 106 CFU mL-1 ) yielded CPM of 23.3 ± 12.9%. When these pathogens were delivered in combination, CPM significantly increased for both the high- (98.3 ± 1.36%) and low-dose combinations (76.7 ± 17.05%) (p < .001). Lysozyme activity was found to be different at 24 and 48 hpc, with the high-dose vAh group demonstrating greater levels than unexposed control fish at each time point. Three proinflammatory cytokines (tnfα, il8, il1b) demonstrated increased expression levels at 48 hpc. These results demonstrate the additive effects on mortality when these two pathogens are combined. The synthesis of these mortality and health metrics advances our understanding of coinfections of these two important catfish pathogens and will aid fish health diagnosticians and channel catfish producers in developing therapeutants and prevention methods to control bacterial coinfections.

Flavobacterium covae is the predominant species of columnaris-causing bacteria impacting the Channel Catfish industry in the southeastern United States.

OBJECTIVE: Columnaris disease is a leading cause of disease-related losses in the catfish industry of the southeastern United States. The term "columnaris-causing bacteria" (CCB) has been coined in reference to the four described species that cause columnaris disease: Flavobacterium columnare, F. covae, F. davisii, and F. oreochromis. Historically, F. columnare, F. covae, and F. davisii have been isolated from columnaris disease cases in the catfish industry; however, there is a lack of knowledge of which CCB species are most prevalent in farm-raised catfish. The current research objectives were to (1) sample columnaris disease cases from the U.S. catfish industry and identify the species of CCB involved and (2) determine the virulence of the four CCB species in Channel Catfish Ictalurus punctatus in controlled laboratory challenges. METHODS: Bacterial isolates or swabs of external lesions from catfish were collected from 259 columnaris disease cases in Mississippi and Alabama during 2015-2019. The DNA extracted from the samples was analyzed using a CCB-specific multiplex polymerase chain reaction to identify the CCB present in each diagnostic case. Channel Catfish were challenged by immersion with isolates belonging to each CCB species to determine virulence at ~28°C and 20°C. RESULT: Flavobacterium covae was identified as the predominant CCB species impacting the U.S. catfish industry, as it was present in 94.2% (n = 244) of diagnostic case submissions. Challenge experiments demonstrated that F. covae and F. oreochromis were highly virulent to Channel Catfish, with most isolates resulting in near 100% mortality. In contrast, F. columnare and F. davisii were less virulent, with most isolates resulting in less than 40% mortality. CONCLUSION: Collectively, these results demonstrate that F. covae is the predominant CCB in the U.S. catfish industry, and research aimed at developing new control and prevention strategies should target this bacterial species. The methods described herein can be used to continue monitoring the prevalence of CCB in the catfish industry and can be easily applied to other industries to identify which Flavobacterium species have the greatest impact.

NEW GENUS AND SPECIES OF CYCLOCOELIDAE STOSSICH, 1902 (PLATYHELMINTHES: DIGENEA) INFECTING THE NASOPHARYNGEAL CAVITY OF CANADA GOOSE, BRANTA CANADENSIS (ANSERIFORMES: ANATIDAE) FROM WESTERN ALABAMA.

While surveying the parasites of birds associated with western Alabama aquaculture ponds, we collected several specimens of Anativermis normdroneni n. gen., n. sp. (Digenea: Cyclocoelidae) from the nasopharyngeal cavity of a Canada goose, Branta canadensis (Linnaeus, 1758) (Anseriformes: Anatidae). These flukes were heat killed and fixed in neutral buffered formalin for morphology or preserved in 95% ethanol for DNA extraction. Anativermis resembles Morishitium (Witenberg, 1928) by having testes that are spheroid with smooth margins and located in the posterior quarter of the body, an anterior testis that is lateral to the midline and abuts the respective cecum, a posterior testis that is medial (testes diagonal) and abuts the cyclocoel, a genital pore that is immediately postpharyngeal, and a vitellarium that is discontinuous posteriorly. The new genus differs from Morishitium and is unique among all other cyclocoelid genera by having the combination of a body that is broadest in the anterior body half, a posterior body end that is more sharply tapered than the anterior body end, an ovary that nearly abuts the posterior testis, a vitellarium that is asymmetrical and distributes from the area immediately posterior to the cecal bifurcation posteriad to approximately the level of the ovary, and uterine loops extending dorsolateral to the ceca and filling the space between the ceca and the respective body margin for nearly the entire body length. The new genus was recovered as a distinct lineage in separate 28S, 18S, and ITS2 phylogenetic analyses. This is the first report of a cyclocoelid infecting the Canada goose and of a cyclocoelid from Alabama.

Investigating the Ability of Edwardsiella ictaluri and Flavobacterium covae to Persist within Commercial Catfish Pond Sediments under Laboratory Conditions.

Two prevalent bacterial diseases in catfish aquaculture are enteric septicemia of catfish and columnaris disease caused by Edwardsiella ictaluri and Flavobacterium covae, respectively. Chronic and recurring outbreaks of these bacterial pathogens result in significant economic losses for producers annually. Determining if these pathogens can persist within sediments of commercial ponds is paramount. Experimental persistence trials (PT) were conducted to evaluate the persistence of E. ictaluri and F. covae in pond sediments. Twelve test chambers containing 120 g of sterilized sediment from four commercial catfish ponds were inoculated with either E. ictaluri (S97-773) or F. covae (ALG-00-530) and filled with 8 L of disinfected water. At 1, 2, 4-, 6-, 8-, and 15-days post-inoculation, 1 g of sediment was removed, and colony-forming units (CFU) were enumerated on selective media using 6 × 6 drop plate methods. E. ictaluri population peaked on Day 3 at 6.4 ± 0.5 log10 CFU g-1. Correlation analysis revealed no correlation between the sediment physicochemical parameters and E. ictaluri log10 CFU g-1. However, no viable F. covae colonies were recovered after two PT attempts. Future studies to improve understanding of E. ictaluri pathogenesis and persistence, and potential F. covae persistence in pond bottom sediments are needed.

The Use of Kaolin as a Prophylactic Treatment to Prevent Columnaris Disease (Flavobacterium covae) in Commercial Baitfish and Sportfish Species.

Aquaculture farms in Arkansas, USA routinely battle columnaris disease caused by Flavobacterium covae. Columnaris is prevalent during stressful events such as feed training and when fish are stocked at high densities in holding vats before sale. Kaolin clay was effective in laboratory trials as a treatment for columnaris in catfish. As a result, fish farmers are interested in applying kaolin products but were hesitant as they feared that the high doses of kaolin clay in vats might negatively affect the gills and overall health of fish. Therefore, we evaluated potential clay concentrations that might be used to prophylactically treat fish in vats. The effects of low to excessively high doses (0, 1, 2, 4, or 8 g/L) of kaolin clay (AkuaProTM, Imerys, GA, USA) were evaluated using a 72 h bioassay conducted in static tanks using Micropterus salmoides, Pomoxis nigromaculatus, Lepomis macrochirus, Ictalurus punctatus, Notemigonus crysoleucas, and Pimephales promelas. Results of these trials revealed a 100% survival rate across all six fish species exposed to kaolin clay at concentrations of up to 8 g/L for 48 h (followed by a 24 h recovery period in clean water) with no adverse effects to eyes, skin, gastrointestinal tract, or liver histology noted at any treatment. In addition, Micropterus salmoides analyzed for heavy metals due to exposure to the clay indicated that concentrations did not differ from control fish.

Investigation of dietary exogenous protease and humic substance on growth, disease resistance to Flavobacterium covae and immune responses in juvenile channel catfish (Ictalurus punctatus).

Many bacterial pathogens impact the US catfish industry, and disease control can be challenging for producers. Columnaris disease in channel catfish, Ictalurus punctatus, is primarily caused by Flavobacterium covae (formerly F. columnare). Immunostimulants may enhance nonspecific immune responses and offer an alternative to antibiotic treatments in catfish. Furthermore, dietary protein sources and inclusions are also essential to fish health and nutrition and may enhance overall fish performance in pond culture. The current project evaluated two immunostimulants: a protease complex (PC) and a humic substance (HS) derived from a reed-sedge peat product. A 60-day trial examined the effects of five diets on growth performance, immune response and resistance to experimental F. covae infection in channel catfish. Diets included a high-quality fishmeal diet (32%; CF32), a high-protein soy-based diet (32%; C32), a low-protein soy-based diet (28%; C28; predominately used in industry), a low-protein soy diet supplemented with C28 + PC at 175 g metric ton-1 and C28 + HS in a low-protein diet at 23 g metric ton-1 . Following feeding for 60 d, juvenile channel catfish were sampled for growth performance and baseline health indicators (n = 3; body mucus, blood for sera, kidney and spleen). A subset of fish was then subjected to an immersion-based in vivo challenge trial with F. covae (ALG-00-530; 106 CFU mL-1 exposure). At 60d post-initiation, there were no dietary differences in the relative growth rate (p = .063) or thermal growth coefficient (p = .055), but the 32% diets generally appeared to perform best. Post-challenge, the C32 group's mortality was higher than the C28 + PC (p = .006) and C28 + HS diets (p = .005). Although not significant, the C28 and CF32 groups also demonstrated higher mortality compared to both PC and HS diets. Sera lysozyme concentration was found to increase following pathogen challenge (p < .001) and in comparison with mock-challenged catfish (p < .001). Elevated expression levels of proinflammatory cytokines (il-1ß, il-8, tnf-α and tgf-ß) were observed at trial midpoint and post-infection when compared to 60d. The C28 treatment was found to have lower tnf-α expression than the C28 + PC (p = .042) and C28 + HS (p = .042) groups following exposure to F. covae. These challenge data suggest that the immunostimulants (PC and HS) in plant-based protein may be beneficial in protecting against F. covae when offered in low-protein channel catfish diets.

The Infection Dynamics of Experimental Edwardsiella ictaluri and Flavobacterium covae Coinfection in Channel Catfish (Ictalurus punctatus).

Edwardsiella ictaluri and Flavobacterium covae are pervasive bacterial pathogens associated with significant losses in catfish aquaculture. Bacterial coinfections have the potential to increase outbreak severity and can worsen on-farm mortality. A preliminary assessment of in vivo bacterial coinfection with E. ictaluri (S97-773) and F. covae (ALG-00-530) was conducted using juvenile channel catfish (Ictalurus punctatus). Catfish were divided into five treatment groups: (1) mock control; (2) E. ictaluri full dose (immersion; 5.4 × 105 CFU mL-1); (3) F. covae full dose (immersion; 3.6 × 106 CFU mL-1); (4) E. ictaluri half dose (immersion; 2.7 × 105 CFU mL-1) followed by half dose F. covae (immersion; 1.8 × 106 CFU mL-1); and (5) F. covae half dose followed by half dose E. ictaluri. In the coinfection challenges, the second inoculum was delivered 48 h after the initial exposure. At 21 days post-challenge (DPC), the single dose E. ictaluri infection yielded a cumulative percent mortality (CPM) of 90.0 ± 4.1%, compared with 13.3 ± 5.9% in the F. covae group. Mortality patterns in coinfection challenges mimicked the single dose E. ictaluri challenge, with CPM of 93.3 ± 5.4% for fish initially challenged with E. ictaluri followed by F. covae, and 93.3 ± 2.7% for fish exposed to F. covae and subsequently challenged with E. ictaluri. Despite similarities in the final CPM within the coinfection groups, the onset of peak mortality was delayed in fish exposed to F. covae first but was congruent with mortality trends in the E. ictaluri challenge. Catfish exposed to E. ictaluri in both the single and coinfected treatments displayed increased serum lysozyme activity at 4-DPC (p < 0.001). Three pro-inflammatory cytokines (il8, tnfα, il1ß) were evaluated for gene expression, revealing an increase in expression at 7-DPC in all E. ictaluri exposed treatments (p < 0.05). These data enhance our understanding of the dynamics of E. ictaluri and F. covae coinfections in US farm-raised catfish.

Persistence of a Wild-Type Virulent Aeromonas hydrophila Isolate in Pond Sediments from Commercial Catfish Ponds: A Laboratory Study.

Virulent Aeromonas hydrophila (vAh) is a major bacterial pathogen in the U.S. catfish industry and is responsible for large-scale losses within commercial ponds. Administering antibiotic feeds can effectively treat vAh infections, but it is imperative to discern new approaches and better understand the mechanics of infection for this bacterium. As such, the persistence of vAh in pond sediments was determined by conducting laboratory trials using sediment from four commercial catfish ponds. Twelve chambers contained sterilized sediment, vAh isolate ML-09-119, and 8 L of water maintained at 28 °C and were aerated daily. At 1, 2, 4, 6, and 8 days, and every 7th day post-inoculation for 28 days, 1 g of sediment was removed, and vAh colony forming units (CFU) were enumerated on ampicillin dextrin agar. Viable vAh colonies were present in all sediments at all sampling periods. The vAh growth curve peaked (1.33 ± 0.26 × 109 CFU g-1) at 96 h post-inoculation. The population plateaued between days 14 and 28. No correlations were found between CFU g-1 and physiochemical sediment variables. This study validated the ability of vAh to persist within pond sediments in a laboratory setting. Further research on environmental factors influencing vAh survivability and population dynamics in ponds is needed.

Effects of Different Feeding Regimes on Growth Rates and Fatty Acid Composition of Largemouth Bass Micropterus nigricans at High Water Temperatures.

As the northern Largemouth bass (LMB) (Micropterus nigricans) industry shifts toward fingerling production, implementing practical feeding strategies to ensure efficient growth during high water temperatures is paramount. Twenty (12.7 ± 0.2 g) (Trial 1) and fifteen (7.2 ± 0.1 g) (Trial 2) LMB fingerlings were stocked in two recirculating systems (each containing nine tanks), acclimated to 30 °C, with one system fed daily rations of 3, 5 and 7% body weight (Trial 1), and the second system fed to satiation daily, every second day, or every third day (Trial 2), for 28 days each. All treatments were triplicated. Multiple growth metrics and lipid composition were analyzed. The 3% treatment yielded the lowest final average weight (36.05 g) and FCR (0.83), with no difference in final biomass in Trial 1 treatments. Fish fed to satiation daily and every second day produced FCRs and biomasses of 0.83 and 356.78 g, and 0.93 and 272.26 g, respectively. There were no differences in total lipid concentration, however, fatty acid profiles differed significantly between all treatments within their respective trials. Feeding LMB fingerlings 3% of total body weight or feeding daily to satiation allows for efficient growth at 30 °C and implements cost-effective feeding strategies.

Effects of Crowding and Water Flow on Golden Shiners Notemigonus crysoleucas, Held in a Flow Tank.

Split-pond systems (SPS) such as those used for catfish are being considered for raising baitfish. When using these systems for baitfish such as golden shiners Notemigonus crysoleucas, an important factor to be considered is how well the species can tolerate crowding, as the design mandates fish be confined to a smaller portion of the pond. Another aspect of the SPS design is the water flow between the two units for at least 10-14 h each day. SPS can be successfully implemented for other species if factors such as crowding, and water flow do not affect growth. Two laboratory studies were conducted each using 12, 40-L tank flow-through system to observe the growth and physiological performance of golden shiners held for 28 days at three crowding densities: 600; 1,200, and 2,400 fish/m3, keeping water flow at 1 cm/s (Experiment 1) and using three flow rates: 1, 2, and 4 cm/s at similar densities (600 fish/m3) (Experiment 2). At the end of the experiments, fish were subject to acute 1-min confinement stress and whole-body cortisol was measured at 30 min intervals for up to 2 h to monitor the secretion pattern and recovery. Results from experiments showed no difference in the final weight, length, feed conversion, and survival among treatment groups; survival rates were lower in the flow study because of columnaris Flavobacterium covae infections. Baseline cortisol was also not different among the treatments. Cortisol increased 30 min after the acute stress and returned to near baseline in 2 h in the crowding study suggesting acclimation to the chronic stressor. However, in the flow study, cortisol remained elevated even after 2 h, and hence a compromised pathophysiological response. Crowding and water flow do not impair feed intake, growth, or survival in golden shiners, and in these aspects may be a suitable species for SPS.

Evaluating Stress-Mediated Microbial Pathogenesis in Golden Shiners, Notemigonus crysoleucas.

Flavobacterium covae (columnaris) is a microbial pathogen of the Golden Shiner (Notemigonus crysoleucas), a principal bait species. We investigated the effects of density and water temperature on the survival of fish subjected to a columnaris challenge and whether flow cytometry (FCM) could be a fast and reliable method to distinguish and enumerate F. covae populations from water and fish in experimental tanks. Juvenile Golden Shiners averaging 2.62 (±0.78 S.D.) g (negative for F. covae) were used in simultaneous trials at 22°C and 28°C in two ultra-low flow-through systems: each consisting of four treatments and five replicates per treatment. Treatments were fish stocked at either 600 fish/m3 or 2,400 fish/m3 and either challenged with F. covae or not; survival was observed for 48 h after challenge. Samples of water and fish tissue were obtained for FCM enumerations and validation by qPCR. No significant differences in survival were recorded between density treatments; however, high temperature and columnaris challenge treatments showed significantly higher mortality. Bacterial enumeration (number/mL) by FCM highly correlated with bacterial counts r = 0.81 (p = 0.001) in the water samples. Higher water temperatures may have increased columnaris infections and mortality in Golden Shiners. Flow cytometry is a reliable method of enumerating F. covae from experimental tank water samples.

A Review of Bacterial Co-Infections in Farmed Catfish: Components, Diagnostics, and Treatment Directions.

Catfish production is a major aquaculture industry in the United States and is the largest sector of food fish production. As producers aim to optimize production yields, diseases caused by bacterial pathogens are responsible for high pond mortality rates and economic losses. The major bacterial pathogens responsible are Edwardsiella ictaluri, Aeromonas spp., and Flavobacterium columnare. Given the outdoor pond culture environments and ubiquitous nature of these aquatic pathogens, there have been many reports of co-infective bacterial infections within this aquaculture sector. Co-infections may be responsible for altering disease infection mechanics, increasing mortality rates, and creating difficulties for disease management plans. Furthermore, proper diagnoses of primary and secondary pathogens are essential in ensuring the correct treatment approaches for antimicrobials and chemical applications. A thorough understanding of the interactions and infectivity dynamics for these warm water bacterial pathogens will allow for the adoption of new prevention and control methods, particularly in vaccine development. This review aims to provide an overview of co-infective pathogens in catfish culture and present diagnostic case data from Mississippi and Alabama to define prevalence for these multiple-species infections better.

Physio-biochemical, metabolic nitrogen excretion and ion-regulatory assessment in largemouth bass (Micropterus salmoides) following exposure to high environmental iron.

Iron overload is a significant water quality issue in many parts of the world. Therefore, we evaluated the potential toxic effects of waterborne elevated iron on largemouth bass (Micropterus salmoides), a highly valued sport and aquaculture fish species. First, a 96 h-LC50 toxicity assay was performed to understand the tolerance limit of this species to iron; and was determined to be 22.07 mg/L (as Fe3+). Thereafter, to get a better insight on the fish survival during long-term exposure to high environmental iron (HEI) (5.52 mg/L, 25% of the determined 96 h-LC50 value), a suite of physio-biochemical, nitrogenous metabolic and ion-regulatory compensatory responses were examined at 7, 14, 21 and 28 days. Results showed that oxygen consumption dropped significantly at 21 and 28 days of HEI exposure. Ammonia excretion rate (Jamm) was significantly inhibited from day 14 and remained suppressed until the last exposure period. The transcript concentration of Rhesus glycoproteins Rhcg2 declined; likely diminishing ammonia efflux out of gills. These changes were also reflected by a parallel increment in plasma ammonia levels. Under HEI exposure, ion-balance was negatively affected, manifested by reduced plasma [Na+] and parallel inhibition in branchial Na+/K+-ATPase activity. Muscle water content was elevated in HEI-exposed fish, signifying an osmo-regulatory compromise. HEI exposure also increased iron burden in plasma and gills. The iron accumulation pattern in gills was significantly correlated with a suppression of Jamm, branchial Rhcg2 expression and Na+/K+-ATPase activity. There was also a decline in the glycogen, protein and lipid reserves in the hepatic tissue from 14 days, 28 days and 21 days, respectively. Overall, we conclude that sub-lethal chronic iron exposure can impair normal physio-biochemical and ion-regulatory functions in largemouth bass. Moreover, this data set can be applied in assessing the environmental risk posed by a waterborne iron overload on aquatic life.

Physiological insights into largemouth bass (Micropterus salmoides) survival during long-term exposure to high environmental ammonia.

Waterborne ammonia is an environmental pollutant that is toxic to all aquatic animals. However, ammonia induced toxicity as well as compensatory mechanisms to defend against high environmental ammonia (HEA) are not well documented at present for largemouth bass (Micropterus salmoides), a high value fish for culture and sport fisheries in the United States. To provide primary information on the sensitivity of this species to ammonia toxicity, a 96 h-LC50 test was conducted. Thereafter, responses at physiological, ion-regulatory and transcript levels were determined to get insights into the underlying adaptive strategies to ammonia toxicity. For this purpose, fish were progressively exposed to HEA (8.31 mg/L representing 25% of 96 h-LC50) for 3, 7, 14, 21 and 28 days. Temporal effects of HEA on oxygen consumption rate (MO2), ammonia and urea dynamics, plasma ions (Na+, Cl- and K+), branchial Na+/K+-ATPase (NKA) and H+-ATPase activity, muscle water content (MWC), energy store (glycogen, lipid and protein) as well as branchial mRNA expression of Rhesus (Rh) glycoproteins were assessed. Probit analysis showed that 96 h-LC50 of (total) ammonia (as NH4HCO3) at 25 °C and pH 7.8 was 33.24 mg/L. Results from sub-lethal end-points shows that ammonia excretion rate (Jamm) was strongly inhibited after 7 days of HEA, but was unaffected at 3, 14 and 21 days. At 28 days fish were able to increase Jamm efficiently and concurrently, plasma ammonia re-established to the basal level. Urea production was increased as evidenced by a considerable elevation of plasma urea, but urea excretion rate remained unaltered. Expression of Rhcg isoform (Rhcg2) mRNA was up-regulated in parallel with restored or increased Jamm, suggesting its ammonia excreting role in largemouth bass. Exposure to HEA also displayed pronounced augmentations in NKA activity, exemplified by a rise in plasma [Na+]. Furthermore, [K+], [Cl-] and MWC homeostasis were disrupted followed by recovery to the control levels. H+-ATPase activity was elevated but NKA did not appear to function preferentially as a Na+/NH4+-ATPase. From 14 days onwards MO2 was depressed, potentially an attempt towards minimizing catabolism. Glycogen content in liver and muscle were temporarily depleted, whereas a remarkable increment in protein was evident at the last exposure period. Overall, these data suggest that ammonia induced toxicity can disturb several biological processes in largemouth bass, however, it can adapt to the long-term sub-lethal ammonia concentrations by activating various components of ammonia excretory, ion-regulatory and metabolic pathways.