Clarias Magur Research Articles

Farmer Friendly Low Cost Breeding and Seed Rearing Technology of Walking Cat Fish (Clarias Magur)

Farmer Friendly Low Cost Breeding and Seed Rearing Technology of Walking Cat Fish (Clarias Magur)

Molecular characterization and ornithine-urea cycle genes expression in air-breathing magur catfish (Clarias magur) during exposure to high external ammonia

The air-breathing magur catfish (Clarias magur) is a potential ureogenic teleost because of its functional ornithine-urea cycle (OUC), unlike typical freshwater teleosts. The ability to convert ammonia waste to urea was a significant step towards land-based life forms from aquatic predecessors. Here we investigated the molecular characterization of some OUC genes and the molecular basis of stimulation of ureogenesis via the OUC in magur catfish. The deduced amino acid sequences from the complete cDNA coding sequences of ornithine transcarbamyolase, argininosuccinate synthase, and argininosuccinate lyase indicated that phylogenetically magur catfish is very close to other ureogenic catfishes. Ammonia exposure led to a significant induction of major OUC genes and the gene products in hepatic and in certain non-hepatic tissues of magur catfish. Hence, it is reasonable to assume that the induction of ureogenesis in magur catfish under hyper-ammonia stress is mediated through the activation of OUC genes as an adaptational strategy.

Non-invasive method for collection of Clarias magur (Hamilton, 1822) spermatozoa: A novel approach for catfish domestication, aquaculture and conservation

The current practice of captive breeding of Clarias magur involves killing male fish to obtain sperm for fertilisation. The sperm cannot be obtained by hand stripping because of the extremely low volume of seminal fluid in the testis. This sacrificial method of seed production has been applied to most catfish species. In this study, we reported an alternative approach that allowed harvesting of C. magur spermatozoa for artificial fertilisation without sacrificing male fish. Sexually mature C. magur males were anaesthetised and placed on the surgery table. A midline incision of 2.5 cm was made on the abdomen, and the testicular lobes were carefully lifted from the coelomic cavity. Using sterilised scissors and forceps, the testicular tissue was partially harvested, weighed to nearest 0.01 g, and macerated using a mortar and pestle in physiological saline. The abdominal incision was stitched using a surgical thread and topically treated with antimycotic ointment; subsequently, the fish were resuscitated in clean oxygenated water. Using the surgical technique, we harvested a maximum of 283 mg of testicular tissue, which corresponded to a count of 9.0 × 108 spermatozoa/mL, from a male fish weighing 210 g. By using the sacrificial approach, a maximum of 500 mg of testicular tissue, corresponding to a count of 16.0 × 108 spermatozoa/mL, was recovered from a male fish weighing 220 g. The surgically obtained spermatozoa were used to inseminate C. magur eggs; the cross resulted in healthy spawn with a fertilisation rate of 80%–98%. The results were similar to those obtained using the conventional sacrificial approach, which indicated the viability of partial surgical harvest of testicular tissue in seed production in C. magur for aquaculture without sacrificing male fish.

Optimal dietary lipid and protein level for growth and survival of catfish Clarias magur larvae

Large scale farming of Clarias magur (Indian walking catfish) is limited due to the non-availability of seed because of high mortality rate occurring during its larval rearing. The aim of the present study was to develop an optimal strategy based on the dietary lipids and proteins for enhancing the larval survival. A 3-week study from 14-35 day post hatching (dph) was conducted to elucidate the effects of dietary lipid and protein level on growth, survival and mRNA expression of growth related genes of C. magur larvae. Significantly (P < 0.05) high growth on average wet weight basis and survival rate was observed in 8% dietary lipid inclusion level followed by 10% and 12% lipid contents from 14-35 dph larval rearing phase. The effect of dietary lipid on the body composition was also reflected on the lipid content of larvae where a significant difference was observed. Similarly, growth-related genes like growth hormone (GH) and insulin-like growth factor I (IGF-I) exhibited a significantly high expression at 8% lipid level compared to 10% and 12% whereas, insulin-like growth factor binding protein3 (IGFBP3) showed a reverse trend. Based on the results, 8% dietary lipid along with the protein combination improved the performances significantly with regard to growth and survival. However, this experiment did not help to differentiate whether the differences observed at different time points are due to dietary protein level or the age of larvae. Hence, another experiment was conducted in which diets were formulated to contain different protein levels (55, 50 and 45%) with a constant 8% level of lipid in all the treatments along with commercial diet as control. The inclusion of 8% lipid and 55% protein was observed to show excellent performance with respect to growth, survival and the mRNA expression of important growth-related genes. Hence, a formulated diet with crude protein of 55% and 8% lipid can be suggested as the optimal microdiet for the larval rearing of C. magur.

Expression and activity of key lipases during the larval development of walking catfish (Clarias magur).

The study was conducted to investigate the expression and activity of key lipolytic enzymes during the ontogenetic development of Clarias magur. After partial characterization, the messenger RNA (mRNA) expression analysis of lipoprotein lipase (LPL), pancreatic triacylglycerol lipase (PL), and bile salt-activated lipase (BAL) genes along with the specific lipase activity were performed in larvae from Day 1 after hatching till 34-day posthatch (dph). Heterogeneous patterns of mRNA expression were shown by the important lipolytic enzymes and were detected before first exogenous feeding during the yolk-sac stage. LPL started increasing from 13 dph and peaked at 16 dph followed by a declining trend till 34 dph. However, the PL observed to be peaking at 9, 22, and 30 dph. Similarly, BAL showed an increasing trend from 11 to 22 dph with a significantly high level of mRNA expression at 16 dph. Later, the specific lipase activity was evaluated which appears at Day 1 after hatching with a progressive increase from 7 to 16 dph and a further declining trend afterwards with a peak at 22 dph. The results indicated the development of exocrine pancreas at 16 dph. Furthermore, the transcript levels and the activity of lipases were regulated with the age. Hence, the present study can be helpful in devising different strategies containing optimum lipid levels at a suitable stage of development for improving the survival during larval rearing. Furthermore, the study could be a baseline for elucidating the optimized dietary lipid levels of this catfish during its larval rearing.

Expression of alpha-amylase and growth-related genes during early larval developmental stages of Clarias magur

To understand the carbohydrate utilization ability during developmental stages of Clarias magur, the mRNA levels and enzyme activity of amylase were studied. Furthermore, the transcript levels of some important growth genes like growth hormone (GH), insulin-like growth factor I (IGF-I) and insulin-like growth factor binding protein (IGFBP) were evaluated. The enzymatic activity and mRNA expression of alpha-amylase along with expression of growth-related genes were examined from hatching until 34 days post-hatching (dph) by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Subsequently, mRNA expression pattern revealed the significantly (P < .05) high transcript level of alpha-amylase during early stages and early peaks were found at 3 to 6 dph, later the expression decreased till the end of experiment. Growth-related genes like GH, IGF-I and IGFBP showed a variable level of mRNA expression pattern from 0 dph to 34 dph. However, there was no significant difference (P > .05) in the mRNA expression of growth-related genes among the different time points of the present study. Moreover, the amylase activity revealed almost same pattern as that of its mRNA expression. In general, the present results suggested that C. magur has the ability to utilize the digestible carbohydrates during the early stages and can be helpful in developing low cost feeds for larvae.

Transcriptome analysis reveals novel insights in air-breathing magur catfish (Clarias magur) in response to high environmental ammonia

The facultative air-breathing magur catfish (Clarias magur) frequently face different environmental challenges, such as hyper-ammonia, and desiccation stresses in their natural habitats. All these stresses lead to higher accumulation of body ammonia, thereby causing various harmful effects to the fish due to its toxicity. Nonetheless, the mechanisms underlying ammonia-induced toxicity is yet not clear. In the present study, we used RNA sequencing and utilized a modified method for de novo assembly of the transcriptome to provide an exhaustive study on the transcriptomic alterations of magur catfish in response to high environmental ammonia (HEA; 25 mM NH4Cl). The final contig assembly produced a total of 311,076 unique transcripts (termed as unigenes) with a GC content of 48.3% and the average length of 599 bp. A considerable number of SSR marker associated with these unigenes were also detected. A total of 279,156 transcripts were successfully annotated by using various databases. Comparative transcriptomic analysis revealed a total of 3453 and 19,455 genes were differentially expressed in the liver and brain tissues, respectively, in ammonia-treated fish compared to the control. Enrichment analysis of the differentially expressed genes (DEGs) showed that several GO and KEGG pathway terms were significantly over-represented. Functional analysis of significantly elevated DEGs demonstrated that ammonia stress tolerance of the magur catfish was associated with quite a few pathways related to immune response, oxidative stress, and apoptosis, as well as few transporter proteins involved with ammonia and urea transport. Both liver and brain tissues showed HEA-mediated oxidative damage with consequent activation of antioxidant machinery. However, elevated ROS levels led to an activation of inflammatory cytokines and thus innate immune response in the liver. Conversely, in the brain ROS-mediated irreversible cell damages activated apoptosis via both p53-Bax-Bcl2 and caspase-mediated pathways. The present study provides a novel understanding of the molecular responses of this air-breathing catfish against the ammonia-induced stressors, which could elucidate the underlying mechanisms of adaptation of this facultative air-breather living under various environmental constraints.

Length–weight relationship and growth performance of different life stages of hatchery‐produced magur,Clarias magur(Hamilton, 1822)

Length–weight relationship (LWR) of 661 numbers of hatchery‐produced magur, Clarias magur of all the life stages such as hatchlings, fry, fingerlings, juveniles and adults, was studied. The LWR of all the life stages of magur was estimated and the equations are as follows, hatchlings W = 0.0052*L¹.²⁸² (r² = 0.8784), fry W = 0.0088*L³.⁰²⁸ (r² = 0.8113), fingerlings W = 0.0065*L³.²²⁶ (r² = 0.8906), juveniles W = 0.0028*L³.⁴²⁰ (r² = 0.8175) and adults W = 0.0017*L³.⁴⁹¹ (r² = 0.8310). A positive allometric growth (b > 3) was observed in all the life stages except hatchlings. The annual growth rate of magur was observed with the body length and weight of 24 ± 1 cm and 115 ± 17 g, respectively, under the commercial production system. This is the first kind of study that describes about the LWR and growth performance of all the life stages of hatchery‐produced C. magur catfish from hatchlings to adults under commercial production system. This study will be more useful to the hatchery managers and fish growers to optimize the resources and husbandry practices to improve the growth, survival, and biomass production.

Role of Taurine in Cellular Volume Regulation in Erythrocytes of Air-Breathing Catfish (Clarias magur) Under Osmotic Stress

The present study investigated the role of taurine in cellular volume regulation of erythrocytes isolated from freshwater air-breathing magur catfish (Clarias magur) under osmotic stress. Exposure of erythrocytes, pre-loaded with or without taurine, to hypotonic medium (− 80 mOsmol/L) led to a significant decrease in taurine level in the erythrocytes due to efflux of taurine through a band 3 transporter protein present in the plasma membrane with a slight increase in cellular volume of erythrocytes by 12–13%, whereas incubation of erythrocytes with hypertonic medium (+ 80 mOsmol/L) with taurine caused a significant uptake of taurine by the erythrocytes through the Na+-dependent pathway but without any loss of taurine from the erythrocytes which was accompanied by a slight decrease in the cellular volume of erythrocytes by 11–12%. Furthermore, a direct correlation between the osmosensitive cellular volume and taurine release could be established in the erythrocytes of magur catfish under hypotonic stress (r = 0.9921). In conclusion, the erythrocytes of air-breathing magur catfish do possess a very efficient taurine-dependent volume regulatory mechanism to resist the changes in cellular volume under anisotonic conditions as a unique adaptational strategy to defend against the osmosensitive changes in cellular volume of erythrocytes.

Antioxidant activity of endogenously produced nitric oxide against the zinc oxide nanoparticle-induced oxidative stress in primary hepatocytes of air-breathing catfish, Clarias magur

The facultative air-breathing magur catfish (Clarias magur) regularly encounter various environmental challenges including the exposure to nanomaterials discarded as industrial wastes in water bodies. The present investigation aimed at determining the possible ZnO NP-induced oxidative stress and also the antioxidant strategy of nitric oxide (NO), generated endogenously, in primary hepatocytes of magur catfish. Exposure of primary hepatocytes to different concentrations of ZnO NPs (5 and 10 μg/mL) led to a sharp rise of intracellular concentrations of hydrogen peroxide (H2O2) and malondialdehyde (MDA) within 6 h, which decreased gradually at later stages. This phenomenon was accompanied by an initial decrease of superoxide dismutase (SOD) and catalase (CAT) activities, the expression of their corresponding genes and the enzyme protein levels, with a subsequent significant increase of all these parameters at later stages. Most interestingly, exposure to ZnO NPs also stimulated the NO production by the primary hepatocytes as a consequence of induction of inducible nitric oxide synthase (iNOS) activity, higher expression of nos2 gene and iNOS protein. Furthermore, when the NO production by the hepatocytes was inhibited by either aminoguanidine (inhibitor for iNOS) or BAY (inhibitor for NFκB) in the presence of ZnO NPs, the intracellular concentrations of H2O2 and MDA was significantly elevated. This elevation was accompanied by a subsequent decrease of sod and cat genes expression, thereby suggesting that the inhibition of NO production leads to oxidative stress. Thus, it is believed that the magur catfish uses the strategy of stimulation of endogenous NO production by inducing the nos2 gene and simultaneous NO-mediated induction of sod and cat genes to defend against the NP-induced oxidative stress. It is the first report of such NO-mediated antioxidant strategy in any teleost fish to defend against the NP-induced oxidative stress and corresponding cellular damages.

Molecular characterization and expression profiling of 17-beta-hydroxysteroid dehydrogenase 2 and spermatogenesis associated protein 2 genes in endangered catfish, Clarias magur (Hamilton, 1822)

The 17-beta-hydroxysteroid dehydrogenase 2 (17β-HSD2) enzyme regulates steroid levels by the inactivation of estrogen and androgens. Spermatogenesis associated protein 2 (SPATA2) plays a vital role in spermatogenesis in vertebrates including fish. We report cloning and characterization of full cds of 17β-HSD2 and SPATA2 genes in Clarias magur. The full-length cDNA sequences of 17β-HSD2 and SPATA2 were 1187 bp (ORF 1125 bp) and 1806 bp (ORF 1524 bp) encoding 375 and 508 amino acids, respectively. Signal peptide analysis revealed SPATA2 is nonsecretory, while 17β-HSD2 is a secretory protein. Hydropathy profiles showed both proteins are hydrophilic in nature. Tissue distribution of both the genes revealed high mRNA level of SPATA2 in all tissues examined indicating its wide range of expression. 17β-HSD2 indicated higher expression in preparatory phase compared to spawning phase in ovary while it was opposite in case of testis. SPATA2 showed significantly higher expression in preparatory phase compared to spawning phase in both ovary and testis. Administration of OvatideTM (GnRH analog) resulted in upregulation of SPATA2 expression at 6 and 16 h post-injection while 17β-HSD2 showed upregulation only at 6 h post-injection. To the best of our knowledge, this is a first report on characterization of 17β-HSD2 and SPATA2 full-length cDNA in catfish.

Molecular identification of two cestodes species parasitizing freshwater fishes in India.

In the present study, molecular identification of two species of cestodes, Lytocestus indicus and Senga lucknowensis infecting freshwater fishes Clarias magur and Channa punctata, respectively in Manipur is carried out. To ascertain the taxonomic status of these helminth parasites, 18S gene marker was used. Phylogenetic analysis of 18S of Lytocestus sp. showed that it claded with L. indicus from Indian Isolate with a sequence similarity index of 99%. In case of Senga sp., the phylogenetic analysis revealed that it formed a separate clade with S. lucknowensis and Senga vishakapatnamensis, and the sequence similarity index showed maximum homogeneity with S. lucknowensis i.e., 99.8%. Thus, molecular characterization revealed that the two species of cestodes belong to L. indicus and S. lucknowensis.

Hyper-ammonia stress causes induction of inducible nitric oxide synthase gene and more production of nitric oxide in air-breathing magur catfish, Clarias magur (Hamilton).

Nitric oxide (NO) is an important signalling molecule that plays diverse physiological functions in several vertebrates including that of adaptation to various stressful stimuli. The air-breathing magur catfish (Clarias magur) is known to tolerate a very high external ammonia (HEA) stress in its natural habitats. We report here the possible induction of inducible nitric oxide (inos) gene and more generation of NO in magur catfish exposed to HEA. Exposure to HEA (25mM NH4Cl) for 14days led to the higher accumulation of NO in different tissues of magur catfish and also more efflux of NO from the perfused liver of NH4Cl-treated fish as a consequence of high build of toxic ammonia in body tissues. More synthesis and accumulation of NO in body tissues was associated with the induction of iNOS activity, which otherwise was not detectable in control fish. The stimulation of iNOS activity in HEA exposed fish was mainly due to induction of inos gene as evidenced by more expression of inos mRNA and also more abundance of iNOS protein in different tissues of magur catfish. Immunocytochemical analysis indicated the zonal specific expression of iNOS protein in different tissues of magur catfish. The augmentation of iNOS in the fish under HEA could be an adaptive strategy of the fish to defend against the ammonia stress through the generation of NO. Therefore, the present finding identifies the potential role of iNOS to enhance the adaptive capacity and survivability of catfish under various adverse environmental and pathological conditions that it faces in its natural habitats.

CmZNF384-like gene in hypoxia-tolerant Indian catfish, Clarias magur (Hamilton 1822) in hypometabolic states associated with acute hypoxia and summer aestivation

In the present study, a novel gene designated as CmZNF384L, of which partial transcript (GenBank accession number JK487591) was identified previously to encode a zinc finger 384-like protein in suppression subtractive hybridization studies, was examined for its genetic feature and gene expression under normoxic and hypoxic conditions in Clarias magur. The largest ORF of assembled contig contains 1365 nucleotide residues and encodes a protein of 454 amino acids with eight Kru¨ppel-type zinc finger C2H2 domains, which is considered as a member of beta-beta-alpha zinc finger superfamily. Bioinformatic analysis of protein-protein interactions by STRING 10.0 yielded the highest score or confidence on its interaction with COL1A1. Gene expression analyses revealed constitutive transcript expression under normoxia in the six tissues examined, while the transcript levels were significantly higher, after both short-term (experimental) and long-term (natural) hypoxia in head kidney and heart tissues, after short-term hypoxia in spleen, and after long-term hypoxia in liver. No significant difference of transcript levels was observed in muscle and brain tissues between normoxic and hypoxic conditions. The pattern, especially increased transcript level in heart to hypoxia, may indicate critical role of CmZNF384L in the molecular mechanism of highly complex hypoxia tolerance in the species.

Chitosan-eurycomanone nanoformulation acts on steroidogenesis pathway genes to increase the reproduction rate in fish

The study was undertaken to explore the molecular mechanism of eurycomanone, a major compound of Eurycoma longifolia plant in increasing the reproductive processes in the male fish model. Chitosan-nanoconjugated eurycomanone nanoparticles with a significant particle size [130 nm (CED1); 144.1 nm (CED2)] and stable zeta potentials (+49.1 mV and +30 mV) were synthesized and evaluated against naked eurycomanone (ED1 and ED2). In present study, short-term and long-term experiments were conducted to evaluate the effect of nano-formulation on expression of endocrine-related genes, circulating hormone concentrations (Follicle stimulating hormone, FSH; luteinizing hormone, LH; progesterone, testosterone and 17-β estradiol) and reproductive capacity of male Clarias magur. In short-term experiment, the sampling of tissues was done on hourly basis after injection of eurycomanone either alone or with chitosan and long-term experiment was carried for 21 days and in this the injection was repeated after 7 days and 14 days. Treatments CED1 and CED2 showed controlled and sustained surge of the transcript level of selected genes (except aromatase) and serum hormones (except 17β-estradiol) compared to ED1 and ED2 groups. The transcript levels of aromatase and serum 17β-estradiol hormone showed the declining trend in the chitosan conjugated groups. The gonadosomatic index (GSI), reproductive capacity, intracellular calcium and selenium and cellular structure of testes were improved in CED1 and CED2 groups compared to other treatments. Furthermore, the effect of chitosan conjugated eurycomanone was evaluated in primary testicular cells and an increase in the mRNA expression level of endocrine-related genes was detected. This is the first report of the use of chitosan conjugated eurycomanone and present study elucidates the molecular mechanism of eurycomanone in increasing the reproductive output in animals.

In Vitro Culture and Characterization of Testis-Derived Cells from Clarias magur (Hamilton, 1822)

A cell-based model offers many potential applications in biology. In vitro culture of testis-originated cells of Clarias magur was carried out using explant method. The cells were grown in different media compositions (including fetal bovine serum), concentrations, and temperatures. The developed culture was grown up to five passages. Leibovitz-15 (L-15) media with 20% FBS was found suitable, and culture conditions between 25 and 28 °C temperatures supported cell growth. The study is further focused on evaluation of the requirement of the other supplements along with the favorable basal media for growth and maintenance of testis-derived cell culture of C. magur. Epithelial-like cells in morphology were observed which were further confirmed with cell-specific markers. Chromosome observation revealed diploid number of 50 chromosomes as reported for the species. The species specificity of cultured cells was confirmed by amplification of two universal markers COI and 16S rRNA. The present study can be useful in the development of testicular cell line of C. magur.

Fabrication of chitosan nanoparticles loaded with aromatase inhibitors for the advancement of gonadal development in Clarias magur (Hamilton, 1822)

Aromatase inhibitors (AIs) block the conversion of testosterone to 17β-estradiol and thereby increase the serum testosterone levels. The present work was aimed for development and characterization of nanoconjugated AIs for studying the effect on the gonadal development in Clarias magur. Two AIs such as Fadrozole and Letrozole were used in this study and conjugated with chitosan nanoparticles. The size and zeta potential of the blank chitosan nanoparticles was 47.6 nm and 29 mV, respectively. The size of chitosan conjugated Letrozole (LZC) was 108.9 nm with a zeta potential of 18.9 mV while chitosan conjugated Fadrozole (FZC) showed the size of 150.5 nm with a zeta potential of 25.6 mV. Entrapment efficiency of LZC and FZC were 86.6% and 84.3%, respectively. The FTIR analysis confirmed the conjugation between chitosan and AIs. Fish receiving an intramuscular injection of LZC showed a higher level of serum testosterone than FZC injected fishes. On the other hand, a lower level of 17β-estradiol was observed in LZC treated fish. These results indicate the higher efficiency of letrozole over fadrozole. The oral delivery of Letrozole alone and conjugated with chitosan nanoparticles was carried out through feed (100 mg/kg of feed) to fingerlings. The LZC treated group showed advancement in the testicular development which was evident in serum hormonal analysis, histological examination of gonads and Gonadosomatic Index (GSI). Thus, the nano-formulation developed in the present study may be useful for improving the reproductive output in fish.

Body weight at harvest and its heritability estimate in Clarias magur (Hamilton, 1822) reared under mono and polyculture systems

&lt;p&gt;The present study was aimed to identify the effect of non-genetic factors on harvest body weight of &lt;em&gt;Clarias magur&lt;/em&gt; (Hamilton, 1822) reared under mono and polyculture systems and to estimate its heritability. Magur fishes from three different geographical regions &lt;em&gt;viz.,&lt;/em&gt; Andhra Pradesh, Assam and West Bengal were procured and stocked at Fresh Water Fish Farm (FWFF) of ICAR-Central Institute of Fisheries Education, Balbhadarpuram, Kakinada, Andhra Pradesh, India. A total of 139 pairs of fishes were mated to produce full-sib families of which only 42 families had either 15 or more offsprings or survived till tagging and of these a total of 1373 fishes were PIT (Passive Integrated Transponder) tagged. Tagged fishes were reared in mono and polyculture systems. In polyculture system, magur were stocked along with rohu &lt;em&gt;Lobeo rohita&lt;/em&gt; fishes. The average harvest body weight of magur reared in monoculture was 146.27±1.36 g and was significantly higher than the average body weight of fishes reared in polyculture (137.33±2.28 g) (p&amp;lt;0.05). Male fishes were significantly heavier than females at harvest. The heritability for harvest body weight from animal model was 0.55±0.76 and from single pair mating it was 0.63±0.10. The heritability for harvest body weight in mono and polyculture systems differed from one another.&lt;/p&gt;

Differential expression of multiple glutamine synthetase genes in air-breathing magur catfish, Clarias magur and their induction under hyper-ammonia stress

The present study demonstrates the unique presence of three different gs genes (cmgs01, cmgs02, and cmgs03) in air-breathing ureogenic magur catfish (Clarias magur), which is otherwise reported to be encoded by a single gene in higher vertebrates. Of these three genes, two (cmgs01and cmgs03) were identified as ‘liver’ form, predominantly expressed in liver cells, and the third one as ‘brain’ form (cmgs02), expressed chiefly in brain cells. Molecular characterization studies have revealed conservation of homologous active site residues in all the three gs genes. In silico analysis, accompanied by GS enzyme assay and Western blot analysis of different GS isoforms in different subcellular fractions indicated the mitochondrial localization of cmGS01 and cmGS03 in liver and kidney cells and cytosolic localization of cmGS02 in brain cells. Further, exposure of magur catfish to high external ammonia (HEA; 25 mM NH4Cl) led to a significant induction of multiple gs genes as evidenced by higher expression of different gs mRNAs at variable levels in different tissues. The cmgs01 and cmgs03 mRNA levels elevated significantly in liver, kidney, muscle, and gills, whereas the cmgs02 mRNA level increased considerably in the brain after 14 days of exposure to HEA. These increases in mRNA levels were associated with a significant rise in cmGS01 and cmGS03 proteins in liver, kidney, muscle, and gills, and the cmGS02 protein in the brain after 14 days of exposure to HEA. Therefore, it can be concluded that the unique differential expression of three gs genes and their induction under high ammonia level probably helps in detoxification of ammonia to glutamine and further to urea via the ornithine-urea cycle in ureogenic as well as non-ureogenic tissues of these magur catfish.

Expression and activity of trypsin and pepsin during larval development of Indian walking catfish (Clarias magur)

The gene expression of the trypsinogen and pepsinogen and activity of trypsin and pepsin in Indian walking catfish (Clarias magur) were investigated during the larval period to study the development of the digestive system. To this end, cDNA encoding the partial trypsinogen and pepsinogen were cloned and sequenced. Subsequently, the mRNA expression of genes was evaluated by quantitative polymerase chain reaction (qRT-PCR) along with the estimation of the activity of trypsin and pepsin. The mRNA expression pattern of trypsin appeared on 3rd-day post-hatching (dph) with a peak at 9 dph and 22 dph, while mRNAs of pepsin although detected at 4 dph but exhibited the significantly (P < 0.05) higher expression at 19 and 22 dph. The specific activity of trypsin increased from 11 dph onwards up to 16 dph. Similarly, the specific activity of pepsin enzyme showed a sudden increase from 13 to 19 dph. Based on these results, we suggest that the weaning time for C. magur larvae could be after 16 dph, as the activities were significantly increased after this stage which marks the functional development of the exocrine pancreas and stomach.