Sub-lethal concentration of arsenic interferes with the proliferation of hepatocytes and induces in vivo apoptosis in Clarias batrachus L

We studied the hepatocellular alterations induced by sub-lethal concentrations (0.50 muM) of arsenic in Indian catfish Clarias batrachus L. Sub-lethal arsenic exposure altered serum aspartate aminotransferase and alkaline phosphatase levels and brought about significant changes in different serum biochemical parameters. Arsenic exposure reduced total hepatocyte protein content and suppressed the proliferation of hepatocytes in a time-dependent manner. Routine histological studies on liver documented arsenic-induced changes characterized by dilated sinusoids, formation of intracellular edema, megalocytosis, vacuolation and appearance of hepatic cells with distorted nuclei. Transmission electron microscopy of hepatocytes further revealed hyperplasia and hypertrophy of mitochondria, development of dilated rough endoplasmic reticulum and changes in peroxisome size with duration of arsenic exposure. Degeneration of mitochondrial cristae and condensation of chromatin was also evident in arsenic-exposed hepatocytes. A significant number of hepatocytes isolated from arsenic-exposed fish stained with annexin V and demonstrated DNA ladder characteristic of apoptosis. Single-cell gel electrophoresis of exposed hepatocytes also revealed the development of comets usually seen in apoptotic cells. Using specific inhibitors it was determined that the arsenic-induced apoptosis of hepatocytes was caspase-mediated, involving the caspase 3 pathway.     

Effect of alkalinity (pH 10) on ureogenesis in the air-breathing walking catfish,Clarias batrachus

Exposure of fish to alkaline conditions inhibits the rate of ammonia excretion, leading to ammonia accumulation and toxicity. The purpose of this study was to determine the role of ureogenesis via the urea cycle, to avoid the accumulation of ammonia to a toxic level during chronic exposure to alkaline conditions, for the air-breathing walking catfish, Clarias batrachus, where a full complement of urea cycle enzyme activity has been documented. The walking catfish can survive in water with a pH up to 10. At a pH of 10 the ammonia excretion rate by the walking catfish decreased by approximately 75% within 6 h. Although there was a gradual improvement of ammonia excretion rate by the alkaline-exposed fish, the rate remained 50% lower, even after 7 days. This decrease of ammonia excretion was accompanied by a significant accumulation of ammonia in plasma and body tissues (except in the brain). Urea-N excretion for alkaline-exposed fish increased 2.5-fold within the first day, which was maintained until day 3 and was then followed by a slight decrease to maintain a 2-fold increase in the urea-N excretion rate, even after 7 days. There was also a higher accumulation of urea in plasma and other body tissues (liver, kidney, muscle and brain). The activity of glutamine synthetase and three enzymes operating in the urea cycle (carbamyl phosphate synthetase, argininosuccinate synthetase, argininosuccinate lyase) increased significantly in hepatic and extra-hepatic tissue, such as the kidney and muscle in C. batrachus, during exposure to alkaline water. A significant increase in plasma lactate concentration noticed during alkaline exposure possibly helped in the maintenance of the acid-base balance. It is apparent that the stimulation of ureogenesis via the induced urea cycle is one of the major physiological strategies adopted by the walking catfish (C. batrachus) during chronic exposure to alkaline water, to avoid the in vivo accumulation of ammonia to a toxic level in body tissues and for the maintenance of pH homeostasis.     

黄颡鱼HSC70基因及其组织表达分析

热休克蛋白70（HSP70）与生物体的抗胁迫能力密切相关。本文采用RACE (Rapid amplification of cDNA ends) 技术,从黄颡鱼Pelteobagrus fulvidraco克隆到一种组成型热休克蛋白（HSC70）基因及其cDNA。该cDNA全长2245bp,包括5′非编码区82bp,3′非编码区225bp,开放阅读框(ORF) 1938bp,编码645个氨基酸组成的蛋白质。黄颡鱼HSC70基因含有8个内含子,与人、鼠、虹鳟和花斑溪鳉的HSC70基因内含子数目相同,位置相似。其中,最长内含子（873bp）位于5′端非编码区,其余内含子（长度在80－251bp之间不等）均在编码区以内。黄颡鱼HSC70基因编码的氨基酸序列与南方鲶的相似度最高,达96.13%,与欧洲银鲫和团头鲂的相似度分别为94.45%和94.14%。RT-PCR检测显示,正常情况下黄颡鱼HSC70在血细胞、心脏、肝、头肾、脾、鳃、肌肉和脑中均有表达,但表达量在鳃中最高,肌肉中最低;统计结果显示,热激后HSC70在血细胞、肝、头肾和脑中的表达量显著上升(p<0.05),而在其余组织中热激前后的表达差异不显著(p>0.05)。     

The gene structure and expression of the non-specific cytotoxic cell receptor protein (NCCRP-1) in Atlantic cod (Gadus morhua L.)

The non-specific cell receptor protein (NCCRP-1) serves an important function in target cell recognition and activation of non-specific cytotoxic cells in teleosts. Atlantic cod NCCRP-1 was identified in a suppression-subtractive cDNA library and NCCRP-1 from Atlantic salmon, rainbow trout, Japanese medaka and fathead minnow was found deposited in the GenBank as EST sequences. The predicted amino acid sequences of these receptors contain the characteristic functional domains representing NCCRP-1, and phylogenetic analyses support the identification of five NCCRP-1 orthologues. Cod NCCRP-1 is shorter and has a different intron/exon organization from the common carp and channel catfish counterparts, but shows high extent of conservation in NCCRP-1 signature motives. Quantitative real-time PCR analyses showed that the gene expression of cod NCCRP-1 was higher in the lymphoid organs, head kidney (90-fold) and spleen (30-fold), compared to the organ with lowest expression. NCCRP-1 gene expression was not induced by in vitro treatment of head kidney cells with polyinosinic polycytidylic acid (poly I:C) or lipopolysaccharide (LPS), or by in vivo injections with poly I:C or formalin killed Vibrio anguillarum. These results show that the cod NCCRP-1 gene is differentially expressed in organs, and that gene expression is not induced by the tested treatments.     

Characterization of a galactose binding serum lectin from the Indian catfish, Clarias batrachus: possible involvement of fish lectins in differential recognition of pathogens

A lectin with molecular mass around 200 kDa was isolated from the serum of the Indian catfish Clarias batrachus. The bioactivity of this serum lectin was Ca2+ and pH dependent. The lectin appeared to be specific for alpha-methyl galactose and sialoglycoproteins like porcine and bovine submaxillary mucin and could agglutinate human, rabbit, mice, rat and chicken erythrocytes. This fish lectin was able to specifically agglutinate different gram negative bacteria. When it was checked against different strains of the fish pathogen Aeromonas sp., it significantly altered the viability and pathogenicity of the bacteria. Binding of the lectin to Aeromonas sp., resulted in a dose dependent increase in the bactericidal activity of fish macrophages. However, when the lectin was checked against different gram positive bacteria it could not agglutinate or affect the viability of those strains and also failed to bring about any significant change in the bactericidal potential of fish macrophages. The lectin was able to induce the proliferation of head kidney lymphocytes of Clarias and helped in the release of 'IL-1' like cytokines from head kidney macrophages.     

Seminoma in hybrid catfish [Clarias batrachus (Linnaeus) female x Clarias gariepinus (Burchell) male

Spontaneous testicular tumors, seminoma, were noticed in four male hybrid catfish (C. batrachus female x C. gariepinus male) after the age of two years. The hybrids showed massive abdominal swelling with catchectic body and free lobulated, encapsulated tumors (> 325 g) within the serosanguinous fluid-filled peritoneal cavities. The tumor cells were large and polyhedral with prominent centrally located nuclei. Other vital organs appeared normal. It seems to be the first report of seminoma in hybrid catfish and possibly of genetic cause.     

Effects of copper, cadmium and chromium cations on the freshwater fish Clarias batrachus L

The data on the effects of cations such as Cu2+, Cd2+ and Cr6+ on the changes in the biochemical parameters in a freshwater fish, Clarias batrachus L., showed an increase of the protein content in the liver, kidney, stomach, intestine, testis and ovary, and a decrease in the muscle after Cu2+ and Cd2+ treatment as compared with control data; but the Cr6+ did not cause any changes of protein concentration in the kidney and testis. The administration of Cu2+ and Cd2+ increased the concentration of free amino acids in all the fish organs, whereas the Cr6+ did not changes this concentration in the muscle. A decrease in dry weight, and an increase in tissue permeability after these treatments were recorded in all the organs studied. In general, the above biochemical parameters of the organs were affected by treatments of the above cations in the following order: Cd greater than Cu greater than Cr over control values of C. batrachus, and their effects were markedly pronounced in the liver and kidney, followed by the intestine, stomach, muscle, testis and ovary in this species.     

Air-breathing catfish, Clarias batrachus upregulates glutamine synthetase and carbamyl phosphate synthetase III during exposure to high external ammonia

We assessed the possible upregulation of glutamine synthetase (GS) and typical 'fish type' carbamyl phosphate synthetase III (CPS III) in detoxification of ammonia in different tissues of the walking catfish (Clarias batrachus) during exposure to 25 mM NH(4)Cl for 7 days. Exogenous ammonia led to an increase in ammonia and urea concentrations in different tissues. The results revealed the presence of relatively high levels of GS activity in the brain, liver and kidney, unexpectedly, also in the muscle, and even higher levels in the intestine and stomach. Exposure to high external ammonia (HEA) caused significant increase of activities of GS, CPS III and CPS I-like enzymes, accompanied with the upregulation of GS and CPS III enzyme proteins in different tissues. Exposure to HEA also led to a sharp rise of plasma cortisol level, suggesting being one of the primary causes of upregulation of GS and CPS III enzymes activity. Liver perfusion experiments further revealed that exposure to HEA enhances the capacity of trapping ammonia to glutamine and urea by the liver of walking catfish. These results suggest that the upregulation of GS and CPS III activity in walking catfish during exposure to HEA plays critical roles to ameliorate the toxic ammonia to glutamine, and also to urea via the induced ornithine-urea cycle possibly through the involvement of cortisol.     

Cyclic AMP level and phosphodiesterase activity during 17alpha,20beta-dihydroxy-4-pregnen-3-one induction and theophylline inhibition of oocyte maturation in the catfish,Clarias batrachus

This study directly tested the hypothesis that the induction of oocyte maturation in the catfish Clarias batrachus is followed by a transient decrease in oocyte cyclic AMP (cAMP) level that is due to an increase in phosphodiesterase (PDE) activity. Further, the PDE inhibitor theophylline was used to investigate the possible role of PDE in the maturation-inducing action of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP), the physiological maturation-inducing steroid of this catfish species. The results obtained from batches of oocytes taken from the same donor at the same time clearly show a close relationship between dose-dependent induction of germinal vesicle breakdown (GVBD) and PDE activity with a concomitant decrease in cAMP in the oocytes treated with different concentrations of 17alpha,20beta-DP. In contrast, theophylline prevents GVBD and inhibits PDE activity by promoting cAMP accumulation in oocytes. A time-dependent decrease in PDE activity and an increase in cAMP content with a marked inhibition of GVBD were recorded even in oocytes pre-stimulated with 1 microgram/ml 17alpha,20beta-DP for 6 h and then treated with 1 mM theophylline for various times. These results suggest that cAMP plays a key role in the regulation of oocyte maturation in C. batrachus which may be mediated by PDE activity.     

Effects of dietary arsenic levels on serum parameters and trace mineral retentions in growing and finishing pigs

This experiment was conducted to investigate the effect of dietary arsenic (As) levels on growth performance, serum biochemistry, and the retention of iron, copper, and zinc in tissues of growing and finishing pigs. Ninety-six crossbred pigs were randomly allotted to four dietary treatments. The corn-soybean basal diets were supplemented with 0, 10, 20, and 30 mg As/kg. Arsenic trioxide was used as the arsenic source. The feeding experiment lasted for 78d. The results showed that the high arsenic diet decreased average daily gain (ADG) (p<0.05) and increased feed gain ratio (F/G) (p<0.05). Arsenic intake significantly increased (p<0.05) serum γ-gultamyltransferase (GGT), glutamic-pyruvic transaminase (GPT), and alkaline phosphatase (ALP) activities, and decreased (p<0.05) total protein, urea nitrogen, creatinine, and triglycerides. Glutamic-oxalacetic transaminase (GOT) activity, albumin, and cholesterol were not affected (p>0.05). Arsenic feeding elevated (p<0.05) liver and kidney copper concentration, but reduced (p<0.05) copper concentration in heat, bile, and lymphaden of intestine mesentery. There were increases in iron levels in liver, bile, spleen, thymus, and pancreas in pigs fed the high As diets (p<0.05), but iron contents in kidney, heart, and serum were decreased by the arsenic treatment (p<0.05). Zinc concentrations were increased (p<0.05) in liver, kidney, and thymus of pigs with arsenic treatment, but decreased (p<0.05) in bile and lymphaden of intestine mesentery. This study suggested that high dietary As levels could alter serum biochemical parameters and the retention of copper, iron, and zinc in the viscera of growing and finishing pigs.     

Viral RNA kinetics is associated with changes in trace elements in target organs of Coxsackie virus B3 infection

Trace elements are pivotal for the host defense, as well as potentially important for viral replication and virulence. Studies of sequential changes in viral replication in target organs of infection are sparse and a possible association with changes in specific trace elements is unknown. In this study Balb/c mice were infected with Coxsackie virus B3 (CVB3). Results indicated that sequential changes in viral replication (RT-PCR) were related to changes in trace element (arsenic, copper, iron, selenium and zinc) concentrations (as determined by ICP-MS) on days 3, 5 and 7 of the infection in serum, heart, lung, liver, pancreas, kidney, spleen, intestine and brain. After an initial viral peak on day 3, viral load drastically decreased in all organs, i.e. by >99% (serum), 97% (lung), 98% (liver), 60% (pancreas), 95% (kidney) and 93% (spleen), except in the heart, intestine and brain in which viral load increased after day 3. Selenium decreased in all organs except the heart while arsenic decreased in all organs except the kidney, spleen and brain. Moreover, selenium was negatively correlated to viral load in serum, liver, pancreas and intestine. To conclude, these findings give evidence that trace elements are directly involved in the replication of CVB3.     

The morphology of the accessory air-breathing organs of the catfish, Clarias batrachus: A SEM Study

The scanning electron microscope (SEM) has been used to study the morphology of the accessory air-breathing organs of the catfish Clarias batrachus . Although the gross morphology of the dendritic organs, the fan organs and the membrane lining the supra-branchial chambers differ, the nature of the respiratory surfaces are similar. The gaseous exchange surfaces of all three organs consist of double rows of paired lamellae, a feature strongly indicative of their common origin from the gills. The surfaces of the epithelial cells from the respiratory organs were seen to have numerous small projections consisting of microvilli and short microridges. This is in contrast to the concentric whorls of micro-ridges on the surface of cells from the interlamella regions of these organs.     

Starvation-induced impairment of metabolism in a freshwater catfish

Starvation induced changes in citrate synthase (CS), glucose-6-phosphate dehydrogenase (G6-PDH), lactate dehydrogenase (LDH), DNA, RNA, RNA/DNA ratio and protein were studied in the freshwater catfish Clarias batrachus. Starvation gradually decreased the activity of CS, G6-PDH and LDH in brain, liver and skeletal muscle of the freshwater catfish. The maximum reduction in these enzyme activities upto 35-45% was observed after 35 days of fasting. This shows substantial decline in aerobic and biosynthetic capacity during starvation period. DNA, RNA, RNA/DNA ratio and protein contents were also reduced from 40-67% which reflects reduction in an overall capacity of the protein synthesis. Starvation-induced macromolecular changes indicate impairment of metabolism in fish.     

Effects of cyclophosphamide on the immune system and disease resistance of Asian catfish Clarias batrachus

Cyclophosphamide (CYP), a multifunctional alkylating agent is known as a potent immunosuppressor in endotherms. Here, an experiment was conducted in an ectothermic Asian catfish species, Clarias batrachus to investigate its effect on non-specific and specific immunity as well as disease resistance against a common bacterial pathogen challenge. CYP was intraperitoneally injected for 3 consecutive days at a rate of 200 mgkg(-1) body weight. After 72 h post-injection, control and CYP-treated fish were screened for superoxide production through nitroblue tetrazolium (NBT) assay, myeloperoxidase (MPO) activity, packed cell volume (PCV) and total protein, lysozyme, alternative complement activity (ACH50) and natural haemagglutinin titre as a measure of non-specific immunity level as well as disease resistance against Aeromonas hydrophila challenge to vaccinated and unvaccinated fish. To study the effect on specific immunity, CYP was injected thrice at an interval of 7 days after bacterin injection and serum antibody titre was measured by bacterial agglutination titre assay. The results showed a significant (P<0.05) decrease in NBT and MPO activities, and percent survival against A. hydrophila challenge (both in unvaccinated and vaccinated fish) in CYP-treated fish, when compared to control fish. The above results support the immunosuppressive action of CYP in freshwater catfish, C. batrachus. The neutrophil activities, as measured through superoxide production and myeloperoxidase levels, might be important contributors during A. hydrophila infection and that treatment with CYP reduces phagocytic killing power and inhibits resistance against aeromoniasis.     

The relative impacts of native and introduced predatory fish on a temporary wetland tadpole assemblage

Understanding the relative impacts of predators on prey may improve the ability to predict the effects of predator composition changes on prey assemblages. We experimentally examined the relative impact of native and introduced predatory fish on a temporary wetland amphibian assemblage to determine whether these predators exert distinct (unique or non-substitutable) or equivalent (similar) impacts on prey. Predatory fish included the eastern mosquitofish (Gambusia holbrooki), golden topminnow (Fundulus chrysotus), flagfish ( Jordanella floridae), and the introduced walking catfish ( Clarias batrachus). The tadpole assemblage included four common species known to co-occur in temporary wetlands in south-central Florida, USA: the oak toad (Bufo quercicus), pinewoods treefrog (Hyla femoralis), squirrel treefrog (Hyla squirella), and eastern narrowmouth toad (Gastrophryne carolinensis). Tadpoles were exposed to different predators in wading pools under conditions similar to those found in surrounding temporary wetlands (particularly in terms of substrate type, the degree of habitat complexity, and temperature). Native predators were similar with respect to predation rate and prey selectivity, suggesting similar energy requirements and foraging behavior. Conversely, native fish predators, especially G. holbrooki, were distinct from the introduced C. batrachus. In contrast to expectations, C. batrachus were less voracious predators than native fish, particularly G. holbrooki. Moreover, survival of G. carolinensis and H. femoralis were higher in the presence of C. batrachus than G. holbrooki. We suggest that C. batrachus was a less efficient predator than native fish because the foraging behavior of this species resulted in low predator-prey encounter rates, and thus predation rate. In combination with a related field study, our results suggest that native predatory fish play a stronger role than C. batrachus in influencing the spatial distribution and abundance of temporary wetland amphibians in the landscape.     

Role of amino acid metabolism in an air-breathing catfish,Clarias batrachus in response to exposure to a high concentration of exogenous ammonia

The air-breathing ureogenic walking catfish (Clarias batrachus) faces various environmental constraints throughout the year leading to the problem of accumulation of toxic ammonia. In the present study, the possible role of conversion of accumulated ammonia to various non-essential free amino acids (FAAs) was tested in this fish under hyper-ammonia stress caused by exposing the fish at 25 mM NH(4)Cl for 7 days. Significant accumulation of ammonia of approximately two- to threefold was observed in different tissues (except in the brain), which was accompanied with the significant accumulation of non-essential FAAs in the NH(4)Cl-exposed fish. There was approximately two- to threefold increase of non-essential FAAs in different tissues and in the plasma of the NH(4)Cl-exposed fish compared to the control fish after 7 days of exposure, which was mainly attributable to the increase of Asp, Ala, Gly, Glu, Gln and taurine (Tau) concentrations in general, with certain tissue-specific variations. This was also accompanied with significant increase of activity of certain amino acid metabolism-related enzymes such as the glutamine synthetase (approx. two- to threefold), glutamate dehydrogenase (ammonia utilizing direction) (approx. twofold), aspartate and alanine aminotransaminases (approx. twofold) mainly in the liver, kidney and muscle of the NH(4)Cl-exposed fish. Thus, it appears that the walking catfish has the capacity of active conversion of accumulated ammonia to non-essential FAAs under condition of high concentrations of external ammonia. However, the increase of urea excretion rate due to active conversion of ammonia to urea via the induced urea cycle appears to be quantitatively much more important pathway than the increase of tissue levels of FAAs in dealing with a severe ammonia load.     

HG (II)-induced changes in some biochemical parameters in the freshwater fish Clarias batrachus

The data on the effect of Hg(II) on changes of biochemical parameters in the freshwater fish, Clarias batrachus L. showed an increased protein content in the liver, kidney, stomach, intestine, testis and ovary, and a decreased content of it in the muscle over control data. A decrease in DNA, RNA and dry weight and an increase in free amino acids, tissue permeability and the activities of protease and RNase were recorded in all the organs by the treatment with Hg(II). In general, the effect of Hg(II) was maximum in the liver and kidney, followed by the intestine, stomach, muscle, testis and ovary of this species.     

Histophathology and electron microscopy of channel catfish virus in infected channel catfish,Ictalurus punctatus (Rafinesque)*

A histologic and electron microscopic study was made on selected organs from channel catfish Ictalurus punctatus (Rafinesque) fingerlings that were experimentally infected with channel catfish virus (CCV). Histopathology was characterized by necrosis and haemorrhage in kidney and liver, and haemorrhage in the spleen and gastrointestinal tract. Virus replication occurred in nuclei of cells in the kidney, liver and spleen. Intranuclear inclusion bodies consisting of geometric crystalline arrays and lamellar structures were associated with virus replication.     

Two morphological types of pineal window in catfish in relation to photophase and scotophase activity: a morphological and experimental study

The pineal window is a transparent/translucent pineal covering on the dorsal surface of the cranium of certain fishes and is associated with light reactions of fish. In the present study, catfish species Clarias batrachus, Heteropneustes fossilis, Mystus vittatus, M. seenghala, and M. cavassius were examined for the type of pineal window present. Two morphologically different types of pineal window were found: an opaque-looking pineal window in C. batrachus and H. fossilis and a translucent type of pineal window in M. vittatus, M. seenghala, and M. cavassius. The distributional pattern of pigments in the melanophores at the pineal window were studied in terms of Melanophore Index (MI). In all of the species studied, a pineal foramen, a subepidermal lens-like tissue, and pineal end vesicle were present. Experiments were carried out on catfish having the opaque pineal window, as it is uncommon in catfish. Catfish with normal and shielded pineal window were exposed to conditions of artificial constant illumination (LL) and darkness (DD) to evaluate the effects of altered photoperiods on the state of pigmentation of melanophores at the pineal window. Recordings of diel activity patterns, which are light dependent in catfish, were carried out under both natural and artificial photoperiods in fish with a normal or shielded window in order to assess its functional nature. The existence of two morphologically and functionally different types of pineal window in a relatively closely related group of catfish has been demonstrated in this study. The nature of the opaque type of pineal window has been reconsidered based on new experimental evidence.