Molecular characterization and expression patterns of Nanog gene validating its involvement in the embryonic development and maintenance of spermatogonial stem cells of farmed carp,Labeo rohita

Background: The homeobox containing transcription factor Nanog plays crucial roles in embryonic development/proliferation and/or maintenance of spermatogonial stem cells(SSCs) via interacting with transcription factors such as Oct4 and Sox2 in mammals. However, knowledge of its exact mechanistic pathways remains unexploited. Very little is known about teleost Nanog. Information on the Nanog gene of farmed rohu carp(Labeo rohita) is lacking. We cloned and characterized the Nanog gene of rohu carp to understand the expression pattern in early developmental stages and also deduced the genomic organization including promoter elements.Results: Rohu Nanog(LrNanog) cDNA comprised an open reading frame of 1,161 nucleotides bearing a structural homeodomain; whereas, the genomic structure contained four exons and three introns suggesting that it is homologous to mammalian counterparts. Phylogenetical y, it was closely related to freshwater counterparts. Protein sequence(386 AA of42.65 kDa) comparison revealed its low similarity with other vertebrate counterparts except that of the conserved homeodomain. Tissue distribution analysis revealed the existence of LrNanog transcripts only in adult gonads. The heightened abundances in the ovary and proliferating spermatogonia suggested its participations in maternal inheritance and male germ cell development. The potentiating abundances from fertilized egg onwards peaking at blastula stage vis-à-vis decreasing levels from gastrula stage onwards demonstrated its role in embryonic stem cell development. We also provided evidence of its presence in SSCs by western blotting analysis. Further, the promoter region was characterized, predicting a basal core promoter and other consensus elements.Conclusion: The molecular characterization of LrNanog and its documented expression profiling at transcript and protein levels are indicative of its functional linkage with embryonic/spermatogonial stem cell maintenance. This is the first report of LrNanog genomic organization including its promoter sequence information with predicted regulatory elements of a large-bodied carp species. This will be useful for elucidating its mechanism expression in future. Nanog could be used as a potential biomarker for proliferating carp SSCs.

Characteristics of dehalogenase from bacteria isolated from the Gut of Pond-reared Rohu (<i>Labeo rohita</i>) Juveniles in Myanmar

Unwarranted accumulation of halogenated compounds in the rivers and streams has in recent years emerged due to the widespread use agricultural pesticides. The presence of these halogenated compounds in the water does not only suppress the immune system of fish but adversely induce serious morbidity and mortality among cultured stocks. Importantly, gradual accumulation of these compounds in the system of cultured and wild freshwater fish species cultured in ponds and floating net-cages in dams and rivers, respectively, poses some risks to humans, the end users. In this study, we attempted to isolate bacteria from the gut of pond-reared rohu (Labeo rohita) in Myanmar, screened the isolated bacteria for dehalogenase gene using molecular technique and tested the ability of these bacteria to degrade halogenated compounds in vitro. The eight bacterial strains studied were identified as Enterobacter mori strain MK- 121001, Enterobacter cloacae strains MK121003, MK-121004, MK121010, Ralstonia solanacearum strain 121002, Acinetobacter baumannii strain MK121007, Chromobacterium violaceum strain MK121009 and Pantoea vagans strain 121011. Only three bacterial strains (MK121002, MK121007 and MK121009) were capable of degrading 2,2-dichloropropionic acid (2,2-DCP) as the sole carbon source up to a final substrate concentration of 20 mM. Their mean growth doubling time ranging from 6-23 hours with the maximum of chloride ion released of 85%. PCR amplifica- tion with oligonucleotide primers designed from group I dehalogenase revealed the presence of deha- logenase genes in all isolates suggesting dehalogenase gene in strains 121001, 121003, 121004, 121010 and 121011 were silenced. In contrast, group II dehalogenase primers did not show any PCR amplification. These results suggest that MK121002, MK121007 and MK121009 only encode a group I dehalogenase and its non-stereoselectivity is in agreement with previoulsly described group I haloacid dehalogenase. The partial gene sequences were blasted but no significant sequence identity was observed. Therefore, it suggest the 2-haloacid dehalogenase of MK121002, MK12-1007 and MK121009 might be a novel group I 2-haloacid dehalogenase. The results indicated a broad distribution of dehalogenation genes in many micro- bial genomes that harbor dehalogenase(s) due to the exposure of the microorganisms to the naturally occurring or man-made halogenated compounds in the environmental systems. So far, microorganisms capable of producing dehalogenases were mainly isolated from soil and scarcely from aquatic animals and their environments. To the authors’ knowledge, this is the first report on the isolation of dehalogenase-producing bacteria from the gut of pond-reared freshwater fish, Labeo rohita, in Myanmar.

Multi-species probiotics enhance survival,growth,intestinal microbiota and disease resistance of rohu(Labeo rohita)larvae

Multi-strain probiotics provide the most dependable approaches to improve health,immune response,and disease resistance in farmed fishes.In the present study,we examined the effects of multi-species probiotics on the survival,growth,immune response,and disease resistance of rohu(Labeo rohita)larvae.Newly hatched larvae from the day of first feeding(average weight of 0.003 g)were reared with multi-species probiotics having a combination of Bacillus subtilis(109 colony forming units(cfu)/mL)and Lactobacillus spp.(Lactobacillus plantarum,Lactobacillus buchneri-1011 cfu/mL)in water containing doses of 0(control-C),0.5 mL/L(treatment 1-T1),and 1.0 mL/L(treatment 2-T2)in triplicates for 90 days.After the experiment,a challenge test was performed to assess the fish's resistance to pathogenic Aeromonas veronii.Significantly higher survival was recorded in larvae of treated groups(87%in T2 and 79%in T1)compared to the control(62%).Significantly higher growth performance(weight gain and specific growth rate—SGR)was shown by the probiotic-treated larval groups compared to the control.Probiotic supplementation resulted in significantly higher counts of total viable colony(TVC)and lactic acid bacteria(LAB)in the intestine.Some immunological parameters(mucosal fold fattening,goblet cell abundance,expansion of lamina propria and enterocytes)of the gut were significantly better in probiotic-treated fish.The liver of treated fish showed irregular shape nuclei turning into regular shape and reducing spaces between the hepatic cells.Probiotic-treated fish had the highest post-challenge survival rate(90%)against A.veronii infection.The erythrocytes of challenged fish treated with probiotics had significantly lower frequencies of various nuclear and cellular abnormalities.These findings suggest that multi-species probiotic supplements could improve the survival,growth,health status,and immune response of rohu in the early stages of its development.

Study on histo-biochemical biomarkers of chromium induced toxicity in Labeo rohita

Aquatic environment gets highly polluted due to the presence of heavy metals,which are usually discharged into water bodies because of rapid industrialization.The main purpose of this study is to evaluate the effects of chromium toxicity on histo-biochemical biomarkers of Labeo rohita.A total of 240 randomly selected fish were distributed into four aquaria(n=20).Three groups designated as T_(1),T_(2) and T_(3) were exposed with 1/3rd,1/5th and 1/10th of LC_(50) of chromium chloride,and the fourth group was chromium-free control(T0).Results showed a significant(p<0.05)decrease in blood parameters such as hematocrit,red blood cells,hemoglobin,mean cell hemoglobin and platelets.In contrast,mean cell volume and white blood cell values increased significantly.The results obtained by comet assay showed that DNA damage was increased significantly(p<0.05)in chromium-exposed groups compared to the control.At the histopathological level,observed alterations in the gill tissues are degeneration of epithelium,fusion of secondary lamellae,lamellar curling,hypertrophy,telangiectasis and oedema,while in the liver is necrosis,dilation of the vein,congestion of blood vessels,hepatocytes degeneration,melano-macrophage centers,pigmentation and hemorrhage.In the kidney,histological alterations such as glomerular destruction,necrosis,damaged blood vessels,oedema and pigmentation were seen.Those results suggest that chromium has the ability to bring variations DNA integrity,histopathology and blood of Labeo rohita.

EPIZOIC ALGAE FROM A TELEOST FISH, Labeo rohita

Epizoic algae growing upon molluscs and turtles were earlier reported by Hutchinson and La Rivers. Recently Leghari (1983) has reported epizoic algae from some molluscs of Sind. Many species of green and blue-green algae have been recorded as growing upon the body of fish. Vinyard found that Cladophora sp. occurred on the operculum of Ictiobus bubalus from lake Texoma, USA. In 1960 Hoffman et al. re-

Effectiveness of oxytetracycline in reducing the bacterial load in rohu fish(Labeo rohita,Hamilton)under laboratory culture condition

Objective:To observe the effectiveness of most widely used antibiotic,oxytetracycline(OTC)in reducing the bacterial load in rohu fish under artificial culture condition in the laboratory.Methods:The experiment was conducted in the Faculty Fisheries,Bangladesh Agricultural University,Mymensingh-2202.The fish were reared in 8 aquaria where fish in 5 aquaria were used for replication of the treatment(experimental group)and fish in remaining 3 aquaria were considered as a control(Control group).OTC was fed to the fish in the experimental aquarium at the rate of 2 g/kg through diet twice daily whereas fish reared under control condition was given feed without antibiotic for 20 d and bacterial content in the aquarium water,gills,skin and intestine of fish were estimated at every alternative day after onset of the experiment.Results:Rearing the fish with OTC treated feed resulted in gradual decrease of bacterial load in the aquarium water,gills,intestine and skin of the fish whereas the content remain unchanged Or little increased in the control group.W ater quality parameters such as dissolved oxygen,pH and total hardness were within the suitable range in the experimental aquarium but not in control aquaria throughout the experimental period.Conclusions:These results suggest that OTC could be a potential antibiotic to reduce the bacterial load in fish and can be used commercially for maintaining the fish health in aquarium conditions.

鲮、麦鲮和野鲮之RAPD的遗传标记

以野生和池塘养殖的64个凌（Cirrhinamonlitorella）、10个麦坡（C．mrigola）和10个野峡（Labeorohita）样本为材料进行了随机扩增多态性DNA（RAPD）遗传分析。通过26个10bp随机引物扩增鲮、麦峻和野峻样品分别得到107、73、87个DNA片断，其多态位点百分率分别为35．5％，39．7％，37．9％。鲮与野鲮，麦鲮与野鲮及鲮与麦鲮的遗传距离分别为0．22，0．58，0．64。利用UPAMA和NJ建立了3种鲮之间分子水平上的亲缘关系图，认为鲮与野鲮亲缘关系比麦鲮与野鲮和鲮与麦鲮更近，与同功酶相近，与传统分类不一致。

Gel-Forming Ability of Rohu as Affected by Egg White Powder Addition

The gel-forming ability of rohu (Labeo rohita) mince with and without egg white powder (EW) was investigated. Gel from washed mince (washed gel) was prepared under two setting conditions: kamaboko (40°C) and modori (60°C). The gel-forming ability of kamaboko and modori gels was improved by the addition of EW at 2%. The autolytic inhibition of kamaboko gel was obtained in gel added with 2% EW, and 1% EW of modori gel. No marked change was observed in the TCA-soluble peptide content of either gel with the addition of EW above 1%. No effect on the whiteness of both gels was shown after the addition of EW. The addition of EW exhibited smaller cavities and a more compact fibrous network in microstructure.

Bioaccumulation of Trace Metals in Tissues of Rohu Fish for Environmental Risk Assessment

In the present study, the two lakes, Vengaiah lake (Lake A-Sewage polluted receiving discharge from storm water drain) and Yellamallappa Chetty lake (Lake B-Industrially polluted) situated near Krishnarajpuram-Hoskote taluk, Bangalore, Karnataka were selected for analysis of trace metals viz., arsenic, aluminium, cadmium, lead, mercury, iron, copper and zinc in water samples. Muscle and gill tissues of freshwater fish Labeo rohita reared in these water bodies were analysed for bioaccumulation of trace metals. Hebbal fish farm was considered as a reference site (Control site) for water and fish samples. Trace metals were analysed by atomic absorption spectroscopy and values were compared with those recommended by FAO/WHO in water and fish samples. Trace metals such as Al, As and Hg were detected in the water sampled from lake B which is attributed to the differences in the sources of pollutants. Fish tissues viz., muscle and gills sampled from Lake B exhibited high concentration of Al, Pb and Cd content showing a positive correlation with their concentration in water samples. The remaining metals as Cu, Zn and Fe were detected in water sampled from all water bodies and also in the fish tissues. Gills exhibited higher concentration of metals in fish from lake B. Bioaccumulation of these trace metals in fish tissues may cause potential danger to human health when consumed on regular basis. Thus necessary remedial measures are required to combat water contamination and its management keeping in view the aquatic ecosystem and public health. Values were statistically significant at P < 0.0001.

Effect of Dietary Crude Protein Level of Feed on Growth and Survival of Rohu Fry

Fry rearing is one of the important stage which aims at obtaining high growth and survival for production of fingerlings required for stocking into grow out ponds as well as rehabilitation in natural habitat. This experiment was conducted with the purpose to test the effect of dietary crude protein level (CP %) of feeds prepared from similar feed ingredients in different ratios on growth performance and survival rate of Rohu fry (Labeorohita). An initial density of 100 fry/m2 was maintained in hapa fixed in the cemented tank. The dietary CP% level of feed tested were 20% CP, 25% CP, 30% CP, and 35% CP fed at 5% body weight. The experiment ran for 53 nursing days. The results showed that there was no significant difference (P > 0.05) in the growth rate (g/day) of fry among treatments. Rather group fed with higher protein level grew comparatively better indicating possibility of increasing need of protein in diets. However, the survival rate (%) of rohu fry was significantly different (P < 0.05) in each tested CP% level of feed. Highest survival (82%) of fry was found in the feed of CP 35% and lowest (56%) in the feed of CP 25%. It was predicted that feed with increasing level of CP % in diet is essential for increasing survival rate.

印度恒河蓝黑鲮与南亚黑鲮杂交个体的分子检测(英文)

利用鉴别性的同工酶位点从印度恒河水系的支流Banganga中检测到了蓝黑鲮(Labeo calbasu)与南亚黑鲮(Labeorohita)的一个杂交个体。线粒体DNA的单倍型模式进一步确认这个杂交个体的母本是河蓝黑鲮。

In vivo immunostimulatory effect of the amoebocyte lysate and plasma of Asian horseshoe crab,Tachypleus gigas in a piscine model

Antimicrobial proteins/peptides are becoming a new generation of immunostimulants for prevention and disease control in human and animals,including aquatic animals.As the haemolymph of horseshoe crabs(Tachypleus)contains broad ranges of bioactive compounds,we have explored the in vivo immunostimulating potential of amoebocyte lysate and plasma using a fish model.Indian major carp,Labeo rohita,yearlings were injected intraperitoneally with two doses of lysate and plasma at 50 and 100µg protein per fish.No abnormalities and/or mortalities were recorded in any group.L.rohita injected with 50µg lysate and 100µg plasma protein showed significant enhancement(P<0.01)of various haematological and immunological parameters.There was a significant rise in the total protein and globulin content,myeloperoxidase and respiratory burst activity following injection with 50µg lysate and 100µg plasma protein.The agglutinating and haemagglutinating activities were increased albeit not significantly(P>0.01)in any groups.On the contrary,a significantly high hemolysin titre was recorded in fish that received 100µg plasma protein.Following challenge with Aeromonas hydrophila,both lysate and plasma protein(s)cross protected the fish after 30 days.The highest survival(50%)was recorded in group injected with 50µg lysate protein,followed by 45%in both 100µg lysate and plasma protein injected groups.