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.

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.

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.

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.

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.

Differential modulation of oxidative stress, antioxidant defense, histomorphology, ion-regulation and growth marker gene expression in goldfish (Carassius auratus) following exposure to different dose of virgin microplastics.

Goldfish (Carassius auratus) juveniles were exposed to virgin polyvinyl chloride microplastics (PVC-MPs) in triplicate at 0, 0.1 or 0.5 mg/L for four days. Afterwards, the histopathology of the gills, liver and intestines were examined, along with various antioxidant enzymes and indicators of oxidative damage (malondialdehyde (MDA) and hydrogen peroxide (H2O2)), in the brain, liver and gills. In addition, we also studied the expression of hepatic insulin-like growth factor-1 (IGF-1), insulin-like growth factor binding protein 1 (IGFBP-1) and growth hormone (GH) receptor, while cortisol receptor (CR) and cytochrome P450 1A (CYP1A) gene expression were assayed in both the liver and gills. Histological analysis revealed PVC-MPs in the intestines at 0.1 and 0.5 mg/L, along with substantially shorter villi. The gills appeared undamaged by PVC-MPs exposure and had limited or no effect to antioxidant activity, Na+/K+-ATPase and H+-ATPase activity or plasma ion levels, but there was a prominent upsurge of the detoxification enzymes glutatione S-transferase (GST) activity and CYP1A expression. Livers showed inflammation and some occurrences of hemorrhaging and necrosis at 0.5 mg/L. While the brain showed some evidence of oxidative damage, the liver was the most susceptible to oxidative damage, based on increased MDA, H2O2 and various antioxidant enzymes. Hepatic expression of IGFBP-1 and GH receptor were significantly downregulated at 0.5 mg/L while CR was upregulated. Results indicate that exposure to environmentally relevant PVC-MP can cause oxidative damage in the brain and liver, adverse histomorphological changes to the intestine and liver and alter the gene expression in goldfish.

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.