Molecular characterization of toll-like receptor 2 (TLR2), analysis of its inductive expression and associated down-stream signaling molecules following ligands exposure and bacterial infection in the Indian major carp, rohu (Labeo rohita)

Toll-like receptors (TLRs) are one of the key components of innate immunity. Among various TLR types, TLR2 is involved in recognizing specific microbial structures such as peptidoglycan (PGN), lipoteichoic acid (LTA), zymosan etc., and after binding them it triggers myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway to induce various cytokines. In this report, TLR2 gene was cloned and characterized in rohu (Labeo rohita), which is highly commercially important fish species in the farming-industry of Indian subcontinent. Full-length rohu TLR2 (rTLR2) cDNA comprised of 2691 bp with a single open reading frame (ORF) of 2379 bp encoding a polypeptide of 792 amino acids (aa) with an estimated molecular mass of 90.74 kDa. Structurally, it comprised of one leucine-rich repeat region (LRR) each at N-terminal (LRR-NT; 44-55 aa) and C-terminal (LRR-CT; 574-590 aa), 21 LRRs in between C and N-terminal, one trans-membrane (TM) domain (595-612 aa), and one TIR domain (645-790 aa). Phylogenetically, rohu TLR2 was closely related to common carp and exhibited significant similarity (93.1%) and identity (88.1%) in their amino acids. During embryogenesis, rTLR2 expression was detected as early as ∼7 h post fertilization indicating its importance in embryonic innate immune defense system in fish. Basal expression analysis of rTLR2 showed its constitutive expression in all the tissues examined, highest was in the spleen and the lowest was in the eye. Inductive expression of TLR2 was observed following zymosan, PGN and LTA exposure and Streptococcus uberis and Edwardsiella tarda infections. Expression of immunoregulatory cytokine interleukin (IL)-8, in various organs was significantly enhanced by ligands exposure and bacterial infections, and was correlated with inductive expression of TLR2. In vitro studies showed that PGN treatment induced TLR2, MyD88 and TRAF6 (TNF receptor associated factor 6) expression, NF-κB (nuclear factor kappa B) activation and IL-8 expression. Blocking NF-κB resulted in down-regulation of PGN mediated IL-8 expression indicating the involvement of NF-κB in IL-8 induction. Together, these findings highlighted the important role of TLR2 in immune surveillance of various organs, and in augmenting innate immunity in fish in response to pathogenic invasion. This study will be helpful in developing preventive measures against infectious diseases in fish.     

Family association between immune parameters and resistance to Aeromonas hydrophila infection in the Indian major carp, Labeo rohita

Seven innate immune parameters were investigated in 64 full-sib families (the offspring of 64 sires and 45 dams) from two year-classes of farmed rohu carp (Labeo rohita). Survival rates were also available from Aeromonas hydrophila infection (aeromoniasis) recorded in controlled challenge tests on a different sample of individuals from the same families. Due to strong confounding between the animal additive genetic effect and the family effects (common environmental + non-additive genetic), reliable additive (co)variance components and hence heritabilities and genetic correlations could not be obtained for the investigated parameters. Therefore, estimates of the association of challenge test survival with the studied immune parameters were obtained as product moment correlations between family least square means. These correlations revealed statistically significant (p < 0.05) negative correlations of survival with bacterial agglutination titre (−0.48), serum haemolysin titre (−0.29) and haemagglutination titre (−0.34); and significant positive correlation with ceruloplasmin level (0.51). The correlations of survival to aeromoniasis with myeloperoxidase activity, superoxide production and lysozyme activity were found to be not significantly different from zero (p > 0.05). Assuming that the negatively correlated candidate traits are not favourable as indirect selection criteria, the results suggest that ceruloplasmin level could potentially be a marker for resistance to aeromoniasis in rohu. The use of this immune parameter as an indirect selection criterion for increased resistance to aeromoniasis in rohu will, however, require that the parameter shows significant additive genetic variation and a significant genetic correlation with survival. Further studies are therefore needed to obtain a reliable heritability estimate for ceruloplasmin and its genetic correlation with survival from aeromoniasis.     

The immunosuppressive effect of alpha-permethrin on Indian major carp, rohu (Labeo rohita Ham.)

The immunosuppressive effects of bath exposure to a sub lethal concentration of the synthetic pyrethroid alpha-permethrin (3.05 x 10(-4) mg l(-1)) in the Indian Major carp, Labeo rohita was studied after 45 days' exposure. In some groups, the effects of alpha-permethrin on non-specific defences and serum enzymes of carp were investigated after challenge with Aeromonas hydrophila. Several nonspecific immune responses and serum enzymes were reduced after exposure of alpha-permethrin. Bactericidal activity of rohu serum was reduced significantly in pesticide and bacteria treated fish. The Glutamic Oxaloacetate Transaminase (GOT) and Glutamic Pyruvate Transaminase (GPT) activity were increased in immunosuppressed fish. Blood glucose level was elevated significantly and Hb% was reduced significantly in pesticide and bacteria treated fishes as compared to the negative control.     

Isolation of enriched carp spermatogonial stem cells from Labeo rohita testis for in vitro propagation

The in vitro culture system for spermatogonial stem cells (SSCs) is a powerful tool for exploring molecular mechanisms of male gametogenesis and gene manipulation. Very little information is available for fish SSC biology. Our aim was to isolate highly pure SSCs from the testis of commercially important farmed carp, Labeo rohita. The minced testis of L. rohita was dissociated with collagenase. Dissociated cells purified by two-step Ficoll gradient centrifugation followed by magnetic activated cell sorting (MACS) using Thy1.2 (CD90.2) antibody dramatically heightened recovery rate for spermatogonial cells. The purified cells were cultured in vitro conditions for more than two months in L-15 media containing 10% fetal bovine serum (FBS), 1% carp serum, and other nutrients. The proliferative cells were dividing as validated by 5-bromo-2′-deoxyuridine (BrdU) incorporation assay and formed colonies/clumps with the typical characteristics of SSCs A majority of enriched cell population represented a Vasa⁺, Pou5f1/pou5f1⁺, Ssea-1⁺, Tra-1-81⁺, plzf⁺, Gfrα1/gfrα1⁻, and c-Kit/c-kit⁻ as detected by immunocytochemical and/or quantitative real-time polymerase chain reaction (RT-PCR) analyses. Thus, Thy1⁺ SSCs were enriched with greater efficiency from the mixed population of testicular cells of L. rohita. A population of enriched spermatogonial cells could be cultured in an undifferentiated state. The isolated SSCs could provide avenue for undertaking research on basic and applied reproductive biology.     

The immunomodulatory effects of tuftsin on the non-specific immune system of Indian Major carp, Labeo rohita

The purpose of this study was to determine if injections of different dosages of tuftsin would enhance the immune response and disease resistance against the infections due to the opportunistic pathogens Aeromonas hydrophila and Edwardsiella tarda in Labeo rohita fingerlings. Hence, four different dosages of tuftsin in PBS suspension at the rate of 0, 5, 10, 15 mg kg(-1) body weight of fish were injected intraperitoneally to the fingerlings of L. rohita at 2-week intervals for four times. After every 2-week interval, different serum biochemical, haematological and immunological parameters of fish were evaluated. Biochemical and haematological parameters including serum total protein content, albumin content, globulin content, albulin:globulin ratio, glucose content, leucocyte counts etc.; cellular immune parameters including superoxide anion production, phagocytic activities, lymphokine production index etc.; humoral immune parameters including lysozyme activity, complement activity, serum bactericidal activity etc., in the fish were evaluated after every 2-week interval. After 56 days, fish were divided into two subgroups under each major treatment group for challenge with two pathogens A. hydrophila and E. tarda. The mortality (%) and agglutinating antibody titre was recorded on 28th day post challenge. Most of the immune parameters including leucocyte count, phagocytic ratio, phagocytic index, lysozyme activity, complement activity, and serum bactericidal activity were significantly (p相似文献   

Isolation and identification of yolk proteins in Indian major carp,Labeo rohita

Two yolk proteins (YP1 and YP2) from the ovaries of Indian major carp, Labeo rohita were isolated by gel filtration and partially characterized by the use of hydroxyapatite ultrogel column in conjunction with native PAGE. On native PAGE YP1 gave a single protein band, whereas YP2 of gel filtration revealed the contamination of YP1, which was removed by adsorption chromatography on hydroxyapatite ultrogel and then the YP2 was the purified one as judged by electrophoresis. Both YP1 and YP2 also stained for lipid and contained alkalilabile phosphorus. Therefore, both yolk proteins were lipophosphoprotein. The molecular weights of YP1 and YP2 were 620 kDa and 225 kDa respectively as determined by gel filtration on Sepharose 4B. When YP1 and YP2 were compared in relation to some physicochemical characteristics with yolk proteins of other oviparous vertebrates including fish, they were lipovitellin like. Antiserum to YP2 crossreacted with YP2 and vitellogenin suggesting that YP2 was the cleaved product of vitellogenin. Anti-YP2 antiserum was not crossreacted with native YP1, whereas reduced and/or denatured YP1 was crossreacted indicating the presence of antigenic determinants in the inner core region of YP1 polypeptide.     

Stage-dependent hatching responses of rohu (Labeo rohita) embryos to different concentrations of cryoprotectants and temperatures

Hatching performances of three embryonic stages of postfertilization rohu (Labeo rohita) (9-, 12-, and 15-h) were examined after treatment with various concentrations (0.5-4.5M) of two cryoprotectants (methanol and propylene glycol) supplemented with 0.1M trehalose. Different lengths of storage (1-48 h) and temperature (-4 degrees C to ambient) were studied. Of the three stages of embryonic development, the 12-h stage proved to be the most suitable stage for low temperature storage, showing the highest percentage of hatch out (72+/-2%) with 2.0M methanol and 0.1M trehalose. Methanol was more useful for storage at higher temperatures and propylene glycol at subzero temperatures. The maximum possible duration of effective storage of 12-h embryos was 31h in 2.0M methanol at 0 degrees C. No hatch out was found beyond 31h of storage with all concentrations of methanol at 0 degrees C. The results of interactions was that the optimal concentration of methanol was 3.0M at 4 degrees C, 2.0M at 0 degrees C, and 1.5M at 4 degrees C. Among three embryonic stages 12-h stage showed better results in trehalose treatment than sucrose. Among all concentrations of trehalose tested 0.1M gave the maximal survival rate of the rohu embryos.     

Development and characterization of three new diploid cell lines from Labeo rohita (Ham.)

Development of cell lines from fish for identifying the pathogenesis of viral diseases and for vaccine production against viral and bacterial diseases is imperative where they are of commercial importance. Three new diploid fish cell lines (RF, RH, and RSB) were developed from fin, heart, and swim bladder of an Indian major carp, Labeo rohita, commonly called Rohu. All the cell lines were optimally maintained at 28°C in Leibovitz‐15 medium supplemented with 10% FBS. The propagation of RH and RSB cells was serum dependent, with a low plating efficiency (<16%), whereas RF cells showed 20% efficiency. The cytogenetic analysis revealed a diploid count of 50 chromosomes. The cells of RF and RSB were found to be epithelial, where as the cells of RH were mostly fibroblastic. The viability of the RF, RH, and RSB cell lines was 75, 70 and 72%, respectively after 6 months of storage in liquid nitrogen. The origin of the cell lines was confirmed by the amplification of 496 and 655 bp fragments of 16S rRNA and Cytochrome Oxidase Subunit I (COI) of mtDNA. The new cell lines would facilitate viral disease diagnosis and genomic studies. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The effect of Euglena viridis on immune response of rohu, Labeo rohita (Ham.)

The study evaluated the effect of dietary doses of Euglena viridis on the immune response and disease resistance of Labeo rohita fingerlings against infection with the bacterial pathogen Aeromonas hydrophila. L. rohita fingerlings were fed with diet containing 0 (Control), 0.1 g, 0.5 g, 1.0 g Euglena powder kg⁻¹ dry diet for 90 days. Biochemical (serum total protein, albumin, globulin, albumin:globulin ratio), haematological (WBC, RBC, haemoglobin content) and immunological (superoxide anion production, lysozyme, serum bactericidal activity) parameters of fish were examined after 30, 60 and 90 days of feeding. Fish were challenged with A. hydrophila 90 days post-feeding and mortalities were recorded over 10 days post-infection. The results demonstrate that fish fed with Euglena showed increased levels of superoxide anion production, lysozyme, serum bactericidal activity, serum protein and albumin (P < 0.05) compared with the control group. Following challenge with A. hydrophila less survivability was observed in the control group (56.65%) than the group fed the experimental diets. The group fed 0.5 g Euglena kg⁻¹ dry diet showed the highest percentage survival (75%). These results indicate that Euglena stimulates the immunity and makes L. rohita more resistant to A. hydrophila infection.     

Expression analysis of heat shock protein genes during Aeromonas hydrophila infection in rohu,Labeo rohita,with special reference to molecular characterization of Grp78

Heat shock protein (Hsp) genes are stress-related genes that activate the host immune system during infection. Hsp genes expression in fish, studied during bacterial infections, is mostly confined to Hsp70 and Hsp90. The present study is an expression analysis of seven Hsp genes: Apg2, Hsp90, Hsp70, glucose-regulated protein 78 (Grp78), heat shock cognate 70 (Hsc70), Grp75, and Hsp30 during Aeromonas hydrophila infection in rohu, Labeo rohita. Forty-eight rohu juveniles were challenged with 3 × 10⁷ cfu bacteria per 20 g of body weight intraperitoneally. The expression of these genes was studied in infected liver, anterior kidney, and spleen tissues of rohu at different time periods: 0, 1, 3, 6, 12, 24, 48, 72 h, 7, and 15 days post-infection by qPCR. The Hsp gene modulation was greater in liver as compared to spleen and kidney tissues. Induced expression of Apg2, Hsp90, Grp78, Grp75, and Hsc70 was noticed during peak periods (3 to 24 h post-challenge) of bacterial infectivity. Hsp70 was found to be down-regulated during the process of infection. In contrast to the other six genes, Hsp30 showed a variable expression pattern in all three tissues. Grp78 was found to be the most highly (1,587-fold) expressed gene in liver at 12 h post-challenge. Further, molecular characterization of Grp78 revealed it to be a highly conserved Hsp gene, essential not only during infection but also during early developmental stages of rohu, and its expression was noticed in all organs studied except in gill tissues, which indicated its potential immune regulatory role during infection process.     

Microsatellite markers from the Indian major carp species,Catla catla

The Indian major carp Catla catla is indigenous to the major river systems of Northern India, Pakistan and Bangladesh, and is an important contributor to aquaculture production in India and Bangladesh. Five polymorphic microsatellite loci, developed from Catla catla are described and their utility in other major carp and cyprinid species is tested.     

Studies on the age and growth of the bata,Labeo bata (Ham.) (Cyprinidae,Teleostei) from the river Kali,India

Age and growth of Labeo bata (Ham.) was studied by the analysis of annuli found on the scale and by length-frequency distribution. The fish attained lengths of 131, 194, 236, 277, 314, 341 and 364 mm at the end of the 1st, 2nd, 3rd, 4th, 5th, 6th and 7th years of life respectively. The increase in length of scale bears a constant relationship with the increase in length of fish, and regression analysis yielded a straight line between scale and body length. Calculated values could be expressed as: Y = –2.534 + 0.064 XThe growth rate of the fish was found high during the 1st and 2nd years and decreased gradually afterwards till the 7th year. Both sexes showed more a or less similar growth rate and attained a similar longevity. Growth trend of the fish confirmed the von Bertalanffy growth equation: Lt = 450(l–e^{–0.2165(t+0.5963))} The seasonal growth curve was chiefly influenced by feeding intensity in fishes of 1st year class while in adults it was affected by feeding intensity as well as by maturation of the gonads.     

Exploring the gut microbiota composition of Indian major carp,rohu (Labeo rohita), under diverse culture conditions

Gut microbiota of freshwater carps are often investigated for their roles in nutrient absorption, enzyme activities and probiotic properties. However, little is known about core microbiota, assembly pattern and the environmental influence on the gut microbiota of the Indian major carp, rohu. The gut microbial composition of rohu reared in different culture conditions was analysed by 16S rRNA amplicon sequencing. There was variation on gut microbial diversity and composition. A significant negative correlation between dissolved oxygen content (DO) and alpha diversity was observed, thus signifying DO content as one of the key environmental factors that regulated the diversity of rohu gut microbial community. A significant positive correlation was observed between phosphate concentration and abundance of Actinobacteria in different culture conditions. Two phyla, Proteobacteria and Actinobacteria along with OTU750868 (Streptomyces) showed significant (p < 0.05) differences in their abundance among all culture conditions. The Non-metric multidimensional scaling ordination (NMDS) analysis using Bray-Curtis distances, showed the presence of unique gut microbiota in rohu compared to other herbivorous fish. Based on niche breadth, 3 OTUs were identified as core generalists, persistent across all the culture conditions whereas the specialists dominated in the rohu gut microbiota assembly. Co-occurrence network analysis revealed positive interaction within core members while mutual exclusion between core and non-core members. Predicted microbiota function revealed that different culture conditions affected the metabolic capacity of gut microbiota of rohu. The results overall indicated the significant effect of different rearing environments on gut microbiota structure, assembly and inferred community function of rohu which might be useful for effective manipulation of gut microbial communities of rohu to promote better health and growth under different husbandry settings.     

Establishment of a leukocyte cell line derived from peritoneal macrophages of fish,Labeo rohita (Hamilton, 1822)

A continuous leukocyte cell line with phagocytic activity was established from peritoneal macrophages of rohu, Labeo rohita (LRPM). LRPM was initiated from adherent mononuclear leukocytes isolated from peritoneal cavity of rohu, without use of any growth factors or feeder cells. These cells exhibited maximum growth at 30 °C in L-15 medium containing 20 % foetal bovine serum, and has been subcultured for more than 60 passages till date. The cells showed 85 % viability after 6 months of storage in liquid nitrogen. The species of origin of the LRPM was confirmed by the amplification and sequencing of 655 bp fragment of cytochrome oxidase subunit I of mitochondrial DNA. Functionally, LRPM showed phagocytic activity of yeast cells and fluorescent latex beads as evaluated by phase contrast and scanning electron microscopy, respectively. Immuno-modulators such as bacterial lipopolysaccharide and phorbol myristate acetate resulted in functional activation of LRPM; and enhanced their microbicidal activity through release of reactive oxygen species and nitric oxide. Culture supernatant from activated cells also revealed lysozyme activity. Cells of LRPM were positive for alpha-naphthyl acetate esterase enzyme indicating macrophage lineage. Our results indicate that this cell line can be a useful in vitro tool to study the role of macrophages in teleost immune system and to evaluate the effects of new aquaculture drugs. The LRPM cell line represents the first reported leukocyte cell line of peritoneal origin from any freshwater species of fish.     

Organization of the olfactory system of the Indian major carp <Emphasis Type="Italic">Labeo rohita</Emphasis> (Ham.): A scanning and transmission electron microscopy study

Catla catla, Labeo rohita, and Cirrhinus mrigala represent important alimentary fish in India. Their reproduction/breeding depends on seasons. Fish perceive external factors-stimuli and chemical signals through the olfactory system that plays the key role in central regulation of reproduction. However, no electron microscopy data are available on organization of olfactory components of these fish. We studied organization of the olfactory organ in male L. rohita using scanning (SEM) and transmission electron microscopy (TEM). This organ consists of olfactory epithelium, a short nerve, and olfactory bulb. The olfactory organ is ovoid in shape and consists of about 47–52 lamellae in adults and about 14–20 lamellae in fingerlings. These lamellae originate from the midline raphe. By SEM, microvillar sensory and ciliated non-sensory cells were observed in the lamellae. TEM revealed microvillar receptor cell with rough endoplasmic reticulum and Golgi apparatus towards apical end. Basal cells were present at the base of receptor cell, supporting cells were located adjacent to the olfactory receptor neurons, while epithelial cells—in the nonsensory part of olfactory epithelium. Mast, blastema, and macrophage cells were also found at the basement membrane. This work is the first publication on ultrastructural organization of the olfactory system of the Indian major carp, which provides information about morphological and ultrastructural organization of the olfactory system and opens new avenues for further investigation of chemical neuroanatomy, sensory signal processing, and neural regulation of reproduction in the Indian major carp.     

Effect of dietary supplementation of probiotic and vitamin C on the immune response of Indian major carp, Labeo rohita (Ham.)

The immunostimulatory effect of probiotics and vitamin C has been established in many systems including fish. An investigation was carried out to study the effect of dietary supplementation of a probiotic bacterium "Bacillus subtilis", vitamin C in the form of ascorbyl polyphosphate and their combination on the immune response of Indian major carp, rohu, (Labeo rohita Ham.) fingerlings fed for a period of 60 days. The total serum protein and globulin content was significantly higher (p<0.05) in probiotic (B. subtilis @ 10(8) CFU/g of the feed) fed group while the respiratory burst activity of blood neutrophils was significantly high in vitamin C (ascorbyl polyphosphate @100 mg per kg diet) fed group. The antibody level was significantly high in Bacillus subtilis treated group followed by the probiotic (B. subtilis @ 10(8) CFU/g of the feed) and ascorbyl polyphosphate (ascorbyl polyphosphate @100 mg per kg diet) combined group. The least percentage of mortality was recorded in B. subtilis treated group (25%) followed by 35 and 40% in ascorbyl polyphosphate treated and B. subtilis and ascorbyl polyphosphate combined groups, respectively.     

Organization of olfactory system of the Indian major carp Labeo rohita (Ham.): a study using scanning and transmission microscopy

Catla catla, Labeo rohita, and Cirrhinus mrigala are important alimentary fish in India. Their reproduction (breeding) depends on season. The fish perceive external factors-stimuli and chemical signals through the olfactory system that plays the key role in the central regulation of reproduction. However, in the available literature, any electron microscopy data on organization of olfactory elements in these fish are absent. We have studied ultrastructure of the olfactory organ in male L. rohita by using scanning (SEM) and transmission electron microscopy (TEM). The olfactory organ consists of olfactory epithelium, a short nerve, and olfactory bulb. The organ has oval shape and consists of approximately 47-52 lamellae in adult fish and of 14-20 lamellae in fish at the stage of fingerling. These lamellae originate from the midline raphe. By using SEM, the presence of microvillar sensory and ciliated non-sensory cells in these lamellae is shown. By using TEM, a microvillar receptor cell is revealed, which has rough endoplasmic reticulum and Golgi apparatus towards the apical end. Basal cells are found at the base of the receptor cell; supporting cells are located adjacent to olfactory receptor neurons, while epithelial cells--in the non-sensory part of olfactory epithelium. Mast, blastema and macrophages cells are also found in the basal lamina. This work is the first publication on structural organization of olfactory system of the Indian major carp, which provides information about morphological and ultrastructural organization of olfactory system and opens new opportunities for study of chemical neuroanatomy, sensory signal processing, and nervous regulation of reproduction of the Indian major carp.     

Passive transfer of maternal antibodies and their existence in eggs, larvae and fry of Indian major carp, Labeo rohita (Ham.)

Lack of immune competence in the early stages of life leads to severe mortality in larval stages of different fish species including Indian major carp (IMC). Investigation through indirect enzyme linked immunosorbent assay (ELISA) and agglutination test revealed a significant increase in specific serum antibody response in the brood fish of Indian major carp, Labeo rohita (Ham.) following immunisation with a virulent Aeromonas hydrophila bacterin 1 month prior to breeding, which was transferred to larvae through the egg. No significant differences (P > 0.05) in mean antibody levels in larvae at the 1st and 2nd weeks post-hatch was recorded while a slight rise in antibody level was observed in 3-week-old fry, perhaps due to exposure to A. hydrophila present in the aquatic environment. Immunised brood fish serum, egg and larval extracts in non-reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent western blot analysis revealed an antibody molecule of approximate molecular weight 210 kDa. On challenge with virulent A. hydrophila, a significant reduction in mortality was recorded in immunised larvae and fry (58.0, 43.75 and 37.14% in the 1st, 2nd and 3rd week, respectively) relative to control fish (87.0, 79.0 and 76.4% in 1st, 2nd and 3rd week, respectively). The present study indicated the role of maternally derived antibody in protection of hatchlings of Indian major carp against specific pathogens.