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.

Tilapia Lake Virus(TiLV)disease:Current status of understanding

Tilapia Lake Virus(TiLV)disease is an emerging and transboundary disease of tilapia cultures,causing mortality up to 90%globally.TiLV is a negative sense single stranded RNA virus belongs to family Amnoonviridae,genus Tilapinevirus and species Tilapia tilapinevirus.The first TiLV outbreak to fishes was reported from Israel followed by other countries viz.,Ecuador,Colombia,Egypt,Thailand,Chinese Taipei,India,Malaysia,Bangladesh,Uganda,Tanzania,Peru,Mexico,Philippines,Indonesia,and USA.All the life stages of Tilapia(belonging to the family Cichlidae)are vulnerable to TiLV infection.However,river barb and giant gourami have also been found susceptible to TiLV infection.The virus infects the vital organs of the fish,including eyes,brain,and liver.The notable pathological finding of this disease includes syncytial cell formation and massive hepatocellular necrosis with pyknotic and karyolytic nuclei in the liver cells of infected fish.The disease is very contagious and spreads through both horizontal and vertical transmission.Several sensitive and rapid molecular diagnostic tools like reverse transcriptase polymerase chain reaction(RT-PCR),RT-quantitative PCR(RT-qPCR),loop-mediated isothermal amplification(LAMP)have been developed for early detection of the virus.Till date,no comprehensive control measures have been developed throughout the globe,although aggressive work on this line is going on.Implementations of strict good management practices,including quarantine protocols,are the only available option to combat the outbreak and spread of the disease.This review emphasizes the etiology,occurrence and distribution,mode of transmission,pathology and pathogenesis,diagnosis,possible control measures,and challenges of TiLV disease.

Revealing localization and regulation of GTPase PmRab7 in lymphoid cells of Penaeus monodon after WSSV infection

Objective:To identify white spot syndrome virus(WSSV)entry into the host-cells of the cultured shrimp Penaeus monodon,we have attempted to localize PmRab7(Ras-related in brain)which is playing a vital role in the WSSV internalization.Methods:In this study,we have cloned PmRab7 and expressed in Escherichia coli,further purified rPmRab7 was used for antibody production,isolation of lysosomal sub-cellular fractions and western blot against lysosomal protein.Moreover,high fold-change in PmRab7 regulation with increasing copy number of WSSV has been studied by using real-time PCR.Results:651 bp amplicon size gene was successfully amplified,ligated amplicon with pTZ T-tail vector confirmed by colony PCR and retriction enzyme digestion on agarose gel.Subcloned(pRSET-B)651 bp gene transformed successfully in Rosetta and after 6 h of induction expressed rPmRab7 was on SDS page,furthermore soluble fraction of rPmRab7(26 kDa)was purified by ni-NTA column.AntiPmRab7 antibody was received by Merk Pvt.Ltd.,and western blot analysis revealed that PmRab7 is present in the lysosomal sub-cellular fraction.Copy number of WSSV was increased 5 fold in 24 h and 20 fold in 72 h of infection and subsequently transcrtipt of PmRab7 was Ct=1.0 to Ct=8.5.Conclusions:Presence of PmRab7 on lysosome clearly indicating PmRab7 participating in lysosomal maturation,other hand WSSV may follow the same route of entry.WSSV internalization has directly linked with regulation of PmRab7.

Use of sewage in split doses to enhance water productivity for fish culture

The problem of sewage disposal has received great attention worldwide.The raw sewage contains a variety of high inorganic and organic matters that affect natural water environment.To mitigate such problem,sewage may be recycled through aquaculture practice.Sewage recycling in aquaculture enhances water productivity through nutrients input.Proper loading of sewage ensures viable aquaculture;otherwise,fish mortality occurs due to poor water quality.To optimize sewage application,two different experiments were conducted,each with four treatments.In both experiments,three fish species namely rohu(Labeo rohita Hamilton,1822),mrigal(Cirrhinus mrigala Hamilton,1822),and bata(Labeo bata Hamilton,1822)were tested in triplicate in FRP(Fibre-Reinforced Plastic)tanks.Different sewage concentrations(0,25%,50%and 75%)used in first experiment were prepared by mixing freshwater,showing Biochemical Oxygen Demand(BOD)2.0±0.4 mg/L,10.8±1.4 mg/L,19.6±1.5 mg/L and 41.6±2.58 mg/L,respectively.After 30 days rearing,results showed≥75%fish survival in sewage concentrations up to 50%with BOD level 19.6±1.5 mg/L.Less than 50%fish survived in 75%sewage concentration,with BOD level as 41.6±2.58 mg/L.The second experiment was conducted for 90 days considering 50%sewage concentration as basal dose with BOD level as 19.6 mg/L as an acceptable limit for fish survival.Split doses of sewage were applied in T_(1),T_(2) and T_(3) treatments fortnightly,weekly and semi-weekly intervals,while single dose was used in C(control)treatment.Application of split doses resulted better hydro-biological changes,including nutrients recovery,in T_(1),T_(2) and T_(3) than that of single dose in control.Fish growth plotted with net primary productivity(NPP),phytoplankton and zooplankton densities exhibited positive correlations in T_(2)(12 doses)and T_(3)(24 doses),considered as optimal doses to ensure better water productivity for desirable fish production than sewage with single dose or limited doses(6 doses).