Differential responses of hepatopancreas transcriptome between fast and slow growth in giant freshwater prawns (Macrobrachium rosenbergii) fed a plant-based diet.

Efficient utilisation of plant-based diets in the giant freshwater prawn, Marcrobrachium rosenbergii, varies according to individual, suggesting that it might be associated with differences in physiological and metabolic responses. Therefore, we aimed to investigate the individual differences in the growth response of shrimp fed to a soybean-based diet (SBM). Two hundred shrimp were fed SBM for 90 days, and specific growth rate (SGR) was determined individually. Fast- and slow-growing shrimp (F-shrimp vs. S-shrimp), with the highest and lowest 5% SGRs, respectively, were sampled to determine haemolymph chemistry and carcass composition. The hepatopancreas of these shrimps were used for transcriptome analysis through RNA sequencing (RNA-Seq). The results showed no significant differences in haemolymph chemistry parameters. In terms of carcass proximate composition, F-shrimp exhibited higher protein composition than did S-shrimp, suggesting that F-shrimp have higher protein anabolism. Using RNA-seq and real-time reverse transcription polymerase chain reaction (qRT-PCR), the expression levels of several genes encoding physiologic and metabolic enzymes were found to be upregulated in F-shrimp compared to in S-shrimp, suggesting that these enzymes/proteins mediated the efficient use of SBM-based diets for growth promotion in shrimp. Various DEGs associated with the immune system were observed, indicating a difference in immune processes between F- and S-shrimp. The expression of several housekeeping genes was found to be upregulated in S-shrimp. Collectively, the upregulated expression of several enzymes associated with physiological and/or metabolic processes and increased protein anabolism may be attributed to the efficient use of SBM for maximal growth in shrimp.

Systemic and mucosal immune responses in red tilapia (Oreochromis sp.) following immersion vaccination with a chitosan polymer-based nanovaccine against Aeromonas veronii.

A mucoadhesive chitosan polymer-based nanoplatform has been increasingly recognized as an effective mucosal vaccine delivery system for fish. The present study aimed to investigate the effectiveness of immersion vaccination with a chitosan polymer-based nanovaccine to elicit an immune response in serum and mucus of red tilapia and evaluate its protective efficacy after immersion challenge with a heterogenous strain of Aeromonas veronii UDRT09. Six hundred red tilapia (22 ± 1.8 g) were randomly allocated into four experimental groups: control, empty-polymeric nanoparticle (PC), formalin-killed vaccine (FKV), and chitosan polymer-based nanovaccine (CS-NV) in triplicate. The specific IgM antibody levels and their bactericidal activity were assessed in serum and mucus for 28 days after immersion vaccination and followed by immersion challenge with A. veronii. The immersion vaccine was found to be safe for red tilapia, with no mortalities occurring during the vaccination procedure. The specific IgM antibody levels and bactericidal activity against A. veronii in both serum and mucus were significantly higher in red tilapia vaccinated with CS-NV compared to the FKV and control groups at all time points. Furthermore, the serum lysozyme activity, ACH50, and total Ig levels demonstrated a significant elevation in the groups vaccinated with CS-NV compared to the FKV and control groups. Importantly, the Relative Percentage Survival (RPS) value of the CS-NV group (71 %) was significantly higher than that of the FKV (15.12 %) and PC (2.33 %) groups, respectively. This indicates that the chitosan polymer-based nanovaccine platform is an effective delivery system for the immersion vaccination of tilapia.

Identification of immune-responsive circular RNAs in shrimp (Litopenaeus vannamei) upon yellow head virus infection.

Circular RNAs (circRNAs) are a subclass of non-coding RNAs (ncRNAs) formed through a process known as back-splicing. They play a crucial role in the genetic regulation of various biological processes. Currently, circRNAs have been identified as participants in the antiviral response within mammalian cells. However, circRNAs in shrimp infected with the yellow head virus (YHV) remain largely unexplored. Therefore, this study aims to identify circRNAs in the hemocytes of Litopenaeus vannamei during YHV infection. We discovered 358 differentially expressed circRNAs (DECs), with 177 of them being up-regulated and 181 down-regulated. Subsequently, eight DECs, including circ_alpha-1-inhibitor 3, circ_CDC42 small effector protein 2, circ_hemicentin 2, circ_integrin alpha V, circ_kazal-type proteinase inhibitor, circ_phenoloxidase 3, circ_related protein rab-8B, and circ_protein toll-like, were randomly selected for analysis of their expression patterns during YHV infection using qRT-PCR. Furthermore, the circRNAs' characteristics were confirmed through PCR, RNase R treatment, and Sanger sequencing, all of which were consistent with the features of circRNAs. These findings contribute to a better understanding of circRNAs' involvement in the antiviral response in shrimp.

Effects of CRISPR/Cas9-mediated dnd1 knockout impairs gonadal development in striped catfish.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology allows for the generation of loss-of-function mutations to enable efficient gene targeting to produce desired phenotypes, such as the production of germ cell-free fish. This technology could provide several applications for aquaculture and conservation of fisheries resources, such as the prevention of overpopulation in fish culture and gene flow from escaped farmed fish into wild populations and the production of germ cell-free recipient larvae for germ cell transplantation. This study aimed to develop CRISPR/Cas9 mediated dead-end 1 (dnd1) knockout techniques for striped catfish (Pangasianodon hypophthalmus). To optimise CRISPR/Cas9-induced dnd1 knockout, three single-guide RNAs (sgRNAs) were designed to target upstream sequences of start codon of the dnd1 gene. A combination of two concentrations of each sgRNA (100 and 200 ng/µl) and three concentrations of Cas9 (100, 250, and 500 ng/µl) was microinjected into fertilised striped catfish eggs. These sgRNAs/Cas9 could induce indel mutations and lower the primordial germ cell (PGC) numbers. Histological analyses indicated that sgRNA3 targeting upstream and nearest to the start codon at 200 ng/µL and Cas9 at 500 ng/µL showed the lowest PGC number. The reduction in PGC number was confirmed by in situ hybridisation using antisense dnd1 and vasa probes. All sgRNA/Cas9 combinations reduced the expression of dnd1, cxcr4b, dazl, nanos1, nanos2, and vasa, and the lowest expression levels were observed in gonads obtained from fish injected with 200 ng/µL sgRNA3 and 500 ng/µL Cas9 (P < 0.05). In addition, at 1 year of age, a significantly lower gonadosomatic index was observed in fish injected with all sgRNA and Cas9 at 500 ng/µL. Moreover, compared to the control fish, the ovaries and testes presented different morphologies in the sgRNA/Cas9-injected fish, that is, few previtellogenic oocytes in the ovary and spermatogonial cell-less testes. In conclusion, CRISPR/Cas 9 targeting dnd1 knockout at the upstream sequences of start codon was achieved, which resulted in the downregulation of dnd1 and lowered PGC number.

Isolation of Potential Probiotic Bacillus spp. from the Intestine of Nile Tilapia to Construct Recombinant Probiotic Expressing CC Chemokine and Its Effectiveness on Innate Immune Responses in Nile Tilapia.

This study aimed to investigate the potential probiotic Bacillus spp. from the intestine of Nile tilapia in order to construct a recombinant probiotic for the enhancement of the Nile tilapia immune response. One hundred bacterial isolates from the intestine of Nile tilapia were characterized for species identification using the 16s ribosomal RNA (rRNA). Only Bacillus isolates with exhibited antagonistic activity were investigated for their biological functions, which included protease-producing capacity, bile salts and pH tolerance, antibiotic susceptibility, and pathogenicity tests. According to the best results, Bacillus isolate B29, as closely related to B. subtilis, was selected to construct a recombinant probiotic for the delivery of CC chemokine protein (pBESOn-CC). The existence of recombinant probiotics was confirmed by Western blotting before the feeding trial. In addition, the CC chemokine mRNA level was quantified in the intestine of fish fed probiotics after 30 days of feeding. Total immunoglobulin, lysozyme activity, alternative complement 50 activity (ACH50), and phagocytic activity of fish fed either wild-type or recombinant probiotics were significantly increased, indicating that probiotics could stimulate the Nile tilapia immune system through different processes. Interestingly, the dietary supplementation of recombinant probiotics has a stronger immune response enhancement than the wild-type strain.

Investigating the Effect of Various Sous-Vide Cooking Conditions on Protein Structure and Texture Characteristics of Tilapia Fillet Using Synchrotron Radiation-Based FTIR.

The effects of various sous-vide (SV) cooking conditions (50-60℃, 30-60 min) on physicochemical properties related to the texture characteristics, protein structure/degradation, and sensory acceptability of tilapia fillet (Oreochromis niloticus) were investigated. With an increasing temperature and processing time of SV cooking, protein degradation (of both myofibrils and connective tissue) was more pronounced, as evaluated by the decrease in water- and salt-soluble proteins, total collagen, as well as the changes in the ratio of secondary protein structures (α-helix, ß-sheet, ß-turn, etc.), which were determined by synchrotron-FTIR (SR-FTIR). These degradations were associated with the improvement of meat tenderness, as estimated by shear force and texture profile analyzer (TPA) results. Among all SV conditions, using 60 ℃ for 45 min seems to be the optimal condition for tilapia meat, since it delivered the best results for texture characteristics and acceptability (p < 0.05). Moreover, principal component analysis (PCA) results clearly demonstrated that the highest texture-liking score of this condition was well associated with the intensity of ß-sheets, which seem to be the crucial component that affected the texture of SV-cooked tilapia more so than other parameters. The findings demonstrated the potential of SR-FTIR to decipher the biomolecular structure, particularly the secondary protein structure, of SV-cooked tilapia. This technique provided essential information for a better understanding of the changes in biomolecules related to the textural characteristics of this product.

Transcriptome-based insights into the regulatory role of immune-responsive circular RNAs in Litopanaeus vannamei upon WSSV infection.

Circular RNAs (circRNAs) are non-coding RNAs (ncRNAs) originating from a post-transcriptional modification process called back-splicing. Despite circRNAs being traditionally considered by-products rather than independently functional, circRNAs play many vital roles, such as in host immunity during viral infection. However, in shrimp, these remain largely unexplored. Therefore, this study aims to identify circRNAs in Litopenaeus vannamei in the context of WSSV infection, one of the most eradicative pathogens threatening shrimp populations worldwide. We identified 290 differentially expressed circRNAs (DECs) in L. vannamei upon WSSV infection. Eight DECs were expressed from their parental genes, including alpha-1-inhibitor-3, calpain-B, integrin-V, hemicentin-2, hemocytin, mucin-17, proPO2, and rab11-FIP4. These were examined quantitatively by qRT-PCR, which revealed the relevant expression profiles to those obtained from circRNA-Seq. Furthermore, the structural and chemical validation of the DECs conformed to the characteristics of circRNAs. One of the functional properties of circRNAs as a miRNA sponge was examined via the interaction network between DECs and WSSV-responsive miRNAs, which highlighted the targets of miRNA sponges. Our discovery could provide insight into the participation of these ncRNAs in shrimp antiviral responses.

Characterization of ddx4 and dnd Homologs in Snakeskin Gourami (Trichopodus pectoralis) and Their Expression Levels during Larval Development and in Gonads of Males and Females.

The purpose of this study was to clone and characterize ddx4 and dnd1 homologs in snakeskin gourami (Trichopodus pectoralis) and to determine their expression levels during larval development and in the gonads of males and females. Both cDNAs contained predicted regions that shared consensus motifs with the ddx4 family in teleosts and the dnd family in vertebrates. Phylogenetic tree construction analysis confirmed that these two genes were clustered in the families of teleosts. Both ddx4 and dnd1 mRNAs were detectable only in the gonads, particularly in germ cells. These two genes were expressed during early larval development. The expression of ddx4 was high during early larval development and decreased with increasing developmental age, whereas dnd1 expression increased with developmental age. In adult fish, the expression levels of both genes were higher in the ovary than in the testis. Overall, these findings provide valuable molecular information on ddx4 and dnd, and can be applied in future reproductive biological studies relating to sex dimorphism in snakeskin gourami.

The interferon-like proteins, Vagos, in Fenneropenaeus merguiensis elicit antimicrobial responses against WSSV and VPAHPND infection.

The Vago interferon-like protein participates in the interplay between interferon regulatory factors and the expression of immune-responsive genes. Vago was initially perceived to participate only in the antiviral activation through JAK/STAT pathway. However, certain isoforms of Vago can stimulate antimicrobial responses. Here we identify Vago isoforms in Fenneropenaeus merguiensis (FmVagos) and how they function in antiviral and antibacterial responses against highly invasive pathogens, including white spot syndrome virus (WSSV) and Vibrio parahaemolyticus (VPAHPND). Three isoforms of FmVagos were identified: FmVago4, FmVago5a, and FmVago5b, and expressed throughout tissues of the shrimp. During infection, FmVago4, FmVago5a, and FmVago5b, were up-regulated after WSSV and VPAHPND challenges at certain time points. Pre-injection of purified recombinant FmVago4 (rVago4), FmVago5a (rVago5a), and FmVago5b (rVago5b) proteins could significantly reduce the mortality of shrimp upon WSSV infection, while the increase of survival rate of VPAHPND-infected shrimp was observed only in rVago4 treatment. The immunity routes that FmVagos might instigate in response to the pathogens were examined by qRT-PCR, revealing that the JAK/STAT pathway was activated after introducing rVago4, rVago5a, and rVago5b, while the Toll/IMD pathway and proPO system, combined with PO activity, were provoked only in the rVago4-treated shrimp. Our finding suggests cross-talk between Vago's antiviral and antimicrobial responses in shrimp immunity. These findings complement previous studies in which Vago and its specific isoform could promote viral and bacterial clearance in shrimp.

Development of Flavor and Taste Components of Sous-Vide-Cooked Nile Tilapia (Oreochromis niloticus) Fillet as Affected by Various Conditions.

This study aims to shed light on the association between non-volatile and volatile compounds related to flavor/taste characteristics as well as sensory acceptability of Nile tilapia fillet (Oreochromis niloticus) cooked by various sous-vide (SV) conditions (50−60 ℃, 30−60 min), with fish cooked with boiling water used as control. Higher temperatures and longer processing times of SV cooking led to greater protein and lipid oxidation as indicated by the increase in total sulfhydryl (-SH), carbonyl, free fatty acid (FFA) contents as well as peroxide values (PV) and thiobarbituric acid reactive substance (TBARS) values. The differences in flavor/taste components including adenosine triphosphate (ATP)-related compounds, free amino acids (FAAs) and volatiles were also obtained, which directly affect sensory acceptability as evaluated by using the hedonic scale. Based on principal component analysis (PCA) results, the acceptability score was strongly correlated with inosine monophosphate (IMP) and acetoin, which seem to be the most crucial flavor enhancers for cooked tilapia. Among all samples, tilapia processed at 60 °C for 45 and 60 min, which contained significantly higher IMP and acetoin (p < 0.05) than others, had significantly higher flavor-liking and overall-liking scores, with a more than 7.5 meaning for high acceptability (p < 0.05), indicating the optimal SV conditions for tilapia fillet. Overall, the present finding indicated that the SV-cooking technique, at the optimal conditions, can improve the meat quality of cooked fish, in terms of flavor/taste characteristics, compared with traditional cooking (control).

Effects of Jerusalem Artichoke (Helianthus tuberosus) as a Prebiotic Supplement in the Diet of Red Tilapia (Oreochromis spp.).

The aim of the present study was to evaluate the effects of a Jerusalem artichoke-supplemented diet on the blood chemistry, growth performance, intestinal morphology, expression of antioxidant-related genes, and disease resistance against Aeromonas veronii challenge in juvenile red tilapia. A completely randomized design (CRD) was followed to feed red tilapias with three experimental diets: control, 5.0 g/kg JA-supplemented (JA5), or 10.0 g/kg JA-supplemented (JA10) diets in triplicates for 4 weeks. The results revealed that the growth performance, weight gain (WG), specific growth rate (SGR), and average daily gain (ADG) of fish fed diets JA5 and JA10 were significantly higher (p < 0.05) than those of fish fed the control diet. Fish fed the control diet had significantly higher T-bilirubin, D-bilirubin, and ALT in blood serum than fish fed JA5 and JA10, as well as higher BUN than fish fed JA5. The number of goblet cells in the proximal and distal parts of the intestine revealed that the number of acid, neutral, and double-staining mucous cells of fish fed diets JA5 and JA10 was significantly higher (p < 0.05) than in fish fed the control diet. The diets including the prebiotic (JA5 and JA10) were associated with a significant increase in the expression of gpx1 and gst antioxidant-related genes and disease resistance against A. veronii in juvenile red tilapia. Therefore, JA5 and JA10 can be employed as promising prebiotics for sustainable red tilapia farming.

Characterization of a vasa homolog in Mekong giant catfish (Pangasianodon gigas): Potential use as a germ cell marker.

For the long-term preservation of the genetic resources of endangered fish species, a combination of germ cell cryopreservation and transplantation can be an effective technique. To optimize these techniques, it is important to identify undifferentiated germ cells possessing transplantability, such as primordial germ cells, type A spermatogonia (ASGs), and oogonia. In this study, a homolog of vasa cDNA in Mekong giant catfish (MGC-vasa) (Pangasianodon gigas), which is an endangered species inhabiting the Mekong river, was cloned and characterized for use as a putative germ cell marker. Results indicate that MGC-Vasa contained all of the consensus motifs, including the arginine-glycine and arginine-glycine-glycine motifs, as well as the nine conserved motifs belonging to the DEAD-box family of proteins. Results from phylogenetic analysis indicated MGC-vasa also grouped with Vasa and was clearly distinguishable from Pl10 in other teleosts. Results from analysis of abundance of mRNA transcripts using reverse transcription-polymerase chain reaction and in situ hybridization performed on immature Mekong giant catfish testis indicated vasa was present specifically in germ cells, with large abundances of the relevant mRNA in spermatogonia and spermatocytes. Sequence similarity and the specific localization of MGC-vasa in these germ cells suggest that the sequence ascertained in this study was a vasa homolog in Mekong giant catfish. Furthermore, vasa-positive cells were detected in prepared smears of testicular cells, indicating that it may be a useful germ cell marker for enzymatically dissociated cells used for transplantation studies.

Early feeding with hyperglucidic diet during fry stage exerts long-term positive effects on nutrient metabolism and growth performance in adult tilapia (Oreochromis niloticus).

The present study aimed to investigate nutritional programming of carbohydrate metabolism in Nile tilapia. Early nutritional intervention stimulus was achieved by feeding fry with high-protein/low-carbohydrate (HP/LC) or low-protein/high-carbohydrate (LP/HC) diet since first feeding for 4 weeks, and the effect of nutritional stimulus on carbohydrate and its related metabolism was evaluated through the adult stage. Our findings indicated that at week 1, LP/HC diet-fed fry had lower levels of mRNA for genes coding gluconeogenesis and amino acid catabolism and higher levels of hk2 (P < 0⋅05). As expected, in adult tilapia, although LP/HC diet-fed fish had poorer growth (end of stimulus), the fish showed compensatory growth. There were permanent effects of early high-carbohydrate (HC) intake on several parameters, including (1) modulating hepatic composition, (2) increased muscle glycogen, (3) lower levels of enzymes involved in amino acid catabolism and (4) higher levels of glycolytic enzymes in glycolysis. Finally, HP/LC diet- and LP/HC diet-fed fish were challenged with different dietary carbohydrate levels. Irrespective of challenging diets, the early HC stimulus had significant effects on adult tilapia by (1) promoting utilisation of glucose, which had protein-sparing effects for better growth, (2) inducting lipogenesis and (3) decreasing amino acid catabolism. Taken together, for the first time, we demonstrated that early HC feeding was effective for positive nutritional programming of metabolism in Nile tilapia (an omnivorous fish). It led to the improvement of growth performance in adult fish associated with early feeding, which is linked to a better ability to use glucose, to induce lipogenesis, and to suppress amino acid catabolism.

Effects of Different Dietary Vegetable Lipid Sources on Health Status in Nile Tilapia (Oreochromis niloticus): Haematological Indices, Immune Response Parameters and Plasma Proteome.

This study aimed to investigate the effects of DLs, including palm oil (PO; an SFAs), linseed oil (LO; n-3 PUFAs) and soybean oil (SBO; n-6 PUFAs) on the health status of Nile tilapia (Oreochromis niloticus) during adulthood. Three experimental diets incorporating PO, LO or SBO were fed to adult Nile tilapia for a period of 90 days, and haematological and innate immune parameters were evaluated. Proteome analysis was also conducted to evaluate the effects of DLs on plasma proteins. The tested DLs had no significant effects on red blood cell (RBC) count, haematocrit, haemoglobin, and total immunoglobulin and lysozyme activity. Dietary LO led to increased alternative complement 50 activity (ACH50), and proteome analysis revealed that PO and SBO enhanced A2ML, suggesting that different DLs promote immune system via different processes. Dietary LO or SBO increased the expression of several proteins involved in coagulation activity such as KNG1, HRG and FGG. Increased HPX in fish fed with PO suggests that SFAs are utilised in heme lipid-oxidation. Overall, DLs with distinct fatty acids (FAs) affect several parameters corresponding to health status in Nile tilapia, and dietary LO and SBO seemed to strengthen health in this species.

Aging- and temperature-related activity of spermatogonial stem cells for germ cell transplantation in medaka.

Spermatogonial transplantation can contribute to developing a novel method of producing seedlings for both aquaculture and biotic conservation. This study's purpose was to investigate aging- and temperature-related changes in the numbers and stem cell functions of type-A spermatogonia (ASG) in the model fish medaka (Oryzias latipes). The ASG numbers in medaka of different ages were quantified via histological observation and enzymatic dissociation of vasa-Gfp medaka testes. The ASG numbers were higher in eight-month-old medaka (maturation) than in four-month-old medaka (the onset of maturation). However, ASG numbers decreased in 18-month-old medaka (senescence). Low water temperature appeared to slow down both testis development and aging processes. To study the effects of aging on ASG stem cell activity, testicular cell suspensions containing GFP-expressed ASG were prepared from vasa-Gfp medaka donors at 4 and 18 months of age and transplanted into recipient hybrid larvae of medaka (O. latipes x O. curvinotus), which provided young stem-cell-niches. The findings revealed no significant differences in ASG colonization rates isolated from medaka of different ages. Each group displayed similar rates of germ-line transmission. Furthermore, water temperature had no significant effects on each ASG's stem cell activity. Taken together, these results indicated that aging and temperature affect ASG numbers. However, ASG isolated from medaka with different ages were transplanted into gonads with a young niche microenvironment, and there was no evidence of donor aging on stem cell activity.

Glucose Injection Into Yolk Positively Modulates Intermediary Metabolism and Growth Performance in Juvenile Nile Tilapia (Oreochromis niloticus).

The aim of this study was to explore for the first time in omnivorous fish the concept of nutritional programming. A nutritional stimulus was accomplished by microinjecting 2 M glucose into yolk reserves during the alevin stage in Nile tilapia (Oreochromis niloticus). At the molecular level in fry, at 1 week post-injection, glucose stimuli were associated with the up-regulation of genes involved in glycolysis (pklr, hk1, hk2, and pkma), glucose transport (glut4) pathways and down-regulation of genes related to gluconeogenesis (g6pca1, g6pca2, and pck1) and amino acid catabolism (asat, alat) (P < 0.05), demonstrating that the larvae well received the glucose stimulus at a molecular level. Moreover, 20 weeks after glucose injection, early glucose stimuli were always linked to permanent effects in juvenile fish, as reflected by a higher level of glycolytic enzymes [gck, hk1 and hk2 at both mRNA and enzymatic levels and pyruvate kinase (PK) activity]. Finally, the effects of the glucose stimulus history were also examined in fish fed with two different dietary carbohydrate/protein levels (medium-carbohydrate diet, CHO-M; high-carbohydrate diet, CHO-H) in juvenile fish (during weeks 20-24). As expected, the CHO-H diet induced the expression of glycolytic and lipogenic genes (gck, pklr, hk1, hk2, fpkma, fasn, and g6pd) and suppressed the expression of gluconeogenic and amino acid catabolism genes (g6pca1, pck1, pck2, asat, alat, and gdh). Nevertheless, the early glucose stimulus led to persistent up-regulation of glycolytic enzymes (gck, pklr, hk1, and hk2) at both the mRNA and enzyme activity levels and glucose transporter glut4 as well as lower gluconeogenic pck1 gene expression (P < 0.05). More interestingly, the early glucose stimulus was associated with a better growth performance of juvenile fish irrespective of the diets. These permanent changes were associated with DNA hypomethylation in the liver and muscles, suggesting the existence of epigenetic mechanisms at the origin of programming. In conclusion, for the first time in tilapia, early glucose stimuli were found to be clearly associated with a positive metabolic programming effect later in life, improving the growth performance of the fish.

Transcriptomic analysis of female and male gonads in juvenile snakeskin gourami (Trichopodus pectoralis).

The snakeskin gourami (Trichopodus pectoralis) exhibits sexual dimorphism, particularly in body size. Since the snakeskin gourami is usually marketed during sexual maturation, the sexual size dimorphism has become an economically important trait. Sex-biased gene expression plays a key role in phenotypic sexual dimorphism. Therefore, using high-throughput RNA sequencing (RNA-seq) technology, we aimed to explore the differentially expressed genes (DEGs) in ovary and testis during sex differentiation in juvenile snakeskin gourami. Our results revealed a number of DEGs were demonstrated to be overexpressed in ovary (11,625 unigenes) and testis (16,120 unigenes), and the top 10 female-biased (rdh7, dnajc25, ap1s3, zp4, polb, parp12, trim39, gucy2g, rtbs, and fdxr) and male-biased (vamp3, nbl1, dnah2, ccdc11, nr2e3, spats1, pih1d2, tekt3, fbxo36, and mybl2) DEGs were suggested to be mainly associated with ovary and testis differentiation, respectively. Additionally, using real-time reverse transcription polymerase chain reaction (qRT-PCR), validation of the differential expression of 21 genes that were previously shown to be related to gonad development was performed (ar, bHLH, cyp19a1, daz, dead-end, esrb, esrrg, gnrhr, gpa, gsg1l, hsd17B, mospd1, nanos-1, nanos-2, p53, piwi-1, piwi-2, rerg, rps6ka, tgf-beta, and VgR). The results showed a significantly positive correlation (0.84; P < 0.001) between the results of RNA-seq and qRT-PCR. Therefore, RNA-seq analysis in our study identified global genes that were associated with ovary and testis differentiation in the juvenile phase of the snakeskin gourami. Our findings provide valuable transcriptomic bioinformation for further investigation of reproductive biology and applications of sex manipulation.

Enrichment of transplantable germ cells in salmonids using a novel monoclonal antibody by magnetic-activated cell sorting.

In the fish germ cell transplantation system, only type A spermatogonia (ASGs) and oogonia are known to be incorporated into the recipient genital ridges, where they undergo gametogenesis. Therefore, high colonization efficiency can be achieved by enriching undifferentiated germ cells out of whole testicular cells. In this study, we used magnetic-activated cell sorting (MACS) for enriching undifferentiated germ cells of rainbow trout using a monoclonal antibody that recognizes a specific antigen located on the germ cell membrane. We screened the antibodies to be used for MACS by performing immunohistochemistry on rainbow trout gonads. Two antibodies, nos. 172 and 189, showed strong signals for ASGs and oogonia. Next, we performed MACS with antibody no. 172 using gonadal cells isolated from vasa-gfp rainbow trout showing GFP in undifferentiated germ cells. We found that GFP-positive cells are highly enriched in antibody no. 172-positive fractions. Finally, to examine the transplantability of MACS-enriched cells, we intraperitoneally transplanted sorted or unsorted cells into recipient larvae. We observed that transplantability of sorted cells, particularly ovarian cells, were significantly higher than that of unsorted cells. Therefore, MACS with antibody no. 172 could enrich ASGs and oogonia and become a powerful tool to improve transplantation efficiency in salmonids.

Characterization and expression of a vasa homolog in the gonads and primordial germ cells of the striped catfish (Pangasianodon hypophthalmus).

The analysis of early gonadogenesis during larval development requires a molecular marker that is specifically expressed in the germ cell lineage, such as the vasa gene. In this study, we cloned and characterized vasa in the striped catfish (Pangasianodon hypophthalmus), and designated this as Phy-vasa. Phy-vasa contained all of the predicted consensus motifs that are shared among the vasa genes in other fish species, including RG and RGG repeats, ATPase motifs, and a DEAD-box, and phylogenetic analysis using various DEAD-box family proteins demonstrated that the Phy-vasa protein clustered within the Vasa family. Reverse transcription polymerase chain reaction (RT-PCR) indicated that Phy-vasa mRNA only occurred in the testis and ovary, and in situ hybridization showed that the gene was expressed only in the germ cells, with strong expression in the spermatogonia and oogonia. To investigate early gonadogenesis in catfish larvae, we undertook histological characterization and in situ hybridization using a Phy-vasa probe, which showed that migration of the primordial germ cells (PGCs) most commonly occurred in larvae at 2-10 days post-fertilization (dpf), the PGCs started to be surrounded by gonadal somatic cells at around 10-20 dpf, and rapid proliferation of the PGCs had begun by 30 dpf. These findings provide a valuable insight into early gonadal development in the striped catfish.

Effects of Microencapsulated Saccharomyces cerevisiae on Growth, Hematological Indices, Blood Chemical, and Immune Parameters and Intestinal Morphology in Striped Catfish, Pangasianodon hypophthalmus.

This study investigated the effects of dietary probiotic Saccharomyces cerevisiae in the striped catfish, Pangasianodon hypophthalmus, which is an important aquaculture species. Freeze-dried microencapsulated probiotic S. cerevisiae with guar gum was performed and used for fish feed supplementation. Striped catfish were fed for 120 days with one of three experimental diets: basal diet (control), basal diet supplemented with 106-CFU S. cerevisiae g-1 diet (S. cerevisiae 106), and basal diet supplemented with 108-CFU S. cerevisiae g-1 diet (S. cerevisiae 108). The S. cerevisiae-supplemented diets significantly improved growth performance including growth rate and feed conversion ratio over 120 days of culture period (P < 0.05). The rate of survival was similar in all experimental groups. Supplementation with S. cerevisiae did not significantly affect whole body proximate composition (P > 0.05). In addition, probiotic S. cerevisiae had no effects on hematological indices and blood chemistry values (glucose, cholesterol, triglycerides, protein, albumin, blood urea nitrogen, chloride, calcium, magnesium, iron, and phosphorus) (P > 0.05). However, dietary S. cerevisiae led to increases in humoral immune parameters including total immunoglobulin, lysozyme, and alternative complement activities (P < 0.05). Dietary S. cerevisiae led to increase intestinal villus height in the anterior part of intestine (P < 0.05). Taken together, while the dietary S. cerevisiae had no detectable effects on hematological indices and several metabolic indicators, significant beneficial probiotic effects were observed on rates of growth, feed conversion ratio, and immune parameters.