Channel Catfish Liver Research Articles

Dietary betulinic acid alleviates high carbohydrate diet-induced hepatic de novo lipogenesis through AMPK signaling and improves liver health in channel catfish (Ictalurus punctatus)

A high carbohydrate diet (HCD) induced excessive hepatic de novo lipogenesis, which caused metabolic disorder and impaired the health of fish species. Betulinic acid (BA) has been applied to ameliorate fatty liver and anti-inflammatory in mammals. Here, we investigated the effects of dietary BA supplementation on HCD-induced hepatic de novo lipogenesis and liver health in channel catfish (Ictalurus punctatus). The catfish were fed with three diets: NCD (18 % carbohydrate), HCD (36 % carbohydrate), HCD+BA (36 % carbohydrate with 150 mg/kg BA). After an 8-week feeding trial, we found that dietary BA decreased hepatosomatic index (HSI) and condition factor (CF) without affecting the growth of channel catfish fed HCD. Dietary BA significantly reduced the HCD-induced excessive lipid accumulation (oil red O area, TG content, and de novo lipogenesis gene expression, P < 0.05). Moreover, we found that dietary BA prevented hepatic lipid accumulation via activating phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and inhibiting sterol regulatory element-binding protein 1 (SREBP1) (P < 0.05) in liver of channel catfish fed HCD. Besides, we also found that dietary BA significantly prevented HCD-induced liver damage, as confirmed by liver H&E staining, reduced inflammation biomarkers (ALT, AST, hydroxyproline, α-sma and tgf-1β), downregulated expression of ER stress-related genes (eif2a, bip, atf4, xbp1) (P < 0.05). Overall, the present study indicated that dietary BA inclusion attenuated hepatic de novo lipogenesis, improved liver inflammation and reduced ER stress in channel catfish fed HCD.

Transcriptomic Association Analysis of the Metabolic Mechanism of Sulfamethoxazole in Channel Catfish (Ictalurus punctatus).

Sulfamethoxazole is a widely used antimicrobial drug used to treat bacterial diseases in aquaculture. To understand the gene expression in channel catfish liver after treatment with sulfamethoxazole, in this study, the treatment group received sulfamethoxazole (100 mg/kg bw), which was administered orally once, and samples were taken at 5 h, 12 h, and 6 d after the administration of sulfamethoxazole, while the control group was orally administered sterile water. To further identify potentially significant genes, a transcriptome analysis using RNA-seq was carried out. More than 50 million high-quality reads were found. After filtering and quality analysis, these reads were identified as 54,169,682, 51,313,865, 51,608,845, and 49,333,491. After counting 23,707 of these transcripts for gene expression, it was discovered that 14,732 of them had genes with differential expression. Moreover, we found that the annotation with the most GO variation was "cellular process" (1616 genes), "metabolic process" (1268 genes), "binding" (1889 genes), and "catalytic activity" (1129 genes). KEGG pathways showed that the "metabolic pathway" was the pathway that was significantly enriched in both experimental groups when comparing the experimental groups: 5 h and 12 h (128 genes); 5 h and 6 d (332 genes); and 12 h and 6 d (348 genes). Also, UDP- glucuronosyltransferase (ugt), which is associated with glucuronidation, and UDP-glucuronosyltransferase 2C1-like (ugt2a1) showed significant upregulation. Carboxylesterase 5A-like (ces3), which promotes fatty acyl and cholesteryl ester metabolism, and the glutathione transferase family were upregulated in the expression of sulfamethoxazole metabolism in the liver, which significantly affected the metabolic effects of the drug. Meanwhile, dypd, uck2b, and rrm2, which are related to nucleotide synthesis and metabolism, were upregulated. Our study extends the knowledge of gene expression in drug metabolism in channel catfish and further provides insight into the molecular mechanism of sulfamethoxazole metabolism.

Artemisinin alters disposition regularities of enrofloxacin and its metabolite ciprofloxacin in channel catfish (Ictalurus punetaus) following combined oral administration

The aim of this investigation was to explore the effect of artemisinin (ART) on pharmacokinetic (PK) and tissue distribution characteristics of enrofloxacin (ENR) and ciprofloxacin (CIP) in channel catfish following combined and single medicine. The channel catfish in ENR group were treated with ENR, and the fish in ENR-ART group were orally co-administrated with ENR and ART. ENR and CIP concentrations in channel catfish were determined using ultra performance liquid chromatography, and PK parameters for ENR and CIP were calculated by WinNonlin software. Moreover, the multi-drug resistance 1 (MDR1) gene expression and cytochrome P-450 3A (CYP 3A) enzyme activities in channel catfish liver were measured. The results showed that lower ENR and CIP levels were observed in ENR-ART group than that in ENR group, and similar regularities in PK parameters were also found that Cmax, AUC and MRT values in ENR group were all greater than those in ENR-ART group. Furthermore, higher MDR1 gene expression and CYP 3A enzyme activities in channel catfish liver were observed in ENR-ART group than those in ENR group. The present investigation indicates that ART can reduce the ENR concentrations, accelerate the excretion of ENR and decrease the antibacterial efficacy of ENR in channel catfish.

Feed Restriction Alleviates Chronic Thermal Stress-Induced Liver Oxidation and Damages via Reducing Lipid Accumulation in Channel Catfish (Ictalurus punctatus).

Caloric restriction is known to suppress oxidative stress in organ systems. However, whether caloric/feed restriction alleviates chronic thermal stress in aquatic animals remains unknown. Here, we set up three feeding rations: 3% BW (3% body weight/day), 2.5% BW (restricted feeding, 2.5% body weight/day) and 2% BW (high restricted feeding, 2% body weight/day), to investigate the effects and mechanism of feed restriction on improving chronic heat-induced (27 to 31 °C) liver peroxidation and damages in channel catfish (Ictalurus punctatus). The results showed that, compared to 3% BW, both 2.5% BW and 2% BW significantly reduced the liver expressions of hsc70, hsp70 and hsp90, but only 2.5% BW did not reduce the growth performance of channel catfish. The 2.5% BW and 2% BW also reduced the lipid deposition (TG) and improved the antioxidant capacity (CAT, SOD, GSH and T-AOC) in the liver of channel catfish. The heat-induced stress response (plasma glucose, cortisol and NO) and peroxidation (ROS and MDA) were also suppressed by either 2.5% BW or 2% BW. Moreover, 2.5% BW or 2% BW overtly alleviated liver inflammation and damages by reducing endoplasmic reticulum (ER) stress (BIP and Calnexin) and cell apoptosis (BAX, Caspase 3 and Caspase 9) in the liver of channel catfish. In conclusion, 2.5% body weight/day is recommended to improve the antioxidant capacity and liver health of channel catfish during the summer season, as it alleviates liver peroxidation and damages via suppressing lipid accumulation under chronic thermal stress.

Effectiveness of Cathelicidin Antimicrobial Peptide against Ictalurid Catfish Bacterial Pathogens.

One of the major goals in aquaculture is to protect fish against infectious diseases as disease outbreaks could lead to economic losses if not controlled. Antimicrobial peptides (AMPs), a class of highly conserved peptides known to possess direct antimicrobial activities against invading pathogens, were evaluated for their ability to protect Channel Catfish Ictalurus punctatus and hybrid catfish (female Channel Catfish × male Blue Catfish I. furcatus) against infection caused by the fish pathogen Aeromonas hydrophila ML09-119. To identify effective peptides, the minimum inhibitory concentrations against bacterial pathogens Edwardsiella ictaluri S97-773, Edwardsiella piscicida E22-10, A. hydrophila ML09-119, Aeromonas veronii 03X03876, and Flavobacterium columnare GL-001 were determined invitro. In general and overall, cathelicidins derived from alligator and sea snake exhibited more potent and rapid antimicrobial activities against the tested catfish pathogens as compared to cecropin and pleurocidin AMPs and ampicillin, the antibiotic control. When the peptides (2.5µg of peptide/g of fish) were injected into fish and simultaneously challenged with A. hydrophila through immersion, increased survival rates in Channel Catfish and hybrid catfish were observed in both cathelicidin (alligator and sea snake) treatments as compared to other peptides and the infected control (P<0.001) with alligator cathelicidin being the overall best treatment. Bacterial numbers in the kidney and liver of Channel Catfish and hybrid catfish also decreased (P<0.05) for cathelicidin-injected groups at 24 and 48h after challenge infection. These results show the potential of cathelicidin to protect catfish against bacterial infections and suggest that an approach overexpressing the peptide in transgenic fish, which is the long-term goal of this research program, may provide a method of decreasing bacterial disease problems in catfish as delivering the peptides via individual injection or feeding would not be economically feasible.

Effect of cold-anesthetization rate on blood biochemical parameters and muscle composition during live channel catfish Ictalurus punctatus waterless preservation

Cold-anesthetization waterless live fish preservation is considered a promising alternative strategy. This work investigates the effects of temperature chilling rates of 2, 4, and 6 °C/h on serum biological parameters and muscular chemical composition of channel catfish Ictalurus punctatus and the influence of waterless preservation time (6, 9, 12, 15, and 18 h) on survival after subjection to three chilling rates. Fish subjected to increasing chilling rates showed lower survival and shorter preservation time. The research results revealed that a temperature gradient drop of 2 °C/h was conducive to maintaining the activity of creatinine (CREA), lactic dehydrogenase (LDH), and pyruvate kinase (PK). The serum aspartic transaminase (AST) and alanine transaminase (ALT) activity dramatically increased after anesthetization. Blood urea nitrogen (BUN) and CREA, whose activity is sensitive to hydropenic conditions, increased significantly after waterless preservation and showed a slight reversal after recovery. Anesthetization caused a significant decrease in protein and fat in muscle, while no obvious trend was observed among different chilling groups. Muscle glycogen (MG) and water-holding capacity (WHC) decreased after anesthetization, then increased after preservation for 12 h, and decreased again after recovery for 24 h. The muscle pH showed the opposite trend. It can be concluded that the whole process resulted in marked changes in the muscle chemical composition. The development of a new green method for anesthetization of live channel catfish for long-term waterless preservation in the present work helps to determine the physiological changes that occur during preservation, providing an effective reference for investigation in other freshwater fish.

Treatment with Streptozotocin (STZ) increases expression of macrophage migration inhibitory factor (MIF) but not metalloproteinase‐13 (MMP‐13) mRNA in the liver of channel catfish

Tissue repair during liver damage depends on the recruitment and activation of macrophages. In rodents, the production of MIF is essential for the recruitment of scar‐forming macrophages. In addition, production of MMP‐13 appears to be critical for successful remodeling and hepatic tissue repair. In our previous studies with channel catfish, STZ treatment induced hypoglycemia and an abnormal gross morphology of the liver and gall bladder. This suggests that the liver is the target organ for STZ in channel catfish and causes acute liver damage. This results in hypoglycemia, necrosis and eventually organ failure. However, whether liver damage induced by the STZ treatment results in recruitment and activation of macrophage in the liver of channel catfish has never been examined. Liver samples were collected from juvenile channel catfish 7 days after intraperitoneal administration of 0, 3.6, 36, 180, or 360 mg/kg STZ on day 0 (n=4 fish/treatment). Expression of genes (MMP‐13 and MIF) were measured using quantitative real‐time PCR. Expression of MIF and MMP‐13 changed in response to increases in STZ dosage (p&lt;0.01, and p=0.05, respectively.) Expression of MMP‐13 mRNA decreased in response to an increase in STZ dosage. Expression of MIF mRNA was significantly higher in fish treated with 360 mg/kg body weight compared to control (p&lt;0.05). Decreased expression of MMP‐13 suggests STZ treatment inhibits macrophage‐mediated tissue repair. Given that MIF has been associated with multiple liver diseases, STZ treatment may activate multiple mechanisms other than those associated with fibrosis to induce liver damage in channel catfish.Support or Funding InformationThis study is supported by Kansas Idea Network of Biomedical Research Excellence (P20GM103418).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Treatment with Streptozotocin (STZ) causes hypoglycemia and alters the stability of reference genes for real‐time PCR analysis in the liver of channel catfish

Genetically selected channel catfish for increased growth often develops phenotype similar to human obesity, suggesting that channel catfish may be a model organism to investigate human metabolic disorders. Streptozotocin (STZ) treatment induces hyperglycemia in mammals and some fish species. However, the effects of STZ treatment on blood glucose level as well as other physiological parameters in channel catfish have never been examined in detail. In this study, we tested the hypothesis that treatment with STZ affects body weight, blood glucose level, and hepatic expression of house‐keeping genes. Juvenile channel catfish (28.3 ± 2g) were treated with 0, 3.6, 36, 180, or 360 mg/kg STZ intraperitoneally on day 0 (n=4 fish/treatment). On day 7, blood glucose level was measured and blood, liver, muscle and brain samples were collected. Expression of five common housekeeping genes (beta‐macroglobulin, alpha‐tubulin, 18s ribosomal RNA, beta‐actin, and GAPDH) were measured using real‐time PCR. The weight of fish did not change between days 0 and 7 among all treatments. Blood glucose levels were lower in fish treated with 180 or 360 mg/kg STZ compared to fish treated with 0 mg/kg STZ. Treatment with STZ at higher doses altered expression of 4 housekeeping genes. Expression of beta‐macroglobulin, alpha‐tubulin, and beta‐actin mRNA was higher in fish treated with higher doses of STZ compared to 0 mg/kg STZ. In contrast, expression of GAPDH mRNA tended to be less in 360 mg/kg STZ than 0 mg/kg STZ. Expression of 18s rRNA was not affected by STZ treatment. Rather than causing hyperglycemia, treatment with high doses of STZ decreased blood glucose level in channel catfish, suggesting a difference in glucose metabolism exists between channel catfish and humans.Support or Funding InformationThis study is supported by APS IOSP fellowship program and Kansas Idea Network of Biomedical Research Excellence (P20GM10348).

Validation of Dual-energy X-Ray Absorptiometry to Predict Body Composition of Channel Catfish, Ictalurus punctatus.

Dual-energy X-ray absorptiometry (DXA) provides a noninvasive way to determine lean tissue mass (LTM), fat mass (FM), bone mineral content (BMC), and bone mineral density (BMD) in humans and small mammals. Live channel catfish (n=74, 78g - 1200 g) were anesthetized and scanned in both a lateral position and a dorsa-ventral position. Six individual fish (300g - 600g) were scanned five times each to determine precision by the coefficient of variation. Precision was good for LTM (0.75-1.06%) and for BMC and BMD (2-2.6%). Precision for FM was not good (27-34%), which was due to the very low FM (0-1g) recorded by the DXA. However, using the predicted values, FM precision improved to 5-5.5%. DXA values for LTM, FM, and BMC were significantly different from chemical analysis (P< 0.001). DXA overestimated LTM and underestimated FM and BMC. However, all three compartments were strongly correlated with carcass values (P <0.0001). Using the prediction equations and the jackknife procedure, predicted values of LTM, FM, and BMC were not significantly different from the carcass values (P >0.05). DXA may also be a valuable tool for evaluating body condition longitudinally in commercial or in threatened or endangered fish species, where non-invasive procedures would be invaluable.

Stearoyl‐CoA Desaturase mRNA in Channel Catfish: A Potential Molecular Marker to Investigate Development of Obesity Using Non‐model Fish Species

Objectives of the present study were to characterize SCD‐1 gene in channel catfish and examine its mRNA expression in various tissues. Genetic selection toward increased growth results in development of obesity‐like phenotype in channel catfish, suggesting that channel catfish may serve as an alternative animal model to study human obesity development. However, the underlying mechanism(s) that results in the development of obesity‐like phenotype, as well as the consequence of developing such a phenotype, is unclear. In mammals, development of obesity leads to changes in expression and function of various enzymes involved in lipid metabolism such as stearoyl‐CoA desaturase‐1 (SCD‐1). Currently, little is known about the role of SCD‐1 in channel catfish physiology. Channel catfish expressed sequence tag clones that correspond to the zebrafish SCD‐1 sequence were identified and used as templates to generate primers for the RT‐PCR. The PCR amplicon (599 bp) was generated from channel catfish liver cDNA and shared high sequence similarity (around 70%) with SCD‐1 mRNA of various fish species. Expression of SCD‐1 mRNA was detected in brain, liver, and kidney. Currently, changes in expression of SCD‐1 mRNA in relation to changes in food intake and genetic selection toward growth are being investigated. This project is supported by the Kansas IDeA Network of Biomedical Research Excellence.

Expression, purification and antibacterial activity of the channel catfish hepcidin mature peptide

Hepcidins are small cysteine-rich cationic antimicrobial peptides. The channel catfish (Ictalurus punctatus) hepcidin cDNA has been characterized, but recombinant protein expression and purification was not reported. I. punctatus hepcidin is comprised of 96 residues, with eight functionally important conserved cysteine residues located in the C-terminal region of the mature peptide, suggesting that this region is responsible for the antibacterial activity. In this study, a cDNA fragment (mCH) encoding the 25 amino acid mature peptide was cloned from channel catfish liver, and inserted into vector pET-32a(+) to produce a construct that expressed a hexahistidine-tagged thioredoxin (trxA) fusion protein that was cleavable using enterokinase. The trxA–mCH fusion protein was expressed in Escherichia coli BL21 (DE3) at 25°C, using 1mM IPTG for induction. Greater than 80% of the fusion protein was expressed solubly, but was not biologically active. Removal of the trxA fusion partner by enterokinase resulted in mCH that exhibited antibacterial activity against two Gram-positive (Listeria monocytogenes and Staphylococcus aureus), and two Gram-negative (E. coli and Pseudomonas aeruginosa) bacteria.

Slow O-demethylation of methyl triclosan to triclosan, which is rapidly glucuronidated and sulfonated in channel catfish liver and intestine

The antibacterial personal care product triclosan is discharged in municipal waste, and converted in part by bacteria in sewage sludge and soil to its more lipid-soluble methyl ether, methyl triclosan. Triclosan and methyl triclosan have been detected in water, sediment, fish and invertebrates near sewage treatment facilities. Understanding the biotransformation of methyl triclosan and triclosan in a model food fish, the channel catfish, will be of value in assessing the likelihood that these compounds will bioaccumulate in exposed fish, and therefore potentially pass up the food chain. We hypothesize that cytochrome P450 will catalyze the O-demethylation of methyl triclosan to yield triclosan, which is likely to undergo glucuronidation or sulfonation of the phenolic hydroxyl group. Conversion of methyl triclosan to triclosan was measured by LC/MS/MS following aerobic incubation of varying concentrations of methyl triclosan with NADPH and hepatic and intestinal microsomes from untreated, 3-methylcholanthrene-treated (10mg/kg, i.p.) or PCB-126-treated (0.1mg/kg, i.p.) channel catfish (n=4 per treatment group). The Km values for methyl triclosan were similar for untreated, 3-methylcholanthrene-treated and PCB-126-treated catfish liver microsomes, ranging from 80 to 250μM. Vmax values for O-demethylation ranged from 30 to 150pmol/min/mg protein, with no significant differences between controls, PCB-126-treated or 3-methylcholanthrene-treated fish, suggesting that methyl triclosan O-demethylation was not a CYP1-catalyzed reaction. Methyl triclosan O-demethylation activities in intestinal microsomes were similar to or lower than those found with liver microsomes. The calculated rate of O-demethylation of methyl triclosan in catfish liver at 1μM, a concentration reported in exposed fish, and 21°C, an early summer water temperature, is 0.10pmol/min/mg protein. This slow rate of metabolism suggests that upon continued exposure, methyl triclosan may bioaccumulate in the channel catfish. Triclosan itself, however, was readily glucuronidated by hepatic and intestinal microsomes and sulfonated by hepatic and intestinal cytosol. Triclosan glucuronidation followed Michaelis-Menten kinetics when rates were measured across a concentration range of 5–1000μM, whereas triclosan sulfonation exhibited substrate inhibition at concentrations above 10–20μM in both intestinal and hepatic cytosol. Based on the enzyme kinetic constants measured in hepatic and intestinal fractions at 21°C, triclosan at 1μM could be glucuronidated at rates of 23 and 3.2pmol/min/mg protein respectively in liver and intestine, and sulfonated at rates of 277 (liver) and 938 (intestine) pmol/min/mg protein. These rates are much higher than the rates of demethylation of methyl triclosan, and suggest that triclosan would be rapidly cleared and unlikely to bioaccumulate in catfish tissues

An in vitro screening method to evaluate chemicals as potential chemotherapeutants to control Aeromonas hydrophila infection in channel catfish

To develop an in vitro screening method to be used for identifying potential effective chemotherapeutants to control Aeromonas hydrophila infections. Using catfish gill cells G1B and four chemicals (hydrogen peroxide, sodium chloride, potassium permanganate and D-mannose), the feasibility of using an in vitro screening method to identify potential effective chemotherapeutants was evaluated in this study. In vitro screening results revealed that, at concentration of 100 mg l⁻¹, H₂O₂ was the only chemical tested that was able to completely abolish the attachment and invasion of Aer. hydrophila to catfish gill cells. In vivo virulence studies using live channel catfish through bath immersion confirmed that H₂O₂ was the only chemical tested that was able to significantly (P < 0·001) reduce the mortality (from 90 or 100% to 0 or 20%) caused by Aer. hydrophila infections. The in vitro screening method using catfish gill cells G1B could be used to initially identify potential effective chemotherapeutants to control Aer. hydrophila. An in vitro screening method using catfish gill cells to identify potential effective chemotherapeutants described here will cut cost in research compared with the method of using live fish to screen lead compounds for fish disease control.

Glucuronidation of Polychlorinated Biphenylols and UDP-Glucuronic Acid Concentrations in Channel Catfish Liver and Intestine

Polychlorinated biphenylols (OH-PCBs) are potentially toxic polychlorinated biphenyl metabolites that can be eliminated by glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGTs). OH-PCBs with a 3,5-dichloro-4-hydroxy substitution pattern have been detected in blood from humans and wildlife, suggesting slow elimination. In this study we assessed the glucuronidation of 4-OH-PCBs with zero, one, or two chlorine atoms flanking the 4-hydroxyl group and zero to four chlorine atoms in the aphenolic ring in microsomes from channel catfish liver and proximal intestine. Product formation was quantitated with [(14)C]UDP-glucuronic acid (UDPGA). Physiological concentrations of UDPGA were measured in preparations of liver and intestine. When the OH-PCB concentrations were varied in the presence of saturating UDPGA concentrations, glucuronidation V(max) values were higher in hepatic than in intestinal microsomes (0.40-3.4 and 0.12-0.78 nmol/min/mg of protein, respectively), whereas the K(m) values were generally lower for intestine (0.042-0.47 mM) than for liver (0.11-1.64 mM). In both tissues V(max) values with 3,5-dichloro-4-OH-PCBs were lower than with the corresponding 3-chloro-4-OH-PCBs. Varying the UDPGA concentrations in the presence of saturating concentrations of OH-PCB showed that the K(m) for UDPGA was lower in intestine (27 microM) than in liver (690 microM). The measured concentration of UDPGA in catfish liver (246-377 nmol/g) was lower than the K(m) for UDPGA, suggesting that in vivo rates of glucuronidation may be suboptimal, whereas in intestine the measured UDPGA concentration (71-258 nmol/g) was higher than the K(m) for UDPGA. Although liver has a greater glucuronidation capacity than proximal intestine, the properties of intestinal UGTs in channel catfish enable them to efficiently glucuronidate low concentrations of OH-PCBs.

Microarray analysis of gene expression in the blue catfish liver reveals early activation of the MHC class I pathway after infection with Edwardsiella ictaluri

The acute nature of disease outbreaks in aquaculture settings has served to emphasize the importance of the innate immune response of fish for survival and led to the recent identification and characterization of many of its components. Catfish, the predominant aquaculture species in the United States, is an important model for the study of the teleost immune system. However, transcriptomic-level studies of disease-related gene expression in catfish have only recently been initiated, and understanding of immune responses to pathogen infections is limited. Here, we have developed and utilized a 28K in situ oligonucleotide microarray composed of blue catfish (Ictalurus furcatus) and channel catfish (Ictalurus punctatus) transcripts. While channel catfish accounts for the majority of commercial production, the closely related blue catfish possesses several economically important phenotypic traits. Microarray analysis of gene expression changes in blue catfish liver after infection with Gram-negative bacterium Edwardsiella ictaluri indicated the strong upregulation of several pathways involved in the inflammatory immune response and potentially in innate disease resistance. A multifaceted response to infection could be observed, encompassing the complement cascade, iron regulation, inflammatory cell signaling, and antigen processing and presentation. The induction of several components of the MHC class I-related pathway following infection with an intracellular bacterium is reported here for the first time in fish. A comparison with previously published expression profiles in the channel catfish liver was also made and the microarray results extended by use of quantitative RT-PCR. Our results add to the understanding of the teleost immune responses and provide a solid foundation for future functional characterization, genetic mapping, and QTL analysis of immunity-related genes from catfish.

Expression analysis of the acute phase response in channel catfish ( Ictalurus punctatus) after infection with a Gram-negative bacterium

The acute phase response (APR) is a set of metabolic and physiological reactions occurring in the host in response to tissue infection or injury and is a crucial component of the larger innate immune response. The APR is best characterized by dramatic changes in the concentration of a group of plasma proteins known as acute phase proteins (APPs) which are synthesized in the liver and function in a wide range of immunity-related activities. Utilizing a new high-density in situ oligonucleotide microarray, we have evaluated the APR in channel catfish liver following infection with Edwardsiella ictaluri, a bacterial pathogen that causes enteric septicemia of catfish. Our catfish microarray design (28K) builds upon a previous 19K channel catfish array by adding recently sequenced immune transcripts from channel catfish along with 7159 unique sequences from closely related blue catfish. The analysis of microarray results using a traditional 2-fold change in gene expression cutoff and a 10% false-discovery rate revealed a well-developed APR in catfish, with particularly high upregulation (>50-fold) of genes involved in iron homeostasis (i.e. intelectin, hemopexin, haptoglobin, ferritin, and transferrin). Other classical APP genes upregulated greater than 2-fold included coagulation factors, proteinase inhibitors, transport proteins, and complement components. Upregulation of the majority of the complement cascade was observed including the membrane attack complex components and complement inhibitors. A number of pathogen recognition receptors (PRRs) and chemokines were also differentially expressed in the liver following infection. Independent testing of a selection of differentially expressed genes with real-time RT-PCR confirmed microarray results.

Evaluation of Bioelectric Impedance to Predict Carcass Yield, Carcass Composition, and Fillet Composition in Farm‐Raised Catfish

Abstract.— Bioelectric impedance analysis (BIA) was evaluated as a method for predicting carcass yield, fat, and moisture in live channel catfish, Ictalurus punctatus (N = 20), and fat and moisture in fillets from channel catfish (N= 20) and channel catfish female × blue catfish male, I. furcatus, hybrids (N= 20). Fish were cultured in ponds, fed a commercial catfish diet (28% protein), and harvested at market weight (450 g‐900 g, ∼19 months post‐hatch). Live channel catfish were tranquilized, weighed, and sexed. Resistance and reactance were measured with a four‐terminal impedance analyzer. Fish were then deheaded, eviscerated, weighed, and carcass yield was calculated. Fillets from channel catfish and channel catfish × blue catfish hybrids were measured for weight, resistance, and reactance. Carcasses and fillets were ground and fat and moisture were determined by chemical analysis. Regression models including total weight, resistance, and reactance as independent variables explained 71%, 75%, and 65% of the variation in carcass yield, fat, and moisture, respectively, in live fish. Regression models with fillet weight, resistance, and reactance as independent variables explained 62% and 41% of the variation in fillet fat and moisture, respectively, in channel catfish, and 53% and 58% of the variation in fillet fat and moisture, respectively, in channel catfish × blue catfish hybrids. Models including resistance and reactance explained significantly more variation in the traits measured than did models containing only whole weight as an independent variable. Improvements in prediction accuracy will be needed to make BIA a useful tool for predicting carcass yield, carcass composition, and fillet composition in farm‐raised catfish.

Isolation and characterization of a novel cytochrome P450 (CYP2 family) isoform from channel catfish

Channel catfish ( Ictalurus punctatus) express at least four isoforms of cytochrome P450 (CYP) that are cross-reactive with anti-CYP2K1 (rainbow trout) antibodies. These proteins appear to be the major constitutive CYPs in the liver of I. punctatus and show distinct sex- and age-specific variations in expression. The objective of the present work was to purify a CYP2-related hepatic CYP from I. punctatus. Hepatic microsomes were prepared from channel catfish liver and solubilized with sodium cholate and polidocanol. CYP proteins were precipitated with polyethyleneglycol and applied to an octylamino-Sepharose CL-4b column. CYP-containing fractions were subsequently subjected to chromatography on DEAE-sepharose, HA-ultragel, and CM-cellulose columns. A single heme protein (ca. 47 kDa), which reacted with anti-CYP2K1 antibodies, was resolved. A sequence for the first 15 amino acids was determined. This sequence showed homology with both CYP2M1 and CP2K1 isoforms. Expression of this band is down regulated by exposure to ethanol and fenitrothion. However, no correlation with several CYP-mediated catalytic activities has been detected as yet.

Purification and Characterization of Hepatic and Intestinal Phenol Sulfotransferase with High Affinity for Benzo[a]pyrene Phenols from Channel Catfish, Ictalurus punctatus

Cytosol from channel catfish liver and intestinal mucosa has high sulfotransferase activity with low concentrations of 3-, 7-, or 9-hydroxybenzo[a]pyrene. To further investigate this conjugation pathway, sulfotransferase activity toward 9-hydroxybenzo[a]pyrene was isolated from catfish intestinal and hepatic cytosol by chromatography on anion exchange and PAP–agarose affinity columns. SDS–PAGE of the active fractions showed that one major band with molecular size of about 41,000 Da was isolated from intestine, while two bands of about 41,000 and 31,000 Da were obtained from liver. Antibodies against human phenol-sulfating sulfotransferase cross-reacted strongly with the 41,000-Da bands from liver and intestine, but weakly with the hepatic 31,000-Da protein. N-Terminal sequence information could not be obtained from the pure proteins. Following digestion, an internal sequence of 20 amino acid residues was obtained from the hepatic 41,000-Da protein, which matched a sequence found in several mammalian sulfotransferases. No fish sulfotransferase sequences were available for comparison. The identity of the hepatic 31,000-Da protein was not established. The purified 41,000-Da proteins had very high activities with 3-, 7-, or 9-hydroxybenzo[a]pyrene, with Km values in the 40–100 nM range and Vmax 125–300 nmol/min/mg of protein. Substrate inhibition was observed when the concentrations of hydroxylated benzo[a]pyrenes were above 0.5 μM. As well as benzo[a]pyrene phenols, the purified 41,000-Da sulfotransferases catalyzed sulfation of 2-naphthol, 4-nitrophenol, 4-methylumbelliferone, 7-(hydroxymethyl)-12-methylbenz[a]anthracene, dehydroepiandrosterone, estrone, and 17β-estradiol. Phenolic compounds were the preferred substrates for the purified enzymes.

Effects of the oxidant potassium permanganate on the expression of gill metallothionein mRNA and its relationship to sublethal whole animal endpoints in channel catfish.

Potassium permanganate is an oxidant heavily used in fish culture. The effects of this compound were examined utilizing molecular (Metallothionein) and whole animal endpoints following an 8-week exposure to nominal concentrations of 0.5 (daily) and 1.0 and 2.0 mg/L (on alternate days) of potassium permanganate (PM). In order to measure MT, a complementary DNA clone of metallothionein (MT) was cloned and sequenced from the liver of channel catfish treated with a single injection of cadmium chloride (10 mg/kg). The cDNA was obtained by reverse transcriptase polymerase chain reaction (RT-PCR), using 3' rapid amplification of cDNA ends (RACE) technique. No significant correlation was observed with gill MT expression or sublethal endpoints indicative of toxicity (weight, length, condition index [CI], or liver somatic index [LSI). MT mRNA expression in gill was significantly reduced only after 8 weeks in the 2.0 mg/L treatment. Decreases in CI were observed in males at all time points after 4 weeks, at the 2.0 mg/L treatment concentration, with a NOEC of 1 mg/L. Reductions in LSI that were not dose dependent were also observed in both males and females throughout the 8-week study and no consistent reduction in weight gain or length was observed. These data demonstrate that minimal changes in sublethal effects occur in fish following 0.5-2.0 mg/L PM treatment after 4 weeks, but recovery from adverse effects is observed by 8 weeks, suggesting that acute (typically less than 1 week) treatment of channel catfish with PM would not significantly affect fish health.