Unveiling the dynamics of embryogenesis and immune genes expression pattern in the amur common carp (Cyprinus carpio haematopterus).

Amur common carp (Cyprinus carpio haematopterus), is a commercially important fish species that has been genetically improved over the years through selective breeding. Despite its significance in aquaculture, limited knowledge exists regarding its embryogenesis and immune genes associated with its early stages of life. This article represents a detailed study of the embryogenesis and innate immune gene expression analysis of the Amur common carp during its ontogenic developments. The entire embryonic developmental process of ∼44 h could be divided into eight periods, beginning with the formation of the zygote, followed by cleavage, morula, blastula, segmentation, pharyngula, and hatching. The segmentation period, which lasted for ∼ 6 h, exhibited the most significant changes, such as muscle contraction, rudimentary heart formation, increased somites number, and the initiation of blood circulation throughout the yolk. The expression of immune-related genes, namely toll-like receptor (TLR)4, nucleotide-binding oligomerization domain (NOD)1, NOD2 and interleukin (IL)-8 showed stage-specific patterns with varying levels of expression across the developmental stages. The TLR4 gene exhibited the highest expression during the neurella stage, while NOD1 and NOD2 peaked during hatching and IL-8 reached its maximum level during the gastrula stage. This is the first report of the innate immune gene expression during the embryogenesis of Amur common carp.

A comprehensive review on the dynamic role of toll-like receptors (TLRs) in frontier aquaculture research and as a promising avenue for fish disease management.

Toll-like receptors (TLRs) represent a conserved group of germline-encoded pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) and play a crucial role in inducing the broadly acting innate immune response against pathogens. In recent years, the detection of 21 different TLR types in various fish species has sparked interest in exploring the potential of TLRs as targets for boosting immunity and disease resistance in fish. This comprehensive review offers the latest insights into the diverse facets of fish TLRs, highlighting their history, classification, architectural insights through 3D modelling, ligands recognition, signalling pathways, crosstalk, and expression patterns at various developmental stages. It provides an exhaustive account of the distinct TLRs induced during the invasion of specific pathogens in various fish species and delves into the disparities between fish TLRs and their mammalian counterparts, highlighting the specific contribution of TLRs to the immune response in fish. Although various facets of TLRs in some fish, shellfish, and molluscs have been described, the role of TLRs in several other aquatic organisms still remained as potential gaps. Overall, this article outlines frontier aquaculture research in advancing the knowledge of fish immune systems for the proper management of piscine maladies.

Red blood cells of Labeo rohita express Toll-like receptors, NOD- like receptors, interleukins, and interferon-I in response to Gram-negative bacterial infections and lipopolysaccharide stimulations.

Red blood cells (RBCs) are the most abundant cell types in the circulatory system of vertebrates. In fish, RBCs retain their nuclei throughout their lifetime and remain transcriptionally and translationally active. While their primary function is typically associated with gas exchange, recent reports indicate that nucleated red blood cells can play a significant role in regulating the body's innate immune response. The current article describes the innate immune role of red blood cells in rohu (Labeo rohita), a freshwater fish species that holds significant commercial importance in India and South-East Asian nations. From the whole blood and mucosal surface RBCs have been isolated through density gradient centrifugation with HiSep™LSM 1077 (density 1.007 ± 0.0010) and their purity has been confirmed by the Giemsa staining followed by microscopical observations. Toll-like receptors (TLR2, 3, 4, 5) and nucleotide oligomerization domain (NOD)-like receptors (NOD1 and NOD2) in RBCs of rohu fingerlings were observed to be significantly activated (P < 0.05) on infection with Aeromonas hydrophila and Edwardsiella tarda. This activation resulted in increased expression of interleukins (IL-8, IL-1ß) and interferon (IFN)-I genes. The activation of TLR4, NOD1 and NOD2, as well as the expression of interleukins and IFN-I genes have been observed in both in vivo and in vitro stimulation of rohu RBCs with lipopolysaccharides. These findings highlight the importance of fish RBCs in enhancing innate immunity against various pathogenic invasions in rohu.

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.

Labeo rohita Mx1 exhibits the critical structural motifs of the family of large GTPases of mammals and is activated by rhabdovirus vaccination and bacterial RNA stimulations.

Myxovirus resistance (Mx) proteins belonging to the dynamin superfamily of high molecular weight GTPases exist in various isoforms and play crucial role in innate immunity. In addition to the isoforms, Mx1 also plays important role in exerting its anti-viral actions against a broad range of animal RNA viruses. In rohu (Labeo rohita), mx1 full-length cDNA sequence consists of 2440 nucleotides (nt) encoding 628 amino acids (aa) polypeptide of 71.289 kDa. Structurally, it belongs to the family of large GTPases with one DYNc domain (13-257aa) comprising of dynamin family motifs (LPRGSGIVTR) and the tripartite GTP-binding motifs (GDQSSGKS, DLPG and TKPD) at the N-terminal and one GED domain (537-628aa) at C-terminus. Rohu Mx1 is closely related to zebrafish Mx1 and is widely expressed in gill, liver, kidney, spleen and blood. In response to rhabdovirus vaccinations, poly I:C stimulation and bacterial infections, mx1 gene expression in rohu was significantly (p < 0.05) induced in majority of the tested organs/tissues. Stimulation of rohu gill cell line with bacterial RNA also induced mx1 gene expression. Together these data suggest the important role of Mx1 in innate immunity in rohu against wide spectrum of fish pathogens.

TLR5-Mediated Reactivation of Quiescent Ranavirus FV3 in Xenopus Peritoneal Macrophages.

Ranaviruses such as frog virus 3 (FV3) are large double-stranded DNA (dsDNA) viruses causing emerging infectious diseases leading to extensive morbidity and mortality of amphibians and other ectothermic vertebrates worldwide. Among the hosts of FV3, some are highly susceptible, whereas others are resistant and asymptomatic carriers that can take part in disseminating the infectious virus. To date, the mechanisms involved in the processes of FV3 viral persistence associated with subclinical infection transitioning to lethal outbreaks remain unknown. Investigation in Xenopus laevis has revealed that in asymptomatic FV3 carrier animals, inflammation induced by heat-killed (HK) Escherichia coli stimulation can provoke the relapse of active infection. Since Toll-like receptors (TLRs) are critical for recognizing microbial molecular patterns, we investigated their possible involvement in inflammation-induced FV3 reactivation. Among the 10 different TLRs screened for changes in expression levels following FV3 infection and HK E. coli stimulation, only TLR5 and TLR22, both of which recognize bacterial products, showed differential expression, and only the TLR5 ligand flagellin was able to induce FV3 reactivation similarly to HK E. coli Furthermore, only the TLR5 ligand flagellin induced FV3 reactivation in peritoneal macrophages both in vitro and in vivo These data indicate that the TLR5 signaling pathway can trigger FV3 reactivation and suggest a role of secondary bacterial infections or microbiome alterations (stress or pollution) in initiating sudden deadly disease outbreaks in amphibian populations with detectable persistent asymptomatic ranavirus.IMPORTANCE This study in the amphibian Xenopus laevis provides new evidence of the critical role of macrophages in the persistence of ranaviruses in a quiescent state as well as in the reactivation of these pathogens into a virulent infection. Among the multiple microbial sensors expressed by macrophages, our data underscore the preponderant involvement of TLR5 stimulation in triggering the reactivation of quiescent FV3 in resident peritoneal macrophages, unveiling a mechanistic connection between the reactivation of persisting ranavirus infection and bacterial coinfection. This suggests a role for secondary bacterial infections or microbiome alterations (stress or pollution) in initiating sudden deadly disease outbreaks in amphibian populations with detectable persistent asymptomatic ranavirus.

Toll-interacting protein in the freshwater fish Labeo rohita exhibits conserved structural motifs of higher eukaryotes and is distinctly expressed in pathogen-associated molecular pattern stimulations and bacterial infections.

Toll-interacting protein (Tollip) is a critical regulator of TOLL- like receptor (TLR)-signaling pathway. It is predominantly associated with TLR2 and TLR4 during acute inflammatory conditions and inhibits the TLR-mediated nuclear factor-kappa activation by suppressing the autophosphorylation of interleukin-1 receptor-associated kinase and its kinase activity. This article describes the Tollip of Labeo rohita (LrTollip), a highly valuable freshwater fish from the Indian subcontinent. The full-length LrTollip complementary DNA (1412 nucleotides) encodes a 276-amino acid (aa) protein, depicting a highly conserved target of the Myb1 (Tom1)-binding domain (TBD; 1-53 aa), conserved core domain 2 (C2; 54-151 aa), and coupling of ubiquitin to endoplasmic reticulum degradation (CUE; 231-273 aa) domains of mouse and human counterparts. The key amino acids exerting the critical functions of Tollip, such as phospholipids recognition and ubiquitination, are present in the C2 and CUE domains of LrTollip, respectively. LrTollip is widely expressed in the kidneys, gills, spleen, liver, and blood, and among these tested tissues, the highest expression is observed in blood. In response to TLR ligands and NOD-like receptor (NLR) ligands stimulations and Aeromonas hydrophila, Edwardsiella tarda, and Bacillus subtilis infections, LrTollip gene expression is induced in various organs/tissues with remarkable difference in their kinetics. These data together suggest the important role of LrTollip in TLR- and NLR-signal transduction pathways and immune-related diseases in fish.

NF-κB signaling induces inductive expression of the downstream molecules and IgD gene in the freshwater carp, Catla catla.

Toll-like receptors (TLRs) in innate immune system act as primary sensors in detecting the microbial components and activate their signaling cascades to induce NF-κB (nuclear factor NF-κB) towards the augmentation of immunoglobulin (Ig) synthesis. To gain insights into the efficacy of NF-κB pathway in immunoglobulin D (IgD) synthesis in the Indian Major Carp Catla catla, cloning and sequencing of TLR-signaling downstream molecules [TRAF3 (TNF receptor-associated factor 3), NEMO (nuclear factor-kappa B essential modulator), NF-κB and BAFF (B cell activating factor)] were performed by infecting the fish with pathogens. mRNA expression analysis of the downstream molecules and IgD showed significant up-regulation of these genes in kidney (P ≤ 0.001) as compared to spleen (P ≤ 0.05). To ascertain the role of NF-κB pathway in IgD synthesis, the primary cell culture of kidney and spleen in monolayer cell suspension was treated with NF-κB inhibitor (BAY 11-7082) and down-regulation of BAFF, NEMO, NF-κB, and IgD gene was observed. These results highlight the importance of NF-κB signaling pathway in augmenting the IgD gene expression in the freshwater carp, Catla catla.

Molecular characterization and expressional quantification of lgp2, a modulatory co-receptor of RLR-signalling pathway in the Indian major carp Labeo rohita following pathogenic challenges and PAMP stimulations.

Lgp2 (laboratory of genetics and physiology 2) is a cytosolic viral sensor of the RLR (retinoic acid-inducible gene 1 like receptor) family member without the caspase recruitment domain, having both inhibitory and stimulatory roles in RLR-signalling pathway. In India, Labeo rohita (rohu) is one of the leading and economically favoured freshwater fish species. Several immunological sentry proteins have been reported in this fish species, but no information is available on the RLR members. This study was aimed at cloning and characterization of full-length lgp2-cDNA (complementary DNA) in rohu and investigation of its expressional modulations following various pathogen-associated molecular pattern stimulations and bacterial infections. The full-length lgp2-cDNA sequence obtained through rapid amplification of cDNA ends-PCR consisted of 2299 nucleotides with an open reading frame of 2034 bp encoding 677 amino acids. In rohu-Lgp2, four conserved domains - a DEAD/DEAH box helicase domain, Pfam type-III restriction enzyme domain, helicase superfamily c-terminal domain and RIG-I C-terminal regulatory domain - have been detected. Within these domains, several important functional motifs, such as ATP-binding site, ATPase motif, RNA unwinding motif and RNA-binding sites, have also been identified. In healthy rohu, lgp2 gene was abundantly expressed in gill, liver, kidney, spleen and blood. In response to both in vitro and in vivo treatments using double-stranded RNA (poly I:C), lgp2 gene expression was significantly (P < 0.05) upregulated in all tested tissues and also in the LRG (Labeo rohita gill) cells. lgp2 gene expression significantly (P < 0.05) increased on stimulation of LRG cells using γ-d-glutamyl-meso-diaminopimelic acid and muramyl dipeptide. In vivo treatment using lipopolysaccharide and Aeromonas hydrophila-derived RNA resulted in both up- and down-regulation of lgp2 gene expression. Upon gram-positive and gram-negative bacterial infections, the expression of the lgp2 gene increased at different times in almost all the tested tissues. These integrated observations in rohu suggest that Lgp2 is an antiviral and antibacterial cytosolic receptor. SIGNIFICANCE STATEMENT: Lgp2, a cytosolic viral sensor of retinoic acid-inducible gene 1 like receptor family member, has been cloned in Labeo rohita. The complete sequence of rohu lgp2-complementary DNA consisted of 2299 nucleotides with an open reading frame of 2034 bp encoding 677 amino acids. It consisted of a DExDc, RES-III, HELICc, Pfam RIG-I_C-RD, ATP-binding site, ATPase motif, RNA unwinding motif and RNA-binding site. Upon bacterial infection, double-stranded RNA and various pathogen-associated molecular pattern stimulations, lgp2 gene expression significantly increased, indicating its role as an antiviral and antibacterial cytosolic receptor.

Effects of dietary α-linolenic acid/linoleic acid ratio on growth performance, tissue fatty acid profile, serum metabolites and Δ6 fad and elovl5 gene expression in silver barb (Puntius gonionotus).

BACKGROUND: Silver barb (Puntius gonionotus) is a medium-sized carp that is promising for freshwater aquaculture in Asia. This study's aim was to investigate the ideal dietary α-linolenic acid (ALA): linoleic acid (LA) ratio for maximizing long-chain polyunsaturated fatty acid (LC-PUFA) synthesis and their deposition in the muscle of silver barb, as that of fish oil based control diet. RESULT: Fish (with an initial body weight of 11.07 ± 0.12 g) were fed for 60 days with five experimental iso-proteinous, iso-lipidic, and iso-caloric diets, supplemented with linseed oil and peanut oil at varying levels to obtain ALA:LA ratios of 0.35, 0.51, 0.91, 2.04, 2.66. A control diet was prepared by supplementing fish oil. The dietary ALA:LA ratio did not influence the growth performance of fish. With increased dietary ALA:LA ratios, LA content decreased and ALA content increased in the muscle and liver of silver barb. The n-3 LC-PUFA level in muscle and liver was not influenced by feeding different ratios of ALA:LA, whereas n-6 LC-PUFA was decreased in the muscle and increased in the liver with increased dietary ALA:LA ratios. Increasing dietary ALA:LA ratio increased the Δ6fad and elovl5mRNA expression in the liver, muscle, brain, and intestinal tissues of silver barbs. CONCLUSION: Silver barb possess the ability to elongate and desaturate ALA and LA to their end products EPA and DHA. The highest level expression of Δ6 fad and elovl5 mRNA at the dietary ALA:LA ratio of 2.66 suggests greater affinity of these enzymes towards ALA than LA in silver barb. © 2019 Society of Chemical Industry.

Molecular characterization and expressional modulation of IRAK1 as downstream signaling adaptor molecule of TLR-signaling pathways in Labeo rohita following PAMPs stimulation and bacterial infections.

Interleukin-1 receptor associated kinase (IRAK1) is one of the crucial signal transduction mediators in TLR/IL-1R signaling pathways in host immune system. To investigate about it in rohu (Labeo rohita), one of the economically important freshwater fish species in the Indian subcontinent, we cloned, characterized and analyzed its expression following bacterial infection and pathogens associated molecular patterns (PAMPs) stimulation. The full-length cDNA of rohu IRAK1 (LrIRAK1) consisted of 2765 nucleotide (nt) having an ORF of 2115 nt encoding a polypeptide of 704 amino acids (aa) with a molecular mass of 70.4 kDa. Structurally, LrIRAK1 consisted of twenty-nine helix, twelve strands and forty one coils making one N-terminal death domain (19-94 aa) and a central serine threonine kinase catalytic domain (or kinase domain) (188-489aa). In addition to these two prominent domains, LrIRAK1 also contained highly conserved amino acids viz., lysine 215 and aspartic acid 314 and threonine 185, 361 which were reported to be important for kinase and phosphorylation activity respectively in other animals. Similar to higher vertebrates, LrIRAK1 also consisted of CDK1 (cyclin-dependent kinase1) at 338-352 aa; NEK2 (NIMA-related kinase 2) at 47-61 aa; NEK6 (NIMA-related kinase 6) at 581-595 aa and AMPK (AMP- activated protein kinase) motif at 518-538 aa. Phylogenetically, LrIRAK1 is closely related to cave fish, common carp exhibiting high similarity (~95%) and identity (~90%). In the uninfected fish, the LrIRAK1 expression was highest in liver (~11.5 fold) and lowest in blood. In response to Aeromonas hydrophila, Edwardsiella tarda and Bacillus subtilis infection and various TLR and NLR-ligands stimulation, the expression of LrIRAK1 was markedly enhanced at various time points in almost all the tested tissues. These results together suggest the key role of LrIRAK1 in pattern recognition receptors (PRRs)-mediated host defense against pathogenic insults.

Molecular characterization and expression analysis of two crucial MAPKs- jnk1 and erk1 as cellular signal transducers in Labeo rohita in response to PAMPs stimulation and pathogenic invasion.

Mitogen-activated protein kinases (MAPKs) are crucial Ser/Thr protein kinases that play important roles in innate immunity by converting extracellular stimuli into a wide range of cellular responses, including the production of cytokines. In this study, two MAPK genes, jnk1 and erk1, were cloned and characterized in rohu (Labeo rohita), a commercially important freshwater fish species in the Indian subcontinent. In healthy rohu, both jnk1 and erk1 gene expressions were highest in the spleen as compared to gill, liver, blood and kidney tissues. In vitro stimulation of the L. rohita gill (LRG) cell line with γ-D-glutamyl-meso-diaminopimelic acid, muramyl dipeptide and polyinosinic: polycytidylic acid (poly I:C) resulted in significantly enhanced expressions of jnk1 and erk1 genes. In the in vivo experiments, jnk1 and erk1 gene expressions were also enhanced in lipopolysaccharides and poly I:C-treatment. Infection of rohu fingerlings with Aeromonas hydrophila and Bacillus subtilis revealed significantly enhanced expressions of the jnk1 and erk1 genes in all of the tested organs/tissues. Together these results imply the important role of jnk1 and erk1 genes in fish during pathogenic invasion and diseases.

Cloning and identification of antimicrobial peptide, hepcidin from freshwater carp, Catla catla on pathogen challenge and PAMPs stimulation.

Hepcidin, a cationic cysteine-rich antimicrobial peptide (AMP) acts in hormone regulation and iron homeostasis in the host body. However, the biological property of hepcidin in immune reaction remains unexplored. In aquatic milieu, environmental and pathogenic stressors cause detrimental infections, which are defended by various immunological cells and antimicrobial peptides. In this study, hepcidin gene has been cloned from freshwater carp, Catla catla. The partially cloned hepcidin consists of 200 bp nucleotide sequence encoding 66 amino acids. Nucleotide sequence showed 97% and 91% similarity with Labeo rohita and Cyprinus carpio, respectively. Expression profile revealed significant up-regulation (P ≤ 0.0001) in liver as compared to other tissues in different conditions. In Aeromonas hydrophila challenged C. catla, liver showed higher expression level of hepcidin at 72 h as compared to other tissues. In skin, hepcidin expression showed significant upraise during 24 h in Streptococcus uberis infection. In Argulus sp. infected fishes, up-regulation of hepcidin expression was noted in liver, intestine and skin. The inactivated viral antigen-stimulated fishes, a substantial rise in liver was observed implying hepcidin as an important molecule in combating the pathogenic infections in freshwater carp, C. catla. Fishes stimulated with pathogen-associated molecular patterns (PAMPs) triggered the increased expression of hepcidin mRNA in liver, kidney and skin. This study indicates the presence of hepcidin as antimicrobial peptide in neutralizing the pathogenic infection in fishes.

Molecular cloning, characterization and expression analysis of MHCI and chemokines CXCR3 and CXCR4 gene from freshwater carp, Catla catla.

The immune system with large number of molecules protects the host against a plethora of continuously evolving microbes. Major histocompatibility complex (MHC) molecules serve as cardinal elements of the adaptive immune system responsible for the activation of the adaptive immunity in the host. The present study reports MHCI molecule in freshwater carp, Catla catla, and its differential expression in immunologically relevant tissues post-infection with Gram-negative and Gram-positive bacteria. The MHCI sequence of C. catla had 502 bp nucleotides encoding putative 146 amino acids. The phylogenetic analysis exhibited its evolutionary conservation within the Cyprinidae family and formed a different clade with the higher vertebrates. Simultaneously, CXCR3 and CXCR4 chemokines were cloned and characterized for their expression in infected tissues. Analysis of immunologically relevant tissues of the infected fish exhibited an increase of MHCI gene expression and the down-regulation of CXCR3 and CXCR4 chemokines, indicating a tricky interaction between the innate and adaptive immune system. It was found that intestine, skin and spleen played a crucial role in the contribution of the defense activity which instigated the self-immunity. These immune activities can provide useful information to understand the interaction of self and non-self- immune system in freshwater fish, Catla catla.

Toll like receptor induces Ig synthesis in Catla catla by activating MAPK and NF-κB signalling.

The molecular crosstalk of proximal innate immune receptor signaling mediated by Toll-like receptors (TLRs) is crucial in generating an adaptive immune response. The extracellular-signal regulated kinases (ERK) participate in propagating intracellular signals initiated by stimulated TLRs to transcription factors eliciting cytokine release. Although ERK signaling has been extensively studied in mammalian counterparts, very little is known about its existence in carps and its role in augmentation of immunoglobulin (Ig) synthesis. Therefore, to gain insights into the efficacy of MAP kinase cascade in orchestrating fish antigen receptor generation, Catla catla fingerlings were induced with various TLR agonists or pathogen associated molecular patterns (PAMPs). Analysis of upstream signaling events revealed that PAMPs stimulated the tissues leading to a significant upregulation (P < 0.001, One-way ANOVA) of different TLRs (TLR2, TLR3, TLR4 and TLR5) followed by activation of MyD88 dependent and independent pathway. Activation of ERK and NF-κB mediated cytokine production consequently triggered the enhanced expression of IgZ and IgM as was evident by qRT-PCR analysis, flow cytometry, immunoblotting and ELISA. Pretreatment with ERK inhibitor (UO126) antagonized PAMPs mediated TLR stimulation, leading to sequential downregulation of MyD88/NF-κB/cytokines via interrupting ERK/NF-κB signaling axis. Together these results demonstrate that TLR stimulation triggers IgZ and IgM production via activation of ERK and NF-κB in C. catla indicating that NF-κB mediated cytokine production and ERK1/2 signaling is not only functional in fish, but may be crucial for generation of Ig repertoire in lower vertebrates.

Influence of dietary lipid levels on growth, nutrient utilization, tissue fatty acid composition and desaturase gene expression in silver barb (Puntius gonionotous) fingerlings.

Silver barb (Puntius gonionotus) is considered as a promising medium-sized carp species for freshwater aquaculture in Asia. This study in silver barb was carried out to evaluate the effects of increasing dietary levels of lipid on growth, nutrient utilization, whole-body composition, tissue fatty acid composition and Δ6 fatty acyl desaturase (Δ6 fad) gene expression. Fish (11.3 ±â€¯0.23 g of initial body weight) was fed for 60 days with five experimental diets: FO-0 (control feed); FO-30; FO-60; FO-90 and FO-120 containing 0, 30, 60, 90 and 120 g fish oil kg-1 diet, respectively. Among the diets, the highest specific growth rate (SGR), protein efficiency ratio (PER) and whole-body lipid content, and the lowest feed conversion ratio (FCR) were recorded with FO-120 diet. The saturated fatty acids (SFA) level in the muscle was significantly (P < .05) increased with the enhanced FO supplementation, whereas monounsaturated fatty acids (MUFA) level decreased. Increased level of fish oil in the diet also enhanced the n-3 PUFA and n-3 LC-PUFA (long-chain polyunsaturated fatty acid) in the muscle and liver. The expression of Δ6 fad gene was downregulated, whereas the serum biochemical constituents were either remain unchanged or enhanced with increased FO supplementation in the diets of silver barb.

Lrcasp9 shares similarity in structural motifs with human caspase-9 and is activated following bacterial infection and anti-viral vaccination.

Among various caspases, caspase-9 plays a crucial role in the initiation phase of apoptotic cascade. To investigate about it in a high-valued freshwater fish species rohu (Labeo rohita), we cloned and characterized full-length caspase-9 cDNA (Lrcasp9) and analyzed its expression following bacterial infections and anti-viral vaccinations. The Lrcasp9 consisted of 1619-bp nucleotides (nt) having an ORF of 1302 nt encoding a polypeptide of 433 amino acids (aa) with a molecular mass of ∼ 48.20 kDa. Structurally, Lrcasp9 comprised of one CARD domain (1-89 aa) and one CASc domain (161-430 aa). The CASc domain consisted of one large subunit (p20) spanning from 168 to 300 aa, and a small sub unit (p10) from 343 to 430 aa. The caspase family signature histidine active motif H233SAYDCCVVIILSHG247, cysteine active motif K287PKLFFIQACGG298 and pentapeptide "QACGG" active sites present in the p20 domain of Lrcasp9 was conserved across fish species, mouse and human caspase-9. Phylogenetically, it was closely related to common carp caspase-9 and exhibited significant similarity (90.1%) and identity (85.3%) in their amino acid sequence. In the uninfected fish, Lrcasp9 gene expression was highest (~ 5.3-fold) in blood and lowest in gill. In response to Aeromonas hydrophila and Edwardsiella tarda infection and rhabdoviral vaccination, Lrcasp9 gene expression was significantly (p > 0.05) enhanced in gill, liver, kidney and spleen, and also in vitro during cell death, suggesting activation of the intrinsic apoptotic pathway in bacterial infections and anti-viral vaccination in Labeo rohita.

LrHMGB1 Shares Structural Similarities with Human HMGB1, and Its Expression Is Induced in Bacterial Infection, Antiviral Vaccination, and Pathogen-Associated Molecular Patterns Stimulation.

The high-mobility group box 1 (HMGB1) protein is a highly conserved nonhistone chromosomal protein ubiquitously present in almost all cell types. In the nucleus, it facilitates DNA repair and replication, V(D)J recombination, stabilization of nucleosome, and in the cytoplasm, it regulates autophagy and apoptosis. In addition to these intracellular functions, HMGB1 also facilitates activation of innate immune responses and plays key roles in host defense. To investigate its role in fish, we cloned and characterized HMGB1 in Labeo rohita (LrHMGB1), the most important freshwater fish species in the Indian subcontinent. The full-length cDNA sequence of LrHMGB1 consisted of 787 bp having an open reading frame of 618 bp encoding 205 amino acids (aa) polypeptide, with an estimated molecular mass of 23.61 kDa and isoelectric point (pI) of 5.96. Motif search of LrHMGB1 revealed two homologous DNA-binding domains, the A-box and B-box comprising 8-78 aa and 94-162 aa, respectively, and a negatively charged acidic C-terminal tail (179-204) that consisted of 26 consecutive aspartic and glutamic acid residues. The amino acids sequence of LrHMGB1 protein and the secondary structure having helix (H) and coils (C) in tandem in the A- and B-box region and only coils in the acidic tail region shared significant similarity with mouse and human HMGB1. In addition to the three prominent motifs (A-box, B-box, and the acidic tail), the site of acetylation (lys27-29), phosphorylation (serine38,41,45,52), methylation (lys43), and oxidation (cysteine44,105) in LrHMGB1 was also conserved across the fish species, mouse, and human. LrHMGB1 was expressed during embryogenesis and was widely expressed in various organs/tissues having highest expression in blood. In response to bacterial infection, antiviral vaccination, and pathogen-associated molecular patterns stimulations, LrHMGB1 gene expression was significantly (p < 0.05) induced in various organs and tissues. These results together suggest an evolutionary conserved structure and function of HMGB1 from fish to human.

Diversity of Immunoglobulin (Ig) Isotypes and the Role of Activation-Induced Cytidine Deaminase (AID) in Fish.

The disparate diversity in immunoglobulin (Ig) repertoire has been a subject of fascination since the emergence of prototypic adaptive immune system in vertebrates. The carboxy terminus region of activation-induced cytidine deaminase (AID) has been well established in tetrapod lineage and is crucial for its function in class switch recombination (CSR) event of Ig diversification. The absence of CSR in the paraphyletic group of fish is probably due to changes in catalytic domain of AID and lack of cis-elements in IgH locus. Therefore, understanding the arrangement of Ig genes in IgH locus and functional facets of fish AID opens up new realms of unravelling the alternative mechanisms of isotype switching and antibody diversity. Further, the teleost AID has been recently reported to have potential of catalyzing CSR in mammalian B cells by complementing AID deficiency in them. In that context, the present review focuses on the recent advances regarding the generation of diversity in Ig repertoire in the absence of AID-regulated class switching in teleosts and the possible role of T cell-independent pathway involving B cell activating factor and a proliferation-inducing ligand in activation of CSR machinery.

Gonadotropin receptors of Labeo rohita: Cloning and characterization of full-length cDNAs and their expression analysis during annual reproductive cycle.

Follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), secreted from pituitary, stimulate gonadal function by binding to their cognate receptors FSH receptor (FSHR), and LH/choriogonadotropin receptor (LHCGR). Rohu (Labeo rohita) is a commercially important seasonal breeder freshwater fish species, but till date, the regulation of expression of gonadotropins and their receptors gene during different phases of annual reproductive cycle has not been investigated. We envisaged the critical role of these molecules during seasonal gonadal development in this carp species. We cloned full- length cDNAs of fshra and lhcgrba from rohu testis using RACE (Rapid amplification of cDNA ends) and analyzed their expression along with fsh and lh by quantitative real time PCR (qRT-PCR) assay at various gonadal developmental stages of the annual reproductive cycle. Full-length rohu fshra and lhcgrba cDNA encodes 670 and 716 amino acids respectively, and in adult fish, they were widely expressed in brain, pituitary, gonad, liver, kidney, head kidney, heart, muscle, gill, fin, eye and intestine. In male, both fsh and fshra transcripts showed high level of expression during spermatogenesis, however, in female, expression level was found to be higher in the fully grown oocyte stages. The expression of rohu lh and lhcgrba mRNA increased with increment of gonadosomatic index and showed highest level during spermiation stage in male and fully matured oocyte stage in female. These results together may suggest the involvement of fshra and lhcgrba in regulating function of seasonal gonadal development in rohu.